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the lowest excited state of singlet oxygen , o2 ( g ) , has become in the last few years a precious chemical . its versatility and physical and chemical characteristics have opened the door to a wide range of application fields . for instance , properties such as its stereoselectivity are exploited for the synthesis of oxygen - containing fine organic chemicals , whereas its extreme reactivity and oxidation power are key to its use in the decontamination of waters or in medical and environmental photodynamic processes including the sterilization of blood or plasma samples and cancer therapy . moreover , the particularly long radiative lifetime of this excited state and its excess of energy relative to the ground state ( gs ) , resonant to iodine atoms , allows its use in laser technology for the production of excited iodine atoms via energy transfer . this great heterogeneity of scenarios has motivated the quest for the search of new methods for its synthesis , specifically adapted to the conditions and requirements of the different contexts where this species needs to be produced . attending to the nature of the force that drives the o2 generation mechanism , these protocols can be classified into physical or chemical . physical processes produce o2 from the direct excitation of molecular oxygen with photons of different wavelengths ( i.e. , radiofrequency or ir ) . chemical methods that can be mediated or not by light , however , involve the participation of other reagents that chemically evolve to o2 or that alternatively act as intermediates for the storage of energy that is then transferred to molecular oxygen in its gs , leading to the desired excited product . decomposition of polyoxygenated species such as ozonides , endoperoxides , peroxyacetyl nitrate , or superoxide ions stand out as important chemical sources of o2 in the absence of light . the following reactions of hydrogen peroxide : i(ref ( 11))ii(ref ( 12))iii(ref ( 13))iv(haber - weiss reaction)or the self - reaction of alkylhydroperoxidesv(russell reaction ) have been also reported as efficient chemical methods for producing o2 in the dark . interestingly , some of these reactions have been as well proposed to be responsible for the generation of o2 in biological systems . in fact , reactions ( i ) and ( iv ) have been postulated as part of the defense strategy occurring during phagocytosis , and lipid hydroperoxides generated along oxidative stress processes connected to diseases such as atherosclerosis were found to decompose with the participation of a metal cation catalyst or enzymes following reaction ( v ) , leading to o2 . conventional o2 reactions initiated by light require the excitation of a sensitizer , showing preferably an important yield for intersystem crossing ( isc ) and characterized by long - lived triplets . once the triplet state of the photosensitizer has been populated , an energy - transfer process occurs during the collision of this species with surrounding gs molecular oxygen molecules that leads to the generation of o2 and the recovery of the sensitizer in its initial gs ( type ii mechanism of photosensitization ) . obviously , keeping the concentration of environmental oxygen molecules constant and high is a key factor for the success of these techniques but can , however , pose problems for particular applications where the oxidant needs to be generated in oxygen depleted conditions . this is for instance the case of solid or vascular damaged tumors where oxygen is not able to reach their interior . in these cases , the efficacy of conventional photosensitizing reactions is expected to be low and thus alternative photosensitive oxygen carriers , able to release singlet oxygen upon their activation with the correct wavelength , have been specifically designed for this purpose . an example of a prototype of oxygen carrier photosensitizer is the tetraantraporphyrazine proposed by freyer and leupold , which combines the virtues of tetrapyrrole standard photosensitizers ( i.e. , absorption maxima in the red / nir region of the electromagnetic spectrum , etc . ) with the possibility to eject o2 regardless the concentration of molecular oxygen in the tissues , thanks to the four anthraceneendoperoxide ( apo ) moieties with which the porphyrazine core is substituted . a model to delve in the understanding of how o2 alone , without considering other reactive oxygen species , participates in cell damaging in photodynamic processes . the photochemistry of endoperoxides has also been investigated in detail from both theoretical and experimental standpoints . it has been demonstrated that o2 is not the only photoproduct that results from the interaction of endoperoxides with uv photons ( see scheme 1 ) . in addition to cycloreversion or the breaking of the pair of c o bonds that leads to the aromatic hydrocarbon + o2 , competing o o homolysis ( recall scheme 1 ) is responsible of diverse photoproducts , such as quinones , acetals , or diepoxides . although the aromatic moiety influences greatly the absorption spectrum of endoperoxides , a common feature to this class of compounds is the nature of the electronic states leading to the two main photochemical decomposition pathways . while * states are responsible for cycloreversion , * states can lead to o o homolysis . this work presents the first full - dimensional dynamical study of a model endoperoxide , the cyclohexadieneendoperoxide ( chdepo , c6h6o2 ) system , which shows an eight - fold degeneracy region ( 8ci ) in the potential energy surface ( pes ) , where four singlets and four triplets are degenerate . this time - resolved picture , complemented with key frames extracted from quantum chemistry calculations , sheds light on the two competing mechanisms that account for o o homolysis and the cleavage of c o bonds , leading to o2 . particular key questions that will be addressed are ( i ) the mechanism by which o2 is produced from endoperoxides , ( ii ) the degree of competition between o2 generation and o o homolysis , and ( iii ) the role played by the triplets in the photochemistry of these systems . stationary points and conical intersections ( ci ) were optimized at the casscf level of theory , employing an ano - s basis set , contracted for h as [ 2s1p ] and for c , o as [ 3s2p1d ] . unless otherwise specified , the active space used throughout the calculations includes the complete valence space ( two pairs of cc/*cc and oo/*oo ) and the orbitals sitting on the oxygens co/*co ( two pairs ) and oo/*oo , necessary to properly account for o o homolysis and cycloreversion ; this comprises altogether a total of 14 electrons distributed into 12 orbitals , ( see figure s1 ) . stationary points in the first singlet potential were optimized using a single root , whereas in the case of cis2/s1 and cis1/s0 ( see below ) state average casscf calculations over three and two roots were used , respectively . condon region was ensured by computing minimum energy paths ( meps ) at the same level of theory as specified above with a 6 - 31 g * basis set and using the minimum number of roots necessary , i.e. , equal to the root numbering of the gradient followed . final energies were calculated as state average over four singlet and four triplet states at ms - caspt2//casscf(14,12)/ano - rcc on the optimized geometries . the contraction scheme for the ano - rcc is h [ 3s2p ] and for c , o as [ 4s3p2d ] . unless otherwise specified , all the calculations were performed with the 76 version of the molcas program . for completeness and consistency , since the basis set and active spaces used herein differ from previous studies , we have recomputed particular regions of the o o homolysis mep already discussed in other works , following the casscf(14,12)/ano - rcc protocol and calculated from the scratch others , necessary to interpret the dynamical results . orbit couplings ( soc ) were simulated using tully s fewest switches surface hopping scheme ( see supporting information for further details ) , as described in the sharc method . nuclei are treated classically and follow newton s equations , whereas electrons are treated quantum mechanically . for the integration of newton s equations the evolution of the probability amplitudes determining the contribution of the different adiabatic states to the total wave function was integrated using the fourth order runge kutta algorithm with a time step of 10 fs . a decoherence correction was applied , as recommended by granucci and persico with the parameters c = 1 and = 0.1 hartree . these parameters ( c and ) rescale the amplitudes of the electronic wavepacket after each nuclear time step , accounting for the evolution of hypothetical trajectories running in other electronic states along other gradients different to that of the current electronic state . previous studies on three - dimensional atom - molecule systems have highlighted the importance of accounting for decoherence effects but have also demonstrated that the precise value of these parameters has a small influence in the dynamics when comparing with quantum approaches . however , other larger systems as polyconjugated organic molecules have been demonstrated to be much more sensitive to their variation . for the simulation of the uv spectrum , a set of 1000 initial uncorrelated geometries and velocities was generated according to a wigner harmonic distribution of the lowest vibrational state of the ground electronic state , taking as input an harmonic frequency calculation at the casscf(14,12)/6 - 31 g * level of theory . casscf / ano - rcc and caspt2 spectra , considering the first four singlet states , were constructed from a superposition of gaussians with the maximum height modified according to the oscillator strength of the transitions and sitting at the position of the vertical excitation energies computed at these two levels of theory ( width of the gaussian = 0.1 ev ) . for comparison , the casscf and ms - caspt2 vertical absorption spectra of chdepo were calculated following the same protocol specified above , using the casscf/6 - 31 g * optimized geometry as a reference . from the initial ensemble of 1000 geometries , a subset of 78 initial conditions , concentrated in an energy window of 0.15 ev centered around the absorption maximum at 4.65 ev , were selected , based on their oscillator strengths . for these trajectories , energies and gradients for the first four singlets and triplets were computed on - the - fly using the casscf(14,12)/ano - rcc protocol as implemented in molcas package . after the hop events the kinetic energy was adjusted with the goal to conserve the total energy of the system , scaling the atom velocities along their current direction . finally , and unless otherwise specified to avoid the artificial elongation of the c h bonds due to the failure of the harmonic approximation , dynamics simulations were performed substituting hydrogen atoms for deuterium , as suggested elsewhere . the vertical casscf and caspt2 absorption spectra of chdepo the casscf method predicts the brightest transitions ( * ) above 8 ev , not shown in table 1 . the low - energy region of the casscf spectrum is in turn dominated by two weaker absorptions at 5.0 and 6.0 ev , showing a mixed oo*cc/*oo*oo character . in contrast to the casscf spectrum , the most intense bands ( * ) are concentrated in the region around 5.5 ev , whereas two transitions governing the lowest energy region of the spectrum are calculated below 5 ev at 3.9 and 4.7 ev , the second four times more intense than the first one . similarly to casscf , caspt2 predicts a strong *oo*cc/*oo*oo mixing for the s1 and s2 electronic states . subtle differences with previous works are attributed to small changes in the reference geometry and the basis set . figure 1 displays the position of the casscf and caspt2 vertical excitations ( black and red vertical lines below 8 and 6 ev , respectively ) , superimposed to the casscf and caspt2 spectra based on the 1000 geometries generated to mimic the nuclear wavepacket ( solid black and red spectra ) . both spectra consist of an intense band , showing a shoulder at higher energies , preceded by a weaker absorption . consistently with what is observed for the vertical spectrum , we find that casscf overestimates by a factor of 0.25 the absorption energies , taking caspt2 as a reference ; compare the position of the least ( 4.8 vs 3.8 ev ) and most energetic bands ( 6.2 vs 4.7 ev ) at casscf and caspt2 . a decomposition analysis of these bands in terms of the contributing states ( see figures s6 and s7 ) reveals that the weakest band mainly results from the first excited state in both spectra , whereas the s2 and s3 states are the responsible for the principal band , increasing the s3 its contribution to the main band after including dynamic correlation . the pathological overestimation of excitation energies by casscf at the fc region , as compared with caspt2 calculations , could be detrimental for the dynamics , leading to undesired photoproducts due to the excess of energy accumulated by the starting ensemble of initial geometries . however , taking into account that caspt2 analytical gradients are not available in molcas and that the use of caspt2 numerical gradients is computationally unaffordable for a study as the one suggested here , we opted for a common solution previously adopted by other authors that consists in the scaling of the energies and gradients ( see supporting information for more details ) . black solid line corresponds to the spectrum calculated using sa4-casscf(14,12)/ano - rcc level of theory , while red solid line represents the results corrected using the ms - caspt2 method . black dotted line outlines the casscf scaled spectrum ( casscf ) . to quantify the impact of such approximation , we have scaled by a factor of 0.75 the casscf spectrum and compare it with the caspt2 one . the almost complete superposition of the red solid line and the dotted black line in figure 1 denotes a very good agreement between the scaled casscf and the caspt2 results at the franck a similar assessment of the impact of the scaling was performed along the mep , taking as a reference the gs equilibrium geometry . guide the interpretation of the dynamical results and to assess the quality of the method used in the on - the - fly electronic structure calculations , we have investigated the mep connected to both o o homolysis and cycloreversion with the casscf method . since the reaction paths are independent of the nuclear masses , all the static calculations were performed on hydrogenated chdepo . more reliable caspt2 calculations were performed at the stationary , critical , and intermediate points along the mep where the comparison with caspt2 benchmark values was found to be critical for the dynamics . interestingly , casscf and caspt2 provide pes in qualitatively good agreement , at least for the regions relevant to the photochemistry of these systems . the scaling of the casscf energies along the two meps was found to reduce the slope of the profiles mimicking the caspt2 result but also to decrease the energy barriers . however , since casscf provides similar reaction barriers as caspt2 and the system has only to face these barriers at the gs pes , the scaling of the energies was suppressed once the trajectory deactivates to the gs . thus , overall , we expect the scaling to have a small effect on the time scales of the two photochemical processes studied but not to influence the mechanism or product distributions . figure 2 shows the scheme proposed for o o homolysis mechanism based on the casscf mep calculated following the gradient of the second root ( i.e. , first excited state ) . similarly to previous calculations for the same and other endoperoxides , the mep from the franck condon region leads barrierlessly to an energetically accessible high degeneracy point of four singlet states ( 4ci ) , among which the gs is included . structurally speaking , this corresponds to a point of the pes where the system presents an internuclear o o distance at which the distribution of six electrons into the oo , *oo , oo and *oo orbitals leads to four different configurations energetically indistinguishable . extrapolating from other previous dynamics works studying nonadiabatic dynamics across more than two state degeneracy points , deactivation through this particular funnel is expected to be achieved on very short time scales due to the occurrence of large regions of seams of three and two degenerate states that would significantly enhance the population transfer toward the gs . global static picture of the o o homolysis mechanism of chdepo based on mep calculations ( from this work and ref ( 21e ) ) . final energies relative to the gs minimum ( in ev ) were calculated at ms - caspt2//sa4-casscf(14,12)/ano - rcc level of theory . this high - order degeneracy point of the pes was previously shown to be connected with four different diradical minima in the gs pes approximately of the same stability . starting from the most stable , minbryy , we have estimated in 0.7 ev the energy barrier , tsh2 , that requires the breaking of the two ch bonds sitting at the endoperoxide carbons , leading to the formation of the benzoquinone and molecular h2 photoproducts . these photoproducts have been identified as the most stable for chdepo and the only ones observed from our dynamics simulations following o o homolysis mechanism , see below . condon region following the gradient of the brighter second excited state ( third root ) . similarly to the mep from the first excited state in figure 2 , this path proceeds showing neither minima nor energy barriers toward a ci with the gs , see figure 3 . on the way to the gs , however , a ci s2/s1 with the second root is also found that might deviate population to the lower lying state . the s1/s0 conical intersection is expected to bifurcate population between two minima in the ground pes , i.e. , the franck condon minimum ( minfc ) and minsw . along the mep coordinate , we observe the stretching of one of the two c o bonds that increases from a 1.472 value at the fc geometry to 3.901 at the position of the minsw , corresponding to the intermediate along the stepwise cleavage of the endoperoxide bridge . logically , the rupture of one of the two c o bonds is concomitant to the progressive recovery of the planarity of the hydrocarbon moiety , due to the redistribution of electronic density among all the c of the ring . o bond still separates the population reaching minsw from the final cycloreversion photoproducts , benzene + o2 ( recall scheme 1 and figure 3 ) . starting from this minimum , the breaking of the second c o bond involves overcoming an energy barrier of 0.3 ev to reach a predissociation minimum , mino2 , where the hydrocarbon and oxygen are weakly interacting through van der waals forces . the height of the barrier ( 0.3 ev ) , much lower than the initial franck condon energy ( 4.72 ev ) , is not expected to prevent the formation of the cycloreversion products along the dynamics . the probability to populate the triplets along both o o homolysis and cycloreversion pathways was evaluated by computing the soc at selected points of the pes . especially relevant are the values of the couplings calculated at the position of the 4ci that amount to 70 cm or at the region of the pes corresponding to minimum mino2 , where the soc increases up to 180 cm . global static picture of the cycloreversion mechanism of chdepo based on mep calculations . final energies relative to the gs minimum ( in ev ) were calculated at ms - caspt2//sa4-casscf(14,12)/ano - rcc level of theory . other information on the mep calculations can be found in the supporting information . although very mixed at the fc region , from the inspection of the fate of the meps constructed along the gradient of the two lowest lying excited states , it could be inferred that the character of the two first excited states is respectively *oo*oo and *oo*cc , which is also consistent with the oscillator strengths computed vertically . in summary , the static picture described above reveals that the two proposed deactivation mechanisms for chdepo , o o homolysis and cycloreversion , are likely to take place simultaneously upon uv excitation . in both scenarios , the system would reach barrierlessly a gs minimum , minbryy / minsw , from which a small energy barrier separates the final photoproducts . the following dynamics simulations will help elucidating additional details on the deactivation mechanisms as well as the final ratio of different photoproducts . figure 4 shows the time evolution of the four singlet ( s0 , s1 , s2 , and s3 ) and four triplet states ( t1 , t2 , t3 , and t4 ) population of the 78 trajectories propagated , created using deuterated chdepo , along 100 fs . although the propagation was done in 16 spin orbit states , arising from the diagonalization of the total hamiltonian , for simplicity the analysis will be done on the spin - free states , where the electronic wave function was projected back into the initial singlet and triplet states . initially , the trajectories are distributed according to a 65:35 ratio between the s2 and s3 electronic states . this translates into a mixture of *oo*oo and *oo*cc states , where the *oo*cc states are dominant ( 85% vs 15% ) . interestingly , 30 fs only after photoexcitation , the population of the initially populated states ( s2 and s3 ) rapidly decays in favor of the s1 and s0 states . this fast decay is perfectly consistent with the steep meps computed for o o homolysis and cycloreversion mechanisms , which do not predict any barrier on the way to the population of lower lying electronic states . at t = 50 fs , the population of the four singlets becomes equal and oscillates for 10 fs around an average value of 0.2 , which is compatible with a situation of the wavepacket exploring the region of the pes corresponding to the 4ci . from 60 fs onward , the population of the s0 grows momentarily slightly larger than for the other singlets to decrease again at the final time of the propagation . at the final time of the simulations , the total population is distributed as 60% in the singlet and 40% in the triplet manifold , with all the electronic states within each manifold carrying approximately the same population . a particularly interesting observation is that the triplet character of the trajectories shows up at very early times of the simulation , i.e. , below 20 fs , and that the maximum total expected population of the triplets is achieved already at a t = 50 fs . this observation is in line with the conclusions drawn in other works that support that isc in organic molecules can be ultrafast even if no heavy elements are present . in order to determine the final distribution of photoproducts derived from o o homolysis and cycloreversion processes , we have followed the evolution of the distance between the centers of mass of the benzene and o2 moieties , dbenz this distance is expected to oscillate around small values for the trajectories evolving via the o o homolysis mechanism , whereas for cycloreversion this value is expected to increase gradually as the two co bonds are cleaved . time evolution of the average quantum probability of singlet ( s0 in red , s1 in green , s2 dark blue , s3 in pink ) and triplet ( t1 in light blue , t2 in yellow , t3 in black , t4 in gray ) states . trajectories leading to o o homolysis , in blue the ones producing b+o2 and other products in red and green . percentages for the different products are specified . according to this criterion , we have classified all the trajectories into four main groups . 2 , which corresponds to the distance between o2 and benzene centers of mass at the optimized casscf gs geometry . the first group of trajectories , denoted in black in figure 5 , is characterized by a progressive diminishing of the dbenz oo distance until reaching the value of zero ; this corresponds to a structure where the two oxygens , although still bonded to their adjacent c atoms , lie at the largest possible distance , coplanar with the rest of the atoms of the ring . after t = 80 fs , this distance again increases until reaching a slightly smaller value than the initial one . these trajectories , which represent a 63% of the total , have been ascribed to the o o homolysis mechanism and perfectly describe the oscillating bending movement that the system experiences as the endoperoxide bond is broken and the 8ci degeneracy point is reached . these trajectories will end up in any of the four theoretically predicted minima , characterized by a dbenz next group of trajectories , distinguished in blue in figure 5 , are characterized by a linear increase in the dbenz oo distance with time , reaching a maximum value of 6 at the final time of the simulation . these trajectories , which amount to 10% , correspond to cycloreversion , leading to benzene and o2 as final products . another 20% of the trajectories , in green in figure 5 , evolve to a structure in which both the endoperoxide and a single c o bonds are dissociated . a similar situation is observed for the remaining 4% of the trajectories , highlighted in red in figure 5 , which show the simultaneous increase of both the o o and the two c o distances . from the very high energy expected for these processes , we could infer that the employed ab initio methodology is not able to correctly describe this region of the pes and significantly underestimates the energy barrier for the dissociation of two or more bonds simultaneously . in principle , the shape of the potential energy profiles for cycloreversion depicted in figure 3 shows energy barriers flanking minsw , that would favor the evolution of the system toward the formation of cycloreversion products , rather than reverting to the initial gs or o o homolysis products , and the structural evolution of the red and green trajectories in figure 5 parallel to the blue group of trajectories would justify directly imputing these later trajectories to cycloreversion ( in blue ) . with this more logical assumption , a final total yield for cycloreversion of ca . 30% is obtained , in line with the experimental observations for the larger endoperoxide , apo . an analysis of the multiplicity at the final point of the propagation for different groups of trajectories reveals that no cycloreversion products are formed in the triplet manifold ( blue trajectories in figure 5 ) . however , ca . 50% of the trajectories leading to o o homolysis end up in a triplet state . this is compatible with the existence of the 8ci along the rupture of the endoperoxide bridge . also interesting is the fact that none of the trajectories revert to the starting point of the simulation , leading to a 100% yield of photoproducts , also in line with the experiments in refs ( 22b and 22c ) . although we have evidence that birradical minima are formed from the o o homolysis mechanism along the dynamics , quite unexpectedly , none of these trajectories was found to lead to the final products , i.e. , benzoquinone + d2 during the 100 fs propagation time . we attribute these results to the combination of dynamic effects and the use of a reduced active space . in principle , the correct and simultaneous description of both o o homolysis and cycloreversion processes would require that the active space includes a pair of co , *co , ch , and *ch orbitals . after including the remaining orbitals from the ring and and orbitals of the endoperoxide , such an active space would necessarily increase its size to 16 orbitals , which is computationally prohibitive for a dynamical study , such as the one proposed here . however , since the inclusion of the ch and *ch orbitals is decisive for a correct description of the tsh2 structure , lacking these orbitals most likely overestimates the energy barrier connected to the loss of h2 , hindering the evolution of the trajectories toward the final photoproducts . further dynamical effects might also influence the output of this product in the dynamics . the formation of h2 involves on the one hand the concerted cleavage of the two ch bonds and , on the other , the in phase bending vibration of the two oc1c2 angles , where c1 and c2 stand for the c atoms holding the endoperoxide bridge . this bending mode would allow the symmetric puckering of the hydrocarbon ring and the h atoms to encounter . in other words , the momenta of the oxygen and h atoms should point in opposite directions to direct the approach between the two h atoms . this precise alignment of the momenta of o and h atoms might need longer time scales than the ones allowed here , partially explaining the absence of trajectories leading to the final products bq + h2 . in fact , an exemplary trajectory propagated during additional 80 fs demonstrates the formation of h2 ( see next section ) . o homolysis and cycloreversion mechanisms will be provided in the next analysis of representative trajectories section . in this section , several trajectories representative for the o o homolysis and cycloreversion photoreactions will be discussed in detail . figures 6 and 7a , respectively , exemplify the generation of o2 and h2 from excited chdepo . for the description of cycloreversion ( figure 6 ) , we have chosen two trajectories with chdepo initially photoexcited to the s3 ( panel a ) and the s2 ( panel b ) . the trajectory in panel a , illustrates the generation of o2 in the excited state followed by gs relaxation , whereas the trajectory in panel b , is representative for the other trajectories where the dissociation of o2 takes place in two sequential stages : the first in the excited state and the second in the gs , in agreement with the static picture described in figure 3 . time evolution of two representative trajectories of deuterated chdepo leading to b + o2 products . singlet states are represented by solid lines , ( s0 in red , s1 in green , s2 in blue , and s3 in pink ) , while triplet states are denoted with dotted lines in light blue t1 , yellow t2 , black t3 , and gray t4 . the trajectory of figure 6a relaxes to the s2 very fast after 10 fs . the cleavage of one of the two c o bonds induces the degeneracy of the s3 and s2 electronic states , promoting the backward hop to the upper electronic state 10 fs later . during the following 40 fs the system evolves in the s3 , where it experiences the dissociation of the second c o bond . in this region of the pes , we observe the degeneracy between the s3 and the second triplet state ( t2 in yellow ) . however , no isc is observed at this point of the propagation . for t = 60 fs , the system hops back to the s2 state , where it remains for 15 fs more and then finally decays to the s0 . this last stage of the mechanism structurally translates into the distancing of the o2 molecule from the hydrocarbon moiety . the second trajectory in figure 6b relaxes as well via internal conversion within the first 10 fs to the immediately below excited state , s1 , where it spends the following 10 fs , until it reaches a new internal conversion funnel to the s0 . the former two interstate crossings can be readily identified with the cis2/s1 and cis1/s0 conical intersections optimized along the static calculations , see figure 3 . at t = 20 fs , the oxygen moiety is bonded to the hydrocarbon through a single c o bond , while the other has been dissociated on the way to the gs . an increase of the potential energy for the s0 is observed upon the dissociation of the remaining c o bond . finally , an additional barrier needs to be overcome for the dissociation of the weak van der waals complex , in agreement again with the topology of the potential energy profiles depicted in figure 3 . from the careful analysis of the electronic structure of the final benzene and oxygen moieties and the degeneracy ( double ) of the singlet electronic states where the system is at the final time of the propagation for all the trajectories leading to o2 , we infer that the hydrocarbon and o2 are generated in their ground and g electronic state , respectively . also interesting is the fact that the electronic state reached at the end of the propagation does not correspond to the most stable , since there is at least another triplet of lower energy ( t1 ) . in spite of the significant soc computed along this mechanism and the occurrence of degeneracy regions between singlets and triplets , no isc was observed along none of the trajectories evolving according to the cycloreversion mechanism . we ascribe this negligible role of the triplets in the cycloreversion mechanism to the topology of the singlet and triplet potentials along the global cycloreversion reaction coordinate . in fact , the only region of the pes where isc is likely to occur , i.e. , where close lying triplet states ( see figure 6b ) and considerable spin - orbit coupling ( 60 cm ) exist , is at the position of the minsw minimum , where the system could be trapped . however , the orientation of the momentum of the trajectories undergoing cycloreversion in the direction of the access to the transition state tso2 prevents the confinement of the trajectories in this region of the pes long enough time for the system to reach the triplet manifold . the trajectory selected for describing o o homolysis ( figure 7 ) was created using hydrogenated chdepo . this trajectory starts from the second excited state , s2 , and evolves very rapidly ( in 20 fs ) following a very steep potential to the 8ci region , consistently with the static results discussed above , recall figure 2 . during this time , the system starts dissociating the endoperoxide bridge . once in the high degeneracy region , the system spends several tens of fs visiting different electronic states , including triplets . the gs is reached for the first time only 25 fs after the trajectory is initiated , supporting the findings of previous work , stating that high order degeneracy points constitute extremely efficient deactivation funnels to the gs . from a structural viewpoint , during its journey along the 8ci , the endoperoxide bond continues dissociating at the same time that the two d atoms sitting at the carbons holding the endoperoxide bridge move closer . since for none of the trajectories computed , the two d atoms succeeded in forming molecular h2 , the opposite movement , restoring the endoperoxide bridge while separating the d atoms , was observed . for some trajectories , this oscillatory movement was repeatedly observed until the final time of the simulations . in order to study the last stages of the o o homolysis mechanism , an additional trajectory based on initial conditions generated using h instead of d was propagated for a longer time . since the short ch distances of the structure for t = 100 fs did not allow introducing the ch and *ch orbitals , we decided to propagate further the trajectory with the smaller active space ( 10,8 ) excluding ch and co orbitals and the 6 - 31 g basis set . this figure also presents the optimized geometry for the tsh2 , accounting for the concerted dissociation of the two ch bonds leading to h2 , that is similar to the geometries recorded at t = 140 and 150 fs . as concluded from the sequence of frames of this trajectory , the generation of h2 from the preceding biradicals requires ca . 100 more fs either to evolve the active space so as to exchange other less important valence orbitals for the ch and *ch orbitals or to put in phase the momenta of h / o atoms to correctly describe h2 dissociation . ( a ) time evolution of a representative trajectory leading to bq + h2 products . singlet states are represented as solid lines , ( s0 in red , s1 in green , s2 in blue , and s3 in pink ) , while triplet states are denoted with dotted lines in light blue t1 , yellow t2 , black t3 , and gray t4 . the vertical dotted line in black in panel a indicates the point when the trajectory reaches the s0 and the energy scaling is switched off . ( b ) snapshots for longer propagation times and the optimized geometry of the tsh2 for comparison . note that for this trajectory deuterium atoms where replaced by hydrogens ( see text ) . this work presents the first complete analysis , from both static and dynamic viewpoints , of the photophysics and photochemistry of an endoperoxide . to this aim o homolysis , leading to benzoquinone + h2 , and cycloreversion , generating benzene and o2 as photoproducts . the static and dynamic results are consistent in describing both pathways consisting of two steps . the first step is a barrierless deactivation to gs intermediates : minsw ( cycloreversion , figure 3 ) or one of the four biradical minima , for instance minbryy , ( o o homolysis , figure 2 ) . the second step to generate the final photoproducts takes place in the gs , where the system needs to overcome an energy barrier that corresponds to the cleavage of the second c o bond , tso2 , in cycloreversion ( figure 3 ) or to the concerted rupture of both ch bonds simultaneously , tsh2 , in o o homolysis ( figure 2 ) . a number of important mechanistic conclusions with implications in several biological and technological applications can be drawn from our study:(1)the generation of o2 takes place through a stepwise mechanism . the breaking of the first c o bond takes place barrierlessly on the way to the gs after going through several conical intersections . the second c o is , however , cleaved once the system is in the gs after overcoming an energy barrier.(2)in agreement with the experimental observations , our dynamics simulations predict that o2 is generated in its lower electronic excited state ( g).(3)according to the present simulations , the triplets do not play a significant role in the o2 generation mechanism . no isc was registered along any of the cycloreversion trajectories in contrast to o o homolysis , where an important population transfer to the triplet manifold was observed . the breaking of the first c o bond takes place barrierlessly on the way to the gs after going through several conical intersections . the second c o is , however , cleaved once the system is in the gs after overcoming an energy barrier . in agreement with the experimental observations , our dynamics simulations predict that o2 is generated in its lower electronic excited state ( g ) . according to the present simulations , the triplets do not play a significant role in the o2 generation mechanism . no isc was registered along any of the cycloreversion trajectories in contrast to o o homolysis , where an important population transfer to the triplet manifold was observed . despite the lack of experimental information on chdepo , it is possible to establish some links between our simulations and the recent femtosecond uv pump probe experiments investigating the dual photochemistry of the larger endoperoxide apo . interestingly , and similar to the experiments , our simulations predict the full transformation of the endoperoxide into its photoproducts ; that is , no trajectories were found to revert to the original gs of chdepo . excitation of apo at 270 or 282 nm , which populates the high - lying spectroscopic state s4 ( * ) , leads to a yield for the cycloreversion reaction oscillating between 25% and 29% , in line with our theoretical results ( 30% ) , starting from the *oo*cc electronic state . also consistent with the experiment , the leading photodeactivation process is o o homolysis with a final yield of 65% . however , and in contrast with the experiment , we do not detect any cycloreversion trajectory evolving along the triplet manifold in chdepo . the role of the triplets seems to be only important along the o o homolysis deactivation pathway . the rationalization of the mechanism for o2 generation and its competition with side pathways leading to photoproducts is expected to inspire the development of new photosensitizers to be used in the many different areas where the production of this oxidant has a leading role .

a synergistic approach combining high - level multiconfigurational static calculations and full - dimensional ab initio surface hopping dynamics has been employed to gain insight into the photochemistry of endoperoxides . electronic excitation of endoperoxides triggers two competing pathways , cycloreversion and o o homolysis , that result in the generation of singlet oxygen and oxygen diradical rearrangement products . our results reveal that cycloreversion or the rupture of the two c o bonds occurs via an asynchronous mechanism that can lead to the population of a ground - state intermediate showing a single c o bond . furthermore , singlet oxygen is directly generated in its most stable excited electronic state 1g . the triplet states do not intervene in this mechanism , as opposed to the o o homolysis where the exchange of population between the singlet and triplet manifolds is remarkable . in line with recent experiments performed on the larger anthracene-9,10-endoperoxide , upon excitation to the spectroscopic * electronic states , the primary photoreactive pathway that governs deactivation of endoperoxides is o o homolysis with a quantum yield of 65% .

Introduction Results and Discussion Conclusions

PMC3703883

in healthy humans , skeletal muscle accounts for ~7080% of the insulin - stimulated glucose uptake , being the major site of glucose disposal and , thus , exerting a key role in regulating whole body glucose homeostasis . accordingly , understanding changes that occur to this tissue during obesity and diabetes development is crucial to elucidate the underlying causes of insulin resistance and to reveal new targets for its treatment . insulin resistance in skeletal muscle has long been recognized as a characteristic feature of type 2 diabetes and plays a major role in the pathogenesis of the disease . although several epidemiological data have shown that the consumption of added sugars as ingredients in processed or prepared foods and caloric beverages has dramatically increased over the last decades , most of the experimental studies investigating the development of insulin resistance in the skeletal muscle have been based on genetic manipulation or use of high fat diets [ 24 ] . in contrast , the molecular mechanisms underlying the detrimental effects of sugar , mainly those on skeletal muscle , are not completely understood . in the present study , we used a previously developed mouse model of chronic exposure to high - fructose corn syrup ( hfcs ) to investigate the deleterious effects of high sugar intake on skeletal muscle . hfcs syrup , used as an ingredient in processed or prepared foods and caloric beverages , is synthesized by refining corn starch , contains 55% fructose and 42% glucose and to date accounts for over 40% of all added caloric sweeteners . the peroxisome proliferator activated receptor ( ppar ) superfamily of transcription factors , that includes the isoforms ppar- , ppar- , and ppar- , has been widely shown to exert crucial roles in energy homeostasis regulation and glucose metabolism . ppar- , one of the most promising pharmacological target implicated in obesity - associated insulin resistance , is highly expressed in skeletal muscle , at 10- and 50-folds higher levels compared with ppar- and ppar- , respectively . however , its potential effects in affecting skeletal muscle glucose intake and insulin sensitivity are only now being elucidated . schuler et al . showed that mice in which ppar- is selectively ablated in skeletal myocytes exhibit fiber - type switching , obesity , and type 2 diabetes . besides , a very recent paper has suggested that the improvement of glucose homeostasis by angiotensin receptor blockers in hypertensive patients involves a selective ppar- activation in the skeletal muscle . so far , the exact molecular mechanisms underlying the observed ppar--induced changes have not yet been determined . hence , the present study aimed to determine whether chronic administration of the selective ppar- agonist gw0742 in hfcs - fed mice may ameliorate the impairment of signaling pathways triggered in skeletal muscles by chronic high sugar intake . several studies have revealed that a large number of muscle - derived secretory cytokines ( collectively termed myokines ) can act locally in an autocrine / paracrine manner , linking skeletal muscle to regulation of physiological processes in other tissues . we have , thus , also investigated whether ppar- agonism may affect the serum levels of the well - known myokine interleukin-6 ( il-6 ) and the muscular expression of a member of the fibroblast growth factor ( fgf ) superfamily , fgf-21 , a recently identified myokine involved in the interorgan communication . to further extend our investigation on the ability of ppar- agonism to modulate inflammatory pathways involved in local insulin resistance pathogenesis , the effects of ppar- activation on nuclear translocation of the transcription nuclear factor - kappab ( nf-b ) and expression of its target genes have also been studied . four - week - old male c57bl6/j mice ( harlan - italy ; udine , italy ) were housed in a controlled environment at 25 2c with alternating 12-h light and dark cycles . they were provided with a piccioni pellet diet ( n. 48 , gessate milanese , italy ) and water ad libitum . all the animals were fed with a normal pellet diet for 1 week prior to the experiment . the animals were then allocated to two dietary regimens , chow diet and normal drinking water ( control ) or a chow diet and 15% ( wt / vol ) hfcs solution in drinking water ( hfcs ) for 30 weeks . the concentration of hfcs solution as well as the period of dietary manipulation was chosen according to previous animal studies investigating the metabolic effects of long - term ( 6 - 7 months ) access to hfcs . animal care was in compliance with italian regulations on the protection of animals used for experimental and other scientific purposes ( dm 116/92 ) , and the experiment was approved by the turin university ethics committee . after the initial period of 14 weeks of dietary manipulation , each of the two diet groups ( control and hfcs diet ) was further subdivided to obtain four different treatment groups : chow diet and normal drinking water ( control , n = 10 ) , chow diet supplemented with gw0742 ( 1 mg / kg / day ) and normal drinking water ( control + gw , n = 6 ) , chow diet and 15% ( wt / vol ) hfcs solution in drinking water ( hfcs , n = 10 ) , and chow diet supplemented with gw0742 ( 1 mg / kg / day ) and 15% ( wt / vol ) hfcs solution in drinking water ( hfcs + gw , n = 10 ) . the drug was daily administered with the food for the last 16 weeks , and the mice were allowed to continue to feed on their respective diets until the end of the study . gw0742 is a highly potent and selective ppar- agonist ( murine ec50 : 28 nm for ppar- ; 8,900 nm for ppar- ; > 10,000 nm for ppar- ) , with an acceptable pharmacokinetic profile and activity in vivo . the dose and the kinetics of administration were chosen based on those we have previously shown to improve glucose tolerance and insulin sensitivity in vivo . one day before the mice were due to be killed , the ogtt was performed after a fasting period of 6 h by administering glucose ( 2 g / kg ) by oral gavage . once before administration and 15 , 30 , 60 , 90 , 120 , and 150 min afterward , blood was obtained from the saphenous vein , and glucose concentration was measured with a conventional glucometer ( accu - check compact kit , roche diagnostics gmbh , mannheim , germany ) . after 16 weeks from the start of the drug treatment ( i.e. , after 30 weeks of dietary manipulation ) , the mice were anaesthetised with i.p . injection ( 30 mg / kg ) of zoletil 100 ( laboratories virbac , france ) and killed by aortic exsanguination . the serum lipid profile was determined by measuring the content of triglycerides , total cholesterol , high - density lipoprotein ( hdl ) , and low density lipoprotein ( ldl ) by standard enzymatic procedures using reagent kits ( hospitex diagnostics , florence , italy ) . plasma leptin and adiponectin levels were measured using enzyme - linked immunosorbent assay ( elisa ) kits ( leptin and adiponectin mouse elisa kits , abcam , cambridge uk ) . the gastrocnemius muscle and epididymal fat were isolated , weighed , rapidly freeze - clamped with liquid nitrogen , and stored at 80c . briefly , gastrocnemius were homogenised at 10%(w / v ) in a potter - elvehjem homogenizer ( wheaton , nj , usa ) using a homogenisation buffer containing 20 mm hepes , ph 7.9 , 1 mm mgcl2 , 0.5 mm edta , 1 mm egta , 1 mm dithiothreitol ( dtt ) , 0.5 mm phenylmethylsulfonyl fluoride ( pmsf ) , 5 g / ml aprotinin , and 2.5 g / ml leupeptin . supernatants were removed and centrifuged at 14000 rpm at 4c for 40 minutes to obtain the cytosolic fraction . the pelleted nuclei were resuspended in an extraction buffer and centrifuged at 14000 rpm for 20 minutes at 4c . the amount of proteins contained in the cytosolic and nuclear fraction was determined using a bca protein assay following the manufacturers ' instructions . skeletal muscle triglycerides were extracted from gastrocnemius homogenates and assayed using reagent kits according to the manufacturer 's instructions ( triglyceride quantification kit , abnova corporation , aachen , germany ) . about 60 g of total proteins was loaded for western blot experiments . proteins were separated by 8% sodium dodecyl sulphate - polyacrylamide gel electrophoresis ( sds - page ) and transferred to a polyvinyldene difluoride ( pvdf ) membrane , which was then incubated with a primary antibody ( rabbit anti - ppar- , dilution 1 : 500 ; rabbit antitotal gsk-3 , dilution 1 : 200 ; goat anti - pgsk-3 ser , dilution 1 : 200 ; rabbit antitotal akt , dilution 1 : 1000 ; mouse anti - pakt ser , dilution 1 : 1000 ; goat anti - icam-1 , dilution 1 : 500 ; rabbit antitotal irs-1 , dilution 1 : 200 ; goat anti - pirs-1 ser , dilution 1 : 200 ; rabbit anti - glut-4 , dilution 1 : 2000 ; rabbit anti - glut-5 , dilution 1 : 100 ; rabbit anti - inos , dilution 1 : 200 ; rabbit anti - nf-b dilution 1 : 1000 ; rabbit antitotal ampk , dilution 1 : 1000 ; rabbit anti - pampk thr , dilution 1 : 1000 ; rabbit antitotal acc , dilution 1 : 500 ; rabbit anti - pacc ser , dilution 1 : 1000 ; rabbit anti - cpt-1 , dilution 1 : 200 ) . blots were then incubated with a secondary antibody conjugated with horseradish peroxidase ( dilution 1 : 10000 ) and developed using the ecl detection system . the immunoreactive bands were visualised by autoradiography and the density of the bands was evaluated densitometrically using gel - pro analyzer 4.5 , 2000 software ( media cybernetics , silver spring , md , usa ) . the membranes were stripped and incubated with tubulin monoclonal antibody ( dilution 1 : 5000 ) and subsequently with an anti - mouse antibody ( dilution 1 : 10000 ) to assess gel - loading homogeneity . for immunodetection of glucose transporter type-4 ( glut-4 ) , sections were incubated overnight with rabbit anti - glut-4 antibody ( abcam ) , dilution 1 : 200 , and for 1 hour with goat anti - rabbit igg - hrp conjugated secondary antibody ( bio - rad laboratories , hercules , ca , usa ) . for detection of glucose transporter type 5 ( glut-5 ) , sections were incubated overnight with rabbit anti - glut-5 antibody ( abcam ) , dilution 1 : 50 , for 1 hour with swine anti - rabbit igg - biotinylated secondary antibody ( dako , glostrup , denmark ) , and for 1 hour with streptavidin hrp conjugate ( southern biotech , birmingham , al , usa ) . the specific staining was detected with diaminobenzidine ( dab , sigma - aldrich ) , and sections were visualized with olympus - bx4i microscope connected by a photographic attachment ( carl zeiss , oberkochen , germany ) . for each antibody , a negative control was included in which the primary antibody was replaced with a nonimmune isotypic control antibody . serum levels of il-6 and fgf-21 levels in gastrocnemius homogenates were measured by elisa according to the manufacturer 's instructions ( mouse fgf-21 and il-6 elisa kits , r&d systems , abingdon , uk ) . unless otherwise stated ( rockford , il , usa ) , and pvdf was from the millipore corporation ( bedford , massachusetts , usa ) . antibodies were from cell - signaling technology ( beverly , ma , usa ) , santa cruz biotechnology ( santa cruz , ca , usa ) , and abcam ( cambridge , cb , uk ) . the anti - mouse , anti - rabbit , and anti - goat horseradish peroxidase - linked antibodies were from santa cruz biotechnology ( santa cruz , ca , usa ) , and luminol ecl was from perkinelmer ( waltham massachusetts , usa ) . all values in both the text and figures are expressed as mean sd for n observations . one - way analysis of variance with dunnett 's post - hoc test was performed using the graphpad prism version 4.02 for windows ( graphpad software , san diego , california , usa ) , and p values below 0.05 were considered as significant . after 30 weeks of dietary manipulation , mice on hfcs group had significantly higher body mass than the control group , with an increase in body mass over 30% ( 36.00 1.41 g versus 32.25 1.67 g ; p < 0.01 ) , whereas gw0742 administration induced a slight but not significant reduction in body mass ( 35.00 2.83 g ) . epididymal fat mass was increased by hfcs manipulation in comparison to control diet ( 4.22 0.35% body mass versus 3.66 0.29% body mass ; p < 0.01 ) , and values were reduced to the control level by drug administration ( 3.86 0.33% body mass ) . in contrast , neither dietary manipulation nor drug treatment affected gastrocnemius mass ( % body mass : 0.97 0.19 control , 0.92 0.09 hfcs , and 0.92 0.06 hfcs + gw ) . as we previously reported , the hfcs diet caused a significant increase in serum triglycerides , total cholesterol , and ldl concentrations and a marked decrease in the hdl levels . interestingly , the hfcs diet significantly affected insulin sensitivity . a significant increase in serum insulin levels was observed in mice of hfcs group compared to control mice ( 2.26 0.51 g / l versus 1.27 0.07 g / l ) , and this increase was almost completely abolished by gw0742 ( 1.51 0.46 g / l ) . fasting glucose concentrations were elevated in serum of hfcs animals in comparison to control animals ( 103.4 12.0 mg / dl versus 78.7 0.3 mg / dl ) and reduced to control levels by gw0742 ( 81.3 10.9 mg / dl ) . moreover , hfcs mice showed a significant impairment in glucose tolerance to exogenously administered glucose . although all the groups reached a glycemic peak at 15 min postglucose challenge ( control : 223.1 12.0 mg / dl ; hfcs : 212.7 7.3 mg / dl ; hfcs + gw : 198.8 10.3 mg / dl ) , glycemic levels at 3060 min post glucose challenge in hfcs - fed mice were higher than those recorded in the control group ( 30 min : 182.9 8.3 mg / dl versus 129.4 8.0 mg / dl , p < 0.05 ; 60 min : 130.7 10.3 mg / dl versus 96.8 4.0 mg / dl , p < 0.05 ) . gw0742 significantly ( p < 0.05 ) improved glucose tolerance in hfcs - fed mice , showing significantly reduced glucose concentrations at both 30 and 60 min postglucose challenge ( 89.7 5.2 mg / dl and 86.7 4.3 mg / dl , resp . ) . data on the effects of dietary manipulation and chronic gw0742 treatment on serum levels of il-6 , adiponectin , and leptin are reported in table 1 . the experimental diet caused a more than fourfold increase in serum il-6 concentrations , whereas gw0742 administration significantly decreased the il-6 levels . adiponectin and leptin showed different patterns : in fact , the level of adiponectin in hfcs mice was lower than in control mice , while that of leptin increased versus controls . when gw0742 was administered to hfcs mice there was a significant increase ( p < 0.01 ) in the adiponectin levels associated with a significant decrease ( p < 0.01 ) in leptin serum concentration . it must be noted that in control mice gw0742 had no significant effect on any of the previously described metabolic parameters . as shown in figure 1 in contrast , daily administration of gw0742 resulted in a twofold increase in expression of its pharmacological target , with maximum effect in the presence of dietary manipulation . the hfcs diet did not alter the protein expression of irs-1 , akt , or gsk-3 compared to the control group . however , hfcs caused a marked increase in ser phosphorylation of irs-1 in parallel with reduced ser phosphorylation of akt ( figures 2(a ) and 2(b ) ) . ser phosphorylation of gsk-3 , a downstream target of akt , was also reduced in the presence of hfcs ( figure 2(c ) ) , suggestive of impaired insulin signaling downstream of irs-1 . most notably , gw0742 significantly attenuated all the effects of hfcs on irs-1 , akt , and gsk-3 phosphorylation , measured at the steady state . in comparison to control animals , glut-4 expression was reduced in the gastrocnemius of hfcs mice , without reaching a statistical significance , and increased following drug treatment ( figure 3(a ) ) . similarly , the hfcs diet markedly reduced glut-5 expression , whereas gw0742 administration resulted in a significant increase in glut-5 expression ( figure 3(c ) ) . notably , gw0742 treatment not only increased carriers expression levels but also induced a significant membrane translocation , thus increasing muscle glucose uptake ( figures 3(b ) and 3(d ) ) . the triglyceride content was doubled in the gastrocnemius of hfcs mice in comparison with control animals , whereas skeletal muscle triglyceride accumulation was significantly reduced by gw0742 administration ( figure 4 ) . changes in the phosphorylation / activation of the amp - activated protein kinase ( ampk)/acetyl - coa carboxylase ( acc ) system , whose central role in the regulation of cellular lipid homeostasis is well known , were evaluated by immunoblotting experiments on gastrocnemius homogenates . as reported in figure 5 , there was no significant effect of hfcs feeding or drug treatment on total ampk and acc protein content . in contrast , chronic hfcs exposure markedly reduced thr phosphorylation of ampk and its substrate ser acc . interestingly , gw0742 treatment led to a robust increase in phosphorylation of both ampk and acc in hfcs - fed mice ( figures 5(a ) and 5(b ) ) . the effects of ppar- agonism on fatty acid oxidation in skeletal muscle cells are further indicated by the significant increase in the protein expression of carnitine palmitoyl transferase-1 ( cpt-1 ) elicited by gw0742 in hfcs - fed mice ( figure 5(c ) ) . to investigate the muscle inflammatory response induced by hfcs - diet and the intracellular signaling pathway(s ) that might be involved in the protective mechanisms evoked by gw0742 when compared to control mice , hfcs mice developed significant increase in the nuclear translocation of the p65 nf-b subunit in the mouse gastrocnemius , indicating the activation of this transcriptional factor ( figure 6(a ) ) . treatment with gw0742 resulted in a significant reduction in nuclear translocation of p65 and , hence , in the activation of nf-b . the intercellular adhesion molecule-1 ( icam-1 ) , whose role in the recruitment of neutrophils is widely accepted , was slightly detected in the gastrocnemius of control animals , whereas its expression was dramatically increased by hfcs diet ( figure 6(b ) ) . as shown in figure 6(c ) , also the expression of the inducible nitric oxide synthase ( inos ) was increased in the presence of dietary manipulation . interestingly , these changes were suppressed by gw0742 administration , as evidenced by densitometric analysis of the related autoradiograms . the beneficial effects of ppar- activation were associated with a dramatic increase of fgf-21 production in the mouse gastrocnemius . as shown in figure 7 , in comparison to control mice ( 46.45 1.13 pg / mg protein ) , hfcs diet did not affect the production of this myokine ( 49.38 4.12 pg / mg protein ) , whereas gw0742 treatment induced a twofold increase ( 87.08 6.37 pg / mg protein ) . in agreement with previous observations [ 14 , 15 ] , the present study shows that chronic exposure to the most widely used added sugar hfcs caused a significant increase in body mass associated with increases in serum levels of triglycerides , ldl - cholesterol , glucose , insulin , il-6 , leptin , and hypoadiponectinemia . using the same experimental protocol here reported , we have recently demonstrated that the molecular mechanism underlying the deleterious effects of hfcs involves the hepatic upregulation of fructokinase , the main fructose - metabolizing enzyme , which may account for the increase in serum levels of free fatty acids and hyperuricemia . we also reported that the documented increase in serum uric acid level significantly contributed to the development of chronic kidney injury . interestingly , we demonstrated that chronic administration of the ppar- ligand gw0742 exerted beneficial effects by preventing the upregulation of fructokinase in the liver and activation of the inflammatory signaling complex nlrp3 inflammasome in the kidney . despite these preliminary data , so far , there are no studies on the effects of long - term exposure to hfcs , the major sweetener added to beverages and food , on skeletal muscle , which is a major site of postprandial glucose disposal and is therefore one of the insulin - sensitive tissues most likely to manifest early signs of insulin resistance . here , we investigated ( i ) the mechanisms underlying metabolic disturbances in skeletal muscle of mice exposed for 30 weeks to high intake of hfcs and ( ii ) the local effects evoked by ppar- chronic activation . the sugar was added to the drink water at a concentration that covers 10% of the daily caloric intake , corresponding to the average energy intake in the form of ingested sweeteners in the western diet . we documented the hfcs - induced alteration in the insulin signal transduction pathway , as shown by the impaired phosphorylation of irs-1 protein as well as of the downstream key insulin signaling molecules , akt , and gsk-3 , an akt substrate . this is in keeping with previous studies showing that irs-1 serine phosphorylation can interfere with subsequent akt and gsk-3 phosphorylation , disrupting insulin signal transduction . in our experimental model , oral administration of the selective ppar- agonist gw0742 was associated with a significant improvement of the defective insulin signaling in the skeletal muscle , which may account at least in part for the changes in serum lipid profile and insulin sensitivity . as the inhibition of gsk-3 evokes an increase in the glycogen synthesis [ 18 , 19 ] , we may speculate that gw0742 administration modulates muscle glycogen storage through inactivation of gsk-3. although no direct evidence of drug treatment on glucose storage has been reported in our study , we documented the effects of chronic ppar- activation on glucose transporters expression and distribution . specifically , we found that ppar- activation by gw0742 evoked an increase in expression of glut-4 , the most abundant glucose transporter isoform in skeletal muscle , and its translocation from intracellular compartments to the plasma membrane , thus facilitating glucose transport . similarly , glut-5 , the fructose carrier with low capacity to transport glucose , was scarcely detectable in the gastrocnemius muscle of hfcs - fed mice , and its expression and membrane translocation were enhanced by chronic ppar- activation . because fructose is highly present in the hfcs diet and contributes to carbohydrate metabolism in muscle , the increase in glut-5 may favor a better utilization of fructose supplied in the diet . in any case , the reduction in glut-4 and glut-5 expression and translocation detected in the hfcs group could represent an adaptation to the chronic exposure to a sugar - enriched diet , while the increases in these carrier isoforms in the presence of the selective ppar- agonist gw0742 may account , at least in part , for the improvement in insulin - induced muscle glucose uptake . recent data have shown that akt inhibitors or dominant - negative akt constructs regulate the translocation , targeting , and fusion of glut-4-containing vesicles [ 23 , 24 ] . similarly , irs-1 phosphorylation has been reported to affect glut-4 translocation and subsequent glucose uptake in mouse skeletal myocytes . we may , thus , speculate that the beneficial effects of the ppar- ligand gw0742 are secondary to activation of the irs-1/akt / gsk-3 pathway . this is also supported by previous findings showing that the chronic administration of gw0742 in rats results in phosphoinositide - dependent kinase phosphorylation and , hence , increased akt phosphorylation . another enzyme complex which could be involved in ppar- metabolic effects within the skeletal muscle is ampk , a serine / threonine protein kinase that has emerged as a key player in both lipid and glucose metabolism in skeletal muscles [ 27 , 28 ] . in mouse c2c12 myotubes , ampk activation potentiated insulin action by reducing irs-1 serine phosphorylation , while reduced muscle ampk activity has been reported to aggravate muscle insulin resistance following a high - fat diet over 30 weeks . in agreement with these observations , we found that gw0742 increased phosphorylation of thr regulatory site on ampk which , in turn , causes acc phosphorylation . when acc is inactive ( phosphorylated form ) , a fall in malonyl - coenzyme a occurs , which disinhibits cpt-1 and increases mitochondrial import and oxidation of long - chain fatty acids . our data demonstrate that chronic exposure to gw0742 administration increased skeletal muscle cpt-1 expression , thus shunting toward fatty acid oxidation , and this effect was associated with reduced triglyceride accumulation . these findings suggest that the beneficial effects evoked by the ppar- agonist gw0742 are at least partially dependent on ampk activation . the direct involvement of ppar- agonism in mediating improvements in muscle insulin sensitivity and lipid metabolism is also confirmed by results showing that ppar- is expressed in the gastrocnemius of both control and hfcs - fed mice and , more importantly , that its expression is substantially increased by chronic administration of gw0742 , thus suggesting that the observed drug effects are due to a ligand - dependent ppar- activation . this is consistent with previous works showing that ppar- activation is involved in regulating myogenesis [ 3235 ] . in search of the mechanism(s ) underlying the protective action of gw0742 , we investigated whether ppar- activation may affect the local inflammatory response associated with muscular metabolic injury . although ppar- has been implicated in the regulation of systemic inflammatory responses associated with metabolic dysregulation , so far , reports on anti - inflammatory effects of ppar activation in skeletal muscle are rather scarce . here , we show for the first time that in the skeletal muscle gw0742 reverts the diet - induced increase in the nuclear translocation of nf-b p65 , a transcriptional factor that plays an important role in regulating the transcription of a number of genes , especially those involved in producing mediators of local and systemic inflammation ( such as cytokines , chemokines , and cell adhesion molecules ) . this observation is in agreement with previous studies reporting that ppar- activation induces the physical interaction between ppar- and the p65 subunit of nf-b and reduced the lps - induced degradation of the inhibitory protein inhibitor of kappa b , thus preventing nf-b activation [ 36 , 37 ] . the reduction of nf-b activation by gw0742 treatment may account for the observed reduction in the serum levels of il-6 , which is known to be mostly released from skeletal muscle , and in the local expression of the nf-b - dependent proteins inos and icam-1 , whose role in the development of insulin resistance has been recently documented [ 38 , 39 ] . as several studies have reported an association between gsk-3 and nf-b activity , we might speculate that ppar- activation phosphorylates , and hence , activates the akt pathway , which in turn phosphorylates , and hence , inhibits gsk-3 , presumably resulting in the inhibition of nf-b and , in turn , nf-b - dependent proinflammatory gene transcription . although this cross talk among different signaling pathways by pharmacological ppar- modulation is intriguing , it must be stressed , however , that the lack of direct experimental evidence of a causal relationship between the improved local and systemic insulin sensitivity and the treatment - induced reduction of nf-b activation in our experimental model limit the interpretation of the molecular mechanism(s ) underlying our findings . one of the most recently identified mediators that facilitates organ cross talk and the related control of impaired glucose homeostasis in metabolic diseases is a member of the fgf family , fgf-21 . fgf-21 has been previously shown to lower blood glucose levels in several diabetic rodent and monkey models [ 41 , 42 ] , to regulate lypolisis in white adipose tissue [ 43 , 44 ] and substrate utilization in the liver . the metabolic effects of fgf-21 on glucose metabolism involve selective regulation by different ppar isoforms . conversely , ppar- activation with feeding promotes fgf-21 production in adipocytes but not in liver [ 48 , 49 ] . to our knowledge , this is the first paper that demonstrates a correlation between ppar- activation and increased fgf-21 levels in skeletal muscle , thus adding an original piece of evidence to the complex mechanisms by which ppar- can regulate several biological functions . our observation is also in keeping with a previous study showing that circulating fgf-21 levels consistently increase in human subjects in response to pharmacological activation of ppar- . although a previous investigation has revealed that ppar- directly regulates expression of the fgf-21 gene through elements located within the 500-bp upstream region of the gene , to date , a consensus sequence of ppar- binding site in the promoter region of the fgf-21 gene has not yet been identified . besides in vitro experiments with fgf-21 , small interfering rna are warranted to clarify whether gw0742 beneficial effects are related to the direct modulation of fgf-21 biological functions . the recent findings on fgf-21 ability to prevent insulin resistance in human myoblasts by inhibiting nf-b activation may also suggest that the muscle - derived fgf-21 acts in an autocrine fashion to amplify ppar- inhibitory effects on the expression of nf-b - dependent inflammatory genes . this further supports the existence of multiple anti - inflammatory pathways involved in the beneficial effects evoked by ppar- activation . in conclusion , we have shown that feeding mice with a hfcs diet for 30 weeks evoked skeletal muscle insulin resistance and lipid accumulation which were associated with activation of inflammatory pathways . in addition , this study , showing a strong induction of fgf-21 in the skeletal muscle after chronic administration of the ppar- ligand gw0742 , enhances our knowledge of the mechanisms of action of ppar- agonism and provides further insights into the role of fgf-21 as mediator of the tissue cross - talk that underlines the integrated control of the metabolic inflammation .

peroxisome proliferator activated receptor ( ppar)- agonists may serve for treating metabolic diseases . however , the effects of ppar- agonism within the skeletal muscle , which plays a key role in whole - body glucose metabolism , remain unclear . this study aimed to investigate the signaling pathways activated in the gastrocnemius muscle by chronic administration of the selective ppar- agonist , gw0742 ( 1 mg / kg / day for 16 weeks ) , in male c57bl6/j mice treated for 30 weeks with high - fructose corn syrup ( hfcs ) , the major sweetener in foods and soft - drinks ( 15% wt / vol in drinking water ) . mice fed with the hfcs diet exhibited hyperlipidemia , hyperinsulinemia , hyperleptinemia , and hypoadiponectinemia . in the gastrocnemius muscle , hfcs impaired insulin and amp - activated protein kinase signaling pathways and reduced glut-4 and glut-5 expression and membrane translocation . gw0742 administration induced ppar- upregulation and improvement in glucose and lipid metabolism . diet - induced activation of nuclear factor-b and expression of inducible - nitric - oxide - synthase and intercellular - adhesion - molecule-1 were attenuated by drug treatment . these effects were accompanied by reduction in the serum concentration of interleukin-6 and increase in muscular expression of fibroblast growth factor-21 . overall , here we show that ppar- activation protects the skeletal muscle against the metabolic abnormalities caused by chronic hfcs exposure by affecting multiple levels of the insulin and inflammatory cascades .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

PMC4062863

cardiovascular diseases ( cvd ) , among them hypertensive disease , is causing significant morbidity and mortality in adult population globally . hypertensive disease is considered in case of untreated systolic blood pressure ( bp ) of greater than or equal to 140 mmhg and/or diastolic bp of greater than or equal to 90 mmhg or one being on antihypertensive medication . the disease is among the leading causes of death and disability [ 3 , 4 ] , and its prevalence is increasing worldwide particularly in developing countries as a result of nutritional transition and westernization . hypertensive disease is a significant contributor [ 6 , 7 ] to the increasing incidence and mortality as a result of cvd globally . sub - saharan africa appears to bear the heaviest burden of this preventable disease as an emerging threat ; for instance , in cameroon , the prevalence rose from 8.3% in 1994 to 20.8% in 2004 among the adult urban dwellers . in kenya , available data shows that the disease is gaining momentum and has been identified as an important cause of morbidity and mortality in urban population amidst high prevalence of communicable diseases . the main characteristics and risk factors for hypertensive disease are well established and appear to be consistent across societies . a study among kenyan muslim males in mombasa identified cigarette smoking , alcohol consumption , and overweight as the main risk factors for the disease . similarly , a study conducted at a leading private hospital in kenya , hypertensive disease was the main risk factor for ischemic stroke . the implications are enormous for a health care system that is already struggling with the burden of tropical and communicable diseases including the impact of hiv / aids and now the rising noncommunicable diseases . kenyan defence forces ( kdf ) personnel are recruited and retained based on stringent healthy criteria ; they undergo routine medical checkups , treatment , and health awareness . therefore , they may have favorable physical and physiological parameters as indicators of healthy cardiovascular system . additionally , military personnels maintain strict physical activity schedules and drills regular medical checkups which may result in physical fitness . on the other hand , military personnels practice lifestyle habits similar to the general population which may predispose them to cvd , such as smoking , alcohol intake , and sedentary lifestyle habits . recently , studies have demonstrated a trend toward increasing hypertensive disease among kdf consistent with other military personnel [ 1214 ] globally . the causation may be multifactorial including occupation - related stress , a potentially important cardiovascular risk factor [ 1517 ] . military personnels are generally under a lot of pressure and work - related stress , which have been implicated for their biologically negative effects and mental strain [ 18 , 19 ] . we hypothesized that physiological , behavioral and , dietary characteristics associated with hypertension among kdf mimic the main risk factors and characteristics for cvd in the developed western societies . this study adopted a cross - sectional design to determine physiological , behavioral , and dietary characteristics associated with hypertension among 340 ( 170 hypertensive and 170 normotensive ) kdf . eligible participants were examined by trained nurses at the military medical clinic in armed forces memorial hospital ( afmh ) , nairobi . this is a referral hospital that provides healthcare services to the kdf personnel and their nuclear families . it has bed capacity of 114 and 8 specialized outpatient clinics among them a medical outpatient clinic ( mopc ) . the clinic runs twice a week attending to an average of 40 patients per clinic day . the eligible participants with hypertension were personnels with essential hypertension , on antihypertensive or nonpharmacological therapy for elevated blood pressure based on who classification ( bp > 140/90 mmhg ) as per the hospital file and were being followed up at afmh mopc . written consent was obtained after comprehensive explanation of study objectives , risks , and benefits . conventional measurements of physiological as well as anthropometric parameters were conducted by trained nurses thereafter . physiological variables . hypertension was defined according to the seventh report of the joint national committee on prevention , detection , evaluation , and treatment of high bp criteria as having an untreated systolic bp of greater than or equal to 140 mmhg or diastolic bp greater than or equal to 90 mmhg or being on medication for hypertension . participants were asked to sit and relax in a silent room for 5 minutes before their arterial pulse and blood pressure were measured in the right arm two times in the sitting or supine position using a calibrated digital instrument , appropriate devices , and cuff sizes . the bp and heart rate were measured with an electronic automatic monitor ( omron m4 , omron healthcare gmbh , hamburg , germany ) . the final pulse and bp were the average of two consecutive readings obtained from the participant . demographic and social life information including age , marital status , level of education , and deployment to peace missions , history of smoking with its frequency were obtained from each individual using a standardized questionnaire given to the participants to complete on their own . food intake and dietary patterns . participants were asked to report types of foods including meat consumption , beverages such as beer , wine , and whiskey , the frequency of their consumption ( daily , weekly , and monthly ) , and the amounts consumed . physical activity . participation in sports or exercises was assessed through a questionnaire , and the respondents were asked how often , and the average duration per session they engaged in each of the common physical activities . individual activities included walking , jogging or running , weight lifting , tennis , soccer , swimming , bicycling , dancing , and other strenuous exercise . the respondents indicated their usual frequency of participation in each of the above activities by choosing one of the following categories : never , less than once per month , 13 times per month , 1 - 2 times per week , 36 times per week , or every day . the average time per episode of the activities included less than 15 , 1630 , 3160 min , or more than 60 min , while intensity was categorized as light , moderate , or vigorous . anthropometric measures including weight , height , body mass index ( bmi ) , waist circumference ( wc ) , and hip circumference ( hc ) were measured by standard methods . wc was determined by measuring waist diameter at midpoint between iliac crest and lower border of tenth rib , with an average of three measurements considered as wc . central obesity was wc > 102 cm and 95 cm as defined for male and female participants , respectively . bmi was calculated based on weight in kilogram divided by square of height in meter ( kg / m ) . waist and hip circumferences were measured using a nonelastic tape measure ( seca , germany ) and were subsequently used to calculate waist to hip ratio ( whr ) . weight and height were obtained using weighing scale with a stadiometer ( seca gmbh , co.kg , germany ) and , thereafter , used to calculate body mass index ( bmi ) . height was measured and recorded to the nearest 0.1 cm , while the waist circumference was measured with a nonstretchable tape measure to the nearest 0.1 cm . we calculated descriptive statistics ( e.g. , means , standard deviations , correlations ) for each of the major study variables and performed paired t tests to determine whether mean scores differed for hypertensive and normotensive participants . demographic characteristics and potential risk factors were compared by t - test for continuous variables and the chi - square ( ) test for categorical variables . association between physiological , dietary patterns , and hypertension by behavioral factors was assessed through subgroup analyses based on different levels of these factors . adjusting for the effect of all the predictors associated with hypertension all tests of statistical significance were two - sided , and the analyses were conducted using spss for windows ( release 17.0 , 2008 ; spss , chicago , il , usa ) . ethical clearance was obtained from the university of nairobi / kenyatta national hospital ethics and research committee ( approval no . p74/03/2011 ) as well as the ministry of education , science and technology ( permit no . authorization to carry out the research at afmh was given by the armed forces memorial hospital medical advisory committee ( approval no . gender . majority ( 91.6% ) of the study participants were male ( table 1 ) . most ( 95% ) of participants with hypertension were males compared to 88% of nonhypertensive participants . on further analyses , there were lower odds of being hypertensive among the female gender ( or = 0.44 ; 95% ci 0.191.01 , p = 0.05 ) . the mean age was 45.1 ( sd 7.7 ) years among participants with hypertensive diseases compared to normotensive 40.8 ( 7.3 ) ( table 1 ) . participants with hypertensive disease were 4.82 years older than nonhypertensive subjects ( p < 0.01 ) . majority of hypertensive participants were aged from 39 to 48 years , compared to 14% among normotensive . the odds of being older among participants with hypertension were highest among 49 to 58 years . additionally , the odds of hypertension in this age group were threefold that of 18 to 28 years ( odds ratio [ or ] = 3.08 ; 95% ci 1.27.94 , p = 0.02 ) age group . we show the relationship between deployment to peace missions and hypertensive disease ( table 2 ) . there was significant ( = 34.40 ; df = 1 ; p < 0.01 ) association between hypertension and participation in peace missions . majority ( 68% ) of subjects who had participated in peace missions were hypertensive compared to 36.08% who had never been to peace missions . educational level . the association between educational level and hypertension is shown ( table 1 ) . majority ( 70% ) of participants with hypertensive disease had secondary level education , while most ( 58.2% ) of the normotensive attained tertiary qualification . the majority ( 87% ) of higher ( 63% ) number of participants with hypertensive disease reported having consumed alcohol compared to 52.07% normotensive subjects ( table 1 ) . additionally , a strong ( = 34.33 , df = 3 ; p < 0.01 ) association between frequency of alcohol consumption and hypertension was established . the most commonly consumed alcoholic drink among hypertensive participants was beer ( 83.04% ) compared to 74.16% among normotensives . furthermore , the odds of having hypertension among participants who consumed alcohol daily and/or 1 to 3 times per week were significant ( or = 6.29 ; 95% ci = 0.66.77 , p < 0.01 ) and ( or = 3.39 , 95% ci = 0.23.43 , p < 0.05 ) , respectively . additionally , the odds of having hypertension among the drinkers of beer or spirits were significant ( or = 1.54 ; 95% ci = 0.31.6 , p < 0.05 ) and ( or = 1.2 ; 95% ci = 0.040.13 , p < 0.05 ) , respectively . smoking was prevalent among participants with hypertensive disease compared to normotensives ( table 1 ) . more ( 32% ) participants with hypertension were ex - smokers compared to 18% of normotensives . a strong ( = 11.72 , df = 2 ; p < 0.01 ) association additionally , t - test analyses showed the smoking duration that is significantly ( p < 0.05 ) longer among participants with hypertension compared to normotensives . the average duration of smoking was 2.66 and 2.14 years among hypertensive and nonhypertensive , respectively . the duration of smoking for ex - smokers was significantly ( p < 0.01 ) longer for hypertensive compared to the nonhypertensive subjects . the average duration of smoking was 2.6 and 1.8 years among the hypertensive and non hypertensive , respectively . there was a significant ( p < 0.05 ) association between consuming red meat and hypertension . the frequency of red meat consumption among hypertensive was higher ( 66.4% daily consumption ) compared to nonnormotensive ( 45.58% daily consumption ) . the majority ( 69% ) of all participants reported that their work involved lifting weights ( table 2 ) . the participants also reported having participated in cardiorespiratory endurance , flexibility , and muscular endurance as well as muscular strength activities . our findings showed that 32% of participants with hypertension compared to 21% normotensives were not involved in physical activities . there was a significant ( = 9.34 , df = 2 ; p = 0.009 ) association between type of physical activity engaged in and subjects hypertensive status . a higher percentage of normotensives respondents ( 81.18% ) reported participating in muscular endurance exercises compared to 62.94% of hypertensive participants . participants with hypertensive disease displayed higher anthropometric measurements compared to normotensives ( table 2 ) . they were more likely to be overweight ( 59.76% versus 28.24% ) or obese ( 19.53% versus 3.53% ) compared to normotensives . the association between bmi - defined obesity and hypertension was statistically significant ( p < 0.01 ) , and only 2.35% of the normotensives had central obesity compared to 32.94% of participants with hypertension ( p < 0.01 ) . the mean heart rate for participants with no hypertension was lower compared to hypertensive subjects . there was significant ( p < 0.01 ) mean difference in heart rate of 6.02 beats / minute between hypertensives ( 79.91 ) compared to normotensives ( 73.89 ) participants . in addition , participants with hypertension had higher mean systolic as well as diastolic bp ( 142.37/84.6 mmhg ) compared to normotensive ( 123.71/76 mmhg ) subjects , and indeed , the measurements of bp were significantly different . our study investigated the physiological , behavioral , and dietary characteristics associated with hypertension among kenyan defence forces . the observed characteristics were consistent with those seen in patients with hypertension in developed western countries and are in line with other studies on civilian population . this is likely to raise a debate on the role of physical activity in maintaining health and specifically the cardiovascular health . ordinarily , the military personnel ought to be physically fit , with healthy cardiovascular parameters because of their stringent healthy criteria , regular physical activity schedule , controlled military weight standards , and regular medical checkups and health awareness . our findings , however , point to the fact that hypertension may be a resultant of interplay between multiple factors . we show that aging was associated with diagnosis of hypertensive disease consistent with other studies . for instance , a study from cameroon found that the mean bp and prevalence of hypertension increased with advanced age . other studies have also implicated the risk of developing hypertension to age , with significant increase occurring among 4574 years age group . diagnosis of hypertension appeared to be lower among participants with tertiary compared to primary and secondary education . this may be attributed to health awareness and practices among participants who attained higher education . studies have shown that hypertension and its risk factors are relatively unknown to people of lower education level . additionally , higher educational achievement has been associated with greater healthcare seeking behavior and awareness that may alleviate the risk factors associated with hypertension . well educated people are more knowledgeable , practice healthy behavior , and have superior nutritional education and practices and strong stress coping mechanism which may synergize in prevention of chronic diseases . these findings are consistent with studies that have established a linear relationship between daily consumption of more than 3 drinks of alcohol and the increased risk of hypertension . recently , among the spaniards , alcohol consumption specifically beer and spirits have been implicated with high incidence of hypertension . alcohol acts to augment the vasoconstriction caused by catecholamines and vasopressin which inhibits endothelium - dependent vasodilatation resulting in high blood pressure , whereas its reduction results in a dose - dependent decline in both systolic and diastolic bp . a direct relationship between current smoking and hypertension was demonstrated with rates among smokers compared to nonsmokers . the findings concur with those of mancia and coworkers who reported that smokers had higher ambulatory bp compared to nonsmokers , but cessation of smoking failed to directly reduce the bp . these findings are consistent with wenzel and colleagues who established a similar pattern among brazilian soldiers , in which prevalence of hypertension was higher among ex - smokers . several other studies have implicated current smoking to increased bp and the risk for hypertension with the underlying mechanism due the role of nicotine in activating sympathetic nervous system and the resultant arterial stiffness [ 30 , 31 ] . for instance , in the dietary approaches to stop hypertension ( dash ) study , diet rich in fruits , vegetables , and low - fat dairy products with reduced saturated fat were shown to reduce systolic bp by 5 mmhg . furthermore , higher intake of fruits and vegetables was related to slow rise in systolic bp and diastolic bp over time regardless of age , weight , and other foods , and high - level consumption of fruits and vegetable has been implicated with lower risk of hypertension . vegetable and fruit dietary patterns are inversely associated with cvd mortality , whereas meat dietary pattern is directly associated with cvd or ischemic heart disease mortality . the relationship between fruit and vegetable dietary pattern and the risk for hypertension may be explained biochemically . flavonoids are structurally diverse compounds that exhibit cardioprotective properties which include antioxidant , anti - inflammatory , and induction of apopttoic effects [ 3639 ] . oxidative stress which is a disturbance in the equilibrium status of prooxidant / antioxidant systems in intact cells has been proposed as the cause of hypertension . the antioxidant activity and increased levels of reactive oxygen species scavenging conferred by flavonoid - rich foods such as fruits and vegetables may therefore be a plausible explanation on their hypertension reducing properties . we have shown that the frequency of red meat consumption among hypertensive participants was slightly higher . the observation is consistent with other findings which have established the role of diet low in fruits , high in fats , and red meat increases chances of hypertension and cvd . increased consumption of red meat and reduced intake of fruits among hypertensive participants in our study point toward the possible association and the risk for hypertensive disease . among cameroonian defense forces , it was also established that consumption of vegetables and fruits , tubers , and legumes was inversely associated with the prevalence of hypertension . similarly , a recent report showed an association between frequent meat and fat consumption with higher prevalence of hypertension in sub - saharan africa . the results showed an association between the type of work performed and the bp status . there was significant association between report on engagement in muscular endurance exercises and reduced chances of hypertension among participants . it is well established that physical inactivity is a risk factor for hypertension and other cvd , , and , therefore , exercise plays a critical role in maintaining optimum blood pressure . regular aerobic activity has been associated with reduced systolic blood pressure by an average of 6.9 mmhg and diastolic blood pressure of 4.9 mmhg . participants with hypertensive disease had higher anthropometric ( bmi and whr ) parameters , consistent with the strong association between higher bmi and increased risk of hypertension . the association has consistently been associated with central obesity ( an indicator of higher waist - to - hip ratio ) and abnormal lipid profiles which are key risk factors in development of hypertension . this may be attributed to stress resulting from change of climatic conditions , combat exposure , and separation from family and friends . combat exposure elicits psychological stress due to adjustment to various sociocultural background , engagement with enemy forces , encounter with wounded civilians or soldiers and maimed bodies , and also loss of colleagues , which is a fact that may explain the high prevalence of hypertension among the armed forces . this is the most recent study to investigate the physiological , behavioral , and dietary characteristics associated with hypertension among kdf . it , therefore , has a great public health importance with potential relevance for other armed forces and disciplined forces in many developing countries . the study was well structured to comprehensively capture sociodemographic , lifestyle characteristics , anthropometric measures , and dietary patterns as well as physical activity levels . we combined both survey questionnaire and objective measurement of physiological and anthropometric parameters designed to take into account complex interactions among different components and their cumulative influences on hypertension among the military personnel . more research is required to establish whether prediction rules and/or risk scores may be used to identify at - risk individuals for preventive strategy as well as targeted specific behavioral interventions among this population . the study population may not be representative of the entire armed forces in kenya because of the small sample . the participants were mainly recruited from military institutions around nairobi through afmh where they had sought treatment . they had participated voluntarily , and this may have captured a population with a greater concern and awareness for their health therefore affecting generalizability . furthermore , the assessment of smoking , alcohol consumption , dietary , and activity levels were retrospective ; therefore , recall bias may not have been completely eliminated . however , such a bias may not have been substantial as this method served to collect information from the entire study population in the same manner , and the data were obtained before the screening . the study failed to compare different variables on the basis of ranks which may have missed critical analysis on which ranks of the military are highly affected by the hypertension , an important guide to intervention . the study also missed to capture salt intake and lipids profiles among kenya military which are critical components in risk assessment . our study design failed to establish whether the combination of smoking , less physical activity , and stressful work condition results in more severe disease . finally , this was a cross - sectional study and because of the design , the causal relationship between characteristics and diagnosis hypertension may not be strongly established . in conclusion , we underscore the role of lifestyle , social habits such as smoking , alcohol consumption , and physical inactivity , higher anthropometric parameters ( bmi and whr ) , and diet rich in red meat and low in fruit for their association with hypertension . the findings of the physiological , behavioral , and dietary characteristics among kenya defence forces mimic the main risk factors and characteristics for cvd among patients in the western developed countries whose lifestyle adoption is happening fast in low and middle - income countries .

background . hypertensive disease is increasing in developing countries due to nutritional transition and westernization . hypertensive disease among kenya military may be lower because of health - focused recruitment , physical activities , routine checkups , and health awareness and management , but the disease has been increasing . purpose . the purpose of this study was to determine physiological , behavioral , and dietary characteristics associated with hypertension among kenyan military . methods . a cross - sectional study involving 340 participants was conducted at armed forces memorial hospital . participants ' history , risk factors assessment , and dietary patterns were obtained by structured questionnaire , while physiological and anthropometric parameters were measured . results . hypertensive participants were likely to have higher age , physiological , and anthropometric measurements , and they participated in peace missions . daily alcohol and smoking , frequent red meat , and inadequate fruits and vegetables were associated with hypertension . conclusions . the findings mimic the main risk factors and characteristics for hypertensive disease in developed countries whose lifestyle adoption is happening fast in low and middle - income countries . whether or not prediction rules and/or risk scores may identify at - risk individuals for preventive strategy for targeted behavioral interventions among this population require investigation .

1. Introduction 2. Methods 3. Statistical Analysis 4. Ethical Consideration 5. Results 6. Discussion

PMC4224518

multivalent drug - polymer conjugates , in which multiple copies of a drug are covalently attached to a polymeric scaffold either directly or through linker chemistry , are a highly studied pathway to improve therapeutic index . in particular , conjugation of methotrexate ( mtx ) to poly(amidoamine ) ( pamam ) dendrimer has been extensively studied , with over 100 related publications since 2002 . acetylated , neutral g5 pamam has been of particular interest as a drug delivery vector because of its narrow polydispersity index ( pdi ) , low toxicity and immunogenicity , and molecular weight ( mw ) of about 30 kda that allows it to solubilize multiple hydrophobic ligands while still being small enough to diffuse through tissue for cell - level targeting . mtx is a structural derivative and competitive inhibitor of folic acid ( fa ) ; therefore , it was proposed that mtx conjugates may also be able to provide increased binding and uptake when multivalently displayed on a polymer scaffold . has shown that multivalent g5 pamam folic acid conjugates ( g5-(cog - fa)n ) bind to fbp via a slow onset , tight - binding interaction in which fa first binds , the protein structure rearranges , and then polymer protein van der waals forces provide the final tight - binding interaction ( figure 1a ) . in other words , the tight - binding of polymer conjugate does not arise from multiple fa - protein interactions , and a single fa - fbp interaction suffices to initiate the polymer interaction by licata and tkachenko , wherein fa serves as a strong key to unlock van der waals polymer / protein interactions . for such protein ligand polymer interactions , a particularly interesting aspect to explore is key strength . when interacting with fbp , mtx is a weak key as compared to fa . as measured by surface plasmon resonance , the kd of mtx to fbp is 2 times greater than that of fa . this difference arises from a 40% decrease in ka coupled with a 30% increase in kd . however , several orders of magnitude in higher concentrations of mtx are required to trigger the observed conformational change of the fbp . mechanism of binding for ( a ) g5-(cog - fa ) conjugates to fbp mimics slow , tight binding . ( b ) g5-(cog - mtx ) conjugates cause less protein conformational change to the polymer and do not irreversibly bind . abbreviations : cog = cyclooct-1-yn-3-glycolic acid , c = polymer conjugate , p = protein . ( 1 ) do the g5 pamam mtx conjugates interact with fbp via the slow - onset , tight binding mechanism ? ( 2 ) can the weaker mtx still initiate the strong polymer protein van der waals interactions ? ( 3 ) can greater polymer size ( i.e. , dimer g5 with mw of 60 000 da vs monomer g5 mw of 30 000 da ) give rise to increased total polymer ( 4 ) does the weaker binding of mtx allow g5-mtxn to achieve multivalent binding , as previously proposed ? ( 5 ) what is the impact of tuning key strength by varying the linker connecting the key to the polymer ? in this work , we examine the individual and combined impacts of vector size variation , linker chemistry , and dendrimer valency by synthesizing monomeric and dimeric g5 conjugates with well - defined numbers of mtx attached to the dendrimer through two linker systems . recent protocols developed to isolate precise ratio ligand - to - dendrimer conjugates from stochastic distributions were employed to create conjugates with narrow , well - defined populations . surface plasmon resonance ( spr ) and isothermal titration calorimetry ( itc ) were employed to measure the impact of these parameters on g5-mtxn to fbp binding . all chemicals and materials were purchased from sigma - aldrich or fisher scientific and used as received unless otherwise specified . ethylene diamine ( eda ) core g5 pamam dendrimer was purchased from dendritech and purified into monomer ( g5 ; average molecular weight of 27.7 kda by gpc , with a range of 2630 kda as determined by mass spectrometry ) and dimer ( d ; average molecular weight of 53 kda by gpc ) samples as previously described . click - easy mfco - n - hydroxysuccinimide was purchased from berry & associates synthetic medicinal chemistry . conjugates were prepared using g5 via edc - nhs coupling . in brief , 240.4 mg of amine - terminated monomer g5 was dissolved to 0.16 m in deionized water ( di ) . a total of 12.0 mg of cog ligand was activated by dissolving to 10.5 m in acetonitrile with 2.65 equiv of edc and 2.78 equiv of n - hydroxysuccinimide and stirring for 2 h. the activated cog solution was added dropwise via syringe pump ( 0.33 ml / min ) to the dendrimer solution and allowed to stir overnight . the product was purified using amicon ultra centrifugal units , 10 kda cutoff membranes , 2 pbs washes , and 4 di washes . the material was fully acetylated ( 100% of remaining primary amines converted to acetyl groups , henceforth termed ac ) by redissolving in anhydrous methanol ( 0.19 m ) , adding 450 equiv of triethylamine and 360 equiv of acetic anhydride , and stirring for 4 h. the product mixture was purified by centrifugation and isolated by lyophillization . g5-cog4.7(avg ) was characterized by rp - uplc and h nmr ( supporting information figures s4s5 ) . click - easy mfco - n - hydroxysuccinimide was activated by dissolving to 10.5 m in acetonitrile and added dropwise via syringe pump ( 0.33 ml / min ) to the dendrimer solution . the product was purified using amicon ultra centrifugal units , 10 kda cutoff membranes , two pbs washes , and four di washes . g5-mfco2.0(avg ) was characterized by rp - uplc and h nmr ( supporting information figures s6s7 ) . g5-g5 dimer cog conjugates were prepared using edc / nhs coupling according to the procedures outlined for g5 pamam monomer . a total of 100.8 mg of dimer and 5 equiv of cog ligand were employed . d - cog4.8(avg ) was characterized by rp - uplc and h nmr ( supporting information figures s8s9 ) . g5 pamam dimer mfco conjugates were prepared using click - easy mfco - n - hydroxysuccinimide according to the procedures outlined for g5 pamam monomer . a total of 115.5 mg of dimer and 6.4 equiv of mfco ligand were employed . d - mfco6.2(avg ) was characterized by rp - uplc and h nmr ( supporting information figures s10s11 ) . g5 pamam monomer and dimer dendrimers with precise ratios of cog or mfco ligands per dendrimer were isolated via rp - hplc according to modified literature procedures . briefly , multiple injections of the stochastic conjugate containing an average ligand / dendrimer ratio were performed with a c18 column and a water / acetonitrile gradient with 0.1% tfa . eluted fractions were collected and combined to obtain g5 pamam monomer samples with precise cog / dendrimer ratio of 05 ( g5-cogx , x = 15 ) or precise mfco / dendrimer ratio of 04 ( g5-mfcox , x = 14 , 5 + ) . g5 pamam dimer samples with precise cog / dimer ratio of 02 ( d - cogx , x = 12 , 3 + ) were also obtained . products were purified using pd-10 desalting protocols with di as the equilibration buffer , dissolved in 10 pbs , and then lyophilized to dry . curve fitting of uplcs by igor pro was performed to provide yield , purity , and hplc mfco and cog averages . ( left ) rp - hplc traces and fractions collected from average conjugations of ( a ) g5-cogx , ( b ) d - cogx , ( c ) g5-mfcox , and ( d ) d - mfcox.(right ) rp - uplc traces of average conjugation ( black ) and each collected fraction . dendrimers with defined numbers of covalently bound methotrexates were synthesized via click reaction of precise ratio g5-cogn , g5-mfcon , d - cogn , or d - mfcon conjugates and -azido - mtx . briefly , dendrimer conjugates were dissolved in dmso to 40 mm with respect to the click ligand , and a 10-fold excess of -azido - mtx ( 40 mm in dmso ) was added . solutions were agitated for 48 h , then diluted to 2.5 ml with 10 pbs and purified using pd-10 desalting columns ( gravity protocols ) . further purification was performed via 10 kda cutoff dialysis against di with 16 media changes . curve fitting of uplc chromatograms provided yield , purity , and mtx averages ( supporting information figures s12s16 ) . isolation of precise ligand / dendrimer ratio conjugates was achieved using a waters 600 controller , waters 2707 autosampler , and waters 2998 photodiode array running empower 2 software , additionally equipped with a waters fraction collector iii on a phenomenex jupiter 300 c18 prep column ( 21.2 150 mm , 5 m particles ) . the weak solvent ( solvent a ) was hplc grade water with 0.1% tfa , and the strong solvent ( solvent b ) was hplc grade acetonitrile with 0.1% tfa . the gradient employed at 16 ml / min was as follows : 2.1 min load step at 95%a/5%b , 3.9 min gradient to 80%a/20%b , 15 min gradient to 65%a/35%b , 5 min gradient to 55%a/45%b , followed by 3 min was at 20%a/80%b , then equilibrating at starting conditions for 5 min before next injection . the stochastically synthesized dendrimer conjugates were dissolved to 20 mg / ml concentration and 910 l injections were used . five - second fractions were collected starting at 9 min 30 s into each run for a total of 120 fractions . analytical chromatograms were collected on a waters acquity uplc equipped with a scaled method using a phenomenex jupiter 4.6 100 mm column . uplc chromatograms were fit with gaussian peaks using igor pro version 6.0.3.1 software . h nmr spectra were obtained used 10 s preacquisition delays and a total of 64 scans . all sample solutions were set to a dendrimer concentration of 5 mg / ml in deuterium oxide . all experiments were performed on a nano itc standard volume from ta instruments ( lindon , ut ) . g5-(mfco - mtx)4.0(avg ) ( 70 , 200 , 400 m with respect to mtx ) , g5-(mfco - mtx)4 + ( 313.3 m with respect to mtx ) , free mtx ( 200 m ) , and fbp ( 4 m ) solutions were prepared in ph 7.4 pbs buffer and then degassed for 25 min . before loading , the reference cell of the itc was refilled with degassed , nanopure water every 2 days . after flushing the sample cell with buffer , the syringe was filled with the mtx solution and the sample cell was filled with the fbp solution . using itcrun software the parameters of the itc were set ( stir rate , 250 rpm ; injection interval , 1000 s ; injection volume , 12 l ; injections , 20 ; temperature , 25 c ) and the instrument was allowed to auto equilibrate before starting the titrations . controls were performed by injecting the same concentration of mtx conjugates into pbs buffer . these control runs were then subtracted from the experimental runs to account for heat from mixing or dilution and were analyzed using ta nanoanalyze software ( ver 2.4.1 ) . the area under each peak was integrated and the resulting data was modeled using an independent model fit with variables h , binding stoichiometry ( n ) , and ka . spr experiments were conducted in a biacore x instrument ( pharmacia biosensor ab ) . an immobilized folate binding protein ( fbp ) chip was prepared following the instrument prompted protocols , using a solution of 0.2 m edc and 0.05 m nhs as an activating solution , 1 mg / ml fbp solution as the immobilization solution , and ethanolamine as the deactivation solution . the chip was characterized using free fa and methotrexate solutions ranging from 0.1 to 2 mm . conjugate samples were dissolved in fresh ph 7.4 hbs - ep buffer ( fisher scientific ) at 60 m and serially diluted to 10 , 5 , 2.5 , and 1.25 m in buffer . the system was allowed to equilibrate at the beginning of each run for no less than 300 s , followed by a 2 min , 30 l ( 50555 via bubble method ) injection . the system was monitored for no less than 500 s per injection . between each run , the chip was washed with a 5 l injection of ph 1.5 buffer to remove bound materials followed by a prime step prior to the next injection . isolation of precise ligand / dendrimer ratio conjugates was achieved using a waters 600 controller , waters 2707 autosampler , and waters 2998 photodiode array running empower 2 software , additionally equipped with a waters fraction collector iii on a phenomenex jupiter 300 c18 prep column ( 21.2 150 mm , 5 m particles ) . the weak solvent ( solvent a ) was hplc grade water with 0.1% tfa , and the strong solvent ( solvent b ) was hplc grade acetonitrile with 0.1% tfa . the gradient employed at 16 ml / min was as follows : 2.1 min load step at 95%a/5%b , 3.9 min gradient to 80%a/20%b , 15 min gradient to 65%a/35%b , 5 min gradient to 55%a/45%b , followed by 3 min was at 20%a/80%b , then equilibrating at starting conditions for 5 min before next injection . the stochastically synthesized dendrimer conjugates were dissolved to 20 mg / ml concentration and 910 l injections were used . five - second fractions were collected starting at 9 min 30 s into each run for a total of 120 fractions . analytical chromatograms were collected on a waters acquity uplc equipped with a scaled method using a phenomenex jupiter 4.6 100 mm column . uplc chromatograms were fit with gaussian peaks using igor pro version 6.0.3.1 software . h nmr spectra were obtained used 10 s preacquisition delays and a total of 64 scans . all sample solutions were set to a dendrimer concentration of 5 mg / ml in deuterium oxide . all experiments were performed on a nano itc standard volume from ta instruments ( lindon , ut ) . g5-(mfco - mtx)4.0(avg ) ( 70 , 200 , 400 m with respect to mtx ) , g5-(mfco - mtx)4 + ( 313.3 m with respect to mtx ) , free mtx ( 200 m ) , and fbp ( 4 m ) solutions were prepared in ph 7.4 pbs buffer and then degassed for 25 min . before loading , the syringe and cell were each rinsed with degassed pbs buffer three times . the reference cell of the itc was refilled with degassed , nanopure water every 2 days . after flushing the sample cell with buffer , the syringe was filled with the mtx solution and the sample cell was filled with the fbp solution . using itcrun software the parameters of the itc were set ( stir rate , 250 rpm ; injection interval , 1000 s ; injection volume , 12 l ; injections , 20 ; temperature , 25 c ) and the instrument was allowed to auto equilibrate before starting the titrations . controls were performed by injecting the same concentration of mtx conjugates into pbs buffer . these control runs were then subtracted from the experimental runs to account for heat from mixing or dilution and were analyzed using ta nanoanalyze software ( ver 2.4.1 ) . the area under each peak was integrated and the resulting data was modeled using an independent model fit with variables h , binding stoichiometry ( n ) , and ka . spr experiments were conducted in a biacore x instrument ( pharmacia biosensor ab ) . an immobilized folate binding protein ( fbp ) chip was prepared following the instrument prompted protocols , using a solution of 0.2 m edc and 0.05 m nhs as an activating solution , 1 mg / ml fbp solution as the immobilization solution , and ethanolamine as the deactivation solution . the chip was characterized using free fa and methotrexate solutions ranging from 0.1 to 2 mm . conjugate samples were dissolved in fresh ph 7.4 hbs - ep buffer ( fisher scientific ) at 60 m and serially diluted to 10 , 5 , 2.5 , and 1.25 m in buffer . the system was allowed to equilibrate at the beginning of each run for no less than 300 s , followed by a 2 min , 30 l ( 50555 via bubble method ) injection . the system was monitored for no less than 500 s per injection . between each run , the chip was washed with a 5 l injection of ph 1.5 buffer to remove bound materials followed by a prime step prior to the next injection . a major challenge to detailed scientific understanding of multivalent dendrimer binding mechanisms has been the presence of trailing generations ( g1g4 ) and oligomers ( dimers , trimers , etc . ) in the dendrimer conjugates studied . recently , we have developed methods for gram - scale isolation of pamam materials as a function of dendrimer size and milligram - scale isolation as a function of conjugate valency . these methods create an opportunity to improve valency control of multivalent conjugates and improve the ability to analyze the binding mechanism of previously developed multicomponent mixtures . here , we directly compare pamam monomer and dimer mtx conjugates using spr- and itc - based measurements of binding characteristics . these experiments probe the effect of vector size both as a design principle for its own sake and to attribute binding behaviors to monomer and dimer , which were present in the materials employed in previous spr studies . in addition to valency , the influence of linker length / flexibility / hydrophobicity for the system was also explored . g5 pamam dendrimer monomer ( g5 ) and dimer ( d ) were isolated from commercially available material as previously described . stochastic conjugations to fluorinated ( mfco ) and nonfluorinated ( cog ) ring strain promoted click chemistry ligands were performed via amide coupling reactions resulting in materials containing a distribution of ligand / dendrimer ratios ranging from 0 to 13 covalently attached ligands per dendrimer . peak fitting of rp - uplc chromatograms was employed to determine the average ligand - to - dendrimer ratio for each sample . these stochastic mixtures were then isolated by semipreparative rp - hplc following previously reported protocols . the purity of the isolated , precise ratio ligand - to - dendrimer conjugates range from 90 to 100% ( as defined by ligand / dendrimer ratio dispersity ) . in addition to the precise ligand / dendrimer ratio materials , samples containing a high average number of mfco ligands ( g5-mfco6.6(avg ) and d - mfco5.3(avg ) ) and a high average of cog ligands ( d - cog6.2(avg ) ) were also obtained . importantly , these samples contain no unfunctionalized ( x = 0 ) or monofunctional ( x = 1 ) materials , making them good controls for the observation of multivalent ( e.g. , chelation or statistical rebinding ) effects . a summary of all isolated materials can be found in table 1 . these rp - hplc methods are versatile and have been successfully employed to isolate precise ratio ligand / dendrimer conjugates for both monomer and dimer pamam and using three different ligands to date . the presumed mechanism allowing this isolation protocol is a favorable interaction between the conjugated , hydrophobic ligand and the reverse - phase c18 column . the mfco ligand , which contains a longer carbon chain in addition to the fluorine on the cyclooctyne , results in significantly higher resolution than the shorter cog ligand . this is illustrated in figure 2 , panels a and c , where g5-mfco2.0(avg ) has baseline resolution between n = 0 and 1 , and n = 1 and 2 samples ( figure 2c ) , whereas g5-cog4.7(avg ) has peak overlap for all peaks , even n = 0 and n = 1 ( figure 2a ) . the difference between the x = 1 and x = 2 peak centers is 0.64 min for the g5-cog conjugates compared to 1.02 min for the mfco conjugates . the improved resolution likely arises from the greater hydrophobicity of the longer chain resulting in greater interaction with the c18 stationary phase , leading to increased retention as a function of number of ligands . the increased resolution is repeated in the dimer conjugates ; however , the resolution of both dimer species is less than the corresponding monomer . as the time difference in peak centers for x = 1 , 2 is nearly identical between the monomer and dimer for both ligands ( 0.59 and 1.04 min for dimer conjugated to cog and mfco , respectively ) , the reduced resolution is the result of the increased peak width of the dimer species ( arising from a broader mw distribution of branching defects ) . better rp - hplc resolution allows for the isolation of higher ratio materials , increased yield of all conjugates , and increased purity of resulting samples . the conjugate samples , containing g5 monomer or dimer conjugated to precise numbers ( x = 03 ) or high average ( 3 + or 4 + ) numbers of cog or mfco , were then allowed to react with -azido - mtx . a detailed analysis of each sample s fractional composition is summarized in table 2 . click efficiencies were approximately equal for monomer and dimer conjugates and for mfco and cog . because of undesired interactions of the mfco conjugates with the spr surface ( vide infra ) , the binding of g5-(mfco - mtx)n conjugates to fbp was measured by itc . the stochastically conjugated monomer g5-(mfco - mtx)4(avg ) sample displayed endothermic binding ( supporting information figure s1a ) . this indicates that binding between the conjugate and protein can occur , but there is an energetic cost . by way of contrast , there was no observable binding between the isolated high average g5-(mfco - mtx)4.4 sample , which contains only dendrimer clicked to 3 or more mtx . one interpretation of these results would be that the flexibility and hydrophobicity of the mfco linker cause it to fold into the interior regions of the g5 dendrimer . consequently , the energetic penalty to hydrate the ligand and allow for mtx / protein interaction is significantly higher , which negatively impacts the enthalpy of binding . because the lower average sample shows some interaction with the protein , it is possible that there is cooperativity within the mfco linkers that further block the mtx from interacting with the protein in the higher average sample . weak , reversible binding for g5-(cog - mtx)n ( n = 0.9 , 1.9 , 2.9 ) to surface immobilized fbp was observed at all concentrations tested . the control samples ( fully acetylated monomer and dimer with no mtx ) had similar binding to both chips , which when subtracted gave relatively flat chromatograms ( figure 3a and e ) . by way of contrast , all three tested valencies for the monomer show both a dendrimer and total mtx concentration dependent binding with the fbp surface . figure 4 reveals that g5-(cog - mtx)n exhibits greater association phase binding ( i.e. , higher signal ) as valency increases ( figure 4a ) . for all injections , the signal initially rose sharply at the beginning of the injection and quickly reached equilibrium between association and dissociation . in other words , in figure 1b ka and kd are both large and rapid as compared to k2 , and strong irreversible binding does not occur . at the end of the injection , all chromatograms quickly return to baseline , confirming the fast dissociation of all bound species . this indicates that even the divalent ( 66% ) and trivalent species ( 13% ) in the g5-(cog - mtx)2.9 experience only monovalent binding to the fbp surface . there is no multivalency effect for increasing the mtx valency ( n ) for g5-(cog - mtx)n . the data were fit to the expression r = rmax[c]/([c ] + kd ) to yield the apparent kd values of 6.3 0.4 10 m , 7.4 0.7 10 m , and 8.6 1.1 10 m for g5-(cog - mtx)0.9 , g5-(cog - mtx)1.9 , and g5-(cog - mtx)2.9 , respectively ( all fits used a common rmax value of 67 , see below ) . however , when the data is plotted as a function of total mtx concentration it fits well to a single binding isotherm ( figure 4b ) . fitting the concentration dependent data for all three samples to a single expression resulted in an rmax value of 67 8 and a kd value of 7.6 1.8 10 m. spr results of all click products . color gradients represent least concentrated ( light ) to most concentrated ( dark ) injections . ( a ) g5 ( acetylated control ) , ( b ) g5-(cog - mtx)0.9 , ( c ) g5-(cog - mtx)1.9 , ( d ) g5-(cog - mtx)2.9 , ( e ) d ( acetylated control ) , ( f ) d-(cog - mtx)1.1 , ( g ) d-(cog - mtx)2.0 , ( h ) d-(cog - mtx)6.0 . saturation of spr signal during the binding phase as a function of ( a ) conjugate concentration for monomer samples , ( b ) total mtx concentration for monomer samples , ( c ) conjugate concentration for dimer samples , and ( d ) total mtx concentration for dimer samples . the data in panels a and b is fit to the expression r = rmax[c]/([c ] + kd ) for an a + b c equilibrium . total concentration of mtx does not explain the trend in the binding phase ( figure 4d ) and a higher average number of mtx ligands per dendrimer actually show less surface binding for a given concentration of mtx . however , there is a binding phase signal dependence based on dendrimer concentration ( figure 4c ) . the data indicate that at the concentrations tested , d-(cog - mtx)n with an mtx valency of at least n = 1 , an association / dissociation equilibrium ( ka and kd as represented schematically in figure 1c ) is not reached during sample injection ( figure 3f h ) . at equilibrium , higher mtx valencies would be expected to have more total binding due to statistical rebinding effects . however , at these concentrations a single mtx / fbp interaction ( figure 3e f ) is sufficient to establish the van der waals interaction ( k2 in figure 1 ) between the dimer and protein . the amount of binding observed is therefore approximately equal for equivalent dendrimer concentrations ( figure 4c ) . with a stronger binder , such as fa ( figure 1a ) , equilibrium is established at lower concentrations and the total binding becomes dependent on total ligand concentration ( vide infra , figures 56 ) . therefore , a dependence on mtx valency can not be established for the d-(cog - mtx)n case . the higher concentrations ( to achieve equilibrium during the injection ) that would have allowed full exploration of the likely sigmoidal functional response were not tested due to conjugate solubility . quantitative fits of the type shown for figure 4a and b are not possible . however , it can be concluded that the interaction must be keyed by conjugated mtx , as there is negligible accumulation observed in the acetylated control ( d ) sample ( figure 3e ) . in this case , possible mtx valency dependent effects on binding , including statistical rebinding and chelation , do not lead to the observed greater overall conjugate binding . comparison of the spr sensograms of a d-(cog - mtx)6.0 sample to two different g5-(cog - fa)n samples , with total signal normalized . amount of bound material d-(cog - mtx)n after 500 s of dissociation is dependent on absolute mtx concentration and fbp density , not mtx valency . as indicated by supporting information figure s3 , both g5-(mfco - mtx)n and d-(mfco - mtx)n have a negative overall signal in injection / association phase as a result of nonspecific interactions with the chip surface in the control flow cell ( fc2 ) . the mtx conjugate data described above can be compared to a study of monomeric g5-(cog - fa)n ( n = 0 , 1 , 1.2 , 1.9 , 2.7 ) conjugates by van dongen , banaszak holl et al . that indicated greatly increased avidity via spr with a significant portion of bound material not dissociating over the time scale of the experiment . because this observation held true for even a purely monovalent ( folic acid - to - dendrimer ratio of 1 ) sample , it was concluded that the observation of increased avidity was due to the sum of weak van der waals interactions between the dendrimer and protein surface ( figure 1a ) . this interaction is initiated by an interaction between a conjugated fa and the fbp , which causes a conformational change in the protein exposing a more hydrophilic surface to interact with the dendrimer ( schematically represented as a color change in figure 1 ) . mtx , as a structurally modified competitive inhibitor of fa , may undergo a similar interaction with the fbp . however , although the experiments here were performed on surfaces with both higher protein densities and higher conjugate concentrations , a similar avidity increase for monomer g5-(cog - mtx)n conjugates was not observed at even the highest valency tested ( n = 3 ) . previous studies indicated only a 2 fold increased binding of free fa as compared to free mtx ; therefore , the significant reduction in binding strength of g5-(cog - mtx)n compared to g5-(cog - fa)n is most likely due to a reduction in polymer / protein interactions . mtx is a weak key to the fbp lock , and the structural rearrangement of the protein is not large enough to establish van der waals interactions for a polymer of the same size . for the mtx case , there are indications of enhanced avidity for the dimer samples ( d-(cog - mtx)n ) . however , when comparing the overall shape of the g5-(cog - fa)n and d-(cog - mtx)6.0(avg ) sensograms ( figure 5 ) it becomes clear that there are some fundamental differences . for the g5-(cog - fa)n samples , nearly all materials that bound ( to a protein density dependent saturation point ) remained permanently attached to the surface . by contrast , a large percentage of the d-(cog - mtx)n material quickly dissociates after the conjugate flow has stopped . this indicates that kd is much larger for the mtx conjugates as compared to the fa conjugates , that k2 is much smaller ( figure 1a and c ) , or that for some binding sites steric contraints prevent achieving the magnitude of polymer / protein interaction that leads to irreversible binding . the d-(cog - mtx)n conjugates show a dendrimer concentration , not mtx concentration , dependent association to the protein surface ( figure 4c ) . the absolute signal , when compared to that of the monomer / mtx conjugates , is approximately 10 higher in the dimer species at equivalent molar concentrations . a 2-fold increase may be expected by this technique due to doubling in mass . this leads to a qualitative assumption that more binding occurs in dimer samples as compared to monomer samples for equivalent total mtx concentrations . association signal is likely caused by the larger mass and radius of the dimer increasing the van der waals interactions between the dendrimer / fbp ( i.e. , an increase in k2 from negligible in the monomer data to contributing for dimer conjugates ) . chelate - type binding between multiple mtx and one or more fbp is not observed , in even the high - valency dimer samples . figure 6 indicates that the amount of irreversibly bound material ( defined as the portion of signal remaining after 500 s of dissociation ) is dependent only on the absolute mtx concentration in solution and not on conjugate valency . if chelate binding was responsible for the irreversible binding , the bivalent and high valent samples would be expected to have enhanced binding as compared to the monovalent samples at similar mtx concentrations . instead , the amount of strongly bound material has a linear dependence on mtx concentration prior to reaching a saturation at 120 response units . previous spr studies on pamam - cog - mtx conjugates ( containing mixtures of dimer and monomer ) indicated both monovalent and multivalent binding occurred for stochastic valencies of n = 5 and n = 10 . the present work indicates that the origin of the portion of binding assigned as multivalent arose from the presence of dimers conjugated to 3 or more mtx . the dendrimer material in previous studies contained 14% dimer and assuming a poisson distribution , the multivalent portion of the n = 5 and 10 samples would be 13% and 14% , respectively . new chromatographic methods have enabled the examination of vector molecular weight effects ( monomer vs dimer ) , linker system effects ( cog vs mfco ) , and valency effects on the highly studied drug delivery system of pamam dendrimer conjugated to methotrexate . the answers to the questions posed at the start of the paper can now be addressed : ( 1 ) the monomer g5 pamam - mtx conjugates show none of the irreversible binding previously attributed to multivalency for stochastic conjugate synthesized using commercial dendrimer containing oligomers of g5 . the dimer g5 pamam - mtx conjugates do show irreversible binding , including d - mtx1 , which can not bind via a multivalent mtx mechanism . the slow - onset , tight - binding mechanism is consistent with these observations . ( 2 and 3 ) although the weaker mtx does not initiate sufficient structural change for the irreversible binding to occur between fbp and monomer g5 , the larger dimer of g5 interacting with the protein is able to generate a large enough interaction for irreversible binding . ( 4 ) it has been suggested that weaker linkers could provide longer surface diffusion times and , thus , potentially allow multivalent ligand binding . ( 5 ) we attempted to tune key strength by varying the linker ; however , this resulted in a number of changes including differential solution binding enthalpy likely related to hydrophobicity and undesired binding to control surfaces . in summary , the preparation of controlled valency monomer g5 and dimer g5g5 conjugates , demonstrate that multivalency is not an active binding mechanism for these conjugates and a slow , onset tight binding mechanism is proposed that is consistent with mechanistic hypotheses for the analogous fa conjugates and the mechanistic proposals of licata and tkachenko .

generation 5 poly(amidoamine ) ( g5 pamam ) methotrexate ( mtx ) conjugates employing two small molecular linkers , g5-(cog - mtx)n , g5-(mfco - mtx)n were prepared along with the conjugates of the g5-g5 ( d ) dimer , d-(cog - mtx)n , d-(mfco - mtx)n . the monomer g5-(cog - mtx)n conjugates exhibited only a weak , rapidly reversible binding to folate binding protein ( fbp ) consistent with monovalent mtx binding . the d-(cog - mtx)n conjugates exhibited a slow onset , tight - binding mechanism in which the mtx first binds to the fbp , inducing protein structural rearrangement , followed by polymer protein van der waals interactions leading to tight - binding . the extent of irreversible binding is dependent on total mtx concentration and no evidence of multivalent mtx binding was observed .

Introduction Experimental Section Methods Results and Discussion Conclusions

PMC4158300

the glomerular filtration rate ( gfr ) is the most widely used indicator of overall renal function . the gfr can be measured by clearance of an ideal , usually exogenous , filtration marker such as inulin , iothalamate , edta , diethylene triamine pentaacetic acid , or iohexol . the clearance of endogenous markers such as creatinine or blood urea nitrogen can overestimate or underestimate the gfr . all these methods have the drawback to be complex and require 24-hour urine collection , which is not always practicable in day - to - day clinical practice . a more pragmatic approach is to estimate gfr from equations based on serum creatinine [ 1 , 2 ] . cystatin c is a nonglycosylated protein with low molecular mass ( 13.3 kda ) generated by all nucleated cells of the body at a constant rate , is freely filtered by the glomerulus , and is not secreted by renal tubules but completely reabsorbed with subsequent degradation by proximal tubular cells [ 4 , 5 ] . for those properties compared with serum creatinine , cystatin c levels are less dependent on ethnicity , sex , age , and muscle mass or protein intake . moreover , as serum creatinine , cystatin c is an independent predictor of cardiovascular and overall mortality . compared to gfr measurement based on the renal clearance of exogenous markers , estimated glomerular filtration rate ( egfr ) is more precise if derived from both cystatin c and creatinine levels in serum . on the other hand , measurement of cystatin c is costly and egfr based on cystatin c needs further validation across a broad spectrum of populations with or without chronic kidney disease . the purpose of our current study was to compare estimates of egfr based on cystatin c and creatinine in randomly recruited uruguayans , using egfr derived from cystatin c as reference . genotipo fenotipo y ambiente de la hipertension en uruguay ( gefa - ht - uy ) is a prospective cohort study started in april 2012 conducted by the unidad de hipertensin arterial , hospital de clnicas dr . the ethics committee of the university hospital approved the study protocol and all participants gave informed written consent . the aim of the study is to explore the relation of blood pressure with genetic and environmental factors in a representative uruguayan population sample . nuclear families were randomly recruited from the inhabitants of a geographically defined area , the juana de amrica housing project , located about 10 km from downtown montevideo . a nuclear family had to include at least one parent and two siblings . family members living at the same address or within a distance of no more than 10 km were eligible . 149 people had participated , of whom we excluded 30 from the present analysis , because either cystatin c or creatinine had not been measured ( n = 22 ) or because they had not yet completed the physical examination ( n = 8) . trained observers administered a standardised questionnaire inquiring into each participant 's medical history , smoking and drinking habits , and intake of medications . after participants had rested for 5 minutes in the sitting position , the observers obtained five consecutive blood pressure readings ( phase v diastolic pressure ) to the nearest 2 mm hg , using mercury sphygmomanometers . standard cuffs had a 12 24 cm inflatable portion , but if upper arm girth exceeded 31 cm , larger cuffs with 15 35 cm bladders were used . hypertension was an office blood pressure of at least 140 mm hg systolic or 90 mm hg diastolic or use of antihypertensive drugs . the observers measured body height to the nearest 0.5 cm with a pliable measurer and the participant standing against the wall . venous blood samples were obtained after at least 12 hours fasting and were kept at 4c and within 2 hours period were analysed for serum levels of cystatin c , creatinine , cholesterol , and glucose . diabetes mellitus was the use of anti - diabetic drugs or a fasting glucose 126 mg / dl ( 7 after the participants had rested 15 min in the supine position , we recorded during an 8 s period the radial waveforms at the right side by applanation tonometry . we used a high - fidelity spc-301 micromanometer ( millar instruments , houston , tx ) interfaced with a computer running sphygmocor software , version 8.2 ( atcor medical , west ryde , new south wales , australia ) . we discarded recordings when the systolic or diastolic variability of consecutive waveforms exceeded 5% or the amplitude of the pulse wave signal was less than 80 mv . we calibrated the radial pulse wave on the brachial blood pressure . from the radial signal , the sphygmocor software calculates the aortic pulse wave by means of a validated generalised transfer function [ 10 , 11 ] . the augmentation index was the ratio of the second to the first peak of the pressure wave expressed as a percentage . aortic pulse wave velocity was measured by sequential ecg - gated recordings of the arterial pressure waveform at the carotid and femoral arteries . we measured the distances from the suprasternal notch to the carotid sampling site ( distance a ) and from the suprasternal notch to the femoral sampling site ( distance b ) . pulse wave travel distance was calculated as distance b minus distance a. pulse transit time was the average of 10 consecutive beats . pulse wave velocity was the distance in meters divided by the transit time in seconds . serum cystatin c was measured by a particle - enhanced turbidimetric immunoassay ( petia ) , ( cobas , roche diagnostics , germany ) . the latex enhanced particles coated with anticystatin c antibodies in the reagent agglutinate with the human cystatin c in the sample . the degree of the turbidity caused by the aggregate was determined turbidimetrically at 546 nm . this assay has a detection limit of 0.4 mg / l and a coefficient of variation of 1.3% . serum creatinine was measured by modified kinetic jaff methods with the modifications described elsewhere [ 13 , 14 ] . the detection limit is 0.17 mg / dl and the coefficient of variation was 1.6% . we use a creatinine method that has calibration traceable to an idms reference measurement procedure according to present recommendations [ 15 , 16 ] . first , we computed egfr from serum cystatin c egfrcys , as described by inker and colleagues . next , we calculated egfr from serum creatinine according to the idms - traceable mdrd study equation ( mdrd ) [ 17 , 18 ] formula ( egfrmdrd ) or the chronic kidney disease epidemiology collaboration ( ckd - epi ) equation ( egfrepi ) . finally , as proposed by inker and colleagues , we also derived egfr from both serum cystatin c and serum creatinine ( egfrmix ) . all aforementioned estimates [ 13 ] account for sex and age and with the exception of egfrcys also consider ethnicity ( black versus nonblack ) . this particular characteristic was irrelevant for our current study as our participants only included whites mainly of european descent . table s1 in the online data supplement provides detailed information on each formula ( see table s1 in supplementary material available online at http://dx.doi.org/10.1155/2014/837106 ) . in our current analyses , we compared findings based on the various methods to estimate gfr against egfrcys as the referent method . low glomerular filtration rate ( l - gfr ) was an egfr < 60 ml / min/1.73 m based on a single determination of each biomarker . for database management and statistical analysis , we used sas software , version 9.3 ( sas institute , cary , nc ) . first , in exploratory analyses , we assessed the characteristics of participants by fourths of the distribution of egfrcys . for comparison of means and proportions , we applied student 's t - test ( or anova ) and the statistic , respectively . we assessed agreement between paired measurements on a continuous scale by bland and altman 's method . to allow comparison with literature data , we also computed correlation coefficients . the national kidney foundation kdoqi guideline proposes a threshold of 60 ml / min/1.73 m to diagnose chronic kidney disease . in categorical analyses , we , therefore , also assessed the agreement between equations to dichotomize subjects in l - gfr or not - l - gfr using cohen 's kappa statistic . a kappa value of 0.20 or less indicates slight agreement , 0.20 to 0.40 fair agreement , 0.41 to 0.60 moderate agreement , 0.61 to 0.80 substantial agreement , and 0.81 to 1.00 almost perfect agreement . we studied the association between the four definitions of egfr dichotomized at 60 ml / min/1.73 m in hypertensive and diabetic subjects by mcnemar 's test for paired comparisons of proportions . because of the low frequencies in some cells , we applied exact statistics in two - by - two tables . finally , we assessed the added capacity of egfrcys to differentiate between normotension versus hypertension or between people with or without diabetes mellitus , using the integrated discrimination improvement ( idi ) and the net reclassification improvement ( nri ) [ 22 , 23 ] . the 119 participants included 68 women ( 57.1% ) and 53 ( 44.5% ) hypertensive patients , of whom 35 ( 66.0% ) were on antihypertensive drug treatment . among 68 women and 51 men , 11 ( 16.2% ) and 5 ( 9.8% ) were smokers ; 21 women ( 30.1% ) and 32 men ( 62.7% ) reported intake of alcohol . in smokers , median tobacco use was 10 cigarettes per day ( interquartile range , 615 ) . in drinkers , the median alcohol consumption was 8 g per day ( interquartile range , 454 ) . in the whole study population , age ( sd ) averaged was 56.5 ( 17.3 ) years and systolic and diastolic blood pressure 126.5 ( 19.6 ) mm hg and 79.6 ( 11.7 ) mm hg , respectively . based on a self - report of the main maternal and paternal background , 37 participants ( 31.1% ) reported a mixture of caucasian , african , or native - american , while 82 participants ( 68.9% ) reported coincident caucasian background . among all participants , serum cystatin c and serum creatinine averaged from 0.99 ( 0.22 ) mg / l to 0.81 ( 0.21 ) mg / dl with no difference between women and men for cystatin c ( 0.99 versus 1.00 mg / l ; p = 0.77 ) , whereas women had lower serum creatinine than men had ( 0.72 versus 0.93 mg / dl ; p < 0.0001 ) . in all participants , mean values were 80.0 ( 23.8 ) ml / min/1.73 m for egfrcys , 89.7 ( 22.5 ) ml / min/1.73 m for egfrmdrd , 91.5 ( 19.0 ) ml / min/1.73 m for egfrepi , and 85.6 ( 20.2 ) ml / min/1.73 m for egfrmix , with no sex differences ( p 0.23 ) . table 1 provides the characteristics of participants by fourths of the distribution of egfrcys , which was used as reference . the prevalence of hypertension ( p = 0.027 ) , age ( p < 0.0001 ) , and systolic blood pressure ( p = 0.0097 ) , but not diastolic blood pressure ( p = 0.79 ) or mean arterial pressure ( p = 0.18 ) increased ( p = 0.027 ) with lower egfrcys category . the central systolic augmentation index and aortic pulse wave velocity also rose ( p < 0.0001 ) across decreasing fourths of the egfrcys distribution . trends in egfrmdrd , egfrepi , and egfrmix ran in parallel with the distribution of egfrcys . figure 1 shows the bland and altman plots comparing egfrmdrd , egfrepi , and egfrmix , with egfrcys as the referent method . table 2 shows the mean deviations of egfrmdrd , egfrepi , and egfrmix from egfrcys . in all participants , egfrmdrd , egfrepi , and egfrmix were , respectively , 9.7 , 11.5 , and 5.6 ml / min/1.73 m higher than egfrcys . the corresponding 2 sd intervals expressed in ml / min/1.73 m ( figure 1 ) ranged from 38.5 to + 57.9 for egfrmdrd , 25.5 to + 48.5 for egfrepi , and 10.2 to + 21.4 for egfrmix , and the corresponding correlation coefficients were 0.04 ( p = 0.69 ) , 0.26 ( p = 0.0046 ) , and 0.40 ( p < 0.0001 ) , respectively . analyses stratified according to sex , age , normotension versus hypertension , and absence versus presence of diabetes mellitus were consistent with those in all participants ( table 2 ) . figure 2 shows that across fourths of the distribution of egfrcys , egfrmdrd , egfrepi , and egfrmix were consistently higher ( p < 0.002 ) than egfrcys except in the highest category of egfrcys ( p = 0.25 ) . the prevalence of l - gfr was the highest for egfrcys , intermediate for egfrmix , and the lowest for egfrmdrd and egfrepi ( table 3 ) . using egfrcys as reference , cohen 's kappa statistic was 0.230 ( 95% confidence interval [ ci ] , 0.036 to 0.427 ; p = 0.0005 ) for egfrmdrd , 0.151 ( ci , 0.021 to 0.322 ; p = 0.032 ) for egfrepi , and 0.587 ( ci , 0.399 to 0.775 ; p < the prevalence of l - gfr was higher ( p < 0.0001 ) if patients were categorized based on egfrcys ( 17 patients , 32.1% ) compared with egfrmdrd ( 4 patients , 7.5% ) , egfrepi ( 3 patients , 5.7% ) , or egfrmix ( 8 patients , 15.1% ) . among 20 diabetic patients , we observed a similar trend . the prevalence of l - gfr was 8 patients ( 40.0% ) based on egfrcys , 2 patients ( 10.0% ) based on egfrmdrd , 2 patients ( 10.0% ) based on egfrepi , and 3 patients ( 15% ) based on egfrmix . however , the differences with egfrcys did not reach formal statistical significance ( p 0.075 ) . finally , we explored whether an egfrcys below 60 ml / min/1.73 m improved the differentiation between normotension versus hypertension or between people without or with diabetes mellitus based on the other estimates of gfr . however , the classification based on egfrcys did not improve idi ( p 0.53 ) or nri ( p 0.24 ) for hypertension or idi ( p 0.37 ) or nri ( p 0.24 ) for diabetes mellitus . in our current analysis , we compared the performance of the equations based on cystatin c and creatinine to estimate gfr in a uruguayan population sample . the uruguayan population has been considered as mainly european descent , with a negligible native american or african contributions . however , based on serological and molecular markers , recent studies demonstrate that native american and african had an important influence in the conformation of the present one [ 2426 ] . the key finding was that egfrcys provides lower estimates in comparison with creatinine based equations ( egfrmdrd and egfrepi ) . mean egfr was 80.0 , 89.7 , 91.5 , and 85.6 ml / min/1.73 m for egfrcys , egfrmdrd , egfrepi , and egfrmix , respectively . thus , the prevalence of l - gfr , was higher when derived from equations involving cystatin c ( egfrcys and egfrmix ) than when derived from creatinine based equations . the prevalence of l - gfr using egfrcys , egfrmdrd , egfrepi , and egfrmix was 21.8% , 5.9% , 3.4% , and 11.8% , respectively . in categorical analysis , the agreement between cystatin - based ( egfrcys ) equations and creatinine - based equations ( egfrmdrd and egfrepi ) to detect egfr under 60 ml / min/1.73 m was low . over the past , many reports highlighted the ability of cystatin c to detect renal disease early in different settings [ 2731 ] . several researchers reported cystatin c outperforms serum creatinine in the early diagnosis of acute kidney injury [ 32 , 33 ] or its prognosis . in line with our results , other investigators reported lower estimates of egfr using cystatin c as biomarker [ 6 , 35 , 36 ] . in contrast , in a belgian population sample [ n = 4,189 ] , delanaye and colleagues report that the prevalence of egfr below 60 ml / min/1.73 m using egfrcys , egfrmdrd , egfrepi , and egfrmix was 4.7% , 13.0% , 9.8% , and 5.0% , respectively . in a recent report , among 394 old ( > 74 y ) subjects and patients from england , kilbride and colleagues describe lower egfrcys ( 55.2 ml / min/1.73 m ) in comparison with creatinine based equations ( egfrmdrd , 57.6 ml / min/1.73 m ; egfrepi , 57.0 ml / min/1.73 m ) . grams and colleagues , analyzing representative subsamples of the adult participants in the national health and nutrition examination surveys ( nhanes ; 19881994 [ n = 15,133 ] and 19992002 [ n = 8,238 ] ) , observed slightly higher egfr using cystatin c than creatinine equations ( 102.9 versus 99.4 ml / min/1.73 m ) . however , in line with our results , the proportion of subjects with egfr below 60 ml / min/1.73 m was larger if derived from cystatin c than creatinine ( 5.5% versus 4.7% ) . more recently , tsai and colleagues reported similar results in diabetic patients enrolled in nhanes . the mean egfrcys was 100.7 ml / min/1.73 m and the egfrepi was 95.1 ml / min/1.73 m while the proportions of patients with egfr below 60 ml / min/1.73 m were 22.0% and 16.5% , respectively . pattaro and colleagues also found similar results in a cross - sectional analysis of a population from three alpine villages . finally , in a recent meta - analysis shlipak and colleagues noticed similar mean egfrcys and egfrepi estimates while the proportion of subjects under the threshold to define ckd was 13.7 and 9.7 ml / min/1.73 m using cystatin and creatinine based equations , respectively . in line with our current results , delanaye described low agreement between creatinine and cystatin c based formulas ( kappa statistic 0.32 [ egfrcys versus egfrmdrd ] and 0.39 [ egfrcys versus egfrepi ] ) . in this belgian study , the percentage of discordant subjects estimated by egfrmdrd , egfrepi , and egfrmix was 11.2% , 8.29% , and 2.71% , taking egfrcys as reference . how to explain the discrepancies between cystatin c and creatinine based equations ? we analysed creatinine in fresh serum samples and cystatin c in samples that were kept frozen at 80c . over a period of 10 years , a decay in cystatin c levels occurs using a particle - enhanced nephelometric assay ( penia ) . such decay is not observed if one uses the more robust particle - enhanced turbidimetric assay ( petia ) . furthermore , our samples were analysed within one year after blood collection and were processed with calibration each time . we used a validated method [ 40 , 41 ] and reagents ( tina - quant cystatin c gen . 2 ) standardized to the international reference material erm - da 471/ifcc , as currently recommended for the use of ckd - epi equations [ 3 , 7 ] . studies of bias ( mean difference from reference method ) usually overestimated gfr compared with the reference when using mdrd ( range 1.0 to + 3.5 ) or ckd - epi ( range 0.23 to + 4.4 ) and underestimated when using cystatin c based equations ( range 5.7 to 1.2 ) [ 38 , 4244 ] . fewer studies showed a lower positive bias of cystatin c than ckd - epi based equations [ 7 , 45 ] . several epidemiological studies showed that cystatin c is a better predictor of outcomes in coronary heart disease , acute coronary syndrome , and heart failure , independently of serum creatinine and gfr estimation [ 6 , 4649 ] . furthermore , peralta et al . demonstrated in a large and ethnically diverse population that subjects with decreased egfrcys had elevated risk of death , cardiovascular disease , and heart failure and had an elevated risk of kidney failure [ 50 , 51 ] . in keeping with these studies , we found a higher prevalence of l - gfr among hypertensive subjects ( p < 0.001 ) if they were categorized based on egfrcys ( 32.1% ) than on egfrepi ( 5.7% ) with a similar but not significant trend among the few diabetics patients . however , when computing idi and nri we did not observe significant differences between the various estimates of egfr for hypertension or diabetes ( p 0.24 ) . in accordance with previous reports [ 48 , 5254 ] , the prevalence of hypertension ( p = 0.27 ) and age ( p < 0.0001 ) , systolic blood pressure ( p < 0.01 ) , cholesterol ( p = 0.004 ) , aortic pulse wave velocity ( p < 0.0001 ) , and the central augmentation index ( p < 0.0001 ) increased with lower categories of egfrcys ( table 1 ) . our results should be interpreted within the limitation of the study . first , we did not have a reliable gold standard due to the variability in 24 h urine collections . performing inulin or iothalamate clearance implies invasive and tedious procedures that are not suitable for our population study . second , participants of the study may not be representative of the uruguayan population , because we randomly sampled a neighbourhood and our participants had a higher cardiovascular risk profile than the general population of uruguay . finally , the small sample size of our population is a limiting factor to analyse specific subgroups of participants . however , the number of participants is large enough to describe the difference between egfr estimating equations . in conclusion , to our knowledge , this is the first report based on a population from south america comparing to the performance of egfr equations based on cystatin c and creatinine . we confirm discrepancies in egfr using equation based on different biomarkers , particularly in the range of gfr under 60 ml / min/1.73 m. generally , the equation based on cystatin c , compared with creatinine , results in lower egfr values and , therefore , higher estimates of the prevalence of egfr below 60 ml / min/1.73 m.

background . estimation of glomerular filtration rate ( egfr ) from biomarkers has evolved and multiple equations are available to estimate renal function at bedside . methods . in a random sample of 119 uruguayans ( 54.5% women ; 56.2 years ( mean ) ) , we used bland and altman 's method and cohen 's kappa statistic to assess concordance on a continuous or categorical ( egfr < 60 versus 60 ml / min/1.73 m2 ) scale between egfrcys ( reference ) and egfr derived from serum creatinine according to the modification of diet in renal disease ( egfrmdrd ) or the chronic kidney disease epidemiology collaboration equations ( egfrepi ) or from both serum cystatin c and creatinine ( egfrmix ) . results . in all participants , egfrmdrd , egfrepi , and egfrmix were , respectively , 9.7 , 11.5 , and 5.6 ml / min/1.73 m2 higher ( p < 0.0001 ) than egfrcys . the prevalence of egfr < 60 ml / min/1.73 m2 was the highest for egfrcys ( 21.8% ) , intermediate for egfrmix ( 11.8% ) , and the lowest for egfrmdrd ( 5.9% ) and egfrepi ( 3.4% ) . using egfrcys as reference , we found only fair agreement with the equations based on creatinine ( cohen 's kappa statistic 0.15 to 0.23 ) . conclusion . using different equations we reached clinically significant differences in the estimation of renal function . egfrcys provides lower estimates , resulting in higher prevalence of egfr < 60 ml / min/1.73 m2 .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

PMC3333768

rhomboid proteases are the largest family of enzymes that hydrolyze peptide bonds within the cell membrane . although discovered to be serine proteases only a decade ago , rhomboid proteases are already considered to be the best understood intramembrane proteases . the presence of rhomboid proteins in all domains of life emphasizes their importance but makes their evolutionary history difficult to chart with confidence . the near ubiquity of rhomboid proteases across evolution suggests broad , organizational roles that are not directly essential for cell survival . functions have been deciphered in only about a dozen organisms and fall into four general categories : initiating cell signaling in animals , facilitating bacterial quorum sensing , regulating mitochondrial homeostasis , and dismantling adhesion complexes of parasitic protozoa . although in no organism has the full complement of rhomboid function yet been elucidated , links to devastating human disease are emerging rapidly , including to parkinson 's disease , type ii diabetes , cancer , and bacterial and malaria infection . rhomboid proteases are unlike most proteolytic enzymes , because they are membrane - immersed ; understanding how the membrane immersion affects their function remains a key challenge . proteolysis within the membrane was discovered in seemingly rare contexts nearly 15 years ago [ 1 - 3 ] . it is now widely appreciated that this fascinating regulatory paradigm permeates most areas of modern cell biology [ 4 - 7 ] . of the three protease families that catalyze intramembrane proteolysis , rhomboid enzymes are the only family that were not discovered from the direct study of human disease . the name ' rhomboid ' has its origin deep in the rich folklore of drosophila genetics . rhomboid emerged from the historic quest to identify all genes required to organize construction of a free - living organism from a single cell . because genes were named after the altered appearance of the mutant larval cuticle , the mis - shaped , rhombus - like head skeleton of the mutant embryo earned rhomboid its name . mutating the growth factor that rhomboid activates yielded indistinguishable head - skeleton defects , and was named spitz ( ' pointed ' in german ) . the rhomboid gene was cloned and sequenced by bier and colleagues in 1990 , revealing a seven transmembrane ( 7tm ) protein with no homology to any sequence known at the time . the spitz sequence was more informative , encoding a clear epidermal growth factor ( egf)-like protein . the fact that rhomboid mirrored spitz phenotypically , and encoded a seven tm protein , led to the proposal that it might be a serpentine receptor for spitz signaling . but as sequencing of genomes from diverse organisms began to reveal rhomboid homologs in every form of cellular life , it became clear that rhomboid proteins may be at the core of very diverse biological regulation . sequence analysis , however , yielded no clues about the underlying biochemical function of rhomboid proteins , and no other homologs were as well studied as drosophila rhomboid . a decade of drosophila genetics had , however , set the stage for a biochemical approach : rhomboid was definitively implicated as an upstream activator of spitz in the signal - sending cell , providing a framework for analyzing its molecular function [ 12 - 15 ] . a focused analysis of spitz activation eventually yielded four key pieces of the puzzle : ( i ) substochiometric levels of rhomboid triggered spitz proteolysis , implying that rhomboid acts enzymatically ; ( ii ) proteolysis depends absolutely only on four rhomboid residues , and their identity is consistent with serine protease catalysis ; ( iii ) spitz proteolysis is blocked only by serine protease inhibitors ; and ( iv ) spitz is cleaved within its tm segment at a depth similar to that of the putative rhomboid catalytic serine . these pieces fit together into a model in which rhomboid acts as an intramembrane serine protease for spitz , which was confirmed 4 years later by reconstituting cleavage with pure proteins [ 17 - 19 ] . the ability to study spitz proteolysis as a direct test of rhomboid activity was used to determine that even distant bacterial homologs are functional intramembrane serine proteases . most bacterial species are now known to encode one rhomboid protease , while some encode two , and very few encode three . rhomboid proteases are also present in many if not most archaea , but the greatest expansion occurred in multicellular organisms and some parasitic protozoa . although the human , mouse and drosophila genomes encode at least seven rhomboid genes , the largest number of rhomboid genes are encoded by plants ( 13 in arabidopsis ) , which do not have egf signaling [ 21 - 23 ] . in many of these diverse organisms , at least one rhomboid has directly been demonstrated to have proteolytic activity ( table 1 ) [ 16,20,23 - 26 ] . known rhomboid protease substrates and functions across evolution only those rhomboid proteases whose proteolytic activity has been detected are included in the table . rhomboid proteases are found in all branches of life , yet the sequence identity across all family members is strikingly low , around only 6% . we suggest that this is not despite rhomboid proteases being so widespread but because of it . this divergence is exacerbated by their sequences being predominantly transmembrane and thus experiencing a different evolutionary pressure . this has made phylogenetic analyses noisy , yielding few incontrovertible conclusions and inevitably fueling debate . of particular intrigue is their evolutionary origin : rhomboid proteins have been argued to be perhaps the most widely distributed membrane proteins in nature ( figure 1 ) . this near ubiquity is instinctively viewed as evidence of an ancient enzyme family that evolved early . although this is likely if the last universal common ancestor already encoded several different rhomboid proteins , phylogenetic analysis has also raised the possibility of a different history in which rhomboid proteins are a later invention of bacteria that rapidly spread to most other organisms . this scenario requires a controversial amount of horizontal gene transfer to populate all kingdoms of life . currently the true nature of rhomboid phylogenetic history remains a point of inconsolable debate , but three observations serve as valuable guiding principles . a subset of 109 rhomboid and derlin family protein reference sequences , retrieved from the ncbi refseq database , was chosen to illustrate their diversity . mega 5.05 was used to align sequences by muscle and construct an unrooted neighbor - joining phylogenetic tree . branches are labeled according to their common characteristics and are shaded or outlined to denote active or inactive protease sequences , respectively . individual sequence names are colored black , blue , or red to indicate a 6tm , 6 + 1tm , or 1 + 6tm arrangement , respectively , with each refseq accession number included within parentheses . despite the tremendous number and diversity of rhomboid proteins , structures of only two 6tm the first organizing principle emerging from sequence analysis is the observation that rhomboid proteases come in three distinct topological flavors ( figure 2 ) . the simplest consists of the 6tm core , which itself is the smallest catalytically active unit . this form predominates in bacteria , but is also represented , albeit more rarely , in eukaryotic organisms , including animals . to this basic unit eukaryotes add a seventh tm segment following the 6tm core ( 6 + 1tm form ) . five of the seven drosophila , human , and mouse rhomboid proteins are of this form lastly , a distinct form of 7tm rhomboid proteases exists in endosymbiotic organelles , adding the seventh tm preceding the 6tm core ( 1 + 6tm form ) . the best studied are those imported into mitochondria [ 28 - 30 ] , although interest in plastid - resident rhomboid proteins has recently been sparked . although the sequence analysis is clear on these three topological distinctions , their functional relevance is unclear . the expectation is that they confer different biochemical properties , although current evidence , albeit limited , indicates that many bacterial 6tm forms and eukaryotic 6 + 1tm forms show similar activity against surrogate substrates , including spitz . the smallest , catalytically active form of a rhomboid protease consists of a 6tm core , with variable amino termini ( dashed lines ) . most eukaryotic and mitochondrial rhomboid proteases have an additional tm segment , added either carboxy - terminally ( eukaryotes , blue ) or amino - terminally ( mitochondria , red ) to the 6tm core , as depicted . catalytic residues are in yellow for nucleophilic chemistry ( hydrolysis ) and white for electrophilic residues ( oxyanion transition state stabilization ) . although protease activity has been reconstituted with both the 6tm and 6 + 1tm rhomboid forms in vitro , it is only a 6tm form called glpg from escherichia coli and haemophilus influenzae that has proven amenable to structural analysis [ 34 - 37 ] . this major breakthrough - the first atomic - resolution structure of any intramembrane protease - not only confirmed that proteolysis is intramembrane and catalyzed by a serine protease apparatus , but revealed an unanticipated and complex architecture . although a thorough description is beyond the scope of the current discussion ( see for a comprehensive review ) , two features are characteristic ( figure 3 ) : although most tm helices are long and run roughly perpendicular to the membrane , the fourth tm segment runs slanted relative to the others and enters the center of the protein as an extended loop , converting to an helix at the catalytic serine . more unexpected was the orientation of the long l1 loop connecting tms 1 and 2 , which forms a lateral hairpin that lies half submerged in the membrane . this feature , which has not been encountered before or since , has major structural implications and results in a highly asymmetric protein . it is assumed that the structure of the other rhomboid forms will be analogous , and recent modeling of the mitochondrial 1 + 6tm form on e. coli glpg hints at an unanticipated level of similarity . the crystal structure of the 6tm core of the e. coli rhomboid protease glpg ( pdb 2nrf molecule a ) is shown from three vantage points ( ' top view ' is looking at the cell from the outside with the membrane in the plane of the page ) . the protein forms a compact helical bundle , with two characteristic features . a short and slanted tm4 ( black ) forms a helix below the catalytic serine ( circled in the ' back ' view ) , but an extended loop ( l3 ) above it . the l1 loop ( purple ) forms a hairpin structure that nestles between tms 1 and 3 and protrudes laterally into the outer leaflet of the membrane ( red dashed lines representing the membrane interface are provided only for reference ) . catalytic dyad residues serine and histidine are in cyan ; putative oxyanion - stabilizing electrophilic asparagine and histidine residues are in red . in addition to the number of tms , two further variations provide potential for additional rhomboid diversity . first , in all three forms , the cytosolic amino termini are highly variable , ranging from large domains to being non - existent . the implications , however , remain unclear , at least partly because achieving well - diffracting crystals required absence of this domain , making its relationship to the catalytic core speculative . on the simplest level two predominant clusters are a distinct 6 + 1tm form in animals , called irhom proteins , and a 6tm form that is represented widely in eukaryotes by the derlin proteins [ 42 - 45 ] . both of these have been implicated in endoplasmic reticulum - associated degradation ( erad ) . derlins have clear sequence homology near the membrane - submerged l1 loop , but also less conspicuously along their entire length , and are thus likely to adopt a glpg - like 6tm structure . although clearly not proteolytic , their potential similarity to other aspects of the rhomboid protease mechanism should not be discounted at this early stage ( but lie beyond the scope of this review ) . the second guiding principle stems from the tremendous diversity of organisms that encode rhomboid enzymes . since these include organisms that do not encode any known forms of cell - to - cell communication , sequence information implies that rhomboid proteins perform an ancient and fundamental role in cell biology . this function is not essential for cell survival , however , because several lineages are missing rhomboid genes entirely , presumably by gene loss . although defining the cellular functions of rhomboid proteases has proven a persistent challenge , focused investigations have succeeded in documenting the function of at least one rhomboid in nearly a dozen organisms ( table 1 ) . these functions are usually regulated by substrate trafficking , and fall into four broad categories ( figure 4 ) . rhomboid-1 is localized in the golgi apparatus , and cleaves spitz ( green ) after it is transported from the endoplasmic reticulum by star ( purple ) . top right : the mitochondrial rhomboid parl cleaves pink1 to reduce parkin recruitment to mitochondria and downregulate mitophagy . bottom right : malaria parasite - encoded rhomboid proteases cleave adhesins to disassemble the junction formed between parasite and host erythrocyte at the end of invasion . note that adhesins ( in black ) , initially held in internal organelles , encounter rhomboid only when they are secreted onto the surface and motored to the posterior of the parasite . bottom left : the providencia rhomboid protease aara activates tata by removing a small amino - terminal extension . this allows tata to assemble into the machinery required for protein ( and presumably quorum - sensing signal ) export . in the left two roles rhomboid cleavage activates a latent factor first , rhomboid proteases initiate animal cell signaling by releasing growth factors from the membrane . this function emerged from detailed genetic study of drosophila development ; rhomboid proteases are localized in the golgi apparatus and act as the signal - generating component by cleaving spitz to initiate the pathway in neighboring cells . although a role in regulating egf signaling is also seen in caenorhabditis elegans vulval development , cerom-1 has a surprisingly minor role as a target of egf signaling that sets up a paracrine loop to amplify and spread the signal . even less is clear in mammals : recent investigations have localized rhomboid proteins to the secretory pathway and cell surface and begun to uncover increased rhomboid expression in cancer cells with potential links to growth factor signaling . however , this is not limited to active rhomboid proteases ; expression of the irhom rhbdf1 , which is localized in the endoplasmic reticulum in human epithelial cancer cells , increased secretion of the egf ligand transforming growth factor- . accordingly , rhbdf1 silencing decreased pathway activation through egf receptor ( egfr ) , erk and akt phosphorylation , and limited tumor growth in mice . the drosophila homolog , however , was recently found to have the opposite effect of decreasing egfr signaling by promoting the erad - mediated degradation of egf ligands . the basis of this remarkable discrepancy is currently unclear ; knockout mouse studies are expected to provide clarity on the physiological roles of rhomboid proteins . recent studies have also placed the mitochondrial rhomboid protease at the nexus of key pathways that govern mitochondrial fusion , mitophagy and apoptosis . all mitochondrial rhomboid proteins are encoded in the nuclear genome , and imported into mitochondria . the main function of the mitochondrial rhomboid pcp1 is to release the dynamin - like gtpase mgm1 from the membrane . because mgm1 is essential for mitochondrial fusion and mgm1 cleavage occurs only in healthy mitochondria , this limits fusion to occurring between healthy organelles . a similar function was described in drosophila , but genetic interactions soon revealed further complexity in metazoans ; the mitochondrial rhomboid dmrho-7 also participates in the parkin / pink1 pathway that malfunctions in parkinson 's disease . it has recently become clear that the human mitochondrial rhomboid parl cleaves pink1 to suppress its ability to recruit the parkin ubiquitin ligase onto mitochondria [ 56 - 58 ] . without parl cleavage a parl knockout mouse suffers tremendous atrophy several months after birth resulting from malformed mitochondria and elevated apoptosis , although without mitochondrial fusion defects . parl has also been implicated in suppressing apoptosis in lymphocytes , potentially through a different substrate , high - temperature regulated a ( htra , also called omi ) . intriguingly , mutations in parl have recently been found in parkinson 's disease patients and diabetes patients , although the significance of these mutations for disease remains speculative . the third category of rhomboid function was revealed in providencia stuartii , a gram - negative bacterial pathogen . genetic screens identified its rhomboid homolog , aara , to be required for production of an unidentified signal for quorum sensing . once the similarity to rhomboid was noted , proteolytic activity of aara was demonstrated against spitz , and aara was found to partially rescue tissue development of drosophila mutant in rhomboid . historically , the intriguing similarity of activating drosophila egf signaling and producing an auto - inducer for bacterial quorum sensing , both by a rhomboid , received much attention . but the similarity proved to be superficial when the substrate was identified to be tata , a component of the twin - arginine translocation machinery . as such , aara removes a short amino - terminal extension , presumably to activate the machinery for signal secretion , rather than activating the signal itself . tata from other bacteria , including e. coli , lacks this short extension and is immediately active , and the aara function is therefore an exception . nevertheless , this is the only known function for a rhomboid protease in any prokaryote , and it dramatically highlights the apparent diversity of rhomboid function even within similar bacteria . this is the only role that was discovered by searching for rhomboid targets using substrate specificity determinants . the adhesins of plasmodium and toxoplasma are necessary for host - cell invasion , making them essential proteins for the survival of these obligate intracellular parasites . these parasites encode six or more rhomboid proteases , two of which in each organism are known to process these adhesins at the end of the invasion program [ 25,26,68 - 70 ] . the precise need for this dismantling is not entirely clear , but has been thought to free the parasite from being tethered to the host plasma membrane . recent knockdown experiments indicate that this processing is important for efficient invasion , although the full extent is incompletely understood and may involve later functions during parasite replication within the host cell . even the non - cell - invasive entamoeba histolytica encodes a highly active rhomboid protease , which is localized to the parasite surface but which relocalizes to phagosomes during feeding and the bud neck during immune evasion , perhaps to shed surface proteins , including lectins . the functions of other plasmodium or toxoplasma rhomboid proteases not involved in invasion are not yet understood , and many other parasites encode rhomboid enzymes whose functions have never been explored . perhaps the most powerful , yet subtle , guiding principle that can be deduced from the near ubiquity of rhomboid proteases is that they possess a biochemical property that is both very rare and highly useful : but what ? solving this riddle requires understanding the enzymatic features of rhomboid proteases , and remarkable progress has been made towards these goals ( reviewed in ) . this includes reconstitution of proteolysis with pure proteins , protease inhibitor profiling , extensive analysis of residues essential for activity , and structural visualization of catalytic residues and with a covalently bound inhibitor [ 34 - 37,78 ] . moreover , the initial paradox of how water is delivered to the membrane - immersed active site for hydrolysis was largely addressed by structural analyses [ 34 - 37 ] : the active site lies submerged about 10 below the presumed membrane surface , but with an open cavity above the active site for water access ( figure 3 ) . structure - function analyses of rhomboid proteases have also revealed several unusual proteolytic properties that make them unlike most serine proteases . these differences are clear evidence of convergent evolution to a serine protease mechanism down an independent path . first , structural analysis indicates that nucleophilic catalysis is achieved by a histidine - serine catalytic pair , rather than the more common aspartate - histidine - serine catalytic triad [ 34 - 37 ] . the identity of the residues that stabilize the oxyanion transition state is uncertain , but this stabilization is most likely mediated by asparagine and/or histidine side - chains ( figure 3 ) . use of an asparagine for oxyanion stabilization is uncommon but strikingly analogous to the mechanism of the conventional serine protease subtilisin . the third unusual catalytic property of rhomboid proteases relates to the direction in which substrates lie across the active site cleft relative to the catalytic residues . although initially thought to be similar to nearly all other serine proteases , identification of the substrate gate on the opposite side of glpg relative to expectation mandated that substrates approach the catalytic residues from the so - called ' si ' face . consistent with this stereochemistry are rhomboid 's resistance to most canonical serine protease inhibitors and a weak but specific sensitivity to monocyclic -lactams . it should be stressed that the definitive evidence for substrate orientation , identity of the oxyanion hole , and the nature of substrate stabilization await a co - structure with a peptide substrate . rhomboid proteases have been studied largely within the framework of an established serine protease precedent as a way to interpret rhomboid mechanism , which is instructive but does not help to understand how they are different . although deciphering the specifics of the catalytic chemistry is essential for designing effective inhibitors , the key functional properties of rhomboid enzymes that are relevant to the cell are unlikely to be determined by its catalytic mechanism . these defining features most likely result from membrane - immersion of the enzyme , and more recent investigations have started to study rhomboid proteases as integral membrane proteins directly . the greatest impact of membrane immersion is on how substrates and rhomboid proteases behave ( as reviewed in ) . the closed ring of tm segments observed in the first crystal structure suggested that something must move to clear a path for lateral substrate entry [ 34 - 37 ] . only mutations that weaken tm5 packing with tm2 were found to enhance protease activity by up to ten - fold , thereby identifying the gate functionally . this dramatic enhancement also revealed that gate opening is the rate - limiting step for intramembrane proteolysis . molecular dynamics simulations and structural analysis in a bicelle also suggest membrane thinning surrounding glpg , but its mechanistic implications remain unclear . investigating the role of the membrane in greater detail promises to reveal the defining features of the rhomboid proteolysis system . perhaps the most powerful , yet subtle , guiding principle that can be deduced from the near ubiquity of rhomboid proteases is that they possess a biochemical property that is both very rare and highly useful : but what ? solving this riddle requires understanding the enzymatic features of rhomboid proteases , and remarkable progress has been made towards these goals ( reviewed in ) . this includes reconstitution of proteolysis with pure proteins , protease inhibitor profiling , extensive analysis of residues essential for activity , and structural visualization of catalytic residues and with a covalently bound inhibitor [ 34 - 37,78 ] . moreover , the initial paradox of how water is delivered to the membrane - immersed active site for hydrolysis was largely addressed by structural analyses [ 34 - 37 ] : the active site lies submerged about 10 below the presumed membrane surface , but with an open cavity above the active site for water access ( figure 3 ) . structure - function analyses of rhomboid proteases have also revealed several unusual proteolytic properties that make them unlike most serine proteases . these differences are clear evidence of convergent evolution to a serine protease mechanism down an independent path . first , structural analysis indicates that nucleophilic catalysis is achieved by a histidine - serine catalytic pair , rather than the more common aspartate - histidine - serine catalytic triad [ 34 - 37 ] . the identity of the residues that stabilize the oxyanion transition state is uncertain , but this stabilization is most likely mediated by asparagine and/or histidine side - chains ( figure 3 ) . use of an asparagine for oxyanion stabilization is uncommon but strikingly analogous to the mechanism of the conventional serine protease subtilisin . the third unusual catalytic property of rhomboid proteases relates to the direction in which substrates lie across the active site cleft relative to the catalytic residues . although initially thought to be similar to nearly all other serine proteases , identification of the substrate gate on the opposite side of glpg relative to expectation mandated that substrates approach the catalytic residues from the so - called ' si ' face . consistent with this stereochemistry are rhomboid 's resistance to most canonical serine protease inhibitors and a weak but specific sensitivity to monocyclic -lactams . it should be stressed that the definitive evidence for substrate orientation , identity of the oxyanion hole , and the nature of substrate stabilization await a co - structure with a peptide substrate . rhomboid proteases have been studied largely within the framework of an established serine protease precedent as a way to interpret rhomboid mechanism , which is instructive but does not help to understand how they are different . although deciphering the specifics of the catalytic chemistry is essential for designing effective inhibitors , the key functional properties of rhomboid enzymes that are relevant to the cell are unlikely to be determined by its catalytic mechanism . these defining features most likely result from membrane - immersion of the enzyme , and more recent investigations have started to study rhomboid proteases as integral membrane proteins directly . the greatest impact of membrane immersion is on how substrates and rhomboid proteases behave ( as reviewed in ) . the closed ring of tm segments observed in the first crystal structure suggested that something must move to clear a path for lateral substrate entry [ 34 - 37 ] . only mutations that weaken tm5 packing with tm2 were found to enhance protease activity by up to ten - fold , thereby identifying the gate functionally . this dramatic enhancement also revealed that gate opening is the rate - limiting step for intramembrane proteolysis . molecular dynamics simulations and structural analysis in a bicelle also suggest membrane thinning surrounding glpg , but its mechanistic implications remain unclear . investigating the role of the membrane in greater detail promises to reveal the defining features of the rhomboid proteolysis system . the rhomboid gene was identified in the drosophila screens of the late 1970s and early 1980s , and it was cloned and sequenced about a decade later . it took another decade , until 2001 , for its biochemical function as an intramembrane serine protease to be revealed . it has now been a decade since that turning point , and advances in the intervening period have culminated in rhomboid proteases becoming widely regarded as the best understood of all intramembrane proteases . biochemical insights and defined roles in parasitic protozoa ( reviewed in ) place rhomboid study on the cusp of becoming applicable in a therapeutic setting . a major lingering obstacle is a rudimentary understanding of its unusual enzymatic mechanism , but these questions are being pursued intensively , and momentum towards a sophisticated understanding is building . by contrast , defining the cellular roles of rhomboid proteases has been a slow process . although even early biochemical insights have led to the identification of substrates that can be cleaved , whether these candidates are indeed physiological targets , and if so , whether they truly represent a major rhomboid function , remain unknown . for example , although the study of human rhbdl2 over the past 7 years has uncovered at least three well - cleaved substrates ( thrombomodulin , b - type ephrins , and egf ) , it is still unclear which , if any , are actual physiological targets , and whether cleavage represents a bona fide contribution to cellular function . perhaps the most humbling example is e. coli glpg , whose atomic details have been revealed in over a dozen structures and countless mutants , yet its cellular function remains a complete mystery . in reality , it is not the ability to find substrate candidates but rather their validation that has proven to be the bottleneck in these studies . the urgent need is for approaches with which to study enzymes under physiological settings on a higher throughput scale . this , in turn , will focus biochemical investigations by providing physiological targets and new functional contexts . work in our laboratory is supported by the howard hughes medical institute , and the david and lucile packard foundation .

summaryrhomboid proteases are the largest family of enzymes that hydrolyze peptide bonds within the cell membrane . although discovered to be serine proteases only a decade ago , rhomboid proteases are already considered to be the best understood intramembrane proteases . the presence of rhomboid proteins in all domains of life emphasizes their importance but makes their evolutionary history difficult to chart with confidence . phylogenetics nevertheless offers three guiding principles for interpreting rhomboid function . the near ubiquity of rhomboid proteases across evolution suggests broad , organizational roles that are not directly essential for cell survival . functions have been deciphered in only about a dozen organisms and fall into four general categories : initiating cell signaling in animals , facilitating bacterial quorum sensing , regulating mitochondrial homeostasis , and dismantling adhesion complexes of parasitic protozoa . although in no organism has the full complement of rhomboid function yet been elucidated , links to devastating human disease are emerging rapidly , including to parkinson 's disease , type ii diabetes , cancer , and bacterial and malaria infection . rhomboid proteases are unlike most proteolytic enzymes , because they are membrane - immersed ; understanding how the membrane immersion affects their function remains a key challenge .

Summary Gene organization and evolutionary history Characteristic structural features Localization and function Mechanism Frontiers Competing interests Acknowledgements

PMC3807517

the howard university environmental justice partnership ( huejp ) consists of several departments within the university that have been addressing environmental issues in impacted communities nationwide . the partnership was established to accomplish the following goals : establishment of a forum for communication and information sharing on environmental activities between howard university departments . development of a mechanism for collaboration among howard university departments on environmental projects achieved through the coordination of field experiences in the practice of disease prevention and health promotion for students of howard university and collaborating institutions . development of a mechanism for the collaboration of howard university with community groups , governmental agencies , professional organizations , hbcus , and other academic institutions , for the development and implementation of environmental programs . the primary mechanisms utilized thus far have been community - based workshops and the development of relevant and culturally competent environmental health instructional materials for health professionals . the workshops have been used to distribute minority health and environmental justice related resource information , instructional materials , and teaching techniques to both health professionals and community residents . a series of community based workshops on the use of geographic information systems ( gis ) as a tool in brownfields and environmental justice priority setting and community analysis have been conducted since 1995 . providing impacted communities with access to a cadre of environmental experts in different fields to take advantage of the multidisciplinary aspects of environmental problems , and to prevent duplication of efforts in recognition of funding limitations and other resources . this goal is achieved through collaborations with government and non - government officials on environmental justice , risk communication , and environmental health matters , when requested by communities as well as collaborations with state and local health departments and community based organizations on environmental justice and minority health issues . this effort assists in the improvement of teaching and learning at the grantee school and the other participating schools . current membership of the howard university ej partnership includes the program in atmospheric sciences , the school of engineering & computer science , college of medicine , the college of pharmacy , nursing , & allied health , and the urban environment institute . the basic approach of the hu - atsdr ( atsdr is the agency for toxic and disease registry ) partnership is to strengthen and develop linkages between : hbcus and other minority - serving institutions , community residents , environmental health researchers , and health care providers who live and work in environmentally impacted communities through several elements . the partnership actively develops strategies for assisting individual communities in identifying the needs of its at - risk populations . it has also developed a comprehensive plan for working with the community to address the problems and issues concerning policies , regulations , and procedures related to the environmental characterization and assessment , cleanup , and reuse of contaminated sites and properties . the first step is the organization of public forums in order to identify and prioritize community environmental health concerns . the central mechanism for this is the formation and development of community - based working groups ( cbws ) that will ultimately resolve the community s desires and needs . a unique feature of these cbws is the environmental health leadership training for community residents and health care providers around the identified hazards . the community s active participation in the development and implementation of this project is vital to the project s success and this partnership seeks to involve the community in a significant way in all possible aspects . the cbws will assist in the development of interventions for health care providers on adverse health effects related to environmental exposures . members of the hu - atsdr partnership technical team will perform statistical analysis of existing health data in order to characterize risks and to identify health data gaps . multidisciplinary teams then conduct mini - campaigns to collect in - situ measurements and perform model - based assessments of the sources of environmental exposures . the field efforts include in - situ measurements , environmental sampling , and laboratory analysis . we believe that this project can serve as a new paradigm for the development of community - university partnership programs that address environmental hazards in minority and underserved communities nationwide . when working with minority and low - income communities who experience adverse health effects that are perceived as being related to environmental pollution , the huejp first listens to the concerns and complaints of the impacted community . most communities with adverse health effects are disappointed in exposure assessments because conclusions and recommendations are many times made without anyone querying the residents about their health effects . the next step is to develop community - university - local government - health providers workgroups with community residents as equal members and participants in the planning and implementation of the outcomes . this approach to disease prevention and health promotion will use the activities logic model recommended by the w.k . a diagrammatic example of the currently employed model is depicted in figure 1 . this type of model links the activities together in a manner that shows how the program will be implemented . a team of faculty , postdoctoral fellows , and graduate students will collect data on the environmental pollutants and sites that may impact the particular communities health . these data along with community input will be used to design an appropriate strategy to educate and inform the residents on what actions they need to take in order to protect themselves from environmental risks . in many circumstances , the community has only anecdotal information about the diseases and/or birth defects in their neighborhood . a pre / post - test is developed to administer to the community at the first meeting and to healthcare providers that serve the targeted community . if requested by the community , huejp will also use the public forums to help develop a community health survey and train local hbcu(s ) or minority serving institutions ( msis ) students on administering the survey . the community - based working group to develop an appropriate action plan uses the results of these studies . the hu urban environment institute ( uei ) will provide legal expertise in superfund and environmental law as to how best funding sources like superfund technical assistance grants ( tag ) and dod community assistance grants ( cag ) can be utilized to achieve the impacted community s goals . week 1 - notification of site , preliminary research team assembled preliminary research assignments distributed , including community survey . week 2 - impacted community representatives and local public health department officials are contacted to schedule a community meeting . the outcomes of this meeting will be a statement of the community s environmental health issues , identification of the membership of the community - based working group . the meeting will be held a time , day , and location that are most convenient for maximum community involvement . week 4 - conduct a follow - up community meeting with students from nearby hbcu(s ) or msi(s ) to assist with documentation ; administer environmental health pre - test . week 5 - student research team will evaluate the meeting notes for health areas where additional information is needed . student researchers in chemistry and environmental engineering will develop an environmental assessment plan to verify hazardous constituents and provide analytical data for community information . community pharmacists are often very helpful in collecting this type of information ; efforts are made to encourage their participation . workgroups meet outside of the larger group meeting and will report their findings to the larger community . efforts are made to get local students involved in both the community health survey and workgroup meetings . workgroup topics include : health effects , outreach and communication , environmental assessment , social effects , law and policy ; and other areas the community wants to address . student research team , with assistance from faculty advisors , takes discussion from community meetings and develops a community health survey . week 10- survey is mailed to community and others who have been participants at the meetings for input and comments and discussion at meeting # 3 . the strategy development phase will be followed by an implementation phase that is guided by the workgroup plans . the huejp will provide assistance in the implementation phase of the project where needs are identified in the overall ej strategy . week 1 - notification of site , preliminary research team assembled preliminary research assignments distributed , including community survey . week 2 - impacted community representatives and local public health department officials are contacted to schedule a community meeting . the outcomes of this meeting will be a statement of the community s environmental health issues , identification of the membership of the community - based working group . the meeting will be held a time , day , and location that are most convenient for maximum community involvement . week 4 - conduct a follow - up community meeting with students from nearby hbcu(s ) or msi(s ) to assist with documentation ; administer environmental health pre - test . week 5 - student research team will evaluate the meeting notes for health areas where additional information is needed . student researchers in chemistry and environmental engineering will develop an environmental assessment plan to verify hazardous constituents and provide analytical data for community information . community pharmacists are often very helpful in collecting this type of information ; efforts are made to encourage their participation . set up workgroups on specific topics related to the site / problem . workgroups meet outside of the larger group meeting and will report their findings to the larger community . efforts are made to get local students involved in both the community health survey and workgroup meetings . workgroup topics include : health effects , outreach and communication , environmental assessment , social effects , law and policy ; and other areas the community wants to address . student research team , with assistance from faculty advisors , takes discussion from community meetings and develops a community health survey . week 10- survey is mailed to community and others who have been participants at the meetings for input and comments and discussion at meeting # 3 . the strategy development phase will be followed by an implementation phase that is guided by the workgroup plans . the huejp will provide assistance in the implementation phase of the project where needs are identified in the overall ej strategy . from january 26 , 1942 until september 30 , 1989 , the department of defense operated the defense distribution depot , memphis , tennessee ( hereafter refereed to as the ddmt ) . during the operation of the ddmt , dunn field , a 64-acre parcel , the site was placed on the national priority list and became a superfund site in october 1992 because of groundwater contamination . the types of hazardous wastes maintained and/or destroyed at the site include chemical warfare materials consisting of several thousand pounds of mustard - filled german bombs , chemical agent identification kits ( caik ) consisting of glass ampoules of mustard and lewisite ( a blistering chemical agent ) , chloroform , chloropicrin 50% , concentrated phosgene , impregnite , and decontaminating agents made up of organic n - chloroamide and 1.1.2.2-tetrachloroethane . additionally , organic chlorinating agents ( n - chloroamide and 1,3,-dichloroo-5,5-dimethlyhydantoin ) were buried with food stocks , paints , acids , herbicides , volatile organic solvents , and medical waste . historic documentation and aerial photographs have been used to determine where the actual disposal sites might be located as neither the department of defense nor the responsible contractors reportedly know all of the burial locations [ 2 , 3 ] . the community living adjacent to dunn field has consistently voiced concerns regarding the possible migration of contaminants from both on - site and off - site burial locations via surface and subsurface routes . there is widespread anecdotal evidence of numerous cancers , deaths , birth defects , and miscarriages in their community that they believe are related to the possible exposures to ddmt contaminants or their byproducts . there is a significant amount of anxiety related to the fears of the community and their lack of direct participation in the remediation and evacuation plans for the ddmt . the community has requested testing of the drainage ditches and residential yards , but their requests have been regarded as unwarranted , even though the exposure assessment of the site was based on historical records , not on actual testing of soil , water , and air . community members further fear that each time the soil on the ddmt is disrupted , especially in dunn field , their neighbors will become sick or die . the ddmt concerned citizens committee has defined the impacted community as being bound by hernando street on the west , pendleton and ketchum streets on the east , kerr street on the north , and interstate 240 and clementine street on the south . figure 2 shows a representation of the demographics of the ddmt residential neighborhoods . according to 1990 us census data , the zip code adjacent to the ddmt ( 38106 ) has approximately 40 , 352 residents . fifty - three ( 53 ) percent of this zip code s households have incomes less than $ 15,000 . thirty - seven ( 37 ) percent of the housing structures were built before 1949 , and seventy ( 70 ) percent of the housing was built before 1969 . based on the understanding that the residential neighborhoods adjacent to the memphis depot had experienced toxic exposures that have resulted in residential contamination and possible adverse health impacts , our objectives were to : characterize the background levels of contaminants in soil and water in the adjacent community , characterize community exposure levels of soil and water toxics , and assess the anecdotal record of mortality and morbidity associated with the ddmt . the air , soil , and water quality data were collected according to epa standards and compared to control communities outside of the mississippi delta region . previous sampling for risk and exposure assessment served as a secondary basis for the sampling plan . however , the primary factors determining the sampling sites were community concerns and the clear identification of access points for transfer of toxics from the ddmt directly into the adjacent community . previous sampling and testing at the ddmt are characterized by two principal shortcomings ( 1 ) the concerns of the community were not directly addressed to the satisfaction of the residents and ( 2 ) there were no significant off - site testing to establish a baseline for the concentrations of any contaminants . the us army contracted lockheed - martin in 1985 to perform a risk assessment for the communities surrounding the ddmt . a formidable amount of work was performed , but the primary concerns of the community were not addressed . no matter how comprehensive a risk assessment is , it will not address the sustained and cumulative exposures over two generations of residents . another issue regarding the previous investigations of contamination from the ddmt is the lack of a refined search for byproducts and breakdown products of the chemical stockpile . lockheed - martin generically lists several classes of contaminants screened including pesticides , metals , and volatile organic compounds ( vocs ) . since the chemical inventory of the ddmt is known , a more refined search is in order . for example , lewisite was one of the chemical warfare agents stored in the ddmt . if one were looking for evidence of lewisite migration and contamination , a search for enhanced arsenic levels in both elemental and molecular forms would be in order . additionally , chlorinated organic compounds and enhanced liberation of chlorine from soil - gas experiments might serve as a more useful tracer than generic tests for pesticides , metals , and volatile organic compounds ( vocs ) . it has been shown in the recent literature that an effective tracer of warfare agents is the presence of organo - phosphorus esters and acids . these species tend to have low vapor pressures and would not be highly susceptible to detection by methods designed for volatile organic compounds . the huejp work plan was designed with the purpose of estimating community exposure levels in order to assess the possibility of significant contamination of off - site locations in the residential communities adjacent to the ddmt . the work plan is composed of four basic components , the assessment phase , the determination phase , the quantification and analysis phase , and the evaluation and response phase . environmental samples were collected , preserved , and analyzed using procedures either specifically detailed in or designed to be consistent with the environmental investigations standard procedures and quality assurance , 1996 ( eisopqa ) manual . all sample types were screened for heavy metals ( without speciation ) including cadmium , chromium , lead , arsenic , and mercury ; total organic carbon ; and several inorganic anions ( sulfate , chloride , fluoride , phosphate , nitrite , and nitrate ) . the samples were collected from four separate sites near the ddmt . each site was chosen based on its mutual proximity to the ddmt and the adjacent residential area as well as the anecdotal record of cancers , deaths , or illnesses . the locations are described below : 1835 rozelle neighborhood : soil samples were collected from a backyard where anecdotal records had indicated off - site burial ( and subsequent removal ) of chemical waste from the ddmt . cane creek high school : water and sediment samples were collected from an open drainage ditch behind and adjacent to the main school building . both anecdotal and site records report that cane creek was routinely used to dispose chemicals and waste . run - off from the ddmt is drained directly into cane creek . cane creek runs throughout the residential community and passes directly underneath the primary air - intake ducts of the high school . it was clear that even during the relatively cool spring ( ambient temperatures were in the range of 45f55f ) the creek retains the distinct and pungent odor that the residents complain about . water and sediment samples from cane creek were also collected at locations upstream and downstream of the high school by approximately one - half mile . corner of ball and mullen : sediment and water samples were collected from an open drainage ditch extending from the western boundary of the ddmt into the adjacent community . storm water runoff drainage ditch : the uncovered storm water run - off is another direct access point for facile migration of contaminants from the ddmt into the community . it is located on the southern boundary of the ddmt site map ( figure 3 ) . we note that while the cane creek samples were collected from a non - stationary point source ( flowing water ) that rarely dries completely it is useful to compare aqueous samples to those of sediment and soil from the vicinity of the cane creek hamilton high school . while there have been no recent reports of dumping from the ddmt into cane creek an evaluation of its current state still gives an indication of the environmental state of this exposed transport pathway . cane creek has historically been and continues to be a venue for recreational activities for children and adults residing in the neighborhoods surrounding the ddmt . a comparison of the nearby soils and sediments from drainage ditches may give an indication of the retention of environmental contaminants and the extent of its transport into the community . one would expect that the sediments in the drainage ditches might be more concentrated sources of contaminants since the water in these areas tend to stand rather than flow continuously . the basic objectives were to examine the water , soil , and sediment samples for evidence of cadmium , chromium , lead , arsenic , mercury , organic carbon ( including polyaromatic hydrocarbons ) , selected herbicides , insecticides , and chemical warfare agents ( e.g. soman , sarin , sulfur mustard , nitrogen mustard ) , and other chemicals that ranked high on the ddmt inventory . our organic analysis focused on water - soluble total organic carbon ( toc ) because of the greater potential risk of exposure to these types of compounds due to their ability to be transported via hydrological pathways . figure 4 shows the results of the total organic carbon analysis averaged over all of the samples at each location . a phoenix 8000 toc analyzer was used to estimate the total organic loading in each environmental sample . the presence of organics in the aqueous extracts was first screened using uv - visible spectroscopy . the cane creek samples exhibited the expected uniformity in values of toc and are represented on the graph as a single data point . although the cane creek samples had more toc than the drainage ditches , none of the samples contained more than 10 ppm toc . there is considerable variability observed in the anion concentrations from site to site . despite this , no single site possessed concentrations of any of the tested species that were higher than the epa standards . the concentration of this species was observed to be the greatest in the cane creek and ball and mullin samples . the retention time for the unknown was consistent enough for it to be identified as the same species in all samples . all samples were tested for a full suite of heavy metals using atomic absorption spectroscopy . lead , arsenic , cadmium and chromium were the heavy metals identified in at least one set of samples . lead and chromium were observed at all sites except for rozelle street . since the observed concentrations were very low and because rozelle street was the only site without a source of running water we feel that the most likely source of lead is the lead piping used in the water distribution system . of the heavy metals surprisingly , the average concentration of chromium observed in the cane creek , ball and mullen , and stormwater runoff was 126 ppm , tripling the epa limit of 40 ppm . additionally , the speciation of the chromium is a critical factor in determining both its environmental toxicity and clear source receptor relationships . since our current methods of analysis do not permit the identification of the oxidation states of the heavy metals , our observations are limited to the identification of total environmental chromium . we note that previous engineering reports on the ddmt do not provide significant insights as to the possible sources of environmental chromium . the excessive concentrations of chromium are noteworthy because of the well - documented associations between environmental chromium and cancer incidences . cane creek is also by far the most accessible exposure pathway as it is largely uncovered , flows throughout the entire community and is easily available for children and domesticated animals . it is noteworthy to add that adolescent cancer incidences in the ddmt vicinity are higher than the national average and community anecdotal records report significant incidences of cancers in pets . a total constituent / contaminant index was derived to summarize the results of the field measurements . the contaminant index was derived by weighing each of the contributions of organics , anions , and heavy metals based equally and then scaling the value of each contaminant observed at each site location by the total amount of each type of contaminant observed . the result of this is presented in figure 7 . while not quantitative , this chart serves to provide a qualitative comparison of contamination at the various sites . from this chart it becomes clear that heavy metal contamination was pervasive at the sites where there was either running or standing water . significant amounts of organic compounds , unidentified anions , and heavy metals ( cr and cd ) have been detected in samples from the cane creek hhs and ball & mullen sites , with a close runner - up at the 1835 rozelle street the ic results showed that there was an unidentified anion detected in high quantities for all sites , with cane creek and ball & mullen ranking the highest . both lead and chromium were found at all sites , except the soil sample sites at 1835 rozelle street . the observed levels of cadmium in the stormwater runoff area were also significant ( more than 65% of the epa limit ) . this site represents a direct pathway for toxicant flow into the community and other similar locations may have had higher concentrations of this toxicant in the past . the results of this study indicate that there has been some presence of contamination with the marked appearance of heavy metals , an unidentified anion , and organic materials . this should provide clear justification for further sampling and detailed analysis of environmental contamination of the adjacent neighborhoods .

an interdisciplinary environmental assessment team from the howard university environmental justice partnership ( huejp ) conducted a site visit and assessment of the defense depot of memphis , tennessee in february of 2000 . this depot was built in the late 1940 s for storage of numerous chemicals and munitions . as the years progressed , many memphis citizens have grown to believe that the activities and chemical stockpile located at this site have negatively affected the health environment of their residents . there is anecdotal evidence and documentation of numerous cancers and other illnesses in those local territories , and specifically , at the memphis depot site . currently , this depot is closed and in remediation by the local government . particularly , citizens of the rozelle community have started a campaign to investigate any signs of exposure pathways to noted health risks . the huejp was contacted and asked to investigate the community concerns . obliging to the request , we aimed to sample at three drainage sites and a residential site , talk to local citizens , and gain any additional information that would be helpful in relieving anxiety in the rozelle community . soil , sediment , and water samples were collected and analyzed for total organic carbon , inorganic anions , and heavy metals . these data show that for the four sites sampled , the highest concentrations of organic compounds and heavy metals were located either within a residential area or in an area with a direct transport pathway to the community . atomic absorption analysis revealed detectable amounts of cadmium , lead and chromium metals at all sites with direct transport pathways into the residential community , with chromium concentrations being far in excess of the epa standard limits .

Introduction Strategies for Disease Prevention and Health Promotion Typical Schedule for Strategy Development Overview of Defense Depot of Memphis, Tennessee Research of Methodology Discussion of Sampling Plan Results and Analysis Summary

PMC4045546

characterisation of drug effects in in vitro brain slice experiments is complicated by the absence of a blood supply to the tissue . this means that drugs must be delivered to the tissue by passive diffusion via the perfusion solution . because substances diffuse at different rates according to their physicochemical properties , it can be difficult to know how long to expose the tissue in order to achieve drug penetration into the healthiest layers of the slice approximately the middle 100 m for a 400 m thick slice . for drugs that diffuse slowly , there is a risk that a negative result will be spuriously attributed to an inactive drug , when in fact it never reached its site of action in the tissue . directly measuring drug concentrations in tissue slicesnecessitates methodologies that are time consuming and complex and can not realistically be applied to each and every drug one may want to test . this motivated us to explore alternative , simpler approaches for estimating drug diffusion in brain slice tissue . to this end , we have borrowed a pharmacological technique known as pharmacokinetic / pharmacodynamic ( pkpd ) modelling , which is used clinically to describe the relationship between the time course of drug effect and the delivered dose . when anesthetic drugs are delivered to the circulation , the length of the delay is related to the time taken for the drug to diffuse from the circulation to the effect site in the brain . by applying fick 's law of diffusion ( stating that the rate of equilibration of a substance is proportional to its concentration gradient ) , it is possible to calculate the equilibration half - time ( t1/2ke0)a measure of the time required for a substance to reach half of its equilibrated concentration at the tissue recording site . in this study we have applied these principles to quantify the rate at which anesthetic drugs diffuse into neocortical slice tissue . one of these ( etomidate ) was chosen because its diffusion characteristics in brain tissue have been carefully investigated , allowing a direct comparison with the results of this study . we also compared the anesthetic drugs to magnesium , a simple ion that we hypothesised should diffuse at a faster rate . all experimental procedures were approved by the waikato ethics committee at waikato university , hamilton , waikato , new zealand ( ethics approval number 836 ) . the solutions were made with double distilled water and stored at 14c for no longer than 7 days . all solutions were saturated with carbogen ( 95% o2 ; 5% co2 ) for at least 15 minutes prior to use . three solutions were used.protective : acsf for brain extraction and tissue slicing , containing 92.7 mm nacl , 3 mm kcl , 19 mm mgcl2 , 0 mm cacl2 , 1.2 mm nah2po4 , 24 mm nahco3 , and 25 mm d - glucose .no - magnesium : acsf lacking magnesium ions , containing 124 mm nacl , 5 mm kcl , 2 mm cacl2 , 1.25 mm nah2po4 , 26 mm nahco3 , and 10 mm d-glucose.normal : acsf containing magnesium ions , composed of 125 mm nacl , 2.5 mm kcl , 1 mm mgcl2 , 2 mm cacl2 , 1.25 mm nah2po4 , 26 mm nahco3 , and 10 mm d - glucose . protective : acsf for brain extraction and tissue slicing , containing 92.7 mm nacl , 3 mm kcl , 19 mm mgcl2 , 0 mm cacl2 , 1.2 mm nah2po4 , 24 mm nahco3 , and 25 mm d - glucose . no - magnesium : acsf lacking magnesium ions , containing 124 mm nacl , 5 mm kcl , 2 mm cacl2 , 1.25 mm nah2po4 , 26 mm nahco3 , and 10 mm d - glucose . normal : acsf containing magnesium ions , composed of 125 mm nacl , 2.5 mm kcl , 1 mm mgcl2 , 2 mm cacl2 , 1.25 mm nah2po4 , 26 mm nahco3 , and 10 mm d - glucose . mouse - neocortical brain slices were obtained from adult wild - type ( 129sv ) mice , both female and male . prior to decapitation and brain dissection , the mice were anesthetised with carbon dioxide ( co2 ) . the cerebrum was removed and placed into ice - cold protective acsf , made according to nowak and bullier . a vibratome ( campden instruments , uk ) was used to slice the brain in 400 m thick coronal sections between bregma 1 mm and 5 mm . each slice was placed into carbogenated no - magnesium acsf solution to recover for at least an hour prior to recording at room temperature ( approximately 28c ) . for experimental recording , each slice was transferred to a recording bath ( tissue recording system , kerr scientific instruments , new zealand ) perfused with carbogenated no - magnesium acsf solution at a gravity - fed flow rate of 6.0 ml / min . extracellular field potentials were recorded from a 50 m teflon - coated tungsten electrode , referenced to a silver / silver - chloride electrode positioned in the recording bath . the recording electrode was positioned at a depth of approximately 200 m ( i.e. , the middle of the slice ) in the cerebral cortex , with no particular cortical location targeted . the data was recorded with a 1000x gain , low- and high - pass filtered at 1000 hz , and 1.0 hz respectively ( model 1800 ac amplifier , a - m systems , usa ) , and sampled at a frequency of 5000 samples / second ( power 1401 , cambridge electronic designs , uk ) . mains filtering at 50 hz was not required because the experiments were conducted within an electrically shielded room . the data was saved for analysis using matlab ( version 7.3.0.267 ( r2006b ) , the mathworks inc . , spontaneous seizure - like event ( sle ) activity was recorded for at least 10 minutes prior to anesthetic delivery . thereafter the anesthetic of choice was delivered to the tissue using the same gravity - fed perfusion system , resulting in a gradual increase in concentration within the recording bath . anesthetic delivery was continued for 15 minutes , followed by washout with drug - free no - magnesium acsf . no - magnesium acsf perfusion was continued until the frequency of sles returned to baseline . two anesthetics were tested : etomidate ( 24 m , n = 14 from 4 animals ) and thiopental ( 250 m , n = 14 from 4 animals ) . drug dosage was based on previous cortical slice experiments using etomidate and the relative clinical potencies of the two agents . the anesthetic solutions were made by adding the appropriate amount of each drug directly to precarbogenated acsf immediately prior to perfusion . for comparison with the anesthetic drugs , we also tested the diffusion of magnesium ions ( n = 13 from 2 animals ) . slices were perfused with magnesium - containing normal acsf ( which eliminates sle activity ) for 15 minutes , followed by washout of magnesium with no - magnesium acsf until sle activity returned to baseline levels . the data was analysed in an identical fashion to the anesthetic agents ( see below ) . the sle time courses were used to plot the relationship between drug effect ( sle frequency ) and drug concentration . the changes in anesthetic and magnesium ion concentrations with time were not measured directly in this study . rather , an estimate of the concentration time course was obtained in a preliminary experiment by measuring the real - time changes in bath solution conductivity during perfusion of an 0.1 mm solution of sodium chloride . the salt solution was perfused into the bath from a starting concentration of 0 mm , using the same flow parameters as for the experimental protocols . because solution conductivity increases approximately linearly with the increase in concentration of salt in solution , the time course of conductivity changes give an estimate of the time - course of concentration change . we made the assumption that the time course for perfusion of the salt solution into the bath would be similar to that for the anesthetics . concentration - effect plots displayed the anticipated hysteresis loops ( figure 1(c ) ) , reflecting ( 1 ) the time delay between delivery of the drug in the perfusion solution and its diffusion to the site of action in the tissue and ( 2 ) the reverse delay between removal of the drug from the perfusion solution and its washout from the tissue . we used a standard pharmacokinetic / pharmacodynamic ( pkpd ) model to collapse this loop ( figure 1(d ) ) in order to obtain the first order rate constant ke0 , according to ( 1)dceffdt = ke0(cpceff ) , where cp is the acsf concentration of the drug , ceff is the concentration at the effect site in the tissue , and ke0 is the first order rate constant for movement of drug into the tissue . the ceff was estimated by iteratively running the aforementioned model with a series of ke0 steps . for each iteration , a nonlinear inhibitory sigmoid emax curve was fitted to the data ( figure 1(d ) ) by the following equation : ( 2)effect = emax(emaxemin)ceffec50+ceff , where effect is the sle frequency , the emax and emin are the maximum and minimum effects for each slice , ec50 is the anesthetic concentration at which effect is midway between this maximum and minimum , and gamma ( ) describes the slope of the concentration - response relationship . ke0 was determined from the iteration yielding the greatest coefficient of determination ( r ) for measured and modelled effect for each slice . ke0 was converted into ke0 half - life ( t1/2ke0 ) , according to the relationship ( 3)t1/2ke0=ln2ke0 . data normality was assessed using the kolmogorov - smirnov test and statistical tests applied accordingly . sles were characterized by an initial large population depolarization , followed occasionally by a 47 hz oscillation of approximately three seconds . the baseline median ( range ) sle frequency for each test condition was 2.5 ( 1.43.2 ) , 3.5 ( 1.16.5 ) , and 2.4 ( 1.03.9 ) events / min for etomidate , thiopental , and magnesium , respectively . in each case , delivery of the test compound reliably effected a reduction in sle frequency to 1.6 ( 0.43.2 ) , 0.9 ( 0.42.6 ) , and 0.5 ( 0.21.6 ) events / min , respectively , before returning to baseline levels after drug washout . there was no statistically significant difference in baseline sle frequency across the three test conditions ( p = 0.13 , kruskal - wallis test ) , and sle frequency following drug washout was similar to baseline in all cases ( p = 0.82 , kruskal - wallis test ) . an example of the pattern of sle activity from one slice is shown in figure 1(a ) . t 1/2 k e0 values calculated for each anesthetic and for the magnesium acsf experiments are shown in table 1 . a value of 83.1 ( 19.4330.1 ) min for etomidate represents its equilibration half - time . in other words , after 83 minutes , etomidate had reached half of its equilibrated concentration at the site of recording ( in the middle of the slice ) . t1/2ke0 values were similar for the two anesthetics but significantly shorter for magnesium ( 26.1 ( 8.677.0 ) min , p < 0.01 for comparison with both thiopental and etomidate , kruskal - wallis test ) . this was consistent with the expectation that magnesium ions would diffuse rapidly into the slice tissue . additional parameters , ec50 and gamma , were derived from the inhibitory emax curves , relating to the potency of the drug and speed of onset / offset , respectively ( table 1 ) . the only difference of note was a higher ec50 value for etomidate compared to the other agents , suggesting that etomidate effected a lesser reduction in event frequency . this is confirmed by the sle frequency data showing that event frequency at maximum etomidate effect was higher compared to both magnesium and thiopental ( p < 0.001 and p < 0.05 , resp . , one - way anova ) . the aim of this study was to determine whether an analytical technique carried over from clinical pharmacological studies could be adapted and applied to cortical slices for estimating drug diffusion times . we were motivated by the need for a simple and practical method of determining the minimum time required for a given drug to reach effective concentrations within the depth of slice tissue . to achieve this , we estimated the rate of equilibration between acsf and the cortical slice effect site by calculating the t1/2ke0 for two anesthetic drugs and magnesium ions . firstly , the long equilibration time calculated in this study for etomidate ( t1/2ke0 of 83 min ) is consistent with analytical studies of the same drug showing that it takes upwards of 100 minutes for etomidate to reach equilibrium in the middle of a 400 m thick brain slice . secondly , values for etomidate and thiopental were not significantly different , which is in agreement with in vivo pkpd studies showing that the equilibration delays for these agents are similar [ 711 ] . thirdly , the much shorter t1/2ke0 for magnesium ( approximately 30 minutes ) matches the expectation that a simple ion should diffuse much faster than the more chemically complex anesthetic drugs . the diffusion time was substantially longer in the slice model compared to in vivo , with t1/2ke0 values for etomidate ( t1/2ke0 1.52.7 min ) and thiopental ( t1/2ke0 1.2 min ) more than an order of magnitude shorter than calculated for the same agents in this study [ 711 ] . this was anticipated because the diffusion distance in the slice is 510 times greater than in vivo . the intercapillary distance in the normal mammalian cerebral cortex is in the order of 5060 m , meaning that the maximum diffusion distance is 2530 m . this compares to a diffusion distance of 100200 m in the 400 m isolated cortical slice ( depending on recording electrode depth ) . a proportion of this variability may be attributed to inconsistencies in inhibitory emax curve fitting due to discrepancies in the shape of the two halves of concentration - effect loops . this can result in slight mismatches between the two arms of the loop when collapsed , resulting in variability in curve fitting from test to test . one cause of such a mismatch could be a discrepancy in the frequency of sle activity at the start of the recording compared to at the end . another possible source of t1/2ke0 variability could be small inconsistencies in electrode depth placement from slice to slice . while every effort was made to position the electrodes in the mid - layer of the slice , it was not possible to precisely measure electrode depth . as shown by benkwitz et al . , the rate of diffusion is faster near the top surface than deeper within the slice . thus , the concentration - time course will be somewhat affected by the depth position of the electrode . on the basis of this variability , a minimum of 10 repeat experiments is indicated in order to obtain a meaningful estimate of equilibration time . it is also important to note that the methods described account only for drug diffusion , not any additional delays to drug effect that could be associated with drug - receptor interactions and/or effect transduction pathways . standard deep anesthetic dose was estimated to be approximately 250 m for thiopental . both anesthetics generated robust , repeatable , and reversible reductions in sle frequency , confirming that the dosing was appropriate . in conclusion , pkpd modelling confirms that the anesthetic drugs etomidate and thiopental diffuse slowly into cortical slice tissue and that this method is a useful tool for investigating drug diffusion characteristics in brain slice pharmacological studies .

pharmacological brain slice experiments are complicated by the need to ensure adequate drug delivery deep into the healthy layers of the tissue . because tissue slices have no blood supply , this is achieved solely by passive drug diffusion . the aim of this study was to determine whether pharmacokinetic / pharmacodynamic ( pkpd ) modeling could be adapted to estimate drug diffusion times in neocortical brain slices . no - magnesium seizure - like event ( sle ) activity was generated in 41 slices ( 400 m ) . two anesthetic agents , etomidate ( 24 m , n = 14 ) and thiopental ( 250 m , n = 14 ) , and magnesium ions ( n = 13 ) were delivered to effect reversible reductions in sle frequency . concentration - effect hysteresis loops were collapsed using a first order rate constant model and equilibrium half - lives ( t1/2ke0 ) derived . the t1/2ke0 values obtained were consistent with expectations . the median ( range ) t1/2ke0 of 83.1 ( 19.4330.1 ) min for etomidate is in keeping with its known slow diffusion into brain slice tissue . values for etomidate and thiopental ( 111.8 ( 27.8198.0 ) min ) were similar , while magnesium had a significantly faster equilibration rate ( t1/2ke0 of 26.1 ( 8.677.0 ) min ) compared to the anesthetics , as expected for a simple ion . in conclusion , pkpd modeling is a simple and practical method that can be applied to brain slice experiments for investigating drug diffusion characteristics .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

PMC4569987

oral cancer is the 11 most common cancer in the world , and death due to it ranks 6 globally . in india , particularly it has the highest incidence of oral cancer globally and accounts for approximately 3040% of all other cancers . malignant transformation of oral leukoplakia ( ol ) is 120% over 130 years while oral lichen planus ( olp ) and oral submucous fibrosis ( osmf ) have malignant transformation of 0.2%/year and 713% respectively . progression of these oral potentially malignant disorders to carcinoma is multi - stage process and is preprogrammed temporarily in the genome . many molecular studies have explored a number of genes that are altered , amplified , dysregulated in expression or deleted in head and neck tumorigenesis . beyond prevention , the quench for early detection of the lesion and its progression is necessary as it increases the possibility of favorable treatment outcome and better prognosis . recently , various tumor markers have received more attention and one among them is cell to cell adhesion molecule , e - cadherin ( e - cad ) , tumor suppressor gene that is expressed in epithelial tissues . e - cad , 120-kda glycoprotein , a calcium - dependent cell - surface adhesion molecule that works as an intercellular adhesion molecule between epithelial cells and is also involved in the transduction of signals controlling various cellular events , including polarity , differentiation , growth and cell migration . in addition , e - cad has the ability to inhibit cell proliferation by up - regulating the p27 via epidermal growth factor receptor . it is encoded by the gene cdh1 located on chromosome 16q-22 and the loss in e - cad expression has been correlated with cancer progression . e - cadherin is also known to express in various carcinomas of head and neck , esophagus , prostrate , pancreas , stomach and uterine cervix and its reduced expression has been correlated with aggressive behavior , high proliferation , invasion , metastasis and poor prognosis of cancer . several researchers hypothesized that e - cad expression is reduced with increase in grade of dysplasia . the reduced expression of e - cad may be a reliable indicator of increase in invasiveness of oral carcinomas . therefore , the objective of the present study is to evaluate the expression of e - cad in oral precancerous lesions and oral carcinomas in comparison with normal oral mucosa ( nom ) by immunohistochemical method , so as to unravel its role in progression and invasion of these conditions . the study was conducted in the department of oral medicine and radiology , sri sai college of dental surgery , vikarabad following the ethical committee clearance . a total sample size of 50 subjects was chosen , with study group consisting of 10 patients in each group diagnosed with ol , osmf , olp and oral squamous cell carcinoma ( oscc ) . any subject with systemic disease ( e.g. : diabetes mellitus , thyroid disorders and liver dysfunction ) was excluded . samples of these study groups were selected after the lesions were confirmed both clinically and histopathologically following informed consent of the patient . clinically , all the samples of ol demonstrated homogenous type of leukoplakia , criteria proposed by who ( 1980 ) , the histological degree of epithelial dysplasia was based on the proportion of the height of the epithelial layer that presents the dysplastic changes with mild to severe dysplastic changes . clinicopathological criteria proposed by kumar et al . were used for osmf classification , all the subjects belong to stage 2 clinically . in this present study , 6 subjects were of reticulate type of olp , two were of atrophic type and two were of erosive type according to andreasen et al . classification criteria as proposed by robbins et al . were considered for oscc in which 2 subjects were well - differentiated , six were of moderately differentiated and remaining two were of poorly differientiated . expression of e - cad was done by immunohistochemistry , and antibodies and reagents used for immunohistochemistry technique were obtained from envision kit , dako , denmark and dako , carpinteria , ca , ( usa ) . primary antibody - mouse anti - human e - cadsecondary antibody - anti - mouse iggperoxidase blockconjugate - horse radish peroxidase ( hrp)chromogen substrate diaminobenzidine tetra hydrochloride ( dab ) . primary antibody - mouse anti - human e - cad secondary antibody - anti - mouse igg conjugate - horse radish peroxidase ( hrp ) chromogen substrate four micron thick sections were taken onto poly - l - lysine adhesive coated slides and incubated for 3 h at 5060 centigrade and procedure of e - cad staining was performed following dewaxing and antigen retrieval procedure by immersing in 10 mm ethylenediaminetetraacetic acid . following which dipped in the sections were then washed in tris - wash buffer for two changes for 5 min each . then sections were incubated with prediluted primary antibody e - cad ( clone nch-38 , 1:200 dilution , dako , carpinteria , ca , usa ) at room temperature for 24 h in a humid chamber . super enhancer of the secondary antibody was added for 30 min and then washed in tris - wash buffer thoroughly for 24 min . then dab ( 1 drop ) + wash buffer ( 1 ml ) was added for 10 min . for visualization the sections were then lightly counterstained using mayer 's hematoxylin for 10 s , after which sections were gently washed in running tap water for 60 s. mounting was done in dpx mounting media ( glycerol ) . assessment of e - cad positive cells was performed using double headed light microscope at 10 and 40 . the criteria used to define e - cad antigen positive cells were brown staining in dysplastic cell membrane ; the pattern was described as normal when staining was exclusively membranous and of similar intensity to adjacent normal epithelium . presence of immunostaining in the cell membrane of various layers of epithelium was evaluated in randomized six fields / intensity of positively stained cells as percentage expression at 40x and graded as 0 ( under 10% positively stained cells ) , 1 + ( 1025% positively stained cells : weak expression ) , 2 + ( 2550% positively stained cells : mild to moderate expression ) , 3 + ( 5075% positive cells : moderate to strong expression ) and 4 + ( over 75% of intensity of positively stained cells : very strong expression ) . the current grading criteria were chosen as defined by simionescu et al . in their study , as grade of dysplasia increased , expression of e - cad was reduced in oscc patients . the expression of e - cad was interpreted by an experienced pathologist who was blinded as to which group the patient belonged , to eliminate bias . interpretation of e - cad expression was done by two independent observers , and the results were tabulated . the inter - examiner reliability in recording expression of e - cad was found to be 0.96 ( intraclass correlation coefficient ) implying a good agreement between the two observers . hence , the study was preceded with a single examiner . following the tabulation of the data , all calculations were performed using ibm spss statistical software package ( spss 14.0 for windows , spss inc . , the study was conducted in the department of oral medicine and radiology , sri sai college of dental surgery , vikarabad following the ethical committee clearance . a total sample size of 50 subjects was chosen , with study group consisting of 10 patients in each group diagnosed with ol , osmf , olp and oral squamous cell carcinoma ( oscc ) . any subject with systemic disease ( e.g. : diabetes mellitus , thyroid disorders and liver dysfunction ) was excluded . samples of these study groups were selected after the lesions were confirmed both clinically and histopathologically following informed consent of the patient . clinically , all the samples of ol demonstrated homogenous type of leukoplakia , criteria proposed by who ( 1980 ) , the histological degree of epithelial dysplasia was based on the proportion of the height of the epithelial layer that presents the dysplastic changes with mild to severe dysplastic changes . clinicopathological criteria proposed by kumar et al . were used for osmf classification , all the subjects belong to stage 2 clinically . in this present study , 6 subjects were of reticulate type of olp , two were of atrophic type and two were of erosive type according to andreasen et al . classification criteria as proposed by robbins et al . were considered for oscc in which 2 subjects were well - differentiated , six were of moderately differentiated and remaining two were of poorly differientiated . expression of e - cad was done by immunohistochemistry , and antibodies and reagents used for immunohistochemistry technique were obtained from envision kit , dako , denmark and dako , carpinteria , ca , ( usa ) . primary antibody - mouse anti - human e - cadsecondary antibody - anti - mouse iggperoxidase blockconjugate - horse radish peroxidase ( hrp)chromogen substrate diaminobenzidine tetra hydrochloride ( dab ) . primary antibody - mouse anti - human e - cad secondary antibody - anti - mouse igg conjugate - horse radish peroxidase ( hrp ) chromogen substrate four micron thick sections were taken onto poly - l - lysine adhesive coated slides and incubated for 3 h at 5060 centigrade and procedure of e - cad staining was performed following dewaxing and antigen retrieval procedure by immersing in 10 mm ethylenediaminetetraacetic acid . following which dipped in the sections were then washed in tris - wash buffer for two changes for 5 min each . then sections were incubated with prediluted primary antibody e - cad ( clone nch-38 , 1:200 dilution , dako , carpinteria , ca , usa ) at room temperature for 24 h in a humid chamber . super enhancer of the secondary antibody was added for 30 min and then washed in tris - wash buffer thoroughly for 24 min . then dab ( 1 drop ) + wash buffer ( 1 ml ) was added for 10 min . for visualization the sections were then lightly counterstained using mayer 's hematoxylin for 10 s , after which sections were gently washed in running tap water for 60 s. mounting was done in dpx mounting media ( glycerol ) . assessment of e - cad positive cells was performed using double headed light microscope at 10 and 40 . the criteria used to define e - cad antigen positive cells were brown staining in dysplastic cell membrane ; the pattern was described as normal when staining was exclusively membranous and of similar intensity to adjacent normal epithelium . presence of immunostaining in the cell membrane of various layers of epithelium was evaluated in randomized six fields / intensity of positively stained cells as percentage expression at 40x and graded as 0 ( under 10% positively stained cells ) , 1 + ( 1025% positively stained cells : weak expression ) , 2 + ( 2550% positively stained cells : mild to moderate expression ) , 3 + ( 5075% positive cells : moderate to strong expression ) and 4 + ( over 75% of intensity of positively stained cells : very strong expression ) . the current grading criteria were chosen as defined by simionescu et al . in their study , as grade of dysplasia increased , expression of e - cad was reduced in oscc patients . the expression of e - cad was interpreted by an experienced pathologist who was blinded as to which group the patient belonged , to eliminate bias . interpretation of e - cad expression was done by two independent observers , and the results were tabulated . the inter - examiner reliability in recording expression of e - cad was found to be 0.96 ( intraclass correlation coefficient ) implying a good agreement between the two observers . hence , the study was preceded with a single examiner . following the tabulation of the data , all calculations were performed using ibm spss statistical software package ( spss 14.0 for windows , spss inc . , the data obtained from the study were compiled , tabulated and subjected to statistical analysis . the results that were obtained are presented in the following manner : table 1 shows the general information of sex - wise distribution in the five groups . each group consisted of 10 subjects , which comprised of 6 ( 60% ) males and 4 ( 40% ) females in ol group , olp group and osmf group whereas in oscc group and control group , it comprised of 5 males ( 50% ) and 5 females ( 50% ) [ graph 1 ] . sex - wise distribution of the groups sex - wise distribution of the groups table 2 shows the general age distribution of all five groups . the mean age and standard deviation ( sd ) of ol was 41.10 12.38 , in osmf 30.90 4.07 , in olp 36.00 8.30 , in oscc 50.10 7.80 and in control group 37.30 9.09 [ graph 2 ] . age - wise distribution of the groups expression of e - cadherin in ol , osf , olp , oscc and normal tissue [ graph 3 ] expression and comparison of e - cadherin in all groups all 10 subjects of normal tissue expressed 4 + ( very strong expression ) . intense staining of e - cad is seen in the spinous layer and basal cell layer , but not at the basal surface of basal cells and superficial layer as a phenomenon of natural desquamation . in 10 subjects of leukoplakia group ( all of were homogenous type ) , 4 subjects scored as 1 + ( weak expression ) , 4 subjects showed as 2 + ( mild to moderate expression ) , 1 subject scored as 3 + ( moderate to strong expression ) and remaining 1 subject scored as 4 + ( very strong expression ) [ figures 1 and 2 ] . histopathological expression of oral leukoplakia using h and e , 10 immunohistochemical expression of e - cadherin in oral leukoplakia at , 40 in 10 subjects of ol , 50% of lesion showed mild dysplasia , of which 1 subject scored as 4 + ( very strong expression ) , 3 subjects scored as 2 + ( mild to moderate expression ) and remaining 1 subject showed as 1 + ( weak expression ) . about 30% of lesions showed moderate dysplasia , in which 1 subject scored as 3 + ( moderate to strong expression ) and 2 subjects scored as 1 + ( weak expression ) . remaining 20% of lesions showed severe dysplasia , in which 1 subject scored as 2 + ( mild to moderate expression ) and another scored as 1 + ( weak expression ) . in 10 subjects of osmf group ( all of were stage 2 ) , 2 subjects ( 20% ) scored as 1 + ( weak expression ) , 4 subjects ( 40% ) scored as 2 + ( mild to moderate expression ) , 3 subjects ( 30% ) scored as 3 + ( moderate to strong expression ) and remaining 1 ( 10% ) subject showed as 4 + ( very strong expression ) [ figures 3 and 4 ] . histopathological expression of oral submucous fibrosis using h and e , 10 immunohistochemical expression of e - cadherin in oral submucous fibrosis at , 40 of 10 osmf subjects , 5 ( 50% ) subjects showed mild stage histopathologically , in which 1 subject scored as 1 + ( weak expression ) , 3 subjects showed as 2 + ( mild to moderate expression ) and 1 subject scored as 4 + ( strong expression ) and remaining 5 ( 50% ) subjects showed moderate stage , in which 1 subject showed as 1 + ( weak expression ) , 1 subject showed as 2 + ( mild to moderate expression ) and other 3 subjects scored as 3 + ( moderate to strong expression ) . in 10 subjects of olp group , 6 subjects ( 60% ) showed 1 + ( weak expression ) , 1 subjects ( 10% ) scored as 2 + ( mild to moderate expression ) and remaining 3 subjects ( 30% ) scored as 3 + ( moderate to strong expression ) [ figures 5 and 6 ] . histopathological expression of oral lichen planus using h and e , 10 immunohistochemical expression of e - cadherin in oral lichen planus at , 40 in correlation with clinical types , of 6 reticular types of olp , 3 subjects ( 30% ) scored as 3 + ( moderate to strong expression ) , 1 subject ( 10% ) scored as 2 + ( mild to moderate expression ) and 2 subjects ( 20% ) showed 1 + ( weak expression ) . of the remaining 4 subjects , 2 subjects demonstrated atrophic type and 2 subjects as erosive type , both types expressed as 1 + ( weak expression ) . in 10 subjects of oscc group , 6 subjects ( 60% ) scored as 1 + ( weak expression ) , 3 subjects ( 30% ) scored as 2 + ( mild to moderate expression ) and remaining 1 subject ( 10% ) scored as 3 + ( moderate to strong expression ) [ figures 7 and 8 ] . histopathological expression of oral squamous cell carcinoma using h and e , 40 immunohistochemical expression of e - cadherin in oral squamous cell carcinoma at , 40 among 10 subjects of oscc , 6 ( 60% ) subjects showed moderately differentiated oscc in which 4 subjects scored as 1 + ( weak expression ) , 1 subject showed as 2 + ( mild to moderate expression ) , 1 subject showed as 3 + ( moderate to strong expression ) . about 20% of oscc are of well - differentiated and another 20% are of poorly differentiated carcinoma in which 1 subject scored as 1 + ( weak expression ) and 1 subject scored as 2 + ( mild to moderate expression ) . majority of the subjects with oscc ( n = 90% ) and olp ( n = 80% ) had grade 1 + expression , while majority of ol ( n = 70% ) scored 1 + and 2 + expression and majority of osmf ( n = 60% ) scored 2 + expression . this difference in expression of e - cad was statistically significant in comparison with normal control groups with p 0.001 . expression of e - cadherin in ol , osf , olp , oscc and normal tissue [ graph 3 ] expression and comparison of e - cadherin in all groups all 10 subjects of normal tissue expressed 4 + ( very strong expression ) . intense staining of e - cad is seen in the spinous layer and basal cell layer , but not at the basal surface of basal cells and superficial layer as a phenomenon of natural desquamation . in 10 subjects of leukoplakia group ( all of were homogenous type ) , 4 subjects scored as 1 + ( weak expression ) , 4 subjects showed as 2 + ( mild to moderate expression ) , 1 subject scored as 3 + ( moderate to strong expression ) and remaining 1 subject scored as 4 + ( very strong expression ) [ figures 1 and 2 ] . histopathological expression of oral leukoplakia using h and e , 10 immunohistochemical expression of e - cadherin in oral leukoplakia at , 40 in 10 subjects of ol , 50% of lesion showed mild dysplasia , of which 1 subject scored as 4 + ( very strong expression ) , 3 subjects scored as 2 + ( mild to moderate expression ) and remaining 1 subject showed as 1 + ( weak expression ) . about 30% of lesions showed moderate dysplasia , in which 1 subject scored as 3 + ( moderate to strong expression ) and 2 subjects scored as 1 + ( weak expression ) . remaining 20% of lesions showed severe dysplasia , in which 1 subject scored as 2 + ( mild to moderate expression ) and another scored as 1 + ( weak expression ) . in 10 subjects of osmf group ( all of were stage 2 ) , 2 subjects ( 20% ) scored as 1 + ( weak expression ) , 4 subjects ( 40% ) scored as 2 + ( mild to moderate expression ) , 3 subjects ( 30% ) scored as 3 + ( moderate to strong expression ) and remaining 1 ( 10% ) subject showed as 4 + ( very strong expression ) [ figures 3 and 4 ] . histopathological expression of oral submucous fibrosis using h and e , 10 immunohistochemical expression of e - cadherin in oral submucous fibrosis at , 40 of 10 osmf subjects , 5 ( 50% ) subjects showed mild stage histopathologically , in which 1 subject scored as 1 + ( weak expression ) , 3 subjects showed as 2 + ( mild to moderate expression ) and 1 subject scored as 4 + ( strong expression ) and remaining 5 ( 50% ) subjects showed moderate stage , in which 1 subject showed as 1 + ( weak expression ) , 1 subject showed as 2 + ( mild to moderate expression ) and other 3 subjects scored as 3 + ( moderate to strong expression ) . in 10 subjects of olp group , 6 subjects ( 60% ) showed 1 + ( weak expression ) , 1 subjects ( 10% ) scored as 2 + ( mild to moderate expression ) and remaining 3 subjects ( 30% ) scored as 3 + ( moderate to strong expression ) [ figures 5 and 6 ] . histopathological expression of oral lichen planus using h and e , 10 immunohistochemical expression of e - cadherin in oral lichen planus at , 40 in correlation with clinical types , of 6 reticular types of olp , 3 subjects ( 30% ) scored as 3 + ( moderate to strong expression ) , 1 subject ( 10% ) scored as 2 + ( mild to moderate expression ) and 2 subjects ( 20% ) showed 1 + ( weak expression ) . of the remaining 4 subjects , 2 subjects demonstrated atrophic type and 2 subjects as erosive type , both types expressed as 1 + ( weak expression ) . in 10 subjects of oscc group , 6 subjects ( 60% ) scored as 1 + ( weak expression ) , 3 subjects ( 30% ) scored as 2 + ( mild to moderate expression ) and remaining 1 subject ( 10% ) scored as 3 + ( moderate to strong expression ) [ figures 7 and 8 ] . histopathological expression of oral squamous cell carcinoma using h and e , 40 immunohistochemical expression of e - cadherin in oral squamous cell carcinoma at , 40 among 10 subjects of oscc , 6 ( 60% ) subjects showed moderately differentiated oscc in which 4 subjects scored as 1 + ( weak expression ) , 1 subject showed as 2 + ( mild to moderate expression ) , 1 subject showed as 3 + ( moderate to strong expression ) . about 20% of oscc are of well - differentiated and another 20% are of poorly differentiated carcinoma in which 1 subject scored as 1 + ( weak expression ) and 1 subject scored as 2 + ( mild to moderate expression ) . altogether , majority of the subjects with oscc ( n = 90% ) and olp ( n = 80% ) had grade 1 + expression , while majority of ol ( n = 70% ) scored 1 + and 2 + expression and majority of osmf ( n = 60% ) scored 2 + expression . this difference in expression of e - cad was statistically significant in comparison with normal control groups with p 0.001 . the shift in cancer research from the histopathological to the molecular and genetic levels was brought about by many new technologies , one among them is immunohistochemical study and is believed that understanding the molecular basis of cancer will lead to precise diagnosis and better treatment . in the oral mucosa , epithelium shows adaption to different mechanical demands and maintains its structure by a process of continuous cell renewal and a lot of factors are involved in maintaining the balance between proliferation and differentiation . one among them is e - cad , localized on the surface of epithelial cells in regions of cell - cell contact known as adherens junctions and regulates the cell aggregations . e - cad is also involved in transduction of signals controlling various cellular events , including polarity , differentiation , growth and cellular migration . in normal epithelium , e - cad is expressed in spinous and basal layer expect for the basal surface of the basal layers and superficial layers as a process of continuous renewal of cells . oral cancer is the most prevalent group of malignancies in indian subcontinent and the development of it is a multistep process , where in involving interactions between several factors such as tobacco - associated carcinogens , areca nut , betel quid and alcohol consumption , and/or viral infections and is a multi - stage process which requires modulation of cell - cell and cell - stromal interactions . loss of e - cad expression has been noted with poorly differentiated morphology in a large number of malignancies such as cervix , esophagus , including head and neck . hence , the present study was conducted to assess the expression of e - cad in ol , osmf , olp and oscc patients in comparison to nom by immunohistochemistry . in the present study , 10 samples of nom as control group were taken with average mean age and sd of 37.30 9.09 . these control tissues showed intense continuous membranous staining of e - cad in all the epithelial cells of spinous layer and to less extent staining is seen in basal cells and less staining or complete absence in superficial layers of cells which is suggestive of normal desquamation . this was consistent with the reported annual age incidence rate of 2.1/1000 in men and 1.3/1000 in women by rajendran and shivapathasundaram in their study . most of the previous studies on ol were also of the opinion that the common site of occurrence is buccal mucosa 76% , then alveolar sulcus 19% followed by tongue 5% by raina et al . , which is consistent to our present study where 9 subjects ( 90% ) of ol lesion are on buccal mucosa ; of these , majority of the subjects that is , 40% showed 2 + ( mild to moderate expression ) . remaining 1 subject ( 10% ) are on tongue that expressed as 1 + ( weak expression ) . in the present study , all the samples were of homogenous type of leukoplakia , with showing mild to severe dysplastic changes . these samples , as shown in table 3a , inferences that , there is no significant association between the degree of dysplasia with down - regulation of e - cad expression . this observation of our study was not consistent to various previous studies conducted by yuwanati et al . , chaw et al . who concluded that , reduction in the e - cad expression was seen as the degree of dysplasia increases from mild to severe , as the severity increases . staining intensities of e - cadherin expression between normal and different subgroups of leukoplakia overall , the majority of subjects in ol group ( n = 80% ) showed weak and mild membranous staining of e - cad in suprabasal and spinous layer of cells as shown in table 3 . hence , there is a reduction in e - cad expression in dysplastic layers of ol . this observation of our study was consistent with the studies conducted by yogesh et al . , kaur et al . who stated that atypical features of dysplasia are strongly correlated to the loss of expression of e - cad . yogesh et al . in his study also explained that , there is a variation in the expression of e - cad in the dysplastic epithelium with varying degrees of dysplasia or severity of dysplasia and location of tissue , would suggest that these alterations are the result of the progression of dysplasia and could be a late event , which is suggestive of change toward a cell phenotype with the ability to invade . all of the subjects of this group belong to the mean average age with sd of 30.90 4.07 , which is in contrast to the previous studies which say that it is a disease of adulthood between the ages of 4560 years . the possible reason for osmf occurring at young age could be that the patients were exposed to the irritants from a very young age . , according to them , the mean age is around 30 years . of 10 osmf samples , all the subjects were demonstrated as stage 2 clinically and histopathologically , 50% samples showed mild stage of osmf in which majority of samples showed mild to moderate expression that scored as 2 + and remaining 50% showed moderate stage , in which majority of the samples showed moderate to strong expression that scored as 3 + as shown in table 3b . in our study , as in shown table 3 , altogether majority of osmf samples ( n = 60% ) showed reduced expression of e - cad with mild to moderate intensity . this finding of our study was consistent with the study conducted by das et al . staining intensities of e - cadherin expression between normal and different stages of osmf the present study of 10 olp samples , 6 were males ( 60% ) and 4 were females ( 40% ) . in this present study , 6 ( 60% ) of the 10 subjects were of reticulate type , which is the most common type to occur according to many previous studies , 2 ( 20% ) were of atrophic type and 2 ( 20% ) were of erosive type . buccal mucosa is the most common site for olp followed by tongue , gingiva that is in consistent with our study where 60% are on buccal mucosa , 20% are on tongue and 20% are on gingiva . in 10 samples of olp group , with respect to clinical types , of 6 reticular types of olp , 3 subjects ( 30% ) scored as 3 + ( moderate to strong expression ) , 1 subject ( 10% ) scored as 2 + ( mild to moderate expression ) and 2 subjects ( 20% ) showed 1 + ( weak expression ) . of remaining 4 subjects , 2 subjects demonstrated atrophic type and 2 subjects as erosive type , both types expressed as 1 + ( weak expression ) . as a whole , majority of our olp samples ( n = 70% ) as in table 3 showed reduced or weak expression of e - cad in basal and suprabasal layer of cells , which is in accordance to study conducted by neppelberg et al . , who stated that , focal loss of e - cad in basal keratinocytes may contribute to loss of cell - to - cell contacts and destruction of basal keratinocytes and thus , this may play a role in the pathogenesis of olp and also concluded in his another study ( 2007 ) that e - cad were not significant sensitive markers to select olp lesions at risk for development of oscc . in another study that was conducted by ebrahimi et al . , also showed decreased expression of e - cad , this finding is consistent with our findings . the author concluded with their study that , mixed expression pattern of olp with variably resembling normal as well as tumor tissue and also with the previous data that , it can not be judged whether olp lesions are at an increased risk of malignant development . the present study consists of 10 oscc samples and of these samples , 5 ( 50% ) were males and 5 ( 50% ) were females . among 10 samples of oscc , majority of samples as shown in table 3c that has shown no significant association between the degree of differentiation of tumor and expression of e - cad . this observation of our study was consistent with the study conducted by shinohara et al . , diniz - freitas et al . , whose studies also showed no significant association between the degree of differentiation of tumor and expression of e - cad . staining intensities of e - cadherin expression between normal and different stages of oscc of 10 samples of oscc groups , altogether majority of samples ( n = 90% ) showed mild to moderate expression , these observation of our study suggest that , there was overall reduction or lost membranous expression of e - cad in the deep invasive tumor margin of primary tumor . a study conducted by hung et al . , concluded that immunoreactivity of e - cad was progressively reduced from normal mucosa followed by oral precancerous lesions and significantly decreased in primary oscc ( 58% ) , which was also coinciding with our findings of e - cad expression . upon assessing the expression of e - cad in ol , osmf , olp and oscc , as majority of the samples with oscc ( 90% ) , ol ( 80% ) , olp ( 70% ) and osmf ( 60% ) samples showed mild to moderate expression of e - cad staining , which was suggestive of reduction in dysplastic cells on comparison to nom cells . this difference in expression and variation of e - cad upon comparison with normal mucosa was statistically significant ( p < 0.001 ) . within the limitation of the study , it could be concluded that there was significant variation of expression of e - cad with the histopathological dysplasia of the oral precancerous lesions and the tumor differentiation of the oral cancers in comparison with nom . however , there was no correlation of the degree of loss of expression of e - cad with the degree of dysplasia or the tumor differentiation of oral cancers . since , there was a variation in the expression of e - cad , but its value as a prognostic marker is questionable . a longitudinal study with more sample size is required to confirm this conclusion .

objective : the aim of the present study was to assess the expression of e - cad in oral precancerous lesions and conditions and oral carcinomas in comparison with normal mucosa.materials and methods : total of 50 samples were selected for the study and were categorized into five groups and 10 samples in each group as group i - oral leukoplakia ( ol ) , group ii - oral lichen planus ( olp ) , group iii - oral submucous fibrosis ( osmf ) , group iv - oral squamous cell carcinoma ( oscc ) and group v - normal oral mucosa ( nom ) as control group . all the samples were assessed for the expression of e - cad by immunohistochemical study.results:upon assessing the expression of e - cad in ol , osmf , olp and oscc , as majority of the samples with oscc ( 90% ) , ol ( 80% ) , olp ( 70% ) and osmf ( 60% ) showed mild to moderate expression of e - cad staining , which was suggestive of reduction in dysplastic cells on comparison to nom cells . this difference in expression and variation of e - cad upon comparison with normal mucosa was statistically significant ( p < 0.001).conclusion : there is significant ( p < 0.001 ) variation of expression of e - cad with the histopathological dysplasia of the oral precancerous lesions and conditions , and the tumor differentiation of the oral cancers . however , there was no correlation of the degree of loss of expression of e - cad with the degree of dysplasia or the tumor differentiation of oral cancers . we conclude with our study that , there is a variation in the expression of e - cad but its value as a prognostic marker is questionable .

INTRODUCTION MATERIALS AND METHODS Study setting Evaluation of the staining for E-cadherin Statistical analysis RESULTS Expression of E-cadherin in normal tissue [ Expression of E-cadherin in oral leukoplakia [ Expression of E-cadherin in oral submucous fibrosis [ Expression of E-cadherin in oral lichen planus [ Expression of E-cadherin in oral squamous cell carcinoma [ DISCUSSION CONCLUSION

PMC4214767

growing up , i was fascinated by logical thinking and math . after bumping by chance into biology for my undergraduate degree , i became increasingly excited about it once i started to do my own experiments in the lab of aldo di leonardo at the university of palermo . what truly triggered my passion was an episode during my phd interview at the european molecular biology laboratory ( embl ) . using time - lapse videos in real time , michael way showed me how the bacterium listeria infects cells . the ability to monitor processes i knew then that a scientific career would provide me with a long fulfilling journey of discovery . i have had the privilege of training in institutions and laboratories where the richness of scientific thinking as well as resources propelled me through a rewarding learning experience . i did my phd ( 19982003 ) with suzanne eaton at the embl and max planck institute . suzanne 's free scientific mind and contagious enthusiasm for scientific discovery provided a stimulating framework for defining the questions that excited me . of great influence also was the open - door policy at my phd institutions . hierarchy was only a formality , and scientific discussions happened freely among different labs and across the hierarchical ladder . this fertile context contributed to my passion for addressing mechanisms of tissue growth in development by live imaging using drosophila . i did my postdoc ( 20032009 ) with elaine fuchs at rockefeller university , studying tissue regeneration using skin hair follicle in mice as a model system . elaine and her laboratory , a group of very talented scientists , provided me with strong training that fostered my independence and taught me approaches for efficiency and productivity . i started my laboratory in 2009 in the genetics department at yale medical school , recruited by two terrific scientists , richard lifton and haifan lin , who believed in my potential and supported me at a time when it was n't clear how things would turn out and who continue to support and inspire me to this date . when i established my lab , i wanted to understand how cells orchestrate growth within a tissue and how hierarchical organization plays a role in cell choices at the level of single cells as well as in integration within a group of cells , resulting in a robust and harmonious process of growth . the challenge in addressing these questions was posed by the fact that these processes are highly dynamic , but the field largely used static analysis to study them . during my doctoral thesis , i had experienced firsthand how live imaging had provided us not only a better understanding of the process we were studying but , especially , allowed us to discover new biology that we had not anticipated . thus , as i began to set up my lab , i addressed the above questions with canonical approaches and invested in a high - risk / high - reward approach to establish live imaging in the mouse skin . after more than one year of troubleshooting and several discouraging roadblocks , we were finally able to visualize and manipulate hair follicle stem cells and their niches in an intact living mouse . for example , we showed that stem cells can be dispensable for tissue regeneration and that other cells can reprogram to adopt their fates during injury . conversely , we demonstrated that the niche is required for hair follicle regeneration ( greco and guo , 2010 ; rompolas et al . , 2012 , 2013 ; rompolas and greco , 2014 ; deschene , myung , et al . , 2014 ; in retrospect , what i had accomplished was combining my passion for visualizing biological processes in vivo with my knowledge on stem cells gained during my postdoc . this allowed me to create a niche for my lab and distinguish myself from my previous mentors . while defining the key questions and the unique angle for my lab was key to establishing my lab , the next challenge was to identify a way to execute them . in that regard , we depend on our lab members and colleagues to carry out our ideas ( i.e. , writing papers and obtaining grants ) . to establish a highly functional lab , i believe that , in addition to defining key exciting questions , the principal investigator ( pi ) must balance two critical components : business and mentoring . and mentoring , describe the challenges junior pis face in embracing them , and conclude by describing some of the strategies i have adopted in my own lab . how do we maximize the creation of ideas and data ? how do we make these good ideas a reality that catches people 's attention ? in this regard , establishing a lab is analogous to setting up a business . in a way , i visualize it as being given a small shop to rent in a big mall . in order for us to be noticed , we need to create a product ( our data ) that people ( our colleagues ) can look at and decide whether it is worth their attention / investment or not . we need to make a brand ( our unique angle for producing data ) , find investors ( therefore excite future potential reviewers / funding agencies ) , and gain visibility by going around and creating publicity ( giving talks ) . i define mentoring as the guidance provided by a more experienced researcher to a less experienced one ( mentee ) that contributes to the mentee 's development as a scientist . this includes teaching trainees how to design experiments and align expectations and how to prepare for talks . all of that should be done within the context of a relationship based on truth and mutual trust . pis are dependent on their students and postdocs for the realization of their ideas and , therefore , for the success of their labs . it is a mutual dependency . while it is clear that the pi 's investment of time and energy in developing the competencies of the mentees are an investment in the business that supports all members involved , it is less clear how to provide good mentoring that feeds both parties , the mentee and the mentor . there are a number of challenges that prevent people ( especially young investigators ) from being proper mentors and getting the most out of their labs . they have to transition from postdoctoral training , in which they had to master benchwork and a working relationship with primarily one person , the pi , to productively managing a team . second , there is a dramatic increase in the number of different tasks that we need to cover , which pull us in several different directions . third , it is not easy to recognize that mentoring is instrumental in maximizing the efforts and the establishment of our lab . 1 ) institutions have to recognize these challenges and provide training to educate junior faculty on how to best manage and mentor a group . 2 ) junior faculty members themselves have to be proactive about acquiring the necessary knowledge from midcareer pis , preferably in groups with open discussion on current challenges . 3 ) pis must educate their mentees on how to be leaders and mentors themselves . there are different models that can be adopted to best mentor a group while trying to feed into creating the products ( papers and grants ) . one model envisions the leader as the one who seeds ideas and leaves the lab members in charge to develop them in practice . an alternative model , not mutually exclusive with the first one , sees the leader as the one who fosters an environment in which people generate ideas . while i naturally lean toward model 2 , it can also be argued that this model has the advantage of 1 ) giving ownership to the mentee for the scientific project , 2 ) engendering continuous reevaluation of the excitement and novelty associated with the project , and 3 ) helping to identify the most practical and fastest way to execute the project . model 1 is perhaps more efficient in the short term , but in the long term , it runs the risks ( among others ) of creating less independent scientists who can not propagate knowledge to the next generation as efficiently or represent the lab at meetings . thus one of my mentoring approaches is to involve my group in the several tasks i need to perform , as this fills two purposes . these tasks include training a lab member to give a talk outside the lab , having a lab member prepare a grant proposal , and so on . what this creates is a supportive , unified group experience that elevates the impact and depth of the science we do , thereby feeding into the lab business as well . specifically , i created the following systems : i set up a number of different forums in addition to the canonical lab meetings and weekly one - on - one meetings . these include brainstorming sessions , when each lab member takes a turn giving a chalk talk to the entire lab over beer and pizza about his or her vision on his or her current project and possible future directions . this is in addition to a broad review of all data with me every six months , when i spend 45 h with each individual , discussing all our goals , aligning them , and discussing all the data produced and the expectations we have moving forward . since these forums have been put in place , these approaches have led to shaping stories earlier than i anticipated and allowing lab members to contribute to one another 's projects more effectively.i seek opportunities for my lab members to give talks outside the lab in order for us to more effectively think about science . every time we start a project , we get attracted to questions that excite us . the process , however , of going from our questions to finding answers is often lengthy and somewhat abstract ( what uri alon in his essay refers to as a cloud ) , a process comparable to creating an object from clay . as it starts , it does n't have a shape , and my mentee and i keep working that material , thinking over time about a product that excites us , is unique , and could be attractive to a broader audience . the way we get there relies strongly on giving talks and especially on the approach used to prepare for talks . to give a practical example , every time a lab member is giving a talk , the preparation follows three steps : 1 ) he / she will build it two weeks before the event , discussing it back and forth with me . this helps both of us start to think hard about the collected data , the best angle for presenting them , and what conclusions can we draw from them . 2 ) the lab member will give a practice talk to the lab one week ahead of the event , with everyone actively participating by constructively criticizing , dismantling , and remolding the entire talk . 3 ) the lab member will give a practice talk to me only few days before the event to finalize it and sharpen all the edges . strikingly , while at first read this may seem to be a lot of work , this has been the best investment of my time from the beginning , because it has , first , allowed me to put together our manuscripts much faster as a result of this intense thinking ; second , it has allowed me to give ownership to the lab member for his or her own project ; and third , it has created a sense of unity that allows everyone to feel protected while pushing hard for their own projects as well as for those of their colleagues . i set up a number of different forums in addition to the canonical lab meetings and weekly one - on - one meetings . these include brainstorming sessions , when each lab member takes a turn giving a chalk talk to the entire lab over beer and pizza about his or her vision on his or her current project and possible future directions . this is in addition to a broad review of all data with me every six months , when i spend 45 h with each individual , discussing all our goals , aligning them , and discussing all the data produced and the expectations we have moving forward . since these forums have been put in place , these approaches have led to shaping stories earlier than i anticipated and allowing lab members to contribute to one another 's projects more effectively . i seek opportunities for my lab members to give talks outside the lab in order for us to more effectively think about science . every time we start a project , we get attracted to questions that excite us . the process , however , of going from our questions to finding answers is often lengthy and somewhat abstract ( what uri alon in his essay refers to as a cloud ) , a process comparable to creating an object from clay . as it starts , it does n't have a shape , and my mentee and i keep working that material , thinking over time about a product that excites us , is unique , and could be attractive to a broader audience . the way we get there relies strongly on giving talks and especially on the approach used to prepare for talks . to give a practical example , every time a lab member is giving a talk , the preparation follows three steps : 1 ) he / she will build it two weeks before the event , discussing it back and forth with me . this helps both of us start to think hard about the collected data , the best angle for presenting them , and what conclusions can we draw from them . 2 ) the lab member will give a practice talk to the lab one week ahead of the event , with everyone actively participating by constructively criticizing , dismantling , and remolding the entire talk . 3 ) the lab member will give a practice talk to me only few days before the event to finalize it and sharpen all the edges . strikingly , while at first read this may seem to be a lot of work , this has been the best investment of my time from the beginning , because it has , first , allowed me to put together our manuscripts much faster as a result of this intense thinking ; second , it has allowed me to give ownership to the lab member for his or her own project ; and third , it has created a sense of unity that allows everyone to feel protected while pushing hard for their own projects as well as for those of their colleagues . thus my mentorship ( lab meetings , brainstorming , six - month review , etc . ) leads to scientific success ( papers ) and , therefore , business success ( funding ) . finally , my mentorship feeds into my business model not only by producing successful science but also by producing a healthy , happy work environment . how do we maximize the creation of ideas and data ? how do we make these good ideas a reality that catches people 's attention ? in this regard , establishing a lab is analogous to setting up a business . in a way , i visualize it as being given a small shop to rent in a big mall . in order for us to be noticed , we need to create a product ( our data ) that people ( our colleagues ) can look at and decide whether it is worth their attention / investment or not . we need to make a brand ( our unique angle for producing data ) , find investors ( therefore excite future potential reviewers / funding agencies ) , and gain visibility by going around and creating publicity ( giving talks ) . i define mentoring as the guidance provided by a more experienced researcher to a less experienced one ( mentee ) that contributes to the mentee 's development as a scientist . this includes teaching trainees how to design experiments and align expectations and how to prepare for talks . all of that should be done within the context of a relationship based on truth and mutual trust . pis are dependent on their students and postdocs for the realization of their ideas and , therefore , for the success of their labs . it is a mutual dependency . while it is clear that the pi 's investment of time and energy in developing the competencies of the mentees are an investment in the business that supports all members involved , it is less clear how to provide good mentoring that feeds both parties , the mentee and the mentor . there are a number of challenges that prevent people ( especially young investigators ) from being proper mentors and getting the most out of their labs . they have to transition from postdoctoral training , in which they had to master benchwork and a working relationship with primarily one person , the pi , to productively managing a team . second , there is a dramatic increase in the number of different tasks that we need to cover , which pull us in several different directions . third , it is not easy to recognize that mentoring is instrumental in maximizing the efforts and the establishment of our lab . 1 ) institutions have to recognize these challenges and provide training to educate junior faculty on how to best manage and mentor a group . 2 ) junior faculty members themselves have to be proactive about acquiring the necessary knowledge from midcareer pis , preferably in groups with open discussion on current challenges . 3 ) pis must educate their mentees on how to be leaders and mentors themselves . there are different models that can be adopted to best mentor a group while trying to feed into creating the products ( papers and grants ) . one model envisions the leader as the one who seeds ideas and leaves the lab members in charge to develop them in practice . an alternative model , not mutually exclusive with the first one , sees the leader as the one who fosters an environment in which people generate ideas . while i naturally lean toward model 2 , it can also be argued that this model has the advantage of 1 ) giving ownership to the mentee for the scientific project , 2 ) engendering continuous reevaluation of the excitement and novelty associated with the project , and 3 ) helping to identify the most practical and fastest way to execute the project . model 1 is perhaps more efficient in the short term , but in the long term , it runs the risks ( among others ) of creating less independent scientists who can not propagate knowledge to the next generation as efficiently or represent the lab at meetings . thus one of my mentoring approaches is to involve my group in the several tasks i need to perform , as this fills two purposes . these tasks include training a lab member to give a talk outside the lab , having a lab member prepare a grant proposal , and so on . what this creates is a supportive , unified group experience that elevates the impact and depth of the science we do , thereby feeding into the lab business as well . specifically , i created the following systems : i set up a number of different forums in addition to the canonical lab meetings and weekly one - on - one meetings . these include brainstorming sessions , when each lab member takes a turn giving a chalk talk to the entire lab over beer and pizza about his or her vision on his or her current project and possible future directions . this is in addition to a broad review of all data with me every six months , when i spend 45 h with each individual , discussing all our goals , aligning them , and discussing all the data produced and the expectations we have moving forward . since these forums have been put in place , these approaches have led to shaping stories earlier than i anticipated and allowing lab members to contribute to one another 's projects more effectively.i seek opportunities for my lab members to give talks outside the lab in order for us to more effectively think about science . every time we start a project , we get attracted to questions that excite us . the process , however , of going from our questions to finding answers is often lengthy and somewhat abstract ( what uri alon in his essay refers to as a cloud ) , a process comparable to creating an object from clay . as it starts , it does n't have a shape , and my mentee and i keep working that material , thinking over time about a product that excites us , is unique , and could be attractive to a broader audience . the way we get there relies strongly on giving talks and especially on the approach used to prepare for talks . to give a practical example , every time a lab member is giving a talk , the preparation follows three steps : 1 ) he / she will build it two weeks before the event , discussing it back and forth with me . this helps both of us start to think hard about the collected data , the best angle for presenting them , and what conclusions can we draw from them . 2 ) the lab member will give a practice talk to the lab one week ahead of the event , with everyone actively participating by constructively criticizing , dismantling , and remolding the entire talk . 3 ) the lab member will give a practice talk to me only few days before the event to finalize it and sharpen all the edges . strikingly , while at first read this may seem to be a lot of work , this has been the best investment of my time from the beginning , because it has , first , allowed me to put together our manuscripts much faster as a result of this intense thinking ; second , it has allowed me to give ownership to the lab member for his or her own project ; and third , it has created a sense of unity that allows everyone to feel protected while pushing hard for their own projects as well as for those of their colleagues . i set up a number of different forums in addition to the canonical lab meetings and weekly one - on - one meetings . these include brainstorming sessions , when each lab member takes a turn giving a chalk talk to the entire lab over beer and pizza about his or her vision on his or her current project and possible future directions . this is in addition to a broad review of all data with me every six months , when i spend 45 h with each individual , discussing all our goals , aligning them , and discussing all the data produced and the expectations we have moving forward . since these forums have been put in place , these approaches have led to shaping stories earlier than i anticipated and allowing lab members to contribute to one another 's projects more effectively . i seek opportunities for my lab members to give talks outside the lab in order for us to more effectively think about science . every time we start a project , we get attracted to questions that excite us . the process , however , of going from our questions to finding answers is often lengthy and somewhat abstract ( what uri alon in his essay refers to as a cloud ) , a process comparable to creating an object from clay . as it starts , it does n't have a shape , and my mentee and i keep working that material , thinking over time about a product that excites us , is unique , and could be attractive to a broader audience . the way we get there relies strongly on giving talks and especially on the approach used to prepare for talks . to give a practical example , every time a lab member is giving a talk , the preparation follows three steps : 1 ) he / she will build it two weeks before the event , discussing it back and forth with me . this helps both of us start to think hard about the collected data , the best angle for presenting them , and what conclusions can we draw from them . 2 ) the lab member will give a practice talk to the lab one week ahead of the event , with everyone actively participating by constructively criticizing , dismantling , and remolding the entire talk . 3 ) the lab member will give a practice talk to me only few days before the event to finalize it and sharpen all the edges . strikingly , while at first read this may seem to be a lot of work , this has been the best investment of my time from the beginning , because it has , first , allowed me to put together our manuscripts much faster as a result of this intense thinking ; second , it has allowed me to give ownership to the lab member for his or her own project ; and third , it has created a sense of unity that allows everyone to feel protected while pushing hard for their own projects as well as for those of their colleagues . thus my mentorship ( lab meetings , brainstorming , six - month review , etc . ) leads to scientific success ( papers ) and , therefore , business success ( funding ) . finally , my mentorship feeds into my business model not only by producing successful science but also by producing a healthy , happy work environment . while generally thought of as independent entities , science business mentorship go hand in hand in my opinion . mentoring brings depth and quality to business , and business brings effectiveness and productivity to mentoring . while everyone naturally enjoys witnessing the accomplishments that our lab members obtain , the process for getting them there is not as intuitive and is sometimes quite intense , which makes us question whether it is the right investment of our energies . because of this , seeking sources of mentorships through established courses and internal resources at our university is paramount for the effectiveness and establishment of junior pi laboratories . investing time in meaningful mentorship fosters a productive and harmonious work environment that results in successful science and , therefore , business .

it is a tremendous honor for my group and me to receive the recognition of the 2014 women in cell biology junior award . i would like to take the opportunity of this essay to describe my scientific journey , discuss my philosophy about running a group , and propose what i think is a generalizable model to efficiently establish an academic laboratory . this essay is about my view on the critical components that go into establishing a highly functional academic laboratory during the current tough , competitive times .

WHAT HOOKED ME ON SCIENCE MY JOURNEY DURING MY PhD AND POSTDOCTORAL TRAINING MY LAB SCIENCE AND PHILOSOPHY: MENTORING AS A BUSINESS MODEL Definition of business Definition of mentoring Challenges in mentoring Example of proposed solutions: this model in the context of a group CONCLUSIONS

PMC3030037

a 65-year - old , 175 cm , 91 kg , male patient with prostate cancer was admitted for laparoscopic prostatectomy . he had an unremarkable medical history without any surgery . on the blood tests , the hemoglobin was 11.3 g / dl and the hematocrit was 35.1% . the liver function , renal function , blood coagulation time and bleeding time were all in the normal ranges . the electrocardiogram ( ekg ) was also normal , but simple chest x - ray showed bronchitis . on the pulmonary function test ( pft ) , the vital signs before anesthetic induction showed a blood pressure ( bp ) of 150/90 mmhg , a heart rate ( hr ) of 65/min , a respiratory rate ( rr ) of 16/min , a body temperature of 36.5 and the oxygen saturation on pulse oximetry ( spo2 ) was 96% . after pre - oxygenation with 100% oxygen , anesthesia was induced with thiopental sodium 250 mg and rocuronium 50 mg . endotracheal intubation was then performed with an 8.0 mm inner diameter ( i d ) endotracheal tube . anesthesia was maintained with o2 and n2o both at 1.5 l / min and sevoflurane at an optimal concentration , while vecuronium was intermittently administered to maintain adequate muscle relaxation . the partial pressure of end - tidal carbon dioxide ( etco2 ) was continuously monitored from the induction of anesthesia , and it was 33 mmhg right after tracheal intubation . after endotracheal intubation , the gastrointestinal tract was suctioned using a levin tube . a central venous pressure ( cvp ) catheter was inserted into the right subclavian vein and a 16-g catheter was placed in the left external jugular vein . a modified allen 's test was performed at the left radial artery and then a catheter was inserted to continuously monitor the arterial pressure . after anesthetic induction , mechanical ventilation was performed with a tidal volume ( tv ) of 550 ml and a rr of 12/min . at that time , the bp was 120/70 mmhg , the hr was 58/min and there was a spo2 of 99% , a cvp of 8 - 9 mmhg , an etco2 of 30 mmhg , a peak airway pressure of 21 cmh2o and a mean airway pressure of 17 cmh2o . the arterial blood gas analysis ( abga ) showed normal findings and the ekg also indicated a regular sinus rhythm . when the operational preparations were completed , the surgeon connected a trocar to a carbon dioxide gas pump and induced a pneumoperitoneum using co2 gas . the abdominal cavity pressure was maintained at 12 mmhg . after the insufflation , the etco2 gradually increased to 40 mmhg ; therefore , the rr increased to 14/min . the patient was placed in trendelenburg 's position at 25 - 30 degrees at the surgeon 's request . at that time , the cvp was 13 mmhg , the peak airway pressure was 31 cmh2o , the mean airway pressure was 23 cmh2o , the etco2 was 34 - 35 mmhg and the spo2 was between 98 and 100% . another abga one hour after the onset of operation showed a ph of 7.42 , a paco2 of 40 mmhg , a pao2 of 165 mmhg , a hco3 of 25.9 meq / l , a base excess of 1.4 mmol / l , a sao2 of 100% and a hematocrit of 32% . it took 5 hours to completely remove the prostatic gland with five trocars under a pneumoperitoneum . after removal of the prostatic gland and local hemostasis was performed under laparoscopic surgery , the operation was interrupted due to the surgeon 's unavoidable reasons . the patient was then repositioned from trendelenburg 's position to the normal position , and the pneumoperitoneum was deflated by ventilating two or three times a large volume of gas and the trocars were not removed from the abdominal cavity . after the deflation of the pneumoperitoneum , the peak airway pressure gradually decreased to 22 cmh2o and the etco2 also gradually decreased to 30 mmhg . at that time , his bp was 110/70 mmhg , the hr was 65/min and the spo2 was 99% , which indicated that his vital signs were stable . up to that moment , the total blood loss was less than 200 ml and the hematocrit was 31% . about 30 minutes later , the patient was repositioned in the trendelenburg position , and pneumoperitoneum with re - insufflation of co2 gas into the abdominal cavity was induced with keeping the intra - abdominal pressure less than 12 mmhg . during insufflation , there was no intra - abdominal bleeding and no other abnormalities detected on the viewing screen of the laparoscope . when the gas insufflation was close to the end , the patient 's bp dropped suddenly from 120/80 mmhg to 60/30 mmhg , and hr increased sharply from 60/min to 100/min with tachyarrhythmia , including premature atrial contractures in a few seconds . later , the hr returned to normal sinus rhythm with a rate of 78/min , and also the spo2 decreased from 99% to 92% . the peak airway pressure increased from 31 cmh2o to 38 cmh2o , and the etco2 also increased from 35 mmhg to 39 mmhg and the cvp increased from 13 mmhg to 18 mmhg . manual ventilation was immediately performed using 100% oxygen , and the gas insufflation was stopped . the breathing sounds were clear on both lung fields and there were no secretions during endotracheal suction . his bp was not increased , and 2 doses of ephedrine 8 mg and phenylephrine 100 ug were additionally injected . however , his bp was not restored and it remained at 70/40 mmhg , and so dopamine was infused at 6 ug / kg / min . under the suspicion of co2 embolism , he was moved into durant 's position ( the left lateral decubitus position with a head - down tilt ) and blood was aspirated via the cvp catheter , but gas bubbles were not detected . about 15 minutes after the onset of hypotension , transesophageal echocardiography ( tee ) was done at the middle of the esophagus for monitoring with a probe to secure a good view in all directions , but no gas bubbles were observed in the 4 chambers . a simple chest x - ray was taken to exclude the possibility of co2 pneumothorax , subcutaneous emphysema and pneumomediastinum , and there were no abnormalities other than atelectasis in both lower lobes . approximately 20 minutes after the sudden drop in bp , his vital signs took a favorable turn : the bp was 100/60 mmhg , the hr was 85/min and the spo2 was 99% . however , the peak airway pressure gradually increased to 39 cmh2o and the etco2 increased to 40 mmhg . at the moment , the tv was maintained at 550 ml and the rr was kept at 14/min . abga showed that the ph was 7.30 , the paco2 was 49 mmhg , the pao2 was 116 mmhg , the hco3 was 24.1 forty minutes after the bp decline , his vital signs were maintained at the bp of 100/60 mmhg , a hr of 80/min , a cvp of 15 cmh2o and an etco2 of 37 mmhg . at that time , the surgeon decided to perform laparotomy because the configuration of the patient 's prostate gland and bladder neck was so abnormal that suturing of the incision site was difficult . yet as the open surgery proceeded , massive bleeding developed , which required a transfusion of blood . abga during the transfusion showed a ph of 7.28 , a paco2 of 46 mmhg , a pao2 of 175 mmhg , a hco3 of 21.6 meq / l , a base excess of -5.1 mmol / l , a sao2 of 99% and a hematocrit of 27% . for fear that an increase in the peak airway pressure and cvp could cause pulmonary edema , 10 mg of furosemide the amount of urine was 500 ml within the first hour and this was followed by approximately 300 ml of urine output per hour . it took 9 hours and 15 minutes for the operation and 10 hours and 10 minutes for anesthesia . the total amount of infused fluid was 9,200 ml , the amount of transfused blood was 1,600 ml , the urine output was 1,080 ml and the estimated blood loss was 2,500 ml . at the end of operation , the patient was transferred to the intensive care unit ( icu ) with the endotracheal tube in place . the chest x - ray conducted one hour after transfer revealed a finding of mild pulmonary edema . with the patient under synchronized intermittent mandatory ventilation ( simv ) , the peak airway pressure was 28 cmh2o and the cvp was 13 cmh2o . three hours later , the peak airway pressure and cvp decreased to 25 cmh2o and 12 cmh2o , respectively . extubation was performed on the second postoperative day ( pod 2 ) , and he was transferred to the general ward on pod 4 . on pod 10 , he was discharged home without any related complications . after the pneumoperitoneum was transiently deflated and the surgery was stopped after removal of the prostate under laparoscopic surgery , when we tried to resume the surgery with creating a pneumoperitoneum by re - insufflation of co2 gas into the abdominal cavity , an abrupt severe cardiovascular collapse emerged . two major pathogeneses have been described for the major causes of cardiovascular collapse and cardiac arrest during laparoscopic surgery : gas embolism caused by gas insufflations and the vagal reflex activated by peritoneal stretch . compared with conventional open surgery , laparoscopic surgery has benefits such as less tissue damage , less post - operative pain , a faster return to everyday living due to the short recovery time , less post - operative scarring , etc . , and so laparoscopic surgery has been widely used in various areas . in most cases , in order to secure the visual field and space for surgery , a pneumoperitoneum is deliberately induced for the surgery . a pneumoperitoneum is achieved by insufflating the abdomen with gas such as carbon dioxide , nitrous oxide , helium , argon , etc . . among them , co2 is largely used because it is chemically inert , colorless , inexpensive , readily available and less explosive than air , and it also has such high solubility in the blood that there is less risk of embolism even though co2 is absorbed fast into the blood . when gas is insufflated into the abdominal cavity , the abdominal pressure increases and the co2 in the abdominal cavity is absorbed into the blood stream , leading to hypercarbia . it also compresses the arterial vessels , so that the afterload and systemic vascular resistance increase , whereas the cardiac output decreases . to minimize such hemodynamic changes the risk of complications during laparoscopic surgery such as hypercarbia , subcutaneous emphysema , pneumothorax and pneumomediastinum may increase according to a longer duration of surgery , a higher etco2 , a greater number of used trocars and an older age of the patient . if an infusion flow rate reaches over 1 l / min or the intra - abdominal pressure passes over 15 mmhg during insufflation of co2 gas , then complications seem to easily develop . in other words , too fast an infusion rate of co2 with excessive pressure is likely to induce embolism , causing the gas lock phenomenon , which interrupts blood flow in the vena cava or the right atrium , and then it decreases the cardiac output and induces cardiovascular collapse . about 60% of the symptomatic cases of co2 embolism occur during the early period of insufflations of co2 , while the majority of the episodes incur when trocars are improperly inserted into the abdominal cavity or they are directly inserted into veins or organs . sometimes , gas embolism may develop without direct infusion of gas when the gas diffuses into the damaged vessels around the surgical site or the abdominal wall . in the present case , sudden and serious hypotension occurred during the induction of pneumoperitoneum , which was performed to resume the operation . co2 embolism was suspected as the primary cause of the hypotension due to the diffusion of carbon dioxide into injured blood vessels that were damaged by the surgical operation . other factors , such as hypovolemia , a vagal reflex caused by sudden peritoneal stretch , co2 pneumothorax , pneumomediastinum , pulmonary emphysema or air embolism could be considered as alternative causes . considering that there was no bleeding caused by the operation until the development of hypotension and that the laparoscopic findings observed since the resumption of pneumoperitoneum did not detect any bleedings in the abdominal cavity , and that the cvp and urine output were properly maintained , hypovolemia was unlikely to be the cause . as for the peritoneal stretch - induced vagal reflex , it could also be ruled out as the cause because the hr during the hypotension was not low enough to be regarded as bradycardia , but it was maintained at 78/min , which was slightly higher than the peri - operative hr . simple chest x - ray helped eliminate the possibility that the hypotension was induced by co2 pneumothorax , pneumomediastinum or pulmonary emphysema . air embolism unusually can occur in open prostate surgery because of the affluent venous vascular plexus in the prostate gland and using trendelenburg 's position . however , in the present case , there was less chance that air entered into the blood circulation because the laparoscopic surgery created airtight conditions and the trocars still remained in the abdominal cavity even though the operation was suspended . therefore , co2 embolism was suspected to be responsible in the current case for such phenomena as the abrupt onset of hypotension , the relative tachycardia following several occurrences of premature atrial contraction , the increase in the cvp , the increase in the etco2 , the decrease in oxygen saturation ( spo2 ) , etc . the symptoms of co2 embolism vary according to the amount of gas absorbed into the blood and the inflow rate of gas into the blood . with the rapid influx of a large amount of gas , a gas - lock takes place , leading to a significant decrease in the cardiac output and an increase of the physiological dead space due to a large amount of effluent gas into the lung . severe embolism may cause hypotension , bradycardia , arrhythmia , an increase in the mean pulmonary artery pressure , an increase in the cvp , a change in the doppler wave form and heart sounds , millwheel murmur , cyanosis , excessive right heart strain seen on ekg , an increase in the peak airway pressure , a change in the etco2 ( an increase in the early stage and a decrease in the late stage ) , etc . the etco2 can increase at the early stage of embolism because co2 flowing into the blood is emitted through the lung , but it can decrease at the latter stage as the cardiac output decreases and physiological dead space increases with the elapse of time . yet in the present case , the etco2 showed a slow increase , but there was no decrease in the latter stage . it might mean that the rate of development or the volume of embolism was not high enough to induce a severe decrease of cardiac output or a big increase of the physiological dead space . furthermore , while the cardiac output did not decrease significantly , the hypotensive condition lasted for a long time in our patient . the reason for the prolonged hypotension was guessed that the volume effect of the embolism exceeded the rate of embolism formation . because co2 has high solubility in blood , a large amount of absorbed co2 may be dissolved into the blood rather than inducing gas - lock or being emitted through the lung , resulting in systemic hypercarbia , which may induce prolonged hypotension by dilating the blood vessels and reducing the peripheral vascular resistance . the symptoms and risks of gas embolism are different not only according to the gas influx volume and rate , but also for the different kinds and solubility of the gas . even a small amount of helium or argon gas embolism can be lethal , while symptoms are rarely seen with a large amount of co2 embolism due to its high solubility . reported that 60% of the patients without any abnormalities of their vital signs revealed co2 shadows on the right atrium and ventricle by tee when the patients had undergone laparoscopic cholecystectomy . in a study by park et al . when making observations , via tee , of air shadows in the right atrium of 20 patients who had undergone laparoscopic cholecystectomy , emboli were detected in 4 patients of 20 patients during insufflation of co2 gas during dissection of the gallbladder . their results would support that laparoscopic surgery using pneumoperitoneum is always vulnerable to the possibility of co2 embolism . monitoring devices such as tee and a doppler probe attached to the epigastric / precordial region or the esophagus , and a pulmonary trunk catheter are well - known for their high sensitivity to detect gas embolism , while spo2 and etco2 are useful for the early detection of hypoxia and embolism as well as for estimation of the embolus . for making the definite diagnosis of embolism , it is 5 to 10 times more sensitive than transthoracic doppler - echocardiography , enough to be able to diagnose paradoxical embolism due to its capacity to visualize even the left heart . paradoxical embolism can be caused by the formation of a right - to - left shunt through a patent foramen ovale ( pfo ) , which is detected in 20 - 30% of the population of normal adults . it is known that tee can detect even as little as 0.5 cm of air and the detected air can be aspirated by a cvp catheter in 50% of the cases . yet in the present case , we could not detect any co2 shadows on tee . for these reasons , it is plausible to assume that the amount of co2 running into the blood was small or the co2 already was dissolved into the blood , or that the co2 migrated from the heart into the pulmonary vessels . considering that tee was not used directly after the onset of hypotension , but it was used more than 15 minutes after it developed , and that the hemodynamic change and etco2 decrease were not significant , the co2 was more likely to have been dissolved into the blood . as soon as co2 embolism is suspected , the insufflation of co2 should be discontinued and the pneumoperitoneum should be deflated , while the patient should be positioned to the durant position ( head - down tilt and left lateral decubitus position ) . the durant position allows air bubbles to migrate towards the apex of the heart , which prevents the formation of a gas - lock by gathering up air into the right ventricular apex . therefore , when a left lateral decubitus position is difficult to apply , it would be helpful to use the head - down tilt position alone . anesthesiologists should stop using n2o and they should alleviate the hypoxia and hypercarbia by performing mechanical controlled hyperventilation using 100% o2 . simultaneously , central venous aspiration of blood intimately mixed with air - bubbles can be performed to withdraw gas directly from the heart . the patient 's vital signs should be promptly improved by applying adjuvant therapy such as administering fluid and inotropics . in our case , ventilation with 100% o2 was performed , the patient was placed in durant 's position , air was aspirated and fluid and inotropics such as ephedrine , phenylephrine , dopamine , etc . were administered immediately at the onset of symptoms . due to excessive embolism , if the patient 's cardiovascular collapse is not restored and the patient does not respond to the adjuvant therapy , then a cardiopulmonary bypass ( cpb ) should be performed . diakun reported a case of severe co2 embolism that was encountered during laparoscopic surgery for the excision of the uterine septum using a hysteroscope . sudden arrhythmia was rapidly followed in turn by cardiac arrest , an increase of the etco2 , a decrease of oxygen saturation and an increase of the peak airway pressure . they administered atropine , bicarbonate , epinephrine and calcium and they applied defibrillation four times for resuscitation . there were no responses , and the cardiac rhythm did not respond to all these measures . then cpb was performed with internal cardiac massage and the patient was successfully resuscitated . in the present case the most highly possible cause was that a large amount of co2 had flowed into the blood through the injured blood vessels that were damaged by the surgical operation , which was caused by the re - induction of the pneumoperitoneum to resume the stopped operation after removal of the prostate gland under laparoscopic surgery . furthermore , the possibility of a gas insufflation rate over to 12 mmhg and that of the patient being placed into the trendelenburg position seem to be other risk factors that are responsible for co2 running into the damaged vascular plexus of the surgical site . kypson and greenville also reported an episode of co2 embolism that developed after gas re - insufflation during a coronary artery bypass graft ( cabg ) . after they harvested the saphenous vein using an endoscope and they completed the cpb , they tried to re - insufflate the co2 gas in order to confirm hemostasis on the surgical site of the saphenous vein . then , a serious co2 embolism occurred and they had to perform cpb again to resuscitate the potentially fatal cardiovascular collapse . therefore , it should be emphasized that re - insufflation of co2 gas after the deflation of pneumoperitoneum is a strong risk factor for co2 embolism because a large amount of gas might flow into the blood through damaged blood vessels . in addition , cottin et al . reported two deaths out of seven patients with gas embolism that developed during laparoscopic surgery . they indicated that a previous history of having received intra - abdominal surgery could be a potential risk factor for gas embolism due to the possibility of vascular injury . in summary , we experienced a case of severe cardiovascular collapse when we tried to resume the stopped operation by re - inducing a pneumoperitoneum with co2 gas during laparoscopic prostatectomy . through the current case , it came to our attention that physicians are apt to overlook the potential risks of severe cardiovascular collapse during laparoscopic surgery even though laparoscopic surgery is widely employed . that is to say , it should be noted that the laparoscopic surgery with co2-induced pneumoperitoneum is not free from the risk of co2 embolism . therefore , surgeons should refrain from re - inducing pneumoperitoneum after deflation during laparoscopic surgery , and they should pay closer attention to the patient 's choice for operation and assess the surgical risk factors such as a previous history of intra - abdominal surgery and also properly manage the intra - abdominal pressure . in addition , anesthesiologists should be thoroughly prepared for the rare , but potentially lethal complications of co2 embolism .

although symptomatic carbon dioxide ( co2 ) embolism is rare , it recognized as a potentially fatal complication of laparoscopic surgery . sudden hemodynamic instability could be a co2 embolism especially during insufflation . a 65-year - old man received laparoscopic prostatectomy for 5 hours under co2 pneumoperitoneum without any problem . after resection of prostate , it was stopped following deflation . thirty minutes later , peumoperitoneum was re - induced to continue the operation . shortly after re - insufflation , the patient revealed hemodynamic instability suggested a co2 embolism ; severe hypotension , tachyarrythmia , hypoxemia , increased cvp , and changed end - tidal co2 . gas insufflation was stopped . he was managed with durant 's position , fluid and cardiotonics for 20 minutes . the residual was completed by open laparotomy . re - insufflation , inducing gas entry through the injured vessels , might be a risk factor for co2 embolism in this case . the risk to the patient may be minimized by the surgical team 's awareness of co2 embolism and continuous intra - operative monitoring of end - tidal co2 .

Case Report Discussion

PMC4738945

many methods are used to investigate the different components of human movement in healthy and pathological conditions including motion capture ( 3 , 4 , 17 , 18 ) and electromyography ( 1 , 2 , 8 , 1214 ) . the control of human movement has been a research focus for many years and investigators have examined many aspects of the central and peripheral nervous systems as they pertain to vertebrate ( 6 , 7 , 15 ) and human movement ( 2 , 6 , 7 , 11 , 12 ) . a popular tool for research and clinical assessment of peripheral nervous however , emg has inherent weaknesses including cross talk and amplitude cancellation ( 9 ) . questions have been raised as to the validity of surface and fine wire emg ( 10 ) . furthermore , the limitations of emg have led to questions pertaining to muscular function during human movement ( 9 , 10 , 16 ) based on the aqueous nature of muscle . in addition to the limitations of the emg signals , many different methods exist to process and present emg data . a few of these methods include the peak root mean squared ( prms ) , mean root mean squared ( mrms ) and integrated emg ( iemg ) . a limiting factor in the interpretation of different emg studies is the difference between the methods used to determine the amplitude of muscle activation . the prms uses a single value , the maximum of the rms smoothed signal , to represent muscle activation ; however , it is not robust against movement artifact and error inherent within the emg signal . the mrms is a robust measure that limits the effects of movement artifact , however is also less sensitive to changes in the emg signal and may mask differences in muscle activation intensity between experimental conditions . the iemg is more robust than prms to movement artifact , but is sensitive to temporal changes in onset and offset of muscle activation . due to the temporal component of the iemg signal , it may not provide an accurate measure of the amplitude of muscle activity in different groups or experimental conditions . though each method properly used will provide useful information pertaining to the neural control of the movement in question , each has inherent limitations . the use of a single method of determining emg amplitude provides internal validity , however it remains unclear as to whether these methods produce the same value for muscle activation intensity . therefore the purpose of this study is to compare three methods of determining the amplitude of muscle activation using surface emg . the null hypothesis was that the three different methods would not yield statistically different emg amplitude values . the alternate hypothesis was that each of the three methods of determining emg amplitude values would be statistically different . twelve subjects ( 6 male ; 6 females ) between the ages of 18 years and 25 years ( age : 22.91.4 yrs , height : 1.690.25 m , mass : 77.918.0 kg ) participated in the current study . subjects were healthy and free of lower extremity injury for the previous six months and had no history of major lower extremity injury or neurological disorder . all participants signed an informed consent statement approved by the university of texas of the permian basin institutional review board . gait velocity was maintained within 10% ( 5% ) of the self - selected velocity determined during three practice trials during data collections . surface electromyography ( 2000hz , bts engineering , bolgona , italy ) was collected from the medial head of the gastrocnemius ( mg ) , tibialis anterior ( ta ) and fibularis longus ( fl ) . mg surface electrodes were placed parallel to the muscle fibers over the belly of the medial head of the gastrocnemius ( 5 ) . surface electrodes used to measure ta muscle activity were placed over the largest area of muscle mass parallel to and just lateral to the longitudinal axis of the anterior aspect of the tibia ( 5 ) . fl muscle activation was assessed using surface electrodes placed over the largest muscle mass of the fibularis longus muscle in parallel with muscle fibers approximately one - fourth of the distance between the head of the fibula and the lateral malleolus ( 5 ) . the skin beneath each electrode placement site was shaved , abraded and cleansed to minimize skin resistance . emg signals from each trial were rectified and smoothed using the root mean squared with a 20ms smoothing window . the magnitude of emg activation was determined using three methods : peak rms ( prms ) , mean rms ( mrms ) and integrated emg ( iemg ) . peak rms was calculated as the maximum value of the rms signal during the stance phase of gait . conversely , mean rms was calculated as the mean of the rms signal during the stance phase of gait . for the integrated emg analysis , emg signals were integrated across the stance phase of gait . all emg values were normalized to the highest emg value within that measurement type for each subject ( i.e. prms / max prms ) . subject means used in statistical analyses were calculated as the mean of the seven trials performed by each subject using the three candidate methods . a 33 ( muscle measurement ) repeated measures analysis of variance ( anova ) with a tukey s post - hoc was used to assess statistical differences between the three methodologies ( spss 16.0 , spss inc . , twelve subjects ( 6 male ; 6 females ) between the ages of 18 years and 25 years ( age : 22.91.4 yrs , height : 1.690.25 m , mass : 77.918.0 kg ) participated in the current study . subjects were healthy and free of lower extremity injury for the previous six months and had no history of major lower extremity injury or neurological disorder . all participants signed an informed consent statement approved by the university of texas of the permian basin institutional review board . gait velocity was maintained within 10% ( 5% ) of the self - selected velocity determined during three practice trials during data collections . surface electromyography ( 2000hz , bts engineering , bolgona , italy ) was collected from the medial head of the gastrocnemius ( mg ) , tibialis anterior ( ta ) and fibularis longus ( fl ) . mg surface electrodes were placed parallel to the muscle fibers over the belly of the medial head of the gastrocnemius ( 5 ) . surface electrodes used to measure ta muscle activity were placed over the largest area of muscle mass parallel to and just lateral to the longitudinal axis of the anterior aspect of the tibia ( 5 ) . fl muscle activation was assessed using surface electrodes placed over the largest muscle mass of the fibularis longus muscle in parallel with muscle fibers approximately one - fourth of the distance between the head of the fibula and the lateral malleolus ( 5 ) . the skin beneath each electrode placement site was shaved , abraded and cleansed to minimize skin resistance . emg signals from each trial were rectified and smoothed using the root mean squared with a 20ms smoothing window . the magnitude of emg activation was determined using three methods : peak rms ( prms ) , mean rms ( mrms ) and integrated emg ( iemg ) . peak rms was calculated as the maximum value of the rms signal during the stance phase of gait . conversely , mean rms was calculated as the mean of the rms signal during the stance phase of gait . for the integrated emg analysis , emg signals were integrated across the stance phase of gait . all emg values were normalized to the highest emg value within that measurement type for each subject ( i.e. prms / max prms ) . subject means used in statistical analyses were calculated as the mean of the seven trials performed by each subject using the three candidate methods . a 33 ( muscle measurement ) repeated measures analysis of variance ( anova ) with a tukey s post - hoc was used to assess statistical differences between the three methodologies ( spss 16.0 , spss inc . , the peak rms measurement produced significantly lower normalized activation intensities than mean rms ( p = 0.001 ) and integrated emg ( p = 0.001 ; table 1 , figure 1 ) measurements . mean rms and integrated emg values were similar in each of the three tested muscles ( p = 0.258 ; table 1 ) . there were no significant muscle ( p = 0.974 ) or muscle by method interactions ( p = 0.535 ) . in biomechanics and exercise science , many methods are used to measure the amplitude of muscle activation ; however differences in methodology can often lead to difficulties in interpreting the findings of research studies . the purpose of the current study was to compare three methods of determining emg amplitude in movement studies to determine their inter - relationships . the research question pertains to the similarity of muscle activation amplitude using each of the three methods . the findings of the current study demonstrate that the measurement method used to determine emg amplitude may affect the research results . the mean rms and integrated emg values were significantly higher than the peak rms values . these differences in normalized emg amplitude , though statistically significant , were not substantially large and would not inhibit interpretation of the research results . given that these three methods were used on the same emg signal , it is interesting to note that the peak rms produced lower activation intensities . the limitation of using peak rms to determine emg signal amplitude is that it does not provide a robust measure regarding movement artifact and signal noise . using the root mean square to smooth and process the emg data limits the effect of outliers and noise ; however , the normalization factor is also measured using peak rms and may lead to erroneous normalized emg values . peak rms may not be the best measure of emg amplitude in the presence of noisy emg data and a more robust measure may be preferred . both mean rms and integrated emg values are more robust measures than peak rms regarding instantaneous noise and movement artifact . however , due to the temporal component of integrated emg care must be taken in normalizing iemg data . in the present study , iemg data were analyzed over the stance phase , which was temporally maintained within 10% ( 5% ) . if the experimental movement or population does not allow for consistency in the temporal component of the movement in question , integrated emg may have limitations in the interpretation of the data . furthermore , it is pertinent to note that if a repeated measures design is used to assess an intervention , the temporal component of the movement must be maintained for the accurate use of integrated emg . the mean rms value is the most robust measure of emg amplitude to movement artifact , signal noise and temporal changes in the movement . in the current study , however , in a repeated measures design which may alter the temporal component of the movement the mean rms would not be affected by the changes in the time component . the amplitude of the emg signal measured by iemg , however , would be affected by the altered time component . moreover , it is less affected by the presence of movement artifact or signal noise than peak rms . these characteristics of the mrms method suggest it is the best method tested for the determination of emg signal amplitude . a limitation of the current study is that data were only collected in a single movement condition and the differences in methodology could not be tested in multiple movement conditions which would alter the temporal component of the data . additionally , the current data were collected in level walking and a more dynamic activity such as running , cutting or landing may have led to a lower quality of data including movement artifact . the addition of a more dynamic movement condition would have tested these three methods of determining emg amplitude more thoroughly . the participants in the current study were healthy , young adults with no movement limitations . a patient population or an elderly population may have movement limitations leading to more erratic data which also would have improved the applicability of the current study . the findings of the study demonstrate that the measure used to determine emg amplitude is an important decision in study design . an important consideration in choosing the appropriate measure of emg amplitude is the robustness of each measure and its inherent resistance to error . in the presence of artifact - free surface emg data , each of these measures is sensitive to repeated measures and would provide internally valid statistical analyses . a further extension of these data would be to provide empirical evidence confirming the internal validity of these data in a repeated measures design .

electromyography is a commonly used method to determine relative effort and neuromuscular drive to skeletal muscle . a limitation of the interpretation of emg within the literature is the many methods used to determine the intensity of muscle activation . in the current study , ten healthy young adults performed a level walking task while emg was recorded from the tibialis anterior , medial gastrocnemius and fibularis longus . the emg data were rectified and smoothed using the root mean squared ( rms ) . peak rms ( prms ) , mean rms ( mrms ) and integrated emg ( iemg ) were normalized to the peak value within the subject and were used to determine emg amplitude . a 33 repeated measures analysis of variance was used to determine significant differences between the methods of determining emg amplitude . the findings of the current study show that prms produced significantly lower emg amplitudes than mrms or iemg values . furthermore , mrms and iemg produced nearly identical normalized emg amplitudes . based on the findings of this study and the components of each measurement of emg amplitude , it is suggested to use mrms to determine emg amplitude .

INTRODUCTION METHOD Participants Protocol Data Processing and Reduction Statistical Analysis RESULTS DISCUSSION

PMC5312675

the term confabulation typically refers to the production of false memories in the context of neurological pathology . however , it has also been used more broadly to describe the production of unintentionally false statements about one 's self or the world , beyond the domain of memory ( deluca , 2000 , feinberg and roane , 1997 , hirstein , 2005 ) . in this broader sense , a clear conceptual distinction between confabulation , delusion and anosognosia ( unawareness of illness ) becomes harder . while maintaining these separate terms therefore has conceptual advantages ( kopelman , 1999 ) , considering confabulation in parallel to other similar symptoms allows for cross - fertilisation between studies on such phenomena ( see fotopoulou , 2010 , hirstein , 2005 , langdon and turner , 2010 ) . in the present article , disturbed sensation of limb ownership ( dso ; baier & karnath , 2008 ) , including some patients further present with a positive ( in the jacksonian sense ; 1932 ) variant termed somatoparaphrenia , whereby disownership is accompanied by delusional beliefs , such as personification of the affected limb and/or attributing it to someone else ( gerstmann , 1942 ; see also feinberg and venneri , 2014 , vallar and ronchi , 2009 , for reviews ) . furthermore , dso can co - occur with an apparent inability to acknowledge or recognise one 's contralesional paralysis , so - called anosognosia for hemiplegia ( ahp ; babinski , 1914 ; see fotopoulou , 2014 , fotopoulou , 2015 for reviews ) . whether anosognosia and body ownership disturbances are caused by common underlying neural and psychological deficits or whether they represent independent disorders , remains debated ( e.g. , compare baier & karnath , 2008 and gandola et al . , 2012 ) . importantly , these disorders offer a unique window of insight into the neurophysiological mechanisms by which the body is consciously experienced ( body awareness ) . these mechanisms have received significant scientific interest in recent decades , including the development of several psychophysical and virtual reality paradigms that can generate subjective , somatic illusions in healthy volunteers ( see kilteni , maselli , kording , & slater , 2015 for a review ) . for instance , pioneering work on illusory ownership of a fake hand , i.e. , the rubber - hand illusion ( rhi ; botvinik & cohen , 1998 ) , has emphasised the contribution of multisensory integration , i.e. , the integration of sensory signals from different modalities , to the sense of body ownership ( holmes and spence , 2005 , stein and standford , 2008 ) . vision has been one of the most studied modalities in this paradigm ( kilteni et al . , 2015 ) , however , these effects tend to be considered in the context of visuo - tactile integration . only a few rhi studies have measured subjective feelings of hand ownership following mere visual exposure to a fake hand , i.e. , independently of tactile manipulations . some of these studies reported no effects ( e.g. , longo , schr , kammers , tsakiris , & haggard , 2008 ; see also rohde , di luca , & ernst , 2011 for anecdotal evidence ) , while others found that mere vision conditions can change feelings of ownership for the fake hand ( pavani et al . visual conditions , rather than on negative findings from the comparison of synchronous versus asynchronous tactile stimulation ; a comparison whose interpretation seems more complex than initially thought ( e.g. , rohde et al . , 2011 ) . moreover , while it is well - established that under certain circumstances , conflicting visual feedback from fake , or virtual , or visually misplaced hands via mirrors and wedge prisms ( see holmes & spence , 2006 for review ) , can override proprioception ( the so - called visual capture of proprioception ) , recent studies have established that position sense recalibrations can be dissociated from the sense of body ownership during the rhi ( abdulkarim and ehrsson , 2016 , rohde et al . , 2011 ; see also makin , holmes , & ehrsson , 2008 for an early review ) . thus , the relation between visual capture of proprioception and subjective ownership feelings remains unclear during mere vision conditions . we will heuristically call this possibility , visual capture of ownership ( hereafter reffered to as voc ; visual ownership capture for brevity ) . to this end , the present study will focus on voc by a fake hand ( independently of any synchronous or asynchronous tactile stimulation ) . to the best of our knowledge , the neural mechanisms of voc remain unexplored in healthy participants , as existing functional neuroimaging studies have not included mere vision conditions ( see makin et al . moreover , such functional neuroimaging studies can only establish correlations , while studies in brain damaged patients can be informative regarding the causal role of some brain areas and their connections . unfortunately , the relation between experimentally - induced conditions of voc and neuropsychological dso has not been systematically explored . moreover , comparisons between the few existing studies are hindered by the vast differences in the conceptualisation and measurement of the observed phenomena . indeed , in a series of studies , berti and her colleagues , have proposed that some right hemisphere patients show what they describe as a monothematic delusion of body ownership , which relies on observing another person 's hand in one 's contralesional ( affected ) side and in egocentric , body - congruent perspective ( garbarini et al . , 2013 , garbarini et al . , 2014 ) . however , the phenomenon may be more general and complex than these studies suggest . in a previous study involving eight hemiplegic patients with right hemisphere lesions , including one patients with dso ( fotopoulou et al . , 2008 ) , all patients immediately accepted as their own a stationary rubber hand placed congruently with their own left hand . thus , voc may be a pervasive phenomenon following right hemisphere damage and it may also be dissociable from dso ( see also zeller et al . this possibility is supported by the only case study in the literature that applied the classic rhi paradigm to the affected ( left ) hand of a patient with dso following damage to the right - hemisphere ( van stralen , van zandvoort , kappelle , & dijkerman , 2013 ) . even though a mere vision condition was not tested , the patient experienced feelings of ownership over the affected hand , in conditions of both synchronous and asynchronous tactile stimulation . importantly , this possibility raises a paradox , which to our knowledge has not been addressed in the literature : if patients with dso are willing to accept as theirs a realistic hand that is visually presented in a congruent position as their own hand , why do they deny the ownership of their own hand when viewed under similar conditions ? such results could only be explained if some other bottom - up deficits ( e.g. , hypoesthesia ) , or damage to top - down mechanisms that would normally allow the integration of such signals with pre - existing models of the body ( see tsakiris , 2010 ) , prevented the patient 's own hand from being recognised as one 's own . although we could not quantify and examine all these possibilities in the present study , our main aim was to systematically assess for the first time whether patients with right - hemisphere damage and dso show voc by a fake hand ( without any synchronous or asynchronous tactile stimulation ) . to this end , we recruited 31 patients with recent right - hemisphere strokes , including seven patients with dso . furthermore , using lesion mapping procedures ( i.e. , the voxel - based lesion - symptom mapping ( vlsm ) approach ; bates et al . , 2003 ) , we examined the lesions associated with ( 1 ) voc versus failure of voc and ( 2 ) proprioceptive deficits in our sample , as a way to characterise the relation between such deficits , own hand ownership and rubber hand ownership . , we expected that ( 1 ) visual capture would elicit ownership of the rubber hand in the majority of our patients ( irrespective of diagnostic group ) and particularly those with greater proprioceptive deficits . as far as neural mechanisms are concerned , we expected : ( a ) proprioceptive deficits to be associated with lesions in primary somatosensory areas in the parietal cortex ; ( b ) voc to be associated with the involvement of further , multi - modal areas in the posterior parietal cortex and mostly in the ventral premotor cortex and the posterior insular cortex , as these brain regions have been linked to ownership feelings of fake hands in previous functional imaging studies on the rhi ( ehrsson et al . , 2004 , tsakiris et al . , 2007 ) ; ( c ) by contrast , failures of voc should be associated with sparing of such cortical areas ( see also zeller et al . , 2011 who only found lesions in the white matter connections of this area ) , in the sense that these patients would show either less proprioceptive deficits , and/or greater monitoring of proprioceptive errors during multisensory integration conditions . finally , ( d ) dso was expected to be selectively associated , with additional cortical lesions to the parietal cortex , such as the temporoparietal junction ( tpj ) that can been linked with filtering signals from the body in relation to pre - existing body models ( tsakiris , 2010 ) . thirty - one patients with right hemisphere lesions due to an ischaemic or haemorrhagic stroke were consecutively recruited from three acute stroke units in london , uk , using the following criteria : ( 1 ) unilateral right - hemisphere lesion as confirmed by clinical neuroimaging ( ct or mri ) , ( 2 ) contralesional hemiplegia , ( 3 ) < 4 months from symptom onset , ( 4 ) no previous history of neurological or psychiatric illness , ( 5 ) > 7 years of education , ( 6 ) no medication with severe cognitive or mood side - effects , ( 7 ) no severe language impairment that precluded completion of the study assessments ; ( 8) willingness and availability to participate in the study . dso were evaluated using a modified version of the cutting questionnaire ( cutting , 1978 ) . the assessment began by distracting the patient , placing their left arm in their right visual field and asking what is this ? , is this your hand ? and does it feel like it belongs to you ? . if the patient denied ownership of the arm , they were then asked , does it feel like it belongs to anyone else ? and anyone in particular ? . the questionnaire was scored on a 3-point scale ( 2 = asomatognosia and/or somatoparaphrenia ; 1 = partial acknowledgment of body ownership ; 0 = full acknowledgment ) . patients with a score of 2 on the modified cutting questionnaire were categorised as dso . patients were examined for ahp via a method previously described ( besharati et al . , 2014 , 2008 ) , based on the berti interview ( berti , ladavas , & della corte , 1996 ) and the feinberg scale ( feinberg , roane , & ali , 2000 ) . the berti interview , which includes specific questions regarding motor ability ( e.g. , can you move your left arm ? ) and confrontation questions ( e.g. , please touch my hand with your left hand ; have you done it ? ) , is scored on a 3-point scale ( 2 = denial of motor impairment and failure to reach examiner hand ; 1 = denial of motor impairment but admits failure to reach examiner hand ; 0 = full acknowledgment of motor impairment ) . the feinberg scale , used as a secondary measure of awareness , consists of 10 items including general self - report items ( e.g. , do you have any weakness anywhere ? ) and task - related items ( e.g. , please try and move your left arm for me ; did you move it ) . each item was scored on a 3-point scale ( 1 = complete unawareness ; .5 = partially unaware ; 0 = completely aware ) to produce an overall score out of 10 ( 10 = completely unaware ; 0 = completely aware ) . patients scoring 1 or 2 on the berti interview and at least 4 on the feinberg scale were categorised as ahp . dso and ahp assessments were conducted during the general clinical and cognitive assessment ( see below ) and directly before the experimental testing to make certain the clinical phenomena were present during the experimental assessment . using these methods , we identified seven right brain injured patients with ahp and dso ( ahp + dso group ; 4 women ; mean age sd : 67.6 12.5 years , range 4178 ; mean education sd : 11.7 1.9 years , range 914 ; mean lesion test interval sd : 7.4 5.6 days , range 116 ) , nine right brain injured patients with ahp , but without dso ( ahp group ; 6 women ; mean age sd : 70.6 16.5 years , range 3688 ; mean education sd : 11.8 2.2 years , range 915 ; mean lesion test interval sd : 14.7 11.6 days , range 435 ) , and fifteen right brain injured control patients without ahp or dso ( hp group ; 6 women ; mean age sd : 66.9 13.1 years , range 4788 ; mean education sd : 12.6 2.6 years , range 918 ; mean lesion test interval sd : 17.5 14.1 days , range 342 ) . all patients were right handed according to the edinburgh inventory ( oldfield , 1971 ) , except one ambidextrous control patient . all participants provided written informed consent and the study was approved by the local nhs ethics committee . in addition to the dso and ahp scales , each patient underwent a standard neurological and neuropsychological examination . motor deficits of the upper and the lower limbs were assessed using the medical research council scale ( mrc ; guarantors of brain , 1986 ) . proprioception was assessed with eyes closed by applying a small vertical movement to three joints ( middle finger , wrist and elbow ) , at three time intervals , according to a method previously described ( vocat , staub , stroppini , & vuilleumier , 2010 ) , scored on a 10-point scale ( 0 = severe proprioceptive deficit ; 9 = no deficit ) . the customary confrontation technique ( bisiach , vallar , perani , papagno , & berti , 1986 ) was administered to test visual fields ( upper and lower quadrants ) and tactile extinction ( upper and lower limbs ) . patients were also assessed using the following standardised tests : ( a ) the mini mental state examination ( mmse ; folstein , folstein , & mchugh , 1975 ) and the montreal cognitive assessment ( moca ; nasreddine et al . , 2005 ) as a measure of global intellectual ability , as well as the wechsler test of adult reading ( wechsler , 2001 ) as a measure of premorbid intelligence ; ( b ) the 5-item test from the moca ( nasreddine et al . , 2005 ) to assess long - term verbal recall and the verbal digit - span task from the wechsler adult intelligence scale iii ( wechsler , 1998 ) to assess verbal working memory ; ( c ) five subtests of the behavioural inattention test ( bit ; wilson , cockburn , & halligan , 1987 ; star cancellation , line bisection , line crossing , copy , and representational drawing of the clock ) as a measure of visuospatial neglect , as well as the one - item test ( bisiach et al . , 1986 ) and the comb / razor test ( mcintosh , brodie , beschin , & roberston , 2000 ) to assess personal neglect ; ( d ) a clinical assessment of left / right disorientation was additional conducted ; ( e ) the frontal assessment battery ( fab ; dubois , slachevsky , litvan , & pillon , 2000 ) and the cognitive estimates test ( shallice & evans , 1978 ) to assess executive and reasoning abilities ; and ( f ) the hospital anxiety and depression scale ( hads ; zigmond & snaith , 1983 ) to assess anxiety and depression . the main experiment assessed whether visual capture of a visible , motionless rubber hand influenced body ownership ( voc ) in three groups of patients : ahp versus ahp + dso versus hp . there were two rubber hand ownership questions : is this [ pointing to the rubber hand ] your left hand ? ( yes / no response ) and to what extent do you feel this is your left hand ? ( scale out of 10 ; not at all = 0 , completely = 10 ) . the yes / no ownership answers were analysed qualitatively only , while the ownership answers based on the scale were used as our primary dependent variable voc and analysed quantitatively in both behavioural and lesion comparisons . subsequently , to test the strength of the effect , patients were instructed to try to move their left hand and we provided congruent visual feedback of rubber hand movement by moving it according to the instruction ( as in fotopoulou et al . , 2008 ) . the same two rubber hand ownership questions were repeated and an ownership change score was calculated as the difference between the ownership scores before and after the movement of the rubber hand by the experimenter . a life - sized rubber model of a left hand was used to create visual capture . a suitable rubber hand was selected for each patient in order to resemble their own real hand in terms of size , shape , and skin tone . the procedure of the experiment was similar to the one used by fotopoulou et al . patients were tested on the stroke ward while sitting upright in their bed or a comfortable chair . at the beginning of the experiment , the main experimenter distracted the patient 's attention with general questions ( e.g. , about past history ) , and an assisting experimenter sat to the left and slightly behind the patient , holding the proximal end of the rubber hand covered by a pillow . while the patient was distracted , the assisting experimenter placed the rubber hand on a second pillow in front of the patient close to their midline , such that the distal end of the rubber hand could be seen protruding from in between the two pillows , in a natural ( canonical ) position and orientation ( fig . 1 ) . the patient 's own left hand was positioned out of sight beneath the pillow and the rubber hand , also at the patient 's midline ( i.e. , at the same orientation as the rubber hand ) . once the rubber hand was in position , the main experimenter instructed the patient to look at the hand ( i.e. , the rubber hand ) in front of them for 15 sec and then tested for ownership of the rubber hand as described above ( i.e. , visual capture effect ) . any spontaneous comments or relevant behaviours were noted by the examiner . in the movement condition , the main experimenter asked the patient to slightly raise his / her left hand immediately following a tap on a table in front of them , and the assisting experimenter lifted the rubber hand accordingly . immediately after the visual feedback of movement , patients had to answer the rubber hand ownership questions described above , as well as a question serving as a manipulation check , namely a movement detection question ( did the arm move ? yes / no response ) . owning to paralysis ( see inclusion criteria ) , there was of course no movement of the patient 's actual left hand in any conditions . after the experiment , patients were debriefed to explain the aim of the experiment and any questions were addressed . wilk tests indicated that the data were not normally distributed in the majority of cases ( p < .05 ) . therefore , we performed group analyses on the epidemiologic data , standardised neuropsychological tests , the experimental measures of voc , and a correlational analysis based on our prediction regarding the relation between voc and proprioceptive deficits , using non - parametric tests and corrections for multiple comparisons , as appropriate ( detailed below ) . the location and extent of brain lesions was mapped in each patient , based on a ct - scan ( or a 1.5 t mri - scan for 5 patients ) obtained within the first week of admission . the scans of three hemiplegic patients ( in the hp group ) were unavailable and these patients were therefore excluded from further imaging analyses . the native structural scan of each patient was not normalised , but reoriented and aligned to match the stereotaxic space of the t1-weighted mri scan template from the mni ( montreal neurological institute ) , provided within the mricron software ( http://www.mccauslandcenter.sc.edu/mricro/mricron/ ; rorden & brett , 2000 ) . the quality of normalisation when working with a sample such as ours of elderly stroke patients presents a range of problems ( e.g. , larger ventricles ) and can greatly disrupt the normalisation process leading to inaccurate spatial normalisation ( brett et al . , 2001 , although techniques have been introduced to help improve the accuracy of automated spatial normalisation of individuals with brain injury ( see brett et al . , 2001 ) , the quality and accuracy of the normalisation is still compromised , with the lesion location often shifting during the normalisation process . therefore , manual lesion demarcation using a normalised brain template is often considered as the preferred method , as it more accurately identifies the lesion location , especially in cases of older patients with brain injury ( rorden & karnath , 2004 ) . thus , all the lesions were drawn onto the mni template , whilst using all available scans to guide the delineation , by a researcher ( sb ) who was blind to the patient grouping and hypothesis of the study . percentage lesion overlay maps for the three groups , ahp , ahp + dso , and hp , were computed and lesion volume was obtained . the vlsm approach ( bates et al . , 2003 ) identifies voxels significantly associated with a cognitive deficit in a group of patients , and involves running a t - test for continuous data , comparing patients ' scores on a neuropsychological test in those patients with versus without a lesion at every voxel . this technique as implemented in the software package npm ( non - parametric mapping ; http://www.mccauslandcenter.sc.edu/mricro/npm/ ; rorden , karnath , & bonilha , 2007 ) was used to identify anatomical regions associated with ( 1 ) the baseline sense of body ownership using a binomial classification ( 0 = dso , 1 = normal ) based on the cutting questionnaire ( cutting , 1978 ) n = 28 ) ; ( 2 ) proprioceptive deficits , using the scale out of 9 , in the three groups ( n = 28 ) ; ( 3 ) visual capture of ownership , using the 11-point scale , in the three groups ( n = 28 ) ; ( 4 ) failures in the visual capture of ownership using the reversed 11-point scale in the three groups ( n = 28 ) . the analyses were restricted to the voxels in which at least two patients had lesions ( 8% and 13% of the sample , respectively ) . owing to the relatively small sample size , results were calculated with the permutated non - parametric brnnel menzel test to correct for multiple comparison and small sample size ( rorden et al . , 2007 ) . permutation testing is appropriate here because it preserves power , relative to bonferroni correction ( rorden et al . , 2007 ) . table 1 shows the demographic characteristics ( age , education , gender and delay between stroke and assessment ) and the neuropsychological performances of the three groups . as expected , ahp + dso showed more dso than both other groups , namely ahp and hp ( z = 3.3 and 3.7 respectively , p 's < .001 ) , as tested by the modified cutting questionnaire . whitney u tests revealed that ahp ( z = 3.9 and 4 respectively , p 's < .001 ) and ahp + dso ( z = 3.6 and 3.7 respectively , p 's < .001 ) were significantly worse than hp on the berti and feinberg scales . wallis tests revealed that the delay , age and educational differences between the three groups ( ahp , ahp + dso and hp ) were not significant ( p 's > .1 ) . all three groups showed a similar profile of neuropsychological impairments in global intellectual ability , memory , executive functions and mood , with no significant differences between groups ( p 's > .05 ; see table 1 ) . however , kruskal wallis tests revealed significant differences between the three groups in personal neglect [ (2 ) = 7.4 , p = .02 ] , visuospatial neglect ( see table 1 ) , and a trend effect in the clock drawing task [ (2 ) = 6.1 , p = .05 ] . bonferroni - corrected post - hoc tests indicated that ahp and ahp + dso patients showed significantly greater visuospatial ( but not personal ) neglect compared with hp ( see table 1 ) . performance of the ahp and ahp + dso patients was equivalent ( p 's > .017 ) . the groups also differed significantly in proprioception [ (2 ) = 12.7 , p = .002 ] , but not in tactile extinction on the left side , especially on the left upper limb ( p 's > .1 ) . there was no difference between the ahp ( mean = 4.3 2.5 ) and ahp + dso ( mean = 4.1 1.7 ) groups ( p = .84 ) , but both were more impaired than the hp group ( mean = 7.4 1 ; z = 2.3 , p = .017 and z = 3.2 , p = .0004 , respectively ) . the majority of patients ( 64.5% ) experienced an immediate feeling that the motionless rubber hand was their own and responded yes to the initial rubber hand ownership question , even in the absence of any tactile stimulation or movement . specifically , 6/7 patients in the ahp + dso group ( 86% ) , 6/9 patients in the ahp group ( 67% ) , and 8/15 patients in the hp group ( 53% ) experienced ownership of the rubber hand from visual capture alone . although these percentages were higher in the two groups with delusions compared with the control group , a kruskal wallis test showed that there were no significant differences between the three groups in voc using the 11-point scale measure described above [ (2 ) = 1.5 , p = .47 ] . additionally , no significant differences between the three groups were found on movement ownership change scores [ (2 ) = 1.1 , p = .6 ] , as expected from the fact that only two out of 31 patients changed their responses , with 6/7 patients in the ahp + dso group ( 86% ) , 7/9 patients in the ahp group ( 77.8% ) , and 7/15 patients in the hp group ( 46.7% ) experienced ownership of the rubber hand following the movement condition . moreover , all ahp + dso patients , all but one of the ahp patients and the majority ( 11/15 , 73.3% ) of the hp patients noticed the rubber hand movement during this condition . using the spearman 's rank correlation coefficient , we found a significant negative correlation between visual capture of ownership and the proprioception scores in the three groups together ( r = .47 , p = .02 ) . this correlation remains significant in the ahp + dso group alone ( r = .85 , p = .03 ) , but not for the other two groups ( p 's > .05 ) , indicating that greater proprioceptive impairment was associated with greater rubber hand ownership , particularly in the group with body ownership delusions . all lesions resulted from a first - ever unilateral stroke within the right middle cerebral artery territory . group - level lesion overlay maps for the ahp group ( n = 9 ) , ahp + dso group ( n = 7 ) and hp group ( n = 12 ) are illustrated in fig . lesion volume ( i.e. , total number of voxels ) was not significantly different between the group who experienced ownership of the rubber hand ( voc ) ( n = 17 , mean = 66068 59666 ) and the group who did not ( n = 11 , mean = 40224 49637 , p = .06 ) . vlsm analysis , looking at brain areas associated with voc ( fig . < .05 ) involving the right superior temporal gyrus ( stg ) in the anterior part , the frontal operculum ( fop ) , the inferior frontal gyrus ( ifg ) and the supramarginal gyrus ( smg ) . by contrast , vlsm analysis , looking at brain areas associated with greater voc failure ( fig . 4 ) , identified significant voxels ( p < .05 ) involving the posterior part of the insula ( pins ) , a small area of the putamen ( put ) , and the posterior limb of the internal capsule . furthermore , vlsm analysis , looking at brain areas associated with greater proprioceptive deficit ( fig . 3c ) , identified significant voxels ( p < .05 ) involving the right superior temporal gyrus ( stg ) in the anterior part , the pallidum ( pal ) , the insula ( ins ) , and the somatosensory cortex , primary ( si ) and secondary ( sii ) . finally , lesion volume ( i.e. , total number of voxels ) was not significantly different between the ahp + dso group ( n = 7 , mean = 85087 60320 ) and the group without dso ( ahp and hp groups ; n = 21 , mean = 46191 53050 , p = .09 ) . vlsm analysis , looking at brain areas associated with worse performance on the dso assessment ( fig . 3a ) , identified significant voxels ( p < .05 ) involving the right superior temporal gyrus ( stg ) , the tpj , the middle frontal gyrus ( mfg ) and the supramarginal gyrus ( smg ) . the majority of our patients with damage to the right hemisphere ( 64.5% overall and up to 86% of the patients with dso experienced strong feelings of ownership over a motionless rubber hand just seconds after seeing it and without any tactile stimulation voc , in accordance with previous reports ( fotopoulou et al . , 2008 ) . moreover , voc was maintained even when the rubber hand was moved in space by the experimenter . in the few studies on healthy controls that have tested similar mere vision conditions , the percentage of people experiencing voc tends to be much lower ( guimmarra et al . , 2010 , longo et al . , 2008 ; see also rohde et al . , 2011 for anecdotal evidence ) . indeed , in previous published ( crucianelli , metcalf , fotopoulou , & jenkinson , 2013 ) and unpublished data ( crucianelli et al . , in preparation ) from our group , we found that voc occurred in 13/70 ( 18% ) of healthy individuals . however , voc tends to increase in virtual reality paradigms that can achieve spatial coincidence between visual and proprioceptive cues rather than mere spatial congruency and plausibility ( maselli & slater , 2013 ; see also kilteni et al . , 2015 for review ) . our data are consistent with these findings , in that we found a positive association between proprioceptive deficits and voc across our patient groups and particularly in the dso group . thus , taken together , these results suggest that when information from proprioception is not available due to damage , or it is not informative ( e.g. , there is no mismatch in the spatial location of the seen and felt body parts ) , visual cues from a realistic body part can be sufficient to generate not only recalibration of hand position ( holmes & spence , 2006 for review ) , but also feelings of fake hand ownership . in our patients , this pathologically exaggerated voc effect occurred when proprioception was impaired by lesions to areas including the primary and secondary somatosensory cortex ( see fig . however , it also appears that such damage is not sufficient for the pathological voc effect , as lesions to cortical areas further up the neurocognitive hierarchy were selectively associated with the voc effect . specifically , voc scores were primarily associated with lesions to the frontal operculum and the inferior frontal gyrus , and to a lesser degree parietal areas ( see fig . interestingly , in functional imaging studies on the rhi , the onset of subjective feelings of ownership for the fake hand and its vividness correlated with activation of similar areas in the frontal operculum and the premotor cortex ( ehrsson et al . , 2004 , thus , when damage to the central , parietal representation of proprioceptive signals is also accompanied by damage to such frontal areas , there is a pathological dominance of vision over proprioception and body ownership feelings of the rubber hand . by contrast , failures of voc were associated mostly with subcortical lesions ( see also zeller et al . , 2011 ) and with the posterior part of the insula . we had expected this area to be instead associated by greater pathological voc in our patients as in the case of frontal operculum , given the association of both of these areas with feelings of ownership during the classic conditions of the rhi ( ehrsson et al . the fact that neurological patients can present with both deficits in body ownership ( failures of voc ) and pathological exaggerations of such feelings ( voc ) , however , suggests a potential separation between the role of these two areas in body ownership . the frontal operculum and the premotor cortex may contribute to feelings of body ownership by monitoring mismatches in multisensory integration in a forward way , i.e. , in a way analogous to how efferent motor signals ( berti et al . , 2005 , frith et al . , 2000 ) or , proprioceptive predictions ( fotopoulou , 2015 ) dominate motor awareness in ahp due to the selective involvement of these areas ( see berti et al . , 2005 , fotopoulou et al . , 2010 ) . by contrast , the posterior insular cortex may instead be related to more fundamental aspects of the sense of ownership , integrating and monitoring different aspects of somatosensation , including interoceptive modalities ( critchley , wiens , rotshtein , ohman , & dolan , 2004 ) . however , further specification of this role would be speculative based on the current data , particularly given the small number of patients in this analysis and the intrinsic limitations of lesion analyses as regards this area ( kodumuri et al . although patients with dso had more proprioceptive deficits than control patients , and showed more voc than the control group , the latter difference was not statistically significant . interestingly , as mentioned in the introduction , it remains paradoxical why patients with dso would deny the ownership of their own arm when they see it , while they are willing to accept as theirs a realistic , congruently placed , fake hand . indeed , our lesion analyses reveals a dissociation between pathological voc and dso , with the latter being selectively associated with more posterior lesions , including the right temporoparietal junction and a large area of the supramarginal gyrus , and to a lesser degree the middle frontal gyrus ( see fig . lesions of the right middle frontal gyrus have previously been correlated with somatoparaphrenia ( feinberg and venneri , 2014 , gandola et al . interestingly , regions close to the medial frontal cortex have also been involved in several self - related functions , such as the sense of an integrated self and the differentiation between the self and the external world ( feinberg , 2013 ) . in our study , the frontal network differs between the sense of body ownership and the rubber hand ownership , involving more the middle frontal gyrus for the former ( fig . the contribution of the right temporoparietal junction ( including the supramarginal gyrus ) in body ownership has been recognised as an early test - of - fit mechanism ( tsakiris , 2010 ) , comparing expectations about the state of the body , with current sensory events . one possibility is therefore that information about the affected arm from a modality other than vision ( as it is their own hand they are visually not recognising ) is generating error signals that can not be predicted by existing top - down , expectations of selfhood ( for the wider theoretical context of this hypothesis see fotopoulou , 2015 ) . we have not been able to test the various candidate modalities in this study , and hence we merely put forward some possible hypotheses for further examination . although the role of defective proprioception is unlikely to be sufficient to explain dso ( as many patients with proprioceptive deficits do not show dso ) , and in the present study many patients with proprioceptive deficits where likely to feel ownership for a rubber hand placed congruently with their own , it remains possible that patients with dso are unable to generate appropriate proprioceptive predictions about their own arm and these affect their ownership . for example , they may experience their own arm to be in a different position than the one tested and hence they may deny its ownership when they are asked to look at this familiar arm ( rather than a rubber hand ) in a different position . more generally , a recent rhi study in paralysed patients has argued that any alteration of the normal flow of signals present during movements may affect feelings of body ownership ( burin et al . , 2015 ) . in addition , although the rubber hand was placed in a position congruent and similar to the patients own arms , it remains possible , particularly given the frequent deficits in personal neglect in dso patients , that patients deny an arm they see in personal space ( i.e. , their own arm ) more often than an arm they see in proximity but in peripersonal space ( i.e. , the rubber hand ) . finally , although we did not find any evidence for this hypothesis based on limited bedside assessments of tactile extinction in the current study , a candidate modality would be somatosensation , including exteroceptive ( tactile perception impairment or hypoesthesia ) and interoceptive variants ( e.g. , pain ) . despite their dso , it is thus possible that damage to the rtpj , does not allow these new sensations to be integrated in patients ' body representation . this hypothesis would need to be tested in future studies , but it is compatible with anecdotal evidence in the field , as captured by this quote from the existing literature ; i know they [ left arm and leg ] look like mine , but i can feel they are not , and i ca n't believe my eyes ( c. w. olson , 1937 , the current study has shown that a fake hand can capture ownership in patients with right - hemisphere damage . however , future studies should manipulate the visuo - semantic and visuo - spatial properties of the presented hand in order to establish the constraints of this phenomenon ( see tsakiris , 2010 , kilteni et al . , 2015 for discussions ) . measurements and manipulations of exteroceptive and interoceptive domains of contralateral somatosensation can further add specificity to our neuroanatomical findings . moreover , although we did not find significant differences in voc between our three groups , larger samples are necessary to exclude a relationship between voc and somatic delusions . moreover , such future studies with larger samples could investigate such factors in groups fully balanced for neuropsychological performance , or with statistical tests allowing for co - variation of various neuropsychological functions that was not possible in the current study . for instance , our lesions analysis included activations in the anterior region of the right superior temporal gyrus that have been elicited in visual - orienting and alertness tasks ( sturm & willmes , 2001 ) , demonstrating a general attention function rather than a specific role in terms of self - processing ( apps & tsakiris , 2014 ) . actually , the same region of the right superior temporal gyrus was found in our three lesion analyses ( fig . , future studies should include a left - hemisphere damage patient group allowing for greater interpretation regarding laterality . lastly , it is important to recognise that interpretation of the neuroanatomical correlates are limited by our relatively small sample size and inherit limitations to our lesion mapping approach ( geva et al . , 2012 , our interpretations do not take into account the structural and functional connectivity between areas and related functional networks . contrary to some functional neuroimaging methods , current voxel - based lesion analyses methods also do not allow for correlations between function and neural activity at the whole brain level . instead , they focus on the most frequently lesioned voxels in a sample of patients with behavioural deficits . this has the advantage of pointing to certain areas that may have a necessary functional role in a network , but it does not tell us much more about that network and its connections . in addition , lesion analyses methods are limited by the fact that certain areas may simply be more frequently damaged following certain types of stroke ( tkodumuri et al . , 2016 ) . nevertheless , all previous lesion mapping studies in such disorders are subject to comparable limitations , with our study being one of the few that has directly compared experimental scores with lesion data . future studies will have to use better structural lesion data and functional mri paradigms to be able to more accurately identify brain areas and importantly also white matter connections related to voc and somatic delusions of ownership . overall , the present data highlight three important findings . first , the majority of patients with right perisylvian fissure lesions experience feelings of ownership over a rubber hand , without any tactile stimulation . second , this visual capture of ownership is associated with proprioceptive deficits and lesions mostly in the frontal operculum and the premotor motor cortex . third , this mechanism seems behaviourally and neurally dissociated from the feelings of disownership experienced by somatoparaphrenic patients . it seems that in the latter patients , feelings of disownership dominate vision in the case of their own arm , while vision dominates their feeling of ownership in the case of others arms . this work was funded by an european research council ( erc ) starting investigator award for the project the bodily self n313755 to a.f . , a neuropsychoanalysis foundation fellowship to p.m.j . , and a commonwealth scholarship , an oppenheimer memorial trust fellowship , and a neuropsychology international fellowship award from the british psychological society in conjunction with the british neuropsychological society to s.b .

the various neurocognitive processes contributing to the sense of body ownership have been investigated extensively in healthy participants , but studies in neurological patients can shed unique light into such phenomena . here , we aimed to investigate whether visual capture by a fake hand ( without any synchronous or asynchronous tactile stimulation ) affects body ownership in a group of hemiplegic patients with or without disturbed sensation of limb ownership ( dso ) following damage to the right hemisphere . we recruited 31 consecutive patients , including seven patients with dso . the majority of our patients ( 64.5% overall and up to 86% of the patients with dso ) experienced strong feelings of ownership over a rubber hand within 15 sec following mere visual exposure , which correlated with the degree of proprioceptive deficits across groups and in the dso group . using voxel - based lesion - symptom mapping analysis , we were able to identify lesions associated with this pathological visual capture effect in a selective fronto - parietal network , including significant voxels ( p < .05 ) in the frontal operculum and the inferior frontal gyrus . by contrast , lesions associated with dso involved more posterior lesions , including the right temporoparietal junction and a large area of the supramarginal gyrus , and to a lesser degree the middle frontal gyrus . thus , this study suggests that our sense of ownership includes dissociable mechanisms of multisensory integration .

Introduction Materials and methods Results Discussion Conclusion Funding

PMC4737663

rhabdomyolysis is a clinical syndrome characterized by muscle necrosis and the release of intracellular muscle constituents into the circulation . it can be caused by burns , shock , acidosis , infections , crush trauma , immobility , malignancy , toxins , drugs , and muscle diseases1 ) . among the various types of rhabdomyolysis , non - traumatic exertional rhabdomyolysis ( exrml ) occurs in individuals with normal muscles and appears when the energy supplied to the muscle is insufficient23 ) . exrml related to military training , marathon running , weight training , and other forms of strenuous exercise have been reported4 ) . spinning is a group indoor cycling exercise , which has become popular recently over the past few years . this stationary cycling program is composed of five core movements and riders may synchronize their pedaling to the rhythm of the music while they are exercising5 ) . we observed that recently there has been an increasing number of patients who were diagnosed with exrml at cha bundang medical center after attending a spinning class . here , we introduced the clinical cases of spinning induced rhabdomyolysis and review the related literature . we found 11 cases of exrml patients who were admitted to cha bundang medical center between july 1 , 2014 and march 31 , 2015 . their mean body mass index ( bmi ) was 23.92.6 kg / m ( ranged from 19.6 to 28.0 ) . two to three days prior to the presentation , the patients had attended a spinning class for the first time . nine of the patients had brown - colored urine within two days and another two patients noticed that their urine had become darker three days after their first spinning class . none of the patients had any significant past medical history and were not on any medication . their thighs were moderately firm and warm to the touch , with reduced range of movement and strength . complete blood - count , urea , creatinine , and electrolyte values were within normal ranges at the initial blood analysis . however , their serum creatinine phosphokinase ( cpk ) level was over 11,000 u / l ( normal range : 38 - 160 ) . additionally , other serum levels of muscle enzymes ( creatine kinase - mb type , lactate dehydrogenase , alanine aminotransferase , aspartate aminotransferase , and myoglobin ) were significantly elevated ( table 1 ) . serum bun and creatinine levels stayed within the normal range and no acute kidney injury ( aki ) had developed during treatment . dipstick testing of the urine was positive ( 3 + ) for blood ; however , subsequent microscopy revealed less than four red blood cells under high power field ( hpf ) microscopy . a bone scan study showed diffuse type of soft tissue uptakes in the damaged muscles including rectus femoris , sartorius , vastus medialis , vastus lateralis , and pectineus ( fig . a diagnosis of rhabdomyolysis was made and the patients were treated with a combination of bed rest and aggressive intravenous fluid ( normal saline ) to maintain a urine output of 200 to 300ml / hour . when the urine ph was less than 6.0 , sodium bicarbonate ( mixed with 5% dextrose water ) was used for urine akalinization . although eight of the patients had mild hypocalcemia , calcium supplementation was unnecessary because no hypocalcemic symptoms were observed ( table 1 ) . in a span of 6 - 12 days , one patient ( number 1 ) was hospitalized for an additional 4 days after her cpk level was markedly reduced ( 4,820 u / l ) on the 8 day along with her complaints of thigh myalgia . exrml has been defined by a combination of findings , including exercise - associated muscle pain , swelling , dark urine , and increased cpk level to at least five times the upper limit of normal , and myoglobinuria6 ) . it should be noted that our patients had cpk levels of nearly 70 times the upper limit of normal ( table 1 ) . also , a urine dipstick that is positive for blood but shows few red blood cells is suggestive of myoglobinuria7 ) . in the pathogenesis of exercise - induced rhabdomyolysis , calcium(ca ) has been suggested as an important key factor3 ) . during exercise , a decreased amount of adenosine triphosphate ( atp ) could cause dysfunction of na - k atpase and an increased level of na in the cells , resulting in the reverse activation of na - ca exchanger mode3 ) . consequently , the increased intracellular calcium leads to the activation of proteases , skeletal muscle cell contraction , mitochondrial dysfunction , reactive oxygen species production , and eventual skeletal muscle cell death23 ) . treatment of rhabdomyolysis involves stopping the continued breakdown of the muscle fibers and preventing the development of aki . the rapid and aggressive infusion of intravenous crystalloid is used to obtain a urinary output of 200 - 300ml / hour . myoglobin is toxic in acidic urine , and most experts advocate the use of intravenous sodium bicarbonate to maintain a urinary ph of 6.5 or greater67 ) . loop diuretics may be given to patients who develop volume overload complications such as pulmonary edema . the initiation of renal replacement therapy in clinical practice depends on the status of renal impairment , with complications such as life - threatening hyperkalemia , anuria , metabolic acidosis , or hyperhydration without response to diuretic therapy89 ) . it is reported that both continuous venovenous hemofiltration with a high permeability membrane and high cutoff membrane hemodiafiltration are effective in removing serum myoglobin ( 17kda)1011 ) . although successful treatment with plasmapheresis to remove serum myoglobin in statin - induced rhabdomyolysis have been reported , its use seems still controversial9 ) . most of the related articles mentioned that the majority of spinning - induced rhabdomyolysis patients were discharged without any complication when they were treated early and aggressively with intravenous fluids7 ) . however , in rare cases , some patients demonstrated severe complications , such as compartment syndrome and aki4 ) . aki in rhabdomyolysis may occur due to renal vasoconstriction , formation of intratubular casts , and direct myoglobin toxicity caused by accumulation of myoglobin in the renal tubules19 ) . compartment syndrome may occur in the leg or forearm due to the edema caused by muscle injury and tissue damage4 ) . cardiac dysrhythmias via electrolyte abnormalities and disseminated intravascular coagulopathy are other possible complications of exrml4 ) . known risk factors of exrml are the exercise experience , intensities or types of exercises37 ) . other possible causes were hyperthermal environments , electrolyte imbalance , malnutrition , creatine supplements and alcohol drinking3 ) . influenza virus infection and genetic deficiency of metabolic factors can be risk factors as well3 ) . it has been reported that men are vulnerable to exrml because estrogen plays a protective role1213 ) . interestingly , all 11 spinning - induced rhabdomyolysis cases in this article occurred in females . it is unclear whether they were malnourished by their diet plan or had little exercise experience and thus were at higher risk of spinning induced rhabdomyolysis . third , exercise should be modified or abstained in the presence of evitable risk factors such as influenza . all 11 patients were treated early and properly without any complication , except a mild non - symptomatic hypocalcemia . however , considering the possibility of severe complications of exrml as aforementioned , and the medical expenses of hospital stay , information and guidelines for preventing spinning - induced rhabdomyolysis have to be provided prior to starting the exercise .

non - traumatic exertional rhabdomyolysis ( exrml ) occurs in individuals with normal muscles when the energy supplied to the muscle is insufficient . here , we report 11 cases of spinning - induced rhabdomyolysis and review related literature . spinning is a kind of indoor bicycle sport . the 11 patients who were diagnosed with exrml and admitted to cha bundang medical center were female and their ages ranged from 15 to 46 years . two to three days prior to the presentation , the patients had attended a spinning class for the first time . all the patients had been otherwise healthy without any known medical illnesses . they were successfully treated without any complications , except mild non - symptomatic hypocalcemia . however , in the literature , severe complications such as compartment syndrome or acute kidney injury had been reported in relation to exrml including spinning - induced rhabdomyolysis . this spinning exercise needs prior guidelines and specific warnings to prevent exertional rhabdomyolysis .

Introduction Case Report Discussion

PMC4238775

target population - the target population consisted of 120 tb patients living in the mam who were ensured by the mexican institute of social security ( imss 2014 ) . the imss provides health care for 83.5% of the patients in nuevo len . during a 16-month period , all patients who visited the units of familiar medicine of the mam with symptoms suggestive of ptb [ 268 ( 100% of the suspicious cases in the mam in the care of the imss ) ] were included in this study . a total of 134 patients had mtb - positive sputum samples and 120 of these patients agreed to participate in this study . the study group was composed of 64% men and 36% women between the ages of 13 - 84 years ( median = 49 years ) . the range of ages was 18 - 68 years and the time of residence in the mam ranged from less than one year to 68 years ( mean standard deviation = 34.06 16.03 years ) . the participants presented clear symptoms of ptb ( who 2014 ) and acid - fast bacilli ( afb ) in their sputum samples . ptb patients attended their respective family medicine clinics or their zone hospitals during the study . in these facilities , afb were detected and reported by a chemist working in the clinical laboratory of each medical facility . collection of data regarding potential tb risk factors - the collection of epidemiological data and the isolation , mtb typing and genotyping were performed in the northeast biomedical research centre ( cibin ) of the imss . the patients with a clinical diagnosis of ptb signed an informed consent form and answered a questionnaire to provide appropriate information regarding the factors that could favour tb transmission ( such as crowded places frequented by the ptb patients ) . familial contacts and human immunodeficiency virus / acquired immune deficiency syndrome status were sought and correlated with the clusters ; additionally , groups of clusters were analysed , but because the data were negative , these results are not presented in this article . social networks and risk factors - the elements of social networks analysed in this work were family medical clinics , zonal general hospitals , municipalities and familial contacts . the risk factors examined were comorbidities , patterns of resistance or sensitivity to first line anti - tubercular drugs , occupation and drug consumption ( alcoholism , tobacco use and psychotropic substance abuse ) . control mtb strain - the mtb h37rv strain ( american type culture collection , catalogue 27294 ) was included as an internal genotyping control . mtb isolates - each participant donated up to three sputum samples from which mycobacteria were isolated and typified . immediately after collection , the samples were transported to our laboratory in the cibin , decontaminated and the bacteria were concentrated by centrifugation . a smear from every sputum sample was prepared and stained using the ziehl - neelsen method and afb were microscopically examined ( ellis & zabrowarny 1993 ) . each of these samples was inoculated onto lowenstein - jensen ( lj ) slants ( becton dickinson , usa ) and incubated in a 5% co2 atmosphere at 37c for two - five weeks . slants with typical mtb colonies were typified ( kent & kubica 1985 , balandrano - campos et al . 1996 ) and stored at -70c until use . recovery of frozen isolates - the frozen mtb isolates were thawed at -20c overnight and then at 4c for 3 - 4 h. mycobacteria were inoculated on lj slants and incubated at 37c until colonies were clearly visible . drug sensitivity tests for first - line anti - mtb drugs - the tests were performed with a bactec 460 radiometric system ( siddiqi 1996 ) . the assayed drugs were isoniazid ( inh ) ( 0.1 g / ml ) , streptomycin ( 2.0 g / ml ) , rifampicin ( rif ) ( 2.0 g / ml ) , ethambutol ( 2.5 g / ml ) and pyrazinamide ( 100 mg / ml ) . extraction of mtb dna - under aseptic conditions , two loops of mycobacteria colonies were transferred to a glass test tube containing 2.5 ml of tris - edta buffer [ 10 mm tris / hcl ph 8) ( amresco , usa ) and 1.0 mm edta ( promega , usa ) ] and five - six glass beads . the suspension was homogenised using a vortex and a 0.5-ml aliquot was transferred to a 1.5-ml capped conical polypropylene tube ( eppendorf , germany ) and inactivated in an 80c water bath ( terlab m40 , mexico ) for 50 min . dna was extracted from mycobacteria according to the ctab ( hexadecyltrimethyl - ammonium bromide ) method ( van soolingen et al . 2001 ) . ( 1997 ) using a commercial kit ( isogen life science , the netherlands ) . ( 1993 ) and miru - vntr analyses were performed according to cowan et al . , the following 15 loci were analysed : miru 4 , 10 , 16 , 26 , 31 and 40 ( cowan et al . 2002 ) as well as mtub 04 , etr c , etr a , qub 11b , mtub21 , qub26 , mtub30 , mtub39 and qub4156 ( supply et al . ( 2013 ) , we defined clusters as those ptb cases with indistinguishable mtb genotypes . the spoligotypes were converted to their corresponding octal codes as described by dale et al . ( 2001 ) and clusters and unique patterns were identified using spss software , v.10.0 . the rflp - is6110 data were analysed with bionumerics software , v.2.0 ( applied maths , belgium ) using the unweighted pair group method with an arithmetical mean and dice s coefficient . the running differences in the gels were normalised using the relative position of the /hind iii and x174/hae iii molecular weight markers ( sigma - aldrich ) using bionumerics software . to determine the best tolerance , when the alignments of all of the reference rflp - is6110 patterns from all the agarose gels matched , the tolerance value was set at 1.9% . the genotypes were considered identical according to the results from the analyses performed by the bionumerics program . because rflp - is6110 codes have not been previously described , we designed a code using the relative migration of each is6110 band ( rf ) of every mtb isolate dna sample ( fig . 1 ) . the description of the rflp - is6110 codes is as follows : the rflp - is6110 codes have two arms of numbers , separated by a dot . the left arm is composed of one or two digits and includes information regarding the number of bands found in each mtb isolate . the right arm contains the rf of each dna band with an is-6110 element . each rf is represented by three numbers . from left to right , the rf values were placed in descending order of dna - molecular weight . the rf values were calculated by dividing the distance between the origin and front of the electrophoresis by the distance travelled by each is6110-band and multiplying by 1 10 . the miru - vntr patterns were analysed with microsoft excel 2002 software ( microsoft co , usa ) . the cluster groups with comparable codes were named as follows to differentiate the clusters from the genotyping methods : spoligotypes , miru - vntr and rflp - is6110 , smr , spoligotypes and rflp - is6110 , sr , spoligotypes and miru - vntr , sm , miru - vntr and rflp - is6110 , mr , spoligotypes , s , rflp - is6110 , r , and miru - vntr , m. the methods are referred to by their corresponding shortened names ( spoligotyping , miru - vntr and rflp - is6110 ) . these were generated by the bionumerics software and show the relationships of clusters and groups of clusters using the rflp - is6110 method exclusively . venn diagram - the venn diagram describes the metrics by which the 120 mtb isolates were organised using spoligotyping , miru - vntr and rflp - is6110 . the diagram was constructed using powerpoint software for windows 7 ( microsoft co ) with the data depicted in table i. statistics - the significance ( p = 0.05 ) for correlation between the aforementioned cluster - groups and the ptb characteristics was calculated by spearman s . statistical correlation referred to the relationships that involved the dependence of code - set belonging to each mtb cluster group having comparable genetic codes and each ptb patient characteristic . ethics - this protocol represented a minor risk for tb patients and was authorised by the scientific and ethical committee of the cibin / imss ( protocol r-2005 - 1908 - 2 ) . 1 shows the rflp - is6110 patterns and dendrogram of the 120 ptb clinical isolates . 2 ) describes the metrics by which the 120 mtb isolates were organised using spoligotyping , miru - vntr and rflp - is6110 . while 70.83% ( 85 ) of the isolates were clustered , 29.17% ( 35 ) of the isolates ( unique or orphan cases ) were not clustered by any genotyping method . the clusters constituted the following groups with respect to the 120 mtb isolates : smr , 5% ( 6 isolates ) , sm , 12.50% ( 15 isolates ) , sr , 1.60% ( 2 isolates ) , m , 5% ( 6 isolates ) and s , 46.67% ( 56 isolates ) . supplementary data , table i shows each case identified by the number assigned by our laboratory and its corresponding s , m or r code . supplementary data , table ii retains the identical organisation of the mtb cases as clusters and groups of clusters , as in supplementary data , table i. supplementary data , table ii depicts the factors that could favour ptb transmission in our target population . five groups were formed with comparable codes for spoligotyping , miru - vntr and rflp - is6110 ( smr ) , a combination of two codes ( sr and sm ) or only one ( s and m ) . group six was formed by orphan cases . additionally , supplementary data , table ii shows that the variables with the highest frequencies were first ( family medicine clinic 35 ) and second level medical facilities ( zonal general hospitals 4 and 17 ) , municipalities ( guadalupe and monterrey ) , comorbidities ( diabetes mellitus type 2 ) , occupation ( car painting , mining and industrial production ) , multidrug resistance ( to at least rif and inh ) and drug consumption ( tobacco use and alcoholism ) . schematic representation by which the 120 mycobacterium tuberculosis - isolates included in this study were organised by spoligotyping , mycobacterial interspersed repetitive units - variable number tandem repeats ( miru - vntr ) and the conventional restriction fragment length polymorphism ( rflp)-is6110 analysis . association of cluster groups with situations favouring tb transmission - tables i , ii show the association of cluster groups with crowded places frequented by ptb patients and risk factors . smr presented a significant correlation with family medicine clinics , municipalities , comorbidities and drug consumption . sr and factors favouring mtb transmission were not calculated because the number of cases was insufficient . no relationship was found between a cluster group and members of the same family or neighbours . we showed that a combination of rflp - is6110 , miru - vntr and spoligotyping is essential to identify mtb clusters , to group the clusters and to discard orphan cases . using these data , we investigated how social networks and risk factors correlated with the cluster groups ( glynn et al . smr was the only group that correlated with two of the three elements of social networks and with two of the four risk factors considered in this study . our data strongly suggest that when tb surveys are performed in a metropolis , such as the mam , the more markers that are considered in performing a correlation analysis with risk factors , the more reliable the connection with a ptb epidemic focus would be . the factors that correlated with smr substantiated a recent mtb transmission . regarding social networks ( family medicine clinics , primary clinics and municipalities ) , clinic waiting rooms are closed environments with recirculated air , in which bacillipherous patients are seated by individuals without ptb . during standard treatment , patients repeat this type of visit several times . no correlation was observed between smr and zonal general hospitals , possibly because tb patients are treated in family medicine clinics by an epidemiologist until the case of tb is cured or the patient is referred to a pulmonologist at a specialty hospital . thus , the risk of ptb transmission in second level facilities is markedly reduced . ptb transmission throughout social networks has been previously reported ; garca - garca et al . ( 2000 ) found an rflp - is6110 cluster that developed after ptb micro - epidemics occurred in a social network formed by assiduous clients attending clandestine bars . in addition to first level medical facilities , workers and housewives typically frequent the same crowded public places , such as schools , markets , stores , cinemas , churches or bars and are transported by urban buses . these factors are recognised as risk factors ( davies 2005 ) and we identified the correlations of diabetes and drug abuse with smr . the application of a combination of spoligotyping , miru - vntr and rflp - is6110 and an analysis of spearman s correlation of these genotypes with factors that could facilitate mtb transmission is required to discover new ptb micro - epidemics that are possible in a metropolitan population . considering the increase in multidrug resistant and extended drug resistant mtb strains that threaten tb control worldwide ( who 2014 ) , the recent development of strains resistant to all first and second line anti - tb drugs , which have been identified as completely mtb resistant ( totally drug - resistant - mtb ) ( rowland 2012 ) , could be useful for applying analyses such as this one to describe future tb surveys or potential epidemic foci of ptb . table iassociation of cluster - groups and social networksgroup level of medical attention clinics of familiar medicinezonal general hospitalsmunicipality spearman s correlationsmr 0.033 0.1660.013srnc ncncsm0.6770.2560.380s0.0780.1050.316m0.7790.5590.261a : groups of clusters having comparable codes;b : not computable ( nc ) because these groups were constituted just by one cluster having two cases . capitals correspond to groups of clusters showing comparable codes of spoligotyping ( s ) , mycobacterial interspersed repetitive units - variable number tandem repeats ( m ) or restriction fragment length polymorphism - is6110 ( r ) . groups of clusters having comparable codes ; : not computable ( nc ) because these groups were constituted just by one cluster having two cases . capitals correspond to groups of clusters showing comparable codes of spoligotyping ( s ) , mycobacterial interspersed repetitive units - variable number tandem repeats table iiassociation of cluster - groups with risk factorsgroupcomorbiditypattern of drug resistanceoccupationdrug consumption spearman s correlationsmr 0.047 0.6431.000 0.012 srnc ncncncsm0.3810.2780.2340.531s0.1960.4000.0670.271 m 0.047 0.4921.0000.391a : not computable ( nc ) because these groups were constituted just by one cluster having two cases . capitals correspond to groups of clusters showing comparable codes of spoligotyping ( s ) , mycobacterial interspersed repetitive units - variable number tandem repeats not computable ( nc ) because these groups were constituted just by one cluster having two cases . capitals correspond to groups of clusters showing comparable codes of spoligotyping ( s ) , mycobacterial interspersed repetitive units - variable number tandem repeats ( m ) or restriction fragment length polymorphism - is6110 ( r ) .

the characteristics of tuberculosis ( tb ) patients related to a chain of recent tb transmissions were investigated . mycobacterium tuberculosis ( mtb ) isolates ( 120 ) were genotyped using the restriction fragment length polymorphism - is6110 ( r ) , spacer oligotyping ( s ) and mycobacterial interspersed repetitive units - variable number of tandem repeats ( m ) methods . the mtb isolates were clustered and the clusters were grouped according to the similarities of their genotypes . spearman s rank correlation coefficients between the groups of mtb isolates with similar genotypes and those patient characteristics indicating a risk for a pulmonary tb ( ptb ) chain transmission were ana- lysed . the isolates showing similar genotypes were distributed as follows : smr ( 5% ) , sm ( 12.5% ) , sr ( 1.67% ) , mr ( 0% ) , s ( 46.67% ) , m ( 5% ) and r ( 0% ) . the remaining 35 cases were orphans . smr exhibited a significant correlation ( p < 0.05 ) with visits to clinics , municipalities and comorbidities ( primarily diabetes mellitus ) . s correlated with drug consumption and m with comorbidities . smr is needed to identify a social network in metropolitan areas for ptb transmission and s and m are able to detect risk factors as secondary components of a transmission chain of tb .

PATIENTS, MATERIALS AND METHODS RESULTS DISCUSSION

PMC4668142

kinesio taping ( kt ) is an alternative taping technique developed in the 1970s by dr . , kt may improve strength , control , and performance ; reduce pain ; prevent injury ; inhibit and facilitate motor activity ; and increase range of motion ( rom)1,2,3 . kt has lately gained popularity among sports professionals for injury prevention and performance enhancement4 . however , the mechanism through which kt exerts its hypothesized effects is unclear , and studies have not confirmed on the effectiveness of the methodology . kt has been shown to support muscle movement and relieve abnormal feeling or pain in the skin . mori et al.5 suggested kt normalizes muscle function , and kase et al.2 showed that the stretch rate and width of the kt affects the fascia and flow of lymph fluid . kt also reduces cervical pain and increases rom 24 hours after whiplash injury6 , and according to slupik et al.7 , it has a preliminary effect on quadriceps peak torque and electromyographic activity in healthy subjects . chang et al . found no significant difference in maximal wrist flexor strength among 10 baseball players who received kt , a placebo , or no tape8 . only a study by lee et al . has reported a positive acute effect of kt on isometric muscle strength9 . although kt is widely used in clinical practice , several studies show no any change in muscle strength with kt application in healthy individuals10,11,12 . a few research studies have evaluated the biomechanical effectiveness of kt during walking or running . howe et al . showed that kt does not change hip or knee kinematics or kinetics in asymptomatic runners compared with the no tape condition13 . indicate that kt does not alter the muscle peak torque generation or total work done in healthy subjects14 . the current investigation compared the effects of kt facilitation and inhibition applications on spatiotemporal knee kinematics and kinetics during walking . all subjects were completely inactive . those who reported active knee pain ; lower limb trauma ; a history of spinal , hip , knee , or foot pathology ; or any neurological impairment were excluded . average age , body weight , and height were 19.7 0.4 years old , 51.5 4.8 kg , and 1.64 3.4 cm , respectively . all participants provided informed consent , and the study was approved by the orthopedic prosthetic and orthotic department review board commission of the university of ankara . the kinesio tape was a hypoallergenic , nonmedicated , waterproof , porous , adhesive tape made of cotton that could be stretched longitudinally up to 3040% of its original length . the subject s skin was shaved , cleaned with alcohol , and dried before the tape was applied . 1.facilitation application ) : y - shaped kt was applied the quadriceps of each subject , according to kenso kase s kinesio taping manual , by the same physical therapist . the two extremities of the subject s knees were taped while the subject lay in the supine position with the hip flexed 30 degrees and the knee flexed 60 degrees . the tape was applied with no tension approximately 5 cm below the origin of the rectus femoris and with moderate tension ( 2550% of available tension ) along the two tails . the tails were placed on the skin without tension when they were approximately 25 cm from the end . 2.inhibitation application ) : kt was applied from muscle insertion to origin using light tension ( 1525% of available tension ) . the tape was a y - shaped strip , with the tail that surrounded the quadriceps muscle held in the stretching position . inhibitation application a three - dimensional quantitative gait evaluation was performed in the motion analysis laboratory at the ankara university department of prosthetics and orthotics using a vicon nexus system ( oxford metrics , oxford , uk ) by the same experienced physiotherapist ( sg ) using six infrared jai cameras at 250 hz and one force plate ( amti ) . the standard plug - in gait marker set was used to capture kinematic data : a total of 16 reflective markers were placed on the anterior superior iliac spine , mid - lateral thigh , lateral knee joint , lateral shank , lateral malleolus , and second metatarsal head , over the posterior calcaneus bilaterally , and on the posterior superior iliac spine . subjects were asked to walk barefoot at a comfortable , self - selected walking speed without tape and then application of facilitation and inhibition kt , respectively . at least five good trials in which the subject s footfalls landed completely on the force plates were captured . the nonparametric wilcoxon test was used to compare baseline measurements and measurements for the two taping applications , respectively , within each subject . spatiotemporal parameters were significantly different among the trials ( table 1table 1.spatiotemporal parametersno tapen=16facilitation ktn=16inhibition ktn=16cadence ( steps / min)110 7.4113 7.4**115 8.5*stride length ( m)1.29 0.071.30 0.06 * 1.27 0.07*walking speed ( m / s)1.20 0.81.23 0.5 * 1.21 0.8 * * comparison of the no tape condition , facilitation kt , and inhibition kt by nonparametric wilcoxon testcadence : the number of steps per minute . step length : the distance along the line of progression from opposite foot contact to current foot contact . walking speed : the speed of the current foot based on the stride time and stride length . ) . cadence walking speed and stride length increased with the facilitation kt application compared with the no tape condition ( p<0.05 ) . cadences significantly increased and stride length decreased with the inhibition kt application compared with the no tape condition ( p < 0.05 ) . * * p < 0.001 ; * p<0.05 . comparison of the no tape condition , facilitation kt , and inhibition kt by nonparametric wilcoxon test cadence : the number of steps per minute . step length : the distance along the line of progression from opposite foot contact to current foot contact . walking speed : the speed of the current foot based on the stride time and stride length . kinematic data are displayed in table 2table 2.sagittal plane joint angles ( degrees)kinematic parameterssagittal planeno tapen=16facilitation ktn=16inhibition ktn=16knee flexion- extension rom ( )60.7 3.461.2 3.860.4 3.9max knee flexion ( )57.1 2.957.1 2.656.2 3.2**max knee extension ( )3.3 3.33.8 4.1*3.6 4.0 * * * p < 0.001 ; * p<0.05 . comparison of the no tape condition , facilitation kt , and inhibition kt by nonparametric wilcoxon test . sagittal plane knee joint kinematics were significantly different with both kt applications , respectively , compared with the no tape condition . the degree of maximal knee flexion was significantly lower with the inhibition kt application , and degree of maximum knee extension was significantly increased with both kt applications compared with the no tape condition . knee joint sagittal plane rom was not different with either kt application . * * p < 0.001 ; * p<0.05 . comparison of the no tape condition , facilitation kt , and inhibition kt by nonparametric wilcoxon test kinetic data are displayed in table 3table 3.sagittal plane moments ( nm / kg ) and powers ( w)kinetic parameterssagittal planeno tapen=16facilitation ktn=16inhibition ktn=16km10.21 0.10.15 0.11 * 0.31 0.0*km20.26 0.160.32 0.15*0.07 0.2kp10.24 0.20.31 0.30.27 0.2kp20.13 0.10.10 0.70.03 0.07kp30.15 0.130.19 0.20.19 0.1*kp41.47 0.571.45 0.50.74 1.1 * * * p < 0.001 ; * p<0.05 . comparison of the no tape condition , facilitation kt , and inhibition kt by nonparametric wilcoxon testkm1 : maximum knee flexion moment ; km2 : maximum knee extension moment ; kp1 : eccentric knee extensor activity during the loading response phase ; kp2 : concentric knee extensor activity during the mid - stance phase ; kp3 : eccentric activity in the rectus femoris during the pre - swing phase ; kp4 : eccentric activity in the hamstrings during terminal swing . knee external flexion moment during the early stance phase ( km1 ) decreased significantly with the facilitation kt ( p<0.05 ) and increased with the inhibition kt ( p<0.05 ) . knee external extension moment during mid - stance phase ( km2 ) increased with the facilitation kt ( p<0.05 ) . knee power parameters , including eccentric knee extensor activity during loading response ( i.e. , eccentric quadriceps activity , kp1 ) and concentric knee extensor activity during mid - stance ( i.e. , concentric quadriceps activity , kp2 ) were not significantly different after either kt application . eccentric activity in the rectus femoris during the pre - swing phase ( kp3 ) was significantly increased with the inhibition kt application ( p < 0.05 ) , while eccentric activity of the hamstrings during the terminal swing of gait ( kp4 ) was decreased ( p < 0.05 ) . * * p < 0.001 ; * p<0.05 . comparison of the no tape condition , facilitation kt , and inhibition kt by nonparametric wilcoxon test km1 : maximum knee flexion moment ; km2 : maximum knee extension moment ; kp1 : eccentric knee extensor activity during the loading response phase ; kp2 : concentric knee extensor activity during the mid - stance phase ; kp3 : eccentric activity in the rectus femoris during the pre - swing phase ; kp4 : eccentric activity in the hamstrings during terminal swing the main purpose of the current study was to determine whether kt had short - term effects on knee kinematics and kinetics parameters using two different kt techniques in the knees of healthy individuals . our study showed significant differences in spatiotemporal parameters , knee kinematics , and kinetics gait analysis parameters with facilitation and inhibition kt applications . facilitation kt application enhanced cadence , stride length , walking speed , and the degree of maximal knee extension compared with the no - tape condition . this application also increased knee extensor moment , which suggests increased quadriceps muscle torque generation , and decreased knee flexor moment . an elevation in the quadriceps lever arm in the taped condition may have augmented knee extensor moment without increasing quadriceps muscle force . a meta - analysis by williams et al.4 indicates that seven out of ten studies they examined showed an increase in muscle strength with kt application ; however , three isokinetic studies did not show a significant effect of kt application on quadriceps muscle strength in healthy subjects . according to lumbroso et al.15 , application of kt to the gastrocnemius increased the muscle s peak force immediately and after application of kt for 2 days , and rom increased in all of the lower - extremity joints measured . kt may increase blood circulation within the taped area , which may influence the muscle and myofascial functions that increase rom within the muscle16 . kt application may stretch or increase pressure applied to the skin , and this external load may stimulate cutaneous mechanoreceptors that impact knee flexion / extension rom . another possibility is that fear of movement is related to pain intensity in patients and that application of kt provides sensory feedback that increases rom6 . kt is used to correct biomechanical movements , allowing individuals to improve performance by increasing the power created through their movements . hsu et al . showed that kt significantly improved scapular orientations compared with a placebo treatment in baseball players with shoulder impingement syndrome17 . showed a 54% increase in vastus medialis muscle activity 24 hours after kt application7 . karatas et al . reported that kt may be an effective method for reducing neck and low back pain and improving functional performance18 . in a study of 40 asymptomatic trained amateur cyclists , nelson found that kt application above the rectus femoris significantly increased knee flexion19 . suggested that kt shortens the time to reach peak torque generation during isometric knee extension , which has important implications for sports performances that require the rapid generation of peak muscular force20 . our results demonstrated that facilitation kt application improved gait performance by increasing cadence , walking speed , and knee extension moment . research has shown that application of kt to healthy tennis players decreased fatigue by maintaining forearm extensor strength21 , and this suggests that kt may have also affected proprioception in addition to improving muscular strength and power in present study . however , other studies have shown that kt did not improve proprioceptive response in the ankle , as determined by reproductive measures of joint position sense1 . the proprioceptive effect of kt is unclear , but kt may stimulate the cutaneous mechanoreceptors by applying pressure to , and stretching , the skin ; thus , an elastic tape such as kinesio tape may cause proprioceptive stimulation while improving joint rom and muscle function , further enhancing cadence22 . our results suggest that facilitation kt application may enhance an individual s gait performance during walking or sportive activities due to its assistance of the terminal stance in the gait cycle . the inhibition kt technique increased knee flexor moment , decreased knee power ( i.e. , eccentric activity of the hamstrings ) during the terminal swing phase , and decreased knee flexion during walking . suggest that the inhibition kt application increases motor unit recruitment in asymptomatic individuals several hours after kt is applied , which would justify the lack of biomechanical changes resulting from this method of kt . we propose that the inhibition kt application technique improves minor soft tissue injury recovery and prevents overuse injuries in the joint of interest . inhibition kt application had a positive effect on knee kinetics and kinematics compared with the no - tape condition in our study . inhibition kt application with physical therapy rehabilitation treatment in the acute phase or with orthotic treatment may be valuable , and it may also help control knee hyperextension in paralytic patients with knee - ankle foot orthosis ( kafo ) without the need for orthotic popliteal straps . also , patients with equinus have prevalent knee hyperextension and may benefit from inhibition kt application along with ankle - foot orthosis ( afo ) . first , the study had small sample sizes , which may limit the translatability of our findings to other populations . second , placebo effects may have contributed to the observed differences because we did not apply placebo taping to eliminate the psychological effect of the tape . further research is recommended to examine the association between sports performance with therapeutic tape and the acute and chronic effects of kt on the function , balance , and neuromuscular performance of patients under a rehabilitation program .

[ purpose ] the application of kinesio taping may improve strength and performance , inhibit and facilitate motor activity , and increase range of motion . the aim of this study was to compare the effects of kinesio taping facilitation and inhibition applications on spatiotemporal knee kinematics and kinetics during walking activity in healthy subjects . [ subjects and methods ] a three - dimensional quantitative gait evaluation was performed without tape and with , facilitation and inhibition kinesio taping application on the knee . sixteen healthy female college students ( age , 19.7 0.4 years ; height , 1.64 3.4 cm ; body mass , 51.5 4.8 kg ) participated in the study . [ results ] spatiotemporal parameters ( cadence , walking speed , stride length ) were significantly different among the trials . knee joint sagittal plane range of motion was not different with either kinesio taping application . knee external flexion moment during the early stance phase decreased significantly with facilitation kinesio taping and increased with the inhibition kinesio taping . knee external extension moment during the mid - stance phase increased with facilitation kinesio taping . knee power parameters , eccentric activity in the rectus femoris during the pre - swing phase was significantly increased with inhibition kinesio taping application , while eccentric activity of the hamstrings during the terminal swing of gait was decreased . [ conclusion ] these findings showed that facilitation kinesio taping application affected the terminal stance phase and that inhibition kinesio taping influenced the terminal swing phase compared with the no tape condition .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC4868241

therefore , moderate exercise or training and appropriate maintenance are critical . for those with impaired limbs , it is more crucial to perform proper reasonable rehabilitation training to restore normal daily function . most human physical actions involve using the hands . according to reports from the u.s . national security agency , one - third of occupationally impaired body functions affect the upper limbs1 . in its functional disability standards , the american medical association identifies that losing one arm is equal to losing 60% of the body mechanisms . the loss of a hand equates to the loss of 90% of the arm function or 54% of the entire body mechanism2 ; having healthy upper limbs is a matter not to be ignored . usually , patients with an upper limb dysfunction need to be trained repeatedly using appropriate rehabilitation equipment to recover . clinically some of the most frequently used equipment include exercise skate of the arm , exercise skate of the hand , vertical tower , incline board , stacking cones and cura motion exercises . there are many therapeutic methods such as mechanical arms ( passive or positive patient training through mechanical structures)3,4,5,6,7,8 , video games ( follow the instructions on the screen to move mechanical arms to help neural rehabilitation)9 , and virtual reality ( integrate and improve sound , video , graphics , and text ) to make users feel they are experiencing it for real10,11,12,13,14 . the shoulder joint is frequently used in daily activities , making it prone to sprain and bruising , causing shoulder rotation or abduction disorders . patients decrease the shoulder joint mobility because of the fear of pain , thereby affecting damaged function reconstruction . shown in fig . 1.traditional training activities is a traditional shoulder training activity called the shoulder finger ladder , which strengthens and increases the shoulder angle movement using finger movements . patients face a wall ( front and side ) and move the fingers of the affected side upward along the ladder to their maximum reach . the arm of the affected side is used to move a plastic piece from the left to right or from the right to left to train the initiation movement . traditional training activities researchers are motivated to introduce virtual reality concepts to traditional rehabilitation training as it increases patient enthusiasm and repeatability . the primary goal of this research was to train the upper limbs . to facilitate participants using this training in daily life , researchers applied the microsoft kinect somatosensory devices ( for windows ) for the 3d human motion capture system . this system detects human skeleton coordinates such as the palms , wrists , and both shoulders to develop unity games . participants can be trained through these games and scenes on the screen without actually having to touch a real entity . this study used mission - oriented training in applying kinect somatosensory device software development with unity 3d games to enhance the training effects . this approach is very convenient and safe because participants only need to touch the assigned virtual objects using their upper extremity . argued that the kinect - based 3d reachable workspace analysis provides sufficiently accurate and reliable results compared to motion capture systems , and that proposed methods could be promising for the clinical evaluation of upper extremity in neurological or musculoskeletal conditions15 . training using video games played on the xbox kinect may be an effective intervention for the rehabilitation of stroke patients16 . unity 3d is a low price , powerful , and intuitive game engine applied widely in industry . even though it can be used to develop games it does not support a somatosensory application . kinect is designed to detect human skeleton information which is the key to developing somatosensory games . the signals are captured and transmitted to unity 3d through microsoft sdk ( kinect for windows sdk ) or open ni ( open nature interaction ) to drive the game character actions . figure 2fig . 2.overall scheme of the rehabilitation system , ( a ) computing angle of shoulder joint shows the overall schematic diagram of this study . human skeletons were displayed in action on a pc using windows sdk , the unity 3d software tool , and the kinect s sensing device . since the kinect senses human skeleton data , this 3d coordinate data must be projected to the corresponding unity3d s virtual scenes on the pc screen . the virtual scenes can be used to construct and plan game scenes or express different design collision effects . the hardware interface used microsoft s product and the kinect hardware for windows to connect to the computer . the advantage of the kinect is that its skeleton recognition technology can be used to determine actions while other relevant action information capture technology is captured through the physical installation of many sensing elements and cables . the development interface and application program part is mainly composed of the installation of drivers for the kinect for windows sdk and unity3d software . common programming language c # between these two was used to write a program , the game application was produced using the compilation and function calls of the kinect sdk through unity3d . this program could be used to measure data while the game was running , as well as recorded the 3d coordinates of the skeleton of the participants during the training process . overall scheme of the rehabilitation system , ( a ) computing angle of shoulder joint to capture human skeleton coordinates via the kinect device , the angles could be measured by the bones that connect to the joints , and the angle differences in movements can help medical workers understand the accuracy of poses and action changes for patients in the process of training activities . figure 2 ( a ) shows the collected data for the shoulder , elbow , and wrist joint coordinates , which were s(sx , sy , sz)e(ex , ey , ez)w(wx , wy , wz ) the elbow angle formula was figure 3(a)fig . 3.design of the motion trajectory ( take right hand as an example ) shows the training activities in the frontal plane the shoulder finger ladder design used as training for upper extremity lifting or measurement purposes . the bottom is the reference ( ref ) , the rectangular areas are reminders for the participants to touch a virtual position ( represented by the letters from a the red rectangular area ( figure marked as a ) is used for participants to touch according to the ref ( the very bottom of the figure ) upon completion of the designated touch order ( shown as a step ) . the patient virtually touched the point with their hands . at the same time , the red rectangular area automatically moves on the screen to show the completion of one round as soon as the participants have completed a design of the motion trajectory ( take right hand as an example ) the following contains the explanation for each ladder planned for the shoulder finger ladder . the subject stands in front of the kinect device at the start with their body captured completely . the captured coordinate data for the head , spine , hip center , elbow , wrist joint were h(hx , hy , hz)s(sx , sy , sz)hc(hcx , hcy , hcz)e(ex , ey , ez)w(wx , wy , wz ) . the ref position is defined as : h(hx , hy* , hz* ) , 0.5 < < 0.95 , 0.75 < < 1 the related positions are defined by the ref on the starting position ( pos_min ) of the ladder : s(sx , sy , sz* ) , 0.75 < < 1 the ending position ( pos_min ) of the ladder is : hc(hcx , hcy , hcz)* ) , 0.75 < < 1 adjacent to the ladder is the gap ( if the area is divided into k ) : figure 3(b ) shows the training activities in the frontal plane the planned design for the single curved shoulder . the subject sequentially touched the objects virtually from the left side of the hand toward the right , clockwise and then in the counterclockwise direction to the original starting point . this activity trained the shoulder , elbow , and forearm and was very helpful for hand - eye coordination and reaction cultivation . patients with frozen shoulders were trained with the shoulder finger ladder and the single curved shoulder training activities combined . point of the shoulder finger ladder activity could be the radius of curvature the single curved shoulder activity which provided better protection for the subject in the safety training process . this experiment was performed in two parts : one for those with normal shoulder function and the other for those with shoulder disability . the participants used their hands according to the indicator points on the screen to virtually complete the exercise ; the computer system recorded the coordinates and performed the statistical analysis , which can be referenced by clinic personnel later on . to confirm the reproducibility and stability of the system development , it was very helpful to understand whether this system was stable enough for clinical training activities for assessing and analyzing the test results from participants of different ages and body types or the same participant with various testing times17 , 18 . in the normal shoulder function group , 8 participants conducted 12 rounds of training and testing with the device developed in this study . the collected training data were analyzed to examine the variations before and after the training to determine whether the system could provide consistent evaluations of the usually used hand , and to make sure that the system was stable with high repeatability . in the shoulder disability group , there were 4 participants conducting training for 6 weeks ( twice a week ) , with 3 rounds of practice and 3 minutes of rest between each round . the ethical committee at the taipei medical university hospital approved the study , and written informed consent was obtained from each participant . eight subjects ( age range 2130 , 3140 , 4150 , 5160 ; 2 subjects in each group ) with normal shoulder function were chosen to perform the shoulder finger ladder and the single curved shoulder exercise 12 times ( 3 rounds in each practice ) of the training and testing to check reproducibility and stability . 4.the performance of subject a-1 ( left handed ) in the shoulder finger ladder test shows the single curved shoulder test screen of the subjects . it shows that the subjects raised their right hand to touch the object ( red ) on the oval . figure 4(b ) shows the performance of subject a-1 ( left handed ) in 12 exercises . the left and right - hand average usage time reached stability at approximately up to 33.5sec and 41.2 seconds in the seventh and ninth test , respectively . the total average usage time for the left and right hand was 35.4 ( sd = 2.35 ) and 42.8 ( sd = 1.60 ) seconds , respectively . the results indicate that dominant side movement is more flexible than the non - dominant side . the performance of subject a-1 ( left handed ) in the shoulder finger ladder test table 1table 1.the performance record of 8 subjects with normal shoulder functionyear groupsubject / genderdominant sideaverage using time ( sec)shoulder finger laddersingle curved shoulderleft handright handleft handright hand2130a-1/male / left hand21.60.823.90.835.42.442.82.6a-2/female / right hand23.90.822.40.743.02.335.12.23140b-1/female / left hand23.40.825.10.938.12.243.92.7b-2/male / right hand25.40.923.10.844.62.837.42.34150c-1/male / left hand25.10.828.60.940.22.648.12.6c-2/female / right hand29.10.925.30.950.12.841.42.75160d-1/male / left hand27.01.032.91.645.23.349.42.9d-2/female / right hand32.21.726.91.051.22.943.63.3 shows the performance record of 8 ( 4 age groups ) subjects with normal shoulder function . the results indicated that the average usage time for the dominant side of the subjects in the same age group were close to the shoulder finger ladder and single curved shoulder activity ; indicating good system stability . the average usage time for both hands indicated that the dominant side movement is more flexible than the weak side . moreover , the flexibility and ability to respond in elders were slightly inferior to that of the younger groups . the study included 4 subjects with impaired shoulders who participated in the test ; the basic information of these subjects are shown in table 2table 2.basic information of the 4 subjects with impaired shouldersubjectagegenderdominant side / affected sidesymptomsp-153maleright / rightsports injuryp-248maleright / rightfrozen shoulderp-355femaleleft / leftfrozen shoulderp-467maleright / lefttraffic accidentp-546femaleright / rightvocational injuryp-663maleleft / righttraffic accident . table 3table 3.result of the shoulder finger ladder and single curved shoulder test on the affect sidetypesubjectaverage using time ( sec)wk 12wk 34wk 56shoulder finger ladderp-132.91.928.41.127.50.1p-235.72.032.11.431.30.4p-336.01.932.61.331.60.3p-439.82.436.52.035.01.0p-533.12.331.91.931.00.2p-639.22.836.22.935.10.9single curved shoulderp-166.519.548.97.647.50.1p-268.918.553.812.352.91.3p-369.217.254.010.353.81.0p-478.718.768.69.967.41.3p-567.619.851.911.151.21.1p-677.117.667.19.166.31.2value are expressed as mean sd displays the result ( average using time ) of the 6 subjects ( p1p6 ) during the entire test period of the shoulder finger ladder and the single curved shoulder presented in a bi - week interval . every subject s performance showed positive results in the mid - stage , and stability in the post - stage . value are expressed as mean sd to assess the average performance time of subjects in the 6 week test trial , researchers set the benchmark as the average score of tests given 4 times biweekly . researchers performed pairwise statistical tests ; see table 4table 4.result of the statistical test ( pairwise)typesubjectbetween the weekswk 12 / wk 34wk 34 / wk 56wk 12 / wk 56shoulder finger ladderp-132.9/28.4 * 28.4/27.532.9/27.5*p-235.7/32.1 * 32.1/31.335.7/31.3*p-336.0/32.6 * 32.6/31.636.0/31.6*p-439.8/36.5 * 36.5/35.039.8/35.0*p-533.1/31.9 * 31.9/31.033.1/31.0*p-639.2/36.2 * 36.2/35.139.2/35.1*single curved shoulderp-166.5/48.9 * 48.9/47.566.5/47.5*p-268.9/53.8 * 53.8/52.968.9/52.9*p-369.2/54.0 * 54.0/53.869.2/53.7*p-478.7/68.6 * 68.6/67.478.7/67.4*p-567.6/51.9 * 51.9/51.267.6/51.2*p-677.1/67.1 * 67.1/66.377.1/66.3**paired - t test , p<0.05 for the results . the first and second weeks were adjusted for the stage for all subjects , however , the performance was not ideal . there was a significant improvement in the third - fourth and the fifth - sixth weeks . the t - test results comparing 12 weeks to 34 week and 12 weeks compared to 56 weeks showed a significant difference . the average performance reached stability after the third week , and there was no significant difference in the t - test results at 34 weeks and 56 weeks . the results indicated that this system was effective in the training of each subject . * the shoulder joint has the largest range of motion , the most complicated action form and is the most frequently used joint in physical activities , resulting in a higher injury frequency . this study used the kinect somatosensory device with unity software to develop 3d situational games for upper extremity training activities . using games as training methods helps improve concentration , patient interest in participation , and helps patients temporarily forget about their body discomfort . the equipment used in this study is inexpensive , easy to obtain and the system is easy to install . our group will continue to recruit more cases with impaired upper limb function to conduct related research and develop suitable rehabilitation training games . to have more effective methods , researchers have introduced games and virtual reality training to help participants train their upper limbs in a relaxed environment .

[ purpose ] researchers imported games and virtual reality training to help participants train their shoulders in a relaxed environment . [ subjects and methods ] this study included the use of kinect somatosensory device with unity software to develop 3-dimensional situational games . the data collected from this training process can be uploaded via the internet to a cloud or server for participants to perform self - inspection . the data can be a reference for the medical staff to assess training effectiveness for those with impairments and plan patient rehabilitation courses . [ results ] in the training activities , 8 subjects with normal shoulder function demonstrated that the system has good stability and reproducibility . six subjects with impaired shoulder underwent 6 weeks of training . during the third week of training , average performance stabilized . the t - test comparing 12 weeks to 34 weeks and 56 weeks showed significant differences . [ conclusion ] using games as training methods improved patient concentration , interest in participation and allowed patients to forget about their body discomfort . the equipment utilized in this study is inexpensive , easy to obtain , and the system is easy to install . people can perform simple self - training both at home or in the office .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC4065202

halogen atoms are commonly found in inorganic , organic , and pharmacological molecules . it has been reported that 50% of compounds in high - throughput drug screening contains halogens . halogen bonds , referring to the noncovalent interactions between halogens and electron - rich atoms , are both strong and tunable . when attached to a strong electron - withdrawing group , halogen atoms may display an electron - depleted region on the outermost portion of the molecular surface , which has been referred to as the -hole . halogen atoms and some elements of groups iv vi with a -hole can thus form strong interactions with negatively charged sites or electron - donor - rich groups ( e.g. , lewis bases , -electrons , and anions ) . such strong interactions are often referred to as halogen bonds ( x - bonds ) and offer alternatives to other common classical interactions , such as hydrogen bonds ( h - bonds ) , that play important roles in the supramolecular chemistry of biosystems and nanomaterials . in addition to areas such as crystal engineering and solid - state materials , ligand design is also increasingly taking advantage of the halogen - bonding phenomenon . for example , a number of recent studies reported halogen - based hiv reverse transcriptase inhibitors . in another biochemical application , ho and co - workers utilized bromine - substituted uracil to promote the assembly of four - stranded dna junctions where the halogen atoms facilitated the noncovalent bonding by acting as electrophilic sites . given the importance of halogens and halogen bonds , it is crucial to develop accurate molecular mechanics ( mm ) models to capture their electronic structure and molecular interactions . however , because of their complicated charge distribution and high polarizability , it is difficult to model the halogen atoms using atomic point charges with spherically symmetric potentials . the partial negative charges typically assigned to halogen atoms make their electrostatic interactions with electron donors repulsive instead of attractive . to deal with such anisotropic charge distributions and the -hole effect , jorgensen et al . introduced off - center charged sites to halogen atoms in the opls - aa force field to capture the halogen bonds , which led to improvement in predictions for the density , heat of vaporization , relative hydration , and binding free energy . similar treatments of halogen electrostatics and the -hole effect have been reported previously . beyond the fixed - charge model , a more physically appealing approach to model the complicated anisotropic electronic structure of halogens is to incorporate higher - order multipole moments . among the available methods , the atomic multipole optimized energetics for biomolecular applications ( amoeba ) force field , which features atomic - based multipole moments ( up to quadrupole moments ) and inducible dipole - based polarizability , ought to be a good platform for the development of a halogen model . the amoeba force field has been developed for water , common single - atom ions , common organic molecules , and proteins . in addition , the importance of an explicit representation of electronic polarization in molecular mechanics models has been demonstrated by various groups over the past decade . in this paper , we investigated a polarizable multipole - based molecular mechanics model for a series of chlorine - substituted methanes , including ccl4 ( carbon tetrachloride ) , chcl3 ( chloroform ) , ch2cl2 ( dichloromethane ) , and ch3cl ( chloromethane ) . these simple chlorine - containing compounds allow us to focus on chlorine itself before investigating more complex chlorine - containing drugs . the classical force field was parametrized by using a combination of ab initio quantum mechanics ( qm ) and experimental data . transferability of the force field was tested by computing the chlorocompounds hydration free energies ( hfe ) and solvent reaction fields on a reference solute . detailed analysis of the energetics of homo- and heterodimers using the resulting force field along with ab initio calculations gives further insight into the intermolecular interactions of halogen atoms . overall , the calculated hydration free energies and solvent electric fields agreed with experiment more satisfactorily for the less substituted compounds , and tetrachloromethane s properties were the most difficult to reproduce . the total energy is given by1the terms in 1 include the valence contributions corresponding , respectively , to the bonds , angles , bond - angle cross couplings , out - of - plane , and torsional energies . the long - range electrostatic interactions , including both permanent and polarizable components , are treated with the particle - mesh ewald ( pme ) algorithm . to derive the amoeba polarizable force field parameters , poltype was used to estimate initial atomic multipoles at the mp2/6 - 311 g * * level of theory using stone s original the resulting multipole moments ( with monopoles fixed ) were then further refined by fitting to the electrostatic potential computed at the mp2/6 - 311++g ( 2d , 2p ) level of theory to overcome the potential instability of directly applying dma to bigger basis sets with diffuse functions . the atomic polarizability of cl ( 2.5 ) was determined by matching to the ab initio qm molecular polarizability tensor for ch3cl ( 5.3 , 3.8 , 3.8 ) . the same cl atomic polarizability was applied to mono- to tetrachloromethanes ( table 1 ) . the van der waals ( vdw ) parameters of c and h have been transferred from the ch- in methanol and methylamine . the cl vdw parameters were first determined by fitting to the ab initio potential energy profiles of molecular dimers evaluated at the mp2/aug - cc - pvqz level of theory . the fitting procedure was carried out using the forcebalance program in conjunction with the tinker 6 package . the geometry of each dimer pair was optimized using mp2/aug - cc - pvtz in gas phase . the potential energy profile for each pair was then generated by displacing the molecules along the cc or co axis . single - point energy calculations at the mp2/aug - cc - pvqz level of theory with basis set superposition error ( bsse ) correction were applied to obtain the interaction energy for each generated structure . because the dispersive energy may contribute significantly to the interaction energy of chloride compounds , the quality of mp2/aug - cc - pvqz interaction energy was also examined by comparing with the ccsd(t)/cbs result for a ch3cl - water dimer . the ccsd(t)/cbs interaction energy was estimated following the extrapolation approach of hobza and co - workers . the mp2 correlation energy was extracted to cbs from aug - cc - pvtz and aug - cc - pvqz , and then the correlation energy was adjusted from the mp2 to the ccsd(t ) level by adding the difference between the two methods using the aug - cc - pvdz basis set . the calculated mp2/aug - cc - pvqz and ccsd(t)/cbs dimer interaction energy values are 1.071 and 1.100 kcal / mol , respectively . in addition , for the purpose of calibrating the classical model , the hf and mp2 energies seemed to converge reasonably well at the aug - cc - pvqz basis set by comparing to the cbs results ( see supporting information ( si ) table s1 ) . thus , mp2/aug - cc - pvqz has been used to obtain all the gas - phase dimer energies in this study . ccl4 , chcl3 , ch2cl2 , and ch3cl as pure liquids and as single molecules solvated in water . for the pure liquid the liquid simulations were first equilibrated through a 50 ps trajectory in the npt ensemble at 298 k and 1 bar , followed by another 500 ps production run for data collection . to estimate the heat of vaporization , we used the equation below:2where eliq is the averaged potential energy of single molecule in the liquid box and egas is the energy of the single molecule in the gas phase and is calculated by running a 500 ps simulation for one molecule using a 0.1 fs time step . by computing the liquid density and heat of vaporization for each chloromethane from molecular dynamics simulations , forcebalance was applied to iteratively optimize the vdw parameters for cl . forcebalance allows the fitting of selected force field parameters to qm cluster energies and/or liquid thermodynamic properties using newton the hfe for each chloromethane was computed using the conventional thermodynamic cycle consisting of discharging , van der waals decoupling , and gas - phase recharging steps . the charging / discharging step handles the polarization effect in the amoeba force field through the scaling of solute atomic polarizability . thus , the final hfe can be expressed as:3where adischarging(aq ) and adecoupling(aq ) are the free energy changes due to turning off the electrostatic and vdw between the solute and environment respectively , and arecharging(vac ) corresponds to the intramolecular electrostatic interactions in vacuum . the vdw annihilation used a soft - core buffered 147 potential , as described in our previous publications . the discharging schedules are = 0 , 0.1 , 0.2 , ... 1 ; decoupling schedules are = 0.0 , 0.2 , 0.4 , 0.5 , 0.55 , 0.6 , 0.625 , 0.65 , 0.7 , 0.75 , 0.8 , 0.9 , 0.95 . the bennett acceptance ratio ( bar ) method was used to evaluate the free energy changes . for each of the chloromethanes that exist as a liquid at room temperature ( all but ch3cl ) , we simulated a solution consisting of 395882 chloromethane molecules ( to fill a 45 cubic box ) and 1 acetophenone molecule and calculated the electric field the solvent exerts onto the c = o bond of acetophenone using methods previously described . the solvent box was equilibrated for 100 ps in an npt ensemble , and production dynamics were carried out for 500 further ps , during which the solvent field was calculated every 10 fs . the calculated electric fields can be compared with experimental values evaluated from vibrational frequencies through the linear vibrational stark equation:4 in eq 4 , obs is the experimental c = o vibrational frequency of acetophenone dissolved in one of the three chloromethane solvents , fsolv is the electric field the chloromethane solvent exerts onto the c = o bond , 0 is a reference frequency associated with zero - electric field , and probe is the c = o vibration s stark tuning rate . 0 and probe correspond to the vibration s gas - phase frequency and difference dipole moment ( measured in stark spectroscopy ) and are also calibrated against a set of reference solvents . to examine the electronic structure of cl atoms and the -hole effect , we evaluated the electrostatic potential surface of each of the four chloromethanes ( figure 1 ) . as suggested by scholfield et al . , the electron - withdrawing ability of the atoms and functional groups that are bonded to cl affect the size of the -hole on cl , which is consistent with our qm calculations . as shown in figure 1d , in ch3cl , the electrostatic potential around cl in ch3cl is mostly negative ( red ) , but the outermost portion of cl s surface facing away from the central carbon is actually neutral ( green ) . this is in accordance with the fact that cl is more electronegative than c and , thus , bears a partial negative charge in a c cl bond . however , the covalent bond pulls the electron distribution toward the center of the bond and leaves a neutral patch on the outermost surface of cl along the c cl axis . from ch2cl2 to ccl4 ( figures 1 ) , this patch becomes larger and increasingly positive ( blue ) as the electron - withdrawing forces generated by additional cl atoms increase . these observations are recapitulated by the amoeba atomic multipole moments derived from qm calculations . in table 2 , the calculated electrostatic charges ( monopoles ) indicate that net charge on cl become less negative going from mono- to tetrachloromethane . furthermore , the z component of the dipole moment on cl ( pointing along the cl c bond vector ) decreases from ch3cl to ccl4 , suggesting diminishing charge separation along the cl c bond , while the positive -hole gets larger . in addition , there are large quadrupole components on the cl atoms , relative to those of c and h ( see the parameters in the si ) . the small root - mean - square difference between the electrostatic potentials calculated from qm and atomic multipoles ( table 2 ) suggests the distributed atomic multipole moments are able to represent these complex charge distributions . in table 1 , qm and force field calculated molecular polarizability values show very good agreement across the four compounds . the atomic polarizability values for c and h were transferred from the existing amoeba parameter set ; the same cl atomic polarizability was used for all four compounds here ( all parameters are given in the si ) . the cl atomic polarizability is almost twice that of the c atom , and the molecular polarizability steadily increases from ch3cl to ccl4 . ab initio molecular electrostatic potential surfaces calculated at the mp2/6 - 311g++ ( 2d , 2p ) level . ( a ) ccl4 , ( b ) chcl3 , ( c ) ch2cl2 , ( d ) ch3cl . the electrostatic potential is mapped on the surface of molecular electron density at 0.001 au contours . coloring scheme : red ( < 12.55 kcal / mol ) , yellow ( 5.02 kcal / mol ) , green ( 0 kcal / mol ) , light blue ( 5.02 kcal / mol ) , and blue ( > 12.55 kcal / mol ) . the atomic multipoles were derived from ab initio calculations at the mp2/6 - 311++g(2d,2p ) level . the rmse is based on a comparison to the ab initio electrostatic potential at the same level . we examined a series of homodimers of ccl4 , chcl3 , ch2cl2 , and chcl3 , as well as heterodimers with a water molecule ( figure 2 ) in which the geometries were randomly chosen near local minima on the dimer energy surface . because poltype already produced the electrostatic and valence parameters based on the qm calculations on the monomers , the qm calculations on dimers allow us to quickly estimate the vdw parameters ( each chloromethane has its own cl parameters ) . the use of heterodimers with water ensures that the parameters are transferable to another environment . geometry optimizations of these dimers were carried out at the mp2/aug - cc - pvtz level . the bsse - corrected association energy at the mp2/aug - cc - pvqz level was obtained at different cc distances for homodimers and different co distances for heterodimers . the forcebalance program was then used to fit the vdw parameters to the gas - phase dimer energy . ( a ) ccl4ccl4 , ( b ) ccl4water , ( c ) chcl3chcl3 , ( d ) chcl3water , ( e ) ch2cl2ch2cl2 , ( f ) ch2cl2water , ( g ) ch3cl ch3cl , ( h ) ch3cl water . carbon , orange ; cl , green ; h , white ; o , red . amoeba and qm calculated pairwise interaction energy for dimers in gas phase . the cl s vdw for ccl4 : d ( diameter ) = 3.62 , = 0.4 kcal / mol , and rmse ( qm vs ameoba ) = 0.51 kcal / mol . the cl s vdw for chcl3 : d = 3.06 , = 1.62 kcal / mol , and rmse = 1.43 kcal / mol . the cl s vdw for ch2cl2 : d = 3.49 , = 0.5 kcal / mol , and rmse = 1.02 kcal / mol . the cl s vdw for ch3cl : d = 3.67 , = 0.20 kcal / mol , and rmse = 1.24 kcal / mol . by optimizing the vdw parameters , ameoba was able to reproduce the ab initio interaction energies of homodimers reasonably well ( figure 3 ) . however , for heterodimers , the agreement is generally worse , particularly for the chcl3h2o dimer . the difference between the qm and amoeba interaction energy is 1.0 kcal / mol at the minimum energy distance . in figure 3 , it was also noticed that neither vdw parameter d nor appears to display a consistent chemical trend going from ccl4 to ch3cl , as observed for the electrostatic parameters ( table 1 and table 2 ) . as noted earlier , these configurations were randomly chosen and might not represent the most important configurations in the liquid phase . for example , in the chcl3h2o dimer ( figure 2d ) , the h atom instead of cl atom of chcl3 is facing the water o atom . although the dimer data is likely insufficient for determining the final vdw parameters , these simple gas - phase calculations provide a set of starting parameters for subsequent examination of liquid state properties using molecular dynamics simulations . the gas - phase qm study is important in the sense that it provides detailed information on electrostatic and intermolecular interactions in an isolated environment , although it is challenging to directly compare these results to experimental measurements . we next optimized the gas - phase qm derived cl s vdw parameters for each of the chloromethanes using the experimental liquid density and heat of vaporization data and the forcebalance method . given that there are two free parameters , it is not surprising that the final amoeba results are in excellent agreement with experimental values for both density and heat of vaporization . from initial optimization , it was found that the resulting cl diameters , d , across all four chloromethane are very similar . thus , we decided to use the same d parameter and reoptimized the vdw energy depth parameter for each cl type in the four chloromethanes . the rmse is 0.01 g / cm for the density and 0.12 kcal / mol for the heat of vaporization . interestingly , the resulting optimal parameter systematically decreases from mono- to tetrachloromethane ( table 3 ) . recall that the cl charge and dipole moment also follow a similar trend ( table 2 ) . in addition to the neat liquid simulation , we evaluated the hydration free energy of each of the chloromethanes to examine the transferability of the resulting parameters and the potential energy model . the hfe of small molecules is an important physical property in many chemical and biological processes , such as protein extensive efforts have been made to improve the fixed - charge force fields to reduce the hfe error of small organic molecules to 1.0 kcal / mol . previously , we have shown that for a group of common organic molecules , the hfe calculated by amoeba is in excellent agreement with experimental data ( rmse 0.4 kcal / mol ) . the computed hfe for each chloromethane using the alchemical free energy methods as described in the computational methods section is shown in table 3 . the errors are well within 1 kcal / mol for mono- , di- , and trichloromethane , indicating reasonable transferability of the model from neat liquid to a water environment . overall , there is a clear trend of increasing error from mono- to tetrachloromethanes . the large error suggests that the model for tetrachloromethanes needs to be further examined for the cause of the transferability issues . density and heat of vaporization data were used in the parameterization ; the hydration results were not . hvap ( kcal / mol ) , heat of vaporization ; ( g / cm ) , liquid density at room temperature ; ahyd ( kcal / mol ) , hydration free energy ; subscript with cal , calculated values ; subscript with expt , experimental reference . this value is measured at 293 k previous work has demonstrated that a carbonyl vibration s frequency reports on the local electric field created by the surrounding solvent according to a simple linear model ( eq 4 ) . the amoeba force field accurately predicts solvent electric fields , and so , by extension , can describe solvent - induced frequency shifts and band - broadening . drawing on this concept , we evaluated the electric fields that the chloromethane solvents exert on the test solute acetophenone by recording the infrared spectrum of acetophenone dissolved in ch2cl2 , chcl3 , and ccl4 ( 10 mm ) and mapping the peak frequency and linewidth to the mean electric field and electric field standard deviation using linear models calibrated against seven nonhalogen - containing reference solvents . the average electric fields for the chloromethane solvents are significantly greater than expected from continuum models ( such as the onsager reaction field or poisson boltzmann equation ) , implying the presence of h - bonds and x - bonds between chloromethane molecules and the c = o vibrational probe . the amoeba model reproduces the average solvent field exerted by ch2cl2 and chcl3 reasonably well , including the small difference between them . agreement among simulations and experimental values for the dispersion of these two solvents electric fields is similarly strong . the good agreement between amoeba and experimentally determined electric fields suggests the electrostatic moments assigned to these molecules , derived from gas phase calculation , give a correct description of their electrostatic interactions in the condensed phase , as well . ccl4 exerts smaller but nonetheless significant electric fields , although md simulations substantially underestimate them . because ccl4 possesses no h - bonding capacity but is expected to donate the strongest x - bonds among the solvents studied , the increased discrepancy is consistent with the possibility that the amoeba model renders electrostatics of x - bonds insufficiently attractive . peak frequency and linewidth ( cm ) , electric field mean , and standard deviation ( mv / cm ) . electric field calculated from = ( 0.484 0.029 ) fsolv + 1703.6 . the general philosophy of the amoeba model is a classical potential energy function applicable in different chemical and physical environments . the introduction of the inducible atomic dipole , which allows the electrostatics of a molecule to respond to its instantaneous environment , is a step forward in the development of a more transferable force field . in the past , for many ionic and organic molecular systems , we were able to achieve such transferability by iteratively optimizing the vdw parameters between the gas - phase cluster energy and liquid experimental properties . in this study , the electrostatic parameters were derived from qm ; vdw parameters were initially derived from qm dimer energetics and optimized in liquid simulations . to examine the transferability of the final parameters back to the gas - phase environment , about 20 new dimer configurations were extracted from the neat liquid and hydration free energy simulation trajectories of ccl4 and ch3cl , respectively . by computing the radial distribution function ( rdf ) for relevant atom pairs ( figure 4a to d ) , we were able to identify the first solvation shell , from which the homo- and heterodimers were chosen randomly for the subsequent qm it is noticed from figure 4f that the closest clo distance in ccl4 solution is in the range of 34 , which is much longer than the c cl covalent bond ( 1.8 ) this observation , together with the evidence that our clasical model can well reproduce the dimer interaction energy as shown next ( figure 5 ) , indicates that the so - called halogen bonds , formed by the positive -hole on cl and electron rich o , are of noncovalent nature and can be modeled by a combination of electrostatic and vdw inteaction as can most hydrogen bonds . ( a ) ccl4 neat liquid simulation , 20 neighboring ccl4 molecules were found around the reference ccl4 within 11 ( second peak ) . ( b ) ccl4 in solvent , with 21 water neighboring molecues around within 10 . ( c ) ch3cl liquid simulation , 21 neighboring ch3cl molecules were found within 7.6 . ( d ) ch3cl in solvent , with 21 neighboring water molecules found within 5.4 . ( e ) clcl distance rdfs for ccl4 and ch3cl in pure liquid simulations . ( open circles ) refers to results from the vdw parameter set optimized using the liquid properties , and gas ( stars ) refers to the results from the parameter set determined from qm dimer energy profile in gas phase ( figure 3 ) . rliquid / gas indicates the correlation coefficient between qm and mm simulations : ( a ) ccl4ccl4 , rmses for liquid and gas are 0.27 and 0.23 kcal / mol , respectively . ( b ) cclh2o , rmses for liquid and gas are 0.16 and 0.20 kcal / mol , respectively . ( c ) ch3clch3cl , rmses for liquid and gas are 0.07 and 0.13 kcal / mol , respectively . ( d ) ch3clh2o , rmses for liquid and gas are 0.13 and 0.22 kcal / mol , respectively . for a total of 83 dimers , we evaluated the association energy by using both qm ( bsse corrected mp2/aug - cc - pvqz ) and the amoeba force field . the correlation between the qm and force field results using the initial gas - phase determined vdw parameters and final , liquid - optimized vdw parameters is shown in figure 5 . by inspecting the rliquid / gas values , it can be seen that the final liquid - optimized parameter set in general yields very good correlation with qm results , with r = 0.850.99 and rmse = 0.070.27 kcal / mol . in addition , the liquid - optimized parameters did better than the gas - phase qm energy - derived parameter set , especially for the ch3cl - ch3cl dimer . during the initial vdw parametrization based on the gas - phase qm dimer energy ( figure 3 ) , one single conformation was chosen randomly for each dimer so that it is possible that we used a local minimum - energy configuration for ch3clch3cl that is uncommon in the liquid state . we further examined a few structures ( figure 6a , b1 , b2 , b3 , d1 , and d2 ) that correspond to the outliers labeled in figure 5 . structures observed in liquid - phase simulations ( figure 6 b1 , b2 , b3 , d1 , and d2 ) are very different from those used in the gas - phase dimer vdw parametrizations ( figure 2 ) . in ccl4ccl4 dimer configuration a ( in figures 5 and 6 ) , the intermolecular clcl distance ( 3.4 ) is similar to that of the gas - phase qm optimized structure ( 3.3 , figure 2a ) . on the other hand , the rdf of ccl4ccl4 in figure 4e shows that there are very few pairs with clcl distances less than 3.5 in the liquid state . in other words , configuration a is a rare case in the liquid state but close to the optimal structure in the gas phase . it is therefore understandable that the liquid - optimized vdw parameter set did worse on this structure than the parameter set directly fitted to the gas - phase dimer energy profile . a similar conclusion can be drawn by comparing d1(co distance 3.2 , similar to the gas - phase structure ) and d2(co distance 4.5 , prevalent in liquid simulation ) . for the b1 , b2 , and b3 configurations extracted from liquids , the h atom of water is facing the cl of ccl4 , and in the gas - phase dimer we used above ( figure 2b ) , the o atom of water is facing the cl . the overall worse performance of the gas - phase parameters on these structures is a reults of the inferior transferability when only one configuration is used in the preliminary parametrization . among all the liquid phase results computed by amoeba , the root of this poor agreement likely lies in the force field s inaccurate description of ccl4 s x - bonding to the o atoms of water and acetophenone . the dielectric constant of ccl4 ( 2.2 ) is close to that of n - hexane ( 1.9 ) , yet it exerts more than twice the electric field ( 25 vs 11 mv / cm ) on acetophenone . x - bonding interactions are responsible for the additional electrostatic attraction , yet this effect is clearly not captured in our electric field simulations ( table 4 ) ; more subtly , it might also explain why the calculated hfe of ccl4 was overly endergonic ( table 3 ) and why ccl4water heterodimer interaction energies were not in strong accordance with qm ( figure 5b ; rliquid = 0.85 ) . strong agreement with ccl4 s experimental heat of vaporization , density ( table 3 ) , and homodimer interaction energies ( figure 5a ) may have been possible because ccl4 does not have a good x - bond acceptor itself , so the effect is muted in neat systems . conformations found in liquid - phase simulations for which the amoeba model has large errors . given the abundance of organochlorine compounds or halogen compounds in general in areas of chemistry , biology , and drug discovery , it is important to have an accurate classical force field for modeling such compounds and their interaction with other molecules . however , the traditional fixed atomic charge force field is inadequate for treating the complex electrostatics in halogen compounds , especially when the -hole , we applied the amoeba model to investigate a series of chloromethanes , including ccl4 , chcl3 , ch2cl2 , and ch3cl . the atomic multipole framework is a natural choice for complicated charge distribution seen on the halogen atoms . the electrostatic parameters were derived from qm calculations of the monomers , and the vdw parameters were optimized against liquid densities and heats of vaporization . the electrostatic potentials of monomers were well described by atomic multipoles , including the large charge separation observed on the cl atom . the qm gas - phase dimer energies were reasonably reproduced by amoeba for homodimers , but less satisfactory for heterodimers formed with a water molecule . the neat liquid density and heat of vaporization , which were used as the vdw parameter optimization targets , were well reproduced by amoeba simulations . the rmse is 0.01 g / cm for the simulated density and 0.12 kcal / mol in heat of vaporization . the hydration free energy and solvent field , which were not used in the parametrization process , agree quite well for the less substituted chloromethanes ; however , the predicted tetrachloromethane hfe showed a larger deviation from the experimental data ( 1.91 kcal / mol ) . by examining a number of dimer configurations from the liquid state simulations , we show that , in general , the amoeba interaction energies are very well correlated with the qm results . h2o heterodimer interaction energies suggest that x - bonds , formed between the positive -hole on cl and the negative charge on o , can be reliably treated by a combination of electrostatic and vdw interactions , just like h - bonds . however , the observation that ccl4 s electric fields were modeled qualitatively incorrectly ( table 4 ) and the problems with ccl4h2o interaction energies probably reflect deeper issues with the amoeba potential function rather than the parametrization . in particular , the problems mentioned hint at a cancellation of error in which electrostatic interactions are made insufficiently attractive while vdw interactions are under - repulsive . in diagnosing this energy decomposition problem , we point out that cl atom has much larger vdw parameters ( diameter and energy well depth ) than the other elements ( c , o , and h ) in this study . the vdw interaction energys for unlike pairs ( e.g. , cl h or cl o ) are computed by using simple combining rules , which are known to be problematic . this problem would be more severe when multiple cl atoms are in close contact with a very dissimilar atom such as o or h in water . on the other hand , it has been shown that the electrostatic energy by point multipoles ( or point charges ) tends to underestimate the electrostatic attraction for large diffuse molecular species . moreover , an inadequate vdw combination rule would prevent molecules from adopting the correct spatial orientations , and electric fields ( especially from quadrupole moments , such as on cl ) depend very sensitively on distances . in developing force fields that can correctly decompose interaction energies into electrostatic and vdw components ( and not rely on error cancelation ) , training data that encode information about specific intermolecular interactions is particularly incisive . to this end , vibrational stark shifts and heterodimer interaction energies appeared to be more discriminating than the other properties analyzed . to the best of our knowledge , this study represents the first example of using vibrational stark measurements to assess the parametrization of a force field , and we expect they will be of further use moving forward , since they isolate the effect of electrostatic interactions from other intermolecular forces . nonetheless , the current parametrization procedure reported herein appears satisfactory for modeling most organochlorine compounds and can be extended to chlorine - containing molecules of interest .

the charge distribution of halogen atoms on organochlorine compounds can be highly anisotropic and even display a so - called -hole , which leads to strong halogen bonds with electron donors . in this paper , we have systematically investigated a series of chloromethanes with one to four chloro substituents using a polarizable multipole - based molecular mechanics model . the atomic multipoles accurately reproduced the ab initio electrostatic potential around chloromethanes , including ccl4 , which has a prominent -hole on the cl atom . the van der waals parameters for cl were fitted to the experimental density and heat of vaporization . the calculated hydration free energy , solvent reaction fields , and interaction energies of several homo- and heterodimer of chloromethanes are in good agreement with experimental and ab initio data . this study suggests that sophisticated electrostatic models , such as polarizable atomic multipoles , are needed for accurate description of electrostatics in organochlorine compounds and halogen bonds , although further improvement is necessary for better transferability .

Introduction Computational Methods Results and Discussion Conclusion

PMC4408389

radical cystectomy ( rc ) with pelvic lymph node dissection is considered to be the gold standard treatment of muscle invasive bladder cancer ( bc ) . in some cases of high grade non muscle invasive disease which is refractory to bcg therapy , radical cystectomy is also a viable option . in the majority of cases , the most popular choice of diversion is either an ileal conduit or an orthotopic neobladder . recently , however , minimally invasive radical cystectomy techniques have been gaining popularity and nowadays in most cases robot assisted technique is used . robot assisted radical cystectomy ( rarc ) has been shown to be equivalent to open radical cystectomy in terms of oncological and functional outcomes , while superior when it comes to the perioperative outcomes such as shorter hospital stay and blood loss [ 15 ] . it has been shown that robotic techniques have comparable oncologic outcomes in terms of nodal yield and positive margin rates compared to the open technique [ 2 , 5 ] . therefore , many surgeons are still adopting the hybrid approach when performing the cystectomy using robotic assistance and completing the urinary diversions extracorporeally to shorten the operating time . wiklund and associates have pioneered the technique of intracorporeal urinary diversion creating both neobladders and ileal conduits completely intracorporeally . the technique is to be credited as an almost identical replication of open surgical principles . recently , the group reported their oncologic and functional outcomes of their rarc cohort showing comparable results to open series . equally , desai and gill presented remarkable outcomes using a modification of the aforementioned technique . these reports have further consolidated that rarc and intracorporeal diversion are technically feasible with good outcomes . however , these observations are limited to very few pioneering centers worldwide and it remains unknown whether rarc and intracorporeal urinary diversions are reproducible by other tertiary centers with robotic expertise . particularly , intracorporeal neobladder formation is regarded a technically challenging and potentially time consuming surgery with steep learning curves [ 9 , 10 ] . herein , we present a homogenous series of patients who underwent rarc and subsequent intracorporeal urinary diversion in the form of ileal conduit and neobladder . we aim to demonstrate that rarc with intracorporeal urinary diversion is safely reproducible and teachable given adequate robotic experience . to our knowledge , this is one of the largest series of rarc with intracorporeal urinary diversion to date . we retrospectively identified 101 patients who underwent rarc and intracorporeal urinary diversion in two academic centers from october 2009 to october 2014 . institutional review board approval ( # 081/2014r ) was obtained before starting data collection and analysis . a total of 300 radical cystectomies were performed in the two centers during the same period . one surgeon from each center performed the rarc with intracorporeal diversions , each experienced in open cystectomy and robotic prostatectomy . a dedicated robotic team comprising of a senior resident and nurse practitioner participated routinely in all the cases . the indications for rarc were muscle invasive transitional carcinoma ( tcc ) and high grade non muscle invasive disease refractory to intravesical bcg treatment . both men and women tcc histology , coagulopathy , pre existing incontinence for patients undergoing neobladder , cerebrovascular disease and severe pulmonary dysfunction rendering robotic surgery impossible . in our series , patients with previous intra abdominal surgeries or radiotherapy were not excluded . data analysis comprised of perioperative variables ( operating time , blood loss , hospital stay ) , standardized complication reporting using the clavien dindo system , pathology reporting and functional as well as oncologic outcomes . furthermore , 3year cancer specific survival ( css ) and overall survival ( os ) were reported . rarc was done using a three arm da vinci s system ( intuitive surgical , sunnyvale , california ) . the optical trocar was placed 2 cm above the umbilicus while a 0 degree telescope was used in all cases . the two remaining robotic trocars were placed a handbreadth lateral to the umbilicus and two more 12 mm assisting trocars were inserted 5 cm above the anterior superior iliac spine . finally , a 5 mm trocar was placed in the right upper abdomen . nerve sparing in both men and women was attempted whenever oncologically permitted . in females , nerve sparing was achieved by careful preservation of a tissues lateral to vagina to avoid damage to autonomic nerves . extended pelvic node dissection included the external , the internal as well as the common iliac nodes , the obturator fossa and the presacral area . the bladder specimen and the lymph nodes both ureters were clipped early and the left ureter was transposed below the sigmoid mesocolon . the robot was redocked and a 50 cm loop of terminal ileum was isolated approximately 20 cm proximal to the ileocecal valve . it is crucial to ensure that the most dependent part can reach down to pelvis easily to ensure a tension free urethro the ileal loop was next discontinued using a 60 mm endo gia stapler ( covidien ) . bowel continuity was then restored using an endo gia stapler by performing a functional end to end anastomosis . the isolated loop was consequently opened according to previously described techniques from wiklund et al . . the posterior plate was then reconstructed using a 3/0 absorbable suture in a running fashion . j stents were placed over guide wire and the ends were advanced through the wall of the reservoir . stents were advanced to the skin through the 12 mm trocar . in the patients who underwent intracorporeal ileal conduit , 15 cm of the terminal ileum 20 cm away from the ileocaecal valve was isolated . the distal end of ileal conduit was then brought out to the right abdominal wall and matured . specimens were ultimately retrieved through a separate incision ( specimen delivered via the vagina in women ) . rarc was done using a three arm da vinci s system ( intuitive surgical , sunnyvale , california ) . the optical trocar was placed 2 cm above the umbilicus while a 0 degree telescope was used in all cases . the two remaining robotic trocars were placed a handbreadth lateral to the umbilicus and two more 12 mm assisting trocars were inserted 5 cm above the anterior superior iliac spine . nerve sparing in both men and women was attempted whenever oncologically permitted . in females , nerve sparing was achieved by careful preservation of a tissues lateral to vagina to avoid damage to autonomic nerves . extended pelvic node dissection included the external , the internal as well as the common iliac nodes , the obturator fossa and the presacral area . the bladder specimen and the lymph nodes both ureters were clipped early and the left ureter was transposed below the sigmoid mesocolon . the robot was redocked and a 50 cm loop of terminal ileum was isolated approximately 20 cm proximal to the ileocecal valve . it is crucial to ensure that the most dependent part can reach down to pelvis easily to ensure a tension free urethro the ileal loop was next discontinued using a 60 mm endo gia stapler ( covidien ) . bowel continuity was then restored using an endo gia stapler by performing a functional end to end anastomosis . the isolated loop was consequently opened according to previously described techniques from wiklund et al . . the posterior plate was then reconstructed using a 3/0 absorbable suture in a running fashion . j stents were placed over guide wire and the ends were advanced through the wall of the reservoir . both ureters were then anastomosed to the afferent limb using a 4/0 absorbable suture . in the patients who underwent intracorporeal ileal conduit , 15 cm of the terminal ileum 20 cm away from the ileocaecal valve was isolated . the uretero the distal end of ileal conduit was then brought out to the right abdominal wall and matured . specimens were ultimately retrieved through a separate incision ( specimen delivered via the vagina in women ) . in total , 101 rarcs with intracorporeal urinary diversion were identified in 82 men and 19 women . out of which , 73 patients ( 57 male , 16 female ) underwent intracorporeal neobladder ; and 28 patients ( 25 male , 3 female ) underwent intracorporeal ileal conduit . the mean body mass index ( bmi ) was 26.5 kg / m ( 1934 ) . in those patients who underwent intracorporeal neobladder , neoadjuvant chemotherapy was administered in 13 patients ( 12.9% ) with presumably advanced stage ( table 1 ) . the preoperative stage and grade of disease is also included in the table . the mean operating time was 402.3 minutes ( 205690 ) and blood loss was 345.3 ml ( 501000 ) while the mean diversion time was 156.6 minutes ( 85300 ) . bilateral nerve sparing was performed in all females and in 52 out of 82 men ( 63.4% ) . the postoperative tumor stage was cis in 7 patients ( 6.9% ) , pt0 in 11 patients ( 10.9% ) , pt1 in 6 patients ( 5.9% ) , pt2 in 33 patients ( 32.7% ) , pt3 in 31 patients ( 30.7% ) , pt4 in 13 patients ( 12.8% and all of them were high grade disease . out of all the patients , 8.9% had positive ureteric margins despite having negative intra concomitant prostate cancer was found in 29 men ( 35.3% ) . the gleason score was 3 + 3 in 18 patients ( 62.1% ) , 3 + 4 in 6 patients ( 20.7% ) and 4 + 3 in 3 patients ( 10.3% ) and gleason 8 disease a total of 3 men ( 10.3% ) had positive prostatic margins ( 2 men from ileal conduit arm and one patient from the neobladder arm ) . there was no preoperative psa performed , as it was not part of routine preoperative investigations . patient 's characteristics bmi body mass index , asa american society of anesthesiologists , cis carcinoma in situ , bcg bacillus calmette gurin perioperative and postoperative outcome pathology characteristics the perioperative complications were grade i in 12 patients ( 11.9% ) , grade ii in 16 patients ( 15.8% ) , grade iii in 28 patients ( 27.7% ) and grade iv in 9 patients ( 8.9% ) . minor ( clavien i&ii ) and major ( clavien iii ) complication rates were 27.7% and 36.6% respectively . early surgery related complications ( < 30 days ) occurred in 50.5% of patients with 6 patients requiring reoperation . out of which , three patients had prolonged ileus , one patient had post operative hemorrhage , one patient had small bowel anastomosis dehiscence and one patient had small bowel sealed perforation treated with small bowel resection . there was no statistical difference between the complication rates of neobladder and ileal conduit patients . the average follow up was 27.5 months ( 152 ) . during the follow ups , one patient ( 1.0% ) who underwent intracorporeal neobladder had developed local recurrence . metastatic disease developed in 25 patients ( 24.8% ) and all of them received chemotherapy . 17 patients have died due to metastatic disease during the follow up period , whereas 9 patients died from other non cancer related disease . the 3year cancer specific survival ( css ) and overall survival ( os ) was 80.2% and 69.8% respectively ( table 4 ) . early ( < 30 days ) and late ( > 30 days ) surgical complications the continence and sexual outcomes were assessed in patients 12 months after surgery . continence is defined as usage of less than 1 pad a day and iief5 score was used to assess the potency of patients . daytime continence could be achieved in 89.2% , while complete nighttime continence was noted in 67.6% of patients who underwent intracorporeal neobladder . erectile dysfunction was reported in 63.5% of men ( table 5 ) . functional and oncologic outcomes the perioperative complications were grade i in 12 patients ( 11.9% ) , grade ii in 16 patients ( 15.8% ) , grade iii in 28 patients ( 27.7% ) and grade iv in 9 patients ( 8.9% ) . minor ( clavien i&ii ) and major ( clavien iii ) complication rates were 27.7% and 36.6% respectively . early surgery related complications ( < 30 days ) occurred in 50.5% of patients with 6 patients requiring reoperation . out of which , three patients had prolonged ileus , one patient had post operative hemorrhage , one patient had small bowel anastomosis dehiscence and one patient had small bowel sealed perforation treated with small bowel resection . late surgery there was no statistical difference between the complication rates of neobladder and ileal conduit patients . the average follow up was 27.5 months ( 152 ) . during the follow ups , one patient ( 1.0% ) who underwent intracorporeal neobladder had developed local recurrence . metastatic disease developed in 25 patients ( 24.8% ) and all of them received chemotherapy . 17 patients have died due to metastatic disease during the follow up period , whereas 9 patients died from other non cancer related disease . the 3year cancer specific survival ( css ) and overall survival ( os ) was 80.2% and 69.8% respectively ( table 4 ) . early ( < 30 days ) and late ( > 30 days ) surgical complications continence is defined as usage of less than 1 pad a day and iief5 score was used to assess the potency of patients . daytime continence could be achieved in 89.2% , while complete nighttime continence was noted in 67.6% of patients who underwent intracorporeal neobladder . erectile dysfunction was reported in 63.5% of men ( table 5 ) . functional and oncologic outcomes rarc has been gaining popularity as a viable alternative to open radical cystectomy for patients with bladder carcinoma . however , it is still not as widely adopted as robotic radical prostatectomy for various reasons . recent reports have confirmed the long term safety and efficacy of laparoscopic rc as well as of rarc . equivalent oncologic outcomes have been shown by using surrogate parameters such as nodal yield and margin status . secondly , rarc especially intracorporeal diversion has always been considered a very lengthy procedure with the potential risk of complications associated with the steep trendelenburg position . this may be true for the initial series ; however , contemporary data has indicated competitive surgical times and other studies have also shown that operating time and patient 's length of stay continues to improve with the learning curve . thirdly , rarc and intracorporeal urinary diversion has been deemed a cumbersome procedure consisting of a robotic part and an open extracorporeal part for urinary diversion . many have hence questioned the point of performing rarc with extracorporeal diversion as this potentially takes away the advantages of minimally invasive surgery such as smaller wound incision and reduced bowel exposure . they followed the principles of open surgery in creating a studer neobladder including proper folding . their approach has evolved over time leading to technical modifications and a reduction of complications and operative times . in a cumulative analysis of 70 patients with a median follow up of 30.3 months , they found negative margins in 98.6% . relevant complications occurred in 31.4% at 30 d and 18.6% at > 30 d. at 90 d , the overall complication rate was 58.5% . the recurrence free , cancer specific , and overall survival at 24 months was 80.7% , 88.9% , and 88.9% . daytime continence and satisfactory sexual function or potency at 12 months ranged between 70% and 90% in both men and women . these results are well in line with contemporary open series . a retrospective comparison to open diversion in the framework of the international robotic cystectomy consortium including 935 patients further confirmed the safety of intracoroporeal diversion by showing lower risk of postoperative complication including gastrointestinal complications in these patients . notably , only 61 patients had undergone intracorporeal neobladder formation in this comparative study . in the u.s . , similar approaches have been described . although shorter follow up with fewer patients , comparable results have been reported . herein , we present our cumulative experience with rarc and intracorporeal urinary diversion performed in two tertiary referral centers with robotic expertise . this is a representation of a real life experience of rarc and intracorporeal urinary diversion outside of the pioneering centers . many findings by the pioneer groups have been corroborated and their results have been widely replicated . hence , rarc and intracorporeal urinary diversion may be safely implemented into clinical routine whenever adequate robotic experience is available . to the best of our knowledge , our report describes the largest series of rarc and intracorporeal diversion apart from the karolinska group . obviously , our surgical times in the initial cases were long due to the learning curve . however , over time average operative time spent could be reduced to less than 6 hours . this is in line with what has been shown by various studies that robotic cystectomy and intracorporeal urinary diversion can be done safely with a structured approach in a high volume center [ 9 , 15 ] . from our experience , another important element to a successful rarc with intracorporeal urinary diversion that provides patient with good oncological , functional and perioperative outcomes is a dedicated robotic team . rarc with intracorporeal diversion is extremely complex and can be intimidating for any young robotic surgeon . it helps to have a dedicated robotic team as familiarization is important not just for the surgeon , but also for the bedside assistants , scrub team as well as the theatre staff . in our practice , a dedicated team comprised of a senior resident and nurse practitioner participates routinely in all the cases . teamwork from all the members of the team involved is crucial to ensure a smooth sailing surgery to minimize complications , conversion rate and shorten the operating time . ileal anastomosis , smaller skin incision for delivery of specimen and , decreased bowel manipulation and exposure to external environment . all these factors can ultimately contribute the better outcomes in terms of better perioperative outcomes and lower risk of postoperative complications [ 15 , 14 ] . our results have shown the safety and reproducibility of this complex technique with comparable operating times , perioperative complications , functional and oncologic outcomes with the pioneer centers . in terms of hospital stay , our series showed a longer hospital stay compared to the contemporary series ; this could be partly attributed to our subsidy system whereby the patients have to stay for 1 week to receive full subsidies . however , we face the similar limitations faced by previous authors such as retrospective nature , limited number of patients and follow up durations . however , current results seem to favor robotic cystectomy with intracorporeal urinary diversion ; a conclusion which can only be established eventually with a randomized study of adequate power . with the advancement of technology and refinement of surgical technique , it is a matter of time before more will embark on robotic cystectomy and intracorporeal urinary diversions . a standardized technique with a dedicated robotic team in a high volume center makes robotic intracorporeal urinary diversion a feasible and reproducible technique .

introductionrobot assisted radical cystectomy ( rarc ) plus intracorporeal urinary diversion is feasible . few centers worldwide demonstrated comparable functional and oncologic outcomes . we reported a large series of rarc and intracorporeal diversion to assess its feasibility and reproducibility.material and methodswe identified 101 rarcs in 82 men and 19 women ( mean age 68.3 years ) from october 2009 to october 2014 . the patients underwent rarc and pelvic lymphadenectomy followed by intracorporeal urinary diversion ( ileal conduit/ neobladder ) . out of the 101 patients , 28 ( 27.7% ) received intracorporeal ileal conduits and 73 ( 72.3% ) intracorporeal neobladders . studer pouch was performed in all the patients who underwent intracorporeal neobladder formation . perioperative , functional and oncologic results including css and os are reported.resultsmean operative time was 402.3 minutes ( 205690 ) and blood loss was 345.3 ml ( 501000 ) . the mean hospital stay was 17.1 days ( 562 ) . all the surgeries were completed with no open conversion . minor complications ( grade i and ii ) were reported in 27.7% of patients while major complications ( grade iii and above ) were reported in 36.6% of patients . the mean nodal yield was 20.6 ( 046 ) . positive ureteric margins were found in 8.9% of patients . the average follow up was 27.5 months ( 152 ) . daytime continence could be achieved in 89.2% of patients who underwent intracorporeal neobladder . the 3year cancer specific survival ( css ) and overall survival ( os ) was 80.2% and 69.8% respectively.conclusionsrarc with intracorporeal diversion is safe and reproducible in non pioneer tertiary centers with robotic expertise having acceptable operative time and complications as well as comparable functional and oncologic outcomes .

INTRODUCTION MATERIAL AND METHODS Surgical technique Intracorporeal neobladder Intracorporeal ileal conduit Statistical analysis RESULTS Complications Oncologic outcomes Functional outcomes DISCUSSION CONCLUSIONS

PMC5022524

extra - adrenal pheochromocytomas ( eaps ) , more accurately known as paragangliomas , are rare tumours that account for 10 - 25% of pheochromocytomas . some 53% of eaps occur in the organs of zuckerkandl , which are a collection of paraganglia situated anterolateral to the distal abdominal aorta between the origin of the inferior mesenteric artery and the aortic bifurcation . the traditional treatment of a paraganglioma of organ of zuckerkandl ( poz ) involves open surgical excision , and only seven previous cases of laparoscopic resection have been reported [ table 1 ] . we describe successful laparoscopic resection of a large poz in a young woman and review the previously reported cases . a slender , 22-year - old woman with a body mass index ( bmi ) of 23 was being treated for hypertension for 2 years but it remained poorly controlled despite three antihypertensive drugs . an abdominal ultrasound showed an 8 cm 7 cm mass on the left side of the abdomen . a contrast - enhanced computed tomograph ( ct ) [ figure 1a ] confirmed a vascular tumour lateral to the aortic bifurcation and extending along the left common iliac artery . haematological and biochemical investigations were normal except for grossly elevated plasma free normetanephrine and free metanephrine levels . a metaiodobenzylguanidine ( mibg ) scan [ figure 1b ] showed intense uptake in the area corresponding to the mass identified on the ct scan , but no other areas of uptake were identified . a work - up for multiple endocrine neoplasia type 2 ( men2 ) was negative . her preoperative preparation included a high - salt diet and alpha blockade with prazosin for 2 weeks , followed by beta blockade with propranolol for 1 week . ( a ) ct scan showing a vascular tumour ( arrow ) adjacent to the aortic bifurcation ( b ) mibg scan showing increased uptake in the area corresponding to the tumour identified on the ct scan a laparoscopic excision was planned with the patient placed in the partial left lateral decubitus position . a 10-mm port was placed in the right iliac fossa for the telescope using an open entry . a 5-mm port was placed to the left side of this port , and another 5 mm port in the midline in the suprapubic area [ figure 2 ] . a steep trendelenburg position allowed the small bowel and omentum to be moved away from the tumour , which was seen protruding through the descending and sigmoid mesocolon [ figure 3a ] . a long horizontal incision was made in the anterior leaf of the mesocolon using an ultrasonic shears ( harmonic scalpel , ethicon endo - surgery , mumbai , mh , india ) . the inferior mesenteric artery lying posterior to the tumour was carefully separated from it and preserved . the mesocolic incision was closed with a running 2/0 polydiaxanone ( pds , johnson and johnson , mumbai , india ) . the specimen was placed in a plastic bag and extracted through a 5-cm pfannenstiel incision that incorporated the incision made for the suprapubic 5-mm port . except for brief periods of hypertension during dissection of the tumour she remained stable throughout surgery . the total operative time was 125 min and intraoperative blood loss was around 40 ml . she was observed in the intensive care unit overnight , started on liquids and diet the day after surgery and discharged on post - operative day 4 . the histopathology report confirmed the tumour to be a paraganglioma with no features of malignancy . she remains normotensive and off her antihypertensive medication at the 18-month follow - up , at the time of writing , and her ct scan and biochemical tests , repeated 1 year after the surgery , were normal . positions of the ports and the suprapubic 5-cm incision used for extraction of the specimen . dotted circle denotes the position of the palpable tumour ( a ) large tumour protruding into the descending mesocolon ( b ) vessel entering the tumour from the inferior mesenteric artery being clipped paraganglia are the main source of catecholamine production in the foetus and regress after 3 years of age . failure of chromaffin cells at the site of paraganglia to regress leads to the development of paragangliomas . the majority of the tumours occurring in the organ of zuckerkandl are benign and non - functional , but malignant and functional tumours are known to occur at this site . nearly half of the paragangliomas are familial and associated with neurofibromatosis type 1 , men2 , von hippel - lindau syndrome or hereditary paraganglioma syndrome . the symptoms of paragangliomas may be related either to excessive catecholamine secretion or pressure symptoms due to a retroperitoneal mass . headache , fainting and dizziness with hypertension are common with norepinephrine - producing tumours , whereas those secreting epinephrine tend to produce irritability , tachycardia , diarrhoea , excessive sweating , flushing and possibly hypertension . yadav et al . reported a paraganglioma producing ureteral obstruction , whereas blecha et al . plasma free metanephrines and urinary fractionated metanephrines carry the highest sensitivity ( 99% and 97% , respectively ) compared to the other plasma or urinary metabolites . a ct scan is usually the first imaging test performed but may not identify lesions smaller than 2 cm . a magnetic resonance imaging ( mri ) scan has higher sensitivity and specificity in detection of paragangliomas , which appear hyperintense on t2-weighted sequences . alpha blockade ( with phenoxybenzamine or prazosin ) followed by beta blockade ( with propranolol or a cardio - selective beta blocker ) as well as adequate hydration are crucial in the preoperative preparation of poz . close intraoperative haemodynamic monitoring and control of hypertensive or hypotensive episodes that may occur by an experienced anaesthetist allows the surgery to proceed smoothly . surgical resection is the treatment of choice for poz , and laparoscopic resection is suited for small ( < 7 cm ) tumours that show no radiological signs of invasion . depending on their location , strategies utilised to approach the tumour included duodenal kocherisation as well as colonic mobilisation . only thaper et al . described a mesocolic approach similar to ours , but they utilised three 10-mm ports , one sited at the umbilicus and the others on the left side of the abdomen . in our patient we chose to place the 10-mm camera port in the right iliac fossa to allow direct , end - on visualisation of the tumour . the original plan of colonic mobilisation to approach the poz was modified to a transmesocolic approach once it was apparent that this would provide early access to the tumour vasculature arising from the aorta and inferior mesenteric artery . the left ureter and the inferior mesenteric artery are structures that need to be safeguarded during resection of a poz , although on occasion the latter may have to be resected . in rare instances of malignant poz invading the aorta or the inferior vena cava , open resection and reconstruction of these vessels as reported by dossett et al . may be warranted . extraction of the specimen in a plastic bag via a low pfannenstiel incision resulted in a cosmetic scar in our young patient . histopathological examination alone does not accurately differentiate benign and malignant paragangliomas , and the diagnosis of malignancy is based on the presence of local invasion and/or presence of metastases . in a collective review of 135 cases of poz found 41% to be malignant . in view of this , long - term follow - up with annual clinical , biochemical and radiological evaluation is mandatory in all patients with poz . in case of local recurrence however , palliative therapy in the form of iodine-131 ( i)-mibg may have to be resorted to if the tumour is unresectable or metastatic . in conclusion , poz is a rare tumour that can be resected safely laparoscopically by experienced surgeons . adequate preoperative preparation , careful intraoperative management as well as post - operative care form the cornerstones of successful outcome in these patients .

paragangliomas are catecholamine - secreting neuroendocrine tumours arising from chromaffin tissue at extra - adrenal sites . the commonest site for a paraganglioma is the organ of zuckerkandl . traditional treatment of paraganglioma of organ of zuckerkandl ( poz ) involves open surgical resection , and only a few cases of laparoscopic approach to this pathology have been reported . we report the successful laparoscopic resection of a large poz in a 22-year - old woman and review the previous cases reporting a laparoscopic approach to this rare tumour .

INTRODUCTION CASE REPORT DISCUSSION CONCLUSION Financial Support and Sponsorship Conflicts of Interest

PMC5146198

oxytocin , a neuropeptide produced in the hypothalamus , has gained considerable attention given its presumed role in prosocial behaviors ( donaldson and young , 2008 ) . in this regard , endogenous oxytocin levels have been associated with trust behaviors ( zak et al . , 2005 , 2009 ) , maternal sensitivity ( feldman et al . , 2012 ) and empathy towards strangers ( barraza and zak , 2009 ) . furthermore , administration of oxytocin by nasal spray promotes enhanced generosity ( zak et al . , 2007 ) , trust ( kosfeld et al . , 2005 ) , empathy ( domes et al . , 2007 ) , positive communication ( ditzen et al . , 2009 ) , and helping behavior ( riem et al . , 2013 ) . through interactions with other biological systems , oxytocin can modulate stress responses , such as cortisol and cytokine reactivity , and may thus be germane to stress - related psychological disorders ( mcquaid et al . , 2014 ) . for instance , intranasal oxytocin attenuated salivary cortisol elevations elicited by a physical stressor ( cardoso et al . , 2013 ) , limited the cortisol rise elicited by social ostracism ( linnen et al . , 2012 ) and that associated with couple conflict ( ditzen et al . , 2009 ) . as encouraging as these data seem , a recent meta - analysis indicated a modest non - significant effect of intranasal oxytocin in attenuating cortisol levels during stressful laboratory tasks . however , a dampening effect of oxytocin on cortisol was apparent in those studies that involved a challenging laboratory task that elicited a robust stimulation of the hpa - axis as well as in studies involving clinical populations ( cardoso et al . , 2014 ) . it is likely that social interactions and social support are important components of the stress - attenuating effects of oxytocin . social support and enhanced connectedness is strongly associated with improved physical ( uchino , 2006 ) and mental health ( cruwys et al . , 2013 ) , and provides an important method of dealing with stressors , thereby attenuating the propensity for illness development ( holt - lunstad et al . , 2008 ) . in this regard , social support diminished cortisol and activation of the dorsal anterior cingulate cortex elicited by a psychosocial stressor ( eisenberger et al . , 2007 ) . furthermore , in males who received social support coupled with intranasal oxytocin prior to a laboratory stressor ( trier social stress test ; tsst ) , a less pronounced cortisol rise occurred compared to individuals who received either social support , oxytocin or neither of these treatments ( heinrichs et al . , 2003 ) . following a social stressor , children who were able to see or hear their mothers , had higher oxytocin levels and cortisol levels were diminished compared to children that had no - contact with their mothers ( seltzer et al . , 2010 ) . to be sure , the effectiveness of social support in attenuating stressor responses may vary as a function of the quality of the support provided , or the closeness and empathy of the person providing support ( holt - lunstad et al . there have been several studies that assessed the influence of intranasal oxytocin on stress responses , but relatively few reports examined the effect of acute stressors on endogenous oxytocin levels in women under ordinary conditions ( i.e. in the absence of lactation and mother - infant bonding ) . moreover , the data that have been reported concerning oxytocin release in response to acute psychosocial stressors have been inconsistent ( taylor et al . , 2006 , ditzen et al . 2012 ) , possibly being related to various procedural differences , including the way in which oxytocin levels were determined . in particular , in some studies oxytocin was measured without first extracting the hormone from blood , and thus molecules in addition to oxytocin might have been tagged , yielding exceptionally high levels , likely reflecting oxytocin together with other factors that were present ( see szeto et al . , 2011 , for a discussion of this topic ) . the overarching aim of the current investigation was to determine , whether plasma oxytocin levels among women , would increase in response to a psychosocial stressor . women were chosen for the current investigation as it has been suggested that plasma oxytocin might be most relevant to women , and vasopressin to men ( taylor et al . , 2010 ) . this may be particularly relevant as estrogen regulates oxytocin functioning ( i.e. transcription of oxytocin as well as its receptor ; choleris et al . , 2008 ) . psychosocial responses and biological variations , including cortisol , oxytocin , and estradiol levels , were assessed in women at baseline and following the tsst . efforts were made to eliminate factors that could confound the potential effects of stressors on oxytocin levels . for instance , in the current study , using females , the effects of a stressor were assessed on circulating oxytocin in properly extracted samples . furthermore , as self - reported menstrual phase may be unreliable , and estrogen can regulate oxytocin functioning ; ( choleris et al . , 2008 ) , estradiol levels were also assessed . as well , in the present study , the impact of support from a close female friend was assessed , considering that social support coming from a male may be less effective ( glynn et al . , 1999 ) . it was hypothesized that having social support from a close female friend would attenuate anxiety , negative mood outcomes as well as cortisol stress responses . however , it was uncertain whether or not oxytocin levels would be expected to rise in response to the stressor or social support manipulation . conflicting findings were reported in this regard , with some indicating that oxytocin either does not increase in response to a stressor irrespective of social support ( taylor et al . , 2006 , ditzen et al . , 2007 ) , increases in response to a stressor if social support is also present ( seltzer et al . , 2010 ) , or increases in response to a stressor in the absence of support ( pierrehumbert et al . , 2012 given these disparate findings , in the present investigation we also examined whether qualities of the individual providing support , including empathy , would be related to the participants stress responses . conceivably , elevated empathy among individuals providing support may affect stress responses among participants , resulting in attenuated stressor effects . the study included female undergraduate students ( n = 67 ) from carleton university ranging in age from 17 to 30 years ( mage = 19.37 , sd = 2.08 ) that were recruited using the online sona system . participants represented an ethnically diverse sample comprising white ( 50.7% , n = 34 ) , black ( 19.4% , n = 13 ) arab / west asian ( 9.0% , n = 6 ) asian ( 6.0% , n = 4 ) , latin american / hispanic ( 4.5% , n = 3 ) , south asian ( 2.0% , n = 3 ) , south east asian ( 1.5% , n = 1 ) , aboriginal ( 1.5% , n = 1 ) , and other ( 1.0% , n in addition to these participants , data relevant to several psychosocial factors were collected from the friends who provided social support during the laboratory session ( n = 18 ) . this group of individuals ranged in age from 17 to 21 years with a mean age of 18.78 ( sd = 1.17 ) and comprised an ethnically diverse sample that included white ( 61.1% , n = 11 ) , black ( 16.7% , n = 3 ) , asian ( 5.6% , n = 1 ) , south asian ( 5.6% , n = 1 ) , south east asian ( 5.6% , n = 1 ) , and other ( 5.6% , n = 1 ) . participants first completed a brief on - line pre - screening questionnaire ( part 1 ) that determined eligibility for the laboratory session that comprised the tsst and a blood draw ( part 2 ) . the on - line pre - screening questionnaire assessed the presence of a number of exclusion criteria , such as medical conditions or medications that may influence hormone functioning , as well as issues surrounding blood sampling ( e.g. a fear of needles , previous history of nausea or fainting during blood collection , or difficulty with veins ) . eligible participants were then randomly assigned to one of three conditions : stress - social support ( participants were asked to bring a friend to a laboratory session , n = 18 ) , stress - no support ( participants arrived alone to the laboratory session , n = 23 ) or controls ( no stress , no friend , n = 26 ) . to avoid a selection bias , all participants who stated they could bring a close females friend were randomly assigned to one of the three conditions , including that in which no friend was present . all procedures in this study were approved by the carleton university ethics committee for psychological research . laboratory sessions were conducted between 1300 and 1730 h , and women were asked not to eat , drink ( with the exception of water ) or smoke for at least an hour before arriving to the session . once informed consent was signed , participants filled out a number of questionnaires assessing demographic information ( including information on oral contraceptive use and menstrual cycle phase ) , depressive symptomatology and a relationship closeness measure ( related to the friend they brought with them to the laboratory session ) . this allowed participants 30 min to habituate to the laboratory environment . upon completion of these measures , a registered nurse inserted a catheter into the participants non - dominant arm for blood collection . participants were then asked to relax for 10 min to habituate to the catheter . following the relaxation period , participants in the stress conditions were instructed that they would be given 10 min to prepare for an employment task comprising a five - minute speech and five - minute mental arithmetic task in front of a panel of graduate student judges . in addition , participants were told they were being videotaped during the psychosocial stressor . during the 10-min preparatory period , participants in the stress - no support condition prepared for the stressor alone , whereas participants within the stress - social support condition had their friend present . the friends were instructed to provide emotional and/or instrumental support to the participant . immediately following the 10 min preparatory period , participants in the two stressor conditions underwent the tsst . participants in the control condition were asked to complete an employment task , which comprised writing down their strengths and past work / volunteer experience on a form . immediately following the tsst or control task , participants completed questionnaires including focused on stressor appraisal , state anxiety and affect scores . furthermore , approximately 30 min following this , the general distrust , and empathy scales were completed . blood , which was continuously being drawn through a dakmed ambulatory pump , was sampled at five time - points during the session , which included 15 min before the tsst or the written employment task ( controls ) , immediately before the stressor / tasks began , and then at five , 15 and 30 min following completion of the tsst or the written employment task . upon arrival to the experimental session , the friend was led to a nearby room in which they signed an informed consent , and were asked to complete a questionnaire booklet assessing basic demographic information , depressive symptomatology , a closeness of relationship measure , and empathy scores . they were told about the stressful nature of the employment task ( their friend would undergo ) , and told that they would be assisting the participant during the 10 min preparatory period . once the preparatory period was complete , participants were asked to return to a nearby room to complete the remainder of the questionnaires . they were not present when the participant was being tested ( instructions adapted from heinrichs et al . the 21-item beck depression inventory ( bdi ) was used as a measure of depressive symptomatology ( beck et al . each item comprises one of four options , ranging from low to high depressive symptomatology . total scores were calculated by summing across all items , ( chronbach 's = .88 ) . inclusion of other in the self scale ( ioss ) ( aron et al . , 1992 ) was used to assess closeness of the friend and the participant . this assessment provides a graphical representation of seven separate venn - like diagrams , in which participants are asked to choose one , where increasing overlap between two circles represents a greater degree of closeness . an 8-item distrust and cynicism scale ( derived from the cook - medley hostility scale ) was used to measure general feelings of cynical distrust ( greenglass and julkunen , 1989 , greenglass and julkunen , 1991 ) . each item can be rated from 0 ( completely disagree ) to 3 ( completely agree ) . a total score was calculated by taking the mean across all items ( = .82 ) . items such as ; it is safer to trust nobody can be found in this scale . the 28 item interpersonal reactivity index ( davis , 1983 ) was used to assess total empathy scores . each item ranges from 0 ( does not describe me well ) to 4 ( describes me well ) , with higher scores reflecting greater empathy . i am often quite touched by things that i see happen, can be found in this scale . total empathy scores were created using a mean across all items ( = .82 ) . the 28-item stress appraisal measure ( sam ) ( peacock and wong , 1990 ) comprises multiple subscales representing appraisals of threat , challenge , centrality , stressfulness , controllability and uncontrollability . in the present study , these items were measured on a five - point scale ranging from 1 ( not at all ) to 5 ( extremely ) , with higher scores indicating higher levels of perceived stress . a mean across all items created a total stress appraisal score ( = .95 ) . state anxiety was assessed using the spielberger state trait anxiety inventory ( stai ) ( spielberger , 1983 ) . the 20-item state anxiety scale was used to measure current feelings of anxiety following the tsst . items ranged from 1 ( not at all ) to 4 ( very much ) , where higher scores indicate greater state anxiety . total scores were obtained by summing across all items ( = .95 ) . the 41-item positive and negative affect schedule ( panas ) ( watson et al . , 1988 ) assessed the presence and severity of positive and negative affective states following the tsst . responses ranged on a six - point scale from 0 ( not at all ) to 6 ( extremely ) . various mood - state subscales were calculated including sadness ( = .91 ) , anger ( = .94 ) , fear ( = .93 ) and shame ( = .96 ) . total negative affect scores were calculated by a mean across items ( = .94 ) . blood samples were collected continuously , at a low draw rate , ( 2.08 ml/5 min ) into chilled edta coated ( for plasma ) and serum separator ( for serum ) vacutainer tubes . separate chilled edta tubes were used for the collection of oxytocin , which contained aprotinin . samples were taken at an increased draw rate ( 6.9 ml/5 min ) 15 min before the tsst or the employment task ( controls ) , immediately before the stressor / tasks began , and then at five , 15 and 30 min post - task . approximately 2.76 ml of plasma and serum were collected for each of the time - points of interest . following collection , samples were centrifuged for 15 min at 4 c and 2100 g , plasma and serum were immediately aliquoted into eppendorf tubes and frozen at 80 c . plasma cortisol was determined in duplicate by a radioimmuno assay ( ria ) using the i kit obtained from icn biomedicals inc . the intra - assay variability was less than 8% and the minimum detectable concentration was .17 g / dl . prior to the assay , oxytocin was extracted as recommended following the procedure manual from enzo life science inc . , ( farmingdale , ny ) . for the extraction procedure , 1 ml of plasma was used and samples were evaporated with nitrogen gas , following which samples were stored at 20 c until the assay . plasma oxytocin concentrations were determined through an elisa kit obtained from enzo life science inc . , according to the manufacturer 's instructions . furthermore , according to the manufacturer 's kit information , cross reactivity for arginine vasopressin was less than .02% and the minimum detectable was 3.75 pg / ml . serum estradiol was determined through an elisa kit obtained from invitrogen ( camarillo , ca ) . the intra - assay variability was less than 5% and the minimum detectable concentration was 5 2pgml . of the 67 participants , there were four individuals from whom blood samples could not be obtained due to complications with small veins . as well , in some instances participants did not have five valid samples for hormone detection ( e.g. , failure of blood collection equipment , collapsed vein or not enough blood could be obtained ) , thus they were removed from any repeated measures analyses and as such the n 's per group will vary . statistical analyses were performed using spss for windows 18.0 ( spss science , chicago , illinois , usa ) . analyses assessing the influence of the tsst on stress appraisals and state anxiety were performed using one - way analyses of variance ( anova ) ( stressor condition : controls , stress - support , stress - no support ) . a manova was used to determine the effects of the tsst condition on mood outcomes , including sadness , anger , fear , shame and negative affect . oxytocin was analyzed using a 3 ( stressor condition ) x 5 ( time : 5 time - points ) mixed measures anova with time serving as the within - group factor . for cortisol assessment , an area under the curve ( auc ) analysis with respect to increase was performed using the formula proposed by pruessner et al . ( 2003 ) with stressor condition and oral contraceptives ( yes vs. no ) serving as the between groups factors . follow - up comparisons comprised t - tests with a bonferonni correction to maintain the alpha level at .05 . additionally , pearson 's correlation coefficients were determined between cortisol , oxytocin , and distrust levels . pearson 's correlation coefficients were also determined between the friends ' psychosocial scores and the participants ' psychosocial and biological scores . as expected , prior to the tsst , there were no differences between individuals in the control , stress - support , or stress - no support conditions on levels of depressive symptoms , f ( 2 , 64 ) = .21 , p = .81 . following the tsst , stress appraisals varied as a function of the stressor condition , f ( 2 , 64 ) = 37.08 , p < .001 , = .54 . as shown in fig . 1a , individuals in both the stress - support and the stress - no support conditions reported elevated stressfulness compared to controls , p < .001 and p < .001 , respectively . state anxiety also differed with the stressor condition , f ( 2 , 64 ) = 9.94 , p < .001 , = .24 , such that individuals who were stressed but did not receive support exhibited elevated state anxiety compared to controls , p < .001 , an effect not apparent among the stressed individuals who received social support p = .15 ( fig . 1b ) . a significant manova revealed differences in affective states following the tsst as a function of the stressor condition , f ( 10 , 122 ) = 4.77 , p < .001 , = .28 . as shown in fig . 2 , there was an overall effect of stress for feelings of shame , f ( 2 , 64 ) = 13.88 , p < .001 , = .30 , such that higher levels of shame occurred among both the stress - no support and stress - support conditions compared to controls , p < .001 and p = .001 respectively . levels of sadness also differed between stressor conditions , f ( 2 , 64 ) = 6.10 , p = .004 , the stressed - no support group displayed elevated sadness compared to controls , p < .001 , whereas individuals who were stressed but received support did not differ from controls , p = .32 . feelings of anger differed between stressor conditions , f ( 2 , 64 ) = 7.90 , p = .001 , = .20 , such that individuals in the stress - no support condition reported more anger compared to controls , p = .001 , an effect not apparent among individuals in the stress - support condition . further , as shown in fig . 2 , anger tended to be somewhat higher in the stress - no support compared to the stress - support condition , although this only approached significance , p = .06 . fear and negative affect followed a similar trend , f ( 2 , 64 ) = 10.21 , p < .001 , = .24 and f ( 2 , 64 ) = 10.54 , p < .001 , = .25 respectively . in these instances , individuals who were stressed and had no support displayed elevated fear and negative affect compared to controls , p 's = .001 and their stress - support counterparts , p 's = .02 . an analysis was conducted to assess the extent to which the participants and the friends they brought to the laboratory shared the closeness they felt toward each other . unexpectedly , the correlation of perceived closeness between the friend and the participant , indicated that friends ' perceptions of closeness and participants ' perceptions of closeness with each other were unrelated , r ( 15 ) = .02 , p = .93 . furthermore , friends ' perceptions of closeness were not related to participants ' psychosocial responses to the tsst , including state anxiety r ( 15 ) = .15 , p = .56 , stress appraisals , r ( 15 ) = .17 , p = .50 , and any of the participants ' biological scores at baseline or in response to the tsst . as well , the participants ' perceptions of closeness also did not significantly correlate with their psychosocial or biological stress responses and similarly , the friends ' level of empathy did not relate to the participants ' state anxiety , r ( 16 ) = .05 , p = .86 , or stress appraisal , r ( 16 ) = .02 , p = .93 scores . interestingly , empathy scores of the friend were inversely related to the participants ' cortisol levels at both baseline , r ( 15 ) = .60 , p = .01 , and following the tsst , r ( 15 ) = .50 , p = .04 . as well , a relationship was found between the friends ' level of empathy and the participants ' basal oxytocin levels , such that higher empathy from the friend was associated with elevated oxytocin levels at baseline among the participants , r ( 15 ) = .56 , p = .02 . it was first of interest to examine the relationships between baseline oxytocin levels with depression and distrust scores . as shown in table 1 , depressive scores were not significantly associated with oxytocin , however , a significant inverse relationship was found between distrust and oxytocin , r ( 59 ) = .28 , p = .03 . as well , oxytocin at baseline was inversely related to baseline cortisol levels , r ( 55 ) = .28 , p = .04 . this correlation was not found to be significant when run separately according to stressor condition . although estrogen is known to influence oxytocin transcription , as shown in table 1 , no relationship was found between levels of oxytocin and estradiol at baseline . almost half of the females in the current experiment reported using oral contraceptives ( n = 30 ) , whereas 36 females were not taking oral contraceptives . as expected , females taking oral contraceptives ( m = 40.31 , se = 8.26 ) displayed reduced serum estradiol levels compared to females not taking oral contraceptives , ( m = 73.84 , se = 8.47 ) , t ( 1 , 52 ) = 2.83 , p = .007 . similar to estradiol , oxytocin levels at baseline also did not vary based on whether females were using oral contraceptives or not , f ( 2 , 59 ) = .35 , p = .55 . therefore , for further oxytocin analyses , neither oral contraceptives nor estrogen were controlled for . there were no initial difference between controls ( m = 10.54 4.98 ) , the stress - support ( m = 10.97 1.11 ) or stress - no support ( m = 12.06 .96 ) groups on baseline levels of oxytocin , f ( 2 , 59 ) = .65 , p = .53 . furthermore , over the course of the tsst or the written employment task ( for controls ) , oxytocin levels did not significantly increase , although there was a trend in this direction , f ( 4 , 180 ) = 2.14 , p = .08 , = .05 . additionally , oxytocin levels in response to the tsst did not vary by stressor condition , f ( 8 , 180 ) = .40 , in addition to this analysis , auc analyses were also conducted for oxytocin with respect to ground ( full area beneath the oxytocin scores , irrespective of whether or not oxytocin rose ) and with respect to increase ( change of oxytocin levels ) . in neither instance oral contraceptive users displayed elevated baseline cortisol levels ( m = 14.19 , se = 1.61 ) compared to individuals not taking oral contraceptives ( m = 8.62 , se = 1.15 ) , t ( 1 , 55 ) = 2.88 , p = .006 . however , cortisol levels did not vary as a function of menstrual phase or smoking . thus , oral contraceptive use was included as a variable in subsequent analyses relating to cortisol . individuals not taking oral contraceptives included , ( control , n = 8) , ( stress - support , n = 9 ) and ( stress - no support , n = 14 ) , and individuals who were taking oral contraceptives included , ( control , n = 10 ) , ( stress - support , n = 7 ) , and ( stress - no support , n = 8) . an analysis of area under the curve ( auc ) indicated that although there was no main effect of stressor condition on cortisol , f ( 2 , 50 ) = 2.22 , p = .12 , = . 08 , there was a main effect of oral contraceptives , f ( 2 , 64 ) = 6.50 , p = .014 , = .12 , with a larger auc being present among individuals who were taking oral contraceptives ( mean difference = 154.27 ) . additionally , cortisol varied as a function of the stressor condition oral contraceptives interaction , f ( 2 , 50 ) = 5.44 , p = .007 , = .18 . as shown in fig . 3 , and confirmed by the follow - up tests of the simple effects comprising this interaction , among individuals not using oral contraceptives ( left hand panel ) , cortisol did not vary in relation to the tsst . however , for individuals using oral contraceptives , the stress - no support group displayed a larger cortisol auc compared to controls ( mean difference = 358.86 ) , p = .004 . further , while there were no differences between controls and individuals who were stressed with support available , the auc tended to be greater in the stress - no support group compared to the stress - support group ( mean difference = 266.77 ) , p = .071 . cortisol was also examined through an individual - peak analysis , which accounts for the fact that individuals display peak cortisol levels at different times following a stressor , and the results were identical . in the current study , the tsst increased stressor appraisals irrespective of social support being available , whereas state anxiety scores following the tsst were only elevated among individuals who were stressed and did not receive support . in essence , although participants in both conditions were equally cognizant of the stressor , social support buffered the anxiety associated with the tsst . similarly , relative to controls , individuals who were stressed and did not receive support displayed high levels of anger , fear , and sadness as well as overall negative affect . in contrast , stressed individuals who received support did not exhibit elevations of these emotions , again pointing to the stress - buffering effects of social support ( cohen and wills , 1985 ) . interestingly , despite the effectiveness of social support in acting against some of the negative emotions promoted by the tsst , feelings of shame were not diminished by support . it is not immediately apparent why shame was not attenuated ; however , such feelings may be a uniquely powerful emotion ( relative to the others measured ) , being a threat to the social self , and might thus not be amenable to being appreciably diminished by social support ( gruenewald et al . , 2004 ) . although the availability of social support was accompanied by reductions of several negative emotions , the independent assessments made by participants and their friends regarding their closeness were not consistent with one another ( i.e. , participants ' perceived closeness to the friend was not necessarily shared ) . likewise , the degree of closeness and/or empathy expressed by the friend was not related to anxiety or negative affect elicited by the tsst among participants , and similarly , the participants ' perceived closeness to the friend was unrelated to their feelings of anxiety or negative affect . it has been predicted that relationship quality is an important component of support efficacy ( holt - lunstad et al . , 2007 ) . however , in the present investigation social support appeared to buffer against anxiety and negative emotionality irrespective of perceived closeness . in effect , simply because individuals did not share their perceived closeness , this may not have had any bearing on the effectiveness of the support provided . the measure of closeness adopted ( inclusion of other in the self ) is one that has been frequently used and is considered to be a valid measure of closeness ( tropp and wright , 2001 ) . given the presumed relationship between oxytocin and prosocial behaviors , and that oxytocin was also implicated in inferring the mood states of others ( domes et al . , 2007 ) , it is interesting that having a friend present with generally high levels of empathy was related to elevated oxytocin levels among participants . although it is tempting to suggest that the higher empathy of the friend caused an oxytocin release , the correlational nature of the data preclude this conclusion . empathy has been found to be related to endogenous oxytocin release , particularly among females ( barraza and zak , 2009 ) , and intranasal oxytocin promotes empathetic behaviors ( domes et al . , 2007 ) , but it has yet to be shown that empathy of one individual may be related to oxytocin levels in a close other . this finding , being the first of this nature to be reported , certainly needs to be replicated . lower baseline oxytocin levels were related to higher levels of distrust , which is consistent with the reported elevated plasma oxytocin levels associated with trust behaviors in a monetary game ( zak et al . , 2005 ) as well as trust - related interactions involved in sharing secrets ( kri et al . , 2009 ) . furthermore , salivary oxytocin correlated positively with trust upon arriving to a laboratory testing condition for a second time ( tops et al . , 2013 ) . consistent with these reports , intranasal oxytocin administration increased trust - related behaviors in a monetary game ( kosfeld et al . , 2005 ) , and trust with confidential information ( mikolajczak et al . , 2010 ) . unlike the studies showing a relationship between endogenous oxytocin and trust ( zak et al . , 2005 , kri et al . , 2009 ) , however , such a relationship has not always been reported ( christensen et al . , 2014 ) . it was suggested that these discrepant results may be due to the procedure in the former studies not including the extraction of oxytocin from samples prior to the assay being conducted and hence the presence of oxytocin may have been contaminated by other factors being assayed concurrently ( christensen et al . , 2014 ) . in the current study , the relation between oxytocin and basal levels of general distrust was found in samples where the hormone was extracted prior to the assay , although this does not imply that oxytocin scores would be linked to trust or distrust following an experimental manipulation . , 2009 , linnen et al . , 2012 , cardoso et al . , 2013 ) , baseline oxytocin was inversely related to circulating cortisol . although oxytocin is thought to contribute to affiliative / social approach behaviors by diminishing fear / anxiety ( taylor , 2006 ) , it is uncertain whether the oxytocin link to cortisol was tied to prosocial behaviors . although studies in animals have indicated that oxytocin elevations are elicited by acute stressors ( neumann , 2002 , danevova et al . , cortisol treatment in humans is known to increase oxytocin levels ( tops et al . , 2012 ) , and thus it might have been expected that a stressor would elicit a similar outcome . in this regard , it was shown that the tsst elicited a rapid increase in salivary ot ( jong et al . , 2015 ) . that said , as oxytocin measurements in the saliva and blood may not be measuring the same thing ( mccullough et al . , 2013 ) , it is premature to base conclusions that incorporate oxytocin variations elicited by stressor in these two different products . however , as previously observed ( taylor et al . , 2006 , plasma oxytocin levels were unaffected by the tsst , and this was the case in the current manuscript regardless of circulating estradiol levels or whether women were using oral contraceptives . it is also important to consider that oxytocin is released in a pulsatile fashion and is a dynamic process ( ludwig and leng , 2006 ) and thus , it is possible that the time of collection did not capture changes in this hormone . this said , blood for each sample was collected over a two minute period , and samples were taken at three post - stressor intervals , thus making this possibility less likely . cortisol profiles among women taking oral contraceptives , as in earlier reports ( kirschbaum et al . , 1999 ) , were distinct from non - oral contraceptive users . compared to females not using oral contraceptives , those taking oral contraceptives in the current study displayed elevated baseline plasma cortisol levels as well as greater plasma cortisol responses to the tsst . indeed , it was only among women using oral contraceptives that the tsst effectively increased plasma cortisol . although it has been reported that oral contraceptive users displayed a blunted cortisol response , this only occurred when cortisol was measured in saliva , whereas an appreciable increase ( 55% ) was apparent in blood ( kirschbaum et al . , 1999 ) . thus , the present findings in blood are consistent with those previously reported , and reinforce the distinction between salivary free cortisol and total cortisol levels in plasma ( which mainly comprises it was suggested that among oral contraceptive users , estrogen stimulates corticosteroid - binding globulin ( cbg ) synthesis , resulting in reduced free bioactive cortisol ( kajantie , 2008 ) and enhanced bound cortisol . this aside , it was surprising that cortisol levels among females not taking oral contraceptives were largely unaffected by the tsst . the high baseline estradiol levels among females in the current study who were not taking oral contraceptives , may have contributed to the lack of cortisol change in response to the tsst . in this regard , it has been observed that women with high estrogen levels ( corresponding to the luteal phase of the cycle ) , display significantly increased free cortisol levels in response to stressors ( i.e. , within saliva ) , but blunted blood cortisol stress responses ( kirschbaum et al . , 1999 ) . furthermore , it may be significant that although the tsst is a potent stressor , its effects on cortisol are typically more pronounced among males ( kajantie and phillips , 2006 ) . this could be related to differences of sex hormones , but it could also be linked to the finding that males tend to display greater responses to achievement - oriented stressors , such as mathematics and verbal tasks , whereas women show a greater reactivity to rejection related stressors ( stroud et al . , 2002 ) . among oral contraceptive users in the present study , social support attenuated the cortisol rise in response to the tsst that was otherwise evident among the individuals who did not receive support . it is thought that one potential mechanism by which social support may buffer cortisol stress responses is through hypothalamic oxytocin release , as treatment with an oxytocin receptor antagonist blocked the social buffering effects in rodents ( smith and wang , 2014 ) . likewise , female children that received some form of support from their mothers displayed elevated oxytocin and attenuated cortisol responses to the tsst ( seltzer et al . , 2010 ) . thus , although it might have been expected in the current investigation that the females receiving social support from a close friend would display elevated oxytocin levels following the stressor , this hormone was unaffected by the social support manipulation . given that plasma oxytocin might not reflect changes in brain oxytocin , our findings do not necessarily suggest that brain oxytocin levels are unimportant in the buffering effects of support ( landgraf and neumann , 2004 , leng and ludwig , 2015 ) . in essence , central oxytocin release could buffer hpa axis responses to stressors , but levels of this hormone in the periphery might not adequately reflect oxytocin changes in brain . the sample size was admittedly modest , but it is unlikely that increasing the sample size would have had any bearing on the lack of an oxytocin rise in response to the stressor or the absence of a cortisol rise among women not using oral contraceptives as in both instances , the stressor did not elicit even a hint of a hormonal change . we can not exclude the possibility that , in this instance , the tsst was not stressful enough to activate the hpa axis among all participants . however , as already indicated , the cortisol measure was determined in blood , and it is uncertain how the stressor and use of oral contraceptives might have interacted in affecting free cortisol in saliva . ideally , cortisol levels should have been determined from both saliva and blood . it may also be the case that the lack of hormonal change was due to the timing of the baseline sample . in this regard , participants ' baseline sample was drawn 10 min following needle insertion ( 40 min after arrival to the laboratory ) and therefore , hormone levels may have already increased as a result of the anxiety associated with the needle prick . ideally , a longer length of time should have transpired between needle insertion and the baseline sample ; however , as the continuous blood draw was already 70 min , this was not considered feasible . in this regard it would have also been advantageous to have extended the blood draw in order to capture the cortisol decline and return to baseline levels , however , once more this was unfortunately not feasible . furthermore , a group was not included in the present study in which support was available in the absence of a stressor manipulation . thus , it was not possible to examine the influence of social support on oxytocin levels over time in the absence of a stressor . likewise , it would have been advantageous to include a manipulation in which a neutral party was present ( as opposed to a friend ) who could have acted as a distractor and thus modified the stress response . in this way it could be determined whether the buffering effects observed with respect to cortisol were linked to a close friend being present prior to the actual tsst or whether simply having another person present could have acted in this capacity , possibly by distracting participants . these caveats notwithstanding , the current study addressed several important issues concerning endogenous oxytocin functioning in relation to stressful experiences . only a limited number of studies have included females in research pertaining to stress and oxytocin , particularly those studies in which intranasal oxytocin was administered ( owing to complications relating to hormonal fluctuations across the menstrual cycle ) . yet , increasing evidence has pointed to differential actions ( or correlations ) of endogenous and exogenous oxytocin on various behaviors among men and women ( taylor et al . , 2010 , hoge et al . , 2014 ) , thus suggesting that this hormone may potentially serve different functions in men and women . the present investigation , in women , suggested that basal oxytocin and cortisol levels are related , and oxytocin is linked to attitudes , such as distrust and empathy . a psychosocial stressor in the form of the tsst , did not affect plasma oxytocin levels , but among women using oral contraceptives the stressor increased circulating cortisol . this outcome could be buffered by social support , and once again this occurred despite oxytocin levels in plasma being unaffected by this manipulation . this is not altogether surprising as plasma oxytocin might not be an accurate reflection of oxytocin functioning within stress - relevant brain regions ( landgraf and neumann , 2004 , leng and ludwig , 2015 ) . certainly , it needs to be determined whether circulating oxytocin is an ideal measure to assess the linkage to the stress - attenuating properties of social support . this said , studies in animals have made it fairly clear that central oxytocin release is fundamental to the positive effects of social support on stress responses ( smith and wang , 2014 ) , but studies in humans have typically relied on plasma oxytocin to provide data relevant to prosocial behaviors . in this regard , efforts have been made to assess the effects of intranasal oxytocin treatment , but it is uncertain how much of the hormone actually reaches the brain . nonetheless , in humans , this approach is a reasonable option at the moment , particularly when coupled with analyses of polymorphisms related to oxytocin or its receptor . this research was supported by the canadian institutes of health research ( cihr ) and the natural sciences and engineering research council of canada ( nserc ) . the funding sources had no further role in the study or in the writing of this article . all authors report no conflicts of interest in the conduct or in the reporting of this research .

stress responses in humans can be attenuated by exogenous oxytocin administration , and these stress - buffering properties may be moderated by social factors . yet , the influence of acute stressors on circulating endogenous oxytocin levels have been inconsistent , and limited information is available concerning the influence of social support in moderating this relationship . in the current investigation , undergraduate women ( n = 67 ) were assessed in the trier social stress test ( tsst ) with either social support available from a close female friend or no social support being available . an additional set of women served as controls . the tsst elicited marked elevations of state anxiety and negative emotions , which were largely attenuated among women who received social support . furthermore , baseline oxytocin levels were inversely related to women 's general feelings of distrust , as well as basal plasma cortisol levels . despite these associations , oxytocin levels were unaffected by the tsst , and this was the case irrespective of oral contraceptive use or estrogen levels . in contrast , plasma cortisol elevations were elicited by the psychosocial stressor , but only in women using oral contraceptives , an effect that was prevented when social support was available . taken together , these data provisionally suggest that changes in plasma oxytocin might not accompany the stress attenuating effects of social support on cortisol levels . moreover , as plasma oxytocin might not reliably reflect brain oxytocin levels , the linkage between oxytocin and prosocial behaviors remains tenuous .

Introduction Methods Statistical analyses Results Discussion Role of funding source Conflict of interest

PMC3297836

one of the most complex problems the visual system has to solve is recognizing objects across a wide range of encountered variations . retinal information about one and the same object can dramatically vary when position , viewpoint , lighting , or distance change , or when the object is partly occluded by other objects . in computer vision , there are a variety of models using alignment , invariant properties , or part - decomposition methods ( roberts , 1965 ; fukushima , 1982 ; marr , 1982 ; ullman et al . , 2001 ; viola and jones , 2001 ; lowe , 2004 ; torralba et al . , 2008 ) , which are able to identify objects across a range of viewing conditions . some computational models are clearly biologically inspired and take for example the architecture of the visual system into account ( e.g. , wersing and krner , 2003 ) , or cleverly adapt the concept of a powerful computer vision algorithm ( e.g. , the fourier - mellin transform ) to a neurobiologically plausible alternative ( sountsov et al . such models can successfully detect objects in sets of widely varying natural images ( torralba et al . , 2008 ) and achieve impressive invariance ( sountsov et al . , 2011 ) . in general however , computer vision models are developed for practical image analysis applications ( handwriting recognition , face detection , etc . ) for which fast and accurate object recognition and not neurobiological validity is pivotal . therefore , these models are generally less powerful in explaining how object constancy arises in the human brain . indeed , models are common ; good theories are scarce as suggested by stevens ( 2000 , p. 1177 ) . humans are highly skilled in object recognition , and they outperform machines in object recognition tasks with great ease ( fleuret et al . , 2011 ) . this is partly because they are able to strategically use semantics and information from context or memory . in addition , they can direct attention to informative features in the image , while ignoring distracting information . such higher cognitive processes are difficult to implement , but improve object recognition performance when taken into account ( lowe , 2000 ) . computer vision models might become more accurate in recognizing objects across a wide range of variations in image input , when implementing algorithms derived from neurobiological observations . reciprocally , our interpretation of such neurobiological findings might be greatly improved by insights in the underlying computational mechanisms . humans can identify objects with great speed and accuracy , even when the object percept is degraded , occluded or presented in a highly cluttered visual scene ( e.g. , thorpe et al . , 1996 ) . however , which computational mechanisms enable such remarkable performance is not yet fully understood . to create a comprehensive theory of human object recognition and how it achieves invariant object recognition , computational mechanisms derived from modeling efforts , we highlight recent developments in object recognition research and put forward a common brain space framework ( cbs ; goebel and de weerd , 2009 ; peters et al . , 2010 ) in which empirical data and computational results can be directly integrated and quantitatively compared . object recognition , discrimination , and identification are complex tasks . different encounters with an object are unlikely to take place under identical viewing conditions , requiring the visual system to generalize across changes . information that is important to retrieve object identity should be effectively processed , while unimportant view - point variations should be ignored . that is , the recognition system should be stable yet sensitive ( marr and nishihara , 1978 ) , leading to inherent tradeoffs . how the visual system is able to accomplish this task with such apparent ease is not yet understood . the first suggests that objects can be recognized by cardinal ( non - accidental ) properties that are relatively invariant to the objects ' appearance ( marr , 1982 ; biederman , 1987 ) . thus , these invariant properties and their spatial relations should provide sufficient information to recognize objects regardless of their viewpoint . however , how such cardinal properties are defined and recognized in an invariant manner is a complex issue ( tarr and blthoff , 1995 ) . the second type of theory suggests that there are no such invariants but that objects are stored in the view as originally encountered ( which , in natural settings encompasses multiple views being sampled in a short time interval ) , thereby maintaining view - dependent shape and surface information ( edelman and blthoff , 1992 ) . recognition of an object under different viewing conditions is achieved by either computing quality matches between the input and stored presentations ( perrett et al . , 1998 ; riesenhuber and poggio , 1999 ) or by transforming input to match the view specifications of the stored representation ( blthoff and edelman , 1992 ) . the latter normalization can be accomplished by interpolation ( poggio and edelman , 1990 ) , mental transformation ( tarr and pinker , 1989 ) , or alignment ( ullman , 1989 ) . view - invariant theories suggest that the visual system recognizes objects using a limited library of non - accidental properties , and neural representations are invariant . evidence for such invariant object representations have been found at final stages of the visual pathway ( quiroga et al . , 2005 ; freiwald and tsao , 2010 ) . in contrast , the second class of theories assumes that neural object representations are view - dependent , with neurons being sensitive to object transformations . clearly , the early visual system is sensitive to object appearance : the same object can elicit completely different , non - overlapping neural activation patterns when presented at different locations in the visual field . so , object representations are input specific at initial stages of processing , whereas invariant representations emerge in final stages . however , how objects are represented by intermediate stages of this processing chain is not yet well understood . likely , multiple different transforms are ( perhaps in parallel ) performed at theses stages . this creates multiple object representations , in line with the various types of information ( such as position and orientation ) that have to be preserved for interaction with objects . moreover , position information aids invariant object learning ( einhuser et al . , 2005 ; li and dicarlo , 2008 , 2010 ) and representations can reflect view - dependent and view - invariant information simultaneously ( franzius et al . , 2011 ) . the following section reviews evidence from monkey neurophysiology and human neuroimaging on how object perception and recognition are implemented in the primate brain . as already alluded to above , the visual system is hierarchically organized in more than 25 areas ( felleman and van essen , 1991 ) with initial processing of low - level visual information by neurons in the thalamus , striate cortex ( v1 ) and v2 ; and of more complex features in v3 and v4 ( carlson et al . , 2011 ) . further processing of object information in the human ventral pathway ( ungerleider and haxby , 1994 ) , involves higher - order visual areas such as the lateral occipital cortex ( loc ; malach , 1995 ) and object selective areas for faces ( ffa ; kanwisher et al . , 1997 ) , bodies ( eba ; downing et al . , 2001 ) , words ( vwfa ; mccandliss et al . , 2003 ) , and scenes ( ppa ; epstein et al . , 1999 ) . the first studies on the neural mechanisms of object recognition were neurophysiological recordings in monkeys . in macaque anterior inferotemporal ( it ) cortex , most of the object - selective neurons are tuned to viewing - position ( logothetis et al . , 1995 ; booth and rolls , 1998 ) , in line with viewpoint - dependent theories . on the other hand , it neurons also turned out to be more sensitive to changes in non - accidental than to equally large pixel - wise changes in other shape features ( metric properties ; kayaert et al . , 2003 ) , providing support for structural description theories ( biederman , 1987 ) . taken together , these studies provide neural evidence for both theories ( see also rust and dicarlo , 2010 ) . however , to which degree object representations are stored in an invariant or view - dependent manner across visual areas , and how these representations arise and are matched to incoming information , remains elusive . the bold signal reflects neural activity at the population rather than single - cell level . the highest functional resolution provided by standard 3 tesla mri scanners is around 2 2 2 mm , which is too coarse to zoom into the functional architecture within visual areas . however , more subtle information - patterns can be extracted using multi - voxel pattern analysis ( mvpa ; haynes et al . , 2007 ) or fmri - adaptation ( fmri - a ; grill - spector and malach , 2001 ) . mvpa can reveal subtle differences in distributed fmri patterns across voxels resulting from small biases in the distributions of differentially tuned neurons that are sampled by each voxel . by using classification techniques developed in machine learning , distributed spatial patterns of different classes ( e.g. , different objects ) can be successfully discriminated ( see fuentemilla et al . for example , changing the position of an object significantly changes patterns in loc , even more than replacing an object ( at the same position ) by an object of a different category ( sayres and grill - spector , 2008 ) . rotating the object ( up to 60 ) did not change loc responses however ( eger et al . , 2008 ) suggesting that loc representations might be view - dependent in only some aspects . fmri - a exploits the fact that the neuronal ( and the corresponding hemodynamic ) response is weaker for repeated compared to novel stimuli ( miller and desimone , 1994 ) . thus , areas are sensitive to view - dependent changes when their bold response returns to its initial level for objects that are presented a second time , but now from a different view - point for example , a recent study observed view - point and size dependent coding at intermediate processing stages ( v4 , v3a , mt , and v7 ) , whereas responses in higher visual areas were view - invariant ( konen and kastner , 2008 ) . remarkably , these view - invariant representations were not only found in the ventral ( e.g. , loc ) , but also in the dorsal pathway ( e.g. , ips ) . the dorsal where / how or perception - for - action pathway is involved in visually guided actions toward objects rather than in identifying objects which is mainly performed by the ventral or what pathway ( goodale and milner , 1992 ; ungerleider and haxby , 1994 ) . for this role , maintaining view - point dependent information in higher dorsal areas seems important , which however was thus not confirmed by the view - invariant results in ips ( but see james et al . , 2002 ) . likewise , another recent study ( dilks et al . , 2011 ) revealed an unexpected tolerance for mirror - reversals in visual scenes in a parahippocampal area thought to play a key role in navigation ( e.g. , janzen and van turennout , 2004 ) and reorientation ( e.g. , epstein and kanwisher , 1998 ) , functions for which view - dependent information is essential . for example , different findings on size , position , and viewpoint - invariant representations in different subparts of the loc have been found ( grill - spector et al . , 1999 ; these divergent findings might be partly related to intricacies inherent to the fmri - a approach ( e.g. , krekelberg et al . , 2006 ) , and its sensitivity to the design used ( grill - spector et al . , 2006 ) and varying attention ( vuilleumier et al . , 2005 ) and task demands ( e.g. , ewbank et al . , 2011 ) . the latter should not be regarded as obscuring confounds however , since they appear to strongly contribute to our skilled performance . object perception is accompanied by cognitive processes supporting fast ( e.g. , extracting the gist of a scene , attentional selection of relevant objects ) and accurate ( e.g. , object - verification , semantic interpretation ) object identification for subsequent goal - directed use of the object ( e.g. , grasping ; tool - use ) . these processes engage widespread memory- and frontoparietal attention - related areas interacting with object processing in the visual system ( corbetta and shulman , 2002 ; bar , 2004 ; ganis et al . , 2007 ) . as the involvement of such top - down processes might be particularly pronounced in humans and weaker or even absent in monkeys and machines respectively efforts to integrate computational modeling with human neuroimaging remain essential ( see tagamets and horwitz , 1998 ; corchs and deco , 2002 for earlier work ) . with the advent of ultra - high field fmri ( 7 tesla scanners ) , both the sensitivity ( due to increases in signal - to - noise ratio linearly dependent on field strength ) and the specificity ( due to a stronger contribution of gray - matter microvasculature compared to large draining veins and less partial volume effects ) of the acquired signal improves significantly , providing data at a level of detail which previously was only available via invasive optical imaging in non - human species . the functional visual system can be spatially sampled in the range of hundreds of microns , which is sufficient to resolve activation at the cortical column ( yacoub et al . , 2008 ; zimmermann et al . , 2011 ) and layer ( polimeni et al . , 2010 ) given that cortical columns are thought to provide the organizational structure forming computational units involved in visual feature processing ( hubel and wiesel , 1962 ; tanaka , 1996 ; mountcastle , 1997 ) , the achievable resolution at ultra - high fields will therefore not only produce more detailed maps , but really has the potential to yield new vistas on within - area operations . the approach we propose is to project the predicted activity in a modeled area onto corresponding cortical regions where empirical data are collected ( figure 1 ) . by interfacing empirical and simulated data in one anatomical direct and quantitative mutual hypothesis testing based on predicted and observed spatiotemporal activation patterns can be achieved . more specifically , modeled units ( e.g. , cortical columns ) are 1-to-1 mapped to corresponding neuroimaging units ( e.g. , voxels , vertices ) in the empirically acquired brain model ( e.g. , cortical gray matter surface ) . as a result , a running network simulation creates spatiotemporal data directly on a linked brain model , enabling highly specific and accurate comparisons between neuroimaging and neurocomputational data in the temporal as well as spatial domain . note that in cbs ( as implemented in neurolator 3d ; goebel , 1993 ) , computational and neuroimaging units can flexibly represent various neural signals ( e.g. , fmri , eeg , meg , fnirs , or intracranial recordings ) . furthermore , both hidden and output layers of the neural network can be projected to the brain model , providing additional flexibility to the framework as predicted and observed activations can be compared at multiple selected processing stages simultaneously ( see figure 2 for an example ) . the common brain space framework : measured neuroimaging data ( left panel ) and simulated data ( right panel ) are projected to the same anatomical brain space via network - brain links ( see figure 2 ) . input : ( a ) visualization of common brain space ( cbs ) in neurolator : each computational unit of a neural network layer is separately connected to a topographically corresponding location on the cortical sheet via a networkbrain link ( nbl ) . in this example , model layers v1 , loc , and ffa are connected to the corresponding brain regions v1 , loc , and ffa on a mesh reconstruction of an individual 's gray - white matter boundary . for this participant , v1 , loc , and ffa were localized using standard retinotopy and related fmri region - of - interest mapping techniques . by connecting a running neural network , activity in the connected layers ( b ) in neurolator , functional mri data can be projected on the cortical mesh , similar to the standard functional - anatomical data co - registration applied in fmri analyses . output : ( c ) depending on display mode , cortical patches ( i.e. , vertices ) either represent the empirical or the simulated fmri data . since the observed and simulated datasets are in the same anatomical space , identical fmri analyses tools can be used to analyze observed and simulated timeseries . to model the human object recognition system , we developed large - scale networks of cortical column units , which dynamics can either reflect the spike activity , integrated synaptic activity , or oscillating activity ( when modeled as burst oscillators ) , resulting from excitatory and inhibitory synaptic input . to create simulated spatiotemporal patterns , each unit of a network layer ( output and/or hidden ) is linked to a topographically corresponding patch on a cortical representation via a so - called network - to - brain link ( nbl ) . via this link , activity of modeling units in the running network is transformed into timecourses of neuroimaging units , spatially organized in an anatomical coordinate system . importantly , when simulated and measured data co - exist in the same representational space , the same analysis tools ( e.g. , mvpa , effective connectivity analysis ) can be applied to both data sets allowing for quantitative comparisons ( figure 2 ) . see peters et al . we propose that such a tight integration of neuroimaging and modeling data allows reciprocal fine - tuning and facilitates hypothesis testing at a mechanistic level as it leads to falsifiable predictions that can subsequently be empirically tested . importantly , there is a direct topographical correspondence between computational ( cortical columnar ) units at the model and brain level . moreover , comparisons between simulated and empirical data are not limited to activity patterns in output stages ( i.e. , object - selective areas in anterior it such as ffa or even more anterior in putative face exemplar regions ; kriegeskorte et al . , 2007 ) , but also at intermediate stages ( such as v4 and loc ) . interpreting the role of feature representations at intermediate stages may be essential for a comprehensive brain model of object recognition ( ullman et al . studying several stages of the visual hierarchy simultaneously , by quantitatively comparing ongoing visual processes across stages both within and between the simulated and empirically acquired dataset , may help to clarify how higher - order invariant representations are created from lower - level features in several ways . incoming percepts might be differently transformed and matched to stored object representations at several stages , with view - dependent matching at intermediate stages and matching of only informative properties ( biederman , 1987 ; ullman et al . monitoring activity patterns at multiple processing stages simultaneously is desirable , given that early stages are influenced by processing in later stages . to facilitate object recognition , invariant information is for example fed back from higher to early visual areas ( williams et al . , 2008 ) , suggesting that object perception results from a dynamic interplay between visual areas . finally , it is important to realize that such top - down influences are not limited to areas within the classical visual hierarchy , but also engage brain - wide networks involved in initial guessing 2006 ) , object selection ( serences et al . , 2004 ) , context integration ( graboi and lisman , 2003 ; bar , 2004 ) , and object verification ( ganis et al . , 2007 ) . such functions should be incorporated in computational brain models to fully comprehend what makes human object recognition so flexible , fast , and accurate . modeling higher cognitive functions is in general challenging , but may be aided by considering empirical observations in object perception studies where the level of top - down processing varies ( e.g. , ganis et al . , 2007 ) . the interactions between the visual pathway and frontoparietal system revealed by such fmri studies can be compared at multiple processing stages to simulations , allowing a more subtle , process - specific fine - tuning of the modeled areas . a number of recent fmri studies applied en- and decoding techniques developed in the field of machine learning and computer vision , to interpret their data ( kriegeskorte et al . , 2011 ; naselaris et al . , 2011 ; see laconte , 2011 for an extention to brain - computer - interfaces ) , showing that both fields are starting to approach each other . for example , by summarizing the complex statistical properties of natural images using a computer vision technique , a visual scene percept could be successfully reconstructed from fmri activity ( naselaris et al . , 2009 ) . the trend to investigate natural vision is noteworthy , given that processing cluttered and dynamic natural visual input rather than artificially created isolated objects poses additional challenges to the visual system ( einhuser and knig , 2010 ) . we believe that now columnar - level imaging is in reach with the advent of high - resolution fmri ( in combination with the recently developed en- and decoding fmri methods ) the time has come to more directly integrate computational and experimental neuroscience , and test which predictions of the various object recognition models are supported by this new type of empirical evidence . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

recent advances in computer vision and experimental neuroscience provided insights into mechanisms underlying invariant object recognition . however , due to the different research aims in both fields models tended to evolve independently . a tighter integration between computational and empirical work may contribute to cross - fertilized development of ( neurobiologically plausible ) computational models and computationally defined empirical theories , which can be incrementally merged into a comprehensive brain model . after reviewing theoretical and empirical work on invariant object perception , this article proposes a novel framework in which neural network activity and measured neuroimaging data are interfaced in a common representational space . this enables direct quantitative comparisons between predicted and observed activity patterns within and across multiple stages of object processing , which may help to clarify how high - order invariant representations are created from low - level features . given the advent of columnar - level imaging with high - resolution fmri , it is time to capitalize on this new window into the brain and test which predictions of the various object recognition models are supported by this novel empirical evidence .

Introduction Exploring invariant object recognition in the human visual system Integration of computational and experimental findings in CBS Conflict of interest statement

PMC5383625

according to a paper by ishizaki et al . of the nasu branch of the zootechnical experiment station in 1949 , although the purpose of selective breeding of horses to develop specific traits had been demonstrated by the army during the period prior to the end of world war ii , a new perspective on selective breeding of draft horses in the postwar era was required . this was especially true in terms of body size , with the need for draft horses that were of smaller size but with sufficient power for doing farm work in japan and with economic efficiency for feeding . therefore , ishizaki performed a series of experiments on the relationship between body size and working power in draft horses . although there were several foreign reports on the relationship between body size and work power in heavy draft horses and it had been suggested that body weight ( bw ) is an important factor determining pulling power , there were few reports on smaller draft horses such as those in japan . in order to determine the most suitable size of draft horses that are fit for japanese farm conditions , the work power of horses weighing from 200 to 500 kg was investigated . in a series of experiments , endurance , endurance tests were conducted by having horses draw carts over 3 successive days . working power was evaluated by having horses plow , draw a cart , pack , or be ridden for 4 hr . maximal pulling power was evaluated by having horses draw carts and wagons with heavy loads for a distance of 600 m. in the first experiment , three korean ponies ( avg . bw 190 kg ) were used as a model of 200 kg horses . the previously described endurance , working power , and maximal pulling power tests were performed . the major findings of the endurance test were as follows : 1 ) korean ponies could draw carts ( 147 kg cart weight and 210 kg load ) 24 km per day for 3 successive days . for this test , the mean draft workload was 20 kg ( 1/9 of the pony s bw ) , and the working time per day was about 7 hr including 2 hr of rest . 2 ) when drawing carts ( 147 kg cart weight and 480520 kg load ) with a mean draft workload of 40 kg , ponies could draw the cart 8 km per day over a working time of about 3 hr including 1 hr of rest . major findings obtained from the working power tests were as follows : 1 ) regarding plowing , korean ponies could plow a 720 m paddy field in about 3.5 hr . the plowing depth was 11 cm , and the mean draft workload was 4245 kg . 2 ) regarding drawing carts , korean ponies could pull carts with a mean draft workload of 20 kg about 13 km in 4 hr including 1 hr of rest . 3 ) regarding packing , korean ponies could carry an 80 kg load and walk 16 km in about 4 hr including 30 min of rest . 4 ) regarding riding : in riding ( proportion of 10 min walking and 5 min trotting ) , korean ponies could go 18 km in about 4 hr including 1 hr of rest . the mean speed was 74 m / min when walking and 162 m / min when trotting . major findings from the maximum pulling power test were as follows : 1 ) when pulling carts ( 147 kg cart weight ) , korean ponies could pull a load of about 1,000 kg . in this case , the mean draft workload was 5864 kg . 2 ) when drawing wagons ( 300 kg wagon weight ) , ponies could pull a load of 675850 kg with a mean draft workload of 5666 kg . from these results , it appeared that there are some suitable types of farm work for these ponies on small japanese farms . because the horses used in this study were not in good condition because of a shortage of feed and the fact that they had never been trained until the experiments , it was concluded that these horses might have reasonable potential for use in farm work if they were fed and trained appropriately . similar experiments were performed on hokkaido ponies weighing about 300 kg , as well as crossbred horses weighing about 500 kg . relationships between work power of draft horses and their bw were evaluated using data from 185 to 622 kg obtained during several power tests and maximal pulling power tests mentioned above . when the horses were made to work pulling carts or wagons with a mean draft workload of about 1/10 of their bw , there was no significant correlation between walking speed and bw . when they plowed a paddy field with a japanese plow , the two factors became significantly correlated , i.e. , the lighter the bw , the slower the speed . however , it was not indicated if the draft workload of the plow was the same percentage of bw for different - sized horses . in a riding test with repeated bouts of walking for 10 min followed by trotting for 5 min , 190 kg korean ponies moved markedly slower than heavier horses . when horses walked freely during the tests , the stride frequency of heavier horses was lower , while their stride length was greater than that of lighter horses ( fig . the authors generated this graph using data in a report by ishizaki et al . . ) . these relationships between body size , stride length , and stride frequency are typical of nearly all mammalian quadrupeds . small mammals can not move their legs fast enough to make up for their short stride length , whereas , large mammals must keep their legs more vertically beneath them to minimize the risk of breaking a leg when extending a stride so far that the bone can not withstand the shearing force . as a result , the fastest mammals ( cheetah , pronghorn antelope , indian blackbuck ) are all about 50 kg in bw . stride frequency and stride length in relation to body weight . the authors generated this graph using data in a report by ishizaki et al . . the plowing of paddy fields with a japanese plow was work too heavy for korean ponies that only weighed about 200 kg . the 300 kg hokkaido pony appeared to be the smallest horse that could easily pull a japanese plow in a paddy field . the correlation coefficient between the area plowed and the bw of the horse was r=0.92 . the maximum weight load that could be carried as well as the load that could be pulled by horses were correlated with body weight , with correlation coefficients of r=0.91 and r=0.98 , respectively . although the maximal pulling power of horses was almost proportional to their bw , it was suggested that pulling power was different from packing ability in terms of bw . the regression coefficient of the percentage of area surrounded by the four hoofs of the horse was quite similar to that of the maximal load that could be packed in relation to bw . from these results , it could be concluded that the area surrounded by the four hoofs had a certain relationship to packing ability . the question as to the size required for deeper plowing , i.e. , to a depth of 1820 cm , was investigated using 13 horses weighing from 306 to 652 kg . the results suggested that horses weighing 498 kg or more could perform deeper plowing for 4 hr , and in terms of body conformation , those horses would be at least 148 cm in height , 177 cm in chest circumference , 19.9 cm in cannon circumference , and 156 cm in body length . relationships between body size and pulling power were also evaluated by another research group at the tohoku agricultural experiment station using 3 percherons ( avg . 650 kg bw ) , 3 crossbred horses ( 500600 kg bw ) , 3 trotters ( 340440 kg bw ) , and 3 hokkaido ponies ( avg . power tests ( 80 kg , 1/6 of bw , and 1/4 of bw ) were performed . the results revealed that pulling power per kg bw was highest in the trotters , then the percherons , hokkaido ponies , and crossbred horses , in that order . and others [ 4 , 23 ] showed that smaller horses raised in japan could perform farm work without being excessively stressed by the workload . however , because a bw effect was observed in some of the power tests , careful attention had to be paid when selecting horses for certain types of work . in order to evaluate workload during exercise , it is necessary to measure energy expenditure . because energy expenditure can be estimated from oxygen consumption ( vo2 ) , measurement of vo2 during exercise is essential for calculation of energy expenditure . although the douglas bag method was a standard method for measuring vo2 of animals , the experiments required that a newly devised face mask and other equipment had to be improved for application in animals exercising in field conditions . there was no study on this topic for horses in japan until tatsumi et al . of the national institute of agricultural sciences tried making measurements as described later in this chapter . to study respiration during draft exercise , sawazaki et al . of the university of tokyo compared respiratory frequency ( rf ) during 3 different types of draft work : plowing , cultivating , and harrowing using the same equipment okabe and sugiyama had used [ 10 , 38 ] . according to their observations , rf increased in a fluctuating manner and reached a given steady state for each exercise . the average draft workloads for plowing and harrowing ( 3035 kg ) were greater than that for cultivating ( 2025 kg ) . because horses had to walk on softer ground when harrowing than when plowing , it is generally considered that harrowing is heavier work than plowing . because the rate of increase in rf tended to be higher when harrowing than plowing and higher when plowing than cultivating , they suggested that rf is higher when performing heavier work . oxygen consumption of horses exercising in various forms of draft work during field exercise was measured using the douglas bag method by tatsumi et al . in the first experiment , energy expenditure during paddy field plowing of 2 crossbred horses weighing about 540 kg with the draft workload set at 50 kg ( about 1/10 of the horse s bws ) was measured . plowing distance , plowing depth , and speed were 368 m , 10 cm , and 6080 m / min , respectively . in the morning , expired gas was collected for 6 min and analyzed to determine resting values before any food had been ingested before the measurement . expired gas was also collected and analyzed throughout 5 min of the main exercise and during recovery for 6 min . the results showed that the net exercise caloric cost per 100 m was 200 calories . gross efficiency , net efficiency , and relative metabolic rate were 18% , 26.5% , and 4.7 , respectively . because this workload was considered to be equivalent to draft work on a flat surface , it was concluded that this work would not be excessive for well - trained draft horses . a second study was done to measure energy metabolism during 4 different kinds of pulling when working crops , including 14-cm - deep plowing ( 55 kg draft workload ) , 18-cm - deep plowing ( 112 kg draft workload ) , soil crushing with a square harrow ( 42 kg draft workload ) , and intertillage using a cultivator ( 48 kg draft workload ) , and the results were compared using 3 crossbred horses . expired gases were collected throughout walking for 360 m and during 10 min of recovery to calculate vo2 . relative metabolic rate was calculated from energy expenditure during exercise by subtracting basal metabolic energy expenditure . since basal metabolic energy expenditure had not yet been measured in horses at that time , resting metabolic energy expenditure measured in early morning was used instead of basal metabolic energy expenditure for the calculation of relative metabolic rate . tatsumi named this variable the relative metabolic rate of animal . according to the results in table 1table 1.energy expenditure during various types of draft work in paddy fields and on farmspaddy field plowing14-cm - deep plowing18-cm - deep plowingcrushing soilintertillagedraft workload ( kg)54.755.31124248velocity ( m / min)71.850.846.35655.9breadth of plowing ( m)2.00.110.11.20.67depth of plowing ( cm)10.013.718.03.79.3work ( cal)44.646.894.635.540.5energy expenditure ( cal)248313359299292net efficiency ( % ) 26.621.736.11721.2relative metabolic rate4.75.46.35.85.2crushing soil and intertillage were performed with a square harrow and cultivator , respectively . , it was presumed that 18-cm - deep plowing was a heavy draft workload but efficient work , while soil crushing was a low draft workload but inefficient work . energy metabolism of 3 different pulling methods including shoulder - trunk traction , chest - trunk traction , and shoulder traction was evaluated by using a sleigh that was weighted to offer a draft workload that was 1/101/9 of the horse s bw during a 400 m unstrained walk . although draft workload , work rate , and net efficiency were almost equal among the 3 methods , the calories used for the work and relative metabolic rate tended to be higher for shoulder traction . from these results , it was concluded that the preferred method for each horse must be chosen by considering its body size , pulling weight , and the pulling condition , because it was assumed that the point of support or point of action of the lever where force was applied might be different among the 3 pulling methods . although the relative metabolic rate calculated from energy expenditure is a useful index for evaluation of workload , several special pieces of equipment , e.g. , a douglas bag or gas analyzer , were needed for the measurements , and the measurement procedure was somewhat complicated . therefore , the relationship between vo2 and pulse rate , which was easier to measure in field conditions than vo2 , was examined in order to use pulse rate as an index of vo2 for exercise tests in field conditions . resting pulse rate was obtained by auscultation and averaged for 2 min , and the pulse rate immediately after exercise was counted from 10 to 40 sec after exercise and converted to beat / minute . seven crossbred horses ( 450550 kg bw ) were used for this study . in the first experiment , 5 horses walked freely 3 times at speeds of 40 , 60 , 80 , 100 , and 120 m / min , respectively . in the second experiment , 3 horses walked 5 times while pulling a draft workload equal to 1/7 of the horse s bws in a sleigh at speeds of 60 , 80 , 100 , and 120 m / min , respectively . vo2 and pulse rate were correlated ( r=0.83 ) , as were relative metabolic rate and pulse rate ( r=0.78 ) . these results indicated that pulse rate measured immediately after exercise could be a useful index of workload . although tatsumi s recommendation that pulse rate should be counted immediately after exercise , e.g. , from 5 to 20 sec afterward , and converted to beat / min was more reasonable than calculating it from 10 to 40 sec after exercise , needless to say , direct measurement of heart rate ( hr ) during exercise is much more preferable . following these studies , a new system for recording an electrocardiogram ( ecg ) during exercise using radiotelemetry of the university of tokyo in order to obtain hr during exercise [ 9 , 30 , 31 ] , and a new simplified device for measurement of hr during draft exercise was also developed . effects of differences in walking speed on energy metabolism were evaluated using 6 crossbred horses ( 400500 kg bw ) [ 52 , 54 ] . as shown in the lower panels in fig . 3fig . [ 52 , 54 ] . , relative metabolic rate and energy consumption per minute increased linearly with increasing speed . on the other hand , the upper panels show that total energy consumption at 80 m / min was lowest and that the values for total energy consumption at speeds of 60 and 100 m / min were lower than those of 40 and 120 m / min . similarly , the values for energy consumption expressed per kg bw and per m at 80 these results indicate that the cost of transport at the speed of 80 m / min was the minimum and that walking faster or slower is less efficient , perhaps because less elastic energy is stored in muscles and tendons that can be recovered during the stride to reduce metabolic energy expenditure . when horses walked freely , total energy consumption and energy calculated per kg bw and distance traveled showed similar low values as in the experiment , suggesting that free walking is the most efficient gait and speed . energy metabolism while walking in water of different depths was evaluated using 2 crossbred horses ( 482 and 542 kg bw ) . horses walked in a circular pool with a 12 m diameter for a total distance of 320 m with 4 different water depths ( 0 , 18 , 36 , and 54 cm ) at a speed of 60 m / min . total energy expenditure and net energy expenditure increased linearly with increasing water depth , and net energy expenditure at a depth of 54 cm was nearly twice that at a depth of 18 cm ( fig . the authors generated this graph using data in a report by tatsumi et al . . ) . the relative metabolic rate increased to 3 times the resting metabolic rate at a depth of 54 cm ; this was almost the same as when walking on the flat ground with a speed of 100 m / min for a horse carrying no load . the authors generated this graph using data in a report by tatsumi et al . . a research project by nomura of the university of tokyo was conducted in order to develop a new perspective on performance testing for selecting better draft horses using heart rate as an index . in 1960 , nomura and tominaga recorded ecg from a draft horse walking on a specially constructed treadmill while drafting 5 different weights : 1/15 , 1/10 , 1/8 , 1/6.5 , and 1/5 of the horse s bw . then , in 1964 , nomura had succeeded in recording ecg during field exercise , including sprint galloping , using a newly constructed radiotelemetry system [ 9 , 30 , 31 , 34 ] . however , this system was large in scale and very expensive , and only one other similar system was available in a research facility at that time in japan . accordingly , nomura developed a beat meter to measure hr of draft horses during heavy draft work . although this device could not record raw ecg , it enabled hr to be measured by counting the number of popping sounds per unit time that were created when the meter detected larger amplitude signals in the ecg , e.g. , the qrs complex . preliminary experiments to establish experimental protocols were conducted using 3 horses raised at the stock farm of the university of tokyo . heart rate was measured using a beat meter during exercise with the horses pulling a sleigh with 4 different weights : 40 , 75 , 100 , and 125 kg . 5.effects of differences in carriage weight ( indicated by different symbols ) on heart rate ( hr ) . heart rate during exercise increased with increasing pulling weight ( draft workload was measured with a dynamometer ) , and there was a tendency for the recovery of hr to be delayed when the weight pulled increased . after another similar experiment , the following basic experimental protocol was proposed : 1 ) the reference draft weight would be set to about 1/4 of the horse s bw , and at least 3 different weights , including upper and lower reference weights , would be used . 2 ) a 300 m straight course would be used to obtain a steady state . heart rate during exercise was measured every 15 sec just after passing the 50 , 150 , and 250 m marks after the start of walking , at about 3045 sec , 90105 sec , and 150165 sec , respectively . heart rate after exercise was measured every 30 sec during the following time intervals 3060 sec , 90120 sec , and 150180 sec . 5 ) workload was calculated as : [ ( draft workload distance ) ( bw time ) ] and given as an absolute number without units when graphs were drawn . 6 ) performance was evaluated using a correlation diagram between workload and total hr during exercise . by drawing 2 straight lines at the upper and lower limits of the sd of the regression line , performance evaluation was assigned to 3 categories : a , the area above the upper limit line ; b , the area between the upper and lower limit lines ; and c , the area below the lower limit line . a was classified as passing , b was classified as good , and c was classified as excellent . effects of differences in carriage weight ( indicated by different symbols ) on heart rate ( hr ) . a total of 22 horses raised at the oh - u national livestock breeding station with daily training ( trained ) and horses without training ( untrained ) were tested using the abovementioned protocols . when total hr during exercise and recovery was plotted on a graph , the relationship between total hr and workload was linear ( fig . the authors generated this graph using data in a report by nomura . ) , and the total hrs of trained horses were lower than those of untrained horses . to determine whether a linear relationship between workload and total hr existed in individual horses , the same test was performed several times using 6 horses . horse a was considered to be an excellent horse , and horses e and f were untrained horses . the authors generated this graph using data in a report by nomura . whether or not the relationship between workload and total hr would remain linear when the workload became too heavy or light was investigated . the effect of a heavier draft workload was investigated at the tokachi national livestock breeding station using 3 different draft workloads ( 142156 kg , 197213 kg , and 243255 kg ) and 22 horses with bws between 649 and 750 kg . the results yielded a graph in which the slope between the workload and total hr became steeper when the workload exceeded 450 . similar experiments were performed using 15 horses of lighter weight than in the previous experiment ( 530650 kg bw ) by setting different draft workloads ( about 100 , 150 , and 200 kg ) . the results were portrayed in a similar graph showing that the slope became steeper when workload exceeded 450 ( fig . 8fig . 8.relationship between total hr ( heart rate during exercise and recovery ) and workload with a heavier draft workload . the authors generated this graph using data in a report by nomura . ) . although nomura did not evaluate his data statistically , according to our statistical analysis , there were significant differences between the slopes of the two regression lines . because the workload exceeded the critical value of 450 when 1/3 or more of a horse s bw was added the workload , nomura concluded that 1/3 of a horse s bw must be the upper limit for the performance test . in another experiment , the effects of lighter draft workloads ( 90 , 130 , and 170 kg ) were investigated at the tohoku agricultural experiment station and iwate national livestock breeding station using 24 horses weighing between 530725 kg . this experiment resulted in a graph showing that the slope became steeper when the workload was 250 or less , and nomura suggested that less than 1/5 of a horse s bw ( a draft workload of about 100 kg ) could not be used for the performance test . based on these experiments , nomura suggested that the draft workload to be used for the performance test should be between about 1/5 and 1/4 of the horse s bw . using the principles that nomura had shown in his experiments , performance tests were performed at the oh - u national livestock breeding station during the period from august 1963 to april 1964 , and the research results were reported in 1965 . relationship between total hr ( heart rate during exercise and recovery ) and workload with a heavier draft workload . in hokkaido , the northern large island of japan , there is a unique form of horse racing called ban - ei the horses race along a 200-m straight sand course that has two hills ( the first is 1-m in height and the second is 1.61.7-m in height ) while pulling a sled with 4601,000 kg of weight . recently , exercise physiological data during draft work in heavy draft horses used in ban - ei were investigated at the national livestock breeding center tokachi station [ 1 , 2 ] . in one of these studies , four percherons and 3 bretons were exercised by pulling weights of 350 , 450 , 550 , 650 , and 750 kg on a 300 m flat sand course . in thoroughbred racehorses , v200 ( running velocity at a hr of 200 beat / min ) is frequently measured and utilized as an index of fitness [ 7 , 8 , 36 , 37 ] . the draft horses were exercised for 300 m [ ( 100 m at a walk ) + ( 100 m at a trot ) + ( 100 m at a maximal run ) ] in order to calculate v200 . running velocity and hr during each exercise were recorded , and v200 was calculated for each weight . as shown in fig . 10.v200 ( running velocity at a heart rate of 200 beat / min ) and blood lactate concentration measured 3 min after exercise in heavy draft horses . the authors generated this graph using data in a report by arakawa .200 decreased as weight increased . although the draft workload was not measured and , therefore , workload as shown in nomura s experiment was not calculated , it seemed that reasonable data were obtained . in these experiments , the blood lactate concentration , which nomura did not measure , the results showed that blood lactate concentration 3 min after exercise increased as pulling weight increased , with the values for 650 and 750 kg being 14.8 and 15.5 mmol / l , respectively ( fig . these results suggest that the workload of a draft horse running 300 m while pulling a weight of 650750 kg is similar to that of a thoroughbred flat racing [ 3 , 26 , 48 ] . v200 ( running velocity at a heart rate of 200 beat / min ) and blood lactate concentration measured 3 min after exercise in heavy draft horses . in addition to the exercise physiological studies mentioned in this article , studies on the biomechanics of draft work were performed during the same period by okabe et al . of the university of tokyo . in these studies , changes in vertical movements of several parts of the body of the horse [ 39 , 41 ] and alterations of the joint angles [ 40 , 42 ] were analyzed using serial photography at 1/25 sec intervals photographed from a vehicle moving at the same speed as the horse , and the effects of different draft weights on stride frequency and stride length were also evaluated . studies on exercise physiology of other animals , e.g. , cattle and goats , were conducted during the same period . although research results obtained from these studies did not have practical significance as much as those on horses , the authors briefly summarize the results of several of those studies . uesaka of kyoto university , who recorded the first ecg of cattle in japan in 1943 , observed the effects of draft work on body temperature , respiration , pulse rate , blood variables , and ecg in cattle during 4,000 m of draft exercise with a draft workload of 25 or 50 kg [ 55 , 57 ] . in order to understand gas metabolism during draft work , habu et al . of kyoto university collected gas samples in cattle at rest and during exercise using a douglas bag and reported that resting vo2 was 0.149 l / kghr and rq was about 0.72 and that vo2 increased 46 times compared with at rest by 4,000 m when doing draft work with a draft workload of 6065 kg [ 5 , 6 ] . the steady states of many physiological variables were investigated using cattle for various pulling times ( 10 , 30 , 60 , and 120 min ) and a draft workload of 12% of their bws [ 44 , 45 ] . the effects of exercise on pulse rate in goats were also investigated [ 58 , 59 ] . the effects of draft exercise on physiological variables in goats [ 20 , 21 ] and cattle were investigated using a treadmill . in the first study on goats , changes in vo2 , carbon dioxide production , heat produced , respiratory quotient , pulse rate , oxygen pulse , respiration rate , pulmonary ventilation rate , tidal air , oxygen decrement , and oxygen debt were measured , and it was concluded that oxygen pulse per kg bw might be used as an index for the level of fatigue . the effects of air temperature , training , work , and metabolic disturbance caused by thyroidectomy in goats were also investigated . the effects of preceding work on resting oxygen pulse immediately before test work and the shape of the pulse rate curve at an early stage of the test work in cattle were also investigated . cattle were exercised on a horizontal treadmill while drafting a draft workload of 15% of their bws for 40 min ( a ) without or ( b ) with preceding work ( the same work as the test work ) . the results showed that the resting oxygen pulse decreased from 0.051 ml / kg ( a ) to 0.047 ml / kg ( b ) and that the pulse rate in the early stage of the test work was higher in b than a. from these findings , the authors suggested that the oxygen pulse per kg bw and the shape of the pulse rate curve at the early stage of the test work could be used as indices of fatigue and work capacity of cattle as well as goats . some of these findings were similar to those of warm - up studies in thoroughbred racehorses [ 27 , 28 ] . after world war ii , the number of horses raised in japan decreased dramatically , mainly due to the big shift in usage of the horse from the role of a warhorse to the role of a draft horse . additionally , the number of draft horses decreased thereafter due to the development and spread of farm machinery after the 1950s . in the present day , the total number of horses raised in japan is about 80,000 , and among them , only about 7,000 draft horses are raised , less than 10% of the total . in the time period when the studies described in this article were performed , about 400,000 horses were still raised in japan . although nomura of the university of tokyo pessimistically described in his report in 1964 that due to social conditions only 200,000 draft horses would be required for breeding in the future , the present situation is far beyond his expectation . in the present day , with there being only a few cases in which draft horses actually work in the field , the research results obtained from the series of studies mentioned in this article do not have realistic applicable meaning , although it is certainly useful to consider these studies in the context of them being valuable for understanding their place in the history of equine exercise physiology in japan . in conclusion , this article traced the rich history of exercise physiological studies of draft horses in japan . furthermore , it also briefly discussed studies conducted on cattle and goats as well as comparisons for some of the results from the draft horse studies . although draft horses are not as important in our modern society as they were in past years , nevertheless , they played an important role in japan s equine heritage and can be appreciated for their former importance to agriculture and society .

abstractalthough the total number of horses raised in japan dramatically decreased after world war ii , because draft horses were still used for farm work in paddy fields and on farms during the period of the 1950s and 1960s , a performance test for selecting better draft horses was needed . in order to determine the most suitable size of draft horses for japanese farm conditions , the working power of horses weighing from 185 to 622 kg was evaluated by performing an endurance test , several kinds of working power tests , and maximum pulling power tests . oxygen consumption during draft exercise was measured by the douglas bag method in order to evaluate effects of draft workload under the conditions of different types of work ( 14- and 18-cm plow depths , cultivator , and tillage ) , traction methods ( shoulder traction , shoulder - trunk traction , and chest - trunk traction ) , walking speeds ( 40 , 60 , 80 , 100 , and 120 m / min ) , and depths of water ( 0 , 18 , 36 , and 54 cm ) on energy expenditure . the relationship between energy consumption and pulse rate during exercise was also evaluated . a study of a performance test for draft horses was conducted to establish a new approach for evaluating draft horse performance using heart rate as an index . for this study , a beat meter for measuring heart rate was developed , and experimental protocols were used to evaluate the relationship between heart rate and workload . although the research results obtained from these studies do not have particular relevance in the current day , these studies are valuable for understanding the history of equine exercise physiology in japan .

Studies on the Relationship between Body Size and Work Power Studies on Energy Expenditure during Exercise Studies on Performance Tests Using Heart Rate as a New Index Studies on Biomechanics of Draft Horses and Exercise Physiology of Other Animals Conclusions

PMC3830152

abdominal haemorrhaging in newborns is a dramatic event that may be caused by several obstetrical and foetal factors including breech or complicated delivery , hepatosplenomegaly , macrosomy and anoxic liver congestion . birth injuries can be divided into two categories based on their aetiology : 1- injuries deriving from hypoxia and ischemia ; 2- injuries deriving from mechanical stress during labour and delivery . in this second group intra - abdominal injuries are rare and involve rupturing or haemorrhaging in the liver , spleen , or adrenal glands . three potential mechanisms lead to intra - abdominal injury : 1- direct trauma ; 2- compression of the chest against the surface of the spleen or liver ; 3- chest compression leading to tearing of the ligamentous insertions of the liver or spleen . clinical symptoms are correlated with the appearance of shock , the intensity of which is proportional to the severity of the trauma . especially in the first hour of life , the clinical signs of impending shock could be misunderstood or mistaken for asphyxia . a central hematocrit may be misleading immediately after acute blood loss and hypotension may also be a late sign . a 3400-gram neonate was delivered by vacuum extractor in a country hospital after a normal pregnancy in a young healthy woman . the baby cried and was put on the mothers abdomen however his clinical status deteriorated unexpectedly and required cardiopulmonary resuscitation ( cpr ) with intubation and external cardiac massage . an umbilical venous catheter ( uvc ) the neonatal transport team transferred the baby to our nicu with a presuntive diagnosis of perinatal asphyxia . at 1 and half hours of age , ph was 7.29 pco2 22.4 eb -14 , arterial haemoglobin 15.5 he was breathing spontaneously , with oxygen saturation levels 92 - 95% , and was administered dopamine . on admission blood gases showed normal respiratory exchange and a severe metabolic acidosis ( ph 7.08 po2 112 pco2 22 eb 21.6 ) . the chest x - ray [ figure 1 ] at the birth hospital showed a slim cardiac silhouette and a mal - positioned uvc . an echocardiogram showed a normal heart , however during the exam , ten minutes after arrival , there was the onset of bradycardia which failed to clear up . at the positioning of a new uvc , the umbilical stump bled profusely and the catheter tip entered too easily and too deeply . an x - ray was requested while the infant was administered three doses of colloide , dopamine , and dobutamine in increasing doses , and bicarbonate through the new catheter . the x - ray showed a white abdomen with the catheter tip , once again in an unusual position [ figure 2 ] . an anaesthesiologist and surgeon were immediately summoned , however the infant , in irreversible shock , died 50 after admission . anteroposterior roentgenogram of chest and abdomen obtained in the delivery room , 30 of age : bowel gas pattern suggests presence of peritoneal fluid . the cardiac silhouette is very slim anteroposterior roentgenogram of chest and abdomen obtained in the intensive care unit , 4 hours of age : bowel gas pattern suggests presence of peritoneal fluid . in the short clinical history of our patient there were several significant features : all our efforts to establish a positive cardiac function were ineffective ; shock was not amenable to fluid replacement therapy ; both the first and second x - rays showed a very slim cardiac silhouette , with a bowel gas pattern suggesting the presence of peritoneal fluid , and a configuration of both the uvcs that was inconsistent with the usual course of the umbilical vein ; the bleeding from the umbilical stump was too profuse . the drop in the haemoglobin level , hypotension and presence of free blood in the peritoneal cavity confirmed the occurrence of a major intra - abdominal haemorrhage . direct or indirect trauma of an abdominal organ during delivery represents the major pathogenetic factor . a central haematocrit , immediately after acute blood loss , may be misleading , so serial hematocrits should be obtained , and hypotension may also be a late sign . perforation of the peritoneum and intra - abdominal haemorrhaging has been described as a complication of umbilical vein catheterizations however in our case the uvc was inserted after deterioration of the clinical status . liver angioma is the most common congenital condition that could lead to abdominal bleeding during birth due to a different tensile strength of the tissue in comparison with the rest of the liver . in our case , disconnection of the splenic vascular pedicle causing hemoperitoneum has also been described as well as rupturing of the liver in term newborns of normal weight after uncomplicated delivery . in our case the infant presented immediate and severe shock , and was totally unresponsive to fluid replacement therapy . from the appearance of both the uvcs positions we believe that both the catheters in our patient were positioned in the peritoneal cavity , outside of the vascular bed . this hypothesis would explain why all our efforts to reinstall a positive blood pressure were ineffective as the infusions were running through the ucv . on reviewing the infants obstetric history , the delivery was complicated by the use of a vacuum extractor and the repeated use of the kristeller manoeuvre . we are aware that a lateral x - ray could have helped in defining the localizations of both the ucvs , moreover , an autopsy would have been decisive in demonstrating the interruption of the vascular pedicle , however we believe that there is a strong evidence of this aetio - pathogenesis in the clinical evolution of our patient . even without evidence of birth trauma , intra - abdominal interruption of the vascular tree of the hypochondriac organs in the case of shock and unresponsiveness to fluid replacement therapy through umbilical vessels must be kept in mind to plan the therapy and select a different infusions route .

we report a case of fatal intra - abdominal bleeding in a term newborn delivered by vacuum extractor and kristeller manouvre . although autopsy was not performed in compliance with the parents wishes , there is strong evidence of a massive abdominal haemorrhage due to injuries of the hypochondriac organs probably leading to disconnection of a vascular pedicle .

INTRODUCTION CASE REPORT DISCUSSION

PMC3521939

endoscopic submucosal dissection ( esd ) is currently accepted as standard treatment strategy for gastrointestinal neoplasms without evidence of lymph node metastasis , for esd allows one - piece resection even in the presence of large or ulcerous lesions.1 - 3 according to the guidelines of the japanese esophageal society , the indications for the diagnosis and treatment of esophageal squamous cell carcinoma are : 1 ) high - grade intraepithelial neoplasms , including noninvasive squamous cell carcinomas and 2 ) intramucosal invasive squamous cell carcinomas , limited to lamina propria mucosae ( m2 ) and without evidence of vessel infiltration.4 furthermore , the extended indications for endoscopic resection of early gastric cancer suggest that differentiated adenocarcinomas of 30 mm or larger in diameter are entirely free of nodal metastasis , when there is a lack of lymphatic - vascular capillary involvement or when the submucosal penetration is 500 m or less.2 more recently , hirasawa et al.5 reported that intramucosal undifferentiated adenocarcinomas of 20 mm or less in diameter , without lymphatic - vascular capillary involvement or ulcerous findings , present a negligible risk of lymph node metastasis . based on these findings , more clinicians are recognizing the importance of accurately determining the depth of invasion prior to treatment . consequently , increased attention has been given to making precise pre - treatment diagnosis to avoid unnecessary invasive procedures , such as surgical resection of mucosal cancers that are , in fact , eligible for endoscopic resection . unfortunately , however , the exact determination of invasion depth of superficial gastrointestinal neoplasms remains challenging , and additional surgical resections are still required in several cases . conventionally , the accurate assessment of invasion depth is achieved through a combination of several diagnostic modalities , for example using endoscopic ultrasound in adjunct to regular endoscopy . the recent developments in endoscopic devices , namely the advent of magnifying endoscopy with narrow - band imaging ( me - nbi ) , have enabled better visualization of fine mucosal and vascular structures under magnified view . in particular , a certain consensus has been reached regarding the effectiveness of me in determining invasion depth for intrapapillary capillary loop ( ipcl ) classification of esophageal cancer6 and pit - pattern classification of colon cancer.7 moreover , me - nbi has proven itself useful for qualitative diagnosis and for determining the range and tissue type of gastric cancer . in this review , we focused on the usefulness of me - nbi in measuring depth of invasion of superficial upper gastrointestinal cancer . the red - green - blue ( rgb ) sequential system and the color charge coupled device ( ccd ) system differ in their magnification abilities . while the rgb sequential system allows optical magnification of images up to 80 times , the color ccd system has digital zooming power of 1.2 to 1.5 times magnification . high definition endoscopes , even the ones using the color ccd system , have physical zoom properties , allowing the tip of the endoscopes to approach the mucosal surface as close as 2 mm without compromising resolution . such combination results in magnifying power of at least 50 times.8 microvasculature and microsurface structures are anatomical structures that are observed when examining the gastrointestinal tract with me . only the microvasculature can be observed when using conventional white light , but when using nbi , both the microvasculature and microsurface structures can be clearly observed . nbi is a novel endoscopic technique that may enhance the accuracy of diagnosis using narrow - band width filters in a rgb sequential illumination system.9 this technique produces different images at distinct levels of the mucosa and increases the contrast between the epithelial surface and the subjacent vascular pattern . nbi may provide the same contrast - enhancement abilities as chromoendoscopy without requiring the use of dye agents.10 the basic principle of nbi is that the depth of penetration into the mucosa depends on the wavelength being used : blue band for superficial layers , red band for deep layers , and green band for intermediate layers.11 because gastrointestinal cancer originates from the mucosa , the use of blue - colored , short - wavelength visible light that can only penetrate into the mucosa , may be helpful in the detection of minute early expressions . in addition , cancers are associated with autonomous angiogenesis - a characteristic responsible for cancer growth and metastasis - and hence , abundant blood vessels . therefore , the recognition of abnormal capillary beds may assist in cancer diagnosis.12 for short - wavelength visible light is mostly absorbed by hemoglobin in blood vessels , a black color is observed when blood vessels are illuminated with such light . thus , the subtle differences in mucosal lesions can be discriminated by distinctive colors , and the capillary bed of the mucosal surface can be inspected more clearly when illuminated with narrow - band blue ( 41515 nm ) and green ( 54015 nm ) wavelengths . the nbi system available in south korea ( evislucera spectrum system , cv-260sl ; olympus medical systems co. , ltd . , tokyo , japan ) has a filter that transmits only 41515 and 54015 nm wavelengths ; when the switch for nbi is activated while observing the upper gastrointestinal system under white light , this filter is inserted , and only a narrow - band around these two wavelengths is transmitted . after the lesions are illuminated , the monochromatic ccd absorbs the reflected light and generates images . because black and white images have a low resolution , the light absorbed by the monochromatic ccd is composited into rgb to generate color images . consequently , the main image colors consist of brown and green , and the lesions can be observed in higher resolution than with black and white images . nbi observation without magnification has the disadvantage of producing conditions that are sometimes too dark to identify morphological and color changes . the reason for dark images is simply because the nbi system uses only two narrow illumination lights , whereas conventional white light imaging uses a broadband visible light . for these reasons , it is important that users understand the technical backgrounds of these newer endoscopic procedures . me without nbi has been used for observation of gastric mucosa for many years.13 unfortunately , modification of gastric mucosa by acid and chronic inflammation caused by helicobacter pylori infection disturbs accurate evaluation of fine mucosal structures on magnified images , and for that reason the developments in this certain technique have been delayed . me - nbi is expected to give maximum performance in making accurate diagnosis , and enhance the contrast detailed morphological changes in the mucosal surface and clearly visualizing the microvascular structures . inoue et al.14,15 first reported the importance of morphological changes of the ipcl in making diagnosis of esophageal squamous cell carcinoma . ipcl is tennis racket - shaped dark - brown capillaries in the mucosa , derived from the side - branch blood vessels in the submucosal layer ( fig . me findings with regard to capillary pattern are classified into five types ( i - v ) according to the degree of change in the ipcl pattern , such as dilatation , tortuosity , caliber change in one ipcl , or various shapes in multiple ipcls.15 classifications include type i ( normal mucosa ) , type ii ( regenerating epithelium or inflammation ) , type iii ( low - grade dysplasia ) , type iv ( high - grade dysplasia ) , and type v ( cancer ) . . 2 , 3 ) is further divided into types v-1 ( m1 ) , v-2 ( m2 ) , v-3 ( m3 , sm1 ) , and vn ( more invasion than sm2 ) . they concluded that the endoscopic diagnosis of flat mucosal lesions was possible by classifying the ipcl pattern in the magnified images . they recommended periodic endoscopic surveillance for the ipcl type iii , while treating lesions of higher than ipcl type iv needs endoscopic mucosal resection / esd . however , it is difficult to assess the depth of invasion using me with a regular system . because nbi has capillary enhancing effects , the microscopic ipcl patterns are readily identified and evaluated by endoscopists with experience in me . based on their study of 263 esophageal cancers , arima et al.16,17 reported that the diagnostic accuracy of me in measuring depth of invasion was 98.8% for m1 and m2 cancers , 68% for m3 and sm1 cancers , and 84.0% for cancers invading into the middle third ( sm2 ) and the lower third ( sm3 ) of submucosal layer . they concluded that magnifying observation facilitated identification of lesion microinvasion , thereby increasing the proportion of correctly diagnosed m3 or sm1 cancers , in comparison with conventional endoscopic diagnosis . kumagai et al.18 also reported that the rate of accurate diagnosis of invasion depth was 83.3% in cases where detailed pictures were obtained using me . they mentioned about the possibility of distinguishing tumors that are shallower than m2 or deeper than m3 . yoshida et al.19 reported that me - nbi improved the accuracy of invasion depth assessments for superficial esophageal cancer . in summary , a number of reports recognizes the practical values me - nbi , and the usefulness of me - nbi in measuring the depth of invasion for esophageal squamous cell carcinoma have been corroborated in several studies . however , because analyzing the ipcl results requires considerable amount of time , energy , and experience , me - nbi can not always be readily employed . me has been shown to be effective in distinguishing benign from malignant tumors , establishing tissue types , and determining the extent of gastric cancer.20 - 23 but its usefulness in measuring gastric cancer invasion depth is yet to be verified . yao et al.20 reported that magnifying observation of microvascular architecture was useful for discriminating flat reddened carcinoma from gastritis . they proposed unique endoscopic findings for intestinal type gastric cancers : 1 ) presence of a demarcation line between the reddish lesion and the surrounding mucosa ; 2 ) disappearance of the regular subepithelial capillary network ; and 3 ) the presence of an irregular microvascular pattern within the flat lesions ( fig . 4).20 these characteristics based on mucosal and microvascular architecture are also reliable markers for differentiation between depressed gastric cancer and benign lesions . nakayoshi et al.21 classified the microvascular pattern of superficial gastric cancers into three groups based on their me - nbi images ( fig . 5 ) : 1 ) fine network pattern ; 2 ) corkscrew pattern ; and 3 ) unclassified pattern . fine network pattern and corkscrew pattern were useful in identifying differentiated adenocarcinoma and undifferentiated adenocarcinoma , respectively . me - nbi was also found to be useful in detecting the lateral extension of superficial gastric cancer.21 this quality is very important in detailed assessment of the safety margin and it enables improved targeting of neoplastic lesions during endoscopic treatment . yagi et al.24 assessed the relationships between microvessels , pits , and histological patterns using me - nbi . they found that the magnified view of the cancerous area showed three types of patterns ( fig . 6 ) : 1 ) a mesh pattern ( consisting of mesh - like connected microvessels ) ; 2 ) a loop pattern ( consisting of loop - like microvessels that were not connected and had tubule - like mucosal structures along them ) ; and 3 ) an interrupted pattern ( consisting of interrupted thick or thin vessel without mucosal structures ) . most ( 94.9% ) lesions showing a mesh or loop pattern were mucosal cancers , whereas 92.3% of lesions showing an interrupted pattern were submucosal differentiated adenocarcinoma . the authors clarified the characteristic features of the magnified view of mucosal differentiated gastric adenocarcinoma and the characteristics of invasive changes , including submucosal invasion.24 kobara et al.25 reported the usefulness of me - nbi in determining gastric cancer invasion depth . they defined the following three findings as diagnostic indicators of sm2 : non - structure , scattery vessels , and multi - caliber vessels ( fig . all three indicators were present more frequently in sm2 than in sm1 ( p<0.05 ) . all depressed gastric cancer with 2 points were sm2 , demonstrating a significant difference in score distribution between sm1 and sm2 ( p<0.05).25 several investigators have reported that sm cancer is associated with the appearance of non - structure areas in fine mucosal structure , irregularly - shaped , dilated , or extended microvessels and evidence of a hypovascular tumor.26,27 in summary , the studies on gastric cancer support the usefulness of me - mbi in cancer differentiation and cancer margin establishment . the studies on the usefulness of me - mbi in measuring invasion depth for gastric cancers have several limitations . for one , the absolute number of reports is too small to reach any kind of significance or consensus . also , only the depressed type cancers and differentiated type cancers have been studied so far . moreover , the lack of standardized parameter makes it difficult to accurately describe the depth of submucosal invasion . however , one can suspect submucosal invasion when there is appearance of non - structure areas , when hypovascularity is recognized and when irregularly shaped and dilated microvessel are present . nbi is becoming a promising endoscopic technology that enables better detection and accurate diagnosis of neoplastic lesions in the gastrointestinal tract . however , most studies conducted so far have been limited to a single center study , carried out by one or a few observers . overcoming the diagnostic discrepancies that occur among clinicians is a task that must be solved in the future . a large scale , prospective , randomized trial should be conducted to obtain more detailed information on me - nbi . the characteristic features of the microvascular pattern and microsurface structure of gastrointestinal neoplasms may allow endoscopists to diagnose gastrointestinal cancer more accurately . if differentiating mucosal cancer from submucosal cancer by me - nbi becomes applicable , this technique will contribute to making clinical decisions regarding the treatment options for gastrointestinal tract neoplasms , avoiding unnecessary operations and ultimately

with the widespread application of endoscopic submucosal dissection for the treatment of gastrointestinal neoplasms , accurate determination of the invasion depth prior to intervention has become an indispensable part of treatment planning . narrow - band imaging ( nbi ) is a novel endoscopic technique that may enhance the accuracy of diagnosis . magnifying endoscopy with nbi has been shown to be effective for determining invasion depth for intrapapillary capillary loop classification of esophageal cancer and microvascular pattern of stomach cancer . such precise pre - treatment staging of early neoplastic lesions in the gastrointestinal tract warrants timely initiation of disease - tailored treatment and , ultimately , better quality of life and improved patient survival .

INTRODUCTION MAGNIFYING ENDOSCOPY NBI ME-NBI ESOPHAGEAL CANCER GASTRIC CANCER CONCLUSIONS

PMC3843332

the diagnosis of extrahepatic biliary atresia ( ehba ) was always delayed and done by exclusion , peroperative cholangiogram , and histopathology . recent advances in imaging have led to increased antenatal prediction of ehba , particularly on high - resolution maternal ultrasonography . prenatal mr studies enhance fetal diagnosis , with many reports of antenatal diagnosis of choledochal cysts ( cdcs ) on ultrasound ( usg ) and magnetic resonance imaging ( mri ) studies published . amongst these , to the best of our knowledge , biliary atresia ( ba ) has not been reported . we report a case of ehba with terminal cystic dilatation of cbd predicted on antenatal and postnatal imaging with operative and histopathologic confirmation . a 25-year - old primigravida presented to our hospital at 22 week of gestation for target scan targetted imaging for fetal anomalies ( tiffa ) . anatomical survey revealed a longitudinally oval , avascular anechoic cyst measuring 14 9 mm in the right upper abdomen , separate from duodenum , showing continuity with hepatic hilum superiorly without identifiable gall bladder ( gb ) . fetal mri was performed 2 weeks later on 1.5 t essenza ( siemens , erlangen , germany ) to investigate the correct anatomical location of the cyst and its relationship with adjacent organs to assist with prognostication and treatment planning for the perinatal period . t2-weighted half - fourier acquisition single - shot turbo spin - echo ( haste , tr / te 900/86 ms , 4-mm slice thickness ) sequences were obtained in the axial , coronal , and sagittal planes with respect to the fetus . sequences were monitored in real time and manipulated to determine optimal imaging planes during the scan , as fetal orientation is unpredictable and often changes during acquisitions . in addition to the standard orthogonal planes , oblique planes were acquired for demonstrating the relationship to surrounding structures . mri showed a 14 9 mm ellipsoid [ figure 1a ] homogenous cystic lesion at liver hilum hyperintense on t2w images . the cyst was longitudinally oval , coursing posteroinferiorly from hepatic hilum appreciated on sagittal images [ figure 1c ] . coronal images revealed hyperintense cord - like structure above the cyst , showing communication with it [ figure 1d ] . the normal morphology of fetal gb was not visualized in the inferior surface of liver in right upper quadrant . ( a ) usg : cystic lesion ( 14 9 mm ) at porta hepatis ( arrow ) separate from duodenum without identifiable gall bladder . ( b - d ) mri : axial images reveal the cyst ( arrow ) posterior to the duodenum ( asterisk ) ( b ) . on sagittal images , the cyst is coursing posterior to the midabdomen ( c ) . on coronal images , hilar structures were replaced by hyperintense cord - like structure above the cyst ( arrow ) ( d ) the imaging characteristics on usg and mri are presented in table 1 . prenatal imaging features follow - up using serial usg scans in pregnancy revealed static size of the cyst with persistent ghost gb . postnatal usg , on day 3 after a 4-h fast , revealed liver extending beyond subcostal margin ; echotexture was homogenous with normal surface contour . there was an echogenic area anterior to the portal vein at porta and measured 3.2 mm [ figure 2a ] . a well - defined , longitudinally oval anechoic cyst was present at the hilum ~14 9 mm . the cyst size , echogenicity , and morphology were the same as seen in prenatal imaging [ figure 2b ] . maximum width and length of gb was 11 2 mm , with irregular , compressed lumen s / o ghost gb [ figure 2c ] . hepatic artery was prominent , running parallel to the right portal vein , and measured 1.7 mm . main portal vein ( mpv ) diameter was obtained at the same level , which measured 3 mm . the ratio of ha / mpv diameter was > 0.5 ( > 0.45 increase ) [ figure 2d ] . ( a ) echogenic area anterior to the portal vein at porta ( arrow):triangular cord sign . ( b ) a well - defined , longitudinally oval anechoic cyst present at the hilum ~14 9 mm . maximum length and width of gb was 11 2 mm with irregular compressed lumen ( arrow ) ( s / o ghost gb ) . ( d ) prominent hepatic artery ( arrow ) seen running parallel to the right portal vein and measured 1.7 mm . the ratio of ha / mpv diameter was > 0.5 ( > 0.45 increased ) the imaging characteristics at porta are presented in table 2 . postnatal usg features liver function tests ( lft ) revealed increased -glutamyl transferase ( 831 u / l ) , elevated alkaline phosphatase ( 698 u / l ) , conjugated hyperbilirubinemia ( 2.8 mg / dl ) , and normal serum glutamate pyruvate transaminase ( sgpt ) 10 u / l , serum glutamate oxaloacetate transaminase ( sgot ) 29 u / l , albumin , and international normalised ratio [ ( inr ) . hepatobiliary scintigraphy was performed on day 24 which showed prompt and good uptake by the liver and absent tracer excretion into the intestines in 24-h delayed images . hepatobiliary scintigraphy showed prompt and good uptake by the liver and absent tracer excretion into the intestines in 24-h delayed images . gallbladder and extrahepatic ducts were not visualized a diagnosis of extrahepatic cystic biliary atresia ( cba ) was made based on imaging . operative findings included firm brownish liver , fibrotic porta inclusive of non - visualized right / left hepatic duct , common hepatic duct , and proximal cbd [ figure 4a ] . corresponding schematic representation is shown in figure 4b and was classified as type 3 ba according to japanese classification system . ( a ) operative findings included firm brownish liver ( l ) , fibrotic porta inclusive of non - visualized right / left hepatic duct , common hepatic duct , and proximal cbd ( arrow ) . cystic structure was seen at the site of terminal part of cbd ( c ) , going behind the duodenum ( d ) , and ending blindly . ( b ) corresponding schematic representation showing japanese classification system with our case classified as type 3 atresia of entire ductal system ( arrow ) with terminal dilatation of cbd ( c ) , ending blindly and being engulfed by pancreatic tissue ( p ) excision of fibrotic hilar structures including cyst en masse with a kasai 's portoenterostomy was performed with ~30 cm of roux - en - y jejunal loop . cba is an uncommon variant of ba in which prognosis may be relatively favorable but liable to misdiagnosis as cdc . it is classified into three types according to the japanese classification system based on the atretic segment of bile duct into type 1 ( distal atresia ) , type 2 ( proximal atresia ) , and type 3 ( complete atresia ) . the cyst can occur anywhere along the atretic segment and is proximal to atresia in types 1 and 2 and distal to it in type 3 . a thorough literature search revealed less than 100 cases of ehba associated with cdcs reported till date of which majority were type 1-ehba ( 76% ) and only 21% were type 3-ehba as in our case , classified according to japanese pathological classification system . the mainstay of confirmatory diagnosis in most of the published reports was surgical exploration and intraoperative cholangiogram . zhou et al . have demonstrated the accuracy of usg diagnosis to be 98 - 100% and evaluated objective differential characteristics of cba and cdcs . the collaborative signs include gb signs , hilar signs , hepatic artery signs , and miscellaneous signs ( increased hepatic subcapsular flow ) on usg . ghost gb is an indirect sign of unfilled atretic gb in ehba described by zhou et al . as gb length < 19 mm , lack of a smooth mucosal lining with an indistinct wall , and irregular or lobular contour . hilar cyst in ba represents fibrotic remnant of the cbd , explaining the constant size and echogenicity . prominent hepatic artery has been a well - known association with ba , making it a possible collaborative imaging sign . the present case showed triangular cord and all the collaborative signs described on postnatal usg as presented in table 2 . , fetal gb presents as a right - sided pear - shaped structure on the inferior surface of liver and is consistently demonstrated from 18 weeks of gestational age onward . before 27 weeks , the signal intensity of fetal bile is exclusively hyperintense on t2w , corresponding to t1w hypointensity . non - visualization of fetal gb with cyst at porta is diagnostic of cba on prenatal imaging . in our case , the normal shape of gb was not visualized in its expected location , thus confirming ghost thus , prenatal mri can improve the pick - up rate of ehba and also differentiate between cba and cdc . to the best of our knowledge , this is the first report of prenatal mr predicted cba . non - visualization of gb and cyst specific to biliary tract on prenatal mri are suggestive of ba . bhatnagar et al . have reported cdc with ba , but the present case differs in that the ba existed both proximally and distally . abnormal pancreas was evident in this case peroperatively where the terminal cystic structure was engulfed by pancreas . kasai 's portoenterostomy has been the universally accepted initial surgical modality to establish biliary drainage . the present case also underwent kasai 's portoenterostomy after the excision of fibrotic extrahepatic biliary ducts . early surgery , as evident in this case that was operated < 8 weeks of life , has yielded satisfactory initial biliary excretion leading to normal serum bilirubin levels . imaging helps in the prediction of ehba and also helps in early postnatal surgical referral , improving the results of kasai 's portoenterostomy .

extrahepatic biliary atresia ( ehba ) is an uncommon cause of neonatal jaundice . antenatal magnetic resonance imaging ( mri ) diagnosis of ehba has not been published to the best of our knowledge till date . ehba with cystic component is likely to be mistaken for choledochal cyst . a case that was antenatally predicted and postnatally confirmed by surgery and histopathology is being reported . all imaging signs are analyzed herewith . imaging helps in the prediction of ehba and also helps in early postnatal surgical referral which in turn improves the results of kasai 's portoenterostomy .

Introduction Case Report Discussion

PMC4058168

despite the promising fact that the consequences of cardiovascular diseases ( cvd ) have decreased considerably during the past two decades due to considering healthier lifestyles and using more efficient treatment regimens , these diseases are still accounting for at least 30% of global deaths , as can be , for example , found in gebel . in addition , these diseases are still the major cause of deaths in developed countries , as reported by lloyd - jones et al . and roger et al . consequently , the costs associated with the diagnosis and treatment of cvd are already heavy and are still increasing every day . therefore , the cvd risk stratification algorithms have recently gained increasing attention [ 4 , 5 ] . in addition , it can be noticed that the majority of serious cardiovascular events occur in subjects or cases at low or intermediate risk [ 6 , 7 ] . therefore , there is an urgent need for more efficient screening tools to identify such vulnerable subjects or cases as early as possible to be able to benefit from considering drug treatment and changes towards healthier lifestyles ( instead of considering surgical operations and other invasive treatment approaches ) . for example , a recent study showed that more than 50% of a group of randomly selected middle - aged individuals ( that were apparently healthy ) were actually suffering from subclinical atherosclerosis in the coronary or carotid arteries ( without knowing about it ) . therefore , screening of the general population , especially middle - aged and elderly subjects , for cardiovascular diseases is proven to be controversial . the degradation or lack of elasticity in arterial vessel walls ( i.e. , their stiffness grade or severity ) affects the blood pressure and flow and is directly or indirectly related to other cardiovascular consequences and serious events . myocardial infarction , left ventricular hypertrophy , and other serious cardiovascular diseases and deficiencies ( as well as other related serious events ) can be caused by arterial wall stiffness ( i.e. , abnormal or lack of elasticity ) . for example , coronary heart disease and its consequences of increased arterial vessel wall stiffness can result in increased systolic blood pressure , increased pulse pressure , and increased mechanical load on the heart [ 1012 ] . another factor which is usually associated with the degradation of elasticity and consequently with increased arterial stiffness is ageing [ 1315 ] . arterial stiffness can be noninvasively evaluated by applanation tonometry which is a validated , reproducible , user - friendly , and low - cost method for this purpose [ 16 , 17 ] . in addition , measuring the pulse wave velocity ( pwv ) at the carotid and femoral arteries is considered as the gold standard measure of arterial stiffness . however , de feyter found a disadvantage in these approaches as they are mainly based on cross - sectional studies , and a more efficient approach , according to de feyter , should also include longitudinal studies . it has been shown in many research works ( e.g. , three examples can be found in [ 1921 ] ) that it is possible to identify the presence of coronary atherosclerosis and predict the risk of adverse coronary events , by using noninvasive imaging modalities for the evaluation of atherosclerotic disease in noncoronary peripheral vessels such as the carotid and the femoral arteries [ 2224 ] . recent research results indicate that degraded arterial vessel wall elasticity usually appears early , even before the appearance of any clinical symptoms or atherosclerotic plaques [ 2527 ] . therefore , most noninvasive approaches , which were proposed for the diagnosis of cardiovascular diseases , were based on evaluating and quantifying the elasticity or stiffness of arterial vessel walls . however , the methods that were proposed for early and accurate diagnosis of cardiovascular diseases had over the years relied mainly upon invasive procedures . in 1990 , parker and jones introduced such a method based on the concept of wave intensity ( wi ) analysis . the wave intensity ( wi ) signal was defined as the product of dp du , where dp and du represent the change in pressure and flow , respectively , inside a given blood vessel . both parameters of the wi signal were measured invasively inside the arterial vessel , and this requirement limited the clinical use of the wi - analysis method . a recently introduced technique by larsson et al . , known as wave intensity wall analysis ( wiwa ) , can be considered as an important development of the wi - analysis concept to make it noninvasive . the changes in pressure ( dp / dt ) and flow ( du / dt ) were estimated noninvasively by approximating the strain rate of the arterial wall in the radial and the longitudinal directions , respectively , from ultrasound image sequences . however , all wi - analysis approaches and variants have several common disadvantages . at first , the basic concept of traditional wi - analysis is based on the simultaneous measurement of the change in blood pressure and the change in blood flow inside the carotid artery , which is a nontrivial task ( parker 2009 ) . in addition , since the wi - analysis approach is defined as the product of two quantities that are highly contaminated with noise , the obtained results are doubly sensitive to noise ( parker ) . the recent wiwa method which estimates these quantities from ultrasound image sequences suffers from inaccuracy due to the difficulty in estimating the longitudinal strain in general and also the difficulty in manually finding the right or most suitable region of the arterial vessel to perform the measurements and get usable and reliable results ( larsson et al . ) . in addition , a serious issue with the wiwa concept is that the measured strain rates can not be claimed to really or exactly correspond to changes in blood pressure and flow . in any case , wi - analysis remains limited in the sense that it can not provide any insight into the ( physical and physiological ) conditions of arterial vessel walls in a local manner , and it is not concerned with visualizing the true wave progression along the vessel walls . therefore , the current study presented in this work suggests simplifying the analysis and limiting it to evaluating and quantifying the elasticity of the walls of the carotid artery by measuring its radial distension locally in small regions along it . many existing methods perform local radial - motion measurements of arterial stiffness by estimating the corresponding distensibility , compliance , elastic modulus , or stiffness index . however , none of these methods is free from limitations , and the validity and reproducibility of the performed measurements show large variation and nonrobustness [ 32 , 33 ] . recently , azar and muhammed used a self - organizing neural network to automatically track the motion of the wall of the carotid artery in ultrasound image sequences . however , a serious disadvantage of that approach was the heavy computational load that was required by the used self - organizing neural network . in addition to that , the goal of this approach was to achieve an algorithm which results in a more efficient and practical wiwa approach . it aimed at automating the task of producing wiwa signatures of good quality , which could help achieve a more accurate and objective diagnosis . therefore , the aim of this work is to develop a method that can overcome the limitations of the existing ones when measuring deviations from normality . a previous attempt to achieve this goal was performed by hamid muhammed and azar , where the best location on the carotid artery wall was automatically identified to be able to obtain a radial distension signal of accepted quality . fourier analysis of the radial distension signals obtained for healthy and pathological cases could help differentiate between healthy and pathological cases . the achieved classification results were promising , showing significant differences between healthy young , healthy elderly , and pathological elderly cases . however , the dataset used for that study was limited and a larger dataset should be used for a more realistic and fair evaluation of that method . therefore , the current work utilizes an extended dataset of that used in to consider a population with higher variation of all three classes or categories of cases . the current paper describes a novel method for visualizing and characterizing the wave propagation along the whole vessel wall in an automated fashion . this approach can reveal and shed light on the wavy motion 's mechanical properties along the carotid artery wall and aid in the accurate diagnosis of the cardiovascular system . the contributions of this paper are as follows.an automated method for efficient and easy - to - understand visualization of wave propagation along arterial vessel walls is proposed . the approach captures both spatial and temporal variations in the radial direction of the vessel walls and reveals the characteristics of these wavy patterns.the proposed method is shown to be able to aid in differentiating between healthy and pathological conditions , by visual inspection , in terms of the shape and the 2d pattern of the generated spatiotemporal map of the wave progression in each case . such visual characteristics and differences between healthy and pathological cases can aid medical specialists in diagnosing the condition of the carotid artery and the cardiovascular system.the fourier transform is applied to the spatiotemporal 2d map to generate the corresponding spatiospectral 2d map . visual inspection of the new map can reveal the spectral characteristics , which are related to the physical and mechanical properties , and consequently the physiological condition along the corresponding arterial vessel wall.a novel approach is proposed to analyze the spatiospectral 2d maps in an automated manner to differentiate between healthy and pathological conditions of the arterial vessel walls.through extensive experiments and testing , the proposed noninvasive method is shown to be a robust , objective , and automated tool that can aid in achieving a reliable and accurate diagnosis of the physiological condition of the arterial vessel walls . an automated method for efficient and easy - to - understand visualization of wave propagation along arterial vessel walls is proposed . the approach captures both spatial and temporal variations in the radial direction of the vessel walls and reveals the characteristics of these wavy patterns . the proposed method is shown to be able to aid in differentiating between healthy and pathological conditions , by visual inspection , in terms of the shape and the 2d pattern of the generated spatiotemporal map of the wave progression in each case . such visual characteristics and differences between healthy and pathological cases can aid medical specialists in diagnosing the condition of the carotid artery and the cardiovascular system . the fourier transform is applied to the spatiotemporal 2d map to generate the corresponding spatiospectral 2d map . visual inspection of the new map can reveal the spectral characteristics , which are related to the physical and mechanical properties , and consequently the physiological condition along the corresponding arterial vessel wall . a novel approach is proposed to analyze the spatiospectral 2d maps in an automated manner to differentiate between healthy and pathological conditions of the arterial vessel walls . through extensive experiments and testing , the proposed noninvasive method is shown to be a robust , objective , and automated tool that can aid in achieving a reliable and accurate diagnosis of the physiological condition of the arterial vessel walls . the results of analyzing the spatiotemporal and spatiospectral 2d maps are compared with the corresponding results obtained using the method proposed and implemented in . finally , the results of the two new approaches proposed in this work can be combined to achieve better results . in this study , three datasets of ultrasound image sequences of the carotid artery were used . each dataset consisted of healthy young cases , healthy elderly cases , and cases suffering from coronary artery disease ( cad ) . significant stenosis was diagnosed when there was a reduction of minimal lumen diameter by more than 50% as compared with the proximal reference . the first dataset consisted of nine healthy young cases ( 3145 years old ) and eight healthy elderly cases ( 6270 years old ) where two of them were suffering from cad . it was provided by the school of technology and health ( sth ) , royal institute of technology ( kth ) , in collaboration with karolinska institutet ( ki ) , stockholm , sweden . the second dataset consisted of 14 healthy elderly cases ( 5669 years old , males and females ) in addition to 20 pathological elderly cases ( 6173 years old , males and females ) suffering from cad . it was provided by the division of cardiology , rafik hariri university hospital , beirut , lebanon . the third dataset was the largest and consisted of 21 healthy young cases ( 3445 years old , males and females ) , 27 healthy elderly cases ( 4967 years old , males and females ) , and 47 pathological elderly cases ( 4877 years old , males and females ) suffering from cad . this dataset was provided by ibn al - bitar hospital for cardiac surgery , ibn al - nafis hospital for cardiovascular diseases , and the iraqi center for heart diseases , baghdad , iraq . in these experimental studies , these experiments were approved by the corresponding local ethics committees in sweden , lebanon , and iraq . extracting the vessel wall in an ultrasound image sequence poses a considerable challenge mainly due to the low signal - to - noise ratio ( snr ) , unclear boundaries , and varying shape , location , and pixels ' intensities across different frames . therefore , the segmentation process should take advantage of both spatial and temporal information in the image sequence in order to segment the carotid artery wall in the images ( i.e. , the frames ) accurately . figure 1 shows a typical image of a case with carotid artery stenosis ( cas ) , which occurs when some parts of this artery 's walls become thicker and consequently get narrower ( as marked by the ellipse in figure 1 ) . at first , each frame , which is a single still image , was clustered using a number ( n ) of certain spatial / textural features , where every pixel became an object or an element in an n - dimensional feature space . n was set to three in our case since the features chosen were the following : the mean , the standard deviation , and the entropy of a square neighborhood around each pixel . based on these features , the k - means clustering algorithm was implemented to segment each frame into three classes , as shown in figure 2 . in figure 2 , it is not easy to differentiate vessel wall boundaries from other boundaries , since the obtained sets of three features of these regions are very similar when considering only one frame . even a human operator may not be able to distinguish or reveal any characteristic differences by processing only one such stationary image . however , when the images are set in a time sequence , the motion allows us to identify the vessel walls , since it is mainly the vessel walls that move while other boundaries and details remain relatively static ( i.e. , not moving ) . therefore , to be able to achieve useful or acceptable results , both spatial and temporal information need to be integrated into the segmentation process . the additional feature , which was utilized for this purpose , was the standard deviation of the pixel values across the frames in the image sequence . at the end , this process ensures that the most prominent one , of the two vessel walls which appear in each frame , will be selected , that is , the one that is the most dynamic and moving more actively across all frames ( by virtue of the standard deviation feature ) and the most visible ( by virtue of the k - means algorithm ) . figure 3 shows a segmented carotid artery wall using all four features mentioned previously ( three spatial statistical features and one temporal statistical feature ) . the inspiration behind the idea of visualizing the wave propagation stems from the need for automated best - region search and selection so as to improve upon manual region selection in larsson et al . as well as azar and hamid muhammed in which the user is given the choice of cropping a region of interest ( roi ) in the first frame , as shown in figure 4 . automated wave tracking is based on the systematic assignment and evaluation of different regions along the vessel wall , as described by hamid muhammed and azar . the method proceeds by , at first , effectively isolating the internal section of the vessel wall that is in contact with blood flow . then , a series of regions of interest ( rois ) are assigned along the longitudinal direction of the vessel wall in a manner such that these rois overlap midway as shown in figure 5 . the basic concept is to obtain , from each region of interest ( roi ) which is extracted as described in the previous section in figure 5 , a time dependent signal per roi showing the radial variation at the corresponding roi . however , in , this type of radial - variation signal was computed for only one roi which is expected to give a usable signal of sufficient quality . if the vessel wall truly moves in a wavy pattern ( as its inner layer is being affected and attracted by the blood flow ) , then it should be possible to track that wave as it moves along the vessel wall , that is , in the longitudinal direction . the systematic assignment of midway overlapping rois at different locations undertaken previously may be utilized exactly for this purpose . the idea is to simply show , in a three - dimensional ( 3d ) plot , the variation in the radial direction ( radial distension ) as a function of both time ( frame number ) and space along the vessel wall ( longitudinal location ) . thus , the separate signals , each showing the variation of the radial thickness ( e.g. , the diameter ) of the vessel as a function of time at a given roi , are stacked beside each other ( or concatenated behind each other ) and presented in a 3d plot as a function of time and longitudinal position . perhaps the only observation that can be noticed , in this 3d plot , is the rapid radial variation across the frames . the idea of the new approach is to show each 3d plot as a two - dimensional ( 2d ) image or map using pseudo colors for the third dimension representing the radial vessel thickness at different locations ( or positions ) and time points . the echocardiography ( ecg ) signal is also utilized to be able to more exactly know when certain wavy patterns appear in the 2d map . figure 7(b ) shows such a 2d map ( in grey scale ) corresponding to the same healthy young case presented in figure 6 . figure 7 gives evidence of how certain regions ( rois ) of the vessel wall move longitudinally in a wavy - like pattern across time . comparing the 2d map in figure 7(b ) with the corresponding ecg signal in figure 7(a ) can help in characterizing this wavy pattern . it is , for example , easy to recognize three pairs of darker blobs on the left side of the image in figure 7(b ) . each such pair of darker blobs ( followed by one light grey blob ) is located within one ecg cycle ( i.e. , one heartbeat ) as it can be noticed from figure 7(a ) . the brighter boundaries of these blobs , which are almost connected , show the longitudinal motion of the carotid artery wall as a function of time . the black arrows in figure 7(c ) show how these boundaries and the corresponding longitudinal motion propagate with time in a zigzag manner , which is a result of the vessel wall boundary moving several times in the longitudinal direction to the right then backward to the left . comparison of this zigzag pattern with the corresponding ecg signal in figure 7(a ) can provide detailed information concerning the progression of the wave and the condition along the vessel wall locally at each roi . it can , for instance , be observed that , within each ecg cycle , the vessel wall performs a longitudinal right - left motion three times . this motion pattern is repeated in almost an identical fashion during all three ecg cycles , as shown in figure 7(c ) . approximately , the same characteristic pattern appears in the spatiotemporal 2d maps of all healthy young cases examined in this study . this pattern is an indication that the vessel wall is nonrigid and flexible enough to exhibit a longitudinal right - left motion three times before completely relaxing to its initial position . therefore , this characteristic pattern can be found in all healthy young cases , while it seems to be absent in all elderly and pathological ( cad ) cases that were included and examined in this study . figure 8 shows a 3d plot for a pathological case ( coronary case 1 , cad1 ) . in this case too , as in the healthy young case presented in figure 6 , it seems that it is not easy to interpret this 3d plot , and the only observation that can be noticed is a rapid radial variation with time . therefore , the corresponding 2d map is generated , as shown in figure 9(b ) , to be used to provide a more easily interpreted spatiotemporal visualization approach . the corresponding ecg signal is also plotted in figure 9(a ) to be utilized to achieve a more accurate interpretation and to consequently obtain a more correct assessment or diagnosis of the physiological condition of the carotid artery . since the plotted ecg signal shows four ecg cycles , it should be expected to recognize four repeated ( almost identical ) patterns in the corresponding 2d map to indicate that the current case is healthy . but , since that is not the case ( as explained in figure 9(c ) ) , it is then possible to draw the conclusion that the case presented in figure 9 does not seem to correspond to a healthy subject . the black arrows in figure 9(c ) indicate approximately how the longitudinal motion occurs . in the current case , the vessel wall performs a right - left motion twice within each ecg cycle . this motion seems to be irregular when comparing the obtained motion patterns within all four consecutive ecg cycles . furthermore , during each ecg cycle , this double right - left pattern of coronary case cad1 is much slower than the triple one obtained for the healthy young case y1 . figures 10 and 11 present another pathological case , coronary case 2 ( cad2 ) . in this case , it can be noticed that the 3d curves in figure 10 are changing much slower with time when compared with the two cases presented previously . the ecg signal presented in figure 11(a ) shows three ecg cycles , and the corresponding image in figure 11(b ) contains two brighter regions on the right side of the image . these two regions , each of which is located within one ecg cycle ( the 2nd and the 3rd cycles ) , represent considerably less prominent patterns ( faded patterns ) than those observed in the previous cases . especially within the 1st ecg cycle , the corresponding pattern is much more faded than the other two . these observations indicate that the motion of the vessel wall in this case is irregular and much slower than in previous cases . here , it can be noted that the 3d curves in figure 12 are changing even much slower with time compared to those of the coronary case cad2 , presented in figure 10 . furthermore , the pattern presented in figure 13(b ) is much more faded than the pattern of coronary case ( 2 ) presented in figure 11(b ) . however , an attempt to mark the pattern ( manually with white arrows ) is presented in figure 13(c ) . this figure shows that the vessel wall seems to perform a right - left motion twice within each ecg cycle . this motion is somewhat regular , but the variation in the corresponding 2d pattern is much slower , much weaker , and clearly different from the triple motion pattern of the healthy young case y1 . this task is based on the transformation of the spatiotemporal wave representation into the frequency domain . the basic idea and purpose of this task is to extract and evaluate some features or parameters that can serve as measures for the characteristics and properties of the mechanical wave signals propagated along the blood vessel wall . this wavy signal or pattern is generated due to changes in blood flow and pressure inside the vessel while pumping blood by the heart . an efficient way to understand and analyze a signal is by looking at the magnitude of its fourier spectrum , because it can show if this signal is periodic or irregular , if it is weak or strong ( depicting small or large variations ) , and if it is changing quickly or slowly ( i.e. , the frequency components obtained ) . another important property of the fourier spectrum is that it is phase - shift invariant , which makes aligning and comparing the properties of a set of signals an easy task . figure 14 presents four spatiospectral 2d maps which correspond to the four cases discussed previously in this section as examples representing the three categories considered in this study . each of these maps is generated by applying the fourier transform to each column in the corresponding spatiotemporal 2d map . as a result , each radial - variation time - signal of an roi is replaced by the magnitude of its fourier spectrum . as an example , figure 5 shows the chosen rois along the wall of the carotid artery for one case . what can be easily observed from this kind of spatiospectral 2d map is whether all rois ' local radial - motion signals have the same spectral properties or not . in case all rois along a vessel wall have similar or almost similar fourier spectra , this indicates homogeneous mechanical properties and physiological quality along the vessel wall , which in turn can indicate a healthy status . in figure 14(a ) , which shows the spatiospectral 2d map of the healthy young case y1 , it can be observed that all rois have almost the same frequency bandwidth and spectral pattern , indicating homogeneous spectral characteristics and mechanical properties along the vessel wall . however , that is not the case in figures 14(b ) , 14(c ) , and 14(d ) , which show the spatiospectral 2d maps of the healthy elderly case ( e1 ) , the pathological elderly case cad1 ( coronary case 1 ) , and the pathological elderly case cad2 ( coronary case 2 ) , respectively . in all these three cases , it is easy to observe nonhomogeneous spectral properties along the vessel walls . the frequency bandwidth is much narrower at a majority of the rois in the healthy elderly case e1 ( i.e. , the motion of these rois is slower and smoother ) , while several rois have much wider frequency bandwidths than the healthy young case y1 ( i.e. , the motion of these rois is faster and not smooth ) . in the coronary cases , cad1 and cad2 , the frequency bandwidth is much wider than the healthy young case y1 at all rois . several rois cover almost the whole frequency bandwidth of the fourier spectra , as in the case of roi number 6 in cad1 ( figure 14(c ) ) and rois numbers 14 and 15 in cad2 ( figure 14(d ) ) . however , it is possible to automatically evaluate the homogeneity of the spectral characteristics and properties of the spatiospectral 2d maps instead of using visual inspection . by this way two straightforward statistical measures can be used for this purpose : a type of mean - value measure in addition to a variance measure across all rois ( along one blood vessel wall ) considered and used for the assessment task . since the spectrum of the wavy motion at each roi is presented as a column in the corresponding spatiospectral 2d map , the average of all columns ( i.e. , average of spectra for all rois ) of the map is calculated and analyzed . in figure 15 , the plots in the left column ( a1 , b1 , c1 , and d1 ) show four spectral curves , each of which is the average of the columns of the spatiospectral 2d map of one of the four cases presented in figure 14 . the normalized value of the sum of the areas under each resulting spectral curve ( i.e. , for each case ) is also calculated and presented . but , inspecting these values shows mainly the difference between young and elderly cases . in other words , these results show that using only the sums under these curves is not enough to differentiate between the three categories considered in the current study . therefore , more advanced analysis methods should be used to achieve this purpose . an efficient approach to reveal the characteristics of the shape of a curve is through extracting its spectral properties by utilizing the fourier transform ( ft ) and computing its spectrum . some results of applying this approach are presented in the right column of figure 15 where four plots are shown ( a2 , b2 , c2 , and d2 ) , denoted as ft curves , because these results are obtained by applying ft twice on the data ( i.e. , ft means computing the ft of the average of fts ) . each one of these four plots ( shown in figures 15(a2 ) , 15(b2 ) , 15(c2 ) , and 15(d2 ) ) presents the result of applying ft to the corresponding spectral curve in the left column of the same figure ( figure 15 ) . normalized values of the sums of the areas under each resulting ft curve are also presented in each subfigure , showing noticeable differences between the three categories of cases : the healthy young case y1 , the healthy elderly case e1 , and the two coronary cases , cad1 and cad2 . for the variance measure , the standard deviation of all columns of the 2d map ( i.e. , the standard deviation of the spectra for all rois ) is calculated and analyzed . therefore , each value of the resulting vector is the standard deviation of one row in the 2d map . in order to simplify the analysis to measure the overall form of a standard deviation curve , it can be normalized and its envelope curve can be computed . furthermore , higher frequencies can be given more importance than the lower ones when evaluating the nonhomogeneity of the spectral properties along the blood vessel wall . as a classification feature , ramp - weighted normalized sums of the areas under the envelope curve of the standard deviation are computed for all cases . figure 16 shows four plots that correspond to the four cases presented in figure 14 . the values of this new feature are obtained and presented in figure 16 , showing that this feature can be used as an efficient classification parameter to differentiate between the three categories considered in the current study . in this section , the methods proposed , discussed , and tested in the previous sections are utilized to classify the three types of cases or categories that are available for the current study . totally , 30 healthy young cases , 47 healthy elderly cases , and 69 pathological elderly cases ( suffering from coronary artery disease ) were analyzed and classified using these methods . figures 17 and 18 summarize the performance of using the normalized sums of the areas under the corresponding ft curve and the standard deviation curve 's envelope for each case as a classification feature or parameter to differentiate between these three categories of cases . in both figures , it is much easier to differentiate the healthy young cases from the elderly ones than differentiating the pathological elderly cases from the healthy elderly ones . therefore , the following analysis will focus on differentiating between pathological and healthy elderly cases . in figure 17 , the feature values are between 21.5 and 32.1 for the pathological elderly cases and between 22.7 and 45 for the healthy elderly cases . when considering the lower horizontal separation line ( at a feature value of 27.3 ) in figure 17 as a threshold to identify pathological elderly cases among all elderly cases in the current study , a sensitivity of 78.3% and a specificity of 74.5% can be obtained . on the other hand , for the 30 healthy young cases , this shows that the healthy young cases can be easily separated from the pathological elderly cases with 100% accuracy . furthermore , the overlap extent , between the two sets of feature values for the healthy young cases and the healthy elderly cases , is much less than the corresponding overlap between healthy and pathological elderly cases . in the same way , in figure 18 , the feature values for the pathological elderly cases and for the healthy elderly cases are between 0.22 and 1.19 and between 1.24 and 0.4 , respectively . when considering the higher horizontal separation line ( at a feature value of 0.1 ) in figure 18 as a threshold to identify pathological elderly cases among all elderly cases , furthermore , the feature values are below 0.5 for all healthy young cases , showing that these cases can be separated with 100% accuracy from the pathological elderly cases . in addition , the overlap between the two sets of feature values of the young and the elderly healthy cases is much less than the overlap between the healthy and the pathological elderly cases . however , to achieve better results , it is possible to combine the results in both figures 17 and 18 so that it is enough for a case to be true positive or true negative in one of these two figures . in other words , by considering the resulting feature values and the chosen classification thresholds presented in these figures , a sensitivity of 92.8% and a specificity of 76.6% can be obtained . the pairwise student 's t - test and the pairwise anova test were performed , at each time , using two sets of 47 pairs of values ( for pathological elderly cases and healthy elderly cases ) for each of the two features discussed and used previously in this work . that is , pairs of the feature values of all 47 healthy elderly cases and a randomly chosen set of 47 feature values of pathological elderly cases were used in these tests . these two statistical analysis tests resulted in p values much less than 0.0001 indicating a significant difference between each of such two sets of 47 feature values . in addition , for comparison , the method proposed in was used to differentiate between the three classes of cases in all datasets available for this work . the results of using the two spectral area measures , proposed in , for normalized frequencies 0 < f 1 ( green curve ) and 0 < f 0.15 ( blue curve ) are presented in figure 19 . the thresholds chosen and used for classification using these two spectral area measures are shown as two green horizontal lines for the approach considering 0 < f 1 as two blue horizontal lines for 0 < f 0.15 . the corresponding sensitivity and specificity measures for this method were 78.3% and 74.5% , respectively , for 0 < f 1 , while these classification quality measures were 85.5% and 78.7% , respectively , for 0 < f 0.15 . what these results reveal is that the method proposed in is optimized for the smaller dataset used in that study . therefore , the results obtained in that study were optimal achieving 100% accuracy , compared to 82.8% when applying the approach considering the normalized frequencies 0 < f 0.15 in the current work . two approaches for visualizing the wave patterns temporally and spatially , as well as spectrally and spatially along the walls of the carotid artery , were presented . a systematic procedure was utilized to divide the vessel wall longitudinally into small regions of the same size and overlapping midway . each of these regions was evaluated in terms of radial variation ( radial distension ) . two means of visualizing these patterns were presented , namely , a 2d spatiotemporal wave - representation map and a 2d spatiospectral wave - representation map . an automated procedure was developed to provide a new method of characterizing the wavy motion of the carotid artery wall . the method involved capturing the progression of the wave patterns that appear both temporally and spatially along the vessel wall by measuring radial distension , at each time point ( i.e. , in each frame of the ultrasonic video ) , within each one of the overlapping small regions . by this way , a 2d spatiotemporal map , using pseudo colors , was generated for the case at hand , which belongs to one of the three categories of cases considered in this study . the spectral characteristics of these wavy patterns , which are related to the physiological properties along the carotid artery wall , were obtained by using the fourier transform to compute the spectrum of the radial - variation signal of each small region . the resulting 2d maps of these two automated visualization methods can be used to aid in the noninvasive diagnosis and detection of pathological conditions in the cardiovascular system in two ways . firstly , for each subject , visual inspection of the corresponding two 2d maps can help the cardiologist understand the physiological condition of the examined arterial wall . in addition , it is also possible to utilize an automated method to compute a number of useful features that can be used to evaluate and classify the physiological condition of the arterial wall . the findings obtained in this study show that there are inherent differences in the way a wave progresses along an arterial vessel wall between healthy and pathological cases . it is evident that , in pathological elderly cases , the wave does not propagate as it would be normally expected as in healthy cases ; for example , it is often skewed and/or damped due to plaques , impediments , and/or stiffness of the artery . the differences that can be observed in the resulting 2d patterns indicate that such spatiotemporal and spatiospectral representations can be utilized to simplify the task of medical specialists in detecting any defects in the condition of the carotid artery through visualizing , inspecting , interpreting , analyzing , and comparing these patterns with those of normal healthy cases . this automated method , which is based on 2d spatiotemporal and spatiospectral visualization , can also be used to assess the progression of a given treatment by inspecting and comparing the corresponding 2d maps of the same case before and after the treatment . the proposed method for automated evaluation of the homogeneity and the variation in the patterns of the spatiospectral maps resulted in a sensitivity of 97.1% and a specificity of 74.5% , compared to 85.5% and 78.7% , respectively , when employing the method proposed in . the specificity of the method presented was moderate because it was based on the general basic principle of studying the characteristics of the wave propagation along the carotid artery . the hypothesis that was suggested and tested in this work was that , in subjects suffering from cad , abnormal physiological function of the cardiovascular system would cause disturbances to the physical properties of the wave propagation process through arterial vessel walls in general and along the carotid artery in particular . pathological wave propagation may give indication to cad and serious vascular problems , and , in this paper , it has been demonstrated that the type of abnormal or disturbed wave propagation in healthy elderly subjects is significantly different than that in elderly subjects with cad . however , the sensitivity and specificity of this novel automated method can be enhanced by defining a new category of suspected cases , with feature values in between those of the pathological elderly and the healthy elderly cases . the reliability of this method is confirmed by the fact that all healthy young cases can be separated from the pathological cases with an accuracy of 100% . the advantage of this approach of presenting the 2d patterns of the wavy motion of the arterial vessel wall is that it can provide much more information ( temporally , spectrally , and spatially ) than the wave intensity signal concerning the progression of the wave and the spatially localized condition of the wall of the carotid artery . however , accurate physiological insight is required to be able to interpret such 2d maps and draw deductions from these types of spatiotemporal and spatiospectral visualization approaches . the automation of the feature extraction process in addition to the automatic classification to differentiate between healthy and pathological cases would significantly simplify the task of medical specialists in identifying abnormal motion of arterial vessel walls and detecting cardiovascular symptoms . this approach has a number of desired properties , such as being noninvasive , fully automated , fast , user friendly , intuitive , straightforward , robust , reproducible , and objective . finally , this approach can be developed further to build a computer - aided decision support system for the automatic classification and diagnosis of different arterial characteristics and physiological conditions among different categories of patients ( e.g. , to target even narrower categorization or early stages of atherosclerosis ) with high accuracy .

a novel method for characterizing and visualizing the progression of waves along the walls of the carotid artery is presented . the new approach is noninvasive and able to simultaneously capture the spatial and the temporal propagation of wavy patterns along the walls of the carotid artery in a completely automated manner . spatiotemporal and spatiospectral 2d maps describing these patterns ( in both the spatial and the frequency domains , resp . ) were generated and analyzed by visual inspection as well as automatic feature extraction and classification . three categories of cases were considered : pathological elderly , healthy elderly , and healthy young cases . automatic differentiation , between cases of these three categories , was achieved with a sensitivity of 97.1% and a specificity of 74.5% . two features were proposed and computed to measure the homogeneity of the spatiospectral 2d map which presents the spectral characteristics of the carotid artery wall 's wavy motion pattern which are related to the physical , mechanical ( e.g. , elasticity ) , and physiological properties and conditions along the artery . these results are promising and confirm the potential of the proposed method in providing useful information which can help in revealing the physiological condition of the cardiovascular system .

1. Introduction 2. Materials and Methods 3. Results 4. Conclusions

PMC4938710

precise regulation of gene expression in response to environmental and endogenous signals is fundamental for reproductive success . the ability to adapt gene expression in response to the changing external conditions is particularly important for plants owing to their sessile nature . in eukaryotes , the organization of genomic dna into chromatin and the ordered regulation of its accessibility to the transcription machinery are central to gene regulation ( li et al . mechanisms for regulation of chromatin structure include atp - dependent chromatin remodeling as well as post - translational histone modifications . atp - dependent chromatin remodeling complexes alter nucleosome composition and positioning , and thus can regulate dna accessibility via chromatin compactness ( li et al . , 2007 , narlikar et al . , 2013 ) . the swr1 complex ( swr1c ) is an evolutionarily conserved swi2/snf2-related atpase - containing chromatin remodeling complex that catalyzes the replacement of h2a by the histone variant h2a.z in nucleosomes ( kobor et al . , 2004 , 2004 ) . through its distinct physicochemical properties , h2a.z influences nucleosome stability , and therefore chromatin structure , to modulate gene expression ( thambirajah et al . , 2006 , these properties along with its incorporation into the chromatin out of mitosis have made h2a.z central to transcriptional regulation underlying development and environmental responses ( talbert and henikoff , 2014 ) . in budding yeast , swr1c has been characterized as a large multi - protein structure with 13 accessory subunits in complex with the atpase , swr1 ( kobor et al . , 2004 , several of the non - catalytic subunits including actin - related protein 6 ( arp6 ) and swr1 complex 6 ( swc6 ) have been shown to be essential for histone replacement . arp6 , swc6 , and swc2 act as a sub - complex , where the proteins are mutually essential for each other 's association and function within the complex ( wu et al . , 2005 ) . in line with the current understanding , swr1c is expected to act as a single complex in plants to catalyze h2a.z incorporation ( figure 1a ) . the components and function of swr1c have been shown to be conserved in arabidopsis ( noh and amasino , 2003 , deal et al . , 2005 , martin - trillo et al . , 2006 , choi et al . , 2007 , march - diaz et al . , 2007 , lzaro et al . , in contrast to other organisms , swr1c and h2a.z are not essential for viability in arabidopsis ( coleman - derr and zilberman , 2012 ) . forward and reverse genetic analyses have identified the swr1 homolog photoperiod - independent early flowering 1 ( pie1 ) as well as arp6 , swc6 , and h2a.z encoding genes ( hta8 , hta9 , hta11 ) ( noh and amasino , 2003 , deal et al . , 2005 , martin - trillo et al . , 2006 , lzaro et al . , 2008 , march - daz et al . , 2008 , coleman - derr and zilberman , 2012 ) . as in other eukaryotes , the non - catalytic subunits of swr1c in arabidopsis are considered to have similar functions and to be essential for h2a.z deposition by the atpase subunit . mutants defective in swr1c are highly pleiotropic ( march - daz et al . , 2008 , coleman - derr and zilberman , 2012 ) . swr1c components and h2a.z have been shown to function in various aspects of plant development , most importantly in the timing of the vegetative to reproductive transition ( noh and amasino , 2003 , deal et al . , 2005 , martin - trillo et al . , 2006 , arp6 has been shown to regulate meiosis during megagametogenesis through the regulation of meiotic gene expression ( qin et al . , 2014 ) . in addition , the role of h2a.z in modulating chromatin structure was recently shown to be also important for crossovers during meiosis . while the chromatin landscape at the crossover hotspots is marked with h2a.z nucleosomes , the arp6 mutant is compromised in meiotic crossovers ( choi et al . , 2013 ) . h2a.z has recently been proposed to be critical for genome stability and dna repair in arabidopsis . the pie1 , arp6 and swc6 mutants show constitutive dna damage and compromised somatic homologous recombination as well as hypersensitivity to genotoxic agents ( rosa et al . , 2013 ) . importantly , swr1c and h2a.z have also been implicated in regulating gene expression in response to environmental signals in general ( coleman - derr and zilberman , 2012 ) . arp6 has been shown to be important for repression of genes involved in the phosphate starvation response ( smith et al . , 2009 ) . temperature - dependent h2a.z nucleosome dynamics have been shown to modulate thermosensory responses in arabidopsis ( kumar and wigge , 2010 ) and in grasses ( boden et al . , 2013 ) . mutations in swr1c components pie1 and swc6 or in h2a.z have been reported to result in constitutive activation of defense responses , demonstrating their importance in biotic interactions as well ( march - daz et al . , 2008 ) . although swr1c and h2a.z have been implicated in immunity in arabidopsis , the respective role of the complex subunits is still not well understood in plant defense . moreover the function of arp6 , a key component of the swr1c subunit in h2a.z deposition , has not been previously studied in this respect . in this study , we have carried out a comprehensive analysis to unravel the role of swr1c components in immunity and gene regulation . while resistance to biotrophic and necrotrophic pathogens is impaired in pie1 , swc6 , and hta9 hta11 mutants , arp6 showed wild - type or increased resistance . loss of pie1 and swc6 but not arp6 , leads to impaired effector - triggered immunity ( eti ) . genome - wide gene expression analyses have further highlighted the potentially specialized roles of h2a.z and swr1c components in gene regulation and thereby in plant defense responses . to study the role of h2a.z in plant defense processes , we analyzed mutants defective in swr1 complex components pie1 , arp6 , and swc6 , as well as those depleted of the histone variant h2a.z . consistent with previous studies , pie1 - 2 ( noh and amasino , 2003 ) , arp6 - 1 ( deal et al . , 2005 ) , and swc6 - 1 ( lzaro et al . , 2008 ) mutants showed pleiotropic growth and developmental phenotypes including early flowering , elongated petioles , and reduced fertility when grown in long photoperiod ( ld ) ( figure 1b ) . the pie1 mutant showed the most severe growth defects and even though it displayed accelerated reproductive transition , bolting was delayed . the arp6 and swc6 mutants phenocopied each other with characteristic early flowering and serrated leaves . this was consistent with the biochemical interaction of arp6 and swc6 in yeast , where their existence in the complex is mutually dependent ( mizuguchi et al . , 2004 ) . the hta9 - 1 hta11 - 1 double mutant ( loss of two major h2a.z encoding genes out of the possible three in arabidopsis ) phenocopied arp6 and swc6 mutants with early flowering and serrated leaves . the severe growth defects in the pie1 mutant have been previously attributed to the de - repression of immune responses characterized by spontaneous cell death and upregulation of defense genes ( march - daz et al . , 2008 ) . growth in short photoperiods ( sd ) largely suppressed some of the growth defects of the pie1 mutant , even though the rosette size remained significantly smaller compared with the other swr1c mutants ( figure 1b ) . all the studies reported here , unless otherwise specified , were performed on plants grown under sd . swr1c components have been proposed to be negative regulators of plant immunity with pie1 , swc6 , and hta9 hta11 mutants reported to display spontaneous cell death and enhanced resistance to virulent bacterial pathogens ( march - daz et al . , 2008 ) . the role of arp6 in this process is still not known and has been expected to be similar to pie1 , swc6 , and h2a.z . in order to investigate this , we characterized the defense responses of these mutants . in our experimental conditions , trypan blue staining revealed no apparent spontaneous cell death in the mutants ( figure 1c ) . we occasionally observed patches of cell death and leaf torsion in some leaves of the pie1 mutant ( supplemental figure 1 ) . to investigate their immunity phenotypes further , we challenged 4-week - old mutants with the hemibiotrophic bacterial pathogen p. syringae pv tomato dc3000 ( pst dc3000 ) by spray inoculation and monitored bacterial growth . we found that pie1 , swc6 , and hta9 hta11 mutants showed more macroscopic disease symptoms and increased susceptibility toward pst dc3000 compared with the wild - type col-0 . however , the arp6 mutant showed increased resistance ( figure 1d and 1e ) . overall , pie1 , swc6 , and hta9 hta11 mutants accommodated 10 to 15 times more bacterial titers at 3 days post inoculation ( dpi ) than the wild type . the pie1 , swc6 , and hta9 hta11 mutants displayed severely compromised resistance similar to that of the enhanced disease susceptibility1 ( eds1 - 2 ) mutant , which was used as a susceptible control ( feys et al . , 2001 ) . there were no differences in the bacterial titers at day 0 ( 2 h post inoculation ) between the different mutants and the wild - type col-0 plants . a transgenic swc6 complemented line showed wild - type resistance to pst dc3000 ( supplemental figure 2 ) , suggesting that the resistance phenotype we observe is caused by the mutation . our observations suggest that h2a.z , pie1 , and swc6 are essential for basal resistance in arabidopsis , whereas arp6 has an opposite function . these results were intriguing as a previous study has shown that pie1 , swc6 , and hta9 hta11 mutants displayed rather constitutive defense activation marked by spontaneous lesions and disease - resistance phenotypes ( march - daz et al . , 2008 ) . as mentioned earlier , the pie1 mutant showed severe growth defects when grown under ld . these phenotypes were partially suppressed by growth under sd ( figure 1b ) . in order to investigate whether the severe growth defects in pie1 under ld were due to constitutive defense activation and therefore to test if pie1 shows photoperiod - dependent defense phenotypes , we assessed its resistance to pst dc3000 in these growth conditions . to bypass early flowering induction by constant growth under ld photoperiods , the mutants along with the wild type were pre - grown for two weeks under sd conditions before being shifted to ld for another two weeks prior to infection . consistent with the results from the sd experiments , pie1 remained increasingly susceptible to pst dc3000 ( supplemental figure 3a ) . moreover , it showed no spontaneous cell death in the leaves under these conditions , similarly to the other mutants ( supplemental figure 3b ) , except for the patches of dead tissue observed as in sd . these results suggest that the growth defects in the pie1 mutant are not necessarily a consequence of enhanced defense activation . the defense phenotypes of arp6 , swc6 , and hta9 hta11 grown in ld were also similar to the results obtained under sd conditions ( supplemental figure 3a ) further confirming that the immunity phenotypes we observed are not due to specific growth conditions , especially the photoperiod . these results also confirm that the contrasting defense phenotypes observed in this study and by march - daz et al . ( 2008 ) are unlikely to be caused by growth conditions such as the photoperiod . during interaction with avirulent pathogens , recognition of effectors by the receptor proteins in the host plant leads to the activation of eti . in arabidopsis , the bacterial effectors avrrps4 and avrrpt2 trigger resistance through recognition by the toll interleukin-1 receptor ( tir)-type and the coiled coil ( cc)-type nucleotide binding - leucine - rich - repeat ( nb - lrr ) proteins rps4/rrs1 and rps2 , respectively ( kunkel et al . 2009 ) . to examine the possible roles of swr1c and h2a.z in eti , we inoculated the corresponding mutants with avirulent pst dc3000 expressing either avrrps4 or avrrpt2 effectors . the pie1 and swc6 mutants are more susceptible compared with wild - type in both interactions ( figure 2a and 2b ) . they were , however , not as hypersusceptible as eds1 or ndr1 ( non - race - specific disease resistance1 ) , used as susceptible controls in response to avrrps4 and avrrpt2 ( century et al . , 1995 ) , respectively . consistent with the earlier results , arp6 displayed full resistance like wild - type plants , suggesting that arp6 mutation does not alter rps4- and rps2-mediated resistance . the hta9 hta11 double mutant showed mild but not significant susceptibility compared with wild - type . in arabidopsis , h2a.z is encoded by three genes hta9 , hta11 , and hta8 . the presence of hta8 , even though generally lowly expressed ( supplemental figure 4 ) , in the hta9 hta11 double mutant could compensate for the lack of hta9 and hta11 . interestingly , arp6 function appears to be dispensable . to understand the basis of the failure to mount eti , we examined the level of salicylic acid ( sa ) accumulation , an important defense hormone in response to biotrophic pathogens and particularly during eti ( robert - seilaniantz et al . , 2011 ) . the basal level of sa did not noticeably differ between the the wild type and the mutants except for arp6 , where it was higher ( figure 2c ) . interestingly , 24 h after infiltration with pst dc3000 avrrps4 , all the mutants showed increased sa accumulation in comparison with col-0 . accumulation of sa in pie1 , swc6 , and hta9 hta11 was 2-fold higher relative to col-0 , and reached 3-fold in arp6 . our results show that in spite of sa accumulation , resistance to virulent and avirulent p. syringae is impaired in pie1 and swc6 mutants , suggesting that swr1c activity ( especially pie1 , swc6 ) and h2a.z are required for sa - dependent downstream signaling processes . these results are further supportive to the resistance phenotypes of the mutants to virulent pst dc3000 ( figure 1d and 1e ) . signaling crosstalks and antagonistic interactions between different defense pathways regulated by hormones have been extensively studied ( robert - seilaniantz et al . , 2011 , sa - mediated defense is activated during interaction with biotrophic pathogens while jasmonic acid ( ja)-mediated defense is active against necrotrophs and herbivores ( glazebrook , 2005 , howe and jander , 2008 ) . as presented earlier , pie1 , swc6 , and hta9 hta11 mutant plants showed impaired sa - mediated defense responses even though they accumulated a high level of the sa hormone in response to pseudomonas infection . in order to investigate whether these mutants are affected in the sa / ja crosstalk , we investigated their resistance to the necrotrophic pathogen botrytis cineria . leaves of the wild - type and mutant plants were drop inoculated with spore suspension , and the growth of lesions was monitored . average lesion size measured 3 dpi was significantly bigger in pie1 , swc6 , and hta9 hat11 . the proportions of outgrowing lesion type iv ( lesion over 6 mm in diameter ) in pie1 , swc6 , and hta9 hat11 were higher compared with arp6 mutant plants that showed mild resistance ( figure 3 and supplemental figure 5 ) . these results show that perturbation of h2a.z incorporation results in a compromised ja / ethylene ( et)-mediated immunity . it also suggests that components of swr1c do not play the same function in this defense pathway , as arp6 showed wild - type resistance to botrytis , as opposed to the rest of the mutants . in order to elucidate the molecular basis of the phenotypes described above , and to dissect the role of h2a.z and swr1c subunits in gene regulation , we carried out a global transcriptome analysis of the wild - type and mutant lines using rna - seq . three replicates of total rna from 2-week - old seedlings from each mutant were sequenced . genes showing at least 2-fold change from the wild type with a p value cut - off of 0.05 were used for further analyses . we found that mutations in swr1c components result in misregulation of a large number of genes ( figure 4a and 4b and supplemental dataset 1 ) . the pie1 mutation resulted in the largest number of gene misregulation , with 2295 genes upregulated and 1051 genes downregulated . the genes misregulated in our study showed statistically significant ( hypergeometric test ) overlap with the previously published data ( march - daz et al . , 2008 , coleman - derr and zilberman , 2012 ) . we found a total of 671 genes to be commonly misregulated among all four mutants ( 473 genes upregulated and 198 genes downregulated ) . this accounts for 20% of the genes misregulated in pie1 , and 22% , 32% , and 27% in arp6 , swc6 , and hta9 hta11 , respectively . moreover , the commonly misregulated genes ( up and down ) showed a statistically significant overlap with h2a.z enrichment in the gene bodies ( coleman - derr and zilberman , 2012 ) . we found that 1262 genes were uniquely upregulated in pie1 accounting for 55% of the total number of genes upregulated in this mutant . in arp6 , swc6 , and hta9 hta11 , however , the proportions of uniquely downregulated genes were 25% in arp6 , 4.5% in swc6 , and 18% in hta9 hta11 . the large proportion of uniquely misregulated genes in pie1 suggests a potential swr1c / h2a.z independent role for pie1 in gene regulation . to explore this further and to elucidate the relationship between individual mutants , we performed principal component analysis ( pca ) of the rna - seq data . the first three principal components ( pc1 , pc2 , and pc3 ) together accounted for 92.1% of the variance in the data ( 44.44% , 30.55% , and 17.11% , respectively ) ( figure 4c ) . it also revealed that the four mutant genotypes segregate into three distinct groups : swc6 and hta9 hta11 mutants co - segregated while pie1 and arp6 mutants were distinct . pc1 clearly separated pie1 from the rest of the group , whereas pc3 defined the contribution of arp6 ( figure 4c and supplemental figure 6 ) . this analysis has further strengthened the possibility of non - overlapping functions for the complex components . in line with the pca analysis , differential gene expression analyses showed a poor correlation of gene misregulation caused by arp6 and pie1 mutations ( r = 0.16 ) ( figure 4d ) , as well as between hta9 hta11 and pie1 ( r = 0.11 ) ( figure 4e ) , which is surprising as pie1 mutation is expected to phenocopy h2a.z depletion to a large extent . the arp6 and swc6 mutants in arabidopsis have similar morphological and developmental phenotypes , especially early flowering and serrated leaves . moreover , arp6 and swc6 have been shown to physically interact in arabidopsis ( march - diaz et al . intriguingly , in our rna - seq data , the transcriptional changes due to these mutations did not show a strong correlation ( r = 0.42 ) ( figure 4f ) . this observation is consistent with the defense phenotypes described above , suggesting that arp6 and swc6 could have distinct functions in arabidopsis . in agreement with the pca analysis , swc6 and hta9 hta11 double mutants displayed a strong correlation of transcriptional misregulation ( r = 0.75 ) , indicating overlapping roles for swc6 and h2a.z in gene regulation ( figure 4 g ) . this is in contrast to arp6 , which showed only a modest correlation ( r = 0.38 ) with hta9 hta11 ( supplemental figure 7 ) . to understand the function of swr1c and h2a.z in gene regulation , particularly in immunity we found a significant over - representation of sa response ( p = 4.46 10 ) and defense ( p = 7.75 10 ) related go terms in the 473 genes commonly misregulated in the mutants ( supplemental dataset 2 ) . this is in accordance with what was reported earlier ( march - daz et al . , the commonly downregulated genes where enriched in triterpene ( thalianol , p = 1.77 10 and tricyclic triterpenoid metabolic process , p = 7.04 10 ) related go terms . in addition , pie1 showed a significant over - representation of several biological processes including auxin metabolic processes , membrane organization , and response to heat , indicating the important role of pie1 in gene expression in general . in swc6 and hta9 hta11 , the go term response to heat was significantly enriched , which is consistent with h2a.z function in response to temperature ( kumar and wigge , 2010 ) . we also observed that swc6 and hta9 hta11 mutants display an enrichment of hydrogen peroxide - related go terms like in arp6 ( supplemental dataset 2 ) . taken together , the results of the transcriptome analysis demonstrate that the components of the swr1c have distinct roles in the regulation of gene expression in arabidopsis . our rna - seq analysis revealed that , in the swr1c mutants , particularly pie1 , genes required for disease resistance were misregulated as previously shown ( march - daz et al . , 2008 ) . genes involved in systemic acquired resistance such as pr1 , pr5 , eds5 , and nimin1 were highly upregulated in pie1 ( figure 5a5d and supplemental dataset 1 ) . only pr5 among these genes was upregulated in swc6 and hta9 hta11 , but not to the same extent as in pie1 . interestingly , the pie1 mutant shows severely compromised resistance in spite of the enhanced expression of defense genes ( as shown above ) . to further understand the molecular basis for this paradox we observed that genes encoding the wrky family of transcription factors are upregulated in pie1 ( figure 5e and supplemental dataset ) . wrky transcription factors are important regulatory components during plant response to pathogen infection and abiotic stresses . moreover , they have been implicated in plant immunity as both positive and negative regulators ( pandey and somssich , 2009 , rushton et al . , wrky38 and wrky62 , in particular , are highly expressed in the pie1 background ( figure 5f and 5 g ) . these two wrky proteins were shown to be negative regulators of resistance to pathogens . in line with these observations , the uniquely upregulated genes in pie1 are also enriched in go terms related to negative regulation of the defense response ( p = 1.02 10 ) . sa is a key regulator of signaling networks involved in defense along with other hormones such as ja ( robert - seilaniantz et al . , 2011 ) . sa- and ja - mediated defense responses are triggered in the plant depending on the nature of the pathogen ( van der does et al . , 2013 ) . these two pathways act antagonistically to modulate defense responses . activation of one pathway usually correlates with attenuation of the other ( thaler et al . , 2012 ) . consistent with this , we found that the sa - inducible glutaredoxin gene grx480 , which has been proposed to antagonize the ja - responsive transcription of pdf1.2 is nearly 10-fold upregulated in pie1 ( figure 6a ) . transgenic plants ectopically expressing grx480 displayed reduced ja - induced gene expression ( ndamukong et al . , 2007 ) . in fact , uniquely upregulated genes in pie1 are enriched in go terms related to the ja metabolic process ( p = 9.12 10 ) and ja - mediated signaling pathway ( p = 5.17 10 ) . for instance , genes involved in ja biosynthesis ( figure 6b6d ) , signaling ( figure 6e and 6f ) , and response ( figure 6g6i ) were upregulated . we further analyzed the expression of ja - induced genes ( nemhauser et al . , 2006 ) in our data and found that these are upregulated in pie1 compared with the other mutants ( figure 6j ) , confirming that ja - responsive pathways are derepressed in spite of the sar genes being upregulated . these observations , together with the upregulation of negative regulators of defense ( e.g. , wrkys ) , could explain the unexpected disease susceptibility phenotypes we observe . they also point to a potential role that pie1 plays in coordinating distinct defense signaling pathways , especially in the sa - ja antagonism . our analyses revealed that mutants of the swr1c components show considerable differences in physiological and molecular phenotypes , leading to the assumption that the complex components may be functionally specialized . it is also likely that the non - enzymatic subunits might not necessarily be essential for complex function , and their depletion or inclusion in the complex could modulate its function . the components could also have functions independent of h2a.z incorporation . to further elucidate the possible specialization and to understand their role in relation to each other , we analyzed double mutant plants . since arp6 and swc6 mutants show similar morphological and developmental phenotypes but contrasting immunity traits , we analyzed arp6 swc6 double mutants to study disease resistance without the confounding effects of contrasting developmental phenotypes . the double mutants did not show any noticeable additional morphological phenotypes than the single mutants suggesting overlapping functions in development . as shown above , arp6 showed resistance to dc3000 while the swc6 mutant showed increased susceptibility compared with wild - type col-0 . interestingly , the arp6 swc6 double mutants displayed increased susceptibility to pst dc3000 phenocopying swc6 ( figure 7 ) . these results indicate that swc6 is epistatic to arp6 in disease - resistance traits . despite the overlapping functions in development , it seems that loss of swc6 significantly compromises the regulation of immunity genes by swr1c more than the loss of arp6 . our gene expression analyses have shown that genes involved in systemic acquired resistance such as pr1 and pr5 were derepressed in the swr1c mutants and therefore showed increased basal expression ( figure 5 ) . interestingly , however , pie1 , swc6 , and hta9 hta11 mutants were severely compromised in resistance to both virulent and avirulent p. syringae strains in spite of the enhanced basal expression of defense genes . to understand the molecular basis for these paradoxical phenotypes , we analyzed defense gene expression in the mutants in response to pst dc3000 . gene expression was analyzed in 4-week - old wild - type col-0 and the mutants by quantitative rt pcr 6 h post inoculation with pst dc3000 or mock treatment ( 10 mm mgcl2 ) ( figure 8) . as expected , pr1 showed very low basal expression in col-0 under control conditions , whereas bacterial infection resulted in a strong induction of gene expression ( figure 8a ) . the arp6 mutant showed derepression of pr1 and a stronger induction in response to pst dc3000 . on the other hand , the pie1 mutant showed a strong constitutive expression of pr1 ; however , inoculation with pst dc3000 inoculation resulted in strong downregulation ( figure 8a ) . in swc6 and hta9 hta11 , where basal expression of pr1 was modestly altered , dc3000 infection resulted in its repression ( figure 8a ) . similarly , analysis of pr5 expression showed that while it was strongly induced in arp6 in response to pst dc3000 , there was little or no induction in pie1 , swc6 , and hta9 hta11 mutants ( figure 8b ) . these results show that induction of pr1 and pr5 was compromised upon pathogen inoculation in pie1 , swc6 , and hta9 hta11 mutants despite the higher basal expression . together , these results show that while it is required to maintain defense genes in repressed state in the absence of pathogen signals , swr1c function , and therefore h2a.z are essential for strong pathogen - induced defense gene activation that is essential for disease resistance . swr1c is an evolutionarily conserved atp - dependent chromatin remodeling complex involved in the post mitotic incorporation of the histone variant h2a.z into nucleosomes . putative swr1c subunits have been identified and shown to be involved in plant growth and development as well as environmental adaptation . although they have been implicated in immunity , their exact role is yet to be understood . above all , the respective roles that each subunit has in gene regulation and their influence in major physiological outcomes remain elusive . we undertook a comprehensive analysis of swr1c and h2a.z mutants to systematically analyze their function in plant immunity and gene regulation . our results indicate that perturbing swr1c function or depleting h2a.z levels results in severely impaired immune responses : pie1 , swc6 , and h2a.z are positive regulators of resistance in arabidopsis against both biotrophic and necrotrophic pathogens . transcriptome analysis revealed further complexities as the swr1c subunits showed overlapping but , more importantly , distinct functions in the regulation of gene expression . in this study , we found that mutations of the swr1c subunits , pie1 and swc6 , and depletion of h2a.z severely impaired immunity suggesting that they are essential for defense responses in arabidopsis . pie1 , swc6 , and h2a.z have been implicated in immunity in an earlier study , where mutations in the respective genes were reported to result in constitutive activation of defense responses including spontaneous cell death and increased resistance to virulent p. syringae ( march - daz et al . , 2008 ) . while the developmental phenotypes we observed were in agreement with the previous reports ( deal et al . , 2005 , 2008 ) , defense phenotypes were contrasting in our experiments . while p. syringae infection assays were performed under the same growth conditions in our study and in that of march - daz et al . we used appropriate genotypes as controls ( eds1 , ndr1 , etc . ) in all our experiments and have shown the expected phenotypes , suggesting that the phenotypic differences are not due to the experimental conditions . we have also extended our analyses to test if the swr1c mutants show photoperiod - dependent defense phenotypes and performed disease resistance assays using pst dc3000 under long photoperiod . strikingly pie1 , swc6 , and hta9 hta11 were all increasingly susceptible independent of the photoperiod , suggesting that the difference in the resistance phenotype is likely not due to growth conditions , particularly day length . it is still likely that other environmental factors such as nutrient availability associated with plant growth conditions could underlie the discrepancy . however , our reproducible results with both virulent and avirulent p. syringae strains , as well as the expected phenotypes for both positive and negative control genotypes , support our conclusions that the swr1c mutants pie1 , swc6 , and hta9 hta11 are compromised in disease resistance . as reported previously ( noh and amasino , 2003 , march - daz et al . , 2008 ) , we found that the pie1 mutant is highly pleiotropic , displaying stunted growth and low fertility . moreover , as described above , it showed increased susceptibility even under long photoperiod conditions , where its growth defects were more severe . these observations suggest that the pleiotropic phenotypes of pie1 are not necessarily a result of constitutive defense activation . it is , however , still likely that defective regulation of certain defense - associated pathways underlies these phenotypes despite the lack of apparent disease resistance . similar examples where defense pathways are turned on without apparent enhanced resistance have been described previously . mutation in cpk28 genes for instance , resulted in growth defects and constitutively enhanced ja accumulation and activation of ja - responsive genes , but not enhanced resistance to necrotrophs ( matschi et al . , 2015 ) . similar to the impaired basal immunity in response to the virulent pathogen p. syringae , pie1 and swc6 mutants showed compromised resistance to pst dc3000 carrying avrrps4 and avrrpt2 effectors , suggesting that pie1 and swc6 are required for rps4- and rps2-mediated immunity . the hta9 hta11 showed only a mild susceptibility , likely due to the compensatory activity of hta8 . arp6 , however , was dispensable for eti as the mutant showed wild - type resistance . these discrepancies in nb - lrr - mediated resistance between the different mutants were not due to an altered expression of either rps4 or rps2 ( supplemental dataset ) and further highlight the functional specialization of the complex subunits . activation of r - mediated defense results in sa accumulation and signaling ( vlot et al . , sa acts as an important intracellular signal for defense gene activation , and mutants impaired in sa accumulation usually display compromised basal defense and eti . although pie1 and swc6 mutants were clearly defective in mounting resistance to pst dc3000 avrrps4 , we notice a robust increase in sa accumulation in response to this effector , suggesting that rps4-mediated recognition and downstream signaling are unlikely to be altered . in response to pst dc3000 avrrps4 compromised eti in pie1 and swc6 in spite of sa accumulation suggests that sa - mediated downstream responses might be affected in these mutant backgrounds . sa - induced gene expression and defense activation are dependent on npr1 ( cao et al . , 1994 , delaney et al . , 1995 ) . similar to the results observed for pie1 and swc6 , the npr1 mutant accumulates a high level of sa but fails to mount sar ( delaney et al . , 1995 , clarke et al . npr1 is also required for the negative feedback regulation of sa production ( delaney et al . , 1995 ) . the hyperaccumulation of sa following infection with pst dc3000 avrrps4 in spite of enhanced susceptibility suggests a compromised npr1 activity in pie1 and swc6 . it is therefore tempting to speculate that swr1c activity and h2a.z might be required for proper functioning of npr1-mediated regulation of immunity . our results showing that pr gene induction following pst dc3000 infection was perturbed in pie1 , swc6 , and hta9 hta11 mutants also support this hypothesis . in addition , pie1 and swc6 could also be important for npr1- and sa - independent processes required for resistance . we found that swr1c and h2a.z are also important for resistance to the necrotrophic pathogen botrytis cineria . depletion of pie1 , swc6 , and h2a.z , not arp6 , in mutants led to increased susceptibility . this was rather intriguing given the antagonistic interaction between sa- and ja - mediated defenses ( robert - seilaniantz et al . the contrasting phenotypes of the mutants show that pie1 , swc6 , and h2a.z , not arp6 have a positive function in ja / et - mediated defense . this result was not expected especially in the case of pie1 , which showed an increase in the ja - responsive genes , while sa - responsive genes are also upregulated . moreover , pie1 mutation also resulted in a higher level of expression of grx480 , which is a negative regulator of ja - mediated immunity . these results indicate that mutations of the swr1c components alter plant resistance and gene expression in general . taken together , our data point to the importance of swr1c function , and the pleiotropic effect of compromised swr1c function leads to misregulation of a large subset of defense genes , including both positive and negative regulators , giving rise to complex physiological outcomes as a consequence of epistatic interactions . further molecular dissection would unravel the precise regulatory role of h2a.z in transcriptional reprogramming in immunity . the discrepant phenotypes observed when arp6 is depleted from swr1c suggest that the complex could regulate different defense pathways depending on the components recruited to it . our double mutant analysis for resistance to p. syringae supports an epistatic interaction of arp6 and swc6 , which have contrasting roles in immunity , as we observed in the mutants . extending the genetic dissection would further elucidate the complex genetic interactions between the complex components in relation to resistance to necrotrophs and the coordination of potentially antagonistic signaling pathways . our study has revealed that the swr1c subunits are functionally specialized and have non - overlapping functions in immunity . the transcriptomic analysis that we performed further established this . in our analyses pie1 is the arabidopsis homolog of swr1 in yeast , the enzymatic subunit of the complex ( noh and amasino , 2003 ) . surprisingly , transcriptional misregulation in pie1 and hta9 hta11 double mutant showed a poor correlation ( figure 4e ) , while hta9 hta11 still has a functional hta8 , likely explaining the poor agreement . a recent study has also shown similar non - overlapping changes while comparing the transcriptome of pie1 with that of h2a.z triple mutant ( coleman - derr and zilberman , 2012 ) . it is likely that pie1 and h2a.z have also independent roles of one another , and that pie1 could functions independently of h2a.z incorporation . consistent with their contrasting effects in plant immunity , arp6 and swc6 mutants clearly maintained distinct molecular phenotypes as supported by the gene expression analysis . in budding yeast , arp6 and swc6 have been shown to be essential subunits in swr1c for h2a.z deposition ( mizuguchi et al . , 2004 , wu et al . , 2005 ) . along with swc2 , another component of the swr1c in yeast , they act as a sub - complex that requires all three proteins for their association with the complex and for histone exchange ( mizuguchi et al . , 2004 , wu et al . , 2005 ) . the contrasting immunity phenotypes and the divergent transcriptome we observed in our analyses suggest that arp6 and swc6 do not necessarily always act as part of the same complex and that they could have independent functions in arabidopsis , either related or not to h2a.z . it has been previously proposed that arp6 could have h2a.z - independent roles in gene expression ( yoshida et al . , 2010 ) . of all the mutant pairs studied , swc6 and hta9 hta11 showed the strongest correlation in gene expression regulation ( figure 4 g ) suggesting that swc6 functions solely as a subunit of swr1c . it remains to be seen how much of the gene expression changes that we observe in the mutants are due to changes in h2a.z incorporation . further investigations are required to fully elucidate the role that each subunit could have in histone exchange and therefore gene regulation . detailed analysis of the rna - seq data revealed that the pie1 mutant showed significant upregulation of both positive and negative regulators of defense . pathogen - induced wrky transcription factors function as positive or negative regulators of immunity ( eulgem and somssich , 2007 , pandey and somssich , 2009 ) , and epistatic interaction between the two types of regulators is required for the proper control and fine tuning of defense . of particular interest was the strong upregulation of wrky38 and wrky62 , which have been shown to be negative regulators of immunity , and their overexpression has led to reduced pr1 gene induction in response to bacterial infection ( kim et al . , 2008 ) . this is consistent with the compromised pathogen - induced pr1 expression in pie1 mutant ( figure 8) . our data have shown that the pie1 mutant showed severely compromised immunity in spite of upregulation of a remarkable number of genes involved in sar such as pr1 . while constitutive activation of sar genes is indicative of pie1 playing a repressive role in sa - mediated gene expression in the absence of pathogens , data shown in figure 2c show that lack of resistance is not due to the inability of pie1 to accumulate sa . in addition , compromised defense gene induction in response to p. syringae ( figure 8) indicates that while pie1 has a clear role in maintaining sar genes repressed in the absence of pathogens , it is essential for proper gene induction and resistance upon infection . a complex network of signaling pathways underlies defense responses in plants . defense signaling elicited by the plant hormones sa and ja are mutually antagonistic and determine defense against biotrophic and necrotrophic pathogens , respectively ( thaler et al . , 2012 ) . ja biosynthesis and ja - responsive genes are highly upregulated in pie1 , suggesting that pie1 is also a negative regulator of ja - mediated responses . remarkably , these results also implicate pie1 activity in regulating sa / ja signaling trade - off . our data suggest that swr1c and h2a.z might be playing an important role in maintaining the fine balance between signaling pathways in plant immunity . therefore , in the context of pie1 , where activating and repressive factors are similarly de - repressed , the physiological outcome will be the consequence of epistatic interactions . in support of this , we found that pie1 is compromised in resistance to both biotrophic and necrotrophic pathogens . compromised defense gene induction in the susceptible mutant backgrounds such as pie1 , swc6 , and hta9 hta11 in response to pst further molecular dissection is needed to elucidate the regulatory dynamics of defense gene expression in the context of chromatin remodeling . we have shown that , although they are as part of the same complex , swr1c subunits have divergent functions in gene regulation and therefore in important biological processes such as immunity . in this comprehensive study , we investigated defense responses and gene expression and have clearly shown the involvement of swr1c and h2a.z in immunity as positive regulators . although the swr1c component proteins are not involved in defense signaling events directly , they exert their roles through gene regulation . the genome - wide transcriptomic analysis of the corresponding mutants is consistent with the early reports showing that swr1c and h2a.z are involved in regulating defense - related genes ( march - daz et al . , 2008 , coleman - derr and zilberman , 2012 ) . this is particularly in line with the observation that responsive genes are enriched in h2a.z on gene bodies ( coleman - derr and zilberman , 2012 ) . chromatin state and architecture have been implicated in the repression of sar and stress - related gene expression in general ( bartsch , 2006 , hamon and cossart , 2008 , van den burg and takken , 2009 , ma et al . , 2011 , our extensive analyses highlight that , despite the evolutionary conservation of fundamental molecular processes such as chromatin remodeling , in arabidopsis swr1c components carry out non - overlapping roles . this can be either due to some of the non - enzymatic subunits having roles outside the swr1c or the potential variation of the degree to which depletion or inclusion of each of the subunits affects the complex function . it is likely that some of the non - enzymatic components such as swc6 and arp6 are non - essential for complex activity and that their depletion will affect the complex differently . however , further molecular dissection is required to comprehensively define the role that each component protein plays in the assembly , recruitment to specific target , and histone exchange activity . importantly , our results that h2a.z and swr1c are positive regulators of plant resistance to different pathogens are particularly significant in the context of environmental modulation of defense responses . elevated temperatures have been shown to suppress plant immunity ( wang et al . , 2009 , nucleosome dynamics have been shown to underlie temperature responses in arabidopsis ( kumar and wigge , 2010 ) . therefore , compromised immunity in the swr1c subunit mutants where h2a.z deposition is perturbed could be reminiscent of the influence of temperature response . further studies will unravel the role of h2a.z - mediated chromatin dynamics in the environmentally modulated defense gene expression . we used the previously described mutants arp6 - 1 ( deal et al . , 2005 ) , pie1 - 2 ( noh and amasino , 2003 ) , swc6 - 1 ( lzaro et al . , 2008 ) , hta9 - 1 , hta11 - 1 ( march - daz et al . , 2008 ) , ndr1 - 1 ( century et al . , 1995 ) , and eds1 - 2 ( bartsch , 2006 ) , and the transgenic complementation line swc6 35s - myc : atswc6 ( choi et al . , 2007 ) in our experiments . for bacterial infection assays , the ros burst assay , and trypan blue leaf staining , arabidopsis plants were grown on soil for 5 weeks in sd conditions ( 8 h light/16 h dark ) in sanyo growth cabinets at 22c and 70% relative humidity . otherwise 2-week - old seedlings grown in half - strength murashige and skoog ( ms ) plates after seed - surface sterilization were used . briefly , spray inoculations were performed with bacterial suspension at 2 10 and 2 10 cfu / ml of p. syringae pv tomato dc3000 and pst dc3000 carrying avrrps4 or avrrpt2 genes , respectively , in 10 mm mgcl2 with 0.04% silwet l-77 . infiltration experiments were performed with bacterial suspensions at 10 cfu / ml in 10 mm mgcl2 using a needless syringe . bacterial titers where determined at day 0 ( 2 h ) and/or at day 3 post inoculation . significant differences to col-0 are expressed using student 's t - test at p < 0.05 . the botrytis cinerea infection assay was performed as previously described in schoonbeek et al . a botrytis spore suspension ( 2.5 10 spore / ml ) prepared in quarter - strength potato dextrose broth ( pdb , 6 g / l ) was used for drop inoculation on the leaves of 5-week - old arabidopsis plants . five leaves were inoculated with one droplet of spores of botrytis cinerea isolate b05.10 . free sa was extracted twice from 10 mg of freeze - dried leaves with 10% methanol and 1% acetic acid in the presence of 125 nm 2-hydroxybenzoic-3,4,5,6-d4 acid as internal standard ( cdn isotopes ) . the samples were run on a xevo tqs tandem mass spectrometer attached to an acquity uplc system . separation was on a 50 2.1 mm 2.7 kinetex xb c18 column ( phenomenex ) running in a gradient of acetonitrile versus 0.1% formic acid in water . for rna - seq analysis , total rna was extracted from 2-week - old seedlings grown on ms solid medium using the rneasy plant mini kit ( qiagen ) according to the manufacturer instructions . the 50-bp - long single - end reads were mapped to the arabidopsis genome ( tair10 ) and quantified to generate rpkm ( reads per kilobase of exon per million fragments mapped ) values . the tophat - cufflinks pipeline was used to align rna - seq reads to arabidopsis thaliana reference genome assembly . differential gene expression and pca analysis was performed with the rpkm values generated using strand ngs 2.0 ( agilent ) . the expression values for the three biological replicates were analyzed for statistical significance using a one - way analysis of variance test applying the benjamini - hochberg false discovery rate ( fdr ) and the multiple testing correction ( benjamini and hochberg , 1995 ) and asymptotic p value computation with an adjusted p value cut - off of p < 0.05 . genes with absolute fold change values of at least two were included in our analysis as differentially expressed genes . for the comparison of our transcriptome data with the published results , we considered the 15 199 genes that passed the fdr cut - off of < 0.05 as the global number . four - week - old wild - type and mutant plants were used for analyzing defense gene expression in response to bacterial inoculation . plants were either spray inoculated with pst dc3000 or were mock treated with 10 mm mgcl2 . total rna was isolated as above , and 1 g of rna was used as a template for reverse transcription with superscript iii reverse transcriptase ( invitrogen ) and oligo dt according to the manufacturer 's instructions . ef1 ( at5g60390 ) transcript levels were used as internal reference ( oligos : 5 tacgccccagttctcgattg 3 and 5 ggcttggttggggtcatctt 3 ) for analyzing pr1 ( oligos : 5 accaggcacgaggagcggta 3 and 5 tccccgtaaggcccaccaga 3 ) and pr5 ( oligos : 5 acccacagcacagagacacaca 3 and 5 tggccataacagcaatgccgc 3 ) expression . quantitative rt pcr experiments were performed in a light cycler lc480 using light cycler 480 sybr green i master ( roche ) . this work was supported by a biotechnology and biological sciences research council ( bbsrc ) grant bb / i019022/1 and institute strategic program grants bb / j004588/1 and bb / j004553/1 . designed experiments ; s.b . performed most of the experiments and data analyses ; s.n.g . performed experiments and data analysis for figures 7 and 8 ; s.b . and s.v.k .

incorporation of the histone variant h2a.z into nucleosomes by the swr1 chromatin remodeling complex is a critical step in eukaryotic gene regulation . in arabidopsis , swr1c and h2a.z have been shown to control gene expression underlying development and environmental responses . although they have been implicated in defense , the specific roles of the complex subunits and h2a.z in immunity are not well understood . in this study , we analyzed the roles of the swr1c subunits , photoperiod - independent early flowering1 ( pie1 ) , actin - related protein6 ( arp6 ) , and swr1 complex 6 ( swc6 ) , as well as h2a.z , in defense and gene regulation . we found that swr1c components play different roles in resistance to different pathogens . loss of pie1 and swc6 function as well as depletion of h2a.z led to reduced basal resistance , while loss of arp6 fucntion resulted in enhanced resistance . we found that mutations in pie1 and swc6 resulted in impaired effector - triggered immunity . mutation in swr1c components and h2a.z also resulted in compromised jasmonic acid / ethylene - mediated immunity . genome - wide expression analyses similarly reveal distinct roles for h2a.z and swr1c components in gene regulation , and suggest a potential role for pie1 in the regulation of the cross talk between defense signaling pathways . our data show that although they are part of the same complex , arabidopsis swr1c components could have non - redundant functions in plant immunity and gene regulation .

Introduction Results Discussion Methods Funding Author Contributions

PMC3268986

the road to predicting future scientific and technological developments is paved with spectacular failures and memorable epigrams . niels bohr , for example , was fond of saying , prediction is very difficult , especially about the future . it is also a corollary of albert einstein s quip that , if we knew what it was we were doing , it would not be called research , would it ? among the most celebrated examples of erroneous predictions are that flying cars and personal jetpacks would be common - place by now [ 1 , 2 ] . the 25th anniversary of an event as notable as the launch of the journal of computer - aided drug design ( jcamd ) compels us to look ahead nonetheless , uncertain though our vision might be . fortunately , it is a testament to human nature that the direction taken by a scientific discipline is generally foreshadowed by where the barriers to its progress lie . here we discuss three of those barriers understanding the role of entropy in ligand binding , dealing with predictive uncertainty and the difficulty of curating large amounts of data in hopes that doing so will provide some indication of where the field might be headed in the next 25 years . our goal here is mainly to raise questions ; providing answers to those questions remains for those who come after . protein interactions was primarily a matter of running sophisticated and long enough molecular dynamics simulations on powerful enough hardware , constrained only by limits on patience and resources . it was no accident that three of the early landmark papers on scoring functions appeared in the journal in the mid-1990 s [ 35 ] . the goal of predicting affinity from ligand and protein structure on a purely mechanistic basis seems less attainable today , though . empirical methods are often competitive with or superior to mechanistic ones when it comes to predicting affinity within chemical series . in some cases and in the hands of some practitioners , empirical methods are superior , as are some empirical ligand - based methods that ignore the structure of the target protein altogether and are completely empirical in nature . indeed , the inventor of comparative molecular field analysis ( comfa)arguably the archetypal mechanistic modeling approach for ligands now advocates topomeric alignments that abandon any pretense of relying on realistic ligand conformations . there is merit in the argument that the performance of mechanistic models is limited by the inherent difficulty of docking a flexible ligand accurately into a flexible protein target , but supplanting a rigid lock and key model of ligand protein interaction with one involving induced fit begs a fundamental question : if the protein is in a lower - energy state before the ligand inserts itself into the active site than it is after , then where does the extra energy needed to turn the key and change the shape of the lock after binding come from ? for the process to be spontaneous , there must be a net decrease in free energy ( g ) above and beyond that gained by the initial insertion of the ligand into the unoccupied binding site . somewhat counterintuitively , a major positive contributor to the entropy of binding ( s ) is increased protein fluidity after a ligand binds in particular , an increase in anharmonic , coupled motions within the complex [ 810 ] . the coupling is presumably mediated at least in part by polar interactions between residues , which may contribute to the apparent ubiquity of enthalpy - entropy compensation in ligand binding . the resulting entanglement of enthalpy and entropy is , in turn , consistent with recent analysis of isothermal calorimetric data for ligand protein complexes for which high - resolution complexes are available , work which underscores just how tightly s is connected to the enthalpy of binding ( h ) : tang and marshall showed that the residual error in models of the total free energy of formation ( g ) for such complexes is lower than the residual error in models of its h and -ts components ( t is the absolute temperature ) . the scope of the challenge of fully accounting for the affinity of a protein for a ligand based on a static structure of their complex is underscored by the huge difference in stability observed between complexes of time - dependent and time - independent inhibitors with prostaglandin h2 synthase ( cox-1 ) . time - independent binding is fast and readily reversible , whereas time - dependent binding involves a subsequent slow step ( minutes ) and is nearly irreversible . nonetheless , x - ray analysis of the complexes shows that the ligands adopt similar conformations and the enzyme adopts it would seem that some sort of dynamic effect must account for the differences between the two , and the sort of anharmonic fluidity cited above is a prime candidate . accounting for such dynamic contributions to s is complicated , however , by their fractal nature ( at least in the case of myoglobin , lysozyme and bovine pancreatic trypsin inhibitor ) , which suggests that they are chaotic or emergent properties of complex systems . if that is true in general , the dynamic contributions will be difficult to deal with using existing mechanistic methodology . appreciating the chaotic nature of the interaction could , however , help molecular designers understand what makes slow , tight - binding ( time dependent ) inhibitors special . regression models of all types have historically been characterized in terms of how well they fit the data upon which they are based , usually by the root mean square error ( rmse ) , where error corresponds to the difference between the observed and predicted response . prospectively estimating how much the predicted response for a new point in the model s descriptor space is likely to deviate from the observed response the predictive uncertainty if a model is based on linear regression and the descriptors are statistically independent variables , classical statistical theory provides tools for calculating how large the deviation from prediction is expected to be for a new observation . unfortunately , those tools are inadequate when the model is non - linear or the descriptors are correlated , and one of those conditions almost always holds when drug molecules and biological responses are involved . the desire for a quantitative estimate of predictive uncertainty is not diminished simply because statistical theory fails to provide rigorous tools for providing one , however . until fairly recently , the only way this need was addressed in quantitative structure activity relationship ( qsar ) analysis was a pragmatic one in which a test set was held back from the model building process and the root mean square error of prediction ( rmsep ) was calculated across the compounds in the test set . the qsar community has recently come to realize that this approach is inadequate when the compound for which a prediction is desired differs too greatly from the compounds in the training set used to build a model or the test set used to evaluate its predictive reliability . that realization has led to the definition of applicability domains , ranges of descriptors ( or , in a few cases , combinations of descriptors ) outside of which a model can not be relied upon to accurately predict responses or properties . the need for a specified and well - defined applicability domain has even moved into the regulatory realm , at least in europe . unfortunately , the need to establish explicit measures of predictive reliability has yet to penetrate mechanistic approaches to activity prediction , i.e. , structure - based molecular design . perhaps it goes without saying that the absence of appropriate force field parameters for a silicon atom would compromise the predictions produced by a mechanistically based docking program . there also seems to be an implicit assumption in the field , however , that exactly how a given set of carbon , hydrogen , nitrogen , oxygen , sulfur , phosphorous and halogen atoms are assembled to form a new compound is at most incidental to predictive success , and that aggregate measures of retrospective performance on test sets are all that is needed . at the extremes this is clearly an absurd assumption , especially for methods that have any empirical component at all . it is unlikely that a program trained solely on proteases will accurately dock a steroid into the estrogen receptor , for example , or that a program that has never seen a sulfonamide will handle one correctly . one response to these concerns is that the current pool of known structures for ligand protein complexes is now so large that it represents a broad enough ( albeit markedly biased ) sampling of biochemically relevant space that addition of a few new chemical classes or new targets is unlikely to affect the overall performance significantly . even were this so , it overlooks the point that the value of a prediction is a direct function of the novelty of the structure for which the prediction is being made , regardless of whether the value in question is social or commercial . hence , the predictive uncertainty at the edge of the training and test set space is more relevant than the average uncertainty across the entire retrospective structural space . this is one reason that many in industry are turning to rolling tests sets blocks of compounds assayed since the model was built to assess the predictive performance of models . but they do so fairly slowly , so the compounds made in the last 3 months tend to be different than those made a year ago ; not enormously so , but enough that some will lie at the edge of the historical applicability domain . this approach also helps address the point that there may be some combinations of descriptors that are relevant for new chemistry but which were inadequately explored in the training and test sets . the ultimate goal should be making reliable predictive uncertainty estimates for individual new compounds . some exploratory work has already been done in this area utilizing sampling and the variance of ensemble predictions [ 16 , 17 ] , but more is sorely needed if modeling is going to remain relevant to drug discovery . most of what has been done to date assumes ( explicitly or implicitly ) that the more similar a candidate molecule is to a molecule from the training set , the smaller the uncertainty in the prediction will be , or that the uncertainty in the prediction is directly related to the prediction errors of training ( or test set ) molecules that are similar to the candidate molecule . the exact meaning of molecular similarity can be very sensitive to context however . molecular similarity , like beauty , is in the eye of the beholder , and the key beholder in this case is a target receptor , not a medicinal chemist and not a computer model . all similarity - based approaches are prone to failure when presented with what might aptly be called a phantom variable . consider , for example , the case of a protein like the pept1 transporter , which has a strong preference for binding di- and tri - peptides composed entirely of l amino acids ( l - peptides ) over peptides that include one or more d amino acid residues ; enantiomeric peptides composed entirely of d amino acids ( d - peptides ) bind even less well . hence a 2d qsar model built using data only for l - peptides is unlikely to correctly predict the behavior of d - peptides . the model will see a d - peptide as being within its applicability domain if the enantiomer is , however , and will predict it with confidence just as high ( or low ) as for the corresponding l - peptide . the same holds true if similarity is based on independent descriptors not used to build the model . fingerprint - based measures of similarity , for example , might place a new d - peptide within the applicability domain of the model , but the transporter is likely to see the situation differently . nor is the problem limited to 2d qsar ; it extends to 3d methods as well . there will almost always be something that distinguishes a given new candidate molecule from the molecules in the training set , and the target may turn out to be sensitive to that difference . just this sort of situation doubtless accounts for some of the activity cliffs that have received so much attention in the recent literature [ 1921 ] . the problem is further exacerbated for applicability domains by the need to use many descriptors to model target properties for very large data sets . the more descriptors are used , the greater is the likelihood that a candidate molecule will fall outside the limits of one or more of these descriptors the curse of dimensionality . this is the reverse of the ( reasonable ) expectation that the larger the training set , the larger a model s applicability domain should be ! it would seem that some measure of molecular similarity that is sensitive to context is needed , as well as recognition that the descriptors needed to construct a model may not always provide enough information to reliably estimate that model s predictive uncertainty for all new candidates . the rapid increase in computing power since 1986 , coupled with the ease of access provided by the world wide web and digitization of data sources , has made it easier than ever before to quickly obtain information on an enormous range of topics . indeed , the authors drew heavily on internet resources to put together this paper . that data is spread thin , however , in the sense that any single primary source generally contains only a few items relevant to any particular topic . the task of pulling dispersed data together once fell to people who wrote review articles . in many areas , that function has been supplanted by databases generated using computer programs . unlike the authors of review articles unlike the conscientious authors , at any rate much of the data contained in any single database is , by its nature , derivative . in many cases , it has been copied over several times since its original abstraction from some primary source . the lack of discrimination involved in automated database construction may be a virtue when it preserves data that might not fit neatly into a human reviewer s preconceptions or might evade their notice , but it becomes a vice when erroneous data fails to be recognized and labeled as such . the structure of gallamine triethiodide is a good illustrative example where many major databases ended up containing the same mistaken datum . until mid-2011 , anyone relying on an internet search would have erroneously concluded that gallamine triethiodide is a tribasic amine . the error resulted from mis - parsing the common name at some point as meaning that the compound is a salt of gallamine and ethiodidic acid , identifying gallamine as the active component and retrieving the relevant structure . in fact , gallamine triethiodide is what you get when you react gallamine with three equivalents of ethyl iodide ( fig . to gallamine triethiodide conversion of gallamine to gallamine triethiodide it is easy for a name - to - structure conversion program to make this mistake with a common name rather than the systematic one , but the triethiodide usage is systematic in its own context . the same is true for stearates , acetates , palmitates , etc . , where esters often get mixed up with salts . when correctly used , such names are correct and unambiguous in context : 4-androstendiol has no basic centers to form a salt with acetic acid , so the problem is that the usage does not conform to the universal standard expected by software that ignores chemical context . ( as it happens , a simple web search fails to make it clear what the correct iupac name for gallamine triethiodide actually is . n , n - triethylethanaminium ) triiodide , whereas wolframalpha gives it as 2-[2,6-bis(2-triethylammonioethoxy)phenoxy]ethyl - triethyl - azanium triiodide . 1,2,3-tris(2-triethylammonioethoxy)benzene tri - iodide is more informative and fully consistent with the example provided as part of iupac rule c-816.3 but is unlikely to be produced by any automated name generation program that starts by searching for a base name using a prioritized look - up table . ) the authors have been particularly sensitized to this issue by time recently spent curating a data set of cytochrome p450 ( cyp ) reactions , where typographical errors and misinterpretation of semi - systematic drug metabolite names were all too common . ironically , the use of such names in the literature ( as opposed to fully systematic iupac nomenclature , which is more prone to interpretive and typographical errors ) often makes it easier to determine the actual structure of substrates and their metabolites . that said , it is the authors experience that there is no substitute for explicit structural depiction in facilitating validation , something which should be strongly encouraged in all publications , especially when new compounds are introduced . it is tempting to automate curation itself by accepting the version of any given factoid that appears most frequently on the web as the correct one . the ease of implementation seems to make the use of such methods inevitable , but the potential for ( false ) positive reinforcement in such a system may do more harm than good . indeed , the origin of the quote attributed to niels bohr at the start of this article has been addressed in just this way a process which readily yields the wrong answer [ 2527 ] . mis - attribution of a quote is arguably inconsequential , but misassignment of structures either base structures ( as for gallamine triethiodide ) or tautomeric and protonation states can severely compromise a model or simulation . this is especially so when the error is systematic , as in the case of steroid esters or ethiodide salts . the extensive use of automated systems for populating public and private databases with standardized structures can severely exacerbate the problem . misprotonation examples include the misrepresentation of piperazines as diprotonated rather than as monoprotonated species and the conversion of amidinium ions into diprotonated aminals ( fig . 2common misrepresentations of piperazines ( left ) and amidinium cations ( right ) common misrepresentations of piperazines ( left ) and amidinium cations ( right ) such unlikely proximal dications are generated when proximity effects context are ignored , and docking such species into a binding site that bears a net negative charge will wildly exaggerate the affinity of the compound in most cases . so long as science progresses at least in part by making constructive mistakes , there will always be a considerable amount of bad data mixed in with the good . curating such large databases effectively is , however , extraordinarily difficult and only rarely appreciated when it does get done . the problem is exacerbated by three facts of grant application life in 2011 : that support is relatively easy to obtain for compiling new cross - linked databases ; that the perceived value of such databases is based more on the amount of data they include than on the reliability of that data ; and that it is very difficult to obtain funding for maintenance or manually supervised curation of existing databases . this is a remarkable state of affairs for a discipline still recovering from the damage inflicted by the notion that high - throughput screening and combinatorial chemistry programs would produce a groundswell of new drugs by sheer force of numbers . that experience showed clearly that the quality of data is more critical than how much of it there is and underscored the sharp differences in value between data , information and knowledge . one hopes that new compilations will soon be expected to provide an indication of where each datum came from , if not the ultimate primary source . the cyp curation effort alluded to above was greatly facilitated by the data sets being well documented in terms of citation of primary sources . it is also important to cite the actual source from which data was obtained , however . one often finds examples in the literature ( and even more often on web pages ) where a primary source c is cited , when , in fact , a secondary source b was used which cited source c. failing to acknowledge such use of indirect data sources readily propagates errors , e.g. , when source b incorrectly copies or misinterprets the information in source c. when garland marshall , andy vinter and hans - dieter hltje launched this journal 25 years ago , it was believed by some in the community that computers would ultimately perhaps sooner rather than later given that fact , they might well have named it the journal of computational molecular design . they opted instead for the journal of computer - aided molecular design , which proved a prescient choice . it is easier now to appreciate the inherent limitations of computer programs as well as their potential for augmenting and extending the reach of molecular designers . the three hurdles highlighted here understanding entropy , estimating uncertainty and effective curation of large databases are daunting but not technically insurmountable . but the psychological and institutional challenges they represent are perhaps equally forbidding : people naturally tend to focus on the direct and familiar ( enthalpy ) rather than on the subtle ( entropy ) ; they resist quantifying how much they do not know ( uncertainty ) ; and they tend to favor increasing quantity at the expense of quality ( curation ) . those aspects of the challenges , too , can be overcome . if the community does so , the fruit of our labors will be evident to our successors 25 years from now when they read the journal of computer - aided molecular design as their cars cruise through the air on autopilot . or perhaps they will prefer to hear it spoken directly into their minds through their brain chip implant as they try out the latest jet - pack .

the computational chemistry and cheminformatics community faces many challenges to advancing the state of the art . we discuss three of those challenges here : accurately estimating the contribution of entropy to ligand binding ; reliably estimating the uncertainties in model predictions for new molecules ; and being able to effectively curate the ever - expanding literature and commercial databases needed to build new models .

Introduction Entropy Uncertainty Curating oceans of data Conclusion

PMC1568575

a polychlorinated biphenyl ( pcb ) and tri- and tetrachlorinated benzene - containing electrical transformer was involved in an explosion and fire in a modern office building in binghamton , new york , on february 5 , 1981 . because of an unusual system of air shafts the entire building and adjacent garage became contaminated with toxic chemicals . polychlorinated dioxins , furans , and biphenylenes were formed as pyrolytic by - products . before the extent of the chemical contamination was appreciated workers were exposed to these chemicals . four years after the explosion and after the expenditure of over $ 22 million for cleaning and other expenses , the building remains closed.imagesfigure 1.figure 2.figure 3.figure 4.figure 5.figure 6.figure 7.figure 8.figure 9.figure 10 .

Images

PMC4006509

constipation as a common disorder in children accounts for more than 3% of visits to a pediatrician and 10 to 25% of visits to pediatric gastroenterologist . functional constipation accounts for 95% of children with chronic constipation while only 5% have an organic cause for their symptoms . chronic functional constipation ( cfc ) is defined as a delay in defecation with less than three times per week existing for more than two weeks and being associated with stool withholding behavior . treatment includes primary stool evacuation , toilet training , high fiber diet and laxative administration . children usually are treated with a combination of toilet training , running a bowel diary , and oral laxatives . in many patients the early difficulty with defecation that causes pain , fear , and refusal to use toilet , often progresses to the formation of vicious cycles of increasing fecal retention and the rectum increases in capacity , then the experience of passing large , hard stools is repeated . there is increasing distress as overflow fecal incontinence compounds the problem for the older child . surgical treatment has been used in children s refractory constipation treatment . however , surgical methods are often regarded to be traumatic . botulinum toxin a , a widely applicable acetyl - cholinesterase inhibitor that causes clinically reversible muscle paralysis , is now a popular drug for treating many conditions associated with smooth or skeletal muscle dysfunction . transcutaneous needle - free injection of botulinum toxin into the anal sphincter is a novel and safe new treatment of chronic idiopathic constipation and anal fissure in children . the purpose of this study was to determine the utility of intra sphincteric injection of botox in the treatment of children with refractory constipation referred to our center . this was a prospective case control study which was done in children s medical center in tehran university of sciences . all children who suffered from chronic constipation for more than three months , and who had not responded to medical treatment , were referred to pediatric surgical clinic for interventions by pediatric gastroenterologist . perineal examination in the first step provided that they did not have macroscopic anatomical abnormalities . in case of accompanying diseases or abnormal anus and perineum , and children with transition zone in barium enema also , manometry and rectal biopsy was performed in all children . those with ganglion cells in their rectal biopsy and who had high threshold recto anal inhibitory reflex ( rair ) or absent rair in manometry , entered the study . botox injection was carried out under general anesthesia in three regions of the anal sphincter in lithotomy position . the botulinum toxin dysport was injected in sphincter in 3 , 6 and 9 oclock . total dysport dosis was 160 units , half of it ( i.e. 80 units ) was injected in 6 oclock and one - fourth ( i.e. 40 u ) in 3 oclock and one - fourth in 9 oclock , injection was done simultaneously in both internal and external anal sphincters . in the first month after injection , all patients were evaluated once a week and were investigated regarding the improvement or recurrence of symptoms and followed up monthly in the first six months . the response to botox injection six months after injection was compared with the control group . for evaluation of the patients condition after the botox injection , patients were asked questions about the presence of the signs of constipation including painful defecation , vomiting , stool s consistency , soiling and defecation intervals . the sample size based on statistical calculations is 88 patients ( 44 cases and 44 controls ) . continuous variables are expressed in the form of mean and standard deviation . for comparing proportions , chi 2 test and for comparing quantitative variables , defecation of painful stool existed in 88% of patients before botox injection and it was reduced to 15% after botox injection . in the control group,90% of patients had painful defecation , which reduced to 86% after medical treatment and the difference was statistically significant ( p=0.0001 ) . stool condensation existed in 80% of patients before botox injection , and it was reduced to 28% after botox injection . in the control group , it existed in 81% of children and reduced to 78% after medical treatment and the difference was statistically significant ( p=0.0001 ) . soiling existed in 62% of patients before botox injection and was reduced to 8% after botox injection . in the control group it reduced from 62% to 42.5% after medical treatment and defection interval more than 3 days existed in 100% of patients before botox injection and it was reduced to 15% after botox which was also statistically significant ( p=0.0001 ) . in the control group , 98% of the patients had defection intervals more than 3 days and it was the same after medical treatment . in average , defecation interval before botox injection was 9.1 days , and after botox injection was reduced to 2.6 days , which is statistically significant . there were 22 boys and 18 girls , the difference was not statistically significant . in table 1 , the symptoms of chronic constipation before and after the botox injection are expressed as percentages . in table 2 , the percentage of decrease in signs has been brought based on the sex of patients in which there was no significant differece between girls and boys , therefore , the effect of botox injection did not have any relation with the sex of patient with chronic idiopathic constipation . symptoms of chronic constipation before and after botox injection percentages of decrease in signs and symptoms of chronic constipation before and after botulinum toxin injection based on the sex of patients in this study , the minimum age of the patients was 2 years and the maximum 12 years , the most frequent age group was 5 years . diagram 1 shows the frequency of age group s distribution in botox injection group . in table 3 the decrease of signs is compared between two groups of above and below 5 year olds , in which a significant difference is not evident . therefore , the effect of botox injection has no relationship with the age of patients with chronic idiopathic constipation . in table 3 , the decrease of signs has been compared between the two groups of above and below 5 year olds , in which a significant difference was also not observed . therefore , the effect of botox injection has no relationship with the age of patients with chronic idiopathic constipation . the age distribution in children with refractory chronic constipation who received botulinum toxin injection signs and symptoms of chronic constipation in two above and below 5 year olds , before and after botulinum toxin injection when constipation becomes chronic and refractory , it produces much distress and problems for the child and family . the exact cause of chronic constipation is not identified in most children , therefore it is diagnosed as functional or idiopathic . anorectal manometry has shown some abnormalities in these patients including higher threshold to rectal distention or abnormal rectal sensation . another cause of chronic and refractory constipation is internal anal sphincter achalasia . internal anal sphincter achalasia ( iasa ) is a chronic constipation that is clinically very similar to hirschsprung s disease . in patients with iasa , ganglion cells are seen in rectal biopsy and in manometric evaluation rectoanal inhibitory reflex is absent . anal achalasia can cause obstruction or constipation in children who have undergone pull through procedure for treatment of hd . anal dilatation might have a role in management of chronic refractory idiopathic constipation in children in preschool age . an alternative approach to treatment of iasa in patients without hd is toxin botulinum injection in anal sphincter . toxin botulinum interferes with acetylcholine release at the neuromuscular junction in anal sphincter after injection . by this mechanism , it produces a chemical denervation of sphincter . therefore intra sphincteric botulinum toxin injection by inhibition of neural stimulation to sphincter can induce a chemical myectomy and it may be useful in treatment of iasa and chronic idiopathic constipation . in one study , toxin botulinum injection in internal anal sphincter had definite effect in improvement of patients who had obstruction or constipation after pull through for treatment of hirschsprung s disease . in these patients , residual aganglionosis and anastomotic stricture were ruled out by rectal biopsy and barium enema and physical examination . in another study injection of botulinum toxin had a transient effect in treatment of most children with anismus and in some patients it showed a long term effect . in this study painful defecation existed in 88% of patients before toxin botulinum injection and it was reduced to 15% after toxin botulinum injection and the difference was statistically significant ( p=0.0001 ) . moreover , stool condensation existed in 80% of these patients before toxin botulinum injection , it was reduced to 28% after toxin botulinum injection , the difference is statistically significant ( p=0.0001 ) . soiling existed in 62% of patients before botox injection and was reduced to 8% after botox injection . in the control group it reduced from 62% to 42.5% after medical treatment and the difference was statistically significant ( p=0.0001 ) . defection interval more than 3 days existed in 100% of patients before botox injection and it was reduced to 15% after botox which is also statistically significant ( p=0.0001 ) . in the control group , 98% had defection intervals more than 3 days and it remained the same after medical treatment . in average defecation interval before botox injection was 9.1 days and after botox injection was reduced to 2.6 days which is also statistically significant ( p=0.0001 ) . injectio of botulinum toxin into the anal sphincter is an effective and safe new treatment of chronic idiopathic constipation in children .

objectiveinjection of botulinum toxin into the anal sphincter is a novel and safe new treatment of chronic idiopathic constipation and anal fissure in children . the purpose of this study was to determine the utility of intra sphincteric injection of botox in the treatment of children with refractory constipation.methodsall children who suffered from chronic constipation for more than three months , and who had not responded to medical treatment , were referred to pediatrics surgical clinic for surgical intervention by pediatric gastroenterologist . the patients were randomly divided into cases and control group . the control group received no injection and was only treated with stool softeners . the case group received this therapy in addition to injection . after the botox injection , patients were asked about the presence of the signs of constipation including painful defecation , vomiting , stool consistence , soiling and defecation interval.findingsdefecation of painful stool existed in 88% of patients before botox injection and it was reduced to 15% after botox injection . in the control group , 90% of patients had painful defecation , which reduced to 86% after medical treatment ( p=0.0001 ) . stool was hard in 80% of patients before was reduced to 28% after botox injection . in the control group , it existed in 81% of children and reduced to 78% after medical treatment ( p=0.0001 ) . soiling existed in 62% of patients before and was reduced to 8% after botox injection , but in the control group it reduced from 62% to 42.5% after medical treatment ( p=0.0001 ) . in the control group , 98% of the patients had defecation intervals more than 3 days and it was the same after medical treatment . in case group , this index before botox injection was 9.1 days , and after botox injection was reduced to 2.6 days ( p=0.0001).conclusionour study results showed that injection of botulinum toxin into anal sphincter is an effective and safe new treatment of chronic idiopathic constipation in children .

Introduction Subjects and Methods Findings Discussion Conclusion Conflict of Interest

PMC3361853

although chronic mania has been described in psychiatry literature for a century now , it has not received the equivalent status as given to chronic depression by the current nosological systems . one of the major limitations of this is the lack of systematic research on this entity . in this report , we present a case of chronic mania and discuss the clinical features of chronic mania reported in the literature , which are helpful in distinguishing chronic mania from acute mania . mr a , 22-years - old , unemployed , and belonged to a nuclear family of lower socioeconomic status , presented with an insidious - onset illness of 5 years duration , with a continuous course , characterized by dysphoric mood , grandiose ability , grandiose associations , grandiose identity , poor self - care , wandering , abusiveness , assultativeness , and aggressive behavior . the patient had been using cannabis off and on for a few days , but use of or abstinence from cannabis did not have much influence on the nature of psychopathology . he also had a history of tobacco dependence syndrome since the age of 15 years . over the period of 3 years , he frequently absconded from home ; whenever found he would be in a disheveled state and when asked about his whereabouts , he would talk about his grandiose abilities , identity , and association and would say that he had been on his special mission of the army . he was treated with some psychotropic medications after continuation of symptoms for a period of 3 years , with which family members perceived only 20% improvement in his psychopathology . later , he was treated with tablets of olanzapine 20 mg / day , lithium 600 mg / day , and valproate 500 mg / day , and he showed a 25% improvement in his symptoms with the above combination . he had been kept in seclusion for few months before being brought to our center . early developmental history did not reveal any abnormality , and there was no history of hyperthymic traits . there was no history suggestive of schneider 's first - rank symptoms , overfamiliarity , depressive features , head injury , and seizures . on examination , he was found to have dysphoric mood and he expressed grandiose delusions of being a general in the army and ability to control people at remote areas . he also had secondary delusions that people are trying to harm him because of his special powers and abilities . his cognitive functions were preserved , but he lacked insight into his illness . with the available information , his routine investigations and a computerized tomography scan of brain did not reveal any abnormality . he was managed with a course of 12 electroconvulsive therapy treatments along with olanzapine 30 mg and benzodiazepines . he showed only marginal improvement in his symptoms with the above combination . following this , he was shifted to tablets of haloperidol 15 mg / day along with lithium 1050 mg / day ( serum lithium level 0.7 meq / l ) . with this combination , over the period of 6 weeks , the patient showed improvement in his overall behavior , delusion of persecution subsided , but the grandiose delusions persisted . his family was asked to shift the patient to a long - stay mental health facility . however , his family decided to keep the patient at home . over the period of next 1 year , he was continued on the combination of haloperidol 20 mg / day and lithium 1050 mg/ day , during which he did not take up any work and continued to harbor the grandiose delusions and had elevated mood . however , he was better in the form of lack of abusiveness and running away behavior . the current nosological systems define mania by a duration of 7 days , and epidemiological studies suggest that untreated mania usually remits within 6 months , though in some cases it may last longer . classically , chronic mania has been defined as the presence of manic symptoms for more than 2 years without remission . however , in recent years , other researchers have used different definitions such as lack of improvement by at least two points from baseline on the clinical global impression - bipolar disorder mania scale at any observation during the 12 months after starting treatment for acute mania . however in literature , chronic mania lasting for about 48 years has also been described . studies that have evaluated patients with bipolar disorders have reported an incidence of 615% for chronic mania among all the patients with bipolar disorders . with regard to symptomatology and associated clinical features , one of the recent studies that compared patients of acute mania and chronic mania suggested that chronic course usually arises in the background of hyperthymic temperament and recurrent mania , with a deteriorative pattern . furthermore , the authors reported that compared to patients with acute mania , patients with chronic mania have significantly a high rate of almost constant euphoria , grandiose delusions , and related delusions and relatively low rates of sleep disturbance , psychomotor agitation , and hypersexuality . another study suggested that compared with treatment responders , patients who do not respond and run a chronic course have a lower severity of mania symptoms at baseline but a higher prevalence of delusions / hallucinations , have a shorter duration of current episode prior to start of the treatment , are less socially active , and have a higher occupational impairment . however , despite the presence of clear diagnostic symptoms of mania , it is often difficult to diagnose a patient with chronic mania because of superimposed cyclothymic or hyperthymic temperament or overlap of symptoms between mania , schizophrenia , and schizoaffective disorders . in general , it is suggested that the grandiosity in chronic mania is associated with an elevated mood , compared to schizophrenia , in which patients exhibit grandiosity associated with the flat effect . with regard to management , it is generally suggested that chronicity denotes poor outcome ; however , reports contrary to this belief also exist . the index case exhibited manic symptoms for 5 years prior to presentation to us , with minimum improvement with treatment . he has been under our care of about 1.5 years , with no change in the core manic symptoms despite good treatment compliance . this clinical picture fits with the clinical description of chronic mania and suggests that in rare patients , mania can run a chronic course . in recent times , there have been a few reports of chronic mania from various other centers in india , and this suggests that chronic mania does exist in clinic population and there is a need to study this clinical entity more meaningfully to understand its biological correlates and treatment outcome .

chronic mania ( defined as the presence of manic symptoms for more than 2 years without remission ) poses significant problems in diagnosis and management . generally it denotes poor outcome , though contrary reports are available . we present a case of chronic mania and discuss the clinical features of chronic mania reported in the literature , which are useful in distinguishing chronic mania from acute mania .

INTRODUCTION CASE REPORT DISCUSSION

PMC3692136

rna is an important biomolecule , as a catalyst ( 1 ) , a director of post - transcriptional modification ( 2 ) and gene regulation ( 3 ) , a target of drugs ( 4,5 ) and also a pharmaceutical ( 6,7 ) . secondary structure is the set of the canonical base pairs , and tertiary structure is the set of additional contacts and the complete three - dimensional structure . because rna folding is generally hierarchical , secondary structure secondary structure prediction can provide a framework for understanding the mechanism of action of rna . secondary structure prediction is also an important consideration in the design of sirna and antisense dna oligonucleotides both to avoid structure in the mrna target of these agents and in avoiding self - structure in agents , either of which prevents them from hybridizing with their targets ( 914 ) . rna structure prediction can also make predictions about which regions of sequence are accessible for interacting with proteins ( 15 ) . finally , secondary structure prediction can be used to identify novel functional rna sequences encoded in genomes ( 1618 ) . rnastructure is a software package for rna secondary structure prediction and analysis ( 19 ) . it was first reported in 1998 as a free energy minimization program for microsoft windows ( 20 ) . it has been expanded to include methods for predicting bimolecular structure ( 12 ) , conserved structures in multiple homologs ( 2124 ) and sirna design ( 9 ) . several methods are available for predicting structures for a single sequence , including maximum expected accuracy ( 25 ) , stochastic sampling ( 26 ) , exhaustive traceback ( 27 ) and pseudoknot prediction ( 28 ) . graphical user interfaces are provided for microsoft windows , macintosh os - x and linux . command line interfaces are also available for these operating systems . finally , the set of underlying c++ classes are available as a library for use by programmers . this report describes a new set of web servers that provide the functionality of rnastructure . for example , to predict a secondary structure for a single sequence , the server called predict a secondary structure can be used to run calculations by multiple programs . this approach is designed to be user - friendly because it bundles all the available methods for addressing a specific problem , and for this reason most users will want to use these servers . table 1.list of individual programs provided by the rnastructure web serverserversreferencesdescriptionallsub(27,29)generate all possible low free energy structures for a nucleic acid sequence.bifold(12)predict the lowest free energy structure for two interacting sequences , allowing intramolecular base pairs.bipartition(30)perform a partition function calculation for two interacting nucleic acid sequences . intramolecular pairs are not allowed.circlecompare(19)compare two structures using overlaid circular plots to emphasize pairing differences and pseudoknots.ct2dot(19)convert a ct - formatted structure into a dot bracket file.dot2ct(19)convert a dot bracket file into a ct file.draw(19)draw the secondary structure of a nucleic acid strand , with or without color annotation.duplexfold(31)predict the lowest free energy structure for two interacting sequences , not allowing intramolecular base pairs.dynalign(17,21,32)calculate the lowest free energy secondary structures common to two unaligned sequences.efn2(33)calculate the folding free energy of structures in a ct file.ensembleenergy(30)calculate the ensemble folding free energy change for a sequence.fold(33,34)predict the lowest free energy structure and a set of low free energy structures for a sequence.maxexpect(25)generate a structure or structures composed of highly probable base pairs.multilign(22)predict low free energy secondary structures common to three or more sequences using progressive iterations of dynalign.oligoscreen(35)predict stability of hybridization and self - structure for a list of oligonucleotides.oligowalk for sirna design(9,11)design efficient sirnas for a given mirna target.partition(30)perform a partition function calculation on a single sequence to calculate base pair probabilities.parts(23,36)predict the common secondary structure , including base pair probabilities , for two unaligned sequences.probablepair(30)generate secondary structures composed of base pairs with probabilities that exceed a specified threshold.probknot(28)predict a secondary structure composed of probable base pairs , which might include pseudoknots.removepseudoknots(19,37)remove pseudoknots from a structure.scorer(31,30)calculate the sensitivity and positive predictive value ( ppv ) for a predicted as compared to the accepted structure.stochastic(26,36)generate a representative sample of structures using stochastic sampling.turbofold/turboknot(24,38)calculate the conserved structures of three or more unaligned sequences using iteratively refined partition functions . table 2.list of rnastructure web servers organized around a problemserversprograms run ( see table 1 for description)predict a secondary structurefold , maxexpect , probknot , partitionpredict a secondary structure common to two sequencesdynalign , partspredict a secondary structure common to three or more sequencesmultilign , turbofold / turboknotpredict a bimolecular secondary structurebifold , duplexfold list of individual programs provided by the rnastructure web server list of rnastructure web servers organized around a problem the structure prediction servers work using either bare sequences pasted into a window , or fasta - formatted files uploaded to the server . the exception to this is the set of servers that predict a structure common to three or more sequences . for these servers , including multilign and turbofold , the sequence window requires multiple fasta - formatted sequences . servers that act on structures , such as circlecompare , draw , efn2 , removepseudoknots or scorer , require an upload of a ct file . another alternative is to use a dot - bracket formatted structure and convert it to ct format using the dot2ct component of the rnastructure server . for each individual server , sample input data can be generated automatically to illustrate format and to provide a test case for the server . for structure prediction servers , clicking a link pastes sample sequences into the sequence windows . for the servers that act on structures , a link is provided to download a sample ct file , and this can be uploaded back to the server as sample data . for fold , partition and predict a secondary structure , structure prediction can be restrained using shape mapping data , when available , to improve the accuracy of structure prediction ( 39 ) . allsub , dynalign , fold , partition and predict a secondary structure accept folding constraints , including the ability for force specific base pairs , forbid specific pairs , force a nucleotide paired , force a nucleotide unpaired , and specify that a nucleotide is accessible to chemical modification ( 33,34 ) . , an e - mail address can be provided as part of the input data . if this is done , an e - mail is sent to the address when the calculation is complete and the results are available . the structure prediction servers display the predicted structure using an svg drawing , which can be rendered by web browsers with an svg plugin ( figure 1 ) . if more than one structure is predicted , i.e. suboptimal structures or a structure sample is present , previous and next buttons are displayed to enable scrolling between structures . for each predicted structure , the structure can be downloaded as a jpeg , svg , pdf , postscript or ct file . each output page displays the rnastructure executable and the command line used to generate the results ( 40 ) . each input file is a link , allowing download of the processed - form data . this makes clear exactly what calculation is performed , and facilitates a user learning the capabilities of the rnastructure software . the server does not rely on javascript running on the client side , although a single exception to this is the previously available oligowalk web server ( 11 ) . the server itself is a computer cluster with a head node and five compute nodes , each with two dual core processors . a queuing system prioritizes the order at which queries are executed , which is generally in the order in which queries were made . the exceptions to this rule are calculations submitted for dynalign , multilign or turbofold , which are submitted to the server as multiple - core calculations . these may need to wait in the queue at times when a number of other time - consuming calculations are already running . this ensures that the resource will be available to the broader community with reasonable wait times . currently , for example , the single sequence structure prediction methods are limited to sequences of 2500 nucleotides or less . turbofold is limited to a maximum of 12 sequences up to 1600 nucleotides in length . if the required calculation exceeds the web server requirements , the software can be downloaded ( http://rna.urmc.rochester.edu/rnastructure.html ) and run locally . the help documents each of the input and output fields , and is organized by individual server . for each server help page , a link is provided to the underlying rnastructure executable ( 19 ) , which provides additional details about the program . a separate help page details the server limitations , as explained above . rnastructure is designed to be a user - friendly software package , accessible to the community of investigators studying rna ( 19 ) . these new web servers continue this tradition , and provide the software to a wider user base . the accuracy of the algorithms has been extensively tested in prior publications . as the algorithms in rnastructure are improved , and new algorithms developed , the web servers will be updated to continue to make rnastructure available to the community . figure 1.sample structure prediction . this is the predicted lowest free energy structure for the sample sequence for the predict a secondary structure server . it is color annotated according to base pairing probability ; more probable pairs are more likely to be correctly predicted than less probable pairs ( 30 ) this is the predicted lowest free energy structure for the sample sequence for the predict a secondary structure server . it is color annotated according to base pairing probability ; more probable pairs are more likely to be correctly predicted than less probable pairs ( 30 ) . national institutes of health ( nih ) [ r01 gm076485 to d.h.m . ] . funding for open access charge : nih [ r01 gm076485 ] .

rnastructure is a software package for rna secondary structure prediction and analysis . this contribution describes a new set of web servers to provide its functionality . the web server offers rna secondary structure prediction , including free energy minimization , maximum expected accuracy structure prediction and pseudoknot prediction . bimolecular secondary structure prediction is also provided . additionally , the server can predict secondary structures conserved in either two homologs or more than two homologs . folding free energy changes can be predicted for a given rna structure using nearest neighbor rules . secondary structures can be compared using circular plots or the scoring methods , sensitivity and positive predictive value . additionally , structure drawings can be rendered as svg , postscript , jpeg or pdf . the web server is freely available for public use at : http://rna.urmc.rochester.edu/rnastructureweb .

INTRODUCTION ORGANIZATION INPUT OUTPUT FORMATS IMPLEMENTATION HELP CONCLUSION FUNDING

PMC4060514

fuelled by the obesity epidemic , there is considerable interest in the developmental origins of white adipose tissue ( wat ) and the stem / progenitor cells from which it arises . while increased visceral fat mass is associated with metabolic dysfunction , increased subcutaneous wat is protective . there are 6 visceral fat depots : perirenal , gonadal , epicardial , retroperitoneal , omental and mesenteric and it is a subject of much debate whether these have common developmental origins and whether this differs from subcutaneous wat . here we show that all 6 visceral wat depots receive a significant contribution from cells expressing wt1 late in gestation . conversely , no subcutaneous wat or brown adipose tissue ( bat ) arises from wt1 expressing cells . postnatally , a subset of visceral wat continues to arise from wt1 expressing cells , consistent with the finding that wt1 marks a proportion of cell populations enriched in wat progenitors . we show all visceral fat depots have a mesothelial layer like the visceral organs with which they are associated and provide several lines of evidence that wt1 expressing mesothelium can produce adipocytes . these results : reveal a major ontogenetic difference between visceral and subcutaneous wat ; pinpoint the lateral plate mesoderm as a major source of visceral wat ; support the notion that visceral wat progenitors are heterogeneous ; and suggest that mesothelium is a source of adipocytes .

Supplementary Material

PMC3482708

it has been used as methylating agents , laboratory reagents , pesticides , and in microscopy . the human exposure to methyl iodide occurs through routes like dermal , inhalation , and oral ingestion . predominantly neuropsychiatric symptoms of acute exposure to monohalomethanes consist of headache , nausea , vomiting , drowsiness , dizziness , giddiness , diarrhea , confusion , ataxia , slurred speech , paralysis , convulsions , delirium , coma , and death.[26 ] one case of suicidal parenteral exposure has been reported . prominent delayed neuropsychiatric manifestations as behavioral disturbances , cognitive deficits , psychoses , and emotional liability have also been noted lasting for months to years . the primary target organs in cases of severe poisoning are lungs , liver , kidney , and brain . animal studies have shown methyl iodide to be a potential carcinogen and teratogen . a case presenting with repeated overexposure producing recurrent attacks of multifocal neurological dysfunction mimicking multiple sclerosis has also been reported . we report two cases who presented to emergency services of tertiary care public hospital , after accidental exposure to methyl iodide for a short duration . a 23-year - old industrial worker , was accidentally exposed to an unknown quantity methyl iodide vapors for 5 to 10 seconds at his place of work . this resulted in loss of consciousness for few minutes . after regaining consciousness , he had altered behavior in the form of muttering to self , running around aimlessly , picking at his clothes and saying fearfully that somebody is coming to kill him . mental status examination further revealed that he was disoriented in time , place , and person ; physical examination showed pulse was 102 per minute and weak and blood pressure was 100/70 mmhg at the time of emergency evaluation . his pupils were dilated and he had a left seventh nerve palsy of lower motor neuron type and depressed gag reflex . the patient reported fearfulness , persecutory delusions , and occasional auditory hallucinations after resolution of neurological symptoms . a 34 year industrial worker , was brought with accidental workplace exposure to methyl iodide for 15 to 30 s. he had been working there for the past 9 years . on his entry to emergency unit , his pulse was 90/min and blood pressure was 110/80 mm of hg and respiratory rate was 30/min . all routine investigations revealed no abnormality except for small pleural effusion on the chest x - ray . gradually , the patient 's sensorium improved over next 2 days , he complained of extreme anxiety , fear , restlessness , and poor sleep . over the same time span , the patient showed complete resolution of the neurological signs . in both these cases no history of any other organic , metabolic , infective , or intoxication causes was found on detailed enquiry . they were given benzodiazepine drugs for symptomatic relief , i.e. to control anxiety and agitation . a 23-year - old industrial worker , was accidentally exposed to an unknown quantity methyl iodide vapors for 5 to 10 seconds at his place of work . this resulted in loss of consciousness for few minutes . after regaining consciousness , he had altered behavior in the form of muttering to self , running around aimlessly , picking at his clothes and saying fearfully that somebody is coming to kill him . mental status examination further revealed that he was disoriented in time , place , and person ; physical examination showed pulse was 102 per minute and weak and blood pressure was 100/70 mmhg at the time of emergency evaluation . his pupils were dilated and he had a left seventh nerve palsy of lower motor neuron type and depressed gag reflex . the patient reported fearfulness , persecutory delusions , and occasional auditory hallucinations after resolution of neurological symptoms . a 34 year industrial worker , was brought with accidental workplace exposure to methyl iodide for 15 to 30 s. he had been working there for the past 9 years . on his entry to emergency unit , his pulse was 90/min and blood pressure was 110/80 mm of hg and respiratory rate was 30/min . all routine investigations revealed no abnormality except for small pleural effusion on the chest x - ray . gradually , the patient 's sensorium improved over next 2 days , he complained of extreme anxiety , fear , restlessness , and poor sleep . over the same time span , the patient showed complete resolution of the neurological signs . in both these cases no history of any other organic , metabolic , infective , or intoxication causes was found on detailed enquiry . they were given benzodiazepine drugs for symptomatic relief , i.e. to control anxiety and agitation . these case reports highlight concurrence of frankly psychotic features and acute confusional state in workers vulnerable to industrial exposure to toxic chemicals . understanding the mechanism of neurotoxicity will perhaps throw some light on co - existence of both psychiatric and neurological symptoms . these have been shown to bind to cytochrome oxidase in the mitochondria and thus disrupt the oxidative pathway of atp generation leading to cell death and neurotoxicity . depletion of gsh has also shown to decrease cell 's ability to combat oxidative stresses and thus the cytotoxicity.[1113 ] further , the presence of psychotic features in such patients does not warrant treatment with antipsychotic drugs and caution should be maintained while doing the same in an already compromised nervous system . no specific antidote or treatment modality has been studied except for efficacy of n - acetylcysteine in a few cases . awareness of these toxic effects at vulnerable work places will lead to timely and appropriate interventions . importance of safety precautions and education of both workers and supervisors can not be overemphasized here . relatively , the indian literature is found to be inadequate regarding safety exposure limits ( sels ) of individual industrial chemicals . occupational safety and health administration ( osha ) , 1989 , and american conference of governmental industrial hygienists ( acgih ) , 1984 - 85 , recommend permissible exposure limit ( pel ) of methyl iodide as ( for 8 h ) 2 ppm . however , timely identification of toxic effects and early intervention will go a long way in these unfortunate accidental events .

methyl iodide is a monohalomethane and with a chemical formula ch3i . acute exposures to methyl iodide have frequently occurred in the workplace . predominantly , neuropsychiatric symptoms of acute exposure to monohalomethanes consist of headache , nausea , vomiting , drowsiness , dizziness , giddiness , diarrhea , confusion , ataxia , slurred speech , paralysis , convulsions , delirium , coma , and death . we report two cases who presented to our emergency services after accidental exposure to methyl iodide for a short duration . these case reports highlighted concurrence of frankly psychotic features and acute confusional state in workers vulnerable to industrial exposure to toxic chemicals . understanding the mechanism of neuro - toxicity will perhaps throw some light on co - existence of both psychiatric and neurological symptoms . awareness of these toxic effects at vulnerable work places will lead to timely and appropriate interventions . importance of safety precautions and education of both workers and supervisors can not be overemphasized here

INTRODUCTION CASE REPORTS Case 1 Case 2 DISCUSSION

PMC2234497

cyclic nucleotide gated ( cng ) channels are members of the voltage - gated channel family and are the subject of many reviews ( finn et al . , 1996 ; pugh , 1996 ; zagotta and siegelbaum , 1996 ; karpen , 1997 ; li et al . , 1997 ; molday and molday , 1998 ; biel et al , 1999 ; broillet and firestein , 1999 ; frings , 1999 ; richards and gordon , 2000 ; bradley et al . , 2001 ; flynn et al . , 2001 ; kaupp and seifert , 2002 ; warren and molday , 2002 ; zimmerman , 2002 ; matulef and zagotta , 2003 ) . cng channels that mediate the light response in retinal rods are nonselective cation channels that are weakly voltage dependent and are thought to be tetramers consisting of one - and three -subunits . they can be modulated by a number of factors , including phosphorylation enzymes , ca / calmodulin , and divalent cations . we recently found that rod cng channels can also be modulated by all - trans - retinal ( atr ) and related compounds ( dean et al . , 2002 ) . atr is a member of a class of compounds called retinoids that includes vitamin a and its derivatives ( nau and blaner , 1999 ) . retinoids regulate a wide variety of physiological processes , including gene transcription , immune responses , and visual transduction . in rod visual transduction , the only direct action of light is the photoisomerization of the retinoid chromophore 11-cis - retinal to atr within opsin , the protein portion of the photopigment rhodopsin , in the rod disk membrane . this isomerization induces a conformational change in rhodopsin , which leads to the activation of a gtp - binding protein , transducin . the resulting decrease in cgmp concentration causes closure of cng ion channels in the rod outer segment plasma membrane , hyperpolarizing the cell , and reducing the release of glutamate onto retinal bipolar cells ( rodieck , 1998 ; pugh and lamb , 2000 ; roof and makino , 2000 ; burns and baylor , 2001 ; fain et al . after light activates the transduction cascade , atr is released from its binding pocket in rhodopsin into the rod disk interior , where it apparently binds an abcr transporter that shuttles it across the disk membrane into the cytosol to be converted by retinol dehydrogenase to atr ( sun et al . , 1999 ; weng et al . , 1999 ; next , all - trans - retinol is transferred from the rod outer segment to the extracellular space , where it may be complexed with interphotoreceptor retinoid binding protein ( irbp ; pepperberg et al . , 1993 ) , which appears to protect it from degradation . all - trans - retinol then enters the surrounding retinal pigment epithelial cells , where it is converted back into 11-cis - retinal , which is eventually returned to the rod to be reinserted into opsin ( crouch et al . , 1996 ; saari , 1999 ) . each of these processes is occurring in the tens of minutes required to reset rhodopsin during dark adaptation after exposure to bright light . at the same time , other components in the transduction cascade are also being reset . this includes production of cgmp by guanylate cyclase , so that cng channels can reopen . approximately 3 mm atr is expected to be released from rhodopsin in bright light , if the rod outer segment is considered as a single compartment ( saari , 1999 ) . since retinoids have been shown to modulate several kinds of ion channels ( sidell and schlichter , 1986 ; bosma and sidell , 1988 ; fukuhara et al . , 1997 ; vellani et al . , 2000 ; zhang and mcmahon , 2000 , 2001 ) , it seemed natural to ask whether they might also modulate rod cng channels . we recently showed that atr can induce long shut states in rod cng channels , and that it acts in the nanomolar range at near physiological cgmp concentrations ( dean et al . , 2002 ) . here we further characterize the inhibition of rod cng channels by atr , and suggest that atr is a closed - state inhibitor that prefers unliganded channels and has profound effects on the channel 's cgmp sensitivity . our findings raise the question of whether atr might contribute to the slow time course of dark adaptation by making it more difficult for channels to reopen despite the increase in cgmp concentration after cessation of light . nm-174278 ) and rod ( cngb1 ; embl / genbank / ddbj accession no . x89626 ) were provided by william n. zagotta ( university of washington , seattle , wa ) ( in the pgemhe plasmid ) and robert s. molday ( university of british columbia , vancouver , canada ) , respectively . the pgemhe plasmid contained the untranslated sequence of the xenopus -globin gene to promote high protein expression in oocytes ( liman et al . , 1992 ) . partial ovariectomies were performed on anesthetized xenopus laevis frogs , and individual oocytes were isolated by treatment with 1 mg / ml collagenase type 1a ( worthington ) in a low - calcium solution ( 82.5 mm nacl , 2.5 mm kcl , 5 mm hepes , 1 mm mgcl2 , at ph 7.6 ) . for coexpression of and subunits , crna for cnga1 and cngb1 were mixed in a ratio of 1:4 before injection . for current measurements from multichannel patches , injected oocytes were incubated at 16c for 112 d before patch clamp experiments . for single - channel studies , the same amount of crna was injected , but the oocytes were kept at 16c overnight and then incubated at 4c ( to slow expression ) for 112 d before patch - clamp experiments . oocytes were stored in a solution containing : 96 mm nacl , 2 mm kcl , 1.8 mm cacl2 , 5 mm hepes , 1 mm mgcl2 , 2.5 mm pyruvic acid , 100 u / ml penicillin , and 100 g / ml streptomycin , at ph 7.6 . the vitelline membrane was removed by mechanical dissection after treatment with a hypertonic solution containing : 100 mm n - methyl - d - glucamine , 2 mm kcl , 10 mm egta , 10 mm hepes , and 1 mm mgcl2 , at ph 7.4 . water - soluble solutions were applied using a 36-solution patch perfusion system , rsc-100 rapid solution changer ( molecular kinetics ) . both sides of the patches were bathed in a low - divalent sodium solution consisting of : 130 mm nacl , 200 m edta , and 2 mm hepes , at ph 7.2 . the solution bathing the intracellular surface of the patch contained various concentrations of cgmp ( sigma - aldrich ) dissolved in the low - divalent solution . niflumic acid ( 500 m ; sigma - aldrich ) was added to the extracellular solution to block ca - activated cl channels endogenous to xenopus oocytes . atr stocks were made in 100% ethanol and kept in amber glass vials covered in aluminum foil and stored at 80c or 20c until use . the purity and stability of the atr stock was checked by measuring the absorption spectrum ( 200800 nm ) with a beckman du640 spectrophotometer . atr was applied to the intracellular or extracellular surface of patches by removing 50% of the bath volume , vigorously mixing the retinoid stock into this solution using a glass pasteur pipette in a glass beaker , and then pouring this solution back into the remaining bath and mixing again . we found that the greatest concentration of ethanol ( 0.1% ) applied to any patch had no effect on cgmp - activated current or on the seal resistance . atr ( sigma - aldrich ) stocks were applied to patches under dim room light conditions . spectroscopic measurements showed no degradation of atr under these conditions ; however , degradation was apparent in brighter room light . standard patch clamp methods were used to record currents from excised , inside - out , and outside - out oocyte patches . pipette openings were typically 0.55 m in diameter with resistances of 1.015 m after fire polishing . macroscopic currents were recorded at voltages ranging from 100 to + 100 mv in 50-mv steps from a holding potential of 0 mv . leak currents were measured in the low divalent solution without cgmp for inside - out patches , and in 5 mm mgcl2 to block the cng channels for outside - out patches . all macroscopic currents from multichannel patches were measured in the steady - state after completion of voltage - dependent gating ( karpen et al . , 1988 ) and before significant ion depletion effects ( zimmerman et al . , 1988 ) . for heteromeric channels , the presence of the ( cngb1 ) subunit was confirmed by exposing the patch to l - cis - diltiazem , which sensitively blocks heteromeric channels but has little effect on homomeric channels ( chen et al . , 1993 ) . single - channel records were obtained as sequential sweeps lasting 8 s each , while membrane voltage was held continuously at + 80 mv . pipettes were coated with dental wax before polishing to reduce the noise from electrode capacitance . atr was added to patches only after allowing for completion of the spontaneous increases in apparent cgmp affinity of the rod channel due to dephosphorylation by endogenous patch - associated phosphatases ( gordon et al . , 1992 ; molokanova et al . , these increases in apparent cgmp affinity took tens of minutes and were monitored in inside - out patches by sampling the current periodically at cgmp concentrations ( typically 10 m ) that were below the k1/2 , while incubating the patch the rest of the time in saturating cgmp ( 2 mm ) to accelerate the process ( molokanova et al . , 1999 ) . since changing the cgmp concentration was not practical for outside - out patches , we simply incubated these patches in 2 mm cgmp for 4060 min before applying atr . for atr dose response curves on inside - out patches , the bath contained either a saturating concentration of cgmp ( 2 mm ) or a low concentration of cgmp that activated 78% of the maximal current at 2 mm cgmp . the current was monitored for about an hour after each addition of atr to ensure that steady - state had been reached . experiments addressing the voltage dependence of atr inhibition were performed at a constant holding potential of either + 50 or 50 mv , with frequent sampling of the current at that holding potential until steady - state had been reached . then the holding potential was jumped to the opposite voltage and again monitored until steady - state was reached . for most patches , the leak was rechecked at the end of the experiment by applying the low divalent solution to the patch through a glass capillary tube anchored in the bath and attached to a syringe . patch currents were recorded using an axopatch 1b or 200 patch - clamp amplifier ( axon instruments , inc . ) with analogue - to - digital converters connected to a macintosh quadra or g4 computer running pulse software ( instrutech ) . the data were low - pass filtered at 2 khz and sampled at 10 or 25 khz . data analysis was performed using the igorpro software package ( wavemetrics ) or qub suite ( www.qub.buffalo.edu ) . the model depicted in fig . 3 and 8 a has the following definitions and equations : o = open , ion - conducting channel . i0 = maximum possible current at a fixed cgmp or camp concentration with [ r ] = 0 . \documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{gather*}\frac{{\mathrm{i}}}{{\mathrm{i}}_{{\mathrm{max}}}}= \\ \frac { \left \left[{\mathrm{o}}\right ] \right } { \left \left[{\mathrm{o}}\right ] \right + \left \left[{\mathrm{c}}_{{\mathrm{1}}}\right ] \right + \left \left[{\mathrm{c}}_{{\mathrm{2}}}\right ] \right + \left \left[{\mathrm{c}}_{{\mathrm{3}}}\right ] \right + \left \left[{\mathrm{c}}_{{\mathrm{1}}}{\mathrm{r}}\right ] \right + \left \left[{\mathrm{c}}_{{\mathrm{2}}}{\mathrm{r}}\right ] \right + \left \left[{\mathrm{c}}_{{\mathrm{3}}}{\mathrm{r}}\right ] \right + \left \left[{\mathrm{c}}_{{\mathrm{4}}}{\mathrm{r}}\right ] \right } = \\ \frac{{\mathrm{l}}}{{\mathrm{l}}+{\mathrm{1}}+ \left \left({\mathrm{k}}_{{\mathrm{4}}}{\mathrm{l}}+{\mathrm{k}}_{{\mathrm{3}}}\right ) \right \left \left[{\mathrm{r}}\right ] \right ^{{\mathrm{n}}}+\displaystyle\frac{{\mathrm{1}}+{\mathrm{k}}_{{\mathrm{1 } } } \left \left[{\mathrm{r}}\right ] \right ^{{\mathrm{n}}}}{{\mathrm{k}}_{{\mathrm{cn}}}^{{\mathrm{2 } } } \left \left[{\mathrm{cgmp}}\right ] \right ^{{\mathrm{2}}}}+\displaystyle\frac{{\mathrm{1}}+{\mathrm{k}}_{{\mathrm{2 } } } \left \left[{\mathrm{r}}\right ] \right ^{{\mathrm{n}}}}{\displaystyle\frac{{\mathrm{1}}}{{\mathrm{2}}}{\mathrm{k}}_{{\mathrm{cn } } } \left \left[{\mathrm{cgmp}}\right ] \right } } \end{gather*}\end{document } \documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{gather*}\frac{{\mathrm{i}}}{{\mathrm{i}}_{{\mathrm{0}}}}= \\ \frac { \left \left({\mathrm{l}}+{\mathrm{1}}+\displaystyle\frac{{\mathrm{1}}}{{\mathrm{k}}_{{\mathrm{cn}}}^{{\mathrm{2 } } } \left \left[{\mathrm{cgmp}}\right ] \right ^{{\mathrm{2}}}}+\displaystyle\frac{{\mathrm{1}}}{\displaystyle\frac{{\mathrm{1}}}{{\mathrm{2}}}{\mathrm{k}}_{{\mathrm{cn } } } \left \left[{\mathrm{cgmp}}\right ] \right } \right ) \right { \mathrm{i}}_{{\mathrm{max } } } } { \left \left\{{\mathrm{l}}+{\mathrm{1}}+ \left \left({\mathrm{k}}_{{\mathrm{4}}}{\mathrm{l}}+{\mathrm{k}}_{{\mathrm{3}}}\right ) \right \left \left[{\mathrm{r}}\right ] \right ^{{\mathrm{n}}}+\displaystyle\frac{{\mathrm{1}}+{\mathrm{k}}_{{\mathrm{1 } } } \left \left[{\mathrm{r}}\right ] \right ^{{\mathrm{n}}}}{{\mathrm{k}}_{{\mathrm{cn}}}^{{\mathrm{2 } } } \left \left[{\mathrm{cgmp}}\right ] \right ^{{\mathrm{2}}}}+\displaystyle\frac{{\mathrm{1}}+{\mathrm{k}}_{{\mathrm{2 } } } \left \left[{\mathrm{r}}\right ] \right ^{{\mathrm{n}}}}{\displaystyle\frac{{\mathrm{1}}}{{\mathrm{2}}}{\mathrm{k}}_{{\mathrm{cn } } } \left \left[{\mathrm{cgmp}}\right ] \right } \right\ } \right { \mathrm{i}}_{{\mathrm{max}}}}\end{gather*}\end{document } \documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{equation*}{\mathrm{i}}_{{\mathrm{0}}}=\frac{{\mathrm{l } } \left \left({\mathrm{i}}_{{\mathrm{max}}}\right ) \right } { { \mathrm{l}}+{\mathrm{1}}+\displaystyle\frac{{\mathrm{1}}}{{\mathrm{k}}_{{\mathrm{cn}}}^{{\mathrm{2 } } } \left \left[{\mathrm{cgmp}}\right ] \right ^{{\mathrm{2}}}}+\displaystyle\frac{{\mathrm{1}}}{\displaystyle\frac{{\mathrm{1}}}{{\mathrm{2}}}{\mathrm{k}}_{{\mathrm{cn } } } \left \left[{\mathrm{cgmp}}\right ] \right } } \end{equation*}\end{document } values of kcn and l for activation of the channel by cgmp and camp were derived from fits of cyclic nucleotide dose 3 , circles ; and for camp , fig . 2 of crary et al . , 2000 ) . these values of kcn and l are comparable to those in the literature ( e.g. , fodor et al . , 1997 ; rosenbaum et al . , 2003 ) and predict an open probability of 0.95 in 2 mm cgmp , which is similar to that measured in our single - channel recordings ( dean et al . , 2002 ) . an open probability of 0.05 in saturating camp is consistent with the ratio of maximum currents obtained for camp and cgmp after the spontaneous dephosphorylation process that shifts the cyclic nucleotide dose response curves of these channels in excised patches ( see materials and methods ; gordon et al . , 1992 ; molokanova et al . , nm-174278 ) and rod ( cngb1 ; embl / genbank / ddbj accession no . x89626 ) were provided by william n. zagotta ( university of washington , seattle , wa ) ( in the pgemhe plasmid ) and robert s. molday ( university of british columbia , vancouver , canada ) , respectively . the pgemhe plasmid contained the untranslated sequence of the xenopus -globin gene to promote high protein expression in oocytes ( liman et al . , 1992 ) . partial ovariectomies were performed on anesthetized xenopus laevis frogs , and individual oocytes were isolated by treatment with 1 mg / ml collagenase type 1a ( worthington ) in a low - calcium solution ( 82.5 mm nacl , 2.5 mm kcl , 5 mm hepes , 1 mm mgcl2 , at ph 7.6 ) . for coexpression of and subunits , crna for cnga1 and cngb1 were mixed in a ratio of 1:4 before injection . for current measurements from multichannel patches , injected oocytes were incubated at 16c for 112 d before patch clamp experiments . for single - channel studies , the same amount of crna was injected , but the oocytes were kept at 16c overnight and then incubated at 4c ( to slow expression ) for 112 d before patch - clamp experiments . oocytes were stored in a solution containing : 96 mm nacl , 2 mm kcl , 1.8 mm cacl2 , 5 mm hepes , 1 mm mgcl2 , 2.5 mm pyruvic acid , 100 u / ml penicillin , and 100 g / ml streptomycin , at ph 7.6 . the vitelline membrane was removed by mechanical dissection after treatment with a hypertonic solution containing : 100 mm n - methyl - d - glucamine , 2 mm kcl , 10 mm egta , 10 mm hepes , and 1 mm mgcl2 , at ph 7.4 . water - soluble solutions were applied using a 36-solution patch perfusion system , rsc-100 rapid solution changer ( molecular kinetics ) . both sides of the patches were bathed in a low - divalent sodium solution consisting of : 130 mm nacl , 200 m edta , and 2 mm hepes , at ph 7.2 . the solution bathing the intracellular surface of the patch contained various concentrations of cgmp ( sigma - aldrich ) dissolved in the low - divalent solution . niflumic acid ( 500 m ; sigma - aldrich ) was added to the extracellular solution to block ca - activated cl channels endogenous to xenopus oocytes . atr stocks were made in 100% ethanol and kept in amber glass vials covered in aluminum foil and stored at 80c or 20c until use . the purity and stability of the atr stock was checked by measuring the absorption spectrum ( 200800 nm ) with a beckman du640 spectrophotometer . atr was applied to the intracellular or extracellular surface of patches by removing 50% of the bath volume , vigorously mixing the retinoid stock into this solution using a glass pasteur pipette in a glass beaker , and then pouring this solution back into the remaining bath and mixing again . we found that the greatest concentration of ethanol ( 0.1% ) applied to any patch had no effect on cgmp - activated current or on the seal resistance . atr ( sigma - aldrich ) stocks were applied to patches under dim room light conditions . spectroscopic measurements showed no degradation of atr under these conditions ; however , degradation was apparent in brighter room light . standard patch clamp methods were used to record currents from excised , inside - out , and outside - out oocyte patches . pipette openings were typically 0.55 m in diameter with resistances of 1.015 m after fire polishing . macroscopic currents were recorded at voltages ranging from 100 to + 100 mv in 50-mv steps from a holding potential of 0 mv . leak currents were measured in the low divalent solution without cgmp for inside - out patches , and in 5 mm mgcl2 to block the cng channels for outside - out patches . all macroscopic currents from multichannel patches were measured in the steady - state after completion of voltage - dependent gating ( karpen et al . , 1988 ) and before significant ion depletion effects ( zimmerman et al . , 1988 ) . for heteromeric channels , the presence of the ( cngb1 ) subunit was confirmed by exposing the patch to l - cis - diltiazem , which sensitively blocks heteromeric channels but has little effect on homomeric channels ( chen et al . , 1993 ) . single - channel records were obtained as sequential sweeps lasting 8 s each , while membrane voltage was held continuously at + 80 mv . pipettes were coated with dental wax before polishing to reduce the noise from electrode capacitance . atr was added to patches only after allowing for completion of the spontaneous increases in apparent cgmp affinity of the rod channel due to dephosphorylation by endogenous patch - associated phosphatases ( gordon et al . , 1992 ; 1997 ) . these increases in apparent cgmp affinity took tens of minutes and were monitored in inside - out patches by sampling the current periodically at cgmp concentrations ( typically 10 m ) that were below the k1/2 , while incubating the patch the rest of the time in saturating cgmp ( 2 mm ) to accelerate the process ( molokanova et al . since changing the cgmp concentration was not practical for outside - out patches , we simply incubated these patches in 2 mm cgmp for 4060 min before applying atr . for atr dose response curves on inside - out patches , the bath contained either a saturating concentration of cgmp ( 2 mm ) or a low concentration of cgmp that activated 78% of the maximal current at 2 mm cgmp . the current was monitored for about an hour after each addition of atr to ensure that steady - state had been reached . experiments addressing the voltage dependence of atr inhibition were performed at a constant holding potential of either + 50 or 50 mv , with frequent sampling of the current at that holding potential until steady - state had been reached . then the holding potential was jumped to the opposite voltage and again monitored until steady - state was reached . for most patches , the leak was rechecked at the end of the experiment by applying the low divalent solution to the patch through a glass capillary tube anchored in the bath and attached to a syringe . patch currents were recorded using an axopatch 1b or 200 patch - clamp amplifier ( axon instruments , inc . ) with analogue - to - digital converters connected to a macintosh quadra or g4 computer running pulse software ( instrutech ) . the data were low - pass filtered at 2 khz and sampled at 10 or 25 khz . data analysis was performed using the igorpro software package ( wavemetrics ) or qub suite ( www.qub.buffalo.edu ) . 3 and 8 a has the following definitions and equations : o = open , ion - conducting channel . i0 = maximum possible current at a fixed cgmp or camp concentration with [ r ] = 0 . \documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{gather*}\frac{{\mathrm{i}}}{{\mathrm{i}}_{{\mathrm{max}}}}= \\ \frac { \left \left[{\mathrm{o}}\right ] \right } { \left \left[{\mathrm{o}}\right ] \right + \left \left[{\mathrm{c}}_{{\mathrm{1}}}\right ] \right + \left \left[{\mathrm{c}}_{{\mathrm{2}}}\right ] \right + \left \left[{\mathrm{c}}_{{\mathrm{3}}}\right ] \right + \left \left[{\mathrm{c}}_{{\mathrm{1}}}{\mathrm{r}}\right ] \right + \left \left[{\mathrm{c}}_{{\mathrm{2}}}{\mathrm{r}}\right ] \right + \left \left[{\mathrm{c}}_{{\mathrm{3}}}{\mathrm{r}}\right ] \right + \left \left[{\mathrm{c}}_{{\mathrm{4}}}{\mathrm{r}}\right ] \right } = \\ \frac{{\mathrm{l}}}{{\mathrm{l}}+{\mathrm{1}}+ \left \left({\mathrm{k}}_{{\mathrm{4}}}{\mathrm{l}}+{\mathrm{k}}_{{\mathrm{3}}}\right ) \right \left \left[{\mathrm{r}}\right ] \right ^{{\mathrm{n}}}+\displaystyle\frac{{\mathrm{1}}+{\mathrm{k}}_{{\mathrm{1 } } } \left \left[{\mathrm{r}}\right ] \right ^{{\mathrm{n}}}}{{\mathrm{k}}_{{\mathrm{cn}}}^{{\mathrm{2 } } } \left \left[{\mathrm{cgmp}}\right ] \right ^{{\mathrm{2}}}}+\displaystyle\frac{{\mathrm{1}}+{\mathrm{k}}_{{\mathrm{2 } } } \left \left[{\mathrm{r}}\right ] \right ^{{\mathrm{n}}}}{\displaystyle\frac{{\mathrm{1}}}{{\mathrm{2}}}{\mathrm{k}}_{{\mathrm{cn } } } \left \left[{\mathrm{cgmp}}\right ] \right } } \end{gather*}\end{document } \documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{gather*}\frac{{\mathrm{i}}}{{\mathrm{i}}_{{\mathrm{0}}}}= \\ \frac { \left \left({\mathrm{l}}+{\mathrm{1}}+\displaystyle\frac{{\mathrm{1}}}{{\mathrm{k}}_{{\mathrm{cn}}}^{{\mathrm{2 } } } \left \left[{\mathrm{cgmp}}\right ] \right ^{{\mathrm{2}}}}+\displaystyle\frac{{\mathrm{1}}}{\displaystyle\frac{{\mathrm{1}}}{{\mathrm{2}}}{\mathrm{k}}_{{\mathrm{cn } } } \left \left[{\mathrm{cgmp}}\right ] \right } \right ) \right { \mathrm{i}}_{{\mathrm{max } } } } { \left \left\{{\mathrm{l}}+{\mathrm{1}}+ \left \left({\mathrm{k}}_{{\mathrm{4}}}{\mathrm{l}}+{\mathrm{k}}_{{\mathrm{3}}}\right ) \right \left \left[{\mathrm{r}}\right ] \right ^{{\mathrm{n}}}+\displaystyle\frac{{\mathrm{1}}+{\mathrm{k}}_{{\mathrm{1 } } } \left \left[{\mathrm{r}}\right ] \right ^{{\mathrm{n}}}}{{\mathrm{k}}_{{\mathrm{cn}}}^{{\mathrm{2 } } } \left \left[{\mathrm{cgmp}}\right ] \right ^{{\mathrm{2}}}}+\displaystyle\frac{{\mathrm{1}}+{\mathrm{k}}_{{\mathrm{2 } } } \left \left[{\mathrm{r}}\right ] \right ^{{\mathrm{n}}}}{\displaystyle\frac{{\mathrm{1}}}{{\mathrm{2}}}{\mathrm{k}}_{{\mathrm{cn } } } \left \left[{\mathrm{cgmp}}\right ] \right } \right\ } \right { \mathrm{i}}_{{\mathrm{max}}}}\end{gather*}\end{document } \documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{equation*}{\mathrm{i}}_{{\mathrm{0}}}=\frac{{\mathrm{l } } \left \left({\mathrm{i}}_{{\mathrm{max}}}\right ) \right } { { \mathrm{l}}+{\mathrm{1}}+\displaystyle\frac{{\mathrm{1}}}{{\mathrm{k}}_{{\mathrm{cn}}}^{{\mathrm{2 } } } \left \left[{\mathrm{cgmp}}\right ] \right ^{{\mathrm{2}}}}+\displaystyle\frac{{\mathrm{1}}}{\displaystyle\frac{{\mathrm{1}}}{{\mathrm{2}}}{\mathrm{k}}_{{\mathrm{cn } } } \left \left[{\mathrm{cgmp}}\right ] \right } } \end{equation*}\end{document } values of kcn and l for activation of the channel by cgmp and camp were derived from fits of cyclic nucleotide dose 3 , circles ; and for camp , fig . 2 of crary et al . , 2000 ) . these values of kcn and l are comparable to those in the literature ( e.g. , fodor et al . , 1997 ; rosenbaum et al . , 2003 ) and predict an open probability of 0.95 in 2 mm cgmp , which is similar to that measured in our single - channel recordings ( dean et al . , 2002 ) . an open probability of 0.05 in saturating camp is consistent with the ratio of maximum currents obtained for camp and cgmp after the spontaneous dephosphorylation process that shifts the cyclic nucleotide dose response curves of these channels in excised patches ( see materials and methods ; gordon et al . , 1992 ; molokanova et al . , 1 , which presents current families from two multi - channel patches in response to a series of voltage pulses in the presence of saturating ( left ) or low ( right ) cgmp , with or without atr . greater inhibition was seen with only 30 nm atr in low cgmp than with ten times as much atr in high cgmp . fig . response curves for patches exposed to low and saturating cgmp . for these data , low cgmp was that required to give 7.4% of the current obtained for each patch with saturating cgmp . because there is some patch - to - patch ( i.e. , channel - to - channel ) variability in apparent cgmp affinity , the exact concentration of cgmp needed to obtain 7.4% imax also varied , but was 1015 m . the atr dose response curves were fit with hill relations with cooperativity constants of 1.41.8 , suggesting that more than one atr molecule may interact with each channel . atr inhibits homomeric ( cnga1 ) rod channels more potently at low than at saturating cgmp concentrations . currents were measured from multichannel , inside - out patches of homomeric ( cnga1 ) rod channels . the raw traces represent families of cgmp - activated currents in response to voltage steps ranging from 100 to + 100 mv in 50-mv increments , from a holding potential of 0 mv . ( left ) current families depicting control and inhibition at saturating cgmp ( 2 mm cgmp ) by 300 nm atr ( 50.6% inhibition ) . ( right ) current families showing control and inhibition by 30 nm atr ( 67.6% inhibition ) at a low cgmp concentration that elicits 7.4% of maximal cgmp induced current . response relations for homomeric ( cnga1 ) and heteromeric ( cnga1 + cngb1 ) rod cng channels . cyclic gmp activated currents were measured at + 100 mv before and after atr addition . currents were monitored for 1 h after atr addition to ensure that the atr inhibition had reached steady - state . averaged data , plotted with sem ( error bars ) were fit with the hill equation ( solid or dashed curves ) : i / i0 = ic50/(ic50 + [ atr ] ) , where i is current remaining at a given [ atr ] , i0 is the current before atr addition , ic50 is the concentration of atr required to achieve half maximal inhibition , and n is the hill ( cooperativity ) coefficient . data points are averaged from 19 patches ; ic50 = 220 nm and n = 1.4 . open triangles , heteromeric ( cnga1 and cngb1 ) rod channel dose response relation for atr inhibition in saturating ( 2 mm ) cgmp . data points are from five patches ; ic50 = 185 nm and n = 1.8 . open circles , homomeric ( cnga1 ) rod channel dose response relation for atr inhibition at cgmp concentrations evoking 7.4% activation . the native rod channel appears to be a heteromer containing one subunit ( cngb1 ) and three subunits ( cnga1 ; weitz et al . , 2002 ; zheng et al . , 2002 ; zhong et al . , 2002 ; 2 , atr was approximately equally effective at inhibiting heteromeric ( cnga1/cngb1 ; open triangles ) and homomeric ( cnga1 ; filled circles ) rod channels expressed in the oocytes . thus , in contrast to channel inhibition by pseudechetoxin ( brown et al . , 1999 ) , calmodulin ( chen et al . , 1994 ) , and l - cis - diltiazem ( chen et al . , 1993 ) , inhibition by atr appears not to be substantially affected by the presence of the subunit ( cngb1 ) . this figure also gives quantitative evidence for the high apparent affinity of cnga1 for atr at low cgmp concentrations ( open circles ) , as introduced in fig . 3 shows that the effect of atr inhibition was to reduce both the apparent affinity of the channel for cgmp and the maximal current at saturating cgmp . the saturating form of the lower curve suggests that atr is not a competitive inhibitor of cgmp binding , since raising cgmp over a wide range does not relieve atr inhibition . the downward shift of the curve with atr suggests that atr either inhibits opening of fully liganded channels and/or partially blocks the open channels , reducing the single - channel conductance . previous single - channel analysis ( dean et al . , 2002 ) suggests that a partial reduction in single - channel conductance is unlikely , and that atr may induce long - lived closed or inactivated states . although a complete block of the pore was not ruled out by those studies , open - pore block is unlikely because atr inhibits more effectively at low than at high cgmp , i.e. , at low , rather than high , open probability . furthermore , the data shown in fig . 3 are well - described by a model ( solid curves ) which assumes that atr interferes with the allosteric opening transition of the channel and does not alter its single - channel conductance ( see materials and methods and fig . 8 for details of the model ) . cgmp dose response relation for homomeric ( cnga1 ) rod channels with and without 400 nm atr . steady - state , cgmp - evoked currents were measured at + 100 mv from several patches with and without 400 nm atr . averaged data were normalized to the maximum current obtained with 2 mm cgmp and no atr , and fit with an allosteric model ( smooth curves ; see materials and methods and fig . 8 for details ) . data points are averaged from 27 patches , plotted with sem ( error bars ) . model parameters : l = 19.8 , kcn = 7,200 m. triangles : cgmp dose response relation with 400 nm atr . model parameters : l = 19.8 , kcn = 7,200 m , k1 = 9.0 10 m , k2 = 4.0 10 m , k3 = 2.8 10 m , k4 = 2.0 10 m , r = 400 nm , and n = 1.43 . inhibition of rod cng channels ( cnga1 ) by atr was slow and often demonstrated two components . a shows a typical time course of inhibition by 400 nm atr applied to the intracellular surface of an excised , inside - out , multichannel patch . this time course was described by two exponentials , with time constants of 119 and 1,667 s ; for 11 patches showing two components , the mean time constants ( sem ) were 110 18.1 s and 2,081 666 s. many patches demonstrated only a single exponential time course with a time constant of hundreds of seconds ( 854 105 s , 12 patches ) . because of the variability in the time course , we were unable to find any correlation between the relative magnitude or time constant of each component and the atr concentration . we wondered whether access of the lipophilic atr to its interaction site(s ) depended on patch geometry , the lipid or protein composition of the membrane , or other variable patch properties ( karpen et al . when the amount of inhibition was changed by jumping voltage ( see fig . 7 below ) or cgmp concentration 6 below ) after loading the patch with atr , changes in inhibition were more rapid and were always described by a single exponential . this supports the notion that the slow , variable , sometimes complex time course observed with atr addition may reflect access problems relating to patch structure . time course of inhibition by 400 nm atr in 2 mm cgmp for inside - out and outside - out , multichannel patches containing homomeric ( cnga1 ) rod channels . ( a ) time course of inhibition for this inside - out patch is best fit with a double exponential ( fast = 119 s and slow = 1,667 s ) , although patches were often fit with a single exponential . ( b ) the time course for outside - out patches was so much slower than for inside - out patches that very little inhibition was measured during the lifetime of the patch ; for this patch , a single - exponential fit gave = 6,748 s , although clearly the current had not reached steady - state by the end of the record . if inhibition by atr is slow because it involves restricted access of atr to a crevice or binding pocket of the channel that is accessible primarily from the intracellular surface , then inhibition might be slower or weaker when atr is applied to the outside surface of the membrane . to test for this possibility , we measured the time course of inhibition when atr was applied to outside - out patches . a typical time course for application of 400 nm atr to an outside - out patch is shown in fig . 4 b. these data demonstrate a single exponential with a time constant of 6,748 s ( 112 min ) . the slower , weaker action of atr from the outside of the membrane would be consistent with a mechanism in which atr exerts its effects via the intracellular surface of the membrane or channel ( see discussion ) . as reported previously ( dean et al . , 2002 ) , reversal of atr inhibition by washout from the chamber was difficult , presumably because of the preference of this lipophilic inhibitor for the membrane or hydrophobic binding sites over water . however , single - channel and multichannel records provide evidence for reversibility of inhibition by atr . 5 shows channel opening after a long shut period characteristic of inhibition by atr ( dean et al . , 2002 ) . this may reflect dissociation of atr from its interaction site(s ) , and therefore reversibility , but we can not rule out the appearance of a new channel in the patch ( however , simultaneous openings of two channels were never seen in several minutes of recording from this patch ) . single - channel recordings in the presence of atr suggest that the channel can reopen after long - lived closed or inactivated states . raw current traces in a c were recorded at a holding potential of + 80 mv from an inside - out patch containing one homomeric ( cnga1 ) channel . the lower solid line represents the zero - current level when the channel was closed . the patch was bathed in saturating ( 2 mm ) cgmp ( a ) , and then 200 nm atr was added to the bath ( b and c ) . ( a ) a representative portion of a control trace depicting normal channel activity in saturating cgmp ; the channel spends the majority of its time open . ( b ) after adding atr , the channel activity remained relatively normal for a time . ( c ) sometime before the start of this sweep , the channel entered a long - lived closed state , but reopening of the channel ( with atr still in the bath ) occurred near the end of the sweep . of the 3.2 s shown , the channel was in a closed state for 2.5 s ; another portion of the record for this patch showed a 6-s closure . these long - lived closed states with atr have been documented previously ( dean et al . , 2002 ) . better evidence for reversibility of atr inhibition was provided by multichannel patches exposed to jumps in either cgmp or voltage . at the start of the experiment in fig . 6 , the addition of 100 nm atr to a patch in low cgmp gave > 99% inhibition . when the cgmp concentration was raised to 5 mm in the continued presence of 100 nm atr , there was an increase in current thought to reflect two processes : ( a ) the opening of channels that were initially associated with little or no cgmp and no atr ; and ( b ) the opening of channels that were initially associated with atr , but lost atr upon binding cgmp . the latter process is thought to account for most of the current increase , since most of the channels are inhibited by atr at low cgmp , as seen in the data in fig . 6 and supported by calculations of the model from fig . current recovered with high cgmp after inhibition by atr in low cgmp . after applying 100 nm atr to a bath solution containing low cgmp ( activating 7.5% of maximal current ) , the patch current was monitored until the inhibition reached a steady - state ( 99.5% inhibition ) . the cgmp concentration in the bath was then raised to 5 mm , with the continued presence of 100 nm atr in the bath . the increase in [ cgmp ] opens some channels that were previously unliganded , and also presumably relieves some of the inhibition by atr , since atr is a less effective inhibitor in high than in low cgmp ( figs . 1 and 2 ) . after a gap representing the time when cgmp was mixed into the chamber , the current recovery was fit by a single - exponential rise with a time constant of 82 s. if atr is a closed - state inhibitor , it should work better at negative than at positive voltages , since the channel is slightly voltage dependent , with depolarization favoring opening . since atr has no net charge , no additional voltage dependence of inhibition is expected unless the atr interaction site moves in the electric field or is obscured by the movement of another region of the channel . thus , in the left panel , applying atr with a holding potential of + 50 mv reduced the current as channels became inhibited , but switching to 50 mv reduced the current even more , as more channels closed and therefore became more susceptible to atr inhibition . reversing this slight voltage dependence was compared quantitatively with the voltage dependence of gating in the absence of atr and was found to be similar . thus , atr did not induce any significant voltage dependence beyond the existing weak voltage dependence of channel gating . interestingly , in both cases , the time course of the voltage - induced jump in current is much faster ( = 44 s for 50 mv and 64 s for + 50 mv ) than that of the initial change in current during application of atr and may reflect the time required for preequilibrated atr to interact with the channel . the initial slow decreases in current shown in fig . 7 are consistent with that depicted in fig . 4 a and appear to reflect complex , patch - dependent parameters , as discussed above . inhibition of homomeric ( cnga1 ) rod channels by 200 nm atr in saturating ( 2 mm ) cgmp showed no significant voltage dependence other than that expected with voltage - dependent changes in open probability . for the top panel , an inside - out , multichannel patch was maintained at a holding potential of + 50 mv in 200 nm atr and 2 mm cgmp and monitored to steady - state . a double exponential provided the best fit to the time course of the inhibition , with fast = 206 s and slow = 3,779 s. after steady - state was achieved , the holding potential was changed to 50 mv . the time course of the resulting decrease in current ( increase in inhibition ) was best fit with a single exponential , with = 44 s. for the lower panel , the same experiment was performed on another patch , except that the initial membrane potential was 50 mv , and it was then switched to + 50 mv . a double exponential provided the best fit to the initial time course of inhibition , with fast = 77 s and slow = 769 s. the time course of the increase in current ( decrease in inhibition ) after switching the voltage to + 50 mv was best fit with a single exponential , with = 64 s. these changes in atr inhibition with voltage are consistent with the expected voltage - dependent changes in open probability and the greater atr inhibition of closed versus open channels . the finding that atr is a much more effective inhibitor at low than at high cgmp ( figs . 1 and 2 ) could reflect a preference of atr either for closed channels or for channels without bound cyclic nucleotide ( unliganded channels ) . one way to distinguish between these two possibilities is with the partial agonist , camp ( gordon and zagotta , 1995 ) . for rod cng channels , a saturating concentration of camp ( 20 mm ) gives only 5% of the maximal activation obtained in saturating cgmp ( 2 mm ) . 8 a compares atr dose response relations for low cgmp , saturating camp and saturating cgmp on homomeric rod cng channels . b ( equations in materials and methods ) approximates all three relations , and assumes that atr prefers closed over open channels , but also unliganded over liganded channels ( k1 > k2 > k3 > > k4 ) . we were not able to fit the data by assuming that all atr equilibrium constants were equal , i.e. , that inhibition depended only on whether the channel was closed , and not on the number of bound ligand molecules . 8 a , which is a prediction for the camp experiment if ligand occupancy does not affect atr inhibition ( i.e. , k1 = k2 = k3 > > 8 b further assumes that when the fully liganded , open rod channel binds atr , it closes and can not reopen with atr bound . the data could not be fit with a model in which the rod channel could open with atr bound . thus , the data and model in fig . 8 suggest that atr prefers unliganded , closed channels over channels that are liganded and/or open . atr appears ( a ) data were measured as described in fig . 2 for atr dose response curves in saturating ( 2 mm ) cgmp ( blue filled circles ) , saturating ( 20 mm ) camp ( red squares ; giving only 5% of the activation obtained with 2 mm cgmp ) , and low ( 1015 m ) cgmp ( green open circles ; giving 7.4% of the activation obtained with 2 mm cgmp ) . error bars are sem values . averaged data points from 27 patches were normalized to io and fit with the model ( b ) described above ( see materials and methods and results ) , where k1 = 9.0 10 m , k2 = 4.0 10 m , k3 = 2.8 10 m , k4 = 2.0 10 m , and n = 1.43 . for saturating and low cgmp , l = 19.8 and kcn = 7,200 m ; for camp , l = 0.055 and kcn = 1,144 m. the red dashed line represents the prediction of the model for camp if atr binding depends only on the open probability and not on ligand occupancy ( i.e. , k1 = k2 = k3 = 9 10 m ) . finally , the model depicted in fig . 8 b also gave a reasonable description of the cgmp dose , we used the same constants as for the curves in fig . 8 a , changing only the concentrations of cgmp and atr to match the experimental conditions . thus , the reduction in current by atr at saturating concentrations of cgmp can be explained simply by an inhibition of channel opening by atr , and it is not necessary to invoke a reduction in single - channel conductance or competition between atr binding and cgmp binding . results presented here suggest that atr is a potent , reversible , closed - state inhibitor of both homomeric ( cnga1 ) and heteromeric ( cnga1/cngb1 ) rod cng channels . atr acts best from the intracellular side of the membrane to decrease the apparent agonist affinity , but it does not seem to compete with cyclic nucleotides at their binding domain . the inhibition is not appreciably voltage dependent , and may involve multiple atr molecules per channel . descriptions of the data with an allosteric model suggest that atr prefers unliganded channels , but is also very effective on closed , liganded channels . the model assumes that atr interacts more weakly with open channels and drives them to close as well . it is not clear whether atr has its effects by interacting directly with the channel protein or some intermediary protein , or by altering bilayer channel interactions . if atr alters bilayer protein interactions , it may disrupt lipid rafts in the membrane and thereby alter the behavior of channels embedded in the rafts ( edidin , 1997 ; brown and london , 1998 ; jacobson and dietrich , 1999 ; martens et al . , 2000 ; brady et al . , 2004 ) , it may form retinoid - rich microdomains ( boeck and zidovetzki , 1988 ) that affect channel function , or it may alter physical properties of the bilayer , such as curvature , at the lipid protein interface ( lundbaek et al . , 1996 ; cantor , 1999 ; lundbaek and andersen , 1999 ) . changes in bilayer curvature , for example , have been shown to have dramatic effects on the dimerization of gramicidin channels ( lundbaek and andersen , 1994 ) . evidence for lipid rafts has been found in rod outer segment membranes ( seno et al . , 2001 ) and in oocytes ( luria et al . , 2002 ) , and a recent report ( brady et al . , 2004 ) shows that disruption of lipid rafts drastically reduces the apparent camp affinity of olfactory cng ( cnga2 ) channels . finally , it should be mentioned that schiff base reactions can occur between atr and lipids ; this offers another potential action of atr via the bilayer . however , this mechanism seems unlikely here in light of our previous finding ( dean et al . , 2002 ) that the rod cng channel is inhibited quite well by all - trans - retinol , which can not form a schiff base . on the other hand , retinoids , including atr , are known to interact directly with proteins , such as rhodopsin , retinoid binding proteins , and transcription factors ( newcomer et al . , 1998 ; nau and blaner , 1999 ; noy , 2000 ; kefalov et al . , 2001 ) . a putative atr binding pocket within the channel protein might include residues at locations that are distant from each other in the primary sequence , but near each other in the three - dimensional structure of the channel ; for example , in rhodopsin , the binding pocket is made up of residues that are contributed by adjacent transmembrane helices . direct interactions with the channel could involve hydrophobic crevices or domains , either among the transmembrane segments or within hydrophobic areas in the cytoplasmic amino- and carboxy - terminal domains . it is also possible that atr acts by way of another protein associated with the channel , rather than directly on the channel ; however , we have found similar inhibition of the channel in the native rod outer segment membrane ( unpublished data ) , where protein ( and lipid ) components are different . our results suggest that atr is a closed - state inhibitor , as are some other lipophilic agents , including tetracaine ( fodor et al . , 1997 ) and dequalinium ( rosenbaum et al . , 2004 ) . however , unlike these inhibitors , atr appears to discriminate not only between closed and open channels , but also between liganded and unliganded channels . this raises the question of whether atr may interact at sites within or near the cyclic nucleotide binding domain . although atr does not appear to be a competitive inhibitor of cyclic nucleotide binding , it may interact with a region whose conformation changes in response to cyclic nucleotide binding ( matulef and zagotta , 1999 ) . a comparison of channel inhibition by extracellularly and intracellularly applied atr may give clues as to the mechanism of its action . retinoids insert into bilayers , and flip - flop across them , very rapidly on a millisecond time scale . however , they take at least seconds to leave a bilayer into an aqueous solution , and retinoid interactions with proteins take seconds to minutes ( noy , 1999 ) . we found that channel inhibition was orders of magnitude slower and less effective when atr was applied to the extracellular surface of the membrane than when it was applied to the intracellular surface . one possible explanation for this result is that the atr interaction site(s ) was accessible only from the intracellular surface of the channel . thus , when atr was applied extracellularly , the channel only sensed the small fraction of the applied atr that had passed through the bilayer and remained near the intracellular surface long enough to find the interaction site(s ) . a bright light sufficient to bleach all rhodopsin molecules in a rod is expected to produce 3 mm atr in the rod outer segment ( saari , 1999 ) . although most of this atr is expected to associate with the disk membranes , some will most likely be in or near the plasma membrane and therefore near the cng channels . to date , the only retinoid binding protein that has been described in rods is irbp , which is produced by the rods and secreted into the extracellular matrix . it is possible that irbp may buffer intracellular , as well as extracellular , atr . furthermore , retinol dehydrogenase , which converts atr to all - trans - retinol after light absorption , would also reduce the amount of atr available to inhibit the cng channels . however , the ic50 for atr inhibition of the channel is 25 nm with 1015 m cgmp , and should be much lower when the free cgmp concentration is in the range expected in vivo : 6 m in the dark and even lower in the light ( nakatani and yau , 1988 ) . thus , it is reasonable to assume that some inhibition is possible in a rod exposed to bright , prolonged light . one potential caveat to this proposal is the fact that rod cng channels are modulated by phosphorylation enzymes , ca - binding proteins , and perhaps other agents ; thus , their sensitivity to atr in vivo may not be the same as that in our excised patches . if atr does indeed inhibit the channels in the intact rod , what role might this inhibition play in visual transduction ? one possibility is that it could contribute to the slow time course of dark adaptation after an exposure to bright and/or prolonged light , since the reduced apparent cgmp affinity of the channels would tend to make them remain closed even when the cgmp levels are recovering . finally , inhibition of the channels by atr may occur in certain disease conditions , such as in stargardt 's disease , in which there is an abnormal accumulation of atr .

rod vision begins when 11-cis - retinal absorbs a photon and isomerizes to all - trans - retinal ( atr ) within the photopigment , rhodopsin . photoactivated rhodopsin triggers an enzyme cascade that lowers the concentration of cgmp , thereby closing cyclic nucleotide gated ( cng ) ion channels . after isomerization , atr dissociates from rhodopsin , and after a bright light , this release is expected to produce a large surge of atr near the cng channels . using excised patches from xenopus oocytes , we recently showed that atr shuts down cloned rod cng channels , and that this inhibition occurs in the nanomolar range ( aqueous concentration ) at near - physiological concentrations of cgmp . here we further characterize the atr effect and present mechanistic information . atr was found to decrease the apparent cgmp affinity , as well as the maximum current at saturating cgmp . when atr was applied to outside - out patches , inhibition was much slower and less effective than when it was applied to inside - out patches , suggesting that atr requires access to the intracellular surface of the channel or membrane . the apparent atr affinity and maximal inhibition of heteromeric ( cnga1/cngb1 ) channels was similar to that of homomeric ( cnga1 ) channels . single - channel and multichannel data suggest that channel inhibition by atr is reversible . inhibition by atr was not voltage dependent , and the form of its dose response relation suggested multiple atr molecules interacting per channel . modeling of the data obtained with camp and cgmp suggests that atr acts by interfering with the allosteric opening transition of the channel and that it prefers closed , unliganded channels . it remains to be determined whether atr acts directly on the channel protein or instead alters channel bilayer interactions .

INTRODUCTION MATERIALS AND METHODS Expression of Channels in Xenopus Oocytes Electrophysiological Solutions and their Application Electrophysiological Recordings and Analysis RESULTS DISCUSSION

PMC3309678

on may 11 , 2009 , an african civet ( civettictis civetta ) displaying clinical signs consistent with rabies was killed by rangers in ikoma ward within serengeti national park ( figure 1 ) . rangers were contacted because the civet had bitten a child on the right leg in an unprovoked attack . the wound was washed with soap and water , and the child received postexposure rabies vaccination but no rabies immunoglobulin . brain samples from the civet were tested multiple times ( as part of a training course ) at the central veterinary laboratory in tanzania . results of the fluorescent antibody test and a direct rapid immunohistochemistry test were positive for lyssavirus - specific antigen . when testing was complete , the samples were sent to the animal health and veterinary laboratories agency ( ahvla , weybridge , uk ) for additional confirmation of results and molecular analysis . blue dot indicates location of ikoma lyssavirus infected african civet within ikoma ward in northwest tanzania . top left , map of africa indicating study area in tanzania ( gray box ) . rna was extracted by using trizol reagent ( invitrogen , paisley , uk ) , and a pan - lyssavirus reverse transcription pcr yielded a specific 606-bp amplicon ( 9 ) . bayesian reconstructions were used for phylogenetic analysis of the nucleoprotein gene region ( 405 bp ) and included representatives of all species from the lyssavirus genus ; results showed that the sequence was unique and most closely related to wcbv ( figure 2 ) . a canine rabv biotype from tanzania and a mongoose rabv biotype from southern africa were included in the dataset . nucleotide comparisons indicated similar divergence from all lyssavirus species ( minimum identity 62.2% australian bat lyssavirus , maximum identity 68.6% wcbv ) , including 12 canine rabv sequences from domestic and wild animals in the serengeti ecosystem ( 64.1%65.1% identity ) . the posterior probabilities indicated that the ikov and wcbv grouping was strongly supported , despite low sequence identity . further phylogenetic analysis of representatives from other rhabdoviruses demonstrated that ikov is a member of the lyssavirus genus ( 41.6%50.9% identity to representative rhabdovirus sequences that are available for this region of the genome ) ( data not shown ) . phylogenetic relationships between all currently identified lyssaviruses compared with ikoma lyssavirus ( ikov ; shown in boldface italics ) , as determined on the basis of partial nucleoprotein gene sequences ( 405 bp ) . relationships are presented as an unrooted phylogram based on bayesian markov chain monte carlo ( mcmc ) analysis . the mcmc analysis was performed in mrbayes ( version 3.1.2 ; http://mrbayes.sourceforge.net/ ) , by using the general - time reversible model with a proportion of invariable sites and a gamma - shaped distribution of rates across sites , treating values for model parameters as unknown variables with uniform priors to be estimated in each analysis . four simultaneous mcmc chains ( 3 heated and 1 cold , according to the default settings ) were run for 10 generations ; trees were sampled every 100 generations . the first 25,000 trees were discarded as the burn - in phase ; the remaining trees were used to estimate consensus phylograms and bayesian posterior probabilities . posterior probability values represent the degree of support for each node on the tree : only values > 0.90 are shown ; values > 0.95 are shown in boldface . scale bar indicates branch length , expressed as the expected number of substitutions per site . arav , aravan virus ; ablv , australian bat lyssavirus ; bblv , bokeloh bat lyssavirus ; duvv , duvenhage virus ; eblv-1 and eblv-2 , european bat lyssavirus type 1 and 2 ; irkv , irkut virus ; khuv , khujand virus ; lbv , lagos bat virus ( lineages a , b , c and d ) ; mokv , mokola virus ; rabv , rabies virus ; shibv , shimoni bat virus ; wcbv , west caucasian bat virus . we describe evidence , based on genomic sequences obtained from the brain sample of an african civet with clinical signs consistent with rabies , for the existence of a novel lyssavirus designated ikov ( ikoma lyssavirus ) . the sample was frozen and thawed several times before being sent to ahvla , which had a detrimental effect on the sample quality and resulted in viral rna degradation and loss in viral viability . the results of confirmatory fats performed at ahvla were inconclusive , and attempts to isolate virus by using the rabies tissue culture inoculation test and the mouse inoculation test were unsuccessful . despite the lack of isolated virus , the pan - lyssavirus sensitivity and specificity of the fluorescent antibody test ( the test prescribed by the world organisation for animal health as the standard for rabies testing ) and direct rapid immunohistochemistry test support the assertion that a novel lyssavirus exists in the region . in addition , despite the poor quality of the sample , molecular techniques identified a lyssavirus - specific amplicon that was confirmed to be unique by phylogenetic analysis and to be highly divergent from known circulating rabv strains . a real - time pcr also detected this unique lyssavirus sequence , confirming that both molecular tests are pan - lyssavirus specific and are sufficient for the detection of highly divergent novel lyssaviruses ( 11 ) . the child who was bitten by the african civet received appropriate wound care and postexposure rabies vaccination . at the time of this report we can not , however , draw any conclusions as to whether the african civet was shedding virus when it bit the child or whether postexposure vaccinations are effective against ikov . this case of rabies in an african civet in the center of serengeti national park was highly unexpected . since 2000 , the park had been free of rabies and no cases had been detected within a 30-km radius . however , subsequent molecular characterization demonstrated that this case of rabies had not been caused by a rabv from a canine source . thus , a breach had not occurred ; instead , a novel lyssavirus with an unknown reservoir had caused the infection . previously published data on lyssavirus infection in african civets ( n = 6 ) was restricted to the rabv mongoose lineage ( 12 ) . although african civets can be infected with rabv and ikov , infrequent detection of lyssaviruses in this species suggests that they are more likely to be incidental hosts . the nocturnal , opportunistic foraging behaviors of african civets imply that contact with bats is possible , particularly at roosts where interactions with a grounded rabid bat are more likely to occur . in the absence of virus surveillance for rabies in bats and other mammals in tanzania and typing of all lyssavirus - positive samples is necessary to determine the distribution and prevalence of ikov . bats in neighboring kenya could be informative , given the strong posterior probability values on the grouping of ikov and wcbv in the bayesian analysis ( figure 2 ) ( 13 ) . additional genomic and evolutionary analysis is underway to support ikov as a new lyssavirus species , potentially grouping with wcbv in phylogroup iii ( 14 ) , and to determine the antigenic diversity of the glycoprotein ( 15 ) . given that ikov is highly distinct from rabv ( more genetically distinct than wcbv from rabv ) and that a human has been bitten by an infected animal , the effectiveness of current rabies vaccines needs to be further investigated .

evidence in support of a novel lyssavirus was obtained from brain samples of an african civet in tanzania . results of phylogenetic analysis of nucleoprotein gene sequences from representative lyssavirus species and this novel lyssavirus provided strong empirical evidence that this is a new lyssavirus species , designated ikoma lyssavirus .

The Study Conclusions

PMC4460670

the dna microarray technology has provided monitoring of thousands of genes simultaneously in a single experiment . however , gene expression data have some characteristics which cause difficulty in analyzing data with many classifiers such as high - dimension - often exceeds more than ten of thousands - in contrast of small - sample size often a few hundred samples and high - noise nature of data . hence , the main challenge is to find a small subset of relevant genes to improve classification accuracy with robustness . using this technology and check - outs the changes in expression levels of genes between samples , can help physicians to have efficient diagnosing as well as effective treatments ( schena , 1996 ) , ( schena et al . 1995 ) , study ( dong ling tong , 2011 ) , developed a hybrid genetic algorithm ( ga ) - neural network model for feature selection on unpreprocessed microarray data . the fitness value ga is based on an accuracy of standard feed - forward artificial neural network ( ann ) . the main point of the genetic algorithm - neural network algorithm is to select highly informative genes by the calculation of the both ga fitness function and the ann weights simultaneously in ( li - yeh chuang , 2011 ) , taguchi - ga and correlation - based feature selection used as a hybrid methods , and the k - nearest neighbor ( k - nn ) served as a classifier and also in paper ( li - yeh chuang et al . , 2011 ) another study based on taguchi binary particle swarm optimization ( pso ) conducted by the same authors . in paper ( bing liu , 2004 ) , a combinational feature selection method with ensemble neural networks was used for classification . rank sum test , principal components analysis ( pca ) , clustering , and t - test are used to extract and select features . in this work , bootstrap technique is used to resample data , and also cooperative and competitive neural networks are tested on data and create the output . in paper ( shen qi , 2007 ) , the combination of modified discrete pso and support vector machines ( svm ) for tumor classification is applied to select genes with the ability of high accuracy classification . in paper ( li - yeh chuang , 2008 ) , improved binary pso is in order to feature selection , and the k - nn method serves as a classifier for gene expression data classification problems . in ( shen qi , 2008 ) , a hybrid pso and tabu search with linear discriminant analysis ( lda ) classification were developed for gene selection and cancer classification . paper ( emmanuel martineza , 2010 ) proposed an algorithm based on swarm intelligence feature selection method in which , the initialization and update of only a subset of particles are happened in the swarm . the most frequent genes are evaluated by the ga / svm again to obtain the most final relevant gene subset . in ( leping li , 2001 ) ga and the k - nn are combined to identify most frequent gene for cancer classification . in ( yang , 2009 ) , a hybrid method based on information gain and gas are proposed for gene selection in microarray data sets . the k - nn method with leave - one - out cross validation served as a classifier for evaluating the fitness function of this hybrids algorithm . in study ( jenny nskog , 2011 ) , classification performance of five normalization methods and three gene selection methods as t - test , relief , paired distance , and eight machine learning methods as a decision tree with gini index and information gain criterion , svm classifier with different kernels and also neural network are compare with each other . in paper ( xiaosheng wang , 2011 ) , use single genes to create classification models and identified the most powerful genes for class discrimination . by these kinds of classifiers , include diagonal lda , k - nn , support vector machine and random forest . then they constructed simple rules for cancer prediction by these single genes . in ( shital shah , 2007 ) , an integrated algorithm involves a ga and correlation - based heuristics for data preprocessing and decision tree , and svm algorithms are used for making predictions . paper ( jinn - yi yeh , 2007 ) , applies gas with an initial solution provided by t - statistics for selecting a subset of genes and the decision tree is used as a classifier to build model on top of these selected genes . in study ( chu , 2005 ) , feature selection methods , such as pca , class separability measure , fisher ratio , and t - test are used for gene selection . and a voting scheme is then applied to do multi - group classification by binary svm . in study ( makoto takahashi a , 2010 ) , an unpaired t - tests with one of the supervised classifiers , study ( khan javed , june 2001 ) , applied a method for classifying cancers using anns on small , round blue cell tumors as a model . t - test and pca are used to reduction dimensionality of data sets . in ( nikhil r pal , 2007 ) , a multilayer networks with online gene selection ability and relational fuzzy clustering was used to identify a small set of biomarkers for accurate classification . in our paper , we use the hybrid of ga and pso algorithm as a feature selection method and the fitness of each gene subset ( chromosome ) is determined by ann classifier 's accuracy . the 10-fold cross validation classification accuracy on the gene subset in the training and evaluation samples is evaluation criteria . the group of gene subset with the highest 10-cv classification accuracy is considered as the optimal gene subset . after we have selected the most frequent genes , we can use them for discrimination of blind test data to see the response of evaluation hybrid system on these kinds of data . at last , we use a decision tree classifier to see the relation between founded biomarkers and rule extraction . this point should be considered that one of our purposes is increasing accuracy of classification problems by selecting the best parameters of the classifier without using any trial and errors of users . using a suitable combination of optimization algorithms for feature selection and also selecting proper classifier can improve the classification results . in this section , we introduce the gene expression datasets which were used in this paper and also propose the modified hybrid algorithm . the first data include 72 samples in two type of classes as acute lymphoblastic leukemia ( all ) and acute myeloid leukemia ( aml ) . these two categories of cancer are quite similar at the microscopic level and have a same behavior over the years . the second are generated by ( alon u , 1999 ) for colon cancer categories . the last data include 49 samples in two class of breast cancers 25 samples are placed in estrogen receptor ( er+ ) class , and 24 of them are placed in er class . the original size of features or genes in these data is 7129 and was generated by ( west , 2001 ) . datasets which used for classification problems for testing the efficiency of proposed method in the following discussion , we introduce the proposed algorithm which is used on gene expression profiles . ga and pso are two optimization algorithms which have many advantages in these kinds of problems . they are computational optimization method that search all part of the solution space with a different kind of solution or a group of feature subsets to find the best answer in each iteration . in ga , the searching process only needs to determine the value of the objective function at different points and also , no additional information like differentiation of function is needed . pso algorithm was developed by kennedy and eberhart in 1995 ( eberhart r , 1995 ) . in pso , each particle moves in the search space with a velocity adjusted by its own memory and its neighbors to find the best solutions . in contrast to pso algorithm , chromosomes in ga algorithm share the information between each other . it performs data processing by providing small processor that are parallel interconnected with each other to form a network to solve a problem . neural networks are used to implement complex functions in various fields , including pattern recognition , identification , classification , speech and image processing , and control systems . after tuning or training the neural network , network behavior is related to the connections between its members . in general , the neural network has three layers of neurons such as an input layer , hidden layers , and output layer . performance of the hidden layers is determined by inputs and weighted vectors between input and hidden layers . weights between input and hidden units have to be determined when a hidden unit is been active . performance of the output layers depends on the weights between the hidden and output units . in multi - layer perceptron networks or feed - forward networks , each layer may be determined by it 's parameter matrices and the network can form by a combination of nonlinear operators . the goal is finding and estimation of the mapping function between input and output spaces . estimation of suitable network is based on a minimization of the error between the desired output and network 's output . in each layer , activation functions can be nonlinear in both layers and also can be different from each other . in these networks , there are two types of weight matrices , such as an intermediate layer or hidden layers and output layer weight matrix . these matrixes sizes depend on the number of neurons in hidden layers and output layer 's neurons . we can write : training neural networks mean selecting the best model of network by the best parameters such as weights , number of neurons based on the cost function . the task of pattern classification in ann is to assign an input pattern as gene expression profile represented by feature vector to one of the introduced classes such as normal or cancer . after providing the best network based on feature vectors and parameters , our model can be able to predict the class of new data based on training . now , in this section , we give a detailed description of our proposed algorithm . we can implement both of these algorithms in hybrid form to benefit the useful advantages of both of them and covered their problems . in this paper , ann is used as a classifier and fitness function of hybrid pso / ga algorithm ( kao , 2008 ) , ( du , 2006 ) , ( juang , 2004 ) , ( robinson , 2002 ) . at first of the implementation the integration of data includes whole genomes of human , so most of the genes in the database are not useful and irrelevant for classification problems . we have eliminate genes that ( 1 ) their expression value is very low , ( 2 ) have little change in expression value in hole samples , ( 3 ) genes that have a low standard deviation and have no impressive changes around the mean of expression value , ( 4 ) genes that have low information entropy . next , we have select top ranked genes by , t - test method and apply them as an input of hybrid pso / ga system . , 10% of data must belong randomly as a blind test data , and also remaining 90% of data can be entering to training and evaluation phase of the algorithm by 10-fold cross validation . the value of parameters such as , size of population , length of chromosome and particles , rate of mutation and crossover in ga , inertia coefficient ( w ) , training factors ( learning factors ) , and maximum velocity is mentioned in table 2 . in addition of creating initial position ( xid ) , initial velocity ( vid ) of every particle should be determined randomly in the population . this stage is related to making the initial population , at first the population with n chromosome create randomly . the length of particles or chromosomes can be explained as , adding number of features which has been selected based on statistical method and 11 additional genes which have been used for determination of optimum parameters of classifier by hybrid algorithm . primary random and binary initialization are taken place first , in such a way that 1 shows the existence of the feature in training system and 0 is meaning of not existing of that feature . now , each chromosome is a word of bits in two main parts . first part is equal to feature dimensions size ( segment 1 ) , and the second part is used for determining and designing classifier parameters . the second part contains three sub - parts , which can be seen with details in table 3 . the second segment of a chromosome ( one bit of chromosome ) determines the number of layers in the network . we assign 5 bit for each layer which converted to the decimal number during training . the fitness values for all particles have to be calculated in order to determine functionality of each particle , which is so - called validation of particles . a sample chromosome of psoga / ann population the velocity and position of the particles have been updated based on equation below : the best particle as x and the best personal memories of each particle as x is updating . in this paper , a binary pso algorithm is used by the authors ( kennedy , 1997 ) . it is important to note that in genetic operators , there is no discussion in speed changes or the best memory of offspring ; hence , we have determined the best memory of offspring based on the best memory of parents which have the best fitness value . after this step , this is the time for running ga , from the solutions which are presented by the pso , the crossover and mutation operators are applied on selected parents . in this paper , we have used roulette - wheel as a selection method . roulette - wheel is a technique which selects parents based on the fitness value on each of them . since the algorithm is binary , we use bit inversion ( set zero to one and vice versa ) method for the mutation operator . in this paper , we are using three crossover methods such as a single point , double point , and uniform crossover by a random probability to be able to use all benefits and advantages of these crossover methods simultaneously . at the end of the progress , the best features with the best parameters of classifier are selected , so we have applied these features and parameters to blind test that has no interference in the training and validation phase at all . on average , biomarkers that have been repeated > 6 times in the best locations are reported . finally , the decision tree 's rules can be found from the best - extracted features . this flowchart shows a summary of how the system works and the relation between feature selection method and classifier . now , in this section , we give a detailed description of our proposed algorithm . we can implement both of these algorithms in hybrid form to benefit the useful advantages of both of them and covered their problems . in this paper , ann is used as a classifier and fitness function of hybrid pso / ga algorithm ( kao , 2008 ) , ( du , 2006 ) , ( juang , 2004 ) , ( robinson , 2002 ) . at first of the implementation the integration of data includes whole genomes of human , so most of the genes in the database are not useful and irrelevant for classification problems . we have eliminate genes that ( 1 ) their expression value is very low , ( 2 ) have little change in expression value in hole samples , ( 3 ) genes that have a low standard deviation and have no impressive changes around the mean of expression value , ( 4 ) genes that have low information entropy . next , we have select top ranked genes by , t - test method and apply them as an input of hybrid pso / ga system . , 10% of data must belong randomly as a blind test data , and also remaining 90% of data can be entering to training and evaluation phase of the algorithm by 10-fold cross validation . the value of parameters such as , size of population , length of chromosome and particles , rate of mutation and crossover in ga , inertia coefficient ( w ) , training factors ( learning factors ) , and maximum velocity is mentioned in table 2 . in addition of creating initial position ( xid ) , initial velocity ( vid ) of every particle should be determined randomly in the population . this stage is related to making the initial population , at first the population with n chromosome create randomly . the length of particles or chromosomes can be explained as , adding number of features which has been selected based on statistical method and 11 additional genes which have been used for determination of optimum parameters of classifier by hybrid algorithm . primary random and binary initialization are taken place first , in such a way that 1 shows the existence of the feature in training system and 0 is meaning of not existing of that feature . now , each chromosome is a word of bits in two main parts . first part is equal to feature dimensions size ( segment 1 ) , and the second part is used for determining and designing classifier parameters . the second part contains three sub - parts , which can be seen with details in table 3 . the second segment of a chromosome ( one bit of chromosome ) determines the number of layers in the network . we assign 5 bit for each layer which converted to the decimal number during training . the fitness values for all particles have to be calculated in order to determine functionality of each particle , which is so - called validation of particles . a sample chromosome of psoga / ann population the velocity and position of the particles have been updated based on equation below : the best particle as x and the best personal memories of each particle as x is updating . in this paper , a binary pso algorithm is used by the authors ( kennedy , 1997 ) . it is important to note that in genetic operators , there is no discussion in speed changes or the best memory of offspring ; hence , we have determined the best memory of offspring based on the best memory of parents which have the best fitness value . after this step , this is the time for running ga , from the solutions which are presented by the pso , the crossover and mutation operators are applied on selected parents . in this paper , we have used roulette - wheel as a selection method . roulette - wheel is a technique which selects parents based on the fitness value on each of them . since the algorithm is binary , we use bit inversion ( set zero to one and vice versa ) method for the mutation operator . in this paper , we are using three crossover methods such as a single point , double point , and uniform crossover by a random probability to be able to use all benefits and advantages of these crossover methods simultaneously . at the end of the progress , the best features with the best parameters of classifier are selected , so we have applied these features and parameters to blind test that has no interference in the training and validation phase at all . on average , biomarkers that have been repeated > 6 times in the best locations are reported . finally , the decision tree 's rules can be found from the best - extracted features . this flowchart shows a summary of how the system works and the relation between feature selection method and classifier . by applying the proposed algorithm to 3 cancer databases , the amounts of accuracy , sensitivity , precision , specificity were computed . our goal is to find the best possible combination and comparison of this modified algorithm with the others methods . proportional to the number of samples in each database , we select top ranked genes ( 5060 ) , and then we apply them to a hybrid algorithm . following a discussion , we introduce the biomarkers which obtained by a hybrid algorithm , then extract rules which are achieved by the decision tree from the biomarkers . the results indicate the good performance of our proposed algorithm in finding small subset of features with high accuracy . furthermore , the results show the good similarities between our biomarkers and the biomarkers that have been introduced in others literature . from the results , we can understand that the hybrids algorithm with this classifier have a better result rather than the result which obtained from individual genetic and pso algorithms . furthermore , we can improve the accuracy of classification by determining its parameters automatically during the feature selection stage with small suitable feature of subsets . the result of applying hybrid algorithm ( pso / ga ) to ann classifier with t - test preprocessing on cancer databases because of more emphasizing on presented pso / ga / ann hybrid algorithm , we do further checks with more details on these results . figure 2 shows the most frequent genes of running algorithm with 10 fold cross validation . in leukemia cancer type ( aml , aml ) , 17 biomarkers are selected with our hybrid algorithm , 20 genes in colon cancer and 12 genes in breast cancer , are selected as the most frequent genes , respectively . all these genes have been repeated > 6 times out of 10 times of running the algorithm . occurrence frequency of genes by hybrid particle swarm optimization / genetic algorithm / artificial neural network algorithm with 10-fold cross validation . figures from left to right are : ( a ) for breast cancer ( b ) colon cancer and ( c ) blood cancer type acute lymphoblastic leukemia and acute myeloid leukemia discovered biomarkers for all groups by pso / ga / ann for more details , we use a heat map showing on discovered biomarkers . the point which is important is that we can view a graphical representation of the changes in genes behavior in cancer data by displaying heat map . it is desirable that the behavior of genes in cancer samples is similar but different from healthy samples . for example , a group of genes have low expression in normal samples in contrast , another group of genes has high expression in the normal sample . hence , the thing which is important is that these genes can interact with each other to separate cancer samples from normal samples correctly . in figure 3 , images show the heat maps of leukemia cancer in two types , colon and breast cancer , respectively . in these heat maps , red color represents values above the mean , black represents the mean , and green represents values below the mean of a gene across all columns samples . heat maps view on three cancer data show the difference behavior of genes in two classes of data . ( a - c ) the result for breast and colon cancer data and leukemia cancer in types acute lymphoblastic leukemia and acute myeloid leukemia , respectively m13241 , u84487 , l22524 , l22524 , u74612 , d76435 have high expression value in er and the others biomarkers have low expression in these groups . d49950 , m55150 , m32304 , m16038 , m62762 , x61587 have high expression in aml groups and m31303 , m65214 , d86967 , d63880 , x59417 , s50223 x97267 , x66401 , u07139 , l07633 , m31211 have low expression in these groups . m76378 , h43887 have low expression value in colon cancer , but the others founded biomarkers have high expression value in cancer sample . at last , we apply decision tree algorithm on biomarkers which obtained by the introduced hybrid approach in table 5 to use for finding rules between them . we use c5.0 , which is one of the decision tree algorithms by spss clementine 12 software . in this work , we find 3 rules with 91% accuracy using 10 fold - cross validations for blood cancer type all , aml . in this type of blood cancer , classification with consideration of these two genes , we found that gene x61587 has high expression in aml samples ; in contrast , gene m31211 has low expression in this cancer type aml . in fact , using these two genes that have different behaviors in two class of sample , and discover rules with decision tree , can help us to have proper classification . the rest of the table 6 shows the rules for the breast cancer and colon cancer . the obtained accuracy for these cancer data and on high ranked genes in occurrence frequency are , 91% , 89% , and 83% , respectively [ table 7 ] . we use a decision tree classifier to for biological point of view in our works . summarizes results and comparison with literatures extracted rules by decision tree on 4 cancer database in the following , we can have some comparison on proposed algorithm with the others works . the first comparison is based on an accuracy of classification which is shown in table 6 . then we perform a comparison between the biomarkers presented in this article and the references articles . the tables show that our algorithm can achieve to high accuracy for the classification problem than others . also in addition of high accuracy rather than other works , the procedures of modeling in references papers are test with different parameters by users , but our algorithm was running without any user 's interference and any trial and errors . in all and aml , the algorithm finds 17 biomarkers that 7 of them are common with reference paper ( golub , 1999 ) . biomarkers which are in common with reference paper on leukemia ( all , aml ) are m62762 , m31303 , m31211 , m16038 , x59417 , s50223 , m55150 . in rest two type of cancer , in breast cancer 4 biomarkers out of 12 biomarkers are the same with ( west , 2001 ) and also 4 genes out of 20 in colon cancer with ( alon , 1999 ) . biomarkers which are in common with reference paper on breast cancer are x58072 , u95740 , l24203 , and s37730 . biomarkers which are in common with reference paper on colon cancer are t57619 , t48804 , x55715 , t61609 . in this paper , we have used hybrid combination of pso and ga algorithm with ann without any trial and user interface in determining the classifier 's parameters such as number of layers and number of neurons in each layer . the main comparison is based on accuracy of classification that is , shown in table 5 . following a discussion and regarding to result the accuracy of 100% is achieved for blood cancer types 96.67% and 96% is achieved for colon and breast cancer data , respectively . this result is better than the individual use of pso and ga algorithm and also the ability of algorithm in determining the training parameters and small feature subsets in databases perfectly with no user interface is another point of work which is proper . niloofar yousefi moteghaed received b.sc . and m.sc degree in biomedical engineering from the science and research branch of islamic azad university university , tehran in 2010 and 2012 respectively . now she is ph.d student in biomedical engineering in shahid beheshti university of medical sciences and health since 2012 . e - mail : [email protected] keivan maghooli has received his b.sc . in electronic engineering from the shahid beheshti university , tehran , iran , m.sc . in biomedical engineering from the tarbiat modaress university , tehran , iran , and ph.d . in biomedical engineering from the research and science branch , azad university , tehran , iran , majoring in data mining , signal processing and artificial intelligence . he has been with the biomedical faculty at research and science branch , azad university , tehran , iran , since 2000 , where he is currently an assistance of professor and head of bioelectric department . e - mail : [email protected] shiva pirhadi received b.sc . and m.sc degree in biomedical engineering from the science and research branch of islamic azad university university , tehran , iran in 2010 and 2012 respectively . now she is ph.d student in biomedical engineering in science and research branch of azad university since 2012 . e - mail : [email protected] masoud garshasbi has received his b.sc . in biology from the ferdowsi university , mashhad , iran ( 2001 ) , and his m.sc . in human genetics from the university of social welfare and rehabilitation ( uswr ) , tehran , iran ( 2003).he obtained his ph.d . ( 2009 ) and post doctoral ( 2011 ) in human molecular genetics from max planck institute , berlin , germany by working on the genes involved in mental retardation . at 2011 he joined as an assistant professor to the department of medical genetics , faculty of medical sciences , tarbiat modares , tehran , iran . he is also founder and head of medical genetic department at the dna laboratory , tehran , iran .

the improvement of high - through - put gene profiling based microarrays technology has provided monitoring the expression value of thousands of genes simultaneously . detailed examination of changes in expression levels of genes can help physicians to have efficient diagnosing , classification of tumors and cancer 's types as well as effective treatments . finding genes that can classify the group of cancers correctly based on hybrid optimization algorithms is the main purpose of this paper . in this paper , a hybrid particle swarm optimization and genetic algorithm method are used for gene selection and also artificial neural network ( ann ) is adopted as the classifier . in this work , we have improved the ability of the algorithm for the classification problem by finding small group of biomarkers and also best parameters of the classifier . the proposed approach is tested on three benchmark gene expression data sets : blood ( acute myeloid leukemia , acute lymphoblastic leukemia ) , colon and breast datasets . we used 10-fold cross - validation to achieve accuracy and also decision tree algorithm to find the relation between the biomarkers for biological point of view . to test the ability of the trained ann models to categorize the cancers , we analyzed additional blinded samples that were not previously used for the training procedure . experimental results show that the proposed method can reduce the dimension of the data set and confirm the most informative gene subset and improve classification accuracy with best parameters based on datasets .

INTRODUCTION MATERIALS AND METHODS Proposed Algorithm RESULT DISCUSSION CONCLUSION BIOGRAPHIES

PMC4664754

the iclip and rna - seq data are available at the following geo series : gse60392 , http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse60392.celltreatmentfeaturereplicatesgeo accession urlhuman flp - in 293untreatedrbfox2 iclip2http://www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc = gsm1477476human flp - in 293untreatedcpsf2 iclip2http://www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc = gsm1477475human flp - in 293non - silencing lentivirus knockdownpaired - end rna - seq2http://www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc = gsm1477603http://www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc = gsm1477604human flp - in 293rbfox2 lentivirus knockdownpaired - end rna - seq2http://www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc = gsm1477601http://www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc = gsm1477602human flp - in 293cpsf2 lentivirus knockdownpaired - end rna - seq2http://www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc = gsm1477599http://www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc = gsm1477600 cells were transduced with lentiviruses expressing shrnas targeting rbfox2 or cpsf2 , or as a control a non - silencing ( ns ) shrna , and then puromycin selected for 4 days . flp - in-293 cells were grown in 10 cm plates and then subjected to uv - c irradiation ( 200 mj / cm , stratalinker 2400 ) . upon removal of phosphate buffered saline ( pbs ) , cells were scraped off and precipitated by centrifugation at 3000 rpm for 10 min . hcl ph 7.4 , 100 mm nacl , 1 mm mgcl2 , 0.1 mm cacl2 , 1% np-40 , 0.1% sds , 0.5% sodium deoxycholate ) . samples were partially digested with rnase i by incubation with 10 l rnase i ( ambion ) ( diluted 1:500 in lysis buffer ) and 5 l turbo dnase ( ambion ) for 5 min at 37 c while shaking at 1100 rpm . cells were centrifuged at 14,000 rpm for 15 min to collect the cross - linked lysate . to prepare the beads for rna purification , 50 l of protein g - coated dynabeads ( invitrogen ) was washed twice with 1 ml lysis buffer and resuspended in 200 l lysis buffer containing 35 g of rbfox2 ( bethyl laboratories , a300 - 864a ) or cpsf2 ( bethyl laboratories , a301 - 580a ) antibody . the beads were rotated at room temperature for 60 min , washed twice with lysis buffer , and added to the cross - linked lysate . after overnight incubation at 4 c , the beads were washed four times with high - salt wash buffer ( 50 mm tris hcl ph 7.4 , 1 m nacl , 1 mm edta , 0.1% sds , 0.5% sodium deoxycholate , 1% np-40 ) and once with pnk wash buffer ( 20 mm tris hcl ph 7.4 , 10 mm mgcl2 , 0.2% tween-20 ) . the beads were resuspended in 5 l 10 pnk buffer , 44 l h2o and 1 l pnk enzyme ( neb ) and incubated at 37 c for 20 min while shaking at 1100 rpm . samples were then washed twice with high - salt wash buffer and once with 900 l pnk wash buffer . to ligate linkers to the 3 end of rnas , 4.5 l of purified rna was mixed with 1 l of 10 m mircat33 adaptor ( integrated dna technologies ) , 1.5 l of rna ligase buffer , 0.5 l of t4 rna ligase 2 , truncated k227q ( neb ) , and 7.5 l of peg 8000 , and incubated at 30 c for 6 h. samples were then end - labeled using pnk enzyme and run on a nupage novex 10% bis tris protein gel ( invitrogen ) with 1 mops running buffer ( invitrogen ) . proteins and covalently bound rnas were then transferred to a nitrocellulose membrane ( whatman ) using a novex wet transfer apparatus ( invitrogen ) . the nitrocellulose membrane was rinsed with 1 pbs , wrapped in plastic wrap and exposed to x - ray film . to isolate the rna protein complex , the region of the nitrocellulose membrane corresponding to the complex size of interest was cut out . cross - linked rnas were then isolated by incubating the nitrocellulose membrane pieces with pk buffer ( 100 mm tris hcl ph 7.5 , 50 mm nacl , 10 mm edta ) containing 2 mg / ml proteinase k ( roche ) for 20 min at 37 c , after which an equal volume of pk buffer containing 7 m urea was added to the tube and the samples were incubated for another 20 min at 37 c . subsequently , 600 l of phenol / chloroform ( ambion ) was added , and the mixture was incubated for 10 min at room temperature . samples were then centrifuged for 10 min at 13,000 rpm at room temperature , and the aqueous phase was transferred into a new microtube and again centrifuged . 400 l of supernatant was mixed with 0.5 l glycoblue ( ambion ) , 40 l 3 m sodium acetate ph 5.5 , and 1 ml 100% ethanol and incubated overnight at 20 c . rnas were precipitated by centrifugation for 30 min at 15,000 rpm at 4 c , washed with 500 l 80% ethanol and resuspended in 30 l h2o . to carry out reverse transcription , 30 l purified rna was mixed with 1 l of either bc2 ( 5-pggcgatgagatcggaagagcgtcgtgtagggaaagagtgt - sp18-ctcg gcattcctgctgaaccgctcttccgatct - ccttggcacccgagaattcca-3 ) or bc3 ( 5-pggcgatgagatcggaagagcgtcgtgtagggaaagagtgt - sp18-ctcg gcattcctgctgaaccgctcttccgatct - ccttggcacccgagaattcca-3 ) prt primer ( 10 m concentration ) and 1.5 l 10 mm dntp mix , and incubated at 65 c for 5 min and then transferred to ice . subsequently , 9 l of 5 first strand synthesis buffer ( invitrogen ) , 2.25 l 0.1 m dtt , and 1.25 l superscript iii reverse transcriptase ( invitrogen ) were added to a final volume of 45 l . the cdnas were size - separated on a 6% tbe urea gel ( invitrogen ) . two size fractions were recovered from the gel by cutting the gel corresponding to a cdna size of 120155 nt and 150225 nt . gel fragments were mixed with 400 l te buffer , crushed with a 1 ml syringe plunger and centrifuged overnight at 1100 rpm at 37 c . gel pieces and the supernatant were separated using a costar spinx column ( corning incorporated ) . 40 l 3 m sodium acetate ph 5.5 and 0.5 l glycogen were added to the supernatant and centrifuged at 13,000 rpm at 20 c overnight to precipitate the reverse transcription product . the cdna samples were then circularized by mixing with 2.0 l 10 circligase buffer ( epicentre ) , 1 l 50 mm mncl2 and 1 l circligase ii ( epicentre ) and incubated for 34 h at 60 c . for high - throughput sequencing , briefly , cdnas were mixed with 1 m each of pe1.0 ( 5-aatgatacggcgaccaccg agatctacactctttccctacacgacgctcttccgatct-3 ) and pe2.0 ( 5-caagc agaagacggcatacgagatcggtctcggcattcctgctgaaccgctcttccgatct ) primers ( 10 m concentration ) and kapa 2 ready mix ( kapa biosystems ) , and pcr amplification was performed as per the manufacturer 's instructions . the pcr product was gel eluted and subjected to sequencing on a hiseq 2000 sequencing system ( illumina ) . two biological replicates of the rbfox2 and cpsf2 iclip libraries were sequenced with a single - end length of 100 bp homopolymer sequences > 10 bp were removed followed by sequence quality assessment using fastqc ( v 0.10.1 ) [ http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ] ( fig . 1 ) . after clean up , the read lengths were in the range of 2172 bp . the fastqc score for all the bases was > 30 , indicating that the reads were of high quality . quality assessment was also performed on raw reads before clean up and similar quality was observed . following cleanup , 2009 ) using bowtie ( v1.0.0 ) with the following parameter setting : -q -e 100 -l 20 -m 1 best strata . finally , the iclip - seq reads were collapsed to remove pcr artifacts using picard tools ( v1.94 ) . reproducibility of the biological replicates was assessed using pearson correlation analysis ( rbfox2 r = 0.825 , p - value < 2.2e 16 ; cpsf2 r = 0.824 , p - value < 2.2e 16 ) ( see fig . rna - seq samples were prepared using a truseq rna library prep kit v2 ( illumina ) according to the manufacturer 's instructions . sequencing was done using an illumina hiseq 2000 with a paired - end length of 100 bp for duplicated ns , rbfox2- knockdown and cpsf2-knockdown samples . the fastqc score for all the bases was > 30 , indicating that the reads were of high quality . 2009 ) using tophat ( v2.0.9 , bowtie2/2.1.0 ) with the following parameter setting : -g [ ucsc_hg19_knowngene ] mate - inner - dist 50 b2-very - sensitive . the ucsc_hg19_knowngene annotation file was downloaded from ucsc table browser and quality of alignment was assessed using samstat ( v 1.09 ) . a pie chart describing the quality of sequence alignment distribution showed that > 97% of the reads have mapq 30 , indicating the high mapping quality of the reads ( fig . gene expression level ( fpkm ) and differential gene expression analysis were performed using cufflinks ( v2.1.1 ) . python script , provided by the dexseq ( v1.10.6 ) package , was used for exon level read count estimation with the following parameter setting : pearson correlation analysis of gene expression levels was performed to evaluate the reproducibility between biological replicates ( ns : r = 0.991 , p - value < 2.2e 16 ; rbfox2 : r = 0.991 , p - value < 2.2e 16 ; cpsf2 : r = 0.992 , p - value < 2.2e 16 ) ( fig . s2a in ) pearson correlation analysis of exon level expression also demonstrated high reproducibility between biological replicates ( ns : r = 0.997 , p - value < 2.2e 16 ; rbfox2 : r = 0.999 , p - value < 2.2e 16 ; cpsf2 : r = 0.989 , p - value < 2.2e 16 ) ( fig . multidimensional scaling ( mds ) plot was generated to visualize the similarity of gene expression between biological replicates and dissimilarity among ns , rbfox2 and cpsf2 knockdown samples using cummerbund package ( v 2.8.2 ) . the results of the mds plot showed that the biological replicates clustered closely while there was a clear segregation among ns , rbfox2 and cpsf2 knockdown samples , indicating that biological replicates were similar to each other and different knockdown groups had different expression profiles ( fig . 2c ) . to visualize the distribution of gene expression level for each sample , a boxplot was generated for each of the samples using log10 transformed fpkm values from cufflinks ( fig . the quartiles and overall range were consistent between biological replicates , indicating that the data were reproducible and of high quality . alternative splicing events were identified using the dexseq ( v1.10.6 ) package , which models the exon counts as the negative binomial distribution and uses generalized linear models to test differential exon - usage using variance estimated from biological replicates . sequence depths were used to normalize the exon counts for each sample followed by dispersion estimation before detecting alternative splicing events . dispersion plots showed that most of the final exon level dispersions follow the fitted line as expected ( fig . exons with a false discovery rate < 0.05 , log2 fold change greater than 1.2 , and a minimum of 10 counts for at least one of the samples were considered differentially expressed . alternative splicing events with chromosome coordinates overlapping with constitutive exons ( ensemble release version 75 ) by at least 1 bp were filtered out using the bioconductor genomicranges ( v 1.18.1 ) package . exons shorter than 12 bp or belonging to differentially expressed genes were filtered out from the list . dexseq creates a set of disjoint exon fragments ( pseudo exon ) from the ensemble exon annotation file . cells were transduced with lentiviruses expressing shrnas targeting rbfox2 or cpsf2 , or as a control a non - silencing ( ns ) shrna , and then puromycin selected for 4 days . flp - in-293 cells were grown in 10 cm plates and then subjected to uv - c irradiation ( 200 mj / cm , stratalinker 2400 ) . upon removal of phosphate buffered saline ( pbs ) , cells were scraped off and precipitated by centrifugation at 3000 rpm for 10 min . hcl ph 7.4 , 100 mm nacl , 1 mm mgcl2 , 0.1 mm cacl2 , 1% np-40 , 0.1% sds , 0.5% sodium deoxycholate ) . samples were partially digested with rnase i by incubation with 10 l rnase i ( ambion ) ( diluted 1:500 in lysis buffer ) and 5 l turbo dnase ( ambion ) for 5 min at 37 c while shaking at 1100 rpm . cells were centrifuged at 14,000 rpm for 15 min to collect the cross - linked lysate . to prepare the beads for rna purification , 50 l of protein g - coated dynabeads ( invitrogen ) was washed twice with 1 ml lysis buffer and resuspended in 200 l lysis buffer containing 35 g of rbfox2 ( bethyl laboratories , a300 - 864a ) or cpsf2 ( bethyl laboratories , a301 - 580a ) antibody . the beads were rotated at room temperature for 60 min , washed twice with lysis buffer , and added to the cross - linked lysate . after overnight incubation at 4 c , the beads were washed four times with high - salt wash buffer ( 50 mm tris hcl ph 7.4 , 1 m nacl , 1 mm edta , 0.1% sds , 0.5% sodium deoxycholate , 1% np-40 ) and once with pnk wash buffer ( 20 mm tris hcl ph 7.4 , 10 mm mgcl2 , 0.2% tween-20 ) . the beads were resuspended in 5 l 10 pnk buffer , 44 l h2o and 1 l pnk enzyme ( neb ) and incubated at 37 c for 20 min while shaking at 1100 rpm . samples were then washed twice with high - salt wash buffer and once with 900 l pnk wash buffer . to ligate linkers to the 3 end of rnas , 4.5 l of purified rna was mixed with 1 l of 10 m mircat33 adaptor ( integrated dna technologies ) , 1.5 l of rna ligase buffer , 0.5 l of t4 rna ligase 2 , truncated k227q ( neb ) , and 7.5 l of peg 8000 , and incubated at 30 c for 6 h. samples were then end - labeled using pnk enzyme and run on a nupage novex 10% bis tris protein gel ( invitrogen ) with 1 mops running buffer ( invitrogen ) . proteins and covalently bound rnas were then transferred to a nitrocellulose membrane ( whatman ) using a novex wet transfer apparatus ( invitrogen ) . the nitrocellulose membrane was rinsed with 1 pbs , wrapped in plastic wrap and exposed to x - ray film . to isolate the rna protein complex , the region of the nitrocellulose membrane corresponding to the complex size of interest was cut out . cross - linked rnas were then isolated by incubating the nitrocellulose membrane pieces with pk buffer ( 100 mm tris hcl ph 7.5 , 50 mm nacl , 10 mm edta ) containing 2 mg / ml proteinase k ( roche ) for 20 min at 37 c , after which an equal volume of pk buffer containing 7 m urea was added to the tube and the samples were incubated for another 20 min at 37 c . subsequently , 600 l of phenol / chloroform ( ambion ) was added , and the mixture was incubated for 10 min at room temperature . samples were then centrifuged for 10 min at 13,000 rpm at room temperature , and the aqueous phase was transferred into a new microtube and again centrifuged . 400 l of supernatant was mixed with 0.5 l glycoblue ( ambion ) , 40 l 3 m sodium acetate ph 5.5 , and 1 ml 100% ethanol and incubated overnight at 20 c . rnas were precipitated by centrifugation for 30 min at 15,000 rpm at 4 c , washed with 500 l 80% ethanol and resuspended in 30 l h2o . to carry out reverse transcription , 30 l purified rna was mixed with 1 l of either bc2 ( 5-pggcgatgagatcggaagagcgtcgtgtagggaaagagtgt - sp18-ctcg gcattcctgctgaaccgctcttccgatct - ccttggcacccgagaattcca-3 ) or bc3 ( 5-pggcgatgagatcggaagagcgtcgtgtagggaaagagtgt - sp18-ctcg gcattcctgctgaaccgctcttccgatct - ccttggcacccgagaattcca-3 ) prt primer ( 10 m concentration ) and 1.5 l 10 mm dntp mix , and incubated at 65 c for 5 min and then transferred to ice . subsequently , 9 l of 5 first strand synthesis buffer ( invitrogen ) , 2.25 l 0.1 m dtt , and 1.25 l superscript iii reverse transcriptase ( invitrogen ) were added to a final volume of 45 l . the cdnas were size - separated on a 6% tbe urea gel ( invitrogen ) . two size fractions were recovered from the gel by cutting the gel corresponding to a cdna size of 120155 nt and 150225 nt . gel fragments were mixed with 400 l te buffer , crushed with a 1 ml syringe plunger and centrifuged overnight at 1100 rpm at 37 c . gel pieces and the supernatant were separated using a costar spinx column ( corning incorporated ) . 40 l 3 m sodium acetate ph 5.5 and 0.5 l glycogen were added to the supernatant and centrifuged at 13,000 rpm at 20 c overnight to precipitate the reverse transcription product . the cdna samples were then circularized by mixing with 2.0 l 10 circligase buffer ( epicentre ) , 1 l 50 mm mncl2 and 1 l circligase ii ( epicentre ) and incubated for 34 h at 60 c . for high - throughput sequencing , briefly , cdnas were mixed with 1 m each of pe1.0 ( 5-aatgatacggcgaccaccg agatctacactctttccctacacgacgctcttccgatct-3 ) and pe2.0 ( 5-caagc agaagacggcatacgagatcggtctcggcattcctgctgaaccgctcttccgatct ) primers ( 10 m concentration ) and kapa 2 ready mix ( kapa biosystems ) , and pcr amplification was performed as per the manufacturer 's instructions . the pcr product was gel eluted and subjected to sequencing on a hiseq 2000 sequencing system ( illumina ) . two biological replicates of the rbfox2 and cpsf2 iclip libraries were sequenced with a single - end length of 100 bp homopolymer sequences > 10 bp were removed followed by sequence quality assessment using fastqc ( v 0.10.1 ) [ http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ] ( fig . the fastqc score for all the bases was > 30 , indicating that the reads were of high quality . quality assessment was also performed on raw reads before clean up and similar quality was observed . following cleanup , 2009 ) using bowtie ( v1.0.0 ) with the following parameter setting : -q -e 100 -l 20 -m 1 best strata . finally , the iclip - seq reads were collapsed to remove pcr artifacts using picard tools ( v1.94 ) . reproducibility of the biological replicates was assessed using pearson correlation analysis ( rbfox2 r = 0.825 , p - value < 2.2e 16 ; cpsf2 r = 0.824 , p - value < 2.2e 16 ) ( see fig . rna - seq samples were prepared using a truseq rna library prep kit v2 ( illumina ) according to the manufacturer 's instructions . sequencing was done using an illumina hiseq 2000 with a paired - end length of 100 bp for duplicated ns , rbfox2- knockdown and cpsf2-knockdown samples . the fastqc score for all the bases was > 30 , indicating that the reads were of high quality . 2009 ) using tophat ( v2.0.9 , bowtie2/2.1.0 ) with the following parameter setting : -g [ ucsc_hg19_knowngene ] mate - inner - dist 50 b2-very - sensitive . the ucsc_hg19_knowngene annotation file was downloaded from ucsc table browser and quality of alignment was assessed using samstat ( v 1.09 ) . a pie chart describing the quality of sequence alignment distribution showed that > 97% of the reads have mapq 30 , indicating the high mapping quality of the reads ( fig . 2a ) . gene expression level ( fpkm ) and differential gene expression analysis were performed using cufflinks ( v2.1.1 ) . python script , provided by the dexseq ( v1.10.6 ) package , was used for exon level read count estimation with the following parameter setting : pearson correlation analysis of gene expression levels was performed to evaluate the reproducibility between biological replicates ( ns : r = 0.991 , p - value < 2.2e 16 ; rbfox2 : r = 0.991 , p - value < 2.2e 16 ; cpsf2 : r = 0.992 , p - value < 2.2e 16 ) ( fig . s2a in ) pearson correlation analysis of exon level expression also demonstrated high reproducibility between biological replicates ( ns : r = 0.997 , p - value < 2.2e 16 ; rbfox2 : r = 0.999 , p - value < 2.2e 16 ; cpsf2 : r = 0.989 , p - value < 2.2e 16 ) ( fig . multidimensional scaling ( mds ) plot was generated to visualize the similarity of gene expression between biological replicates and dissimilarity among ns , rbfox2 and cpsf2 knockdown samples using cummerbund package ( v 2.8.2 ) . the results of the mds plot showed that the biological replicates clustered closely while there was a clear segregation among ns , rbfox2 and cpsf2 knockdown samples , indicating that biological replicates were similar to each other and different knockdown groups had different expression profiles ( fig . 2c ) . to visualize the distribution of gene expression level for each sample , a boxplot was generated for each of the samples using log10 transformed fpkm values from cufflinks ( fig . the quartiles and overall range were consistent between biological replicates , indicating that the data were reproducible and of high quality . alternative splicing events were identified using the dexseq ( v1.10.6 ) package , which models the exon counts as the negative binomial distribution and uses generalized linear models to test differential exon - usage using variance estimated from biological replicates . sequence depths were used to normalize the exon counts for each sample followed by dispersion estimation before detecting alternative splicing events . dispersion plots showed that most of the final exon level dispersions follow the fitted line as expected ( fig . exons with a false discovery rate < 0.05 , log2 fold change greater than 1.2 , and a minimum of 10 counts for at least one of the samples were considered differentially expressed . alternative splicing events with chromosome coordinates overlapping with constitutive exons ( ensemble release version 75 ) by at least 1 bp were filtered out using the bioconductor genomicranges ( v 1.18.1 ) package . exons shorter than 12 bp or belonging to differentially expressed genes were filtered out from the list . dexseq creates a set of disjoint exon fragments ( pseudo exon ) from the ensemble exon annotation file . here , we described rbfox2 and cpsf2 iclip datasets and their effects on alternative splicing at internal exons and introns . our results show that cpsf along with sympk is involved in promoting alternative splicing at internal introns and exons and shed light on a new role of mrna 3 end formation factors .

alternative splicing is a key mechanism for generating proteome diversity , however the mechanisms regulating alternative splicing are poorly understood . using a genome - wide rna interference screening strategy , we identified cleavage and polyadenylation specificity factor ( cpsf ) and symplekin ( sympk ) as cofactors of the well - known splicing regulator rbfox2 . to determine the role of cpsf in alternative splicing on a genome - wide level , we performed paired - end rna sequencing ( rna - seq ) to compare splicing events in control cells and rbfox2 or cpsf2 knockdown cells . we also performed individual - nucleotide resolution uv cross - linking and immunoprecipitation ( iclip ) to identify direct binding targets of rbfox2 and cpsf2 . here , we describe the experimental design , and the quality control and data analyses that were performed on the dataset . the raw sequencing data have been deposited in ncbi 's gene expression omnibus and are accessible through geo series accession number gse60392 .

Direct link to deposited data Experimental design, materials and methods Cell lines Individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) High-throughput sequencing and bioinformatic analysis of iCLIP data High-throughput sequencing and bioinformatic analysis of RNA-seq data Discussion Conflict of interest

PMC4877497

long - term potentiation ( ltp ) , a form of brain plasticity characterized by a long - lasting increase in synaptic coupling of neurons , has been suggested as a candidate mechanism mediating processes of learning and memory in the nervous system [ 1 , 2 ] . first characterized in the hippocampal formation , ltp has now been shown to occur at synapses throughout the nervous system , including cortical sensory areas such as the primary visual ( v1 ) , auditory , and somatosensory cortices [ 46 ] . in v1 , ltp has been successfully induced under both in vitro and in vivo conditions , with work in slice preparations indicating that ltp is limited to a well - defined time window during early postnatal life , after which v1 synapses become resistant to ltp induction [ 712 ] . interestingly , under in vivo conditions , ltp is readily induced in the fully matured v1 of adult rodents [ 1315 ] , indicative of some fundamental differences in the induction of ltp in v1 between in vivo and in vitro conditions . an important aspect of ltp regulation lies in the role of various neuromodulators present in the extracellular environment . for example , acetylcholine ( ach ) exerts a powerful , modulatory effect by enhancing ltp in v1 , an effect that is apparent under both in vitro and in vivo conditions and for a number of different ( e.g. , weak and strong ) ltp induction protocols [ 14 , 16 , 17 ] . a similar , facilitating effect on ltp is also seen with histamine application directly in v1 of rats in vivo , highlighting the importance of a variety of neuromodulators as gating mechanism for the induction of plasticity at cortical synapses [ 1921 ] . the central serotonergic ( 5-hydroxytryptamine , 5-ht ) system has also been implicated in the modulation of cortical synaptic plasticity . serotonergic fibers originating in the dorsal and median raphe nuclei provide a dense innervation of the entire forebrain , including v1 [ 2225 ] . the presence of 5-ht receptors in v1 has been confirmed by radioligand - binding experiments [ 26 , 27 ] and in situ hybridization techniques , implying functionality of the 5-ht input to v1 . previous work on the role of 5-ht in the regulation of ltp in v1 has yielded inconsistent results . in v1 slices obtained from kittens ( 4080 days old ) , 5-ht application facilitated ltp induction in layer 4 neurons , effects that were absent in older ( more than 120 days ) animals [ 8 , 9 ] . similarly , in the immature rat v1 in vitro , ltp of layer 2/3 neurons elicited by layer 4 stimulation was impaired by 5-ht depletion or bath application of antagonists of 5-ht1a or 5-ht2 receptors . together , these observations suggest a facilitating role for 5-ht in ltp induction in the immature v1 , findings that have recently been extended to more mature ( 810 weeks old ) rats . surprisingly , others have reported an inhibition of ltp in layers 2/3 of v1 slices obtained from juvenile rats following bath application of 5-ht [ 12 , 30 ] . these results have led to the suggestion that the developmentally increasing serotonergic tone in v1 is responsible for the loss of ltp throughout cortical maturation [ 10 , 31 ] , at least under in vitro conditions ( see above ) . however , it is unclear how to reconcile this assumption with data demonstrating serotonergic facilitation of ltp in the more mature v1 , as well as observations that both ltp and ocular dominance plasticity in v1 can be reinstated in adult rodents following treatment with the selective serotonin reuptake inhibitor ( ssri ) fluoxetine . the above summary of prior work suggests that the role of 5-ht in the regulation of ltp in v1 is not fully understood and may also depend on the age and developmental status of the animal . further , there is a clear lack of information regarding serotonergic effects on ltp assessed in the intact brain in vivo , which appears important , given the differences in ltp induction in the mature v1 between in vivo and in vitro preparations ( see above ) . to clarify some of these unresolved issues , we examined the role of 5-ht and some 5-ht receptors in the induction of ltp in the v1 of juvenile and adult rats using intact , urethane - anesthetized animals , an experimental preparation that continues to express ltp well into adulthood [ 1315 ] . all procedures adhered to the guidelines of the canadian council on animal care and were approved by the queen 's university animal care committee . experiments were conducted on adult ( 300550 g ; > 70 days old ) or juvenile ( 190240 g ; mean age : 45 days , age range : 4248 days ) male long - evans rats ( charles river laboratories , saint - constant , qc , canada ) . the animals were housed in a colony room under a reversed 12 : 12-h light cycle ( lights on at 19:00 h ) , with water and food access ad libitum . all efforts were made in order to minimize animal suffering and the number of animals employed for these experiments . each animal was deeply anesthetized with 2.0 g / kg urethane ( sigma - aldrich , oakville , on , canada ) , administered intraperitoneally ( i.p . ) as four doses of 0.5 g / kg each , given every 20 min . fifteen minutes prior to the start of surgery , the local analgesic bupivacaine ( marcaine ; hospira healthcare corporation , montreal , qc , canada ) was administered subcutaneously to the skin and tissue along the incision line over the skull ( two or three injections ; total of 5 mg / kg ) . throughout the surgical procedure and experiment , body temperature was monitored with a rectal thermometer and maintained at 37 1c by means of an electrical heating pad and fleece insulating blankets surrounding the body . after anesthesia induction , a rat was placed in a stereotaxic apparatus , the skull bone was exposed , and small holes were drilled overlying the following areas ( all measurements taken from bregma and the skull surface ) : lateral geniculate nucleus ( lgn ) , anterior - posterior 4.1 mm , lateral + 4.1 mm , and ventral 4.8 to 5.1 mm ; v1 , anterior - posterior 7.6 mm , lateral + 3.6 mm , and ventral 0.8 to 1.2 mm . two additional holes were drilled in the bone overlying the left and right cerebellum to secure jewelry screws , which served as ground and reference connections . a concentric , bipolar stimulation electrode ( sne-100 ; rhodes medical instruments , david kopf instruments , tujunga , ca ) was lowered into the lgn , while a monopolar recording electrode ( 125 m diameter teflon - insulated stainless steel wire ) was placed in the superficial layers of v1 . the final , ventral depth of both electrodes was adjusted to yield maximal amplitude field postsynaptic potentials ( fpsps ) recorded in v1 in response to single - pulse lgn stimulation . stimulation of the lgn ( single 0.2 ms pulses ) was achieved by connecting the stimulation electrode to a stimulus isolation unit ( ml 180 stimulus isolator ; ad instruments , toronto , on , canada ) providing a constant current output . the fpsps in v1 were recorded differentially , with the recording electrode referenced against a screw in the bone overlying the cerebellum . the v1 signal was amplified ( half - amplitude filters at 0.3 hz to 1 khz ) , digitized ( 10 khz ) by an a / d converter ( powerlab 4/s system running scope software v. 3.6.5 ; ad instruments ) , and stored for offline analysis . for each rat , an input - output curve was established by stimulating the lgn at increasing intensities ( 0.11.0 ma in 0.1 ma increments ) and the intensity yielding approximately 5060% of the maximal fpsp amplitude was then used for the remainder of the experiment ( see figure 1 ) . cortical fpsps were recorded every 30 s until 30 min of stable baseline recordings were achieved ( 5% difference between successive data points for fpsps averaged over 10 min epochs ) . subsequently , theta burst stimulation ( tbs ) was delivered to the lgn , consisting of five single pulses ( at 100 hz ) per burst , with bursts repeated at 5 hz for a total of 10 bursts ( pulse intensity and duration were the same as stated above ) . recordings of fpsps ( every 30 sec ) continued for 2 h following tbs delivery . in all experiments , spontaneous electrocorticographic ( ecog ) activity was also recorded through the same v1 electrode used for fpsp recordings . for the ecog , the cortical signal was digitized ( 200 hz ) , band - pass filtered ( 0.350 hz ) , and analyzed offline for peak power in the main frequency bands ( low delta , 0.51 hz ; delta , 14 hz ; theta , 48 hz ; alpha , 812 hz ; beta , 1220 hz ; and gamma , 2040 hz ) . the ecog was sampled ( 5 sec epochs ) prior to the onset of fpsp baseline recordings and at the end of experiment , that is , 120 min after tbs . to investigate the roles of 5-ht and different 5-ht receptors , independent groups of animals received one of the following drug treatments : 5-hydroxytryptamine hydrochloride ( 5-ht ; 0.1 or 10 mm ; sigma - aldrich ) ; n-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-n-2-pyridinylcyclohexanecarboxamide maleate ( way 100635 ; 1 mm ; tocris bioscience , ellisville , mo , usa ) ; 1-[10,11-dihydro-8-(methylthio ) dibenzo(z)[b , f]thiepin-10-yl]-4-methylpiperazine maleate ( methiothepin ; 1 mm ; tocris bioscience ) ; 8-hydroxy-2-(di - n - propylamino)-tetralin hydrobromide ( 8-oh - dpat ; 1 mm ; tocris bioscience ) . way 100635 and 8-oh - dpat act as potent 5-ht1a receptor antagonist and agonist , respectively [ 3234 ] , and 8-oh - dpat also exhibits an affinity for 5-ht7 receptors ( see ) . methiothepin is a potent 5-ht2 receptor antagonist but also acts as an antagonist at 5-ht1 , 5-ht5a , 5-ht5b , 5-ht6 , and 5-ht7 receptors [ 36 , 37 ] . all compounds were dissolved in artificial cerebrospinal fluid ( acsf ) , consisting of 118.3 mm nacl , 4.4 mm kcl , 1.2 mm mgso4 , 1.0 mm nah2po4 , 2.5 mm cacl2 , 22.1 mm nahco3 , and 10.0 mm glucose , with the exception of methiothepin , which was dissolved in either a mixture of acsf and dimethyl sulfoxide ( dmso ; n = 5 ) , saline ( n = 4 ) or acsf and distilled water ( n = 4 ; there were no significant differences in ltp among these different vehicle solutions and rats were combined into a single methiothepin group ) . ( mab 6.14.2 , 15,000-da cut - off polyether sulfone membrane , outer diameter 0.6 mm ; s.p.e . limited , north york , on , canada ) was mounted immediately adjacent to the v1 recording electrode , with the probe tip extending approximately 1 mm past the electrode tip . drug concentrations reaching the brain are estimated to be ~10% of the acsf content within the vicinity of the probe membrane ( about 1 mm ; [ 38 , 39 ] ) . the dialysis probe was connected to a 2.5ml hamilton syringe using fep microtubing ( s.p.e . the syringe was driven by a microdialysis pump ( cma 402 ; cma microdialysis , solna , sweden ) at a flow rate of 1 l / min , with perfusion beginning 20 min prior to the acquisition of baseline fpsp recordings and continuing throughout the entire experiment . at the conclusion of electrophysiological data acquisition , all animals received a supplementary dose ( 1.0 ml ) of urethane and , after 510 min , were perfused through the heart with 0.9% saline ( ~50 ml ) followed by 10% formalin ( ~100 ml ) . the brains were removed and stored in 10% formalin for a minimum of 24 h before sectioning ( 40 m slices ) with a cryostat . slices were then mounted onto microscope slides and inspected with a digital microscope to verify electrode placements . histological inspections and decisions on the accuracy of electrode placements were made by an experimenter who was blind to the results of individual animals . data from inaccurate placements were omitted from this study ( 77 and 39 rats included and rejected due to missed placements , resp . ) . cortical fpsps were analyzed with scope software ( v. 3.6.5 ; ad instruments ) . with the electrode configuration employed in the present study , fpsps elicited in v1 consisted of a predominant , large amplitude , negative - going component . the amplitude of this component was automatically detected and computed with scope software ( using the data pad function ) by measuring the voltage difference between the maximal fpsp negativity and the baseline voltage sampled immediately prior to the stimulation artifact . once individual fpsps were analyzed in this manner , they were averaged over successive 10 min intervals ( i.e. , 20 fpsps / interval ) and then normalized by dividing them by the average baseline ( pre - tbs ) amplitude of each animal . amplitude data are presented as mean standard error of the mean ( sem ) and were analyzed with a repeated - measures analysis of variance ( anova ) and followed up , where statistically appropriate , with bonferroni post hoc tests or unpaired student 's t - tests using spss software ( v. 15.0 ; spss , colorado springs , co , usa ) . the analysis was conducted with time ( 10 min averaging epochs ) as the repeated / within - subjects factor and drug group as the between - subjects factor . five - second epochs of ecog activity were analyzed using chart software ( v. 5.5.6 ; ad instruments ) . for each epoch , spectral analysis was used to determine the % of total power contained in each of the following frequency bands : 0.51 hz , 14 hz , 48 hz , 812 hz , 1220 hz , and 2040 hz . data from individual experiments were averaged across treatment groups and statistically analyzed by anova using spss software . in urethane - anesthetized animals , single - pulse stimulation of the lgn reliably elicited fpsps in the ipsilateral v1 ( figure 1 ) . in agreement with previous work using this experimental preparation [ 14 , 15 , 18 ] , fpsps were composed mainly of a large amplitude ( up to 0.5 mv ) , negative component , with a latency to peak of 1618 ms following lgn stimulation ( figure 1 ) . these fpsps appear to reflect , for the most part , excitatory current sinks originating in layers 2/3 of v1 . levels of ltp may be influenced by differences in baseline ( i.e. , prior to ltp induction ) synaptic strength . thus , we initially computed and compared the baseline amplitude of fpsps in all experimental groups prior to the delivery of tbs to induce ltp . as shown in figure 2 , for all groups , baseline fpsp amplitudes were in the range of 0.37 to 0.47 mv . application of the high concentration of 5-ht ( 10 mm ) resulted in a modest , but significant ( p < 0.05 , t - test ) suppression of fpsp amplitude relative to rats receiving acsf application in v1 . none of the other pharmacological treatments produced a significant change in fpsp amplitude relative to the acsf condition ( figure 2 ) . to determine the effects of 5-ht application on ltp , separate groups of adult animals received application of either acsf or 5-ht at 0.1 mm or 10 mm . application occurred locally in v1 by means of reverse microdialysis , with the dialysis probe situated immediately adjacent to the cortical recording electrode . during acsf application ( n = 8) , tbs of the lgn reliably induced ltp , with fpsp amplitude increasing to 121 4% of baseline ( figure 3 ; all values reported here are averages of the last 30 min of the experiment , i.e. , from 91 to 120 min after tbs delivery ) . during application of 0.1 mm ( n = 5 ) or 10 mm of 5-ht ( n = 7 ) , fpsp amplitude reached 131 5% and 133 4% of the baseline , respectively ( figure 3 ) . these values appeared higher than those observed during acsf application , but this difference did not reach statistical significance , as revealed by the anova showing a significant main effect of time , but not of group ( f2,17 = 0.5 , p > 0.6 ) or a time group interaction ( f10,88 = 0.91 , p > 0.5 ) . thus , under the present , experimental conditions , 5-ht did not exert a significant influence on ltp induction in the thalamocortical visual system of adult rats . to examine a possible effect of 5-ht blockade on ltp , we initially applied the broad - acting 5-ht receptor antagonist methiothepin ( 1.0 mm ) in a separate group of adult animals ( n = 13 ) . in these rats , tbs of the lgn resulted in an increase of fpsp amplitude to 149 3% of baseline during the last 30 min of the experiment ( figure 4 ) . given that methiothepin was dissolved in several , different vehicle solutions ( see section 2.4 . ) a new group of control rats was also examined ( n = 11 ; consisting of acsf / dmso , n = 4 ; saline , n = 3 ; and acsf / h20 , n = 4 ) . in these rats , fpsp amplitude reached 130 4% of baseline during the final 30 min of the experiment ( figure 4 ) . an anova revealed that methiothepin enhanced ltp levels , as indicated by significant main effects of time ( f5,116 = 38.8 , p < 0.001 ) and group ( f1,22 = 6.38 , p < 0.02 ) , as well as a significant time group interaction ( f3,116 = 3.47 , p 0.005 ) . in order to further characterize the ltp enhancement seen with 5-ht receptor blockade , we tested the effect of v1 application of way 100635 ( 1 mm , n = 10 ) , a highly selective antagonist at 5-ht1a receptors [ 31 , 32 ] . in this group of rats , tbs increased fpsp amplitude to 146 4% of baseline ( figure 5 ) . this level of ltp was significantly higher than that in rats receiving application of acsf ( the same group as in figure 3 ; 121 4% of the baseline , n = 8) , as indicated by the main effects of time ( f5,88 = 22.9 , p < 0.001 ) and group ( f1,16 = 10.51 , p < 0.005 ) , and by a time group interaction ( f5,88 = 2.75 , p 0.02 ) . based on the results reported above , it appears that 5-ht1a receptors exert an inhibitory effect over ltp induction in the mature v1 . to further examine the role of these receptors , we applied the highly selective 5-ht1a receptor agonist 8-oh - dpat in v1 ( 1 mm , n = 8) . in these animals , tbs elicited ltp , with fpsp amplitude reaching 123 3% of baseline during the last 30 min of the experiment ( figure 6 ) . these values were not significantly different from those in rats receiving acsf application ( same group as in figure 3 ) , as indicated by a significant main effect of time ( f5,68 = 20.8 , p < 0.001 ) , but not of group ( f1,14 = 0.7 , p = 0.4 ) , and no significant time group interaction ( f5,68 = 0.8 , p = 0.6 ) . thus , blockade of 5-ht1a receptors facilitates ltp , but 5-ht1a receptor activation does not result in further inhibition of ltp , similar to the effects noted above with direct 5-ht application in v1 . considerable evidence from in vitro experiments suggest that 5-ht plays a role in developmental plasticity , including the timing and closure of the critical period for plasticity in the rodent v1 [ 8 , 9 , 12 , 17 , 23 , 27 , 29 , 31 ] . thus , we also investigated the effects of 5-ht1a receptor blockade by way 100635 application ( 1 mm ) in a group of juvenile animals ( age range : 4248 days ) . in juvenile control animals ( n = 7 ; mean age 44 days ) receiving acsf application in v1 , tbs resulted in ltp , with fpsp amplitude at 130 7% of the baseline during the final 30 min of the experiment ( figure 7 ) . surprisingly , in the presence of way 100635 , juvenile rats ( n = 8 ; mean age 44.9 days ) showed reduced ltp , with fpsp amplitude at 110 6% of baseline during the last 30 min of the experiment ( figure 7 ) . levels of ltp in these two groups of juvenile rats were different , as highlighted by a main effect of time ( f4,47 = 14.4 , p < 0.001 ) and a significant time group interaction ( f4,47 = 2.984 , p < 0.03 ) , even though the main effect of group only approached statistical significance ( f1,13 = 4.3 , p = 0.058 ) . together , these observations suggest that there is a developmental switch in the role of 5-ht1a receptors in regulating v1 plasticity , with 5-ht1a receptor activation promoting plasticity in the juvenile v1 , while assuming an inhibitory role in v1 of adult , fully matured animals . in addition to sampling evoked fpsps , we also recorded ecog activity in v1 to assess potential effect of 5-ht or drug application on spontaneous , oscillatory activity of the cortex . for all experiments , ecog activity was assessed before the onset of baseline fpsp recordings ( i.e. , 20 min after the onset of drug application ) and at the end of the experiment . in adult animals , the ecog was dominated by large amplitude , slow oscillations , with peak power concentrated in the low delta ( 0.51 hz ) and delta ( 14 hz ) frequency bands , as determined by power spectral analyses ( figure 8) . power in all frequency bands ( 0.51 hz , 14 hz , 48 hz , 812 hz , 1220 hz , and 2040 hz ) remained quite stable over the course of the experiment when acsf was applied to v1 ( figure 8) . further , application of 5-ht , methiothepin , or way 100635 did not result in any significant changes in ecog activity over the course of the experiment when compared with acsf application ( figure 8 ; all group effects and group by time effects nonsignificant , p 's > 0.05 ) . similar observations were made for ecog activity in juvenile rats , which also exhibited peak power in the low delta ( 0.51 hz ) and delta ( 14 hz ) frequency bands that was not significantly altered by application of way 100635 ( data not shown ) . these data suggest that changes in spontaneous , oscillatory activity in v1 do not account for the effects of methiothepin and way 100635 to alter ltp in v1 following tbs of the lgn in urethane - anesthetized animals . the present set of experiments examined the role of 5-ht and 5-ht1a receptors in gating thalamocortical plasticity between lgn and v1 of juvenile and adult rats studied under urethane anesthesia . application of 5-ht ( 0.1 to 10 mm ) in v1 did not affect the induction or maintenance of ltp in adult animals under the present , experimental conditions . however , v1 application of the broad - acting 5-ht receptor antagonist methiothepin or the selective 5-ht1a receptor antagonist way 100635 resulted in a clear facilitation of ltp in adult rats , suggestive of a suppression of ltp by endogenous 5-ht release and 5-ht receptor activation . in contrast , way 100635 reduced ltp when tested in a group of juvenile rats ( mean age 45 days ) . none of the pharmacological treatments that altered ltp induction exerted significant effects on baseline synaptic transmission ( fpsp amplitude prior to ltp induction ; note that 5-ht at 10 mm suppressed baseline fpsp amplitude but did not affect ltp ) . similarly , none of the pharmacological treatments altered spontaneous ecog activity recorded in v1 over the course of the experiment . together , these results indicate that 5-ht1a receptors play an important , age - dependent role in gating plasticity in the thalamocortical visual system of rats , with 5-ht1a receptor activation facilitating ltp in juveniles , but inhibiting ltp in the brains of adult animals . we were surprised that , in our experiments , 5-ht application exerted no detectable effect on ltp induction in v1 . a considerable amount of in vitro work has shown that 5-ht can alter ltp in v1 slice preparations , even though there are considerable inconsistencies in the specific results among various studies . in v1 slices of kittens , 5-ht has been shown to enhance ltp recorded in layer 4 , effects that were absent in slices obtained from adult animals [ 8 , 9 ] . depletion of 5-ht ( in combination with noradrenaline depletion ) has been shown to impair ltp in layers 2/3 of the immature rat v1 in vitro , an effect that was mimicked by application of either 5-ht1a or 5-ht2 receptor antagonists . recently , these findings have been extended by showing that bath application of 5-ht enhances ( in fact , reinstates ) ltp in layers 2/3 of v1 of adult ( 810 weeks old ) rats , confirming that 5-ht can exert a facilitating effect on the induction of long - lasting plasticity in v1 . in clear contrast , other studies have indicated that 5-ht application results in a pronounced suppression of ltp at synapses between layers 4 and 2/3 of v1 of rats ( 35 weeks old ) , an effect that is mediated by both 5-ht1a and 5-ht2 receptors [ 10 , 30 , 40 , 41 ] . in fact , in v1 slices of 5-week - old rats , ltp could no longer be induced ; however , acute bath application of the 5-ht receptor antagonist methysergide , acute 5-ht depletion , or neurotoxic ablation of serotonergic neurons restored ltp [ 10 , 31 ] . a similar , inhibitory effect of 5-ht application has also been demonstrated for the induction of long - term depression in v1 in vitro . interestingly , 5-ht levels in v1 show a significant increase ( up to 3.5-fold from 3 to 5 weeks of age ) over postnatal development [ 10 , 31 ] . these observations have led to the hypothesis that 5-ht , in concert with other neurochemical changes ( particularly the maturation of gabaergic circuits [ 43 , 44 ] ) , contributes to the loss of v1 plasticity over postnatal life , resulting in the closure of the critical or sensitive period of v1 development [ 10 , 31 , 42 ] . our data are consistent with the hypothesis that the release of endogenous 5-ht exerts a tonic , inhibitory influence on ltp induction , at least in adult rats . cortical 5-ht is detectable in urethane - anesthetized rats , even during periods of ecog synchronization . we speculate that , under the present conditions , this effect of endogenous 5-ht is maximal , making the application of exogenous 5-ht ineffective in further suppressing ltp . however , future studies using 5-ht - depleted animals are necessary to examine this hypothesis . the presumed , inhibitory effect of endogenous 5-ht is relieved by methiothepin and way 100635 , indicative of a role of 5-ht1a receptors , even though we do not rule out the involvement of other 5-ht receptor types ( e.g. , 5-ht7 receptors , which are blocked by methiothepin and have recently been implicated in processes of learning , memory , and synaptic plasticity ) . in v1 slices of young ( 5 weeks old ) rats , both 5-ht1a and 5-ht2 receptors mediate the effect of 5-ht to suppress ltp induction [ 10 , 40 , 41 ] . it is noteworthy , however , that way 100635 and methiothepin application resulted in very similar levels of ltp enhancement , indicating that 5-ht1a receptor blockade alone is sufficient to result in a substantial disinhibition of ltp induction mechanisms under the present , experimental conditions . in contrast to the enhancement of ltp in adult animals , application of way 100635 in juvenile animals ( 4248 days ) suppressed ltp , indicative of an age - related switch in the role of 5-ht1a receptors in gating v1 plasticity . a similar phenomenon has previously been described for direct 5-ht application , albeit in a direction opposite to that revealed by the current set of experiments . park et al . observed that , in v1 slices obtained from juvenile rats ( 5 week old ) , 5-ht suppressed ltp , while 5-ht enhanced ltp in slices of adult animals ( 810 weeks old ) . at present , it is not clear why this pattern contradicts the data obtained in our experiments . were mediated by 5-ht2 receptors , while out data clearly indicate a role for 5-ht1a binding sites in v1 ( see above ) . thus , it is possible that different receptors populations do , indeed , exert opposing effects on plasticity gating in v1 , an assumption that requires a critical assessment with further investigations ( also see for a differential effect of 5-ht1a and 5-ht2 receptor activation on inhibitory transmission in v1 ) . to the best of our knowledge , the current experiments are the first to assess the role of 5-ht in gating ltp in the rodent v1 under in vivo conditions . work conducted in vitro clearly offers significant advantages in terms of delineating microscopic and mechanistic properties of synaptic transmission and plasticity , such as the concurrent analysis of changes in inhibitory and excitatory synapses in v1 during ltp induction . at the same time , there appear to be some important differences between ltp studied in vivo compared to in vitro conditions . numerous studies have shown that v1 synapses studied in vitro become increasingly resistant to ltp induction with postnatal maturation , and slices harvested from adult animals do not show ltp with standard induction protocols [ 7 , 8 , 1012 , 31 , 48 , 49 ] . in sharp contrast , ltp is readily induced and maintained in v1 when studied in adult , intact - animal ( anesthetized ) preparations [ 1316 , 18 , 21 ] . also of interest , protocols that effectively elicit ltd in v1 in vitro often fail to do so when tested under in vivo conditions [ 50 , 51 ] . thus , there clearly are some fundamental differences in the mechanisms and/or conditions that govern the induction of long - lasting plasticity at v1 synapses between in vitro and in vivo conditions , which might explain some of the apparent discrepancies among studies ( see above ) . the use of tissue harvested from very young animals ( 20 days old or less ) , the routine use of some pharmacological agents in the bath solution ( e.g. , gabaergic antagonists ) , the removal of long - range ( corticocortical , thalamocortical , and subcortical ) projections , and the loss neuromodulatory inputs in slice preparations all introduce conditions that are very different from those present in vivo ( see for a detailed discussion of the advantages and disadvantages of electrophysiological work conducted in vivo and in vitro ) . these questions and discrepancies highlight the need for further work , in particular in intact animals , which preserve the anatomical connectivity and complex , physiological interactions among cortical and subcortical networks that regulate activity and plasticity of synapses in v1 and elsewhere . importantly , anesthetized and nonanesthetized preparations should also be compared to assess whether the systemic effects of urethane or other anesthetics alter the plasticity properties of v1 synapses [ 51 , 52 ] . the results of the present experiments indicate that 5-ht1a receptors exert a tonic , inhibitory influence over ltp in adult animals but facilitate ltp in the juvenile v1 . prior work has shown that 5-ht1a receptors are located ( albeit in different densities ) on both interneurons and pyramidal cells of v1 . thus , direct effects on principal neurons and/or the modulation of inhibitory tone in v1 are likely candidate mechanisms for the effects noted in our experiments . the observation that 5-ht1a receptor activation can suppress nmda receptor functions in principle v1 cells suggests a relatively direct action , but this does not preclude an involvement of other mechanisms ( e.g. , disinhibition or changes in the excitatory - inhibitory balance [ 47 , 53 ] ) in the effects observed here . for the present experiments , we employed drug concentrations that are higher than those used for typical in vitro experiments . there are several reasons why we decided to use these higher concentrations : ( a ) pharmacological agents were applied by means of reverse microdialysis ; it is generally assumed that only about 10% of drug molecules will diffuse across the dialysis probe membrane and into the surrounding , neural tissue [ 38 , 39 ] ; ( b ) drugs applied under in vivo conditions by reverse dialysis ( but also during direct infusions ) undergo extensive degradation , due to interactions with the probe membrane and lipophilic molecules , as well as diffusion and continuous enzymatic breakdown [ 38 , 55 ] . thus , as has been pointed out by others [ 55 , 56 ] , there is a clear discrepancy in terms of effective drug concentrations when comparing experimental in vivo and in vitro approaches . nevertheless , we do acknowledge that it will be important for future work to establish whether the effects reported here can be elicited with drug concentrations that are lower than those employed for the present set of experiments . there is a growing body of evidence that 5-ht plays an important role in shaping plasticity of the developing and mature nervous system and that alterations in 5-ht transmission can result in neurodevelopmental and psychiatry disorders [ 57 , 58 ] . prior work has shown that ltp in v1 , in addition to its important role in ocular dominance plasticity [ 6 , 11 , 59 ] , may also mediate processes of visual ( perceptual , recognition ) learning and memory storage [ 59 , 60 ] . based on these hypotheses and the results of the present investigation , we anticipate that altering serotonergic transmission in v1 exerts profound , age - dependent effects on visual processing and learning . for example , blockade of 5-ht1a receptors in v1 may enhance perceptual learning in adults , when 5-ht acts to stabilize synaptic connectivity ; opposite behavioral effects would be expected in juvenile animals , when 5-ht1a receptors act to facilitate plasticity induction in v1 . clearly , investigations that involve a combination of behavioral , pharmacological , and electrophysiological approaches are required to characterize the role of 5-ht in visual behavior and directly test some of the hypothesis stated above . finally , the modulatory effects of 5-ht on activity- and experience - dependent plasticity also are of relevance to the potential treatment of various nervous system disorders . for example , chronic treatment with a selective serotonin reuptake inhibitor has been shown to reinstate ocular dominance plasticity ( and ltp ) in the v1 and allow recovery from amblyopic visual deficits in adult rodents , highlighting the therapeutic potential of serotonergic manipulations that alter the plasticity potential of cortical circuits ( but see ) . it remains to be established whether the effects following chronic , serotonergic manipulations relate to the role of 5-ht in acute plasticity gating described with the present set of experiments .

the rodent primary visual cortex ( v1 ) is densely innervated by serotonergic axons and previous in vitro work has shown that serotonin ( 5-ht ) can modulate plasticity ( e.g. , long - term potentiation ( ltp ) ) at v1 synapses . however , little work has examined the effects of 5-ht on ltp under in vivo conditions . we examined the role of 5-ht on ltp in v1 elicited by theta burst stimulation ( tbs ) of the lateral geniculate nucleus in urethane - anesthetized ( adult and juvenile ) rats . thalamic tbs consistently induced potentiation of field postsynaptic potentials ( fpsps ) recorded in v1 . while 5-ht application ( 0.110 mm ) itself did not alter ltp levels , the broad - acting 5-ht receptor antagonists methiothepin ( 1 mm ) resulted in a clear facilitation of ltp in adult animals , an effect that was mimicked by the selective 5-ht1a receptor antagonist way 100635 ( 1 mm ) . interestingly , in juvenile rats , way 100635 application inhibited ltp , indicative of an age - dependent switch in the role of 5-ht1a receptors in gating v1 plasticity . analyses of spontaneous electrocorticographic ( ecog ) activity in v1 indicated that the antagonist - induced ltp enhancement was not related to systematic changes in oscillatory activity in v1 . together , these data suggest a facilitating role of 5-ht1a receptor activation on ltp in the juvenile v1 , which switches to a tonic , inhibitory influence in adulthood .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

PMC4499966

music is often used to ameliorate the monotony of an activity , as a stimulus when performing exercises , or simply as background music1 , 2 . music can affect physical activity by producing psychological effects that influence mood , emotion , affect ( i.e. , feeling of pleasure or displeasure ) , cognition , and behavior as well as psychophysiological effects associated with subjective perceived exertion and fatigue2,3,4 . although music can increase motivation and affective components associated with high levels of effort , its effects on low- to moderate - intensity activities such as walking are more pronounced5 . many sedentary individuals with little experience in physical activities , especially women , have adopted walking as an exercise to initiate regular physical activity6 . walking is a popular form of exercise that is easy and has a low risk of injury ; besides promoting physical fitness , walking can aid the loss and maintenance of body weight7 . the effect of music on walking is attributed to psychophysiological and ergogenic changes that mainly affect the final performance of the exercise and perceptual responses8 . many attempts have been made to provide greater psychological effects to help beginners adhere to an exercise program9,10,11 . self - selected intensity may provide a tolerable , motivating , and effective exercise intensity for the development and maintenance of physical aptitude in individuals with low fitness levels10 . moreover , music can help beginners during self - selected walking by maximizing motivation and affect as well as acting as a distractor from non - pleasurable stimuli12 , 13 . although music and self - selected walking can help individuals follow a regular physical exercise regime , few studies have evaluated the effect of musical stimulation on self - selected walking pace exercise . therefore , this study assessed the effects of musical tempo on physiological , affective , and perceptual responses as well as the performance of self - selected walking pace . the purpose and procedure of the study were explained to each subject , who then voluntarily signed an informed consent agreement before participating in the experiment . all subjects were classified as having a sedentary lifestyle , i.e. , performing less than 30 min of moderate physical activity three or more days a week . the inclusion criteria were as follows : ( a ) 2951 years of age ; ( b ) ability to participate in regular physical exercise ; ( c ) negative responses to all questions in the physical activity readiness questionnaire ( par - q ) ; ( d ) body mass index ( bmi ) from 18.527 kgm ; and ( e ) a personal statement of not having smoked in the last 12 months . meanwhile , the exclusion criteria were the presence of cardiovascular , metabolic , or orthopedic disease or any other contraindications as determined by medical history in the preceding 12 months . this study was approved by the research ethics committee of the department of health sciences at the federal university of parana ( ufpr ) in curitiba , brazil . the subjects were divided into three treatment groups : the control group , which was not subjected to musical stimulation , musical stimulation at 90 beats per minute ( bpm ) , and musical stimulation at 140 bpm . the genre of music used was pop , and the songs were listened to through headphones . the music volume was adjusted to a level considered pleasurable by the subject before the start of the walk . all subjects completed three exercise sessions : ( 1 ) sample screening and familiarization , ( 2 ) an incremental test to exhaustion to determine physiological variables , and ( 3 ) a 30-min walk on the treadmill at a self - selected pace . all experiments were conducted in the morning ( between 08:00 and 12:00 h ) under similar environmental conditions ( 21 c and 55% relative humidity ) . the subjects were advised not to consume alcohol , caffeine , or perform vigorous physical activity 24 h prior to each test . physiological responses , rating of perceived exertion ( rpe ) , and affective responses were recorded during walking at a self - selected pace . thus , the independent variable was musical stimulus ( i.e. , control , or musical stimulation at 90 or 140 bpm ) , whereas the dependent variables were physiological responses , rpe , affective responses , and performance at a self - selected walking pace . to facilitate understanding of the experimental procedures , the subjects performed a familiarization session , during which they were taught to correctly use the scales and resources required to perform the test procedures . the incremental test to exhaustion on a treadmill was conducted using the standard protocol proposed by bruce15 , with 3-min stages for the evaluation of vo2 max . the subjects were verbally encouraged to continue the exercise to the point of exhaustion . the criteria required to achieve vo2 max were as follows : ( a ) a plateau of vo2 ( changes < 150 mlmin ) , ( b ) respiratory exchange ratio 1.10 , and ( c ) heart rate ( hr ) within 10 bpm of the maximum level expected for the subject s age16 . hr ( bpm ) was measured every 5 s using a polar monitoring system ( polar electro , oy , finland ) . a metabolic open - circuit breathing system ( true max 2400 , parvo medics , salt lake city , ut , usa ) was used to measure vo2 , carbon dioxide produced ( vco2 ) , and pulmonary ventilation ( ve ) every 15 s throughout the test . each time prior to the determination of vo2 max , the gas analyzer was calibrated with known concentrations of gases . oxygen consumption at the ventilatory threshold ( vo2lv ) was determined as described by caiozzo et al17 . vo2lv was identified as the point at which the ratio of minute ventilation plotting of oxygen consumption ( ve / vo2 ) versus the ratio of minute ventilation co2 production ( ve / vco2 ) deviates from normalcy . we initially conducted a 5-min warm - up at 1.11 m / s without inclination . the subjects were subsequently instructed to self - select a walking pace for the next 30 min as described by pintar et al10 . adjustments were made during the first minute of walking and then at 5 , 10 , 15 , 20 , and 25 min . the physiological variables were evaluated using the same instruments and procedures used in the incremental treadmill test ; however , only values obtained at 10 , 15 , 20 , 25 , and 30 min18 were considered . the data recorded during last 2 min in 15-s intervals of the respective stages were averaged , and the overall averages were subsequently calculated using these values to represent the physiological responses during the 30-min walk . perceived exertion was determined using the rpe omni - res scale19 . this instrument basically consists of a 10-point likert scale in which 0 indicates extremely easy and 10 indicates extremely difficult . subjects were instructed how to use the scale and perform docking procedures before the tests as described by utter et al19 . the instrument was administered during the last 15 s of each stage in a minute and during the 30-min walk test . the values obtained at 10 , 15 , 20 , 25 and 30 min were used in the analysis . affective responses were determined according to the feeling scale proposed by hardy and rejeski20 . this instrument basically consists of an 11-point scale with single bipolar items ranging from + 5 ( very good ) to 5 ( very bad ) with 0 indicating neutral . the scale was used during the last 15 s of each stage of 3 min , the maximal incremental test , and 30-min walk test at a self - selected pace . the values obtained at 10 , 15 , 20 , 25 , and 30 min were analyzed . the subjects were asked to indicate their feelings of pleasure or displeasure at the abovementioned time points . standardized definitions of affection were presented before the maximal incremental test and 30-min walk test at a self - selected pace . one - way anova was used to analyze the physiological anthropometric variables ( i.e. , maximal test ) and final results of the 30-min walk test . meanwhile , multivariate analysis was used to verify the physiological , positive perceptual , and affective responses during the 30-min walk test at a self - selected pace . all data were analyzed using spss for windows version 17.0 ( spss inc . , chicago , il , usa ) . one - way anova was used to analyze the physiological anthropometric variables ( i.e. , maximal test ) and final results of the 30-min walk test . meanwhile , multivariate analysis was used to verify the physiological , positive perceptual , and affective responses during the 30-min walk test at a self - selected pace . all data were analyzed using spss for windows version 17.0 ( spss inc . , chicago , il , usa ) . physiological and anthropometric measurements with respect to age are shown in table 1table 1.anthropometric and physiological characteristics of the subjectsvariables90 bpm140 bpmcontroln1099age ( y)42.4 4.542.7 6.641.7 5.0weight ( kg)61.1 8.959.9 4.662.7 11.6height ( cm)1.57 0.041.58 0.051.58 0.05bmi ( kgm)24.5 2.724.0 2.025.1 4.0body fat ( % ) 26.8 4.725.1 3.325.8 6.4hrmax ( bpm)176.1 9.0182.8 8.9174.9 13.8hrvt ( bpm)143.0 14.2154.8 10.3139.6 20.4vo2max ( mlkg min)29.7 6.129.2 4.327.2 4.6vo2vt ( mlkgmin)24.2 3.223.9 1.721.4 4.0bmi : body mass index ; vo2max : maximal oxygen consumption ; vo2vt : oxygen consumption in ventilatory threshold ; hrvt : heart rate in ventilatory threshold ; hrmax : maximal heart rate ; bpm : beats per minute . data are expressed as mean sd .. no significant differences were observed among groups . bmi : body mass index ; vo2max : maximal oxygen consumption ; vo2vt : oxygen consumption in ventilatory threshold ; hrvt : heart rate in ventilatory threshold ; hrmax : maximal heart rate ; bpm : beats per minute . data are expressed as mean sd . the physiological , perceptual and affective responses of the groups obtained during the 30-min walk test at a self - selected pace are shown in table 2table 2.physiological , perceptual , and affective responses to 30 minutes of walking at a self - selected paceaffect ( 5 to + 5)10 min15 min20 min25 min30 min90 bpm3.2 1.13.2 1.42.6 1.81.6 2.01.6 1.3140 bpm2.9 2.02.2 2.01.9 2.31.7 2.41.5 2.3control3.7 0.93.2 1.13.3 0.73.1 0.82.4 1.0% hrmax10 min15 min20 min25 min30 min90 bpm57.9 7.360.3 6.962.4 7.863.9 5.865.9 6.6140 bpm66.6 9.468.7 1070.3 10.171.0 10.473.5 10control62.0 9.864.6 10.967.0 11.567.7 13.070.3 14.4% vo2max10 min15 min20 min25 min30 min90 bpm39.8 8.840.5 10.741.4 13.642.1 12.542.9 11.8140 bpm45.4 13.147.1 13.150.0 13.751.7 13.252.9 14.3control39.9 12.141.2 12.243.0 14.246.1 13.248.3 13.2rpe ( 010)10 min15 min20 min25 min30 min90 bpm2.2 0.62.8 1.13.5 1.44.3 1.54.2 1.5140 bpm3.6 1.9 * 4.3 1.7 * 5.2 1.3 * 5.7 1.0 * 6.1 1.5*control2.0 1.02.3 1.12.6 1.02.9 0.93.0 1.1 * significant differences between 140 bpm group and control group ( p < 0.00 ) . bpm , beats per minute .. there were no significant differences among groups with respect to % hr or % vo2 max . although no significant differences in the affective response were noted , the rpe of the groups determined at 10 min ( f(2 , 25 ) = 4.092 , p = 0.029 , np = 0.247 ) , 15 min ( f(2 , 25 ) = 5.435 , p = 0.011 , np = 0.303 ) , 20 min ( f(2 , 25 ) = 10.233 , p = 0.001 , np = 0.450 ) , 25 min ( f(2 , 25 ) = 12.406 , p = 0.000 , np = 0.498 ) , and 30 min ( f(2 , 25 ) = 10.985 , p = 0.000 , np = 0.468 ) exhibited significant differences . however , there was no difference in the rpe ( f(2 , 25 ) = 10.377 , p = 0.001 ) between the control group and the group exposed to musical stimulation at 90 bpm . * significant differences between 140 bpm group and control group ( p < 0.00 ) . the average walking performance after 30 min of self - selected walking is shown in table 3table 3.average speed during 30 minutes of walking at a self - selected pacetreadmill speed ( mmh)90 bpm140 bpmcontrol81.6 8.391.6 8.3 * 80.0 6.6 * significant differences between 140 bpm group and control group ( p < 0.029 ) . bpm , beats per minute .. there were significant differences in the average walking speed among the groups ( f ( 2 , 25 ) = 3.897 , p = 0.034 ) . * significant differences between 140 bpm group and control group ( p < 0.029 ) . in the present study , different musical conditions did not promote sufficient stimuli to change exercise intensity ; in particular , there were no significant differences in physiological responses ( i.e. , hr and vo2 ) during 30 min of self - selected walking . these findings are similar to those reported by brownley et al.12 , who investigated the effect of music on trained and untrained subjects . likewise , other studies also found no effect of music on hr or vo213 , 21,22,23 . because of a lack of methodological standards , some studies indicate the effects of music on the rpe are inconclusive13 , 24 ; however , a previous study identified that different musical stimuli can influence perceptual responses during exercise25 , 26 . potteiger et al.21 observed that subjects who exercised listening to fast , classical , or self - selected music exhibited lower rpe than those who exercised without music . nakamura et al.23 found that subjects who exercised while listening to music of their choice covered more distance and exhibited lower perceptual responses than those who exercised while listening to non - preferred songs . in the present study , music improved fatigue tolerance ( i.e. , increased rpe ) during walking at a self - selected pace , and fast music ( 140 bpm ) promoted greater performance at the end of the walk . according to karageorghis et al.2 , rpe tends to decrease during low - intensity exercise but not moderate - intensity exercise . during self - selected activity the association between self - selected walking and fast music can have allowed the subjects to dissociate exertional perceptions from walking performance27 . regarding walking performance , the present results are consistent with the literature , affirming that music can induce an ergogenic effect and improve exercise performance , especially in low- to moderate - intensity activity2 , 8 , 13 . this phenomenon may be related to the potential role of music in motivating and distracting people in monotonous and/or unpleasant situations2 , 5 . however , few studies have reported the effects of music on exercise - related affective responses ; although these investigations were not based on self - selected exercise , their results are consistent with those of the present study , showing no significant differences in music - related affective responses to exercise12 , 13 . more importantly , in the present study , even though the rpe was higher with musical stimuli , no affective changes were observed among the three groups examined . karageorghis et al.28 and harmon and kravitz29 suggest this is because fast music can capture the attention and temporarily distract the subject from sensations related to fatigue . intervention studies should be conducted to observe the long - term effects of listening to music during exercise on perceptual , physiological , and affective responses . in conclusion , fast music ( 140 bpm ) increases rpe and performance in walking pace without significantly altering hr , vo2 , or affective responses . hence , listening to fast music during self - selected walking could help sedentary people or novices perform regular exercise , distract them from tiredness and fatigue , and create a better sense of wellbeing while performing the activity .

[ purpose ] this study investigated the effects of musical tempo on physiological , affective , and perceptual responses as well as the performance of self - selected walking pace . [ subjects ] the study included 28 adult women between 29 and 51 years old . [ methods ] the subjects were divided into three groups : no musical stimulation group ( control ) , and 90 and 140 beats per minute musical tempo groups . each subject underwent three experimental sessions : involved familiarization with the equipment , an incremental test to exhaustion , and a 30-min walk on a treadmill at a self - selected pace , respectively . during the self - selected walking session , physiological , perceptual , and affective variables were evaluated , and walking performance was evaluated at the end . [ results ] there were no significant differences in physiological variables or affective response among groups . however , there were significant differences in perceptual response and walking performance among groups . [ conclusion ] fast music ( 140 beats per minute ) promotes a higher rating of perceived exertion and greater performance in self - selected walking pace without significantly altering physiological variables or affective response .

INTRODUCTION SUBJECTS AND METHODS Statistical analysis RESULTS DISCUSSION

PMC5225776

extracorporeal membrane oxygenation ( ecmo ) is an essential part of life support and acts as the last resort for severe cardiorespiratory failure neonates without response to aggressive conventional treatment . ecmo improves oxygenation and organ perfusion and saves precious time for failed heart and lung to recover . venous - venous ecmo and venous - arterial ecmo are two most common ecmo modes . since the first case of successful neonatal ecmo for a severe meconium aspiration syndrome ( mas ) neonate in 1976 , ecmo has been used and saved thousands of neonates suffering from respiratory and/or cardiac failure world widely . the most common candidate diseases for neonatal ecmo are mas , persistent pulmonary hypertension of neonate , congenital diaphragmatic hernia , severe sepsis , etc . neonatal ecmo is still at the underdeveloped stage in mainland china , in comparison with other countries . the current paper reported the first neonate with early - onset group b streptococcus ( gbs ) sepsis saved by v - a ecmo in mainland china , and we also conducted a comprehensive literature review of neonatal ecmo utility in mainland china . a term neonate with gestational age of 39 weeks and birth weight of 3180 g was born by spontaneous vaginal delivery with unremarkable maternal history ; however , the gbs status was unknown . she did not need aggressive resuscitation in the delivery room , had good apgar score of 10 and 10 at 1 and 5 min , respectively . eight hours later , she developed respiratory distress and needed to be intubated and received mechanical respiratory support ; the antibiotics were also started empirically after blood culture sampling . she was transported to our neonate intensive care unit ( nicu ) at the age of 15 h. in nicu , she developed cardiac and respiratory failure with hypotension and hypoxemia , even though she received an aggressive fluid replacement and inotropic agents including epinephrine and large dose of dopamine . she was put on high - frequency oscillatory ventilation with high mean airway pressure of 17 cmh2 o , and high fio2 of 1.0 , she was given inhaled nitric oxide as well due to pulmonary hypertension confirmed by bedside echocardiography . however , the blood gas was poor as following : ph 7.24 , pao2 35.5 mmhg , paco2 40.8 mmhg , and base deficit 9.0 mmol / l . the oxygenation index ( oi = map after ruling out neonatal ecmo contraindications of intracranial hemorrhage , unrepairable congenital heart defects ( chds ) , she was put on v - a ecmo at the age of 23 h. in addition , the blood culture sent in referral hospital reported after was positive for gbs and confirmed the diagnosis of early onset gbs sepsis complicated by cardiorespiratory failure with no response to maximal conventional treatment . medtronic minimax plus oxygenator and bio - console 560 centrifugal blood pump was used in this patient . the v - a ecmo cannulation was achieved by right jugular vein and common carotid artery insertion . the positions of the catheters were guided by bedside echo . during the 273 h ecmo procedure , the blood gas , lactate level , and glucose level were monitored every 2 h , c - reactive protein , and chest x - ray was done daily . the ecmo flow was maintained at 100130 ml / kg / min based on hemodynamic monitoring . she tolerated ecmo well without major complications , apart from mild bleeding at the sites of cannulation , thrombocytopenia , etc . and , she was decannulated at the age of 12 days after 273 h ecmo and extubated the day after . neurologic evaluations including cranial magnetic resonance imaging and electroencephalogram were normal before discharge at the age of 36 days . until july 2015 , there were totally 69,114 patients registered in the extracorporeal life support organization ( elso ) received ecmo treatment worldwide based on the data of elso . and , more than half of the cases were neonatal cases which consisted 51.4% ( 35505/69114 ) of all , and the survive rate were 74% and 41% in respiratory failure and cardiac failure cases , respectively . in comparison with the advanced and sophisticated ecmo utility in critical patients in developed countries , the ecmo utility was still at the underdeveloped stage , especially neonatal ecmo , in mainland china . and , survive rate remains much lower than developed countries . to know the current state of neonatal ecmo in mainland , china , we conducted a comprehensive literature review through searching neonatal ecmo in chinese databases , including china knowledge resource integrated database and wanfang database ; we also used the keywords of ( ecmo ) and china [ affiliation ] as searching strategy in pubmed for papers published by authors from mainland , china . after abstract reviewing , 8 papers were recruited in the full paper assessment . based on the clinical information , we deleted duplicated cases from the same institutions . after that , we collected 22 reported neonatal cases with the birth weight of 2.84.0 kg . fourteen of the 22 patients were patients with chds , while the other 8 patients were non - chd patients , including one mas , 4 neonatal respiratory distress syndrome ( nrds ) , 2 pulmonary dysplasia , 1 cardiac arrest secondary to hydronephrosis and electrolytes disturbance . v - a ecmo was utilized in 21 of 22 patients ; the duration of ecmo running was 64 42 h ( range from 7 to 173 h ) . complications occurred in 13 patients , and the survive rate was 41% ( 9/22 ) . of note , the survive rate in non - chd patients was only 25% ( 2/8 ) , and none of the patients with congenital anomalies , such as pulmonary dysplasia and hydronephrosis survived . the current case is the first neonate case with severe gbs sepsis saved by v - a ecmo in mainland , china . reported neonatal ecmo cases done in mainland , china in this article , we reported the first neonatal ecmo for gbs sepsis neonate in mainland china , and the ecmo duration of 273 h was the longest among all reported cases . among 22 reported neonatal ecmo cases in previous papers , there were only 2 non - chd patients survived . the ecmo running durations in our case were much longer than the 48 h in the mas case and 50 h in the nrds case . according to the available evidence , gbs could cause a severe inflammatory reaction in the patients , which is also manifested in our case by extremely elevated c - reactive protein levels . this kind of inflammation recovers much slower than the self - limited lung diseases such as mas and nrds . that could be the underlying reason for a long duration of ecmo running in our case . of note , because of low survive rate in patients with pulmonary dysplasia ; the administration of neonatal ecmo in such patients should be cautious . in mainland china , the first reported neonatal ecmo case was a patient with left hypoplastic heart in 2006 , and the first reported non - chd case was a neonate with mas in 2009 . the previous 22 cases reported by chinese authors were done only by big hospitals located in beijing , shanghai , hangzhou , and guangdong , these 4 megacities with the highest economical level in mainland china . the high fee of ecmo not covered by chinese medicare is the biggest hurdle for developing neonatal ecmo in china . the neonatal ecmo was still underdeveloped in mainland china ; however , because of the remarkable effectiveness of ecmo in treating critical patients without response to conventional treatment , ecmo does provide a chance of survival for neonates who have a grave prognosis by conventional treatment .

we report the first successful treatment of extracorporeal membrane oxygenation ( ecmo ) in a neonate with group b streptococcus ( gbs ) sepsis and cardiorespiratory failure , and further conduct a literature review in the experience of neonatal ecmo utility in mainland china . a term neonate with cardiorespiratory failure secondary to gbs sepsis was put on venous - arterial ecmo at 23 h of age . after 273 h of ecmo running , the patient was saved and without major complications . the comprehensive literature review demonstrated that there were 22 neonates received ecmo previously in mainland china , 14 of 22 of the patients are cases with congenital heart defects . the overall survival rate was 41% ( 9/22 ) . neonatal ecmo was underdeveloped in mainland , china . moreover , it does provide a chance of survival for neonates who have a grave prognosis by conventional treatment .

Introduction Case Report Discussion Conclusion None Financial support and sponsorship Conflicts of interest

PMC4158265

however , mgp has other properties that may relate to its biological role . it is synthesized in a wide variety of tissues during embryonic life [ 15 ] . mgp is synthesized in embryonic kidney tubules and mgp protein and vitronectin colocalize at focal sites . mgp is highly expressed in cancers of the ovary , testes , kidney , prostate , and glioblastomas but its function in neoplastic cells is unknown [ 1215 ] . mgp is a migration - promoting protein for glioblastoma , suggesting that it can promote the cancer spreading . vitronectin binding suggested a mechanism to anchor mgp in the extracellular matrix where it may bind to bmps and/or calcium to prevent calcification . mgp has at least 2 functional domains , a vitronectin extracellular matrix binding domain in the c - terminal and a calcification inhibitory domain in the n - terminal half of the protein . mutations in human mgp that prevent synthesis of functional mgp suffer from keutel syndrome , with excessive calcification of cartilage and pulmonary artery stenosis [ 5 , 18 , 19 ] . excessive calcification occurs if mgp is absent , but the mechanism of calcification inhibition is unknown . a postulated mechanism of calcification inhibition is regulation of bone morphogenetic protein-2 ( bmp-2 ) , a cytokine / morphogen in the transforming growth factor beta superfamily [ 10 , 2023 ] . a role in transport of calcium - phosphate complexes has also been suggested , because mgp binds calcium phosphate crystals and forms a serum complex with fetuin - a and calcium phosphate [ 24 , 25 ] . calcification inhibition by mgp is connected to the gla residues contained in the n - terminal half of mgp which are involved in binding of bmp-2 , hydroxyapatite , and fetuin - a calcium phosphate complexes [ 22 , 2426 ] . the gla domain includes the vitamin k - dependent , gamma - carboxyglutamic acids ( gla ) , and all 3 phosphoserines [ 2 , 27 , 28 ] . the functional role of gla and the gla domain is supported by calcification defects that occur after treatment with warfarin , an inhibitor of gla formation [ 29 , 30 ] . the present studies show mgp binds fibronectin and augments cell adhesion and spreading on fibronectin . mgp localizes with fibronectin in many embryonic tissues , possibly augmenting cell matrix interaction in development . one aspect of mgp function is mediated by binding fibronectin and increasing cell interactions with fibronectin . matrix gla protein was purified as described from bovine bone , with the final step being reversed phase purification on a c18 column . after purification on hplc , mgp is a relatively insoluble protein with an approximate water solubility of 10 g / ml ; purified mgp was kept at 20c in a solution of approximately 40% acetonitrile and 0.06% trifluoroacetic acid in water . a typical purification peak contained 0.2 to 0.4 mg / ml . the final concentration of mgp was achieved by dilution into aqueous buffer at 10 g / ml or lower . concentration of mgp was estimated by absorbance at 280 nm , using an extinction coefficient of 1.0 mg / ml = 1.0 absorbance units . rabbit antibodies to bovine mgp and the purified igg fraction were produced as described using pierce immunopure columns [ 6 , 31 ] . the mgp peptide corresponding to amino acids 6177 in the secreted human protein nh2-eryamvygynaaynryf - cooh was synthesized by the molecular resource center of the university of tennessee , hsc . bovine serum albumin , human plasma fibronectin , and its proteolytic fragments , including the iii1-c polypeptide ( anastellin ) , guinea pig liver tissue transglutaminase ( ttg ) , human fibrinogen , gelatin , pooled normal rabbit serum , were purchased from sigma . the reducible crosslinking chemical dithiobis ( succinimidyl proprionate ) ( dsp ) and the immunopure igg purification kit were purchased from pierce . protein binding was assessed by the ability of filter immobilized target proteins to bind to iodinated mgp in an overlay buffer . the methods for assessing mgp binding by nitrocellulose transferred proteins from sds polyacrylamide gels ( sds - page ) and electroblotting were described previously [ 6 , 3133 ] . briefly , proteins were run in an sds - page and then transferred to shleicher and schuell bas - nc reinforced nitrocellulose membranes by electroblotting on a hoefer electroblot apparatus . in some experiments proteins were transferred to membranes by dot - blotting on a bio - rad bio - dot apparatus as described in . the membranes are incubated in 1% bovine serum albumin ( bsa ) , 10 mm phosphate , 140 mm nacl , and ph 7.4 ( bsa - pbs ) overnight at 4c . the filter overlay is performed in bsa - pbs with 0.1% tween 20 buffers containing radiolabeled mgp for 4 hours at ambient temperature and then washed in incubation buffer . the membrane is exposed to kodak biomax ms film in a kodak biomax transcreen - he intensifying screen and developed on an automatic film developer . fibronectin ( fn ) , fibrinogen ( fg ) , and vitronectin ( vn ) were mixed with tissue transglutaminase ( ttg ) and iodinated mgp , respectively , in buffer containing 0.05 m tris , 2.5 mm calcium , and 1 mm dithiothreitol ( tcd buffer ) at 37 degrees . final concentrations of 0.18 mg / ml fn , 0.18 mg / ml fg , or 0.18 mg / ml vn were incubated with 0.0125 mg / ml ttg and trace amounts of i - mgp ( 2.5 ng / ml ) containing 30,000 dpm . to determine the effect of antibodies to mgp , 31 g / ml rabbit anti - mgp igg was included during incubation ( see figure 2(c ) ) . after incubation , samples were processed immediately for sds page in sample buffer containing 2% sds and 2% 2-mercaptoethanol treated at 100 degrees for 2 min . the electrophoretic gels were processed by wrapping in plastic film and exposure to kodak biomax ms film with he enhancing screen for the appropriate time at 80c . after exposure 0.5 mg / ml fibronectin iii1-c polypeptide , 0.5 mg / ml fn , 0.5 mg / ml vn , or 0.5 mg / ml ovalbumin were incubated with trace amounts of -i - mgp ( 2.5 ng / ml , 30,000 dpm ) at ambient temperature for 2 hours in 5 mm phosphate , 75 mm nacl , 2.5 mm edta , and 0.05 mg / ml gelatin ph 7.4 . after 2 hours , either 1 mg / ml dsp or an equal volume of dmso for controls the reaction was quenched for 15 min at ambient temperature with tris ph 8 to react with free dsp the frozen at 20 degrees for sds gel electrophoresis or gel filtration chromatography . for sds - page , samples are diluted with 2% sds sample buffer ph 6.8 with or without 2% 2-mercaptoethanol and boiled for 2 min . for gel filtration chromatography , samples are diluted with 4 m guanidine hcl , 0.1 m tris , 0.1% tween 20 , and 0.5 mg / ml gelatin ph 8.0 . some samples were reduced with 2% 2-mercaptoethanol for 10 min at ambient temperature to break dsp crosslinks before dilution with buffer . gel filtration chromatography was performed on an ap ( pharmacia - lkb ) sephacryl hr s200 packed in a bio - rad column ( 1.6 19 cm ) equilibrated with 4 m guanidine , 0.1 m tris , and 0.1% tween 20 , ph 8.0 . constant volume fractions ( 30 drops / fraction ) were collected and counted on a packard auto - gamma counter to determine the elution position of radioactive mgp . controls were mgp and ovalbumin incubated with dsp , mgp alone , proteins incubated without dsp crosslinking , and dsp treated samples reduced with mercaptoethanol to reduce and break crosslinks . to determine whether mgp affected cell attachment to fibronectin , hela cells were used as an immortal cancer derived cell line that is popular as a model of cell behaviors such as attachment , spreading , and integrin action [ 3437 ] . initial studies demonstrated they had a dose - dependent cell attachment response to increasing concentrations of the matrix proteins . short term attachment studies within 1 hour after cell release from feeder plates were performed . hela cells are grown in liebovitz l-15 medium supplemented with 10% fetal bovine serum ( fbs ) with gentamicin and amphotericin b added at the recommended concentrations . 8095% confluent cells are harvested by release with 5 mm edta in calcium - free pbs ph 7.4 , washed with 10% fbs l-15 and resuspended to 250,000 cells / ml in serum - free l-15 . the cell suspension is used immediately for cell attachment assays below . in a typical cell attachment assay , 96-well plates are coated with the indicated concentrations of fibronectin in 50 mm sodium bicarbonate buffer ph 9.6 ( coat buffer ) . for control with no fibronectin the well is treated with coat buffer alone . after incubating overnight at 4c covered with parafilm to prevent evaporation , fibronectin solutions are removed and replaced with the indicated amount of mgp in coat buffer or coat buffer alone as control . after incubating at 37c in a humidified atmosphere for 3 hours , the second mgp or control solutions are removed and replaced with blocking solution made with 1% bsa in pbs ph 7.4 which has been heat inactivated at 65c for 30 min . after 1.5 hours at 37c in a humidified atmosphere , the wells are emptied and 25,000 hela cells added in 0.1 ml of serum - free l-15 medium using an 8-well multiwell pipet . vitronectin mediated cell attachment experiments were performed with the same procedure , except that vitronectin replaces fibronectin in the plate coating . the wells are immediately washed twice with serum - free l-15 medium and then fixed with 96% ethanol for 10 min and then stained with 0.1% crystal violet dye in distilled water for 30 min at ambient temperature . the dye is removed and the wells washed gently with 0.375 ml of distilled water 3 times . after wells have dried completely , 0.1 ml / well of 0.2% triton x100 is added and incubated at ambient temperature overnight covered with parafilm to prevent evaporation . the plates are read at 595 nm on a molecular devices spectramax 2500 multiwell plate reader . data is entered as replicates ( usually 6 but a minimum of 4 replicates for each data point ) into prism 2.0 for analysis , nonlinear regression plotting , and statistical analysis . in some experiments the first fibronectin coat is replaced by a first mgp coat and then followed by a second coat of fibronectin to ascertain the effect of switching the order of addition of fibronectin and mgp . in some experiments after an initial coat of fibronectin the remaining nonspecific protein binding sites on the plate are blocked with bsa - pbs before addition of mgp to see if mgp effects required nonspecific protein binding sites on wells . in other experiments the effect of rabbit anti - mgp antibody on mgp enhancement of fibronectin - cell binding was assessed by adding 31 g / ml ( 0.0312 a280/ml ) of anti - mgp igg in the second coat step containing mgp . hela cells are grown and harvested nonenzymatically as described above and , after washing once with l-15 supplemented with 10% fbs , cells are resuspended in serum - free l-15 medium to 15000 cells / ml . 9000 hela cells/0.6 ml are added to each chamber of lab - tek 4 chamber glass slides . lab - tek chamber slides are previously coated with 0.0 , 0.2 , or 0.4 g / ml of fibronectin in coat buffer overnight at 4c and then a second coat of 3 g / ml mgp for 3 hours at 37c and then blocked with heat inactivated bsa - pbs for 1.5 h at 37c as described above . cells attach for 2 hours and then are rinsed gently twice with serum - free l-15 medium . freshly made 1% paraformaldehyde in pbs is added to fix cells for 10 min at ambient temperature , washed pbs for 10 min , and then treated with 0.1% triton x-100 in pbs for 5 min to permeabilize cells . the bsa - pbs is then replaced with 1 : 40 dilution of phalloidin alexafluor488 in heat inactivated bsa - pbs for 20 min . after one wash with heat inactivated bsa - pbs the chambers are washed twice with pbs . the slides are placed in a 10 cm petri dish immersed in pbs and then imaged using 10x or 40x water immersion objectives on a zeiss axiophot fluorescence microscope equipped with a digital camera . 1416 random microscopic field images taken on the 10x objective were analyzed , with a minimum of 100 cells analyzed for each condition . the procedures in nih image are as follows : an introduction and tutorial were used to determine cell areas . briefly , low power images of cells taken with the 10x objective were modified by autocontrast and grayscale conversion in photoshop . the images were opened in nih image , scaled to fit the window , and modified with the lutz tool to turn cells red then under analyze , analyze particle . after examination to manually remove clumped / adherent cells from analysis , the results are copied into excel as area / cell . the cell spreading analysis is similar to that used in a previous publication measuring cell attachment and spreading on titanium surfaces . paraffin embedded sections of formalin fixed day 19 embryos were obtained as described previously [ 4 , 6 ] . the sections were deparaffinized with xylene , rehydrated , and boiled for 2 min in 0.01 m sodium citrate , ph 6 for antigen retrieval as described previously . sections were blocked with buffer containing normal horse serum at 1.5% then incubated with rabbit anti - mgp at 20 g / ml or mouse antifibronectin monoclonal antibody ( clone ist-3 sigma ) at 1 : 5000 dilution . after an overnight 4c incubation and washing , biotinylated horse antiuniversal igg ( vector ) is added . images were collected on a zeiss axiophot microscope equipped with a pc and 6.3x and 40x objectives . as seen in figure 1(a ) , overlay of dot - blotted proteins with radioactive mgp demonstrates that mgp binds to fibronectin and fibronectin fragment iii1-c ( anastellin ) . mgp did not bind ovalbumin or other fragments of fibronectin with the exception of the 110 kilodalton fibronectin fragment that weakly binds to mgp . mgp binds to the sds - page band of fibronectin iii1-c ( figure 1(b ) ) . we previously reported that mgp binds to vitronectin and fibronectin but not laminin , type ii collagen , osteocalcin , tissue transglutaminase , chondroitin sulfate glycosaminoglycan , biglycan , beta - casein , ovalbumin , or bovine serum albumin . figure 1(c ) shows that mgp61 - 77 also binds to the first type iii fibronectin domain . ttg was incubated in mixtures of i - mgp with fibronectin , fibrinogen , or vitronectin , respectively . the products were analyzed by autoradiographs of samples run on sds - page to identify shifts in radioactive mgp ( figure 2(a ) ) . radioiodinated mgp was incorporated into higher fibronectin ( fn ) and fibrinogen ( fg ) bands . there was no mgp incorporated into higher molecular weight bands for mgp incubated with fn or fg the absence of ttg . transglutaminase shifted a small amount of mgp to higher molecular weight but not to the size or extent found with either fibrinogen or fibronectin . the band at the bottom of the figure 2(a ) is free radioiodinated mgp . figure 2(b ) shows the coomassie blue stained gel exposed to film in figure 2(a ) . ttg converted only a small fraction of the fibronectin ( fn ) to multimers as there is little change in lanes with and without ttg . ttg was more efficient at crosslinking fibrinogen ( fg ) to multimers which can be as additional bands when ttg is present but not in samples without ttg ( figure 2(b ) ) . note that the coomassie blue stained band in all lanes is bsa , a component of the buffer used to separate radioiodinated mgp from unincorporated iodine during iodination . mgp in the ttg crosslinked fibronectin lane comigrated at the stacking gel surface or the top of the running gel . the presence of anti - mgp polyclonal antibodies blocked the ability of tissue transglutaminase to incorporate mgp into a fibronectin multimer ( figure 2(c ) ) . increasing the concentration of ttg increased the amount of mgp shifted to higher molecular weight with fibronectin . transglutaminase could incorporate mgp into larger complexes of fibronectin but not with vitronectin , ovalbumin , or bovine serum albumin . chemical crosslinking could confirm that the mgp and iii1-c are in close proximity in solution . i - mgp and iii1-c , vitronectin , or ovalbumin was incubated under associative conditions then reacted with dsp , a disulfide containing homobifunctional crosslinking agent . the prediction is that interacting proteins would become covalently crosslinked in the presence of a crosslinking agent . if dsp is added to a mixture of fibronectin iii1-c and i - mgp , a higher molecular weight crosslinked species is observed on autoradiographs after sds - page ( figure 3(a ) , iiic , 2me , and + dsp lane ) . if samples are reduced with 2-mercaptoethanol ( 2me ) , the dsp crosslink is reduced and larger bands disappear ( figure 3(a ) iiic , + 2me , and + dsp lane ) . no additional higher molecular weight bands are present for iiic in the absence of dsp crosslinker ( iiic , 2me , and dsp lane ) . the lack of higher molecular weight species is due to disruption of protein interactions in 2% sds buffer heated to 100c . mgp and either vitronectin or fibronectin was crosslinked by dsp although not to the same extent as seen with iii1-c ( figure 3(a ) , vn and fn , 2me , and + dsp lanes ) . it is possible that the increased crosslinking of iii1-c and mgp results from aggregation of the bound complex that increases crosslinking efficiency . ovalbumin is a negative control for binding ; mgp and ovalbumin were not crosslinked by dsp . if the products of dsp crosslinked i - mgp and fibronectin iii1-c are denatured in 4 m guanidine containing buffer then analyzed by sephacryl s200 gel filtration , an elution profile with mgp shifted to a peak near the excluded volume , ve , is the result ( figure 3(b ) , filled circles ) . if the crosslinked sample is reduced with mercaptoethanol prior to column loading , the elution profile is similar to controls , for example , i - mgp alone ( figure 3(b ) , open circles ) . the i mgp peak normally elutes around fraction 37 with a minor peak at fraction 28 ( figure 3(b ) , open triangles ) . control reactions with i - mgp with dsp , i - mgp + iii1-c in the absence of dsp were identical in gel filtration elution profile with mgp alone or the reduced crosslink ( open diamonds or squares , resp . ) . a four - fold decreased amount of iii1-c in the mixture with labeled mgp and dsp resulted in a smaller s200 column ve peak ( data not shown ) . the data show that iii1-c and the crosslinking agent were necessary to produce the shift in the mgp peak and are consistent with a close interaction between fibronectin iii1-c and mgp . figure 4(a ) shows that cell attachment increases with increasing fibronectin until a maximum is reached at fibronectin coating concentrations between 0.8 and 3.2 g / ml ( solid line with closed circles ) . mgp enhanced cell attachment to fibronectin ( open circles with the dashed line , figure 4(a ) ) . the cell attachment curve for equal amounts of fibronectin is shifted to the left if mgp is added at 3 g / ml . the result is analogous to increasing the apparent affinity of cells for equivalent amounts of fibronectin ( figure 4(a ) ) . in addition , mgp did not increase cell attachment beyond the maximum observed for fibronectin alone . the lack of inherent cell attachment activity for mgp is in agreement with the absence of cell binding activity previously reported . in contrast to mgp augmenting cell attachment to fibronectin , mgp did not augment attachment of cells to vitronectin . there was a small inhibitory effect of mgp on cell binding to vitronectin , but in some experiments there was no significant effect . under the same conditions and cell line , mgp consistently augments cell attachment on fibronectin but not on vitronectin . antibody to mgp inhibited the ability of mgp to augment cell attachment ( figure 4(b ) ) . anti - mgp rabbit igg abolished the ability of mgp to enhance cell attachment to fibronectin ( mgp compared to mgp + antimgp , p .05 ) , but an equal amount of nonspecific rabbit igg did not . the presence of igg at the concentrations used did not affect cell attachment to fibronectin , because anti - mgp igg or control igg added in the absence of mgp was identical to the control fibronectin alone ( control , figure 4(b ) ) . increasing amounts of mgp dose dependently enhanced cell attachment to fibronectin ( figure 4(c ) ) . a dose response curve of increasing mgp ( 0.0 , 0.8 , 3.0 , and 6.0 g / ml ) reveals a change in apparent affinity with an increasing shift of the binding curve to the left indicating increased cell attachment at each concentration of fibronectin . again , maximum binding of cells to fibronectin was not affected ( figure 4(c ) ) . there was no inherent mgp cell attachment activity , because cells did not attach even at the highest amounts of mgp ( 6.0 g / ml ) if fibronectin was not present . cells attach and spread on fibronectin as integrins and other cell adhesive proteins form focal adhesions which connect to the actin cytoskeleton . to determine whether mgp affects cell spreading on fibronectin , the average area of cells attached to fibronectin alone or fibronectin plus mgp was determined by calculation of average cell area from over 100 cells as described in experimental procedures . mgp induced significantly greater cell spreading at each fibronectin concentration ( figure 5(a ) , fn versus fn + mgp , p .0001 indicated by asterisk ) . there were too few cells attached in the mgp alone wells to measure spreading ( mgp does not have cell adhesive activity on its own ) . the appearance of selected microscopic fields of cells adherent on 0.4 g / ml fibronectin coated surfaces is shown is in figures 5(b ) and 5(c ) . the appearance of selected microscopic fields of cells on the same concentration of fibronectin plus mgp is shown in figures 5(d ) and 5(e ) . it was previously shown that mgp in the rat embryonic kidney localizes in the developing ureter and the collecting tubules . fibronectin is widely distributed in the rat embryonic kidney , including in the ureter and collecting tubules ( figures 6(a ) , 6(b ) , and 6(c ) ) . localization of mgp in a serial section reveals that fibronectin is present in some of the same collecting tubules and ureter epithelium as mgp ( figures 6(d ) , 6(e ) , and 6(f ) ) . arrows in figures 6(b ) , 6(c ) , 6(e ) , and 6(f ) indicate similar structures in serial sections with both fibronectin and mgp . fibronectin has a wider distribution within the developing kidney , being present in other tubules with little or no mgp , in connective tissue areas , and in forming glomeruli ( figures 6(a)6(c ) ) . developing glomeruli has little or no mgp at this stage of development ( figure 6(d ) ) . this confirms the results from a previous publication that showed that mgp binds to fibronectin , vitronectin , and fibrinogen but not tissue transglutaminase , type ii collagen , fibromodulin , osteocalcin , osteonectin , heparin , decorin , casein , or ovalbumin . the current study shows that mgp binds to a region of fibronectin made up by its first type iii repeat called iii1-c or anastellin . mgp did not bind the 30 , 45 kilodalton fragments of fibronectin that do not contain a type iii repeat [ 40 , 41 ] . mgp showed some affinity for a 110 kilodalton fragment of fibronectin that is comprised of many type iii repeats [ 40 , 41 ] . a region of mgp comprised of amino acids 6177 was proposed as an extracellular matrix binding region . the current study shows that mgp6177 peptide binds to fibronectin and also to the fibronectin iii1-c fragment . for example , human mgp is originally 84 amino acids long but has 7 amino acids removed from the c - terminus , so that the mature protein is 77 amino acids long and ends with the peptide sequence identical to the mgp6177 peptide used for this study . while bovine mgp was used in this study , the results imply that the human mgp will bind to extracellular matrix proteins via a sequence at its carboxyl terminal . the proteolytic processing of the mgp c - terminal to remove amino acids 7884 may relate to activating a protein interaction site . the crosslinked multimerized fibronectin formed by incubation with ttg is analogous to the insoluble matrix fibronectin present in many tissues . the close association of mgp and fibronectin suggested that mgp may become incorporated into fibronectin crosslinked multimers . indeed , addition of mgp during crosslinking of fibronectin by ttg resulted in its incorporation into a larger multimer form of fibronectin . the i mgp remained with the fibronectin multimer after boiling in 2% sds that dissociates the mgp and fibronectin interaction . it is possible that transglutaminase crosslinks mgp to fibronectin , although further studies are necessary to prove crosslinking occurs and determine which amino acids of mgp and fibronectin are involved . the shift of smaller amounts of mgp to higher molecular weight by ttg suggested it may become crosslinked to itself or to ttg , but we were unable to directly establish that mgp is a substrate of ttg . to prove mgp was a substrate for ttg we attempted incorporation of radiolabeled putrescine as described . the association of mgp with crosslinked fibronectin confirms a close association of mgp with fibronectin . although mgp binds to vitronectin , ttg did not incorporate mgp into a high molecular weight component with vitronectin . the results suggest that mgp may become a component of crosslinked fibronectin and fibrinogen in vivo . this has been established by overlay assays and by dsp crosslinking of mgp to the fibronectin iii1-c polypeptide . overlay assays show that the c - terminal mgp 6177 amino acid peptide can bind to fibronectin and to the iii1-c polypeptide . a noncarboxylated form of mgp ( ucmgp or glu - mgp ) accumulates in calcified atherosclerotic plaque [ 22 , 30 ] . further studies will be necessary to determine whether glu - mgp and mgp share binding for fibronectin and the iii1-c domain . this is not simply an additive effect of providing more adhesive protein , because mgp by itself has no adhesive activity . the lack of inherent cell adhesion activity suggests that mgp acts by altering the ability of cells to bind fibronectin . mgp binding of fibronectin seems to be the key , because the enhanced cell attachment was observed in bsa - coated surfaces in which mgp - fibronectin binding was the only way that the enhancement could occur . mgp enhanced cell attachment to fibronectin but not to vitronectin , another extracellular matrix protein bound by mgp . these studies explored the mechanism of mgp binding to fibronectin and the effect of exogenous fibronectin and mgp on cell attachment and spreading . hela cells are a well characterized cell line that is a popular model for cell attachment . hela cells express the integrins for fibronectin and vitronectin binding [ 3437 ] . the goal was to characterize the effect of mgp for fibronectin binding and cell - fibronectin interactions in a short , 1 hour period when cellular synthesis and secretion of fibronectin , mgp , and other attachment proteins could be minimized . there has been a positive correlation of mgp expression with tumor progression and poor prognosis in glioma [ 15 , 16 , 45 ] , but a negative correlation of mgp expression with tumor progression and metastasis in renal and prostate carcinoma and decreased mgp has been found in colon carcinoma . future studies should determine whether mgp has the effect in multiple normal and neoplastic cell types . other studies could determine the effect of mgp for migration and invasion assays in hela compared to other cancer cell types , but longer term studies need to consider the ability of cells to synthesize fibronectin , mgp , integrins , or metalloproteinases that affect migration and invasion . the expression of mgp mrna is very high in the embryonic rat kidney [ 1 , 4 ] . we demonstrated that mgp protein is also synthesized during this time and that localization of mgp mrna was strongest in the developing ureter and collecting tubules on embryonic days 1719 . immunolocalization studies showed mgp localized in embryonic ureters and collecting tubules also contained abundant fibronectin . calcification inhibition and bmp-2 binding requires posttranslational carboxylation to produce gla in a vitamin k - dependent step [ 22 , 26 , 29 ] . the phosphorylation of 3 serine residues at the amino terminal end of mgp is necessary for its secretion to inhibit deposition of hydroxyapatite in the extracellular matrix [ 2 , 47 ] . the amino terminal 154 amino acids of mgp contains the phosphoserines and gla necessary for calcification inhibition , whereas a c - terminal peptide mgp6177 has been shown previously to bind vitronectin and in the current studies to bind fibronectin [ 6 , 26 ] . it is not clear whether the binding of fibronectin or modulating the cell response to fibronectin is related to the calcification inhibitory activity of mgp . it is more likely that this mgp property is related to migration - promoting activity that is demonstrated for glioblastoma cells . it is possible that mgp is a multifunctional protein that can act to modify cell - matrix interactions during embryonic life or in cancer cells and also as a calcification inhibitor in selected adult tissues . this could help explain why many tissues that contain mgp synthesizing cells do not become calcified in mgp knockout animals and in keutel syndrome , a human disease characterized by loss of mgp function . mgp binds via a 6177 amino acid sequence present at the physiologic c - terminus . mgp can become part of transglutaminase crosslinked multimers of fibronectin , suggesting it may be a component of fibronectin matrices . cells attach and spread better on fibronectin and mgp coated surfaces than to fibronectin alone , even though mgp itself has no cell attachment activity . in tissues , mgp localizes near fibronectin , suggesting that interactions between the proteins are possible in vivo . the ability of mgp to alter cell interactions with fibronectin is a potential reason for cancer cells and certain embryonic cells to overexpress mgp .

background . matrix gla protein ( mgp ) is a vitamin k - dependent , extracellular matrix protein . mgp is a calcification inhibitor of arteries and cartilage . however mgp is synthesized in many tissues and is especially enriched in embryonic tissues and in cancer cells . the presence of mgp in those instances does not correlate well with the calcification inhibitory role . this study explores a potential mechanism for mgp to bind to matrix proteins and alter cell matrix interactions . methods . to determine whether mgp influences cell behavior through interaction with fibronectin , we studied mgp binding to fibronectin , the effect of mgp on fibronectin mediated cell attachment and spreading and immunolocalized mgp and fibronectin . results . first , mgp binds to fibronectin . the binding site for mgp is in a specific fibronectin fragment , called iii1-c or anastellin . the binding site for fibronectin is in a mgp c - terminal peptide comprising amino acids 6177 . second , mgp enhances cell attachment and cell spreading on fibronectin . mgp alone does not promote cell adhesion . third , mgp is present in fibronectin - rich regions of tissue sections . conclusions . mgp binds to fibronectin . the presence of mgp increased cell - fibronectin interactions .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

PMC4310883

cystectomy and urinary diversion is a procedure frequently ( 1023% incidence ) complicated by postoperative ileus [ 17 ] . ileus can result in pain limiting mobility , vomiting with increased risk of dehiscence and aspiration , threatened nutritional status , and is associated with a greater risk of additional complications and death . recovery of bowel function governs length of stay in many bowel surgeries but also in cystectomy and urinary diversion . numerous interventions to prevent postoperative ileus and hasten recovery of bowel function have been practiced and studied including early feeding , ambulation , avoidance of nasogastric tube , omission of mechanical bowel prep , chewing gum [ 12 , 13 ] , and optimized fluid management . multimodal perioperative pathways have been developed to achieve early recovery from surgery , focusing on perioperative nutrition , minimizing mechanical bowel preparation , and analgesic alternatives to systemic opioids [ 15 , 16 ] . the food and drug administration approved alvimopan for accelerating upper and lower gastrointestinal tract recovery after partial large bowel or small bowel resection with primary anastomosis . several studies have examined the efficacy in reducing length of stay and readmission rates of patients receiving alvimopan who have undergone bowel resection [ 1821 ] as well as the cost effectiveness [ 22 , 23 ] . one smaller retrospective study showed a diminished length of stay in patients treated with alvimopan as part of a multimodal postoperative pathway in patients undergoing cystectomy . however , no studies have evaluated the cost effectiveness of alvimopan alone in a patient cohort undergoing cystectomy and urinary diversion in a real world setting of moderate volume center and epidural analgesia . between january 2008 and january 2012 , 98 consecutive patients underwent cystectomy and urinary diversion for benign and oncologic indications at a single academic institution by 7 surgeons . twelve of these patients suffered clavien 3b or greater complications ( death ( 2 ) , laparotomy for dehiscence / evisceration ( 2 ) , cardiogenic shock ( 2 ) , stroke requiring carotid endarterectomy , ischemic leg requiring embolectomy , laparotomy for small bowel obstruction , laparotomy for delayed rectal injury , status epilepticus , pulmonary hemorrhage ) thus making los independent from bowel function and were eliminated from analysis as their los was unlikely to be governed by bowel function . patients with muscle invasive bladder cancer were referred to oncology to discuss neoadjuvant chemotherapy . preoperatively , bowel preparation was performed with 4 liters of polyethylene glycol and two days of clear liquid diet . radical or supratrigonal cystectomy was performed with ileal conduit , indiana pouch , transverse colon conduit , or mainz neobladder . cystectomy technique was performed through a low midline incision or via a robotic assisted laparoscopic approach but all diversion was performed extracorporally . the adoption of a robotic assisted technique occurred during this study period . a blake drain and 8 french ureteral catheters patients received care in the surgical intensive care unit or the acute care floor postoperatively . patients were kept nil per os ( npo ) until passage of flatus in most cases , but in younger , healthier patients this was determined by surgeon discretion . in 2010 , all surgeons agreed to institute alvimopan . alvimopan was administered with a sip of water at a 12 mg dose preoperatively and then 12 mg every 12 hours for up to 15 doses or until discharge . all patients were eligible for the drug ; with no patients with stage 5 chronic kidney disease or severe hepatic impairment ( child pugh class c ) . the medical center is a participant in the entereg access support and education program for risk evaluation and monitoring of safety for alvimopan . discharge criteria were : tolerating solid food without nausea or vomiting , pain controlled on oral pain medications , ambulatory , laboratory values and vital signs within normal limits , and urinary diversion teaching performed . most patients had a bowel movement prior to discharge , but only a flatus was a firm discharge criteria . the cost analysis was from the institutional perspective with hospital cost data obtained from the medical center finance department . total costs ( not charges ) were obtained and included direct variable , overhead , and fixed variable costs . all costs were adjusted to 2012 us dollars using the medical care component of the consumer price index . the wald test was used in a poisson regression to determine alvimopan administration effect on los in univariate analysis . multivariate analysis was performed using poisson regression by taking into account variables with p < 0.05 due to high number of variables relative to the number of subjects . poisson regression based on quartiles of propensity scores on variables with p < 0.1 . for all testing , p value of < 0.05 was deemed to be significant . between january 2008 and january 2012 , 98 consecutive patients underwent cystectomy and urinary diversion for benign and oncologic indications at a single academic institution by 7 surgeons . twelve of these patients suffered clavien 3b or greater complications ( death ( 2 ) , laparotomy for dehiscence / evisceration ( 2 ) , cardiogenic shock ( 2 ) , stroke requiring carotid endarterectomy , ischemic leg requiring embolectomy , laparotomy for small bowel obstruction , laparotomy for delayed rectal injury , status epilepticus , pulmonary hemorrhage ) thus making los independent from bowel function and were eliminated from analysis as their los was unlikely to be governed by bowel function . patients with muscle invasive bladder cancer were referred to oncology to discuss neoadjuvant chemotherapy . preoperatively , bowel preparation was performed with 4 liters of polyethylene glycol and two days of clear liquid diet . radical or supratrigonal cystectomy was performed with ileal conduit , indiana pouch , transverse colon conduit , or mainz neobladder . cystectomy technique was performed through a low midline incision or via a robotic assisted laparoscopic approach but all diversion was performed extracorporally . the adoption of a robotic assisted technique occurred during this study period . a blake drain and 8 french ureteral catheters were left in all patients . patients received care in the surgical intensive care unit or the acute care floor postoperatively . patients were kept nil per os ( npo ) until passage of flatus in most cases , but in younger , healthier patients this was determined by surgeon discretion . alvimopan was administered with a sip of water at a 12 mg dose preoperatively and then 12 mg every 12 hours for up to 15 doses or until discharge . all patients were eligible for the drug ; with no patients with stage 5 chronic kidney disease or severe hepatic impairment ( child pugh class c ) . the medical center is a participant in the entereg access support and education program for risk evaluation and monitoring of safety for alvimopan . discharge criteria were : tolerating solid food without nausea or vomiting , pain controlled on oral pain medications , ambulatory , laboratory values and vital signs within normal limits , and urinary diversion teaching performed . most patients had a bowel movement prior to discharge , but only a flatus was a firm discharge criteria . the cost analysis was from the institutional perspective with hospital cost data obtained from the medical center finance department . total costs ( not charges ) were obtained and included direct variable , overhead , and fixed variable costs . test for amount billed , amount billed minus drug cost , and insurance paid . cost was adjusted for pre and perioperative characteristics in regression analysis . all costs were adjusted to 2012 us dollars using the medical care component of the consumer price index . the wald test was used in a poisson regression to determine alvimopan administration effect on los in univariate analysis . multivariate analysis was performed using poisson regression by taking into account variables with p < 0.05 due to high number of variables relative to the number of subjects . poisson regression based on quartiles of propensity scores on variables with p < 0.1 . for all testing , p value of < 0.05 was deemed to be significant . after removal of the clavien iiib or higher complications , there were 36 patients in the alvimopan group and 50 patients in the pre , we found that , although ordered , patients did not always receive the drug . the entire cohort was 77% male and had a mean age of 63.5 years and a mean bmi of 28.4 kg / m . preoperative variables trends that increased over the time period between the pre alvimopan and alvimopan era included a shift from increased epidural catheters ( 82% vs. 60% , p = 0.032 ) to patient controlled analgesia ( 7% vs. 31% , p = 0.004 ) . there was increased disposition of patients from the or to the intensive care unit in the alvimopan group ( 44% vs. 66% , p = 0.048 ) . additionally , use of robotic technique increased markedly in the alvimopan group ( 8% vs. 25% , p = 0.030 ) . perioperative characteristics of estimated blood loss and or time were no different between the cohorts . with regards to outcomes , readmission for ileus ( 4.4% pre time to clear liquid diet was 5.3 and 3.7 days in the pre alvimopan and alvimopan groups , respectively ( p = 0.058 ) . alvimopan cohort at 7.1 days compared to 4.8 days in the treatment group ( p = 0.020 ) . length of stay was reduced by 1.9 days ( 18% reduction in los ) in the alvimopan group ( 10.5 vs. 8.6 days , p = 0.005 , 95% ci 0.63.3 ) ( see table 2 ) . while most patients stayed 8 to 10 days , the alvimopan group had one third of patients leaving at 7 days or earlier , while the pre alvimopan group los was 11 days or greater in 40% of patients ( figure 1 ) . no dose response effect was noted when comparing the number of doses administered versus los beyond a single dose . on multivariate analysis taking into account robotic technique , or time , ebl , icu disposition , epidural use , and patient controlled analgesia , there was a 16% reduction in los ( p = 0.048 , 95% ci 0.129% ) . importantly , the robotic technique did not affect los and thus could not account for the difference of los between the pre and post alvimopan cohorts . using propensity scoring poisson regression to take into account additional variables , the magnitude of los reduction was again 16% ( 95% ci 0.030% , p = 0.050 ) . costs were lower in the alvimopan group in the 35 patients with available cost data versus the 47 in the pre alvimopan cohort . alvimopan group ( 2012 usd ) compared to the alvimopan group mean of $ 32,443 ( p < 0.001 ) for a savings of $ 8,161 or 20% reduction of cost . when adjusting for the significantly different preoperative and perioperative variables of epidural , pca , indication , robotic technique , or time , ebl , and icu disposition , the mean costs remained lower in the alvimopan group by $ 7,062 ( p = 0.003 ) ( figure 2 ) . after removal of the clavien iiib or higher complications , there were 36 patients in the alvimopan group and 50 patients in the pre , we found that , although ordered , patients did not always receive the drug . the entire cohort was 77% male and had a mean age of 63.5 years and a mean bmi of 28.4 kg / m . preoperative variables trends that increased over the time period between the pre alvimopan and alvimopan era included a shift from increased epidural catheters ( 82% vs. 60% , p = 0.032 ) to patient controlled analgesia ( 7% vs. 31% , p = 0.004 ) . there was increased disposition of patients from the or to the intensive care unit in the alvimopan group ( 44% vs. 66% , p = 0.048 ) . additionally , use of robotic technique increased markedly in the alvimopan group ( 8% vs. 25% , p = 0.030 ) . perioperative characteristics of estimated blood loss and or time were no different between the cohorts . with regards to outcomes , readmission for ileus ( 4.4% pre alvimopan vs. 0% alvimopan , p = 0.26 ) was similar in the groups . time to clear liquid diet was 5.3 and 3.7 days in the pre alvimopan and alvimopan groups , respectively ( p = 0.058 ) . alvimopan cohort at 7.1 days compared to 4.8 days in the treatment group ( p = 0.020 ) . length of stay was reduced by 1.9 days ( 18% reduction in los ) in the alvimopan group ( 10.5 vs. 8.6 days , p = 0.005 , 95% ci 0.63.3 ) ( see table 2 ) . while most patients stayed 8 to 10 days , the alvimopan group had one third of patients leaving at 7 days or earlier , while the pre alvimopan group los was 11 days or greater in 40% of patients ( figure 1 ) . no dose response effect was noted when comparing the number of doses administered versus los beyond a single dose . on multivariate analysis taking into account robotic technique , or time , ebl , icu disposition , epidural use , and patient controlled analgesia , there was a 16% reduction in los ( p = 0.048 , 95% ci 0.129% ) . importantly , the robotic technique did not affect los and thus could not account for the difference of los between the pre and post alvimopan cohorts . using propensity scoring poisson regression to take into account additional variables , the magnitude of los reduction was again 16% ( 95% ci 0.030% , p = 0.050 ) . costs were lower in the alvimopan group in the 35 patients with available cost data versus the 47 in the pre alvimopan cohort . the mean cost was $ 40,604 in the pre alvimopan group ( 2012 usd ) compared to the alvimopan group mean of $ 32,443 ( p < 0.001 ) for a savings of $ 8,161 or 20% reduction of cost . when adjusting for the significantly different preoperative and perioperative variables of epidural , pca , indication , robotic technique , or time , ebl , and icu disposition , the mean costs remained lower in the alvimopan group by $ 7,062 ( p = 0.003 ) ( figure 2 ) . second , this reduction in los does translate to reduction in costs . finally , this demonstrates a real world implementation of alvimopan administration which realizes the same advantages as a randomized controlled trial . innumerable interventions have been studied to reduce postoperative ileus with mixed results . chewing gum administered early in the perioperative period after bowel surgery has been evaluated in meta analyses with the conclusion that it is a low cost safe modality that decreases time to flatus and bowel movement but not los . cystectomy and urinary diversion is a surgery fraught with complications with an inpatient complication rate of 4964% and a 30 day readmission rate around 25% . gi complications , specifically postoperative ileus account for the plurality of this morbidity . interventions to decrease postoperative ileus can be in the pre , peri , and post operative period . forgoing mechanical bowel preparation has been shown to reduce prolonged ileus and los after cystectomy by shafii et al . . intraoperatively , transesophageal doppler optimized fluid management yielded a marked reduction in postoperative ileus as well as a two day reduction in time to flatus and bowel movement but no change in los . pruthi and colleagues observed that institution of routine chewing gum postoperatively led to reduction in time to flatus and bowel movement by about half a day each . similarly , choi and colleagues demonstrated in a randomized trial of 50 patients that chewing gum reduces time to flatus by 18% and time to bowel movement by 18% . brodner and colleagues demonstrated that compared to traditional management , epidural analgesia , forced mobilization , and early enteral feeding leads to earlier time to first bowel movement after radical cystectomy . wallen and colleagues describe their perioperative pathway for reducing los and hastening bowel recovery with metoclopramide , ketorolac , chewing gum , and early oral feeding as the cornerstones of their care plan . similar multimodal pathways spanning the pre , peri , and postoperative period have been described with success in decrease time to flatus or bowel movement and mixed results in diminishing los . alvimopan was first reported in 2001 in 14 healthy volunteers to attenuate the delayed enteric transit time but not the analgesic effects of morphine . wolff reported in 2004 in a randomized controlled study that alvimopan 6 mg and 12 mg doses reduced time to return bowel function and the 12 mg dose reduced time to discharge order by almost 1 full day in 510 patients undergoing laparotomy for bowel resection or hysterectomy . it has been shown to decrease los in small and large bowel surgery by 1 or 2 days . of note , the promise of alvimopan for chronic opioid use associated bowel dysfunction was cut short by a study revealing 3 cardiac events in a chronically dosed alvimopan group which could neither be deemed causal nor were reproducible . no cardiac toxicities have been noted with the short term use of alvimopan described in this study . vora and colleagues demonstrated in 50 patients that alvimopan administration decreases times to discharge order , flatus , bowel movement , clear liquid diet , and regular diet with magnitude of effect ( 2 days ) similar to the current study . however , nasogastric decompression , an intervention fraught with increased complications and delayed gi recovery , was utilized in the control group but not the alvimopan group , thus confounding the findings . hilton et al . performed a sensitivity analysis assuming a reduction in rate of postoperative ileus by 50% which revealed the conditions under which alvimopan would be cost effective . however , this was a purely theoretical study and did not include novel patient data . they estimate an incremental cost of a hospital day is $ 1110 before taking into account additional tests , nutrition , or procedures . the authors recognized the cost effectiveness of alvimopan would be contingent upon the rate of postoperative ileus and the degree to which alvimopan reduces the rate of postoperative ileus . unfortunately , postoperative ileus tends to be a nebulous term which essentially means extended cessation of bowel function beyond the period of the expected transient cessation of bowel function . in studies assessing prevention of postoperative ileus definitions it is for these very reasons that we chose to exclude this as an endpoint in favor of more objective outcomes such as los , time to clear liquid diet , time to regular diet , use of parenteral nutrition , and readmission for ileus . furthermore , these endpoints are of more utility in a retrospective study where time to flatus and time to first bowel movement are not always well documented . the strength of our study is related to the results of a multicenter rct confirming reduction of los by a mean 2.6 days . our experience contributes to the literature by demonstrating the advantage of alvimopan in decreasing los by 1.9 days even in the setting of epidural analgesia and when all 15 doses of alvimopan were not received . while a new intervention was implemented empirically based on translation from a different discipline , this was not performed for study purposes . the sequential rather than randomized nature thus limits interpretation of this data due to nonrandom biases that are due to chronology of the cases . this is addressed with multivariate analysis to reduce the impact of known variables such as robotic technique and epidural analgesia . interestingly , contrary to most contemporary literature , there was not a diminished los in the robotic technique in this series . this likely reflects the early end of the learning curve in our robotic cystectomy experience and mandates future study . , alvimopan is associated with decrease los by almost 2 days after cystectomy and urinary diversion . furthermore , this is reflected in cost of stay with a 20% reduction in cost . alvimopan is an effective adjunct to early ambulation , epidural analgesia , optimized fluid management and early feeding to combat postoperative ileus after cystectomy and urinary diversion .

introductionwe sought to evaluate the cost effectiveness of perioperative use of alvimopan in cystectomy and urinary diversion . a recent randomized controlled trial demonstrated the efficacy of alvimopan in reducing postoperative ileus and length of stay in cystectomy ; however , a major limitation was the exclusion of epidural analgesia.materials and methodseighty six cystectomy and urinary diversion procedures performed by seven surgeons were analyzed between january 2008 and april 2012 . the first 50 patients did not receive alvimopan perioperatively , while the subsequent 36 received a single dose of 12 mg preoperatively and then 12 mg every 12 hours for 15 doses or until discharge.resultsthe groups were equal with respect to age , gender , indication , surgeon , and type of diversion . patients who received alvimopan experienced a shorter length of stay ( los ) versus those in who did not receive alvimopan ( 10.5 vs. 8.6 days , p = 0.005 , 95% ci 0.63.3 ) . readmission for ileus was low in both alvimopan and control groups ( 0% and 4.4% , respectively ) . costs were significantly lower in the alvimopan group than the control groups ( 2012 usd 32,443 vs. 40,604 p < 0.001 ) . this difference stood up to multivariate analysis with a $ 7,062 difference in hospital stay.conclusionsuse of alvimopan in the routine perioperative care of our cystectomy and urinary diversion patients has decreased los by 1.9 days . additionally , institution of routine perioperative alvimopan has reduced costs by $ 7,062 per admission ( 20% reduction ) . this demonstrates a real world application of alvimopan at a moderate volume center .

INTRODUCTION MATERIAL AND METHODS Subjects Operative details Intervention Outcomes Statistical analisys RESULTS Pre and perioperative characteristics Efficacy Costeffectiveness DISCUSSION CONCLUSIONS

PMC4297118

orthopedic surgeons and researchers worldwide are continuously faced with the challenge of regenerating articular cartilage defects ( fig . 1 ) . the poor healing capacity of articular cartilage due to avascularity is the main motivation for this elaborate and ambitious topic of research . despite the efforts that have been made within this subject , current repair strategies are not able to mimic the biological and biochemical properties of articular cartilage . past tissue engineering approaches for articular cartilage repair were based on the development of rigid scaffolds in which cell seeding and penetration were not perfectly achieved . in addition to the use of rigid scaffolds , hydrogels , cell therapies , and scaffold - free approaches have been studied to overcome large cartilage defects . in the last decade , matrix - associated autologous chondrocyte transplantation was established as a new approach for cartilage repair . large idiopathic cartilage lesion on the medial femoral condyle in a 36-year - old male patient . ( a , b ) preoperative and ( c ) intraoperative . in this review , furthermore , a brief discussion of the state of the art of current cell printing procedures mimicking native cartilage is offered in light of their use as future alternatives for cartilage tissue engineering . articular cartilage has an elaborate compartmental structure that must be understood before the development of a mimicking structure can occur . the main functions of articular cartilage are the transmission of loads from the joint to the underlying subchondral bone , the absorption of impact forces , and the promotion of a smooth , low - friction and gliding movement of the joint . these functions compromise the rheological viscoelastic properties of cartilage when it is subjected to a constant load . the behavior of cartilage is time dependent , decreasing its eminence by approximately half of the initial size over a lifetime . furthermore , when cartilage begins to degenerate or undergoes trauma , the recovery process is very slow or nonexistent . articular cartilage lacks a self - healing capacity mainly because of its lack of vascularity and low chondrocyte activity . articular cartilage is a connective tissue composed of the extracellular matrix ( ecm ) containing collagen , proteoglycans , and water . in the matrix of mature cartilage , the remaining constituents are small amounts of collagen types v , vi , ix , x , and xi , interconnected by proteoglycans and hyaluronan , which constitute about 10% of the total weight of articular cartilage . the mechanical properties of cartilage , for example , tensile strength and stiffness , are provided by type ii collagen fibers , which exhibit a triple - helix structure , restraining and immobilizing proteoglycans within the ecm . due to their affinity for water this depends on water pressurization , which determines its permeability based on the concentration of proteoglycans . furthermore , the domains of proteoglycans repel each other , allowing a larger area to be occupied and consequently contributing to greater strength and stiffness of the tissue . hyaluronan is a nonsulfated glycosaminoglycan that exists as a hydrophilic coat around each chondrocyte . its rheological properties , similar to other proteoglycans , help provide mechanical resistance to compression of articular cartilage . its physicochemical properties provide a temporary hydrated environment conducive to cell migration by facilitating cell detachment . water comprises up to 80% of the total weight of articular cartilage , contributing actively to joint lubrication and elasticity as well as the transport of nutrients . the only cell type present within articular cartilage is the chondrocyte , occupying approximately 2% of the total volume . however , articular cartilage can be organized into 4 major zones : superficial ( 10%-20% ) , middle ( 40%-60% ) , lower ( 30% ) , and calcified . as a result , there is a highly coordinated cell distribution within these zones , which can be distinguished on the basis of morphological criteria such as cell shape , size , and arrangement as well as collagen , proteoglycans , and hyaluronan expression . naturally , these chondrocytes differ in their phenotype , genotype , and functions they perform . figure 2 shows the zonal compartments of articular cartilage . in the superficial zone , chondrocytes are more elongated and flattened , collagen fibers are aligned parallel with the surface , and there is only a low amount of proteoglycans . gradually through the middle zone , chondrocytes become rounded , the presence of proteoglycans is greater , and there is a random arrangement of collagen . particularly in the lower zone , the major cell type self - assembles in columns , and collagen fibers align perpendicular to the bone . the closer to the calcified zone they are , the more chondrocytes tend to express various types of collagen , and the more ecm is produced . growth factors , matrix composition , electrical fields , hydrostatic pressures , and mechanical loads affect chondrocyte metabolism . besides , their metabolism is aerobic in an environment with low oxygen concentration . cartilage regeneration and remodeling are dependent upon articular chondrocytes and their metabolism , including synthesis of ecm molecules such as collagen , proteoglycans , and degradative enzymes . depending in which compartment these cells are located , there is an evident variation in their morphology and functions they perform . articular cartilage tissue engineering is a combination of different fields of research , such as molecular biology , materials science , and biomedical engineering . in the future , an approach for mimicking cartilage tissue will need to exhibit characteristics of these distinct areas , that is , a combination of a natural and/or synthetic material , cells ( chondrocytes , stem cells ) , and signaling molecules ( growth factors , extracellular molecules ) . figure 3 illustrates the idea that the inclusion of these topics is essential to obtaining an adequate articular cartilage substitute . starting with the input of a patient s computed tomography ( ct ) data into a computerized tool system , it will be possible to precisely print a combination of resources , resulting in a 3-dimensional ( 3-d ) construction mimicking native articular cartilage tissue . in this review , , we present the biomaterials used for articular cartilage repair during the past years as well as current concepts of cell printing converging with cartilage research . biomaterials can be characterized and subdivided into different categories such as origin , mechanical properties , and viscoelasticity . the biomaterials presented in table 1 that are used as substitutes of articular cartilage are grouped into 3 main areas : 1 ) natural , 2 ) synthetic , and 3 ) composites of natural / synthetic materials . representative scheme of a current concept for engineering articular cartilage : encapsulation of cells combined with signaling molecules into hydrogels to be printed . overview of studied biomaterials for articular cartilage repair note : hmscs = human mesenchymal stem cells ; szcs = superficial zonal chondrocytes ; mdzcs = middle / deep zonal chondrocytes ; hescs = human embryonic stem cells ; mg-63 = human osteosarcoma cell line ; gag = glycosaminoglycan ; gp = glycerol phosphate ; pla = polylactide acid ; pegt - pbt = poly(ethylene glycol)-terephthalate - poly(butylene terephethalate ) ; pva = poly(vinyl alcohol ) ; peg - pmma = poly(ethylene glycol)-poly(methyl methacrylate ) ; pcl = polycaprolactone ; dpclpc = dihydroxypolycaprolactone phosphorylcholine ; daps = 1,2-dihydroxy - n , n - dimethylamino - propane sulfonate ; mdpclt = monohydroxy dimethylacrylate polycaprolactone triol ; cap = calcium phosphate ; tcp = tri(calcium phosphate ) ; cas = calcium sulfate ; gcap = gag - calcium phosphate ; cpbta = chitosan - poly(butylene terephthalate adipate ) ; ha - co - hdpe = hyaluronic acid - co - high density polyethylene ; plcl = poly(l - lactide - co - caprolactone ) . commonly used natural materials in cartilage research are agarose , alginate , chitosan , collagen , fibrin , and hyaluronan . via specific surface receptors , these biomaterials interact with cells to contribute to cell migration , production of extracellular molecules , and consequently proliferation . several studies have shown that chondrocytes differentiate when entrapped in agarose , which stimulates an expression of the original phenotype . in addition , a positive production of glycosaminoglycan ( gag ) by chondrocytes in vivo and in vitro corroborates their application for cartilage research . awad et al . compared the chondrogenic differentiation of adipose - derived adult stem cells seeded in agarose and alginate hydrogels , resulting in the synthesis of proteoglycan and sulfate gag in the presence of transforming growth factor beta 1 ( tgf-1 ) . moreover , mouw et al . studied the gag fine structure from different scaffolds in which agarose constructs had both the highest gag - to - dna ratio and fraction of disulfated residues , converting this material into the most similar to native articular cartilage . furthermore , dynamically loaded cell - seeded agarose hydrogels reached the young modulus of canine knee cartilage . a biomimetic woven composite scaffold in which a cell - agarose hydrogel was studied relative to its load - bearing potential as well as its biological support was proposed by moutos et al . , proving the possibility of inducing initial engineered properties similar to those of native articular cartilage . more recently , tan et al . encapsulated primary immature bovine articular chondrocytes in an agarose hydrogel exposed to a mechanical overload , concluding that these constructs exhibit a reparative ability , contrary to native cartilage . alginate is a polysaccharide extracted from brown algae , which is extensively investigated as a cartilage substitute , serving as supporting scaffold for cell growth . alginate - based scaffolds exhibit biocompatibility as well as great gelling properties , consequently supporting chondrocyte phenotype . furthermore , alginate interacts with the cells via specific surface receptors , similar to agarose , facilitating cell migration and the proliferation and production of extracellular molecules . used an alginate hydrogel for the repair of a chondral defect , demonstrating the feasibility of an in situ additive manufacturing technique with this natural resource . moreover , a porous polyvinyl alcohol hydrogel scaffold combined with alginate microspheres was manufactured by scholten et al . , suggesting its potential for the replacement of cartilage defects due to the possibility of controlling mechanical properties while promoting cellular migration . cultured articular chondrocytes in alginate hydrogels and studied their mechanical properties at different points in time . an increase of the young modulus was observed over time , and the mechanical stiffness reached properties of natural hyaline cartilage . chitosan is composed of glucosamine and n- acetylglucosamine monomers and , being a natural polysaccharide extracted by deacetylation of chitin , is an unlimited resource . similarly to native cartilage , chitosan contains gag and hyaluronic acid ( ha ) , justifying its wide use within the cartilage tissue engineering field . moreover , it has proved to be biocompatible and biodegradable as well as inert and noncytotoxic . however , chitosan lacks fast gelling properties , leading to the possibility that it will flow out of the joint when applied , forming cartilage - like tissue ectopically . thus , hoemann et al . developed a chitosan solution that is space filling , gels within minutes , and adheres to cartilage in situ . this solution has been shown to support the in vitro and in vivo accumulation of cartilage matrix by primary chondrocytes and , more importantly , persisted into chondral defects at least up to 1 week in vivo . in addition , park et al . used a new chitosan - pluronic hydrogel as an injectable cell carrier system for cartilage regeneration . the proliferation of chondrocytes and the synthesis of gags showed the potential of this new scaffold system . chitosan / poly(3-caprolactone ) ( pcl ) scaffolds in different solutions have also become increasingly important within the field of articular cartilage tissue repair . the largest neocartilage formation of chondrocytes / scaffold constructs was observed in scaffolds consisting of 75 wt% chitosan and 25 wt% pcl . cell - seeded hydrogels were transplanted into articular cartilage defects in vivo and analyzed after 12 and 24 weeks . the cell - seeded chitosan hydrogels filled the cartilage defect completely within 24 weeks , thereby demonstrating its capability for cartilage tissue engineering . furthermore , alves da silva et al . investigated the effect of synovial fluid flow on chondrogenic differentiation of human mesenchymal stem cells ( hmscs ) seeded onto chitosan - poly(butylene terephthalate adipate ) mesh scaffolds when cultured in a flow perfusion bioreactor . after 28 days , ecm and collagen type ii production was observed , suggesting a beneficial effect on the chondrogenic differentiation of cells , induced by the flow shear stress in a bioreactor . tested several properties of chitosan , such as molecular weight , deacetylation degree , and calcium content within osteochondral scaffolds implanted in rabbit knees for a period of 3 months . their results revealed that chitosan scaffolds with mineral contents of approximately 18 wt% , low molecular weight ( 11.49 kda ) , and low deacetylation degree ( 83% ) show structured subchondral bone as well as cartilage tissue regeneration . chitosan / blood implants were studied in several studies of articular cartilage tissue repair after microfracturing in vivo . chitosan stabilized the blood clot and inhibited its shrinkage , thereby completely filling the defect . fourteen days after surgery , a higher density of hmscs was observed in chitosan / blood clots as compared to blood clots in control defects . thirty - five days after surgery , the chitosan constructs were better integrated within the defect and showed more mature chondrocyte foci in comparison with chitosan - free blood clots . in addition to extensive cellular growth in chitosan scaffolds , the mechanical properties of chitosan are also promising . additional chitosan nanofibers increased the ultimate tensile deformation and the elastic modulus of collagen type i scaffolds . it is a natural protein with a triple - helix structure , which can physically form a thermally reversible gel with good cell adhesion properties . the development of a stabilized type i collagen hydrogel was attempted by mueller - rath et al . and was also seeded with human articular chondrocytes . mechanical compression and filtration were applied on the scaffolds , with the aim of improving the loading capacity . indeed , cells within condensed collagen scaffolds were able to proliferate and produce extracellular molecules , suggesting that higher forces carrying capacity are important for 3-d autologous chondrocyte implantation . as described earlier , jancari et al . analyzed the mechanical properties of different modified collagen scaffolds . combining collagen with hydroxyapatite particles or chitosan nanofibers led to increased elastic moduli . chen et al . evaluated the feasibility of using bone marrow mesenchymal stem cell seeded type ii collagen scaffolds implanted in rabbits for articular cartilage repair . after 8 weeks , chondrocyte - like cells and extracellular molecules were found in the newly formed tissue with no signs of inflammation , suggesting that collagen may be a suitable supporting material as a substitute for cartilage defects . more recently , kon et al . developed a collagen / hydroxyapatite - based novel , nanocomposite multilayered biomaterial to replace cartilage and subchondral bone , which was tested in patients for up to 24 months . as a result , young and active patients underwent a fast recovery in contrast to older patients or patients with previous surgery , who exhibited worse results . nevertheless , this study proved the safety and potential of graded biomimetic collagen - based scaffolds for promoting cartilage and bone restoration with good 2-year follow - up results , confirming the potential of collagen as a natural resource for replacing the osteochondral unit . human fibrin gels are approved by the food and drug administration ( fda ) and were extensively studied as a potential support for cartilage tissue engineering . when cultured with chondrocytes , fibrin hydrogels stimulate the production of gag and thus the adequate formation of the ecm . have performed a review of the use of fibrin hydrogels for articular cartilage repair , primarily focusing on mice studies and culminating in an applied swine model study . their results suggest that the combination of autologous chondrocytes and allogenic devitalized cartilage matrices suspended in fibrin glue allows the formation of cartilage - like tissue . directed a study in 5 patients with large articular cartilage defects in which autologous bone marrow mesenchymal stem cells were expanded in culture and placed into platelet - rich fibrin glue intraoperatively . after 12 months , mri of 3 patients showed a complete defect fill , contrary to the other two , which revealed incomplete congruity with native cartilage . also recently , a fibrin / ha composite hydrogel scaffold was used in an in vivo study for chondrocyte seeding and pig knee cartilage regeneration by rampichov et al . the quality of the healing process was dependent on the initial chondrocyte concentration : scaffolds containing 9 10 cells / ml were lined on both sides by a thin noncellular transient zone toward the joint cartilage . type ii collagen positive staining was found within these noncellular layers as well as adjacent to replaced fibrocartilaginous tissue . in an in vitro study , swine chondrocytes were cultured in fibrin hydrogels and showed enhanced synthetic activity and notable matrix deposition . on the other hand , mechanical analysis after 5 weeks did not show properties close to native cartilage . hyaluronan is a gag present in native cartilage , being one essential component of the cartilage ecm . similarly to other natural materials , hyaluronan is also a target resource for cartilage research due to its ability to entrap living cells , supporting their proliferation and differentiation . hyaff-11 ( fidia advanced biopolymer , abano terme , italy ) is a hyaluronan - based commercially available biodegradable polymer , which was investigated as a support for hmsc chondrogenesis differentiation by lisignoli et al . in this study , cellular differentiation towards chondrocytes was induced via a range of tgf-1 concentrations , and it was found that without the growth factor stimulation , hmscs did not survive . an increased expression of collagen type ii was found , whereas collagen type i was down - regulated . these results indicate a first achievement in bringing the cells into close contact , favoring interactions with other extracellular molecules and representing an important natural material source for cartilage tissue engineering . in addition , chondrocytes cultured in hyaluronan hydrogels synthesized with a methacrylated form of hyaluronan showed a uniform cell distribution and matrix deposition . unfortunately , oliveira et al . proposed a new biomaterial for cartilage repair derived from microbial fermentation of the sphingomonas paucimobilis micro - organism ; gellan gum is a polysaccharide commonly used in food and in the pharmaceutical industry . this attracted attention due to the material s properties of dissolving easily in water , and when heated in solution with divalent cations , it forms a gel upon lowering the temperature under controlled conditions . in this work , gellan gum hydrogels were mechanically and rheologically evaluated , revealing excellent properties as cartilage substitutes ( compressive storage and sol - gel transition approximately 40 kpa at 36 c , respectively ) . histological analysis of human nasal chondrocytes cultured into gellan gum hydrogels showed positive cell morphology results , supporting the potential of this new biomaterial for cartilage regeneration . in addition to naturally resourced materials , synthetic materials also constitute a large pool for cartilage research . current synthetic materials used within this field are poly(ethylene glycol ) ( peg ) , poly(n - isopropylacrylamide ) ( nipaam ) , polylactide acid ( pla ) and derivates ( plla , plga , pdla ) , polyurethane ( pu ) , and poly(vinyl alcohol ) ( pva ) . these polymers offer relative ease of processing as well as mechanical properties suitable for this type of application ( young modulus of native cartilage is approximately 0.2 - 0.3 gpa ) . in the particular case of hydrogels , they exhibit a high potential to entrap living cells as well as providing a highly hydrated environment , facilitating nutrient diffusion , and serving as biological stimuli for migration , proliferation , and differentiation of cells . peg is a polyether extensively used as material support in cartilage research studies . under hydrogel or rigid scaffold form , it has proven to support the attachment , viability , proliferation , and production of the ecm of seeded chondrocytes . although peg was investigated as a cartilage substitute alone , most of the studies reveal an improved strength and compression modulus when used in combination with other natural or synthetic materials , such as albumin , collagen mimetic peptide ( cmp ) , poly(methyl methacrylate ) ( pmma ) , and poly(butylene terephthalate ) ( pbt ) . characterized peg hydrogels and amphiphilic interpenetrating polymer networks ( ipns ) of peg combined with pmma , testing different molecular weights , cross - link densities , and pmma volume fraction . they showed that lower molecular weight values , higher cross - link densities , and a higher pmma fraction lead to a higher equilibrium modulus and lower water content . however , ipns enhanced the hydrogels strength , converting them into materials suitable as cartilage substitutes . recently , scholz et al . evaluated an injectable peg - albumin hydrogel supplemented with ha for its impact on angiogenesis . native healthy articular cartilage lacks vascularization , whereas pathological blood vessel formation enhances its degeneration . therefore , human chondrocytes were encapsulated into the peg - albumin hydrogel and subcutaneously implanted in immunodeficient mice . after 2 weeks , no formation of blood vessels was detected inside the hydrogel , and at the same time , cells maintained their characteristic genotype expressing type i and ii collagen and aggrecan , promising that peg - albumin hydrogels are a beneficial implant support for chondrocytes . a new approach to chondrogenic differentiation of hmscs to neocartilage was attempted by liu et al . , which synthesized a cmp containing a gfoger sequence flanked by gpo repeat units ( ( gpo)4gfoger(gpo)4gcg , cmp ) incorporated into a peg hydrogel . histological analysis revealed an increased accumulation of collagen type ii and aggrecan in cells within peg - cmp hydrogels . moreover , the presence of an activation of cartilage - specific genes and enhanced ecm accumulation was shown , which indicates that a peg - cmp hydrogel is a promising hmsc carrier to injured cartilage for cartilage tissue engineering . pnipaam is an inverse thermosensitive polymer derived from polyacrylic acid , which has a phase transition above its low critical solution temperature ( lcst ) of around 32 c . a copolymer can be achieved through copolymerization of pnipaam with acrylic acid ( aac ) , resulting in pnipaam - co - aac , which gels at 37 c and becomes liquid at lower temperatures , thus displaying great potential for a cartilage supporting matrix . a pnipaam - based engineered cartilage embedded with chondrocytes was developed by ibusuki et al . to study the usefulness of suturing cartilage defects in rabbits with 2 different covering materials ( periosteum and collagen ) . a histological evaluation 5 weeks after implantation showed no inflammation or vascularization formation . whether the covering material used was periosteum or collagen , type ii collagen was produced by chondrocytes throughout the transplant . these results revealed the suitability of pnipaam - based hydrogels for the reconstruction of cartilaginous tissue with minimal surface deformation and no leakage of the transplant . an injectable gellable poly(n - isopropylacrylamide)-grafted gelatin ( pnipaam - gelatin ) scaffold was analyzed by ibusuki et al . interestingly , biological factors shown by total collagen and s - gag as well as mechanical characteristics reached values of native cartilage over the culture time . furthermore , a chitosan - pnipaam injectable hydrogel was synthesized by chen et al . , which revealed an lcst of around 30 c . when chondrocytes are entrapped within this hydrogel , both the vitality of cells and their phenotypic morphology are preserved . in addition , lower nh2/cooh ratio copolymers show faster sol - gel phase transition as well as improved mechanical strength over pnipaam hydrogels , therefore being suitable as a scaffold for tissue engineering of cartilage . more recently , park et al . evaluated pnipaam - co - vinylimidazole - p(nipaam - co - vi ) hydrogel constructs composed of rabbit chondrocytes and tgf-1 heparinized nanoparticles for cartilage replacement , suggesting that a higher cell number is accompanied by the maintenance of phenotype , therefore providing a suitable model for tissue engineering of cartilage . pla is a biodegradable polyester that has been investigated as a support matrix for cell carrying within cartilage tissue engineering research . related polymers are poly(d - lactide ) ( pdla ) , poly(lactic - co - glycolic acid ) ( plga ) , and poly(l - lactide ) ( plla ) . tanaka et al . compared several polylactide and related polymer scaffolds ( plla , plga , pla / cl , pdla ) with different porosities ( 80%-95% ) and pore sizes ( 0.3 - 2.0 mm ) administered with a chondrocyte / atelocollagen mixture , which were implanted subcutaneously in nude mice . after 2 months of implantation , the scaffolds were macroscopically and histologically studied , showing that their 3-d shape was maintained throughout the probing period , with the exception of the control , which was a transplant of a chondrocyte / atelocollagen mixture without a scaffold support . moreover , levels of type i and type ii collagen production as well as gag were higher for plla and plga scaffold transplants , suggesting their higher affinity as cartilage substitutes . furthermore , the number of macrophages surrounding the scaffolds was quantified , revealing once again the greater results for plla and plga ; that is , fewer macrophages were present within these scaffolds compared to the resting ones , demonstrating that these are adequate articular cartilage substitutes . a bioactive collagen - grafted plla membrane for tissue engineering of cartilage was examined by chen et al . , which was subject to dc - pulsed oxygen plasma treatment to enhance cell attachment and growth . chondrocytes seeded into this membrane revealed positive proliferation , vitality , and differentiation rates , corroborated by the secretion of collagen and gag . moreover , these membranes were able to maintain chondrocyte morphology and structure , suggesting their potential for cartilage research . more recently , the same authors suggested an improvement of their previous work , combining plla membranes with cationized gelatin , which revealed enhanced expression of characteristic markers such as type ii collagen , aggrecan , and sox-9 . formation of ectopic cartilage was detected 28 days after subcutaneous implantation using histology and immunostaining . found a compressive modulus of approximately 6 mpa in plla scaffolds with a porous microstructure . combined with fibrin gel , mechanical properties increased , and higher cell proliferation and gag were found . nanofiber - based plga scaffolds with different lactic acid / glycolic acid ratios were analyzed in another study by shin et al . tensile modulus , ultimate tensile stress , and corresponding strain of these scaffold types nearly reached values of human cartilage . in addition , cell proliferation and ecm deposition revealed the capability of these scaffold types for cartilage tissue engineering . polyurethanes exhibit several advantages for use as articular cartilage substitutes , such as ease of processing as injectable gels or pastes , the possibility of in situ polymerization , as well as adequate mechanical properties . porous polyurethane networks containing the zwitterionic components dihydroxypolycaprolactone phosphorylcholine ( dpclpc ) and 1,2-dihydroxy - n , n - dimethylamino - propane sulfonate ( daps ) were developed by adhikari et al . the polymers were mixed with hydrated gelatin beads , which conferred compression strength appropriate for their use as articular cartilage repair scaffolds . histologically , 2 months after subcutaneous implantation in rats , the polymers showed a mild degradation ( 10%-15% ) , which was enhanced after 14 months to 35% and 60% for daps and dpclpc polymers , respectively , with approximately 60% of the dpclpc implant containing fibroblast infiltration . hence , dpclpc - containing polymers appear to be useful in delivering cells and growth factors for cartilage tissue engineering . more recently , the same research group proposed a urethane - based polymer resulting from the polymerization of diisocyanato poly(ethylene glycol ) and monohydroxy dimethacrylate poly(-caprolactone ) triol for articular cartilage repair . in vivo studies revealed a micro - sized capsule formation and a mild host tissue response . in vitro , chondrocytes were seeded into these constructs and maintained in static and dynamic culture for up to 8 weeks , exhibiting cell viability , proliferation , migration , and ecm production . under dynamic culture , the chondrocyte - seeded polymers behaved more similarly to native articular cartilage , producing type ii and type iv collagen as well as keratin sulfate . grad et al . analyzed cell - seeded porous polyurethane scaffolds and showed an increase of the young modulus over the culture period . polyvinyl alcohol is a water - soluble synthetic polymer with excellent adhesive properties , which , because it is able to entrap living cells , appears to have great potential for engineering synthetic articular cartilage . charlton et al . attempted a semidegradable pva - plga scaffold aiming to mimic the mechanical properties of native cartilage . recently , the capability of pva hydrogels strengthened with ultrahigh molecular weight polyethylene was analyzed by holloway et al . however , the potential of this stabilized scaffold system is limited due to its nondegradability . scaffolds with varying plga percentages were studied , and those with higher plga content were found to be more suitable as a cartilage substitute ; that is , larger pores are derived from higher plga content , which encourages the migration of chondrocytes into the construct . engineered a pva - alginate hydrogel to evaluate the neocartilage - forming potential of human nasal septum chondrocytes . the seeded scaffolds were exposed to a bioreactor culture for up to 10 days and further implanted into the dorsum of nude mice for 6 weeks . histological results revealed abundant gag deposition as well as type ii collagen abundant intensity compared with the tissue throughout the pva . constructs within the bioreactor culture showed an approximately 20% higher compressive equilibrium modulus compared with scaffolds implanted immediately without pre - exposure to a bioreactor , thus demonstrating their potential as native cartilage substitutes . besides the use of scaffolds and cell sources for engineering articular cartilage , growth factors may also contribute to a better mimicking construct . therefore , mohan et al . proposed a combination of a pva - poly(caprolactone ) scaffold seeded with mesenchymal stem cells and variations of tgf-1 , tgf-3 , and bone morphogenetic protein ( bmp ) 2 . the presence of growth factors proved to influence stem cells morphology , differentiation , distribution , and secretion of ecm molecules . these authors suggested a combination of tgf-3 and bmp-2 , which promoted better cell differentiation into chondrocytes compared to the others . although several combinations of scaffolds , cells , and growth factors were mainly studied by experts worldwide , tran et al . suggested a scaffold - free approach for engineered articular cartilage constructs . a high amount of tissue - engineered cartilage was created from porcine chondrocytes using a bioreactor , firstly centrifuging a high - density chondrocyte cell suspension onto an agarose layer and afterwards transferring it into the bioreactor for up to 4 weeks . after 1 week of static culture , the constructs were firm , could be easily handled and manipulated with forceps , and did not adhere to the agarose layer . an ecm rich in proteoglycans was found among dynamic culture as compared to static culture constructs . furthermore , these results suggest that the use of a bioreactor is able to improve both the biochemical and biomechanical properties of engineered cartilage . cartilage tissue engineering has attracted much attention , and currently , natural and synthetic biomaterials are in clinical use for cartilage replacement . despite the number of biomaterials available and their review in numerous in vitro studies and multiple in vivo animal studies , only a few are in clinical use . however , the efficacy of these biomaterials has to be proven through long - term clinical outcomes for further evaluation . collagen i / iii scaffolds are mainly used in matrix - associated autologous chondrocyte transplantation . the 5-year follow - up results of this procedure showed significantly improved postoperative values as compared to the preoperative values . in addition to collagen scaffolds , other clinical studies have analyzed the function of hyaluronan scaffolds for cartilage reconstruction . interestingly , a second arthroscopy was performed after 12 months and mri after 24 months . in accordance with the above - mentioned studies , the patients experienced clinical and functional improvements . bioseed - c ( biotissue technologies , freiburg , germany ) , a polyglycolic / polylactic acid and polydioxane based scaffold , was evaluated as a treatment option for cartilage defects . forty patients were re - evaluated after 2 years and showed significant improvement in pain reduction and quality of life . of 79 patients at the beginning , 14 underwent second - look arthroscopy , and 5 patients underwent repeat surgery . taken together , these biomaterials showed significant improvements in the patient postoperative values , thereby demonstrating their capability for cartilage replacement . all of the studies mentioned used autologous chondrocytes in their procedures , and therefore , 2 surgeries were performed . during a first surgery , autologous chondrocytes were harvested and cultured before being implemented in a 3-d matrix . during a second step , the cell - scaffold components were transplanted . for these reasons , new approaches are emerging , which can be more comfortable for the patient . cell printing technologies , for example , are also important current alternatives that are discussed in the following section . commonly used natural materials in cartilage research are agarose , alginate , chitosan , collagen , fibrin , and hyaluronan . via specific surface receptors , these biomaterials interact with cells to contribute to cell migration , production of extracellular molecules , and consequently proliferation . several studies have shown that chondrocytes differentiate when entrapped in agarose , which stimulates an expression of the original phenotype . in addition , a positive production of glycosaminoglycan ( gag ) by chondrocytes in vivo and in vitro corroborates their application for cartilage research . awad et al . compared the chondrogenic differentiation of adipose - derived adult stem cells seeded in agarose and alginate hydrogels , resulting in the synthesis of proteoglycan and sulfate gag in the presence of transforming growth factor beta 1 ( tgf-1 ) . moreover , mouw et al . studied the gag fine structure from different scaffolds in which agarose constructs had both the highest gag - to - dna ratio and fraction of disulfated residues , converting this material into the most similar to native articular cartilage . furthermore , dynamically loaded cell - seeded agarose hydrogels reached the young modulus of canine knee cartilage . a biomimetic woven composite scaffold in which a cell - agarose hydrogel was studied relative to its load - bearing potential as well as its biological support was proposed by moutos et al . , proving the possibility of inducing initial engineered properties similar to those of native articular cartilage . more recently , tan et al . encapsulated primary immature bovine articular chondrocytes in an agarose hydrogel exposed to a mechanical overload , concluding that these constructs exhibit a reparative ability , contrary to native cartilage . alginate is a polysaccharide extracted from brown algae , which is extensively investigated as a cartilage substitute , serving as supporting scaffold for cell growth . alginate - based scaffolds exhibit biocompatibility as well as great gelling properties , consequently supporting chondrocyte phenotype . furthermore , alginate interacts with the cells via specific surface receptors , similar to agarose , facilitating cell migration and the proliferation and production of extracellular molecules . used an alginate hydrogel for the repair of a chondral defect , demonstrating the feasibility of an in situ additive manufacturing technique with this natural resource . moreover , a porous polyvinyl alcohol hydrogel scaffold combined with alginate microspheres was manufactured by scholten et al . , suggesting its potential for the replacement of cartilage defects due to the possibility of controlling mechanical properties while promoting cellular migration . cultured articular chondrocytes in alginate hydrogels and studied their mechanical properties at different points in time . an increase of the young modulus was observed over time , and the mechanical stiffness reached properties of natural hyaline cartilage . chitosan is composed of glucosamine and n- acetylglucosamine monomers and , being a natural polysaccharide extracted by deacetylation of chitin , is an unlimited resource . similarly to native cartilage , chitosan contains gag and hyaluronic acid ( ha ) , justifying its wide use within the cartilage tissue engineering field . moreover , it has proved to be biocompatible and biodegradable as well as inert and noncytotoxic . however , chitosan lacks fast gelling properties , leading to the possibility that it will flow out of the joint when applied , forming cartilage - like tissue ectopically . thus , hoemann et al . developed a chitosan solution that is space filling , gels within minutes , and adheres to cartilage in situ . this solution has been shown to support the in vitro and in vivo accumulation of cartilage matrix by primary chondrocytes and , more importantly , persisted into chondral defects at least up to 1 week in vivo . in addition , park et al . used a new chitosan - pluronic hydrogel as an injectable cell carrier system for cartilage regeneration . the proliferation of chondrocytes and the synthesis of gags showed the potential of this new scaffold system . chitosan / poly(3-caprolactone ) ( pcl ) scaffolds in different solutions have also become increasingly important within the field of articular cartilage tissue repair . the largest neocartilage formation of chondrocytes / scaffold constructs was observed in scaffolds consisting of 75 wt% chitosan and 25 wt% pcl . cell - seeded hydrogels were transplanted into articular cartilage defects in vivo and analyzed after 12 and 24 weeks . the cell - seeded chitosan hydrogels filled the cartilage defect completely within 24 weeks , thereby demonstrating its capability for cartilage tissue engineering . furthermore , alves da silva et al . investigated the effect of synovial fluid flow on chondrogenic differentiation of human mesenchymal stem cells ( hmscs ) seeded onto chitosan - poly(butylene terephthalate adipate ) mesh scaffolds when cultured in a flow perfusion bioreactor . after 28 days , ecm and collagen type ii production was observed , suggesting a beneficial effect on the chondrogenic differentiation of cells , induced by the flow shear stress in a bioreactor . tested several properties of chitosan , such as molecular weight , deacetylation degree , and calcium content within osteochondral scaffolds implanted in rabbit knees for a period of 3 months . their results revealed that chitosan scaffolds with mineral contents of approximately 18 wt% , low molecular weight ( 11.49 kda ) , and low deacetylation degree ( 83% ) show structured subchondral bone as well as cartilage tissue regeneration . chitosan / blood implants were studied in several studies of articular cartilage tissue repair after microfracturing in vivo . chitosan stabilized the blood clot and inhibited its shrinkage , thereby completely filling the defect . fourteen days after surgery , a higher density of hmscs was observed in chitosan / blood clots as compared to blood clots in control defects . thirty - five days after surgery , the chitosan constructs were better integrated within the defect and showed more mature chondrocyte foci in comparison with chitosan - free blood clots . in addition to extensive cellular growth in chitosan scaffolds , the mechanical properties of chitosan are also promising . additional chitosan nanofibers increased the ultimate tensile deformation and the elastic modulus of collagen type i scaffolds . it is a natural protein with a triple - helix structure , which can physically form a thermally reversible gel with good cell adhesion properties . the development of a stabilized type i collagen hydrogel was attempted by mueller - rath et al . and was also seeded with human articular chondrocytes . mechanical compression and filtration were applied on the scaffolds , with the aim of improving the loading capacity . indeed , cells within condensed collagen scaffolds were able to proliferate and produce extracellular molecules , suggesting that higher forces carrying capacity are important for 3-d autologous chondrocyte implantation . as described earlier , jancari et al . analyzed the mechanical properties of different modified collagen scaffolds . combining collagen with hydroxyapatite particles or chitosan nanofibers led to increased elastic moduli . chen et al . evaluated the feasibility of using bone marrow mesenchymal stem cell seeded type ii collagen scaffolds implanted in rabbits for articular cartilage repair . after 8 weeks , chondrocyte - like cells and extracellular molecules were found in the newly formed tissue with no signs of inflammation , suggesting that collagen may be a suitable supporting material as a substitute for cartilage defects . more recently , kon et al . developed a collagen / hydroxyapatite - based novel , nanocomposite multilayered biomaterial to replace cartilage and subchondral bone , which was tested in patients for up to 24 months . as a result , young and active patients underwent a fast recovery in contrast to older patients or patients with previous surgery , who exhibited worse results . nevertheless , this study proved the safety and potential of graded biomimetic collagen - based scaffolds for promoting cartilage and bone restoration with good 2-year follow - up results , confirming the potential of collagen as a natural resource for replacing the osteochondral unit . human fibrin gels are approved by the food and drug administration ( fda ) and were extensively studied as a potential support for cartilage tissue engineering . when cultured with chondrocytes , fibrin hydrogels stimulate the production of gag and thus the adequate formation of the ecm . have performed a review of the use of fibrin hydrogels for articular cartilage repair , primarily focusing on mice studies and culminating in an applied swine model study . their results suggest that the combination of autologous chondrocytes and allogenic devitalized cartilage matrices suspended in fibrin glue allows the formation of cartilage - like tissue . furthermore , haleem et al . directed a study in 5 patients with large articular cartilage defects in which autologous bone marrow mesenchymal stem cells were expanded in culture and placed into platelet - rich fibrin glue intraoperatively . after 12 months , mri of 3 patients showed a complete defect fill , contrary to the other two , which revealed incomplete congruity with native cartilage . also recently , a fibrin / ha composite hydrogel scaffold was used in an in vivo study for chondrocyte seeding and pig knee cartilage regeneration by rampichov et al . the quality of the healing process was dependent on the initial chondrocyte concentration : scaffolds containing 9 10 cells / ml were lined on both sides by a thin noncellular transient zone toward the joint cartilage . type ii collagen positive staining was found within these noncellular layers as well as adjacent to replaced fibrocartilaginous tissue . in an in vitro study , swine chondrocytes were cultured in fibrin hydrogels and showed enhanced synthetic activity and notable matrix deposition . on the other hand , mechanical analysis after 5 weeks hyaluronan is a gag present in native cartilage , being one essential component of the cartilage ecm . similarly to other natural materials , hyaluronan is also a target resource for cartilage research due to its ability to entrap living cells , supporting their proliferation and differentiation . fidia advanced biopolymer , abano terme , italy ) is a hyaluronan - based commercially available biodegradable polymer , which was investigated as a support for hmsc chondrogenesis differentiation by lisignoli et al . in this study , cellular differentiation towards chondrocytes was induced via a range of tgf-1 concentrations , and it was found that without the growth factor stimulation , hmscs did not survive . an increased expression of collagen type ii was found , whereas collagen type i was down - regulated . these results indicate a first achievement in bringing the cells into close contact , favoring interactions with other extracellular molecules and representing an important natural material source for cartilage tissue engineering . in addition , chondrocytes cultured in hyaluronan hydrogels synthesized with a methacrylated form of hyaluronan showed a uniform cell distribution and matrix deposition . unfortunately , proposed a new biomaterial for cartilage repair derived from microbial fermentation of the sphingomonas paucimobilis micro - organism ; gellan gum is a polysaccharide commonly used in food and in the pharmaceutical industry . this attracted attention due to the material s properties of dissolving easily in water , and when heated in solution with divalent cations , it forms a gel upon lowering the temperature under controlled conditions . in this work , gellan gum hydrogels were mechanically and rheologically evaluated , revealing excellent properties as cartilage substitutes ( compressive storage and sol - gel transition approximately 40 kpa at 36 c , respectively ) . histological analysis of human nasal chondrocytes cultured into gellan gum hydrogels showed positive cell morphology results , supporting the potential of this new biomaterial for cartilage regeneration . in addition to naturally resourced materials , synthetic materials also constitute a large pool for cartilage research . current synthetic materials used within this field are poly(ethylene glycol ) ( peg ) , poly(n - isopropylacrylamide ) ( nipaam ) , polylactide acid ( pla ) and derivates ( plla , plga , pdla ) , polyurethane ( pu ) , and poly(vinyl alcohol ) ( pva ) . these polymers offer relative ease of processing as well as mechanical properties suitable for this type of application ( young modulus of native cartilage is approximately 0.2 - 0.3 gpa ) . in the particular case of hydrogels , they exhibit a high potential to entrap living cells as well as providing a highly hydrated environment , facilitating nutrient diffusion , and serving as biological stimuli for migration , proliferation , and differentiation of cells . peg is a polyether extensively used as material support in cartilage research studies . under hydrogel or rigid scaffold form , it has proven to support the attachment , viability , proliferation , and production of the ecm of seeded chondrocytes . although peg was investigated as a cartilage substitute alone , most of the studies reveal an improved strength and compression modulus when used in combination with other natural or synthetic materials , such as albumin , collagen mimetic peptide ( cmp ) , poly(methyl methacrylate ) ( pmma ) , and poly(butylene terephthalate ) ( pbt ) . characterized peg hydrogels and amphiphilic interpenetrating polymer networks ( ipns ) of peg combined with pmma , testing different molecular weights , cross - link densities , and pmma volume fraction . they showed that lower molecular weight values , higher cross - link densities , and a higher pmma fraction lead to a higher equilibrium modulus and lower water content . however , ipns enhanced the hydrogels strength , converting them into materials suitable as cartilage substitutes . recently , scholz et al . evaluated an injectable peg - albumin hydrogel supplemented with ha for its impact on angiogenesis . native healthy articular cartilage lacks vascularization , whereas pathological blood vessel formation enhances its degeneration . therefore , human chondrocytes were encapsulated into the peg - albumin hydrogel and subcutaneously implanted in immunodeficient mice . after 2 weeks , no formation of blood vessels was detected inside the hydrogel , and at the same time , cells maintained their characteristic genotype expressing type i and ii collagen and aggrecan , promising that peg - albumin hydrogels are a beneficial implant support for chondrocytes . a new approach to chondrogenic differentiation of hmscs to neocartilage was attempted by liu et al . , which synthesized a cmp containing a gfoger sequence flanked by gpo repeat units ( ( gpo)4gfoger(gpo)4gcg , cmp ) incorporated into a peg hydrogel . histological analysis revealed an increased accumulation of collagen type ii and aggrecan in cells within peg - cmp hydrogels . moreover , the presence of an activation of cartilage - specific genes and enhanced ecm accumulation was shown , which indicates that a peg - cmp hydrogel is a promising hmsc carrier to injured cartilage for cartilage tissue engineering . pnipaam is an inverse thermosensitive polymer derived from polyacrylic acid , which has a phase transition above its low critical solution temperature ( lcst ) of around 32 c . a copolymer can be achieved through copolymerization of pnipaam with acrylic acid ( aac ) , resulting in pnipaam - co - aac , which gels at 37 c and becomes liquid at lower temperatures , thus displaying great potential for a cartilage supporting matrix . a pnipaam - based engineered cartilage embedded with chondrocytes was developed by ibusuki et al . to study the usefulness of suturing cartilage defects in rabbits with 2 different covering materials ( periosteum and collagen ) . a histological evaluation 5 weeks after implantation showed no inflammation or vascularization formation . whether the covering material used was periosteum or collagen , type ii collagen was produced by chondrocytes throughout the transplant . these results revealed the suitability of pnipaam - based hydrogels for the reconstruction of cartilaginous tissue with minimal surface deformation and no leakage of the transplant . an injectable gellable poly(n - isopropylacrylamide)-grafted gelatin ( pnipaam - gelatin ) scaffold was analyzed by ibusuki et al . interestingly , biological factors shown by total collagen and s - gag as well as mechanical characteristics reached values of native cartilage over the culture time . furthermore , a chitosan - pnipaam injectable hydrogel was synthesized by chen et al . , which revealed an lcst of around 30 c . when chondrocytes are entrapped within this hydrogel , both the vitality of cells and their phenotypic morphology are preserved . in addition , lower nh2/cooh ratio copolymers show faster sol - gel phase transition as well as improved mechanical strength over pnipaam hydrogels , therefore being suitable as a scaffold for tissue engineering of cartilage . more recently , park et al . evaluated pnipaam - co - vinylimidazole - p(nipaam - co - vi ) hydrogel constructs composed of rabbit chondrocytes and tgf-1 heparinized nanoparticles for cartilage replacement , suggesting that a higher cell number is accompanied by the maintenance of phenotype , therefore providing a suitable model for tissue engineering of cartilage . pla is a biodegradable polyester that has been investigated as a support matrix for cell carrying within cartilage tissue engineering research . related polymers are poly(d - lactide ) ( pdla ) , poly(lactic - co - glycolic acid ) ( plga ) , and poly(l - lactide ) ( plla ) . tanaka et al . compared several polylactide and related polymer scaffolds ( plla , plga , pla / cl , pdla ) with different porosities ( 80%-95% ) and pore sizes ( 0.3 - 2.0 mm ) administered with a chondrocyte / atelocollagen mixture , which were implanted subcutaneously in nude mice . after 2 months of implantation , the scaffolds were macroscopically and histologically studied , showing that their 3-d shape was maintained throughout the probing period , with the exception of the control , which was a transplant of a chondrocyte / atelocollagen mixture without a scaffold support . moreover , levels of type i and type ii collagen production as well as gag were higher for plla and plga scaffold transplants , suggesting their higher affinity as cartilage substitutes . furthermore , the number of macrophages surrounding the scaffolds was quantified , revealing once again the greater results for plla and plga ; that is , fewer macrophages were present within these scaffolds compared to the resting ones , demonstrating that these are adequate articular cartilage substitutes . a bioactive collagen - grafted plla membrane for tissue engineering of cartilage was examined by chen et al . , which was subject to dc - pulsed oxygen plasma treatment to enhance cell attachment and growth . chondrocytes seeded into this membrane revealed positive proliferation , vitality , and differentiation rates , corroborated by the secretion of collagen and gag . moreover , these membranes were able to maintain chondrocyte morphology and structure , suggesting their potential for cartilage research . more recently , the same authors suggested an improvement of their previous work , combining plla membranes with cationized gelatin , which revealed enhanced expression of characteristic markers such as type ii collagen , aggrecan , and sox-9 . formation of ectopic cartilage was detected 28 days after subcutaneous implantation using histology and immunostaining . found a compressive modulus of approximately 6 mpa in plla scaffolds with a porous microstructure . combined with fibrin gel , mechanical properties increased , and higher cell proliferation and gag were found . nanofiber - based plga scaffolds with different lactic acid / glycolic acid ratios were analyzed in another study by shin et al . tensile modulus , ultimate tensile stress , and corresponding strain of these scaffold types nearly reached values of human cartilage . in addition , cell proliferation and ecm deposition revealed the capability of these scaffold types for cartilage tissue engineering . polyurethanes exhibit several advantages for use as articular cartilage substitutes , such as ease of processing as injectable gels or pastes , the possibility of in situ polymerization , as well as adequate mechanical properties . porous polyurethane networks containing the zwitterionic components dihydroxypolycaprolactone phosphorylcholine ( dpclpc ) and 1,2-dihydroxy - n , n - dimethylamino - propane sulfonate ( daps ) were developed by adhikari et al . the polymers were mixed with hydrated gelatin beads , which conferred compression strength appropriate for their use as articular cartilage repair scaffolds . histologically , 2 months after subcutaneous implantation in rats , the polymers showed a mild degradation ( 10%-15% ) , which was enhanced after 14 months to 35% and 60% for daps and dpclpc polymers , respectively , with approximately 60% of the dpclpc implant containing fibroblast infiltration . hence , dpclpc - containing polymers appear to be useful in delivering cells and growth factors for cartilage tissue engineering . more recently , the same research group proposed a urethane - based polymer resulting from the polymerization of diisocyanato poly(ethylene glycol ) and monohydroxy dimethacrylate poly(-caprolactone ) triol for articular cartilage repair . in vivo studies revealed a micro - sized capsule formation and a mild host tissue response . in vitro , chondrocytes were seeded into these constructs and maintained in static and dynamic culture for up to 8 weeks , exhibiting cell viability , proliferation , migration , and ecm production . under dynamic culture , the chondrocyte - seeded polymers behaved more similarly to native articular cartilage , producing type ii and type iv collagen as well as keratin sulfate . grad et al . analyzed cell - seeded porous polyurethane scaffolds and showed an increase of the young modulus over the culture period . polyvinyl alcohol is a water - soluble synthetic polymer with excellent adhesive properties , which , because it is able to entrap living cells , appears to have great potential for engineering synthetic articular cartilage . charlton et al . attempted a semidegradable pva - plga scaffold aiming to mimic the mechanical properties of native cartilage . recently , the capability of pva hydrogels strengthened with ultrahigh molecular weight polyethylene was analyzed by holloway et al . however , the potential of this stabilized scaffold system is limited due to its nondegradability . scaffolds with varying plga percentages were studied , and those with higher plga content were found to be more suitable as a cartilage substitute ; that is , larger pores are derived from higher plga content , which encourages the migration of chondrocytes into the construct . engineered a pva - alginate hydrogel to evaluate the neocartilage - forming potential of human nasal septum chondrocytes . the seeded scaffolds were exposed to a bioreactor culture for up to 10 days and further implanted into the dorsum of nude mice for 6 weeks . histological results revealed abundant gag deposition as well as type ii collagen abundant intensity compared with the tissue throughout the pva . constructs within the bioreactor culture showed an approximately 20% higher compressive equilibrium modulus compared with scaffolds implanted immediately without pre - exposure to a bioreactor , thus demonstrating their potential as native cartilage substitutes . besides the use of scaffolds and cell sources for engineering articular cartilage , growth factors may also contribute to a better mimicking construct . therefore , mohan et al . proposed a combination of a pva - poly(caprolactone ) scaffold seeded with mesenchymal stem cells and variations of tgf-1 , tgf-3 , and bone morphogenetic protein ( bmp ) 2 . the presence of growth factors proved to influence stem cells morphology , differentiation , distribution , and secretion of ecm molecules . these authors suggested a combination of tgf-3 and bmp-2 , which promoted better cell differentiation into chondrocytes compared to the others . although several combinations of scaffolds , cells , and growth factors were mainly studied by experts worldwide , tran et al . suggested a scaffold - free approach for engineered articular cartilage constructs . a high amount of tissue - engineered cartilage was created from porcine chondrocytes using a bioreactor , firstly centrifuging a high - density chondrocyte cell suspension onto an agarose layer and afterwards transferring it into the bioreactor for up to 4 weeks . after 1 week of static culture , the constructs were firm , could be easily handled and manipulated with forceps , and did not adhere to the agarose layer . an ecm rich in proteoglycans was found among dynamic culture as compared to static culture constructs . furthermore , these results suggest that the use of a bioreactor is able to improve both the biochemical and biomechanical properties of engineered cartilage . cartilage tissue engineering has attracted much attention , and currently , natural and synthetic biomaterials are in clinical use for cartilage replacement . despite the number of biomaterials available and their review in numerous in vitro studies and multiple in vivo animal studies , only a few are in clinical use . however , the efficacy of these biomaterials has to be proven through long - term clinical outcomes for further evaluation . collagen i / iii scaffolds are mainly used in matrix - associated autologous chondrocyte transplantation . the 5-year follow - up results of this procedure showed significantly improved postoperative values as compared to the preoperative values . in addition to collagen scaffolds , other clinical studies have analyzed the function of hyaluronan scaffolds for cartilage reconstruction . interestingly , a second arthroscopy was performed after 12 months and mri after 24 months . in accordance with the above - mentioned studies bioseed - c ( biotissue technologies , freiburg , germany ) , a polyglycolic / polylactic acid and polydioxane based scaffold , was evaluated as a treatment option for cartilage defects . forty patients were re - evaluated after 2 years and showed significant improvement in pain reduction and quality of life . of 79 patients at the beginning , 14 underwent second - look arthroscopy , and 5 patients underwent repeat surgery . taken together , these biomaterials showed significant improvements in the patient postoperative values , thereby demonstrating their capability for cartilage replacement . all of the studies mentioned used autologous chondrocytes in their procedures , and therefore , 2 surgeries were performed . during a first surgery , autologous chondrocytes were harvested and cultured before being implemented in a 3-d matrix . during a second step , the cell - scaffold components were transplanted . for these reasons cell printing technologies , for example , are also important current alternatives that are discussed in the following section . the majority of the studies presented share as their main concern the biological stimuli provided by the supporting natural or synthetic material matrix as well as the improvement of the mechanical and rheological properties for cell encapsulation . new articular cartilage tissue engineering techniques are currently emerging , such as inkjet cell printing , controlled deposition cell printing tools , and laser cell printing , which will provide a faster and autologous 3-d tissue replacement of cartilage defects . repairing knee and hip articular cartilage defects the treatment of large cartilage defects in osteoarthritis is becoming one of the most performed procedures ; for example , 8.5 million people in the united kingdom are affected by joint pain , which may be related to osteoarthritis . however , several experts are focusing extensively on the development of cell printing technologies aimed at the production of 3-d engineered tissues , for cardiovascular and urinary tract applications , for example . focused cell printing for repairing cartilage defects was performed by cohen et al . a development technique for directly fabricating articular chondrocyte seeded alginate constructs in arbitrary geometries with multi - axial zonal organization the crescent - shaped piece mimicking native articular cartilage was drawn using a computer - aided design ( cad ) program , placed into the control software of the printer , and printed . moreover , a cad model of an ovine meniscus was constructed from a ct scan and printed . their results showed an increased young modulus of the samples after manufacturing : 1.8 0.1 kpa ( n = 6 ) . biological evaluation revealed an increased gag content in printed samples to 18.9 4.2 g/g dna after 3 weeks of incubation . using this technique , a chondrocyte - seeded alginate hydrogel was deposited within arbitrary geometries without the requirement of molds to manufacture an implant , proving its suitability for engineering articular cartilage . more recently , shanjani et al . proposed a powder - based solid free - form technique for printing calcium polyphosphate constructs , using pva as a polymeric binder that was eliminated afterwards during the annealing process . the porous , solid free - form structures showed an average compressive strength of 34 mpa , which is 57% higher compared to calcium polyphosphate structures made using conventional sintering . histologically , these constructs were able to support cartilage formation in vitro , based on chondrocyte growth within the surfaces of the material . in which a computer - aided biofabrication technique was used based on laser - induced forward transfer ( lift ) . this study showed that the cells maintained their vitality after the printing process , and differentiation of chondrocytes was allowed by the high cell densities used with lift . thus , the fabrication of lift 3-d scaffold - free autologous tissues has proven to maintain their predefined structure , suggesting the potential of this approach for developing new engineered articular cartilage tissue . nowadays , cell printing is not seen entirely as a new concept for tissue engineering approaches . several research groups have been working on the development of printing technologies in recent years to meet the challenge of generating newly formed tissue , including engineered articular cartilage . however , the investigation of novel hydrogels or copolymerization of hydrogels as support matrices for cell encapsulation will still be a topic of discussion over the next several years . moreover , turning these printable hydrogels into smarter materials would require the presence of signaling molecules , growth factors , or ecms , such as tgfs , soxs , and bmps , and therefore , extensive research still needs to be done . cell - seeded hydrogels are already able to survive the printing process as well as maintain their potential to differentiate , thus producing the ecm , which confers the mechanical support to the printed tissue . concerning the materials referred to in this review , natural materials exhibit an improved cytocompatibility , whereas synthetic materials appear to have better mechanical properties ( table 2 ) . although synthetic materials could represent promising candidates for articular cartilage substitute supports , natural materials such as chitosan appear to be a more appropriate choice to be made . its degradation products such as hyaluronan , sulfates , and collagen are involved in the formation of cartilage and are thus beneficial for achieving a mimicking tissue . among the cell types , both mesenchymal stem cells and primary isolated human articular chondrocytes have shown positive results when encapsulated into the hydrogels . due to the easier isolation and more comfortable procedure for the patient moreover , hmscs are chondrocyte progenitors that have the advantage of forming stroma and ecms inside the constructs when already committed into one lineage . advantages and disadvantages of studied biomaterials for articular cartilage repair note : peg = poly(ethylene glycol ) ; pla = polylactide acid ; pva = poly(vinyl alcohol ) ; ecm = extracellular matrix . in this review , we have presented the latest developments using natural and synthetic materials serving as cartilage substitutes as well as the in vitro / in vivo and preclinical / clinical state of the art of articular cartilage repair . three main parameters should be carefully addressed : 1 ) the choice of a supporting material , 2 ) the choice of cell source , and 3 ) the choice of method for developing articular cartilage substitutes . to our knowledge , a natural material , namely chitosan , has been shown to have satisfactory compression and tensile strength , that is , good mechanical properties , which in combination with facilitating the adhesion of cells , allowing their migration and differentiation , appears to be an excellent choice as a supporting material ( table 3 ) . gels such as agarose , collagen , or hyaluronan alone do not demonstrate sufficient mechanical strength for this type of application ; therefore , their use is not entirely suitable for newly formed cartilage . the preferred cell source to be used for cartilage tissue engineering is hmscs for several reasons already mentioned in this review . the main advantage is that it is a more comfortable procedure for the patient as well as avoiding complications or even tissue rejection after the second surgery . also very important is the fact that hmscs can coexist in different stages of their differentiation , allowing several phenotypes and genotypes to be present within the same tissue , which would mimic more precisely native articular cartilage . relative to the choice of the method , we suggest in this review bioprinting or biofabrication techniques , such as 2-photon polymerization , lift , or direct free - form fabrication . the precision of these techniques would make it possible to selectively print cells and gels mimicking native cartilage more than past approaches , that is , to print more cells on the surface and fewer cells in the lower zone of cartilage . nonbiofabricated gels containing cells have a homogeneous cell distribution , not ideal for this type of application . in native cartilage , heterogeneous cell morphology , distribution , migration , and differentiation occur gradually from the surface to the calcified bone ( fig . qualitative evaluation of biomechanical and biological properties of supporting materials for neocartilage tissue note : peg = poly(ethylene glycol ) ; pla = polylactide acid ; pva = poly(vinyl alcohol ) . changoor et al . compared the formation of neocartilage tissue on the interface of a biomaterial with normal and degenerated cartilage , suggesting that there are still a few differences that can be addressed . most important is to mimic the orientation of collagen fibers , which plays a crucial role in native tissue . while the fiber diameter in the middle and lower zones of neocartilage in contact with a biomaterial is very similar to the normal cartilage , in the superficial zone , it is still critical . the design of zonal compartments can be achieved more easily using bioprinting , where chondrocytes and collagen fibers are directly organized and printed as in the native form . these biofabrication techniques allow the optimization of tissue - engineered cartilage constructs , contrary to former techniques where cells were distributed randomly . moreover , the possibility of designing tissue - engineered cartilage would help to improve the mechanical strength and compression modulus of the final product as well as guiding chondrocytes for migration , differentiation , and production of the ecm differently depending on their zonal location . in table 4 , several advantages and disadvantages of biofabrication techniques are listed . to our understanding , the direct free - form printing technique is more advantageous compared to the others listed in this table because it enables the simultaneous printing of gels with cells and signaling molecules . for example , 2-photon polymerization also allows this combination with cells , but it is limited to the use of photosensible polymers . direct freeform , however , is lacking in structural support during the printing process , which is not very critical due to the relatively simple patterning of cartilage defects . moreover , cells are expected to self - assemble and rapidly produce the ecm , which will confer the most important support to the neotissue . in all , the key concept for achieving an optimized articular cartilage substitute is not to see the cartilaginous tissue as a construct but as a compartmentalized tissue with differently shaped cells that exhibit various functions . advantages and disadvantages of current printing techniques suitable for articular cartilage tissue engineering after developing the first step of cell - gel printing , keeping the cells alive with nutrients and appropriate co2 levels is also of importance . because there is no vascularization present in cartilage tissue , this process needs to be assured via diffusion . therefore , the use of a bioreactor is recommended , most importantly due to the fact of exposing the cells to shear stress , a physical stimulation that is essential for their migration and differentiation . the chondrocytes need to feel where they are located . also for this reason a brief state of the art relative to in vitro / in vivo preclinical / clinical studies is also addressed in this review . while clinical studies with printed articular cartilage tissue were not yet investigated , in vitro studies are already running . , using the direct free - form printing technique , proved that articular chondrocytes are viable after the printing steps and able to proliferate . apart from the printing context , materials such as collagen and hyaluronan have already been clinically tested and are known for their encouraging results . however , these studies were based mainly on matrix - associated chondrocyte implantation techniques , which still have the disadvantage of requiring the harvesting of chondrocytes similar to the old - fashioned autologous chondrocyte implantation method . in all , the development of mimetic hydrogels with specific biological properties related to articular cartilage native tissue will help to discover improved , functional , and novel engineered tissue for clinical application . over the last decades , we have advanced in the study of newly developed articular cartilage substitutes . we have achieved a good understanding of the morphology , chemistry , and physics of native articular cartilage , and we have made forward strides , namely in the use of cutting - edge printing technologies for mimicking articular cartilage tissue . however , the research world must reach a simple , accessible , and reasonably priced solution to the healing of articular cartilage defects ; otherwise , surgeons may adhere to the old - fashioned methods for healing their patients chondral defects .

orthopedic surgeons and researchers worldwide are continuously faced with the challenge of regenerating articular cartilage defects . however , until now , it has not been possible to completely mimic the biological and biochemical properties of articular cartilage using current research and development approaches . in this review , biomaterials previously used for articular cartilage repair research are addressed . furthermore , a brief discussion of the state of the art of current cell printing procedures mimicking native cartilage is offered in light of their use as future alternatives for cartilage tissue engineering . inkjet cell printing , controlled deposition cell printing tools , and laser cell printing are cutting - edge techniques in this context . the development of mimetic hydrogels with specific biological properties relevant to articular cartilage native tissue will support the development of improved , functional , and novel engineered tissue for clinical application .

1. Introduction 2. Native Articular Cartilage 3. Biomaterials and Cartilage Repair 3.1. Natural Materials 3.2. Synthetic Materials 4. Future Outlook: Applied Cell Printing for Articular Cartilage Repair 5. Discussion 6. Conclusions

PMC4460240

the two most frequently used traditional methods for taxonomic identification of algae beyond the genus level are by microscopic examination and genotyping [ 15 ] . microscopic examination methods require skill and experience as they involve a thorough knowledge of the thousands of possible variations in the shape and morphology of the algae as well as familiarity with the algae ecology and reproduction . to go beyond the genus level to species or substrains of a species can require a scanning electron microscope ( sem ) or other more complicated tests and techniques such as in the case of diatoms . even at this level , there may still be some ambiguity and discussion among the experts in the field of phycology . while giving excellent results , it requires skill and takes time and if hired out to a laboratory that specializes in sequencing , it can be costly . genotyping requires primers that can isolate and amplify a portion of the genome of the species of interest , typically the 16s rdna gene [ 810 ] for prokaryotic algae ( cyanobacteria or blue - green algae ) and the 18s rdna gene for eukaryotic algae . these genes are both highly conserved among algae but have regions of variability that can be used for species identification [ 2 , 1113 ] . the sequence of these regions can then be compared to known sequences and the most likely species matching sequence can be determined . while sequencing is the gold standard method for evaluation of sequence variants , alternative methods with more rapid analysis times and lower expense are also being evaluated for their potential . one such method is capillary electrophoresis single - strand conformational polymorphisms ( ce - sscp ) . sscp entails heat denaturing the double stranded dna until the hydrogen bonds holding the double helix backbone together disassociate and the dna becomes two single strands in a dilute solution . these single strands of dna are snap - cooled on ice causing the single strand to fold back on itself in ways that are dictated by its unique nucleotide sequence into something called a conformer . under conditions of low temperature and nondenaturing polymer the single - stranded dna conformers will migrate according to their shape and will have a distinct mobility . sscp performed on a capillary electrophoresis instrument gives the advantages of automated loading of samples and the use of laser - induced fluorescence detection , which allows for sensitive detection at low concentrations and repeatable results . ce - sscp has been used to successfully differentiate between mutated and nonmutated genes [ 9 , 1618 ] and also to identify and differentiate bacterial species from such diverse samples such as blood , cheese , and soil [ 14 , 1921 ] . in addition , herrera - suplveda used ce - sscp to monitor activities of harmful algal blooms of the baja california sur coastal waters and noted significant advantages of this method over having to keep on hand an expert taxonomist . in this research capillary electrophoresis single - strand conformational polymorphism ( ce - sscp ) was explored to identify different algal species . six pure species level cultures and two genus level cultures were obtained from carolina biological supply company ( burlington , nc , usa ) . the eight different cultures represented three different types of algae including green algae , brown algae , and cyanobacteria . table 1 gives a list of the various types of algal species used for the fingerprint database . dna was extracted using either the powerplant pro dna isolation kit from mo bio laboratories ( carlsbad , ca , usa ) or the dneasy plant mini kit from qiagen ( germantown , md , usa ) . the powerplant and the dneasy plant mini kit protocols were followed to obtain clean dna samples for pcr . the pcr reagents 5x gotaq flexi buffer , 25 mm mgcl2 , 10 mm dntp , and taq polymerase were obtained from promega ( madison , wi , usa ) and all of the pcr primers used were obtained from invitrogen ( carlsbad , ca , usa ) . forward ( sense ) primers were fluorescently labeled with fam ( absorption 494 nm , emission 522 nm ) and reverse ( antisense ) primers were fluorescently labeled with hex ( absorption 535 nm , emission 553 nm ) to enhance the ease of identification between the forward and reverse single - strand peaks in the electropherograms . once the primers were obtained , they were diluted to a stock concentration of 100 m and this solution was used to make a working solution of 20 m for the pcr reactions . after preparing the pcr mixture , it was placed in an apollo atc401 thermocycler ( ramsey , mn , usa ) . a fragment of either the 16s or 18s gene variable regions was amplified using the primer pairs shown in table 2 . the appropriate fluorescently labeled primer pairs were used in a pcr reaction mixture ( 20 l ) consisting of 14 ng of dna , 1x gotaq flexi buffer , 0.2 mm dntp , 20 m primer ( forward and reverse ) , 2.0 mm mgcl2 , and 2.5 u of gotaq polymerase ( promega corp . , madison , wi , usa ) . after gently pipetting to mix and spinning the mixture for ~15 seconds to remove bubbles , the pcr mixture was subjected to the following amplification cycles on an apollo atc401 thermocycler : 95c for 25 minutes , 3035 cycles each of 95c for 3045 seconds , 5c below the melting temperature of the primer with the lowest temperature for 3045 seconds , and 72c for 4560 seconds , followed by one cycle of final extension step at 72c for 520 minutes , with temperature , time , and cycle ranges based on an optimization of the program and the melting temperatures of the different primer pairs . amplified dna fragments were purified using the qiaquick - spin pcr purification kit ( qiagen inc . , quantification of purified dna was determined with a nanodrop 1000 spectrophotometer ( thermo scientific , wilmington , de , usa ) based on the absorbance of dna at 260 nm . the purified samples were used as a stock solution for preparing sscp samples and were stored at 20c . the fluorescently labeled purified dna stock solutions were diluted to 1 ng/l with 10 mm tris - hcl ten l of the diluted , purified dna was loaded per well into 4 wells of a 96-well plate followed by a denaturing step at 95c for 3 minutes on the atc 401 thermocycler . the samples were immediately snap - cooled on ice for 3 minutes before loading onto the abi 3130 genetic analyzer ( applied biosystems , foster city , ca , usa ) which has a four - capillary array . the capillaries were 36 cm in length with an inner diameter of 50 m . the capillaries were loaded with the 3.5% pdma polymer for 2 to 3 min and preelectrophoresis was performed for 3 min at 417 v / cm . the dna samples were injected at 333 v / cm for 15 seconds and separated at 417 v / cm ( 15 kv ) with an associated current of 37.5 ma at 25c . the abi 3130 utilizes lif detection with a 488 nm showa laser ( 25 mw , 7.5 amps ) and has an electrokinetic injection method . polydimethylacrylamide ( pdma ) polymer was synthesized using the method of albarghouthi et al . except with a decrease in the nitrogen bubbling time ( from 16 hours to 4 hours ) . after completion of the reaction , the polymer was purified by dialysis in deionized water using spectra - por cellulose ester dialysis membranes ( spectrum , gardena , ca , usa ) , having a molecular mass cutoff of 1000 da . after dialysis was complete , the polymer was frozen at 80c for 48 hours and then recovered by lyophilization ( labconco , kansas city , ks , usa ) . dry polymers were dissolved in the running buffer which was 1x tbe buffer + 10% glycerol ( 89 mm tris - base , 89 mm boric acid , 2 mm edta , and 10% glycerol ) to obtain polymer concentrations of 3.5% ( w / v ) . for each type of algae ( prokaryotic or eukaryotic ) , two different variable regions were analyzed by ce - sscp . for the prokaryotic algae ( cyanobacteria ) , variable regions 2 and 3 of the 16s rdna gene were analyzed . figure 1 shows the electropherograms of the sscp products of variable region 2 of the 16s rdna gene of two prokaryotic algae species anabaena inaequalis ( a. inaequalis ) and nostoc muscorum ( n. muscorum ) . since multiple peaks were detected over a significant range of migrations times , the electropherograms were split into two different graphs . it should be noted while migration times can vary slightly from run to run due to the injection of fresh polymer , for a set of runs , the rsd value was 0.39% . the peak patterns in figure 1(a ) show a marked difference between the two species . there are three very strong forward ( blue ) and two reverse ( red ) peaks between the 12- to 15-minute migration time for n. muscorum , while a. inaequalis had only one very small forward and reverse peak during this same migration time . figure 1(b ) is a continuation of this graph and shows the later migrating single - stranded dna conformers . n. muscorum generated ten small forward peaks and five small reverse peaks , while a. inaequalis had eight larger forward peaks and three reverse peaks . both n. muscorum and a. inaequalis demonstrated multiple peaks which were often in clusters . in an ideal ce - sscp electropherogram , there would be a single peak for each species . multiple peaks that are very close together , as seen in figure 1(b ) especially , could indicate that the pure culture sample was actually multiple subspecies with slight variations in the sequence . alternative , multiple peaks have been previously detected for pure dna samples where the sequences allowed for multiple highly favorable conformations to be formed during the snap - cooling process . the same amount of primer was used in each reaction , so the amplification efficiencies of the different algae dna templates may have resulted in different electropherogram peak intensities . there is also the possibility of multiple and/or different copies of the same 16s rdna gene in some species . this could explain the differences in intensities and/or the presence of multiple peaks in the algae samples . the presence of multiple peaks is not a result of the simultaneous amplification using two fluorescently labeled primers . the electropherograms produced from amplifications with one labelled and one nonlabelled primer were identical to those produced from each strand in the dual labeled samples . the ce - sscp electropherograms for the second 16s rdna gene variable region examined ( variable region 3 ) are shown in figure 2 . figure 2(a ) shows the early migrating peaks ( between 10 to 20 min ) ; there are two very small forward peaks and one reverse peak for n. muscorum while a. inaequalis has no discernable peaks . in figure 2(b ) , n. muscorum has four larger forward peaks and five reverse peaks . the reverse strands of both n. muscorum and a. inaequalis are at approximately the same migration time , but n. muscorum has a small peak at the 22-minute mark . these two peaks at 21.5 minutes look very similar to the same peaks for n. muscorum . however , the first set of peaks for a. inaequalis at the 20.8-minute time has a difference in the migration time giving a slightly different pattern between the two species . patterns produced by these two cyanobacteria are extremely stable and the differences between them are repeated in all of the duplicated ce - sscp runs ( n 4 ) . therefore , these fingerprint differences clearly demonstrate how the electropherogram pattern could be used to differentiate between these two species . however , while the distinct pattern of peaks was very repeatable , the multiple peaks can create difficulties involved in interpreting these patterns as they can mask separate peaks and make the pattern difficult to read . two different regions of each gene were examined to ensure verification of separate patterns for each species . for these two prokaryotic algae , the 16s rdna gene variable region 2 provides the clearest differences in ce - sscp patterns between the two species , although both variable regions could be used to differentiate them . after comparing two species of cyanobacteria using two variable regions of the 16s rdna gene the eukaryotic algae are of a higher cell complexity and have an 18s rdna gene in their nucleus . therefore the dna sample is from the 18s rdna gene variable regions 7 and 9 , respectively . figure 3 provides a comparison of six species shown in the same graph to highlight the differences between both similar and dissimilar species of algae and how ce - sscp can be used to distinguish between them . four of these eukaryotic species belong to the phylum chlorophyta and two belong to the class chlorophyceae which is one of the classes of green algae , while one is in the phylum charophyta ( a conjugating algae ) , and one is in the phylum ochrophyta ( a brown algae ) . as with the cyanobacterium , there are distinct differences in the patterns between the algal species . in figure 3(a ) , the brown algae ectocarpus sp . shows multiple small peaks immediately followed by a single large peak at 13 minutes and multiple smaller peaks at 13.8 minutes . mougeotia sp . has a small peak at 12.5 minutes followed by multiple larger peaks at 13.5 minutes , while chlorella vulgaris ( c. vulgaris ) has a single small forward peak just before the 14-minute migration time , and ulothrix fimbriata ( u. fimbriata ) has only a very small peak at 15.5-minute migration time . scenedesmus quadricauda ( s. quadricauda ) has a small peak at 13 minutes and oedogonium foveolatum ( o. foveolatum ) has multiple very small peaks from 13 to 15 minutes . interestingly , the early migrating peaks are almost all for the forward strand of dna . in figure 3(b ) both mougeotia sp . and c. vulgaris have a large forward strand peak very close to the 23-minute migration point , as does the brown algae ectocarpus sp . , while u. fimbriata has two smaller peaks at 21.5 and 24 minutes . s. quadricauda has two slightly larger forward peaks at 22 and 23 minutes , and o. foveolatum has two small forward peaks at 20 and 21.5 and a larger forward peak at 24 minutes . in the reverse dna strand , o. foveolatum , u. fimbriata , c. vulgaris , and mougeotia sp . all have a similar peak around 26 minutes , while s. quadricauda and ectocarpus sp . the forward and reverse strands of the dna give a pattern that is repeatable but in some cases may not differentiate enough between very similar species . this is the reasoning behind using at least two different variable regions ; if one region can not differentiate decisively between the patterns a second region may be examined . in figure 4 , a different variable region ( variable region 9 ) was explored for these same six algae . in figure 4(a ) , ectocarpus sp . has a reverse peak at 13.5 minutes , and c. vulgaris and o. foveolatum have small reverse peaks around 12 minutes . there are no early migrating peaks for s. quadricauda or u. fimbriata . in figure 4(b ) , ectocarpus sp . c. vulgaris has a large reverse peak at 18.5 minutes , while u. fimbriata has small multiple peaks at just past 19 minutes . s. quadricauda has three small reverse peaks just before 18 minutes , and o. foveolatum has two larger peaks at 19 minutes . c. vulgaris has a larger peak at 18.5 minutes followed closely by a triple peak pattern ( somewhat obscured by the reverse peak overlaid on top of them ) at just before the 19 minutes . u. fimbriata has small multiple peaks just after the 18-minute mark and s. quadricauda has four small forward peaks through 17 to 18 , while o. foveolatum has a larger triple peak around 18 minutes . the advantages of ce - sscp are the rapid analysis and clear differences in peak patterns . these changes in pattern could be used to detect changes in an algal population over time , effectively giving a snapshot of changes in either a closed or open system . the pattern is highly repeatable ; in other words , the nucleotide sequence determines the shape and therefore the mobility of the conformers and this shape does in fact give a specific pattern that can be considered a fingerprint . using the two different variable regions , we were able to show differences in the peak patterns of both prokaryotes and eukaryotes . even very similar algal species had distinct , repeatable patterns . while the pattern or fingerprint is unique to the algal dna sequence , these peaks could indicate that the pure cultures are not genetically pure or more likely represent different conformers from the same sequence . ce - sscp is a relatively fast , easy , and inexpensive method for determining changes in a small section of dna such as a variable region of the 16s or 18s rdna gene of the small subunit of the ribosome . in this work , ce - sscp demonstrated the ability to differentiate between two different blue - green algae , five different green algae , and one brown algal species . the fingerprint patterns obtained through ce - sscp of two variable regions from the 16s rdna gene and two variable regions from the 18s rdna gene were highly repeatable and unique to each species . this technique shows significant potential for being differentiating algae and could be used to monitor an algal system over time .

capillary electrophoresis single - strand conformational polymorphism ( ce - sscp ) was explored as a fast and inexpensive method to differentiate both prokaryotic ( blue - green ) and eukaryotic ( green and brown ) algae . a selection of two blue - green algae ( nostoc muscorum and anabaena inaequalis ) , five green algae ( chlorella vulgaris , oedogonium foveolatum , mougeotia sp . , scenedesmus quadricauda , and ulothrix fimbriata ) , and one brown algae ( ectocarpus sp . ) were examined and ce - sscp electropherogram fingerprints were compared to each other for two variable regions of either the 16s or 18s rdna gene . the electropherogram patterns were remarkably stable and consistent for each particular species . the patterns were unique to each species , although some common features were observed between the different types of algae . ce - sscp could be a useful method for monitoring changes in an algae species over time as potential shifts in species occurred .

1. Introduction 2. Materials and Methods 3. Results 4. Conclusions

PMC4660523

since the 19 century , different types of lid margin rotation have been used to correct the upper eyelid cicatricial entropion.1 anterior approach procedures are typically performed with a through - and - through incision placed 4 mm from the lash line . variations of this procedure have been described by green , pannas , hotz12 and recently by wies3 and ballen.4 the world health organization has renamed the wies / ballen procedure bilamellar lid margin rotation and recommended it as standard modality to correct trachomatous cicatricial entropion.5 trabut6 in 1949 was , likely , the first to use a different approach to manage cicatricial entropion . a conjunctival incision is used to divide the tarsal plate into two fragments . after creating a plane between the orbicularis and both tarsal fragments , the distal tarsus is advanced over the marginal tarsal fragment with sutures exiting on the lash line . this simple procedure creates a downward vector on the marginal portion of the tarsus , which rotates the margin upward . neither of these two basic lid margin rotation procedures addresses other common lid problems such as dermatochalasis , retraction , or ptosis . in this study , we use a lid crease incision to combine both basic mechanisms of lid margin rotation and if necessary , to concomitantly correct a variety of lid abnormalities . if desired , a standard blepharoplasty skin or skin + muscle resection can be outlined the lid is then infiltrated with a local anesthetic solution and a 4 - 0 silk traction suture is inserted through the tarsal edge of the lid margin . a pretarsal skin muscle flap is raised exposing the whole tarsal plate to the level of the lash roots [ figure 1a ] . the lid is everted over a cotton - tipped applicator with care to place the applicator under and not over the skin - muscle flap . using a number 15 bard - parker scalpel blade and westcott scissors , a curved incision parallel to the lid margin is performed through the full thickness of the tarsus 3 mm posteriorly to the margin [ figure 1b ] . three double - armed 6 - 0 polyglactin ( vicryl ) sutures are then passed through half thickness of the central , medial , and lateral aspects of the distal cut edge of tarsus [ figure 1c ] and attached to the orbicularis near the lash line [ figure 1d ] . the noncentral sutures should be placed in a radial fashion similar to corneal transplant stitches . as the sutures are tied , the distal portion of the tarsus is advanced over the marginal tarsus [ figure 1e ] , and the marginal orbicularis is pushed backward rotating outward both lamellae of the lid margin [ figure 1f ] . if necessary , the levator aponeurosis can be recessed or advanced to correct any retraction or ptosis . main surgical steps of the lid crease approach to upper lid margin rotation : ( a ) using a standard blepharoplasty incision a pretarsal skin - muscle flap is raised exposing the whole tarsal flap until the lash roots are visualized , ( b ) tarsotomy is performed with a conjunctival incision 34 mm from the lid margin , ( c ) a 6 - 0 vycril suture is inserted first through the distal segment of the tarsus , ( d ) the suture is passed on the orbicularis muscle close to the lash roots , ( e ) as the suture is tied the distal tarsal fragments is slightly advanced over the marginal tarsus and traction is exerted on the orbicularis , ( f ) final aspect of the lid rotation we have used the lid crease approach to manage trachomatous cicatricial entropion at the king khaled eye specialist hospital on a regular basis . we have treated 60 upper lids of 40 patients ( 23 females and 17 males ) with a mean age of 70.9 13.01 years . the follow - up ranged from 1 to 12 months ( mean 3.0 months 2.71 ) . forty percent of the patients ( 24 lids ) had > 3 months of follow - up . trichiasis was corrected in all but one lid , with two inward lashes touching the cornea medially despite adequate margin position . this patient had cicatricial entropion , trichiasis , and a loose pretarsal anterior lamella with no clear lid crease preoperatively [ figure 2 ] . it should be noted that the lid crease approach is not new but has been largely overlooked . in a pioneering article sadiq and pai7 documented their approach of combining the traditional lid margin rotation with a blepharoplasty incision . we believe that the technique we have described has several advantages compared to the classic wies / ballen surgery.34 first , the anterior lamella incision is not on the same level as the tarsotomy . with this approach , the vascularization of the marginal portion of the orbicularis is not compromised , and the chances of ischemia and necrosis of the margin are significantly reduced . second , as the tarsal plate is completely exposed , it is easy for the surgeon to distribute the forces of the sutures arranging the lateral and medial stitches radially . it is well known that one of the most common complications of lid margin rotation is contour abnormality.8 as recommended in the manual of the so - called bilamellar lid rotation ( wies / ballen surgery ) published by the world health organization , the anterior lamella incision follows a straight line.5 in this setting , the external sutures tends to be parallel and not radially distorting the central portion of the margin . as the marginal orbicularis is completely dissected up to the lash follicles and the distal tarsal fragment is advanced over the marginal , a downward vector moves the edge of the marginal tarsus downward , which rotates the margin upward . finally , incising the anterior lamella on the lid crease position allows the surgeon to address a variety of common lid problems such as aponeurotic ptosis , lid retraction , laxity of the pretarsal lamella , dermatochalasis , and so on .

lid margin rotational procedures have been used to correct cicatricial trachomatous entropion since the 19th century . there are two basic types of surgeries used for lid margin rotation . the first type is based on through - and - through approach combining tarsotomy and the use of sutures on the anterior lamella . the second type of surgery was suggested by trabut , who proposed a tarsal advancement by posterior approach . we demonstrate that using a lid crease incision combines the basic mechanisms of the anterior and posterior approaches and in addition , addresses a variety of lid problems commonly found in the aged population with cicatricial entropion . after tarsal plate exposure , a tarsotomy through conjunctiva is performed as described by trabut . then , instead of using external sutures secured by bolsters , internal absorbable sutures can be used to simultaneously advance the distal tarsal fragment and exert strong tension on the marginal orbicularis muscle . sixty lids of 40 patients underwent surgery with a lid crease incision . the follow - up ranged from 1 to 12 months ( mean 3.0 months 2.71 ) . forty percent of the patients ( 24 lids ) had more than 3 months of follow - up . adequate margin rotation was achieved in all lids but one that showed a medial eyelash touching the cornea .

INTRODUCTION SURGICAL TECHNIQUE RESULTS DISCUSSION Financial support and sponsorship Conflicts of interest

PMC5410916

the aim of radiation therapy is the application of ionizing radiation to treat or control the growth of cancer cells forming a tumor ( 1 ) . in comparison to external beam radiation therapy ( ebrt ) , brachytherapy allows the exposure to radiation to be concentrated on certain organs , but this comes at the expense of a more invasive treatment ( 2 ) . there are problems associated with this method , like the chance of radioactive seed escaping the treatment zone and affecting the neighboring tissues ( 3 ) . another challenge concerning this method is that dose distribution on the area of interest is quite ambiguous ( 4 ) , and dose rate and depth - dose profile of each radioisotope source are constant ( 5 ) , also when using radionuclide - based source , there is no way to turn off radiation production whether it is needed or not ( 6 ) . in the 1980s , collaboration between alan sliski and his team at photoelectron and mit resulted in the development of an electronic brachytherapy system ( ebs ) ( 7 ) , which managed to address some of the above problems by using an alternative point source radiation treatment approach ( 8) . the advantages of ebs in comparison to radionuclide - based brachytherapy include : unlike radionuclide - based sources , in ebs radiation is generated by an x - ray tube ( 9 ) ; the therapy dose is adjustable as it can be altered by adjusting the current and voltage of x - ray tube ( 10 ) ; the use of lower energies in ebs means increased radiobiological effectiveness and that surrounding environment can be better protected by less extensive shielding ( 11 ) ; the dose distributions can be controlled more accurately with the tube s kvp adjustment ( 12 ) ; the use of ebs allows dose distribution to be targeted more accurately and the dose fall - off rate to be better controlled , and this can prevent unnecessary delivery of dose to marginal healthy tissues ( 13 ) ; in ebs , the tube potential is adjustable , which gives better control over radiation quality and leads to a better dose conformity ( 2 ) . the zeiss intrabeam radiotherapy system ( irs ) is a 50 kv x - ray unit designed specifically for interstitial brachytherapy ( 14 ) . however , considering the problems associated with the use of brachytherapy isotopes , including iridium-192 used for treatment of breast cancer via mammosite method , in 2005 , xoft company introduced the miniature x - ray tube as a response to these issues . axxent electronic brachytherapy system delivers focused , conformal doses of radiation at high dose rates using a novel miniature x - ray source operating at 40 , 45 or 50 kvp ( 15 ) . characteristics of this system make it particularly useful for treatment of cancer developed in the breast and the inner surface of a body cavity like the bed of an excised tumor ( 16 ) . in 2013 , all three , the zeiss intrabeam system ( carl zeiss , oberkochen , germany ) , the axxent system ( xoft , fremont , ca ) and the esteya electronic brachytherapy system ( elekta ab - nucletron , stockholm , sweden ) mentioned above , use particular photon emitting sources , for which task group-186 of the american association of physicists in medicine ( aapm ) have developed a number of guidelines ( 17 ) . the aapm has also provided exact definitions for the miniature electronic brachytherapy x - ray sources ( mebxs ) emitting a spectrum of bremsstrahlung and characteristic x - ray photons ( 18 ) . the work of task group 152 of aapm has resulted in a model regulation for electronic brachytherapy , where the term electronic brachytherapy has been defined as a method of radiation therapy using electrically generated x - rays to deliver a radiation dose at a distance of up to a few centimeters by intracavitary , intraluminal , or interstitial application , or by applications with the source in contact with the body surface or very close to the body surface ( 19 ) . through these developments , electronic brachytherapy system has emerged as one of the most rapidly disseminating medical devices in the world . however , the core component of these systems is still the miniature tube that allows the low energy x - ray to be produced at high dose rates ( 20 ) . the x - ray spectrum produced by this tube has a continuous distribution ( bremsstrahlung effect ) with a line spectrum characteristic ( 21 ) , and has an extensive use in medical physics ( 22 ) . there are several concerns regarding the electronic brachytherapy , and one of these concerns is the manner in which the x - ray dose is distributed . this issues a major concern because for the treatment to be efficient , the dose should be delivered uniformly into the cavity or area of interest . as a result , the geometry and the structure of the target used in the x - ray tube of ebs plays a central role in the delivered dose distribution ( 9 ) . thus , the aim of the study was to compare the optimal target thickness , the energy spectrum at different positions , presence of beryllium window , and the relative dose of hemispherical and hemispherical - conical sources using the functions of mcnpx code ( 2.6.0 ) . the utility of monte carlo ( mc ) simulation for the study of physics of nuclear medicine , radiology , and radiation therapy is a dominantly accepted fact ( 23 ) . the ability of monte carlo codes to model the full geometric detail of real world systems has also been proven ( 24 ) . the transport of neutron , photon and electron though a three - dimensional medium with arbitrary material and geometry , can be simulated with a general - purpose monte carlo n - particle code ( 25 ) . one tool for such simulation is the mcnpx code , which is a combination of mcnp4c code and the code developed by lahet ( los alamos high - energy transport ) for the transport and interaction of nucleons , pions , muons and light ions in complex geometries . all desirable features of mcnp4c and mcnp5 have been incorporated into the mcnpx code , but in addition , this code covers the transport of 34 new types of particles ( protons , light ions , etc . ) , and so expands the particle - energy range that can be simulated ( 26 ) . this general - purpose radiation transport code can cover continuous - energy transport up to one tev , and provides several sources and tally options . in addition , mcnpx utilize interactive graphics , and can be run with both sequential and parallel processors ( 27 ) . the version 2.6 of mcnpx requires even less memory than the previous versions , and this allows it to simulate larger voxel phantoms . however , voxel - based phantoms of large matrix sizes still can not be simulated by mcnp5 ( 28 ) . in this study , the code mcnpx ( version 2.6.0 ) was used to simulate the target thickness , the generated x - ray photon spectra and the dose delivered through 2 targets of different material . a file containing cell card , surface card , and data card was written based on the geometry of hemispherical and hemispherical - conical sources , and was then used for simulation with mcnpx ( 2.6.0 ) . simulations were based on cylinders with internal radius ( the target ) , middle radius ( the output window ) , and outer radius ( the water cooling sheath ) of 1.1 , 1.6 , and 2.7 mm , respectively . densities of gold , tungsten , beryllium , and water were assumed to be 19.32 , 19.3 , 1.848 , 0.998 , g / cm respectively ( 29 ) . considering the necessity of bremsstrahlung production for extracting an output , all stages of coupled electron / photon transport was simulated with the mode : p e card . this study employed the energy cut - off approach , which is a simple variance reduction method , where any particle whose energy is lower than a threshold will be disregarded . simulating these cut - off particles can give more ( yet unnecessary ) information , but eliminating them can improve computation time without significant consequences ( 28 ) . in all stages of simulations , the cut - off energy was assumed to be 1 kev for both electrons and photons . in the simulations , the length of the source ( 15 mm ) was modeled along the z - axis with its bottom ( start ) coinciding with the origin . the source was considered to be symmetric about the z - axis to facilitate the calculation dose delivery with the mc code . the reason behind the selection of this configuration was the presence of its - style in mcnp . its - style employs the transport parameters that belong to the energy step of the nearest interruption , and leads to better results than the mcnp - style ( 30 ) . as a result library cards plib = 04p and elib = 03e were also used in the input file in accordance with the mcnpx ( 2.6.0 ) manual . the materials such as tungsten , molybdenum , and rhodium with different atomic numbers and densities , can be good candidates for the x - rays targets . currently , the intrabeam system ( hemispherical source ) uses a gold target ( z=79 ) because of good heat conductance , but the reason behind choosing tungsten for hemispherical - conical is that it has a high atomic number and a high melting point ( 3422c ) ( 31 ) , which ensure not only an efficient x - ray production but also a good tolerance for high power deposition ( 32 ) . it should also be noted that x - ray production efficiency is correlated with the square of the atomic number of source ( z ) , and so the atomic numbers of tungsten ( z=74 ) and gold ( z=79 ) make them a good choice for this purpose ( 33 ) . tungsten also has good strength and ductility and a low rate of vaporization in vacuum ( 34 ) . the miniature sources simulated with monte carlo code mcnpx ( 2.6.0 ) and their specific input files are shown in figure 1 . in this part of the study , as figure 2 ( a , b , and c ) shows , the targets of different thicknesses ( without the output window and water cooling sheath ) and with sizes specified in the input file , were tested for energy levels of 40 , 45 and 50 kev using mcnpx visual editor software with electron beam particle display . the electron penetration ( into the thickness of the target ) and particle track plots were used to determine a rough range for the desirable thickness , and thus provide a ground for quantitative and more accurate determination of size of ot using the code . the process of determining the optimal angle of the hemispherical - conical source was started by setting up the interface of two cones with two hemispheres . the outer hemisphere had a fixed radius of 0.4 mm and the other one had a variable radius . the outer cone was cut by a cylinder with a radius of 1.1 mm and the other one was cut by a thinner cylinder with variable radius . the bottom end of the latter cylinder was cut by the plane that was the measure of distance between the target and the electron source . once this slightly complex structure was set up , the desired solid disc - shaped electron source was applied using the monte carlo code , and the optimal angle of the hemispherical - conical source was determined to be in the range of 33 to 34 degrees depending on the used energy level . note that any change in any of the described variables alters the apex of the cone , and changes the obtained angle . the optimized angles obtained for the hemispherical - conical and the rough approximations obtained for tt were used along with the 0.8 micron disk source ( 15 ) , to simulate two sources with 30 thicknesses ranging from 0.1 microns to 3 microns ( increasing in 0.1 steps ) , and with energy levels of 40 , 45 , and 50 kev ( increasing in 5 kev steps ) by running 180 instances of 300 minute programs . programs were run in groups of 6 on a computer with intel ( r ) core i 7 - 4790k [email protected] , and 16.0 gb ram . for each target , the surface with which particles ( namely electrons and photons ) were colliding was named the input surface and the surface in which bremsstrahlung was being produced was named the f2 were considered for electrons colliding with the input surface , and the tallies * f1 and * f2 were considered for electrons in the output surface . the distance between electron source and the surface that cuts the lower end of the innermost cylinder was 10 mm . in all runs , the error of the process was less than 2% and in some tallies it reached down to a hundredth of one percent . in mcnpx , physical parameters expressed by the cards phys : e and phys : p can be used to improve the efficiency of electron / photon transport computations . this task is achieved by simplifying the less important physical aspects of the model ( 35 ) . however , emax was assumed to be 1 kev higher than what is defined in the electron source profile and specified in the data card . for example , when the energy of electron source was 50 kev , the emax of the photon - electron phys card was assumed to be 51 kev to avoid calculation errors . the ring detector tally has been defined as a point detector tally where the location of the point detector is variable but can be sampled from some location on the ring . the use of ring detector leads to improved performance of point detector for those problems in which geometrical structures have rotational symmetry with respect to one axis ( 36 ) . considering the good features of the ring tally f5 and symmetry of miniature sources about z - axis , this tally was used for the calculation of energy spectra . the other issue that needed to be considered was the material of 0.5 mm thick output window ( substrate ) . because of the lack of accurate knowledge about the material of this component in the miniature sources of xoft company , this component was assumed to be made of beryllium . this material was selected because of features such as good strength to weight ratio , good thermal strength , transparency to shorter wavelengths , good availability ( 37 ) , low atomic number and its ability to let through even the lower energy x - rays without absorption ( 33 ) , and also the fact that the zeiss company usually uses beryllium as the substrate or heat buffer . to calculate the spectra of sources , one time - just the target alone was used ( figure 2 , b ) , and another time - the target , beryllium window , and water cooling sheath again ( figure 1 ) were used . this means that spectra were calculated both inside and outside the target and beyond and within the water cooling sheath . figures 3 and 4 show the obtained spectra . in the course of calculating the spectra , the energy peaks were determined with the help of bin card and with a step of 0.1 kev . also , the miniature sources were assumed to be in a sphere of air with a radius of 20 cm . in this part of the work , tallies to be used along with a mesh tally to calculate the absorbed dose were selected . in mcnp ( 2.6.0 ) , tallies commonly used for dose calculation are f6 , * f4 , and * f8 . the use of each of these tallies for dose calculation has some advantages and drawbacks . however , considering the negligible range of secondary electrons in solid or liquid media at photon energies lower than 200 kev ( 38 ) , and the presence of negligible radiative losses ( 39 ) , and since total kerma has about the same magnitude as the collision kerma ( 38 ) , the absorbed dose can be approximated by collision kerma ( 39 ) and therefore f6 tally was recognized as the best option for this calculation . this tally is known as the track length estimate of energy deposition , or as the heating or energy deposition tally , and is faster than other tallies of dose calculation . considering the symmetry of source about the z - axis , the dose on this axis was calculated using cylindrical cells . this dose was calculated in the presence of beryllium output window and also with the use of water instead of beryllium . given the sensitivity of the work , a benchmark with different thicknesses and radii was conducted to assess the setting . in the end , 10 consecutive cylindrical cells with identical height and radius were used to calculate the axial dose . the average particle records run with f6 tally to calculate the axial dose in both presence and absence of beryllium and all three energy levels for gold and tungsten targets were , respectively , 16310 and 17610 . the radial dose too was calculated with the assumption of symmetry of source around the z - axis . radial ( transverse ) dose was calculated using 19 concentric cylindrical cells with identical thickness , but with radii of 015 mm , 1530 mm , and so on until the last cell . in this part of work , the radial dose at all energy levels was calculated using average particle records of 30110 and 28210 respectively for the hemispherical - conical and the hemispherical source . here , the running time of the program tasked with calculating the radial dose of each source was 1000 minutes . it should be mentioned that calculation of axial and radial dose was carried out for a sphere of water with a radius of 20 cm . also , to prevent overlap between the cells , they were assumed to be a miniscule distance apart . in this study , each 6 programs were run concurrently by a computer with the mentioned specifications , and the average statistical error of these programs along both axes ranged from hundredth of a percent to 2.7% . one of the obviously important features of mcnpx ( 2.6.0 ) is the presence of various types of mesh tallies with specific applications . the type-1 mesh tally can be used to calculate and display quantities such as tracks , flux , dose , and pedep ( photon energy deposition ) in the desired part of the structure . in the mesh tally method , the cards tmesh and endmd determine the start and end of the mesh tally frame and the cards cora , corb , and corc determine the mesh size . the mesh can be defined with cartesian , cylindrical or spherical coordinates based on existing structure . to calculate the dose via a mesh tally method , after preparing the input file , the type-1 mesh tally card was inserted at the end of this input file to calculate the pedep ( equivalent to f6 tally ) . considering the symmetry of source about the z - axis , the radial dose was calculated using the cylindrical coordinates in the form of cmesh1 : p pedep , cora1 0 4i 3 , corb1 2.25 5i 2.55 , corc1 0.01 11i 360 ( mesh tally frame is independent of the problem geometry ) . the execution of mcnpx code ( 2.6.0 ) created a binary mdata file , which was then converted with the gridconv to a format that could be read with tecplot . tecplot software was then used to convert the data into a mesh plot with calculated dose values presented over the geometry the utility of monte carlo ( mc ) simulation for the study of physics of nuclear medicine , radiology , and radiation therapy is a dominantly accepted fact ( 23 ) . the ability of monte carlo codes to model the full geometric detail of real world systems has also been proven ( 24 ) . the transport of neutron , photon and electron though a three - dimensional medium with arbitrary material and geometry , can be simulated with a general - purpose monte carlo n - particle code ( 25 ) . one tool for such simulation is the mcnpx code , which is a combination of mcnp4c code and the code developed by lahet ( los alamos high - energy transport ) for the transport and interaction of nucleons , pions , muons and light ions in complex geometries . all desirable features of mcnp4c and mcnp5 have been incorporated into the mcnpx code , but in addition , this code covers the transport of 34 new types of particles ( protons , light ions , etc . ) , and so expands the particle - energy range that can be simulated ( 26 ) . this general - purpose radiation transport code can cover continuous - energy transport up to one tev , and provides several sources and tally options . in addition , mcnpx utilize interactive graphics , and can be run with both sequential and parallel processors ( 27 ) . the version 2.6 of mcnpx requires even less memory than the previous versions , and this allows it to simulate larger voxel phantoms . however , voxel - based phantoms of large matrix sizes still can not be simulated by mcnp5 ( 28 ) . in this study , the code mcnpx ( version 2.6.0 ) was used to simulate the target thickness , the generated x - ray photon spectra and the dose delivered through 2 targets of different material . a file containing cell card , surface card , and data card was written based on the geometry of hemispherical and hemispherical - conical sources , and was then used for simulation with mcnpx ( 2.6.0 ) . simulations were based on cylinders with internal radius ( the target ) , middle radius ( the output window ) , and outer radius ( the water cooling sheath ) of 1.1 , 1.6 , and 2.7 mm , respectively . densities of gold , tungsten , beryllium , and water were assumed to be 19.32 , 19.3 , 1.848 , 0.998 , g / cm respectively ( 29 ) . considering the necessity of bremsstrahlung production for extracting an output , all stages of coupled electron / photon transport was simulated with the mode : p e card . this study employed the energy cut - off approach , which is a simple variance reduction method , where any particle whose energy is lower than a threshold will be disregarded . simulating these cut - off particles can give more ( yet unnecessary ) information , but eliminating them can improve computation time without significant consequences ( 28 ) . in all stages of simulations , the cut - off energy was assumed to be 1 kev for both electrons and photons . in the simulations , the length of the source ( 15 mm ) was modeled along the z - axis with its bottom ( start ) coinciding with the origin . the source was considered to be symmetric about the z - axis to facilitate the calculation dose delivery with the mc code . the reason behind the selection of this configuration was the presence of its - style in mcnp . its - style employs the transport parameters that belong to the energy step of the nearest interruption , and leads to better results than the mcnp - style ( 30 ) . as a result library cards plib = 04p and elib = 03e were also used in the input file in accordance with the mcnpx ( 2.6.0 ) manual . the materials such as tungsten , molybdenum , and rhodium with different atomic numbers and densities , can be good candidates for the x - rays targets . currently , the intrabeam system ( hemispherical source ) uses a gold target ( z=79 ) because of good heat conductance , but the reason behind choosing tungsten for hemispherical - conical is that it has a high atomic number and a high melting point ( 3422c ) ( 31 ) , which ensure not only an efficient x - ray production but also a good tolerance for high power deposition ( 32 ) . it should also be noted that x - ray production efficiency is correlated with the square of the atomic number of source ( z ) , and so the atomic numbers of tungsten ( z=74 ) and gold ( z=79 ) make them a good choice for this purpose ( 33 ) . tungsten also has good strength and ductility and a low rate of vaporization in vacuum ( 34 ) . the miniature sources simulated with monte carlo code mcnpx ( 2.6.0 ) and their specific input files are shown in figure 1 . in this part of the study , as figure 2 ( a , b , and c ) shows , the targets of different thicknesses ( without the output window and water cooling sheath ) and with sizes specified in the input file , were tested for energy levels of 40 , 45 and 50 kev using mcnpx visual editor software with electron beam particle display . the electron penetration ( into the thickness of the target ) and particle track plots were used to determine a rough range for the desirable thickness , and thus provide a ground for quantitative and more accurate determination of size of ot using the code . the process of determining the optimal angle of the hemispherical - conical source was started by setting up the interface of two cones with two hemispheres . the outer hemisphere had a fixed radius of 0.4 mm and the other one had a variable radius . the outer cone was cut by a cylinder with a radius of 1.1 mm and the other one was cut by a thinner cylinder with variable radius . the bottom end of the latter cylinder was cut by the plane that was the measure of distance between the target and the electron source . once this slightly complex structure was set up , the desired solid disc - shaped electron source was applied using the monte carlo code , and the optimal angle of the hemispherical - conical source was determined to be in the range of 33 to 34 degrees depending on the used energy level . note that any change in any of the described variables alters the apex of the cone , and changes the obtained angle . the optimized angles obtained for the hemispherical - conical and the rough approximations obtained for tt were used along with the 0.8 micron disk source ( 15 ) , to simulate two sources with 30 thicknesses ranging from 0.1 microns to 3 microns ( increasing in 0.1 steps ) , and with energy levels of 40 , 45 , and 50 kev ( increasing in 5 kev steps ) by running 180 instances of 300 minute programs . programs were run in groups of 6 on a computer with intel ( r ) core i 7 - 4790k [email protected] , and 16.0 gb ram . for each target , the surface with which particles ( namely electrons and photons ) were colliding was named the input surface and the surface in which bremsstrahlung was being produced was named the output surface. the tallies f1 and f2 were considered for electrons colliding with the input surface , and the tallies * f1 and * f2 were considered for electrons in the output surface . the distance between electron source and the surface that cuts the lower end of the innermost cylinder was 10 mm . in all runs , the error of the process was less than 2% and in some tallies it reached down to a hundredth of one percent . in mcnpx , physical parameters expressed by the cards phys : e and phys : p can be used to improve the efficiency of electron / photon transport computations . this task is achieved by simplifying the less important physical aspects of the model ( 35 ) . however , emax was assumed to be 1 kev higher than what is defined in the electron source profile and specified in the data card . for example , when the energy of electron source was 50 kev , the emax of the photon - electron phys card was assumed to be 51 kev to avoid calculation errors . the ring detector tally has been defined as a point detector tally where the location of the point detector is variable but can be sampled from some location on the ring . the use of ring detector leads to improved performance of point detector for those problems in which geometrical structures have rotational symmetry with respect to one axis ( 36 ) . considering the good features of the ring tally f5 and symmetry of miniature sources about z - axis , this tally was used for the calculation of energy spectra . the other issue that needed to be considered was the material of 0.5 mm thick output window ( substrate ) . because of the lack of accurate knowledge about the material of this component in the miniature sources of xoft company , this component was assumed to be made of beryllium . this material was selected because of features such as good strength to weight ratio , good thermal strength , transparency to shorter wavelengths , good availability ( 37 ) , low atomic number and its ability to let through even the lower energy x - rays without absorption ( 33 ) , and also the fact that the zeiss company usually uses beryllium as the substrate or heat buffer . to calculate the spectra of sources , one time - just the target alone was used ( figure 2 , b ) , and another time - the target , beryllium window , and water cooling sheath again ( figure 1 ) were used . this means that spectra were calculated both inside and outside the target and beyond and within the water cooling sheath . figures 3 and 4 show the obtained spectra . in the course of calculating the spectra , the energy peaks were determined with the help of bin card and with a step of 0.1 kev . also , the miniature sources were assumed to be in a sphere of air with a radius of 20 cm . in this part of the work , tallies to be used along with a mesh tally to calculate the absorbed dose were selected . in mcnp ( 2.6.0 ) , tallies commonly used for dose calculation are f6 , * f4 , and * f8 . the use of each of these tallies for dose calculation has some advantages and drawbacks . however , considering the negligible range of secondary electrons in solid or liquid media at photon energies lower than 200 kev ( 38 ) , and the presence of negligible radiative losses ( 39 ) , and since total kerma has about the same magnitude as the collision kerma ( 38 ) , the absorbed dose can be approximated by collision kerma ( 39 ) and therefore f6 tally was recognized as the best option for this calculation . this tally is known as the track length estimate of energy deposition , or as the heating or energy deposition tally , and is faster than other tallies of dose calculation . considering the symmetry of source about the z - axis , the dose on this axis was calculated using cylindrical cells . this dose was calculated in the presence of beryllium output window and also with the use of water instead of beryllium . given the sensitivity of the work , a benchmark with different thicknesses and radii was conducted to assess the setting . in the end , 10 consecutive cylindrical cells with identical height and radius were used to calculate the axial dose . the average particle records run with f6 tally to calculate the axial dose in both presence and absence of beryllium and all three energy levels for gold and tungsten targets were , respectively , 16310 and 17610 . the radial dose too was calculated with the assumption of symmetry of source around the z - axis . radial ( transverse ) dose was calculated using 19 concentric cylindrical cells with identical thickness , but with radii of 015 mm , 1530 mm , and so on until the last cell . in this part of work , the radial dose at all energy levels was calculated using average particle records of 30110 and 28210 respectively for the hemispherical - conical and the hemispherical source . here , the running time of the program tasked with calculating the radial dose of each source was 1000 minutes . it should be mentioned that calculation of axial and radial dose was carried out for a sphere of water with a radius of 20 cm . also , to prevent overlap between the cells , they were assumed to be a miniscule distance apart . in this study , each 6 programs were run concurrently by a computer with the mentioned specifications , and the average statistical error of these programs along both axes ranged from hundredth of a percent to 2.7% . one of the obviously important features of mcnpx ( 2.6.0 ) is the presence of various types of mesh tallies with specific applications . the type-1 mesh tally can be used to calculate and display quantities such as tracks , flux , dose , and pedep ( photon energy deposition ) in the desired part of the structure . in the mesh tally method , the cards tmesh and endmd determine the start and end of the mesh tally frame and the cards cora , corb , and corc determine the mesh size . the mesh can be defined with cartesian , cylindrical or spherical coordinates based on existing structure . to calculate the dose via a mesh tally method , after preparing the input file , the type-1 mesh tally card was inserted at the end of this input file to calculate the pedep ( equivalent to f6 tally ) . considering the symmetry of source about the z - axis , the radial dose was calculated using the cylindrical coordinates in the form of cmesh1 : p pedep , cora1 0 4i 3 , corb1 2.25 5i 2.55 , corc1 0.01 11i 360 ( mesh tally frame is independent of the problem geometry ) . the execution of mcnpx code ( 2.6.0 ) created a binary mdata file , which was then converted with the gridconv to a format that could be read with tecplot . tecplot software was then used to convert the data into a mesh plot with calculated dose values presented over the geometry figure 5 shows graphs that by using them , the size of the optimum thicknesses of miniature sources was obtained for energies 40 , 45 , and 50 kg , values of 0.99 , 1.08 , and 1.34 for tungsten target , the values of 0.92 , 1.01 , and 1.06 respectively for the gold target . figure 3 shows the spectrums by their use , the production of the photon flux in tungsten and gold targets were compared . figure 5 shows the spectrum that by their use , the values of 8.9 , 10.2 and 11.6 kev were obtained for energy peaks characteristic for tungsten target , and values of 10.4 , 11.8 and 13.8 kev respectively for the gold target . in this study , axial and radial doses were computed for tungsten and gold targets for the applied energies , and comparison between them is according to figures 68 . in the simulation of this study , two types of thin transmission targets made of tungsten and gold with different atomic numbers , but with the same thickness and energy levels ( and almost the same density ) are subjected to electron bombardment , so factors determining the produced x - rays ( bremsstrahlung and characteristic x - ray ) and optimal thickness of the target are the energy of incident electron , and atomic number , thickness and shape of the target . when accelerated , electrons moving with certain energy , collide with tungsten and gold targets , the beam broadening in the target is under heavy influence of the atomic number of target material , and increases with the increase of atomic number ( because of more collisions in the target ) . so overall , the lower atomic number of tungsten , as compared to gold , means that the ot ( electron range ) of tungsten target rises faster than the gold one . also note that when the target is thinner than the range of incident electron , some electrons will manage to cross the full thickness of the target and this leads to partial and sub - optimal conversion of electron energy to x - ray ( 40 ) ( a in figure 2 ) . so it can be argued that lower thicknesses have a lower attenuation effect and greater thicknesses have a greater attenuation effect on the x - rays produced in the target material . however , in an excessively thick target , photons produced in the first section of the target will be attenuated by the remaining part of the material ( 41 ) . it can also be argued that using less than the optimum thicknesses decreases the intensity of outgoing photons and average spectrum energy , the use of optimum thickness leads to the maximum intensity of photons , and the use of greater than ots again decreases the intensity of photons and the average spectrum energy , because the target surface may reflect the electrons ( c in figure 2 ) . as a result , there must be an optimum thickness ( b in figure 2 ) based on which most electrons would be converted to photons and the maximum light intensity would be produced . overall , given that monte carlo simulation of both targets was conducted with similar conditions , optimum thickness and x - ray production are affected not only by the energy , target thickness , and atomic number but also by the shape of the target . the plots shown in figures 3 and 4 illustrate these important effects . according to these figures , for the gold target , the optimum thicknesses of 45 and 50 kev energy levels are relatively close ( their corresponding curves are a match and the numerical values are also close ) , but for the tungsten target , corresponding curves are apart which point to the presence of a different mechanism in tungsten as compared to gold . as the energy level increases , so does the optimal tt , and curve peaks and maxima shift forward , and this demonstrates the effectiveness of all mentioned factors . figure 9 shows the diagram of the relationship between electron range with the release of energy within the target until it is stopped , so that the optimum thickness is less than the electron ranges and sources with larger optimum thickness , the electron range within them is far greater than the other sources . the line tangent to these curves crosses the x - axis ( tt ) at the point which represents the range of electrons on that energy level , and this range can be used to determine the ot for the used energy . the curves depicted in figure 4 show the results of optimization of thickness of tungsten and gold targets with monte carlo code mcnpx ( 2.6.0 ) . these curves show that as target thickness increases , at first , the values of surface flux and flow of electrons at input level increase , but after reaching a maximum value at a certain thickness of surface , flux of electrons at input surface and energy flux and flow of electrons at the output surface remain constant . at output surface , as target thickness increases beyond its optimal value , self - absorption factor increases with thickness and as a result , the surface flux and the surface flow of electrons at the output surface decrease . at the input layer , flow and flux of electrons increase until the same point of ot . the plots also show that , in line with discussions of section 4.1 , surface flux and flow production in the tungsten target are far greater than that in the gold target . figure 5 shows a view of the x - ray spectra produced inside ( figure a ) and outside ( figure b ) the gold and tungsten targets . at low energy levels , spectra produced inside the gold and tungsten targets had energy peaks of , respectively , 1.7 kev and 2.1 kev and spectra produced outside the gold and tungsten targets had peaks of , respectively , ( 3 , 1.7 ) and ( 3 , 2.1 ) . the mean values of the highest energy peak for gold and tungsten targets were 6.1210 and 5.4910 , respectively . based on the mean values calculated for energy levels of 40 , 45 , and 50 kev , the energy produced inside the tungsten target is , respectively , 10% , 9.7% , and 8.7% greater than the gold target . the energy flux outside the gold target at energy levels of 40 , 45 , and 50 kev was estimated to be 11% , 10% , and 9% lower than the tungsten target . the statistical error in the calculation of these spectra started at 1% in the energy level of 1.5 kev , reached to a hundredth of a percent in the middle of the spectrum , and increased to 3.5% in the energy level of 46 kev . the errors obtained for energy levels lower than 1.5 or higher than 46 kev were higher than these values . figure 4 shows the spectra produced in water ( a ) and air ( b ) for different levels of the used energy . these spectra show that , the amount of energy obtained for tungsten target water / air ratio ( 0.92% , 1.4% , and 1.45% ) and gold target air / water ( 0.34% , 0.31% , and 0.29% ) is more at energy levels of 40 , 45 , and 50 kev , respectively . furthermore , the spectra depicted in figure 4 show that the highest energy peak in tungsten and gold targets are , respectively , 6.3410 and 9.6610 . the error in calculation of these spectra started at 2% in the energy level of 4.4 kev , reached down to a hundredth of a percent in the middle of the spectrum , and increased to 3 % in the energy level of 48 kev ; meanwhile , the errors obtained for energy levels lower than 4.4 or higher than 48 kev were higher than these values . in this stage of analysis , the running time of each program was 3,827 minutes . examination of spectra depicted in figures 3 and 4 show continuous x - ray spectra resulting from the electron bombardment of the surface of ots obtained for hemispherical - conical and hemispherical miniature sources . it can also be stated that x - ray spectrum produced inside the target depends on the energy of incident electron ; the spectrum of output beam ( its intensity ) after the target and before the output window depends on the target geometry ( shape and thickness ) ; and the spectrum of output beam ( its intensity ) after the beryllium window depends on thickness , density , and an atomic number of materials of the output window ( here beryllium ) . but the important point regarding the spectrum of the output beam is that , unlike the bremsstrahlung , the beamed characteristic x - rays have discontinuous energies and as figures 3 and 4 show , the peaks of spectrum imply the transition of electrons from upper shells to the l - shell . the analysis of dose calculation plots was carried out with two objectives : i ) comparing the axial dose in the presence of beryllium as the material of output window with the result obtained with the use of water instead of beryllium , and ii ) comparing the radial doses obtained with f6 tally and mesh tally method . figure 6 shows the plots used for calculation of axial dose with f6 tally . to assess the axial dose , the results of cells with the same number ( there were a total of 10 dose calculation cells ) but in the models with different energy levels ( 40 , 45 , 50 kev ) , source shapes ( hemispherical and hemispherical - conical ) and output window materials ( beryllium and water ) were compared , and the average differences were determined as a percentage . in the presence of beryllium , the doses of hemispherical source at energy levels of 40 , 45 , and 50 kev were , respectively , 12.83% , 8.94% and 13.15% higher than those of the hemispherical - conical source , while in the absence of beryllium , the dose differences were 9.1% , 6.2% , and 8.2% ( in the same order ) . figures 7 and 8 show the radial dose calculated with f6 tally and type-1 mesh tally . f6 tally reports the dose of each cell directly under the label of the averaged over a cell kerma ( in mev / gr per photon ) . type-1 mesh tally however operates as pedep ( photon energy deposition ) equivalent to f6 tally , and can be used to calculate the energy deposition per unit volume ( mev / cm3 per particle ) . the difference of mesh tally with f6 tally is that mesh tally gives the dose by converting the flux , while f6 tally gives the dose directly . also , mesh tally method requires the density of the medium ( here , water ) to be applied . at this stage of work , the doses calculated with f6 tally and a mesh tally was compared . the greatest dose calculated with mesh tally at energy level was used as reference , and the radial doses calculated with f6 tally were compared with these reference values . this comparison showed that for the hemispherical - conical source , the doses calculated at energy levels of 40 , 45 , and 50 kev using the mesh tally were , respectively , 7.5% , 2.5% , and 1.6% higher than those calculated with f6 tally . for the hemispherical source , the doses obtained at energy levels of 40 , 45 , and 50 kev with mesh tally were 2.5% , 6.5% , and 3.5% higher than those obtained with f6 tally . this difference can be attributed to the difference in the density of the medium ( water ) and difference in the origin of the coordinate system used for mesh tally frame with the one defined for the cells of f6 tally . despite adjustments , this error proved to be persistent . in any case , the axial and radial doses calculated for hemispherical miniature source , were greater than those calculated for hemispherical - conical source . the axial and radial doses calculated for both sources showed a high gradient near the source and a decrease with distance from the source , at a rate of 1/r3 . data analysis also showed that the presence of beryllium energy window has a greater impact on the hemispherical source than on the hemispherical - conical source . in the simulation of this study , two types of thin transmission targets made of tungsten and gold with different atomic numbers , but with the same thickness and energy levels ( and almost the same density ) are subjected to electron bombardment , so factors determining the produced x - rays ( bremsstrahlung and characteristic x - ray ) and optimal thickness of the target are the energy of incident electron , and atomic number , thickness and shape of the target . when accelerated , electrons moving with certain energy , collide with tungsten and gold targets , the beam broadening in the target is under heavy influence of the atomic number of target material , and increases with the increase of atomic number ( because of more collisions in the target ) . so overall , the lower atomic number of tungsten , as compared to gold , means that the ot ( electron range ) of tungsten target rises faster than the gold one . also note that when the target is thinner than the range of incident electron , some electrons will manage to cross the full thickness of the target and this leads to partial and sub - optimal conversion of electron energy to x - ray ( 40 ) ( a in figure 2 ) . so it can be argued that lower thicknesses have a lower attenuation effect and greater thicknesses have a greater attenuation effect on the x - rays produced in the target material . however , in an excessively thick target , photons produced in the first section of the target will be attenuated by the remaining part of the material ( 41 ) . it can also be argued that using less than the optimum thicknesses decreases the intensity of outgoing photons and average spectrum energy , the use of optimum thickness leads to the maximum intensity of photons , and the use of greater than ots again decreases the intensity of photons and the average spectrum energy , because the target surface may reflect the electrons ( c in figure 2 ) . as a result , there must be an optimum thickness ( b in figure 2 ) based on which most electrons would be converted to photons and the maximum light intensity would be produced . overall , given that monte carlo simulation of both targets was conducted with similar conditions , optimum thickness and x - ray production are affected not only by the energy , target thickness , and atomic number but also by the shape of the target . the plots shown in figures 3 and 4 illustrate these important effects . according to these figures , for the gold target , the optimum thicknesses of 45 and 50 kev energy levels are relatively close ( their corresponding curves are a match and the numerical values are also close ) , but for the tungsten target , corresponding curves are apart which point to the presence of a different mechanism in tungsten as compared to gold . as the energy level increases , so does the optimal tt , and curve peaks and maxima shift forward , and this demonstrates the effectiveness of all mentioned factors . figure 9 shows the diagram of the relationship between electron range with the release of energy within the target until it is stopped , so that the optimum thickness is less than the electron ranges and sources with larger optimum thickness , the electron range within them is far greater than the other sources . the line tangent to these curves crosses the x - axis ( tt ) at the point which represents the range of electrons on that energy level , and this range can be used to determine the ot for the used energy . the curves depicted in figure 4 show the results of optimization of thickness of tungsten and gold targets with monte carlo code mcnpx ( 2.6.0 ) . these curves show that as target thickness increases , at first , the values of surface flux and flow of electrons at input level increase , but after reaching a maximum value at a certain thickness of surface , flux of electrons at input surface and energy flux and flow of electrons at the output surface remain constant . at output surface , as target thickness increases beyond its optimal value , self - absorption factor increases with thickness and as a result , the surface flux and the surface flow of electrons at the output surface decrease . at the input layer , flow and flux of electrons increase until the same point of ot . the plots also show that , in line with discussions of section 4.1 , surface flux and flow production in the tungsten target are far greater than that in the gold target . figure 5 shows a view of the x - ray spectra produced inside ( figure a ) and outside ( figure b ) the gold and tungsten targets . at low energy levels , spectra produced inside the gold and tungsten targets had energy peaks of , respectively , 1.7 kev and 2.1 kev and spectra produced outside the gold and tungsten targets had peaks of , respectively , ( 3 , 1.7 ) and ( 3 , 2.1 ) . the mean values of the highest energy peak for gold and tungsten targets were 6.1210 and 5.4910 , respectively . based on the mean values calculated for energy levels of 40 , 45 , and 50 kev , the energy produced inside the tungsten target is , respectively , 10% , 9.7% , and 8.7% greater than the gold target . the energy flux outside the gold target at energy levels of 40 , 45 , and 50 kev was estimated to be 11% , 10% , and 9% lower than the tungsten target . the statistical error in the calculation of these spectra started at 1% in the energy level of 1.5 kev , reached to a hundredth of a percent in the middle of the spectrum , and increased to 3.5% in the energy level of 46 kev . the errors obtained for energy levels lower than 1.5 or higher than 46 kev were higher than these values . figure 4 shows the spectra produced in water ( a ) and air ( b ) for different levels of the used energy . these spectra show that , the amount of energy obtained for tungsten target water / air ratio ( 0.92% , 1.4% , and 1.45% ) and gold target air / water ( 0.34% , 0.31% , and 0.29% ) is more at energy levels of 40 , 45 , and 50 kev , respectively . furthermore , the spectra depicted in figure 4 show that the highest energy peak in tungsten and gold targets are , respectively , 6.3410 and 9.6610 . the error in calculation of these spectra started at 2% in the energy level of 4.4 kev , reached down to a hundredth of a percent in the middle of the spectrum , and increased to 3 % in the energy level of 48 kev ; meanwhile , the errors obtained for energy levels lower than 4.4 or higher than 48 kev were higher than these values . in this stage of analysis , the running time of each program was 3,827 minutes . examination of spectra depicted in figures 3 and 4 show continuous x - ray spectra resulting from the electron bombardment of the surface of ots obtained for hemispherical - conical and hemispherical miniature sources . it can also be stated that x - ray spectrum produced inside the target depends on the energy of incident electron ; the spectrum of output beam ( its intensity ) after the target and before the output window depends on the target geometry ( shape and thickness ) ; and the spectrum of output beam ( its intensity ) after the beryllium window depends on thickness , density , and an atomic number of materials of the output window ( here beryllium ) . but the important point regarding the spectrum of the output beam is that , unlike the bremsstrahlung , the beamed characteristic x - rays have discontinuous energies and as figures 3 and 4 show , the peaks of spectrum imply the transition of electrons from upper shells to the l - shell . the analysis of dose calculation plots was carried out with two objectives : i ) comparing the axial dose in the presence of beryllium as the material of output window with the result obtained with the use of water instead of beryllium , and ii ) comparing the radial doses obtained with f6 tally and mesh tally method . figure 6 shows the plots used for calculation of axial dose with f6 tally . to assess the axial dose , the results of cells with the same number ( there were a total of 10 dose calculation cells ) but in the models with different energy levels ( 40 , 45 , 50 kev ) , source shapes ( hemispherical and hemispherical - conical ) and output window materials ( beryllium and water ) were compared , and the average differences were determined as a percentage . in the presence of beryllium , the doses of hemispherical source at energy levels of 40 , 45 , and 50 kev were , respectively , 12.83% , 8.94% and 13.15% higher than those of the hemispherical - conical source , while in the absence of beryllium , the dose differences were 9.1% , 6.2% , and 8.2% ( in the same order ) . figures 7 and 8 show the radial dose calculated with f6 tally and type-1 mesh tally . f6 tally reports the dose of each cell directly under the label of the averaged over a cell kerma ( in mev / gr per photon ) . type-1 mesh tally however operates as pedep ( photon energy deposition ) equivalent to f6 tally , and can be used to calculate the energy deposition per unit volume ( mev / cm3 per particle ) . the difference of mesh tally with f6 tally is that mesh tally gives the dose by converting the flux , while f6 tally gives the dose directly . also , mesh tally method requires the density of the medium ( here , water ) to be applied . at this stage of work , the doses calculated with f6 tally and a mesh tally was compared . the greatest dose calculated with mesh tally at energy level was used as reference , and the radial doses calculated with f6 tally were compared with these reference values . this comparison showed that for the hemispherical - conical source , the doses calculated at energy levels of 40 , 45 , and 50 kev using the mesh tally were , respectively , 7.5% , 2.5% , and 1.6% higher than those calculated with f6 tally . for the hemispherical source , the doses obtained at energy levels of 40 , 45 , and 50 kev with mesh tally were 2.5% , 6.5% , and 3.5% higher than those obtained with f6 tally . this difference can be attributed to the difference in the density of the medium ( water ) and difference in the origin of the coordinate system used for mesh tally frame with the one defined for the cells of f6 tally . despite adjustments , this error proved to be persistent . in any case , the axial and radial doses calculated for hemispherical miniature source , were greater than those calculated for hemispherical - conical source . the axial and radial doses calculated for both sources showed a high gradient near the source and a decrease with distance from the source , at a rate of 1/r3 . data analysis also showed that the presence of beryllium energy window has a greater impact on the hemispherical source than on the hemispherical - conical source . it was found that , before being absorbed , the x - ray resulted from the electron bombardment of target must traverse a path which puts its spectrum under the influence of factors such as the energy of incident electron , cross sectional area of produced bremsstrahlung , target shape , the atomic number of material of target , and density of material constituting the energy window . this study concluded that the presence of beryllium window has a greater impact on the hemispherical source than on the hemispherical - conical source , which is reflected in the higher absorbed dose . this could be the reason behind the selection of beryllium for the energy window of intrabeam system ; and the lower impact of beryllium on tungsten is perhaps the reason why axxent system uses a combination of beryllium and yttrium

introductionsince the heart of the electronic brachytherapy system is a tube of a miniature x - ray and due to the increasing use of electronic brachytherapy , there is an urgent need for acquiring knowledge about the x - ray spectrum produced , and distribution of x - ray dose . this study aimed to assess the optimal target thickness ( tt ) , the x - ray source spectrum , and the absorbed dose of two miniature sources of hemispherical and hemispherical - conical used in electronic brachytherapy systems , through a monte carlo simulation.methodsconsidering the advantages of mcnpx code ( 2.6.0 ) , two input files corresponding to the characteristics of the investigated miniature sources were prepared for this code and then were used for simulation . the optimal thickness ( ot ) of gold and tungsten targets was determined for the energy levels of 40 , 45 , and 50 kilo-electron-volts.resultsin this study , the values of the size of the optimal thickness of 0.92 , 1.01 and 1.06 for gold target and values of 0.99 , 1.08 and 1.34 for tungsten target were obtained for energies 40 , 45 and 50 kev that using these values , the optimum thickness of 0.92 , x - ray spectrum within and outside targets , axial and radial doses for the used energy were calculated for two miniature sources.conclusionit was found that the energy of incident electron , target shape , cross - sectional area of the produced bremsstrahlung , atomic number of materials constituting of the target and output window are the factors with the greatest impacts on the produced x - ray spectrum and the absorbed dose .

1. Introduction 2. Material and Methods 2.1. Simulation of miniature X-ray sources 2.2. Computation of optimal thickness of Targets 2.3. Computation of spectra resulting from the miniature sources 2.4. Calculation of absorbed dose 3. Results and discussion 3.1. Evaluation of the factors affecting the optimal target thickness 3.2. Exanimation of the obtained spectra and plots 3.3. Analysis of the absorbed dose based on the type of tally and mesh tally 4. Conclusions

PMC3820233

fall - related injuries are high in the developed countries , and in the us . they are recorded as the second largest source of unintentional fatal injuries and the main source of non fatal injuries1,2,3 . in 2005 , the national center of injury prevention and control ( ncipc ) of korea reported elderly females to be more likely than men to have injuries due to falling . in 2005 , kannus4 found that over 80% of elderly patients were admitted to hospital due to falls , and roach5 report that over 50% of these patients died within 1 year after they were hospitalized . it is well known that besides the acute injuries sustained , the long - term effects of hospitalization are more serious as they can prevent the recovery of a patient 's muscle strength , reducing their ability to be self sufficient . there are many factors that can cause falls by older adults : joint instability and muscle imbalance6,7,8,9 , reduction in joint range of motion , reduction in the ability to ambulate proficiently10 , and deficiencies in lower body strength11,12,13,14 . a decreased ability to ambulate proficiently arises from a loss of muscle mass and strength which is attributable to aging2 , 15 . other factors that can also cause serious falls are medicinal side effects , and chronic diseases associated with aging , e.g. arthritis . ultimately , preventing falls is better than cure , and kavanagh16 and many researchers have concluded older adults should participate in exercise programs to prevent falls and maintain self - sufficiency , which is linked to a high quality life . berg and his colleagues17 state that older adults should participate in regular exercise to prevent falls . it is recommended that a fall prevention exercise program should strengthen the lower body18 , rectify muscle imbalances16 , incorporate balance exercises19 and increase flexibility20 . wolf21 implemented tai chi as a falling prevention exercise program and reported that after 15 weeks of tai chi , the frequency of falls was reduced by 47.5% . dayhoff22 implemented an elastic band exercise for 14 weeks and reported an increase in the subjects ' preferred gait speed . skeleton23 had participants perform resistance training for 10 weeks and reported that subjects ' muscle strength and gait speed both increased . however , since older adults are susceptible to arthritis , lower range of motion and chronic diseases exercise training programs should be carefully developed so as not to exacerbate any pre - existing problems . aqua aerobic exercise has been shown by many studies to be highly effective at reducing pain for patients with arthritis and disabled populations24 , as well as improving flexibility , strength , and balance disorders25 and has a significant positive psychological effect26 . kim27 reported that aqua therapy increases cardiovascular endurance , flexibility , muscle strength , muscle balance and reduces the percentage of body fat . * indicates a significant interaction between time and group , p<0.05 . * * indicates a significant difference between before and after , p<0.05 . subjects ' bone density was measured by whole body dual energy x - ray absoptiometry ( dexa ; ma ) . twenty of the subjects had a score below 1 , and they were excluded from this study due to the high impact nature of the forced perturbation . twenty subjects commenced the aqua aerobic therapy training , and they were divided randomly into two groups of 10 . during the 12 weeks of aqua therapy exercise 5 subjects dropped out leaving only 7 in the control group and 8 in the experimental group ( table 1 ) . as in rikli and jones study19 , subjects ' flexibility , agility , balance and muscle strength were measured and recorded ( table 4 ) . subjects ' muscle strength was measured by a cybex ( 770 , new york ) . both knee flexion and extension were measured at two speeds ; 60/s and 180/s . muscle power was defined as the peak torque generated at a constant velocity of 180/s . similarly muscle strength was defined as the peak torque generated at a constant velocity of 60/s . each trial lasted 5 seconds and was repeated three times with three minutes of rest between each trial , as recommended by rikli and jones19 . the peak torque was recorded for knee flexion strength and knee extension power , was testing was performed one week before the gait experiment . therefore this current study investigated the effects that an aqua exercise therapy has on the physiological and biomechanical factors of gait . a secondary aim was to examine if improvements in gait variables would improve older adults ' responses in a perturbed gait condition . during gait observations , kinematics at 60 hz were recorded by 8 oqus 500 cameras ( qualisys , sweden ) , and kinetics on by 2 amti force platforms ( amti , usa ) were recorded at 1200 hz by qualisys track manager ( qtm , qualisys sweden ) . after explanation of the testing procedures and obtaining subjects ' informed consent , the experiment began . subjects strapped into an overhead harness for safety . for the dynamic trials the participants walked at a self - selected pace three times down a walkway . the participants were then escorted out of the room while the walkway was raised and the perturbation device was set up . next , the participants were instructed to walk along the walkway and then step down . the participants were informed that this step - down procedure would have to be done three times . after each trial the participants were escorted back outside the laboratory and the perturbation device was reset . during these final 3 trials the perturbation platform would open up randomly and perturbation data was collected once for each participant as they became apprehensive of walking on the walkway after the perturbation . the aqua therapy exercise program was carried out three times per week for one hour in each session ( table 2 ) . there were three main parts to the exercise session : including warm up , aqua aerobics , and cool down . the indoor pool was 25 m long , 4 m wide and 1.3 m deep , and had a water temperature of 281c and a room temperature of 26c . after the participants began to feel warmer a slow aqua aerobics pace was set which was then increased steadily . the aqua therapy consisted of aquatic adaption time , breathing techniques , floating and posturing . equipment were introduced to maintain the participants ' enjoyment , and as each piece of equipment could be used to train difference aspects of fitness , they were used for overall variation and stimulus of different muscles . the cool down gave the participants time to do stretching and have some free time . the intensity of the aqua therapy was set according to the ratings of the borg perceived exertion ( rpe ) scale set at an intensity level of 711 for the first 3 weeks then the intensity was increased to 1213 . after labeling of the data and interpolation in qtm , the data were exported to a c3d file , and modeling and manipulation were performed with visual3d ( c - motion , usa ) . a 2 2 ( group time ) anova with repeated measures were performed to compare the data between the two groups according to the measurement time . independent t tests were used to verify that there were no significant differences between the control and experimental group at the initial stage of the study . further , significant differences were investigated using the paired t - test to investigate the differences between pre and post treatment . participants ' weight , muscle mass , fat mass and mineral mass for both the control and experiment groups before and after the aqua aerobic therapy were measured using an inbody 720 . there was no significant interaction between groups and time for the muscle mass and the mineral mass , but there were significant reductions in weight ( p<0.05 ) and body fat mass ( p<0.05 ) ( table 3 ) . * * indicates a significant interaction between time and group , p<0.05 . there were no significant differences between before and after the intervention in muscle strength and power , but there were significant interaction effects ( p<0.05 ) . for flexibility and agility , there were significant interactions . there were significant increases in muscle strength , power , flexibility , agility and balance of the participants in the aqua therapy group , whereas the control group demonstrated either reductions or no change ( table 4 ) . * * indicates a significant interaction between time and group , p<0.05 . to investigate how participants recovered after the perturbation various gait - related variables were measured and calculated . there were no significant differences between the experiment and control groups before and after the intervention in support time , step length and jerk cost . there were statistical differences and interactions in stride and step times between the groups . these results demonstrate that the experiment group participants reduced their stride time , which shows that they recovered more quickly after the perturbation ( table 5 ) . the kinetic variables calculated were the maximum moments of the ankles , knees and hips , and the propulsion force of the perturbed foot after landing . there were both significant main effects ( p<0.05 ) and interaction effects ( p<0.01 ) of the maximum ankle moment . similarly for the maximum knee moment there were both significant main ( p<0.05 ) and interaction ( p<0.05 ) effects . there were no significant effects found for the maximum hip moment and the propulsion impulse . to summarize the kinetic data , the participants in the experiment group were able to develop more ankle and knee moment to prevent their body falling and recover . the hip moments did not seem to affect the recovery of the participants ( table 6 ) . the increasing risk of falls increases with aging , and the aims of this study were to investigate whether aqua exercise therapy can help the elderly by preventing falls and improving their gait patterns after a perturbation . it was our hypothesis that aqua exercise training would increase the elderly participants ' physical strength , flexibility , and balance which would in turn help improve a subjects ' ability to react to a perturbation . our results are similar to those of previous studies which have reported that 12 weeks aqua therapy exercise improves lower body strength , power , flexibility , agility and balance16 , 19 , 20 . also , due to the training effect the weight and body fat mass was reduced , whereas no significant changes were seen in these variables in the control group . kinematics and kinetics were recorded and compared to evaluate subjects ' reaction to perturbation . our data show that the older adults of the experiment group managed the perturbation more efficiently and had a more stable gait pattern . oddsson and his colleagues29 demonstrated that recovery after a perturbation takes about 3 or 4 steps depending on limb strength and age . the present study found no significant differences in the support time , step time , step length and jerk cost after the perturbation . however , recovery started at the second step , shown by the significant difference in stride times between before and after the perturbation . the maximum joint moment of the landing leg show that subjects due to an increase in strength were able to create more ankle and knee moment which helped their quick recovery of balance after the perturbation . one of the main limitations of this study was the danger associated with perturbed gait , especially with older adult subjects . subjects with a high bone density were selected due to the risks of perturbed gait , and they might not have been representative of the general older adult female population , as it has been shown that exercising has a positive effect on the bone density of older adults aged in their sixties to eighties30 . to conclude , aqua exercise therapy may be used as a method of training for older female adults to prevent falls . we recommend further study of older adults ' reaction to a perturbation , includingthe measurement of emg as well as more detailed kinematic and kinetic data analysis of the first few steps after the perturbation .

[ purpose ] the purpose of the present study was to examine the effects of an aqua aerobic therapy exercise for older adults on biomechanical and physiological factors affecting gait . [ subjects ] a total of 15 subjects participated in this study and they were randomly divided into the experimental and the control group . [ methods ] physiological variables , leg strength , power and flexibility , and biomechanical variables , both kinematic and kinetic , were measured before and after the aqua aerobic therapy exercise . each subject was instructed to walk along an elevated walkway and during the trials a trapdoor opened at random to create a 10 cm falling perturbation . full body motion and kinetics was gathered during the gait . [ results ] there were significant reductions in body weight , and body fat mass , and stride time after the perturbation . significant increases in leg strength corresponded to the maximum joint moment of the landing leg showing that the subjects ' ability for recovery of balance after the perturbation improved . [ conclusion ] as the results showed significant improvements in gait pattern and recovery time after perturbed gait , we conclude that aqua aerobic therapy is an effective exercise method for training older adults to reduce their risk of falling .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC3589687

smartphones are widely - used and their market share increases constantly [ http://www.w3schools.com/browsers/browsers_mobile.asp ] . an increase of traffic by 15 and a triplication of the devices within the next five years is forcasted for germany . apple s presentation of the revolutionary iphone in 2007 contributed largely to this as it combined a simple handling with a huge variety of function for the first time and has been awarded therefore many times [ http://www.jdpower.com/consumer-ratings/electronics/ratings/909201396/2012-wireless+consumer+smartphone+customer+satisfaction+study/index.htm ] . apple shares the market leadership in the smartphone market with samsung and nokia , . the increasing market share of smartphones and the ability to access websites mobile easily change the requirements of the users . up until today websites have been displayed on laptops and desktops only and optimisation focused on those two formats . recent upgrowth of smartphones demand a change with respect to the optimisation of content for much smaller display sizes this is often referred to as responsive webdesign [ https://en.wikipedia.org/wiki/responsive_web_design ] . the changes begin with reshuffling of single elements to improve the legibility ( e.g. alternative navigation ) , can be enhanced by images with smaller file size to reduce traffic and ultimately can be as neat as a complete simulation of a native application ( applications that have been developed using the manufacturer s developing kit ( sdk ) ) which integrates into the operating system intuitively ( see figure 1 ( fig . davies and his colleagues investigated the learning and study habits of students and questioned how smartphones are used . they found out that smartphones are mostly used to answer a question right on the spot ( timely access to key facts learning in context ) and that students would use smartphones to repeat learned topics . due to the author s interest in smartphones as a learning platform and his fascination for the development of own applications a prototype was designed to find out if a smartphone can be more than a reference book . the leading question was whether and how the application can be realised and if there are any limitations ? an questionnaire was designed to get an insight into the student s thoughts about mobile learning . hangman [ https://de.wikipedia.org/wiki/galgenm%c3%a4nnchen ] is a classical game for two or more players in which one player thinks of a word and the other tries to guess it by suggesting letters . the idea of this game has been adapted within the project mediman so that medical terms must be guessed . at the end of each game the term is defined and explained to produce a benfit for the student ( see figure 2 ( fig . 2 ) ) . before starting the actual development of the application a decision regarding the programming language had to be reached . the author decided to program a native html website in combination with the javascript framework jquery mobile [ http://jquerymobile.com/ ] as it can be used to build native looking applications on one hand but also supports many mobile devices out of the box [ http://jquerymobile.com/ ] . jquery mobile is widely spread , possible alternatives with similar features are jqtouch and sencha touch . mediman uses a mysql database to store the data and ajax ( asynchronus javascript and xml ) to increase usability , e.g. no page reload is necessary to start a new game round as data is loaded in the background . during the development simulation programs where used that the benefit is that the developer does not actually has to own all the devices to run tests with them . to start the application the user needs to type in the internet adress ( url ) if the website is additionally added to the home screen a defined icon appears and the difference between a native application and a web site simulating it vanishes even more ( see figure 1 ( fig . 1 ) and figure 2 ( fig . subsequent to the development process a questionnaire with nine items was desigend . if not declared otherwise a likert scalling was used . test users ( n=20 ) were asked to submit the online forms between june and juliy 2012 after they received an invitation via e - mail . all test users are students ( third year or above ) attending faculty of medicine of the university of munich and took part voluntarily . they aged 25 years in average and most of them were male ( 63,6% ) . the majority of the users used products of the apple iphone family , followed by devices by samsung . one user used a nokia n8 , an other a apple ipod touch 2 g ( category other ) . when it comes to usage frequency of the devices for learning the majority of the users stated that they use it more than once a week or at least at times ( < 5/month ) . only three of the test users use it rarely ( < 1/month ) or never ( see figure 4 ( fig . 5 ) indicates that only few test users think that many learning applications for smartphones exist . in contrast most of the users stated that smartphones are an ideal plattform they are modern , widely spread and easy to use ( see figure 6 ( fig . the evaluation of the developed application is dominated by the impression that the idea is interesting and well realised . usabilty feels comfortable , a learning progress is not distinctive ( see table 1 ( tab . ( one should not need to write or type in too much that wo nt work for smartphones very well ) and aim must be to reach goal with a few clicks . smartphones are widely spread devices with plenty of possible applications due to their connection to the internet . the faculty of medicine of the university of stanford for example picked up this trend and equip every new medical student with an apple ipad since 2010 . parallel to the introduction the faculty established a portal with learning material ( presentations , videos , manuals and more ) which can be accessed by the students for free using a app . this allows the students to access the needed material when they want and how often they want . so far , available e - learning programs , e.g. casus , work within a desktop environment and they do their job mostly fine . those programs are therefore designed and optimised for desktop computers ( with internet browsers ) , big screens and a computer mouse for interaction . the e - learning programs can be used with modern smartphones aswell but they are always suboptimal with respect to usability and legibility . the growing role smartphones play in our daily life due to their increasing market share demands an adaptation to the changed needs of the users . the analysis of the questionnaire showes that the test users think that smartphones are an ideal learning environment because most of them carry one with them all the time . this is comprehensible as for the first time ever more smartphones than desktop computers have been sold last year [ http://www.canalys.com/newsroom/smart-phones-overtake-client-pcs-2011 ] . the survey showed aswell that not enough learning applications specially designed for smartphones exist ( see figure 2 ( fig . 2 ) ) , the desire for them though is high ( see table 2 ( tab . however do not allow the conclusion that existing learning applications should simply be optimised for the smaller display sizes . the demands of the users seem to be more differentiated as they seem to look for short sessions explicitly . a test user submited the free text item covering the needs of the users with the comment that he or she would like to start sessions while travelling betwen two university locations or even when sitting in the train . short texts and optimised cases for the repetition of knowledge seem the be more important than continuous text . applications generally speaking can be produced as native applications for a chosen manufacturer or operating system ( e.g. apple ios , android ) and can then advertised and sold through the manufacturer s online store . apple , for example , will check each version before deploying it to the online store and can even refuse to publish it . instead of developing the application for the various operating systems individually the author used programming languages that are normally used to program websites ( html , css , javascript ) in combination with a javascript framework called jquery mobile . this framework simulates a native application optically and because the necessary skills already existed no further longsome familiarisation with sdks was necessary . this ensured a short development time , the list of supported devices of the framework is long and checks by the manufacturers prior to the aproval of an application to their online store does not exist . as a sort of hybrid technique phonegap ( http://phonegap.com ) must be mentioned ( web sites will be transformed and compiled into native apps ) but was not chosen for the development of mediman as it would have required training . offering applications through the online stores means automatic avertising but web applications allow faster publication due to no certivication process . the results of the survey show that learning applications designed as a web site with an ideal integration into the smartphone simulating a native application seems to work for the test users . we did not find any relevant limitations using web site programming languages as no device specific function , e.g. motion sensor , was used . uncertainty exists insofar as the analysis of the submited data of the test users did not show a clear trend if an active internet connection may be a requirement for running the learning application ( see table 2 ( tab . the anxiety that this is explicitly a problem of the used programming languages used in the example project mediman can be qualified as version 5 of html supports storing data locally [ http://diveintohtml5.info/storage.html ] . apart from the technical aspect the author thinks that mobile plans with unlimited traffic are common . today , only few learning applications designed to work seamlessly on smartphones exist this is reflected in the usage frequency . it does not seem to be the one and only solution to adapt existing e - learning programs to fit into the small screen sizes because the needs of users is different . the survey showed that dealing with learning applications for smartphones will be an important topic in the future . mediman , the example project that was used mainly to demonstrate the potential and feasibility of the realisation of a small and simple learning application for smartphones was rated well within the questionnaire . as this was one of the main aims the project succeeded in our opinion and further devlopments should be considered . due to the small number of participants we should not draw a general conclusion out of the given data but as a high relevance can be derived further research on this topic must be seeked .

mobile devices with a connection to the internet smartphones are seen all over the place since the popular introduction of the apple iphone . similar products existed but no company managed to combine simplicity and functionality so seamlesly . their market share increases constantly and web sites get optimised for the small display sizes ( often referred to as responsive webdesign ) otherwise the usability lacks . students seem to like smartphones aswell and a good question is if and to what extend those devices can play a role in e-learning.mediman , an adaptation of the common game hangman has been developed for smartphones . test users asked to complete an online questionnaire . so far , only few e - learning applications for smartphones seem to exist . this is reflected in the low usage frequency . especially the fact that most of the test users wear a smartphone with them all the time makes it an ideal learning plattform . short learning sessions were rated more important than continuous text . the majority of the 11 test users rated mediman as well developed . the foremost question whether a smartphone e - learning application is feasible must be answered positive acceptance in the test user group was shown . e - learning applications on smartphones will be an important topic in the future as market shares increase constantly . further studies are required due to the small number of partitipants in our survey .

Introduction Methods Results Discussion Competing interests

PMC4648500

proteins fold into their biologically functional 3d structures by forming cooperative networks of weak interactions that compete against the entropy of the flexible polypeptide chain . these complex interaction networks hold the key to folding mechanisms and the rational design of new protein folds . this is because proteins reside in their native or unfolded states for long periods of time ( up to days ) , but the transitions between these two states , and thus the formation and disassembly of the interaction network , seem to occur almost instantaneously . indeed , advanced analysis of single - molecule experiments has recently shown that at least some folding transitions take place over periods on the order of a few microseconds , a time scale that is also equivalent to previous empirical estimates of the folding speed limit . a comprehensive understanding of folding interaction networks would thus entail the characterization of rare events in individual protein molecules at an atomistic level of detail , and with sub - microsecond resolution . both experiments and simulations have led to significant advances in our understanding of the protein folding process , and their respective capabilities and limitations are such that a combination of the two may lead to insights and cross - validation that could not be obtained using either paradigm alone . experimental methods can reach atomic - level resolution when investigating the millisecond time scale , but can access sub - microsecond time scales only using coarse - grained spectroscopic probes . molecular dynamics ( md ) simulations , on the other hand , can generate continuous , atomistically detailed folding and unfolding trajectories , but are computationally demanding , and rely on physical approximations whose range of applicability has not yet been fully ascertained . here we use a combination of nuclear magnetic resonance ( nmr ) experiments and long - time - scale md simulations to elucidate key elements of the folding process of the single - domain protein gpw . gpw is a 62-residue entire gene product that folds into an antiparallel + topology in microseconds . its ultrafast folding unfolding relaxation rate places gpw in the fast exchange nmr regime over the relevant temperature range , and makes it an attractive target for long md simulations . a combination of thermodynamic and kinetic criteria suggests this moderate - sized domain folds over a low free energy barrier . moreover , gpw exhibits distinctly sigmoidal equilibrium thermal unfolding with well - resolved pre- and post - transition baselines that should facilitate the accurate analysis of its unfolding thermodynamics at the atomic level . in experimental studies of the atom - by - atom thermal unfolding behavior of gpw , we observe a multilayered process in which the large - scale structural changes characterizing the global , two - state - like unfolding transition are superimposed on a more intricate set of atomic- and residue - level structural changes . we observe a similar level of underlying complexity in coordinated computational studies of the equilibrium unfolding of gpw . the simulations thus support our experimental results and are also consistent with previous computational studies performed on other proteins . moreover , from our combined experimental and computational analysis , we infer that the complex structural changes that we observe at the residue level with both methods are intimately connected to the protein interaction network that ultimately determines the folding mechanism . nmr is a powerful tool for investigating protein conformational changes with atomic resolution . nmr relaxation dispersion methods , for example , render high - resolution structural information on transient , low - populated folding intermediate states ( i.e. , invisible states ) . in principle , time resolution limits the application of these methods to proteins with a somewhat slow folding rate ( < 3000 s ) . recently , this limit has been successfully pushed forward to investigate unfolding fluctuations of native gpw at very low temperature ( 273 k ) , taking advantage of the slow down in gpw folding rate at this temperature . an alternative approach comes from work on the one - state downhill folding scenario . one - state downhill folders are single - domain proteins that fold unfold in microseconds by diffusing down a barrier - less free energy surface at all experimental conditions . in thermodynamic terms , these domains unfold through a gradual , minimally cooperative unfolding process that results in a broad distribution of structure - specific equilibrium denaturation behaviors . such remarkable thermodynamic features have been exploited to infer key aspects of the folding interaction network from the cross - correlations among hundreds of atomic unfolding curves obtained by nmr in equilibrium denaturation experiments . however , one - state downhill folding domains are not widespread , and , in principle , their folding mechanisms could be different from those of other proteins . the important question is whether the same nmr approach can be extended to the more general case of folding over free energy barriers in which the equilibrium unfolding process is also distinctly more cooperative . this is an important fundamental question because it has been generally assumed that folding of small proteins follows a two - state mechanism , in which case all probes that effectively report on the integrity of the native structure exhibit the same unfolding behavior and there is no net gain in probing the process with atomic resolution . in fact , the two - state assumption has been used as justification for interpreting any deviations from the global unfolding behavior that might be observable by nmr as arising from processes unrelated to folding . therefore , observing unfolding heterogeneity by nmr that is systematically larger than experimental uncertainty , yet fully consistent with the global unfolding process , would provide important experimental evidence that protein folding cooperativity is , in general terms , finite and limited , as predicted by theory and often observed in simulations . in the current study , we performed the nmr analysis of gpw thermal unfolding at ph 3.5 to slow proton amide exchange and thus ensure obtaining full chemical shift ( cs ) assignments when the protein is unfolded at high temperature . at ph 3.5 the thermal denaturation of gpw measured by far - uv circular dichroism ( cd ) , a low - resolution backbone - sensitive technique , is sigmoidal and exhibits a rather sharp transition . analysis with the simplest two - state model ( ignoring changes in heat capacity , which are small for gpw and do not result in any signs of cold denaturation at this ph ) produces a tm = 329 k and h = 135 kj / mol . relative to neutral ph , the mildly acidic conditions used here decrease the denaturation midpoint of gpw by about 11 but keep the unfolding enthalpy essentially unchanged ( figure s1 ) . the slightly lower stability is convenient because it centers the unfolding process within the experimental range ( 273373 k ) and makes the determination of baselines more accurate . to check whether the mildly acidic conditions induce structural changes in the gpw native state , we determined the 3d structure at ph 3.5 ( pdb : 2l6r ) and compared it with that obtained previously at neutral ph . the comparison between the lowest energy structures for both conditions renders a backbone rmsd of 0.7 ( figure 1a ) , which is comparable to the variability between pairs of structures within the ph 3.5 ensemble ( table s1 ) . therefore , the structural changes induced by ph are minimal . in addition , the slight destabilization observed by cd seems to arise from an overall increase in net positive charge at this ph , rather than from changes in specific interactions . first , overall screening of the electrostatic repulsions at ph 3.5 by addition of 1 m salt results in complete recovery of the unfolding behavior observed at neutral ph ( figure s1 ) . second , the aliphatic carbon cs values for the side - chains of the 11 basic residues in gpw ( 7 r and 4 k ) are essentially identical at both phs . the cs insensitivity of the r and k side chains to the protonation status of their acidic counterparts suggests the absence of specific electrostatic interactions in the gpw native structure . third , we determined the pka values for all the carboxylic residues in gpw and the lone histidine using nmr ( figure s2 ) . the titration shows that all these residues have pka values ( 4.5 0.14 for the five e in the sequence , 3.66 0.15 for the three d , and 6.7 for h ) that are very close to the standard values of the reference amino acids in unstructured peptides . the lack of significant pka shifts indicates that there is no significant coupling between the charge status of those residues and the folding process of gpw . for the nmr analysis of thermal unfolding we used the n amide , c and c cs for the 62 residues in the protein ( a total of 180 atomic unfolding curves ) . relative to protons , the cs for these atoms offer far superior performance for the high - resolution analysis of protein unfolding because they are easier to interpret in structural terms , exhibit minimal temperature dependence , and ring - current effects are less significant . the nmr spectra recorded around the denaturation midpoint ( where changes in populations are maximal ) did not show significant line broadening , consistent with gpw being in the fast nmr exchange regime . interestingly , the cs denaturation profiles proved to be very heterogeneous ( all cs versus t data are given in table s2 ) . a large set of the curves ( 102 ) was two - state - like ( 2sl ) , thus showing single transitions , 35 showed two apparent transitions ( three - state - like , 3sl ) , and others showed even more complex patterns ( cp ) . fitting the 2sl curves to a two - state model rendered high variability in tm and h that goes well beyond experimental uncertainty ( (h ) = 62 kj / mol and (tm ) = 9.3 k relative to the 17 kj / mol and 1.6 k that are the respective median fitting errors at 68% confidence ) . the variability in thermodynamic parameters observed for the 2sl curves results in a distribution of native probabilities that exhibits maximal heterogeneity at the denaturation midpoint . this observation is qualitatively similar to what was originally reported for the one - state downhill folder bbl . for gpw the distribution of probabilities is comparatively much sharper , in line with its distinctly sigmoidal global unfolding process . this result confirms that the atomic heterogeneity is proportional to the overall unfolding broadness of the protein , as has been proposed before , and not an artifact from poorly defined high or low temperature baselines . three - state fits of the 3sl curves also produced high variability ( the parameters for all two - state and three - state fits are shown in tables s3 and s4 ) . however , the global unfolding obtained as the signal - weighted average of all the cs data is a simple sigmoidal curve that overlaps perfectly with the denaturation profile obtained by a low - resolution backbone - sensitive technique such as cd ( figure 1b ) . this important control demonstrates that the observed variability in cs represents the true gpw thermal unfolding , which is simple in global terms but inherently complex at the atomistic level . experimental nmr analysis of the equilibrium thermal unfolding of gpw at atomic resolution . ( a ) superposition of the lowest - energy structure from the nmr ensembles obtained at ph 3.5 ( blue ) and previously determined at ph 6.5 ( cyan ) . ( b ) the global nmr thermal unfolding behavior represented by the second component from the singular value decomposition ( svd ) of the 180 cs ( blue , left scale ) is compared to the unfolding curve measured at low resolution by circular dichroism ( green , right scale ) . ( c ) the 15 different types of atomic unfolding behaviors obtained for gpw from cluster analysis . clusters 18 have a single transition ( 2sl ) , clusters 913 have two apparent transitions ( 3sl ) , and clusters 14 and 15 include curves with more complex patterns ( cp ) . each panel shows a representative experimental cs curve as example ( colored circles ) , the number of cluster elements , and the expected global behavior for reference ( black curve ) . the latter was calculated fixing the thermodynamic parameters to those of the two - state fit to the blue curve in panel b ( tm = 329 k and h = 133 kj / mol ) and fitting the native and unfolded baselines for each probe . interestingly , the distribution of atomic unfolding behaviors throughout the protein is seemingly random . for example , grouping the 102 simplest curves ( 2sl ) by atom type only reveals weak trends : backbone - reporting probes ( n and c ) unfold on average more cooperatively ( higher h ) than side - chain - sensitive c , which tend to exhibit less cooperative curves and also slightly higher tm ( table s5 ) . a more detailed analysis using data - clustering tools produced 15 different clusters ( figure 1c ) , each with its own distinctive properties ( tables s6s8 ) . all these clusters contribute to the overall gpw unfolding signal as they all contain significant numbers of curves ( three or more ) and display comparable changes in cs upon unfolding . the most characteristic one is cluster 5 , which is largest in size and reproduces the global unfolding behavior closely . in fact , considered alone , cluster 5 could be construed as solid evidence of two - state equilibrium unfolding behavior . however , such an argument implies discarding 80% of the curves and 71% of the total cs signal . the elements of the different clusters are spread around the gpw sequence without revealing any obvious structural or topology - based pattern ( figure 2 ) . for example , elements from cluster 5 are found in both -helices , the -hairpin , and the short loops connecting them . what figure 2 reveals , however , is that cs curves from the same residue are often classified in the same or a similar cluster , indicating that multiple atomic probes provide consistent information on unfolding at the residue level . another general feature that emerges from figure 2 is the large concentration of cp curves on both tails , which is consistent with looser coupling of the tails to the rest of the protein . overall , the entire cs data set provides a close look at atomistic complexities of the gpw equilibrium unfolding process that are difficult to obtain using other currently available experimental methods , and which demonstrate that nmr can expose a great deal of structural detail behind the rather sharp equilibrium unfolding process of a barrier - crossing fast - folding protein . we thus conclude that the atom - by - atom nmr approach is extensible from the one - state downhill folding regime to a more general scenario , confirming experimentally what had been proposed before by theory and providing an excellent opportunity to compare experiments and simulations at the atomic level . the elements of the 15 clusters obtained from the analysis of the 180 nmr cs curves are overlaid on a schematic representation of the gpw native structure . md simulations offer the potential for direct examination of folding and unfolding trajectories at an atomic level of detail . until recently , however , even the longest such simulations did not reach the time scale on which protein folding takes place . in recent years , the development of fast kinetic experiments has led to the discovery of many proteins that approach the s folding speed limit , while special - purpose hardware has extended the reach of continuous , all - atom , explicit - solvent md simulations to the millisecond time scale . these advances have allowed the execution of single equilibrium simulations encompassing the repeated folding and unfolding of a number of small proteins , shedding light on various aspects of the folding process . such simulations , however , are based on imperfect physical models ( force fields ) of the interatomic interactions that underlie the dynamics of real biomolecular systems . the ability to compare the results of gpw simulations with corresponding experimental measurements is thus useful not only for developing a deeper understanding of the process of protein folding , but for assessing and improving the accuracy of current force fields . gpw is indeed a particularly suitable protein model for such purposes , as it has been recently demonstrated by comparing , at the chemical shift level , the properties of a partially unfolded species detected by nmr relaxation dispersion and predicted by coarse - grained simulations . in the work reported here , we performed a number of md simulations in parallel with the experiments described above . using anton , a special - purpose supercomputer designed for md simulations , we performed ( i ) a 250 s simulation of the reversible ( un)folding of gpw at 340 k ( the approximate tm in the force field ) to characterize the folding kinetics and ( ii ) four independent 200 s simulated tempering ( st ) simulations to calculate temperature - dependent properties equivalent to those investigated experimentally by nmr . from the comparison between the five runs , we could assess the simulation convergence and the statistical errors of the calculated quantities . all simulations were started from an extended conformation , and transitions from disordered conformations to an ensemble of structures consistent with the native state ( c-rmsd below 1 from the nmr structure ) were observed multiple times in each simulation . visual inspection of the c-rmsd time series for the 340 k simulation ( figure 3a ) reveals a high degree of structural heterogeneity , strongly suggesting that gpw samples several metastable states in addition to the native and unfolded states . a kinetic - clustering analysis is indeed consistent with the presence of several metastable states that interconvert on the microsecond time scale ( figure 3b ) . near the denaturation midpoint , the native state appears to be in fast equilibrium with a folding intermediate ( i2 ) in which the two helices and the native hydrophobic core are well formed , but where the -hairpin is largely unstructured . ( a ) time series of the c-rmsd from the first structure of pdb entry 2l6q obtained from a 250 s equilibrium md simulation at 340 k started from an extended conformation . ( b ) markov model of the folding free - energy surface obtained from a kinetic - cluster analysis of all the simulation trajectories combined . the rates of interconversion between clusters , estimated from the equilibrium md simulation at 340 k , are also reported . ( c ) population of the metastable states identified in the cluster analysis as a function of temperature . statistical errors for the cluster populations are estimated from comparison of the two independent st simulations . the cluster populations observed in the equilibrium md simulation at 340 k are reported as circles . i2 is in slightly slower kinetic exchange with a more unstructured state in which only helix 1 is formed ( i1 ) . the cluster analysis identifies two additional , partly folded ensembles that are stable on the microsecond time scale : one in which the -hairpin is formed but the helices are disorganized ( m1 ) , and another in which helix 1 is formed and interacts in non - native fashion with a partially formed helix 2 ( m2 ) . m1 and m2 can be considered misfolded kinetic traps , and have populations of 15% at 340 k. this model is consistent with a folding mechanism in which helix 1 is formed early on the folding pathway , while the -hairpin is the last structural motif to consolidate . from the st simulations , we calculated the changes in population of each metastable state as a function of temperature ( figure 3c ) . the populations at 340 k extracted from st and the constant - temperature md simulation ( circles in figure 3c ) agree to within 10% , with the exception of the unfolded state , which is less populated in the latter . given the larger amount of data collected , the better convergence properties of st over md simulations , and the good agreement between the different st simulations , this discrepancy ( 1 rt error in relative free energy ) is likely to reflect a larger statistical error in the constant - temperature md simulation . as expected , the population of the fully disordered unfolded state ( u ) increases steadily with temperature , and dominates at the highest temperature . at low temperature , i2 and the native ensemble are the most populated states , but there is some structural heterogeneity with a substantial population of m2 . i1 and m1 reach maximal population near the denaturation midpoint . the relative thermal stability of the different structural clusters reflects their relative order of appearance along the folding pathway , similarly to what has been previously observed for other fast - folding proteins , thus indicating that useful mechanistic information can in this case be obtained from thermal denaturation data . the structural ensemble obtained from the md simulations at 300 k conforms with the noes measured experimentally at the same temperature ( average violation 0.02 ; only 2 noe restraints are violated by more than 1.5 ) . the native conformations in the simulations are thus very similar to the experimental nmr structure ( figure 4a ) . we thus measured the folding - unfolding kinetics of gpw at ph 3.5 using nanosecond infrared t - jump experiments . the folding relaxation of gpw could be well fit to a single exponential decay for all temperatures . at the same temperature of the standard md simulation ( 340 k ) the relaxation rate measured experimentally is 1/(8.8 s ) ( blue in figure 4b ) , which is essentially identical to the relaxation rate previously measured at neutral ph . for the simulations , we used the number of helical residues as a proxy for the ir signal and calculated its autocorrelation function over the 250 s equilibrium md simulation ( red in figure 4b ) . the obtained decay could not be properly fitted to a single exponential decay , but could be well fitted to a double - exponential function with rates of 1/(0.5 s ) and 1/(4.6 s ) . the fast phase corresponds to helix - melting and -forming events that seem to occur within metastable states in the simulation , and it is thus not associated with folding . the slow process , which accounts for most of the amplitude and corresponds to the overall folding process in the simulations , is in good agreement with the experimental midpoint relaxation rate measured here at ph 3.5 ( figure 4b ) and in a previous work at ph 6 . the agreement further improves if we consider the low water viscosity of the tip3p model , which typically results in roughly a 2-fold speedup of the folding kinetics . from a coarse - grained viewpoint , the md simulations thus appear to provide an excellent description of the global features of gpw folding . comparing long - time - scale simulations of gpw folding with experiments . ( a ) structural comparison showing the superposition of one representative conformation of the gpw folded state from st simulations ( red ) and the lowest - energy structure from the nmr ensemble at ph 3.5 ( blue ) . the experimental ir relaxation kinetics to a final t = 340 k is shown in blue , and its fit to a single exponential function in cyan . the simulation kinetics is shown in red , its fit to a double exponential function in yellow , and the resulting amplitudes of the fast and slow phases in gray . the average atomic unfolding signal is obtained from the singular value decomposition of the 180 cs experimental or simulation curves , and then normalized by fitting to a two - state model . the red circles show the simulation curve and the blue line the nmr experimental curve of figure 1b . the temperature scale for the simulation ( top ) is compressed by a factor of 2.97 and shifted by 7 degrees relative to the experimental scale ( bottom ) . the upper bar shows the color - coded specific segments in gpw and the vertical lines the segment tm . in addition to customary comparisons of native structure and global quantities like folding rate and melting temperature , the combination of the nmr experiments ( figure 1 ) and the st simulations offer the opportunity for a more detailed comparison between simulation and experiment with regard to the thermodynamic and structural properties of gpw as a function of temperature . to achieve this , we calculated the average n nh , c , and c cs for all the conformations observed in the st simulations at each temperature using both camshift and sparta+ . such average computed chemical shifts are equivalent to the experimental values , which for a protein in the fast nmr exchange regime are also population averages . the first important observation is that individual computed cs curves are also very heterogeneous . the cluster analysis classified the data in 13 clusters with more than three curves , which display significant differences in cooperativity and denaturation midpoint ( figure s3 ) . in general , the computed data recapitulates the heterogeneity observed experimentally with the exception of the most complex behaviors ( cp curves ) . figure s3 also shows that the ensemble variability in cs for each temperature is small ( although it increases somewhat at the lowest temperatures due to poorer sampling ) relative to the overall changes resulting from unfolding . as observed before for the nmr experiments , however , direct comparison reveals that the simulated unfolding is about 3 times broader than its experimental counterpart , whereas the agreement in denaturation temperature is good ( within 7 k ) . the much broader melting reflects a substantial underestimation of the unfolding enthalpy in simulations a phenomenon that appears to be a general ( though not yet completely characterized ) feature of current biomolecular force fields . when the simulated temperature scale is compressed accordingly , however , the average cs curves overlay almost perfectly ( figure 4c ) . the agreement with experiment the computed cs curves can not be fitted to two- or three - state models as accurately as the experiments because they have higher data point fluctuations and a more poorly defined low temperature baseline . as a way to surmount this limitation , we classified the computed and experimental cs curves by protein segments to compare the average unfolding properties of the various secondary structure elements in gpw . in this analysis the experimental data shows that the second -strand , which has no tertiary contacts beyond the hairpin , is intrinsically less stable ( midpoint 5 k below the global tm ) . all other segments have tm values closer to the global process , with the first -strand exhibiting the lowest tm among them and the beginning of helix 2 the highest one ( figure 4d ) . at this level , most of the atomic heterogeneity is thus averaged out , indicating that the various secondary structure elements unfold almost concertedly , with the exception of strand 2 . the simulations draw a slightly different picture in which there is more variability among protein segments ( figure 4e ) . the simulations do pick up the low stability of the second strand , but extend it to the whole hairpin region ( 1-2 and l2 ) . moreover , in the simulation helix 1 is more stable than helix 2 . to investigate the unfolding of gpw in more depth this method was originally devised for inferring the interaction network of one - state ( global ) downhill folding proteins from equilibrium nmr unfolding experiments . the degree of coupling between every pair of residues is thus obtained from the similarities among the unfolding patterns of all the possible cs - curve pairs ( see the methods section for details ) . a strong coupling between two residues is interpreted as indicative of concerted unfolding , whereas a weaker coupling implies uncorrelated unfolding events ( e.g. , residues with very different tm ) . primary couplings are between residues interacting in the native structure , and secondary couplings occur when the two residues are indirectly connected through mutual interactions with others . the approach is directly extensible to the nmr data of gpw because it is atomistically complex ( figure 1 , table s2 ) , and is also transferable to the simulations , since we have computed cs thermal unfolding curves for all the relevant atoms . figure 5 shows the thermodynamic coupling matrices resulting from the nmr experiments ( left ) and simulations ( right ) . both the experimental and simulation - based matrices exhibit a complex pattern of high ( red ) , intermediate ( yellow ) , and low ( blue ) couplings , indicating that at the residue level there still is significant variation in unfolding behaviors . as first step in this analysis , we take advantage of the md simulation results to better understand and test the mechanistic connection between the thermodynamic coupling matrix and the underlying folding interaction network . this is an important issue that could not be examined before because the nmr experimental data does not provide the structural information about the folding mechanism that is readily obtained from the simulated trajectories . along these lines , we find that the thermodynamic information encoded in the simulated coupling matrix ( figure 5 , right ) agrees with the mechanistic information obtained from the kinetic clustering of the simulations ( figure 3b ) . kinetic clustering shows that the -hairpin is flexible in the native state , and unstructured in all other clusters ( the only exception is m1 , in which the hairpin is the only formed structure ) . consistently , the hairpin is virtually uncoupled to everything else ( figure 5 , right ) and has the lowest melting temperature ( figure 4e ) . thus , the structural element with lowest thermodynamic stability in the simulations forms last during folding and is also the least coupled to other protein regions in the matrix . mapping the folding interaction network of gpw from the residue upper panels show the experimental ( left column ) and simulation - based ( right column ) residue residue thermodynamic coupling matrix ( highest coupling in red , lowest in dark blue ) , with native contacts identified using hollow black circles . the bottom right triangle shows the entire coupling matrix , while the upper left triangle shows only primary couplings ( interacting residues ) . the structural and sequence distribution of the overall degree of coupling ( defined as the sum of the couplings of a given residue to all other residues ) is shown in the intermediate and bottom panels . residues were classified in six levels of coupling ( from low to high : dark blue , light blue , cyan , yellow , orange , red ) using data clustering tools . the two helices can be either formed or frayed in various clusters , but when they are formed , they tend to be formed for their full length ( see i1 , i2 , and m2 in figure 3 ) , in agreement with their strong local coupling ( figure 5 , right ) . helix 1 , which is better defined structurally than helix 2 in all clusters , exhibits stronger secondary couplings . the presence of interactions between the end of helix 1 and beginning of helix 2 in multiple clusters ( native , i2 , and to a lesser extent , i1 ) is consistent with the strong interhelix coupling in the matrix , while the very transient interactions between the protein ends are consistent with their weak coupling observed in the matrix . residue coupling matrices as an analytical tool for probing protein folding interaction networks . moreover , the computed coupling matrix provides a picture of the folding process in simulation , in which individual helices can form independently of global folding , that is consistent with the simulated ir relaxation profiles ( figure 4b ) and the kinetic clustering analysis ( figure 3b ) . this consistency further supports the use of equilibrium denaturation cs measurements as a way to obtain residue this exercise is highly instructive from the viewpoint of evaluating and enhancing force field accuracy . figure 5 shows that the simulated and experimental matrices do indeed share their main features . as in the simulation - based coupling matrix , for example , the experimental matrix identifies strong coupling between the end of helix 1 and the loop and the beginning of helix 2 , as well as marginal coupling between the protein ends and between the -hairpin and the rest of the protein . moreover , high local coupling is found in the helical regions . at the residue level the similarities are notable to the extent that the simulations correctly capture the polarized distribution of strongly and weakly coupled residues along the length of the two helices and the hairpin ( see the color patterns plotted on the gpw structure ; figure 5 middle ) . such similarities indicate that the simulations reproduce the major structural and mechanistic aspects of gpw ( un)folding . certain differences between the experimental and simulation - based coupling matrices emerge , however , when inspected in more depth . in the experiment , the residues with high overall coupling are more scattered throughout the sequence ( figure 5 , bottom ) whereas in the simulation , coupling tends to concentrate in the two helices ( figure , bottom ) , consistent with the observation of a less cooperative unfolding process ( figure 4d , e ) . in particular , the simulations do not capture a critical structural node that includes the hinge connecting the hairpin with helix 2 ( l2 , residues 3541 ) and the middle of helix 1 ( h1 m , residues 715 ) . for instance , l2 and h1 m couple with the other hinge ( residues 1924 ) and the beginning of helix 2 ( residues 4148 ) through primary contacts . strong secondary couplings ( red - orange squares with no contacts ) then link both segments to the remainder of both helices and to some residues in the first half of the hairpin , thus expanding the network over most of the protein . in summary , while the simulations and the nmr experiments give consistent results with regard to the main aspects of the folding process , with early formation of helices and late formation of the hairpin , the simulated thermal unfolding process is more decoupled at the level of secondary structure elements than that demonstrated experimentally , providing a structural basis for the decreased cooperativity observed in simulations ( figures 3 and 4 ) . we speculate that the higher stability of isolated secondary structure elements in simulation may result in a free energy surface containing more metastable states than observed experimentally . a possible reason for this discrepancy is that current force fields and water models tend to produce unfolded states that are too collapsed , and this collapse may lead to overstabilization of individual secondary structure elements , making the folding transition less cooperative . theory and simulations have suggested that complex folding mechanisms may still result in global two - state kinetics , but obtaining experimental evidence for this type of conjecture has been challenging , since it requires the use of a method that provides a level of structural detail comparable to that of atomistic simulations . the nmr experiments reported here constitute a step in that direction , revealing that the apparently simple and cooperative global unfolding behavior of the protein gpw , which is in many ways consistent with two - state folding ( and thus in principle an all - or - none process ) , hides a far richer process at the atomic level . the combination of experiments and simulations demonstrates that analysis of such atomistic details obtained from nmr data in terms of residue residue coupling matrices , a technique that was originally developed for the investigation of one - state downhill folding mechanisms , provides important information related to the folding interaction network and mechanism of gpw . moreover , the inherent features of gpw folding indicate that the experimental determination of residue residue coupling matrices by nmr should be broadly applicable to fast folding proteins , even if they fold by crossing a free energy barrier . practically , the reach of the method will be determined by the interplay between two interrelated parameters . first , the folding rate of the protein must be considerably faster than the chemical shift time scale ( i.e. , rates higher than 1/(200 s ) ) to ensure fast - exchange nmr conditions . the second parameter is the intrinsic degree of folding cooperativity , because above a certain threshold the atomic heterogeneity might be indistinguishable from experimental uncertainty . as the rate slows down , folding cooperativity will concomitantly increase , and thus the nmr analysis of sharper , more challenging unfolding transitions will also encounter decreased resolution due to line broadening . nevertheless , there is nowadays an ample collection of single - domain proteins that have been experimentally identified to fold in microseconds and which contains examples from all major structural classes . this nmr experimental procedure thus emerges as a new approach to the characterization of folding interaction networks and as a complement to md simulations in addressing the longstanding challenge of characterizing the folding and unfolding of proteins with atomic resolution . a sequence containing residues 162 of the original gpw gene described by davidson and co - workers was subcloned into the expression vector pbat . both unlabeled and uniformly n- and c - labeled gpw protein were produced using the same procedures previously described . thermal unfolding experiments monitored by far - uv circular dichroism were performed on a jasco j-815 spectropolarimeter equipped with peltier thermal control using a 1 mm path - length cuvette . the samples were prepared at a protein concentration of 30 m in 20 mm citrate buffer adjusted to ph 3.5 . nmr samples of c , n - labeled gpw were prepared in a shigemi tube at 1 mm concentration in 20 mm glycine buffer , 0.1 mm nan3 , ph 3.5 , 5% d2o / h2o and 100% d2o . under these conditions nmr experiments were acquired at 294 k in a bruker avance iii 600 mhz spectrometer equipped with a triple resonance z - axial - gradient probe . sequence backbone chemical shift assignments were obtained from the following experiments : [ h n ] hsqc , hnco , hncacb , and cbca(co)nh . side chain h , n , and c assignments were obtained from hbha(co)nh , h(cco)nh , c(co)nh , and hcch - tocsy . noe data were obtained from 3d n - hsqc - noesy and 4d c - hmqc - noesy using a mixing time of 110 ms . all experiments were processed with nmrpipe and analyzed with pipp . for the structure determination of gpw , experimental restraints derived from noe cross - peaks , hydrogen bond distances , and dihedral angles were obtained . only unambiguous interproton distance restraints were used , and errors of 25% of the distances were applied to obtain lower and upper distance limits . hydrogen bond distance restraints ( rnh o = 1.92.5 , rn o = 2.83.4 ) were defined according to the experimentally determined secondary structure of the protein . the program talos+ was used to obtain 94 and backbone torsion angle constraints for those residues with statistically significant predictions . structure calculations were performed with the program x - plor - nih 2.16.0 by minimizing a target function that includes an harmonic potential for experimental distance restraints , a quadratic van der waals repulsion term for the nonbonded contacts , and a square potential for torsion angles . starting structures were calculated and heated to 3000 k and cooled in 30 000 steps of 0.002 ps during simulated annealing . the final ensemble of 20 nmr structures was selected based on lowest energy and no restraint - violation criteria . these conformers had no distance restraint violations and no dihedral angle violations greater than 0.3 and 5 , respectively . structures were validated using procheck - nmr and molprobity , which show that the family of 20 structures are of considerable high quality in terms of geometry ( 92.6% of the residues populating the most favored regions of the ramachandran plot for the whole ensemble ) and side chain packing . coordinates were deposited in the protein data bank with accession code 2l6r and chemical shifts were deposited in the biological magnetic resonance bank ( bmrb code 17322 ) . we obtained the chemical shift values of the side chain co carbons of all asp and glu residues in gpw by analyzing a series of h(ca)co spectra , a 2d version of the hcaco experiment . the h1 chemical shifts of the his15 were determined from a series of [ h c ] hsqc spectra . all the spectra were recorded on an 1 mm c , n - double labeled gpw sample , 0.1 mm dss in d2o at different phs ranging from 1.5 to 8.1 ( 8.11 , 7.16 , 6.01 , 5.09 , 4.08 , 3.57 , 3.02 , 1.98 and 1.53 ) and 294 k. nmr spectra were analyzed with sparky . the pka values were obtained by a nonlinear least - squares fit of the experimental ph titration curves to the following equation : where 1 and 2 are the chemical shift values at the lowest and highest ph , respectively . data fitting was done with origin pro 8.0 ( originlab ) . as a first step , the reversibility of gpw thermal unfolding at the protein concentrations required for multidimensional nmr experiments was assessed using a nmr sample consisting of 1 mm n - labeled gpw in 20 mm glycine buffer , 0.1 mm nan3 , ph 3.5 , 5% d2o / h2o . this sample was heated up to 100 c for a period of 2 h and then cooled back down . the perfect cross - peaks superimposition of the two [ h n ] hsqc spectra recorded before and after heating , indicated full reversibility in the relevant conditions . equilibrium thermal unfolding was investigated using multidimensional nmr experiments to monitor the changes in h , n , and c chemical shifts in the 273371 k temperature range . for this purpose we used a bruker 5 mm txi - probe with z - axis gradient able to stand up to 423 k and a bto temperature control unit . accurate temperature calibration was carried out monitoring the ethylene glycol cs at every experimental temperature using a sample of ethylene glycol prepared in a regular 5 mm diameter nmr tube and using an air flow - rate of 535 l / h and following the procedure described by amman et al . briefly , the neat ethylene glycol sample was equilibrated for 30 min at each temperature before acquiring the spectrum . standard one - pulse 1d h nmr experiments were used to monitor the changes in proton frequency of ch2 and oh with temperature . these changes were converted to temperature using equation : t(k ) = 466.5 102.00 . for the thermal unfolding experiments we employed a nmr sample at 1 mm concentration of c , n - labeled gpw in 20 mm glycine buffer , 0.1 mm nan3 , 0.01 dss , ph 3.5 , 5% d2o / h2o . in these experiments the water resonance was used as spectral reference since the observation of the standard dss signal is precluded in multidimensional nmr experiments because of isotope filtering . thus , before recording the set of multidimensional experiments , a 1d h nmr experiment was recorded and employed to calibrate the position of the water signal relative to dss at each temperature . pressurizable tubes manufactured by new era enterprises ( u.s . ) with 0.77 mm thick glass were used to minimize evaporation of the sample . we measured the ph of nmr samples at the different experimental temperatures using a thermal block and a ph - meter recalibrated at each temperature . we observed that the ph of the samples was held constant at a value of 3.5 0.1 over the whole temperature range . protein backbone amide n , h , c , and c chemical shifts were assigned with the following set of experiments : [ h n ] hsqc , cbca(co)nh , and hncacb . gpw folds in fast exchange regime relative to the nmr chemical shift time scale , and thus , each atom produces a single chemical shift corresponding to the dynamically averaged conformational ensemble at every condition . the ensemble average chemical shifts for all relevant atoms were fully assigned in the 273371 k temperature range at fixed intervals of 5 k. at temperature conditions within the unfolding transition region ( slightly below and above the tm ) the temperature interval for nmr experiments was reduced to 3 k to increase the data density in the region of largest change in cs . all experiments were processed with nmrpipe and analyzed with pipp to obtain accurate chemical shift determinations using a multilevel contour averaging procedure . at ph 3.5 and high temperatures gpw experienced slow proteolysis at specific positions in the primary sequence involving glutamine residues . we thus monitored protein integrity by comparing [ h n ] hsqc spectra recorded before and after every set of triple - resonance nmr experiments . at temperatures higher than 345 k the gpw proteolysis rate was higher than 1/(5 days ) so we used a freshly prepared sample for recording the suite of triple resonance nmr experiments at each temperature above 345 k. folding - unfolding relaxation kinetics of gpw was measured using a nanosecond laser - induced t - jump apparatus equipped with infrared detection . samples were prepared at 4 mg / ml concentration in 20 mm deuterated glycine buffer , after multiple cycles of liophilization and dilution in d2o to achieve complete deuteration of the exchangeable amide protons . the buffer and sample were adjusted to pd = 3.5 , correcting for the isotope effect on the glass electrode readout . the relaxation kinetics of gpw after a jump of 10 k to a final temperature of 328 k were recorded on a custom - built infrared laser - induced temperature apparatus described previously . briefly , the fundamental wavelength of a nd : yag laser ( continuum surelite i ) operating at a repetition rate of 4 hz is shifted , by passing through a 1 m path raman cell ( lightage inc . ) filled with a high pressure mixture of h2 and ar , to 1907 nm , resonant with the infrared absorption of d2o . in this way pulses < 10 ns with energy of up to 30 mj were obtained , that induced a local temperature jump of about 1012 when focused onto the sample . the folding - unfolding relaxation kinetics of gpw was observed using a quantum cascade laser ( daylight solutions ) tuned to 1632 cm to match the absorption of the helix signal within the gpw amide i band . the light transmitted from the sample was recorded using a fast mct detector ( kolmar technologies ) coupled with an oscilloscope ( tektronix dpo4032 ) . samples were held in a cell formed by two mgf2 windows separated by a 50 m teflon spacer and thermostated at the proper base temperature using two peltier thermoelectric coolers ( te technology inc . ) in a custom - built sample holder . the transmission of d2o was used as an internal thermometer to measure the amplitude of the temperature jump . molecular dynamics ( md ) simulations of gpw were initiated in an extended conformation . the 62-amino acids sequence mvrqeelaaaraalhdlmtgkrvatvqkdgrrveftatsvsdlkkyiaelevqtgmtqrrrg was solvated in a cubic 60 60 60 box , containing 6345 water molecules and 15 mm nacl . asp , glu , arg , lys , and his residues were treated as charged . the charmm - h force field ( see som ) was used to represent the system . the system was initially equilibrated at 340 k and 1 bar in the npt ensemble . production runs were performed with the anton specialized hardware using a 2.5 fs time step . bonds involving hydrogen atoms were restrained to their equilibrium lengths using the m - shake algorithm . nonbonded interactions were truncated at 10 , and an ewald method was used to describe the long - range electrostatic interactions . four simulated tempering simulations of 200 s each , were performed in the npt ensemble . two simulations used 18 temperature intervals between 278 and 398 k , and two used 21 temperature intervals between 278 and 438 k. exchanges between temperatures were attempted every 20 ps of simulation . to gain additional kinetic information , we also performed a 258 s equilibrium md simulation at 340 k in the nvt ensemble . a kinetic clustering analysis was used to identify the most relevant metastable states on the free - energy surface through a fit of the autocorrelation functions of 400 cc contacts on time scales ranging from 0.5 to 50 s . in order to obtain consistent clustering , helical residues for the analysis of the autocorrelation function were identified according to the stride definition , as implemented in vmd . as a first step , all obtained experimental cs curves were classified in three groups according to the unfolding behavior : ( 1 ) 102 curves were classified as 2-state - like ( 2sl ) curves because they had a distinctly sigmoidal trend characterized by a single transition and a single peak in their derivative ; ( 2 ) 35 curves were classified as 3-state - like ( 3sl ) because they exhibited two apparent transitions and thus a bisigmoidal shape and two peaks in their derivative ; and ( 3 ) 43 curves did not fit any of the other two categories ( whether because they exhibited multiple transitions or no obvious transition ) , and were thus included in the complex - pattern ( cp ) group . the cs curves classified as 2sl and 3sl were fitted to a thermodynamic two - state or a three - state unfolding model equation , respectively , using the procedures described previously . the data set containing all of the experimental cs curves was analyzed globally using data clustering methods . cs curves belonging to the 2sl and 3sl groups were clustered according to both the thermodynamic parameters obtained in their fits ( tm and h for 2sl and tm1 , tm2 , h1 , and h2 for 3sl ) or according to the probability of the native state as a function of temperature obtained from the two and three fits . both methods rendered equivalent results . cs curves included in the cp group were clusterized according to their similarity after normalization of the signal using the z - score procedure . all clustering routines were performed with the kmeans algorithm by running 10 000 trials to guarantee convergence and selecting the solution with minimum residuals . the 102 2sl curves were clustered into 12 clusters , out of which 8 clusters had at least 3 elements , whereas the other 4 clusters had only 1 or 2 elements . clustering of the 35 3sl curves produced 5 clusters with at least 3 elements . cp curves were grouped in 10 clusters by kmeans according to their zscored shape , out of which only 2 clusters had 3 elements or more . using these procedures , a total of 15 characteristic clusters were identified with at least three elements containing in total 132 atomic unfolding curves . the thermodynamic coupling index ( tci ) for each pair of residues in a protein is calculated from the pairwise comparison between all the cs curves of the first residue in the pair with all the curves of the second residue . the thermodynamic coupling index of residues x and y is then calculated by summing all the possible pairwise comparisons of atomic folding curves as reported in the following equation : where pm and pn are row vectors from a matrix including all the zscored cs curves in the protein . the numerator thus corresponds to the mean rmsd for all cs curves in the data set . px , i and py , j are the vectors from the same matrix that correspond to the cs curves of residues x and y , respectively ( i runs over all curves of residue x and j over all curves of residue y ) . the tci for residues x and y is positive when the average euclidean distance between all their cross - pairs is smaller than the mean rmsd for all cs curves in the protein and negative otherwise . the tci matrix is constructed by repeating the same procedure over all possible residue pairs . this procedure was applied to both the experimental cs curves measured by nmr and the synthetic cs curves computed with either camshift or sparta+ from all the conformations observed at each temperature in the st simulations .

the integration of atomic - resolution experimental and computational methods offers the potential for elucidating key aspects of protein folding that are not revealed by either approach alone . here , we combine equilibrium nmr measurements of thermal unfolding and long molecular dynamics simulations to investigate the folding of gpw , a protein with two - state - like , fast folding dynamics and cooperative equilibrium unfolding behavior . experiments and simulations expose a remarkably complex pattern of structural changes that occur at the atomic level and from which the detailed network of residue residue couplings associated with cooperative folding emerges . such thermodynamic residue residue couplings appear to be linked to the order of mechanistically significant events that take place during the folding process . our results on gpw indicate that the methods employed in this study are likely to prove broadly applicable to the fine analysis of folding mechanisms in fast folding proteins .

Introduction Results and Discussion Conclusions Methods

PMC4075200

arterial variations of upper limb are very common and different variations have been described in the literature , first mentioned by von haller in 18 century . as such these variations are of not much importance in normal individuals , but we here mention a rare case of such a variation in which the presence of an anomalous superficial ulnar artery in addition to the normal radial and ulnar arteries was used to our advantage to harvest a pedicle flap to cover an elbow defect thus avoiding the morbidity and cosmetic deformity associated with the radial artery forearm flap . we in this case report want to suggest that such vascular anomalies can be of advantage if we are prepared to think out of the box ! a 60 year old right handed male presented with the complaints of swelling in the left lower arm over the medial aspect for the past 6 months and progressively increasing in size . examination revealed a 7 cm 5 cm swelling with central ulceration on the anteromedial aspect of the left lower arm [ figure 1a ] . magnetic resonance imagingrevealed a well - defined lobulated lesion measuring 5 4 4.2 seen involving the subcutaneous tissue of the medial aspect of the lower arm . the patient was treated with pre - operative external beam radiotherapy to the left elbow , using cobalt 60 , 50 gy/25#/37 days . later he was planned for surgical excision of the lesion and cover with pedicled radial artery forearm flap / ulnar artery perforator flap . ( a ) pre - operative photo , ( b ) defect , ( c ) access incision exposing the superficial ulnar artery , ( d ) flap incision committed for a superficial ulnar artery based flap , ( e ) normal deep radial and ulnar arteries , ( f ) final flap inset , ( g ) follow - up at 6 weeks following wide excision , there was an 8 cm 9 cm defect exposing the biceps tendon , brachial artery , median and ulnar nerve , all the structures were uninjured . an access incision made over the middle of the forearm for the planned pedicled radial artery flap / ulnar artery perforator flap , on incising the skin , there was an anomalous superficial branch arising from the brachial artery at the level of elbow running up to the distal third forearm [ figure 1c ] , multiple perforators were visualised arising from this vessel and going into the dermis.from the same incision radial and ulnar vessels were visualised.in view of visible perforators and uninjured vessels up to the brachial artery and venae comitantes , decision was taken to base an islanded flap on this anomalous vessel with skin paddle dimension of 10 cm 8 cm [ figure 1d].no significant perforators from ulnar artery were seen during the further dissection.as the vessel was arising in arm from brachial artery the arc of rotation was less and flap could be raised from the muscular part of the forearm ( c.f . radial artery forearm flap ) with potentially less chance of donor site morbidity.both major vessels of forearm were spared [ figure 1e].flap bleed was excellent [ figure 1f ] . multiple perforators were visualised arising from this vessel and going into the dermis . from the same incision radial and ulnar vessels uninjured vessels up to the brachial artery and venae comitantes , decision was taken to base an islanded flap on this anomalous vessel with skin paddle dimension of 10 cm 8 cm [ figure 1d ] . . as the vessel was arising in arm from brachial artery the arc of rotation was less and flap could be raised from the muscular part of the forearm ( c.f . radial artery forearm flap ) with potentially less chance of donor site morbidity . both major vessels of forearm were spared [ figure 1e ] . patient had an uneventful post - operative recovery with 100% graft take [ figure 1 g ] . various studies have provided a classification of the arterial variations in the upper limb , as well as a model of arterial development based on the study of large anatomical and embryological samples . arterial variations in the upper limb have been the subject of much controversy since von haller mentioned their existence for the first time in the 18 century . the classic theory of singer , stated that the adult pattern develops from a main axial trunk , which extends from the axilla to the fingers and represents the brachial and interrosseus artery . the different arteries arise via sprouting angiogenesis : first the median artery , then the ulnar then the superficial brachial and finally , an anastomosis at elbow level between the brachial and superficial brachial arteries , which eventually become the initial part of the radial artery , whereas the proximal segment regresses to form the definitive adult pattern . the new findings by rodriguez - niedenfuhr et al . , suggest that the arterial pattern of the upper limb develops from an initial capillary plexus by a proximal to distal differentiation ( in the forearm with a posterior anterior polarity ) due to the maintenance , enlargement and differentiation of certain capillary vessels and the regression of others . it is suggested that the persistence , enlargement and differentiation of capillaries forming the initial capillary plexus , which would normally remain in a capillary state or even regress , gives rise to arterial variations of the definitive arterial pattern , rather than sprouting of aberrant vessels . variations located exclusively in the arm : superficial brachial artery : incidence 4.8% , which instead of coursing deep to the median nerve runs in front of itarterial variations located along the arm and forearm : the brachioradial artery is the most frequent one with incidence of 14%.it originated most frequently from the upper third of the brachial artery coursing superficial to the median nerve but deep to the brachial fascia and passed behind the bicipital aponeurosis and did not anastomose with the normal brachial artery . in the forearm they course deep to the fascia and reached their usual positionsarterial variations located exclusively in the forearmthe median artery has been shown to present two different patterns , the antebrachial pattern ending in the forearm and the palmar one reaching the palm to supply the fingers.the superficial radial artery is a radial artery coursing over the tendons defining the snuff box with incidence of 0.4% . variations located exclusively in the arm : superficial brachial artery : incidence 4.8% , which instead of coursing deep to the median nerve runs in front of it arterial variations located along the arm and forearm : the brachioradial artery is the most frequent one with incidence of 14% . it originated most frequently from the upper third of the brachial artery coursing superficial to the median nerve but deep to the brachial fascia and passed behind the bicipital aponeurosis and did not anastomose with the normal brachial artery . in the forearm they course deep to the fascia and reached their usual positions arterial variations located exclusively in the forearm the median artery has been shown to present two different patterns , the antebrachial pattern ending in the forearm and the palmar one reaching the palm to supply the fingers . the superficial radial artery is a radial artery coursing over the tendons defining the snuff box with incidence of 0.4% . vascular anomalies in the upper limb are of interest in raising flaps for reconstruction , one needs to be aware of such possible arterial variations in order to prevent complications and also to use such anomalous vessels for the reconstructive purposes thereby avoiding the sacrifice of major limb vessels . variations located exclusively in the arm : superficial brachial artery : incidence 4.8% , which instead of coursing deep to the median nerve runs in front of itarterial variations located along the arm and forearm : the brachioradial artery is the most frequent one with incidence of 14%.it originated most frequently from the upper third of the brachial artery coursing superficial to the median nerve but deep to the brachial fascia and passed behind the bicipital aponeurosis and did not anastomose with the normal brachial artery . in the forearm they course deep to the fascia and reached their usual positionsarterial variations located exclusively in the forearmthe median artery has been shown to present two different patterns , the antebrachial pattern ending in the forearm and the palmar one reaching the palm to supply the fingers.the superficial radial artery is a radial artery coursing over the tendons defining the snuff box with incidence of 0.4% . variations located exclusively in the arm : superficial brachial artery : incidence 4.8% , which instead of coursing deep to the median nerve runs in front of it arterial variations located along the arm and forearm : the brachioradial artery is the most frequent one with incidence of 14% . it originated most frequently from the upper third of the brachial artery coursing superficial to the median nerve but deep to the brachial fascia and passed behind the bicipital aponeurosis and did not anastomose with the normal brachial artery . in the forearm they course deep to the fascia and reached their usual positions arterial variations located exclusively in the forearm the median artery has been shown to present two different patterns , the antebrachial pattern ending in the forearm and the palmar one reaching the palm to supply the fingers . the superficial radial artery is a radial artery coursing over the tendons defining the snuff box with incidence of 0.4% . vascular anomalies in the upper limb are of interest in raising flaps for reconstruction , one needs to be aware of such possible arterial variations in order to prevent complications and also to use such anomalous vessels for the reconstructive purposes thereby avoiding the sacrifice of major limb vessels . the usefulness of the flap lies in it uncomplicated dissection , preservation of both major vessels of forearm thus avoiding any vascular compromise to the extremity . its advantage also lies in the fact that the donor site healing is uneventful and cosmetically acceptable when covered with a skin graft . the superficial ulnar artery has been previously reported to as a trap while raising a radial forearm flap .

upper limb shows a large number of arterial variations . this case report describes the presence of additional superficial ulnar artery which was used to raise a pedicle flap to cover an arm defect thus avoided using the main vessel of the forearm - radial or ulnar artery . vascular anomalies occurring in the arm and forearm tend to increase the likelihood of damaging the superficial anomalous arteries during surgery . superficial ulnar or radial arteries have been described to originate from the upper third of the brachial artery ; here we report the origin of the anomalous superficial ulnar artery originating from the brachial artery at the level of elbow with the concomitant presence of normal deep radial and ulnar arteries .

INTRODUCTION CASE REPORT OPERATIVE FINDINGS DISCUSSION Incidence of arterial variations CONCLUSION

PMC3914190

laser in situ keratomileusis is approved by the us food and drug administration ( fda ) for the treatment of myopia , hyperopia , and astigmatism and has been a popular choice among refractive surgeons for almost 2 decades now [ 1 , 2 ] . hyperopic lasik consists of an annular zone of ablation to cause a relative flattening of the corneal periphery and a concomitant relative steepening of the center ( optical zone ) to achieve the desired refractive effect . early attempts to correct hyperopia included techniques such as hexagonal keratotomy , thermokeratoplasty , keratophakia , and keratomileusis but have met with only limited success . with the advent of the excimer laser technology , reshaping of the corneal surface to a desired contour with submicron precision and reproducibility became a reality . with the prototype ophthalmic excimer lasers , the visual and refractive outcomes of lasik for hyperopia were less predictable due to challenges arising from the need to use larger ablation diameters and the relative difficulty to deliver uniformly distributed laser energy in ablation zones larger than 6 mm . newer models have permitted larger ablation diameters and the development of various microkeratomes facilitated the construction of larger corneal flaps [ 1 , 3 ] . despite this technology , predictability and stability are markedly reduced with treatment , especially of high levels of hyperopia [ 47 ] . with the success of excimer lasers , the desire to develop a solid - state laser with an output wavelength of 213 nm was born . this resulted from the problems associated with gas lasers , including the use of the toxic gas fluorine , which is a safety issue in the clinical environment . in recent years , this nd : yag laser has gained recognition as a possible alternative to the long - established 193 nm excimer laser system . in addition to the absence of toxic gas , recent evidences have been discussed and demonstrated the advantages of the solid - state laser over the traditional excimer laser . it has been shown to create a smoother ablation surface by the accurate transfer of energy onto the corneal stroma [ 811 ] . a smaller spot size of 0.6 mm , which is 2.5 times smaller than a typical excimer laser spot size , contributes less mechanical stress on the cornea causing less damage to the corneal structure [ 8 , 12 ] . histopathologic evidence has demonstrated a more favorable response after irradiation with 213 nm wavelength which may provide a more predictable wound healing and refractive response . furthermore , the 213 nm wavelength solid - state laser has been proven to be less sensitive to corneal hydration , limiting its effect on the final refractive outcome . the purpose of this study was to evaluate the long - term efficacy , accuracy , stability , and safety of laser in situ keratomileusis using a 213 nm wavelength solid - state laser for the treatment of hyperopia . this prospective consecutive case series was done at an outpatient refractive surgery center in manila , philippines . all patients with hyperopia who consulted at the center without any evidence of other ocular pathology that might affect the final visual and refractive outcomes were included . the preoperative and postoperative examinations included uncorrected distance visual acuity ( udva ) , subjective manifest refraction , corrected distance visual acuity ( cdva ) , cycloplegic refraction , slit lamp biomicroscopy , and keratometry ( k ) . manifest refractions ( mr ) were converted to manifest refractive spherical equivalent ( mrse ) . patients with any of the following conditions were not considered candidates for surgery : suspected ectasia or keratoconus , active ocular disease such as glaucoma and uveitis , significant cataract or retinal pathology , thin cornea ( < 500 m ) autoimmune disease , diabetes , or any other serious medical conditions . patients who are engaged in sports and military activities , breastfeeding and pregnant women , and those who did not consent for the procedure two surgeons ( a. l. d. agahan , r. p. evangelista ) performed the laser procedures from september 2006 to september 2010 . the target for 32 eyes was emmetropia while 2 eyes were overcorrected to 1.50 d beyond the target correction . preoperatively , all patients received 2 - 3 drops of topical proparacaine and 1 drop of povidone - iodine 5% solution . the suction ring was applied to the limbus and a 9.5 mm corneal flap was created using the hansatome microkeratome ( bausch and lomb surgical ) . the ablation was then carried out using the pulzar z1 213 nm wavelength solid - state laser ( customvis ) with centration based on the line of sight ( line joining the point of fixation to the center of the pupil ) . the ablation profile had an annular shape with 5.5 mm optical zone and 8.0 mm treatment zone . after the ablation was completed , the corneal flap was repositioned and the edges were dried with weck - cel sponges . the retractor was removed and one drop of prednisolone acetate 1% was given . the patients were instructed to instill moxifloxacin 0.3% 1 drop 4x / day , prednisolone acetate 1% 1 drop 4x / day , and carboxymethylcellulose 1 drop 4x / day and were discontinued after 2 weeks . follow - up visits were at 1 day , 1 week , 1 , 3 , and 6 months , and 1 , 2 , 3 , and 4 years . examination included ucva , cdva , manifest and cycloplegic refraction , slit lamp examination , applanation tonometry , and keratometry . outcome measures were accuracy ( postoperative mrse , attempted versus achieved correction ) , efficacy ( postoperative udva and cdva compared with preoperative cdva ) , stability ( average preoperative and postoperative mrse at each follow - up visit ) , and safety ( change in snellen lines of cdva ) . other outcome measures were flap- and ablation - related complications and pre- and postoperative keratometry readings . table 1 shows the baseline characteristics and demographics of all patients included in the study . twenty - eight eyes were followed up for at least 1 year , 20 eyes for at least 2 years , 14 eyes for at least 3 years , and 6 eyes for 4 years . the mean udva improved from logmar 0.6 0.25 ( ~20/80 ) preoperatively to logmar 0.17 0.13 ( ~20/30 ) at the last followup examination . figure 1 shows the cumulative udva and cdva after lasik compared with the preoperative cdva . at the last follow - up visit ( mean follow - up : 25.18 13.79 months ) , 9 eyes ( 26.47% ) 32 eyes ( 94.12% ) can see 20/40 or better without correction and all eyes can see 20/40 or better with correction . the postoperative udva of 17 eyes ( 50% ) , 25 eyes ( 73.53% ) , and 34 eyes ( 100% ) were within 1 , 2 , and 3 lines of the preoperative cdva , respectively . at the end of the followup , the postoperative mrse was within 0.50 d in 20 eyes ( 58.82% ) and within 1.00 d in 31 eyes ( 91.18% ) . none of the eyes had an mrse beyond 1.25 d of emmetropia ( figure 2 ) . the postoperative defocus spherical equivalent was within 0.50 d in 13 eyes ( 38.24% ) , within 1.0 d in 25 eyes ( 73.53% ) , and within 1.5 d in 32 eyes ( 94.12% ) . no eye had a defocus spherical equivalent of greater than 2 d at the end of followup ( figure 3 ) . the mean attempted hyperopic correction was 3.22 1.55 d and the mean achieved hyperopic correction was 3.10 1.48 d. these were shown to be correlated ( r = 0.812 ) with 19 eyes ( 55.88% ) within 0.50 d of the target refraction and 29 eyes ( 85.30% ) within 1.00 d. no eye had a final mrse > 1.25 d from the target refraction ( figure 5 ) . the linear regression equation in figure 4 also shows a general tendency towards undercorrection ( slope = 0.857 , intercept = 0.334 ) . refractive stability was attained at the first postoperative month and the mean residual refractive error remained relatively stable at each follow - up visit . there was regression of 0.3 d after the third postoperative year with an average of 0.025 d per month . more than half of the eyes ( 19 eyes , 55.88% ) achieved a postoperative cdva equal to the preoperative cdva . figure 8 shows the percentage of eyes with various amounts of astigmatism in the preoperative and postoperative period . the mean preoperative cylinder was 0.82 0.92 d and the mean postoperative cylinder was 0.60 0.50 d. the mean change in cylindrical power was 0.22 0.9 d ( range : 3 d to + 1 d ) at the end of the followup . the mean induced astigmatism was 0.66 0.23 d greater than the baseline value in 11 eyes . no eye developed astigmatism greater than 1.0 d. the mean keratometry ( k ) value was 42.81 1.81 d preoperatively and 45.53 2.01 d postoperatively . the mean difference between the preoperative and postoperative k value was 2.71 1.79 d. the mean difference between k1 and k2 was 1.23 0.66 d preoperatively and 1.04 0.47 d postoperatively . based on the k values , the mean change in cylindrical power was 0.19 0.83 d ( range : 2.25 to + 1.25 d ) and the mean induced astigmatism was 0.6 0.32 d at the end of the followup . the concept of lamellar corneal surgery to alter the refractive state of the eye was first reported by barraquer in 1949 . he suggested the possibility to manipulate the curvature of the air / tear film interface by adding or removing corneal tissue . this concept has undergone a long history of evolution , the most recent of which is the laser in situ keratomileusis . this technique combines the wound healing advantages of lamellar procedures with the precision of excimer laser . several reports have been published regarding its predictability and stability especially for low - to - moderate hyperopic refractive errors [ 2 , 5 , 7 , 1419 ] . the success of the excimer laser technology has led to the emergence of another laser technology , an nd : yag laser with an output wavelength of 213 nm . it has been acknowledged as a substitute for the excimer laser because it eliminates the use of the toxic gas fluorine , thus increasing its safety profile . our study evaluated the long - term efficacy , accuracy , safety , and stability of laser in situ keratomileusis using a 213 nm solid - state laser for the treatment of hyperopia . thirty - four eyes of 17 patients with preoperative mrse ranging from + 0.75 d to + 7.00 d ( + 2.81 1.44 d ) were included . all eyes underwent lasik using a 213 nm solid - state laser with target refraction ranging from emmetropia up to 1.50 d of overcorrection and were followed up to 4 years postoperatively ( mean followup : 25.18 13.79 months ) . at the last visit , 26.47% of eyes attained a udva of 20/20 and 94.12% had a udva of 20/40 or better . ( 20/20 , 34.1% ; 20/40 , 87.8% ) , and gker et al . ( 20/20 , 70.1% ; 20/40 , 99.3% ) , and nepomuceno et al . ( 20/20 , 44.4% ; 20/40 , 88.9% ) were better than those achieved in our study . the low proportion of eyes having a ucva of 20/20 or better at the end of followup was attributed to the relatively significant number of eyes having preoperative astigmatism of 0.50 d ( 0.5 d : 14.71% , 0.75 d : 2.94% , 1.0 d : 17.65% , 1.5 d : 14.71% , 2.25 d 2.94% , 3.0 d 8.82% ) . two of the study eyes had a preoperative mrse of + 7.0 d and at this higher degree of hyperopia , the result of lasik becomes less predictable [ 2 , 5 , 7 , 16 , 17 ] . this could also be contributory to the lower percentage of eyes having a final udva of 20/20 . in our study , the mean attempted hyperopic correction was 3.22 1.55 d and the mean achieved hyperopic correction was 3.10 1.48 d. linear regression analysis showed a good correlation between the attempted and achieved amount of hyperopic correction with r value of 0.812 . mrse at the last follow - up visit of 55.88% and 85.30% of the study eyes was within 0.50 d and 1.0 d of the intended mrse , respectively , which reflects the refractive accuracy of hyperopic lasik using the 213 nm laser . this is in accordance with other studies using the conventional excimer laser [ 2 , 4 , 5 , 7 , 14 , 15 , 1821 ] . the mean difference between the preoperative and postoperative k values of 2.71 d , which is close to the attempted hyperopic correction of 3.22 d , further mirrors its accuracy in terms of ablation profile . in addition , none of the study eyes required retreatment , which ascertains the accuracy even more . hyperopic regression of 0.3 d was observed between the third and fourth postoperative year with an average of 0.025 d per month . other published works reported refractive stability after the first month , at 3 months , and at 6 months [ 15 , 22 ] . regression was also noted at various postoperative periods , starting at 1 month , 3 months , and 1 year [ 5 , 16 ] . in the beaver dam eye study , the mean hyperopic shift over a 5-year period was + 0.12 d in a population consisting of individuals aged 4384 years . most of the hyperopic change occurred in those younger than 65 years , with a mean positive shift of 0.22 d. likewise , in the blue mountains eye study where participants of 49 years or older were enrolled , the mean hyperopic shift over a 5-year period was + 0.19 d. the largest hyperopic change was noted in persons aged 49 to 54 years at baseline , recorded at + 0.41 d. in those younger than 65 years , the mean positive change was 0.36 d. since our study consisted of patients younger than 65 years , the regression that occurred during the 4-year period reflects the normal age - related physiologic change in refraction . zadok et al . reported complications such as free cap , irregular cut , and irregular astigmatism with 1 eye losing 3 lines of bcva and 2 eyes losing 2 lines of bcva , and all were in the high hyperopia group . in our study , more than half ( 55.88% ) had no change in cdva throughout followup . the loss of cdva lines observed could be due to the high amount of corneal steepening which induced significant degree of optical aberration leading to degradation of image quality . the predictability of high hyperopic refractive errors compared with myopic refractive errors of similar degree is much poorer . it was determined that the use of a larger optical and treatment zone offers good predictability and refractive stability for higher degrees of hyperopia by diminishing undercorrection and regression associated with a smaller effective optical zone . although the upper limit of hyperopic lasik is frequently set at + 5.0 d , we do not have enough data to conclude the outcome of this study for higher degrees of hyperopia . the incidence of induced astigmatism is relatively higher than previous studies [ 16 , 18 ] , but this did not affect the postoperative cdva . possible causes of induced cylinder in lasik are alteration of the hinge placement and change in the thickness and shape of the lamellar flap . in our study , no correlation was seen between the hinge ( placed superiorly ) and the axis of the induced cylinder . we used an optical zone of 5.5 mm with a treatment zone of 8.0 mm which is slightly smaller than the currently recommended larger optical and blend zones . argento and cosentino demonstrated better accuracy and stability with the use of optical zones larger than 5.9 mm evidenced by superior visual acuity and lower residual refractive error compared to smaller optical zones . likewise compared the ablation profiles of 5 9 mm , 5.5 8.5 mm , and 6 9 mm . he noted a 2.5% increase in spherical equivalent correction for every 0.5 mm increase in optical zone size resulting in a tendency toward undercorrection and overcorrection using a 5 9 mm and a 6 9 mm ablation profile , respectively . he favored the use of a larger inner zone which should produce a lager functional optical zone . the percentage of 1-line bscva loss was the lowest in the 6 9 mm group showing a better safety profile . lastly , el - helw and emarah concluded that increasing the optical zone diameter is more predictable and stable in the correction of hyperopia . in our study , the rate of hyperopic regression was not intensified by the use of a smaller oz , contrary to the mentioned reports . the present study used a 213 nm solid - state laser which has gained popularity because of its good safety profile . the fundamental wavelength of this laser is 1064 nm , and 3 nonlinear optical crystals can be used to produce the fifth harmonic wavelength of 213 nm . in vivo studies have shown that the 213 nm laser provides advantages in the form of a more controlled ablation site and better stability in cell numbers , implying a more predictable ablation outcome . in vitro , higher levels of superoxide dismutase ( sod ) in corneal keratocytes irradiated with 213 nm suggest a better endogenous protection against free radicals induced by laser surgery . enucleated rabbit eyes that underwent myopic prk using both excimer laser and solid - state laser systems showed a smooth ablation surface with no edema or distortion to adjacent corneal tissue , indicating absence of thermal damage . the combination of an extremely small spot ( 0.6 mm ) , a uniform intensity beam distribution , a fast pulse rate , and an ultrafast tracking / scanning system contribute to the accuracy of this laser system . the fact that the 193 nm laser system is more sensitive to corneal hydration and environmental humidity is also well known . absorption at the 213 nm wavelength is 1 to 4 times less sensitive to corneal hydration , thus the refractive correction becomes less sensitive to changes in corneal hydration . the 213 nm solid - state laser system does not require the use of fluoride gas , making it a safer alternative to the 193 nm excimer laser . the laser system has also been shown to be efficient at producing less heat with a maximum temperature of 38.4 generated compared with that of the excimer laser , which is at 52.9. it operates on a standard wall current without special wiring requiring a lower maintenance and running cost . the study 's small sample size , the wide variability in the amount of hyperopia , and the difference in the period of followup are its major limitations . additionally , corneal topographic changes , patient satisfaction , and quality of vision are also important parameters to be taken into consideration . in conclusion , our results show that the 213 nm solid - state laser system is safe , effective , accurate , and predictable for the treatment of hyperopia .

purpose . to evaluate the long - term efficacy , accuracy , stability , and safety of hyperopic laser in situ keratomileusis ( lasik ) using a 213 nm wavelength solid - state laser . methods . this prospective noncomparative case series consisted of 34 eyes of 17 patients which underwent hyperopic lasik using a 213 nm solid - state laser ( pulzar z1 , customvis ) at an outpatient refractive surgery center in manila , philippines . the preoperative and postoperative examinations included uncorrected distance visual acuity ( udva ) , subjective manifest refraction , corrected distance visual acuity ( cdva ) , cycloplegic refraction , slitlamp biomicroscopy , and keratometry ( k ) . main outcome measures . accuracy , efficacy , stability , and safety of the refractive procedure . results . mean follow - up was 25.18 13.79 months . at the end of follow - up , 26.47% had a udva of 20/20 and 94.12% had a udva of 20/40 . manifest refractive spherical equivalent ( mrse ) was within 0.50 d of the target refraction in 55.88% and within 1.0 d in 85.30% of the study eyes . refractive stability was noted in the 1st postoperative month while hyperopic regression was noted after the 3rd postoperative year . no eye lost more than 2 lines of cdva . conclusion . our results show that the 213 nm solid state laser system is safe , effective , accurate , and predictable for the treatment of hyperopia .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

PMC4537617

since 1985 , terms like physical activity , exercise , fitness training , training , fitness and physical fitness are often confused with one another and sometimes they are used interchangeably ( 14 ) . the rapid socio - economic and demographic mutations , the need to benefit from the natural dimension has led to a diversification of the application of physical exercise / sport that today it presents itself with different objectives and with the research of different forms of satisfaction . thus decreasing the demand of organized sport , competitive racing and the increase of individualized physical activities , aimed to achieve different objectives such as inner balance or psychophysical well - being . the phenomenon of physical exercises or commonly defined as fitness , exercise , conditioning , resistance training or better fitness training , however , is a very complex reality . indeed , with the term fitness , it is possible to identify a range of activities that are conducted every day in fitness centers ( gyms ) and we can regroup them into gym resistance training activities ; group fitness activities ; functional fitness activities . in the last decade , the activities that undergo the term fitness have evolved . initially aerobics , step aerobics , jogging , conditioning and bodybuilding were the most common gym activities that enabled population to be fit , now we may found more diversified and more careful to the needs of active and non - active population activities . some examples are : funky , zumba , fit box , cycling activities , calisthenics based training , cross fit , suspension training , kettlebell training , total body conditioning , core training , boot camp , functional training , pilates , yoga , stretching . it is clear that including such activities under the more generic term fitness or fitness training , a psychological process starts in people s mind that day after day mutates the meaning of the term itself ( 57 ) . there are various definitions of fitness ( 8) , that go from an ability to perform daily activities with vigor , to a demonstration of traits and capacities that are associated with a low risk of premature development of hypokinetic diseases ( e.g. , those associated with physical inactivity ) . although , when we speak about fitness we include the state of wellness that is defined as a multidimensional state of being describing the existence of positive health in an individual as exemplified by quality of life and a sense of well - being ( 911 ) . consistent with this definition , there is no doubt that there is a strong relationship between physical fitness and many components of wellness . there is considerable evidence that physical fitness , and the behaviors that build it , can reduce risk of illness and early death ( 5 , 10 , 12 , 13 ) . in addition , fitness can lead to enhanced cognitive functioning and can enhance one s ability to participate in leisure , often a satisfying social experience . poor health can occur even in highly fit people because of factors beyond personal control such as hereditary conditions or conditions caused by bacterial / viral infections . this is a risk factor for various diseases such as coronary diseases , diabetes , metabolic syndrome , hypertension and hypercholesterolemia . although , inactive people develop a rr due to chd that is similar to those who smoke cigarettes , have hypertension and have hypercholesterolemia ( 14 , 15 ) . this is just one of the countless examples that confirm that regular exercise represents an important factor in prevention against disease , that are most often associated with death in industrialized countries . analyzing various populations that go from youth to elderly , from health to pathological conditions , from specific to recreational fitness users , we will try to answer to some questions such as what benefit fitness training have on health ? or which is its preventive role ? and witches are fitness s major implications ? and more in general , what is fitness training ? the review follows the 27 key points stated in the prisma statement ( 16 , 17 ) . all the studies included in the review were fitness or health based and had to provide specific information regarding all the questions that this review is trying to answer and updating the original review of the authors . the majority of the cited studies were published from 2006 to 2014 and the ones with a previous date than 2006 were included only if scientific literature did not provide , for that period , pertinent evidence . we also searched on scopus elsevier , cochrane , doaj , caspur , scielo , and plosone . the standardized search strategy included the use of the terms exercise , exercise training , fitness training , health , and fitness in the title , abstract , and keyword field . we searched also the most common diseases related to physical inactivity ( diabetes , metabolic syndrome , stroke , atherosclerosis , hypertension and obesity ) . the overall research has produced 7737 results but the inclusion criterion has skimmed results to 117 items . of this after careful analysis , only 92 were inserted in text . the 25 studies not included even if matched the inclusion criteria ( year , relevance ) were evaluated as off - topic in respect to this search . all the selected articles were then divided in 4 major categories : 1 ) children and adolescents fitness levels ; 2 ) fitness training in elderly ; 3 ) pathology prevention through fitness training ; 4 ) training through fitness activities . all the studies included in the review were fitness or health based and had to provide specific information regarding all the questions that this review is trying to answer and updating the original review of the authors . the majority of the cited studies were published from 2006 to 2014 and the ones with a previous date than 2006 were included only if scientific literature did not provide , for that period , pertinent evidence . we also searched on scopus elsevier , cochrane , doaj , caspur , scielo , and plosone . the standardized search strategy included the use of the terms exercise , exercise training , fitness training , health , and fitness in the title , abstract , and keyword field . we searched also the most common diseases related to physical inactivity ( diabetes , metabolic syndrome , stroke , atherosclerosis , hypertension and obesity ) . the overall research has produced 7737 results but the inclusion criterion has skimmed results to 117 items . of this after careful analysis , only 92 were inserted in text . the 25 studies not included even if matched the inclusion criteria ( year , relevance ) were evaluated as off - topic in respect to this search . all the selected articles were then divided in 4 major categories : 1 ) children and adolescents fitness levels ; 2 ) fitness training in elderly ; 3 ) pathology prevention through fitness training ; 4 ) training through fitness activities . the american physical therapy association sop ( 18 ) has developed a task force to summarize fitness guidelines for children and adolescents . these state that to promote overall fitness , youth should participate in 60 minutes or more of physical activity every day , and that physical therapists should apply research relevant to health - related physical fitness when treating youth , promoting fitness , health , and wellness ( 19 , 20 ) . a high fitness level , in this specific case cardiovascular fitness , can improve quality of life and make ordinary tasks such as street crossing easier ( 21 ) . a number of 13 highly fit children and 13 low fit children aged 8- to 10-yr - old were tested on a treadmill that was integrated with an immersive virtual world . child pedestrians crossed the street while undistracted , listening to music , or conversing on a hands - free cellular phone . cell phones impaired street crossing success rates compared with the undistracted or music conditions for all participants , a result that supports previous research . higher - fit children maintained street crossing success rates across all three conditions , whereas lower - fit children showed decreased success rates when on the phone , relative to the undistracted and music conditions . the results suggest that higher levels of childhood aerobic fitness may attenuate the impairment typically associated with multitasking during street crossing ; these evidences are also confirmed earlier ( 21 , 22 ) . furthermore , a low level of fitness in youth can lead to obesity - insulin resistance and diabetes type 2 ( 22 , 23 ) . authors ( 24 ) have investigated on this phenomenon in 13.01.9 years old subjects , and tried to understand the involved mechanisms . fasting laboratory studies ( insulin , glucose , lipid profile ) and assessments of fitness , body composition , skeletal muscle oxidative phosphorylation and intramyocellular lipid content ( imcl ) , were performed at baseline and study completion . change in fitness was related to change in insulin resistance in response to lifestyle modification and exercise in obese children . imcl increased with exercise in these obese children , which may reflect greater muscle lipid oxidative capacity . other studies have investigated on the relationship between aerobic fitness and the adiposity in 8 and 12 year old overweight boys . the results confirm the previous analyzed study s findings ; those with a lower percentage of adiposity had a greater level of aerobic fitness ( 25 , 26 ) . further intervention has been performed on 99 african and hispanic girls and their mothers ( 27 ) . the girls attended a daily exercise , nutrition education , and counseling sessions from 9:00 a.m. to 5:00 p.m. mothers attended 2-h weekly exercise , nutrition , and counseling sessions . findings indicated statistically significant reductions in percent body fat , abdominal fat , and 1-mile run / walk minutes indicating the effectiveness of increasing fitness level even in young girls . these findings let us understand that not only there is an improvement on insulin sensitivity or muscle lipid capacity , but this leads to a reduction of body fat that is an important factor for quality of life and self - esteem ( 28 , 29 ) . two decades ago changes in eating habits does not seem to have influenced the prevalence of obesity in the uk population whereas there seems to be a correlation between hours of television a week and obesity , so it is evident an interesting link between obesity and hypokinesia ( 30 ) . this section supports the idea that the improvement of physical fitness during childhood is determinant for the future pathologies prevention . as we will see more forward on this review , the fitness training may represent valid support against inactivity , overweight and obesity ( fig . intracellular transport mechanisms of glucose in 1993 , the cdc and acsm stated that people should not carry out intensive training to improve their health . the joint committee of the two organizations concluded that a moderate level of activity ( walking , doing housework or gardening , playing with the kids ) took place during the day , providing the sufficient amount of exercise to improve their health ( 6 , 7 , 31 ) . since then this approach has been widely adopted by many governments and associations . currently , there is no uniformity of opinions among researchers that deal with exercise and recommendations and/or guidelines . in fact , these discrepancies are a challenge to the political part of the world of health . recommend a too vigorous training means to scare people ; on the other hand , a too bland training may not induce the expected benefits . it is a dilemma that we find in many areas of medical research , from ageing to diet , to exercise physiology , where scientific data is not absolutely discriminatory ( 7 , 32 ) . according to the mrfit study and the scholar blair , people with low levels of fitness had a higher probability of death during the eight years of the study , and also the two studies suggest that a moderate activity reduces this risk . the bias of the blair s study , as observed by other researchers , is that the level of fitness is related to genetics and if we wanted to use exercise as a drug other data show that the risk for cardiovascular disease decreases linearly with the increase in physical activity ( 35 , 36 ) and only those who perform strenuous physical activity showed a reduction in the risk of heart attack . although many authors agree that physical activity is necessary to reduce many of the risks related to health . there is still no unanimity of opinions on the amount and intensity of exercise necessary ( 2 ) . the acsm had recently recommended a moderate aerobic exercise most ( though not all ) of the days of the week with resistance exercises twice a week . to most people this may seem a prohibitive operating frequency but thinking that the goal of spending 150 kcal per day may be achieved even with frequent walks added to the individual caloric expenditure ( 6 , 7 ) , although not all authors agree on the limited amount of exercise and the fact that splitting the same amount in more sessions leads to the same effects ( 5 , 37 ) . a recent study has compared two different exercise programs in elderly : resistance training vs aerobic training . resistance training group 1 ) performed 6 exercises of resistance training twice a week , and aerobic group 2 ) participated in walking activity for 30 minutes twice a week . functional assessment was measured by the short physical performance battery , flexibility test , and the six - minute walking test . mean age was 68.8 years in the resistance training group and 69.1 years in the aerobic exercise group . no statistical difference was seen when groups were compared in the short physical performance battery , flexibility , and six - minute walking test . this is an interesting result that shows how important physical activity is , besides its typology ( 38 ) . further studies ( 37 , 3943 ) have also shown that different typologies of resistance training , like classical vs functional training have given to elderly the same benefits in terms of strength and that besides the volume of training an improvement in physical fitness gives improvements in systolic and diastolic blood pressure with a reduction of 3.6% and 1.2% , respectively , body mass index by 1.1% , and peripheral blood glucose was reduced by 2.5% . only low vs. high velocity training has shown differences in terms of power ( 37 ) appearing that in older men there may be a significantly greater improvement in functional performance and muscular power with power training versus low velocity resistance training . even short - term resistance training improves fitness in elderly ( 41 ) . a program of only 6 weeks was sufficient to enhance muscle quality of the knee extensors in elderly women , which resulted in beneficial changes in functional capacity . fitness benefits in the elderly do not stop just in improving quality of life , improving functional capacity or strength that will be used in everyday tasks but pushes its self - further . physical activity enhances bone mineral density ( 42 ) and more a recent evidence confirms that the resistance training is an effective intervention strategy to slow down sarcopenia ( 44 , 45 ) . the role of exercise on sarcopenia in the elderly is also properly discussed in a recent study ( 46 ) . authors discussed about the principles governing the prescription of physical activity for older people with sarcopenia and published some specific advices for how to engage older adults in appropriate training programs ( 46 ) . another important aspect is related to the fact that the overall fitness is a significant mortality predictor in older adults , independent of overall or abdominal adiposity ( 43 ) . according to what stated in this section , clinicians should consider the importance of preserving functional capacity by recommending regular physical activity for older individuals , normal - weight and overweight alike . there are many well - qualified evidence on the relationship between physical inactivity and diabetes ( 47 , 48 ) . some reports were built based on the observation of the prevalence of this disease in populations with different lifestyles and reflected the increase in incidence in people who had left their native country for moving in more industrialized countries ( 49 ) . physical inactivity is significantly associated with the development of igt ( impaired glucose tolerance ) and diabetes ( 5052 ) . physical activity has a protective role against the development of diabetes ( 24 , 53 , 54 ) even when there is a family history of this disease that can also cause significant metabolic and anthropometric modifications in young healthy subjects ( 55 , 56 ) . exercise is a potent stimulator of glucose transport and insulin independent at the same time enhances the action of insulin , and this effect persists even after days after the exercise . it seems that muscle contraction stimulates translocation of glut4 on the membrane through mechanisms different from insulin , even if the two stimuli can be added . the hypothesis is that muscle contraction stimulates glucose transport through the increase of cytoplasmatic ca ( 57 ) . this is likely to enhance both functional as a mean of preventing the onset of diabetes also through the control of body weight and as a mean to improve the clinical condition of the patient ( 58 , 59 ) . there is also an increase of the number of people with diabetes that also have a low level of fitness that are so at major risk in developing cardiovascular disease as secondary consequences . a comparison between aerobic training and resistance training has been done in the above - mentioned population ( 60 ) . what was found was that structured exercise training , especially the aerobic training component , was associated with a greater number of participants moving above established thresholds , indicative of low cardio respiratory fitness . these results have public health and clinical implications for the growing number of patients with type 2 diabetes at high risk for cardiovascular disease . other major consequence of physical inactivity or in general a low fitness level is the metabolic syndrome that is highly prevalent in populations around the world , regardless of the definition used ( 61 ) . physical inactivity and obesity are two of the major modifiable risk factors for the metabolic syndrome ( 62 ) . cross - sectional and prospective studies have generally found that levels of physical activity and fitness are inversely related to the prevalence of this syndrome . research that is more recent has also suggested that sedentary behaviors , such as excessive time spent watching television or using a computer , are significantly associated with an increased risk for this syndrome . separate but complementary approaches that encourage increased participation in physical activity and discourage sedentary behaviors , both at the individual and population level , may be useful in reducing the prevalence of this syndrome ( 2 , 63 ) . further evidences show the positive influence of cardio respiratory fitness in both , moderate and vigorous , and metabolic syndrome ( 64 ) . in an experimental group of 18 non - diabetic subjects with metabolic syndrome , that underwent 8 weeks of increasing intensity stationary cycle training , even though there was an absence of weight loss , it resulted in enhanced mitochondrial biogenesis , and increased expression of insulin receptors and glut4 in muscle ( 65 ) . application of metabolomics to nutrition and health research is increasing and while much effort has been invested in understanding factors that influence the metabolomic profile there is relatively little known about the impact of fitness levels ( 66 ) . a reduced excretion of amino acids in adults is associated with increased fitness levels and an increased fat oxidation rate during exercise . interestingly , higher levels of branched chain amino acids were associated with lower fitness levels and higher insulin resistance ( 66 ) . when talking about fitness and pathology prevention a great association must be done with cardiovascular diseases . most of the studies of cvd have verified the influence of exercise on cvd mortality . it seems from the most recent data based on the direct estimation of the cardio respiratory fitness ( crf ) that there is a clear inverse relationship between crf and reduction of cvd mortality and that this reduction is proportional to an increased level of fitness and / or an increased amount of physical activity ( 67 , 68 ) . early intensive aerobic exercise training could be considered in subacute stroke patients not only to enhance their cardiovascular fitness but also to maximize their functional recovery ( 69 ) . physical fitness training is known to be beneficial for persons with a number of comorbid conditions or risk factors for stroke ( 70 , 71 ) . a study conducted on 10 chronic stroke survivors ( 72 ) wanted to understand what kind of training would be beneficial for these subjects . patients trained 3 days / wk for a total of 12 weeks , with four sets of four repetitions at 85%95% one repetition maximum in unilateral leg press and plantarflexion with an emphasis on maximal mobilization of force in the concentric phase . after training , leg press strength improved by 30.6 kg ( 75% ) and 17.8 kg ( 86% ) ; plantarflexion strength improved by 35.5 kg ( 89% ) and 28.5 kg ( 223% ) for the unaffected and affected limbs , respectively . the 6-min walk test improved by 13.9 m , and the timed up and go test time improved by 0.6 secs . maximal strength training improved muscle strength in the most affected as well as in the no affected leg in chronic stroke survivors ( 72 ) . other important cvd is hypertension that is defined from a medical point of view as a short course situation characterized by very high values of blood pressure ( 73 ) . the benefits of physical activity and increased fitness levels are well known in preventing and/or reducing hypertension ( 68 , 7476 ) . the first that in both cases either in continuous and interval group blood pressure decreased and the general cardio respiratory fitness level increased and that between the two groups the interval training showed the major results ( 77 , 78 ) . it is so advisable to perform this type of training though is less time consuming and beneficial in hypertension or non - hypertension conditions ( 79 ) . fitness also finds utility in rehab of individuals undergoing residential treatment for methamphetamine ( ma ) dependence ( 80 ) . this study assesses the feasibility and efficacy of an 8-week endurance and resistance - training program on fitness measures . individuals recovering from ma dependence showed substantial improvements in aerobic exercise performance , muscle strength and endurance , and body composition with fitness training . at the end of this section , we may state that these findings demonstrate the feasibility of physical exercise interventions in particular conditions and show an excellent responsiveness to a different kind of exercise stimulus resulting in physiological changes that might enhance recovery from drug dependence and certainly could translate into a quality of life improvements ( fig . characteristics of the metabolic syndrome raising awareness , that fitness improves the quality of life , has pushed a greater number of people to practice physical activities . these have evolved over the years , giving rise to several studies focused on exercise prescription guidelines(7 ) , progression models in resistance training for healthy adults ( 81 ) , program variables and key training principles ( 82 ) and new disciplines ( 83 ) . having already seen that fitness training has positive effects body , we are going to clarify what are the differences between some of these fitness activities ( 84 ) . an immediate comparison between traditional training vs. functional training on strength development in adults ( 85 ) has shown that there are no statistical differences between the two protocols , but these become evident if we stratify by gender , evincing a greater strength increase due to traditional training in women compared to functional training . another important consideration must be done when considering the training frequency : this must not be neither too bland nor too frequent , advising then a frequency of 3 times a week to maximize improvements ( 86 ) . other comparison was done between mission essential fitness circuit - style training program and a standard army physical readiness training on fitness , physiological , and body composition changes ( 87 ) . active duty army personnel were randomly assigned to two groups ( mef = 34 or aprt = 33 ) for 8 weeks of training ( 15 sessions each ) . the mef program included functional movements focused on strength , power , speed , and agility . baseline and post - test measures included the army physical fitness test , physiological indicators , body composition , and additional fitness indicators . the mef participants significantly increased their push - ups , bench press , and flexibility and significantly decreased their 2-mile run and step test heart rate compared to participants doing aprt . other authors compared 7-week physical training program with traditional army physical fitness training in improving the selected measures of physical fitness and military task performance . subjects performed a 30-meter rush wearing a fighting load , a simulated casualty recovery wearing a fighting load , a one - repetition maximum ( 1rm ) bench press , a maximum repetition pull - up test , a medicine ball put , a vertical jump . the participants were assigned by block randomization to either traditional army physical training ( tt ) of calisthenics and running or a novel program ( nt ) of calisthenics , resistance , aerobic , speed , power , and agility training . nt was superior to tt in improving bench press medicine ball put , 30-m rush times and casualty recovery times . these findings suggest that a short - term physical training program is effective in improving strength , power , and speed among previously conditioned men . novel approach as seen is useful both for civil and military population in strength and fitness development ( 85 , 88 ) . in recent years , even kettlebells have re - emerged as a popular training modality for strength and conditioning in fitness activities ( 89 ) . this type of training is usually considered part of resistance training but recent evidence has shown that can even considerably increase vo2 max . this could be a useful tool for coaches in implementing cardiovascular fitness in a non - conventional way ( 90 ) . it must be also said that kettlebell training in comparison to weightlifting training even if both are effective in increasing strength and power , do nt generate strength gains like weightlifting movements ( 91 ) . talking about non - conventional fitness training even though scientific literature has still a lack in this matter of research , cross fit is gaining interest around the world ( 92 ) . cross fit can be defined as a high intensity power training for resistance and aerobic training . a study conducted amongst 23 males and 20 female over a 10 week training program wanted to investigate the effects of cross fit on aerobics and body composition . results of this study showed significant improvements of vo2 max in males and females as well as decreased body fat percentage in males and females . significant correlations between absolute oxygen consumption and oxygen consumption relative to body weight was found in both men and women indicating cross fit improved vo2 max scaled to body weight independent of changes to body composition . these data suggest that cross fit significantly improves vo2 max and body composition in subjects of both genders across all levels of fitness ( 92 ) . this section pointed out the fact that nowadays there is a large variety of fitness activities available within gyms and fitness centers ( table 1 ) . even though they significantly differ with each other , the common aim they have is the well - being of the people through the improvement of the physical fitness components and the psychological balance . the most common fitness activities performed nowadays within fitness centers and gyms weight loss ( also called fat - loss ) maximal aerobic capacity ( also called endurance ) intensive cardiovascular fitness interval trainingextensive cardiovascular fitness interval trainingincremental cftconstant cft intensive cardiovascular fitness interval training extensive cardiovascular fitness interval training the american physical therapy association sop ( 18 ) has developed a task force to summarize fitness guidelines for children and adolescents . these state that to promote overall fitness , youth should participate in 60 minutes or more of physical activity every day , and that physical therapists should apply research relevant to health - related physical fitness when treating youth , promoting fitness , health , and wellness ( 19 , 20 ) . a high fitness level , in this specific case cardiovascular fitness , can improve quality of life and make ordinary tasks such as street crossing easier ( 21 ) . a number of 13 highly fit children and 13 low fit children aged 8- to 10-yr - old were tested on a treadmill that was integrated with an immersive virtual world . child pedestrians crossed the street while undistracted , listening to music , or conversing on a hands - free cellular phone . cell phones impaired street crossing success rates compared with the undistracted or music conditions for all participants , a result that supports previous research . higher - fit children maintained street crossing success rates across all three conditions , whereas lower - fit children showed decreased success rates when on the phone , relative to the undistracted and music conditions . the results suggest that higher levels of childhood aerobic fitness may attenuate the impairment typically associated with multitasking during street crossing ; these evidences are also confirmed earlier ( 21 , 22 ) . furthermore , a low level of fitness in youth can lead to obesity - insulin resistance and diabetes type 2 ( 22 , 23 ) . authors ( 24 ) have investigated on this phenomenon in 13.01.9 years old subjects , and tried to understand the involved mechanisms . fasting laboratory studies ( insulin , glucose , lipid profile ) and assessments of fitness , body composition , skeletal muscle oxidative phosphorylation and intramyocellular lipid content ( imcl ) , were performed at baseline and study completion . change in fitness was related to change in insulin resistance in response to lifestyle modification and exercise in obese children . imcl increased with exercise in these obese children , which may reflect greater muscle lipid oxidative capacity . other studies have investigated on the relationship between aerobic fitness and the adiposity in 8 and 12 year old overweight boys . the results confirm the previous analyzed study s findings ; those with a lower percentage of adiposity had a greater level of aerobic fitness ( 25 , 26 ) . further intervention has been performed on 99 african and hispanic girls and their mothers ( 27 ) . the girls attended a daily exercise , nutrition education , and counseling sessions from 9:00 a.m. to 5:00 p.m. mothers attended 2-h weekly exercise , nutrition , and counseling sessions . findings indicated statistically significant reductions in percent body fat , abdominal fat , and 1-mile run / walk minutes indicating the effectiveness of increasing fitness level even in young girls . these findings let us understand that not only there is an improvement on insulin sensitivity or muscle lipid capacity , but this leads to a reduction of body fat that is an important factor for quality of life and self - esteem ( 28 , 29 ) . two decades ago changes in eating habits does not seem to have influenced the prevalence of obesity in the uk population whereas there seems to be a correlation between hours of television a week and obesity , so it is evident an interesting link between obesity and hypokinesia ( 30 ) . this section supports the idea that the improvement of physical fitness during childhood is determinant for the future pathologies prevention . as we will see more forward on this review , the fitness training may represent valid support against inactivity , overweight and obesity ( fig . in 1993 , the cdc and acsm stated that people should not carry out intensive training to improve their health . the joint committee of the two organizations concluded that a moderate level of activity ( walking , doing housework or gardening , playing with the kids ) took place during the day , providing the sufficient amount of exercise to improve their health ( 6 , 7 , 31 ) . since then this approach has been widely adopted by many governments and associations . currently , there is no uniformity of opinions among researchers that deal with exercise and recommendations and/or guidelines . in fact , these discrepancies are a challenge to the political part of the world of health . recommend a too vigorous training means to scare people ; on the other hand , a too bland training may not induce the expected benefits . it is a dilemma that we find in many areas of medical research , from ageing to diet , to exercise physiology , where scientific data is not absolutely discriminatory ( 7 , 32 ) . according to the mrfit study and the scholar blair , people with low levels of fitness had a higher probability of death during the eight years of the study , and also the two studies suggest that a moderate activity reduces this risk . the bias of the blair s study , as observed by other researchers , is that the level of fitness is related to genetics and if we wanted to use exercise as a drug other data show that the risk for cardiovascular disease decreases linearly with the increase in physical activity ( 35 , 36 ) and only those who perform strenuous physical activity showed a reduction in the risk of heart attack . although many authors agree that physical activity is necessary to reduce many of the risks related to health . there is still no unanimity of opinions on the amount and intensity of exercise necessary ( 2 ) . the acsm had recently recommended a moderate aerobic exercise most ( though not all ) of the days of the week with resistance exercises twice a week . to most people this may seem a prohibitive operating frequency but thinking that the goal of spending 150 kcal per day may be achieved even with frequent walks added to the individual caloric expenditure ( 6 , 7 ) , although not all authors agree on the limited amount of exercise and the fact that splitting the same amount in more sessions leads to the same effects ( 5 , 37 ) . a recent study has compared two different exercise programs in elderly : resistance training vs aerobic training . resistance training group 1 ) performed 6 exercises of resistance training twice a week , and aerobic group 2 ) participated in walking activity for 30 minutes twice a week . functional assessment was measured by the short physical performance battery , flexibility test , and the six - minute walking test . mean age was 68.8 years in the resistance training group and 69.1 years in the aerobic exercise group . no statistical difference was seen when groups were compared in the short physical performance battery , flexibility , and six - minute walking test . this is an interesting result that shows how important physical activity is , besides its typology ( 38 ) . further studies ( 37 , 3943 ) have also shown that different typologies of resistance training , like classical vs functional training have given to elderly the same benefits in terms of strength and that besides the volume of training an improvement in physical fitness gives improvements in systolic and diastolic blood pressure with a reduction of 3.6% and 1.2% , respectively , body mass index by 1.1% , and peripheral blood glucose was reduced by 2.5% . only low vs. high velocity training has shown differences in terms of power ( 37 ) appearing that in older men there may be a significantly greater improvement in functional performance and muscular power with power training versus low velocity resistance training . a program of only 6 weeks was sufficient to enhance muscle quality of the knee extensors in elderly women , which resulted in beneficial changes in functional capacity . fitness benefits in the elderly do not stop just in improving quality of life , improving functional capacity or strength that will be used in everyday tasks but pushes its self - further . physical activity enhances bone mineral density ( 42 ) and more a recent evidence confirms that the resistance training is an effective intervention strategy to slow down sarcopenia ( 44 , 45 ) . the role of exercise on sarcopenia in the elderly is also properly discussed in a recent study ( 46 ) . authors discussed about the principles governing the prescription of physical activity for older people with sarcopenia and published some specific advices for how to engage older adults in appropriate training programs ( 46 ) . another important aspect is related to the fact that the overall fitness is a significant mortality predictor in older adults , independent of overall or abdominal adiposity ( 43 ) . according to what stated in this section , clinicians should consider the importance of preserving functional capacity by recommending regular physical activity for older individuals , normal - weight and overweight alike . there are many well - qualified evidence on the relationship between physical inactivity and diabetes ( 47 , 48 ) . some reports were built based on the observation of the prevalence of this disease in populations with different lifestyles and reflected the increase in incidence in people who had left their native country for moving in more industrialized countries ( 49 ) . physical inactivity is significantly associated with the development of igt ( impaired glucose tolerance ) and diabetes ( 5052 ) . physical activity has a protective role against the development of diabetes ( 24 , 53 , 54 ) even when there is a family history of this disease that can also cause significant metabolic and anthropometric modifications in young healthy subjects ( 55 , 56 ) . exercise is a potent stimulator of glucose transport and insulin independent at the same time enhances the action of insulin , and this effect persists even after days after the exercise . it seems that muscle contraction stimulates translocation of glut4 on the membrane through mechanisms different from insulin , even if the two stimuli can be added . the hypothesis is that muscle contraction stimulates glucose transport through the increase of cytoplasmatic ca ( 57 ) . this is likely to enhance both functional as a mean of preventing the onset of diabetes also through the control of body weight and as a mean to improve the clinical condition of the patient ( 58 , 59 ) . there is also an increase of the number of people with diabetes that also have a low level of fitness that are so at major risk in developing cardiovascular disease as secondary consequences . a comparison between aerobic training and resistance training has been done in the above - mentioned population ( 60 ) . what was found was that structured exercise training , especially the aerobic training component , was associated with a greater number of participants moving above established thresholds , indicative of low cardio respiratory fitness . these results have public health and clinical implications for the growing number of patients with type 2 diabetes at high risk for cardiovascular disease . other major consequence of physical inactivity or in general a low fitness level is the metabolic syndrome that is highly prevalent in populations around the world , regardless of the definition used ( 61 ) . physical inactivity and obesity are two of the major modifiable risk factors for the metabolic syndrome ( 62 ) . cross - sectional and prospective studies have generally found that levels of physical activity and fitness are inversely related to the prevalence of this syndrome . research that is more recent has also suggested that sedentary behaviors , such as excessive time spent watching television or using a computer , are significantly associated with an increased risk for this syndrome . separate but complementary approaches that encourage increased participation in physical activity and discourage sedentary behaviors , both at the individual and population level , may be useful in reducing the prevalence of this syndrome ( 2 , 63 ) . further evidences show the positive influence of cardio respiratory fitness in both , moderate and vigorous , and metabolic syndrome ( 64 ) . in an experimental group of 18 non - diabetic subjects with metabolic syndrome , that underwent 8 weeks of increasing intensity stationary cycle training , even though there was an absence of weight loss , it resulted in enhanced mitochondrial biogenesis , and increased expression of insulin receptors and glut4 in muscle ( 65 ) . application of metabolomics to nutrition and health research is increasing and while much effort has been invested in understanding factors that influence the metabolomic profile there is relatively little known about the impact of fitness levels ( 66 ) . a reduced excretion of amino acids in adults is associated with increased fitness levels and an increased fat oxidation rate during exercise . interestingly , higher levels of branched chain amino acids were associated with lower fitness levels and higher insulin resistance ( 66 ) . when talking about fitness and pathology prevention a great association must be done with cardiovascular diseases . most of the studies of cvd have verified the influence of exercise on cvd mortality . it seems from the most recent data based on the direct estimation of the cardio respiratory fitness ( crf ) that there is a clear inverse relationship between crf and reduction of cvd mortality and that this reduction is proportional to an increased level of fitness and / or an increased amount of physical activity ( 67 , 68 ) . early intensive aerobic exercise training could be considered in subacute stroke patients not only to enhance their cardiovascular fitness but also to maximize their functional recovery ( 69 ) . physical fitness training is known to be beneficial for persons with a number of comorbid conditions or risk factors for stroke ( 70 , 71 ) . a study conducted on 10 chronic stroke survivors ( 72 ) wanted to understand what kind of training would be beneficial for these subjects . patients trained 3 days / wk for a total of 12 weeks , with four sets of four repetitions at 85%95% one repetition maximum in unilateral leg press and plantarflexion with an emphasis on maximal mobilization of force in the concentric phase . after training , leg press strength improved by 30.6 kg ( 75% ) and 17.8 kg ( 86% ) ; plantarflexion strength improved by 35.5 kg ( 89% ) and 28.5 kg ( 223% ) for the unaffected and affected limbs , respectively . the 6-min walk test improved by 13.9 m , and the timed up and go test time improved by 0.6 secs . maximal strength training improved muscle strength in the most affected as well as in the no affected leg in chronic stroke survivors ( 72 ) . other important cvd is hypertension that is defined from a medical point of view as a short course situation characterized by very high values of blood pressure ( 73 ) . the benefits of physical activity and increased fitness levels are well known in preventing and/or reducing hypertension ( 68 , 7476 ) . the first that in both cases either in continuous and interval group blood pressure decreased and the general cardio respiratory fitness level increased and that between the two groups the interval training showed the major results ( 77 , 78 ) . it is so advisable to perform this type of training though is less time consuming and beneficial in hypertension or non - hypertension conditions ( 79 ) . fitness also finds utility in rehab of individuals undergoing residential treatment for methamphetamine ( ma ) dependence ( 80 ) . this study assesses the feasibility and efficacy of an 8-week endurance and resistance - training program on fitness measures . individuals recovering from ma dependence showed substantial improvements in aerobic exercise performance , muscle strength and endurance , and body composition with fitness training . at the end of this section , we may state that these findings demonstrate the feasibility of physical exercise interventions in particular conditions and show an excellent responsiveness to a different kind of exercise stimulus resulting in physiological changes that might enhance recovery from drug dependence and certainly could translate into a quality of life improvements ( fig . raising awareness , that fitness improves the quality of life , has pushed a greater number of people to practice physical activities . these have evolved over the years , giving rise to several studies focused on exercise prescription guidelines(7 ) , progression models in resistance training for healthy adults ( 81 ) , program variables and key training principles ( 82 ) and new disciplines ( 83 ) . having already seen that fitness training has positive effects body , we are going to clarify what are the differences between some of these fitness activities ( 84 ) . an immediate comparison between traditional training vs. functional training on strength development in adults ( 85 ) has shown that there are no statistical differences between the two protocols , but these become evident if we stratify by gender , evincing a greater strength increase due to traditional training in women compared to functional training . another important consideration must be done when considering the training frequency : this must not be neither too bland nor too frequent , advising then a frequency of 3 times a week to maximize improvements ( 86 ) . other comparison was done between mission essential fitness circuit - style training program and a standard army physical readiness training on fitness , physiological , and body composition changes ( 87 ) . active duty army personnel were randomly assigned to two groups ( mef = 34 or aprt = 33 ) for 8 weeks of training ( 15 sessions each ) . the mef program included functional movements focused on strength , power , speed , and agility . baseline and post - test measures included the army physical fitness test , physiological indicators , body composition , and additional fitness indicators . the mef participants significantly increased their push - ups , bench press , and flexibility and significantly decreased their 2-mile run and step test heart rate compared to participants doing aprt . other authors compared 7-week physical training program with traditional army physical fitness training in improving the selected measures of physical fitness and military task performance . subjects performed a 30-meter rush wearing a fighting load , a simulated casualty recovery wearing a fighting load , a one - repetition maximum ( 1rm ) bench press , a maximum repetition pull - up test , a medicine ball put , a vertical jump . the participants were assigned by block randomization to either traditional army physical training ( tt ) of calisthenics and running or a novel program ( nt ) of calisthenics , resistance , aerobic , speed , power , and agility training . nt was superior to tt in improving bench press medicine ball put , 30-m rush times and casualty recovery times . these findings suggest that a short - term physical training program is effective in improving strength , power , and speed among previously conditioned men . this functional novel approach as seen is useful both for civil and military population in strength and fitness development ( 85 , 88 ) . in recent years , even kettlebells have re - emerged as a popular training modality for strength and conditioning in fitness activities ( 89 ) . this type of training is usually considered part of resistance training but recent evidence has shown that can even considerably increase vo2 max . this could be a useful tool for coaches in implementing cardiovascular fitness in a non - conventional way ( 90 ) . it must be also said that kettlebell training in comparison to weightlifting training even if both are effective in increasing strength and power , do nt generate strength gains like weightlifting movements ( 91 ) . talking about non - conventional fitness training even though scientific literature has still a lack in this matter of research , cross fit is gaining interest around the world ( 92 ) . cross fit can be defined as a high intensity power training for resistance and aerobic training . a study conducted amongst 23 males and 20 female over a 10 week training program wanted to investigate the effects of cross fit on aerobics and body composition . results of this study showed significant improvements of vo2 max in males and females as well as decreased body fat percentage in males and females . significant correlations between absolute oxygen consumption and oxygen consumption relative to body weight was found in both men and women indicating cross fit improved vo2 max scaled to body weight independent of changes to body composition . these data suggest that cross fit significantly improves vo2 max and body composition in subjects of both genders across all levels of fitness ( 92 ) . this section pointed out the fact that nowadays there is a large variety of fitness activities available within gyms and fitness centers ( table 1 ) . even though they significantly differ with each other , the common aim they have is the well - being of the people through the improvement of the physical fitness components and the psychological balance . the most common fitness activities performed nowadays within fitness centers and gyms weight loss ( also called fat - loss ) maximal aerobic capacity ( also called endurance ) intensive cardiovascular fitness interval trainingextensive cardiovascular fitness interval trainingincremental cftconstant cft intensive cardiovascular fitness interval training extensive cardiovascular fitness interval training evidence has shown that practicing and improving fitness has positive effects on human body . in children and adolescents , the effects of practicing regular physical activity can give a positive base for a healthy future life . this physiological condition is essential in the prevention of various diseases such as diabetes , metabolic syndrome and more in general the major cardiovascular disease . furthermore having a high level of fitness helps even an in ordinary task that in general makes life easier . even in elderly it is possible to prevent the physiological strength decrease with a proper fitness resistance training protocol that is important because has known , strength is the most important motor skill , and having a good level of strength can prevent early hospitalization . furthermore , especially in elderly women a regular fitness based program can improve or prevent osteoporosis , a dramatic condition that with the increase of physical inactivity is spreading worldwide . beyond age , regular physical activity and a healthy lifestyle ( so an increased fitness level ) can help individuals in being has previously shown , regular physical activity increases insulin sensitivity and this enables the body to use glucose and other nutrients in a better way than sedentary . this condition is determinant in pathologies such as diabetes that are positively correlated to physical inactivity . even cvd are linked with fitness especially with aerobic fitness and this reduces morbidity for secondary pathologies . therefore , a positive healthier condition is doubly beneficial : in first instance for the gain in health and secondary for the reduced medical costs that a healthy individual has to sustain . further considerations although have to be done not only on the health related consequences of fitness , but should be observed in the performance aspects that everyday gym , and not only , practitioners are used too . during the last decade , has shown , various fitness disciplines have emerged , these are valid and useful in improving fitness compared to traditional training . the evolution of society in maximization production and reducing application time is also reflecting in fitness and performance : we can see this in the massive number of high intensity protocols that have been developed . examples like hiit , cross - fit , boot camp training , functional training routines and many more are the proof of this evolution . this means that fitness training is evolving dynamically in accordance to the needs of people . in conclusion , the world of fitness , gyms and fitness centers is a big , diversified and specialized community in continuous evolution , also online through thousands of www pages . the needs of society and the will to increase health and promote positive lifestyles is making fitness a pivot around which everything revolves . children , adolescents , healthy and not healthy adults can all benefit from practicing fitness training and although these activities according to personal characteristics can be recreational and fun . a minimum of 60 minutes per day of physical activity is recommended , but seen , how said , this world is dynamically evolving , it is important to stay up to date with new findings from scientific literature . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc ) have been completely observed by the authors .

background : this review based upon studies searched from the major scientific libraries has the objective of clarifying what is fitness training in modern days , the implications that it has on health in both youth and elderly and finally discuss fitness training practical implications.methods:the prisma statement was partially adopted and a number of 92 items were selected , according to the inclusion criteria . results were discussed in 4 main sections : 1 . children and adolescents fitness levels ; 2 . fitness training in the elderly ; 3 . pathology prevention through fitness training ; 4 . training through fitness activities.results:this review pointed out the fact that nowadays there is a large variety of fitness activities available within gyms and fitness centers . even though they significantly differ with each other , the common aim they have is the wellbeing of the people through the improvement of the physical fitness components and the psychological balance.conclusion:fitness instructors recommendations should be followed in gym context and should be contingent upon an individual s objectives , physical capacity , physical characteristics and experience .

Introduction Methods Eligibility criteria Information sources Results Children and adolescents fitness levels Fitness training in elderly Pathology prevention through fitness training Training through Fitness activities Discussion Conclusion Ethical considerations

PMC3579455

tobacco smoking is a major cause of morbidity and mortality in developed countries . there are more than 4500 identified chemicals and over 60 potential or probable human carcinogens in cigarette smoke . the complex mixture of cigarette smoke has been classified by iarc ( the international agency for research on cancer ) as a group 1 known human carcinogen . the institute of medicine ( iom ) in 2000 , at the request of the food and drug administration ( fda ) , considered whether harm reduction approaches through reducing toxin exposures in smoke via modified - risk tobacco products ( mrtps ) could feasibly enhance tobacco control for smokers who will not or could not quit . recently , the fda has been given legislative authority over tobacco products , including the ability to establish performance standards ( e.g. , regulating the amount of carcinogen emissions and exposure to smokers through product design changes ) and evaluating manufacturers health claims of mrtps . in the last several years , there has been a renewed interest by the tobacco companies to manufacture these . while tobacco product design changes developed by the manufacturers or mandated by the fda can be screened in the laboratory for changes in smoke chemical constituents and toxicology , ultimately the impact of such changes must be evaluated in humans using biomarkers . however , there are only a limited number of biomarkers that assess exposure , and none that have been sufficiently validated for lung cancer risk . given the wide variety of tobacco toxicants , a broad range of biomarkers needs to be developed and validated to assess the impact of cigarette design changes on human exposure and health . the best available cigarette smoke biomarkers are chemically specific , reflecting only a narrow range of known toxicants . these target polycyclic aromatic hydrocarbons ( e.g. , benzo(a)pyrene ) , tobacco - specific nitrosamines ( e.g. , nnn , nnk ) , aromatic amines , and volatile hydrocarbons ( e.g. , benzene , 1,3-butadiene and acrolein ) . among these , polycyclic aromatic hydrocarbons ( pahs ) and tobacco - specific nitrosamines ( tsnas ) are considered the major causative agents in cigarette smoke contributing to lung cancer , and for cancer risk overall . acrolein is a suspected human carcinogen and carries the highest level of risk for respiratory effects in experimental animals , while benzene is a known human leukemogen . metabolomics is an omics technology that provides a simultaneous assessment of numerous molecules ( i.e. , the metabolome ) allowing for the quantification of individual metabolites and the identification of a phenotypic profile for clusters of metabolites . it provides information about metabolites from exogenous toxin exposures and those from cellular endogenous pathways as a result of the toxic exposures . in contrast to currently available smoke exposure biomarkers that are mostly chemically specific , metabolomics can provide information allows for broader phenotype assessments incorporating profiles within and across disease pathways . separately , metabolomics provide phenotypic information about the cell s environment and mechanistic pathways that genomics and transcriptomics do not . metabolomics is becoming an important component of systems biology , especially in determining the global metabolic profile by detecting thousands of small and large molecules in various media ranging from cell cultures to human biological fluids such as urine , saliva , and blood . more and more modern instrumentations and technologies are emerging for the metabolomics research , such as gas chromatography mass spectrometry ( gc ms ) , liquid chromatography mass spectrometry ( lc ms ) , high - performance liquid chromatography coupled with electrochemical coulometric array ( lceca ) , and nuclear magnetic resonance ( nmr)-based study . each platform has its specific strengths and weaknesses . for example , gc ms has great separation efficiency and resolution but analyzes only the volatile metabolic compounds ; lceca has great reproducibility and sensitivity but is low - throughput and provides only limited chemical structure information ; nmr is a fast and reliable nondestructive detector but has poor resolving power and poor sensitivity requiring larger amount of analyte compared to mass spectrometry . lc ms is an important tool with great flexibility in metabolomics compared to the other methods . it is often used to identify the low - abundance metabolites for a targeted study or to obtain the largest metabolomics profile for a global approach . with the development of ultrahigh pressure liquid chromatography ( uplc ) , better chromatographic resolution and peak capacity compared to hplc is achieved , and therefore it has become the platform of choice in our study . there has been a great increase in the research of metabolomics , and a number of studies have shown the utility in assessing human disease risk . however , while some studies show the utility in urine samples , little has been shown to validate the reproducibility of global metabolomics profile using lc thus , this study assesses ultra performance liquid chromatography coupled to quadrupole with time - of - flight mass spectrometry ( uplc qtof - ms ) method for use in metabolomics and applies the procedure to human samples from smokers and nonsmokers . all reagents and solvents were of hplc grade . 4-nitrobenzoic acid ( 4-nba ) , debrisoquine ( as debrisoquine sulfate ) , cotinine , ( )-nicotine , 1,11-undecanedicarboxylic acid and 3-hydroxycoumarin were purchased from sigma - aldrich ( st . louis , mo ) ; pseudooxynicotine , 3-hydroxycotinine , and cotinine n - oxide were purchased from toronto research chemicals ( north york , on , canada ) ; acetonitrile ( acn ) and water were purchased from fisher optima grade ( fisher scientific , waltham , ma ) . i , in order to test the variations of uplc qtof - ms assay , eight smokers plasma were split into two aliquots ( 5 l ) , and one aliquot was sampled consecutively 5 times , immediately followed by the second aliquot sampled three times . the eight subjects were sampled consecutively , followed by a repeat of the experiment for the first two subjects . experiment ii takes advantage of a quality control procedure used for ensuring assay consistency during the course of a large sample set analysis . here , a pooled sample from 7 nonsmoker subjects undergoing therapeutic phlebotomy were placed as every tenth sample among a sample set of 613 independent samples from 160 smokers ; 62 injections of the pooled control were analyzed . experiment iii was intended to determine if there was a metabolomic result difference between smokers and nonsmokers . there were nine smokers and ten nonsmokers ; the latter recruited from a three - arm study validating biomarkers in smokers , former smokers , and nonsmokers . smokers : blood samples were obtained from 160 cross - sectional study of smokers intended to characterize cigarette smoke exposure and the development and validation of biomarkers . as an eligibility criterion , they had a stable smoking pattern for at least 6 months of 10 cigarettes per day or more . there were 613 samples from these subjects that were analyzed in a single session . the samples in the first group , used for the laboratory validation studies for experiment ii , were nonsmokers who were undergoing therapeutic phlebotomy for hemachromatosis or myeloproliferative diseases . their plasma was pooled and aliquots were interspersed with the smokers samples in every tenth aliquot . the second group , used to assess metabolomic differences between smokers and nonsmokers for experiment iii , used nonsmokers from a study of biomarker validation in smokers , former smokers , and nonsmokers from the tobacco product assessment consortium ( tobprac ) . these were healthy persons who smoked less than 100 cigarettes in their lifetime and recruited by irb - approved local media . demographics of all the participants are listed in supplementary tables 1 to 3 in the supporting information . blood was collected as serum or plasma ( heparin green top tubes ) as indicated . sample aliquots were mixed with 195 l of 66% acn containing the internal standards debrisoquine and 4-nba . the samples were centrifuged at 16000 g for 10 min at 4 c to remove particulates and precipitated proteins . one - hundred fifty microliters of the supernatant was then transferred into an autosampler vial , followed by uplc samples were injected onto a reverse - phase 50 2.1 mm acquity 1.7-m c18 column ( waters , milford , ma ) using an acquity uplc system ( waters , milford , ma ) with a gradient mobile phase consisting of 2% acn in water containing 0.1% formic acid ( a ) and 2% water in acn containing 0.1% formic acid ( b ) . each sample was resolved for 10 min at a flow rate of 0.5 ml / min . the gradient consisted of 100% a for 0.5 min then a ramp of curve 6 to 60% b from 0.5 to 4.0 min , then a ramp of curve 6 to 100% b from 4.0 to 8.0 min , hold at 100% b until 9.0 min , then a ramp of curve 6 to 100% a from 9.0 to 9.2 min , followed by a hold at 100% a until 10 min . mass spectrometry was performed on a q - tof premier ( waters , milford , ma ) operating in either negative - ion ( esi ) or positive - ion ( esi+ ) electrospray ionization mode with a capillary voltage of 3200 v and a sampling cone voltage of 20 v in negative mode and 35 v in positive mode . the desolvation gas flow was set to 800 l / h and the temperature was set to 350 c . the cone gas flow was 25 l / h , and the source temperature was 120 c . accurate mass was maintained by introduction of lockspray interface of sulfadimethoxine ( 311.0814 [ m + h ] or 309.0658 [ m h ] ) at a concentration of 250 pg/l in 50% aqueous acetonitrile and a rate of 150 l / min . data were acquired in centroid mode from 50 to 850 m / z in ms scanning . the metabolite identifications were confirmed by comparing the retention time under the same chromatographic conditions and by matching the fragmentation pattern of the parent ion from the biological sample to that of the standard metabolite using tandem mass spectrometry ( uplc qtof - ms / ms ) . the raw data from uplc qtof instrument were converted to network common data format ( netcdf ) files . they were then preprocessed using xcms for peak detection , retention time correction and peak matching to obtain a peak list in which each peak is represented by its m / z value , retention time and intensities ( peak area ) across samples . preprocessed data sets were analyzed using matlab ( mathworks , natick , ma ) and metaboanalyst ( www.metaboanalyst.ca ) to perform scatter plot , hierarchical clustering analysis and principal component analysis ( pca ) . r was used for anova ( analysis of variance ) and coefficients of variation analysis in experiment i and random forest classification . significant features were searched against the madison - qingdao metabolomic consortium database ( mmcd ) and the human metabolome database ( hmdb ) with the mass accuracy of 10 parts per million to identify putative metabolite identifications in experiment iii . for the metabolomic experiments to provide sound biological insights into pathobiology , it is imperative to demonstrate that the variability of metabolomics measurements is within acceptable limits . ms platform for the metabolomic analysis of urine samples has been examined from unspecified healthy subjects , which indicated that the within - day reproducibility of uplc qtof - ms system is sufficient to ensure data quality in global metabolomic studies , after sufficient equilibration of the system . in this study , the reproducibility of lc ms - based metabolomic experiments using blood samples of smokers and nonsmokers is examined . the use of blood , while more difficult to collect compared to urine , is conceptually a better matrix for biomarkers because it is not dependent on renal excretion , for example , a need to adjust for urinary creatinine or stability of a metabolite in urine in vivo and ex vivo . for blood , there is a choice of serum or plasma ; the former will include metabolites that result from coagulation and blood processing , and so includes compounds not existing in vivo . significant differences for plasma versus serum have been noted by wedge et al . , where they found correlations of many metabolites including glycerophosphocholines , creatinine , erythritol and glutamine in the biological pathways with the prognosis of small cell lung cancer in plasma but not in serum , indicating the clinical feasibility of plasma for metabolomics study . a recent study comparing metabolite profiles in both biofluids has reported a higher concentration of metabolites in serum , and a better reproducibility in plasma . however , the overall reproducibility was good in both biofluids . for smoke exposure in particular , more nicotine - related metabolites were seen in serum samples while better stability were observed in the plasma . the choice of serum versus plasma in our study was dictated by the availability of large volumes of sample needed for the numerous replicate experiments . the data indicate , however , that the choice for serum versus plasma was not important for the purposes of validation and reproducibility . the reproducibility analysis is performed in three parts : the first utilizes plasma from 8 smokers in experiment i to analyze the reproducibility of the lc ms - based metabolomics data over a short period of time . the various factors that affect the quality of the data are examined and their respective contributions to the data variability are analyzed . as part of experiment i , immediately following the multiple injections of the 8 smokers , the samples from the first two subjects were reinjected 5 times and 3 times . thus , the replicates followed the initial 64 chromatographic runs and 13 h in time . results of these latter two subjects are compared against the first analyses to examine the effect of running time and potential carry - over on the data . ms platform over multiple days is examined using experiment ii through a qc analysis similar to the approach by gika , et al . after establishing the reproducibility of lc ms - based metabolomics data for plasma samples , experiment iii was conducted to validate the use of metabolomics for the assessment of smoke exposure , as recommended by hatsukami , et al . ms - based proteomics experiments , it is assumed that the variability of the intensity of a peak can be attributed to either sample preparation variation or machine measurement . for the two aliquots of each si , i = 1 , 2, ... ,8 in experiment i , their difference is mainly due to the sample preparation variation . and for the repeated injections of each aliquot , their difference is derived from the measurement noise , which results from variability in the chromatography and the mass spectroscopic measurements . we refer to the variability due to biological differences among subjects as inter - individual variation . for the first 8 biological subjects s1s8 , it can be shown that there is minimal sample preparation variability and measurement noise but large differences due to the interindividual variation . the intensity of each peak from the peak - wise variation in the metabolomics data can be modeled as1where yijk is the log transformation of the observed intensity of the ith biological subject ( i = 1 , 2 , ... , 8) , the jth aliquot ( j = 1 , 2 ) , and the kth injection ( k = 1,2, ... ,5 if j = 1 , k = 1,2,3 if j = 2 ) ; represents the average concentration of each peak ; i , j(i ) and k(ij ) follow the normal distribution with mean zero and variances b2 , s2 , and m2 respectively , and i , j(i ) and k(ij ) are mutually independent . a random effects analysis of variance ( anova ) model was used to estimate the variations b2 , s2 , and m2 , which characterize the contributions of the interindividual difference , sample preparation difference and measurement noise to the variability of the data . the variability due to chromatography and machine measurement error was first assessed by visually inspecting the total ion chromatograms ( tics ) of the repeated runs from the same aliquot . , the assessment of the variability will not be affected by different choices of preprocessing schemes . in figure 1 , the tics of the five runs from the first aliquot of s1 are compared with each other . it was found that there was perfect qualitative reproducibility for all five runs from the same biological subject and aliquot . . then the peak list ( list of peaks ) from preprocessing was used to assess the variability among the repeated injections from an aliquot . for the first aliquot of biological subject s1 to s8 , the logarithmic intensities of all the peaks from the first injection were compared against the logarithmic intensities of all the peaks from the other injections using scatter plots in figure 2 . the scatter plots of the peak intensities from two injections are largely on the diagonal line and exhibits a high correlation ( pearson correlation = 0.98 ; p < 0.001 ) , indicating a high resemblance between the repeated injections . since the five repeated injections span a time of 57.5 min , it is evident that the lc scatter plots of logarithmic intensities of the five injections of the first aliquot of s1 to s8 represents ( a ) first injections vs second injections , ( b ) first injections vs third injections , ( c ) first injections vs fourth injections , and ( d ) first injections vs fifth injections . the variance calculated from the anova model and the coefficient of variation were compared along with m / z values and retention times to see if the measurement error of the lc ms system is more significant over a particular region of chromatogram and mass spectrum , as sample preparation variation and interindividual variation should not be affected by chromatogram or mass measurement . generally , the largest variation appears at the beginning ( less than 25 seconds ) of the chromatogram run ( figure 3 ) . the reason for the large variation at the beginning of the chromatogram may be attributed to the high aqueous gradient of the reverse - phase chromatography that can not retain the very polar compounds or hydrophilic metabolites in the beginning of the elution . if this area is considered to be important , it likely can be resolved by using hilic ( hydrophilic interaction liquid chromatography ) . the variation toward the end of the chromatogram is often observed partly due to baseline shift and can result in an overestimate of the intensities of the analytes . thus , we have discarded the last two minutes of the chromatography during the preprocessing of the data . as shown in figure 3 , the retention time of peaks only go up to 480 s , where the variability of data is moderate . the variation caused by the measurement noise shows a significant negative correlation ( spearman correlation = 0.66 ; p < 0.001 , see supplementary figure 1 , supporting information ) with peak intensity . it is understandable as the noise effect is less severe on high - intensity peaks and it is relatively easy to detect and quantify high - intensity peaks . overall , the mean cv for the measurement noise was 0.02 , which ranged from 0.003 to 0.297 . similar to the above , the greatest cvs were in the areas at the beginning of the chromatogram . the correlation coefficient for the cv in relation to peak intensities is shown in figure 4 ( spearman correlation = 0.785 ; p < 0.001 ) . those metabolites with cv more than 10% have been removed from the analysis for biomarker discovery . distribution of the estimated measurement error in cv ( % ) over m / z values and retention times . the dot size and color corresponds to the cv value : the larger the dot , and the brighter the color of the dot , the larger the cv value of the individual peak . scatter plot of the estimated measurement noise in coefficient of variation ( % ) versus the mean intensities of peaks over s1 to s8 . the scatter plot also was used to examine the variability due to sample preparation . the repeated injections from each aliquot were averaged to obtain the mean intensity of each peak for an aliquot . the logarithmic intensities of all peaks of the first aliquot of the eight biological subjects were compared with the logarithmic intensities of peaks of the second aliquot of the biological subjects . the two aliquots show a high degree of resemblance as the intensities are largely on the diagonal line ( supplementary figure 2 , supporting information ) and exhibits a high correlation ( pearson correlation = 0.99 ; p < 0.001 ) . the results demonstrate that the sample preparation does not significantly affect the quality of the lc ms data . hierarchical clustering of the 8 subjects each with 8 injections ( five plus three injections ) from experiment i is shown in figure 5a . the result confirms that the variability due to sample preparation is generally equal to or smaller than the measurement error , so it is sometimes masked by the measurement error . hierarchical clustering of all metabolites ( a ) from subject 1 ( s1 ) to subject 8 ( s8 ) and ( b ) from s1 to s8 plus the analytical replicates s1 and s2. heat map colors represent relative values , in which red represents values above the mean , black represents the mean , and green represents values below the mean of a row ( metabolite ) across all columns ( samples ) . in experiment i , the first two biological subjects s1 and s2 were repeated at the end of the experiment as s1 and s2 , with separate sample preparations and injections . between s1 ( s2 ) and s1 ( s2 ) , there were 56 other sample runs , and they were more than 10 h separated in time . the effects of run time and carry - overs on data variability can be evaluated by comparing s1 vs s1 and s2 vs s2. the logarithmic intensities of the four aliquots from s1 and s2 were compared with those of the four aliquots from s1 and s2. a high degree of resemblance is evident between s1 , s2 and s1 , s2 ( supplementary figure 3 , supporting information ) and exhibits a high correlation ( pearson correlation = 0.97 ; p < 0.001 ) , which showcases that the lc ms platform is stable after a moderate time period ( more than 10 h ) . we then analyzed a new hierarchical clustering adding experimental replicates ( s1 and s2 ) that were run at the end of the experiment , and the result showed that the same sample s analytical replicates were clustered together ( s1 to s1 , and s2 to s2 , see figure 5b ) . thus , the increased running time and carry - overs of the sample analyzed between them does not significantly affect the data reproducibility . the estimated variances are shown as the box plot in figure 6 and the variance and coefficient variation are summarized in table 1 . the results confirm that the variations between measurement error and sample preparation variation are comparable , while they are smaller than the interindividual variations , even if these replicates were analyzed 13 h apart . box - plot of the estimated variances due to interindividual variation , sample preparation and measurement noise . from the model equation ( 1 ) , we can deduce that the variance of the mean intensity of all the biological subjects is2or more general3where i is number of biological subjects , j is the number of aliquots per subject and k is the number of injections per aliquot . this means that the variability of the mean of the observed peak intensity is dominated by the variation because of interindividual difference . thus , the most effective way to reduce the variability in lc ms data is to increase the number of biological subjects under investigation , given the total number of sample runs . because of this understanding , in experiment ii , only one aliquot per subject and one injection per aliquot are used for the lc ms - based metabolomics analysis of 613 biological subjects . experiment ii provides the quality control data from a large metabolomics study of smokers ( n = 160 ) with up to four separate blood draws before and after two cigarettes each ( 613 samples ) . for the quality control , 62 pooled healthy control samples were prepared by mixing equal volumes ( 400 l ) of plasma from seven nonsmokers and were inserted as every 10th sample of the run . the pooled aliquot is assumed to be homogeneous and repeatedly injected across the entire lc ii , 62 pooled quality control samples were injected during the analysis of 675 samples , including the 62 pooled samples . the analyses were conducted uninterrupted over 138 h. the peak list is acquired after peak detection and alignment using xcms . the preprocessing may help to reduce the variation in data due to instrument noise as only peak regions are considered in the analysis . this assesses reproducibility of the chromatography , quantitation of the spectroscopy , and run - to - run carryover and cross - contamination while running as a batch in a chromatographic system . the reproducibility of the quality control replicates was analyzed through unsupervised principal component analysis ( pca ) by metaboanalyst . pca has been shown to be an effective approach to visualize high - dimensional data by projecting the data point into a low - dimension space . if a certain degree of platform stability has been attained , the qc samples should cluster tightly together in the pca score plot . pca of the experimental samples and qc samples has revealed a pattern as shown in figure 7 , which gives an indication about the reproducibility of the data . although there are some variations among the qc samples , they occupy a relatively constrained space in the pca score plot . when we compare our study ( 675 injections with 62 qcs , total run time 138 h ) with the validation guideline for urine samples from gika et al . ( 130 injections with 16 qcs , total run time 29 h ) , the performance of our qc samples showed little variation in tight clustering until samples run later in time . the deviation from the qc cluster might be due to the time - related drift in instrumental performance over a long time span in the large scale study , or the matrix effect due to the largely peptide / protein - based plasma samples compare to urine . figure 8 is the hierarchical clustering analysis of samples and qcs shown as a heatmap using pearson s correlation for the similarity measure ( distance ) , and clustering algorithms using ward s linkage ( clustering to minimize the sum of squares of any two clusters ) . the result demonstrated that our platform is capable of discriminating the metabolome from clusters of qc samples and experimental samples , which again provides confidence in the quality of our pooled healthy controls throughout the run . scores plot between the selected principle components ( pcs ) showed difference between samples and qcs in their metabolomic profiles . clustering result shown as heatmap ( distance measure using pearson , and clustering algorithm using ward ) of the pooled qc samples and experimental samples . there is wide interindividual variation for smoking behavior , which is affected by several factors such as race , gender , psychological factors and genetic background . metabolomic profiling may identify differences in exposure and response , reflecting inherent biological differences . metabolomics has the power to simultaneously detect carcinogen metabolites and endogenous metabolites affected by smoke exposure . thus , both exposure and effect can be assessed . to examine the feasibility of a metabolomic study in assessing smoke exposure , experiment iii of 9 smokers and 10 nonsmokers random forests analysis was used to perform supervised classification and feature selection , and top 50 important features were selected with 100% accuracy by multidimensional scaling plot ( figure 9a ) and can be visualized according to their rank importance ( figure 9b ) . to preliminarily identify the metabolites in experiment iii , the madison - qingdao metabolomic consortium database and the human metabolome database were searched ; 169 putative features from the positive mode and 53 from the negative mode were identified . then , 12 candidate metabolites were manually selected based on their putative identifications and availability of chemical standards for comparisons , as listed in table 2 . validation for these candidate metabolites were then done by acquiring ms / ms spectra . as expected , the ms / ms spectra of nicotine , cotinine , 3-hydroxycotinine and cotinine n - oxide were verified and matched well with those from authentic compounds in our serum samples ( figure 10 and supplementary figure 4 , supporting information ) . , it is primarily metabolized to cotinine by cytocrome p450 2a6 ( cyp2a6 ) enzyme and further metabolized by the same enzyme to 3-hydroxycotinine . thus , the ratio of cotinine and 3-hydroxycotinine reflect a stable cyp2a6 metabolic activity for nicotine metabolism . pseudooxynicotine , 1,11-undecanedicarboxylic acid and 3-hydroxycoumarin were also verified by ms / ms identification . pseudooxynicotine is an amino ketone product of nicotine by soil bacteria and was reported to be the direct precursor to the tobacco - specific lung carcinogen nnk in the bacterial systems . that the incubation of nicotine with human liver microsomes produced pseudooxynicotine through 2-hydroxylation of nicotine but has not been identified in humans . since microflora has been found to play a crucial role in human metabolome , tobacco - related metabolites metabolized by bacteria could have contributions to carcinogenesis that had nt been thought of before . nicotine - related metabolites including nicotine , cotinine , 3-hydroxycotinine , cotinine n - oxide ( a minor metabolite of nicotine ) and pseudooxynicotine validated by ms / ms were all significantly higher in intensity among smokers compare to virtually zero among nonsmokers ( figure 11 ) . in our results , 3-hydroxycoumarin and 11-undecanedicarboxylic acid were seen in both groups but higher in peak areas among nonsmokers compare to smokers ( figure 11 ) . 3-hydroxycoumarin is the metabolic product of the natural compound coumarin which can be found in plants and spices . metabolism of coumarin in humans is mainly carried out by cyp2a6 to 7-hydroxycoumarin but also can be metabolized by cyp3a4 to 3-hydroxycoumarin . nicotine is mainly metabolized by cyp2a6 enzyme , and thus could increase bioactivation of cyp2a6 and the 7-:3-hydroxycoumarin ratio among smokers . however , most of the reports on 3-hydroxycoumarin were done in rodent models and thus needs further clarification in human . 11-undecanedicarboxylic acid is a dicarboxylic acid with a 13-carbon dibasic acid ( tridecanedioic ) occurring in plant and animal tissues . the relationship of 11-undecanedicarboxylic acid to cigarette smoke is still unknown , maybe through altering the carboxylic acid metabolism in the tobacco leaf or during the manufacture of cigarettes . ( a ) forest accuracies were calculated and the sample classifications were presented by multidimensional scaling ( mds ) plot . in this plot , nonsmokers ( red ) and smokers ( blue ) were well separated in serum samples . ( b ) visualization of the top metabolites across all samples identifies the rank importance of the ions . ms / ms spectrum from authentic compounds of cotinine ( a1 ) , hydroxycotinine ( b1 ) , pseudooxynicotine ( c1 ) , and 1,11-undecanedicarboxylic acid ( d1 ) . ms / ms spectrum obtained from serum samples of smoker ( a2 , b2 , c2 ) or nonsmoker ( d2 ) were also presented for comparison . box plots of peak areas for seven candidate metabolites among smokers and nonsmokers . our results further show the feasibility and potential of the metabolomics profiling to identify new biomarkers of cigarette smoke exposure and lung cancer risk . given that cigarette smoke is a complex mixture , it is possible that metabolic profiles will provide additional information for tobacco - related disease risk different from existing chemically specific biomarkers , such as those reported by hecht et al . also , broad detection methods such as used here can screen for unanticipated changes in smoke exposure as cigarette designs change . for example , the technology used to decrease some constituents might increase those of others ( e.g. , for the eclipse cigarette , acrolein and co increased ) . another reason for needing a broad biomarker such as a metabolomics profile of exposure and risk is to help tailor prevention and early detection strategies for former and current smokers . while this study will not directly assess biomarkers for lung cancer risk , the first step in developing these is the development of biomarkers of smoke exposures . metabolomics provides information about the metabolic status of living systems and can provide phenotypic information about the cell s environment and mechanistic pathways , as well as having clinical utility including as a risk biomarker . it is inexpensive and high - throughput , and has the potential to identify new biomarkers of cigarette smoke exposure and the consequent disease risk . such biomarkers will assist in the evaluation of tobacco products and performance standards , and for identifying smokers at risk for lung cancer . upon the basis of our study using uplc qtof - ms , low variability is observed in measurement variation and sample preparation variation . when applying qc samples to the large scale study , qc clustering presents high stability of the platform but not the ones run later in time . various factors including the stability and maintenance of individual instrument , sample preparation techniques , and the choice of column could affect the performance of the chromatography result . therefore , we recommend that for future metabolomics studies , especially large scale epidemiological studies , a separate experiment to assess the variability of the platform before applying precious human samples is needed so as not to exceed the results reported herein . after determining the required sample size , the number of aliquots and injections needed according to the variability of the specific platform , a pooled qc sample set to be run in between the experimental samples throughout the run is crucial in order to further assess the repeatability of the experiment . after preprocessing and analyzing the data , metabolites with cv no more than 1015% should be considered for the biomarker discovery . metabolomics can potentially be used to better characterize smoking - related disease risks to enhance prevention and early detection methods , and by the fda in the regulation of tobacco products . the recent authority to the fda over tobacco products allows the fda to mandate product performance standards governing smoke exposure to toxic constituents and also requires the fda to evaluate manufacture s health claims for modified - tobacco products of purported reduced exposure . the fda decisions , however , will need to be supported by scientific studies and a better understanding of cigarette smoke toxicology . it also will require support through human studies and biomarkers that reflect the complex exposure to tobacco smoke . today , though , there are only a few biomarkers of exposure and no validated biomarkers of cancer risk . as proof of principle , several new biomarkers were identified herein that have not thus far considered to be biomarkers of tobacco smoke exposure . thus , metabolomics has the potential to develop a metabolic phenotype in healthy smokers of smoke exposure and disease risk .

metabolomics is likely an ideal tool to assess tobacco smoke exposure and the impact of cigarette smoke on human exposure and health . to assess reproducibility and feasibility of this by uplc qtof - ms , three experiments were designed for the assessment of smokers blood . experiment i was an analysis of 8 smokers with 8 replicates . experiment ii was an analysis of 62 pooled quality control ( qc ) samples from 7 nonsmokers plasma placed as every tenth sample among a study of 613 samples from 160 smokers . finally , to examine the feasibility of metabolomic study in assessing smoke exposure , experiment iii consisted of 9 smokers and 10 nonsmokers serum to evaluate differences in their global metabolome . there was minimal measurement and sample preparation variation in all experiments , although some caution is needed when analyzing specific parts of the chromatogram . when assessing qc samples in the large scale study , qc clustering indicated high stability , reproducibility , and consistency . finally , in addition to the identification of nicotine metabolites as expected , there was a characteristic profile distinguishing smokers from nonsmokers . metabolites selected from putative identifications were verified by ms / ms , showing the potential to identify metabolic phenotypes and new metabolites relating to cigarette smoke exposure and toxicity .

Introduction Materials and Methods Results and Discussion Conclusions

PMC2800019

blunt traumatic aortic rupture ( tar ) has traditionally been treated with early open surgical repair . recently , endovascular stent - grafting of tar has been described as a less invasive and possibly more favorable option . a 73-yr - old woman presented with multiple trauma and chest pain after a traffic accident . traumatic aortic injury was suggested by a chest radiography , which showed mediastinal widening and left pleural effusion . a spiral computed tomography ( ct ) scan revealed an extraluminal hematoma in the distal aortic arch with a contained aortic rupture ( fig . however , the patient had concomitant comminuted fractures in the right tibia , left femur , and the radius bilaterally , which required surgery . surgery for the tar was therefore delayed for about 1 month to allow orthopedic postoperative recovery . to allow a minimally invasive surgery , a hybrid aortic stent - grafting procedure was performed , consisting of a left subclavian artery ( lsca ) debranching procedure combined with proximal descending thoracic aortic stenting . median sternotomy for the debranching procedure was avoided by transposing the lsca to the left common carotid artery ( lcca ) through a 5 cm left supraclavicular incision . the lsca and lcca were dissected and freed from their surrounding attachments after moderate systemic heparinization with an intravenous ( iv ) of 5,000 iu . the proximal lsca was clamped and transected , and the proximal stump was closed with a continuous running suture of 4 - 0 polypropylene . the lcca was partially clamped and opened longitudinally , and the transected distal portion of the lsca was attached in an end - to - side manner . on postoperative day 5 , the interventional radiologist inserted a thoracic aortic stent percutaneously using the seldinger technique . a 5f - calibrated angiographic catheter was advanced into the aortic arch via the contralateral common femoral artery to determine the morphology and extent of the aortic rupture ( fig . the delivery system was advanced under fluoroscopy after iv injection of 5,000 iu heparin until the tip reached the origin of the lcca . finally a seal endoprosthesis ( s & g biotech , korea ) was inserted into the aortic arch to complete the procedure . a series of aortograms were performed during deployment to ensure accurate proximal and distal positioning . the proximal bare stent was placed precisely in the middle of the ascending aorta , allowing free blood flow to both arch vessels . intraoperative angiography was used for post - procedural assessment of stent - graft positioning and endoleak ( fig . 2c ) showed successful exclusion of the rupture site , with no any signs of endoleak or graft - related complications . at the first visit to the out - patient department , the patient was doing well and showed good healing of the supraclavicular incisional wound ( fig . emergency open thoracotomy for traumatic aortic ruptures is associated with a peri - procedural mortality rate ranging between 17% and 21% , and a paraplegia rate as high as 7% ( 1 , 2 ) , due mainly to the large proportion of patients who present with multiple concomitant co - morbid conditions . although delaying surgical repair of tar may overcome the high mortality and morbidity risks associated with emergency surgery , delaying open surgery has been found to have an in - hospital mortality risk of 2 - 5% ( 3 , 4 ) . recent results have suggested that endovascular stent - grafting may be a viable , ' minimally invasive ' alternative , which can achieve the same results as emergency open thoracotomy but with lower mortality and morbidity risks ( 5 - 7 ) . endovascular stent - grafting , however , has several drawbacks that may limit its broader application . for example , most tars occur at the aortic isthmus level , so the proximal landing zone in these patients is usually short . furthermore , obligate coverage of the lsca ostium is often required to achieve complete sealing of the rupture site , so it may cause ischemia or thrombosis ( 8 , 9 ) . proximal fixation was enhanced in our patient by deploying a graft with an extended bare proximal portion . this approach could ensure adequate anchorage without aortic arch branch occlusion , even in patients with short landing zones . furthermore , by transposing the lsca onto the lcca through the small supraclavicular incision , a full sternotomy could be avoided while preserving blood flow to the lsca . occlusion of the lsca during unprotected stent insertion is frequently regarded as inconsequential . in summary , this report presents our first experience with hybrid tar , combining supra - aortic branch transposition with endovascular stent - grafting . currently available techniques for stent - grafting of the descending aorta , combined with minimally invasive debranching procedures , may lead to the further development of safe and effective strategies that will avoid the high risks associated with conventional open surgery .

emergency surgical repair for acute traumatic aortic ruptures has been associated with a high peri - procedural mortality rate . endovascular stent - grafting , as a less invasive procedure , has shown encouraging results . this report describes a patient with a short landing zone , who was treated by transposing the supra - aortic branch without sternotomy , followed by covered stent - grafting with an extended proximal bare portion to enhance fixation .

INTRODUCTION CASE REPORT DISCUSSION

PMC4228587

nuclear medicine ( nm ) is one of the fastest growing specialties in the world and has evolved as a tool for individualization of patient care and for improvement of health care outcomes . clinical nm has progressed beyond its own boundary into new domains of diagnostic radiology and oncology with the ever - increasing use of integrated structure - function imaging . there has been a considerable shift in the morbidity and mortality profile of the population across the world in recent times , wherein the leading causes of mortality include chronic and noncommunicable diseases , especially cardiovascular diseases and cancer . the changes in lifestyle coupled with increasing life expectancies and corresponding increases in the aging population have brought the developing world even closer to the developed world in terms of the incidence of health problems . demographic changes coupled with the rising impact of chronic diseases , is widening the role of nm in management of the diverse group of diseases in various parts of the world . conducting cost analysis help hospitals the information obtained from cost analysis also help the organizations operate more cost effectively as well as monitor and control costs where necessary . it also helps hospitals to improve quality and efficiency of care they provide and manage their resources better . however , published studies on costing of nm procedures are few in number , pointing to a lacuna in knowledge on this issue . resource management and optimal resource utilization in hospitals has gained a significant importance in today 's global economy , resulting in the traditional accounting systems becoming inadequate and sub - optimal for managing hospital resources and accurately determining service costs . traditional cost accounting uses broad averages for assigning the cost of resources uniformly to procedures or services , when individual procedures may be using these resources in a nonuniform manner . while traditional costing allocates overhead and indirect costs in proportion to production volume or to direct costs , such costs are directly traced in activity based costing ( abc ) techniques to activities or services through tracing methods , resulting in a more accurate estimate of unit costs than the traditional costing technique . this study was conducted to ascertain the cost of performing different diagnostic procedures by the nm department of a tertiary care government teaching hospital by utilizing abc methodology of costing . this study is an observational , descriptive and cross - sectional study carried out over a period of 12 months at a nm centre of tertiary care multi - specialty government teaching hospital , where patients are investigated and treated for free . the center under study has two fulltime nm physicians , one fulltime nm technician and one part - time safaiwala . the study entails classifying costs , identifying cost drivers and tracing all costs related to various diagnostic procedures performed at the nm centre by using abc costing method . initially , the patients undergoing diagnostic procedures and the staff administering these procedures were observed by the study team to develop adequate process literacy . ethical clearance for the study was obtained from the institutional ethics committee of the study hospital . time motion study was conducted to estimate the direct labor hours , that is , the time required by the staff for performing each type of procedure and equipment run time was calculated on the basis of the mean time required for conducting various diagnostic procedures using various equipments available with the center . cumulative labor hours and cumulative machine hours were thus calculated by adding the relevant observed times for each type of procedures [ table 1 ] . the various procedures and their relevant details the cost of technetium various records were perused for ascertaining specific input costs , e.g. staff salary slips were used for estimation of salary and wages and departmental and hospital inventory books for estimation of the inventory . various rates of hospital engineering services were used for estimation of equipment maintenance and repair charges . the municipal corporation water rates and state electricity boards tariff rates were used for estimation of water and electricity usage charges and prevalent land utilization rates straight - line depreciation was utilized to estimate the depreciated value of equipments , whereas comprehensive annual maintenance contract rates were accepted towards the calculation of cost for equipment maintenance . the cost of various supplies and consumables was estimated from the receipt vouchers and the rate contract documents . prevalent market rates were taken for calculation and ascertaining certain overhead expenses such as stationery , laundry , and various supply items like clothing and furniture . the various costs calculated were thereby traced to their respective cost drivers , cost driver being a variable , such as level of activity or volume , that usually affects costs over a given time span and has a cause and effect relationship between a change in level of activity or volume and a change in the level of costs and total expenses under various heads were calculated [ table 2 ] . the costs thus obtained from the study were compared with that prevailing at a private tertiary care hospital located in the same city and central government health scheme ( cghs ) rates . tracing of various costs to suitable cost drivers idle time cost of professionals , cost of biomedical , radiological and general waste disposal and apportioned cost of administration and management of the center were not included in the study . a total of 2076 investigations were conducted at the nm department during the study period , which included newly registered cases as well as follow - up of old cases . the majority of procedures were observed to be cardiac myocardial perfusion imaging ( mpi ) scans followed by renal and bone scans , respectively [ figure 1 ] , the total number of procedures being 9.23% more than that of the previous year . major investigations carried out from january 01 , 2012 to december 31 , 2012 the total expenses towards operating this nm department , subsequent to tracing of each cost driver were found to be rs . the major cost centers contributing to the total expenses were observed to be staff salaries , equipment run and maintenance costs and costs of medicines and consumables [ figure 2 ] . the cost per unit procedure for various diagnostic procedures varied from rs . 869 for a thyroid scan to rs . 11,230 for a meta - iodo - benzyl - guanidine ( mibg ) allocation of all costs to cost centers costs of the most common investigations conducted at this center the cost of major procedures carried out in the nm department being studied was then compared with that of a private tertiary care hospital located in the same city and cghs rates and found to be competitive in respect of prevailing market rates [ figure 3 ] . activity - based costing is an approach to service costing that differs from traditional costing methods because of the way it assigns costs . traditional costing methods assigns costs using an allocation process that has little bearing to the reason costs were incurred initially . instead of collecting costs by category ( e.g. people costs , occupancy , supplies ) and then arbitrarily allocating them to cost centers or products , abc technique identifies various activities performed , traces costs to these activities and then uses various cost drivers to trace the cost of activities to the final service , cost drivers being factors that reflect consumption of activities by product or service . a total of 2076 investigations were carried out during the study period out of which nearly 31% were mpi scans followed by renal and bone scans , pointing toward the increasing morbidity load because of noncommunicable diseases . as per j. r. ballinger , the two widely used nm studies were found to be mpi and bone scintigraphy . the cost of unit procedures being conducted by the nm department varied as per different resource inputs for each procedure , thyroid scans costing rs . the total expenses incurred towards the salary and wages of the staff was rs . 2,810,000 , which amounted to nearly 39% of the total expenses and provides a direction towards future efforts towards cost - containment by optimizing direct labor utilization . significant expenditure was being incurred towards procurement of consumables by this center , amounting to approximately 29% of the total expenditure . a significant reduction can be achieved in the expenses towards consumables with the adoption of certain practices like proper scheduling of patients and adherence to such schedule , sharing of radioactivity with other local nm centers for overflow optimization among such centers , indigenization of equipments and consumables , cost awareness program for clinicians and utilization reviews to prevent wastage of radioactivity . the cost of equipment maintenance ( 8.38% ) is prohibitively high , which can be brought down substantially through training of the staff in the first line maintenance of equipments , leading to reduction in the cost of comprehensive annual maintenance contract . similarly , energy conservation and regular audit will lead to saving of electricity , which is a major contributor towards procedure costing . when the cost of diagnostic procedures calculated was compared with the market rates being charged by private hospitals located in the same city as well as cghs rates , unit cost of procedures like stress thallium , renogram scans and mpi scan were found to be significantly lesser and very competitive . the unit costs of various diagnostic procedures were found to be competitive , when compared against prevalent market rates . the most cost effective investigation was found to be stress thallium scan with a unit cost of rs . 4113.00 ( usd 68.55 ) in this center , whereas the same was priced at rs . respectively at the private hospital and cghs rates , respectively . another significant difference in price was found with the mibg scan , which was estimated at rs . 11230.00 ( usd 187.17 ) per procedure , whereas the same was priced at rs . 3835.00 ( usd 63.92 ) , which was more than the rates of cghs ( rs . the advantage of using abc costing technique is that it provides management with logical and precise cost information and once available , this information can be used to chart a plan of action . when incorporated into critical management systems , it can serve as a powerful tool for continuously rethinking and dramatically improving products and services , processes , and market strategies . costing of various diagnostic procedures conducted at the nm department in the present study provides a precise costing knowledge that can be utilized for establishing a profitable pricing policy as well as institute measures towards cost containment of prominent cost drivers of the study hospital , thus making the hospital the best process owner in the market . an annual increase of 9.23% was observed in the number of diagnostic procedures being performed by the center . the benefits of diagnostic imaging are immense and the increased sophistication and clinical efficacy of imaging have revolutionized the practice of medicine , resulting in dramatic growth over the past quarter century . the hazards associated with expanding use of imaging modalities using ionizing radiation can be minimized by preventing the inappropriate use of such imaging and by optimizing studies that are performed to obtain the best image quality with the lowest radiation dose possible . the principal attributes of ensuring patient safety are clinical acumen , mastery of technology and dedication to safety and quality . efforts such as education for all stakeholders in the principles of radiation safety , appropriate utilization of imaging to minimize any associated radiation risk , standardization of radiation dose data to be achieved during imaging and identification of alternative imaging of patients who may have already reached threshold levels of estimated exposure from diagnostic imaging will be necessary in future for enhancing patient safety in the operations of nm procedures . this study suffers from certain limitations in ignoring certain overhead costs incurred towards security and general administration . moreover , a note of caution is mandated due to the complicated and time consuming methodology of abc costing , which may not be easy to perform by the uninitiated . nuclear medicine procedures not only carries a high cost but also subjects the patients to a risk of radiation exposure , thus raising the all - important question of economics and patient safety . utilizing abc costing routinely in the nm department will provide precise costing of various diagnostic procedures being performed by such departments , leading to effective cost containment measures . moreover , managerial strategies towards maximizing efficiency , optimizing resources and minimizing waste will go a long way in making nm procedures affordable and accessible to all sections of the indian society , thus pointing to the necessity of periodic costing exercises in every nm department .

context : escalating health care expenses pose a new challenge to the health care environment of becoming more cost - effective . there is an urgent need for more accurate data on the costs of health care procedures . demographic changes , changing morbidity profile , and the rising impact of noncommunicable diseases are emphasizing the role of nuclear medicine ( nm ) in the future health care environment . however , the impact of emerging disease load and stagnant resource availability needs to be balanced by a strategic drive towards optimal utilization of available healthcare resources.aim:the aim was to ascertain the cost of diagnostic procedures conducted at the nm department of a tertiary health care facility by employing activity based costing ( abc ) method.materials and methods : a descriptive cross - sectional study was carried out over a period of 1 year . abc methodology was utilized for ascertaining unit cost of different diagnostic procedures and such costs were compared with prevalent market rates for estimating cost effectiveness of the department being studied.results:the cost per unit procedure for various procedures varied from rs . 869 ( usd 14.48 ) for a thyroid scan to rs . 11230 ( usd 187.16 ) for a meta - iodo - benzyl - guanidine ( mibg ) scan , the most cost - effective investigations being the stress thallium , technetium-99 m myocardial perfusion imaging ( mpi ) and mibg scan . the costs obtained from this study were observed to be competitive when compared to prevalent market rates.conclusion:abc methodology provides precise costing inputs and should be used for all future costing studies in nm departments .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

PMC2905945

neuroblastoma is the most common pediatric solid tumor , deriving from immature or precursor cells of the ganglionic lineage of the sympathetic nervous system [ 1 , 2 ] endowed with remarkable heterogeneity with regard to histology and clinical behavior [ 3 , 4 ] . the neuroblastoma cell lines derived from the fresh tumors show various degrees of differentiation , chromosomal alterations , and morphology and consequently , a great variability in the gene expression profile . hypoxia is a condition of low oxygen tension that characterizes many pathological tissues and that is a critical determinant of tumor cell growth , susceptibility to apoptosis , and resistance to radio and chemotherapy [ 68 ] . the general response to hypoxia involves activation of biochemical pathways leading to alternative ways to generate energy that becomes scant in low oxygen . hypoxia modulates gene expression through the activation of several transcription factors , among which the hypoxia - inducible transcription factor-1 ( hif-1 ) [ 7 , 10 ] , and -2 ( hif-2 ) are the most studied . rapidly expanding neuroblastoma tumors present areas of hypoxia and it has been reported that hif-2 expression correlates with poor prognosis [ 13 , 14 ] suggesting a central role of hypoxia in tumor progression . hifs transactivate the hypoxia - responsive element ( hre ) present in the promoter or enhancer elements of many genes encoding angiogenic , metabolic , and metastatic factors [ 8 , 15 , 16 ] . however , neuroblastoma cell lines respond differently to hypoxia and the nature of the modulated genes depends strongly on the type and genetic makeup of the cell . furthermore , amplification and/or overexpression of mycn oncogene , occurring in poor prognosis tumors , influence the transcriptional response to hypoxia of neuroblastoma cell lines . the identification of molecular markers capable of discriminating the hypoxic status of the tumor may result in the discovery of new risk factors for neuroblastoma patients ' stratification and potential targets for tumor therapy . to this end , we were interested in identifying hypoxia signatures that discriminate the hypoxia status of neuroblastoma cell lines . unsupervised analysis of gene expression profile could not be applied to this system because the overwhelming effect of mycn amplification on the transcriptome masked the response to hypoxia . the application of a supervised approach represented by regularization with double optimization on microarray data , an embedded feature selection technique proposed by zou and hastie and studied by de mol et al . these results raise the question as to whether this signature is the only possible outcome of the l1-l2 regularization algorithm , and hence the only source of neuroblastoma hypoxia markers , or whether additional signatures , with similar characteristics of performances and robustness can be derived from the experimental data set . hypoxia induces massive transcriptional changes in the cell [ 2022 ] and it is possible that additional signatures may be found by the l1-l2 algorithm under appropriate conditions . the l1-l2 regularization algorithm has to deal with heterogeneity of the response of each cell line and with the background noise that is enhanced by the high dimensionality of the system composed by a low number of samples ( n = 18 in this work ) relative to the large number of the expression values for each sample ( p = 54 , 613 ) . the n p scenario is a common issue in signal processing and machine learning [ 23 , 24 ] . furthermore , the strong response of each cell line to alteration of the genetic makeup ( e.g. , mycn rearrangement ) tends to overcame and mask the response to hypoxia . here , we explore the possibility that l1-l2 feature selection algorithm may generate new hypoxia signatures following prior knowledge - based data filtering techniques as a preprocessing step to feature selection . most dimensionality reduction methods , such as pca and other unsupervised learning methods , rely only on the input data and may be driven by strong concurrent signals which are unrelated with , and somehow hide , the problem under study . alternative strategies of data filtering are based on some form of prior knowledge of the biology of the system . the information collected by gene ontology ( go ) , a project having the aim of classifying gene products in terms of their associated biological processes , cellular and molecular components can help identifying the pathways related to hypoxia and restricts the analysis to smaller sets of data . in this paper , we demonstrate that l1-l2 regularization applied separately to probesets representing genes belonging to selected go ontologies has the capability to generate robust hypoxia signatures , equivalent to that generated by the whole data set yielding biologically relevant information in a fraction of computer time . microarray data were downloaded from the gene expression omnibus public repository at national center for biotechnology information database ( accession number gse15583 ) . these data represent the gene expression profile of nine neuroblastoma cell lines cultured under normoxic ( 20% o2 ) or hypoxic ( 1% o2 ) conditions for 18 hours as detailed in , to obtain a total of 18 samples . affymetrix hg - u133 plus 2.0 genechip ( affymetrix , santaclara , ca ) were used for this study . gene expressions were then extracted from cel files and normalized using the robust multichip average ( rma ) method by running an r script using the bioconductor package affy . comparative analysis of hypoxic relative to normoxic expression profiles for each cell line was conducted on genespring 7.3 software ( agilent technologies ) . gene expression data were normalized using per chip normalization and per gene normalization first , each signal was normalized based upon the median signal in that chip ( per chip normalization ) . function by which each normalized value is corrected based upon the median of the measurements for that gene in all samples . finally , only genes that were modulated by at least 2-fold between hypoxic and normoxic cells were considered differentially expressed . the biological groups were obtained from the literature and they were divided into three main categories depending on the biological characteristics of our experimental system ( see table 2 ) : ( i ) hypoxia related groups [ 9 , 17 , 29 ] ; ( ii ) mycn related groups [ 3032 ] ; ( iii ) neuroblastoma related groups [ 31 , 33 , 34 ] . the selected functional groups were then filtered to avoid overlapped or duplicated categories and were defined according to predetermined pathways and functional categories annotated by the gene ontology project . the core of our approach is the l1-l2 regularization originally presented in and further developed and studied in [ 35 , 36 ] . to describe such method we first fix some notation in the learning framework . assume we are given a collection of n examples / subjects , each represented by a p - dimensional vector x of gene expressions . each sample is associated with a binary label y , assigning it to a class ( e.g. , patient or control ) . the dataset is therefore represented by a n p matrix x , where p n and y is the n - dimensional labels vector . we consider a linear model f(x ) = x , . note that = 1 , , p is a vector of weight coefficients and each probeset is associated to one coefficient . a classification rule can be then defined taking sign ( f(x ) ) = sign ( x , ) . if is sparse , that is some of its entries are zero , then some genes will not contribute in building the estimator . the estimator defined by l1-l2 regularization solves the following optimization problem : ( 1)l1l2=argmin [ ||yx||22+[||||1+||||22 ] ] , where the least square error is penalized with the l1 and l2 norm of the coefficient vector . the least square term ensures fitting of the data whereas adding the two penalties allows avoiding overfitting . the relative weight of the two terms is controlled by the parameter . the role of the two penalties is different , the l1 term ( sum of absolute values ) enforces the solution to be sparse while the l2 term ( sum of the squares ) preserves correlation among the genes . this approach guarantees consistency of the estimator and enforces the sparsity of the solution by the l1 term , while preserving correlation among input variables with the l2 term . differently to we follow the approach proposed in , where the solution l1l2 , computed through the simple iterative soft thresholding , is followed by regularized least squares ( rlss ) to estimate the classifier on the selected features . the parameter in the l1-l2 regularization is fixed a priori and governs the amount of correlation . by tuning in ( 0 , + ) we obtain a one - parameter family of solutions which are all equivalent in terms of prediction accuracy , but differ on the degree of correlation among the selected features . in practice , has an upper bound , max , such that for > max selection does not change , because all correlated features were already selected with = max . by setting = 100 , the maximal value , the maximal gene list , which is correlation aware , is obtained . conversely , the minimal list is obtained for values of equal to or lower than 1 . the training for selection and classification requires the choice of the regularization parameters for both l1-l2 regularization and rls denoted with * and * , respectively . hence , statistical significance and model selection is performed within double - selection bias - free cross - validation loops ( see for details ) . the classification performance of the system is measured by the leave - one - out error that is the percentage of misclassified samples . in other words , leave - one - out error is equal to one minus accuracy . in order to assess a common list of probesets , we based ours on the frequency , that is , we decided to promote as relevant variables the most stable probesets across the lists . the correlation among the probesets selected by the l1-l2 algorithm was performed as previously described in . briefly , we build blocks of correlated probesets using a variation of well - known agglomerative clustering techniques based on pearson distance . we first examine the minimal list , which genes are clustered via hierarchical clustering with correlation distance and average linkage . since no objective algorithm , other than heuristics , is available for establishing the number of clusters , for each go class the cut of the hierarchical graph determining the number of clusters is chosen following visual examination of the graph . in particular we set the cut at 0.75 of the maximum linkage value in the dendrogram . for each go class the cut of the hierarchical graph determining the number of clusters is chosen following visual examination of the graph . each probeset in the maximal list is then assigned to the cluster which average correlation with the given probeset is the highest . in this way we populate the clusters built from the minimal list with all the probesets coming from the maximal list . we mapped the hre elements in the promoter regions of the genes represented in the affymetrix hg - u133 plus 2.0 genechip . we downloaded the annotation file for the hg - u133 plus 2.0 from netaffx analysis center ( http://www.affymetrix.com/ ) and the dataset was restricted to the known mrna sequences listed in the ensembl database v56 . promoter the first 2,000 base pairs upstream the transcription initiation site and generated a dataset containing the promoters of the genes coding for the mrnas spotted on the chip . the hre matrix has been obtained from 69 experimental validated human hre sequences with matdefine tool ( genomatix software gmbh ) . hre consensus elements [ ( g|c|a|t ) ( c|g|t|a ) ( g|c|a|t ) ( t|g|c|a ) ( a|g ) ( cgtg ) ( c|g|t|a ) ( g|c|a|t ) ( g|c|t|a ) ( c|g|t|a ) ] were searched in the promoter sequences with matinspector software ( genomatix ) with core similarity = 1 and optimized matrix similarity . was used to evaluate the significance of the hre frequency in the promoter regions of genes belonging to the different signatures . we studied nine neuroblastoma cell lines heterogeneous with respect to mycn amplification and morphology ( table 1 ) . the cell lines were cultured under normoxic and hypoxic conditions for 18 hours and the total rna was tested for gene expression profiling using the affymetrix hg - u133 plus 2.0 platform . the response to hypoxia of each individual cell line was first analyzed by measuring the fold change as the ratio of the expression level between hypoxic and normoxic samples . we found that the response of each neuroblastoma cell line to hypoxia is characterized by a high number of modulated genes ranging from 855 to 1609 for the upregulated and from 758 to 1317 for the downregulated probesets ( table 1 ) . however , the modulated genes changed from cell line to cell line ( data not shown ) and only the application of a strong feature selection technique , represented by the l1-l2 regularization , allowed to identify a single signature of 11 probesets ( all - chip signature ) discriminating the normoxic and the hypoxic status . the large amount of hypoxia - modulated genes suggests that additional hypoxia signatures may be identified if we reduce the background noise of the system . to this end we restricted our analysis to the genes known to be involved in the hypoxic response on the bases of our reading of the literature and comprised in the biological processes according to the gene ontology ( go ) classification . the selection of the go classes was based on the reports of hypoxia modulated genes without attempting to distinguish the various cell types under investigation . 13 biological processes that are involved in hypoxia response were selected ( table 2 ) . we reasoned that this approach would restrict the analysis to the probesets that have a high impact on the hypoxic response while potentially eliminating the noisy features . to explore the potential interference from mycn status in the classification process , we selected and tested 7 biological processes involved in mycn activity ( table 2 ) . finally , we selected a third group of go processes related to the neuroblastoma biology as a control . for each of the 38 classes shown in table 2 , the l1-l2 algorithm selected a list of hypoxia discriminating probesets and calculated the corresponding classification leave - one - out error . the output of the l1-l2 regularization algorithm depends on the parameter that governs the amount of correlation allowed among the probesets . we set = 100 , the maximal value , to obtain the most comprehensive signature maximizing the number of correlated probesets to be included in the output . the validation has been performed by leave - one - out cross - validation on the 18 samples . then , a unique list is obtained as the union of the probesets included in the 18 lists , with a frequency score calculated as the frequency of each probeset in the 18 lists generated by the cross validation loops . stable probesets were defined as those characterized by a frequency score equal to , or higher than , 50% as previously reported in . the use of cross validation allows the selection protocol to generate an unbiased and objective output beyond the theoretical results that guarantee the robustness of the core algorithm . a leave - one - out error of 20% was chosen as the cutoff level for the classification performance . the leave - one - out error of the all - chip signature is 17% . the only classes characterized by a list of selected probesets capable of generating a leave one - out error lower than the 20% cutoff were apoptosis ( 17% ) , glycolysis ( 11% ) , and oxidative phosphorylation ( 11% ) ( table 2 ) , all of them belonging to the hypoxia biological group . these results demonstrate that , within each of the above classes , there is a list of probesets capable of discriminating the condition of the cell lines thereby defining three new neuroblastoma hypoxia signatures , named apoptosis signature , glycolisis signature , and oxidative phosphorylation signature . as expected , there were no go classes belonging to the mycn or neuroblastoma biological groups that generated hypoxia signatures , supporting the validity of our choice of hypoxia - related go functional classes . although mycn represents a strong signal that drives major transcriptome difference in neuroblastoma cell lines , our results show that there are no enough discriminatory genes in the mycn - related processes . these results demonstrate that the feature selection method applied is capable of revealing the differences occurring among hypoxic and normoxic neuroblastoma cell lines by filtering out strong competing signals , such as mycn amplification status . the list of the probesets comprising the 11 probesets ( all - chip signature ) and the newly identified signatures is shown in table 3 and consists of 10 probesets for apoptosis signature , 3 for glycolysis - signature , and 32 for the oxidative phosphorylation signature . the new signatures highlight 41 probesets that were not previously included in the all - chip signature and contribute to the discrimination of the hypoxic status . furthermore , the 32 probesets of the oxidative phosphorylation signature does not overlap with the all - chip signature , demonstrating that the increased resolution generated by data filtering allows the identification of previously discarded relevant go processes . the hypoxia signatures present in the literature show different sizes and gene composition [ 9 , 4346 ] . since different cell types respond heterogeneously to hypoxia by modulating different set of genes , we decided to compare our results with the published hypoxic gene signatures obtained from neuroblastoma cell lines ( table 4 ) . in order to make the comparison feasible , in fact , an overlap is present in all - chip ( 3/8 genes ) , apoptosis ( 2/4 genes ) , and glycolysis ( 2/2 genes ) signatures . interestingly , there is no overlap ( 0/24 genes ) among the results published by jgi et al . and the oxidative phosphorylation signature . about 33% of the genes spotted on the chip present a hre sequence in the promoter region . we investigated whether there was enrichment in hre containing promoter in the genes composing our signatures . we found that all the signatures are significantly enriched ( p < .01 ) in genes containing hre ( table 4 ) . in particular , all the genes included in all - chip , apoptosis , and glycolysis signatures contain at least one hre , while hre containing genes constitute 91% of the oxidative phosphorylation signature . these results support the idea that our signatures are associated with the hypoxia status . the whole signature , rather than individual genes , is important for discriminating the hypoxic status . for example , vegf is a gene whose expression is strongly related to hypoxia and is part of the apoptosis and angiogenesis classes , both of which are part of the hypoxia biological group . however , the contribution of vegf probesets is not sufficient to reach the discriminatory power required to generate a significant signature out of the angiogenesis class as opposed to the apoptosis class . the strong discriminatory power of the signatures can be visualized by a 3-dimensional representation of the probesets projected on their 3 principal components . l1-l2 algorithm produces a multigene model but the multidimensional representation can be well approximated by the tridimensional picture when the number of probesets is not too large . figure 1 depicts the separation of probesets belonging to the glycolysis and shows that the two classes of normoxic and hypoxic cell lines are clearly separated in the multidimensional space . in conclusion , we demonstrate that , upon data reduction the l1-l2 algorithm can identify new hypoxia signatures that have equivalent discriminatory power relative to that obtained by the analysis of the whole transcriptome . from the computational stand point , this process allows to reduce the computer time by approximately 10 times , from days to hours with an average machine , facilitating the analysis of the data . finally , it is important to highlight the possibility of applying our method to different experimental settings by choosing appropriate selection of go processes . our prior knowledge - based method produces nested lists of relevant probesets but does not highlight the correlation among them , and it should be completed by a postprocessing step depicting the correlation structure . we computed a distance matrix based on the expression values of the probesets and subdivided it into 8 modules by hierarchical clustering . these modules represent subgroups of correlated probesets that are positively or negatively associated to the hypoxic status . this information is important to pick the correct probesets in order to assess the expression of these markers in the in vivo setting . furthermore , these data lend themselves to the tuning of the parameter that is part of the l1-l2 algorithm . the output of the l1-l2 regularization algorithm depends on the free parameter that governs the amount of correlation allowed among the probesets and selects the amount of probesets to be included in the signature . by setting = 100 , the maximal value , we can obtain a comprehensive signature more descriptive of the biology of the system . by setting = 1 , we can obtain an equally discriminating signature with fewer genes thereby more effective in identifying critical biomarkers for diagnostic applications . the results are shown in table 5 , where the probesets selected by l1-l2 regularization with both = 1 and = 100 for oxidative phosphorylation are listed . the results demonstrated that the reduction in is associated with a smaller signature ( from 32 to 16 probesets ) as expected by the fact that correlated probesets tend to be discarded . the identification of signatures discriminating the hypoxic status of the tumor cell may be important for our understanding of the biology of neuroblastoma tumors and for the stratification of the patients in risk groups . one way to generate a robust and reliable hypoxic signature is the application of a supervised approach represented by l1-l2 regularization that generates an 11 probesets signature discriminating the hypoxic status of our panel of nine neuroblastoma cell lines . here , we demonstrate that l1-l2 feature selection algorithm generates new and robust hypoxia signatures following prior knowledge - based data filtering techniques as a preprocessing to feature selection . these new signatures have the same discriminatory power as that generated by the whole data set and yield biologically relevant information in a fraction of computer time . the data filtering is based upon the use of the prior information contained in go and the literature , and it allows restricting the analysis to smaller data sets . this process filters out not only many noisy probesets but also the probesets selected from the all - chip analysis whose strong relation with hypoxia hid some weaker but important genes . l1-l2 regularization algorithm following data filtering selects probesets that were not the first chosen when all the probesets were considered . the prior knowledge utilized in setting up the filter , comes from the current literature from which we derived the molecular pathways that are important for the response of the cell to the hypoxic environment . interestingly , the new signatures were found only in this group and not in other collections of go pathways like those related to the effects of the mycn oncogene or to the neuroblastoma biology . in general , the identification of the go classes related to the phenomenon under investigation may be an empirical , but effective way to target the potential source of signatures to be fed to the l1-l2 regularization . we speculate that this approach could be used to address questions that go beyond the hypoxic status and may find signatures characterizing other pathophysiological situations provided that there is a relevant cellular model and there are sufficient insights in the underlying molecular mechanisms . the nested structure of the selected gene lists allows the choice of the desired level of complexity , which is the magnitude of signature , maintaining all the information extracted from the data . for example , the minimal list may be preferable when interested in finding biomarkers to be used on large - scale diagnostic tests due to potential constrains on time , cost , and resources . finally , working on a limited number of probesets has a major impact on the computational time required for the analysis that changes from days to hours , thereby allowing more leeway to the study of the dataset .

hypoxia is a condition of low oxygen tension occurring in the tumor and negatively correlated with the progression of the disease . we studied the gene expression profiles of nine neuroblastoma cell lines grown under hypoxic conditions to define gene signatures that characterize hypoxic neuroblastoma . the l1-l2 regularization applied to the entire transcriptome identified a single signature of 11 probesets discriminating the hypoxic state . we demonstrate that new hypoxia signatures , with similar discriminatory power , can be generated by a prior knowledge - based filtering in which a much smaller number of probesets , characterizing hypoxia - related biochemical pathways , are analyzed . l1-l2 regularization identified novel and robust hypoxia signatures within apoptosis , glycolysis , and oxidative phosphorylation gene ontology classes . we conclude that the filtering approach overcomes the noisy nature of the microarray data and allows generating robust signatures suitable for biomarker discovery and patients risk assessment in a fraction of computer time .

1. Background 2. Materials and Methods 3. Results and Discussion 4. Conclusions

PMC4451154

in recent years , owing to the rapid economic development and changes in lifestyle , the number of patients with diabetes mellitus in china has increased dramatically . the age - standardized prevalence rates of total diabetes and prediabetes in china are reportedly 9.7% and 15.5% . among the patients with type 2 diabetes mellitus , epidemiologic studies have demonstrated that dyslipidemia increases the risk of cardiovascular diseases [ 24 ] . because dyslipidemia is asymptomatic , it is difficult to diagnose and manage , and many clinicians ignore the importance of dyslipidemia in patients with diabetes . a previous study based on the chinese national nutrition and health survey in 2002 investigated the prevalence of dyslipidemia in 14252 chinese adults and found that the overall prevalence of dyslipidemia was 18.6% ( 22.2% in men and 15.9% in women ) . in addition , although numerous studies have shown that serum lipid levels are high in diabetes patients , data on serum lipid and lipoprotein levels in patients newly diagnosed with type 2 diabetes mellitus are scarce , especially in chongqing , a midlevel developed city in southwest china with living conditions representative of the majority of chinese cities and thus a fitting representative of the general chinese population . the aim of the study was to analyze the trends in serum lipid levels and the prevalence of patients with dyslipidemia , coronary heart disease ( chd ) , hypertension , and cerebrovascular disease in adults with newly diagnosed type 2 diabetes mellitus in the southwest chinese han population during 20032012 . overall , 994 inpatients newly diagnosed with type 2 diabetes mellitus between january 2003 and december 2012 were collected from 3 different hospitals including the second affiliated hospital of chongqing medical university , bishan district people 's hospital of chongqing , china , and fuling district people 's hospital of chongqing , china . all participants were han ethnic . none of these patients received prescribed lipid or hormone replacement therapy , and those taking lipid - lowering medication were excluded . all subjects had complete data regarding total cholesterol ( tc ) , low - density lipoprotein cholesterol ( ldl - c ) , high - density lipoprotein cholesterol ( hdl - c ) , and serum triglyceride ( tg ) levels from each study year . this project was approved by the human research ethics committee of chongqing medical university . because our study is retrospective , patient information was anonymized and deidentified when data were collected and analyzed , thus exempting the requirement for informed consent . according to national cholesterol education program adult treatment panel ( ncep atp ) iii , the diagnosis of dyslipidemia for patients with diabetes is based on the presence of one or more of the following criteria : high tc ( 5.18 mmol / l ) , low hdl cholesterol ( 1.04 mmol / l for men and 1.30 mmol / l for women ) , and high tg ( 1.70 mmol / l ) levels . hypertension was defined as systolic and diastolic blood pressure 140 and 90 mmhg , respectively , and/or current treatment with antihypertensive medications . chd was diagnosed on the basis of a history of acute coronary syndrome or was confirmed by computed tomography angiography , or coronary angiography . cerebrovascular disease , including ischemic stroke and hemorrhagic stroke , was diagnosed using brain computed tomography or brain magnetic resonance imaging . the trends of the mean serum lipid levels were analyzed by comparing the data of 3089-year - old adults obtained from 5 studies : 2003 - 2004 , 2005 - 2006 , 2007 - 2008 , 2009 - 2010 , and 2011 - 2012 . these 5 sets of data were obtained independent of each other . at the time of diagnosis , blood was drawn after an 8 h fast for measuring tg , tc , hdl - c , ldl - c , and hemoglobin a1c ( hba1c ) levels . the same methods of lipid level measurements were used in all 5 examinations . the gpo - pap method and cod - ce - pap assay hdl and ldl cholesterol levels were measured using the cat method ( maker biotechnology co. , ltd . , the body mass index ( bmi ) was calculated by dividing the weight ( kg ) by the square of the height ( m ) . information about the diagnosis of chd , hypertension , and cerebrovascular disease was collected from the patients ' medical records . the results are expressed as mean standard deviation for tc , hdl - c , and ldl - c . geometric means and standard errors of geometric means are presented for serum triglyceride levels because the distribution is highly skewed . the prevalence of dyslipidemia and the morbidity rates of chd , hypertension , and cerebrovascular disease are presented as percentages . we calculated the differences between the time periods using analysis of variance ( anova ) for continuous variables ; kruskal - wallis test was used for nonnormally distributed variables ; to test whether the percentage of patients with dyslipidemia , hypertension , chd , and cerebrovascular disease changed over the years , the chi - square test was used . , chicago , il , usa ) , and p values less than 0.05 were considered statistically significant . the medical data of 994 subjects ( 550 men and 444 women ; age range : 3089 years ) were analyzed . the time trends in the mean age ( p = 0.723 ) , bmi level ( p = 0.052 ) , and haemoglobin a1c ( hba1c ) level ( p = 0.391 ) of the patients with newly diagnosed type 2 diabetes were not of statistical significance during the study period ( table 1 ) . the mean tc level of 3089-year - old adults increased from 4.92 1.15 mmol / l in 2003 - 2004 to 5.30 1.17 mmol / l in 2011 - 2012 ( p = 0.039 ; figure 1 ) . the mean serum tc level was higher in women than in men in each period ( table 2 ) . the mean ldl - c levels gradually increased between 2003 - 2004 and 2011 - 2012 ( p = 0.004 ; figure 1 ) . a significant increase was observed in the ldl - c levels in men ( from 2.64 0.80 mmol / l in 2003 - 2004 to 3.09 1.08 mmol / l in 2011 - 2012 ; p = 0.001 ; table 2 ) . in women , mmol / l in 2003 - 2004 to 3.18 1.26 mmol / l in 2011 - 2012 , but this increase was not statistically significant ( p = 0.280 ; table 2 ) . in each period , the mean ldl - c level was higher in women than in men ( table 2 ) . the mean serum triglyceride levels of all patients increased till 2005 - 2006 and then decreased till 2011 - 2012 ( p = 0.148 ; figure 1 ) . mmol / l in 2003 - 2004 to 1.06 0.24 mmol / l in 2011 - 2012 ( p < 0.001 ; figure 1 ) . in men , the hdl cholesterol level showed a significant ( p < 0.001 ) downward linear trend from 1.19 0.36 mmol / l in 2003 - 2004 to 1.02 0.22 mmol / l in 2011 - 2012 ( table 2 ) , whereas , in women , the hdl - c levels showed no significant change . the mean serum hdl - c level was lower in men than in women in each period ( table 2 ) . the percentage of patients with a serum total cholesterol level of at least 5.18 mmol / l increased gradually from 40.7% in 2003 - 2004 to 55.6% in 2011 - 2012 ( p = 0.001 ; figure 2 ) . the prevalence of hypo - hdl - cholesterolemia for men increased greatly from 32.8% in 2003 - 2004 to 71.2% in 2009 - 2010 and then decreased to 53.7% in 2011 - 2012 ( p < 0.001 ; figure 2 ) . in women the percentage of hypo - hdl - cholesterolemia increased from 54.8% to 72.1% during the study period ( p = 0.008 ; figure 2 ) . no significant differences were observed in the prevalence of hyper - ldl - cholesterolemia and hypertriglyceridemia between 2003 and 2012 . but we observed that majority of patients newly diagnosed with type 2 diabetes mellitus have high ldl cholesterol levels . the prevalence rates of hypertension , chd , and cerebrovascular disease at the time of diagnosis of type 2 diabetes mellitus are shown in figure 3 . in this study , we found that both the tc levels and the percentage of patients with hypercholesterolemia increased gradually , and the same trend was observed for ldl cholesterol . the ncep atp iii constantly monitors the elevated levels of ldl cholesterol , which is a major risk factor contributing to chd . the recommended ldl cholesterol levels for individuals at high risk for cardiovascular disease and for those without overt cardiovascular disease are < 1.8 mmol / l and the lowest mean ldl cholesterol level for all participants was 2.72 0.83 mmol / l observed in 2003 - 2004 and not only was higher than the recommended levels , but was also found to have increased since 2003 - 2004 , reaching a high of 3.11 1.09 mmol / l in 2011 - 2012 . hence , the management of high ldl cholesterol level in diabetes patients is vital for reducing the risk of cardiovascular events . although lowering ldl cholesterol level is the major focus in this field of study , benefits associated with targeting other lipids have also been previously demonstrated . for example , hdl cholesterol level is a strong negative indicator for cardiovascular events , with high levels of hdl cholesterol having been suggested to protect against atherosclerotic manifestations . on the basis of several epidemiological studies , when hdl cholesterol levels are increased by 1.0 mg / dl , the occurrence of chd simultaneously decreases by 2 - 3% . in our study , unfavorable upward trends were observed in hdl cholesterol levels and the prevalence of low hdl cholesterol for men and women . accordingly , several studies have shown that hdl cholesterol levels were markedly reduced in both men and women with diabetes mellitus compared with those in nondiabetic individuals . therefore , management of hdl cholesterol levels should be essential in patients with newly diagnosed type 2 diabetes mellitus . although no significant changes were observed in the serum triglycerides levels during 20032012 , we speculate that this trend may have been influenced by differences in the diets of the patients . according to the values of ncep atp iii classification , both men and women had elevated triglyceride levels in each time period . being overweight , sedentary , or a cigarette smoker is associated with elevated triglyceride levels ; thus , positive lifestyle changes can help lower triglyceride levels . in our study , we found that , at the time of diagnosis of diabetes , elevated triglyceride levels , high ldl cholesterol levels , and low hdl cholesterol levels were commonly noted , and these are all characteristic features of diabetic dyslipidemia . moreover , we found that the mean total and ldl cholesterol levels were higher in women than in men . one possible reason for this observation is that hormonal changes after menopause cause a rapid increase in lipid levels , which become higher than those in men . in the past several years , total serum cholesterol levels have declined in most western populations , such as in australia , north america , and europe ; however , they have increased in the east , southeast asia , and the pacific regions [ 14 , 15 ] . in china , the mean levels of serum lipids have been reported to be much higher in areas with rapid economic growth , such as shanghai and beijing , compared to those in rural areas . remarkable socioeconomic development along with a rise in the living standards of chinese han population in the past few decades may explain the unfavorable trends in the lipid profiles observed herein . numerous studies have indicated that lifestyle factors such as high calorie intake , population - wide sedentary lifestyle , decreased physical activities , and increased obesity are related to increased serum lipids levels [ 17 , 18 ] . although we did not investigate changes in the eating habits and physical activities of individuals in this study , other studies have reported that the eating habits of the chinese population have changed dramatically from the traditional dietary pattern ( plant - based , low - fat diet ) to the western dietary pattern ( high - fat , low - carbohydrate diet ) , which is characterized by excess calorie intake . another study reported that china is experiencing rapidly escalating rates of overweightness and obesity in recent years . in china , the rate of overweightness has doubled from 13.5% in 1991 to 26.7% in 2006 , and the number of obese individuals has tripled . the classifications of overweightness and obesity in chinese individuals are based on the bmi cutoffs of 24 kg / m and 28 kg / m , respectively , and in our study , the average bmi was higher than 24 kg / m in each period , indicating that most of the patients with newly diagnosed type 2 diabetes mellitus were overweight . another possible factor responsible for the changes in serum lipid levels is a decrease in physical activity . a sedentary lifestyle is now becoming widespread , especially among urban residents , with the average weekly physical activity among adults having decreased by more than a third between 1991 and 2006 . increasing reliance on automobiles and increasing availability of buses have replaced the traditional ways of transportation such as biking or walking , and the increased use of television and computers has replaced the traditional means of recreation such as swimming and running [ 24 , 25 ] . thus , lifestyle and behavioral changes need to be addressed in order to reduce the incidence of dyslipidemia . the united kingdom prospective diabetes study provided conclusive evidence that , with effective control of hyperglycemia in diabetes patients , morbidity due to microvascular complications can be significantly reduced . however , intensive treatment of hyperglycemia did not significantly reduce macrovascular disease . in our study , in accordance with the increase in serum lipid levels , the trends of chd and cerebrovascular disease in people newly diagnosed with diabetes also continuously increased during the study period . first , when we classified the study participants into different ages , no statistical significance was observed , and this could be because we did not use a very large enough sample size . second , this was a cross - sectional analysis and we could investigate factors affecting the prevalence of dyslipidemia , such as dietary intake and physical activities . hence , further research is needed to assess the effects of lifestyle factors on serum levels of lipids . third , the patients taking lipid - lowering medication were excluded ; the percentages of people with dyslipidemia did not include these patients . the results of this study indicated unfavorable upward trends in serum lipid levels and in the prevalence of dyslipidemia , chd , and cerebrovascular disease among patients with newly diagnosed type 2 diabetes mellitus in the southwest chinese han population . widespread promotions of ways to lower lipid levels and of treatment strategies should be intensified to reduce cardiovascular morbidity and mortality . in conclusion , dyslipidemia is preventable and now is the ideal time for implementing appropriate strategies to battle this epidemic .

objective . to determine the trends of serum lipid levels and dyslipidemia in adults newly diagnosed with type 2 diabetes mellitus during 20032012 in southwest china . methods . serum lipid measurements of 994 adults were obtained from 5 independent , cross - sectional studies ( 2003 - 2004 , 2005 - 2006 , 2007 - 2008 , 2009 - 2010 , and 2011 - 2012 ) . the main outcome measures were mean serum total cholesterol , low - density lipoprotein cholesterol , high - density lipoprotein cholesterol , and triglyceride levels ; body mass index ; hemoglobin a1c level ; and the percentages of patients with dyslipidemia , hypertension , coronary heart disease , and cerebrovascular disease . results . the mean total cholesterol and low - density lipoprotein cholesterol levels increased from 4.92 1.15 to 5.30 1.17 mmol / l ( p = 0.039 ) and 2.72 0.83 to 3.11 1.09 mmol / l ( p = 0.004 ) , respectively , and the mean hdl cholesterol level declined from 1.22 0.30 to 1.06 0.24 mmol / l ( p < 0.001 ) . the percentages of patients with dyslipidemia increased gradually . the incidence of coronary heart and cerebrovascular diseases increased from 8.2% to 19.1% and 6.6% to 15.3% , respectively ( p < 0.05 ) . conclusion . unfavorable upward trends were observed in serum lipid levels and the prevalence of dyslipidemia , coronary heart disease , and cerebrovascular disease in adults newly diagnosed with type 2 diabetes mellitus in southwest china during 20032012 .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion

PMC4357875

all over india there are more than 20,000 anaesthesiologists practicing the art and science of anaesthesiology . the anaesthesiologists practice in different setups , such as teaching institutes , corporate hospitals and smaller nursing homes . the techniques , equipments and overall circumstances are different in every level of anaesthesia practice . however all of them have a similar kind of apprehension , as far as the legal issues are concerned . the legal issues are discussed in many conferences , continuing medical educations and in informal meetings among fellow anaesthesiologists . there is a need to address this issue at national , state and city levels , through specialist bodies such as the indian society of anaesthesiologists ( isa ) . with these considerations in mind , it was decided to conduct a survey to elicit the existing knowledge , perception and opinion of the individual anaesthesiologists in regard to legal issues in anaesthesia practice . the survey was performed by asking 875 anaesthesiologists about their views regarding the legal issues , if they have faced any such problems and how are they placed regarding the legal problems . the investigator met them individually , over a period from may 2012 to april 2014 , during various scientific meets of the anaesthesiologists . in the survey , only the opinions of anaesthesiologists , who were in anaesthesia practice for more than 3 years were taken in the account . the questions were asked in the informal group talk and the results are entered by the investigator in the data sheets specially prepared after analysing the responses of individual anaesthesiologist towards various issues [ table 2 ] . different category wise questions asked to individual anaesthesiologist in the informal talk data sheet specially prepared for survey at different scientific meets during the period of may 2012 to april 2014 , the author attended 13 scientific programs related to anaesthesiology . during these scientific meets attended by anaesthesiologists , table 3 shows the various scientific programmes covered and the number of anaesthesiologists interviewed according to their type of practice . the responses gathered from the anaesthesiologists were analysed and the same are depicted in table 4 . scientific programs covered for the survey and the number of anaesthesiologists interviewed , according to their type of practice the responses obtained from the anaesthesiologists for the different questions asked the medico - legal aspects are always of immense importance from the practicing anaesthesiologists point of view . this is why every conference or meeting of anaesthesiologists leads to the discussion on legal issues . hence , it was decided to study the nature of apprehension amongst anaesthesiologists regarding implications of the law related to anaesthesia practice . the response was very poor and it was not possible to extract replies from any of those anaesthesiologists . the questionnaire was decided to be applied as group administered questionnaire , an approved method of survey . it is easier to approach more number of respondents and during discussion interviewer can clarify the questionnaire if needed . as the freelance practitioners are generally more worried , being without an institutional cover , the number of freelance practitioners was more in the survey . whenever and wherever medico - legal issues are discussed , the freelance practitioners are always in a good number . the opinion of anaesthesiologists who has at least 3 years standing in the profession was taken into account . this excluded younger specialists and students who were yet to get proper experience in the specialty . such exclusion was decided as opinions after actual practical experience , and facing problems will be more helpful for my study . many textbooks of anaesthesia and professional bodies of anaesthesiologists give ample importance to this aspect of anaesthesiology . but it is a common observation that many of the patients are referred to physicians for their opinion on medical illness , who ultimately come back to the anaesthesiologist with a fitness certificate ( physician 's fitness ) . majority of anaesthesiologists dislike the fitness certificate from a physician , but do insist on sending patients for physician 's opinion pre - operatively for assessment of medical illness if present . the majority ( 701/875 80.11% ) few ( 80/875 9.14% ) were not bothered who gives fitness , while few colleagues did not mind giving this responsibility to physicians ( 73/875 8.34% ) and surgeons ( 21/875 2.4% ) . this opinion was with a comment that it does not matter who gives fitness certificate , as ultimately it is anaesthesiologist 's discretion to administer anaesthesia . as a safety cover against litigations , the majority of anaesthesiologists felt that asking for fewer investigations may be interpreted as negligence and hence insisted on battery of investigations before accepting patient for anaesthesia . there are discussions and guidelines available in the textbook of anaesthesia regarding the quality and quantity of investigations to be obtained before anaesthesia . but as individual anaesthesiologist , everyone decides to err on safer side by doing maximum possible investigations before administration of anaesthesia . in this survey , the majority ( 646/875 - - 73.84% ) opined that we are doing less investigations than ideal while only 229 out of 875 ( 26.17% ) opined that enough investigations were done for pre - operative evaluation . the reason offered for doing less investigations was the compromising attitude of anaesthesiologists because of economic constraints . the anaesthesiologists working in corporate setups could order investigations to their satisfaction ( 91 of 122 ) . kumar and srivastav in their review article on role of routine laboratory investigations in pre - operative evaluation state that , 30 60% tests continue to be greatly in excess of that recommended . the reasons the authors quote for doing more number of laboratory tests are , difficulty in changing previous work pattern or behaviour , fear of cancellation of surgery , missing some important information during evaluation , and institutional requirement . in the present survey , the reason could be the same as those quoted by the authors ; the clinicians firmly believed that more the investigations better is the safety . on other words , the anaesthesiologists believe that more investigations must be done because of the reasons quoted , but are not doing because of the economic constraint and doing less laboratory investigations may be taken as negligence . many of the anaesthesiologists want isa to prepare guidelines regarding pre - operative investigations pertaining to our setups . actual pre - operative optimization of the patient is done in the surgical wards only . when asked who should undertake the task of optimization of the patient , majority of anaesthesiologists ( 690 of 875 78.85% ) said that it must be done in surgical wards only . surgeons need to take help of any specialist to make the patient fit for anaesthesia . few ( 136/875 15.54% ) opined that physicians are better for the purpose while even less ( 49/875 5.6% ) thought that it is surgeon 's duty , but nobody expressed that the anaesthesiologist had responsibility for preparation of patient for anaesthesia . the anaesthesiologists did complain that the drugs started by the attending doctor pre - operatively were not suitable for the technique of anaesthesia they were going to offer and the anaesthesiologists had to modify the technique or had to struggle significantly for the safe conduct of anaesthesia . the american board of anaesthesiology defines anaesthesiology as practice of medicine dealing with assessment of , consultation for and preparation of patient for anaesthesia . however , nobody likes to take up the responsibility of pre - operative optimization of the patient or preparation of the patient for anaesthesia on themselves . this important issue is many a times left to a clerical staff of the hospital . in present survey , majority of anaesthesiologists did agree that the job is done by some class 3 or 4 employees of the hospital . 829 of 875 ( 94.74% ) committed that consent was taken by hospital staff only , while few ( 5.26% ) said that it was surgeon 's responsibility . majority protocols of anaesthesia practice have ensured entry of consent in the list but are taken as only checking the consent taken by hospital staff . but every anaesthesiologist felt concerned about the consent and opined that the consent form must be appropriate . due to non - availability of a standardised consent format , many anaesthesiologists try to prepare their own format of consent form , taking help of any and every model he comes across . the consent is always an informed consent , but there is a lot of controversy and confusion regarding the information offered to the patient . very few anaesthesiologists agreed that they conveyed information of anaesthesia risk ( 74 of 875 8.45% ) , whereas 160 of 875 ( 18.28% ) convey multi - factorial risk to the patient . only 10 out of 875 ( 1.14% ) explained the death risk to the patients for routine surgery and anaesthesia . risk to life is one such controversial issue , majority felt that although every anaesthesia procedure carries only thing that is informed in many consent forms is about possibility of shifting to higher centres if need arises . every anaesthesiologist 's nightmare is death of the patient on table . for patients of american society of anaesthesiologists physical status v ( e ) , many a times it is difficult to avoid death on table and majority of anaesthesiologists think that for medico - legal safety , as a rule , informed consent we must take on table death consent from the relatives of moribund patients . how far this is useful in preventing legal litigations is of doubtful value . there is no such entity as table death consent in literature , but many anaesthesiologist colleagues ( 779/875 - 89.02% ) insist that for moribund patients such consent must be taken . 10.98% had no opinion on this issue , but no one resisted the idea of table death consent . consent is actually a bilateral contract between two parties but in my survey i noted that majority of anaesthesiologists ( 696/875 79.54 ) never signed the consent form themselves , while ( 172 out of 875 19.65% ) signed the consent form sometimes . even the surgeons also do not counter sign the consent form in majority of places . the majority of anaesthesiologists opined that at many places they were working in compromised operating rooms . on direct questioning everyone replied that the operation theatres they were working in were adequately equipped , but overall they were still worried about medico - legal implications of inadequate theatre equipments . a little detailed probing revealed that defibrillators were not available in many operating rooms . only 165 of 875 ( 18.85% ) maintained that the theatres they were working were fully equipped and the number was more from the corporate sector ( 98 of 122 8.03% ) . regarding the role of isa , majority ( 831 of 875 94.97% ) felt that isa should prepare guidelines regarding theatre equipment that could be applicable to every type of operation theatre . paradoxically , few of the anaesthesiologists ( 44 5.02% ) were concerned that since theatres at peripheral smaller nursing homes were very much compromised and if isa publishes a list of minimum mandatory operating room standards , then anaesthesiologists working at periphery may face litigations . the guidelines are generally expected to protect patients , as well as the practicing anaesthesiologists . it not only helps in postoperative management of the patient but also saves anaesthesiologists from legal issues . in spite of the importance of record keeping only 360 of 875 ( 41.14% ) informed that they kept record of all cases , while 389 ( 44.45% ) wrote notes for very few of their cases . 126 ( 14.4% ) did not write any kind of anaesthesia notes . the teaching institutes and corporate hospitals were better placed regarding this aspect , as the protocol of those institutes made everyone to write notes ( 133/163 - 81.59 and 119/122 97.54% respectively ) . one - liner record. majority of anaesthesiologists ( 668 76.34% ) were not happy with the present methodology of anaesthesia charts , labeling it cumbersome . the electronic record keeping was the main hope of the better record keeping for many anaesthesiologists . any professional body of anaesthesiologists advice to have an insurance cover for anaesthesia practice . in the survey , majority ( 858/875 - 98.05% ) of anaesthesiologists had their indemnity insurance policy running . irrespective of the place where they worked , everyone opted for indemnity insurance , but the amount for which cover was taken varied individually . the anaesthesiologists who were not covered presently ascribed it to defaulting in renewing the insurance . about the services of the insurance company , the reasons expressed were that the companies were not much interested in promoting the indemnity schemes and such schemes did not fall in the priority category of the company plans . regarding few medico - legal cases revealed during the survey , the concerned anaesthesiologists were not happy with company 's service . there was a common appeal from all anaesthesiologists to isa to look in this matter and start our own indemnity insurance scheme . actual number of anaesthesiologists facing legal litigations is very low ; 7 that is 0.008% had faced or were facing medico - legal problems , lesser than western data but overall fear of medico - legal litigations was 100% . every one practicing this vital branch of medicine is worried about medico - legal litigations . the various suggestions made by colleague anaesthesiologist were : good record keeping ( 752 85.94%)observing minimum mandatory operating room standards ( 763 87.20%)rapport with patients , surgeons and hospital staff ( 290 33.14%)preparing protocols for every action or procedure ( 312 35.65% ) . good record keeping ( 752 85.94% ) observing minimum mandatory operating room standards ( 763 87.20% ) rapport with patients , surgeons and hospital staff ( 290 33.14% ) preparing protocols for every action or procedure ( 312 35.65% ) . the freelance practitioner does not believe in protocols , but the teaching faculty members ( 66.87% ) do insist on protocols . few other suggestions from many colleagues were , regular updating of knowledgerapport with fellow colleague , anaesthesiologistsoptimum preoperative investigations . regarding the litigations every anaesthesiologist thinks that whatever happens in the theatre is a collective responsibility of surgeon , anaesthesiologist and the hospital administrator . regarding indications of surgery every anaesthesiologist felt that it was the prerogative of the surgeon only and anaesthesiologist was not in a position to influence that aspect of treatment . the majority of anaesthesiologists felt that isa should have medico - legal cell at every city , district , state , zone and national level to help members . inspite of textbooks advising to curtail investigations the practicing colleagues think that investigations are necessary to wipe out negligence tags . this survey is based on a group administered questionnaire , which has few limitations ; like privacy of the respondent is not totally ensured and they may not get enough time to think and respond . so also influence of dominating nature of a single or multiple individuals in the group can bias few opinions . but inspite of these limitations we can expect better reflection of opinions in this survey because the population surveyed is elite and well educated anaesthesiologists a survey of anaesthesiologists was done to access perception of law related to their clinical practice . all types of practitioners were included in the study but the apprehension depicted by every one of them was similar in nature . the present operation theatre conditions need to improve and anaesthesiologists want isa to take lead in specifying operation theatre standards , pre - operative investigations and consent strategy . a suggestion of setting of medico - legal cell at all levels will alleviate apprehension . uniformity at national level regarding all these factors can defuse the apprehension amongst anaesthesiologists for legal issues . the professional indemnity insurance also needs streamlining from insurance companies and isa also needs to support the members .

background and aims : the anaesthesiologists understanding and knowledge of law has an important bearing on issues , related to anaesthesia practice . lack of such knowledge could also lead to misperceptions affecting both patient care and professional practices . the aim of the study was to find out exact nature of apprehension of the anaesthesia providers towards the implementation of the law related to anaesthesia practice.methods:a prospective survey of 875 anaesthesia specialists was conducted during informal meetings personally by the investigator and the responses to a standard set of queries were noted . also included were the remarks and suggestions related to each query . three different set of practitioners depending on the place of work ( teaching hospital , corporate hospital and small establishments ) were queried.results:the opinions of all three groups of anaesthesiologists did not differ materially . the opinions and views were similar in many aspects and controversy existed in few areas . major confusion was about how much information should be provided to patients before anaesthesia to obtain informed consent . opinions were offered for prevention of litigations and how to face litigations but there was the lack of clarity on these issues.conclusions:the anaesthesiologists are invariably confused about how exactly they should prevent litigations or respond to them . the majority expressed need for intervention by indian society of anaesthesiologists ( isa ) to prepare protocols and set up medico - legal cells .

INTRODUCTION METHODS RESULTS DISCUSSION CONCLUSION

PMC4271717

they would like to know the origin of their cancer , the prognosis for the cancer , and whether their children are at risk of getting cancer . thus , the need for genetic counseling is increasing all the time.13 when whole gene exon maps and the complete genetic makeup are available to everyone , genetic counseling would be needed to explain the implications and importance of the findings . there is also a potential risk that mutation carriers may be subject to different forms and degrees of genetic discrimination.4 genetic responsibility for oneself and others is a strongly debated implication of genetic testing for cancer and demands broad consideration of the boundaries between individual and community rights.4 similarly , until gene maps are available from primary tumors and from each relapse , it would not be possible to provide a picture of the cancer or of the genes involved its development . even when the gene maps are available , it will still always have to be supplemented by the clinical events connected to genes . all the data need to be understood and interpreted for the benefit of a patient seeking help . genetic knowledge can be perceived as enhancing the control that individuals have over their lives or as paralyzing the decision process of an individual who may feel predestined to suffer a serious disease.5 as research continues to unveil more drug gene and disease gene associations and clinical practice moves toward the concept of personalized medicine , it is critical that clinicians understand which pharmacogenetic assays are available to identify differences between individuals , as they predict the safety and efficacy of anticancer drugs.6 the mapping of the human genome and technological developments in dna sequencing , gene expression profiling , and proteomics have raised expectations of implementing genotype phenotype data into the clinical decision process and have also multiplied the complex interaction of genetic and other laboratory parameters that can be used for therapy adjustments.7 new data produced for clinical purposes , including genetic testing , differ from old data that lack genetic testing.3 this increases the gap between old and new drugs , which is based on the availability of dna sequencing data . however , with efficient research efforts , it is possible to reach the required level of knowledge with existing chemotherapy drugs . this makes it possible to rely on old drugs and continue using them in the course of therapy decisions , which are increasingly based on new data from gene maps . all old clinical trial materials should be reassessed , and if samples exist , the trials should be rerun to extract gene - related information . new , faster , and cheaper methods , eg , the exome variation analyzer , make broadly focused whole genome sequencing possible.8 such an action would probably rewrite some of the major phase 3 trial results . if this is not done , there may not be enough data to determine the best way to use existing drugs , and so will use them according to the old treatment guidelines that lack gene - related elements . in that case , only the new trials with new targeting agents will have these essential new elements , and this will extremely widen the gap between old and new trials . this drug companies find it difficult enough to get their new molecules through approval processes and will not have a financial incentive to accomplish something with old , non - patentable , low - priced alternative products in their drug portfolios.9 re - running the clinical trials would be highly beneficial for ordinary clinicians , and thus , governments should take the responsibility for doing so . furthermore , it should be the role of the government to define the financing required to analyze biomaterials with the complete patient records already available . international research teams supported by public funding agencies , such as the national institutes of health , and by private foundation , such as the wellcome trust , are rapidly enlarging the catalog of genetic changes associated with neoplasia and other ailments , using ever faster , ever cheaper sequencing methods and heavy - duty bioinformatics.10 the main inhibitory element is not the low volume of samples available , but poor or non - existent clinical data combining clinical cases with findings in tumor samples . this problem is well recognized from chemotherapy trials and this is one of the reasons why open - label phase iii studies are so strongly acknowledged in clinical oncology . retrospective data , especially routinely written patient records , are imperfect sources of background information on gene results . clinical patient records should be redesigned to have a structure that supports automatic data extraction and to make it possible to have real - life reference material for particular therapy options . this article will discuss new elements related to genetic testing focusing on defining changes and evaluating what they would mean for practicing physicians . this review is based on a pubmed search , which was performed with the terms medical decision and gene ( table 1 ) . publications in languages other than english and trials involving non - human subjects were excluded . abstracts of 171 publications were reviewed and the classification was performed with the predetermined variables listed in tables 2 and 3 . a search of the term gene on pubmed produces almost two million hits ( 1,971,971 ) . within clinical oncology , there were 189,328 hits , a number that then decreased dramatically , when the search was conducted for both clinical oncology and gene or for genome terms ( table 1 ) . a search for terms including both gene and clinical oncology offers relatively few hits ( 9,224 ) and a search for medical decision offers even fewer hits ( 1,253 ) . combining clinical oncology , medical decision , and gene in a search term produced 171 hits ( table 2 ) . in fact , 154 articles were about clinical situations , and 17 articles , despite the presence of the search term hits , were preclinical papers . of the original articles with all indications , abstract analysis revealed 25 articles dealing with driver mutations , 25 with multiple gene analysis including 9 on 21-gene assay , and 16 on the oncotype dx test . gene profiling , either dna or rna , was dealt in 17 abstracts.1127 the following cancer topics were the subject of fewer than seven articles : colon ( 3 ) , leukemia ( 5 ) , brain ( 3 ) , prostate ( 2 ) , bladder ( 1 ) , thyroid ( 1 ) , liver ( 2 , her-2 ( 6 ) , kras ( 2 ) , egfr ( 2 ) , radiotherapy ( 2 ) , and pediatric ( 2 ) . in 17 articles , there were 68 review articles , of which 23 mainly focused on driver mutations , while profiles were addressed in 26 articles . the indications most frequently examined in articles were breast ( 24 ) , colorectal ( 10 ) , and lung cancer ( 7 ) , and other cancers were addressed just a few times ; these included ovarian ( 3 ) , prostate ( 3 ) , sarcoma ( 2 ) , gastric ( 1 ) , and testis ( 1 ) . gene profiles were central to 26 review articles , with proteomics in three and large - scale genomics in three , and multiple gene tests were reviewed in 17 articles , 21-gene assays in five , the oncotype dx test in three , and the metagene test in one . early articles largely focused on counseling , but there is a marked trend toward the publication of articles on gene profile work . the fact that we are still at the beginning of a new era of cancer treatment can be seen from the number of trials registered under the term individualized medicine . the clintrials.com web site reports 149 protocols listed under the term individualized medicine and cancer as at november 2014 . the spread of trials is quite uniform over different cancer areas , as can be seen in table 4 . the activity reported focused primarily on big indications : lung , breast , and colorectal cancer . however , there were also quite a lot of activities in the field of renal cancer , which speak of the importance of individualized treatment in small indications , eg , renal cancer . currently , there is no general process common to all pathology departments to prepare samples for molecular testing . similarly , such testing is done only in some institutions and usually in clinical trials , or otherwise on a limited number of patients . normally , these samples are used to provide histological diagnosis with only a few driver mutation tests , eg , her-2 amplification , and mutations of ras , braf and egfr genes . our clinical data should come from clinical trials that produce complete clinical data , whereas ordinary clinical data are not complete and may be missing various pieces of information , resulting in difficult to derive full benefit from that data . before investing in full - scale exon testing for all patients , in this study , literature was searched using pubmed research with a defined set of terms : clinical oncology and medical decision and gene . by doing this , it was possible to follow the development of the area , as reflected in a number of articles ( table 3 ) . the bibliographical approach was utilized to give a solid perspective and overview of an area that is developing so rapidly . of course , one set of search terms can not pick up everything in a broad research field , but it may illuminate the study area . the search term used provided 171 hits , which represent only 0.0000867% of pubmed articles defined under the term gene ( table 1 ) . a review of articles spanning 20 years reveals a move from individual driver mutation articles and counseling papers toward multiple genes and gene profile papers ( table 3 ) . however , in the past five years , only 17 original papers published could be classified as focused on gene profiles.1127 a similar slow development can be seen in the number of clinical trial protocols dealing with individualized medicine and cancer ( table 4 ) . it was concluded in 2012 that as a rule the molecular data on gene - driven therapies and specifically on breast cancer gene profiles were not sufficiently mature to include them in decision - making algorithms determining treatment recommendations for individual patients.18 in many ways , the promise of a new era of gene - driven therapies relies on all tumor genes being taken into account at one time and making a difference to patient therapy.28 however , development is proceeding slowly and is likely to advance stepwise through the application of the classical logic of the natural sciences . this process is typified by focusing on one question at a time , and obtaining a near complete picture depends on first resolving a sufficient number of sub questions . the clinical necessity of considering one patient at a time and collecting all the problems of that patient in a table at once present a considerable challenge for genetic science . it was suggested that the discovery of the magic bullet was close and that once identified it would resolve the clinical question of how to successfully treat metastasized cancer . monoclonal antibodies offer the promise of cancer - specific drug targeting ( paul ehrlich s magic bullet ) , but a modest number of anticancer antibodies have been approved for clinical use only during the past decade.23 it took three decades to get bioscience knowledge to the level where it was possible to announce some magic bullets , the magical antibody era has not yet occurred . instead , antibody technology has provided similar answers to other technology areas , and the promise of a rapid revolution has failed to materialize . at the time of writing , there are still more than 400 such antibody molecules awaiting clinical trials , so the revolution is far from becoming reality.29 gene - driven therapies have been proposed to have a major impact on future drug developments and also on oncologists therapy decisions . this time , the possibility of radical change happening in a short space of time is stronger than it proved to be with antibodies . the simplest change occurs when a certain driver mutation known to be a predictive factor is recognized . luckily , there are now a few drugs that will work on limited patient populations , eg , her-2 and trastuzumab30 and kras wild - type and cetuximab.31 the availability of these drugs will offer new treatment options . traditionally , treatments are defined to suit the largest number of people , usually resulting in 1040% of treated patients receiving some benefit , while the remainder is actually treated for nothing . the fundamental rationale has been to generalize the treatments offered in clinical trials to all patients . the current demand to demonstrate efficacy in the form of large phase iii trials the limitations are understood , as it is not always possible to access the same patient profile in real life as it is in clinical trials . however , the new thinking provides an opportunity to enrich the treatment effect by selecting patients based on genetic alterations . instead of giving a standard consensus treatment to everyone , gene - driven therapy assesses the patient s gene status to select treatments to suit that individual patient . success in cancer treatment should no longer be measured in terms of a percentage of patients responding to a certain treatment , but should extend to include the overall success in terms of the number of healed patients given various treatments guided by gene alterations . of course , this new decision - making culture is just started to form , and to significantly advance , it will require access to data covering clinically usable treatments . the idea would be to use data on certain types of patients with definite genes and gene variations relating to predictable treatment results . doing so would mean that all individually crafted treatments could be based on some , if not many , pieces of data from treatment already carried out . dna analysis of the patient will give information on probable drug interactions and possible metabolic issues before starting a patient on medication . during the past 10 years , cancer medicine has experienced technological advances , now allowing rapid and inexpensive sequencing of the entire human genome.32 the adoption of genome - wide association studies on responses to drugs , genome sequencing from drug development , and treatment programs are the most striking short - term opportunity to improve the drug candidate pipeline and to boost the efficacy of medication already in use.33 on the other hand , patient dna would be needed to establish the differences in tumor dna and could also be used in the evaluation of the therapeutic index , for example , in measuring the pharmacokinetic characteristics of the patient . tumor samples will provide prognostic information on a particular cancer type ; moreover , multiple genes tools are objective and reproducible in terms of the analysis of proliferation , but these approaches may still overlook the biological heterogeneity with tumors evidenced by distinct cell subpopulations with different genomic patterns and functions.34 in addition , there could also be a predictive side discovering how the efficacy of a therapy is related to gene alterations over the course of the follow up . this body of information is expanding all the time.35 these new diagnostic tools are adding prognostic and predictive elements to the ordinary diagnosis ; in particular , they address the relationship between clinical judgment and clinical decision - making , as the biological and clinical components are realigned.36 predictive connections need solid and accurate clinical data , which are not normally available from patient records . the use of patient records retrospectively will be constrained by considerable volumes of missing data and could markedly reduce the value of large sample banks . it is becoming increasingly apparent that it may not be the volume of samples but that the completeness of patient clinical data will define the actual value of a sample bank . the u.s . federal drug administration has approved a 2,000 gene test for working diagnoses and management of metastatic and poorly differentiated cancers , which encompasses over 4% of cancers of unknown origin . following the test results , the recommendation for guideline - consistent chemotherapy increased from 42 to 65% of patients , and the recommendation for non - guideline- consistent regimens declined from 28 to 13%.37 for patients with difficult - to - diagnose cancers , the test changed the working diagnosis and given therapy for the majority of patients.37 it can be stated that there is a conceptual limitation to the current genome decision - making , as it is based on whether a certain individual factor is found or not . therefore , single exon changes termed driving mutations are currently in focus , while a complete gene cluster analysis or complete gene profiling for everyone could only be performed experimentally and theoretically.38 clinical data combining gene changes and clinical patient data are still lacking , and it is not possible to take into account more than a small fraction of gene alterations . however , future data will be based primarily on the extraction of problems and focusing on a single gene mutation at a time . this will restrict results , and it is likely that a considerable amount will be missed . validated molecular tests assessing tumor tissue or patient germ line dna already inform decision - making ; however , many theoretical and regulatory challenges must be overcome before the promise of personalized molecular medicine can be fully realized.39 an analysis capable of including all genes is still some way in the future , and a complete genome analysis , which could also take into account introns and dna remaining outside known gene structures , is even further away . it is calculated that only around 5% of dna could be defined as genes and the remainder is noncoding or junk dna.40 it may well be that only 20,00030,000 genes affect the functioning of the dna , but the whole dna molecule may function together to produce the end effect.41 this means that introns and junk dna may have a distinct but as yet unknown role in dna . it is not unreasonable to think that dna , despite being constructed from simple pieces ( including four nucleosides ) , will possess multilayer complexity with interactions and network effects . many genes can be involved when dna works , but they exert their effect through sum effects in which all genes operate as a network , and only when all genes have had their input is the direction in which the dna function moves determined . the gene network is complex and creates a sum effect , so that all genes have an impact on the end function but in a variable way . there are in fact several examples of somatic mutations in tumors driving therapies by predicting responses . this has sparked the active development of targeted therapies with predictive factors . there is also a realization that germ line dna variants can help optimize cancer drug dosing and predict the susceptibility of patients to the adverse side effects of these drugs knowledge that can ultimately be used to improve the benefit / risk ratio of cancer treatment for individual patients.42 the number of pharmacogenetic assays available is continuously expanding as more molecularly targeted anticancer drugs begin their clinical development . analysis of germ line dna mutations can often help to predict pharmacokinetics and pharmacodynamic responses , whereas somatic dna mutations are particularly useful in predicting tumor response . it is considered essential for clinicians to understand the molecular pathways for anticancer drugs , the therapeutic implications of mutations within these pathways , and the clinical assays available to test for pharmacogenetic differences.6 the use of individualized medicine may increase differences among institutions , as there would be no single correct treatment that could be used universally . in principle , treatment guidelines will only define approaches to how gene alterations could be interpreted to understand their usefulness in a patient s therapy . massive databanks will have records of gene - based therapy decisions , and physicians will track their treatments and success against a certain patient s cancer . however , the unifying feature will be the number of healed patients with a number of treatments . finally , patient populations will be acknowledged to be different , and instead of the patients , the treatment focus will be on the genes used that drove the therapy . the canadian regulatory body , the medical advisory secretariat ( mas ) , has begun work on evidence - based reviews of published literature and published a thorough review of gene expression profiling to guide adjuvant chemotherapy decisions in women with early breast cancer . it was an evidence - based economic analysis , following which the mas concluded that there are methodological and statistical limitations that affect both the generalizability of the currently available evidence , as well as the magnitude and statistical strength of the observed effect sizes.43 they found low - quality evidence of the prognostic value of an oncotype - dx test and only very low - quality evidence of its predictive value . the test was not recommended for widespread use , but the mas did request more evidence be sought . an ordinary patient visit process to an oncologist in the future might involve the patient presenting a gene card , which would be scanned and all specific information transferred to a server to make it available to the oncologist . the doctor would then check for recorded interactions between the patient s genes and the available drugs , and also screen the risk levels revealed by the family s medical history and predisposing genes found in the dna of family members . the tumor and metastases would already have been analyzed in gene analysis centers , producing genome exon results ready to be inserted into programs linking existing data to the therapy options . following a clinical examination and diagnosis , the doctor would decide on the medicine to be administered . the integrated gene program would propose cytostats and biostats ( targeted biological agents with moderate treatment effect ) according to published data in descending order of calculated efficacy , while simultaneously screening for side effects against the patient s medical history and family gene records . only once that process is completed would a final proposal for treatment be made by the computer program . despite the upcoming changes in decision - making related to gene - driven therapies , the doctor patient relationship will undoubtedly remain and must be supported by an overarching sense of trust . doctors can not hide behind perfectly functioning test programs : patients want to know that their own doctor is responsible for the decisions involving the treatment of their cancer . that element of there being one doctor interpreting the patient s situation and treating them is what will remain . for an oncologist , this genomic workout will provide the most efficient tool to support the understanding of the development of a particular cancer and its peculiar characteristics and to prioritize therapies accordingly .

a new era of medicine is rapidly approaching , which will change not only pathological diagnosis but also medical decision - making . this paper raises the question of how well prepared doctors are to address the new issues that will soon confront them . the human genome has been completely sequenced and general understanding about cancer biology has increased enormously with understanding that unregulated gene function and complicated changes in signal pathways are related to uncontrolled cell growth . thus , gene - driven therapy involving alterations to genes are recognized to present new therapy options . this advance will necessitate major changes to the decision - making aspect of physicians . this article focuses on defining the pertinent changes and addressing what they mean for practicing physicians .

Introduction Methods Results Discussion

PMC4586955

large randomized controlled trials ( rcts ) are frequently considered the reference standard for producing high levels of scientific evidence to guide clinical practice ( 1 , 2 ) . in an attempt to overcome inconsistencies throughout the world , the guideline for good clinical practice ( gcp ) was approved on 17 july 1996 and implemented for clinical trials from 17 january 1997 ( 3 , 4 ) . the gcp was followed by a large number of decrees and circulars , increasing the regulatory complexity for the authorization and conduct of clinical trials . the increase in regulatory issues went hand in hand with the refinement of methodologies and research techniques . the inclusion of fewer patients than is required usually results in extended trial periods and raised costs . hence , one of the most obvious challenges in a trial is to optimize the recruitment of the required number of patients in a timely and efficient manner ( 5 ) . excellent leadership and negotiation and communication skills optimize managing project deliverables , patient recruitment practices , and compliance with regulatory requirements . on the other hand , a poorly managed clinical trial can lead to product approval delays and increased costs . moreover , it must not be forgotten that the development of a new drug from synthesis to marketing takes on average 12 years and costs more than 250 million usd ( 6 ) , and of more than 10000 molecules synthesized only 1 or 2 will hit the market ( 7 ) . if it is true that a well - structured protocol can answer clinical questions , it is equally true that this is not enough to ensure the success of a study at risk of failure due to inept management ( 8) . in the last decades , although a systematic approach to project management has been applied in engineering and construction industries , there is a dearth of data on project management in the context of clinical research . the purpose of this review is to provide an overview of the published literature on how the knowledge of project management can increase the chance of the success of clinical trial projects . we searched published studies in the english language from 2002 to 2012 in the cochrane database of systematic reviews , medline , google scholar , and embase . we reviewed the titles of articles for relevant content , discarded unrelated articles , subjected the remaining articles to abstract review , and subsequently reviewed the selected relevant papers in detail . finally , we hand searched the reference lists of key studies and reviews and citations thereof to identify any additional articles not captured by the main search strategy . a clinical trial has many characteristics in common with other types of business projects as is defined in the project management domain ( 8) . these characteristics include but not limited to the following : - there are pre - defined objectives to be achieved . - identified resources are necessary to achieve these objectives . - activities should be planned to achieve the objectives . - the work should be monitored constantly . - the criteria for evaluating the results should be defined . - the results should be evaluated . - the project is closed when the objectives have been achieved or when the objectives will not or can not be met , or when the need for the project no longer exists . clinical trial management is most simply defined as the process that an organization follows to ensure that quality ( defined as minimized risks and clean data ) is delivered efficiently and punctually ( 9 ) . it refers to a standards - driven process that a project manager initiates and follows in order to successfully manage clinical trial sites , clinical research associates , and workflow by using clinical trial management tools or software ( 9 ) . prolonged timelines and heavy costs related to large trials have prompted a new focus on more efficient clinical trial management ( 10 ) . it is possible to dramatically reduce the total cost of a clinical trial by 60% - 90% without compromising the scientific validity of the results ( 11 ) . given the modest effect of the most beneficial treatments and the dangers inherent in failing to identify risks before the marketing of cardiovascular drugs , in particular , the importance of clinical trial expenses is increasing in cardiovascular medicine , where the emphasis is on large trials with adequate sample sizes in order to detect the clinically relevant differences ( 12 ) . the reduction in the sample size would decrease expenses , but it is not likely to provide adequate power to determine the risks and benefits of a new drug . similar to many projects in general that are not completed on time , on budget ( 13 - 15 ) , and to the expected quality ( 16 ) , many clinical trials fail to deliver due to the absence of a structured , practical , businesslike approach to trial management . there is no clearly defined code for the management practice of a clinical trial ( 16 ) . although many successful rcts have been previously produced and have changed clinical practice , the knowledge and expertise achieved on how to deliver such trials have not been broadly published ( 16 ) . lengthy approaches for reviewing the literature , developing a protocol , attracting the fund , and designing data collection forms usually exist ; however , rarely does the same approach exist for the management of clinical trial projects ( 17 ) . each trial requires the development of a work plan ( or scope - of - work document ) that can be used to clearly define which department is responsible for which task when the study is being conducted by multiple departments out of a single institution . likewise , the same document can be drawn upon to define responsibilities if multiple institutions ( e.g. , a coordinating center and a statistical center ) are involved . the more the detail included in this document in advance , the more likely it is that the important tasks will not be missed and that each group is not assuming that someone else is responsible for the task . a scope - of - work document can also be utilized to help with budgeting for the different members of the team . another critical document to create as part of the initial planning requirements is a timeline with realistic milestones . there are many tools that can be exploited to create a timeline , including microsoft project software . appropriate gantt charts and resource planning are part of microsoft project , but unfortunately not all trial managers either have this readily available resource or know how to input the needed data to make it a useful tool . the timeline , in conjunction with the scope - of - work document , will collectively provide the roadmap for the overall project . a more accurate control , regardless of the therapeutic area or trial stages ( all the way through from preclinical phase 1 to post - approval phase 4 studies ) , is ensured by typically breaking down the life cycle of each clinical trial project into 4 phases : conceptual , planning , implementation , and analysis ( figure 1 ) . a project is based on input , which is normally supplied by the sponsor of the project and generates the output ( 2 ) . an input is the objective to be achieved in a specific predetermined time ( enrollment of x evaluable subjects in x months , the sponsor inclusion in the database of data collected in x days , etc . ) and is generally described in the contract . a clinical study produces an output that will ultimately help provide answers to the study objectives ; these results are described in a clinical study report that states formally the end of the study . as we move through the implementation phase , the amount of work and number of players involved increase significantly and this is where project management becomes more critical . monitoring is an essential element for project management and is constantly done throughout the duration of the project . it allows managers to collect measures and disseminate performance information in order to identify areas that need special attention and to implement any action improvement ( 18 ) . a phase of a project usually ends with a review of the tasks and monitoring of the objectives ( 1 ) . icf , informed consent form ; mop , manual of procedures ; crf , case report form ; irb , institutional review board ; sae , serious adverse event ; and ctr , clinical technical report . a protocol is a document that describes the purpose , design , methodology , statistical considerations , and organization of a study and provides basic information and rationale for the clinical study . the protocol writing is a task for one person , usually the principal investigator , not a committee ( 19 ) . the principal investigator should also be supportive and committed , and be available to take the lead on clinical or scientific issues . however , it is not necessary for this person to be involved in the day - to - day running of the trial . it is the responsibility of a trial manager to check how the protocol will be put into practice on a day - to - day basis ( 19 ) . although a well - structured protocol can answer scientific methodological questions during the study , projects may fail even with well - written protocols if it is not meticulously considered how the protocol will be put into practice on a day - to - day basis . the principal investigator and the trial manager , therefore , have overall responsibility for delivering the trial . the project manager is the person responsible for managing the project in order to achieve the objectives in respect of cost and time as well as the technical quality of the result . while trial managers are not required to complete all the tasks , they are responsible for ensuring that all aspects of the project are planned , implemented , monitored , and controlled on a daily basis , and project outputs and outcomes are obtained ( 20 ) . unfortunately , the responsibilities of this role are complex and the body of knowledge available to guide trial managers is still very limited . as farrell et al . ( 8) suggested : it is time to produce standard trial management guidelines and develop robust methods of evaluation . ( 21 ) conducted in australia , at the completion of the project , the researchers were asked via a questionnaire to express their opinion about the application of project management for medical research projects . the responders agreed that project management contributes substantially to the research and emphasized that trial manager characteristics are of essential importance within the team . a perfect project plan will not ensure the success of a project without its implantation by a strong clinical trial coordinator or manager . the requirements of a clinical project manager are similar to those of any other project manager ( communication and presentation skills , ability to organize and motivate others , enthusiasm , drive innovation and leadership , ability to manage the budget of the study and monitor costs , strategic and operational skills to plan and conduct a study , capacity to mediate between the various figures ) , but also add to these scientific competence and motivation ( 8) . the manager can detect and correct errors that are sometimes severe ( e.g. , protocol deviations , deviations in consent ) , emphasizing the importance of monitoring throughout the conduct of the study ( 22 ) . regular and constant complete communication between the trial manager with the principal investigator and other stakeholders is essential to make sure all aspects remain on track as per the original plan . the figures of a good clinical trial coordinator may be summarized as 30% research knowledge and technical skills and 70% communication skills ( 9 ) . clinical trials all need the same coordinated processes and systems , irrespective of the size , scope , costs , or period . the key challenge is then to implement and maintain effective management systems and techniques in response to the needs of the trial project ( 23 ) . drug and biologics development projects have to contend with several factors , including inability to control patient registration , unpredictability of clinical outcomes ( e.g. , adverse events ) , and reliance on regulatory agencies ( e.g. , the fda in the united states ) , for the approval of a new drug , which influences schedule and budget as the key performance indicators of project management . as a result , even with the application of project management , the best we can do is to improve our predictions for the launch dates of new products into market . another apparent challenge in clinical trials is to register the patient sample size as timely and efficiently as possible ( 5 ) . each clinical trial is unique because one tiny difference in the protocol needs between two trials may lead to drastic changes in yielding recruitment lifecycle and register timing parameters . many factors such as inclusion / exclusion criteria , visit schedule , recruitment tactics , placebo allotment , development phase , and patient motivation can potentially affect the final number of patients who enroll in the trial . a lower number of available patients than is expected usually results in an extended length of time to recruit a certain number of patients and increased costs . multicenter trials provide high - yielding recruitment strategies that can be advantageous if a large number of patients is required and also offer the advantage of higher generalizability of the findings ( 24 , 25 ) . however , performing international multicenter trials requires a complex organization , a dedicated and experienced methods center , and motivated participating investigators across the different sites ( 8 , 26 ) . in the strategies for trial enrollment and participation study ( steps ) ( 27 ) , an analysis of 114 multicenter trials demonstrated that 45% failed to reach 80% of the pre - specified sample size . less than one - third enrolled their original target number of participants within the time originally specified , and around one - third had extensions in time and resources . among the factors observed more often in trials that recruited successfully was having a dedicated trial manager ( odds ratio : 3.80 , 95% ci : 0.79 to 36.14 ; p = 0.087 ) ( 27 ) . identifying appropriate clinical sites and setting up realistic enrollment expectations are also challenges in multicenter studies . because site principal investigators often lack the time to spend on administrative tasks , it is crucial to have a trial coordinator who could adopt much of the time . when making a decision to participate , sites find it more important to have sufficient support than to be offered a financial compensation for participation ( 28 , 29 ) . in addition , the presence of a trial manager will facilitate an appropriate study infrastructure for the daily management of the study ( 30 ) . finally , achieving ethics approval is often a lengthy process because procedures , documents , and legislation may vary among participating sites in different countries ( 31 ) . it is , therefore , recommended to have dedicated and well - trained trial staff to accomplish these administrative tasks ( 32 - 34 ) . the steps collaborators ( 27 ) proposed that the dimensions of conducting a successful trial comprise marketing , sales , and ongoing client management and identified that in the recruitment stage of a trial , the most demanding activity is to establish and implement a range of effective management techniques parallel to those used in a successful business . they also suggested that future trials consider the different needs at different phases in the life of trials and focus on the process of actually performing trials ( 27 ) . a complete patient follow - up and high - quality data collection in a large multicenter or multinational trial require the establishment of a relatively complex organization ( 35 ) . individual sites generally arrange their local ethics procedures and trial logistics and receive per - patient payments as compensation . alternatively , a single trial manager may handle all trial - related tasks for multiple sites in a certain geographic area . the fixation using alternative implants for the treatment of hip fractures ( faith ) trial ( 36 ) aimed to assess how these two different strategies affect the performance of the trial . in this study , trial progression in the netherlands , where a central trial manager performed most tasks for multiple centers in a defined geographical area , was better than that in canada and the united states , where local research assistants were appointed at individual sites . although the collection of follow - up data was equally adequate in both systems , the central trial management system was related to a shorter trial start - up time , higher inclusion rate , and higher inclusion percentage ( 36 ) . furthermore , the availability of the central trial manager in the netherlands made it possible for smaller community hospitals to participate in the faith multicenter trial . these sites generally lack infrastructure for proper study conduct or the local site s coordinator is normally unable or reluctant to participate in such trials . be that as it may , these centers treat more patients from the targeted population ( e.g. , femoral neck fracture patients in the faith trial ) than large university hospitals . thus , the availability of a central trial manager can positively influence the recruitment rate . clinical trial management is most simply defined as the process that an organization follows to ensure that quality ( defined as minimized risks and clean data ) is delivered efficiently and punctually ( 9 ) . it refers to a standards - driven process that a project manager initiates and follows in order to successfully manage clinical trial sites , clinical research associates , and workflow by using clinical trial management tools or software ( 9 ) . prolonged timelines and heavy costs related to large trials have prompted a new focus on more efficient clinical trial management ( 10 ) . it is possible to dramatically reduce the total cost of a clinical trial by 60% - 90% without compromising the scientific validity of the results ( 11 ) . given the modest effect of the most beneficial treatments and the dangers inherent in failing to identify risks before the marketing of cardiovascular drugs , in particular , the importance of clinical trial expenses is increasing in cardiovascular medicine , where the emphasis is on large trials with adequate sample sizes in order to detect the clinically relevant differences ( 12 ) . the reduction in the sample size would decrease expenses , but it is not likely to provide adequate power to determine the risks and benefits of a new drug . similar to many projects in general that are not completed on time , on budget ( 13 - 15 ) , and to the expected quality ( 16 ) , many clinical trials fail to deliver due to the absence of a structured , practical , businesslike approach to trial management . there is no clearly defined code for the management practice of a clinical trial ( 16 ) . although many successful rcts have been previously produced and have changed clinical practice , the knowledge and expertise achieved on how to deliver such trials have not been broadly published ( 16 ) . lengthy approaches for reviewing the literature , developing a protocol , attracting the fund , and designing data collection forms usually exist ; however , rarely does the same approach exist for the management of clinical trial projects ( 17 ) . each trial requires the development of a work plan ( or scope - of - work document ) that can be used to clearly define which department is responsible for which task when the study is being conducted by multiple departments out of a single institution . likewise , the same document can be drawn upon to define responsibilities if multiple institutions ( e.g. , a coordinating center and a statistical center ) are involved . the more the detail included in this document in advance , the more likely it is that the important tasks will not be missed and that each group is not assuming that someone else is responsible for the task . a scope - of - work document can also be utilized to help with budgeting for the different members of the team . another critical document to create as part of the initial planning requirements is a timeline with realistic milestones . there are many tools that can be exploited to create a timeline , including microsoft project software . appropriate gantt charts and resource planning are part of microsoft project , but unfortunately not all trial managers either have this readily available resource or know how to input the needed data to make it a useful tool . the timeline , in conjunction with the scope - of - work document , will collectively provide the roadmap for the overall project . a more accurate control , regardless of the therapeutic area or trial stages ( all the way through from preclinical phase 1 to post - approval phase 4 studies ) , is ensured by typically breaking down the life cycle of each clinical trial project into 4 phases : conceptual , planning , implementation , and analysis ( figure 1 ) . a project is based on input , which is normally supplied by the sponsor of the project and generates the output ( 2 ) . an input is the objective to be achieved in a specific predetermined time ( enrollment of x evaluable subjects in x months , the sponsor inclusion in the database of data collected in x days , etc . ) and is generally described in the contract . a clinical study produces an output that will ultimately help provide answers to the study objectives ; these results are described in a clinical study report that states formally the end of the study . as we move through the implementation phase , the amount of work and number of players involved increase significantly and this is where project management becomes more critical . monitoring is an essential element for project management and is constantly done throughout the duration of the project . it allows managers to collect measures and disseminate performance information in order to identify areas that need special attention and to implement any action improvement ( 18 ) . a phase of a project usually ends with a review of the tasks and monitoring of the objectives ( 1 ) . icf , informed consent form ; mop , manual of procedures ; crf , case report form ; irb , institutional review board ; sae , serious adverse event ; and ctr , clinical technical report . a protocol is a document that describes the purpose , design , methodology , statistical considerations , and organization of a study and provides basic information and rationale for the clinical study . the protocol writing is a task for one person , usually the principal investigator , not a committee ( 19 ) . the principal investigator should also be supportive and committed , and be available to take the lead on clinical or scientific issues . however , it is not necessary for this person to be involved in the day - to - day running of the trial . it is the responsibility of a trial manager to check how the protocol will be put into practice on a day - to - day basis ( 19 ) . although a well - structured protocol can answer scientific methodological questions during the study , projects may fail even with well - written protocols if it is not meticulously considered how the protocol will be put into practice on a day - to - day basis . the principal investigator and the trial manager , therefore , have overall responsibility for delivering the trial . the project manager is the person responsible for managing the project in order to achieve the objectives in respect of cost and time as well as the technical quality of the result . while trial managers are not required to complete all the tasks , they are responsible for ensuring that all aspects of the project are planned , implemented , monitored , and controlled on a daily basis , and project outputs and outcomes are obtained ( 20 ) . unfortunately , the responsibilities of this role are complex and the body of knowledge available to guide trial managers is still very limited . as farrell et al . ( 8) suggested : it is time to produce standard trial management guidelines and develop robust methods of evaluation . in a research project , alcohol and pregnancy project , ( 21 ) conducted in australia , at the completion of the project , the researchers were asked via a questionnaire to express their opinion about the application of project management for medical research projects . the responders agreed that project management contributes substantially to the research and emphasized that trial manager characteristics are of essential importance within the team . a perfect project plan will not ensure the success of a project without its implantation by a strong clinical trial coordinator or manager . the requirements of a clinical project manager are similar to those of any other project manager ( communication and presentation skills , ability to organize and motivate others , enthusiasm , drive innovation and leadership , ability to manage the budget of the study and monitor costs , strategic and operational skills to plan and conduct a study , capacity to mediate between the various figures ) , but also add to these scientific competence and motivation ( 8) . the manager can detect and correct errors that are sometimes severe ( e.g. , protocol deviations , deviations in consent ) , emphasizing the importance of monitoring throughout the conduct of the study ( 22 ) . regular and constant complete communication between the trial manager with the principal investigator and other stakeholders is essential to make sure all aspects remain on track as per the original plan . the figures of a good clinical trial coordinator may be summarized as 30% research knowledge and technical skills and 70% communication skills ( 9 ) . clinical trials all need the same coordinated processes and systems , irrespective of the size , scope , costs , or period . the key challenge is then to implement and maintain effective management systems and techniques in response to the needs of the trial project ( 23 ) . drug and biologics development projects have to contend with several factors , including inability to control patient registration , unpredictability of clinical outcomes ( e.g. , adverse events ) , and reliance on regulatory agencies ( e.g. , the fda in the united states ) , for the approval of a new drug , which influences schedule and budget as the key performance indicators of project management . as a result , even with the application of project management , the best we can do is to improve our predictions for the launch dates of new products into market . another apparent challenge in clinical trials is to register the patient sample size as timely and efficiently as possible ( 5 ) . each clinical trial is unique because one tiny difference in the protocol needs between two trials may lead to drastic changes in yielding recruitment lifecycle and register timing parameters . many factors such as inclusion / exclusion criteria , visit schedule , recruitment tactics , placebo allotment , development phase , and patient motivation can potentially affect the final number of patients who enroll in the trial . a lower number of available patients than is expected usually results in an extended length of time to recruit a certain number of patients and increased costs . multicenter trials provide high - yielding recruitment strategies that can be advantageous if a large number of patients is required and also offer the advantage of higher generalizability of the findings ( 24 , 25 ) . however , performing international multicenter trials requires a complex organization , a dedicated and experienced methods center , and motivated participating investigators across the different sites ( 8 , 26 ) . in the strategies for trial enrollment and participation study ( steps ) ( 27 ) , an analysis of 114 multicenter trials demonstrated that 45% failed to reach 80% of the pre - specified sample size . less than one - third enrolled their original target number of participants within the time originally specified , and around one - third had extensions in time and resources . among the factors observed more often in trials that recruited successfully was having a dedicated trial manager ( odds ratio : 3.80 , 95% ci : 0.79 to 36.14 ; p = 0.087 ) ( 27 ) . identifying appropriate clinical sites and setting up realistic enrollment expectations are also challenges in multicenter studies . because site principal investigators often lack the time to spend on administrative tasks , it is crucial to have a trial coordinator who could adopt much of the time . when making a decision to participate , sites find it more important to have sufficient support than to be offered a financial compensation for participation ( 28 , 29 ) . in addition , the presence of a trial manager will facilitate an appropriate study infrastructure for the daily management of the study ( 30 ) . finally , achieving ethics approval is often a lengthy process because procedures , documents , and legislation may vary among participating sites in different countries ( 31 ) . it is , therefore , recommended to have dedicated and well - trained trial staff to accomplish these administrative tasks ( 32 - 34 ) . the steps collaborators ( 27 ) proposed that the dimensions of conducting a successful trial comprise marketing , sales , and ongoing client management and identified that in the recruitment stage of a trial , the most demanding activity is to establish and implement a range of effective management techniques parallel to those used in a successful business . they also suggested that future trials consider the different needs at different phases in the life of trials and focus on the process of actually performing trials ( 27 ) . a complete patient follow - up and high - quality data collection in a large multicenter or multinational trial require the establishment of a relatively complex organization ( 35 ) . individual sites generally arrange their local ethics procedures and trial logistics and receive per - patient payments as compensation . alternatively , a single trial manager may handle all trial - related tasks for multiple sites in a certain geographic area . the fixation using alternative implants for the treatment of hip fractures ( faith ) trial ( 36 ) aimed to assess how these two different strategies affect the performance of the trial . in this study , trial progression in the netherlands , where a central trial manager performed most tasks for multiple centers in a defined geographical area , was better than that in canada and the united states , where local research assistants were appointed at individual sites . although the collection of follow - up data was equally adequate in both systems , the central trial management system was related to a shorter trial start - up time , higher inclusion rate , and higher inclusion percentage ( 36 ) . furthermore , the availability of the central trial manager in the netherlands made it possible for smaller community hospitals to participate in the faith multicenter trial . these sites generally lack infrastructure for proper study conduct or the local site s coordinator is normally unable or reluctant to participate in such trials . be that as it may , these centers treat more patients from the targeted population ( e.g. , femoral neck fracture patients in the faith trial ) than large university hospitals . thus , the availability of a central trial manager can positively influence the recruitment rate . the numerous and complex regulatory issues introduced into the management of clinical trials according to the gcp as well as medical science advances in methodologies and procedures have rendered the field of clinical research extremely complex . this complexity , however , has conferred a tremendous benefit for both parties involved in the studies : for patients by providing a guarantee for their safety and respecting their rights and for investigators by providing guidelines that , if carefully followed , guarantee valid results . in addition , investigators , who are employed to implement the gcp in the conduct of clinical studies , have also begun to apply them to clinical practice with clear benefits for patients . this has significantly increased the workload of investigators and the need for having a project management with scientific background , knowledge , and regulatory skills in conducting clinical research . the clinical project manager is a professional who applies the definition of project management to the field of clinical research to ensure that all stages of a clinical trial are properly managed , that the objectives of the trial are achieved on time , on budget , and according to the gcp , and that the safety of the subjects participating in the clinical trial and the quality of collected data are guaranteed . one of the most obvious challenges in trials is to register an efficient sample size of patients in a timely fashion . another challenge in a multicenter study is to identify appropriate clinical sites and set up realistic recruitment expectations . finally , the key challenge is to implement and maintain effective management systems and techniques in response to the needs of the trial project . project management in health and medical research may substantially benefit both the managerial and scientific aspects of medical projects . however , there is still no randomized controlled trial to compare trial projects progressed with and without the use of project management . we recommend such a comparison to provide a high level of evidence in this regard .

context : a well - structured protocol for a clinical trial may be able to answer clinical questions , but it can not be deemed enough to ensure success in the face of incompetent management of time as well as human and economic resources . to address this problem , in this article , we present our literature review on evidence as to how a good knowledge of proper management among researchers can enhance the likelihood of the success of clinical trial projects.evidence acquisition : using multiple search strategies , we conducted a literature review on published studies in the english language from 2002 to 2012 by searching the cochrane database of systematic reviews , medline , google scholar , and embase.results:our review suggests that a successful trial requires a work plan or work scope as well as a timeline . the trial manager should subsequently manage the study in accordance with the plan and the timeline . many research units have called for a clinical project manager with scientific background and regulatory skills to effect coordination among various aspects of a clinical trial.conclusions:project management may benefit both the managerial and scientific aspects of medical projects and reduce fund waste . however , little has been written to date on project management in the context of clinical research . the suggestions represent the views of the individual authors . to provide a high level of evidence in this regard , we recommend that a randomized controlled trial be performed to compare trial projects progressed with and without the use of project management .

1. Context 2. Evidence Acquisition 3. Results 3.1. Clinical Trials Project Management 3.2. The Clinical Trial Project Plans 3.3. The Life Cycle of a Clinical Trial 3.4. The Clinical Trial Protocols 3.5. Roles and Key Characteristics of a Trial Manager 3.6. Challenges of Project Management in Clinical Trials 3.7. Central Management Versus Local Management 4. Conclusions

PMC2129154

during bouts of endurance exercise lasting longer than 90 minutes , fatigue generally coincides with low muscle glycogen content , suggesting that simply ingesting carbohydrates during exercise and having glucose available in the blood is not enough to sustain exercise for an extended period of time . this notion led researchers to believe that it may be necessary to load one 's body with glucose prior to the long exercise bout . carbohydrate loading ( > 6 g / kg / d ) prior to participation in an endurance exercise competition has been shown to help delay the onset of fatigue by approximately 20% during endurance events lasting longer than 90 minutes [ 2 - 4 ] . one exception to this rule is a study conducted by burke et al . , where seven trained cyclists ingesting 69 g / kg / d of carbohydrate showed no improvement in performance in a 100 kilometer timed trial , despite significantly increased muscle glycogen concentrations . nutritional recommendations for endurance athletes directly prior to competition have traditionally included an intense glycogen depleting training period of approximately two days followed by a glycogen loading period for 34 days , ingesting approximately 6070% of total energy intake as carbohydrates . these recommendations were constructed based on several studies conducted for performance enhancement for this type of athlete [ 7 - 9 ] . however , most of these studies were conducted using only male subjects and the nutritional recommendations have since been used for populations including both males and females . this idea of normality across genders has recently proved to be incorrect as research has shown that there are many metabolic differences between genders that are stemmed from inherent hormonal differences . specifically , the role of estradiol appears to be the mediator of these metabolic differences and could therefore affect the ability of a female to store , breakdown , and utilize carbohydrates in the same manner as a male [ 10 - 13 ] . the scope of this literature review will include a brief description of the role of estradiol in relation to metabolism and gender differences seen in carbohydrate metabolism and loading . is secreted cyclically by ovaries , peaking at the time of ovulation ( see figure 1 ) . in addition to assisting with development , estradiol has also been linked with various enzymes that play a role in energy metabolism . decreased circulating levels of adipocyte lipoprotein lipase ( lpl ) has been correlated with high estradiol levels , which may result in enhanced triglyceride use in skeletal muscle at varying time points during the menstrual cycle [ 16 - 19 ] . some studies have also shown lipid utilization to be higher during the luteal phase of the menstrual cycle . in addition , there is no evidence that indicates variations in muscle glycogen concentrations throughout the menstrual cycle . note the increase in estradiol during the follicular phase just before ovulation , and more even levels during the luteal phase after ovulation . prior to examining gender related differences in carbohydrate loading , gender differences between carbohydrate metabolism must be examined . there appears to be no difference between genders in basal levels of muscle glycogen [ 13,22 - 24 ] , skeletal muscle glut-4 , or hexokinase . however , females do appear to have an enhanced sensitivity to insulin in skeletal muscle , which would theoretically result in increased muscle glycogen storage , as well as enhanced fat storage , but the gender differences in insulin sensitivity are beyond the scope of this paper . in addition , there have yet to be any studies conducted assessing differences in glycogen synthase activity or branching enzyme . knowledge of changes of these enzymes could assist in the understanding of inherent metabolic differences that exist between genders . while there appears to be no inherent difference at basal levels of muscle glycogen , there does appear to be a gender related difference in the actual breakdown and metabolism of carbohydrates ( table 1 provides a summary of specific studies ) . there is evidence that females tend to oxidize less total carbohydrate than men ; however , the mechanism behind this phenomenon remains unclear . one possible explanation could be that women have a lower rate of glucose appearance than that of men during endurance exercise [ 27 - 29 ] . males and females were matched according to peak oxygen consumption ( vo2peak ) per kg lean body mass , training history , and physical activity level . all females were tested during mid - follicular phase of the menstrual cycle to avoid possible elevated lipid utilization that has been shown during the luteal phase . results of the study showed no gender differences for utilization of fatty acids , blood glucose , and glycogen , and that females oxidized more myocellular triglycerides than males , making up 25% of total vo2peak for females and only 5% of total vo2peak for males . this indicates that while there were no gender differences in the relative contribution of carbohydrates and lipids , there does seem to be a difference in the utilization of various lipid sources . this coincides with observations seen at rest , indicating greater utilization of fatty acids in the skeletal muscle rather than those derived from the adipose tissue [ 16 - 19 ] . . conducted two similar studies evaluating glycogen depletion in the vastus lateralis during endurance exercise . the 1990 study showed that women had significantly less glycogen depletion than men during treadmill running , but the 1995 study showed no gender difference in glycogen depletion during submaximal cycling . despite no difference in glycogen depletion , the study did show that women oxidized significantly more lipid and less carbohydrate and protein compared to men during an exercise bout at 75% vo2peak . these data concur with previous observations of greater lipid oxidation of females during submaximal endurance exercise , but the source of fatty acids differ . it appears as though females tend to utilize more fatty acids from adipose tissue during submaximal exercise , whereas the main source of increased fatty acid utilization at rest is from skeletal muscle [ 16 - 19 ] . romijin and colleagues also addressed intensity in relation to gender differences in substrate utilization in rats . in both studies , the participants exercised at intensities of 25 , 65 , and 85% of vo2max . the 1993 study showed that in males muscle triglyceride lipolysis was stimulated only at higher intensities and that at 65% vo2max muscle glycogen and triglyceride oxidation decreased . the 2000 study showed that in females carbohydrate oxidation increased progressively with exercise intensity , and that the highest rate of fat oxidation was during exercise at 65% of vo2max . when comparing the two studies , the authors concluded that after correction for differences in lean body mass , there were no differences between these results and previously reported data in endurance - trained men studied under the same conditions , except for slight differences in glucose metabolism during low - intensity exercise . it is important to note that not all of these studies controlled for variations of lipid metabolism during the menstrual cycle , thus the observed differences between rest and exercise may simply be due to measurement of fatty acid utilization during different phases of the menstrual cycle . tarnopolsky also assessed glycogen use in the vastus lateralis via muscle biopsy over the course of a 31-day endurance cycling training protocol and found no gender difference in glycogen sparing . a possible explanation of this contradiction with previous literature could be from different muscle recruitment between running and cycling . however , two other studies found that men use more glycogen than women during cycle exercise , but these two studies assessed glycogen use via a stable isotope method rather than muscle biopsy . conducted a study to assess gender differences in fuel metabolism during long - duration exercise . fuel oxidation was measured using indirect calorimetry and blood samples were drawn for circulating substrate and hormone levels . results indicated that females expended more total energy from fat oxidation ( 50.9% ) than that of men ( 43.7% ) , but less total energy from carbohydrates ( 45.7% for women and 53.1% for men ) . in addition to differences in fuel metabolism , males also had higher circulating levels of catecholamines . these results suggest that females may be more sensitive to the lipolytic actions of catecholamines than men . with the knowledge that females tend to oxidize a greater amount of fatty acids than males , researchers then assessed the effects of estradiol administration to males . results indicated that with the addition of 17--estradiol to male rats , breakdown of muscle tissue was not diminished during endurance exercise . in fact , administration of 17--estradiol to males and oophorectomized female rats resulted in hepatic and muscle glycogen sparing during endurance exercise , increased intramuscular triglyceride content , and decreased adipocyte lpl . similar results have been observed in human studies with 17--estradiol administration to males and ammenorrheic females resulting in a lower rate of glucose disappearance . the addition of 17--estradiol also appears to increase the activity of enzymes in fat oxidation pathways such as carnotine - palmitoyl transferase-1 ( cpt-1 ) . the role of cpt-1 is to transfer the fatty acyl group from coa to carnitine on the cytosolic side of the inner membrane . together , these findings suggest that the gender - related differences in carbohydrate metabolism and glycogen use in skeletal muscle may be due to both hepatic glycogen sparing , as well as enhanced muscle triglyceride utilization . increased dietary carbohydrate intake can result in enhanced endurance exercise performance by increasing muscle glycogen stores , but may not in all instances as displayed by burke et al . most of the early studies proving this performance enhancing strategy were conducted with predominantly male subjects [ 7 - 9 ] . the need to assess gender differences with carbohydrate loading and glycogen storage stems from altered glycogen storing ability at different phases of the menstrual cycle and the influence of estradiol on glycogen utilization . one of the first studies to evaluate a possible gender difference in glycogen storage after carbohydrate loading was conducted by tarnopolsky et al . in 1995 . in this study , male and female runners were asked to increase carbohydrate intake for four days , manipulating carbohydrate intake from 55% to 75% of total energy intake . the results of the study showed that men increased muscle glycogen content 41% and improved performance time 45% following a one - hour cycling bout , whereas women showed no increase in muscle glycogen and improved performance time by only 5% . the authors speculated that a possible reason for this gender - related difference could be that the increase in dietary carbohydrate intake may not have been enough to elicit glycogen super - compensation . the female participants in this particular study ingested 6.4 g / kg body weight of carbohydrate , while the men ingested 8.2 g / kg body weight of carbohydrate . however , several studies suggest that there is a " carbohydrate loading threshold , " of 810 g / kg that is necessary to achieve the ergogenic benefits of carbohydrate loading [ 7 - 9,42 ] . with this knowledge of a " carbohydrate loading threshold , " . also conducted a study to assess these gender differences , but rather than a moderate increase in dietary carbohydrate intake , participants ingested a carbohydrate level of 12 g / kg of fat free mass per day . james found that by regulating for fat free mass in conjunction with cessation of daily physical training , women and men were able to achieve similar levels of glycogen super - compensation . after the " carbohydrate loading threshold " was determined , three other studies assessed the loading ability of females at this level of dietary carbohydrate ingestion and found that in order to achieve this intake , women would need to increase their total energy intake by 34% during the carbohydrate loading period . by increasing energy intake 34% , females were able to achieve similar concentrations of glycogen as males , and there were no gender differences in hexokinase activity . however , one study found that even with this increase in carbohydrate ingestion , the females were only able to achieve an increase in glycogen stores that was 50% of what was observed in males . therefore , for a female to carbohydrate load and achieve benefits comparable to those of a male , the female must consume extra calories rather than simply increasing the percentage of dietary carbohydrate load . specifically , a female needs to consume about 30% more daily energy for four days to ensure that carbohydrate intake achieves levels higher than 8 g / kg / d . for a 55 kg distance runner , this would be 440 g of carbohydrate , equaling about 1760 calories daily . if this runner is active and consuming 2500 calories per day , this would represent approximately 70% of the total daily energy intake from carbohydrate , which is in concurrence with current recommendations , and is only 5% higher than the 4565% american daily recommendation for carbohydrate . one possible option that may assist with increased carbohydrate consumption and increased carbohydrate utilization is to employ both a loading method and carbohydrate supplementation prior to competition . andrews et al . showed that females used significantly more carbohydrates during submaximal performance following carbohydrate loading and supplementation compared to females who either only supplemented carbohydrates or ingested a placebo . despite many questions that remain to be answered in regards to gender differences in carbohydrate metabolism during endurance exercise , it appears as though female athletes do have the capacity for glycogen super - compensation at levels comparable to males when fed comparable amounts of carbohydrates relative to lean body mass . in order to enhance glycogen - storing ability and obtain peak performance from female endurance athletes , it is necessary for future studies to control for menstrual cycle phase . in addition , future studies should assess the influence of estradiol on energy substrate utilization at rest and during various submaximal bouts of endurance exercise in relation to glycogen storage . with this research and knowledge , female athletes could not potentially erase physiological gender differences , but gender differences in performance as well .

prior to endurance competition , many endurance athletes participate in a carbohydrate loading regimen in order to help delay the onset of fatigue . the " classic " regimen generally includes an intense glycogen depleting training period of approximately two days followed by a glycogen loading period for 34 days , ingesting approximately 6070% of total energy intake as carbohydrates , while the newer method does not consist of an intense glycogen depletion protocol . however , recent evidence has indicated that glycogen loading does not occur in the same manner for males and females , thus affecting performance . the scope of this literature review will include a brief description of the role of estradiol in relation to metabolism and gender differences seen in carbohydrate metabolism and loading .

Introduction The Role of Estradiol in Metabolism Carbohydrate Metabolism Differences in Substrate Utilization Effects of Estradiol Administration Carbohydrate Loading Conclusion and Future Recommendations

PMC4327634

one of the goals of root canal treatment is to eliminate bacteria , bacterial products and debris from the root canal system21 . most bacteria found in the canal space may be removed by the mechanical action of endodontic instruments . however , in several situations , due to the complex anatomy of the root canal system , organic residues and bacteria lodged deep inside the dentinal tubules can not be reached even after careful mechanical instrumentation6,25 . in these cases , the use of irrigating solutions is essential to ensure bacterial minimization12,14 and elimination of organic tissue remnants29 . numerous products are currently used as endodontic irrigants , such as sodium hypochlorite ( naocl ) , chlorhexidine gluconate , calcium hydroxide and saline1,2,4,5,10,1214,18,19,21,24,2628 . each product has different properties and several studies have compared their antimicrobial effect , chemical properties and biocompatibility to establish an ideal solution to be used as an adjuvant to root canal treatment . it is highly desirable that the chemical agents selected as endodontic irrigants possess favorable properties , such as antimicrobial activity and dissolution of organic tissues , assist in root canal system debridement and induce a favorable reaction in the periapical tissues24,27 . naocl is one of the most popular and widely used endodontic irrigants due to its antibacterial activity and capacity of dissolving necrotic tissue remanants23 . the antimicrobial effect of naocl results from the formation of hypochlorous acid ( hocl ) , when in contact with organic debris . hocl exerts its effect by oxidation of sulphydryl groups within bacterial enzyme systems , thereby disrupting the microbial metabolism23 . although it is an effective antibacterial agent , naocl is harmful outside the root canal and causes damage when in contact with the periradicular tissues3,11,27 . chlorhexidine gluconate has also been suggested as an effective irrigating solution and should be used as an alternative to naocl17 . this solution , by attaching to bacterial cytoplasmic membranes , disrupts the osmotic balance , resulting in leakage of intracellular material10 . it also binds to hydroxyapatite and soft tissues , changing their electrical field to compete with bacterial binding15 . in addition , chlorhexidine gluconate presents a residual antibacterial effect on the infected canals , which is a favorable characteristic4,5,12 . however , this antibacterial agent has always been used in a liquid presentation and its inability to dissolve pulp rests has been a problem in some situations . some attempts have been made to overcome this deficiency by the combined use of naocl and chlorhexidine14 or the use of chlorhexidine gel , which has been shown to present an effective antibacterial effect1,69,13,18,28 and a good performance as an intracanal medication21,22 . nevertheless , little data is available about tissue reaction to chlorhexidine gel and further research should be undertaken to validate its use as an irrigating solution during endodontic treatment . thus , the purpose of the present study was to compare the reaction of rat subcutaneous connective tissue to 0.9% sterile saline , 2.5% naocl , 5.25% naocl , 2% chlorhexidine gluconate solution and 2% chlorhexidine gluconate gel . the cytotoxic effects of the root canal irrigants on the subcutaneous tissue of rats were examined using previously described methodologies19,27,30 . twenty - four male wistar rats weighing 250 - 270 g were used for in vivo experiments . the animals were housed in a temperature - controlled environment with water and food ad libitum . all experiments were conducted in accordance with the national institute of healthy ( nih ) guidelines on the welfare of experimental animals and after approval by the ethics in research committee of the dental school of araatuba , so paulo state university . subcutaneous rat tissue reaction to the following irrigating solutions was evaluated : 0.9% sterile saline ; 2.5% naocl ; 5.25% naocl ; 2.0% chlorhexidine gluconate solution ; 2.0% chlorhexidine gluconate gel . under general anesthesia with xylazine ( 10 mg / kg body weigth ) and 5% ketamine hydrochloride ( 25 mg / kg body weigth ) , the dorsal skin of the animals was shaved and cleaned with 10% iodine solution . using a glass template , 6 circles were demarcated on the dermis of each rat leaving 2 cm between each circle . using a syringe , 0.1 ml of each root canal irrigant was injected subcutaneously into 5 circles . for the control group , the needle of an empty syringe was introduced in the 6th circle , but no irrigant was injected . evaluations were made 2 h , 48 h , 14 days and 30 days after injection . in each examination period , 6 animals from experimental groups were sacrificed by anesthetic overdose . the dorsal skin was shaved and tissue specimens were excised with a scalpel , stored in 10% formalin solution for 48 h , washed in running water to remove traces of the fixating solution and thereafter embedded in paraffin blocks using standard procedures . three - micrometer - thick sections were obtained from the paraffin - embedded specimens and stained with hematoxylin and eosin13 . from each tissue sample , 5 sections presenting the greatest inflammatory reaction were examined with a light microscope ( olympus corporation ina plant , ina , japan ) . the areas of inflammatory reaction were evaluated quantitatively and the number of inflammatory cells was counted under x25 magnification . to standardize the results , the inflammatory cells ( neutrophils , eosinophils , lymphocytes , macrophages , giant cells ) were counted using specific software ( leica qwin v3 ; leica microsystems imaging solutions ltd , . statistical analysis was performed by anova and tukey 's test ( graph prism 3.0 ; graph pad software , san diego , ca , usa ) at 5% significance level . in the present investigation , the control group ( figure 1 ) showed few or no inflammatory reaction areas . no significant relationship was observed among the inflammatory reactions at the 4 different evaluation periods for this group ( p>0.05 ) . it was observed a significantly higher inflammatory reaction at the sites injected with the irrigants compared to the site where no irrigant was injected ( control ) ( table 1 ) . tissue reaction to the solutions over time - rows ( p<0.05 ; different letters indicate statistically significant difference at 5% ) . comparison among the solutions at the evaluation periods - columns ( p<0.05 ; different indicate statistically significant difference at 5% ) . the sterile saline group ( figure 2 ) presented a milder inflammatory response than that of the other experimental groups ( table 1 ) . there was a significant increase ( p<0.05 ) in inflammatory response at 48 h compared to the initial period . a significant decrease in mean number of inflammatory cells was observed at 14 and 30 days , with no statistically significant difference in relation to the initial 2-h period ( p>0.05 ) . the 2.5% naocl ( figure 3 ) and 2.0% chlorhexidine gluconate solution groups ( figure 4 ) presented similar results and inflammatory response pattern ( table 1 ) . in both cases , it was observed a mild inflammatory reaction at 2 h. the mean number of inflammatory cells reached the peak at 48 h remaining moderate up to the 14-day period , with statistically significant difference compared to initial response ( p<0.05 ) . at 30 days , it was observed a significantly decrease in the inflammatory response , with no significant difference compared to the 2-h period ( p>0.05 ) . in the 5.25% naocl group ( figure 5 ) , the mean inflammatory reaction values were elevated ( table 1 ) . the inflammatory response was considered statistically significant at 48 h and 14 days , compared to the 2-h period , and the mean number of inflammatory cells was maintained up to the 30- day period ( p<0.05 ) . the 2.0% chlorhexidine gluconate gel group ( figure 6 ) presented a mild initial inflammatory response that increased significantly ( p<0.05 ) at 48 h and 14 days ( table 1 ) . at 30 days , it was observed a decrease in the mean number of inflammatory cells ( p<0.05 ) . comparing all groups ( table 1 ) , it was possible to observe that at 2 h only the 5.25% naocl group showed a higher inflammatory response ( p<0.05 ) compared to the control group . at 48 hours , all experimental groups showed a statistically significant increase of inflammatory cells when compared to control group ( p<0.05 ) . at 14 days , only the 0.9% sterile saline group showed a similar result to that of the control group ( p>0.05 ) . at this time , the 2.5% naocl , 5.25% naocl and 2.0% chlorhexidine gel groups remained with a higher mean number of inflammatory cells compared to the control group ( p<0.05 ) . the 2.0% chlorhexidine solution group presented a milder inflammatory response but still higher than that of the control group ( p<0.05 ) . at 30 days , only the 5.25% naocl group showed a significant mean number of inflammatory cells compared to the control group . in the present investigation , the control group ( figure 1 ) showed few or no inflammatory reaction areas . no significant relationship was observed among the inflammatory reactions at the 4 different evaluation periods for this group ( p>0.05 ) . it was observed a significantly higher inflammatory reaction at the sites injected with the irrigants compared to the site where no irrigant was injected ( control ) ( table 1 ) . tissue reaction to the solutions over time - rows ( p<0.05 ; different letters indicate statistically significant difference at 5% ) . comparison among the solutions at the evaluation periods - columns ( p<0.05 ; different indicate statistically significant difference at 5% ) . the sterile saline group ( figure 2 ) presented a milder inflammatory response than that of the other experimental groups ( table 1 ) . there was a significant increase ( p<0.05 ) in inflammatory response at 48 h compared to the initial period . a significant decrease in mean number of inflammatory cells was observed at 14 and 30 days , with no statistically significant difference in relation to the initial 2-h period ( p>0.05 ) . the 2.5% naocl ( figure 3 ) and 2.0% chlorhexidine gluconate solution groups ( figure 4 ) presented similar results and inflammatory response pattern ( table 1 ) . in both cases , it was observed a mild inflammatory reaction at 2 h. the mean number of inflammatory cells reached the peak at 48 h remaining moderate up to the 14-day period , with statistically significant difference compared to initial response ( p<0.05 ) . at 30 days , it was observed a significantly decrease in the inflammatory response , with no significant difference compared to the 2-h period ( p>0.05 ) . in the 5.25% naocl group ( figure 5 ) , the mean inflammatory reaction values were elevated ( table 1 ) . the inflammatory response was considered statistically significant at 48 h and 14 days , compared to the 2-h period , and the mean number of inflammatory cells was maintained up to the 30- day period ( p<0.05 ) . the 2.0% chlorhexidine gluconate gel group ( figure 6 ) presented a mild initial inflammatory response that increased significantly ( p<0.05 ) at 48 h and 14 days ( table 1 ) . at 30 days , it was observed a decrease in the mean number of inflammatory cells ( p<0.05 ) . comparing all groups ( table 1 ) , it was possible to observe that at 2 h only the 5.25% naocl group showed a higher inflammatory response ( p<0.05 ) compared to the control group . at 48 hours , all experimental groups showed a statistically significant increase of inflammatory cells when compared to control group ( p<0.05 ) . at 14 days , only the 0.9% sterile saline group showed a similar result to that of the control group ( p>0.05 ) . at this time , the 2.5% naocl , 5.25% naocl and 2.0% chlorhexidine gel groups remained with a higher mean number of inflammatory cells compared to the control group ( p<0.05 ) . the 2.0% chlorhexidine solution group presented a milder inflammatory response but still higher than that of the control group ( p<0.05 ) . at 30 days , only the 5.25% naocl group showed a significant mean number of inflammatory cells compared to the control group . several microorganisms found in the root canal system may be removed by the mechanical action of endodontic instruments . nevertheless , due to the complexity of root canal anatomy , even after meticulous mechanical procedures , microorganisms located deeply in the dentinal tubules can not be removed . for this reason , a range of substances have been used as irrigating solutions during root canal preparation to remove debris and necrotic pulp tissue remnants and to eliminate microorganisms that can not be reached by mechanical instrumentation . an ideal endodontic irrigant must present favorable characteristics including maximum tissue dissolving capacity and antibacterial activity , and must induce mild or no inflammatory response in the tissues14,25,27 . therefore , the evaluation of tissue inflammatory response is important because inflammation is the first part of the healing process , which justifies the study of the inflammatory progression after different stimuli , including irrigating solutions20 . the importance of these studies can be attributed to the fact that irrigant extrusion into surrounding periodontal tissues can occur in case of perforations and improper techniques or even in teeth with fully developed roots . it was found that the control group ( no injection ) induced mild or no reaction in the subcutaneous tissue and that this process did not change at the different experimental periods . likewise , other studies have reported only a very mild inflammatory reaction at the site of penetration with the needle of an empty syringe19,27 , suggesting that this reaction occurred due to a mechanical trauma caused by the puncture itself . in the present study , the 0.9% sterile saline group showed a mild inflammatory response with a significant increase in the mean number of inflammatory cells only at 48 h. these findings are in agreement with those reported elsewhere27 , 30 , suggesting that saline promoted a favorable reaction of connective tissues and can be considered as material biocompatible since tissue repair occurred 14 and 30 days . the 2.0% naocl and 2.0% chlorhexidine gluconate groups presented a moderate connective tissue inflammatory reaction . although the mean number of inflammatory cells was considered significant at 48 h and 14 days , it could be observed a decrease in the number of inflammatory cells at 14 and 30 days , suggesting an ongoing connective tissue repair process . a similar outcome was observed at 14 days in other studies , which evaluated 0.12% chlorhexidine gluconate as an irrigating solution27,30 . in these cases , the inflammatory response was at the highest level 48 h later , whereas the level of inflammation dropped by the end of the second week27,30 . on the other hand , another study27 found that 1% chlorhexidine gluconate was less toxic , less irritating and induced a faster regeneration than 0.5% naocl . it has also been demonstrated that chlorhexidine gluconate had no less antibacterial effect than naocl and that , because of its lower toxicity , should be preferred in root canal therapy , especially in cases of immature teeth12 . although chlorhexidine gluconate has been indicated as an effective irrigating solution due to its favorable properties4,5,12,15,17 , the literature has suggested that , in some situations , this solution is not able to dissolve pulp tissues6,14 . therefore , to solve this deficiency , some authors have recommended the combined use of naocl and chlorhexidine14 or the use of chlorhexidine gel1,69,13,18,22,28 . the use of a viscous irrigant has been suggested because viscosity would compensate for the incapacity of chlorhexidine to dissolve pulp tissue by promoting a better mechanical cleansing of the root canal and removing dentin debris and tissue remnants . it would also promote a better lubricating action and improve the antimicrobial property6,26 . in the present study , it was also found that 2.0% chlorhexidine gluconate gel presented a mild initial inflammatory response that increased at 48 h and 14 days and decreased at 30 days . these results can be explained by the fact that the viscous bases used in these irrigants have little solubility in water . thus , this finding suggests that chlorhexidine gel can promote a higher inflammatory response in the connective tissue because its clearance by the tissue occurs slower than the liquid presentation of the same substance . chlorhexidine gluconate gel has been extensively used in dentistry with good results in caries control by reducing streptococcus mutans and lactobacillus species , and as an adjuvant in periodontal therapy by controlling gram- positive and gram - negative bacterial growth2 . the use of chlorhexidine gel in endodontics has been suggested in few studies , with a good performance as an intracanal medication13,22 or as an irrigant6,9,18,28 . despite the lack of studies evaluating the biocompatibility of this material , chlorhexidine gel has been shown to present a great potential for use as a routine endodontic irrigant as it has low toxicity and a wide antimicrobial spectrum6 . it has been reported that 5.25% naocl solution promotes an irritating effect on the periapical tissues and that , at the end of the second week of evaluation , foreign body granuloma formation occurred30 . in the same way as observed in the present investigation , other studies have reported that the number of inflammatory cells remained elevated at the sites treated with 5.25%naocl even 14 days after injection , and that complete healing was not observed19,27,30 . also evaluating the toxic effect of 5.25% naocl , a previous study showed that in sites injected with this solution , tissue regeneration occurred at a slower rate when compared to the sites injected with 2.0% chlorhexidine gluconate . it has also been reported that 2.0% chlorhexidine gluconate displayed residual antibacterial activity and was more powerful and less toxic than 5.25% naocl19 . taken together , these results support the idea that 5.25% naocl promotes a toxic effect on connective tissue and should not be considered as an ideal root canal irrigant , especially when compared to other chemical agents that have favorable properties , such as biocompatibility . in conclusion , the results of the present study indicate that 5.25% naocl was the most toxic endodontic irrigant to rat subcutaneous connective tissue and the other solutions had a similar behavior at the end of the 30-day evaluation period .

the purpose of this study was to compare the reaction of rat subcutaneous connective tissue to 0.9% sterile saline , 2.5% sodium hypochlorite ( naocl ) , 5.25% naocl and 2% chlorhexidine gluconate solution or gel . six circles were demarcated on the dorsal skin of 24 male wistar rats , leaving 2 cm between each circle . using a syringe , 0.1 ml of each root canal irrigant was injected subcutaneously into 5 circles . in the 6th circle , the needle of an empty syringe was introduced into the skin , but no irrigant was injected ( control group ) . evaluations were undertaken at 2 h , 48 h , 14 days and 30 days post - procedure . tissue samples were excised , embedded in paraffin blocks and 3-m - thick sections were obtained and stained with hematoxylin and eosin . the areas of inflammatory reaction were evaluated and analyzed statistically by anova and tukey 's test . the control group showed few or no inflammatory reaction areas in the subcutaneous tissue . 0.9% saline solution , 2.0% chlorhexidine solution and 2.5% naocl showed a good biocompatibility , as very mild inflammatory reaction was detected at 14 days and tissue repair occurred at 30 days . 5.25% naocl was the most toxic irrigant , as the number of inflammatory cells remained elevated at 14 and 30 days . the group treated with 2.0% chlorhexidine gluconate gel presented a moderate inflammatory response at 14 days , which decreased at 30 days , being considered similar to that of the control group , 0.9% saline solution , 2.0% chlorhexidine solution and 2.5% naocl at this experimental period .

INTRODUCTION MATERIALS AND METHODS RESULTS Tissue Reaction to the Solutions over Time Comparison among the Solutions at the Evaluation Periods DISCUSSION CONCLUSIONS

PMC4532838

many nations have invested in the standardisation of healthcare information technologies ( hit ) . healthcare information exchanges ( hie ) or sharing of clinical data is simply not feasible without appropriate standardisation . without having the hie , we can not expect to establish and operate a cost effective and sustainable healthcare system that has high quality of care . therefore , many countries have put significant effort in hit standardisation and are adhering to certain standardisation processes . although each nation has those interests in hit standardisation , the structure and process of the standardisation are a little bit different . in the united states , there are many standard developing organizations ( sdos ) , such as the american society for testing and materials ( astm ) , the american society of mechanical engineers ( asme ) , health level seven ( hl7 ) , and so on . for example , astm accepts ideas and reviews and creates standards , which then become the national standards . regarding hie , the continuity of care record ( ccr ) is a key standard from astm for the purpose of defining a standard summary record at a point in time to facilitate clinical communication between providers . hl7 developed the clinical document architecture ( cda ) , which defines a standard structured definition of a signed clinical document to facilitate document - based hie between electronic medical record ( emr)/electronic health record ( ehr ) systems . in 2007 , the continuity of care document was created with convergence of cda and ccr by the two sdos . the american national standard institute ( ansi ) is a non - profit organization founded in 1981 . it credits various sdos and approves standards of the private sector 's efforts and outputs . the office of national coordinator for health information technology under the department of health and human services was founded in 2001 to support the private sector and to facilitate the adoption of developed standards in the healthcare industry . thus , the private hit sector is taking a dominant role in the area of standards development and adoption in the united states , and the government is actively supporting this . the national standards body of the united kingdom is the british standards institute ( bsi ) founded in 1901 . bsi has established the vast majority of standards at the national level , including hit . in australia , standards australia ( sa ) is in charge of developing and approving all kinds of standards including hit . sa has several internal parts , such as the accreditation board for standards development organizations , standards australia council , and the standards development committee . in contrast , the government of new zealand is directly involved in standards development in hit and runs the health information standards organisation ( hiso ) . it may be more effective because several empirical studies have shown that government power usually has a strong influence on the healthcare industry . the government of new zealand has already established governmental organizations regarding hit and has created various hit standards , which it updates regularly . those guidelines on hit standardisation the objective of this study was to review the hit standardisation and progress of new zealand . specifically , this paper describes how governmental organizations for healthcare ict are organised , how they work , and how they transfer current hit standardisation to the healthcare industry . this kind of governmental involvement has various political implications on how healthcare politicians document policies of hit and presents as an opportunity to provide a benchmark for other nations . thus , this study could provide many other countries with meaningful implications regarding hit standardisation . we reviewed reports regarding the hit standards and standardisation processes published by new zealand government agencies , including the ministry of health , health it board , and hiso . then , we summarised these materials focusing on governance structures , standardisation processes , and their outcomes . finally , we investigated several important issues regarding the current hit standardisation status of new zealand . the governmental department supporting the national healthcare service system in new zealand is the ministry of health operating through the national health board ( nhb ) ( figure 1 ) . the former has direct responsibility for delivering public healthcare to the people and supports the latter . the nhb is an advisory board for the former and is responsible for overseeing various activities of district health boards ( dhbs ) including funding , monitoring , and planning . the national health it board ( nhitb ) under the nhb is the governing body for health information standards in new zealand . it has a sub - committee called the hiso which provides important advice to the nhitb regarding the development and adoption of national hit standards . hiso collaborates with the health sector architects group , comprising about 30 experts from various sectors of the healthcare setting . the hiso 's responsibilities include providing advice to the nhitb about the development of health information standards , identifying and adopting relevant international standards , managing and ensuring standard development process with an open and consistent way , and ratifying new standards and withdrawing old ones . they have some obligations to comply with hiso standards under the parliamentary funding agreement called the operational policy framework ( opf ) . the opf 2014/15 section 11.2.3.c requires : " dhbs to proactively support the development and adoption of health information standards organization ( hiso ) standards by : adhering to and meeting the requirements of all published hiso standards , identifying and reporting on needs or opportunities for new standards and updates to existing standards , participating in the development or enhancement of standards , supporting trial implementation of interim hiso standards ( p.103 ) . " . new zealand is divided into four health regions , each with a number of public hospitals and primary health organizations . under the national health it plan , regions are responsible for delivering a regional hospital patient administration system , regional hospital / community clinical workstation / portal and regional clinical data repository . hospitals operate the usual clinical specialty and clinical support systems , including radiology information system / picture archiving and communication system ( pacs ) , laboratory information system , and pharmacy management . hospitals are implementing the same software for medicines reconciliation , medication management and e - discharge . hospitals tend to operate best - of - breed solutions rather than integrated product suites . however , the sector places a lot of importance on systems interoperability , and that is where much of governmental standardisation effort is directed . all general practitioners ( gp ) use an emr system , called practice management systems , and many now offer a patient portal . access by hospital emergency departments and other providers to gp - held patient data is enabled by provider portals , which are also now in rollout around the country . in addition to regional and primary care solutions , there are national solutions , including the national health index ( patients ) and health provider index . new zealand has implemented a national clinical assessment application based on the comprehensive clinical assessment for aged care ( interrai ) methodology , and the government is rolling out a centrally hosted maternity clinical information system . hl7 version 2.x message - based hie has been operational for many years in the area of lab results , discharge summaries , primary care medical records and e - referrals . while this has been very successful , new zealand is now moving towards web service - based hie for new applications . the interoperability reference architecture was authored by an expert group in 2011 , and the essential parts were standardised as health information exchange architecture building blocks and ratified as an interim standard by the hiso . this comprises three pillars : 1 ) hiso 10040.1 is about clinical data repositories and their interfaces . it states requirements for a particular set of document - sharing services based on the ihe xds integration profile . xds defines methods to store , locate and retrieve information from multiple sources or repositories ( as in regional and national levels ) . the central mechanism used to achieve 2 ) hiso 10040.2 is about establishing a single content model for supporting the semantics of information exchange . it is based on ccr specification and prescribes a set of detailed clinical models expressed as openehr archetypes . 3 ) finally , hiso 10040.3 is about the use of hl7 cda structured documents as the common currency of exchange - the payload . this new service - oriented hie approach is underpinned by a virtual private network called connected health , which is provided by a number of telecommunication providers . the new model of information sharing is to use regional clinical data repositories to make shared clinical documents available to distributed care teams , using a record locator service to index the collective content of all repositories and make content findable by clinical workstation and patient portal systems . new zealand is also implementing a national e - prescription broker system for prescribing and dispensing medications in the community . this system links gps and community pharmacies , and it will also be used to convey hospital discharge prescriptions . government has developed a systematized nomenclature of medicine - clinical terms ( snomed ct)-based national medicines terminology and a national medicines formulary to enabler e - prescribing in the hospital and community . regarding hit standardisation , full standardisation of systems , hardware and software across the entire health sector is not the ultimate goal of the nhitb . the snomed - ct has been accepted by hiso and has an endorsed standard status . the hiso plays a critical role in creating hit standards from their inception to their adoption . figures 2 and 3 provide an overview of the development , review and maintenance of hiso standards . during this time the hiso ( through the national health it board ) is running a standards adoption and compliance program . current efforts are researching and establishing a detailed knowledge of the present situation in the health provider community . critical priorities are those agreed by sector leaders and the national health it board , which take precedence over all other new it priorities . these critical priorities include the new zealand electronic prescription service ( nzeps ) , electronic medicines reconciliation ( emr ) , clinical workstation ( cws ) , and clinical data repository ( cdr ) . important priorities are determined based on the existence of strong local or regional needs or requirements , and these include the electronic prescribing and administration ( epa ) of medicines and the new zealand universal list of medicines ( nzulm ) , ereferrals and edischarges . emerging priorities are the areas of emerging work so health organisations can make an early start on them such as eenrolment , telehealth , and so on . regions have autonomy to implement patient administration , clinical data repository , clinical specialty and clinical support systems of their own choosing . regional systems are required to integrate with the national health index and health provider index systems . there are nationally endorsed choices in certain classes of software , including clinical workstation and medications management . the pervasiveness of this software and the desirability of a common user interface for the highly mobile clinical workforce were behind these choices . the governmental department supporting the national healthcare service system in new zealand is the ministry of health operating through the national health board ( nhb ) ( figure 1 ) . the former has direct responsibility for delivering public healthcare to the people and supports the latter . the nhb is an advisory board for the former and is responsible for overseeing various activities of district health boards ( dhbs ) including funding , monitoring , and planning . the national health it board ( nhitb ) under the nhb is the governing body for health information standards in new zealand . it has a sub - committee called the hiso which provides important advice to the nhitb regarding the development and adoption of national hit standards . hiso collaborates with the health sector architects group , comprising about 30 experts from various sectors of the healthcare setting . the hiso 's responsibilities include providing advice to the nhitb about the development of health information standards , identifying and adopting relevant international standards , managing and ensuring standard development process with an open and consistent way , and ratifying new standards and withdrawing old ones . they have some obligations to comply with hiso standards under the parliamentary funding agreement called the operational policy framework ( opf ) . the opf 2014/15 section 11.2.3.c requires : " dhbs to proactively support the development and adoption of health information standards organization ( hiso ) standards by : adhering to and meeting the requirements of all published hiso standards , identifying and reporting on needs or opportunities for new standards and updates to existing standards , participating in the development or enhancement of standards , supporting trial implementation of interim hiso standards ( p.103 ) . " . new zealand is divided into four health regions , each with a number of public hospitals and primary health organizations . under the national health it plan , regions are responsible for delivering a regional hospital patient administration system , regional hospital / community clinical workstation / portal and regional clinical data repository . hospitals operate the usual clinical specialty and clinical support systems , including radiology information system / picture archiving and communication system ( pacs ) , laboratory information system , and pharmacy management . hospitals are implementing the same software for medicines reconciliation , medication management and e - discharge . hospitals tend to operate best - of - breed solutions rather than integrated product suites . however , the sector places a lot of importance on systems interoperability , and that is where much of governmental standardisation effort is directed . all general practitioners ( gp ) use an emr system , called practice management systems , and many now offer a patient portal . access by hospital emergency departments and other providers to gp - held patient data is enabled by provider portals , which are also now in rollout around the country . in addition to regional and primary care solutions , there are national solutions , including the national health index ( patients ) and health provider index . new zealand has implemented a national clinical assessment application based on the comprehensive clinical assessment for aged care ( interrai ) methodology , and the government is rolling out a centrally hosted maternity clinical information system . hl7 version 2.x message - based hie has been operational for many years in the area of lab results , discharge summaries , primary care medical records and e - referrals . while this has been very successful , new zealand is now moving towards web service - based hie for new applications . the interoperability reference architecture was authored by an expert group in 2011 , and the essential parts were standardised as health information exchange architecture building blocks and ratified as an interim standard by the hiso . this comprises three pillars : 1 ) hiso 10040.1 is about clinical data repositories and their interfaces . it states requirements for a particular set of document - sharing services based on the ihe xds integration profile . xds defines methods to store , locate and retrieve information from multiple sources or repositories ( as in regional and national levels ) . the central mechanism used to achieve this is a registry providing a consolidated index over all content within the ecosystem . 2 ) hiso 10040.2 is about establishing a single content model for supporting the semantics of information exchange . it is based on ccr specification and prescribes a set of detailed clinical models expressed as openehr archetypes . 3 ) finally , hiso 10040.3 is about the use of hl7 cda structured documents as the common currency of exchange - the payload . this new service - oriented hie approach is underpinned by a virtual private network called connected health , which is provided by a number of telecommunication providers . the new model of information sharing is to use regional clinical data repositories to make shared clinical documents available to distributed care teams , using a record locator service to index the collective content of all repositories and make content findable by clinical workstation and patient portal systems . new zealand is also implementing a national e - prescription broker system for prescribing and dispensing medications in the community . this system links gps and community pharmacies , and it will also be used to convey hospital discharge prescriptions . government has developed a systematized nomenclature of medicine - clinical terms ( snomed ct)-based national medicines terminology and a national medicines formulary to enabler e - prescribing in the hospital and community . regarding hit standardisation , full standardisation of systems , hardware and software across the entire health sector is not the ultimate goal of the nhitb . the snomed - ct has been accepted by hiso and has an endorsed standard status . the hiso plays a critical role in creating hit standards from their inception to their adoption . figures 2 and 3 provide an overview of the development , review and maintenance of hiso standards . during this time the hiso ( through the national health it board ) is running a standards adoption and compliance program . current efforts are researching and establishing a detailed knowledge of the present situation in the health provider community . critical priorities are those agreed by sector leaders and the national health it board , which take precedence over all other new it priorities . these critical priorities include the new zealand electronic prescription service ( nzeps ) , electronic medicines reconciliation ( emr ) , clinical workstation ( cws ) , and clinical data repository ( cdr ) . important priorities are determined based on the existence of strong local or regional needs or requirements , and these include the electronic prescribing and administration ( epa ) of medicines and the new zealand universal list of medicines ( nzulm ) , ereferrals and edischarges . emerging priorities are the areas of emerging work so health organisations can make an early start on them such as eenrolment , telehealth , and so on . regions have autonomy to implement patient administration , clinical data repository , clinical specialty and clinical support systems of their own choosing . regional systems there are nationally endorsed choices in certain classes of software , including clinical workstation and medications management . the pervasiveness of this software and the desirability of a common user interface for the highly mobile clinical workforce were behind these choices . this study reviewed the governance structure , standardisation processes , and current standards of hit in new zealand . through this study , we could verify that the government of new zealand plays an important role in hit standardisation . first , the government of new zealand , unlike those of other countries , has had specific hit bodies to drive and support standardisation , including the national health it board , the hiso and the health sector architects group , which actively participate in the standardisation processes . they collect good ideas from the healthcare industry and the public sector and then develop various hit standards . third , we also could see that there were various specific outcomes of healthcare hit , such as e - referral systems , e - prescribing systems , and so on . new zealand 's governmental structures and processes may be a direct and efficient way of achieving the benefits of hit standardisation because government power has a strong influence on markets , and many healthcare organizations are under government control , such as public and tertiary hospitals . several empirical and theoretical studies have shown that government power and influence is the strongest driving force . firstly , we wish to investigate whether there would be any beneficial effect of hit standards on healthcare outcomes , such as minimising errors in healthcare organization . we are also interested to see how other countries could benchmark with the achievements of hit standardisation as in new zealand . conferences or international cooperation for sharing experiences and information with lagging countries could lead to valuable outcomes . finally , we believe this kind of active government involvement could bring about many benefits in terms of reducing errors and improving the quality of care at the national level . we expect that this study will provide a benchmark for those lagging countries regarding ict standards and standardisation processes .

objectivesmany countries try to efficiently deliver high quality healthcare services at lower and manageable costs where healthcare information and communication technologies ( ict ) standardisation may play an important role . new zealand provides a good model of healthcare ict standardisation . the purpose of this study was to review the current healthcare ict standardisation and progress in new zealand.methodsthis study reviewed the reports regarding the healthcare ict standardisation in new zealand . we also investigated relevant websites related with the healthcare ict standards , most of which were run by the government . then , we summarised the governance structure , standardisation processes , and their output regarding the current healthcare ict standards status of new zealand.resultsnew zealand government bodies have established a set of healthcare ict standards and clear guidelines and procedures for healthcare ict standardisation . government has actively participated in various enactments of healthcare ict standards from the inception of ideas to their eventual retirement . great achievements in ehealth have already been realized , and various standards are currently utilised at all levels of healthcare regionally and nationally . standard clinical terminologies , such as international classification of diseases ( icd ) and systematized nomenclature of medicine - clinical terms ( snomed - ct ) have been adopted and health level seven ( hl7 ) standards are actively used in health information exchanges.conclusionsthe government to new zealand has well organised ict institutions , guidelines , and regulations , as well as various programs , such as e - medications and integrated care services . local district health boards directly running hospitals have effectively adopted various new ict standards . they might already be benefiting from improved efficiency resulting from healthcare ict standardisation .

I. Introduction II. Methods III. Results 1. Governance of HIT Standards in New Zealand 2. Use of EMR/EHR Systems in Hospitals and Primary Care Settings 3. Current Standards on HIE in New Zealand 4. Process of the HIT Standardisation 5. Programs of HISO for National Health ICT Standardisation 6. Nationally Standardised EMR or EHR Systems IV. Conclusion

PMC5133725

rest is important for natural repair of damaged tissue and has many potential benefits for ill humans , such as conserve metabolic resource for healing and recovery , reduce oxygen consumption by muscles and reduce harmful falls . however , for patients in the intensive care unit ( icu ) , prolonged bed rest would lead to disuse of musculoskeletal system and cause systemic problems to other body systems[2 - 4 ] . the lack of mechanical stimulation caused decrease in bone mass and strength , and induced muscle atrophy . prolonged bed rest demonstrated to decrease bone mineral density ( bmd ) and increase calcium excretion[5 - 7 ] . the lack of mechanical loading decreased the tendon stiffness and affected the development of the bone - tendon insertion ( bti ) , a unique structure that serves as an interface for force transmission from tendon to bone and acts as a stress absorber during mechanical loading . furthermore , the available individual studies only focused on the deterioration of a single specific tissue type in the musculoskeletal system , either bone loss , or muscle atrophy , instead of investigating on the systemic adaptation of the whole musculoskeletal system during the period of disuse . this study was performed to investigate whether disuse has a systemic effect on all involved musculoskeletal tissues and whether there are differences in sensitivity within a single type of tissues . patients in the intensive care unit ( icu ) or patients who require prolonged bed rest , such as those who suffer from hip fracture , are encouraged to have early mobilization and weight - bearing . reported that cycling for 20 min / day enhanced recovery of functional exercise capacity , self - perceived functional status , and muscle force at hospital discharge of icu patients with a cardiorespiratory condition . compare with non - weight - bearing exercise , weight - bearing exercise has further improved hip extensor strength in elder inpatients . therefore , weight - bearing exercises might provide greater mechanical stimulation on the body and protect the musculoskeletal system from disuse - induced musculoskeletal deterioration . daily short - duration of weight - bearing ( wb ) can be easily applied clinically because the duration of the therapy is relatively short and does not require extra equipment . we hypothesized that daily short - duration of wb would be sufficient to prevent disuse - induced bone loss , muscle atrophy , and bti deterioration . to simulate the effect of disuse on the musculoskeletal system during prolonged bed rest , a well - established tail - suspension model in rats was used because this model is well tolerated by the animal with minimal evidence of stress . the current study evaluated systemic deterioration of musculoskeletal system , including bone , muscle , and bti complex , and investigated the effect of wb on prevention of musculoskeletal system deterioration in an established tail - suspension model in rats . twenty - four , three months old , male sprague - dawley rats were obtained from the china astronaut research and training center , beijing , pr china . the rats were divided into three groups : control group ( cg ) , tail - suspension group ( tg ) , and weight - bearing group ( wbg ) . cg did not receive any intervention while tg and wbg underwent standard tail - suspension to simulate the effect of disuse on the hindlimbs according to the established protocol[21 - 23 ] . the rats were suspended in a head - down position and formed a 30 angle between the floor and the torso of the rat . the hindlimbs of the rats were not allowed to touch the side of the cage and the floor . the rats were also allowed free - cage movement and access to water and standard rat chow ad libitum with their forelimbs . on the second day of tail - suspension , daily cage activity was set for 20 min / day , 5 days / week in wbg while tg did not receive any treatment . both hind limbs of the rats were harvested for isolating relevant tissues , including bone , muscle , and bti complex for various evaluations ( table 1 ) . soleus muscle , which contained predominantly slow twitch fibers , and extensor digitorum longus muscle ( edl ) , which contained predominantly fast twitch fibers , were harvested and weighted before specified evaluations . f : femur ; t : tibia ; edl : extensor digitorum longus ; sol : soleus muscle ; ppt : patella - patellar tendon ; ibmd : integral bone mineral density ; tbmd : trabecular bone mineral density ; csa : cross - section area ; csmi : cross - sectional moment of inertia ; n / a : not applicable . the femur and tibia were isolated and placed on a custom - made plastic holder for pqct measurement ( densiscan 2000 , scanco medical , brttisellen , switzerland ) . the integral bmd ( ibmd ) and trabecular bmd ( tbmd ) of the femur and tibia were measured according to our established protocol . ibmd is the bmd of the whole bone within the region of interest ( roi ) , which includes both trabecular bone and cortical bone . the geometrical parameters of the femur and tibia , including the cross - sectional area ( csa ) of the bony tissue and cross - sectional moment of inertia ( csmi ) , were also measured . csmi is calculated as csmi=(pi - c)areap where pi is the position of the i pixel , c is the position of the center pixel ( the centric pixel of the identified bone ) , and areap is the area of a pixel . the csa of the muscles at mid tibia , an indicator of muscle strength , was measured ( table 1 ) . the tensile properties of the patella - patellar tendon ( ppt ) insertion were evaluated according to our published protocol . briefly , the quadriceps - patella - patellar tendon - tibia ( qpptt ) complex was isolated from the right hindlimbs . the qpptt complex was mounted onto a custom - made fixator and attached to a mechanical testing machine ( h25k - s , hounsfield test equipment ltd , surrey , uk ) with a 250 n load cell ( figure 1 ) . the thickness and width of the ppt insertion were measured with a fine caliper and the cross - sectional area of the insertion was calculated . a testing speed of 100 mm / min up to failure of the qpptt complex was used based on our previous published work . a : a custom - made jig for tensile test for the quadriceps - patellapatellar tendon - tibia ( qpptt ) complex . b : the testing jig consisted of an upper clamp that secured the patella together with the distal quadriceps , and a lower clamp that gripped the proximal tibia . a piece of sand paper was placed between the clamps to increase the friction between the clamp and the patella . coefficient of variation ( cv ) was calculated by dividing the standard deviation by the mean . statistical analysis was performed using spss 16.0 for windows ( ibm , ny , usa ) . one - way analysis of variance ( one - way anova ) with bonferroni post hoc test was performed to evaluate the differences among different groups . twenty - four , three months old , male sprague - dawley rats were obtained from the china astronaut research and training center , beijing , pr china . the rats were divided into three groups : control group ( cg ) , tail - suspension group ( tg ) , and weight - bearing group ( wbg ) . cg did not receive any intervention while tg and wbg underwent standard tail - suspension to simulate the effect of disuse on the hindlimbs according to the established protocol[21 - 23 ] . the rats were suspended in a head - down position and formed a 30 angle between the floor and the torso of the rat . the hindlimbs of the rats were not allowed to touch the side of the cage and the floor . the rats were also allowed free - cage movement and access to water and standard rat chow ad libitum with their forelimbs . on the second day of tail - suspension , daily cage activity was set for 20 min / day , 5 days / week in wbg while tg did not receive any treatment . both hind limbs of the rats were harvested for isolating relevant tissues , including bone , muscle , and bti complex for various evaluations ( table 1 ) . soleus muscle , which contained predominantly slow twitch fibers , and extensor digitorum longus muscle ( edl ) , which contained predominantly fast twitch fibers , were harvested and weighted before specified evaluations . f : femur ; t : tibia ; edl : extensor digitorum longus ; sol : soleus muscle ; ppt : patella - patellar tendon ; ibmd : integral bone mineral density ; tbmd : trabecular bone mineral density ; csa : cross - section area ; csmi : cross - sectional moment of inertia ; n / a : not applicable . the femur and tibia were isolated and placed on a custom - made plastic holder for pqct measurement ( densiscan 2000 , scanco medical , brttisellen , switzerland ) . the integral bmd ( ibmd ) and trabecular bmd ( tbmd ) of the femur and tibia were measured according to our established protocol . ibmd is the bmd of the whole bone within the region of interest ( roi ) , which includes both trabecular bone and cortical bone . the geometrical parameters of the femur and tibia , including the cross - sectional area ( csa ) of the bony tissue and cross - sectional moment of inertia ( csmi ) , were also measured . csmi is calculated as csmi=(pi - c)areap where pi is the position of the i pixel , c is the position of the center pixel ( the centric pixel of the identified bone ) , and areap is the area of a pixel . the csa of the muscles at mid tibia , an indicator of muscle strength , was measured ( table 1 ) . the tensile properties of the patella - patellar tendon ( ppt ) insertion were evaluated according to our published protocol . briefly , the quadriceps - patella - patellar tendon - tibia ( qpptt ) complex was isolated from the right hindlimbs . the qpptt complex was mounted onto a custom - made fixator and attached to a mechanical testing machine ( h25k - s , hounsfield test equipment ltd , surrey , uk ) with a 250 n load cell ( figure 1 ) . the thickness and width of the ppt insertion were measured with a fine caliper and the cross - sectional area of the insertion was calculated . a testing speed of 100 mm / min up to failure of the qpptt complex was used based on our previous published work . a : a custom - made jig for tensile test for the quadriceps - patellapatellar tendon - tibia ( qpptt ) complex . b : the testing jig consisted of an upper clamp that secured the patella together with the distal quadriceps , and a lower clamp that gripped the proximal tibia . a piece of sand paper was placed between the clamps to increase the friction between the clamp and the patella . coefficient of variation ( cv ) was calculated by dividing the standard deviation by the mean . statistical analysis was performed using spss 16.0 for windows ( ibm , ny , usa ) . one - way analysis of variance ( one - way anova ) with bonferroni post hoc test was performed to evaluate the differences among different groups . twenty - four , three months old , male sprague - dawley rats were obtained from the china astronaut research and training center , beijing , pr china . the rats were divided into three groups : control group ( cg ) , tail - suspension group ( tg ) , and weight - bearing group ( wbg ) . cg did not receive any intervention while tg and wbg underwent standard tail - suspension to simulate the effect of disuse on the hindlimbs according to the established protocol[21 - 23 ] . the rats were suspended in a head - down position and formed a 30 angle between the floor and the torso of the rat . the hindlimbs of the rats were not allowed to touch the side of the cage and the floor . the rats were also allowed free - cage movement and access to water and standard rat chow ad libitum with their forelimbs . on the second day of tail - suspension , daily cage activity was set for 20 min / day , 5 days / week in wbg while tg did not receive any treatment . both hind limbs of the rats were harvested for isolating relevant tissues , including bone , muscle , and bti complex for various evaluations ( table 1 ) . soleus muscle , which contained predominantly slow twitch fibers , and extensor digitorum longus muscle ( edl ) , which contained predominantly fast twitch fibers , were harvested and weighted before specified evaluations . f : femur ; t : tibia ; edl : extensor digitorum longus ; sol : soleus muscle ; ppt : patella - patellar tendon ; ibmd : integral bone mineral density ; tbmd : trabecular bone mineral density ; csa : cross - section area ; csmi : cross - sectional moment of inertia ; n / a : not applicable . the femur and tibia were isolated and placed on a custom - made plastic holder for pqct measurement ( densiscan 2000 , scanco medical , brttisellen , switzerland ) . the integral bmd ( ibmd ) and trabecular bmd ( tbmd ) of the femur and tibia were measured according to our established protocol . ibmd is the bmd of the whole bone within the region of interest ( roi ) , which includes both trabecular bone and cortical bone . the geometrical parameters of the femur and tibia , including the cross - sectional area ( csa ) of the bony tissue and cross - sectional moment of inertia ( csmi ) , were also measured . csmi is calculated as csmi=(pi - c)areap where pi is the position of the i pixel , c is the position of the center pixel ( the centric pixel of the identified bone ) , and areap is the area of a pixel . the csa of the muscles at mid tibia , an indicator of muscle strength , was measured ( table 1 ) . the tensile properties of the patella - patellar tendon ( ppt ) insertion were evaluated according to our published protocol . briefly , the quadriceps - patella - patellar tendon - tibia ( qpptt ) complex was isolated from the right hindlimbs . all periarticular connective soft tissues and soft tissues around the knee were removed . the qpptt complex was mounted onto a custom - made fixator and attached to a mechanical testing machine ( h25k - s , hounsfield test equipment ltd , surrey , uk ) with a 250 n load cell ( figure 1 ) . the thickness and width of the ppt insertion were measured with a fine caliper and the cross - sectional area of the insertion was calculated . a testing speed of 100 mm / min up to failure of the qpptt complex was used based on our previous published work . a : a custom - made jig for tensile test for the quadriceps - patellapatellar tendon - tibia ( qpptt ) complex . b : the testing jig consisted of an upper clamp that secured the patella together with the distal quadriceps , and a lower clamp that gripped the proximal tibia . a piece of sand paper was placed between the clamps to increase the friction between the clamp and the patella . coefficient of variation ( cv ) was calculated by dividing the standard deviation by the mean . statistical analysis was performed using spss 16.0 for windows ( ibm , ny , usa ) . one - way analysis of variance ( one - way anova ) with bonferroni post hoc test was performed to evaluate the differences among different groups . after four weeks of tail - suspension and different treatments , ts ( 76.89% of cg , p<0.05 ) , and wbg ( 69.85% of cg , p<0.05 ) showed significant decrease in weight compared with the cg . however , no significant difference was detected among ts and wbg ( figure 2 ) . for both femur and tibia , cg showed significantly higher ibmd , tbmd , bone csa , cortical csa , and csmi than tg and wbg ( p<0.05 ) ( table 2 ) . all pqct - measured parameters did not show significant difference between tg and wbg in either femur or tibia ( p>0.05 ) . ibmd of femur and tibia in tg and wbg showed the least percentage of decrease ( 82.2% and 90.3% of cg ) while csmi showed the greatest percentage decrease ( 43.0 - 76.2% of cgs ) . note : the percentage in the brackets represent the percentage of current assessment corresponds to cg . pqct : peripheral quantitative computed tomography ; ibmd : integral bone mineral density ( bmd ) ; tbmd : trabecular bmd ; csa : cross - sectional area ; csmi : cross - sectional moment of inertia ; number in brackets represent the percentage of the value compared to that of cg ; wbg ( 47.7% of cg , p<0.05 ) showed significantly lower muscle mass in the soleus muscle than that of cg ( figure 3 ) . tg ( 78.7% of cg , p<0.05 ) and wbg ( 72.4% of cg , p<0.05 ) showed significantly lower muscle mass in edl than that in cg ( figure 4 ) . moreover , tg ( 63.9% of cg , p<0.05 ) and wbg ( 62.4% of cg , p<0.05 ) showed significantly lower csa of tibia muscle than that of cg ( figure 5 ) . cg showed significantly higher weight than those of tg and wbg . * : p<0.05 . the ppt samples failed with an avulsion of bone at the bti complex during mechanical testing . the tensile failure strength of ppt insertion was the highest in cg ( figure 6 ) . tg ( 76.1% of cg , p<0.05 ) and wbg ( 70.6% of cg , p<0.05 ) showed significantly lower failure strength than that of cg . tensile strength of the patella - patellar tendon ( ppt ) complex of different groups . cg showed significantly higher failure strength than that of tg and wbg . * : p<0.05 . after four weeks of tail - suspension and different treatments , ts ( 76.89% of cg , p<0.05 ) , and wbg ( 69.85% of cg , p<0.05 ) showed significant decrease in weight compared with the cg . however , no significant difference was detected among ts and wbg ( figure 2 ) . for both femur and tibia , cg showed significantly higher ibmd , tbmd , bone csa , cortical csa , and csmi than tg and wbg ( p<0.05 ) ( table 2 ) . all pqct - measured parameters did not show significant difference between tg and wbg in either femur or tibia ( p>0.05 ) . ibmd of femur and tibia in tg and wbg showed the least percentage of decrease ( 82.2% and 90.3% of cg ) while csmi showed the greatest percentage decrease ( 43.0 - 76.2% of cgs ) . note : the percentage in the brackets represent the percentage of current assessment corresponds to cg . pqct : peripheral quantitative computed tomography ; ibmd : integral bone mineral density ( bmd ) ; tbmd : trabecular bmd ; csa : cross - sectional area ; csmi : cross - sectional moment of inertia ; number in brackets represent the percentage of the value compared to that of cg ; wbg ( 47.7% of cg , p<0.05 ) showed significantly lower muscle mass in the soleus muscle than that of cg ( figure 3 ) . tg ( 78.7% of cg , p<0.05 ) and wbg ( 72.4% of cg , p<0.05 ) showed significantly lower muscle mass in edl than that in cg ( figure 4 ) . moreover , tg ( 63.9% of cg , p<0.05 ) and wbg ( 62.4% of cg , p<0.05 ) showed significantly lower csa of tibia muscle than that of cg ( figure 5 ) . cg showed significantly higher weight than those of tg and wbg . * : p<0.05 . the ppt samples failed with an avulsion of bone at the bti complex during mechanical testing . the tensile failure strength of ppt insertion was the highest in cg ( figure 6 ) . tg ( 76.1% of cg , p<0.05 ) and wbg ( 70.6% of cg , p<0.05 ) showed significantly lower failure strength than that of cg . tensile strength of the patella - patellar tendon ( ppt ) complex of different groups . cg showed significantly higher failure strength than that of tg and wbg . * : p<0.05 . after four weeks of tail - suspension and different treatments , ts ( 76.89% of cg , p<0.05 ) , and wbg ( 69.85% of cg , p<0.05 ) showed significant decrease in weight compared with the cg . however , no significant difference was detected among ts and wbg ( figure 2 ) . for both femur and tibia , cg showed significantly higher ibmd , tbmd , bone csa , cortical csa , and csmi than tg and wbg ( p<0.05 ) ( table 2 ) . all pqct - measured parameters did not show significant difference between tg and wbg in either femur or tibia ( p>0.05 ) . ibmd of femur and tibia in tg and wbg showed the least percentage of decrease ( 82.2% and 90.3% of cg ) while csmi showed the greatest percentage decrease ( 43.0 - 76.2% of cgs ) . note : the percentage in the brackets represent the percentage of current assessment corresponds to cg . pqct : peripheral quantitative computed tomography ; ibmd : integral bone mineral density ( bmd ) ; tbmd : trabecular bmd ; csa : cross - sectional area ; csmi : cross - sectional moment of inertia ; number in brackets represent the percentage of the value compared to that of cg ; wbg ( 47.7% of cg , p<0.05 ) showed significantly lower muscle mass in the soleus muscle than that of cg ( figure 3 ) . tg ( 78.7% of cg , p<0.05 ) and wbg ( 72.4% of cg , p<0.05 ) showed significantly lower muscle mass in edl than that in cg ( figure 4 ) . moreover , tg ( 63.9% of cg , p<0.05 ) and wbg ( 62.4% of cg , p<0.05 ) showed significantly lower csa of tibia muscle than that of cg ( figure 5 ) . cg showed significantly higher weight than those of tg and wbg . * : p<0.05 . the cross - sectional area ( csa ) of muscle at the mid tibia . the ppt samples failed with an avulsion of bone at the bti complex during mechanical testing . the tensile failure strength of ppt insertion was the highest in cg ( figure 6 ) . tg ( 76.1% of cg , p<0.05 ) and wbg ( 70.6% of cg , p<0.05 ) showed significantly lower failure strength than that of cg . tensile strength of the patella - patellar tendon ( ppt ) complex of different groups . cg showed significantly higher failure strength than that of tg and wbg . * : p<0.05 . the tail - suspension disuse model demonstrated systemic deterioration of the musculoskeletal system , including bone , muscle , and bti complex after 4 weeks of tail - suspension . there were variations in musculoskeletal tissue deterioration , which depended on type of tissues for evaluation . the current study was the first to report the deterioration of the bti in the tail - suspension model . bone strength is contributed by several factors , including the quality of the bone matrix , which consists of bmd and extracellular proteins , and bone geometry , such as the bone csa . the product of bmd and bone csa is also known as the bone strength index , which is a noninvasive measurement for predicting the bone bending strength . our result demonstrated that there was a decrease in bmds , including ibmd and tbmd , and bone structural measurements , including bone csa , cortical csa , and axial csmis . this suggested that the bending strength of the bone in the hindlimbs was weakened after prolonged duration of disuse . there was a difference in the rate of bone loss between cortical bone and trabecular bone due to disuse . our results showed that tbmd had greater decrease than ibmd , a bmd measurement that includes both tbmd and cortical bone bmd . demonstrated that , bone loss occurred in the cortical compartment during the first 60 days of bed rest while trabecular bone loss might be more significantly for longer periods . vico et al . found that cortical bone loss was less significant than that of trabecular bone at the weight - bearing tibia after six months of spaceflight . in addition to bmd , the bone resorption markers , such as urinary concentration of c - telopeptide of type i collagen ( ctx - i ) and deoxypridinoline ( dpd ) , were elevated after bed rest and suggested an increase in bone resoprtion . therefore , after prolonged disuse , there is imbalance of bone formation and bone resorption . furthermore , the distribution of calcium and phosphorus in bone was affected by prolonged disuse , which might also weaken the material properties of bone . therefore , disuse affects both the structural characteristics and the material properties of bone and hence the skeletal mechanical properties . muscle atrophy is characterized by decreased muscle size , such as the muscle fiber csa and protein content , and muscle functions , such as reduced force , and increased insulin resistance . the current study showed that muscle mass and csa were decreased in the rats of tg . this muscle atrophy might be caused by the reduction in protein synthesis , which was triggered by unloading and decrease in mechanical strains imposed on the muscles . furthermore , disuse and unloading also induced alterations of muscle cell phenotype from slow fibers ( myosin heavy chain type i ) to fast fiber ( myosin heavy chain type ii ) . this changes in muscle fiber type would be most significant in soleus muscle because it was predominately composed of slow - twitch muscle fibers . the atrophic muscles were also reported to show increase in insulin resistance . in general , insulin is responsible for stimulation of muscle protein synthesis when intramuscular amino acid availability is sufficient . therefore , insulin resistance might further affect the performance and change the metabolic adaptations of the muscles . tail suspension - induced deterioration of bti was reflected by significant decrease in tensile failure strength of bti complex , with typical avulsion of bone at patellar tendon insertion in tg in rats . results from our current study and previous studies from others demonstrated that hindlimb suspension of rats induced bone and muscle atrophy . atrophy of adjacent muscles near the bti caused decrease in mechanical loading to the corresponding bti and tendon , which may contribute to the deterioration of the bti . variations in activity level might lead to differences in mechanical stimulation and loading that might also lead to large variation in the measurements . however , from the pqct result of the current study , the variations of the measurements for tg and wbg were 5 - 15% and 17% for failure strength , which were comparable to the variations of the measurements for cg ( 5 - 15% for pqct and 13% for failure strength ) . therefore , the variation in activity level might be smaller than biological variations among individual rats . soleus muscle , which consists of slow twitch fibers , would suffer more pronounced atrophy than edl after prolonged period of disuse . based on the result of the current study , soleus muscle did not show more pronounced atrophy than that of edl muscle . this can be explained by no significant difference was found between cg and tg for soleus muscle while significant difference was detected in edl muscle . even though the mean percentage changes of edl and soleus between cg and tg were comparable ( 77.6% for edl and 72.9% for soleus ) , the variation in the mass of the soleus muscle ( cv=50% ) was larger than the variation in that of the edl ( cv=16% ) . this might be due to the fact that the rats would intrinsically contract the edl for support during hindlimb suspension , which might vary the deterioration rate of the edl . riley et al . reported that eccentric contraction - like lesions appeared in hindlimb suspension unloading rats after 15 - 60 min of reloading . the atrophic fibers were more susceptible to sarcomere reloading damage as the filaments force per filament was found increased . therefore , this muscle damage might lead to further decrease in muscle weight in wbg but not in tg . weight - bearing activities are defined as activities during which the body works against the force of gravity and the feet and legs carry one s body weight . the current treatment protocol for short - duration weight - bearing exercise did not prevent the disuse - induced , systemic deterioration of the musculoskeletal system . tg and wbg did not show significant difference among each other with respect to the measured variables on the musculoskeletal system . zhang et al . reported that four hours of daily standing only partially relieved the deterioration in femur while prevented mass reduction and histomorphometric changes in the soleus muscle . reported that , for anabolic stimulation on bone , the addition of weight - bearing exercise was more effective than extending duration of the exercise . umemura et al . reported that five jumps per day was sufficient to increase bone mass and breaking force in rats . therefore , the duration of the weight - bearing exercise might not be the determining factor compared with the inclusion of weight - bearing exercises . in order to minimize the duration of the stimulation therapy , exercises that are higher intensity than weight - bearing standing maybe necessary for maintenance of the musculoskeletal system . however , typical exercises , such as swimming and jumping which are reported to prevent bone loss , may not be appropriate for the patients with prolonged bed - rest . the amount of space is limited in the icu and icu patients often attached with a wide - ranging instruments and sensors which would also limit their movement . other exercise modalities that act on the whole body may be applied to stimulate the musculoskeletal system . low - magnitude high - frequency vibration is an exercise modality that provides mechanical loading and was shown effective on different types of tissues of the musculoskeletal system , including bone , muscle , and tendon . therefore , this exercise modality might have potential for prevention of disuse - induced musculoskeletal deterioration . there were a few limitations in the current experimental study , including : 1 ) the function of quadriceps muscle was not assessed that might be related to the tensile properties of qpptt complex . the quadriceps consists of a mix of muscle fiber types and earlier studies have demonstrated that unloading and disuse caused atrophy of quadriceps muscle in human and rats . therefore , the quadriceps muscles might also be affected and led to decrease in tensile strength ppt complex . 2 ) as an important component of the joint , articular cartilage might also be affected by the microgravity or disuse that should also be evaluated in the future studies . future studies would also benefit from examining tissues , including muscle , tendon , and bone within the same region of the body ( e.g. tibia , calf muscle and achilles tendon insertion ) to observe the changes in relation to each tissue . disuse caused systemic deterioration in the musculoskeletal system , including the bone , muscle , and bti complex . the present regime for short duration of weight - bearing treatment did not show significant effect on prevention of deterioration of the musculoskeletal system resulted from prolonged disuse .

objectives : to investigate deterioration of musculoskeletal system due to prolonged disuse and the potential of daily short - duration weight - bearing as countermeasures.methods:twenty-four adult male sprague - dawley rats were divided into control group ( cg , no intervention ) , tail - suspension group ( tg , tail - suspension without treatment ) , and weight - bearing group ( wbg , tail - suspension with 20 min / day , 5 days / week body weight loading ) . after four weeks of treatment , femur and tibia , soleus and extensor digitorum longus were evaluated for bone and muscle quality respectively . tensile properties of bone - tendon insertion ( bti ) were evaluated using patella - patellar tendon complex.results:disuse induced deterioration on bone , muscle , and bti after four weeks . compared with cg , tg and wbg showed significant decrease in bone mineral density ( bmd ) of trabecular bone in distal femur ( 4.3 - 15.2% ) , muscle mass ( 31.3 - 52.3% ) , muscle cross - sectional area ( 29.1 - 35% ) , and failure strength of bti ( 23.9 - 29.4% ) . tensile test showed that the failure mode was avulsion of bone at the bti . no significant difference was detected between tg and wbg for all assessments on bone , muscle , and bti.conclusions:disuse caused deterioration of bone , muscle , and bti while daily short - duration of weight - bearing did not prevent this deterioration . mechanical stimulation with higher intensity and longer duration may be necessary to prevent musculoskeletal deterioration resulted from prolonged disuse .

Introduction Methods None Tail-suspension model in rats peripheral Quantitative Computed Tomography (pQCT) Mechanical testing for bone tendon insertion (BTI) complex Statistics Results None Body weight Evaluations on bone mineral status Evaluations on muscle mass and CSA Evaluations on bone-tendon insertion tensile property Discussion Conclusion

PMC4897452

three - fourths of the earth 's surface is covered with water . out of this three - fourths , the rest is found in the form of salt water in oceans , which can not be directly used for human consumption . the world 's population is expected to reach 8.1 billion by the end of 2025 . with depleting fresh water sources , additional water supplies will be important in determining the future of humans on this planet . like fossil fuel resources , the need for water is likely to be a major factor for world stability in the coming few years [ 3 , 4 ] . there are technological innovations being made , as well as new water sources being explored , to cater to the growing water demand around the world . if used efficiently , the concept of desalination is a very viable option for addressing the problem of water scarcity . the worldwide desalination industry is expected to grow 140 percent over the next decade , with 25 billion united states dollar ( usd ) in capital investment by 2010 and 56 billion usd by 2015 . thermal methods of desalination such as multistage flash ( msf ) and multiple - effect distillation ( med ) have been found successful in converting seawater into pure water , but the energy required for this process makes it unviable to be used on a large scale . membrane technology , such as reverse osmosis ( ro ) , suffers from membrane fouling and a high rate of brine production . therefore , research is continually being carried out in order to find out the sustainable way of desalting seawater . it is found to be highly efficient with a low rate of brine production ; fo is an area of active research . if adopted , this technique might be the answer to all of our water problems . , 9 indian states and 4 union territories are surrounded by seawater along one or more sides , desalination can play a major role in augmenting the fresh water requirements of the country if technically and economically efficient desalination technology is implemented for obtaining pure water from sea water in these regions . additionally , areas such as tamil nadu , gujarat , and andhra pradesh have a huge problem of water scarcity due to poor river water availability , low ground water levels , and high water demand . since the launch of the first five - year plan in 1951 , 1105 billion indian rupees have been spent to provide safe drinking water , yet it continues to be a major issue . thus , it is imperative that india focuses its attention on seawater in order to meet the water demands of the growing population . of all the desalination techniques available , in this paper , we focus our attention on fo . the key part of this paper is the comparison of fo with all of the other prevalent , as well as emerging , techniques in india . then we discuss the application of fo in seawater desalination along with its advantages and disadvantages . in the final part of the review , we look at the challenges of bringing fo into the indian market . due to limitations on literature on the topic , a complete comparison of the techniques on all 14 parameters india represents 16 percent of the world 's population and at its present growth rate is expected to hit the 1.5 billion mark within the next four decades . the government of india 's planning commission estimates the water demand to increase to 1180 bcm ( billion cubic meters ) by the year 2050 . this represents a nearly 66 percent increase from the known 710 bcm of water demand in 2010 . with the depleting water sources and increase in the demand , it becomes imperative to switch to seawater to meet the water requirements in india . suryanarayanan says that desalination technologies are still evolving in india and indo - asian news service report suggests that the indian desalination water market is expected to grow at a compounded annual growth rate ( cagr ) of 30 percent over a period of five years from 2013 to 2018 . there has been a sudden increase in the capacity of plants desalinating saline water from year 2006 as shown in figure 1 . in addition , the desalination capacity in india is seen at 5.35 million m / d by 2018 , which is 342.1% increase from its capacity in 2013 . thus , it can be concluded that desalination water market in india is expected to grow at a rapid rate . thermal processes include multistage flash ( msf ) , multiple - effect distillation ( med ) , and vapour compression ( vc ) , whereas membrane processes include reverse osmosis ( ro ) , electrodialysis ( ed ) , and forward osmosis ( fo ) . figure 2 shows the distribution of the desalination technologies adopted in india in the financial year 2012 - 13 . since membrane based technologies are considered to be 23 percent cheaper than thermal based technologies for generating desalinated water , plants in india are mostly membrane based . figure 3 illustrates the breakup of the desalination plants currently operating in india by capacity . it can be seen that currently desalination plants in india are mostly of sizes up to 10,000 cubic meters per day ( m / d ) . gujarat and tamil nadu have emerged as desalination hubs due to poor water availability , low ground water levels , and high demand . desalination is also used in the coastal regions of maharashtra , andhra pradesh , karnataka , and west bengal . reverse osmosis ( ro ) is a membrane separation process in which the seawater permeates through a membrane by applying a pressure larger than the osmotic pressure of the seawater . the membrane is permeable for water but not for the dissolved salts . in this way , a separation between a pure water fraction ( the product ) and a concentrated fraction ( the retentate or concentrate ) is obtained . a typical ro desalting plant consists of three sections , namely , pretreatment section , membrane section , and posttreatment section . development of ro membranes of very high rejection , while maintaining high permeability , has the potential to reduce energy consumption . bhabha atomic research centre erected desalination plants in andhra pradesh , gujarat , rajasthan , tamil nadu , port blair , and lakshadweep . bharat heavy electricals limited ( bhel ) operated 12 desalination plants using ro technology in tamil nadu . multistage flash ( msf ) starts with saline feed water being passed through a series of tubes which preheat the water . the heated water is then passed through a vessel , called the stage , with pressure lower than that of the brine heater , which results in flashing of the saline water . only a small percentage of the heated water is converted into steam , depending upon the pressure maintained at the next stage . the remaining water is then introduced to a subsequent stage , which has a lower pressure . barc built a 425 kld msf pilot plant in bombay and a 4.5 mld msf unit as a part of 6.3 mld hybrid msf - ro plants . multieffect distillation ( med ) takes place in a series of vessels or effects and uses the principle of evaporation and condensation at reduced ambient pressures . the feed water , after being preheated in the final condenser , is fed in equal proportions to the various vessels . the water is sprayed on the evaporator surface ( tubes ) after being heated to its boiling point . the steam comes from the steam turbines of a power plant , or boiler , where the steam inside condenses as the water sprayed on the evaporators vaporizes . the vapours from the first effect are used to heat the surfaces of the succeeding effects . the vapours produced in the last effect are condensed in the final condenser cooled by incoming saline feed water to yield pure water . at reliance industries ltd . ( ril ) in india , four med plants have been in operation since 1998 , each with a nominal production of 12,000 m / d . a fifth med unit of 14,400 m / d capacity was operational in february 2005 . vapour compression ( vc ) is a highly reliable and efficient process which uses mechanical energy to desalinate water . heat is pumped from a reservoir which is at a low temperature to a reservoir which is at higher temperature . the basic system consists of an evaporator - condenser and a compressor where water vapours act as a working fluid . this results in a rise of the saturation temperature and pressure , which is then condensed to form condensate releasing latent heat of condensation in the process . the latent heat is transferred across the heat transfer surface into the water layer side causing further evaporation . in solar humidification - dehumidification ( shdh ) , solar energy is used to heat sea water , which results in the humidification of air . the humidified air rises up to form clouds , which upon dehumidification pours down as rain . the man - made version of this cycle is called the humidification - dehumidification desalination cycle . the electrodialysis reversal ( edr ) process is based on the principle that most dissolved salts are positively and negatively charged and are attracted towards an electrode of the opposite charge . selective membranes allow either passage of anions or cations , leading to the separation of ions . anion selective membranes allow the passage of anions but block cations , whereas cation selective membranes allow the passage of cations but block anions . polarity of the electrode is frequently reversed to free up accumulated charge on the membrane surface , thus minimizing membrane fouling . the process of freezing desalination ( fd ) is based on the principle that the formation of ice crystals leads to the exclusion of dissolved solids . during freezing , the frozen water is rinsed to remove the remaining salts adhering to the ice crystals . ( nf ) , a pressure driven membrane separation process , the separation takes place across a semipermeable membrane . the pressure difference between the feed and filtrate side of membrane leads to the separation of salt from water . lower operation pressure , higher flux , and higher retention of multivalent anion salts provide it an advantage over ro . forward osmosis ( fo ) is a natural phenomenon where a difference in osmotic pressure drives the solvent from a region of lower concentration to a region of higher concentration across a selectively permeable membrane . the low - concentration solution is called the feed solution and the high - concentration solution is called the draw solution . feed solution is generally seawater or brackish water that needs to be desalinated , while the draw solution is prepared in a laboratory using a solute of high osmotic pressure . osmotic pressure difference between the feed and draw solution causes the water to flow into the draw solution from the feed solution . regeneration of the draw solution is the most expensive stage in the fo desalination process . nanofiltration , reverse osmosis , or other thermal processes are proposed for the regeneration of draw solution . table 1 provides a detailed comparison of the traditional techniques as well as techniques being researched for desalination . it can be seen that fo requires less energy per m of water than the other thermal and membrane techniques . solar energy is in abundance , but the technical knowledge to harness it efficiently is still primitive . nanofilters have also shown early promise but there is still time before its theoretical knowledge can be efficiently applied to desalination . stand - alone nanofilters face many problems , such as fouling , and therefore need to be incorporated with another suitable technique as a hybrid system . the cost required to construct an fo plant is considerably less than most of the other thermal technologies , even reverse osmosis . compared to ro , fo has less membrane fouling , scaling , and brine discharge . even though the product water quality varies over a wider range than the thermal methods , this is not of much importance since the dissolved solid content is well under the specified world health organization ( who ) guidelines . greenhouse gas ( ghg ) emissions are also considerably less in membrane techniques compared to the thermal techniques . however , the draw solutions and membranes in fo are still areas of active research in order to improve upon the process . additionally , though fo membranes suffer from fouling which other techniques besides membrane techniques do not , the advantages of fo outweigh its disadvantages . therefore , if efficiently used , fo can prove to be an efficient way of desalinating seawater . membrane development : an appropriate membrane should be able to reduce the effect of concentration polarization , fouling , and reverse solute diffusion . values of fo flux calculated using solution diffusion theory tend to have higher values than experimental values . this is attributed to the presence of internal and external concentration polarization which reduces the osmotic force.draw solution development : a suitable draw solution should have high osmotic pressure , be easily recoverable , and exhibit minimal internal concentration polarization . factors such as low cost , zero toxicity , and low fouling are also important.membrane fouling : membranes having low fouling are still being researched . membranes would have a longer working life leading to reduction in capital and operational costs.brine discharge : disposal of brine produced is a major issue since it causes environmental problems . however , not only is it a challenge for fo , but also it is a challenge for every other desalination technology.fragmentation : creating an industrial standard would be a problem because most of the indian market is fragmented with many regional players .bureaucratic hurdles : new technology adopted has to be approved by the government before it can be introduced into the market . slow execution and bureaucratic hurdles may prove to be time consuming and cause problems before the technology can be adopted .improper training : as the technology is new , lack of adequate training of the staff handling the plant would be a major problem for the industry . membrane development : an appropriate membrane should be able to reduce the effect of concentration polarization , fouling , and reverse solute diffusion . values of fo flux calculated using solution diffusion theory tend to have higher values than experimental values . this is attributed to the presence of internal and external concentration polarization which reduces the osmotic force . draw solution development : a suitable draw solution should have high osmotic pressure , be easily recoverable , and exhibit minimal internal concentration polarization . factors such as low cost , zero toxicity , and low fouling are also important . membranes would have a longer working life leading to reduction in capital and operational costs . brine discharge : disposal of brine produced is a major issue since it causes environmental problems . however , not only is it a challenge for fo , but also it is a challenge for every other desalination technology . fragmentation : creating an industrial standard would be a problem because most of the indian market is fragmented with many regional players . bureaucratic hurdles : new technology adopted has to be approved by the government before it can be introduced into the market . slow execution and bureaucratic hurdles may prove to be time consuming and cause problems before the technology can be adopted . improper training : as the technology is new , lack of adequate training of the staff handling the plant would be a major problem for the industry . 50 leading scientists and professionals working in the field of desalination in india were surveyed on the potential fo setup that can be viably operated in the indian market . according to the results of the survey , the fo membranes which can be easily synthesized using the existing setup in india are ( a ) cellulose triacetate membrane and ( b ) polybenzimidazole . the possible draw solutions which can be used in the indian market are ( a ) sulphur dioxide solution , ( b ) glucose solution , ( c ) ammonium carbonate solution , ( d ) sodium chloride solution , and ( e ) potassium nitrate + sulphur dioxide solution . also , for regeneration purposes , ro or any thermal process can be employed , as the technical knowhow of these processes is very good in india considering the data presented in figures 2 and 3 . it is proposed that the concentrate from the process is disposed of using any of the following three techniques independently or in combination of each other : ( a ) sewer discharge , ( b ) evaporation ponds , and ( c ) land application . the costs of desalination vary significantly depending on the size and type of the desalination plant , the source and quality of incoming feed water , the plant location , site conditions , qualified labour , energy costs , and plant lifetime . the capital cost includes the purchase cost of major equipment , auxiliary equipment , land , construction , management overheads , and contingency costs . annual running costs consist of costs for energy , labour , chemicals , consumables , and spare parts . as is evident from figure 2 , approximately 88% of the desalination plants operating in india are based on ro , and the cost analysis of ro and fo has been done . we have used the model developed by ch2 m hill ( a global environmental consulting company ) , which estimates the cost involved in setting up an fo plant and ro plant . the model has been found useful in estimating costs involved up to 15 mgd capacity of recovered water from impaired solution . the important assumptions on which cost model of both swro and fo depends are number of hours per day the plant operates24 hours , number of days per year the plant operates329 days , membrane replacement frequency6 years , number of membrane replacements in 20 years3.membrane replacement costs , cartridge filter replacement costs , labour , and chemical costs are included in the annual operation and maintenance cost in both swro and fo models . number of hours per day the plant operates24 hours , number of days per year the plant operates329 days , membrane replacement frequency6 years , number of membrane replacements in 20 years3 . table 2 shows the estimated costs involved in setting up both fo and ro plants of design capacity 1 mgd . a plant operating on fo technology can be constructed at 90% of the construction cost and operated at 80% of the operation cost of an swro plant , with the current options of fo membranes and draw solution available . extensive research is being carried out to develop fo membranes offering better flux performance and draw solutions which can be much more easily regenerated . as and when better membranes and draw solutions are available , the construction and operating cost of fo plants is expected to reduce further . most of the present desalination techniques are currently areas of active research , which is being performed to constantly find ways of improving the technologies and reducing the capital cost as well as increasing the efficiency . early signs show fo to be the most promising technique that can be used efficiently to meet india 's water demand . fo requires low capital input and results in lesser membrane fouling , and the product water is of high quality . low capital cost is going to act as an incentive , resulting in increased expenditure for the adoption of this technique . however , lack of trained personnel will prove to be a hindrance and so there is still time before this technique proves to be a major player in the indian market .

with an increase in demand of freshwater and depleting water sources , it is imperative to switch to seawater as a regular source of water supply . however , due to the high total dissolved solid content , it has to be desalinated to make it drinkable . while desalination technologies have been used for many years , mass deployment of such technologies poses a number of challenges like high energy requirements as well as high negative environmental impact through side products and co2 emissions . the purpose of this paper is to present a sustainable technology for desalination . forward osmosis , an emerging technology , is compared with the other commonly used technologies worldwide , namely , multieffect distillation , multistage flash distillation , and reverse osmosis as well as other emerging technologies like vapour compression , solar humidification dehumidification , nanofiltration , and freezing desalination . as energy consumption and associated greenhouse gas emissions are one of the major concerns of desalination , this paper concludes that forward osmosis is an emerging sustainable technology for seawater desalination . this paper then presents the challenges involved in the application of forward osmosis in india and presents a plant setup . in the end , the cost comparison of a forward osmosis and reverse osmosis plant has been done and it was concluded that forward osmosis is economically better as well .

1. Introduction 2. Desalination in India 3. Desalination Techniques 4. Comparison between the Desalination Technologies 5. Challenges in Settingup of FO Plant 6. Proposed FO Setup in India 7. Cost Analysis 8. Conclusion

PMC5421978

public health physical activity guidelines ( pag ) established in 2008 ( 1 ) are now being reviewed with proposed release in 2018 . the pag provide information about the amount and intensity of physical activity ( pa ) needed to positively influence health at all stages of life . for children and adolescents , there is evidence that pa and health - related physical fitness ( hrf ; e.g. , cardiorespiratory fitness [ crf ] and muscular strength and endurance ) are reciprocally related and indirectly influence each other . previous research has found a relationship between pa and higher levels of crf , stronger muscles and bones , and lower body fatness ( 12 , 19 ) . santos and colleagues ( 19 ) reported that youth who were classified as meeting the pag and engaged in low levels of sedentary behavior ( based on the median value for sedentary time by age and gender ) were more likely to achieve a criterion - referenced standard for crf . although previous research findings from observational and experimental studies indicates an association between pa and hrf , more information is needed regarding the exact amount of activity ( e.g. , number of days with 60 minutes of aerobic pa ) that is associated with healthier levels of hrf . thus , additional evidence on the doseresponse relationship between the amount of pa and hrf outcomes is needed ( 9 , 16 ) . the current pag for children and adolescents is 60 minutes of daily pa , which should include 3 days of musculoskeletal and bone - enhancing activities . strong and colleagues ( 23 ) provided support for this recommendation after conducting a systematic review of 850 articles . strong et al.s recommendation for 60 minutes of daily pa was largely based on the results of intervention studies with overweight and obese children that revealed 3045 minute bouts of pa on 35 days per week improved various health outcomes ( 9 ) . sixty minutes of daily pa was chosen to help account for inter- and intra - individual differences in response to pa in free - living situations ( 9 , 23 ) . strong and colleagues found strong evidence regarding the amount of activity youth needed to improve health and behavioral outcomes , but dietz ( 4 ) indicated that there were still important gaps in the literature . dietz emphasized that investigating different types ( e.g. , aerobic pa ) and doses ( e.g. , five times weekly ) of pa would likely have varying effects on adverse health outcomes . addressing these gaps is important as the pag are currently being reviewed and potentially modified . while there are different types of pa , aerobic pa was the primary focus of the current study because aerobic activities are the most common and have the broadest physiological and health effects ( 8) . previous research has indicated that aerobic pa can improve different aspects of hrf ( 14 ) , but limited information is available regarding the exact number of days of aerobic pa needed for youth to achieve healthier hrf levels and how each additional day of aerobic pa affects fitness outcomes . further investigation will help identify the actual minimum number of days needed to achieve a fitness level sufficient to accrue health benefits . ortega and colleagues ( 15 ) assessed the relationship between pa and crf and found that 60 minutes or more of daily pa was associated with higher crf levels in youth . however , the participants were placed into dichotomous groups based on those who achieved 60 minutes or more of daily pa and those who did not over a 4-day period . thus , there is currently a need for more information regarding the number of days of aerobic pa that is associated with adolescents achieving hrf . the purpose of this study was to expand on previous research using aerobic pa by investigating the association between the number of days of aerobic pa and physical fitness achievement based on the fitnessgram assessment ( i.e. , progressive aerobic capacity endurance run [ pacer ] laps , push - ups , curl - ups , trunk lift , and body mass index [ bmi ] ) . it was hypothesized that there would be a positive association between the number of days of aerobic pa and each component of hrf ( i.e. , crf , body composition , and muscular fitness ) . data were collected from 36 physical education classes at six different schools in a southwestern urban school district . this resulted in a final sample of 4448 middle school students in grades 68 . the students ranged in age from 10 to 16 years old ( mage = 12.52 , sd = 1.02 ) . approximately 55% of the sample was white , 28% was hispanic , 13% was african american , 3% was asian , and 1% was american indian , which was reflective of the demographics of the entire district . students responded to a single item for aerobic pa that was based on pa items from the fitnessgram and the cdc ( 14 ) youth risk behavior surveillance system . respondents self - reported the number of days they were physically active ( i.e. , increased heart rate and breathing hard some of the time ) for 60 minutes during the past 7 days . these activities included moderate ( e.g. , walking , slow bicycling , or outdoor play ) or vigorous ( e.g. , jogging , active games , or active sports such as basketball , tennis , or soccer ) activity . the fitnessgram assessments of crf ( i.e. , pacer ) , body composition ( i.e. , bmi ) , and muscular fitness ( i.e. , curl - ups , 90 pushups , and trunk lift ) were used to determine hrf . the pacer is a multistage test adapted from the 20-meter shuttle run ( 17 ) . participants run as long as possible across a 20-meter course at a specified pace that gets faster each minute . their vo2max is predicted based on the number of laps they completed ( 17 ) . body composition was estimated using bmi , which is an indicator of a person s weight relative to their height [ i.e. , bmi = weight ( kg ) / height ( m ) ; 17 ] . for curl - ups , participants attempted to complete up to 75 curl - ups at a specified pace ( i.e. , 1 curl for every 3 seconds ; 17 ) . they lied flat on their backs with their knees bent and arms extended with their palms on the mat . participants slid their fingers from one side of a measuring strip to the other side and then curled back down . participants completed the trunk lift by laying prone on a mat with toes pointed and hands placed under their thighs . participants then lifted their upper body off the floor in a slow , controlled manner to a maximum height of 12 inches . lastly , participants completed as many 90 pushups as possible at a rhythmic pace ( i.e. , one push - up for every three seconds ; 17 ) . participants started in a prone position and pushed off the ground until their arms were straight while also keeping their back and legs straight . they lowered their bodies until their elbows were bent at a 90 angle and then pushed off the ground again . for each test , participants were classified as either in the healthy fitness zone ( hfz ) or needs improvement zones ( niz ) based on criterion - referenced standards for gender and age ( 11 , 14 ) , which are the minimal level of fitness necessary to experience health benefits and to protect against diseases associated with sedentary living ( 17 , 18 ) . the crf and bmi assessments now use two levels for niz ( i.e. , ni and ni - health risk ; 24 ) . however , participants in either level were placed in the niz because the data were collected before the new classifications were established . written parental consent and child assent were obtained once the school district and principals at the six middle schools gave permission to conduct the study . trained research assistants assisted the physical education instructors during data collection at each school for one week to administer the fitnessgram , and students responded to the item that assessed their aerobic pa . because not all physical education teachers were trained equally to conduct the fitnessgram assessment , a certified fitnessgram administrator supervised all the testing procedures . the presence of a certified administrator has been found to improve accuracy ( 9 ) and schoolteachers have been found to record accurate results ( 13 ) . additional individual demographic information was provided by the school district including age , sex , race , and socioeconomic status ( ses ) . age was determined according to each participant s fitnessgram completion date , and they were labeled as being either white or nonwhite for race . ses level was based on federal guidelines for determining which students qualified for free or reduced lunches at the school based on family income ( www.fns.usda.gov/cnd/lunch/ ) . participants were categorized into one of the following two groups for ses : receiving a free or reduced lunch or not receiving any assistance . no other identifying information was collected , and students at each school were entered into a drawing for cash prizes as an incentive to participate . descriptive statistics included demographic information ( age , sex , race , ses , and school site ) and the prevalence of meeting the 2008 guideline for daily aerobic pa . descriptive discriminant analysis ( dda ) was conducted as the primary analysis to determine whether differences existed in the dependent variables ( i.e. , of pacer laps , push - ups , curl - ups , height of trunk lift , and bmi ) as a function of independent variable ( i.e. , the number of days with 60 minutes of self - reported aerobic pa during the past 7 days ) . two models were used to investigate this relationship , and alpha level was set at .05 . model 1 assessed the direct relationship between aerobic pa and the dependent variables , and model 2 examined the same relationship while controlling for age , sex , race , ses , and school site . these covariates were used because previous research has found evidence of these variables influencing health - related fitness ( 2 , 3 , 5 , 20 , 25 ) . secondary analysis included five chisquare tests to investigate the relationship between days of aerobic pa and healthy fitness zone ( hfz ) status for each of the five components of the fitnessgram . this included tests for aerobic capacity ( i.e. , pacer ) , body composition ( i.e. , bmi ) , and muscular fitness ( mf ; i.e. , push - up , curl - up , and trunk lift ) . data were collected from 36 physical education classes at six different schools in a southwestern urban school district . this resulted in a final sample of 4448 middle school students in grades 68 . the students ranged in age from 10 to 16 years old ( mage = 12.52 , sd = 1.02 ) . approximately 55% of the sample was white , 28% was hispanic , 13% was african american , 3% was asian , and 1% was american indian , which was reflective of the demographics of the entire district . students responded to a single item for aerobic pa that was based on pa items from the fitnessgram and the cdc ( 14 ) youth risk behavior surveillance system . respondents self - reported the number of days they were physically active ( i.e. , increased heart rate and breathing hard some of the time ) for 60 minutes during the past 7 days . these activities included moderate ( e.g. , walking , slow bicycling , or outdoor play ) or vigorous ( e.g. , jogging , active games , or active sports such as basketball , tennis , or soccer ) activity . the fitnessgram assessments of crf ( i.e. , pacer ) , body composition ( i.e. , bmi ) , and muscular fitness ( i.e. , curl - ups , 90 pushups , and trunk lift ) were used to determine hrf . the pacer is a multistage test adapted from the 20-meter shuttle run ( 17 ) . participants run as long as possible across a 20-meter course at a specified pace that gets faster each minute . their vo2max is predicted based on the number of laps they completed ( 17 ) . body composition was estimated using bmi , which is an indicator of a person s weight relative to their height [ i.e. , bmi = weight ( kg ) / height ( m ) ; 17 ] . for curl - ups , participants attempted to complete up to 75 curl - ups at a specified pace ( i.e. , 1 curl for every 3 seconds ; 17 ) . they lied flat on their backs with their knees bent and arms extended with their palms on the mat . participants slid their fingers from one side of a measuring strip to the other side and then curled back down . participants completed the trunk lift by laying prone on a mat with toes pointed and hands placed under their thighs . participants then lifted their upper body off the floor in a slow , controlled manner to a maximum height of 12 inches . lastly , participants completed as many 90 pushups as possible at a rhythmic pace ( i.e. , one push - up for every three seconds ; 17 ) . participants started in a prone position and pushed off the ground until their arms were straight while also keeping their back and legs straight . they lowered their bodies until their elbows were bent at a 90 angle and then pushed off the ground again . for each test , participants were classified as either in the healthy fitness zone ( hfz ) or needs improvement zones ( niz ) based on criterion - referenced standards for gender and age ( 11 , 14 ) , which are the minimal level of fitness necessary to experience health benefits and to protect against diseases associated with sedentary living ( 17 , 18 ) . the crf and bmi assessments now use two levels for niz ( i.e. , ni and ni - health risk ; 24 ) . however , participants in either level were placed in the niz because the data were collected before the new classifications were established . written parental consent and child assent were obtained once the school district and principals at the six middle schools gave permission to conduct the study . trained research assistants assisted the physical education instructors during data collection at each school for one week to administer the fitnessgram , and students responded to the item that assessed their aerobic pa . because not all physical education teachers were trained equally to conduct the fitnessgram assessment , a certified fitnessgram administrator supervised all the testing procedures . the presence of a certified administrator has been found to improve accuracy ( 9 ) and schoolteachers have been found to record accurate results ( 13 ) . additional individual demographic information was provided by the school district including age , sex , race , and socioeconomic status ( ses ) . age was determined according to each participant s fitnessgram completion date , and they were labeled as being either white or nonwhite for race . ses level was based on federal guidelines for determining which students qualified for free or reduced lunches at the school based on family income ( www.fns.usda.gov/cnd/lunch/ ) . participants were categorized into one of the following two groups for ses : receiving a free or reduced lunch or not receiving any assistance . no other identifying information was collected , and students at each school were entered into a drawing for cash prizes as an incentive to participate . descriptive statistics included demographic information ( age , sex , race , ses , and school site ) and the prevalence of meeting the 2008 guideline for daily aerobic pa . descriptive discriminant analysis ( dda ) was conducted as the primary analysis to determine whether differences existed in the dependent variables ( i.e. , of pacer laps , push - ups , curl - ups , height of trunk lift , and bmi ) as a function of independent variable ( i.e. , the number of days with 60 minutes of self - reported aerobic pa during the past 7 days ) . two models were used to investigate this relationship , and alpha level was set at .05 . model 1 assessed the direct relationship between aerobic pa and the dependent variables , and model 2 examined the same relationship while controlling for age , sex , race , ses , and school site . these covariates were used because previous research has found evidence of these variables influencing health - related fitness ( 2 , 3 , 5 , 20 , 25 ) . secondary analysis included five chisquare tests to investigate the relationship between days of aerobic pa and healthy fitness zone ( hfz ) status for each of the five components of the fitnessgram . this included tests for aerobic capacity ( i.e. , pacer ) , body composition ( i.e. , bmi ) , and muscular fitness ( mf ; i.e. , push - up , curl - up , and trunk lift ) . demographic and descriptive data ( i.e. , meeting the guidelines for aerobic pa , sex , race , ses , and school site ) are presented in table 1 . means and standard deviations for each component of the fitnessgram are presented in table 2 . to estimate the effect of being more physically active , an effect size was calculated comparing 7 days to 0 days for each fitness variable ( table 2 ) . the effect sizes for pacer , push - ups , curl - ups , and trunk lift ranged from 0.330.59 , whereas the effect size for bmi was 0.10 . dda ( n = 4329 ) was conducted to determine if differences existed among the number of days that students reported 60 minutes of aerobic pa ( 0 through 7 days ) and a created composite variable for physical fitness ( i.e. , number of pacer laps , push - ups , curl - ups , height of trunk lift , and bmi ) . similar to manova , the composite variable created in dda maximizes group differences and is analogous to predicted scores in regression ( 22 ) . only results for model 1 are presented here because model 2 ( n = 3235 ) , controlling for age , sex , race , ses , and school site , explained only 1% of additional variance and the interpreted results did not change . the first step for a dda was to determine if the sample data met the assumption for homogeneity of variance / covariance using log determinants ( 7 ) . the greatest difference between log determinants was .87 , indicating that the assumption was met . the second step was to determine how many of the discriminant functions were worth interpreting . only the full model test of functions 1 to 5 was statistically significant , wilks lambda = .94 , (35 ) = 270.61 , p < .01 , and the squared canonical correlation ( i.e. , variance accounted for effect size ) between the grouping variable and the composite was .06 . to determine which variables contributed to group differences , squared structure coefficients ( rs2 ; i.e. , squared correlation between each dependent variable and the composite variable ) and standardized coefficients ( i.e. , relative importance of each dependent variable ) were examined , which are in table 3 . group differences were primarily due to the number of pacer laps ( rs2=.72 ) , push - ups ( rs2=.45 ) , curl - ups ( rs2=.36 ) , and trunk lift ( rs2=.21 ) . bmi explained only 4% of the variance in the composite variable and did not contribute to group differences on the composite . however , both push - ups and curl - ups had lower standardized coefficients compared to their respective structure coefficients , indicating that some of the variance in the composite was being credited to at least one of the other dependent variables . there was a statistically significant ( p < .01 ) correlation between push - ups and pacer laps ( r = .57 ) , curl - ups ( r = .48 ) , and bmi ( r = .32 ) . the standardized coefficient for pacer was also lower than its structure coefficient , but its standardized coefficient was not affected as much because pacer was more strongly related with the composite than the other variables . furthermore , bmi had a low standardized coefficient because the little variance that bmi shared with the composite was also shared with the other dependent variables . this was indicated by the significant correlation between bmi and pacer laps ( r = .35 ) , curl - ups ( r = .22 ) , and push - ups ( r = .32 ) . the group centroids ( i.e. , mean of each group on the composite ) were examined to determine if there was a statistically significant difference among the groups ( table 4 ) . the group centroids for 5 , 6 , and 7 days of aerobic pa were not significantly different from each other ( p .11 ) , but these three group centroids were significantly higher than the group centroids for 4 days ( p < .01 ; cohen s d = .18 to .29 ) . the group centroid for 4 days of aerobic pa was significantly higher than those for 2 days ( p < .01 ; cohen s d = .27 to .48 ) , but it was not significantly different from 3 days ( p < .09 ) . three days was significantly higher than the group centroids for 2 days ( p = .01 ; cohen s d = .16 to .38 ) and lower than the group centroids for 5 days ( p < .01 ; cohen s d = .29 to .40 ) . two days was not significantly different than 1 day , and 0 days had a significantly lower group centroid than all the other groups ( p < .01 ; cohen s d = .21 to .76 ) . since the group centroids for 5 , 6 , and 7 days of aerobic pa were not significantly different from each other , the percentage of students who reported at least 5 days of aerobic pa and less than 5 days of aerobic pa was also reported in table 1 to compare with the percentage of students who met the 2008 pag . specifically , 41.8% of students reported at least 5 days of aerobic pa , whereas only 16.8% of students reported 60 minutes of daily aerobic pa lastly , five chi - square tests of independence were conducted to examine the relation between the number of days of aerobic pa and hfz status ( i.e. , being in either the hfz or niz ) for each component of the fitnessgram . these tests were performed because theoretically one group could have significantly lower fitness scores on a component than another group while a similar percentage in both groups could be in the hfz . the results indicated that the relationship between aerobic pa and pacer laps , [ (7 , n = 4426 ) = 146.93 , p < .01 ] , push - ups , [ (7 , n = 4423 ) = 95.97 , p < .01 ] , curl - ups [ (7 , n = 4416 ) = 60.26 , p < .01 ] , trunk lift [ (7 , n = 4431 ) = 46.35 p < .01 ] , and bmi [ (7 , n = 4448 ) = 40.76 , p < .01 ] was statistically significant . the percentage of students in hfz for each component can be found in table 5 . adolescents in the current study who reported 5 , 6 , or 7 days with 60 minutes of aerobic pa per week had similar scores on measures of hrf . in addition , those who self - reported 4 days of aerobic pa generally had lower fitness scores than those with 5 days , and those with 0 days had the lowest fitness scores . these differences also existed when the control variables were used , which indicated that individual differences due to sex , age , race , receiving a reduced lunch ( i.e. , low ses ) , or school site had a small impact on fitness scores . except for the trunk lift test , the chi - square analysis indicated that the highest percentages of students in the hfz reported at least 5 days of aerobic pa . generally , the percentage of students achieving the hfz presented in table 4 illustrates a linear increase in performance for each fitness variable across the days of reported pa with a plateauing at 5 days . overall , the 2008 pag were supported because a large percentage of students achieving 60 minutes of daily aerobic pa were in the hrf , but 7 days was not necessarily better than 5 or 6 days . to determine the minimum and optimal amount of aerobic pa , dose - response studies are useful for understanding the relation between pa and health outcomes , such as hrf ( 9 ) however , this relationship has not been thoroughly investigated in children and adolescents ( 16 ) . janssen ( 9 ) argued that this lack of research has hampered the development of pa guidelines because existing pa recommendations for children and youth are not based on adequate evidence . as a result , it is difficult to set a range of minimal and maximal pa for youth . based on the findings of the present study , a positive association was found between days of aerobic pa and hrf , but students who reported 5 or 6 days with 60 minutes of aerobic pa did not have better scores on the fitnessgram assessments than those who reported 7 days . janssen and leblanc conducted a systematic review of 86 studies that investigated the relation between physical activity , fitness , and health in school - aged children and youth ( 10 ) . they reported that an average of 30 minutes per day of pa lead to some health benefits , whereas additional health benefits were generally achieved at the higher end of the pa spectrum ( 10 ) . thus , those who achieved 5 days of 60 minutes of aerobic pa in the current study were still reporting more pa than 30 minutes per day . in addition , it is important to recognize that the type and dose of pa needed to improve different health outcomes might vary ( 4 ) . for example , dietz ( 4 ) emphasized that the amount of pa needed to improve health outcomes for cardiovascular disease may differ from that for impacting obesity . thus , while the current results indicate that 5 days of 60 minutes of aerobic pa is sufficient to improve hrf compared to < 5 days , more information is still needed for hrf and other health outcomes . first , possible confounding variables were controlled in the dda analysis , including sex , age , race , ses , and school site . similar results were found when no covariates were included , which strengthened the finding that at least 5 days of aerobic pa were needed for children to have a statistically significant impact on their overall fitness . second , the fitness measures from the fitnessgram have previously been found to be valid and reliable ( 10 , 14 ) . third , the demographics of the sample improved the generalizability of the results to the student population in the district . in addition , the sample was comparable to a national sample from the cdc in regards to the number of days of 60 minutes of pa per week ( 6 ) . despite the strengths of this study , there are limitations . first , the data analysis and the cross - sectional design of the study does not allow for causal relationships to be determined . second , social desirability bias can influence self - report data ( e.g. , wanting to appear to be more physically active ) . to limit the influence of social desirability , pa items were completed after the fitness testing and answered on the reverse side of participants fitness results cards to prevent other students from seeing their data . third , there has been limited research using a single - item pa measure with youth . however , a recent study by scott and colleagues ( 21 ) investigated the reliability and validity of a single - item pa measure with adolescents . similar to the current study , their pa item assessed the number of days that the participants reported at least 60 minutes of pa over the past 7 days . their findings indicated that the single - item measure had similar test - retest reliability and concurrent validity against accelerometers as another existing pa measure ( 21 ) . thus , while the current item does not measure all aspects of pa ( e.g. , intensity and time of day ) , there is evidence that a single - item measure of pa can be used in adolescent populations , especially when recruiting large sample sizes or dealing with budgetary restrictions . lastly , while the survey item was intended to measure aerobic pa , it is possible that some activities may include both aerobic and muscle - strengthening activities ( e.g. , climbing and gymnastics ) . the 2008 physical activity guidelines ( 1 , 13 ) also suggest children and youth engage in muscle - strengthening and bone - strengthening activities at least 3 days per week . future research would benefit from investigating the role of both muscle- and bone - strengthening exercises along with aerobic pa . although these activities are not always mutually exclusive ( e.g. , running is an aerobic and bone - strengthening activity ) , these three types of pa account for the recommended 60 minutes of daily pa . in addition , more experimental research with youth is needed to determine the causal relationship between the number of days with 60 minutes of aerobic pa and hrf outcomes . experimental research would provide additional dose - response evidence regarding the amount of pa that youth need to improve hrf . finally , it may also be beneficial to consider the role of sedentary behavior during adolescence ( e.g. , watching tv and using smartphones ) and whether decreasing time spent in sedentary behaviors can lead to improvements in hrf in adolescents . this study adds to the literature on dose - response studies in important ways , particularly as decisions are made regarding modifications in the current pag . it represents one of the few studies to investigate the relationship between aerobic pa and achieving a criterion fitness standard . limited research has investigated the dose - response relationship with children and adolescents , which signifies a need for identifying the minimal pa targets for children and adolescents on specific health outcomes , such as hrf . importantly , results illustrate that the current daily pa recommendation is sufficient but not necessary to achieve health - related fitness . this has implications for the upcoming review and possible modification of the public health guidelines for americans . this study fulfills the united states department of health and human services request for additional research on the 2008 pag and provides initial evidence that the impact of aerobic pa on health - related fitness plateaus at 5 days per week .

public health guidelines for physical activity ( pa ) behaviors are being updated with 2018 as a proposed release date . currently , 60 minutes of daily pa are recommended for youth . thus , the purpose of this study was to investigate the association between reported days of aerobic pa and health - related fitness ( hrf ) . participants included 4448 students in grades 68 . self - reported days of 60 minutes of aerobic pa was obtained . hrf was assessed with fitnessgram . discriminant analysis indicated that weekly days of aerobic pa related to hrf . adolescents who participated in 5 days of weekly aerobic pa generally had better fitness results than those with 4 days . chi - square analyses indicated the highest percentages of adolescents in the fitnessgram healthy fitness zone ( hfz ) for each test item were those with 5 days of aerobic pa . these findings provided initial support that the impact of aerobic pa on hrf plateaus at 5 days per week . thus , adolescents may be able to improve hrf even if they do not meet the currently recommended guidelines for daily pa .

INTRODUCTION METHODS Participants Protocol Statistical Analysis RESULTS DISCUSSION

PMC4540883

late - stage diabetes is always associated with multiple chronic complications that lead to extremely high rates of mortality and disability . the disease has recently been regarded as a chronic low - grade inflammatory disease whose onset is characterized with persistent low - grade inflammation1 . the latest studies confirm the significant elevation of tumor necrosis factor - alpha ( tnf- ) , interleukin ( il)-6 , c - reactive protein ( crp ) , etc . in diabetes2 , 3 . in addition , diabetes patients exhibit abnormal expression of adipokines such as leptin , resistin , and adiponectin , indicating chronic low - grade inflammation3 . furthermore , inflammatory factors involved in the genesis of insulin resistance ( ir ) also contribute to the pathogenesis and progression of diabetes . those findings strongly indicate a close association between low - grade inflammations and type 2 diabetes mellitus ( t2 dm ) . regular exercise improves glycolipid metabolism and ir , thereby reducing diabetes - associated complications . exercise can reduce systemic low - grade inflammation and thus effectively improve chronic inflammatory diseases such as obesity , metabolic syndrome , diabetes mellitus , ageing , etc.5 , 6 . it can decrease the levels of inflammatory factors such as tnf- , il-6 , crp7 , 8 , and leptin9 while increasing the levels of anti - inflammatory factors such as il-4 , il-10 , and adiponectin in diabetes patients10 , 11 . exercise also reduces the inflammation level and improves ir by enhancing anti - stress and anti - oxidative effects . nevertheless , the anti - inflammatory mechanism induced by exercise is complex and incompletely understood . toll - like receptor 4 ( tlr4 ) is a major member of tlr family , which acts as a core mediator of innate immunity . tlr4 is an important receptor for the chronic inflammatory response because it is closely related to the inflammatory response . it substantially increases the expressions of inflammatory factors such as tnf- , il-6 , and il-1 by regulating the activities of transcription factors such as nf-b12 ; this creates a persistent low - level chronic inflammatory response and ir in vivo . under oxidative stress , nf-b also activates its inhibitor and increases the serine phosphorylation of insulin receptors and insulin receptor substrate , leading to ir13 . thus , tlr4 and nf-b play pivotal roles in generating an inflammatory response in vivo . therefore , they might play critical roles in the pathogenesis and progression of diabetes . accordingly , the present study investigated the long - term effects of a combination of aerobic and resistance exercises for 12 weeks on tlr4 , nf-b , and inflammatory factors in diabetes patients . thus , we aimed to provide a theoretical basis to encourage exercise therapy for patients with diabetes . from march 2012 to march 2014 , 42 patients with non - insulin - dependent t2 dm were selected from among the outpatients visiting the department of cardiovascular rehabilitation , xiangya hospital , central south university , changsha , china . the patients were randomized to the conventional therapy group ( ct group , n = 20 ) or the intensive therapy group ( it group , n = 22 ) . the ct group received regular drug treatment and diet guidance , while the it group additionally performed aerobic and resistance exercises for 12 weeks . the inclusion criteria were as follows : ( 1 ) meeting the diagnostic criteria proposed by the world health organization in 1999 , patients who presented diabetes symptoms , serum glucose level 11.1 mmol / l when detected randomly , fasting blood glucose 7.0 mmol / l , and oral glucose tolerance test ( ogtt ) showing a 2-hour postprandial blood glucose ( pbg ) 11.1 mmol / l ; ( 2 ) no severe complications such as acute infectious diseases , numbness of limbs , decreased visual acuity , and diabetic nephropathy , retinopathy , or foot ; ( 3 ) no severe cardiovascular or cerebrovascular diseases such as severe arrhythmia , or acute myocardial infarction or stroke , or severe joint disease leading to poor exercise tolerance ; and ( 4 ) no frequent exercise ( exercise frequency less than twice per week for less than 15 minutes ) . the study procedures were approved by the institutional review board of central south university , changsha , china . cardiopulmonary exercise testing ( cpet ) was performed on all participants before the intervention in a laboratory with 5055% humidity and at a temperature of 2425c . the maximum oxygen consumption ( vo2max ) was defined as the highest value of oxygen consumption measured during the exercise period . using an incremental and symptom - limited protocol , participants made their efforts to their maximal capacity on a cycle ergometer ( schiller cs-200 , switzerland ) . during the exercise , the initial workload was 1020 w , and then increased by 1020 w / min . finally , the test was terminated when any of the following symptoms occured , such as fatigue , dyspnea , angina , adverse changes in blood pressure , musculoskeletal complaint , and electrocardiographic evidence of ischemia or arrhythmia . participants were also asked to self report the intensity of exercise by the borg scale of rate of perceived exertion ( rpe)14 . all the subjects in the it group received a combined home - based exercise program and supervised exercise training at the department of cardiovascular rehabilitation , xiangya hospital , central south university . these subjects took part in at least 310 supervised training sessions at hospital at the beginning of the program . each subject received in - person instructions on how to exercise at a moderate - intensity level , how to monitor heart rate , how to use the resistant band , and how to acquire an individualized home - based exercise program . the intervention was gradually shifted to home - based exercise for the rest time of total 12 weeks , as the subjects were familiar with the whole exercise procedure . the exercise program mainly consisted of the aerobic exercise and the resistance training . aerobic exercise was conducted for a total of 4060 minutes per session , 3 sessions per week . the exercise intensity for each patient set at 4060% of his / her vo2 max . as the weeks progressed , the exercise intensity was increased to 60%vo2max , while the duration was increased to 30 minutes over the period of 1 month . the aerobic exercise session generally consisted of a 10-minute warm - up period and a 15-minute cool - down period . rpe ( 1213 on the borg scale ) was also used to assist in estimating the moderate exercsie intensity16 . the patients also performed a moderate - intensity ( i.e. , 5060% of the one repetition maximum , 1rm ) resistance training program 23 times per week . the subjects performed all resistance exercises with 810 repetitions in two sets by using an elastic band ( thera - band ; hygenic corporation , akron , oh , usa ) . resistance training intensity commenced at 50% of 1rm and increased to 60% in the fourth week of the program . in addition , the rpe ( 1114 on the borg scale ) was also used to monitor the training intensity . the patients were instructed to avoid the valsalva maneuver during the resistance training program . in addition to the training , routine diet guidance and psychological counseling were provided . all the subjects had to visit the hospital to make sure the right home - based exercise program every month . furthermore , the telephone consultation was applied to enhance the compliance of these subjects and check out the individual feedback during the exercise procedure . the telephone consultation lasted for 510 minutes and was offered once a week for 3 months18 . the ogtt was performed before and after the intervention , and the results were recorded in detail . plasma glucose concentrations were analyzed by glucose oxidase method , and insulin levels were measured using ria kit ( beijing north institute , beijing , china ) . serum total cholesterol ( tc ) , low - density lipoprotein ( ldl ) , high density lipoprotein ( hdl ) , and triglyceride ( tg ) concentrations were determined by using a cobas fara robot ( roche , switzerland ) . insulin resistance index ( iri ) was calculated using the method of homeostatic model assessment ( homa)19 . venous blood ( 15 ml ) was drawn from each participant and subjected to ficoll - hypaque density gradient centrifugation in order to separate mononuclear cells ; 5 ml venous blood was used for quantitative polymerase chain reaction ( qpcr ) to detect the expression levels of tlr4 and nf-b mrna , while the remaining 10 ml was used to detect the expression levels of tlr4 and nf-b proteins . sera were analyzed by elisa to determine the expression levels of il-18 , il-33 , ptx3 , and ykl40 . the mononuclear cells were treated with lysis buffer , and 30 g total protein was loaded and separated on a 10% sds - page gel before transfer to a nitrocellulose membrane . -actin was used as a loading control . after incubating with the recommended horseradish peroxidase secondary antibodies , chemiluminescence fluorescence image analysis system was used to scan fluorescence images and various optical density value ( od value ) was calculated by bandscan image analysis software . the relative expression of target genes was determined by the the ratio of od value of the target genes to that of internal reference gene . total rna was extracted from the mononuclear cells using trizol reagent ( invitrogen , waltham , ma , usa ) according to the manufacturer s instructions . the expressions of tlr4 and nf-bp65 mrna were detected by sybr green qpcr assay ( bio - rad , hercules , ca , usa ) . the specic primers were as follows : tlr4 forward : 5- tgggtaaggaatgagctagtaaa -3 , reverse:5- gctgggacaccacaacaa -3 ; -actin forward : 5- catcctgcgtctggacctgg-3 , reverse : 5-taatgtcacgcacgatttcc-3 ; nf-bp65 forward:5- -cccatcgggttcccataaag-3 , reverse : 5-gcctgaagcaaatgttggcgta-3. all reactions were performed in triplicate . the biochemical analyses of serum il-18 , il-33 , ptx3 , and ykl40 were performed by elisa ( abcam , ma , usa ) ; human il-18 , il-33 , ptx3 , and ykl40 were detected quantitatively . all factors were quantified using polyclonal antibodies that recognized native human il-18 , il-33 , ptx3 , or ykl40 and a series of plates containing wells coated with predetermined amounts of recombinant human il-18 , il-33 , ptx3 , or ykl40 . continuous variables are expressed as mean standard deviation and were compared using the student - newman - keuls test . the insulin resistance index ( iri ) showed a skewed distribution and a natural logarithmic transformation was performed befor statistical analysis . the diabetes groups did not differ significantly from the normal control group with respect to age ( 52.59 11.43 vs. 51.20 11.34 years ) or gender ( f30/m12 vs. f13/m7 ) . compared to the ct group , fasting blood glucose ( fbg ) , postprandial blood glucose ( pbg ) , fasting plasma insulin ( fin ) , postprandial plasma insulin ( pin ) , hba1c , ln homa - ir , tg , tc , hdl , and ldl levels were not significantly different between the ct and it groups ( all p > 0.05 , table 1table 1.changes of serum glucose , insulin , homa - ir , and blood lipidsvariablesct group ( n = 20 ) it group ( n = 22 ) control group ( n = 20)pre - treatmentpost - treatmentpre - treatmentpost - treatmentfbg ( mmol / l)7.14 1.976.37 1.40**6.84 1.425.78 0.48**5.65 0.41pbg ( mmol / l)11.08 2.639.00 1.91**11.30 2.177.30 0.986.26 1.04fin ( u / ml)12.81 6.1611.65 5.2613.40 5.119.04 3.19**9.19 3.73pin ( u / ml)94.54 46.0569.67 31.30**83.66 30.7536.16 18.2634.50 11.74hba1c ( % ) 6.88 0.766.44 0.65**6.85 1.145.89 0.615.73 0.31homa - ir4.23 2.993.32 1.734.11 1.982.33 0.922.30 0.94ln homa - ir1.26 0.601.08 0.501.33 0.400.77 0.400.74 0.43tg ( mmol / l)1.99 1.071.75 0.762.20 1.271.17 0.35 * * 1.13 0.31tc ( mmol / l)4.62 1.314.45 1.125.53 1.654.77 1.03 * 3.91 0.56hdl ( mmol / l)1.27 0.311.31 0.301.38 0.291.43 0.261.38 0.19ldl ( mmol / l)2.58 1.002.46 1.023.22 1.472.54 1.22 p < 0.05 , * * p < 0.01 vs. pre - treatment ; p < 0.05 , p < 0.01 vs. conventional therapy group . fbg : fasting blood glucose ; fin : fasting insulin ; hba1c : glycated hemoglobin ; homa - ir : the index for homeostasis model assessment of insulin resistance ; ldl : low - density lipoprotein ; pbg : postprandial blood glucose ( 2-h ) ; pin : postprandial insulin ( 2-h ) ; tc : total cholesterol ; tg : triglycerides ) . data are mean sd . * p < 0.05 , * * p < 0.01 vs. pre - treatment ; p < 0.05 , p < 0.01 vs. conventional therapy group . fbg : fasting blood glucose ; fin : fasting insulin ; hba1c : glycated hemoglobin ; homa - ir : the index for homeostasis model assessment of insulin resistance ; ldl : low - density lipoprotein ; pbg : postprandial blood glucose ( 2-h ) ; pin : postprandial insulin ( 2-h ) ; tc : total cholesterol ; tg : triglycerides in the ct group , fbg , pbg , pin , and hba1c decreased significantly after treatment ( p < 0.05 , < 0.01 ) . meanwhile , fbg , pbg , fin , pin , hba1c , and ln homa - ir decreased significantly in the it group , ( all p < 0.01 ) . pbg , pin , hba1c , and ln homa - ir were significantly lower in the it group than the ct group ( p < 0.01 , < 0.05 , table 1 ) . before treatment , the mrna and protein expression levels of tlr4 and nf-bp65 , and serum il-18 level were significantly higher in the ct and it groups than the control group , while il-33 level were significantly lower in the ct and it groups ( p < 0.05 , < 0.01 ) . after the exercise intervention , the mrna and protein expressions of tlr4 and nf-bp65 were significantly lower in the it group than the ct group ( p < 0.05 , < 0.01 ) . the melting and amplification curves of tlr4 and nf-b mrna detected by qpcr are shown in fig . serum il-18 level was significantly lower in the it group than the ct group ( p < 0.05 ) . meanwhile , the serum il-33 level was significantly higher in the it group than the ct group but lower than that in the control group ( p < 0.05 ) . there was no significant difference in the expression levels of ptx3 or ykl40 among the three groups ( table 2table 2.effects of exercise intervention on the inflammatory factors in diabetes patientsitemsct group ( n = 20)it group ( n = 22)control group ( n = 20)pre - treatmentpost - treatmentpre - treatmentpost - treatmenttlr4 mrna1.68 0.29**0.77 0.181.85 0.31**0.54 0.63**1.05 0.18nf-bp65mrna1.28 0.26**1.01 0.111.41 1.77**0.86 0.660.86 0.12tlr4 protein0.95 0.34**0.67 0.33 * 0.86 0.25**0.41 0.160 . . 28 0.090.27 0.11il-180.80 0.12**0.72 0.12 * 0.81 0.09**0.51 0.100.59 0.31ptx30.63 0.130.63 0.140.64 0.150.58 0.080.54 0.78il-330.40 0.09**0.65 0.12 * 0.42 0.14**0.85 0.14 * 1.38 0.19data are mean sd , * p < 0.05 , * * p < 0.01 vs. control group ; p < 0.05 , p < 0.01 vs. conventional therapy group . ct group : the conventional therapy group ; it group : the intensive therapy group ; il-18 : interleukin-18 ; il-33 : interleukin-33 ; nf-bp65 : nuclear factor-bp65 ; ptx3 : pentraxin - related protein 3 ; tlr4 : toll - like receptor 4 ; ykl40 : human cartilage glycoprotein 39 and fig . 2fig . 2.changes in the protein expressions of tlr4 and nf-bp65 before and after the intervention . a : control group ; b : conventional therapy group before intervention ; c : conventional therapy group after intervention ; d : intensive - therapy group before intervention ; e : intensive therapy group after intervention . nf-bp65 : nuclear factor-bp65 ; lr4 : toll - like receptor 4 ) . melting and amplification curves of tlr4 mrna and nf-b mrna detected by qpcr . : toll - like receptor 4 data are mean sd , * p < 0.05 , * * p < 0.01 vs. control group ; p < 0.05 , p < 0.01 vs. conventional therapy group . ct group : the conventional therapy group ; it group : the intensive therapy group ; il-18 : interleukin-18 ; il-33 : interleukin-33 ; nf-bp65 : nuclear factor-bp65 ; ptx3 : pentraxin - related protein 3 ; tlr4 : toll - like receptor 4 ; ykl40 : human cartilage glycoprotein 39 changes in the protein expressions of tlr4 and nf-bp65 before and after the intervention . a : control group ; b : conventional therapy group before intervention ; c : conventional therapy group after intervention ; d : intensive - therapy group before intervention ; e : intensive therapy group after intervention . many studies show aerobic exercise including home - based exercise decreases blood glucose , lipid , and hba1c levels as well as improves insulin sensitivity , hypercoagulability state , quality of life , and physical condition in t2 dm patients20,21,22,23,24,25 . in the present study , t2 dm patients performed a combination of aerobic exercises and resistance training for 12 weeks . the results show significant improvements in the fasting and postprandial blood glucose , hba1c , tg , tc , ldl cholesterol , and ir in the intensive - therapy group after 12 weeks of therapy compared to conventional therapy . balducci et al.26 report that compared to conventional therapies , a 1 year exercise intervention significantly reduced the blood glucose , tg , tc , and ldl cholesterol levels of patients with t2 dm while significantly increasing hdl cholesterol level . tokmakidis et al.27 report that a 4-month exercise intervention significantly reduced the fasting and ( 2-hour ) postprandial blood glucose , insulin , and hba1c levels of diabetes patients . thus , the present results are concordant with those of balducci et al . and tokmakidis et al . among diabetes patients , regular exercise has a better blood glucose - controlling effect , because it improves insulin sensitivity . moreover , regular exercise enhances blood pressure and blood lipid control , thus reducing the risks of large vascular and microvascular complications . despite the attention of many researchers for a long time , the latest studies indicate a close association between the pathogenesis of diabetes mellitus and a chronic low - grade inflammatory response . in the present study , the mrna and protein expression levels of tlr4 and nf-b in mononuclear cells as well as serum levels of the inflammatory factor il-18 were higher in t2 dm patients than healthy controls . however , the expression level of the protective cytokine il-33 was lower in t2 dm patients than control group . meanwhile , the serum inflammatory factors ptx3 and ykl40 did not differ significantly between t2 dm patients and controls . these results indicate tlr4 , nf-b , il-18 , and il-33 may play important roles in the pathogenesis of diabetes mellitus . previous studies report that patients with diabetes mellitus have higher tlr4 mrna and protein expression in mononuclear cells and that these changes are closely associated with blood glucose and hba1c levels28 . nf-b subsequently activates a series of inflammatory factors including tnf- , il-1 , il-6 , and il-8 and induces the expressions of several adherence factors and chemokines by mononuclear macrophages and endothelial cells30 . this cascade ultimately results in ir and secretary dysfunction of insulin in the body . il-18 is mainly secreted by mononuclear macrophages and may be important for generating an inflammatory response31 . some studies report diabetes patients have elevated plasma il-18 expression , which is closely associated with fasting blood glucose and hba1c levels32 . in addition , elevated il-18 expression is reported to be an independent risk factor for the development of diabetes mellitus33 . il-33 , a member of the il-1 cytokine family , is released after cell damage or as a negative regulator of nf-b gene transcription ; it not only enhances cytokine production by t helper cells , but also reduces the activities of natural killer cells and the generation of oxidation products ; therefore , it is recognized as a protective factor against ir , diabetes mellitus , and obesity . elevated il-33 expression improves fasting blood glucose and glucose tolerance34 , 35 . on the contrary , the removal of il-33 may increase the cytotoxicity of natural killer cells and trigger inflammatory factors such as tnf- , possibly resulting in ir36 . although some studies show ptx3 and ykl40 are associated with the onset and progression of diabetes37 , 38 , we found no significant increases of their expression levels . this discrepancy is probably due to the variation in the duration and type of disease of our patients . the present results indicate diabetes patients exhibit increased expressions of some inflammatory factors and decreased expressions of protective cytokines , indicating a close relationship between diabetes mellitus and a chronic low - grade inflammatory response . long - term regular exercise can reduce chronic low - level inflammation in humans , and an exercise - containing lifestyle may also delay the genesis and development of t2dm3 , 39 . there was research also demontrated that the home - based exercise improved the metabolic asset and reduced the systemic inflammation of sedentary people40 . another study proved that home - based exercise could improve clinical features and systemic inflammation of patients with chronic obstructive pulmonary disease41 . in the present study , the intensive intervention program for diabetes patients included a 12-week moderate - intensity workout program consisting of aerobic exercise and resistance training ( i.e. , the it group ) . after exercise training , the it group had significantly lower tlr4 and nf-b mrna and protein expressions as well as serum il-18 levels than the ct group . meanwhile , the serum level of il-33 ( a protective cytokine ) was significantly higher in the it group than the ct group but lower than that in the control group . exercise can significantly reduce tlr4 expression on the surface of mononuclear cells in non - diabetes patients42 , 43 . lambert et al . also report a similar reduction in elderly obese patients following long - term exercise therapy44 . other studies also demonstrate that exercise reduces il-18 levels in elderly people and patients with metabolic syndrome45 , 46 . furthermore , regular exercise increases the expressions of heat shock proteins and il-33 by vascular endothelial cells . the present study suggests the anti - inflammatory mechanism of long - term medium - intensity exercise may be associated with decreased mrna and protein expressions of tlr4 and nf-b , and the inflammatory factor il-18 as well as elevated levels of the anti - inflammatory factor il-33 , all of which improve ir and the outcome of diabetes . as the sample size was relatively small , our results may be affected by some biases . moreover , the mechanism by which exercise intervention alters the gene expressions of inflammatory factors remains unclear . therefore , further animal and cellular experiments must be conducted to clarify the underlying mechanism of exercise intervention . in conclusion , the present study demonstrates the existence of low - grade chronic inflammation in diabetes patients and that long - term medium - intensity exercise can not only decrease the mrna and protein expressions of tlr4 and nf-b , but can also reduce the level of the inflammatory factor il-18 and increase the level of the anti - inflammatory factor il-33 . thus , long - term moderate - intensity exercise can resolve inflammatory response , improve ir , and achieve better blood glucose control in t2 dm patients .

[ purpose ] to investigate the effects of combined aerobic and resistance training on glycolipid metabolism and inflammation levels in type 2 diabetes mellitus patients . [ subjects and methods ] forty - two diabetes patients were randomized to the conventional therapy group ( n = 20 ) or intensive therapy group ( n = 22 ) . the control group contained 20 healthy people . the conventional therapy group received routine drug therapy and diet control , while the intensive therapy group additionally underwent combined aerobic and resistance training for 12 weeks . the oral glucose tolerance test and cardiopulmonary exercise testing were performed . toll - like receptor 4 and nf-bp65 protein and mrna expressions were determined by qpcr and western blotting . elisa was used to determine the expression levels of interleukin-18 , interleukin-33 , pentraxin - related protein 3 , and human cartilage glycoprotein 39 . [ results ] after exercise training , the intensive therapy group had significantly lower postprandial blood glucose , postprandial insulin , and glycated hemoglobin level and insulin resistance index than the conventional therapy group . the intensive therapy group had significantly lower toll - like receptor 4 and nf-bp65 protein and mrna expressions , and serum interleukin-18 levels but significantly higher serum interleukin-33 levels . [ conclusion ] combined aerobic and resistance training can improve glycolipid metabolism and reduce low - grade inflammation in patients with diabetes mellitus patients .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC4872488

urinary tract infection ( uti ) is the most common bacterial diseases in children.1 the prevalence of uti has been estimated that 7% of girls and 2% of boys will be experienced at least one episode of uti before 6 years of age.2 moreover , escherichia coli are responsible for about 80% of febrile and afebrile utis.3 this disease is running - in with kidney , urinary tract , and bladder.4 its clinical symptoms are dysuria , frequency and urgency abdominal and flanks pain with or without fever.5 uti sometimes do not show specific signs in children unlike adults and occurs with unusual signs such as weight loss , failure to thrive , anorexia , jaundice , and fever with the unknown resource . diagnosis and proper treatment are very important because of early complications such as sepsis , bacteremia , and late complications such as hypertension and chronic renal failure ( rf).6 despite of close relation between urinary system and the outside circumstances of the body and the entrance of pathogenic bacteria into the urinary system , there are several factors to clean up and defend against these pathogens.1 ros interferes in host defense mechanisms and causes cell damage . producing ros is low typically , and this low - levels are necessary for several biologic processes including intracellular differentiation , cell production , and arrest of cell growth , immunity , and defense against micro - organisms.7825 in human body , there is a fine and precise balance between the production and elimination of ros.6 external antioxidants such as vitamins e and c and endogenous antioxidants such as superoxide dismutase ( sod ) , catalase ( cat ) , and glutathione peroxidase have role in inhibition of some oxidants such as hydroxyl radicals , superoxide.7 increase of ros with or without a reduction in antioxidant defenses is defined as oxidative stress ( os ) and causing the destruction of biologic macromolecules means that the pathogenesis of many involved.5 in other words , os is resulted by an imbalance between oxidant and antioxidants.7 to reduce os in body , antioxidants act as cleaning - free radicals and are thought to have a role in complete safety.7 this study aimed to evaluate the antioxidant capacity of plasma levels of total antioxidant capacity ( tac ) and oxidant - antioxidant balance in children with uti in comparison with controls . this case control study was conducted on children < 15-year - old who were referred to pediatric nephrology clinic due to the complaints of uti symptoms . sample size was determined as 125 in which 50 patients and 1.5 times for controls.7 the local ethical committee ( code number 5795 ) of zahedan university of medical sciences approved the study protocol , and written informed consent was obtained from the parents . for suspected cases , patients with the following criteria were included in the study : positive reaction for leukocyte esterase in urine test strip and/or pyuria ( microscopic examination of urine sediment : 10 white blood cells ) . uti was diagnosed when there was significant bacteriuria ( > 100.000 colony - forming units / ml ) in midstream flow by clean exclusion criteria included of refusing cooperation received antibiotics in the last week and anti - inflammatory drug during the 3 days and vitamins within a week , underlying rheumatic and inflammatory diseases and urinary tract malformation . the control groups were selected from those who referred for check up to the same health centers . about 3 ml of blood sample was collected from each individual test tube without anticoagulant . to evaluate the oxidant - antioxidant balance and tac of serum in children following methods were used : total antioxidant assay based on ferric reducing ability of plasma by method benzie and strain.9 total oxidant assay by using the ferric - xylenol orange method,13 os index ( osi ) , which is an indicator of the degree of os , was calculated as the percent ratio total oxidants to total antioxidants osi = ( tox [ m]/tao [ m ] ) 100 and prooxidant antioxidant balance assay was performed according to sharifian , et al.4 spss 20 ( ibm corp . released 2011 . armonk , ny : ibm corp ) was used with t - test when the distribution of data was normal and if not , mann whitney u - test was used to compare the means or ranks in two groups . in our study , 125 children were entered in the division of 50 and 75 for patients and control groups , respectively . in both groups , the variables of age , gender , weight , serum total oxidant , serum total antioxidant , oxidant - antioxidant balances , and osi were measured . the case group consisted of 50 patients , 13 ( 26% ) male and 37 ( 74% ) were female . the control group included 75 patients , 35 ( 47% ) males and 40 ( 53% ) were female ( pearson chi - square = 17.415 , p < 0.001 ) . the mean age of patients was 4.36 3.58 years compared to 4.77 2.90 years in control group and observed that the difference was not statistically significant ( p = 0.144 ) . the means of weight were 15.83 9.02 kg and 15.25 5.1 kg for case and control groups , respectively , with no statistically significant difference ( p = 0.41 ) . all variables have normal distribution accordance to kolmogorov smirnov z - test and p value of ( 1.210 , 0.051 ) , ( 0.949 , 0.329 ) , ( 0.519 , 0.950 ) , and ( 0.960 , 0.315 ) for oxidant , antioxidant , balance of oxidant - antioxidant , and osi , respectively . the results of one sample of kolmogorov smirnov z - test for normality the results of comparison in parameters in the study revealed that the oxidant , any oxidat means of tac plasma level were 615.57 213.80 m and 814.92 217.05 m in the groups of case and control , respectively , which was statistically significant ( p < 0.001 ) observed [ table 2 ] . results of comparison in parameter the mean plasma levels of total oxidant in patients were 93.28 31.6 when in the control group was 82.53 16.84 , which was statistically significant ( p = 0.014 ) [ table 3 ] . demographic characteristics the means of oxidant -antioxidant balance in patients and in control were 70.60 20.46 and 62.7 16.35 , respectively . osi in patients was 9.37 3.04 in the control group 7.51 2.62 , which was statistically significant ( p = 0.001 ) . this study aimed to assess the balance of oxidant - antioxidant serum and antioxidant capacity in children with uti . to achieve this goal , the total oxidant , tac , oxidant - antioxidant balance , and osi in children with uti and control groups were measured . serum antioxidant statuses in patients have decreased equilibrium between oxidants and antioxidants in favor of oxidants and osi and were higher in patients than controls . in a relative study by kurutas et al . , the status of os urinary infection was evaluated and concluded that the levels of lipid peroxidation level were increased whereas cat and sod activity were decreased in positive urine culture compared to negative culture . in addition , it was observed that uti causes os and increased lipid , and antioxidant enzymes may lead to insufficiency of antioxidant enzymes.5 in urine samples of both patients and controls , the levels of cat , sod , and malondialdehyde ( mda ) were measured and compared and significant differences ( p < 0.01 ) were found . the level of cat and sod was increased , when antioxidant mda levels were decreased.5 although this study was conducted on urine samples , it showed that os in uti leading to insufficiency of antioxidant enzymes . measured antioxidant status in urine and serum by tac and plasma vitamin c levels as well oxidative statuses with total peroxide and osi.7 the means of parameters were compared , and the relationships between them were determined . total peroxide and osi levels were found to be lower , and tac was found to be higher in the urine of patients with uti . in this study , it was observed that plasma vitamin c concentration and tac of serum were lower ( both with p < 0.001 ) and total peroxide and osi levels were higher in patients than controls ( p < 0.001 ) . we also concluded similar results in comparison with vitamin c in which we did not use it . in a study , urinary levels of 8-hydroxy-2-deoxyguanosine ( 8-oxodg ) and tac were checked as markers of os and antioxidant capacity in children with uti . patients with positive dimercaptosuccinic acid ( dmsa ) had higher levels of urinary 8-oxodg ( p = 0.003 ) and tac ( p = 0.001 ) compared to patients with normal dmsa.14 it was included that pregnancy may aggravate os1516 various diseases accompany many inflammatory conditions and influence the endogenous antioxidant . studied the dynamics of os in the blood and urine of children with kidney diseases such as glomerulonephritis , pyelonephritis ( pyn ) , rf , and lower utis . they confirmed that products of lipid peroxidation and enzyme antioxidant capacity were proper test in combination with clinical parameters for the dynamics of os and markers of intoxication in children with inflammatory and immunological active parenchymal kidney disorders.17 ece et al . studied the level of total antioxidants , peroxides , total peroxide , and osi in patients with nephrotic syndrome and reported an increase in osi . although our study was performed on other illness , results were same.18 petrovic et al . reported that the parameters total antioxidant status ( tas ) and osi change with the duration of inflammation while only osi reflected changing in the oxidative - antioxidant balance at discharge from the hospital . however , none of the examined parameters were changed according to the acute changes in kidney function.24 hashemi et al . concluded that tac showed a significant decrease in the relative deficit balance resulting from oxidant - antioxidant in patients with nephrotic syndrome.8 although our study population was not the patients with nephrotic syndrome , the tac was significantly lower in the group with uti . showed that os in pyelonephritic kidney was accompanied by a reduced level of mitochondrial b - cell lymphoma 2 . importantly , renal cell death and animal mortality were both alleviated by mitochondria - targeted antioxidant 10 ( 6-plastoquinonyl ) decyl rhodamine 19 ( skqr1 ) . they suggested that pyn can be treated by reducing mitochondrial ros and thus by protecting mitochondrial integrity and lowering kidney damage.19 gorukac evaluated the effects of rolipram , a phosphodiesterase four enzyme inhibitor , on e. coli - induced renal oxidative damage in an acute pyn rat model . tissue mda and nitric oxide levels and sod and cat activities were significantly increased in the kidneys from the pyn groups . however , rolipram administration reduced renal mda , nitric oxide levels , and enhanced sod and cat activities . the histopathology examinations demonstrated that rolipram treatment reduced the inflammation grade in the kidney specimens.20 the activities of the antioxidant enzymes , cat , sod , glutathione peroxidase ( gsh - px ) , and xanthine oxidase ( xo ) were also elevated by e. coli . however , caffeic acid phenethyl ester ( cape ) ( phosphodiesterase four inhibitors ) administration reduced mda and no levels as well as xo activity when it is a cause of increasing sod and gsh - px activities . they recommended cape administrations decrease the oxidative damage occurring in pyn and , therefore , could be used for medical management of bacterial nephropathy.21 some treatments applied to decrease oxidative damage in uti . acute renal inflammatory injury can be prevented much more effectively by combination therapy rather than by conventional therapy alone . grr s , et al . investigated the involvement of os in the pathogenesis of acute pyn and evaluated the impact of meloxicam and/or l - carnitine in addition to conventional antibiotic treatment . l - carnitine and meloxicam alleviated os , probably by decreasing lipid peroxidation and enforcing antioxidant defense system.20 treatment by thymoquinone ( tq ) before or during e. coli inoculation prevents oxidative damage in acute pyn in an ascending obstructive rat model . the oxidative damage occurred in pyn and , therefore , it could be used as a supportive agent to protect the kidneys from oxidative damage caused by pyn.2223 the limitations of our study were small size of population and indirect measures of renal parenchymal inflammation . employing direct assessment with upper urinary tract imaging studies such as scintigraphy , association between the oxidative status parameters , direct assessment of renal inflammation , and subsequent scarring is necessary . equilibrium between oxidants and antioxidants is in favor of oxidants , and the osi was higher in patients compared to their counterparts in control group . uti may causes os and increases lipid peroxidation level that leading to insufficiency of antioxidant enzymes . many studies revealed that the majority of antioxidants such as rom , tos , bap , tas and ttp reducing oxidative stress.101112 we can conclude that tas and osi , as markers of os during uti , are sensitive in accompanying inflammatory conditions . however , none of the examined parameters were sensitive to acute changes in kidney function . further investigations are needed to evaluate whether tas , tos , and osi could be used to monitor disease severity in children with uti . given that there are no adequate studies in this area ; therefore , more studies are recommended to be done first on a comprehensive sample from some different societies with various circumstances . comprehensive and general results would be the tools for acquired decisions by planners in the field of health policies . second , a few studies are needed to be done on the status of os and antioxidant balance before and after the consumption of some antioxidants such as vitamin c and vitamin e. perhaps , in the future , we can suggest that patients with uti may benefit from antioxidant treatment as additives drug .

background : urinary tract infection ( uti ) is the most common bacterial infections in children . this studyaimed to investigate the oxidative and antioxidate status of plasma in patients with uti and to compare them with those of the controls.methods:this case control study of 5075 children in the given order was performed in 2013 at the pediatric clinic of infections in zahedan hospital of ali ibn abi talib . the antioxidative status of plasma were evaluated by measuring the total antioxidant capacity ( tac ) the oxidative status of samples was assessed by measuring the total peroxide and the oxidative stress index ( osi ) levels . the means of the parameters were compared and the relationship among them was determined . data were analyzed using spss 20 ( ibm corp . released 2011 . ibm spss statistics for windows , version 20.0 . armonk , ny : ibm corp ) . student 's t - test and mann whitney u - test were applied in various situations of our questions ; 95% confidence interval was considered for the level of significance.results:the results showed that total oxidant serum status in uti patients was higher compared to controls when total antioxidant serum was lower . the balance of oxidant - antioxidant serum was in favor of oxidant serum and this term was confirmed by osi.conclusion:our results showed that the plasma levels of tac in patients with uti were decreased compared to controls , and oxidant - antioxidant balance and osi caused increased os in patients .

INTRODUCTION METHODS RESULTS DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest

PMC3980213

barrett s oesophagus ( bo ) was endoscopically defined by the presence of at least 2 to 3 cm of columnar epithelium in the lower oesophagus . recently , the global evidence - based consensus of montreal defined barrett oesophagus as columnar metaplasia lining the distal oesophagus , with specialized intestinal metaplasia ( i m ) with goblet cells , or gastric metaplasia with cardial type or fundi - coxyntic type mucosa . the main feature of intestinal metaplasia is the presence of alcian blue - positive mucin - producing goblet cells . moreover , in clinical practice , the finding of cardial and/or fundic mucosa without intestinal metaplasia in the distal oesophagus is frequent and it has been reported as columnar - lined oesophagus ( clo ) . however , it probably represents an early reflux - lesion preceding an evident barrett phenotype with goblet cells , in keeping with previous studies showing that ck7 is a marker related to early phase of gastroesophageal reflux , and it is present also in clo biopsies lacking of i m . bo with i m represents an important risk factor for oesophageal adenocarcinoma , in keeping with the progression sequence metaplasia - dysplasia - adenocarcinoma . hypothesized that patients with cardial metaplasia could potentially progress towards dysplasia and adenocarcinoma through i m . instead , few and controversial data are reported in the literature about the progression risk of clo lacking of i m , giving rise to a question about the clinical management of this lesion . on this basis , we studied a series of oesophageal biopsies from patients with endoscopic abnormalities related to columnar metaplasia , by performing some double and triple immunohistochemical / histo - chemical stains ( ck7/alcian / pas , p53/alcian/ pas , aurora a / alcian / pas ) in order to simultaneously assess the mucinous histochemical features of the cells with aberrant expression of such antigens . the aim of the study was to evaluate if some immunophenotypic changes were present in clo cases without i m and if these changes could precede the appearance of the goblet cells or may be present independently of them . aurora a maps in 20q13.2 , a chromosomal region frequently amplified and over - expressed in several human malignancies ( breast , bladder , prostate , ovary , colon and pancreas ) . recently its expression was evidenced in barrett adenocarcinoma and in clo . in experimental studies , the over - expression of aurora a was oncogenic and constantly associated with mutation of the gene tp53 that , becoming unable to block the kinase activity of the aurora a , leads to a strong accumulation of extra - copies of centrosomes . cells without a functional p53 and with high levels of aurora a are characterized by repeated cell cycles , until they become aneuploid . these observations identify the protein aurora a as a prognostic marker , being itself an accomplice of carcinogenesis . to our knowledge , this is the first study in which a combined immunohistochemical/ histochemical method has been applied in the study of barrett pathology . this is a retrospective study performed on 57 biopsies obtained from h. pylori - free patients showing columnar metaplasia of the lower oesophagus at the endoscopy . the cases were retrieved from broader clinical records , consisting of 819 oesophageal biopsies collected between june 2006 and june 2012 and comprising 228 cases ( 27.8% ) of columnar metaplasia ( 135 with bo and 93 with clo ) and 26 cases of adenocarcinoma ( 3.1% ) . formalin - fixed , paraffin - embedded specimens were retrieved from the archives of the department of pathology , university of palermo ( italy ) . for each patient , 4-quadrant biopsy specimens have been taken every 2 cm and stained with hematoxylin - eosin and alcian - pas . the patients were divided into three groups : i ) group a , 20 bo cases consisting of i m , with alcian blue - positive goblet cells ; ii ) group b , 17 cases of clo consisting of pas+ columnar metaplasia without alcian blue positive goblet cells ; iii ) group c , 20 cases with intermediate features of transitional metaplasia ( tm ) , consisting of pas+ columnar mucosa with only few cells showing initial production of acid - alcian blue positive - mucins , but devoid of goblet cell morphology . the combined staining method consists of two steps : in the first step an immunohistochemical staining was performed , according to the manufacturer s instructions ( universal lsaab kit , dako ) . primary antibodies used in this study were : ck7 ( clone sp52 ; 1:100 ; ventana ) , p53 ( clone do7 ; 1:200 ; ventana ) and aurora a ( clone ep1008y ; 1:100 ; epitomics ) . immunohisto -chemical positivity was considered significant when it was cytoplasmic for ck7 , nuclear for p53 and nuclear or nuclear / cytoplasmic for aurora a. as positive control for aurora a and p53 , we referred to a case of adenocarcinoma arising in the lower oesophagus ( barrett adenocarcinoma ) . for aurora a and p53 , the positivity in at least 3% of the nuclei was considered significant . for ck7 , cytoplasmic staining in at least one gland negative controls , lacking primary antibody incubations , were included for each immunohistochemical run . in the second step , a histochemical staining was performed on the same sections previously subjected to the immunohistochemical assay . at first , alcian blue staining was performed and the results of this double immunohistochemical / histochemical staining were observed . subsequently , pas staining was added ( triple staining ) and the results were observed again . the histochemical stainings were performed with only minor modifications according to poletti et al . for alcian blue staining , the sections were stained with alcian blue solution ( ph 2.5 ) for 30 min at room temperature , washed in running tap water for 10 min , rinsed in di water , counterstained in mayer s hematoxylin for 5 min and washed in di water . the acetic acid as mordant was not used because it has no effect on the final results . for pas staining , sections were oxidized in 0,5% periodic acid solution for 5 min , rinced in di water , placed in schiff reagent for 15 min , washed in tap water for 5 min , counterstained in mayer s hematoxylin for 1 min and washed in di water . finally , the slides were dehydrated , cleared and mounted . differentiation with hydrochloric acid was omitted because it markedly reduces tissue antigenicity and causes fading of the chromogenic reaction when the immunohistochemical staining is performed before pas staining . a case of adenocarcinoma of the lower oesophagus ( barrett adenocarcinoma ) is showed in figure 1a . the tumor exhibited strong nuclear positivity for p53 and aurora a ( figure 1b ) and it was used as positive control for the immunohistochemical stains with these antibodies . ck7 was positive in all the three groups ; it was more diffusely and strongly expressed in group a , while focally and more weakly in groups b and c. group a. both aurora a and p53 immunostainings were positive in the nuclei of alcian blue+ goblet cells in 6 of 20 cases ( figure 2a ) . noteworthy , they were positive also in nuclei of pas+/alcian blue negative columnar cells , whereas adjacent alcian blue+ goblet cells were negative [ figure 2b ] . group b. eight of 17 cases showed focal nuclear positivity for aurora a and p53 in pas+/alcian blue negative columnar cells ( figure 3 b , c ) . noteworthy , ck7 was expressed just in the same aurora a / p53 positive areas ( figure 3d ) . group c. four of 20 cases showed positive immunostaining both for aurora a and p53 . aurora a was positive both in alcian blue - negative cells ( figure 4a ) and in cells with initial production of alcian blue - positive mucins , but lacking of goblet cell morphology ( cells with intermediate features between i m and clo ) ( figure 4b ) . as in the previous group , p53 and ck7 were expressed in the same areas , despite the absence of i m . noteworthy , the sequential histochemical steps of the double and triple staining did not affect the immunohistochemical results obtained in the previous step . the combined use of immunohistochemical / histochemical staining methods has rarely been reported in the literature . wong et al . used the double alcian blue / chromogranin a and alcian - blue / mib-1 staining in specimen of gastric mucosa with intestinal metaplasia and neuroendocrine hyperplasia , to distinguish the proliferating cellular component . to our knowledge , a double histochemical / immunohistochemical method has not been applied to the study of oesophageal pathology . in our study , all the cases stained positively , at least focally , for ck7 , considered an early marker of gastroesophageal reflux because it is usually absent in normal fundic and cardial mucosa , but present in columnar metaplastic mucosa of the oesophagus , including cases lacking of i m . furthermore , our study showed nuclear expression of aurora a kinase in columnar metaplasia lacking of goblet cells ( group b ) , often correlating with p53 nuclear expression . the aurora a kinase belongs to serine / threonine group of kinases that operate as key regulators of mitosis and it has a role in the cell cycle progression . several studies showed that a deregulation in the expression and activity of this protein leads to defects in the assembly of the centrosome and of the mitotic spindle , aneuploidy , genetic instability and tumourigenesis . nuclear positivity has been reported in hormone - related ( breast , ovary and prostate ) human cancers associated with a poor outcome , but not in non - hormone responsive conditions , such as in the oesophageal columnar metaplasia and in the barrett adenocarcinoma . previously , we evidenced the overexpression of aurora a with rt - pcr in cases of columnar mucosa with and without i m . in the present study , we evidenced aurora a immunohistochemical overexpression in the nuclei of pas+ cells . in the group a we found nuclear aurora a - positive staining in some alcian blue+ goblet cells of barrett mucosa , but , noteworthy , other goblet cells did not stain for aurora a , whereas the adjacent pas+ columnar cells stained positively . this is in keeping with the findings of group b cases , lacking of i m , in which aurora a was present in the nuclei of pas+ columnar cells . the over - expression of aurora a in non - goblet cells may support the recently introduced conception of bo without intestinal metaplasia , in contrast with the definition in which the goblet cells represented an essential prerequisite for the diagnosis of bo and an obliged step in the cancer progression . in fact , the definition of bo is still confusing and ambiguous . according to the american college of gastroenterology and to the american gastroeneterological association , intestinal metaplasia is the only type of oesophageal columnar epithelium that clearly predisposes to malignancy and it is still required for the diagnosis of bo . furthermore , our findings are in keeping with kelty et al . , who observed that patients with clo lacking of i m on biopsy have a similar cancer risk to those with specialized i m , so they conclude that i m is not essential for cancer risk . of course , since the carcinogenesis is a multistep process consisting of many overlapping mutations , the over - expression of aurora a in the columnar mucosa , with or without i m , should not be considered a condition necessarily leading to the tumour , but a potentially reversible condition after stopping or decreasing of the stimulus . the appearance of aurora a and p53 nuclear positivity , together with the reflux - related ck7 cytoplasmic positivity , in areas lacking of i m , could suggest another way besides the traditional im - dysplasia - adenocarcinoma sequence . in keeping with our immunophenotypical findings , we hypothesize a cancer sequence , in which ck7/ auroraa / p53/pas+ columnar mucosa could represent an intermediate step of the carcinogenesis , independently from the presence of intestinal alcian blue+ goblet cells ( figure 5 ) . recently , in fact , adenocarcinoma of gastroesophageal junction arising from cardial type metaplasia have been described , suggesting again the hypothesis of a double pathway previously reported by us . noteworthy , in our study , consisting of 819 oesophageal biopsies collected between june 2006 and june 2012 , we found 26 cases of adenocarcinoma ( 3.1% ) , 25 of which aroused in patients without a known clinical history of bo , lacking previous endoscopy and never treated with gerd therapy . on the contrary , none of 228 patients with endoscopic and histologic columnar - lined oesophagus ( 135 with bo and 93 with clo ) , treated with constant therapy for gerd symptoms , developed a high - grade dysplasia / adenocarcinoma , during a mean follow - up of 5 years ( range 1 - 12 ) with bi - annual endoscopy and biopsy . only one case of clo , untreated , progressed to bo with dysplasia ( figure 6 ) . given our results , we think that the immunophenotypical alterations ( such as aurora a and p53 expression ) in some cells of clo cases could be by themselves responsible of a risk progression , despite the absence of i m . therefore , in our view , also clo patients require an appropriate management , because the outcome is not ab initio predictable . of course , in consideration of the low risk of cancer progression , both in clo and bo patients treated with ppi therapy , and of the necessity to refine the surveillance program to obtain a good cost / benefit ratio , we think that a more long - term interval of endoscopies ( e.g. , a 3-year interval ) could be enough if bo and clo cases are properly treated . ppi therapy , in fact , could be useful not only for the gerd symptoms but also to reduce the risk of cancer progression . in conclusion , the combined staining method allowed us to simultaneously evidence the type of mucins produced by the columnar cells and the immunophenotypical expression of aurora a , p53 and ck7 that may be indicative of an early phase of the carcinogenetic pathway independently from the i m step .

intestinal metaplasia in barrett s oesophagus ( bo ) represents an important risk factor for oesophageal adenocarcinoma . instead , few and controversial data are reported about the progression risk of columnar - lined oesophagus without intestinal metaplasia ( clo ) , posing an issue about its clinical management . the aim was to evaluate if some immunophenotypic changes were present in clo independently of the presence of the goblet cells . we studied a series of oesophageal biopsies from patients with endoscopic finding of columnar metaplasia , by performing some immunohistochemical stainings ( ck7 , p53 , auroraa ) combined with histochemistry ( alcian - blue and alcian / pas ) , with the aim of simultaneously assess the histochemical features in cells that shows an aberrant expression of such antigens . we evidenced a cytoplasmic expression of ck7 and a nuclear expression of aurora a and p53 , both in goblet cells of bo and in non - goblet cells of clo , some of which showing mild dysplasia . these findings suggest that some immunophenotypic changes are present in clo and they can precede the appearance of the goblet cells or can be present independently of them , confirming the conception of bo as the condition characterized by any extention of columnar epithelium . this is the first study in which a combined immunohistochemical / histochemical method has been applied to barrett pathology .

Introduction Materials and Methods Results Discussion

PMC3218703

organized preventive screening programs for antenatal care were first introduced in western europe in the twentieth century with the hope that routine antenatal care would contribute to a reduction in maternal and infant mortality rates . figures on maternal mortality in the developed world show that the risk of death as a result of pregnancy and child birth is approximately 1 in 7000 compared with 1 in 23 for women living in parts of africa where antenatal care is poor or nonexistent.1 the human immunodeficiency virus ( hiv ) pandemic is one of the most serious health crises faced by the world today . an estimated 33.4 million people were living with hiv / acquired immunodeficiency syndrome as at 2009.2 the prevalence of hiv in nigeria has risen from 1.8% to 5.0% in 2003.3 a disproportionate burden has been placed on women and children , who in many settings continue to experience high rates of new hiv infection and hiv - related illness and death . most children living with hiv acquire the infection through mother - to - child transmission ( mtct ) , which can occur during pregnancy , labor , delivery , or breastfeeding . in the absence of any intervention breastfeeding by an infected mother increases the risk by 5%20% to a total of 20%45%.4 the risk of mtct can be reduced to less than 2% by interventions that include antiretroviral prophylaxis given to women during pregnancy and labor and to the infant in the first weeks of life , during elective cesarean delivery ( prior to the onset of labor and rupture of membranes ) , and avoidance of breastfeeding.5 with these interventions , new hiv infections in children are becoming increasingly rare in many parts of the world , particularly in high - income countries . in many resource - constrained settings , elective cesarean delivery is seldom feasible,7 and it is often neither acceptable nor safe for mothers to refrain from breastfeeding . however , recent guidelines from the world health organization6 recommend that mothers known to be hiv - infected ( and whose infants are hiv - uninfected or of unknown hiv status ) should exclusively breastfeed their infants for the first six months of life , introducing appropriate complementary foods after that . breastfeeding should then be stopped only when a nutritionally adequate and safe diet without breast milk can be provided . in these settings , efforts to prevent hiv infection in infants initially focused on reducing mtct around the time of labor and delivery . the unknown hiv status of these unbooked women presenting to clinic for the first time in the third trimester of pregnancy poses a risk not only to the patient and her baby , but also to the staff caring for them in the peripartum period . the baby , who is the most critical element in vertical transmission , would invariably not benefit from the prevention of mother - to - child transmission ( pmtct ) hiv program , and eventually add to the bulk of pediatric hiv patients resulting in a heavy burden on their parents , health facilities , and community . in this first prospective study from the niger delta in nigeria we have investigated the prevalence of hiv and birth outcomes in women who do not access any antenatal care . all pregnant women in labor admitted to the isolation ward at the university of port harcourt teaching hospital with pregnancy - related complications , or in the immediate puerperium , were counseled and written informed consent was obtained to allow for blood taking and for retroviral screening . an unbooked woman was defined as a woman presenting to clinic for the first time in the third trimester of pregnancy . the university of port harcourt teaching hospital is a 500-bed tertiary hospital providing specialist obstetrics and gynecology services to women in the cosmopolitan city of port harcourt and other surrounding states of the niger delta in nigeria . the hospital is one of the centers running the pmtct program sponsored by the federal government of nigeria . a total of 118 women were enlisted for the study . the data collected included biodata , mode of delivery , pregnancy outcome , parity , and intrapartum complications . hiv screening was carried out using a double enzyme - linked immunosorbent assay ( elisa ) method , as provided in the commercially available second - generation genscreen ( bio - rad , france ) and immunocomb elisa test for the qualitative and differential diagnosis of hiv ( orgenics , israel ) . data were entered and analyzed using statistical package spss version 9 ( spss inc . , chicago , il ) . statistical analysis included descriptive analysis of mean , standard deviation , and chi - square analysis . a p value of < 0.05 was considered to be statistically significant in all statistical analyses . data were entered and analyzed using statistical package spss version 9 ( spss inc . , chicago , il ) . statistical analysis included descriptive analysis of mean , standard deviation , and chi - square analysis . a p value of < 0.05 was considered to be statistically significant in all statistical analyses . of the 118 pregnant women enlisted for this study , 30 ( 25.4% ) were positive for hiv . gestational age of subjects at presentation was 2840 weeks and mean age was 35.04 8.06 years . the prevalence of hiv was significantly higher among pregnant women with no formal education ( n = 14 , 11.9% ) than for those with primary ( n = 9 , 7.6% ) , secondary ( n = 5 , 4.2% ) , or tertiary ( n = 2 , 1.7% ) education , as shown in figure 1 . most of the deliveries were spontaneous live births ( n = 83 , 70.3% ) , with some still births ( n = 2 , 17% ) and some intrauterine fetal deaths ( n = 15 , 12.7% ) , as shown in figure 2 . vaginal delivery was the predominant mode of delivery ( n = 89 , 75.5% ) among the pregnant women studied , while 29 ( 24.5% ) had emergency cesarean section . among the occupational groups , the prevalence of hiv was significantly ( p = 0.04 ) higher among traders ( n = 14 , 11.9% ) than in career women ( n = 5 , 4.2% ) as shown in figure 3 . the majority of subjects were primigravidas 66 ( 55.9% ) , while 52 ( 44.1% ) were multigravidas . multigravid women were more susceptible to hiv infection ( n = 17 , 14.4% ) compared with primigravid women ( n = 13 , 11.0% ) as shown in figure 4 . in this study we investigated the prevalence of hiv and pregnancy outcomes in an unbooked obstetric population in the niger delta . our observed prevalence is significantly higher than that observed among booked antenatal subjects ( 5.0% ) studied in a 2003 seroprevalence sentinel survey in nigeria.3 counseling and detection of women infected with hiv is a difficult issue in low resource settings , where there is a high tendency for out - of - hospital births , home births , and parallel antenatal care.4,5 the pool of unbooked patients often report to appropriate health facilities late during their pregnancy with difficult labor or other disease conditions complicating their pregnancy . the prevalence of hiv was significantly higher among unbooked patients with no formal education than in better educated subjects . the question has often arisen concerning the nature of women who are most likely to be unbooked . previous studies found that women who are homeless and addicted to illicit substances,8 with a low level of education,9 low income,10 and low socioeconomic status11 are more likely to access antenatal care late or be unbooked . there may be several reasons for this association , including better educated people generally having greater access to information than those who have less formal education , and are more likely to make informed decisions and act on information given . in addition , better educated people generally have better jobs and greater access to money and other resources which can help them lead healthier lives . previous reports9,12 have suggested that a number of interrelated sociodemographic factors , including high parity , are a reason for late or poor access to antenatal services . similarly , shaffer in a previous report on barriers to access of antenatal care , particularly among ethnic minorities , marginalized groups and socially deprived populations with high parity has suggested that cultural issues relating to language and antenatal staff insensitivity are important , and can deter some women from accessing antenatal care early or regularly.13 easily overlooked details , such as gender of the consulting obstetrician , can make a big difference to women s perception of antenatal services , particularly in highly religious populations . a study of islamic women living in australia , tsiankasas , and liamputtong14 found that the prospect of being given an ultrasound by a male doctor , rather than a female doctor , caused them to cancel antenatal appointments . another study reported that hispanic women living in the us failed to return for antenatal appointments because they felt that staff members were too rushed or simply unwilling to answer their questions.15 these cultural oversights may be viewed as disrespectful by women from various ethnic groups and have the potential to generate feelings of frustration and resentment for women in need of antenatal care.16 we observed that the majority of subjects in this study presented at a gestational age between 28 and 40 weeks , and with significantly poor birth outcomes , particularly high rates of still births ( 17% ) , intrauterine fetal death ( 12.7% ) , and emergency cesarean section ( 24.5% ) . there is an increasing body of evidence that prenatal care in pregnant women living with hiv improves perinatal as well as maternal outcomes , particularly in facilities where there is a comprehensive pmtct program . herbst et al17 examined the perinatal outcomes for women who had not accessed prenatal care and those who did . they found that not having any prenatal care increased the rate of preterm birth , low birth weight babies , and more morbidity for the mothers . unbooked women had more cesarean sections for fetal distress , and their babies were more predisposed to respiratory distress , intraventricular hemorrhage , and death before discharge . a previous study in port moresby general hospital , papua , new guinea , observed that unbooked mothers have a perinatal death rate which is four times that of those who attended antenatal clinics before their delivery.18 similarly treacy et al19 examined the outcomes of 101 unbooked women at the rotunda hospital in dublin and observed that unbooked women had a significantly worse perinatal outcome . the unknown hiv status of this category of patients poses a risk not only to the patient , her baby , but also for the health staff caring for them in the peripartum period.20 however , the baby is the most critical element in vertical transmission , and would invariably be left unattended and eventually add to the number of pediatric hiv patients , resulting in a heavy burden on their parents , health facilities , and the community . the gestational age at presentation to hospital reflects the booking habits of this high - risk group of women who appear or present to the labor ward after a failed attempt at home delivery with the help of traditional birth attendants . most of the patients had a spontaneous vaginal delivery , having being admitted in established labor . this trend leaves little or no time for antiretroviral therapy to be administered under the pmtct program , with the hope of reducing transmission . this trend also exposes surgeons , nurses , and midwives to the possible risk of transmission . counseling and detection of pregnant women infected with hiv is a difficult issue , particularly in low resource settings where there is a high tendency for out - of - hospital births , home births , and other parallel nonhospital - based antenatal care.21 in this study , the seroprevalence rate amongst unbooked women was significantly higher than in booked patients in previous studies . these findings are very pertinent to health care delivery in our environment , because a critical number of unbooked patients may not be benefiting from the pmtct program , thus increasing the pediatric hiv burden in our environment . there is the need to develop innovative ways to engage with these hard to reach groups who may not access conventional antenatal services , by engaging in widespread community health education to raise awareness of voluntary counseling and testing , antenatal care , and the pmtct program .

despite recent advances in the prevention of transmission of human immunodeficiency virus ( hiv ) infection from mother to child during pregnancy , infants continue to be born and infected with hiv , particularly in africa . this study was undertaken to determine the seroprevalence of hiv infection among unbooked pregnant women in the niger delta of nigeria . one hundred and eighteen consecutively recruited unbooked subjects presenting to the isolation ward at the university of port harcourt teaching hospital were screened for hiv . among the 118 subjects studied , 30 ( 25.4% ) were positive for hiv . hiv-1 was the predominant viral strain . gestational age of subjects at presentation was 2840 weeks and mean age was 35.04 8.06 years . the majority of subjects were primigravidas 66 ( 55.9% ) , while 52 ( 44.1% ) were multigravidas . the prevalence of hiv was significantly higher among unbooked pregnant women with less formal education : 14 ( 11.9% ) compared with 9 ( 7.6% ) , 5 ( 4.2% ) , and 2 ( 1.7% ) for those with primary , secondary , and tertiary education , respectively ( p = 0.01 ) . among the occupational groups , the prevalence of hiv was significantly higher among traders 14 ( 11.9% ) than in career women 5 ( 4.2% , p = 0.04 ) . multigravid women were more susceptible to hiv infection 17 ( 14.4% ) than primigravid women . perinatal mortality and emergency cesarean section was high among unbooked pregnant women . the prevalence of hiv observed amongst unbooked antenatal subjects in this study is significantly higher than those of booked patients in previous studies . these findings are very pertinent to health care delivery , because this pool of unbooked patients may not be benefiting from the prevention of maternal to child transmission program , thus increasing the pediatric hiv burden in our environment .

Introduction Materials and methods Statistical analysis Results Discussion

PMC3276078

vascular smooth muscle cell ( smc ) differentiation is an essential component of vascular development . a variety of human vascular diseases can be traced to a defect in smooth muscle development or proliferation [ 13 ] . in mice with defective smooth muscle development , they include mouse neural crest stem cell line monc-1 , mouse embryonic fibroblast 10t1/2 and mouse embryonic stem cells . under certain culture conditions , these cells express smc - specific markers that indicate smc differentiation , which include contractile apparatus - associated proteins such as calponin-1 and smooth muscle -actin ( sm -actin ) . however , several problems arise when using these cell models for the study of smc differentiation . first , because most of the cells used in smc differentiation studies are of mouse origin , there may be important interspecies differences in the differentiation environment , intracellular molecules involved in the differentiation process and underlying signaling mechanisms promoting smc differentiation . second , some cell lines are not naive cells but immortalized cell lines derived from a primary culture . the regulating mechanisms involved in smc differentiation may be changed in the immortalized cell lines . as an alternative , simper et al . have described smc progenitor cells in human peripheral blood mononuclear cells . however , based on the experience of our lab and several other labs , progenitor cells isolated from human peripheral blood can hardly proliferate under in vitro culture condition and usually deteriorate after 3 weeks , and as a result , it is impossible to maintain the cell line for serial analysis and meaningful comparisons . also , the cell samples isolated from donors can only be used one time due to the lack of proliferative properties . the variation in blood samples from donors of different ethnicities , ages , genders and medical backgrounds multi - lineage progenitor cell lines ( mlpcs ) are karyotypically normal multi - potent progenitor cells obtained from post - partum human umbilical cord blood . it is possible to continue to expand these cells , and they can differentiate down specific lineages beyond 20 passages . their high purity and proliferative features make mlpc an ideal tool for the study of smc differentiation . transforming growth factor 1 ( tgf-1 ) is thought to play a key role in smc differentiation and is known to coordinately upregulate a variety of smc differentiation markers in cultured smc from mature blood vessels as well as pluri - potential stem cells . in this study , we investigated the differentiation of mlpc into smc lineage cells , which was induced by tgf-1 . we found that the mrna levels of a variety of smc - specific markers were increased during this process , whereas endothelial cell - specific markers and epc marker cd34 were decreased in tgf-1-treated mlpc . recombinant human tgf-1 was obtained from r&d systems ( minneapolis , mn , usa ) . the mesenchymal stem cell medium bulletkit was obtained from cambrex - walker ( walkersville , md , usa ) . iq sybr green super - mix kit was obtained from bio - rad ( hercules , ca , usa ) . all of the primers were synthesized by sigma genosys ( the woodlands , tx , usa ) . mlpcs were cultured in mesenchymal stem cell basic medium to maintain their undifferentiated status and were then subcultured by using trypsin - edta reagent in the regular way . mlpcs at passage 4 to passage 6 were used in the experiment . to induce cell differentiation , mlpcs were seeded at 1.510 in each well on a 6 well plate containing mesenchymal stem cell basic medium plus 1% fbs with or without tgf-1 ( 1 ng / ml or 5 ng / ml ) . mlpcs were cultured for 7 days , and cell rna was then harvested for pcr analysis . the genomic dna contamination in rna preparation was removed by using the dna - free kit ( ambion , austin , tx ) , and a lack of detectable genomic dna was confirmed by pcr . total rna ( 0.5 g ) was reverse - transcribed into cdna using the iscipt cdna synthesis kit ( bio - rad ) following the manufacturer s instruction . primers for all tested genes were designed using the beacon designer 2.1 software ( bio - rad ) . the quality of individual pairs of primers was confirmed by running conventional pcr before real - time pcr to make sure there were no detectable primer dimers or non - specific products yielded . the real - time pcr reaction mixture included the following : 250 nm primers , 50 ng cdna , and iq sybr green supermix ( 0.2 mm of each dntp , 25 u / ml itaq dna polymerase , sybr green i , 10 nm fluorescein , 3 mm mgcl2 , 50 mm kcl , and 20 mm tris - hcl ) . using the icycler iq real - time pcr detection system ( bio - rad ) , pcr cycling conditions were set as follows : 95c for 90 seconds , 40 cycles at 95c for 20 seconds , and 60c for 1 minunte . melting curve analysis was performed on the icycler over the range 5595c by monitoring iq sybr green fluorescence with increasing temperature ( 0.5c increment changes at 10 seconds intervals ) . sample cycle threshold ( ct ) values were determined from plots of relative fluorescence units ( rfu ) versus pcr cycle number during exponential amplification so that sample measurement comparison was possible . standard curves for all primer amplifications were generated by plotting average ct values against the logarithm starting quantity of target template molecules ( series dilution of cdna template : 50 , 10 , 2 , 0.4 , and 0.08 ng ) , followed by a sum of least squares regression analysis . the gene expression in each sample was normalized to gapdh expression as [ 2^(ctgapdhctgene ) ] . data from the control and tgf-1-treated groups were analyzed using a paired student s t test ( one tail , minitab software , sigma breakthrough technologies , inc . recombinant human tgf-1 was obtained from r&d systems ( minneapolis , mn , usa ) . the mesenchymal stem cell medium bulletkit was obtained from cambrex - walker ( walkersville , md , usa ) . iq sybr green super - mix kit was obtained from bio - rad ( hercules , ca , usa ) . all of the primers were synthesized by sigma genosys ( the woodlands , tx , usa ) . mlpcs were cultured in mesenchymal stem cell basic medium to maintain their undifferentiated status and were then subcultured by using trypsin - edta reagent in the regular way . mlpcs at passage 4 to passage 6 were used in the experiment . to induce cell differentiation , mlpcs were seeded at 1.510 in each well on a 6 well plate containing mesenchymal stem cell basic medium plus 1% fbs with or without tgf-1 ( 1 ng / ml or 5 ng / ml ) . mlpcs were cultured for 7 days , and cell rna was then harvested for pcr analysis . the genomic dna contamination in rna preparation was removed by using the dna - free kit ( ambion , austin , tx ) , and a lack of detectable genomic dna was confirmed by pcr . total rna ( 0.5 g ) was reverse - transcribed into cdna using the iscipt cdna synthesis kit ( bio - rad ) following the manufacturer s instruction . primers for all tested genes were designed using the beacon designer 2.1 software ( bio - rad ) . the quality of individual pairs of primers was confirmed by running conventional pcr before real - time pcr to make sure there were no detectable primer dimers or non - specific products yielded . the real - time pcr reaction mixture included the following : 250 nm primers , 50 ng cdna , and iq sybr green supermix ( 0.2 mm of each dntp , 25 u / ml itaq dna polymerase , sybr green i , 10 nm fluorescein , 3 mm mgcl2 , 50 mm kcl , and 20 mm tris - hcl ) . using the icycler iq real - time pcr detection system ( bio - rad ) , pcr cycling conditions were set as follows : 95c for 90 seconds , 40 cycles at 95c for 20 seconds , and 60c for 1 minunte . melting curve analysis was performed on the icycler over the range 5595c by monitoring iq sybr green fluorescence with increasing temperature ( 0.5c increment changes at 10 seconds intervals ) . sample cycle threshold ( ct ) values were determined from plots of relative fluorescence units ( rfu ) versus pcr cycle number during exponential amplification so that sample measurement comparison was possible . standard curves for all primer amplifications were generated by plotting average ct values against the logarithm starting quantity of target template molecules ( series dilution of cdna template : 50 , 10 , 2 , 0.4 , and 0.08 ng ) , followed by a sum of least squares regression analysis . the gene expression in each sample was normalized to gapdh expression as [ 2^(ctgapdhctgene ) ] . data from the control and tgf-1-treated groups were analyzed using a paired student s t test ( one tail , minitab software , sigma breakthrough technologies , inc . , san marcos , tx ) . after 7 days of exposure to tgf-1 , several smc - specific markers were dramatically increased . the addition of tgf-1 ( 1 ng / ml ) to mesenchymal stem cell medium significantly increased the mrna levels of sm22 , calponin-1 , sm -actin , caldesmon , tropomyosin and myosin light chain kinase ( mlck ) to 1215.5% , 1974.6% , 567% , 429.7% , 567% and 162.8% , respectively , when compared to controls ( medium only ) ( p<0.05 , figure 1a f ) . tgf-1 ( 5 ng / ml ) also increased the mrna levels of these markers significantly ( p<0.05 ) , but the effect was less than that of the tgf-1 treatment at 1 ng / ml ( figure 1a f ) . in addition , we investigated the expression level of cd105 ( a tgf- accessory receptor ) in mlpcs . we found that cd105 is expressed at a high level in mlpcs under control conditions ( relative mrna level is 0.156 , data not shown ) . after 7 days , tgf-1 at 1 ng / ml and 5 ng / ml led to a significant downregulation of cd105 mrna levels to 61.7% and 70.8% of that of controls , respectively ( p<0.05 , figure 2 ) . two ec - specific markers , ve - cadherin and vegfr-2 , were detected in mlpcs under control conditions . after 7 days of exposure to tgf-1 at 1 ng / ml , there was a significant reduction in ve - cadherin and vegfr-2 mrna levels to 15.4% and 70.8% of control levels , respectively . tgf-1 at 5 ng / ml also significantly decreased ve - cadherin and vegfr-2 ; mrna levels were 21.8% and 66.1% of controls , respectively ( p<0.05 , figure 3a and b ) . we investigated the expression of cd34 , a marker of endothelial progenitor cells , in mlpcs . cd34 was expressed at a low level in mlpcs under control conditions ( relative mrna level is 2.34e-05 , data not shown ) . after 7 days , the addition of tgf-1 at 1 ng / ml and 5 ng / ml to the mesenchymal stem cell culture media significantly decreased the mrna level of cd34 to 28.7% and 25.1% of control levels , respectively ( p<0.05 , figure 4 ) . the mrna level of cd146 was low in mlpcs under control conditions ( relative mrna level is 3.8e-05 , data not shown ) . after 7 days , tgf-1 at 1 ng / ml and 5 ng / ml significantly increased the mrna level of cd146 to 2430% and 2605% of that of the controls , respectively ( p<0.05 , figure 5 ) . after 7 days of exposure to tgf-1 , several smc - specific markers were dramatically increased . the addition of tgf-1 ( 1 ng / ml ) to mesenchymal stem cell medium significantly increased the mrna levels of sm22 , calponin-1 , sm -actin , caldesmon , tropomyosin and myosin light chain kinase ( mlck ) to 1215.5% , 1974.6% , 567% , 429.7% , 567% and 162.8% , respectively , when compared to controls ( medium only ) ( p<0.05 , figure 1a f ) . tgf-1 ( 5 ng / ml ) also increased the mrna levels of these markers significantly ( p<0.05 ) , but the effect was less than that of the tgf-1 treatment at 1 ng / ml ( figure 1a f ) . in addition , we investigated the expression level of cd105 ( a tgf- accessory receptor ) in mlpcs . we found that cd105 is expressed at a high level in mlpcs under control conditions ( relative mrna level is 0.156 , data not shown ) . after 7 days , tgf-1 at 1 ng / ml and 5 ng / ml led to a significant downregulation of cd105 mrna levels to 61.7% and 70.8% of that of controls , respectively ( p<0.05 , figure 2 ) . two ec - specific markers , ve - cadherin and vegfr-2 , were detected in mlpcs under control conditions . after 7 days of exposure to tgf-1 at 1 ng / ml , there was a significant reduction in ve - cadherin and vegfr-2 mrna levels to 15.4% and 70.8% of control levels , respectively . tgf-1 at 5 ng / ml also significantly decreased ve - cadherin and vegfr-2 ; mrna levels were 21.8% and 66.1% of controls , respectively ( p<0.05 , figure 3a and b ) . we investigated the expression of cd34 , a marker of endothelial progenitor cells , in mlpcs . cd34 was expressed at a low level in mlpcs under control conditions ( relative mrna level is 2.34e-05 , data not shown ) . after 7 days , the addition of tgf-1 at 1 ng / ml and 5 ng / ml to the mesenchymal stem cell culture media significantly decreased the mrna level of cd34 to 28.7% and 25.1% of control levels , respectively ( p<0.05 , figure 4 ) . the mrna level of cd146 was low in mlpcs under control conditions ( relative mrna level is 3.8e-05 , data not shown ) . after 7 days , tgf-1 at 1 ng / ml and 5 ng / ml significantly increased the mrna level of cd146 to 2430% and 2605% of that of the controls , respectively ( p<0.05 , figure 5 ) . mlpcs originate from human umbilical cord blood , are karyotypically normal , demonstrate proliferative features , and are highly pure . in the present study , we observed the differentiation of mlpcs to smc lineage in the presence of tgf-1 as evidenced by an upregulation of the mrna levels of both early and late phase smc - specific markers and adhesion molecule cd146 . two ec markers , as well as epc marker cd34 and tgf-1 cell surface accessory protein cd105 , were decreased in tgf-1 treated mlpcs . as such , these data suggest that mlpcs represent a promising tool for the study of smc differentiation . quiescent vascular smcs exhibit a phenotype characterized by the expression of several contractile apparatus - associated proteins ; these are used as markers to identify smc lineage differentiation . we checked both early phase markers ( sm -actin , sm22 ) and late phase markers ( mlck , calponin-1 , caldesmon , and tropomyosin ) for smc differentiation in mlpcs . we found that treatment with tgf-1 led to a dramatic increase in the mrna levels of all the tested markers in mlpcs after 7 days of culture ; this strongly indicates the differentiation of mlpcs into smc lineage in the presence of tgf-1 . chen s et al . reported that tgf-1 ( 5 ng / ml ) increases the expression of sm -actin , sm22 , calponin-1 and myosin in the neural crest stem cell line monc-1 in vitro . . also found that tgf-1 ( 2 ng / ml ) induces sm -actin , sm22 and smooth muscle myosin heavy chain ( smmhc ) in 10t1/2 mesenchymal cells in vitro . in our study , the mrna levels of all the tested smc markers in mlpcs were upregulated to a comparable level with tgf-1 treatment when compared to those found in neural crest stem cell line ( monc-1 ) or 10t1/2 mesenchymal cells . the signaling pathway underlying tgf-1-induced smc - specific marker expression in mlpcs was not investigated in this study . investigated differential tgf--smad signaling for early versus late smc marker expression ; sm -actin promoter activity was found to be dependent on both smad 2 and smad 3 whereas smooth muscle myosin heavy chain ( smmhc ) activity is smad2 dependent in mouse embryonic stem cell . chen s et al . also found that tgf- increased sm -actin and sm22 in the neural crest stem cell line through activation of smad2 and smad3 . in addition , rhoa was reported to be essential in smad signaling during tgf--induced sm a - actin , sm22 and calponin expression in the neural crest stem cell line monc-1 . meanwhile , the pi3 kinase / akt pathway was found to be involved in tgf-1-induced sm -actin , sm22 and smmhc expression in 10t1/2 mesenchymal cells . cd105 is a transmembrane accessory receptor for tgf-. by forming a heteromeric complex with distinct tgf- receptors , it modulates the access of tgf- to the signaling complex and the postponed cellular responses to tgf- . we found that cd105 is expressed at a high level in mlpcs in the control condition ( relative mrna level is 0.156 ) , which may facilitate tgf-1/tgf- receptor signaling transduction . over a long duration of tgf-1 treatment ( 7 days ) , expression of cd105 was decreased , which may be due to a negative feedback mechanism . the specific signaling mechanism involved in tgf-1-induced differentiation of mlpcs into smc will need to be further elucidated . progenitor cells are primitive cells that possess the capacity to differentiate into multiple lineages under different conditions . evidence has shown that progenitor cells from human peripheral blood can differentiate into ec when stimulated by shear stress and into smcs in conditions of cyclic strain . hence , we investigated the expression of two ec specific markers to confirm that the potential for ec lineage differentiation by the mlpcs was suppressed under the designated conditions of this study . we found that the mrna levels of ve - cadherin and vegfr-2 were significantly decreased by tgf-1 on day 7 of culture . vegfr-2 is the pivotal receptor mediating the mitogenic action of vegf ; it plays an essential role in angiogenesis , neovascularization and epc differentiation . ve - cadherin is another ec - specific marker which has been shown to play important role in vasculogenesis and angiogenesis . the downregulated ve - cadherin and vegfr-2 observed in the mlpcs provides further confirmation that tgf-1 induced smc lineage differentiation in the mlpcs . similarly , chen s et al . found that tgf-1 reduced epithelial markers while inducing smc - specific markers in the neural crest stem cell line monc-1 . reported that tgf- inhibited proliferation and sheet formation of embryonic stem cell ( esc)-derived ecs . stimulation of esc - derived ecs with tgf- resulted in phosphorylation of both smad2 and smadl/5 . the specific signaling mechanism underlying the tgf-1-induced reduction in ec markers in mlpc need to be further studied . in addition to ec markers , we tested the epc marker cd34 in mlpcs . cd34 has been used as a hematopoietic stem cell marker ; endothelial progenitor cells are characterized by the expression of cd34 , cd133 and vegfr-2 . in this study , cd34 was expressed at a low level in mlpc under the control conditions , and expression was further reduced by tgf-1 treatment ; this suggests that the tendency for ec lineage differentiation by the mlcps was suppressed under the designated conditions . this in accordance with a previous finding that cd34 expression was downregulated during tgf-1 induced myofibroblast differentiation . cd146 , melanoma cell adhesion molecule , is also expressed in smc and plays an important role in vasculogenesis and embryo development . cd146 , when activated , induces association of p59 with cd146 , resulting in the phosphorylation of p125 and its binding with paxillin ; this finding suggests that cd146 is involved in outside - in signaling and may contribute to focal adhesion assembly , reorganization of the cytoskeleton , intercellular interaction , maintenance of cell shape , and control of cell migration and proliferation . in this study , cd146 was significantly increased by tgf-1 treatment in mlpcs , which may facilitate signaling transduction by tgf-1 and contribute to the differentiation of mlpcs into mature smc . in conclusion , a variety of smc - specific markers , including early and late phase markers , were dramatically increased in mlcp treated with tgf-1 . meanwhile , two ec - specific markers as well as the epc marker cd34 were significantly decreased . these data strongly indicate that mlcps differentiate into smc lineage cells in the presence of tgf-1 . mlpcs are karyotypically normal , non - transformed , non - immortalized cells that are obtained from post - partum human umbilical cord blood . because they have been expanded from a single cell and have the capacity to differentiate into multiple lineages , they are highly pure and proliferative . mlpcs offer significant advantages over other currently used cell models , such as 10t1/2 cells , the neural crest stem cell line monc-1 and smc progenitor cells from human peripheral blood for the study of smc differentiation . this study demonstrates a novel cell model for smc lineage differentiation analysis , which may increase our understanding of smc differentiation and help contribute to the field of vascular repair and tissue engineering .

summarybackgroundvascular smooth muscle cell ( smc ) differentiation is an essential component of vascular repair and tissue engineering . however , currently used cell models for the study of smc differentiation have several limitations . multi - lineage progenitor cells ( mlpcs ) originate from human umbilical cord blood and are cloned from a single cell . the object of this study was to investigate whether mlpcs could differentiate into smcs in vitro with induction by transforming growth factor 1 ( tgf-1).material / methodsmlpcs were treated without or with tgf-1 ( 1 and 5 ng / ml ) in mesenchymal stem cell media plus 1% fbs for 7 days . total rna was isolated from the mlpcs , and semi - quantitative real - time pcr was performed to test the following mrna levels : early and late phase smc - specific markers , two endothelial cell ( ec)-specific markers , endothelial progenitor cell ( epc ) marker cd34 , tgf-1 accessory protein cd105 , and adhesion molecule cd146.resultstgf-1 ( 1 ng / ml ) significantly increased the mrna levels of smc - specific markers sm22 , calponin-1 , sm -actin , caldesmon , tropomyosin and mlck as well as adhesion molecule cd146 . the mrna levels of ec - specific markers ve - cadherin and vegfr-2 , epc marker cd34 and tgf-1 accessory protein cd105 were decreased significantly , after mlpc were treated with tgf-1 ( 1 ng / ml ) . tgf-1 at 5 ng / ml showed similar effect on the expression of these genes.conclusionsthis study demonstrates that in the presence of tgf-1 , mlpcs undergo smc lineage differentiation indicating that mlpcs are a promising cell model for smc lineage differentiation studies , which may contribute to advances in vascular repair and tissue engineering .

Background Material and Methods Chemicals and reagents Cell culture Real-time PCR Statistical analysis Results TGF-1 increased the expression of SMC markers in MLPCs TGF-1 decreased the expression of endothelial cell-specific markers in MLPCs L2 TGF- TGF-1 increased the expression of adhesion molecule CD146 in MLPCs Discussion Conclusions

PMC2238938

homologs inherit similarities from their common ancestor , while structural analogs converge to similar structures due to a limited number of energetically favorable ways to pack secondary structural elements ( sses ) ( 1,2 ) . homology is inferred from sequence , structure and functional similarities , and several databases exist to catalog homologous proteins , e.g. pfam ( 3 ) , scop ( 4 ) and cath ( 5 ) . in contrast , analogy is more difficult to assess , and currently , no database exists that is devoted to structural analogs . traditionally , studies aimed at discriminating homologs and analogs from their structures use domains classified in the same scop superfamily as homologs and domains classified in the same scop fold but different superfamilies as analogs ( 68 ) . however , scop authors do not explicitly state that members of different superfamilies are necessarily analogous . domains are grouped in scop superfamilies when convincing evidence for their homology exists . when additional evidence comes to light , for instance , through newly determined structures , many studies argue for homology between certain scop superfamilies or even folds ( 1013 ) . hence , using domains in the same scop fold but different superfamilies as structural analogs is problematic because some domains in this category may be homologous . in an attempt to assemble a reliable set of structural analogs , we construct the malisam ( manual alignments for structurally analogous motifs ) database . by definition thus , to identify structural analogs convincingly , we need to minimize the possibility of homology . to this end , we explore three situations where we are more confident that the structural similarities have arisen in the absence of evolutionary relationships . in the first situation , a hybrid motif , formed by an insertion together with part of the evolutionary core of one domain family , may fortuitously match the core motif in another evolutionarily unrelated domain family . second , an interface motif , formed by part of each domain or subunit contacting along a domain domain or subunit subunit interface , may happen to be structurally similar to the core motif of another unrelated domain family . and third , an artificial protein , obtained through selection from random peptides or sequence design experiments not biased by sequences of a particular homologous family , is a bona fide analog to a structurally similar , natural protein . to determine if a structural motif is a hybrid motif , an interface motif , or a core motif , we use the descriptions of the involved fold , superfamily or family in the scop database , in particular , scop definitions of fold cores ( 4 ) . currently , malisam does not include examples of structural analogy between stand - alone , compact domains because , for stand - alone domains , we can not argue for analogy convincingly by ruling out homology , especially when the domains share stronger structural similarity . it is our hope that , by studying the reliable analogs in malisam , researchers can gain a better understanding of structural analogy and develop better approaches to discriminating between remote homologs and analogs . in this work , a structural motif refers to a set of sses with a specific spatial arrangement ( architecture ) and sequential connectivity ( topology ) , allowing for circular permutations ( 14 ) . two structural motifs are regarded as analogs if they come from evolutionarily distinct domain families , but share the same architecture and topology , allowing for circular permutations . a domain refers to a possible evolutionary unit with a well - defined hydrophobic core . domain. for instance , scop has a whole multi - domain protein class , in which , by definition , a scop in addition , our domains frequently correspond to duplicated structural repeats in scop , when such repeats constitute evolutionary units and form a well - defined hydrophobic core within each repeat with fewer interactions between repeats . the 130 pairs of analogous motifs in malisam are organized in the following three categories corresponding to the three situations discussed in introduction section . in evolution , a domain family usually preserves a common core structure while accumulating insertions and deletions in the periphery ( 15 ) . a core motif is composed entirely of sses belonging to the evolutionary core of a domain family , while a hybrid motif is composed of both core elements and peripheral insertions . a hybrid motif in one family may happen to be structurally similar to the core motif in another evolutionarily unrelated family . for instance , a hybrid -grasp motif found in formate dehydrogenase is formed by part of the ferredoxin core and a large c - terminal extension . this hybrid -grasp motif is analogous to the core -grasp motif in protein l ( 16 ) . this structural phenomenon is a special case of a general property called gregariousness ( 17 ) , i.e. proteins displaying partial structural similarity to many , frequently unrelated proteins . consider two ancient domain families with evolutionary cores denoted as ab and bc , respectively . as represented by b and b , these two families show partial similarity between their cores . although most members in the first family have structure ab , some members carry an additional part c and have structure abc . the bc motif in these proteins is structurally similar to the core motif bc in the second family . to justify analogy between bc and bc , it is necessary to demonstrate that c is an insertion rather than a deletion , i.e. the first family evolved from ab to abc instead of from abc to ab . if c is not present in the majority of the family members and is restricted to a particular phylogenetic or functional group within the family , it is most likely that proteins with structure ab are ancient and proteins with structure abc are comparatively new . in other words , ab is the ancient core of the family , and c is a recent insertion . thus , though ab and bc show partial structural similarity , they are not homologous , both being ancient and having different cores . since ab and abc are homologous , abc and bc can not be homologous . therefore , the hybrid motif bc and the core motif bc are structural analogs . figure 1 illustrates an analogous pair comprised of a hybrid motif and a core motif . glyoxalase / bleomycin resistance protein / dihydroxybiphenyl dioxygenase consists of four -strands and one -helix with the sequential connection of . one representative of this superfamily , the first domain in the antibiotic resistance protein fosa from pseudomonas aeruginosa ( 18 ) , is diagramed in figure 1a . in this domain ( termed structural repeat having glyoxalase fold in scop ) , the motif is a core motif , and no other sses are present . figure 1b depicts another domain that contains a motif , namely , the first domain in the oligopeptide - binding protein oppa from salmonella typhimurium ( 19 ) , which belongs to the scop superfamily periplasmic - binding protein - like ii. the core of this superfamily includes five -strands and their connecting helices , as illustrated in figure 1c by a more typical member in this superfamily , the first domain in lysine / arginine / ornithine - binding protein ( lao ) from s. typhimurium ( 20 ) . although fosa and oppa are classified in different scop superfamilies , folds and even classes , the two motifs boxed in figure 1a and b are similar in both architecture and topology . a comparison of figure 1b and c suggests that the -hairpin highlighted in red , which is present in oppa but absent in lao , is an insertion . indeed , a manual inspection of the structures in this superfamily shows that the -hairpin is present in only 4 out of the superfamily 's 32 proteins . moreover , these four proteins form a more closely related subgroup as indicated by their higher sequence identities to one another than to other proteins in this superfamily . all these observations suggest that the -hairpin does not belong to the ancient core of this superfamily and is instead an insertion that appeared later in evolution . thus , the motif in oppa ( figure 1b ) has a hybrid origin : the red -hairpin is an insertion while the remaining two strands and one helix are core elements . therefore , the hybrid motif in oppa and the core motif in fosa ( figure 1a ) define a pair of analogs . figure 1.an analogous pair composed of a hybrid motif and a core motif . ( a ) the first domain in fosfomycin resistance protein a ( fosa ) from p. aeruginosa ( pdb code 1nki ) . the core region is colored from deep blue ( n - terminus ) to pale blue ( c - terminus ) . ( b ) the first domain in oligopeptide - binding protein ( oppa ) from s. typhimurium ( pdb code 1b05 ) . the core region is colored from deep green ( n - terminus ) to pale green ( c - terminus ) , and the inserted -hairpin is colored in red . ( c ) the first domain of lysine / arginine / ornithine - binding protein ( lao ) from s. typhimurium ( pdb code 1lst ) . the core region is colored from deep green ( n - terminus ) to pale green ( c - terminus ) . the pdb code , chain identifier and starting and ending residue numbers are given for each sequence . ( a ) the first domain in fosfomycin resistance protein a ( fosa ) from p. aeruginosa ( pdb code 1nki ) . the core region is colored from deep blue ( n - terminus ) to pale blue ( c - terminus ) . ( b ) the first domain in oligopeptide - binding protein ( oppa ) from s. typhimurium ( pdb code 1b05 ) . the core region is colored from deep green ( n - terminus ) to pale green ( c - terminus ) , and the inserted -hairpin is colored in red . ( c ) the first domain of lysine / arginine / ornithine - binding protein ( lao ) from s. typhimurium ( pdb code 1lst ) . the core region is colored from deep green ( n - terminus ) to pale green ( c - terminus ) . the pdb code , chain identifier and starting and ending residue numbers are given for each sequence . we use the following procedure to find analogous pairs consisting of a hybrid motif and a core motif . first , we represent the most commonly found structure motifs ( 21,22 ) or their circularly permuted forms by matrices and use these matrices as queries in prosmos ( 23 ) searches to find scop domains that contain the query motif . we inspect one randomly selected representative domain from each superfamily to determine whether the query motif occurring in this superfamily is a hybrid or not , according to scop description of the core of this superfamily . sses that do not belong to the core are considered to be insertions , and a hybrid motif should contain both core elements and inserted elements . the coordinates of a hybrid motif are extracted from the original pdb file and used as a query in dali ( 24 ) searches against a culled pdb database , in which any two proteins share < 50% sequence identity . the hits are sorted by dali z score and inspected manually to select the most similar core motif(s ) to the query . the hybrid motif and the selected core motif are then added to the database as a pair of analogs . currently , there are 91 pairs of such analogs in malisam . an interface motif is formed by part of each domain or subunit contacting along a domain domain or subunit subunit interface . in compact multi - domain proteins or obligate multi - chain complexes , where domains or subunits are closely associated , an interface motif may be well defined enough to mimic the structure and topology of a core motif , which is composed entirely of sses belonging to the evolutionary core of a domain family . for the interface motif and the core motif to be a pair of analogs , they should come from evolutionarily unrelated domain families . particularly , the interface motif should not cover the complete core of any of the contributing domains or subunits because in that case , the core motif might be a duplicated or elaborated version of that completely covered domain or subunit . figure 2 shows an analogous pair comprised of an interface motif and a core motif . the actin filament capping protein capz ( figure 2a ) is a heterodimer of two homologous subunits and ( 25 ) . scop divides each subunit into three domains , with the n - terminal domain being a 3-helical bundle ( diagramed in ribbons in figure 2a ) . the two 3-helical bundle domains from the two subunits make many hydrophobic contacts at the dimer interface , forming a 4-helical bundle ( framed in red ) . this 4-helical bundle defines an interface motif , consisting of part of the 3-helical bundle from the subunit followed by part of the 3-helical bundle from the subunit . figure 2b depicts one monomer in the conserved hypothetical protein xcc0516 homopentamer from xanthomonas campestris ( 26 ) . this 4-helical bundle in xcc0516 comprises the whole polypeptide chain and is a core motif . the interface 4-helical bundle in capz ( boxed in red in figure 2a ) shares the same overall structure and topology with the upper portion of the core 4-helical bundle in xcc0516 ( boxed in red in figure 2b ) . yet the interface motif comes from a 3-helical bundle domain family , while the core motif comes from an evolutionarily unrelated 4-helical bundle domain family . therefore , the interface motif in capz and the core motif in xcc0516 can be regarded as a pair of analogs . ( a ) the actin filament capping protein capz from gallus gallus ( pdb code 1izn ) . the three domains in each subunit are represented as a ribbon diagram , a pentagon and a rectangle , respectively . ( b ) the conserved hypothetical protein xcc0516 from x. campestris ( pdb code 2gsc ) . the polypeptide chain is colored in rainbow from n - terminus ( blue ) to c - terminus ( orange ) . the part that is aligned to the hybrid 4-helical bundle motif in capz is in red frame . the disordered region between the 2nd and the 3rd helices is represented by a dotted curve . the helices in the red frames in ( a ) and ( b ) are diagramed as cylinders in the same colors above or below their corresponding sequences . the pdb code , chain identifier and starting and ending residue numbers are given for each sequence . ( a ) the actin filament capping protein capz from gallus gallus ( pdb code 1izn ) . the three domains in each subunit are represented as a ribbon diagram , a pentagon and a rectangle , respectively . ( b ) the conserved hypothetical protein xcc0516 from x. campestris ( pdb code 2gsc ) . the polypeptide chain is colored in rainbow from n - terminus ( blue ) to c - terminus ( orange ) . the part that is aligned to the hybrid 4-helical bundle motif in capz is in red frame . the disordered region between the 2nd and the 3rd helices is represented by a dotted curve . the helices in the red frames in ( a ) and ( b ) are diagramed as cylinders in the same colors above or below their corresponding sequences . the pdb code , chain identifier and starting and ending residue numbers are given for each sequence . the same procedure described above to find hybrid motifs is used to identify interface motifs . in addition , we manually inspected the scop entries in the multi - domain proteins class or with duplication in their annotations in a search for interface motifs . the coordinates of an interface motif are extracted from the original pdb file and used as a query in dali ( 24 ) searches against the aforementioned , culled pdb database to find the most similar core motif(s ) for it . note that , before running dali , the coordinates file of a subunit subunit interface motif is preprocessed so that it contains only one chain . currently , malisam stores 33 pairs of analogs that are composed of an interface motif and a core motif . an artificial protein and a natural protein we analyze the entries in the scop class designed proteins and select the ones with de novo sequences and reasonably complex structures to serve as queries in dali searches against the aforementioned representative pdb database . we pay attention to whether an artificial protein is designed without a sequence bias towards a particular homologous family , e.g. the artificially designed ww domain ( pdb 1e0 m ) will not work as an analog to natural ww domains , because it is based largely on the consensus sequence of ww domain family . by inspecting top dali hits , we select the most similar natural proteins for an artificial protein query . currently , malisam contains six pairs of analogs that consist of an artificial protein and a natural protein , but we expect this number to increase due to active research in the field of protein sequence design . each analogous pair is aligned manually by visual inspection and automatically by three programs [ dali ( 28 ) , tm - align ( 29 ) and fast ( 30 ) ] . in constructing the manual alignment for a pair , we first superimpose the two motifs in the insight ii software according to their corresponding sses , then transform the structural superposition into sequence alignment following these general rules : ( i ) corresponding sses are aligned and loops are frequently ignored ; ( ii ) h - bonding networks in -sheets are followed , i.e. if two residues are aligned , their respective h - bond partners are also aligned and ( iii ) gap openings are avoided as much as possible . different aligners , manual or automatic , may give different structural alignments for the same pair of structures . for example , since -strands are repeats of two - residue units , one can superimpose two -strands in several ways or registers by shifting one strand relative to the other by two residues at a time . such kind of ambiguity in structural alignments has been discussed in the literature ( 31,32 ) . to quantify the similarity between a manual alignment and an automatic alignment , we compute an agreement by dividing the number of positions aligned the same way in the two alignments by the total number of aligned positions in the manual alignment . the distribution of these agreements is shown in figure 3 . out of the three programs used frequently , the disagreements between different aligners result from the aforementioned register or shifting problem . the horizontal axis corresponds to the ranges of the agreement bins , and the vertical axis corresponds to the number of pairs that fall into each bin . the horizontal axis corresponds to the ranges of the agreement bins , and the vertical axis corresponds to the number of pairs that fall into each bin . to characterize a manual or automatic alignment between two analogous motifs , we compute six alignment - based scores , namely , aligned length , sequence identity , c rmsd , gdt_ts ( 33 ) , compass - like score and consensus . sequence identity and compass - like score measure how similar the two motifs are in sequence by comparing two single sequences or two multiple sequence alignments , respectively . in calculating the compass - like score , two sequence profiles , one for each motif , are generated by running psi - blast ( 34 ) , aligned according to the manual or automatic structure alignment , and then compared and scored by the scoring function used in the compass program ( 35 ) . in calculating the consensus score , we compare the four alignments generated by the four aligners ( manual , dali , tm - align and fast ) , assuming that similarities captured by different aligners are more likely to be correct . for each aligner , the consensus score equals the percent of its alignment that is agreed by at least one of the other three aligners . ( 36 ) or by clicking a score name on a pair - specific page at the malisam website . table 1 shows the mean and the standard error of each score and each aligner over the 130 pairs in malisam . manual alignments are typically shorter than dali and tm alignments , but longer than fast alignments . also , manual alignments have the best average rmsd , gdt_ts and consensus score , implying their higher overall quality than the automatic alignments . table 1.mean and standard error of various scores for each alignerdalitm - alignfastmanualaligned length ( a.a.)60.96 1.0461.12 1.2350.83 1.0256.69 1.00sequence identity ( % ) 8.21 0.328.06 0.318.61 0.358.54 0.36rmsd ( )3.11 0.053.06 0.053.23 0.222.92 0.06gdt_ts ( % ) 59.05 0.5960.24 0.6357.18 0.9460.53 0.73compass- like11.46 0.7412.08 0.7610.45 0.7610.75 0.72consensus ( % ) 67.02 2.4847.41 2.9066.84 2.7673.93 2.37the mean and the standard error for each score and each aligner . for rmsd , a smaller value indicates higher similarity ; for all other scores , a larger value means higher similarity . mean and standard error of various scores for each aligner the mean and the standard error for each score and each aligner . for rmsd , a smaller value indicates higher similarity ; for all other scores , a larger value means higher similarity . in evolution , a domain family usually preserves a common core structure while accumulating insertions and deletions in the periphery ( 15 ) . a core motif is composed entirely of sses belonging to the evolutionary core of a domain family , while a hybrid motif is composed of both core elements and peripheral insertions . a hybrid motif in one family may happen to be structurally similar to the core motif in another evolutionarily unrelated family . for instance , a hybrid -grasp motif found in formate dehydrogenase is formed by part of the ferredoxin core and a large c - terminal extension . this hybrid -grasp motif is analogous to the core -grasp motif in protein l ( 16 ) . this structural phenomenon is a special case of a general property called gregariousness ( 17 ) , i.e. proteins displaying partial structural similarity to many , frequently unrelated proteins . consider two ancient domain families with evolutionary cores denoted as ab and bc , respectively . as represented by b and b , these two families show partial similarity between their cores . although most members in the first family have structure ab , some members carry an additional part c and have structure abc . the bc motif in these proteins is structurally similar to the core motif bc in the second family . to justify analogy between bc and bc , it is necessary to demonstrate that c is an insertion rather than a deletion , i.e. the first family evolved from ab to abc instead of from abc to ab . if c is not present in the majority of the family members and is restricted to a particular phylogenetic or functional group within the family , it is most likely that proteins with structure ab are ancient and proteins with structure abc are comparatively new . in other words , ab is the ancient core of the family , and c is a recent insertion . thus , though ab and bc show partial structural similarity , they are not homologous , both being ancient and having different cores . since ab and abc are homologous , abc and bc can not be homologous . therefore , the hybrid motif bc and the core motif bc are structural analogs . figure 1 illustrates an analogous pair comprised of a hybrid motif and a core motif . glyoxalase / bleomycin resistance protein / dihydroxybiphenyl dioxygenase consists of four -strands and one -helix with the sequential connection of . one representative of this superfamily , the first domain in the antibiotic resistance protein fosa from pseudomonas aeruginosa ( 18 ) , is diagramed in figure 1a . in this domain ( termed structural repeat having glyoxalase fold in scop ) , the motif is a core motif , and no other sses are present . figure 1b depicts another domain that contains a motif , namely , the first domain in the oligopeptide - binding protein oppa from salmonella typhimurium ( 19 ) , which belongs to the scop superfamily periplasmic - binding protein - like ii. the core of this superfamily includes five -strands and their connecting helices , as illustrated in figure 1c by a more typical member in this superfamily , the first domain in lysine / arginine / ornithine - binding protein ( lao ) from s. typhimurium ( 20 ) . although fosa and oppa are classified in different scop superfamilies , folds and even classes , the two motifs boxed in figure 1a and b are similar in both architecture and topology . a comparison of figure 1b and c suggests that the -hairpin highlighted in red , which is present in oppa but absent in lao , is an insertion . indeed , a manual inspection of the structures in this superfamily shows that the -hairpin is present in only 4 out of the superfamily 's 32 proteins . moreover , these four proteins form a more closely related subgroup as indicated by their higher sequence identities to one another than to other proteins in this superfamily . all these observations suggest that the -hairpin does not belong to the ancient core of this superfamily and is instead an insertion that appeared later in evolution . thus , the motif in oppa ( figure 1b ) has a hybrid origin : the red -hairpin is an insertion while the remaining two strands and one helix are core elements . therefore , the hybrid motif in oppa and the core motif in fosa ( figure 1a ) define a pair of analogs . ( a ) the first domain in fosfomycin resistance protein a ( fosa ) from p. aeruginosa ( pdb code 1nki ) . the core region is colored from deep blue ( n - terminus ) to pale blue ( c - terminus ) . ( b ) the first domain in oligopeptide - binding protein ( oppa ) from s. typhimurium ( pdb code 1b05 ) . the core region is colored from deep green ( n - terminus ) to pale green ( c - terminus ) , and the inserted -hairpin is colored in red . the hybrid motif is framed in yellow . ( c ) the first domain of lysine / arginine / ornithine - binding protein ( lao ) from s. typhimurium ( pdb code 1lst ) . the core region is colored from deep green ( n - terminus ) to pale green ( c - terminus ) . the pdb code , chain identifier and starting and ending residue numbers are given for each sequence . ( a ) the first domain in fosfomycin resistance protein a ( fosa ) from p. aeruginosa ( pdb code 1nki ) . the core region is colored from deep blue ( n - terminus ) to pale blue ( c - terminus ) . ( b ) the first domain in oligopeptide - binding protein ( oppa ) from s. typhimurium ( pdb code 1b05 ) . the core region is colored from deep green ( n - terminus ) to pale green ( c - terminus ) , and the inserted -hairpin is colored in red . ( c ) the first domain of lysine / arginine / ornithine - binding protein ( lao ) from s. typhimurium ( pdb code 1lst ) . the core region is colored from deep green ( n - terminus ) to pale green ( c - terminus ) . the pdb code , chain identifier and starting and ending residue numbers are given for each sequence . we use the following procedure to find analogous pairs consisting of a hybrid motif and a core motif . first , we represent the most commonly found structure motifs ( 21,22 ) or their circularly permuted forms by matrices and use these matrices as queries in prosmos ( 23 ) searches to find scop domains that contain the query motif . we inspect one randomly selected representative domain from each superfamily to determine whether the query motif occurring in this superfamily is a hybrid or not , according to scop description of the core of this superfamily . sses that do not belong to the core are considered to be insertions , and a hybrid motif should contain both core elements and inserted elements . the coordinates of a hybrid motif are extracted from the original pdb file and used as a query in dali ( 24 ) searches against a culled pdb database , in which any two proteins share < 50% sequence identity . the hits are sorted by dali z score and inspected manually to select the most similar core motif(s ) to the query . the hybrid motif and the selected core motif are then added to the database as a pair of analogs . an interface motif is formed by part of each domain or subunit contacting along a domain domain or subunit subunit interface . in compact multi - domain proteins or obligate multi - chain complexes , where domains or subunits are closely associated , an interface motif may be well defined enough to mimic the structure and topology of a core motif , which is composed entirely of sses belonging to the evolutionary core of a domain family . for the interface motif and the core motif to be a pair of analogs , they should come from evolutionarily unrelated domain families . particularly , the interface motif should not cover the complete core of any of the contributing domains or subunits because in that case , the core motif might be a duplicated or elaborated version of that completely covered domain or subunit . figure 2 shows an analogous pair comprised of an interface motif and a core motif . the actin filament capping protein capz ( figure 2a ) is a heterodimer of two homologous subunits and ( 25 ) . scop divides each subunit into three domains , with the n - terminal domain being a 3-helical bundle ( diagramed in ribbons in figure 2a ) . the two 3-helical bundle domains from the two subunits make many hydrophobic contacts at the dimer interface , forming a 4-helical bundle ( framed in red ) . this 4-helical bundle defines an interface motif , consisting of part of the 3-helical bundle from the subunit followed by part of the 3-helical bundle from the subunit . figure 2b depicts one monomer in the conserved hypothetical protein xcc0516 homopentamer from xanthomonas campestris ( 26 ) . this 4-helical bundle in xcc0516 comprises the whole polypeptide chain and is a core motif . the interface 4-helical bundle in capz ( boxed in red in figure 2a ) shares the same overall structure and topology with the upper portion of the core 4-helical bundle in xcc0516 ( boxed in red in figure 2b ) . yet the interface motif comes from a 3-helical bundle domain family , while the core motif comes from an evolutionarily unrelated 4-helical bundle domain family . therefore , the interface motif in capz and the core motif in xcc0516 can be regarded as a pair of analogs . figure 2.an analogous pair composed of an interface motif and a core motif . ( a ) the actin filament capping protein capz from gallus gallus ( pdb code 1izn ) . the three domains in each subunit are represented as a ribbon diagram , a pentagon and a rectangle , respectively . ( b ) the conserved hypothetical protein xcc0516 from x. campestris ( pdb code 2gsc ) . the polypeptide chain is colored in rainbow from n - terminus ( blue ) to c - terminus ( orange ) . the part that is aligned to the hybrid 4-helical bundle motif in capz is in red frame . the disordered region between the 2nd and the 3rd helices is represented by a dotted curve . the helices in the red frames in ( a ) and ( b ) are diagramed as cylinders in the same colors above or below their corresponding sequences . the pdb code , chain identifier and starting and ending residue numbers are given for each sequence . ( a ) the actin filament capping protein capz from gallus gallus ( pdb code 1izn ) . the three domains in each subunit are represented as a ribbon diagram , a pentagon and a rectangle , respectively . ( b ) the conserved hypothetical protein xcc0516 from x. campestris ( pdb code 2gsc ) . the polypeptide chain is colored in rainbow from n - terminus ( blue ) to c - terminus ( orange ) . the part that is aligned to the hybrid 4-helical bundle motif in capz is in red frame . the disordered region between the 2nd and the 3rd helices is represented by a dotted curve . the helices in the red frames in ( a ) and ( b ) are diagramed as cylinders in the same colors above or below their corresponding sequences . the pdb code , chain identifier and starting and ending residue numbers are given for each sequence . the same procedure described above to find hybrid motifs is used to identify interface motifs . in addition , we manually inspected the scop entries in the multi - domain proteins class or with duplication in their annotations in a search for interface motifs . the coordinates of an interface motif are extracted from the original pdb file and used as a query in dali ( 24 ) searches against the aforementioned , culled pdb database to find the most similar core motif(s ) for it . note that , before running dali , the coordinates file of a subunit subunit interface motif is preprocessed so that it contains only one chain . currently , malisam stores 33 pairs of analogs that are composed of an interface motif and a core motif . an artificial protein and a natural protein are a pair of analogs if they are structurally similar . we analyze the entries in the scop class designed proteins and select the ones with de novo sequences and reasonably complex structures to serve as queries in dali searches against the aforementioned representative pdb database . we pay attention to whether an artificial protein is designed without a sequence bias towards a particular homologous family , e.g. the artificially designed ww domain ( pdb 1e0 m ) will not work as an analog to natural ww domains , because it is based largely on the consensus sequence of ww domain family . by inspecting top dali hits currently , malisam contains six pairs of analogs that consist of an artificial protein and a natural protein , but we expect this number to increase due to active research in the field of protein sequence design . each analogous pair is aligned manually by visual inspection and automatically by three programs [ dali ( 28 ) , tm - align ( 29 ) and fast ( 30 ) ] . in constructing the manual alignment for a pair , we first superimpose the two motifs in the insight ii software according to their corresponding sses , then transform the structural superposition into sequence alignment following these general rules : ( i ) corresponding sses are aligned and loops are frequently ignored ; ( ii ) h - bonding networks in -sheets are followed , i.e. if two residues are aligned , their respective h - bond partners are also aligned and ( iii ) gap openings are avoided as much as possible . different aligners , manual or automatic , may give different structural alignments for the same pair of structures . for example , since -strands are repeats of two - residue units , one can superimpose two -strands in several ways or registers by shifting one strand relative to the other by two residues at a time . such kind of ambiguity in structural alignments has been discussed in the literature ( 31,32 ) . to quantify the similarity between a manual alignment and an automatic alignment , we compute an agreement by dividing the number of positions aligned the same way in the two alignments by the total number of aligned positions in the manual alignment . the distribution of these agreements is shown in figure 3 . out of the three programs used frequently , the disagreements between different aligners result from the aforementioned register or shifting problem . the horizontal axis corresponds to the ranges of the agreement bins , and the vertical axis corresponds to the number of pairs that fall into each bin . the horizontal axis corresponds to the ranges of the agreement bins , and the vertical axis corresponds to the number of pairs that fall into each bin . to characterize a manual or automatic alignment between two analogous motifs , we compute six alignment - based scores , namely , aligned length , sequence identity , c rmsd , gdt_ts ( 33 ) , compass - like score and consensus . sequence identity and compass - like score measure how similar the two motifs are in sequence by comparing two single sequences or two multiple sequence alignments , respectively . in calculating the compass - like score , two sequence profiles , one for each motif , are generated by running psi - blast ( 34 ) , aligned according to the manual or automatic structure alignment , and then compared and scored by the scoring function used in the compass program ( 35 ) . in calculating the consensus score , we compare the four alignments generated by the four aligners ( manual , dali , tm - align and fast ) , assuming that similarities captured by different aligners are more likely to be correct . for each aligner , the consensus score equals the percent of its alignment that is agreed by at least one of the other three aligners . ( 36 ) or by clicking a score name on a pair - specific page at the malisam website . table 1 shows the mean and the standard error of each score and each aligner over the 130 pairs in malisam . manual alignments are typically shorter than dali and tm alignments , but longer than fast alignments . also , manual alignments have the best average rmsd , gdt_ts and consensus score , implying their higher overall quality than the automatic alignments . table 1.mean and standard error of various scores for each alignerdalitm - alignfastmanualaligned length ( a.a.)60.96 1.0461.12 1.2350.83 1.0256.69 1.00sequence identity ( % ) 8.21 0.328.06 0.318.61 0.358.54 0.36rmsd ( )3.11 0.053.06 0.053.23 0.222.92 0.06gdt_ts ( % ) 59.05 0.5960.24 0.6357.18 0.9460.53 0.73compass- like11.46 0.7412.08 0.7610.45 0.7610.75 0.72consensus ( % ) 67.02 2.4847.41 2.9066.84 2.7673.93 2.37the mean and the standard error for each score and each aligner . for rmsd , a smaller value indicates higher similarity ; for all other scores , a larger value means higher similarity . mean and standard error of various scores for each aligner the mean and the standard error for each score and each aligner . for rmsd , a smaller value indicates higher similarity ; for all other scores , a larger value means higher similarity . clicking a pair name redirects the browser to that pair 's specific page ( figure 4 ) , which contains a summary table , a short justification for analogy , a table of alignment - based scores and structure - based sequence alignments . the summary table provides basic information about the two motifs in that pair : pdb code , chain identifier , residue ranges and scop code and classification . the structural superposition reconstructed from a manual or automatic alignment can be viewed in pymol ( http://pymol.sourceforge.net/ ) by selecting the aligned motifs in pymol link or can be downloaded by selecting the pdb format superposition link . a compressed file of the entire database with alignments of all pairs can be downloaded from ftp://iole.swmed.edu/pub/cheng/analogs/analogs.tar . the malisam database is a compilation of manual as well as automatic alignments between structurally analogous motifs . to the best of our knowledge , malisam may be used in protein - folding studies , for the similarities between analogs result from folding and packing constraints rather than evolutionary relatedness . a reliable set of structural analogs provided in malisam could be used in the training of remote homology detection methods , since these methods frequently involve discrimination between homologs and structural analogs . additionally , malisam manual alignments should be useful for the development of better automatic structure alignment techniques .

malisam ( manual alignments for structurally analogous motifs ) represents the first database containing pairs of structural analogs and their alignments . to find reliable analogs , we developed an approach based on three ideas . first , an insertion together with a part of the evolutionary core of one domain family ( a hybrid motif ) is analogous to a similar motif contained within the core of another domain family . second , a motif at an interface , formed by secondary structural elements ( sses ) contributed by two or more domains or subunits contacting along that interface , is analogous to a similar motif present in the core of a single domain . third , an artificial protein obtained through selection from random peptides or in sequence design experiments not biased by sequences of a particular homologous family , is analogous to a structurally similar natural protein . each analogous pair is superimposed and aligned manually , as well as by several commonly used programs . applications of this database may range from protein evolution studies , e.g. development of remote homology inference tools and discriminators between homologs and analogs , to protein - folding research , since in the absence of evolutionary reasons , similarity between proteins is caused by structural and folding constraints . the database is publicly available at http://prodata.swmed.edu/malisam .

INTRODUCTION CONTENTS AND METHODS A hybrid motif and a core motif An interface motif and a core motif An artificial protein and a natural protein Alignments and scores WEB INTERFACE CONCLUSION

PMC4153285

all experiments complied with eu council directive 91/630/eec , which lays down minimum standards for the protection of pigs , and eu council directive 98/58/ec , which concerns the protection of animals kept for farming purposes . . closely related f1 gilts ( large white landrace ) were selected as replacement breeding stock at birth on a commercial breeding company 's ( hermitage ai ) multiplier farm and exposed to the same group housing and feeding regimens up to final selection at 22 weeks of age . at this time , 158 gilts were selected on their backfat levels at the p2-site ( 79 mm or 1014 mm , respectively ) and categorised as thin ( n 78 ) or fat ( n 80 ) . backfat depth was measured at the last rib and 65 mm from the backbone of the gilt using an ultrasound scanner ( lean - meater , renco corporation ) on both the left and right side and the mean was recorded . the difference in backfat between groups was nutritionally accentuated until service ( 32 weeks ) when the backfat depths of thin and fat gilts were 12 ( sem 06 ) and 19 ( sem 06 ) mm , respectively . between 22 and 30 weeks of age , thin gilts were fed 18 kg / d dry sow diet ( 619 g / kg lysine , 130 mj digestible energy ( de)/kg ) and the fat gilts were provided with ad libitum access to a gilt developer diet ( 585 g / kg lysine , 143 mj de / kg ) ( table 1 ) . for 4 weeks before planned mating , gilts were provided with constant boar contact by introducing a boar into an adjoining pen . at 2 weeks before service , all gilts were provided with ad libitum access to a lactation sow diet for the flushing effect ( table 1 ) . a quantity of 9 ml of regumate equine ( altrenogest , 2.2 g / ml ; intervet productions s.a . ) per gilt was added to the lactation sow diet daily for 6 d to synchronise gilts to oestrus . gilts were provided with 10 min boar contact twice daily to aid oestrus detection once they came off the regumate . gilts were artificially inseminated at onset of standing oestrus and again 24 h later using semen pooled from eight closely related hylean large white boars ( hermitage ai ) . immediately after service , gilts were moved to individual gestation pens ( 24 m 06 m ; o'donovan engineering ) where they were fed , once per d , the dry sow diet ( table 1 ) until day 110 of gestation . each gilt was fed 18 kg / d ( 234 mj de / d ) for the first 25 d of gestation . on day 25 of gestation , gilts from each body condition group ( fat or thin ) were blocked according to weight and expected farrowing date and allocated at random to one of three feeding levels : ( a ) restricted ( 18 kg / d ) , ( b ) control ( 25 kg / d ) or ( c ) high feed level ( 35 kg / d ) , until day 90 of gestation . in total there were six treatment groups : thin restricted , thin control , thin high feed level , fat restricted , fat control and fat high feed level . after day 90 of gestation all gilts were fed 25 kg / d through to day 110 . 110 gilts were moved to individual farrowing pens and liquid fed ( big dutchman ) the lactation diet ( table 1 ) until farrowing at an allocation of 203 kg / d ( 288 mj de / d ) . gilts were accommodated in farrowing rooms with ten gilts per room in national pig development company type farrowing crates ( o'donovan engineering ) with hinged bottom bars . after farrowing , gilts were scale fed the lactation diet using a lactation feed curve increasing from 288 mj de / d at day 0 to 1127 mj de / d at day 28 post - farrowing . room temperature was maintained at 20c except at farrowing when the temperature was increased to 24c for 48 h. at farrowing litter weight , total born , born alive , stillbirths and mummies were recorded as well as individual piglet birth weights . litter size was standardized at farrowing to approximately twelve pigs per litter by cross - fostering within treatment groups within 24 h of birth . the offspring were then kept with the gilt and suckled by her for the first 28 d of their life . in addition , a creep feed ( 165 mj / de , lysine 16 % ; startrite 88 ; nutec ) was fed to all litters from day 12 postpartum to weaning at approximately day 28 postpartum . table 1.composition of experimental diets ( on an air dry basis ; g / kg)*gilt developer dietdry sow dietlactation dietwheat4239barley83298929350soyabean meal ( 50 % crude protein)7575160soyabean oil701040mineral and vitamins151515lysine hcl050520dl - methionine0007l - threonine0008di - calcium phosphate555limestone flour111112salt444phytase010101chemical compositiondm870871873crude protein153116158crude fat852639crude fibre323430ash393643lysine5856291digestible energy ( mj / kg)143130142*dry sow diet and lactation diet provided ( mg / kg completed diet ) : cu , 30 mg ; fe , 70 mg ; mn , 62 mg ; zn , 80 mg ; i , 06 mg ; se , 02 mg ; vitamin a as retinyl acetate , 3 mg ; vitamin d3 , as cholecalciferol , 25 g ; vitamin e as dl--tocopheryl acetate , 100 mg ; vitamin k , 2 mg ; vitamin b12 , 15 g ; riboflavin , 5 mg ; nicotinic acid , 12 mg ; pantothenic acid , 10 mg ; choline chloride , 500 mg ; biotin , 200 g ; folic acid , 5 mg ; thiamin , 2 mg ; pyridoxine , 3 mg. synthetic amino acids. sow diets contained 500 phytase units ( ftu ) per kg finished feed from natuphos 5000 ( basf). calculated from standard book values for ingredients . composition of experimental diets ( on an air dry basis ; g / kg ) * dry sow diet and lactation diet provided ( mg / kg completed diet ) : cu , 30 mg ; fe , 70 mg ; mn , 62 mg ; zn , 80 mg ; i , 06 mg ; se , 02 mg ; vitamin a as retinyl acetate , 3 mg ; vitamin d3 , as cholecalciferol , 25 g ; vitamin e as dl--tocopheryl acetate , 100 mg ; vitamin k , 2 mg ; vitamin b12 , 15 g ; riboflavin , 5 mg ; nicotinic acid , 12 mg ; pantothenic acid , 10 mg ; choline chloride , 500 mg ; biotin , 200 g ; folic acid , 5 mg ; thiamin , 2 mg ; pyridoxine , 3 mg . synthetic amino acids . sow diets contained 500 phytase units ( ftu ) per kg finished feed from natuphos 5000 ( basf ) . calculated from standard book values for ingredients . at weaning , gilts were moved to individual pens in the service area and provided with ad libitum access to the lactation diet in pellet form until service . the weaning to oestrus interval and feed intake during this period gilts were returned to the dry sow accommodation and provided with the dry sow diet in liquid form ( 30 mj de / d ( about 23 kg / d on a fresh weight basis ) ) through to the subsequent farrowing . gilts were weighed and backfat measurements were taken at about 22 weeks , at service ( about 32 weeks ) on day 25 , 50 , 80 and 110 of gestation , at weaning and at the subsequent oestrus . feed allocation is presented on a meal equivalent fresh weight ( kg / d ) basis for each period ( table 1 ) . to allow time for the gilts to adapt to their housing and their respective feeding levels , a cotton swab ( salivette plain ) was attached to a pair of surgical tongs and placed in the mouth of the gilt at three time points during the day : 09.30 ( prior to feeding ) , 12.30 and 15.30 hours . gilts were allowed to chew on the cotton swab for approximately 30 s , or until saturated . swabs were then returned to the container and centrifuged at 1000 g for 2 min at room temperature within 1 h of collection . saliva samples were assayed for cortisol levels in duplicate by a salivary cortisol eia kit ( salimetrics ) . cortisol concentration was quantified by interpolating absorbance readings from a standard curve generated in the same assay . a colostrum sample ( about 5 ml ) colostrum samples were manually collected from teats at anterior , middle and posterior locations of the udder , pooled and immediately frozen at 20c for subsequent analysis . colostrum samples were analysed in duplicate for igg levels using specific igg pig - elisa kits ( bethyl laboratories inc . ) . the igg levels were quantified according to the manufacturer 's instructions by interpolating absorbance readings from standard curves generated in the same assay . milk samples ( about 15 ml ) were collected from gilts after their morning meal on day 21 of lactation which represents peak lactation . piglets were removed from the gilt in the morning in order to facilitate refill of the mammary gland before sampling at noon . milk samples were manually collected from teats at anterior , middle and posterior locations of the udder , after a 1 ml ( 10 iu ) intramuscular injection of oxytocin ( eurovet animal health ) to induce milk let down . milk samples were thawed at 4c and preservatives were added to prolong shelf life ( broad spectrum microtabs ii ; d&f control systems inc . ) . milk samples were diluted with distilled water prior to analysis to ensure that sample composition fell within the validated calibration range for the infrared analyser . samples were heated to 40c and analysed in duplicate for percentage fat , protein and lactose concentrations on an infrared analyser ( milkoscan ft 6000 ; foss electric ) . protein content was validated using the kjeldahl method ( result r 09396 ) according to iso . gottlieb technique ( result r 09313 ) according to james . following extraction , 30 mg of the respective milk fat were dissolved in hexane and transesterfied at room temperature by the addition of 200 l of 2 m - methanolic koh . after 5 min the reaction was terminated using 05 g of sodium hydrogen sulphate monohydrate and the resulting fatty acid methyl esters ( fame ) were analysed by glc ( 3400 ; varian ) as previously described by childs et al . . feed samples for analysis were collected before feeding at intervals throughout the experiment and were pooled for analysis . dm , crude ash , crude protein , crude fat and crude fibre were analysed by the methods described previously . data were analysed using mixed models in sas ( sas institute , inc . ) . for gilt production performance , the fixed effects were body condition ( thin or fat ) , feed level ( restricted , control and high ) , block and the body condition feed level interaction . for the data describing body weight of the gilt , the weight of the gilts at day 25 of gestation was included as a covariate . for the milk and colostrum results , body condition , feed level and the interactions between body condition and feed level time was included as the repeated statement , and gilt ( feed level ) included as the subject . results were considered statistically significant when p < 005 and were considered as trends when p 010 . the ingredient composition and the nutrient content of the gilt diets are presented in table 1 . the final number of gilts included in the analysis for production parameters was sixty - eight thin and seventy - two fat gilts , due to sixteen repeating and two deaths . the effect of maternal body condition and feed level during gestation on gilt weight , backfat levels , lactation performance , length of pregnancy and subsequent reproductive performance is presented in table 2 . table 2.influence of the main effects , maternal body condition ( thin or fat ) and gestation feed level ( restricted : 18 kg / d ; control : 25 kg / d ; or high feed level : 35 kg / d ) on sow performance during gestation , during lactation and on subsequent reproductive performance(adjusted mean values with their pooled standard errors)treatmentthinfat sem p*restrictedcontrolhigh feed sem p * n 6872444848sow body weight ( kg)day 25 of gestation141715851610001149714991507204092day 50 of gestation1610176616300011605167617832070001day 80 of gestation1826198016400011759189020612080001day 110 of gestation1986213517600011919204322202200001farrowing weight1712184516500011622177519392000001weaning weight167617382010051616170118042510001sow backfat ( mm)day 25 of gestation1221890320001156155155041096day 50 of gestation13420103100011581651800400001day 80 of gestation14820203700011571701990470001day 110 of gestation14419003400011491651870430001weaning backfat11213803100011161221360390001daily lactation feed intake ( kg / d)498484012404354346946101540001lactation body weight change ( % ) 10359410400001 + 0073946581284001length of pregnancy ( d)11551155019098115511541156024086lactation length ( d)277277050081280277274050047weaning to oestrus ( d)*52560120025545553015050subsequent performancetotal pigs born / litter124124049097132120120062030total pigs born dead / litter0611024020070811020055 mean values within a row with unlike superscript letters were significantly different ( p < there was no significant body condition feed level interaction for any of the variables tested. estimated value : empty farrowing weight = ( sow weight at day 110 ( total born 228 ) ) . the value of 228 kg is an estimate of the increased weight in the gravid uterus and in mammary tissue attributed to each pig in a litter . lactation body weight change ( % ) = ( sow weaning weight ( sow weight at day 110 ( total born 228)))/(sow weight day 110 ( total born 228 ) ) 100 . influence of the main effects , maternal body condition ( thin or fat ) and gestation feed level ( restricted : 18 kg / d ; control : 25 kg / d ; or high feed level : 35 kg / d ) on sow performance during gestation , during lactation and on subsequent reproductive performance ( adjusted mean values with their pooled standard errors ) mean values within a row with unlike superscript letters were significantly different ( p < 005 ) ( tukey kramer adjusted ) . * there was no significant body condition feed level interaction for any of the variables tested . estimated value : empty farrowing weight = ( sow weight at day 110 ( total born 228 ) ) . the value of 228 kg is an estimate of the increased weight in the gravid uterus and in mammary tissue attributed to each pig in a litter . lactation body weight change ( % ) = ( sow weaning weight ( sow weight at day 110 ( total born 228)))/(sow weight day 110 ( total born 228 ) ) 100 . the body weight of pre - selected thin and fat gilts was different from day 25 of gestation onwards , with thin gilts being lighter throughout the trial ( p < 005 ) . at day 25 , body weights for restricted , control and high feed level gilts were similar ( p > 005 ) . from day 50 onwards , however , feed level influenced body weight of the animals , with the restricted fed gilts being lighter than control fed gilts , and control fed gilts being lighter than high feed level gilts ( p < 0001 ) . thin gilts had lower backfat levels than fat gilts at day 25 and throughout the trial ( p < 0001 ) . at day 25 of gestation , backfat depths were similar for restricted , control and high feed level gilts ( p > 005 ) . from day 50 onwards , however , feed level influenced the backfat depth ( p < 0001 ) . restricted gilts had a higher average daily feed intake during lactation than control and high feed level animals ( p < 0001 ) . there was no difference in daily feed intake for thin and fat gilts ( p > 005 ) . during lactation , fat gilts lost more weight than thin gilts ( p < 0001 ) . gilts restricted during gestation did not lose weight during lactation , while control and high feed level gilts did ( p < 001 ) . body condition or feed level did not influence the length of pregnancy ( p > 005 ) or lactation length ( p > 005 ) . there was a tendency towards a body condition feed level interaction for the weaning to oestrus interval . fat control gilts tended to have a longer weaning to oestrus interval than thin control gilts ( 591 v. 505 d ; sem 0212 d ; p = 006 ) . thin gilts had a shorter weaning to oestrus interval than fat gilts ( p < 005 ) . there was no effect of the feed level or body condition during the first gestation on the number of pigs born alive ( p > 005 ) or born dead ( p > 005 ) at the second parity . the main effects of maternal body condition and feed level during gestation on piglet growth up to weaning ( day 28 ) are presented in table 3 . restricted gilts had higher numbers of piglets born alive than gilts on the control feed level with both groups having similar values to that of high gilts ( p < 005 ) . restricted gilts gave birth to lighter piglets than gilts on the high feed level with both groups having similar values to that of control gilts ( p < 005 ) . the mean weaning weight of the piglets was influenced by the body condition of the gilt , with piglets from the fat gilts being heavier than piglets from thin gilts ( p < 005 ) . piglets from fat gilts also had higher average daily gain from birth to weaning than piglets from thin gilts ( p < 005 ) . table 3.influence of the main effects , maternal body condition ( thin or fat ) and gestation feed level ( restricted : 18 kg / d ; control : 25 kg / d ; or high feed level : 35 kg / d ) on litter size , piglet performance at birth and weaning(adjusted mean values with their pooled standard errors)treatmentthinfat sem p*restrictedcontrolhigh feed sem p * n 6872444848litter sizelitter birth weight ( kg)165173049025171165170062072total number born121127038064130119123048020number born alive / litter114119038089124111114049004number born dead / litter07608001300590640800900165051number of pre - weaning deaths per litter17919802290521872141640287043total number weaned / litter101990220171049799027021piglet body weightmean piglet birth weight ( kg)14714900280671411501530036005cv of birth weight ( % ) 1836194407050231905188718780884099mean weaning weight ( kg)70374301400047217257220174091cv of weaning weight ( % ) 1709181508210331623181518491029023adg ( g / d)20242143421005209020952066528091cv of adg2040204510780981849209721821276015adg , average daily gain . mean values within a row with unlike superscript letters were significantly different ( p < 005 ) ( tukey kramer adjusted ) * there was no body condition feed level interaction for any of the variables tested. litter size was standardised at farrowing to approximately twelve pigs within treatment groups. within - litter cv values . influence of the main effects , maternal body condition ( thin or fat ) and gestation feed level ( restricted : 18 kg / d ; control : 25 kg / d ; or high feed level : 35 kg / d ) on litter size , piglet performance at birth and weaning ( adjusted mean values with their pooled standard errors ) adg , average daily gain . mean values within a row with unlike superscript letters were significantly different ( p < 005 ) ( tukey kramer adjusted ) * there was no body condition feed level interaction for any of the variables tested . litter size was standardised at farrowing to approximately twelve pigs within treatment groups . within - litter cv values . there was a time effect , with salivary cortisol concentrations being highest in the morning prior to feeding ( fig . salivary cortisol concentrations were 986 , 504 and 533 nmol / l ( sem 0419 nmol / l ; p < 0001 ) at 09.30 , 12.30 and 15.30 hours , respectively . thin gilts tended to have higher mean cortisol levels than fat gilts ( 734 v. 615 nmol / l ; p < 0001 ) ) than those for gilts fed the high feed level ( 500 nmol / l ) and tended to be higher ( sem 0611 nmol / l ; p < 010 ) than those for gilts fed the control feed level ( 673 , control fed gilts tended to have higher ( p = 010 ) salivary cortisol concentrations than gilts fed the high feed level . correlations between the average birth weight and maternal morning cortisol levels were calculated . there was a weak negative correlation ( 02651 ; p < 002 ) between maternal morning cortisol levels and the average birth weight of the piglets . however , when maternal body condition or feed level was analysed together with morning cortisol their correlations with average birth weight were : thin , 00548 ( p > 005 ) ; fat , 04771 ( p > 0003 ) ; restricted , 03755 ( p > 005 ) ; control , 01177 ( p > 005 ) ; and high feed level , 03625 ( p = 010 ) . fig . 1.effect of maternal body condition ( thin or fat ) and gestation feed level ( restricted , 18 kg / d ; control , 25 kg / d ; or high feed level , 35 kg / d ) on saliva cortisol levels at three different time points ( 09.30 , 12.30 and 15.30 hours ) . values are adjusted means , with their pooled standard errors represented by vertical bars . , thin restricted ; , thin control ; , thin high feed level ; , fat restricted ; , fat control ; , fat high feed level . table 4.effect of maternal body condition ( thin or fat ) and gestation feed level ( restricted : 18 kg / d ; control : 25 kg / d ; or high feed level : 35 kg / d ) on saliva cortisol levels at day 80 of pregnancy , and igg levels in colostrum at parturition and day 21 milk composition(adjusted mean values with their pooled standard errors)thinfatp*treatment18 kg25 kg35 kg18 kg25 kg35 kg sem body conditionfeed level n 212324232524cortisol ( nmol / l)927703579778644421091200810001 n 91414121513milk composition day 21protein ( % ) 56585660566101304750881fat ( % ) 75615877819103100030760lactose ( % ) 61596160605700704870613 * there was no significant body condition feed level interaction for cortisol , colostrum or milk composition . effect of maternal body condition ( thin or fat ) and gestation feed level ( restricted , 18 kg / d ; control , 25 kg / d ; or high feed level , 35 kg / d ) on saliva cortisol levels at three different time points ( 09.30 , 12.30 and 15.30 hours ) . values are adjusted means , with their pooled standard errors represented by vertical bars . , thin restricted ; , thin control ; , thin high feed level ; , fat restricted ; , fat control ; , fat high feed level . effect of maternal body condition ( thin or fat ) and gestation feed level ( restricted : 18 kg / d ; control : 25 kg / d ; or high feed level : 35 kg / d ) on saliva cortisol levels at day 80 of pregnancy , and igg levels in colostrum at parturition and day 21 milk composition ( adjusted mean values with their pooled standard errors ) * there was no significant body condition feed level interaction for cortisol , colostrum or milk composition . the result of colostrum analysis at parturition for igg levels is shown in table 4 . there were no differences between the colostrum igg levels of thin ( 1107 mg / ml ) and fat ( 1362 mg / ml ) gilts ( sem 1523 mg / ml ; p > 005 ) . in addition , feed level did not influence colostrum igg levels for restricted ( 1388 mg / ml ) , control ( 1115 mg / ml ) and high ( 1198 mg / ml ) feed level gilts ( sem 1351 mg / ml ; the effects of maternal body condition and gestation feed level on milk composition at day 21 postpartum are presented in table 4 . fat gilts had a higher milk fat percentage compared with thin gilts ( 83 v. 65 % fat ; sem 042 % ; p < 0001 ) . milk fat levels were not influenced by the level of feeding during gestation , being 76 , 71 , and 75 % ( sem 055 % ; p > 005 ) for restricted , control and high feed level , respectively . gestation feeding level or body condition had no effect on protein and lactose levels in the milk . thin gilts had higher levels of total saturated fat ( 3934 g/100 g of fame ) in their milk than fat gilts ( 3691 g/100 g of fame ; sem 0736 g/100 g of fame ; p < 001 ) ( table 5 ) . fat gilts had higher levels of unsaturated fat in their milk ( 6201 g/100 g of fame ) than thin gilts ( 5972 g/100 g of fame ; sem 0715 g/100 g of fame ; p < 005 ) . the unsaturated : saturated fat ratio was 155:1 and 173:1 ( sem 0056 ; p < 005 ) for thin and fat gilts , respectively . there was no effect of feed level during gestation on saturated or unsaturated fat concentration in milk . there was no effect of maternal body condition or feed level during gestation on the composition of n-3 and n-6 pufa . feed level during gestation significantly affected the concentration of c10 : 0 , c12 : 0 , c14 : 0 , c14 : 1c9 , c16 : 1c9 , c18 : 0 , c18 : 1c9 and c20 : 2 and tended to influence c6 : 0 and c15 : 0 concentrations . body condition significantly affected the concentration of c10 : 0 , c12 : 0 , c14 : 0 , c14 : 1c9 , c15 : 0 , c16 : 0 , c16 : 1c9 , c17 : 1c7 , c18 : 0 , c18 : 1c9 and c20 : 3n-6 and tended to affect the concentrations of c10 : 1 , c14 : 1t9 , c9 , t11 conjugated linoleic acid and c20 : 0 ( see table 5 ) . table 5.effect of maternal body condition ( thin or fat ) and gestation feed level ( restricted : 18 kg / d ; control : 25 kg / d ; or high feed level : 35 kg / d ) on fatty acid composition of milk at day 21 of lactation(adjusted mean values with their pooled standard errors)thinfatptreatment18 kg25 kg35 kg18 kg25 kg35 kg sem body conditionfeed level n 91414121513fatty acid composition ( g/100 g of fame ) of milk day 21*c4 : 0011006005005008002004104270395c10 : 0018015014017012010002000350005c12 : 0026022022025018017002300100006c14 : 0341304313319269242021600040014c14 : 1 c9032022023023017018003300050018c15 : 00100010200920097008000710009500140099c16 : 0324031123142313629472708146100180113c16 : 1 c91197944936947837784101800230052c17 : 0020024023021023021001803880144c17 : 1 c7031034031029031024003000500187c17 : 1 c10033035047043035028010405840886c18 : 0306391382357402449029500450006c18 : 1 total25428630027930333918500200015c18 : 2 t9 , t12 n-6163179173173167180009708880656c18 : 2n-616416115816817516509001590637c18 : 3n-6006009007008008012002402810571c18 : 3n-3162155155165171162009101310784c9 , t11 cla005006005007007007000900770680c20 : 2019027027021026038004502000010c20 : 3n-6021030027032033038004400090286c20 : 4n-6038051039045047050004802290181c22 : 5n-3013015015013016015001906270366c22 : 6n-30011003700210026002400280007505510185c24 : 0006005005006008005002107330609total unsaturated591960115985600061926409143300170154total saturated398538963922390237023469147500140146total n-3181183178190197187010601410697total n-6187018801827194020091925095801200669ratio unsaturated : saturated152158156158171189011300220192ratio n-6:n-3103510351028102410261033019307080992fame , fatty acid methyl esters ; cla , conjugated linoleic acid . mean values within a row with unlike superscript letters were significantly different ( p < 005 ) ( tukey kramer adjusted ) . * only fatty acids of > 005 g/100 g of fame are shown ( c6 : 0 , c8 : 0 , c9 : 0 , c10 : 1 , c14 : 1 t9 , iso c16 : 0 , c16 : 1 t9 , c20 : 0 , c20 : 1 , c20 : 3n-3 , c20 : 5n-3 , c21 : 5 , c24 : 0 have been excluded for values > 005 g/100 g of fame). there was a body condition feed level interaction for c20 : 3n-3 ( p = 005 ) and a trend on c6 : 0 ( p = 006 ) , c9 : 0 ( p = 009 ) and c22 : 6n-6 ( p = 009 ) . effect of maternal body condition ( thin or fat ) and gestation feed level ( restricted : 18 kg / d ; control : 25 kg / d ; or high feed level : 35 kg / d ) on fatty acid composition of milk at day 21 of lactation ( adjusted mean values with their pooled standard errors ) fame , fatty acid methyl esters ; cla , conjugated linoleic acid . mean values within a row with unlike superscript letters were significantly different ( p < 005 ) ( tukey kramer adjusted ) . * only fatty acids of > 005 g/100 g of fame are shown ( c6 : 0 , c8 : 0 , c9 : 0 , c10 : 1 , c14 : 1 t9 , iso c16 : 0 , c16 : 1 t9 , c20 : 0 , c20 : 1 , c20 : 3n-3 , c20 : 5n-3 , c21 : 5 , c24 : 0 have been excluded for values > 005 g/100 g of fame ) . there was a body condition feed level interaction for c20 : 3n-3 ( p = 005 ) and a trend on c6 : 0 ( p = 006 ) , c9 : 0 ( p = 009 ) and c22 : 6n-6 ( p = 009 ) . the present study demonstrates that gestation feeding level affects the numbers of offspring born alive per litter and offspring birth weight , while maternal body condition affects weaning weight and growth of offspring . this is , to our knowledge , the first study combining feed level and body condition interactions during pregnancy to determine their influences on postnatal piglet growth . restricted gilts had higher levels of salivary cortisol than those provided with high feed levels during gestation . piglets from fat gilts had a higher average daily gain between birth and weaning than piglets from thin gilts . day 21 milk from lactating fat gilts had a higher percentage of fat , less saturated fat and higher unsaturated fat than milk from thin gilts . we selected gilts at 22 weeks of age based on their backfat and for this reason we can not rule out the possibility that the offspring in our study may have been genetically predisposed , as opposed to programmed in utero , to the body condition influences observed . on the other hand , closely related f1 gilts were selected to minimise the genetic variation within the trial and indeed the backfat differences used for selection purposes at 22 weeks were small . piglets born to fat gilts had higher average daily gain between birth and weaning and were heavier at weaning than piglets born to thin gilts . this increased postnatal growth rate may be attributable to ( a ) the higher milk fat percentage of the fat gilts , ( b ) the difference in milk fat composition and/or ( c ) it could be due to other factors not measured such as appetite , which can be affected by programming or possibly the uptake of nutrients in the gastrointestinal tract . a high fat content in sow milk is desirable as it promotes weight gain and fat deposition in piglets that functions as an insulation layer . although milk fat and fatty acid composition levels are largely determined by the level and source of dietary fat used in the diet , revell et al . also observed that fat levels in the milk increased by 21 % for fat sows ( 340 g of body fat / kg body weight ) compared with thin ( 280 g of body fat / kg body weight ) sows . interestingly , in our study the fat gilts on a high feed level during gestation had the highest ratio ( 189:1 ) of unsaturated : saturated fat in their milk . milk tag come from two sources : biosynthesis of fatty acids within the mammary gland ( de novo synthesis ) and uptake from the plasma by the mammary gland . saturated fat contains mainly scfa that arise predominately from de novo synthesis in the mammary gland , while longer - chain fatty acids arise directly from blood lipids from dietary fatty acids . as all gilts were given the same lactation diet and there was no difference in lactation feed intake , the milk fat difference between thin and fat gilts observed may have arisen from differences in rates of de novo synthesis . in addition , the thin restricted gilts had higher levels of fat in their milk than the thin control and thin high feed level gilts , suggesting different pathways of energy distribution for maintaining high fat levels in the milk in these groups . it is probable that for the thin gilts the main fat source for milk production came from the feed , contrary to the fat gilts where the primary source could have been a mixture of endogenous and exogenous sources . although the main limiting factor for intake in piglets is milk volume , the differences found in piglet growth between thin and fat gilts could also be influenced by the differences in milk fatty acid composition . for example , the amount of oleic acid ( c18 : 1cis9 ) was higher in the milk from fat gilts and increased with gestation feeding level . in vitro cellular models altering the conformation of a c18 : 1 double bond from cis to trans ( oleic acid to elaidic acid ) decreases cholecystokinin secretion , a satiety hormone involved in appetite regulation . oleic acid also provides a signal of nutrient abundance which switches fuel sources from carbohydrates to lipids . this would indicate that offspring from fat gilts were receiving a healthier milk composition , with regard to appetite regulation . of the n-3 and n-6 pufa , only c20 : 3n-6 , a precursor for the synthesis of prostaglandins and other eicosanoids , was increased in the milk of fat gilts . an alteration in the balance of eicosanoid synthesis can cause chronic inflammation , arterial hypertension , chd , atherosclerosis and diabetes mellitus later in life . saturated fats c10 : 0 ( capric acid ) , c12 : 0 ( lauric acid ) and c14 : 0 ( myristic acid ) increased in milk both from restricted fed gilts and from thin gilts . the importance of the lactation period has been demonstrated in rats where postnatal factors overcame both genetic predisposition and prenatal factors in determining the development of adiposity , insulin sensitivity and brain pathways that mediate these functions . feeding regimens and body condition did not influence the n-6 to n-3 pufa levels , indicating that supplementation with different dietary fats would be required to change n-6:n-3 ratios . restricted gilts had the highest level of salivary cortisol , most likely due to undernutrition and suboptimal gut fill being stressful to the pregnant animal . high levels of cortisol during pregnancy may lead to in utero growth restriction as thin restricted gilts gave birth to piglets with the lowest birth weights . undernutrition per se can reduce birth weight in offspring due to the reduced nutrient supply to the fetus as a consequence of suboptimal feeding of the mother . for example , a reduction to 30 % of ad libitum intake in pregnant rats resulted in newborns with a 25 % reduced birth weight compared with the control . however , in addition there is also substantial evidence that maternal glucocorticoid levels , such as cortisol , affect offspring birth weight and glucose metabolism . monkeys subjected to a mild stressor in the form of noise and removal from their cage during pregnancy gave birth to lower birth weight offspring . furthermore , glucocorticoid administration during pregnancy reduces birth weight by 9 % in human subjects , and by 15 % in response to one dose of 05 mg / kg betamethasone in sheep . in addition , pregnant sows treated orally with hydrocortisone - acetate gave birth to piglets with lower birth weight ( 15 kg ) compared with piglets born to control sows ( 165 kg ) . surprisingly , restricted gilts gave birth to the highest number of piglets born alive . this could be because of differences in placental attachment and blood flow due to treatment . . showed that oral administration of hydrocortisone - acetate to sows during pregnancy ( day 21 to 110 ) increased the number of piglets born alive . they hypothesised that this could be due to enhanced maturation of the organs of the piglets born to cortisol - treated sows . mullan & williams also found that total birth weights were lower for gilts restricted to 15 kg feed per d during pregnancy . one suggested pathway is that low placental 11-hydroxy - steroid dehydrogenase type 2 activity correlates with lower birth weight because of increased transplacental passage of active maternal glucocorticoids . as cortisol is an inhibitor of the growth hormone insulin - like growth factor 1 axis , this elevated level might suppress insulin - like growth factor 1 actions , causing growth retardation . our results suggest that even though restrictive feeding increased the number of piglets born alive per litter , a fixed uterine capacity resulted in lighter individual piglets in these larger litters . restricting feed intake during pregnancy did not increase the total number of piglets born per litter , indicating that despite the lack of a significant effect of feed level on the number of piglets born dead , a numerical reduction in the number of piglets born dead was in part responsible for the increase in the number of piglets born alive from restricted gilts . in conclusion , few maternal body condition gestation feed level interaction effects were observed for offspring growth . during gestation , feed - restricted gilts had higher cortisol levels and gave birth to lighter piglets . weaning weights were heavier and average daily gain was greater in piglets born to fat gilts . furthermore , body condition of gilts and feed level during gestation altered the milk fat percentage and profile , with thin gilts having higher levels of saturated fat than fat gilts .

the fetal and early postnatal environment can have a long - term influence on offspring growth . using a pig model , we investigated the effects of maternal body condition ( thin or fat ) and maternal gestation feeding level ( restricted , control or high ) on maternal stress , milk composition , litter size , piglet birth weight and pre - weaning growth . a total of sixty - eight thin ( backfat depth about 8 mm ) and seventy - two fat ( backfat depth about 12 mm ) gilts were selected at about 22 weeks . this backfat difference was then accentuated nutritionally up to service at about 32 weeks . during gestation , individual gilts from within each group were randomly allocated to a gestation diet at the following feed allowances : 18 kg / d ( restricted ) ; 25 kg / d ( control ) and 35 kg / d ( high ) until day 90 of gestation . during gestation restricted gilts had higher levels of cortisol than high and control fed animals . piglets born to fat gilts had higher average daily gain during the lactation period and higher weaning weights at day 28 than piglets born to thin gilts . gilts on a high feed level had heavier piglets than those provided with restricted and control allocations . fat gilts had less saturated fat in their milk at day 21 of lactation and higher unsaturated fat levels . no differences were found in the n-6:n-3 pufa ratio in the milk between thin and fat gilts . in conclusion , maternal body condition influenced the daily weight gain of offspring up to weaning ( day 28 ) and milk fat composition . furthermore , maternal feed level during gestation alters maternal cortisol levels and milk fat composition .

Materials and methods Results Discussion

PMC3853449

the study included 80 unrelated joag patients presenting in the glaucoma clinic at our tertiary eye care center from 2005 to 2010 . the study was approved by the ethics committee of our institute . an informed consent to participate in the study was obtained from all patients and from parents when the patients were < 18 years of age and the study conformed to the tenets of the declaration of helsinki . following were considered as the diagnostic criteria for joag primary open angle glaucoma ( poag ) patients presenting between 10 years and 40 years of ageiop > 22 mmhg in at least one eye on more than two occasionsglaucomatous optic neuropathy in one or both eyes with visual field loss consistent with optic nerve damageopen angles on gonioscopy in both eyes . primary open angle glaucoma ( poag ) patients presenting between 10 years and 40 years of age iop > 22 mmhg in at least one eye on more than two occasions glaucomatous optic neuropathy in one or both eyes with visual field loss consistent with optic nerve damage open angles on gonioscopy in both eyes . exclusion criteria were : history of corticosteroid use , presence of any other retinal or neurological pathology , evidence of secondary causes of raised iop ( pigment dispersion , pseudoexfoliation or trauma ) , those with any pathology detected on gonioscopy such as angle recession , pigmentation of the angle greater than grade 3 or peripheral anterior synechia , those with corneal diameter > 12 mm and those who had any ocular surgery or laser trabeculoplasty in the past . joag patients were administered a questionnaire and details pertaining to their socio - economic and demographic status were obtained . the information as provided by the parents was relied upon for those < 18 years . among the clinical attributes as ascertained from their records , age at diagnosis , gender , presence of a family history , baseline iop , and the visual field defect at presentation were assessed . a family history of glaucoma was considered to be present if 2 or more first degree relatives were affected including the proband . following socio - economic and demographic parameters were assessed to look for an association with the severity at presentation . the patients were classified into different socio - economic classes ( sec ) depending upon their urban or rural background respectively . scoring was based on the total family income , educational level , per capita income , occupation of the head of a family member that has been described , and validated previously in population studies . depending upon their educational level , the patients were classified as : illiteratematriculatehigh schoolgraduate / post - graduate graduate / post - graduate general awareness regarding glaucoma among patients was assessed using the following broad questions : if they had previously heard of glaucomaif they were aware of disease symptomatologyif they were aware of glaucoma running in familiesif they knew about the role of intra ocular pressure in causing glaucomaif the visual loss due to glaucoma is irreversible or notif they were aware of any treatment modalities available for glaucoma . if they had previously heard of glaucoma if they were aware of disease symptomatology if they were aware of glaucoma running in families if they knew about the role of intra ocular pressure in causing glaucoma if the visual loss due to glaucoma is irreversible or not if they were aware of any treatment modalities available for glaucoma . awareness was accordingly classified as either complete or incomplete depending upon the responses of the patients . upon answering each of the questions correctly , assessment of accessibility to an ophthalmologist was carried out by enquiring about the distance ( in kilometers ) from the nearest ophthalmologist , availability of easy transport , and history of prior consultation with an ophthalmologist . all patients with a private transport were classified under easy availability whereas , those using public transport were further enquired about the time and expenditure involved in arranging transport and were then classified upon their subjective ease in getting health - care access . severity of the disease was graded on the visual field score using the 30 - 2 visual fields of the humphrey visual field analyzer of the worse eye . on the basis of advanced glaucoma intervention study scoring system , patients presenting with a worse eye scores greater than 12 were classified as severe disease whereas , those with scores less than 12 as non - severe . any patient with vision < 3/60 in one eye was classified as severe glaucoma . in patients with a unilateral glaucomatous optic neuropathy , these were graded as : mild : < visual field loss in one eyemoderate : > visual field loss in one eye or < visual field loss in both eyessevere : > visual field defect in both eyes or complete visual field loss in one eye . mild : < visual field loss in one eye moderate : > visual field loss in one eye or < visual field loss in both eyes severe : > visual field defect in both eyes or complete visual field loss in one eye . statistical analysis was carried out to study the association of various socio - economic and clinical attributes with disease severity using spss version 16.5 ( chicago il , usa ) . a descriptive analysis of the entire cohort was carried out , and data were represented as means and frequency percentages . chi - square test and an independent t-test were applied to compare categorical and continuous data respectively . then multivariate binomial logistic regression analysis of the factors associated with severity at presentation was performed . the disease severity at presentation using a worse eye vfd and with binocular field defects at presentation was analyzed separately . the patients were classified into different socio - economic classes ( sec ) depending upon their urban or rural background respectively . scoring was based on the total family income , educational level , per capita income , occupation of the head of a family member that has been described , and validated previously in population studies . depending upon their educational level , the patients were classified as : illiteratematriculatehigh schoolgraduate / post - graduate graduate / post - graduate general awareness regarding glaucoma among patients was assessed using the following broad questions : if they had previously heard of glaucomaif they were aware of disease symptomatologyif they were aware of glaucoma running in familiesif they knew about the role of intra ocular pressure in causing glaucomaif the visual loss due to glaucoma is irreversible or notif they were aware of any treatment modalities available for glaucoma . if they had previously heard of glaucoma if they were aware of disease symptomatology if they were aware of glaucoma running in families if they knew about the role of intra ocular pressure in causing glaucoma if the visual loss due to glaucoma is irreversible or not if they were aware of any treatment modalities available for glaucoma . awareness was accordingly classified as either complete or incomplete depending upon the responses of the patients . upon answering each of the questions correctly , assessment of accessibility to an ophthalmologist was carried out by enquiring about the distance ( in kilometers ) from the nearest ophthalmologist , availability of easy transport , and history of prior consultation with an ophthalmologist . all patients with a private transport were classified under easy availability whereas , those using public transport were further enquired about the time and expenditure involved in arranging transport and were then classified upon their subjective ease in getting health - care access . severity of the disease was graded on the visual field score using the 30 - 2 visual fields of the humphrey visual field analyzer of the worse eye . on the basis of advanced glaucoma intervention study scoring system , patients presenting with a worse eye scores greater than 12 were classified as severe disease whereas , those with scores less than 12 as non - severe . any patient with vision < 3/60 in one eye was classified as severe glaucoma . in patients with a unilateral glaucomatous optic neuropathy , these were graded as : mild : < visual field loss in one eyemoderate : > visual field loss in one eye or < visual field loss in both eyessevere : > visual field defect in both eyes or complete visual field loss in one eye . mild : < visual field loss in one eye moderate : > visual field loss in one eye or < visual field loss in both eyes severe : > visual field defect in both eyes or complete visual field loss in one eye . statistical analysis was carried out to study the association of various socio - economic and clinical attributes with disease severity using spss version 16.5 ( chicago il , usa ) . a descriptive analysis of the entire cohort was carried out , and data were represented as means and frequency percentages . chi - square test and an independent t-test were applied to compare categorical and continuous data respectively . odds ratio was calculated with 95% confidence interval . a univariate and then multivariate binomial logistic regression analysis of the factors associated with severity at presentation was performed . the disease severity at presentation using a worse eye vfd and with binocular field defects at presentation was analyzed separately . the mean age at diagnosis of patients was 25.4 8.7 years with an equal distribution of patients in the age groups of 10 - 20 years ( 32% ) , 21 - 30 years ( 34% ) and 31 - 40 years ( 34% ) . mean age at presentation among females was 21 10 years compared to males 26 8.25 years ; p = 0.045.65 ( 81% ) patients were male . no , statistically significant difference was found in the age at diagnosis and severity of visual field defect . the mean age at diagnosis was 25 8.7 years and 25.9 8.9 years among patients with and without severe visual field defect at presentation ( p = 0.644 ) . patients with a positive family history presented 4 years earlier ( p = 0.045 , ci : 0.09 - 8.8 ) compared to those without a family history . no statistically significant difference was found in the worse eye mean deviation scores or the highest untreated ( baseline ) iop among patients with or without a family history of glaucoma [ table 1 ] . however , considering the binocular vfd it was found that those with a positive family history were 10 times less likely to present with severe binocular vfd [ table 2 ] . association of family history of glaucoma with age at diagnosis , gender and severity of visual field defect in the worse eye socio - economic and clinical correlates with severity of disease based on advanced glaucoma intervention study scores in the worse eye the mean highest untreated iop at presentation among our joag patients was 39 8 mmhg . 1 shows the distribution of patients with regard to the highest untreated iop at presentation . distribution of patients with regard to baseline intraocular pressure socio - economic evaluation revealed 44 ( 55% ) patients belonged to upper and upper middle classes and 36 ( 45% ) to the lower middle and upper lower classes . upper and upper middle were clubbed together into broader upper class and the lower middle and upper lower into lower sec for statistical analysis . out of 80 , 10 ( 12.5% ) were illiterate , 30 ( 37.5% ) had completed education up to or less than matriculation level , 10 ( 12.5% ) had completed high school education , and the rest 30 ( 37.5% ) were graduate or post - graduate . only 18 ( 22.5% ) patients in our series had complete awareness regarding their disease . among those with a positive family history , only 6 ( 28.5% ) patients had complete awareness regarding glaucoma while among those who did not have a family history of glaucoma , 12 ( 20% ) had complete awareness about glaucoma ( p = 0.438 , ci : 0.5 - 4.89 ) . no statistically significant association between gender and glaucoma awareness was found in the study ( p = 0.346 , ci : 0.43 - 10.43 ) . most of the patients in our study had no difficulty in accessing health - care facilities with more than two - third of patients having an easy access to an ophthalmologist . forty three ( 54% ) patients in our study presented with a severe visual field defect . using the binocular vfd for grading severity of glaucoma , 53% of the joag patients had severe vfd , 10% had moderate field defects and 37% had mild defect . there was no correlation between the age at diagnosis with the severity of vfd ( r = 0.23 ; p = 0.4 ) . joag patients presenting at a younger age were as much likely to have a severe disease as those presenting later [ fig . correlation of worse eye mean deviation ( md ) score with age at diagnosis patients were categorized into those with highest untreated iop > 35 mmhg and 35 mmhg . a univariate binary logistic regression analysis showed a statistically significant association between the highest untreated iop of > 35 mmhg , lower socio - economic status and educational level below matriculation among joag patients with a worse eye vfd scores . using a multivariate analysis only baseline untreated iop > 35 mmhg was found to be most significantly associated with severe disease at presentation ; adjusted odds ratio ( aor ) : 3.8 , p = 0.022 [ table 3 ] . socio - economic and clinical correlates with severity of disease based on binocular visual field defects table 3 highlights the association of various factors with the disease severity based on the binocular vfd . analysis revealed 7 times and approximately 6 times higher likelihood of presenting with binocularly severe vfd among those with baseline iop > 35 mmhg ( aor : 7 p = 0.003 ) and lower sec respectively ( aor : 5.7 p = 0.01 ) . the mean age at diagnosis of patients was 25.4 8.7 years with an equal distribution of patients in the age groups of 10 - 20 years ( 32% ) , 21 - 30 years ( 34% ) and 31 - 40 years ( 34% ) . mean age at presentation among females was 21 10 years compared to males 26 8.25 years ; p = 0.045.65 ( 81% ) patients were male . no , statistically significant difference was found in the age at diagnosis and severity of visual field defect . the mean age at diagnosis was 25 8.7 years and 25.9 8.9 years among patients with and without severe visual field defect at presentation ( p = 0.644 ) . patients with a positive family history presented 4 years earlier ( p = 0.045 , ci : 0.09 - 8.8 ) compared to those without a family history . no statistically significant difference was found in the worse eye mean deviation scores or the highest untreated ( baseline ) iop among patients with or without a family history of glaucoma [ table 1 ] . however , considering the binocular vfd it was found that those with a positive family history were 10 times less likely to present with severe binocular vfd [ table 2 ] . association of family history of glaucoma with age at diagnosis , gender and severity of visual field defect in the worse eye socio - economic and clinical correlates with severity of disease based on advanced glaucoma intervention study scores in the worse eye the mean highest untreated iop at presentation among our joag patients was 39 8 mmhg . 1 shows the distribution of patients with regard to the highest untreated iop at presentation . socio - economic evaluation revealed 44 ( 55% ) patients belonged to upper and upper middle classes and 36 ( 45% ) to the lower middle and upper lower classes . upper and upper middle were clubbed together into broader upper class and the lower middle and upper lower into lower sec for statistical analysis . out of 80 , 10 ( 12.5% ) were illiterate , 30 ( 37.5% ) had completed education up to or less than matriculation level , 10 ( 12.5% ) had completed high school education , and the rest 30 ( 37.5% ) were graduate or post - graduate . only 18 ( 22.5% ) patients in our series had complete awareness regarding their disease . among those with a positive family history , only 6 ( 28.5% ) patients had complete awareness regarding glaucoma while among those who did not have a family history of glaucoma , 12 ( 20% ) had complete awareness about glaucoma ( p = 0.438 , ci : 0.5 - 4.89 ) . no statistically significant association between gender and glaucoma awareness was found in the study ( p = 0.346 , ci : 0.43 - 10.43 ) . most of the patients in our study had no difficulty in accessing health - care facilities with more than two - third of patients having an easy access to an ophthalmologist . forty three ( 54% ) patients in our study presented with a severe visual field defect . using the binocular vfd for grading severity of glaucoma , 53% of the joag patients had severe vfd , 10% had moderate field defects and 37% had mild defect . there was no correlation between the age at diagnosis with the severity of vfd ( r = 0.23 ; p = 0.4 ) . joag patients presenting at a younger age were as much likely to have a severe disease as those presenting later [ fig . 2 ] . correlation of worse eye mean deviation ( md ) score with age at diagnosis patients were categorized into those with highest untreated iop > 35 mmhg and 35 mmhg . a univariate binary logistic regression analysis showed a statistically significant association between the highest untreated iop of > 35 mmhg , lower socio - economic status and educational level below matriculation among joag patients with a worse eye vfd scores . using a multivariate analysis only baseline untreated iop > 35 mmhg was found to be most significantly associated with severe disease at presentation ; adjusted odds ratio ( aor ) : 3.8 , p = 0.022 [ table 3 ] . socio - economic and clinical correlates with severity of disease based on binocular visual field defects table 3 highlights the association of various factors with the disease severity based on the binocular vfd . analysis revealed 7 times and approximately 6 times higher likelihood of presenting with binocularly severe vfd among those with baseline iop > 35 mmhg ( aor : 7 p = 0.003 ) and lower sec respectively ( aor : 5.7 p = 0.01 ) . patients of joag are known to present with high iop and severe glaucomatous damage at presentation compared to adult poag patients . factors associated with severity at presentation among adult glaucoma patients include the lower socio - economic status , presence of a family history , poor health literacy and ethnicity . unfortunately , there are no studies among young patients with early onset glaucoma that have looked into the factors associated with severity at presentation . apart from a higher baseline iop our study confirmed that poor socio - economic status was associated with the greater disease severity at presentation among joag patients . joag patients in our study with a family history of glaucoma tended to present earlier and thus had less severe disease , probably because they were detected earlier . this is supported by landers et al . who also found positive family history to be associated with better visual fields when patients under 50 years of age were considered . other studies however , found the presence of a positive family history as a significant risk factor for severity among poag patients . juvenile glaucoma patients with a positive family history in our study were 10 times less likely to have severe binocular vfd at presentation . probably affection of one of the eyes and awareness regarding disease among those with a positive family history could be the contributory factors to their seeking care and early detection . patients with a family history of glaucoma are more aware of the disease . and increasing awareness among the family members of patients could be responsible for earlier presentation . in our study too , joag patients with a positive family history tended to present at least 4 years earlier compared to those who did not have a family history of glaucoma . lack of awareness about glaucoma across all categories of patients in india had been alluded to before though in the same study , it was found that the younger patients awareness was more compared to the older patients . however , in our study , we did not find an association between the awareness of the disease and the severity at presentation , which could be because most patient 's awareness was poor . another reason could be not using a grading system for awareness in the present study and it is possible that the awareness among those with familial glaucoma may be of a higher grade compared to those without a family history . poor health literacy has a significant association with severe disease at presentation among glaucoma patients and also contributes to the lack of compliance to therapy . glaucoma awareness has been evaluated in the past using the questionnaires that included understanding of their overall glaucoma care , medications , treatment , prevention of its consequences , and the benefits of lowering eye pressure . a study conducted in india evaluated awareness regarding glaucoma by asking questions related to the iop , progressive nature of the disease , irreversibility of visual loss and importance of medical treatment and compliance . our study revealed that juvenile glaucoma patients belonging to lower sec were 6 times more likely to present with a severe disease . a similar association of lower sec and late presentation was evident in a previous study from india where the authors concluded that glaucoma was a disease that most severely affected the underprivileged . socio - economic deprivation is well - known to be associated with severe disease at presentation even in developed countries . there was no association with the age at diagnosis and severity of visual field defect at presentation . younger patients , as well as those diagnosed after 35 years were likely to present with the severe vfd . studies have shown certain genetic mutations to be associated with younger age at diagnosis and more severe disease among early onset glaucoma patients . however , we believe , from our series , the influence of lower socio- economic status overwhelms most other influences on the severity of the disease though it is probable that a strong influence of certain genetic alterations may explain why some patients present with much higher iop than other joag patients and this need to be further investigated . one of the limitations of the study relates to the study being conducted at a tertiary eye care center introducing a berksonian bias , which was evident in that majority of patients presenting to us had a severe disease . a population based study ; however , is difficult due to the rarity of joag in the general population . furthermore , since most joag patients in the study presented with advanced glaucomatous optic neuropathy in at least one eye , we were unable to evaluate severity based on the optic nerve head parameters and had to rely on vfd in grading the severity . from the findings of the study , we can conclude that the severity of vfd among patients with early onset glaucoma , at presentation , is greater among those without a family history of glaucoma , and those from a lower socio - economic class compounded by higher baseline iop . thus , both clinical and socio - economic factors play a significant role in determining the severity at presentation among joag patients .

aim : the aim of this study was to evaluate the clinical , socio - economic , and demographic factors associated with the severity at presentation among juvenile primary open angle glaucoma ( joag ) patients.materials and methods : age at diagnosis , family history , baseline intraocular pressure ( iop ) , access to health - care , socio - economic status , and glaucoma awareness among 80 unrelated joag patients presenting between 10 years and 40 years of age were analyzed for their association with the severity at presentation . severity at presentation was graded based on worse eye visual field using the advanced glaucoma intervention study score and on binocular visual field defects at presentation.results:patients with a positive family history presented 4 years earlier ( p = 0.045 , confidence interval [ ci ] : 0.09 - 8.8 ) compared to those without a family history . lower socio - economic status ( odds ratio [ or ] 5.7 , p = 0.01 , ci : 1.5 - 22 ) , and higher baseline iop ( or 7 , p = 0.003 , ci : 1.9 - 26 ) were associated with severe glaucomatous visual field defect at presentation . a negative family history was associated with a 10 times likelihood of presenting with a severe glaucomatous field defect ( or 0.1 , p = 0.007 , ci : 0 - 0.5).conclusions : clinical , socio - economic , and demographic factors are contributory to the severity at presentation among young patients with early onset glaucoma . presence of a family history is associated with an earlier presentation among these patients and a reduced risk of the severe presentation .

Materials and Methods Socio-economic status Educational level Glaucoma awareness Health care accessibility Grading severity of glaucoma Statistical analysis Results Clinical features Socio-economic and demographic features Discussion

PMC3904728

the relationship of the anatomical structures in the retromolar region makes any procedures executed in this region very complex , the extraction of the mandibular third molar being the most frequent surgical intervention accomplished there . many times , this procedure is executed by professionals who are not specialists in oral and maxillofacial surgery , which puts the integrity of the lingual nerve ( ln ) at risk . the ln , one of the mandibular nerve branches , third division of the trigeminal nerve , presents fibres of the chorda tympani nerve , a branch of the facial nerve , responsible for the sensitive innervations of the anterior two thirds of the tongue and for the parasympathetic innervations of the sublingual and submandibular glands . according to fign , the chorda tympani nerve is an extensive communicated branch , between the facial and lns , which has sensory taste fibres , relating to the anterior two thirds of the tongue and from the palatal vault , and parasympathetic secretor fibres going to the submandibular and sublingual glands . these anastomoses explain the loss of taste in the lesions of the ln . in the infratemporal fossa , the lingual and inferior alveolar nerves are crossed by the maxillary artery , which runs horizontally , around the medial condyles head , followed by the maxillary vein . the mandibular third molar is situated at the end of the body of the mandible where it is connected with the relatively thin ramus . so , there is a high risk of ln damage using the lingual split technique or elevating the third molar flap medially to the distoangular recess . furthermore , injury to the nerve might occur iatrogenically by direct injury caused by the tooth dislocation to the submandibular region during extraction or for excision of the nerve during the removal of a tumor of the submandibular gland , followed by trauma , orthognathic surgery , third molar extraction due to the intubation procedures of general anesthesia , endodontic treatment of the posterior teeth , tooth implants , intra - osseous lesions curettage , partial or total resection of the tongue or mandibular and periodontal procedures [ 4 - 8 ] beyond local anesthesia . the present study aimed to describe the anatomic relationship of the ln with the nearby structures in the retromolar region and in the third molar socket , disclosing its pathway from the retromolar region until the point that it runs along the floor of the mouth , crossing with the submandibular gland duct . the third molar had completely erupted and it was not possible to determine the gender , since the samples were detached from the body . the exploration of the ln was accomplished through a four centimeter incision in the retromolar region until the point that it adopted a horizontal pathway moving away from the lingual plate , at the point that it crosses with the submandibular duct . the point at the oblique line in the retromolar region was defined as point a. the central point in the lingual gingival margin that corresponds to the third molar socket was identified as point b. the point at which the submandibular duct intersects the ln in the sublingual region was identified as point c. the proper nerve at the retromolar region was defined as point d. the point of the ln , at the level of the third molar mandibular socket , nearest the lingual cortical and that intersects an imaginary line parallel to the long axis of the socket was defined as point e. the following measurements were taken : the retromolar region ( a ) to the point nearest the ln ( d ) ; the third molar socket ( b ) to the ln point in the retromolar region ( d ) ; the distance between the alveolar rim ( b ) and the point at which the submandibular duct intersects the ln in the oblique direction ( c ) ; the distance from the third molar socket ( b ) to the ln ( e ) in a horizontal direction and vertical direction . the measurements were taken from the most superior aspect of the ln ( figure 1 ) . project representing the reference points adopted for the measures : a = retromolar region ; b = third molar socket ; c = intersection point between ln and submandibular duct , d = ln at retromolar region and e = the point of ln , at the level of the third molar mandibular socket , nearest lingual cortical and that intersects an imaginary line paralled to the long axis of the socket . all of the measurements were realized by the same examiner through a manual caliper and analyzed by the spss 16.0 program , through a descriptive analysis via the measurement of the medium - position and the measure of the standard deviation ( sd ) variability . the mean distance between point a to point d was 7.2 mm ( sd 1.7 mm ) . the b to d distance , on average , was 9.4 mm ( sd 1.8 mm ) . point b to point c was , on average , 22.6 mm ( sd 6.6 mm ) . the relationship of point b to point e , in a horizontal direction , which represents the distance of the lingual plate to the nerve was 4.4 mm ( sd 2.4 mm ) , ranging from 11 mm to 2 mm . the distance between points b and e , in the vertical direction , which represents the relationship between the nerve and the lingual alveolar rim , was 16.8 mm ( sd 5.7 mm ) , ranging from 29 mm to 12 mm . at the point that the ln intersects the submandibular duct , there was no constant position relative to the nerve and the duct . in this study , 15 side heads presented the submandibular duct ( wartons duct ) and passed over the ln , and in 9 cases , the duct crossed below the nerve . from the 24 parts analyzed in this study , seven presented healthy mandibular third molar teeth , 12 parts also had the inferior second molar , and 3 parts also presented the maxillary third molar . in this study , the distance between the retromolar region ( a ) and the ln ( d ) , in the retromolar region , was 7.2 mm ( sd 1.7 mm ) and the distance between the ln ( d ) in the retromolar region , and the third molar socket ( b ) was 9.4 mm ( sd 1.8 mm ) ( figure 1 ) . this distance does not provide security or comfort to the surgeon to accomplish large incision maneuvers and detachment of the nearby tissue , since some cases require extensive detachment , and point a limits the ln position in this region . according to the pell and gregory classification , this distance tends to be closer in the level 3 position - the mandibular third molar in the mandibular ramus in which the nerve runs nearest to the retromolar region , presenting vulnerability during surgical procedures in this region . pogrel et al . found one case where the nerve passed above the mandibular lingual plate . in this position , the ln is vulnerable to trauma , may suffer injury during gingival detachment in the retromolar region and is difficult to reach even in procedures that dont involve sharpened instruments in the lingual plate of the mandible . the distance between the alveolar rim , at the level of the third molar region ( b ) , and the sublingual region at the intersection point between the ln and the submandibular gland duct ( c ) was found to be 22.6 mm ( sd 6.6 mm ) ( table 1 and figure 1 ) , and was located in the premolars region . it is important to observe the relationship between the nerve and the duct of the submandibular gland ( wartons duct ) , because the position of both structures is not always maintained the same . this relationship is very important during surgical procedures involving the submandibular gland . in this study , in 62.5% of the cases , the duct passed above the nerve and in 37.5% the nerve passed below the duct . hlzle and wolff investigated the relationship of the ln with the adjacent structures in the atrophic jaws . they found that in most cases the wartons duct passed above the ln , and of a total of 34 samples , they found in 4 cases the wartons duct running deeply in the floor of the mouth , which they did not find with the ln ( figure 2 ) . average and standard deviation by type of measure of the distances between lingual nerve ( ln ) and the adjacent structures in the inferior third molar region image showing the intersection point between ln ( yellow ) and the submandibular gland duct ( black ) . with respect to the horizontal distance ( be ) , 4.4 mm ( sd 2.4 mm ) was found between the ln and the mandibular plate , at the level of the third molar socket ( figure 3 and table 1 ) . ( 2.06 mm ; sd 1.1 mm ) and by hlzle and wolff ( 0.86 mm ; sd 1 mm ) . kiesselbach and chamberlain found a horizontal distance of 0.59 mm ( sd 0.9 mm ) between the ln and the mandibular lingual plate . . found this same relationship with 2.53 mm ( sd 1.48 mm ) . this can be justified for the size of our sample , since in the behnia et al . study it involved 669 lns in 430 corpses with a maximum of 24h post - mortem . our specimens were conserved by immersion in 10% formaldehyde , with a time of immersion varying from 2 to 5 years , which might cause changes in the tissue volume of the analyzed parts . diagram of the vertical and horizontal relationship between ln and lingual plate of the mandible , measured from the most external surface of the nerve . the vertical average distance found was 16.8 mm ( sd 5.7 mm ) and the horizontal one was 4.4 mm ( sd 2.4 mm ) . another fact that might have contributed to the divergence of our result in the horizontal measure was the method used . in the research of pogrel and in the hlzle and wolff studies , the ln was measured taking the retromolar region as a reference , and not the third molar socket , as was done in our study . the measure found in this study highlights the relationship between the ln and the mandibular lingual plate - 4.4 mm , representing great vulnerability to the nerve . during dental sectioning , this surgical step must be completed by the introduction of a straight extractor and the dental division is achieved by movements of rotation inside the tooth . when the vertical relationship between the ln ( e ) and the mandibular lingual plate in the third molar region ( b ) was analyzed , a distance of 16.8 mm ( sd 5.7 mm ) was found ( figure 3 and table 1 ) . ( 8.32 mm ; sd 4.05 mm ) , by behnia et al . ( 3.01 mm ; sd 0.42 mm ) , by hlzle and wolff ( 7.83 mm ; sd 1.65 mm ) , by kiesselbach and chamberlain ( 2.28 mm ; sd 0.9 mm ) and miloro et al . and hlzle and wolff carried out measures with the retromolar region as a reference and not the third molar socket . the study executed by behnia et al . involved the greatest sample in the literature , totaling 669 nerves . they found that in 14.05% of the cases the nerve passed above the mandibular lingual plate and in one case it ran in the retromolar region accurately on the mandibular surface . in 85.8% of the cases , the nerve was in its typical position and in 149 cases ( 23.27% ) it was in direct contact with the lingual plate of the jaw in the alveolar process . they affirm that there were no difference in the ln position in males and females and no correlation between the ln positions at each side , with both presenting independent topographies . bataineh investigated the sensory impairment of the lingual and inferior alveolar nerves after removal of impacted mandibular third molars under local anesthesia and found 2.6% of the patients presenting paresthesia in the ln region , with increased incidence associated with increased lingual flap . fielding and reck registered a case of permanent bilateral anesthesia of the ln after removal of the third molar and the most probable cause was the anomalous position of the ln . robinson and smith defend that avoiding lingual retraction reduces the incidence of temporary and permanent damage . the incidence of damage to the ln was reduced to 0.2% when the lingual flap is avoided and when the lingual plate is preserved . current protocols in the eua emphasize the increased buccal flap , avoiding involvement of the lingual tissue to reduce the risks of impairment to the ln [ 21 - 23 ] . this instrument , with a double - ended shaped to fit the lingual contour of the mandible of the third molar region , would be inserted between the lingual periosteum and the lingual plate of the jaw , providing more space for the surgeon to work , better visibility and lingual flap protection . this technique would be used for procedures of dental sectioning and bone removal in the distal region to the third molar . using this instrument in only 1.6% of the cases presented temporary paresthesia and no case of permanent damage . many surgeons prefer to use some instruments to perform the nerve protection , as with the molt n 9 instrument . nevertheless , there are cases of ln injury by compression , because of its sharp edges and because it did not supply sufficient control during insertion in the depth of the molt n 9 . chossegros et al . evaluated the protection of the ln with a periosteum rising ( molt or similar ) . they concluded that the ln protection , in these cases , is totally unnecessary , which has no advantage for ln protection . many times it is impossible to accomplish the mandibular third molar removal or other procedures involving this same region without manipulating the lingual mucosa , which already enables injury to the nerve , even temporarily . this complication must be discussed with the patient to orientate him at the consultation before surgery . blackburn and bramley affirmed that the bone must be removed in order to facilitate visualization and the drill must be advanced / directed by the surgeon . in every case there are cases in the literature of damages to the ln following endotracheal intubation for procedures that do not involve the oral structures [ 4 - 8 ] . in a recent study , the incidence of damage to the ln was higher in mandibular third molar surgeries under general anesthesia ( 18% ) than 3% under local anesthesia . the etiology of ln damage associated with general anesthesia is multifactorial : complicated and forced laryngoscope , several attempt of laryngoscope , anterior displacement of the jaw for a long period , compression of the tongue , macroglossia , placing a drain plug in the throat and mandibular retraction . niemi et al . affirmed that the deterioration of the function of the ln becomes more difficult with tongue control in anterior - posterior movements . this involvement in the voice production might be the result of the motor impairment , suggesting that the damage also results in interference in the proprioceptive answer of the muscular fibres of the tongue . repairs with or without grafts done immediately after trauma have better results , with progressive worsening when done 3 , 6 , 9 or 12 months after the injury . although there have been several attempts , there is not yet an attendance protocol for these cases . in addition , the long - term outcome of microsurgery is very variable . maybe , the difficult access and the constant mobility in the surgery area ( to eat , to swallow and to speak ) can contribute to the low success rate . this study showed a close relationship of the lingual nerve and the mandibular lingual cortical , presenting a distance of 4.4 mm horizontally and 16.8 mm vertically . as a result , unless adequate protection of the lingual nerve is acquired by following an adequate surgical technique , the lingual nerve will always be vulnerable to damage during surgical intervention or manipulation in this region .

abstractobjectivesthis study evaluated the relationship of the lingual nerve with the adjacent anatomical structures of the mandibular third molar region , influencing the dentist to be aware of the variability of these relationships.material and methodssamples of 24 human corpse half - heads were selected and divided according with the presence or absence of the mandibular third molars . the lingual nerve ( ln ) was explored , showing its run from the oblique line until its crossing with the submandibular gland duct . the measurements along the ln and the adjacent anatomical structures were taken at the retromolar , molar and sublingual region with the use of a digital caliper.resultsthe distance from the ln and the third molar socket , which represents the horizontal distance of the lingual plate to the nerve , on average , was 4.4 mm ( sd 2.4 mm ) . the distance from the ln and the lingual alveolar rim , which represents the vertical relationship between the nerve and the lingual alveolar rim of the third molar socket , on average , was 16.8 mm ( sd 5.7 mm ) . the ln has a varied topography that leaves it very vulnerable during any procedure executed in this region.conclusionsunless adequate protection of the lingual nerve is acquired by following an adequate surgical technique , the lingual nerve will always be vulnerable to damage during surgical intervention or manipulation in this region .

INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGMENTS AND DISCLOSURE STATEMENTS

PMC5013110

this cadaveric study aimed to assess four different techniques of amputation using equipment , which may be carried by a prehospital doctor and the uk fire service . the fire service hacksaw could be used if a gigli saw was not available to cut bone . the holmatro dedicated cutter is a viable option for a life - threatening entrapment where only firefighters can safely access the patient . emergency amputation is a very rare but potentially life - saving intervention in the prehospital setting . it may be required due to entrapment in the wreckage of a road traffic collision , agricultural or industrial machinery . the indications to perform prehospital amputation include : an immediate and real risk to the patient 's life due to a scene safety emergencya deteriorating patient physically trapped by a limb when they will almost certainly die during the time taken to secure extricationa completely mutilated non - survivable limb retaining minimal attachment , which is delaying extrication and evacuation from the scene in a non - immediate life - threatening situationthe patient is dead and their limbs are blocking access to potentially live casualties.1 an immediate and real risk to the patient 's life due to a scene safety emergency a deteriorating patient physically trapped by a limb when they will almost certainly die during the time taken to secure extrication a completely mutilated non - survivable limb retaining minimal attachment , which is delaying extrication and evacuation from the scene in a non - immediate life - threatening situation the patient is dead and their limbs are blocking access to potentially live casualties.1 the majority of prehospital doctors in the uk have not been formally or practically trained to undertake emergency prehospital amputation although most organisations have standard operating procedures in place for this eventuality . a gigli saw is most commonly used for an emergency prehospital amputation but this is not carried by every prehospital practitioner ( personal communication ) . the role of hydraulic equipment carried by the uk fire service has been considered as a potential last resort method for entrapped peri - arrest patients . review of the literature identified one study which assessed the role of a guillotine surgical amputation with a bone saw versus the holmatro jaws of life at different levels using porcine and human cadaveric lower limbs.2 the aim of this study was to observe the results of lower limb amputation using a surgical technique with a gigli saw ; a surgical technique with a hacksaw ; immediate amputation with a reciprocating saw and immediate amputation with a holmatro device . the study was carried out in the west midlands surgical training centre , located at university hospitals coventry & warwickshire national health service ( nhs ) trust , which is licensed by the human tissue authority ( license 30019 ) . ethical approval was not required as research involving previously collected , non - identifiable tissue samples in accordance with the terms of donor consent is excluded from nhs research ethics approval review.3 permission was obtained from west midlands fire service to use hydraulic equipment and a full risk assessment was conducted prior to the study . personal protective equipment , including helmet with visor , face mask and heavy - duty gloves , was worn by the firefighter operators and a 3 m boundary was established for the observers . two fresh frozen self - donated cadavers with no preservative or arterial treatment , and of similar weight and size , were selected for the study . a point on the distal femur 10 cm above the superior border of the patella was measured and marked on each of the four limbs . this level was selected as an appropriate site to simulate a patient severely trapped by their lower limbs in a motor vehicle . the circumference of the thighs at this point ranged between 43.1 and 45.0 cm in the four lower limbs . a combat application tourniquet ( c - a - t ) was applied , proximal to the marked site , and tightened firmly with the windlass to simulate application for haemorrhage control . two surgical procedures of guillotine amputation were performed by a single trauma surgeon with experience of emergency amputation in the field . these were : ( 1 ) using scalpel to cut the skin and fascia , paramedic shears to divide the muscles and gigli saw to cut the femoral bone ( figure 1 ) , followed by paramedic shears to cut the remaining posterior soft tissue and ( 2 ) using scalpel , paramedic shears and a hacksaw from the fire service equipment ( figure 2 ) with a similar technique to above . the surgical operator was assisted by a critical care paramedic ( not previously involved in an amputation ) who passed equipment and protected sharps . a further two procedures were performed by the fire service personnel direct to skin using the dewalt reciprocating saw ( figure 3 ) and the holmatro dedicated cutter ( figure 4 ) : both are routinely carried on local fire service appliances . the ranks of the fire service operators were watch commander and crew commander , respectively , and both had significant experience of using the equipment in operational duties . the firefighters were briefed by the first author just before the procedure , but no advice was provided during the timed experiment . immediately after the procedure and 1 month later , the firefighters were contacted to ensure there were no adverse psychological reactions to being involved in the study . the dewalt dc305 is a cordless handheld 3.7 kg reciprocating saw with a 36 v rechargeable battery . it has an adjustable saw shoe at the base of the saw , which leaves an exposed blade length of 200 mm , and the stroke length is 28.5 mm . the device used had been fully charged but did not have a new blade fitted and had previously been used for cutting motor vehicles in extrication training . the holmatro dedicated cutter cu 3020 has specially developed blades , which are normally used for cutting vehicle roof , door and window posts . the cutting force in the centre of the blades is 188 kn ( 19 tonnes ) . the weight of the device is 12.5 kg and the blades open to an internal distance of 159 mm or an external width of 256 mm . deadman 's handle principle , which means when the operating handle is released , the blades return to the neutral position and tool movement ceases . the holmatro dedicated cutter can be used under water and down to temperatures of 20c . the device is normally connected via a dual core single hose line to a hydraulic pump running with unleaded fuel but for the purposes of the indoor experiment an electric pump was used to avoid fumes . the primary outcome measures were the time from knife to skin to full amputation with the limb disconnected ; the number of attempts and the perceived risks to the rescuer or patient during the procedure . postprocedure , the secondary outcomes of quality of the skin , soft tissue and bone cut were assessed by inspection of the proximal stump . no credible grading system was found to score the quality of cut so a system of grading was devised ( grade 1=very poor quality , very ragged cut , unsalvageable damage ; 2=poor quality , ragged cut , significant damage ; 3=average quality , acceptable damage ; 4=good quality , moderately smooth cut , minimal damage and grade 5 was excellent quality , smooth cut , no obvious damage ) . six clinical raters independently analysed the procedure and observed the quality of the incisions immediately after the procedure . the raters were not blinded to the technique used and were experienced consultants from a prehospital background . this experiment was conducted during the west midlands pre - hospital emergency surgical skills course . amputations were repeated , with the gigli saw and hacksaw , by 14 other prehospital ( non - surgical ) doctors on the course but not graded . the scans were reported by an experienced consultant radiologist who specialises in musculoskeletal trauma ; who did not see the cadaveric specimens and was blinded to the cutting technique . results of amputation techniques the time to set up the equipment was not formally recorded but was negligible with all methods . amputation was completed by all techniques in less than 91 s. the hacksaw jammed on the bone and required resiting , which accounted for the three cuts . two different angles were required by the reciprocating saw to complete the amputation . with the holmatro device , two cuts were required , allowing time for the jaws to open and close again , and a small amount of posterior bridging soft tissue remained , which was cut by the firefighters using paramedic shears . there was no fracture propagation of the proximal bone or comminution of the cut bone with any technique . the quality of the holmatro bone cut on ct appeared to be superior to the grading given by the observers . the bone cut was more angled with the gigli saw and transverse with the hacksaw surgical technique . from the reported ct images , there was 5 cm more proximal soft tissue injury using the holmatro technique . the firefighters reported that there was no feedback , vibration or resistance when operating the holmatro or dewalt reciprocating saw . this is the first published study to explore the feasibility of more than one method of emergency prehospital lower limb amputation using fire service equipment . the results found that all methods of emergency amputation were viable and could be executed within 91 s in study conditions . where a skilled prehospital doctor can access the patient , the technique of using a scalpel , paramedic shears and then either a gigli saw or a fire service hacksaw are both effective rapid methods producing a good quality proximal stump . if only a firefighter could access the patient due to hazards ( car on fire , rising flood water or chemical contamination ) , the reciprocating saw direct to skin was the most rapid technique but carried significant risks to the rescuer of blood splattering , aerosolation of tissue and bone and cutting through the surface under the limb . there was no obvious significant vibration transmitted from the reciprocating saw and the cut was effortless . the firefighters reported that they would have concerns that the battery might fail during the procedure and that the technique involved a definitive action by the firefighter . however , they felt that this was the quickest technique and therefore preferred for a very extreme situation such as a vehicle on fire . the dewalt cordless saw is a smaller device and therefore may be easier to use in a confined space with no trailing cable within the cordon . if no other equipment was available for cutting through the bone , a viable solution would be for a doctor to use scalpel and paramedic shears for the skin and soft tissue and the reciprocating saw for the bone . a risk would be the continuation of the cut through the bone and into the soft tissue , but the ct results from this single example indicate that this will not necessarily impact on the proximal stump quality . in the amputation with the holmatro device , the operator had to wait for the jaws to open and close again to cut deeper tissue and then still had to use the paramedic shears to cut through the posterior soft tissue of the thigh , which delayed the amputation . it was expected that the holmatro cutting device would fragment the bone and there was indeed a loud splintering sound using the jaws , which would be unpleasant for bystanders . however , there was no evidence of bone fragmentation of the proximal stump on ct . . however , 5 cm may not be significant , as a more proximal level will be made for all patients during definitive terminalisation of the stump in hospital . an added advantage of the holmatro device is that it can work underwater so it may be a valid option for the trapped immersed patient where other surgical techniques are impossible . the simulation was not true to life in respect of taking place in a training centre with adequate lighting ; the cadavers were not trapped and positioned supine on a table and there were no other clinical demands or emotive element to the procedure . the access to a trapped casualty in a road traffic collision ( rtc ) may be more complex , especially when attempting to angle the reciprocating saw or to place the blades of the holmatro device around the femur . the surgical operator was highly skilled in amputation : this is not representative of the average consultant level prehospital doctor . when the procedure was repeated with the gigli saw and hacksaw by non - surgical doctors on the course , it was found to be technically similar but took up to a minute longer . the gigli saw was found to be most difficult to use by non - surgical operators and the authors recommend that prehospital practitioners practise the technique before needing to use the skill . this could be done by using a gigli saw to cut through a small fixed tree branch . the skill of the firefighters using the equipment for amputation is likely to be generalisable to other firefighters in the real - life setting . the lower limbs were selected as being a maximum of 19 mm difference in circumference but were from two different cadaveric bodies so may have had different tissue and bone strengths . the cadaveric specimens were elderly and may have had osteoporotic bone compared with a younger trapped prehospital patient . the preservation method may have had an effect on the pliability and retractability of the muscle after cutting . there was a low resting blood volume in the cadaveric specimens : this meant that haemorrhage could not be reliably assessed during the techniques . despite application of a proximal tourniquet , there was a very small amount of blood loss detected from all limbs and a significant amount of blood splatter from the reciprocating saw . in real - life , the practitioner may well apply two tourniquets proximally and this would be recommended for future studies . only one attempt was made with each of the four methods ( due to the availability of cadaveric specimens ) providing a very small sample size . the different techniques were only trialled on the distal femur : chosen as a likely site for the entrapped rtc patient . other sites such as the knee joint , the tibia or the ankle could be explored in future studies . this evaluation found different results compared with the above knee amputations in the previous published study . the mcnicholas study used an orthopaedic bone saw which is not available in the prehospital environment ; the holmatro hydraulic device was similar . the surgical techniques used medial and lateral longitudinal incisions for soft tissue flaps : this would not be indicated in the emergency setting and explains why the time to amputation was slightly longer . the holmatro above knee amputation in the mcnicholas study took half the time of the cut in this study with the same number of cutting actions , and it is unclear why this might be . the results of above knee amputation with the holmatro device found 40 mm of proximal fracture propagation on visual inspection : this was not seen in our study with the benefit of ct images . the raters measuring the subjective outcomes worked individually without conferring , but had seen the technique performed , creating potential observer bias . the radiologist reporting the ct scans of the proximal femurs was blinded to the technique used and reported that the results were very obvious with low risk of interobserver variability . this study has not considered how the fire service equipment would be decontaminated following emergency medical use on human tissue or the psychological impact of firefighters being involved in a surgical amputation . emergency prehospital guillotine amputation of the distal femur can effectively be performed using scalpel and paramedic shears with bone cuts by the gigli saw or fire service hacksaw . the reciprocating saw could be used to cut bone if no other equipment was available but carries risks of blood splattering and deeper uncontrolled continuation of the cut . the holmatro cutting device is a viable option for a life - threatening entrapment where only firefighters can safely access the patient , but would not be a recommended primary technique for medical staff .

objectiveprehospital emergency amputation is a rare procedure , which may be necessary to free a time - critical patient from entrapment . this study aimed to evaluate four techniques of cadaveric lower limb prehospital emergency amputation.methoda guillotine amputation of the distal femur was undertaken in fresh frozen self - donated cadavers . a prehospital doctor conducted a surgical amputation with gigli saw or hacksaw for bone cuts and firefighters carried out the procedure using the reciprocating saw and holmatro device . the primary outcome measures were time to full amputation and the number of attempts required . the secondary outcomes were observed quality of skin cut , soft tissue cut and ct assessment of the proximal bone . observers also noted the potential risks to the rescuer or patient during the procedure.resultsall techniques completed amputation within 91 s. the reciprocating saw was the quickest technique ( 22 s ) but there was significant blood spattering and continuation of the cut to the surface under the leg . the holmatro device took less than a minute . the quality of the proximal femur was acceptable with all methods , but 5 cm more proximal soft tissue damage was made by the holmatro device.conclusionsemergency prehospital guillotine amputation of the distal femur can effectively be performed using scalpel and paramedic shears with bone cuts by the gigli saw or fire service hacksaw . the reciprocating saw could be used to cut bone if no other equipment was available but carried some risks . the holmatro cutting device is a viable option for a life - threatening entrapment where only firefighters can safely access the patient , but would not be a recommended primary technique for medical staff .

What is already known on this subject? What might this study add? Introduction Methods Results Discussion Conclusion

PMC4305606

walking is the most frequent movement in the daily lives of humans , and stable walking is a basic element of a healthy and pleasant life1 . maintaining stability is most important in moving the body forward through repeated use of the legs2 . the muscles of the hip joint are responsible for stabilizing the pelvic and lumbar regions and play an important role in kinematic control during walking3 . the increased incidence of a sedentary lifestyle and the lack of exercise among modern people has resulted in an increased incidence of back pain , and a habitual sedentary lifestyle decreases abdominal muscle strength and increases the degree of body imbalance4 . an excessive biomechanical load on the lumbar area can induce pain in the lumbar region , due to abdominal muscle atrophy and weakening , and spinal joint instability , and decrease joint endurance , flexibility , and range of motion5 . lumbar muscle weakness and imbalance are major factors in reduced exercise performance , and strengthening the muscles in the lumbar region reportedly facilitates efficient functioning of the upper and lower body6 . back pain decreases an individual s walking speed and pain - related gait disturbances typically appear association with in back pain7 . the walking characteristics of patients with back pain include decreased walking speed and an asymmetrical gait posture8 . to prevent and treat these conditions , strength training and stabilization exercises are performed , including mat , ball , and sling exercises , and plyometric and circuit training . ultimately , the purpose of stabilization exercises is to recover movement control and balance by increasing spinal and pelvic stability during functional posture and movement9 . to increase the exercise performance rate , an exercise that can easily be performed by anyone walking is a simple exercise with a low probability of injury that requires no special technique and can be performed by anyone10 . however , walking is a representative aerobic exercise , and improper walking form with body imbalance may cause pain . therefore , in this study , we investigated the effects of running in place accompanied by the abdominal maneuver , a basic stabilization exercise , on gait characteristics . this study selected 30 male and female college students who were randomly assigned to either the training group ( tg ) ( male , 2 ; female , 13 ) , which performed a walking in place exercise , or the control group ( cg ) ( male , 2 ; female , 13 ) , which did not perform the walking in place exercise . the selection criteria included people with no spinal abnormality , lumbar pain , or neurological disorders , who did not drink before the experiment , and who were neither overweight nor taking medicine . people who regularly performed weight training or other exercises were excluded , because these exercises may affect gait characteristics . this study was approved by the korea nazarene university s institutional review board and the subjects were safely protected throughout the experiment . all of the subjects understood the purpose of this study and provided their written informed consent prior to participating in accordance with the ethical principles of the declaration of helsinki . the mean age , height , and weight of the tg participants in this study were 21.50.5 years , 162.47.1 cm , and 55.46.7 kg , respectively , while those of the cg participants were 20.50.8 years , 163.34.9 cm , and 57.69.2 kg , respectively . sex data were analyzed using the test , while age , height , and weight data were analyzed using the independent t - test . there were no intergroup differences ( p>0.05 ) . to maintain posture during the running in place exercise , the students were placed at the very center of a marked 30-cm square space . the jaw was withdrawn to maintain the cervical spine in the neutral position and the transverses abdominis and multifidus muscles were contracted using the abdominal drawing - in maneuver to maintain the waist and pelvis in the neutral position . the participants feet were positioned 10 cm apart and one knee was bent 90 and elevated to reach the hip joint ; the elbow opposite to the elevated leg was bent 90 and elevated to eye level . participants ran with arms and legs crossed in the sagittal plane , and the procedure was repeated for a set number of times . while exercising , the subjects were advised to maintain their posture with minimum deviation in all directions ( left and right or to and fro ) within the restricted space . a total of three exercise sets were performed , and each set consisted of 20 times running in place , a 15-s rest , 20 times running in place , a 15-s rest , 30 times running in place , a 20-s rest , 30 times running in place , a 20-s rest , and 30 times running in place . the rest between sets was 3 min long and one performance time was defined as when both feet touched the ground . each participant made 17 running motions every 10 s. to apply the optimal exercise intensity setting for the students and to increase the load during the total 6-week exercise program , two sets were performed in the first and second week , and three sets were performed in the third to sixth weeks . one 30-min round of exercise was performed three times a week for 6 weeks . the cg participants did not perform any particular exercise and they were assessed twice at the beginning and end of the exercise intervention . gait analysis was performed using the optogait gait analysis system ( microgate italy , bolzano - bozen , italy ) , a device consisting of an optical detection system . the transmitter bar has 96 light - emitting diodes ( leds ) that communicate in the infrared spectrum , the receiver bar , which is positioned opposite it , has the same number of leds . the transmitter and receiver bars of the optogait were installed on both sides of a treadmill . communication between the bars is blocked by the subjects movements during walking , and the interference is used to derive the step length difference ( sld ) , stance phase difference ( stpd ) , swing phase difference ( swpd ) , single support difference ( ssd ) , and step time difference ( std ) are evaluated . using a data capture rate of 1,000 transmissions / receptions per second , running , a series of jumping tests , or the time on the ground or in the air can be accurately measured . from these basic data , the software analyzes the data measured in real time in a series of movements . the data collected by the optogait uses the differences in gait variables between the left and right legs ; the smaller the difference , the greater the stability and balancing ability . the collected data were statistically processed using spss 12.0 ko ( spss , chicago , il , usa ) . the paired t - test was used to test the significance of the differences between before and after the experiment in each group , and the independent t - test was used to test the significance of the differences between the two groups . all data are shown as means and standard deviation , and the significance level , , was chosen as 0.05 . when pre- and post - intervention values of tg and cg were compared , sld , swpd , ssd , and std of subjects in the tg were significantly different , but no significant difference in any parameter was found in the cg ( p<0.05 ) ( table 1table 1.comparison of pre- and post - intervention sld , stpd , swpd , ssd and std in each group ( meansd ) ( unit : sld - cm , stpd , swpd , ssd , std-%)categorygrouppre - interventionpost - interventionsldtraining group*3.41.51.60.8control group3.53.83.22.1stpdtraining group2.72.91.10.9control group2.21.52.61.4swpdtraining group*3.01.81.80.8control group2.71.73.22.9ssdtraining group*3.32.31.51.0control group2.81.83.02.1stdtraining group*3.21.91.91.0control group3.41.63.01.9 * p<0.05 ; sld , step length difference ; stpd , stance phase difference ; swpd , swing phase difference ; ssd , single support difference ; std , step time difference ) . * p<0.05 ; sld , step length difference ; stpd , stance phase difference ; swpd , swing phase difference ; ssd , single support difference ; std , step time difference when pre - intervention , post - intervention , and the change between pre- and post - intervention of tg and cg were compared , there were no significant differences in any of the pre - intervention parameters ; however , post - intervention sld , stpd , and ssd values were significantly different , and the pre- and post - intervention changes in stpd , swpd , and ssd values were also significantly different between the two groups ( p<0.05 ) ( table 2table 2.comparison of sld , stpd , swpd , ssd and std between the training group and control group ( meansd ) ( unit : sld - cm , stpd , swpd , ssd , std-%)categorytraining groupcontrol grouppre - interventionsld3.41.53.53.8stpd2.72.92.21.5swpd3.01.82.71.7ssd3.32.32.81.8std3.21.93.41.6post - interventionsld*1.60.83.22.1stpd*1.10.92.61.4swpd1.80.83.22.9ssd*1.51.03.02.1std1.91.03.01.9change between pre- and post - interventionsld1.71.90.33.8stpd*1.53.10.31.6swpd*1.11.90.42.5ssd*1.82.60.12.0std1.21.90.42.5 * p<0.05 ) . , it enables the body to assume a new posture with smooth movement of the center in preparation for the movement of the arms and legs against gravity11 . in particular , when there is a problem in the lumbar region , the walking speed decreases and an asymmetric gait posture develops8 , suggesting that stability of the body center has a large impact on limb movement . judge et al.12 reported that muscle strengthening exercises , stretching , and balance exercises improve muscle strength and walking speed . brill et al.13 reported that the walking time and number of steps after exercise improved by 3.9% and 13.6% in elderly individuals , respectively . the abdominal drawing - in method effectively stabilizes muscles in the lumbar region , including the transversus abdominis14 , 15 , and running in place , which is similar to plyometric exercise , effectively activates the muscles around the hip joint3 . in this study , we observed a remarkable reduction in the differences in the gait characteristics of the left and right legs in tg compared with those in the cg , which suggests that the combination of dynamic lumbar stabilization exercises and upper and lower extremity movements improved subjects stability and balancing ability . panjabi16 emphasized the importance of lumbar stabilization exercise in correcting a disturbed gait , and topp et al.17 asserted that a muscle strengthening program effectively improves stable walking speed and balancing capability . in addition , approximately 80% of the time used for walking is occupied by the body being supported on one foot . the alternating style of one - foot standing seems to be an important factor in balancing the trunk , and grabiner et al.18 reported that trunk muscle strength is an important factor in gait stability . in conclusion , running in place accompanied by the abdominal drawing - in technique helps lumbar stabilization , strengthens and activates the muscles around the pelvis and hip joints , and seems to have a positive effect on gait symmetry .

[ purpose ] this study was conducted to investigate the effects of running in place accompanied by abdominal drawing - in on the gait characteristics of healthy adults . [ subjects ] the total number of subjects was 30 , and 15 were randomly placed in the training group ( tg ) and 15 in the control group ( cg ) . [ methods ] to determine the gait characteristics of tg and cg , step length difference ( sld ) , stance phase difference ( stpd ) , swing phase difference ( swpd ) , single support difference ( ssd ) , and step time difference ( std ) were evaluated using optogait , a gait analysis system . [ results ] when the pre - intervention and post - intervention results of tg and cg were compared , statistically significant differences in sld , swpd , ssd , and std of tg were found . [ conclusion ] running in place accompanied by abdominal drawing - in might help reduce the deviation between left and right gait variables during walking .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC4393941

carmine is a natural red pigment extracted from dried scale insects ( cochineal , dactylopius coccus ) . the main chemical component of this pigment is carminic acid ( 7--d - glucopyranosyl-9,10-dihydro-3,5,6,8-tetrahydroxy-1-methyl-9,10-dioxoanthracene carboxylic acid , figure 1 ) . carmine is widely used for coloring food products , cosmetics , and medicines [ 2 , 3 ] . in 2012 , the japanese consumer affairs agency published a report on carmine detailing its potential links to dyspnea and anaphylactic reactions . this report detailed approximately 20 articles from 1960s about anaphylaxis resulting from the use of the cosmetics or consumption of food containing carmine . carminic acid is not the cause of these allergic reactions ; they are rather caused by protein contaminants in the carmine [ 5 , 6 ] . some of the allergenic protein contaminants have been identified , and one of them has high homology with phospholipase a , which is a well - known allergen in vespidae venom . hence , the use of carmine in foods is regulated both in japan and european union . for consumer safety , the general method for detection of protein contaminants in carmine involves suspension of the pigment in an acidic solution ( 1% aqueous solution of phosphoric acid ) and then separation of any protein contaminants from the solution by hplc . this method is time - consuming because about ten hplc analyses are required to obtain a sufficient quantity of protein for sds - page analysis . therefore , an alternative method that is rapid , simple , and has high reproducibility is needed . in this study , we developed a rapid and simple method for detection of protein contaminants in carmine with high reproducibility . sample b was manufactured by kishi kasei company ( carminic acid , lot number 174302 ) , and we carried out further purification of sample b as follows . firstly , sample b was dissolved in 5% of phosphoric acid and loaded on a spherical polymerized divinylbenzene resin . then bound carminic acid was eluted by 50% ethanol and dried ( sample c ) . to separate the protein fraction from the carminic acid , we used an ultrafiltration device with a molecular weight cut - off of 3,000 da . two ultrafiltration devices were tested ; one was v - shaped , with a regenerated cellulose membrane ( amicon ultra-0.5 ml 3 k , millipore , billerica , ma ) , and the other was cylindrical , with a modified polyethersulfone membrane ( nanosep 3 k omega , pall corporation , port washington , ny ) . the initial sample volume was 500 l , and the filtration was done by centrifugation until the samples were concentrated to about 0.40.7 of their initial volume . after these filtration steps , the concentration of carminic acid in each sample would be diluted by at least 6 10 times mathematically . electrophoresis was carried out using precast gels ( 15% choju gel , oriental instruments , sagamihara , japan ) and stained using silver stain ms kit ( wako chemicals , osaka , japan ) . each carmine sample was dispersed in phosphoric acid ( 1% ( mass fraction ) aqueous solution ) at a final concentration of 50 mg / ml using ultrasonication . a white precipitate of aggregated protein contaminants was observed when sample a ( cochineal extract , lot number m008363 , biocon ) was dispersed in phosphoric acid ( figure 2 ) . we had confirmed low reproducibility of hplc separations , such as disappearance and shift of a peak from contaminant proteins ( data not shown ) . the aggregation of protein contaminants in the phosphoric acid would reduce the reproducibility of protein separation . as a logical approach to prevention of aggregation of protein contaminants , we made use of protein denaturant first . therefore , in the present method , urea ( 8 mol / l ) and guanidine hydrochloric acid ( 6 detection of proteins is usually performed by colorimetric methods such as biuret method and ninhydrin reaction . free carminic acid interferes with the detection of allergenic proteins in the carmine by colorimetric analysis , and it needs to be removed before analysis . in addition , to detecting the allergenic proteins , protein fractions of > 6 kda need to be collected , and determining the molecular weight of protein contaminants is also important . both sds - page and mass spectrometric analysis are generally used methods for determination of molecular weight of proteins . bound carminic acid on protein shift in molecular weight is found in mass spectrometric analysis , and a smear band pattern is also acquired from sds - page . consequently , in the present study , two ultrafiltration devices were compared for separating the protein fraction from the carmine solution . both of these had molecular weight cut - offs of 3,000 da and are described in section 2 . the initial sample volume was 500 l , and the filtration was done by centrifugation until the samples were concentrated to about 0.40.7 of their initial volume . 50 , 100 , and 200 mg / ml concentrations of carmine were suspended in each solution . at 100 and 200 mg / ml of carmine , carminic acid suspensions were difficult to remove by both ultrafiltration devices , and a concentration of 50 mg / ml was used in subsequent experiments . after filtration , the concentration of carminic acid in each sample was diluted by at least 6 10 times . the amicon ultra-0.5 v - shaped ultrafiltration device could not quantitatively remove the carminic acid , and thus the sds - page results were obscured by remaining pigment ( figure 3(a ) ) . however , the cylindrical ultrafiltration device did remove sufficient carminic acid to allow the protein contaminant band to be clearly observed at around 60 kda . although the cylindrical ultrafiltration device provided more effective removal of carminic acid than the v - shaped device , the detection of low molecular weight proteins ( < 20 kda ) was low ( figure 3(b ) ) . these results indicate that the nanosep 3 k omega ultrafiltration device is better than the amicon ultra-0.5 for the removal of carminic acid , but protein recovery may be an issue . the loss of the protein could be caused by damage to the ultrafiltration device from the high specific gravity solution used for dispersion of the carmine ( e.g. , 8 mol / l urea and 6 mol / l guanidine hydrochloric acid ) . therefore , sds - page running buffer , 25 mmol / l tris(hydroxyamino)methane , 200 mmol / l glycine , and 1% ( mass fraction ) sds , was trialed for dispersion of the carmine samples . this buffer has the following benefits to our analysis : it contains sds , which is a surfactant having strong protein - denaturing ability ; the specific gravity is much lower than that of 8 mol / l urea and 6 mol / l guanidine hydrochloric acid ; and it does not interfere with protein migration in acrylamide gel . the three carmine samples were each dispersed at a final concentration of 50 mg / ml in sds - page running buffer by ultrasonication ( figure 4(a ) ) . the nanosep 3 k omega was used to remove carminic acid , and this was followed by sds - page and silver staining ( figure 4(b ) ) . for sample a , not all of the carminic acid was removed by ultrafiltration , but the remaining carminic acid had little effect on the electrophoresis . several protein bands were observed at 5070 kda for all of the carmine samples . the intensities of these bands were highest in sample a , followed by sample b , and lowest in sample c. protein bands were also observed in a lower molecular weight region ( < 20 kda ) for the partially purified carmine . we confirmed the results of the rank order of intensities of protein bands ' exhibited high reproducibility . the amicon ultra-0.5 was also tested for removing carminic acid from a suspension of carmine in sds - page running buffer . compared with the nanosep 3 k omega device , much of time was required ( several days ) and more of the carminic acid remained in the solution after amicon ultra-0.5 filtration . remaining carminic acid was able to be confirmed by view of acrylamide gel before staining ( figure 5(a ) ) and the electrophoretic pattern of protein contaminants showed more smearing by binding carminic acid on proteins ( figure 5(b ) ) . based on the sds - page results , the dependence on purity of carmine was obvious with the nanosep 3 k omega protocol . although the detection limit of the low molecular weight proteins ( < 20 kda ) seemed to be lower with the nanosep 3 k omega than with the amicon ultra-0.5 , all of the bands were clearly visible still . also the proteins detected at low molecular weight ( < 20 kda ) by silver staining with the amicon ultra-0.5 were also possible artifact due to the effect of the remaining carminic acid . to examine the effect of the shape of the ultrafiltration device on the isolation and detection of the protein contaminants , we also tested a microsep advance centrifugal device 3 k ( pall corporation ) . like the nanosep 3 k omega however , this device is v - shaped rather than cylindrical , and the membrane is 10 times larger than that in the nanosep ; filtration with this device took a long time ( several days ) . the loss of low molecular weight proteins was similar to that with the nanosep 3 k omega ( data not shown ) . these results suggest that the cylindrical filtration device is more suitable for removal of carminic acid than the v - shaped ultrafiltration device . carminic acid can form strong bonds with proteins through both electrostatic and hydrophobic interactions . therefore the method commonly used for detection of protein contaminants in carmine involves suspension of the carmine in an acidic solution . the acidic solution results in protein denaturation and protein aggregation , which cause analytical and separation difficulties . in this study , we found that sds - page running buffer worked as an effective solvent for dispersion of carmine for removal of protein contaminants . the ultrafiltration method used in the present study had sufficient sensitivity and precision . this method is simpler and less expensive than hplc separation and requires only a centrifuge . when selecting an ultrafiltration device for the separation of proteins from carminic acid , a cylindrical device containing a modified polyethersulfone membrane gives good results , and the protein contaminants can also be detected by silver staining sds - page . this method could potentially be applied to other natural pigments and to the removal of unreacted dyes used for protein labeling . a rapid and simple method was developed for the analysis of protein contaminants from carmine . method 's development focused on preventing protein aggregation under acidic protein denaturation conditions and the use of an ultrafiltration device . to prevent protein aggregation , sds - page running buffer was used for dispersion of the carmine . a cylindrical ultrafiltration device containing a modified polyethersulfone membrane provided the best separation of protein contaminants , which could then be observed as bands by sds - page .

protein contaminants in carmine can cause dyspnea and anaphylactic reactions in users and consumers of products containing this pigment . the method generally used for detection of proteins in carmine has low reproducibility and is time - consuming . in this study , a rapid , simple , and highly reproducible method was developed for the detection of protein contaminants in carmine . this method incorporates acidic protein denaturation conditions and ultrafiltration . to prevent protein aggregation , sodium dodecyl sulfate containing gel electrophoresis running buffer was used for dispersing the carmine before filtration . an ultrafiltration device was used to separate the protein contaminants from carminic acid in the carmine solution . two ultrafiltration devices were compared , and a cylindrical device containing a modified polyethersulfone membrane gave the best results . the method had high reproducibility .

1. Introduction 2. Materials and Methods 3. Results and Discussion 4. Conclusion

PMC4280825

injection of angiotensin iv ( ang iv , val - tyr - ile - his - pro - phe ) , an angiotensin ii metabolite , improved memory and learning in rats . the positive impact of ang iv on cognition was subsequently confirmed in a series of animal models in both rats and mice.[25 ] structurally related analogues to ang iv , such as the endogenous lvv - hemorphin-7 ( leu - val - val - tyr - pro - trp - thr - glu - arg - phe ) , in which a tyrosine residue is attached to lipophilic amino acid residues , as in ang iv , exhibited similar outcomes . these abilities to improve processes related to memory and learning have attracted considerable interest in recent years.[7 , 8 ] a specific binding site for ang iv was identified in 1992,[9 , 10 ] and high densities of the binding sites were found in areas of the brain that are associated with cognitive , sensory , and motor functions , including the hippocampus . insulin - regulated aminopeptidase ( irap ) , a high - affinity receptor for ang iv , was purified in 2001 . irap has been identified as cystinyl aminopeptidase ( cap , ec 3.4.11.3 ) , placental leucine aminopeptidase ( p - lap , soluble human homologue ) , oxytocinase , gp160 , or vp165.[1214 ] irap , a single - spanning transmembrane zinc - metallopeptidase that belongs to the m1 family of aminopeptidases , has attracted awareness as a potential target for pharmaceuticals aimed at the treatment of cognitive disorders.[8 , 1618 ] it is hypothesized that the inhibition of the catalytic activity of irap by the hexapeptide ang iv results in longer half - lives of endogenous substrates to irap , such as vasopressin and oxytocin , which are macrocyclic disulfides and are known to exert favorable influences on cognitive parameters in the brain , although several alternative and relevant hypotheses explaining the action of ang iv have been proposed.[20 , 21 ] we recently reported the syntheses and irap inhibition data from a series of constrained macrocycles derived from ang iv and with structural similarities to , for example , oxytocin in the n terminus.[2224 ] among those , ha-08 ( 1 ) , which has a 3htyr residue in a 13-membered macrocyclic system is 20 times more potent than ang iv as an irap inhibitor . although further optimization and application of the metathesis reaction delivered very potent and more metabolically stable macrocyclic carba analogues , these inhibitors , designed from structures of cyclic substrates , are still too peptidic and not likely to be able to enter the brain . discovered a series of drug - like irap inhibitors encompassing a benzopyran system.[7 , 25 , 26 ] these inhibitors were identified after in silico screening of 1.5 million commercially available compounds against an irap model structure . administration of one of these inhibitors , hfi-419 ( 2 ) , a pronounced enhancement of memory in two memory paradigms in rat was observed . we were encouraged by these findings and performed a screening campaign of a library of 10 500 low - molecular - weight compounds and measured the inhibition of the catalytic activity of irap from chinese hamster ovary ( cho ) cells . after subsequent hit confirmation experiments , we identified sulfonamide 3 as one of the most promising compounds . we herein report the syntheses of a series of analogues of 3 , their inhibitory capacity , and structure activity relationships of this new class of irap inhibitors . in the primary high - throughput screen ( hts ) , analogues of 3 , with a carboxylate group rather than the bioisosteric tetrazole in the ortho , meta , or para positions of the aromatic ring , were all found to be devoid of capacity to inhibit or be very weak inhibitors of irap , suggesting that an acidic function is not a sufficient criterion to achieve inhibition . hence , the ic50 values for the meta carboxylate and the corresponding ethyl ester were determined to be greater than 125 m in the hit confirmation experiments . furthermore , the regioisomer of 3 , with the tetrazole ring positioned in the para position rather than in the meta position , was inactive according to the preliminary data . to examine the basic structure activity relationships , compounds 3 , 722 , 25 , and 27 were synthesized and evaluated as inhibitors in an irap enzyme assay with a special emphasis to assess whether the thiophene ring , sulfonamide function , and the acidic nh of the tetrazole are prerequisites for binding to irap . the target compounds 3 , 722 , 25 , and 27 were synthesized as shown in schemes 13 . compound 3 , 722 were synthesized from 3-amino phenyltetrazole ( 4 ) or 3-amino - n - methylbenzamide ( 5 ) reacting with the corresponding aryl / heteroaryl sulfonyl chloride to give good to excellent yields . n - methylated arylsulfonamide 25 was synthesized from 3-amino benzonitrile ( 23 ) as shown in scheme 2 . first , the sulfonylation of 4-bromo-5-chlorothiophene-2-sulfonyl chloride with 23 gave the desired product , 4-bromo-5-chloro - n-(3-cyanophenyl)thiophene-2-sulfonamide , which was used without purification for further treatment with methyl iodide in presence of sodium hydride to afford compound 24 in 78 % overall yield . the tetrazole ring formation starting from cyanophenyl sulfonamide ( 24 ) was successfully achieved via the reported method of koguro et al . by using sodium azide and triethylammonium hydrochloride salt in dry toluene to deliver product 25 in 71 % yield . for the synthesis of compound 27 , 4-bromo-5-chlorothiophene-2-carboxylic acid was coupled with 3-amino phenyltetrazole via standard peptide coupling conditions using benzotriazol-1-yl - oxytripyrrolidinophosphonium hexafluorophosphate ( pybop ) as a peptide coupling reagent for 18 hours at 25 c with dimethylformamide ( dmf ) as the solvent ( scheme 3 ) . reagents and conditions : a ) ch2cl2 , pyridine , 25 c , 14 h. synthesis of irap inhibitor 25 . reagents and conditions : a ) 4-bromo-5-chlorothiophene-2-sulfonyl chloride , ch2cl2 , pyridine , 25 c , 14 h ; b ) thf , nah , mei , 0 c to 25 c , 3 h , 78 % ; c ) nan3 , et3nhcl , toluene reflux , 12 h , 71 % . synthesis of irap inhibitors 27 . reagents and conditions : a ) dmf , pybop , et3n , 25 c , 18 h , 81 % . the biological data from the evaluation of 3 , 722 , 25 , and 27 as inhibitors of irap are presented in table 1 . the nh group on the tetrazole ring seems essential to the inhibitory activity since methylation of the nitrogen affords inactive compound 7 . to find out whether a considerably less acidic nh is sufficient for activity , apparently a tetrazole with an acidic hydrogen but neither a carboxylate nor amide in the meta position of the aromatic ring results in irap inhibitory activity . biological evaluation of compounds 3 , 722 , 25 , and 27 in the irap inhibition assay ic50 is defined as the compound concentration required to inhibit irap activity by 50 % . values represent the mean standard deviation of best - fit values from individual test occasions ( carried out on different days ) . no values above 125 m are reported as this was the highest compound concentration tested . classic bacteriostatic arylsulfonamides , such as sulfamethoxazole and sulfadiazine , which are structurally similar to 3 , are weak acids . the presence of the moderately acidic nh is important to retain activity since methylation , leading to 25 , rendered a considerably less potent inhibitor . furthermore , displacement of the sulfonamide linker for an amide provided the weak inhibitor 27 . the loss of most of the inhibitory potency of 27 could be attributed to 1 ) the restricted rotation of the amide bond not allowing adoption of an optimal binding to irap , 2 ) the loss of an acidic nh , or alternatively 3 ) the sulfonamide oxygen(s ) being crucial for binding . substitution of the 4-bromo for a 4-chloro group to provide 9 did not result in an improvement in the inhibitory activity , but substitution of the thiophene for a benzene ring resulted in a small improvement ( c.f . furthermore , an additional chloro substituent in the ortho position rendered an inhibitor with a good inhibitory capacity ( 11 ) . a fluoro group in the ortho position of a para bromo derivative ( 12 ) provided a potent inhibitor while with two ortho substituents , as in compound 13 , a decline in potency was observed . compound 14 with two methyl groups located in the meta and para positions exhibited good potency , but biphenyl compound 15 was found to be more than ten times less active ( ic50=3.11.8 vs 443.3 m ) . the observation that a chloro or fluoro substituent was accepted in the ortho position by the enzyme prompted us to make the more bulky annelated benzooxadiazole derivative ( 16 ) , which acted as a potent irap inhibitor . benzothiophenes 17 and 18 and methylindole derivative 19 were approximately 10 times less active as inhibitors . it is notable that the nonsubstituted thiophene , benzene , and pyridine derivatives 20 , 21 , and 22 , respectively , exhibited all very poor abilities to inhibit the protease . furthermore , irap inhibitors 10 , 14 , and 16 exhibited a more than 10-fold preference for irap than for the protein homologue aminopeptidase n ( apn ) ( unpublished data ) . in an attempt to rationalize the observed activities of the synthesized compounds , a docking study of the series was conducted using glide ( version 5.8 ; for details , see experimental section ) . to date , no crystal structure of irap has been reported . in order to model the binding of the inhibitors , we utilized apn for which several high - resolution protein ligand co - crystal structures have been reported . twelve of the sixteen amino acids that are found in the catalytic site of apn are conserved in irap , where the catalytic site is defined as within 3 of val and tyr in ang iv when co - crystallized in apn ( pdb code 4fys ) ; see supporting information for sequence alignment . since apn and irap have a high sequence identity in proximity to the catalytic zinc , where we hypothesize that the modeled ligands are binding , we find it reasonable to assume that models of the binding modes found in the catalytic region of apn can be extended to irap . the docking produced several possible binding modes but all with rather poor glide docking scores . however , by visual inspection , we identified a potential binding mode of the series that to some extent accounts for the observed structure activity relationships . figure 1 shows this binding mode illustrated using compound 3 . in the proposed binding mode , the negatively charged tetrazole of 3 is involved in zinc binding and , in addition , is stabilized in the catalytic site by a hydrogen bond to tyr 477 ( irap : tyr 549 ) . this tyr residue is highly conserved in the m1 family of metalloproteases and is indicated to be important for binding and stabilization of the catalytic transition state . furthermore , the compound is stacked between phe 472 ( irap : phe 544 ) and phe 896 ( irap : tyr 961 ) in the active site . the stacking interaction with phe 544 in irap has previously been reported as a key interaction for ligand and substrate binding.[31 , 32 ] two of the amino acids in contact with compound 3 differ between apn and irap . phe 896 is replaced by tyr in irap , and gln 213 is replaced by glu . we anticipate that the difference between the protein structures should not have a significant impact on the present binding mode model of 3 as the aromatic stacking interaction should remain largely the same changing from phe to tyr and since gln 213 is not involved in any specific interaction with the ligand . potential binding mode of 3 shown in the homologue aminopeptidase n ( apn ) active site ( pdb code 4fys ) . the catalytic zinc is depicted as a grey bullet , and amino acids discussed in the text are highlighted ; gln 213 and phe 896 are displayed in pink sticks , and phe 472 is displayed in light grey sticks . this binding mode model fits nicely with the inactivity of the methylated tetrazole found in 7 , which can not produce a complimentary negative charge to the catalytic zinc . the model can possibly also account for the preference for tetrazole over carboxylic acid by the different directionality of their zinc binding moiety . replacing the tetrazole with a carboxylic acid , the carboxylate interacts with the catalytic zinc in the least common anti interaction . furthermore , n - methylation of the sulfonamide in 25 would , in this binding mode , generate a steric clash with the enzyme , thus possibly explaining the decrease in activity caused by this structural modification . however , the decreased activity of compounds extended in the phenyl para position is not accounted for properly by this model . in order to fully conclude the binding mode of this series of compounds it is possible that a crystal structure of irap would reveal differences in structure that are hard to predict using the approach applied herein , and thus fully account for the observed structure activity relationships . new improved enhancers of cognitive functions are persistently desired for the treatment of the cognitive decline associated with alzheimers disease , brain trauma , and cerebral ischemia , since the clinical studies of the cholinesterase inhibitors and n - methyl - d - aspartate ( nmda ) receptor antagonists used today have been mostly disappointing.[34 , 35 ] it is therefore not surprising that the receptor(s ) involved in the beneficial effects of ang iv has emerged as a relevant new target for drug intervention . high - throughput screening of 10 500 low - molecular - weight compounds resulted in a series of hits , one of which was sulfonamide 3 . as deduced from an analysis of analogues to 3 , for example the corresponding carboxylic acid that was assayed in the primary screen and found to be inactive , and from a small structure activity relationship study involving compounds 3 , 722 , 25 , and 27 , it is concluded that the sulfonamide function and a tetrazole ring in the meta position of the aromatic ring are crucial for irap inhibition . further optimization , together with characterization of their mechanism of action and modes of binding are required to make these compounds attractive as research tools for in vivo studies and as potential cognitive enhancers . purification was performed on column chromatography using silica gel ( 60120 mesh size ) and then preparative thin - layer chromatography ( tlc ) or preparative reversed - phase high - performance liquid chromatography ( rp - hplc ) ( uv - triggered ( 254 nm ) fraction collection with a dionex ultimate 3000 hplc system , using an agilent prepht zorbax sb - c8 column ( 21.2150 mm , 5 m particle size ) or a macherey nagel nucleodur c18 column ( 21125 mm , 5 m particle size ) , and h2o / ch3cn/0.05 % hcooh as eluents in a gradient ( 3090 % , 10 ml min over 10 min ) . analytical hplc - mass spectrometry ( hplc - ms ) was performed on a dionex ultimate 3000 hplc system with a bruker amazon sl ion trap mass spectrometer and detection by uv ( diode array detector ) and ms ( electrospray ionization , esi or esi+ ) , using a phenomenex kinetex c18 column ( 503.0 mm , 2.6 m particle size , 100 pore size ) and a flow rate of 1.5 ml min . infrared ( ir ) spectra were obtained on a varian 1000 ft - ir spectrometer ; absorbances are reported in cm . h and c nmr spectra were obtained on a varian mercury spectrometer ( h : 400 mhz , c : 101 mhz ) using cdcl3 , cd3od , [ d6]acetone , or [ d6]dmso as the solvent . high - resolution mass spectra ( hrms ) were recorded on a micromass q - tof2 mass spectrometer equipped with an electrospray ion source . general procedure for the synthesis of 3 , 722 : to a solution of appropriate amine ( 4 or 5 ) ( 1.0 mmol ) in anhydrous ch2cl2 ( 5 ml ) , under an atmosphere of nitrogen , was added appropriate sulfonyl chloride ( 1.2 mmol ) and pyridine ( 3.0 mmol ) . the resulting reaction mixture was stirred at rt overnight and then quenched with saturated aq nh4cl ( 20 ml ) ; the ph was adjusted to around 4 by addition of 1 m aq hcl . the combined organic phase was dried over na2so4 , filtered , and evaporated under reduced pressure . the residue obtained was purified by silica gel flash column chromatography ( ch2cl2/meoh , 98:290:10 ) to give the corresponding product . n-(3-(1h - tetrazol-5-yl)phenyl)-4-bromo-5-chlorothiophene-2-sulfonamide ( 3 ) : white solid ( 358 mg , 85 % ) ; mp : 211213 c ; h nmr ( 400 mhz , [ d6]dmso ) : =7.89 ( t , j=1.9 hz , 1 h ) , 7.837.75 ( m , 1 h ) , 7.69 ( s , 1 h ) , 7.56 ( t , j=8.0 hz , 1 h ) , 7.35 ppm ( ddd , j=8.2 , 2.2 , 1.0 hz , 1 h ) ; c nmr ( 101 mhz , [ d6]dmso ) : =155.93 , 138.47 , 138.11 , 134.13 , 132.85 , 131.11 , 126.18 , 123.89 , 123.47 , 119.31 , 112.03 ppm ; hrms ( esi+ ) : m / z [ m+h ] calcd for c11h8brcln5o2s : 419.8991 , found : 419.8981 ; ir ( neat):=3247 , 1624 , 1583 , 1572 , 1479 , 1403 , 1372 , 1336 , 1314 , 1158 , 1040 , 1022 , 935 cm . 4-bromo-5-chloro - n-(3-(1-methyl-1h - tetrazol-5-yl)phenyl)thiophene-2-sulfonamide ( 7 ) : white solid ( 387 mg , 89 % ) ; mp : 8991 c ; h nmr ( 400 mhz , [ d6]dmso ) : =11.08 ( s , 1 h ) , 7.73 ( s , 1 h ) , 7.64 ( dt , j=6.7 , 1.6 hz , 2 h ) , 7.617.55 ( m , 1 h ) , 7.437.39 ( m , 1 h ) , 4.13 ppm ( s , 3 h ) ; c nmr ( 101 mhz , [ d6]acetone ) : =154.42 , 139.22 , 138.50 , 134.76 , 133.96 , 131.31 , 126.63 , 126.51 , 124.40 , 122.20 , 112.18 , 35.64 ppm ; hrms ( esi+ ) : m / z [ m+h ] calcd for c12h10brcln5o2s2 : 433.9148 , found : 433.9150 ; ir ( neat ) : = 3057 , 1738 , 1592 , 1485 , 1404 , 1342 , 1214 , 1154 , 1023 , 941 cm . 3-(4-bromo-5-chlorothiophene-2-sulfonamido)-n - methylbenzamide ( 8) : white solid ( 344 mg , 84 % ) ; mp : 195197 c ; h nmr ( 400 mhz , [ d6]acetone ) : =7.86 ( td , j=1.8 , 0.6 hz , 1 h ) , 7.81 ( s , 1 h ) , 7.67 ( dt , j=7.3 , 1.5 hz , 1 h ) , 7.48 ( s , 1 h ) , 7.487.39 ( m , 2 h ) , 2.90 ppm ( d , j=4.6 hz , 3 h ) ; c nmr ( 101 mhz , [ d6]acetone ) : =167.13 , 139.58 , 138.20 , 137.20 , 134.41 , 133.59 , 130.28 , 124.64 , 124.48 , 121.59 , 112.00 , 26.81 ppm ; hrms ( esi+ ) : m / z [ m+h ] calcd for c12h9brcln2o2s2 : 408.9083 , found : 408.9093 ; ir ( neat):=3424 , 2929 , 1739 , 1641 , 1550 , 1472 , 1401 , 1336 , 1220 , 1147 , 1009 cm . n-(3-(1h - tetrazol-5-yl)phenyl)-4,5-dichlorothiophene-2-sulfonamide ( 9 ) : white solid ( 312 mg , 89 % ) ; mp : 212214 c ; h nmr ( 400 mhz , [ d6]acetone ) : =8.10 ( ddd , j=2.3 , 1.6 , 0.5 hz , 1 h ) , 7.95 ( ddd , j=7.7 , 1.6 , 1.1 hz , 1 h ) , 7.58 ( t , j=7.8 hz , 1 h ) , 7.55 ( s , 1 h ) , 7.50 ppm ( ddd , j=8.1 , 2.3 , 1.1 hz , 1 h ) ; c nmr ( 101 mhz , [ d6]acetone ) : =157.54 , 138.67 , 138.04 , 132.25 , 131.80 , 131.44 , 127.40 , 125.40 , 124.89 , 124.51 , 120.55 ppm ; hrms ( esi+ ) : m / z [ m+h ] calcd for c11h10cl2n5o2s2 : 375.9496 , found : 375.9498 ; ir ( neat):=3237 , 1740 , 1573 , 1478 , 1402 , 1324 , 1230 , 1151 , 1024 , 933 , 882 cm . n-(3-(1h - tetrazol-5-yl)phenyl)-3,4-dichlorobenzenesulfonamide ( 10 ) : white solid ( 300 mg , 81 % ) ; mp : 184186 c ; h nmr ( 400 mhz , [ d6]acetone ) : =9.49 ( s , 1 h ) , 8.05 ( ddd , j=2.2 , 1.7 , 0.5 hz , 1 h ) , 7.98 ( dd , j=2.0 , 0.5 hz , 1 h ) , 7.89 ( ddd , j=7.7 , 1.6 , 1.1 hz , 1 h ) , 7.827.72 ( m , 2 h ) , 7.52 ( td , j=7.9 , 0.5 hz , 1 h ) , 7.43 ppm ( ddd , j=8.2 , 2.3 , 1.1 hz , 1 h ) ; c nmr ( 101 mhz , [ d6]acetone ) : =157.48 , 140.64 , 139.09 , 137.85 , 133.77 , 132.43 , 131.36 , 129.82 , 127.82 , 127.27 , 124.44 , 124.23 , 120.22 ppm ; hrms ( esi+ ) : m / z [ m+h ] calcd for c13h10cl2n5o2s : 369.9932 , found : 369.9930 ; ir ( neat):=3178 , 1740 , 1564 , 1484 , 1373 , 1329 , 1156 , 1093 , 1031 , 949 cm . n-(3-(1h - tetrazol-5-yl)phenyl)-2,4,5-trichlorobenzenesulfonamide ( 11 ) : white solid ( 312 mg , 77 % ) ; mp : 250254 c ; h nmr ( 400 mhz , cd3od ) : =10.08 ( br s , 1 h ) , 8.33 ( m ( s ) , 1 h ) , 8.16 ( m , 1 h ) , 8.0 ( m ( s ) , 1 h ) , 7.94 ( m , 1 h ) , 7.647.56 ppm ( m , 2 h ) ; c nmr ( 101 mhz , acetone ) : =159.19 , 137.79 , 137.66 , 136.87 , 133.38 , 132.93 , 131.45 , 130.75 , 130.61 , 123.51 , 122.80 , 118.84 , 118.78 ppm ; hrms ( esi+ ) : m / z [ m+mecn+h ] calcd for c15h12cl3n6o2s : 444.9808 , found : 444.9832 ; ir ( neat):=3237 , 3093 , 2360 , 1891 , 1566 , 1173 , 930 , 687 cm . n-(3-(1h - tetrazol-5-yl)phenyl)-4-bromo-2-fluorobenzenesulfonamide ( 12 ) : white solid ( 283 mg , 71 % ) ; mp : 196198 c ; h nmr ( 400 mhz , [ d6]acetone ) : =8.07 ( ddd , j=2.2 , 1.6 , 0.6 hz , 1 h ) , 7.907.79 ( m , 2 h ) , 7.63 ( dd , j=9.7 , 1.8 hz , 1 h ) , 7.57 ( ddd , j=8.4 , 1.9 , 0.8 hz , 1 h ) , 7.50 ( ddd , j=8.1 , 7.4 , 0.6 hz , 1 h ) , 7.45 ppm ( ddd , j=8.2 , 2.2 , 1.3 hz , 1 h ) ; c nmr ( 101 mhz , [ d6]acetone ) : =160.63 , 158.05 , 138.89 , 132.94 , 131.27 , 129.13 , 129.09 , 127.30 , 124.23 , 123.64 , 121.74 , 121.50 , 119.67 ppm ; hrms ( esi+ ) : m / z [ m+h ] calcd for c13h10brfn5o2s : 397.9723 , found : 397.9725 ; ir ( neat):=3235 , 1705 , 1563 , 1468 , 1409 , 1368 , 1331 , 1268 , 1231 , 1170 , 1059 , 915 cm . n-(3-(1h - tetrazol-5-yl)phenyl)-4-bromo-2,6-difluorobenzenesulfonamide ( 13 ) : white solid ( 325 mg , 78 % ) ; mp : 210213 c ; h nmr ( 400 mhz , cd3od ) : =7.89 ( td , j=1.7 , 0.8 hz , 1 h ) , 7.71 ( ddd , j=7.7 , 1.7 , 1.1 hz , 1 h ) , 7.46 ( ddd , j=8.2 , 7.6 , 0.5 hz , 1 h ) , 7.407.34 ppm ( m , 3 h ) ; c nmr ( 101 mhz , cd3od ) : =159.40 ( dd , jcf=261.6 , jcf=5.0 hz ) , 156.27 , 137.90 , 130.15 , 127.83 ( t , jcf=12.8 hz ) , 125.63 , 123.11 , 122.41 , 118.48 , 116.82 ( dd , jcf=26.9 , jcf=3.9 hz ) , 116.34 ppm ( t , jcf=16.1 hz ) ; hrms ( esi+ ) : m / z [ m+mecn+h ] calcd for c15h12brf2n6o2s : 456.9894 , found : 456.9904 ; ir ( neat):=3251 , 3087 , 2993 , 2910 , 2844 , 2613 , 2552 , 2481 , 1877 , 1595 , 1558 , 1471 , 1420 , 1174 , 1031 , 918 , 854 cm . n-(3-(1h - tetrazol-5-yl)phenyl)-3,4-dimethylbenzenesulfonamide ( 14 ) : white solid ( 273 mg , 83 % ) ; mp : 221223 c ; h nmr ( 400 mhz , cd3od ) : =7.94 ( m , 1 h ) , 7.77 ( m , 1 h ) , 7.63 ( m , 1 h ) , 7.52 ( m , 1 h ) , 7.39 ( m , 1 h ) , 7.32 ( m , 1 h ) , 2.35 ppm ( br s , 6 h ) ; c nmr ( 101 mhz , cd3od ) : =156.23 , 142.61 , 139.20 , 137.81 , 136.75 , 129.99 , 129.79 , 127.62 , 125.24 , 124.55 , 122.94 , 122.51 , 118.76 , 18.52 , 18.44 ppm ; hrms ( esi+ ) : m / z [ m+mecn+h ] calcd for c17h19n6o2s : 371.1290 , found : 371.1290 ; ir ( neat):=3246 , 2360 , 1576 , 1481 , 1326 , 1151 , 932 cm . n-(3-(1h - tetrazol-5-yl)phenyl)-[1,1-biphenyl]-4-sulfonamide ( 15 ) : white solid ( 306 mg , 81 % ) ; mp : 206210 c ; h nmr ( 400 mhz , cd3od ) : =8.037.99 ( m , 3 h ) , 7.867.79 ( m , 3 h ) , 7.727.68 ( m , 2 h ) , 7.587.51 ( m , 3 h ) , 7.497.44 ppm ( m , 2 h ) ; c nmr ( 101 mhz , cd3od ) : =156.14 , 145.81 , 139.10 , 139.06 , 138.20 , 130.11 , 128.77 , 128.23 , 127.53 , 127.30 , 126.96 , 125.47 , 123.07 , 122.75 , 118.95 ppm ; hrms ( esi+ ) : m / z [ m+mecn+h ] calcd for c21h19n6o2s : 419.1290 , found : 419.1295 ; ir ( neat):=3239 , 2857 , 2717 , 2466 , 1588 , 1480 , 1400 , 1368 , 1325 , 1157 , 933 , 680 cm . n-(3-(1h - tetrazol-5-yl)phenyl)-7-chlorobenzo[c]oxadiazole-4-sulfonamide ( 16 ) : yellow solid ( 287 mg , 76 % ) ; mp : 168170 c ; h nmr ( 400 mhz , [ d6]acetone ) : =8.20 ( d , j=7.4 hz , 1 h ) , 8.05 ( td , j=1.8 , 0.8 hz , 1 h ) , 7.897.77 ( m , 2 h ) , 7.537.37 ppm ( m , 2 h ) ; c nmr ( 101 mhz , [ d6]acetone ) : =157.46 , 149.94 , 146.03 , 138.54 , 136.80 , 131.20 , 131.06 , 128.11 , 127.87 , 127.15 , 124.32 , 123.93 , 119.86 ppm ; hrms ( esi+ ) : m / z [ m+h ] calcd for c13h9cln7o3s : 378.0176 , found : 378.0174 ; ir ( neat):=3289 , 1692 , 1597 , 1493 , 1427 , 1346 , 1275 , 1228 , 1153 , 1040 , 939 cm . n-(3-(1h - tetrazol-5-yl)phenyl)benzo[c]thiophene-1-sulfonamide ( 17 ) : white solid ( 261 mg , 73 % ) ; mp : 238240 c ; h nmr ( 400 mhz , [ d6]acetone ) : =8.56 ( s , 1 h ) , 8.31 ( ddd , j=8.1 , 1.4 , 0.7 hz , 1 h ) , 8.067.99 ( m , 2 h ) , 7.78 ( dt , j=7.4 , 1.5 hz , 1 h ) , 7.607.33 ppm ( m , 4 h ) ; c nmr ( 101 mhz , acetone ) : =156.41 , 140.34 , 138.62 , 136.37 , 133.49 , 132.87 , 130.21 , 126.09 , 125.73 , 125.58 , 123.16 , 123.00 , 122.74 , 122.24 , 118.27 ppm ; hrms ( esi+ ) : m / z [ m+mecn+h ] calcd for c17h15n6o2s2 : 399.0698 , found : 399.0710 ; ir ( neat):=3422 , 3096 , 2814 , 1569 , 1481 , 1150 , 935 , 744 , 587 cm . n-(3-(1h - tetrazol-5-yl)phenyl)benzo[b]thiophene-2-sulfonamide ( 18 ) : white solid ( 243 mg , 68 % ) ; mp : 205208 c ; h nmr ( 400 mhz , cd3od ) : =7.93 ( ddd , j=2.2 , 1.7 , 0.5 hz , 1 h ) , 7.897.83 ( m , 3 h ) , 7.72 ( ddd , j=7.7 , 1.7 , 1.1 hz , 1 h ) , 7.507.35 ppm ( m , 3 h ) ; c nmr ( 101 mhz , cd3od ) : =156.26 , 141.61 , 140.09 , 138.50 , 137.48 , 129.98 , 129.63 , 127.08 , 125.49 , 125.35 , 125.17 , 123.21 , 123.07 , 122.26 , 119.17 ppm ; hrms ( esi+ ) : m / z [ m+mecn+h ] calcd for c17h15n6o2s2 : 399.0698 , found : 399.0698 ; ir ( neat):=3602 , 3520 , 3249 , 2912 , 2762 , 2642 , 2428 , 1592 , 1562 , 1484 , 1421 , 1358 , 1152 , 873 cm . n-(3-(1h - tetrazol-5-yl)phenyl)-1-methyl-1h - indole-6-sulfonamide ( 19 ) : white solid ( 269 mg , 76 % ) ; mp : 235240 c ; h nmr ( 400 mhz , cd3od ) : =8.21 ( m , 1 h ) , 7.95 ( m , 1 h ) , 7.747.70 ( m , 2 h ) , 7.52 ( m , 1 h ) , 7.46 ( m , 1 h ) , 7.39 ( ddd , j=8.2 , 2.2 , 1.1 hz , 1 h ) , 7.36 ( d , j=3.2 hz , 1 h ) , 6.63 ( dd , j=3.2 , 0.9 hz , 1 h ) , 3.87 ppm ( s , 3 h ) ; c nmr ( 101 mhz , cd3od ) : =156.22 , 139.52 , 138.57 , 131.48 , 129.87 , 129.59 , 127.81 , 125.15 , 122.86 , 122.30 , 121.08 , 119.44 , 118.74 , 109.49 , 102.02 , 31.79 ppm ; hrms ( esi+ ) : m / z [ m+h ] calcd for c16h15n6o2s : 355.0977 , found : 355.0981 ; ir ( neat):=3245 , 3014 , 2360 , 1575 , 1480 , 1151 , 1049 , 934 cm . n-(3-(1h - tetrazol-5-yl)phenyl)thiophene-2-sulfonamide ( 20 ) : white solid ( 243 mg , 79 % ) ; mp : 175180 c ; h nmr ( 400 m , cd3od ) : =7.99 ( m , 1 h ) , 7.877.78 ( m , 2 h ) , 7.67 ( dd , j=3.8 , 1.4 hz , 1 h ) , 7.57 ( m , 1 h ) , 7.45 ( ddd , j=8.1 , 2.2 , 1.1 hz , 1 h ) , 7.15 ppm ( dd , j=5.0 , 3.8 hz , 1 h ) ; c nmr ( 101 mhz , cd3od ) : =156.23 , 139.99 , 138.83 , 132.53 , 132.51 , 130.06 , 127.08 , 125.38 , 123.38 , 123.02 , 119.20 ppm ; hrms ( esi+ ) : m / z [ m+mecn+h ] calcd for c13h13n6o2s2 : 349.0541 , found : 349.0538 ; ir ( neat):=3237 , 2360 , 1574 , 1480 , 1326 , 1156 , 1039 , 931 , 717 cm . n-(3-(1h - tetrazol-5-yl)phenyl)benzenesulfonamide ( 21 ) : white solid ( 214 mg , 71 % ) ; mp : 179183 c ; h nmr ( 400 mhz , cd3od ) : =7.997.88 ( m , 3 h ) , 7.78 ( ddd , j=7.7 , 1.7 , 1.0 hz , 1 h ) , 7.65 ( m , 1 h ) , 7.617.54 ( m , 2 h ) , 7.51 ( ddd , j=8.2 , 7.7 , 0.5 hz , 1 h ) , 7.39 ppm ( ddd , j=8.2 , 2.2 , 1.0 hz , 1 h ) ; c nmr ( 101 mhz , cd3od ) : =156.24 , 139.55 , 139.00 , 132.79 , 130.04 , 128.86 , 126.88 , 125.31 , 123.15 , 122.72 , 118.97 ppm ; hrms ( esi+ ) : m / z [ m+mecn+h ] calcd for c15h15n6o2s 343.0977 , found : 343.0974 ; ir ( neat):=3239 , 2898 , 2360 , 1576 , 1481 , 1233 , 1161 , 1089 , 1040 , 932 , 683 cm . n-(3-(1h - tetrazol-5-yl)phenyl)pyridine-3-sulfonamide ( 22 ) : white solid ( 154 mg , 51 % ) ; mp : 219223 c ; h nmr ( 400 mhz , cd3od ) : =8.92 ( m , 1 h ) , 8.70 ( m , 1 h ) , 8.19 ( m , 1 h ) , 7.86 ( m , 1 h ) , 7.74 ( m , 1 h ) , 7.54 ( m,1 h ) , 7.46 ( ddd , j=8.2 , 7.7 , 0.5 hz , 1 h ) , 7.31 ppm ( m , 1 h ) ; c nmr ( 101 mhz , cd3od ) : =156.31 , 152.83 , 147.12 , 138.22 , 136.46 , 135.24 , 130.16 , 125.73 , 124.11 , 123.32 , 123.19 , 119.19 ppm ; hrms ( esi+ ) : m / z [ m+h ] calcd for c12h11n6o2s : 303.0664 , found : 303.0670 ; ir ( neat):=2896 , 2746 , 2360 , 1581 , 1470 , 1358 , 1166 , 1112 cm . 4-bromo-5-chloro - n-(3-cyanophenyl)-n - methylthiophene-2-sulfonamide ( 24 ) : to a solution of 3-aminobenzonitrile ( 23 ) ( 1.0 mmol ) in anhydrous ch2cl2 ( 5 ml ) , under an atmosphere of nitrogen , was added 4-bromo-5-chlorothiophene-2-sulfonyl chloride ( 1.2 mmol ) and pyridine ( 3.0 mmol ) . the resulting reaction mixture was stirred at rt overnight and then quenched with saturated aq nh4cl ( 20 ml ) . the combined organic phase was dried over na2so4 , filtered and evaporated under reduced pressure . the crude product ( 91 % purity determined by lcms ) obtained nah ( 60 % dispersion in mineral oil ) ( 1.2 mmol ) was added under nitrogen atmosphere . ch3i ( 1.5 mmol ) was then added dropwise , and the solution was stirred at 25 c for 3 h. the reaction mixture was quenched with saturated aq nh4cl ( 20 ml ) , and the aqueous phase was extracted with ch2cl2 ( 310 ml ) . the combined organic phase was dried over na2so4 , filtered and evaporated under reduced pressure to afford crude product , which was purified by silica gel flash column chromatography ( isohexane / etoac , 90:1080:20 ) to give product 24 as a white solid ( 306 mg , 78 % ) ; mp : 138140 c ; h nmr ( 400 mhz , [ d6]dmso ) : =7.84 ( dd , j=6.0 , 1.9 hz , 2 h ) , 7.71 ( d , j=0.8 hz , 1 h ) , 7.697.58 ( m , 2 h ) , 3.26 ppm ( s , 3 h ) ; c nmr ( 101 mhz , [ d6]dmso ) : =141.48 , 135.29 , 133.89 , 133.59 , 132.38 , 132.12 , 130.93 , 130.50 , 118.40 , 112.84 , 112.53 , 38.19 ppm ; hrms ( esi+ ) : m / z [ m+h ] calcd for c12h9brcln2o2s2 : 390.8977 , found : 390.8976 ; ir ( neat):=3088 , 3070 , 2223 , 1603 , 1580 , 1482 , 1455 , 1436 , 1401 , 1355 , 1311 , 1270 , 1196 , 1158 , 1140 , 1070 , 1018 , 933 cm . n-(3-(1h - tetrazol-5-yl)phenyl)-4-bromo-5-chloro - n - methylthiophene-2-sulfonamide ( 25 ) : the mixture of nitrile 24 ( 1.0 mmol ) , nan3 ( 2.0 mmol ) , and et3nhcl ( 2.0 mmol ) in dry toluene ( 5 ml ) was heated at reflux for 12 h with constant stirring . after cooling , the reaction mixture was acidified ( ph 2 ) using 2 n aq hcl and extracted with ch2cl2 ( 320 ml ) . the combined organic phase was dried over na2so4 , filtered and evaporated under reduced pressure to afford the crude product , which was purified by silica gel flash column chromatography ( ch2cl2/meoh , 95:0590:10 ) to give product 25 as a white solid ( 309 mg , 71 % ) ; mp : 152154 c ; h nmr ( 400 mhz , [ d6]acetone ) : =8.14 ( ddd , j=7.8 , 1.7 , 1.1 hz , 1 h ) , 8.06 ( ddd , j=2.2 , 1.7 , 0.5 hz , 1 h ) , 7.65 ( ddd , j=8.2 , 7.8 , 0.5 hz , 1 h ) , 7.53 ( ddd , j=8.1 , 2.3 , 1.1 hz , 1 h ) , 7.49 ( s , 1 h ) , 3.42 ppm ( s , 3 h ) ; c nmr ( 101 mhz , [ d6]acetone ) : =156.40 , 141.79 , 134.76 , 134.29 , 133.34 , 130.22 , 129.01 , 126.37 , 126.20 , 125.32 , 111.72 , 37.79 ppm ; hrms ( esi+ ) : m / z [ m+h ] calcd for c12h10brcln5o2s2 : 433.9148 , found : 433.9145 ; ir ( neat):=1712 , 1558 , 1454 , 1398 , 1354 , 1154 , 1067 , 1018 , 909 cm . n-(3-(1h - tetrazol-5-yl)phenyl)-4-bromo-5-chlorothiophene-2-carboxamide ( 27 ) : 4-bromo-5-chlorothiophene-2-carboxylic acid ( 26 ) ( 1.0 mmol ) was dissolved in dry dmf , and pybop ( 1.5 mmol ) was added . 3-(1h - tetrazol-5-yl)aniline ( 4 ) ( 2.0 mmol ) and et3n ( 2.0 mmol ) were then added , and the stirring continued for an additional 18 h. the reaction mixture was then diluted with water ( 10 ml ) and extracted with etoac ( 310 ml ) . the combined organic phase was dried over na2so4 , filtered and evaporated under reduced pressure to afford the crude product , which was purified by silica gel flash column chromatography ( ch2cl2/meoh , 97:0390:10 ) to give product 27 as a white solid ( 312 mg , 81 % ) ; mp : 263265 c ; h nmr ( 400 mhz , [ d6]dmso ) : =10.61 ( s , 1 h ) , 8.49 ( t , j=1.9 hz , 1 h ) , 8.15 ( d , j=0.6 hz , 1 h ) , 7.91 ( ddd , j=8.2 , 2.2 , 1.0 hz , 1 h ) , 7.77 ( ddd , j=7.7 , 1.7 , 1.0 hz , 1 h ) , 7.60 ppm ( t , j=8.0 hz , 1 h ) ; c nmr ( 101 mhz , [ d6]dmso ) : =158.12 , 155.68 , 139.09 , 138.06 , 131.52 , 131.05 , 130.02 , 124.94 , 122.55 , 122.49 , 118.47 , 110.96 ppm ; hrms ( esi+ ) : m / z [ m+h ] calcd for c12h8brcln5os : 383.9321 , found : 383.9319 ; ir ( neat):=3284 , 2883 , 1739 , 1631 , 1591 , 1539 , 1407 , 1303 , 1178 , 1079 , 1028 cm . the enzymatic assay applied for screening purposes as well as follow - up dose response characterization was based on the use of membrane preparations from cho cells as a source of enzymatic activity . these cells are known to abundantly express irap and contain minimal amounts of contaminating apn activities . the cho cell membrane preparation was conducted based on the procedure described by demaegt et al . with lysis of washed cells being done by means of ultrasonication followed by multiple strokes ( > 20 ) with a dounze homogenizer and a centrifugation ( 30 000g for 30 min at 4 c ) procedure for pelleting and washing of the membranes . the extent of enzymatic activity in each sample was quantified in a microtiter - based screening assay using the peptide - like substrate l - leucine - p - nitroanilide ( l - leu - pna ; sigma l9125 ) , which upon irap - mediated cleavage produces para - nitroaniline that absorbs at 405 nm . the screening campaign was performed at chemical biology consortium sweden ( cbcs ) using a primary screening set of 10 500 compounds ( majority of compounds donated by biovitrum ab , stockholm , sweden ) . the screen was conducted in a 384-well format , whereas follow - up dose response experiments were conducted in a transparent 96-well plate ( nunc , product no . the final assay volume was 200 l in an assay buffer consisting of 50 mm tris - hcl , 150 mm nacl , and 0.1 mm phenylmethanesulfonylfluoride ( pmsf ) at ph 7.4 . the assays were conducted in the presence of a final concentration of 1 mm l - leu - pna and homogenized cho cell membranes from 50 000200 000 cells per well , depending on test occasion and membrane batch . the protocol for running the dose response assay started with a serial dilution of the compound stock solutions at 10 mm in 99.9 % anhydrous dmso ( sigma aldrich ) by a factor of 1/3 in columns 111 of the 96-well plates . column 12 was reserved for controls : the equivalent amount of dmso was added to wells a12d12 ( negative controls representing 0 % inhibition of the enzyme ) , whereas a 10 mm stock solution of 3 was placed in wells e12h12 serving as controls representing 100 % inhibition of the enzyme activity . the dmso solutions were then diluted in assay buffer and transferred to the transparent assay plates in triplicate ( 50 l to each well both for samples and controls ) , followed by addition of diluted homogenized cho cell membranes ( 50 l ) and substrate ( 100 l ) to initiate the enzymatic reaction . the plates were covered and then incubated at rt until a statistically significant difference was observed ( 612 h ) between the controls ( z > 0.8 ) . care was taken to ensure the readings were taken at a time point when the absorbance signal still increased linearly with time . raw data were then imported into microsoft excel for conversion to % inhibition data based on the controls on each plate . after data from triplicate samples were averaged , the curves were fitted to a four - parameter dose response model within xlfit ( model 205 ) to obtain best - fit values for the ic50 , hill slope , and the upper and lower limits of the dose response curve . docking was conducted using glide ( version 5.8 , schrdinger , llc , new york , ny , 2012 ) sp mode.[3941 ] the crystal structure 4fyt was downloaded from the protein data bank ( pdb ) and prepared using the protein preparation wizard ( schrdinger suite 2012 protein preparation wizard ; epik version 2.2 , schrdinger , llc , new york , ny , 2012 ; impact version 5.7 , schrdinger , llc , new york , ny , 2012 ; prime version 2.3 , schrdinger , llc , new york , ny , 2012 ) in maestro ( version 9.3 , schrdinger , llc , new york , ny , 2012 ) . all water molecules were deleted , hydrogen bond assignments were optimized , and a minimization of the hydrogens was carried out . the structures were prepared for docking using ligprep ( version 2.5 , schrdinger , llc , new york , ny , 2012 ) generating different protonation states . purification was performed on column chromatography using silica gel ( 60120 mesh size ) and then preparative thin - layer chromatography ( tlc ) or preparative reversed - phase high - performance liquid chromatography ( rp - hplc ) ( uv - triggered ( 254 nm ) fraction collection with a dionex ultimate 3000 hplc system , using an agilent prepht zorbax sb - c8 column ( 21.2150 mm , 5 m particle size ) or a macherey nagel nucleodur c18 column ( 21125 mm , 5 m particle size ) , and h2o / ch3cn/0.05 % hcooh as eluents in a gradient ( 3090 % , 10 ml min over 10 min ) . analytical hplc - mass spectrometry ( hplc - ms ) was performed on a dionex ultimate 3000 hplc system with a bruker amazon sl ion trap mass spectrometer and detection by uv ( diode array detector ) and ms ( electrospray ionization , esi or esi+ ) , using a phenomenex kinetex c18 column ( 503.0 mm , 2.6 m particle size , 100 pore size ) and a flow rate of 1.5 ml min . infrared ( ir ) spectra were obtained on a varian 1000 ft - ir spectrometer ; absorbances are reported in cm . h and c nmr spectra were obtained on a varian mercury spectrometer ( h : 400 mhz , c : 101 mhz ) using cdcl3 , cd3od , [ d6]acetone , or [ d6]dmso as the solvent . high - resolution mass spectra ( hrms ) were recorded on a micromass q - tof2 mass spectrometer equipped with an electrospray ion source . general procedure for the synthesis of 3 , 722 : to a solution of appropriate amine ( 4 or 5 ) ( 1.0 mmol ) in anhydrous ch2cl2 ( 5 ml ) , under an atmosphere of nitrogen , was added appropriate sulfonyl chloride ( 1.2 mmol ) and pyridine ( 3.0 mmol ) . the resulting reaction mixture was stirred at rt overnight and then quenched with saturated aq nh4cl ( 20 ml ) ; the ph was adjusted to around 4 by addition of 1 m aq hcl . the combined organic phase was dried over na2so4 , filtered , and evaporated under reduced pressure . the residue obtained was purified by silica gel flash column chromatography ( ch2cl2/meoh , 98:290:10 ) to give the corresponding product . n-(3-(1h - tetrazol-5-yl)phenyl)-4-bromo-5-chlorothiophene-2-sulfonamide ( 3 ) : white solid ( 358 mg , 85 % ) ; mp : 211213 c ; h nmr ( 400 mhz , [ d6]dmso ) : =7.89 ( t , j=1.9 hz , 1 h ) , 7.837.75 ( m , 1 h ) , 7.69 ( s , 1 h ) , 7.56 ( t , j=8.0 hz , 1 h ) , 7.35 ppm ( ddd , j=8.2 , 2.2 , 1.0 hz , 1 h ) ; c nmr ( 101 mhz , [ d6]dmso ) : =155.93 , 138.47 , 138.11 , 134.13 , 132.85 , 131.11 , 126.18 , 123.89 , 123.47 , 119.31 , 112.03 ppm ; hrms ( esi+ ) : m / z [ m+h ] calcd for c11h8brcln5o2s : 419.8991 , found : 419.8981 ; ir ( neat):=3247 , 1624 , 1583 , 1572 , 1479 , 1403 , 1372 , 1336 , 1314 , 1158 , 1040 , 1022 , 935 cm . 4-bromo-5-chloro - n-(3-(1-methyl-1h - tetrazol-5-yl)phenyl)thiophene-2-sulfonamide ( 7 ) : white solid ( 387 mg , 89 % ) ; mp : 8991 c ; h nmr ( 400 mhz , [ d6]dmso ) : =11.08 ( s , 1 h ) , 7.73 ( s , 1 h ) , 7.64 ( dt , j=6.7 , 1.6 hz , 2 h ) , 7.617.55 ( m , 1 h ) , 7.437.39 ( m , 1 h ) , 4.13 ppm ( s , 3 h ) ; c nmr ( 101 mhz , [ d6]acetone ) : =154.42 , 139.22 , 138.50 , 134.76 , 133.96 , 131.31 , 126.63 , 126.51 , 124.40 , 122.20 , 112.18 , 35.64 ppm ; hrms ( esi+ ) : m / z [ m+h ] calcd for c12h10brcln5o2s2 : 433.9148 , found : 433.9150 ; ir ( neat ) : = 3057 , 1738 , 1592 , 1485 , 1404 , 1342 , 1214 , 1154 , 1023 , 941 cm . 3-(4-bromo-5-chlorothiophene-2-sulfonamido)-n - methylbenzamide ( 8) : white solid ( 344 mg , 84 % ) ; mp : 195197 c ; h nmr ( 400 mhz , [ d6]acetone ) : =7.86 ( td , j=1.8 , 0.6 hz , 1 h ) , 7.81 ( s , 1 h ) , 7.67 ( dt , j=7.3 , 1.5 hz , 1 h ) , 7.48 ( s , 1 h ) , 7.487.39 ( m , 2 h ) , 2.90 ppm ( d , j=4.6 hz , 3 h ) ; c nmr ( 101 mhz , [ d6]acetone ) : =167.13 , 139.58 , 138.20 , 137.20 , 134.41 , 133.59 , 130.28 , 124.64 , 124.48 , 121.59 , 112.00 , 26.81 ppm ; hrms ( esi+ ) : m / z [ m+h ] calcd for c12h9brcln2o2s2 : 408.9083 , found : 408.9093 ; ir ( neat):=3424 , 2929 , 1739 , 1641 , 1550 , 1472 , 1401 , 1336 , 1220 , 1147 , 1009 cm . n-(3-(1h - tetrazol-5-yl)phenyl)-4,5-dichlorothiophene-2-sulfonamide ( 9 ) : white solid ( 312 mg , 89 % ) ; mp : 212214 c ; h nmr ( 400 mhz , [ d6]acetone ) : =8.10 ( ddd , j=2.3 , 1.6 , 0.5 hz , 1 h ) , 7.95 ( ddd , j=7.7 , 1.6 , 1.1 hz , 1 h ) , 7.58 ( t , j=7.8 hz , 1 h ) , 7.55 ( s , 1 h ) , 7.50 ppm ( ddd , j=8.1 , 2.3 , 1.1 hz , 1 h ) ; c nmr ( 101 mhz , [ d6]acetone ) : =157.54 , 138.67 , 138.04 , 132.25 , 131.80 , 131.44 , 127.40 , 125.40 , 124.89 , 124.51 , 120.55 ppm ; hrms ( esi+ ) : m / z [ m+h ] calcd for c11h10cl2n5o2s2 : 375.9496 , found : 375.9498 ; ir ( neat):=3237 , 1740 , 1573 , 1478 , 1402 , 1324 , 1230 , 1151 , 1024 , 933 , 882 cm . n-(3-(1h - tetrazol-5-yl)phenyl)-3,4-dichlorobenzenesulfonamide ( 10 ) : white solid ( 300 mg , 81 % ) ; mp : 184186 c ; h nmr ( 400 mhz , [ d6]acetone ) : =9.49 ( s , 1 h ) , 8.05 ( ddd , j=2.2 , 1.7 , 0.5 hz , 1 h ) , 7.98 ( dd , j=2.0 , 0.5 hz , 1 h ) , 7.89 ( ddd , j=7.7 , 1.6 , 1.1 hz , 1 h ) , 7.827.72 ( m , 2 h ) , 7.52 ( td , j=7.9 , 0.5 hz , 1 h ) , 7.43 ppm ( ddd , j=8.2 , 2.3 , 1.1 hz , 1 h ) ; c nmr ( 101 mhz , [ d6]acetone ) : =157.48 , 140.64 , 139.09 , 137.85 , 133.77 , 132.43 , 131.36 , 129.82 , 127.82 , 127.27 , 124.44 , 124.23 , 120.22 ppm ; hrms ( esi+ ) : m / z [ m+h ] calcd for c13h10cl2n5o2s : 369.9932 , found : 369.9930 ; ir ( neat):=3178 , 1740 , 1564 , 1484 , 1373 , 1329 , 1156 , 1093 , 1031 , 949 cm . n-(3-(1h - tetrazol-5-yl)phenyl)-2,4,5-trichlorobenzenesulfonamide ( 11 ) : white solid ( 312 mg , 77 % ) ; mp : 250254 c ; h nmr ( 400 mhz , cd3od ) : =10.08 ( br s , 1 h ) , 8.33 ( m ( s ) , 1 h ) , 8.16 ( m , 1 h ) , 8.0 ( m ( s ) , 1 h ) , 7.94 ( m , 1 h ) , 7.647.56 ppm ( m , 2 h ) ; c nmr ( 101 mhz , acetone ) : =159.19 , 137.79 , 137.66 , 136.87 , 133.38 , 132.93 , 131.45 , 130.75 , 130.61 , 123.51 , 122.80 , 118.84 , 118.78 ppm ; hrms ( esi+ ) : m / z [ m+mecn+h ] calcd for c15h12cl3n6o2s : 444.9808 , found : 444.9832 ; ir ( neat):=3237 , 3093 , 2360 , 1891 , 1566 , 1173 , 930 , 687 cm . n-(3-(1h - tetrazol-5-yl)phenyl)-4-bromo-2-fluorobenzenesulfonamide ( 12 ) : white solid ( 283 mg , 71 % ) ; mp : 196198 c ; h nmr ( 400 mhz , [ d6]acetone ) : =8.07 ( ddd , j=2.2 , 1.6 , 0.6 hz , 1 h ) , 7.907.79 ( m , 2 h ) , 7.63 ( dd , j=9.7 , 1.8 hz , 1 h ) , 7.57 ( ddd , j=8.4 , 1.9 , 0.8 hz , 1 h ) , 7.50 ( ddd , j=8.1 , 7.4 , 0.6 hz , 1 h ) , 7.45 ppm ( ddd , j=8.2 , 2.2 , 1.3 hz , 1 h ) ; c nmr ( 101 mhz , [ d6]acetone ) : =160.63 , 158.05 , 138.89 , 132.94 , 131.27 , 129.13 , 129.09 , 127.30 , 124.23 , 123.64 , 121.74 , 121.50 , 119.67 ppm ; hrms ( esi+ ) : m / z [ m+h ] calcd for c13h10brfn5o2s : 397.9723 , found : 397.9725 ; ir ( neat):=3235 , 1705 , 1563 , 1468 , 1409 , 1368 , 1331 , 1268 , 1231 , 1170 , 1059 , 915 cm . n-(3-(1h - tetrazol-5-yl)phenyl)-4-bromo-2,6-difluorobenzenesulfonamide ( 13 ) : white solid ( 325 mg , 78 % ) ; mp : 210213 c ; h nmr ( 400 mhz , cd3od ) : =7.89 ( td , j=1.7 , 0.8 hz , 1 h ) , 7.71 ( ddd , j=7.7 , 1.7 , 1.1 hz , 1 h ) , 7.46 ( ddd , j=8.2 , 7.6 , 0.5 hz , 1 h ) , 7.407.34 ppm ( m , 3 h ) ; c nmr ( 101 mhz , cd3od ) : =159.40 ( dd , jcf=261.6 , jcf=5.0 hz ) , 156.27 , 137.90 , 130.15 , 127.83 ( t , jcf=12.8 hz ) , 125.63 , 123.11 , 122.41 , 118.48 , 116.82 ( dd , jcf=26.9 , jcf=3.9 hz ) , 116.34 ppm ( t , jcf=16.1 hz ) ; hrms ( esi+ ) : m / z [ m+mecn+h ] calcd for c15h12brf2n6o2s : 456.9894 , found : 456.9904 ; ir ( neat):=3251 , 3087 , 2993 , 2910 , 2844 , 2613 , 2552 , 2481 , 1877 , 1595 , 1558 , 1471 , 1420 , 1174 , 1031 , 918 , 854 cm . n-(3-(1h - tetrazol-5-yl)phenyl)-3,4-dimethylbenzenesulfonamide ( 14 ) : white solid ( 273 mg , 83 % ) ; mp : 221223 c ; h nmr ( 400 mhz , cd3od ) : =7.94 ( m , 1 h ) , 7.77 ( m , 1 h ) , 7.63 ( m , 1 h ) , 7.52 ( m , 1 h ) , 7.39 ( m , 1 h ) , 7.32 ( m , 1 h ) , 2.35 ppm ( br s , 6 h ) ; c nmr ( 101 mhz , cd3od ) : =156.23 , 142.61 , 139.20 , 137.81 , 136.75 , 129.99 , 129.79 , 127.62 , 125.24 , 124.55 , 122.94 , 122.51 , 118.76 , 18.52 , 18.44 ppm ; hrms ( esi+ ) : m / z [ m+mecn+h ] calcd for c17h19n6o2s : 371.1290 , found : 371.1290 ; ir ( neat):=3246 , 2360 , 1576 , 1481 , 1326 , 1151 , 932 cm . n-(3-(1h - tetrazol-5-yl)phenyl)-[1,1-biphenyl]-4-sulfonamide ( 15 ) : white solid ( 306 mg , 81 % ) ; mp : 206210 c ; h nmr ( 400 mhz , cd3od ) : =8.037.99 ( m , 3 h ) , 7.867.79 ( m , 3 h ) , 7.727.68 ( m , 2 h ) , 7.587.51 ( m , 3 h ) , 7.497.44 ppm ( m , 2 h ) ; c nmr ( 101 mhz , cd3od ) : =156.14 , 145.81 , 139.10 , 139.06 , 138.20 , 130.11 , 128.77 , 128.23 , 127.53 , 127.30 , 126.96 , 125.47 , 123.07 , 122.75 , 118.95 ppm ; hrms ( esi+ ) : m / z [ m+mecn+h ] calcd for c21h19n6o2s : 419.1290 , found : 419.1295 ; ir ( neat):=3239 , 2857 , 2717 , 2466 , 1588 , 1480 , 1400 , 1368 , 1325 , 1157 , 933 , 680 cm . n-(3-(1h - tetrazol-5-yl)phenyl)-7-chlorobenzo[c]oxadiazole-4-sulfonamide ( 16 ) : yellow solid ( 287 mg , 76 % ) ; mp : 168170 c ; h nmr ( 400 mhz , [ d6]acetone ) : =8.20 ( d , j=7.4 hz , 1 h ) , 8.05 ( td , j=1.8 , 0.8 hz , 1 h ) , 7.897.77 ( m , 2 h ) , 7.537.37 ppm ( m , 2 h ) ; c nmr ( 101 mhz , [ d6]acetone ) : =157.46 , 149.94 , 146.03 , 138.54 , 136.80 , 131.20 , 131.06 , 128.11 , 127.87 , 127.15 , 124.32 , 123.93 , 119.86 ppm ; hrms ( esi+ ) : m / z [ m+h ] calcd for c13h9cln7o3s : 378.0176 , found : 378.0174 ; ir ( neat):=3289 , 1692 , 1597 , 1493 , 1427 , 1346 , 1275 , 1228 , 1153 , 1040 , 939 cm . n-(3-(1h - tetrazol-5-yl)phenyl)benzo[c]thiophene-1-sulfonamide ( 17 ) : white solid ( 261 mg , 73 % ) ; mp : 238240 c ; h nmr ( 400 mhz , [ d6]acetone ) : =8.56 ( s , 1 h ) , 8.31 ( ddd , j=8.1 , 1.4 , 0.7 hz , 1 h ) , 8.067.99 ( m , 2 h ) , 7.78 ( dt , j=7.4 , 1.5 hz , 1 h ) , 7.607.33 ppm ( m , 4 h ) ; c nmr ( 101 mhz , acetone ) : =156.41 , 140.34 , 138.62 , 136.37 , 133.49 , 132.87 , 130.21 , 126.09 , 125.73 , 125.58 , 123.16 , 123.00 , 122.74 , 122.24 , 118.27 ppm ; hrms ( esi+ ) : m / z [ m+mecn+h ] calcd for c17h15n6o2s2 : 399.0698 , found : 399.0710 ; ir ( neat):=3422 , 3096 , 2814 , 1569 , 1481 , 1150 , 935 , 744 , 587 cm . n-(3-(1h - tetrazol-5-yl)phenyl)benzo[b]thiophene-2-sulfonamide ( 18 ) : white solid ( 243 mg , 68 % ) ; mp : 205208 c ; h nmr ( 400 mhz , cd3od ) : =7.93 ( ddd , j=2.2 , 1.7 , 0.5 hz , 1 h ) , 7.897.83 ( m , 3 h ) , 7.72 ( ddd , j=7.7 , 1.7 , 1.1 hz , 1 h ) , 7.507.35 ppm ( m , 3 h ) ; c nmr ( 101 mhz , cd3od ) : =156.26 , 141.61 , 140.09 , 138.50 , 137.48 , 129.98 , 129.63 , 127.08 , 125.49 , 125.35 , 125.17 , 123.21 , 123.07 , 122.26 , 119.17 ppm ; hrms ( esi+ ) : m / z [ m+mecn+h ] calcd for c17h15n6o2s2 : 399.0698 , found : 399.0698 ; ir ( neat):=3602 , 3520 , 3249 , 2912 , 2762 , 2642 , 2428 , 1592 , 1562 , 1484 , 1421 , 1358 , 1152 , 873 cm . n-(3-(1h - tetrazol-5-yl)phenyl)-1-methyl-1h - indole-6-sulfonamide ( 19 ) : white solid ( 269 mg , 76 % ) ; mp : 235240 c ; h nmr ( 400 mhz , cd3od ) : =8.21 ( m , 1 h ) , 7.95 ( m , 1 h ) , 7.747.70 ( m , 2 h ) , 7.52 ( m , 1 h ) , 7.46 ( m , 1 h ) , 7.39 ( ddd , j=8.2 , 2.2 , 1.1 hz , 1 h ) , 7.36 ( d , j=3.2 hz , 1 h ) , 6.63 ( dd , j=3.2 , 0.9 hz , 1 h ) , 3.87 ppm ( s , 3 h ) ; c nmr ( 101 mhz , cd3od ) : =156.22 , 139.52 , 138.57 , 131.48 , 129.87 , 129.59 , 127.81 , 125.15 , 122.86 , 122.30 , 121.08 , 119.44 , 118.74 , 109.49 , 102.02 , 31.79 ppm ; hrms ( esi+ ) : m / z [ m+h ] calcd for c16h15n6o2s : 355.0977 , found : 355.0981 ; ir ( neat):=3245 , 3014 , 2360 , 1575 , 1480 , 1151 , 1049 , 934 cm . n-(3-(1h - tetrazol-5-yl)phenyl)thiophene-2-sulfonamide ( 20 ) : white solid ( 243 mg , 79 % ) ; mp : 175180 c ; h nmr ( 400 m , cd3od ) : =7.99 ( m , 1 h ) , 7.877.78 ( m , 2 h ) , 7.67 ( dd , j=3.8 , 1.4 hz , 1 h ) , 7.57 ( m , 1 h ) , 7.45 ( ddd , j=8.1 , 2.2 , 1.1 hz , 1 h ) , 7.15 ppm ( dd , j=5.0 , 3.8 hz , 1 h ) ; c nmr ( 101 mhz , cd3od ) : =156.23 , 139.99 , 138.83 , 132.53 , 132.51 , 130.06 , 127.08 , 125.38 , 123.38 , 123.02 , 119.20 ppm ; hrms ( esi+ ) : m / z [ m+mecn+h ] calcd for c13h13n6o2s2 : 349.0541 , found : 349.0538 ; ir ( neat):=3237 , 2360 , 1574 , 1480 , 1326 , 1156 , 1039 , 931 , 717 cm . n-(3-(1h - tetrazol-5-yl)phenyl)benzenesulfonamide ( 21 ) : white solid ( 214 mg , 71 % ) ; mp : 179183 c ; h nmr ( 400 mhz , cd3od ) : =7.997.88 ( m , 3 h ) , 7.78 ( ddd , j=7.7 , 1.7 , 1.0 hz , 1 h ) , 7.65 ( m , 1 h ) , 7.617.54 ( m , 2 h ) , 7.51 ( ddd , j=8.2 , 7.7 , 0.5 hz , 1 h ) , 7.39 ppm ( ddd , j=8.2 , 2.2 , 1.0 hz , 1 h ) ; c nmr ( 101 mhz , cd3od ) : =156.24 , 139.55 , 139.00 , 132.79 , 130.04 , 128.86 , 126.88 , 125.31 , 123.15 , 122.72 , 118.97 ppm ; hrms ( esi+ ) : m / z [ m+mecn+h ] calcd for c15h15n6o2s 343.0977 , found : 343.0974 ; ir ( neat):=3239 , 2898 , 2360 , 1576 , 1481 , 1233 , 1161 , 1089 , 1040 , 932 , 683 cm . n-(3-(1h - tetrazol-5-yl)phenyl)pyridine-3-sulfonamide ( 22 ) : white solid ( 154 mg , 51 % ) ; mp : 219223 c ; h nmr ( 400 mhz , cd3od ) : =8.92 ( m , 1 h ) , 8.70 ( m , 1 h ) , 8.19 ( m , 1 h ) , 7.86 ( m , 1 h ) , 7.74 ( m , 1 h ) , 7.54 ( m,1 h ) , 7.46 ( ddd , j=8.2 , 7.7 , 0.5 hz , 1 h ) , 7.31 ppm ( m , 1 h ) ; c nmr ( 101 mhz , cd3od ) : =156.31 , 152.83 , 147.12 , 138.22 , 136.46 , 135.24 , 130.16 , 125.73 , 124.11 , 123.32 , 123.19 , 119.19 ppm ; hrms ( esi+ ) : m / z [ m+h ] calcd for c12h11n6o2s : 303.0664 , found : 303.0670 ; ir ( neat):=2896 , 2746 , 2360 , 1581 , 1470 , 1358 , 1166 , 1112 cm . 4-bromo-5-chloro - n-(3-cyanophenyl)-n - methylthiophene-2-sulfonamide ( 24 ) : to a solution of 3-aminobenzonitrile ( 23 ) ( 1.0 mmol ) in anhydrous ch2cl2 ( 5 ml ) , under an atmosphere of nitrogen , was added 4-bromo-5-chlorothiophene-2-sulfonyl chloride ( 1.2 mmol ) and pyridine ( 3.0 mmol ) . the resulting reaction mixture was stirred at rt overnight and then quenched with saturated aq nh4cl ( 20 ml ) . the combined organic phase was dried over na2so4 , filtered and evaporated under reduced pressure . the crude product ( 91 % purity determined by lcms ) obtained nah ( 60 % dispersion in mineral oil ) ( 1.2 mmol ) was added under nitrogen atmosphere . ch3i ( 1.5 mmol ) was then added dropwise , and the solution was stirred at 25 c for 3 h. the reaction mixture was quenched with saturated aq nh4cl ( 20 ml ) , and the aqueous phase was extracted with ch2cl2 ( 310 ml ) . the combined organic phase was dried over na2so4 , filtered and evaporated under reduced pressure to afford crude product , which was purified by silica gel flash column chromatography ( isohexane / etoac , 90:1080:20 ) to give product 24 as a white solid ( 306 mg , 78 % ) ; mp : 138140 c ; h nmr ( 400 mhz , [ d6]dmso ) : =7.84 ( dd , j=6.0 , 1.9 hz , 2 h ) , 7.71 ( d , j=0.8 hz , 1 h ) , 7.697.58 ( m , 2 h ) , 3.26 ppm ( s , 3 h ) ; c nmr ( 101 mhz , [ d6]dmso ) : =141.48 , 135.29 , 133.89 , 133.59 , 132.38 , 132.12 , 130.93 , 130.50 , 118.40 , 112.84 , 112.53 , 38.19 ppm ; hrms ( esi+ ) : m / z [ m+h ] calcd for c12h9brcln2o2s2 : 390.8977 , found : 390.8976 ; ir ( neat):=3088 , 3070 , 2223 , 1603 , 1580 , 1482 , 1455 , 1436 , 1401 , 1355 , 1311 , 1270 , 1196 , 1158 , 1140 , 1070 , 1018 , 933 cm . n-(3-(1h - tetrazol-5-yl)phenyl)-4-bromo-5-chloro - n - methylthiophene-2-sulfonamide ( 25 ) : the mixture of nitrile 24 ( 1.0 mmol ) , nan3 ( 2.0 mmol ) , and et3nhcl ( 2.0 mmol ) in dry toluene ( 5 ml ) was heated at reflux for 12 h with constant stirring . after cooling , the reaction mixture was acidified ( ph 2 ) using 2 n aq hcl and extracted with ch2cl2 ( 320 ml ) . the combined organic phase was dried over na2so4 , filtered and evaporated under reduced pressure to afford the crude product , which was purified by silica gel flash column chromatography ( ch2cl2/meoh , 95:0590:10 ) to give product 25 as a white solid ( 309 mg , 71 % ) ; mp : 152154 c ; h nmr ( 400 mhz , [ d6]acetone ) : =8.14 ( ddd , j=7.8 , 1.7 , 1.1 hz , 1 h ) , 8.06 ( ddd , j=2.2 , 1.7 , 0.5 hz , 1 h ) , 7.65 ( ddd , j=8.2 , 7.8 , 0.5 hz , 1 h ) , 7.53 ( ddd , j=8.1 , 2.3 , 1.1 hz , 1 h ) , 7.49 ( s , 1 h ) , 3.42 ppm ( s , 3 h ) ; c nmr ( 101 mhz , [ d6]acetone ) : =156.40 , 141.79 , 134.76 , 134.29 , 133.34 , 130.22 , 129.01 , 126.37 , 126.20 , 125.32 , 111.72 , 37.79 ppm ; hrms ( esi+ ) : m / z [ m+h ] calcd for c12h10brcln5o2s2 : 433.9148 , found : 433.9145 ; ir ( neat):=1712 , 1558 , 1454 , 1398 , 1354 , 1154 , 1067 , 1018 , 909 cm . n-(3-(1h - tetrazol-5-yl)phenyl)-4-bromo-5-chlorothiophene-2-carboxamide ( 27 ) : 4-bromo-5-chlorothiophene-2-carboxylic acid ( 26 ) ( 1.0 mmol ) was dissolved in dry dmf , and pybop ( 1.5 mmol ) was added . 3-(1h - tetrazol-5-yl)aniline ( 4 ) ( 2.0 mmol ) and et3n ( 2.0 mmol ) were then added , and the stirring continued for an additional 18 h. the reaction mixture was then diluted with water ( 10 ml ) and extracted with etoac ( 310 ml ) . the combined organic phase was dried over na2so4 , filtered and evaporated under reduced pressure to afford the crude product , which was purified by silica gel flash column chromatography ( ch2cl2/meoh , 97:0390:10 ) to give product 27 as a white solid ( 312 mg , 81 % ) ; mp : 263265 c ; h nmr ( 400 mhz , [ d6]dmso ) : =10.61 ( s , 1 h ) , 8.49 ( t , j=1.9 hz , 1 h ) , 8.15 ( d , j=0.6 hz , 1 h ) , 7.91 ( ddd , j=8.2 , 2.2 , 1.0 hz , 1 h ) , 7.77 ( ddd , j=7.7 , 1.7 , 1.0 hz , 1 h ) , 7.60 ppm ( t , j=8.0 hz , 1 h ) ; c nmr ( 101 mhz , [ d6]dmso ) : =158.12 , 155.68 , 139.09 , 138.06 , 131.52 , 131.05 , 130.02 , 124.94 , 122.55 , 122.49 , 118.47 , 110.96 ppm ; hrms ( esi+ ) : m / z [ m+h ] calcd for c12h8brcln5os : 383.9321 , found : 383.9319 ; ir ( neat):=3284 , 2883 , 1739 , 1631 , 1591 , 1539 , 1407 , 1303 , 1178 , 1079 , 1028 cm . the enzymatic assay applied for screening purposes as well as follow - up dose response characterization was based on the use of membrane preparations from cho cells as a source of enzymatic activity . these cells are known to abundantly express irap and contain minimal amounts of contaminating apn activities . the cho cell membrane preparation was conducted based on the procedure described by demaegt et al . with lysis of washed cells being done by means of ultrasonication followed by multiple strokes ( > 20 ) with a dounze homogenizer and a centrifugation ( 30 000g for 30 min at 4 c ) procedure for pelleting and washing of the membranes . the extent of enzymatic activity in each sample was quantified in a microtiter - based screening assay using the peptide - like substrate l - leucine - p - nitroanilide ( l - leu - pna ; sigma l9125 ) , which upon irap - mediated cleavage produces para - nitroaniline that absorbs at 405 nm . the screening campaign was performed at chemical biology consortium sweden ( cbcs ) using a primary screening set of 10 500 compounds ( majority of compounds donated by biovitrum ab , stockholm , sweden ) . the screen was conducted in a 384-well format , whereas follow - up dose response experiments were conducted in a transparent 96-well plate ( nunc , product no . the final assay volume was 200 l in an assay buffer consisting of 50 mm tris - hcl , 150 mm nacl , and 0.1 mm phenylmethanesulfonylfluoride ( pmsf ) at ph 7.4 . the assays were conducted in the presence of a final concentration of 1 mm l - leu - pna and homogenized cho cell membranes from 50 000200 000 cells per well , depending on test occasion and membrane batch . response assay started with a serial dilution of the compound stock solutions at 10 mm in 99.9 % anhydrous dmso ( sigma aldrich ) by a factor of 1/3 in columns 111 of the 96-well plates . column 12 was reserved for controls : the equivalent amount of dmso was added to wells a12d12 ( negative controls representing 0 % inhibition of the enzyme ) , whereas a 10 mm stock solution of 3 was placed in wells e12h12 serving as controls representing 100 % inhibition of the enzyme activity . the dmso solutions were then diluted in assay buffer and transferred to the transparent assay plates in triplicate ( 50 l to each well both for samples and controls ) , followed by addition of diluted homogenized cho cell membranes ( 50 l ) and substrate ( 100 l ) to initiate the enzymatic reaction . the plates were covered and then incubated at rt until a statistically significant difference was observed ( 612 h ) between the controls ( z > 0.8 ) . care was taken to ensure the readings were taken at a time point when the absorbance signal still increased linearly with time . raw data were then imported into microsoft excel for conversion to % inhibition data based on the controls on each plate . after data from triplicate samples were averaged , the curves were fitted to a four - parameter dose response model within xlfit ( model 205 ) to obtain best - fit values for the ic50 , hill slope , and the upper and lower limits of the dose response curve . docking was conducted using glide ( version 5.8 , schrdinger , llc , new york , ny , 2012 ) sp mode.[3941 ] the crystal structure 4fyt was downloaded from the protein data bank ( pdb ) and prepared using the protein preparation wizard ( schrdinger suite 2012 protein preparation wizard ; epik version 2.2 , schrdinger , llc , new york , ny , 2012 ; impact version 5.7 , schrdinger , llc , new york , ny , 2012 ; prime version 2.3 , schrdinger , llc , new york , ny , 2012 ) in maestro ( version 9.3 , schrdinger , llc , new york , ny , 2012 ) . all water molecules were deleted , hydrogen bond assignments were optimized , and a minimization of the hydrogens was carried out . the structures were prepared for docking using ligprep ( version 2.5 , schrdinger , llc , new york , ny , 2012 ) generating different protonation states . as a service to our authors and readers , this journal provides supporting information supplied by the authors . such materials are peer reviewed and may be re - organized for online delivery , but are not copy - edited or typeset . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors .

the inhibition of insulin - regulated aminopeptidase ( irap , ec 3.4.11.3 ) by angiotenesin iv is known to improve memory and learning in rats . screening 10 500 low - molecular - weight compounds in an enzyme inhibition assay with irap from chinese hamster ovary ( cho ) cells provided an arylsulfonamide ( n-(3-(1h - tetrazol-5-yl)phenyl)-4-bromo-5-chlorothiophene-2-sulfonamide ) , comprising a tetrazole in the meta position of the aromatic ring , as a hit . analogues of this hit were synthesized , and their inhibitory capacities were determined . a small structure activity relationship study revealed that the sulfonamide function and the tetrazole ring are crucial for irap inhibition . the inhibitors exhibited a moderate inhibitory potency with an ic50=1.10.5 m for the best inhibitor in the series . further optimization of this new class of irap inhibitors is required to make them attractive as research tools and as potential cognitive enhancers .

Introduction Results and Discussion Conclusions Experimental Section Chemistry Biology Computational methods Supporting Information

PMC4203393

polymerases are critical to the replication and repair of dna . while replication of dna is an essential first step for cell division , repair of dna is needed when insults such as uv rays , environmental toxins , and some drugs chemically modify dna . these modifications can yield a diverse array of mutations . to understand the mechanisms of dna replication and repair , it is crucial to understand how a polymerase processes dna lesions . as part of ongoing carcinogenesis research and to understand the mechanisms of dna mutation and repair , we have been studying how the bulky and mutagenic arylamine dna lesions ( figure 1a ) interact with a polymerase or a repair protein . using f nmr , microcalorimetric , and other biophyisical methods , we have shown that the arylamine lesions adopt three unique conformations : base - displaced stacked ( s ) , major groove b - type ( b ) , and minor - groove wedge ( w ) , depending on the location of the lesion ( figure 1b ) . the relative populations of s- , b- , and w - conformers depend on the nature of attachment on the central nitrogen ( n - acetyl vs n - deacetylated ) and the hydrophobic carcinogen ring moiety ( planar vs twisted ) as well as the base sequences ( flanking vs near long - range ) surrounding the lesion . ( b ) major ( upper image ) and minor ( lower image ) groove views of the prototype b- , s- , and w - conformers of arylamine dg lesions in cpk model with the dna duplex in gray surface ( color code : arylamine lesion , red ; modified - dg , cyan ; dc opposite the lesion site , green ) . note that the arylamine lesion ( red ) in w - conformation is wedged in the narrow minor groove . it has been shown that most replicative polymerases easily bypass the planar and n - deacetylated aminofluorene ( af ) adducts after a brief stall at the lesion site . on the other hand , the bulkier n-(2-deoxyguanosin-8-yl)-2-acetylaminofluorene ( aaf ) analogues can not be readily bypassed and thus stall dna synthesis . in in vitro studies with x - family polymerase , aaf adducts lead to 2 base deletion mutations , while af extends full - length primers . a recent study via single - molecule fluorescence spectroscopy showed that high - fidelity polymerases can not extend a primer whose terminus occurs across from aaf . in e. coli , aaf adducts result mostly in frameshift mutations , while both af and aaf adducts cause point mutations . in mammals , this difference in mutagenic profiles has been attributed to the presence of a bulky acetyl group on the central nitrogen , which causes the aaf adduct to adopt a syn conformation . in contrast , the af adduct adopts an anti-/syn - conformation , while the n-(2-deoxyguanosin-8-yl)-4-fluoro-4-aminobiphenyl ( fabp ) adduct adopts exclusively an anti - conformation . other factors influencing adduct - induced mutations include topology , insertion of the nucleotide opposite the lesion site , and the characteristics of the polymerase . numerous crystal structure and kinetic analysis studies are available and provide information on actions of native and damaged dna with various polymerases . however , only few examples of replicative polymerases complexed with bulky arylamine - modified dna are available with atomic resolution details , presumably due to difficulties with obtaining crystals . high - resolution solution nmr can offer dynamic information alternative to that of static crystallography . however , some bulky dna lesions cause conformational variation in the dna that can also occur upon binding with a polymerase , which introduces additional challenges to the use of this method . as a result , most nmr studies thus far are limited to adducted dna without the full presence of polymerases and repair proteins . theoretical / molecular dynamic simulations in conjunction with limited nmr and crystal data have been useful . other available techniques for biomolecular interactions such as electrophoretic mobility ( gel shift or gel retardation assay ) and filter - binding assays provide valuable information on binding affinity . however , these approaches contribute either little or no insight on the kinetic parameters underlying complex formation . moreover , these techniques require strenuous work to determine binding parameters . in addition , gel assays do not allow the samples of interest to be in chemical equilibrium due to fast dissociation rate during electrophoresis ; thus , it is difficult to measure proper binding kinetics and thermodynamics . finally , microcalorimetry , such as isothermal titration calorimetry ( itc ) , is a fast and robust method that certainly could be used to characterize binding interactions and the thermodynamics of polymerase dna interactions in free solution , but low - affinity interactions would require higher protein concentrations . surface plasmon resonance ( spr ) is a powerful , chip - based , and label - free solution technology that can provide real - time information on kinetics and thermodynamics . spr relies on changes in the refractive index that are due to changes in mass and can thus measure a small difference in binding ( kd ) at the subnanomolar level . we have recently communicated our initial spr work on the binding affinities of kf - exo to arylamine dna lesions . subsequently , a similar study was conducted to elucidate how faf lesions affect the active site conformation of the human repair enzyme pol and how the structure and sequence of the dna affects its ability to be repaired . in the present study , we are providing a complete set of spr data on the binding of kf - exo or pol to faaf and fabp lesions in two different sequences ( cg*a and tg*a ) . to complement the spr binding results , we also conducted dynamic f nmr as well as steady - state nucleotide insertion kinetics . the results are discussed in terms of adduct - induced conformational heterogeneity , the effect of the 5-flanking base sequence , substrate specificity , and the nature of a polymerase . the purpose of the present article is 2-fold : ( 1 ) to give the full details of our previous spr work and ( 2 ) to introduce spr to the chemical toxicology community as a powerful alternative to existing techniques for investigating protein dna interactions . as a result , the choice of polymerases used in the present study was based largely on the experimental systems in our previous work . obviously , future spr studies should be expanded to a range of y - family bypass polymerases , which are more likely to be involved in replication of bulky dna lesions . dna sequences containing 5-biotin - labeled 31-mer oligonucleotides , phosphorylated 52-mer hairpin , and 21-mer complementary sequences ( figure 2b , c ) were purchased from operon ( eurofin , huntsville , al ) in desalted form and purified by reverse - phase high - performance liquid chromatography ( rp - hplc ) . the hplc system consisted of a hitachi ezchrom elite hplc system with an l2450 diode array detector and a clarity column ( 10 mm 150 mm , 3 m ) ( phenomenex , torrance , ca ) . the mobile phase system involved a 20 min linear gradient profile from 3 to 16% ( v / v ) acetonitrile with 100 mm ammonium acetate buffer ( ph 6.5 ) at a flow rate of 2.0 ml / min . kf - exo ( d424a ) and pol were received as gifts from dr . catherine joyce ( yale university , new haven , ct ) and dr . william beard ( niehs , research triangle park , nc ) . primer oligonucleotide constructs for ( b ) kf - exo and ( c ) pol . approximately 70 m of a faaf or fabp dg modified 16-mer template was annealed with a 9-mer primer in a 1:1 molar ratio to produce a ds / ss junction containing duplexes ( figure 3 ) . the samples were lyophilized and dissolved in 300 l of typical ph 7.0 nmr buffer containing 10% d2o/90% h2o with 100 mm nacl , 10 mm sodium phosphate , and 100 m edta . all f nmr spectra were recorded using a dedicated 5 mm f / h dual probe on a varian 500 mhz spectrometer operating at 476.5 mhz , using acquisition parameters described previously . the spectra were acquired in the h - decoupled mode and referenced relative to that of cfcl3 by assigning external c6f6 in c6d6 at 164.9 ppm . f nmr spectra were measured at two different temperatures , 5 and 25 c . single - nucleotide / full - length extension experiments for both fabp and faaf dg adducts in kf - exo were performed as described previously . briefly , the 9-mer primer was 5-radiolabeled using [ -p]atp and t4 polynucleotide kinase ( t4 pnk ) following the manufacturer s protocol . the p - labeled primer ( 50 pmol ) was annealed with either an unmodified or adducted template oligonucleotide ( 60 pmol ) by heating at 95 c for 5 min and then slowly cooling to room temperature in 3 h. for pol assays , a 1 nt gap was generated by adding a downstream 9-mer primer with 5-phosphate group while annealing with radiolabeled primer ( 9-mer ) and template ( 19-mer ) . the ds / ss primer template sequence ( 20 nm ) was incubated with kf - exo ( 0.5 or 1.0 nm ) for 5 min to form a binary complex in tris buffer ( tris , 50 mm , ph 7.4 ; bsa , 50 g / ml ; 5% ( v / v ) glycerol ) . the reaction was initiated by adding a dntp ( 100 m)/mgcl2 ( 5 mm ) solution to a binary mixture and was incubated at 22 c for 10 min . the reaction was arrested with gel loading buffer ( containing 50 mm edta ( ph 8.0)/95% formamide solution ) . the quenched sample was heated at 95 c for 5 min and immediately cooled on ice . the products were resolved with a denaturing polyacrylamide gel ( 20% polyacryamide ( w / v)/7 m urea ) , electrophoresed at 2500 v for 4 h. the gel was exposed on a kodak phosphor imaging screen overnight and scanned with a typhoon 9410 variable mode imager . to determine the efficiency of dctp insertion opposite the adducted site , steady - state kinetic parameters for incorporation of the nucleotide opposite the unmodified and fabp - modified templates were determined by using the reported literature procedures . the reactions were performed with pol ( 0.5 nm ) and oligonucleotide ( 20 nm ) at 22 c . for the unmodified sequence , reactions were performed in a shorter time period of 0.510 min for nucleotide incorporation and up to 30 min in the case of modified templates . the percentage of primer extended in kinetic assays was determined by taking the ratio of extended primer to the total amount of primer ( unextended + extended primer ) . the modification of 5-biotin cga / tga sequences ( 31-mer ) was carried out using the previously reported procedures ( figure 2 ) , and the modified products were purified by rp - hplc and characterized by maldi - tof mass spectrometry . biotinylated unmodified ( 20 m ) or modified 31-mer ( 20 m ) was annealed with 20 m of 52-mer hairpin by heating at 95 c for 5 min and cooling to room temperature ( figure 2 ) . the annealed mixture was ligated by using 4000 u t4 dna ligase in 1 ligase buffer for 16 h at room temperature . the ligated 83-mer oligonucleotide was purified by 10% denaturing polyacrylamide gel ( figure s1 ) and extracted using the crush and soak method . the desalted oligonucleotide was incubated with 2,3-dideoxy - thymidine-5-triphosphate ( ddttp ) ( 1 mm ) in the presence of kf - exo ( 1 m ) and 5 mm mgcl2 for 12 h. the dideoxy - terminus dna was purified by rp - hplc ( figure s2 ) after precipitation of protein using phenol either a biotinylated 31- , 83- , or 84-mer dna sequence ( 100 pmol ) was mixed with 2 l of matrix containing 1 l of 3-hydroxy picolinic acid ( 3-hpa ) ( 50 mg / ml dissolved in acetonitrile / water 50% v / v ) and 1 l of diammonium hydrogen citrate ( dahc ) ( 50 mg / ml dissolved in acetonitrile / water 50% v / v ) . the mass spectrometric measurement of 31-mer oligonucleotides was carried out in a reflectron positive mode . the calibration of the instrument in reflectron positive mode was performed using low - molecular - weight oligonucleotide or peptide standard calibration kit . for high - molecular - weight oligonucleotides ( > 10 000 da ) , calibration was done in a linear negative mode using 52- , 80- , 90- , and 100-mer standards with laser power 120 in order to enhance the signal intensity . the spectral data was processed by using shimadzu biotech maldi - ms software with processing parameters as follows : smoothing filter width as 20 channels , baseline filter width as 80 channels , and double threshold . a carboxymethylated dextran - coated cm5 chip supplied by ge healthcare was used to immobilize streptavidin ( sa ) via the amine coupling kit on flow cells by following the previously reported literature . the edc / nhs mixture was injected over the surface for 7 min followed by sa ( 50 g / ml dissolved in sodium acetate buffer , ph 4.5 ) . the unreacted reactive esters were blocked with 1 m ethanolamine for 7 min . the running buffer used for immobilization was 1 hbs - ep buffer containing 10 mm hepes ( ph 7.4 ) , 150 mm nacl , 3 mm edta , and 0.05% nonionic surfactant p20 . after sa immobilization , the surface was washed with 50 mm naoh for 60 s pulse , which was repeated five times to remove the free sa until the change in response unit reached below 20 ru . the surface was stabilized by injecting ( 34 times ) running buffer followed by equilibration with running buffer for 1 h. the 84-mer biotinylated dna - hairpin sequences of either unmodified or adducted dna ( 0.250.3 nm ) were injected over the flow cells 2 or 4 for 60120 s individually to achieve 0.73.5 ru . the flow cells were washed with running buffer to remove the unbound dna and to stabilize the surface . before conducting kinetics experiments , 1 mm ddttp in the presence of kf - exo ( 1 m ) and 5 mm mgcl2 was injected over the surface for 5 min followed by 0.05% sds to remove the polymerase . for pol experiments , a 1 nt gap was created by using the same dna coating approach , and , in addition , a corresponding downstream complementary sequence ( 21-mer ) containing a 5-phosphate group ( 2 nm ) was injected over the surface for 5 min . kf - exo was injected with or without dntps ( 100 m ) over the dna surface in random order ( neither ascending nor descending concentrations ) . each concentration was repeated twice . for the binary system , varying concentrations of kf - exo ( 010 nm ) prepared in running buffer containing 1 hbs - p along with 100 g / ml bovine serum albumin ( bsa ) and 5 mm mgcl2 was used . the polymerase was injected for 30 s at a flow rate of 100 l / min followed by dissociation of polymerase . the surface was regenerated using 0.05% sds at a flow rate of 100 l / min , and the injection time was 30 s followed by an extra wash with running buffer . after regeneration of the surface , the surface was stabilized with running buffer for 15 min . initially , three startup steps with running buffer and four zero - concentration injections were performed to condition the surface . for the ternary system , individual dntp ( 100 m ) was mixed with varying concentrations of kf - exo and injected over the surface . the binding affinity constants ( kd ) for binary and ternary systems were calculated using steady - state affinity analysis in biaevaluation software v1.0 as the association rate for the ternary system , particularly as dctp and unmodified dg are near the diffusion limit . the sensorgrams for binary systems were globally fitted with biasimulation basic kinetics module software by using experimental ka and kd values ( figure s7 ) . similar experiments were carried out for pol interaction studies with adduct present both at the nongapped duplex dna and 1 nt gap dna . single - nucleotide ( 1 nt)-gapped dna was generated by annealing a corresponding downstream primer . for nongapped dna , the concentration of pol was varied up to 1000 nm , whereas for 1 nt gap , it was 0100 nm depending on the dg adduct embedded . an overall scheme for the construction of the biotinylated hairpin - based template primer strands is depicted in figure 2a . fabp- or faaf - modified biotin-31-mer oligonucleotides were prepared according to published procedures . the 52-mer hairpin dna was annealed and ligated to the biotinylated 31-mer ( figure 2b , c ) . the hairpin structure was created to improve the thermal stability of the oligonucleotide constructs on a gold chip during kinetics experiments . as a result , the same oligonucleotide constructs could be used multiple times with different polymerases and buffer conditions . finally , the lesion was positioned at the 22nd base , with 21 bases on the 5-side and 28 bases on the 3-side , in order to avoid close contact between the polymerase and the chip surface . the resulting template / primer strands , containing the biotinylated 84-mer hairpin , were purified by denaturing polyacrylamide gel ( figure s1 ) and used for further study . maldi - tof spectrum , obtained in reflectron mode , of the faaf - modified biotin - tg*a-31-mer sequence is shown in figure s3 . a distinctive peak at 9841.30 da is in close agreement with theory ( 9839.90 da , m / z : + 1.40 ) , and the inset is a linear negative mode spectrum . the inset b at 25925.76 da corresponds to the 83-mer strand consisting of the biotin-31-mer - tg[faaf]a- and the 52-mer hairpin in the absence of ddt at the primer terminus ( theoretical 25923.00 da , m / z : + 2.76 ) . the inset c at 26206.70 da corresponds to the 84-mer strand formed by adding ddt to the primer terminus of the 83-mer strand ( 26211.00 da , m / z : 4.30 ) . the corresponding tg[fabp]a , cg[faaf]a , and cg[fabp]a sequences were similarly characterized ( figure s4s6 ) . all of the calculated and experimental m / z values are shown in table s1 . to examine lesion - induced conformational heterogeneity , we measured f nmr spectra of modified 16/9-mer template / primer duplexes . as shown in figure 3 , the f nmr spectra of fabp- and faaf - modified duplexes in the cg*a and tg*a sequences are compared at 25 c . fabp duplexes exhibited a single peak at 116.4 ppm in both sequences , which is consistent with the chemical shift range observed previously for the anti - b - type fabp conformer . the bulky faaf displayed three f signals , with two prominent peaks of similar intensity at around 114 to 116 ppm , for both sequences . we have previously reported the chemical shift ranges that correspond to the b- , s- , and w - conformers of faaf - modified duplexes , i.e. , 115.0 to 115.5 ppm for the b - conformer , 115.5 to 117.0 ppm for the s - conformer , and 117.0 to 118.0 ppm for the w - conformer . hence , the present faaf - induced heterogeneity could be a variation of the b / s / w heterogeneity . in contrast to the aforementioned study , however , the f signals in the present study are derived from the lesions at the ds / ss junction , not fully paired double helical duplexes . the relative shielding of f signals and the narrow chemical shift range ( 2 ppm ) in the present work are probably due to the flexible lesions at the ds / ss junction . as a result , we could not unequivocally assign the signals to the b- , s- , or w - conformer . f nmr spectra of fabp and faaf adducts in the cga and tga duplexes at a ds / ss junction at 25 c . single - nucleotide incorporation was carried out using the e. coli exonuclease - deficient klenow fragment ( kf - exo ) and the human base excision repair polymerase ( pol ) ( figure 4 ) . like any other high - fidelity replicative polymerase , kf - exo prefers the ds / ss replication fork as a template / primer dna substrate . in the unmodified dna control , the primer was immediately elongated to full length in the presence of all four nucleotides and kf - exo ( data not shown ) . with the fabp - modified template , however , primer elongation was largely stalled at the lesion site , with some insertion of the correct dctp opposite the lesion ( figure 4a ) . assays of full - length and single - nucleotide incorporation into fabp - adducted cg*a and tg*a sequences with ( a ) kf - exo and ( b ) pol . unlike kf - exo , pol prefers a single - nucleotide gap as a substrate . with pol , there was no full extension of either the unmodified ( not shown ) or fabp - modified template ( figure 4b ) . as for faaf , no nucleotide insertion was observed with either kf - exo or pol , even at high enzyme concentrations or with a longer incubation period ( data not shown ) , because the lesion had completely blocked elongation . we conducted steady - state experiments to investigate the impact of conformational heterogeneity on nucleotide insertion kinetics . the results for kf - exo and pol are summarized in tables 1 and 2 , respectively . to examine the influence of lesions , we used the relative insertion efficiency fins , which was defined as ( kcat / km)modified or mismatched /(kcat / km)unmodified . with kf - exo , the fins of dctp opposite -cg[fabp]a- was 500-fold lower than that of the unmodified control ( table 1 ) this is contrasted with -tg[fabp]a- , which was reduced only 33-fold . in the pol assay ( table 2 ) , the fins of dctp opposite fabp in the cga sequence was 142-fold lower than that of the control , while in the tga sequence , the fins was 59-fold lower than that of the control . these results indicate that the nucleotide insertion efficiency is consistently greater in the tga sequence compared to that in the cga sequence , regardless of the polymerase structure . we were unable to perform similar steady - state kinetics experiments for faaf because this lesion caused a major blockage at the replication fork . after activation with streptavidin ( sa ) , flow cells 1 and 3 were retained as blank references , and dna was coated on the sa surface of flow cells 2 and 4 . surface testing , regeneration buffer scouting , and the mass transport limitation test were performed before the kinetics experiments , as described previously . dna coating at 0.7 resonance units ( ru ) did not show any influence of mass transport ; an increase in flow rate of the analyte did not alter the association rate . however , at 10 ru , mass transport became a limiting factor , as the association rate deviated with the flow rate of the analyte ( data not shown ) . on the basis of this study of mass transport limitation , all of the experiments were carried out in the dna coating range between 0.7 and 3.5 ru . the sensorgrams for the binary binding between kf - exo and the unmodified tga controls or the modified tg*a oligonucleotide constructs are shown in figure 5a . we performed steady - state affinity analysis of the binary and ternary complexes in the presence of four dntps ( figure 6 ) . a similar set of results for the cga sequence have been reported previously , and the results on the binding affinity of kf - exo to both tga and cga sequences are summarized in table 3 . sensorgrams of binary complexes of ( a ) kf - exo and ( b ) pol with unmodified and modified tga sequences ( 1:1 binding fitted curves are overlaid as red lines ) . steady - state affinity analysis of interaction of kf - exo with ( a ) -tg[faaf]a- and ( b ) -tg[fabp]a- sequences . as for the unmodified controls , kf - exo binds tightly in both sequences in the presence of the correct dctp . the affinity of binding for the cga sequence was reduced by 30- , 62- , 264- , and 34-fold in binary , datp , dgtp , and dttp , respectively , compared to that for the correct dctp binding ( table 3 ) . similar results were obtained for tga , where the binding affinity was reduced by 15- , 39- , 180- , and 40-fold in binary , datp , dgtp , and dttp , respectively ( table 3 ) . these results are consistent with those of the nucleotide insertion assay , which showed preferential insertion of the correct dctp . kf - exo bound strongly to the modified tg*a templates . in the tg*a sequence , the kd value for fabp was 4.9-fold greater than that of the control , and the kd value for faaf was 8.8-fold greater than that of the control . similar changes were observed in the cg*a sequence , where the kd for fabp was 10.8-fold larger than that for the control , and the kd for faaf was 7.2-fold larger than that for the control . these differences are primarily due to the much slower dissociation rates observed for the modified template / primer for both the cg*a sequence ( faaf kd : 0.02 s ; fabp kd : 0.01 s ) and the tg*a sequence ( faaf kd : 0.01 s ; fabp kd : 0.01 s ) . the net stabilization energies were positive and ranged from 1.10 to 1.47 kcal / mol ( table s2 ) . kd for the correct nucleotide was 0.190.25 nm with faaf and 0.290.30 nm with fabp , while for the incorrect nucleotide , kd was 0.280.67 nm with faaf and 0.310.66 nm with fabp . for pol , binding assays were performed on two distinct substrates : nongapped ds / ss and 1 nt gap . the results for the binary and ternary systems on both cga and tga sequences are summarized in table 4 . weak binding was observed for the nongapped dna , with kd values of 0.8 m ( data not shown ) . in contrast , the binding affinity of pol increased 1000-fold with the 1 nt gap . kd values are in nanomolar ( nm ) . for nongapped duplex dna ( ds - dna ) with pol , kd values exceeds 0.8 m . as for the unmodified controls , pol binds to the correct dctp more tightly . the binding affinity for the dctp is 2.7-fold higher in the tga sequence and 4.5-fold higher in the cga sequence ( table 4 ) . in contrast to kf - exo , the binding in the binary complex between the modified template and pol is less tight than that in the complex containing the unmodified template , where the differences in binding are approximately 3-fold for fabp and 5- to 6-fold for faaf , respectively . similar to kf - exo , the binary complex with faaf showed slower off rates ( kd : 0.01 s ) with pol in both sequences . faaf ( figure 5b ) are relatively poor ; however , the residual plots for the dg faaf / pol binary complex ( figure s10 ) indicate a good curve fit within 1% values of rmax . the complexes with b - conformeric fabp exhibited unusually faster dissociation rates for both the cg*a and tg*a sequences , where the kd values were 0.76 and 0.40 s , respectively , and the negative net stabilization energy was 1.04 and 0.27 kcal / mol , respectively ( table s2 ) . figure s9 shows the sensorgrams for the ternary complexes between pol and the faaf- and fabp - modified cg*a constructs . we have recently reported a similar set of binding results for the n - deacetylated faf . with the correct nucleotide dctp , the pol binds 2.7-fold more tightly in the ternary complex than that in the binary complex and 3000-fold more tightly than to that of nongapped dna . the binding affinity to the incorrect nucleotide was 4- to 5-fold lower than that to the correct dctp . the lesion in the 1 nt gap reduced the binding affinity of pol by 6-fold for faaf and 3-fold for fabp , virtually eliminating the nucleotide selectivity of pol at the lesion site . the affinity for pol binding decreased in the order dg > fabp > faaf . in the present study , we have employed spr to investigate the binary and ternary binding interactions of kf - exo and pol to two prototype arylamine dna lesions ( fabp and faaf ) in the context of two different sequences ( cg*a and tg*a ) . kf - exo is a 68 kda high - fidelity replicative a - family bacterial dna polymerase , which carries polymerase and 35 exonuclease activities and has been used extensively as a model enzyme for studying adduct - induced dna synthesis . pol is the smallest ( 39 kda ) eukaryotic polymerase , belonging to the x - family of base excision repair dna polymerases , and has been characterized extensively . with pol , primer extension past aaf adduct was blocked , but full - length products were shown to contain exclusively 2 deletion mutations . although its role is limited in base excision repair , pol has been additionally implicated in the replication of various dna damage . for example , deregulation of pol may enhance the genetic instability induced by bulky lesions such as cisplatin and uv radiation . fabp and faaf are c8-substituted dg adducts that contain structurally unique arylamine structures , i.e. , n - acetylated / coplanar - fluorene and n - deacetylated / twisted - biphenyl , respectively ( figure 1a ) . finally , the two sequences ( cg*a vs tg*a ) were selected because of their marked difference in the s / b population ratios observed with the n - deacetylated faf . the spr results , along with data from f nmr and steady - state primer kinetics , elucidate how lesion - induced conformational heterogeneity alters the binding capacity of a polymerase and thus its nucleotide insertion efficiency . we constructed the 84-mer hairpin - based oligonucleotides for spr ( figure 2 ) based on the following considerations . first , the incorporation of ddt at the 3-end of the primer prevents the usual nucleophilic attack of the 3-hydroxyl to the incoming dntp and thus blocks the formation of a phosphodiester bond . this ensures the stability of the ternary complex polymerase / template - primer / dntp for spr measurements . previous assays using gel electrophoresis , single - molecule fret , or crystallography have consistently shown that the absence of 3-oh at the primer terminus does not affect the affinity with which polymerases bind to binary and ternary complexes of dna . second , while kf - exo requires a minimum of 11 bases , because it covers approximately 5 bases downstream from the primer / template junction and 6 to 7 bases upstream of the 3-primer s terminus , pol can operate on any length of dna containing a 1 nt gap . we observed very tight binding of kf - exo with native unmodified dg in the presence of the correct incoming nucleotide dctp . this system exhibited high nucleotide selectivity , with kd values increasing in the order dctp dttp datp dgtp ( table 3 ) . the spr results are in agreement with nucleotide insertion assays , which showed exclusive insertion of the correct dctp over other dntps . 2:1 and higher order complexes have also been observed in solution by various biochemical and biophysical methods . as shown in figure s11 , comparison between theoretical and experimental rmax for pol and kf - exo are in good agreement , indicating a 1:1 complex . initially , we carried out a spr binding assay of pol using the nongapped ds / ss junction replication fork . the binding was very weak , with kd values in the micromolar concentration range . however , upon introduction of the 1 nt gap ( figure 2c ) , the dna binding affinity of pol increased 200- to 1000-fold . these results indicate that the presence of 5-po4 enhances the binding affinity of the 8 kda lyase domain as well as the 31 kda catalytic domain . the observed differences in binding affinity are consistent with previous reports in which the lyase domain in the duplex ( nongapped ) dna was flexible . introduction of the 1 nt gap enhances the binding affinity of the polymerase to dna . the results are also in agreement with gel assays , which have previously shown that addition of the correct dctp opposite unmodified dna enhances the binding affinity of polymerase compared to that with other nucleotides by an induced - fit model adopted by pol . an unusually greater binding of kf - exo was observed for modified dg , where the kd of this interaction was 511-fold higher than the kd for interaction with the unmodified native dna substrate . the binary binding affinity decreased in the order fabp > faaf > dg for the cg*a sequence and faaf > fabp > dg for the tg*a sequence ( table 3 ) . previous studies have also shown tighter binary binding of kf - exo with the aaf adduct . using gel - retardation assays , dzantiev and romano showed that the bulky and hydrophobic aaf interacts with nearby hydrophobic amino acid residues , strengthening its binding to the active site of kf - exo . the authors suggested that such lesion - induced conformational adjustment may block the conformational change required to properly accommodate an incoming nucleotide . it is well - established that the n - deacetylated fluorinated analogue faf adducts ( figure 1 ) adopt sequence - dependent equilibrium between b- and s - conformers . fabp is similarly n - deacetylated , but it lacks a methylene bridge , resulting in a bulky twisted biphenyl moiety . in other words , fabp may behave like faaf at the replication fork of the template in the active site of a polymerase . in contrast to the unmodified control , modified adducts displayed a significant decrease ( 713-fold ) in dissociation rate , with positive net stabilization energy ( table s2 ) . the markedly slower off rates are consistent with single - molecule fret studies as well as gel shift assays in which the presence of the bulky dna adduct stabilizes the binary complex and does not induce dissociation before nucleotide incorporation . in contrast to kf - exo , pol exhibited significantly less binary binding affinity to the modified templates . furthermore , the modified sequences exhibited significantly faster dissociation rates and more negative net stabilization energies . as in the ds / ss situation discussed above , it is likely that faaf promotes conformational heterogeneity in a sequence containing a 1 nt gap . such heterogeneity may hinder the interaction of that sequence with key amino acids in the polymerase , thus preventing the polymerase from undergoing conformational change that is necessary for strong binding . nucleotide selectivity was low in the ternary complexes with kf - exo , where the kd values indicate poor discrimination between the correct ( kd 0.190.30 nm ) and incorrect ( kd 0.280.67 nm ) nucleotides . this poor selectivity does not depend on the nature of the lesion ( fabp vs faaf ) or the 5-flanking base ( cg*a vs tg*a ) . the lack of nucleotide selectivity appears to be in agreement with the results of tryptic digestion studies , in which the aaf polymerase complex maintains an unstable noncatalytic open conformation in the presence of any dntp . in other words , aaf modification did not stabilize the complexes in relation to the incoming nucleotide . this is contrasted with native dna , to which the polymerase binds very tightly in the presence of the correct nucleotide dctp and is insensitive to digestion . our f nmr results ( figure 3 ) indicate a complex conformational heterogeneity of the bulky faaf at the ds / ss templating position , which may prevent the polymerase from properly accommodating an incoming dntp . this reasoning is in accord with the weak electron densities observed for the arylamine base in the active site of t7 dna polymerase , where the authors of the previous study also concluded that conformational heterogeneity may hinder the insertion of an incoming nucleotide . the low selectivity for incoming nucleotides could also arise from the high stability of the binary complex , which may hinder the polymerase s ability to recognize the incoming nucleotide . no crystal structures or high - resolution nmr structures are currently available for complexes between any dna polymerase and abp or the fluorinated fabp . in the present study , fabp in both sequences exhibited a single f signal , possibly for a b- or a b / s - conformational mix , owing to the presumed conformational flexibility at the ds / ss junction . these nmr data , albeit in the absence of a polymerase , are in agreement with the gel - based kinetics data , which reveal a preference toward inserting the correct nucleotide over other nucleotides ( table 1 ) . in the case of kf - exo , the tg*a sequence favored the insertion of dctp more efficiently than the cg*a sequence . the relative insertion efficiency fins of dctp opposite fabp was significantly lower in the cg*a ( 500-fold ) and tg*a ( 33-fold ) sequences compared to that of the unmodified controls ( table 1 ) . this 15-fold difference in fins is puzzling because fabp at the ds / ss junction exhibited a single f signal in both sequences ( figure 3 ) . however , we have shown previously that faf in the duplex setting displayed a greater s - conformer in the cg*a duplex ( 50% ) relative to that in tg*a ( 38% ) . as mentioned above , it is likely that the absence of coplanarity in fabp would embrace intermediate structures between faaf and faf , as observed from f nmr , gel , and spr assays . the spr results with pol ( table 4 ) indicated that a modified templating base weakens the polymerase s binding affinity and the nucleotide selectivity ( figure s9 , table 4 ) . the reduced binding affinity of pol to the modified template dna could be related to the lesion - induced conformational heterogeneity in the active site of the polymerase . in the closed conformation , key amino acids such as lys 234 and tyr 271 interact with the minor groove of the primer strand , while arg 283 interacts with the template strand of dna . as mentioned above , it is possible that the faaf at the 1 nt gap may hinder the active site geometry and thus prevent the conformational change necessary to form the catalytic ternary complex . we previously observed similar conformational heterogeneity caused by faf bound to 1 nt gap dna in both the absence and presence of pol . the results are also consistent with translesion synthesis studies in which the minor groove conformation benzo[a]pyrene diol epoxide n - dg adducts creates steric clash with the active site of pol , thereby reducing the insertion rate . these results are in agreement with the steady - state kinetics data that show significant reductions in the fins of dctp opposite fabp in the cg*a and tg*a sequences ( 142- and 59-fold , respectively ) , relative to that of the corresponding unmodified controls . the question is how to reconcile the apparent lack of discrimination between dntp at the binding step ( figure 7 and tables 3 and 4 ) with the clear preference for accurate insertion of dctp ( figure 4 and tables 1 and 2 ) . we have recently shown that the af adduct can change its binding characteristics at the replication fork or in a single - nucleotide gap in the active sites of dna polymerases . similarly , it is plausible that the dynamics of fabp- and faaf - induced conformational heterogeniety could be altered to accommodate an incoming dntp within the active site of polymerases in a way that favors the incorporation of the correct base dctp . plots of nucleotide specificity ratio ( kd - binary / kd ) with ( a , b ) kf - exo and ( c , d ) pol for unmodified and modified tg*a and cg*a dna templates . kd - binary - dg represents kd of unmodified dna - polymerase binary complex , and denominator kd represents the ternary complex of unmodified dna ( or ) binary and ternary complexes of adducted dna . in the present study , we have taken advantage of the sensitivity of spr , which allowed us to probe the delicate interaction between polymerases and dna strands containing arylamine dna lesions at the binary and ternary complex levels . we found that 0.73.5 ru of dna coating was sufficient , with no significant interference from mass transport limitation . the binding specificity ratios ( kd of the control binary complex over the kd of a ternary complex ) in the presence of dntps for the unmodified ( dg ) and faaf- and fabp - modified lesions are plotted in figure 7 . we observed highly specific binding between kf - exo and the native dna substrates in the presence of the correct dctp ( green ) opposite a dg templating base ( figure 7a , b ) . this is consistent with the polymerase undergoing a conformational change , from open to closed , to form watson kf - exo binds weakly with the incorrect dntps , probably retaining the catalytically incompetent open conformation . the binding of dgtp ( pink ) with kf - exo was particularly poor . similar binding results were obtained with pol ( figure 7c , d ) , although the affinities for modified ternary complexes were generally weaker than those with kf - exo . in both enzymes , however , we observed no discernible nucleotide specificity ( dntps ) and sequence effects ( cg*a vs t*ga ) . kd values for the ternary complexes for unmodified dna were determined using affinity analysis because the association rate ( ka ) reaches the near - diffusion limit in native dna . this procedure allowed for the monitoring of interactions between unmodified or adducted dna with different polymerases on a single chip . the present work also demonstrates the utility of spr in distinguishing the substrate preference of different polymerases ( e.g. , ds / ss vs 1 nt gap for pol ) . to our knowledge , this is the first comprehensive use of spr to probe nucleotide insertion kinetics during the action of a polymerase . furthermore , the present spr work advances the limits of spr technology , demonstrating that spr can measure subnanomolar affinity differences between incoming nucleotides and the active site of a polymerase . in conclusion , we have characterized the spr binding affinity of the mutagenic fabp and faaf lesions bound to kf - exo and pol . kf - exo binds strongly to ds / ss template / primer dna , whereas pol prefers gapped dna . tighter binding was observed between unmodified dg and kf - exo or pol . the systems exhibited nucleotide selectivity , with kd values increasing in the order of dctp dttp datp dgtp . unlike pol , kf - exo binds tightly to both faaf and fabp lesions in the binary systems . the relative insertion efficiency fins of dctp opposite fabp was significantly higher in the tg*a sequence compared to that in the cg*a sequence and the unmodified controls . while the lesion effect was not significant in kf - exo , the active site of pol is sensitive to the faaf - induced conformational heterogeneity . our spr data are complemented by primer steady - state kinetics and f nmr data and provide valuable insights into how lesion - induced conformational heterogeneity in dna alters the action of polymerases and thus affects the nucleotide insertion efficiency and coding potential .

surface plasmon resonance ( spr ) was used to measure polymerase - binding interactions of the bulky mutagenic dna lesions n-(2-deoxyguanosin-8-yl)-4-fluoro-4-aminobiphenyl ( fabp ) or n-(2-deoxyguanosin-8-yl)-7-fluoro-2-acetylaminofluorene ( faaf ) in the context of two unique 5-flanking bases ( cg*a and tg*a ) . the enzymes used were exo - nuclease - deficient klenow fragment ( kf - exo ) or polymerase ( pol ) . specific binary and ternary dna binding affinities of the enzymes were characterized at subnanomolar concentrations . the spr results showed that kf - exo binds strongly to a double strand / single strand template / primer junction , whereas pol binds preferentially to double - stranded dna having a one - nucleotide gap . both enzymes exhibited tight binding to native dna , with high nucleotide selectivity , where the kd values for each base pair increased in the order dctp dttp datp dgtp . in contrast to that for pol , kf - exo binds tightly to lesion - modified templates ; however , both polymerases exhibited minimal nucleotide selectivity toward adducted dna . primer steady - state kinetics and 19f nmr results support the spr data . the relative insertion efficiency fins of dctp opposite fabp was significantly higher in the tg*a sequence compared to that in cg*a . although kf - exo was not sensitive to the presence of a dna lesion , faaf - induced conformational heterogeneity perturbed the active site of pol , weakening the enzyme s ability to bind to faaf adducts compared to fabp adducts . the present study demonstrates the effectiveness of spr for elucidating how lesion - induced conformational heterogeneity affects the binding capability of polymerases and ultimately the nucleotide insertion efficiency .

Introduction Materials and Methods Results Discussion

PMC5245927

most published floating clavicle report a dislocation or fracture of one or both ends of the clavicle . we reported a new framework of this injury in a young triathlon athlete ; medial - end displaced fracture with co - existent double disruption of the superior shoulder suspensory complex ( sssc ) with the anterior shoulder arch wholly disconnected from the nearby structure . the infrequent publications and the rarity of this type of injuries do nt support the surgeon about the choice of the best possible treatment . however , if they are involved patients with high functional demands , the authors suggest the surgical management of medial - end clavicle fractures followed by restoration of sssc complex if damaged on more than two locations . injuries of the lateral aspect of the shoulder are common . in most cases the force applied to the shoulder girdle is mild , getting an isolated lesions of the acromion - clavicular joint or of the clavicle . high - energy hit trauma that laterally impacts the shoulder is less common and usually it is often associated to other major damages ( such as ribs fractures , thoracic injuries , vascular or brachial plexus impairment ) . it is also reported an unusual type of fracture - dislocation named floating clavicle with dislocation of both ends , or with a sternoclavicular dislocation and fracture of the middle - third or the lateral end of the clavicle [ 1 - 5 ] . ( medial - end displaced clavicle fracture with co - existent double disruption of the sssc ) in young triathlete , as a result of a high - velocity sport fall . a 33-year - old male triathlon athlete , during a bicycle training fell on his right side , hitting his shoulder on the ground , but with no other trauma associated . he presented an altered sternoclavicular skin profile with a swollen and bruised area on the lateral surface of his shoulder . he suffered pain in the sternoclaviular joint and in the shoulder , while the abduction and extension of the upper limb was impossible ( pre - operative dash score : 90.9 ) . the initial radiographs showed an extrarticular displaced fracture of the medialend of the clavicle ( robinson type 1b.1 ) , acromion ( kuhn type ii ) and coracoid process ( ogawa type i ) fractures . these bone lesions were also confirmed by a computed tomography ( ct ) scan with 3d reconstruction . the images showed a displaced comminuted extrarticular medial - end clavicle fracture with anterior dislocation , an undisplaced fracture of the coracoid and acromion processes ( fig . 1 and 2 ) . x - ray of the displaced medial - end clavicle fracture a. ct - scan shows the displaced medial - end clavicle fracture and co - existent double distruption of sssc . on the 3rd post - injury day , prior to the fixation of sssc injuries , we treated the clavicle . under general anesthesia , with the patient in beach chair position with mayfield headrest , we performed a skin incision directly on the medial - end of the clavicle , respecting the sternoclavicular joint . we exposed the displaced fracture and fixed bone fragments with a t - shape plate 2.5 mm with 5 locking screws ( intrauma ) . after the clavicle fixation , we performed percutaneous acromion fracture fixation with two cancellous bone screws with washer ( asnis , stryker 5 mm x 55 and 60 mm length ) ( fig . we achieved a good clavicle and acromial fixation , while the coracoid process was satisfactorily stable without any other surgical step . thus , the day after surgery , he started active and passive movements of the elbow , wrist and hand . one month post - operatively , the patient underwent x - ray that showed fractures in healing . the patient started water rehabilitation with isometric shoulder exercise , active and passive abduction , adduction , flexion and extension shoulder movement , wall crawl , and elastic resistance exercise . he referred no difficulty in performing activities which required little efforts , mild difficulty to performing overhead movements and severe difficulty in performing activities which needed high efforts of the shoulder and the arm ( dash score : 52.3 ) . the patient recovered full rom of the shoulder without stiffness and reported no pain in performing active movements , mild efforts of the shoulder and during sport activities , such as running and riding a bike , but on the other hand , he reported persisted moderate pain during swimming training ( dash score : 29.5 ) . the clinical evaluation at the follow up 12 months after the operation showed the patient had recovered full shoulder function ( no limitation if compared with contralateral side ) and was able to perform triathlon races with no pain or limitations ( dash score : 11.4 ) most reports of a floating clavicle comprise of a dislocation of both ends of the clavicle , while other authors document a different clinical presentation : thomas et al.1 ) document a patient with a sternoclavicular dislocation associated to the fracture of the middle - third , while serra et al . report an anterior sternoclavicular dislocation with a nondisplaced fracture of the lateral end of the clavicle . our case is an exceptional injury in that it was the result of a high - velocity fall with a displaced medial - end clavicle fracture and a co - existent double disruption of sssc ( acromion and coracoid processes ) without other major lesions . usually it is due to a direct trauma with an impact occurred on the lateral side of shoulder and it is of ten associated to other major damages : ribs fractures , thoracic injuries , vascular or brachial plexus impairment [ 2 - 4 ] . ordinarily a low - energy trauma directed anteriorly may strike only the acromionclavicular joint or clavicle , but if the vector of the force steers posteriorly the acromion , glenoid or scapular spine may be involved . however , high - energy lateral impaction forces can disrupt both the anterior and the posterior arches and usually causes internal lesions[4 , 5 ] . as suggested by scarlat , in our case the trauma action began laterally on the shoulder with a vector directed from an external to an internal direction . at the time of the fall the first one displayed an anterior and superior trend ( antero - superior force : asf ) involving the anterior shoulder arch , thus causing the medial - end clavicle fracture , while the second one took a posterior - inferior direction ( posteror - inferior force : pif ) arousing the acromion , and the base of coracoid process lesions ( co - existent double distruption of sssc ) [ 2 , 5 ] . the coracoacromial ligament , the acromion - clavicular joint and the connection between coracoid and clavicle ( conoid and trapezoid ligaments ) remained undamaged . then , the anterior shoulder arch was wholly disconnected from the nearby structure ; in short , it was just floating . to explain the absence of other major lesions and the soft tissue damage , we hypothesized the damping effect played by the lateral shoulder hypertrophic muscles ( deltoid and rotator cuff muscles ) in the young triathlon athlete and secondly the direction and the partition of the trauma forces . furthermore , we hypothesized that that the absence of the ribs fractures was due to the angle of the asf and pif vectors ; they resulted justly tangential to the chest wall , splitting the injury s energy towards the costoclavicular and the scapulothoracic spaces . the infrequent publications and the rarity of this type of injuries did nt support the surgeon about the choice of the best possible treatment [ 1 , 6 ] . non - operative treatment has been recommended by many authors for close fractures , though others recommend only clavicular osteosynthesis as a stabilizing procedure ( especially in case of open fractures ) , whereas various papers support a double osteosynthesis of clavicular and scapular fractures , [ 7 , 8 ] . some authors believe that the stability of the shoulder girdle depends on the integrity of the superior shoulder suspensory complex ( sssc ) . if sssc is intact and the shoulder girdle is not medially displaced more than 20 mm , a nonsurgical treatment for scapula and all ipsilateral clavicle fractures may be recommended . however , studies report a worse outcome for patients with sssc double lesion treated with non - surgical management and other authors speak of a better recovery in those who were operated for a lesion of the medial end of the clavicle . in this specific case in order to restore biomechanical function and to ensure the best and fastest recovery of the young triathlon athlete s shoulder it was mandatory to maintain the length of the anterior and the posterior arches . we chose a stable internal fixation in order to stabilize the displaced medial clavicle fracture and the acromion . the reason for a surgical management of the clavicle at first was due to avoid possible common sequelae from unstable clavicular fractures , such as nowak et al stated . we decided not to manage the coracoid process and scapular fractures after evaluating their stability . although the management of medial - end clavicle fractures is an open debate , most of the authors supports the surgical indication for displaced fractures and in the case of neurovascular compromise ; even in the case of a complete sssc breakage on more than two locations in structures with the ring concept ( such as the pelvis ) some authors agree that it causes instability , and surgical intervention is usually indicated [ 8 - 10 ] . in this paper we reported a variant type of this injury ( medial - end displaced fracture with co - existent double disruption sssc ) in a young triathlon athlete . the infrequent publications and the rarity of this type of injuries do nt support the surgeon about the choice of the best possible treatment . the clinical results described in this case report encourage further studies to draw a standardized surgical approach for the medial clavicle fracture . in case of medial - end clavicle fracture and complete sssc breakage in more than two locations ( the ring concept , such as the pelvis ) the shoulder is unstable and surgical intervention is indicated .

introduction : most published floating clavicle report a dislocation or fracture of one or both ends of the clavicle.case presentation : we reported a new framework of this injury in a young triathlon athlete ; medial - end displaced fracture with co - existent double disruption of the superior shoulder suspensory complex ( sssc ) with the anterior shoulder arch wholly disconnected from the nearby structure.conclusion:the management of these complex fractures remains an open debate . the infrequent publications and the rarity of this type of injuries do nt support the surgeon about the choice of the best possible treatment . however , if they are involved patients with high functional demands , the authors suggest the surgical management of medial - end clavicle fractures followed by restoration of sssc complex if damaged on more than two locations .

Introduction: Case Presentation: Conclusion: Introduction Case Report Discussion Conclusion

PMC5084478

overexpression of human epidermal growth factor receptor 2 ( her2 ) is reported approximately in 1520% primary human breast cancers . her2 and other member of her superfamily , consist of tyrosine kinase receptors which have roles in the proliferation 's regulation and epithelial cells ' survival . it is one of the members of her family and has four plasma membrane - bound receptors of tyrosine kinases that transmit extracellular signals to initiate cellular signaling pathways via mitogen - activated protein kinase , phosphoinositide 3-kinase , phospholipase c , protein kinase c , and signal transducer and activator of transcription . recently , there are some reports that her2 is expressed in many tissues , including the breast , gastrointestinal tract , kidney , and heart . it has been suggested that possession of her2 lead to cell proliferation and also apoptosis suppression . patients who suffer her2-positive breast cancer had poor outcome ; however , after introducing trastuzumab , a recombinant humanized monoclonal antibody that binds to the extracellular subdomain of her2 , the destinies have changed . it is worth noting that not all the patients take advantages of trastuzumab administration , approximately 15% of patients relapse after treatment , indicating trastuzumab resistance eventually emerges in the patients . several resistance mechanisms have been noted to be responsible including changes in receptor antibody interaction , induction of the downstream pathways due to increasing in signaling from either other members of the her family or other receptors , and finally constitutive activation of downstream elements . resistance to trastuzumab as a her2 receptor antagonist , causing concerns , and in recent years , great efforts were applied in developing therapeutic agents to either potentiate the effect of trastuzumab or target cells which have become resistant to trastuzumab . ado - trastuzumab emtansine ( t - dm1 ) , is an antibody - drug conjugate , in which trastuzumab linked to the fungal cytotoxic agent , mertansine . antibody - drug conjugate such as t - dm1 are composed of antigen - specific antibodies bounds to cytotoxic agents in order to selectively transfer drugs to tumor cells and minimize systemic toxicity . t - dm1 consists of the monoclonal antibody trastuzumab linked to a potent microtubule inhibitor ( emtansine ) , leading to a targeted delivery of chemotherapy agent to cells that overexpress her2 . a derivative of maytansine , emtansine ( also known as dm1 ) , is a cytotoxic agent that its mechanism of action is , microtubule inhibition similar to vinca alkaloids . off note , emtansine are more potent than vincristine and vinblastine in vitro and failed to show therapeutic effect at tolerable doses . t - dm1 binds to the her2 receptors , and makes the her2 receptor and drug - antibody conjugate internalized via endocytosis . hence , emtansine binds to its target , tubulin , and causes cell cycle arrest and apoptosis . the role of trastuzumab is targeted delivery of emtansine and inhibition of her2 signaling pathways . t - dm1 has primarily been evaluated as a single agent in patients with metastatic her2-positive breast cancer . there are several phase ii studies on which t - dm1 was administered every 3 weeks as a single agent . afterwards , the phase iii clinical trial , trastuzumab emtansine for her2-positive advanced breast cancer ( emilia ) , was released . this study was the only competed phase iii clinical study , in which t - dm1 administration was randomly compared to lapatinib plus capecitabine in patients with her2-positive , unresectable , locally advanced , or metastatic breast cancer who were previously treated with trastuzumab and a taxane . the results were in favor of the positive effects of t - dm1 administration in overall survival and median duration of response . according to the package labeling , appropriate candidates for t - dm1 include patients who have received prior therapy for metastatic disease or those who have developed recurrent disease during or within 6 months of completing adjuvant therapy . the national comprehensive cancer network has recommended t - dm1 as a preferred agent for patients with her2-positive metastatic breast cancer and prior trastuzumab therapy . according to promising and hopeful results of t - dm1 administration in patients with her2-positive metastatic breast cancer , this agent attracts attention in using in other her2-positive non - breast cancers . new investigation unleashed the presence of her2 receptors in other malignant organs such as bladder , gastric , and urogenital carcinosarcomas . in this article , we reviewed the available published data from preclinical and clinical trials over the use of t - dm1 in her2-positive non - breast cancers . this review focuses on encouraging researchers and clinicians to perform future investigation in this era . one of the most frequent and fatal human malignancies all over the world is gastric cancer . since it is asymptomatic in preliminary phase , many patients suffered from gastric cancer are diagnosed when cancer develop beyond curative strategy . by recent development in molecular biology of her2 and analyses in genomics and proteomics finding , it has now been discovered that her2 is implicated in other severe forms of cancer , especially gastric cancer . the frequency of her2-positive overexpression in gastric cancer estimated around 20% and is associated with poor clinical outcome . hence , inhibiting the her family signal transduction is likely contribute to improved survival of patients surfed from gastric cancer . one of the milestone studies over the use of anti - her2 antibody in the treatment of gastric cancer is trastuzumab for gastric cancer ( toga ) study . toga was an open - label , international , phase iii , randomized controlled trial undertaken in 122 centers worldwide . in this study , patients with gastric or gastro - esophageal junction cancer who have overexpression of her2 protein were included . participants were randomly assigned to receive a standard chemotherapy regimen or chemotherapy in combination with intravenous trastuzumab . the toga trial is a landmark in the treatment of gastric cancer , since following the successful completion of the study , trastuzumab , in combination with chemotherapy , approved for this indication . results showed that median overall survival was 138 months ( 95% confidence interval [ 95% ci ] 1216 ) in those received trastuzumab plus chemotherapy compared to 111 months in those received chemotherapy alone ( hazard ratio 074 ; 95% ci 060091 ; p = 00046 ) . prolonged overall survival in trastuzumab administrated group , led to the guidelines recommendation that her2 testing should be routinely performed in patients with metastatic or recurrent gastric cancer . due to promising results by trastuzumab combination therapy in her2-positive gastric cancer , consequently t - dm1 became the corn stone of consideration . in gastric cancer , t - dm1 has been evaluated in several preclinical , in vitro and in vivo , her2 positive cell culture . for the first time in 2011 , barok et al . studied the effects of t - dm1 on her2-positive human gastric cancer cells and xenograft tumors . in vitro study , four her2-positive gastric cancer cell lines including ( n-87 , oe-19 , snu-216 , and mkn-7 ) were applied . results showed that except in snu-216 cells , t - dm1 has superior inhibitory activity against cancer cells in comparison to trastuzumab . in vivo study , using same cell lines in xenograft tumors , similar positive antitumor effects was found by t - dm1 . in this study , t - dm1 has a promising antitumor effect in her2-positive gastric cancer cell lines by direct effects including apoptotic cell death and improper mitosis as well as antibody - mediated cellular cytotoxicity , even in tumors which had developed resistance to trastuzumab . in another preclinical study , t - dm1 as a single agent showed statistically significant antitumor activity in three her2-rich expression tumor models evaluated ( nci - n87 , sch and 4 - 1st ) . in addition , the combination of t - dm1 and pertuzumab , a her2 dimerization inhibitor , in xenografted gastric tumors induced significant tumor regression . the possible mechanisms involved in tumor regression of combination treatment are enhancement of antibody - dependent and increase in cellular cytotoxicity by akt , v - akt murine thymoma viral oncogene homolog 1 , and signal inhibition . nowadays , in a clinical trial which is underway , the efficacy and safety of t - dm1 compared with standard taxane therapy in patients with her2-positive advanced gastric cancer was examined . in this multicenter , randomized , adaptive phase ii / iii study , patients will be randomized to one of the three arms , 3.6 mg / kg t - dm1 every 3 weeks , 2.4 mg / kg t - dm1 every week , or taxane therapy ( docetaxel or paclitaxel per investigator choice ) , for at least four cycles ( 12 weeks ) . patients will receive study protocol treatment until disease progression , unacceptable toxicity , or withdrawal . considered endpoints of the study are overall survival , progression free survival , duration of response , and time to gastric cancer symptom progression , as well as safety . using t - dm1 in her2-positive gastric cancer is in infancy , lots of well - designed randomized control trials must be handled to clarify the real indication , proper doses , and duration of treatment by this agent . urothelial bladder carcinoma is a major global health concern responsible for 165,000 deaths annually . over the two past decades , there has been no statistically significant improvement in 5-year patients ' survival rates for locally advanced and metastatic condition . in recent study , several mutations in the extracellular and tyrosine kinases domains of her2 , was reported in urothelial bladder cancers . it has been suggested that having her2 expression correlated with inferior recurrence - free survival . furthermore , identifying of her2 mutations and the role of this pathway in bladder carcinogenesis is desirable . more heavily reason back to the use of anti - her2 agents , ( e.g. , trastuzumab , lapatinib , ado - trastuzumab emtansine , and pertuzumab ) in the bladder carcinoma 's treatment . in a phase ii study done by hussain et al . , the safety and efficacy ( response rates , time to disease progression , survival ) of trastuzumab , carboplatin , gemcitabine , and paclitaxel in advanced urothelial carcinoma with her2 over - expression were evaluated . these patients achieved a remarkable response rate of 70% ( 31 out of 44 patients ; 5 complete responses , and 26 partial responses ) , and a median time to progression and overall survival of 9.3 and 14.1 months , respectively . the primary end point of the study was cardiac toxicity that was higher than projected . today , it is believed that novel strategies targeting her2 lead to outcomes of her2 positive bladder cancer 's improvement . an encouraging preliminary study was designed by the aim of evaluating the effect of t - dm1 compared to trastuzumab in different cell lines and xenograft models of her2 over expressing in bladder cancer . in this study , rt4v6 cell line was used as a highest her2 expressing bladder cancer cell line . in comparison to trastuzumab t - dm1 made apoptosis of rt4v6 cells after g2/m arrest on cell cycle analysis . the most interesting finding was that her2 expression was higher in cell lines with acquired cisplatin resistance 's cell line . moreover , in an orthotopic bladder cancer xenograft model , tumor growth of cisplatin resistant cells , rt112 , was significantly inhibited by t - dm . these inhibitory effects are suggested to be via the induction of apoptosis compared to treatment with control igg or trastuzumab . taken together , although there is a paucity of data around the administration of her2-possitive bladder cancer , but when we want to looking for emerging strategy , in bladder cancer treatment , targeting this pathway must be considered . however , t - dm1 showed promising antitumor effects in preclinical models of her2 over expressing bladder cancer , but running several well - designed clinical trial is warranted to prove the potential positive effects of t - dm in affected patients . carcinosarcoma , also characterized as malignant mixed mllerian tumors , are one of the rarest and challenging tumors among all gynecological malignancies . these tumors compromise in any genital tract anatomical organs of the female body such as endometrium , ovary , cervix , vulva , vagina or fallopian tube . but among that , endometrium is the most common affected site . on the other hands , uterine sarcoma accounts for 35% of all corpus uteri malignancies with an incidence in the united states of 14/100,000 women . on the other hands , ovarian carcinosarcoma has a worse prognosis than uterine carcinosarcoma , with a median survival rate of 832 months and recurrence rates of 50100% . this high rate of tumor recurrence , resistance to therapy , and the poor median survival of patients suffered from carcinosarcoma propose a need to identify more effective treatment modalities . by identifying carcinomatous component - induced tumor growth , it is suggested that therapeutic interventions targeting cellular pathways that drive epithelial cell proliferation may potentially be beneficial against these kind of tumor . it has been reported that her2/neu is overexpressed and amplification in uterine carcinosarcoma ranged from 17% to 43% . having her2 , raising the possibility that anti - her2 therapies may be an effective modalities in the treatment of carcinosarcoma . for the first time , english et al . evaluated the t - dm1 against her2-positive uterine serous carcinoma in vitro followed by developing a supportive carcinosarcoma in vivo model . in this study , fifteen primary uterine serous carcinoma cell lines were assessed for her2 protein expression by the aim of immunohistochemistry and flow - cytometry . they found that near 33% of uterine serous carcinoma were positive for her2 gene amplification and protein overexpression . in vitro , t - dm1 , and trastuzumab cytostatic and apoptotic activities were evaluated by flow - cytometry - based proliferation assays and showed that t - dm1 was notably more effective than trastuzumab in inhibiting cell proliferation and inducing apoptosis in her2 overexpression 's cell line . moreover , in mice model , t - dm1 treatment led to highly reduction in tumor size and longer survival comparing to trastuzumab and control group . furthermore , t - dm1 showed effective antitumor activity in both xenograft and cell lines models . it may represent a novel treatment option for her2-positive uterine serous carcinoma patients with disease refractory to trastuzumab and traditional chemotherapy . the positive effects of t - dm1 against primary her2 positive and her2 negative carcinosarcoma cell lines were documented in another report . nicoletti et al . in 2015 , in a similar experimental study noted that t - dm1 was dramatically more effective than trastuzumab in inhibiting cell proliferation and in inducing g2/m phase cell cycle arrest in the her2 expressing cell lines in vitro and in vivo . according to new and promising finding from these two study , we can conclude that t - dm1 at least may represent a novel treatment option for the subset of her2 positive carcinosarcoma patients with disease refractory to chemotherapy . antibody - mediated therapy for the treatment of various human malignancies has proved efficacy in the past 30 years , and it is now one of the most successful strategies for targeted treatment of patients suffering from hematological malignancies and solid tumors . not so far days , when trastuzumab , as a recombinant humanized monoclonal antibody , has been introduced to her2 positive breast cancer treatment , hope opens door for these group of poor prognosis cancers . but todays concerns were raised along to trastuzumab resistance that was induced by malignant cells . evidence suggests that combinations of her2-directed agents by other cytotoxic ones may show additive or synergistic effects and lead to improving outcome . by developing the antibody drug conjugates technique , the opportunity came up to take advantage of selectively delivery of potent cytotoxic drugs by monoclonal antibodies to antigen - expressing tumor cells . base on the success results from experimental studies , recently , food and drug administration was approved brentuximab vedotin ( adcetris ) and t - dm1 ( kadcyla ) , for clinical applications of cancer treatment . the clinical studies demonstrated that t - dm1 is associated with improved efficacy and safety compared with traditional chemotherapy . several clinical trials showed the beneficiary effects of t - dm1 in the her2-positive metastatic setting and in patients with prior exposure to trastuzumab and lapatinib . the use of t - dm1 in other malignances with her2 overexpression and gene - amplification except than breast cancer is of key interest . new technical development has demonstrated that her2 overexpression is seen in some other malignancies such as gastric cancer , bladder cancer , uterine and ovarian carcinosarcomas . it has suggested that up to 20% , 9% , 30% of gastric , bladder , and uterine carcinosarcomas , respectively , overexpress her2 and is associated with significantly worse outcomes . in comparison to her2 positive breast cancer by near 30% overexpression , these quantities are large enough to be considering in treatment modalities . by developing the new concern around trastuzumab resistance and on the other hand , documented efficacy of t - dm1 as better therapeutic option , t - dm1 is now in the spotlight . this review has discussed just some of the limited data showing the preclinical benefits of t - dm1 for treating her2-positive non - breast cancer . all in vitro and in vivo study confirmed that t - dm1 administration led to prolonging overall survival and progression - free survival . hence , by considering these promising results , it is time to assessed clinical responses by designing phase i / ii clinical trials . if future clinical results can demonstrate the similar results as preclinical data , then new era in the treatment of any her2-positive non - breast cancer and survival prolongation will be created . in march 2011 , based on the results of the toga trial , trastuzumab was intensively approved in gastric cancer . the introduction of trastuzumab made new term in gastric cancer entitled her2-positive gastric cancer , similar to her2-positive breast cancer . this discovery gives us a clue that her2-positive non - breast cancers are developing and identifying . finding new agents for treatment of them are important future issues . as an example , new advancing in the knowledge of urothelial carcinoma pathophysiology and underlying molecular mechanisms showed the most relevant molecular pathways that might demonstrate therapeutic potential . the most important relevant overactive signaling networks are fibroblast growth factor receptor , pi3k / akt / mtor , and her2 . any development in identifying the signaling networks on each cancer types leads to use of another possible effective treatment modality in the future . furthermore , if clinical trials reach to same prominent results as we reviewed . in the near future , we will see the routine use of trastuzumab conjugated agents such as t - dm1 in chemotherapy regime of other her2-positive non - breast malignancies . we strongly recommend managing well - designed control trials to clarified future additional data , over the more precise number and roles of her2 receptors as a therapeutic target in her2-positive non - breast malignancies . we need to know many details about the best administrated regime in her2-positive non - breast malignancies . while we are in the beginning of way and it is soon for any important decision . azadeh moghaddas was responsible for literature review , data collection , and preparing and editing the manuscript draft . a part of this study was presented in 18th international conference on non communicable diseases ( icncd 2016 ) which was held in amsterdam , the netherlands . a part of this study was presented in 18th international conference on non communicable diseases ( icncd 2016 ) which was held in amsterdam , the netherlands .

the national comprehensive cancer network ( nccn ) has recommended ado - trastuzumab emtansine ( t - dm1 ) as a preferred agent for patients with human epidermal growth factor receptor-2 ( her2)-positive metastatic breast cancer and prior trastuzumab therapy . overexpression of her2 was reported in other cancer types such as bladder , gastric and urogenital carcinosarcomas similar to what is discovered in breast cancer . some preclinical studies demonstrated the potential anti - tumor effects of t - dm1 in her2-positive non - breast cancers . there is a paucity of data over the clinical evaluation of t - dm1 in human studies of non - breast cancer patients . we review some preclinical and ongoing clinical studies that assessed the efficacy of t - dm1 administration in the treatment of non - breast her2 positive malignancies . performing large and well - designed trials in this area is matter of interest and highly recommended .

INTRODUCTION ADO-TRASTUZUMAB EMTANSINE IN HUMAN EPIDERMAL GROWTH FACTOR RECEPTOR 2-POSITIVE GASTRIC CANCER ADO-TRASTUZUMAB EMTANSINE IN HUMAN EPIDERMAL GROWTH FACTOR RECEPTOR 2-POSITIVE BLADDER CANCER ADO-TRASTUZUMAB EMTANSINE IN HUMAN EPIDERMAL GROWTH FACTOR RECEPTOR 2-POSITIVE UTERINE AND OVARIAN CARCINOSARCOMAS CONCLUSION AUTHORS' CONTRIBUTION Financial support and sponsorship Conflicts of interest

PMC3689565

entrainment in nature is accomplished by exposure of animals to the cycles of day and night , which consist of three powerful photic cues : the daylight , twilight transitions and the natural dim scotopic illumination . although the dim illumination failed to evoke any phase shifts or suppress the melatonin secretion in nocturnal rodents , it was regarded as a persuasive photic contributor capable of altering the basic properties of the circadian pacemaker . for example , the dimly lit nights modified the waveform of activity rhythms of the white - footed mice , bats and owl - monkeys . dim nocturnal illumination in hamsters extended the range of entrainment , increased the incident of dissociation of locomotor activity rhythm and showed the phase - dependent effects on reentrainment and masking . moreover , the continuous dim green light in hamsters lengthened the period of free - running rhythm and increased the duration of activity by ~3 h when compared with the hamsters in continuous complete darkness . the dim nocturnal irradiance of ~0.2 lx was reported to accelerate the reentrainment in the young and old hamsters following simulated advance and delay jetlags of 4 and 8 h. dimly - lit nights modified the basic properties of the pacemakers controlling the pupal eclosion and adult locomotor activity rhythms of a few insects too . for example , the dim natural and artificial nighttime illuminations altered the attributes of entrainment and free - running rhythm of the pupal eclosion of d. jambulina . in the same species , the nocturnal illumination at 0.0006 lx advanced the activity onset by several hours , extended the activity duration , enhanced the activity level and shortened the period of free - running rhythm . varying durations ( 915 h ) of the dim scotophase at 0.0006 lx modulated the fundamental properties of the circadian pacemakers that controlled the adult locomotor activity rhythm of d. jambulina . dim nocturnal irradiance in d. melanogaster also advanced the morning peak but delayed the evening peak of activity that rendered the flies completely nocturnal moreover , it also altered the clock protein rhythms in the pacemaker neurons of the brain . dim illumination even during the daytime had profound influence on the locomotor activity rhythms of a few insects . for instance , the dim photophase at 1 lx delayed the onset of the locomotor activity rhythm of the onion fly , delia antiqua and lengthened the period of free - running rhythm in continuous dim light . the dim photophase contrary to the bright one , enhanced the activity level and stimulated the behavioral components like the resting , grooming and feeding in d. melanogaster . thus , these studies revealed that the dim naturalistic light during the night or even during the day had multitude of effects on the circadian features of several mammals and insects . nevertheless , the influence of the dimly lit nights in accelerating the reentrainment following simulated jetlags has not been reported yet in any diurnal animal model . the objective of the present study was to examine the efficacy of the dimly lit nights in accelerating the reentrainment of the circadian rhythm of the adult locomotor activity of drosophila biarmipes , which is predominantly a diurnal insect in nature . this report is the part of ongoing investigation in the ability of the photic zeitgebers to influence the reentrainment following the simulated jetlags in d. biarmipes . recently , the authors have demonstrated that the bright photophase at 300 lx as compared with the dim photophase at 30 lx , speeded up the reentrainment in this species following the phase advance and delay of light - dark cycles . even the duration of photophase influenced the rate of reentrainment in d. biarmipes , however , it was dependent on the direction of the shifts in light - dark cycles . the short photophase of 9 h and the long photophase of 15 h accelerated the reentrainment following simulated advance and delay jetlags , respectively . the stock culture of the wild - type strain designated as md-101 of drosophila biarmipes was maintained at 21 0.5c and ~60% relative humidity ( r.h . ) in light - dark ( ld ) cycles of 12 h of white light at 300 lx and 12 h of complete darkness which are regarded as the standard ld cycles . the broad spectrum ( 365810 nm ) white light obtained from the uv - free white light emitting diodes ( 6 vdc-0.02w , the angle of light emission 2030 , ligitek electronics co. ) was used during the photophase at 300 lx as well as during the dim scotophase at 0.03 lx . the computerized photoelectric method for recording the adult locomotor activity rhythm of drosophila has been described in details . however , the method in brief is as follows . three units of the chronobiology kit ( stanford software systems , release version 1c , 19982004 - 62-channel ) were used for recording the activity rhythm and for the analysis of the circadian parameters . one - day old males were individually introduced in an activity recording glass tube ( 100 mm long 7 mm outer diameter ) . one end of the tube was inserted in 5 g of culture medium kept in a plastic container ( vol . 10 ml ) which served as the source of food and water for the fly . the other end of the tube was closed with a loose cotton - plug for aeration . this arrangement allowed the uninterrupted recording of the activity for ~10 d. thereafter , the fly was transferred to a fresh tube to continue the data recording . each tube was placed in the path of an infrared beam ( peak transmission of 738 nm ) of an activity recording device . twenty such activity recording devices were housed in each of 35 light - proof wooden - enclosures ( 60 80 90 cm ) kept in a room maintained at 21 0.5c and ~60% r.h . efficacy of the dim nocturnal illumination in accelerating the reentrainment was examined by subjecting the flies to two types of lighting schedules wherein the 12 h photophase was at 300 lx for all flies but the 12 h scotophase was at 0 l ( d ) for the control flies and 0.03 lx , i.e. , the artificial starlight ( s ) for the experimental flies . flies were maintained for two generations in each lighting schedule to avoid the after - effects of the standard ld cycles in which they were bred for several generations . two experiments were performed on the one - day old males ( n = 49 ) of the third generation in the same lighting regime in which they were maintained . the first experiment examined whether the synchronization of the control and experimental flies by ld 12:12 and ls 12:12 cycles , respectively , was the circadian entrainment or the masking effects of the scotopic illumination . this was accomplished by subjecting the flies to each lighting schedule for 10 d and thereafter , transferring them to continuous darkness ( dd ) that commenced at the lights - on phase of the day 11 . five parameters of entrainment were evaluated : the phase - angle difference between the activity onset ( aon ) phase of the morning ( m ) peak and the lights - on transition ( o ) , the phase - angle difference between the activity offset ( aoff ) phase of the evening ( e ) peak and the lights - off transition ( e ) , the phase - relationship between the aoff phase of the m peak and the aon phase of the e peak ( m - e ) , the duration of activity ( ) , and the duration of the rest ( ) . the mean activity profile during entrainment of 10 d in each lighting regime was presented in 0.25 h bins ( mean sd , n = 49 flies ) to distinguish the circadian peak ( the expected peak appearing before the lights - on or lights - off transition ) from the masking peak ( the short activity burst in response to the lights - on or lights - off transition ) . the entrainment was comprehensively determined by considering the three forms of stable phase relationships ( the o , m - e and e ) in each lighting schedule and the predictable phase ( the aon phase of the m peak of the last day of the entrainment ) from which the free - run commenced upon transfer to dd . activity level ( al ) was defined as the average number of activity passes per fly per day from the pooled data of 49 flies during the entrainment and the stable free - running state in dd . the period of free - running rhythm ( ) in dd was computed by the chi - square periodogram analysis ( step - size = 5 min ) . the second experiment examined the efficacy of the dim nocturnal irradiance in influencing the reentrainment . initially , the control and experimental flies were entrained for 10 d by ld 12:12 and ls 12:12 cycles , respectively . on day 11 , the lighting regime was abruptly phase advanced or phase delayed by shortening and lengthening of the scotophase by 8 h , respectively . each shifted lighting schedule was continued for 1823 d until the stable reentrainment was instituted . it took several days for the flies to re - entrain and the reentrainment was regarded to have accomplished when the three forms of the previous phase relationships ( the o , m - e and the e ) were reestablished in the shifted lighting schedule . transients were the temporary oscillatory states of the aon and aoff phases between the initial entrainment and the subsequent stable reentrainment . advancing and/or delaying transients endured by the aon and aoff phases were individually counted to distinguish the different rate of reentrainment of these two phase markers . there was always a single activity bout during 14 transients immediately following the advance or delay shift in the lighting schedule , therefore the offset of such single activity bout was regarded as the aoff phase of the prospective e peak of the rhythm . furthermore , two forms of transients were distinguished : the orthodromic form in which the transients were in a direction of the shifted ld cycle , and the antidromic form in which the transients were in a direction opposite to the shifted ld cycle . values of the parameters of entrainment and free - running rhythmicity were the means ( sd , n = 49 flies ) . the wilcoxon - mann - whitney test was performed on the activity data of both experiments to see any significant effect of the dim scotopic irradiance . the figure 1a and c show the actograms of the representative control and experimental flies of d. biarmipes entrained by the ld 12:12 and ls 12:12 cycles , respectively . all flies exhibited the bimodal activity pattern with the major m peak and the minor e peak as shown in their activity profiles ( fig . the control flies were characterized by the circadian m and e peaks as well as the masking peaks at the lights - on and lights - off transitions ( fig . the experimental flies too showed the circadian m and e peaks , in addition to the lights - on induced masking peak , nonetheless , the lights - off induced masking peak was perpetually missing ( fig . moreover , the aoff phase of the e peak always occurred just before the lights - off transitions since the activity was never extended in the early part of the dimly lit night . after - effects of the nocturnal illumination on were examined by transferring the flies from each lighting regime to dd that initiated the robust free - running rhythmicity following 2.4 0.8 delaying and 4.4 1.1 advancing transients in the control and experimental flies , respectively ( n = 49 flies in each lighting schedule ) . the bimodal activity pattern was never observed in any group of the flies once transferred to dd . the free - running rhythmicity was initiated from the aon phase of the last m peak of the entrained rhythm and not from the lights - on phase of the lighting schedule . moreover , the free - running rhythmicity was generated by the participation of the m peak only , as the e peak was clearly left out . all parameters of the entrainment and free - running rhythmicity of the experimental flies were significantly different from that of the control flies ( p < 0.001 ) ( table 1 ) . the actograms of the representative control and experimental males of d. biarmipes which were entrained by ld 12:12 ( a ) and ls 12:12 ( c ) cycles , respectively , and then transferred to dd on the day 11 ( oblique arrow ) . the mean activity profiles of 49 control ( b ) and experimental ( d ) males during the entrainment are showing the circadian peaks ( dark arrows ) , the masking peaks ( open arrows ) and the phase relationship between the aoff of the m peak and aon of the e peak ( m - e ) . the dark and open time bars denote the photophase ( 300 lx ) and scotophase ( 0 lx ) , respectively , the long dark time bars denote dd and the hatched portion of the time - bars denotes the dim nocturnal irradiance at 0.03 lx . values are given in hours ( mean sd , n = 49 flies ) . o , the phase angle difference between the activity onset and lights - on ; e , the phase angle difference between the activity offset and lights - off ; m - e , the phase relationship ( i.e. , the interval ) between the morning and evening activity peaks ; -ent , duration of activity during entrainment ; -ent , duration of rest during entrainment ; dd , duration of activity during free - run in dd ; -dd , duration of rest during free - run in dd ; , the period of free - running rhythm in dd ; al - dd , the activity level in dd . the reentrainment in both groups of the flies following the phase advance as well as phase delay of the lighting schedules was also mediated by the m peak only as the e peak was never involved ( fig . 2 ) . the actograms of the representative control and experimental flies following the phase advance of 8 h are presented in the figure 2a and b , respectively . the reentrainment of the aon and aoff phases of both groups of the flies differed with respect to the direction and number of transients ( fig . 3a ) . in both groups of the flies , the aon phase reentrained consistently by the orthodromic advancing transients , whereas the aoff phase initially endured the orthodromic advancing transients , and then the antidromic delaying transients ( fig . the actograms of the four representative males of d. biarmipes which were subjected to 8 h phase advance and delay of ld 12:12 cycles ( a and c , respectively ) and ls 12:12 cycles ( b and d , respectively ) . transients after advance and delay jetlags . advancing and delaying transients ( mean s.d . , n = 49 flies ) endured by the activity onsets ( aon - ld ) and offsets ( aoff - ld ) of the control males and that of the experimental males ( aon - ls and aoff - ls ) of d. biarmipes following the advance ( a ) and delay shift ( b ) in the lighting schedules . the asterisks denote the statistically significant effects of the dim nocturnal illumination ( p < 0.001 ) . the actograms of the representative control and experimental flies after the phase delay of 8 h are present in the figure 2c and d , respectively . both , the aon and aoff phases of the control and experimental flies reentrained consistently by the orthodromic delaying transients ( fig . the decompression of in all flies occurred when the morning activity bout was dissociated into two components . the first component reestablished its customary phase relationship with the lights - on phase , whereas the second component was delayed until it achieved the usual phase position with the lights - off event ( fig . the transients experienced by the aon and aoff phases of the experimental flies after the advance as well as the delay shifts in the lighting schedules were significantly less than that of the control flies ( p < 0.001 ) ( fig . moreover , the total number of transients endured by the aon phases of all flies of each group were always significantly less than the total number of transients endured by the aoff phases after the advance or delay shift in the lighting regime ( p < 0.001 ) . the synchronization of the activity rhythm of the control and experimental flies of d. biarmipes by the 24 h lighting schedules with completely dark nights ( fig . 1c ) , was the circadian entrainment indeed and not the masking effects of the scotopic lighting conditions . this could be corroborated by the distinctive o and e in each lighting regime , the origin of the free - running rhythmicity from the aon phase of the rhythm and not from the lights - on phase of the light - dark cycles , the undeniable presence of the transients following the transfer from the lighting regime to dd , and the characteristic in dd as the after - effect of the different photic ambience of the prior scotophase . the authors have also demonstrated such influence of the extremely dim nocturnal illumination at 0.0006 lx on the attributes of entrainment and the free - running rhythmicity of the locomotor activity of d. jambulina . although such nocturnal illumination was 50-times dimmer than that used in the present study , it advanced the activity onset in the ls 12:12 cycles and shortened the in subsequent dd . it would be interesting to examine the efficacy of such extremely dim nocturnal illumination in influencing the process of reentrainment in d. biarmipes , d. jambulina or a diurnal mammalian experimental model like the indian palm - squirrel . the present study comprehensively assessed the reentrainment by using five basic but often overlooked components of the entrainment : the aon and aoff phases as the rhythm markers , the m - e , and . except for the two studies on reentrainment , the activity onset phase had been used as a sole rhythm marker to assess the reentrainment of locomotor activity rhythm , however , this approach fell short of projecting the accurate behavior of the underlying pacemakers during the reentrainment process . these studies apparently overlooked the complex organization of the waveform of the locomotor activity rhythm with five indispensable components , which are prone to be modified by various features of the lighting regime . if the aon phase is regarded as the exclusive phase maker in the present study , then the reentrainment would be much faster than demonstrated here ( figs . 2 and 3 ) . melatonin ameliorates the jetlag - symptoms only after the phase advance of the ld cycle , whereas , the dim nocturnal illumination indisputably accelerates the reentrainment in d. biarmipes following both , the phase advance and delay of the lighting schedules ( fig . 2 ) . these results are in agreement with the acceleration of reentrainment in hamsters by the dim nocturnal irradiance . the possible mechanism for the acceleration of reentrainment in d. biarmipes could be the photic or non - photic . the photic mechanism envisaged the direct effect of the dimly lit nights on the pacemakers . dim scotopic illumination is documented to alter the circadian waveform of the activity rhythm by modulating the and thereby changing the coupling mechanism between the m and e oscillators . the dim nighttime illumination unequivocally altered the waveform of the activity rhythm of d. biarmipes as the of the experimental flies during both , the entrained and free - running states was considerably longer than that of the control flies ( table 1 ) . such change in the waveform of the activity rhythm apparently augmented the flexibility of the underlying pacemakers resulting in minimizing the desynchrony among the constituent oscillators following the abrupt shifts in the ls cycle . that obviously reduced the number of transients endured by the aon and aoff phases of the experimental flies . the existence of such a desynchrony among molecular components of the pacemakers controlling the locomotor activity rhythm of rodents was shown to prolong the reentrainment . the non - photic mechanism for accelerating the reentrainment in d. biarmipes would involve the indirect effects of the dim nights on the pacemakers . a remarkable increment in the activity level caused by the dimly lit nights might act as the potential non - photic input to the pacemaker . such a non - photic input although not induced by the dim light speeded up the resynchronization in rodents too . for instance , the activity increment immediately following the phase advance of the ld cycles resulted in an impressive acceleration of reentrainment in the hamsters . an increment in the activity level caused by the dim scotopic irradiance was demonstrated to act through a non - photic mechanism too that influenced the entrainment and phase resetting in hamsters . the dim scotopic illumination apparently promoted the dissociation of the activity rhythm by facilitating the non - photic mechanism as well as the photic mechanism in the hamsters subjected to the ldld 7:5:7:5 cycles . furthermore , the dim nocturnal irradiance influenced the reentrainment in the hamsters by altering the magnitude of the phase resetting caused either by the bright light or the non - photic input such as the cage - changes . the dim nocturnal illumination also augmented the activity level of the young hamsters but the possibility of its role as a non - photic zeitgeber to speed up the resynchronization was ruled out . since the old hamsters exposed to the dimly - lit nights reentrained always faster than those exposed to the dark - nights , despite the both groups of hamsters had a comparable activity level . that prompted the authors to investigate the influence of the activity increment caused by the physical exercise schedule on the rate of reentrainment in d. biarmipes ( sinam et al . . an impressive increment of about 29% in the activity level was observed in the dark - night exposed males ( n = 69 ) of d. biarmipes when subjected to 1 h bout of physical exercise immediately following the 6 h advance shift in the ld cycles . however , such increment in the activity level failed to accelerate the reentrainment . therefore , the effectiveness of the activity increment induced by the dim - nights as a non - photic input to the pacemakers to accelerate the reentrainment in d. biarmipes should be negated in the present study . other features of the photic zeitgeber were documented to accelerate the reentrainment of d. biarmipes , too . for instance , the bright photophase akin to the dim nocturnal illumination in the present study , indiscriminately accelerated the reentrainment in d. biarmipes , following shifts in ld cycles in both the directions . these results were ascribed to the enhanced zeitgeber strength of the bright photophase that reinforced the coupling mechanism of the aon and aoff phases with the lights - on and lights - off transitions , respectively . the duration of photophase also influenced the reentrainment in this species , nevertheless , it depended on the direction of the ld cycle shift . the short photophase accelerated the reentrainment only after the phase advance , whereas the long photophase accelerated the reentrainment only after the phase delay of the ld cycles . these results were attributed to the early aon phase and short of the flies exposed to the short photophase , and the opposite was true for the flies exposed to the long photophase . such studies may have the practical application as a non - drug treatment to minimize the jetlag problems , especially , among the frequent flyers like the cabin - crew , sportspersons , etc .

jetlag results from the misalignment between the endogenous circadian timing and the civil timing after a transmeridian flight . efficacy of the dim nocturnal illumination ( 0.03 lx ) in accelerating the reentrainment following simulated jetlags in drosophila biarmipes was examined by subjecting the flies to 24 h light - dark cycles in which the 12 h photophase was at 300 lx for all flies but the scotophase was at 0 and 0.03 lx for the control and experimental flies , respectively . reentrainment was always faster in the experimental flies than the control ones . moreover , unlike melatonin , the dimly lit nights accelerated the reentrainment following both , the phase advance and delay of the light - dark cycles . this study might have potential application as a non - drug jetlag treatment .

Introduction Methods Results Discussion

PMC4514266

according to the authors of a 2004 article published in nature , humans were born to run . bramble and lieberman have suggested that our body structure was significantly influenced by the fact that we needed to run for survival . a growing contingency believes that we were designed with all we need in our feet to be able to run without shoes or with minimal shoes that mimic the barefoot running striking pattern . in fact , there has been a suggestion that running without the assistance of modern running shoes might lead to a reduction in the incidence of running injuries . barefoot running has been resurgent in recent years as well as running in minimalistic shoes . research into different patterns of foot - strike and the kinematics of lower limb of barefoot and shod running has similarly proliferated [ 2,46 ] . habitual barefoot runners run with a fore - foot strike ( ffs ) or mid - foot strike ( mfs ) , compared to habitually shod runners who tend to run with a rear - foot strike ( rfs ) . decreased collision forces created with ffs or mfs patterns in relation to rfs might justify the anecdotal reports of decreased injuries in barefoot runners . kinematics and kinetics analysis of simulating barefoot running ( sbr ) are apparently analogous to the barefoot condition of habitual barefoot runners . findings from several studies of kinematic and kinetics differences between barefoot and shod running vary according to the population under investigation . at typical velocities of endurance running ( 3.334.5 m / s ) , habitually shod runners tend to land with a dorsiflexed ankle and heel - strike pattern . on the contrary , at similar velocities , habitual barefoot runners tend to run with a ffs or mfs patterns , landing with a more plantarflexed ankle at initial contact than do habitually shod runners . therefore , training will induce motor changes influencing the foot - strike patterns ( fsp ) and the kinematics of running . however , most studies have evaluated the acute changes in kinematics , kinetics , spatiotemporal variables , or oxygen cost during shod and barefoot running without an opportunity to habituate to the barefoot running [ 1012 ] . this is understandable , as the required amount of time for a safe transition into barefoot running is not established yet , and the transition to simulated barefoot running , by itself , might involve higher risk of injury . neuromuscular control during running is influenced by landing pattern and type of shoes worn . in rfs running pattern , the ta muscle is considered to be the muscle of specific interest . the ta muscle has 2 major functions during initial contact : it dorsiflexes the ankle before heel - strike , and it decrease the plantar - flexion moment created around the ankle joint due to the heel - strike . the positioning of the foot requires a concentric contraction of the ta to assume the dorsiflexed position during heel - strike , whereas the reduction of the foot - slapping movement is associated with eccentric contraction of the ta to control the plantar flexion moment created during heel - strike . consequently , we can postulate that the activity of the ta muscle should be considerably different in the pre- and post - heel - strike phase and should be changed while running with an ffs versus rfs . ground reaction force ( grf ) is an important factor in the study of the kinetics of the lower extremities during running . the muscular activity of the lower limb is altered in response to ground reaction forces . during running one such input is the grf , which occurs during the ground contact phase of each stride . one possible reaction is the modification of the muscle activity patterns in response to that force . it has been speculated that there is a requirement for the muscles to control and , thus , minimize soft - tissue vibrations during locomotion and , thus , that there will be a change in muscle activity patterns in response to different vibration loadings on the lower extremity . impact forces in heel - toe running are forces resulting from the collision of the heel with the ground , reaching their maximum ( the impact peak ) earlier than 50 ms after first contact . the rate at which the impact peak is reached is termed the loading rate and is a correlate of the major frequency of the impact peak . impact forces have frequency contents of 1020 hz and should be expected to produce vibrations of the soft tissues of the body . changes in the myoelectric patterns of the lower extremities of the muscle activities have been shown to respond to frequencies of applied continuous vibrations of different impact forces . a major limitation of all those prior works was that the subjects recruited were not particularly experienced in barefoot running . runners not accustomed to running barefoot could have their natural foot structure weakened by long - term footwear use and their proprioceptive sensitivity may be reduced ; therefore , they could be less effective in adapting their running style when running in this condition . a thorough search of the current scientific literature revealed that there is no published research investigating differences in habituated and non - habituated subjects , as most studies have used initial responses of habitually barefoot runners for their investigations ; however , several studies showed that habitually shod runners run differently from habitually barefoot runners . still , it remains uncertain how long the habituation period should be for habitually shod runners to make a safe transition into simulated barefoot running . previous studies attempted changing the running motor patterns and running kinematics through strength , neuromuscular interventions , or plyometric , within 69 weeks in duration . a 6-week program was selected for our study to allow preliminary adaptation of musculoskeletal structures to different impact forces , with the purpose of decreasing the risk of injury from too - rapid transition . subsequently , higher sbr training loads could be gradually introduced to induce a training effect . therefore , the current study investigated the effects of a 6-week transition program of sbr on the stance - phase kinetics in habitually shod female runners when compared with the same group in a non - habituated state , and thereby to investigate acute and the chronic changes in this group . a total of 12 female runners mean ( sd ) age 25.73.4 years ; height 162.27.7 cm ; body weight 59.46.9 kg , and body mass index 22.51.2 kg / m volunteered to participate in the study . inclusion criteria were no prior history of joint injury or surgery , and no medical conditions affecting the connective tissue . all subjects were heel - strikers free of any obvious mal - alignment or injuries at the time of data collection . running in standard cushioned shoes prior to the beginning of the study involved the neutral and anti - pronation type models . all our subjects were recreational runners accustomed to running 35 days per week and an average 25 km per week for at least for the last 6 weeks , with the intention of remaining at a similar intensity for the following 6 weeks . subjects were excluded if they had any lower - limb injuries that had prevented them from running in the last 6 months ; had been treated in a rehabilitation program for the lower limb ; or had experienced minimalistic or sbr running . electromyography ( emg ) activity was measured from the tibialis anterior ( ta ) and the lateral gatstroceniums ( gas ) . circular pre - gelled 10-mm bipolar ag - agcl surface electrodes ( el503 ; biopac systems , inc . , goleta , ca ) were placed in parallel on the belly of each muscle in alignment with the direction of the muscle fibers and the distal tendon of each muscle with a 20-mm inter - electrode distance ( according to standards provided by seniam.org ) . for the ta muscle , the electrodes were placed about 15 cm below the center of the kneecap on the upper third of the ta muscle . the gas electrode location was centrally placed in a lateral fashion distal from the midpoint of the belly to the tendinous junction . electrodes and telemetry amplifiers were secured to the skin using medical tape to minimize movement artifacts and to prevent the electrodes from losing surface contact due to sweating . the mvc tests for the ta and lg muscles were performed while the subjects were in a sitting position with the knee flexed at 90. the subject was instructed to perform three 5-s maximum voluntary isometric contractions for each selected muscle against the resistance of the same tester and was given verbal encouragement while doing so . the electromyography was recorded using a sampling rate of 2000 hz through a 24-bit a / d converter . the raw data were processed using a band - pass filter ( 15150 hz ) . the emg was integrated then divided by the maximum voluntary contraction ( mvc ) to normalize the emg activity of very participant . , in the following conditions : ( a ) the pre - activation phase : 50 ms before foot landing until foot landing , ( b ) the impact phase , and ( c ) the peak push - off phase ( figure 1a , 1b ) . the emg activity of the selected group of muscles were synchronized with a high frame rate camera ( cam - hfr - a ) svhs sony video camera ( basler , biopac systems , inc . , goleta , ca ) to capture the running phases as series of videos at 100 fps ( 640480 resolution ) . the camera was mounted on a tripod placed 2 m from the treadmill and aligned so the plane of the camera was parallel to the treadmill . the camera was leveled using the bubble level attached to the tripod and set to the height of the subject s knee during running . runners ran on an instrumented treadmill ( zebris fdm ; zebris medical gmbh , allgu , germany ) at 10 km / h . the treadmill had an embedded pressure mat containing more than 15 000 pressure sensors from which data were integrated to produce the vertical ground reaction force . once the runners demonstrated a stable running pattern , data were sampled at 100 hz for 60 s. the variables of interest vertical impact peak ( ip ) , active peak ( ap ) , vertical instantaneous loading rate ( vilr ) , and vertical average loading rate ( valr ) were extracted from the processed data and were obtained by the method described by crowell and davis .these early impact variables were chosen for their demonstrated association with various running injuries [ 2830 ] . the ip was the local maximum between foot - strike and maximum force on the vertical ground reaction force curve ; it usually occurred within the first 50 ms of stance phase ( figure 2 ) . the vilr was the maximum slope of the vertical ground reaction force curve between successive data points in the region from 20% to 80% of the vip ( figure 2 ) . this was the most linear portion of the curve in the early part of stance . the valr was the slope of the line through the 20% point and the 80% point . all stance phases were extracted from data and transferred to matlab for processing using a custom - written matlab program ( v8.3 r2014a , math works , inc . , temporal information for heel - toe latency was used to compute the gait attributes ( ip , ap , valr , and vilr ) for each stance phases , and averages were computed for data analysis . to visualize , the grf data were normalized to 0100% of the stance phase ( figure 2 ) . subjects were evaluated pre- and post - intervention while running at ( 10 km / h ) on a conventional instrumented treadmill ( zebris fdm ; zebris medical gmbh , allgu , germany ) in both simulated barefoot and shod conditions . this velocity was selected to represent a comfortable running pace for recreational runners , and to compare with the results of other studies that had evaluated barefoot and shod running kinematics at similar velocities [ 46 ] . all subjects came to our laboratory for 3 identical testing sessions separated by the 6-week habituation period in addition to the training sessions . test conditions were the same in all conditions and took place indoors in a temperature - controlled area with artificial lighting . subjects avoided strenuous exercise in the 24 h pre - test and warmed - up according to their usual routines . after the placement of the emg electrodes , subjects ran at a self - selected velocity for at least 4 min to feel comfortable running on a treadmill . after 4 min , treadmill velocity was increased to 10 km / h before a data collection period ( duration 60 s ) . data was collected for 60 s at the 5 min of running , allowing enough time above the 4 min that has been suggested to be required to optimize leg stiffness and running technique , depending on surface and shoe hardness . given that endurance running involves repetitive impacts , a long sample period of 60 s was selected to more adequately represent average loading over a longer period of time . stride frequency was calculated by the number of steps that occurred on the right foot during the 60 s. the entire testing protocol was repeated again after a single training session in non - habituated sbr condition and following the 6-week habituation period of sbr ( post - tests ) . during the post - tests , subjects were reminded before testing commenced to concentrate on running technique , but were given no feedback while running , in order to maintain technical consistency . all subjects expressed comfort with treadmill running with the attached emg electrode before data collection and were not aware of when kinematic data was being captured . the intervention is this study was instruction and training to adopt a forefoot strike running technique . fivefinger bikila ls ( vff ; vibram , ma , usa ) minimal footwear . immediately after pre - tests , each subject was provided with a structured progression of sbr over a 6-week habituation period and relevant injury prevention exercises . running technique guidelines were also provided based on current findings in the literature ( table 1 ) . the program incorporated sbr running into the subject s normal training routines ( increasing from ~10% to ~25% ) , which required that the sbr running took place at the beginning of any training session , and then subjects were allowed to continue their normal training load in their own preferred conventional running footwear . thus , subjects would gradually increase exposure to sbr during this period , while also maintaining the remainder of their training schedule in conventional running shoes . each subject was provided with detailed guidelines , including a structured progression of sbr over the 6-week habituation period ( tables 1 , 2 ) . the program , which included visual feedback and instruction on technique , simply asked subjects to run in the simulated barefoot condition at a comfortable velocity and to include specific training drills and exercises designed to teach forefoot striking consisting of weight shifting , falling forward , foot tapping , and high hopping , as described previously . additional emphasis included using the hamstrings muscle group to pull the foot from the ground versus push the foot off the ground using the gastrocnemius and soleus muscles . the subjects also practiced running barefoot and were provided with verbal cueing to run quietly to eliminate the tendency to heel strike upon ground contact . a video camera was used to record individual running form to demonstrate forefoot technique running errors ( e.g. , heel - striking , over - striding ) . exercise instruction was conducted 3 times per week for approximately 25 min each session for the first week . a typical training session during the first week consisted of approximately 15 to 20 min of the specific training drills , followed by forefoot running practice for distances of 0.25 km . the verbal cueing and video camera were used during the running practice time . the rationale for adopting this approach was to prepare the lower extremity for safe transition for a forefootfoot stike pattern because most of the typical deficits encountered were weak calf , reduced subtalar joint dorsiflexion , and inhibition/ weakness of foot intrinsic muscles . a power analysis was conducted for expected outcomes with a type i error probability of 0.05 and a power of 0.8.this analysis indicated that n=12 would provide a statistical power of ~80% ( g*power v3.0.10 free software ) . descriptive statistics for variables and measures of central tendency for continuous variables were calculated to summarize the data . a kolmogorov - smirnov normality test proved all variables to be normally distributed . repeated - measures anova was used to evaluate the primary outcome variables of the emg activities . the post hoc bonferroni test was used to analyze differences between pre - intervention , non - habituated sbr and after 6 weeks habituation sbr . a paired t test was used to evaluate the difference in the biomechanical variables ( stride length , stride frequency , vertical grf , and rates of loading ) between the shod and habituated simulated barefoot running conditions . a total of 12 female runners mean ( sd ) age 25.73.4 years ; height 162.27.7 cm ; body weight 59.46.9 kg , and body mass index 22.51.2 kg / m volunteered to participate in the study . inclusion criteria were no prior history of joint injury or surgery , and no medical conditions affecting the connective tissue . all subjects were heel - strikers free of any obvious mal - alignment or injuries at the time of data collection . running in standard cushioned shoes prior to the beginning of the study involved the neutral and anti - pronation type models . all our subjects were recreational runners accustomed to running 35 days per week and an average 25 km per week for at least for the last 6 weeks , with the intention of remaining at a similar intensity for the following 6 weeks . subjects were excluded if they had any lower - limb injuries that had prevented them from running in the last 6 months ; had been treated in a rehabilitation program for the lower limb ; or had experienced minimalistic or sbr running . electromyography ( emg ) activity was measured from the tibialis anterior ( ta ) and the lateral gatstroceniums ( gas ) . circular pre - gelled 10-mm bipolar ag - agcl surface electrodes ( el503 ; biopac systems , inc . , goleta , ca ) were placed in parallel on the belly of each muscle in alignment with the direction of the muscle fibers and the distal tendon of each muscle with a 20-mm inter - electrode distance ( according to standards provided by seniam.org ) . for the ta muscle , the electrodes were placed about 15 cm below the center of the kneecap on the upper third of the ta muscle . the gas electrode location was centrally placed in a lateral fashion distal from the midpoint of the belly to the tendinous junction . electrodes and telemetry amplifiers were secured to the skin using medical tape to minimize movement artifacts and to prevent the electrodes from losing surface contact due to sweating . the mvc tests for the ta and lg muscles were performed while the subjects were in a sitting position with the knee flexed at 90. the subject was instructed to perform three 5-s maximum voluntary isometric contractions for each selected muscle against the resistance of the same tester and was given verbal encouragement while doing so . the electromyography was recorded using a sampling rate of 2000 hz through a 24-bit a / d converter . the raw data were processed using a band - pass filter ( 15150 hz ) . the emg was integrated then divided by the maximum voluntary contraction ( mvc ) to normalize the emg activity of very participant . , in the following conditions : ( a ) the pre - activation phase : 50 ms before foot landing until foot landing , ( b ) the impact phase , and ( c ) the peak push - off phase ( figure 1a , 1b ) . the emg activity of the selected group of muscles were synchronized with a high frame rate camera ( cam - hfr - a ) svhs sony video camera ( basler , biopac systems , inc . , goleta , ca ) to capture the running phases as series of videos at 100 fps ( 640480 resolution ) . the camera was mounted on a tripod placed 2 m from the treadmill and aligned so the plane of the camera was parallel to the treadmill . the camera was leveled using the bubble level attached to the tripod and set to the height of the subject s knee during running . runners ran on an instrumented treadmill ( zebris fdm ; zebris medical gmbh , allgu , germany ) at 10 km / h . the treadmill had an embedded pressure mat containing more than 15 000 pressure sensors from which data were integrated to produce the vertical ground reaction force . once the runners demonstrated a stable running pattern , data were sampled at 100 hz for 60 s. the variables of interest vertical impact peak ( ip ) , active peak ( ap ) , vertical instantaneous loading rate ( vilr ) , and vertical average loading rate ( valr ) were extracted from the processed data and were obtained by the method described by crowell and davis .these early impact variables were chosen for their demonstrated association with various running injuries [ 2830 ] . the ip was the local maximum between foot - strike and maximum force on the vertical ground reaction force curve ; it usually occurred within the first 50 ms of stance phase ( figure 2 ) . the vilr was the maximum slope of the vertical ground reaction force curve between successive data points in the region from 20% to 80% of the vip ( figure 2 ) . this was the most linear portion of the curve in the early part of stance . the valr was the slope of the line through the 20% point and the 80% point . all stance phases were extracted from data and transferred to matlab for processing using a custom - written matlab program ( v8.3 r2014a , math works , inc . , temporal information for heel - toe latency was used to compute the gait attributes ( ip , ap , valr , and vilr ) for each stance phases , and averages were computed for data analysis . to visualize , the grf data were normalized to 0100% of the stance phase ( figure 2 ) . subjects were evaluated pre- and post - intervention while running at ( 10 km / h ) on a conventional instrumented treadmill ( zebris fdm ; zebris medical gmbh , allgu , germany ) in both simulated barefoot and shod conditions . this velocity was selected to represent a comfortable running pace for recreational runners , and to compare with the results of other studies that had evaluated barefoot and shod running kinematics at similar velocities [ 46 ] . all subjects came to our laboratory for 3 identical testing sessions separated by the 6-week habituation period in addition to the training sessions . test conditions were the same in all conditions and took place indoors in a temperature - controlled area with artificial lighting . subjects avoided strenuous exercise in the 24 h pre - test and warmed - up according to their usual routines . all subjects wore standard , cushioned shoes for the shod running . after the placement of the emg electrodes , subjects ran at a self - selected velocity for at least 4 min to feel comfortable running on a treadmill . after 4 min , treadmill velocity was increased to 10 km / h before a data collection period ( duration 60 s ) . data was collected for 60 s at the 5 min of running , allowing enough time above the 4 min that has been suggested to be required to optimize leg stiffness and running technique , depending on surface and shoe hardness . given that endurance running involves repetitive impacts , a long sample period of 60 s was selected to more adequately represent average loading over a longer period of time . stride frequency was calculated by the number of steps that occurred on the right foot during the 60 s. the entire testing protocol was repeated again after a single training session in non - habituated sbr condition and following the 6-week habituation period of sbr ( post - tests ) . during the post - tests , subjects were reminded before testing commenced to concentrate on running technique , but were given no feedback while running , in order to maintain technical consistency . all subjects expressed comfort with treadmill running with the attached emg electrode before data collection and were not aware of when kinematic data was being captured . the intervention is this study was instruction and training to adopt a forefoot strike running technique . immediately after pre - tests , each subject was provided with a structured progression of sbr over a 6-week habituation period and relevant injury prevention exercises . running technique guidelines were also provided based on current findings in the literature ( table 1 ) . the program incorporated sbr running into the subject s normal training routines ( increasing from ~10% to ~25% ) , which required that the sbr running took place at the beginning of any training session , and then subjects were allowed to continue their normal training load in their own preferred conventional running footwear . thus , subjects would gradually increase exposure to sbr during this period , while also maintaining the remainder of their training schedule in conventional running shoes . each subject was provided with detailed guidelines , including a structured progression of sbr over the 6-week habituation period ( tables 1 , 2 ) . the program , which included visual feedback and instruction on technique , simply asked subjects to run in the simulated barefoot condition at a comfortable velocity and to include specific training drills and exercises designed to teach forefoot striking consisting of weight shifting , falling forward , foot tapping , and high hopping , as described previously . additional emphasis included using the hamstrings muscle group to pull the foot from the ground versus push the foot off the ground using the gastrocnemius and soleus muscles . the subjects also practiced running barefoot and were provided with verbal cueing to run quietly to eliminate the tendency to heel strike upon ground contact . a video camera was used to record individual running form to demonstrate forefoot technique running errors ( e.g. , heel - striking , over - striding ) . exercise instruction was conducted 3 times per week for approximately 25 min each session for the first week . a typical training session during the first week consisted of approximately 15 to 20 min of the specific training drills , followed by forefoot running practice for distances of 0.25 km . the verbal cueing and video camera were used during the running practice time . the rationale for adopting this approach was to prepare the lower extremity for safe transition for a forefootfoot stike pattern because most of the typical deficits encountered were weak calf , reduced subtalar joint dorsiflexion , and inhibition/ weakness of foot intrinsic muscles . a power analysis was conducted for expected outcomes with a type i error probability of 0.05 and a power of 0.8.this analysis indicated that n=12 would provide a statistical power of ~80% ( g*power v3.0.10 free software ) . descriptive statistics for variables and measures of central tendency for continuous variables were calculated to summarize the data . a kolmogorov - smirnov normality test proved all variables to be normally distributed . repeated - measures anova was used to evaluate the primary outcome variables of the emg activities . the post hoc bonferroni test was used to analyze differences between pre - intervention , non - habituated sbr and after 6 weeks habituation sbr . a paired t test was used to evaluate the difference in the biomechanical variables ( stride length , stride frequency , vertical grf , and rates of loading ) between the shod and habituated simulated barefoot running conditions . emg amplitudes of the tibialis anterior and gastrocnemius during the pre - activation phase showed a significant difference between foot - striking pattern in both shod running and habituated sbr ( table 3 , figure 3 ) . amplitudes of the gas in the pre - activation and stance phases showed significant higher activity in habituated sbr compared to shod condition ( 60.0211.32 vs. 18.634.70 , respectively ) . no statistical difference was observed concerning the ta muscles for the pre - activation amplitudes between the shod condition and non - habituated sbr . however , a significant difference was detected in the pre - activation amplitude of the gas between the shod condition and non - habituated sbr . moreover , concerning the stance and push - off phases , there was no significant statistical difference between the sbr and the sr for both groups of muscles ( table 3 , figure 3 ) . a comparison of the pre - intervention and post - intervention results revealed that both valr and vilr were significantly reduced during habituated sbr running compared to shod running . the rate of loading is calculated as 2080% of the impact transient ( when present ) or to ( 312% ) of stance phase when impact transient is absent . the average vertical loading rate for habituated sbr runners was ( 24.274.09 ) body weights per second , which was significantly lower than that of shod runners ( 38.335.01 , p<0.001 ) ( table 4 , figure 4 ) . the impact force was 0.600.14 body weight in barefoot runners , which was significantly lower than the 1.390.47 ( p<0.001 ) body weight in shod runners ( table 4 ) . a significant difference of the spatiotemporal variables ( stride length , stride frequency , step time , contact time , and flight time ) was detected between shod and habituated sbr conditions . the stride length was significantly lower during sbr compared to shod condition ( 1.590.21 vs. 1.760.19 m , p<0.001 ) . the stride frequency in habituated sbr was significantly greater than shod running ( 2.800.10 vs. 2.690.13 steps / push - off sec , p<0.001 ) . emg amplitudes of the tibialis anterior and gastrocnemius during the pre - activation phase showed a significant difference between foot - striking pattern in both shod running and habituated sbr ( table 3 , figure 3 ) . amplitudes of the gas in the pre - activation and stance phases showed significant higher activity in habituated sbr compared to shod condition ( 60.0211.32 vs. 18.634.70 , respectively ) . no statistical difference was observed concerning the ta muscles for the pre - activation amplitudes between the shod condition and non - habituated sbr . however , a significant difference was detected in the pre - activation amplitude of the gas between the shod condition and non - habituated sbr . moreover , concerning the stance and push - off phases , there was no significant statistical difference between the sbr and the sr for both groups of muscles ( table 3 , figure 3 ) . a comparison of the pre - intervention and post - intervention results revealed that both valr and vilr were significantly reduced during habituated sbr running compared to shod running . the rate of loading is calculated as 2080% of the impact transient ( when present ) or to ( 312% ) of stance phase when impact transient is absent . the average vertical loading rate for habituated sbr runners was ( 24.274.09 ) body weights per second , which was significantly lower than that of shod runners ( 38.335.01 , p<0.001 ) ( table 4 , figure 4 ) . the impact force was 0.600.14 body weight in barefoot runners , which was significantly lower than the 1.390.47 ( p<0.001 ) body weight in shod runners ( table 4 ) . a significant difference of the spatiotemporal variables ( stride length , stride frequency , step time , contact time , and flight time ) was detected between shod and habituated sbr conditions . the stride length was significantly lower during sbr compared to shod condition ( 1.590.21 vs. 1.760.19 m , p<0.001 ) . the stride frequency in habituated sbr was significantly greater than shod running ( 2.800.10 vs. 2.690.13 steps / push - off sec , p<0.001 ) . to the best of our knowledge , few authors have examined barefoot running and none of them have reported a biomechanical and muscular analysis of the differences between shod running and barefoot running after habituation . this is the first study to investigate the effect of 6-week habituation of sbr training on running kinetics . the results of the current study showed that the 6-week intervention of controlled sbr training was enough to induce significant changes in kinetics of the lower limb during barefoot running . studies over the past 2 decades have provided strong evidence that continued practice of a task ( training ) facilitates neuromuscular adaptations , which are characterized by more skilled control of movement and muscle recruitment patterns . training - induced adaptations of descending motor commands reflect learning within the cns and can be represented by changes in muscle electromyography ( emg ) function ( motor recruitment ) . like training , passive interventions such as shoes and in - shoe orthoses , it is well - acknowledged that the innervation activity during running cycle consists of 3 phases : the pre - activation phase ( starting before ground contact ) , the activation phase ( during weight - acceptance ) , and the innervation phase ( during push - off ) . the main finding in the present study is that 6 weeks of habituation to sbr induces neuromuscular changes in ta and gas muscles activation pattern during different phases of running . the emg activity of the ta was found to be significantly more activated during the stance phase of shod condition when compared to the habituated sbr condition . the integrated emg value during the 50 ms prior to foot - strike was highest during pre - activation shod running and the lowest during habituated sbr . additionally , a significant increase in pre - activation of the gastrocnemius muscle was observed during habituated sbr compared to shod running , which supports the reduction of heel impact observed by changing to a forefoot strike pattern . our findings support the results of the previous literature that reported a greater recruitment of ga in the pre - activation phase in the shod condition when compared to non - habituated barefoot running condition . however , our results did not show a significant statistical difference in the emg activities of the ta for the pre - activation , stance , and push - off phases between the non - habituated sbr and shod conditions . in agreement with our findings , reported no significant difference in pre - activation levels of the tibialis anterior when comparing non - habituated barefoot and shod running . emg activity of the ta showed a high recruitment of muscular activity before and after heel strike . ta emg intensities for shod running showed a greater activation 50 ms before impact compared to the weight - acceptance phase of shod running . the activity before heel strike keeps the ankle dorsiflexed and tunes the muscle for the anticipated impact . indeed , emg activity before heel strike is pre - programmed based on the anticipated impact shock . just before ground contact , muscle activity has a crucial role in preparing the locomotor system for the landing and the subsequent ground impact . the required protection from repeated shock of the muscular skeletal structure could lead to a higher pre - activation of plantar flexor muscles . it is also worth noting that higher active pre - stretch levels , as well as the decrease of contact time seen during sbr , could improve the stretch - shortening cycle behavior of the plantar flexor muscles and therefore possibly allow better storage and restitution of elastic energy compared to shod running . the most favorable difference between sbr and shod running is the significant reduction in impact transient and the loading rate in the sbr barefoot condition . this is deemed significant because the magnitude of this impact transient has been correlated with the risk of running injuries . in the present experiment , sbr running was characterized by decreased loading rates and impact forces . the significant lower values of impact forces and loading variables ( vilr , valr , vip and ap ) observed in sbr compared to the shod running conditions are in line with previous studies . in contrast , no significant difference was observed by de wit et al . for running speed 3.5 and 5.5 m / s and by dickinson et al . in 6 subjects running across a force plate . in both of those works , firstly , the subjects of the previous studies were not accustomed to running barefoot and this could have reduced their ability to dampen the forces elicited at impact while barefoot . according to robbins et al . furthermore , the subjects run in a lab runway and a limited number of steps were analyzed . as suggested by divert el al , it is possible that when data are collected on a limited number of steps , runners are able to sustain and then maintain high impact forces . in contrast , a habituation period , as experienced by our subjects , would lead the runners to adopt strategies to reduce stress under the heel . when running barefoot , step duration , stride length , and flight time were significantly shorter , and stride frequency was significantly higher , than in the shod conditions . stride frequency is inversely proportional to step duration multiplied by velocity , and velocity was controlled in this study . thus , it would be expected that if one variable increased , the other should decrease , and vice versa , as was recorded in the present study . an increased contact time when shod might be may be partially attributable to the mass of the traditional shoe . stride frequency when running barefoot has been compared with running in traditional shoes by divert et al . and squadrone and gallozzi . our study and that of squadrone and gallozzi both reported significantly higher stride frequencies in the barefoot condition . we hypothesized that the biomechanical adjustment observed in stride kinematics could help to limit the larger impact forces that should be absorbed by the muscular skeletal system at each step . the hypothesis that further changes would occur in barefoot kinetics was supported by significant changes in the emg activities of the ta and gas after 6 weeks of sbr . of particular note is that the validity of the previous body of work is compromised by the lack of evaluation after habituation , or re - training , of previously shod rearfoot - striking runners to barefoot forefoot - striking running styles . while future research may standardize the transition protocol , this should be done carefully because individuals progress differently , and higher injury rates may result from enforcing progression . consequently , one should be cautious of generalizing the results to highly trained athletes , whose running mechanics may be extremely consistent . care should also be taken when comparing kinetic data from this study with that of studies in which subjects experienced running at higher velocities . . however , treadmill usage allowed subjects to be familiarized with each condition and reach a stable state in their stride pattern before data acquisition occurred . not knowing when data would be acquired also had other potential benefits when compared with over - ground testing and use of force - plates . the lack of standardization of the traditional shoes used may have impacted the results in the shod condition . participants were tested in their regular training shoes to most accurately represent the normal running mechanics exhibited in training . for the transition program , sbr training protocol was prescribed according to the total training volume to simplify the protocol and encourage compliance . this , however , makes it harder to quantify the dose of the intervention received by each subject or the amount of sbr training as an amount of total training capacity for each subject . treadmill gait and over - ground gait are not identical . however , treadmill usage allowed subjects to be familiarized with each condition and reach a stable state in their stride pattern before data acquisition occurred . not knowing when data would be acquired also had other potential benefits when compared with over - ground testing and use of force - plates . the lack of standardization of the traditional shoes used may have impacted the results in the shod condition . participants were tested in their regular training shoes to most accurately represent the normal running mechanics exhibited in training . for the transition program , sbr training protocol was prescribed according to the total training volume to simplify the protocol and encourage compliance . this , however , makes it harder to quantify the dose of the intervention received by each subject or the amount of sbr training as an amount of total training capacity for each subject . the findings of this study showed that changes in motor patterns in habitually shod runners are possible and can be achieved within 6 weeks . six weeks of habituated sbr led to significantly decreased activity of the tibialis anterior in the pre - activation and absorptive phase of stance and may reduce higher risk of running injuries ( e.g. , chronic exertional compartment syndrome ) . habitually shod runners did not automatically alter their landing patterns from heel strike to a non - heel strike pattern during early exposure to barefoot running . during non - habituated sbr , therefore , a gradual transitioning program with real - time kinetic feedback and evaluation is recommended .

backgroundthe aim of this study was to examine the effects of a 6-week training program of simulated barefoot running ( sbr ) on running kinetics in habitually shod ( wearing shoes ) female recreational runners.material/methodstwelve female runners age 25.73.4 years gradually increased running distance in vibram fivefingers minimal shoes over a 6-week period . the kinetic analysis of treadmill running at 10 km / h was performed pre- and post - intervention in shod running , non - habituated sbr , and habituated sbr conditions . spatiotemporal parameters , ground reaction force components , and electromyography ( emg ) were measured in all conditions.resultspost-intervention data indicated a significant decrease across time in the habituation sbr for emg activity of the tibialis anterior ( ta ) in the pre - activation and absorptive phase of running ( p<0.001 ) . a significant increase was denoted in the pre - activation amplitude of the gastrocnemius ( gas ) between the shod running , unhabituated sbr , and habituated sbr . six weeks of sbr was associated with a significant decrease in the loading rates and impact forces . additionally , sbr significantly decrease the stride length , step duration , and flight time , and stride frequency was significantly higher compared to shod running.conclusionsthe findings of this study indicate that changes in motor patterns in previously habitually shod runners are possible and can be accomplished within 6 weeks . non - habituation sbr did not show a significant neuromuscular adaptation in the emg activity of ta and gas as manifested after 6 weeks of habituated sbr .

Background Material and Methods Subjects Electromyography Ground reaction force Protocol Interventions Data analysis Results Electromyography Ground reaction force Spatiotemporal variables Discussion Limitations Conclusions

PMC2880844

percutaneous laser disc decompression ( pldd ) is a minimally invasive treatment option for vertebral disc herniation refractory to conservative treatment . food and drug administration in 1991 1 . based on the reduced risk associated with less invasive procedures , pldd has increased in popularity , with reportedly over 30,000 pldd procedures performed in 2001 2 . pldd is performed under local anesthesia via a laser fiber percutaneously inserted into the nucleus pulposus . laser energy is applied through the fiber , resulting in vaporization of nucleus pulposus contents 3 . improvement in discogenic pain with pldd is based upon laser - induced evaporation of water within the disc ; this results in a very slight decrease in disc size due to water loss . because the intervertebral disc is essentially a closed hydraulic system , a small decrease in volume leads to a significantly larger decrease of intradiscal pressure ; in vitro experiments confirm this 4,5 . the short term decrease in pressure is due to evaporation of water content within the nucleus pulposus ; long term effects are thought to be due to protein denaturation , which limits the ability of the nucleus to resorb additional water and reduces stiffness of the disc 3,6,7 . this hypothetically results in a more even distribution of weight across the intervertebral disc 8 . consistent with a lumbar location for the majority of intervertebral disc herniations , most published studies have focused on the use of pldd for the treatment of lumbar disc disease . thoracic discogenic pain or herniation causing neural impingement is less common than that in the cervical or lumbar regions . certain impact injuries , such as parachute landings , can result in thoracic disc damage . invasive treatment of such injuries often involves a thoracotomy procedure with either a discectomy or fusion implantation . although many studies have been done on lumbar and cervical pldd procedures , few have been done on the thoracic region . in order to assess the efficacy and safety of pldd for the treatment of thoracic disc disease , we performed a study of ten patients with thoracic discogenic pain who were unresponsive to conservative intervention . we performed a prospective study of ten patients ( 8 male and 2 female ) with an age range of 35 - 73 years . all patients presented with mid - thoracic axial ( n=7 ) or radicular ( n=1 ) pain that failed to improve with conservative management , which included typical modalities such as physical therapy , pain medication , and epidural steroid injections . physical examination revealed localized thoracic pain without recreation of symptoms with palpation . all the patients had negative facet injections , to evaluate for the possibility of facet joint pain as the underlying cause . the patients had positive discograms that correlated to their pain ; in the case of the individual with radicular symptoms , she had total relief of her pain with a thoracic nerve root block , confirming this as the source of her pain . all patients were diagnosed with thoracic discogenic pain based on mri and provocative discogram results . magnetic resonance imaging ( mri ) findings that were considered abnormal included changes described as irregular nuclear shape , reduced disc height , hypo - intense disc signal , annular tears , high intensity zones , endplate changes , and modic changes 9,10,11 . all patients underwent diagnostic injections to confirm the source of their pain ; these injections included either thoracic nerve root blocks for radicular symptoms and/or provocative discograms 12 . the provocative discograms involved low - pressure injections of at least three disc levels and one of the levels was utilized as a control . thoracic discography has been utilized as a controversial confirmatory test for discogenic pain for some time with debatable results 13,14 . the authors identified only two studies by the same authors for inclusion , each over 10 years old . due to the lack of established accuracy of discography , we required the combination of an abnormal mri and a positive provocative discogram to diagnose intervertebral discs as the source of pain and to identify potential study participants . it was felt that gross herniations producing significant cord compression would be better treated with a laminotomy approach , and that not enough disc material would be removed to resolve the stenosis in such situations . thus only patients with contained disc protrusions were considered for pldd and patients with gross herniation were removed from the study and sent for laminotomy or conventional discectomy . patients ' chosen for the procedure reported their thoracic pain using the visual analog scale ( vas ) pain scores at baseline . posttreatment , patients were evaluated every six months via telephone call or direct patient contact for at least 18 months . the patient was specifically asked to address the pain level of the thoracic spine region and not the body as a whole . a double - blinded study would have been preferable with a sham group but was not possible at our facility . cefofloxin was utilized unless there was an allergy , in which case ciprofloxin was used . mild sedation was used during the procedure and the patient was able to converse with the surgeon in order to express any unusual pain . fluoroscopy was utilized during the procedure for proper count of the thoracic vertebrae and to determine the entry site . the entry site was similar in position to a typical thoracic discogram and was approximately 3 inches lateral to the midline of the spine . caution was necessary due to the lung fields being close to the needle entry site , increasing the risk of pneumothorax . once the entry site was determined , the skin and deeper tissues were anesthetized with a mixture of 0.25% bupivacaine and 1% lidocaine with epinephrine via a 27-gauge needle . a 15 blade was used to create a stab incision of approximately inch . through the incision , an 18-gauge , 3.5-inch spinal needle was inserted and the needle was guided into the middle of the disc using fluoroscopy . once properly placed , a direct firing holmium laser ( diameter 0.5 mm ) at 20 watts and 10 repetitions per second ( 6030 - 10405 joules , mean 7633 ) was utilized in short bursts to vaporize the inside of the disc . in most cases these burst were of ten second intervals ; the patient usually complained of burning mid back pain which limited the time per lasing period . after the lasing periods , the disc was cooled with normal saline ( at least 100 ml ) mixed with cefazolin ( unless there was an allergy ) . saline irrigation was performed after , not simultaneously with , laser application due to the size of the diameter of the working environment and because a running irrigant would reduce the laser 's effectiveness and thus increase operating times . total lasing time was approximately 3 minutes , but varied from 80 seconds to 300 seconds . in most cases , the end point was when the pressure in the disc was reduced and injection of the normal saline occurred without any resistance . at this point the needle was removed and either the next disc was commenced or the procedure was finished . expected possible complications included those seen with thoracic discogram and laser usage such as infection or discitis , pneumothorax , nerve injury , and burn injuries 14 . each patient had a post procedural chest x - ray to rule out pneumothorax ; no pneumothorax was seen . length of follow - up ranged from 18 to 31 months ( mean : 24.2 months ) . patients were asked to assess their thoracic pain via a vas score preoperatively and at final follow - up visit . median vas score pretreatment was 8.5 ( range : 5 - 10 ) and median vas at final follow - up was 3.8 ( range : 0 - 9 ) . six of ten patients ' scores improved by at least 6 points ; one patient improved by 1 point and three patients ' scores did not improve . . however , certain types of impact , such as parachute landing , carry an increased risk of thoracic disc injury . optimal treatment of thoracic discogenic pain is identical to that of disease of other spinal regions , i.e. aggressive medical management with anti - inflammatory medication , hot compresses , and physical therapy . although epidural steroid injections are questionably proven to help with such cases , they are commonly utilized and are thus considered as a conservative therapy . patients who fail to improve with conservative treatments have several invasive options , including conventional thoracotomy procedures for discectomy and fusion , posterior fusion , laminotomy , and disc decompression . high energy laser carriers the risk of tissue burns , but low energy lasers may be insufficient to adequately induce vaporization 1 . lasers near the infrared region currently used in pldd include neodymium : ytrium - aluminum garnet laser [ nd : yag ] , holmium : ytrium- aluminum - garnet laser [ ho : yag ] , and diode laser . lasers with visible green radiation include double - frequency nd : yag laser and potassium - titanyl - phosphate [ ktp ] laser . most lasers use a 3 mm outer cannula combined with a fiberoptic viewing cable 17 . there is no clear consensus regarding the most effective and safe laser or the ideal wavelength that should be used 1 . pldd is a minimally invasive technique that reduces intradiscal pressure by vaporization of a small volume of water within the nucleus pulposus . this results in decreased overall pressure and a more even distribution of weight across the disc , with subsequent relief of discogenic pain . pldd is performed most commonly for lumbar disc disease , and published reports on the efficacy of pldd in thoracic discogenic pain are lacking . the majority of pldd studies are of small size and observational in nature ; thus the true efficacy of this technique is uncertain 18 . multiple case series have reported success with pldd for the treatment of lumbar discogenic pain 15,19 - 30 ; however , no randomized controlled trials have been performed . a systematic review by singh et al . reported that conclusive evidence of efficacy is lacking , and large scale , comparative trials are warranted given the potential benefit of pldd 18 . the majority of authors report fair to good improvement in approximately 75% of patients , most commonly based upon the mcnab scale . rates of complication are low , the most common being septic or aseptic discitis , disc rupture , epidural hematoma , and nerve root damage 24,30 - 32 . a recent study on thoracic pldd procedures by hellinger et al . reported improvement in 41 of 42 patients six weeks after percutaneous laser decompression and nucleotomy ( pldn ) 35 . the authors reported three adverse events : one occurrence each of pneumothorax , pleurisy , and spondylodiscitis . long - term outcome was not reported and thus it is unknown if their results extended beyond the study period of 6 weeks . in our study , six of ten patients reported significant pain relief based on visual analog scale pain scores concerning their thoracic pain issue . utilizing a paired student 's t - test , the differences between the pre- and post - treatment groups showed greater than a 99% confidence interval confirming that the improvement was indeed significant . the 60% improvement level noted is slightly lower , although still in agreement , with published reports of pldd in patients with lumbar disc disease 15,19,34 . of interest , patients appeared to fall into two main groups : those gaining significant improvement and those receiving little or no improvement at all . in reviewing the patients who failed treatment , we could not distinguish any specific features , such as mri findings or other clinical data , which could be utilized to screen these potential failed patients in the future . intervention for the treatment of thoracic disc disease carries a risk of pneumothorax , and particular concern was given to this possibility during the intervention . no patient developed pneumothorax , and no evidence of discitis , infection , or nerve injury was noted . this is one of the first reports of the successful application of pldd for the treatment of thoracic discogenic pain . although the study group is small with only ten patients , six out of the ten patients reported significant improvement at long - term ( greater than 18 month ) follow - up , and no adverse events were reported . pldd could be considered a viable option with a low risk of complication for the treatment of thoracic discogenic pain that does not resolve with conservative treatment . nonetheless , due to the small study size , we recommend a larger double - blinded study to confirm our results .

background : discogenic pain or herniation causing neural impingement of the thoracic vertebrae is less common than that in the cervical or lumbar regions . treatment of thoracic discogenic pain usually involves conservative measures . if this fails , conventional fusion or discectomy can be considered , but these procedures carry significant risk.objectives : to assess the efficacy and safety of percutaneous laser disc decompression ( pldd ) for the treatment of thoracic disc disease.methods : ten patients with thoracic discogenic pain who were unresponsive to conservative intervention underwent the pldd procedure . thoracic pain was assessed using the visual analog scale ( vas ) scores preoperatively and at 6-month intervals with a minimum of 18-months follow - up . patients were diagnosed and chosen for enrollment based on abnormal mri findings and positive provocative discograms . patients with gross herniations were not included.results : length of follow - up ranged from 18 to 31 months ( mean : 24.2 mo ) . median pretreatment thoracic vas score was 8.5 ( range : 5 - 10 ) and median vas score at final follow - up was 3.8 ( range : 0 - 9 ) . postoperative improvement was significant with a 99% confidence interval . of interest , patients generally fell into two groups , those with significant pain reduction and those with little to no improvement . although complications such as pneumothorax , discitis , or nerve damage were possible , no adverse events occurred during the procedures.limitations : the study is limited by its small size and lack of a sham group . larger controlled studies are warranted.conclusions : with further clinical evidence , pldd could be considered a viable option with a low risk of complication for the treatment of thoracic discogenic pain that does not resolve with conservative treatment .

Background and objectives Methods Results Discussion Conclusion

PMC4779376

the clinical proteomic tumor analysis consortium ( cptac ) was charged with establishing proteome characterization centers to measure protein differences across large numbers of tumors from the cancer genome atlas ( tcga ) . to ensure that the differences they found were reproducible , each center conducted many replicate analyses for a pair of patient - derived xenograft tissues as a comparative reference material ( compref ) , first as a preliminary validation of their workflows and later interspersed among the tcga samples to ensure ongoing validation of their workflows . ms / ms experiments , represent a unique opportunity to evaluate the variability of protein differentiation technologies across multiple platforms . variability can enter proteomic experiments through sample handling and proteolysis , prefractionation , liquid chromatography , mass spectrometry configuration , and bioinformatics . at the start of the program in 2011 , cptac evaluated ischemia time in the context of global proteomes and phosphotyrosine signaling to evaluate potential impacts for proteomic changes after blood supply to a tissue is lost but before the sample is frozen . at the same time , cptac conducted platform validation for each proteome characterization center by distributing aliquots of the compref xenografts ; data from each site could then be used to assess the variability contributed by analytical methodology . most sites opted to modify their platforms in response to these data , most commonly by switching to brplc fractionation from scx or ief fractionation techniques . patient - derived tumor xenografts are a technology by which tumors can be induced in mice via cells taken from human tumors , affording better latitude for experimentation . when tissue is harvested from a xenograft , it will contain both human and mouse proteins since the tumor cells are from a different species than the host environment . the xenografts employed in this study were whim2 ( basal ) and whim16 ( luminal - b ) , drawn from a larger breast cancer study at washington university in st . both xenografts produced large tumors ; they were grown in a sufficient number of mice to generate a pool of protein that was large enough to serve all of the cptac proteome characterization centers . while the differences between basal and luminal - b tumors have been studied at length , the proteomic differences between them can not be scored against a comprehensive answer key . these experiments with whim2 and whim16 represent the three chief techniques used for differentiating proteins in discovery proteomics experiments . itraq is one of the most common isobaric labeling strategies in use at present ; after digestion with trypsin , peptides from each sample are labeled with different chemicals of nearly identical mass on their n - termini and lysine side chains , and then peptides from different samples can be mixed together for lc ms / ms analysis . ions of a particular peptide from multiple samples will be isolated and fragmented together , each producing a characteristic reporter ion at low m / z in the ms / ms scan . the intensities of these reporter ions reflect the quantity of that peptide in the source samples . spectral counting is a label - free strategy by which the number of spectra attributed to a particular protein from an individual sample can be compared to the number of spectra observed for that protein in another sample of similar complexity . alternatively , data of this type may be analyzed by integrating the extracted ion chromatograms for intact peptide ions drawn from successive ms scans . a protein that matches to intense ions in the first experiment but less intense ions in a second experiment both analyses of label - free data , however , rely on the ability to control for higher overall identification rates in one sample than in another . measuring the reproducibility of differential proteomics a team from surromed evaluated the stability of peptide intensities measured from many replicates of a reference plasma sample in lc this work was extended to encompass isotopic labeling strategies with an ab sciex qstar xl by kim et al . in 2007 . old et al . found that spectral counting methods detected differences better and that peptide intensities estimated enrichment ratios better from thermofinnigan lcq deca data using scx or gel exclusion fractionation in 2005 . a similar 2005 investigation by zybailov et al . found that spectral counting methods and isotope enrichment methods produced highly correlated results , but spectral counting was more reproducible across their replicates . in 2006 and 2007 , the phillip c. wright laboratory published two investigations into the reproducibility of itraq quantitation , producing a coefficient of variation of 0.09 and probing sources of variations in itraq reporter ion intensities and documenting the value of replicates in these studies . patel et al . applied itraq labeling and label - free mass spectrometry - based proteomics approaches to the proteome of the bacterium methylocella silvestris , and the results showed good agreement between the itraq experiment and the label - free approach for relative quantification . in 2012 , wang et al . reported a comparative study of itraq and label - free lc - based quantitative proteomics approaches using two chlamydomonas reinhardtii strains the comparison indicated that the label - free method provided better quantitation accuracy for high fold change ratios and identified 40% more proteins , but the itraq method showed better quantitation accuracy and reproducibility . in a 2013 study of human adenovirus infections by trinh et al . , a label - free method showed higher levels of protein up- or downregulation in comparison to itraq - labeled samples , and data suggested that the label - free method was more accurate than the itraq method . compared label - free and label - based strategies for proteome analysis of hepatoma cell lines , showing that the label - free approach outperformed tmt methods regarding proteome coverage , but the label - free method was found to be less accurate than tmt approaches . in addition to these comparative studies , the literature is also rich with papers on the advantages and disadvantages of different strategies of quantitative mass spectrometry in proteomics . in general , these technology assessments have been limited in the number of replicates and instruments they include , and the time frame from first to last replicate has been relatively short . the cptac compref experiments , on the other hand , span six different instruments and range up to 10 months in duration . because the same pair of pooled samples is represented in each experiment , the data are informative for technical replicate variation rather than biological replicate variation . the data supporting this study were generally drawn from interstitial experiments ; the whim2 and whim16 samples were run as periodic quality controls in a longer series of tcga samples . this study evaluates these data to answer the following key questions:are differential proteomics experiments repeatable for a particular workflow?are difference lists reproducible in comparing two different workflows?do the various differential proteomics technologies see the same biology ? are differential proteomics experiments repeatable for a particular workflow ? are difference lists reproducible in comparing two different workflows ? patient - derived xenograft tumors from established basal ( whim2 ) and luminal - b ( whim16 ) breast cancer intrinsic subtypes were raised subcutaneously in 8 week old nod.cg-prkdc il2rg / szj mice ( jackson laboratories , bar harbor , me ) as previously described . have significantly different gene expression and proteomic signatures that are related to their intrinsic biology and endocrine signaling . tumors from each animal were harvested by surgical excision at approximately 1.5 cm with minimal ischemia time by immediate immersion in a liquid nitrogen bath . the tumor tissues were then placed in precooled tubes on dry ice and stored at 80 c . a tissue pool of cryopulverized tumors was prepared in order to generate sufficient material that could be reliably shared and analyzed among multiple laboratories . briefly , tumor pieces were transferred into precooled covaris tissue - tube 1 extra ( tt01xt ) bags ( covaris no . 520007 ) and processed in a covaris cryoprep cp02 device using different impact settings according to the total tumor tissue weight : < 250 mg = 3 ; 250350 mg = 4 ; 350440 mg = 5 ; and 440550 mg = 6 . 1131 - 436 ) on dry ice , and the tissue was thoroughly mixed with a metal spatula precooled in liquid nitrogen . the tissue powder was then partitioned ( 100 mg aliquots ) into precooled cryovials ( corning no . ( note that cryopulverized tissue will melt if it is transferred to a plastic weighing boat . ) all procedures were carried out on dry ice to maintain tissue in a powdered , frozen state . each site processed the tissue powder by independent protocols ; protein denaturation and digestion were not controlled by a cptac - wide standard operating procedure . ms / ms experiments from the whim2 and whim16 samples ; thermo fisher ltq orbitrap velos , orbitrap elite , and q - exactive models were included . three instruments ( ovelos@10 , ovelos@45 , and qexac@56 , where the number represents an anonymized cptac institution ) produced itraq 4plex experiments . in each 4plex , two channels represented each of the two samples . three other instruments ( ovelos@65 , oelite@65 , and qexac@98 ) produced label - free experiments in which whim2 and whim16 were analyzed separately . all sites except site 65 employed hcd for data - dependent ms / ms production , with orbitrap measurement of fragment ions . both ovelos@65 and oelite@65 the resulting tables are available as a microsoft excel spreadsheet in the supporting information . instrument - specific details are provided in supporting information method a. to maximize information yield from these data , three different search engines were applied to each lc ms / ms experiment ( figure 1 ) . myrimatch 2.1.138 , ms - gf+ versions 9630 and 9979 , and comet version 2014.01 searched a fasta sequence database containing ncbi refseq human ( 32 799 sequences , downloaded sept 7 , 2011 ) , ncbi refseq mouse ( 29 617 sequences , downloaded march 4 , 2011 ) , and the porcine trypsin sequence . each sequence was reversed in silico by the search engines for target - decoy estimation of error rates ; the decoy sequences from all three search engines were denoted by an xxx prefix . fully tryptic and semitryptic peptides were allowable matches . in all cases , a precursor mass tolerance of 20 ppm was applied , allowing for an error of one neutron in monoisotope selection . site 65 data were measured in the ion trap , so fragment ions were allowed to vary by up to 0.5 m / z in myrimatch or within a one m / z bin by comet and ms - gf+ . for all other sites , fragments were required to fall within 20 ppm of expected m / z by myrimatch , within a 0.01 m / z bin by comet , or within the hcd model of ms - gf+ . post - translational modifications expected a mass shift of 57.021464 da on all cys , and all algorithms allowed met to gain 15.994915 da through oxidation as a dynamic modification . ms - gf+ also allowed for acetylation of n - termini ( + 42.010565 da ) . up to two missed cleavages a mass shift of 144.102063 da was assumed to be found at both n - termini and lys residues ( and the itraq protocol was applied in ms - gf+ ) . comet and myrimatch results were exported to pepxml files , whereas ms - gf+ results were written to mzidentml format . label - free sets were processed once by a spectral counts method and once by extracted ion chromatograms . while instrument - specific assemblies of psms were used for repeatability analysis , an all - instrument assembly was analyzed for reproducibility and biological pathway and network enrichment . identification scores were translated to q - values in idpicker 3.1 , build 599 . the psm fdr ( false discovery rate ) was limited to 0.5% , with the fdr being estimated by doubling the number of decoy hits and dividing by the total passing a threshold . the number of spectra required per protein was then increased from two until the empirical protein fdr had fallen below 5% . for each site , the number of spectra required per protein was as follows : ovelos@10 = 7 , ovelos@45 = 3 , qexac@56 = 19 , ovelos@65 = 4 , oelite@65 = 2 , and qexac@98 = 4 . idpicker assembled protein groups using parsimony rules that eliminated subset and subsumable proteins from the list and grouped indiscernible proteins . idpicker matched each protein name from refseq to an hgnc ( hugo gene nomenclature committee ) gene symbol for human or to a mgi ( mouse gene informatics ) gene symbol for mouse . if neither was available for a protein name , then the software used the name of the protein preceded by unmatched . the software allows for graphical or script - driven extraction of quantities in the form of spectral counts ( spc ) or intensities , organized for this study by gene rather than protein groups . if spectra could be attributed to multiple transcripts of a particular gene , then reporting by gene would consolidate all of the psms to the single gene source , thus reducing the number of hypotheses to be tested in finding expression differences . two kinds of intensities can be used : itraq reporter ion values can be summed across all psms for each channel for each gene or extracted precursor ion chromatograms ( xic ) in label - free experiments can be integrated and summed across all psms for each gene . in itraq , idpicker computes sums rather than ratios , though users can subsequently compute ratios from the summed intensity for each gene . this places a greater weight on genes with intense psms than on those with weak signals . when inferring xic intensities , the software was not directed to integrate precursor intensity in the absence of a confident psm through retention time mapping due to the large numbers of lc the software exported these quantitative tables by the command line idpquery tool from the idpicker sqlite databases . for cross - site and biological pathway and network analysis , a conjoint assembly of data from all sites ms / ms experiments , the conjoint assembly spanned 3285 pepxml / mzidentml files . as in the individual instrument assemblies , a psm fdr of 0.5% was applied . requiring 20 spectra per gene pushed the empirical protein fdr below 5% . the raw abundance data , either spectral counts or itraq and xic intensities data , need to be normalized before any statistical analysis . variation in sampling handling and sample loading across experiments can lead to variation in the gene abundance and thus introduce systematic biases into the differentiation study . to correct for the overall experiment - wise difference , the spectral counts were normalized by the overall abundance of each experiment using the total spectral counts in an experiment . itraq and xic intensities data were normalized by the median intensities in an experiment . though the qspec method was originally employed in label - free proteomics data , the modeling framework can be similarly applied to model the reporter ion intensities from itraq data . to compare the log ratios between whim2 and whim16 , a linear mixed effect regression model was constructed for xic and itraq intensities and a generalized linear mixed effect model for spectral counts . a detailed description of the method is presented in supporting information method b. fortran source code for this differentiation is available at http://homepages.uc.edu/~wang2x7/research.htm . the estimation of gene variance was a key factor differentiating the per - instrument and cross - instrument analyses ( figure 1 ) . in one - versus - one replicate comparison , the normal distribution was assumed for log xic and itraq intensities . all of the nondifferential genes were assumed to have the same variance , and the constant variance was inferred from the pool of nondifferential genes detected by the model . this was a strong assumption that the variances are the same for all intensity levels , but a check on the fitted model confirmed that the equal variance assumption is approximately satisfied ( see supporting information figure s1 ) . for spectral count data , the poisson distribution was assumed , and thus the variance was set to equal the mean . a check on the data showed that overdispersion may have affected the data , leading to an underestimate of variance . a negative binomial model was fitted , and its results were very similar to those under a poisson assumption . by contrast , in the multiple replicates comparison used for the cross - instrument reproducibility evaluation , gene variances were estimated individually for both the xic / itraq intensities and the spectral count cases . for all genes observed from the xenograft samples , only genes for which the geneid contained at least one human gene name were considered . , we set up a requirement of minimum evidence for a gene to be included in statistical differentiation analysis . following bing zhang s minimal spectral count requirement , we required a minimum of 1.4 spectra , on average , for multiple replicate comparison . for example , in ovelos@65 ( a j ) , 28 spectra were needed before a gene was evaluated for differentiation ( 1.4 10 replicates 2 samples ) . in one - versus - one comparison , we set the threshold as 2 spectra per replicate . additionally , handling zero values for spectral counts , xics , or itraq intensities deserves special consideration as a zero may be interpreted as either a missing value ( nas ) or a measured zero . genes that had only zero spectral counts in either whim2 or whim16 were excluded ( this may eliminate some number of genuinely infinite differences , though most will be low - information genes ) . these spectral count requirements were applied prior to itraq or xic differentiation as well . for genes with a mixture of zero and positive values in abundance , the handling of spectra counts data differs from that of itraq or xic data . in modeling of spectral counts , for the continuous itraq and xic intensity data , we noticed that zeroes mostly likely came from sources other than biological reasons , such as intensities below the detection limit or failure in peak detection . treating zeros the same as positive values in the modeling may severely violate the log - normal distribution assumptions on the continuous data . we thus treated zeroes in itraq and xic as missing values ; they were not used in differentiation analysis . the difference in modeling zero values may lead to contradictory differentiation results from using spectral count data as noted in the results and discussions ; one potential solution is to model both zero and positive parts in differentiation analysis using zero - inflated models . determining the intersection of differential genes in the context of biology emphasized a subset of the overall data . the analysis included these data sets : ovelos@10 ( itraq , 6 replicates ) , ovelos@45 ( itraq , 5 replicates ) , qexac@56 ( itraq , 5 replicates ) , ovelos@65 ( spc , first 10 replicates ) , ovelos@65 ( xic , first 10 replicates ) , qexac@98 ( spc , first 4 replicates ) , and qexac@98 ( xic first 4 replicates ) . the oelite@65 data were excluded because the instrument was known to be in deficient operating order at the time the 4 replicates were collected . the last 6 replicates of ovelos@65 were excluded because they were interstitial with a different type of sample than the first 10 replicates . the final 6 replicates of qexac@98 were excluded because ion transmission through the quadrupole was considerably lower during data collection . to perform pathway enrichment , we eliminated genes that were mapped only to the mouse genome . when both human and mouse gene names were associated with spectra , only the human name was retained . in cases where expression could be attributed to multiple isoforms , we chose to keep the first isoform listed , and when a natural read - through event was indicated ( two gene symbols separated by a hyphen ) , only the first symbol was retained . though these heuristic filters could conceivably introduce biases , we note that these rules were applied only to 20 multiple isoform proteins and 6 read - through proteins of the 8126 genes considered ( 0.25% ) . fisher s exact test was used to examine the statistical enrichment of those genes called as more highly expressed in whim2 or in whim16 for each of the 188 pathways relative to all those genes not in the specified pathway . the genes upregulated for each of the two xenografts were handled separately to avoid losing statistical power . hochberg procedure , and we considered multiple - test - corrected p values below 0.05 to be significant . we also interpreted the genes expressed significantly highly in whim2 or in whim16 against the context of a global protein we first identified the hierarchical modules from the iref network based on the netsam algorithm . then , based on fisher s exact test , we calculated the p values of enrichment for all modules in each differentially expressed gene set identified by the seven data sets . hochberg procedure . applying an fdr limit of 5% , we finally identified whim2- or whim16-specific enriched modules for each of seven data sets . all of the above procedures were implemented in the netgestalt web tool ( http://www.netgestalt.org ) . we also used netgestalt to covisualize the enriched modules of seven data sets for identification of the functional consistency . the six instruments included in this study varied considerably in labeling and fractionation strategy , lc gradients and mass spectrometry instruments , and replicate schedule and data replacement policy . correspondingly , large differences appeared in rates of ms / ms collection . the two q - exactive instruments in the study produced high maximum sustained rates of ms / ms collection : 9.10 hz for qexac@56 and 9.28 hz for qexac@98 , exceeding the best rate of acquisition in orbitraps for ms / ms scans ( 4.35 hz for cid in ovelos@65 or 3.89 hz for hcd in ovelos@45 ) . translating these spectra to identified psms produced a less clear distinction based on instrument type ( figure 2 ) . while ovelos@65 produced relatively low sensitivity in terms of distinct peptides , averaging 58 028 identified spectra ( 26 250 distinct peptide sequences ) from its 15 fractions per sample , it showed an encouraging degree of consistency across 10 months of continuous operation . qexac@98 used essentially the same amount of time per sample , though split to only five fractions , and its first four experiments for the two samples identified an average of 99 916 spectra ( 53 045 distinct peptide sequences ) ; unfortunately , this rate tapered off considerably , and by the time replicates six through ten were collected , identification rates were much lower . the instrument operator reported that the ion transmission through the quadrupole worsened significantly in later runs . viewing these results instead in terms of discernible protein groups flattened the differences among instruments . the cumulative counts of distinct proteins at < 5% empirical protein fdr were ovelos@10 = 11 428 , ovelos@45 = 11 603 , qexac@56 = 15 655 , ovelos@65 = 8640 , oelite@65 = 4199 , and qexac@98 = 10 435 . note that these counts included both mouse and human proteins since a xenograft incorporates proteins from both species . when data from each instrument were assembled separately with 0.5% psm fdr and < 5% empirical protein fdr , identification sensitivity varied considerably by site . ailing instrument oelite@65 yielded the lowest sensitivity by far , whereas qexac@56 produced a remarkable number of identifications . label - free instruments ran whim2 and whim16 separately , whereas itraq instruments combined these samples into a single 4plex . rather than emphasize distinguishable protein groups , we projected psms to the genes from which those peptides derived . this projection ensured that spectra associated with peptides shared among multiple isoforms transcribed from the same gene would be counted only once . in addition , reporting spectra per gene makes it simple to relate proteomics data to biological pathways and networks that are described by the use of hgnc identifiers . when multiple genes represented exactly the same sets of peptides , they were reported as an indiscernible gene group . repeatability of identification scaled with the numbers of spectra identified for each gene group . first , the genes were separated into exponentially sized bins : 47 , 815 , 16 - 31 , 3263 , 64127 , 128255 , 256511 , and 5121023 spectra across all replicates of both whim2 and whim16 . this excluded 585 proteins with disproportionately high spectral counts , such as the maximum for mouse albumin : 63 734 . an average of 998 genes fell into each bin , with the highest spectral count bin including 610 genes . for each bin , we computed the average number of replicates in which the genes produced any identifiable spectra ( out of 16 replicates ) . for whim2 , the averages were 2.57 , 4.96 , 8.29 , 11.71 , 13.98 , 15.18 , 15.58 , and 15.83 . whim16 produced very similar averages : 2.12 , 3.96 , 7.00 , 10.37 , 13.23 , 15.12 , 15.72 , and 15.93 . of the 8566 genes ovelos@65 identified , 3616 were identified universally in whim2 and 3306 were identified universally in whim16 . a total of 2730 genes were identified universally across both whim2 and whim16 . when a similar analysis was performed for the itraq experiments of ovelos@10 , the bins began at 7 spectra because that was the minimum number required to control the protein fdr . even in the set of genes with only 713 spectra in evidence , the identification repeatability rate was quite high : 5.12 of 6 replicates . once genes had produced 28 or more spectra in aggregate , the average identification repeatability was close to 6.00 . of the 11 284 genes that ovelos@10 identified , 9822 were identified in all six replicates . reports from idpicker were used to find two sets of differential genes from each label - free replicate , one based on spectral counts ( spc ) and another based on extracted ion chromatograms ( xics ) and to find one set of differential genes from each itraq replicate ( see experimental procedures : statistical differentiation ) . this part of the study sought to determine the reproducibility of differentiation for individual experiments rather than across multiple replicates . for label - free experiments , this would mean comparing the spectral counts or precursor intensities from a set of fractions representing one analysis of whim2 to the comparable data from a set of fractions representing one analysis of whim16 ( analyses comparing multiple replicates are performed below ) . in these itraq experiments , each experiment inherently produced duplicate information for whim2 and for whim16 ; this analysis , however , compares data from a single channel of whim2 to a single channel of whim16 . to determine differences , we chose strategies that have been evaluated in the peer - reviewed literature . we opted to employ a pooled variance model for the single - replicate comparisons ; similar strategies have been implemented as local - pooled - error , empirical bayes , and other tests for microarray experiments . the approach embodied in idpicker for extracting precursor ion intensity has been published separately , including an evaluation of the rate of false attribution of intensity to a peptide . because the volume of data was so extensive , we opted not to take advantage of the ability to align retention time for fractions across replicates . as a result , a peptide that was present in both replicates contributed a spectrum count or precursor intensity when it was identified in a replicate but contributed no count or intensity when it was not identified in a replicate . in all analyses , evidence is summed at the level of the gene that gave rise to proteoforms to which counts or intensity were identified . working with ratios is certainly more common in itraq analysis , but idpicker 3.1 was capable of intensity output only , not ratios , summing the precursor intensities for all psms supporting the gene identification . submitted work by jian - ying zhou demonstrated that this method is equivalently resistant to false positives while producing high data set correlations . , who concluded that our results demonstrate that the sum of intensities and total least squares algorithms provided the most accurate estimates of protein abundance for a wide range of simulated and experimental conditions . the commonly used average of ratios algorithm consistently provided estimates with the highest errors . the fraction of observed genes found to be differential at a 5% fdr varied strongly by instrument . the itraq instruments yielded low average differential gene fractions : ovelos@10 = 1.6% , ovelos@45 = 3.4% , and qexac@56 = 2.5% ; our investigation showed that the bayesian model expected a high degree of variance in each gene s reporter ion intensities when no biological difference was present ( the model assumed that at least half of genes would not differ between the two xenografts ) . label - free data sets were analyzed twice : once solely on the basis of spectral counts ( spc ) and once solely on the basis of extracted ion current ( xic ) from precursor ions in ms scans . xic analysis found somewhat higher fractions of genes to be differential : ovelos@65 = 5.0% , oelite@65 = 3.5% , and qexac@98 = 7.7% . qexac@98 produced a wide range of differential gene fractions , reflecting the drop in identification sensitivity ( figure 2 ) ; the first four replicates averaged 12.9% differential genes , whereas the last six averaged 4.3% . shifting to spc analysis instead ( which shifts to a poisson expectation of variance in the statistical model ) altered the differential gene fractions : ovelos@65 = 9.8% , oelite@65 = 3.5% , and qexac@98 = 5.3% ( 8.2% for the first four replicates , and 3.4% for the last six ) . we note that the fraction of genes found to be differential in the ovelos@65 set was larger under spc analysis , whereas the fraction was higher in qexac@98 under xic analysis ; this may reflect the optimizations in methodology applied at each site . two different strategies were applied to determine the repeatability of differential gene detection among experiments from a given instrument . the first simply asked which differences are consistent with those from at least one other replicate experiment on the same instrument ; these overlaps were also evaluated through cohen s kappa statistic . the second compared the rankings of genes on an axis from highly upregulated in whim2 to unchanging to highly upregulated in whim16 . both analyses compared data from a single itraq channel to those from another single itraq channel , despite the collection of duplicate data in itraq 4plexes . this decision was intended to reflect the realities of large - scale itraq experiments , where duplicate data collection is rare . figure 3 visualizes the set of differential genes from each replicate on each instrument . differential genes were called common and colored blue if they were also found as differences in other experiments from the same instrument . they were labeled unique and colored orange if they were found in only one replicate from this instrument . all four possible comparisons were generated from the duplicate itraq experiments ; both the low and high m / z channels for whim2 were compared to both the low and high m / z channels for whim16 . agreement in itraq experiments was defined somewhat more stringently than in label - free experiments to reflect the use of this technology in experiments spanning many 4plexes . consider the case where whim2 was presented in duplicate on channels 114 and 115 , while whim16 was reported on the 116 and 117 channels . a comparison between channels 114 and 116 has mutual information with a comparison between channels 114 and 117 . even a comparison of 114/116 versus 115/117 , however , will have shared information because they would be based on the same set of psms . as a result , common differential genes from itraq experiments were required to be confirmed by altogether separate lc how many of the differential genes from each experiment are confirmed by at least one other replicate experiment ? blue differential genes are found in common by another experiment in this instrument , whereas orange ones are unique to a single replicate . for itraq , note that instruments that produced more replicates were likely to have a higher proportion of common differential genes by random chance . the extent to which figure 3 is dominated by common differential genes is an encouraging development for this field ; the average percentage of differences in common for each instrument was as follows : ovelos@10 = 88% , ovelos@45 = 81% , qexac@56 = 87% , ovelos@65-xic = 78% , oelite@65-xic = 61% , qexac@98-xic = 63% , ovelos@65-spc = 93% , oelite@65-spc = 88% , and qexac@98-spc = 91% . the ailing oelite@65 generated a large proportion of unique differences , but the overall numbers of differential genes were quite low , and only three other replicates could confirm any particular difference . qexac@98 also presented a fairly large number of unique differential genes , but it seems clear that the falloff in sensitivity for the final six replicates was costly . the high degree of agreement seen in the latter replicates for this instrument ( e j ) was reassuring in that while reduced sensitivity led to shorter lists of differences it did not compromise reproducibility . a more restrictive analysis , based on cohen s kappa , compared the genes declared to be differential in pairs of experiments drawn from each instrument . essentially , this is built around a contingency table ; how many genes are differential in a and differential in b , how many genes are differential in neither a nor b , and how many genes are found to be differential in one but not the other . in computing this metric , we included only those genes that appeared in both experiments . the itraq instruments produced median kappas in the range of 0.49 to 0.57 , with maximum values universally higher than 0.88 resulting from comparisons with a channel in common . for all three label - free instruments , the kappa values were substantially higher for spc analysis than for xic analysis ( a difference of 0.18 to 0.34 ) , with median kappas of 0.50 for qexac@98-spc , 0.58 for ovelos@65-spc , and 0.74 for oelite@65-spc . signed log posterior probabilities were produced for each gene in each replicate , with the magnitude of the negative log posterior probabilities indicating the significance with which a gene may be called differential and the sign marking the orientation of that potential difference . the ranks of genes on these lists can then be compared in a spearman rank correlation . figure 4 shows four example plots of correlation coefficients for signed log posterior probabilities from each replicate against the values from each other replicate . the average number of genes included in each instrument for differentiation was as follows : ovelos@10 = 6571 , ovelos@45 = 5877 , qexac@56 = 8880 , ovelos@65-spc = 2381 , oelite@65-spc = 1344 , and qexac@98-spc = 2643 ( xic counts were only slightly different ) . rank correlations compare the ordering of genes by signed posterior probabilities . ovelos@10 illustrates the extent to which differential probabilities within an itraq 4plex are more similar than those across multiple 4plexes . the qexac@56 shows an exception to this behavior in replicate b. spectral count - based differentiation produces similar overall correlations to itraq , though without the benefit of being able to compare within lc ms / ms experiments . the declining sensitivity of identification in qexac@98 gave low correlation values other than the one between the first two replicates . data from ovelos@10 differentiate the correlation values within a 4plex from those between different 4plex replicates . within a 4plex correlation coefficients between 4plexes , however , never rose as high as 0.8 but rarely fell below 0.7 . this disparity may result in large part from the fact that intensities within a 4plex are drawn from exactly the same set of psms for a given gene , rendering them more comparable than a case where intensities are drawn from entirely different psms ( and likely distinct sets of peptide sequences ) as disparate sets of lc qexac@56 generally follows the same pattern of behavior , with an even greater difference between internal - to-4plex and external comparisons . the behavior for replicate b , however , bears closer attention . the data from channels 114 and 115 were considerably different , and the external correlations for channel 114 were lower against all other replicates . the result is a reminder that a great many different factors must all go well for reproducible proteomic differentiation experiments . ( note , however , that the internal data analysis pipeline by site 56 did not show a reduced correlation for this channel . the internal pipeline used a different search engine , a peptide ratio - based assessment , and a pearson correlation on log fold change . ) label - free experiments may be simpler to conduct at the bench , but they are also subject to variation . the 16 replicates from ovelos@65 in figure 4 reveal a general decrease in correlation for replicate g. looking back to figure 3 reveals an increased proportion of unique differential genes for this replicate ; replicate g also produced the lowest kappa metric for this instrument in both spc and xic analysis . qexac@98 was subject to considerable variation in identification sensitivity ( figure 2 ) , and its correlation values were consistently lower than those for other instruments . all of the correlation plots can be found in supporting information figure s3 . comparing across instruments made it possible to take advantage of the replicates produced for each instrument . instead of building differential gene lists from individual instrument assemblies , the cross - instrument evaluation was developed from an assembly that spanned all of the data ( figure 1 and experimental procedures : bioinformatics and biostatistics : protein identification ) . at an empirical protein fdr of 4.58% , the assembly included 12 741 protein groups supported by a total of 6 980 499 psms . the bayesian model was able to estimate variance per gene through the use of replicates , improving its discrimination power . the number of gene groups tested for differentiation in each instrument was as follows : ovelos@10 = 6570 , ovelos@45 = 5744 , and qexac@56 = 8081 . single - replicate analysis had shown a maximum of 3.4% differential genes for these three instruments , but those percentages rose significantly when the software was able to estimate variance using replicate measurements for each gene : ovelos@10 = 31.7% , ovelos@45 = 9.4% , and qexac@56 = 39.3% . when the first three and last three replicates for ovelos@10 were considered separately , the number of differential genes plummeted to 13.3 and 14.2% for the early and late sets , respectively ; doubling from three to six replicates greatly improved the statistical power for detecting differential genes . ovelos@45 , by contrast , identified the fewest overall genes and yielded the least differentiation among the itraq instruments . examining the underlying data revealed higher typical variance per gene than that for other sites , and the operators of this instrument noted that each replicate was a full process replicate ( repeating digestion , labeling , and mixing ) rather than a technical replicate that simply repeated lc an internal analysis by this site showed good consistency in the genes found to be differential for each replicate ( see supporting information figure s4 ) . the label - free data from ovelos@65 and qexac@98 provided enough replicates to allow for subset comparisons , whereas oelite@65 lagged enough in performance that it was not examined in detail . the first 10 and last 6 replicates from ovelos@65 were split to two sets , reflecting that the former were interstitial with tcga samples and the latter were interstitial with normal tissue . similarly , the data from qexac@98 for the first 4 replicates were separated from the last 6 replicates to reflect the different schedules for collecting these two sets . p = 3219 , oelite@65 = 1896 , qexac@98a d = 4893 , and qexac@98e j = 2758 . the counts for label - free instruments were taken from the spc analysis ; the number of genes compared by xic was slightly lower since some precursor ions were not matched . the differential gene fractions by spc analysis were 64.2 and 48.7% for the early and late sets for ovelos@65 , whereas the qexac@98 percentages were 34.1 and 37.0% ( note , though , that the later set from this instrument included substantially fewer genes ) . analyzing the same data by xic , though , yielded 51.9 and 59.6% differential genes for ovelos@65 , demonstrating that having more replicates is not a guarantee for a higher fraction of differential genes . the xic analysis for qexac@98 produced 32.3% differential genes in the high - sensitivity early set but 39.8% in the later , less sensitive runs for this instrument . since spc and xic analyses are generated from the same underlying psms , one might expect that their lists of differences are highly similar . however , the sum of precursor ion intensity observed for a given gene may be stable while spectral counts vary between two cohorts , or vice versa . difference lists from three data sets were interrogated for this purpose : ovelos@65a j , ovelos@65k p , and qexac@98a d . oliveros ) compared the lists of up - in - whim16 and up - in - whim2 genes for both xic and spc analysis ( see supporting information figure s5 ) . of all genes named as up - in - whim2 in these three sets , from 47 to 64% of all genes named as up - in - whim16 in the three sets , from 53 to 75% were named in both analyses . the two techniques contradicted each other in only one category : genes named as up - in - whim2 by spc but up - in - whim16 by xic . the first 10 replicates of ovelos@65 produced two genes in this conflict , while the last 6 replicates produced another 9 . examining underlying data from these conflicts yielded examples of three phenomena . in the first case , the numbers of identified spectra and the sums of precursor intensity simply point in opposite directions . in the second case , zero counts were treated as observations in spc analysis but as missing data in xic analysis , placing greater weight on replicates that reported nonzero intensities . in the third case , sorting genes by log2 ratios for fdr determination introduced noise that could be avoided by sorting on regression coefficients instead . given that the number of differential genes in a list ranged from 928 to 1328 , these conflicts never rose above 1% , which we judged to be tolerable in this study . venn analysis of differential genes from the three itraq instruments shows that almost every difference found by ovelos@45 was also found by the other two instruments ( see supporting information figure s6 for these cross - instrument images ) . among the 1403 up - in - whim2 genes , the bulk of the gene differences was found by qexac@56 only ( 58% ) , by ovelos@10 only ( 18% ) , or by both instruments ( 18% ) . a similar pattern appears in the 2269 up - in - whim16 genes , with 33% found by qexac@56 only , 9% by ovelos@10 only , 38% found by both these instruments , and 19% found by all three instruments . the data for label - free instruments showed great consistency between early and late sets of ovelos@65 data along with a substantial number of differences found only by qexac@98 ( presumably due to its high sensitivity ) . whereas more spc differences were observed by ovelos@65a j , the xic analysis favored the later ovelos@65k p set instead . figure 5 visualizes the extent of agreement for differential genes found in one set of replicates versus those found in others . whether analyzing the data by spc or by xic , many of the differences found in the first four replicates of qexac@98 were not reproduced by other label - free sets ( perhaps because of the increased sensitivity achieved in these experiments ) . a similar phenomenon arose from qexac@56 ; this instrument produced by far the highest psm sensitivity of the study , and the many additional differential genes inferred from its data could not be confirmed by other data sets . the low number of overall differences found in the ovelos@45 set reflects a relatively low overall gene sensitivity and a low degree of differential discrimination ; by contrast , the differences found by this instrument were almost entirely consistent with the differences produced by others . to what extent are the differential genes found for each data set confirmed by other data sets ? those represented by blue were found to be differential in common with another data set in that graph panel , as well . orange genes , on the other hand , were unique to a particular data set . the fraction of differential genes confirmed by another instrument in the fourth panel of figure 5 ranged from 67% for ovelos@65a j by spc to 99% for ovelos@45 . however , biological insight is often derived by projecting data at the level of the functional pathway or subnetwork . to examine the stability of biological insight that could be derived from data generated on each platform , we calculated the statistical enrichment of genes identified as more highly expressed in whim2 or in whim16 in each kegg pathway and each network module generated from the iref protein protein interaction network ( see experimental procedures ) using fisher s exact test with benjamini overall , many more proteins were identified as more highly expressed in whim16 than were identified as more highly expressed in whim2 . four kegg pathways ( antigen processing and presentation , arginine / proline metabolism , glutathione metabolism , and glycolysis / gluconeogenesis ) were enriched in up - in - whim16 lists from all platforms . the top 10 most significant pathways from each platform showed consistency , though these sets varied ( table 1 ) . focal adhesion was ranked as first or second most significant by the three itraq data sets , and it was in the top 10 most significant pathways for both partitions of the ovelos@65 data under both spc and xic methods , but it was not determined to be significant by qexac@98 by either analysis . similarly , five network modules were identified as the top 10 most significant modules enriched with up - in - whim16 lists from at least four platforms ( table 2 and figure 6 ) . only two modules were enriched for the qexac@98 data under the spc method , and no module was enriched for the qexac@98 data under the xic method ( figure 6 ) . specially , modules level_2_module_3 ( response to wounding ) and level_2_module_11 ( immune system processes ) were ranked as first or second most significant modules by four platforms . the up - in - whim2 genes were less consistent in both pathway and network analyses , but this was largely due to the fact that far fewer genes were observed to be significantly differential in whim2 by any platform , and pathway enrichment overall was much lower . indicates that the pathway was in the top 10 most significant pathways for that data set . + indicates that the pathway was significant ( corrected p value < 0.05 ) in that data set . enriched function is the most significant go ( gene ontology ) biological process term enriched with genes in the module . indicates that the pathway was in the top 10 most significant pathways for that data set . netgestalt - based analysis of functional consistency for up - in - whim16 genes from seven data sets . ( a ) proteins in the iref network are placed in a linear order together with the hierarchical modular organization of the network . ( b g ) for each data set , the network modules found to be enriched are colored light red . within these modules , we also evaluated the possibility of permutation - based fdr estimation for pathway enrichment at a 10% threshold . under these circumstances , qexac@98 resembled the other instruments to a greater degree , with significant hits occurring in an additional 10 pathways appearing in table 1 by spc analysis . the permutation analysis also found 5 additional pathways to be significant in table 1 by xic analysis . at this time , netgestalt has not yet gained the ability to perform permutation - based fdr correction . differential proteomic technologies are complex , and that complexity opens the door to many potential sources of variability . just as proteomic inventory experiments sample from a large potential pool of peptides , differential proteomics experiments sample from a large potential pool of differential proteins / genes . given that the underlying inventories from these six instruments ranged more than an order of magnitude in the number of distinct peptides they identified ( figure 2 ) , the degree of conformity in the differential gene lists across instruments was rather surprising ( figure 5 ) . projecting into kegg pathways or iref biological networks further reinforced this agreement among instruments . quality control methods and standard operating procedures are a necessary part of clinical proteomics , and sharing results for common samples between laboratories can be a powerful means to evaluate performance in both proteomic inventories and differentiation . the association of biomolecular resource facilities has been particularly influential in such cross - laboratory studies . when qc methods highlight an lc ms / ms experiment from a fractionated set as deficient , however , the ideal response is to rerun the entire fractionated set rather than replacing a subset of the fractions . to replace only the problematic fractions can increase the variability of the set by adding in the drift of the instrument as a function of time . figure 4 illustrates an important principle for isobaric labeling studies : intensity values within an itraq 4plex replicate were more comparable than values between different sets of replicates because they are drawn from the same set of psms . the speed and capacity of these methods clearly exceed those of label - free approaches as a result of multiplexing . current isobaric reagents can support analysis of the greater than 100 sample comparisons carried out by the cptac data production centers through the use of a common pooled sample included in each 4plex or through a randomized block design . the degree of multiplexing possible through use of current isobaric labeling reagents is at 10plex through use of the tandem mass tag ( tmt , thermo ) reagents , and further increases in the degree of multiplexing are anticipated as new chemistries and encoding strategies are developed . to return to the questions that motivated this study , it appears clear that repeatable performance in differential proteomics is feasible , whether isobaric labels or label - free methods are employed . the repeatability of ovelos@65 over 10 months of nearly continuous data acquisition was quite high , though the sixth of 16 replicates was somewhat questionable in the correlation analysis of figure 4 . the diminished performance in the final six replicates of qexac@98 is a clear reminder that instrument performance needs to be comparable for differentiation to be repeatable . the large increase in differential genes for the cross - instrument reproducibility analysis reminds us of the value of multiple replicates for differential proteomics . while this study did not take advantage of full biological replicates for its two samples , these are certainly valuable in most experiments . using the information from technical replicates for this study greatly increased the number of differential genes by enabling per - gene variability estimates . technical variation remains a fact of life for all proteomics laboratories ; this study , like many others in the field , might have benefited from the use of randomization and blocking designs in addition to its use of replication . if laboratories deploy different methodologies to analyze the differences between the same two complex samples , then they will assuredly see differences in the gene or protein lists produced by the two technologies . when label - free data were analyzed by spectral counting rather than precursor intensity , the differences yielded a high degree of overlap . when itraq rather than label - free methods are deployed , the differential genes were again quite similar . these overlaps suggest a degree of maturity in proteomic methods that has grown through years of development along multiple tracks . at base , biologists need to know that differential proteomics technologies can produce meaningful results . our assessment showed that biological pathway and network analysis is highly consistent across instruments . which subset of genes is observed to be differentially regulated may be sampled from a larger set associated with a pathway or network . the pattern of these differences yields a stronger signal in combination . with these questions addressed , we hope to see wider deployment of differential proteomics in support of clinical and biological studies .

the nci clinical proteomic tumor analysis consortium ( cptac ) employed a pair of reference xenograft proteomes for initial platform validation and ongoing quality control of its data collection for the cancer genome atlas ( tcga ) tumors . these two xenografts , representing basal and luminal - b human breast cancer , were fractionated and analyzed on six mass spectrometers in a total of 46 replicates divided between itraq and label - free technologies , spanning a total of 1095 lc ms / ms experiments . these data represent a unique opportunity to evaluate the stability of proteomic differentiation by mass spectrometry over many months of time for individual instruments or across instruments running dissimilar workflows . we evaluated itraq reporter ions , label - free spectral counts , and label - free extracted ion chromatograms as strategies for data interpretation ( source code is available from http://homepages.uc.edu/~wang2x7/research.htm ) . from these assessments , we found that differential genes from a single replicate were confirmed by other replicates on the same instrument from 61 to 93% of the time . when comparing across different instruments and quantitative technologies , using multiple replicates , differential genes were reproduced by other data sets from 67 to 99% of the time . projecting gene differences to biological pathways and networks increased the degree of similarity . these overlaps send an encouraging message about the maturity of technologies for proteomic differentiation .

Introduction Experimental Procedures Results and Discussion Conclusions

PMC2174399

from october 2000 to july 2002 , we recruited male newborns registered at the san cecilio university hospital ( one of the two reference public hospitals serving granada province in southern spain ) , excluding delivering mothers with serious chronic diseases , such as diabetes , hypertension , or thyroid disease , those who developed any pregnancy complication that could affect fetal growth or development , and nonresidents in the hospital referral area . a total cohort of 702 mother all boys with urogenital malformations ( cryptorchidism and/or hypospadias ) born in the study period were included . this study was approved by the institutional ethical committee of the san cecilio university hospital , and all participating mothers signed informed consent . all boys in the cohort were examined at birth ( within 2 days ) , and those diagnosed with cryptorchidism and/or hypospadias were reexamined at 1 month of age . the examination technique and definition of cryptorchidism and hypospadias followed recommendations of a danish finnish study ( boisen et al . 2004 ) developed by scorer ( 1964 ) . in brief , all examinations were done by two physicians in warm conditions ( room temperature 2024c ) with the child in supine position . testicular position was recorded after manipulation of the testis to the most distal position along the pathway of normal descent using firm traction . fifty boys were diagnosed with cryp - torchidism and/or hypospadias ; two of these were excluded from the investigation because their parents refused to participate . for each case , three controls were selected by gestational age ( 1 week ) , date of birth ( 7 days ) , and parity ( primiparous / multiparous ) . although the case control ratio was 1:3 , only 114 controls met the matching criteria . cases were 27 boys born with undescended testes ( 19 unilaterally , 8 bilaterally ) that persisted until 30 days of age , 19 boys born with hypospadias , and 2 boys born with both malformations . among cryptorchidism , 17 were severe and 12 were mild cases , classifying non - palpable inguinal and/or suprascrotal cases as severe and high scrotal cases as mild . information on potential confounding variables related to parents , pregnancy and delivery , and activities with potential for pesticide exposure were gathered from structured face - to - face interviews with the mother held within the first 48 hr after delivery . both mother and interviewer were blinded to the case or control status of the child . occupational exposure was derived from questions on paid employment and jobs focusing on chemicals with possible endocrine activity or previously described as male reproductive toxicants ( van tongeren et al . questionnaires were completed for 47 ( 96% ) cases and 105 ( 92% ) controls . child cohort and those selected as controls in the case control study , except for a small difference in father s age ( cohort , 33.6 5.7 years vs. controls , 31.8 5.3 years ; p = 0.01 ) and in gestational age in days ( cohort , 276.6 9.2 days vs. controls , 272.6 11.6 days ; p = 0.002 ) , although not when expressed in weeks ( 39.5 1.3 weeks vs. 38.9 1.6 weeks , p = 0.9 ) . samples of placenta without deciduas basalis and chorionic plate were collected at the time of delivery from 36 ( 75% ) cases and 109 ( 95.6% ) controls and sent to the laboratory of medical investigation for analysis . bioaccumulated compounds were extracted from samples by a previously described method ( fernandez et al . briefly , 1.6 g placenta homogenate were dissolved in hexane and eluted in a glass column filled with alumine merck 90 ( 70230 mesh no . 1097 ; merck , darmstadt , germany ) that had been dried at 600c for 4 hr , followed by the addition of 5% water . the eluate obtained was concentrated at reduced pressure under nitrogen stream to a volume of 800 l and then injected ( 4 200 l ) into the preparative high - pressure liquid chromatography ( hplc ) ( waters model 501 millipore apparatus with ultraviolet / visible detector model 490 ; millipore , marlborough , ma , usa ) . one microliter of hplc chromatography fraction was dried , dissolved in n - hexane , and then injected into a gas chromatography apparatus . the presence of aldrin , dieldrin , endrin , lindane , methoxychlor , endosulphan i and ii , mirex , o , p-ddt ( dichlorodiphenyl - trichloroethane ) , p , p-ddt , o , p-ddd ( dichlorodiphenyldichloroethane ) , p , p-dde , endosulfan diol , sulfate , lactone , and ether was determined by gas chromatography with electron - capture detection , using p-dichloro - benzophenone as internal standard , and mass spectrometry . standard solutions of organo - chlorine compounds were analyzed previously to determine retention times and calibration curves of these chemicals . the recovery of studied chemicals , measured by spiking placenta samples with pure chemicals , ranged from 84 to 102% . the operational quality control and the limit of detection ( lod ) ranged from 0.1 to 3 ng / ml . walnut creek , ca , usa ) with millennium chromatography manager software was equipped with a methyl silicone column ( length 30 m ) and dual ni-63 electron capture detectors . the gas chromatography - mass spectrometry apparatus ( saturn 2000 ; varian inc . ) with automatic injector was equipped with a db5-ms capillary column ( 30 m 0.25 mm ) . we estimated the total effective xenoestro - gen burden ( texb ) to measure the exposure to xenoestrogens in placenta extracts . previously , an hplc method was developed to separate natural estrogens ( fraction ) from more lipophilic xenoestrogens ( fraction ) without destroying either ( fernandez et al . extensive testing demonstrated that the pesticides ddt and metabolites and dieldrin , aldrin , and lindane , among other organochlorines , all elute in the hplc -fraction , along with other chlorinated and/or brominated organohalogenated chemicals . the estrogenicity of the -fraction , which contains no endogenous sex - hormones , can be considered a marker of the texb of environmental organohalogenated estrogens ( ibarluzea et al . the combined estrogenic effect is analyzed from its proliferative effect on mcf-7 human breast cancer cells and expressed as estradiol equivalent units . duplicated dry pooled and fractions ( eluted from 0 to 11 min and from 13 to 30 min , respectively ) were resuspended in charcoal - dextran serum and tested in the e - screen bioassay for estrogenicity using a slight modification of the originally described technique ( soto et al . each sample was assayed in triplicate with a negative ( vehicle ) and positive ( estradiol ) control in each plate . the proliferative effect of the fractions was referred to the maximal effect obtained with estradiol transformed into estradiol equivalent units ( eeq ) , by reading from a dose response curve prepared using estradiol ( concentration range 0.1 pm to 10 nm ) , and was expressed as total effective xenoestrogen burden ( texb- and texb- ) in eeq per gram of tissue and per gram of lipid ( ibarluzea et al . we performed statistical analysis to determine associations between predictors of interest and the presence of cryp - torchidism and/or hypospadias and between exposure to environmental pesticides and these urogenital malformations . all statistical analyses were performed using spss statistical software for windows version 11.0 ( spss inc . , we computed odds ratios ( ors ) for malformation and 95% confidence interval ( ci ) by unconditional and conditional logistic regression . potential confounding variables were selected on the basis of their significant association with outcomes in the univariate models . the modifying effect of these variables and their association with organochlo - rine levels and texb values were investigated . in the final adjusted model , differences between groups were tested with pearsons chi - square test or fishers exact test , when appropriate . from october 2000 to july 2002 , we recruited male newborns registered at the san cecilio university hospital ( one of the two reference public hospitals serving granada province in southern spain ) , excluding delivering mothers with serious chronic diseases , such as diabetes , hypertension , or thyroid disease , those who developed any pregnancy complication that could affect fetal growth or development , and nonresidents in the hospital referral area . a total cohort of 702 mother all boys with urogenital malformations ( cryptorchidism and/or hypospadias ) born in the study period were included . this study was approved by the institutional ethical committee of the san cecilio university hospital , and all participating mothers signed informed consent . all boys in the cohort were examined at birth ( within 2 days ) , and those diagnosed with cryptorchidism and/or hypospadias were reexamined at 1 month of age . the examination technique and definition of cryptorchidism and hypospadias followed recommendations of a danish finnish study ( boisen et al . 2004 ) developed by scorer ( 1964 ) . in brief , all examinations were done by two physicians in warm conditions ( room temperature 2024c ) with the child in supine position . testicular position was recorded after manipulation of the testis to the most distal position along the pathway of normal descent using firm traction . fifty boys were diagnosed with cryp - torchidism and/or hypospadias ; two of these were excluded from the investigation because their parents refused to participate . for each case , three controls were selected by gestational age ( 1 week ) , date of birth ( 7 days ) , and parity ( primiparous / multiparous ) . although the case control ratio was 1:3 , only 114 controls met the matching criteria . cases were 27 boys born with undescended testes ( 19 unilaterally , 8 bilaterally ) that persisted until 30 days of age , 19 boys born with hypospadias , and 2 boys born with both malformations . among cryptorchidism , 17 were severe and 12 were mild cases , classifying non - palpable inguinal and/or suprascrotal cases as severe and high scrotal cases as mild . information on potential confounding variables related to parents , pregnancy and delivery , and activities with potential for pesticide exposure were gathered from structured face - to - face interviews with the mother held within the first 48 hr after delivery . both mother and interviewer were blinded to the case or control status of the child . occupational exposure was derived from questions on paid employment and jobs focusing on chemicals with possible endocrine activity or previously described as male reproductive toxicants ( van tongeren et al . questionnaires were completed for 47 ( 96% ) cases and 105 ( 92% ) controls . child cohort and those selected as controls in the case control study , except for a small difference in father s age ( cohort , 33.6 5.7 years vs. controls , 31.8 5.3 years ; p = 0.01 ) and in gestational age in days ( cohort , 276.6 9.2 days vs. controls , 272.6 11.6 days ; p = 0.002 ) , although not when expressed in weeks ( 39.5 1.3 weeks vs. 38.9 1.6 weeks , p = 0.9 ) . samples of placenta without deciduas basalis and chorionic plate were collected at the time of delivery from 36 ( 75% ) cases and 109 ( 95.6% ) controls and sent to the laboratory of medical investigation for analysis . bioaccumulated compounds were extracted from samples by a previously described method ( fernandez et al . briefly , 1.6 g placenta homogenate were dissolved in hexane and eluted in a glass column filled with alumine merck 90 ( 70230 mesh no . 1097 ; merck , darmstadt , germany ) that had been dried at 600c for 4 hr , followed by the addition of 5% water . the eluate obtained was concentrated at reduced pressure under nitrogen stream to a volume of 800 l and then injected ( 4 200 l ) into the preparative high - pressure liquid chromatography ( hplc ) ( waters model 501 millipore apparatus with ultraviolet / visible detector model 490 ; millipore , marlborough , ma , usa ) . one microliter of hplc chromatography fraction was dried , dissolved in n - hexane , and then injected into a gas chromatography apparatus . the presence of aldrin , dieldrin , endrin , lindane , methoxychlor , endosulphan i and ii , mirex , o , p-ddt ( dichlorodiphenyl - trichloroethane ) , p , p-ddt , o , p-ddd ( dichlorodiphenyldichloroethane ) , p , p-dde , endosulfan diol , sulfate , lactone , and ether was determined by gas chromatography with electron - capture detection , using p-dichloro - benzophenone as internal standard , and mass spectrometry . standard solutions of organo - chlorine compounds were analyzed previously to determine retention times and calibration curves of these chemicals . the recovery of studied chemicals , measured by spiking placenta samples with pure chemicals , ranged from 84 to 102% . the operational quality control and limits of detection and quantification were previously reported ( lopez - espinosa et al . the limit of detection ( lod ) ranged from 0.1 to 3 ng / ml . ca , usa ) with millennium chromatography manager software was equipped with a methyl silicone column ( length 30 m ) and dual ni-63 electron capture detectors . the gas chromatography - mass spectrometry apparatus ( saturn 2000 ; varian inc . ) with automatic injector was equipped with a db5-ms capillary column ( 30 m 0.25 mm ) . we estimated the total effective xenoestro - gen burden ( texb ) to measure the exposure to xenoestrogens in placenta extracts . previously , an hplc method was developed to separate natural estrogens ( fraction ) from more lipophilic xenoestrogens ( fraction ) without destroying either ( fernandez et al . extensive testing demonstrated that the pesticides ddt and metabolites and dieldrin , aldrin , and lindane , among other organochlorines , all elute in the hplc -fraction , along with other chlorinated and/or brominated organohalogenated chemicals . the estrogenicity of the -fraction , which contains no endogenous sex - hormones , can be considered a marker of the texb of environmental organohalogenated estrogens ( ibarluzea et al . the combined estrogenic effect is analyzed from its proliferative effect on mcf-7 human breast cancer cells and expressed as estradiol equivalent units . duplicated dry pooled and fractions ( eluted from 0 to 11 min and from 13 to 30 min , respectively ) were resuspended in charcoal - dextran serum and tested in the e - screen bioassay for estrogenicity using a slight modification of the originally described technique ( soto et al . each sample was assayed in triplicate with a negative ( vehicle ) and positive ( estradiol ) control in each plate . the proliferative effect of the fractions was referred to the maximal effect obtained with estradiol transformed into estradiol equivalent units ( eeq ) , by reading from a dose response curve prepared using estradiol ( concentration range 0.1 pm to 10 nm ) , and was expressed as total effective xenoestrogen burden ( texb- and texb- ) in eeq per gram of tissue and per gram of lipid ( ibarluzea et al . we performed statistical analysis to determine associations between predictors of interest and the presence of cryp - torchidism and/or hypospadias and between exposure to environmental pesticides and these urogenital malformations . all statistical analyses were performed using spss statistical software for windows version 11.0 ( spss inc . , we computed odds ratios ( ors ) for malformation and 95% confidence interval ( ci ) by unconditional and conditional logistic regression . potential confounding variables were selected on the basis of their significant association with outcomes in the univariate models . the modifying effect of these variables and their association with organochlo - rine levels and texb values were investigated . in the final adjusted model , differences between groups were tested with pearsons chi - square test or fishers exact test , when appropriate . considered separately , the prevalence of cryptorchidism at 1 month of age was 1.1% , and the prevalence of hypospadias at delivery was 0.74% . mean age ( sd ) of the mothers was 29 4.96 years , 9.2% of the mothers were illiterate , and 14.5% had university studies . more than half of the women lived in rural areas ( 53.9% ) , but only 13.2% stated work in agriculture . one of four of the women were overweight or obese before pregnancy , according to world health organization criteria ( world health organization 1998 ) [ body mass index ( bmi ) > 25 ] . table 1 shows the relationships between selected characteristics and urogenital malformations . after testing a large number of more complex stratifications , we categorized maternal occupation as agricultural or other . an almost 3.5-fold increase in risk for urogenital malformation was observed when the mother reported taking part in agricultural activities . in contrast , when the father s work was categorized as agricultural or other , no association was found . however , when a different approach was taken and fathers were asked about specific work tasks and chemical exposures , a 2.98-fold increased risk of urogenital malformation was found for the highest versus lowest occupational exposure level . effects of an urban or rural setting on mother child exposure were examined by categorizing their place of residence as having a population of more ( urban ) or less ( rural ) than 10,000 inhabitants , but no association was found . a history of spontaneous abortion was associated with an increased risk of cryp - torchidism and/or hypospadias ( or= 4.20 ; 95% ci , 1.1116.66 ) . other maternal characteristics that were studied but showed no association with cryptorchidism or hypospadias were problems during pregnancy ( i.e. , hyper - emesis , episodes of bleeding , and loss of amni - otic fluid ) , use of oral contraceptive before pregnancy , and smoking habit ( table 1 ) . no association was seen with maternal weight , although when bmi was categorized below and above the mean value of the study population ( 23 kg / m ) , a higher bmi was borderline significantly associated with risk of cryp - torchidism and/or hypospadias in the conditional regression analysis adjusted for maternal age and birth weight [ adjusted or ( aor ) = 1.89 ; 95% ci , 0.844.26 ] . a weight gain of 12 kg during pregnancy appeared to have a protective effect ( or = 0.56 ; 95% ci , 0.261.06 ) . interestingly , mothers of cases gained significantly ( p = 0.07 ) less weight during pregnancy ( 11.3 4.7 kg ) compared with mothers of controls ( 13.9 5.5 kg ) ( table 1 ) . effects on risk of urogenital anomaly of type of delivery , fetal presentation , weeks of gestation , child weight and length , head size , presence of other malformation , and season of birth are also shown in table 1 . the risk increased with decreasing birth length ( p < 0.05 ) but not with decreasing birth weight ( or = 2.54 ; 95% ci , 0.699.17 ) , although the latter was close to statistical significance ( p = 0.1 ) . interestingly , the mean weight of cases ( 3110.63 614.67 g ) was significantly ( p = 0.03 ) lower than the mean weight of controls ( 3304.39 449.13 g ) . weeks of gestation , with category boundary established at 37 weeks , was associated with risk of malformation , with a more frequent presence of cryptorchidism and/or hypospadias in the group of lower gestational age ( or = 1.98 ; 95% ci , 0.884.46 ) , and this risk was higher in the conditional logistic analysis [ crude or ( cor ) = 4.18 ; 95% ci , 0.7523.34 ; aor = 2.29 ; 95% ci , 0.3813.74 ] . delivery by cesarean was more frequent in the cryptorchidism / hypospadias group ( or = 2.44 ; 95% ci , 0.5111.62 ) . the season of birth had an effect in our study population . taking spring as category reference , more boys with malformations were born in another season , especially in winter ( cor = 1.28 ; 95% ci , 1.199.12 ) , although without statistical significance in the conditional logistic analysis adjusted for maternal age and birth weight ( aor = 5.42 ; 95% ci , 0.5615.42 ) . finally , the risk of cryptorchidism and/or hypospadias was associated with presence of minor disorders of the urogenital tract such as phymosis , hydrocele , epispadia , and/or micropenis ( or = 1.83 ; 95% ci , 0.913.67 ) . all placentas studied were positive for at least one chemical ( mean residue number 7.6 3.9 ) . detectable concentrations of p , p-dde were found in 84.8% of placenta samples , with a mean value of 9.21 ng / g of placenta . lindane and endosulfan- , and ether followed dde in frequency of presence and were detected in 61.4 , 52.4 , and 53.1% , respectively , although at lower concentrations ( table 2 ) . a higher number of pesticides were detected in cases than in controls ( 9.3 3.2 vs. 6.9 3.1 , respectively ; p = 0.002 ) , but there were no statistically significant differences between cases and controls in the mean concentration of any pesticide except for dieldrin ( 1.19 vs. 0.33 ng / g placenta , p < 0.05 ) and lindane ( 0.72 vs. 0.36 ng / g placenta , p = 0.007 ) . residues of o , p-ddt , p , p-ddt , lindane , endosulfan- , and mirex were more frequently detected in cases than in controls , and the unconditional regression analysis associated malformation risk with the presence of these same chemicals , as follows : o , p-ddt ( or = 2.25 ; 95% ci , 1.034.89 ) ; p , p-ddt ( or = 2.63 ; 95% ci , 1.215.72 ) , lindane ( or = 3.38 ; 95% ci , 1.368.38 ) , mirex ( or = 2.85 ; 95% ci , 1.226.66 ) , and endosulfan- ( or = 2.19 ; 95% ci , 0.994.82 ) . table 3 lists these findings and results of the conditional regression analysis with crude and adjusted ors for malformations in relation to the presence of these five selected pesticide residues . seventy - two percent of placentas from cases and 54% of those from controls were positive for estrogenicity of the fraction . mean values of estrogenicity , measured as the texb of the fraction , were 2.92 eeq pm / g of placenta for cases versus 1.45 eeq pm / g of placenta for controls . in the unconditional regression analysis , the texb of the fraction was borderline associated with the risk of malformation , with an or of 2.02 ( 95% ci , 0.844.80 ) that increased to an or of 2.82 ( 95% ci , 1.107.24 ) in the conditional regression analysis adjusted for maternal age and birth weight ( table 3 ) . the texb of the fractions was more frequently positive and higher in cases versus controls but without reaching statistical significance . neither the texb- nor - fraction showed association with any of the 16 pesticides quantified . the relationship between the estrogenicity of fractions ( texb- ) and the variables most frequently related to malformations was investigated . the variables associated with texb- levels were maternal age , educational level , bmi before pregnancy , and parent occupation . all placentas from mothers working in agriculture were positive ( lod ) . when variables were simultaneously examined in a multivariate regression model , the main predictors of urogenital malformations were pregnancy weight gain , dieldrin levels , and presence of mirex or lindane ( table 4 ) . we used a novel method to measure exposure of the embryo - fetus to xenoestrogens by estimating the estrogenicity resulting from the combined effect of chemicals extracted from placentas ( texb measurement ) ( fernandez et al . three of four placentas from boys with cryptorchidism and/or hypospadias and one of two placentas from control boys had a measurable level of estrogenicity due to xenoestrogens ( texb of the fraction ) . we detected a statistically sig - nificant relationship between the risk of malformation and xenoestrogen exposure in the study population . therefore , we are able to report the first demonstration of a significant relationship between male urogenital malformation and the estrogenicity of the -fraction the estrogenicity due to bioaccumulated organohalogenated xenoestrogens . compared with controls , cases had an or for detectable versus nondetectable texb- of 2.82 ( 95% ci , 1.107.24 ) . this process is known to depend on a balanced steroid ratio and is therefore highly sensitive to disruption by exogenous estrogens ; exposure to edcs would provoke an unbalanced androgen / estrogen ratio , leading to an inadequate maturation of sertoli and leydig cells ( de kretser 2003 ; skakkebaek et al . disagreements in the scientific community about adverse trends in male reproductive health may be partly related to difficulties in comparing studies across time , to the selection of study populations , and to differences in clinical definitions and diagnostic procedures for these diseases . skakkebaek and co - workers ( 2001 ) proposed that sperm counts , demand for assisted reproduction , testicular cancer , hypospadias , and undescended testes are all symptoms of a single underlying entity , the so - called testicular dysgenesis syndrome . they concluded that environment and lifestyle factors are among the most likely causes , with minor participation of the genomic background . following proposals of separate genetic but common environmental components of risk for cryptorchidism and hypospadias ( akre et al . 1999 ) , cryp - torchidism and hypospadias were considered comparable entities in terms of their etiology , grouping together boys with one or both malformations in the statistical analysis . the prevalence of cryptorchidism and/or hypospa - dias was 1.8% in our series , within the range reported by studies in other geographic areas ( boisen et al . 2004 , 2005 ; paulozzi 1999 ; preiksa et al . 2005 ; toppari et al . 2001 ) . when the two malformations were considered separately , the prevalence of cryptorchidism at 1 month of age was 1.1% , and the prevalence of hypospadias at delivery was 0.74% . ( 2005 ) detected a lower frequency of cryp - torchidism at birth in lithuanian boys ( 5.7% ) in comparison with danes ( 9.0% ) and a higher frequency in comparison with finns ( 2.4% ) . highly variable prevalences of cryp - torchidism have been reported in western countries on the basis of registry studies ( boisen et al . however , comparisons in the incidence rate of urogenital malformations among countries may be limited by differences in study populations ( registry versus cohort studies ) , case definitions , and examination techniques . the spanish collaborative study of congenital malformations ( ecemc ) reported ( martnez - frias et al . 2004 ) that the frequency of hypospadias in spain was 1 of 284 male births ( 0.35% ) and had remained at this level for the past few decades until after 1996 , when a decreasing frequency of severe forms was recorded . the authors suggested that this effect was probably caused by a radical change in exposure that affected the whole country . surprisingly , this change was not observed in the prevalence of any other congenital malformations . all placentas had measurable concentrations of at least one of the 16 organochlorine pesticides quantified , reflecting the ubiquity of exposure in the population , although the number of quantifiable residues was significantly higher in cases than in controls . no single chemical could be positively and significantly associated with the biologic effect measured by texb-. there may be several reasons for this lack of concordance . thus , the estrogenic effect measured in the e - screen bioassay is a consequence of the combined effect of measured organohalogens ; and the estrogenic effect may be caused by other chemicals not quantified , either other organochlorine pesticides or other lipophilic compounds . over the past three decades , the highest concentrations detected in placentas in our study were those of ddt , isomers , and metabolites , followed by endosulfans , with p , p-dde showing both highest mean concentration and frequency . these findings are far below the mean values reported in placenta tissue of women in a neighboring region of southeast spain ( falcon et al . 2004 ) . in the latter series , recruited from 1998 to 2000 , the mean total ddt value found was similar to findings in placenta in a romanian population in 19961997 ( hura the present p , p-dde results are similar to those recently reported in placentas collected from 1997 to 2001 in finland ( shen et al . 2005 ) . in our view , caution must be taken in comparing concentrations of lipophilic endocrine disrup - tor chemicals in adipose tissue with data derived from lipid - adjusted concentrations in blood / serum or placenta . nevertheless , our results showed an excess risk of malformation associated with the presence of five pesticides ( o , p-ddt , p , p-ddt , endosulfan- , lindane , and mirex ) . the ors were higher than unity , reaching 9.48 ( 95% ci , 2.4336.96 ) for lindane in the conditional regression analysis adjusted for maternal age and birth weight . in the multivariate regression model , a significant association was observed between maternal involvement in agriculture activities and an increased frequency of congenital malformations in the offspring , although agricultural employment by fathers was not associated with a significant increase in risk . other authors have reported a higher risk of congenital anomalies at birth according to occupation ( dolk et al . in fact , pesticide exposure is likely in agricultural work even when direct handling of chemicals is not reported ( garcia et al . thus , entry into a greenhouse after spraying can be a major source of exposure , as can be the use of fertilizers and crop - preserving chemicals . moreover , factors related to residence in an agricultural setting may be an important source of inadvertent exposure to pesticides ( olea et al . no effect of parental age on urogenital malformations was observed , in agreement with most ( preiksa et al . an increased risk of these malformations was , however , significantly associated with lower birth weight , as previously documented ( berkowitz et al . 1999 ) , and also appeared to be related to low gestational age although signifi - cance was not reached , unlike in some other reports ( berkowitz et al . ( 2006 ) found that a higher number of organochlorine pesticide residues in placenta was associated with lower birth weight , con - firming these observations . a 4-fold increased risk of malformation in the sons of women with a history of stillbirth was noted ( or = 4.2 ; 95% ci , 1.1116.66 ) , confirming previous observations ( weidner et al . although cryptorchidism and hypospadias have frequently been described in associated congenital malformations ( mayr et al . 1999 ) , only minor concomitant diseases of the urogenital tract were observed in our series , confirming findings by other authors ( biggs et al . 2001 ) , but no signifi - cant association was observed in the present study ( or = 2.44 ; 95% ci , 0.5111.62 ) . seasonal cyclicity in the month of delivery / conception in relation to these malformations has been reported . in agreement with some of these authors ( e.g. , mayr et al . 1999 ) , a seasonal cyclicity was also observed in the present study , with a peak of cases born between january and march ( cor = 1.28 ; 95%ci , 1.199.12 ) . if the first trimester is considered the most vulnerable time for the embryo - fetus , and if spring is the season during which most of the pesticides were applied , this additional information may strengthen the results . unfortunately , we do not have accurate data on the application of pesticides in spring versus other seasons . finally , maternal weight before pregnancy , categorized below and above the median value of the bmi ( 23 kg / m ) , showed close to a sig - nificant association . we have no explanation for this finding , but it would be interesting to consider it together with the observation of a lower body weight increase during pregnancy in mothers of cases versus mothers of control boys ( 11.3 4.7 kg vs. 13.9 5.5 kg ) . in conclusion , although the complexity of human biology makes it very difficult to establish a relationship between edc exposure and male congenital malformations , our data suggest that environmental chemicals with estrogenic activity play a role in the risk of cryptorchidism and/or hypospadias . unfortunately , few comparable studies have addressed this issue ( e.g. , vidaeff and sever 2005 ) , and these were limited to a small number of edcs ( hosie et al . however , the present study illustrates the utility for exposure assessment of a biomarker that evaluates the combined effects of bioaccumulated xenoestrogens in placentas .

backgroundexposure to xenoestrogens during pregnancy may disturb the development and function of male sexual organs.objectivein this study we aimed to determine whether the combined effect of environmental estrogens measured as total effective xenoestrogen burden ( texb ) is a risk factor for male urogenital malformations.methodsin a case control study , nested in a mother child cohort ( n = 702 ) established at granada university hospital , we compared 50 newborns with diagnosis of cryptorchidism and/or hypospadias with 114 boys without malformations matched by gestational age , date of birth , and parity . controls did not differ from the total cohort in confounding variables . texb and levels of 16 organochlorine pesticides were measured in placenta tissues . characteristics of parents , pregnancy , and birth were gathered by questionnaire . we used conditional and unconditional regression models to estimate odds ratios ( ors ) and 95% confidence intervals ( cis).resultstexb from organohalogenated compounds was detectable in 72% and 54% of case and control placentas , respectively . compared with controls , cases had an or for detectable versus non - detectable texb of 2.82 ( 95% ci , 1.107.24 ) . more pesticides were detected in cases than in controls ( 9.34 3.19 vs. 6.97 3.93 ) . ors for cases with detectable levels of pesticides , after adjusting for potential confounders in the conditional regression analysis , were o , p-ddt ( or = 2.25 ; 95% ci , 1.034.89 ) , p , p-ddt ( or = 2.63 ; 95% ci , 1.215.72 ) , lindane ( or = 3.38 ; 95% ci , 1.368.38 ) , mirex ( or = 2.85 ; 95% ci , 1.226.66 ) , and endosulfan alpha ( or = 2.19 ; 95% ci , 0.994.82 ) . engagement of mothers in agriculture ( or = 3.47 ; 95% ci , 1.339.03 ) , fathers occupational exposure to xenoestrogens ( or = 2.98 ; 95% ci , 1.118.01 ) , and history of previous stillbirths ( or = 4.20 ; 95% ci , 1.1116.66 ) were also associated with risk of malformations.conclusionswe found an increased risk for male urogenital malformations related to the combined effect of environmental estrogens in placenta .

Methods Design and participants Laboratory analyses Statistical analysis Results Discussion

PMC3134861

nanoparticle drug carriers improve the therapeutic benefit of chemotherapeutic agents by exploiting the enhanced permeability and retention ( epr ) of solid tumours to improve localised chemotherapeutic drug accumulation ( , as reviewed in ) . one such drug carrier is the nanoparticle which forms through the self - association of amphiphilic diblock copolymers in aqueous environments . the hydrophobic core of these nanoparticles provides a reservoir which can serve as an in vivo carrier of hydrophobic drugs , and the hydrophilic shell confers compatibility with intravenous injection [ 3 , 4 ] . drug delivery effectiveness is dependent on circulation stability of the vehicle , the degree of retention of drug by the vehicle in the circulation , and on the level of accumulation of encapsulated drug in the targeted tumour . retention of encapsulated drug in the plasma during the early distribution phase of the carrier is crucial for maximising drug delivery to solid tumours since particles must remain in the circulation for at least 6 h to achieve the epr effect . importantly , delivery is also tumour dependent given that the epr effect is influenced by characteristics of the tumour such as the size and number of discontinuities in the endothelium , the interstitial tumour fluid pressure , and the tumour size ( as reviewed in ) . understanding the in vivo stability of the nanoparticle is vital to optimise nanoparticle formulation variables [ 1 , 5 ] . chemical characteristics of the stabiliser , size of the polymer , the size of nanoparticles , the drug / polymer ratio , and compatibility between the drug the nanoparticle core all have the potential to impact nanoparticle stability [ 7 , 8 ] . for instance , since self - associations between the core - forming hydrophobic portion of the copolymer contributes to the stability of the nanoparticle , incorporating drugs into the core can impact these stabilising forces [ 9 , 10 ] . potentially , too high a drug / polymer ratio could increase the rate of release of drug from the nanoparticle in the circulation and thereby reduce amount of encapsulated drug reaching the tumour . there are many in vitro assays of nanoparticle stability ; however these assays correlate poorly with in vivo nanoparticle circulation longevity and nanoparticle drug retention . to understand the effect of our formulation variables on drug release in vivo and on the accumulation of the nanoparticle at the tumour site and on the release of entrapped agent at the tumour we needed to track the nanoparticle carrier and the entrapped agents simultaneously in vivo . one indication of particle stability can be made by simply following the level of the entrapped drug circulating in the plasma over time . since agents entrapped in vehicles are protected against metabolism all drug in the plasma can be considered to be drug retained in the carrier . however loss of drug from the circulation does not distinguish between release of the drug from the carrier and drug cleared entrapped within the carrier . historically , diblock copolymer nanoparticles have been followed by direct chemical labelling of the unit copolymer concomitant with demonstration of continued association of the labelled copolymer unit with the assembled nanoparticle in plasma samples [ 12 , 13 ] . following a single nanoparticle in this manner can be prohibitively labour intensive and thus tracing the plasma circulation and biodistribution of the carrier portion of the nanoparticle has rarely been done . arguably an agent that is employed in such minute quantities as to not impact the stability of the core , that is very well retained by intact particles and that does not exchange into plasma lipids or proteins could prove useful to track nanoparticles in vivo . cholesteryl hexadacyl ether ( [ h ] che ) is a nonmetabolizable hydrophobic compound widely used to radioactively label liposomal nanoparticles and not known to exchange between membranes . here we have modified this approach by incorporating che into diblock copolymer nanoparticles and we demonstrate that monitoring the che provided credible and valuable information which aided in formulating diblock copolymer nanoparticles . in a previous report , we described a series of paclitaxel prodrugs , propac 1 to propac 9 , and detailed the in vitro stability , plasma circulation characteristics , and antitumour efficacy of nanoparticles formulations of these prodrugs . propac 7 , is a prodrug in which paclitaxel is conjugated via a diglycolate linker to a c22 acyl chain . the nanoparticles were comprised of a stabiliser , 2.5k peg-3k ps , a costabilising lipid ( popc ) , and a prodrug and the nanoparticles formed through flash coprecipitation . the lipid tail of the costabilising lipid inserts into the hydrophobic core of the nanoparticle while the polar headgroup localises the lipid to the interface between the hydrophobic core and the hydrophobic shell of the nanoparticle . the costabilising lipid is necessary to prevent hydrolytic cleavage of the prodrug during storage . in order to optimise our carrier formulation we employed che to monitor nanoparticles prepared with a variety of diblock copolymers stabilisers and at a number of different component ratios . we then used this information to modify the nanoparticle drug retention characteristics and to investigate the impact of nanoparticle formulation on accumulation of the nanoparticles in tissues or in tumours . the simplicity of this approach has allowed us to generate a rapid and simple screening method which we used to assess the behaviour of many polymer formulations without necessitating the generation of specific covalently linked markers . the following copolymer stabilisers were obtained from polymer source ( dorval , canada ) ; polyethylene 2.5k glycol - polystyrene 3k ( ps ) , polyethylene glycol 2k - poly ( dl)-lactide 3.9k ( pla ) , and polyethylene glycol 5k - polycaprolactone 6k ( pcl ) . the pegylated lipid 2-distearoyl - sn - glycero-3-phosphoethanolamine - n[amino(polyethylene glycol)-2000 ] ( dspe - peg2k ) was purchased from avanti polar lipids ( alabaster al , usa ) . palmitoyl oleoyl phosphatidylcholine ( popc ) was purchased from northern lipids , burnaby , canada . [ h ] cholesteryl hexadecyl ether , 45 ci / mmol , ( [ h ] che ) was obtained from perkin elmer life and analytical sciences , inc . ultima gold scintillation fluid was purchased by perkinelmer life and analytical sciences ( boston , massachusetts ) . foxn1 athymic nude mice ( 7 - 8 weeks ) were obtained from harlan ( indianapolis in , usa ) . paclitaxel - prodrug , 2-o-(5-o - docosanyldiglycoloyl)-paclitaxel ( propac 7 ) was synthesised as previously described . briefly , nanoparticles without drugs were prepared by dissolving 40 mg of polymer , ps , pla , pcl , or dspe - peg2k , in 2 ml of ethanol : tetrahydrofuran ( 4 : 1 v / v ) . this solution was mixed with water using an impinging jet mixer with flow rates set at 6 ml / min ( solvent ) and 59 ml / min ( water ) using two harvard apparatus phd 2000 syringe pumps . to prepare prodrug loaded nanoparticles propac 7 , popc , and ps were combined at the indicated component ratio in ethanol : tetrahydrofuran ( 4 : 1 v / v ) and mixed with water as for the drug - free nanoparticles . [ h ] che - labelled nanoparticles were prepared by adding che to the solvent mixture prior to nanoparticle formation . for plasma clearance studies , 2.5 ci [ h ] che/40 mg of polymer was added ( 0.0000030 che / copolymer ( w / w ) ) . solvent was removed by dialysis in spectrum laboratories 3500 mw cut - off dialysis tubing ( spectrum laboratories , rancho dominguez ca , usa ) against water . nanoparticles were concentrated in 300 mm sucrose by crossflow dialysis using a 100 kd ( 0.5 mm lumen , 60 cm path length ) midgee hoop cartridge ( ge healthcare life sciences , piscataway nj , usa ) . nanoparticle size characteristics were determined by dynamic light scattering in water using a malvern zetasizer nano - zs ( malvern instruments , worcestershire , uk ) . plasma samples or nanoparticle suspensions ( 50 l ) were mixed with 150 l of diluents ( methanol : acetonitrile , 2 : 1 v / v ) by vigorous vortexing and propac 7 was recovered in the 10000 g supernatant . the mobile phase consisted of a linear gradient from methanol/10 mm sodium acetate buffer ( ph 5.6 ) ( 70 : 30 v / v ) to 100% methanol at a flow rate of 1 ml / min . a 20 l sample was injected onto the column and peak area was monitored by uv detection at 227 nm . separation of nanoparticles from the bulk protein fraction was performed on a 20 cm 1 cm sepharose 4b ( ge healthcare life sciences , piscataway nj , usa ) column equilibrated within hepes buffered saline , ph 7.4 at a flow rate of 0.5 ml / min . plasma samples ( 200 l ) were loaded onto the column and 150 l fractions were collected and aliquots of each fraction were analyzed for [ h ] che in ultimagold scintillation fluid ( perkin elmer , norwalk ct , usa ) or counted on a beckman - coulter ls 6500 scintillation counter and for protein using pierce microbca assay ( thermoscientific rockford il , usa ) and read using a victor multilabel plate reader ( perkin elmer , norwalk ct ) . all animal experiments were conducted according to protocols approved by the university of british columbia 's animal care committee and in accordance with the current guidelines established by the canadian council of animal care . nanoparticles were sterile filtered through 0.22 micron filters then injected into mice via the tail vein at a dose of 10 l / g body weight up to a maximum of 250 l . whole blood was collected into edta coated microtainer tubes ( bd biosciences bedford , ma , usa ) . plasma was collected by centrifugation of the tubes at 2800 rpm for 10 minutes and stored 20c until analysis . the quantity of propac 7 in ( 50 l ) a second 50 l aliquot of each sample was measured for nanoparticle carrier by monitoring the [ h ] che by liquid scintillation counting with a ls 6500 multi - purpose scintillation counter ; beckman coulter canada inc . we determined the plasma half - life , the time to reach 1/2 of the initial propac or 1/2 of the initial nanoparticle concentration , from the plasma concentration versus time curve . we have previously determined the plasma volume of foxn1 mice averages 0.043 ml per gram of body weight up to a body weight of up to 25 g. athymic nude foxn1 mice were injected s.c . with 2 10 colorectal carcinoma hct-116 cells . / ps 1 : 1 : 2 ( w / w / w ) were injected when tumours reached an average size of 200 mm . tumours , tissue , and blood were collected at 1 , 4 , 8 , 16 , 24 , and 48 hours post injection . blood was centrifuged and the plasma fraction was recovered and stored frozen at 20c until analysis . tissue samples were digested in solvable ( ge health ) at 60c overnight then decolourised with h202 . data values are reported as mean standard deviation ( sd ) . a standard one - way analysis of variance ( anova ) we have previously described paclitaxel prodrug nanoparticles generated through flash coprecipitation . when the initial total concentration of the prodrug , colipid , and diblock copolymer stabiliser in the solvent was less than 40 mg / ml , this procedure produced small , homogeneous nanoparticles with a prodrug trapping efficiency of > 90% . propac 7/popc / ps 1 : 1 : 2 ( w / w / w ) nanoparticles had a mean diameter of 2030 nm and unimodal size distributions suggesting a single population of nanoparticles . the prodrug stability was monitored by hplc and the particle physical stability over time was monitored by dls . when maintained at 4c for 11 weeks , less than 5% of propac 7 was hydrolysed and nanoparticle size increased by less than 15% . we used column chromatography to demonstrate the strong retention of che in nanoparticles during both in vitro and in vivo exposures to plasma and we provide examples of each in figure 1 . we prepared a 20 cm sepharose 4b column and established that nanoparticles eluted before the bulk protein fraction on this column . nanoparticles incubated with mouse plasma at 37c for one hour in vitro gave the same [ h ] elution profile as control nanoparticles ( in water ) ( data not shown ) . figure 1(a ) shows data from an in vivo experiment in which [ h ] che labelled propac 7/popc / ps 1 : 1 : 2 ( w / w / w ) nanoparticles were injected i.v . into mice and plasma plasma was immediately chromatographed on the column and aliquots of the collected fractions were assayed for protein or [ h ] . the [ h ] chromatographic elution profile of nanoparticles recovered from the plasma were unchanged from that of the control nanoparticles . these data suggest that [ h ] che remained associated with our nanoparticles in plasma and did not exchange out of the nanoparticles and associate with plasma lipoproteins in vivo or in vitro . we strengthened the contention that che does not transfer to plasma proteins or lipids by incubating relatively unstable dspe - peg2k nanoparticles with plasma in vitro . dspe - peg2k nanoparticles have transition temperature of 15c , and thus the lipid chains assume a disorder state well below physiological temperatures . in the presence of bovine serum albumin protein or human plasma , dspe - peg2k nanoparticles were chromatographed on a sepharose 4b column and the elution profile of the radioactivity is shown in figure 1(b ) . the [ h ] associated with dspe - peg2k nanoparticles elutes as a single sharp peak . we then incubated dspe - peg2k nanoparticles in mouse plasma for one hour at room temperature and chromatographed this sample on the same sepharose 4b column . aliquots of these collected fractions were assayed for protein or [ h ] and the elution profile is also shown in figure 1(b ) . the bulk of the [ h ] coelutes with the nanoparticles ; however a trailing shoulder peak appears in the serum treated chromatograph is indicative of some degree of nanoparticle disruption . importantly radioactivity is not found associated with the bulk protein fraction implying that the che did not redistribute to the lipoproteins . we have found [ h ] che generally applicable to correlating nanoparticle formulation variables and in vivo plasma circulation including varying the stabilisers . we prepared [ h ] che - labelled nanoparticles with various stabilisers , in this instance , without colipid or prodrug ( table 1 ) and the plasma circulation characteristics of these nanoparticles are shown in figure 2 . the data from ps nanoparticles is shown in figure 2(a ) . approximately 50% of the [ h ] label remained 24 hours after injection , suggesting very stable , long circulating nanoparticles are formed with this copolymer . the more rapid clearance of [ h ] associated with pla and pcl ( figures 2(b ) and 2(c ) , resp . ) finally , we included dspe - peg2k nanoparticles , which have been demonstrated to be unstable in plasma in vitro [ 17 , 18 ] . we contend that the in vivo stability will also be poor for these nanoparticles and should clear very rapidly from the circulation . we also predicted that if che from dspe - peg2k nanoparticles exchanged into plasma , we would observe a contradictorily prolonged [ h ] circulation . the rapid in vivo clearance of [ h ] associated with the dspe - peg2k nanoparticles figure 2(d ) suggested that [ h ] che lost from these nanoparticles did not exchange to plasma lipids or lipoproteins . to develop an assay to monitor the effect of formulation parameters on prodrug release in vivo we prepared [ h ] che - labelled ps nanoparticles with prodrug and colipid ; propac 7/popc / ps 1 : 1 : 2 ( w / w / w ) . the circulation profiles of the [ h ] and the prodrug are shown in figure 3 . to aid with the interpretation of data , we have reproduced the data for the nanoparticles without colipid or prodrug ( figure 2(a ) ) . first , by comparing the circulation of [ h ] associated with nanoparticles with and without prodrug , we were able to gauge the effect of encapsulating agents into the core of the nanoparticle . long [ h ] circulation times were observed when [ h ] che was incorporated into nanoparticles without prodrug ( t1/2 > 24 hours ) . a significant shortening of [ h ] circulation time was observed when colipid and propac 7 were introduced into the nanoparticles ( t1/2 14 hours ) . we attributed the enhanced clearance of [ h ] associated with prodrug loaded nanoparticles to a less stable nanoparticle formed when the prodrug was incorporated into the nanoparticle . secondly , by simultaneously monitoring [ h ] and propac 7 in prodrug loaded nanoparticles we were able to demonstrated prodrug clearance was faster than particle clearance . this suggested that the prodrug clearance occurred through both release of the prodrug from the nanoparticle and nanoparticle clearance . it is noteworthy that we have not always observed prodrug in plasma clearing faster than [ h ] . in a previous report we observed prodrug cleared at a rate equal to that of the [ h ] . this occurred when nanoparticles were loaded with a nonmetabolisable paclitaxel drug conjugate , suggesting that in this instance all prodrug was being cleared still associated with the nanoparticle . in a further set of experiments we demonstrated that we could monitor the effect of formulation composition on particle clearance . we prepared a series of [ h ] che - labelled propac 7/popc / ps nanoparticles with component weight ratios of 1 : 1 : 4 , 1 : 1 : 2 , 1 : 1 : 1 , and 2 : 2 : 1 ( w / w / w ) ( table 2 ) . the mean diameters of the nanoparticles increased as the prodrug / polymer ratio increased . the data for [ h ] plasma clearance following injection of these nanoparticles the particles with the lowest amount of prodrug relative to polymer , propac 7/popc / ps 1 : 1 : 4 ( w / w / w ) had the smallest particle sizes and long [ h ] che - circulation lifetimes . increasing the prodrug / polymer ratio in propac 7/popc / ps 1 : 1 : 2 ( w / w / w ) generated nanoparticles with 30% larger diameters , however the plasma clearance of the [ h ] was unchanged relative to the propac 7/popc / ps 1 : 1 : 4 ( w / w / w ) nanoparticles . this suggests that in vivo stability of these particles was not impacted by the increase in prodrug . nanoparticles with propac 7/popc / ps 1 : 1 : 1 and 2 : 2 : 1 ( w / w / w ) formed larger nanoparticles and had faster [ h ] che - plasma clearances . these results suggested that the nanoparticles formed with this diblock copolymer stabiliser have a tolerance limit for the amount of prodrug which can be incorporated into the hydrophobic core . this information was used to determine the maximum nanoparticle drug content obtainable with minimal impact of nanoparticle stability . in addition to varying component ratios and stabilisers , we have used [ h ] che labelling to evaluate the role of alternate costabilisers ( vitamin e - succinate or phosphatidylglycerol ) , and for nanoparticles loaded with different paclitaxel prodrugs or with hydrophobic prodrugs of other chemotherapeutic agents ( gemcitabine and doxorubicin ) ( not shown ) . the loading capacity of nanoparticles is likely unique for each diblock copolymer and different for every drug . the ability to trace the nanoparticle in vivo is a valuable tool for choosing stabilisers and determining optimal loading ratios . importantly , labelling nanoparticles by physically incorporating che is technically quite simple . to further evaluate the utility of this nanoparticle labelling technique we monitored plasma clearance and tumour accumulation of [ h ] che - labelled nanoparticles . the goal was to try to understand the effect of formulation variables and dosing schedule on efficacy . to demonstrate that we could monitor nanoparticle accumulation in tissues and tumours , [ h ] che - labelled propac 7/popc / ps nanoparticles 1 : 1 : 2 ( w / w / w ) were injected into hct-116 tumour - bearing mice . the plasma and tissues were harvested and the [ h ] levels were measured at various times . the results in figure 5 shows plasma clearance of [ h ] associated with prodrug loaded nanoparticles ( figure 5(a ) ) and accumulation of [ h ] in the organs and the tumours of the same mice over a 48-hour period ( figures 5(b)5(f ) ) . notably , [ h ] accumulated in hct-116 tumours for more than 48 hours underscoring the need for stable nanoparticles to maximise chemotherapeutic efficacy . we have employed [ h ] che - labelled nanoparticles in order to compare accumulations in large and small tumours of same cell origin and to assess nanoparticle accumulation in different types of tumour ( not shown ) . the reliance of nanoparticle tumour accumulation on the epr effect makes tumour accumulation subject to variables such as the degree of tumour vascularization . the accumulation of che - labelled nanoparticles was useful to optimise dosing schedule and to understand the impact of nanoparticle composition on tumour efficacy . the in vivo stability of controlled release formulations critically impacts biodistribution and the pharmacokinetics of the encapsulated agent . it is imperative to characterise the effect of formulation variables on carrier stability and drug retention . unfortunately in vitro assays of nanoparticle stability do not predict the stability of nanoparticles in vivo and labelling of diblock copolymers can be quite labour intensive . we have used entrapped radioactive agents to label our nanoparticles for in vivo assessment of nanoparticle stability . we have found that this label provided relevant data which accelerated screening of many variables and which aided in nanoparticle component selection and optimising component ratios . furthermore , we further test selected formulations in efficacy experiments to substantiate our formulation choices made using plasma clearance data . as a caution , one must confirm that a noncovalently incorporated nanoparticle label remains associated with the nanoparticle in plasma . we verified this by running size exclusion chromatographic columns which separated our nanoparticles from the bulk protein of the plasma and in every instance , all the [ h ] co - eluted from the column with nanoparticles . one other potential flaw is an underestimation of nanoparticle stability if the [ h ] che is rapidly released and clearance of the nanoparticles themselves remain intact .

diblock copolymer nanoparticles encapsulating a paclitaxel prodrug , propac 7 , have been used to demonstrate the usefulness of a nonmetabolizable radioactive marker , cholesteryl hexadecyl ether ( che ) , to evaluate nanoparticle formulation variables . since che did not exchange out of the nanoparticles , the rate of clearance of the che could be used as an indicator of nanoparticle stability in vivo . we simultaneously monitored prodrug circulation and carrier circulation in the plasma and the retention of che relative to the retention of prodrug in the plasma was used to distinguish prodrug release from nanoparticle plasma clearance . nanoparticles labelled with che were also used to evaluate accumulation of nanoparticles in the tumour . this marker has provided relevant data which we have applied to optimise our nanoparticle formulations .

1. Introduction 2. Materials and Methods 3. Results and Discussion 4. Conclusion

PMC5391820

lacquer cracks are considered to be healed mechanical fissures in the retinal pigment epthelium ( rpe)-bruch s membrane choriocapillaris complex and are seen ophthalmoscopically as yellowish linear lesions.1,2 we have reported the characteristics of lacquer cracks in the spectral - domain optical coherent tomographic ( sd - oct ) , fluorescein angiographic ( fa ) , indocyanine green angiographic ( icga ) , and fundus autofluorescence ( faf ) images.3 all the findings were compatible with the suggested pathophysiology of healed mechanical fissures in the rpe - bruch s membrane choriocapillaris complex . lacquer cracks generally occur in the posterior pole of the fundus , and to date , there has been only one case report on lacquer cracks that appeared in the peripheral fundus.4 however , the description of these lacquer cracks was limited . we report a patient with high myopia with lacquer cracks observed in the peripheral fundus . we present the characteristics of the cracks observed in the fa and faf images and also the three - dimensional magnetic resonance images ( 3d mri ) of both eyes of this patient . a 37-year - old man visited the high myopia clinic of the tokyo medical and dental university hospital with a complaint of decreased vision in both eyes . his visual acuity was 20/20 in both eyes , and the refractive error was 14.5 d od ( right eye ) and 14.0 d os ( left eye ) . dilated fundus examination showed that the optic disk cups were enlarged in both eyes , but there were no abnormalities in the peripheral region ophthalmoscopically ( figure 1 ) . he was suspected to have glaucoma in both eyes , and the goldmann perimetry was performed . a slight reduction in the sensitivity in the nasal visual field of the left eye was found . fa showed multiple arch - shaped , hyperfluorescent linear lesions running circumferentially in the peripheral fundus of both eyes . some of these lesions were observed to run parallel to each other ( figure 2 ) . faf showed hypoautofluorescence at the corresponding sites ( figure 3 ) . from these findings , we diagnosed these lesions as lacquer cracks in the peripheral fundus . however , the entire posterior region of the globe was enlarged in both eyes ( figure 4 ) . written informed consent was obtained from the patient for publication of this report and any accompanying images . the local committee of tokyo medical and dental university hospital judged that the ethical review of this case report was unnecessary . we reported earlier that lacquer cracks appeared hyperfluorescent during the entire angiographic phase of fa because of the window defect in the early phase and tissue filling in the late phase . these findings corresponded with the pathophysiology of lacquer cracks that are considered to be healed mechanical fissures in the rpe - bruch s membrane choriocapillaris complex.3 in the present case , fa showed hyperfluorescent , arch - shaped linear lesions running circumferentially in the peripheral fundus during the entire angiographic phase , and faf showed hypoautofluorescence at the corresponding sites . these linear lesions need to be distinguished from angioid streaks , pigmented peripheral streaks,5 and choroidal rupture . their ophthalmoscopic , fa , and faf appearances were completely different from these lesions , and the patient had no history of trauma to both eyes . although the direction and the area of the lesions were not typical of lacquer cracks , the fa and faf findings were the same as those of lacquer cracks in the posterior fundus . thus , these lesions were most likely also due to ruptures of the rpe - bruch s membrane choriocapillaris complex . lacquer cracks generally occur in the posterior pole of the fundus and are caused by an elongation of the globe.6 in the present case , 3d mri did not show any protrusions of the globe indicating a posterior staphyloma , but instead the entire posterior globe was expanded . it might be possible that the expansion of the peripheral area was the cause of the lacquer cracks occurring in the peripheral fundus . with increasing time , lacquer cracks progress to other myopic fundus changes including patchy atrophy , diffuse atrophy , and choroidal neovascularization.7 there have not been reports about peripheral myopic choroidal neovascularization before , and it is not certain whether peripheral lacquer cracks would cause myopic choroidal neovascularization in the future . however , we occasionally notice atrophic regions in relatively peripheral fundus in highly myopic eyes . these lesions might have resulted from peripheral lacquer cracks . therefore , cases with peripheral lacquer cracks should be followed regularly with attention paid to whether the myopic fundus changes develop not only in the posterior pole but also in the peripheral region . we report a case with peripheral linear lesions that have properties of the lacquer cracks found in the posterior pole of the eye . although peripheral lacquer cracks are rare and are sometimes difficult to be detected by ophthalmoscopy , they should be considered in the differential diagnosis of peripheral linear lesions showing hyperfluorescence in fa .

we report a case with lacquer cracks observed in the peripheral fundus . a 37-year - old patient with bilateral high myopia who visited our clinic was examined by fluorescein angiography ( fa ) and fundus autofluorescence ( faf ) to determine whether there were myopic fundus lesions . fa showed many arch - shaped , hyperfluorescent linear lesions running circumferentially in the peripheral fundus . faf showed hypo - autofluorescence at the corresponding sites . these characteristics were very similar to the lacquer cracks present in the posterior fundus in highly myopic eyes . although lacquer cracks in the peripheral fundus are rare , they should be considered in the differential diagnosis of peripheral linear lesions showing hyperfluorescence in fa .

Introduction Case report Discussion Conclusion

PMC3134024

fractures of the thoracic and lumbar spine in pediatric patients are relatively uncommon as compared to adults.1 the incidence of complete and incomplete neurological injuries is almost equal.2 the recovery of the neurologic function following severe traumatic spinal cord injury occurs with a significantly greater incidence in children than adults , and these improvements can occur over a prolonged postinjury period.3 although specific studies have not been reported in children , conus medullaris lesions have greater chances of recovery compared to the spinal cord injuries.4 the posterior dislocation of the spine is a rare injury in children with most of the cases reported in adults.5 only one case of posterior dislocation at the l1l2 level5 and another at the lumbosacral junction6 have been reported in the pediatric spine . we report a rare case of a complete posterior dislocation ( retrospondyloptosis ) of the first lumbar vertebra over the second with complete neurological deficit . a 7-year - old boy presented to the emergency department of our hospital after 1 week of injury to back . the patient had been grazing cattle and had wrapped the rope of a cow around his trunk . the cow started running suddenly and as a result the child sustained a sudden jerk to the trunk and was dragged to some distance [ figure 1 ] . the injury was associated with pain , deformity over back , and a complete loss of sensations and movements in both lower limbs . cowherd 's injury in this case on examination , there was kyphotic deformity in the back with few abrasions . a neurological examination revealed grade 0 power of all muscles around all the joints of both lower limbs . plantar , knee , ankle , and bulbocavernosus reflexes were absent ( frankel grade a paraplegia ) . a radiographic examination revealed complete posterior translation of the first lumbar vertebra over the second ( retrospondyloptosis ; figure 2a and b ) . mr imaging revealed the translation to be between the first lumbar body and the intervertebral disc between the first and second lumbar vertebra . anteroposterior ( a ) and lateral ( b ) radiographs of the dorsolumbar spine showing retrospondyloptosis of l1 over l2 . ( c ) ct scan ( axial section ) showing a double vertebra sign ; reconstructed sagittal section ( d ) showing retrospondyloptosis of l1 over l2 . the t2-weighted mri section showing the l1l2 disc attached to l1 as seen in both axial ( e ) and saggital ( f ) cuts ; intervertebral disc and l1 vertebra visualized in the same transverse section open reduction and internal fixation of the dislocation was planned . a spinous process of l2 was grasped by a towel clip to facilitate reduction [ figure 3a and b ] . interspinous ligaments were removed and space was created for the passage of 18 gauge stainless steel wire loops around the laminae ; intraoperative neurological monitoring was not available . stabilization was done using 5-mm loop rectangle and sublaminar wires with two levels above and two below the affected segment . the choice of the implant was dictated by the socioeconomic status and the availability of the implant . the posterior spinal fusion at four segments was performed with decortication of posterior elements and allograft . postoperative radiographs revealed loss of reduction probably due to the breakage of the wire loop [ figure 3c and d ] . a plaster of paris spinal jacket was applied for 6 weeks followed by mobilization with taylor 's spinal brace . intraoperative photographs demonstrating the deformity in the spine and the reduction maneuver done ( a and b ) . postoperative anteroposterior and lateral radiographs of the dorsolumbar spine depicting the loss of preoperative reduction obtained probably due to the breakage of the wire loop ( c and d ) . lateral radiograph ( e ) and sagittal ct section of the dorsolumbar region ( f ) without any further deterioration in the anteroposterior vertebral alignment at 15-month follow - up showing remodeling of the spinal canal the patient showed neurological recovery in the form of paresthesias in both lower limbs after 1 week of surgery . gradually , the sensations improved further and at 9 weeks , the patient had complete sensory recovery . bladder and bowel sensations were regained at 2 weeks and in the following 2 weeks , the patient had complete control of bowel / bladder functions . the motor function was last to return with flickering movements of toes at 5 weeks . the functions improved gradually over the next 4 weeks in an asymmetrical fashion with more recovery on the left side . the power was grade 3 at the hip and knee and grade 2 at the ankle by 9 weeks after trauma [ figure 4 ] . the motor functions continued to improve and at 15 months , the patient is a community ambulator with a walking frame ( frenkel grade d ) . radiographs and ct scan of the sagittal section showed remodeling of the spinal canal [ figure 3e and f ] . clinical radiograph showing power at hip ( flexion ) and knee ( extension ) at fifteen months follow - up spinal injuries are relatively rare in children . fracture dislocations of the lumbar spine are further uncommon in comparison to cervical spine injuries . retrolisthesis of the lumbar spine is a rare injury with very few cases described in adults and only two in pediatric spines.56 one of the reported cases had retrolisthesis at the lumbosacral junction and the other a traumatic retrolisthesis of the first lumbar vertebra . the elasticity of the spinal column and ligament laxity predisposes the children to have a spinal cord injury in the absence of an obvious injury of the spinal column . however , the recovery of neurological deficits occurs with a higher incidence and neurological recovery appears over a delayed period of time.3 among the two cases of lumbar spine retrolisthesis reported in the literature , one had a complete neurological deficit which did not recover on follow up6 while the other had an incomplete deficit which recovered completely at 6-month follow up.5 it is difficult to comment on spinal shocks in injuries at the level of conus because the absence of the bulbocavernosus reflex can be a part of the traumatic conus medullaris syndrome itself as was in our case.7 fracture dislocations are very unstable injuries and require operative stabilization whether they are associated with a complete or an incomplete neurological injury . in patients with a complete neurological injury , the stabilization aids in rehabilitation . at least two levels above and two levels below should be instrumented.1 yazici et al.5 did a four - level instrumentation with a modified luque frame while verhelst et al.6 performed four - level posterior pedicle screw fixation . the child 's spine suffered a sudden jerk ( because of the cow pulling the rope ) which carried with it the second lumbar vertebra and the spine inferior to it [ figure 1 ] . the fixation was done using a loop rectangle with sublaminar wires at two vertebrae above and two vertebrae below the lesion . posterior fusion was done using allograft . however , there was loss of reduction in the postoperative period indicating that the number of levels instrumented with the rectangle was inadequate . with very little literature available regarding the options of treatment in such injuries , we wish to highlight that fixation required in such severe injuries should be more secure than is normally recommended for spinal injuries . the case reported by us is the second of its kind reported in the literature . the first reported by yazici et al . had an incomplete neurological injury which recovered completely at 6-month follow - up.5 in our case , the patient had a complete neurological deficit but demonstrated sequential recovery after surgery .

traumatic retrolisthesis of the first lumbar vertebra is a rare injury and only one case has been documented in the literature . we report a case of traumatic retrolisthesis of the first lumbar vertebra in a 7-year old child . he was injured after being dragged by a cow and presented with frenkel grade a paraplegia . his plain radiographs revealed complete retrolisthesis of the first lumbar vertebra over the second . the patient was treated surgically with open reduction and sublaminar wire loop rectangle fixation . the patient showed frankle grade d ( frankle grade ) neurological recovery in the postoperative period over a period of 15 months . this case is reported in view of rarity and mechanism of injury is described .

I C D

PMC4706962

within the last decades radiotelemetry became a widely used and highly recognized methodological in vivo approach for measuring a variety of behavioral and physiological parameters in conscious , unrestrained animals of various sizes , for instance , in the context of electroencephalogram ( eeg ) , electrocardiogram ( ecg ) , electromyogram ( emg ) , blood pressure , body core temperature , or activity measurements [ 17 ] . virtually any species can be analyzed using radiotelemetry from laboratory rodents such as mice and rats to cats , dogs , pigs , and primates [ 3 , 8 ] . even fish , reptiles , and amphibians are subject to radiotelemetric investigation . in the past , it has proven to be valuable in the characterisation of various transgenic animal models of human diseases , such as epilepsies , sleep disorders , and neurodegenerative and neuropsychiatric disorders [ 7 , 1012 ] . a number of methods collecting physiological data including biopotentials from mice and rats have been described . a special focus had been given on physical restraint methods , tethered systems , worn in jacket recorder systems , or nonimplanted radiotransmitters [ 13 , 14 ] . in general , telemetry recordings from conscious animals are thought to be superior to those from restrained or anesthetized animals , since they represent the normal physiological and behavioral state and are more predictive of the results that would be achieved in humans [ 1 , 3 ] . restraining methods can induce stress artefacts and severely influence basic physiological parameters , such as heart rate , blood pressure , body core temperature , and food intake . a classical restraining approach in eeg recording is the use of tethered systems [ 13 , 14 ] in which electrodes are connected to a miniature socket anchored to the skull and exposed for attachment of a cable . although tethered systems allow relatively free movement of the animal , one of its major disadvantages is the fact that it is still semirestraining and that there is an increased risk of infection at the electrode implantation site . although wireless radiotelemetry technology for monitoring larger laboratory animals has existed for some time , it has only recently become affordable , reliable , and relatively easy - to - use [ 10 , 15 , 16 ] , even for mice and rats . smaller transmitters are now commercially available ( e.g. , ta10eta - f20 , eta - f10 ; data sciences international , dsi , usa ) , which can be implanted in mice greater than 20 g ( ~10 weeks ) , so that animal size is no longer an absolute limitation . indeed , transmitter size continuously decreases and companies develop systems with even more enhanced features . implantable transmitter systems are capable of minimizing most disadvantages related to potential recording - associated stress by restraining systems . mice are able to show a complete repertoire of physiological behavior including resting , locomotor activity ( exploration ) , and sleep ( rem , slow - wave sleep ) [ 17 , 18 ] . currently , there is an intense discussion on how to limit the number of experimental animals in science and to reduce their suffering . clearly , animal experimentation and animal models of human and animal diseases are essential for our understanding of the pathophysiology and subsequent progress in therapy . furthermore , animal experiments are a paramount in drug research and development and substantially contribute to preclinical / toxicological studies in drug licensing . no alternatives are yet available to animal experimentation to understand the complex interdependence of different neuronal subsystems which would be otherwise impossible to be illuminated . at the same time , the 3r , that is , replacement , reduction , and refinement strategy in the eu and the usa , strongly encourages research into complementary and alternative methods . radiotelemetry is an example of a successful 3r strategy as it can reduce the number of experimental animals and their suffering compared to other techniques [ 3 , 19 , 20 ] . this is the first contiguous , detailed description of a successful , quick , and simple surgical procedure for s.c . pouch implantation of a radiofrequency transmitter in mice and rats and subsequent lead placement in both epidural and deep intracerebral positions . we discuss numerous commercial and self - assembled approaches in radiotelemetry and the pros and cons of different implantation techniques . in addition , we provide valuable information on equipment , fields of application , for example , spontaneous eeg recordings , circadian influence on central rhythmicity , sleep studies , that is , spontaneous sleep , pharmacologically induced sleep , sleep deprivation , and seizure analysis , and the combination of radiotelemetry with other techniques such as pump systems or auditory brainstem - evoked potential recordings . furthermore , examples of high - quality electrocorticograms ( ecogs ) as well as deep intracerebral recordings from mice and rats are displayed and special attention is paid to detecting , analyzing , and avoiding eeg artefacts . we also comment on frequent experimental pitfalls in radiotelemetry and strategies for reduction of trauma and optimization of pain management during postoperative recovery . quantitatively , most radiotelemetric studies are carried out in rodents , that is , mice and rats . transgenic mouse models that fulfil the requirements of homology , isomorphism , and predictability related to a specific human disease entity are a primary subject of radiotelemetric methodology , particularly in cardiovascular and neurological research . besides basic science , radiotelemetry has also become the method of choice in drug research and development including preclinical toxicological and mutagenicity studies . radiotelemetry has been applied in a vast majority of mammals including cats , dogs , pigs , and primates , but also in other animal taxa such as fish , amphibians , or reptiles [ 7 , 9 , 2123 ] . in this paper it should be noted that the miscellaneous mouse and rat strains available can severely differ in basic physiological and pathophysiological characteristics that have to be considered or evaluated prior to performing subsequent electrophysiological experiments [ 2426 ] . mouse strains , for example , can exhibit dramatic discrepancies in their responding to applicable dosages of specific anesthetics [ 27 , 28 ] necessitating pilot experiments to evaluate the most effective and secure anesthetic approach . in addition , it is known that mouse strains can display clear differences in sleep architecture and seizure susceptibility [ 29 , 30 ] , two fields that radiotelemetry is often applied to . another nonnegligible issue is gender : the oestrous cycle can strongly affect central rhythmicity , its circadian dependency , and sleep as well as seizure activity [ 3133 ] . in general , it seems recommendable to perform gender - specific analyses , or if financial and experimental capacity is limited to restrict to male mice as they are lacking confounding effects of the estrous cycle . it should also be tested whether a gender - specific ( re)analysis ( post hoc analysis ) of recorded data is possible . as electrophysiological in vivo studies using mice and rats are highly susceptible to a variety of confounders / disruptive factors , controllable parameters such as age and body weight should display small interindividual variability within a given study group . the same holds true for the housing conditions ( see below ) . within an animal facility , mice and rats are normally housed in filter - top cages or even better in individually ventilated cages . we first transfer mice from the animal facility to ventilated cabinets that are placed in special lab rooms exclusively dedicated to house implanted animals and their subsequent recording . we use ventilated cabinets , such as model 9av125p ( tecniplast , germany ) and uniprotect cabinet ( bioscape , germany ) . however , other cabinet models , for example , from scanbur ( denmark ) , alesco ( germany ) , and so forth , are also commercially available and enable temperature maintenance of 21 2c , 5060% relative humidity , and a conventional 12 h light / dark cycle . in general , they offer a wide range of configurations and complimentary equipment and can be run with positive and negative pressure . most importantly , ventilated cabinets reduce the time of exposure to microorganisms , dust , allergens , and other contaminants for animals as well as for personnel . as long as the ventilated cabinet is closed , such exposure is eliminated as all supply and exhaust air is cleaned through the highly efficient filters . as a result , the risk of infection of the animals inside the cabinet when the doors are opened is reduced . this risk can further be eliminated by transferring the cages to a laminar flow cabin for handling procedures . doors of ventilated cabinets are transparent made of polycarbonate with magnetic closure and can be covered with red transparency film so that the circadian rhythm remains undisturbed from external light . this is of central relevance as noise is a severe confounding element in almost any experimental setting using transgenic models [ 3538 ] . thus , standardized housing conditions are mandatory for actually all scientific questions , particularly for studies where circadian rhythmicity plays a major role , such as analysis of sleep architecture [ 39 , 40 ] . mice 12 weeks of age are used for most experimental settings ; this , of course , depends on the scientific questions to be addressed . prior to implantation ( see below ) mice are housed in groups of 3 - 4 in clear macrolon cages type ii ( 26.7 cm 20.7 cm 14.0 cm , area 410 cm ) with ad libitum access to drinking water and standard food pellets . clear macrolon cages type iii ( 42.5 cm 26.6 cm 18.5 cm , area 800 cm ) are used for rats . it should be noted that even the cage bottom structure , namely , wire - bottom cages or bottom bedding can eventually affect heart rate , body weight , and locomotor activity due to stress reaction . animals should not be separated and placed individually at this stage as isolation could also cause stress influencing experimental results . however , following radiotransmitter implantation , animals should be housed separately as the animals tend to manipulate wound stiches / sutures or metal clips ( see below ) . clearly , open housing conditions do not seem to be appropriate for most scientific questions due to environmental disturbances of mice and rats based on , for example , running lab equipment , air - conditioning , or working lab staff . open housing conditions are judged inappropriate for sleep studies , as they can result in a sleep deprivation setting rather than recording of physiological spontaneous sleep . whenever possible , mice and rats should be housed in separate rooms and separate ventilated cabinets . there should also be mouse and rat specific equipment so that neither mice nor rats can sense the presence of each other as this poses stress to the animals . finally , we would like to comment on the evolving issue of environmental enrichment and its relevance for radiotelemetry . environmental enrichment exposes laboratory animals to novelty and complexity through alterations in the physical and social environment , which can lead to enhanced sensory , cognitive , and physical stimulation . it should be noted that enriched housing strategies are a highly recommended practice by governing bodies regulating animal welfare . this is mainly due to the fact that enrichment causes benefits on rodents ' well - being based on the more naturalistic environment . in females , the impact of maternal enrichment on both offspring and maternal behavior and physiology also has to be considered . studies revealed that the environment has significant impact : for example , smaller testing chambers and a sensory attenuation cubicle around the chamber reduced spontaneous physical activity in mice . one major limitation of current experimental approaches is that most studies are conducted on unstimulated , sedentary animals that have unlimited access to food in the home cage . interestingly , environmental enrichment which enhances cognitive activity , sensory stimulation , and physical exercise has been shown to induce severe effects on central nervous system ( cns ) activity and behavior in both wild - type and transgenic rodent models , relative to standard - housed littermate controls [ 45 , 46 ] . in addition , environmental enrichment has beneficial effects outside the cns , such as a reduction in excess body fat . it has been widely recommended that positive study results , for example , therapeutic interventions , which are found to show promise in standard - housed preclinical animal models should be subsequently tested also under conditions of enhanced environmental enrichment . this strategy helps to identify therapeutics which continue to show efficacy in housing contexts of superior environmental construct validity . fascinatingly , it has been demonstrated that exposure to environmental enrichment significantly improves learning and memory in aged mice and reduces the abundance of 5-hydroxymethylcytosine , a gene - environment interaction mediated at the epigenetic level [ 47 , 48 ] . in general , it must be considered that recordings under enriched conditions might be less standardized and exhibit increased data variability . in many cases the administration of preoperative medication preoperative stress and pain can be relieved , for example , by benzodiazepines such as diazepam or midazolam , which serve as minor tranquilizers . all animal experimentation has to be performed according to the guidelines of the local council on animal care . in most cases , researchers further have to certify that all animal experimentation was carried out in accordance with superior legislation , for example , the european communities council directive of 24 november 1986 ( 86/609/eec ) or individual regional or national legislation . specific effort has to be made to minimize the number of animals used and their suffering . in this regard , radiotelemetry plays an important role in the 3r , that is , replacement , reduction , and refinement strategy . several telemetry systems , for example , dsi , tse systems ( usa ) , indus instruments ( usa ) , and millar inc . ( usa ) , are commercially available and slightly differ in their functional and technical specifications . the system that is finally chosen by the user should depend on lab specific requirements and current and future research goals and experiments . one should also consider whether the telemetry setup is compatible with other electrophysiological systems so that both might be connected or extended ( see connection of dsi system and the auditory recording system from tucker davis technologies ( tdt , usa ) below ) . amazingly , a literature search revealed a total of 29 publications claiming the development of a self - made radiotelemetry device of substantial novelty that best meets the requirements of the users [ 4977 ] . the most relevant advantage of self - made system as brain - machine interfaces pertains to their low costs . however , it has to be considered that one has to invest time and effort in developing self - made systems including complex analysis software later on . in most cases , a commercially available system is more expensive but might be more straightforward and is definitely time - saving . the telemetry system used in our lab is obtained from dsi . as a standard telemetry implant we use the one - channel physiotel transmitter ta10eta - f20 ( technical specification : 3.9 g , 1.9 cc , input voltage range 2.5 mv ; channel bandwidth ( b ) 1200 hz , nominal sampling rate ( f ) 1000 hz ( f = 5b ) , temperature operating range 3441c , warranted battery life 4 months , on - off mechanism magnetically actuated , dsi , figure 1 ) capable of measuring biopotentials , that is , eeg , ecg , emg , and physical activity and temperature . in addition m2-f20eet ( technical specifications : 3.9 g , 1.9 cc , input voltage range 1.25 mv , channel bandwidth ( b ) 150 hz , nominal sampling rate ( f ) 250 hz ( f = 5b ) , temperature operating range 34.0 to 41.0c , warranted battery life 1.5 month , on - off mechanism magnetically actuated ) . these two extra - small implants can be used in mice , hamsters , gerbils , and rats . it should be noted that a number of transmitter types are available that differ in the number of recording channels , their transmitter bandwidth and nominal sampling rate , physical dimensions , battery life span , and in situ battery exchangeability . finally , the size of the chosen transmitter determines the minimum animal weight . for the ta10eta - f20 and tl11 m2-f20-eet the minimum animal weight for an s.c . thus , specific care has to be taken in choosing the correct transmitter type for a specific scientific question to be addressed . there are multiple examples in the literature of transmitter type applications where transmitters were used out of the range of their technical specifications , for example , high - frequency gamma pseudo - recording by a transmitter that was not able to record this activity . at the worst the signal entities are transmitted to a receiver , for example , rpc-1 ( dsi ) , which picks up the telemetered data from the implant and forwards these data to a data exchange matrix ( dsi ) , the latter serving as a multiplexer . in the following a detailed description of radiofrequency transmitter implantation and electrode positioning will be provided . the total duration for the implantation and electrode placement clearly depends on the number of eeg deflections and whether recordings are to be performed from the cortex or deep intracerebral brain structures . well - trained scientists will manage to do surgery within 30 to 45 min . it turned feasible for beginners to practice implantation using euthanized mice or rats in order to speed up the implantation procedure and to get used to computerized stereotaxic implantation . a long - lasting implantation procedure , particularly long - lasting narcosis poses specific stress to the animal and leads to subsequent impairment of recovery with potential loss of the animal . small rodents are predisposed to hypothermia due to their high ratio of body surface ( mouse , 10.5 ( weight in g ) ; rats , 10.5 ( weight in g ) ) to body volume . thus , supplemental warmth has to be given during and after surgery using recirculating warm water blankets , electrical warming plates , heat lamps , forced warm air units , or pocket warmers to maintain body core temperature . in addition , to avoid corneal desiccation , eyes are to be covered with petroleum - based artificial tear ointment or dexpanthenol ( bepanthen , bayer , germany ) during the whole implantation period and early recovery until the blinking reflex is totally restored . further supplies include disinfectant , 2% glutaraldehyde solution ( sigma , germany ) , 0.3%/3% hydrogen peroxide solution ( sigma ) , sterile drapes , tapes , sterile scalpel blades with handle , sterile gloves , sterile pads , sterile 0.9% saline ( nacl ) or lactated ringer 's solution ( prewarmed for injection ) , sterile cotton tip applicators , and nonabsorbable surgical suture material ( 5 - 0/6 - 0 ) for lead ligation . the following instruments , for example , from fst ( germany ) , turned out to be helpful , that is , tissue forceps 1 2 teeth ( 12 cm length ) , standard pattern forceps ( 12 cm and 14.5 cm length ) , lexar baby scissors ( 10 cm ) , tungsten carbide iris scissors ( 11.5 cm ) , halsey micro needle holder ( 15.5 cm ) , iris scissors extra thin ( 10.5 cm ) , olsen - hegar needle holder - extra delicate , graefe forceps - curved , serrated , and graefe forceps - curved , with teeth and bulldog clamps . an elegant and fast way is using a heat - based surgical instrument sterilizer with glass beats ( fsn , germany ) . a binocular surgical magnification microscope and a cold light source , for example , kl2500 lcd ( schott , germany ) , for intense illumination via flexible or self - supporting the experimenter should wear a clean laboratory coat , a facemask , a head cover , and sterile gloves . clearly , optimal supplies and instruments vary from lab to lab and must meet lab - specific and institutional requirements . rats and particularly mice present an anesthetic challenge because of their small size and varied responses to anesthetic drugs within strains and between genders [ 27 , 28 , 79 ] . even animals of the same age can severely vary in body weight , fat distribution , and fat proportion , thus posing specific requirements to the dosage of injectable and volatile anesthetics due to differences in the distribution volume [ 80 , 81 ] . this paragraph provides basic information for injectable anesthesia and inhalant anesthesia for both mice and rats . a common and well - established anesthetic injection regime consists of a combination of esketaminhydrochloride ( ketanest , parke - davis / pfizer , germany , 100 mg / kg ) and xylazine hydrochloride ( rompun 2% bayer , germany , 10 mg / kg ) . whereas ketamine as a predominant glutamate receptor antagonist severely dampens brain activity causing loss of consciousness [ 82 , 83 ] although this dosage is usually sufficient for the implantation duration it turned out that different mouse and rat strains and in particular different transgenic mouse lines can exhibit totally different susceptibility towards ketamine / xylazine injection . some ( transgenic ) mouse lines display an extremely narrow therapeutic anesthetic window , probably related to primary or secondary effects of transgenesis leading to either insufficient narcosis or overdosage and death , the latter often based on xylazine mediated lethal respiratory depression or cardiac arrest . other injectable anesthetics in rodents include pentobarbital , ketamine / xylazine / acepromazine , ketamine / medetomidine , or tribromoethanol . according to our experience , volatile anesthetics can be helpful in strains and lines that display increased and critical sensitivity to injectable anesthetics . inhalation narcosis , for example , using isoflurane , sevoflurane , or halothane , can be controlled easily , particularly with regard to initiation , maintenance , and termination , that is , emergence / arousal of / from anesthesia [ 80 , 81 , 84 ] . the fast and almost immediate respiratory elimination of isoflurane ( respiratory rate : mouse 150220 breaths / min ; rat 70115 breaths / min ; minute ventilation : mouse 24 ml , rat 200 ml ) guarantees fast recovery . the delivery of volatile anesthetics to rodents is usually done using a silicone facemask since in small size rodents endotracheal intubation is more difficult to perform than in larger species . the advantage of endotracheal intubation is that the animal can be artificially ventilated in the event of cardiac or respiratory arrest . however , it should be noted that attempts to intubate rodents , even by experienced personnel , often traumatize the oropharynx , larynx , trachea , and at times the oesophagus . in general , endotracheal intubation and artificial respiration are not required for mouse and rat anesthesia . using volatile anesthetics has a number of advantages over injectable anesthetics , for example , minimal animal handling , large margin of safety , ease of anesthetic control , low cost of the anesthetic drug , and use of no controlled drugs . inhalation anesthesia has thus become the standard method of general anesthesia for mice and rats in biomedical research . drawbacks of this approach are mainly restricted to the high initial costs of equipment including isoflurane vaporizer , supply gas ( carbogen ) and its regulator , a flowmeter , an induction chamber , tubing , face masks , and an isoflurane absorber ( scavenging method ) . we use isoflurane baxter ( 100%v / v ) in a matrix tm vip 3000 calibrated vaporizer and a scavenger system from harvard apparatus ( usa ) . for initiation of isoflurane anesthesia there are various providers that offer a huge variety of transmitter types , not only for biopotential recordings such as eeg , ecg , and emg but also for pressure measurements ( blood pressure and respiratory pressure ) , and other applications . most transmitter types also provide information on body temperature ( normal mouse body core temperature is 36.538.0c and normal rat body core temperature is 37.038.0c ) and activity . with regard to body temperature subcutaneous pouch implantation of the radiofrequency transmitter ( see below ) results in slightly decreased temperature values . nevertheless , it provides valuable information particularly during the postsurgical recovery period . as the lifespan of a typical radiofrequency transmitter is limited it is recommended to make notes about the individual recording times so that one is aware when the battery is going to cease . it should also be considered that many radiofrequency transmitters are turned on via a magnetic switch ; thus , even if they are not used the storage time is limited . as outlined in section 2.3 the technical specifications of each transmitter type also have to be considered carefully and must fit the experimental and analytical tasks . special attention has to be paid to the transmitter bandwidth ( b ) and nominal sampling rate ( f ) . following proper anesthesia it is recommendable to remove body hair from the scalp using a shaver ( aesculap , eickemeyer , germany ) . the shaved areas can be scrubbed with mercurochrome ( merbromin , germany ) or 70% ethanol as disinfectant . it should be noted that transgenic mice in particular might not tolerate disinfectants well and might exhibit skin irritation or inflammation of variable degree . depilation cream should not be applied due to skin irritation and possible wound healing impairments later on . incisions sites can also be cleaned with disinfectant betaisodona standard solution ( mundipharma , germany ) . the animal will be placed in a sterile field on a warming surgery table to avoid hypothermia of the animal during the surgical procedure . for good fixation usage of an operation microscope might be helpful during the whole procedure . using surgical scissors a 2 cm midline incision of the scalp from the forehead to the neck once the pouch is established , the transmitter is placed subcutaneously inside the pocket with the sensing leads oriented cranially . pouch on the back is a potential reduction in signal strength ( e.g. , monitored by the dataquest a.r.t . software , dsi ) due to the increased distance of the transmitter from the receiver plate . therefore , the transmitter is implanted subcutaneously at the flank close to the ventral abdominal region and fixed at the skin using the transmitter 's suture tab and a single stich to avoid postoperative movement of the transmitter . in this position , a reduction in signal strength is not observed compared to the i.p . implantation procedure . though temperature values are below body core values , the recording allows for monitoring the temporal development of s.c . temperature . undoubtedly , the s.c . pouch implantation of the radiofrequency transmitter is the most recommendable one . the intraperitoneal implantation starts with a 1.52 cm midline abdominal incision , that is , laparotomy . the abdominal skin and wall are opened and both incision edges are held aside using a 5 mm wound retractors together with elastomers , fixators , and a base frame ( fst , germany ) . next , the transmitter is placed into the peritoneal cavity on top of the gastrointestinal tract . anatomical forceps are to be preferred to surgical forceps , as they produce less damage to the tissue , particularly when manipulating the bowel . in rare cases , local bleeding from the abdominal wound edges can occur which can easily be staunched using a thermocauter ( heiland , germany ) . next , both leads are tunneled through the abdominal wall at the cranial part of the incision using a 14-gauge needle . a trocar together with a plastic sleeve can be tunnelled subcutaneously along the lateral thoracic wall to the intended position at the neck , where a second skin incision was made before . the trocar can be withdrawn from the sleeve and the leads are tunnelled through it . alternatively , a thin trocar with a short silicon tubing attached at its tip can be used to tunnel the leads subcutaneously to the neck . in this case , the sensing electrodes can be tied mechanically to the silicon tubing or by attaching them with glue . leads are secured on the underlying muscle layer using a nonabsorbable interrupted stay suture ( ethilonii , 4 - 0 , m-2 , ethicon , germany ) . if not tacked , the intrinsic elasticity of the electrodes will make implantation of the electrodes more difficult later . the transmitter is fixed in the peritoneal cavity using its suture tab to avoid intra - abdominal movement and possible impingement . finally , the abdominal wall is closed using nonabsorbable suture material ( ethilon ii , 4 - 0 , m-2 , ethicon , germany ) . however , the ventral skin incision is closed using wound clips ( michel , 7.5 1.75 mm , heiland , germany ) , as mice tend to manipulate and bite sutures . wound generally , the intra - abdominal implantation is much more difficult to perform and takes longer time even for an experienced surgeon than the s.c . approach and in addition poses specific risks of pressure necrosis of the gut or even blockage of the stomach or bladder . implantation can size - dependently depress activity , reduce voluntary running , and lead to inflammation of the diaphragm , large intestine , and duodenum . implantation it has also been reported that increased intra - abdominal pressure can reduce the venous blood flow back to the heart and increase vagal activity . 2004 analyzed the effects of body- ( b- ) to - transmitter ( t ) ( b / t ) size ratio on growth and circadian rhythmicity . a large b / t ratio was associated with a minimization of the adverse effects of i.p . it becomes obvious that in small experimental animals such as mice implanted transmitters can alter activity , behavior , circadian rhythmicity , and sleep in a strain dependent manner . we use two special , fully equipped stereotaxic setups for mice and rats ( figure 2 ) from stoelting ( usa ) and neurostar ( germany ) . the stereotaxic device includes the stereotaxic frame with ear bars and tooth clamp size - adapted for mice and rats , respectively . in addition , the stereotaxic frame also includes a gas anesthetic mask ( stoelting , usa ) with connections to the isoflurane evaporator and the isoflurane scavenger module . the system serves as a computerized 3d stereotaxic setup with a specific software ( stereodrive ; neurostar , germany ) including a user interface for navigation and 3d atlas , allowing axial , coronal , and sagittal views . the setup enables reproducible and precise electrode placement according to stereotaxic coordinates . as in our case the high - speed precision drill is mounted on the vertical arm of the stereotaxic frame . in some cases , researchers mount pencils or pens on the vertical arm leaving a tiny mark at the coordinates of choice on top of the skull . afterwards the animal is removed from the stereotaxic frame and holes are drilled manually . clearly , stereotaxic drilling is much more precise and mandatory in small rodents like mice . manual handling of a high - speed drill might be possible in rats but is not precise and dangerous . one should be aware that mice and rats severely differ in the neurocranial bone thickness . the thickness of the murine cranial bones depends strongly on the localisation ( os frontale : midline section : 320390 m , lateral section : 300430 m ; os parietale : midline section : 210250 m , lateral section : 200210 m ; os occipitale : midline section : 600730 m , lateral section : 380420 m ) . damage of the inner ear can be avoided by covering ear bars with cotton balls . for some experimental purposes , like screening for audiogenic seizures or seizure susceptibility , this is of especially high importance . this avoids a tonic applanation of the skull , which may result in a sudden breakthrough of the drill head and potential damage mainly in the cortical field . for craniotomy , we use the zeppelin neurosurgical high - speed precisionmotor drill system zmm-100e from adeor medical technologies ( germany ) . a midline skin incision , 10 mm on the head and 510 mm down the neck , is made and the s.c . the superficial thin tissue layer of the skull is pretreated with 0.3% h2o2 for the mouse skull and 3% for the rat skull . this pretreatment removes any tissue from the skull and assures strong contact of the glass ionomer cement ( kent dental , kent express ltd . , uk ) which is used later on . it also accentuates the sutures as well as bregma and lambda landmarks of the skull , which are essential for precise stereotaxic lead placement ( figures 3(a)3(d ) ) . it should be noted that this procedure can produce severe oxidative damage to the surrounding tissue . as pointed out before thus , care must be taken not to incubate h2o2 for too long as this can lead to penetration of h2o2 through the skull and oxidative damage of the underlying cortex . following the bleaching of the skull , burred holes are drilled at the coordinates of choice using a high - speed drill with typical drill head diameter of 0.30.5 mm . the diameter of the holes might be smaller depending on the electrode diameter . as a general rule , the smaller the diameter , the less damage is produced . haemorrhage reflecting intraosseous bleeding from the cut edge of the skull can be easily staunched with a cotton tampon and is self - limiting in general . fatal bleeding was never observed as positioning of the electrodes should differ from the localisation of dural sinuses . however , once a dural sinus has been hit , the survival rate of the animals is dramatically reduced . the surface electrodes are shortly bend at the tip and placed directly on the dura mater for epidural lead placement , for example , in a bipolar deflection or ( pseudo ) unipolar deflection . as an example , a surface motor cortex m1/m2 electrode is positioned at : cranial 1 mm , lateral 1.5 mm ( left hemisphere ) . an epidural reference electrode can be placed on the cerebellar cortex , for example , bregma 6 mm , lateral of bregma 1 mm ( left hemisphere ) or bregma 6 mm , and lateral of bregma 1 mm ( right hemisphere ) , respectively ( figure 3(d ) ) , as the cerebellum serves electroencephalographically as a silent region . it is often claimed in the literature that this procedure can be carried out without damage of the dura . next , electrodes were fixed with glass ionomer cement ( kent dental , kent express ltd . , uk ) , which is extremely hard and gives strong adhesion to the underlying neurocranium . after the cement has dried ( 5 min ) , the scalp is closed using over - and - over sutures with nonabsorbable 6 - 0 suture material ( ethilon polyamid , ethicon , germany ) ( figure 1(d ) ) . following implantation and fixation of the tip of the sensing lead using glass ionomer cement , no severe inflammatory reactions either of the cerebrum , the cerebellum , the meninges , or skin have been observed . in rare cases , increased pressure caused by the underlying cement resulted in skin necrosis during the observation period of 46 weeks ; thus , an optimum amount of cement should be used to guarantee best electrode fixation and lowest s.c . pressure . it should be noted that it is also possible to place cortical screws and attach the sensing leads of the radiofrequency transmitter afterwards . the scalp and skull of the animal are pretreated as described above . for deep electrode implantation holes special attention has to be paid to the electrode material characteristics and its connection to the transmitters sensing leads . we use stainless steel or tungsten electrodes which are parylene coated ( 14.5 cm original length , 250 m diameter , 50100 kohm tip impedance , epoxylite insulation metal microelectrodes , fhc inc . , usa ) . first , the electrodes have to be shortened to the required length . an additional part of the electrode is required to connect it to the stainless steel helix of the transmitter lead . this square angle is important so that the transmitter lead can divert horizontally from the skull towards the transmitter . it reduces pressure on the skin and helps to fixate the electrode and the lead to the skull . next , the stainless steel helix of the transmitter lead is exposed by removing a short section of the silicone isolation at the tip of the transmitter lead using a sterile scalpel blade . by rewiring the lead of the transmitter to the deep brain electrode , a suitable connection of both components is ensured . in general , soldering is not recommended as this can result in severe noise in the recordings . once the solid electrode is connected to the transmitter lead , the electrode can be attached to the vertical arm of the stereotaxic device to lower it to the target brain region . when the target position is reached the electrodes the ca1 region is one example of a deep electrode target . in order to reach this hippocampal region of interest , ca1 holes are drilled and positioned at the following coordinates referring to the bregma : caudal 2 mm , lateral 1.5 mm ( right hemisphere ) , and dorsoventral ( depth ) 2 mm ( see figures 3(d)3(f ) ) . an epidural reference electrode can be placed on the cerebellar cortex , for example , bregma 6 mm , lateral of bregma 1 mm ( left or right hemisphere ) ( figure 3(d ) ) . once the electrodes are placed and attached to the sensing leads , the scalp is closed using over and over sutures ( ethilon , 6 - 0 ) . in addition , there is intraindividual variability in the anatomical localisation of the major craniometric landmarks bregma and lambda [ 88 , 89 ] . the differential electrode is placed at the localisation of choice whereas the reference electrode can be placed on a silent region of the brain , for example , the cerebellum . special care has to be taken when using bipolar deflections as these can result in extinction of recorded eeg activity when activity of high bilateral synchronicity is present . this holds true for spontaneous absence like activity with characteristic spike - wave discharges or spike - wave discharge activity that has been induced pharmacologically , for example , via gbl ( ghb ) [ 90 , 91 ] . after implantation , animals are placed back into their home cage and within the first 3 - 4 days after surgery body core temperature is maintained using a thermal lamp , warming plate , or heating pad . as the ratio of body surface to body weight is high in small rodents ( 36 cm/20 g in mice ) , external temperature support was not removed until mice were able to maintain physiological body core temperature themselves . this can be monitored easily using the transmitter temperature sensing function if technically available . for pain management several injectable analgesics are available : narcotic opioids , opioid agonists / antagonists , 2 agonists , local anesthesia , and nonsteroidal anti - inflammatory drugs ( nsaid ) , that is , buprenorphine , butorphanol , tramadol , flunixin , ketoprofen , metamizole , meloxicam , carprofen , acetaminophen , and lidocaine [ 80 , 81 ] . for example , metamizole - sodium ( novaminsulfon - ratiopharm 1 , ratiopharm , germany ) , which is used for postoperative pain management at 100 mg / kg body weight for four days after surgery can be administered intraperitoneally in ringer solution or 0.9% nacl . one of the disadvantages of metamizole is that it has to be administered repetitively due to its short half - life . thus , we normally use 50 mg / ml carprofen ( rimadyl , pfizer , germany ) diluted in ringer solution or 0.9% nacl for postoperative analgesia . food consumption ( ~15 g/100 g / d ; ~5 g/24 h ) and water consumption ( ~15 ml/100 g / d ; ~5 ml/24 h ) should be monitored carefully . no matter which transmitter implantation site is chosen , the maximum loss in body weight is around days 4 - 5 after surgery . in addition , the animals should be closely monitored for the return of their normal postures and behaviors . systemic administration of antibiotics such as enrofloxacin or trimethoprim - sulphonamides [ 80 , 81 ] is often recommended but is not performed in our studies and no inflammatory signs of meningitis or encephalitis at the sites of implantations were detected after surgery or at postmortem examination of any mouse implanted . in general , animals recover quickly and are given 10 to 14 additional days to fully recover before starting eeg recordings for further analysis . this recovery period is based on the observation that 10 days after surgery no difference in physiological parameters between transmitter implanted , nonimplanted , and sham - operated animals could be detected . it should be noted however that special experimental settings , for example , behavioral tasks , demand for longer recovery periods , especially when performing i.p . it is highly recommendable to use an animal implantation and documentation sheet to document animal recovery . a maximum reduction in body weight was observed around days 4 - 5 after surgery followed by a slight , but steady increase of weight during a 1014-day recovery period after which it remained constant . the time pattern of recovery is in line with the postoperative development of body weight following i.p . implantation for ecg recording . to verify the correct electrode placement targeting , for example , the ca1 region , brains are extirpated postmortem and fixed in 4% paraformaldehyde . afterwards , brains are cut to 60 m slices using a vibroslice tissue cutter ems 5000-mz ( campden instruments limited , uk ) and hematoxylin or nissl stained for visualization of the branch canal . thus , it is strongly recommended not to cut the sensing leads to the minimum length right from the beginning . instead , the flexible , full - length leads should be placed subcutaneously as they do not impair the animal . special care has to be taken not to impinge or damage the sensing lead insolation . using a biodetergent , for example , neodisher ( medizym , germany ) , for removing tissue debris ( 24 h at rt ) and 2% glutaraldehyde ( roth , germany ) for resterilisation ( at least 2 h at rt ) , the transmitters are prepared for reimplantation . for removing all traces of chemical sterilant , especially glutaraldehyde , the transmitters are temporarily stored overnight in sterile saline or water . the lifespan of the radiofrequency transmitter battery restricts the time of continuous recordings . in some systems , the battery and the transmitting unit of the radiofrequency transmitter are connected via a socket meaning that once the battery is discharged it can be replaced by a new one following a small cutaneous longitudinal incision . in another system an external recharge via , high - quality eegs can be reliably recorded for up to 8 weeks at least . ossification from the drilled holes has the capability to lift up the electrodes with time resulting in emg and ecg contamination , thus limiting optimum recording duration to a few months . eeg recording systems making use of a tethering system require special connectors to attach the sensing leads to the implanted wire and might thereby introduce significant noise to the system . radiotelemetry systems using screw electrodes or isolated stainless steel wires for implantation might therefore exhibit similar issues . in our recordings following implantation the signal - to - noise ratio ranged from 100 nv to 1 v . as the eeg signal is weak , it is highly susceptible towards ecg and emg contamination , both of which can evolve into a severe problem ( figure 8) . this often occurs if the sensing lead is not properly secured or isolated from extracranial tissues by glass ionomer cement or if the silicone insulation of the sensing leads is damaged during the implantation procedure or later on due to manipulation by the animal . ecg contamination can be easily identified due to its very regular pattern ( r - spikes , i.e. , heart rate in mice 300650 beats / min ( 510.8 hz ) and 250370 beats / min ( 46.2 hz ) in rats . typical respiratory rates in mice are 150220 breaths / min ( 2.53.7 hz ) and 70115 breaths / min ( 1.21.9 hz ) in rats . ten days after radiotransmitter implantation , simultaneous video - eeg recordings from the motor cortex ( m1 ) and the hippocampal ca1 region are performed for long - term recordings , for example , 48 h in all animals from both study groups using dataquest art 32 bit operating system with dataquest a.r.t . 4.31 gold version , data exchange matrix and a video network camera system ( axis p1343 video camera including day / night capability , sweden ) . different types of recordings can be performed depending on the scientific question to be addressed . thus , recordings should either be done at the same day- ( or night- ) time or be preceded by an entire circadian analysis of rhythmicity . sleep analysis does not only include spontaneous ( at least 24 h ) recordings , but also include pharmacologically induced sleep , for example , via urethane and sleep deprivation studies ( figure 4 ) . furthermore , a predominant field of application is seizure analysis and recording of evoked potentials . in many experimental settings one potential approach would be daily drug application via s.c . or i.p . injection . osmotic pumps are prepared 24 h in advance under sterile conditions and stored in saline solution ( 0.9% ) at 37c until implantation . animals are anesthetized using isoflurane ( 2 - 3% ) and a small incision is made on the back of the animal . for rats , small s.c . pouches are made on both sides of the cut edge adding up to the size of the pump plus additional space for movability . for mice it is recommendable to make a small cutaneous incision between the shoulders so that the animal can not reach the stiches with its teeth . the pouch is flushed with sterile saline solution , the osmotic pump placed inside , and the cut sewn with a few stitches . if the animal has the chance to manipulate the stiches with its teeth , wound clips should be used instead . as for radiofrequency transmitter implantation , rimadyl ( 5 mg / kg ) is administered for pain relief . besides osmotic pumps , mechanical pumps are also available , for example , smp-200 from iprecio ( usa ) . auditory evoked potentials ( aeps ) from specific brain areas , one can use an auditory stimulation unit , for example , a rz6 multi i / o processor system of tdt and the biosigrz software ( tdt ) . the stimulus presentation and equipment control are coordinated using the rz6 system and biosigrz software ( tdt ) ( figure 5 ) . the stimulus is presented via a loudspeaker ( mf1 multi - function speaker , tdt ) placed centrally above the cage . a second output signal is transferred to an oscilloscope ( dpo 3012 , tektronix , inc . , the oscilloscope is used as a trigger unit to exhibit a rectangular signal each time an acoustic stimulus is presented . the trigger signal is transferred to an a / d converter ( c11v ; dsi ) and forwarded to the dsi data exchange matrix via a local area network type cable . the data exchange matrix multiplexes data signals from the active receiver ( rpc-1 , dsi ) and the a / d converter combines both signals and sends the signal stream to the data acquisition system ( dataquest a.r.t . all auditory recordings should be performed in a sound - attenuated recording chamber placed inside a faraday cage for electrical isolation . illustrating all potential experimental combinations with eeg radiotelemetry would be beyond the scope of this presentation . however , we want to point out that various other valuable settings are possible , including , for example , the combination with behavior and cognition test regimes or intracerebroventricular cannulation and application settings . it is generally recommendable to perform long - term baseline recordings following a 1014-day recovery period . baseline recordings , for example , allow for detection of potential implantation - related artifacts , the description of spontaneous time - frequency characteristics of surface or deep eeg recordings , the circadian influence on rhythmicity , and spontaneous or induced seizure activity [ 9597 ] . epilepsy models include acute and chronic pharmacological models as well as genetic models of epilepsy . in the following some pharmacological seizure models administration of r / s - baclofen ( sigma , germany ) , for example , at 20 mg / kg and bicuculline methobromide ( bmb , sigma , germany ) , for example , at 10 mg / kg . both substances , baclofen , acting pharmacodynamically as a gaba(b ) receptor agonist , as well as bicuculline , which exerts antagonistic effects on gaba(a ) receptors , can provoke spikes and spike waves in the ecog based on the induction and maintenance of hypersynchronisation processes within the thalamocortical - corticothalamic circuitry . systemic administration of 4-aminopyridine ( 4-ap ) , for example , at a dosage of 10 mg / kg i.p . or pentylenetetrazole ( ptz ) , in mice can provoke generalized tonic - clonic seizures . shortly after the injection , mice are hypoactive , followed by a mild , partial myoclonus mainly affecting the face , for example , vibrissal twitching , the head , and/or forelimbs . this state can evolve into a generalised clonus characterised by loss of upright posture , whole body clonus involving all four limbs , jumping , wild running , and , finally , a tonic extension of the hindlimbs . figure 6(a ) illustrates a characteristic recording following 4-ap administration ( 10 mg / kg ) . at early stages of seizure development ( myoclonus of the head , face , and forelimbs ) the eeg contains only marginal emg contamination . following sporadic spike activity ( ) the generalised clonus initiates with a characteristic deflection ( 1 ) and a subsequent episode of continuous spike activity . although this period is characterised by massive muscle activity due to the whole body clonus , spike activity of the brain can clearly be determined and emg contamination is surprisingly low , indicating that the implantation procedure is capable of exhibiting electroencephalographic signals selectively even under generalised seizure conditions , when eeg signals might be expected to be masked by emg artefacts . the first generalised clonus is followed by a typical short postictal depression ( 2 - 3 ) which is followed by a second generalized clonus which ended in a tonic extension of hindlimbs ( 4 ) . clearly , dose - effectiveness studies should be performed to identify the optimum dosage for a specific scientific question . in particular , optimum dosages can severely differ between genders , strains , and transgenic mouse lines . control recordings should be performed prior to injection and control groups that have been administered a drug - free vehicle also have to be included . hippocampal seizure activity can be induced by kainic acid ( ka ) or n - methyl - d - aspartate ( nmda , sigma ) . the non - nmda receptor agonist ka is generally administered intraperitoneally at a dose of 1030 mg / kg . as outlined above , pentylenetetrazole and 4-ap r / s - baclofen and -hydroxybutyrolactone are normally used to induce nonconvulsive absence like activity in mice . hippocampal or complex partial seizures represent a third important seizure subgroup that can be acutely induced by various glutamate receptor agonists . administration of ka , for example , causes a well - characterized hippocampal seizure syndrome ( figure 6(d ) ) that can be analyzed according to a slightly modified seizure score from : stage 1 , no behavioral change ; stage 2 , facial clonus ; stage 3 , forelimb clonus ; stage 4 , rearing ; stage 5 , falling ; stage 6 , status epilepticus ; stage 7a , jumping , tonic seizure > 30 s ; stage 7b , jumping , tonic seizure 3060 s ; stage 8 , maximum generalized seizure activity , respiratory arrest , and death . it should be noted that various seizure scores have been described in the literature that slightly differ from each other . besides ka administration of nmda at a dose of 150 mg / kg . in nmda treated mice , seizures developed through a sequence of paroxysmal scratching , hypermotility and circling , tonic - clonic convulsions , and , occasionally , death . the following semiquantitative scale can be used for the examination of seizure severity slightly modified according to : stage 0 , no response ; stage 1 , excessive grooming and paroxysmal scratching ; stage 2 , mild hypermotility ; stage 3 , extensive hypermotility and circling ; stage 4 , forepaw clonus and tail hypertonus ; stage 5 , generalized tonic - clonic convulsions ; stage 6 , status epilepticus ; stage 7 , death . one of the most popular chronic seizure models is the pilocarpine model of mesial temporal lobe epilepsy ( mtle ) in rats . various pilocarpine administration regimes have been described in the literature that lead to a more or less stable and life - long hippocampal seizure phenotype . figure 7 illustrates examples of cortical and hippocampal eegs in the rat pilocarpine model ( see also supplementary video in supplementary material available online at http://dx.doi.org/10.1155/2016/8213878 ) . it should be noted that eeg artefacts can sometimes mimic ictiform discharges ( figure 8) . thus , special attention has to be paid to reduce ecg , emg , and externally induced eeg signal disturbance . animal models provide a means to investigate the fundamental mechanisms of abnormal electrical discharge that occur during seizure activity . understanding the pathogenesis of seizures and epilepsy , therapy development has greatly benefited from these models . however , it should be noted that reductionist approaches also exist , making use of models in drosophila melanogaster , caenorhabditis elegans , and zebrafish danio rerio . studies of seizures / epilepsy in mutant mice provide a framework for understanding the critical features of the brain that regulate excitability and hyperexcitability . these , and as yet undiscovered , mouse mutants will continue to serve as the foundation for basic epilepsy research when displaying the principles of homology , isomorphism , and predictability regarding human diseases . a considerable level of effort is still devoted to the discussion of animal models of epilepsy and consensus on which models best reflect the human condition is difficult to achieve . clearly , emerging models are pushing our perspective towards novel genetic , cellular , and anatomical hypotheses for the etiopathogenesis of epilepsies . about 100 genetically modified mouse models were reported to exhibit an epileptic phenotype in the current literature . in the past decade , the number of genetically modified mice exhibiting seizures or reduced sensitivity to convulsant manipulations has risen exponentially , with no clear endpoint in sight . to a large extend , these mutant mice feature a general inactivation or knockout of a specific gene and confirm much of what has already been known about the sophisticated excitation - inhibition balance in the cns . new genetically engineered mice provide additional insights into cellular mechanisms underlying seizure generation , genetic interactions that exacerbate seizure phenotype , and neurodevelopmental influences . from these mice candidate genes of ion channels , postsynaptic receptors , and genes required for neuronal migration eeg radiotelemetry has been one of the most effective techniques in the in vivo characterization of these mouse models as it does not only allow for screening for spontaneous seizure activity but also following pharmacological provocation . in addition , eeg radiotelemetry enables the recording under special experimental , that is , behavioral settings under which seizures might occur and thus proofs to be a most valuable tool in the field . analytical approaches in eeg radiotelemetry are a sophisticated field and are beyond the scope of this presentation . similar to self - made transmitter systems many scientists have written their own home - made programs to analyze eeg data . it is also possible to analyze data collected with self - made recording systems using well - established analysis software depending on the data format . most commercially available telemetry systems offer specific modules for frequency methods like fast fourier transformation ( fft ) analysis , automated seizure detection , or automated sleep analysis . telemetry enables researches to acquire huge amounts of eeg data and automated analysis systems provide an easy and elegant way to analyze these data sets efficiently . however , special attention has to be paid to the configuration settings of automated analysis systems and it seems recommendable to compare software based analysis with visual / manual analyses of eeg data . although tools for fft - based eeg analysis are generally provided , special scientific questions might require the development of new analytical tools . it illustrates the establishment of a novel analytical tool to detect highly synchronous hippocampal theta oscillations using deep intrahippocampal ca1 recordings following administration of urethane . data segments with a length of 30 min each are extracted from the preinjection phase and the posturethane phase . data segments are analyzed using complex morlet wavelets to calculate both frequency and amplitude of oscillations . the complex morlet wavelet is defined by (x ) = ( b)(1/2)exp(2icx)exp(x / b ) , where b is the bandwidth parameter , c the center frequency , and i the imaginary unit . this wavelet or similar ones have often been applied in the literature to study eeg data , as they guarantee optimal resolution in both frequency and time . in our case , the bandwidth parameter and center frequency are both set to 3 in order to particularly weight the frequency resolution to distinguish frequency differences on the 0.1 hz level , but not to neglect a sufficient time resolution . eeg data are analyzed in the frequency range of 0.212 hz with a step size of 0.1 hz , thus including the typical delta , theta , and alpha frequency ranges . in order to apply the wavelet technique for extraction of theta - oscillatory segments this theta detection method imitates the standard visual inspection of theta oscillations and is mainly based on a complex elaboration of the frequency architecture of theta activity . the theta oscillation detection criterion is equal to the quotient of the maximum amplitude in the theta frequency range ( 3.58.5 hz ) and the maximum amplitude in the upper delta frequency range ( 23.4 hz ) for a time window of 2.5 s. with a ratio above 1.5 , that is , the maximum theta amplitude is at least 50% higher than the amplitude in the upper delta band , the related 2.5 s eeg segment is classified as a theta oscillatory epoch . an interval of 2.5 s represents a minimal duration for a theta oscillation and prevents from false negative detections of certain noisy epochs and lies within the range definitions of . the upper delta frequency range serves as a control frequency band because physiological relevant delta activity appears during nontheta epochs , for example , during slow - wave sleep , which is highly damped during theta activity . eeg radiotelemetry has turned out to be a most valuable tool in basic research and drug research and development , particularly in neuropsychiatric , neurodegenerative , and epilepsy research . eeg radiotelemetry is per se a most sensitive tool harboring tremendous analytical potentials . as an example , complex analysis of eeg time - frequency patterns might result in predictive biomarkers in neurodegenerative diseases in the future . however , it is always mandatory to pay specific attention to potential pitfalls of this sophisticated technique and to factors that might influence radiotelemetric results and which might cause sporadic or systematic bias in telemetry data .

eeg radiotelemetry plays an important role in the neurological characterization of transgenic mouse models of neuropsychiatric and neurodegenerative diseases as well as epilepsies providing valuable insights into underlying pathophysiological mechanisms and thereby facilitating the development of new translational approaches . we elaborate on the major advantages of nonrestraining eeg radiotelemetry in contrast to restraining procedures such as tethered systems or jacket systems containing recorders . whereas a main disadvantage of the latter is their unphysiological , restraining character , telemetric eeg recording overcomes these disadvantages . it allows precise and highly sensitive measurement under various physiological and pathophysiological conditions . here we present a detailed description of a straightforward successful , quick , and efficient technique for intraperitoneal as well as subcutaneous pouch implantation of a standard radiofrequency transmitter in mice and rats . we further present computerized 3d - stereotaxic placement of both epidural and deep intracerebral electrodes . preoperative preparation of mice and rats , suitable anaesthesia , and postoperative treatment and pain management are described in detail . a special focus is on fields of application , technical and experimental pitfalls , and technical connections of commercially available radiotelemetry systems with other electrophysiological setups .

1. Introduction 2. Materials and Methods 3. EEG Radiotelemetry: Practical Application 4. Analysis of Telemetric EEG Recordings 5. Concluding Remarks

PMC5298012

with the recent advances in climate modeling and computational capabilities , general circulation models ( gcms ) can produce simulations of the future climate at increasing spatial resolutions . this leads to representation of weatherscale phenomena , which produce highly structured gcm output over local features ( e.g. , orography ) . the resolution of weatherscale features in climate models represents a significant advance in the scientific foundation of climate simulations and also improves the potential of models to be relevant to climatechange adaptation applications . the resolution of weatherscale features in climate models helps to link weather science and climate science and motivates the development of new gcm evaluation techniques . the comparison of modeled fields to observations and/or reanalysis products can be regarded as the primary method to analyze the realism of models by identification of biases and variability and descriptions of the uncertainties . widely used quantification methods for such comparison are visual analysis of select parameters of model simulations , and summary statistics ( e.g. , root mean squared error ) calculated on a grid pointbygrid point basis [ endris et al . , 2013 ; haslinger et al . , 2013 ; landgren et al . , 2014 ; martynov et al . , 2013 ; sillmann et al . , 2013 ; sylla et al . , such approaches are important since they are informative of the temporal and geographical distribution of model characteristics . however , they fail to address the source of a particular bias within a model , which is crucial to production of more realistic simulations of the climate system . the main components of a gcm are the dynamical core [ williamson , 2007 ] , which solves the governing fluid and thermodynamic equations on resolved scales , and the physical parameterizations , which represent subgridscale processes and other processes not included in the dynamical core such as radiative transfer [ rood , 2011 ] . these components can be composed of several subcomponents ( e.g. , cloud macro and microphysics , tracer transport , etc . ) communicating with each other through couplers or mediators . the complexity of gcm structure contributes to the difficulty of determining the causality of model errors . model errors can be local , produced by a particular component or by the combination of multiple components . analysis methods using summary statistics likely average out such errors , losing knowledge on causality . this study aims to address this problem by adopting an objectbased approach to quantify the bias , describe the uncertainty , and identify their sources within a gcm ( explained in the next section ) . by the application of a classification tree algorithm , we aim to partition the precipitation field to isolate the different types of precipitation features ( as identified by yorgun and rood ) and analyze them separately with the expectation that different features ( originating from different underlying processes ) will exhibit different statistical characteristics . additionally , with the partitioning of the precipitation field , we are also motivated by differences between the directly forced precipitation and the precipitation that is produced internally , their characteristics , and organization at intermediate and small scales ( i.e. , crossing the scales from grid to fully resolved ) . capturing and analyzing these differences is valuable in terms of pinpointing the shortcomings of different models and their causes . we focus on a comparative study of the eulerian spectral and the finite volume ( fv ) [ lin and rood , 1996 , 1997 ] dynamical core components of the national center for atmospheric research ( ncar ) community atmosphere model ( cam ) version 5.0 [ neale et al . , [ yorgun and rood , 2014 , 2015 ] , we focused on the monthly mean simulations and quantified the differences between the two dynamical cores . in this study , we add the time dimension to the analysis by analyzing daily simulations . it should be noted that this is not a model verification study , but rather a modeltomodel comparison in order to quantify the differences between cam eulerian and cam fv dynamical cores in their simulation of the precipitation over sierra nevada [ yorgun and rood , 2014 ] . an object is defined as a coherent system with an associated set of measurable parameters [ douglass , 2000 ] . in an objectbased model evaluation approach , features such as fronts , rain bands , clouds in deep convection , etc . are defined and isolated as objects [ posselt et al . , 2012 ; xu et al . , process and objectbased evaluation preserves information in quantitative analyses by avoiding the need for extensive spatial and temporal averaging . objectbased evaluation has been used in evaluation of weather forecasts and climate simulations [ byna et al . , 2011 ; davis et al . , 2006 ; ebert and mcbride , 2000 ; micheas et al . , 2007 ; skok et al . , 2013 ; wernli et al . , 2008 ; wick et al . , 2013 ] . in objectbased methods , the meteorological features are identified / detected in the domain of interest , and then isolated and compared with their analogs between modeled and/or observed fields . this makes it possible to evaluate processes in models without needing to reproduce the time and location of , for example , a particular observed cloud system or a cyclone . the detection of an object is traditionally done by setting a threshold for a variable and evaluating the grid points in the field relative to that threshold ( i.e. , is the grid value above or below the threshold ? ) . this evaluation is done to identify if the grid point ( or observation ) belongs to the class of the feature to be detected . the main disadvantages of these methods are the subjective decisions in thresholding ( user defined fixedvalues ) , which leads to misses in detection . the results of the current detection methods are highly dependent on these fixed threshold values and even the use of multiple thresholds [ xu et al . , 2005 ; this study involves development of a more efficient detection strategy that will alleviate the fixed userdefined threshold problem using a pattern recognition algorithm ( classification trees ) . the algorithm has the flexibility to use information from other fields . in our problem , orographic precipitation features are detected and isolated by using information both from precipitation rates and the topography . our analysis starts with the selection of orographic precipitation features ( study features ) that have consistent differences in controlled gcm simulations with the cam eulerian spectral core and the cam fv core . a brief summary of the significant results of our previous timeaveraged analysis is given in section 2 of this paper ; however , the detailed results can be found in yorgun and rood and yorgun and rood . once the study features are analyzed qualitatively , the identification and detection algorithm with classification trees ( explained in section 3 ) are applied . the features are isolated from the whole precipitation field as objects and matched with their analogs between cam eulerian spectral and cam fv experimental model runs . the comparisons of the statistical characteristics ( i.e. , peak value , mean value , and variance ) of these objects are presented in section 4 . an object is defined as a coherent system with an associated set of measurable parameters [ douglass , 2000 ] . in an objectbased model evaluation approach , features such as fronts , rain bands , clouds in deep convection , etc . are defined and isolated as objects [ posselt et al . , 2012 ; xu et al . , process and objectbased evaluation preserves information in quantitative analyses by avoiding the need for extensive spatial and temporal averaging . objectbased evaluation has been used in evaluation of weather forecasts and climate simulations [ byna et al . , 2011 ; davis et al . , 2006 , 2007 ; skok et al . , 2013 ; wernli et al . , 2008 ; wick et al . , the meteorological features are identified / detected in the domain of interest , and then isolated and compared with their analogs between modeled and/or observed fields . this makes it possible to evaluate processes in models without needing to reproduce the time and location of , for example , a particular observed cloud system or a cyclone . the detection of an object is traditionally done by setting a threshold for a variable and evaluating the grid points in the field relative to that threshold ( i.e. , is the grid value above or below the threshold ? ) . this evaluation is done to identify if the grid point ( or observation ) belongs to the class of the feature to be detected . the main disadvantages of these methods are the subjective decisions in thresholding ( user defined fixedvalues ) , which leads to misses in detection . the results of the current detection methods are highly dependent on these fixed threshold values and even the use of multiple thresholds [ xu et al . , 2005 ; xu , 2009 ; posselt et al . , 2012 ; skok et al . , this study involves development of a more efficient detection strategy that will alleviate the fixed userdefined threshold problem using a pattern recognition algorithm ( classification trees ) . the algorithm has the flexibility to use information from other fields . in our problem , orographic precipitation features are detected and isolated by using information both from precipitation rates and the topography . our analysis starts with the selection of orographic precipitation features ( study features ) that have consistent differences in controlled gcm simulations with the cam eulerian spectral core and the cam fv core . a brief summary of the significant results of our previous timeaveraged analysis is given in section 2 of this paper ; however , the detailed results can be found in yorgun and rood and yorgun and rood . once the study features are analyzed qualitatively , the identification and detection algorithm with classification trees ( explained in section 3 ) are applied . the features are isolated from the whole precipitation field as objects and matched with their analogs between cam eulerian spectral and cam fv experimental model runs . the comparisons of the statistical characteristics ( i.e. , peak value , mean value , and variance ) of these objects are presented in section 4 . the comparison of the 21 year january mean precipitation on the west coast of united states between global precipitation climatology project ( gpcp ) observations [ beck et al . , 2005 ] and the atmospheric model intercomparison project ( amip ) runs with the eulerian spectral and the fv dynamical cores reveal significant differences [ yorgun and rood , 2014 ] . for example , the fv model with 0.5 resolution successfully simulates the dry region between the coast ranges and the sierra nevada , whereas the eulerian spectral model with t170 triangular truncation merges the precipitation features related to the two mountain ranges to create a single precipitation feature [ yorgun and rood , 2014 ] . we selected these features as our study features and conducted experimental model runs with ncar cam 5.0 with eulerian spectral t85 ( 1.4 ) and t170 ( 0.7 ) triangular truncations , and fv 1 and 0.5 resolutions in order to reproduce and further evaluate these features . three topography configurations were created using gaussian bellshaped mountains ranging from a single mountain peak toward a more structured setup resembling the coast ranges and the sierra nevada ( table 1 ) . the mountain specifications for three experimental setups the treatment of the topography is different between the cam eulerian and cam fv dynamical cores as discussed in yorgun and rood , and this difference is kept as is in the experimental model runs in this study in order to analyze the standard model configurations . the physics / dynamics timesteps are also different , i.e. , the physics and dynamics time steps for the cam eulerian spectral model are identical , and are 1800 and 300 s for t85 and t170 , respectively . for the cam fv model , the physics time steps are 1800 and 600 , and the dynamics time steps are 180 and 60 s for 1 and 0.5 , respectively . the physical parameterization is set to a simplified moist parameterization suite called simplephysics suite [ reed and jablonowski , 2012 ] for both cam fv and the cam eulerian spectral dynamical cores . the suite allows physical processes that are important for orographic precipitation , which are largescale condensation , boundary layer turbulence of horizontal momentum , temperature and specific humidity , and surface fluxes of horizontal momentum , evaporation ( specific humidity ) , and sensible heat ( temperature ) from the surface to the lower atmosphere . moisture ( specific humidity ) is transported to the mountains via zonal and meridional winds , followed by orographic lift and subsequent largescale condensation and precipitation . although the cam 5.0 includes the subgridscale gravity wave drag parameterization especially to account for the deceleration of the zonal wind above the surface [ neale et al . , 2010 ] , the simple physics package does not have such parameterization thus the solutions presented in this study are not affected by it . the simulations start with a purely westerly wind and they evolve to have northerly , southerly , and vertical components as the simulations continue . three types of precipitation features are simulated in all three topographical setups , which are shown in the monthly mean plot of the double mountain setup ( figure 1 ) . 30 day mean total precipitation ( mm / d ) , surface geopotential ( topography ) for double mountain setup of experimental test cases , simulated by ( a ) cam eulerian spectral t85 , ( b ) cam eulerian spectral t170 , ( c ) cam fv 1 , and ( d ) cam fv 0.5 dynamical cores . surface geopotential contours are 500 , 2000 , 5000 , and 11000 ms , with the peak contour at the interior of the mountain . the numbers 1 , 2 , and 3 on figure 1b indicate the largescale feature due to stable upslope ascent , smallscale feature due to local evaporation and leeside convergence , and the feature due to leeward baroclinic waves , respectively . the features as indicated by their corresponding numbers in the cam eulerian spectral t170 simulation ( figure 1b ) are : largescale features due to stable upslope ascentsmallscale features due to local evaporation and leeside convergencefeature due to leeward baroclinic waves largescale features due to stable upslope ascent smallscale features due to local evaporation and leeside convergence feature due to leeward baroclinic waves the largescale features ( 1 ) appear in the windward side of the mountains early in the simulation ( day 1 ) covering a relatively large area whereas the smallscale features ( 2 ) manifest later in the simulation ( around day 15 ) closer to the peaks of the mountains . the peak precipitation values within the largescale features are less than that of the smallscale features . the features due to the baroclinic waves also appear early in the simulation and they consistently producing light precipitation with their shape resembling the wave structure . these features are different in their origins and evolutions , thus it is important to identify and evaluate them as separate objects . in yorgun and rood , we focused on the largescale stable upslope ascent features ( # 1 in figure 1b ) . in that study , we made an analysis of the monthly mean simulations of these features and quantified the differences between the two dynamical cores using kmeans cluster analysis for identification , and variography for comparison of the features . in this study , we add the time dimension to the analysis by looking at daily simulations of both the largescale stable upslope ascent and the smallscale features due to local evaporation and leeside convergence ( # 2 in figure 1b ) . this way we aim to quantify the spatiotemporal differences in the representation of these features between the cam eulerian spectral and the cam fv dynamical cores . the features due to leeward baroclinic waves ( # 3 in figure 1b ) are away from the topography , therefore they are excluded from current analysis and the focus is kept on the features that are over the topography in this study . we focus on the double mountain , and the realistic topographical setups ( table 1 ) using classification trees for the detection and isolation of the precipitation features . a classification tree [ breiman et al . , 1984 ] algorithm was implemented for detection and isolation of precipitation features simulated in idealized test cases . classification trees is a machinelearning method for constructing prediction models from data by recursively partitioning the data space and fitting a simple model into each partition . , k , and p predictor variables ( x 1 , x 2 , , x p ) , a classification strategy aims to construct a model to predict the class value of y from values of x. the solution is a partition of the x space into k disjoint sets s1 , s2 , , sk , such that the predicted value of y is j if x belongs to sj for j = 1 , 2 , , k. classification trees yield rectangular sets sj by recursively partitioning the data field on x [ loh , 2011 ] . the data field ( in our case the precipitation rate ) is partitioned into rectangular boxes that are called nodes . the initial field ( the root node ) is split into a series of parent and leaf nodes , where each parent node is split into two leaf nodes . the splitting is done until a stopping criterion is met for a leaf node in which case the leaf node is called a terminal node . the measure of node impurity ( explained below ) is based on the distribution of the observed y values in the node . our algorithm finds a subset over all x values for the split that minimizes the difference between the impurity of the node and the impurity of its two leaf nodes . an example classification tree of a precipitation field simulated by cam fv 0.5 is given in figure 2 . an example classification tree with two rectangular splits at day 15 total precipitation rate for the double mountain case , simulated by cam fv 0.5. th is threshold . the root node is the initial precipitation field for the double mountain case simulated by cam fv 0.5. the first split is done over a longitude line , which partitions the initial field such that the largescale upslope ascent and the smallscale local evaporation and leeside convergence features remain in one node ( westward of the split boundary ) , and the feature due to the leeward waves remain in the other ( eastward of the split boundary ) . the resulting two nodes of split 1 are both leaf nodes of the root node ( i.e. , the combination of the two leaf nodes gives the root node ) . in this example , it is assumed that the impurity measure of the node containing the feature due to the leeward baroclinic wave meets the stopping criterion ; therefore , it can also be labeled as a terminal node . a further split is done over the node containing the large and smallscale features ; therefore , it can also be labeled as a parent node . the second split is done over a latitude line on the parent node resulting from split 1 , which separates the smallscale features in one node , and the largescale features on the other . both nodes are the leaf nodes of their parent node ; however , there are no more splits in this example so they can also be labeled as terminal nodes . as can be seen in figure 2 , there are two threshold values employed for each split ( th1 and th2 ) . although these threshold values are fixed values , they are determined objectively after each split by the characteristics of the field thus they are not userdefined fixed values for the whole application . the decision of these threshold values , the decision of the longitude ( or latitude ) line as the split boundary due to an impurity measure , and the decision of stopping criteria after each split are the key factors in the formation of such a classification tree . these factors are explained in detail by looking at the first split given in figure 3 . day 15 total precipitation rate , surface geopotential ( topography ) for double mountain case , simulated by fv 0.5. the longitudinal split boundary is shown with the red line . in figure 3 , the whole precipitation field is the root node and is split into two leaf nodes at longitude 104 w. for each partition , the algorithm calculates the impurity of each node via a measure called gini index : ( 1)gininode=y=1ypy(1py)where py is the frequency of occurrence of gridpoints belonging to class y. we defined two classes ( y = 1 , 2 ) in our calculations , one being the rain ( i.e. , the gridpoint that belongs to the object of interest ) and the other being the nonrain ( i.e. , any gridpoint outside objects ) . the classes and the corresponding node impurities are defined via thresholds . the decision of the split is done via maximizing the qsplit parameter : ( 2)qsplit = impurityparentnnode1nparentimpuritynode1+nnode2nparentimpuritynode2where nparent , nnode1 , and nnd2 are the total number of gridpoints included in the parent node and subsets of leaf nodes ( nodes 1 and 2 ) , respectively . each impurity value ( i.e. , the impurity values for parent , node1 , and node2 ) in equation ( 2 ) is calculated by the gini index given in equation ( 1 ) ( i.e. , impurity(node ) = gini(node ) ) . the split in figure 3 is achieved via maximizing the qsplit with the threshold value of 0.5 mm / d ( i.e. , if a gridpoint value is above 0.5 mm / d , it is considered rain class , if it is below this threshold , it is considered nonrain class ) . after this first split , the algorithm continues to split each leaf node until all the resulting nodes become terminal , which is decided when the impurity of nodes drops below a certain value ( i.e. , until they become pure ) . this value is taken as 0.05 in this study , which ensures very low impurity in the resulting nodes . the splitting is done sequentially in latitudes and longitudes ( if the first split is in longitudes , then the second is in latitudes , third is in longitudes etc . ) ; however , this sequence can be changed depending on the user 's choice . this nature of classification trees that allow stepbystep splitting of the root node into terminal leaf nodes allows the resetting of the threshold value according to the characteristics of each parent node . in figure 3 , node 1 includes two types of features discussed in section 2 , namely the largescale upslope ascent feature and the smallscale feature due to local evaporation and leeside convergence , whereas node 2 includes the feature due to the leeward waves . these features are different in their manifestations , characteristics , and evolutions in time ; therefore , they should not be classified with the same threshold . the algorithm objectively calculates the threshold before each split by looking at the statistics of the parent node to be split . in this study , the threshold is defined by adding half of the standard deviation of the node to the mean value of the node . this measure is selected empirically based on the qualitative analysis on the precipitation features we have previously conducted [ yorgun and rood , 2014 ] and can be changed according to the nature of the problem . the types , shapes , and structures of the precipitation features were analyzed and visually differentiated in that analysis , and that knowledge is used as validation of the resulting isolated objects by the algorithm in this study . we used several different statistical combinations ( e.g. , mean , 2 x mean , mean + standard deviation , etc . ) and observed that the mean + 0.5 x standard deviation results matched the expected shape / structure of the different precipitation features better . as indicated previously , the decision tree algorithm partitions the initial field into rectangular boxes . depending on the threshold value , the algorithm may split a precipitation feature into more than one rectangle by chopping off the edges of the curved feature . a postprocessing was conducted to overcome the issue where we reconstructed the split features if any into the original form . the qualitative analysis [ yorgun and rood , 2014 ] gave information about the locations of each type of feature , therefore the terminal nodes , which are exactly over the peak of mountains are aggregated to form the smallscale features due to local evaporation and leeside convergence . the other two types of features are formed in the same manner by measuring the node distances to mountain peaks and the locations relative to the mountain peaks ( i.e. , the euclidian distance between the center of mass of the object within a node to the mountain peak ) . the similar distances in the windward sides of the mountains are merged to form the largescale stable upslope features , whereas the similar distances in the leeward side ( of the eastward mountain in double mountain case ) are merged to form the features due to baroclinic waves . this flexibility of the algorithm also allows the usage of other information ( e.g. , moisture flux convergence , winds ) if needed . the detection and extraction of the features with the classification tree algorithm was applied to daily precipitation results for a total model run time of 30 days . once the features were extracted , some simple statistical analyses ( e.g. , mean values , peak values , variances ) were conducted and the results were compared for all four simulations , which are discussed in the next section . the classification tree algorithm is applied to the daily simulations of the double mountain and the realistic setups of the idealized simulations . our comparison focuses on the largescale upslope ascent features and the smallscale features due to local evaporation and leeside convergence simulated by cam fv 1 and 0.5 , and cam eulerian spectral t85 and t170 triangular truncation resolutions . an example of how the algorithm partitions the precipitation field simulated by cam fv 0.5 for the double mountain setup is given in figure 4 . the algorithm was able to identify and isolate the largescale upslope ascent feature ( figure 4b ) and the smallscale local evaporation and leeside convergence feature ( figure 4c ) . the largescale features in front of the windward mountain and the smallscale features on the leeward mountain are selected to be included in our evaluation . the analysis on the largescale feature in front of the leeward mountain and the smallscale feature on the windward mountain produced similar results to those included in this study ; therefore , they are excluded to avoid repetitive results ( they are classified as the same with these two types of features ) . the algorithm was also applied to cam fv 1 and the cam eulerian spectral t85 and t170 simulations of the double mountain case and the precipitation features were successfully isolated . then the peak value , the mean value , and the variance for the isolated features were calculated within the enclosed object boundaries for each day over 30 days of simulation time . day 24 total precipitation rate , surface geopotential ( topography ) for the double mountain case , simulated by ( a ) cam fv 0.5. the isolated largescale stable upslope ascent feature on the windward mountain and the smallscale feature on the leeward mountain are shown in b and c , respectively . the results for the largescale stable upslope ascent feature ( figures 5a , 5c , and 5e ) show close agreement between cam fv 1 and 0.5 , and the cam eulerian spectral t170 models ; however , there is a disagreement for cam eulerian spectral t85 results ( the dotted line ) . cam eulerian spectral t85 simulated this largescale feature with lower intensity , which is in agreement with our qualitative analysis in yorgun and rood . this lower intensity was directly related to the spectral filtering of the topography to control aliasing . figure 5e shows that cam eulerian spectral t85 also produced lower values of variance whereas the other simulations exhibit high variances in the beginning and between day 5 and day 10 . note that the variance is calculated over the values within the boundaries of the objects determined by the classification tree algorithm ; therefore , low values of variance indicate a smoother feature without a distinct peak of precipitation . a peak precipitation is expected for a stable upslope precipitation feature given the nature of such features [ roe , 2005 ] , where a peak value is observed together with lower values around it . the variance results also show how both cam fv resolutions ( dashed lines ) agree closely with each other yielding slightly higher values than that of cam eulerian spectral t170 . the results start to become more similar for all models after day 15 for the largescale stable upslope feature as it loses intensity and the amount of rain is reduced . these results suggest that the largescale stable upslope precipitation is resolved by both schemes , with the spectral scheme revealing strong sensitivity to the topographical filtering required to control aliasing and noise [ yorgun and rood , 2014 ] . the peak value , mean value , and the variance for the largescale stable upslope ascent feature on the windward mountain ( a , c , e ) , and the smallscale local evaporation and leeside convergence feature on the leeward mountain ( b , d , f ) for the double mountain setup , simulated by all four models . the agreement of model simulations for the largescale upslope ascent feature is not seen for the smallscale local evaporation and leeside convergence feature ( figures 5b , 5d , and 5f ) . cam eulerian spectral t170 starts simulating this object the earliest , with low peak and variance values ( figures 5b and 5f ) followed by an abrupt increase afterward and an oscillatory behavior until the end of the simulation with significantly higher values compared to both cam fv resolutions . on the other hand , cam fv 1 and 0.5 resolution simulations ( starting on day 24 and 15 , respectively ) exhibit relatively stable behavior , keeping similar peak , mean , and variance values throughout the simulations . cam eulerian spectral t85 did not simulate this feature within the 30 day simulation time ; however , a 45 day run was made and it was observed that spectral t85 simulated this feature starting at day 37 . the mean values for the smallscale feature ( figure 5d ) for cam eulerian spectral t170 are stable throughout the simulation time . the mean values are calculated such that the precipitation rate is summed over all grid points and divided by the number of grid points within the boundaries of the object . a qualitative examination of the isolated smallscale local evaporation and leeside convergence features shows that cam eulerian spectral t170 simulates these features over a larger spatial extent , therefore the mean values of cam eulerian spectral t170 are comparable to cam fv 0.5 even though cam eulerian spectral t170 produces higher peak precipitation rates . high variance values ( i.e. , high and low values enclosed within the object ) for cam eulerian spectral t170 also support the agreement in the mean values between cam eulerian spectral and cam fv . the agreement of mean values , but the disagreement of the variance between the models suggest that there is a level of agreement between models in terms of the amount of rain they produce within a given object , however there are significant differences in the variability of the simulated precipitation . it should also be noted that the evolution of the mean and variance for the whole field ( as opposed to the partitioned field presented in this study ) resembles the behavior of the largescale features ( figures 5c and 5e ) . this shows the utility of the objectbased technique used in this study , i.e. , partitioning of the whole field led us understand the behavior of the smallscale features which would otherwise be lost . the results shown in figure 5 give a quantitative picture of how scales of precipitation features differ between the cam fv and cam eulerian spectral dynamical cores . the results for the more realistic idealized setup with smaller scales in the horizontal provide the next level of complexity in analyzing how these dynamical cores simulate both largescale and smallscale precipitation . figure 6 shows the day 20 total precipitation rate simulated by the cam eulerian spectral t85 and t170 , cam fv 1 and fv 0.5 for the realistic idealized setup . as discussed in yorgun and rood , there are significant differences in the simulated precipitation between cam eulerian spectral and cam fv models with the increased complexity of the underlying topography . we focus on the largescale upslope ascent and the smallscale local evaporation and leeside convergence features simulated on the leeward mountain by finer resolution simulations ( cam eulerian spectral t170 and cam fv 0.5 ) . as seen in figures 6b and 6d , the precipitation features simulated on the leeward mountain are more complex than in the double mountain case . it is also worth noting that the much higher leeward mountain range does not behave like the single mountain experiment . that is , the lower windward range has a strong influence on the circulation and the precipitation . both the high and lowresolution fv experiments have a region of no rain between the mountains . the lower resolution cam eulerian spectral ( figure 6a ) does not even represent the twomountain structure after the spectral filter has smoothed the topography to be consistent with the t85 resolution . the higherresolution cam eulerian spectral ( figure 6d ) joins the precipitation from two mountain ranges . day 20 total precipitation rate ( mm / d ) , surface geopotential ( topography ) for the realistic case , simulated by ( a ) cam eulerian spectral t85 , ( b ) cam eulerian spectral t170 , ( c ) cam fv 1 , and ( d ) cam fv 0.5. surface geopotential contours are 500 , 2000 , 5000 , and 11000 ms , with the peak contour at the interior of the mountain . we note that for the realistic case the mountains are only separated by 3 longitude , as opposed to 7 longitude for the double mountain case . this offers special challenges for the global basis functions of the spectral method in terms of the ability to resolve the gap ( valley ) between the mountains . though the fv dynamics do not formally resolve features of the spatial scale of the valley between the ranges , the local nature of the scheme allows the physics to function at a scale that increases the meteorological realism of the wetdry contrast . focusing on the objects , and the possible differentiation of largescale upslope and smallscale leeside precipitation , the features are not obviously distinct over the leeward mountain in figures 6b and 6d . however , the classification tree algorithm is able to isolate each feature successfully by the precipitation value patterns , without the need of additional information from other fields such as winds or moisture flux convergence . as we have shown in yorgun and rood , the positive moisture flux convergence ( mfc ) is a good indication of precipitation occurring on smaller scales . figure 7 shows the mfc plots ( figures 7a and 7b ) and precipitation ( figures 7c and 7d ) . the smallscale , leeside precipitation features were objectively isolated from the whole precipitation field ( figures 6b and 6d ) by the classification tree algorithm for cam fv 0.5 and cam eulerian spectral t170 , respectively . the surface evaporation leads to an influx of moisture , which combined with the smallscale circulation caused by the complex topography , leads to a convergence of moisture leading to precipitation . therefore , these features are not related to stable upslope ascent but rather the local evaporation and leeside convergence features the classification tree algorithm isolated from the whole precipitation field . day 20 moisture flux convergence ( 10 kg ms ) by ( a ) cam fv 0.5 , ( b ) cam eulerian spectral t170 and their related precipitation ( mm / d ) objects ( c , d ) isolated by the classification tree algorithm . surface geopotential contours are 500 , 2000 , 5000 , and 11000 ms , with the peak contour at the interior of the mountain . the peak and the mean values as well as the variances are calculated for these isolated features ( figure 8) . in this case , there is better agreement between the cam fv 0.5 and cam eulerian spectral t170 simulated smallscale local evaporation and leeside convergence features compared to their analogs in the double mountain setup ( figures 5b , 5d , and 5f ) . in the realistic case , the onset of the smallscale feature is earlier for cam fv 0.5 , which was not the case for double mountain setup . the mean values for cam fv 0.5 are higher than that of cam eulerian spectral t170 ( figure 8b ) , which is also not the case for the double mountain setup ( figure 5d ) . the mean values and the variance for cam eulerian spectral t170 exhibit an abrupt increase right after the onset of precipitation . the statistical analysis given in figure 8 shows that , unlike in the double mountain case , both cam eulerian spectral t170 and cam fv 0.5 simulated similar smallscale local evaporation and leeside convergence features . ( a ) the peak value , ( b ) mean value , and ( c ) the variance for the smallscale local evaporation and leeside convergence features for the realistic setup shown in figure 7 , simulated by cam fv 0.5 and cam eulerian spectral t170 . the fv simulation is aligned with the topography , and more confined to the leeward side of the mountain . the largest quantitative difference is the spatial extent , where cam eulerian spectral t170 bulges due to the shape of topography ( figure 7d ) . though , statistically similar in this case , the relation of the spatial structure to the topography suggests different behavior of the two dynamical cores . the results , thus far , establish that when well resolved , with comparable lift , the two dynamical cores are consistent in their representation of stable , upslope precipitation . the issue of topographical lift is not trivial , as the filtering to control aliasing and noise in the spectral method leads to large differences at different resolutions . the smallerscale precipitation , which might be framed as that associated with internal as opposed to forced dynamics , is quite different between the two dynamical cores . the first is associated with dynamical structure , e.g. , waves , that are formed after the flow encounters the mountains . the second type is numerical ; that is , a numerical artifact causes condensation and precipitation . the first type is expected to show consistency with the moisture flux and its convergence . the statistics and spatial structure of the smallerscale precipitation objects suggest different behavior between the two dynamical cores . in yorgun and rood , we noted the smallscale features were highly sensitive to the specification of the seasurface temperature ( sst ) . numerical experiments show that when increasing the sst above 288 k the smallscale precipitation features become dominant not just over the mountains , but , also , away from the mountains . as a sensitivity experiment , therefore , we decrease the surface temperature of the experimental configuration by 1k ( i.e. , 287 k instead of 288 k ) for the double mountain configuration . the statistics for the largescale and the smallscale objects are shown in figure 9 , calculated as differences from the control experiment with the original surface temperature . for the stable , upslope simulation , the differences between the two experiments are small for both dynamical cores ( figures 9a and 9c ) . for the smallscale precipitation , the 1k difference of seasurface temperature causes much larger changes in the spectral simulation than in the fv simulation ( figures 9b and 9d ) . 287 k sst runs ) for ( a , c ) the largescale stable upslope ascent feature on the windward mountain , and ( b , d ) the smallscale local evaporation and leeside convergence feature on the leeward mountain for the double mountain setup , simulated by cam eulerian spectral t170 and cam fv 0.5. it is reasonable to postulate that the gridscale noise structure of the two dynamical cores has an important impact on precipitation . notionally , dispersion errors , especially in water vapor transport [ lin and rood , 1996 ; rood , 1987 ] , can cause numerical initiation of precipitation . however , to determine cause and effect in simulations of this complexity is more difficult . the condensation of moisture is a discontinuous process in time , which introduces sharp gradients that are especially problematic for spectral models . these processes are candidates to produce the gibbs phenomenon ; thereby reducing the realism of spectral model simulations [ geil and zeng , 2015 ; navarra et al . , it is natural in model studies of precipitation to focus on where it is raining and the characteristics of the rain events . however , to understand the sensitivity of precipitation to a dynamical core , it is proposed that examination of gridscale structure , which can initiate precipitation , is better founded . to suggest the potential value of this approach , the whole extent , rather than the boundaries determined by the classification tree algorithm , of the features on the western ( nominally , windward ) mountain for day 16 the color scheme of figure 10 ( 00.1 mm / d ) is adjusted to show how the gridscale precipitation differs between fv 0.5 and cam eulerian spectral t170 . not only are there scalloped edges of the rain border , but the structure of the gradient of increasing precipitation on the western side of the mountain has lobes of higher and lower precipitation . this suggests a strategy of removing the objects from the field in order to expose the gridscale information , which will be the subject of future studies . day 16 total precipitation rate for the smallscale features on the windward mountain , surface geopotential ( topography ) for double mountain case , simulated ( a ) by fv 0.5 and ( b ) by cam eulerian spectral t170 . the statistics and spatial structure of the smallerscale precipitation objects suggest different behavior between the two dynamical cores . in yorgun and rood , we noted the smallscale features were highly sensitive to the specification of the seasurface temperature ( sst ) . numerical experiments show that when increasing the sst above 288 k the smallscale precipitation features become dominant not just over the mountains , but , also , away from the mountains . as a sensitivity experiment , therefore , we decrease the surface temperature of the experimental configuration by 1k ( i.e. , 287 k instead of 288 k ) for the double mountain configuration . the statistics for the largescale and the smallscale objects are shown in figure 9 , calculated as differences from the control experiment with the original surface temperature . for the stable , upslope simulation , the differences between the two experiments are small for both dynamical cores ( figures 9a and 9c ) . for the smallscale precipitation , the 1k difference of seasurface temperature causes much larger changes in the spectral simulation than in the fv simulation ( figures 9b and 9d ) . the peak value , and the variance difference ( 288 k 287 k sst runs ) for ( a , c ) the largescale stable upslope ascent feature on the windward mountain , and ( b , d ) the smallscale local evaporation and leeside convergence feature on the leeward mountain for the double mountain setup , simulated by cam eulerian spectral t170 and cam fv 0.5. it is reasonable to postulate that the gridscale noise structure of the two dynamical cores has an important impact on precipitation . notionally , dispersion errors , especially in water vapor transport [ lin and rood , 1996 ; rood , 1987 ] , can cause numerical initiation of precipitation . however , to determine cause and effect in simulations of this complexity is more difficult . the condensation of moisture is a discontinuous process in time , which introduces sharp gradients that are especially problematic for spectral models . these processes are candidates to produce the gibbs phenomenon ; thereby reducing the realism of spectral model simulations [ geil and zeng , 2015 ; navarra et al . , 1994 ] . it is natural in model studies of precipitation to focus on where it is raining and the characteristics of the rain events . however , to understand the sensitivity of precipitation to a dynamical core , it is proposed that examination of gridscale structure , which can initiate precipitation , is better founded . to suggest the potential value of this approach , the whole extent , rather than the boundaries determined by the classification tree algorithm , of the features on the western ( nominally , windward ) mountain for day 16 the color scheme of figure 10 ( 00.1 mm / d ) is adjusted to show how the gridscale precipitation differs between fv 0.5 and cam eulerian spectral t170 . not only are there scalloped edges of the rain border , but the structure of the gradient of increasing precipitation on the western side of the mountain has lobes of higher and lower precipitation . this suggests a strategy of removing the objects from the field in order to expose the gridscale information , which will be the subject of future studies . day 16 total precipitation rate for the smallscale features on the windward mountain , surface geopotential ( topography ) for double mountain case , simulated ( a ) by fv 0.5 and ( b ) by cam eulerian spectral t170 . daily simulations of cam eulerian spectral t170 and t85 , cam fv 1 and 0.5 resolutions for the double mountain and the realistic setups were analyzed in an objectbased framework . the treatment of the topography as well as the physics / dynamics timesteps are different between cam eulerian and cam fv models used in this study ( as indicated in section 2 ) in order to keep the standard model configurations . a classification tree algorithm that allows flexible thresholding though these idealized simulations have simple , smooth topography , complexity in the precipitation fields develops quickly . the classification tree algorithm using objective thresholding proved successful in sensible isolation of precipitation features even as the complexity of the precipitation field increased . the isolation of like objects in different simulations let us focus on local phenomena and quantify the differences between cam eulerian spectral and cam fv models in simulation of orographic precipitation . we documented higher sensitivity of the spectral simulations , compared to the fv simulations , to resolution as well as to changes in the surface temperature of the aquaplanet . generally , it is sought to minimize the diffusion needed to assure accurate diffusion . at comparable levels of diffusion to manage numerical dispersion errors and gridscale noise , the spectral methods continue to show signatures of spectral ringing , oscillations , which leave signatures in the precipitation fields . the results of this study suggest that even for a climate model , investigation of precipitation events on daily of even hourly output can provide information about model biases . we were able to quantify the differences between the fv and the spectral models ( e.g. , the erratic behavior of spectral models , especially at the onset of the smallscale precipitation features ) by looking at the daily evolution of simulated precipitation features . averaging in the case of monthlymean analysis would lose this information . smallscale features such as the ones identified due to localevaporation carry significant information about the bias originated by their corresponding physical parameterizations and numerical schemes . local scales where significant bias can manifest in gcm simulations , which can not be quantified with summary statistics due to averaging out . our study showed that analyzing local features rather than a whole precipitation field gives more accurate insight about the nature of bias . two different types of features over the mountains of the same model run ( double mountain case ) show significant difference in their evolution over time . these differences are large enough to contribute to biases in the means , partitioning in the scales of precipitation , and , especially in complex topography , provision of precipitation to watersheds . the smallscale feature has a completely different mechanism of manifestation ( i.e. , local evaporation and leeside convergence ) than the largescale , upslope precipitation , and isolating these features gives insight about the bias related to that mechanism , how it is parameterized , and how the parameterization is coupled with dynamics . one prominent conclusion of this study is that the resolvable spatial scales of precipitation features to be simulated play a crucial role in how they will be simulated by gcms . this is naturally related to the scales of the mountains involved in the simulation . throughout the discussion of the results , it has been clear that the simulation of largescale features due to stable upslope ascent was in agreement between cam eulerian spectral and cam fv model with the exception of cam eulerian spectral t85 . however , the quantitative characteristics of the simulated smallscale features by cam fv and cam eulerian spectral models were not in agreement . an important reason for this difference , or in other words , the reason why the cam eulerian spectral models failed to simulate precipitation features in agreement with cam fv models and observations as seen in the amip runs [ yorgun and rood , 2014 ] is the bias introduced in smallscale phenomena due to the spectral transform method and the spectral filtering applied to topography in the cam eulerian spectral as discussed in yorgun and rood . our results suggest that the spectral scheme exhibits strong sensitivity to the topographical filtering required to control aliasing and noise . in bala et al . [ 2008a ] , it was discovered that the default configuration of the cam applied the spectralfiltered topography to both the fv and eul dynamical cores . a filter consistent with the fv numerical formulation was introduced ; it was strongly focused on gridscale ( twice the grid length ) waves . in global simulations , comparing the two filters , surface pressure patterns and upper air temperatures , for example , were quite similar . differences in precipitation near topography , for example , the summertime monsoonal flow in mexico , the andes , and the west coast of north america were immediately discernible . the spectralfiltered topography was far smoother than required for the fv numerical attributes , and it was concluded that a specification of topography that was consistent with the dispersion characteristics of the dynamical core was scientifically correct and an integral part of the dynamical core . in the original specification of the mountain wave test , it was suggested that the test be run at 1 resolution as it was determined to be converged at this resolution ( c. jablonowski , 2015 , personal communication ) . with the introduction of the double mountain and the realistic topography , furthermore , we have moved our attention from the convergence of resolved fluid dynamical parameters to parameters dependent on gridscale physics . as discussed in yorgun and rood , for the double mountain case the t85 has effectively 5 grid points between the mountain peaks and the t170 has 10 grid points . for the realistic topography , t85 merges the peaks and t170 has only five grid points . our results suggest that with the spatial scale of the topography at five grids points the spectral simulation degrades badly . as the spatial scale of the topography approaches 10 grid points , the fv and eul solutions start to converge . [ 2008b ] and rood discuss that because the fv core has a highly localized computational stencil , as compared to the global stencil of the spectral basis functions , fv has benefit near steep topography for many variables associated with the model physics . the procedure for the coupling of dynamics and physics in cam 5.0 plays an important role in the manifestation of this type of bias in spectral dynamical core . in cam 5.0 , there are two types of coupling strategies , namely time split and process split . in the time split coupling , dynamics and physics are calculated sequentially , each based on the state produced by other . whereas in the process split coupling the calculations of dynamics and physics are both based on the same past state [ neale et al . , 2010 ] . between the two coupling strategies , process split is more suitable for spectral transform models ( and it is the default in cam 5.0 ) since the time split requires extra spectral transforms to convert the momentum variables provided by the physical parameterizations . the formulation of the cam spectral model is such that every physical process is calculated and updated on the gaussian grid , and then their contribution to the dynamics is transformed to spectral space . this procedure creates problems when dealing with discontinuous physical processes that occur in smallscale such as the ones highlighted in this study ( smallscale features in figure 1 ) . as indicated throughout this paper and quantified in figure 9 , the manifestation of the smallscale features due to local evaporation and leeside convergence have significant contribution from the surface flux parameterization of the simple physics suite [ reed and jablonowski , 2012 ] and a key component of this evaporation is the sst value of the aquaplanet setting . once the gaussian grid precipitation value is updated by surface flux parameterizations , it introduces a relatively high value of precipitation on the grid level and causes a jump between the previous and current time states . this jump is not handled accurately with the spectral transform method and numerical noise is introduced . this noise is clearly observed in figures 5b and 5f where we quantified the initial oscillatory behavior of double mountain test case smallscale feature simulated by the cam eulerian spectral model immediately after its genesis . as the simulation proceeds , the oscillations become less severe since the grid values become similar between the previous and current time states and consequently the spectral transform method produces lesser numerical noise . the cam eulerian spectral model also simulated higher precipitation values compared to fv throughout the simulation which shows the overrepresentation of precipitation by spectral models shown by williamson and rasch . however , we also showed that such noise is not as prominent for the same type of feature in the realistic test case ( figure 8) . the planetary boundary layer ( pbl ) diffusion in simplephysics suite is parameterized such that the physical processes below 850 hpa are affected by it [ reed and jablonowski , 2012 ] . in the double mountain case for the spectral model , the peak mountain height is below 850 hpa ( the smoothing of the topography by filtering also contributes to that ) therefore the smallscale scale features simulated over the mountain are affected by pbl parameterization . whereas the leeward mountain in the realistic case ( where the smallscale features occur ) is sufficiently higher so that the pbl parameterization has lesser effect . therefore , there is more contribution from physics in gaussian grid level in the double mountain case , which results in considerable amount of bias when the values are transformed to the spectral space . our analysis also suggests that when the simulations are physical with less numerical noise ( as in the case of the realistic setup in this study ) , it is observed that there is a correlation between the resolution and the onset time of the smallscale features , i.e. , the higher the resolution , the earlier the onset .

abstractan objectbased evaluation method using a pattern recognition algorithm ( i.e. , classification trees ) is applied to the simulated orographic precipitation for idealized experimental setups using the national center of atmospheric research ( ncar ) community atmosphere model ( cam ) with the finite volume ( fv ) and the eulerian spectral transform dynamical cores with varying resolutions . daily simulations were analyzed and three different types of precipitation features were identified by the classification tree algorithm . the statistical characteristics of these features ( i.e. , maximum value , mean value , and variance ) were calculated to quantify the difference between the dynamical cores and changing resolutions . even with the simple and smooth topography in the idealized setups , complexity in the precipitation fields simulated by the models develops quickly . the classification tree algorithm using objective thresholding successfully detected different types of precipitation features even as the complexity of the precipitation field increased . the results show that the complexity and the bias introduced in smallscale phenomena due to the spectral transform method of cam eulerian spectral dynamical core is prominent , and is an important reason for its dissimilarity from the fv dynamical core . the resolvable scales , both in horizontal and vertical dimensions , have significant effect on the simulation of precipitation . the results of this study also suggest that an efficient and informative study about the biases produced by gcms should involve daily ( or even hourly ) output ( rather than monthly mean ) analysis over local scales .

Introduction ObjectBased Approach Selection of the Study Features and the Experimental Model Runs for Orographic Precipitation Method Discussion of the Results Sensitivity to Sea Surface Temperature (SST) GridScale Noise Structure Conclusions

PMC5008637

albuterol delivered by conventional press - and - breathe metered - dose inhalers ( mdis ) is the most common symptomatic and rescue therapy for managing reversible bronchospasm in asthma and chronic obstructive pulmonary disease ( copd ) . although dose counters can reliably monitor inhaler use,15 they are not currently integrated into some rescue mdis , making it difficult for patients to know when those inhalers are empty . in one study , only 8% of patients reported counting the number of actuations and replacing their inhalers at or before the manufacturer s specified maximum number of actuations had been reached.6 beyond the labeled number of mdi actuations , the amount of active medication available per actuation can vary , resulting in a lower than therapeutic dose.2,7 surveys of patients with asthma who use an mdi indicate that > 50% of patients do not know the number of therapeutic doses remaining in their inhalers.6,7 furthermore , a recent review concluded that up to 40% of patients actually using an empty or nearly empty mdi believe they are taking their asthma medication as prescribed.8 in a recent survey of 224 pediatric and adult patients with asthma or copd , 62% of patients reported feeling anxious about not knowing the quantity of medication remaining in their inhalers.2 of the patients surveyed , 72% reported shaking their inhalers to assess the quantity of remaining medication , and almost one - half ( 42% ) waited until they thought their mdi was no longer working before replacing it . the addition of an integrated dose counter to mdis relieved anxiety about running out of medication for two - thirds of 272 adolescent and adult patients with asthma or copd in a subsequent study.9 in a cohort study of 1,095 adult patients who utilized the emergency department ( ed ) for asthma , 324 ( 30% ) ran out of their inhaled short - acting -agonist ( saba ) or corticosteroid medication during the week before their index ed visit.10 asthma prevalence remains high , affecting almost 26 million americans in 2010,11 with approximately two million asthma ed visits made each year.1113 in addition , an estimated 13.7 million adults in the us reported having a copd diagnosis in 2011,14 and ed visits for copd exacerbations numbered 1.8 million in 2011 , an increase from 1.5 million in 2006.15 this retrospective database analysis using claims data was designed to investigate the impact of integrated dose counter albuterol inhalers on the incidence of respiratory - related ed visits among patients with asthma , copd , or both . we hypothesized that the dose information provided by an integrated dose counter could lead to a reduction in the number of respiratory - related ed visits by decreasing the use of empty or near - empty canisters . data for this study were extracted from the clinformatics data mart retrospective claims database ( optuminsight life sciences , eden prairie , mn , usa ) , which includes anonymized data from an employed , commercially insured united states population , and collected between january 2006 and september 2012 . recorded data include medical claims ( primary care and secondary care ) , pharmacy claims , and laboratory test results . the study comprised a 1-year baseline period for patient characterization and confounder definition and 1-year outcome period for the effectiveness evaluation , with an index date defined as the date of the first prescription for albuterol ( figure 1 ) . the study data were de - identified , thus written informed consent was not possible , and ethics committee review was not sought or deemed necessary by the authors . included patients were aged 464 years at the index date and had a diagnosis of asthma and/or copd and/or exercise - induced bronchoconstriction recorded at any time and at least one prescription for albuterol . all patients had a first recorded prescription for albuterol ( index date ) between january 1 , 2010 , and september 30 , 2011 , and continuous insurance coverage during the study period . patients prescribed ventolin hfa ( glaxosmithkline inc . , research triangle park , nc , usa ) were assigned to the dose counter cohort ; and patients prescribed proair hfa ( teva respiratory llc , horsham , pa , usa ) or proventil hfa ( merck & co. , inc . , whitehouse station , nj , usa ) were assigned to the non - dose - counter cohort . each of these inhalers delivers 108 g of albuterol sulfate ( 90 g of albuterol base ) from the mouthpiece per actuation . both the fine - particle mass and the plume vary only slightly among the three different inhalers and are monitored by the manufacturer and the us food and drug administration as part of the product s commercial release . the exclusion criteria were use of proair hfa ( teva respiratory llc ) or proventil hfa ( merck & co. , inc . ) ( for the dose counter cohort ) or the use of ventolin hfa ( glaxosmithkline inc . ) ( for the non - dose - counter cohort ) during the outcome period and the use of any other saba during the study period ( all patients ) . the asthma subpopulation was defined as patients who had at least one consultation , inpatient admission , or ed visit for asthma recorded at any time during the study period ; the copd subpopulation was defined as those who had at least one consultation , inpatient admission , or ed visit for copd recorded at any time during the study period . the concomitant asthma and copd subpopulation was defined as those patients having at least one consultation , inpatient admission , or ed visit for asthma and copd and/or codiagnoses recorded at any time during the study period . the primary end point was the incidence rate of respiratory - related ed visits , defined as ed visits associated with a lower respiratory diagnostic code . secondary end points for patients with asthma were the incidence rate of severe exacerbations ( defined as respiratory - related inpatient admissions / ed visits or initiation of acute oral corticosteroids16 ) and rate of acute respiratory events ( defined as occurrence of respiratory - related inpatient admission / ed visits or acute use of oral corticosteroids or antibiotics prescribed following a general practitioner visit for lower respiratory tract infection ) . for patients with copd , exacerbations were defined as occurrence of respiratory - related inpatient admission / ed visits or acute use of oral corticosteroids or antibiotics prescribed following a general practitioner visit for lower respiratory tract infection . additional end points included the average daily saba dose and the probability of achieving asthma control ( asthma subpopulation only ) as risk - domain asthma control ( defined as absence of acute respiratory events ) and overall asthma control ( defined as risk - domain asthma control and average daily albuterol dose 180 g ) . data were prepared for analysis by investigating outliers and the type and reason for missing data ; skewed data were categorized if appropriate . because of outliers , all patients with > 10 ed visits were assigned a value of 10 . we evaluated potential confounders , including those that were significantly different at baseline ( independent sample t - test , mann whitney u - test , test , p<0.10 ) and baseline predictors of outcomes ( full multivariable model , p0.05 ) . a multivariable model was used with stepwise reduction to derive the best - fitting model of noncollinear predictors ( p<0.05 ) . the incidence rate of ed visits was calculated using a reduced zero - inflated poisson regression model . exacerbation rates were compared using a reduced zero - inflated poisson regression model , and the odds of achieving asthma control were analyzed using a logistic regression model . average daily saba use was analyzed using a reduced ordinal logistic regression model to determine the odds ratio ( or ) for a higher categorized daily dose compared with a lower categorized daily dose ( 100 , 101200 , 201400 , 401800 , or 800 g albuterol ) . subanalyses were performed for patients with asthma only , copd only , or concomitant asthma and copd because of differences in prescribing indications and outcomes definitions . data for this study were extracted from the clinformatics data mart retrospective claims database ( optuminsight life sciences , eden prairie , mn , usa ) , which includes anonymized data from an employed , commercially insured united states population , and collected between january 2006 and september 2012 . recorded data include medical claims ( primary care and secondary care ) , pharmacy claims , and laboratory test results . the study comprised a 1-year baseline period for patient characterization and confounder definition and 1-year outcome period for the effectiveness evaluation , with an index date defined as the date of the first prescription for albuterol ( figure 1 ) . the study data were de - identified , thus written informed consent was not possible , and ethics committee review was not sought or deemed necessary by the authors . included patients were aged 464 years at the index date and had a diagnosis of asthma and/or copd and/or exercise - induced bronchoconstriction recorded at any time and at least one prescription for albuterol . all patients had a first recorded prescription for albuterol ( index date ) between january 1 , 2010 , and september 30 , 2011 , and continuous insurance coverage during the study period . patients prescribed ventolin hfa ( glaxosmithkline inc . , research triangle park , nc , usa ) were assigned to the dose counter cohort ; and patients prescribed proair hfa ( teva respiratory llc , horsham , pa , usa ) or proventil hfa ( merck & co. , inc . , whitehouse station , nj , usa ) were assigned to the non - dose - counter cohort . each of these inhalers delivers 108 g of albuterol sulfate ( 90 g of albuterol base ) from the mouthpiece per actuation . both the fine - particle mass and the plume vary only slightly among the three different inhalers and are monitored by the manufacturer and the us food and drug administration as part of the product s commercial release . the exclusion criteria were use of proair hfa ( teva respiratory llc ) or proventil hfa ( merck & co. , inc . ) ( for the dose counter cohort ) or the use of ventolin hfa ( glaxosmithkline inc . ) ( for the non - dose - counter cohort ) during the outcome period and the use of any other saba during the study period ( all patients ) . the asthma subpopulation was defined as patients who had at least one consultation , inpatient admission , or ed visit for asthma recorded at any time during the study period ; the copd subpopulation was defined as those who had at least one consultation , inpatient admission , or ed visit for copd recorded at any time during the study period . the concomitant asthma and copd subpopulation was defined as those patients having at least one consultation , inpatient admission , or ed visit for asthma and copd and/or codiagnoses recorded at any time during the study period . the primary end point was the incidence rate of respiratory - related ed visits , defined as ed visits associated with a lower respiratory diagnostic code . secondary end points for patients with asthma were the incidence rate of severe exacerbations ( defined as respiratory - related inpatient admissions / ed visits or initiation of acute oral corticosteroids16 ) and rate of acute respiratory events ( defined as occurrence of respiratory - related inpatient admission / ed visits or acute use of oral corticosteroids or antibiotics prescribed following a general practitioner visit for lower respiratory tract infection ) . for patients with copd , exacerbations were defined as occurrence of respiratory - related inpatient admission / ed visits or acute use of oral corticosteroids or antibiotics prescribed following a general practitioner visit for lower respiratory tract infection . additional end points included the average daily saba dose and the probability of achieving asthma control ( asthma subpopulation only ) as risk - domain asthma control ( defined as absence of acute respiratory events ) and overall asthma control ( defined as risk - domain asthma control and average daily albuterol dose 180 g ) . data were prepared for analysis by investigating outliers and the type and reason for missing data ; skewed data were categorized if appropriate . because of outliers , all patients with > 10 ed visits were assigned a value of 10 . we evaluated potential confounders , including those that were significantly different at baseline ( independent sample t - test , mann whitney u - test , test , p<0.10 ) and baseline predictors of outcomes ( full multivariable model , p0.05 ) . a multivariable model was used with stepwise reduction to derive the best - fitting model of noncollinear predictors ( p<0.05 ) . the incidence rate of ed visits was calculated using a reduced zero - inflated poisson regression model . exacerbation rates were compared using a reduced zero - inflated poisson regression model , and the odds of achieving asthma control were analyzed using a logistic regression model . average daily saba use was analyzed using a reduced ordinal logistic regression model to determine the odds ratio ( or ) for a higher categorized daily dose compared with a lower categorized daily dose ( 100 , 101200 , 201400 , 401800 , or 800 g albuterol ) . subanalyses were performed for patients with asthma only , copd only , or concomitant asthma and copd because of differences in prescribing indications and outcomes definitions . a total of 93,980 patients ages 464 years were included in the study ( dose counter albuterol cohort : n=67,251 [ 72% ] ; non - dose - counter albuterol cohort : n=26,729 [ 28% ] ) ( figure 2 ) . the dose counter and non - dose - counter cohorts were broadly similar at baseline ( table 1 ) . during the outcome period , 341 ( 0.5% ) patients in the dose counter cohort overall had a respiratory - related ed visit , a significantly smaller proportion than in the non - dose - counter cohort ( 304 [ 1.1% ] ; p<0.001 for the comparison ; table 2 ) . the adjusted rate ratio ( rr ) for respiratory - related ed visits , relative to the non - dose - counter cohort , was 0.55 ( 95% confidence interval [ ci ] : 0.470.65 ; adjusted for baseline respiratory - related ed visits , respiratory - related inpatient admissions , and asthma consultations ; gastroesophageal reflux disease diagnosis , rhinitis diagnosis , short - acting muscarinic antagonist use , and -blocker use ) . a total of 75,787 ( 81% ) patients had a diagnosis of asthma , including 53,964 ( 80% ) of those in the dose counter cohort and 21,823 ( 82% ) of those in the non - dose - counter cohort ( table 1 ) . the treatment cohorts were comparable at baseline in terms of demographic characteristics , comorbidities , and medication use ; a statistically significant difference in median age was not clinically significant ( table 1 ) . respiratory - related ed visits were recorded during the outcome period for 270 ( 0.5% ) and 244 ( 1.1% ) patients with asthma in the dose counter and non - dose - counter cohorts , respectively ( p<0.001 ; table 2 ) . those in the dose counter cohort had 51% lower incidence of respiratory - related ed visits ( adjusted rr : 0.49 ; 95% ci : 0.410.59 ) ( table 2 , figure 3 ) . average daily saba dose , severe exacerbation rates , and acute respiratory event rates were similar between treatment cohorts ( tables 3 and 4 , figure 3 ) . however , corresponding with the higher number of ambulatory visits resulting in antibiotic prescriptions ( table 3 ) , patients with asthma in the dose counter cohort had 4% lower odds of achieving risk - domain or overall asthma control than those in the non - dose - counter cohort ( table 4 , figure 3 ) . a total of 6,687 ( 7% ) patients had copd , including 4,953 ( 74% ) in the dose counter cohort and 1,734 ( 26% ) in the non - dose - counter cohort ( table 1 ) . the treatment cohorts were comparable for patients with copd with regard to demographic characteristics , comorbidities , and therapy . respiratory - related ed visits were recorded during the outcome period for 16 ( 0.3% ) and 23 ( 1.3% ) patients with copd in the dose counter and non - dose - counter cohorts , respectively ( p<0.001 ; table 2 ) . patients with copd in the dose counter cohort had 60% lower adjusted respiratory - related ed visit rates versus patients in the non - dose - counter cohort ( adjusted rr : 0.40 ; 95% ci : 0.220.75 ) ( table 2 ) . other copd outcomes were comparable between cohorts , including copd exacerbation rates ( adjusted rr : 1.05 ; 95% ci : 0.971.13 ) and average daily saba dose ( adjusted or : 1.08 ; 95% ci : 0.941.25 ) ( table 4 ) . a total of 6,425 ( 7% ) patients had a codiagnosis of asthma and copd , including 4,730 ( 7% ) of those in the dose counter cohort and 1,695 ( 6% ) of those in the non - dose - counter cohort ( table 1 ) . the treatment cohorts were comparable with regard to demographic characteristics , comorbidities , and therapy . there was no significant difference between the two cohorts in the unadjusted rate of outcome respiratory - related ed visit rates for patients with asthma and copd ( reported for 42 [ 0.9% ] and 23 [ 1.4% ] in dose counter and non - dose - counter cohorts , respectively ; p=0.058 ; table 2 . outcomes were comparable between treatment cohorts in terms of exacerbation rates ( adjusted rr : 1.06 [ 0.971.16 ] ) and average daily saba dose ( adjusted or : 1.05 ; 95% ci : 0.921.20 ; table 4 ) . a total of 75,787 ( 81% ) patients had a diagnosis of asthma , including 53,964 ( 80% ) of those in the dose counter cohort and 21,823 ( 82% ) of those in the non - dose - counter cohort ( table 1 ) . the treatment cohorts were comparable at baseline in terms of demographic characteristics , comorbidities , and medication use ; a statistically significant difference in median age was not clinically significant ( table 1 ) . respiratory - related ed visits were recorded during the outcome period for 270 ( 0.5% ) and 244 ( 1.1% ) patients with asthma in the dose counter and non - dose - counter cohorts , respectively ( p<0.001 ; table 2 ) . those in the dose counter cohort had 51% lower incidence of respiratory - related ed visits ( adjusted rr : 0.49 ; 95% ci : 0.410.59 ) ( table 2 , figure 3 ) . average daily saba dose , severe exacerbation rates , and acute respiratory event rates were similar between treatment cohorts ( tables 3 and 4 , figure 3 ) . however , corresponding with the higher number of ambulatory visits resulting in antibiotic prescriptions ( table 3 ) , patients with asthma in the dose counter cohort had 4% lower odds of achieving risk - domain or overall asthma control than those in the non - dose - counter cohort ( table 4 , figure 3 ) . a total of 6,687 ( 7% ) patients had copd , including 4,953 ( 74% ) in the dose counter cohort and 1,734 ( 26% ) in the non - dose - counter cohort ( table 1 ) . the treatment cohorts were comparable for patients with copd with regard to demographic characteristics , comorbidities , and therapy . respiratory - related ed visits were recorded during the outcome period for 16 ( 0.3% ) and 23 ( 1.3% ) patients with copd in the dose counter and non - dose - counter cohorts , respectively ( p<0.001 ; table 2 ) . patients with copd in the dose counter cohort had 60% lower adjusted respiratory - related ed visit rates versus patients in the non - dose - counter cohort ( adjusted rr : 0.40 ; 95% ci : 0.220.75 ) ( table 2 ) . other copd outcomes were comparable between cohorts , including copd exacerbation rates ( adjusted rr : 1.05 ; 95% ci : 0.971.13 ) and average daily saba dose ( adjusted or : 1.08 ; 95% ci : 0.941.25 ) ( table 4 ) . a total of 6,425 ( 7% ) patients had a codiagnosis of asthma and copd , including 4,730 ( 7% ) of those in the dose counter cohort and 1,695 ( 6% ) of those in the non - dose - counter cohort ( table 1 ) . the treatment cohorts were comparable with regard to demographic characteristics , comorbidities , and therapy . there was no significant difference between the two cohorts in the unadjusted rate of outcome respiratory - related ed visit rates for patients with asthma and copd ( reported for 42 [ 0.9% ] and 23 [ 1.4% ] in dose counter and non - dose - counter cohorts , respectively ; p=0.058 ; table 2 . outcomes were comparable between treatment cohorts in terms of exacerbation rates ( adjusted rr : 1.06 [ 0.971.16 ] ) and average daily saba dose ( adjusted or : 1.05 ; 95% ci : 0.921.20 ; table 4 ) . in this investigation of the impact of an integrated dose counter in albuterol inhalers , the incidence rate of respiratory - related ed visits during the outcome year was estimated to be 45% lower in the dose counter cohort than in the non - dose - counter cohort . this result remained significant after splitting the population by single diagnosis : a 51% lower incidence rate was seen in patients with asthma and a 60% lower rate in those with copd . there was no statistically significant difference in ed visit incidence rate between cohorts for the small subpopulation of patients with codiagnosis of asthma and copd . using a dose counter device was not associated with changes in indicators of disease control , including average daily saba dose , which was not significantly different between the dose counter and non - dose - counter cohorts , for either the overall population or the subpopulations . we can speculate that the lack of difference in exacerbations may be because ed visits were relatively low in number as compared with the other components of the composite exacerbation variables , which included inpatient admissions and oral corticosteroid courses in the case of asthma exacerbations , and also antibiotics prescribed following a general practitioner visit for lower respiratory tract infection in the case of copd exacerbations . moreover , patients in the dose counter cohort of the asthma subpopulation had 4% lower odds of achieving overall asthma control in the outcome period . this was because they had a higher number of ambulatory visits for lower respiratory tract infections with antibiotic prescriptions , a component of the composite asthma control measures . our data suggest that dose counters may enable patients to directly know when their rescue medication is empty and thus avoid using empty inhalers during exacerbations . this knowledge may help to reduce the need for acute health care interventions such as ed visits . however , although this significant difference ( halving ) in ed visits has important clinical consequences , ed visits were a relatively rare event ( absolute rates were low ) in a relatively mild disease population ( those receiving first prescribed rescue medication in the study period and likely to be newly diagnosed patients ) . for patients with more severe disease , such a reduction in costly ed visits may be more important because higher health care costs are associated with greater asthma severity,17 and a substantial proportion of the health care costs associated with copd accrue from patients with frequent ed visits for acute copd exacerbations.18 prior studies of patients with asthma or copd have reported high levels of patient satisfaction ( > 90% ) with the use of dose counters on mdis containing rescue medication , specifically with regard to the ability to know when the inhaler should be replaced.1,2 in a recent online survey of 590 adults and children with asthma , many of whom were found to have empty or expired reliever inhalers , the addition of a dose counter was named most frequently as a means of improving satisfaction with their reliever inhalers.19 in the future , newer technologies may improve patient engagement with their therapy , and gains in disease management may be possible if the rescue dosing data are better integrated into practice . for example , in a recent study , telemonitoring of saba use via a patient - facing smart - phone app , with dose reporting to providers , was associated with decreased use of rescue medication and improved asthma control among those adults initially lacking asthma control.20 an important limitation of this study is the nonrandom treatment assignment , an issue common to all observational studies.21,22 although adjustment for potential confounders was performed wherever feasible , there remains the possibility of unrecognized bias or confounding . for example , we could not determine whether patients with both asthma- and copd - related claims should have been defined as having asthma - copd overlap syndrome.23 moreover , information on spacer use would have been of interest to consider . strengths of the study include , 1 ) the large patient population , 2 ) the fact that all patients were members of an employed , commercially insured population ( thus of similar socioeconomic status ) , and 3 ) the fact that the two cohorts ( dose counter and non - dose - counter ) were in the same insurance plan ( thus affordability was likely similar for all ) . however , the generalizability of study findings is limited to patients receiving a first prescription for albuterol and to employed , commercially insured patients such as those who were included in the database . further studies are needed to explore the use of integrated dose counters for other patient populations . we found that the integration of dose counters into rescue inhaler devices is associated with decreased ed visit frequency . the presence of integrated dose counters on rescue inhalers can help patients avoid using an empty or near - empty inhaler during exacerbations , thereby ensuring available medication for relief of their symptoms . the integration of dose counters on rescue mdis could represent a simple and effective tool to improve clinical outcomes during exacerbations , with a potential for cost savings to health care systems .

backgroundusing a metered - dose inhaler ( mdi ) beyond the labeled number of actuations may result in inadequate dosing of medication , which can lead to poor clinical outcomes . this study compared respiratory - related emergency department ( ed ) visit rates in patients with asthma , chronic obstructive pulmonary disease , or both when they used albuterol mdis with versus without dose counters.methodsthis retrospective study used us claims data to identify patients ( ages 464 years ) with asthma , chronic obstructive pulmonary disease , or both , using albuterol mdis with or without an integrated dose counter . the study comprised a 1-year baseline period for patient characterization and confounder definition and a 1-year outcome period following the first albuterol prescription . the primary end point was the incidence rate of respiratory - related ed visits , compared using a reduced zero - inflated poisson regression model . we also compared severe exacerbation rates and rescue medication use.resultsa total of 93,980 patients were studied , including 67,251 ( 72% ) in the dose counter cohort and 26,729 ( 28% ) in the non - dose - counter cohort . the cohorts were broadly similar at baseline ( 55,069 [ 59% ] female patients ; median age , 37 years ) . the incidence rate of respiratory - related ed visits during the outcome year was 45% lower in the dose counter cohort than in the non - dose - counter cohort ( adjusted rate ratio : 0.55 ; 95% confidence interval : 0.470.64 ) . exacerbation rates and short - acting -agonist use were similar between cohorts.conclusionthese findings suggest that dose counter integration into albuterol mdis is associated with decreased ed visit rates . the presence of integrated dose counters on rescue inhalers can help patients avoid using an empty or near - empty inhaler during exacerbations , thereby ensuring available medication for relief of their symptoms . integrated dose counters on rescue mdis could represent a simple and effective tool to improve clinical outcomes during exacerbations , with a potential for cost savings to health care systems .

Introduction Methods Study design Patients Outcomes Statistical analysis Results Asthma subpopulation COPD subpopulation Asthma and COPD subpopulation Discussion Conclusion

PMC3850614

the relationships between obesity and cardiovascular disease ( cvd ) and type 2 diabetes mellitus ( t2 dm ) are currently understood in terms of systemic changes that excess adipose tissue can induce in the physiologic and metabolic risk markers for these diseases . adipose tissue products are involved in the pathogenesis of essential hypertension ; moreover , as an endocrine organ , through its release of various adipokines , adipose tissue can influence the transport and metabolism of lipids and lipoproteins [ 2 , 3 ] , glucose metabolism , and insulin sensitivity [ 4 , 5 ] and can promote subclinical inflammation . variation in regional adipose tissue distribution may significantly affect risk markers for t2 dm and cvd and risk of these diseases , with stronger associations for central obesity than for generalised obesity [ 712 ] . elucidation of relationships between adiposity and physiologic and metabolic variables is important for our understanding of the role of increasing adiposity in health and disease , and evaluation of relationships between adiposity and risk markers is an important aspect of cvd and t2 dm risk evaluation . choice of adiposity measure is clearly a consideration in such investigations , and this can be informed by knowledge of the strength of relationship between a given adiposity measure and established cvd and t2 dm risk markers . therefore , it is important to establish whether simpler measures of adiposity can provide as much information as measures that are more complex and more theoretically rigorous . here , we test the hypothesis that , in a cohort restricted to white males , body mass index ( bmi ) , a frequently used , simple measure of net adiposity , will correlate with risk markers relating to blood pressure , lipid and lipoprotein metabolism , insulin sensitivity , and subclinical inflammation as strongly as more sophisticated measures . these include a measure of body fat mass derived from four skinfold thickness measures and direct measures of total and regional fat mass derived using dual energy x - ray absorptiometry ( dxa ) . the heart disease and diabetes risk indicators in a screened cohort ( hddrisc ) study is an open cohort study of 1192 white males recruited as part of a company health screening program . skinfold thicknesses were measured from the beginning of the study to 1996 and dxa body fat masses from 1989 to the end of the study . the majority of those who had skinfolds measured underwent an oral glucose tolerance test ( ogtt ) and a minority underwent an intravenous glucose tolerance test ( ivgtt ) . among those who had dxa measurements of body fat mass , very few underwent an ogtt , but the majority underwent an ivgtt . each dataset included participants who were nondiabetic ( fasting plasma glucose , fpg , < 7.0 mmol / l ) and not grossly obese ( bmi < 35.0 kgm , corresponding to 3sd above the mean for baseline bmi measurements in the entire cohort ) and received measurements of bmi , blood pressure , fasting serum total , low - density lipoprotein ( ldl ) and high - density lipoprotein ( hdl ) cholesterol , triglycerides , fasting plasma glucose and insulin , and laboratory measures related to inflammation . datasets were then distinguished as follows : ( 1 ) the 349 men who received measurement of skinfold thicknesses and assessment of insulin sensitivity by ogtt ( bmi / sf group studied between 1972 and 1991 ) ; ( 2 ) the 269 men who received measurement of dxa fat masses and assessment of insulin sensitivity by ivgtt ( bmi / dxa group studied between 1989 and 1997 ) ; ( 3 ) the 156 men who received measurement of both skinfold thicknesses and dxa fat masses and assessment of insulin sensitivity by ivgtt ( bmi / sf / dxa group studied between 1989 and 1996 ) . the majority of participants in the hddrisc study group were senior executives of a large company . the study received local ethics committee approval , and each participant gave their written informed consent . all participants fasted overnight ( only water ) and refrained from smoking and alcohol prior to attending a dedicated metabolic ward . here , a full medical history was taken , including details of smoking habits , alcohol intake , and exercise behaviour . weight and height were measured in light clothing without shoes . among those receiving skinfold measurements , briefly , three measurements were taken , using harpenden callipers , at biceps , triceps , iliac , and subscapular sites . all readings were measured to the nearest mm , unless values were particularly low ( 5 mm ) in which case the nearest 0.5 mm was recorded . among those undergoing measurement of total and regional body fat tissue by dxa , scans were carried out on a lunar radiation corporation ( madison , wi , usa ) dpx scanner with total body scanning carried out at a transverse speed of 16 cm / s or 8 cm / s in men who weighed > 90 kg , with subjects receiving radiation doses of 0.05 or 0.1 gy , respectively . after 15-minute rest , systolic and diastolic blood pressures ( bp ) were taken using a mercury sphygmomanometer . then , with participants in a semirecumbent position , an indwelling cannula was inserted in the antecubital vein of the nondominant arm for blood sampling . blood samples were taken for routine haematology and clinical biochemistry measurements , including white blood cell count ( wbc ) , erythrocyte sedimentation rate ( esr ) , serum globulin and albumin concentrations , serum lipid and lipoprotein concentrations ( cholesterol , triglycerides , and hdl cholesterol ) , and plasma glucose and insulin concentrations ( fpg and fpi , resp . ) . a second sample for fpg and fpi concentration measurements for the ogtt , a glucose load of 1 g / kg body weight was given , with plasma glucose and insulin measured at 30 , 60 , 90 , 120 , 150 , and 180 minutes , following consumption of the glucose solution . for the ivgtt , as previously described , a second cannula was inserted , in this case in the antecubital vein in the arm opposite to the sampling arm , through which a glucose load of 0.5 g / kg body weight was given as 50 percent dextrose . blood sampling was at 3 , 5 , 7 , 10 , 15 , 20 , 30 , 45 , 60 , 75 , 90 , 120 , 150 , and 180 minutes following injection of glucose , and there was no augmentation of accompanying insulin concentrations . these measurements were made using standard laboratory methodology in our own laboratories or local hospital chemical pathology laboratories . plasma glucose and insulin and serum total , hdl cholesterol , and triglyceride concentrations were measured in our own laboratories as described previously . quality control was monitored with commercially available lyophilized sera and by participation in national schemes . between - batch assay coefficients of variation were plasma glucose < 3% ; plasma insulin < 6% ; serum cholesterol and triglycerides < 2% ; serum hdl cholesterol < 4% ; serum globulin and albumin concentrations < 6% . throughout the course of the hddrisc study , changes in measurement methodology were accompanied by detailed comparisons between methods , with application of correction factors if necessary to maintain continuity of measurement standardization . on completion of data collection for the study , detailed checks were also made to exclude the possibility of laboratory drift significantly affecting results . the mean of the three skinfold thickness measurements was calculated , and percent body fat ( sf % fat ) was calculated from the individual means for the four skinfold thickness measures , according to the method of durnin and womersley . body fat mass ( sf total fat ) was then calculated as body weight ( sf % fat/100 ) . the dxa - derived measurement of total body fat mass ( dxa total fat ) was expressed as a percentage of body weight ( dxa % fat ) . proprietary software was used to distinguish upper body or android fat ( dxa android ) and lower body or gynoid fat ( dxa gynoid ) in the dxa scan . the region representative of android fat was defined by an upper horizontal border underneath the chin with vertical borders passing down beside the ribs and a lower border formed by diagonal lines passing through the hip joints and meeting at the perineum . the region representative of gynoid fat was defined as the tissue below these diagonal lines . android or gynoid fat was expressed as a percentage of dxa total fat ( dxa % android and dxa % gynoid , resp . ) . the cv for dxa fat was 1.8% , while cvs for the regional measurements were all < 5% . current cigarette smoking was categorised as 0 , < 5 , 514 , 1524 , and > 24 cigarettes per day alcohol intake as 0 , < 28 , 2856 , and > 56 units per week and exercise habit as no regular , regular nonaerobic , and regular aerobic exercise . serum low density lipoprotein ( ldl ) cholesterol concentration was calculated by the friedewald method . the homeostasis model assessment index of insulin resistance ( homa - ir ) derived from fpg and fpi was calculated . an ogtt - derived index of insulin sensitivity was provided by the matsuda index ( matsuda - si ) . an ivgtt - derived measure of insulin sensitivity , si , ( ivgtt - si ) was provided by minimal model analysis of ivgtt glucose and insulin concentrations , implemented according to an optimised algorithm , as described previously . all data was analysed using stata 8 for windows ( stata , college station , tx , usa ) . summary measures were median and interquartile range for continuous variables and percentages for categorical variables . for parametric analyses , anova for continuous variables and chi - square for categorical variables were used to identify significant variation in bmi and risk marker measures between the groups studied . dummy variables were assigned for each cigarette smoking , alcohol intake , and exercise category . relationships between adiposity measures and between each adiposity measure and each risk marker were explored by partial correlation , taking into account covariation with age , cigarette smoking , alcohol intake , and exercise habit . a significance level of p < 0.05 was adopted with no correction for multiple testings , each correlation undertaken being strongly weighted by existing evidence , thus rendering the universal null hypothesis inapplicable . these analyses were repeated with exclusion of measurements lying > 3sd outside the mean ( theoretically excluding 0.3% of the data ) to minimise any leverage due to outliers . the magnitude and differences in the strengths of the relationships between measures of total body fat and risk markers were explored by comparing confidence intervals for regression coefficients derived from regression analyses incorporating age , cigarette smoking , alcohol intake , and exercise . ages ranged between 26 and 79 years and bmi between 18.4 and 34.9 kg / m . of the 349 men in the bmi / sf group , 65 percent were nonsmokers and 55 percent had an alcohol intake less than 28 u / wk , 1.2 percent were taking blood pressure - lowering agents and 0.3 percent lipid - lowering agents . there was significant variation between the groups in age ( p < 0.001 ) , those in the bmi / sf group having a median age of 47.2 years in contrast with 49.4 in the bmi / dxa group and 54.2 in the bmi / sf / dxa group . 0.001 ) although median bmi in the three groups only varied between 25.1 and 25.5 kgm . smoking ( p < 0.001 ) , alcohol intake ( p < 0.001 ) , and exercise ( p < 0.01 ) also varied between the groups , with heavier smoking , greater alcohol intake , and more exercise evident in the bmi / sf group than in the bmi / dxa or bmi / sf / dxa groups . ( p < 0.001 ) were higher in the bmi / sf group , and , on the basis of higher levels of esr and globulin and lower albumin levels ( p < 0.001 ) , there was evidence of greater inflammation in the bmi / sf group . bmi was strongly associated with sf total fat , sf % fat , dxa total fat , and dxa % fat ( partial correlations 0.680.84 , p < 0.001 ) . bmi was also strongly associated with dxa android and gynoid fat ( partial correlations 0.680.81 , p < 0.001 ) . somewhat weaker associations were seen between bmi and individual skinfold thicknesses ( partial correlations 0.370.69 , p < 0.001 ) , and the weakest associations were seen between bmi and dxa % android and dxa % gynoid fat ( partial correlations 0.30 and 0.30 , resp . , p < 0.001 ) . dxa % android and dxa % gynoid fat were inversely correlated with an r value of 0.91 ( p < 0.001 ) . adiposity measures lying outside 3sd from the mean from these analyses made only very minor differences to the associations observed . our three study groups represent three samples of relationships between bmi and risk factors and two samples of relationships between skinfold thickness - derived measures and dxa - derive measures and risk factors . relative strengths of association according to each adiposity measure are only considered for those risk factors for which each sample showed a significant relationship . associations for sf and dxa percent fat and risk factors did not differ from those for sf total and dxa total fat and risk factors . associations between adiposity measures and total and ldl cholesterol , wbc , globulin , and albumin only exhibited isolated significances that were inconsistent between samples . measures lying outside 3sd from the mean from these analyses made only very minor differences to the associations observed . associations in the bmi / sf / dxa group , independent of age , cigarette smoking , alcohol intake , and exercise habit , between adiposity measures and the key risk factors for which the strongest associations were apparent , ivgtt - si , triglycerides , and systolic bp , are illustrated by standardised regression coefficients in figure 1 . associations between systolic and diastolic bp and bmi and skinfolds in the bmi / sf group were relatively weak ( r < 0.20 ) , the most consistent associations being seen for diastolic bp , with little differences in strength of association between the different adiposity measures ( table 2 ) . stronger associations were apparent in the bmi / dxa group ( r = 0.31 , p < 0.001 for bmi with both systolic and diastolic bp : table 3 ) and in the bmi / sf / dxa group ( r = 0.34 and 0.29 , p < 0.001 for systolic and diastolic bp , resp . , the strongest associations between blood pressure and adiposity measures were seen with bmi . in each of the three groups studied , significant associations were apparent between triglyceride concentrations and all measures of adiposity except triceps skinfold and dxa gynoid fat in the bmi / sf / dxa group ( tables 24 ) . the strongest associations between triglycerides and adiposity measures were seen with bmi ( bmi / sf group , r = 0.24 ; bmi / dxa group , r = 0.41 ; bmi / sf / dxa group , r = 0.34 , all p < 0.001 ) . similar associations were apparent between hdl cholesterol and adiposity measures , but weaker and in the opposite direction . in the bmi / sf group , significant negative associations were apparent between matsuda si and all measures of adiposity , with the strongest association being seen with bmi ( r = 0.33 , p < 0.001 , table 2 ) . similar associations were apparent between homa - ir , fpi , and adiposity measures , but weaker and in the opposite direction . in the bmi / dxa group significant negative associations were apparent between ivgtt - si and all measures of adiposity , with the strongest association being seen with dxa android fat ( r = 0.47 , p < 0.001 , table 3 ) . strong associations were also seen with bmi and dxa total fat ( r = 0.42 and 0.46 , resp . , p < 0.001 ) . similar associations were apparent between homa - ir , fpi , and adiposity measures , but weaker and in the opposite direction , and associations similar to those in the bmi / dxa group were seen in the bmi / sf / dxa group . the only consistent associations between inflammation - related measures and measures of adiposity were between esr and dxa total and android fat ( r = 0.160.19 , p < 0.05 , tables 3 and 4 ) . to explore whether there was any contribution of regional body fat to risk marker variation independent of variation in total fat , each skinfold or dxa measure of regional fat this analysis was restricted to the bmi / sf / dxa group , in which the strongest associations were seen between adiposity measures and risk markers . associations between risk markers and sf total fat were relatively unaffected by pairing with triceps , biceps , and supra - iliac skinfold measures , each of which showed few significant associations when paired with sf total fat . there was , however , evidence of an independent contribution of the subscapular skinfold to variation triglycerides and colinearity between sf total fat and subscapular skinfold with respect to ivgtt - si ( results not shown ) . with inclusion of dxa android fat with dxa total fat ( table 5 ) , both dxa total fat and dxa android fat ceased to be significantly associated with sbp , hdl cholesterol , and fpg , suggesting strong colinearity between dxa total fat and dxa android fat . there was , however , evidence of an independent effect of dxa android fat on measures of insulin sensitivity . with inclusion of dxa gynoid fat with dxa total fat ( table 5 ) , both dxa total fat and dxa gynoid fat remained associated with variation in serum triglycerides and insulin sensitivity - related measures , but the direction of association with dxa gynoid fat was reversed , rendering its associations similar to those seen with dxa % gynoid fat . dxa android and gynoid fat were also entered in a paired analysis of risk marker variation ( table 5 ) . in this analysis , dxa android fat remained significantly associated with a range of risk markers , particularly serum triglycerides and insulin sensitivity - related measures , but associations between dxa gynoid fat and risk markers were eliminated for all risk markers except dbp , triglycerides , and albumin . our findings confirm our hypothesis that , in this group of adult males , bmi , a simple measure of overall adiposity , can account for variation in a broad range of adiposity - related risk markers as effectively as more sophisticated measures derived from skinfold or dxa measurements . to the best of our knowledge , this is the first study to compare in a single investigation bmi , skinfolds , and dxa as correlates of risk marker variation in white adult males . one other study , that is of steinberger and colleagues , combined bmi , skinfold , and dxa adiposity measures , but in 72 adolescent males and 58 adolescent females of white or black race and with insulin sensitivity being measured by the euglycaemic hyperinsulinaemic clamp . despite these differences , their conclusions agree with our finding that bmi is as effective at accounting for risk marker variation as skinfold or dxa - derived measures . other studies , that have included dxa but not skinfold thickness measurements , have reported little difference in strength of association between simple anthropometric and dxa measures of adiposity with respect to risk marker variation [ 2428 ] , and there is also evidence that this is the case with regard to studies including bmi and skinfold but not dxa measurements [ 29 , 30 ] . moreover , there is even evidence that risk marker associations with simple , anthropometric measures of adiposity can be comparable to those with specific fat depots quantified by magnetic resonance imaging ( mri ) . nevertheless , a number of other studies in which specific fat depots have been measured by mri or computed tomography have raised the possibility that the visceral fat depot , with its circulation draining into the hepatic portal vein , is particularly associated with risk marker variation [ 3234 ] . it has been proposed that variation specifically in the visceral fat depot can account for discrepancies in associations between risk markers and overall adiposity , with some obese individuals being metabolically healthy and some individuals with apparently normal levels of adiposity exhibiting a metabolic phenotype typical of obesity . however , a number of other studies have found risk marker variation to be equally associated with variation in visceral or subcutaneous fat depots [ 3640 ] . the relative importance of the visceral and subcutaneous fat depots in risk marker variation could vary according to age , race , or gender and may be affected by variation in the strengths of relationship according to degree of overweight , with the visceral fat depot assuming more importance in overt obesity [ 41 , 42 ] . recruitment was in the context of a long - running , open cohort study and the participants and the periods during which data was being collected differed between the three groups studied . consequently , results were based on three cross - sectional samples , which differed in demographic and risk factor characteristics . it should be noted , however , that relative strengths of association between measures of adiposity and risk factors were generally comparable between cross - sections , which lends support to their validity . a further potential limitation of our study was that waist and hip circumferences were not measured . various anthropometric measures of centrally concentrated adiposity , including waist circumference , waist hip ratio , and waist height ratio , have been explored in previous studies in relation to clinical outcomes and have generally been found to predict clinical outcomes more strongly than bmi [ 9 , 43 ] , although the differential in improvement may be small [ 8 , 44 ] . it is questionable , however , whether anthropometric measures of centrality would have provided any more information than that provided by the dxa measurements of android fat included in our study . nevertheless , given that central fat measures are more predictive of clinical outcomes and that the central fat measure in our study showed little difference from bmi in the strengths of its relationships with the risk factors we measured , it may be inferred that there are factors associated with variation in central fat that we did not measure but which impact strongly on clinical outcomes [ 45 , 46 ] . a particular advantage of our analysis was the relatively low use of medications likely to affect risk marker variation , particularly use of lipid - lowering agents , the majority of measurements having been carried out before the inception of the current , widespread use of these agents . a further advantage was the broad range of risk markers available for analysis , which allowed for strong confirmation of the relative strengths of association between risk markers and adiposity measures . the three indices of total body fat , bmi , sf total fat , and dxa total fat were closely correlated , as was subscapular skinfold with sf total fat and dxa android and gynoid with dxa total fat . risk marker associations suggested that subscapular skinfold alone might predict risk marker variation as effectively as a measure of total adiposity derived from all four skinfolds . as mentioned , dxa android fat conveyed as much if not more information in this respect as did dxa total fat and risk marker associations with dxa % android followed closely on those with dxa android fat . however , the pattern of associations with dxa gynoid fat was markedly different in that dxa % gynoid fat showed opposite directions of association with risk markers to those seen with dxa gynoid fat mass . the strong positive association between dxa gynoid and dxa total fat masses suggests that the dxa gynoid measure incorporates a strong component of variation that simply reflects net adiposity and this could account for the unfavourable associations seen between risk markers and dxa gynoid fat . rendering dxa gynoid as a percentage of body fat , however , distinguished variation in gynoid fat from variation in total fat . as a result of making this distinction , it was apparent that there may be a component of variation in gynoid or lower body fat that has a favourable effect on risk marker variation , as has been suggested by previous studies [ 4751 ] . however , dxa % gynoid fat was also strongly , inversely , associated with dxa % android fat , and it could be argued that a high percentage of gynoid fat merely reflects a low percentage of android fat ( and vice versa ) ; the favourable associations seen between risk markers and dxa % gynoid are then merely secondary to variation in android fat . the analyses we present in table 5 , in which risk marker associations with dxa gynoid fat largely disappeared with inclusion of dxa android fat , which supports the possibility that the favourable associations seen between risk markers and dxa % gynoid are merely secondary to variation in android fat . if dxa gynoid fat had been having an underlying , independent , favourable effect on risk marker variation , these favourable associations would have been expected to remain despite inclusion of android fat in the analysis . overall , the strongest associations seen with variation in any measure of adiposity were with measures of insulin sensitivity , followed by serum triglyceride , hdl cholesterol concentrations , and then blood pressure . serum cholesterol and ldl cholesterol concentrations varied inconsistently with adiposity [ 52 , 53 ] . consistent relationships between adiposity and indices of inflammation were restricted to positive associations between dxa total and android fat and esr . in contrast to our findings , significant associations between white cell count and bmi have been reported , but their magnitude can be relatively small . adiposity - related inflammation relates to macrophage infiltration and activation and is associated with elevated levels of specific cytokines including interleukin-6 , tumour necrosis factor - alpha , and acute phase proteins such as c - reactive protein . it is possible that , had we measured these , stronger associations would have been apparent . it is , nevertheless , noteworthy that in this cohort the inflammation markers we evaluated are significantly related to clinical outcomes [ 56 , 57 ] . in conclusion , bmi can account for variation in risk markers in white , predominantly middle - aged males as well as more sophisticated measures derived from skinfold thickness measurements or dxa scanning .

background . choice of adiposity measure may be important in the evaluation of relationships between adiposity and risk markers for cardiovascular disease and diabetes . aim . we explored the strengths of risk marker associations with bmi , a simple measure of adiposity , and with measures provided by skinfold thicknesses and dual energy x - ray absorptiometry ( dxa ) . subjects and methods . we evaluated in three subgroups of white males ( n = 156349 ) , participating in a health screening program , the strengths of relationship between measures of total and regional adiposity and risk markers relating to blood pressure , lipids and lipoproteins , insulin sensitivity , and subclinical inflammation . results . independent of age , smoking , alcohol intake , and exercise , the strongest correlations with adiposity measures were seen with serum triglyceride concentrations and indices of insulin sensitivity , with strengths of association showing little difference between bmi and skinfold and dxa measures of total and percent body fat ( r = 0.200.46 , p < 0.01 ) . significant but weaker associations with adiposity were seen for serum hdl cholesterol and only relatively inconsistent associations with adiposity for total and ldl cholesterol and indices of subclinical inflammation . conclusions . bmi can account for variation in risk markers in white males as well as more sophisticated measures derived from skinfold thickness measurements or dxa scanning .

1. Introduction 2. Subjects and Methods 3. Results 4. Discussion

PMC3467303

the normal development of the male gametophyte of some plant species can be redirected into embryogenic development , resulting in the formation of embryos able to differentiate into haploid plants ( reynolds , 1997 ) . pollen embryogenesis is of importance in the breeding of certain crop species as it simplifies the fixation of meiotic recombination events via the doubling of the chromosome complement of haploid regenerants . barley is particularly amenable to pollen embryogenesis , a feature which has helped achieve the crop s status as the leading model temperate cereal ( maraschin et al . , 2005a ) . defining reliable structural markers for pollen embryogenesis , which has proved to be difficult to date ( pechan and smykal , 2001 ) , requires the in vivo microscopy of cultured immature pollen ( krens et al . , 1998 ) . previous efforts to track the fate of individual pollen ( bolik and koop , 1991 ; kumlehn and lrz , 1999 ; indrianto et al . , 2001 ; maraschin et al . , 2005b , 2008 ) suffered from the inadequate delivery of signalling molecules produced by co - cultivated embryogenic pollen , a process which is inevitably compromised when the target pollen grains have to be immobilized in a gel matrix . signalling has been shown to be essential for the definition of cell identity and proliferation in plant cell cultures ( kumlehn and lrz , 1999 ; kumlehn et al . , 1999 ) . in addition , these experiments have had to rely on the acquisition of a series of images , in which the time - interval between their capture has been of the order of several hours ; such a long separation can be expected to fail to record a number of important cellular events . a method whereby immature barley pollen , arranged in a monolayer within a liquid medium , develops normally and its growth can be followed over a period of several weeks is demonstrated here . the automated capture of z - stacks of images allowed for an unprecedented level of temporal resolution to be achieved . the method should prove valuable for elucidating the ultrastructural features and the molecular machinery important for the initiation of pollen embryogenesis and other cellular processes occurring in developing pollen . ( 2005 ) was applied to raise barley ( hordeum vulgare l. ) cv . igri ( saatzucht ackermann , irlbach , germany ) plants as a source of immature pollen amenable to pollen embryogenesis . construction of a live - cell chamber was based on a two - well chambered coverglass ( thermo scientific - cat . 155380 ) in combination with an 0.4 m hydrophilic , diaphanous ptfe membrane of a 12 mm millicell cell culture insert ( millipore - cat . picm01250 ) ( hereafter referred to as a ptfe membrane ) and a plastic mask fabricated from a countess cell counting chamber slide ( invitrogen , cat . 1e ) adjusted in size to fit precisely within the well of the chambered coverglass ( fig . . a square hole of size approximately 77 mm was cut in its centre using a heated scalpel blade ( fig . one of the ptfe membranes was then placed over the square hole and , using a pair of heated forceps , the edge of the membrane was fused to the plastic mask ( fig . ( b ) a 12 mm millicell cell culture insert with its 0.4 m hydrophilic ptfe membrane . ( c ) a ptfe membrane in the process of being detached by forceps . 200 000ml immature barley pollen was concentrated 10-fold by removing 900 l of medium from a 1ml volume using a 30 m mesh - coated 1ml pipette tip ( fig . approximately 100 l concentrated cell suspension was pipetted onto the lid of a plastic petri dish ( fig . 2c ) , which ensured that the droplet took on a convex shape . the droplet was left undisturbed for 35min to allow dead cells and detritus to precipitate while the viable cells accumulate at the surface of the droplet . then , a ptfe membrane was brought into contact with the droplet for 23 s , allowing a thin layer of medium containing viable cells to adhere . the membrane was blotted gently with a filter paper to remove as much of the culture medium as possible , and was then placed face down in the well of the chambered coverglass ( fig . 2d ) , avoiding the formation of air bubbles between the membrane and the cover slip . this created a monolayer of immature pollen cells in direct contact with the cover slip ( fig . 2d , 2j ) . 50 l of starvation medium ( smb1 ) ( coronado et al . , 2005 ) semi - solidified by the addition of 6 mg ml agarose ( type i , low eeo ; sigma - aldrich , steinheim , germany ) ( fig . after a second plastic mask was placed above the agarose layer ( fig . 2f , 2 g ) , 1ml of embryogenic pollen culture was pipetted into the chamber to ensure a sufficient supply of signalling molecules for the test pollen ( fig . 2i ) . the combination of agarose and the second mask ensured that the optical focal plane was free of contamination from the feeder cells ( fig . in order to avoid interference from condensation on the inner surface of the chamber lid , a smooth edged 1515 mm gap was cut into the lid of the chamber and covered with a sterile cover slip , which was replaced every day ( fig . the set - up ensured that the test pollen remained immersed in liquid culture medium , thereby providing suitable culture conditions . after 2 d , the starvation medium was withdrawn using a 30 m mesh - coated 1ml pipette tip ( fig . 1h ) , and replaced by 1ml kbp nutrient medium ( coronado et al . , this step had to be done with care to avoid mechanical disturbance of the test pollen . schematic illustrations of the home - made live - cell chamber cover slip used for time - lapse recording of pollen embryogenesis . ( c ) a droplet of highly concentrated pollen suspension on the hydrophobic lid of a plastic petri dish . ( d ) a monolayer of pollen adhering to a ptfe membrane positioned , along with a plastic mask , within a chamber coverglass . ( e ) covering the ptfe membrane with smb1 medium semi - solidified with agarose . ( i ) the addition of 1ml pollen suspension and the covering of the hole with a cover slip ; the live - cell chamber slide is now ready for monitoring . ( j ) the pollen monolayer is sandwiched between the bottom slide of the live - cell chamber and a ptfe membrane . ip , immature pollen . in a series of preliminary trials , in which non - immobilized cultured pollen were used as a control , it was established that neither 5min nor 3min interval recordings had any detrimental effect on pollen vitality and development . reducing the time interval to only 2min , however , caused the cultures to die between 35 d after the start of the recordings . therefore the 3min time - interval was selected as the standard to perform live - cell imaging of pollen embryogenesis . z - stacks of nine images with a total scanning time of 40 s were acquired every 3min . pollen development was monitored for a period of up to 28 d using a hene 633 nm laser line . the optical interval of the z - stacks was gradually increased from 2 m to 4 m over the course of the monitoring period to take account of the expansion of the test pollen . in vivo microscopy was performed using a zeiss lsm 510 meta ( carl zeiss , jena , germany ) equipped with a 20 objective ( na 0.75 ) . the microscope was installed in a dark room kept at a constant 25 c to avoid focus drift . from every individual z - stack finally , 5069 images were used to compile a time - lapse movie depicting the course of pollen development ( see supplementary video s1 at jxb online ) . ( 2005 ) was applied to raise barley ( hordeum vulgare l. ) cv . igri ( saatzucht ackermann , irlbach , germany ) plants as a source of immature pollen amenable to pollen embryogenesis . construction of a live - cell chamber was based on a two - well chambered coverglass ( thermo scientific - cat . 155380 ) in combination with an 0.4 m hydrophilic , diaphanous ptfe membrane of a 12 mm millicell cell culture insert ( millipore - cat . picm01250 ) ( hereafter referred to as a ptfe membrane ) and a plastic mask fabricated from a countess cell counting chamber slide ( invitrogen , cat . 1e ) adjusted in size to fit precisely within the well of the chambered coverglass ( fig . 1 g ) . a square hole of size approximately 77 mm was cut in its centre using a heated scalpel blade ( fig . one of the ptfe membranes was then placed over the square hole and , using a pair of heated forceps , the edge of the membrane was fused to the plastic mask ( fig . ( b ) a 12 mm millicell cell culture insert with its 0.4 m hydrophilic ptfe membrane . ( c ) a ptfe membrane in the process of being detached by forceps . 200 000ml immature barley pollen was concentrated 10-fold by removing 900 l of medium from a 1ml volume using a 30 m mesh - coated 1ml pipette tip ( fig . approximately 100 l concentrated cell suspension was pipetted onto the lid of a plastic petri dish ( fig . 2c ) , which ensured that the droplet took on a convex shape . the droplet was left undisturbed for 35min to allow dead cells and detritus to precipitate while the viable cells accumulate at the surface of the droplet . then , a ptfe membrane was brought into contact with the droplet for 23 s , allowing a thin layer of medium containing viable cells to adhere . the membrane was blotted gently with a filter paper to remove as much of the culture medium as possible , and was then placed face down in the well of the chambered coverglass ( fig . 2d ) , avoiding the formation of air bubbles between the membrane and the cover slip . this created a monolayer of immature pollen cells in direct contact with the cover slip ( fig . 2d , 2j ) . 50 l of starvation medium ( smb1 ) ( coronado et al . , 2005 ) semi - solidified by the addition of 6 mg ml agarose ( type i , low eeo ; sigma - aldrich , steinheim , germany ) ( fig . after a second plastic mask was placed above the agarose layer ( fig . 2f , 2 g ) , 1ml of embryogenic pollen culture was pipetted into the chamber to ensure a sufficient supply of signalling molecules for the test pollen ( fig . 2i ) . the combination of agarose and the second mask ensured that the optical focal plane was free of contamination from the feeder cells ( fig . in order to avoid interference from condensation on the inner surface of the chamber lid , a smooth edged 1515 mm gap was cut into the lid of the chamber and covered with a sterile cover slip , which was replaced every day ( fig . the set - up ensured that the test pollen remained immersed in liquid culture medium , thereby providing suitable culture conditions . after 2 d , the starvation medium was withdrawn using a 30 m mesh - coated 1ml pipette tip ( fig . 1h ) , and replaced by 1ml kbp nutrient medium ( coronado et al . , this step had to be done with care to avoid mechanical disturbance of the test pollen . schematic illustrations of the home - made live - cell chamber cover slip used for time - lapse recording of pollen embryogenesis . ( c ) a droplet of highly concentrated pollen suspension on the hydrophobic lid of a plastic petri dish . ( d ) a monolayer of pollen adhering to a ptfe membrane positioned , along with a plastic mask , within a chamber coverglass . ( e ) covering the ptfe membrane with smb1 medium semi - solidified with agarose . ( i ) the addition of 1ml pollen suspension and the covering of the hole with a cover slip ; the live - cell chamber slide is now ready for monitoring . ( j ) the pollen monolayer is sandwiched between the bottom slide of the live - cell chamber and a ptfe membrane . ip , immature pollen . in a series of preliminary trials , in which non - immobilized cultured pollen were used as a control , it was established that neither 5min nor 3min interval recordings had any detrimental effect on pollen vitality and development . reducing the time interval to only 2min , however , caused the cultures to die between 35 d after the start of the recordings . therefore the 3min time - interval was selected as the standard to perform live - cell imaging of pollen embryogenesis . z - stacks of nine images with a total scanning time of 40 s were acquired every 3min . pollen development was monitored for a period of up to 28 d using a hene 633 nm laser line . the optical interval of the z - stacks was gradually increased from 2 m to 4 m over the course of the monitoring period to take account of the expansion of the test pollen . in vivo microscopy was performed using a zeiss lsm 510 meta ( carl zeiss , jena , germany ) equipped with a 20 objective ( na 0.75 ) . the microscope was installed in a dark room kept at a constant 25 c to avoid focus drift . from every individual z - stack finally , 5069 images were used to compile a time - lapse movie depicting the course of pollen development ( see supplementary video s1 at jxb online ) . the movie shown here is representative of a total of eight individual trials lasting between 14 d and 28 d that were performed to prove the usefulness of the system ( see supplementary video s1 at jxb online ) . every trial comprised between 4 and 15 observed pollen and showed similar patterns of pollen development and similar pollen types were identified . without the possibility of agitating the culture medium , the rate of development was significantly delayed to the extent that it took as long as 28 d for embryogenic microcalli to emerge from the exine . in conventionally grown embryogenic pollen , exine breakage typically occurs in the third week of cultivation . nevertheless , the cultures appeared to retain a high level of viability which was not compromised by the recordings . tracking of four pre - mitotic pollen over the course of 28 d resulted in a set of 91 242 images . the first mitotic division occurred in pollen # 1 after 2 d ( fig . the nuclear division took place at the cell periphery opposite the pollen aperture and produced two equal - sized nuclei . over the course of the following days , a synchronized series of mitotic divisions occurred , generating a multicellular structure ( fig . despite this mitotic activity , the dimensions of this pollen remained constant over the first 14 d ( fig . h ) , but expanded considerably thereafter . in pollen # 2 , the first pollen mitosis occurred after 3 d ( fig . 3b ) , following this the large vacuole remained for approximately 10 d ( fig . 3c , 3d ) . thereafter , the pair of nuclei migrated towards the centre , so that the cytoplasm assumed a star - like pattern . no further mitoses were observed , and this pollen retained a constant size over the entire period of observation . starting at day 15 , detectable starch granules began to accumulate within the cells ( fig . pollen # 3 did not develop at all and collapsed on day seven ( fig . e ) before the first pollen mitosis took place at the cell periphery on day seven , resulting in the generation of a pair of similar - sized nuclei ( fig . several further synchronized series of mitoses subsequently converted this pollen grain into an expanding multicellular structure ( fig . none of the four test pollen described above had its exine ruptured by day 28 , although rupture was observed in some pollen grains of other trials that were run under the same conditions . for comparison , conventionally cultured embryogenic pollen ( non - immobilized , incubated in 35 mm petri dishes ) the results revealed that there are no significant differences between non - immobilized and immobilized pollen with respect to viability , developmental pattern , and ratio of identified cell types ( dead pollen , starch accumulated pollen , multicellular structures ) . representative example of in vivo microscopy of cultured immature pollen over a 28 d period . ( a i ) individual pollen # 1 and # 4 underwent pollen embryogenesis and generated a multicellular structure . ( e , f ) after just one mitotic division , development was arrested in pollen # 2 , and starch granules began to accumulate . ( f i ) pollen # 3 did not show any sign of development and died after 7 d. n , nucleus . bar=10 m . the entire series of images from which those shown in fig . 3 have been selected is available online in the form of a time - lapse video ( see supplementary video s1 at jxb online ) . the experimental set - up described here provided appropriate growing conditions while simultaneously permitting high - quality imaging of cellular events . it also allowed for the culture medium to be completely exchanged , which was not possible in previous attempts to perform time - lapse imaging where the targets were immobilized within a solidified medium ( kumlehn and lrz , 1999 ; indrianto et al . , 2001 ; maraschin et al . , 2005b ) and were thus subject to sub - optimal conditions since they had limited access to the signalling molecules produced by freely growing feeder cells . the presence of the ptfe membrane ensured that the test pollen were trapped within the liquid medium , while at the same time allowing for the free flow of nutrients and signalling molecules . since the test pollen were in close contact to the cover slip of the chamber coverglass , it was possible to use high numerical aperture objectives to acquire high quality images . it has been shown previously that well - spaced cells undergoing pollen embryogenesis can be successfully observed by light microscopy , since spacing avoids the problems of cell overgrowth and optical interference from non - target cells ( kumlehn and lrz , 1999 ; indrianto et al . , this requirement was satisfied by separating the monolayer of observed pollen from a highly concentrated ( 200 000 pollen ml ) feeder population , using a thin layer of solidified medium sandwiched between two permeable ptfe membranes . previous observations of the in vitro development of wheat ( indrianto et al . , 2001 ) and barley ( bolik and koop , 1991 ; kumlehn and lrz , 1999 ; maraschin et al . , 2005a , b , 2008 ) embryogenic pollen invariably used time - intervals of one to several hours . although this level of temporal resolution may be sufficient to identify major events , such long time - intervals are not well suited for the documentation of the full detail of events associated with pollen embryogenesis . the temporal resolution was increased here to 3min and our set - up has proved to be able to maintain the test pollen in a viable state for at least 28 d. cultured immature pollen characteristically develops at a variable rate , as illustrated by the differential performance of the four individual pollen shown in fig . 3 . three contrasting fates , as also outlined by maraschin et al . ( 2005a , b ) , were observed : some cells underwent proliferation , and subsequently formed embryogenic microcalli ; some were arrested in development after initial rounds of mitosis had occurred ; and some failed to develop at all . ( 2005a , b ) reported that , prior to the first mitosis of pollen - embryogenesis - competent pollen of wheat and barley , the nucleus migrates to a central position , leading to the cytoplasm taking on a star - like appearance within the highly vacuolated pollen . this feature has been considered to be a predictive marker for the initiation of pollen embryogenesis . by contrast , in both pollen # 1 and # 4 ( as well as in many other pollen not shown here ) , the first mitosis took place while the nucleus was still at the cell periphery . a star - like pattern did occur after the first pollen mitosis in pollen # 2 ( fig . 3e , 3f ) , which went on to accumulate starch rather than switch to embryogenic development . in our view , therefore , the formation of the star - like cytoplasm is not a reliable morphological marker for embryogenic pollen development . the goal of the 28 d study was a test - case to establish the technical limits of our set - up . in a typical experiment , such long running times are not necessary for detailed scientific observations focusing on the initiation of pollen embryogenesis but could be useful to study cell proliferation and embryo formation . we suggest that the improvement in efficiency and temporal resolution achieved by the cell culture method described here will facilitate a more comprehensive analysis of the key events in pollen embryogenesis , leading potentially to the identification of molecular triggers . in particular , its application to transgenic material expressing fluorophore - labelled cellular structures such as nuclei , chromatin or components of the cytoskeleton will generate a wealth of detailed information concerning the process of pollen embryogenesis . supplementary data can be found at jxb online . supplementary video s1 . a time - lapse movie ( 5069 images , 7 pictures per second ) depicting the course of pollen development over 28 d.

pollen embryogenesis provides exciting opportunities in the areas of breeding and biotechnology as well as representing a convenient model for studying the process of plant cell proliferation in general and embryogenesis in particular . a cell culture system was devised in which immature barley pollen could be cultured as a monolayer trapped between the bottom glass - cover slip of a live - cell chamber and a diaphanous ptfe membrane within a liquid medium over a period of up to 28 d , allowing the process of embryogenesis to be tracked in individual pollen . z - stacks of images were automatically captured every 3min , starting from the unicellular pollen stage up to the development of multicellular , embryogenic structures . the method should prove useful for the elucidation of ultrastructural features and molecular processes associated with pollen embryogenesis .

Introduction Materials and methods Plant material and the induction of pollen embryogenesis Construction and use of a live-cell chamber cover slip Results Discussion Supplementary data

PMC3003976

aging is often associated with reductions in bone mineral density ( bmd ) and muscle mass and , consequently , with higher risk for falls and bone fractures .currently , bone demineralization ( osteopenia ) and muscle loss ( sarcopenia ) affect over 34 million people which results in more than 2 million bone fractures a year in the united states ( nih / niams , 2009 ) . with aging , while both sexes lose bone , women exhibit loss rates substantially higher than men , suffering a greater risk for osteoporosis after menopause [ 35 ] . some studies suggest that the reduced ovarian function and serum hormones levels accelerate the rate of bone loss and may also be involved in the decline of muscle mass and strength . basic exercise guidelines recommended by the american college of sports medicine for healthy adults and elderly people emphasize that training programs consist of resistance , strength , aerobic , and flexibility exercises . resistive training ( rt ) is an exercise modality that imposes heavy loads upon the skeleton and , consequently , should increase both body strength and muscle mass in older women . similar to other studies [ 1 , 7 , 8 ] our group demonstrated that intense rt promotes several benefits in postmenopausal women such as improved body composition parameters and muscular strength with a significant preservation of bmd . however , the literature remains equivocal , since other investigators did not report such findings [ 1012 ] . furthermore , the effectiveness of resistive training on the bone health simultaneously evaluated with functional fitness tests in postmenopausal women who are not using drugs that affect bone or muscle metabolism , is unknown . thus the aim of this study was to evaluate the influences of moderate resistive training on the bmd , as well as the efficacy to improve functional fitness parameters in postmenopausal women without hormone replacement therapy . thirty volunteer women ( age of 57 to 75 years ) were recruited from the regional community adult day care facilities located around the unifesp hospital . all participants had medical examinations and completed questionnaires regarding medical history , and protocols were performed within accordance with the ethical standards of the helsinki declaration ( 1964 , amended in 1975 and 1983 ) . inclusion criteria were age of more than 55 years old , completed menopause for at least two years before , and no use of hormone replacement therapy , or drugs and vitamins that could alter calcium or bone metabolism . exclusion criteria were as follows : participation in current or previous regular exercise training in the last 6 months ; recent hospitalization ; symptomatic cardiorespiratory disease ; hypertension or metabolic syndrome ; severe renal or hepatic disease ; cognitive impairment or progressive and debilitating conditions ; marked obesity with inability to exercise ; recent bone fractures or any other medical contra - indications to training . according to the exclusion criteria , 5 women could not participate . consequently , 25 women were randomized for untrained ( un ; n = 12 ) or resistive trained ( rt ; n = 13 ) group . for the rt group , participation in less than 90% of the stipulated exercise program all of the subjects were instructed to maintain their previously controlled normal dietary intake , to inform any new prescribed medication , and finally to not participate in any other type of physical activity . height ( m ) and weight ( kg ) were measured to calculate body mass index ( bmi = weight / height ) . dual - energy x - ray absorptiometry ( dexa ) was used to scan the lumbar spine ( l1l4 ) and the proximal femoral neck . dtx 200 ( osteometer meditech ) , blind to the subject group . the bruce treadmill protocol ( increments in both speed and grade in 3-minute intervals ) a one - minute warm - up at 1.0 mph without treadmill inclination preceded the bruce treadmill protocol . the subjective level of exertion was assessed using the borg 6-to-20 scale , and the exercise test was stopped due to exhaustion or according to previously described criteria . test was considered satisfactory if the participants reached the age - graded maximal recommended heart rate ( 220age in years ) without stoppage criteria . successive attempts were made with 1 to 2 minutes rest between attempts until failure as previously described . this evaluation was composed by six tests previously reported in the literature to assess physical performance parameters concerning mobility and balance in older adults [ 1518 ] . the arm curl test was used to evaluate upper limb fitness , with the analyzed score as the total number of hand weight curls through the full range of motion ; the chair stand test was used to evaluate lower limbs strength , scored by the number of standing up executed correctly within 30 seconds . agility was evaluated by the 8-foot up - and - go test , and the score was considered as the shortest time to rise from a seated position , walk eight feet , turn back and return to the seated position . the sit and reach test was used to evaluate the lower body flexibility scored by the shortest distance achieved between the extended fingers and the toe when seated with extended leg and the heel resting on the floor . the back scratch test assessed the upper body flexibility and the score was considered the shortest distance achieved between the extended middle fingers when reaching behind the head with one hand and behind the back with the other hand . static balance was assessed by having subjects stood up on just one leg for a maximum of 30 seconds on each side . the score was taken considering the time of quiet standing up , allowing only minimal fluctuations of ankle position or obvious toe clawing , without hopping or upper limbs movement . the test was stopped after 30 seconds if hopping occurred , the ankle movement was excessive , or the hanged foot touched the floor or contacted the stance leg / foot . the resistive training program consisted of one - hour exercise sessions 3 times a week in nonconsecutive days for 24 weeks . each session included the following isotonic exercises : leg press , leg extension , leg curl , chest press , elbow flexion , elbow extension , upper back row , and abdominal flexion . on the first week all subjects from rt group started the exercise program at 40% of 1 rm for each given exercise . the load was gradually increased until the subjects could perform three sets of 1012 repetitions for the given exercise at 60%70% of 1 rm , considered as moderate intensity . when necessary ; adjustments of loads were made every two weeks to promote muscle strength gains . the participants alternated between upper and lower body exercises to minimize fatigue , with rest between sets for 1 minute but no pauses between repetitions . analysis of comparisons between groups along the time periods were performed with 2-way anova with repeated measures , followed by bonferroni 's post - hoc test . comparisons between groups concerning relative changes in variables after 24-weeks were performed by unpaired student 's t - test . statistical analyses were performed with graphpad prism software ( version 4.0 , san diego , ca , usa ) . for all the evaluated parameters , baseline values were similar between un and rt group with satisfactory subject homogeneity . after 24 weeks , there were no significant changes in body weight , bmi , and body fat percentage of the un group , while the rt group exhibited body weight reductions and discreet , but not significant , increments of lean mass . in the un group there was a significant reduction in lumbar spine ( figure 1(d ) , before : 0.883 0.007 versus after : 0.875 0.008 g / cm , p < .05 ) and femoral neck bmd ( figure 1(a ) , before : 0.704 0.006 versus after 0.693 0.005 however , both bmd of lumbar spine ( figure 1(e ) ) and femoral neck ( figure 1(b ) ) did not change significantly in rt group ( ls before : 0.881 0.002 versus after : 0.882 0.004 g / cm and fn before : 0.701 0.004 versus after : 0.004 g / cm ) . as a result , the percentage of bmc decrease in the lumbar spine and femoral neck was significantly different between un and rt groups ( figures 1(f ) and 1(c ) ) , indicating a maintenance of the bone mineral content after resistive training program . concerning muscle strength evaluated by the 1 rm test , the trained group demonstrated improvements of 23% in lower limb strength ( legs ) ( before : 36 4 kg versus after : 47 3 kg , p < .001 ) and 32% in upper body strength ( before : 21 5 kg versus after : 31 8 kg , p < .001 ) . nevertheless , in the un group , no changes were observed either on the lower limbs ( before : 37 7 kg versus after : 38 4 kg , p > .05 ) or on the arms strength ( before : 22 8 kg versus after : 22 9 kg , p > .05 ) . baseline and 24-week evaluation of maximum aerobic power ( vo2 max ) and functional fitness parameters are presented in table 2 . vo2 max remained unchanged after 24 weeks in both groups , and no difference was found between un and rt . nevertheless , the resistive training resulted in a substantial improvement of upper limb and lower body muscular strength as evaluated by the arm curl and chair stand tests , respectively . although agility was unchanged by this exercise program , the rt group exhibited a significant increment on the lower and upper body flexibility , as well as on the static balance . on the other hand , no changes occurred to the un group for any functional fitness parameters . the benefits of resistive training on the bone demineralization in postmenopausal women remain uncertain . therefore , the main contribution of the present study was to reinforce evidence that moderate intensity rt can contribute positively to bone health , muscle strength , and functional fitness of postmenopausal women in the absence of hormone replacement therapy . this maintenance of bmd is clinically very important for postmenopausal women for whom the risk of falls and bone fractures are significantly higher due to advancing age and muscular and bone weakness . taken together , these conditions often lead elderly people to functional dependence and impairment of quality of life . as mentioned before , some authors observed no differences in bmd of lumbar spine [ 1012 ] and femoral neck [ 11 , 12 ] in trained older women compared to untrained group . on the other hand , several studies demonstrated positive effects of rt on the bmd [ 1 , 7 , 8 , 20 ] and similar results were reported in our previous study using high intensity rt ( 85% by 1 rm ) . further than maintenance , it was described a slight increase in bmd of the femoral neck and lumbar spine after one year of high intensity strength training in postmenopausal women life . the exact physiologic mechanism for which resistive training preserves the bmd is not completely understood . nevertheless , bone strain was suggested as playing a key role as mediator of the relationship between loading forces and bone remodeling . the piezoelectric effect in bone could justify the maintenance increase of mineral content in these cases . by this mechanism , actions like compression , tension , sprain , or shear stresses can generate differences on electric potential and create a magnetic field on the specific sites of the bone , stimulating cellular activity and consequently mineral deposition in the stress points . other important aspects should be considered for this bmd improvement , such as the magnitude of the stimuli , which has been described as more important than the frequency of the stimuli . thus , strength training is more effective in increasing or maintaining bmd when compared to running , already known as a good osteogenic enhancer , especially in anatomical sites where both activities produce mechanical stress , such as the femur neck . this difference is often attributed to the relationship between bone metabolism and muscle strength levels . we recently suggested that emphasis in eccentric muscle action influences positively the outcomes of bone mineralization . in fact , this type of muscle training has shown capable to promote larger osteogenic stimulus when compared to concentric muscle actions . to date , no study has evaluated the simultaneous effects of a resistive training on bmd and functional fitness at postmenopausal women . according to our results , the exercise program not only coursed with the preservation of bmd , but also significantly improved the functional fitness of the trained group . falls are responsible by approximately 90% of bone fractures in older women and undoubtedly represent an important public health issue . in the elderly , falls are not random events and occur , at least in part , due to physiological dysfunction such as impaired balance , muscular weakness , and impaired reaction time [ 28 , 29 ] . furthermore , postmenopausal women suffer more fall - related fractures due to greater impairments in balance and muscular strength combined with osteopenia or osteoporosis . fall prevention in older people , especially in women , is mainly searched through nonpharmacological strategies such as evaluating vision and hearing , promoting safety hazards at home , and finally , inducing exercise practice [ 5 , 30 , 31 ] . in fact , exercise modalities which improve agility , strength , and balance may significantly reduce the risk of falls and subsequent fracture , independent of any increase in bone density . despite no increment identified on the vo2 max of rt group , our results corroborate the standpoint of the american college of sports medicine supporting the idea that participation in regular exercise programs is directly related to improvements of muscle strength , flexibility , and range of motion ( acsm , 1998 ) . in addition , it is recommended that exercise should be adapted to maximize muscular strength , and that resistive training is safe and well tolerated in the elderly , eliciting adaptive neuromuscular changes to physiological variables associated with the risk of fall and disability . in the current study this data agrees with one study that found strong association between bmd maintenance with strength improvement to anatomically related bone structures . muscular strength increases are imperative to enhance the ability to carry out daily tasks , such as standing up from a chair or carrying a box . moreover , there was considerable improvement of the static balance of trained women . thus , the moderate resistive training led to significant improvement of lower body and limb strength combined with improved static balance , which has been defined as an independent contributor to fall prevention . interestingly , although were not performed specific activities for joint flexibility , we identified improvement of both the lower and upper body flexibility . this result reinforces the helpfulness of a moderate intensity program , since flexibility lost are associated with development of musculoskeletal impairment and progressive disabilities in old age . taken together , enhancement of all these functional parameters induced by rt should be useful in reducing the risk of falls . contrasting with , we have found no difference on the scores for agility test after rt in older women . possible reasons for this divergence are the different intensity and duration of the training program as well as the agility component of exercise training used by those authors . in the present study , this is a relatively small - sampled and short - term study without long - term follow - up data . moreover , the identified positive outcomes with averaged age of 66 in women do not allow these findings to be generalized to any aging population of both genders . finally , there are some pitfalls in estimating the maximal aerobic power ( indirect measurement of vo2 max ) instead of precisely measuring it through the cardiopulmonary gas exchange approach , although these inconveniences certainly involved both the baseline exercise capacity and that changes in exercise capacity after the rt program . the present study suggests moderate resistive training is able to restrain bmd declines of lumbar spine and femur neck in postmenopausal women , even in the absence of hormone replacement therapy , by preventing or attenuating the bone loss . notably , the exercise program also improved several parameters of functional fitness , including strength , flexibility , and static balance . thus , despite inherent limitations , our data reinforces the safety and utility of this exercise modality for use in larger trials as well as an adjunctive approach for fall and fractures reduction in this population .

twenty five subjects were randomized to untrained ( un ) and resistive - trained ( rt ) groups . the rt group exercised three sessions per week at 60%70% of the load according to individual 1rm test during 24 weeks . both groups were evaluated before and after protocol period assessing lumbar spine ( ls ) and femoral neck ( fn ) bmd by dual - energy x - ray absorptiometry , vo2 max , and neuromuscular fitness . after 24 weeks , there were significant reductions in ls ( 0.89 0.16% loss ) and fn bmd ( 1.54 0.35% loss ) for un but no change was found in the tr ( ls : 0.01 0.12% and fn : 0.04 0.05% loss ) . the un group had no changes in neuromuscular performance . however , rt exhibited a significant improvement on the functional fitness parameters evaluated , with the exception of agility . our results indicate rt suppresses the decline in bmd and simultaneously improves the functional fitness of postmenopausal women without hormone replacement therapy , which may reduce fall risk and related bone fractures .

1. Introduction 2. Material and Methods 3. Results 4. Discussion 5. Conclusion

PMC4501642

the zygomatic body and arch are located in the mid - face , which often affects the proportion of the entire face . aesthetic concepts regarding the facial contour greatly differ due to different ethnic and cultural backgrounds . in asia , in contrast , westerners think people with high , prominent malar are beautiful . among various facial contours , the oval or melon seed facial shape of asian women therefore , reduction malarplasty is one of the most widespread aesthetic surgeries performed in the asian population . a variety of methods for reduction malarplasty have been described since onizuka et al . first reported the technique by chiseling of the zygoma via an intraoral incision in 1983 . according to the surgical routes , these methods can be divided into the following 3 categories : intraoral [ 2,79 ] , coronal [ 1012 ] , and intraoral combined with pre - auricular approaches . considering the basic principles , these techniques mainly consist of bone shaving , complete i - type , l - type , or wedge - section osteotomy on the zygomatic body , and the infracture procedure on the zygomatic body . different infracture techniques through a temporal pre - auricular incision have been introduced for the reduction of the prominent zygomatic body and arch [ 5,1519 ] , but these methods have limitations and are primarily effective for improvement of lateral protrusion of the zygomatic arch . to overcome this shortcoming , we developed and used a novel l - shaped infracture method in 700 patients . post - operative follow - up and x - ray imaging demonstrated this technique to be an effective and safe reduction malarplasty method . seven hundred consecutive patients with prominent malar complexes ( 65 males and 635 females ; 1939 years of age ) were treated with the infracture technique between january 2004 and october 2013 . patients who had contraindications to cosmetic surgery , such as severe diabetes , hypertension , or mental disabilities , were excluded from this study . all patients underwent routine physical and laboratory examinations , including hemograms , urinalyses , liver and renal function testing , coagulation profiles , electrocardiograms , chest x - rays , and cranial x - rays ( waters and submentovertex views ) . facial photographs from the frontal , oblique , and submentovertical views were obtained . while in a sitting position , anterior oblique profiles of the patients were evaluated , the facial proportion and symmetry were assessed , and the dominant area of protrusion was marked on the corresponding skin . when general anesthesia was established , 0.6% lidocaine containing epinephrine ( 1:100,000 ) was injected along the incision line , the upper labiobuccal sulcus , the periosteum of the zygomatic body , and the periauricular incision . after 10 min , a 4-cm intraoral incision was made down to the periosteum on the buccal vestibule . the zygomaticomaxillary and zygomatic bodies to the lateral orbital margin were exposed through subperiosteal dissection . meticulous dissection techniques were used to protect the infraorbital nerve and minimize detachment of the origin of the masseter to the zygomatic arch to prevent post - operative cheek drooping . according to the pre - operative mark on the skin , the outer cortex of the protruding zygomatic process was shaved with sufficient exposure using a zygomatic retractor , followed by an l - shaped osteotomy . with an oscillating saw , the oblique osteotomy was first made from the zygoma - orbital concavity ( concavity between the zygomatic arc and the lateral orbital wall ) to the zygomaticomaxillary suture , and a vertical osteotomy was made from the zygomaticomaxillary suture or the lateral side of the zygomaticomaxillary suture , which mainly depended on the severity of the malar prominence . at this point , it was crucial to maintain a distance of approximately 2 mm between the 2 perpendicular osteotomy lines on the outer cortex and approximately 3 mm on the inner cortex . after the osteotomy procedures , a 1.5-cm vertical pre - auricular incision was made at the posterior margin of the sideburn ( figures 1 , 2 ) . using a mosquito clamp , blunt dissection was performed from the subcutaneous tissue and extended to the periosteum of the zygoma root . a chisel was inserted for a vertical bony cut following the subperiosteal dissection around the osteotomy line . the posterior cutting line was approximately 1 cm anterior to the articular tubercle to avoid affecting post - operative function of the temporomandibular joint . then , the proximal edge of the cut arch was lightly tapped with a hammer under direct vision , which would displace the zygomatic complex by making an incomplete fracture anteriorly and a complete fracture posteriorly , with the infraction point acting as a hinge . the amount of the inward rotation was determined according to the severity and symmetry of the malar prominence , ranging from 5 to 15 mm . if bilateral asymmetry was observed , more inward rotation was performed on the more prominent side . after hemostasis was assured , the intraoral and pre - auricular incisions were closed in a single layer using running sutures without drainage . seven hundred consecutive patients with prominent malar complexes ( 65 males and 635 females ; 1939 years of age ) were treated with the infracture technique between january 2004 and october 2013 . patients who had contraindications to cosmetic surgery , such as severe diabetes , hypertension , or mental disabilities , were excluded from this study . all patients underwent routine physical and laboratory examinations , including hemograms , urinalyses , liver and renal function testing , coagulation profiles , electrocardiograms , chest x - rays , and cranial x - rays ( waters and submentovertex views ) . facial photographs from the frontal , oblique , and submentovertical views were obtained . while in a sitting position , anterior oblique profiles of the patients were evaluated , the facial proportion and symmetry were assessed , and the dominant area of protrusion was marked on the corresponding skin . when general anesthesia was established , 0.6% lidocaine containing epinephrine ( 1:100,000 ) was injected along the incision line , the upper labiobuccal sulcus , the periosteum of the zygomatic body , and the periauricular incision . after 10 min , a 4-cm intraoral incision was made down to the periosteum on the buccal vestibule . the zygomaticomaxillary and zygomatic bodies to the lateral orbital margin were exposed through subperiosteal dissection . meticulous dissection techniques were used to protect the infraorbital nerve and minimize detachment of the origin of the masseter to the zygomatic arch to prevent post - operative cheek drooping . according to the pre - operative mark on the skin , the outer cortex of the protruding zygomatic process was shaved with sufficient exposure using a zygomatic retractor , followed by an l - shaped osteotomy . with an oscillating saw , the oblique osteotomy was first made from the zygoma - orbital concavity ( concavity between the zygomatic arc and the lateral orbital wall ) to the zygomaticomaxillary suture , and a vertical osteotomy was made from the zygomaticomaxillary suture or the lateral side of the zygomaticomaxillary suture , which mainly depended on the severity of the malar prominence . at this point , it was crucial to maintain a distance of approximately 2 mm between the 2 perpendicular osteotomy lines on the outer cortex and approximately 3 mm on the inner cortex . after the osteotomy procedures , a 1.5-cm vertical pre - auricular incision was made at the posterior margin of the sideburn ( figures 1 , 2 ) . using a mosquito clamp , blunt dissection was performed from the subcutaneous tissue and extended to the periosteum of the zygoma root . a chisel was inserted for a vertical bony cut following the subperiosteal dissection around the osteotomy line . the posterior cutting line was approximately 1 cm anterior to the articular tubercle to avoid affecting post - operative function of the temporomandibular joint . then , the proximal edge of the cut arch was lightly tapped with a hammer under direct vision , which would displace the zygomatic complex by making an incomplete fracture anteriorly and a complete fracture posteriorly , with the infraction point acting as a hinge . the amount of the inward rotation was determined according to the severity and symmetry of the malar prominence , ranging from 5 to 15 mm . if bilateral asymmetry was observed , more inward rotation was performed on the more prominent side . after hemostasis was assured , the intraoral and pre - auricular incisions were closed in a single layer using running sutures without drainage . among the 700 patients enrolled in this clinical trial , 200 ( 10 males ) underwent mandibular angle hypertrophy in combination with mandibular angle reduction or genioplasty simultaneously through intraoral and submental incisions . the post - operative management was implemented as follows : a liquid food diet was introduced 68 hours post - operatively ; and soft diet was recommended 35 days post - operatively . the follow - up ranged from 2 to 24 months , and the preoperative and the postoperative frontal and lateral views were recorded ( figure 3 ) . cranial x - rays , including waters and submentovertex views , and facial photographs were obtained to observe the bony changes and assess the aesthetic effects . satisfactory results were achieved in most patients . compared with pre - operative images , the x - ray images 2 weeks post - operatively showed that the contour of the midface was clearly narrowed . three months post - operatively , the bone around the zygomatic arc osteotomy line was remodeled . partial bone posterior to the articular tubercle of the zygomatic arch was absorbed , causing a depression of the root of the arc and a natural transition of the osteotomy line bilaterally , making the midface appear more slender . the anterior bone developed new bone , which made the malar complex more stable ( figure 4 ) . one patient ( 0.14% ) developed maxillary sinusitis 2 weeks post - operatively , which completely resolved after systemic antibiotics and local drainage for 1 week . skin necrosis approximately 1 cm in diameter was noted in 1 case ( 0.14% ) after undergoing zygoma area liposuction , which was treated conservatively . a unilateral forehead wrinkle appeared in 1 patient ( 0.14% ) , which completely resolved within 3 weeks of surgery . thirteen patients ( 1.9% ) presented with slight asymmetry ; 3 patients underwent secondary operations , but 10 patients declined further surgery . other complications , such as hematomas , permanent facial nerve damage , recurrence of malar prominence , temporomandibular joint dysfunction , bone non - union , and cheek drooping , were not observed in the subsequent follow - up . among the 700 patients enrolled in this clinical trial , 200 ( 10 males ) underwent mandibular angle hypertrophy in combination with mandibular angle reduction or genioplasty simultaneously through intraoral and submental incisions . the post - operative management was implemented as follows : a liquid food diet was introduced 68 hours post - operatively ; and soft diet was recommended 35 days post - operatively . the follow - up ranged from 2 to 24 months , and the preoperative and the postoperative frontal and lateral views were recorded ( figure 3 ) . cranial x - rays , including waters and submentovertex views , and facial photographs were obtained to observe the bony changes and assess the aesthetic effects . compared with pre - operative images , the x - ray images 2 weeks post - operatively showed that the contour of the midface was clearly narrowed . three months post - operatively , the bone around the zygomatic arc osteotomy line was remodeled . partial bone posterior to the articular tubercle of the zygomatic arch was absorbed , causing a depression of the root of the arc and a natural transition of the osteotomy line bilaterally , making the midface appear more slender . the anterior bone developed new bone , which made the malar complex more stable ( figure 4 ) . one patient ( 0.14% ) developed maxillary sinusitis 2 weeks post - operatively , which completely resolved after systemic antibiotics and local drainage for 1 week . skin necrosis approximately 1 cm in diameter was noted in 1 case ( 0.14% ) after undergoing zygoma area liposuction , which was treated conservatively . a unilateral forehead wrinkle appeared in 1 patient ( 0.14% ) , which completely resolved within 3 weeks of surgery . thirteen patients ( 1.9% ) presented with slight asymmetry ; 3 patients underwent secondary operations , but 10 patients declined further surgery . other complications , such as hematomas , permanent facial nerve damage , recurrence of malar prominence , temporomandibular joint dysfunction , bone non - union , and cheek drooping , were not observed in the subsequent follow - up . asians and westerners have different facial skeletal shapes ; specifically , asians have mesocephalic skull shapes , whereas westerners have dolichocephalic skull contours . as a result , asians tends to have flatter and shorter faces compared with westerners . in addition , malar prominence will accentuate these features , which in turn might make people appear unattractive and older than their actual ages . thus , reduction malarplasty has been utilized as one of the most popular procedures for aesthetic purposes in asians . among multiple methods applied for reduction malarplasty , osteotomy and repositioning are generally considered to be most reliable and effective , such as complete i - type , l - type , or wedge - section osteotomy on the zygomatic body and infracture procedure on the zygomatic body . most of these methods , however , required rigid interosseous fixation with a mini - plate or wire ; incomplete and inappropriate fixation can result in bone non - union or malunion from an external rotation force through the action of the masseter muscle [ 2123 ] . in addition , despite essentially no foreign body reaction with such materials , many patients are reluctant to use any metallic objects because it is visible on radiographs , and the patients have no intention of letting others know that they had undergone an operation . moreover , the fixation is difficult via the intraoral approach due to the narrow surgical field . indeed the infracture technique was introduced for reduction malarplasty by uhm et al . and yang et al . . a temporal preauricular incision can provide precise manipulation ; however , a temporal preauricular incision may be inappropriate for young patients because of the long operative time , large scalp scar , potential risk of injury to the facial nerve , marked local swelling , and long recovery time . hwang et al . modified the method proposed by yang and park by cutting the outer cortex of the zygoma vertically from the level of the orbital rim outer margin , and repositioning the zygomatic arch inward after 2-point fracturing ( a green - stick fracture anteriorly and complete osteotomy posteriorly ) . also described the technique of an oblique shape greenstick fracture of the zygomatic body and complete osteotomy of the zygomatic arch . the aforementioned infracture methods have limitations , and are only effective for subjects with lateral protrusion of the zygomatic arch ; patients with severe protrusion of the zygomatic body and arch are not suitable candidates . described a blind technique through a minimally invasive intraoral approach using a new instrument , but the technique was difficult to control and not appropriate for surgeons without experience with the technique . we have developed a new l - shape infracture method for treatment of severe protrusion of the zygomatic body and arch . compared with the i - shape infracture technique of the malar body , our technique is more suitable to achieve adequate reduction and efficiently alter the main protrusion area of the zygomatic body if the osteotomy line originates from the same point of the lateral orbit wall and on the premise of not destroying the orbital floor and maxillary sinus . through the pre - auricular sideburn incision , the dissection area of the zygomaticomaxillary and zygomatic bodies can be confined to the lateral orbital margin to prevent the incidence of post - operative cheek drooping . in addition , an osteotomy of the zygomatic arch is performed under direct vision and the angle of the inward rotation could be properly controlled , which facilitates bilateral symmetry , especially for patients with pre - operative facial asymmetry . some authors have suggested that a pre - auricular incision might result in conspicuous scars ; however , the sideburn scar is almost invisible . kim and choi suggested that the infraction technique could probably destroy the natural curve between the zygomatic body and zygomatic arch , but the infracture technique utilized in the current study reduces this risk by preserving the natural transition , and accentuates the malar highlight area at a more posterosuperior site . currently , complete fracture of the posterior arch has been widely utilized because it can significantly reduce facial width ; however , some authors consider post - operative facial depression in the pre - auricular area to be a common problem because the arch is replaced medially and may generate a step - off . in order to avoid this drawback , many authors advocate an oblique osteotomy of the zygoma arch root . x - ray images revealed that the bone surrounding the zygomatic arch osteotomy line can be remodeled and partial bone posterior to the articular tubercle of the zygomatic arch is absorbed , which might lead to an depression of the root of the arch and a natural transition of the 2 sides of the osteotomy line , making the midface more slender . it is not necessary to worry about post - operative facial depression in the pre - auricular area , even using a vertical osteotomy at the zygomatic arch root . we postulate that a pattern of self - regulation might occur in the bone segments and muscles , such as the masseter and temporalis muscles . the operation time is shortened to approximately 0.5 hour , and the time of recovery and swelling is decreased . a few surgical complications are induced using the infracture technique . in the current study , 1 patient had maxillary sinusitis 2 weeks post - operatively , probably due to the damages to the maxillary sinus during the surgery ; however , opinions regarding the damages to the maxillary sinus greatly differ . kim and seul noted that maxillary sinus damage was rarely a serious problem when bone segments were firmly fixed , if damaged . skin necrosis approximately 1 cm in diameter occurred in 1 patient undergoing zygoma area liposuction , thus caution should be exercised to perform the 2 procedures simultaneously because it probably affects the blood supply of the local skin . the unilateral forehead wrinkle resolved in 1 patient ; perhaps the forehead wrinkle was caused by dragging damage to the frontal branch of the facial nerve . kim and seul reported there is possibility of facial nerve damage when surgeons use a pre - auricular incision . although we had carefully assessed the pre - operative conditions of the patients and made adjustments during the operation , slight asymmetry was still observed in 13 patients , probably resulting due to the following reasons : ( 1 ) the osteotomy line was not identical on the 2 sides ; ( 2 ) the soft tissue thickness in the zygomatic region is different on the 2 sides ; and ( 3 ) the pre - operative asymmetry of the bone had not been properly corrected . even though asymmetry can occur with other techniques , pre - operative evaluation and adjustment during surgery other complications , such as hematomas , permanent facial nerve damage , recurrence of the malar prominence , temporomandibular joint dysfunction , bone non - union , and cheek drooping , were not observed in the follow - up period . to achieve good results , mandibular angle reduction or genioplasty the entire face should be regarded as a single unit and a coordinated proportion is crucial for facial beauty . if the patients have mandibular hypertrophy and/or a short chin , the above procedures should be recommended . this technique has a limitation in that it is a demanding procedure , especially for neophytes , to properly control the distance between the 2 osteotomy lines to make a greenstick fracture . moreover , the l - shaped infracture technique has many advantages , such as simple manipulation , no need for internal fixation , short operation and recovery times , and mild surgical complications .

backgroundreduction malarplasty is one of the most common surgical procedures performed in the asian population for aesthetic purposes . although multiple methods have been developed for reduction malarplasty , including a variety of infracture techniques , most of the current procedures have limitations . in the current study we created a new infracture method to circumvent these shortcomings.material/methodsbetween january 2004 and october 2013 , we applied this novel infracture technique in 700 patients . the highest area of the zygomatic body was marked pre - operatively and ground intra - operatively through an intraoral incision . an l - shaped incomplete osteotomy of the zygomatic body was performed with a reciprocating saw , and then a complete perpendicular osteotomy ( 1 cm anterior to the articular tubercle of the zygomatic arch ) was made through a pre - auricular incision . light pressure on the posterior part of the arch produced a greenstick fracture of the anterior osteotomy site , resulting in posterior - inward repositioning of the malar complex . internal fixation was not required.resultssatisfactory aesthetic results and good post - operative stability were achieved . three months post - operatively , the bone around the zygomatic arc osteotomy line was remodeled . the bone posterior to the articular tubercle of the zygomatic arch was partially absorbed , leading to a depression of the root of the arc and a natural transition on both sides of the osteotomy line , making the midface more slender . instead , the anterior bone presented with new bones , making the malar complex more stable.conclusionsthis new method has multiple advantages , including simple manipulation , no need for internal fixation , short operative and recovery times , and few complications . x - ray images showing the bony changes demonstrated that the infracture technique is an effective and ideal method for reduction malarplasty .

Background Material and Methods Patient data Pre-operative examinations Surgical procedures Post-operative management Results Surgical strategies X-ray imaging Post-operative complications Discussion Conclusions

PMC3872627

endodontic retreatment of the teeth with intra - radicular posts is usually a difficult situation for clinicians , increasing the risk of perforations , fractures and weakness of the remaining structure of the teeth . removing such posts is performed with different methods and instruments including the masserann technique , pivots - extracting forceps post excavator , post puller , gonon extractor and other techniques using the rotary instruments or ultrasound . . these instruments especially are effective when zinc phosphate or glass ionomer cements are used . the force needed to remove the cemented posts has shown to be related to the post type ( cast or prefabricated ) , the post design ( tapered or parallel , smooth , serrated or threaded ) , the post length and the type of cement used . studies on the effect of ultrasound on removal of posts cemented with resin cements have shown conflicting results . while some studies showed that these vibrations had no effect on retention of posts , bergeron et al . demonstrated that using ultrasound increased the retention of stainless steel posts cemented with resin cement . however , other studies expressed that application of ultrasound decreased the retention of cast posts cemented with resin cements . all the above studies except one used panavia cement ( kuraray medical , japan ) , which is a self - etch resin cement . considering inferior shear bond strength of self - adhesive resin cements to dentin , it may be hypothesized that these cements may have different characteristics when exposed to ultrasonic vibrations . the purpose of this study was to assess the effect of ultrasonic vibrations from a piezoelectric device on retention of prefabricated metal posts cemented with self - etch and self - adhesive resin cements . in this in vitro study , forty eight extracted premolars were selected according to the form and length of the root ( straight with single canal roots and approximately 13 mm in length and round cross - section ) . the crowns were cut - off using a diamond disk under running water , leaving a 13 mm long root . the root canals were instrumented with k - file ( mani , japan ) up to file # 35 and flared using gates - glidden drills # 1 to # 4 ( mani , japan ) . apical 5 mm of the root canals were obturated with gutta - percha ( gapadent , china ) and ah26 sealer ( dentsply , germany ) employing vertical condensation method . the roots were placed in an incubator ( behdad , iran ) for 48 h in 37c and 100% humidity . the remaining 8 mm of the canal space was prepared by peeso reamers # 2 and # 3 ( mani , japan ) for placing a long dentorama prefabricated post # 2 ( svenska dentorama ab , sweden ) . the peeso reamer # 3 and long dentorama prefabricated post # 2 have the same diameter ( 1.1 mm ) . the posts were examined to have an inactive fit in the canals by placing 8 mm inside the canal without having any mobility in the place . specimens that did not have suitable fitness for a long dentorama post # 2 were eliminated . the roots were embedded in self - curing acrylic resin blocks ( marlic medical ind . canals were cleaned with 97% ethanol and dried with paper cones ( ariadent , iran ) . the specimens were randomly assigned to 4 groups ; in groups 1 and 2 the posts were cemented in the canals using panavia f 2.0 cement ( kuraray medical , japan ) and in groups 3 and 4 using maxcem elite cement ( kerr , usa ) . each cement was prepared according to its manufacturer 's instruction . a black - banded lentulo spiral ( ffdm - pneumat , france ) was used to introduce cement into the preparation and coat the walls of the canal . the posts were coated with cement and inserted in the canals to the prepared depth and held in place for 1 min to ensure complete seating according to manufacturer 's instruction . after initial setting of the cements , all specimens were placed in thermocycling machine ( vafaei , iran ) to simulate the thermal changes of the oral cavity ( 5 - 55c , 500 cycles , 20 s dwell time and 10 s transfer time ) . the posts in groups 2 and 4 received ultrasonic vibrations for 4 min using a piezoelectric unit with the blunt - ended tip ( je 27000 , juya electronic , iran ) with maximum power and minimum water as coolant . for transferring the vibrations to the specimens , the tip of the instrument was placed on buccal , lingual , mesial and distal aspects between the post and dentine ( on cement surface ) , 30 s for each . then , the tip of the instrument touched the post and rotated around it for 2 min . the specimens were fixed in a custom made holder [ figure 1 ] and placed in a universal testing machine ( dartec , england ) . tensile force was introduced to each post along the long axis of the root with a crosshead speed of 1 mm / min until the post came out of the canal . custom made holder for tensile force testing statistical analysis was performed using spss software ( version 11.5 , spss inc . kruskal - wallis test was used to assess the differences between groups at a 5% level of significance . a total of 8 specimens were missed during the traction test as their roots came out of the acrylic block before the posts dislodged , 4 specimens in group 1 , 3 specimens in group 3 and 1 specimen in group 4 . means and standard deviations of dislodgement forces in each group are summarized in table 1 . statistical analysis showed no significant difference between the 4 groups ( p = 0.488 ) ; hence , neither cement type nor ultrasonic vibrations affected the dislodgement force significantly . when a post is to be removed from the root canal , ultrasound may be used to make some fractures in cement until the post comes out with less extraction force . this study shows that ultrasound did not reduce the retention of the posts cemented with panavia or maxcem elite resin cements . these findings are in agreement with previous reports on resistance of resin cements to ultrasonic vibrations . this may be due to the viscoelastic nature of resin cements , which tends to soften the vibrations and absorb the energy transmitted to the post . . this variety could be due to differences in method of the study : type of the post ( custom versus prefabricated ) , type , duration and method of using ultrasonic devices as well as using or not using water . in the study , which showed increase in the retention of the panavia cemented posts by ultrasound application great standard deviation was considered as a main cause of this increase . in the majority of studies , which used custom posts ultrasound decreased the retention of panavia cemented posts except the study by gomes et al . which showed the neutral effect of ultrasound on retention of custom posts . this variety is in duration of using ultrasound , location of the ultrasonic tip on the post , using water or not as a coolant , type of the tip of ultrasonic instrument and etc . about the time period of using ultrasound , some issues have been proposed on how to use ultrasonic instruments including duration of time and water cooling . some authors have recommended 8 - 10 min of ultrasonic application to effectively dislodge cemented posts , and hauman et al . reported that 16 min is not enough for the posts cemented with resin cements . in the current study , smith reported that the average time of ultrasound application needed to extract the post in clinical conditions is about 25% of the time at in vitro conditions . he demonstrated that the leakage of restorative materials , cementation under unfavorable conditions and the forces induced to the posts soon after cementation are the main causes of this difference . in this study , regarding water cooling , it has been shown that ultrasonic vibrations with water coolant is less effective on post removal than without it . this can be caused by the fact that resin cements are not friable and there is no tendency to micro fracture in the cement ; unlike zinc phosphate cement , which is friable . furthermore , due to high viscoelastic characteristic of the resin cement , which are categorized as a plastic material , it tends to absorb energy from posts . however , it can not be assumed that application of ultrasound without water can decrease the retention of resin cemented posts since it can be caused by the generated heat by the ultrasonic device . watanabe et al . found that attachment capacity of resin cements decreased gradually by thermal cycles . therefore , by increasing temperature , resins expand and lose their adhesive characteristic and as a result , their retention . in this study , we used minimum water cooling to simulate clinical situation where high temperatures should be avoided . our results showed no significant differences between removal forces of posts cemented with either panavia ( a self - etch resin ) or maxcem elite ( a self - etch self - adhesive resin ) cements . although it has been shown that self - etch self - adhesive resin cements have inferior bonding strength to dentin , it seems that this does not affect retention of posts in the root canal , where a well - fitting post is to be retained inside a cylindrical preparation . further research seems necessary to confirm the effect of ultrasound on retention of different cements in clinical situations where the materials are subject to different thermal and loading conditions . within the limitations of the current study , the efficacy of ultrasound on reducing the retention of prefabricated metal posts cemented with panavia or maxcem elite resin cements is in doubt .

background : ultrasonic vibrations are used to remove a cemented post from a root canal requiring endodontic retreatment . various results have been reported from the studies that evaluated the effect of ultrasonic instruments in removing the posts cemented with resin cements . the aim of this study was to evaluate the effect of ultrasonic energy on the retention of prefabricated metal post cemented with panavia or maxcem elite cements.materials and methods : in this in vitro study , forty eight extracted single root premolars were decoronated with a diamond disc leaving a 13 mm long root and endodontically treated . the root canals were obturated by gutta - percha up to 5 mm with vertical condensation method and the 8 mm post - space was prepared to receive a no . 2 long dentorama post . the roots were placed in an incubator for 48 h in 37c and 100% humidity . after mounting the teeth in acrylic blocks , posts were cemented in the root canals using panavia f2.0 in 24 specimens and maxcem elite in 24 others . for half of the specimens in each subgroup , an ultrasonic device was applied for 4 min . universal testing machine was used to measure the force needed to remove the posts with a crosshead speed of 1 mm / min until the post came out of the canal . kruskal - wallis test was used for statistical analysis at 5% level of significance.results:the removal force was not significantly different among the groups ( p > 0.05).conclusion : ultrasonic energy did not decrease the retention of posts cemented with panavia or maxcem elite cements . furthermore , it seems that there is no significant difference between removal force of self - etch ( panavia ) and the self - etch self - adhesive ( maxcem elite ) resin cements .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION