Arterioles have a larger lumen diameter than venules.

The centrosome cycle: Centriole biogenesis, duplication and inherent asymmetries

Centrosomes are microtubule-organizing centres of animal cells. They influence the morphology of the microtubule cytoskeleton, function as the base for the primary cilium and serve as a nexus for important signalling pathways. At the core of a typical centrosome are two cylindrical microtubule-based structures termed centrioles, which recruit a matrix of associated pericentriolar material. Cells begin the cell cycle with exactly one centrosome, and the duplication of centrioles is constrained such that it occurs only once per cell cycle and at a specific site in the cell. As a result of this duplication mechanism, the two centrioles differ in age and maturity, and thus have different functions; for example, the older of the two centrioles can initiate the formation of a ciliary axoneme. We discuss spatial aspects of the centrosome duplication cycle, the mechanism of centriole assembly and the possible consequences of the inherent asymmetry of centrioles and centrosomes.

Endothelium-mediated relaxation of porcine collateral-dependent arterioles is improved by exercise training.

BACKGROUND Endothelium-dependent modulation of coronary tone is impaired in the collateral-dependent coronary microcirculation. We used a porcine model of chronic coronary occlusion and collateral development to evaluate the hypothesis that exercise training enhances endothelium-mediated relaxation and increases endothelial nitric oxide synthase (ecNOS) mRNA levels of collateral-dependent microvasculature. METHODS AND RESULTS Adult female miniature swine were subjected to chronic, progressive ameroid occlusion of the proximal left circumflex coronary artery (LCx); after 2 months, animals were randomly exposed to 16-week exercise-training (EX group; treadmill running) or sedentary (SED group; cage confinement) protocols. After completion of EX or SED programs, coronary arterioles ( approximately 100 microm in diameter) were isolated from collateral-dependent LCx (distal to occlusion) and nonoccluded left anterior descending coronary artery (LAD) regions of each heart. Arterioles were studied by in vitro videomicroscopy or frozen for ecNOS mRNA analysis (RT-PCR techniques). Relaxation to the endothelium-dependent vasodilator bradykinin was decreased (P<0.05) in arterioles isolated from collateral-dependent LCx versus nonoccluded LAD regions of SED animals. Bradykinin-mediated relaxation, however, was not different in LCx versus LAD arterioles isolated from EX animals. Nitroprusside-induced relaxation was unaffected by either chronic occlusion or exercise. Importantly, ecNOS mRNA expression was significantly decreased in arterioles isolated from LCx versus LAD regions of SED animals. After training, ecNOS mRNA expression was not different between LAD and LCx arterioles. CONCLUSIONS These data indicate that exercise training enhances bradykinin-mediated relaxation of collateral-dependent LCx arterioles isolated after chronic coronary occlusion, most likely because of effects on ecNOS mRNA expression and increased production of NO.

Mechanosensitivity in the enteric nervous system

The enteric nervous system (ENS) autonomously controls gut muscle activity. Mechanosensitive enteric neurons (MEN) initiate reflex activity by responding to mechanical deformation of the gastrointestinal wall. MEN throughout the gut primarily respond to compression or stretch rather than to shear force. Some MEN are multimodal as they respond to compression and stretch. Depending on the region up to 60% of the entire ENS population responds to mechanical stress. MEN fire action potentials after mechanical stimulation of processes or soma although they are more sensitive to process deformation. There are at least two populations of MEN based on their sensitivity to different modalities of mechanical stress and on their firing pattern. (1) Rapidly, slowly and ultra-slowly adapting neurons which encode compressive forces. (2) Ultra-slowly adapting stretch-sensitive neurons encoding tensile forces. Rapid adaptation of firing is typically observed after compressive force while slow adaptation or ongoing spike discharge occurs often during tensile stress (stretch). All MEN have some common properties: they receive synaptic input, are low fidelity mechanoreceptors and are multifunctional in that some serve interneuronal others even motor functions. Consequently, MEN possess processes with mechanosensitive as well as efferent functions. This raises the intriguing hypothesis that MEN sense and control muscle activity at the same time as servo-feedback loop. The mechanosensitive channel(s) or receptor(s) expressed by the different MEN populations are unknown. Future concepts have to incorporate compressive and tensile-sensitive MEN into neural circuits that controls muscle activity. They may interact to control various forms of a particular motor pattern or regulate different motor patterns independently from each other.

Aortic and large artery compliance in end-stage renal failure.

Pulse wave velocity (PWV) was measured in the aorta, right leg and arm of 90 control subjects (CS) and 92 hemodialysis patients (HD) of the same age and mean arterial pressure (MAP). Blood chemistry, including blood lipids, and echographic dimensions of the aorta, were measured in all subjects. Presence of aortic calcification was evaluated by abdominal X-ray and echography. Whereas femoral and brachial PWV were only slightly increased in HD (P less than 0.05), the aortic PWV was significantly elevated (1113 +/- 319 cm/sec) in comparison with CS (965 +/- 216 cm/sec; P = 0.0016). Aortic diameters were larger in HD, both at the root of aorta (32.7 +/- 4 vs. 28.2 +/- 2.8 mm; P less than 0.0001) and aortic bifurcation (16.9 +/- 3.1 vs. 14.6 +/- 2.2 mm; P less than 0.0001). Although the MAP was similar in HD (109.9 +/- 19.3 mm Hg) and CS (110.2 +/- 17.2 mm Hg), the pulse pressure was significantly increased in HD patients (76.6 +/- 23.7 vs. 63.9 +/- 22 mm Hg; P = 0.007). In the two populations, aortic PWV was found to increase with age (P less than 0.0001) and MAP (P less than 0.0001). The presence of aortic calcification showed only a borderline relationship with the increase in aortic PWV (P = 0.050 in CS and P = 0.069 in HD). As change in PWV is directly related to change in distensibility, and the aortic diameters were increased in HD, these results indicate that aortic wall compliance is decreased in HD, resulting in an increase in the pulsatile component of arterial pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

Multifunctional in vivo vascular imaging using near-infrared II fluorescence

In vivo real-time epifluorescence imaging of mouse hind limb vasculatures in the second near-infrared region (NIR-II) is performed using single-walled carbon nanotubes as fluorophores. Both high spatial (∼30 μm) and temporal (<200 ms per frame) resolution for small-vessel imaging are achieved at 1–3 mm deep in the hind limb owing to the beneficial NIR-II optical window that affords deep anatomical penetration and low scattering. This spatial resolution is unattainable by traditional NIR imaging (NIR-I) or microscopic computed tomography, and the temporal resolution far exceeds scanning microscopic imaging techniques. Arterial and venous vessels are unambiguously differentiated using a dynamic contrast-enhanced NIR-II imaging technique on the basis of their distinct hemodynamics. Further, the deep tissue penetration and high spatial and temporal resolution of NIR-II imaging allow for precise quantifications of blood velocity in both normal and ischemic femoral arteries, which are beyond the capabilities of ultrasonography at lower blood velocities.

The binding of maize DHN1 to lipid vesicles. Gain of structure and lipid specificity.

Dehydrins (DHNs; late embryogenesis abundant D-11) are a family of plant proteins induced in response to abiotic stresses such as drought, low temperature, and salinity or during the late stages of embryogenesis. Spectral and thermal properties of these proteins in purified form suggest that they are "intrinsically unstructured. " However, DHNs contain at least one copy of a consensus 15-amino acid sequence, the "K segment," which resembles a class A2 amphipathic alpha-helical, lipid-binding domain found in other proteins such as apolipoproteins and alpha-synuclein. The presence of the K segment raises the question of whether DHNs bind lipids, bilayers, or phospholipid vesicles. Here, we show that maize (Zea mays) DHN DHN1 can bind to lipid vesicles that contain acidic phospholipids. We also observe that DHN1 binds more favorably to vesicles of smaller diameter than to larger vesicles, and that the association of DHN1 with vesicles results in an apparent increase of alpha-helicity of the protein. Therefore, DHNs, and presumably somewhat similar plant stress proteins in the late embryogenesis abundant and cold-regulated classes may undergo function-related conformational changes at the water/membrane interface, perhaps related to the stabilization of vesicles or other endomembrane structures under stress conditions.

Vascular endothelial cadherin controls VEGFR-2 internalization and signaling from intracellular compartments

Receptor endocytosis is a fundamental step in controlling the magnitude, duration, and nature of cell signaling events. Confluent endothelial cells are contact inhibited in their growth and respond poorly to the proliferative signals of vascular endothelial growth factor (VEGF). In a previous study, we found that the association of vascular endothelial cadherin (VEC) with VEGF receptor (VEGFR) type 2 contributes to density-dependent growth inhibition (Lampugnani, G.M., A. Zanetti, M. Corada, T. Takahashi, G. Balconi, F. Breviario, F. Orsenigo, A. Cattelino, R. Kemler, T.O. Daniel, and E. Dejana. 2003. J. Cell Biol. 161:793–804). In the present study, we describe the mechanism through which VEC reduces VEGFR-2 signaling. We found that VEGF induces the clathrin-dependent internalization of VEGFR-2. When VEC is absent or not engaged at junctions, VEGFR-2 is internalized more rapidly and remains in endosomal compartments for a longer time. Internalization does not terminate its signaling; instead, the internalized receptor is phosphorylated, codistributes with active phospholipase C–γ, and activates p44/42 mitogen-activated protein kinase phosphorylation and cell proliferation. Inhibition of VEGFR-2 internalization reestablishes the contact inhibition of cell growth, whereas silencing the junction-associated density-enhanced phosphatase-1/CD148 phosphatase restores VEGFR-2 internalization and signaling. Thus, VEC limits cell proliferation by retaining VEGFR-2 at the membrane and preventing its internalization into signaling compartments.

Sex-related differences in the presentation, treatment and outcomes among patients with acute coronary syndromes: the Global Registry of Acute Coronary Events.

OBJECTIVE To assess whether sex differences exist in the angiographic severity, management and outcomes of acute coronary syndromes (ACS). METHODS The study comprised 7638 women and 19 117 men with ACS who underwent coronary angiography and were included in GRACE (Global Registry of Acute Coronary Events) from 1999-2006. Normal vessels/mild disease was defined as <50% stenosis in all epicardial vessels; advanced disease was defined as >or=one vessel with >or=50% stenosis. RESULTS Women were older than men and had higher rates of cardiovascular risk factors. Men and women presented equally with chest pain; however, jaw pain and nausea were more frequent among women. Women were more likely to have normal/mild disease (12% vs 6%, p<0.001) and less likely to have left-main and three-vessel disease (27% vs 32%, p<0.001) or undergo percutaneous coronary intervention (65% vs 68%, p<0.001). Women and men with normal and mild disease were treated less aggressively than those with advanced disease. Women with advanced disease had a higher risk of death (4% vs 3%, p<0.01). After adjustment for age and extent of disease, women were more likely to have adverse outcomes (death, myocardial infarction, stroke and rehospitalisation) at six months compared to men (odds ratio 1.24, 95% confidence interval 1.14 to 1.34); however, sex differences in mortality were no longer statistically significant. CONCLUSIONS Women with ACS were more likely to have cardiovascular disease risk factors and atypical symptoms such as nausea compared with men, but were more likely to have normal/mild angiographic coronary artery disease. Further study regarding sex differences related to disease severity is warranted.

In vivo microtubules are copolymers of available beta-tubulin isotypes: localization of each of six vertebrate beta-tubulin isotypes using polyclonal antibodies elicited by synthetic peptide antigens

beta-Tubulin is encoded in the genomes of higher animals by a small multigene family comprising approximately seven functional genes. These genes produce a family of closely related, but distinct polypeptide isotypes that are distinguished principally by sequences within the approximately 15 carboxy-terminal amino acid residues. By immunizing rabbits with chemically synthesized peptides corresponding to these variable domain sequences, we have now prepared polyclonal antibodies specific for each of six distinct isotypes. Specificity of each antiserum has been demonstrated unambiguously by antibody binding to bacterially produced, cloned proteins representing each isotype and by the inhibition of such binding by preincubation of each antiserum only with the immunizing peptide and not with heterologous peptides. Protein blotting of known amounts of cloned, isotypically pure polypeptides has permitted accurate quantitative measurement of the amount of each beta-tubulin isotype present in the soluble and polymer forms in various cells, but has not revealed a bias for preferential assembly of any isotype. Localization of each isotype in three different cell types using indirect immunofluorescence has demonstrated that in vivo each class of microtubules distinguishable by light microscopy is assembled as copolymers of all isotypes expressed in a single cell.

Prevalence and size of directly detected patent foramen ovale in migraine with aura.

BACKGROUND Transcranial contrast Doppler studies have shown an increased prevalence of right-to-left shunts in patients with migraine with aura compared with controls. The anatomy and size of these right-to-left shunts have never been directly assessed. METHODS In a cross-sectional case-control study, the authors performed transesophageal contrast echocardiography in 93 consecutive patients with migraine with aura and 93 healthy controls. RESULTS A patent foramen ovale was present in 44 (47% [95% CI 37 to 58%]) patients with migraine with aura and 16 (17% [95% CI 10 to 26%]) control subjects (OR 4.56 [95% CI 1.97 to 10.57]; p < 0.001). A small shunt was equally prevalent in migraineurs (10% [95% CI 5 to 18%]) and controls (10% [95% CI 5 to 18%]), but a moderate-sized or large shunt was found more often in the migraine group (38% [95% CI 28 to 48%] vs 8% [95% CI 2 to 13%] in controls; p < 0.001). The presence of more than a small shunt increased the odds of having migraine with aura 7.78-fold (95% CI 2.53 to 29.30; p < 0.001). Besides patent foramen ovale prevalence and shunt size, no other echocardiographic differences were found between the study groups. Headache and baseline characteristics did not differ in migraine patients with and without shunt. CONCLUSIONS Nearly half of all patients with migraine with aura have a right-to-left shunt due to a patent foramen ovale. Shunt size is larger in migraineurs than controls. The clinical presentation of migraine is identical in patients with and without a patent foramen ovale.

Classification, functions, and clinical relevance of extracellular vesicles.

Both eukaryotic and prokaryotic cells release small, phospholipid-enclosed vesicles into their environment. Why do cells release vesicles? Initial studies showed that eukaryotic vesicles are used to remove obsolete cellular molecules. Although this release of vesicles is beneficial to the cell, the vesicles can also be a danger to their environment, for instance in blood, where vesicles can provide a surface supporting coagulation. Evidence is accumulating that vesicles are cargo containers used by eukaryotic cells to exchange biomolecules as transmembrane receptors and genetic information. Because also bacteria communicate to each other via extracellular vesicles, the intercellular communication via extracellular cargo carriers seems to be conserved throughout evolution, and therefore vesicles are likely to be a highly efficient, robust, and economic manner of exchanging information between cells. Furthermore, vesicles protect cells from accumulation of waste or drugs, they contribute to physiology and pathology, and they have a myriad of potential clinical applications, ranging from biomarkers to anticancer therapy. Because vesicles may pass the blood-brain barrier, they can perhaps even be considered naturally occurring liposomes. Unfortunately, pathways of vesicle release and vesicles themselves are also being used by tumors and infectious diseases to facilitate spreading, and to escape from immune surveillance. In this review, the different types, nomenclature, functions, and clinical relevance of vesicles will be discussed.

A 3D Map of the Yeast Kinetochore Reveals the Presence of Core and Accessory Centromere-Specific Histone

The budding yeast kinetochore is ~68 nm in length with a diameter slightly larger than a 25 nm microtubule. The kinetochores from the 16 chromosomes are organized in a stereotypic cluster encircling central spindle microtubules. Quantitative analysis of the inner kinetochore cluster (Cse4, COMA) reveals structural features not apparent in singly attached kinetochores. The cluster of Cse4-containing kinetochores is physically larger perpendicular to the spindle axis relative to the cluster of Ndc80 molecules. If there was a single Cse4 (molecule or nucleosome) at the kinetochore attached to each microtubule plus end, the cluster of Cse4 would appear geometrically identical to Ndc80. Thus, the structure of the inner kinetochore at the surface of the chromosomes remains unsolved. We have used point fluorescence microscopy and statistical probability maps to deduce the two-dimensional mean position of representative components of the yeast kinetochore relative to the mitotic spindle in metaphase. Comparison of the experimental images to three-dimensional architectures from convolution of mathematical models reveals a pool of Cse4 radially displaced from Cse4 at the kinetochore and kinetochore microtubule plus ends. The pool of displaced Cse4 can be experimentally depleted in mRNA processing pat1Δ or xrn1Δ mutants. The peripheral Cse4 molecules do not template outer kinetochore components. This study suggests an inner kinetochore plate at the centromere-microtubule interface in budding yeast and yields information on the number of Ndc80 molecules at the microtubule attachment site.

A new era in ovulation induction.

OBJECTIVE To evaluate the efficacy of aromatase inhibitors in ovulation induction, superovulation, and IVF. DESIGN A literature search was conducted with the key words "aromatase inhibitor," "letrozole," "anastrazole," "ovulation induction," "ovulation," and "superovulation" in MEDLINE, EMBASE, and the Cochrane Database of systematic reviews. RESULT(S) Ovulation induction with letrozole is associated with an ovulation rate of 70%-84% and a pregnancy rate of 20%-27% per cycle. In one study, ovulation and pregnancy rates with letrozole seemed to be higher than those of anastrazole. In superovulation, letrozole is associated with few developing follicles and thick endometrium. The use of letrozole for superovulation is associated with a pregnancy rate higher than with the use of clomiphene citrate (CC) (16.7% vs. 5.6%). The addition of letrozole to FSH treatment leads to a decreased FSH requirement. The pregnancy rate for treatment with letrozole and FSH was similar to that for FSH alone. CONCLUSION(S) Aromatase inhibitors are as effective as or superior to CC in ovulation induction and in superovulation. Unlike CC, they do not carry an antiestrogenic effect on the endometrium. Given the advantages of aromatase inhibitors, they can be used to replace CC as ovulation-inducing drugs. Their role in IVF remains to be determined.

Autoradiography of chromosomal DNA fibers from Chinese hamster cells.

Ignorance of the true length of the DNA molecules in the chromosomes of higher organisms has always been a major obstacle to understanding chromosome structure. Consequently, attempts have been made, usually with the aid of electron microscopy, to estimate the size of DNA in higher organisms. Solari (1) has reported the longest such DNA measured before now-a DNA fiber from a sea urchin sperm at least 93 μ, long.

Impact of aortic stiffness on survival in end-stage renal disease.

BACKGROUND Damage to large arteries is a major factor in the high cardiovascular morbidity and mortality of patients with end-stage renal disease (ESRD). Increased arterial stiffness and intima-media thickness, together with increased pulse pressure, are the principal arterial alterations. Whether increased aortic pulse-wave velocity (PWV), a classic marker of increased arterial stiffness, may predict all-cause and/or cardiovascular mortality has never been investigated. METHODS AND RESULTS A cohort of 241 patients with ESRD undergoing hemodialysis was studied between April 1987 and April 1998. The mean duration of follow-up was 72+/-41 months (mean+/-SD). Mean age at entry was 51.5+/-16.3 years. Seventy-three deaths occurred, including 48 cardiovascular and 25 noncardiovascular fatal events. At entry, together with standard clinical and biochemical analyses, patients underwent echocardiography and aortic PWV measured by Doppler ultrasonography. On the basis of Cox analyses, 2 factors emerged as predictors of all-cause and cardiovascular mortality: age and aortic PWV. Hemoglobin and low diastolic pressure interfered to a smaller extent. After adjustment for all the confounding factors, an OR for PWV >12. 0 versus <9.4 m/s was 5.4 (95% CI, 2.4 to 11.9) for all-cause mortality and 5.9 (95% CI, 2.3 to 15.5) for cardiovascular mortality. For each PWV increase of 1 m/s in our study population, all-cause mortality-adjusted OR was 1.39 (95% CI, 1.19 to 1.62). CONCLUSIONS These results provide the first direct evidence that in patients with ESRD, increased aortic stiffness determined by measurement of aortic PWV is a strong independent predictor of all-cause and mainly cardiovascular mortality.

Role of soluble guanylate cyclase in dilator responses of the cerebral microcirculation.

Responses of cerebral blood vessels to nitric oxide (NO) are mediated by soluble guanylate cyclase (sGC)-dependent and potentially by sGC-independent mechanisms. One sGC-independent mechanism by which NO may produce vasodilatation is inhibition of formation of a vasoconstrictor metabolite produced through the cytochrome P450 pathway. In these experiments, we examined the hypothesis that dilatation of cerebral microvessels in response to NO is dependent on activation of sGC. Diameters of cerebral arterioles (baseline diameter=94+/-5 micrometers, mean+/-S.E.) were measured using a closed cranial window in anesthetized rabbits. Under control conditions, YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole], an NO-independent activator of sGC, produced vasodilation that was blocked by ODQ (1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one)(10 microM), an inhibitor of sGC. These findings indicate that sGC is functionally important in cerebral arterioles. In addition, acetylcholine (which stimulates endogenous production of NO by endothelium) produced dilatation of cerebral arterioles that was inhibited by ODQ. For example, 1 microM acetylcholine dilated cerebral arterioles by 34+/-7 and 5+/-1% in the absence and presence of ODQ (10 microM), respectively. Increases in arteriolar diameter in response to sodium nitroprusside (1 microM, an NO donor) were inhibited by approximately 80% by ODQ, but were not affected by 17-ODYA (10 microM) or clotrimazole (10 microM), inhibitors of the cytochrome P450 pathway. Thus, dilatation of the cerebral microcirculation in response to exogenously applied and endogenously produced NO is dependent, in large part, on activation of sGC.

Size separation of circulatory DNA in maternal plasma permits ready detection of fetal DNA polymorphisms.

BACKGROUND Analysis of fetal DNA in maternal plasma has recently been introduced as a new method for noninvasive prenatal diagnosis, particularly for the analysis of fetal genetic traits, which are absent from the maternal genome, e.g., RHD or Y-chromosome-specific sequences. To date, the analysis of other fetal genetic traits has been more problematic because of the overwhelming presence of maternal DNA sequences in the circulation. We examined whether different biochemical properties can be discerned between fetal and maternal circulatory DNA. METHODS Plasma DNA was examined by agarose gel electrophoresis. The fractions of fetal and maternal DNA in size-fractionated fragments were assayed by real-time PCR. The determination of paternally and maternally inherited fetal genetic traits was examined by use of highly polymorphic chromosome-21-specific microsatellite markers. RESULTS Size fractionation of circulatory DNA indicated that the major portion of cell-free fetal DNA had an approximate molecular size of <0.3 kb, whereas maternally derived sequences were, on average, considerably larger than 1 kb. Analysis of size-fractionated DNA (</=0.3 kb) from maternal plasma samples facilitated the ready detection of paternally and maternally inherited microsatellite markers. CONCLUSIONS Circulatory fetal DNA can be enriched by size selection of fragment sizes less than approximately 0.3 kb. Such selection permits easier analysis of both paternally and maternally inherited DNA polymorphisms.

Atherosclerotic plaque rupture--pathologic basis of plaque stability and instability.

Time for primary reveiw 27 days   Atherosclerosis continues to be one of the main subjects in pathology research. The intriguing complexity of its pathogenesis as well as the importance of its clinical sequelae provide a rationale for this [1]. A large number of diseases with totally different clinical presentations are basically atherosclerosis related, and among these, myocardial infarction, stroke, abdominal aneurysms and lower limb ischemia determine to a large extent the morbidity and mortality in Western style populations. But, despite this broad spectrum of clinical disease, most of the acute manifestations of atherosclerosis share a common pathogenetic feature: rupture of an atherosclerotic plaque [2–4]. Plaque disruptions may vary greatly in extent from tiny fissures or erosions of the plaque surface to deep intimal tears which extend into the soft lipid core of lesions; in all these instances, at least some degree of thrombus formation occurs [5, 6]. The abdominal aorta is the arterial site most prominently involved in the process of plaque formation, and also of plaque complications. In this large diameter vessel the process of plaque disruption and thrombosis is not ended by luminal occlusion, and may lead to extensive surface ulcerations comprising large areas of the aortic wall, as can be observed in many autopsy cases at older age. Apart from the undisputable role of atherosclerosis in abdominal aneurysm formation [7], mural thrombosis leads to a surprisingly low rate of clinically significant complications in these patients, although cholesterol emboli can be regularly found in their kidneys and skin at autopsy. Still, it is presently unclear what impact the various biologically active mediators released from eroded aortic surfaces may have on the human body. In contrast, in small diameter vessels such as coronary arteries, occlusive thrombosis is a frequent and often fatal complication of plaque …   * Corresponding author. Tel.: +31-20-5665-633; fax: +31-20-914-738; e-mail a.c.vanderwal@amc.uva.nl

Pharmacokinetic, pharmacodynamic and clinical profile of novel antiplatelet drugs targeting vascular diseases.

Platelet inhibitors are the mainstay treatment for patients with vascular diseases. The current 'gold standard' antiplatelet agent clopidogrel has several pharmacological and clinical limitations that have prompted the search for more effective platelet antagonists. The candidates include various blockers of the purinergic P2Y12 receptor such as prasugrel, an oral irreversible thienopyridine; two adenosine triphosphate analogues that bind reversibly to the P2Y12 receptor: ticagrelor (oral) and cangrelor (intravenous); elinogrel, a direct-acting reversible P2Y12 receptor inhibitor (the only antiplatelet compound that can be administered both intravenously and orally); BX 667, an orally active and reversible small-molecule P2Y12 receptor antagonist; SCH 530348, SCH 205831, SCH 602539 and E5555, highly selective and orally active antagonists on the protease-activated receptor 1. A number of drugs also hit new targets: terutroban, an oral, selective and specific inhibitor of the thromboxane receptor; ARC1779, a second-generation, nuclease resistant aptamer which inhibits von Willebrand factor-dependent platelet aggregation; ALX-0081, a bivalent humanized nanobody targeting the GPIb binding site of von Willebrand factor and AJW200, an IgG4 monoclonal antibody of von Willebrand factor. The pharmacology and clinical profiles of new platelet antagonists indicate that they provide more consistent, more rapid and more potent platelet inhibition than agents currently used. Whether these potential advantages will translate into clinical advantages will require additional comparisons in properly powered, randomized, controlled trials.

Scaffold-based three-dimensional human fibroblast culture provides a structural matrix that supports angiogenesis in infarcted heart tissue.

BACKGROUND We have developed techniques to implant angiogenic patches onto the epicardium over regions of infarcted cardiac tissue to stimulate revascularization of the damaged tissue. These experiments used a scaffold-based 3D human dermal fibroblast culture (3DFC) as an epicardial patch. The 3DFC contains viable cells that secrete angiogenic growth factors and has previously been shown to stimulate angiogenic activity. The hypothesis tested was that a viable 3DFC cardiac patch would stimulate an angiogenic response within an area of infarcted cardiac tissue. METHODS AND RESULTS A coronary occlusion of a branch of the left anterior descending coronary artery was performed by thermal ligation in severe combined immunodeficient mice. 3DFCs with or without viable cells were sized to the damaged area, implanted in replicate mice onto the epicardium at the site of tissue injury, and compared with animals that received infarct surgery but no implant. Fourteen and 30 days after surgery, hearts were exposed and photographed, and tissue samples were prepared for histology and cytochemistry. Fourteen and 30 days after surgery, the damaged myocardium receiving viable 3DFC exhibited a significantly greater angiogenic response (including arterioles, venules, and capillaries) than nonviable and untreated control groups. CONCLUSIONS In this animal model, viable 3DFC stimulates angiogenesis within a region of cardiac infarction and can augment a repair response in damaged tissue. Therefore, a potential use for 3DFC is the repair of myocardial tissue damaged by infarction.

Extracellular vesicles from human cardiac progenitor cells inhibit cardiomyocyte apoptosis and improve cardiac function after myocardial infarction.

AIMS Recent evidence suggests that cardiac progenitor cells (CPCs) may improve cardiac function after injury. The underlying mechanisms are indirect, but their mediators remain unidentified. Exosomes and other secreted membrane vesicles, hereafter collectively referred to as extracellular vesicles (EVs), act as paracrine signalling mediators. Here, we report that EVs secreted by human CPCs are crucial cardioprotective agents. METHODS AND RESULTS CPCs were derived from atrial appendage explants from patients who underwent heart valve surgery. CPC-conditioned medium (CM) inhibited apoptosis in mouse HL-1 cardiomyocytic cells, while enhancing tube formation in human umbilical vein endothelial cells. These effects were abrogated by depleting CM of EVs. They were reproduced by EVs secreted by CPCs, but not by those secreted by human dermal fibroblasts. Transmission electron microscopy and nanoparticle tracking analysis showed most EVs to be 30-90 nm in diameter, the size of exosomes, although smaller and larger vesicles were also present. MicroRNAs most highly enriched in EVs secreted by CPCs compared with fibroblasts included miR-210, miR-132, and miR-146a-3p. miR-210 down-regulated its known targets, ephrin A3 and PTP1b, inhibiting apoptosis in cardiomyocytic cells. miR-132 down-regulated its target, RasGAP-p120, enhancing tube formation in endothelial cells. Infarcted hearts injected with EVs from CPCs, but not from fibroblasts, exhibited less cardiomyocyte apoptosis, enhanced angiogenesis, and improved LV ejection fraction (0.8 ± 6.8 vs. -21.3 ± 4.5%; P < 0.05) compared with those injected with control medium. CONCLUSION EVs are the active component of the paracrine secretion by human CPCs. As a cell-free approach, EVs could circumvent many of the limitations of cell transplantation.

ARNO regulates VEGF-dependent tissue responses by stabilizing endothelial VEGFR-2 surface expression.

AIMS The vascular endothelial growth factor (VEGF) stimulates angiogenesis by induction of vessel permeability, proliferation, and migration of endothelial cells, an important process in ischaemic diseases. ADP-ribosylation factor (ARF) nucleotide-binding site opener (ARNO) (cytohesin-2) is a guanine exchange factor important for cellular signalling through ARF GTPases. However, a role for ARNO in VEGF-dependent endothelial processes has so far not been documented. Therefore, we investigated whether ARNO has a role in VEGF-dependent activation of endothelial cells and thus vessel permeability. METHODS AND RESULTS ARNO expression was observed in endothelial cells in vitro by RT-PCR, western blotting, and immunofluorescence as well as ex vivo by immunohistochemical staining of mouse aorta. Treatment with the cytohesin inhibitor SecinH3 or with an ARNO siRNA prevented VEGF-dependent Akt activation, assessed by detection of phosphorylated Akt, and proliferation of endothelial cells in vitro, measured by methylthiazoletetrazolium (MTT) reduction. In addition, ARNO suppression reduced VEGF-induced permeability in vessels of the mouse (C57BL/6) cremaster muscle in vivo, as measured by extravasation of fluorescein isothiocyanate (FITC)-dextran. Moreover, ARNO knock-down accelerated ligand-induced reduction in vascular endothelial growth factor receptor-2 (VEGFR-2) surface expression, internalization, and degradation, as assessed by flow cytometry and western blotting, respectively. CONCLUSION Our findings indicate an important and novel role for endothelial ARNO in VEGF-dependent initiation of angiogenesis by regulation of VEGFR-2 internalization in endothelial cells, resulting in the activation of the Akt pathway, vessel permeability, and ultimately endothelial proliferation. Thus, ARNO may be a new essential player in endothelial signalling and angiogenesis.

Regulation of immune responses by extracellular vesicles

Extracellular vesicles, including exosomes, are small membrane vesicles derived from multivesicular bodies or from the plasma membrane. Most, if not all, cell types release extracellular vesicles, which then enter the bodily fluids. These vesicles contain a subset of proteins, lipids and nucleic acids that are derived from the parent cell. It is thought that extracellular vesicles have important roles in intercellular communication, both locally and systemically, as they transfer their contents, including proteins, lipids and RNAs, between cells. Extracellular vesicles are involved in numerous physiological processes, and vesicles from both non-immune and immune cells have important roles in immune regulation. Moreover, extracellular vesicle-based therapeutics are being developed and clinically tested for the treatment of inflammatory diseases, autoimmune disorders and cancer. Given the tremendous therapeutic potential of extracellular vesicles, this Review focuses on their role in modulating immune responses, as well as their potential therapeutic applications.

Interplay between microtubule bundling and sorting factors ensures acentriolar spindle stability during C. elegans oocyte meiosis

In many species, oocyte meiosis is carried out in the absence of centrioles. As a result, microtubule organization, spindle assembly, and chromosome segregation proceed by unique mechanisms. Here, we report insights into the principles underlying this specialized form of cell division, through studies of C. elegans KLP-15 and KLP-16, two highly homologous members of the kinesin-14 family of minus-end-directed kinesins. These proteins localize to the acentriolar oocyte spindle and promote microtubule bundling during spindle assembly; following KLP-15/16 depletion, microtubule bundles form but then collapse into a disorganized array. Surprisingly, despite this defect we found that during anaphase, microtubules are able to reorganize into a bundled array that facilitates chromosome segregation. This phenotype therefore enabled us to identify factors promoting microtubule organization during anaphase, whose contributions are normally undetectable in wild-type worms; we found that SPD-1 (PRC1) bundles microtubules and KLP-18 (kinesin-12) likely sorts those bundles into a functional orientation capable of mediating chromosome segregation. Therefore, our studies have revealed an interplay between distinct mechanisms that together promote spindle formation and chromosome segregation in the absence of structural cues such as centrioles.

Arteriolar function in visceral adipose tissue is impaired in human obesity.

OBJECTIVE The purpose of this study was to characterize the relationship between adipose tissue phenotype and depot-specific microvascular function in fat. METHODS AND RESULTS In 30 obese subjects (age 42±11 years, body mass index 46±11 kg/m(2)) undergoing bariatric surgery, we intraoperatively collected visceral and subcutaneous adipose tissue and characterized depot-specific adipose phenotypes. We assessed vasomotor function of the adipose microvasculature using videomicroscopy of small arterioles (75-250 μm) isolated from different fat compartments. Endothelium-dependent, acetylcholine-mediated vasodilation was severely impaired in visceral arterioles, compared to the subcutaneous depot (P<0.001 by ANOVA). Nonendothelium dependent responses to papaverine and nitroprusside were similar. Endothelial nitric oxide synthase inhibition with N(ω)-nitro-l-arginine methyl ester reduced subcutaneous vasodilation but had no effect on severely blunted visceral arteriolar responses. Visceral fat exhibited greater expression of proinflammatory, oxidative stress-related, hypoxia-induced, and proangiogenic genes; increased activated macrophage populations; and had a higher capacity for cytokine production ex vivo. CONCLUSIONS Our findings provide clinical evidence that the visceral microenvironment may be intrinsically toxic to arterial health providing a potential mechanism by which visceral adiposity burden is linked to atherosclerotic vascular disease. Our findings also support the evolving concept that both adipose tissue quality and quantity may play significant roles in shaping cardiovascular phenotypes in human obesity.

Effects of Abciximab on the architecture of platelet-rich clots in patients with acute myocardial infarction undergoing primary coronary intervention.

BACKGROUND Abciximab plus aspirin improves the TIMI 3 flow rate of the infarct-related artery in patients treated with either percutaneous coronary intervention or thrombolysis. The present study investigated whether the reperfusion efficacy of abciximab relates to modifications of clot architecture in patients admitted for acute myocardial infarction (AMI). METHODS AND RESULTS A total of 23 AMI patients in the Abciximab before Direct angioplasty and stenting in Myocardial Infarction Regarding Acute and Long term follow-up (ADMIRAL) trial received, in a double-blind fashion, either abciximab (n=13) or placebo (n=10) before primary stenting. Viscoelastic (G' in dyne/cm(2)) and morphological (mean platelet aggregate surface area [SAG] in micrometer(2)) indexes of ex vivo platelet-rich clots (PRC) were assessed in a double-blind fashion before and after the bolus administration of abciximab or placebo. G' and SAG reflect the mechanical and morphological impact of activated platelets on the PRC fibrin network, respectively. Abciximab administration reduced G' by 63% (P=0.0001) and SAG by 65% (P=0.0007), and no effect was seen in the placebo group. These abciximab-related changes increased fibrin exposure as a consequence of the platelet-aggregate surface reduction and may have improved endogenous fibrinolysis. These effects were identified in all patients, independent of previous heparin administration. CONCLUSIONS Abciximab dramatically reduces platelet aggregate size and increases the fibrin accessibility of ex vivo PRC in AMI patients. These modifications could participate in the better coronary artery patency observed with abciximab.

Disruption of tumor necrosis factor-alpha gene diminishes the development of atherosclerosis in ApoE-deficient mice.

Inflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha) have been implicated in atherogenesis. However, the precise role of TNF-alpha in atherogenesis is still unclear. To examine the effect of TNF-alpha on atherogenesis, we generated compound-deficient mice in apolipoprotein E (apoE) and TNF-alpha (apoE-/-/TNF-alpha-/-) and compared them with apoE-/- mice. Although serum total cholesterol levels were markedly elevated in both apoE-/-/TNF-alpha-/- and apoE-/- mice compared to wild-type mice, no differences were observed between apoE-/-/TNF-alpha-/- and apoE-/- mice. The atherosclerotic plaque area in the aortic luminal surface of apoE-/-/TNF-alpha-/- mice (n=8, 3.1+/-0.4%) was significantly smaller than that of apoE-/- mice (n=7, 4.7+/-0.4%, p<0.001) despite the lack of difference in serum cholesterol levels. The atherosclerotic lesion size in the aortic sinus of apoE-/-/TNF-alpha-/- mice (n=10, 5.1+/-0.3 x 10(5)microm(2)) was also significantly smaller than that of apoE-/- mice (n=11, 7.0+/-0.3 x 10(5)microm(2), p<0.0001). RT-PCR analysis indicated that the expression levels of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) were significantly higher in apoE-/- than apoE-/-/TNF-alpha-/- mice. Macrophages from apoE(-/-) mice showed higher uptake level of oxidized LDL and increased expression level of scavenger receptor class A (SRA) compared to those from apoE-/-/TNF-alpha-/- mice. These results indicate that TNF-alpha plays an atherogenic role by upregulating the expressions of ICAM-1, VCAM-1 and MCP-1 in the vascular wall, and by inducing SRA expression and oxidized LDL uptake in macrophages.

Apoptosis in breast cancer: relationship with other pathological parameters.

Apoptosis is a frequent phenomenon in breast cancer and it can be detected by light microscopy in conventional histopathological sections or by special staining techniques. The number of apoptotic cells as a percentage of cells present, or the number of apoptotic cells per square millimetre of neoplastic tissue, is usually described as the apoptotic index (AI). In breast cancer, the AI is not related to tumour size, axillary lymph node metastasis or distant metastasis at diagnosis. It is greater in invasive ductal carcinomas than in other histological types. High AI is also related to high histological grade, high nuclear grade, comedo-type necrosis, lack of tubule formation, and dense infiltration of the tumour by lymphocytes. Sex steroid receptor-negative tumours have greater AIs than the sex steroid receptor-positive ones. Aneuploid breast cancers with high S-phase fractions (SPFs) also have high AI values compared with diploid tumours with low SPFs. p53-Positive breast cancers have high AIs, whereas tumours that are Bcl-2 positive have low AIs. The AI shows a strong positive correlation to all direct or indirect indicators of cell proliferation, such as mitotic index and Ki67 immunolabelling. Univariate survival analyses show that a high AI is linked with unfavourable disease outcome in axillary lymph node-negative and -positive breast cancer, but multivariate analyses indicate that AI is not an independent prognostic factor. In conclusion, a high AI is related to malignant cellular features and indicators of invasiveness and cell proliferation in breast cancer.

Subcellular steroid/nuclear receptor dynamics.

Steroid hormones, thyroid hormones, retinoic acids, and vitamin D bind to their receptors, which are now called steroid/nuclear receptors, and liganded receptors translocate either intracellularly or intranuclearly and form large protein complexes with cofactors to induce or repress gene transcription. Therefore, steroid/nuclear receptors are ligand-dependent transcription factors. With the advent of green fluorescent protein (GFP) and its color variants, the subcellular distribution of many steroid/nuclear receptors has been found to be much more dynamic than previously thought, with some of the receptors shuttling between the cytoplasm and nucleus. Steroid/nuclear receptors can be divided into three categories based on their unliganded distribution: those that are primarily in the nucleus, those in the cytoplasm, and those with mixed cytoplasmic and nuclear distributions. However, in all cases, the addition of a ligand leads to almost complete nuclear translocation of the receptors. Hormonal stimulation induces intranuclear receptor distribution from a homogeneous pattern to a heterogeneous dot-like image. Ligand binding to steroid/nuclear receptors leads to the recruitment of many proteins including cofactors to provoke the redistribution of receptor complexes in the nucleus. This focal organization could involve more complex events than simple DNA binding sites for transcription. Protein activities and interactions of steroid/nuclear receptors can be imaged and localized in a single cell.

Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms.

In patients with spinal cord injury, the primary or mechanical trauma seldom causes total transection, even though the functional loss may be complete. In addition, biochemical and pathological changes in the cord may worsen after injury. To explain these phenomena, the concept of the secondary injury has evolved for which numerous pathophysiological mechanisms have been postulated. This paper reviews the concept of secondary injury with special emphasis on vascular mechanisms. Evidence is presented to support the theory of secondary injury and the hypothesis that a key mechanism is posttraumatic ischemia with resultant infarction of the spinal cord. Evidence for the role of vascular mechanisms has been obtained from a variety of models of acute spinal cord injury in several species. Many different angiographic methods have been used for assessing microcirculation of the cord and for measuring spinal cord blood flow after trauma. With these techniques, the major systemic and local vascular effects of acute spinal cord injury have been identified and implicated in the etiology of secondary injury. The systemic effects of acute spinal cord injury include hypotension and reduced cardiac output. The local effects include loss of autoregulation in the injured segment of the spinal cord and a marked reduction of the microcirculation in both gray and white matter, especially in hemorrhagic regions and in adjacent zones. The microcirculatory loss extends for a considerable distance proximal and distal to the site of injury. Many studies have shown a dose-dependent reduction of spinal cord blood flow varying with the severity of injury, and a reduction of spinal cord blood flow which worsens with time after injury. The functional deficits due to acute spinal cord injury have been measured electrophysiologically with techniques such as motor and somatosensory evoked potentials and have been found proportional to the degree of posttraumatic ischemia. The histological effects include early hemorrhagic necrosis leading to major infarction at the injury site. These posttraumatic vascular effects can be treated. Systemic normotension can be restored with volume expansion or vasopressors, and spinal cord blood flow can be improved with dopamine, steroids, nimodipine, or volume expansion. The combination of nimodipine and volume expansion improves posttraumatic spinal cord blood flow and spinal cord function measured by evoked potentials. These results provide strong evidence that posttraumatic ischemia is an important secondary mechanism of injury, and that it can be counteracted.

Genetic variants rs1994016 and rs3825807 in ADAMTS7 affect its mRNA expression in atherosclerotic occlusive peripheral arterial disease

AIM Peripheral artery disease (PAD) is a vascular disease affecting peripheral circulation. Recently, genome-wide association studies revealed a relationship between single nucleotide polymorphisms (SNPs) in ADAMTS7 (a disintegrin and metalloprotease with thrombospondin motif 7) and atherosclerosis. In this study, we aimed to determine ADAMTS7 expression in peripheral blood mononuclear cells (PBMCs) and the frequency of ADAMTS7 rs1994016 and rs3825807 polymorphisms in a sample of Turkish patients with PAD, and to evaluate the association of matrix metalloproteinase (MMP) levels with PAD development. METHODS In this case-control study, ADAMTS7mRNA and protein expression was determined using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and western blot, respectively, and rs1994016 and rs3825807 variants in ADAMTS7 were determined by real-time PCR in 115 PAD patients and 116 healthy controls. Plasma levels of nine MMPs were determined using a multiplex immunoassay system. RESULTS ADAMTS7mRNA levels were significantly higher in PAD patients than in controls (t=-2.75, P=.007). There was no significant difference in the frequencies of rs1994016 and rs3825807 between PAD patients and controls (P>.05). In PAD patients, ADAMTS7mRNA levels were significantly increased for the CC genotype of rs1994016 (t=-2.31, P=.026) and TT genotype of rs3825807 (t=-2.23, P=.032). Furthermore, plasma levels of MMP-1, MMP-3, MMP-7, MMP-10, MMP-12, and MMP-13 were significantly higher in PAD patients than in controls (P<.05). CONCLUSION This is the first report of the relationship between PAD and ADAMTS7 expression and the effects of the rs1994016 and rs3825807 variants on PAD development. ADAMTS7 may be associated with PAD development.

Frontal stroke syndromes.

The frontal lobe is the largest lobe of the brain, and it is thus commonly involved in stroke. Moreover, almost one in five strokes is limited to the prerolandic areas. This high frequency of anatomical involvement is in sharp contrast with the apparent rarity of clinical frontal dysfunction in stroke. It is remarkable that frontal behavioral syndromes have been rather uncommonly reported in patients with stroke as compared to patients with other diseases, such as brain tumor. This fact is paradoxical, because an acute process (stroke) is expected to yield more clinical dysfunction than a more chronic disease (tumor). A volume effect may be the main factor leading to this phenomenon. Another interesting aspect of frontal strokes is the contribution of so-called 'silent' strokes, the recurrence of which may nevertheless lead to intellectual decline and compromise recovery from another stroke with more specific neurologic dysfunction. The contribution of stroke to understanding of frontal lobe dysfunction is important, because of the focal nature of this disease, and great opportunity for clinical-topographic classification correlations. One of the first modern attempts to develop a clinical-topographic classification of frontal lobe lesions came from the school of Luria, who tried to delineate three main types of frontal lobe syndromes (premotor syndrome, prefrontal syndrome, medial-frontal syndrome). Recent anatomic correlates using MRI make it possible to improve this classification. We suggest considering six main clinical-anatomic frontal stroke syndromes: (1) prefrontal; (2) premotor; (3) superior medial; (4) orbital-medial; (5) basal forebrain; (6) white matter. Finally, another fascinating topic relates to frontal lobe symptomatology due to stroke sparing the frontal cortex or white matter. This occurs mainly in three instances: lenticulo-capsular stroke, caudate stroke, and thalamic stroke. Studies using blood flow or metabolism measurements suggest that diaschisis (frontal lobe dysfunction from a remote lesion) may play a role. We believe that this is more likely to be related to dynamic interruption of complex circuitry than to static frontal lobe deactivation.

Effectiveness of statin-eluting stent on early inflammatory response and neointimal thickness in a porcine coronary model.

BACKGROUND Drug-eluting stent (DES) implantation is routine during coronary revascularization because DES significantly reduce rates of restenosis and target lesion revascularization compared with bare metal stent (BMS). However, available DES have limitations, such as late thrombosis because of delayed healing with poorer endothelialization and persistent local inflammation. Statins can inhibit cell proliferation, inflammation, and restore endothelial function. The present study evaluated the ability of stent-based cerivastatin delivery to reduce stent-induced inflammatory responses and adverse effects on endothelial function, and to inhibit neointimal hyperplasia in a porcine coronary model. METHODS AND RESULTS Pigs were randomized into groups in which the coronary arteries (9 pigs, 18 coronaries in each group) had either a cerivastatin-eluting stent (CES) or a BMS. All animals survived without any adverse effects. Inflammatory cell infiltration evaluated using scanning electron microscopy on day 3 after stenting was significantly decreased in the treated vessels (inflammation score: 1.15+/-0.12 vs 2.43+/-0.34, p<0.0001). At day 28, endothelial function with intracoronary infusion of bradykinin was preserved in both the CES and BMS groups. Volumetric intravascular ultrasound images revealed decreased intimal volume in the CES group (28.3+/-5.4 vs 75.9+/-4.2 mm3, p<0.0001). Histomorphometric analysis showed reduced neointimal area (1.74+/-0.45 vs 3.83+/-0.51 mm2, p<0.0001) in the CES group despite similar injury scores (1.77+/-0.30 vs 1.77+/-0.22, p=0.97). CONCLUSION In porcine coronary arteries CES significantly decreased neointimal hyperplasia with a decreased early inflammatory response and without endothelial dysfunction.

Improved prognosis of thoracic aortic aneurysms: a population-based study.

CONTEXT Managing thoracic aortic aneurysms identified incidentally by increased use of computed tomography, echocardiography, and magnetic resonance imaging is problematic, especially in the elderly. OBJECTIVE To ascertain whether the previously reported poor prognosis for individuals with thoracic aortic aneurysms has changed with better medical therapies and improved surgical techniques that can now be applied to aneurysm management. DESIGN Population-based cohort study. SETTING AND PATIENTS All 133 patients with the diagnosis of degenerative thoracic aortic aneurysms among Olmsted County, Minnesota, residents between 1980 and 1994 compared with a previously reported cohort of similar patients between 1951 and 1980. MAIN OUTCOME MEASURES The primary clinical end points were incidence, cumulative rupture risk, rupture risk as a function of aneurysm size, and survival. RESULTS In contrast to abdominal aortic aneurysms, for which men are affected predominately, 51% of thoracic aortic aneurysms were identified in women who were considerably older at recognition than men (mean age, 75.9 vs 62.8 years, respectively; P= .01). The overall incidence rate of 10.4 per 100000 person-years (95% confidence interval [CI], 8.6-12.2) between 1980 and 1994 was more than 3-fold higher than the rate from 1951 to 1980. The cumulative risk of rupture was 20% after 5 years. Seventy-nine percent of ruptures occurred in women (P= .01). The 5-year risk of rupture as a function of aneurysm size at recognition was 0% for aneurysms less than 4 cm in diameter, 16% (95% CI, 4%-28%) for those 4 to 5.9 cm, and 31% (95% CI, 5%-56%) for aneurysms 6 cm or more. Overall 5-year survival improved to 56% (95% CI, 48%-66%) between 1980 and 1994 compared with only 19% between 1951 and 1980 (P<.01). CONCLUSIONS In this population, elderly women represent an increasing portion of all patients with clinically recognized thoracic aortic aneurysms and constitute the majority of patients whose aneurysm eventually ruptures. Overall survival for thoracic aortic aneurysms has improved significantly in the past 15 years.

Murine Dishevelled 3 Functions in Redundant Pathways with Dishevelled 1 and 2 in Normal Cardiac Outflow Tract, Cochlea, and Neural Tube Development

Dishevelled (Dvl) proteins are important signaling components of both the canonical beta-catenin/Wnt pathway, which controls cell proliferation and patterning, and the planar cell polarity (PCP) pathway, which coordinates cell polarity within a sheet of cells and also directs convergent extension cell (CE) movements that produce narrowing and elongation of the tissue. Three mammalian Dvl genes have been identified and the developmental roles of Dvl1 and Dvl2 were previously determined. Here, we identify the functions of Dvl3 in development and provide evidence of functional redundancy among the three murine Dvls. Dvl3(-/-) mice died perinatally with cardiac outflow tract abnormalities, including double outlet right ventricle and persistent truncus arteriosis. These mutants also displayed a misorientated stereocilia in the organ of Corti, a phenotype that was enhanced with the additional loss of a single allele of the PCP component Vangl2/Ltap (LtapLp/+). Although neurulation appeared normal in both Dvl3(-/-) and LtapLp/+ mutants, Dvl3(+/-);LtapLp/+ combined mutants displayed incomplete neural tube closure. Importantly, we show that many of the roles of Dvl3 are also shared by Dvl1 and Dvl2. More severe phenotypes were observed in Dvl3 mutants with the deficiency of another Dvl, and increasing Dvl dosage genetically with Dvl transgenes demonstrated the ability of Dvls to compensate for each other to enable normal development. Interestingly, global canonical Wnt signaling appeared largely unaffected in the double Dvl mutants, suggesting that low Dvl levels are sufficient for functional canonical Wnt signals. In summary, we demonstrate that Dvl3 is required for cardiac outflow tract development and describe its importance in the PCP pathway during neurulation and cochlea development. Finally, we establish several developmental processes in which the three Dvls are functionally redundant.

Functional specialization of Piwi proteins in Paramecium tetraurelia from post-transcriptional gene silencing to genome remodelling

Proteins of the Argonaute family are small RNA carriers that guide regulatory complexes to their targets. The family comprises two major subclades. Members of the Ago subclade, which are present in most eukaryotic phyla, bind different classes of small RNAs and regulate gene expression at both transcriptional and post-transcriptional levels. Piwi subclade members appear to have been lost in plants and fungi and were mostly studied in metazoa, where they bind piRNAs and have essential roles in sexual reproduction. Their presence in ciliates, unicellular organisms harbouring both germline micronuclei and somatic macronuclei, offers an interesting perspective on the evolution of their functions. Here, we report phylogenetic and functional analyses of the 15 Piwi genes from Paramecium tetraurelia. We show that four constitutively expressed proteins are involved in siRNA pathways that mediate gene silencing throughout the life cycle. Two other proteins, specifically expressed during meiosis, are required for accumulation of scnRNAs during sexual reproduction and for programmed genome rearrangements during development of the somatic macronucleus. Our results indicate that Paramecium Piwi proteins have evolved to perform both vegetative and sexual functions through mechanisms ranging from post-transcriptional mRNA cleavage to epigenetic regulation of genome rearrangements.

Blood stem cells emerge from aortic endothelium by a novel type of cell transition

The ontogeny of haematopoietic stem cells (HSCs) during embryonic development is still highly debated, especially their possible lineage relationship to vascular endothelial cells. The first anatomical site from which cells with long-term HSC potential have been isolated is the aorta-gonad-mesonephros (AGM), more specifically the vicinity of the dorsal aortic floor. But although some authors have presented evidence that HSCs may arise directly from the aortic floor into the dorsal aortic lumen, others support the notion that HSCs first emerge within the underlying mesenchyme. Here we show by non-invasive, high-resolution imaging of live zebrafish embryos, that HSCs emerge directly from the aortic floor, through a stereotyped process that does not involve cell division but a strong bending then egress of single endothelial cells from the aortic ventral wall into the sub-aortic space, and their concomitant transformation into haematopoietic cells. The process is polarized not only in the dorso-ventral but also in the rostro-caudal versus medio-lateral direction, and depends on Runx1 expression: in Runx1-deficient embryos, the exit events are initially similar, but much rarer, and abort into violent death of the exiting cell. These results demonstrate that the aortic floor is haemogenic and that HSCs emerge from it into the sub-aortic space, not by asymmetric cell division but through a new type of cell behaviour, which we call an endothelial haematopoietic transition.

Nonlinear Elasticity in Biological Gels

Unlike most synthetic materials, biological materials often stiffen as they are deformed. This nonlinear elastic response, critical for the physiological function of some tissues, has been documented since at least the 19th century, but the molecular structure and the design principles responsible for it are unknown. Current models for this response require geometrically complex ordered structures unique to each material. In this Article we show that a much simpler molecular theory accounts for strain stiffening in a wide range of molecularly distinct biopolymer gels formed from purified cytoskeletal and extracellular proteins. This theory shows that systems of semi-flexible chains such as filamentous proteins arranged in an open crosslinked meshwork invariably stiffen at low strains without the need for a specific architecture or multiple elements with different intrinsic stiffnesses.

Sperm function tests and fertility.

Traditionally, the diagnosis of male infertility has depended upon a descriptive evaluation of human semen with emphasis on the number of spermatozoa that are present in the ejaculate, their motility and their morphology. The fundamental tenet underlying this approach is that male fertility can be defined by reference to a threshold concentration of motile, morphologically normal spermatozoa that must be exceeded in order to achieve conception. Many independent studies have demonstrated that this fundamental concept is flawed and, in reality, it is not so much the absolute number of spermatozoa that determines fertility, but their functional competence. In the light of this conclusion, a range of in vitro tests have been developed to monitor various aspects of sperm function including their potential for movement, cervical mucus penetration, capacitation, zona recognition, the acrosome reaction and sperm-oocyte fusion. Such functional assays have been found to predict the fertilizing capacity of human spermatozoa in vitro and in vivo with some accuracy. Recent developments in this field include the introduction of tests to assess the degree to which human spermatozoa have suffered oxidative stress as well as the integrity of their nuclear and mitochondrial DNA. Such assessments not only yield information on the fertilizing capacity of human spermatozoa but also their ability to support normal embryonic development.

Direct in vivo assessment of microcirculatory dysfunction in severe falciparum malaria.

BACKGROUND This study sought to describe and quantify microcirculatory changes in the mucosal surfaces of patients with severe malaria, by direct in vivo observation using orthogonal polarization spectral (OPS) imaging. METHODS The microcirculation in the rectal mucosa of adult patients with severe malaria was assessed by use of OPS imaging, at admission and then daily. Comparison groups comprised patients with uncomplicated falciparum malaria, patients with bacterial sepsis, and healthy individuals. RESULTS Erythrocyte velocities were measured directly in 43 adult patients with severe falciparum malaria, of whom 20 died. Microcirculatory blood flow was markedly disturbed, with heterogeneous obstruction that was proportional to severity of disease. Blocked capillaries were found in 29 patients (67%) and were associated with concurrent hyperdynamic blood flow (erythrocyte velocity, >750 mm/s) in adjacent vessels in 27 patients (93%). The proportion of blocked capillaries correlated with the base deficit in plasma and with the concentration of lactate. Abnormalities disappeared when the patients recovered. In healthy individuals and in patients with uncomplicated malaria or sepsis, no stagnant erythrocytes were detected, and, in patients with sepsis, hyperdynamic blood flow was prominent. CONCLUSION Patients with severe falciparum malaria show extensive microvascular obstruction that is proportional to the severity of the disease. This finding underscores the prominent role that microvascular obstruction plays in the pathophysiology of severe malaria and illustrates the fundamental difference between the microvascular pathophysiology of malaria and that of bacterial sepsis.

Exploiting lymphatic transport and complement activation in nanoparticle vaccines

Antigen targeting and adjuvancy schemes that respectively facilitate delivery of antigen to dendritic cells and elicit their activation have been explored in vaccine development. Here we investigate whether nanoparticles can be used as a vaccine platform by targeting lymph node–residing dendritic cells via interstitial flow and activating these cells by in situ complement activation. After intradermal injection, interstitial flow transported ultra-small nanoparticles (25 nm) highly efficiently into lymphatic capillaries and their draining lymph nodes, targeting half of the lymph node–residing dendritic cells, whereas 100-nm nanoparticles were only 10% as efficient. The surface chemistry of these nanoparticles activated the complement cascade, generating a danger signal in situ and potently activating dendritic cells. Using nanoparticles conjugated to the model antigen ovalbumin, we demonstrate generation of humoral and cellular immunity in mice in a size- and complement-dependent manner.

Large deformation of red blood cell ghosts in a simple shear flow.

Red blood cells are known to change shape in response to local flow conditions. Deformability affects red blood cell physiological function and the hydrodynamic properties of blood. The immersed boundary method is used to simulate three-dimensional membrane-fluid flow interactions for cells with the same internal and external fluid viscosities. The method has been validated for small deformations of an initially spherical capsule in simple shear flow for both neo-Hookean and the Evans-Skalak membrane models. Initially oblate spheroidal capsules are simulated and it is shown that the red blood cell membrane exhibits asymptotic behavior as the ratio of the dilation modulus to the extensional modulus is increased and a good approximation of local area conservation is obtained. Tank treading behavior is observed and its period calculated.

α-Catenin as a tension transducer that induces adherens junction development

Adherens junctions (AJs), which are organized by adhesion proteins and the underlying actin cytoskeleton, probably sense pulling forces from adjacent cells and modulate opposing forces to maintain tissue integrity, but the regulatory mechanism remains unknown at the molecular level. Although the possibility that α-catenin acts as a direct linker between the membrane and the actin cytoskeleton for AJ formation and function has been minimized, here we show that α-catenin recruits vinculin, another main actin-binding protein of AJs, through force-dependent changes in α-catenin conformation. We identified regions in the α-catenin molecule that are required for its force-dependent binding of vinculin by introducing mutant α-catenin into cells and using in vitro binding assays. Fluorescence recovery after photobleaching analysis for α-catenin mobility and the existence of an antibody recognizing α-catenin in a force-dependent manner further supported the notion that α-catenin is a tension transducer that translates mechanical stimuli into a chemical response, resulting in AJ development.

Posttranslational Acetylation of α-Tubulin Constrains Protofilament Number in Native Microtubules

BACKGROUND Microtubules are built from linear polymers of α-β tubulin dimers (protofilaments) that form a tubular quinary structure. Microtubules assembled from purified tubulin in vitro contain between 10 and 16 protofilaments; however, such structural polymorphisms are not found in cells. This discrepancy implies that factors other than tubulin constrain microtubule protofilament number, but the nature of these constraints is unknown. RESULTS Here, we show that acetylation of MEC-12 α-tubulin constrains protofilament number in C. elegans touch receptor neurons (TRNs). Whereas the sensory dendrite of wild-type TRNs is packed with a cross-linked bundle of long, 15-protofilament microtubules, mec-17;atat-2 mutants lacking α-tubulin acetyltransferase activity have short microtubules, rampant lattice defects, and variable protofilament number both between and within microtubules. All-atom molecular dynamics simulations suggest a model in which acetylation of lysine 40 promotes the formation of interprotofilament salt bridges, stabilizing lateral interactions between protofilaments and constraining quinary structure to produce stable, structurally uniform microtubules in vivo. CONCLUSIONS Acetylation of α-tubulin is an essential constraint on protofilament number in vivo. We propose a structural model in which this posttranslational modification promotes the formation of lateral salt bridges that fine-tune the association between adjacent protofilaments and enable the formation of uniform microtubule populations in vivo.

Angina with "normal" coronary arteries: sex differences in outcomes.

BACKGROUND Several studies have demonstrated that women with nonobstructive coronary disease have a high rate of subsequent investigations, rehospitalizations for recurrent chest pain, and repeat coronary angiography. The sex specificity of this finding is unclear. We therefore undertook an evaluation of sex differences in rehospitalization for acute coronary syndrome (ACS) or chest pain in patients with "angiographically normal" coronaries. METHODS A retrospective cohort study using prospectively collected angiographic and clinical data on all patients in British Columbia, Canada, presenting for their first cardiac catheterization with suspected ischemic heart disease but angiographically normal coronaries. RESULTS Among 32,856 patients, 7.1% of men versus 23.3% of women were angiographically normal (P < .001). Among angiographically normal patients, women were older and more likely to present with hypertension, prior stroke, chronic obstructive pulmonary disease, and peripheral vascular disease than men, but Canadian Cardiovascular Society class of angina did not vary by sex. Within 1 year, 1.0% died, (19 women, 18 men, P = .27) and 0.6% had a stroke (13 women, 9 men, P = .91). Readmission to hospital for ACS or chest pain requiring catheterization was significantly higher in women compared to men (adjusted OR 4.06; 95% CI 1.15-14.31). CONCLUSIONS In a contemporary, population-based cohort presenting for cardiac catheterization for suspected ischemia, women with angiographically normal coronaries were >4 times more likely to be readmitted to hospital for ACS/chest pain within 180 days compared to men. The observed sex difference has important social and economic implications and suggests that traditional diagnostic methods may not be optimal for women.

Large scale replication and meta-analysis of variants on chromosome 4q25 associated with atrial fibrillation.

AIMS A recent genome-wide association study identified a haplotype block on chromosome 4q25 associated with atrial fibrillation (AF). We sought to replicate this association in four independent cohorts. METHODS AND RESULTS The Framingham Heart Study and Rotterdam Study are community-based longitudinal studies. The Vanderbilt AF Registry and German AF Network (AFNet) are case-control studies. Participants with AF (n = 3508) were more likely to be male and were older than referent participants (n = 12 173; Framingham 82 +/- 10 vs. 71 +/- 13 years; Rotterdam 73 +/- 8 vs. 69 +/- 9 years; Vanderbilt 54 +/- 14 vs. 57 +/- 14 years; AFNet 62 +/- 12 vs. 49 +/- 14 years). Single nucleotide polymorphism (SNP) rs2200733 was associated with AF in all four cohorts, with odds ratios (ORs) ranging from 1.37 in Rotterdam [95% confidence interval (CI) 1.18-1.59; P = 3.1 x 10(-5)] to 2.52 in AFNet (95% CI 2.22-2.8; P = 1.8 x 10(-49)). There also was a significant association between AF and rs10033464 in Framingham (OR 1.34; 95% CI 1.03-1.75; P = 0.031) and AFNet (OR 1.30; 95% CI 1.13-1.51; P = 0.0002), but not Vanderbilt (OR 1.16; 95% CI 0.86-1.56; P = 0.33). A meta-analysis of the current and prior AF studies revealed an OR of 1.90 (95% CI 1.60-2.26; P = 3.3 x 10(-13)) for rs2200733 and of 1.36 (95% CI 1.26-1.47; P = 6.7 x 10(-15)) for rs10033464. CONCLUSION The non-coding SNPs rs2200733 and rs10033464 are strongly associated with AF in four cohorts of European descent. These results confirm the significant relations between AF and intergenic variants on chromosome 4.

Midlife and Late‐Life Vascular Risk Factors and White Matter Microstructural Integrity: The Atherosclerosis Risk in Communities Neurocognitive Study

BACKGROUND Diffusion tensor imaging measures of white matter (WM) microstructural integrity appear to provide earlier indication of WM injury than WM hyperintensities; however, risk factors for poor WM microstructural integrity have not been established. Our study quantifies the association between vascular risk factors in midlife and late life with measures of late-life WM microstructural integrity. METHODS AND RESULTS We used data from 1851 participants in ARIC (Atherosclerosis Risk in Communities Study) who completed 3-T magnetic resonance imaging, including diffusion tensor imaging, as part of the ARIC Neurocognitive Study (ARIC-NCS). We quantified the association among lipids, glucose, and blood pressure from the baseline ARIC visit (1987-1989, ages 44-65, midlife) and visit 5 of ARIC (2011-2013, ages 67-90, late life, concurrent with ARIC-NCS) with regional and overall WM mean diffusivity and fractional anisotropy obtained at ARIC visit 5 for ARIC participants. We also considered whether these associations were independent of or modified by WM hyperintensity volumes. We found that elevated blood pressure in midlife and late life and elevated glucose in midlife, but not late life, were associated with worse late-life WM microstructural integrity. These associations were independent of the degree of WM hyperintensity, and the association between glucose and WM microstructural integrity appeared stronger for those with the least WM hyperintensity. There was little support for an adverse association between lipids and WM microstructural integrity. CONCLUSIONS Hypertension in both midlife and late life and elevated glucose in midlife are related to worse WM microstructural integrity in late life.

The shunt problem: control of functional shunting in normal and tumour vasculature

Networks of blood vessels in normal and tumour tissues have heterogeneous structures, with widely varying blood flow pathway lengths. To achieve efficient blood flow distribution, mechanisms for the structural adaptation of vessel diameters must be able to inhibit the formation of functional shunts (whereby short pathways become enlarged and flow bypasses long pathways). Such adaptation requires information about tissue metabolic status to be communicated upstream to feeding vessels, through conducted responses. We propose that impaired vascular communication in tumour microvascular networks, leading to functional shunting, is a primary cause of dysfunctional microcirculation and local hypoxia in cancer. We suggest that anti-angiogenic treatment of tumours may restore vascular communication and thereby improve or normalize flow distribution in tumour vasculature.

Patient Outcomes with Teaching Versus Nonteaching Healthcare: A Systematic Review

Background  Extensive debate exists in the healthcare community over whether outcomes of medical care at teaching hospitals and other healthcare units are better or worse than those at the respective nonteaching ones. Thus, our goal was to systematically evaluate the evidence pertaining to this question. Methods and Findings  We reviewed all studies that compared teaching versus nonteaching healthcare structures for mortality or any other patient outcome, regardless of health condition. Studies were retrieved from PubMed, contact with experts, and literature cross-referencing. Data were extracted on setting, patients, data sources, author affiliations, definition of compared groups, types of diagnoses considered, adjusting covariates, and estimates of effect for mortality and for each other outcome. Overall, 132 eligible studies were identified, including 93 on mortality and 61 on other eligible outcomes (22 addressed both). Synthesis of the available adjusted estimates on mortality yielded a summary relative risk of 0.96 (95% confidence interval [CI], 0.93–1.00) for teaching versus nonteaching healthcare structures and 1.04 (95% CI, 0.99–1.10) for minor teaching versus nonteaching ones. There was considerable heterogeneity between studies (I2 = 72% for the main analysis). Results were similar in studies using clinical and those using administrative databases. No differences were seen in the 14 studies fully adjusting for volume/experience, severity, and comorbidity (relative risk 1.01). Smaller studies did not differ in their results from larger studies. Differences were seen for some diagnoses (e.g., significantly better survival for breast cancer and cerebrovascular accidents in teaching hospitals and significantly better survival from cholecystectomy in nonteaching hospitals), but these were small in magnitude. Other outcomes were diverse, but typically teaching healthcare structures did not do better than nonteaching ones. Conclusions  The available data are limited by their nonrandomized design, but overall they do not suggest that a healthcare facility's teaching status on its own markedly improves or worsens patient outcomes. Differences for specific diseases cannot be excluded, but are likely to be small.

Endocrine control of spermatogenesis: Role of FSH and LH/ testosterone.

Evaluation of testicular functions (production of sperm and androgens) is an important aspect of preclinical safety assessment and testicular toxicity is comparatively far more common than ovarian toxicity. This chapter focuses (1) on the histological sequelae of disturbed reproductive endocrinology in rat, dog and nonhuman primates and (2) provides a review of our current understanding of the roles of gonadotropins and androgens. The response of the rodent testis to endocrine disturbances is clearly different from that of dog and primates with different germ cell types and spermatogenic stages being affected initially and also that the end-stage spermatogenic involution is more pronounced in dog and primates compared to rodents. Luteinizing hormone (LH)/testosterone and follicle-stimulating hormone (FSH) are the pivotal endocrine factors controlling testicular functions. The relative importance of either hormone is somewhat different between rodents and primates. Generally, however, both LH/testosterone and FSH are necessary for quantitatively normal spermatogenesis, at least in non-seasonal species.

Polyphenols of Cocoa: Inhibition of Mammalian 15-Lipoxygenase

Abstract Some cocoas and chocolates are rich in ()epicatechin and its related oligomers, the procyanidins. Fractions of these compounds, isolated from the seeds of Theobroma cacao, caused dosedependent inhibition of isolated rabbit 15-lipoxygenase-1 with the larger oligomers being more active; the decamer fraction revealed an IC 50 of 0.8 M. Among the monomeric flavanols, epigallocatechin gallate (IC 50 = 4 M) and epicatechin gallate (5 M) were more potent than ()epicatechin (IC50 = 60 M). ()Epicatechin and procyanidin nonamer also inhibited the formation of 15-hydroxyeicosatetraenoic acid from arachidonic acid in rabbit smooth muscle cells transfected with human 15-lipoxygenase-1. In contrast, inhibition of the lipoxygenase pathway in J774A.1 cells transfected with porcine leukocytetype 12- lipoxygenase (another representative of the 12/15- lipoxygenase family) was only observed upon sonication of the cells, suggesting a membrane barrier for flavanols in these cells. Moreover, epicatechin (IC50 approx. 15 M) and the procyanidin decamer inhibited recombinant human platelet 12-lipoxygenase. These observations suggest general lipoxygenase inhibitory potency of flavanols and procyanidins that may contribute to their putative beneficial effects on the cardiovascular system in man. Thus, they may provide a plausible explanation for recent literature reports indicating that procyanidins decrease the leukotriene/prostacyclin ratio in humans and human aortic endothelial cells.

Sympathetic neural mechanisms in normal and hypertensive pregnancy in humans.

BACKGROUND Direct recordings from peripheral sympathetic nerves have shown an increased sympathetic drive in pregnancy-induced hypertension (PIH) and preeclampsia (PE). It is unknown whether sympathetic drive is altered in normal pregnancy, when arterial blood pressure can be normal or relatively low. The aim of this study was to measure and compare peripheral sympathetic discharge, its vasoconstrictor effect and its baroreceptor control, during pregnancy and postpartum in women with normal pregnancy (NP) and PIH and in normotensive nonpregnant (NN) women. METHODS AND RESULTS Twenty-one women with NP, 18 women with PIH, and 21 NN women had muscle sympathetic nerve activity assessed from multiunit discharges (MSNA) and from single units with defined vasoconstrictor properties (s-MSNA). The s-MSNA in NP (38+/-6.6 impulses/100 beats) was greater (P<0.05) than in NN women (19+/-1.8 impulses/100 beats) despite similar age and body weight but less than in PIH women (P<0.001) (146+/-23.5 impulses/100 beats). MSNA followed a similar trend. Cardiac baroreceptor reflex sensitivity (BRS) was impaired in NP and PIH women relative to NN. After delivery, sympathetic activity decreased to values similar to those obtained in NN, and there was an increase in BRS. In women with NP, the decrease in sympathetic output occurred despite an insignificant change in blood pressure. CONCLUSIONS Central sympathetic output was increased in women with normal pregnancy and was even greater in the hypertensive pregnant group. The findings suggest that the moderate sympathetic hyperactivity during the latter months of normal pregnancy may help to return the arterial pressure to nonpregnant levels, although when the increase in activity is excessive, hypertension may ensue.

Cortical Dynein Controls Microtubule Dynamics to Generate Pulling Forces that Position Microtubule Asters

Dynein at the cortex contributes to microtubule-based positioning processes such as spindle positioning during embryonic cell division and centrosome positioning during fibroblast migration. To investigate how cortical dynein interacts with microtubule ends to generate force and how this functional association impacts positioning, we have reconstituted the 'cortical' interaction between dynein and dynamic microtubule ends in an in vitro system using microfabricated barriers. We show that barrier-attached dynein captures microtubule ends, inhibits growth, and triggers microtubule catastrophes, thereby controlling microtubule length. The subsequent interaction with shrinking microtubule ends generates pulling forces up to several pN. By combining experiments in microchambers with a theoretical description of aster mechanics, we show that dynein-mediated pulling forces lead to the reliable centering of microtubule asters in simple confining geometries. Our results demonstrate the intrinsic ability of cortical microtubule-dynein interactions to regulate microtubule dynamics and drive positioning processes in living cells.

Directed Actin Polymerization Is the Driving Force for Epithelial Cell–Cell Adhesion

We have found that epithelial cells engage in a process of cadherin-mediated intercellular adhesion that utilizes calcium and actin polymerization in unexpected ways. Calcium stimulates filopodia, which penetrate and embed into neighboring cells. E-cadherin complexes cluster at filopodia tips, generating a two-rowed zipper of embedded puncta. Opposing cell surfaces are clamped by desmosomes, while vinculin, zyxin, VASP, and Mena are recruited to adhesion zippers by a mechanism that requires alpha-catenin. Actin reorganizes and polymerizes to merge puncta into a single row and seal cell borders. In keratinocytes either null for alpha-catenin or blocked in VASP/Mena function, filopodia embed, but actin reorganization/polymerization is prevented, and membranes cannot seal. Taken together, a dynamic mechanism for intercellular adhesion is unveiled involving calcium-activated filopodia penetration and VASP/Mena-dependent actin reorganization/polymerization.

Gene expression during gonadogenesis in the chicken embryo.

Genes implicated in vertebrate sex determination and differentiation were studied in embryonic chicken gonads using reverse transcription and the polymerase chain reaction (RT-PCR). Expression profiles were obtained during gonadal sex differentiation for AMH, SOX9, SOX3, the Wilm's Tumour gene, WT1, and the orphan nuclear receptor genes, SF1 and DAX1. Some of these genes showed sexually dimorphic expression profiles during gonadal development, whereas others were expressed at similar levels in both sexes. The gene encoding Anti-Müllerian hormone (AMH) was expressed in both sexes prior to and during sexual differentiation of the gonads, with levels of expression consistently higher in males than in females. SOX9 expression was male-specific, and was up-regulated after the detection of AMH transcripts. SOX3 expression was observed prior to clear SOX9 expression and was up-regulated in both sexes at the onset of gonadal sex differentiation (but declined later in development). The WT1 gene was highly expressed in both sexes, whereas SF1 expression was clearly higher in developing ovaries compared to testes. DAX1 transcripts were observed in both sexes at all stages examined, but expression appeared somewhat higher in developing ovaries. These expression profiles are analysed in terms of current theories of vertebrate sex determination.

Aging, habitual exercise, and dynamic arterial compliance.

BACKGROUND A reduction in compliance of the large-sized cardiothoracic (central) arteries is an independent risk factor for the development of cardiovascular disease with advancing age. METHODS AND RESULTS We determined the role of habitual exercise on the age-related decrease in central arterial compliance by using both cross-sectional and interventional approaches. First, we studied 151 healthy men aged 18 to 77 years: 54 were sedentary, 45 were recreationally active, and 53 were endurance exercise-trained. Central arterial compliance (simultaneous B-mode ultrasound and arterial applanation tonometry on the common carotid artery) was lower (P:<0.05) in middle-aged and older men than in young men in all 3 groups. There were no significant differences between sedentary and recreationally active men at any age. However, arterial compliance in the endurance-trained middle-aged and older men was 20% to 35% higher than in the 2 less active groups (P:<0.01). As such, age-related differences in central arterial compliance were smaller in the endurance-trained men than in the sedentary and recreationally active men. Second, we studied 20 middle-aged and older (53+/-2 years) sedentary healthy men before and after a 3-month aerobic exercise intervention (primarily walking). Regular exercise increased central arterial compliance (P:<0.01) to levels similar to those of the middle-aged and older endurance-trained men. These effects were independent of changes in body mass, adiposity, arterial blood pressure, or maximal oxygen consumption. CONCLUSIONS Regular aerobic-endurance exercise attenuates age-related reductions in central arterial compliance and restores levels in previously sedentary healthy middle-aged and older men. This may be one mechanism by which habitual exercise lowers the risk of cardiovascular disease in this population.

Prediction of coronary artery disease by a systemic atherosclerosis score index derived from whole-body MR angiography

BACKGROUND Whole-body magnetic resonance angiography (WB-MRA) has shown its potential for the non-invasive assessment of nearly the entire arterial vasculature within one examination. Since the presence of extra-cardiac atherosclerosis is associated with an increased risk of coronary events, our goal was to establish the relationship between WB-MRA findings, including a systemic atherosclerosis score index, and the presence of significant coronary artery disease (CAD). METHODS WB-MRA was performed on a 1.5T scanner in 50 patients scheduled to undergo elective cardiac catheterization for suspected CAD. In each patient, 40 extra-cardiac vessel segments were evaluated and assigned scores according to their luminal narrowing. The atherosclerosis score index (ASI) was generated as the ratio of summed scores to analyzable segments. RESULTS ASI was higher in patients with significant (> 50% stenosis) CAD (n = 27) vs. patients without CAD (n = 22; 1.56 vs. 1.28, p = 0.004). ASI correlated with PROCAM (R = 0.57, p < 0.001) and Framingham (R = 0.36, p = 0.01) risk scores as estimates of the 10-year risk of coronary events. A ROC derived ASI of > 1.54 predicted significant CAD with a sensitivity of 59%, specificity of 86% and a positive predictive value of 84%. Logistic regression revealed ASI > 1.54 as the strongest independent predictor for CAD with a 11-fold increase in likelihood to suffer from significant coronary disease. On the contrary, while 15/27 (55%) of patients with CAD exhibited at least one extra-cardiac stenosis > 50%, only 3/22 (14%) of those patients without CAD did (p = 0.003). The likelihood for an extra-cardiac stenosis when CAD is present differed between vascular territories and ranged from 15% for a carotid stenosis to 44% for a stenosis in the lower extremities. CONCLUSION This study provides important new evidence for the close association of extra-cardiac and coronary atherosclerosis. The novel findings that a WB-MRA derived systemic atherosclerosis score index is not only associated with established cardiovascular risk scores but is also predictive of significant CAD suggest its potential prognostic implications and underline the importance to screen for coronary disease in patients with extra-cardiac manifestations of atherosclerosis.

Cellular adaptation to mechanical stress: role of integrins, Rho, cytoskeletal tension and mechanosensitive ion channels.

To understand how cells sense and adapt to mechanical stress, we applied tensional forces to magnetic microbeads bound to cell-surface integrin receptors and measured changes in bead displacement with sub-micrometer resolution using optical microscopy. Cells exhibited four types of mechanical responses: (1) an immediate viscoelastic response; (2) early adaptive behavior characterized by pulse-to-pulse attenuation in response to oscillatory forces; (3) later adaptive cell stiffening with sustained (>15 second) static stresses; and (4) a large-scale repositioning response with prolonged (>1 minute) stress. Importantly, these adaptation responses differed biochemically. The immediate and early responses were affected by chemically dissipating cytoskeletal prestress (isometric tension), whereas the later adaptive response was not. The repositioning response was prevented by inhibiting tension through interference with Rho signaling, similar to the case of the immediate and early responses, but it was also prevented by blocking mechanosensitive ion channels or by inhibiting Src tyrosine kinases. All adaptive responses were suppressed by cooling cells to 4 degrees C to slow biochemical remodeling. Thus, cells use multiple mechanisms to sense and respond to static and dynamic changes in the level of mechanical stress applied to integrins.

Orientationally invariant indices of axon diameter and density from diffusion MRI.

This paper proposes and tests a technique for imaging orientationally invariant indices of axon diameter and density in white matter using diffusion magnetic resonance imaging. Such indices potentially provide more specific markers of white matter microstructure than standard indices from diffusion tensor imaging. Orientational invariance allows for combination with tractography and presents new opportunities for mapping brain connectivity and quantifying disease processes. The technique uses a four-compartment tissue model combined with an optimized multishell high-angular-resolution pulsed-gradient-spin-echo acquisition. We test the method in simulation, on fixed monkey brains using a preclinical scanner and on live human brains using a clinical 3T scanner. The human data take about one hour to acquire. The simulation experiments show that both monkey and human protocols distinguish distributions of axon diameters that occur naturally in white matter. We compare the axon diameter index with the mean axon diameter weighted by axon volume. The index differs from this mean and is protocol dependent, but correlation is good for the monkey protocol and weaker, but discernible, for the human protocol where greater diffusivity and lower gradient strength limit sensitivity to only the largest axons. Maps of axon diameter and density indices from the monkey and human data in the corpus callosum and corticospinal tract reflect known trends from histology. The results show orientationally invariant sensitivity to natural axon diameter distributions for the first time with both specialist and clinical hardware. This demonstration motivates further refinement, validation, and evaluation of the precise nature of the indices and the influence of potential confounds.

No Pasaran! Role of the axon initial segment in the regulation of protein transport and the maintenance of axonal identity.

The transmission of information in the brain depends on the highly polarized architecture of neurons. A number of cellular transport processes support this organization, including active targeting of proteins and passive corralling between compartments. The axon initial segment (AIS), which separates the somatodendritic and axonal compartments, has a key role in neuronal physiology, as both the initiation site of action potentials and the gatekeeper of the axonal arborization. Over the years, the AIS main components and their interactions have been progressively unraveled, as well as their role in the AIS assembly and maintenance. Two mechanisms have been shown to contribute to the regulation of protein transport at the AIS: a surface diffusion barrier and an intracellular traffic filter. However, a molecular understanding of these processes is still lacking. In the view of recent results on the AIS cytoskeleton structure, we will discuss how a better knowledge of the AIS architecture can help understanding its role in the regulation of protein transport and the maintenance of axonal identity.

Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis.

New blood vessel formation (angiogenesis) is a fundamental event in the process of tumor growth and metastatic dissemination. Hence, the molecular basis of tumor angiogenesis has been of keen interest in the field of cancer research. The vascular endothelial growth factor (VEGF) pathway is well established as one of the key regulators of this process. The VEGF/VEGF-receptor axis is composed of multiple ligands and receptors with overlapping and distinct ligand-receptor binding specificities, cell-type expression, and function. Activation of the VEGF-receptor pathway triggers a network of signaling processes that promote endothelial cell growth, migration, and survival from pre-existing vasculature. In addition, VEGF mediates vessel permeability, and has been associated with malignant effusions. More recently, an important role for VEGF has emerged in mobilization of endothelial progenitor cells from the bone marrow to distant sites of neovascularization. The well-established role of VEGF in promoting tumor angiogenesis and the pathogenesis of human cancers has led to the rational design and development of agents that selectively target this pathway. Studies with various anti-VEGF/VEGF-receptor therapies have shown that these agents can potently inhibit angiogenesis and tumor growth in preclinical models. Recently, an anti-VEGF antibody (bevacizumab), when used in combination with chemotherapy, was shown to significantly improve survival and response rates in patients with metastatic colorectal cancer and thus, validate VEGF pathway inhibitors as an important new treatment modality in cancer therapy.

Pericyte differentiation.

Pericytes are defined in vivo by their location: They are embedded within the basement membrane of microvessels. They form an integral part of the microvascular wall and are believed to participate in angiogenesis, although their precise role is not clear. Pericytes derived from the retinal microvasculature have been cultured and identified by a series of phenotypic characteristics that clearly distinguishes them from other stromal cells such as smooth muscle cells. Pericytes in vitro form multicellular nodules rich in extracellular matrix. This matrix becomes mineralized in the presence of growth medium containing serum, without exogenous beta-glycerophosphate. These results indicate that pericytes represent primitive mesenchymal cells able to differentiate into an osteogenic phenotype. Pericyte differentiation also is defined by alterations in their response to transforming growth factor beta 1 and changes in the synthesis and/or deposition of various extracellular matrix proteins such as laminin, Type IV collagen, tenascin, Type X collagen osteonectin, and thrombospondin-1. Angiogenesis is associated commonly with mineralization. These data suggest that pericytes may contribute to mineralization in vivo.

Canonical Wnt signaling regulates organ-specific assembly and differentiation of CNS vasculature.

Every organ depends on blood vessels for oxygen and nutrients, but the vasculature associated with individual organs can be structurally and molecularly diverse. The central nervous system (CNS) vasculature consists of a tightly sealed endothelium that forms the blood-brain barrier, whereas blood vessels of other organs are more porous. Wnt7a and Wnt7b encode two Wnt ligands produced by the neuroepithelium of the developing CNS coincident with vascular invasion. Using genetic mouse models, we found that these ligands directly target the vascular endothelium and that the CNS uses the canonical Wnt signaling pathway to promote formation and CNS-specific differentiation of the organ's vasculature.

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In higher eukaryotes, introns are spliced out of protein-coding mRNAs by the spliceosome, a massive complex comprising five non-coding RNAs (ncRNAs) and about 200 proteins. By comparing the differences between spliceosomal proteins from many basal eukaryotic lineages, it is possible to infer properties of the splicing system in the last common ancestor of extant eukaryotes, the eukaryotic ancestor. We begin with the hypothesis that, similar to intron length (that appears to have increased in multicellular eukaryotes), the spliceosome has increased in complexity throughout eukaryotic evolution. However, examination of the distribution of spliceosomal components indicates that not only was a spliceosome present in the eukaryotic ancestor but it also contained most of the key components found in today's eukaryotes. All the small nuclear ribonucleoproteins (snRNPs) protein components are likely to have been present, as well as many splicing-related proteins. Both major and trans-splicing are likely to have been present, and the spliceosome had already formed links with other cellular processes such as transcription and capping. However, there is no evidence as yet to suggest that minor (U12-dependent) splicing was present in the eukaryotic ancestor. Although the last common ancestor of extant eukaryotes appears to show much of the molecular complexity seen today, we do not, from this work, infer anything of the properties of the earlier "first eukaryote. "

Margination of white blood cells in microcapillary flow.

Margination of white blood cells (WBCs) towards vessel walls is an essential precondition for their efficient adhesion to the vascular endothelium. We perform numerical simulations with a two-dimensional blood flow model to investigate the dependence of WBC margination on hydrodynamic interactions of blood cells with the vessel walls, as well as on their collective behavior and deformability. We find WBC margination to be optimal in intermediate ranges of red blood cell (RBC) volume fractions and flow rates, while, beyond these ranges, it is substantially attenuated. RBC aggregation enhances WBC margination, while WBC deformability reduces it. These results are combined in state diagrams, which identify WBC margination for a wide range of flow and cell suspension conditions.

Blockade of adrenoreceptors inhibits the splenic response to stroke.

Recent studies have highlighted the involvement of the peripheral immune system in delayed cellular degeneration after stroke. In the permanent middle cerebral artery occlusion (MCAO) model of stroke, the spleen decreases in size. This reduction occurs through the release of splenic immune cells. Systemic treatment with human umbilical cord blood cells (HUCBC) 24 h post-stroke blocks the reduction in spleen size while significantly reducing infarct volume. Splenectomy 2 weeks prior to MCAO also reduces infarct volume, further demonstrating the detrimental role of this organ in stroke-induced neurodegeneration. Activation of the sympathetic nervous system after MCAO results in elevated catecholamine levels both at the level of the spleen, through direct splenic innervation, and throughout the systemic circulation upon release from the adrenal medulla. These catecholamines bind to splenic alpha and beta adrenoreceptors. This study examines whether catecholamines regulate the splenic response to stroke. Male Sprague-Dawley rats either underwent splenic denervation 2 weeks prior to MCAO or received injections of carvedilol, a pan adrenergic receptor blocker, prazosin, an alpha1 receptor blocker, or propranolol, a beta receptor blocker. Denervation was confirmed by reduced splenic expression of tyrosine hydroxylase. Denervation prior to MCAO did not alter infarct volume or spleen size. Propranolol treatment also had no effects on these outcomes. Treatment with either prazosin or carvedilol prevented the reduction in spleen size, yet only carvedilol significantly reduced infarct volume (p < 0.05). These results demonstrate that circulating blood borne catecholamines regulate the splenic response to stroke through the activation of both alpha and beta adrenergic receptors.

Vanishing cerebellar infarcts in a migraine patient.

Recent population-based studies have suggested that migraine is a risk factor for the development of infarct-like lesions in the territory of the posterior circulation. These lesions are thought to be true vascular infarcts based on their size, location and magnetic resonance imaging (MRI) characteristics. However, as there are no postmortem studies identifying the pathology of these MRI findings, their true aetiology is unknown. A case patient with migraine is presented, who developed what appeared to be cerebellar infarcts on MRI, but these lesions vanished on repeat imaging 16 days later, questioning their aetiology as vascular ischaemic based infarcts.

A map of human circular RNAs in clinically relevant tissues

Cellular circular RNAs (circRNAs) are generated by head-to-tail splicing and are present in all multicellular organisms studied so far. Recently, circRNAs have emerged as a large class of RNA which can function as post-transcriptional regulators. It has also been shown that many circRNAs are tissue- and stage-specifically expressed. Moreover, the unusual stability and expression specificity make circRNAs important candidates for clinical biomarker research. Here, we present a circRNA expression resource of 20 human tissues highly relevant to disease-related research: vascular smooth muscle cells (VSMCs), human umbilical vein cells (HUVECs), artery endothelial cells (HUAECs), atrium, vena cava, neutrophils, platelets, cerebral cortex, placenta, and samples from mesenchymal stem cell differentiation. In eight different samples from a single donor, we found highly tissue-specific circRNA expression. Circular-to-linear RNA ratios revealed that many circRNAs were expressed higher than their linear host transcripts. Among the 71 validated circRNAs, we noticed potential biomarkers. In adenosine deaminase-deficient, severe combined immunodeficiency (ADA-SCID) patients and in Wiskott-Aldrich-Syndrome (WAS) patients' samples, we found evidence for differential circRNA expression of genes that are involved in the molecular pathogenesis of both phenotypes. Our findings underscore the need to assess circRNAs in mechanisms of human disease. KEY MESSAGES: circRNA resource catalog of 20 clinically relevant tissues. circRNA expression is highly tissue-specific. circRNA transcripts are often more abundant than their linear host RNAs. circRNAs can be differentially expressed in disease-associated genes.

Multiple roles of vascular endothelial growth factor (VEGF) in skeletal development, growth, and repair.

Publisher Summary This chapter discusses the developmental roles of vascular endothelial growth factor (VEGF) in skeletal morphogenesis, speculates on the future directions of research in this area, and describes some of the challenges in the field. VEGF regulates osteoclastic differentiation, migration, and activity. VEGF is, therefore, a key coordinator of the entire process. VEGF is necessary for osteoclastic activity both at the stage when the primary ossification center is established and later during bone growth. A number of studies have led to the identification of VEGF as a critical factor for the survival of chondrocytes. Several factors with important roles in regulating bone formation also induce the expression of VEGF by osteoblasts. Prostaglandins E1 and E2, BMP-4, BMP-6, BMP-7, FGF-2, TGF-β, endothelin-1, IGF-1, and vitamin D3 can all induce VEGF expression in osteoblasts by activating a variety of signaling pathways. Bone fractures can heal in two divergent ways, similar to the two ways of forming bone during embryonic development. Stabilized fractures heal by intramembranous ossification, and unstable fractures undergo endochondral ossification. The similarity between the embryonic bone development and repair of fractured bones, coupled with the finding that VEGF is expressed at sites of bone fracture, suggests that VEGF is involved in bone repair as it is in bone development.

CLASPs link focal adhesion-associated microtubule capture to localized exocytosis and adhesion site turnover

Turnover of integrin-based focal adhesions (FAs) with the extracellular matrix (ECM) is essential for coordinated cell movement. In collectively migrating human keratinocytes, FAs assemble near the leading edge, grow and mature as a result of contractile forces and disassemble underneath the advancing cell body. We report that clustering of microtubule-associated CLASP1 and CLASP2 proteins around FAs temporally correlates with FA turnover. CLASPs and LL5β (also known as PHLDB2), which recruits CLASPs to FAs, facilitate FA disassembly. CLASPs are further required for FA-associated ECM degradation, and matrix metalloprotease inhibition slows FA disassembly similarly to CLASP or PHLDB2 (LL5β) depletion. Finally, CLASP-mediated microtubule tethering at FAs establishes an FA-directed transport pathway for delivery, docking and localized fusion of exocytic vesicles near FAs. We propose that CLASPs couple microtubule organization, vesicle transport and cell interactions with the ECM, establishing a local secretion pathway that facilitates FA turnover by severing cell-matrix connections.

Inflammatory signaling in human Tuberculosis granulomas is spatially organized

Granulomas are the pathological hallmark of tuberculosis (TB). However, their function and mechanisms of formation remain poorly understood. To understand the role of granulomas in TB, we analyzed the proteomes of granulomas from subjects with tuberculosis in an unbiased manner. Using laser-capture microdissection, mass spectrometry and confocal microscopy, we generated detailed molecular maps of human granulomas. We found that the centers of granulomas have a pro-inflammatory environment that is characterized by the presence of antimicrobial peptides, reactive oxygen species and pro-inflammatory eicosanoids. Conversely, the tissue surrounding the caseum has a comparatively anti-inflammatory signature. These findings are consistent across a set of six human subjects and in rabbits. Although the balance between systemic pro- and anti-inflammatory signals is crucial to TB disease outcome, here we find that these signals are physically segregated within each granuloma. From the protein and lipid snapshots of human and rabbit lesions analyzed here, we hypothesize that the pathologic response to TB is shaped by the precise anatomical localization of these inflammatory pathways during the development of the granuloma.

Left–right asymmetry in the vertebrate embryo: from early information to higher-level integration

Although vertebrates seem to be essentially bilaterally symmetrical on the exterior, there are numerous interior left–right asymmetries in the disposition and placement of internal organs. These asymmetries are established during embryogenesis by complex epigenetic and genetic cascades. Recent studies in a range of model organisms have made important progress in understanding how this laterality information is generated and conveyed to large regions of the embryo. Both commonalities and divergences are emerging in the mechanisms that different vertebrates use in left–right axis specification. Recent evidence also provides intriguing links between the establishment of left–right asymmetries and the symmetrical elongation of the anterior–posterior axis.

Plasma lipids and prolactin in patients with breast cancer.

In a comparative study of pre- and postmenopausal women with benign and malignant breast disease, a number of differences were observed in circulating plasma prolactin and lipid concentrations. Plasma lipids, phospholipids, triglycerides, cholesterol and free fatty acids were all higher in blood obtained from breast cancer patients prior to surgery. HDL-Cholesterol levels were significantly lower in these patients. These differences remained when the patient groups were sub-divided according to menopausal status. Plasma prolactin concentrations were also found to be higher in cancer compared with non-cancer patients, this effect being more marked in premenopausal than in postmenopausal patients. Premenopausal patients with invasive or poorly differentiated disease had significantly higher prolactin levels than those with non-invasive disease. No correlations were found between plasma prolactin and any of the lipid fractions.

Smallest angiosperm genomes found in lentibulariaceae, with chromosomes of bacterial size.

Nuclear holoploid genome sizes (C-values) have been estimated to vary about 800-fold in angiosperms, with the smallest established 1C-value of 157 Mbp recorded in Arabidopsis thaliana. In the highly specialized carnivorous family Lentibulariaceae now three taxa have been found that exhibit significantly lower values: Genlisea margaretae with 63 Mbp, G. aurea with 64 Mbp, and Utricularia gibba with 88 Mbp. The smallest mitotic anaphase chromatids in G. aurea have 2.1 Mbp and are thus of bacterial size (NB: E. coli has ca. 4 Mbp). Several Utricularia species range somewhat lower than A. thaliana or are similar in genome size. The highest 1C-value known from species of Lentibulariaceae was found in Genlisea hispidula with 1510 Mbp, and results in about 24-fold variation for Genlisea and the Lentibulariaceae. Taking into account these new measurements, genome size variation in angiosperms is now almost 2000-fold. Genlisea and Utricularia are plants with terminal positions in the phylogeny of the eudicots, so that the findings are relevant for the understanding of genome miniaturization. Moreover, the Genlisea-Utricularia clade exhibits one of the highest mutational rates in several genomic regions in angiosperms, what may be linked to specialized patterns of genome evolution. Ultrasmall genomes have not been found in Pinguicula, which is the sister group of the Genlisea-Utricularia clade, and which does not show accelerated mutational rates. C-values in Pinguicula varied only 1.7-fold from 487 to 829 Mbp.

Evaluation of individuals with pulmonary nodules: when is it lung cancer? Diagnosis and management of lung cancer, 3rd ed: American College of Chest Physicians evidence-based clinical practice guidelines.

OBJECTIVES The objective of this article is to update previous evidence-based recommendations for evaluation and management of individuals with solid pulmonary nodules and to generate new recommendations for those with nonsolid nodules. METHODS We updated prior literature reviews, synthesized evidence, and formulated recommendations by using the methods described in the "Methodology for Development of Guidelines for Lung Cancer" in the American College of Chest Physicians Lung Cancer Guidelines, 3rd ed. RESULTS We formulated recommendations for evaluating solid pulmonary nodules that measure > 8 mm in diameter, solid nodules that measure ≤ 8 mm in diameter, and subsolid nodules. The recommendations stress the value of assessing the probability of malignancy, the utility of imaging tests, the need to weigh the benefits and harms of different management strategies (nonsurgical biopsy, surgical resection, and surveillance with chest CT imaging), and the importance of eliciting patient preferences. CONCLUSIONS Individuals with pulmonary nodules should be evaluated and managed by estimating the probability of malignancy, performing imaging tests to better characterize the lesions, evaluating the risks associated with various management alternatives, and eliciting their preferences for management.

Ticagrelor vs. clopidogrel in patients with acute coronary syndromes and diabetes: a substudy from the PLATelet inhibition and patient Outcomes (PLATO) trial

AIMS patients with diabetes mellitus (DM) have high platelet reactivity and are at increased risk of ischaemic events and bleeding post-acute coronary syndromes (ACS). In the PLATelet inhibition and patient Outcomes (PLATO) trial, ticagrelor reduced the primary composite endpoint of cardiovascular death, myocardial infarction, or stroke, but with similar rates of major bleeding compared with clopidogrel. We aimed to investigate the outcome with ticagrelor vs. clopidogrel in patients with DM or poor glycaemic control. METHODS AND RESULTS we analysed patients with pre-existing DM (n = 4662), including 1036 patients on insulin, those without DM (n = 13 951), and subgroups based on admission levels of haemoglobin A1c (HbA1c; n = 15 150). In patients with DM, the reduction in the primary composite endpoint (HR: 0.88, 95% CI: 0.76-1.03), all-cause mortality (HR: 0.82, 95% CI: 0.66-1.01), and stent thrombosis (HR: 0.65, 95% CI: 0.36-1.17) with no increase in major bleeding (HR: 0.95, 95% CI: 0.81-1.12) with ticagrelor was consistent with the overall cohort and without significant diabetes status-by-treatment interactions. There was no heterogeneity between patients with or without ongoing insulin treatment. ticagrelor reduced the primary endpoint, all-cause mortality, and stent thrombosis in patients with HbA1c above the median (HR: 0.80, 95% CI: 0.70-0.91; HR: 0.78, 95% CI: 0.65-0.93; and HR: 0.62, 95% CI: 0.39-1.00, respectively) with similar bleeding rates (HR: 0.98, 95% CI: 0.86-1.12). CONCLUSION ticagrelor, when compared with clopidogrel, reduced ischaemic events in ACS patients irrespective of diabetic status and glycaemic control, without an increase in major bleeding events.

Phylogenetic analysis of mRNA polyadenylation sites reveals a role of transposable elements in evolution of the 3′-end of genes

mRNA polyadenylation is an essential step for the maturation of almost all eukaryotic mRNAs, and is tightly coupled with termination of transcription in defining the 3'-end of genes. Large numbers of human and mouse genes harbor alternative polyadenylation sites [poly(A) sites] that lead to mRNA variants containing different 3'-untranslated regions (UTRs) and/or encoding distinct protein sequences. Here, we examined the conservation and divergence of different types of alternative poly(A) sites across human, mouse, rat and chicken. We found that the 3'-most poly(A) sites tend to be more conserved than upstream ones, whereas poly(A) sites located upstream of the 3'-most exon, also termed intronic poly(A) sites, tend to be much less conserved. Genes with longer evolutionary history are more likely to have alternative polyadenylation, suggesting gain of poly(A) sites through evolution. We also found that nonconserved poly(A) sites are associated with transposable elements (TEs) to a much greater extent than conserved ones, albeit less frequently utilized. Different classes of TEs have different characteristics in their association with poly(A) sites via exaptation of TE sequences into polyadenylation elements. Our results establish a conservation pattern for alternative poly(A) sites in several vertebrate species, and indicate that the 3'-end of genes can be dynamically modified by TEs through evolution.

The prognostic impact of the platelet distribution width-to-platelet count ratio in patients with breast cancer

Activated platelets promote tumor cell growth, angiogenesis, and invasion. Platelet activity can be inferred by platelet volume indices (PVIs), which include platelet distribution width (PDW), mean platelet volume (MPV), platelet distribution width-to-platelet count ratio (PDW/P), and mean platelet volume-to-platelet count ratio. Platelets and platelet-related markers, such as the platelet-to-lymphocyte ratio, have been found to be significant prognostic factors in patients with breast cancer. However, the role of PVIs for predicting survival in breast cancer remains unknown; hence, we performed this retrospective analysis of 275 patients with breast cancer. PVIs were compared with clinicopathological variables, and were assessed to identify independent indicators associated with disease-free survival (DFS) using the Cox proportional hazards model. An elevated PDW/P significantly correlated with age and HER2 status. Univariate analysis revealed that elevated PDW, MPV, and PDW/P as well as tumor size, nuclear grade, and lymph node involvement were significantly associated with inferior DFS rates (tumor size: p<0.01; nuclear grade, lymph node involvement, PDW, MPV, and PDW/P: p<0.05). On multivariate analysis, a large tumor size and elevated PDW/P were significant prognostic factors for DFS, with hazard ratios of 3.24 (95% confidence interval [CI]: 1.24-8.47) and 2.99 (95% CI: 1.18-7.57), respectively (p<0.05). Our study is the first to reveal that an elevated PDW/P significantly reduces DFS in patients with breast carcinoma. Measuring the PDW/P is simple, relatively inexpensive, and almost universally available using routine blood counts; this makes it an attractive biomarker for improved risk assessment.

CLIP-170 interacts with dynactin complex and the APC-binding protein EB1 by different mechanisms.

CLIP-170 is a "cytoplasmic linker protein" implicated in endosome-microtubule interactions and in control of microtubule dynamics. CLIP-170 localizes dynamically to growing microtubule plus ends, colocalizing with the dynein activator dynactin and the APC-binding protein EB1. This shared "plus-end tracking" behavior suggests that CLIP-170 might interact with dynactin and/or EB1. We have used site-specific mutagenesis of CLIP-170 and a transfection/colocalization assay to address this question in mammalian tissue culture cells. Our results indicate that CLIP-170 interacts, directly or indirectly, with both dynactin and EB1. We find that the CLIP-170/dynactin interaction is mediated by the second metal binding motif of the CLIP-170 tail. In contrast, the CLIP-170/EB1 interaction requires neither metal binding motif. In addition, our experiments suggest that the CLIP-170/dynactin interaction occurs via the shoulder/sidearm subcomplex of dynactin and can occur in the cytosol (i.e., it does not require microtubule binding). These results have implications for the targeting of both dynactin and EB1 to microtubule plus ends. Our data suggest that the CLIP-170/dynactin interaction can target dynactin complex to microtubule plus ends, although dynactin likely also targets MT plus ends directly via the microtubule binding motif of the p150(Glued) subunit. We find that CLIP-170 mutants alter p150(Glued) localization without affecting EB1, indicating that EB1 can target microtubule plus ends independently of dynactin.

The molecular requirements for cytokinesis.

After anaphase onset, animal cells build an actomyosin contractile ring that constricts the plasma membrane to generate two daughter cells connected by a cytoplasmic bridge. The bridge is ultimately severed to complete cytokinesis. Myriad techniques have been used to identify proteins that participate in cytokinesis in vertebrates, insects, and nematodes. A conserved core of about 20 proteins are individually involved with cytokinesis in most animal cells. These components are found in the contractile ring, on the central spindle, within the RhoA pathway, and on vesicles that expand the membrane and sever the bridge. Cytokinesis involves additional proteins, but they, or their requirement in cytokinesis, are not conserved among animal cells.

Glucose tolerance and blood pressure: long term follow up in middle aged men.

OBJECTIVE to investigate the role of glucose tolerance in the development of hypertension. DESIGN Retrospective analysis of the results of a health check up in a group of clinically healthy middle aged men in the late 1960s (median year 1968). The subjects were invited to enter into a primary prevention trial for cardiovascular disease in 1974, when they underwent clinical examination for risk factors. The trial was completed in 1979, when the men were re-examined. Follow up was in 1986. SETTING Institute of Occupational Health, Helsinki, Finland and second department of medicine, University of Helsinki. SUBJECTS In all, 3490 men born during 1919-34 participated in a health check up in the late 1960s. In 1974, 1815 of these men who were clinically healthy were entered into a primary prevention trial for cardiovascular disease. On clinical examination 1222 of the men were considered at high risk of cardiovascular disease. Of these, 612 received an intervention and were excluded from the study. A total of 593 men were without risk factors. The study comprised all of the men who did not have an intervention (n = 1203). In 1979, 1120 men were re-examined, and in 1986 945 men attended follow up. There were two groups for analysis: one comprising all subjects and the other comprising only men who were normotensive in 1968 and for whom complete information was available. INTERVENTIONS By 1979, 103 men were taking antihypertensive drugs, and by 1986, 131 were taking antihypertensive drugs and 12 were taking drugs for hyperglycaemia. MAIN OUTCOME MEASURES Blood glucose concentration one hour after a glucose load, blood pressure, and body weight were measured in 1968, 1974, and 1979. In 1986 blood pressure and body weight were recorded. RESULTS Men who were hypertensive in 1986 had significantly higher blood pressures (p less than 0.0001) and (after adjustment for body mass index and alcohol intake) significantly higher blood glucose concentrations one hour after a glucose load at all examinations than those who were normotensive in 1986. Regression analysis showed that the higher the blood glucose concentration after a glucose load in 1968 the higher the blood pressure during the following years. Those men between the second and third tertiles of blood glucose concentration in 1968 had a significantly higher risk of developing hypertension (odds ratio 1.71, 95% confidence interval 1.05 to 2.77) compared with those below the first tertile. CONCLUSION In this study men who developed hypertension tended to have shown an increased intolerance to glucose up to 18 years before the clinical manifestation of their disorder. Blood glucose concentration one hour after a glucose load was an independent predictor of future hypertension.

In vivo electroporation in the embryonic mouse central nervous system

This protocol describes a basic method for in vivo electroporation in the nervous system of embryonic mice. Delivery of electric pulses following microinjection of DNA into the brain ventricle or the spinal cord central canal enables efficient transfection of genes into the nervous system. Transfection is facilitated by forceps-type electrodes, which hold the uterus and/or the yolk sac containing the embryo. More than ten embryos in a single pregnant mouse can be operated on within 30 min. More than 90% of operated embryos survive and more than 90% of these survivors express the transfected genes appropriately. Gene expression in neurons persists for a long time, even at postnatal stages, after electroporation. Thus, this method could be used to analyze roles of genes not only in embryonic development but also in higher order function of the nervous system, such as learning.

VEX1 controls the allelic exclusion required for antigenic variation in trypanosomes.

Allelic exclusion underpins antigenic variation and immune evasion in African trypanosomes. These bloodstream parasites use RNA polymerase-I (pol-I) to transcribe just one telomeric variant surface glycoprotein (VSG) gene at a time, producing superabundant and switchable VSG coats. We identified trypanosome VSG exclusion-1 (VEX1) using a genetic screen for defects in telomere-exclusive expression. VEX1 was sequestered by the active VSG and silencing of other VSGs failed when VEX1 was either ectopically expressed or depleted, indicating positive and negative regulation, respectively. Positive regulation affected VSGs and nontelomeric pol-I-transcribed genes, whereas negative regulation primarily affected VSGs. Negative regulation by VEX1 also affected telomeric pol-I-transcribed reporter constructs, but only when they contained blocks of sequence sharing homology with a pol-I-transcribed locus. We conclude that restricted positive regulation due to VEX1 sequestration, combined with VEX1-dependent, possibly homology-dependent silencing, drives a "winner-takes-all" mechanism of allelic exclusion.

Mechanical modulation of receptor-ligand interactions at cell-cell interfaces.

Cell surface receptors have been extensively studied because they initiate and regulate signal transduction cascades leading to a variety of functional cellular outcomes. An important class of immune receptors (e.g., T-cell antigen receptors) whose ligands are anchored to the surfaces of other cells remain poorly understood. The mechanism by which ligand binding initiates receptor phosphorylation, a process termed "receptor triggering", remains controversial. Recently, direct measurements of the (two-dimensional) receptor-ligand complex lifetimes at cell-cell interface were found to be smaller than (three-dimensional) lifetimes in solution but the underlying mechanism is unknown. At the cell-cell interface, the receptor-ligand complex spans a short intermembrane distance (15 nm) compared to long surface molecules (LSMs) whose ectodomains span >40 nm and these LSMs include phosphatases (e.g., CD45) that dephosphorylate the receptor. It has been proposed that size-based segregation of LSMs from a receptor-ligand complex is a mechanism of receptor triggering but it is unclear whether the mechanochemistry supports such small-scale segregation. Here we present a nanometer-scale mathematical model that couples membrane elasticity with the compressional stiffness and lateral mobility of LSMs. We find robust supradiffusive segregation of LSMs from a single receptor-ligand complex. The model predicts that LSM redistribution will result in a time-dependent tension on the complex leading to a decreased two-dimensional lifetime. Interestingly, the model predicts a nonlinear relationship between the three- and two-dimensional lifetimes, which can enhance the ability of receptors to discriminate between similar ligands.

Identification and characterization of subpopulations in undifferentiated ES cell culture.

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass (ICM) and the epiblast, and have been suggested to be a homogeneous population with characteristics intermediate between them. These cells express Oct3/4 and Rex1 genes, which have been used as markers to indicate the undifferentiated state of ES cells. Whereas Oct3/4 is expressed in totipotent and pluripotent cells in the mouse life cycle, Rex1 expression is restricted to the ICM, and is downregulated in pluripotent cell populations in the later stages, i.e. the epiblast and primitive ectoderm (PrE). To address whether ES cells comprise a homogeneous population equivalent to a certain developmental stage of pluripotent cells or a heterogeneous population composed of cells corresponding to various stages of differentiation, we established knock-in ES cell lines in which genes for fluorescent proteins were inserted into the Rex1 and Oct3/4 gene loci to visualize the expression of these genes. We found that undifferentiated ES cells included at least two different populations, Rex1(+)/Oct3/4(+) cells and Rex1(-)/Oct3/4(+) cells. The Rex1(-)/Oct3/4(+) and Rex1(+)/Oct3/4(+) populations could convert into each other in the presence of LIF. In accordance with our assumption that Rex1(+)/Oct3/4(+) cells and Rex1(-)/Oct3/4(+) cells have characteristics similar to those of ICM and early-PrE cells, Rex1(+)/Oct3/4(+) cells predominantly differentiated into primitive ectoderm and contributed to chimera formation, whereas Rex1(-)/Oct3/4(+) cells differentiated into cells of the somatic lineage more efficiently than non-fractionated ES cells in vitro and showed poor ability to contribute to chimera formation. These results confirmed that undifferentiated ES cell culture contains subpopulations corresponding to ICM, epiblast and PrE.

Beta interferon controls West Nile virus infection and pathogenesis in mice.

Studies with mice lacking the common plasma membrane receptor for type I interferon (IFN-αβR(-)(/)(-)) have revealed that IFN signaling restricts tropism, dissemination, and lethality after infection with West Nile virus (WNV) or several other pathogenic viruses. However, the specific functions of individual IFN subtypes remain uncertain. Here, using IFN-β(-)(/)(-) mice, we defined the antiviral and immunomodulatory function of this IFN subtype in restricting viral infection. IFN-β(-)(/)(-) mice were more vulnerable to WNV infection than wild-type mice, succumbing more quickly and with greater overall mortality, although the phenotype was less severe than that of IFN-αβR(-)(/)(-) mice. The increased susceptibility of IFN-β(-)(/)(-) mice was accompanied by enhanced viral replication in different tissues. Consistent with a direct role for IFN-β in control of WNV replication, viral titers in ex vivo cultures of macrophages, dendritic cells, fibroblasts, and cerebellar granule cell neurons, but not cortical neurons, from IFN-β(-)(/)(-) mice were greater than in wild-type cells. Although detailed immunological analysis revealed no major deficits in the quality or quantity of WNV-specific antibodies or CD8(+) T cells, we observed an altered CD4(+) CD25(+) FoxP3(+) regulatory T cell response, with greater numbers after infection. Collectively, these results suggest that IFN-β controls WNV pathogenesis by restricting infection in key cell types and by modulating T cell regulatory networks.

Exercise training-induced adaptations in the coronary circulation.

Aerobic exercise training induces an increase in coronary blood flow capacity that is associated with altered control of coronary vascular resistance and, therefore, coronary blood flow. The relative importance of metabolic, myogenic, endothelium-mediated, and neurohumoral control systems varies throughout the coronary arterial tree, and these control systems contribute in parallel to regulating coronary vascular resistance to differing degrees at each level in the coronary arterial tree. In addition to this nonuniformity of the relative importance of vascular control systems in the coronary arterial tree, it appears that exercise training-induced adaptations are also distributed spatially, in a nonuniform manner throughout the coronary tree. As a result, it is necessary to examine training-induced adaptations throughout the coronary arterial tree. Adaptations in endothelium-mediated control play a role in training-induced changes in control of coronary vascular resistance, and there is evidence that the effects of training may be different in large coronary arteries than in the microcirculation. Also, there is evidence that the mode, frequency, and intensity of exercise training bouts and duration of training may influence the adaptive changes in endothelial function. Exercise training has also been shown to induce changes in responses of coronary vascular smooth muscle to vasoactive agents and alterations in the cellular-molecular control of intracellular Ca2+ in coronary vascular smooth muscle of conduit coronary arteries and to enhance myogenic reactivity of coronary resistance arteries. Exercise training also appears to have different effects on vascular smooth muscle in large coronary arteries than in the microcirculation. For example, adenosine sensitivity is increased in conduit coronary arteries and large resistance arteries after training but is not altered in small coronary resistance arteries of trained animals. Although much remains to be studied, evidence clearly indicates that chronic exercise alters the phenotype of coronary endothelial and vascular smooth muscle cells and that plasticity of these cells plays a role in adaptation of the cardiovascular system in exercise training.

Local Increases in Mechanical Tension Shape Compartment Boundaries by Biasing Cell Intercalations

Mechanical forces play important roles during tissue organization in developing animals. Many tissues are organized into adjacent, nonmixing groups of cells termed compartments. Boundaries between compartments display a straight morphology and are associated with signaling centers that are important for tissue growth and patterning. Local increases in mechanical tension at cell junctions along compartment boundaries have recently been shown to prevent cell mixing and to maintain straight boundaries. The cellular mechanisms by which local increases in mechanical tension prevent cell mixing at compartment boundaries, however, remain poorly understood. Here, we have used live imaging and quantitative image analysis to determine cellular dynamics at and near the anteroposterior compartment boundaries of the Drosophila pupal abdominal epidermis. We show that cell mixing within compartments involves multiple cell intercalations. Frequency and orientation of cell intercalations are unchanged along the compartment boundaries; rather, an asymmetry in the shrinkage of junctions during intercalation is biased, resulting in cell rearrangements that suppress cell mixing. Simulations of tissue growth show that local increases in mechanical tension can account for this bias in junctional shrinkage. We conclude that local increases in mechanical tension maintain cell populations separate by influencing junctional rearrangements during cell intercalation.

Relationship between reduced forced expiratory volume in one second and the risk of lung cancer: a systematic review and meta-analysis.

BACKGROUND Individuals with severely impaired lung function have an increased risk of lung cancer. Whether milder reductions in forced expiratory volume in 1 second (FEV(1)) also increase the risk of lung cancer is controversial. Moreover, there is little consensus on whether men and women have similar risks for lung cancer for similar decreases in FEV(1). METHODS A search was conducted of PubMed and EMBASE from January 1966 to January 2005 and studies that examined the relationship between FEV1 and lung cancer were identified. The search was limited to studies that were population based, employed a prospective design, were large in size (> or = 5000 participants), and adjusted for cigarette smoking status. RESULTS Twenty eight abstracts were identified, six of which did not report FEV1 and eight did not adjust for smoking. Included in this report are four studies that reported FEV1 in quintiles. The risk of lung cancer increased with decreasing FEV1. Compared with the highest quintile of FEV1 (> 100% of predicted), the lowest quintile of FEV1 (< approximately 70% of predicted) was associated with a 2.23 fold (95% confidence interval (CI) 1.73 to 2.86) increase in the risk for lung cancer in men and a 3.97 fold increase in women (95% CI 1.93 to 8.25). Even relatively small decrements in FEV1 ( approximately 90% of predicted) increased the risk for lung cancer by 30% in men (95% CI 1.05 to 1.62) and 2.64 fold in women (95% CI 1.30 to 5.31). CONCLUSION Reduced FEV1 is strongly associated with lung cancer. Even a relatively modest reduction in FEV1 is a significant predictor of lung cancer, especially among women.

Ultrasound imaging for lumbar punctures and epidural catheterisations: systematic review and meta-analysis.

OBJECTIVE To determine whether ultrasound imaging can reduce the risk of failed lumbar punctures or epidural catheterisations, when compared with standard palpation methods, and whether ultrasound imaging can reduce traumatic procedures, insertion attempts, and needle redirections. DESIGN Systematic review and meta-analysis of randomised controlled trials. DATA SOURCES Ovid Medline, Embase, and Cochrane Central Register of Controlled Trials up to May 2012, without restriction by language or publication status. REVIEW METHODS Randomised trials that compared ultrasound imaging with standard methods (no imaging) in the performance of a lumbar puncture or epidural catheterisation were identified. RESULTS 14 studies with a total of 1334 patients were included (674 patients assigned to the ultrasound group, 660 to the control group). Five studies evaluated lumbar punctures and nine evaluated epidural catheterisations. Six of 624 procedures conducted in the ultrasound group failed; 44 of 610 procedures in the control group failed. Ultrasound imaging reduced the risk of failed procedures (risk ratio 0.21 (95% confidence interval 0.10 to 0.43), P<0.001). Risk reduction was similar when subgroup analysis was performed for lumbar punctures (risk ratio 0.19 (0.07 to 0.56), P=0.002) or epidural catheterisations (0.23 (0.09 to 0.60), P=0.003). Ultrasound imaging also significantly reduced the risk of traumatic procedures (risk ratio 0.27 (0.11 to 0.67), P=0.005), the number of insertion attempts (mean difference -0.44 (-0.64 to -0.24), P<0.001), and the number of needle redirections (mean difference -1.00 (-1.24 to -0.75), P<0.001). CONCLUSIONS Ultrasound imaging can reduce the risk of failed or traumatic lumbar punctures and epidural catheterisations, as well as the number of needle insertions and redirections. Ultrasound may be a useful adjunct for these procedures.

Human monocyte characteristics are altered in hypercholesterolaemia.

Peripheral blood monocytes are involved during atherogenesis in adhering to endothelium, migrating into the subendothelial space and taking-up lipoproteins to become macrophage/foam cells. We have assessed whether peripheral blood monocyte characteristics are altered in human hyperlipidaemia in age/sex/smoking status matched pairs of patients and controls. Monocytes from the hypercholesterolaemic patients, as opposed to the controls, were more sensitive to stimulation by the agonist, N-formyl-methionyl-leucyl-phenylalanine, with respect to chemokinesis (stimulation index 1.48 +/- 0.17 vs. 1.10 +/- 0.14), chemotaxis (4.05 +/- 0.55 vs. 2.72 +/- 0.24) and adhesion to porcine aortic endothelial monolayers (1.26 +/- 0.05 vs. 1.17 +/- 0.06). The patients' monocyte total surface expression of the adhesion glycoprotein CD11b/CD18 (37.5 +/- 7.1 vs. 36.0 +/- 7.1), but not CD11c/CD18 (31.6 +/- 7.2 vs. 31.4 +/- 6.8), was increased; however, the monocytes in hyperlipidaemia were larger (9.15 +/- 0.11 microns vs. 8.98 +/- 0.11 microns) such that the surface density of CD11b/CD18 was not altered (0.144 +/- 0.029 vs. 0.142 +/- 0.029). The data suggest that circulating monocytes are functionally different in hypercholesterolaemia. This may explain the increased involvement by monocytes in hypercholesterolaemia-related atherogenesis.

Cell death and immune privilege.

The host response to pathogens involves complex inflammatory responses and immune reactions. While these are central to host defense and vital to clearing infections, they are often accompanied by injury to surrounding tissue. Most organ systems can tolerate these responses without permanent consequences. However, there are sites that limit the spread of inflammation because it can threaten organ function. The most prominent examples of these are the eye, brain, and reproductive organs (testis, ovary), where even minor bouts of inflammation can have long-term consequences for the survival of the organism. In these organs immune responses either do not proceed, or proceed in a manner different from other areas; thus, they are called "immunologically privileged. " Here a functioning immune response can be the culprit that leads to disease.

Serum plant and other noncholesterol sterols, cholesterol metabolism and 22-year mortality among middle-aged men.

OBJECTIVE To evaluate long-term prognostic effect of serum noncholesterol sterols, including plant sterols, in middle-aged men with high cardiovascular disease (CVD) risk, without statins at baseline. METHODS This was a prospective study of 232 men (mean age 60 years) at high risk of CVD in 1985-1986. Most were hypercholesterolemic, 29 (12%) had a history of CVD or cancer, 6 (3%) had diabetes, and 46 (20%) had metabolic syndrome (MS). Measured noncholesterol sterols (expressed as absolute concentrations or ratios to serum cholesterol to standardize for cholesterol concentrations) included lathosterol and desmosterol (reflect cholesterol synthesis), and plant sterols (campesterol and sitosterol) and cholestanol (reflect cholesterol absorption). Main outcome measure was total mortality. RESULTS At baseline, markers of cholesterol synthesis and absorption showed expected inverse associations. During the 22-year follow-up 101 men (43%) died. At baseline, nonsurvivors smoked more, exercised less and had more components of MS (although not filling strict criteria), whereas traditional risk factors of CVD were not significantly different. Of the noncholesterol sterols (either absolute or ratio), only sitosterol was significantly higher in survivors than in nonsurvivors (P=0.02). In multivariable analyses, highest sitosterol-to-cholesterol tertile was associated with significantly lower mortality risk (HR 0.51, 95% CI 0.30-0.87) as compared to lowest tertile. Other associations were nonsignificant, although a "global" index of cholesterol metabolism (desmosterol-to-sitosterol ratio) suggested higher cholesterol synthesis and lower absorption to be associated with higher total and CVD mortality. CONCLUSION Higher serum plant sterol levels in middle-aged men predicted lower long-term mortality risk, possibly reflecting an association between higher synthesis/lower absorption of cholesterol and mortality.

Angiotensin II Evokes Angiogenic Signals within Skeletal Muscle through Co-ordinated Effects on Skeletal Myocytes and Endothelial Cells

Skeletal muscle overload induces the expression of angiogenic factors such as vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-2, leading to new capillary growth. We found that the overload-induced increase in angiogenesis, as well as increases in VEGF, MMP-2 and MT1-MMP transcripts were abrogated in muscle VEGF KO mice, highlighting the critical role of myocyte-derived VEGF in controlling this process. The upstream mediators that contribute to overload-induced expression of VEGF have yet to be ascertained. We found that muscle overload increased angiotensinogen expression, a precursor of angiotensin (Ang) II, and that Ang II signaling played an important role in basal VEGF production in C2C12 cells. Furthermore, matrix-bound VEGF released from myoblasts induced the activation of endothelial cells, as evidenced by elevated endothelial cell phospho-p38 levels. We also found that exogenous Ang II elevates VEGF expression, as well as MMP-2 transcript levels in C2C12 myotubes. Interestingly, these responses also were observed in skeletal muscle endothelial cells in response to Ang II treatment, indicating that these cells also can respond directly to the stimulus. The involvement of Ang II in muscle overload-induced angiogenesis was assessed. We found that blockade of AT1R-dependent Ang II signaling using losartan did not attenuate capillary growth. Surprisingly, increased levels of VEGF protein were detected in overloaded muscle from losartan-treated rats. Similarly, we observed elevated VEGF production in cultured endothelial cells treated with losartan alone or in combination with Ang II. These studies conclusively establish the requirement for muscle derived VEGF in overload-induced angiogenesis and highlight a role for Ang II in basal VEGF production in skeletal muscle. However, while Ang II signaling is activated following overload and plays a role in muscle VEGF production, inhibition of this pathway is not sufficient to halt overload-induced angiogenesis, indicating that AT1-independent signals maintain VEGF production in losartan-treated muscle.

Microenvironment rigidity modulates responses to the HER2 receptor tyrosine kinase inhibitor lapatinib via YAP and TAZ transcription factors.

Stiffness is a biophysical property of the extracellular matrix that modulates cellular functions, including proliferation, invasion, and differentiation, and it also may affect therapeutic responses. Therapeutic durability in cancer treatments remains a problem for both chemotherapies and pathway-targeted drugs, but the reasons for this are not well understood. Tumor progression is accompanied by changes in the biophysical properties of the tissue, and we asked whether matrix rigidity modulated the sensitive versus resistant states in HER2-amplified breast cancer cell responses to the HER2-targeted kinase inhibitor lapatinib. The antiproliferative effect of lapatinib was inversely proportional to the elastic modulus of the adhesive substrata. Down-regulation of the mechanosensitive transcription coactivators YAP and TAZ, either by siRNA or with the small-molecule YAP/TEAD inhibitor verteporfin, eliminated modulus-dependent lapatinib resistance. Reduction of YAP in vivo in mice also slowed the growth of implanted HER2-amplified tumors, showing a trend of increasing sensitivity to lapatinib as YAP decreased. Thus we address the role of stiffness in resistance to and efficacy of a HER2 pathway-targeted therapeutic via the mechanotransduction arm of the Hippo pathway.

A Mechanism Linking Extra Centrosomes to Chromosomal Instability

Chromosomal instability (CIN) is a hallmark of many tumours and correlates with the presence of extra centrosomes. However, a direct mechanistic link between extra centrosomes and CIN has not been established. It has been proposed that extra centrosomes generate CIN by promoting multipolar anaphase, a highly abnormal division that produces three or more aneuploid daughter cells. Here we use long-term live-cell imaging to demonstrate that cells with multiple centrosomes rarely undergo multipolar cell divisions, and the progeny of these divisions are typically inviable. Thus, multipolar divisions cannot explain observed rates of CIN. In contrast, we observe that CIN cells with extra centrosomes routinely undergo bipolar cell divisions, but display a significantly increased frequency of lagging chromosomes during anaphase. To define the mechanism underlying this mitotic defect, we generated cells that differ only in their centrosome number. We demonstrate that extra centrosomes alone are sufficient to promote chromosome missegregation during bipolar cell division. These segregation errors are a consequence of cells passing through a transient 'multipolar spindle intermediate' in which merotelic kinetochore-microtubule attachment errors accumulate before centrosome clustering and anaphase. These findings provide a direct mechanistic link between extra centrosomes and CIN, two common characteristics of solid tumours. We propose that this mechanism may be a common underlying cause of CIN in human cancer.

Long-term follow-up of cervical disease in women screened by cytology and HPV testing: results from the HART study

BACKGROUND Several studies have shown that testing for high-risk human papillomavirus (HPV) types results in an improved sensitivity for CIN2+, compared with cytology, although with a somewhat lower specificity. METHODS We obtained follow-up results, with at least one smear after participation in the HART study, which compared HPV testing (HC-II) with cytology as a primary screening modality. RESULTS With a median follow-up of 6 years, 42 additional cases of CIN2+ were identified; women who were HPV positive at baseline were more likely to develop CIN2+ than those who were HPV negative (hazard ratio (HR) 17.2; 95% confidence interval (CI) (9.3-31.6)) and the risk increased with increasing viral load. Compared with HPV-negative women (relative light unit (RLU) <1), the HR (95% CI) was 5.4 (1.6, 18.2) for 1-10 RLU and 25.5 (13.6, 47.9) for RLU > or = 10. Positive cytology (borderline or worse compared with negative) was also predictive of developing CIN2, although to a lesser extent (HR 8.7; 95% CI (4.5-17.1)). Only one case of CIN3 and three cases of CIN2 were found in women who showed a positive cytology result but were HPV negative at baseline. CONCLUSION After 5 years of follow-up, CIN2+ occurred in 0.23% of women who were HPV negative at baseline compared with 0.48% of women who showed a negative cytology result, indicating a much longer low-risk interval for CIN2+ after HPV testing.

Human placental lactogen and unconjugated estriol concentrations in twin pregnancy: monitoring of fetal development in intrauterine growth retardation and single intrauterine fetal death.

Human placental lactogen and unconjugated estriol concentrations in maternal serum were evaluated in 100 uneventful twin pregnancies, and these values were compared with those observed in 16 twin pregnancies associated with intrauterine growth retardation or single intrauterine fetal death. In pregnancies associated with intrauterine growth retardation (n = 8), human placental lactogen levels were at the lower limit of normal range for singleton pregnancies, whereas estriol levels were normal in most cases. When one of the fetuses had died before week 33 of pregnancy (n = 5), both human placental lactogen and estriol levels were low and they were almost at the levels in singleton pregnancy. When intrauterine fetal death occurred after week 36 of pregnancy (n = 3), both hormone levels remained normal until term. Thus human placental lactogen rather than estriol is a good indicator of intrauterine growth retardation in twin pregnancy. Both human placental lactogen and estriol are useful for the monitoring of the surviving fetus in the case of single intrauterine fetal death.

The surgical anatomy of the conduction tissues.

On the basis of our collective experience we have reviewed the disposition of the cardiac conduction tissues as they might be observed by the surgeon in both normal and abnormal hearts. The sinus node lies subepicardially in the terminal sulcus; because of its variable blood supply the entire superior cavoatrial junction is a potential danger area. There are no morphologically discrete tracts extending through the atrial tissues between sinus and atrioventricular nodes. The atrioventricular node, the atrial extent of the atrioventricular conduction axis, is contained exclusively within the triangle of Koch. The axis penetrates through the central fibrous body and branches on the muscular ventricular septum immediately beneath the interventricular component of the membranous septum. The landmarks to these structures are described as they might be seen through the right atrium, left atrium, and aorta. Consideration is then given to the surgical anatomy of the abnormal muscular atrioventricular connections that underscore the ventricular pre-excitation syndromes. Finally, rules are developed whereby the disposition of the conduction tissues can be predicted with accuracy in congenitally malformed hearts, in the settings of both normal and abnormal chamber connections. The most important variables in this respect are alignment between the atrial and ventricular septal structures and the pattern of ventricular architecture present.

Dynein pulls microtubules without rotating its stalk.

Dynein is a microtubule motor that powers motility of cilia and flagella. There is evidence that the relative sliding of the doublet microtubules is due to a conformational change in the motor domain that moves a microtubule bound to the end of an extension known as the stalk. A predominant model for the movement involves a rotation of the head domain, with its stalk, toward the microtubule plus end. However, stalks bound to microtubules have been difficult to observe. Here, we present the clearest views so far of stalks in action, by observing sea urchin, outer arm dynein molecules bound to microtubules, with a new method, "cryo-positive stain" electron microscopy. The dynein molecules in the complex were shown to be active in in vitro motility assays. Analysis of the electron micrographs shows that the stalk angles relative to microtubules do not change significantly between the ADP.vanadate and no-nucleotide states, but the heads, together with their stalks, shift with respect to their A-tubule attachments. Our results disagree with models in which the stalk acts as a lever arm to amplify structural changes. The observed movement of the head and stalk relative to the tail indicates a new plausible mechanism, in which dynein uses its stalk as a grappling hook, catching a tubulin subunit 8 nm ahead and pulling on it by retracting a part of the tail (linker).