The inositol hexakisphosphate kinases (IP6Ks) are the principal enzymes that generate inositol pyrophosphates. There are three IP6Ks (IP6K1, 2, and 3). Functions of IP6K1 and IP6K2 have been substantially delineated, but little is known of IP6K3's role in normal physiology, especially in the brain. To elucidate functions of IP6K3, we generated mice with targeted deletion of IP6K3. We demonstrate that IP6K3 is highly concentrated in the brain in cerebellar Purkinje cells. IP6K3 physiologically binds to the cytoskeletal proteins adducin and spectrin, whose mutual interactions are perturbed in IP6K3-null mutants. Consequently, IP6K3 knock-out cerebella manifest abnormalities in Purkinje cell structure and synapse number, and the mutant mice display deficits in motor learning and coordination. Thus, IP6K3 is a major determinant of cytoskeletal disposition and function of cerebellar Purkinje cells.
SIGNIFICANCE STATEMENT We identified and cloned a family of three inositol hexakisphosphate kinases (IP6Ks) that generate the inositol pyrophosphates, most notably 5-diphosphoinositol pentakisphosphate (IP7). Of these, IP6K3 has been least characterized. In the present study we generated IP6K3 knock-out mice and show that IP6K3 is highly expressed in cerebellar Purkinje cells. IP6K3-deleted mice display defects of motor learning and coordination. IP6K3-null mice manifest aberrations of Purkinje cells with a diminished number of synapses. IP6K3 interacts with the cytoskeletal proteins spectrin and adducin whose altered disposition in IP6K3 knock-out mice may mediate phenotypic features of the mutant mice. These findings afford molecular/cytoskeletal mechanisms by which the inositol polyphosphate system impacts brain function.
Inositol phosphates are major signaling molecules with inositol 1, 4, 5-trisphosphate (IP3) best known as a second messenger molecule releasing intracellular calcium. During the past decade higher inositol polyphosphates have been characterized, most notably diphosphoinositol-pentakisphosphate (PP-IP5), which contains an energetic pyrophosphate bond and is typically referred to as IP7 (Stephens et al., 1993). IP7 is synthesized by a family of three inositol hexakisphosphate kinases (IP6K;Voglmaier et al., 1996; Saiardi et al., 1999; Saiardi et al., 2001), and, to a lesser extent, by the VIP group of enzymes (Mulugu et al., 2007). The three IP6Ks generate the same isomer of IP7 with the pyrophosphate bond at C-5. However, they derive from three distinct genes and appear to mediate different functions (Saiardi et al., 2001; Chakraborty et al., 2010; Rao et al., 2014). Mice with targeted deletion of IP6K1 display male sterility, enhanced Akt signaling, and resistance to diabetes (Chakraborty et al., 2010). IP6K2 appears to be a mediator of cancer cell migration and tumor metastasis (Rao et al., 2015) and is required for p53-associated apoptosis (Koldobskiy et al., 2010). However, little is known about the function of IP6K3.
Adducins and spectrins are ubiquitously expressed cytoskeletal proteins. Adducin has three subtypes: α, β, and γ. α- and γ-Adducins are expressed similarly in most tissues, whereas β-adducin is most abundant in the brain (Joshi et al., 1991; Dong et al., 1995). All three adducin proteins contain an N-terminal globular head domain, a neck domain, and a C-terminal tail domain with a highly conserved MARCKS motif at the end, which is required for association with spectrin (Matsuoka et al., 2000). α- and β-Adducins form heterodimers in the brain.
Spectrins are comprised of α- and β-subunits, which associate to form heterotetramers. Human spectrin contains two α-subunits (α1 and α2) and five β-subunits (β1, β2, β3, β4, and β5; Bennett and Baines, 2001). α1/β1-Spectrins are expressed in erythrocytes, while α2/β2, α2/β3, and α2/β4 spectrins predominate in nonerythroid tissues. α-Spectrin incorporates triple helical repeat domains, an src homology domain 3 (SH3), and a C-terminal calmodulin-related domain. β-Spectrin contains an N-terminal calponin homology (CH) domain, which binds adducin, a triple helical repeat, and a C-terminal pleckstrin homology (PH) domain region (Bennett and Baines, 2001).
Adducin is localized to and binds spectrin-actin junctions, promoting the association of spectrin with actin (Gardner and Bennett, 1987). Binding of the two proteins is mediated by the C-terminal tail and MARCKS domain of adducin (Li et al., 1998) and the N-terminal CH domain of β-spectrin (Li and Bennett, 1996).
Morphologic alterations in neuronal dendritic spines underlie synaptic plasticity, processes that are especially well exemplified in Purkinje cells of the cerebellum. The cytoskeleton of dendritic spines is comprised of actin and actin-regulating proteins, including adducins and spectrins (Matsuoka et al., 1998; Gao et al., 2011; Nestor et al., 2011). The shape of Purkinje cell dendritic spines is determined by the arrangements and attachments of their cytoskeletal elements (Landis and Reese, 1983). β-Spectrins regulate the morphology and functional dynamics of dendritic spines (Gao et al., 2011; Nestor et al., 2011). Cells with deficiencies of spectrin/adducin interaction display disrupted morphology (Hu et al., 1995).
In the present study we report that IP6K3 is abundantly expressed in cerebellar Purkinje cells and physically binds adducin/spectrin. Mutual interactions of adducin and spectrin are severely disrupted in IP6K3-null mice cerebellum. Mice with targeted deletion of IP6K3 manifest abnormalities in cerebellar Purkinje cell structure as well as reduced numbers of synapses. IP6K3 mutant mice manifest disorders of motor learning and coordination. IP6K3 noncatalytically influences the disposition of the spectrin-adducin complex, which can account for its influences upon cerebellar Purkinje cells and motor behavior.
Chemicals were from Sigma-Aldrich. Anti-IP6K1, IP6K2, IP6K3, anti-GST (HRP conjugate), and anti-Flag-tag antibodies were from Sigma-Aldrich. Anti-GAD65, GAPDH, β-actin antibodies, and rabbit anti-calbindin antibody were from Cell Signaling Technology. Anti-VGlut-1 and anti-VGlut-2 antibodies were from Millipore. Anti-gephyrin, vesicular GABA transporter (VGAT), and Homer1 antibodies were from Synaptic Systems. Anti-α-adducin, β-adducin, α2-spectrin, normal rabbit IgG, and normal mouse IgG antibodies and mouse anti-calbindin antibody were from Santa Cruz Biotechnology. Anti-β2-spectrin antibody was from Bethyl Laboratories. Alexa 488 goat anti-rabbit and Alexa 568 goat anti-mouse IgG were from Life Technologies. IP6K3 shRNA and control shRNA plasmids were from Sigma-Aldrich.
Eight-week-old male mice were used. The wild-type and IP6K3 KO animals were littermates from heterozygous parents. Animal breeding and procedures were conducted in strict accordance with the NIH Guide for Care and Use of Laboratory Animals.
Standard methods for cell lysis, immunoprecipitation, SDS-PAGE, and Western blot were as described previously (Zhu et al., 2011). Briefly, mouse brain tissue or cultured cells were lysed on ice in buffer containing 20 mm Tris-HCl, pH 7.5; 150 mm NaCl; 1% NP-40; 1% sodium deoxycholate; 1 mm Na2EDTA; 1 mm EGTA; protease inhibitors; and phosphatase inhibitors. Insoluble cell debris was removed by centrifugation for 10 min at 16,000 g. For immunoprecipitation, cell lysates were precleared by protein A/G beads for 1 h at 4°C, then treated with the primary antibody overnight at 4°C, followed by pull-down with protein A/G beads for 1 h at 4°C. Beads were washed six times with lysis buffer on ice.
Cell culture and lentiviral transduction.
HEK293 and HEK293T cells were cultured under standard conditions. Lentiviral production and transduction were generated as previously reported (Fu et al., 2013). IP6K3 knock-down HEK293 cells were generated by lentiviral transduction and maintained in DMEM with 5 μg/ml puromycin.
The actin-binding (CH) domain (1–275 aa), repeat (RE) domain (303–525 aa), and PH domain (2197–2307 aa) of β2-spectrin (Li and Bennett, 1996) and the wild-type IP6K3 and kinase-dead mutant IP6K3 (K217A) were cloned into the pGEX-6P-2 vector (GE Healthcare Life Sciences). PreScission Protease was used to cleave the wild-type IP6K3 and kinase-dead mutant IP6K3 (K217A) from the fusion protein. The Flag-tag β-adducin tail and MARCKS domain (437–726 aa; Li et al., 1998) were cloned into pT7-MAT-1 for in vitro protein expression.
Immunofluorescence staining and electron microscopy.
Immunofluorescence staining and electron microscopy were performed as previously reported (Fu et al., 2010). The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the NIH. For immunofluorescence staining, animals were perfused with 4% paraformaldehyde and brain slices were cut in sagittal section and incubated with 10% goat serum and then with primary antibodies at 4°C overnight. The fluorescence-labeled secondary antibodies were incubated for 1 h at room temperature. Nuclei were counterstained with DAPI. Slices were mounted with ProLong Gold Antifade Mountant. Pictures were taken under confocal microscope (Zeiss LSM 700; NIH Award S10 OD016374). The range indicator on the microscope shows no "red" or no oversaturated pixels to prevent artifacts caused by overexposure. Synapses were identified by double staining of presynaptic and postsynaptic markers (VGAT and gephyrin for GABAergic synapses, VGlut1 and Homer1 for parallel fiber synapses, and VGlut2 and Homer1 for climbing fiber synapses). The primary antibodies were anti-VGAT (rabbit polyclonal; Synaptic Systems), anti-gephyrin (mouse monoclonal; Synaptic Systems), anti-VGlut1 (mouse monoclonal; Millipore), anti-VGlut2 (mouse monoclonal; Millipore), and anti-Homer1 (rabbit polyclonal; Synaptic Systems). The primary antibodies were diluted in PBS at a ratio of 1:100. The fluorescence-labeled secondary antibodies were Alexa 488 goat anti-rabbit IgG (Life Technology) and Alexa 568 goat anti-mouse IgG (Life Technology). The secondary antibodies were diluted in PBS at a ratio of 1:300. Volocity software (V6.3; PerkinElmer) was used to quantify the density of synapses, determined by colocalization of both presynaptic and postsynaptic markers. Synapse density was determined by counting the number of synapses per unit area. For electron microscopy, animals were perfused with 2% glutaraldehyde and 2% paraformaldehyde, and the samples were embedded into EPON resin. Slides were cut in sagittal and coronal sections with focus on the molecular layer of cerebellum, where the synapses on Purkinje cell dendrites predominate. Sections were stained with uranyl acetate and lead citrate and viewed by transmission electron microscopy. Synapses were counted from at least six different views of pictures.
Golgi stain and H&E stain.
The Golgi stain was performed using the FD Rapid GolgiStain Kit (FD Neurotechnologies). The H&E stain followed standard protocols. Mice were anesthetized and briefly perfused with PBS followed by 4% paraformaldehyde. For Golgi stain, mice brains were immersed in impregnation solution at room temperature for 3 weeks in the dark. Tissues were then transferred into Solution C (FD Rapid GolgiStain Kit) and stored in the dark at 4°C for 1 week. Sections (200 μm in thickness) were cut on a cryostat at −20°C. Sections were stained and, following dehydration and clearing, they were mounted with Permount Mounting Medium.
Mouse behavioral tests were conducted on 8-week-old males at the Johns Hopkins University Brain Science Institute Behavioral Core. Wild-type and IP6K3-null mice were littermates of heterozygous parents, 12 mice per group. Animals were housed in ventilated racks and given ad libitum access to food and water. Animals were moved to their final house location 2 weeks before testing. Starting 1 week before testing, animals were handled daily up until the first day of behavior testing. All testing occurred during the animals' normal light phase (7:30 A.M.–9:30 P.M.). The order of testing was as follows: open field, rotarod, and gait analysis with only one test done per week. All procedures were approved by The Johns Hopkins University Animal Care and Use Committee.
General locomotor activity in IP6K3 knock-out mice was assessed by open field analysis (Photobeam Activity System; San Diego Instruments). Mice were placed in the center of an enclosed acrylic chamber and allowed to explore freely for 30 min. Photobeams tracked horizontal movements and rearing behavior in the x and y directions.
Following open field studies, motor learning and coordination were examined in IP6K3-null mice by the rotarod test (Rotamex-5; Columbus Instruments). Testing was conducted over a 3 d period, where each mouse was given five trials per day and latency to fall off the rotarod was measured as it accelerated from 4 to 99 rpm over a 5 min period. Before the start of testing on the first 2 d, each mouse was given a habituation trial by being placed on the rotarod, which was rotating at a constant speed of 4 rpm for 10 min. The highest and lowest latencies of the trials were discarded and the average taken from those remaining.
The CatWalk XT system (Noldus Information Technologies) was used to study walking sequence and patterns of IP6K3 mutant mice. Before testing, mice were given four training days to acclimate to the testing apparatus. On the testing day, animals were placed on the glass plate until they completed three valid trials. A valid trial was one in which the mouse walked the length of the tunnel in <10 s with <60% variation in speed. Data were recorded using a high-speed camera fixed directly beneath the glass plate and were analyzed using CatWalk 9.0 XT software (Noldus Information Technologies). Sequence of movement was used to identify stride length, step width, and percentage of forepaw-hindpaw overlap. Averages were taken across valid trials on testing day. All trials marked as valid by the software were then reviewed manually to ensure that animals did not turn around or stop during the trial.
Quantitative data are expressed as means with SEM. Data were analyzed by one-way ANOVA, unpaired Student's t test, and Pearson's correlation coefficient. Open field and rotarod data were analyzed by two-way repeated-measures ANOVA. The volume of Purkinje cells was quantified by using ImageJ. The Purkinje cell number was quantified by counting the cell number per unit length. P < 0.05 was considered statistically significant. For nonquantitative data, results were representative of at least three independent experiments.
To evaluate rigorously physiologic functions of IP6K3, we developed mice with targeted deletion of IP6K3 (Fig. 1A). IP6K3 knock-outs breed fairly normally, though they produce fewer progeny than wild-type mice (wild-type 7 ± 1 vs KO 5 ± 1; six pairs of breeding). IP6K3 immunostaining reveals substantial levels in cortex, hippocampus, thalamus, hypothalamus, and cerebellum with virtually total loss of the enzyme in null mice (Fig. 1B). Staining is somewhat patchy in midbrain and cortex. At this low level of resolution, the basis for the patchiness and cellular localizations cannot be discriminated. The IP6K3-null mice do not display gross abnormalities of brain structure (Fig. 1C–E). We explored motor behavior of mutant mice (Fig. 1F–H). Overall activity in an open field does not appear impaired in the knock-out mice (Fig. 1F); however, motor learning and coordination are disturbed, as is evident in rotarod performance (Fig. 1G). Moreover, gait analysis indicates defects in the overlap of hindpaws and forepaws but no significant difference in stride length and step width (Fig. 1H). Motor deficits manifested by the mutant mice resemble disabilities of mice with Purkinje cell dysfunction. Interestingly, in cerebellar molecular layer, IP6K3 is most highly expressed in Purkinje cells with substantial levels as well in interneurons (Fig. 1B,I,J).
IP6K3 is highly expressed in cerebellar Purkinje cells, defects of motor learning, and coordination in IP6K3 knock-outs. A, Schematic representation of the mouse IP6K3 gene with coding regions shaded black, noncoding regions in white, and the exon number indicated on top. A close-up view of exons 5 and 6 in the wild-type, targeted, and knock-out alleles is shown. The knock-out allele loses the splice site, coding region, and the 3UTR of exon 6. B, Immunostaining of IP6K3 in sagittal sections of wild-type and IP6K3 KO mice brain (top) reveals abundant levels of IP6K3 in the cortex and cerebellar molecular layer. C–E, H&E staining on wild-type and IP6K3 KO mice brain sagittal section. C, Whole brain. D, Hippocampus. E, Cerebellum. F, Open field behavioral analysis reveals negligible alterations in IP6K3 KO mice; y-axis, total activity (beams breaks); x-axis, 5 min intervals. G, Rotarod tests reveal reduced motor learning and coordination in IP6K3 KO mice; y-axis, the latency to falling in second; x-axis, days of testing. H, Gait analysis shows significantly decreased overlap of hindpaws and forepaws in IP6K3 KO mice; y-axis, the percentage of forepaw-hindpaw overlap, with the averages taken across trials on testing day. The stride length and step width do not display notable differences. I, Immunostaining of IP6K3 and calbindin in cerebellar molecular layer from wild-type and IP6K3 KO mice. IP6K3 is expressed mainly in Purkinje cells. Scale bar, 20 μm. J, Immunostaining of IP6K3 and calbindin in wild-type cerebellar molecular layer. IP6K3 is most highly expressed in Purkinje cells (arrow) with substantial levels as well in interneurons (top arrowhead, basket cell; bottom arrowhead, stellate cell). Data are represented as mean ± SEM, *p < 0.05, ***p < 0.001, 8-week-old male mice. B–E, I, J, n = 5; F–H, n = 12.
Much less is known about protein–protein interactions of IP6K3 compared with IP6K1 or IP6K2. To gain insight into functions of IP6K3, we sought potential interacting proteins. Accordingly, we immunoprecipitated IP6K3 and explored proteins that coprecipitate. Mass spectrometric analysis of the most prominently interacting proteins identifies spectrins and adducins as major binding partners for IP6K3 (Fig. 2A).
IP6K3 binds spectrins and adducins in the cerebellum. A, Immunoprecipitation (IP) of IP6K3 from wild-type mice cerebellum with rabbit IgG as a control. Silver staining and mass spectrometric analysis identify spectrins and adducins pulled down by IP6K3. B, Immunoprecipitation of IP6K3, α-adducin, and β-adducin from wild-type mice cerebellum with mouse IgG and rabbit IgG as controls. Western blot indicates that IP6K3 binds to α-adducin and β-adducin. C, Immunoprecipitation of IP6K3, α2-spectrin, and β2-spectrin from wild-type mice cerebellum with mouse IgG and rabbit IgG as controls. Western blot shows IP6K3 binding to α2-spectrin and β2-spectrin. D, Immunoprecipitation of IP6K1, IP6K2, and IP6K3 from wild-type mice cerebellum. Silver staining and mass spectrometric analysis indicates pull-down of spectrins and adducins by IP6K3 but not IP6K1 or IP6K2. E, Immunoprecipitation of IP6K1, IP6K2, and IP6K3 from wild-type mice cerebellum. Western blot shows that IP6K3 but not IP6K1 or IP6K2 binds to α-adducin, β-adducin, α2-spectrin, and β2-spectrin.
We examined in more detail interactions of IP6K3, adducins, and spectrins (Fig. 2B,C). IP6K3 coprecipitates with α- and β-adducin, while precipitation of the two adducins leads to coprecipitation of IP6K3 and the complementary adducin isoforms (Fig. 2B). Similarly, precipitation of IP6K3 leads to coprecipitation of α2- and β2-spectrin, while each of these spectrins coprecipitates with IP6K3 as well as the other spectrin isoform (Fig. 2C). Thus, IP6K3 occurs in a complex with the adducins and spectrins.
We wondered whether interactions with spectrins and adducins are selective for IP6K3 or might be evident with other IP6 kinase isoforms. Immunoprecipitation with antibodies to IP6K3 coprecipitates spectrins and adducins, while these proteins do not coprecipitate with IP6K1 or IP6K2 (Fig. 2D). In the same experiments we conducted Western blots focusing on subtypes of spectrins and adducins that bind IP6 kinase isoforms (Fig. 2E). IP6K3 coprecipitates with α2- and β2-spectrin and α- and β-adducin (Fig. 2E). Spectrins and adducins do not coprecipitate with IP6K1 or IP6K2.
Spectrin and adducin are well known to interact biochemically and to colocalize in cells (Li et al., 1998). We wondered whether IP6K3 impacts such interactions. Accordingly, we deleted IP6K3 by RNA interference in HEK293 cells (Fig. 3A). In the IP6K3-deleted cells coprecipitation of α2- and β2-spectrin with α-adducin is markedly diminished (Fig. 3B). Similarly coprecipitation of α- and β-adducin with β2-spectrin is much lower in the mutants (Fig. 3B). Thus, physiologic interactions of spectrins and adducins are substantially impaired with loss of IP6K3, indicating an important role for IP6K3 in maintaining the spectrin-adducin complexes.
Impaired adducin/spectrin interaction in IP6K3-deleted cells. A, IP6K3 is deleted in HEK293 cells by shRNA lentiviral transduction. B, Knockdown of IP6K3 in HEK293 cells reduces adducin/spectrin binding. C, Immunostaining in IP6K3-deleted HEK293 cells of β-adducin and β2-spectrin (top two lanes) as well as F-actin and β2-spectrin (bottom two lanes). Colocalization of β-adducin with β2-spectrin is significantly reduced in IP6K3 knockdown cells, as is colocalization of F-actin with β2-spectrin. Data are presented as mean ± SEM, ***p < 0.001. IP, immunoprecipitation; Ctrl, control.
Similarly, colocalizations of β-adducin and β2-spectrin monitored microscopically depend upon IP6K3 (Fig. 3C). In control HEK293 cells β-adducin, β2-spectrin, and F-actin are similarly localized in the vicinity of the plasma membrane. Colocalization of β-adducin and β2-spectrin is reduced almost 60% in IP6K3 knock-outs with a similar reduction in the colocalization of β2-spectrin and F-actin.
Using IP6K3 knock-out mice, we confirm the importance of IP6K3 for the disposition of spectrins and adducins (Fig. 4). IP6K3 deletion does not change cerebellar protein levels of spectrins and adducins (Fig. 4A) but markedly alters their interaction (Fig. 4D). Coprecipitation of spectrins and adducins with IP6K3 is verified (Fig. 4B,C). Coprecipitation of β2-spectrin with α-adducin or β-adducin is greatly diminished in IP6K3 knock-outs. Similarly, coprecipitation of α- and β-adducin with β2-spectrin is virtually abolished in IP6K3 mutants (Fig. 4D).
Adducin/spectrin binding is substantially reduced in IP6K3-null mice cerebellum. A, Expression of α-adducin, β-adducin, α2-spectrin, and β2-spectrin is normal in IP6K3 KO mice cerebellum. B, C, Immunoprecipitation (IP) verifies IP6K3 binding to α-adducin, β-adducin, α2-spectrin, and β2-spectrin in mouse cerebellum. D, Immunoprecipitation of α-adducin, β-adducin, α2-spectrin, and β2-spectrin from cerebellum of both wild-type and IP6K3 KO mice. Binding of α-adducin and β-adducin with β2-spectrin is reduced in IP6K3 KO mice cerebellum.
To explore molecular mechanisms for IP6K3/adducin/spectrin interactions, we obtained bacterially purified forms of these proteins (Li and Bennett, 1996; Li et al., 1998; Fig. 5A). Utilizing purified proteins, we show that IP6K3 binds to β-adducin and to β2- spectrin's CH domain directly but not to the repeat or PH domains of spectrin (Fig. 5B,C). Binding of spectrin to adducin is substantially enhanced in the presence of IP6K3 (Fig. 5D). We wondered whether catalytic activity of IP6K3 is required for interactions with spectrin/adducin. Catalytically inactive IP6K3 (K217A) binds spectrin/adducin similarly to the wild-type enzyme (Fig. 5E–G). Moreover, physiological concentrations of IP6 or IP7 fail to influence spectrin-adducin binding, consistent with catalytic activity of IP6K3 not being required for spectrin/adducin interactions (Fig. 5H).
IP6K3 facilitates the binding of adducins with spectrins; kinase activity of IP6K3 is not required. A, In vitro expression and purification of proteins. Coomassie blue staining of the control GST, β2-spectrin CH domain (1–275 aa), β2-spectrin repeat domain (303–525 aa), β2-spectrin PH domain (2197–2307 aa), β-adducin (437–726 aa), wild-type IP6K3, and kinase-dead mutant IP6K3 (K217A). GST, GST control; CH, β2-spectrin CH domain (1–275 aa); RE, β2-spectrin repeat domain (303–525 aa); PH, β2-spectrin PH domain (2197–2307 aa). B, In vitro binding of IP6K3 with β2-spectrin domains and β-adducin (437–726 aa). Immunoprecipitation (IP) of IP6K3, which binds to β2-spectrin CH domain and β-adducin (437–726 aa). The upper band for RE is the full-length band. C, In vitro binding of β2-spectrin domains and β-adducin (437–726 aa) with IP6K3. Immunoprecipitation of β2-spectrin domains (pull-down GST) and β-adducin (437–726 aa, pull-down flag tag). Western blot shows that IP6K3 binds to β2-spectrin CH domain and β-adducin (437–726 aa). D, In vitro binding of β2-spectrin domains with β-adducin (437–726 aa). Immunoprecipitation of β2-spectrin domains (pull-down GST) reveals that β-adducin (437–726 aa) binds only to β2-spectrin CH domain, and the binding is significantly increased by IP6K3. E, In vitro binding of wild-type IP6K3 and kinase-dead mutant IP6K3 (K217A) with β2-spectrin CH domain and β-adducin (437–726 aa). Immunoprecipitation of β2-spectrin CH domain (pull-down GST) and β-adducin (437–726 aa, pull-down flag-tag); Western blot shows kinase-dead mutant IP6K3 (K217A) binds with β-adducin (437–726 aa) and β2-spectrin CH domain similarly to wild-type IP6K3. F, In vitro binding of kinase-dead mutant IP6K3 (K217A) with β-adducin (437–726 aa) and β2-spectrin CH domain. Immunoprecipitation of β2-spectrin CH domain (pull-down GST) indicates that kinase-dead mutant IP6K3 (K217A) binding to β2-spectrin CH domain facilitates the binding of β2-spectrin CH domain with β-adducin (437–726 aa). G, In vitro binding of β-adducin (437–726 aa) and β2-spectrin CH domain with kinase-dead mutant IP6K3 (K217A). Immunoprecipitation of β-adducin (437–726 aa, pull-down flag tag). Western blot shows that binding of kinase-dead mutant IP6K3 (K217A) to β-adducin (437–726 aa) facilitates the binding of β2-spectrin CH domain with β-adducin (437–726 aa). H, In vitro binding of β-adducin (437–726 aa) with β2-spectrin domains. Immunoprecipitation of β2-spectrin domains (pull-down GST); Western blot shows that physiological concentrations of IP6 (20 μm) or IP7 (5 μm) do not affect the binding of β-adducin with β2-spectrin.
We sought potential alterations of overall cerebellar structure in the mutant mice. In mutants, the width of the molecular layer is diminished, whereas width of the granule cell layer is normal as are the number of Purkinje cells (Fig. 6A). Examination of the IP6K3 mutants at high-power magnification reveals alterations in Purkinje cell structure with withered dendritic trees (Fig. 6B,D). The morphologic abnormalities of the mutant mice appear similar in 8-week-old and 12-month-old animals, suggesting that the disorder is developmental rather than degenerative. This conclusion is supported by our failure to detect any signs of inflammation in the mutant mouse brains. The cell size and spine density of mutant Purkinje cells are also decreased (Fig. 6B,C). In contrast, the morphology of granule cells appears normal. Golgi cells, basket cells, and stellate cells of the mutants possess modestly shorter dendrites but overall normal morphology (Fig. 6E).
IP6K3 KO mutant mice manifest altered Purkinje cell structure and reduced synapses. A, H&E staining in cerebellum from wild-type and IP6K3 KO mice (8 weeks old). Width of the molecular layer is decreased in IP6K3 KO mice. The Purkinje cell number and the width of granule layer do not show any significant difference. B, Golgi staining reveals withered dendritic trees of Purkinje cells from 8-week-old IP6K3 KO mice cerebellum. Cell volume is significantly decreased in IP6K3 KO Purkinje cells. C, Golgi staining for the Purkinje cell dendrites from wild-type and IP6K3 KO mice cerebellum (8 weeks old). Spine number is significantly decreased in IP6K3 KO Purkinje cells. D, Golgi staining reveals withered dendritic trees of Purkinje cells from 12-month-old IP6K3 KO mice cerebellum. E, Golgi staining of granule cells, basket cells, Golgi cells, and stellate cells reveals shorter dendritic length (22 ± 5% less for basket cell, 15 ± 4% less for Golgi cell, and 25 ± 8% less for stellate cell) but no significant morphological change in the IP6K3 KOs (8 weeks old). We quantified the length of an average of five dendrites per cell, six cells per mouse. F, Electron microscopy of the cerebellar molecular layer from wild-type and IP6K3 KO mice (8 weeks old). Densities of both symmetric and asymmetric synapses are markedly decreased in IP6K3 KOs. The ultrastructure of synapses is relatively normal in the KOs. Data are represented as mean ± SEM, three mice per group, **p < 0.01, ***p < 0.001.
Excitatory synapses contain asymmetric synaptic elements, whereas inhibitory synapses are symmetric (Harris and Weinberg, 2012). At the electron microscopic level we quantified numbers of symmetric and asymmetric synapses in the cerebellar molecular layer of IP6K3 knock-outs (Fig. 6F). Both symmetric and asymmetric synapses are reduced ∼40–50% in the mutant mice. The synaptic size, presynaptic vesicle number, and synaptic cleft thickness, however, do not show any notable differences.
We stained for markers of GABA and glutamate containing neurons (Fig. 7). IP6K3 knock-out cerebella display reduced levels of GAD65, the GABA synthesizing enzyme and also a marker for GABAergic synapses (Fig. 7A). Staining for VGlut1, a marker for parallel fibers, is also diminished in the mutants (Fig. 7B) as is VGlut2, marking climbing fibers (Fig. 7C).
IP6K3 KO mice display significantly fewer synapses on cerebellar Purkinje cells. A, Immunostaining of GAD65 and calbindin in cerebellar Purkinje cells from wild-type and IP6K3 KO mice. The density of GAD65 is substantially less in IP6K3 KOs. B, Immunostaining of VGlut1 and calbindin in cerebellar Purkinje cells from wild-type and IP6K3 KO mice. The density of VGlut1 is markedly reduced in IP6K3 KOs. C, Immunostaining of VGlut2 and calbindin in cerebellar Purkinje cells from wild-type and IP6K3 KO mice. The density of VGlut2 is notably diminished in IP6K3 KOs. D, Immunostaining of gephyrin and VGAT in cerebellar Purkinje cells from wild-type and IP6K3 KO mice. The density of double-stained GABAergic synapses is significantly reduced in IP6K3 KOs. E, Immunostaining of VGlut1 and Homer1 in cerebellar Purkinje cells from wild-type and IP6K3 KO mice. The density of double-stained parallel fiber synapses is significantly reduced in IP6K3 KOs. F, Immunostaining of VGlut2 and Homer1 in cerebellar Purkinje cells from wild-type and IP6K3 KO mice. The density of double-stained climbing fiber synapses is much less in IP6K3 KOs. G, Statistical analysis from D–F indicates that GABAergic, parallel fiber, and climbing fiber synapses are significantly reduced in IP6K3 KOs. Scale bar, 20 μm. Data are presented as means ± SEM, ***p < 0.001. Mice were 8-week-old males, n = 5.
We explored properties of synapses that might be altered by diminution in these GABA and glutamate markers. We assessed GABA synapses in terms of the juxtaposition of VGAT, reflecting presynaptic GABA neurons, and gephyrin, marking postsynaptic elements of GABA synapses (Tyagarajan and Fritschy, 2014). The IP6K3 knock-outs display a 40% reduction in unit area for GABA synapses (Fig. 7D,G). We examined parallel fiber synapses by costaining for VGlut1, indicating parallel fibers, and Homer1, reflecting postsynaptic elements of excitatory synapses. Numbers of parallel fiber synapses are diminished by ∼40% in the mutant mice (Fig. 7E,G). We assessed climbing fiber synapses by monitoring colocalizations of VGlut2, marking climbing fiber processes, and Homer1, which labels postsynaptic elements. The density of climbing fiber synapses appears reduced ∼50% in IP6K3 knock-outs (Fig. 7F,G). In summary, the principal types of synapses in the cerebellum are substantially diminished in IP6K3 knock-out mice.
In the present study we demonstrate that IP6K3 is highly expressed in cortex and cerebellum. There is no gross difference of brain structure in the mutant mice. The IP6K3 KO mice display impaired motor learning and coordination in rotarod and gait analysis but relative normal performance in open field study. The mutant mice may compensate for gait/rotarod deficits leading to normal open field performance, which incorporates a wide range of motor skills. Motor deficits manifested by the mutant mice are reminiscent of disabilities of mice with Purkinje cell dysfunction. Intriguingly, IP6K3 is most highly expressed in Purkinje cells in cerebellum. IP6K3 acting noncatalytically physiologically regulates interactions of spectrin and adducin. Spectrins and adducins appear to be the principal interactors of IP6K3, which is required for their physiologic association. Deletion of IP6K3 abrogates spectrin-adducin binding and impacts the morphology of cerebellar Purkinje cells as well as their synaptic disposition and motor activity. The dendritic trees of the mutant mice are withered, and the size and spine density of Purkinje cells are reduced along with a substantial decrease in the major forms of cerebellar synapses. Loss of Purkinje dendritic branches and decreased numbers of dendritic spines have been reported to cause decreased motor learning and coordination (Clark et al., 1997). Morphologic changes of Purkinje dendritic trees result in motor defects (Donald et al., 2008). The IP6K3-null Purkinje cells also have disordered dendritic trees, which may mediate the deficits of motor learning and coordination.
The physical binding of IP6K3 with adducins and spectrins to influence their function affords a molecular mechanism to account for cytoskeletal alterations of Purkinje cells in the IP6K3 mutants. Abundant literature establishes a role for β-adducin in the assembly of synapses in the brain and regulation of memory in mammals (Bednarek and Caroni, 2011) and in Drosophila (Pielage et al., 2011). In Drosophila adducin mediates assembly of synapses at the neuromuscular junction (Pielage et al., 2011), and mutations of spectrin impact presynaptic neurotransmitter release (Featherstone et al., 2001). There are multiple subtypes of spectrins. Our study focused upon β2-spectrins, which are robustly coimmunoprecipitated by IP6K3. The β1 subtype of spectrin also regulates the morphological and functional dynamics of dendritic spines (Nestor et al., 2011). The β3 subtype of spectrin has been implicated in synaptogenesis with mutations leading to ataxia (Stankewich et al., 2010). In the cerebellum β3-spectrin mediates development of Purkinje cell dendritic trees and morphogenesis of spines (Gao et al., 2011). Assembly of the spectrin-actin membrane skeleton is regulated by adducin/spectrin interactions (Gardner and Bennett, 1987), whose deficiency disrupts cell morphology (Hu et al., 1995). The disruption of adducin/spectrin interactions in IP6K3 KOs may mediate the morphologic alteration as well as the decreased synapse formation of Purkinje cells. Decreased synapses in the IP6K3 KO mice may also affect electrophysiological properties.
IP6K3 physically binds to adducin's C-terminal domain and to β-spectrin's CH domain, which are the binding sites for adducin/spectrin interactions (Li and Bennett, 1996; Li et al., 1998). Adducin, protein 4.1, and dematin localize at the spectrin–actin junctional complex (Derick et al., 1992) and associate with spectrin to facilitate the spectrin–actin interaction (Correas et al., 1986; Gardner and Bennett, 1987; Koshino et al., 2012). Dematin and protein band 3 act as scaffolds to link adducin with spectrin (Drenckhahn et al., 1985; Khan et al., 2008; Anong et al., 2009; Koshino et al., 2012). Our study provides evidence that IP6K3 serves as a scaffold to link adducin and spectrin, a function that does not require IP6K3's catalytic activity. In the inositol phosphate kinase family there is precedence for noncatalytic actions. Inositol polyphosphate multikinase (IPMK) physiologically generates IP5 but also is a PI3 kinase that forms phosphatidylinositol (3,4,5)-trisphosphate (Resnick et al., 2005). Additionally, acting noncatalytically, IPMK stabilizes the mTOR complex (Kim et al., 2011) and acts as a transcriptional coactivator for several genes (Xu et al., 2013a, b). Our present findings are the first to establish noncatalytic functions for an IP6 kinase.
The ability of IP6K3 to regulate synaptic disposition noncatalytically via influences upon specific cytoskeletal proteins provides a new functional dimension for the IP6 kinases. This action, together with the noncatalytic activities of IPMK (Xu et al., 2013a, b), indicates that the class of inositol phosphate kinases may display myriad cellular activities not mediated by catalytic formation of inositol phosphates. Other classes of enzymes also manifest important noncatalytic activities. Known examples include kinase-dead Braf and oncogenic Ras, which cooperate to induce melanomas (Heidorn et al., 2010). The USP13 enzyme regulates Siah2 ligase stability and activity via noncatalytic ubiquitin-binding domains (Scortegagna et al., 2011). Moreover, a triosephosphate isomerase noncatalytically influences behavior and longevity (Roland et al., 2013).
The most obvious deficit showed by IP6K3 KO mice is the reduction in motor learning and coordination, which indicates cerebellar dysfunction. In the current study, we focus on these phenotypes and show that IP6K3 regulates the morphology and synaptic formation of cerebellar Purkinje cells via direct binding to spectrin/adducin. IP6K3 has also been found in several other regions of the brain, which may mediate diverse functions.
This work was supported by United States Public Health Service Grant DA000266 to S.H.S. We thank members of the Snyder laboratory for reagents and discussions.
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Contemporary Healthcare Capital has focused on providing financing solutions solely to the seniors housing and post-acute care industries for nearly 20 years, which enable us to have a deep understanding of trends and challenges facing the industry at any given time. We are more relationship driven than transactional as evidenced by multi-year clients who continue to utilize our services and products year after year. We don't adhere to "box lending" and uniquely tailor our loans and investments to meet the mutual needs of both our provider/borrower clients as well as our lending partners.
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Drumming (photo book)
English - €30,00 EUR French - €30,00 EUR
With Drumming (1998), choreographer Anne Teresa De Keersmaeker returned to two of her great loves as a choreographer: pure dance and the minimalistic music of Steve Reich. After an impressive artistic journey of almost twenty years, this resulted in an iconic creation to Reich's pulsating score, pushing to the extreme everything which gradually had become part of De Keersmaeker's artistic signature: the mathematical figures, the sustained repetition, the geometric occupation of the space, and the art of continuous variation.
Drumming is characterised by a unification of a very refined structure with virtuosity and the sheer joy of dancing. Not only De Keersmaeker's idiom of energetic movements, but also Dries Van Noten's costumes and Jan Versweyveld's scenography contribute to the strength of the visual composition.
Since 1998, Drumming has been performed continuously on stages all around the globe. This process was followed from up close by photographers Herman Sorgeloos and Anne Van Aerschot. Their elaborate archives offer an exceptional view into the unique aesthetics of this piece.
Their work is accompanied by texts written by Noé Soulier, Tessa Hall and Julia Rubies Subiros and also includes an interview with Anne Teresa De Keersmaeker by Gilles Amalvi.
Available in English and French
128 PAGES • 33.5 x 24 CM • 56 ILL. • SOFTCOVER • €30.00 • EN / ISBN 978-94-6230-268-6 • EN / ISBN 978-0-300-25398-6 (YALE UP) • FR / ISBN 978-94-6230-260-0
Embodying an abstraction / Anne Teresa De Keersmaeker at the Collège de France
A Choreographer's Score: Fase, Rosas danst Rosas, Elena's Aria, Bartók (softcover edition)
Photo book: Anne Teresa De Keersmaeker / Rosas 2007–2017
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Home›Law›Are Divine Laws and regulations Of World Absolute or Shifting?
Everything relating to this world is much more intelligent than we give credit for it truly is a stunning coherence running our way of life. The greater we quit to pressure outcome and straightforward maintain the flow, the greater magic our existence is supposed to be, can look.
Our world is controlled by laws and regulations we have conspired with GOD to produce! These laws and regulations are absolute, all pervading, consistent, accurate, and neutral, which may be known as a precise (applied) science. We might deny any understanding from the intricate workings of those laws and regulations. However I would emphasise that every one people is and try to are interacting and taking advantage of these laws and regulations within our existence, each waking as well as sleeping moment, whether we know about them or otherwise. I might acutely state that we know these divine laws and regulations. I've attempted to describe the Laws and regulations in each and every day statements that people make consciously or subconsciously, or might have heard our parent, teacher, protector, family members say. I understand these statements will resonate on a single level or any other together with your inner knowing.
We know them we rely on them within our every single day speech!
Where our attention goes, it attracts – Loa is easily the most amazing law.
Where our focus is, it manifests – Law of Intention and need is easily the most natural law.
When we give we obtain – Law of Giving is easily the most balanced law.
What circles appears – Law of Compensation(also referred to as Law of Karma) is easily the most effective law.
Be still and know that i'm GOD – Law of Unity or Oneness (God's versions) is easily the most essential law.
Ignore it and when it comes down back, it had been ours – Law of Detachment is easily the most liberating law.
Do less and achieve more – Law of Easy Living may be the simplest law.
We're unique! (in six billion souls) – Law of Uniqueness (also referred to as Law of Purpose) is easily the most authentic law.
When these unconditional Divine Laws and regulations are knowingly utilized, it'll result in a paradigm transfer of our awareness like a "Being". Our Condition to be attracts ALL creation into OUR existence from the formless energy exhibiting vibrant existence and health.
Creation works the following. We focus through our five senses which create thinking, which triggers visualisation, which in turn causes discerning or knowing, which produces feeling, and inspires the doing. This complete creation process results in a condition to be quite simply, it puts in position the machine to get and experience what we should are coming up with within our Being.


The motorsport world's newest single-seater racing series has just been announced. It's called W Series, and there's a crucial difference between it and other junior Formulas: the championship is exclusively for female entrants.
The series will be taking to tracks around Europe for the debut 2019 season, expanding to circuits elsewhere in the world in the years that follow. It's also free to enter, so long as the entrant can pass a "Rigorous pre-selection programme/examination involving on-track testing, simulator appraisal, technical engineering tests, fitness trials," the series organisers say.
So, getting a seat in one of the F3-spec cars will be based on driving talent and driving talent alone, without the ability to snare sponsorship being a factor. There's also a $1.5 million prize pot for competitors, with the champion claiming a third of that.
However, the idea has dramatically split opinion in the racing world on the day of the announcement, with many from the sport - women racing drivers included - voicing support and concerns for the series.
I've won and been on the podium in every series I've raced in. So what's stopping me reaching the very top?
Not my ability. It's lack of Money.
W Series claims that a large part of its mission is to get more women interested in entering the male-dominated sport in the first place. "At the heart of W Series' DNA is the firm belief that women can compete equally with men in motorsport. However, an all-female series is essential in order to force greater female participation," it says.
At this stage, it's hard to predict how the concept will pan out. We'll certainly be watching with interest.


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text:
Treuman Katz Center for Pediatric Bioethics
Case-Based Teaching Guides
Truthfulness in the Physician-Patient Relationship
Case Discussion: Truthfulness
Case Discussion: Truthfulness in the Physician-Patient Relationship - Physician Fallibility, Medical Error and Disclosure
A 9-month-old boy, Andre, comes to the clinic with his mother for his well-child visit. His mother specifically wants to address his immunization status and make sure he is up to date. She hands you his state immunization record. You see that he hasn't received his third Haemophilus influenzaetype B (Hib) or inactivated poliovirus (IPV) immunization yet.
Because his mother is in a hurry to get to her own doctor's appointment, you help expedite Andre's catch-up shots by asking your medical assistant (MA) to give them as you leave the patient's room.
Ten minutes later, as you begin to write a note in Andre's chart, you notice a note written three months earlier, when Andre saw one of your partners. The note states that Andre received Hib#3 and IPV#3 as part of that visit. It occurs to you that the immunization card was probably not completed after Andre's last visit. You run to see if you can stop the MA from administering the shots, but it's too late: you meet her coming out of Andre's room just having given them.
Your patient has received an immunization that he shouldn't have. Do you think that having given him the shot twice, instead of once, should be considered a medical error?
Yes, this should be considered a medical error. There are several definitions of what constitutes medical error, but one commonly cited is from the Institute of Medicine (IOM): failure of a planned action to be completed as intended or the use of a wrong plan to achieve an aim. Relevant examples of medical errors using this definition include wrong-site surgery, chemotherapy dosing error and, as in our case, the mistaken administration of a vaccine.
Despite the IOM definition, medical error can be difficult to define. For instance, is it considered medical error if a planned action is successfully completed, but just not on time? Consider the case of an inpatient with sepsis who is scheduled to receive an IV antibiotic every six hours but receives a dose three hours late because his nurse was busy with another patient. There is no consensus on whether this is considered a medical error. Because the definition of medical error can be problematic, empirical data for the prevalence of medical error can vary quite dramatically, depending on how inclusive or exclusive one makes the definition.
The patient likely will not suffer any harm from this second dose of vaccine. Does that make you think differently as to whether this was an error?
A medical error does not necessarily require that a patient has been harmed. Although the definition of medical error can vary, it is widely recognized that medical error is different from an adverse event. An adverse event is an injury caused by medical management rather than by the underlying disease or condition of the patient (IOM). Therefore, while an adverse event involves an injury, there does not have to be harm experienced by the patient for a medical error to have occurred.
Imagine that you are not only the physician in this case but that you are early in your training, such as an intern or resident. How easy or difficult is it to explain your mistake to your attending?
Most people have difficulty talking about an error they have made. It may be even more difficult for physicians. It has been long recognized that physicians are trained within a culture of perfectionism (Leape). This culture teaches that mistakes are unacceptable and are due to a failure of character, leading most trainees to believe that they must pursue perfection in order to be a good physician. Similarly, when an error occurs, most physicians feel that it occurred because of their negligence. The expectation that a doctor is infallible is often held by patients as well (Hilfiker).
This culture makes the disclosure of mistakes difficult. Acknowledging fallibility in the face of a culture that expects, and can make career judgments upon, infallibility, is very hard to do. Fortunately, there is a growing medical literature that calls for the realization that humans make mistakes and that we must design systems in the workplace that prevent human error (Leape). However, lingering expectations for physicians, and especially physicians-in-training, remain, and it is important to recognize these as potential barriers to one's ability to disclose mistakes.
Should you disclose your mistake to the patient's mother? Why or why not?
Yes, this mistake should be disclosed to the patient's mother. Several principles support the disclosure of medical error.
Honesty and fidelity
The American Medical Association's (AMA) Principles of Medical Ethics states that a physician "shall…be honest in all professional interactions." Many other specialty academies have similar stances. Disclosure is an important part of many professional physician codes because it serves the purpose of developing trust and cooperation essential for a successful therapeutic relationship. Nondisclosure can undermine public trust in medicine by being construed as deceitful as well as disrespectful of the well-being of patients (Matlow).
Patients may be caused avoidable harm if they are injured further by the failure to disclose. An example of this would be failing to disclose that a surgical sponge was left in a patient after abdominal surgery. This most certainly leads to further harm if it is not immediately removed (Matlow, Hebert).
When a patient is harmed, they should be able to seek appropriate restitution or recompense (Hebert).
This principle supports the notion that patients have a claim to information that might be of importance to them and relevant to making future decisions. When not told the truth, the patient's autonomy may be restricted in the sense that they are deprived of information that might be useful in making decisions (e.g., whether to change physicians, be more attentive to record keeping in the future, etc.). One might also argue that nondisclosure is disrespectful - one is making a decision for another person that he or she does not want to know or doesn't need to know this information (Matlow).
Most patients want even harmless errors disclosed. One recent study (Gallagher) revealed that nearly all patients want disclosure of all harmful errors along with information about what happened, why the error occurred, how the error's consequences will be mitigated and how recurrences will be prevented. They also want emotional support from their physician and an apology.
Honest disclosure of medical error should be the rule, and violating that principle would require justification. What are some of the arguments people might have against disclosing this probably harmless error?
Some may argue that there is no need to disclose in this case because the error was minor and very unlikely to lead to any harm. The parents may not realize an error has occurred, and telling them about it will just make things worse by undermining their trust in the medical system. But this argument rests on several assumptions that cannot be guaranteed. First, errors frequently are discovered after the fact, at which time it becomes clear that the error was not disclosed. Second, there is always the potential that this child will suffer a side effect in the next couple days. Third, a mistake has been made, so "protecting" the family from this knowledge in order to maintain their trust in the medical system is flawed reasoning. Failing to disclose in order to maintain the fiction that mistakes do not occur does not really protect patients and their families. It seeks to protect physicians and healthcare providers. Finally, it is worth considering the cost to the provider. Nondisclosure is a form of dishonesty, and being dishonest has a corrosive effect on a physician's character.
Physicians commonly worry about legal liability in admitting a mistake and disclosing an error. There are several issues worth mentioning in this regard. First, in this case, in all likelihood no harm has occurred. Medical malpractice requires a demonstration of harm. Even if harm had occurred, however, there is evidence in the literature that patients and families might be less likely to seek legal action when a physician has dealt with them honestly and forthrightly. In contrast, among the most common reasons families give for filing medical malpractice suits are the following:
They do not feel they have been told everything about their care or why there was a bad outcome.
They feel they have been lied to.
They feel there has been a cover-up.
Physicians also worry that the patient or family may lose trust in them and perhaps find another physician. This is not sufficient reason to justify nondisclosure. Families are owed an honest explanation of their care, regardless of the consequences to the physician. Even if trust is lost, and the family chooses to find a new physician, that is their choice to make. In reality, trust in the physician is more likely to be maintained when the physician has demonstrated a willingness to be honest even under difficult circumstances, rather than when a family discovers that the physician has withheld information from them.
Do physicians, by virtue of the physician-patient relationship, have a duty to disclose all medical errors?
No. Just as in routine patient care, we typically don't tell patients, for example, the percentage of eosinophils that are in their CBC differential, as this is largely technical and insignificant.
So, what errors should we disclose?
There are a couple of approaches here:
A good rule of thumb can be found in the informed-consent process, where the accepted standard is the concept of the "reasonable person." This concept maintains that a provider should disclose what a reasonable person in the patient's position would want to know in order to make an intelligent and informed treatment decision. In the context of medical error, then, a physician should share with their patients the information a reasonable person in the patient's position would want to know about his or her medical condition, including the diagnosis, prognosis, course and outcomes of treatment, complications and errors in their care (Moskop).
Critics of the above approach say that it doesn't seem helpful (what is reasonable, anyway?), and instead propose that errors that don't reach the patient need not be disclosed. The argument supporting this is that in these cases, the "system" in place was robust enough such that the error was recognized and corrected before it reached the patient. In other words, the system worked and did what it was designed to do. If we acknowledge that human error is inevitable, and the systems humans work in should be constructed to minimize or altogether prevent those human errors from reaching the patient, then errors in the healthcare setting that don't reach the patient are exactly what is hoped for.
Beware of the position of "therapeutic privilege," which allows the physician to judge that the disclosure of certain information will in itself be highly detrimental to the patient. Before embarking on this route, one should have well-thought-out reasons that far outweigh the benefits of disclosure.
Beyond disclosing an error to the patient, is there any reason for reporting a medical error to other individuals?
If errors are not brought to the attention of a patient-safety committee or quality-assurance office, efforts to improve the safety of medical practice may be undermined. Quality-improvement techniques designed to prevent the recurrence of errors are thwarted when it is unknown if an error even occurred. Singer states that "the lesson to all of us is that we should learn to love mistakes because they carry in them the kernel of their own elimination."
How should we approach medical error in practice?
Growing evidence shows that patients prefer complete candor and want to be told about all errors that occur in their medical care (adverse events or not). Specifically in the pediatric setting, 99% of parents wanted to be told of the error in the care of their child, regardless of its severity (Hobgood). Other results from this study show that a parent's desire for disclosure does not differ by race, gender, age or insurance status. Also, as mentioned previously, parents are more likely to seek legal action if an error is not disclosed and they learn of its occurrence through other means.
Despite this evidence, and more showing similar results, physicians favor nondisclosure more often than do patients. As stated above, physicians cite a variety of misguided reasons for justifying nondisclosure, including the wish not to upset the patient, fear of disciplinary or legal action, or aversion to implicating colleagues. Some physicians state that they don't disclose errors because they simply don't know how (Hebert, Matlow, Hobgood, Sheldon).
Might it ever be appropriate to deceive a patient?
In the context of a medical error that reaches the patient, no. If the medical error does not reach the patient, as discussed earlier, it is acceptable not to divulge the fact that an error occurred. However, if asked about the error directly by the patient, disclosure, not deception, is encouraged.
In medicine in general, however, there are rare situations in which deception may be appropriate. One must always keep in mind the perspective of the person from whom information is withheld (Bok). Sheldon discusses the case of a physician who finds herself caring for a woman in very critical condition after a car accident that has killed one of the patient's four children and severely injured another. If the woman, in the course of the physician's attempts to stabilize her tenuous medical condition, asks about her children, it may be appropriate to evade a direct response and "never actually indicate that one child is dead" (Sheldon). This seems to be a case in which harm to the mother might be significant enough to warrant temporary deception. In this case, however, it is important to note that the physician fully intends to disclose the truth eventually but has chosen not to do so at this time. In other words, the physician has made a decision about the appropriate timing of the disclosure, rather than a decision not to disclose at all.
Is disclosure of the error enough? Do you have any other obligation to the family?
Disclosure is only the beginning. There are three parts to an ethical response to a mistake (Crigger):
Disclosure: Saying honestly and accurately what has happened
Apology: Taking responsibility for what happened and expressing regret (one does not have to admit fault to take responsibility and apologize)
Amends: Taking appropriate steps to minimize any harm done by the mistake
How might we disclose errors to patients?
Here are some guidelines (Matlow, Hebert):
Disclose promptly what you know about the event. Concentrate on what happened and the possible consequences.
Take the lead in disclosure; don't wait for the patient to ask.
Be prepared for strong emotions.
Accept responsibility for outcomes. Don't act defensively or evasively.
Apologize with a simple "I'm sorry." Patients often appreciate this form of acknowledgement and empathy, which can sometimes even strengthen your relationship with the family.
How do you think you will feel about your mistake?
It is normal to feel inadequacy, uncertainty, anger, humiliation and guilt after making a mistake. One also may be concerned about the many times in the future where there will be repeated opportunities to make the same mistake. These can all take a toll on one's emotional health and be exacerbated by the fact that the culture of medicine is still struggling with how to address these effects. In the 1980s, Hilfiker wrote that "there is no permission given to talk about errors, no way of venting emotional responses." In the 1990s, Leape echoed that concern: "Seldom is there a process to evaluate the circumstances of a mistake and to provide support and emotional healing for the fallible physician." In 2000, Wu described the physician who makes a mistake as the "second victim" because of the emotional trauma that results. Although the patient has long been recognized as the victim, this perspective helps us realize that most physicians also suffer - though in a different way - after making a mistake.
Errors should be seen as opportunities for growth and learning. It is important to recognize the need for support during these times, and to find trusted colleagues with whom you can discuss your response to what has happened.
Conclusion with Suggestions
There is an ethical imperative to disclose medical errors based on the principles of justice, nonmaleficence and respect for persons. In addition, many professional codes in medicine require disclosure. There is also empirical evidence that patients want to be told when an error has occurred in their care. All of these reasons create a firm obligation on the part of the physician for disclosure.
Despite the codes and principles above, there is little guidance as to how and when to report medical errors. Furthermore, there may even be medical errors that are acceptable not to disclose. For example, a good argument can be made that errors that do not reach the patient (e.g., an order for 10 times the dose is caught by the nurse or pharmacist) do not need to be disclosed to patients or their families. In those cases, the system worked, even if one part of that system failed. Because the system worked, the error did not reach the patient: the order was corrected before the wrong dose was given. Humans make mistakes, and physicians are humans; the system in which we work should have processes designed that make errors less likely, and when the system works to catch and correct errors, there is no need to disclose these "close calls" to families.
Making a mistake, particularly one that harms a patient, is often accompanied by emotional turmoil and feelings of guilt, inadequacy and shame. It is important to recognize the need for support during these times and to use errors as an opportunity for personal growth and learning.
The culture of medicine is slowly changing to incorporate systems thinking and trust in order to promote a blame- and shame-free environment for physicians.
When disclosing an error, be timely, tell the patient and family what happened, take responsibility and apologize.
1. Instructor's Guide 2. Student's Guide 3. Case Discussion
This instructor's guide was developed by Douglas J. Opel, MD, senior fellow, Clinical Bioethics, and Douglas S. Diekema, MD, MPH, director of education, Treuman Katz Center for Pediatric Bioethics, Seattle Children's Hospital.
In addition to the copyright notice set forth in the link below, permission to display, cache and print unlimited copies of the Case-Based Teaching Guides referred to on this page is hereby granted, solely for educational purposes, without charge (other than charges solely to cover the costs of copying), and without alteration of the Materials in any way.
Instructor's Guide: Truthfulness
Student's Guide: Truthfulness
Instructor's Guide (PDF)


The 20' Event Tent - Full Print provides a durable hand crank system, along with an easy to transport frame that simply pops up and puts you into business in just minutes. This full color dye-sublimated graphic allows you to be the star of the show during any outdoor fair, farmers market, trade show, or festival. Add weights for additional balance, or full graphic printed side walls for the extra exposure you deserve!


text:
Prairie's pane distrutto, which transforms stale bread into a thing of beauty. (Eric Wolfinger)
A Compelling View of Italy, from Anthony Strong's New Restaurant, Prairie
After more than a decade studying the rules of traditional Italian cuisine, chef Anthony Strong is beginning to bend them, deliciously.
Nick Czap
Considering how long and eagerly I'd been anticipating the debut of Anthony Strong's new restaurant, Prairie, I had surprisingly little in the way of preconceptions about the food. It would have something to do with Italy, I surmised, but beyond that I couldn't quite imagine what the dishes would look like, or the ingredients and flavors with which they might be laced.
About the only thing of which I was certain was that my expectations were high. Strong, after all, is a highly capable chef, a man who does his due diligence, and someone, as I would learn, who can turn something as unprepossessing as a hunk of stale bread into a thing of beauty.
Chef Anthony Strong. (Photo: Aubrie Pick)
I first met Strong in 2011 when preparing to review Locanda—Craig and Annie Stoll's newly opened Roman-inspired restaurant—where he had just begun his tenure as executive chef. Recently returned from a period of intensive work and study in Lazio, Strong came across as something of an autodidact in a conversation that turned from the first-century epicure Apicius, to the geographical nexus of garum production, to the challenges of reverse-engineering the recipe for carciofo alla giudia, a fried artichoke dish peculiar to the Roman Ghetto.
Struck by Strong's enthusiasm and depth of knowledge, I was equally taken by the food at Locanda, where, in the years that followed, I found myself going with compulsive regularity to lose myself in the flavors of the above-mentioned artichokes, whorls of tonnarelli cacio e pepe, and other delicacies. Meanwhile, as Strong continued to explore Italy's food traditions on his annual tours of duty in that country, he was gradually picking up on a curious trend.
"I've gone to Italy a dozen times," said Strong, "from a week to a couple of months, to dig in and cook and work. And I kept noticing there were Japanese stages in the kitchen." While working at an osteria in a town on the outskirts of Rome, he struck up a friendship with a fellow stage from Sapporo with whom he found himself swapping stories about Italian restaurants in the States and in Japan. "After work there was nothing to do," said Strong, "so we'd have a Peroni and talk about Italian food—his version, our version. I'm interested in how a cuisine translates to another region or culture, and it planted the seed for wanting to see Italian food outside of Italy and the U.S."
Strong's guanciale-wrapped mochi was inspired by a classic izakaya dish. (Photo: Aubrie Pick)
Toward the end of his term at Locanda and the Delfina group, Strong took a six-week sabbatical to see and taste for himself how chefs in Japan approached Italian cooking. Stylistically, he said, "the execution tends to be very meticulous, very disciplined. If there's one thing I noticed in Japan, it's that some of the Italian food is so perfectly made, it's kind of lacking that soul." At the same time, he was intrigued by dishes seasoned with traditional Japanese ingredients like kombu and various kinds of dried fish, whose rich, umami flavors are not unlike that of bottarga—the dried, salted roe typically of mullet or tuna—which is used as a condiment on pastas and in other dishes in Sardinia, Sicily, and elsewhere in Italy.
Intrigued by what he saw in Japan, and on subsequent travels in Europe, South America and Mexico, Strong began to envision a different approach to Italian cuisine—one that incorporated ingredients from other culinary traditions, while staying true to Italy's traditional flavors and sensibilities. Recalling his thought process at the time, Strong made an algebraic analogy. "X is sort of like Y," he said. "What if we tweak it a little bit, using tea leaves, or mochi wrapped in guanciale, an Italian take on a classic izakaya dish? I started to see that it was possible to make something that registered as very Italian, and at the same time open up the pantry a bit."
At the end of 2016, Strong left the Delfina group. After a dalliance with an experimental delivery-only restaurant called Young Fava—which, while successful, left him missing human contact—he organized a series of pop-up events at Local Mission Market, where he began testing recipes and a menu whose larger-format entrées lent themselves to sharing. By the time I went to one of his pop-ups—which I remember for an especially yummy squid ink spaghettini with uni—Strong already had his eye on a permanent space in the Mission, the soon-to-be-former home of Hog and Rocks, at the corner of 19th Street and San Carlos.
When I arrived at the appointed time on a mid-October evening, Prairie was packed to the gills. The restaurant—its name an homage to the landscape of Iowa, where Strong spent much of his childhood—had been open precisely six days. Word had gotten out, apparently. My always reliable friend, who had done such justice to the tasting menu at Eight Tables, joined me at a two-top so cozy with its neighbors it gave the proceedings a convivial, communal feel.
The cocktail list was refreshingly short and to the point. I ordered the Sparkling Negroni, bracingly strong due to the standard mix of gin, red vermouth and Campari, yet with a pleasingly round, fruity dimension thanks to an injection of house-carbonated Salice Salentino, a wine from an appellation near Lecce, in Puglia. The wine list, which Strong curates, is similarly compact, just 21 bottles, mostly Italian but with a handful from France. I began my explorations there with a Nebbiolo rosato—lightly floral on the nose with a lovely rose hip tang and an ever-so-slight effervescence—from the Piedmontese winery Ioppa.
The Prairie Sour. (Photo: Aubrie Pick)
Burrata with spring onion, a playful take on the flavors of sour cream and onion dip. (Photo: Aubrie Pick)
We proceeded to dig into a raft of appetizers, beginning with pane distrutto (literally, destroyed bread): giant croutons torn from a day-old levain batard, which, though drenched in olive oil and dunked in tomato pulp intensely seasoned with garlic and chiles, stayed improbably crispy from the first eye-opening bite to the last.
Shiro (white miso) might come as a surprise on an Italian menu, but Strong made it work, at least on the level of taste, by pureeing it with pine nuts and chickpeas into a creamy fluff garnished with a drizzle of urfa biber (a sweet-smoky Turkish pepper), bloomed in water and vinegar. While I found myself struggling somewhat to square miso with the notion of traditional Italian food ways— due to a certain psychological rigidity stemming from the still-fresh trauma of being reprimanded by an eight-year-old Sicilian boy for the transgression of embellishing a Caprese salad with figs—my taste buds had less difficulty homing in on the faint but persistent flavor of pinoli pervading the concoction, which we scooped up and devoured with tiny, halved cucumbers.
One should never say no to burrata, nor did we. Strong gets his from Belfiore in Berkeley, crushes it up with spring onions cooked in olive oil with lemon peel, adds a bit of crème fraîche, and garnishes it with olive oil and dried, powdered spring onion—the flavors of the crème fraîche and the allium a playfully subtle allusion to sour cream and onion dip. The dipping utensils in this case were wafer-thin crostini, toasted to a fare-thee-well on a mesquite-fired grill.
The menu is designed for sharing. (Photo: Aubrie Pick)
Aside from a sleek induction range, Prairie's kitchen is a wood-fired operation, the grill complemented by a futuristic-looking charcoal-burning oven made by the Barcelona-based company Josper. As a result, virtually every dish is infused with a smoky, barbecued deliciousness.
Case in point, a Piedmontese-esque dish of cauliflower, in which a whole head of the stuff is cooked in the Josper until charred on the outside, soft on the inside, then seasoned bagna cauda-style with an emulsion of cream, olive oil, garlic and anchovy, as well as shavings of cured bonito, a core ingredient in dashi, the soup stock that is one of the prime constituents of Japanese cooking. Never in my life had I encountered a cruciferous vegetable that read so much like a charbroiled steak, or that made me want to run out and drop fifteen grand on a Catalan kitchen appliance.
The restaurant's own house-cured guanciale, meanwhile, wrapped around fat little fingers of hot mochi, transported me right back to Emilia-Romagna, where, on a recent trip, every meal had begun with plates of the traditional fried bread, gnocco fritto, served piping hot and topped with mounds of Prosciutto di Parma.
Gulf shrimp and burrata tortelli, at Prairie. (Photo: Aubrie Pick)
From the menu's concise yet eclectic pasta section, we went with the Gulf shrimp and burrata tortelli, a variation on a dish I'd had several times at Locanda and was curious to experience in a new iteration. The tortelli were big and bold, real mouthfuls, stuffed with a mix of burrata, mozzarella, and delectably meaty shrimp, and dressed in a creamy-tangy-spicy sauce made of shrimp stock, fermented chiles, a little milk, and a splash of white wine.
Moving on to the mains, we continued in a maritime theme with a creation akin to moules marinières. The mussels—impeccably fresh and sweet little numbers from Saltspring Island in British Columbia—were steamed with prosecco rather than Breton cider, and mounted with butter seasoned with the beguiling spiciness of nasturtiums, the remnants of which we mopped up with more of those deliciously smoky crostini.
For the last savory dish, it was back to the land, or rather land with a hint of the sea. Among other things, Strong loves dim sum and all the seasonings that go along with it, including XO sauce. Invented in Hong Kong in the 1980s, and used in many different types of Chinese cooking, XO sauce is traditionally made from Jinhua ham, dried scallops, dried shrimp, chiles, garlic, ginger, soy sauce and oil. Strong imagined an Italian version in which he uses 'nduja, an ultra-soft and fatty Calabrian salami, in place of Jinhua ham; Calabrian chiles rather than standard-issue chile flakes; and a bit of anchovy in addition to the dried scallops and dried shrimp.
Given the way he rhapsodizes about this intensely savory-umami-spicy substance, I imagine you'll see it in all sorts of different dishes to come, but on this particular night it was slathered on some wonderfully flavorsome Berkshire spare ribs grilled to maximum juiciness, with an equally luscious side of grilled fennel and garnished, Roman-style, with a scattering of fresh mint.
With the possible exception of a single appetizer, the miso, which didn't quite fit in with my notions of what Italian food is or can be—perhaps a version of tortellini in brodo, made with a miso broth or dashi, would have been easier to wrap my head around—Strong's dishes were not only consistently, excitingly delicious, but possessed of a certain soundness as well.
Considering this, I recalled a conversation with Strong, in which we discussed the question of why, unlike their Italian counterparts who are for the most part tradition-bound and leery of novelty, so many American chefs seem driven to innovate.
"It's an interesting thing," said Strong. "We're the melting pot, right? It's the Wild West and anything goes. There aren't centuries of tradition that we feel beholden to, which is for better or for worse. I think that cooking in America can be such a free-for-all that it can be disjointed at times. It's important to me to go to the source and dig into things as much as possible. That's why I spent so much time in Italy, and that's why I'm not going to start throwing around ingredients from all different cultures without knowing why. The creative aspect is so important, but it also comes with a responsibility to not lose sight of all the traditional things that are beautiful in their own right."
Strong designed Prairie's dining room, whose walls are finished with an indigo dye known in Japan as aizome. (Photo: Aubrie Pick)
// Prairie, 3431 19th St. (Mission), prairiesf.com
At Prairie, Anthony Strong hopes that the perfect Italian dish is ... ›
PRAIRIE Restaurant - San Francisco, CA | OpenTable ›
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There is an article in yesterday's Irish Times on the Large Hadron Collider (LHC), the massive new particle accelerator at CERN, the European Organisation for Nuclear Research.
Unfortunately, journalist Derek Scally repeats the concern that the experiments might create a giant black hole that will swallow the earth, a concern that is completely unfounded.
More sensibly, Mr Scally points out that the participation of Irish scientists in the historic LHC experiments will be severely limited by the fact that Ireland, almost uniquely among 20 major European nations, is not a member of CERN. For decades now, Ireland has baulked at joining this most successful of European scientific collaborations. The omission has decimated Irish research in elementary particle physics, a field of fundamental importance in science, and sits awkwardly with our efforts to become a world leader in science and technology. It has also cost us financially, with Irish engineers and scientists unable to bid for large international contracts in high-tech software and hardware projects.
All this from a country whose only Nobel Prize in science was for splitting the atom!
Nice website – is the comment section activated?
Hi Cormac, everything looks well, keep up the good work.
Great site Cormac, very impressed with the topics that are included.
Thank goodness some bloggers can write. My thanks for this post..


One of my favorite places to find nail polish is at Forever 21 stores. On a recent trip I found Lavender, which is a jelly that builds in 2-3 coats.
It applied like butter, no streaks or gooping. I think it'd be awesome to use for a jelly sammich sometime!
What's your favorite place to find polish?


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Place milk and oats in a saucepan and bring to the boil.
Simmer for 6 minutes until thick and creamy.
Mix in Sweet Freedom Choc Shot Orange Spice.
Top with grated chocolate and clementine slices.
Thank you Libby @babytoddlerfoods for a delicious recipe.


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ARIZONA: Republican Lawmaker Intros $20 Internet Porn Tax With Proceeds To Fund Trump's Border Wall
January 23, 2019	Crackpots, Politics
The Arizona Mirror reports:
A GOP lawmaker has a novel idea for how a border wall with Mexico should be funded: Force every Arizonan who wants to access pornographic material on an electronic device to pay $20.
Republican Rep. Gail Griffin, R-Hereford, has introduced House Bill 2444, which would make "distributors" of devices that allow access to the internet install software to make the offending material not viewable. To remove the blocking software, a person would have to pay the state $20.
Those distributors, which include both manufacturers and retailers, would also be able to impose their own fees for removing the blocker. "It's pretty clearly unconstitutional," said Mike Stabile, a spokesman for the Free Speech Coalition. The Free Speech Coalition is a non-profit that lobbies on behalf of the adult entertainment industry.
Republicans in more than a dozen states have previously floated similar "porn tax" bills, but this is the first that would direct the proceeds to a border wall.
Griffin's bill, you'll be shocked to learn, is the work of notorious nutjob Chris Sevier, who has sued to end same-sex marriage in multiple states by claiming that he has the right to marry his computer. Earlier this month Sevier lost a Texas lawsuit to end Drag Queen Story Hour at a public library.
Tags Arizona border wall Chris Sevier GOP porn
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Editor's note: The author's family lived in several different Afghan provinces where schools were closed while she was growing up during the Taliban years. In 2016, schools in many provinces of Afghanistan are now closed again.
This is a wonderful poem that speaks to how powerful the desire to learn is in you and others. It is distressing that schools are closing again but I hope that you will continue to find ways to learn amidst the increasing threat from ISIS and Taliban. Thank you for writing this.
Thank you for your encouragement words and sharing my poem with your wonderful students.
Hello, Sharifa! Your poem makes me think of how too many in North America take even basic education for granted – or even act as if they could live well without it. If only we all could love learning as you do (and your peers, I am sure).
I hope you can continue your education. Everyone deserves one. Remember: the pen is mightier than the sword.
This poem really reminds me how much people go through in the world. This is such a beautiful poem and I really enjoyed it.
The love of learning and the resilience in getting an education that you write about in this poem is inspiring. I feel as though my own education (and the many opportunities I have been fortunate to be blessed with) is put into perspective, and has made me appreciate it even more. Thank you for sharing this poem with us all.
Dear Sharifa, thank you for sharing this beautiful poem. I admire your dedication to learning all the lessons life has to offer and am inspired by your courage to not let the challenges you have faced define you or prevent you from fighting for your right to receive an education.
The love and desire you have to learn is evident in this poem. It is a beautiful thing to read.
Never let your pursuit of knowledge falter. You poems are beautiful, and speak to my soul. We are worlds apart, but in this poem, I see you.
Your words are quintessential, Sharifa. Your pursuit of knowledge serves as a great inspiration. Never stop filling your cup. Lead by example. Thank you for the outstanding poetry.
This was an interesting poem. It really shows the harshness and brutality of life and how unfair it is for you to get what you really want. An education is something everyone deserves and the fact that someone is taking away your opportunity is just egregious.
Sharifa, I am truly sad that you have had to go through such horrifying experiences in your life. I am also sorry that, your right to an education is being unjustly taken away from you. On another note, your poem was beautiful and very powerful. Your poem, brought out the beauty of education, while still bringing out the horrors of having taken away. I hope that one day, everyone around the world will be able to get a quality education, despite the conditions that exist in their environment. I wish you the best of luck, and hope that you can continue your educational journey.


Poultry Producers: Example of an incinerator that a producer installed at a broiler farm. This incinerator was sized for the operation by our NRCS Technician Alan McGhee. The producer participated in our EQIP program.
Individuals & other entities engaged in livestock and/or crop production are eligible to participate in EQIP. Eligible land includes cropland, pasture, private non-industrial forest land, and other farm/ranch lands. In Arkansas, EQIP is specifically designed to address the resource concerns of water quality issues related to animal manure management & sediment; improved management of irrigation water and reduction in ground water use; reduction of erosion; and improvement of wildlife habitat. All applications will be evaluated for funding based on a state developed procedure to optimize environmental benefits. Applications ranking highest in a funding category will be funded subject to availablity. Our office accepts applications year around.


Dennis is ISA's newest member, and is presently an instructor candidate.
A Dignitary Protection Specialist fully accredited by the Virginia Department of Criminal Justice, he holds the Personal Protection Specialist (PPS) credential and is pending his private investigators license in Maryland.
Dennis is an active EP agent who has served as a detail member, driver, and team leader for celebrity's and diplomats.
Dennis is a United States Marine Corp veteran with ten years of honorable service, and is presently completing a business management degree with Prince George's Community College.
Dennis speaks English and Arabic.