Children with 22q11.2 deletion syndrome (22q11DS) are at risk for social-behavioural and neurocognitive sequelae throughout development. The current study examined the impact of family environmental characteristics on social-behavioural and cognitive outcomes in this pediatric population.
Guardians of children with 22q11DS were recruited through two medical genetics clinics. Con senting guardians were asked to complete several questionnaires regarding their child's social, emotional and behavioural functioning, as well as family social environment and parenting styles. Children with 22q11DS were asked to undergo a cognitive assessment, including IQ and achievement testing, and measures of attention, executive function and memory.
Modest associations were found between aspects of the family social environment and parenting styles with social-behavioural and cognitive/academic outcomes. Regression models indicated that physical punishment, socioeconomic status, parental control and family organisation significantly predicted social-behavioural and cognitive outcomes in children with 22q11DS.
Characteristics of the family social environment and parenting approaches appear to be associated with functional outcomes of children with 22q11DS. Understanding the impact of environmental variables on developmental outcomes can be useful in determining more effective targets for intervention. This will be important in order to improve the quality of life of individuals affected by 22q11DS.
chromosome 22q11.2 deletion syndrome; DiGeorge syndrome; family environment; parenting; social-behavioural functioning; velocardiofacial syndrome
High throughput screening technologies such as acoustic droplet ejection (ADE) greatly increase the rate at which X-ray diffraction data can be acquired from crystals. One promising high throughput screening application of ADE is to rapidly combine protein crystals with fragment libraries. In this approach, each fragment soaks into a protein crystal either directly on data collection media or on a moving conveyor belt which then delivers the crystals to the X-ray beam. By simultaneously handling multiple crystals combined with fragment specimens, these techniques relax the automounter duty-cycle bottleneck that currently prevents optimal exploitation of third generation synchrotrons. Two factors limit the speed and scope of projects that are suitable for fragment screening using techniques such as ADE. Firstly, in applications where the high throughput screening apparatus is located inside the X-ray station (such as the conveyor belt system described above), the speed of data acquisition is limited by the time required for each fragment to soak into its protein crystal. Secondly, in applications where crystals are combined with fragments directly on data acquisition media (including both of the ADE methods described above), the maximum time that fragments have to soak into crystals is limited by evaporative dehydration of the protein crystals during the fragment soak. Here we demonstrate that both of these problems can be minimized by using small crystals, because the soak time required for a fragment hit to attain high occupancy depends approximately linearly on crystal size.
Bisphosphonates reduce skeletal loss and fracture risk, but their use has been limited in patients with chronic kidney disease. This study shows skeletal benefits of zoledronic acid in an animal model of chronic kidney disease.
Bisphosphonates are routinely used to reduce fractures but limited data exists concerning their efficacy in non-dialysis chronic kidney disease. The goal of this study was to test the hypothesis that zoledronic acid produces similar skeletal effects in normal animals and those with kidney disease.
At 25 weeks of age, normal rats were treated with a single dose of saline vehicle or 100 µg/kg of zoledronic acid while animals with kidney disease (approximately 30 % of normal kidney function) were treated with vehicle, low dose (20 µg/kg), or high dose (100 µg/kg) zoledronic acid, or calcium gluconate (3 % in the drinking water). Skeletal properties were assessed 5 weeks later using micro-computed tomography, dynamic histomorphometry, and mechanical testing.
Animals with kidney disease had significantly higher trabecular bone remodeling compared to normal animals. Zoledronic acid significantly suppressed remodeling in both normal and diseased animals yet the remodeling response to zoledronic acid was no different in normal and animals with kidney disease. Animals with kidney disease had significantly lower cortical bone biomechanical properties; these were partially normalized by treatment.
Based on these results, we conclude that zoledronic acid produces similar amounts of remodeling suppression in animals with high turnover kidney disease as it does in normal animals, and has positive effects on select biomechanical properties that are similar in normal animals and those with chronic kidney disease.
Anti-remodeling agents; Bisphosphonate; Bone mechanics; Remodeling suppression
Effects of context on the perception of, and incidental memory for, real-world objects have predominantly been investigated in younger individuals, under conditions involving a single static viewpoint. We examined the effects of prior object context and object familiarity on both older and younger adults’ incidental memory for real objects encountered while they traversed a conference room. Recognition memory for context-typical and context-atypical objects was compared with a third group of unfamiliar objects that were not readily named and that had no strongly associated context. Both older and younger adults demonstrated a typicality effect, showing significantly lower 2-alternative-forced-choice recognition of context-typical than context-atypical objects; for these objects, the recognition of older adults either significantly exceeded, or numerically surpassed, that of younger adults. Testing-awareness elevated recognition but did not interact with age or with object type. Older adults showed significantly higher recognition for context-atypical objects than for unfamiliar objects that had no prior strongly associated context. The observation of a typicality effect in both age groups is consistent with preserved semantic schemata processing in aging. The incidental recognition advantage of older over younger adults for the context-typical and context-atypical objects may reflect aging-related differences in goal-related processing, with older adults under comparatively more novel circumstances being more likely to direct their attention to the external environment, or age-related differences in top-down effortful distraction regulation, with older individuals’ attention more readily captured by salient objects in the environment. Older adults’ reduced recognition of unfamiliar objects compared to context-atypical objects may reflect possible age differences in contextually driven expectancy violations. The latter finding underscores the theoretical and methodological value of including a third type of objects–that are comparatively neutral with respect to their contextual associations–to help differentiate between contextual integration effects (for schema-consistent objects) and expectancy violations (for schema-inconsistent objects).
To examine whether interference with FRNK targeting to focal adhesions (FAs) affects its inhibitory activity and tyrosine phosphorylation.
Methods and Results
Focal adhesion kinase and its autonomously expressed C-terminal inhibitor, focal adhesion kinase–related nonkinase (FRNK), regulate vascular smooth muscle cell (VSMC) signaling and migration. FRNK-paxillin binding was reduced by a point mutation in its FA targeting domain (L341S-FRNK). Green fluorescent protein–tagged wild type and L341S-FRNK were then adenovirally expressed in VSMCs. L341S-FRNK targeted to VSMC FAs, despite previous studies in other cell types. L341S-FRNK affected FA binding kinetics (assessed by total internal reflection fluorescnece [TIRF] microscopy and fluorescence recovery after photobleaching [FRAP]) and reduced its steady-state paxillin interaction (determined by coimmunoprecipitation). Both wt-FRNK and L341S-FRNK lowered basal and angiotensin II–stimulated focal adhesion kinase, paxillin, and extracellular signal–regulated kinase 1/2 phosphorylation. However, the degree of inhibition was significantly reduced by L341S-FRNK. L341S-FRNK also demonstrated significantly greater migratory activity compared with wt-FRNK–expressing VSMCs. Angiotensin II–induced Y168 phosphorylation was Src dependent, as evident by a significant reduction in Y168 phosphorylation by the Src family kinase inhibitor PP2 is 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Surprisingly, Y168 phosphorylation was unaffected by its targeting. Furthermore, Y232 phosphorylation increased approximately 3-fold in L341S-FRNK, which was less sensitive to PP2.
FRNK inhibition of VSMC migration requires both FA targeting and Y168 phosphorylation by Src family kinases. FRNK-Y232 phosphorylation occurs outside of FAs, probably by a PP2-insensitive kinase.
vascular biology; vascular muscle; Src; cell migration; mechanotransduction; tyrosine phosphorylation; vascular remodeling
The most accepted means of evaluating the response of a patient with cervical spondylomyelopathy (CSM) to treatment is subjective and based on the owner and clinician's perception of the gait.
To establish and compare kinetic parameters based on force plate gait analysis between normal and CSM-affected Dobermans.
Nineteen Doberman Pinschers: 10 clinically normal and 9 with CSM.
Force plate analysis was prospectively performed in all dogs. At least 4 runs of ipsilateral limbs were collected from each dog. Eight force platform parameters were evaluated, including peak vertical force (PVF) and peak vertical impulse (PVI), peak mediolateral force (PMLF) and peak mediolateral impulse, peak braking force and peak braking impulse, and peak propulsive force (PPF) and peak propulsive impulse. In addition, the coefficient of variation (CV) for each limb was calculated for each parameter. Data analysis was performed by a repeated measures approach.
PMLF (P = .0062), PVI (P = .0225), and PPF (P = .0408) were found to be lower in CSM-affected dogs compared with normal dogs. Analysis by CV as the outcome indicated more variability in PVF in CSM-affected dogs (P = 0.0045). The largest difference in the CV of PVF was seen in the thoracic limbs of affected dogs when compared with the thoracic limbs of normal dogs (P = 0.0019).
Conclusions and Clinical Importance
The CV of PVF in all 4 limbs, especially the thoracic limbs, distinguished clinically normal Dobermans from those with CSM. Other kinetic parameters less reliably distinguished CSM-affected from clinically normal Dobermans.
Cervical vertebral instability; Dog; Kinetic; Wobbler
A method is presented for screening fragment libraries using acoustic droplet ejection to co-crystallize proteins and chemicals directly on micromeshes with as little as 2.5 nl of each component. This method was used to identify previously unreported fragments that bind to lysozyme, thermolysin, and trypsin.
Acoustic droplet ejection (ADE) is a powerful technology that supports crystallographic applications such as growing, improving and manipulating protein crystals. A fragment-screening strategy is described that uses ADE to co-crystallize proteins with fragment libraries directly on MiTeGen MicroMeshes. Co-crystallization trials can be prepared rapidly and economically. The high speed of specimen preparation and the low consumption of fragment and protein allow the use of individual rather than pooled fragments. The Echo 550 liquid-handling instrument (Labcyte Inc., Sunnyvale, California, USA) generates droplets with accurate trajectories, which allows multiple co-crystallization experiments to be discretely positioned on a single data-collection micromesh. This accuracy also allows all components to be transferred through small apertures. Consequently, the crystallization tray is in equilibrium with the reservoir before, during and after the transfer of protein, precipitant and fragment to the micromesh on which crystallization will occur. This strict control of the specimen environment means that the crystallography experiments remain identical as the working volumes are decreased from the few microlitres level to the few nanolitres level. Using this system, lysozyme, thermolysin, trypsin and stachydrine demethylase crystals were co-crystallized with a small 33-compound mini-library to search for fragment hits. This technology pushes towards a much faster, more automated and more flexible strategy for structure-based drug discovery using as little as 2.5 nl of each major component.
in situ X-ray data collection; acoustic droplet ejection; fragment screening; drug discovery; chemical biology; protein crystallization; synchrotron radiation
Increased sexual risk behaviour in participants enrolled in HIV prevention trials has been a concern. The HVTN 503/Phambili study, a phase 2B study of the Merck Ad-5 multiclade HIV vaccine in South Africa, suspended enrollment and vaccinations following the results of the Step study. Participants were notified of their treatment allocation and continue to be followed. We investigated changes in risk behaviour over time and assessed the impact of study unblinding.
801 participants were enrolled. Risk behaviors were assessed with an interviewer-administered questionnaire at 6-month intervals. We assessed change from enrolment to the first 6-month assessment pre-unblinding and between enrolment and at least 6 months post-unblinding on all participants with comparable data. A one-time unblinding risk perception questionnaire was administered post-unblinding.
A decrease in participants reporting unprotected sex was observed in both measured time periods for men and women, with no differences by treatment arm. At 6 months (pre-unblinding), 29.6% of men and 35.8% of women reported changing from unprotected to protected sex (p <0.0001 for each).Men (22%) were more likely than women (14%) to report behavior change after unblinding (p=0.009). Post-enrolment, 142 (45%) of 313 previously uncircumcised men underwent medical circumcision.
663 participants completed the unblinding questionnaire. More vaccine (24.6%) as compared to placebo recipients (12.0%) agreed that they were more likely to get HIV than most people (p<0.0001), and attributed this to receiving the vaccine.
We did not find evidence of risk compensation during this clinical trial. Some risk behaviour reductions including male circumcision were noted irrespective of treatment allocation.
A description of the upgraded beamline X25 at the NSLS, operated by the PXRR and the Photon Sciences Directorate serving the Macromolecular Crystallography community, is presented.
Beamline X25 at the NSLS is one of the five beamlines dedicated to macromolecular crystallography operated by the Brookhaven National Laboratory Macromolecular Crystallography Research Resource group. This mini-gap insertion-device beamline has seen constant upgrades for the last seven years in order to achieve mini-beam capability down to 20 µm × 20 µm. All major components beginning with the radiation source, and continuing along the beamline and its experimental hutch, have changed to produce a state-of-the-art facility for the scientific community.
beamline; mini-κ; Pilatus 6M; PXRR; macromolecular crystallography; wBPM
Heart failure (HF) is associated with excessive extracellular matrix (ECM) deposition and abnormal ECM degradation leading to cardiac fibrosis. Connective Tissue Growth Factor (CTGF) modulates ECM production during inflammatory tissue injury, but available data on CTGF gene expression in failing human heart and its response to mechanical unloading are limited.
Methods and Results
LV tissue from patients undergoing cardiac transplantation for ischemic (ICM; n=20) and dilated (DCM; n=20) cardiomyopathies, and from nonfailing (NF; n=20) donor hearts were examined. Paired samples (n=15) from patients undergoing LV assist device (LVAD) implantation as “bridge to transplant” (34-1145 days) were also analyzed. There was more interstitial fibrosis in both ICM and DCM compared to NF hearts. Hydroxyproline concentration was also significantly increased in DCM relative to NF samples. The expression of CTGF,TGFB1, COL1-A1, COL3-A1, MMP2 and MMP9 mRNAs in ICM and DCM were also significantly elevated as compared to NF controls. Although TGFB1, CTGF, COL1-A1, and COL3-A1 mRNA levels were reduced by unloading, there was only a modest reduction in tissue fibrosis and no difference in protein-bound hydroxyproline concentration between pre- and post-LVAD tissue samples. The persistent fibrosis may be related to a concomitant reduction in MMP9 mRNA and protein levels following unloading.
CTGF may be a key regulator of fibrosis during maladaptive remodeling and progression to HF. Although mechanical unloading normalizes most genotypic and functional abnormalities, its effect on ECM remodeling during HF is incomplete.
Remodeling; Heart-assist device; Gene expression; collagens
The explosion of the Deepwater Horizon drilling platform created the largest marine oil spill in U.S. history. As part of the Natural Resource Damage Assessment process, we applied an innovative modeling approach to obtain upper estimates for occupancy and for number of manatees in areas potentially affected by the oil spill. Our data consisted of aerial survey counts in waters of the Florida Panhandle, Alabama and Mississippi. Our method, which uses a Bayesian approach, allows for the propagation of uncertainty associated with estimates from empirical data and from the published literature. We illustrate that it is possible to derive estimates of occupancy rate and upper estimates of the number of manatees present at the time of sampling, even when no manatees were observed in our sampled plots during surveys. We estimated that fewer than 2.4% of potentially affected manatee habitat in our Florida study area may have been occupied by manatees. The upper estimate for the number of manatees present in potentially impacted areas (within our study area) was estimated with our model to be 74 (95%CI 46 to 107). This upper estimate for the number of manatees was conditioned on the upper 95%CI value of the occupancy rate. In other words, based on our estimates, it is highly probable that there were 107 or fewer manatees in our study area during the time of our surveys. Because our analyses apply to habitats considered likely manatee habitats, our inference is restricted to these sites and to the time frame of our surveys. Given that manatees may be hard to see during aerial surveys, it was important to account for imperfect detection. The approach that we described can be useful for determining the best allocation of resources for monitoring and conservation.
We used satellite telemetry to study behavior at foraging sites of 40 adult female loggerhead sea turtles (Caretta caretta) from three Florida (USA) rookeries. Foraging sites were located in four countries (USA, Mexico, the Bahamas, and Cuba). We were able to determine home range for 32 of the loggerheads. One turtle moved through several temporary residence areas, but the rest had a primary residence area in which they spent all or most of their time (usually >11 months per year). Twenty-four had a primary residence area that was <500 km2 (mean = 191). Seven had a primary residence area that was ≥500 km2 (range = 573–1,907). Primary residence areas were mostly restricted to depths <100 m. Loggerheads appeared to favor areas with larger-grained sediment (gravel and rock) over areas with smaller-grained sediment (mud). Short-term departures from primary residence areas were either looping excursions, typically involving 1–2 weeks of continuous travel, or movement to a secondary residence area where turtles spent 25–45 days before returning to their primary residence area. Ten turtles had a secondary residence area, and six used it as an overwintering site. For those six turtles, the primary residence area was in shallow water (<17 m) in the northern half of the Gulf of Mexico (GOM), and overwintering sites were farther offshore or farther south. We documented long winter dive times (>4 h) for the first time in the GOM. Characterizing behaviors at foraging sites helps inform and assess loggerhead recovery efforts.
Previous work has shown that the effect of opioid-receptor blockade on memory modulation is critically dependent upon the intensity of stress. The current study determined the effect of adrenergic-receptor blockade on memory modulation under varied levels of stress and then compared the effect of adrenergic-receptor blockade under intense stress to that of a) opioid-receptor blockade and b) concurrent opioid- and adrenergic-receptor blockade. In the first experiment, the β-adrenergic-receptor blocker propranolol impaired retention in the passive-avoidance procedure when administered immediately after exposure to intense stress (passive-avoidance training followed by swim stress) but not mild stress (passive-avoidance training alone). In the second experiment, while separate administration of either propranolol or the opioid-receptor blocker naloxone immediately after exposure to intense stress impaired retention, the combined administration of propranolol and naloxone failed to do so. These findings demonstrate that the effect of β-adrenergic-receptor blockade or opioid-receptor blockade on memory modulation in the passive-avoidance procedure is dependent upon the intensity of stress, and suggest that concurrent inactivation of endogenous adrenergic- and opioid-based memory modulation systems under stressful conditions is protective of memory.
Propranolol; Naloxone; Forced swim; Passive-avoidance training; Stress; Memory modulation
The opiate-receptor antagonist naloxone was administered to rats after passive-avoidance training either alone or in combination with forced-swim stress. A retention test revealed that while naloxone enhanced retention when administered alone, it impaired retention when administered in combination with forced-swim stress. The findings provide evidence for a “protective” endogenous opioid-based system that, when not blocked pharmacologically, limits enhancement or impairment of retention under conditions of mild and intense stress, respectively.
Naloxone; Memory modulation; Opioid; Stress; Forced-swim; Adrenergic
Multiple endocrine neoplasia type 1 (MEN1) is an autosomal syndrome caused by mutations in the MEN1 tumor suppressor gene. While the protein product of MEN1, menin, is ubiquitously expressed, somatic loss of the remaining wildtype MEN1 allele results in tumors primarily in parathyroid, pituitary, and endocrine pancreas. To understand the endocrine specificity of the MEN1 syndrome, we evaluated biallelic loss of Men1 by inactivating Men1 in pancreatic progenitor cells utilizing the Cre-lox system. Men1 deletion in progenitor cells that differentiate into exocrine and endocrine pancreas did not affect normal pancreas morphogenesis and development. However, mice having homozygous inactivation of the Men1 in pancreas developed endocrine tumors with no exocrine tumor manifestation, recapitulating phenotypes seen in the MEN1 patients. In the absence of menin, the endocrine pancreas showed increase in cell proliferation, vascularity and abnormal vascular structures; such changes were lacking in exocrine pancreas. Further analysis revealed that these endocrine manifestations were associated with upregulation in VEGF expression in both human and mouse MEN1 pancreatic endocrine tumors. Together these data suggest the presence of cell-specific factors for menin and a permissive endocrine environment for MEN1 tumorigenesis in endocrine pancreas. Based on our analysis, we propose that menin’s ability to maintain cellular and microenvironment integrity might explain the endocrine restrictive nature of the MEN1 syndrome.
MEN1; pancreatic neuroendocrine tumor; VEGF; tumor suppressor gene; Pdx1
Cardiac muscle cells are known to adapt to their physical surroundings, optimizing intracellular organization and contractile function for a given culture environment. A previously developed in vitro model system has shown that the inclusion of discrete microscale domains (or microrods) in three dimensions (3D) can alter long-term growth responses of neonatal ventricular myocytes. The aim of this work was to understand how cellular contact with such a domain affects various mechanical changes involved in cardiac muscle cell remodeling. Myocytes were maintained in 3D gels over 5 days in the presence or absence of 100 – μm-long microrods, and the effect of this local heterogeneity on cell behavior was analyzed via several imaging techniques. Microrod abutment resulted in approximately twofold increases in the maximum displacement of spontaneously beating myocytes, as based on confocal microscopy scans of the gel xy-plane or the myocyte long axis. In addition, microrods caused significant increases in the proportion of aligned myofibrils (≤20° deviation from long axis) in fixed myocytes. Microrod-related differences in axial contraction could be abrogated by long-term interruption of certain signals of the RhoA-/Rho-associated kinase (ROCK) or protein kinase C (PKC) pathway. Furthermore, microrod-induced increases in myocyte size and protein content were prevented by ROCK inhibition. In all, the data suggest that microdomain heterogeneity in 3D appears to promote the development of axially aligned contractile machinery in muscle cells, an observation that may have relevance to a number of cardiac tissue engineering interventions.
Mechanobiology; Muscle hypertrophy; Digital image correlation; Microenvironment; Finite element; Mechanotransduction
Iron deposition has been shown to occur following myocardial infarction (MI). We investigated whether such focal iron deposition within chronic MI lead to electrical anomalies.
Two groups of dogs (ex-vivo (n = 12) and in-vivo (n = 10)) were studied at 16 weeks post MI. Hearts of animals from ex-vivo group were explanted and sectioned into infarcted and non-infarcted segments. Impedance spectroscopy was used to derive electrical permittivity () and conductivity (). Mass spectrometry was used to classify and characterize tissue sections with (IRON+) and without (IRON-) iron. Animals from in-vivo group underwent cardiac magnetic resonance imaging (CMR) for estimation of scar volume (late-gadolinium enhancement, LGE) and iron deposition (T2*) relative to left-ventricular volume. 24-hour electrocardiogram recordings were obtained and used to examine Heart Rate (HR), QT interval (QT), QT corrected for HR (QTc) and QTc dispersion (QTcd). In a fraction of these animals (n = 5), ultra-high resolution electroanatomical mapping (EAM) was performed, co-registered with LGE and T2* CMR and were used to characterize the spatial locations of isolated late potentials (ILPs).
Compared to IRON- sections, IRON+ sections had higher, but no difference in. A linear relationship was found between iron content and (p<0.001), but not (p = 0.34). Among two groups of animals (Iron (<1.5%) and Iron (>1.5%)) with similar scar volumes (7.28%±1.02% (Iron (<1.5%)) vs 8.35%±2.98% (Iron (>1.5%)), p = 0.51) but markedly different iron volumes (1.12%±0.64% (Iron (<1.5%)) vs 2.47%±0.64% (Iron (>1.5%)), p = 0.02), QT and QTc were elevated and QTcd was decreased in the group with the higher iron volume during the day, night and 24-hour period (p<0.05). EAMs co-registered with CMR images showed a greater tendency for ILPs to emerge from scar regions with iron versus without iron.
The electrical behavior of infarcted hearts with iron appears to be different from those without iron. Iron within infarcted zones may evolve as an arrhythmogenic substrate in the post MI period.
Intracortical microstructure influences crack propagation and arrest within bone cortex. Genetic variation in intracortical remodeling may contribute to mechanical integrity and, therefore, fracture risk. Our aim was to determine the degree to which normal population-level variation in intracortical microstructure is due to genetic variation. We examined right femurs from 101 baboons (74 females, 27 males; aged 7–33 years) from a single, extended pedigree to determine osteon number, osteon area (On.Ar), haversian canal area, osteon population density, percent osteonal bone (%On.B), wall thickness (W.Th), and cortical porosity (Ct.Po). Through evaluation of the covariance in intracortical properties between pairs of relatives, we quantified the contribution of additive genetic effects (heritability [h2]) to variation in these traits using a variance decomposition approach. Significant age and sex effects account for 9 % (Ct.Po) to 21 % (W.Th) of intracortical microstructural variation. After accounting for age and sex, significant genetic effects are evident for On.Ar (h2 = 0.79, p = 0.002), %On.B (h2 = 0.82, p = 0.003), and W.Th (h2 = 0.61, p = 0.013), indicating that 61–82 % of the residual variation (after accounting for age and sex effects) is due to additive genetic effects. This corresponds to 48–75 % of the total phenotypic variance. Our results demonstrate that normal, population-level variation in cortical microstructure is significantly influenced by genes. As a critical mediator of crack behavior in bone cortex, intracortical microstructural variation provides another mechanism through which genetic variation may affect fracture risk.
Primate; Osteoporosis; Biomechanics; Population studies; Bone histomorphometry
On the basis of positive preclinical data, we evaluated the safety and immunogenicity of an alphavirus replicon HIV-1 subtype C gag vaccine (AVX101), expressing a nonmyristoylated form of Gag, in two double-blind, randomized, placebo-controlled clinical trials in healthy HIV-1-uninfected adults. Escalating doses of AVX101 or placebo were administered subcutaneously to participants in the United States and Southern Africa. Because of vaccine stability issues, the first trial was halted prior to completion of all dose levels and a second trial was implemented. The second trial was also stopped prematurely due to documentation issues with the contract manufacturer. Safety and immunogenicity were evaluated through assessments of reactogenicity, reports of adverse events, and assessment of replication-competent and Venezuelan equine encephalitis (VEE) viremia. Immunogenicity was measured using the following assays: enzyme-linked immunosorbent assay (ELISA), chromium 51 (51Cr)-release cytotoxic T lymphocyte (CTL), gamma interferon (IFN-γ) ELISpot, intracellular cytokine staining (ICS), and lymphoproliferation assay (LPA). Anti-vector antibodies were also measured. AVX101 was well tolerated and exhibited only modest local reactogenicity. There were 5 serious adverse events reported during the trials; none were considered related to the study vaccine. In contrast to the preclinical data, immune responses in humans were limited. Only low levels of binding antibodies and T-cell responses were seen at the highest doses. This trial also highlighted the difficulties in developing a novel vector for HIV.
Previous studies of tactile acuity on the fingertip using passive touch have demonstrated an age-related decline in spatial resolution for both sighted and blind subjects. We have re-examined this age dependence with two newly designed tactile-acuity charts requiring active exploration of the test symbols. One chart used dot patterns similar to Braille and the other used embossed Landolt rings. Groups of blind Braille readers and sighted subjects, ranging in age from 12 to 85 years, were tested in two experiments. We replicated previous findings for sighted subjects by showing an age related decrease in tactile acuity by nearly 1% per year. Surprisingly, the blind subjects retained high acuity into old age showing no age-related decline. For the blind subjects, tactile acuity did not correlate with braille reading speed, the amount of daily reading, or the age at which braille was learned. We conclude that when measured with active touch, blind subjects retain high tactile acuity into old age, unlike their aging sighted peers. We propose that blind people's use of active touch in daily activities, not specifically Braille reading, results in preservation of tactile acuity across the lifespan.
touch; tactile perception; tactile acuity; aging; blindness; impaired vision
Glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) and its paralogues were implicated in late-onset Alzheimer’s disease (LOAD), although the strength and direction of association have not been consistent. We genotyped three previously reported SNPs (rs3741916-GAPDH 5’UTR, rs2029721-pGAPD and rs4806173-GAPDHS) in three case-control series (2112 cases and 3808 controls). Rs3741916 showed the strongest LOAD association (p=0.003). The minor allele of rs3741916 showed a protective effect in our combined series (OR=0.87, 95% confidence interval (CI)=0.79–0.96). This is consistent with results from the two published follow-up studies and in opposite direction of the original report. Meta-analysis of the published series with ours suggests presence of heterogeneity (Breslow-Day p<0.0001). Meta-analysis of only the follow-up series including ours revealed a significant protective effect for the minor allele of rs3741916 (OR=0.85, 95% CI=0.76–0.96, p=0.009). Our results support the presence of LOAD variants and heterogeneity at the GAPDH locus. The most promising rs3741916 variant is unlikely to be functional given opposing effects in different series. Identification of functional variant(s) in this region likely awaits deep sequencing.
Alzheimer's disease; Association studies in genetics; Case control studies
Background: Talin is an integrin-actin linker essential for integrin activation.
Results: Talin1 has distinct developmental and postnatal expression in heart versus Talin2. Cardiac-myocyte specific Talin1 deletion alters physiological and molecular responses of the myocardium to stress.
Conclusion: Talin1 has a unique mechanotransductive role in the cardiomyocyte.
Significance: Reduction of talin1 in cardiomyocytes may have beneficial effects in the stressed myocardium.
Integrins are adhesive, signaling, and mechanotransduction proteins. Talin (Tln) activates integrins and links it to the actin cytoskeleton. Vertebrates contain two talin genes, tln1 and tln2. How Tln1 and Tln2 function in cardiac myocytes (CMs) is unknown. Tln1 and Tln2 expression were evaluated in the normal embryonic and adult mouse heart as well as in control and failing human adult myocardium. Tln1 function was then tested in the basal and mechanically stressed myocardium after cardiomyocyte-specific excision of the Tln1 gene. During embryogenesis, both Tln forms are highly expressed in CMs, but in the mature heart Tln2 becomes the main Tln isoform, localizing to the costameres. Tln1 expression is minimal in the adult CM. With pharmacological and mechanical stress causing hypertrophy, Tln1 is up-regulated in CMs and is specifically detected at costameres, suggesting its importance in the compensatory response to CM stress. In human failing heart, CM Tln1 also increases compared with control samples from normal functioning myocardium. To directly test Tln1 function in CMs, we generated CM-specific Tln1 knock-out mice (Tln1cKO). Tln1cKO mice showed normal basal cardiac structure and function but when subjected to pressure overload showed blunted hypertrophy, less fibrosis, and improved cardiac function versus controls. Acute responses of ERK1/2, p38, Akt, and glycogen synthase kinase 3 after mechanical stress were strongly blunted in Tln1cKO mice. Given these results, we conclude that Tln1 and Tln2 have distinct functions in the myocardium. Our data show that reduction of CM Tln1 expression can lead to improved cardiac remodeling following pressure overload.
Cardiac Hypertrophy; Cardiomyopathy; Heart; Integrins; Mechanotransduction; Talin
Activation of γ-globin gene expression in adults is known to be therapeutic for sickle cell disease. Thus, it follows that the converse, alleviation of repression, would be equally effective, since the net result would be the same: an increase in fetal hemoglobin. A GATA-1-FOG-1-Mi2 repressor complex was recently demonstrated to be recruited to the −566 GATA motif of the Aγ-globin gene. We show that Mi2β is essential for γ-globin gene silencing using Mi2β conditional knockout β-YAC transgenic mice. In addition, increased expression of Aγ-globin was detected in adult blood from β-YAC transgenic mice containing a T>G HPFH point mutation at the −566 GATA silencer site. ChIP experiments demonstrated that GATA-1 is recruited to this silencer at day E16, followed by recruitment of FOG-1 and Mi2 at day E17 in wild-type β-YAC transgenic mice. Recruitment of the GATA-1–mediated repressor complex was disrupted by the −566 HPFH mutation at developmental stages when it normally binds. Our data suggest that a temporal repression mechanism is operative in the silencing of γ-globin gene expression and that either a trans-acting Mi2β knockout deletion mutation or the cis-acting −566 Aγ-globin HPFH point mutation disrupts establishment of repression, resulting in continued γ-globin gene transcription during adult definitive erythropoiesis.
Sickle cell disease (SCD) is one of the most common genetic diseases, affecting millions of people worldwide. SCD affects red blood cells' shape and renders them ineffective, resulting in anemia along with attendant complications. The disease is caused by a single point mutation in the coding sequence of the adult β-globin gene that changes normal adult hemoglobin (HbA) to sickle hemoglobin (HbS). Scientific evidence has demonstrated that continued expression of the fetal γ-globin genes (fetal hemoglobin, HbF), which are normally silenced after birth, is the best treatment for SCD, since the pathophysiology is largely ameliorated. Our therapeutic goal is to reactivate the γ-globin genes to substitute for the defective adult β-globin gene. We identified a novel γ-globin gene silencer sequence and demonstrated that a GATA-1-FOG-1-Mi2 repressor complex binds to this sequence and silences γ-globin synthesis. However, data regarding the requirement of Mi2 for silencing is controversial. We demonstrate that γ-globin synthesis increases as Mi2 expression decreases. We also show that repressor complex components assemble sequentially during development; completion of assembly coincides with γ-globin gene silencing. Disruption of either the repressor complex or mutation of its binding site induces γ-globin. Understanding this mechanism will reveal potential new targets for treating SCD.
Tyrosine-phosphorylated focal adhesion kinase (FAK) is required for the hypertrophic response of cardiomyocytes to growth factors and mechanical load, but the role of FAK serine phosphorylation in this process is unknown. The aims of the present study were to characterize FAK serine phosphorylation in cultured neonatal rat ventricular myocytes (NRVM), analyse its functional significance during hypertrophic signalling, and examine its potential role in the pathogenesis of human dilated cardiomyopathy (DCM).
Methods and results
Endothelin-1 (ET-1) and other hypertrophic factors induced a time- and dose-dependent increase in FAK-S910 phosphorylation. ET-1-induced FAK-S910 phosphorylation required ETAR-dependent activation of PKCδ and Src via parallel Raf-1 → MEK1/2 → ERK1/2 and MEK5 → ERK5 signalling pathways. Replication-deficient adenoviruses expressing wild-type (WT) FAK and a non-phosphorylatable, S910A-FAK mutant were then used to examine the functional significance of FAK-S910 phosphorylation. Unlike WT-FAK, S910A-FAK increased the half-life of GFP-tagged paxillin within costameres (as determined by total internal reflection fluorescence microscopy and fluorescence recovery after photobleaching) and increased the steady-state FAK–paxillin interaction (as determined by co-immunoprecipitation and western blotting). These alterations resulted in reduced NRVM sarcomere reorganization and cell spreading. Finally, we found that FAK was serine-phosphorylated at multiple sites in non-failing, human left ventricular tissue. FAK-S910 phosphorylation and ERK5 expression were dramatically reduced in patients undergoing heart transplantation for end-stage DCM.
FAK undergoes S910 phosphorylation via PKCδ and Src-dependent pathways that are important for cell spreading and sarcomere reorganization. Reduced FAK-S910 phosphorylation may contribute to sarcomere disorganization in DCM.
Signal transduction; Src; Protein kinase C; Costamere; Dilated cardiomyopathy