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1.  Controlled Trial of Transfusions for Silent Cerebral Infarcts in Sickle Cell Anemia 
The New England journal of medicine  2014;371(8):699-710.
BACKGROUND
Silent cerebral infarcts are the most common neurologic injury in children with sickle cell anemia and are associated with the recurrence of an infarct (stroke or silent cerebral infarct). We tested the hypothesis that the incidence of the recurrence of an infarct would be lower among children who underwent regular blood-transfusion therapy than among those who received standard care.
METHODS
In this randomized, single-blind clinical trial, we randomly assigned children with sickle cell anemia to receive regular blood transfusions (transfusion group) or standard care (observation group). Participants were between 5 and 15 years of age, with no history of stroke and with one or more silent cerebral infarcts on magnetic resonance imaging and a neurologic examination showing no abnormalities corresponding to these lesions. The primary end point was the recurrence of an infarct, defined as a stroke or a new or enlarged silent cerebral infarct.
RESULTS
A total of 196 children (mean age, 10 years) were randomly assigned to the observation or transfusion group and were followed for a median of 3 years. In the transfusion group, 6 of 99 children (6%) had an end-point event (1 had a stroke, and 5 had new or enlarged silent cerebral infarcts). In the observation group, 14 of 97 children (14%) had an end-point event (7 had strokes, and 7 had new or enlarged silent cerebral infarcts). The incidence of the primary end point in the transfusion and observation groups was 2.0 and 4.8 events, respectively, per 100 years at risk, corresponding to an incidence rate ratio of 0.41 (95% confidence interval, 0.12 to 0.99; P = 0.04).
CONCLUSIONS
Regular blood-transfusion therapy significantly reduced the incidence of the recurrence of cerebral infarct in children with sickle cell anemia. (Funded by the National Institute of Neurological Disorders and Stroke and others; Silent Cerebral Infarct Multi-Center Clinical Trial ClinicalTrials.gov number, NCT00072761, and Current Controlled Trials number, ISRCTN52713285.)
doi:10.1056/NEJMoa1401731
PMCID: PMC4195437  PMID: 25140956
2.  Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening 
An acoustic high-throughput screening method is described for harvesting protein crystals and combining the protein crystals with chemicals such as a fragment library.
Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s−1) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening.
doi:10.1107/S1399004714013728
PMCID: PMC4304690  PMID: 25615864
macromolecular crystallography; acoustic droplet ejection; crystal mounting; drug discovery; chemical biology; high-throughput screening
3.  An International Ki67 Reproducibility Study 
Background
In breast cancer, immunohistochemical assessment of proliferation using the marker Ki67 has potential use in both research and clinical management. However, lack of consistency across laboratories has limited Ki67’s value. A working group was assembled to devise a strategy to harmonize Ki67 analysis and increase scoring concordance. Toward that goal, we conducted a Ki67 reproducibility study.
Methods
Eight laboratories received 100 breast cancer cases arranged into 1-mm core tissue microarrays—one set stained by the participating laboratory and one set stained by the central laboratory, both using antibody MIB-1. Each laboratory scored Ki67 as percentage of positively stained invasive tumor cells using its own method. Six laboratories repeated scoring of 50 locally stained cases on 3 different days. Sources of variation were analyzed using random effects models with log2-transformed measurements. Reproducibility was quantified by intraclass correlation coefficient (ICC), and the approximate two-sided 95% confidence intervals (CIs) for the true intraclass correlation coefficients in these experiments were provided.
Results
Intralaboratory reproducibility was high (ICC = 0.94; 95% CI = 0.93 to 0.97). Interlaboratory reproducibility was only moderate (central staining: ICC = 0.71, 95% CI = 0.47 to 0.78; local staining: ICC = 0.59, 95% CI = 0.37 to 0.68). Geometric mean of Ki67 values for each laboratory across the 100 cases ranged 7.1% to 23.9% with central staining and 6.1% to 30.1% with local staining. Factors contributing to interlaboratory discordance included tumor region selection, counting method, and subjective assessment of staining positivity. Formal counting methods gave more consistent results than visual estimation.
Conclusions
Substantial variability in Ki67 scoring was observed among some of the world’s most experienced laboratories. Ki67 values and cutoffs for clinical decision-making cannot be transferred between laboratories without standardizing scoring methodology because analytical validity is limited.
doi:10.1093/jnci/djt306
PMCID: PMC3888090  PMID: 24203987
4.  Mitophagy Is Required for Acute Cardioprotection by Simvastatin 
Antioxidants & Redox Signaling  2014;21(14):1960-1973.
Abstract
Aims: We have shown that autophagy and mitophagy are required for preconditioning. While statin's cardioprotective effects are well known, the role of autophagy/mitophagy in statin-mediated cardioprotection is not. In this study, we used HL-1 cardiomyocytes and mice subjected to ischemia/reperfusion to elucidate the mechanism of statin-mediated cardioprotection. Results: HL-1 cardiomyocytes exposed to simvastatin for 24 h exhibited diminished protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling, increased activation of unc-51-like kinase 1, and upregulation of autophagy and mitophagy. Similar findings were obtained in hearts of mice given simvastatin. Mevalonate abolished simvastatin's effects on Akt/mTOR signaling and autophagy induction in HL-1 cells, indicating that the effects are mediated through inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Simvastatin-treated HL-1 cells exhibited mitochondrial translocation of Parkin and p62/SQSTM1, fission, and mitophagy. Because Parkin is required for mitophagy and is expressed in heart, we investigated the effect of simvastatin on infarct size in Parkin knockout mice. Simvastatin reduced infarct size in wild-type mice but showed no benefit in Parkin knockout mice. Inhibition of HMG-CoA reductase limits mevalonate availability for both cholesterol and coenzyme Q10 (CoQ) biosynthesis. CoQ supplementation had no effect on statin-induced Akt/mTOR dephosphorylation or macroautophagy in HL-1 cells, but it potently blocked mitophagy. Importantly, CoQ supplementation abolished statin-mediated cardioprotection in vivo. Innovation and Conclusion: Acute simvastatin treatment suppresses mTOR signaling and triggers Parkin-dependent mitophagy, the latter which is required for cardioprotection. Coadministration of CoQ with simvastatin impairs mitophagy and cardioprotection. These results raise the concern that CoQ may interfere with anti-ischemic benefits of statins mediated through stimulation of mitophagy. Antioxid. Redox Signal. 21, 1960–1973.
doi:10.1089/ars.2013.5416
PMCID: PMC4208607  PMID: 23901824
5.  Multipronged CD4 T cell effector and memory responses cooperate to provide potent immunity against respiratory virus 
Immunological reviews  2013;255(1):149-164.
Summary
Over the last decade, the known spectrum of CD4 T cell effect or subsets has become much broader and it has become clear that there are multiple dimensions by which subsets with a particular cytokine commitment can be further defined, including their stage of differentiation, their location and most importantly, their ability to carryout discrete functions. Here we focus on our studies that highlight the synergy among discrete subsets, especially those defined by helper and cytotoxic function, in mediating viral protection and on distinctions between CD4 T cell effectors located in spleen, draining lymph node, and in tissue sites of infection. What emerges is a surprising multiplicity of CD4 T cell functions that indicate a large arsenal of mechanisms by which CD4 T cells act to combat viruses.
doi:10.1111/imr.12088
PMCID: PMC4206082  PMID: 23947353
6.  Solvent minimization induces preferential orientation and crystal clustering in serial micro-crystallography on micro-meshes, in situ plates and on a movable crystal conveyor belt 
Journal of Synchrotron Radiation  2014;21(Pt 6):1231-1239.
Strategies are described for optimizing the signal-to-noise of diffraction data, and for combining data from multiple crystals. One challenge that must be overcome is the non-random orientation of crystals with respect to one another and with respect to the surface that supports them.
X-ray diffraction data were obtained at the National Synchrotron Light Source from insulin and lysozyme crystals that were densely deposited on three types of surfaces suitable for serial micro-crystallography: MiTeGen MicroMeshes™, Greiner Bio-One Ltd in situ micro-plates, and a moving kapton crystal conveyor belt that is used to deliver crystals directly into the X-ray beam. 6° wedges of data were taken from ∼100 crystals mounted on each material, and these individual data sets were merged to form nine complete data sets (six from insulin crystals and three from lysozyme crystals). Insulin crystals have a parallelepiped habit with an extended flat face that preferentially aligned with the mounting surfaces, impacting the data collection strategy and the design of the serial crystallography apparatus. Lysozyme crystals had a cuboidal habit and showed no preferential orientation. Preferential orientation occluded regions of reciprocal space when the X-ray beam was incident normal to the data-collection medium surface, requiring a second pass of data collection with the apparatus inclined away from the orthogonal. In addition, crystals measuring less than 20 µm were observed to clump together into clusters of crystals. Clustering required that the X-ray beam be adjusted to match the crystal size to prevent overlapping diffraction patterns. No additional problems were encountered with the serial crystallography strategy of combining small randomly oriented wedges of data from a large number of specimens. High-quality data able to support a realistic molecular replacement solution were readily obtained from both crystal types using all three serial crystallography strategies.
doi:10.1107/S1600577514017731
PMCID: PMC4211130  PMID: 25343789
in situ X-ray data collection; crystallography; acoustic droplet ejection; serial crystallography
7.  A Cell-Based High-Throughput Screen for Novel Chemical Inducers of Fetal Hemoglobin for Treatment of Hemoglobinopathies 
PLoS ONE  2014;9(9):e107006.
Decades of research have established that the most effective treatment for sickle cell disease (SCD) is increased fetal hemoglobin (HbF). Identification of a drug specific for inducing γ-globin expression in pediatric and adult patients, with minimal off-target effects, continues to be an elusive goal. One hurdle has been an assay amenable to a high-throughput screen (HTS) of chemicals that displays a robust γ-globin off-on switch to identify potential lead compounds. Assay systems developed in our labs to understand the mechanisms underlying the γ- to β-globin gene expression switch during development has allowed us to generate a cell-based assay that was adapted for a HTS of 121,035 compounds. Using chemical inducer of dimerization (CID)-dependent bone marrow cells (BMCs) derived from human γ-globin promoter-firefly luciferase β-globin promoter-Renilla luciferase β-globin yeast artificial chromosome (γ-luc β-luc β-YAC) transgenic mice, we were able to identify 232 lead chemical compounds that induced γ-globin 2-fold or higher, with minimal or no β-globin induction, minimal cytotoxicity and that did not directly influence the luciferase enzyme. Secondary assays in CID-dependent wild-type β-YAC BMCs and human primary erythroid progenitor cells confirmed the induction profiles of seven of the 232 hits that were cherry-picked for further analysis.
doi:10.1371/journal.pone.0107006
PMCID: PMC4165891  PMID: 25225870
8.  Acoustic methods for high-throughput protein crystal mounting at next-generation macromolecular crystallographic beamlines 
Journal of Synchrotron Radiation  2013;20(Pt 5):805-808.
A new system has been developed and tested at the National Synchrotron Light Source with the goal of enabling rapid protein crystal mounting at next-generation macromolecular crystallographic beamlines. The system uses an acoustic ejector to deposit nanoliter-volume droplets containing crystals onto an X-ray transparent conveyor belt, which then moves the droplets into position for cryo-cooling and data collection. The acoustic ejector is capable of operating at a rate of several hundred droplet ejections per second.
To take full advantage of advanced data collection techniques and high beam flux at next-generation macromolecular crystallography beamlines, rapid and reliable methods will be needed to mount and align many samples per second. One approach is to use an acoustic ejector to eject crystal-containing droplets onto a solid X-ray transparent surface, which can then be positioned and rotated for data collection. Proof-of-concept experiments were conducted at the National Synchrotron Light Source on thermolysin crystals acoustically ejected onto a polyimide ‘conveyor belt’. Small wedges of data were collected on each crystal, and a complete dataset was assembled from a well diffracting subset of these crystals. Future developments and implementation will focus on achieving ejection and translation of single droplets at a rate of over one hundred per second.
doi:10.1107/S0909049513020372
PMCID: PMC3747951  PMID: 23955046
acoustic droplet ejection; conveyor belt; crystal mounting; high throughput; X-ray diffraction; macromolecular crystallography
9.  Association of the family environment with behavioural and cognitive outcomes in children with chromosome 22q11.2 deletion syndrome 
Background
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.
Method
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.
Results
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.
Conclusion
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.
doi:10.1111/jir.12054
PMCID: PMC4086857  PMID: 23742203
chromosome 22q11.2 deletion syndrome; DiGeorge syndrome; family environment; parenting; social-behavioural functioning; velocardiofacial syndrome
10.  A Linear Relationship between Crystal Size and Fragment Binding Time Observed Crystallographically: Implications for Fragment Library Screening Using Acoustic Droplet Ejection 
PLoS ONE  2014;9(7):e101036.
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.
doi:10.1371/journal.pone.0101036
PMCID: PMC4079544  PMID: 24988328
11.  Skeletal effects of zoledronic acid in an animal model of chronic kidney disease 
Summary
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.
Introduction
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1007/s00198-012-2103-x
PMCID: PMC4063946  PMID: 22907737
Anti-remodeling agents; Bisphosphonate; Bone mechanics; Remodeling suppression
12.  Incidental Memory of Younger and Older Adults for Objects Encountered in a Real World Context 
PLoS ONE  2014;9(6):e99051.
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).
doi:10.1371/journal.pone.0099051
PMCID: PMC4062423  PMID: 24941065
13.  FRNK Inhibition of Focal Adhesion Kinase–Dependent Signaling and Migration in Vascular Smooth Muscle Cells 
Objective
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.
Conclusion
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.
doi:10.1161/ATVBAHA.110.212761
PMCID: PMC4058887  PMID: 20705914
vascular biology; vascular muscle; Src; cell migration; mechanotransduction; tyrosine phosphorylation; vascular remodeling
14.  Force Plate Gait Analysis in Doberman Pinschers with and without Cervical Spondylomyelopathy 
Background
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.
Objective
To establish and compare kinetic parameters based on force plate gait analysis between normal and CSM-affected Dobermans.
Animals
Nineteen Doberman Pinschers: 10 clinically normal and 9 with CSM.
Methods
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.
Results
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.
doi:10.1111/jvim.12025
PMCID: PMC4025962  PMID: 23278957
Cervical vertebral instability; Dog; Kinetic; Wobbler
15.  Hitting the target: fragment screening with acoustic in situ co-crystallization of proteins plus fragment libraries on pin-mounted data-collection micromeshes 
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.
doi:10.1107/S1399004713034603
PMCID: PMC4014116  PMID: 24816088
in situ X-ray data collection; acoustic droplet ejection; fragment screening; drug discovery; chemical biology; protein crystallization; synchrotron radiation
16.  Does participation in an HIV vaccine efficacy trial affect risk behaviour in South Africa? 
Vaccine  2013;31(16):2089-2096.
Background
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1016/j.vaccine.2013.01.031
PMCID: PMC3942784  PMID: 23370155
17.  Macromolecular crystallography beamline X25 at the NSLS 
Journal of Synchrotron Radiation  2014;21(Pt 3):627-632.
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.
doi:10.1107/S1600577514003415
PMCID: PMC3998817  PMID: 24763654
beamline; mini-κ; Pilatus 6M; PXRR; macromolecular crystallography; wBPM
18.  Regulation of Connective Tissue Growth Factor Gene Expression and Fibrosis in Human Heart Failure 
Journal of cardiac failure  2013;19(4):283-294.
Background
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.
Conclusions
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.
doi:10.1016/j.cardfail.2013.01.013
PMCID: PMC3643143  PMID: 23582094
Remodeling; Heart-assist device; Gene expression; collagens
19.  Estimating Upper Bounds for Occupancy and Number of Manatees in Areas Potentially Affected by Oil from the Deepwater Horizon Oil Spill 
PLoS ONE  2014;9(3):e91683.
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.
doi:10.1371/journal.pone.0091683
PMCID: PMC3966779  PMID: 24670971
20.  Long-term behavior at foraging sites of adult female loggerhead sea turtles (Caretta caretta) from three Florida rookeries 
Marine Biology  2014;161(6):1251-1262.
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.
doi:10.1007/s00227-014-2415-9
PMCID: PMC4033788  PMID: 24882883
21.  Stress-dependent impairment of passive-avoidance memory by propranolol or naloxone 
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.
doi:10.1016/j.pbb.2011.03.005
PMCID: PMC3902981  PMID: 21402095
Propranolol; Naloxone; Forced swim; Passive-avoidance training; Stress; Memory modulation
22.  Stress-dependent enhancement and impairment of retention by naloxone: Evidence for an endogenous opioid-based modulatory system protective of memory 
Behavioural brain research  2009;205(1):290-293.
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.
doi:10.1016/j.bbr.2009.06.007
PMCID: PMC3902997  PMID: 19523491
Naloxone; Memory modulation; Opioid; Stress; Forced-swim; Adrenergic
23.  Recapitulation of Pancreatic Neuroendocrine Tumors in Human Multiple Endocrine Neoplasia Type I (MEN1) Syndrome via Pdx1-directed Inactivation of Men1 
Cancer research  2009;69(5):10.1158/0008-5472.CAN-08-3662.
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.
doi:10.1158/0008-5472.CAN-08-3662
PMCID: PMC3879686  PMID: 19208834
MEN1; pancreatic neuroendocrine tumor; VEGF; tumor suppressor gene; Pdx1
24.  Microdomain heterogeneity in 3D affects the mechanics of neonatal cardiac myocyte contraction 
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.
doi:10.1007/s10237-012-0384-9
PMCID: PMC3407350  PMID: 22407215
Mechanobiology; Muscle hypertrophy; Digital image correlation; Microenvironment; Finite element; Mechanotransduction
25.  Iron Deposition following Chronic Myocardial Infarction as a Substrate for Cardiac Electrical Anomalies: Initial Findings in a Canine Model 
PLoS ONE  2013;8(9):e73193.
Purpose
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.
Methods
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).
Results
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.
Conclusion
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.
doi:10.1371/journal.pone.0073193
PMCID: PMC3774668  PMID: 24066038

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