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1.  Inflammatory Markers of the Systemic Capillary Leak Syndrome (Clarkson Disease) 
Objectives
The Systemic Capillary Leak Syndrome (SCLS) is a rare and potentially fatal disorder resembling systemic anaphylaxis that is characterized by transient episodes of hypotensive shock and peripheral edema. The pathogenesis of SCLS is unknown, and triggers for attacks are apparent only in a minority of patients. We introduce a clinical algorithm for the diagnosis of SCLS, and we investigated potential serum biomarkers of acute SCLS episodes.
Methods
We analyzed serum cytokines in a cohort of 35 patients with an established diagnosis of SCLS and characterized the effects of SCLS sera on endothelial cell function. We investigated the cellular source(s) of CXCL10, a chemokine that was significantly elevated in both basal and acute SCLS sera, by flow cytometry.
Results
Several cytokines were elevated in acute SCLS sera compared to baseline or sera from healthy controls, including CXCL10, CCL2, IL-1β, IL-6, IL-8, IL-12 and TNFα. The majority of acute sera failed to activate endothelial cells as assessed by surface adhesion marker expression. Monocytes appear to be the major source of serum CXCL10, and the percentage of CXLC10+ monocytes in response to IFNγ stimulation was increased in SCLS subjects compared to controls.
Conclusions
The presence of proinflammatory cytokines in acute SCLS sera suggests that inflammation or infection may have a role in triggering episodes. The enhanced capacity of monocytes from SCLS patients to produce CXCL10 suggests a new therapeutic avenue for SCLS.
doi:10.4172/2155-9899.1000213
PMCID: PMC4232957  PMID: 25405070
Systemic capillary leak syndrome; Inflammation; Cytokines; CXCL10; Monocytes
2.  The real-life effectiveness of palivizumab for reducing hospital admissions for respiratory syncytial virus in infants residing in Nunavut 
Respiratory syncytial virus (RSV) infection is the leading cause of hospitalization among infants, and is the cause of considerable morbidity in the Artic regions. Although a safe and effective RSV vaccine remains elusive, palivizumab has shown considerable promise as a prophylactic agent in previous randomized controlled trials. Prompted by the lack of published data involving Inuit or Aboriginal infants, this prospective, observational study aimed to estimate the effectiveness of palivizumab against RSV in this population.
BACKGROUND/OBJECTIVE:
Nunavut has the highest hospitalization rates for respiratory syncytial virus (RSV) worldwide, with rates of 166 per 1000 live births per year <1 year of age. Palivizumab was implemented in Nunavut primarily for premature infants, or those with hemodynamically significant cardiac or chronic lung disease; however, the effectiveness of the program is unknown. The objective of the present multisite, hospital-based surveillance study was to estimate the effectiveness of palivizumab in infants <6 months of age in Nunavut for the 2009 and 2010 RSV seasons.
METHODS:
Infants identified as palivizumab candidates who were <6 months of age were compared with all admissions for lower respiratory tract infection through multisite, hospital-based surveillance documenting the adequacy of palivizumab prophylaxis, admission for lower respiratory tract infection and the results of RSV testing. The OR for RSV admission in unprophylaxed infants was compared with those who were prophylaxed, and the effectiveness of palivizumab was estimated.
RESULTS:
Within the study cohort (n=101) during the two RSV seasons, five of the 10 eligible infants who did not receive adequate prophylaxis were admitted with RSV while two of the 91 infants <6 months of age eligible for palivizumab who were adequately prophylaxed were hospitalized with RSV (OR 22.3 [95% CI 3.8 to 130]; P=0.0005). The estimated effectiveness of palivizumab for the cohort was as high as 96%. Eight eligible infants were missed by the program and did not receive prophylaxis.
CONCLUSION:
Palivizumab was highly effective in reducing hospitalizations due to RSV infection in Nunavut. Further efforts need to be made to ensure that all eligible infants are identified.
PMCID: PMC4128465  PMID: 24367792
Effectiveness; Inuit; Nunavut; Palivizumab; Respiratory syncytial virus
3.  Interphotoreceptor matrix-poly(ϵ-caprolactone) composite scaffolds for human photoreceptor differentiation 
Journal of Tissue Engineering  2014;5:2041731414554139.
Tissue engineering has been widely applied in different areas of regenerative medicine, including retinal regeneration. Typically, artificial biopolymers require additional surface modification (e.g. with arginine–glycine–aspartate-containing peptides or adsorption of protein, such as fibronectin), before cell seeding. Here, we describe an alternative approach for scaffold design: the manufacture of hybrid interphotoreceptor matrix-poly (ϵ-caprolactone) scaffolds, in which the insoluble extracellular matrix of the retina is incorporated into a biodegradable polymer well suited for transplantation. The incorporation of interphotoreceptor matrix did not change the topography of polycaprolactone film, although it led to a slight increase in hydrophilic properties (water contact angle measurements). This hybrid scaffold provided sufficient stimuli for human retinal progenitor cell adhesion and inhibited proliferation, leading to differentiation toward photoreceptor cells (expression of Crx, Nrl, rhodopsin, ROM1). This scaffold may be used for transplantation of retinal progenitor cells and their progeny to treat retinal degenerative disorders.
doi:10.1177/2041731414554139
PMCID: PMC4221930  PMID: 25383176
Retina; photoreceptors; interphotoreceptor matrix; polycaprolactone
4.  Application of an ex vivo cellular model of circadian variation for bipolar disorder research: a proof of concept study 
Bipolar disorders  2013;15(6):694-700.
Objectives
Disruption of circadian function has been observed in several human disorders, including bipolar disorder (BD). Research into these disorders can be facilitated by human cellular models that evaluate external factors (zeitgebers) that impact circadian pacemaker activity. Incorporating a firefly luciferase reporter system into human fibroblasts provides a facile, bioluminescent readout that estimates circadian phase, while leaving the cells intact. We evaluated whether this system can be adapted to clinical BD research and whether it can incorporate zeitgeber challenge paradigms.
Methods
Fibroblasts from patients with bipolar I disorder (BD-I) (n = 13) and controls (n = 12) were infected ex vivo with a lentiviral reporter incorporating the promoter sequences for Bmal1, a circadian gene to drive expression of the firefly Luciferase gene. Following synchronization, the bioluminescence was used to estimate period length. Phase response curves (PRC) were also generated following forskolin challenge and the phase response patterns characterized.
Results
Period length and PRCs could be estimated reliably from the constructs. There were no significant case–control differences in period length, with a nonsignificant trend for differences in PRCs following the phase setting experiments.
Conclusions
An ex vivo cellular fibroblast-based model can be used to investigate circadian function in BD-I. It can be generated from specific individuals and this could usefully complement ongoing circadian clinical research.
doi:10.1111/bdi.12095
PMCID: PMC3762935  PMID: 23782472
biorhythm; bipolar disorder; BmalI; circadian; fibroblast
5.  Sex differences in the inference and perception of causal relations within a video game 
The learning of immediate causation within a dynamic environment was examined. Participants encountered seven decision points in which they needed to choose, which of three possible candidates was the cause of explosions in the environment. Each candidate was firing a weapon at random every few seconds, but only one of them produced an immediate effect. Some participants showed little learning, but most demonstrated increases in accuracy across time. On average, men showed higher accuracy and shorter latencies that were not explained by differences in self-reported prior video game experience. This result suggests that prior reports of sex differences in causal choice in the game are not specific to situations involving delayed or probabilistic causal relations.
doi:10.3389/fpsyg.2014.00926
PMCID: PMC4141458  PMID: 25202293
causal inference; causal perception; learning; video games; sex differences
6.  Engineering retinal progenitor cell and scrollable poly(glycerol-sebacate) composites for expansion and subretinal transplantation 
Biomaterials  2009;30(20):3405-3414.
Retinal degenerations cause permanent visual loss and affect millions world-wide. Presently, a novel treatment highlights the potential of using biodegradable polymer scaffolds to induce differentiation and deliver retinal progenitor cells for cell replacement therapy. In this study, we engineered and analyzed a micro-fabricated polymer, poly(glycerol sebacate) (PGS) scaffold, whose useful properties include biocompatibility, elasticity, porosity, and a microtopology conducive to mouse retinal progenitor cell (mRPC) differentiation. In vitro proliferation assays revealed that PGS held up to 86,610 (±9993) mRPCs per square millimeter, which were retained through simulated transplantations. mRPCs adherent to PGS differentiated toward mature phenotypes as evidenced by changes in mRNA, protein levels, and enhanced sensitivity to glutamate. Transplanted composites demonstrated long-term mRPC survival and migrated cells exhibited mature marker expression in host retina. These results suggest that combining mRPCs with PGS scaffolds for subretinal transplantation is a practical strategy for advancing retinal tissue engineering as a restorative therapy.
doi:10.1016/j.biomaterials.2009.02.046
PMCID: PMC4109162  PMID: 19361860
Biodegradation; Cell adhesion; Elastomer; Stem cell; Nerve tissue engineering; Ophthalmology
7.  Oleoyl Coenzyme A Regulates Interaction of Transcriptional Regulator RaaS (Rv1219c) with DNA in Mycobacteria* 
The Journal of Biological Chemistry  2014;289(36):25241-25249.
Background: RaaS mediates mycobacterial survival in nonpermissive growth conditions by controlling expression of ATP-dependent efflux pumps.
Results: Oleoyl-CoA regulates binding of the RaaS transcription factor to DNA and thus expression of the RaaS regulon and RaaS-mediated persistence.
Conclusion: The activity of bacterial efflux is regulated by metabolites that are produced during active growth.
Significance: Dysregulation of efflux pumps results in killing of persisting mycobacteria with low metabolic activity.
We have recently shown that RaaS (regulator of antimicrobial-assisted survival), encoded by Rv1219c in Mycobacterium tuberculosis and by bcg_1279c in Mycobacterium bovis bacillus Calmette-Guérin, plays an important role in mycobacterial survival in prolonged stationary phase and during murine infection. Here, we demonstrate that long chain acyl-CoA derivatives (oleoyl-CoA and, to lesser extent, palmitoyl-CoA) modulate RaaS binding to DNA and expression of the downstream genes that encode ATP-dependent efflux pumps. Moreover, exogenously added oleic acid influences RaaS-mediated mycobacterial improvement of survival and expression of the RaaS regulon. Our data suggest that long chain acyl-CoA derivatives serve as biological indicators of the bacterial metabolic state. Dysregulation of efflux pumps can be used to eliminate non-growing mycobacteria.
doi:10.1074/jbc.M114.577338
PMCID: PMC4155686  PMID: 25012658
ABC Transporter; Antibiotics; Fatty Acid; Ligand-binding Protein; Mycobacteria; Transcription Repressor
8.  Evidence of Climate-Induced Range Contractions in Bull Trout Salvelinus confluentus in a Rocky Mountain Watershed, U.S.A 
PLoS ONE  2014;9(6):e98812.
Many freshwater fish species are considered vulnerable to stream temperature warming associated with climate change because they are ectothermic, yet there are surprisingly few studies documenting changes in distributions. Streams and rivers in the U.S. Rocky Mountains have been warming for several decades. At the same time these systems have been experiencing an increase in the severity and frequency of wildfires, which often results in habitat changes including increased water temperatures. We resampled 74 sites across a Rocky Mountain watershed 17 to 20 years after initial samples to determine whether there were trends in bull trout occurrence associated with temperature, wildfire, or other habitat variables. We found that site abandonment probabilities (0.36) were significantly higher than colonization probabilities (0.13), which indicated a reduction in the number of occupied sites. Site abandonment probabilities were greater at low elevations with warm temperatures. Other covariates, such as the presence of wildfire, nonnative brook trout, proximity to areas with many adults, and various stream habitat descriptors, were not associated with changes in probability of occupancy. Higher abandonment probabilities at low elevation for bull trout provide initial evidence validating the predictions made by bioclimatic models that bull trout populations will retreat to higher, cooler thermal refuges as water temperatures increase. The geographic breadth of these declines across the region is unknown but the approach of revisiting historical sites using an occupancy framework provides a useful template for additional assessments.
doi:10.1371/journal.pone.0098812
PMCID: PMC4045800  PMID: 24897341
9.  Outcome Probability versus Magnitude: When Waiting Benefits One at the Cost of the Other 
PLoS ONE  2014;9(6):e98996.
Using a continuous impulsivity and risk platform (CIRP) that was constructed using a video game engine, choice was assessed under conditions in which waiting produced a continuously increasing probability of an outcome with a continuously decreasing magnitude (Experiment 1) or a continuously increasing magnitude of an outcome with a continuously decreasing probability (Experiment 2). Performance in both experiments reflected a greater desire for a higher probability even though the corresponding wait times produced substantive decreases in overall performance. These tendencies are considered to principally reflect hyperbolic discounting of probability, power discounting of magnitude, and the mathematical consequences of different response rates. Behavior in the CIRP is compared and contrasted with that in the Balloon Analogue Risk Task (BART).
doi:10.1371/journal.pone.0098996
PMCID: PMC4044015  PMID: 24892657
10.  Genome-wide SNP analysis of the Systemic Capillary Leak Syndrome (Clarkson disease) 
Rare diseases (Austin, Tex.)  2013;1(1):e27445.
The Systemic Capillary Leak Syndrome (SCLS) is an extremely rare, orphan disease that resembles, and is frequently erroneously diagnosed as, systemic anaphylaxis. The disorder is characterized by repeated, transient, and seemingly unprovoked episodes of hypotensive shock and peripheral edema due to transient endothelial hyperpermeability. SCLS is often accompanied by a monoclonal gammopathy of unknown significance (MGUS). Using Affymetrix Single Nucleotide Polymorphism (SNP) microarrays, we performed the first genome-wide SNP analysis of SCLS in a cohort of 12 disease subjects and 18 controls. Exome capture sequencing was performed on genomic DNA from nine of these patients as validation for the SNP-chip discoveries and de novo data generation. We identified candidate susceptibility loci for SCLS, which included a region flanking CAV3 (3p25.3) as well as SNP clusters in PON1 (7q21.3), PSORS1C1 (6p21.3), and CHCHD3 (7q33). Among the most highly ranked discoveries were gene-associated SNPs in the uncharacterized LOC100130480 gene (rs6417039, rs2004296). Top case-associated SNPs were observed in BTRC (rs12355803, 3rs4436485), ARHGEF18 (rs11668246), CDH13 (rs4782779), and EDG2 (rs12552348), which encode proteins with known or suspected roles in B cell function and/or vascular integrity. 61 SNPs that were significantly associated with SCLS by microarray analysis were also detected and validated by exome deep sequencing. Functional annotation of highly ranked SNPs revealed enrichment of cell projections, cell junctions and adhesion, and molecules containing pleckstrin homology, Ras/Rho regulatory, and immunoglobulin Ig-like C2/fibronectin type III domains, all of which involve mechanistic functions that correlate with the SCLS phenotype. These results highlight SNPs with potential relevance to SCLS.
doi:10.4161/rdis.27445
PMCID: PMC4009617  PMID: 24808988
systemic capillary leak syndrome; genetics, genome-wide SNP study; cell junction; cell adhesion; cytoskeleton; vascular permeability
11.  Sensitivity to Changing Contingencies in an Impulsivity Task 
Using a video-game-based escalating interest task, participants repeatedly encountered a reward that gradually increased in value over a 10-second interval. Responding early in the interval netted less immediate reward than responding later in the interval. Each participant experienced four different reward contingencies for waiting. These contingencies were changed three times as the experiment proceeded. Behavior tracked these changing contingencies, but wait times reflected long-term carryover from the previously assigned contingencies. Both the tendency to respond slowly and the optimality of behavior were affected by the order of contingencies experienced. Demographic variables only weakly predicted behavior, and delay discounting rate in a hypothetical money choice task predicted choice only when the contingencies in the game were weaker.
doi:10.1002/jeab.24
PMCID: PMC3900408  PMID: 23658118
12.  Antimicrobial Treatment Improves Mycobacterial Survival in Nonpermissive Growth Conditions 
Antimicrobials targeting cell wall biosynthesis are generally considered inactive against nonreplicating bacteria. Paradoxically, we found that under nonpermissive growth conditions, exposure of Mycobacterium bovis BCG bacilli to such antimicrobials enhanced their survival. We identified a transcriptional regulator, RaaS (for regulator of antimicrobial-assisted survival), encoded by bcg1279 (rv1219c) as being responsible for the observed phenomenon. Induction of this transcriptional regulator resulted in reduced expression of specific ATP-dependent efflux pumps and promoted long-term survival of mycobacteria, while its deletion accelerated bacterial death under nonpermissive growth conditions in vitro and during macrophage or mouse infection. These findings have implications for the design of antimicrobial drug combination therapies for persistent infectious diseases, such as tuberculosis.
doi:10.1128/AAC.02774-13
PMCID: PMC3993263  PMID: 24590482
13.  Magnitude Effects for Experienced Rewards at Short Delays in the Escalating Interest Task 
Psychonomic bulletin & review  2013;20(2):302-309.
A first-person shooter video game was adapted for the study of choice between smaller sooner and larger later rewards. Participants chose when to fire a weapon that increased in damage potential over a short interval. When the delay to maximum damage was shorter (5 – 8 s), people showed greater sensitivity to the consequences of their choices than when the delay was longer (17 – 20 s). Participants also evidenced a magnitude effect by waiting proportionally longer when the damage magnitudes were doubled for all rewards. The experiment replicated the standard magnitude effect with this new video game preparation over time scales similar to those typically used in nonhuman animal studies and without complications due to satiation or cost.
doi:10.3758/s13423-012-0350-7
PMCID: PMC3594385  PMID: 23188742
14.  Multiphoton Imaging with a Direct-diode Pumped Femtosecond Ti:sapphire Laser 
Journal of microscopy  2012;249(2):83-86.
A direct-diode pumped Ti:sapphire femtosecond oscillator is used to perform multiphoton imaging for the first time.
doi:10.1111/j.1365-2818.2012.03688.x
PMCID: PMC3553270  PMID: 23189919
15.  Use of a Synthetic Xeno-Free Culture Substrate for Induced Pluripotent Stem Cell Induction and Retinal Differentiation 
The purpose of this study was to determine whether a proprietary xeno-free synthetic culture surface could be used to aid in the production and subsequent retinal-specific differentiation of clinical-grade induced pluripotent stem cells (iPSCs). It was found that Synthemax cell culture surfaces provide an ideal surface for the xeno-free production, culture, and differentiation of adult somatic cell-derived iPSCs. These findings demonstrate the potential utility of these surfaces for the production of clinical-grade retinal neurons for transplantation and induction of retinal regeneration.
The purpose of this study was to determine whether a proprietary xeno-free synthetic culture surface could be used to aid in the production and subsequent retinal-specific differentiation of clinical-grade induced pluripotent stem cells (iPSCs). iPSCs were generated using adult somatic cells via infection with either a single cre-excisable lentiviral vector or four separate nonintegrating Sendai viruses driving expression of the transcription factors OCT4, SOX2, KLF4, and c-MYC. Retinal precursor cells were derived via targeted differentiation of iPSCs with exogenous delivery of dkk-1, noggin, insulin-like growth factor-1, basic fibroblast growth factor, acidic fibroblast growth factor, and DAPT. Phase contrast microscopy, immunocytochemistry, hematoxylin and eosin staining, and reverse transcription-polymerase chain reaction were used to determine reprogramming efficiency, pluripotency, and fate of undifferentiated and differentiated iPSCs. Following viral transduction, cells underwent prototypical morphological changes resulting in the formation of iPSC colonies large enough for manual isolation/passage at 3–4 weeks postinfection. Both normal and disease-specific iPSCs expressed markers of pluripotency and, following transplantation into immune-compromised mice, formed teratomas containing tissue comprising all three germ layers. When subjected to our established retinal differentiation protocol, a significant proportion of the xeno-free substrate-derived cells expressed retinal cell markers, the number of which did not significantly differ from that derived on traditional extracellular matrix-coated dishes. Synthetic cell culture substrates provide a useful surface for the xeno-free production, culture, and differentiation of adult somatic cell-derived iPSCs. These findings demonstrate the potential utility of these surfaces for the production of clinical-grade retinal neurons for transplantation and induction of retinal regeneration.
doi:10.5966/sctm.2012-0040
PMCID: PMC3659741  PMID: 23283489
Induced pluripotent stem cells; Stem cell culture; Retina; Reprogramming; Pluripotent stem cells; Clinical translation
16.  Functional and morphological analysis of the subretinal injection of human retinal progenitor cells under Cyclosporin A treatment 
Molecular Vision  2014;20:1271-1280.
Purpose
The purpose of this study is to evaluate the functional and morphological changes in subretinal xenografts of human retinal progenitor cells (hRPCs) in B6 mice treated with Cyclosporin A (CsA; 210 mg/l in drinking water).
Methods
The hRPCs from human fetal eyes were isolated and expanded for transplantation. These cells, with green fluorescent protein (GFP) at 11 passages, were transplanted into the subretinal space in B6 mice. A combination of invasive and noninvasive approaches was used to analyze the structural and functional consequences of the subretinal injection of the hRPCs. The process of change was monitored using spectral domain optical coherence tomography (SDOCT), histology, and electroretinography (ERG) at 3 days, 1 week, and 3 weeks after transplantation. Cell counts were used to evaluate the survival rate with a confocal microscope. ERGs were performed to evaluate the physiologic changes, and the structural changes were evaluated using SDOCT and histological examination.
Results
The results of the histological examination showed that the hRPCs gained a better survival rate in the mice treated with CsA. The SDOCT showed that the bleb size of the retinal detachment was significantly decreased, and the retinal reattachment was nearly complete by 3 weeks. The ERG response amplitudes in the CsA group were less decreased after the injection, when compared with the control group, in the dark-adapted and light-adapted conditions. However, the cone-mediated function in both groups was less affected by the transplantation after 3 weeks than the rod-mediated function.
Conclusions
Although significant functional and structural recovery was observed after the subretinal injection of the hRPCs, the effectiveness of CsA in xenotransplantation may be a novel and potential approach for increasing retinal progenitor cell survival.
PMCID: PMC4168833  PMID: 25352736
17.  Defining mental illnesses: can values and objectivity get along? 
BMC Psychiatry  2013;13:346.
Background
The creation of each edition of the Diagnostic and Statistical Manual (DSM) of psychiatry has proven enormously controversial. The current effort to revise the ‘bible’ of disorder definitions for the field of mental health is no exception. The controversy around DSM-5 reached a crescendo with the announcement from National Institute of Mental Health (NIMH) that the institute would focus efforts on the development of their own psychiatric nosology, the Research Domain Criteria (RDoC) (NIMH, 2013).
Discussion
The RDoC seem to be structured around the concern that the only way to find objectivity in the classification of diseases or disorders in psychiatry is to begin with biology and work back to symptoms. Values infuse medical categories in various ways and drive practical considerations about where and how to divide up constellations of already agreed upon symptoms.
Summary
We briefly argue that all nosologies are infused with values and, while we should continue to sharpen the psychiatric nosology, normativity will permeate even the strictest biologically based taxonomy; this need not be a bad thing.
doi:10.1186/1471-244X-13-346
PMCID: PMC3877989  PMID: 24365131
18.  Cyclic Amp-Dependent Resuscitation of Dormant Mycobacteria by Exogenous Free Fatty Acids 
PLoS ONE  2013;8(12):e82914.
One third of the world population carries a latent tuberculosis (TB) infection, which may reactivate leading to active disease. Although TB latency has been known for many years it remains poorly understood. In particular, substances of host origin, which may induce the resuscitation of dormant mycobacteria, have not yet been described. In vitro models of dormant (“non-culturable”) cells of Mycobacterium smegmatis (mc2155) and Mycobacterium tuberculosis H37Rv were used. We found that the resuscitation of dormant M. smegmatis and M. tuberculosis cells in liquid medium was stimulated by adding free unsaturated fatty acids (FA), including arachidonic acid, at concentrations of 1.6–10 µM. FA addition enhanced cAMP levels in reactivating M. smegmatis cells and exogenously added cAMP (3–10 mM) or dibutyryl-cAMP (0.5–1 mM) substituted for FA, causing resuscitation of M. smegmatis and M. tuberculosis dormant cells. A M. smegmatis null-mutant lacking MSMEG_4279, which encodes a FA-activated adenylyl cyclase (AC), could not be resuscitated by FA but it was resuscitated by cAMP. M. smegmatis and M. tuberculosis cells hyper-expressing AC were unable to form non-culturable cells and a specific inhibitor of AC (8-bromo-cAMP) prevented FA-dependent resuscitation. RT-PCR analysis revealed that rpfA (coding for resuscitation promoting factor A) is up-regulated in M. smegmatis in the beginning of exponential growth following the cAMP increase in lag phase caused by FA-induced cell activation. A specific Rpf inhibitor (4-benzoyl-2-nitrophenylthiocyanate) suppressed FA-induced resuscitation. We propose a novel pathway for the resuscitation of dormant mycobacteria involving the activation of adenylyl cyclase MSMEG_4279 by FAs resulted in activation of cellular metabolism followed later by increase of RpfA activity which stimulates cell multiplication in exponential phase. The study reveals a probable role for lipids of host origin in the resuscitation of dormant mycobacteria, which may function during the reactivation of latent TB.
doi:10.1371/journal.pone.0082914
PMCID: PMC3871856  PMID: 24376605
19.  Genome-wide SNP analysis of the Systemic Capillary Leak Syndrome (Clarkson disease) 
Rare Diseases  2013;1:e27445.
The Systemic Capillary Leak Syndrome (SCLS) is an extremely rare, orphan disease that resembles, and is frequently erroneously diagnosed as, systemic anaphylaxis. The disorder is characterized by repeated, transient, and seemingly unprovoked episodes of hypotensive shock and peripheral edema due to transient endothelial hyperpermeability. SCLS is often accompanied by a monoclonal gammopathy of unknown significance (MGUS). Using Affymetrix Single Nucleotide Polymorphism (SNP) microarrays, we performed the first genome-wide SNP analysis of SCLS in a cohort of 12 disease subjects and 18 controls. Exome capture sequencing was performed on genomic DNA from nine of these patients as validation for the SNP-chip discoveries and de novo data generation. We identified candidate susceptibility loci for SCLS, which included a region flanking CAV3 (3p25.3) as well as SNP clusters in PON1 (7q21.3), PSORS1C1 (6p21.3), and CHCHD3 (7q33). Among the most highly ranked discoveries were gene-associated SNPs in the uncharacterized LOC100130480 gene (rs6417039, rs2004296). Top case-associated SNPs were observed in BTRC (rs12355803, 3rs4436485), ARHGEF18 (rs11668246), CDH13 (rs4782779), and EDG2 (rs12552348), which encode proteins with known or suspected roles in B cell function and/or vascular integrity. 61 SNPs that were significantly associated with SCLS by microarray analysis were also detected and validated by exome deep sequencing. Functional annotation of highly ranked SNPs revealed enrichment of cell projections, cell junctions and adhesion, and molecules containing pleckstrin homology, Ras/Rho regulatory, and immunoglobulin Ig-like C2/fibronectin type III domains, all of which involve mechanistic functions that correlate with the SCLS phenotype. These results highlight SNPs with potential relevance to SCLS.
doi:10.4161/rdis.27445
PMCID: PMC4009617  PMID: 24808988
systemic capillary leak syndrome; genetics, genome-wide SNP study; cell junction; cell adhesion; cytoskeleton; vascular permeability
20.  The Spectral Properties of (-)-Epigallocatechin 3-O-Gallate (EGCG) Fluorescence in Different Solvents: Dependence on Solvent Polarity 
PLoS ONE  2013;8(11):e79834.
(-)-Epigallocatechin 3-O-gallate (EGCG) a molecule found in green tea and known for a plethora of bioactive properties is an inhibitor of heat shock protein 90 (HSP90), a protein of interest as a target for cancer and neuroprotection. Determination of the spectral properties of EGCG fluorescence in environments similar to those of binding sites found in proteins provides an important tool to directly study protein-EGCG interactions. The goal of this study is to examine the spectral properties of EGCG fluorescence in an aqueous buffer (AB) at pH=7.0, acetonitrile (AN) (a polar aprotic solvent), dimethylsulfoxide (DMSO) (a polar aprotic solvent), and ethanol (EtOH) (a polar protic solvent). We demonstrate that EGCG is a highly fluorescent molecule when excited at approximately 275 nm with emission maxima between 350 and 400 nm depending on solvent. Another smaller excitation peak was found when EGCG is excited at approximately 235 nm with maximum emission between 340 and 400 nm. We found that the fluorescence intensity (FI) of EGCG in AB at pH=7.0 is significantly quenched, and that it is about 85 times higher in an aprotic solvent DMSO. The Stokes shifts of EGCG fluorescence were determined by solvent polarity. In addition, while the emission maxima of EGCG fluorescence in AB, DMSO, and EtOH follow the Lippert-Mataga equation, its fluorescence in AN points to non-specific solvent effects on EGCG fluorescence. We conclude that significant solvent-dependent changes in both fluorescence intensity and fluorescence emission shifts can be effectively used to distinguish EGCG in aqueous solutions from EGCG in environments of different polarity, and, thus, can be used to study specific EGCG binding to protein binding sites where the environment is often different from aqueous in terms of polarity.
doi:10.1371/journal.pone.0079834
PMCID: PMC3838354  PMID: 24278192
21.  PD5: A General Purpose Library for Primer Design Software 
PLoS ONE  2013;8(11):e80156.
Background
Complex PCR applications for large genome-scale projects require fast, reliable and often highly sophisticated primer design software applications. Presently, such applications use pipelining methods to utilise many third party applications and this involves file parsing, interfacing and data conversion, which is slow and prone to error. A fully integrated suite of software tools for primer design would considerably improve the development time, the processing speed, and the reliability of bespoke primer design software applications.
Results
The PD5 software library is an open-source collection of classes and utilities, providing a complete collection of software building blocks for primer design and analysis. It is written in object-oriented C++ with an emphasis on classes suitable for efficient and rapid development of bespoke primer design programs. The modular design of the software library simplifies the development of specific applications and also integration with existing third party software where necessary. We demonstrate several applications created using this software library that have already proved to be effective, but we view the project as a dynamic environment for building primer design software and it is open for future development by the bioinformatics community. Therefore, the PD5 software library is published under the terms of the GNU General Public License, which guarantee access to source-code and allow redistribution and modification.
Conclusions
The PD5 software library is downloadable from Google Code and the accompanying Wiki includes instructions and examples: http://code.google.com/p/primer-design
doi:10.1371/journal.pone.0080156
PMCID: PMC3836914  PMID: 24278254
22.  Controls on Water Use for Thermoelectric Generation: Case Study Texas, U.S. 
Environmental Science & Technology  2013;47(19):11326-11334.
Large-scale U.S. dependence on thermoelectric (steam electric) generation requiring water for cooling underscores the need to understand controls on this water use. The study objective was to quantify water consumption and withdrawal for thermoelectric generation, identifying controls, using Texas as a case study. Water consumption for thermoelectricity in Texas in 2010 totaled ∼0.43 million acre feet (maf; 0.53 km3), accounting for ∼4% of total state water consumption. High water withdrawals (26.2 maf, 32.3 km3) mostly reflect circulation between ponds and power plants, with only two-thirds of this water required for cooling. Controls on water consumption include (1) generator technology/thermal efficiency and (2) cooling system, resulting in statewide consumption intensity for natural gas combined cycle generators with mostly cooling towers (0.19 gal/kWh) being 63% lower than that of traditional coal, nuclear, or natural gas steam turbine generators with mostly cooling ponds (0.52 gal/kWh). The primary control on water withdrawals is cooling system, with ∼2 orders of magnitude lower withdrawals for cooling towers relative to once-through ponds statewide. Increases in natural gas combined cycle plants with cooling towers in response to high production of low-cost natural gas has greatly reduced water demand for thermoelectric cooling since 2000.
doi:10.1021/es4029183
PMCID: PMC3805314  PMID: 23937226
24.  The intramembrane protease Sppl2a is required for B cell and DC development and survival via cleavage of the invariant chain 
The protease Sppl2a cleaves the N-terminal fragment of invariant chain (CD74) and is required for efficient B cell development and function.
B cell development requires tight regulation to allow for the generation of a diverse repertoire while preventing the development of autoreactive cells. We report, using N-ethyl-N-nitrosourea (ENU)–induced mutagenesis, the identification of a mutant mouse (chompB) with a block in early B cell development. The blockade occurs after the transitional 1 (T1) stage and leads to a decrease in mature B cell subsets and deficits in T cell–dependent antibody responses. Additionally, chompB mice have decreases in myeloid dendritic cells (DCs). The mutation was mapped to the intramembrane protease signal peptide peptidase-like 2a (Sppl2a), a gene not previously implicated in immune cell development. Proteomic analysis identified the invariant chain (CD74) as a key substrate of Sppl2a and suggests that regulated intramembrane proteolysis of CD74 by Sppl2a contributes to B cell and DC survival. Moreover, these data suggest that modulation of Sppl2a may be a useful therapeutic strategy for treatment of B cell dependent autoimmune disorders.
doi:10.1084/jem.20121072
PMCID: PMC3549714  PMID: 23267013
25.  Updated structure of Drosophila cryptochrome 
Nature  2013;495(7441):E3-E4.
doi:10.1038/nature11995
PMCID: PMC3694752  PMID: 23518567

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