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1.  Leishmania major inhibits IL-12 in macrophages by signaling through CR3 (CD11b/CD18) and downregulation of ETS-mediated transcription 
Parasite immunology  2013;35(12):409-420.
Leishmania major is an etiological agent of cutaneous leishmaniasis. The parasite primarily infects immune sentinel cells, specifically macrophages and dendritic cells, in the mammalian host. Infection is receptor mediated and is known to involve parasite binding to cell surface protein complement receptor 3 (CR3, Mac-1, CD11b/CD18). Engagement of CR3 by various ligands inhibits production of interleukin-12 (IL-12), the cytokine that drives anti-leishmanial T helper 1-type immune responses. Likewise, L. major infection inhibits IL-12 production and activation of host macrophages. Our data indicate that in the absence of CR3, L. major-infected bone marrow-derived macrophages produce more IL-12 and nitric oxide compared to WT cells upon LPS stimulation. We therefore investigated multiple signaling pathways by which L. major may inhibit IL-12 transcription through CR3 ligation. We demonstrate that L. major infection does not elicit significant NFκB p65, MAPK, IRF-1, or IRF-8 activation in WT or CD11b deficient macrophages. Furthermore, infection neither inhibits LPS-induced MAPK or NFκB activation, nor blocks IFN-γ-activated IRF-1 and IRF-8. ETS-mediated transcription, however, is inhibited by L. major infection independently of CR3. Our data indicate that L. major mediated inhibition of IL-12 occurs through CR3 engagement, however the mechanism of inhibition is independent of NFκB, MAPK, IRF, and ETS.
PMCID: PMC4001918  PMID: 23834512
CR3; Leishmania major; Signal transduction; ETS
2.  Predictions of heading date in bread wheat (Triticum aestivum L.) using QTL-based parameters of an ecophysiological model 
Journal of Experimental Botany  2014;65(20):5849-5865.
Highlight text
QTL-based parameters of an ecophysiological model, calibrated on an association genetics panel of 210 genotypes, allowed prediction of heading dates of 80 independent genotypes in six independent experiments with a median prediction error of 5.6 days.
Prediction of wheat phenology facilitates the selection of cultivars with specific adaptations to a particular environment. However, while QTL analysis for heading date can identify major genes controlling phenology, the results are limited to the environments and genotypes tested. Moreover, while ecophysiological models allow accurate predictions in new environments, they may require substantial phenotypic data to parameterize each genotype. Also, the model parameters are rarely related to all underlying genes, and all the possible allelic combinations that could be obtained by breeding cannot be tested with models. In this study, a QTL-based model is proposed to predict heading date in bread wheat (Triticum aestivum L.). Two parameters of an ecophysiological model (V sat and P base, representing genotype vernalization requirements and photoperiod sensitivity, respectively) were optimized for 210 genotypes grown in 10 contrasting location × sowing date combinations. Multiple linear regression models predicting V sat and P base with 11 and 12 associated genetic markers accounted for 71 and 68% of the variance of these parameters, respectively. QTL-based V sat and P base estimates were able to predict heading date of an independent validation data set (88 genotypes in six location × sowing date combinations) with a root mean square error of prediction of 5 to 8.6 days, explaining 48 to 63% of the variation for heading date. The QTL-based model proposed in this study may be used for agronomic purposes and to assist breeders in suggesting locally adapted ideotypes for wheat phenology.
PMCID: PMC4203124  PMID: 25148833
Association genetics; ecophysiological model; gene-based modelling; heading date; phenology; wheat.
4.  Nutrition Prescription to Achieve Positive Outcomes in Chronic Kidney Disease: A Systematic Review 
Nutrients  2014;6(1):416-451.
In Chronic Kidney Disease (CKD), management of diet is important in prevention of disease progression and symptom management, however evidence on nutrition prescription is limited. Recent international CKD guidelines and literature was reviewed to address the following question “What is the appropriate nutrition prescription to achieve positive outcomes in adult patients with chronic kidney disease?” Databases included in the search were Medline and CINAHL using EBSCOhost search engine, Embase and the Cochrane Database of Systematic Reviews published from 2000 to 2009. International guidelines pertaining to nutrition prescription in CKD were also reviewed from 2000 to 2013. Three hundred and eleven papers and eight guidelines were reviewed by three reviewers. Evidence was graded as per the National Health and Medical Research Council of Australia criteria. The evidence from thirty six papers was tabulated under the following headings: protein, weight loss, enteral support, vitamin D, sodium, fat, fibre, oral nutrition supplements, nutrition counselling, including protein and phosphate, nutrients in peritoneal dialysis solution and intradialytic parenteral nutrition, and was compared to international guidelines. While more evidence based studies are warranted, the customary nutrition prescription remains satisfactory with the exception of Vitamin D and phosphate. In these two areas, additional research is urgently needed given the potential of adverse outcomes for the CKD patient.
PMCID: PMC3916870  PMID: 24451311
chronic kidney disease; dietetics; evidence based practice; diet therapy; nutrition prescription
5.  Can the Wolf Motor Function Test Be Streamlined? 
To assess upper extremity (UE) capabilities following stroke, the Wolf Motor Function Test (WMFT) measures time to complete 15 UE tasks and 2 strength tasks, but takes 30 to 45 minutes for the clinician to complete.
In an effort to streamline the WMFT, this study evaluated the association between the magnitude of improvement on any timed task of the WMFT and the change score on all other tasks among participants in the Extremity Constraint Induced Therapy Evaluation (EXCITE) trial.
This association was evaluated using regression methods according to chronicity and controlling for key covariates (functional level, gender, concordance) for log mean WMFT scores.
After controlling for covariates, 6 tasks (hand to table (front), hand to box (front), reach and retrieve, lift can, lift pencil, and fold towel) influenced the overall WMFT score for survivors meeting EXCITE criteria and treated within 3 to 9 months poststroke. Six different tasks (extend elbow weight, hand to box (front), lift can, lift pencil, turn key in lock, and fold towel) influenced the overall WMFT score for those receiving constraint-induced movement therapy (CIMT) 1 year later. The importance of certain tasks relative to others may best represent overall UE function, but this streamlining enables the clinician to prioritize these tasks in the evaluation.
The delineation of those tasks depends on the time poststroke from enrollment to CIMT. This study demonstrates that the WMFT can be streamlined from 17 to 6 tasks.
PMCID: PMC3754424  PMID: 19276293
Wolf Motor Function Test; Streamlined; CIMT therapy; Stroke; Rehabilitation
6.  Coordinated regulation of accessory genetic elements produces cyclic di-nucleotides for Vibrio cholerae virulence 
Cell  2012;149(2):358-370.
The function of the Vibrio 7th pandemic island-1 (VSP-1) in cholera pathogenesis has remained obscure. Utilizing ChIP-seq and RNA-seq to map the regulon of the master virulence regulator ToxT, we identify a TCP island encoded small RNA that reduces the expression of a previously unrecognized VSP-1 encoded transcription factor termed VspR. VspR modulates the expression of several VSP-1 genes including one that encodes a novel class of di-nucleotide cyclase (DncV), which preferentially synthesizes a previously undescribed hybrid cyclic AMP-GMP molecule. We show that DncV is required for efficient intestinal colonization and downregulates V. cholerae chemotaxis, a phenotype previously associated with hyperinfectivity. This pathway couples the actions of previously disparate genomic islands, defines VSP-1 as a pathogenicity island in V. cholerae and implicates its occurrence in 7th pandemic strains as a benefit for host adaptation through the production of a regulatory cyclic di-nucleotide.
PMCID: PMC3620040  PMID: 22500802
7.  Designing Insurance to Promote Use of Childhood Obesity Prevention Services 
Journal of Obesity  2013;2013:379513.
Childhood obesity is a recognized public health crisis. This paper reviews the lessons learned from a voluntary initiative to expand insurance coverage for childhood obesity prevention and treatment services in the United States. In-depth telephone interviews were conducted with key informants from 16 participating health plans and employers in 2010-11. Key informants reported difficulty ensuring that both providers and families were aware of the available services. Participating health plans and employers are beginning new tactics including removing enrollment requirements, piloting enhanced outreach to selected physician practices, and educating providers on effective care coordination and use of obesity-specific billing codes through professional organizations. The voluntary initiative successfully increased private health insurance coverage for obesity services, but the interviews described variability in implementation with both best practices and barriers identified. Increasing utilization of obesity-related health services in the long term will require both family- and provider-focused interventions in partnership with improved health insurance coverage.
PMCID: PMC3649320  PMID: 23691284
8.  Phytoplankton-Specific Response to Enrichment of Phosphorus-Rich Surface Waters with Ammonium, Nitrate, and Urea 
PLoS ONE  2013;8(1):e53277.
Supply of anthropogenic nitrogen (N) to the biosphere has tripled since 1960; however, little is known of how in situ response to N fertilisation differs among phytoplankton, whether species response varies with the chemical form of N, or how interpretation of N effects is influenced by the method of analysis (microscopy, pigment biomarkers). To address these issues, we conducted two 21-day in situ mesocosm (3140 L) experiments to quantify the species- and genus-specific responses of phytoplankton to fertilisation of P-rich lake waters with ammonium (NH4+), nitrate (NO3−), and urea ([NH2]2CO). Phytoplankton abundance was estimated using both microscopic enumeration of cell densities and high performance liquid chromatographic (HPLC) analysis of algal pigments. We found that total algal biomass increased 200% and 350% following fertilisation with NO3− and chemically-reduced N (NH4+, urea), respectively, although 144 individual taxa exhibited distinctive responses to N, including compound-specific stimulation (Planktothrix agardhii and NH4+), increased biomass with chemically-reduced N alone (Scenedesmus spp., Coelastrum astroideum) and no response (Aphanizomenon flos-aquae, Ceratium hirundinella). Principle components analyses (PCA) captured 53.2–69.9% of variation in experimental assemblages irrespective of the degree of taxonomic resolution of analysis. PCA of species-level data revealed that congeneric taxa exhibited common responses to fertilisation regimes (e.g., Microcystis aeruginosa, M. flos-aquae, M. botrys), whereas genera within the same division had widely divergent responses to added N (e.g., Anabaena, Planktothrix, Microcystis). Least-squares regression analysis demonstrated that changes in phytoplankton biomass determined by microscopy were correlated significantly (p<0.005) with variations in HPLC-derived concentrations of biomarker pigments (r2 = 0.13–0.64) from all major algal groups, although HPLC tended to underestimate the relative abundance of cyanobacteria. Together, these findings show that while fertilisation of P-rich lakes with N can increase algal biomass, there is substantial variation in responses of genera and divisions to specific chemical forms of added N.
PMCID: PMC3547936  PMID: 23349705
9.  Is leaf dry matter content a better predictor of soil fertility than specific leaf area? 
Annals of Botany  2011;108(7):1337-1345.
Background and Aims
Specific leaf area (SLA), a key element of the ‘worldwide leaf economics spectrum’, is the preferred ‘soft’ plant trait for assessing soil fertility. SLA is a function of leaf dry matter content (LDMC) and leaf thickness (LT). The first, LDMC, defines leaf construction costs and can be used instead of SLA. However, LT identifies shade at its lowest extreme and succulence at its highest, and is not related to soil fertility. Why then is SLA more frequently used as a predictor of soil fertility than LDMC?
SLA, LDMC and LT were measured and leaf density (LD) estimated for almost 2000 species, and the capacity of LD to predict LDMC was examined, as was the relative contribution of LDMC and LT to the expression of SLA. Subsequently, the relationships between SLA, LDMC and LT with respect to soil fertility and shade were described.
Key Results
Although LD is strongly related to LDMC, and LDMC and LT each contribute equally to the expression of SLA, the exact relationships differ between ecological groupings. LDMC predicts leaf nitrogen content and soil fertility but, because LT primarily varies with light intensity, SLA increases in response to both increased shade and increased fertility.
Gradients of soil fertility are frequently also gradients of biomass accumulation with reduced irradiance lower in the canopy. Therefore, SLA, which includes both fertility and shade components, may often discriminate better between communities or treatments than LDMC. However, LDMC should always be the preferred trait for assessing gradients of soil fertility uncoupled from shade. Nevertheless, because leaves multitask, individual leaf traits do not necessarily exhibit exact functional equivalence between species. In consequence, rather than using a single stand-alone predictor, multivariate analyses using several leaf traits is recommended.
PMCID: PMC3197453  PMID: 21948627
Ellenberg numbers; functional traits; leaf density; leaf nitrogen; leaf size; leaf thickness; relative growth rate (RGR); shade tolerance; variation in trait expression
10.  MetR-Regulated Vibrio cholerae Metabolism Is Required for Virulence 
mBio  2012;3(5):e00236-12.
LysR-type transcriptional regulators (LTTRs) are the largest, most diverse family of prokaryotic transcription factors, with regulatory roles spanning metabolism, cell growth and division, and pathogenesis. Using a sequence-defined transposon mutant library, we screened a panel of V. cholerae El Tor mutants to identify LTTRs required for host intestinal colonization. Surprisingly, out of 38 LTTRs, only one severely affected intestinal colonization in the suckling mouse model of cholera: the methionine metabolism regulator, MetR. Genetic analysis of genes influenced by MetR revealed that glyA1 and metJ were also required for intestinal colonization. Chromatin immunoprecipitation of MetR and quantitative reverse transcription-PCR (qRT-PCR) confirmed interaction with and regulation of glyA1, indicating that misregulation of glyA1 is likely responsible for the colonization defect observed in the metR mutant. The glyA1 mutant was auxotrophic for glycine but exhibited wild-type trimethoprim sensitivity, making folate deficiency an unlikely cause of its colonization defect. MetJ regulatory mutants are not auxotrophic but are likely altered in the regulation of amino acid-biosynthetic pathways, including those for methionine, glycine, and serine, and this misregulation likely explains its colonization defect. However, mutants defective in methionine, serine, and cysteine biosynthesis exhibited wild-type virulence, suggesting that these amino acids can be scavenged in vivo. Taken together, our results suggest that glycine biosynthesis may be required to alleviate an in vivo nutritional restriction in the mouse intestine; however, additional roles for glycine may exist. Irrespective of the precise nature of this requirement, this study illustrates the importance of pathogen metabolism, and the regulation thereof, as a virulence factor.
Vibrio cholerae continues to be a severe cause of morbidity and mortality in developing countries. Identification of V. cholerae factors critical to disease progression offers the potential to develop or improve upon therapeutics and prevention strategies. To increase the efficiency of virulence factor discovery, we employed a regulator-centric approach to multiplex our in vivo screening capabilities and allow whole regulons in V. cholerae to be interrogated for pathogenic potential. We identified MetR as a new virulence regulator and serine hydroxymethyltransferase GlyA1 as a new MetR-regulated virulence factor, both required by V. cholerae to colonize the infant mouse intestine. Bacterial metabolism is a prerequisite to virulence, and current knowledge of in vivo metabolism of pathogens is limited. Here, we expand the known role of amino acid metabolism and regulation in virulence and offer new insights into the in vivo metabolic requirements of V. cholerae within the mouse intestine.
PMCID: PMC3448163  PMID: 23015737
11.  Do Ligand Binding and Solvent Exclusion Alter the Electrostatic Character Within the Oxyanion Hole of an Enzymatic Active Site? 
Journal of the American Chemical Society  2007;129(40):12104-12105.
We report the site-specific incorporation of a thiocyanate vibrational probe into the active site oxyanion hole of ketosteroid isomerase (KSI) to test the effect of hydrophobic steroid binding and solvent exclusion on the local electrostatic environment at this position. While binding of an uncharged ground state steroid analog shifts the observed –CN vibrational frequency by +0.4 cm−1 relative to unliganded KSI, binding of an intermediate steroid analog containing localized negative charge results in a +2.8 cm−1 shift. Based on a Stark tuning rate of 0.7 cm−1/(MV/cm), this shift indicates a fivefold larger change in the projection of the local electric field along the –CN bond in the presence of the charged ligand. Binding of a single ring phenolate with oxyanion charge localization equivalent to the intermediate steroid analog but lacking distal hydrocarbon rings results in an identical –CN peak shift. We conclude that solvent exclusion and replacement by hydrophobic steroid rings negligibly alter the electrostatic environment within the KSI oxyanion hole. Development of localized negative charge analogous to that of the dienolate intermediate during steroid isomerization dramatically increases the magnitude of the local electric field. This increase reflects field contributions from the localized negative charge itself as well as possible increased ordering of active site dipoles in response to charge localization.
PMCID: PMC3171184  PMID: 17854190
12.  Rab11 Is Required for Epithelial Cell Viability, Terminal Differentiation, and Suppression of Tumor-Like Growth in the Drosophila Egg Chamber 
PLoS ONE  2011;6(5):e20180.
The Drosophila egg chamber provides an excellent system in which to study the specification and differentiation of epithelial cell fates because all of the steps, starting with the division of the corresponding stem cells, called follicle stem cells, have been well described and occur many times over in a single ovary.
Methodology/Principal Findings
Here we investigate the role of the small Rab11 GTPase in follicle stem cells (FSCs) and in their differentiating daughters, which include main body epithelial cells, stalk cells and polar cells. We show that rab11-null FSCs maintain their ability to self renew, even though previous studies have shown that FSC self renewal is dependent on maintenance of E-cadherin-based intercellular junctions, which in many cell types, including Drosophila germline stem cells, requires Rab11. We also show that rab11-null FSCs give rise to normal numbers of cells that enter polar, stalk, and epithelial cell differentiation pathways, but that none of the cells complete their differentiation programs and that the epithelial cells undergo premature programmed cell death. Finally we show, through the induction of rab11-null clones at later points in the differentiation program, that Rab11 suppresses tumor-like growth of epithelial cells. Thus, rab11-null epithelial cells arrest differentiation early, assume an aberrant cell morphology, delaminate from the epithelium, and invade the neighboring germline cyst. These phenotypes are associated with defects in E-cadherin localization and a general loss of cell polarity.
While previous studies have revealed tumor suppressor or tumor suppressor-like activity for regulators of endocytosis, our study is the first to identify such activity for regulators of endocytic recycling. Our studies also support the recently emerging view that distinct mechanisms regulate junction stability and plasticity in different tissues.
PMCID: PMC3100332  PMID: 21629779
13.  Stomatal vs. genome size in angiosperms: the somatic tail wagging the genomic dog? 
Annals of Botany  2010;105(4):573-584.
Background and Aims
Genome size is a function, and the product, of cell volume. As such it is contingent on ecological circumstance. The nature of ‘this ecological circumstance’ is, however, hotly debated. Here, we investigate for angiosperms whether stomatal size may be this ‘missing link’: the primary determinant of genome size. Stomata are crucial for photosynthesis and their size affects functional efficiency.
Stomatal and leaf characteristics were measured for 1442 species from Argentina, Iran, Spain and the UK and, using PCA, some emergent ecological and taxonomic patterns identified. Subsequently, an assessment of the relationship between genome-size values obtained from the Plant DNA C-values database and measurements of stomatal size was carried out.
Key Results
Stomatal size is an ecologically important attribute. It varies with life-history (woody species < herbaceous species < vernal geophytes) and contributes to ecologically and physiologically important axes of leaf specialization. Moreover, it is positively correlated with genome size across a wide range of major taxa.
Stomatal size predicts genome size within angiosperms. Correlation is not, however, proof of causality and here our interpretation is hampered by unexpected deficiencies in the scientific literature. Firstly, there are discrepancies between our own observations and established ideas about the ecological significance of stomatal size; very large stomata, theoretically facilitating photosynthesis in deep shade, were, in this study (and in other studies), primarily associated with vernal geophytes of unshaded habitats. Secondly, the lower size limit at which stomata can function efficiently, and the ecological circumstances under which these minute stomata might occur, have not been satisfactorally resolved. Thus, our hypothesis, that the optimization of stomatal size for functional efficiency is a major ecological determinant of genome size, remains unproven.
PMCID: PMC2850795  PMID: 20375204
Stomatal size; genome size; seed size; life history; photosynthesis; allometry; ecology; evolution; SLA; leaf structure; CAM; C4
14.  DNA Damage and Reactive Nitrogen Species are Barriers to Vibrio cholerae Colonization of the Infant Mouse Intestine 
PLoS Pathogens  2011;7(2):e1001295.
Ingested Vibrio cholerae pass through the stomach and colonize the small intestines of its host. Here, we show that V. cholerae requires at least two types of DNA repair systems to efficiently compete for colonization of the infant mouse intestine. These results show that V. cholerae experiences increased DNA damage in the murine gastrointestinal tract. Agreeing with this, we show that passage through the murine gut increases the mutation frequency of V. cholerae compared to liquid culture passage. Our genetic analysis identifies known and novel defense enzymes required for detoxifying reactive nitrogen species (but not reactive oxygen species) that are also required for V. cholerae to efficiently colonize the infant mouse intestine, pointing to reactive nitrogen species as the potential cause of DNA damage. We demonstrate that potential reactive nitrogen species deleterious for V. cholerae are not generated by host inducible nitric oxide synthase (iNOS) activity and instead may be derived from acidified nitrite in the stomach. Agreeing with this hypothesis, we show that strains deficient in DNA repair or reactive nitrogen species defense that are defective in intestinal colonization have decreased growth or increased mutation frequency in acidified nitrite containing media. Moreover, we demonstrate that neutralizing stomach acid rescues the colonization defect of the DNA repair and reactive nitrogen species defense defective mutants suggesting a common defense pathway for these mutants.
Author Summary
Studies on intracellular bacterial pathogens have shown the need for maintaining genomic fidelity to promote colonization. Loss of DNA repair functions often leads to attenuation and rapid clearing of the invading pathogen. However, for some pathogens, an increased mutation rate has been shown to be beneficial for promoting host colonization, presumably by allowing the pathogen to rapidly adapt to adverse host conditions. We asked if the non-invasive pathogen V. cholerae experienced increased DNA damage during infection and if so, how the increased damage influenced host colonization and from where the source of the damage was derived. Our results demonstrate that V. cholerae experiences increased DNA damage during infection in the infant mouse model and that loss of ability to repair this damage results in attenuation of virulence. We specifically show that V. cholerae requires both base excision repair and mismatch repair for efficient intestinal colonization. Furthermore, we present evidence that the source of the DNA damage is derived from reactive nitrogen species (RNS) formed by acidified nitrite in the mouse gut and in doing so we identify a new RNS defense protein found in V. cholerae and several other pathogenic bacteria.
PMCID: PMC3040672  PMID: 21379340
15.  DNA Media Storage 
In 1994, University of Southern California computer scientist Dr. Leonard Adelman solved the Hamiltonian Path Problem using DNA as a computational mechanism. He proved the principle that DNA computing could be used to solve computationally complex problems. Because of the limitations in discovery time, resource requirements, and sequence mismatches, DNA computing has not yet become a commonly accepted practice. However, advancements are continually being discovered that are evolving the field of DNA Computing. Practical applications of DNA are not restricted to computation alone. This research presents a novel approach in which DNA could be used as a means of storing files. Through the use of Multiple Sequence Alignment combined with intelligent heuristics, the most probabilistic file contents can be determined with minimal errors.
PMCID: PMC2901528  PMID: 20622994
16.  Toward DNA-based Security Circuitry: First Step – Random Number Generation 
DNA-based circuit design is an area of research in which traditional silicon-based technologies are replaced by naturally occurring phenomena taken from biochemistry and molecular biology. Our team investigates the implications of DNA-based circuit design in serving security applications. As an initial step we develop a random number generation circuitry. A novel prototype schema employs solid-phase synthesis of oligonucleotides for random construction of DNA sequences. Temporary storage and retrieval is achieved through plasmid vectors.
PMCID: PMC2861576  PMID: 20436783
Index Terms; DNA-based circuit design; Security; Random Number Generation; Oligonucleotide Synthesis; Microarray Fabrication
17.  Differential patterns of reactive oxygen species and antioxidative mechanisms during atrazine injury and sucrose-induced tolerance in Arabidopsis thaliana plantlets 
BMC Plant Biology  2009;9:28.
Besides being essential for plant structure and metabolism, soluble carbohydrates play important roles in stress responses. Sucrose has been shown to confer to Arabidopsis seedlings a high level of tolerance to the herbicide atrazine, which causes reactive oxygen species (ROS) production and oxidative stress. The effects of atrazine and of exogenous sucrose on ROS patterns and ROS-scavenging systems were studied. Simultaneous analysis of ROS contents, expression of ROS-related genes and activities of ROS-scavenging enzymes gave an integrative view of physiological state and detoxifying potential under conditions of sensitivity or tolerance.
Toxicity of atrazine could be related to inefficient activation of singlet oxygen (1O2) quenching pathways leading to 1O2 accumulation. Atrazine treatment also increased hydrogen peroxide (H2O2) content, while reducing gene expressions and enzymatic activities related to two major H2O2-detoxification pathways. Conversely, sucrose-protected plantlets in the presence of atrazine exhibited efficient 1O2 quenching, low 1O2 accumulation and active H2O2-detoxifying systems.
In conclusion, sucrose protection was in part due to activation of specific ROS scavenging systems with consequent reduction of oxidative damages. Importance of ROS combination and potential interferences of sucrose, xenobiotic and ROS signalling pathways are discussed.
PMCID: PMC2661893  PMID: 19284649
19.  Expression of Groucho/TLE proteins during pancreas development 
The full-length mammalian homologs of groucho, Tle1, 2, 3, and 4, act as transcriptional corepressors and are recruited by transcription factors containing an eh1 or WRPW/Y domain. Many transcription factors critical to pancreas development contain a Gro/TLE interaction domain and several have been shown to require Gro/TLE interactions for proper function during neuronal development. However, a detailed analysis of the expression patterns of the Gro/TLE proteins in pancreas development has not been performed. Moreover, little is known about the ability of Gro/TLE proteins to interact with transcription factors in the pancreas.
We describe the expression of Gro/TLE family members, and of 34 different transcription factors that contain a Gro/TLE interaction motif, in the pancreas utilizing nine SAGE libraries created from the developing and adult pancreas, as well as the GenePaint database. Next, we show the dynamic expression of Tle1, 2, 3, 4, 5 and 6 during pancreas development by qRT-PCR. To further define the cell-type specificity of the expression of these proteins we use immunofluorescence to co-localize them with Pdx1 at embryonic day 12.5 (E12.5), Ngn3 at E14.5, Pdx1, Nkx2-2, Insulin, Glucagon, Pancreatic polypeptide and Somatostatin at E18.5, as well as Insulin and Glucagon in the adult. We then show that Tle2 can interact with Nkx2-2, Hes1, Arx, and Nkx6-1 which are all critical factors in pancreas development. Finally, we demonstrate that Tle2 modulates the repressive abilities of Arx in a β-cell line.
Although Tle1, 2, 3, and 4 show overlapping expression in pancreatic progenitors and in the adult islet, the expression of these factors is restricted to different cell types during endocrine cell maturation. Of note, Tle2 and Tle3 are co-expressed with Gro/TLE interaction domain containing transcription factors that are essential for endocrine pancreas development. We further demonstrate that Tle2 can interact with several of these factors and that Tle2 modulate Arx's repressive activity. Taken together our studies suggest that Gro/TLE proteins play a role in the repression of target genes during endocrine cell specification.
PMCID: PMC2551604  PMID: 18778483
20.  Random number generation for DNA-based security circuitry 
BMC Bioinformatics  2008;9(Suppl 7):P13.
PMCID: PMC3313176
21.  Identification of transcripts with enriched expression in the developing and adult pancreas 
Genome Biology  2008;9(6):R99.
The expression profile of different developmental stages of the murine pancreas and predictions of transcription factor interactions, provides a framework for pancreas regulatory networks and development.
Despite recent advances, the transcriptional hierarchy driving pancreas organogenesis remains largely unknown, in part due to the paucity of comprehensive analyses. To address this deficit we generated ten SAGE libraries from the developing murine pancreas spanning Theiler stages 17-26, making use of available Pdx1 enhanced green fluorescent protein (EGFP) and Neurog3 EGFP reporter strains, as well as tissue from adult islets and ducts.
We used a specificity metric to identify 2,536 tags with pancreas-enriched expression compared to 195 other mouse SAGE libraries. We subsequently grouped co-expressed transcripts with differential expression during pancreas development using K-means clustering. We validated the clusters first using quantitative real time PCR and then by analyzing the Theiler stage 22 pancreas in situ hybridization staining patterns of over 600 of the identified genes using the GenePaint database. These were then categorized into one of the five expression domains within the developing pancreas. Based on these results we identified a cascade of transcriptional regulators expressed in the endocrine pancreas lineage and, from this, we developed a predictive regulatory network describing beta-cell development.
Taken together, this work provides evidence that the SAGE libraries generated here are a valuable resource for continuing to elucidate the molecular mechanisms regulating pancreas development. Furthermore, our studies provide a comprehensive analysis of pancreas development, and insights into the regulatory networks driving this process are revealed.
PMCID: PMC2481431  PMID: 18554416
22.  Role of Iron in Human Serum Resistance of the Clinical and Environmental Vibrio vulnificus Genotypes▿  
Applied and Environmental Microbiology  2007;73(23):7501-7505.
We recently reported a simple PCR procedure that targets a sequence variation of the virulence-correlated gene locus vcg. It was found that 90% of all clinical isolates possessed the vcgC sequence variant, while 93% of all environmental isolates possessed the vcgE sequence variant. Here we report that the clinical genotype of Vibrio vulnificus is significantly better able to survive in human serum than is the environmental genotype. The presence of a siderophore-encoding gene, viuB, influenced serum survivability among all isolates of V. vulnificus tested. Those strains positive for viuB (all C-type strains but very few E-type strains) showed greater serum survivability than those lacking viuB (most E-type strains). The addition of iron (in the form of ferric ammonium citrate) to human serum restored the survival of E-type strains lacking viuB to levels not significantly different from those of C-type and E-type strains that possess viuB. These findings suggest that viuB may dictate serum survival in both C- and E-type strains of V. vulnificus and may explain why some strains (C- and E-type strains) are pathogenic and others (predominately E-type strains) are not. Additionally, C-type strains exhibited a cross-protective response against human serum, not exhibited by E-type strains, after incubation under nutrient and osmotic downshift conditions that mimicked estuarine waters. This suggests that the nutrient/osmotic environment may influence the survival of V. vulnificus following entry into the human body, leading to selection of the C genotype over the E genotype.
PMCID: PMC2168077  PMID: 17933938
23.  Expertise of French Laboratories in Detection, Genotyping, and Quantification of Hepatitis C Virus RNA in Serum 
Journal of Clinical Microbiology  2004;42(5):2027-2030.
Before initiating new large-scale therapeutic trials for hepatitis C virus (HCV)-infected patients, the French Health Authorities for HCV research decided to organize an evaluation of the expertise of laboratories that could be engaged to undertake molecular biology assays in such trials; 21 experienced laboratories participated in this national evaluation of laboratory expertise, which was performed in two successive rounds. The first round evaluated the laboratories for their abilities to detect HCV RNA in serum, determine genotypes, and quantify HCV RNA loads. The results observed by qualitative assays for HCV RNA detection were 100% sensitivity and 100% specificity for all laboratories. The genotyping results were 100% concordant for 9 laboratories and greater than 90% for 10 laboratories. By contrast, large coefficients of variation were observed for quantitative determination of HCV RNA loads, leading to a second round with standardized quantitative assays only. The dispersion of the results was larger by the AMPLICOR HCV Monitor assay than by the branched-DNA assay (mean coefficients of variation, 57.4 and 16.9%, respectively). In the majority of cases, discrepancies between the results of the two tests were found for samples with high viral loads. These results indicate the usefulness of validating, by controlling for expertise, both the reliabilities of laboratories involved in multicenter work and the standardized assays chosen for use in the evaluation of the biological impacts of new therapies.
PMCID: PMC404593  PMID: 15131165
24.  Lactobacillus paracasei Continuous Ambulatory Peritoneal Dialysis-Related Peritonitis and Review of the Literature 
Journal of Clinical Microbiology  2003;41(6):2783-2784.
We describe the first case of continuous ambulatory peritoneal dialysis (CAPD)-related peritonitis due to Lactobacillus paracasei. It occurred in a 65-year-old patient with recurrent episodes of peritonitis while he was receiving a prolonged course of intraperitoneal vancomycin. L. paracasei should be considered in the differential diagnosis of pathogens in CAPD-related peritonitis, especially in patients receiving prolonged vancomycin or glycopeptide treatment.
PMCID: PMC156530  PMID: 12791931

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