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1.  Insulin Resistance and Glucose and Lipid Concentrations in a Cohort of Perinatally HIV-Infected Latin American Children 
We measured glucose, insulin, and lipids in 249 perinatally HIV-infected Latin American children. Only one subject had impaired fasting glucose; 6.8% had insulin resistance. Abnormalities in total, LDL and HDL cholesterol, and triglycerides were reported for 13%, 13%, 21%, and 34%, respectively. Continued follow up of this populationis necessary to characterize the evolution and clinical consequences of these findings.
PMCID: PMC3695016  PMID: 23360832
2.  Surfing in tortoises? Empirical signs of genetic structuring owing to range expansion 
Biology Letters  2013;9(3):20121091.
Much of our current knowledge about the genetic dynamics in range expansions originates from models, simulations and microcosm experiments that need to be corroborated by field data. Here, we report a neutral genetic pattern that matches the predictions of the genetic surfing theory. Genetic surfing occurs when repeated founding events and genetic drift act on the wave of advance of an expanding population, promoting strong spatial structure. In the range expansion of the tortoise Testudo graeca from North Africa to southeastern Spain, we found several genetic signatures consistent with surfing: a decrease of genetic diversity with distance from the initial founder area, clinal patterns in allele frequencies, rare African alleles which have become common at distal sites in the Spanish range, and stronger spatial differentiation in the expanded range than in the original one. Our results provide support for the theory that genetic drift can be an important force in shaping the genetic structure of expanding populations.
PMCID: PMC3645018  PMID: 23554278
genetic drift; rare alleles; founding events; isolation-by-distance; spur-thighed tortoise
3.  Cell-free biology: exploiting the interface between synthetic biology and synthetic chemistry 
Current opinion in biotechnology  2012;23(5):672-678.
Just as synthetic organic chemistry once revolutionized the ability of chemists to build molecules (including those that did not exist in nature) following a basic set of design rules, cell-free synthetic biology is beginning to provide an improved toolbox and faster process for not only harnessing but also expanding the chemistry of life. At the interface between chemistry and biology, research in cell-free synthetic systems is proceeding in two different directions: using synthetic biology for synthetic chemistry and using synthetic chemistry to reprogram or mimic biology. In the coming years, the impact of advances inspired by these approaches will make possible the synthesis of non-biological polymers having new backbone compositions, new chemical properties, new structures, and new functions.
PMCID: PMC4038125  PMID: 22483202
4.  A Comparison of Multiple Methods for Estimating Parasitemia of Hemogregarine Hemoparasites (Apicomplexa: Adeleorina) and Its Application for Studying Infection in Natural Populations 
PLoS ONE  2014;9(4):e95010.
Identifying factors influencing infection patterns among hosts is critical for our understanding of the evolution and impact of parasitism in natural populations. However, the correct estimation of infection parameters depends on the performance of detection and quantification methods. In this study, we designed a quantitative PCR (qPCR) assay targeting the 18 S rRNA gene to estimate prevalence and intensity of Hepatozoon infection and compared its performance with microscopy and PCR. Using qPCR, we also compared various protocols that differ in the biological source and the extraction methods. Our results show that the qPCR approach on DNA extracted from blood samples, regardless of the extraction protocol, provided the most sensitive estimates of Hepatozoon infection parameters; while allowed us to differentiate between mixed infections of Adeleorinid (Hepatozoon) and Eimeriorinid (Schellackia and Lankesterella), based on the analysis of melting curves. We also show that tissue and saline methods can be used as low-cost alternatives in parasitological studies. The next step was to test our qPCR assay in a biological context, and for this purpose we investigated infection patterns between two sympatric lacertid species, which are naturally infected with apicomplexan hemoparasites, such as the genera Schellackia (Eimeriorina) and Hepatozoon (Adeleorina). From a biological standpoint, we found a positive correlation between Hepatozoon intensity of infection and host body size within each host species, being significantly higher in males, and higher in the smaller sized host species. These variations can be associated with a number of host intrinsic factors, like hormonal and immunological traits, that require further investigation. Our findings are relevant as they pinpoint the importance of accounting for methodological issues to better estimate infection in parasitological studies, and illustrate how between-host factors can influence parasite distributions in sympatric natural populations.
PMCID: PMC3990604  PMID: 24743340
5.  Shuttle vectors for facile gap repair cloning and integration into a neutral locus in Candida albicans 
Microbiology  2013;159(Pt 3):565-579.
Candida albicans is the most prevalent fungal pathogen of humans. The current techniques used to construct C. albicans strains require integration of exogenous DNA at ectopic locations, which can exert position effects on gene expression that can confound the interpretation of data from critical experiments such as virulence assays. We have identified a large intergenic region, NEUT5L, which facilitates the integration and expression of ectopic genes. To construct and integrate inserts into this novel locus, we re-engineered yeast/bacterial shuttle vectors by incorporating 550 bp of homology to NEUT5L. These vectors allow rapid, facile cloning through in vivo recombination (gap repair) in Saccharomyces cerevisiae and efficient integration of the construct into the NEUT5L locus. Other useful features of these vectors include a choice of three selectable markers (URA3, the recyclable URA3-dpl200 or NAT1), and rare restriction enzyme recognition sites for releasing the insert from the vector prior to transformation into C. albicans, thereby reducing the insert size and preventing integration of non-C. albicans DNA. Importantly, unlike the commonly used RPS1/RP10 locus, integration at NEUT5L has no negative effect on growth rates and allows native-locus expression levels, making it an ideal genomic locus for the integration of exogenous DNA in C. albicans.
PMCID: PMC3709822  PMID: 23306673
6.  Mutations in the Escherichia coli Ribosomal Protein L22 Selectively Suppress the Expression of a Secreted Bacterial Virulence Factor 
Journal of Bacteriology  2013;195(13):2991-2999.
Mutations in the ribosomal protein L22 that impair peptide-mediated translation arrest in Escherichia coli have been shown to reduce the expression of several genes, including secA, which encodes an ATPase that drives protein export via the Sec pathway. Here, we used a comparative proteomic approach to obtain insight into the global effects of the L22(Δ82-84) mutation on gene expression and protein synthesis. While the mutation did not affect or modestly affected the level of most soluble proteins, it dramatically reduced the level of antigen 43 (Ag43), a secreted virulence factor that promotes autoaggregation. The reduced protein concentration correlated with a sharp decrease in the abundance and stability of Ag43 mRNA. We found that the overexpression of secA or the inactivation of genes that encode presecretory and membrane proteins restored Ag43 production in the L22 mutant strain. Furthermore, impairment of the Sec pathway in a wild-type strain reduced Ag43 production but did not significantly affect the synthesis of other presecretory proteins. Taken together, these results indicate that Ag43 gene expression is exquisitely sensitive to the status of the Sec machinery and strongly suggest that the L22 mutation decreases the Ag43 concentration indirectly by reducing secA expression. Our results imply the existence of a novel regulatory mechanism in which the efficiency of protein export is coupled to gene expression and help to explain the modulation of SecA synthesis that has been observed in response to secretion stress.
PMCID: PMC3697531  PMID: 23625843
7.  The Effect of Terminal Cleaning on Environmental Contamination Rates of Multidrug-Resistant Acinetobacter baumannii 
American journal of infection control  2012;40(10):10.1016/j.ajic.2012.05.027.
We evaluated the prevalence of multidrug-resistant Acinetobacter baumannii environmental contamination before and after discharge cleaning in rooms of infected/colonized patients. 46.9% of rooms and 15.3% of sites were found contaminated at baseline. 25% of rooms, 5.5% of sites, were found contaminated post cleaning. Cleaning significantly decreased environmental contamination of A. baumannii, however, persistent contamination represents a significant risk factor for transmission. Further studies on this and more effective cleaning methods are needed.
PMCID: PMC3855251  PMID: 23199726
8.  Cryptic Speciation Patterns in Iranian Rock Lizards Uncovered by Integrative Taxonomy 
PLoS ONE  2013;8(12):e80563.
While traditionally species recognition has been based solely on morphological differences either typological or quantitative, several newly developed methods can be used for a more objective and integrative approach on species delimitation. This may be especially relevant when dealing with cryptic species or species complexes, where high overall resemblance between species is coupled with comparatively high morphological variation within populations. Rock lizards, genus Darevskia, are such an example, as many of its members offer few diagnostic morphological features. Herein, we use a combination of genetic, morphological and ecological criteria to delimit cryptic species within two species complexes, D. chlorogaster and D. defilippii, both distributed in northern Iran. Our analyses are based on molecular information from two nuclear and two mitochondrial genes, morphological data (15 morphometric, 16 meristic and four categorical characters) and eleven newly calculated spatial environmental predictors. The phylogeny inferred for Darevskia confirmed monophyly of each species complex, with each of them comprising several highly divergent clades, especially when compared to other congeners. We identified seven candidate species within each complex, of which three and four species were supported by Bayesian species delimitation within D. chlorogaster and D. defilippii, respectively. Trained with genetically determined clades, Ecological Niche Modeling provided additional support for these cryptic species. Especially those within the D. defilippii-complex exhibit well-differentiated niches. Due to overall morphological resemblance, in a first approach PCA with mixed variables only showed the separation between the two complexes. However, MANCOVA and subsequent Discriminant Analysis performed separately for both complexes allowed for distinction of the species when sample size was large enough, namely within the D. chlorogaster-complex. In conclusion, the results support four new species, which are described herein.
PMCID: PMC3851173  PMID: 24324611
9.  Glucocorticoid-induced hyperglycemia is prevalent and unpredictable for patients undergoing cancer therapy: an observational cohort study 
Current Oncology  2013;20(6):e532-e538.
Patients with cancer are often treated with glucocorticoids (gcs) as part of therapy, which may cause hyperglycemia. We sought to define the prevalence of, and risk factors for, hyperglycemia in this setting.
Adult patients taking gc as part of therapy protocols for primary brain tumour or metastasis, for lymphoma, or for bone marrow transplant (bmt) were screened with random glucometer measurements taken at least 3 hours after the last dose gcs.
We screened 90 patients [44.4% women, 55.6% men; mean age: 59.6 years (range: 25–82 years); mean body mass index (bmi): 26.4 kg/m2 (range: 15.8–45.3 kg/m2)] receiving gc as part of cancer treatment. Mean total daily gc dose in the group was 238.5 mg (range: 30–1067 mg) hydrocortisone equivalents. Hyperglycemia (glucose ≥ 8.0 mmol/L) was found in 58.9% (53 of 90), and diabetes mellitus (dm)–range hyperglycemia (glucose ≥ 11.1 mmol/L) in 18.9% (17 of 90). The mean time from gc ingestion to glucometer testing was 5.5 hours (range: 3–20 hours). Presence of hyperglycemia did not correlate with traditional dm risk factors such as age, sex, bmi, and personal or family history of dm. A longer interval from gc dose to testing (p < 0.05), a higher gc dose (p = 0.04), and a shorter interval between the preceding meal and testing (p = 0.02) were risk factors for hyperglycemia in some patient groups.
Glucocorticoid-induced hyperglycemia is common in patients undergoing cancer treatment and cannot be predicted by traditional risk factors for dm. We recommend that all cancer patients receiving gc be screened for hyperglycemia at least 4–6 hours after gc administration.
PMCID: PMC3851349  PMID: 24311953
Hyperglycemia; glucocorticoids; glucocorticoid-induced diabetes; diabetes mellitus
10.  Origin and Length Distribution of Unidirectional Prokaryotic Overlapping Genes 
G3: Genes|Genomes|Genetics  2013;4(1):19-27.
Prokaryotic unidirectional overlapping genes can be originated by disrupting and replacing of the start or stop codon of one protein-coding gene with another start or stop codon within the adjacent gene. However, the probability of disruption and replacement of a start or stop codon may differ significantly depending on the number and redundancy of the start and stop codons sets. Here, we performed a simulation study of the formation of unidirectional overlapping genes using a simple model of nucleotide change and contrasted it with empirical data. Our results suggest that overlaps originated by an elongation of the 3′-end of the upstream gene are significantly more frequent than those originated by an elongation of the 5′-end of the downstream gene. According to this, we propose a model for the creation of unidirectional overlaps that is based on the disruption probabilities of start codon and stop codon sets and on the different probabilities of phase 1 and phase 2 overlaps. Additionally, our results suggest that phase 2 overlaps are formed at higher rates than phase 1 overlaps, given the same evolutionary time. Finally, we propose that there is no need to invoke selection to explain the prevalence of long phase 1 unidirectional overlaps. Rather, the overrepresentation of long phase 1 relative to long phase 2 overlaps might occur because it is highly probable that phase 2 overlaps are retained as short overlaps by chance. Such a pattern is stronger if selection against very long overlaps is included in the model. Our model as a whole is able to explain to a large extent the empirical length distribution of unidirectional overlaps in prokaryotic genomes.
PMCID: PMC3887535  PMID: 24192837
overlapping genes; prokaryotic genomes; unidirectional genes; overlap length distribution
11.  Antiretroviral Adherence During Pregnancy and Postpartum in Latin America 
AIDS Patient Care and STDs  2012;26(8):486-495.
Adherence to antiretrovirals by pregnant women (and postpartum women if breastfeeding) is crucial to effectively decrease maternal viral load and decrease the risk of mother-to-child transmission of HIV. Our objectives were to describe self-reported adherence to antiretrovirals during the antepartum (after 22 weeks of pregnancy) and postpartum periods (6–12 weeks and 6 months), and identify predictors of adherence among HIV-infected women enrolled and followed in a prospective cohort study from June 2008 to June 2010 at multiple sites in Latin America. Adherence was evaluated using the number of missed and expected doses during the 3 days before the study visit. At the pre-delivery visit, 340 of 376 women (90%) reported perfect adherence. This rate significantly decreased by 6–12 weeks (171/214 [80%]) and 6 months postpartum (163/199 [82%], p<0.01). The odds for less than perfect adherence at the pre-delivery visit was significantly higher for pregnant women with current tobacco use (odds ratio [OR]=2.9, 95% confidence interval [CI]: 1.46–6.14; p=0.0029). At 6–12 weeks postpartum, the probability of non-perfect adherence increased by 6% for each 1 year increase in age (OR=1.06, 95% CI: 1.00–1.12, p=0.0497). At 6 months postpartum, the odds of nonperfect adherence was higher for those who were currently using alcohol (OR=3.04, 95% CI: 1.34–6.90; p=0.0079). Although a self-report measure of adherence based on only 3 days may lead to overestimation of actual adherence over time, women with perfect adherence had lower viral loads and higher CD4 counts. Adherence to antiretrovirals decreased significantly postpartum. Interventions should target women at high risk for lower adherence during pregnancy and postpartum, including tobacco and alcohol users.
PMCID: PMC3462409  PMID: 22663185
12.  Low Bone Mass in Behaviorally HIV-Infected Young Men on Antiretroviral Therapy: Adolescent Trials Network Study 021B 
We report evidence of low bone mass in behaviorally human immunodeficiency virus (HIV)–infected young men on antiretroviral therapy with a relatively recent diagnosis of HIV infection, compared with seronegative controls.
Background. Peak bone mass is achieved in adolescence/early adulthood and is the key determinant of bone mass in adulthood. We evaluated the association of bone mass with human immunodeficiency virus (HIV) infection and antiretroviral therapy (ART) during this critical period among behaviorally HIV–infected young men and seronegative controls.
Methods. HIV-positive men (N = 199) and HIV-negative controls (N = 53), ages 14–25 years, were studied at 15 Adolescent Trials Network for HIV/AIDS Interventions sites. HIV-positive participants were recruited on the basis of ART status: ART-naive (N = 105) or on a regimen containing a nonnucleoside reverse transcriptase inhibitor (NNRTI; N = 52) or protease inhibitor (PI; N = 42). Bone mineral density (BMD) and content (BMC) and body composition were measured by dual-energy X-ray absorptiometry (DXA). Results were compared across groups by linear modeling. Bone results were adjusted for race, body mass index (BMI), and type of DXA (Hologic/Lunar).
Results. The HIV-positive and HIV-negative groups had comparable median age (21 years) and racial/ethnic distribution. Median times since HIV diagnosis were 1.3, 1.9, and 2.2 years in the ART-naive, NNRTI, and PI groups, respectively (P = .01). Total and regional fat were significantly lower in the ART-naive group compared with seronegative controls. Mean BMD and Z scores were generally lower among HIV-positive participants on ART, particularly in the PI group. Average Z scores for the spine were below zero in all 4 groups, including controls.
Conclusions. Young men on ART with a relatively recent diagnosis of HIV infection have lower bone mass than controls. Longitudinal studies are required to determine the impact of impaired accrual or actual loss of bone during adolescence on subsequent fracture risk.
PMCID: PMC3491777  PMID: 22573848
13.  Persistence across Pleistocene ice ages in Mediterranean and extra-Mediterranean refugia: phylogeographic insights from the common wall lizard 
Pleistocene climatic oscillations have played a major role in structuring present-day biodiversity. The southern Mediterranean peninsulas have long been recognized as major glacial refugia, from where Northern Europe was post-glacially colonized. However, recent studies have unravelled numerous additional refugia also in northern regions. We investigated the phylogeographic pattern of the widespread Western Palaearctic lizard Podarcis muralis, using a range-wide multilocus approach, to evaluate whether it is concordant with a recent expansion from southern glacial refugia or alternatively from a combination of Mediterranean and northern refugia.
We analyzed DNA sequences of two mitochondrial (cytb and nd4) and three nuclear (acm4, mc1r, and pdc) gene fragments in individuals from 52 localities across the species range, using phylogenetic and phylogeographic methods. The complex phylogeographic pattern observed, with 23 reciprocally monophyletic allo- parapatric lineages having a Pleistocene divergence, suggests a scenario of long-term isolation in multiple ice-age refugia across the species distribution range. Multiple lineages were identified within the three Mediterranean peninsulas – Iberia, Italy and the Balkans - where the highest genetic diversity was observed. Such an unprecedented phylogeographic pattern - here called “refugia within all refugia” – compasses the classical scenario of multiple southern refugia. However, unlike the southern refugia model, various distinct lineages were also found in northern regions, suggesting that additional refugia in France, Northern Italy, Eastern Alps and Central Balkans allowed the long-term persistence of this species throughout Pleistocene glaciations.
The phylogeography of Podarcis muralis provides a paradigm of temperate species survival in Mediterranean and extra-Mediterranean glacial refugia. Such refugia acted as independent biogeographic compartments for the long-term persistence of this species, for the differentiation of its genetic lineages, and for the short-distance post-glacial re-colonization of neighbouring areas. This finding echoes previous findings from recent phylogeographic studies on species from temperate ecoregions, thus suggesting the need for a reappraisal of the role of northern refugia for glacial persistence and post-glacial assembly of Holarctic biota.
PMCID: PMC3711914  PMID: 23841475
Podarcis muralis; Phylogeography; Western Palaearctic; Glacial refugia; Mediterranean peninsulas; Genetic diversity; Temperate species
14.  Evaluation of viral load thresholds for predicting new WHO Stage 3 and 4 events in HIV-infected children receiving highly active antiretroviral therapy 
This study evaluated a wide range of viral load (VL) thresholds to identify a cut-point that best predicts new clinical events in children on stable highly-active antiretroviral therapy (HAART).
Cox proportional hazards modeling was used to assess the adjusted risk of World Health Organization stage 3 or 4 clinical events (WHO events) as a function of time-varying CD4, VL, and hemoglobin values in a cohort study of Latin American children on HAART ≥ 6 months. Models were fit using different VL cut-points between 400 and 50,000 copies/mL, with model fit evaluated on the basis of the minimum Akaike Information Criterion (AIC) value, a standard model fit statistic.
Models were based on 67 subjects with WHO events out of 550 subjects on study. The VL cutpoints of > 2600 copies/mL and > 32,000 copies/mL corresponded to the lowest AIC values and were associated with the highest hazard ratios [2.0 (p = 0.015) and 2.1 (p = 0.0058), respectively] for WHO events.
In HIV-infected Latin American children on stable HAART, two distinct VL thresholds (> 2,600 copies/mL and > 32,000 copies/mL) were identified for predicting children at significantly increased risk of HIV-related clinical illness, after accounting for CD4 level, hemoglobin level, and other significant factors.
PMCID: PMC3360833  PMID: 22343177
Pediatric HIV infection; viral load monitoring; viral load threshold; Latin America
15.  Methods and recommendations for evaluating and reporting a new diagnostic test 
No standardized guidelines exist for the biostatistical methods appropriate for studies evaluating diagnostic tests. Publication recommendations such as the STARD statement provide guidance for the analysis of data, but biostatistical advice is minimal and application is inconsistent. This article aims to provide a self-contained, accessible resource on the biostatistical aspects of study design and reporting for investigators. For all dichotomous diagnostic tests, estimates of sensitivity and specificity should be reported with confidence intervals. Power calculations are strongly recommended to ensure that investigators achieve desired levels of precision. In the absence of a gold standard reference test, the composite reference standard method is recommended for improving estimates of the sensitivity and specificity of the test under evaluation.
PMCID: PMC3661219  PMID: 22476385
16.  Comparison of culture media for detection of Acinetobacter baumannii in surveillance cultures of critically-ill patients 
The objective of this study was to evaluate the performance of CHROMagar Acinetobacter when compared to sheep blood agar, MacConkey agar and MacConkey agar with 6 µg/ml of imipenem for the detection of A. baumannii in surveillance cultures of hospitalized patients. We utilized peri-anal swabs and sputum samples from patients admitted to the University of Maryland Medical Center ICUs from December 7 through December 21, 2009. Samples were plated onto four media in the following order: (1) 5% sheep blood agar (SBA), (2) MacConkey agar, (3) MacConkey agar with 6 µg/ml of imipenem, and (4) CHROMagar Acinetobacter (CHROMagar). SBA was the gold standard to which all media was compared. There were 165 samples collected during the study period. SBA and CHROMagar detected 18 of 18 (100%) Acinetobacter and 11 of 11 (100%) MDR-A. baumannii. MacConkey agar detected 16 of 18 (89%) Acinetobacter and 10 of 11 (91%) MDR- A. baumannii while MacConkey agar with 6 µg/ml imipenem detected 9 of 11 (82%) MDR-A. baumannii. CHROMagar did not differentiate MDR- A. baumannii from non-MDR-A. baumannii. CHROMagar may be useful for rapid detection of patients with MDR-A. baumannii if improved upon to better select for MDR-A. baumannii.
PMCID: PMC3660032  PMID: 21487763
17.  First-in-man phase I trial of two schedules of the novel synthetic tetrahydroisoquinoline alkaloid PM00104 (Zalypsis) in patients with advanced solid tumours 
British Journal of Cancer  2012;106(8):1379-1385.
PM00104 binds guanines at DNA minor grooves, impacting DNA replication and transcription. A phase I study was undertaken to investigate safety, dose-limiting toxicities (DLTs), recommended phase II dose (RP2D), pharmacokinetics (PKs) and preliminary antitumour activity of PM00104 as a 1- or 3-h infusion three-weekly.
Patients with advanced solid tumours received PM00104 in a dose escalation trial, as guided by toxicity and PK data.
A total of 47 patients were treated; 27 patients on the 1-h schedule (0.23–3.6 mg m−2) and 20 patients on the 3-h schedule (1.8–3.5 mg m−2). Dose-limiting toxicities comprised reversible nausea, vomiting, fatigue, elevated transaminases and thrombocytopenia, establishing the 1-h schedule RP2D at 3.0 mg m−2. With the 3-h schedule, DLTs of reversible hypotension and neutropenia established the RP2D at 2.8 mg m−2. Common PM00104-related adverse events at the RP2D comprised grade 1–2 nausea, fatigue and myelosuppression. In both schedules, PKs increased linearly, but doses over the 1-h schedule RP2D resulted in higher than proportional increases in exposure. A patient with advanced urothelial carcinoma had RECIST shrinkage by 49%, and three patients had RECIST stable disease ⩾6 months.
PM00104 is well tolerated, with preliminary evidence of antitumour activity observed. The 1-h 3-weekly schedule is being assessed in phase II clinical trials.
PMCID: PMC3326684  PMID: 22491421
cytotoxic; novel marine-derived compound; phase I; PM00104
18.  An interesting rare case of double volvulus 
BMJ Case Reports  2011;2011:bcr1020103464.
A 71-year-old female presented with recurrent sigmoid volvulus. In the current admission, her symptoms were not settling on conservative measures and subsequently went on to have laparotomy. During laparotomy, along with the sigmoid volvulus, there was associated gallbladder torsion. About 500 cases of gallbladder volvulus have been published in literature, however, in our literature search, the authors did not find any similar published case presenting with volvulus involving the gallbladder and the sigmoid colon at the same time. This patient went onto have cholecystectomy and sigmoid colectomy and had a good postoperative recovery and was discharged on the tenth postoperative day.
At 6-week postoperative follow-up, she was doing well with no specific concerns.
PMCID: PMC3062870  PMID: 22707549
19.  Molecular Basis for Activation of a Catalytic Asparagine Residue in a Self-Cleaving Bacterial Autotransporter 
Journal of molecular biology  2011;415(1):128-142.
Autotransporters are secreted proteins produced by pathogenic Gram-negative bacteria. They consist of a membrane embedded β-domain and an extracellular passenger domain that is sometimes cleaved and released from the cell surface. We solved the structures of three non-cleavable mutants of the autotransporter EspP to examine how it promotes asparagine cyclization to cleave its passenger. We found that cyclization is facilitated by multiple factors. The active site asparagine is sterically constrained to conformations favorable for cyclization while electrostatic interactions correctly orient the carboxamide group for nucleophilic attack. During molecular dynamics simulations, water molecules were observed to enter the active site and form hydrogen bonds favorable for increasing the nucleophilicity of the active site asparagine. When the activated asparagine attacks its main chain carbonyl carbon the resulting oxyanion is stabilized by a protonated glutamate. Upon cleavage, this proton could be transferred to the leaving amine group helping overcome a significant energy barrier. Together these findings provide insight into factors important for asparagine cyclization, a broadly used mechanism for protein cleavage.
PMCID: PMC3230255  PMID: 22094314
EspP; autocleavage; outer membrane protein; crystal structure; asparagine cyclization
20.  Phylogeography and diversification history of the day-gecko genus Phelsuma in the Seychelles islands 
Lying in a shallow continental shelf cyclically affected by oscillating sea levels since the Miocene, the Seychelles islands are particularly interesting for evolutionary studies. Recent molecular studies are generating an emerging picture of the origin of its biota, yet very little is known regarding their phylogeographic structure or on the factors promoting diversification within the archipelago. Here we aimed to obtain a detailed depiction of the genetic structure and evolution of one of the most widespread vertebrate groups in the archipelago: the day-geckos of the genus Phelsuma. In parallel, we aimed to infer divergence times between species and subspecies, testing a long-standing hypothesis that argues for different time since sympatry between species as the cause of their different morphological differentiation across the archipelago.
Molecular data corroborated the existence of two main lineages, corresponding to the two currently recognized species. Divergences between species likely date back to the Mio-Pliocene, while more recent, Pleistocenic, divergences are suggested within each species. Populations from outer islands share mtDNA haplotypes with inner island populations, suggesting very recent dispersals (or introductions). We found no evidence of current gene flow between species, but results pointed to the possibility of gene flow between (now allopatric) subspecies. Time estimates suggest a synchronous divergence within each species (between island groups).
The geographic patterns of genetic variation agree with previous taxonomic subdivisions within each species and the origin of outer islands populations is clearly tracked. The similar intraspecific divergence time estimates obtained suggest that the differential body-size differentiation between species within each group of islands may be driven by factors other than character displacement proportional to time since sympatry, as previously suggested. These factors could include different habitats/resources available within each island group, niche differentiation and/or character displacement. We also bring again into consideration the hypothesis of body size being influenced by the distribution of native vegetation and social systems within this group, although it remains to be tested. Our results highlight not only the necessity of clarifying the role of ecology and interspecific interactions in this group’s morphological diversification and community assemblage, but also the importance of co-evolutionary mechanisms and their importance for appropriate conservation of island biodiversity. Further, we provide a detailed description of the phylogeographic structure of these taxa across these islands, which still remain poorly characterized in this respect.
PMCID: PMC3598968  PMID: 23289814
Phelsuma; Seychelles; Phylogeography; Species-trees; Diversification; Morphological evolution; Character displacement; Biogeography
21.  Do contact precautions cause depression? A two-year study at a tertiary care medical centre 
The Journal of Hospital Infection  2011;79(2):103-107.
Contact precautions, used to reduce the transmission of infectious diseases, include the wearing of gowns and gloves for room entry. Previous small studies have shown an association between contact precautions and increased symptoms of depression and anxiety. A retrospective cohort of all patients admitted to a tertiary care centre over two years was studied to assess the relationship between contact precautions and depression or anxiety. During the two-year period, there were 70 275 admissions including 28 564 unique non-intensive-care-unit (ICU), non-psychiatric admissions. After adjusting for potential confounders, contact precautions were associated with depression [odds ratio (OR) 1.4, 95% confidence interval (CI) 1.2–1.5] but not with anxiety (OR 0.8, 95% CI 0.7–1.1) in the non-ICU population. Depression was 40% more prevalent among general inpatients on contact precautions.
PMCID: PMC3331706  PMID: 21664000
22.  Extreme genetic diversity in the lizard Atlantolacerta andreanskyi (Werner, 1929): A montane cryptic species complex 
Atlantolacerta andreanskyi is an enigmatic lacertid lizard that, according to the most recent molecular analyses, belongs to the tribe Eremiadini, family Lacertidae. It is a mountain specialist, restricted to areas above 2400 m of the High Atlas Mountains of Morocco with apparently no connection between the different populations. In order to investigate its phylogeography, 92 specimens of A. andreanskyi were analyzed from eight different populations across the distribution range of the species for up to 1108 base pairs of mitochondrial DNA (12S, ND4 and flanking tRNA-His) and 2585 base pairs of nuclear DNA including five loci (PDC, ACM4, C-MOS, RAG1, MC1R).
The results obtained with both concatenated and coalescent approaches and clustering methods, clearly show that all the populations analyzed present a very high level of genetic differentiation for the mitochondrial markers used and are also generally differentiated at the nuclear level.
These results indicate that A. andreanskyi is an additional example of a montane species complex.
PMCID: PMC3492105  PMID: 22946997
Atlantolacerta andreanskyi; Lacertidae; Mountain specialist; High Atlas Mountains; Phylogeography; Morocco
23.  Correlates of HIV Testing History among Urban Youth Recruited through Venue-Based Testing in 15 US Cities 
Sexually transmitted diseases  2011;38(8):691-696.
Adolescents and young adults comprise disproportionately high percentages of individuals living with HIV and those with undiagnosed HIV. Our objective was to determine factors associated with history of HIV testing and receipt of results among a sample of urban, high-risk, sexually active adolescents in 15 U.S. cities.
20–30 sexually active youth, aged 12–24 years, were recruited to participate in an anonymous survey and HIV antibody testing at 2–3 venues per city identified by young men who have sex with men, young women of color, or intravenous drug users.
Of the 1457 participants, 72% reported having been previously tested for HIV (89% of whom were aware of their test results). Our sample was diverse in terms of gender, race/ethnicity, and sexual orientation. Factors found to be predictive of testing typically reflect high risk for HIV, except for some high risk partner characteristics, including having had a partner that made the youth have sex without a condom or had a partner with unknown HIV status. Factors associated with knowledge of serostatus are reported. HIV testing appears to be tied more to STI testing services than to primary care.
More strategies are needed that increase testing, including targeting partners of high-risk individuals, insuring receipt of test results, and increasing testing in primary care settings.
PMCID: PMC3155007  PMID: 21758020
24.  The translational regulatory function of SecM requires the precise timing of membrane targeting 
Molecular microbiology  2011;81(2):540-553.
In E. coli, secA expression is regulated at the translational level by an upstream gene (secM) that encodes a presecretory protein. SecM contains a C-terminal sequence motif that induces a transient translation arrest. Inhibition of SecM membrane targeting prolongs the translation arrest and increases SecA synthesis by concomitantly altering the structure of the secM-secA mRNA. Here we show that the SecM signal peptide plays an essential role in this regulatory process by acting as a molecular timer that coordinates membrane targeting with the synthesis of the arrest motif. We found that signal peptide mutations that alter targeting kinetics and insertions or deletions that change the distance between the SecM signal peptide and the arrest motif perturb the balance between the onset and release of arrest that is required to regulate SecA synthesis. Furthermore, we found that the strength of the interaction between the ribosome and the SecM arrest motif is calibrated to ensure the release of arrest upon membrane targeting. Our results strongly suggest that several distinctive features of the SecM protein evolved as a consequence of constraints imposed by the ribosome and the Sec machinery.
PMCID: PMC3134173  PMID: 21635582
membrane targeting; ribosome; SecM; signal peptide; translational regulation
25.  The double life of a bacterial lipoprotein 
Molecular microbiology  2011;79(5):1128-1131.
It has been known for many years that the small lipoprotein Lpp, which is the most abundant protein in E. coli, exists in two forms. The “bound” form of the protein is tethered to the outer membrane (OM) by its N-terminal lipid moiety and covalently attached to the cell wall by its C-terminal lysine residue. The exact location of the “free” form, however, has never been determined. In this issue of Molecular Microbiology, Cowles et al. demonstrate that the free form of Lpp is an integral OM protein whose C-terminus is exposed on the cell surface. The new study provides the first example of a lipoprotein that has a dual localization and adds to a growing body of evidence that lipoproteins can span the OM despite the lack of an obvious transmembrane segment. Furthermore, the new results raise intriguing questions about the assembly of both lipoproteins and other types of OM proteins.
PMCID: PMC3088913  PMID: 21338414

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