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1.  Analysis of the Outer Membrane Proteome and Secretome of Bacteroides fragilis Reveals a Multiplicity of Secretion Mechanisms 
PLoS ONE  2015;10(2):e0117732.
Bacteroides fragilis is a widely distributed member of the human gut microbiome and an opportunistic pathogen. Cell surface molecules produced by this organism likely play important roles in colonization, communication with other microbes, and pathogenicity, but the protein composition of the outer membrane (OM) and the mechanisms used to transport polypeptides into the extracellular space are poorly characterized. Here we used LC-MS/MS to analyze the OM proteome and secretome of B. fragilis NCTC 9343 grown under laboratory conditions. Of the 229 OM proteins that we identified, 108 are predicted to be lipoproteins, and 61 are predicted to be TonB-dependent transporters. Based on their proximity to genes encoding TonB-dependent transporters, many of the lipoprotein genes likely encode proteins involved in nutrient or small molecule uptake. Interestingly, protease accessibility and biotinylation experiments indicated that an unusually large fraction of the lipoproteins are cell-surface exposed. We also identified three proteins that are members of a novel family of autotransporters, multiple potential type I protein secretion systems, and proteins that appear to be components of a type VI secretion apparatus. The secretome consisted of lipoproteins and other proteins that might be substrates of the putative type I or type VI secretion systems. Our proteomic studies show that B. fragilis differs considerably from well-studied Gram-negative bacteria such as Escherichia coli in both the spectrum of OM proteins that it produces and the range of secretion strategies that it utilizes.
PMCID: PMC4319957  PMID: 25658944
2.  Leveraging practice-based research networks to accelerate implementation and diffusion of chronic kidney disease guidelines in primary care practices: a prospective cohort study 
Four practice-based research networks (PBRNs) participated in a study to determine whether networks could increase dissemination, implementation, and diffusion of evidence-based treatment guidelines for chronic kidney disease by leveraging early adopter practices.
Motivated practices from four PBRNs received baseline and periodic performance feedback, academic detailing, and weekly practice facilitation for 6 months during wave I of the study. Each wave I practice then recruited two additional practices (wave II), which received performance feedback and academic detailing and participated in monthly local learning collaboratives led by the wave I clinicians. They received only monthly practice facilitation. The primary outcomes were adherence to primary care-relevant process-of-care recommendations from the National Kidney Foundation Kidney Disease Outcomes Quality Initiative Guidelines. Performance was determined retrospectively by medical records abstraction. Practice priority, change capacity, and care process content were measured before and after the interventions.
Following the intervention, wave I practices increased the use of ACEIs/ARBs, discontinuation of NSAIDs, testing for anemia, and testing and/or treatment for vitamin D deficiency. Most were able to recruit two additional practices for wave II, and wave II practices also increased their use of ACEIs/ARBs and testing and/or treatment of vitamin D deficiency.
With some assistance, early adopter practices can facilitate the diffusion of evidence-based approaches to other practices. PBRNs are well-positioned to replicate this process for other evidence-based innovations.
Electronic supplementary material
The online version of this article (doi:10.1186/s13012-014-0169-x) contains supplementary material, which is available to authorized users.
PMCID: PMC4245828  PMID: 25416998
Implementation; Diffusion; Primary care; Practice-based research network; Chronic kidney disease
3.  Type V Secretion: the Autotransporter and Two-Partner Secretion Pathways 
EcoSal Plus  2010;2010:10.1128/ecosalplus.4.3.6.
PMCID: PMC4229083  PMID: 25401112
4.  The Inversion of the Control Region in Three Mitogenomes Provides Further Evidence for an Asymmetric Model of Vertebrate mtDNA Replication 
PLoS ONE  2014;9(9):e106654.
Mitochondrial genomes are known to have a strong strand-specific compositional bias that is more pronounced at fourfold redundant sites of mtDNA protein-coding genes. This observation suggests that strand asymmetries, to a large extent, are caused by mutational asymmetric mechanisms. In vertebrate mitogenomes, replication and not transcription seems to play a major role in shaping compositional bias. Hence, one can better understand how mtDNA is replicated – a debated issue – through a detailed picture of mitochondrial genome evolution. Here, we analyzed the compositional bias (AT and GC skews) in protein-coding genes of almost 2,500 complete vertebrate mitogenomes. We were able to identify three fish mitogenomes with inverted AT/GC skew coupled with an inversion of the Control Region. These findings suggest that the vertebrate mitochondrial replication mechanism is asymmetric and may invert its polarity, with the leading-strand becoming the lagging-strand and vice-versa, without compromising mtDNA maintenance and expression. The inversion of the strand-specific compositional bias through the inversion of the Control Region is in agreement with the strand-displacement model but it is also compatible with the RITOLS model of mtDNA replication.
PMCID: PMC4182315  PMID: 25268704
5.  Reconstitution of bacterial autotransporter assembly using purified components 
eLife  2014;3:e04234.
Autotransporters are a superfamily of bacterial virulence factors consisting of an N-terminal extracellular (‘passenger’) domain and a C-terminal β barrel (‘β’) domain that resides in the outer membrane (OM). The mechanism by which the passenger domain is secreted is poorly understood. Here we show that a conserved OM protein insertase (the Bam complex) and a molecular chaperone (SurA) are both necessary and sufficient to promote the complete assembly of the Escherichia coli O157:H7 autotransporter EspP in vitro. Our results indicate that the membrane integration of the β domain is the rate-limiting step in autotransporter assembly and that passenger domain translocation does not require the input of external energy. Furthermore, experiments using nanodiscs strongly suggest that autotransporter assembly is catalyzed by a single copy of the Bam complex. Finally, we describe a method to purify a highly active form of the Bam complex that should facilitate the elucidation of its function.
eLife digest
Disease-causing bacteria release molecules called virulence factors to help them infect their host. These virulence factors need to pass through the membrane that surrounds the cell. Indeed, some bacteria, such as Escherichia coli, have two membranes, so some virulence factors need to pass through an extra membrane.
One group of virulence factors found in E. coli are called autotransporters. These proteins have two sections: the passenger domain, which is the main part of the virulence factor, and the β domain, which anchors the autotransporter in the outer membrane. Once the passenger domain is outside the cell, the link to the β domain can be broken to release the virulence factor. However, we do not know how the passenger domain passes through the outer membrane.
By studying an E. coli autotransporter called EspP, Roman-Hernandez et al. have now identified the other proteins that are required for the β domain to insert into an artificial membrane, and allow the passenger domain to pass through the membrane. These other proteins are a group of proteins called the Bam complex and a chaperone protein called SurA. The experiments also show that an external source of energy is not needed to drive this process, and they suggest that the passenger domain moves through a hole in the outer membrane formed by the β domain and/or the Bam complex. Roman-Hernandez et al. also developed a new way to purify the Bam complex that should help all researchers working on this set of proteins.
PMCID: PMC4174580  PMID: 25182416
membrane proteins; protein folding; protein translocation; virulence factors; E. coli
6.  Undervaccination of Perinatally HIV-Infected and HIV-Exposed Uninfected Children in Latin America and the Caribbean 
Perinatally HIV-infected children (PHIV) may be at risk of undervaccination. Vaccination coverage rates among PHIV and HIV-exposed uninfected children (HEU) in Latin America and the Caribbean were compared.
All PHIV and HEU children born from 2002–2007 that were enrolled in a multi-site observational study conducted in Latin America and the Caribbean were included in this analysis. Children were classified as up to date (UTD) if they had received the recommended number of doses of each vaccine at the appropriate intervals by 12 and 24 months of age. Fisher’s exact test was used to analyze the data. Covariates potentially associated with a child’s HIV status were considered in multivariable logistic regression modeling.
Of 1156 eligible children, 768 (66.4%) were HEU and 388 (33.6%) were PHIV. HEU children were significantly (p<0.01) more likely to be UTD by 12 and 24 months of age for all vaccines examined. Statistically significant differences persisted when the analyses were limited to children enrolled prior to 12 months of age. Controlling for birth weight, sex, primary caregiver education and any use of tobacco, alcohol or illegal drugs during pregnancy did not contribute significantly to the logistic regression models.
PHIV children were significantly less likely than HEU children to be UTD for their childhood vaccinations at 12 and 24 months of age, even when limited to children enrolled before 12 months of age. Strategies to increase vaccination rates in PHIV are needed.
PMCID: PMC3717191  PMID: 23860480
Pediatric HIV infection; vaccination; Latin America
7.  Exploration of the Effect of Tobacco Smoking on Metabolic Measures in Young People Living with HIV 
AIDS Research and Treatment  2014;2014:740545.
We conducted cross-sectional, multicenter studies in HIV-positive young women and men to assess metabolic and morphologic complications from tobacco smoking in 372 behaviorally infected HIV-positive youth, aged 14–25 years. Measurements included self-reported tobacco use, fasting lipids, glucose, fat distribution, and bone mineral density (BMD; dual-energy X-ray absorptiometry scans). Overall, 144 (38.7%) self-reported smoking tobacco and 69 (47.9%) of these reported smoking greater than five cigarettes per day. Smokers versus nonsmokers had lower mean total cholesterol (146.0 versus 156.1 mg/dL; P < 0.01) and lower mean total body fat percent (24.1% versus 27.2%, P = 0.03). There was no difference between smokers and nonsmokers in fasting glucose or BMD. There appear to be only minimal effects from tobacco smoking on markers of cardiac risk and bone health in this population of HIV-positive youth. While these smokers may not have had sufficient exposure to tobacco to detect changes in the outcome measures, given the long-term risks associated with smoking and HIV, it is critical that we encourage HIV-positive youth smokers to quit before the deleterious effects become apparent.
PMCID: PMC4119894  PMID: 25114801
8.  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
9.  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
10.  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
11.  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
12.  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
13.  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
14.  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
15.  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
16.  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
17.  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
18.  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
19.  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
20.  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
21.  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
22.  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
23.  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
24.  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
25.  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

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