Subcellular Localization of Proteins in the Anaerobic Sulfate Reducer Desulfovibrio vulgaris via SNAP-tag Labeling and Photoconversion

Systems Biology studies the temporal and spatial 3D distribution of macromolecular complexes with the aim that such knowledge will allow more accurate modeling of biological function and will allow mathematical prediction of cellular behavior. However, in order to accomplish accurate modeling precise knowledge of spatial 3D organization and distribution inside cells is necessary. And while a number of macromolecular complexes may be identified by its 3D structure and molecular characteristics alone, the overwhelming number of proteins will need to be localized using a reporter tag. GFP and its derivatives (XFPs) have been traditionally employed for subcelllar localization using photoconversion approaches, but this approach cannot be taken for obligate anaerobic bacteria, where the intolerance towards oxygen prevents XFP approaches. As part of the GTL-funded PCAP project (now ENIGMA) genetic tools have been developed for the anaerobe sulfate reducer Desulfovibrio vulgaris that allow the high-throughput generation of tagged-protein mutant strains, with a focus on the commercially available SNAP-tag cell system (New England Biolabs, Ipswich, MA), which is based on a modified O6-alkylguanine-DNA alkyltransferase (AGT) tag, that has a dead-end reaction with a modified O6-benzylguanine (BG) derivative and has been shown to function under anaerobic conditions. After initial challenges with respect to variability, robustness and specificity of the labeling signal we have optimized the labeling. Over the last year, as a result of the optimized labeling protocol, we now obtain robust labeling of 20 out of 31 SNAP strains. Labeling for 13 strains were confirmed at least five times. We have also successfully performed photoconversion on 5 of these 13 strains, with distinct labeling patterns for different strains. For example, DsrC robustly localizes to the periplasmic portion of the inner membrane, where as a DNA-binding protein localizes to the center of the cell, where the chromosome is located. Two other proteins - Thiosulfate reductase and ATP binding protein were found to be cytoplasmically distributed, whereas a molybdenum transporter was found to locate to the cell periphery. We judge labeling outcome by (1) SDS gel electrophoresis, followed by direct fluorescence imaging of the gel to address specificity of labeling/confirm expected molecular weight, and subsequent Coomassie analysis to ensure comparable protein levels (2) fluorescence intensity of culture by plate reader for statistical sampling (after adjustment for respective cell numbers) and (3) fluorescence microscopy for addressing cell-to-cell signal variation and potential localization patterns. All three assays were usually found to be consistent with one another. While we have been able to improve the efficacy of photoconversion by drastically reducing (eliminating) non-specific binding with our altered labeling protocol, we are currently working on reducing non-specific photoconversion reaction arising occasionally in non-labeled cells. In addition, we have confirmed the presence of SNAP tagged constructs in three recently cloned E.coli strains under promotor control, and are in the process of utilizing them for evaluating the sensitivity of the photoconversion protocol. Fluorescent Activated Cell Sorting was successfully applied to labeled E.coli cells containing SNAP tagged AtpA protein. Different batches of sorted cells, representing low and high labeling intensity, were re-grown and re-labeled and displayed a labeling efficiency similar to the starter culture, supporting the notion that cell-to-cell differences in labeling reflect difference in protein expression, rather then genetic differences

Disparities and risks of sexually transmissible infections among men who have sex with men in China: a meta-analysis and data synthesis.

BACKGROUND: Sexually transmitted infections (STIs), including Hepatitis B and C virus, are emerging public health risks in China, especially among men who have sex with men (MSM). This study aims to assess the magnitude and risks of STIs among Chinese MSM. METHODS: Chinese and English peer-reviewed articles were searched in five electronic databases from January 2000 to February 2013. Pooled prevalence estimates for each STI infection were calculated using meta-analysis. Infection risks of STIs in MSM, HIV-positive MSM and male sex workers (MSW) were obtained. This review followed the PRISMA guidelines and was registered in PROSPERO. RESULTS: Eighty-eight articles (11 in English and 77 in Chinese) investigating 35,203 MSM in 28 provinces were included in this review. The prevalence levels of STIs among MSM were 6.3% (95% CI: 3.5-11.0%) for chlamydia, 1.5% (0.7-2.9%) for genital wart, 1.9% (1.3-2.7%) for gonorrhoea, 8.9% (7.8-10.2%) for hepatitis B (HBV), 1.2% (1.0-1.6%) for hepatitis C (HCV), 66.3% (57.4-74.1%) for human papillomavirus (HPV), 10.6% (6.2-17.6%) for herpes simplex virus (HSV-2) and 4.3% (3.2-5.8%) for Ureaplasma urealyticum. HIV-positive MSM have consistently higher odds of all these infections than the broader MSM population. As a subgroup of MSM, MSW were 2.5 (1.4-4.7), 5.7 (2.7-12.3), and 2.2 (1.4-3.7) times more likely to be infected with chlamydia, gonorrhoea and HCV than the broader MSM population, respectively. CONCLUSION: Prevalence levels of STIs among MSW were significantly higher than the broader MSM population. Co-infection of HIV and STIs were prevalent among Chinese MSM. Integration of HIV and STIs healthcare and surveillance systems is essential in providing effective HIV/STIs preventive measures and treatments. TRIAL REGISTRATION: PROSPERO NO: CRD42013003721

Development of SimCells as a novel chassis for functional biosensors

This work serves as a proof-of-concept for bacterially derived SimCells (Simple Cells), which contain the cell machinery from bacteria and designed DNA (or potentially a simplified genome) to instruct the cell to carry out novel, specific tasks. SimCells represent a reprogrammable chassis without a native chromosome, which can host designed DNA to perform defined functions. In this paper, the use of Escherichia coli MC1000 ∆minD minicells as a non-reproducing chassis for SimCells was explored, as demonstrated by their ability to act as sensitive biosensors for small molecules. Highly purified minicells derived from E. coli strains containing gene circuits for biosensing were able to transduce the input signals from several small molecules (glucarate, acrylate and arabinose) into the production of green fluorescent protein (GFP). A mathematical model was developed to fit the experimental data for induction of gene expression in SimCells. The intracellular ATP level was shown to be important for SimCell function. A purification and storage protocol was developed to prepare SimCells which could retain their functions for an extended period of time. This study demonstrates that SimCells are able to perform as 'smart bioparticles' controlled by designed gene circuits

Biomarker-Based HIV Incidence in a Community Sample of Men Who Have Sex with Men in Paris, France

BACKGROUND: Population-based estimates of HIV incidence in France have revealed that men who have sex with men (MSM) are the most affected population and contribute to nearly half of new infections each year. We sought to estimate HIV incidence among sexually active MSM in Paris gay community social venues. METHODOLOGY/ PRINCIPAL FINDINGS: A cross-sectional survey was conducted in 2009 in a sample of commercial venues such as bars, saunas and backrooms. We collected a behavioural questionnaire and blood sample. Specimens were tested for HIV infection and positive specimens then tested for recent infection by the enzyme immunoassay for recent HIV-1 infection (EIA-RI). We assessed the presence of antiretroviral therapy among infected individuals to rule out treated patients in the algorithm that determined recent infection. Biomarker-based cross-sectional incidence estimates were calculated. We enrolled 886 MSM participants among which 157 (18%) tested HIV positive. In positive individuals who knew they were infected, 75% of EIA-RI positive results were due to ART. Of 157 HIV positive specimens, 15 were deemed to be recently infected. The overall HIV incidence was estimated at 3.8% person-years (py) [95%CI: 1.5-6.2]. Although differences were not significant, incidence was estimated to be 3.5% py [0.1-6.1] in men having had a negative HIV test in previous year and 4.8% py [0.1-10.6] in men having had their last HIV test more than one year before the survey, or never tested. Incidence was estimated at 4.1% py [0-8.3] in men under 35 years and 2.5% py [0-5.4] in older men. CONCLUSIONS/ SIGNIFICANCE: This is the first community-based survey to estimate HIV incidence among MSM in France. It includes ART detection and reveals a high level of HIV transmission in sexually active individuals, despite a high uptake of HIV testing. These data call for effective prevention programs targeting MSM engaged in high-risk behaviours

Evolution of a supergene that regulates a trans-species social polymorphism

Supergenes are clusters of linked genetic loci that jointly affect the expression of complex phenotypes, such as social organization. Little is known about the origin and evolution of these intriguing genomic elements. Here we analyse whole-genome sequences of males from native populations of six fire ant species and show that variation in social organization is under the control of a novel supergene haplotype (termed Sb), which evolved by sequential incorporation of three inversions spanning half of a 'social chromosome'. Two of the inversions interrupt protein-coding genes, resulting in the increased expression of one gene and modest truncation in the primary protein structure of another. All six socially polymorphic species studied harbour the same three inversions, with the single origin of the supergene in their common ancestor inferred by phylogenomic analyses to have occurred half a million years ago. The persistence of Sb along with the ancestral SB haplotype through multiple speciation events provides a striking example of a functionally important trans-species social polymorphism presumably maintained by balancing selection. We found that while recombination between the Sb and SB haplotypes is severely restricted in all species, a low level of gene flux between the haplotypes has occurred following the appearance of the inversions, potentially mitigating the evolutionary degeneration expected at genomic regions that cannot freely recombine. These results provide a detailed picture of the structural genomic innovations involved in the formation of a supergene controlling a complex social phenotype

Metacarpal trabecular bone varies with distinct hand-positions used in hominid locomotion

Trabecular bone remodels during life in response to loading and thus should, at least in part, reflect potential variation in the magnitude, frequency and direction of joint loading across different hominid species. Here we analyse the trabecular structure across all non-pollical metacarpal distal heads (Mc2-5) in extant great apes, expanding on previous volume of interest and whole-epiphysis analyses that have largely focussed on only the first or third metacarpal. Specifically, we employ both a univariate statistical mapping and a multivariate approach to test for both inter-ray and interspecific differences in relative trabecular bone volume fraction (RBV/TV) and degree of anisotropy (DA) in Mc2-5 subchondral trabecular bone. Results demonstrate that while DA values only separate Pongo from African apes (Pan troglodytes, Pan paniscus, Gorilla gorilla), RBV/TV distribution varies with the predicted loading of the metacarpophalangeal (McP) joints during locomotor behaviours in each species. Gorilla exhibits a relatively dorsal distribution of RBV/TV consistent with habitual hyper-extension of the McP joints during knuckle-walking, whereas Pongo has a palmar distribution consistent with flexed McP joints used to grasp arboreal substrates. Both Pan species possess a disto-dorsal distribution of RBV/TV, compatible with multiple hand postures associated with a more varied locomotor regime. Further inter-ray comparisons reveal RBV/TV patterns consistent with varied knuckle-walking postures in Pan species in contrast to higher RBV/TV values toward the midline of the hand in Mc2 and Mc5 of Gorilla, consistent with habitual palm-back knuckle-walking. These patterns of trabecular bone distribution and structure reflect different behavioural signals that could be useful for determining the behaviours of fossil hominins

Exopolysaccharide Microchannels Direct Bacterial Motility and Organize Multicellular Behavior

The myxobacteria are a family of soil bacteria that form biofilms of complex architecture, aligned multilayered swarms or fruiting body structures that are simple or branched aggregates containing myxospores. Here, we examined the structural role of matrix exopolysaccharide (EPS) in the organization of these surface-dwelling bacterial cells. Using time-lapse light and fluorescence microscopy, as well as transmission electron microscopy and focused ion beam/scanning electron microscopy (FIB/SEM) electron microscopy, we found that Myxococcus xanthus cell organization in biofilms is dependent on the formation of EPS microchannels. Cells are highly organized within the three-dimensional structure of EPS microchannels that are required for cell alignment and advancement on surfaces. Mutants lacking EPS showed a lack of cell orientation and poor colony migration. Purified, cell-free EPS retains a channel-like structure, and can complement EPS- mutant motility defects. In addition, EPS provides the cooperative structure for fruiting body formation in both the simple mounds of M. xanthus and the complex, tree-like structures of Chondromyces crocatus. We furthermore investigated the possibility that EPS impacts community structure as a shared resource facilitating cooperative migration among closely related isolates of M. xanthus