Role and visualization of the single-stranded and double-stranded DNA in the biofilm of Neisseria gonorrhoeae

Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhoea, is a Gram-negative human-adapted diplococcus. 80% of the clinical N. gonorrhoeae isolates encode an unusual Type IV secretion system (T4SS) within the horizontally acquired region - the Gonococcal Genetic Island (GGI). Next to the Single Stranded DNA binding protein (SSB) encoded on the chromosome, a second SSB, SsbB, is encoded within the GGI. SSBs are highly conserved, essential proteins found in all kingdoms of life. They bind single stranded DNA (ssDNA) with high affinity, have low sequence specificity and are involved in DNA recombination, DNA replication and DNA repair. A second copy of SSBs can be encoded on the chromosome or on a plasmid. These SSBs can be involved in diverse mechanisms like natural competence, plasmid segregation and DNA transport. We analyzed the physiological role of SsbB and characterized its function biochemically. We found that close homologs of SsbB are located within a conserved genetic cluster found in genetic islands of different proteobacteria. This cluster encodes DNA processing enzymes such as the ParA and ParB partitioning proteins, the TopB topoisomerase and four conserved hypothetical proteins. The SsbB homologs found in these clusters form a family separated from other ssDNA binding proteins. Remarkably, in contrast to most other SSBs, SsbB did not complement the Escherichia coli ssb deletion mutant. Purified SsbB formed a stable tetramer. Electrophoretic mobility shift assays, fluorescence titration assays, as well as atomic force microscopy demonstrated that SsbB binds ssDNA specifically with high affinity. SsbB binds single stranded DNA with minimal binding frames of 15 and 70 nucleotides for one or two SsbB tetramersrespectively. The binding mode was independent of increasing Mg2+ or NaCl concentrations. No role of SsbB in ssDNA secretion or DNA uptake could be identified, but SsbB strongly stimulated Topoisomerase I activity. We propose that these novel SsbBs play an unknown role in the maintenance of genetic islands. Remarkably the T4SS of N.gonorrhoeae was shown to secrete ssDNA directly into the medium. Currently nothing is known about the exact function of the secreted DNA. Studies have shown that not only exopolysaccharides but also extracellular DNA (eDNA) can play an important role in the initial establishment of biofilms. The composition and the origin of the eDNA are not completely understood. N. gonorrhoeae biofilms contain large amounts of extracellular DNA which play an important role in biofilm formation. To study the role of ssDNA in biofilm formation, the development of biofilms of N. gonorrhoeae strain MS11 was compared with a MS11 ΔtraB strain, which is impaired in ssDNA secretion and the MS11 ΔtraB::traB complementation strain in which ssDNA secretion is restored. Furthermore, the role of ssDNA in biofilm formation was studied by treating biofilms with Exonuclease I which specifically degrades ssDNA. These experiments demonstrated that the secreted ssDNA strongly stimulated biofilm formation especially during initial attachment. Furthermore, we developed a unique technique to separately detect ssDNA and dsDNA. To visualize ssDNA, SSB proteins, which specifically bind ssDNA with high affinity in a sequence-independent manner, were employed. The highly stable SSB protein from Thermoanaerobacter tengcongensis, and different cysteine mutants within this protein were purified to homogeneity. The different cysteines containing proteins were labelled with environmentally sensitive fluorescent probes. Specific combinations of cysteine mutants and fluorescent probes were selected to obtain proteins that showed a strongly increased fluorescence upon binding of ssDNA. To visualize dsDNA the thermostable double stranded DNA binding protein Sac7d of Sulfolobus acidocadarius was used. Both proteins were applied to visualize single- and double-stranded DNA in biofilms and planktonic cultures. Remarkably, only dsDNA could be detected in N. gonorrhoeae biofilms using this approach. We conclude that ssDNA plays an important role in biofilm formation, but that the amount of ssDNA necessary is much lower than the amount of dsDNA found in mature biofilms

Tumour Cannabinoid CB1 Receptor and Phosphorylated Epidermal Growth Factor Receptor Expression Are Additive Prognostic Markers for Prostate Cancer

BACKGROUND: In cultured prostate cancer cells, down-regulation of epidermal growth factor receptor (EGFR) has been implicated in mediating the antiproliferative effect of the endogenous cannabinoid (CB) ligand anandamide. Using a well-characterised cohort of prostate cancer patients, we have previously reported that expression levels of phosphorylated EGFR (pEGFR-IR) and CB(1) receptor (CB(1)IR) in tumour tissue at diagnosis are markers of disease-specific survival, but it is not known whether the two markers interact in terms of their influence on disease severity at diagnosis and disease outcome. METHODOLOGY/PRINCIPAL FINDINGS: Data from a cohort of 419 patients who were diagnosed with prostate cancer at transurethral resection for voiding difficulties was used. Scores for both tumour CB(1)IR and pEGFR-IR were available in the database. Of these, 235 had been followed by expectancy until the appearance of metastases. For patients scored for both parameters, Cox proportional-hazards regression analyses using optimal cut-off scores indicated that the two measures provided additional diagnostic information not only to each other, but to that provided by the tumour stage and the Gleason score. When the cases were divided into subgroups on the basis of these cut-off scores, the patients with both CB(1)IR and pEGFR-IR scores above their cut-off had a poorer disease-specific survival and showed a more severe pathology at diagnosis than patients with high pEGFR-IR scores but with CB(1)IR scores below the cut-off. CONCLUSIONS/SIGNIFICANCE: These data indicate that a high tumour CB(1) receptor expression at diagnosis augments the deleterious effects of a high pEGFR expression upon disease-specific survival

Entering a new era of body indices: the feasibility of a body shape index and body roundness index to identify cardiovascular health status.

BACKGROUND: The Body Mass Index (BMI) and Waist Circumference (WC) are well-used anthropometric predictors for cardiovascular diseases (CVD), but their validity is regularly questioned. Recently, A Body Shape Index (ABSI) and Body Roundness Index (BRI) were introduced as alternative anthropometric indices that may better reflect health status. OBJECTIVE: This study assessed the capacity of ABSI and BRI in identifying cardiovascular diseases and cardiovascular disease risk factors and determined whether they are superior to BMI and WC. DESIGN AND METHODS: 4627 Participants (54±12 years) of the Nijmegen Exercise Study completed an online questionnaire concerning CVD health status (defined as history of CVD or CVD risk factors) and anthropometric characteristics. Quintiles of ABSI, BRI, BMI, and WC were used regarding CVD prevalence. Odds ratios (OR), adjusted for age, sex, and smoking, were calculated per anthropometric index. RESULTS: 1332 participants (27.7%) reported presence of CVD or CVD risk factors. The prevalence of CVD increased across quintiles for BMI, ABSI, BRI, and WC. Comparing the lowest with the highest quintile, adjusted OR (95% CI) for CVD were significantly different for BRI 3.2 (1.4-7.2), BMI 2.4 (1.9-3.1), and WC 3.0 (1.6-5.6). The adjusted OR (95% CI) for CVD risk factors was for BRI 2.5 (2.0-3.3), BMI 3.3 (1.6-6.8), and WC 2.0 (1.6-2.5). No association was observed for ABSI in both groups. CONCLUSIONS: BRI, BMI, and WC are able to determine CVD presence, while ABSI is not capable. Nevertheless, the capacity of BRI as a novel body index to identify CVD was not superior compared to established anthropometric indices like BMI and WC

Characterization of the Single Stranded DNA Binding Protein SsbB Encoded in the Gonoccocal Genetic Island

Background: Most strains of Neisseria gonorrhoeae carry a Gonococcal Genetic Island which encodes a type IV secretion system involved in the secretion of ssDNA. We characterize the GGI-encoded ssDNA binding protein, SsbB. Close homologs of SsbB are located within a conserved genetic cluster found in genetic islands of different proteobacteria. This cluster encodes DNA-processing enzymes such as the ParA and ParB partitioning proteins, the TopB topoisomerase, and four conserved hypothetical proteins. The SsbB homologs found in these clusters form a family separated from other ssDNA binding proteins. Methodology/Principal Findings: In contrast to most other SSBs, SsbB did not complement the Escherichia coli ssb deletion mutant. Purified SsbB forms a stable tetramer. Electrophoretic mobility shift assays and fluorescence titration assays, as well as atomic force microscopy demonstrate that SsbB binds ssDNA specifically with high affinity. SsbB binds single-stranded DNA with minimal binding frames for one or two SsbB tetramers of 15 and 70 nucleotides. The binding mode was independent of increasing Mg 2+ or NaCl concentrations. No role of SsbB in ssDNA secretion or DNA uptake could be identified, but SsbB strongly stimulated Topoisomerase I activity

Toward a socio-economics of the brain drain and distributed human capital

[EN] This paper derives from our joint interest in understanding how scientific mobility affects developing countries. Many authors have addressed the topic previously, both from an economic and from a sociological perspective. However, recent literature evinces dissatisfaction with both analytical frameworks and the framing of public policies addressing the brain drain problematic. This paper is a contribution to understanding the historical and theoretical foundations of the “brain drain” debate. We aim to improve conceptual clarity regarding the itinerancy of human beings and the mobilization of human capital. We develop a critical review of the economics of the brain drain, highlighting the work of some key early thinkers and pointing out the way in which subsequent work has taken up selected aspects of their approaches leaving other challenges aside. We then consider the diaspora networks literature, which is characterized as taking a “connectionist” approach to the brain drain. We identify two fundamental problems: the sidelining of complementarity and context dependency as basic properties of human capital; and a failure to adequately disentangle the concepts of human resources for science and technology (HRST) and human capital in academic and policy discourse about the brain drain.Preliminary versions of this paper were presented at the International Workshop “Mobility, Science and Culture” (Braga, February 2010), the 8th GLOBELICS International Conference (Kuala Lumpur, November 2010) and the Macquarie University Skilled Labour and Migration Group (Sydney, November 2010). The authors would like to thank the colleagues and discussants that provided feedback in these fora. In particular, they are grateful to Claudia de Fuentes and to Jordi Molas for their careful reading of the draft and their useful comments. The authors were grateful recipients of funding from the International Science Linkages Program of the Academy of the Social Sciences in Australia and the Australian Department of Industry, Science and Research, which directly supported this work.Cañibano Sánchez, C.; Woolley, RD. (2015). Toward a socio-economics of the brain drain and distributed human capital. International Migration. 53(1):115-130. https://doi.org/10.1111/imig.12020S11513053