Key mechanisms in the formation of proteinaceous biofilm matrices in Staphylococcus aureus and Staphylococcus epidermidis

Staphylococcus aureus and Staphylococcus epidermidis are major human pathogens and are the leading causes of implant associated infections. After insertion into the body, implants become coated in host proteins, which S. aureus and S. epidermidis use to establish infections. They utilise many surface and secreted proteins that interact with these host proteins to increase attachment to implant surfaces, increase biofilm accumulation, and evade the immune system. S. aureus secretes two coagulases, Coagulase (Coa) and von Willebrand factor binding protein (vWbp), which hijack the host coagulation cascade and trigger the formation of a fibrin network that is a key structure in S. aureus biofilms and shields bacteria from the immune system. We explored which factors cause coagulase expression, the localisation and dynamics of fibrin formation in growing biofilms, and cell-cell variation in fibrin binding using coagulation assays, time lapse confocal microscopy, and single molecule imaging of Coa:SNAP and vWbp:CLIP fusion proteins. Host factors increased coagulase production and loosely associated Coa and vWbp to cell surfaces. Coa mainly localised to cell surfaces to produce a surface attached fibrin pseudocapsule, but could also form fibrin in the wider biofilm matrix. vWbp produced matrix-associated fibrin in the absence of Coa, but associated to cell surfaces to accelerate pseudocapsule production when Coa was also present. These findings indicate a more collaborative role between Coa and vWbp in building the fibrin network than previously suggested. Not all bacteria appeared to contribute to forming fibrin: we identified a slowly- or non-dividing subpopulation of bacteria that did not form a pseudocapsule. We speculated that these bacteria either lack the surface proteins required to bind fibrin, do not produce coagulases and therefore cannot produce fibrin, or both. Extracellular matrix binding protein (Embp) is a giant surface protein expressed by S. epidermidis that attaches biofilms to fibronectin coated surfaces. We aimed to visualise Embp in S. epidermidis biofilms to further investigate its biological role in biofilm formation by constructing fusion proteins with the SNAP tag and monomeric superfolder GFP (msfGFP), but the fusion proteins could not be visualised. This was likely due to improper placement of the protein tags, which were placed before a putative cleavage site after the signal peptide. However, we demonstrated that msfGFP could be successfully secreted by S. aureus, either when fused to a Sec signal peptide or to Coa, which demonstrates that it is a good candidate for labelling extracellular proteins. We also visualised the SNAP tag when secreted at the cross wall during cell division by S. aureus, the same mechanism used to secrete Embp, and therefore envision that it is possible to visualise Embp with one of these two protein tags if the tag is placed after the cleavage site instead

GFP fusions of Sec-routed extracellular proteins in Staphylococcus aureus reveal surface-associated coagulase in biofilms

Staphylococcus aureus is a major human pathogen that utilises many surface-associated and secreted proteins to form biofilms and cause disease. However, our understanding of these processes is limited by challenges of using fluores-cent protein reporters in their native environment, because they must be ex-ported and fold correctly to become fluorescent. Here, we demonstrate the feasibility of using the monomeric superfolder GFP (msfGFP) exported from S. aureus. By fusing msfGFP to signal peptides for the Secretory (Sec) and Twin Arginine Translocation (Tat) pathways, the two major secretion pathways in S. aureus, we quantified msfGFP fluorescence in bacterial cultures and cell-free supernatant from the cultures. When fused to a Tat signal peptide, we detect-ed msfGFP fluorescence inside but not outside bacterial cells, indicating a fail-ure to export msfGFP. However, when fused to a Sec signal peptide, msfGFP fluorescence was present outside cells, indicating successful export of the msfGFP in the unfolded state, followed by extracellular folding and maturation to the photoactive state. We applied this strategy to study coagulase (Coa), a secreted protein and a major contributor to the formation of a fibrin network in S. aureus biofilms that protects bacteria from the host immune system and increases attachment to host surfaces. We confirmed that a genomically inte-grated C-terminal fusion of Coa to msfGFP does not impair the activity of Coa or its localisation within the biofilm matrix. Our findings demonstrate that msfGFP is a good candidate fluorescent reporter to consider when studying proteins secreted by the Sec pathway in S. aureus

Rif1 maintains telomeres and mediates DNA repair by encasing DNA ends

In yeast, Rif1 is part of the telosome, where it inhibits telomerase and checkpoint signaling at chromosome ends. In mammalian cells, Rif1 is not telomeric, but it suppresses DNA end resection at chromosomal breaks, promoting repair by nonhomologous end joining (NHEJ). Here, we describe crystal structures for the uncharacterized and conserved ∼125-kDa N-terminal domain of Rif1 from Saccharomyces cerevisiae (Rif1-NTD), revealing an α-helical fold shaped like a shepherd's crook. We identify a high-affinity DNA-binding site in the Rif1-NTD that fully encases DNA as a head-to-tail dimer. Engagement of the Rif1-NTD with telomeres proved essential for checkpoint control and telomere length regulation. Unexpectedly, Rif1-NTD also promoted NHEJ at DNA breaks in yeast, revealing a conserved role of Rif1 in DNA repair. We propose that tight associations between the Rif1-NTD and DNA gate access of processing factors to DNA ends, enabling Rif1 to mediate diverse telomere maintenance and DNA repair functions

Public Acceptability in the UK and USA of Nudging to Reduce Obesity: The Example of Reducing Sugar-Sweetened Beverages Consumption.

BACKGROUND: "Nudging"-modifying environments to change people's behavior, often without their conscious awareness-can improve health, but public acceptability of nudging is largely unknown. METHODS: We compared acceptability, in the United Kingdom (UK) and the United States of America (USA), of government interventions to reduce consumption of sugar-sweetened beverages. Three nudge interventions were assessed: i. reducing portion Size, ii. changing the Shape of the drink containers, iii. changing their shelf Location; alongside two traditional interventions: iv. Taxation and v. Education. We also tested the hypothesis that describing interventions as working through non-conscious processes decreases their acceptability. Predictors of acceptability, including perceived intervention effectiveness, were also assessed. Participants (n = 1093 UK and n = 1082 USA) received a description of each of the five interventions which varied, by randomisation, in how the interventions were said to affect behaviour: (a) via conscious processes; (b) via non-conscious processes; or (c) no process stated. Acceptability was derived from responses to three items. RESULTS: Levels of acceptability for four of the five interventions did not differ significantly between the UK and US samples; reducing portion size was less accepted by the US sample. Within each country, Education was rated as most acceptable and Taxation the least, with the three nudge-type interventions rated between these. There was no evidence to support the study hypothesis: i.e. stating that interventions worked via non-conscious processes did not decrease their acceptability in either the UK or US samples. Perceived effectiveness was the strongest predictor of acceptability for all interventions across the two samples. CONCLUSION: In conclusion, nudge interventions to reduce consumption of sugar-sweetened beverages seem similarly acceptable in the UK and USA, being more acceptable than taxation, but less acceptable than education. Contrary to prediction, we found no evidence that highlighting the non-conscious processes by which nudge interventions may work decreases their acceptability. However, highlighting the effectiveness of all interventions has the potential to increase their acceptability.The study was funded by the UK Department of Health Policy Research Programme (Policy Research Unit in Behaviour and Health) (Grant ID: PRUN-0409-10109)This is the final version of the article. It first appeared from the Public Library of Science via http://dx.doi.org/10.1371/journal.pone.015599

Marriage, Sex, and Hydrocele: An Ethnographic Study on the Effect of Filarial Hydrocele on Conjugal Life and Marriageability from Orissa, India

Lymphatic filariasis, the second leading cause of permanent and long-term disability, affects 120 million people globally. Hydrocele, an accumulation of fluid in the scrotum that causes it to swell, is one of the chronic manifestations of LF among men and there are about 27 million men with hydrocele worldwide. We conducted ethnographic interviews and discussions with patients, women whose husbands have hydrocele, and the general public in a rural community of eastern India. The study describes how hydrocele impacts patients' sexual and marital life. It reveals the most worrisome effect of hydrocele for patients and their wives due to the inability to have a satisfactory sexual life. Patients expressed their incapacity during sexual intercourse. A majority of hydrocele patients' wives reported that their married life became burdened and couples were not living happily. This study also highlights the impact on marriageability, and some women expressed that a hydrocele patient is the “last choice”. In some cases, the patients were persuaded by their wives to remove hydrocele by surgery (hydrocelectomy). Hence, access to hydrocelectomy has to be strengthened under the Global Programme to Eliminate Lymphatic Filariasis, which is operational in several endemic areas in the world. Also, this activity may be integrated with primary healthcare services and interventions of other neglected tropical diseases

Hepatocyte Permissiveness to Plasmodium Infection Is Conveyed by a Short and Structurally Conserved Region of the CD81 Large Extracellular Domain

Invasion of hepatocytes by Plasmodium sporozoites is a prerequisite for establishment of a malaria infection, and thus represents an attractive target for anti-malarial interventions. Still, the molecular mechanisms underlying sporozoite invasion are largely unknown. We have previously reported that the tetraspanin CD81, a known receptor for the hepatitis C virus (HCV), is required on hepatocytes for infection by sporozoites of several Plasmodium species. Here we have characterized CD81 molecular determinants required for infection of hepatocytic cells by P. yoelii sporozoites. Using CD9/CD81 chimeras, we have identified in CD81 a 21 amino acid stretch located in a domain structurally conserved in the large extracellular loop of tetraspanins, which is sufficient in an otherwise CD9 background to confer susceptibility to P. yoelii infection. By site-directed mutagenesis, we have demonstrated the key role of a solvent-exposed region around residue D137 within this domain. A mAb that requires this region for optimal binding did not block infection, in contrast to other CD81 mAbs. This study has uncovered a new functionally important region of CD81, independent of HCV E2 envelope protein binding domain, and further suggests that CD81 may not interact directly with a parasite ligand during Plasmodium infection, but instead may regulate the function of a yet unknown partner protein

Hiding from the Moonlight: Luminosity and Temperature Affect Activity of Asian Nocturnal Primates in a Highly Seasonal Forest

The effect of moonlight and temperature on activity of slow lorises was previously little known and this knowledge might be useful for understanding many aspects of their behavioural ecology, and developing strategies to monitor and protect populations. In this study we aimed to determine if the activity of the pygmy loris (Nycticebus pygmaeus) is affected by ambient temperature and/or moonlight in a mixed deciduous forest. We radio-collared five females and five males in the Seima Protection Forest, Cambodia, in February to May, 2008 and January to March, 2009 and recorded their behaviour at 5 minutes intervals, totalling 2736 observations. We classified each observation as either inactive (sleeping or alert) or active behaviour (travel, feeding, grooming, or others). Moon luminosity (bright/dark) and ambient temperature were recorded for each observation. The response variable, activity, was binary (active or inactive), and a logit link function was used. Ambient temperature alone did not significantly affect mean activity. Although mean activity was significantly affected by moonlight, the interaction between moonlight and temperature was also significant: on bright nights, studied animals were increasingly more active with higher temperature; and on dark nights they were consistently active regardless of temperature. The most plausible explanation is that on bright cold nights the combined risk of being seen and attacked by predators and heat loss outweigh the benefit of active behaviours

Hormone Therapy and the Risk of Breast Cancer in BRCA1 Mutation Carriers

Background: Hormone therapy (HT) is commonly given to women to alleviate the climacteric symptoms associated with menopause. There is concern that this treatment may increase the risk of breast cancer. The potential association of HT and breast cancer risk is of particular interest to women who carry a mutation in BRCA1 because they face a high lifetime risk of breast cancer and because many of these women take HT after undergoing prophylactic surgical oophorectomy at a young age. Methods: We conducted a matched case-control study of 472 postmenopausal women with a BRCA1 mutation to examine whether or not the use of HT is associated with subsequent risk of breast cancer. Breast cancer case patients and control subjects were matched with respect to age, age at menopause, and type of menopause (surgical or natural). Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated with conditional logistic regression. Statistical tests were two-sided. Results: In this group of BRCA1 mutation carriers, the adjusted OR for breast cancer associated with ever use of HT compared with never use was 0.58 (95% CI = 0.35 to 0.96; P =. 03). In analyses by type of HT, an inverse association with breast cancer risk was observed with use of estrogen only (OR = 0.51, 95% CI = 0.27 to 0.98; P =. 04); the association with use of estrogen plus progesterone was not statistically significant (OR = 0.66, 95% CI = 0.34 to 1.27; P =. 21). Conclusion: Among postmenopausal women with a BRCA1 mutation, HT use was not associated with increased risk of breast cancer; indeed, in this population, it was associated with a decreased risk

HIV testing within general practices in Europe : A mixed-methods systematic review

Funding Information: This work was supported by IWT (Belgium) and the ANRS (France) through the framework of HIVERA JTC 2014. Publisher Copyright: © 2018 The Author(s).Background: Late diagnosis of HIV infection remains a key challenge in Europe. It is acknowledged that general practitioners (GPs) may contribute greatly to early case finding, yet there is evidence that many diagnostic opportunities are being missed. To further promote HIV testing in primary care and to increase the utility of available research, the existing evidence has been synthesised in a systematic review adhering to the PRISMA guidelines. Methods: The databases PubMed, Scopus and Embase were searched for the period 2006-2017. Two authors judged independently on the eligibility of studies. Through a mixed-methods systematic review of 29 studies, we provide a description of HIV testing in general practices in Europe, including barriers and facilitators. Results: The findings of the study show that although various approaches to target patients are used by GPs, most tests are still carried out based on the patient's request. Several barriers obstruct HIV testing in general practice. Included are a lack of communication skills on sexual health, lack of knowledge about HIV testing recommendations and epidemic specificities, difficulties with using the complete list of clinical HIV indicator diseases and lack of experience in delivering and communicating test results. The findings also suggest that the provision of specific training, practical tools and promotion programmes has an impact on the testing performance of GPs. Conclusions: GPs could have an increased role in provider-initiated HIV-testing for early case finding. To achieve this objective, solutions to the reported barriers should be identified and testing criteria adapted to primary healthcare defined. Providing guidance and training to better identify priority groups for HIV testing, as well as information on the HIV epidemic's characteristics, will be fundamental to increasing awareness and testing by GPs.publishersversionPeer reviewe

On the Role of the Difference in Surface Tensions Involved in the Allosteric Regulation of NHE-1 Induced by Low to Mild Osmotic Pressure, Membrane Tension and Lipid Asymmetry

The sodium-proton exchanger 1 (NHE-1) is a membrane transporter that exchanges Na+ for H+ ion across the membrane of eukaryotic cells. It is cooperatively activated by intracellular protons, and this allosteric regulation is modulated by the biophysical properties of the plasma membrane and related lipid environment. Consequently, NHE-1 is a mechanosensitive transporter that responds to osmotic pressure, and changes in membrane composition. The purpose of this study was to develop the relationship between membrane surface tension, and the allosteric balance of a mechanosensitive transporter such as NHE-1. In eukaryotes, the asymmetric composition of membrane leaflets results in a difference in surface tensions that is involved in the creation of a reservoir of intracellular vesicles and membrane buds contributing to buffer mechanical constraints. Therefore, we took this phenomenon into account in this study and developed a set of relations between the mean surface tension, membrane asymmetry, fluid phase endocytosis and the allosteric equilibrium constant of the transporter. We then used the experimental data published on the effects of osmotic pressure and membrane modification on the NHE-1 allosteric constant to fit these equations. We show here that NHE-1 mechanosensitivity is more based on its high sensitivity towards the asymmetry between the bilayer leaflets compared to mean global membrane tension. This compliance to membrane asymmetry is physiologically relevant as with their slower transport rates than ion channels, transporters cannot respond as high pressure-high conductance fast-gating emergency valves