saeRS and sarA Act Synergistically to Repress Protease Production and Promote Biofilm Formation in Staphylococcus aureus

Mutation of the staphylococcal accessory regulator (sarA) limits biofilm formation in diverse strains of Staphylococcus aureus, but there are exceptions. One of these is the commonly studied strain Newman. This strain has two defects of potential relevance, the first being mutations that preclude anchoring of the fibronectin-binding proteins FnbA and FnbB to the cell wall, and the second being a point mutation in saeS that results in constitutive activation of the saePQRS regulatory system. We repaired these defects to determine whether either plays a role in biofilm formation and, if so, whether this could account for the reduced impact of sarA in Newman. Restoration of surface-anchored FnbA enhanced biofilm formation, but mutation of sarA in this fnbA-positive strain increased rather than decreased biofilm formation. Mutation of sarA in an saeS-repaired derivative of Newman (P18L) or a Newman saeRS mutant (ΔsaeRS) resulted in a biofilm-deficient phenotype like that observed in clinical isolates, even in the absence of surface-anchored FnbA. These phenotypes were correlated with increased production of extracellular proteases and decreased accumulation of FnbA and/or Spa in the P18L and ΔsaeRS sarA mutants by comparison to the Newman sarA mutant. The reduced accumulation of Spa was reversed by mutation of the gene encoding aureolysin, while the reduced accumulation of FnbA was reversed by mutation of the sspABC operon. These results demonstrate that saeRS and sarA act synergistically to repress the production of extracellular proteases that would otherwise limit accumulation of critical proteins that contribute to biofilm formation, with constitutive activation of saeRS limiting protease production, even in a sarA mutant, to a degree that can be correlated with increased enhanced capacity to form a biofilm. Although it remains unclear whether these effects are mediated directly or indirectly, studies done with an sspA::lux reporter suggest they are mediated at a transcriptional level

Mannose Binding Lectin Is Required for Alphavirus-Induced Arthritis/Myositis

Mosquito-borne alphaviruses such as chikungunya virus and Ross River virus (RRV) are emerging pathogens capable of causing large-scale epidemics of virus-induced arthritis and myositis. The pathology of RRV-induced disease in both humans and mice is associated with induction of the host inflammatory response within the muscle and joints, and prior studies have demonstrated that the host complement system contributes to development of disease. In this study, we have used a mouse model of RRV-induced disease to identify and characterize which complement activation pathways mediate disease progression after infection, and we have identified the mannose binding lectin (MBL) pathway, but not the classical or alternative complement activation pathways, as essential for development of RRV-induced disease. MBL deposition was enhanced in RRV infected muscle tissue from wild type mice and RRV infected MBL deficient mice exhibited reduced disease, tissue damage, and complement deposition compared to wild-type mice. In contrast, mice deficient for key components of the classical or alternative complement activation pathways still developed severe RRV-induced disease. Further characterization of MBL deficient mice demonstrated that similar to C3−/− mice, viral replication and inflammatory cell recruitment were equivalent to wild type animals, suggesting that RRV-mediated induction of complement dependent immune pathology is largely MBL dependent. Consistent with these findings, human patients diagnosed with RRV disease had elevated serum MBL levels compared to healthy controls, and MBL levels in the serum and synovial fluid correlated with severity of disease. These findings demonstrate a role for MBL in promoting RRV-induced disease in both mice and humans and suggest that the MBL pathway of complement activation may be an effective target for therapeutic intervention for humans suffering from RRV-induced arthritis and myositis

Epistatic Relationships between sarA and agr in Staphylococcus aureus Biofilm Formation

Background: The accessory gene regulator (agr) and staphylococcal accessory regulator (sarA) play opposing roles in Staphylococcus aureus biofilm formation. There is mounting evidence to suggest that these opposing roles are therapeutically relevant in that mutation of agr results in increased biofilm formation and decreased antibiotic susceptibility while mutation of sarA has the opposite effect. To the extent that induction of agr or inhibition of sarA could potentially be used to limit biofilm formation, this makes it important to understand the epistatic relationships between these two loci. Methodology/Principal Findings: We generated isogenic sarA and agr mutants in clinical isolates of S. aureus and assessed the relative impact on biofilm formation. Mutation of agr resulted in an increased capacity to forma biofilmin the 8325-4 laboratory strain RN6390 but had little impact in clinical isolates S. aureus. In contrast, mutation of sarA resulted in a reduced capacity to form a biofilm in all clinical isolates irrespective of the functional status of agr. This suggests that the regulatory role of sarA in biofilm formation is independent of the interaction between sarA and agr and that sarA is epistatic to agr in this context. This was confirmed by demonstrating that restoration of sarA function restored the ability to form a biofilm even in the corresponding agr mutants. Mutation of sarA in clinical isolates also resulted in increased production of extracellular proteases and extracellular nucleases, both of which contributed to the biofilm-deficient phenotype of sarA mutants. However, studies comparing different strains with and without proteases inhibitors and/or mutation of the nuclease genes demonstrated that the agr-independent, sarA-mediated repression of extracellular proteases plays a primary role in this regard. Conclusions and Significance: The results we report suggest that inhibitors of sarA-mediated regulation could be used to limit biofilm formation in S. aureus and that the efficacy of such inhibitors would not be limited by spontaneous mutation of agr in the human host

3 years of liraglutide versus placebo for type 2 diabetes risk reduction and weight management in individuals with prediabetes: a randomised, double-blind trial

Background: Liraglutide 3·0 mg was shown to reduce bodyweight and improve glucose metabolism after the 56-week period of this trial, one of four trials in the SCALE programme. In the 3-year assessment of the SCALE Obesity and Prediabetes trial we aimed to evaluate the proportion of individuals with prediabetes who were diagnosed with type 2 diabetes. Methods: In this randomised, double-blind, placebo-controlled trial, adults with prediabetes and a body-mass index of at least 30 kg/m2, or at least 27 kg/m2 with comorbidities, were randomised 2:1, using a telephone or web-based system, to once-daily subcutaneous liraglutide 3·0 mg or matched placebo, as an adjunct to a reduced-calorie diet and increased physical activity. Time to diabetes onset by 160 weeks was the primary outcome, evaluated in all randomised treated individuals with at least one post-baseline assessment. The trial was conducted at 191 clinical research sites in 27 countries and is registered with ClinicalTrials.gov, number NCT01272219. Findings: The study ran between June 1, 2011, and March 2, 2015. We randomly assigned 2254 patients to receive liraglutide (n=1505) or placebo (n=749). 1128 (50%) participants completed the study up to week 160, after withdrawal of 714 (47%) participants in the liraglutide group and 412 (55%) participants in the placebo group. By week 160, 26 (2%) of 1472 individuals in the liraglutide group versus 46 (6%) of 738 in the placebo group were diagnosed with diabetes while on treatment. The mean time from randomisation to diagnosis was 99 (SD 47) weeks for the 26 individuals in the liraglutide group versus 87 (47) weeks for the 46 individuals in the placebo group. Taking the different diagnosis frequencies between the treatment groups into account, the time to onset of diabetes over 160 weeks among all randomised individuals was 2·7 times longer with liraglutide than with placebo (95% CI 1·9 to 3·9, p<0·0001), corresponding with a hazard ratio of 0·21 (95% CI 0·13–0·34). Liraglutide induced greater weight loss than placebo at week 160 (–6·1 [SD 7·3] vs −1·9% [6·3]; estimated treatment difference −4·3%, 95% CI −4·9 to −3·7, p<0·0001). Serious adverse events were reported by 227 (15%) of 1501 randomised treated individuals in the liraglutide group versus 96 (13%) of 747 individuals in the placebo group. Interpretation: In this trial, we provide results for 3 years of treatment, with the limitation that withdrawn individuals were not followed up after discontinuation. Liraglutide 3·0 mg might provide health benefits in terms of reduced risk of diabetes in individuals with obesity and prediabetes. Funding: Novo Nordisk, Denmark

NOx emission characteristics of gas-fired radiant tube flames: The role of partial premixing

An experimental study of a laboratory natural gas-fired radiant heating tube with quartz walls and a practical burner geometry reveals that confinement of a jet flame in a tube affects its behavior dramatically. Qualitative observations, colors, and visible flame heights demonstrate that the flame burns either as a long, luminous, orange flame or as a very short, blue flame. Wall temperature profiles, global radiation measurements, and an overall energy balance delineate differences in the radiant tube performance between the orange and the blue modes. Measurements of local species concentrations reveal that the confined flame transitions from non-premixed behavior through various levels of partial premixing, affecting exhaust NO\rm\sb{X} amounts favorably or unfavorably depending on the operating condition. Motivated by changes in the NO\rm\sb{X} emission index with the confinement-induced mixing levels, a more fundamental study of NO\rm\sb{X} formation in partially premixed flames was performed. In particular, hydrocarbon-nitrogen chemistry was explored as a possible explanation for a previously observed NO\rm\sb{X} emission index minimum in partially premixed coflow jet flames. A representative hydrocarbon species, CH radical, was studied experimentally in laminar CH\sb4/air flames using chemiluminescence detection and absorption spectroscopy. Temperatures were measured using thin filament pyrometry. Computations of partially premixed flames were performed using the Sandia National Laboratories flame code Oppdif with the GRI-Mech 2.11 chemical kinetic model. Experimental results provide information about the thermal structure of these flames and reveal that the global CH radical concentration increases with increased partial premixing. It is recognized that experimental measurements of a single chemical species are not adequate for assessing the relative importance of competing reaction pathways to the formation of NO. The present computations reveal more detailed information for this purpose. Computational results from low strain rate flames reveal that the CH radical and other hydrocarbon species exhibit unique double concentration peaks in partially premixed flames, resulting in triple NO reaction zones. The hydrocarbon behavior causes widened reburn zones and reduced rates of the reactions which form and consume NO, and thus provide a possible explanation for the NO\rm\sb{X} behavior of partially premixed flames

Effect of Varied Air Flow on Flame Structure of Laminar Inverse Diffusion Flames

The structure of laminar inverse diffusion flames (IDFs) of methane and ethylene was studied using a cylindrical co-flowing burner. Several flames of the same fuel flow-rate yet various air flow-rates were examined. Heights of visible flames were obtained using measurements of hydroxyl (OH) laser-induced fluorescence (LIF) and visible images. Polycyclic aromatic hydrocarbon (PAH) LIF and soot laser-induced incandescence (LII) were also measured. In visible images, radiating soot masks the blue region typically associated with the flame height in normal diffusion flames (NDFs). Increased air flow-rates resulted in longer flames. PAH LIF and soot LII indicated that PAH and soot are present on the fuel side of the flame and that soot is located closer to the reaction zone than PAH. Ethylene flames produced significantly higher PAH LIF and soot LII signals than methane flames, which is consistent with the sooting propensity o

Flame Height Measurement of Laminar Inverse Diffusion Flames

Flame heights of co-flowing cylindrical ethylene-air and methane-air laminar inverse diffusion flames were measured. The luminous flame height was found to be longer than the height of the reaction zone determined by planar laser-induced fluorescence (PLIF) of hydroxyl radicals (OH) because of luminous soot above the reaction zone. However, the location of the peak luminous signals along the centerline agreed very well with the OH flame height. Flame height predictions using Roper’s analysis for circular port burners agreed with measured reaction zone heights when using values for the characteristic diffusion coefficient and/or diffusion temperature somewhat different from those recommended by Roper. The fact that Roper’s analysis applies to inverse diffusion flames is evidence that inverse diffusion flames are similar in structure to normal diffusion flames

Laser Extinction in Laminar Inverse Diffusion Flames

Measurements of line-of-sight laser extinction in a co-annular ethylene-air laminar inverse diffusion flame (IDF) were made to determine soot concentration. Extinction has frequently been used in the literature to measure soot concentration in normal diffusion flames (NDFs), but it has rarely been applied to IDFs. A coflow IDF contains a primary air flow surrounded by a fuel annulus. Soot particles form on the outside of IDFs, advect upward, and eventually quench without being oxidized. It has been proposed in the literature that IDFs will produce less near-flame soot than NDFs because, for flames of comparable fuel, size and flow rates, movement of soot outward into cool regions of an IDF limits its simultaneous exposure to the high temperatures and fuel pyrolysis products needed for soot growth. A two-dimensional soot concentration map of an IDF using experimental data confirms this hypothesis by showing integrated soot volume fractions to be an order of magnitude lower than those reported for NDFs in the literature. Computer simulations of particle temperature histories in an NDF and IDF of similar height lend support to these results