Treponema denticola Major Outer Sheath Protein Induces Actin Assembly at Free Barbed Ends by a PIP2-Dependent Uncapping Mechanism in Fibroblasts

The major outer sheath protein (Msp) of Treponema denticola perturbs actin dynamics in fibroblasts by inducing actin reorganization, including subcortical actin filament assembly, leading to defective calcium flux, diminished integrin engagement of collagen, and retarded cell migration. Yet, its mechanisms of action are unknown. We challenged Rat-2 fibroblasts with enriched native Msp. Msp activated the small GTPases Rac1, RhoA and Ras, but not Cdc42, yet only Rac1 localized to areas of actin rearrangement. We used Rac1 dominant negative transfection and chemical inhibition of phosphatidylinositol-3 kinase (PI3K) to show that even though Rac1 activation was PI3K-dependent, neither was required for Msp-induced actin rearrangement. Actin free barbed end formation (FBE) by Msp was also PI3K-independent. Immunoblotting experiments showed that gelsolin and CapZ were released from actin filaments, whereas cofilin remained in an inactive state. Msp induced phosphatidylinositol (4,5)-bisphosphate (PIP2) formation through activation of a phosphoinositide 3-phosphatase and its recruitment to areas of actin assembly at the plasma membrane. Using a PIP2 binding peptide or lipid phosphatase inhibitor, PIP2 was shown to be required for Msp-mediated actin uncapping and FBE formation. Evidently, Msp induces actin assembly in fibroblasts by production and recruitment of PIP2 and release of the capping proteins CapZ and gelsolin from actin barbed ends

Identification of potential CepR regulated genes using a cep box motif-based search of the Burkholderia cenocepacia genome

BACKGROUND: The Burkholderia cenocepacia CepIR quorum sensing system has been shown to positively and negatively regulate genes involved in siderophore production, protease expression, motility, biofilm formation and virulence. In this study, two approaches were used to identify genes regulated by the CepIR quorum sensing system. Transposon mutagenesis was used to create lacZ promoter fusions in a cepI mutant that were screened for differential expression in the presence of N-acylhomoserine lactones. A bioinformatics approach was used to screen the B. cenocepacia J2315 genome for CepR binding site motifs. RESULTS: Four positively regulated and two negatively regulated genes were identified by transposon mutagenesis including genes potentially involved in iron transport and virulence. The promoter regions of selected CepR regulated genes and site directed mutagenesis of the cepI promoter were used to predict a consensus cep box sequence for CepR binding. The first-generation consensus sequence for the cep box was used to identify putative cep boxes in the genome sequence. Eight potential CepR regulated genes were chosen and the expression of their promoters analyzed. Six of the eight were shown to be regulated by CepR. A second generation motif was created from the promoters of these six genes in combination with the promoters of cepI, zmpA, and two of the CepR regulated genes identified by transposon mutagenesis. A search of the B. cenocepacia J2315 genome with the new motif identified 55 cep boxes in 65 promoter regions that may be regulated by CepR. CONCLUSION: Using transposon mutagenesis and bioinformatics expression of twelve new genes have been determined to be regulated by the CepIR quorum sensing system. A cep box consensus sequence has been developed based on the predicted cep boxes of ten CepR regulated genes. This consensus cep box has led to the identification of over 50 new genes potentially regulated by the CepIR quorum sensing system

Dose-response effects of light at night on the reproductive physiology of great tits (Parus major): integrating morphological analyses with candidate gene expression

Artificial light at night (ALAN) is increasingly recognized as a potential threat to wildlife and ecosystem health. Among the ecological effects of ALAN, changes in reproductive timing are frequently reported, but the mechanisms underlying this relationship are still poorly understood. Here, we experimentally investigated these mechanisms by assessing dose‐dependent photoperiodic responses to ALAN in the great tit (Parus major). We individually exposed photosensitive male birds to one of three nocturnal light levels (0.5, 1.5, and 5 lux), or to a dark control. Subsequent histological and molecular analyses on their testes indicated a dose‐dependent reproductive response to ALAN. Specifically, different stages of gonadal growth were activated after exposure to different levels of light at night. mRNA transcript levels of genes linked to the development of germ cells (stra8 and spo11) were increased under 0.5 lux compared to the dark control. The 0.5 and 1.5 lux groups showed slight increases in testis size and transcript levels associated with steroid synthesis (lhr and hsd3b1) and spermatogenesis (fshr, wt1, sox9, and cldn11), although spermatogenesis was not detected in histological analysis. In contrast, all birds under 5 lux had 10 to 30 times larger testes than birds in all other groups, with a parallel strong increase in mRNA transcript levels and clear signs of spermatogenesis. Across treatments, the volume of the testes was generally a good predictor of testicular transcript levels. Overall, our findings indicate that even small changes in nocturnal light intensity can increase, or decrease, effects on the reproductive physiology of wild organisms

Body composition and habitual and match-day dietary intake of the FNB Maties Varsity Cup rugby players

Background. Rugby is a physically demanding body contact sport. Optimising dietary intake and body composition can positively affect the performance of rugby athletes.Objectives. To determine the body composition, habitual and game-specific nutritional practices of FNB Maties Varsity Cup (MVC) rugby players.Methods. A descriptive, cross-sectional study with an analytical component was conducted. Of all the MVC rugby players (N=35), 18 completed the sections on body composition and match-day dietary intake, while 11 completed the habitual dietary intake section. Body composition data were collected by an International Society for the Advancement of Kinanthropometry-accredited biokineticist. Habitual dietary intake data (via a self-administered 7-day food record) and match-day dietary strategies (via telephonic 24-hour recall interview) were collected and compared with nutritional requirements reported by the International Olympic Committee, the American Dietetic Association, the American College of Sports Medicine and the International Society of Sport Nutrition.Results. Forwards had significantly higher weight (p=0.01), sum of seven skinfolds (p=0.01), percentage body fat (p=0.02), fat mass (p=0.01) and fat-free mass (p=0.01) than backs. Compared with current recommendations, group habitual dietary intake (mean (stand­ard deviation)) was inadequate for total energy (45.4 (9.0) kcal/kg body weight (BW)), carbohydrate (4.3 (0.4) g/kg BW), polyunsaturated fatty acids (6.2 (1.7)% of total energy (TE)), calcium:protein ratio (6.5:1 (3.5:1)) and copper (2.3 (0.4) mg), while displaying higher-than-recommended intakes for total protein (2.4 (0.7) g/kg BW), fibre (37.7 (7.3) g/day), total fat (33.8 (4.3)% TE), saturated fatty acids (11.2 (13.1)% TE), cholesterol (766.3 (371.8) mg) and niacin (45.2 (6.9) µg). Habitual supplement use was high at 91% (n=10/11). Nutritional match-day strategies were excessive for protein (1.2 (0.6) g/kg BW) and fat (0.9 (0.4) g/kg BW) in the pre-event meal, inadequate for energy and carbohydrate during the game and excessive for alcohol (54.4 (59.9) g) after the game.Conclusion. Forwards and backs differed significantly in various body composition measurements. In relation to observed practices, hab­it­ual dietary intake and nutritional match-day strategies were suboptimal, with high reported supplement use. Players in this sport potentially could benefit from specialist input to optimise dietary strategies and body composition in order to enhance performance

Structure-Based Optimization of Covalent, Small-Molecule Stabilizers of the 14-3-3σ/ERα Protein-Protein Interaction from Nonselective Fragments

The stabilization of protein-protein interactions (PPIs) has emerged as a promising strategy in chemical biology and drug discovery. The identification of suitable starting points for stabilizing native PPIs and their subsequent elaboration into selective and potent molecular glues lacks structure-guided optimization strategies. We have previously identified a disulfide fragment that stabilized the hub protein 14-3-3σ bound to several of its clients, including ERα and C-RAF. Here, we show the structure-based optimization of the nonselective fragment toward selective and highly potent small-molecule stabilizers of the 14-3-3σ/ERα complex. The more elaborated molecular glues, for example, show no stabilization of 14-3-3σ/C-RAF up to 150 μM compound. Orthogonal biophysical assays, including mass spectrometry and fluorescence anisotropy, were used to establish structure-activity relationships. The binding modes of 37 compounds were elucidated with X-ray crystallography, which further assisted the concomitant structure-guided optimization. By targeting specific amino acids in the 14-3-3σ/ERα interface and locking the conformation with a spirocycle, the optimized covalent stabilizer 181 achieved potency, cooperativity, and selectivity similar to the natural product Fusicoccin-A. This case study showcases the value of addressing the structure, kinetics, and cooperativity for molecular glue development. </p

Efficacy and Safety of High Potent P2Y12 Inhibitors Prasugrel and Ticagrelor in Patients With Coronary Heart Disease Treated With Dual Antiplatelet Therapy: A Sex-Specific Systematic Review and Meta-Analysis

Background Sex differences in efficacy and safety of dual antiplatelet therapy remain uncertain because of the underrepresentation of women in cardiovascular trials. The aim of this study was to perform a sex-specific analysis of the pooled efficacy and safety data of clinical trials comparing a high potent P2Y12 inhibitor+aspirin with clopidogrel+aspirin in patients with acute coronary syndrome. Methods and Results A systematic literature search was performed. Randomized clinical trials that compared patients following percutaneous coronary intervention/acute coronary syndrome who were taking high potent P2Y12 inhibitors+aspirin versus clopidogrel+aspirin were selected. Random effects estimates were calculated and relative risks with 95% CIs on efficacy and safety end points were determined per sex. We included 6 randomized clinical trials comparing prasugrel/ticagrelor versus clopidogrel in 43 990 patients (13 030 women), with a median follow-up time of 1.06 years. Women and men had similar relative risk (RR) reduction for major cardiovascular events (women: RR, 0.89 [95% CI, 0.80-1.00; men: RR, 0.84 [95% CI, 0.79-0.91) (P for interaction=0.39). Regarding safety, women and men had similar risk of major bleeding by high-potency dual antip

Estimating the prevalence of breast cancer using a disease model: data problems and trends

BACKGROUND: Health policy and planning depend on quantitative data of disease epidemiology. However, empirical data are often incomplete or are of questionable validity. Disease models describing the relationship between incidence, prevalence and mortality are used to detect data problems or supplement missing data. Because time trends in the data affect their outcome, we compared the extent to which trends and known data problems affected model outcome for breast cancer. METHODS: We calculated breast cancer prevalence from Dutch incidence and mortality data (the Netherlands Cancer Registry and Statistics Netherlands) and compared this to regionally available prevalence data (Eindhoven Cancer Registry, IKZ). Subsequently, we recalculated the model adjusting for 1) limitations of the prevalence data, 2) a trend in incidence, 3) secondary primaries, and 4) excess mortality due to non-breast cancer deaths. RESULTS: There was a large discrepancy between calculated and IKZ prevalence, which could be explained for 60% by the limitations of the prevalence data plus the trend in incidence. Secondary primaries and excess mortality had relatively small effects only (explaining 17% and 6%, respectively), leaving a smaller part of the difference unexplained. CONCLUSION: IPM models can be useful both for checking data inconsistencies and for supplementing incomplete data, but their results should be interpreted with caution. Unknown data problems and trends may affect the outcome and in the absence of additional data, expert opinion is the only available judge

From Tethered to Freestanding Stabilizers of 14-3-3 Protein-Protein Interactions through Fragment Linking

Small-molecule stabilization of protein-protein interactions (PPIs) is a promising strategy in chemical biology and drug discovery. However, the systematic discovery of PPI stabilizers remains a largely unmet challenge. Herein we report a fragment-linking approach targeting the interface of 14-3-3 and a peptide derived from the estrogen receptor alpha (ERα) protein. Two classes of fragments—a covalent and a noncovalent fragment—were co-crystallized and subsequently linked, resulting in a noncovalent hybrid molecule in which the original fragment interactions were largely conserved. Supported by 20 crystal structures, this initial hybrid molecule was further optimized, resulting in selective, 25-fold stabilization of the 14-3-3/ERα interaction. The high-resolution structures of both the single fragments, their co-crystal structures and those of the linked fragments document a feasible strategy to develop orthosteric PPI stabilizers by linking to an initial tethered fragment.</p