SpoT Induces Intracellular Salmonella Virulence Programs in the Phagosome.

Guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), together named (p)ppGpp, regulate diverse aspects of Salmonella pathogenesis, including synthesis of nutrients, resistance to inflammatory mediators, and expression of secretion systems. In Salmonella, these nucleotide alarmones are generated by the synthetase activities of RelA and SpoT proteins. In addition, the (p)ppGpp hydrolase activity of the bifunctional SpoT protein is essential to preserve cell viability. The contribution of SpoT to physiology and pathogenesis has proven elusive in organisms such as Salmonella, because the hydrolytic activity of this RelA and SpoT homologue (RSH) is vital to prevent inhibitory effects of (p)ppGpp produced by a functional RelA. Here, we describe the biochemical and functional characterization of a spoT-Δctd mutant Salmonella strain encoding a SpoT protein that lacks the C-terminal regulatory elements collectively referred to as "ctd." Salmonella expressing the spoT-Δctd variant hydrolyzes (p)ppGpp with similar kinetics to those of wild-type bacteria, but it is defective at synthesizing (p)ppGpp in response to acidic pH. Salmonella spoT-Δctd mutants have virtually normal adaptations to nutritional, nitrosative, and oxidative stresses, but poorly induce metal cation uptake systems and Salmonella pathogenicity island 2 (SPI-2) genes in response to the acidic pH of the phagosome. Importantly, spoT-Δctd mutant Salmonella replicates poorly intracellularly and is attenuated in a murine model of acute salmonellosis. Collectively, these investigations indicate that (p)ppGpp synthesized by SpoT serves a unique function in the adaptation of Salmonella to the intracellular environment of host phagocytes that cannot be compensated by the presence of a functional RelA.IMPORTANCE Pathogenic bacteria experience nutritional challenges during colonization and infection of mammalian hosts. Binding of the alarmone nucleotide guanosine tetraphosphate (ppGpp) to RNA polymerase coordinates metabolic adaptations and virulence gene transcription, increasing the fitness of diverse Gram-positive and Gram-negative bacteria as well as that of actinomycetes. Gammaproteobacteria such as Salmonella synthesize ppGpp by the combined activities of the closely related RelA and SpoT synthetases. Due to its profound inhibitory effects on growth, ppGpp must be removed; in Salmonella, this process is catalyzed by the vital hydrolytic activity of the bifunctional SpoT protein. Because SpoT hydrolase activity is essential in cells expressing a functional RelA, we have a very limited understanding of unique roles these two synthetases may assume during interactions of bacterial pathogens with their hosts. We describe here a SpoT truncation mutant that lacks ppGpp synthetase activity and all C-terminal regulatory domains but retains excellent hydrolase activity. Our studies of this mutant reveal that SpoT uniquely senses the acidification of phagosomes, inducing virulence programs that increase Salmonella fitness in an acute model of infection. Our investigations indicate that the coexistence of RelA/SpoT homologues in a bacterial cell is driven by the need to mount a stringent response to a myriad of physiological and host-specific signatures

A multidimensional program including standing exercises, health education, and telephone support to reduce sedentary behavior in frail older adults:Randomized clinical trial

Objectives: The primary aim of this study was to evaluate the effect of a multidimensional program including home-based standing exercises, health education, and telephone support for the reduction of sedentary behavior in community-dwelling frail older adults. The secondary aim of this study was to evaluate the safety and adherence of the program. Study design: A single-blind, randomized controlled trial. Methods: A total of 43 frail older adults were randomly assigned to the intervention and control groups. The intervention consisted of combined strategies including home-based standing exercises, health education, and telephone support for 16 weeks for frail older adults. The control group received orientation regarding the harmful effects of a sedentary lifestyle. Sedentary behavior was evaluated by total sedentary time, accumulated sedentary time in bouts of at least 10 min, and by the break in sedentary time, measured by an accelerometer used for at least 600 min/day for 4 days. Safety was assessed by self-reporting of possible adverse events. Adherence was assessed based on the number of days in which standing exercises were performed by the participants. Repeated measures ANOVA and Tukeys post hoc test were used to analyze the collected data. Results: The intervention group reduced the sedentary time by 30 min/day (p= 0.048), but without significant maintenance after 30 days of the program. Of the total number of participants, 82% (n = 14) of the intervention group participants showed more than 70% adherence to the program. The main adverse effects faced by the intervention group participants were tiredness (53%; n = 9) and lower limb pain (47%; n = 8). Conclusions: The multidimensional program reduced sedentary behavior, was safe, and showed satisfactory adherence in frail older adults

Polarized localization of phosphatidylserine in the endothelium regulates Kir2.1

Lipid regulation of ion channels is largely explored using in silico modeling with minimal experimentation in intact tissue; thus, the functional consequences of these predicted lipid-channel interactions within native cellular environments remain elusive. The goal of this study is to investigate how lipid regulation of endothelial Kir2.1 - an inwardly rectifying potassium channel that regulates membrane hyperpolarization - contributes to vasodilation in resistance arteries. First, we show that phosphatidylserine (PS) localizes to a specific subpopulation of myoendothelial junctions (MEJs), crucial signaling microdomains that regulate vasodilation in resistance arteries, and in silico data have implied that PS may compete with phosphatidylinositol 4,5-bisphosphate (PIP2) binding on Kir2.1. We found that Kir2.1-MEJs also contained PS, possibly indicating an interaction where PS regulates Kir2.1. Electrophysiology experiments on HEK cells demonstrate that PS blocks PIP2 activation of Kir2.1 and that addition of exogenous PS blocks PIP2-mediated Kir2.1 vasodilation in resistance arteries. Using a mouse model lacking canonical MEJs in resistance arteries (Elnfl/fl/Cdh5-Cre), PS localization in endothelium was disrupted and PIP2 activation of Kir2.1 was significantly increased. Taken together, our data suggest that PS enrichment to MEJs inhibits PIP2-mediated activation of Kir2.1 to tightly regulate changes in arterial diameter, and they demonstrate that the intracellular lipid localization within the endothelium is an important determinant of vascular function

Clinical risk factors and atherosclerotic plaque extent to define risk for major events in patients without obstructive coronary artery disease: the long-term coronary computed tomography angiography CONFIRM registry.

AimsIn patients without obstructive coronary artery disease (CAD), we examined the prognostic value of risk factors and atherosclerotic extent.Methods and resultsPatients from the long-term CONFIRM registry without prior CAD and without obstructive (≥50%) stenosis were included. Within the groups of normal coronary computed tomography angiography (CCTA) (N = 1849) and non-obstructive CAD (N = 1698), the prognostic value of traditional clinical risk factors and atherosclerotic extent (segment involvement score, SIS) was assessed with Cox models. Major adverse cardiac events (MACE) were defined as all-cause mortality, non-fatal myocardial infarction, or late revascularization. In total, 3547 patients were included (age 57.9 ± 12.1 years, 57.8% male), experiencing 460 MACE during 5.4 years of follow-up. Age, body mass index, hypertension, and diabetes were the clinical variables associated with increased MACE risk, but the magnitude of risk was higher for CCTA defined atherosclerotic extent; adjusted hazard ratio (HR) for SIS >5 was 3.4 (95% confidence interval [CI] 2.3-4.9) while HR for diabetes and hypertension were 1.7 (95% CI 1.3-2.2) and 1.4 (95% CI 1.1-1.7), respectively. Exclusion of revascularization as endpoint did not modify the results. In normal CCTA, presence of ≥1 traditional risk factors did not worsen prognosis (log-rank P = 0.248), while it did in non-obstructive CAD (log-rank P = 0.025). Adjusted for SIS, hypertension and diabetes predicted MACE risk in non-obstructive CAD, while diabetes did not increase risk in absence of CAD (P-interaction = 0.004).ConclusionAmong patients without obstructive CAD, the extent of CAD provides more prognostic information for MACE than traditional cardiovascular risk factors. An interaction was observed between risk factors and CAD burden, suggesting synergistic effects of both

Coronary atherosclerosis scoring with semiquantitative CCTA risk scores for prediction of major adverse cardiac events: Propensity score-based analysis of diabetic and non-diabetic patients.

AIMS:We aimed to compare semiquantitative coronary computed tomography angiography (CCTA) risk scores - which score presence, extent, composition, stenosis and/or location of coronary artery disease (CAD) - and their prognostic value between patients with and without diabetes mellitus (DM). Risk scores derived from general chest-pain populations are often challenging to apply in DM patients, because of numerous confounders. METHODS:Out of a combined cohort from the Leiden University Medical Center and the CONFIRM registry with 5-year follow-up data, we performed a secondary analysis in diabetic patients with suspected CAD who were clinically referred for CCTA. A total of 732 DM patients was 1:1 propensity-matched with 732 non-DM patients by age, sex and cardiovascular risk factors. A subset of 7 semiquantitative CCTA risk scores was compared between groups: 1) any stenosis ≥50%, 2) any stenosis ≥70%, 3) stenosis-severity component of the coronary artery disease-reporting and data system (CAD-RADS), 4) segment involvement score (SIS), 5) segment stenosis score (SSS), 6) CT-adapted Leaman score (CT-LeSc), and 7) Leiden CCTA risk score. Cox-regression analysis was performed to assess the association between the scores and the primary endpoint of all-cause death and non-fatal myocardial infarction. Also, area under the receiver-operating characteristics curves were compared to evaluate discriminatory ability. RESULTS:A total of 1,464 DM and non-DM patients (mean age 58 ± 12 years, 40% women) underwent CCTA and 155 (11%) events were documented after median follow-up of 5.1 years. In DM patients, the 7 semiquantitative CCTA risk scores were significantly more prevalent or higher as compared to non-DM patients (p ≤ 0.022). All scores were independently associated with the primary endpoint in both patients with and without DM (p ≤ 0.020), with non-significant interaction between the scores and diabetes (interaction p ≥ 0.109). Discriminatory ability of the Leiden CCTA risk score in DM patients was significantly better than any stenosis ≥50% and ≥70% (p = 0.003 and p = 0.007, respectively), but comparable to the CAD-RADS, SIS, SSS and CT-LeSc that also focus on the extent of CAD (p ≥ 0.265). CONCLUSION:Coronary atherosclerosis scoring with semiquantitative CCTA risk scores incorporating the total extent of CAD discriminate major adverse cardiac events well, and might be useful for risk stratification of patients with DM beyond the binary evaluation of obstructive stenosis alone

ApoA-I binding protein (AIBP) decreases mechanical hypersensitivity after plantar incision in a rat model of post-operative pain

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