Ca(2+)-dependent translocation of cytosolic proteins to isolated granule subpopulations and plasma membrane from human neutrophils.

In order to identify cytosolic proteins involved in control of granule exocytosis in human neutrophils, subcellular fractions enriched in each of the 3 major granule subsets were incubated with cytosol from neutrophils in the presence or absence of Ca2+. After washing, proteins were eluted from the organelles by EGTA. Annexins I, II, IV and VI were found to bind to all organelles studied. In addition, a 28-kDa protein was found to bind exclusively to plasma membranes and secretory vesicles, the most readily exocytosed organelle of neutrophils. Ca(2+)-dependent association of cytosolic proteins to different granule subsets may control differential exocytosis of granules.     

VsENBP1 regulates the expression of the early nodulin PsENOD12B

A DNA-binding protein, VsENBP1, previously isolated from Vicia sativa was shown to bind in a sequence-specific manner to the early nodulin ENOD12 gene promoter from Pisum sativum. Here, the functional importance of the VsENBP1 binding sites on the PsENOD12B promoter has been studied in vivo. A promoter-gusA fusion in which a mutation was introduced at the putative target sequence, AATAA, was inactive in nodules of transgenic Vicia hirsuta roots. Gel retardation assays showed that VsENBP1 does not bind to the mutated promoter segment, suggesting that VsENBP1 activates the PsENOD12B expression in nodules through its interaction with its target sequence. In the presence of the 35S enhancer, an ENOD12 promoter-GUS construct gave expression in root vascular tissue in addition to the root nodules. Overexpression of Vsenbp1 in transgenic V. hirsuta roots reduced the leaky expression in root vascular tissue in contrast to nodules in which a small increase in GUS expression was observed. The results indicate that VsENBP1 acts as a repressor of ENOD12 expression in root tissue.     

Use of low‐dose quetiapine increases the risk of major adverse cardiovascular events: results from a nationwide active comparator‐controlled cohort study

At standard doses used for schizophrenia or bipolar disorder, quetiapine has been associated with weight gain and increased levels of triglycerides, to­tal cholesterol and low‐density lipoprotein (LDL) cholesterol, which are risk factors for cardiovascular morbidity and mortality. However, this drug is also commonly used off‐label at low doses for anxiolytic or hypnotic purposes, and its cardiovascular safety at these doses is unknown. We aimed to assess the risk of major adverse cardiovascular events with use of low‐dose quetiapine compared to use of Z‐drug hypnotics in a nationwide, active comparator‐controlled cohort study. The cohort included new users of either drugs in Denmark from 2003 to 2017, aged 18‐85 years, without history of ischemic stroke, myocardial infarction, cancer, and severe mental illness. The main outcome was the occurrence of major adverse cardiovascular events, defined as non‐fatal myocardial infarction or ischemic stroke, or death from cardiovascular causes. Selective serotonin reuptake inhibitors (SSRIs) were used as an alternative comparator in sensitivity analyses. Altogether, we compared 60,566 low‐dose quetiapine users with 454,567 Z‐drug users, followed for 890,198 person‐years in intent‐to‐treat analysis, and 330,334 person‐years in as‐treated analysis. In intention‐to‐treat analysis, low‐dose quetiapine was associated with an increased risk of major adverse cardiovascular events (adjusted hazard ratio, aHR=1.13, 95% CI: 1.02‐1.24, p=0.014) and cardiovascular death (aHR=1.26, 95% CI: 1.11‐1.43, p<0.001). In as‐treated analysis, continuous low‐dose quetiapine use was associated with increased risk of major adverse cardiovascular events (aHR=1.52, 95% CI: 1.35‐1.70, p<0.001), non‐fatal ischemic stroke (aHR=1.37, 95% CI: 1.13‐1.68, p=0.002) and cardiovascular death (aHR=1.90, 95% CI: 1.64‐2.19, p<0.001). The risk of major adverse cardiovascular events was greater in women (aHR=1.28, p=0.02) and those aged ≥65 years at initiation (aHR=1.24, p<0.001). Compared to SSRIs, low‐dose quetiapine use was associated with an increased risk of major adverse cardiovascular events (aHR=1.42, p<0.001), non‐fatal ischemic stroke (aHR=1.27, p=0.0028) and cardiovascular death (aHR=1.72, p<0.001). So, we conclude that the use of low‐dose quetiapine is associated with an increased risk of major adverse cardiovascular events, especially in women and the elderly. On the basis of these findings, we suggest that use of off‐label low‐dose quetiapine for sedative or hypnotic purposes should be discouraged.     

Endospores of thermophilic bacteria as tracers of microbial dispersal by ocean currents

Microbial biogeography is influenced by the combined effects of passive dispersal and environmental selection, but the contribution of either factor can be difficult to discern. As thermophilic bacteria cannot grow in the cold seabed, their inactive spores are not subject to environmental selection. We therefore conducted a global experimental survey using thermophilic endospores that are passively deposited by sedimentation to the cold seafloor as tracers to study the effect of dispersal by ocean currents on the biogeography of marine microorganisms. Our analysis of 81 different marine sediments from around the world identified 146 species-level 16S rRNA phylotypes of endospore-forming, thermophilic Firmicutes. Phylotypes showed various patterns of spatial distribution in the world oceans and were dispersal-limited to different degrees. Co-occurrence of several phylotypes in locations separated by great distances (west of Svalbard, the Baltic Sea and the Gulf of California) demonstrated a widespread but not ubiquitous distribution. In contrast, Arctic regions with water masses that are relatively isolated from global ocean circulation (Baffin Bay and east of Svalbard) were characterized by low phylotype richness and different compositions of phylotypes. The observed distribution pattern of thermophilic endospores in marine sediments suggests that the impact of passive dispersal on marine microbial biogeography is controlled by the connectivity of local water masses to ocean circulation.     

Sensitivity analysis of periprosthetic healing to cell migration,growth factor and post-operative gap using a mechanobiological model

A theoretical rationale, which could help in the investigation of mechanobiological factors affecting periprosthetic tissue healing, is still an open problem. We used a parametric sensitivity analysis to extend a theoretical model based on reactive transport and computational cell biology. The numerical experimentation involved the drill hole, the haptotactic and chemotactic migrations, and the initial concentration of an anabolic growth factor. Output measure was the mineral fraction in tissue surrounding a polymethymethacrylate (PMMA) canine implant (stable loaded implant, non-critical gap). Increasing growth factor concentration increased structural matrix synthesis. A cell adhesion gradient resulted in heterogeneous bone distribution and a growth factor gradient resulted in homogeneous bone distribution in the gap. This could explain the radial variation of bone density from the implant surface to the drill hole, indicating less secure fixation. This study helps to understand the relative importance of various host and clinical factors influencing bone distribution and resulting implant fixation.     

Training increases the concentration of [3H]ouabain-binding sites in rat skeletal muscle

Exercise is associated with a net loss of K+ from the working muscles and an increased plasma K+ concentration, indicating that the capacity for intracellular reaccumulation of K+ is exceeded. Training reduces the exercise-induced rise in plasma K+, and an increased plasma [K+] may interfere with physical performance. Since the clearing of K+ from the extracellular space depends on the capacity for active K+ uptake in skeletal muscle, the effects of training and inactivity on the total concentration of (Na+ + K+)-ATPase was determined. Following 6 weeks of swim training, the concentration of [3H]ouabain-binding sites in rat hindlimb muscles was up to 46% (P less than 0.001) higher than in those obtained from age-matched controls. Whereas muscle Na+, K+ contents remained unchanged, the concentration of citrate synthase increased by up to 76% (P less than 0.001). Training induced no change in the [3H]ouabain-binding-site concentration in the diaphragm, but in the heart ventricles, the K+-dependent 3-O-methylfluorescein phosphatase activity increased by 20% (P less than 0.001). Muscle inactivity induced by denervation, plaster immobilisation or tenotomy reduced the [3H]ouabain-binding-site concentration by 20-30% (P less than 0.02-0.001) within 1 week. In conclusion, training leads to a significant and reversible rise in the concentration of (Na+ + K+)-ATPase in muscle cells. This may be of importance for the beneficial effects on physical performance by improving the maximum capacity for K+ clearance.     

Verminephrobacter aporrectodeae sp. nov. subsp. tuberculatae and subsp. caliginosae, the specific nephridial symbionts of the earthworms Aporrectodea tuberculata and A. caliginosa

Clone library-based studies have shown that almost all lumbricid earthworm species harbour host-specific symbiotic bacteria belonging to the novel genus Verminephrobacter in their nephridia (excretory organs). To date the only described representative from this genus is Verminephrobacter eiseniae, the specific symbiont of the earthworm Eisenia fetida. In this study two novel rod-shaped, non-endosporeforming, betaproteobacterial symbionts were isolated from the nephridia of two closely related earthworm species. Both isolates were affiliated with the genus Verminephrobacter by 16S rRNA gene sequence analysis. Similarly to V. eiseniae, the two isolates grew aerobically with a preference for low oxygen concentrations on a range of sugars, fatty acids and amino acids and fermentatively on glucose and pyruvate. These phenotypes match well with the conditions reported or inferred for the nephridial environment. Based on 16S rRNA gene similarity, DNA–DNA hybridization value and phenotypic characteristics the two isolates are clearly distinct from V. eiseniae. Phenotypic characteristics could not clearly differentiate the two strains as separate species but a low DNA–DNA hybridization value of 57.3%, their earthworm host specificity, differing temperature ranges and pH optima suggest that they represent two subspecies of a novel species of Verminephrobacter. For this species, the name V. aporrectodeae sp. nov. is proposed, with the two subspecies V. aporrectodeae subsp. tuberculatae (type strain, At4^T = DSM 21361^T = LMG 25313^T) and V. aporrectodeae subsp. caliginosae (type strain, Ac9^T = DSM 21895^T = LMG 25312^T) isolated from the nephridia of the earthworms Aporrectodea tuberculata and A. caliginosa, respectively.     

Two Types of Endosymbiotic Bacteria in the Enigmatic Marine Worm Xenoturbella bocki

Two types of endosymbiotic bacteria were identified in the gastrodermis of the marine invertebrate Xenoturbella bocki (Xenoturbellida, Bilateria). While previously described Chlamydia-like endosymbionts were rare, Gammaproteobacteria distantly related to other endosymbionts and pathogens were abundant. The endosymbionts should be considered when interpreting the poorly understood ecology and evolution of Xenoturbella.Xenoturbella bocki is a benthic marine worm first described in 1949 and only found at a few geographical locations at apparently low population densities. It has a very simple body plan (Fig. ​(Fig.1A)1A) with a blind gut (cul-de-sac) and lacks coelomic cavities, a brain, and reproductive and excretory organs (35). Xenoturbella was originally classified as a flatworm, but molecular analyses have placed it within its own animal phylum, with various affiliations (reviewed in reference 34). The most recent phylogenetic analyses using mitochondrial genome and phylogenomic data indicate a position either within Deuterostomia (5) or at the base of Bilateria (14). Thus, the evolutionary history of Xenoturbella remains elusive and it is unclear whether its simple body plan represents a plesiomorphic character or, alternatively, is the result of secondary loss (34). In addition, the ecology of Xenoturbella is not well understood, leaving, e.g., its food source unresolved (4, 18).Open in a separate windowFIG. 1.(A) Schematic drawing of the simple body plan of Xenoturbella (redrawn from reference 35). (B) Cells of the gammaproteobacterial endosymbiont (red) detected by probe XenoGam441-CY3 at high magnification (scale bar, 2 μm). (C) Double hybridization of gammaproteobacterial and chlamydial endosymbionts with probes XenoGam441-CY3 (red) and Cps-1353mod-CY5 (displayed in yellow for better contrast, indicated by the arrow); blue-green structures, autofluorescent tissue recorded in the fluorescein isothiocyanate channel (scale bar, 5 μm). (D) FISH detection of gammaproteobacterial endosymbionts with probe XenoGam441-CY3 (red) in a cross section of an X. bocki specimen. The image is composed of five consecutive image stacks, spanning from the gut (to the very left) to the epidermis (to the very right) (magnification, ×400; scale bar, 50 μm). Dense clusters of gammaproteobacterial endosymbionts (red) are confined to the gastrodermis; 4′,6-diamidino-2-phenylindole (DAPI)-stained cell nuclei and mitochondria of X. bocki are displayed in blue; the green and yellow structures are autofluorescent tissue recorded in the fluorescein isothiocyanate channel. (E and F) TEM images of bacterial cells in sections of germinal tissue from a spermatid cluster (E) and of gastric tissue (F) of X. bocki. Arrows point to examples of different putative bacterial morphotypes. Scale bars, 0.5 μm. n, spermatid nucleus; m, spermatid mitochondrion.Symbiotic bacteria are found in a remarkable number of diverse marine invertebrates (11), where they affect both the ecology and the evolution of their hosts. The first indications of intracellular endosymbionts in Xenoturbella were published by Israelsson (17) and confirmed by electron microscopy of a Xenoturbella spermatid cluster (Fig. ​(Fig.1B);1B); however, our first molecular analyses identified the putative endosymbionts as Gammaproteobacteria, whereas Israelsson (17) had reported a group of endosymbiotic Chlamydiae. The objectives of this study were therefore (i) to identify the putative endosymbionts by 16S rRNA gene analysis, (ii) to localize them inside the host by fluorescence in situ hybridization (FISH), and (iii) to specifically test for the prevalence of the gammaproteobacterial endosymbionts in Xenoturbella.     

Genome-wide association study of blood pressure extremes identifies variant near UMOD associated with hypertension

Hypertension is a heritable and major contributor to the global burden of disease. The sum of rare and common genetic variants robustly identified so far explain only 1%–2% of the population variation in BP and hypertension. This suggests the existence of more undiscovered common variants. We conducted a genome-wide association study in 1,621 hypertensive cases and 1,699 controls and follow-up validation analyses in 19,845 cases and 16,541 controls using an extreme case-control design. We identified a locus on chromosome 16 in the 5′ region of Uromodulin (UMOD; rs13333226, combined P value of 3.6×10⁻¹¹). The minor G allele is associated with a lower risk of hypertension (OR [95%CI]: 0.87 [0.84–0.91]), reduced urinary uromodulin excretion, better renal function; and each copy of the G allele is associated with a 7.7% reduction in risk of CVD events after adjusting for age, sex, BMI, and smoking status (H.R. = 0.923, 95% CI 0.860–0.991; p = 0.027). In a subset of 13,446 individuals with estimated glomerular filtration rate (eGFR) measurements, we show that rs13333226 is independently associated with hypertension (unadjusted for eGFR: 0.89 [0.83–0.96], p = 0.004; after eGFR adjustment: 0.89 [0.83–0.96], p = 0.003). In clinical functional studies, we also consistently show the minor G allele is associated with lower urinary uromodulin excretion. The exclusive expression of uromodulin in the thick portion of the ascending limb of Henle suggests a putative role of this variant in hypertension through an effect on sodium homeostasis. The newly discovered UMOD locus for hypertension has the potential to give new insights into the role of uromodulin in BP regulation and to identify novel drugable targets for reducing cardiovascular risk.