An acto-myosin II constricting ring initiates the fission of activity-dependent bulk endosomes in neurosecretory cells

Activity-dependent bulk endocytosis allows neurons to internalize large portions of the plasma membrane in response to stimulation. However, whether this critical type of compensatory endocytosis is unique to neurons or also occurs in other excitable cells is currently unknown. Here we used fluorescent 70 kDa dextran to demonstrate that secretagogue-induced bulk endocytosis also occurs in bovine chromaffin cells. The relatively large size of the bulk endosomes found in this model allowed us to investigate how the neck of the budding endosomes constricts to allow efficient recruitment of the fission machinery. Using time-lapse imaging of Lifeact–GFP-transfected chromaffin cells in combination with fluorescent 70 kDa dextran, we detected acto-myosin II rings surrounding dextran-positive budding endosomes. Importantly, these rings were transient and contracted before disappearing, suggesting that they might be involved in restricting the size of the budding endosome neck. Based on the complete recovery of dextran fluorescence after photobleaching, we demonstrated that the actin ring-associated budding endosomes were still connected with the extracellular fluid. In contrast, no such recovery was observed following the constriction and disappearance of the actin rings, suggesting that these structures were pinched-off endosomes. Finally, we showed that the rings were initiated by a circular array of phosphatidylinositol(4,5)bisphosphate microdomains, and that their constriction was sensitive to both myosin II and dynamin inhibition. The acto-myosin II rings therefore play a key role in constricting the neck of budding bulk endosomes before dynamin-dependent fission from the plasma membrane of neurosecretory cells

Secretagogue stimulation of neurosecretory cells elicits filopodial extensions uncovering new functional release sites

Regulated exocytosis in neurosecretory cells relies on the timely fusion of secretory granules (SGs) with the plasma membrane. Secretagogue stimulation leads to an enlargement of the cell footprint (surface area in contact with the coverslip), an effect previously attributed to exocytic fusion of SGs with the plasma membrane. Using total internal reflection fluorescence microscopy, we reveal the formation of filopodia-like structures in bovine chromaffin and PC12 cells driving the footprint expansion, suggesting the involvement of cortical actin network remodeling in this process. Using exocytosis-incompetent PC12 cells, we demonstrate that footprint enlargement is largely independent of SG fusion, suggesting that vesicular exocytic fusion plays a relatively minor role in filopodial expansion. The footprint periphery, including filopodia, undergoes extensive F-actin remodeling, an effect abolished by the actomyosin inhibitors cytochalasin D and blebbistatin. Imaging of both Lifeact-GFP and the SG marker protein neuropeptide Y-mCherry reveals that SGs actively translocate along newly forming actin tracks before undergoing fusion. Together, these data demonstrate that neurosecretory cells regulate the number of SGs undergoing exocytosis during sustained stimulation by controlling vesicular mobilization and translocation to the plasma membrane through actin remodeling. Such remodeling facilitates the de novo formation of fusion sites

Activity-driven relaxation of the cortical actomyosin II network synchronizes Munc18-1-dependent neurosecretory vesicle docking

In neurosecretory cells, secretory vesicles (SVs) undergo Ca2(+)-dependent fusion with the plasma membrane to release neurotransmitters. How SVs cross the dense mesh of the cortical actin network to reach the plasma membrane remains unclear. Here we reveal that, in bovine chromaffin cells, SVs embedded in the cortical actin network undergo a highly synchronized transition towards the plasma membrane and Munc18-1-dependent docking in response to secretagogues. This movement coincides with a translocation of the cortical actin network in the same direction. Both effects are abolished by the knockdown or the pharmacological inhibition of myosin II, suggesting changes in actomyosin-generated forces across the cell cortex. Indeed, we report a reduction in cortical actin network tension elicited on secretagogue stimulation that is sensitive to myosin II inhibition. We reveal that the cortical actin network acts as a 'casting net' that undergoes activity-dependent relaxation, thereby driving tethered SVs towards the plasma membrane where they undergo Munc18-1-dependent docking

Development and validation of the DIabetes Severity SCOre (DISSCO) in 139 626 individuals with type 2 diabetes: a retrospective cohort study

OBJECTIVE: Clinically applicable diabetes severity measures are lacking, with no previous studies comparing their predictive value with glycated hemoglobin (HbA1c). We developed and validated a type 2 diabetes severity score (the DIabetes Severity SCOre, DISSCO) and evaluated its association with risks of hospitalization and mortality, assessing its additional risk information to sociodemographic factors and HbA1c. RESEARCH DESIGN AND METHODS: We used UK primary and secondary care data for 139 626 individuals with type 2 diabetes between 2007 and 2017, aged ≥35 years, and registered in general practices in England. The study cohort was randomly divided into a training cohort (n=111 748, 80%) to develop the severity tool and a validation cohort (n=27 878). We developed baseline and longitudinal severity scores using 34 diabetes-related domains. Cox regression models (adjusted for age, gender, ethnicity, deprivation, and HbA1c) were used for primary (all-cause mortality) and secondary (hospitalization due to any cause, diabetes, hypoglycemia, or cardiovascular disease or procedures) outcomes. Likelihood ratio (LR) tests were fitted to assess the significance of adding DISSCO to the sociodemographics and HbA1c models. RESULTS: A total of 139 626 patients registered in 400 general practices, aged 63±12 years were included, 45% of whom were women, 83% were White, and 18% were from deprived areas. The mean baseline severity score was 1.3±2.0. Overall, 27 362 (20%) people died and 99 951 (72%) had ≥1 hospitalization. In the training cohort, a one-unit increase in baseline DISSCO was associated with higher hazard of mortality (HR: 1.14, 95% CI 1.13 to 1.15, area under the receiver operating characteristics curve (AUROC)=0.76) and cardiovascular hospitalization (HR: 1.45, 95% CI 1.43 to 1.46, AUROC=0.73). The LR tests showed that adding DISSCO to sociodemographic variables significantly improved the predictive value of survival models, outperforming the added value of HbA1c for all outcomes. Findings were consistent in the validation cohort. CONCLUSIONS: Higher levels of DISSCO are associated with higher risks for hospital admissions and mortality. The new severity score had higher predictive value than the proxy used in clinical practice, HbA1c. This reproducible algorithm can help practitioners stratify clinical care of patients with type 2 diabetes

New insights into lithology and hydrogeology of the northern Newark Rift Basin

The marginal facies of the Triassic rift basins in the eastern United States are poorly documented, particularly on the hinge or hanging wall margins. This study presents a lithological description and multiscale petrophysical analysis of basement rocks, overlying marginal facies of the early synrift strata, and the basal contact of the Palisade Sill that were drilled and cored in the northeastern part of the Newark Basin, near its terminus. The expression of the Stockton Formation differs from that in the central basin in having thinner layers, with uncertain temporal relationship to the type area. The bottom 50 m is lithologically distinct with brick-red to dark-purple mudstones and sandstones, abundant gypsum-filled fractures, and a thin zone with anomalously high uranium concentration, not associated with organic-rich mudstones as other occurrences in the basin. The crystalline basement is apparently Fordham gneiss, overlain by a thin sandstone layer and a dark-purple hydrophilic mudstone. Despite the abundance of coarse-grained strata and multiple sets of tectonic fractures, hydraulically transmissive zones are sparse, and do not uniquely correlate to fracture and/or matrix characteristics. Enhanced transmissivity may exist along intrusion boundaries due to enhanced thermal fracturing, but more hydraulic data are needed to verify it. Comparison of petrophysical data in two boreholes ∼210 m apart shows no direct correlation of individual lithological units and their hydraulic properties, although the overall formation characteristics are similar. The results highlight challenges for outcrop correlation at the marginal edges of the rift basins and estimating reservoir properties of these heterogeneous formations

Ethnicity and differences between clinic and ambulatory blood pressure measurements.

BACKGROUND: This study investigated the relationship of ethnicity to the differences between blood pressure (BP) measured in a clinic setting and by ambulatory blood pressure monitoring (ABPM) in individuals with a previous diagnosis of hypertension (HT) and without a previous diagnosis of hypertension (NHT). METHODS: A cross-sectional comparison of BP measurement was performed in 770 participants (white British (WB, 39%), South Asian (SA, 31%), and African Caribbean (AC, 30%)) in 28 primary care clinics in West Midlands, United Kingdom. Mean differences between daytime ABPM, standardized clinic (mean of 3 occasions), casual clinic (first reading on first occasion), and last routine BP taken at the general practitioner practice were compared in HT and NHT individuals. RESULTS: Daytime systolic and diastolic ABPM readings were similar to standardized clinic BP (systolic: 128 (SE 0.9) vs. 125 (SE 0.9) mm Hg (NHT) and 132 (SE 0.7) vs. 131 (SE 0.7) mm Hg (HT)) and were not associated with ethnicity to a clinically important extent. When BP was taken less carefully, differences emerged: casual clinic readings were higher than ABPM, particularly in the HT group where the systolic differences approached clinical relevance (131 (SE 1.2) vs. 129 (SE 1.0) mm Hg (NHT) and 139 (SE 0.9) vs. 133 (SE 0.7) mm Hg (HT)) and were larger in SA and AC hypertensive individuals (136 (SE 1.5) vs. 133 (SE 1.2) mm Hg (WB), 141 (SE 1.7) vs. 133 (SE 1.4) mm Hg (SA), and 142 (SE 1.6) vs. 134 (SE 1.3) mm Hg (AC); mean differences: 3 (0-7), P = 0.03 and 4 (1-7), P = 0.01, respectively). Differences were also observed for the last practice reading in SA and ACs. CONCLUSIONS: BP differences between ethnic groups where BP is carefully measured on multiple occasions are small and unlikely to alter clinical management. When BP is measured casually on a single occasion or in routine care, differences appear that could approach clinical relevance.This report presents independent research funded by the National Institute for Health Research (NIHR).The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of HealthThis is a pre-copyedited, author-produced PDF of an article accepted for publication in American Journal of Hypertension following peer review. The version of record, Am J Hypertens (2014) doi: 10.1093/ajh/hpu211, is available online at: http://ajh.oxfordjournals.org/content/early/2014/11/18/ajh.hpu211.long

Haploinsufficiency of RPS14 in 5q− syndrome is associated with deregulation of ribosomal- and translation-related genes

We have previously demonstrated haploinsufficiency of the ribosomal gene RPS14, which is required for the maturation of 40S ribosomal subunits and maps to the commonly deleted region, in the 5q− syndrome. Patients with Diamond-Blackfan anaemia (DBA) show haploinsufficiency of the closely related ribosomal protein RPS19, and show a consequent downregulation of multiple ribosomal- and translation-related genes. By analogy with DBA, we have investigated the expression profiles of a large group of ribosomal- and translation-related genes in the CD34+ cells of 15 myelodysplastic syndrome (MDS) patients with 5q− syndrome, 18 MDS patients with refractory anaemia (RA) and a normal karyotype, and 17 healthy controls. In this three-way comparison, 55 of 579 ribosomal- and translation-related probe sets were found to be significantly differentially expressed, with approximately 90% of these showing lower expression levels in the 5q− syndrome patient group. Using hierarchical clustering, patients with the 5q− syndrome could be separated both from other patients with RA and healthy controls solely on the basis of the deregulated expression of ribosomal- and translation-related genes. Patients with the 5q− syndrome have a defect in the expression of genes involved in ribosome biogenesis and in the control of translation, suggesting that the 5q− syndrome represents a disorder of aberrant ribosome biogenesis

SAXS4COLL: an integrated software tool for analysing fibrous collagen-based tissues

This article provides an overview of a new integrated software tool for reduction and analysis of small-angle X-ray scattering (SAXS) data from fibrous collagen tissues, with some wider applicability to other cylindrically symmetric scattering systems. SAXS4COLL combines interactive features for data pre-processing, bespoke background subtraction, semi-automated peak detection and calibration. Both equatorial and meridional SAXS peak parameters can be measured, and the former can be deconstructed into cylinder and lattice contributions. Finally, the software combines functionality for determination of collagen spatial order parameters with a rudimentary orientation plot capability