A thin layer angiogenesis assay: a modified basement matrix assay for assessment of endothelial cell differentiation

BACKGROUND: Basement matrices such as Matrigel™ and Geltrex™ are used in a variety of cell culture assays of anchorage-dependent differentiation including endothelial cell tube formation assays. The volumes of matrix recommended for these assays (approximately 150 μl/cm(2)) are costly, limit working distances for microscopy, and require cell detachment for subsequent molecular analysis. Here we describe the development and validation of a thin-layer angiogenesis (TLA) assay for assessing the angiogenic potential of endothelial cells that overcomes these limitations. RESULTS: Geltrex™ basement matrix at 5 μl/cm(2) in 24-well (10 μl) or 96-well (2 μl) plates supports endothelial cell differentiation into tube-like structures in a comparable manner to the standard larger volumes of matrix. Since working distances are reduced, high-resolution single cell microscopy, including DIC and confocal imaging, can be used readily. Using MitoTracker dye we now demonstrate, for the first time, live mitochondrial dynamics and visualise the 3-dimensional network of mitochondria present in differentiated endothelial cells. Using a standard commercial total RNA extraction kit (Qiagen) we also show direct RNA extraction and RT-qPCR from differentiated endothelial cells without the need to initially detach cells from their supporting matrix. CONCLUSIONS: We present here a new thin-layer assay (TLA) for measuring the anchorage-dependent differentiation of endothelial cells into tube-like structures which retains all the characteristics of the traditional approach but with the added benefit of a greatly lowered cost and better compatibility with other techniques, including RT-qPCR and high-resolution microscopy. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12860-014-0041-5) contains supplementary material, which is available to authorized users

Th e role of prescribed burning in maintenance of an endangered plant species, Lomatium bradshawii.

Abstract. Responses of a federally listed endangered plant species, Lomatium bradshawii, to the use of fire as a management tool for maintaining remnant wetland prairies were evaluated at two public land areas in the Willamette Valley of western Oregon. Areas containing L. bradshawii were treated with two or three fall season prescribed burns during a nine-year period. Foliar crown area, height, umbellets, and schizocarps of 150 L. bradshawii at Rose Prairie and 250 at Fisher Butte and the recruitment and density of L. bradshawii in 2-m 2 plots at both sites were documented during 1988-1996. When both sites were considered together, crown area, height, umbellets, and schizocarps per plant initially responded positively to burn treatments, but increases were not consistent across years or sites. Crown area tended to increase and then decline after each burn. Burning initially enhanced schizocarp production at both sites; schizocarps declined one or two years after burning but remained much higher in the burn treatments than in controls until 1996. Seedling production was not correlated with schizocarp production at either site. Umbellet and schizocarp production were not correlated with January-June temperatures or precipitation at the nearest weather station. Burning accentuated differences in size and reproductive capacity of L. bradshawii at the two sites and differentially affected recruitment and density. Random resampling of L. bradshawii in 1997 indicated that effects of repeated burning during the previous eight years were hard to detect. At Rose Prairie, foliar crown area, height, number of leaves, umbellets, and schizocarps in 1997 were similar or lower with burning than in unburned controls. At Fisher Butte, L. bradshawii in the two burn treatment were similar to control plants, but three burns significantly increased foliar crown area, number of leaves, and schizocarps. Monitoring recovery for one or two years after a burn may only capture the initial stimulation provided by burning and may foster unrealistically high expectations concerning the viability of an endangered plant population

Hypoglycemia and Clinical Outcomes in Patients With Diabetes Hospitalized in the General Ward

OBJECTIVE: Hypoglycemia is associated with adverse outcomes in mixed populations of patients in intensive care units. It is not known whether the same risks exist for diabetic patients who are less severely ill. In this study, we aimed to determine whether hypoglycemic episodes are associated with higher mortality in diabetic patients hospitalized in the general ward. RESEARCH DESIGN AND METHODS: This retrospective cohort study analyzed 4,368 admissions of 2,582 patients with diabetes hospitalized in the general ward of a teaching hospital between January 2003 and August 2004. The associations between the number and severity of hypoglycemic (≤50 mg/dl) episodes and inpatient mortality, length of stay (LOS), and mortality within 1 year after discharge were evaluated. RESULTS: Hypoglycemia was observed in 7.7% of admissions. In multivariable analysis, each additional day with hypoglycemia was associated with an increase of 85.3% in the odds of inpatient death (P = 0.009) and 65.8% (P = 0.0003) in the odds of death within 1 year from discharge. The odds of inpatient death also rose threefold for every 10 mg/dl decrease in the lowest blood glucose during hospitalization (P = 0.0058). LOS increased by 2.5 days for each day with hypoglycemia (P < 0.0001). CONCLUSIONS: Hypoglycemia is common in diabetic patients hospitalized in the general ward. Patients with hypoglycemia have increased LOS and higher mortality both during and after admission. Measures should be undertaken to decrease the frequency of hypoglycemia in this high-risk patient population

The cytokine language of monocytes and macrophages in systemic sclerosis

Many important observations suggest monocyte/macrophage involvement in systemic sclerosis (SSc). A high concentration of immune mediators, such as IL-6, IL-10 and IL-13, the infiltration of mononuclear cells in affected organs and the production of autoantibodies suggest that immune system dysfunction drives SSc pathogenesis. The recently reported study by Higashi-Kuwata and colleagues, in light of other observations, provides further insight into activation of macrophages/monocytes in SSc patients, suggesting that these cells undergo distinct activation pathways. These results emphasize the need for more detailed analyses of the several markers now defined in SSc peripheral blood mononuclear cells and tissues to better define the cytokine language speaking to monocytes/macrophages in SSc that promote vascular injury and tissue fibrosis

A polygenic and phenotypic risk prediction for polycystic ovary syndrome evaluated by phenomewide association studies

Context: As many as 75% of patients with polycystic ovary syndrome (PCOS) are estimated tobe unidentified in clinical practice. Objective: Utilizing polygenic risk prediction, we aim to identify the phenome-widecomorbidity patterns characteristic of PCOS to improve accurate diagnosis and preventivetreatment.Design, Patients, and Methods: Leveraging the electronic health records (EHRs) of 124 852individuals, we developed a PCOS risk prediction algorithm by combining polygenic risk scores(PRS) with PCOS component phenotypes into a polygenic and phenotypic risk score (PPRS). Weevaluated its predictive capability across different ancestries and perform a PRS-based phenomewide association study (PheWAS) to assess the phenomic expression of the heightened risk ofPCOS.Results: The integrated polygenic prediction improved the average performance (pseudo-R2)for PCOS detection by 0.228 (61.5-fold), 0.224 (58.8-fold), 0.211 (57.0-fold) over the null modelacross European, African, and multi-ancestry participants respectively. The subsequent PRSpowered PheWAS identified a high level of shared biology between PCOS and a range ofmetabolic and endocrine outcomes, especially with obesity and diabetes: "morbid obesity","type 2 diabetes", "hypercholesterolemia", "disorders of lipid metabolism", "hypertension",and "sleep apnea" reaching phenome-wide significance.Conclusions: Our study has expanded the methodological utility of PRS in patient stratificationand risk prediction, especially in a multifactorial condition like PCOS, across different geneticorigins. By utilizing the individual genome-phenome data available from the EHR, our approachalso demonstrates that polygenic prediction by PRS can provide valuable opportunities todiscover the pleiotropic phenomic network associated with PCOS pathogenesis.Abbreviations: AA, African ancestry; ANOVA, analysis of variance; BMI, body mass index; EA,European ancestry; EHR, electronic health records; eMERGE, electronic Medical Records andGenomics Network; GWAS, genome-wide association study; IBD, identity-by-descent; ICDCM, International Classification of Diseases, Clinical Modification; LD, linkage disequilibrium;MA, multi-ancestry; MAF, minor allele frequency; NIH, National Institutes of Health; PCA,principal component analysis; PheWAS, phenome-wide association study; PCOS, polycysticovary syndrome; PPRS, polygenic and phenotypic risk score; PRS, polygenic risk sc

Polygenic inheritance of paclitaxel-induced sensory peripheral neuropathy driven by axon outgrowth gene sets in CALGB 40101 (Alliance)

Peripheral neuropathy is a common dose-limiting toxicity for patients treated with paclitaxel. For most individuals there are no known risk factors that predispose patients to the adverse event, and pathogenesis for paclitaxel-induced peripheral neuropathy is unknown. Determining whether there is a heritable component to paclitaxel induced peripheral neuropathy would be valuable in guiding clinical decisions and may provide insight into treatment of and mechanisms for the toxicity. Using genotype and patient information from the paclitaxel arm of CALGB 40101 (Alliance), a phase III clinical trial evaluating adjuvant therapies for breast cancer in women, we estimated the variance in maximum grade and dose at first instance of sensory peripheral neuropathy. Our results suggest that paclitaxel-induced neuropathy has a heritable component, driven in part by genes involved in axon outgrowth. Disruption of axon outgrowth may be one of the mechanisms by which paclitaxel treatment results in sensory peripheral neuropathy in susceptible patients

Variation in LPA Is Associated with Lp(a) Levels in Three Populations from the Third National Health and Nutrition Examination Survey

The distribution of lipoprotein(a) [Lp(a)] levels can differ dramatically across diverse racial/ethnic populations. The extent to which genetic variation in LPA can explain these differences is not fully understood. To explore this, 19 LPA tagSNPs were genotyped in 7,159 participants from the Third National Health and Nutrition Examination Survey (NHANES III). NHANES III is a diverse population-based survey with DNA samples linked to hundreds of quantitative traits, including serum Lp(a). Tests of association between LPA variants and transformed Lp(a) levels were performed across the three different NHANES subpopulations (non-Hispanic whites, non-Hispanic blacks, and Mexican Americans). At a significance threshold of p<0.0001, 15 of the 19 SNPs tested were strongly associated with Lp(a) levels in at least one subpopulation, six in at least two subpopulations, and none in all three subpopulations. In non-Hispanic whites, three variants were associated with Lp(a) levels, including previously known rs6919246 (p = 1.18×10−30). Additionally, 12 and 6 variants had significant associations in non-Hispanic blacks and Mexican Americans, respectively. The additive effects of these associated alleles explained up to 11% of the variance observed for Lp(a) levels in the different racial/ethnic populations. The findings reported here replicate previous candidate gene and genome-wide association studies for Lp(a) levels in European-descent populations and extend these findings to other populations. While we demonstrate that LPA is an important contributor to Lp(a) levels regardless of race/ethnicity, the lack of generalization of associations across all subpopulations suggests that specific LPA variants may be contributing to the observed Lp(a) between-population variance

Satellite quantification of methane emissions and oil–gas methane intensities from individual countries in the Middle East and North Africa: implications for climate action

We use 2019 TROPOMI satellite observations of atmospheric methane in an analytical inversion to quantify methane emissions from the Middle East and North Africa at up to ∼25 km × 25 km resolution, using spatially allocated national United Nations Framework Convention on Climate Change (UNFCCC) reports as prior estimates for the fuel sector. Our resulting best estimate of anthropogenic emissions for the region is 35 % higher than the prior bottom-up inventories (+103 % for gas, +53 % for waste, +49 % for livestock, −14 % for oil) with large variability across countries. Oil and gas account for 38 % of total anthropogenic emissions in the region. TROPOMI observations can effectively optimize and separate national emissions by sector for most of the 23 countries in the region, with 6 countries accounting for most of total anthropogenic emissions including Iran (5.3 (5.0–5.5) Tg a−1; best estimate and uncertainty range), Turkmenistan (4.4 (2.8–5.1) Tg a−1), Saudi Arabia (4.3 (2.4–6.0) Tg a−1), Algeria (3.5 (2.4–4.4) Tg a−1), Egypt (3.4 (2.5–4.0) Tg a−1), and Turkey (3.0 (2.0–4.1) Tg a−1). Most oil–gas emissions are from the production (upstream) subsector, but Iran, Turkmenistan, and Saudi Arabia have large gas emissions from transmission and distribution subsectors. We identify a high number of annual oil–gas emission hotspots in Turkmenistan, Algeria, and Oman and offshore in the Persian Gulf. We show that oil–gas methane emissions for individual countries are not related to production, invalidating a basic premise in the construction of activity-based bottom-up inventories. Instead, local infrastructure and management practices appear to be key drivers of oil–gas emissions, emphasizing the need for including top-down information from atmospheric observations in the construction of oil–gas emission inventories. We examined the methane intensity, defined as the upstream oil–gas emission per unit of methane gas produced, as a measure of the potential for decreasing emissions from the oil–gas sector and using as reference the 0.2 % target set by the industry. We find that the methane intensity in most countries is considerably higher than this target, reflecting leaky infrastructure combined with deliberate venting or incomplete flaring of gas. However, we also find that Kuwait, Saudi Arabia, and Qatar meet the industry target and thus show that the target is achievable through the capture of associated gas, modern infrastructure, and the concentration of operations. Decreasing methane intensities across the Middle East and North Africa to 0.2 % would achieve a 90 % decrease in oil–gas upstream emissions and a 26 % decrease in total anthropogenic methane emissions in the region, making a significant contribution toward the Global Methane Pledge.</p