Local Cortical Tension by Myosin II Guides 3D Endothelial Cell Branching

SummaryA key feature of angiogenesis is directional control of endothelial cell (EC) morphogenesis and movement [1]. During angiogenic sprouting, endothelial “tip cells” directionally branch from existing vessels in response to biochemical cues such as VEGF or hypoxia and migrate and invade the surrounding extracellular matrix (ECM) in a process that requires ECM remodeling by matrix metalloproteases (MMPs) [2–4]. Tip EC branching is mediated by directional protrusion of subcellular pseudopodial branches [5, 6]. Here, we seek to understand how EC pseudopodial branching is locally regulated to directionally guide angiogenesis. We develop an in vitro 3D EC model system in which migrating ECs display branched pseudopodia morphodynamics similar to those in living zebrafish. Using this system, we find that ECM stiffness and ROCK-mediated myosin II activity inhibit EC pseudopodial branch initiation. Myosin II is dynamically localized to the EC cortex and is partially released under conditions that promote branching. Local depletion of cortical myosin II precedes branch initiation, and initiation can be induced by local inhibition of myosin II activity. Thus, local downregulation of myosin II cortical contraction allows pseudopodium initiation to mediate EC branching and hence guide directional migration and angiogenesis

Low prevalence of hepatitis C co-infection in recently HIV-infected minority men who have sex with men in Los Angeles: a cross-sectional study.

BackgroundGeographic and sociodemographic characterization of hepatitis C virus (HCV) transmission amongst men who have sex with men (MSM) has been limited. Our aim was to characterize HCV prevalence, risk factors for HCV co-infection, and patterns of HIV and HCV co-transmission and transmitted drug resistance mutations (DRMs) in newly HIV-diagnosed Los Angeles MSM.MethodsViral RNA was extracted from stored plasma samples from a Los Angeles cohort of newly diagnosed HIV-infected MSM with well-characterized substance use and sexual behavioral characteristics via computer-assisted self-interviewing surveys. Samples were screened for HCV by qPCR. HCV E1, E2, core, NS3 protease and NS5B polymerase and HIV-1 protease and reverse transcriptase regions were amplified and sequenced. Phylogenetic analysis was used to determine relatedness of HCV and HIV-1 isolates within the cohort and viral sequences were examined for DRMs.ResultsOf 185 newly HIV-diagnosed MSM, the majority (65%) were of minority race/ethnicity and recently infected (57.8%), with median age of 28.3 years. A minority (6.6%) reported injection drug use (IDU), whereas 96 (52.8%) reported recent substance use, primarily cannabis or stimulant use. High risk sexual behaviors included 132 (74.6%) with unprotected receptive anal intercourse, 60 (33.3%) with group sex, and 10 (5.7%) with fisting. Forty-five (24.3%) had acute gonorrhea or chlamydia infection. Only 3 (1.6%) subjects had detectable HCV RNA. Amongst these subjects, HIV and HCV isolates were unrelated by phylogenetic analysis and none possessed clinically relevant NS3 or NS5B HCV DRMs.ConclusionsPrevalence of HCV co-infection was low and there was no evidence of HIV-HCV co-transmission in this cohort of relatively young, predominantly minority, newly HIV-diagnosed MSM, most with early HIV infection, with high rates of high risk sexual behaviors, STI, and non-IDU. The low HCV prevalence in a group with high-risk behaviors for non-IDU HCV acquisition suggests an opportune time for targeted HCV prevention measures

The structure of a GH149 β‐(1 → 3) glucan phosphorylase reveals a new surface oligosaccharide binding site and additional domains that are absent in the disaccharide‐specific GH94 glucose‐β‐(1 → 3)‐glucose (laminaribiose) phosphorylase

Glycoside phosphorylases (GPs) with specificity for β-(1 → 3)-gluco-oligosaccharides are potential candidate biocatalysts for oligosaccharide synthesis. GPs with this linkage specificity are found in two families thus far—glycoside hydrolase family 94 (GH94) and the recently discovered glycoside hydrolase family 149 (GH149). Previously, we reported a crystallographic study of a GH94 laminaribiose phosphorylase with specificity for disaccharides, providing insight into the enzyme's ability to recognize its' sugar substrate/product. In contrast to GH94, characterized GH149 enzymes were shown to have more flexible chain length specificity, with preference for substrate/product with higher degree of polymerization. In order to advance understanding of the specificity of GH149 enzymes, we herein solved X-ray crystallographic structures of GH149 enzyme Pro_7066 in the absence of substrate and in complex with laminarihexaose (G6). The overall domain organization of Pro_7066 is very similar to that of GH94 family enzymes. However, two additional domains flanking its catalytic domain were found only in the GH149 enzyme. Unexpectedly, the G6 complex structure revealed an oligosaccharide surface binding site remote from the catalytic site, which, we suggest, may be associated with substrate targeting. As such, this study reports the first structure of a GH149 phosphorylase enzyme acting on β-(1 → 3)-gluco-oligosaccharides and identifies structural elements that may be involved in defining the specificity of the GH149 enzymes

Measuring Economic Disadvantage During Childhood: A Group-Based Modeling Approach

Recent research suggest that child well-being and subsequent status attainment are influenced not only by the overall magnitude of exposure to family economic disadvantage during childhood, but also by the age of exposure and significant changes in family economic circumstances. Unfortunately, traditional measures of children's economic deprivation, such as permanent and transitory income, persistent or cumulative poverty, and the number and length of poverty spells, fail to differentiate between exposure to disadvantage at different stages in childhood and largely ignore how family economic circumstances are changing over time. In this paper, the authors propose a new method for assessing economic disadvantage during childhood that captures both children's overall levels of exposure to economic disadvantage and their patterns of exposure. This new method, which takes advantage of recent advances in finite mixture modeling, uses a longitudinal latent class model to classify children into a limited number of groups with similar histories of exposure to family economic disadvantage. Using this new methodology, group membership can be related to both family background characteristics and achievement in childhood and early adulthood, making it possible both to assess how family characteristics affect patterns of exposure to disadvantage during childhood and directly test alternative theories about the effect of different patterns of exposure on achievement. In this paper, the relationship between background factors, such as race, parental education, and family structure, and group membership is investigated, as is the association between group membership and achievement in early adulthood. The use of this technique is demonstrated using data from the Panel Study of Income Dynamics (PSID)

Research Note: A comparison of media for the recovery of Campylobacter spp. from long term storage at -80° C

Publication history: Accepted - 23 May 2017; Published - 2019.Pure cultures of Campylobacter spp. can be stored at -80°C for extended periods, however they eventually lose viability. To maintain cultures regular resuscitation and subculturing of strains needs to be undertaken but this requires that staff and media are available for this purpose. Financial pressures on many institutes have had the consequence that regular sub-culturing has become a financial burden. Accordingly this study was undertaken to compare the ability of inexpensive media to recover campylobacters from storage of up to 12 years at -80°C. Brain heart infusion agar and modified charcoal cefoperazone deoxycholate agar base were compared with blood agar. Overall, blood agar was found to be the best medium for this purpose and is to be recommended.This project was funded by the Society for Applied Microbiology, under the auspices of the Students and Graduates into Work Programme

Rac1-Dependent Phosphorylation and Focal Adhesion Recruitment of Myosin IIA Regulates Migration and Mechanosensing

SummaryBackgroundCell migration requires coordinated formation of focal adhesions (FAs) and assembly and contraction of the actin cytoskeleton. Nonmuscle myosin II (MII) is a critical mediator of contractility and FA dynamics in cell migration. Signaling downstream of the small GTPase Rac1 also regulates FA and actin dynamics, but its role in regulation of MII during migration is less clear.ResultsWe found that Rac1 promotes association of MIIA with FA. Live-cell imaging showed that, whereas most MIIA at the leading edge assembled into dorsal contractile arcs, a substantial subset assembled in or was captured within maturing FA, and this behavior was promoted by active Rac1. Protein kinase C (PKC) activation was necessary and sufficient for integrin- and Rac1-dependent phosphorylation of MIIA heavy chain (HC) on serine1916 (S1916) and recruitment to FA. S1916 phosphorylation of MIIA HC and localization in FA was enhanced during cell spreading and ECM stiffness mechanosensing, suggesting upregulation of this pathway during physiological Rac1 activation. Phosphomimic and nonphosphorylatable MIIA HC mutants demonstrated that S1916 phosphorylation was necessary and sufficient for the capture and assembly of MIIA minifilaments in FA. S1916 phosphorylation was also sufficient to promote the rapid assembly of FAs to enhance cell migration and for the modulation of traction force, spreading, and migration by ECM stiffness.ConclusionsOur study reveals for the first time that Rac1 and integrin activation regulates MIIA HC phosphorylation through a PKC-dependent mechanism that promotes MIIA association with FAs and acts as a critical modulator of cell migration and mechanosensing

Transcriptome Profiling of Layer 5 Intratelencephalic Projection Neurons From the Mature Mouse Motor Cortex

The mature cortex contains hugely diverse populations of pyramidal projection neurons (PNs), critical to normal forebrain circuits. In order to understand the healthy cortex, it is essential to characterize this neuronal complexity. We recently demonstrated different identities for Fezf2-positive (Fezf2+ve) and Fezf2-negative (Fezf2−ve) intratelencephalic-PNs (IT-PNs) from layer 5 of the motor cortex (M1). Comparatively, each IT-PN type has a distinct electrophysiological phenotype and the Fezf2+ve IT-PNs display a unique apical dendritic tuft. Here, we aimed to expand our understanding of the molecular underpinnings defining these unique IT-PN types. Using a validated Fezf2-GFP reporter mouse, retrograde labeling techniques and fluorescence activated cell sorting (FACS), combined with a novel approach for low-input RNA-sequencing, we isolated mature Fezf2+ve and Fezf2−ve IT-PNs for transcriptome profiling. Through the comparison of Fezf2+ve and Fezf2−ve IT-PN gene expression profiles, we identified significant enrichment of 81 genes in the Fezf2+ve IT-PNs and 119 genes in the Fezf2−ve IT-PNs. Term enrichment analysis of these enriched genes demonstrated significant overrepresentation of the calcium-binding EF-hand domain in Fezf2+ve IT-PNs, suggesting a greater importance for calcium handling in these neurons. Of the Fezf2−ve IT-PN enriched genes an unexpected and unique enrichment of genes, previously associated with microglia were identified. Our dataset identifies the molecular profiles of two unique IT-PN types in the mature M1, providing important targets to investigate for their maintenance in the healthy mature brain

The Physical Characteristics of a CO2 Seeping Fault: the implications of fracture permeability for carbon capture and storage integrity

To ensure the effective long-term storage of CO2 in candidate geological storage sites, evaluation of potential leakage pathways to the surface should be undertaken. Here we use a series of natural CO2 seeps along a fault in South Africa to assess the controls on CO2 leakage to the surface. Geological mapping and detailed photogrammetry reveals extensive fracturing along the mapped fault trace. Measurements of gas flux and CO2 concentration across the fracture corridor give maximum soil gas measurements of 27% CO2 concentration and a flux of 191 g m−2 d−1. These measurements along with observations of gas bubbles in streams and travertine cones attest to CO2 migration to the surface. Permeability measurements on the host rock units show that the tillite should act as an impermeable seal to upward CO2 migration. The combined permeability and fracture mapping data indicate that fracture permeability creates the likely pathway for CO2 migration through the low permeability tillite to the surface. Heterogeneity in fracture connectivity and intensity at a range of scales will create local higher permeability pathways along the fracture corridor, although these may seal with time due to fluid-rock interaction. The results have implications for the assessment and choice of geological CO2 storage sites, particularly in the assessment of sub-seismic fracture networks