Structures of enveloped virions determined by cryogenic electron microscopy and tomography : Advances in Virus Research

Enveloped viruses enclose their genomes inside a lipid bilayer which is decorated by membrane proteins that mediate virus entry. These viruses display a wide range of sizes, morphologies and symmetries. Spherical viruses are often isometric and their envelope proteins follow icosahedral symmetry. Filamentous and pleomorphic viruses lack such global symmetry but their surface proteins may display locally ordered assemblies. Determining the structures of enveloped viruses, including the envelope proteins and their protein-protein interactions on the viral surface, is of paramount importance. These structures can reveal how the virions are assembled and released by budding from the infected host cell, how the progeny virions infect new cells by membrane fusion, and how antibodies bind surface epitopes to block infection. In this chapter, we discuss the uses of cryogenic electron microscopy (cryo-EM) in elucidating structures of enveloped virions. Starting from a detailed outline of data collection and processing strategies, we highlight how cryo-EM has been successfully utilized to provide unique insights into enveloped virus entry, assembly, and neutralization.Peer reviewe

OM-101 Decreases the Fibrotic Response Associated with Proliferative Vitreoretinopathy

Purpose. This study aimed to investigate the effect of OM-101 on the fibrotic response occurring in proliferative vitreoretinopathy (PVR) in an animal model. Methods. Antifibrotic effect of OM-101 was investigated in vivo. As control, eight weeks old c57black mice underwent intravitreal injection with Hepes (group A) or dispase (0.3 units), to induce retinal detachment (RD) and PVR. The dispase-injected mice were randomly divided into two groups B and C (N=25 mice); in group C, the eyes were treated with intravitreal injection of OM-101 (3 μl), and group B with PBS, as a control. After additional five days, mice were injected with the same initial treatment. Three days later, mice were euthanized, and the eyes were enucleated and processed for histological analysis. Results. Intravitreal injection of dispase caused RD in 64% of the mice in group B, and 93% of those mice had PVR. Only 32% of mice treated with OM-101 and dispase (group C) developed RD, and only 25% of those developed PVR. Conclusions. OM-101 was found effective in reducing the incidence of RD and PVR maintaining the normal architecture of the retina. This study suggests that OM-101 is a potentially effective and safe drug for the treatment of PVR patients

TGF-β1 induced transdifferentiation of rpe cells is mediated by TAK1.

Proliferative vitreoretinopathy (PVR) is an active process that develops as a complication upon retinal detachment (RD), accompanied by formation of fibrotic tissue. The main cells involved in the development of fibrotic tissue during PVR are the retinal pigment epithelial (RPE) cells. The RPE cells undergo epithelial-mesenchymal transition (EMT) which leads to complex retinal detachment and loss of vision. Transforming growth factor-β1 (TGF-β1) is considered as the main player in the EMT of RPE cells, even though the mechanism is not fully understood. This study was performed to determine the possible involvement of transforming growth factor β activated kinase 1 (TAK1) in the EMT process of the RPE cells.ARPE-19 Cells were treated with 5Z-7 oxozeaenol (TAK1 inhibitor) or SB431542 (TGF-β1 receptor kinase inhibitor) followed by TGF-β1 stimulation. Immunofluorescence, scratch assay Real time PCR and collagen contraction assay assessed the EMT features. The phosphorylation of Smad2/3 and p38 was examined using western blots analysis.This study demonstrates that stimulation of RPE cells with TGF-β1 increases α-SMA expression, cell migration and cell contractility, all of which are EMT features. Remarkably, addition of TAK1 inhibitor abolishes all these processes. Furthermore, we show hereby that TAK1 regulates not only the activation of the non-canonical cascade of TGF-β1 (p38), but also the canonical cascade, the Smad2/3 activation. Thus, the outcome of the TGF-β response in RPE cells is TAK1 dependent.This work demonstrated TAK1, a component of the non-canonical pathway of TGF-β1, is a key player in the EMT process, thus provides deep insight into the pathogenesis of PVR. The ability to halt the process of EMT in RPE cells may reduce the severity of the fibrotic response that occurs upon PVR, leading to a better prognosis and increase the probability of success in RD treatment

Mapping of the Lassa virus LAMP1 binding site reveals unique determinants not shared by other old world arenaviruses

<div><p>Cell entry of many enveloped viruses occurs by engagement with cellular receptors, followed by internalization into endocytic compartments and pH-induced membrane fusion. A previously unnoticed step of receptor switching was found to be critical during cell entry of two devastating human pathogens: Ebola and Lassa viruses. Our recent studies revealed the functional role of receptor switching to LAMP1 for triggering membrane fusion by Lassa virus and showed the involvement of conserved histidines in this switching, suggesting that other viruses from this family may also switch to LAMP1. However, when we investigated viruses that are genetically close to Lassa virus, we discovered that they cannot bind LAMP1. A crystal structure of the receptor-binding module from Morogoro virus revealed structural differences that allowed mapping of the LAMP1 binding site to a unique set of Lassa residues not shared by other viruses in its family, illustrating a key difference in the cell-entry mechanism of Lassa virus that may contribute to its pathogenicity.</p></div

Structural Basis for a Convergent Immune Response against Ebola Virus

Ebola virus disease is a severe health problem in Africa. Vaccines that display the Zaire ebolavirus glycoprotein spike complex are a prime component for the effort to combat it. The V(H)3-15/V(lambda)1-40-based class of antibodies was recently discovered to be a common response in individuals who received the Ebola virus vaccines. These antibodies display attractive properties, and thus likely contribute to the efficacy of the vaccines. Here, we use cryo-EM to elucidate how three V(H)3-15/V(lambda)1-40 antibodies from different individuals target the virus and found a convergent mechanism against a partially conserved site on the spike complex. Our study rationalizes the selection of the V(H)3-15/V(lambda)1-40 germline genes for specifically targeting this site and highlights Ebolavirus species-specific sequence divergences that may restrict breadth of V(H)3-15/V(lambda)1-40-based humoral response. The results from this study could help develop improved immunization schemes and further enable the design of immunogens that would be efficacious against a broader set of Ebolavirus species

Six month-follow up of laparoscopic sleeve gastrectomy

Background: The rising prevalence of obesity in today populations has led obese individuals to seek medical interventions. Aside from special diets, routine exercise and in some cases, medical treatment, most of the obese patients, favoring those with morbid or super obesity can benefit from bariatric surgery to lose weight. Laparoscopic sleeve gastrectomy (LSG) is relatively new method to limit the compliance of stomach. The consequent quick satiety during each meal results in gradual weight loss in patients. We investigated the efficacy and safety of this method among a group of our patients. Materials and Methods: This cross-sectional study was conducted in Isfahan, Iran, from January 2012 to January 2013. Thirty-five cases of obesity that had undergone LSG were enrolled and their baseline data of weight, body mass index (BMI), blood sugar, lipid profile, liver function indexes and blood pressure were collected. The patients were followed up for 6 months. The 6-month results were analyzed. Results: There was significant reduction in BMI, weight, blood sugar, blood pressure, liver enzymes and lipid profile components (P < 0.05), except for alkaline phosphatase (ALP) (P = 0.3). The average of excess weight loss percentage after 6 months was 69.2 ± 20.9%. No mortality occurred. Two of the patients had micro anastomotic leaks that were treated with nonoperative management. A case of gross leakage was treated with tube jejunostomy. Conclusion: Our study confirmed the efficacy and safety of LSG as a single surgical intervention for body weight reduction in morbidly and super obese patients

Inhibition of TAK1 abolishes the activation of TGF-β cascades.

<p><b>A</b>: Serum-starved RPE cells were pretreated with or without with 5Z-7-oxozeaenol (1μM) for 1 hour and then with TGF-β (2.5ng/ml) for the indicated times. Total protein extracts were analyzed by western blot using the indicated antibodies. The blot shows a representative result of four independent experiments. <b>B:</b> Levels of phospho-Smad2/3 were quantified and normalized to total Smad2/3 <b>C:</b> Statistical analysis of p-p38 and p-Smad3 activation normalized to p38 and Smad3 respectively, with or without TGF-β stimulation. The histograms present results of 5 independent experiments. Statistics were computed using student t-test (Two tailed distribution equal variance). Data is expressed as the Mean±SD.</p

TAK1 is activated upon TGF-β1 stimulation in RPE cells.

<p><b>A:</b> RPE cells were treated with TGF-β1 (2.5ng/ml) for the indicated times or left untreated. The cells were then immunostained with phospho-Thr 187 TAK1 antibodies (green) and DAPI (blue) as described in Materials and Methods. Representative photographs of three independent experiments. Scale bar is 10μm for all images. <b>B</b>: The histogram demonstrating pixel intensity, measured using Image-J software, is based on three independent experiments. (Number of cells: Control 0 = 24, Control 4 hours = 28, control 24 hours = 21, control 48 hours = 25; TGF-β1 4 hours = 26, TGF-β1 24 hours = 21, TGF-β1 48 hours = 22). Statistics were computed using student t-test (Two tailed distribution equal variance). Data is expressed as the Mean±SD.</p

TAK1 is a general regulator of the EMT process in RPE cells.

<p><b>A:</b> RPE cells were pre-treated with or without 5Z-7-oxozeaenol (1μM) and seeded in collagen lattices in full medium. The experiments were performed in triplicates. Lattices were photo-documented after 24 hours and measured using Image-J software. <b>B:</b> The histogram demonstrates the percentage of lattice area (marked by white line) relative to initial gel area, based on three independent experiments. Statistics were computed using student t-test (Two tailed distribution equal variance). Data is expressed as the Mean±SD.</p