Remodeling of endothelial adherens junctions by Kaposi's sarcoma-associated herpesvirus

Vascular endothelial cadherin (VE-cadherin) connects neighboring endothelial cells (ECs) via interendothelial junctions and regulates EC proliferation and adhesion during vasculogenesis and angiogenesis. The cytoplasmic domain of VE-cadherin recruits α- and β-catenins and γ-catenin, which interact with the actin cytoskeleton, thus modulating cell morphology. Dysregulation of the adherens junction/cytoskeletal axis is a hallmark of invasive tumors. We now demonstrate that the transmembrane ubiquitin ligase K5/MIR-2 of Kaposi's sarcoma-associated herpesvirus targets VE-cadherin for ubiquitin-mediated destruction, thus disturbing EC adhesion. In contrast, N-cadherin levels in K5-expressing cells were increased compared to those in control cells. Steady-state levels of α- and β-catenins and γ-catenin in K5-expressing ECs were drastically reduced due to proteasomal destruction. Moreover, the actin cytoskeleton was rearranged, resulting in the dysregulation of EC barrier function as measured by electric cell-substrate impedance sensing. Our data represent the first example of a viral protein targeting adherens junction proteins and suggest that K5 contributes to EC proliferation, vascular leakage, and the reprogramming of the EC proteome during Kaposi's sarcoma tumorigenesis.     

Aging but not dietary restriction alters the activation-induced apoptosis in rat T cells

The aim of this study was to determine if aging or dietary restriction (DR) alters activation-induced cell death, which is known to regulate cell proliferation and eliminate the high number of activated cells during an immune response. Splenic T cells were isolated from young (4-6 months) and old (25-26 months) Fischer 344 rats that had free access to food, ad libitum (AL), and from dietary-restricted (DR) old (25-26 months) rats that beginning at 6 weeks of age were fed 60% (40% food-restricted) of the diet consume by the AL rats. T cells were incubated with anti-CD3 antibody, or staphylococcal enterotoxin B (primary stimulus) for 72-96 h, followed by restimulation with anti-CD3 (secondary stimulus) for 72 h. Activation-induced apoptosis was assessed by DNA fragmentation and the expression of Fas/CD95 receptor and Fas ligand (Fas-L) was measured by flow cytometry. We found that the amount of DNA fragmentation was significantly (P<0.05) higher in the stimulated and restimulated T cells from AL old rats and DR old rats compared to young rats. The increase in DNA fragmentation with age was paralleled by an increase in the proportion of the cells expressing Fas and Fas-L. However, DR had no significant effect on the age-related increase in DNA fragmentation or the expression of Fas or Fas-L. We also measured the levels of Bcl-2 and Bax protein and found that the level of Bcl-2 decreased and Bax increased with age and that DR had no effect on the age-related changes in the level of Bcl-2 or Bax protein. These results demonstrate that aging but not DR alters activation-induced apoptosis in rat T cells.     

Hepatitis C in the setting of HIV co-infection

Hepatitis C virus (HCV) co-infection is common among HIV-infected individuals and can lead to increased morbidity and mortality in this population. HIV adversely impacts the natural history of HCV disease with higher rates of liver disease progression but the effect of HCV on the natural history of HIV is disputed. Additionally, presence of HCV may decrease tolerability of highly active antiretroviral regimens for HIV treatment due to a potential increase in hepatotoxicity. Currently there is limited information available regarding HCV therapy in the setting of HIV co-infection but the HCV virologic response to interferon regimens appears to be similar to those individuals with HCV infection alone. However, additional information is required to assess the efficacy and safety of HCV therapy including possible interaction of HCV and HIV anti-viral medications in these co-infected individuals.     

Melatonin fails to modulate immune parameters influenced by calorie restriction in aging Fischer 344 rats

The aim of this study was to determine if long-term treatment with melatonin (MEL), a purported anti-aging agent, was as effective as calorie restriction (CR) in modulating immune parameters in aging Fischer 344 male rats. Splenic lymphocytes were isolated from 17-month-old rats that, beginning at 6 weeks of age, were treated with MEL (4 or 16 microg/ml in drinking water) and from 17-month-old rats fed ad libitum (AL) or rats fed a CR diet (55% of AL intake). The number of splenic T cell populations and T cell subsets was measured by flow cytometry, the proliferative response of splenocytes to Concanavalin A (Con A) and lipopolysaccharide (LPS) was measured by [(3)H]thymidine incorporation, and the induction of cytokine production (IL-2 and IFN-gamma) was measured by ELISA assay. In addition, the level of the natural killer (NK) cell activity was assessed by fluorimetric assay. CR rats had a higher number of lymphocytes expressing the na?ve T cell marker (CD3 OX22) than AL rats (P < 0.05). CR rats also showed greater induction of proliferative response, IL-2 and IFN-gamma levels following Con A simulation, and NK cell activity than AL rats (P < 0.05). MEL-treated rats did not differ from AL rats in any of these parameters or in any other measurement. These results indicate that MEL treatment is unable to modulate immune function in a manner comparable with that of CR.     

The PHD/LAP-domain protein M153R of myxomavirus is a ubiquitin ligase that induces the rapid internalization and lysosomal destruction of CD4

The genomes of several poxviruses contain open reading frames with homology to the K3 and K5 genes of Kaposi's sarcoma-associated herpesvirus (KSHV) and the K3 gene of murine gammaherpesvirus 68, which target major histocompatibility complex class I (MHC-I) as well as costimulatory molecules for proteasomal or lysosomal degradation. The homologous gene product of myxomavirus (MV), M153R, was recently shown to reduce the cell surface expression of MHC-I. In addition, normal MHC-I surface expression was observed in cells infected with MV lacking M153R (J. L. Guerin, J. Gelfi, S. Boullier, M. Delverdier, F. A. Bellanger, S. Bertagnoli, I. Drexler, G. Sutter, and F. Messud-Petit, J. Virol. 76:2912-2923, 2002). Here, we show that M153R also downregulates the T-cell coreceptor CD4 and we study the molecular mechanism by which M153R achieves the downregulation of CD4 and MHC-I. Upon M153R expression, CD4 was rapidly internalized and degraded in lysosomes, whereas deletion of M153R from the genome of MV restored CD4 expression. The downregulation of both CD4 and MHC-I was dependent on the presence of lysine residues in their cytoplasmic tails. Increased ubiquitination of CD4 was observed upon coexpression with M153R in the presence of inhibitors of lysosomal acidification. Surface expression of CD4 was restored upon overexpression of Hrs, a ubiquitin interaction motif-containing protein that sorts ubiquitinated proteins into endosomes. Moreover, the purified PHD/LAP zinc finger of M153R catalyzed the formation of multiubiquitin adducts in vitro. Our data suggest that M153R acts as a membrane-bound ubiquitin ligase that conjugates ubiquitin to the cytoplasmic domain of substrate glycoproteins, with ubiquitin serving as a lysosomal targeting signal. Since a similar mechanism was recently proposed for KSHV K5, it seems that members of the unrelated families of gamma-2 herpesviruses and poxviruses share a common immune evasion mechanism that targets host cell immune receptors.     

Influence of aging and caloric restriction on activation of Ras/MAPK, calcineurin, and CaMK-IV activities in rat T cells

The signaling cascade mediated by Ras (p21ras) and MAPK (mitogen-activated protein kinase) and calcium/calmodulin regulating enzymes, calcineurin (CaN) and CaMK-IV, are considered to be essential for T-cell growth and function. In the present study, the effect of aging and caloric restriction (CR) on the induction of Ras and MAPK activation by concanavalin A (ConA) was studied. Splenic T cells were isolated from young (4-6 months) and old (22-24 months) rats that had free access to food (control group), and from caloric restricted old (22-24 months) rats that beginning at 6 weeks of age were fed 60%(40% caloric restriction) of the diet consumed by the control rats. We found that the induction of Ras activity in T cells isolated from control old rats was lower (P<0.001) than that in control young rats. However, the levels of Ras activity in T cells isolated from CR old rats were similar to the levels in the age-matched control rats. The induction of MAPK activity in T cells isolated from control old rats and CR old rats was significantly less than in T cells isolated from control young rats, and caloric restriction significantly (P<0.05) reduced the age-related decline in MAPK activation. We also measured the induction of CaN and CaMK-IV activities by ConA in T cells from control young and old and CR old rats. The induction of both CaN and CaMK-IV activity decreased with age. Caloric restriction significantly (P<0.05) reduced the age-related decline in CaN activity, but had no significant effect on CaMK-IV activity. The changes in Ras/MAPK activation and in CaN and CaMK-IV activity with age or with CR were not associated with alterations in their corresponding protein levels. Thus, caloric restriction has a differential effect on the activation of the upstream signaling molecules that are altered with age.     

Breaking down the wall: fractionation of mycobacteria

Mycobacterium spp. possess a complex cell envelope that consists of a plasma membrane, a peptidoglycan-arabinogalactan complex which in turn is esterified by mycolic acids that form with other non-bound lipids an asymmetric permeability barrier and an outer layer, also called a capsule in the case of pathogenic species. In order to investigate the functional roles of the cell envelope components, especially those of the major pathogens Mycobacterium tuberculosis and Mycobacterium leprae, it is necessary to fractionate the envelope by breaking the unusual wall that covers these bacteria. To this aim we first compared the efficiency of high pressure (cell disrupter/French press) with those of pathogen-compatible breakage methods such as sonication, bead beater and lysozyme treatment using the non-pathogenic Mycobacterium smegmatis. When the distribution of various specific markers of the cell envelope compartments, which include mycolic acids, arabinose, NADH oxidase activity, cell wall and cytosolic proteins, were determined sonication combined with lysozyme treatment was found to be the best option. The protocol of subcellular fractionation was then validated for pathogenic species by applying the method to Mycobacterium bovis BCG cells, an attenuated strain of the M. tuberculosis complex.     

Discovery of Buprestidae rock art in central Iran suggests historical relationship between jewel beetles and mankind

An extensive field survey was undertaken by a group of archaeologists and entomologists to discover arthropod‐like petroglyphs in the center of Iran during 2017 to 2018. A remarkable motif approximately 13 × 5 cm in size was found in the Teymareh rock art site, located in Markazi province, which has yet to be reported. The single rock art distinctly represents a hexapod organism with small antenna, a streaked narrow body and a tapering abdomen. Based on distinguishing aspects of the inspected motif and morphological information of species within the class Insecta, we identified it as a jewel beetle (Coleoptera: Buprestidae). We propose that the buprestid drawing was derived from a historic artist who admired the aesthetic value of the jewel beetle. The jewel beetle's characters within Persian Plateau folklore are also briefly discussed.     

Identification of Hepatotropic Viruses from Plasma Using Deep Sequencing: A Next Generation Diagnostic Tool

We conducted an unbiased metagenomics survey using plasma from patients with chronic hepatitis B, chronic hepatitis C, autoimmune hepatitis (AIH), non-alcoholic steatohepatitis (NASH), and patients without liver disease (control). RNA and DNA libraries were sequenced from plasma filtrates enriched in viral particles to catalog virus populations. Hepatitis viruses were readily detected at high coverage in patients with chronic viral hepatitis B and C, but only a limited number of sequences resembling other viruses were found. The exception was a library from a patient diagnosed with hepatitis C virus (HCV) infection that contained multiple sequences matching GB virus C (GBV-C). Abundant GBV-C reads were also found in plasma from patients with AIH, whereas Torque teno virus (TTV) was found at high frequency in samples from patients with AIH and NASH. After taxonomic classification of sequences by BLASTn, a substantial fraction in each library, ranging from 35% to 76%, remained unclassified. These unknown sequences were assembled into scaffolds along with virus, phage and endogenous retrovirus sequences and then analyzed by BLASTx against the non-redundant protein database. Nearly the full genome of a heretofore-unknown circovirus was assembled and many scaffolds that encoded proteins with similarity to plant, insect and mammalian viruses. The presence of this novel circovirus was confirmed by PCR. BLASTx also identified many polypeptides resembling nucleo-cytoplasmic large DNA viruses (NCLDV) proteins. We re-evaluated these alignments with a profile hidden Markov method, HHblits, and observed inconsistencies in the target proteins reported by the different algorithms. This suggests that sequence alignments are insufficient to identify NCLDV proteins, especially when these alignments are only to small portions of the target protein. Nevertheless, we have now established a reliable protocol for the identification of viruses in plasma that can also be adapted to other patient samples such as urine, bile, saliva and other body fluids.     

Influence of cell adhesion and spreading on impedance characteristics of cell-based sensors

Impedance measurements of cell-based sensors are a primary characterization route for detection and analysis of cellular responses to chemical and biological agents in real time. The detection sensitivity and limitation depend on sensor impedance characteristics and thus on cell patterning techniques. This study introduces a cell patterning approach to bind cells on microarrays of gold electrodes and demonstrates that single-cell patterning can substantially improve impedance characteristics of cell-based sensors. Mouse fibroblast cells (NIH3T3) are immobilized on electrodes through a lysine-arginine-glycine-aspartic acid (KRGD) peptide-mediated natural cell adhesion process. Electrodes are made of three sizes and immobilized with either covalently bound or physically adsorbed KRGD (c-electrodes or p-electrodes). Cells attached to c-electrodes increase the measurable electrical signal strength by 48.4%, 24.2%, and 19.0% for three electrode sizes, respectively, as compared to cells attached to p-electrodes, demonstrating that both the electrode size and surface chemistry play a key role in cell adhesion and spreading and thus the impedance characteristics of cell-based sensors. Single cells patterned on c-electrodes with dimensions comparable to cell size exhibit well-spread cell morphology and substantially outperform cells patterned on electrodes of other configurations.