22 research outputs found

    Effect of early vasopressin vs norepinephrine on kidney failure in patients with septic shock. The VANISH Randomized Clinical Trial

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    IMPORTANCE: Norepinephrine is currently recommended as the first-line vasopressor in septic shock; however, early vasopressin use has been proposed as an alternative. OBJECTIVE: To compare the effect of early vasopressin vs norepinephrine on kidney failure in patients with septic shock. DESIGN, SETTING, AND PARTICIPANTS: A factorial (2×2), double-blind, randomized clinical trial conducted in 18 general adult intensive care units in the United Kingdom between February 2013 and May 2015, enrolling adult patients who had septic shock requiring vasopressors despite fluid resuscitation within a maximum of 6 hours after the onset of shock. INTERVENTIONS: Patients were randomly allocated to vasopressin (titrated up to 0.06 U/min) and hydrocortisone (n = 101), vasopressin and placebo (n = 104), norepinephrine and hydrocortisone (n = 101), or norepinephrine and placebo (n = 103). MAIN OUTCOMES AND MEASURES: The primary outcome was kidney failure-free days during the 28-day period after randomization, measured as (1) the proportion of patients who never developed kidney failure and (2) median number of days alive and free of kidney failure for patients who did not survive, who experienced kidney failure, or both. Rates of renal replacement therapy, mortality, and serious adverse events were secondary outcomes. RESULTS: A total of 409 patients (median age, 66 years; men, 58.2%) were included in the study, with a median time to study drug administration of 3.5 hours after diagnosis of shock. The number of survivors who never developed kidney failure was 94 of 165 patients (57.0%) in the vasopressin group and 93 of 157 patients (59.2%) in the norepinephrine group (difference, -2.3% [95% CI, -13.0% to 8.5%]). The median number of kidney failure-free days for patients who did not survive, who experienced kidney failure, or both was 9 days (interquartile range [IQR], 1 to -24) in the vasopressin group and 13 days (IQR, 1 to -25) in the norepinephrine group (difference, -4 days [95% CI, -11 to 5]). There was less use of renal replacement therapy in the vasopressin group than in the norepinephrine group (25.4% for vasopressin vs 35.3% for norepinephrine; difference, -9.9% [95% CI, -19.3% to -0.6%]). There was no significant difference in mortality rates between groups. In total, 22 of 205 patients (10.7%) had a serious adverse event in the vasopressin group vs 17 of 204 patients (8.3%) in the norepinephrine group (difference, 2.5% [95% CI, -3.3% to 8.2%]). CONCLUSIONS AND RELEVANCE: Among adults with septic shock, the early use of vasopressin compared with norepinephrine did not improve the number of kidney failure-free days. Although these findings do not support the use of vasopressin to replace norepinephrine as initial treatment in this situation, the confidence interval included a potential clinically important benefit for vasopressin, and larger trials may be warranted to assess this further. TRIAL REGISTRATION: clinicaltrials.gov Identifier: ISRCTN 20769191

    An Assay to Monitor HIV-1 Protease Activity for the Identification of Novel Inhibitors in T-Cells

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    The emergence of resistant HIV strains, together with the severe side-effects of existing drugs and lack of development of effective anti-HIV vaccines highlight the need for novel antivirals, as well as innovative methods to facilitate their discovery. Here, we have developed an assay in T-cells to monitor the proteolytic activity of the HIV-1 protease (PR). The assay is based on the inducible expression of HIV-1 PR fused within the Gal4 DNA-binding and transactivation domains. The fusion protein binds to the Gal4 responsive element and activates the downstream reporter, enhanced green fluorescent protein (eGFP) gene only in the presence of an effective PR Inhibitor (PI). Thus, in this assay, eGFP acts as a biosensor of PR activity, making it ideal for flow cytometry based screening. Furthermore, the assay was developed using retroviral technology in T-cells, thus providing an ideal environment for the screening of potential novel PIs in a cell-type that represents the natural milieu of HIV infection. Clones with the highest sensitivity, and robust, reliable and reproducible reporter activity, were selected. The assay is easily adaptable to other PR variants, a multiplex platform, as well as to high-throughput plate reader based assays and will greatly facilitate the search for novel peptide and chemical compound based PIs in T-cells

    The cellular source for APOBEC3G's incorporation into HIV-1

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    <p>Abstract</p> <p>Background</p> <p>Human APOBEC3G (hA3G) has been identified as a cellular inhibitor of HIV-1 infectivity. Viral incorporation of hA3G is an essential step for its antiviral activity. Although the mechanism underlying hA3G virion encapsidation has been investigated extensively, the cellular source of viral hA3G remains unclear.</p> <p>Results</p> <p>Previous studies have shown that hA3G forms low-molecular-mass (LMM) and high-molecular-mass (HMM) complexes. Our work herein provides evidence that the majority of newly-synthesized hA3G interacts with membrane lipid raft domains to form Lipid raft-associated hA3G (RA hA3G), which serve as the precursor of the mature HMM hA3G complex, while a minority of newly-synthesized hA3G remains in the cytoplasm as a soluble LMM form. The distribution of hA3G among the soluble LMM form, the RA LMM form and the mature forms of HMM is regulated by a mechanism involving the N-terminal part of the linker region and the C-terminus of hA3G. Mutagenesis studies reveal a direct correlation between the ability of hA3G to form the RA LMM complex and its viral incorporation.</p> <p>Conclusions</p> <p>Together these data suggest that the Lipid raft-associated LMM A3G complex functions as the cellular source of viral hA3G.</p

    Protein Expression Redirects Vesicular Stomatitis Virus RNA Synthesis to Cytoplasmic Inclusions

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    Positive-strand and double-strand RNA viruses typically compartmentalize their replication machinery in infected cells. This is thought to shield viral RNA from detection by innate immune sensors and favor RNA synthesis. The picture for the non-segmented negative-strand (NNS) RNA viruses, however, is less clear. Working with vesicular stomatitis virus (VSV), a prototype of the NNS RNA viruses, we examined the location of the viral replication machinery and RNA synthesis in cells. By short-term labeling of viral RNA with 5′-bromouridine 5′-triphosphate (BrUTP), we demonstrate that primary mRNA synthesis occurs throughout the host cell cytoplasm. Protein synthesis results in the formation of inclusions that contain the viral RNA synthesis machinery and become the predominant sites of mRNA synthesis in the cell. Disruption of the microtubule network by treatment of cells with nocodazole leads to the accumulation of viral mRNA in discrete structures that decorate the surface of the inclusions. By pulse-chase analysis of the mRNA, we find that viral transcripts synthesized at the inclusions are transported away from the inclusions in a microtubule-dependent manner. Metabolic labeling of viral proteins revealed that inhibiting this transport step diminished the rate of translation. Collectively those data suggest that microtubule-dependent transport of viral mRNAs from inclusions facilitates their translation. Our experiments also show that during a VSV infection, protein synthesis is required to redirect viral RNA synthesis to intracytoplasmic inclusions. As viral RNA synthesis is initially unrestricted, we speculate that its subsequent confinement to inclusions might reflect a cellular response to infection

    Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.

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    BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700

    Pan-retroviral Nucleocapsid-Mediated Phase Separation Regulates Genomic RNA Positioning and Trafficking

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    The duality of liquid-liquid phase separation (LLPS) of cellular components into membraneless organelles defines the nucleation of both normal and disease processes including stress granule (SG) assembly. From mounting evidence of LLPS utility by viruses, we discover that HIV-1 nucleocapsid (NC) protein condenses into zinc-finger (ZnF)-dependent LLPSs that are dynamically influenced by cytosolic factors. ZnF-dependent and Zinc (Zn(2+))-chelation-sensitive NC-LLPS are formed in live cells. NC-Zn(2+) ejection reverses the HIV-1 blockade on SG assembly, inhibits NC-SG assembly, disrupts NC/Gag-genomic RNA (vRNA) ribonucleoprotein complexes, and causes nuclear sequestration of NC and the vRNA, inhibiting Gag expression and virus release. NC ZnF mutagenesis eliminates the HIV-1 blockade of SG assembly and repositions vRNA to SGs. We find that NC-mediated, Zn(2+)-coordinated phase separation is conserved among diverse retrovirus subfamilies, illustrating that this exquisitely evolved Zn(2+)-dependent feature of virus replication represents a critical target for pan-antiretroviral therapies

    The PKR activator, PACT, becomes a PKR inhibitor during HIV-1 replication

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    Background: HIV-1 translation is modulated by the activation of the interferon (IFN)-inducible Protein Kinase RNA-activated (PKR). PKR phosphorylates its downstream targets, including the alpha subunit of the eukaryotic translation Initiation Factor 2 (eIF2α), which decreases viral replication. The PKR Activator (PACT) is known to activate PKR after a cellular stress. In lymphocytic cell lines, HIV-1 activates PKR only transiently and not when cells replicate the virus at high levels. The regulation of this activation is due to a combination of viral and cellular factors that have been only partially identified. Results: PKR is transiently induced and activated in peripheral blood mononuclear cells after HIV-1 infection. The addition of IFN reduces viral replication, and induces both the production and phosphorylation of PKR. In lymphocytic Jurkat cells infected by HIV-1, a multiprotein complex around PKR contains the double-stranded RNA binding proteins (dsRBPs), adenosine deaminase acting on RNA (ADAR)1 and PACT. In HEK 293T cells transfected with an HIV-1 molecular clone, PACT unexpectedly inhibited PKR and eIF2α phosphorylation and increased HIV-1 protein expression and virion production in the presence of either endogenous PKR alone or overexpressed PKR. The comparison between different dsRBPs showed that ADAR1, TAR RNA Binding Protein (TRBP) and PACT inhibit PKR and eIF2α phosphorylation in HIV-infected cells, whereas Staufen1 did not. Individual or a combination of short hairpin RNAs against PACT or ADAR1 decreased HIV-1 protein expression. In the astrocytic cell line U251MG, which weakly expresses TRBP, PACT mediated an increased HIV-1 protein expression and a decreased PKR phosphorylation. In these cells, a truncated PACT, which constitutively activates PKR in non-infected cells showed no activity on either PKR or HIV-1 protein expression. Finally, PACT and ADAR1 interact with each other in the absence of RNAs. Conclusion: In contrast to its previously described activity, PACT contributes to PKR dephosphorylation during HIV-1 replication. This activity is in addition to its heterodimer formation with TRBP and could be due to its binding to ADAR1. HIV-1 has evolved to replicate in cells with high levels of TRBP, to induce the expression of ADAR1 and to change the function of PACT for PKR inhibition and increased replication.</p

    The PKR activator, PACT, becomes a PKR inhibitor during HIV-1 replication

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    Background: HIV-1 translation is modulated by the activation of the interferon (IFN)-inducible Protein Kinase RNA-activated (PKR). PKR phosphorylates its downstream targets, including the alpha subunit of the eukaryotic translation Initiation Factor 2 (eIF2α), which decreases viral replication. The PKR Activator (PACT) is known to activate PKR after a cellular stress. In lymphocytic cell lines, HIV-1 activates PKR only transiently and not when cells replicate the virus at high levels. The regulation of this activation is due to a combination of viral and cellular factors that have been only partially identified. Results: PKR is transiently induced and activated in peripheral blood mononuclear cells after HIV-1 infection. The addition of IFN reduces viral replication, and induces both the production and phosphorylation of PKR. In lymphocytic Jurkat cells infected by HIV-1, a multiprotein complex around PKR contains the double-stranded RNA binding proteins (dsRBPs), adenosine deaminase acting on RNA (ADAR)1 and PACT. In HEK 293T cells transfected with an HIV-1 molecular clone, PACT unexpectedly inhibited PKR and eIF2α phosphorylation and increased HIV-1 protein expression and virion production in the presence of either endogenous PKR alone or overexpressed PKR. The comparison between different dsRBPs showed that ADAR1, TAR RNA Binding Protein (TRBP) and PACT inhibit PKR and eIF2α phosphorylation in HIV-infected cells, whereas Staufen1 did not. Individual or a combination of short hairpin RNAs against PACT or ADAR1 decreased HIV-1 protein expression. In the astrocytic cell line U251MG, which weakly expresses TRBP, PACT mediated an increased HIV-1 protein expression and a decreased PKR phosphorylation. In these cells, a truncated PACT, which constitutively activates PKR in non-infected cells showed no activity on either PKR or HIV-1 protein expression. Finally, PACT and ADAR1 interact with each other in the absence of RNAs. Conclusion: In contrast to its previously described activity, PACT contributes to PKR dephosphorylation during HIV-1 replication. This activity is in addition to its heterodimer formation with TRBP and could be due to its binding to ADAR1. HIV-1 has evolved to replicate in cells with high levels of TRBP, to induce the expression of ADAR1 and to change the function of PACT for PKR inhibition and increased replication.</p
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