Description: Background— Endothelial senescence represents one of the major characteristics of vascular aging and promotes the development of atherosclerosis. Sirtuin-1 (SIRT1) is an NAD-dependent deacetylase possessing antiaging activities. During the occurrence of endothelial senescence, both the expression and activity of SIRT1 are downregulated. The present study was designed to investigate the molecular mechanisms contributing to the loss-of-SIRT1 function in senescent endothelial cells. Methods and Results— After repetitive passages, primary cultures of porcine aortic endothelial cells exhibited a severe senescence phenotype. Western blotting revealed that phosphorylation of SIRT1 at serine 47 (S47) was significantly enhanced in senescent endothelial cells. S47 phosphorylation was stimulated by agents promoting senescence and attenuated by drugs with antisenescence properties. Mutation of S47 to nonphosphorable alanine (S47A) enhanced whereas replacing S47 with phospho-mimicking aspartic acid (S47D) abolished the antisenescent, growth-promoting, and LKB1-downregulating actions of SIRT1. Phosphorylation at S47 was critically involved in the nuclear retention of SIRT1 but abolished its association with the telomeric repeat-binding factor 2–interacting protein 1. Cyclin-dependent kinase 5 (CDK5) was identified as an SIRT1 kinase modulating S47 phosphorylation. Knockdown or inhibition of CDK5 reduced the number of senescent endothelial cells, promoted nuclear exportation of SIRT1, and attenuated the expression of inflammatory genes in porcine aortic endothelial cells. The truncated regulatory subunit of CDK5, P25, accumulated in senescent porcine aortic endothelial cells and atherosclerotic aortas. Long-term treatment with roscovitine, a CDK5 inhibitor, blocked the development of cellular senescence and atherosclerosis in aortas of hypercholesterolemic apolipoprotein E–deficient mice. Conclusion— CDK5-mediated hyperphosphorylation of SIRT1 facilitates the development of endothelial senescence and atherosclerosis.

Description: The distribution and prevalence of H3 subtype influenza viruses in avian and mammalian hosts constitutes a potential threat to both human and avian health. We report a complete genome sequence of a novel reassortant H3N2 avian influenza virus. Phylogenetic analysis showed that HA and NA showed the highest sequence homologies with those of A/white-backed munia/Hong Kong/4519/2009 (H3N2). However, the internal genes had the highest sequence homologies with those of H6 and H7 subtypes. The data provide further evidence of the existence of a natural reassortant H3N2 strain in southern China.

Description: Background and Purpose— We observed that microRNA-424 (miR-424) significantly decreased in an miRNA profile of circulating lymphocytes of patients with ischemic stroke. The present study focused on the potential and mechanism of miR-424 in protecting ischemic brain injury in mice. Methods— Cerebral ischemia was induced by middle cerebral artery occlusion in C57/BL6 mice. Cerebral infarction volume, neuronal apoptosis, and microglia activation were determined by 2,3,5-triphenyltetrazolium chloride staining, immunofluorescence, and Western blot. BV2 microglial cell activity, cell cycle, mRNA, and protein levels of miR-424 targets were accessed by enzyme-linked immunosorbent assay, flow cytometry, real-time polymerase chain reaction, and Western blot, respectively. Results— MiR-424 levels were decreased in the plasma of patients with acute ischemic stroke, as well as in mouse plasma and ipsilateral brain tissue at 4, 8, and 24 hours after ischemia, likewise, in the cortex, hippocampus, and basal ganglia, respectively, after 8-hour ischemia. Interestingly, pre- and post-treatment with overexpression of miR-424 both decreased cerebral infarction size and brain edema after middle cerebral artery occlusion. Meanwhile, lentiviral overexpression of miR-424 inhibited neuronal apoptosis and microglia activation, including suppressing ionized calcium binding adaptor molecule-1 immunoreactivity and protein level, and reduced tumor necrosis factor-α production. In vitro study demonstrated that miR-424 mimics caused G1 phase cell-cycle arrest, inhibited BV2 microglia activity, and reduced the mRNA and protein levels of CDC25A, cyclin D1, and CDK6 in BV2 microglial cells, which were upregulated in brain of middle cerebral artery occlusion mice. Conclusions— MiR-424 overexpression lessened the ischemic brain injury through suppressing microglia activation by translational depression of key activators of G1/S transition, suggesting a novel miR-based intervention strategy for stroke.

Description: Background Extracorporeal membrane oxygenation (ECMO) unloads the heart, providing a bridge to recovery in children after myocardial stunning. ECMO also induces stress which can adversely affect the ability to reload or wean the heart from the circuit. Metabolic impairments induced by altered loading and/or stress conditions may impact weaning. However, cardiac substrate and amino acid requirements upon weaning are unknown. We assessed the hypothesis that ventricular reloading with ECMO modulates both substrate entry into the citric acid cycle (CAC) and myocardial protein synthesis. Methods and Results Sixteen immature piglets (7.8 to 15.6 kg) were separated into 2 groups based on ventricular loading status: 8-hour ECMO (UNLOAD) and postwean from ECMO (RELOAD). We infused into the coronary artery [2- 13 C]-pyruvate as an oxidative substrate and [ 13 C 6 ]-L-leucine as an indicator for amino acid oxidation and protein synthesis. Upon RELOAD, each functional parameter, which were decreased substantially by ECMO, recovered to near-baseline level with the exclusion of minimum dP/dt. Accordingly, myocardial oxygen consumption was also increased, indicating that overall mitochondrial metabolism was reestablished. At the metabolic level, when compared to UNLOAD, RELOAD altered the contribution of various substrates/pathways to tissue pyruvate formation, favoring exogenous pyruvate versus glycolysis, and acetyl-CoA formation, shifting away from pyruvate decarboxylation to endogenous substrate, presumably fatty acids. Furthermore, there was also a significant increase of tissue concentrations for all CAC intermediates (80%), suggesting enhanced anaplerosis, and of fractional protein synthesis rates (〉70%). Conclusions RELOAD alters both cytosolic and mitochondrial energy substrate metabolism, while favoring leucine incorporation into protein synthesis rather than oxidation in the CAC. Improved understanding of factors governing these metabolic perturbations may serve as a basis for interventions and thereby improve success rate from weaning from ECMO.

Description: Abnormal glucocorticoid and neurotrophin signaling has been implicated in numerous psychiatric disorders. However, the impact of neurotrophic signaling on glucocorticoid receptor (GR)-dependent gene expression is not understood. We therefore examined the impact of brain-derived neurotrophic factor (BDNF) signaling on GR transcriptional regulatory function by gene expression profiling in primary rat cortical neurons stimulated with the selective GR agonist dexamethasone (Dex) and BDNF, alone or in combination. Simultaneous treatment with BDNF and Dex elicited a unique set of GR-responsive genes associated with neuronal growth and differentiation and also enhanced the induction of a large number of Dex-sensitive genes. BDNF via its receptor TrkB enhanced the transcriptional activity of a synthetic GR reporter, suggesting a direct effect of BDNF signaling on GR function. Indeed, BDNF treatment induces the phosphorylation of GR at serine 155 (S155) and serine 287 (S287). Expression of a nonphosphorylatable mutant (GR S155A/S287A) impaired the induction of a subset of BDNF- and Dex-regulated genes. Mechanistically, BDNF-induced GR phosphorylation increased GR occupancy and cofactor recruitment at the promoter of a BDNF-enhanced gene. GR phosphorylation in vivo is sensitive to changes in the levels of BDNF and TrkB as well as stress. Therefore, BDNF signaling specifies and amplifies the GR transcriptome through a coordinated GR phosphorylation-dependent detection mechanism.

Description: While simian immunodeficiency viruses (SIVs) are generally nonpathogenic in their natural hosts, dramatic increases in pathogenicity may occur upon cross-species transmission to new hosts. Deciphering the drivers of these increases in virulence is of major interest for understanding the emergence of new human immunodeficiency viruses (HIVs). We transmitted SIVsab from the sabaeus species of African green monkeys (AGMs) to pigtailed macaques (PTMs). High acute viral replication occurred in all SIVsab-infected PTMs, yet the outcome of chronic infection was highly variable, ranging from rapid progression to controlled infection, which was independent of the dynamics of acute viral replication, CD4 + T cell depletion, or preinfection levels of microbial translocation. Infection of seven PTMs with plasma collected at necropsy from a rapid-progressor PTM was consistently highly pathogenic, with high acute and chronic viral replication, massive depletion of memory CD4 + T cells, and disease progression in all PTMs. The plasma inoculum used for the serial passage did not contain adventitious bacterial or viral contaminants. Single-genome amplification showed that this inoculum was significantly more homogenous than the inoculum directly derived from AGMs, pointing to a strain selection in PTMs. In spite of similar peak plasma viral loads between the monkeys in the two passages, immune activation/inflammation levels dramatically increased in PTMs infected with the passaged virus. These results suggest that strain selection and a massive cytokine storm are major factors behind increased pathogenicity of SIV upon serial passage and adaptation of SIVs to new hosts following cross-species transmission. IMPORTANCE We report here that upon cross-species transmission and serial passage of SIVsab from its natural host, the sabaeus African green monkey (AGM), to a new host, the pigtailed macaque (PTM), viral adaptation and increased pathogenicity involve strain selection and a massive cytokine storm. These results permit the design of strategies aimed at preventing cross-species transmission from natural hosts of SIVs to humans in areas of endemicity. Furthermore, our study describes a new animal model for SIV infection. As the outcomes of SIVsab infection in PTMs, African green monkeys, and rhesus macaques are different, the use of these systems enables comparative studies between pathogenic, nonpathogenic, and elite-controlled infections, to gain insight into the mechanisms of SIV immunodeficiency and comorbidities.

Description: Genetic modifications of bacterial chromosomes are important for both fundamental and applied research. In this study, we developed an efficient, easy-to-use system for genetic modification of the Escherichia coli chromosome, a two-plasmid method involving lambda Red (-Red) recombination and I-SceI cleavage. An intermediate strain is generated by integration of a resistance marker gene(s) and I-SceI recognition sites in or near the target gene locus, using -Red PCR targeting. The intermediate strain is transformed with a donor plasmid carrying the target gene fragment with the desired modification flanked by I-SceI recognition sites, together with a bifunctional helper plasmid for -Red recombination and I-SceI endonuclease. I-SceI cleavage of the chromosome and the donor plasmid allows -Red recombination between chromosomal breaks and linear double-stranded DNA from the donor plasmid. Genetic modifications are introduced into the chromosome, and the placement of the I-SceI sites determines the nature of the recombination and the modification. This method was successfully used for cadA knockout, gdhA knock-in, seamless deletion of pepD , site-directed mutagenesis of the essential metK gene, and replacement of metK with the Rickettsia S -adenosylmethionine transporter gene. This effective method can be used with both essential and nonessential gene modifications and will benefit basic and applied genetic research.

Description: The role of the adenosine (ADO) pathway in human immunodeficiency virus type 1/simian immunodeficiency virus (HIV-1/SIV) infection remains unclear. We compared SIVsab-induced changes of markers related to ADO production (CD39 and CD73) and breakdown (CD26 and adenosine deaminase) on T cells from blood, lymph nodes, and intestine collected from pigtailed macaques (PTMs) and African green monkeys (AGMs) that experience different SIVsab infection outcomes. We also measured ADO and inosine (INO) levels in tissues by mass spectrometry. Finally, we assessed the suppressive effect of ADO on proinflammatory cytokine production after T cell receptor stimulation. The baseline level of both CD39 and CD73 coexpression on regulatory T cells and ADO levels were higher in AGMs than in PTMs. Conversely, high INO levels associated with dramatic increases in CD26 expression and adenosine deaminase activity were observed in PTMs during chronic SIV infection. Immune activation and inflammation markers in the gut and periphery inversely correlated with ADO and directly correlated with INO. Ex vivo administration of ADO significantly suppressed proinflammatory cytokine production by T cells in both species. In conclusion, the opposite dynamics of ADO pathway-related markers and contrasting ADO/INO levels in species with divergent proinflammatory responses to SIV infection support a key role of ADO in controlling immune activation/inflammation in nonprogressive SIV infections. Changes in ADO levels predominately occurred in the gut, suggesting that the ADO pathway may be involved in sparing natural hosts of SIVs from developing SIV-related gut dysfunction. Focusing studies of the ADO pathway on mucosal sites of viral replication is warranted. IMPORTANCE The mechanisms responsible for the severe gut dysfunction characteristic of progressive HIV and SIV infection in humans and macaques are not completely elucidated. We report that ADO may play a key role in controlling immune activation/inflammation in nonprogressive SIV infections by limiting SIV-related gut inflammation. Conversely, in progressive SIV infection, significant degradation of ADO occurs, possibly due to an early increase of ADO deaminase complexing protein 2 (CD26) and adenosine deaminase. Our study supports therapeutic interventions to offset alterations of this pathway during progressive HIV/SIV infections. These potential approaches to control chronic immune activation and inflammation during pathogenic SIV infection may prevent HIV disease progression.

Description: We report that GRL-0519, a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI) containing tris -tetrahydrofuranylurethane ( tris -THF) and a sulfonamide isostere, is highly potent against laboratory HIV-1 strains and primary clinical isolates (50% effective concentration [EC 50 ], 0.0005 to 0.0007 μM) with minimal cytotoxicity (50% cytotoxic concentration [CC 50 ], 44.6 μM). GRL-0519 blocked the infectivity and replication of HIV-1 NL4-3 variants selected by up to a 5 μM concentration of ritonavir, lopinavir, or atazanavir (EC 50 , 0.0028 to 0.0033 μM). GRL-0519 was also potent against multi-PI-resistant clinical HIV-1 variants isolated from patients who no longer responded to existing antiviral regimens after long-term antiretroviral therapy, highly darunavir (DRV)-resistant variants, and HIV-2 ROD . The development of resistance against GRL-0519 was substantially delayed compared to other PIs, including amprenavir (APV) and DRV. The effects of nonspecific binding of human serum proteins on GRL-0519's antiviral activity were insignificant. Our analysis of the crystal structures of GRL-0519 (3OK9) and DRV (2IEN) with protease suggested that the tris -THF moiety, compared to the bis -THF moiety present in DRV, has greater water-mediated polar interactions with key active-site residues of protease and that the tris -THF moiety and paramethoxy group effectively fill the S2 and S2' binding pockets, respectively, of the protease. The present data demonstrate that GRL-0519 has highly favorable features as a potential therapeutic agent for treating patients infected with wild-type and/or multi-PI-resistant variants and that the tris -THF moiety is critical for strong binding of GRL-0519 to the HIV protease substrate binding site and appears to be responsible for its favorable antiretroviral characteristics.

Description: The mitochondrial antiviral signaling protein MAVS (IPS-1, VISA, or Cardif) plays an important role in the host defense against viral infection by inducing type I interferon. Recent reports have shown that MAVS is also critical for virus-induced apoptosis. However, the mechanism of MAVS-mediated apoptosis induction remains unclear. Here, we show that MAVS binds to voltage-dependent anion channel 1 (VDAC1) and induces apoptosis by caspase-3 activation, which is independent of its role in innate immunity. MAVS modulates VDAC1 protein stability by decreasing its degradative K48-linked ubiquitination. In addition, MAVS knockout mouse embryonic fibroblasts (MEFs) display reduced VDAC1 expression with a consequent reduction of the vesicular stomatitis virus (VSV)-induced apoptosis response. Notably, the upregulation of VDAC1 triggered by VSV infection is completely abolished in MAVS knockout MEFs. We thus identify VDAC1 as a target of MAVS and describe a novel mechanism of MAVS control of virus-induced apoptotic cell death.