Description: In livers of susceptible but self-curing C57BL/6 mice, intracellular Leishmania donovani infection enhanced Toll-like receptor 4 (TLR4) and TLR2 gene expression. In the liver, infected TLR4 –/– mice showed reduced gamma interferon (IFN-), tumor necrosis factor (TNF), and inducible nitric oxide synthase (iNOS) mRNA expression, higher-level and slowly resolving infection, delayed granuloma formation, and little response to low-dose chemotherapy; in serum, the ratio of IFN- to interleukin 10 (IL-10) activity was decreased by 50%. In contrast, in TLR2 –/– mice, control of liver infection, parasite killing, and granuloma assembly were accelerated and chemotherapy's efficacy enhanced. In livers of infected TLR2 –/– mice, mRNA expression was not increased for inflammatory cytokines or iNOS or decreased for IL-10; however, the serum IFN-/IL-10 ratio was increased 6.5-fold and minimal responses to IL-10 receptor blockade suggested downregulated IL-10. In established infection in wild-type mice, blockading TLR2 induced parasite killing and triggering TLR4 strengthened resistance and promoted chemotherapy's effect. Thus, in experimental L. donovani infection in the liver, TLR4 signaling upregulates and TLR2 signaling downregulates macrophage antileishmanial activity, making both receptors potential therapeutic targets in visceral leishmaniasis for engagement (TLR4) or blockade (TLR2).

Description: Orthopoxviruses (OPVs), which include the agent of smallpox (variola virus), the zoonotic monkeypox virus, the vaccine and zoonotic species vaccinia virus, and the mouse pathogen ectromelia virus (ECTV), form two types of infectious viral particles: the mature virus (MV), which is cytosolic, and the enveloped virus (EV), which is extracellular. It is believed that MVs are required for viral entry into the host, while EVs are responsible for spread within the host. Following footpad infection of susceptible mice, ECTV spreads lymphohematogenously, entering the liver at 3 to 4 days postinfection (dpi). Afterwards, ECTV spreads intrahepatically, killing the host. We found that antibodies to an MV protein were highly effective at curing mice from ECTV infection when administered after the virus reached the liver. Moreover, a mutant ECTV that does not make EV was able to spread intrahepatically and kill immunodeficient mice. Together, these findings indicate that MVs are sufficient for the spread of ECTV within the liver and could have implications regarding the pathogenesis of other OPVs, the treatment of emerging OPV infections, as well as strategies for preparedness in case of accidental or intentional release of pathogenic OPVs.

Description: In cardiac ischemia-reperfusion injury, reactive oxygen species (ROS) generation and upregulation of the hypoxia-inducible protein BNIP3 result in mitochondrial permeabilization, but impairment in autophagic removal of damaged mitochondria provokes programmed cardiomyocyte death. BNIP3 expression and ROS generation result in upregulation of beclin-1, a protein associated with transcriptional suppression of autophagy-lysosome proteins and reduced activation of transcription factor EB (TFEB), a master regulator of the autophagy-lysosome machinery. Partial beclin-1 knockdown transcriptionally stimulates lysosome biogenesis and autophagy via mTOR inhibition and activation of TFEB, enhancing removal of depolarized mitochondria. TFEB activation concomitantly stimulates mitochondrial biogenesis via PGC1α induction to restore normally polarized mitochondria and attenuate BNIP3- and hypoxia-reoxygenation-induced cell death. Conversely, overexpression of beclin-1 activates mTOR to inhibit TFEB, resulting in declines in lysosome numbers and suppression of PGC1α transcription. Importantly, knockdown of endogenous TFEB or PGC1α results in a complete or partial loss, respectively, of the cytoprotective effects of partial beclin-1 knockdown, indicating a critical role for both mitochondrial autophagy and biogenesis in ensuring cellular viability. These studies uncover a transcriptional feedback loop for beclin-1-mediated regulation of TFEB activation and implicate a central role for TFEB in coordinating mitochondrial autophagy with biogenesis to restore normally polarized mitochondria and prevent ischemia-reperfusion-induced cardiomyocyte death.

Description: Defective interfering RNAs (DI-RNAs) of the viral genome can form during infections of negative-strand RNA viruses and outgrow full-length viral genomes, thereby modulating the severity and duration of infection. Here we document the frequent de novo generation of copy-back DI-RNAs from independent rescue events both for a vaccine measles virus (vac2) and for a wild-type measles virus (IC323) as early as passage 1 after virus rescue. Moreover, vaccine and wild-type C-protein-deficient (C-protein-knockout [C KO ]) measles viruses generated about 10 times more DI-RNAs than parental virus, suggesting that C enhances the processivity of the viral polymerase. We obtained the nucleotide sequences of 65 individual DI-RNAs, identified breakpoints and reinitiation sites, and predicted their structural features. Several DI-RNAs possessed clusters of A-to-G or U-to-C transitions. Sequences flanking these mutation sites were characteristic of those favored by adenosine deaminase acting on RNA-1 (ADAR1), which catalyzes in double-stranded RNA the C-6 deamination of adenosine to produce inosine, which is recognized as guanosine, a process known as A-to-I RNA editing. In individual DI-RNAs the transitions were of the same type and occurred on both sides of the breakpoint. These patterns of mutations suggest that ADAR1 edits unencapsidated DI-RNAs that form double-strand RNA structures. Encapsidated DI-RNAs were incorporated into virus particles, which reduced the infectivity of virus stocks. The C KO phenotype was dominant: DI-RNAs derived from vac2 with a C KO suppressed the replication of vac2, as shown by coinfections of interferon-incompetent lymphatic cells with viruses expressing different fluorescent reporter proteins. In contrast, coinfection with a C-protein-expressing virus did not counteract the suppressive phenotype of DI-RNAs. IMPORTANCE Recombinant measles viruses (MVs) are in clinical trials as cancer therapeutics and as vectored vaccines for HIV-AIDS and other infectious diseases. The efficacy of MV-based vectors depends on their replication proficiency and immune activation capacity. Here we document that copy-back defective interfering RNAs (DI-RNAs) are generated by recombinant vaccine and wild-type MVs immediately after rescue. The MV C protein interferes with DI-RNA generation and may enhance the processivity of the viral polymerase. We frequently detected clusters of A-to-G or U-to-C transitions and noted that sequences flanking individual mutations contain motifs favoring recognition by the adenosine deaminase acting on RNA-1 (ADAR1). The consistent type of transitions on the DI-RNAs indicates that these are direct substrates for editing by ADAR1. The ADAR1-mediated biased hypermutation events are consistent with the protein kinase R (PKR)-ADAR1 balancing model of innate immunity activation. We show by coinfection that the C-defective phenotype is dominant.

Description: Brain heart infusion agar containing 3 mg/liter vancomycin (BHI-V3) was used to screen for heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA). There was markedly greater biofilm formation by isolates that grew on BHI-V3 than by strains that did not grow on BHI-V3. Increased biofilm formation by hVISA may be mediated by FnbA- and polysaccharide intercellular adhesin-dependent pathways, and upregulation of atlA and sarA may also contribute to enhanced biofilm formation by hVISA upon prolonged exposure to vancomycin.

Description: Inflammation may be maladaptive to the control of viral infection when it impairs interferon (IFN) responses, enhancing viral replication and spread. Dysregulated immunity as a result of inappropriate innate inflammatory responses is a hallmark of chronic viral infections such as, hepatitis B virus and hepatitis C virus (HCV). Previous studies from our laboratory have shown that expression of an IFN-stimulated gene (ISG), ubiquitin-like protease (USP)18 is upregulated in chronic HCV infection, leading to impaired hepatocyte responses to IFN-α. We examined the ability of inflammatory stimuli, including tumor necrosis factor alpha (TNF-α), lipopolysaccharide (LPS), interleukin-6 (IL-6) and IL-10 to upregulate hepatocyte USP18 expression and blunt the IFN-α response. Human hepatoma cells and primary murine hepatocytes were treated with TNF-α/LPS/IL-6/IL-10 and USP18, phosphorylated (p)-STAT1 and myxovirus (influenza virus) resistance 1 (Mx1) expression was determined. Treatment of Huh7.5 cells and primary murine hepatocytes with LPS and TNF-α, but not IL-6 or IL-10, led to upregulated USP18 expression and induced an IFN-α refractory state, which was reversed by USP18 knockdown. Liver inflammation was induced in vivo using a murine model of hepatic ischemia/reperfusion injury. Hepatic ischemia/reperfusion injury led to an induction of USP18 expression in liver tissue and promotion of lymphocytic choriomeningitis replication. These data demonstrate that certain inflammatory stimuli (TNF-α and LPS) but not others (IL-6 and IL-10) target USP18 expression and thus inhibit IFN signaling. These findings represent a new paradigm for how inflammation alters hepatic innate immune responses, with USP18 representing a potential target for intervention in various inflammatory states. IMPORTANCE Inflammation may prevent the control of viral infection when it impairs the innate immune response, enhancing viral replication and spread. Blunted immunity as a result of inappropriate innate inflammatory responses is a common characteristic of chronic viral infections. Previous studies have shown that expression of certain interferon-stimulated genes is upregulated in chronic HCV infection, leading to impaired hepatocyte responses. In this study, we show that multiple inflammatory stimuli can modulate interferon stimulated gene expression and thus inhibit hepatocyte interferon signaling via USP18 induction. These findings represent a new paradigm for how inflammation alters hepatic innate immune responses, with the induction of USP18 representing a potential target for intervention in various inflammatory states.

Description: Conversion of mesenchymal stem cells into terminally differentiated adipocytes progresses sequentially through regulated transcriptional steps. While it is clear that the late phases of adipocyte maturation are governed by the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR), less is known about the transcriptional control of the initial stages of differentiation. To identify early regulators, we performed a small interfering RNA (siRNA) screen of Forkhead-box genes in adipocytes and show here for the first time that the winged helix factor Foxa3 promotes adipocyte differentiation by cooperating with C/EBPβ and - to transcriptionally induce PPAR expression. Furthermore, we demonstrate that mice with genetic ablation of Foxa3 have a selective decrease in epididymal fat depot and a cell-autonomous defect to induce PPAR specifically in their visceral adipocytes. In obese subjects, FOXA3 is differentially expressed in visceral and subcutaneous adipose depots. Overall, our study implicates Foxa3 in the regulation of adipocyte differentiation and depot-selective adipose tissue expansion.

Description: Staphylococcus epidermidis is one of the most frequent causes of device-associated infections, because it is known to cause biofilms that grow on catheters or other surgical implants. The persistent increasing resistance of S. epidermidis and other coagulase-negative staphylococci (CoNS) has driven the need for newer antibacterial agents with innovative therapeutic strategies. Thanatin is reported to display potent antibiotic activities, especially against extended-spectrum-beta-lactamase-producing Escherichia coli . The present study aimed to investigate whether a shorter derivative peptide (R-thanatin) could be used as a novel antibacterial agent. We found that R-thanatin was highly potent in vitro against coagulase-negative staphylococci, such as S. epidermidis , S. haemolyticus , and S. hominis , and inhibited biofilm formation at subinhibitory concentrations. Properties of little toxicity to human red blood cells (hRBCs) and human umbilical vein endothelial cells, a low incidence of resistance, and relatively high stability in plasma were confirmed. Excellent in vivo protective effects were also observed using a methicillin-resistant S. epidermidis (MRSE)-induced urinary tract infection rat model. Electron microscopy and confocal laser-scanning microscopy analyses suggested that R-thanatin disturbed cell division of MRSE severely, which might be the reason for inhibition of MRSE growth. These findings indicate that R-thanatin is active against the growth and biofilm formation of MRSE in vitro and in vivo . R-thanatin might be considered as a specific drug candidate for treating CoNS infections.

Description: In this study, the regulator MalR (Saci_1161) of the TrmB family from Sulfolobus acidocaldarius was identified and was shown to be involved in transcriptional control of the maltose regulon (Saci_1660 to Saci_1666), including the ABC transporter ( malEFGK ), α-amylase ( amyA ), and α-glycosidase ( malA ). The malR deletion mutant exhibited a significantly decreased growth rate on maltose and dextrin but not on sucrose. The expression of the genes organized in the maltose regulon was induced only in the presence of MalR and maltose in the growth medium, indicating that MalR, in contrast to its TrmB and TrmB-like homologues, is an activator of the maltose gene cluster. Electrophoretic mobility shift assays revealed that the binding of MalR to malE was independent of sugars. Here we report the identification of the archaeal maltose regulator protein MalR, which acts as an activator and controls the expression of genes involved in maltose transport and metabolic conversion in S. acidocaldarius , and its use for improvement of the S. acidocaldarius expression system under the control of an optimized maltose binding protein ( malE ) promoter by promoter mutagenesis.

Description: Kaposi's sarcoma-associated herpesvirus (KSHV) is causally linked to several AIDS-related malignancies, including Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease. The interaction of human immunodeficiency virus type 1 (HIV-1) and KSHV has a central role in promoting the aggressive manifestations of AIDS-KS. We have previously shown that negative factor (Nef), a secreted HIV-1 protein, synergizes with KSHV viral interleukin-6 (vIL-6) to promote angiogenesis and tumorigenesis by activating the AKT pathway (X. Zhu, et al., Oncogene, 22 April 2013, http://dx.doi.org/10.1038/onc.2013.136 ). Here, we further demonstrated the role of soluble and ectopic Nef in the regulation of KSHV latency. We found that both soluble Nef protein and ectopic expression of Nef by transfection suppressed the expression of KSHV viral lytic mRNA transcripts and proteins and the production of infectious viral particles. MicroRNA (miRNA) microarray analysis identified a number of Nef-regulated miRNAs. Bioinformatics and luciferase reporter analyses showed that one of the Nef-upregulated miRNAs, cellular miRNA 1258 (hsa-miR-1258), directly targeted a seed sequence in the 3' untranslated region (UTR) of the mRNA encoding the major lytic switch protein (RTA), which controls KSHV reactivation from latency. Ectopic expression of hsa-miR-1258 impaired RTA synthesis and enhanced Nef-mediated inhibition of KSHV replication, whereas repression of hsa-miR-1258 has the opposite effect. Mutation of the seed sequence in the RTA 3'UTR abolished downregulation of RTA by hsa-miR-1258. Collectively, these novel findings demonstrate that, by regulating cellular miRNA, Nef may inhibit KSHV replication to promote viral latency and contribute to the pathogenesis of AIDS-related malignancies. IMPORTANCE This study found that Nef, a secreted HIV-1 protein, suppressed KSHV lytic replication to promote KSHV latency. Mechanistic studies indicated that a Nef-upregulated cellular miRNA, hsa-miR-1258, inhibits KSHV replication by directly targeting a seed sequence in the KSHV RTA 3'UTR. These results illustrate that, in addition to viral miRNAs, cellular miRNAs also play an important role in regulating the life cycle of KSHV. Overall, this is the first study to report the involvement of Nef in KSHV latency, implying its likely important role in the pathogenesis of AIDS-related malignancies.