Comparative proteomics identifies Schlafen 5 (SLFN5) as a herpes simplex virus restriction factor that suppresses viral transcription.

Intrinsic antiviral host factors confer cellular defence by limiting virus replication and are often counteracted by viral countermeasures. We reasoned that host factors that inhibit viral gene expression could be identified by determining proteins bound to viral DNA (vDNA) in the absence of key viral antagonists. Herpes simplex virus 1 (HSV-1) expresses E3 ubiquitin-protein ligase ICP0 (ICP0), which functions as an E3 ubiquitin ligase required to promote infection. Cellular substrates of ICP0 have been discovered as host barriers to infection but the mechanisms for inhibition of viral gene expression are not fully understood. To identify restriction factors antagonized by ICP0, we compared proteomes associated with vDNA during HSV-1 infection with wild-type virus and a mutant lacking functional ICP0 (ΔICP0). We identified the cellular protein Schlafen family member 5 (SLFN5) as an ICP0 target that binds vDNA during HSV-1 ΔICP0 infection. We demonstrated that ICP0 mediates ubiquitination of SLFN5, which leads to its proteasomal degradation. In the absence of ICP0, SLFN5 binds vDNA to repress HSV-1 transcription by limiting accessibility of RNA polymerase II to viral promoters. These results highlight how comparative proteomics of proteins associated with viral genomes can identify host restriction factors and reveal that viral countermeasures can overcome SLFN antiviral activity.

Identifying the World Health Organization's fifth moment for hand hygiene: Infection prevention in the operating room.

BACKGROUND: The World Health Organization have designed the fifth of their '5 moments' for hand hygiene to account for microbial transfer from patients to equipment in a narrow area around that patient, known as the patient zone. The study was prompted by emerging local confusion about application of the patient zone in the operating room (OR). AIM/OBJECTIVES: In two phases, we aimed to create a '5 moments' style poster displaying an OR patient zone: phase 1, quantify equipment, in direct contact with the patient and, touched by non-scrubbed staff immediately after touching the patient; and phase 2, categorise equipment identified in phase 1 into patient zone and healthcare zone. An objective is to produce a '5 moments' poster for the OR. METHODS: The first phase used non-participant direct overt observation. In phase 2, phase 1 data were collaboratively assigned to patient zone or healthcare zone. Photography and graphic design were used to produce the OR '5 moments' poster. RESULTS: In 11 full-length surgeries, 20 pieces of equipment were in direct contact with the patient and 57 pieces of equipment were touched. In phase 2, a '5 moments' poster showing an OR patient zone was designed. DISCUSSION: Content of the patient zone was identified and displayed in a novel resource. Having shared understanding of the patient zone has potential to sustain hand hygiene compliance and equipment cleaning in the OR. CONCLUSION: Limitations in methods were balanced by collaboration with frontline staff. The study has been used as a teaching tool in the OR and similar settings.

Liver-specific ablation of integrin-linked kinase in mice results in enhanced and prolonged cell proliferation and hepatomegaly after phenobarbital administration.

We have recently demonstrated that disruption of extracellular matrix (ECM)/integrin signaling via elimination of integrin-linked kinase (ILK) in hepatocytes interferes with signals leading to termination of liver regeneration. This study investigates the role of ILK in liver enlargement induced by phenobarbital (PB). Wild-type (WT) and ILK:liver-/- mice were given PB (0.1% in drinking water) for 10 days. Livers were harvested on 2, 5, and 10 days during PB administration. In the hepatocyte-specific ILK/liver-/- mice, the liver:body weight ratio was more than double as compared to 0 h at day 2 (2.5 times), while at days 5 and 10, it was enlarged three times. In the WT mice, the increase was as expected from previous literature (1.8 times) and seems to have leveled off after day 2. There were slightly increased proliferating cell nuclear antigen-positive cells in the ILK/liver-/- animals at day 2 as compared to WT after PB administration. In the WT animals, the proliferative response had come back to normal by days 5 and 10. Hepatocytes of the ILK/liver-/- mice continued to proliferate up until day 10. ILK/liver-/- mice also showed increased expression of key genes involved in hepatocyte proliferation at different time points during PB administration. In summary, ECM proteins communicate with the signaling machinery of dividing cells via ILK to regulate hepatocyte proliferation and termination of the proliferative response. Lack of ILK in the hepatocytes imparts prolonged proliferative response not only to stimuli related to liver regeneration but also to xenobiotic chemical mitogens, such as PB.

Enhanced liver regeneration following changes induced by hepatocyte-specific genetic ablation of integrin-linked kinase.

UNLABELLED: Following liver regeneration after partial hepatectomy, liver grows back precisely to its original mass and does not exceed it. The mechanism regulating this "hepatostat" is not clear and no exceptions have been found to date. Although pathways initiating liver regeneration have been well studied, mechanisms involved in the termination of liver regeneration are unclear. Here, we report that integrin-linked kinase (ILK) (involved in transmission of the extracellular matrix [ECM] signaling by way of integrin receptors) and/or hepatic adaptations that ensue following ILK hepatocyte-targeted removal are critical for proper termination of liver regeneration. Following partial hepatectomy (PHx), mice with a liver-specific ILK ablation (ILK-KO-Liver) demonstrate a termination defect resulting in 58% larger liver than their original pre-PHx mass. This increase in post-PHx liver mass is due to sustained cell proliferation driven in part by increased signaling through hepatocyte growth factor (HGF), and the beta-catenin pathway and Hippo kinase pathways. CONCLUSION: The data indicate that ECM-mediated signaling by way of ILK is essential in proper termination of liver regeneration. This is the first evidence of a defect leading to impaired termination of regeneration and excessive accumulation of liver weight following partial hepatectomy.

Liver-specific ablation of integrin-linked kinase in mice results in abnormal histology, enhanced cell proliferation, and hepatomegaly.

UNLABELLED: Hepatocyte differentiation and proliferation are greatly affected by extracellular matrix (ECM). Primary hepatocytes cultured without matrix dedifferentiate over time, but matrix overlay quickly restores differentiation. ECM also is critical in liver regeneration where ECM degradation and reconstitution are steps in the regenerative process. Integrin-linked kinase (ILK) is a cell-ECM-adhesion component implicated in cell-ECM signaling by means of integrins. We investigated the role of ILK in whole liver by using the LoxP/Cre model system. ILK was eliminated from the liver by mating homozygous ILK-floxed animals with mice expressing Cre-recombinase under control of the alpha fetoprotein enhancer and albumin promoter. After ablation of ILK, animals are born normal. Soon after birth, however, they develop histologic abnormalities characterized by disorderly hepatic plates, increased proliferation of hepatocytes and biliary cells, and increased deposition of extracellular matrix. Cell proliferation is accompanied by increased cytoplasmic and nuclear stabilization of beta-catenin. After this transient proliferation of all epithelial components, proliferation subsides and final liver to body weight ratio in livers with ILK deficient hepatocytes is two times that of wild type. Microarray analysis of gene expression during the stage of cell proliferation shows up-regulation of integrin and matrix-related genes and a concurrent down-regulation of differentiation-related genes. After the proliferative stage, however, the previous trends are reversed resulting in a super-differentiated phenotype in the ILK-deficient livers. CONCLUSION: Our results show for the first time in vivo the significance of ILK and hepatic ECM-signaling for regulation of hepatocyte proliferation and differentiation.

Yeast histone 2A serine 129 is essential for the efficient repair of checkpoint-blind DNA damage.

Cells maintain genomic stability by the coordination of DNA-damage repair and cell-cycle checkpoint control. In replicating cells, DNA damage usually activates intra-S-phase checkpoint controls, which are characterized by delayed S-phase progression and increased Rad53 phosphorylation. We show that in budding yeast, the intra-S-phase checkpoint controls, although functional, are not activated by the topoisomerase I inhibitor camptothecin (CPT). In a CPT-hypersensitive mutant strain that lacks the histone 2A (H2A) phosphatidylinositol-3-OH kinase (PI(3)K) motif at Ser 129 (h2a-s129a), the hypersensitivity was found to result from a failure to process full-length chromosomal DNA molecules during ongoing replication. H2A Ser 129 is not epistatic to the RAD24 and RAD9 checkpoint genes, suggesting a non-checkpoint role for the H2A PI(3)K site. These results suggest that H2A Ser 129 is an essential component for the efficient repair of DNA double-stranded breaks (DSBs) during replication in yeast, particularly of those DSBs that do not induce the intra-S-phase checkpoint.

Metal-dependent inhibition of HIV-1 integrase.

Human immunodeficiency virus type 1 integrase (HIV-1 IN) is an essential enzyme for effective viral replication. Therefore, IN inhibitors are being sought for chemotherapy against AIDS. We had previously identified a series of salicylhydrazides as potent inhibitors of IN in vitro (Neamati, N.; et al. J. Med. Chem. 1998, 41, 3202-3209.). Herein, we report the design, synthesis, and antiviral activity of three novel mercaptosalicylhydrazide (MSH) derivatives. MSHs were effective against the IN catalytic core domain and inhibited IN binding to HIV LTR DNA. They also inhibited catalytic activities of IN in IN-DNA preassembled complexes. Site-directed mutagenesis and molecular modeling studies suggest that MSHs bind to cysteine 65 and chelate Mg(2+) at the active site of HIV-1 IN. Contrary to salicylhydrazides, the MSHs are 300-fold less cytotoxic and exhibit antiviral activity. They are also active in Mg(2+)-based assays, while IN inhibition by salicylhydrazides is strictly Mn(2+)-dependent. Additionally, in target and cell-based assays, the MSHs have no detectable effect on other retroviral targets, including reverse transcriptase, protease, and virus attachment, and exhibit no detectable activity against human topoisomerases I and II at concentrations that effectively inhibit IN. These data suggest that MSHs are selective inhibitors of HIV-1 IN and may serve as leads for antiviral therapeutics.