RNASEK is required for internalization of diverse acid-dependent viruses

Viruses must gain entry into cells to establish infection. In general, viruses enter either at the plasma membrane or from intracellular endosomal compartments. Viruses that use endosomal pathways are dependent on the cellular factors that control this process; however, these genes have proven to be essential for endogenous cargo uptake, and thus are of limited value for therapeutic intervention. The identification of genes that are selectively required for viral uptake would make appealing drug targets, as their inhibition would block an early step in the life cycle of diverse viruses. At this time, we lack pan-antiviral therapeutics, in part because of our lack of knowledge of such cellular factors. RNAi screening has begun to reveal previously unknown genes that play roles in viral infection. We identified dRNASEK in two genome-wide RNAi screens performed in Drosophila cells against West Nile and Rift Valley Fever viruses. Here we found that ribonuclease kappa (RNASEK) is essential for the infection of human cells by divergent and unrelated positive- and negative-strand-enveloped viruses from the Flaviviridae, Togaviridae, Bunyaviridae, and Orthomyxoviridae families that all enter cells from endosomal compartments. In contrast, RNASEK was dispensable for viruses, including parainfluenza virus 5 and Coxsackie B virus, that enter at the plasma membrane. RNASEK is dispensable for attachment but is required for uptake of these acid-dependent viruses. Furthermore, this requirement appears specific, as general endocytic uptake of transferrin is unaffected in RNASEK-depleted cells. Therefore, RNASEK is a potential host cell Achilles’ heel for viral infection.Viral pathogens are quite diverse in their replication strategies; however, all viruses must enter cells to initiate their replication cycles. The first step involves binding of virus particles to the cell surface. Such interactions can involve attachment factors, which have low affinity but concentrate viruses on the surface of cells, and receptors that intricately interact with viral envelope glycoproteins, which, in addition to binding, promote other aspects of infection such as internalization. Although a plethora of receptors and pathways can be used, most viruses take advantage of the cellular endocytic machinery and penetrate from within the cytosol (reviewed in refs. 1–3). Clathrin-mediated endocytosis, macropinocytosis, and caveolin-mediated endocytosis are the best-studied forms of uptake used by viruses. Clathrin-mediated endocytosis is the most common mechanism used by small viruses, as clathrin-coated vesicles have a diameter of 60–200 nm and can be enlarged to fit even larger particles (4, 5). This pathway is constitutive on most cells, and some viruses use preexisting clathrin-coated pits for entry (e.g., dengue virus), whereas others induce the formation of these structures (e.g., influenza virus) (6, 7). Macropinocytosis is an actin-dependent endocytic process for the nonselective uptake of nutrients in response to receptor engagement. It is the predominant pathway for many larger viruses, including vaccinia virus, but is also used by others, including influenza virus under some conditions (8–10). It also remains unclear which pathways are used by some viruses, including Rift Valley Fever virus (RVFV) (11–13).The molecular mechanisms involved in these uptake mechanisms are complex and rely on key molecules and organelles that are essential for cellular viability, as these uptake mechanisms bring nutrients and other metabolites into the cytosol for cellular growth and survival. Indeed, these processes and proteins involved are highly conserved from yeast to humans (14, 15). Depending on the virus entry requirements, some viruses fuse within early endosomal vesicles, whereas others traffic to more acidic compartments or macropinosomes for entry. Because many viruses are dependent on these endosomal trafficking pathways for entry, much effort has been made in identifying the specific cellular genes required for viral entry (16). Therapeutics targeting entry are appealing because it is the first step in the infection cycle, and many viruses use common pathways; thus, inhibition may be broadly antiviral, rather than active against only a specific virus. Furthermore, many viruses have high mutation rates and rapidly evolve resistance to therapeutics targeting virally encoded genes. Conversely, therapeutics against host encoded targets would likely be more difficult for the virus to evade.Recent advances in functional genomic technologies have facilitated the use of unbiased genome-wide RNAi screens to identify cellular genes required for viral infection (17). Such approaches allow for the discovery of otherwise unknown genes that play essential roles in infection. We recently performed such screens in insect cells against two disparate insect-borne human pathogens: the flavivirus West Nile virus (WNV) and the bunyavirus RVFV (18, 19). These are both arthropod-borne human pathogens for which there are no vaccines or therapeutics. Furthermore, these viruses are quite divergent: WNV is a flavivirus that is a globally important cause of encephalitis (20), and RVFV is a bunyavirus that causes significant morbidity and mortality in livestock and humans in Africa (21). In our screens, there were only three genes that promoted infection by both viruses: dRAB5, dSTX7, and dRNASEK (CG40127). The functions of RAB5 and STX7 have been described, but little is known about ribonuclease kappa (RNASEK). Both RAB5 and STX7 are involved in endosomal transport and have roles in viral entry (22, 23). Both WNV and RVFV are enveloped RNA viruses that require an acidic compartment for entry (12, 13, 24). RNASEK is a single-copy, 137-aa protein conserved from insects to humans (25–27) with an unknown function. We set out to determine the role of RNASEK in viral infection and found that RNASEK is required for internalization of a diverse panel of viruses of medical concern.     

Tourniquet application only during cement fixation in total knee arthroplasty: a double-blind, randomized controlled trial

Background: The functional benefits of tourniquet application for short periods compared with standard duration applications during total knee arthroplasty surgery have not been well explored. We aimed to compare functional outcomes between tourniquet application of short duration (during cement fixation only) and tourniquet application of longer duration (from skin incision to just after cement fixation). Methods: We planned to randomize 230 patients to short and long duration groups. The primary outcome was Oxford Knee Score at 10 weeks post‐surgery. In‐hospital blood transfusion rate was also a primary safety measure. Serial measures of knee function were taken together with knee range, quadriceps lag and timed stair tests. Results: The trial was discontinued after randomization of 65 patients. Interim analysis indicated a higher risk of transfusion (odds ratio 7.38, P= 0.015) in the short duration group. No significant difference was observed in Oxford Knee Score at 10 weeks. There were no between‐group differences in rate of recovery up to 52 weeks for any outcome. Conclusions: Restricting tourniquet application to the period of cementing is associated with a significantly higher risk of transfusion. This approach is impractical if it is not offset by gains in functional recovery.     

The colonization of Peyer's patches by a strain of Salmonella typhimurium cured of the cryptic plasmid

We cured a strain of Salmonella typhimurium of its cryptic plasmid and confirmed that orally administered cured strains lost virulence for mice. Loss of the cryptic plasmid rendered the S. typhimurium strain sensitive to the bactericidal action of normal human serum. However, loss of the plasmid did not change the ability of the strain to associate with HeLa cells in tissue culture. Furthermore, when administered orally to mice, both the plasmid-containing and plasmid-free strains invaded the Peyer's patches of the small intestine to the same extent, and both were capable of inducing resistance to oral challenge with virulent S. typhimurium. When injected intraperitoneally, the cured strain was eliminated rapidly, whereas the parental strain persisted. We also showed that the cured strain did not contain a plasmid copy in the chromosome. We propose that although the plasmid-cured strain of S. typhimurium is able to colonize Peyer's patches, it cannot survive when administered intraperitoneally because it is susceptible to elimination by macrophages.     

地理信息系统应用于血吸虫病的监测 Ⅰ.应用预测模型的可能性

目的：建立血吸虫病地理信息系统，利用气象参数建立模型来探讨预测血吸虫病的流行区的可能性。方法：以世界粮农组织出版的FAOCLIM数据库中的数据为基础，以血吸虫发育扩散关系密切的温度和潜在蒸发指数（地面水平衡系统）为基础的改良Malone公式计算血吸虫传播指数，结合AVHRR卫星图片资料，获得校正植被指数（NDVI）和第4频道地面温度指数、高程分布图，在ArcView3．0a和ERDAS软件支持下，进行GIS数据空间分析和地图重叠分析，以某一类别值显示出流行区的地理分布图。结果：血吸虫传播指数（指数值大于900）的分布基本上与中国南部地区的血吸虫病流行区相吻合。多层重叠分布图显示了红色区域的高危地区与长江流域的血吸虫病高发地区基本一致。结论：血吸虫病的流行范围与温度、高程、雨量等因素密切相关。利用气象资料模型和卫星遥感资料对预测血吸虫病的潜在流行区具有可能。准确、快速地利用AVHRR遥感资料来预测、预报血吸虫病流行范围和强度具有应用前景，值得作进一步探讨。     

Thrombopoietin gene transfer-mediated enhancement of angiogenic responses to acute ischemia

The development of new blood vessels is a complex process, likely requiring the synergy of multiple angiogenic mediators. This study focuses on the proximal angiogenic response using the platelet as a complex carrier of critical mediators of angiogenesis. Platelet levels are controlled by circulating levels of thrombopoietin (TPO) functioning to activate megakaryocyte differentiation and platelet release through the c-mpl receptor. We hypothesized that TPO gene transfer should enhance correction of experimental ischemia by providing increased levels of platelets and hence platelet-derived mediators of angiogenesis. To evaluate this hypothesis, we dissected the role of the TPO-c-mpl-megakaryocyte-platelet pathway in the angiogenic response using a model of acute hindlimb ischemia of wild-type, TPO(-/-), and c-mpl(-/-) mice. The data demonstrate that infusion of platelets will enhance the angiogenic response in wild-type mice and that the endogenous angiogenic response is blunted in TPO(-/-) and c-mpl(-/-) mice. Consistent with this observation, adenovirus (Ad)-mediated transfer of TPO (AdTPO) enhanced the correction of ischemia in wild-type and TPO(-/-), but not c-mpl(-/-), mice. Local versus systemic administration of AdTPO showed that the effect of TPO gene transfer was systemic, not local, and it could be replaced by gene transfer of VEGF, one of the many mediators of angiogenesis carried by the platelets, even in the absence of components in the TPO-c-mpl-megakaryocyte-platelet pathway.     

Effectiveness of multiple bolus administration of tissue-type plasminogen activator in acute myocardial infarction

The feasibility and possible advantages of intravenous bolus administration of recombinant tissue-type plasminogen activator (rt-PA) were investigated in 26 consecutive patients with early (less than 6 hours) evolving acute myocardial infarction. Either an intravenous infusion of 40 clot-lysis megaunits (cIMU) double-chain rt-PA over 1.5 hours followed by 20 cIMU over 5 hours (infusion group, n = 12) or 4 intravenous bolus injections of 10 cIMU at 20 minute intervals (bolus group, n = 14) were randomly administered. Coronary arteriography was performed before and at regular predefined intervals up to 90 minutes from the start of rt-PA administration, and at 24 hours. Acute recanalization of the infarct-related coronary artery was demonstrated in 7 of 12 patients (58%; 95% confidence interval 28 to 85%) in the infusion group and 11 of 14 patients (79%; 95% confidence interval 49 to 95%) in the bolus group (difference not significant). Two patients in the bolus group had reoccluded by 24 hours. Mean time from the start of rt-PA to patency of the infarct-related coronary artery was 39 +/- 6 (standard error of the mean) minutes in the infusion group and 28 +/- 6 minutes in the bolus group (p = 0.2). There were no significant differences in the minimum infarct-related coronary artery luminal diameter measured by computerized quantitative arteriography between the infusion group and the bolus group at 90 minutes or at 24 hours.(ABSTRACT TRUNCATED AT 250 WORDS)     

Infection of endothelium with E1(-)E4(+), but not E1(-)E4(-), adenovirus gene transfer vectors enhances leukocyte adhesion and migration by modulation of ICAM-1, VCAM-1, CD34, and chemokine expression

Intravascular introduction of replication-deficient adenoviral vectors (Advectors) provides an ideal model of delivery of transgenes for the treatment of various vascular abnormalities. On the basis of the knowledge that Advectors can induce inflammatory responses after intravascular administration, we speculated that cellular activation by Advector infection could directly modulate the endothelial cell (EC) adhesion molecule/chemokine expression repertoire. Infection of human umbilical vein ECs or bone marrow microvascular ECs with an E1(-)E4(+) Advector resulted in the upregulation of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and CD34, but not E-selectin, P-selectin, CD36, CD13, CD44, HLA-DR or PECAM. Upregulation of ICAM-1, VCAM-1, and CD34 was apparent 12 hours after infection and persisted for weeks after infection. Selective induction of adhesion molecules was mediated by the presence of the E4 gene in the Advector, because infection of ECs with an E1(-)E4(-) Advector had no effect on adhesion molecule expression. ECs infected with E1(-)E4(+) Advector, but not those infected with E1(-)E4(-) Advector, supported the adhesion of leukocytes. Monoclonal antibodies to ICAM-1 and VCAM-1 inhibited adhesion of leukocytes to E1(-)E4(+)-infected ECS: Infection of the ECs with E1(-)E4(+) Advector, but not E1(-)E4(-) Advector, resulted in downregulation of expression of chemocytokines, including interleukin-8, MCP-1, RANTES, and GM-CSF. Nonetheless, a large number of leukocytes migrated through ECs infected with E1(-)E4(+), but not those infected with E1(-)E4(l-), in response to exogenous chemokines. These results demonstrate that infection of ECs with E1(-)E4(+) Advectors, but not E1(-)E4(-) Advectors, may directly augment inflammatory responses by upregulating expression of adhesion molecules and enhancing migration through Advector-infected ECs and suggest that E1(-)E4(-) Advectors may be a better choice for gene-transfer strategies directed to the ECS: