Crystal structures of murine norovirus-1 RNA-dependent RNA polymerase in complex with 2-thiouridine or ribavirin

AbstractMurine norovirus-1 (MNV-1) shares many features with human norovirus (HuNoV) and both are classified within the norovirus genus of Caliciviridae family. MNV-1 is used as the surrogate for HuNoV research since it is the only form that can be grown in cell culture. HuNoV and MNV-1 RNA dependent RNA polymerase (RdRp) proteins with the sequence identity of 59% show essentially identical conformations. Here we report the first structural evidence of 2-thiouridine (2TU) or ribavirin binding to MNV-1 RdRp, based on the crystal structures determined at 2.2Å and 2.5Å resolutions, respectively. Cellular and biochemical studies revealed stronger inhibitory effect of 2TU on the replication of MNV-1 in RAW 264.7 cells, compared to that of ribavirin. Our complex structures highlight the key interactions involved in recognition of the nucleoside analogs which block the active site of the viral RNA polymerase

High fat diet-induced obesity leads to proinflammatory response associated with higher expression of NOD2 protein

Obesity has been reported to be associated with low grade inflammatory status. In this study, we investigated the inflammatory response as well as associated signaling molecules in immune cells from diet-induced obese mice. Four-week-old C57BL mice were fed diets containing 5% fat (control) or 20% fat and 1% cholesterol (HFD) for 24 weeks. Splenocytes (1 × 107 cells) were stimulated with 10 µg/mL of lipopolysaccharide (LPS) for 6 or 24 hrs. Production of interleukin (IL)-1β, IL-6, and TNF-α as well as protein expression levels of nucleotide-binding oligomerization domain (NOD)2, signal transducer and activator of transcription (STAT)3, and pSTAT3 were determined. Mice fed HFD gained significantly more body weight compared to mice fed control diet (28.2 ± 0.6 g in HFD and 15.4 ± 0.8 g in control). After stimulation with LPS for 6 hrs, production of IL-1β was significantly higher (P = 0.001) and production of tumor necrosis factor (TNF)-α tended to be higher (P < 0.064) in the HFD group. After 24 hrs of LPS stimulation, splenocytes from the HFD group produced significantly higher levels of IL-6 (10.02 ± 0.66 ng/mL in HFD and 7.33 ± 0.56 ng/mL in control, P = 0.005) and IL-1β (121.34 ± 12.72 pg/mL in HFD and 49.74 ± 6.58 pg/mL in control, P < 0.001). There were no significant differences in the expression levels of STAT3 and pSTAT3 between the HFD and the control groups. However, the expression level of NOD2 protein as determined by Western blot analysis was 60% higher in the HFD group compared with the control group. NOD2 contributes to the induction of inflammation by activation of nuclear factor κB. These findings suggest that diet-induced obesity is associated with increased inflammatory response of immune cells, and higher expression of NOD2 may contribute to these changes

JNK pathway is involved in the inhibition of inflammatory target gene expression and NF-kappaB activation by melittin

<p>Abstract</p> <p>Background</p> <p>Bee venom therapy has been used to treat inflammatory diseases including rheumatoid arthritis in humans and in experimental animals. We previously found that bee venom and melittin (a major component of bee venom) have anti-inflammatory effect by reacting with the sulfhydryl group of p50 of nuclear factor-kappa B (NF-κB) and IκB kinases (IKKs). Since mitogen activated protein (MAP) kinase family is implicated in the NF-κB activation and inflammatory reaction, we further investigated whether activation of MAP kinase may be also involved in the anti-inflammatory effect of melittin and bee venom.</p> <p>Methods</p> <p>The anti-inflammatory effects of melittin and bee venom were investigated in cultured Raw 264.7 cells, THP-1 human monocytic cells and Synoviocytes. The activation of NF-κB was investigated by electrophoretic mobility shift assay. Nitric oxide (NO) and prostaglandin E₂(PGE₂) were determined either by Enzyme Linked Immuno Sorbent Assay or by biochemical assay. Expression of IκB, p50, p65, inducible nitric oxide synthetase (iNOS), cyclooxygenase-2 (COX-2) as well as phosphorylation of MAP kinase family was determined by Western blot.</p> <p>Results</p> <p>Melittin (0.5–5 μg/ml) and bee venom (5 and 10 μg/ml) inhibited lipopolysaccharide (LPS, 1 μg/ml) and sodium nitroprusside (SNP, 200 μM)-induced activation of c-Jun NH2-terminal kinase (JNK) in RAW 264.7 cells in a dose dependent manner. However, JNK inhibitor, anthra [1,9-cd]pyrazole-6 (2H)-one (SP600215, 10–50 μM) dose dependently suppressed the inhibitory effects of melittin and bee venom on NF-κB dependent luciferase and DNA binding activity via suppression of the inhibitory effect of melittin and bee venom on the LPS and SNP-induced translocation of p65 and p50 into nucleus as well as cytosolic release of IκB. Moreover, JNK inhibitor suppressed the inhibitory effects of melittin and bee venom on iNOS and COX-2 expression, and on NO and PGE₂generation.</p> <p>Conclusion</p> <p>These data show that melittin and bee venom prevent LPS and SNP-induced NO and PGE₂production via JNK pathway dependent inactivation of NF-κB, and suggest that inactivation of JNK pathways may also contribute to the anti-inflammatory and anti-arthritis effects of melittin and bee venom.</p

Right Ventricular Outflow Tract Stenting in a Low Birth Weight Infant Born With Tetralogy of Fallot and Prostaglandin E1 Dependency

Surgical skill and strategy for the correction of tetralogy of Fallot (TOF) have improved and resulted in satisfactory outcomes. However, prematurity and low birth weight continue to remain risk factors for poor outcomes. We present a case of a 2,150 g neonate born with TOF, in whom palliation was achieved with right ventricular outflow tract (RVOT) stenting. Seventy-seven days after the procedure, stenosis of RVOT below the stent was identified. At that time his body weight was 4.9 kg and total corrective surgery was deemed feasible. Eight months following surgical repair, the patient remained well without medical intervention. RVOT stenting may be a viable interim procedure while waiting for a low birth weight neonate born with TOF and prostaglandin E1 dependency to reach optimal weight to undergo corrective surgery

Three Cases of Moraxella osloensis Meningitis: A Difficult Experience in Species Identification and Determination of Clinical Significance

We had three cases of Moraxella osloensis meningitis. The species identification was impossible by conventional and commercial phenotypic tests. However, we could identify the species using the 16S rRNA gene sequencing. Determination of clinical significance was difficult in one patient. All three patients recovered by appropriate antimicrobial therapy

Obstructive Fibrinous Tracheal Pseudomembrane After Tracheal Intubation: A Case Report

Obstructive fibrinous tracheal pseudomembrane is a rare, but potentially fatal complication associated with endotracheal intubation. It has been known that the formation of tracheal pseudomembrane is related with intracuff pressure during endotracheal intubation or infectious cause. But in the patient described in this case, pseudomembrane formation in the trachea was associated with subglottic epithelial trauma or caustic injuries to the trachea caused by aspirated gastric contents during intubation rather than tracheal ischemia due to high cuff pressure. We report a patient with obstructive fibrinous tracheal pseudomembrane after endotracheal intubation who presented with dyspnea and stridor and was treated successfully with mechanical removal using rigid bronchoscopy

Comparison of Factors Associated with Atypical Symptoms in Younger and Older Patients with Acute Coronary Syndromes

Patients with acute coronary syndromes (ACS) who are accompanied by atypical symptoms are frequently misdiagnosed and under-treated. This study was conducted to examine and compare the factors associated with atypical symptoms other than chest pain in younger (<70 yr) and older (≥70 yr) patients with first-time ACS. Data were obtained from the electronic medical records of the patients (n=931) who were newly diagnosed as ACS and hospitalized from 2005 to 2006. The 7.8% (n=49) of the younger patients and 13.4% (n=41) of the older patients were found to have atypical symptoms. Older patients were more likely to complain of indigestion or abdominal discomfort (P=0.019), nausea and/or vomiting (P=0.040), and dyspnea (P<0.001), and less likely to have chest pain (P=0.007) and pains in the arm and shoulder (P=0.018). A logistic regression analysis showed that after adjustment made for the gender and ACS type, diabetes and hyperlipidemia significantly predicted atypical symptoms in the younger patients. In the older patients, the co-morbid conditions such as stroke or chronic obstructive pulmonary disease were positive predictors. Health care providers need to have an increased awareness of possible presence of ACS in younger persons with diabetes and older persons with chronic concomitant diseases when evaluating patients with no chest pain

The effect of alpha lipoic acid in a porcine in-stent restenosis model

SummaryBackgroundThe aim of this study was to investigate the effect of alpha lipoic acid (α-LA) on a porcine in-stent restenosis (ISR) model.MethodsIn protocol 1, porcine vascular smooth muscle cells (PVSMC) were stimulated by granulocyte-colony stimulating factor (G-CSF) in the presence or absence of α-LA. MTT (3-[4,5-dimethylthiazole-2-yl] 2,5-diphenyl tetrazolium bromide) assay and western blotting were used to determine the cell growth inhibitory rate and anti-inflammatory effect associated with nuclear factor-κb (NF-κb) and extracellular signal-regulated kinase (ERK). In protocol 2, 28 days after balloon overdilation injuries, 24 bare metal stents were placed in coronary artery of 12 pigs. The pigs were randomly divided to receive control diet with or without α-LA (100mg/kg). In protocol 3, 8 control stents and 8 α-LA coated stents were randomly implanted in 2 coronary arteries of 8 pigs and follow-up coronary angiogram and histopathologic assessment were performed 4 weeks after stenting.ResultsProtocol 1. The proliferation of PVSMC was inhibited and protein expression of NF-κb and ERK were attenuated by α-LA pretreatment. Protocol 2. On histopathologic analysis, the neointimal area (4.0±1.0mm2 vs. 1.5±0.7mm2, p<0.001) and histopathologic area of stenosis (66.7±10.7% vs. 24.2±9.7%, p<0.001) were reduced in the α-LA feeding group compared to controls. Protocol 3. On histopathologic analysis, the neointimal area (3.9±0.8mm2 vs. 1.0±0.4mm2, p<0.001), and the histopathologic area of stenosis (67.1±8.8% vs. 17.4±10.0%, p<0.001) were reduced in the α-LA coated stent group compared to the control stent group.Conclusionsα-LA feeding and α-LA coated stents inhibit neointimal hyperplasia in porcine ISR, possibly through inhibiting the activation of NF-κb pathway and proliferation of PVSMC

The first reported hepatitis E outbreak in a food manufacturing factory: Korea, 2022

Objectives On February 16, 2022, 12 cases of hepatitis E virus (HEV) infection were reported in a food manufacturing factory in Korea. The aim of this study was to identify additional cases and to determine the source of this HEV outbreak. Methods This study was an in-depth investigation of 12 HEV immunoglobulin M (IgM)-positive cases and their demographic, clinical, and epidemiological characteristics. On-site specimens were collected from the environment and from humans, and a follow-up investigation was conducted 2 to 3 months after the outbreak. Results Among 80 production workers in the factory, 12 (15.0%) had acute HEV infection, all of whom were asymptomatic. The follow-up investigation showed that 3 cases were HEV IgM-positive, while 6 were HEV IgG-positive. HEV genes were not detected in the HEV IgM-positive specimens. HEV genes were not detected in the food products or environmental specimens collected on-site. HEV was presumed to be the causative pathogen. However, it could not be confirmed that the source of infection was common consumption inside the factory. Conclusion This was the first domestic case of an HEV infection outbreak in a food manufacturing factory in Korea. Our results provide information for the future control of outbreaks and for the preparation of measures to prevent domestic outbreaks of HEV infection

Loss of Autophagy Diminishes Pancreatic β Cell Mass and Function with Resultant Hyperglycemia

SummaryAutophagy is a cellular degradation-recycling system for aggregated proteins and damaged organelles. Although dysregulated autophagy is implicated in various diseases including neurodegeneration, its role in pancreatic β cells and glucose homeostasis has not been described. We produced mice with β cell-specific deletion of Atg7 (autophagy-related 7). Atg7 mutant mice showed impaired glucose tolerance and decreased serum insulin level. β cell mass and pancreatic insulin content were reduced because of increased apoptosis and decreased proliferation of β cells. Physiological studies showed reduced basal and glucose-stimulated insulin secretion and impaired glucose-induced cytosolic Ca2+ transients in autophagy-deficient β cells. Morphologic analysis revealed accumulation of ubiquitinated protein aggregates colocalized with p62, which was accompanied by mitochondrial swelling, endoplasmic reticulum distension, and vacuolar changes in β cells. These results suggest that autophagy is necessary to maintain structure, mass and function of pancreatic β cells, and its impairment causes insulin deficiency and hyperglycemia because of abnormal turnover and function of cellular organelles