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The influenza A virus RNA-dependent RNA polymerase complex consists in three subunits, PB2, PB1 and PA, that perform transcription and replication of the viral genome through very distinct mechanisms. Biochemical and structural studies have revealed that the polymerase can adopt multiple conformations and form oligomers. However so far it remained unclear whether the available oligomeric crystal structures represent a functional state of the polymerase. Here we gained new insights into this question, by investigating the incompatibility between non-cognate subunits of influenza polymerase brought together through genetic reassortment. We observed that a 7:1 reassortant virus whose PB2 segment derives from the A/WSN/33 (WSN) virus in an otherwise A/PR/8/34 (PR8) backbone is attenuated, despite a 97% identity between the PR8-PB2 and WSN-PB2 proteins. Independent serial passages led to the selection of phenotypic revertants bearing distinct second-site mutations on PA, PB1 and/or PB2. The constellation of mutations present on one revertant virus was studied extensively using reverse genetics and cell-based reconstitution of the viral polymerase. The PA-E349K mutation appeared to play a major role in correcting the initial defect in replication (cRNA -> vRNA) of the PR8xWSN-PB2 reassortant. Strikingly the PA-E349K mutation, and also the PB2-G74R and PB1-K577G mutations present on other revertants, are located at a dimerization interface of the polymerase. All three restore wild-type-like polymerase activity in a minigenome assay while decreasing the level of polymerase dimerization. Overall, our data show that the polymerase subunits co-evolve to ensure not only optimal inter-subunit interactions within the heterotrimer, but also proper levels of dimerization of the heterotrimer which appears to be essential for efficient viral RNA replication. Our findings point to influenza polymerase dimerization as a feature that is controlled by a complex interplay of genetic determinants, can restrict genetic reassortment, and could become a target for antiviral drug development. [ABSTRACT FROM AUTHOR]

Cathepsin L (CTSL) has been proved to help contain leishmaniasis and mycoplasma infection in mice by supporting cellular immune responses, but the regulatory functions of CTSL on mucosal immune responses haven’t been tested and remain undefined. Here, we investigated the effects of CTSL on SIgA responses and invariant chain (Ii) degradations in the co-cultured swine dendritic cells (DCs) and B cells system in vitro. When the cells system were transfected with vector CTSL-GFP or incubated with recombinant CTSL (rCTSL) before they were infected with Mycoplasma hyopneumoniae (M.hp), SIgA significantly increased and Ii chain was degraded into smaller intermediates, while SIgA decreased when CTSL was knockdown or inhibited with E64. To confirm the SIgA responses promoted by CTSL contribute to the resistance to mycoplasma pneumonia, pigs injected with rCTSL before they were challenged with M.hp, showed milder clinical symptoms and histopathological damage of lungs, less mycoplasma burden together with higher secretion of SIgA, percentages of CD4+ T cells and level of MHC II molecules comparing with the group without rCTSL. Collectively, these results suggested that rCTSL could provide effective protection for piglets against mycoplasma pneumonia by enhancing M.hp-specific mucosal immune responses through its role in antigen presentation by processing the invariant chain. [ABSTRACT FROM AUTHOR]

Proteome analysis of serum from type 2 diabetics with complications may lead to the discovery of diagnostic or prognostic biomarkers. To circumvent the principal barrier of serum proteomics, our investigation aimed to evaluate whether a study of post-translational modification enriched serum proteins could be valuable for the discovery of biomarkers or metabolic pathways related to type 2 diabetes pathogenesis. Type 2 diabetes was induced from high-fat diet fed Sprague Dawley rats with streptozotocin injection. Once diabetic status was confirmed, serum samples from either fasted healthy or diabetic rats were pooled and profiled by two-dimensional difference gel electrophoresis or comparative 2D electrophoresis after protein enrichments using immobilized metal ion, concanavalin A, and lentil affinity chromatography, respectively. Differential expressed proteins were identified and the associated networks were established by an Ingenuity Pathway Analysis. As a result, induced rats became severe diabetic and accompanied by hyperlipidemia, fatty liver, and glomerular hypertrophy. There were 3 total, 14 phosphorylated and 23 glycosylated protein targets differentially expressed. Proteins could be linked to HNF4A, HNF1A, and NFκB transcriptional factors and antigen presentation, humoral immune response, and inflammatory response pathways. Predicted organ toxicity in kidney, heart, and liver matched with our histopathological results. In conclusion, post-translational modification based serum protein enrichment could be a valuable approach to enhance the resolution of serum proteomics without depleting potentially valuable abundant proteins. Our results also indicated the potential association of the hepatic secretome and hepatocyte nuclear factors in the pathogenesis of type 2 diabetes and its complications. [ABSTRACT FROM AUTHOR]

Object: Traumatic intracranial hemorrhage (TICH) patients with acute kidney injury (AKI) were reported to have a high mortality rate. Renal replacement therapy (RRT) is indicated for patients with a severe kidney injury. This study aimed to compare the effects of different RRT modalities regarding chronic dialysis rate among adult TICH patients with AKI. Methods: A retrospective search of computerized hospital records from 2000 to 2010 for patients with a discharge diagnosis of TICH was conducted to identify the index cases. We collected the data of TICH patients with increased intracranial pressure combined with severe AKI who received intermittent hemodialysis (IHD) or continuous veno-venous hemofiltration (CVVH) as RRT. The outcome was dialysis dependence between 2000 and 2010. Results: From a total of 310 patients who were enrolled in the study, 134 (43%) received CVVH and 176 (57%) received IHD. The risk of dialysis dependency was significantly lower in the CVVH group than in the IHD group (adjusted hazard ratio: 0.368, 95% CI, 0.158–0.858, P = 0.034). Diabetes mellitus and coronary artery disease were risk factors for dialysis dependency. CVVH compared with IHD modality was associated with lower dialysis dependency rate in TICH patients combined with AKI and diabetes mellitus and those with an injury severity score (ISS) ≥16. Conclusion: CVVH may yield better renal outcomes than IHD among TICH patients with AKI, especially those with diabetes mellitus and an ISS ≥16. The beneficial impact of CVVH on TICH patients needs to be clarified in a large cohort study in future. [ABSTRACT FROM AUTHOR]

Background: We evaluated the risk of osteoporosis in patients with primary biliary cirrhosis (PBC) using a nationwide population-based dataset. Methods: In a cohort study of 986,713 individuals, we selected 2,493 PBC patients who were aged 18 years or older and had been diagnosed with PBC, based on the International Classification of Disease (ICD-9-CM) codes 571.6, during 20002010. The control cohort comprised 9,972 randomly selected, propensity matched patients (by age, gender, and index date), without PBC. Using this adjusted data, a possible association between PBC and the risk of developing osteoporosis was estimated using a Cox proportional hazard regression model. Results: During the follow-up period, osteoporosis was diagnosed in 150 (6.02%) patients in the PBC cohort and in 539 (5.41%) patients in the non-PBC cohort. After adjusting for covariates, osteoporosis risk was found to be 3.333 times greater in the PBC cohort than in the non-PBC cohort when measured over 6 years after PBC diagnosis. Stratification revealed that the use of ursodeoxycholic acid (UDCA) had no significance in decreasing the risk of osteoporosis when comparing the PBC cohorts with the non-PBC cohorts (P = 0.124). Additionally, osteoporosis risk was significantly higher in PBC patients with steroid use (aHR: 6.899 vs 3.333). Moreover, when comparing the PBC cohorts to the non-PBC cohorts, the non-cirrhotic patients were prone to osteoporosis at a younger age compared to those in the cirrhotic cohorts. We also found that the associated risk of fractures is only prominent for vertebral and wrist fractures in the PBC cohort compared to that in the non-PBC cohort. Conclusion: A significant association exists between PBC and subsequent risk for osteoporosis. Therefore, PBC patients, particularly those treated with steroids, should be evaluated for subsequent risk of osteoporosis. [ABSTRACT FROM AUTHOR]

Background: Hypertriglyceridemia is the third most common cause of acute pancreatitis, but whether the level of triglyceride (TG) is related to severity of pancreatitis is unclear. Aim: To evaluate the effect of TG level on the severity of hypertriglyceridemic pancreatitis (HTGP). Design: Retrospective cohort study. Methods: We reviewed the records of 144 patients with HTGP from 1999 to 2013 at Tri-Service General Hospital. Patients with possible etiology of pancreatitis, such as gallstones, those consuming alcohol or drugs, or those with infections were excluded. The classification of severity of pancreatitis was based on the revised Atlanta classification. We allocated the patients into high-TG and low-TG groups based on the optimal cut-off value (2648 mg/dL), which was derived from the receiver operating characteristic (ROC) curve between TG level and severity of HTGP. We then compared the clinical characteristics, pancreatitis severity, and mortality rates of the groups. Results: There were 66 patients in the low-TG group and 78 patients in the high-TG group. There was no significant difference in the age, sex ratio, body mass index, and comorbidity between the 2 groups. The high-TG group had significantly higher levels of glucose (P = 0.022), total cholesterol (P = 0.002), and blood urea nitrogen (P = 0.037), and lower levels of sodium (P = 0.003) and bicarbonate (P = 0.002) than the low-TG group. The incidences of local complication (P = 0.002) and severe and moderate form of pancreatitis (P = 0.004) were significantly higher in the high-TG group than in the low-TG group. The mortality rate was higher in the high-TG group than in the low-TG group (P = 0.07). Conclusions: Higher TG level in patients with HTGP may be associated with adverse prognosis, but randomized and prospective studies are needed in the future verify this relationship. [ABSTRACT FROM AUTHOR]

Background and Purpose: Timely intravenous (IV) thrombolysis for acute ischemic stroke is associated with better clinical outcomes. Acute stroke care implemented with “Stroke Code” (SC) may increase IV tissue plasminogen activator (tPA) administration. The present study aimed to investigate the impact of SC on thrombolysis. Methods: The study period was divided into the “pre-SC era” (January 2006 to July 2010) and “SC era” (August 2010 to July 2013). Demographics, critical times (stroke symptom onset, presentation to the emergency department, neuroimaging, thrombolysis), stroke severity, and clinical outcomes were recorded and compared between the two eras. Results: During the study period, 5957 patients with acute ischemic stroke were admitted; of these, 1301 (21.8%) arrived at the emergency department within 3 h of stroke onset and 307 (5.2%) received IV-tPA. The number and frequency of IV-tPA treatments for patients with an onset-to-door time of <3 h increased from the pre-SC era (n = 91, 13.9%) to the SC era (n = 216, 33.3%) (P<0.001). SC also improved the efficiency of IV-tPA administration; the median door-to-needle time decreased (88 to 51 min, P<0.001) and the percentage of door-to-needle times ≤60 min increased (14.3% to 71.3%, P<0.001). The SC era group tended to have more patients with good outcome (modified Rankin Scale ≤2) at discharge (49.5 vs. 39.6%, P = 0.11), with no difference in symptomatic hemorrhage events or in-hospital mortality. Conclusion: The SC protocol increases the percentage of acute ischemic stroke patients receiving IV-tPA and decreases door-to-needle time. [ABSTRACT FROM AUTHOR]