Expression in animal cells and characterization of the hepatitis E virus structural proteins

Hepatitis E virus (HEV) is a major human pathogen in much of the developing world. It is a positive-strand RNA virus with a 7.5-kb polyadenylated genome consisting of three open reading frames (ORFs). In the absence of an in vitro culture system, the replication and expression strategy of HEV and the nature of its encoded polypeptides are not well understood. We have expressed the two ORFs constituting the structural portion of the HEV genome in COS-1 cells by using simian virus 40-based expression vectors and in vitro by using a coupled transcription-translation system. We show here that the major capsid protein, encoded by ORF2, is an 88-kDa glycoprotein which is expressed intracellularly as well as on the cell surface and has the potential to form noncovalent homodimers. It is synthesized as a precursor (ppORF2) which is processed through signal sequence cleavage into the mature protein (pORF2), which is then glycosylated (gpORF2). The minor protein, pORF3, encoded by ORF3 is a 13.5-kDa nonglycosylated protein expressed intracellularly and does not show any major processing. pORF3 interacts with a cellular protein of about 18 kDa which we call 3IP, the pORF3-interacting protein. The significance of these findings are discussed in light of an existing model of HEV genome replication and expression

Rescue of a genotype 4 human hepatitis E virus from cloned cDNA and characterization of intergenotypic chimeric viruses in cultured human liver cells and in pigs

Hepatitis E virus (HEV) is an important but extremely understudied human pathogen. Genotypes 1 and 2 are restricted to humans, whereas genotypes 3 and 4 are zoonotic, infecting both humans and pigs. This report describes, for the first time, the successful rescue of infectious HEV in vitro and in vivo from cloned cDNA of a genotype 4 human HEV (strain TW6196E). The complete genomic sequence of the TW6196E virus was determined and a full-length cDNA clone (pHEV-4TW) was assembled. Capped RNA transcripts from the pHEV-4TW clone were replication competent in Huh7 cells and infectious in HepG2/C3A cells. Pigs inoculated intrahepatically with capped RNA transcripts from pHEV-4TW developed an active infection, as evidenced by faecal virus shedding and seroconversion, indicating the successful rescue of infectious genotype 4 HEV and cross-species infection of pigs by a genotype 4 human HEV. To demonstrate the utility of the genotype 4 HEV infectious clone and to evaluate the potential viral determinant(s) for species tropism, four intergenotypic chimeric clones were constructed by swapping various genomic regions between genotypes 1 and 4, and genotypes 1 and 3. All four chimeric clones were replication competent in Huh7 cells, but only the two chimeras with sequences swapped between genotypes 1 and 4 human HEVs produced viruses capable of infecting HepG2/C3A cells. None of the four chimeras was able to establish a robust infection in pigs. The availability of a genotype 4 HEV infectious clone affords an opportunity to delineate the molecular mechanisms of HEV cross-species infection in the future

Cognitive and Functional Outcome among Hospitalized Intracerebral Haemorrhage Patients in West Java’s Top Referral Hospital

Background: Stroke is the highest cause of disability in adults. Disability and cognitive function impairment cause dependency and decreasing quality of life. The objectives of this study was to describe the outcome of functional and cognitive function among intracerebral haemorrhage patients admitted to Dr. Hasan Sadikin General Hospital Bandung in 2013. Methods: This study was a quantitative descriptive study, conducted from October 2016 to August 2017. Data were collected retrospectively with total sampling method from medical records of intracerebral haemorrhage patients admitted to Dr. Hasan Sadikin General Hospital in 2013 assessed with the Montreal Cognitive Assessment-Indonesian Version (MoCA-INA) and Barthel Index. Patients with incomplete medical records , severe aphasia, severe sensoric-motoric impairment, and decreased consciousness were excluded in this study. Data were analyzed using Microsoft Office Excel 2010 and presented in percentage. Results: Out of the 26 subjects, 11 were men and 15 women , who were in the age range of 45–54 years (42.3%), with low education level (61.5%), and unemployed (61.5%) had the highest prevalence on subjects studied. Hypertension was the most common risk factor (78%). Most subjects had cognitive function impairment (69.2%) with delayed memory function as the most common impairment domain found (84.6%), followed by visuospatial/executive function (69.2%). Most subjects had a high score of dependence according to the Barthel test (61.5%). Conclusions: Two third of intracerebral haemorrhage patients have cognitive function impairment and functional dependence. Memory function is the most common impaired cognitive functional domain

Assessment of Intratumoral Heterogeneity in Isolated Human Primary High-Grade Glioma: Cluster of Differentiation 133 and Cluster of Differentiation 15 Double Staining of Glioblastoma Subpopulations

BACKGROUND: Gliomas are the most common primary brain tumors, representing 50–60% of malignant primary brain tumors. Gliomas are highly heterogeneous with marked inter- and intratumoral diversity. Gliomas heterogeneity is a challenging issue in the development of personalized treatment. The simplest method for studying heterogeneity is using ex vivo cell cultures; in our case, the cell lines were isolated from patient with glioblastomas. AIM: Here, we reported distinct cell subpopulations heterogeneity in glioblastoma cells. METHODS: Human glioblastoma cells isolation is conducted by enzymatic method with combination of collagenase I, hyaluronidase, and trypsin enzyme in proportional amount from patient. Immunostaining was performed to assess glial fibrillary acidic protein (GFAP), Ki-67, isocitrate dehydrogenase-1 (IDH-1) status, and program death ligand-1 (PD-L1) expression. Primary glioblastoma cell line was characterized by flow cytometry (fluorescence-activated cell sorting) analysis based on cluster of differentiation (CD) 133 and CD15 marker expression. U87MG and CGNH-89 cell lines were used as control. Distinct subpopulation analysis was performed by double staining of CD133 and CD15 in isolated primary glioblastoma cell line and its comparative control cells. RESULTS: Our isolated glioblastoma cells morphology was adherent cells which were able to form spheres depending on environment. Immunostaining confirmed GFAP, Ki-67, IDH-1 mutants, and PD-L1 expression. Our isolated glioblastoma cells expressed CD133 and CD15, coexpressed CD133/CD15 in different patterns. The highest subpopulation in primary glioblastoma was CD133+/CD15+. CONCLUSION: Glioblastoma cells can be isolated using enzymatic methods. Isolated glioblastoma cells consist of four different subpopulations distinguished by CD133/CD15 double staining. Intratumoral heterogeneity exists and directly or indirectly depends on their microenvironment

SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

BACKGROUND We have previously identified kinase suppressor of ras-1 (KSR1) as a potential regulatory gene in breast cancer. KSR1, originally described as a novel protein kinase, has a role in activation of mitogen-activated protein kinases. Emerging evidence has shown that KSR1 may have dual functions as an active kinase as well as a scaffold facilitating multiprotein complex assembly. Although efforts have been made to study the role of KSR1 in certain tumour types, its involvement in breast cancer remains unknown. METHODS A quantitative mass spectrometry analysis using stable isotope labelling of amino acids in cell culture (SILAC) was implemented to identify KSR1-regulated phosphoproteins in breast cancer. In vitro luciferase assays, co-immunoprecipitation as well as western blotting experiments were performed to further study the function of KSR1 in breast cancer. RESULTS Of significance, proteomic analysis reveals that KSR1 overexpression decreases deleted in breast cancer-1 (DBC1) phosphorylation. Furthermore, we show that KSR1 decreases the transcriptional activity of p53 by reducing the phosphorylation of DBC1, which leads to a reduced interaction of DBC1 with sirtuin-1 (SIRT1); this in turn enables SIRT1 to deacetylate p53. CONCLUSION Our findings integrate KSR1 into a network involving DBC1 and SIRT1, which results in the regulation of p53 acetylation and its transcriptional activity

Cardiovascular Outcomes of Transradial Versus Transfemoral Percutaneous Coronary Intervention in End-Stage Renal Disease: A Regression-Based Comparison

Background: Limited data is available on the comparison of outcomes of transradial (TR) and transfemoral (TF) access for percutaneous coronary intervention (PCI) in patients with end-stage stage renal disease (ESRD). Methods: Online databases were queried to compare cardiovascular outcomes among TR. and TF in ESRD patients. The outcomes assessed included differences in mortality, cerebrovascular accidents (CVA), periprocedural myocardial infarction (MI), bleeding, transfusion, and periprocedural cardiogenic shock (CS). Unadjusted odds ratios (OR) were calculated using a random-effect effect model. Results: A total of 6 studies including 7,607 patients (TR-PCI = 1,288; TF-PCI = 6,319) were included. The overall mean age was 67.7 years, while the mean age for TR-PCI and TF-PCI was 69.7 years and 67.9 years, respectively. TR-PCI was associated with lower incidence of mortality (OR 0.46 95 % CI 0.30-0.70, p \u3c 0.05, I2 0.00 %), bleeding (OR 0.45 95 % CI 0.29, 0.68, p \u3c 0.05, I2 3.48 %), and transfusion requirement (OR 0.52 95 % CI 0.40, 0.67, p \u3c 0.05, I2 0.00 %) (Fig. 1). There were no differences among TR-PCI and TF-PCI for periprocedural MI, periprocedural CS, and CVA outcomes. Conclusion: TR access was associated with lower mortality, bleeding, and transfusion requirement as compared to TF access in patients with ESRD undergoing PCI

Prevalence of Hepatitis E Virus in Swine Fed on Kitchen Residue

The aim of this study was to investigate the prevalence of swine hepatitis E virus (HEV) in pigs fed different feedstuffs (kitchen residue or mixed feeds) and genetic identification of HEV isolated in Hebei province, China. Serum and fecal samples were collected from adult swine. Anti-HEV antibody was evaluated by double sandwich antigen enzyme immunoassay. HEV RNA was extracted from fecal samples and amplified by nested RT-PCR. The reaction products were sequenced, and the sequence analyzed. Virus-like particles were distinguishable by negative staining in the electron microscope. Histopathological observation and immunohistochemical localization were used in the animal models. Overall, the anti-HEV positive percentage of serum samples from pigs fed on kitchen residue was 87.10% (27/31), and 53.06% (130/245) from pigs fed on complete feed. The HEV RNA positivity rate of fecal samples from pigs fed on kitchen residue was 61.54% (8/13), but zero for pigs fed on complete feed. Sequence analysis of these eight samples and comparison with the published sequence showed that there were eight groups that belonged to genotype 4 d and the nucleotide identity was 95.6–99.3%. swHE11 is most closely related to strain CCC220, and the other seven HEV isolates were most closely related to strains swGX40, SwCH189 and V0008ORF3, which are isolates from human and pigs. Histopathological observation showed that there was liver damage in the experimental group, and immunohistochemistry indicated that the HEV antigens were strongly positive at 7 days after infection. The results demonstrated that the prevalence of HEV in pigs fed on kitchen residue was higher than in those fed on complete feed (P<0.05)

Analysis of the Functional Independence Measure Value of Cervical Spine Injury Patients with Conservative Management

Cervical spine injury is one of the most common spinal cord injuries in trauma patients. From 100,000 spinal cord injury cases reported in&nbsp; the United States of America (2008), sixty seven percent involve cervical spine injury. American Spinal Cord Injury Association (ASIA) impairment score is used as an initial assessment but not enough attention prognostic outcome of these patients was paid to. The objective of this study is to analyze the value of functional independence measure (FIM) cervical spine injury patients with conservative management and its correlation with age, sex, type of trauma, onset of trauma, cervical abnormalities, type of cervical spine lesion and ASIA impairment score. A prospective cohort study was performed to all patients with cervical spine injury treated in Neurosurgery Department of Dr. Hasan Sadikin Hospital Bandung that fullfiled the inclusion criteria. The subjects were classified based on age, sex, single/multiple trauma, acute/chronic, cervical abnormalities, complete/incomplete lesion and ASIA impairment score. The FIM examination was performed in Outpatient clinic of Neurosurgery. T-test and chi-square test was done to analyze the data. There were 17&nbsp; cervical spine injury patients treated in Neurosurgery Department of Dr. Hasan Sadikin Hospital during April 2009&ndash;April 2010. The average FIM value of cervical spine injury in those patients is 4+1.63 by cohort prospective study. There were no correlation between FIM value with age, sex, type of trauma, onset of trauma and cervical abnormalities. Significant correlations were found between FIM value with type of cervical spine lesion and ASIA impairment score in cervical spine patients. Type of cervical spine lesion and ASIA impairment score have significant correlation with FIM value of patients in 6 months after cervical injury