Altered expression of cell cycle and apoptotic proteins in chronic hepatitis C virus infection.

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.BACKGROUND: A disrupted cell cycle progression of hepatocytes was reported in chronic hepatitis C virus (HCV) infection, which can contribute significantly in the associated pathogenesis. The present study aimed to further elaborate these disruptions by evaluating the expression of key cell cycle and apoptotic proteins in chronic HCV infection with particular reference to genotype 3. Archival liver biopsy specimens of chronic HCV-infection (n = 46) and normal histology (n = 5) were analyzed by immunohistochemistry using antibodies against proliferation marker Mcm-2, G1 phase marker Cyclin D1, S phase marker Cyclin A, cell cycle regulators p21 (CDK inhibitor) and p53 (tumor suppressor protein), apoptotic protein Caspase-3 and anti-apoptotic protein Bcl-2. RESULTS: Elevated Mcm-2 expression was observed in hepatocytes in chronic HCV infection, indicating increased cell cycle entry. Cyclin D1 expression was higher than cyclin A, which suggests a slow progression through the G1 phase. Expression of cell cycle regulators p21 and p53 was elevated, with no concordance between their expressions. The Mcm-2 and p21 expressions were associated with the fibrosis stage (p = 0.0001 and 0.001 respectively) and that of p53 with the inflammation grade (p = 0.051). Apoptotic marker, Caspase-3, was mostly confined to sinusoidal lining cells with little expression in hepatocytes. Anti-apoptotic protein, Bcl-2, was negligible in hepatocytes and detected principally in infiltrating lymphocytes. Expression of all these proteins was unrelated to the HCV genotype and were detected only rarely in the hepatocytes of normal liver. CONCLUSION: The results showed an arrested cell cycle state in the hepatocytes of chronic HCV infection, regardless of any association with genotype 3. Cell cycle arrest is characterized by an increased expression of p21, in relation to fibrosis, and of p53 in relation to inflammation. Furthermore, expression of p21 was independent of the p53 expression and coincided with the reduced expression of apoptotic protein Caspase-3 in hepatocytes. The altered expression of these cell cycle proteins in hepatocytes is suggestive of an impaired cell cycle progression that could limit the regenerative response of the liver to ongoing injury, leading to the progression of disease

Rapid, Precise and Affordable Estimation of Venlafaxine and Its Metabolites in Highly Polluted Effluent Waters: Proof-of-Concept for Methodology

Widespread presence of pharmaceuticals and their metabolites in the environment of industrialized countries is an emerging global concern. Potential contamination of the soil and water by such pharmacologically active substances poses serious ecotoxicological implications. Several studies assessing the long-term ecological risks of pharmaceutical contaminants mainly focus on the risk assessment of the parent drug, while the potential contributions of their metabolites is often neglected. Presence of selective serotonin and norepinephrine reuptake inhibitor venlafaxine, an antidepressant drug, and its metabolites is a matter of serious concern for aquatic systems, since they are difficult to remove by traditional wastewater treatment processes. The concentration of VEN present in water is reportedly one of the highest among pharmaceuticals; however, the long-term effects of its metabolites have not yet been systematically studied. Given the consideration to complex and time-consuming effluent treatment, and realizing the importance of levels of venlafaxine and its metabolites, a simple and accurate analytical method for quick determination is needed. We designed a selective colorimetric method by using oxidative coupling of drug molecules with 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) reagent, to quantify the presence of venlafaxine and its metabolites in aquatic samples, with special emphasis on effluent. The method was validated for selectivity, specificity and robustness as per the ICH Q2 guidelines to assess its suitability in pharmaceutical samples, as well. Highly sensitive and green economical analytical method was successfully established for estimation of venlafaxine and its metabolites in aquatic samples. The method was quick, as it involved minimum processing steps. The method was accurate and linear in the range of 0.5 to 80 ppm and could successfully detect lowest concentration of 1.3 ppm, thus qualifying its applicability for the desired purpose to check the presence of trace levels of VEN or its metabolites in aquatic samples or in pharmaceutical formulations

Severe Acute Respiratory Syndrome Coronavirus Protein 6 Accelerates Murine Hepatitis Virus Infections by More than One Mechanism▿

The severe acute respiratory syndrome coronavirus (SARS-CoV) encodes numerous accessory proteins whose importance in the natural infection process is currently unclear. One of these accessory proteins is set apart by its function in the context of a related murine hepatitis virus (MHV) infection. SARS-CoV protein 6 increases MHV neurovirulence and accelerates MHV infection kinetics in tissue culture. Protein 6 also blocks nuclear import of macromolecules from the cytoplasm, a process known to involve its C-terminal residues interacting with cellular importins. In this study, protein 6 was expressed from plasmid DNAs and accumulated in cells prior to infection by wild-type MHV. Output of MHV progeny was significantly increased by preexisting protein 6. Protein 6 with C-terminal deletion mutations no longer interfered with nuclear import processes but still retained much of the capacity to augment MHV infections. However, some virus growth-enhancing activity could be ascribed to the C-terminal end of protein 6. To determine whether this augmentation provided by the C terminus was derived from interference with nuclear import, we evaluated the virus-modulating effects of small interfering RNAs (siRNAs) directed against importin-β mRNAs, which down-regulated classical nuclear import pathways. The siRNAs did indeed prime cells for enhanced MHV infection. Our findings indicated that protein 6-mediated nuclear import blocks augmented MHV infections but is clearly not the only way that this accessory protein operates to create a milieu conducive to robust virus growth. Thus, the SARS-CoV protein 6 accelerates MHV infections by more than one mechanism

A detailed analysis of synonymous codon usage in human bocavirus

AbstractHuman bocavirus (HBoV) is a recently discovered parvovirus associated with respiratory and gastroenteric infections inchildren. To date, four distinct subtypes have been identified worldwide. HBoV1 is the most frequently detected bocavirusin clinical samples derived from the respiratory tract. HBoV has a single-stranded DNA genome, which encodes two nonstructuralproteins, NS1 and NP1, and two structural proteins, VP1 and VP2. Despite a large number of available HBoVsequences, the molecular evolution of this virus remains enigmatic. Here, we applied bioinformatic methods to measure thecodon usage bias in 156 HBoV genomes and analyzed the factors responsible for preferential use of various synonymouscodons. The effective number of codons (ENC) indicates a highly conserved, gene-specific codon usage bias in the HBoVgenome. The structural genes exhibit a higher degree of codon usage bias than the non-structural genes. Natural selectionemerged as dominant factor influencing the codon usage bias in the HBoV genome. Other factors that influence the codonusage include mutational pressure, gene length, protein properties, and the relative abundance of dinucleotides. The resultspresented in this study provide important insight into the molecular evolution of HBoV and may serve as a primer for HBoVgene expression studies and development of safe and effective vaccines to prevent infection

Pancreatin-Cetyl Pyridinium Chloride Digestion and Decontamination Method; A Novel, Sensitive, Cost-Effective Method for Culturing Mycobacterium tuberculosis

The present study evaluated the performance of newly developed pancreatin-cetylpyridinium chloride (pancreatin-CPC) digestion and decontamination method (DDM) with N-acetyl L-Cysteine-sodium hydroxide (NALC-NaOH) DDM for isolation of Mycobacteria from clinically suspected pulmonary tuberculosis (PTB) patients. For the study, sputum samples (n = 613) obtained from clinically suspected PTB cases were subjected to direct microscopy, pretreatment with NALC-NaOH DDM (reference method), and pancreatin-CPC DDM followed by culture, and the data were analyzed. The direct microscopy illustrated diagnostic accuracies of 60.4% (sensitivity), 99.77% (specificity), 98.9% (positive predictive value) and 88.3% (negative predictive value), respectively (against culture) for the detection of Mycobacterial species. The pancreatin-CPC DDM showed competitive diagnostic accuracies (against NALC-NaOH DDM) of 99.32% (sensitivity), 94.07% (specificity), 85.05% (positive predictive value), and 99.76% (negative predictive value), respectively, for the isolation of Mycobacterial species. In conclusion, pancreatin-CPC DMM was a highly sensitive, technically simple, and cost-effective method, suggesting its competence to substitute the currently used NALC-NaOH DDM

New hepatitis C virus drug discovery strategies and model systems

INTRODUCTION: Hepatitis C virus is a major cause of liver disease worldwide and the leading indication for liver transplantation in the United States. Current treatment options are expensive, not effective in all patients and are associated with serious side effects. While pre-clinical anti-HCV drug screening is still hampered by the lack of readily infectable small animal models, the development of cell culture HCV experimental model systems has driven a promising new wave of HCV antiviral drug discovery. AREAS COVERED: This review contains a concise overview of current HCV treatment options and limitations with a subsequent in-depth focus on the available experimental models and novel strategies that have and continue to enable important advances in HCV drug development. EXPERT OPINION: With a large cohort of chronically HCV infected patients progressively developing liver disease that puts them at risk for hepatocellular carcinoma and hepatic decompensation, there is an urgent need to develop effective therapeutics that are well-tolerated and effective in all patients and against all HCV genotypes. Significant advances in HCV experimental model development have expedited drug discovery; however, additional progress is needed. Importantly, the current trends and momentum in the field suggests that we will continue to overcome critical experimental challenges to reach this end goal

Altered expression of cell cycle and apoptotic proteins in chronic hepatitis C virus infection-1

and 4 (F4) (Mayer hematoxylin, magnification 400×).<p><b>Copyright information:</b></p><p>Taken from "Altered expression of cell cycle and apoptotic proteins in chronic hepatitis C virus infection"</p><p>http://www.biomedcentral.com/1471-2180/8/133</p><p>BMC Microbiology 2008;8():133-133.</p><p>Published online 5 Aug 2008</p><p>PMCID:PMC2518161.</p><p></p

Specific inhibition of HIV-1 replication by short hairpin RNAs targeting human cyclin T1 without inducing apoptosis

AbstractRNA interference (RNAi), a sequence-specific RNA degradation mechanism mediated by small interfering RNA (siRNA), can be used not only as a research tool but also as a therapeutic strategy for viral infection. We demonstrated that intracellular expression of short hairpin RNA (shRNA) targeting human cyclin T1 (hCycT1), a cellular factor essential for transcription of messenger and genomic RNAs from the long terminal repeat promoter of provirus of human immunodeficiency virus type 1 (HIV-1), could effectively suppress the replication of HIV-1. We also showed that downregulation of hCycT1 did not cause apoptotic cell death, therefore, targeting cellular factor hCycT1 by shRNAs may provide an attractive approach for genetic therapy of HIV-1 infection in the future

Altered expression of cell cycle and apoptotic proteins in chronic hepatitis C virus infection-4

<p><b>Copyright information:</b></p><p>Taken from "Altered expression of cell cycle and apoptotic proteins in chronic hepatitis C virus infection"</p><p>http://www.biomedcentral.com/1471-2180/8/133</p><p>BMC Microbiology 2008;8():133-133.</p><p>Published online 5 Aug 2008</p><p>PMCID:PMC2518161.</p><p></p