Hepatitis C virus p7: molecular function and importance in hepatitis C virus life cycle and potential antiviral target

p7, a 63‐residue peptide encoded by hepatitis C virus (HCV), a major pathogen associated with a risk of developing severe liver disease, is involved in ion channel activity in lipid bilayer membranes both in in vitro and cell‐based assays. p7 protein consists of two transmembrane α‐helices, TM1 and TM2 connected by a loop oriented towards the cytoplasm. HCV relies on p7 function in addition to ion channel formation for efficient assembly, release and production of infectious progeny virions from liver cells. p7 activity is strictly sequence specific as mutation analysis showed the loss of ion channel function. Moreover, p7 ion channel activity can be specifically inhibited by different drugs suggesting the protein as a new target for future antiviral chemotherapy. In the present review, we focused to bring together the recent development to explore the potential role of p7 protein in HCV infection and its inhibition as a therapy.     

Hypoxic-preconditioning induces neuroprotection against hypoxia-ischemia in newborn piglet brain

Preconditioning-induced ischemic tolerance has been documented in the newborn brain, however, the signaling mechanisms of this preconditioning require further elucidation. The aims of this study were to develop a hypoxic-preconditioning (PC) model of ischemic tolerance in the newborn piglet, which emulates important clinical similarities to human situation of birth asphyxia, and to characterize some of the molecular mechanisms shown to be implicated in PC-induced neuroprotection in rodent models. One day old piglets were subjected to PC (8% O2/92% N2) for 3 h and 24 h later were exposed to hypoxia-ischemia (HI) produced by a combination of hypoxia (5% FiO2) for a period of 30 min and ischemia induced by a period of hypotension (10 min of reduced mean arterial blood pressure; ≤70% of baseline). Neuropathologic analysis and unbiased stereology, conducted at 24 h, 3 and 7 days of recovery following HI, indicated a substantial reduction in the severity of brain damage in PC piglets compared to non-PC piglets (P<0.05). PC significantly increased the mRNA expression of hypoxia-inducible factor-1α (HIF-1α) and its target gene, vascular endothelial growth factor (VEGF) at 0 h, 6h, 24 h, 3 and 7 days of recovery. Immunoblot analysis demonstrated that PC resulted in HIF-1α protein stabilization and accumulation in nuclear extracts of cerebral cortex of newborn piglet brain compared to normoxic controls. Protein levels of VEGF increased in a time-dependent manner in both cortex and hippocampus following PC. Double-immunolabeling indicated that VEGF is mainly expressed in neurons, endothelial cells and astroglia. Our study demonstrates for the first time the protective efficacy of PC against hypoxic-ischemic injury in newborn piglet model, which recapitulates many pathophysiological features of asphyxiated human neonates. Furthermore, as has been shown in rodent models of preconditioning, our results suggest that PC-induced protection in neonatal piglets may involve upregulation of VEGF.     

Erratum to: Bicarbonate therapy in the treatment of septic shock: a second look

The use of supplemental sodium bicarbonate for the treatment of patients with septic shock and elevated blood lactate levels remains a controversial therapy. We conducted a retrospective study between March 2004 and February 2009 of 36 consecutive patients diagnosed with septic shock who received continuous infusion of bicarbonate therapy. A control group was matched 1:1 for age, site of infection, and predicted mortality by APACHE II. All patients were managed according to standard protocols. The median time until reversal of shock did not achieve statistical significance between the bicarbonate group (44.5 h [95% confidence interval [CI] 34–54] and the control group (55.0 h [95% CI 39–60] (p = 0.09). The median time to liberation of mechanical ventilation was significantly reduced in the bicarbonate group (10 days [95% CI 5.0–13.0] compared to the control group (14 days [95% CI 9.0–19.0], p = 0.02). The length of intensive care unit (ICU) stay was also shorter in the surviving patients who received bicarbonate compared to controls (median 11.5 days (95% CI 6.0–16.0) vs. 16.0 days (95% CI 13.5–19.0), respectively; p = 0.01). However, there was no difference in 28-day mortality between the two study groups (28%; 95% CI 14–45% vs. 33%; 95% CI 19–51%, respectively; p = 0.79). Infusion of sodium bicarbonate in septic patients with arterial hyperlactatemia may facilitate weaning from mechanical ventilation and reduce length of ICU stay.     

Standardized analysis for the quantification of Vbeta CDR3 T-cell receptor diversity

Assessment of the diversity of the T-cell receptor (TCR) repertoire is often determined by measuring the frequency and distribution of individually rearranged TCRs in a population of T cells. Spectratyping is a common method used to measure TCR repertoire diversity, which examines genetic variation in the third complementarity-determining region (CDR3) region of the TCR Vbeta chain using RT-PCR length-distribution analysis. A variety of methods are currently used to analyze spectratype data including subjective visual measures, qualitative counting measures, and semi-quantitative measures that compare the original data to a standard, control data set. Two major limitations exist for most of these approaches: data files become very wieldy and difficult to manage, and current analytic methods generate data which are difficult to compare between laboratories and across different platforms. Here, we introduce a highly efficient method of analysis that is based upon a normal theoretical Gaussian distribution observed in cord blood and recent thymic emigrants. Using this analysis method, we demonstrate that PBMC obtained from patients with various diseases have skewed TCR repertoire profiles. Upon in vitro activation with anti-CD3 and anti-CD28 coated beads (Xcyte Dynabeads) TCR diversity was restored. Moreover, changes in the TCR repertoire were dynamic in vivo. We demonstrate that use of this streamlined method of analysis in concert with a flexible software package makes quantitative assessment of TCR repertoire diversity straightforward and reproducible, enabling reliable comparisons of diversity values between laboratories and over-time to further collaborative efforts. Analysis of TCR repertoire by such an approach may be valuable in the clinical setting, both for prognostic potential and measuring clinical responses to therapy.     

BRAF inactivation drives aneuploidy by deregulating CRAF

Aspartate-594 is the third most common BRAF residue mutated in human cancer. Mutants of this residue are kinase inactive, and the mechanism(s) by which they contribute to cancer has remained perplexing. Using a conditional knock-in mouse model, we show that the (D594A)Braf mutant does not drive tumor development per se but is able to induce aneuploidy in murine splenocytes and mouse embryonic fibroblasts and contributes to immortalization through the propagation of aneuploid cells. (D594A)Braf lacks kinase activity but induces the related gene product Craf as well as the mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK pathway. Here, we show that the aneuploid phenotype is dependent on Craf. Treatment with the MEK inhibitor U0126 did not attenuate the emergence of aneuploidy but prevented the growth of aneuploid cells. These results provide a previously unidentified link between Craf and chromosomal stability, with important implications for our understanding of the development of cancers with driver mutations that hyperactivate Craf.