Correction: Permissivity of Primary Human Hepatocytes and Different Hepatoma Cell Lines to Cell Culture Adapted Hepatitis C Virus.

[This corrects the article DOI: 10.1371/journal.pone.0070809.].

Signaling lymphocytic activation molecules Slam and cancers: friends or foes?

Signaling Lymphocytic Activation Molecules (SLAM) family receptors are initially described in immune cells. These receptors recruit both activating and inhibitory SH2 domain containing proteins through their Immunoreceptor Tyrosine based Switch Motifs (ITSMs). Accumulating evidence suggest that the members of this family are intimately involved in different physiological and pathophysiological events such as regulation of immune responses and entry pathways of certain viruses. Recently, other functions of SLAM, principally in the pathophysiology of neoplastic transformations have also been deciphered. These new findings may prompt SLAM to be considered as new tumor markers, diagnostic tools or potential therapeutic targets for controlling the tumor progression. In this review, we summarize the major observations describing the implications and features of SLAM in oncology and discuss the therapeutic potential attributed to these molecules.

Modeling of HBV and HCV hepatitis with hepatocyte-like cells.

Chronic liver diseases caused by either hepatitis B or C viruses are a major health problem around the world. Despite major advances accomplished in recent years in understanding the physiology of both viruses using in vitro and/or in vivomodels, there is no vaccine for HCV available. Moreover, susceptibility to acute and chronic infection and the response to treatments are different between HBV or HCV infected patients. Crucial information can be collected using a robust cell model that permits the culture of clinical isolates along with the investigation of the virus-host interaction. The recent progress in the field of cell reprogramming and differentiation has opened new opportunities in viral hepatitis research raising the hopes of developing new improved therapeutics. In this review, we discuss current models for hepatitis B and C studies and their limitations, and also the iPSC model, and its relevance to the viral host cell interactions.

Inactivation of viruses in platelet and plasma products using a riboflavin-and-UV-based photochemical treatment.

BACKGROUND: Multilayered blood safety programs reduce the risk of transfusion-transmitted diseases; however, there remains a risk of window period transmission of screened viruses and transmission of unscreened and emerging viruses from asymptomatic donors. To reduce this risk, a riboflavin-and-UV-light-based pathogen reduction process was evaluated against eight viral agents. STUDY DESIGN AND METHODS: Riboflavin and UV light was evaluated against the following eight viral agents: encephalomyocarditis virus (EMC), hepatitis A virus (HAV), hepatitis C virus (HCV), influenza A (FLUAV), La Crosse virus (LACV), pseudorabies virus (PRV), sindbis virus (SINV), and vesicular stomatitis virus (VSV). Before treatment, a sample was removed to determine the product's initial viral load. After treatment the product's viral load was reevaluated and the log reduction was calculated. RESULTS: Virus reduction after treatment with riboflavin and UV light is equivalent in platelet (PLT) and plasma units, as demonstrated by a 3.2-log reduction of EMC in plasma, PLTs, and PLT additive solution containing 35% plasma. Additionally, the following viral reductions values were observed: HAV 1.8 log, HCV at least 4.1 log, FLUAV at least 5.0 log, LACV at least 3.5 log, PRV 2.5 log, SINV 3.2 log, and VSV at least 6.3 log. CONCLUSIONS: The results observed in this study suggest that treating PLT and plasma products with a riboflavin-and-UV-light-based pathogen reduction process could potentially eliminate window period transmission of screened viruses and greatly reduce the risk of transfusion transmission of unscreened viruses.

microRNAs are dysregulated in the cerebral microvasculature of CKD mice.

Vascular calcification arises during chronic kidney disease (CKD), and increases the risk of cardiovascular mortality. In CKD, alterations of cerebral circulation were linked with an increase in ischemic strokes and behavioral troubles. Studying pathophysiological mechanisms of calcifications and detecting new biomarkers in the cerebral circulation is thus an important issue. microRNAs are small non-coding, single-stranded RNAs that regulate messenger RNAs at the post-transcriptional level. They are involved in numerous pathologies and represent new opportunities to develop disease predictors. We used RT-qPCR to quantify endothelial-specific microRNAs in cerebral arterioles from WT mice and from pathological models of CKD. We used four mice groups: WT SHAM, WT CKD, Apolipoprotein E Knock-Out (ApoE-KO) SHAM, ApoE-KO CKD. Brains were removed after two and ten weeks of uremia and RNA from cerebral arterioles was extracted. miR-17 and miR-126 were the most dysregulated in the pathological conditions, at both the second week and tenth week of uremia. Our results suggest that miR-17 and miR-126 are potential new biomarkers of cerebral troubles of CKD patients and new therapeutic targets for innovative treatments.

Possible involvement of microRNAs in vascular damage in experimental chronic kidney disease.

Chronic kidney disease (CKD) is associated with vascular calcifications and atherosclerosis. There is a need for novel predictors to allow earlier diagnosis of these disorders, predict disease progression, and improve assessment of treatment response. We focused on microRNAs since they are implicated in a variety of cellular functions in cardiovascular pathology. We examined changes of microRNA expression in aortas of CKD and non-CKD wild type mice and apolipoprotein E knock-out mice, respectively. Both vascular smooth muscle-specific miR-143 and miR-145 expressions were decreased in states of atherosclerosis and/or CKD or both, and the expression level of protein target Myocardin was increased. The inflammatory miR-223 was increased in more advanced stages of CKD, and specific protein targets NFI-A and GLUT-4 were dramatically decreased. Expression of miR-126 was markedly increased and expression of protein targets VCAM-1 and SDF-1 was altered during the course of CKD. The drug sevelamer, commonly used in CKD, corrected partially these changes in microRNA expression, suggesting a direct link between the observed microRNA alterations and uremic vascular toxicity. Finally, miR-126, -143 and -223 expression levels were deregulated in murine serum during the course of experimental CKD. In conclusion, these miRNAs could have role(s) in CKD vascular remodeling and may therefore represent useful targets to prevent or treat complications of CKD.

Permissivity of primary human hepatocytes and different hepatoma cell lines to cell culture adapted hepatitis C virus.

Significant progress has been made in Hepatitis C virus (HCV) culture since the JFH1 strain cloning. However, developing efficient and physiologically relevant culture systems for all viral genotypes remains an important goal. In this work, we aimed at producing a high titer JFH1 derived virus to test different hepatic cells' permissivity. To this end, we performed successive infections and obtained a JFH1 derived virus reaching high titers. Six potential adaptive mutations were identified (I599V in E2, R1373Q and M1611T in NS3, S2364P and C2441S in NS5A and R2523K in NS5B) and the effect of these mutations on HCV replication and infectious particle production was investigated. This cell culture adapted virus enabled us to efficiently infect primary human hepatocytes, as demonstrated using the RFP-NLS-IPS reporter protein and intracellular HCV RNA quantification. However, the induction of a strong type III interferon response in these cells was responsible for HCV inhibition. The disruption of this innate immune response led to a strong infection enhancement and permitted the detection of viral protein expression by western blotting as well as progeny virus production. This cell culture adapted virus also enabled us to easily compare the permissivity of seven hepatoma cell lines. In particular, we demonstrated that HuH-7, HepG2-CD81, PLC/PRF/5 and Hep3B cells were permissive to HCV entry, replication and secretion even if the efficiency was very low in PLC/PRF/5 and Hep3B cells. In contrast, we did not observe any infection of SNU-182, SNU-398 and SNU-449 hepatoma cells. Using iodixanol density gradients, we also demonstrated that the density profiles of HCV particles produced by PLC/PRF/5 and Hep3B cells were different from that of HuH-7 and HepG2-CD81 derived virions. These results will help the development of a physiologically relevant culture system for HCV patient isolates.

MicroRNAs and hepatitis C virus: toward the end of miR-122 supremacy.

The most common etiologic agents causing chronic hepatitis are hepatitis C and B viruses (HCV and HBV, respectively). Chronic infection caused by HCV is considered one of the major causative agents of liver cirrhosis and hepatocellular carcinoma worldwide. In combination with the increasing rate of new HCV infections, the lack of a current vaccine and/or an effective treatment for this virus continues to be a major public health challenge. The development of new treatments requires a better understanding of the virus and its interaction with the different components of the host cell. MicroRNAs (miRNAs) are small non-coding RNAs functioning as negative regulators of gene expression and represent an interesting lead to study HCV infection and to identify new therapeutic targets. Until now, microRNA-122 (miR-122) and its implication in HCV infection have been the focus of different published studies and reviews. Here we will review recent advances in the relationship between HCV infection and miRNAs, showing that some of them emerge in publications as challengers against the supremacy of miR-122.

Chronic renal failure alters endothelial function in cerebral circulation in mice.

We examined structure, composition, and endothelial function in cerebral arterioles after 4 wk of chronic renal failure (CRF) in a well-defined murine model (C57BL/6J and apolipoprotein E knockout female mice). We also determined quantitative expression of endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (on serine 1177 and threonine 495), and caveolin-1; quantitative expression of markers of vascular inflammation or oxidative stress [Rock-1, Rock-2, VCAM-1, and peroxisome proliferator-activated receptor-γ (PPARγ)]; and the plasma concentration of L-arginine and asymmetric dimethylarginine (ADMA). Our hypothesis was that endothelial function would be impaired in cerebral arterioles during CRF following either a decrease in NO production (through alteration of eNOS expression or regulation) or an increase in NO degradation (due to oxidative stress or vascular inflammation). Endothelium-dependent relaxation was impaired during CRF, but endothelium-independent relaxation was not. CRF had no effect on cerebral arteriolar structure and composition. Quantitative expressions of eNOS, eNOS phosphorylated on serine 1177, caveolin-1, Rock-1, Rock-2, and VCAM-1 were similar in CRF and non-CRF mice. In contrast, quantitative expression of PPARγ (which exercises a protective role on blood vessels) was significantly lower in CRF mice, whereas quantitative expression of eNOS phosphorylated on the threonine 495 (the inactive form of eNOS) was significantly higher. Lastly, the plasma concentration of ADMA (a uremic toxin and an endogenous inhibitor of eNOS) was elevated and plasma concentration of L-arginine was low in CRF. In conclusion, endothelial function is impaired in a mouse model of early stage CRF. These alterations may be related (at least in part) to a decrease in NO production.

Cerebral arterioles preparation and PECAM-1 expression in C57BL/6J and ApoE-/- mice.

Exploration of molecular mechanisms involved in alterations of cerebral blood vessels in pathology such as chronic kidney disease implies cerebral vessel isolation for proteins and mRNAs quantification. We describe an updated method to isolate brain microvessels from wild type (WT) mice at 14 weeks of age. The quality of cerebral arterioles preparation was determined by measuring the enzymatic activity of gamma-glutamyl transpeptidase, an enzyme that is especially expressed in cerebral microvessels, and the quantitative expression (Western Blot) of platelet endothelial cell adhesion molecule-1 (PECAM-1), alpha-actin and gamma-enolase as markers of respectively endothelial cells, smooth muscle cells and neurons. We then assessed PECAM-1 expression in 14 and 20 weeks old ApoE(-/-) and WT mice. PECAM-1 expression was increased in 14 weeks old ApoE(-/-) mice compared to age matched WT mice. This difference in PECAM-1 expression disappeared at 20 weeks of age. These findings indicate that the present method of mice cerebral arterioles isolation is a valid method. Moreover, PECAM-1 expression, a marker of endothelial cells, changes with age in cerebral arterioles of ApoE(-/-) mice.

Alternative mechanism of activation of the epithelial na+ channel by cleavage.

We examined activation of the human epithelial sodium channel (ENaC) by cleavage. We focused on cleavage of alphaENaC using the serine protease subtilisin. Trimeric channels formed with alphaFM, a construct with point mutations in both furin cleavage sites (R178A/R204A), exhibited marked reduction in spontaneous cleavage and an approximately 10-fold decrease in amiloride-sensitive whole cell conductance as compared with alphaWT (2.2 versus 21.2 microsiemens (microS)). Both alphaWT and alphaFM were activated to similar levels by subtilisin cleavage. Channels formed with alphaFD, a construct that deleted the segment between the two furin sites (Delta175-204), exhibited an intermediate conductance of 13.2 microS. More importantly, alphaFD retained the ability to be activated by subtilisin to 108.8 +/- 20.9 microS, a level not significantly different from that of subtilisin activated alphaWT (125.6 +/- 23.9). Therefore, removal of the tract between the two furin sites is not the main mechanism of channel activation. In these experiments the levels of the cleaved 22-kDa N-terminal fragment of alpha was low and did not match those of the C-terminal 65-kDa fragment. This indicated that cleavage may activate ENaC by the loss of the smaller fragment and the first transmembrane domain. This was confirmed in channels formed with alphaLD, a construct that extended the deleted sequence of alphaFD by 17 amino acids (Delta175-221). Channels with alphaLD were uncleaved, exhibited low baseline activity (4.1 microS), and were insensitive to subtilisin. Collectively, these data support an alternative hypothesis of ENaC activation by cleavage that may involve the loss of the first transmembrane domain from the channel complex.

Indirect activation of the epithelial Na+ channel by trypsin.

We tested the hypothesis that the serine protease trypsin can indirectly activate the epithelial Na(+) channel (ENaC). Experiments were carried out in Xenopus oocytes and examined the effects on the channel formed by all three human ENaC subunits and that formed by Xenopus epsilon and human beta and gamma subunits (epsilonbetagammaENaC). Low levels of trypsin (1-10 ng/ml) were without effects on the oocyte endogenous conductances and were specifically used to test the effects on ENaC. Addition of 1 ng/ml trypsin for 60 min stimulated the amiloride-sensitive human ENaC conductance (g(Na)) by approximately 6-fold. This effect on the g(Na) was [Na(+)]-independent, thereby ruling out an interaction with channel feedback inhibition by Na(+). The indirect nature of this activation was confirmed in cell-attached patch clamp experiments with trypsin added to the outside of the pipette. Trypsin was comparatively ineffective at activating epsilonbetagammaENaC, a channel that exhibited a high spontaneous open probability. These observations, in combination with surface binding experiments, indicated that trypsin indirectly activated membrane-resident channels. Activation by trypsin was also dependent on catalytic activity of this protease but was not accompanied by channel subunit proteolysis. Channel activation was dependent on downstream activation of G-proteins and was blocked by G-protein inhibition by injection of guanyl-5'-yl thiophosphate and by pre-stimulation of phospholipase C. These data indicate a receptor-mediated activation of ENaC by trypsin. This trypsin-activated receptor is distinct from that of protease-activated receptor-2, because the response to trypsin was unaffected by protease-activated receptor-2 overexpression or knockdown.

The A-kinase anchoring protein 15 regulates feedback inhibition of the epithelial Na+ channel.

Protein kinase A anchoring proteins or AKAPs regulate the activity of many ion channels. Protein kinase A (PKA) is a well-recognized target of AKAPs, with other kinases now emerging as additional targets. We examined the roles of epithelial-expressed AKAPs in regulating the epithelial Na+ channel (ENaC). Experiments used heterologous expression with AKAP15, AKAP-KL, and AKAP79 in Xenopus oocytes. Experiments were carried out under high and low Na+ conditions, as Na+ loading is known to affect the baseline activity of ENaC in a PKC-dependent mechanism. ENaC activity was unaffected by AKAP79 and AKAP-KL expression. However, oocytes coexpressing AKAP15 exhibited an 80% and 91% reduction in the amiloride-sensitive, whole-cell conductance in high and low Na+ conditions, respectively. The reduced channel activity was unaffected by PKA activation or inhibition, indicating a PKA-independent mechanism. Expression with a membrane-targeting domain, mutant form of AKAP15 (AKAP15m) prevented the decrease of ENaC activity, but only under low Na+ conditions. In high sodium conditions, coexpression with AKAP15m led to an increase of ENaC activity to levels similar to those observed under low Na+. These results indicate that membrane-associated AKAP15 reduces ENaC activity whereas the cytoplasmically associated one may participate in the channel's feedback inhibition by intracellular Na+, a process known to involve PKC. This hypothesis was further confirmed in coexpression experiments, which demonstrated functional and physical interaction between AKAP15 and PKCalpha. We propose that AKAP15 regulates ENaC via a novel PKA-independent pathway.

Survivin is not only a death encounter but also a survival protein for invading tumor cells.

Cell proliferation and cell death pathways meet at a pivotal crossroad, crucial to maintain normal homeostasis and to eliminate dangerous cells before they start dividing. Survivin (SVV) is an intriguing and fascinating protein at this crossroad that interfaces life and death, through its dual role in facilitating cell division and encountering apoptosis. SVV's prominent expression in essentially all human malignancies, and low or no expression in most normal tissues, suggests that it would be an ideal target for cancer-directed therapy. However, SVV has been recently described as a target for fine tuning by alternative splicing mechanism generating five defined splice variants and a number of other uncharacterized/bizarre isoforms. This diversity indicates that SVV, in addition to its known functions in tumorgenesis, angiogenesis and cardiovascular diseases, might be associated with other unknown functions. Intriguingly, new accumulating evidence from our own work and others, suggest a novel role for SVV in the mechanisms of tumor invasion and metastasis. The SVV pathway has now provided tangible opportunities for targeted, rational cancer therapy. It is therefore an attractive and promising therapeutic target not only for cancer but also for other diseases. Although a number of studies utilizing SVV as an anti-cancer strategy are well underway, further investigation into the exact molecular interactions underpinning its functions is critical for the success of such trials. Impeding development of safe and effective SVV antagonists for clinical use is due to a lack of understanding the molecular mechanisms by which SVV differentially affects apoptosis and cell division in both normal and malignant cells. In this report, in addition to reviewing the SVV known functions, we discuss the newly proposed mechanisms by which SVV might serve as a survival tool for invading tumor cells.

A simple in vivo method for assessing changes of membrane-bound ion channel density in Xenopus oocytes.

Heterologous expression systems, such as Xenopus oocytes, are widely used to study the regulation and the structure function relationship of ion channels and transporters. In the case of ion channels, activity can be easily measured by conventional two-electrode voltage clamping. However, this method only measures the sum of the activity of all plasma membrane-bound channels. Therefore, this measurement cannot discriminate between effects on channel density and individual channel activity. To address this shortcoming, we have developed a simple assay to detect changes of membrane-bound channel density in intact oocytes. This nonradioactive assay relies on specific antibody binding in whole live cells utilizing a simple spectrophotometric measurement. This assay is linear over a wide range of channel expression levels and provides a simple cost-effective way of monitoring changes of membrane-bound channel density. Moreover, when the heterologous proteins poorly express at the plasma membrane, this method becomes advantageous to complex biochemical cell fractionation.

Nonselective cation transport in native esophageal epithelia.

Rabbit esophageal epithelia actively transport Na(+) in a manner similar to that observed in classic electrically tight Na(+)-absorbing epithelia, such as frog skin. However, the nature of the apical entry step is poorly understood. To address this issue, we examined the electrophysiological and biochemical nature of this channel. Western blotting experiments with epithelial Na(+) channel (ENaC) subunit-specific antibodies revealed the presence of all three ENaC subunits in both native and immortalized esophageal epithelial cells. The amino acid sequence of the rabbit alpha-ENaC cloned from native rabbit esophageal epithelia was not significantly different from that of other published alpha-ENaC homologs. To characterize the electrophysiological properties of this native apical channel, we utilized nystatin permeabilization to eliminate the electrical contribution of the basolateral membrane in isolated native epithelia mounted in Ussing-type chambers. We find that the previously described apical Na(+) channel is nonselective for monovalent cations (Li(+), Na(+), and K(+)). Moreover, this channel was not blocked by millimolar concentrations of amiloride. These findings document the presence of a nonselective cation channel in a native Na(+) transporting epithelia, a finding that hereto has been thought to be limited to artificial culture conditions. Moreover, our data are consistent with a potential role of ENaC subunits in the formation of a native nonselective cation channel.

Role of PKCalpha in feedback regulation of Na(+) transport in an electrically tight epithelium.

It has long been known that Na(+) channels in electrically tight epithelia are regulated by homeostatic mechanisms that maintain a steady state and allow new levels of transport to be sustained in hormonally challenged cells. Little is known about the potential pathways involved in these processes. In addition to short-term effect, recent evidence also indicates the involvement of PKC in the long-term regulation of the epithelial Na(+) channel (ENaC) at the protein level (40). To determine whether stimulation of ENaC involves feedback regulation of PKC levels, we utilized Western blot analysis to determine the distribution of PKC isoforms in polarized A6 epithelia. We found the presence of PKC isoforms in the conventional (alpha and gamma), novel (delta, eta, and epsilon), and atypical (iota, lambda, and zeta) groups. Steady-state stimulation of Na(+) transport with aldosterone was accompanied by a specific decrease of PKCalpha protein levels in both the cytoplasmic and membrane fractions. Similarly, overnight treatment with an uncharged amiloride analog (CDPC), a procedure that through feedback regulation causes a stimulation of Na(+) transport, also decreased PKCalpha levels. These effects were additive, indicating separate mechanisms that converge at the level of PKCalpha. These effects were not accompanied by changes of PKCalpha mRNA levels as determined by Northern blot analysis. We propose that this may represent a novel regulatory feedback mechanism necessary for sustaining an increase of Na(+) transport.

Alanyl-tRNA synthetase gene of the extreme acidophilic chemolithoautotrophic Thiobacillus ferrooxidans is highly homologous to alaS genes from all living kingdoms but cannot be transcribed from its promoter in Escherichia coli.

The alaS gene of Thiobacillus ferrooxidans has been cloned and sequenced and its expression in Escherichia coli and T. ferrooxidans analysed. The same genomic organization to that in E. coli (recA-recX-alaS) has been found in T. ferrooxidans. The recA and alaS genes cannot be transcribed from their own promoters in E. coli. In addition to the well-known homology at the protein level between AlaS proteins from various organisms, a strong homology was found between all the known alaS genes from bacteria, archaea and eucarya. Two regions, one of which corresponds to the catalytic core, are particularly well-conserved at the nucleotide sequence level, a possible indication of strong constraints during evolution on these parts of the genes.