Strong Differential Photoion Circular Dichroism in Strong-Field Ionization of Chiral Molecules.

We investigate the differential ionization probability of chiral molecules in the strong-field regime as a function of the helicity of the incident light. To this end, we analyze the fourfold ionization of bromochlorofluoromethane (CHBrClF) with subsequent fragmentation into four charged fragments and different dissociation channels of the singly ionized methyloxirane. By resolving for the molecular orientation, we show that the photoion circular dichroism signal strength is increased by 2 orders of magnitude.

Lentiviral hepatitis B pseudotype entry requires sodium taurocholate co-transporting polypeptide and additional hepatocyte-specific factors.

Hepatitis B virus (HBV) is one of the world's major unconquered infections, resulting in progressive liver disease, and current treatments rarely cure infection. A limitation to discovering new therapies is our limited knowledge of HBV entry and dissemination pathways that hinders the development of in vitro culture systems. To address this gap in our understanding we optimized the genesis of infectious lentiviral pseudoparticles (HBVpps). The recent discovery that the bile salt transporter sodium taurocholate co-transporting polypeptide (NTCP) acts as a receptor for HBV enabled us to assess the receptor dependency of HBVpp infection. HBVpps preferentially infect hepatoma cells expressing NTCP, whereas other non-liver cells engineered to express NTCP do not support infection, suggesting that additional hepatocyte-specific factors are required for HBVpp internalization. These results highlight the value of the HBVpp system to dissect the pathways of HBV entry and dissemination.

Prevention of hepatitis C virus infection by adoptive allogeneic immunotherapy using suicide gene-modified lymphocytes: an in vitro proof-of-concept.

Hepatitis C virus (HCV)-induced, end-stage liver disease is a major indication for liver transplantation, but systematic graft reinfection accelerates liver disease recurrence. Transplantation recipients may be ineligible for direct-acting antivirals, owing to toxicity, resistance or advanced liver disease. Adoptive immunotherapy with liver graft-derived, ex vivo-activated lymphocytes was previously shown to prevent HCV-induced graft reinfections. Alternatively, the applicability and therapeutic efficacy of adoptive immunotherapy may be enhanced by 'ready for use' suicide gene-modified lymphocytes from healthy blood donors; moreover, conditional, prodrug-induced cell suicide may prevent potential side effects. Here, we demonstrate that allogeneic suicide gene-modified lymphocytes (SGMLs) could potently, dose- and time-dependently, inhibit viral replication. The effect occurs at effector:target cell ratios that exhibits no concomitant cytotoxicity toward virus-infected target cells. The effect, mediated mostly by CD56+ lymphocytes, is interleukin-2-dependent, IFN-γ-mediated and, importantly, resistant to calcineurin inhibitors. Thus, post-transplant immunosuppression may not interfere with this adoptive cell immunotherapy approach. Furthermore, these cells are indeed amenable to conditional cell suicide; in particular, the inducible caspase 9 suicide gene is superior to the herpes simplex virus thymidine kinase suicide gene. Our data provide in vitro proof-of-concept that allogeneic, third-party, SGMLs may prevent HCV-induced liver graft reinfection.

Complete photoionization experiments via ultrafast coherent control with polarization multiplexing.

Photoelectron angular distributions (PADs) obtained from ionization of potassium atoms using moderately intense femtosecond IR fields (∼10^{12} W cm^{-2}) of various polarization states are shown to provide a route to "complete" photoionization experiments. Ionization occurs by a net three-photon absorption process, driven via the 4s→4p resonance at the one-photon level. A theoretical treatment incorporating the intrapulse electronic dynamics allows for a full set of ionization matrix elements to be extracted from 2D imaging data. 3D PADs generated from the extracted matrix elements are also compared to experimental, tomographically reconstructed, 3D photoelectron distributions, providing a sensitive test of their validity. Finally, application of the determined matrix elements to ionization via more complex, polarization-shaped, pulses is demonstrated, illustrating the utility of this methodology towards detailed understanding of complex ionization control schemes and suggesting the utility of such "multiplexed" intrapulse processes as powerful tools for measurement.

Self-referencing circular dichroism ion yield measurements for improved statistics using femtosecond laser pulses.

The combination of circular dichroism with laser mass spectrometry via the measurement of ion yields is a powerful tool in chiral recognition, but the measured anisotropies are generally weak. The method presented in this contribution reduces the measurement error significantly. A common path optical setup generates a pair of counter-rotating laser foci in the interaction region of a time-of-flight spectrometer. As the space focus condition is fulfilled for both foci individually, this becomes a twin-peak ion source with well separated and sufficiently resolved mass peaks. The individual control of polarization allows for in situ correction of experimental fluctuations measuring circular dichroism. Our robust optical setup produces reliable and reproducible results and is applicable for dispersion sensitive femtosecond laser pulses. In this contribution, we use 3-methyl-cyclopentanone as a prototype molecule to illustrate the evaluation procedure and the measurement principle.

[Tropism of hepatitis C virus for leukocytes-- importance of the analysis of viral E1 and E2 envelope glycoprotein genes by sequencing]. / Tropisme leucocytaire du virus de l'hépatite C - intérêt de l'analyse des séquences des gènes des glycoprotéines d'enveloppe virales E1 et E2.

The ability of hepatitis C virus (HCV) to infect leukocytes could favour HCV pathogenesis. Although viral infection of these immunocompetent cells is poorly (or not) productive, the impact on their immunomodulatory functions could be important. Viral envelope glycoproteins E1 and E2, because of their crucial role in the recognition of viral receptors on permissive cells, could contribute to viral leukocytic tropism and, as a consequence, to the pathophysiology of HCV chronic infection.

Impact of microRNAs for pathogenesis and treatment of hepatitis C virus infection.

The discovery of RNA interference (RNAi), and of all related RNA silencing processes, was one of the major breakthroughs and is currently changing our understanding of liver physiology and pathogenesis of liver disease. Furthermore, recent studies indicate that microRNAs (miRNAs) are a promising therapeutic target. Plant and insect organisms use RNAi as a major antiviral pathway, whereas mammalian viruses interfere with or even usurp the cellular miRNA repertoire. One remarkable example of such usurpation is provided by hepatitis C virus (HCV), which recruits the liver-specific miR-122 to enhance its abundance. In the HCV-infected patient, the impact of miRNAs for pathogenesis is more complex: whereas miR-122 expression shows no correlation with viral load, decreased pretreatment miR-122 levels are associated with nonresponse during IFN therapy. Following-up on these investigations, miRNA-122 has recently been shown to be a target for antiviral intervention. Treatment of chronically HCV-infected chimpanzees with a novel miR-122 antagonist leads to suppression of HCV viremia. The prolonged virological response to miRNA-based treatment holds promise of a new antiviral therapy with a potentially higher barrier to resistance. This review summarizes recent key discoveries of the impact of miRNAs for pathogenesis and treatment of HCV infection.

New aspects of an anti-tumour drug: sorafenib efficiently inhibits HCV replication.

BACKGROUND AND AIMS: Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and is associated with significant morbidity and mortality. Since there is evidence for an interaction of NS5A with c-Raf we studied whether the c-Raf inhibitor sorafenib affects HCV replication. METHODS: HCV replicating HuH7.5 cells were treated with sorafenib and examined for HCV RNA titres by northern blotting or real time polymerase chain reaction (PCR), for core, NS3 and NS5A expression by immunostaining, and for replication by luciferase reporter assays. RESULTS: Here we demonstrate that in cells replicating infectious HCV particles, NS5A recruits c-Raf to the replicon complex resulting in the activation of c-Raf. Therefore, we studied the effect of inhibition of c-Raf on HCV replication using the anti-tumour drug sorafenib that is known to inhibit c-Raf with high specificity. Sorafenib efficiently blocks HCV replication and viral gene expression. In addition, in HCV-replicating cells sorafenib decreased the hyperphosphorylated form of NS5A and resulted in the formation of additional hypophosphorylated forms. Further, sorafenib caused a rapid dissociation of lipid droplets. We provide evidence that the antiviral effect of sorafenib indeed is caused by inhibition of c-Raf. By contrast, inhibition of targets downstream of c-Raf or inhibition of tyrosine kinases by sunitinib did not affect HCV replication. CONCLUSION: Our data demonstrate that the well-characterised anti-tumour drug sorafenib efficiently blocks HCV replication in vitro. This novel effect of sorafenib should be further explored as an antiviral strategy for patients with chronic HCV infection.

Conservation of hepatitis C virus nonstructural protein 3 amino acid sequence in viral isolates during liver transplantation.

As a consequence of selective pressure exerted by the immune response during hepatitis C virus (HCV) infection, a high rate of nucleotide mutations in the viral genome is observed which leads to the emergence of viral escape mutants. The aim of this study was to evaluate the evolution of the amino acid (aa) sequence of the HCV nonstructural protein 3 (NS3) in viral isolates after liver transplantation. Six patients with HCV-induced liver disease undergoing liver transplantation (LT) were followed up for sequence analysis. Hepatitis C recurrence was observed in all patients after LT. The rate of synonymous (dS) nucleotide substitutions was much higher than that of nonsynonymous (dN) ones in the NS3 encoding region. The high values of the dS/dN ratios suggest no sustained adaptive evolution selection pressure and, therefore, absence of specific NS3 viral populations. Clinical genotype assignments were supported by phylogenetic analysis. Serial samples from each patient showed lower mean nucleotide genetic distance when compared with samples of the same HCV genotype and subtype. The NS3 samples studied had an N-terminal aa sequence with several differences as compared with reference ones, mainly in genotype 1b-infected patients. After LT, as compared with the sequences before, a few reverted aa substitutions and several established aa substitutions were observed at the N-terminal of NS3. Sites described to be involved in important functions of NS3, notably those of the catalytic triad and zinc binding, remained unaltered in terms of aa sequence. Rare or frequent aa substitutions occurred indiscriminately in different positions. Several cytotoxic T lymphocyte epitopes described for HCV were present in our 1b samples. Nevertheless, the deduced secondary structure of the NS3 protease showed a few alterations in samples from genotype 3a patients, but none were seen in 1b cases. Our data, obtained from patients under important selective pressure during LT, show that the NS3 protease remains well conserved, mainly in HCV 3a patients. It reinforces its potential use as an antigenic candidate for further studies aiming at the development of a protective immune response.

[Neutralizing antibodies in hepatitis C virus infection]. / Anticorps neutralisants dirigés contre le virus de l'hépatite C.

Hepatitis C virus (HCV) results in persistent infection in more than 70% of infected individuals despite the development of humoral and cellular immune responses. Following infection, although antibodies targeting epitopes of both structural and non structural proteins are elicited, the virus evades antibody-mediated neutralization. Studies of host neutralizing responses against HCV have been limited by the lack of a convenient tissue culture system for HCV infection. In the past five years in vitro models have been developed to characterize interaction of HCV glycoproteins with host cell entry factors and detect antibodies interfering with HCV entry and infection. These models have been used to characterize targets of neutralizing responses and better understand their impact on the pathogenesis of infection.

Two core promotor mutations identified in a hepatitis B virus strain associated with fulminant hepatitis result in enhanced viral replication.

Viral mutations have been implicated in alteration of the biological phenotype of hepatitis B virus (HBV). We recently cloned and sequenced the viral genome of an HBV strain associated with an outbreak of fulminant hepatitis (FH strain). The FH strain contained numerous mutations in all genomic regions and was functionally characterized by a more efficient encapsidation of pregenomic RNA leading to highly enhanced replication. To define the responsible mutation(s) for the enhanced replication, we introduced individual mutations of the FH strain into a wild-type construct by oligonucleotide-directed mutagenesis. Analysis of viral replication showed that two adjacent mutations in the HBV core promotor (C to T at nucleotide 1768 and T to A at nucleotide 1770) led to high level replication. Similar to the FH strain, this mutant displayed the phenotype of enhanced encapsidation of pregenomic RNA. Functional studies in an encapsidation assay demonstrated that the identified mutations resulted in a minor increase of pregenomic RNA transcription (two- to threefold) and a major transcription-independent enhancement (> 10-fold) of viral encapsidation. Our results demonstrate that the two adjacent mutations in the HBV core promotor region are responsible for the enhanced replication of the FH strain. These two mutations, outside the previously described encapsidation signal, core, and polymerase polypeptides, appeared to affect a novel genetic element involved in viral encapsidation.

Control of ionization processes in high band gap materials via tailored femtosecond pulses.

Control of two basic ionization processes in dielectrics i.e. photo ionization and electron-electron impact ionization on intrinsic time and intensity scales is investigated experimentally and theoretically. Temporally asymmetric femtosecond pulses of identical fluence, spectrum and pulse duration result in different final free electron densities. We found that an asymmetric pulse and its time reversed counterpart address two ionization processes in a different fashion. This results in the observation of different thresholds for surface material modification in sapphire and fused silica. We conclude that control of ionization processes with tailored femtosecond pulses is suitable for robust manipulation of breakdown and thus control of the initial steps of laser processing of high band gap materials.

Composite vector formulation for multiple siRNA delivery as a host targeting antiviral in a cell culture model of hepatitis C virus (HCV) infection.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and cancer worldwide. RNA interference (RNAi)-based gene therapies have emerged recently as a promising tool to treat chronic viral infections. Indeed, small interfering RNAs (siRNAs) provide an opportunity to target host factors required for the viral life cycle. In this study, we evaluated a novel nanovector-based approach for siRNA delivery in a model of chronically infected hepatic cells. We designed original composite nanoparticles by coating the calcium phosphate core with siRNAs targeting HCV host-factors and pyridylthiourea-grafted polyethyleneimine (πPEI). Using combinations of different siRNAs, we observed an efficient and prolonged decrease of HCV replication. Moreover, we showed that the layer-by-layer technique of coating applied to our nanoparticles triggers a sequential release of siRNAs acting on different steps of the HCV life cycle. Together, our results demonstrate the efficacy of these nanoparticles for siRNA delivery and open new perspectives for antiviral therapies.

Genetic variants of hepatitis B virus and their clinical relevance.

Infection with hepatitis B virus (HBV) leads to a wide spectrum of clinical presentations ranging from an asymptomatic carrier state to self-limited acute or fulminant hepatitis to chronic hepatitis with progression to cirrhosis and hepatocellular carcinoma (HCC). Infection with HBV is one of the most common viral diseases affecting man. Both viral factors as well as the host immune response have been implicated in the pathogenesis and clinical outcome of HBV infection. Evidence has been accumulating that HBV mutants are associated with certain clinical disease manifestations, may affect the natural course of the infection and confer resistance to antivirals. Naturally occurring mutations have been identified in the structural and non-structural genes as well as regulatory elements of the virus. The best characterized mutants comprise the pre-core (pre-C) stop codon mutation resulting in a loss of hepatitis B e antigen (HBeAg), defined clusters of mutations in the core promotor resulting in enhanced viral replication and mutations in the hepatitis B core and surface antigens (HBcAg and HBsAg) altering the antigenicity of the virus. More recently, several mutations in the reverse transcriptase/polymerase gene have been identified conferring resistance to antivirals used for the treatment of chronic hepatitis B. In this review, we will focus on the biological phenotype of HBV genetic variants and discuss their clinical relevance for the pathogenesis of HBV-induced liver disease.

Metabolic inactivation of leukotrienes.

The metabolic inactivation of the cysteinyl leukotrienes LTC4 and LTD4 and of the chemotactic LTB4 was studied in the rat in vivo and in hepatocyte suspensions, respectively. 1. Deactivation of LTC4 via LTD4 to LTE4 was a most active process in the blood circulation, catalyzed mainly by ectoenzymes located on the internal wall of blood vessels. Uptake of cysteinyl leukotrienes by hepatocytes and kidney cells contributed to the rapid elimination of these potent mediators whenever they were released into the blood circulation. The initial half-life of LTC4 in vivo was 12 seconds. 2. omega-Oxidation leads to the formation of omega-hydroxylated and omega-carboxylated cysteinyl leukotrienes which were detected in bile and urine. Biliary metabolites included those formed by stepwise beta-oxidative degradation of omega-carboxy-N-acetyl-LTE4, yielding the dinor, tetranor, and hexanor derivative. 3. The peroxisome proliferator clofibrate strongly increased the degradation of LTE4 by omega-oxidation and subsequent beta-oxidation in vivo. The generation of new polar metabolites was detected by HPLC methods and by the use of 3H8-labeled cysteinyl leukotrienes in comparison with the 3H2-labeled precursor. 4. The metabolic degradation and inactivation of cysteinyl leukotrienes in vivo and of LTB4 in isolated hepatocytes was potently inhibited by ethanol. The site of inhibition was the oxidation of omega-hydroxy-N-acetyl-LTE4 and of omega-hydroxy-LTB4 to the respective omega-carboxylated metabolite. This inhibition led to an accumulation of the biologically active LTB4 and of N-acetyl-LTE4. The interference of leukotriene inactivation in the liver may provide a novel explanation for the ethanol-induced inflammatory reaction in acute alcoholic liver disease.

Robust photon locking.

We experimentally demonstrate a strong-field coherent control mechanism that combines the advantages of photon locking (PL) and rapid adiabatic passage (RAP). Unlike earlier implementations of PL and RAP by pulse sequences or chirped pulses, we use shaped pulses generated by phase modulation of the spectrum of a femtosecond laser pulse with a generalized phase discontinuity. The novel control scenario is characterized by a high degree of robustness achieved via adiabatic preparation of a state of maximum coherence. Subsequent phase control allows for efficient switching among different target states. We investigate both properties by photoelectron spectroscopy on potassium atoms interacting with the intense shaped light field.