Tracking the Evolution of Multiple In Vitro Hepatitis C Virus Replicon Variants under Protease Inhibitor Selection Pressure by 454 Deep Sequencing

Resistance to hepatitis C virus (HCV) inhibitors targeting viral enzymes has been observed in in vitro replicon studies and during clinical trials. The factors determining the emergence of resistance and the changes in the viral quasispecies population under selective pressure are not fully understood. To assess the dynamics of variants emerging in vitro under various selective pressures with TMC380765, a potent macrocyclic HCV NS3/4A protease inhibitor, HCV genotype 1b replicon-containing cells were cultured in the presence of a low, high, or stepwise-increasing TMC380765 concentration(s). HCV replicon RNA from representative samples thus obtained was analyzed using (i) population, (ii) clonal, and (iii) 454 deep sequencing technologies. Depending on the concentration of TMC380765, distinct mutational patterns emerged. In particular, culturing with low concentrations resulted in the selection of low-level resistance mutations (F43S and A156G), whereas high concentrations resulted in the selection of high-level resistance mutations (A156V, D168V, and D168A). Clonal and 454 deep sequencing analysis of the replicon RNA allowed the identification of low-frequency preexisting mutations possibly contributing to the mutational pattern that emerged. Stepwise-increasing TMC380765 concentrations resulted in the emergence and disappearance of multiple replicon variants in response to the changing selection pressure. Moreover, two different codons for the wild-type amino acids were observed at certain NS3 positions within one population of replicons, which may contribute to the emerging mutational patterns. Deep sequencing technologies enabled the study of minority variants present in the HCV quasispecies population present at baseline and during antiviral drug pressure, giving new insights into the dynamics of resistance acquisition by HCV.Chronic hepatitis C virus (HCV) infection can lead to liver fibrosis, cirrhosis, hepatocellular carcinoma, and ultimately liver failure. Approximately 170 million people worldwide are infected with HCV (54a). The current standard of care consists of pegylated alpha interferon (Peg-IFN) plus ribavirin (RBV), providing limited efficacy for genotype 1-infected patients, i.e., a sustained virological response (SVR) in 40 to 50% of the patients. Moreover, Peg-IFN/RBV therapy is associated with significant adverse events (9). Therefore, direct antiviral agents (DAA) (previously also known as “specifically targeted antiviral therapies for hepatitis C” or STAT-C) have been a major focus of drug discovery efforts over the last 2 decades. Several NS3/4A (protease), NS5A, and NS5B (polymerase) inhibitors either alone or in combination with Peg-IFN/RBV have recently shown potent antiviral effects in HCV-infected patients (22, 36). However, viral resistance to these novel agents can occur rapidly when they are dosed as monotherapy (43, 49).Because of the high mutation rate of the HCV polymerase (10⁻³ to 10⁻⁵ misincorporations per nucleotide copied [11]) and the high viral production rates in vivo (approximately 10¹² viruses per patient per day [37]), it can be assumed that HCV exists as a diverse population of nonidentical but closely related viral genomes, referred to as a quasispecies (10). A viral quasispecies is characterized by a dominant nucleotide sequence, called a master sequence, and a surrounding mutant spectrum, which can harbor minority subpopulations (42). Although in theory all single and double mutants are produced daily in an infected person (6, 40), it is important to note that mutation rates are not equally distributed over the entire genome and that additional factors, such as viral fitness and the replication environment, determine whether a mutation becomes fixed in a viral quasispecies population (12). The diversity of the viral variants present in an infected individual facilitates the adaptation of the quasispecies to external pressure, such as antiviral treatment, improving the survival chances of the population (53). The speed of such adaptation depends mainly on the turnover of the viral nucleic acid acting as a source of new viral genomes. Whereas in HIV the rapid turnover of infected CD4⁺ T lymphocytes is responsible for the rapid turnover of nucleic acids, in HCV rapid turnover is explained by the short half-life (∼10 h) of HCV RNA strands in the hepatocyte (47). However, if mutation rates exceed a certain limit, called the error threshold, deleterious mutations will accumulate and the viral population will become extinct (4).Recent reports have demonstrated that mutations known to affect the activities of DAA compounds in vitro are present in some treatment-naive patients as either dominant or minority species (6, 13, 19, 21, 27). With the eradication of variants susceptible to the antiviral drugs, resistant viruses initially present as minority species may expand to occupy the freed replicative space, thus becoming the dominant master sequence (1); this may lead to failure of the antiviral regimen. In HIV it has been shown that minority species can play an important role in the accelerated evolution toward resistance to antiretroviral drugs (5). The extent to which preexisting HCV variants may compromise treatment with DAAs, however, is not yet fully understood (3). Depending on the concentration of the antiviral agent, different resistance profiles seem to emerge. In clinical studies, a correlation was noted between the plasma trough levels of the NS3/4A inhibitor telaprevir, the virological response, and the mutations responsible for the drug-resistant phenotype (43). In patients with a low exposure to telaprevir, variants carrying mutations with low resistance to telaprevir in vitro were observed, while higher drug levels were associated with variants conferring a greater degree of resistance in vitro. Correlations between the inhibitor concentration and the mutation profile were also described in in vitro studies (44, 50, 51).HCV replicon cell culture systems have been widely used to characterize resistance against antiviral inhibitors and to assess the impact of resistance mutations on drug susceptibility and replication fitness in vitro (8, 15). Although the information on resistance mutations observed with DAA during clinical trials is still limited, mutations identified in vitro appear to be predictive for those mutations that may emerge in patients (17, 20). In addition, analysis of the genetic variability and diversity of a long-term HCV replicon-containing cell culture has shown that mutations accumulate over time at rates comparable to those observed in vivo: (3.5 to 4.8) × 10⁻³ in vitro versus (1.4 to 1.9) × 10⁻³ in vivo base substitutions/site/year (16). Hence, HCV replicon systems are considered a useful and relevant surrogate system for analyzing the evolutionary dynamics and variations of HCV in response to selection pressure.The detailed study of the dynamics of viral variants present in a quasispecies population has long been hampered by the lack of sensitive sequencing methods. The recent development of deep sequencing technologies may facilitate a better understanding of the genetic composition and natural evolution of viral quasispecies in the presence of antiviral drugs (30, 34, 54). Indeed, studies of HIV suggest that these more-sensitive sequencing technologies detect additional minority variants for both treatment-naive and treatment-experienced patients which could impact the clinical outcome of antiretroviral therapy and may provide important information for treatment planning (2, 23, 41, 46).TMC380765 (Fig. ​(Fig.1)1) is a macrocyclic inhibitor of the HCV NS3/4A protease and a potent inhibitor of HCV RNA replication in vitro, with median 50% effective concentration (EC₅₀) and 90% effective concentration (EC₉₀) values of 35 nM and 106 nM, respectively, in the Huh7-Luc replicon using a luciferase readout (25, 39). Other examples of macrocyclic NS3/4A inhibitors include BILN-2061, ITMN-191, MK7009, and TMC435. To assess the effect of its selective pressure on the composition of the replicon population, selection experiments were performed with different concentrations of TMC380765 and sequence changes were determined with population, clonal, and 454 deep sequencing technologies.Open in a separate windowFIG. 1.Structural formulae of TMC380765.     

ProteomIQ blue, a potent post-stain for the visualization and subsequent mass spectrometry based identification of fluorescent stained proteins on 2D-gels

Manual spot excision for protein identification from fluorescent stained two-dimensional (2-D) gels is hard to accomplish. Here, we explore the use of ProteomIQ Blue as a post-stain method for the visualization of fluorescent stained/labeled proteins. We show that ProteomIQ Blue post-staining is almost as sensitive as staining with SYPRO Ruby or cyanine dyes alone. More than 90% of the protein spots that are stained with the fluorescent stains are still detectable with ProteomIQ Blue. In protein identification by mass spectrometry, ProteomIQ Blue post-stained spots provide high sensitivity and high protein sequence coverage of the peptide mass maps in both MALDI-TOF-MS and ESI-MS/MS analyses. In conclusion, post-staining of fluorescent stained gels with ProteomIQ Blue provides a facile and a powerful method to achieve quantitative protein analysis as well as protein identification in the same semianalytical gel without requiring sophisticated/expensive robotic equipment.     

Enhanced circadian ACTH release in obese premenopausal women: reversal by short-term acipimox treatment

Several studies suggest that the hypothalamo-pituitary-adrenal (HPA) axis is exceedingly active in obese individuals. Experimental studies show that circulating free fatty acids (FFAs) promote the secretory activity of the HPA axis and that human obesity is associated with high circulating FFAs. We hypothesized that HPA axis activity is enhanced and that lowering of circulating FFAs by acipimox would reduce spontaneous secretion of the HPA hormonal ensemble in obese humans. To evaluate these hypotheses, diurnal ACTH and cortisol secretion was studied in 11 obese and 9 lean premenopausal women (body mass index: obese 33.5 +/- 0.9 vs. lean 21.2 +/- 0.6 kg/m(2), P < 0.001) in the early follicular stage of their menstrual cycle. Obese women were randomly assigned to treatment with either acipimox (inhibitor of lipolysis, 250 mg orally four times daily) or placebo in a double-blind crossover design, starting one day before admission until the end of the blood-sampling period. Blood samples were taken during 24 h with a sampling interval of 10 min for assessment of plasma ACTH and cortisol concentrations. ACTH and cortisol secretion rates were estimated by multiparameter deconvolution analysis. Daily ACTH secretion was substantially higher in obese than in lean women (7,950 +/- 1,212 vs. 2,808 +/- 329 ng/24 h, P = 0.002), whereas cortisol was not altered (obese 36,362 +/- 5,639 vs. lean 37,187 +/- 4,239 nmol/24 h, P = 0.912). Acipimox significantly reduced ACTH secretion in the obese subjects (acipimox 5,850 +/- 769 ng/24 h, P = 0.039 vs. placebo), whereas cortisol release did not change (acipimox 33,542 +/- 3,436 nmol/24 h, P = 0.484 vs. placebo). In conclusion, spontaneous ACTH secretion is enhanced in obese premenopausal women, whereas cortisol production is normal. Reduction of circulating FFA concentrations by acipimox blunts ACTH release in obese women, which suggests that FFAs are involved in the pathophysiology of this neuroendocrine anomaly.     

Absence of linkage between transcobalamin II and ABO

Summary In a linkage analysis of 29 pedigrees with a total of 196 individuals, absence of close linkage between transcobalamin II (TC2) and ABO is demonstrated. Recombination fraction values of 相似文献   

Recent methodological advances in the analysis of nitrite in the human circulation: nitrite as a biochemical parameter of the L-arginine/NO pathway

Nitric oxide (NO) plays a pivotal role in the modulation of multiple physiological processes. It acts as a messenger molecule within the cardiovascular system. NO is a highly unstable free radical in circulating blood and is oxidized rapidly to nitrite and nitrate. Recent studies suggest that nitrite has the potential to function as a surrogate of NO production under physiological and pathophysiological conditions and could therefore be of high relevance as a biochemical parameter in experimental and clinical studies. Under hypoxic conditions nitrite is reduced to bioactive NO by deoxyhemoglobin. This mechanism may represent a dynamic cycle of NO generation to adapt the demand and supply for the vascular system. Because of these potential biological functions the concentration of nitrite in blood is thought to be of particular importance. The determination of nitrite in biological matrices represents a considerable analytical challenge. Methodological problems often arise from pre-analytical sample preparation, sample contamination due to the ubiquity of nitrite, and from lack of selectivity and sensitivity. These analytical difficulties may be a plausible explanation for reported highly diverging concentrations of nitrite in the human circulation. The aim of this article is to review the methods of quantitative analysis of nitrite in the human circulation, notably in plasma and blood, and to discuss pre-analytical and analytical factors potentially affecting accurate quantification of nitrite in these human fluids.     

An optimized procedure for whole-mount in situ hybridization on mouse embryos and embryoid bodies

Determining the precise expression pattern of a gene of interest at various stages of development is essential to understanding its biological function in embryology. This protocol describes a sensitive method for whole-mount in situ hybridization (WISH) to mouse embryos, using cRNA probes. Adaptations are provided that allow the protocol to be applied to embryonic stages ranging from blastocysts to postimplantation stage embryos, and to embryoid bodies. We also describe an in situ method for differential detection of two probes. Probe labeling and dissection and preparation of the embryos can be performed in 2 d. The actual WISH procedure can be completed in another 3 d.     

Declines in moth populations stress the need for conserving dark nights

Given the global continuous rise, artificial light at night is often considered a driving force behind moth population declines. Although negative effects on individuals have been shown, there is no evidence for effects on population sizes to date. Therefore, we compared population trends of Dutch macromoth fauna over the period 1985–2015 between moth species that differ in phototaxis and adult circadian rhythm. We found that moth species that show positive phototaxis or are nocturnally active have stronger negative population trends than species that are not attracted to light or are diurnal species. Our results indicate that artificial light at night is an important factor in explaining declines in moth populations in regions with high artificial night sky brightness. Our study supports efforts to reduce the impacts of artificial light at night by promoting lamps that do not attract insects and reduce overall levels of illumination in rural areas to reverse declines of moth populations.     

Prey preference of the phytoseiid miteTyphlodromus pyri 2. Electrophoretic diet analysis

The prey selection of the phytoseiid miteTyphlodromus pyri Scheuten was studied by using polyacrylamide gel electrophoresis to analyse the diet of field-collected predators. The predators were obtained from an orchard where the phytophagous prey mitesPanonychus ulmi (Koch) andAculus schlechtendali (Nalepa) were present in various density ratios.Esterases of both prey species were identified in the predators, but on all sampling datesP. ulmi esterases were discovered much more frequently than those ofA. schlechtendali. The data show thatT. pyri hardly fed onA. schlechtendali over a range of prey density ratios. It is therefore concluded thatT. pyri prefersP. ulmi toA. schlechtendali.     

A paradoxical pro-apoptotic effect of thrombin on smooth muscle cells

Whereas thrombin (below 10 nM) is a potent mitogen, recent studies report that exposure to higher doses of thrombin could lead to apoptosis of neurons and tumor cells. Our results show that prolonged exposure (> or = 24 h) to thrombin (50-100 nM) exerts a pro-apoptotic effect on cultured vascular smooth muscle cells (VSMCs). This phenomenon depends on thrombin serine-protease activity but is independent of PAR-1 and -4 activation and subsequent signaling. The parallel occurrence of cell retraction and cleavage of fibronectin suggests that thrombin-induced apoptosis is consecutive to pericellular proteolysis. These data point to a new potential action of thrombin in the cardiovascular system.