Residue analysis, dissipation patterns of chlorfenapyr, diafenthiuron and their corresponding metabolites in tea trees, and dietary intake risk assessment.

BACKGROUND: Recently, chlorfenapyr and diafenthiuron have been widely used to prevent and control diseases and pests in tea production. However, rare studies have investigated the dissipation patterns of chlorfenapyr, diafenthiuron and their metabolites simultaneously in tea matrices. Here, we established an analytical method to investigate the degradation patterns of five target compounds in tea shoots and made tea samples. Moreover, the dietary intake risk assessment of chlorfenapyr-diafenthiuron mixture among Chinese populations was evaluated based on the supervised field experiment. RESULTS: The mean recoveries of the primary analytes at five spiking levels were between 95.6% and 112.6% in tea shoots and made tea, respectively, and the values of RSD (relative standard deviation) were lower than 9.7% for all the target analytes. The field trial results showed that the half-lives of chlorfenapyr and diafenthiuron based on the residue definition were 10.0-12.4 days and 4.3-5.9 days, respectively, in tea shoots. For the dietary intake risk assessment, the risk quotient (RQ) values in made tea ranged from 30.4% to 73.9% at the pre-harvest interval of 14 days, which were significantly less than 100%. CONCLUSION: The accuracy and precision of the developed method were satisfied by the measurement requirements according to the validation results. The dynamic dissipation experiments suggested that diafenthiuron was much easier to dissipate than chlorfenapyr. Moreover, the existence of tralopyril made the half-life of chlorfenapyr significantly increase, indicating that practical application of chlorfenapyr should take careful consideration of its metabolite. Finally, the potential chronic dietary risks of the chlorfenapyr-diafenthiuron mixture to human communities were within the acceptable range. © 2022 Society of Chemical Industry.

Further Insights into the Oxidative Pathway of Thiocarbonyl-Type Antitubercular Prodrugs: Ethionamide, Thioacetazone, and Isoxyl.

A chemical activation study of the thiocarbonyl-type antitubercular prodrugs, ethionamide (ETH), thioacetazone (TAZ), and isoxyl (ISO), was performed. Biomimetic oxidation of ethionamide using H2O2 (1 equiv) led to ETH-SO as the only stable S-oxide compound, which was found to occur in solution in the preferential form of a sulfine (ETH═S═O vs the sulfenic acid tautomer ETH-S-OH), as previously observed in the crystal state. It was also demonstrated that ETH-SO is capable of reacting with amines, as the putative sulfinic derivative (ETH-SO2H) was supposed to do. Unlike ETH, oxidation of TAZ did not allow observation of the mono-oxygenated species (TAZ-SO), leading directly to the more stable sulfinic acid derivative (TAZ-SO2H), which can then lose a SOxH group after further oxidation or when placed in a basic medium. It was also noticed that the unstable TAZ-SO intermediate can lead to the carbodiimide derivative as another electrophilic species. It is suggested that TAZ-SOH, TAZ-SO2H, and the carbodiimide compound can also react with NH2-containing nucleophilic species, and therefore be involved in toxic effects. Finally, ISO showed a very complex reactivity, here assigned to the coexistence of two mono-oxygenated structures, the sulfine and sulfenic acid tautomers. The mono- and dioxygenated derivatives of ISO are also highly unstable, leading to a panel of multiple metabolites, which are still reactive and likely contribute to the toxicity of this prodrug.

Development of phenylthiourea derivatives as allosteric inhibitors of pyoverdine maturation enzyme PvdP tyrosinase.

Infections caused by Pseudomonas aeruginosa become increasingly difficult to treat because these bacteria have acquired various mechanisms for antibiotic resistance, which creates the need for mechanistically novel antibiotics. Such antibiotics might be developed by targeting enzymes involved in the iron uptake mechanism because iron is essential for bacterial survival. For P. aeruginosa, pyoverdine has been described as an important virulence factor that plays a key role in iron uptake. Therefore, inhibition of enzymes involved in the pyoverdine synthesis, such as PvdP tyrosinase, can open a new window for the treatment of P. aeruginosa infections. Previously, we reported phenylthiourea as the first allosteric inhibitor of PvdP tyrosinase with high micromolar potency. In this report, we explored structure-activity relationships (SAR) for PvdP tyrosinase inhibition by phenylthiourea derivatives. This enables identification of a phenylthiourea derivative (3c) with a potency in the submicromolar range (IC50 = 0.57 + 0.05 µM). Binding could be rationalized by molecular docking simulation and 3c was proved to inhibit the bacterial pyoverdine production and bacterial growth in P. aeruginosa PA01 cultures.

Sublethal effects of methylthio-diafenthiuron on the life table parameters and enzymatic properties of the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae).

The diamondback moth (DBM), Plutella xylostella (L.), is a major pest affecting cruciferous vegetables, and seriously affects the quality and yield of these vegetables. Diafenthiuron is a traditional thiourea-based insecticide, but it is rarely used to control pests on cruciferous vegetables due to its phytotoxicity on these vegetables under high temperature and light conditions. Thus, there is an ongoing need for more effective pesticides that can be used on cruciferous vegetables, possibly including new formulations of diafenthiuron. A new thiourea insecticide, methylthio-diafenthiuron, is intended to optimize the structure of diafenthiuron not only to preserve its insecticidal bioactivity but also to overcome its phytotoxicity to cruciferous vegetables, aiming to control insect pests on cruciferous vegetables. In this study, we compared the toxicity of methylthio-diafenthiuron to some frequently used insecticides on the third-instar larvae of DBM. The parental pupal duration was significantly longer under the treatment than in the control, but the pupal weight, fecundity, and hatching rate significantly decreased. By studying the changes in three detoxifying enzymes within 72 h after treatment with a sublethal concentration, the activity of CarE and ODM in the treatment group significantly increased at first and then decreased. In addition, methylthio-diafenthiuron clearly inhibited three kinds of ATPases in the DBM and significantly reduced the eclosion rate of the pupae. This research provides valuable information for the assessment and rational application of methylthio-diafenthiuron for the control of pests on cruciferous vegetables.

Disubstituted 4-Chloro-3-nitrophenylthiourea Derivatives: Antimicrobial and Cytotoxic Studies.

4-Chloro-3-nitrophenylthioureas 1⁻30 were synthesized and tested for their antimicrobial and cytotoxic activities. Compounds exhibited high to moderate antistaphylococcal activity against both standard and clinical strains (MIC values 2⁻64 µg/mL). Among them derivatives with electron-donating alkyl substituents at the phenyl ring were the most promising. Moreover, compounds 1⁻6 and 8⁻19 were cytotoxic against MT-4 cells and various other cell lines derived from human hematological tumors (CC50 ≤ 10 µM). The influence of derivatives 11, 13 and 25 on viability, mortality and the growth rate of immortalized human keratinocytes (HaCaT) was observed.

Mechanism of N-Acylthiourea-mediated activation of human histone deacetylase 8 (HDAC8) at molecular and cellular levels.

We reported previously that an N-acylthiourea derivative (TM-2-51) serves as a potent and isozyme-selective activator for human histone deacetylase 8 (HDAC8). To probe the molecular mechanism of the enzyme activation, we performed a detailed account of the steady-state kinetics, thermodynamics, molecular modeling, and cell biology studies. The steady-state kinetic data revealed that TM-2-51 binds to HDAC8 at two sites in a positive cooperative manner. Isothermal titration calorimetric and molecular modeling data conformed to the two-site binding model of the enzyme-activator complex. We evaluated the efficacy of TM-2-51 on SH-SY5Y and BE(2)-C neuroblastoma cells, wherein the HDAC8 expression has been correlated with cellular malignancy. Whereas TM-2-51 selectively induced cell growth inhibition and apoptosis in SH-SY5Y cells, it showed no such effects in BE(2)-C cells, and this discriminatory feature appears to be encoded in the p53 genotype of the above cells. Our mechanistic and cellular studies on HDAC8 activation have the potential to provide insight into the development of novel anticancer drugs.

Susceptibility of field populations of the diamondback moth, Plutella xylostella, to a selection of insecticides in Central China.

The diamondback moth (DBM), Plutella xylostella (L.) (Lepidoptera: Plutellidae), is a globally distributed and important economic pest. Chemical control is the primary approach to regulate populations of this pest. However, resistance to insecticides evolves following heavy and frequent use. Therefore, the insecticide resistance in field populations of P. xylostella collected from Central China from 2013 to 2014 was determined with a leaf-dipping method. Based on the results of the monitoring, P. xylostella has developed high levels of resistance to beta-cypermethrin (resistance ratio=69.76-335.76-fold), Bt (WG-001) (RR=35.43-167.36), and chlorfluazuron (RR=13.60-104.95) and medium levels of resistance to chlorantraniliprole (RR=1.19-14.26), chlorfenapyr (RR=4.22-13.44), spinosad (RR=5.89-21.45), indoxacarb (RR=4.01-34.45), and abamectin (RR=23.88-95.15). By contrast, the field populations of P. xylostella remained susceptible to or developed low levels of resistance to diafenthiuron (RR=1.61-8.05), spinetoram (RR=0.88-2.35), and cyantraniliprole (RR=0.4-2.15). Moreover, the LC50 values of field populations of P. xylostella were highly positively correlated between chlorantraniliprole and cyantraniliprole (r=0.88, P=0.045), chlorantraniliprole and spinosad (r=0.66, P=0.039), spinosad and diafenthiuron (r=0.57, P=0.0060), and chlorfenapyr and diafenthiuron (r=0.51, P=0.016). Additionally, the activities of detoxification enzymes in field populations of P. xylostella were significantly positively correlated with the log LC50 values of chlorantraniliprole and spinosad. The results of this study provide an important base for developing effective and successful strategies to manage insecticide resistance in P. xylostella.

A common mechanism of inhibition of the Mycobacterium tuberculosis mycolic acid biosynthetic pathway by isoxyl and thiacetazone.

Isoxyl (ISO) and thiacetazone (TAC), two prodrugs once used in the clinical treatment of tuberculosis, have long been thought to abolish Mycobacterium tuberculosis (M. tuberculosis) growth through the inhibition of mycolic acid biosynthesis, but their respective targets in this pathway have remained elusive. Here we show that treating M. tuberculosis with ISO or TAC results in both cases in the accumulation of 3-hydroxy C(18), C(20), and C(22) fatty acids, suggestive of an inhibition of the dehydratase step of the fatty-acid synthase type II elongation cycle. Consistently, overexpression of the essential hadABC genes encoding the (3R)-hydroxyacyl-acyl carrier protein dehydratases resulted in more than a 16- and 80-fold increase in the resistance of M. tuberculosis to ISO and TAC, respectively. A missense mutation in the hadA gene of spontaneous ISO- and TAC-resistant mutants was sufficient to confer upon M. tuberculosis high level resistance to both drugs. Other mutations found in hypersusceptible or resistant M. tuberculosis and Mycobacterium kansasii isolates mapped to hadC. Mutations affecting the non-essential mycolic acid methyltransferases MmaA4 and MmaA2 were also found in M. tuberculosis spontaneous ISO- and TAC-resistant mutants. That MmaA4, at least, participates in the activation of the two prodrugs as proposed earlier is not supported by our biochemical evidence. Instead and in light of the known interactions of both MmaA4 and MmaA2 with HadAB and HadBC, we propose that mutations affecting these enzymes may impact the binding of ISO and TAC to the dehydratases.

ACH-806, an NS4A antagonist, inhibits hepatitis C virus replication by altering the composition of viral replication complexes.

Treatment of hepatitis C patients with direct-acting antiviral drugs involves the combination of multiple small-molecule inhibitors of distinctive mechanisms of action. ACH-806 (or GS-9132) is a novel, small-molecule inhibitor specific for hepatitis C virus (HCV). It inhibits viral RNA replication in HCV replicon cells and was active in genotype 1 HCV-infected patients in a proof-of-concept clinical trial (1). Here, we describe a potential mechanism of action (MoA) wherein ACH-806 alters viral replication complex (RC) composition and function. We found that ACH-806 did not affect HCV polyprotein translation and processing, the early events of the formation of HCV RC. Instead, ACH-806 triggered the formation of a homodimeric form of NS4A with a size of 14 kDa (p14) both in replicon cells and in Huh-7 cells where NS4A was expressed alone. p14 production was negatively regulated by NS3, and its appearance in turn was associated with reductions in NS3 and, especially, NS4A content in RCs due to their accelerated degradation. A previously described resistance substitution near the N terminus of NS3, where NS3 interacts with NS4A, attenuated the reduction of NS3 and NS4A conferred by ACH-806 treatment. Taken together, we show that the compositional changes in viral RCs are associated with the antiviral activity of ACH-806. Small molecules, including ACH-806, with this novel MoA hold promise for further development and provide unique tools for clarifying the functions of NS4A in HCV replication.

Point mutations within the fatty acid synthase type II dehydratase components HadA or HadC contribute to isoxyl resistance in Mycobacterium tuberculosis.

The mechanism by which the antitubercular drug isoxyl (ISO) inhibits mycolic acid biosynthesis has not yet been reported. We found that point mutations in either the HadA or HadC component of the type II fatty acid synthase (FAS-II) are associated with increased levels of resistance to ISO in Mycobacterium tuberculosis. Overexpression of the HadAB, HadBC, or HadABC heterocomplex also produced high-level resistance. These results show that the FAS-II dehydratases are involved in ISO resistance.

Adapted J6/JFH1-based Hepatitis C virus recombinants with genotype-specific NS4A show similar efficacies against lead protease inhibitors, alpha interferon, and a putative NS4A inhibitor.

To facilitate studies of hepatitis C virus (HCV) NS4A, we aimed at developing J6/JFH1-based recombinants with genotype 1- to 7-specific NS4A proteins. We developed efficient culture systems expressing NS4A proteins of genotypes (isolates) 1a (H77 and TN), 1b (J4), 2a (J6), 4a (ED43), 5a (SA13), 6a (HK6a), and 7a (QC69), with peak infectivity titers of ∼3.5 to 4.5 log10 focus-forming units per ml. Except for genotype 2a (J6), growth depended on adaptive mutations identified in long-term culture. Genotype 1a, 1b, and 4a recombinants were adapted by amino acid substitutions F772S (p7) and V1663A (NS4A), while 5a, 6a, and 7a recombinants required additional substitutions in the NS3 protease and/or NS4A. We demonstrated applicability of the developed recombinants for study of antivirals. Genotype 1 to 7 NS4A recombinants showed similar responses to the protease inhibitors telaprevir (VX-950), boceprevir (Sch503034), simeprevir (TMC435350), danoprevir (ITMN-191), and vaniprevir (MK-7009), to alpha interferon 2b, and to the putative NS4A inhibitor ACH-806. The efficacy of ACH-806 was lower than that of protease inhibitors and was not influenced by changes at amino acids 1042 and 1065 (in the NS3 protease), which have been suggested to mediate resistance to ACH-806 in replicons. Genotype 1a, 1b, and 2a recombinants showed viral spread under long-term treatment with ACH-806, without acquisition of resistance mutations in the NS3-NS4A region. Relatively high concentrations of ACH-806 inhibited viral assembly, but not replication, in a single-cycle production assay. The developed HCV culture systems will facilitate studies benefitting from expression of genotype-specific NS4A in a constant backbone in the context of the complete viral replication cycle, including functional studies and evaluations of the efficacy of antivirals.

Phenylethyl butyrate enhances the potency of second-line drugs against clinical isolates of Mycobacterium tuberculosis.

Ethionamide (ETH) is a second-line drug for the treatment of tuberculosis. As a prodrug, ETH has to be activated by EthA. ethA is controlled by its repressor EthR. 2-Phenylethyl-butyrate (2-PEB) inhibits EthR binding, enhances expression of EthA, and thereby enhances the growth-inhibitory effects of ethionamide, isoxyl, and thiacetazone in Mycobacterium tuberculosis strains with resistance to ETH due to inhA promoter mutations but not ethA mutations.

Diafenthiuron residue and decline in pakchoi and soil under field application.

A simple analytical method based on QuEChERs was established for diafenthiuron residues in packhoi and soil. The residue levels and diaaipation rates of diafenthiuron in packhoi and soil were detected by HPLC-MS. And ultrasonic extraction was employed in the study to improve extraction effectiveness. At three fortification levels of 0.02, 0.1 and 1 mg/kg in packhoi and soil, recoveries were in the range 74.0 percent-100 percent, with relative standard deviations (RSD) of 6.1-14.8 percent. The limit of quantification (LOQ) of method was 0.02 mg/kg for packhoi and soil. In the supervised field trials, the half-lives of diafenthiuron in packhoi and soil were 1.27 and 5.94 day, respectively. The final residue levels of diafenthiuron could not be detected in soil, while only trace amount of diafenthiuron residues were detected in pakchoi.

Stearoyl-CoA desaturase is an essential enzyme for the parasitic protist Trypanosoma brucei.

Trypanosoma brucei, the etiologic agent of sleeping sickness, is exposed to important changes in nutrients and temperature during its life cycle. To adapt to these changes, the fluidity of its membranes plays a crucial role. This fluidity, mediated by the fatty-acid composition, is regulated by enzymes named desaturases. We have previously shown that the oleoyl desaturase is essential for Trypanosoma cruzi and T. brucei. In this work, we present experimental support for the relevance of stearoyl-CoA desaturase (SCD) for T. brucei's survival, in both its insect or procyclic-form (PCF) and bloodstream-form (BSF) stages. We evaluated this essentiality in two different ways: by generating a SCD knocked-down parasite line using RNA interference, and by chemical inhibition of the enzyme with two compounds, Isoxyl and a thiastearate with the sulfur atom at position 10 (10-TS). The effective concentration for 50% growth inhibition (EC(50)) of PCF was 1.0 ± 0.2 µM for Isoxyl and 5 ± 2 µM for 10-TS, whereas BSF appeared more susceptible with EC(50) values 0.10 ± 0.03 µM (Isoxyl) and 1.0 ± 0.6 µM (10-TS). RNA interference showed to be deleterious for both stages of the parasite. In addition, T. brucei-infected mice were fed with Isoxyl, causing a reduction of the parasitemia and an increase of the rodents' survival.

Estimation of inhibitory quotient using a comparative equilibrium dialysis assay for prediction of viral response to hepatitis C virus inhibitors.

The relationship of inhibitory quotient (IQ) with the virologic response to specific inhibitors of human hepatitis C virus (HCV) and the best method to correct for serum protein binding in calculating IQ have not been addressed. A common method is to determine a fold shift by comparing the EC(50) values determined in cell culture in the absence and presence of human serum (fold shift in EC(50) ), but this method has a number of disadvantages. In the present study, the fold shifts in drug concentrations between 100% human plasma (HP) and cell culture medium (CCM) were directly measured using a modified comparative equilibrium dialysis (CED) assay for three HCV protease inhibitors (PIs) and for a novel HCV inhibitor GS-9132. The fold shift values in drug concentration between the HP and CCM (CED ratio) were ∼1 for SCH-503034, VX-950 and GS-9132 and 13 for BILN-2061. These values were ∼3-10-fold lower than the fold shift values calculated from the EC(50) assay for all inhibitors except BILN-2061. Using the CED values, a consistent pharmacokinetic and pharmacodynamic relationship was observed for the four HCV inhibitors analysed. Specifically, an approximate 1 log(10) reduction in HCV RNA was achieved with an IQ close to 1, while 2-3 and greater log(10) reductions in HCV RNA were achieved with IQ values of 3-5 and greater, respectively. Thus, use of CED to define IQ provides a predictive and quantitative approach for the assessment of the in vivo potency of HCV PIs and GS-9132. This method provides a framework for the evaluation of other classes of drugs that are bound by serum proteins but require the presence of serum for in vitro evaluation.

Histone deacetylase activators: N-acetylthioureas serve as highly potent and isozyme selective activators for human histone deacetylase-8 on a fluorescent substrate.

We report, for the first time, that certain N-acetylthiourea derivatives serve as highly potent and isozyme selective activators for the recombinant form of human histone deacetylase-8 in the assay system containing Fluor-de-Lys as a fluorescent substrate. The experimental data reveals that such activating feature is manifested via decrease in the K(m) value of the enzyme's substrate and increase in the catalytic turnover rate of the enzyme.

Structural requirement of phenylthiourea analogs for their inhibitory activity of melanogenesis and tyrosinase.

Effect of a series of 1-phenylthioureas 1a-k and 1,3-disubstituted thioureas 2a-k were evaluated against melanin formation in melanoma B16 cell line and mushroom tyrosinase. Inhibitory activity of tyrosinase of 1-phenylthioureas 1a-k is parallel to their melanogenic inhibition. Thus, the melanogenic inhibition in melanoma B16 cells of 1-phenylthioureas could be the result of inhibition of tyrosinase. However, 1,3-diaryl or 1-phenyl-3-alkylthioureas, 2a-k, appears as melanogenic inhibitor without inhibition of tyrosinase. The molecular docking study of 1e and 2b to binding pocket of tyrosinase provided convincing explanation regarding the necessity of direct connection of planar phenyl to thiourea unit without N'-substitution of phenylthioureas 1 as tyrosinase inhibitor and 2 as non-tyrosinase inhibitor.

Silicon switch approach in TRPV1 antagonist MK-056 and its analogues.

In searching for opportunities to exploit the benefits of silicon in TRPV1 research, we tried to investigate the pharmacological effects of sila-substitution (C/Si exchange) of tert-butyl group in the MK-056 series. Compound 13a, with a 4-positioned trimethylsilanyl group on the B ring in place of tert-butyl group, exhibited the most potent antagonist activity with IC(50) values of 0.15 microM, which is almost equipotent with that of MK-056. This is the first example that tert-butyl group on MK-056 series can be replaced to the other substituent without loss of activity.

Effect of Treatment With 3-Octylthio-1,1,1-Trifluoropropan-2-One in the Diamondback Moth (Lepidoptera: Plutellidae) to the Toxicity of Diafenthiuron, Indoxacarb, and Bacillus thuringiensis.

The diamondback moth, Plutella xylostella (L.), is a worldwide insect pest of cruciferous crops. Although insecticides have long been used for its control, diamondback moth rapidly evolves resistance to almost any insecticide. In insects, juvenile hormone (JH) is critically involved in almost all biological processes. The correct activity of JH depends on the precise regulation of its titer, and juvenile hormone esterase (JHE) is the key regulator. Thus, JH and JHE have become important targets for new insecticide development. Trifluoromethyl ketones are specific JHE inhibitors, among which 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP) has the highest activity. The interaction effects between pretreatment with or combination of OTFP and the insecticides diafenthiuron, indoxacarb, and Bacillus thuringiensis (Bt) were investigated in diamondback moth larvae to determine OTFP's potential as an insecticide synergist. In third-instar larvae, both pretreatment and combination treatment with OTFP decreased or antagonized the toxicities of diafenthiuron, indoxacarb, and Bt at all set concentrations. In fourth-instar larvae, combination treatment with OTFP decreased or antagonized the toxicities of diafenthiuron and indoxacarb at all set concentrations. However, it increased or synergized the toxicity of Bt at lower concentrations despite the limited effect at higher concentrations. Our results indicated that the effect of OTFP on the toxicities of insecticides varied with the type and concentration, larval stage, and treatment method. These findings contribute to the better use of OTFP in diamondback moth control.

Naturally occurring dominant resistance mutations to hepatitis C virus protease and polymerase inhibitors in treatment-naïve patients.

UNLABELLED: Resistance mutations to hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease inhibitors in <1% of the viral quasispecies may still allow >1000-fold viral load reductions upon treatment, consistent with their reported reduced replicative fitness in vitro. Recently, however, an R155K protease mutation was reported as the dominant quasispecies in a treatment-naïve individual, raising concerns about possible full drug resistance. To investigate the prevalence of dominant resistance mutations against specifically targeted antiviral therapy for HCV (STAT-C) in the population, we analyzed HCV genome sequences from 507 treatment-naïve patients infected with HCV genotype 1 from the United States, Germany, and Switzerland. Phylogenetic sequence analysis and viral load data were used to identify the possible spread of replication-competent, drug-resistant viral strains in the population and to infer the consequences of these mutations upon viral replication in vivo. Mutations described to confer resistance to the protease inhibitors Telaprevir, BILN2061, ITMN-191, SCH6 and Boceprevir; the NS5B polymerase inhibitor AG-021541; and to the NS4A antagonist ACH-806 were observed mostly as sporadic, unrelated cases, at frequencies between 0.3% and 2.8% in the population, including two patients with possible multidrug resistance. Collectively, however, 8.6% of the patients infected with genotype 1a and 1.4% of those infected with genotype 1b carried at least one dominant resistance mutation. Viral loads were high in the majority of these patients, suggesting that drug-resistant viral strains might achieve replication levels comparable to nonresistant viruses in vivo. CONCLUSION: Naturally occurring dominant STAT-C resistance mutations are common in treatment-naïve patients infected with HCV genotype 1. Their influence on treatment outcome should further be characterized to evaluate possible benefits of drug resistance testing for individual tailoring of drug combinations when treatment options are limited due to previous nonresponse to peginterferon and ribavirin.