Compartmentalization of Resistance-Associated Substitutions in HIV/HCV-Infected Patients: Possible Correlation with Infecting HCV Genotype.

Resistance-associated substitutions (RASs) may exist prior to treatment and contribute to the failure of treatment with direct-acting antivirals (DAAs). As the major site of HCV replication, naturally occurring variants with RASs may segregate into the liver. In the present study, we performed viral population sequencing to retrospectively investigate the NS3 and NS5A RAS profiles in 34 HIV/HCV coinfected patients naïve to anti-HCV treatment who underwent diagnostic liver biopsy between 2000 and 2006 and had liver and plasma samples available. Sixteen were infected by HCV genotype (GT) 1a, 11 by GT3a, and 7 by GT4d. The analysis of the NS3 domain in GT1a showed a difference in strain between the liver and plasma in three cases, with a preponderance of specific RASs in the liver compartment. In GT4d samples, 6/7 coupled liver and plasma samples were concordant with no RASs. Sequence analysis of the NS5A domain showed the presence of RASs in the livers of 2/16 patients harboring GT1a but not in the corresponding plasma. In GT4d, NS5A RASs were detected in 7/7 liver tissues and 5/7 plasma samples. NS3 domain and NS5A domain were found to be conserved in plasma and livers of patients infected with GT3a. Thus, RASs within GT1a and GT4d more likely segregate into the liver and may explain the emergence of resistant strains during DAA treatment.

Substitution of I222L-E119V in neuraminidase from highly pathogenic avian influenza H7N9 virus exhibited synergistic resistance effect to oseltamivir in mice.

That the high frequency and good replication capacity of strains with reduced susceptibility to neuraminidase inhibitors (NAIs) in highly pathogenic avian influenza H7N9 (HPAI H7N9) virus made it a significance to further study its drug resistance. HPAI H7N9 viruses bearing NA I222L or E119V substitution and two mutations of I222L-E119V as well as their NAIs-sensitive counterpart were generated by reverse genetics for NA inhibition test and replication capability evaluation in vitro. The attenuated H7N9/PR8 recombinant viruses were developed to study the pathogenicity and drug resistance brought by the above substitutions to mice. The IC50 fold change of oseltamivir to HPAI H7N9 with NA222L-119V is 306.34 times than that of its susceptible strain, and 3.5 times than the E119V mutant virus. HPAI H7N9 bearing NA222L-119V had good replication ability with peak value of more than 6log10 TCID50/ml in MDCK cells. H7N9/PR8 virus bearing NA222L-119V substitutions leaded to diffuse pneumonia, significant weight loss and fatality in mice. NA E119V made H7N9/PR8 virus resistant to oseltamivir, and I222L-E119V had synergistic resistance to oseltamivir in mice. Due to the good fitness of drug resistant strains of HPAI H7N9 virus, it is necessary to strengthen drug resistance surveillance and new drug research.

GCG inhibits SARS-CoV-2 replication by disrupting the liquid phase condensation of its nucleocapsid protein.

Lack of detailed knowledge of SARS-CoV-2 infection has been hampering the development of treatments for coronavirus disease 2019 (COVID-19). Here, we report that RNA triggers the liquid-liquid phase separation (LLPS) of the SARS-CoV-2 nucleocapsid protein, N. By analyzing all 29 proteins of SARS-CoV-2, we find that only N is predicted as an LLPS protein. We further confirm the LLPS of N during SARS-CoV-2 infection. Among the 100,849 genome variants of SARS-CoV-2 in the GISAID database, we identify that ~37% (36,941) of the genomes contain a specific trio-nucleotide polymorphism (GGG-to-AAC) in the coding sequence of N, which leads to the amino acid substitutions, R203K/G204R. Interestingly, NR203K/G204R exhibits a higher propensity to undergo LLPS and a greater effect on IFN inhibition. By screening the chemicals known to interfere with N-RNA binding in other viruses, we find that (-)-gallocatechin gallate (GCG), a polyphenol from green tea, disrupts the LLPS of N and inhibits SARS-CoV-2 replication. Thus, our study reveals that targeting N-RNA condensation with GCG could be a potential treatment for COVID-19.

d-Amino acid substitutions and dimerization increase the biological activity and stability of an IL-15 antagonist peptide.

Interleukin (IL)-15 plays an important role in several inflammatory diseases. We have previously identified an IL-15 antagonist called P8 peptide, which binds specifically to IL-15 receptor alpha subunit. However, the P8 peptide rapidly degraded by proteases, limiting its therapeutic application. Thus, we replaced each P8 peptide l-amino acid by its corresponding d-isomers. First, we determined the biological activity of the resulting peptides in a proliferation assay by using CTLL-2 cells. The substitution of l-Ala by d-Ala ([A4a]P8 peptide) increased the inhibitory effect of the P8 peptide in CTLL-2 cells in five-fold. In addition to that, the [A4a]P8 peptide dimer showed the most inhibitory effect. To protect the [A4a]P8 peptide and its dimer against exopeptidase activity, we acetylated the N-terminal of these peptides. At least a three-fold reduction in antagonist activity of acetylated peptides was exhibited. However, the substitution of the N-terminal l-Lys residue of [A4a]P8 peptide and its dimer by d-Lys ([K1k;A4a]P8 peptide) did not affect the antagonist effect of the aforementioned peptides. The [K1k;A4a]P8 peptide dimer was stable to the degradation of trypsin, chymotrypsin, and pepsin up until 48 min. Also, the safety and immunogenicity studies in healthy BALB/c mice demonstrated that the administration of this peptide did not affect the clinical parameters of the animals nor generated antipeptide antibodies. Our findings reveal that two distinct d-amino acid substitutions and dimerization increase the biological activity and stability of P8 peptide. The resulting peptide constitutes a novel IL-15 antagonist with potential applicability in inflammatory diseases.

Targeted Amino Acid Substitutions in a Trichoderma Peptaibol Confer Activity against Fungal Plant Pathogens and Protect Host Tissues from Botrytis cinerea Infection.

Fungal species belonging to the Trichoderma genus are commonly used as biocontrol agents against several crop pathogens. Among their secondary metabolites, peptaibols are helical, antimicrobial peptides, which are structurally stable even under extreme pH and temperature conditions. The promise of peptaibols as agrochemicals is, however, hampered by poor water solubility, which inhibits efficient delivery for practical use in crop protection. Using a versatile synthetic strategy, based on green chemistry procedures, we produced water-soluble analogs of the short-length peptaibol trichogin. Although natural trichogin was inactive against the tested fungal plant pathogens (Botrytis cinerea, Bipolaris sorokiniana, Fusarium graminearum, and Penicillium expansum), three analogs completely inhibited fungal growth at low micromolar concentrations. The most effective peptides significantly reduced disease symptoms by B. cinerea on common bean and grapevine leaves and ripe grape berries without visible phytotoxic effects. An in-depth conformational analysis featuring a 3D-structure-activity relationship study indicated that the relative spatial position of cationic residues is crucial for increasing peptide fungicidal activity.

Fitness-associated substitutions following failure of direct-acting antivirals assessed by deep sequencing of full-length hepatitis C virus genomes.

BACKGROUND: In hepatitis C virus (HCV) infection, treatment failure is generally associated with the selection of resistance-associated substitutions (RAS) conferring reduced susceptibility to direct-acting antiviral (DAA) drugs. Resistant variants continue to replicate after the end of treatment with potential for transmission. This may result from the selection of "fitness-associated substitutions". AIM: To characterise potential "fitness-associated substitutions" in patients infected with genotype 3a failing DAA drugs METHODS: By means of shotgun metagenomics, we sequenced full-length HCV genomes at treatment initiation and at virological relapse in eight patients infected with genotype 3a with cirrhosis failing sofosbuvir and an NS5A inhibitor. The impact of amino acid changes occurring outside of DAA target regions selected in at least two patients were assessed on the in vitro susceptibility to an NS5A inhibitor and replication capacity. RESULTS: At treatment failure, besides selection of known NS5A RASs, especially Y93H, a large number of amino acid changes was observed outside of DAA target regions. We identified four amino acid positions at which observed changes substantially improved in vitro replication capacity without affecting NS5A inhibitor susceptibility. CONCLUSIONS: This is the first in vivo observation combined with in vitro confirmation of selection of phenotypically characterised "fitness-associated substitutions" together with RASs at the time of sofosbuvir-NS5A inhibitor treatment failure in patients infected with genotype 3a with cirrhosis. Our findings may explain the persistence of resistant HCV variants after treatment in patients who did not achieve sustained virological remission.

Acquisition of Cabozantinib-Sensitive MET D1228N Mutation During Progression on Crizotinib in MET-Amplified Triple-Negative Breast Cancer.

BACKGROUND: Targeting of somatic MET mutations using crizotinib has led to strong clinical responses, most frequently in patients with lung cancer, raising the possibility of adopting similar treatment strategies in patients with MET alterations in other cancer types. PATIENT AND METHODS: We describe a patient with advanced triple-negative breast cancer with a 30-fold amplification of MET. Next-generation sequencing of pre- and postprogression biopsies was performed to identify the resistance mechanism emerging after an initial exceptional response to crizotinib. The response of the resistance mutant to type I and II MET inhibitors was assessed in cultured cells. RESULTS: After progressing on crizotinib, a MET-D1228N mutation was detected, which is located in the crizotinib-binding region of the MET kinase domain. Experimental studies demonstrated that this mutation confers complete resistance to crizotinib yet retains cabozantinib sensitivity. Treatment of the patient with cabozantinib led to a subjective improvement in clinical symptoms, but the patient progressed after 7 weeks. CONCLUSION: Although MET mutations are rare in breast cancer, these patients may experience substantial clinical benefit from crizotinib treatment. Nevertheless, drug resistance owing to on-target MET mutations will likely be frequently encountered and comprehensive mechanistic studies to assess sensitivity of these mutants to a series of potential second-line therapies may help guide subsequent treatment for these patients.

Rapid detection of an I38T amino acid substitution in influenza polymerase acidic subunit associated with reduced susceptibility to baloxavir marboxil.

BACKGROUND: The novel cap-dependent endonuclease inhibitor baloxavir marboxil was approved in February 2018 for the treatment of influenza virus infection in Japan. In vitro studies have revealed that an I38T substitution in the polymerase acidic subunit (PA) is associated with reduced susceptibility of influenza viruses to baloxavir. OBJECTIVES: Development of a rapid and simple method for monitoring influenza A(H1N1)pdm09, A(H3N2), and B viruses possessing the I38T substitution in PA. METHODS: Three assays were developed based on RNase H2-dependent PCR (rhPCR) and named A/H1pdm PA_I38T rhPCR, A/H3 PA_I38T rhPCR, and B PA_I38T rhPCR. The assays were evaluated using cDNAs synthesized from in vitro-transcribed PA gene RNA controls, RNAs purified from viruses isolated in the 2017/2018 and 2018/2019 influenza seasons, and RNAs purified from clinical specimens collected in the 2018/2019 influenza season. RESULTS: The assays developed in this study accurately discriminated PA I38 and PA T38 with high sensitivity. CONCLUSIONS: Our assays should be considered a powerful tool for monitoring the emergence of baloxavir-resistant influenza viruses.

Kinetic Profile and Molecular Dynamic Studies Show that Y229W Substitution in an NDM-1/L209F Variant Restores the Hydrolytic Activity of the Enzyme toward Penicillins, Cephalosporins, and Carbapenems.

The New Delhi metallo-ß-lactamase-1 (NDM-1) enzyme is the most common metallo-ß-lactamase identified in many Gram-negative bacteria causing severe nosocomial infections. The aim of this study was to focus the attention on non-active-site residues L209 and Y229 of NDM-1 and to investigate their role in the catalytic mechanism. Specifically, the effect of the Y229W substitution in the L209F variant was evaluated by antimicrobial susceptibility testing, kinetic, and molecular dynamic (MD) studies. The Y229W single mutant and L209F-Y229W double mutant were generated by site-directed mutagenesis. The Km , kcat, and kcat/Km kinetic constants, calculated for the two mutants, were compared with those of (wild-type) NDM-1 and the L209F variant. Compared to the L209F single mutant, the L209F-Y229W double mutant showed a remarkable increase in kcat values of 100-, 240-, 250-, and 420-fold for imipenem, meropenem, benzylpenicillin, and cefepime, respectively. In the L209F-Y229W enzyme, we observed a remarkable increase in kcat/Km of 370-, 140-, and 80-fold for cefepime, meropenem, and cefazolin, respectively. The same behavior was noted using the antimicrobial susceptibility test. MD simulations were carried out on both L209F and L209F-Y229W enzymes complexed with benzylpenicillin, focusing attention on the overall mechanical features and on the differences between the two systems. With respect to the L209F variant, the L209F-Y229W double mutant showed mechanical stabilization of loop 10 and the N-terminal region. In addition, Y229W substitution destabilized both the C-terminal region and the region from residues 149 to 154. The epistatic effect of the Y229W mutation jointly with the stabilization of loop 10 led to a better catalytic efficiency of ß-lactams. NDM numbering is used in order to facilitate the comparison with other NDM-1 studies.

Assessing baloxavir susceptibility of influenza viruses circulating in the United States during the 2016/17 and 2017/18 seasons.

The anti-influenza therapeutic baloxavir targets cap-dependent endonuclease activity of polymerase acidic (PA) protein. We monitored baloxavir susceptibility in the United States with next generation sequencing analysis supplemented by phenotypic one-cycle infection assay. Analysis of PA sequences of 6,891 influenza A and B viruses collected during 2016/17 and 2017/18 seasons showed amino acid substitutions: I38L (two A(H1N1)pdm09 viruses), E23G (two A(H1N1)pdm09 viruses) and I38M (one A(H3N2) virus); conferring 4-10-fold reduced susceptibility to baloxavir.

Wnt5a causes ROR1 to complex and activate cortactin to enhance migration of chronic lymphocytic leukemia cells.

Chronic lymphocytic leukemia cells (CLL) migrate between the blood and lymphoid tissues in response to chemokines. Such migration requires structured cytoskeletal-actin polymerization, which may involve the protein cortactin. We discovered that treatment of CLL cells with Wnt5a causes Receptor tyosin kinase-like orphan receptor 1 (ROR1) to bind cortactin, which undergoes tyrosine phosphorylation at Y421, recruits ARHGEF1, and activates RhoA, thereby enhancing leukemia-cell migration; such effects could be inhibited by cirmtuzumab, a humanized mAb specific for ROR1. We transfected the CLL-cell-line MEC1 with either full-length ROR1 or various mutant forms of ROR1 to examine the structural features required for binding cortactin. We found that the proline-rich domain (PRD) was necessary for ROR1 to recruit cortactin. We generated MEC1 cells that each expressed a mutant form of ROR1 with a single amino-acid substitution of alanine (A) for proline (P) in potential SH3-binding sites in the ROR1-PRD at positions 784, 808, 826, 841, or 850. In contrast to wild-type ROR1, or other ROR1P=>A mutants, ROR1P(841)A failed to complex with cortactin or ARHGEF1 in response to Wnt5a. Moreover, Wnt5a could not induce MEC1-ROR1P(841)A to phosphorylate cortactin or enhance CLL-cell F-actin polymerization. Taken together, these studies show that cortactin plays an important role in ROR1-dependent Wnt5a-enhanced CLL-cell migration.

Characterization of Influenza B Virus Variants with Reduced Neuraminidase Inhibitor Susceptibility.

Treatment options for influenza B virus infections are limited to neuraminidase inhibitors (NAIs), which block the neuraminidase (NA) glycoprotein on the virion surface. The development of NAI resistance would therefore result in a loss of antiviral treatment options for influenza B virus infections. This study characterized two contemporary influenza B viruses with known resistance-conferring NA amino acid substitutions, D197N and H273Y, detected during routine surveillance. The D197N and H273Y variants were characterized in vitro by assessing NA enzyme activity and affinity, as well as replication in cell culture compared to those of NAI-sensitive wild-type viruses. In vivo studies were also performed in ferrets to assess the replication and transmissibility of each variant. Mathematical models were used to analyze within-host and between-host fitness of variants relative to wild-type viruses. The data revealed that the H273Y variant had NA enzyme function similar to that of its wild type but had slightly reduced replication and transmission efficiency in vivo The D197N variant had impaired NA enzyme function, but there was no evidence of reduction in replication or transmission efficiency in ferrets. Our data suggest that the influenza B virus variant with the H273Y NA substitution had a more notable reduction in fitness compared to wild-type viruses than the influenza B variant with the D197N NA substitution. Although a D197N variant is yet to become widespread, it is the most commonly detected NAI-resistant influenza B virus in surveillance studies. Our results highlight the need to carefully monitor circulating viruses for the spread of influenza B viruses with the D197N NA substitution.

Resistance Analysis of a 3-Day Monotherapy Study with Glecaprevir or Pibrentasvir in Patients with Chronic Hepatitis C Virus Genotype 1 Infection.

Glecaprevir (an NS3/4A protease inhibitor) and pibrentasvir (an NS5A inhibitor) are potent and pangenotypic hepatitis C virus (HCV) direct-acting antivirals. This report describes the baseline polymorphisms and treatment-emergent substitutions in NS3 or NS5A detected in samples from HCV genotype 1-infected patients receiving 3-day monotherapy of glecaprevir or pibrentasvir, respectively. None of the NS3 polymorphisms detected in the 47 baseline samples collected prior to glecaprevir monotherapy conferred reduced susceptibility to glecaprevir. The NS3 A156T substitution, which conferred resistance to glecaprevir but had low replication efficiency, emerged in one genotype 1a-infected patient among the 35 patients with available post-baseline sequence data. Baseline NS5A polymorphisms were detected in 12 of 40 patients prior to pibrentasvir monotherapy; most polymorphisms were single-position NS5A amino acid substitutions that did not confer resistance to pibrentasvir. Among the 19 patients with available post-baseline NS5A sequence data, 3 had treatment-emergent NS5A substitutions during pibrentasvir monotherapy. All treatment-emergent NS5A substitutions were linked multiple-position, almost exclusively double-position, substitutions that conferred resistance to pibrentasvir. Replicons engineered with these double-position substitutions had low replication efficiency. In conclusion, resistance-conferring substitutions emerged in a small number of genotype 1-infected patients during glecaprevir or pibrentasvir monotherapy; unlike other NS5A inhibitors, pibrentasvir did not select single-position NS5A substitutions during monotherapy.

New Locus of Drug Resistance in the Human Cytomegalovirus UL56 Gene Revealed by In Vitro Exposure to Letermovir and Ganciclovir.

Letermovir is a human cytomegalovirus (CMV) terminase inhibitor recently approved as prophylaxis in stem cell transplant recipients. In further studies of emerging drug resistance, a baseline laboratory CMV strain was serially propagated in cell culture under a combination of letermovir and ganciclovir. In eight experiments, UL56 terminase gene mutations were detected beginning at 10 passages and included novel amino acid substitutions V236A, L328V, and A365S in a region previously associated with letermovir resistance. Outside this region, the UL56 substitution C25F was detected at moderate drug concentrations in two experiments as either the first detected mutation or an addition to a preexisting V231L substitution. In all cases, mutation at UL56 codon 325 conferring absolute letermovir resistance eventually developed at a median of 20 passages. No UL97 kinase or UL54 DNA polymerase mutations relevant to ganciclovir resistance were detected until many passages after the first detection of the UL56 mutations. UL56 substitutions V236A, L328V, and A365S were shown to confer borderline or low-grade letermovir resistance, while C25F conferred a 5.4-fold increase in letermovir resistance (50% effective concentration [EC50]) by itself and a 46-fold increase in combination with V231L. The evolution of resistance mutations sooner in UL56 than in UL54 or UL97 is consistent with prior in vitro observations, and UL56 codon 25 is a genetic locus for letermovir resistance distinct from loci previously described.

Mutations in Gene fusA1 as a Novel Mechanism of Aminoglycoside Resistance in Clinical Strains of Pseudomonas aeruginosa.

Resistance of clinical strains of Pseudomonas aeruginosa to aminoglycosides can result from production of transferable aminoglycoside-modifying enzymes, of 16S rRNA methylases, and/or mutational derepression of intrinsic multidrug efflux pump MexXY(OprM). We report here the characterization of a new type of mutant that is 4- to 8-fold more resistant to 2-deoxystreptamine derivatives (e.g., gentamicin, amikacin, and tobramycin) than the wild-type strain PAO1. The genetic alterations of three in vitro mutants were mapped on fusA1 and found to result in single amino acid substitutions in domains II, III, and V of elongation factor G (EF-G1A), a key component of translational machinery. Transfer of the mutated fusA1 alleles into PAO1 reproduced the resistance phenotype. Interestingly, fusA1 mutants with other amino acid changes in domains G, IV, and V of EF-G1A were identified among clinical strains with decreased susceptibility to aminoglycosides. Allelic-exchange experiments confirmed the relevance of these latter mutations and of three other previously reported alterations located in domains G and IV. Pump MexXY(OprM) partly contributed to the resistance conferred by the mutated EF-G1A variants and had additive effects on aminoglycoside MICs when mutationally upregulated. Altogether, our data demonstrate that cystic fibrosis (CF) and non-CF strains of P. aeruginosa can acquire a therapeutically significant resistance to important aminoglycosides via a new mechanism involving mutations in elongation factor EF-G1A.

A Single Amino Acid Substitution in the Third Transmembrane Region Has Opposite Impacts on the Selectivity of the Parasiticides Fluralaner and Ivermectin for Ligand-Gated Chloride Channels.

Fluralaner (Bravecto) is a recently marketed isoxazoline ectoparasiticide. This compound potently inhibits GABA-gated chloride channels (GABACls) and less potently glutamate-gated chloride channels (GluCls) in insects. The mechanism underlying this selectivity is unknown. Therefore, we sought to identify the amino acid residues causing the low potency of fluralaner toward GluCls. We examined the fluralaner sensitivity of mutant housefly (Musca domestica) GluCls in which amino acid residues in the transmembrane subunit interface were replaced with the positionally equivalent amino acids of Musca GABACls. Of these amino acids, substitution of an amino acid (Leu315) in the third transmembrane region (TM3) with an aromatic amino acid dramatically enhanced the potency of fluralaner in the GluCls. In stark contrast to the enhancement of fluralaner potency, this mutation eliminated the activation of currents and the potentiation but not the antagonism of glutamate responses that are otherwise all elicited by the macrolide parasiticide ivermectin (IVM). Our findings indicate that the amino acid Leu315 in Musca GluCls plays significant roles in determining the selectivity of fluralaner and IVM for these channels. Given the high sequence similarity of TM3, this may hold true more widely for the GluCls and GABACls of other insect species.

Nonsynonymous Polymorphisms in the Human AS3MT Arsenic Methylation Gene: Implications for Arsenic Toxicity.

Arsenic methylation, the primary biotransformation in the human body, is catalyzed by the enzyme As(III) S-adenosylmethionine (SAM) methyltransferases (hAS3MT). This process is thought to be protective from acute high-level arsenic exposure. However, with long-term low-level exposure, hAS3MT produces intracellular methylarsenite (MAs(III)) and dimethylarsenite (DMAs(III)), which are considerably more toxic than inorganic As(III) and may contribute to arsenic-related diseases. Several single nucleotide polymorphisms (SNPs) in putative regulatory elements of the hAS3MT gene have been shown to be protective. In contrast, three previously identified exonic SNPs (R173W, M287T, and T306I) may be deleterious. The goal of this study was to examine the effect of single amino acid substitutions in hAS3MT on the activity of the enzyme that might explain their contributions to adverse health effects of environmental arsenic. We identified five additional intragenic variants in hAS3MT (H51R, C61W, I136T, W203C, and R251H). We purified the eight polymorphic hAS3MT proteins and characterized their enzymatic properties. Each enzyme had low methylation activity through decreased affinity for substrate, lower overall rates of catalysis, or lower stability. We propose that amino acid substitutions in hAS3MT with decreased catalytic activity lead to detrimental responses to environmental arsenic and may increase the risk of arsenic-related diseases.

Retreatment of patients with treatment failure of direct-acting antivirals: Focus on hepatitis C virus genotype 1b.

The recent development of direct-acting antiviral agents (DAAs) against hepatitis C virus (HCV) infection could lead to higher sustained virological response (SVR) rates, with shorter treatment durations and fewer adverse events compared with regimens that include interferon. However, a relatively small proportion of patients cannot achieve SVR in the first treatment, including DAAs with or without peginterferon and/or ribavirin. Although retreatment with a combination of DAAs should be conducted for these patients, it is more difficult to achieve SVR when retreating these patients because of resistance-associated substitutions (RASs) or treatment-emergent substitutions. In Japan, HCV genotype 1b (GT1b) is founded in 70% of HCV-infected individuals. In this minireview, we summarize the retreatment regimens and their SVR rates for HCV GT1b. It is important to avoid drugs that target the regions targeted by initial drugs, but next-generation combinations of DAAs, such as sofosbuvir/velpatasvir/voxilaprevir for 12 wk or glecaprevir/pibrentasvir for 12 wk, are proposed to be potential solution for the HCV GT1b-infected patients with treatment failure, mainly on a basis of targeting distinctive regions. Clinicians should follow the new information and resources for DAAs and select the proper combination of DAAs for the retreatment of HCV GT1b-infected patients with treatment failure.

Effect of Cysteamine on Mutant ASL Proteins with Cysteine for Arginine Substitutions.

INTRODUCTION: Cysteamine is used to treat cystinosis via the modification of cysteine residues substituting arginine in mutant proteins. OBJECTIVES: We investigated the effect of cysteamine on mutant argininosuccinate lyase (ASL), the second most common defect in the urea cycle. METHODS: In an established mammalian expression system, 293T cell lysates were produced after transfection with all known cysteine for arginine mutations in the ASL gene (p.Arg94Cys, p.Arg95Cys, p.Arg168Cys, p.Arg379Cys, and p.Arg385Cys), allowing testing of the effect of cysteamine over 48 h in the culture medium as well as for 1 h immediately prior to the enzyme assay. RESULTS: Cysteamine at low concentrations showed no effect on 293T cell viability, ASL protein expression, or ASL activity when applied during cell culture. However, incubation of transfected cells with 0.05 mM cysteamine immediately before the enzyme assay resulted in increased ASL activity of p.Arg94Cys, p.Arg379Cys, and p.Arg385Cys by 64, 20, and 197 %, respectively, and this result was significant (p < 0.01). Cell lysates carrying p.Arg385Cys and treated with cysteamine recover enzyme activity that is similar to the untreated designed mutation p.Arg385Lys, providing circumstantial evidence for the assumed cysteamine-induced change of a cysteine to a lysine analogue. CONCLUSION: Since 12 % of all known genotypes in ASL deficiency are affected by a cysteine for arginine mutation, we conclude that the potential of cysteamine or of related substances as remedy for this disease should be investigated further.