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.

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.

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.

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.

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.

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.

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.

High error rates in selenocysteine insertion in mammalian cells treated with the antibiotic doxycycline, chloramphenicol, or geneticin.

Antibiotics target bacteria by interfering with essential processes such as translation, but their effects on translation in mammalian cells are less well characterized. We found that doxycycline, chloramphenicol, and Geneticin (G418) interfered with insertion of selenocysteine (Sec), which is encoded by the stop codon, UGA, into selenoproteins in murine EMT6 cells. Treatment of EMT6 cells with these antibiotics reduced enzymatic activities and Sec insertion into thioredoxin reductase 1 (TR1) and glutathione peroxidase 1 (GPx1). However, these proteins were differentially affected due to varying errors in Sec insertion at UGA. In the presence of doxycycline, chloramphenicol, or G418, the Sec-containing form of TR1 decreased, whereas the arginine-containing and truncated forms of this protein increased. We also detected antibiotic-specific misinsertion of cysteine and tryptophan. Furthermore, misinsertion of arginine in place of Sec was commonly observed in GPx1 and glutathione peroxidase 4. TR1 was the most affected and GPx1 was the least affected by these translation errors. These observations were consistent with the differential use of two Sec tRNA isoforms and their distinct roles in supporting accuracy of Sec insertion into selenoproteins. The data reveal widespread errors in inserting Sec into proteins and in dysregulation of selenoprotein expression and function upon antibiotic treatment.

Combination treatment with hepatitis C virus protease and NS5A inhibitors is effective against recombinant genotype 1a, 2a, and 3a viruses.

With the development of directly acting antivirals, hepatitis C virus (HCV) therapy entered a new era. However, rapid selection of resistance mutations necessitates combination therapy. To study combination therapy in infectious culture systems, we aimed at developing HCV semi-full-length (semi-FL) recombinants relying only on the JFH1 NS3 helicase, NS5B, and the 3' untranslated region. With identified adaptive mutations, semi-FL recombinants of genotypes(isolates) 1a(TN) and 3a(S52) produced supernatant infectivity titers of ~4 log(10) focus-forming units/ml in Huh7.5 cells. Genotype 1a(TN) adaptive mutations allowed generation of 1a(H77) semi-FL virus. Concentration-response profiles revealed the higher efficacy of the NS3 protease inhibitor asunaprevir (BMS-650032) and the NS5A inhibitor daclatasvir (BMS-790052) against 1a(TN and H77) than 3a(S52) viruses. Asunaprevir had intermediate efficacy against previously developed 2a recombinants J6/JFH1 and J6cc. Daclatasvir had intermediate efficacy against J6/JFH1, while low sensitivity was confirmed against J6cc. Using a cross-titration scheme, infected cultures were treated until viral escape or on-treatment virologic suppression occurred. Compared to single-drug treatment, combination treatment with relatively low concentrations of asunaprevir and daclatasvir suppressed infection with all five recombinants. Escaped viruses primarily had substitutions at amino acids in the NS3 protease and NS5A domain I reported to be genotype 1 resistance mutations. Inhibitors showed synergism at drug concentrations reported in vivo. In summary, semi-FL HCV recombinants, including the most advanced reported genotype 3a infectious culture system, permitted genotype-specific analysis of combination treatment in the context of the complete viral life cycle. Despite differential sensitivity to lead compound NS3 protease and NS5A inhibitors, genotype 1a, 2a, and 3a viruses were suppressed by combination treatment with relatively low concentrations.

Chronic beryllium disease, HLA-DPB1, and the DP peptide binding groove.

Multiple epidemiologic studies demonstrate associations between chronic beryllium disease (CBD), beryllium sensitization (BeS), and HLA-DPB1 alleles with a glutamic acid residue at position 69 (E69). Results suggest that the less-frequent E69 variants (non-*0201/*0202 alleles) might be associated with greater risk of CBD. In this study, we sought to define specific E69-carrying alleles and their amino acid sequences in the DP peptide binding groove, as well as their relationship to CBD and BeS risk, using the largest case control study to date. We enrolled 502 BeS/CBD subjects and 653 beryllium-exposed controls from three beryllium industries who gave informed consent for participation. Non-Hispanic white cases and controls were frequency-matched by industry. HLA-DPB1 genotypes were determined using sequence-specific primer PCR. The E69 alleles were tested for association with disease individually and grouped by amino acid structure using logistic regression. The results show that CBD cases were more likely than controls to carry a non-*02 E69 allele than an *02 E69, with odds ratios (95% confidence interval) ranging from 3.1 (2.1-4.5) to 3.9 (2.6-5.9) (p < 0.0001). Polymorphic amino acids at positions 84 and 11 were associated with CBD: DD versus GG, 2.8 (1.8-4.6), p < 0.0001; GD versus GG, 2.1 (1.5-2.8), p < 0.0001; LL versus GG, 3.2 (1.8-5.6), p < 0.0001; GL versus GG, 2.8 (2.1-3.8), p < 0.0001. Similar results were found within the BeS group and CBD/BeS combined group. We conclude that the less frequent E69 alleles confer more risk for CBD than does *0201. Recent studies examining how the composition and structure of the binding pockets influence peptide binding in MHC genes, as well of studies showing the topology of the TCR to likely bind DPB1 preferentially, give plausible biological rationale for these findings.

Euryhaline pufferfish NBCe1 differs from nonmarine species NBCe1 physiology.

Marine fish drink seawater and eliminate excess salt by active salt transport across gill and gut epithelia. Euryhaline pufferfish (Takifugu obscurus, mefugu) forms a CaCO(3) precipitate on the luminal gut surface after transitioning to seawater. NBCe1 (Slc4a4) at the basolateral membrane of intestinal epithelial cell plays a major role in transepithelial intestinal HCO(3)(-) secretion and is critical for mefugu acclimation to seawater. We assayed fugu-NBCe1 (fNBCe1) activity in the Xenopus oocyte expression system. Similar to NBCe1 found in other species, fNBCe1 is an electrogenic Na(+)/HCO(3)(-) cotransporter and sensitive to the stilbene inhibitor DIDS. However, our experiments revealed several unique and distinguishable fNBCe1 transport characteristics not found in mammalian or other teleost NBCe1-orthologs: electrogenic Li(+)/nHCO(3)(-) cotransport; HCO(3)(-) independent, DIDS-insensitive transport; and increased basal intracellular Na(+) accumulation. fNBCe1 is a voltage-dependent Na(+)/nHCO(3)(-) cotransporter that rectifies, independently from the extracellular Na(+) or HCO(3)(-) concentration, around -60 mV. Na(+) removal (0Na(+) prepulse) is necessary to produce the true HCO(3)(-)-elicited current. HCO(3)(-) addition results in huge outward currents with quick current decay. Kinetic analysis of HCO(3)(-) currents reveals that fNBCe1 has a much higher transport capacity (higher maximum current) and lower affinity (higher K(m)) than human kidney NBCe1 (hkNBCe1) does in the physiological range (membrane potential = -80 mV; [HCO(3)(-)] = 10 mM). In this state, fNBCe1 is in favor of operating as transepithelial HCO(3)(-) secretion, opposite of hkNBCe1, from blood to the luminal side. Thus, fugu-NBCe1 represents the first ortholog-based tool to study amino acid substitutions in NBCe1 and how those change ion and voltage dependence.

Clinical course and predictive factors of virological response in long-term lamivudine plus adefovir dipivoxil combination therapy for lamivudine-resistant chronic hepatitis B patients.

The aims of this study were to assess the long-term efficacy of lamivudine (LAM) plus adefovir dipivoxil (ADV) combination therapy in patients with chronic hepatitis B resistant to LAM, to identify predictive factors of complete viral response (HBV-DNA <2.6 log copies/ml at 12 months of combination therapy), and to analyze amino acid substitutions associated with treatment resistance in the hepatitis B virus (HBV) genome. Seventy-two patients who received ADV in addition to LAM for breakthrough hepatitis were enrolled. Undetectable HBV-DNA was observed in 61%, 74%, 81%, 84%, and 85% at 12, 24, 36, 48, and 60 months of combination therapy, respectively. On multivariate analysis, undetectable HBV-DNA during the preceding LAM monotherapy (P < 0.0001), alanine aminotransferase value ≥ the upper limit of normal × 6 (P = 0.006) and HBV-DNA level < 6.0 log copies/ml at the initiation of combination therapy (P = 0.007) were independent significant predictors of complete viral response. The cumulative rate of undetectable HBV-DNA was significantly higher in patients with response to the preceding LAM monotherapy than in those with poor response to it. Breakthrough hepatitis occurred in three patients without complete viral response and with poor response to the preceding LAM monotherapy, and rtA181A/V substitution was detected in one of the three patients. In conclusion, undetectable HBV-DNA during the LAM monotherapy was the strongest independent predictor of complete viral response to the following combination therapy. The efficacy of LAM plus ADV combination therapy may be determined by viral response to the preceding LAM monotherapy.

Two novel fusion inhibitors of human respiratory syncytial virus.

To search for novel drugs against human respiratory syncytial virus (RSV), we have screened a diversity collection of 16,671 compounds for anti-RSV activity in cultures of HEp-2 cells. Two of the hit compounds, i.e., the N-(2-hydroxyethyl)-4-methoxy-N-methyl-3-(6-methyl[1,2,4]triazolo[3,4-a]phthalazin-3-yl)benzenesulfonamide (designated as P13) and the 1,4-bis(3-methyl-4-pyridinyl)-1,4-diazepane (designated as C15), reduced the virus infectivity with IC50 values of 0.11 and 0.13µM respectively. The concentration of P13 and C15 that reduced the viability of HEp-2 cells by 50% was 310 and 75µM respectively. Both P13 and C15 exhibited no direct virucidal activity or inhibitory effects on the virus attachment to cells. However, to inhibit formation of RSV-induced syncytial plaques P13 and C15 had to be present during the virus entry into the cells and the cell-to-cell transmission of the virus. The RSV multiplication in HEp-2 cells in the presence of P13 or C15 resulted in rapid selection of viral variants that were ∼1000 times less sensitive to these drugs than original virus. Sequencing of resistant viruses revealed presence of amino acid substitutions in the F protein of RSV, i.e., the D489G for C15-selected, and the T400I and N197T (some clones) for the P13-selected virus variants. In conclusion, we have identified two novel fusion inhibitors of RSV, and the detailed understanding of their mode of antiviral activity including selection for the drug resistant viral variants may help to develop selective and efficient anti-RSV drugs.

Enhanced anticancer effect of the combination of BIBW2992 and thymidylate synthase-targeted agents in non-small cell lung cancer with the T790M mutation of epidermal growth factor receptor.

Most non-small cell lung cancer (NSCLC) tumors with activating mutations of the epidermal growth factor receptor (EGFR) are initially responsive to first-generation, reversible EGFR tyrosine kinase inhibitors (TKI) such as gefitinib, but they subsequently develop resistance to these drugs through either acquisition of an additional T790M mutation of EGFR or amplification of the proto-oncogene MET. We have now investigated the effects of combination treatment with thymidylate synthase (TS)-targeting drugs and the second-generation, irreversible EGFR-TKI BIBW2992 on the growth of NSCLC cells with the T790M mutation. The effects of BIBW2992 on EGFR signaling and TS expression in gefitinib-resistant NSCLC cells were examined by immunoblot analysis. The effects of BIBW2992 and the TS-targeting agents S-1 (or 5-fluorouracil) or pemetrexed on the growth of gefitinib-resistant NSCLC cells were examined both in vitro and in vivo. The combination of BIBW2992 with 5-fluorouracil or pemetrexed synergistically inhibited the proliferation of NSCLC cells with the T790M mutation in vitro, whereas an antagonistic interaction was apparent in this regard between gefitinib and either of these TS-targeting agents. BIBW2992 induced downregulation of TS in the gefitinib-resistant NSCLC cells, implicating depletion of TS in the enhanced antitumor effect of the combination therapy. The combination of BIBW2992 and either the oral fluoropyrimidine S-1 or pemetrexed also inhibited the growth of NSCLC xenografts with the T790M mutation to an extent greater than that apparent with either agent alone. The addition of TS-targeting drugs to BIBW2992 is a promising strategy to overcome EGFR-TKI resistance in NSCLC with the T790M mutation of EGFR.

Surfactant protein A2 mutations associated with pulmonary fibrosis lead to protein instability and endoplasmic reticulum stress.

Rare heterozygous mutations in the gene encoding surfactant protein A2 (SP-A2, SFTPA2) are associated with adult-onset pulmonary fibrosis and adenocarcinoma of the lung. We have previously shown that two recombinant SP-A2 mutant proteins (G231V and F198S) remain within the endoplasmic reticulum (ER) of A549 cells and are not secreted into the culture medium. The pathogenic mechanism of the mutant proteins is unknown. Here we analyze all common and rare variants of the surfactant protein A2, SP-A2, in both A549 cells and in primary type II alveolar epithelial cells. We show that, in contrast with all other SP-A2 variants, the mutant proteins are not secreted into the medium with wild-type SP-A isoforms, form fewer intracellular dimer and trimer oligomers, are partially insoluble in 0.5% Nonidet P-40 lysates of transfected A549 cells, and demonstrate greater protein instability in chymotrypsin proteolytic digestions. Both the G231V and F198S mutant SP-A2 proteins are destroyed via the ER-association degradation pathway. Expression of the mutant proteins increases the transcription of a BiP-reporter construct, expression of BiP protein, and production of an ER stress-induced XBP-1 spliced product. Human bronchoalveolar wash samples from individuals who are heterozygous for the G231V mutation have similar levels of total SP-A as normal family members, which suggests that the mechanism of disease does not involve an overt lack of secreted SP-A but instead involves an increase in ER stress of resident type II alveolar epithelial cells.

Isoleucine/leucine2 is essential for chemoattractant activity of beta-defensin Defb14 through chemokine receptor 6.

Beta-defensins are both antimicrobial and able to chemoattract various immune cells including immature dendritic cells and CD4 T cells through CCR6. They are short, cationic peptides with a highly conserved six-cysteine motif. It has been shown that only the fifth cysteine is critical for chemoattraction of cells expressing CCR6. In order to identify other residues essential for functional interaction with CCR6 we used a library of peptide deletion derivatives based on Defb14. Loss of the initial two amino acids from the Defb14-1C(V) derivative destroys its ability to chemoattract cells expressing CCR6. As the second amino acid is an evolutionarily conserved leucine, we make full-length Defb14-1C(V) peptides with substitution of the leucine(2) for glycine (L2G), lysine (L2K) or isoleucine (L2I). Defb14-1C(V) L2G and L2K and are unable to chemoattract CCR6 expressing cells but the semi-conservative change L2I has activity. By circular dichroism spectroscopy we can see no evidence for a significant change in secondary structure as a consequence of these substitutions and so cannot attribute loss of chemotactic activity with disruption of the N-terminal helix. We conclude that isoleucine/leucine in the N-terminal alpha-helix region of this beta-defensin is essential for CCR6-mediated chemotaxis.

Impact of amino acid substitutions in the hepatitis C virus genotype 1b core region on liver steatosis and hepatic oxidative stress in patients with chronic hepatitis C.

BACKGROUND: Liver steatosis and hepatic oxidative stress are the histopathological features of chronic hepatitis C. Hepatitis C virus (HCV) genotype 1 core protein induces hepatic steatosis and reactive oxygen species production in transgenic mice. The amino acid substitutions in the HCV core region appear to be related to hepatocarcinogenesis. AIMS: The aim of this study was to clarify the impact of mutations in the HCV core region on oxidative stress and lipid metabolism in patients with chronic hepatitis C. METHODS: Sixty-seven patients (35 men, 32 women; mean age, 58.4 +/- 10.2 years) with chronic hepatitis C with high titres (>5 log IU/ml) were enrolled. Substitutions in amino acids 70, 75 and 91 of the HCV genotype 1b core region, the percentage of hepatic steatosis, and hepatic 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels were investigated in all patients. Urinary 8-OHdG levels were measured in 35 patients. RESULTS: Body mass index, alanine aminotransferase, gamma-glutamyl transferase, and triglyceride levels and substitutions of amino acid 70/Q (glutamine) were significantly associated with the presence of steatosis on univariate analysis. Multivariate analysis showed that substitution of amino acid 70 of glutamine and triglyceride levels were the independent factors related to liver steatosis. Hepatic and urinary 8-OHdG levels were significantly higher in patients with methionine at amino acid 91 of the HCV core region than in those with leucine. CONCLUSION: Substitutions in the amino acids of the HCV genotype1b core region are associated with hepatic steatosis and oxidative stress in patients with chronic hepatitis C.