Ubiquilin and p97/VCP bind erasin, forming a complex involved in ERAD

Loss of ubiquilin or erasin activates ER stress, increases accumulation of polyubiquitinated proteins, and shortens lifespan in worms

DEB025 (Alisporivir) Inhibits Hepatitis C Virus Replication by Preventing a Cyclophilin A Induced Cis-Trans Isomerisation in Domain II of NS5A

DEB025/Debio 025 (Alisporivir) is a cyclophilin (Cyp)-binding molecule with potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. It is currently being evaluated in phase II clinical trials. DEB025 binds to CypA, a peptidyl-prolyl cis-trans isomerase which is a crucial cofactor for HCV replication. Here we report that it was very difficult to select resistant replicons (genotype 1b) to DEB025, requiring an average of 20 weeks (four independent experiments), compared to the typically <2 weeks with protease or polymerase inhibitors. This indicates a high genetic barrier to resistance for DEB025. Mutation D320E in NS5A was the only mutation consistently selected in the replicon genome. This mutation alone conferred a low-level (3.9-fold) resistance. Replacing the NS5A gene (but not the NS5B gene) from the wild type (WT) genome with the corresponding sequence from the DEB025res replicon resulted in transfer of resistance. Cross-resistance with cyclosporine A (CsA) was observed, whereas NS3 protease and NS5B polymerase inhibitors retained WT-activity against DEB025res replicons. Unlike WT, DEB025res replicon replicated efficiently in CypA knock down cells. However, DEB025 disrupted the interaction between CypA and NS5A regardless of whether the NS5A protein was derived from WT or DEB025res replicon. NMR titration experiments with peptides derived from the WT or the DEB025res domain II of NS5A corroborated this observation in a quantitative manner. Interestingly, comparative NMR studies on two 20-mer NS5A peptides that contain D320 or E320 revealed a shift in population between the major and minor conformers. These data suggest that D320E conferred low-level resistance to DEB025 probably by reducing the need for CypA-dependent isomerisation of NS5A. Prolonged DEB025 treatment and multiple genotypic changes may be necessary to generate significant resistance to DEB025, underlying the high barrier to resistance

Effects of antibiotic resistance, drug target attainment, bacterial pathogenicity and virulence, and antibiotic access and affordability on outcomes in neonatal sepsis: an international microbiology and drug evaluation prospective substudy (BARNARDS)

Background Sepsis is a major contributor to neonatal mortality, particularly in low-income and middle-income countries (LMICs). WHO advocates ampicillin–gentamicin as first-line therapy for the management of neonatal sepsis. In the BARNARDS observational cohort study of neonatal sepsis and antimicrobial resistance in LMICs, common sepsis pathogens were characterised via whole genome sequencing (WGS) and antimicrobial resistance profiles. In this substudy of BARNARDS, we aimed to assess the use and efficacy of empirical antibiotic therapies commonly used in LMICs for neonatal sepsis. Methods In BARNARDS, consenting mother–neonates aged 0–60 days dyads were enrolled on delivery or neonatal presentation with suspected sepsis at 12 BARNARDS clinical sites in Bangladesh, Ethiopia, India, Pakistan, Nigeria, Rwanda, and South Africa. Stillborn babies were excluded from the study. Blood samples were collected from neonates presenting with clinical signs of sepsis, and WGS and minimum inhibitory concentrations for antibiotic treatment were determined for bacterial isolates from culture-confirmed sepsis. Neonatal outcome data were collected following enrolment until 60 days of life. Antibiotic usage and neonatal outcome data were assessed. Survival analyses were adjusted to take into account potential clinical confounding variables related to the birth and pathogen. Additionally, resistance profiles, pharmacokinetic–pharmacodynamic probability of target attainment, and frequency of resistance (ie, resistance defined by in-vitro growth of isolates when challenged by antibiotics) were assessed. Questionnaires on health structures and antibiotic costs evaluated accessibility and affordability. Findings Between Nov 12, 2015, and Feb 1, 2018, 36 285 neonates were enrolled into the main BARNARDS study, of whom 9874 had clinically diagnosed sepsis and 5749 had available antibiotic data. The four most commonly prescribed antibiotic combinations given to 4451 neonates (77·42%) of 5749 were ampicillin–gentamicin, ceftazidime–amikacin, piperacillin–tazobactam–amikacin, and amoxicillin clavulanate–amikacin. This dataset assessed 476 prescriptions for 442 neonates treated with one of these antibiotic combinations with WGS data (all BARNARDS countries were represented in this subset except India). Multiple pathogens were isolated, totalling 457 isolates. Reported mortality was lower for neonates treated with ceftazidime–amikacin than for neonates treated with ampicillin–gentamicin (hazard ratio [adjusted for clinical variables considered potential confounders to outcomes] 0·32, 95% CI 0·14–0·72; p=0·0060). Of 390 Gram-negative isolates, 379 (97·2%) were resistant to ampicillin and 274 (70·3%) were resistant to gentamicin. Susceptibility of Gram-negative isolates to at least one antibiotic in a treatment combination was noted in 111 (28·5%) to ampicillin–gentamicin; 286 (73·3%) to amoxicillin clavulanate–amikacin; 301 (77·2%) to ceftazidime–amikacin; and 312 (80·0%) to piperacillin–tazobactam–amikacin. A probability of target attainment of 80% or more was noted in 26 neonates (33·7% [SD 0·59]) of 78 with ampicillin–gentamicin; 15 (68·0% [3·84]) of 27 with amoxicillin clavulanate–amikacin; 93 (92·7% [0·24]) of 109 with ceftazidime–amikacin; and 70 (85·3% [0·47]) of 76 with piperacillin–tazobactam–amikacin. However, antibiotic and country effects could not be distinguished. Frequency of resistance was recorded most frequently with fosfomycin (in 78 isolates [68·4%] of 114), followed by colistin (55 isolates [57·3%] of 96), and gentamicin (62 isolates [53·0%] of 117). Sites in six of the seven countries (excluding South Africa) stated that the cost of antibiotics would influence treatment of neonatal sepsis

HCV core residues critical for infectivity are also involved in core-NS5A complex formation.

Hepatitis C virus (HCV) infection is a major cause of liver disease. The molecular machinery of HCV assembly and particle release remains obscure. A better understanding of the assembly events might reveal new potential antiviral strategies. It was suggested that the nonstructural protein 5A (NS5A), an attractive recent drug target, participates in the production of infectious particles as a result of its interaction with the HCV core protein. However, prior to the present study, the NS5A-binding site in the viral core remained unknown. We found that the D1 domain of core contains the NS5A-binding site with the strongest interacting capacity in the basic P38-K74 cluster. We also demonstrated that the N-terminal basic residues of core at positions 50, 51, 59 and 62 were required for NS5A binding. Analysis of all substitution combinations of R50A, K51A, R59A, and R62A, in the context of the HCVcc system, showed that single, double, triple, and quadruple mutants were fully competent for viral RNA replication, but deficient in secretion of viral particles. Furthermore, we found that the extracellular and intracellular infectivity of all the mutants was abolished, suggesting a defect in the formation of infectious particles. Importantly, we showed that the interaction between the single and quadruple core mutants and NS5A was impaired in cells expressing full-length HCV genome. Interestingly, mutations of the four basic residues of core did not alter the association of core or NS5A with lipid droplets. This study showed for the first time that basic residues in the D1 domain of core that are critical for the formation of infectious extracellular and intracellular particles also play a role in core-NS5A interactions

Mifepristone Treatment in Four Cases of Primary Bilateral Macronodular Adrenal Hyperplasia (BMAH).

CONTEXT: Primary bilateral macronodular adrenal hyperplasia (BMAH) is a rare form of adrenal Cushing syndrome conventionally treated with adrenalectomy. Medical treatment is often reserved for patients not eligible for surgery. However, to date, there have been few studies about the efficacy of mifepristone for the treatment of BMAH associated with hypercortisolism. OBJECTIVE: To describe a series of patients with hypercortisolism due to BMAH treated with mifepristone. DESIGN: We retrospectively assessed 4 patients treated with mifepristone from multiple medical practices with hypercortisolism due to BMAH had who either failed unilateral adrenalectomy, declined surgery, or were poor surgical candidates. RESULTS: Mifepristone induced clinical improvement and remission of the signs and symptoms of hypercortisolism in all the described patients with BMAH. The median treatment duration at the time of efficacy response assessment was 5 months (range: 3-18 months). Improvement in cardiometabolic parameters was observed as early as 2 weeks after treatment was started. All patients achieved improvements in glycemic control and hypertension and had significant weight loss. The most common adverse event observed with mifepristone therapy was fatigue. Increases in thyroid-stimulating hormone level occurred in 2 patients. CONCLUSION: Mifepristone can be an effective alternative to surgery in patients with hypercortisolism due to BMAH

Mutants with core substitutions R50A, K51A, R59A and R62A do not produce infectious HCV particles.

<p>A) Extra- and intracellular infectivity of stable cell lines expressing JFH1-Luc/Neo-wt, its quadruple core mutant (R50A/K51A/R59A/R62A), JFH1-Luc/Neo-Core-Flag and its given mutants was determined by a focus-forming assay. Levels of extra- and intracellular infectivity were expressed as log₁₀ of focus-forming units (ffu) per ml of supernatant or cell lysate, respectively. Mean values of triplicates and standard errors are presented. B) Representative light microscopic pictures of infectious foci in naïve Huh7.5.1 cells exposed to extra- and intracellular HCVcc particles from the experiment described above (A). Cells were counter stained with hematoxylin to visualize the nuclei. The magnification is 20x. C) Stable cell lines expressing JFH1-Luc/Neo-wt, its quadruple core mutant (R50A/K51A/R59A/R62A), JFH1-Luc/Neo-Core-Flag and its quadruple core mutant (Flag-R50A/K51A/R59A/R62A) were seeded in 96-well plate and the standard immunostaining procedure for a focus-forming assay was performed directly on them. Cells were counter stained with hematoxylin to visualize the nuclei. The magnification is 20×.</p

Domain I of NS5A binds to core.

<p>A) NS5A is composed of three domains (Domain I, II, and III) separated by low-complexity sequences (LCSI and LCSII). Domain I of NS5A is composed of a 32-amino-acid N-terminal amphipathic helix (AH), subdomain IA (33–100) and subdomain IB (101–213). B) Mapping of NS5A regions required for binding to core. GST and GST-Core122-170-His (negative controls) or GST-Core1-170-His were used as bait to pulldown domain I NS5A-Flag, domain II NS5A-Flag and domain III NS5A-Flag (left panel) or amphipathic helix NS5A-Flag, subdomain IA NS5A-Flag, and subdomain IB NS5A-Flag (right panel). Captured proteins and 5% of input were analyzed by Western blotting using anti-Flag antibodies. C) GST and truncation forms of GST-Core-His were used as bait to pulldown domain I NS5A-Flag and subdomain IB NS5A-Flag. Captured proteins and 5% of input were analyzed by Western blotting using anti-Flag antibodies.</p

Analysis of extra- and intracellular core in stable cell lines expressing mutant full-length JFH1 genomes.

<p>A) Extracellular core levels of stable cell lines expressing JFH1-Luc/Neo (wt), its quadruple core mutant (R50A/K51A/R59A/R62A), JFH1-Luc/Neo-Core-Flag and its given core mutants were quantified by ELISA. Cells were seeded in 6-well plates in triplicates at the concentration of 10⁵ cells per well and grown for 72 h in 3 ml of complete DMEM. Levels of extracellular core were expressed as log₁₀ of pg/ml of cell culture medium. Mean values of triplicates and standard errors are presented. B) Intracellular core levels were quantified by ELISA using cells plated in the experiment described above (A). Cells were lysed with 0.3 ml of Cell Culture Lysis Reagent. Intracellular core levels were expressed as log₁₀ of pg/ml of cell lysate. Mean values of triplicates and standard errors are presented. C) A long-term stability analysis of core in stable cell lines expressing JFH1-Luc/Neo-Core-Flag, its quadruple core mutant (Flag-R50A/K51A/R59A/R62A) as well as JFH1-Luc/Neo (wt). Cells were kept under neomycin selection for a month and passaged every 3 days. Cell lysates from the first passage (3 d, three days), the sixth passage (18 d, eighteen days) and the tenth passage (30 d, thirty days) were analyzed by Western blotting with anti-Core and anti-Flag antibodies.</p

NS5A-binding site is located in the D1 domain of core.

<p>A) Flag co-immunoprecipitations in Huh7 cells transfected with expression vectors for 3xFlag-Core and NS5A (top panel) or in SGR-JFH1 (Huh7 subgenomic JFH1 replicon) cells transfected with an expression vector for 3xFlag-Core (bottom panel). After immunoprecipitation with anti-Flag antibodies, bound material was eluted with 3xFlag peptide and analyzed by Western blotting with anti-NS5A and anti-Core antibodies. Input of whole-cell lysate (2%) used for each co-immunoprecipitation was probed with anti-NS5A and anti-Core antibodies. B) Mapping of core regions required for NS5A binding. GST (negative control), GST-CypA (positive control) or truncated forms of GST-Core-His were used as bait to pulldown full-length NS5A-His. Captured proteins were analyzed by Western blotting using anti-NS5A antibodies. Input (5%) used for each GST pulldown was probed with anti-NS5A and anti-GST antibodies. C) Schematic representation of core regions required for binding to NS5A. The strongest interaction was expressed as (+++), less strong as (++), weak as (+) and no interaction as (−). The amount of NS5A bound by truncated forms of GST-Core-His was estimated relative to the amount of NS5A bound by GST-Core1-170-His, that showed the highest binding capacity in spite of the lowest level of input. D) Recombinant proteins (GST, GST-CypA, GST-Core1-170-His and NS5A-His) were treated with RNase and DNase (left panel) or benzonase nuclease (right panel) to remove contaminating nucleic acids before pulldown assays. GST-CypA/NS5A-His mixtures were used as controls because this interaction has been shown to be direct. Captured proteins were analyzed by Western blotting using anti-NS5A antibodies. E) Recombinant GST, GST-CypA, GST-Core1-170-His and NS5A-His proteins were mixed with different concentrations of the NS5A inhibitor BMS-790052 (left panel) or the cyclophilin inhibitor CsA (right panel). Captured proteins were analyzed by Western blotting using anti-NS5A antibodies.</p

Schematic representation of the core protein and the constructs used in the study.

<p>A) The precursor core of 191 amino acids is processed by a signal peptide peptidase, giving a mature protein of around 170 amino acids that is composed of two domains, D1 and D2. Based on the charge distribution, the D1 domain can be subdivided into three basic clusters: BD1 (basic domain 1), BD2 (basic domain 2) and BD3 (basic domain 3). B) Two recombinant proteins were used to determine the requirement of basic residues R50, K51, R59 and R62 in core for NS5A binding. GST-Core-BD2-His was used as bait in pulldown assays, while full-length 3xFlag-Core was used in co-immunoprecipitations in SGR-JFH1 cells. A sequence of wild-type BD2 as well as a series of single, double, triple and quadruple alanine substitutions in its context is listed below. C) A scheme of luciferase reporter full-length JFH1 genomes (genotype 2a) used in the study. Previously described Luc-JFH1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088866#pone.0088866-Wakita1" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088866#pone.0088866-Koutsoudakis1" target="_blank">[61]</a> was used to generate JFH1-Luc/Neo construct by insertion of a neomycin-resistant gene (black box) after the luciferase cassette (white box), but before EMCV-IRES (E-I) and all structural and nonstructural HCV proteins (shadow boxes). The JFH1-Luc/Neo-Core-Flag construct was created by an insertion of a Flag tag (DYKDDDDK) with a short linker (SGS) between the amino acids S2 and T3 of core. Single, double, triple and quadruple alanine substitutions of core residues R50, K51, R59 and R62 listed above (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088866#pone-0088866-g001" target="_blank">Figure 1B</a>) were introduced into the JFH1-Luc/Neo-Core-Flag. Additionally, the quadruple core mutant was created in the context of wild-type JFH1-Luc/Neo.</p