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This article presents a systematic investigation of the knitted fabrics made from various blends of intrinsically antimicrobial poly (hydroxybutyrate-co-hydroxyvalerate)/polylactide acid filaments and cotton staple fibers. The effects of blend yarn, fabric structures, and distributions of fibers on antimicrobial properties of resultant yarns and knitted fabrics were studied. The relationships among fiber distribution, blend ratio, and anti-microbial properties were experimentally determined for three blend yarns made by sirofil, wrap-spun, and core-spun spinning technologies. The fabrics made from the sirofil-spun and wrap-spun yarns show better anti-microbial effects against Staphylococcus aureus, Klebsiella pneumoniae, and Candida albicans than those of the core-spun yarns, according to the standard AATCC100-2012 Antibacterial Finishes on Textile Materials (American Association of Textile Chemists and Colorists, 2012). An alternative blending method of co-knitting of the pure poly (hydroxybutyrate-co-hydroxyvalerate)/polylactide acid yarns and cotton yarns achieved excellent antimicrobial effects. Furthermore, a wearing trial of underwear made from the blended knitted fabrics was conducted in a nursing home. The wearing comfort of the garments, low-stress mechanical and surface properties of fabrics were evaluated objectively by the Kawabata Evaluation System of Fabric (KESF) system and subjectively by a questionnaire survey to users.

Introduction: Voters facing illness or disability are disproportionately under-represented in terms of voter turnout. Earlier research has indicated that enfranchisement of these populations may reinforce the implementation of policies improving health outcomes and equity. Due to the confluence of the coronavirus 2019 (COVID-19) pandemic and the 2020 election, we aimed to assess emergency absentee voting processes, which allow voters hospitalized after regular absentee deadlines to still obtain an absentee ballot, and election changes due to COVID-19 in all 50 states. Methods: We performed a cross-sectional study collecting 34 variables pertaining to emergency voting processes and COVID-19-related election changes, including deadlines, methods of submission for applications and ballots, and specialized services for patients. Data were obtained from, in order of priority, state boards of elections websites, poll worker manuals, application forms, and state legislation. We verified all data through direct correspondence with state boards of elections. Results: Emergency absentee voting processes are in place in 39 states, with the remaining states having universal vote-by-mail (n = 5) or extended regular absentee voting deadlines (n = 6). The emergency absentee period most commonly began within 24 hours following the normal absentee application deadline, which was often seven days before an election (n = 11). Unique aspects of emergency voting processes included patients designating an “authorized agent” to deliver their applications and ballots (n = 38), electronic ballot delivery (n = 5), and in-person teams that deliver ballots directly to patients (n = 18). Documented barriers in these processes nationwide include unavailable online information (n = 11), restrictions mandating agents to be family members (n = 7), physician affidavits or signatures (n = 9), and notary or witness signature requirements (n = 15). For the November 2020 presidential election, 12 states expanded absentee eligibility to allow COVID-19 as a reason to request an absentee ballot, and 18 states mailed absentee ballot applications or absentee ballots to all registered voters. Conclusion: While 39 states operate emergency absentee voting processes for hospitalized voters, there are considerable areas for improvement and heterogeneity in guidelines for these protocols. For future election cycles, information on emergency voting and broader election reforms due to COVID-19 may be useful for emergency providers and patients alike to improve the democratic participation of voters experiencing illness. [ABSTRACT FROM AUTHOR] Copyright of Western Journal of Emergency Medicine: Integrating Emergency Care with Population Health is the property of Western Journal of Emergency Medicine: Integrating Emergency Care with Population Health and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Introduction: This study reviews malpractice, also called medical professional liability (MPL), claims involving adult patients cared for in emergency departments (ED) and urgent care settings. Methods: We conducted a retrospective review of closed MPL claims of adults over 18 years, from the Medical Professional Liability Association’s Data Sharing Project database from 2001–2015, identifying 6,779 closed claims. Data included the total amount, origin, top medical specialties named, chief medical factors, top medical conditions, severity of injury, resolution, average indemnity, and defense costs of closed claims. Results: Of 6,779 closed claims, 65.9% were dropped, withdrawn, or dismissed. Another 22.8% of claims settled for an average indemnity of $297,709. Of the 515 (7.6%) cases that went to trial, juries returned verdicts for the defendant in 92.6% of cases (477/515). The remaining 7.4% of cases (38/515) were jury verdicts for the plaintiff, with an average indemnity of $816,909. The most common resulting medical condition cited in paid claims was cardiac or cardiorespiratory arrest (10.4%). Error in diagnosis was the most common chief medical error cited in closed claims. Death was the most common level of severity listed in closed (38.5%) and paid (42.8%) claims. Claims reporting major permanent injury had the highest paid-to-closed ratio, and those reporting grave injury had the highest average indemnity of $686,239. Conclusion: This retrospective review updates the body of knowledge surrounding medical professional liability and represents the most recent analysis of claims in emergency medicine. As the majority of emergency providers will be named in a MPL claim during their career, it is essential to have a better understanding of the most common factors resulting in MPL claims.

Fluorescence imaging is an indispensable method for studying biological processes non-invasively in cells and transparent organisms. Extension into the shortwave infrared (SWIR, 1000-2000nm) region of the electromagnetic spectrum has allowed for imaging in mammals with unprecedented depth and resolution for optical imaging. In this review, we summarize recent advances in imaging technologies, dye scaffold modifications, and incorporation of these dyes into probes for SWIR imaging in mice. Finally, we offer an outlook on the future of SWIR detection in the field of chemical biology.

Benthic marine organisms rely on the dispersal and recruitment of propagules to replenish depleted populations following disturbances. Yet, ecological interactions between colonizing larvae and benthic competitors that become established following a disturbance can be a primary driver of recruitment success. On some coral reefs, local and global stressors have led to a proliferation of macroalgae that can inhibit the recruitment and recovery of corals. The brown macroalga Lobophora is considered a particularly strong inhibitor of coral recruitment, yet there is little information on how the alga affects demographic bottlenecks across early life-history stages. In the present study, we conducted a series of experiments to determine the effects of Lobophora sp. on three distinct life-history stages of the coral Acropora digitifera: larval settlement, early post-settlement survival, and survival and growth of small nubbins (1 cm) created from adult colonies. Our results demonstrate a high sensitivity of coral larvae to Lobophora, with settlement decreasing 16-fold when the alga was present on tiles compared to controls. Moreover, larvae did not settle on tiles when Lobophora cover > 50%. A negative, albeit minor, effect of Lobophora on the early post-settlement survival was evident, despite few recorded incidents of direct competition between settled corals and Lobophora because of low larval settlement in proximity to the alga. Conversely, there was no effect of Lobophora on the growth and survival of coral nubbins. Our results indicate that Lobophora most heavily impacts coral recruitment by inhibiting larval settlement, with the impact of the alga on recruitment decreasing through later life-history stages. These findings are concurrent with recent studies that demonstrate the ability of particular macroalgal species to deter coral larvae from settling on degraded reefs, likely through the release of chemical compounds, thereby impacting the recovery of coral populations following disturbances.

DNA damage is ubiquitous and can arise from endogenous or exogenous sources. DNA-damaging alkylating agents are present in environmental toxicants as well as in cancer chemotherapy drugs and are a constant threat, which can lead to mutations or cell death. All organisms have multiple DNA repair and DNA damage tolerance pathways to resist the potentially negative effects of exposure to alkylating agents. In bacteria, many of the genes in these pathways are regulated as part of the SOS reponse or the adaptive response. In this work, we probed the cellular responses to the alkylating agents chloroacetaldehyde (CAA), which is a metabolite of 1,2-dichloroethane used to produce polyvinyl chloride, and styrene oxide (SO), a major metabolite of styrene used in the production of polystyrene and other polymers. Vinyl chloride and styrene are produced on an industrial scale of billions of kilograms annually and thus have a high potential for environmental exposure. To identify stress response genes in E. coli that are responsible for tolerance to the reactive metabolites CAA and SO, we used libraries of transcriptional reporters and gene deletion strains. In response to both alkylating agents, genes associated with several different stress pathways were upregulated, including protein, membrane, and oxidative stress, as well as DNA damage. E. coli strains lacking genes involved in base excision repair and nucleotide excision repair were sensitive to SO, whereas strains lacking recA and the SOS gene ybfE were sensitive to both alkylating agents tested. This work indicates the varied systems involved in cellular responses to alkylating agents, and highlights the specific DNA repair genes involved in the responses.

The claustrum is a thin sheet of neurons that is densely connected to many cortical regions and has been implicated in numerous high-order brain functions. Such brain functions arise from brain states that are influenced by neuromodulatory pathways from the cholinergic basal forebrain, dopaminergic substantia nigra and ventral tegmental area, and serotonergic raphe. Recent revelations that the claustrum receives dense input from these structures have inspired investigation of state-dependent control of the claustrum. Here, we review neuromodulation in the claustrum—from anatomical connectivity to behavioral manipulations—to inform future analyses of claustral function. Ministry of Education (MOE) Published version This work was supported by the Singapore Ministry of Education under its Singapore Ministry of Education Academic Research Fund Tier 3 (MOE2017-T3-1-002).

The COVID-19 pandemic challenges safe and equitable voting in the United States' 2020 elections, and in response, several states including Rhode Island (RI) have made significant changes to election policy. In addition to increasing accessibility of mail-in voting by mailing applications to all registered voters, RI has suspended their notary/witness requirement for both the primary and general election. However, RI's "emergency" voting process still plays a crucial role in allowing voters who missed the mail-in ballot application deadline, such as those unexpectedly hospitalized in the days leading up to the election, to still cast their ballot. COVID-19 has also forced RI to modify its emergency voting procedures, most notably allowing healthcare workers to serve on bipartisan ballot delivery teams. This commentary highlights these salient updates to voting procedures and serves as a primer as to how interested health care workers may navigate this process alongside patients and lead in the arena of patient voting rights.

The claustrum is a poorly understood but intriguing part of the brain: a new study has found that it plays an important role in drug reward by providing incentive salience to the location where the drug is administered.

Although its dense connections with other brain areas suggests that the claustrum is involved in higher-order brain functions, little is known about the properties of claustrum neurons. Using whole-cell patch clamp recordings in acute brain slices of mice, we characterized the intrinsic electrical properties of more than 300 claustral neurons and used unsupervised clustering of these properties to define distinct cell types. Differences in intrinsic properties permitted separation of interneurons (INs) from projection neurons (PNs). Five subtypes of PNs could be further identified by differences in their adaptation of action potential (AP) frequency and amplitude, as well as their AP firing variability. Injection of retrogradely transported fluorescent beads revealed that PN subtypes differed in their projection targets: one projected solely to subcortical areas while three out of the remaining four targeted cortical areas. INs expressing parvalbumin (PV), somatostatin (SST), or vasoactive intestinal peptide (VIP) formed a heterogenous group. PV-INs were readily distinguishable from VIP-INs and SST-INs, while the latter two were clustered together. To distinguish IN subtypes, an artificial neural network was trained to distinguish the properties of PV-INs, SST-INs, and VIP-INs, as independently identified through their expression of marker proteins. A user-friendly, machine-learning tool that uses intrinsic electrical properties to distinguish these eight different types of claustral cells was developed to facilitate implementation of our classification scheme. Systematic classification of claustrum neurons lays the foundation for future determinations of claustrum circuit function, which will advance our understanding of the role of the claustrum in brain function. Agency for Science, Technology and Research (A*STAR) Ministry of Education (MOE) Published version This work was supported by Singapore Ministry of Education Grants MOE2015-T2-2-095 and MOE2017-T3-1-002 and a National Science Scholarship awarded to A.N. by the Singapore Agency of Science, Technology and Research.

BACKGROUND: Twitter-based chat groups (tweetchats) structured as virtual journal clubs have been demonstrated to provide value to learners. In order to promote topics in medical toxicology, we developed the #firesidetox tweetchat as a virtual journal club to discuss and disseminate topics in medical toxicology. METHODS: A group of medical toxicologists from the American College of Medical Toxicology (ACMT) Public Affairs Committee and editorial board of the Journal of Medical Toxicology (JMT) developed a quarterly one hour tweetchat featuring JMT manuscripts. We gathered basic twittergraphics and used a healthcare hashtag aggregator to measure the number of impressions, participants, and tweets per tweetchat session. A qualitative analysis of important themes from #firesidetox was also completed. RESULTS: During five tweetchats over 12 months, we attracted a mean of 23 participants generating a mean of 150 tweets per #firesidetox tweetchat. Tweets generated a mean of 329,200 impressions (unique user views): these impressions grew by 300% from the first through fifth #firesidetox. The majority of participants self-identified as medical toxicologists or physician learners. Although most were from the USA, participants also came from Australia, Poland, and Qatar. Most tweets centered on medical education and 7.9% tweets were learner-driven or questions asking for a medical toxicologist expert opinion. CONCLUSION: The #firesidetox attracted a diverse group of toxicologists, learners, and members of the public in a virtual journal club setting. The increasing number of impressions, participants, and tweets during #firesidetox demonstrates the tweetchat model to discuss pertinent toxicology topics is feasible and well received among its participants.

Alisporivir is a cyclophilin inhibitor with pan-genotypic anti–hepatitis C virus (HCV) activity and a high barrier to viral resistance. The VITAL-1 study assessed alisporivir as interferon (IFN)-free therapy in treatment-naive patients infected with HCV genotype 2 or 3. Three hundred forty patients without cirrhosis were randomized to: arm 1, alisporivir (ALV) 1,000 mg once-daily (QD); arm 2, ALV 600 mg QD and ribavirin (RBV); arm 3, ALV 800 mg QD and RBV; arm 4, ALV 600 mg QD and pegylated IFN (Peg-IFN); or arm 5, Peg-IFN and RBV. Patients receiving IFN-free ALV regimens who achieved rapid virological response (RVR) continued the same treatment throughout, whereas those with detectable HCV RNA at week 4 received ALV, RBV, and Peg-IFN from weeks 6 to 24. Overall, 300 patients received ALV-based regimens. In arm 1 to arm 4, the intent-to-treat rates of sustained virological response (SVR) 24 weeks after treatment (SVR24) were from 80% to 85%, compared with 58% (n = 23 of 40) with Peg-IFN/RBV. Per-protocol analysis showed higher SVR24 rates in patients who received ALV/RBV, IFN-free after RVR (92%; n = 56 of 61) than with ALV alone after RVR (72%; n = 13 of 18) or with Peg-IFN/RBV (70%; n = 23 of 33). Both RVRs and SVRs to ALV IFN-free regimens were numerically higher in genotype 3– than in genotype 2–infected patients. Viral breakthrough was infrequent (3%; n = 7 of 258). IFN-free ALV treatment showed markedly better safety/tolerability than IFN-containing regimens. Conclusions: ALV plus RBV represents an effective IFN-free option for a proportion of patients with HCV genotype 2 or 3 infections, with high SVR rates for patients with early viral clearance. Further investigations of ALV in IFN-free combination regimens with direct-acting antiviral drugs deserve exploration in future trials. (Hepatology 2015;62:1013-1023)

A small yet promising body of research has been conducted on the use of remote sensing data to retrieve vegetation chlorophyll content for heterogeneous ecosystems at the leaf level; however, the extent to which leaf chlorophyll contents can be estimated from reflectance measurements at the canopy and landscape scales remain uncertain. The goal of this study was to develop and evaluate a species percent cover-based chlorophyll content scaling up procedure that aims to accurately estimate chlorophyll content at canopy or landscape level. Using both field and QuickBird data collected in a heterogeneous tall grassland located in Ontario, Canada, this study calculated vegetation chlorophyll content at canopy and landscape levels, and it correlated chlorophyll data at leaf, canopy, and landscape levels with a red-edge spectral index. Results indicated that the relationships between the red-edge index and vegetation chlorophyll content (e.g., chlorophyll a, chlorophyll b, chlorophyll a + b) were significant at ...

EDP-239, a potent and selective hepatitis C virus (HCV) nonstructural protein 5A (NS5A) inhibitor developed for the treatment of HCV infection, has been investigated in vitro and in vivo . This study sought to characterize genotypic changes in the HCV NS5A sequence of genotype 1 (GT1) replicons and to compare those changes to GT1 viral RNA mutations isolated from clinical trial patients. Resistance selection experiments in vitro using a subgenomic replicon identified resistance-associated mutations (RAMs) at GT1a NS5A amino acid positions 24, 28, 30, 31, and 93 that confer various degrees of resistance to EDP-239. Key RAMs were similarly identified in GT1b NS5A at amino acid positions 31 and 93. Mutations F36L in GT1a and A92V in GT1b do not confer resistance to EDP-239 individually but were found to enhance the resistance of GT1a K24R and GT1b Y93H. RAMs were identified in GT1 patients at baseline or after dosing with EDP-239 that were similar to those detected in vitro . Baseline RAMs identified at NS5A position 93 in GT1, or positions 28 or 30 in GT1a only, correlated with a reduced treatment response. RAMs at additional positions were also detected and may have contributed to reduced EDP-239 efficacy. The most common GT1a and GT1b RAMs found to persist up to weeks 12, 24, or 48 were those at NS5A positions 28, 30, 31, 58 (GT1a only), and 93. Those RAMs persisting at the highest frequencies up to weeks 24 or 48 were L31M and Q30H/R for GT1a and L31M and Y93H for GT1b. (This study has been registered at ClinicalTrials.gov under identifier NCT01856426.)

GS-9451, a novel hepatitis C virus (HCV) nonstructural 3/4a (NS3/4a) protease inhibitor, is highly active in patients infected with HCV genotype 1 (GT 1). The aim of this study is to characterize the clinical resistance profile of GS-9451 in GT 1 HCV-infected patients in a phase 1, 3-day monotherapy study. The full-length NS3/4A gene was population sequenced at baseline, on the final treatment day, and at follow-up time points. NS3 protease domains from patient isolates with emerging mutations were cloned into an NS3 shuttle vector, and their susceptibilities to GS-9451 and other HCV inhibitors were determined using a transient replication assay. No resistance mutations at NS3 position 155, 156, or 168 were detected in any of the baseline samples or in viruses from patients treated with 60 mg of GS-9451 once daily. Among patients who received 200 mg and 400 mg of GS-9451, viruses with mutations at position D168 (D168E/G/V) and R155 (R155K), which confer high-level resistance to GS-9451, were detected in those with GT 1b and GT 1a virus, respectively. Viruses with D168 mutations were no longer detected in any GT 1b patient at day 14 and subsequent time points. In GT 1a patients, R155K mutants were replaced by the wild type in 57% of patients at week 24. These NS3 clinical mutants were sensitive to NS5B and NS5A inhibitors, as well as alpha interferon (IFN-α) and ribavirin. The lack of cross-resistance between GS-9451 and other classes of HCV inhibitors supports the utility of combination therapy.

With the exception of nucleoside analogs, few direct acting antivirals in clinical development are active across the six major hepatitis C virus genotypes. We report novel consensus sequence chimeras for genotypes 2b, 3a, 4a, 5a, and 6a NS5B and show variable susceptibilities over a panel of NS5B inhibitors. Tegobuvir (GS-9190) had EC50s of 100nM for other genotypes tested here. An NS5B F445C mutation engineered into the GT3a, 4a, and 6a chimeric replicons lowered the tegobuvir EC50 to levels comparable to those for genotype 1a, but did not considerably alter the EC50 of site 2 or nucleoside analog inhibitors. These data support the use of HCV chimeras in profiling direct acting antivirals across genotypes and specifically determines the impact of the C445F NS5B polymorphism on tegobuvir potency against genotypes 3a, 4a, and 6a.

Purpose Infection with hepatitis C virus is the leading cause of infectious disease mortality in the United States. BZF961 is a novel small molecule inhibitor of the hepatitis C virus NS3-4A protease. Here we present the results of a randomized, double-blinded, placebo-controlled, multicentered study in treatment-naive patients with chronic hepatitis C virus genotype-1 infection. Methods Patients were enrolled sequentially in 2 parts and treated for 3 days. BZF961 was administered as monotherapy (500 mg BID for 3 days) or in combination with the cytochrome P450 3A4 inhibitor ritonavir to boost its exposure (BZF961 10, 20, or 50 mg QD or BID). Findings BZF961 was safe and well tolerated in the patients studied with no serious adverse events. There were no appreciable differences in adverse events among patients who received BZF961, BZF961 with ritonavir, or placebo. There was a significant, clinically meaningful reduction in viral load from baseline in patients treated either with BZF961 500 mg every 12 hours alone or BZF961 50 mg every 12 hours in combination with ritonavir. Activity against the hepatitis C virus of the lower-dose regimens was apparent but more modest. There were no relevant changes from baseline viral loads in placebo-treated patients. Implications Coadministration of ritonavir with BZF961 boosted BZF961 exposure (including Cmin, which is the clinically relevant parameter associated with antiviral activity) in a therapeutic range with less variability compared with BZF961 alone. For strategic reasons, BZF961 is no longer under development.

The 5′-AMP-activated protein kinase (AMPK) is a master sensor for cellular metabolic energy state. It is activated by a high AMP/ATP ratio and leads to metabolic changes that conserve energy and utilize alternative cellular fuel sources. The kinase is composed of a heterotrimeric protein complex containing a catalytic α-subunit, an AMP-binding γ-subunit, and a scaffolding β-subunit thought to bind directly both the α- and γ-subunits. Here, we use coimmunoprecipitation of proteins in transiently transfected cells to show that the α2-subunit binds directly not only to the β-subunit, confirming previous work, but also to the γ1-subunit. Deletion analysis of the α2-subunit reveals that the C-terminal 386-552 residues are sufficient to bind to the β-subunit. The γ1-subunit binds directly to the α2-subunit at two interaction sites, one within the catalytic domain consisting of α2 amino acids 1-312 and a second within residues 386-552. Binding of the α2 and the γ1-subunits was not affected by 400 μm AMP or ATP. Furthermore, we show that the β-subunit C terminus is essential for binding to the α2-subunit but, in contrast to previous work, the β-subunit does not bind directly to the γ1-subunit. Taken together, this study presents a new model for AMPK heterotrimer structure where through its C terminus the β-subunit binds to the α-subunit that, in turn, binds to the γ-subunit. There is no direct interaction between the β- and γ-subunits.

The NF-B family of transcription factors is activated by a wide variety of signals to regulate a spectrum of cellular processes. The proper regulation of NF-B activity is critical, since abnormal NF-B signaling is associated with a number of human illnesses, such as chronic inflammatory diseases and cancer. We report here that PIAS1 (protein inhibitor of activated STAT1) is an important negative regulator of NF-B. Upon cytokine stimulation, the p65 subunit of NF-B translocates into the nucleus, where it interacts with PIAS1. The binding of PIAS1 to p65 inhibits cytokine-induced NF-B-dependent gene activation. PIAS1 blocks the DNA binding activity of p65 both in vitro and in vivo. Consistently, chromatin immunoprecipitation assays indicate that the binding of p65 to the promoters of NF-B-regulated genes is significantly enhanced in Pias1/ cells. Microarray analysis indicates that the removal of PIAS1 results in an increased expression of a subset of NF-B-mediated genes in response to tumor necrosis factor alpha and lipopolysaccharide. Consistently, Pias1 null mice showed elevated proinflammatory cytokines. Our results identify PIAS1 as a novel negative regulator of NF-B. A large variety of signals, such as proinflammatory cytokines (tumor necrosis factor alpha [TNF-] and interleukin-1 [IL-1]) and bacterial lipopolysaccharide (LPS), activate the NF-B signaling pathway. NF-B is a family of dimeric transcription

Alisporivir (ALV), a cyclophilin inhibitor, is a host-targeting antiviral (HTA) with multigenotypic anti-hepatitis C virus (HCV) activity and a high barrier to resistance. Recent advances have supported the concept of interferon (IFN)-free regimens to treat chronic hepatitis C. As the most advanced oral HTA, ALV with direct-acting antivirals (DAAs) represents an attractive drug combination for IFN-free therapy. In this study, we investigated whether particular DAAs exhibit additive, synergistic, or antagonistic effects when combined with ALV. Drug combinations of ALV with NS3 protease, NS5B polymerase, and NS5A inhibitors were investigated in HCV replicons from genotypes 1a, 1b, 2a, 3, and 4a (GT1a to -4a). Combinations of ALV with DAAs exerted an additive effect on GT1 and -4. A significant and specific synergistic effect was observed with ALV-NS5A inhibitor combination on GT2 and -3. Furthermore, ALV was fully active against DAA-resistant variants, and ALV-resistant variants were fully susceptible to DAAs. ALV blocks the contact between cyclophilin A and domain II of NS5A, and NS5A inhibitors target domain I of NS5A; our data suggest a molecular basis for the use of these two classes of inhibitors acting on two distinct domains of NS5A. These results provide in vitro evidence that ALV with NS5A inhibitor combination represents an attractive strategy and a potentially effective IFN-free regimen for treatment of patients with chronic hepatitis C. Due to its high barrier and lack of cross-resistance, ALV could be a cornerstone drug partner for DAAs.

As a class, nucleotide inhibitors (NIs) of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase offer advantages over other direct-acting antivirals, including properties, such as pangenotype activity, a high barrier to resistance, and reduced potential for drug-drug interactions. We studied the in vitro pharmacology of a novel C -nucleoside adenosine analog monophosphate prodrug, GS-6620. It was found to be a potent and selective HCV inhibitor against HCV replicons of genotypes 1 to 6 and against an infectious genotype 2a virus (50% effective concentration [EC 50 ], 0.048 to 0.68 μM). GS-6620 showed limited activities against other viruses, maintaining only some of its activity against the closely related bovine viral diarrhea virus (EC 50 , 1.5 μM). The active 5′-triphosphate metabolite of GS-6620 is a chain terminator of viral RNA synthesis and a competitive inhibitor of NS5B-catalyzed ATP incorporation, with K i / K m values of 0.23 and 0.18 for HCV NS5B genotypes 1b and 2a, respectively. With its unique dual substitutions of 1′-CN and 2′- C -Me on the ribose ring, the active triphosphate metabolite was found to have enhanced selectivity for the HCV NS5B polymerase over host RNA polymerases. GS-6620 demonstrated a high barrier to resistance in vitro . Prolonged passaging resulted in the selection of the S282T mutation in NS5B that was found to be resistant in both cellular and enzymatic assays (>30-fold). Consistent with its in vitro profile, GS-6620 exhibited the potential for potent anti-HCV activity in a proof-of-concept clinical trial, but its utility was limited by the requirement of high dose levels and pharmacokinetic and pharmacodynamic variability.

GS-5885 is a novel hepatitis C virus (HCV) NS5A inhibitor. In a 3-day monotherapy study in treatment-naive genotype 1a (GT1a) and GT1b HCV-infected subjects, median viral load reductions ranged from 2.3 to 3.3 log 10 HCV RNA IU/ml across dosing cohorts (1, 3, 10, 30, or 90 mg once daily). Here, we report viral sequencing and phenotypic analysis of clinical isolates from this study. Detection of baseline NS5A amino acid substitutions at positions 28, 30, 31, or 93 in GT1a was associated with a reduced treatment response. In the GT1b cohort, Y93H was detected in 100% of subjects at day 4 or 14. In the Gt1a cohort, population sequencing detected NS5A resistance-associated mutations at day 4 or 14 for 3/10 subjects at the 1-mg dose and for all subjects dosed at ≥3 mg. A subset of mutants that confer a low level of reduced susceptibility to GS-5885 was not detected by population sequencing at the 30- and 90-mg doses. Subject-derived M28T, Q30R, L31M, and Y93C mutations all conferred >30-fold reductions in GS-5885 and daclatasvir susceptibilities in vitro . Site-directed NS5A mutants also showed reduced susceptibility to GS-5885. However, all NS5A mutants tested remained fully susceptible to other classes of direct-acting antivirals (DAAs), interferon alpha, and ribavirin. Importantly, the nonoverlapping resistance profile and high potency of GS-5885 support its further development with other direct-acting antivirals for the treatment of chronic HCV. (This study has been registered at ClinicalTrials.gov under registration number NCT01193478.)

Background: Cervical spondyloptosis is defined as the dislocation of the spinal column most often caused by trauma. Due to compression or transection of the spinal cord, severe neurological deficits are common. Here, we review the literature and report a case of traumatic C5–6 spondyloptosis that was successfully treated using an anterior-only surgical approach. Methods: The patient presented with quadriplegia and absent sensation distal to the C5 dermatome following a rollover motor vehicle accident. The preoperative American Spinal Injury Association Impairment Scale was A. Computed tomography of the cervical spine revealed C5–6 spondyloptosis, lamina fractures on the right side at the C3–4 level, and widened facet joint on the right side at C6–7. Results: The patient underwent cervical traction and anterior cervical discectomy and fusion at the C5–6, C6–7 levels; no 360° fusion was warranted. Six months postoperatively, the patient remained quadriplegic below the C5 level. Conclusion: Presently, no consensus is present regarding the best treatment for spondyloptosis. Worldwide, the 360° approach is the most commonly used (45%), followed by anterior-only surgery (31%) and posterior-only surgery (25%). The surgical choice depends upon patient-specific features but markedly varies among geographical regions.

Background & Aims GS-5885 is an inhibitor of the hepatitis C virus (HCV) NS5A protein and exhibits potent suppression of genotype 1 HCV replicons. The safety, tolerability, pharmacokinetics, antiviral activity, and resistance profile of once-daily GS-5885 doses of 1–90mg were evaluated in patients with chronic genotype 1 HCV. Methods Genotype 1 HCV-infected patients were randomized to 3days of once-daily (QD) dosing with placebo (n=12) or GS-5885 1mg (n=10), 3mg (n=10), 10mg (n=20), 30mg (n=10), or 90mg (n=10). Plasma samples for pharmacokinetics, HCV RNA, and NS5A sequencing were collected through day 14. Results GS-5885 was well tolerated and resulted in median maximal reductions in HCV RNA ranging from 2.3 log 10 IU/ml (1mg QD) to 3.3 log 10 IU/ml (10mg QD in genotype 1b and 30mg QD). E max modeling indicated GS-5885 30mg was associated with>95% of maximal antiviral response to HCV genotype 1a. HCV RNA reductions were generally more sustained among patients with genotype 1b vs. 1a. Three of 60 patients had a reduced response and harbored NS5A-resistant virus at baseline. NS5A sequencing identified residues 30 and 31 in genotype 1a, and 93 in genotype 1b as the predominant sites of mutation following GS-5885 dosing. Plasma pharmacokinetics was consistent with QD dosing. Conclusions During 3days of monotherapy, low doses of GS-5885 demonstrated significant antiviral activity in genotype 1a and 1b HCV-infected patients. GS-5885 is currently being evaluated in combination with direct antiviral regimens with and without peginterferon.

In order to assess the natural variation in susceptibility to hepatitis C virus (HCV) NS3 protease inhibitors (PIs) among untreated HCV patient samples, the susceptibilities of 39 baseline clinical isolates were determined using a transient-replication assay on a panel of HCV PIs, including two α-ketoamides (VX-950 and SCH-503034) and three macrocyclic inhibitors (MK-7009, ITMN-191, and TMC-435350). Some natural variation in susceptibility to all HCV PIs tested was observed among the baseline clinical isolates. The susceptibility to VX-950 correlated strongly with the susceptibility to SCH-503034. A moderate correlation was observed between the susceptibilities to ITMN-191 and MK-7009. In contrast, the phenotypic correlations between the α-ketoamides and macrocyclic inhibitors were significantly lower. This difference is partly attributable to reduced susceptibility of the HCV variants containing the NS3 polymorphism Q80K (existing in 47% of genotype 1a isolates) to the macrocyclic compounds but no change in the sensitivity of the same variants to the α-ketoamides tested. Our results suggest that the natural variation in baseline susceptibility may contribute to different degrees of antiviral response among patients in vivo , particularly at lower doses.

Background The purpose of this study was to describe asthma medication adherence behavior and to identify predictors of inhaled corticosteroid (ICS) underuse and inhaled beta-agonist (IBA) overuse. Methods Self-reported medication adherence, spirometry, various measures of status, and blood for immunoglobulin E measurement were collected on 158 subjects from a larger cohort of adults with asthma and rhinitis who were prescribed an ICS, an IBA, or both. Results There was a positive association between ICS underuse and higher forced expiratory volume in one second percent (FEV1%) predicted ( P = .01) and a negative association with lower income ( P = 0.04). IBA overuse was positively associated with greater perceived severity of asthma ( P = 0.004) and negatively with higher education level ( P = 0.02). Conclusions Nonadherence to prescribed asthma therapy seems to be influenced by socioeconomic factors and by perceived and actual severity of disease. These factors are important to assess when trying to estimate the degree of medication adherence and its relationship to clinical presentation.

NF-κB and STATs regulate multiple cellular processes through the transcriptional activation of genes with diversified functions. Although the molecular mechanisms that can turn on/off the overall NF-κB/STAT signaling have been extensively studied, how NF-κB/STAT-target genes can be differentially regulated is poorly understood. Here we report that PIASy, a member of the PIAS (for protein inhibitor of activated STAT) protein family, is a physiologically important transcriptional repressor of NF-κB and STAT1. Piasy deletion in dendritic cells resulted in enhanced expression of a subset of NF-κB and STAT1-dependent genes in response to LPS or IFN-γ treatment, respectively. Consistently, Piasy null mice are hypersensitive to the LPS-induced endotoxic shock. Furthermore, PIASy and PIAS1 display specific as well as redundant effects on the regulation of NF-κB/STAT1 signaling. Pias1 −/− Piasy −/− embryos died before day 11.5. The disruption of one allele of Pias1 in the Piasy −/− background significantly enhanced the effect of Piasy deletion on the transcriptional induction of NF-κB/STAT1-dependent genes, and vice versa. Our results demonstrate that PIASy cooperates with PIAS1 to regulate the specificity and magnitude of NF-κB/STAT1-mediated gene activation.

The 5'-AMP-activated protein kinase (AMPK) is a master sensor for cellular metabolic energy state. It is activated by a high AMP/ATP ratio and leads to metabolic changes that conserve energy and utilize alternative cellular fuel sources. The kinase is composed of a heterotrimeric protein complex containing a catalytic alpha-subunit, an AMP-binding gamma-subunit, and a scaffolding beta-subunit thought to bind directly both the alpha- and gamma-subunits. Here, we use coimmunoprecipitation of proteins in transiently transfected cells to show that the alpha2-subunit binds directly not only to the beta-subunit, confirming previous work, but also to the gamma1-subunit. Deletion analysis of the alpha2-subunit reveals that the C-terminal 386-552 residues are sufficient to bind to the beta-subunit. The gamma1-subunit binds directly to the alpha2-subunit at two interaction sites, one within the catalytic domain consisting of alpha2 amino acids 1-312 and a second within residues 386-552. Binding of the alpha2 and the gamma1-subunits was not affected by 400 mum AMP or ATP. Furthermore, we show that the beta-subunit C terminus is essential for binding to the alpha2-subunit but, in contrast to previous work, the beta-subunit does not bind directly to the gamma1-subunit. Taken together, this study presents a new model for AMPK heterotrimer structure where through its C terminus the beta-subunit binds to the alpha-subunit that, in turn, binds to the gamma-subunit. There is no direct interaction between the beta- and gamma-subunits.

Protein inhibitor of activated STAT Y (PIASy) is the shortest member of the PIAS family and has been reported to modulate the transcriptional activities of STAT1, lymphoid enhancer factor 1 (LEF-1), and the androgen receptor. PIAS proteins have also been identified as E3 ligases for the small ubiquitin-like modifier (SUMO) proteins. PIASy in particular has been reported to mediate SUMO-2/3 modification of LEF-1, sequestering it into nuclear bodies, and SUMO-1 ligation to c-Myb, modulating its transcriptional activation properties. We have cloned murine Piasy and a splice variant which omits exon 6, containing the nuclear retention PINIT motif. Cell culture studies indicate that both the full length and the splice variant are localized in the nucleus but differentially enhance SUMO ligation. To further understand the functions of PIASy, we have generated PIASy-deficient mice. Surprisingly, Piasy(-/-) mice appear phenotypically normal. Activation of STAT1 is not significantly perturbed in Piasy(-/-) cells, and sumoylation patterns for SUMO-1 or SUMO-3 modification are similar when comparing tissues and embryonic fibroblasts from wild-type and knockout mice. Our study demonstrates that at steady state, PIASy is either dispensable or compensated for by other PIAS family members or by other mechanisms when deleted.

Interferon (IFN) activates the signal transducer and activator of transcription (STAT) pathway to regulate immune responses. The protein inhibitor of activated STAT (PIAS) family has been suggested to negatively regulate STAT signaling. To understand the physiological function of PIAS1, we generated Pias1(-/-) mice. Using PIAS1-deficient cells, we show that PIAS1 selectively regulates a subset of IFN-gamma- or IFN-beta-inducible genes by interfering with the recruitment of STAT1 to the gene promoter. The antiviral activity of IFN-gamma or IFN-beta was consistently enhanced by Pias1 disruption. Pias1(-/-) mice showed increased protection against pathogenic infection. Our data indicate that PIAS1 is a physiologically important negative regulator of STAT1 and suggest that PIAS1 is critical for the IFN-gamma- or IFN-beta-mediated innate immune responses.

BACKGROUND: VITAL-1 study assessed safety, tolerability and antiviral activity of the cyclophilin inhibitor alisporivir (ALV) as monotherapy or combination with ribavirin (RBV) compared to IFN/RBV and ALV/IFN in treatment-naive GT2/3 HCV patients. We describe genotypic and phenotypic changes in patients from this study who experienced a viral breakthrough (VB). METHODS: VITAL-1 study patients on IFN-free regimens that did not achieve RVR (, 25 U/ml) at week 4 were administered IFN/RBV/600 mg QD ALV starting at week 6 (IFN-add-on). VB defined as an increase of HCV RNA .1 log10 above nadir. The HCV NS5A gene was analyzed by population sequencing. The entire NS5A region isolated at baseline and at VB was cloned into a JFH1subgenomic shuttle vector for phenotypic analyses. Selected mutations were engineered into a wild type replicon and their impact on replication fitness and susceptibility to ALV and direct acting antiviral drugs were determined. RESULTS: The VB rate with IFN-free or IFN-add-on regimens was 2.7% (7/ 260). Low ALV drug level (Cmin , 20% of group mean; , 100 ng/mL) was observed (5/ 7 VB patients). Genotypic analysis of the NS5A gene revealed amino acid changes in these patients at the time of VB compared to baseline. Replicons bearing NS5A from patients at breakthrough conferred 1.2-16.9-fold shift over baseline EC50 values; 5/7 had impaired replication fitness. No cross-resistance to other classes of HCV inhibitors was observed. Most frequently identified mutations were D320E (D316E in G2), R347W and A349V in Domain II of NS5A, which by themselves decreased ALV susceptibility by 5-, 4, and 8-fold, respectively, in vitro when engineered into wild-type replicon. Combination of multiple mutations decreased susceptibility further, but generally at the cost of replication fitness. CONCLUSION: In treatment-naive G2/3 patients with viral breakthrough, mutations in NS5A domain II were selected, which individually conferred ≤ 8-fold reduced susceptibility to ALV in vitro. The combination of multiple mutations was required to further reduce susceptibility which is consistent with the high barrier to resistance for host-targeting ALV. Observed genotypic changes did not confer cross-resistance to other classes of HCV inhibitors, supporting the use of ALV in future drug combination therapies.