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In a randomized clinical trial, we compare early neutralizing antibody responses after boosting with bivalent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) messenger RNA (mRNA) vaccines based on either BA.1 or BA.4/BA.5 Omicron spike protein combined with wild-type spike. Responses against SARS-CoV-2 variants exhibited the greatest reduction in titers against currently circulating Omicron subvariants for both bivalent vaccines.

In a randomized clinical trial, we compare early neutralizing antibody responses after boosting with bivalent SARS-CoV-2 mRNA vaccines based on either BA.1 or BA.4/BA.5 Omicron spike protein combined with wildtype spike. Responses against SARS-CoV-2 variants exhibited the greatest reduction in titers against currently circulating Omicron subvariants for both bivalent vaccines.

Direct‐acting antiviral therapies (DAAs) may improve kidney function and proteinuria in certain patients with hepatitis C infection (HCV) and chronic kidney disease (CKD). To improve our understanding of HCV‐mediated kidney dysfunction, we aimed to evaluate the baseline predictors of improvement in proteinuria after DAAs in a single‐arm, pilot, clinical trial of ledipasvir 90 mg/sofosbuvir 400 mg once daily for patients with HCV genotype 1 or 4 infection and proteinuric CKD (≥300 mg proteinuria per gram creatinine). Plasma biomarkers of complement system (C3 and C4) and urinary kidney injury biomarkers were measured at baseline, 8 weeks on treatment, 12 weeks following treatment, and 1 year following treatment. We then conducted a retrospective cohort study of patients at Partners Healthcare who had baseline complement component 4 (C4) measured before DAAs for HCV and evaluated the change in estimated glomerular filtration rate (eGFR) before and after therapy. Ten patients with HCV and proteinuric CKD were enrolled in the trial. The mean age was 64 years, 70% male, 70% white, and 30% black. Baseline creatinine was 1.25 mg/dL (SD 0.44), eGFR was 65 mL/min/1.73 m2 (SD 29), and proteinuria was 0.98 g/g creatinine (SD 0.7). Sustained virologic response at 12 weeks was achieved by 80% of patients. Patients with low baseline C4 had improved proteinuria, urinary neutrophil gelatinase‐associated lipocalin, and interleukin‐18 after ledipasvir and sofosbuvir treatment. The retrospective study included 50 patients with CKD and HCV. Twenty patients (40%) had low baseline C4; these patients significantly improved their eGFR (+3.4 ± 11.2 mL/min/1.73 m2) compared to those with normal baseline C4 (−4.4 ± 12.2 mL/min/1.73 m2; P = 0.028). Conclusion: Low C4 may be a marker of kidney dysfunction that improves with DAA therapy.

In COVID-19 the development of severe viral pneumonia that is coupled with systemic inflammatory response triggers multi-organ failure and is of major concern. Cardiac involvement occurs in nearly 60% of patients with pre-existing cardiovascular conditions and heralds worse clinical outcome. Diagnoses carried out in the acute phase of COVID-19 rely upon increased levels of circulating cardiac injury biomarkers and transthoracic echocardiography. These diagnostics, however, were unable to pinpoint the mechanisms of cardiac injury in COVID-19 patients. Identifying the main features of cardiac injury remains an urgent yet unmet need in cardiology, given the potential clinical consequences. Cardiovascular magnetic resonance (CMR) provides an unparalleled opportunity to gain a deeper insight into myocardial injury given its unique ability to interrogate the properties of myocardial tissue. This endeavor is particularly important in convalescent COVID-19 patients as many continue to experience chest pain, palpitations, dyspnea and exertional fatigue, six or more months after the acute illness. This review will provide a critical appraisal of research on cardiovascular damage in convalescent adult COVID-19 patients with an emphasis on the use of CMR and its value to our understanding of organ damage.

Objective The aim of the study was to present: (1) physiatric care delivery amid the SARS-CoV-2 pandemic, (2) challenges, (3) data from the first cohort of post-COVID-19 inpatient rehabilitation facility patients, and (4) lessons learned by a research consortium of New York and New Jersey rehabilitation institutions. Design For this clinical descriptive retrospective study, data were extracted from post-COVID-19 patient records treated at a research consortium of New York and New Jersey rehabilitation inpatient rehabilitation facilities (May 1-June 30, 2020) to characterize admission criteria, physical space, precautions, bed numbers, staffing, employee wellness, leadership, and family communication. For comparison, data from the Uniform Data System and eRehabData databases were analyzed. The research consortium of New York and New Jersey rehabilitation members discussed experiences and lessons learned. Results The COVID-19 patients (N = 320) were treated during the study period. Most patients were male, average age of 61.9 yrs, and 40.9% were White. The average acute care length of stay before inpatient rehabilitation facility admission was 24.5 days; mean length of stay at inpatient rehabilitation facilities was 15.2 days. The rehabilitation research consortium of New York and New Jersey rehabilitation institutions reported a greater proportion of COVID-19 patients discharged to home compared with prepandemic data. Some institutions reported higher changes in functional scores during rehabilitation admission, compared with prepandemic data. Conclusions The COVID-19 pandemic acutely affected patient care and overall institutional operations. The research consortium of New York and New Jersey rehabilitation institutions responded dynamically to bed expansions/contractions, staff deployment, and innovations that facilitated safe and effective patient care.

Many countries in sub-Saharan Africa have experienced lower COVID-19 caseloads and fewer deaths than countries in other regions worldwide. Under-reporting of cases and a younger population could partly account for these differences, but pre-existing immunity to coronaviruses is another potential factor. Blood samples from Sierra Leonean Lassa fever and Ebola survivors and their contacts collected before the first reported COVID-19 cases were assessed using enzyme-linked immunosorbent assays for the presence of antibodies binding to proteins of coronaviruses that infect humans. Results were compared to COVID-19 subjects and healthy blood donors from the United States. Prior to the pandemic, Sierra Leoneans had more frequent exposures than Americans to coronaviruses with epitopes that cross-react with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), SARS-CoV, and Middle Eastern respiratory syndrome coronavirus (MERS-CoV). The percentage of Sierra Leoneans with antibodies reacting to seasonal coronaviruses was also higher than for American blood donors. Serological responses to coronaviruses by Sierra Leoneans did not differ by age or sex. Approximately a quarter of Sierra Leonian pre-pandemic blood samples had neutralizing antibodies against SARS-CoV-2 pseudovirus, while about a third neutralized MERS-CoV pseudovirus. Prior exposures to coronaviruses that induce cross-protective immunity may contribute to reduced COVID-19 cases and deaths in Sierra Leone.

INTRODUCTION AND OBJECTIVE Prune Belly Syndrome (PBS) is characterized by bladder dysmyogenesis, yielding a dysfunctional compliant thick wall with excess collagen deposition. To dissect the cellular heterogeneity and gene expression networks altered in PBS, we report the cell type composition and transcriptional activity of PBS human bladder by using single cell RNA sequencing (scRNA-seq). METHODS Using IRB-approved methods, bladder dome from 2 PBS and 6 non-PBS control (CO) males underwent fresh single-cell digestion. scRNA-seq was performed and 5277 and 31828 bladder cells from PBS and CO patients was detected, respectively. Cell type clusters were graphically displayed by Uniform Manifold Approximation and Projection (UMap) plot and differentially expressed genes (DEGs) were generated to assign each cluster identity. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis was performed for PBS affected genes. RESULTS We identified 17 distinct bladder cell clusters, including 6 fibroblast (1, 2, 3, and 4, immunofibroblast, myofibroblast), 1 smooth muscle (SM), and 2 urothelial (umbrella and basal+intermediate) clusters (Fig 1A-B). Counts of individual cell types were expressed as relative proportions, identifying significant PBS fibroblast enrichment, (67% PBS vs 40% CO). Five of 6 PBS fibroblast sub-types are proportionately fewer in number than in CO. The exception is a dominant fibroblast sub-type we label as fibroblast 4, (61% of all PBS fibroblasts vs

The emergence of the COVID-19 epidemic in the United States (U.S.) went largely undetected due to inadequate testing. New Orleans experienced one of the earliest and fastest accelerating outbreaks, coinciding with Mardi Gras. To gain insight into the emergence of SARS-CoV-2 in the U.S. and how large-scale events accelerate transmission, we sequenced SARS-CoV-2 genomes during the first wave of the COVID-19 epidemic in Louisiana. We show that SARS-CoV-2 in Louisiana had limited diversity compared to other U.S. states, and that one introduction of SARS-CoV-2 led to almost all of the early transmission in Louisiana. By analyzing mobility and genomic data, we show that SARS-CoV-2 was already present in New Orleans before Mardi Gras, and the festival dramatically accelerated transmission. Our study provides an understanding of how superspreading during large-scale events played a key role during the early outbreak in the U.S. and can greatly accelerate epidemics., Genomic and epidemiological analyses provide a clearer picture of one of the earliest SARS-CoV-2 superspreader events in the United States in accelerating transmission, with a single introduction of the virus being responsible for most cases during this period.

We settle the case of equality for the relative isoperimetric inequality outside any arbitrary convex set with not empty interior.

Recent research suggests that SARS-CoV-2-infected individuals can be highly infectious while asymptomatic or pre-symptomatic, and that an infected person may infect 5.6 other individuals on average. This situation highlights the need for rapid, sensitive SARS-CoV-2 diagnostic assays capable of high-throughput operation that can preferably utilize existing equipment to facilitate broad, large-scale screening efforts. We have developed a CRISPR-based assay that can meet all these criteria. This assay utilizes a custom CRISPR Cas12a/gRNA complex and a fluorescent probe to amplify target amplicons produced by standard RT-PCR or isothermal recombinase polymerase amplification (RPA), to allow sensitive detection at sites not equipped with real-time PCR systems required for qPCR diagnostics. We found this approach allowed sensitive and robust detection of SARS-CoV-2 positive samples, with a sample-to-answer time of ∼50 min, and a limit of detection of 2 copies per sample. CRISPR assay diagnostic results obtained nasal swab samples of individuals with suspected COVID-19 cases were comparable to paired results from a CDC-approved qPCR assay performed in a state testing lab, and superior to those produced by same assay in a clinical lab, where the qPCR assay exhibited multiple invalid or inconclusive results. Our assay also demonstrated greater analytical sensitivity and more robust diagnostic performance than other recently reported CRISPR-based assays. Based on these findings, we believe that a CRISPR-based fluorescent application has potential to improve current COVID-19 screening efforts., Highlights • Integrated CRISPR for COVID-19 clinical diagnosis. • Rapidly identify SARS-CoV-2-specific RNA signatures at an ultra-low concentration. • Detection environment friendly and compatible for high throughput COVID-19 screening in hospital. • Successful development and validation with swab samples from patients. • Improve diagnosis of individuals with suspected COVID-19 infections.

Introduction Background cross-reactivity with other coronaviruses may reduce the specificity of COVID-19 rapid serologic tests. The vast majority of women attend prenatal care, which is a unique source of population-based blood samples appropriate for validation studies. We used stored 2018 serum samples from an existing pregnancy cohort study to evaluate the specificity of COVID-19 serologic rapid diagnostic tests. Methods We randomly selected 120 stored serum samples from pregnant women enrolled in a cohort in 2018 in Tegucigalpa, Honduras, at least 1 year before the COVID-19 pandemic. We used stored serum to evaluate four lateral flow rapid diagnostic tests, following manufacturers’ instructions. Pictures were taken for all tests and read by two blinded trained evaluators. Results We evaluated 120, 80, 90, and 90 samples, respectively. Specificity for both IgM and IgG was 100% for the first two tests (95% confidence intervals [CI] 97.0–100 and 95.5–100, respectively). The third test had a specificity of 98.9% (95% CI 94.0–100) for IgM and 94.4% (95% CI 87.5–98.2) for IgG. The fourth test had a specificity of 88.9% (95% CI 80.5–94.5) for IgM and 100% (95% CI 96.0–100) for IgG. Discussion COVID-19 serologic rapid tests are of variable specificity. Blood specimens from sentinel prenatal clinics provide an opportunity to validate serologic tests with population-based samples.

IntroductionBackground cross-reactivity with other coronaviruses may reduce the specificity of COVID-19 rapid serologic tests. Blood collected during prenatal care is a unique source of population-based samples appropriate for validation studies. We used stored 2018 serum samples from an existing pregnancy cohort study to evaluate the specificity of COVID-19 serologic rapid diagnostic tests. MethodsWe randomly selected 120 stored serum samples from pregnant women enrolled in a cohort in 2018, at least one year before the COVID-19 pandemic. We used stored serum to evaluate four lateral flow rapid diagnostic tests, following manufacturers’ instructions. Pictures were taken for all tests and read by two blinded trained evaluators. Results We evaluated 120, 80, 90, and 90 samples, respectively. Specificity for both IgM and IgG was 100% for the first two tests. The third test had a specificity of 98.9% for IgM and 94.4% for IgG. The fourth test had a specificity of 88.9% for IgM and 100% for IgG.Discussion COVID-19 serologic rapid tests are of variable specificity. Blood specimens from sentinel prenatal clinics provide an opportunity to validate serologic tests with population-based samples.

Background The developing field of osteoimmunology supports importance of an interferon (IFN) response pathway in osteoblasts. Clarifying osteoblast-IFN interactions is important because IFN is used as salvage anti-tumor therapy but systemic toxicity is high with variable clinical results. In addition, osteoblast response to systemic bursts and disruptions of IFN pathways induced by viral infection may influence bone remodeling. ZIKA virus (ZIKV) infection impacts bone development in humans and IFN response in vitro. Consistently, initial evidence of permissivity to ZIKV has been reported in human osteoblasts. Hypothesis Osteoblast-like Saos-2 cells are permissive to ZIKV and responsive to IFN. Methods Multiple approaches were used to assess whether Saos-2 cells are permissive to ZIKV infection and exhibit IFN-mediated ZIKV suppression. Proteomic methods were used to evaluate impact of ZIKV and IFN on Saos-2 cells. Results Evidence is presented confirming Saos-2 cells are permissive to ZIKV and support IFN-mediated suppression of ZIKV. ZIKV and IFN differentially impact the Saos-2 proteome, exemplified by HELZ2 protein which is upregulated by IFN but non responsive to ZIKV. Both ZIKV and IFN suppress proteins associated with microcephaly/pseudo-TORCH syndrome (BI1, KI20A and UBP18), and ZIKV induces potential entry factor PLVAP. Conclusions Transient ZIKV infection influences osteoimmune state, and IFN and ZIKV activate distinct proteomes in Saos-2 cells, which could inform therapeutic, engineered, disruptions.

Axillary web syndrome (AWS) is a complication of surgical procedures in breast cancer (BC) patients. This condition with poorly understood incidence and etiology is characterized by the locoregional development of scar tissue, leading to subcutaneous cording, motion impairment and pain. The early identification of patients at risk for AWS would improve their clinical management. Here, we sought to characterize the prevalence of and the risk factors associated with AWS in BC women after surgery.All patients with BC that underwent axillary surgery referred to an Outpatient Service for Oncological Rehabilitation were retrospectively collected. These women were assessed two weeks after the surgical procedure for their clinicopathologic features, type of therapeutic interventions, and AWS presence, laterality, pain, localization, cords type, and number of cords.Altogether, 177 patients (mean aged 60.65 ± 12.26 years) were included and divided into two groups: AWSPOS (n=52; 29.4%) and AWSNEG (n=125; 70.6%). Patients with tumor N ≥1 (OR=3.7; p0.001), subjected to mastectomy, axillary lymph node dissection (ALND) and chemotherapy showed significant correlations with AWS onset (p0.05). The range of shoulder motion limitation (OR=11.2; p0.001) and the presence of breast cancer related lymphedema (OR=3.5; p=0.020) were associated with AWS.Mastectomy, ALND, chemotherapy, low staging tumors, shoulder range of motion limitations, and BCRL represent risk factors for AWS onset. Realizing new strategies for assessing the individual risk of AWS is a crucial clinical need to improve the health-related quality of life of BC survivors.

Introduction Providing feedback is a fundamental principle in medical education; however, as educators, our community lacks the necessary skills to give meaningful, impactful feedback to those under our supervision. By improving our feedback-giving skills, we provide concrete ways for trainees to optimize their performance, ultimately leading to better patient care. Methods In this faculty development workshop, faculty groups used six feedback video vignettes scripted, enacted, and produced by residents to arrive at a shared mental model of feedback. During workshop development, we used qualitative analysis for faculty narratives combined with the findings from a focused literature review to define dimensions of feedback. Results Twenty-three faculty (physical medicine and rehabilitation and neurology) participated in seven small-group workshops. Analysis of group discussion notes yielded 343 codes that were collapsed into 25 coding categories. After incorporating the results of a focused literature review, we identified 48 items grouped into 10 dimensions of feedback. Online session evaluation indicated that faculty members liked the workshop's format and thought they were better at providing feedback to residents as a result of the workshop. Discussion Small faculty groups were able to develop a shared mental model of dimensions of feedback that was also grounded in medical education literature. The theme of specificity of feedback was prominent and echoed recent medical education research findings. Defining performance expectations for feedback providers in the form of a practical and psychometrically sound rubric can enhance reliable scoring of feedback performance assessments and should be the next step in our work.

HIV/HCV co-infection accelerates progressive liver fibrosis, however the mechanisms remain poorly understood. HCV and HIV independently induce profibrogenic markers TGFβ1 (mediated by reactive oxygen species (ROS)) and NFκB in hepatocytes and hepatic stellate cells (HSC) in monoculture, however, they do not account for cellular cross-talk that naturally occurs. We created an in vitro co-culture model and investigated the contributions of HIV and HCV to hepatic fibrogenesis. GFP reporter cell lines driven by functional ROS (ARE), NFκB, and SMAD3 promoters were created in Huh7.5.1 and LX2 cells, using a transwell to generate co-cultures. Reporter cells lines were exposed to HIV, HCV or HIV/HCV. Activation of the 3 pathways were measured, and compared according to infection status. Extracellular matrix products (Col1A1 and TIMP1) were also measured. Both HCV and HIV independently activate TGFβ1 signaling via ROS (ARE), NFκB, and SMAD3 in both cell lines in co-culture. Activation of these profibrotic pathways was additive following HIV/HCV co-exposure. This was confirmed when examining Col1A1 and TIMP1, where mRNA and protein levels were significantly higher in LX2 cells in co-culture following HIV/HCV co-exposure compared with either virus alone. In addition, expression of these profibrotic genes was significantly higher in the co-culture model compared to either cell type in monoculture, suggesting an interaction and feedback mechanism between Huh7.5.1 and LX2 cells. We conclude that HIV accentuates an HCV-driven profibrogenic program in hepatocyte and HSC lines through ROS, NFκB and TGFβ1 upregulation. Furthermore, co-culture of hepatocyte and HSC lines significantly increased expression of Col1A1 and TIMP1. Our novel co-culture reporter cell model represents an efficient and more authentic system for studying transcriptional fibrosis responses, and may provide important insights into hepatic fibrosis. This article is protected by copyright. All rights reserved.

Resume Notre but etait d’evaluer la prise en charge peri-operatoire des patients diabetiques par les medecins anesthesistes et verifier sa conformite aux recommandations professionnelles, enquete menee conjointement dans un Centre hospitalier universitaire (CHU) et un Centre hospitalier prive (CHP). La population etudiee a concerne des patients majeurs diabetiques connus, qu’ils soient non-insulines (type 2 [DT2]), ou insulino-traites (type 1 [DT1], ou DT2 insulines), devant beneficier d’une intervention chirurgicale programmee sur le CHU Pontchaillou de Rennes ou le CHP de Cesson-Sevigne. Le recueil des donnees a ete effectue pendant une periode de 3 mois a l’aide d’un questionnaire standardise, renseigne par l’anesthesiste ou l’interne en charge du patient. Les questionnaires etaient completes lors de la consultation anesthesique, au bloc operatoire, puis a la sortie du patient. Entre janvier et mars 2014, les questionnaires concernant 161 patients diabetiques ont ete recueillis : 117 patients DT2, et 44 patients DT1 ou DT2 insulines. L’exhaustivite globale, evaluee sur les donnees PMSI, est de 74%, mais avec une disparite selon les specialites chirurgicales. Quels que soient le type de diabete et la duree attendue du jeune postoperatoire, une majorite de patients ont une prise en charge conforme aux recommandations (suspension de la metformine, poursuite des autres antidiabetiques oraux et de l’insuline, apports glucoses, relais eventuel par insulinotherapie per- et post-operatoire). Ainsi, si la prise en charge anesthesique des patients diabetiques est complexe, parfois difficile, elle est globalement satisfaisante. Notre etude montre que les recommandations sont relativement bien appliquees par les anesthesistes-reanimateurs, qui peuvent s’appuyer sur des protocoles therapeutiques solides et largement diffuses dans les differents services.

Understanding the control of viral infections is of broad importance. Chronic hepatitis C virus (HCV) infection causes decreased expression of the iron hormone hepcidin, which is regulated by hepatic bone morphogenetic protein (BMP)/SMAD signalling. We found that HCV infection and the BMP/SMAD pathway are mutually antagonistic. HCV blunted induction of hepcidin expression by BMP6, probably via tumour necrosis factor (TNF)-mediated downregulation of the BMP co-receptor haemojuvelin. In HCV-infected patients, disruption of the BMP6/hepcidin axis and genetic variation associated with the BMP/SMAD pathway predicted the outcome of infection, suggesting that BMP/SMAD activity influences antiviral immunity. Correspondingly, BMP6 regulated a gene repertoire reminiscent of type I interferon (IFN) signalling, including upregulating interferon regulatory factors (IRFs) and downregulating an inhibitor of IFN signalling, USP18. Moreover, in BMP-stimulated cells, SMAD1 occupied loci across the genome, similar to those bound by IRF1 in IFN-stimulated cells. Functionally, BMP6 enhanced the transcriptional and antiviral response to IFN, but BMP6 and related activin proteins also potently blocked HCV replication independently of IFN. Furthermore, BMP6 and activin A suppressed growth of HBV in cell culture, and activin A inhibited Zika virus replication alone and in combination with IFN. The data establish an unappreciated important role for BMPs and activins in cellular antiviral immunity, which acts independently of, and modulates, IFN.

A 76-year-old Cambodian man co- infected with hepatitis B virus (HBV) and hepatitis C virus (HCV) 6c-1 presented for care. HBV DNA was intermittently detectable despite anti-HBs levels being above the protective threshold. During treatment for HCV, HBV DNA levels increased. Sequencing revealed multiple mutations including vaccine escape mutation and mutations predicted to enhance fitness. This case represents exacerbation of an HBV vaccine escape mutant during a direct-acting antiviral therapy.

Hepatitis C virus (HCV) infection has been shown to regulate microRNA 130a (miR-130a) in patient biopsy specimens and in cultured cells. We sought to identify miR-130a target genes and to explore the mechanisms by which miR-130a regulates HCV and hepatitis B virus (HBV) replication. We used bioinformatics software, including miRanda, TargetScan, PITA, and RNAhybrid, to predict potential miR-130a target genes. miR-130a and its target genes were overexpressed or were knocked down by use of small interfering RNA (siRNA) or clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 guide RNA (gRNA). Selected gene mRNAs and their proteins, together with HCV replication in OR6 cells, HCV JFH1-infected Huh7.5.1 cells, and HCV JFH1-infected primary human hepatocytes (PHHs) and HBV replication in HepAD38 cells, HBV-infected NTCP-Huh7.5.1 cells, and HBV-infected PHHs, were measured by quantitative reverse transcription-PCR (qRT-PCR) and Western blotting, respectively. We selected 116 predicted target genes whose expression was related to viral pathogenesis or immunity for qPCR validation. Of these, the gene encoding pyruvate kinase in liver and red blood cell (PKLR) was confirmed to be regulated by miR-130a overexpression. miR-130a overexpression (via a mimic) knocked down PKLR mRNA and protein levels. A miR-130a inhibitor and gRNA increased PKLR expression, HCV replication, and HBV replication, while miR-130a gRNA and PKLR overexpression increased HCV and HBV replication. Supplemental pyruvate increased HCV and HBV replication and rescued the inhibition of HCV and HBV replication by the miR-130a mimic and PKLR knockdown. We concluded that miR-130a regulates HCV and HBV replication through its targeting of PKLR and subsequent pyruvate production. Our data provide novel insights into key metabolic enzymatic pathway steps regulated by miR-130a, including the steps involving PKLR and pyruvate, which are subverted by HCV and HBV replication. IMPORTANCE We identified that miR-130a regulates the target gene PKLR and its subsequent effect on pyruvate production. Pyruvate is a key intermediate in several metabolic pathways, and we identified that pyruvate plays a key role in regulation of HCV and HBV replication. This previously unrecognized, miRNA-regulated antiviral mechanism has implications for the development of host-directed strategies to interrupt the viral life cycle and prevent establishment of persistent infection for HCV, HBV, and potentially other viral infections.

The elongation factor Tu GTP binding domain-containing protein 2 (EFTUD2) was identified as an anti-hepatitis C virus (HCV) host factor in our recent genome-wide small interfering RNA (siRNA) screen. In this study, we sought to further determine EFTUD2's role in HCV infection and investigate the interaction between EFTUD2 and other regulators involved in HCV innate immune (RIG-I, MDA5, TBK1, and IRF3) and JAK-STAT1 pathways. We found that HCV infection decreased the expression of EFTUD2 and the viral RNA sensors RIG-I and MDA5 in HCV-infected Huh7 and Huh7.5.1 cells and in liver tissue from in HCV-infected patients, suggesting that HCV infection downregulated EFTUD2 expression to circumvent the innate immune response. EFTUD2 inhibited HCV infection by inducing expression of the interferon (IFN)-stimulated genes (ISGs) in Huh7 cells. However, its impact on HCV infection was absent in both RIG-I knockdown Huh7 cells and RIG-I-defective Huh7.5.1 cells, indicating that the antiviral effect of EFTUD2 is dependent on RIG-I. Furthermore, EFTUD2 upregulated the expression of the RIG-I-like receptors (RLRs) RIG-I and MDA5 to enhance the innate immune response by gene splicing. Functional experiments revealed that EFTUD2-induced expression of ISGs was mediated through interaction of the EFTUD2 downstream regulators RIG-I, MDA5, TBK1, and IRF3. Interestingly, the EFTUD2-induced antiviral effect was independent of the classical IFN-induced JAK-STAT pathway. Our data demonstrate that EFTUD2 restricts HCV infection mainly through an RIG-I/MDA5-mediated, JAK-STAT-independent pathway, thereby revealing the participation of EFTUD2 as a novel innate immune regulator and suggesting a potentially targetable antiviral pathway. IMPORTANCE Innate immunity is the first line defense against HCV and determines the outcome of HCV infection. Based on a recent high-throughput whole-genome siRNA library screen revealing a network of host factors mediating antiviral effects against HCV, we identified EFTUD2 as a novel innate immune regulator against HCV in the infectious HCV cell culture model and confirmed that its expression in HCV-infected liver tissue is inversely related to HCV infection. Furthermore, we determined that EFTUD2 exerts its antiviral activity mainly through governing its downstream regulators RIG-I and MDA5 by gene splicing to activate IRF3 and induce classical ISG expression independent of the JAT-STAT signaling pathway. This study broadens our understanding of the HCV innate immune response and provides a possible new antiviral strategy targeting this novel regulator of the innate response.

Occipital neuralgia (ON) is a neuropathic condition characterized by paroxysmal shooting, lancinating, and stabbing pain episodes in the distribution of the occipital nerve dermatome (Cesmebasi et al., Clin Anat 28: 101–8, 2015; Dougherty, Curr Pain Headache Rep 18: 1–5, 2014). Symptoms are commonly attributed to nerve entrapment, inflammation, or trauma (Dougherty, Curr Pain Headache Rep 18: 1–5, 2014; Vanelderen et al., Pain Pract 10: 137–44, 2010). The etiology of ON remains unknown and its diagnosis is complicated by the similarity of its symptoms to those of other primary headache disorders (Dougherty, Curr Pain Headache Rep 18: 1–5, 2014; Hoppenfeld, Curr Pain Headache Rep 14: 418–23, 2010). ON is most commonly diagnosed clinically (Dougherty, Curr Pain Headache Rep 18: 1–5, 2014). A multimodal approach to treatment, including physical therapy, antiepileptics, tricyclic antidepressants, and greater occipital neuromodulation via anesthetic blockade, is often successful in ameliorating the symptoms of ON (Dougherty, Curr Pain Headache Rep 18: 1–5, 2014; Hoppenfeld, Curr Pain Headache Rep 14: 418–23, 2010).

AIM To characterize the understanding of hepatitis B virus (HBV) and determine if outreach improves HBV understanding among Greater Boston Area immigrants. METHODS Six outreach sessions were held in various community venues in the Greater Boston Area. Verbal consent was obtained from participants prior to starting each session. Each session included a pre-session questionnaire, followed by a teaching session, and then a post-session questionnaire. In person interpreters were present for translation during the teaching session and assistance for questionnaire completion when needed. The questions were developed based on the HBV clinical experience of physicians who serve largely immigrant populations. Questionnaires included Likert-type scale, open-ended, and true-false questions. All results were anonymous. RESULTS One hundred and one people participated in this study. Participants were 30% male with ages ranging from 19 to 87 years. The study population included immigrants from 21 countries, as well as seven United States-born participants. The greatest numbers of participants were from Somalia (44%), Morocco (10%), and Cameroon (8%). Pre session questionnaires revealed that 42% of participants were unaware that HBV can cause cancer, and 50% were unaware that therapies for HBV exist. Our brief teaching intervention led to improved scores on post session questionnaires. For example, at baseline, 58% of participants responded correctly to the question “HBV infection can cause scarring of the liver and liver cancer”, whereas 79% of participants responded correctly after the teaching session (P = 0.01). Furthermore, the mean of total correct answers in the true or false portion of the questionnaire increased from 5.5 to 7.6 (P < 0.001). CONCLUSION A teaching session targeting Boston Immigrants at-risk for HBV helped improve scores on HBV knowledge questionnaires. Outreach may empower at-risk patients to pro-actively seek HBV care.

Flaviviral infections including dengue virus are an increasing clinical problem worldwide. Dengue infection triggers host production of the type 1 IFN, IFN alpha, one of the strongest and broadest acting antivirals known. However, dengue virus subverts host IFN signaling at early steps of IFN signal transduction. This subversion allows unbridled viral replication which subsequently triggers ongoing production of IFN which, again, is subverted. Identification of downstream IFN antiviral effectors will provide targets which could be activated to restore broad acting antiviral activity, stopping the signal to produce endogenous IFN at toxic levels. To this end, we performed a targeted functional genomic screen for IFN antiviral effector genes (IEGs), identifying 56 IEGs required for antiviral effects of IFN against fully infectious dengue virus. Dengue IEGs were enriched for genes encoding nuclear receptor interacting proteins, including HELZ2, MAP2K4, SLC27A2, HSP90AA1, and HSP90AB1. We focused on HELZ2 (Helicase With Zinc Finger 2), an IFN stimulated gene and IEG which encodes a promiscuous nuclear factor coactivator that exists in two isoforms. The two unique HELZ2 isoforms are both IFN responsive, contain ISRE elements, and gene products increase in the nucleus upon IFN stimulation. Chromatin immunoprecipitation-sequencing revealed that the HELZ2 complex interacts with triglyceride-regulator LMF1. Mass spectrometry revealed that HELZ2 knockdown cells are depleted of triglyceride subsets. We thus sought to determine whether HELZ2 interacts with a nuclear receptor known to regulate immune response and lipid metabolism, AHR, and identified HELZ2:AHR interactions via co-immunoprecipitation, found that AHR is a dengue IEG, and that an AHR ligand, FICZ, exhibits anti-dengue activity. Primary bone marrow derived macrophages from HELZ2 knockout mice, compared to wild type controls, exhibit enhanced dengue infectivity. Overall, these findings reveal that IFN antiviral response is mediated by HELZ2 transcriptional upregulation, enrichment of HELZ2 protein levels in the nucleus, and activation of a transcriptional program that appears to modulate intracellular lipid state. IEGs identified in this study may serve as both (1) potential targets for host directed antiviral design, downstream of the common flaviviral subversion point, as well as (2) possible biomarkers, whose variation, natural, or iatrogenic, could affect host response to viral infections.

AIM To characterize the role of apolipoprotein B100 (apoB100) in hepatitis C viral (HCV) infection. METHODS In this study, we utilize a gene editing tool, transcription activator-like effector nucleases (TALENs), to generate human hepatoma cells with a stable genetic deletion of APOB to assess of apoB in HCV. Using infectious cell culture-competent HCV, viral pseudoparticles, replicon models, and lipidomic analysis we determined the contribution of apoB to each step of the viral lifecycle. We further studied the effect of mipomersen, an FDA-approved antisense inhibitor of apoB100, on HCV using in vitro cell-culture competent HCV and determined its impact on viral infectivity with the TCID50 method. RESULTS We found that apoB100 is indispensable for HCV infection. Using the JFH-1 fully infectious cell-culture competent virus in Huh 7 hepatoma cells with TALEN-mediated gene deletion of apoB (APOB KO), we found a significant reduction in HCV RNA and protein levels following infection. Pseudoparticle and replicon models demonstrated that apoB did not play a role in HCV entry or replication. However, the virus produced by APOB KO cells had significantly diminished infectivity as measured by the TCID-50 method compared to wild-type virus. Lipidomic analysis demonstrated that these virions have a fundamentally altered lipidome, with complete depletion of cholesterol esters. We further demonstrate that inhibition of apoB using mipomersen, an FDA-approved anti-sense oligonucleotide, results in a potent anti-HCV effect and significantly reduces the infectivity of the virus. CONCLUSION ApoB is required for the generation of fully infectious HCV virions, and inhibition of apoB with mipomersen blocks HCV. Targeting lipid metabolic pathways to impair viral infectivity represents a novel host targeted strategy to inhibit HCV.

HCV replication disrupts normal endoplasmic reticulum (ER) function and activates a signaling network called the unfolded protein response (UPR). UPR is directed by three ER transmembrane proteins including ATF6, IRE1 and PERK. HCV increases TGF-β1 and oxidative stress, which play important roles in liver fibrogenesis. HCV has been shown to induce TGF-β1 through the generation of reactive oxygen species (ROS) and p38 MAPK, JNK, ERK1/2 and NFκB-dependent pathways. However, the relationship between HCV-induced ER stress and UPR activation with TGF-β1 production has not been fully characterized. In this study, we found that ROS and JNK inhibitors block HCV up-regulation of ER stress and UPR activation. ROS, JNK and IRE1 inhibitors blocked HCV-activated NFκB and TGF-β1 expression. ROS, ER stress, NFκB and TGF-β1 signaling were blocked by JNK specific siRNA. Knockdown IRE1 inhibited JFH1-activated NFκB and TGF-β1 activity. Knockdown of JNK and IRE1 blunted JFH1 HCV up-regulation of NFκB and TGF-β1 activation. We conclude that HCV activates NFκB and TGF-β1 through ROS production and induction of JNK and the IRE1 pathway. HCV infection induces ER stress and the UPR in a JNK-dependent manner. ER stress and UPR activation partially contribute to HCV-induced NF-κB activation and enhancement of TGF-β1.

Liver failure due to hepatitis C virus (HCV) infection is among the leading causes of death in individuals infected with human immunodeficiency virus (HIV) [1]. Nevertheless, HCV infection leads to cirrhosis in a minority of patients and only over a period of decades, even in those coinfected with HIV [2]. Historically, most coinfected patients acquired both HCV and HIV parenterally, and due to the higher infectivity of HCV parenterally, that infection was acquired first [3]. These coinfected patients have a modestly accelerated course of fibrosis progression compared to patients with HCV alone, with a mean time to stage 4 fibrosis (histologic cirrhosis) of 26 years, compared to 34 years in non–HIV-infected patients [4]. Once histologic cirrhosis develops, liver failure and death do not typically occur for another 5–10 years, resulting in an HCV disease course of 3–4 decades or longer. Recently, an international epidemic of HCV infection among HIV-infected men who have sex with men (MSM) has occurred [5, 6]. These men acquired both HIV and HCV sexually, and partly due to the higher infectivity of HIV through a sexual route, that infection was acquired first. Because fibrosis does not progress rapidly after primary HCV infection in patients without underlying HIV infection [7, 8], we were surprised to find that 9 of 11 HIV-infected men with newly acquired HCV had developed moderate liver fibrosis (stage 2 of 4 [9]) shortly after diagnosis of their primary HCV infection [10]. We therefore proposed that there is a rapid onset of HCV-induced fibrosis due to the immunocompromise that results from an established HIV infection. Our findings were corroborated by a group in Belgium who found that 22 of 37 (59%) HIV-infected men who underwent liver biopsy a median of 7 months after the diagnosis of primary HCV infection had stage 2 or 3 fibrosis [11]. We then expanded our biopsy series to 29 men with longer follow-up (up to 2 years after primary HCV infection) and found significantly higher stages of fibrosis in men who underwent liver biopsy later in their HCV course [12], demonstrating that fibrosis is progressive and does not spontaneously resolve after the primary HCV infection period. Neither we nor others [13], however, were able to determine the long-term natural history of the liver disease as the follow-up time was short and most men in these studies were subsequently cured of their HCV infection. So even with corroboration of our findings by Bottieau et al [11], some continued to suggest that early-onset fibrosis might not progress further at a clinically important rate [13]. We have continued to follow a cohort of HIV-infected men with subsequent primary HCV infection that was not cured and now report the cases of 4 men who progressed to decompensated cirrhosis in 17 months to 6 years after primary HCV infection. These cases demonstrate that the early-onset fibrosis due to primary HCV infection in HIV-infected men cannot be considered benign, and can continue to progress rapidly to cirrhosis, liver failure, and death in some men.

Responses to alpha interferon (IFN-α)-based treatment are dependent on both host and viral factors and vary markedly among patients infected with different hepatitis C virus (HCV) genotypes (GTs). Patients infected with GT3 viruses consistently respond better to IFN treatment than do patients infected with GT1 viruses. The mechanisms underlying this difference are not well understood. In this study, we sought to determine the effects of HCV NS5A proteins from different genotypes on IFN signaling. We found that the overexpression of either GT1 or GT3 NS5A proteins significantly inhibited IFN-induced IFN-stimulated response element (ISRE) signaling, phosphorylated STAT1 (P-STAT1) levels, and IFN-stimulated gene (ISG) expression compared to controls. GT1 NS5A protein expression exhibited stronger inhibitory effects on IFN signaling than did GT3 NS5A protein expression. Furthermore, GT1 NS5A bound to STAT1 with a higher affinity than did GT3 NS5A. Domain mapping revealed that the C-terminal region of NS5A conferred these inhibitory effects on IFN signaling. The overexpression of HCV NS5A increased HCV replication levels in JFH1-infected cells through the further reduction of levels of P-STAT1, ISRE signaling, and downstream ISG responses. We demonstrated that the overexpression of GT1 NS5A proteins resulted in less IFN responsiveness than did the expression of GT3 NS5A proteins through stronger binding to STAT1. We confirmed that GT1 NS5A proteins exerted stronger IFN signaling inhibition than did GT3 NS5A proteins in an infectious recombinant JFH1 virus. The potent antiviral NS5A inhibitor BMS-790052 did not block NS5A-mediated IFN signaling suppression in an overexpression model, suggesting that NS5A's contributions to replication are independent of its subversive action on IFN. We propose a model in which the binding of the C-terminal region of NS5A to STAT1 leads to decreased levels of P-STAT1, ISRE signaling, and ISG transcription and, ultimately, to preferential GT1 resistance to IFN treatment.