Diversity of CFTR variants across ancestries characterized using 454,727 UK biobank whole exome sequences.

BACKGROUND: Limited understanding of the diversity of variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene across ancestries hampers efforts to advance molecular diagnosis of cystic fibrosis (CF). The consequences pose a risk of delayed diagnoses and subsequently worsened health outcomes for patients. Therefore, characterizing the spectrum of CFTR variants across ancestries is critical for revolutionizing molecular diagnoses of CF. METHODS: We analyzed 454,727 UK Biobank (UKBB) whole-exome sequences to characterize the diversity of CFTR variants across ancestries. Using the PanUKBB classification, the participants were assigned into six major groups: African (AFR), American/American Admixed (AMR), Central South Asia (CSA), East Asian (EAS), European (EUR), and Middle East (MID). We segregated ancestry-specific CFTR variants, including those that are CF-causing or clinically relevant. The ages of certain CF-causing variants were determined and analyzed for selective pressure effects, and curated phenotype analysis was performed for participants with clinically relevant CFTR genotypes. RESULTS: We detected over 4000 CFTR variants, including novel ancestry-specific variants, across six ancestries. Europeans had the most unique CFTR variants [n = 2212], while the American group had the least unique variants [n = 23]. F508del was the most prevalent CF-causing variant found in all ancestries, except in EAS, where V520F was the most prevalent. Common EAS variants such as 3600G > A, V456A, and V520, which appeared approximately 270, 215, and 338 generations ago, respectively, did not show evidence of selective pressure. Sixteen participants had two CF-causing variants, with two being diagnosed with CF. We found 154 participants harboring a CF-causing and varying clinical consequences (VCC) variant. Phenotype analysis performed for participants with multiple clinically relevant variants returned significant associations with CF and its pulmonary phenotypes [Bonferroni-adjusted p < 0.05]. CONCLUSIONS: We leveraged the UKBB database to comprehensively characterize the broad spectrum of CFTR variants across ancestries. The detection of over 4000 CFTR variants, including several ancestry-specific and uncharacterized CFTR variants, warrants the need for further characterization of their functional and clinical relevance. Overall, the presentation of classical CF phenotypes seen in non-CF diagnosed participants with more than one CF-causing variant indicates that they may benefit from current CFTR modulator therapies.

Stromal Interaction Molecule 1 Maintains ß-Cell Identity and Function in Female Mice Through Preservation of G-Protein-Coupled Estrogen Receptor 1 Signaling.

Altered endoplasmic reticulum (ER) Ca2+ signaling has been linked with ß-cell dysfunction and diabetes development. Store-operated Ca2+ entry replenishes ER Ca2+ through reversible gating of plasma membrane Ca2+ channels by the ER Ca2+ sensor, stromal interaction molecule 1 (STIM1). For characterization of the in vivo impact of STIM1 loss, mice with ß-cell-specific STIM1 deletion (STIM1Δß mice) were generated and challenged with high-fat diet. Interestingly, ß-cell dysfunction was observed in female, but not male, mice. Female STIM1Δß mice displayed reductions in ß-cell mass, a concomitant increase in α-cell mass, and reduced expression of markers of ß-cell maturity, including MafA and UCN3. Consistent with these findings, STIM1 expression was inversely correlated with HbA1c levels in islets from female, but not male, human organ donors. Mechanistic assays demonstrated that the sexually dimorphic phenotype observed in STIM1Δß mice was due, in part, to loss of signaling through the noncanonical 17-ß estradiol receptor (GPER1), as GPER1 knockdown and inhibition led to a similar loss of expression of ß-cell maturity genes in INS-1 cells. Together, these data suggest that STIM1 orchestrates pancreatic ß-cell function and identity through GPER1-mediated estradiol signaling. ARTICLE HIGHLIGHTS: Store-operated Ca2+ entry replenishes endoplasmic reticulum (ER) Ca2+ through reversible gating of plasma membrane Ca2+ channels by the ER Ca2+ sensor, stromal interaction molecule 1 (STIM1). ß-Cell-specific deletion of STIM1 results in a sexually dimorphic phenotype, with ß-cell dysfunction and loss of identity in female but not male mice. Expression of the noncanonical 17-ß estradiol receptor (GPER1) is decreased in islets of female STIM1Δß mice, and modulation of GPER1 levels leads to alterations in expression of ß-cell maturity genes in INS-1 cells.

Durability of sustained virological response to glecaprevir/pibrentasvir and resistance development: A long-term follow-up study.

BACKGROUND AND AIMS: This study aimed to determine durability of sustained virologic response (SVR) in hepatitis C virus-infected participants treated with glecaprevir- and/or pibrentasvir-containing regimens. METHODS: M13-576, a rollover study, evaluated the durability of SVR in a follow-up period of approximately 3 years after hepatitis C virus genotype 1-6-infected participants received a glecaprevir- and/or pibrentasvir-containing regimen in previous phase 2/3 clinical trials. The primary efficacy endpoint was the percentage of participants maintaining SVR and the percentage of participants experiencing relapse or reinfections. Resistance-associated substitutions and safety outcomes related to liver progression were also assessed. RESULTS: Of 384 participants enroled, 377 participants were included in the as-observed population and 287 participants completed the study. In prior studies, 99.7% (376/377) of participants achieved SVR12; of those, an observed 99.5% (374/376) and 100% (286/286) completing the study, maintained SVR. After non-responder imputation of missing data, 286/376 participants (76%) maintained SVR. The participant previously not achieving SVR was a treatment-experienced male with compensated cirrhosis who had NS3 and NS5A substitutions at enrolment, which remained detectable for 12 months. Of the two participants not maintaining SVR, one was re-infected and one experienced late relapse at post-treatment week 60. Five participants (all with a fibrosis stage ≥F3) had hepatocellular carcinoma. No events were deemed related to glecaprevir/pibrentasvir. CONCLUSIONS: Glecaprevir/pibrentasvir demonstrated long-term durability of efficacy after SVR12 was achieved. Hepatic-related decompensation events were not seen. Owing to low incidence of virologic failure, conclusions were not drawn on persistence of resistance-associated substitutions.

Periodontal approach in the management of alveolar cleft.

The alveolar cleft is a bone-related developmental defect in the alveolar process of the maxillae, which is termed as cleft alveolus. The deformity occurs in 75% of the cleft palate and lip patients. Reconstructive surgery can provide both functional and esthetic benefits to such individuals. Conflicting opinions exist on the management of alveolar cleft, and these affect the treatment planning. We present the case of a 19-year-old female patient with a complaint of mobile teeth in the left frontal region of the upper jaw. On clinical examination, unilateral cleft alveolus was observed between the left lateral incisor and the canine region. A multidisciplinary approach was adopted, orthodontic treatment was started, and periodontal regenerative surgery was planned. This report also discusses the substitution of autogenous bone grafts with other materials such as allogenic grafts (demineralized freeze-dried bone allograft), platelet-rich plasma, platelet-rich fibrin membranes, and amnion membranes, which could serve as a new line of treatment for the condition.

Discovery of 2-aminoisobutyric acid ethyl ester (AIBEE) phosphoramidate prodrugs for delivering nucleoside HCV NS5B polymerase inhibitors.

Our HCV research program investigated novel 2'-dihalogenated nucleoside HCV polymerase inhibitors and identified compound 1, a 5'-phosphoramidate prodrug of 2'-deoxy-2'-α-bromo-ß-chloro uridine. Although 1 had a favorable in vitro activity profile in HCV replicons, oral dosing in dog resulted in low levels of the active 5'-triphosphate (TP) in liver. Metabolism studies using human hepatocytes provided a simple assay for screening alternative phosphoramidate prodrug analogs. Compounds that produced high TP concentrations in hepatocytes were tested in dog liver biopsy studies. This method identified 2-aminoisobutyric acid ethyl ester (AIBEE) phosphoramidate prodrug 14, which provided 100-fold higher TP concentrations in dog liver in comparison to 1 (4 and 24 h after 5 mg/kg oral dose).

Synthesis and evaluation of 2'-dihalo ribonucleotide prodrugs with activity against hepatitis C virus.

Hepatitis C virus (HCV) nucleoside inhibitors have been a key focus of nearly 2 decades of HCV drug research due to a high barrier to drug resistance and pan-genotypic activity profile provided by molecules in this drug class. Our investigations focused on several potent 2'-halogenated uridine-based HCV polymerase inhibitors, resulting in the discovery of novel 2'-deoxy-2'-dihalo-uridine analogs that are potent inhibitors in replicon assays for all genotypes. Further studies to improve in vivo performance of these nucleoside inhibitors identified aminoisobutyric acid ethyl ester (AIBEE) phosphoramidate prodrugs 18a and 18c, which provide high levels of the active triphosphate in dog liver. AIBEE prodrug 18c was compared with sofosbuvir (1) by co-dosing both compounds by oral administration in dog (5 mg/kg each) and measuring liver concentrations of the active triphosphate metabolite at both 4 and 24 h post dosing. In this study, 18c provided liver triphosphate concentrations that were 6-fold higher than sofosbuvir (1) at both biopsy time points, suggesting that 18c could be a highly effective agent for treating HCV infected patients in the clinic.

Loss of Diurnal Oscillatory Rhythms in Gut Microbiota Correlates with Changes in Circulating Metabolites in Type 2 Diabetic db/db Mice.

Our hypothesis is that diabetes leads to loss of diurnal oscillatory rhythms in gut microbiota altering circulating metabolites. We performed an observational study where we compared diurnal changes of the gut microbiota with temporal changes of plasma metabolites. Metadata analysis from bacterial DNA from fecal pellets collected from 10-month old control (db/m) and type 2 diabetic (db/db) mice every 4 h for a 24-h period was used for prediction analysis. Blood plasma was collected at a day and night time points and was used for untargeted global metabolomic analysis. Feeding and activity behaviors were recorded. Our results show that while diabetic mice exhibited feeding and activity behavior similar to control mice, they exhibited a loss of diurnal oscillations in bacteria of the genus Akkermansia, Bifidobacterium, Allobaculum, Oscillospira and a phase shift in the oscillations of g.Prevotella, proteobacteria, and actinobacteria. Analysis of the circulating metabolites showed alterations in the diurnal pattern of metabolic pathways where bacteria have been implicated, such as the histidine, betaine, and methionine/cysteine pathway, mitochondrial function and the urea cycle. Functional analysis of the differential microbes revealed that during the day, when mice are asleep, the microbes of diabetic mice were enriched in processing carbon and pyruvate metabolic pathways instead of xenobiotic degradation as was observed for control mice. Altogether, our study suggests that diabetes led to loss of rhythmic oscillations of many gut microbiota with possible implications for temporal regulation of host metabolic pathways.

Glecaprevir/Pibrentasvir in patients with chronic HCV genotype 3 infection: An integrated phase 2/3 analysis.

Glecaprevir coformulated with pibrentasvir (G/P) is approved to treat hepatitis C virus (HCV) infection and was highly efficacious in phase 2 and 3 studies. Treating HCV genotype (GT) 3 infection remains a priority, as these patients are harder to cure and at a greater risk for liver steatosis, fibrosis progression and hepatocellular carcinoma. Data were pooled from five phase 2 or 3 trials that evaluated 8-, 12- and 16-week G/P in patients with chronic HCV GT3 infection. Patients without cirrhosis or with compensated cirrhosis were either treatment-naïve or experienced with interferon- or sofosbuvir-based regimens. Safety and sustained virologic response 12 weeks post-treatment (SVR12) were assessed. The analysis included 693 patients with GT3 infection. SVR12 was achieved by 95% of treatment-naïve patients without cirrhosis receiving 8-week (198/208) and 12-week (280/294) G/P. Treatment-naïve patients with cirrhosis had a 97% (67/69) SVR12 rate with 12-week G/P. Treatment-experienced, noncirrhotic patients had SVR12 rates of 90% (44/49) and 95% (21/22) with 12- and 16-week G/P, respectively; 94% (48/51) of treatment-experienced patients with cirrhosis treated for 16 weeks achieved SVR12. No serious adverse events (AEs) were attributed to G/P; AEs leading to study drug discontinuation were rare (<1%). G/P was well-tolerated and efficacious for patients with chronic HCV GT3 infection, regardless of cirrhosis status or prior treatment experience. Eight- and 12-week durations were efficacious for treatment-naïve patients without cirrhosis and with compensated cirrhosis, respectively; 16-week G/P was efficacious in patients with prior treatment experience irrespective of cirrhosis status.

The Challenges and Opportunities in the Clinical Application of Noncoding RNAs: The Road Map for miRNAs and piRNAs in Cancer Diagnostics and Prognostics.

Discoveries on nonprotein-coding RNAs have induced a paradigm shift in our overall understanding of gene expression and regulation. We now understand that coding and noncoding RNA machinery work in concert to maintain overall homeostasis. Based on their length, noncoding RNAs are broadly classified into two groups-long (>200 nt) and small noncoding RNAs (<200 nt). These RNAs perform diverse functions-gene regulation, splicing, translation, and posttranscriptional modifications. MicroRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs) are two classes of small noncoding RNAs that are now classified as master regulators of gene expression. They have also demonstrated clinical significance as potential biomarkers and therapeutic targets for several diseases, including cancer. Despite these similarities, both these RNAs are generated through contrasting mechanisms, and one of the aims of this review is to cover the distance travelled since their discovery and compare and contrast the various facets of these RNAs. Although these RNAs show tremendous promise as biomarkers, translating the findings from bench to bedside is often met with roadblocks. The second aim of this review therefore is to highlight some of the challenges that hinder application of miRNA and piRNA as in guiding treatment decisions.

Breast cancer associated germline structural variants harboring small noncoding RNAs impact post-transcriptional gene regulation.

Copy Number Variants (CNVs) are a class of structural variations of DNA. Germline CNVs are known to confer disease susceptibility, but their role in breast cancer warrants further investigations. We hypothesized that breast cancer associated germline CNVs contribute to disease risk through gene dosage or other post-transcriptional regulatory mechanisms, possibly through tissue specific expression of CNV-embedded small-noncoding RNAs (CNV-sncRNAs). Our objectives are to identify breast cancer associated CNVs using a genome wide association study (GWAS), identify sncRNA genes embedded within CNVs, confirm breast tissue (tumor and normal) expression of the sncRNAs, correlate their expression with germline copy status and identify pathways influenced by the genes regulated by sncRNAs. We used an association study design and accessed germline CNV data generated on Affymetrix Human SNP 6.0 array in 686 (in-house data) and 495 (TCGA data) subjects served as discovery and validation cohorts. We identified 1812 breast cancer associated CNVs harboring miRNAs (n = 38), piRNAs (n = 9865), snoRNAs (n = 71) and tRNAs (n = 12) genes. A subset of CNV-sncRNAs expressed in breast tissue, also showed correlation with germline copy status. We identified targets potentially regulated by miRNAs and snoRNAs. In summary, we demonstrate the potential impact of embedded CNV-sncRNAs on expression and regulation of down-stream targets.

Efficacy and safety of glecaprevir/pibrentasvir in HCV-infected Japanese patients with prior DAA experience, severe renal impairment, or genotype 3 infection.

BACKGROUND: Once-daily, orally administered, co-formulated glecaprevir (NS3/4A protease inhibitor) and pibrentasvir (NS5A inhibitor) (G/P) demonstrated pangenotypic activity and high sustained virologic response (SVR) rates in studies outside Japan. Here we report safety and efficacy in a subset of Japanese patients with chronic HCV infection who received G/P 300/120 mg in a phase 3, open-label, multicenter study (CERTAIN-1). METHODS: This analysis focuses on three difficult-to-treat subgroups: HCV GT1/2-infected patients who failed to achieve SVR after treatment with a direct acting antiviral (DAA)-containing regimen; GT1/2-infected patients with severe renal impairment (estimated glomerular filtration rate < 30 mL/min/1.73 m2); and GT3-infected patients. Patients in the renal impairment and GT3 cohorts were treatment-naive or interferon treatment-experienced. Noncirrhotic GT1/2-infected, DAA-naïve patients in the renal impairment cohort received G/P for 8 weeks; all other patients were treated for 12 weeks. Primary outcome was SVR (HCV RNA < 15 IU/mL) 12 weeks post-treatment (SVR12). RESULTS: The study enrolled 33 GT1/2-infected patients who failed previous DAA treatment (four with cirrhosis); 12 GT1/2-infected patients with severe renal impairment (two with cirrhosis); and 12 GT3-infected patients (two with cirrhosis). SVR12 was achieved by 31/33 (93.9%), 12/12 (100%), and 10/12 (83.3%) patients, respectively. One serious adverse event (fluid overload, not related to G/P) occurred in a patient on chronic intermittent hemodialysis. CONCLUSIONS: G/P achieved high SVR12 rates and was well tolerated in three difficult-to-treat patient subgroups with limited treatment options in Japan (DAA-experienced patients, patients with severe renal impairment, and GT3-infected patients). These results support the potential suitability of this regimen for these special populations in Japan.

In Vitro Antiviral Activity and Resistance Profile of the Next-Generation Hepatitis C Virus NS5A Inhibitor Pibrentasvir.

Pibrentasvir (ABT-530) is a novel and pan-genotypic hepatitis C virus (HCV) NS5A inhibitor with 50% effective concentration (EC50) values ranging from 1.4 to 5.0 pM against HCV replicons containing NS5A from genotypes 1 to 6. Pibrentasvir demonstrated similar activity against a panel of chimeric replicons containing HCV NS5A of genotypes 1 to 6 from clinical samples. Resistance selection studies were conducted using HCV replicon cells with NS5A from genotype 1a, 1b, 2a, 2b, 3a, 4a, 5a, or 6a at a concentration of pibrentasvir that was 10- or 100-fold over its EC50 for the respective replicon. With pibrentasvir at 10-fold over the respective EC50, only a small number of colonies (0.00015 to 0.0065% of input cells) with resistance-associated amino acid substitutions were selected in replicons containing genotype 1a, 2a, or 3a NS5A, and no viable colonies were selected in replicons containing NS5A from other genotypes. With pibrentasvir at 100-fold over the respective EC50, very few colonies (0.0002% of input cells) were selected by pibrentasvir in genotype 1a replicon cells while no colonies were selected in other replicons. Pibrentasvir is active against common resistance-conferring substitutions in HCV genotypes 1 to 6 that were identified for other NS5A inhibitors, including those at key amino acid positions 28, 30, 31, or 93. The combination of pibrentasvir with HCV inhibitors of other classes produced synergistic inhibition of HCV replication. In summary, pibrentasvir is a next-generation HCV NS5A inhibitor with potent and pan-genotypic activity, and it maintains activity against common amino acid substitutions of HCV genotypes 1 to 6 that are known to confer resistance to currently approved NS5A inhibitors.

Genome-wide profiling of transfer RNAs and their role as novel prognostic markers for breast cancer.

Transfer RNAs (tRNAs, key molecules in protein synthesis) have not been investigated as potential prognostic markers in breast cancer (BC), despite early findings of their dysregulation and diagnostic potential. We aim to comprehensively profile tRNAs from breast tissues and to evaluate their role as prognostic markers (Overall Survival, OS and Recurrence Free Survival, RFS). tRNAs were profiled from 11 normal breast and 104 breast tumor tissues using next generation sequencing. We adopted a Case-control (CC) and Case-Only (CO) association study designs. Risk scores constructed from tRNAs were subjected to univariate and multivariate Cox-proportional hazards regression to investigate their prognostic value. Of the 571 tRNAs profiled, 76 were differentially expressed (DE) and three were significant for OS in the CC approach. We identified an additional 11 tRNAs associated with OS and 14 tRNAs as significant for RFS in the CO approach, indicating that CC alone may not capture all discriminatory tRNAs in prognoses. In both the approaches, the risk scores were significant in the multivariate analysis as independent prognostic factors, and patients belonging to high-risk group were associated with poor prognosis. Our results confirmed global up-regulation of tRNAs in BC and identified tRNAs as potential novel prognostic markers for BC.

Profiling of Small Nucleolar RNAs by Next Generation Sequencing: Potential New Players for Breast Cancer Prognosis.

One of the most abundant, yet least explored, classes of RNA is the small nucleolar RNAs (snoRNAs), which are well known for their involvement in post-transcriptional modifications of other RNAs. Although snoRNAs were only considered to perform housekeeping functions for a long time, recent studies have highlighted their importance as regulators of gene expression and as diagnostic/prognostic markers. However, the prognostic potential of these RNAs has not been interrogated for breast cancer (BC). The objective of the current study was to identify snoRNAs as prognostic markers for BC. Small RNA sequencing (Illumina Genome Analyzer IIx) was performed for 104 BC cases and 11 normal breast tissues. Partek Genomics Suite was used for analyzing the sequencing files. Two independent and proven approaches were used to identify prognostic markers: case-control (CC) and case-only (CO). For both approaches, snoRNAs significant in the permutation test, following univariate Cox proportional hazards regression model were used for constructing risk scores. Risk scores were subsequently adjusted for potential confounders in a multivariate Cox model. For both approaches, thirteen snoRNAs were associated with overall survival and/or recurrence free survival. Patients belonging to the high-risk group were associated with poor outcomes, and the risk score was significant after adjusting for confounders. Validation of representative snoRNAs (SNORD46 and SNORD89) using qRT-PCR confirmed the observations from sequencing experiments. We also observed 64 snoRNAs harboring piwi-interacting RNAs and/or microRNAs that were predicted to target genes (mRNAs) involved in tumorigenesis. Our results demonstrate the potential of snoRNAs to serve (i) as novel prognostic markers for BC and (ii) as indirect regulators of gene expression.

Piwi-interacting RNAs and PIWI genes as novel prognostic markers for breast cancer.

Piwi-interacting RNAs (piRNAs), whose role in germline maintenance has been established, are now also being classified as post-transcriptional regulators of gene expression in somatic cells. PIWI proteins, central to piRNA biogenesis, have been identified as genetic and epigenetic regulators of gene expression. piRNAs/PIWIs have emerged as potential biomarkers for cancer but their relevance to breast cancer has not been comprehensively studied. piRNAs and mRNAs were profiled from normal and breast tumor tissues using next generation sequencing and Agilent platforms, respectively. Gene targets for differentially expressed piRNAs were identified from mRNA expression dataset. piRNAs and PIWI genes were independently assessed for their prognostic significance (outcomes: Overall Survival, OS and Recurrence Free Survival, RFS). We discovered eight piRNAs as novel independent prognostic markers and their association with OS was confirmed in an external dataset (The Cancer Genome Atlas). Further, PIWIL3 and PIWIL4 genes showed prognostic relevance. 306 gene targets exhibited reciprocal relationship with piRNA expression. Cancer cell pathways such as apoptosis and cell signaling were the key Gene Ontology terms associated with the regulated gene targets. Overall, we have captured the entire cascade of events in a dysregulated piRNA pathway and have identified novel markers for breast cancer prognostication.

Next generation sequencing profiling identifies miR-574-3p and miR-660-5p as potential novel prognostic markers for breast cancer.

BACKGROUND: Prognostication of Breast Cancer (BC) relies largely on traditional clinical factors and biomarkers such as hormone or growth factor receptors. Due to their suboptimal specificities, it is challenging to accurately identify the subset of patients who are likely to undergo recurrence and there remains a major need for markers of higher utility to guide therapeutic decisions. MicroRNAs (miRNAs) are small non-coding RNAs that function as post-transcriptional regulators of gene expression and have shown promise as potential prognostic markers in several cancer types including BC. RESULTS: In our study, we sequenced miRNAs from 104 BC samples and 11 apparently healthy normal (reduction mammoplasty) breast tissues. We used Case-control (CC) and Case-only (CO) statistical paradigm to identify prognostic markers. Cox-proportional hazards regression model was employed and risk score analysis was performed to identify miRNA signature independent of potential confounders. Representative miRNAs were validated using qRT-PCR. Gene targets for prognostic miRNAs were identified using in silico predictions and in-house BC transcriptome dataset. Gene ontology terms were identified using DAVID bioinformatics v6.7. A total of 1,423 miRNAs were captured. In the CC approach, 126 miRNAs were retained with predetermined criteria for good read counts, from which 80 miRNAs were differentially expressed. Of these, four and two miRNAs were significant for Overall Survival (OS) and Recurrence Free Survival (RFS), respectively. In the CO approach, from 147 miRNAs retained after filtering, 11 and 4 miRNAs were significant for OS and RFS, respectively. In both the approaches, the risk scores were significant after adjusting for potential confounders. The miRNAs associated with OS identified in our cohort were validated using an external dataset from The Cancer Genome Atlas (TCGA) project. Targets for the identified miRNAs were enriched for cell proliferation, invasion and migration. CONCLUSIONS: The study identified twelve non-redundant miRNAs associated with OS and/or RFS. These signatures include those that were reported by others in BC or other cancers. Importantly we report for the first time two new candidate miRNAs (miR-574-3p and miR-660-5p) as promising prognostic markers. Independent validation of signatures (for OS) using an external dataset from TCGA further strengthened the study findings.

Disease-associated polyglutamine stretches in monomeric huntingtin adopt a compact structure.

Abnormal polyglutamine (polyQ) tracts are the only common feature in nine proteins that each cause a dominant neurodegenerative disorder. In Huntington's disease, tracts longer than 36 glutamines in the protein huntingtin (htt) cause degeneration. In situ, monoclonal antibody 3B5H10 binds to different htt fragments in neurons in proportion to their toxicity. Here, we determined the structure of 3B5H10 Fab to 1.9 Å resolution by X-ray crystallography. Modeling demonstrates that the paratope forms a groove suitable for binding two ß-rich polyQ strands. Using small-angle X-ray scattering, we confirmed that the polyQ epitope recognized by 3B5H10 is a compact two-stranded hairpin within monomeric htt and is abundant in htt fragments unbound to antibody. Thus, disease-associated polyQ stretches preferentially adopt compact conformations. Since 3B5H10 binding predicts degeneration, this compact polyQ structure may be neurotoxic.

Identifying polyglutamine protein species in situ that best predict neurodegeneration.

Polyglutamine (polyQ) stretches exceeding a threshold length confer a toxic function to proteins that contain them and cause at least nine neurological disorders. The basis for this toxicity threshold is unclear. Although polyQ expansions render proteins prone to aggregate into inclusion bodies, this may be a neuronal coping response to more toxic forms of polyQ. The exact structure of these more toxic forms is unknown. Here we show that the monoclonal antibody 3B5H10 recognizes a species of polyQ protein in situ that strongly predicts neuronal death. The epitope selectively appears among some of the many low-molecular-weight conformational states assumed by expanded polyQ and disappears in higher-molecular-weight aggregated forms, such as inclusion bodies. These results suggest that protein monomers and possibly small oligomers containing expanded polyQ stretches can adopt a conformation that is recognized by 3B5H10 and is toxic or closely related to a toxic species.

Identification of host genes involved in hepatitis C virus replication by small interfering RNA technology.

UNLABELLED: Hepatitis C virus (HCV) replication is highly dependent on host cell factors. Identification of these host factors not only facilitates understanding of the biology of HCV infection but also enables the discovery of novel targets for anti-HCV therapy. To identify host genes important for HCV RNA replication, we screened a library of small interfering RNA (siRNA) that targets approximately 4,000 human genes in Huh7-derived EN5-3 cells harboring an HCV subgenomic replicon with the nonstructural region NS3-NS5B from the 1b-N strain. Nine cellular genes that potentially regulate HCV replication were identified in this screen. Silencing of these genes resulted in inhibition of HCV replication by more than 60% and exhibited minimal toxicity. Knockdown of host gene expression by these siRNAs was confirmed at the RNA level and, in some instances, at the protein level. The level of siRNA silencing of these host genes correlated well with inhibition of HCV. These genes included those that encoded a G-protein coupled receptor (TBXA2R), a membrane protein (LTbeta), an adapter protein (TRAF2), 2 transcription factors (RelA and NFkappaB2), 2 protein kinases (MKK7 and SNARK), and 2 closely related transporter proteins (SLC12A4 and SLC12A5). Of interest, some of these genes are members of the tumor necrosis factor/lymphotoxin signaling pathway. CONCLUSION: Findings of this study may provide important information for understanding HCV replication. In addition, these cellular genes may constitute a novel set of targets for HCV antiviral therapy.

Replication efficiency of chimeric replicon containing NS5A-5B genes derived from HCV-infected patient sera.

A transient subgenomic replicon-based shuttle vector system has been developed to investigate how genetic heterogeneity affects HCV replication efficiency. Individual NS5A or NS5B genes or cassettes containing both NS5A and NS5B genes were amplified from "quasispecies" pools derived from HCV genotype 1a or 1b patient sera using RT-PCR and cloned into their respective shuttle vectors. All shuttle vectors containing NS5A or NS5A-5B genes were constructed with the S2204I "adaptive" mutation because replicons lacking the S2204I mutation replicated poorly. Gene sequences of the quasispecies pools within either genotype 1a or 1b patient samples ranged from 94 to 95% in identity. The replication capacity of 1b shuttle vectors containing patient-derived NS5A or NS5B genes averaged 67 and 75%, respectively, relative to the laboratory-optimized 1b replicon. In contrast, the replication efficiencies of both 1a and 1b shuttle vectors containing patient-derived NS5A-5B gene cassettes averaged around 2% relative to the respective laboratory-optimized replicon. All patient-derived replicons were tested in a transient assay for their sensitivity to either interferon-alpha (IFN-alpha) or to the polymerase inhibitor A-782759. Despite the differences in replication efficiency, IC(50) values measured for most of the patient-derived replicons were equivalent to the respective values measured in the control laboratory strain replicons. These results demonstrate that patient sequence heterogeneity affects replication efficiency whenever patient-derived NS5A-5B genes are inserted into the laboratory-optimized replicon. The findings also demonstrate the utility of the shuttle vector system to test patient-derived gene sequences for sensitivity to IFN-alpha and to small molecule inhibitors.