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Background Micro RNA-122 (miR-122) is an important host factor for hepatitis C virus replication. Administration of miravirsen, an anti-miR-122 oligonucleotide, resulted in a dose dependent and prolonged decrease in HCV RNA levels in chronic hepatitis C patients. Aim To assess the plasma level of various mi RNAs in patients dosed with miravirsen. Methods We included 16 of 36 chronic hepatitis C patients who received five injections of either 3 mg/kg ( n = 4), 5 mg/kg ( n = 4), 7 mg/kg ( n = 4) miravirsen or placebo ( n = 4) over a 4-week period in a double-blind, randomised phase 2a study. Plasma levels of 179 mi RNAs were determined by qPCR and compared between patients dosed with miravirsen or placebo. Results Median plasma miR-122 level at baseline in patients receiving miravirsen was 3.9 × 103 compared to 1.3 × 104 copies/4 μL in placebo-dosed patients ( P = 0.68). At week 1, 4, 6 and 10/12, patients dosed with miravirsen had respectively a median 72-fold, 174-fold, 1109-fold and 552-fold lower expression of miR-122 than at baseline ( P = 0.001, as compared to patients receiving placebo). At week 4 of dosing, mi RNA-profiling demonstrated a significant lower expression of miR-210 and miR-532-5p compared to baseline (3.0 and 4.7-fold lower respectively). However, subsequent longitudinal analysis showed no significant differences in miR-210 and miR-532-5p plasma levels throughout the study period. Conclusions We demonstrated a substantial and prolonged decrease in plasma miR-122 levels in patients dosed with miravirsen. Plasma levels of other mi RNAs were not significantly affected by antagonising miR-122. [ABSTRACT FROM AUTHOR]

Purpose: To determine the minimal number of repeated measurements required for precise Nidek Tonoref II autokeratometry., Methods: This prospective, non-intervention study was performed at the Department of Ophthalmology, Randers, Denmark. We used the Nidek Tonoref II autokeratometer to perform 10 successive measurements on 100 right eyes of cooperative individuals. Each keratometry was converted to the spherical equivalent power (SE), while the net astigmatism was converted to polar values along zero (KP(0)) and 45 degrees (KP(45)). All units were in dioptres (D). The precision was calculated as the mean absolute difference between paired measurements, using one or the average of two, three, four or five autokeratometries. Statistical assessment was performed with Dunn's test for repeated measurements with a Bonferroni correction., Results: The precision of SE, KP(0) and KP(45) increased statistically significantly from one to three measurements, with no significant improvement for autokeratometries based on four or five measurements. There was no significant precision difference between one and two measurements., Conclusion: A single keratometry is inadequate, but the vector average of three measurements is sufficient for precise autokeratometry with the Tonoref II device. The consistent use of three keratometries with this device may increase the precision of spherical and toric IOL calculation., (© 2020 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

RG7834 is a potent, orally bioavailable small-molecule inhibitor of hepatitis B virus (HBV) gene expression that belongs to the dihydroquinolizinone (DHQ) chemical class and uniquely blocks production of both viral DNA and antigens. In this study, we used DHQ compounds as tools in a compound-based adaptation version of the yeast three-hybrid screen to identify the cognate cellular protein targets, the non-canonical poly(A) RNA polymerase associated domain containing proteins 5 and 7 (PAPD5 and PAPD7). Interaction with RG7834 was mapped to the catalytic domains of the two cellular enzymes. The role of PAPD5 and PAPD7 in HBV replication was confirmed by oligonucleotide-mediated knockdown studies that phenocopied the result seen with RG7834-treated HBV-infected hepatocytes. The greatest effect on HBV gene expression was seen when PAPD5 and PAPD7 mRNAs were simultaneously knocked down, suggesting that the two cellular proteins play a redundant role in maintaining HBV mRNA levels. In addition, as seen previously with RG7834 treatment, PAPD5 and PAPD7 knockdown led to destabilization and degradation of HBV mRNA without impacting production of viral RNA transcripts. Conclusion: We identify PAPD5 and PAPD7 as cellular host factors required for HBV RNA stabilization and as therapeutic targets for the HBV cure., (© 2018 by the American Association for the Study of Liver Diseases.)

Chronic hepatitis B infection (CHB) is an area of high unmet medical need. Current standard-of-care therapies only rarely lead to a functional cure, defined as durable hepatitis B surface antigen (HBsAg) loss following treatment. The goal for next generation CHB therapies is to achieve a higher rate of functional cure with finite treatment duration. To address this urgent need, we are developing liver-targeted single-stranded oligonucleotide (SSO) therapeutics for CHB based on the locked nucleic acid (LNA) platform. These LNA-SSOs target hepatitis B virus (HBV) transcripts for RNase-H-mediated degradation. Here, we describe a HBV-specific LNA-SSO that effectively reduces intracellular viral mRNAs and viral antigens (HBsAg and HBeAg) over an extended time period in cultured human hepatoma cell lines that were infected with HBV with mean 50% effective concentration (EC 50 ) values ranging from 1.19 to 1.66 μM. To achieve liver-specific targeting and minimize kidney exposure, this LNA-SSO was conjugated to a cluster of three N-acetylgalactosamine (GalNAc) moieties that direct specific binding to the asialoglycoprotein receptor (ASGPR) expressed specifically on the surface of hepatocytes. The GalNAc-conjugated LNA-SSO showed a strikingly higher level of potency when tested in the AAV-HBV mouse model as compared with its non-conjugated counterpart. Remarkably, higher doses of GalNAc-conjugated LNA-SSO resulted in a rapid and long-lasting reduction of HBsAg to below the detection limit for quantification, i.e., by 3 log10 (p < 0.0003). This antiviral effect depended on a close match between the sequences of the LNA-SSO and its HBV target, indicating that the antiviral effect is not due to non-specific oligonucleotide-driven immune activation. These data support the development of LNA-SSO therapeutics for the treatment of CHB infection., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Miravirsen is a β-D-oxy-locked nucleic acid-modified phosphorothioate antisense oligonucleotide targeting the liver-specific microRNA-122 (miR-122). Miravirsen demonstrated antiviral activity against hepatitis C virus (HCV) genotype 1b replicons with a mean 50% effective concentration (EC50) of 0.67 μM. No cytotoxicity was observed up to the highest concentration tested (>320 μM) in different cell culture models, yielding a therapeutic index of ≥ 297. Combination studies of miravirsen with interferon α2b, ribavirin, and nonnucleoside (VX-222) and nucleoside (2'-methylcytidine) inhibitors of NS5B, NS5A (BMS-790052), or NS3 (telaprevir) indicated additive interactions. Miravirsen demonstrated broad antiviral activity when tested against HCV replicons resistant to NS3, NS5A, and NS5B inhibitors with less than 2-fold reductions in susceptibility. In serial passage studies, an A4C nucleotide change was observed in the HCV 5' untranslated region (UTR) from cells passaged in the presence of up to 20 μM (40-fold the miravirsen EC50 concentration) at day 72 of passage but not at earlier time points (up to 39 days of passage). Likewise, a C3U nucleotide change was observed in the HCV 5'UTR from subjects with viral rebound after the completion of therapy in a miravirsen phase 2 clinical trial. An HCV variant constructed to contain the A4C change was fully susceptible to miravirsen. A C3U HCV variant demonstrated overall reductions in susceptibility to miravirsen but was fully susceptible to all other anti-HCV agents tested. In summary, miravirsen has demonstrated broad antiviral activity and a relatively high genetic barrier to resistance. The identification of nucleotide changes associated with miravirsen resistance should help further elucidate the biology of miR-122 interactions with HCV. (The clinical trial study has been registered at ClinicalTrials.gov under registration no. NCT01200420)., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

This case presents a 42-year-old homosexual man with weight loss, urticaria type rash, tinnitus/phonophobia, dizziness and blurred vision and scotoma on the left side. Visual acuity was affected and a left papillitis was present. The patient was tested positive for antibodies against Treponoma pallidum. This case illustrates that symptoms of the syphilis disease can come from different organ systems and cross medical specialties. We encourage clinicians to more readily think of syphilis whenever there is a sexual active patient with complaints from different parts of the body.

Antisense oligonucleotides that recruit RNase H and thereby cleave complementary messenger RNAs are being developed as therapeutics. Dose-dependent hepatic changes associated with hepatocyte necrosis and increases in serum alanine-aminotransferase levels have been observed after treatment with certain oligonucleotides. Although general mechanisms for drug-induced hepatic injury are known, the characteristics of oligonucleotides that determine their hepatotoxic potential are not well understood. Here, we present a comprehensive analysis of the hepatotoxic potential of locked nucleic acid-modified oligonucleotides in mice. We developed a random forests classifier, in which oligonucleotides are regarded as being composed of dinucleotide units, which distinguished between 206 oligonucleotides with high and low hepatotoxic potential with 80% accuracy as estimated by out-of-bag validation. In a validation set, 17 out of 23 oligonucleotides were correctly predicted (74% accuracy). In isolation, some dinucleotide units increase, and others decrease, the hepatotoxic potential of the oligonucleotides within which they are found. However, a complex interplay between all parts of an oligonucleotide can influence the hepatotoxic potential. Using the classifier, we demonstrate how an oligonucleotide with otherwise high hepatotoxic potential can be efficiently redesigned to abate hepatotoxic potential. These insights establish analysis of sequence and modification patterns as a powerful tool in the preclinical discovery process for oligonucleotide-based medicines.

Protein ubiquitination provides an efficient and reversible mechanism to regulate cell cycle progression and checkpoint control. Numerous regulatory proteins direct the addition of ubiquitin to lysine residues on target proteins, and these are countered by an army of deubiquitinating enzymes (DUBs). BRCA1-associated protein-1 (Bap1) is a ubiquitin carboxy-terminal hydrolase and is frequently mutated in lung and sporadic breast tumors. Bap1 can suppress growth of lung cancer cells in athymic nude mice and this requires its DUB activity. We show here that Bap1 interacts with host cell factor 1 (HCF-1), a transcriptional cofactor found in a number of important regulatory complexes. Bap1 binds to the HCF-1 beta-propeller using a variant of the HCF-binding motif found in herpes simplex virus VP16 and other HCF-interacting proteins. HCF-1 is K48 and K63 ubiquitinated, with a major site of linkage at lysines 1807 and 1808 in the HCF-1(C) subunit. Expression of a catalytically inactive version of Bap1 results in the selective accumulation of K48 ubiquitinated polypeptides. Depletion of Bap1 using small interfering RNA results in a modest accumulation of HCF-1(C), suggesting that Bap1 helps to control cell proliferation by regulating HCF-1 protein levels and by associating with genes involved in the G(1)-S transition.

Huntington's disease is a dominant autosomal neurodegenerative disorder caused by an expansion of polyglutamines in the huntingtin (Htt) protein, whose cellular function remains controversial. To gain insight into Htt function, we purified epitope-tagged Htt and identified Argonaute as associated proteins. Colocalization studies demonstrated Htt and Ago2 to be present in P bodies, and depletion of Htt showed compromised RNA-mediated gene silencing. Mouse striatal cells expressing mutant Htt showed fewer P bodies and reduced reporter gene silencing activity compared with wild-type counterparts. These data suggest that the previously reported transcriptional deregulation in HD may be attributed in part to mutant Htt's role in post-transcriptional processes.

VP16 is a virion phosphoprotein of herpes simplex virus and a transcriptional activator of the viral immediate-early (IE) genes. We identified four novel VP16 phosphorylation sites (Ser18, Ser353, Ser411, and Ser452) at late times in infection but found no evidence of phosphorylation of Ser375, a residue reportedly phosphorylated when VP16 is expressed from a transfected plasmid. A virus carrying a Ser375Ala mutation of VP16 was viable in cell culture but with a slow growth rate. The association of the mutant VP16 protein with IE gene promoters and subsequent IE gene expression was markedly reduced during infection, consistent with prior transfection and in vitro results. Surprisingly, the association of Oct-1 with IE promoters was also diminished during infection by the mutant strain. We propose that Ser375 is important for the interaction of VP16 with Oct-1, and that the interaction is required to enable both proteins to bind to IE promoters.

This study was designed to compare the changes in canal configuration resulting from instrumentation by either Profile or Naviflex instruments. Forty mesial canals in extracted human molar teeth were embedded and sectioned at two root levels. Reassembled teeth were instrumented with a modified crown-down technique as described in the Profile training video for Profile files and in a similar manner for Naviflex instruments. Superimposed pre- and postinstrumented cross-sectional root images were projected, traced, and scanned into a computer for analysis. Canal movement, in relation to the furca, and canal area change were recorded. The results showed no significant difference in canal center movement or canal area change between the Profile or Naviflex groups. The degree of canal curvature had no effect on canal center movement or canal area change.

Acetone-fixed cryostat sections of 79 breast carcinomas were immunostained with antibodies to basic fibroblast growth factor (bFGF) and urokinase-type plasminogen activator (uPA). Staining intensity was then compared with microvessel density assessed by manually counting vascular spaces highlighted by immunostaining vascular basal lamina (Type IV) collagen. Extensive (2+) bFGF immunoreactivity was present in neoplastic cells of 30 tumors (38%) and in host-derived stromal cells of 29 cases (37%). Disease recurrence correlated with bFGF staining: 0 to 1+ stromal staining, 30% recurred versus 2+ stromal staining, 73% recurred (P = 0.001) (54-mo median follow-up). Neither stromal nor epithelial bFGF staining correlated significantly with microvessel count; however, there was a statistically significant association between stromal cell bFGF staining and uPA staining of peritumor host cells: absent bFGF--0% 2+ uPA versus weak bFGF--9% 2+ uPA versus 2+ bFGF--29% uPA (P = 0.01). We conclude that elevated expression of bFGF in breast carcinomas is associated with aggressive clinical behavior. Its biologic significance, however, appears more closely related to extracellular matrix remodeling than to induction of prominent neovascularization per se.

A 2-color (PI, cytokeratin--FITC) multi-parametric analysis of intact cells was used to reveal diploid-range epithelial populations by flow cytometry in 108 consecutive DNA aneuploid breast carcinomas. Thirty-eight tumors (35%) contained a significant diploid range epithelial population, defined as cytokeratin-positive cells having a DNA content indistinguishable from that of endogenous lymphocytes and comprising at least 20% of all cytokeratin-positive cells. An additional 23 cases (21%) contained a minor diploid range epithelial population having a normal DNA content and comprising only 5-20% of all cytokeratin-positive cells. Multiple DNA aneuploid stemlines were present in 24 cases (22%). Diploid-range populations were more frequent (91%) in tetraploid cases than in hyperdiploid (32%), hypodiploid (17%) or hypertetraploid cases (20%). The presence of diploid epithelial populations and/or multiple aneuploid stemlines correlated with histologic parameters, including an extensive intraductal component (unimodal--4% vs. multi-modal--57%, P = 0.001), heterogeneous differentiation (unimodal--0% vs. multi-modal--52%, P = 0.001), and multi-focal growth with residual interspersed benign tissue (unimodal--8% vs. multimodal--57%, P = 0.01). These data show that diploid-range epithelial cells are frequent in aneuploid breast carcinomas analyzed by flow cytometry. In some tumors, these populations undoubtedly reflect the presence of residual benign epithelium. The numerical dominance of other histograms by near-diploid measurements suggests the presence of diploid-range neoplastic stemlines which would be 'hidden' by contaminating host-derived cells in single parameter DNA histograms. Finally, the correlation of DNA content heterogeneity with distinctive histologic patterns of breast neoplasia implies that co-existing stemlines may have biological significance in the progression of some tumors.

The 72-kDa (MMP-2, gelatinase A) and the 92-kDa (MMP-9, gelatinase B) matrix metalloproteinases have been associated with tumor cell invasion and metastasis. Immunohistological staining of MMP-2 and MMP-9, basal lamina collagen IV and TIMP-2 were performed on frozen sections of 83 invasive breast carcinomas. MMP-2 and MMP-9 were associated with neoplastic cell plasma membrane in 72% of cases and exhibited inter-tumoral variability of staining intensity. MMP-2 and MMP-9 staining was not correlated with presence of metastases at time of diagnosis or with disease outcome. TIMP-2 was detected in the peri-tumoral stroma and was present in 87% of cases. Residual benign breast tissue was negative for TIMP-2 staining. Neoplasms with diffuse TIMP-2 staining (24%) recurred significantly more frequently (75% recurred) than cases with focal (42% recurred) or absent (27% recurred) TIMP-2. Presence of collagen IV was negatively correlated with gelatinase staining. We conclude that up-regulation of MMP-2 and MMP-9 expression in breast tumor cells is reciprocally correlated to collagen IV staining. Clinical outcome, however, is more closely related to the presence of TIMP-2 than the corresponding MMPs. Enhanced TIMP-2 expression, therefore, may denote a stromal response to tumor invasion, indicative of aggressive behavior in a subset of breast carcinomas.

Inflammatory cell populations were quantitated in 76 consecutive mechanically dissociated clinical breast carcinoma specimens using multiparameter, two-color (PI-cytokeratin/FITC, PI-LCA/FITC) flow cytometric analysis. The percent LCA-positive events varied from 1.3-62.5% (mean = 11%) and correlated with degree of histologic inflammatory cell infiltrate (mild-5.8% LCA+events vs. marked-35.9% LCA + events, P < 0.001), abnormal DNA content (diploid range--4.6% LCA + events vs. aneuploid--13.3% LCA + events, P = 0.003) and poor tumor differentiation (well-moderate-6.5% LCA+events vs. poor--19.9% LCA + events, P = 0.001). Synthesis phase fractions in LCA-positive populations were uniformly less than the corresponding cytokeratin (CK)-positive cells (mean LCA + SPF = 4.4% vs. mean CK + SPF = 15.5%) and varied from 3-11%. Proliferation among inflammatory populations did not correlate statistically with the total percent of LCA or CK-positive events, nor with the SPF in epithelial populations. However, proliferative activity of inflammatory components was greater in tumors with predominately intratumoral, vs. peritumoral, inflammatory cell distribution (4.6% vs. 3.3%, P = 0.03) and in tumors with greater numbers of large, 'transformed' lymphocytes (few = 3.25% vs. many = 6.5% LCA + SPF, P = 0.001). We conclude multiparameter flow cytometric analysis of mechanically-dissociated breast tumors is representative of tumor infiltrating lymphoid populations in breast tumors and provides a potentially useful means of studying biologically relevant tumor-host interactions.

Immunostaining of two invasion-associated proteolytic enzymes, cathepsin D (CD) and urokinase-type plasminogen activator (uPA), was assessed in cryostat sections of 86 stage-heterogeneous breast carcinomas using monoclonal antibodies. Most tumors displayed a focal and/or heterogeneous staining pattern. Overall, staining was more frequent in host-derived stromal and inflammatory cells (uPA 54%, CD 89%) than neoplastic epithelium per se (uPA 24%, CD 70%). Intense (i.e., 2+) stromal, but not neoplastic, CD was significantly correlated with nodal or systematic metastases (node negative--10% versus node positive/systemic--33%, p = 0.04). Further, cumulative staining of more than one enzyme (CD + uPA) or more than one tumor component (stroma + epithelium) correlated with metastatic disease (no metastases--35% versus metastatic--72%, p = 0.005). Neither stromal nor epithelial CD alone was significantly correlated with short-term recurrence free survival, however additive CD staining (i.e., stromal + epithelial) was strongly predictive, overall (both + -75% recurred versus both weak/negative--16% recurred, p = 0.0004) and in node positive patients (p = 0.02). We conclude that (a) enzymes putatively mediating extracellular matrix dissolution may be derived from multiple sources and (b) the metastatic capacity and/or clinical aggressiveness of breast carcinomas may reflect overall proteolytic enzyme expression, suggesting that cooperative enzyme interaction may be required for invasive growth and/or metastasis.