Cancer‐associated somatic DICER1 hotspot mutations cause defective miRNA processing and reverse‐strand expression bias to predominantly mature 3p strands through loss of 5p strand cleavage

Our group recently described recurrent somatic mutations of the miRNA processing gene DICER1 in non‐epithelial ovarian cancer. Mutations appeared to be clustered around each of four critical metal‐binding residues in the RNase IIIB domain of DICER1. This domain is responsible for cleavage of the 3′ end of the 5p miRNA strand of a pre‐mRNA hairpin. To investigate the effects of these cancer‐associated 'hotspot' mutations, we engineered mouse DICER1‐deficient ES cells to express wild‐type and an allelic series of the mutant DICER1 variants. Global miRNA and mRNA profiles from cells carrying the metal‐binding site mutations were compared to each other and to wild‐type DICER1. The miRNA and mRNA profiles generated through the expression of the hotspot mutations were virtually identical, and the DICER1 hotspot mutation‐carrying cells were distinct from both wild‐type and DICER1‐deficient cells. Further, miRNA profiles showed that mutant DICER1 results in a dramatic loss in processing of mature 5p miRNA strands but were still able to create 3p strand miRNAs. Messenger RNA (mRNA) profile changes were consistent with the loss of 5p strand miRNAs and showed enriched expression for predicted targets of the lost 5p‐derived miRNAs. We therefore conclude that cancer‐associated somatic hotspot mutations of DICER1, affecting any one of four metal‐binding residues in the RNase IIIB domain, are functionally equivalent with respect to miRNA processing and are hypomorphic alleles, yielding a global loss in processing of mature 5p strand miRNA. We further propose that this resulting 3p strand bias in mature miRNA expression likely underpins the oncogenic potential of these hotspot mutations. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Deletion of the D domain of the human parainfluenza virus type 3 (HPIV3) PD protein results in decreased viral RNA synthesis and beta interferon (IFN-β) expression

Parvoviridae is a family of small non-enveloped viruses and divided into two subfamilies. The family members infect a wide range of organisms from insects to humans and some of the members (e.g., nonpathogenic adeno-associated viruses) are effective gene therapy delivery vectors. We detailed the synonymous codon usage pattern of Parvoviridae family from the available 58 sequenced genomes through multivariate statistical methods. Our results revealed that nine viruses showed some degree of strong codon bias, and the others possessed a general weak trend of codon bias. ENc-plot and neutrality plot results showed that selective pressure dominated over mutation in shapes coding sequence’s composition. The overall GC content and GC content at the third synonymous codon position were the principal determinants behind the variations within the codon usage patterns, as they both significantly correlated with the first axis of correspondence analysis. In addition, gene length had no direct influence on the codon usage pattern. Densovirinae subfamily and Parvovirinae subfamily possessed nine identical preferred codons, though most of the two subfamilies codon usage frequencies were significantly different. The result of cluster analysis based on synonymous codon usage was discordant with that of taxonomic classification. Adeno-associated viruses formed a separated clade far from other Parvoviridae members in the dendrogram. Thus, we concluded that natural selection rather than mutation pressure accounts for the main factor that affects the codon bias in Parvoviridae family.     

Acute surgical unit safely reduces unnecessary after-hours cholecystectomy

Introduction

The acute surgical model has been trialled in several institutions with mixed results. The aim of this study was to determine whether the acute surgical model provides better outcomes for patients with acute biliary presentation, compared with the traditional emergency surgery model of care.

Methods

A retrospective review was carried out of patients who were admitted for management of acute biliary presentation, before and after the establishment of an acute surgical unit (ASU). Outcomes measured were time to operation, operating time, after-hours operation (6pm – 8am), length of stay and surgical complications.

Results

A total of 342 patients presented with acute biliary symptoms and were managed operatively. The median time to operation was significantly reduced in the ASU group (32.4 vs 25.4 hours, p=0.047), as were the proportion of operations performed after hours (19.5% vs 2.5%, p<0.001) and the median length of stay (4 vs 3 days, p<0.001). The median operating time, rate of conversion to open cholecystectomy and wound infection rates remained similar.

Conclusions

Implementation of an ASU can lead to objective differences in outcomes for patients who present with acute cholecystitis. In our study, the ASU significantly reduced time to operation, the number of operations performed after hours and length of stay.     

Influence of hydroxyurea on fetal hemoglobin production in vitro

Cytotoxic drugs increase circulating fetal hemoglobin levels. We examined the mechanism by measuring the fetal hemoglobin produced per BFU-E-derived erythroblast following hydroxyurea treatment in vivo and in vitro. Treatment of four sickle cell patients increased the percentage of circulating F reticulocytes. The frequencies of bone marrow or peripheral blood BFU-E or CFU-E-derived colonies and their fetal hemoglobin content were unaffected. In all cases, the number of erythroid cells/progenitor-derived colony increased. To explore further the effect of hydroxyurea on fetal hemoglobin production, we added 50 mumol/L hydroxyurea to cultures of peripheral blood BFU-E-derived erythroblasts on 1 of 9 days (day 5 through 13) to nine samples. These BFU-E were derived from the peripheral blood of normal donors, sickle trait donors, and sickle cell anemia patients and from the bone marrows of monkeys. This concentration of hydroxyurea was selected so that the frequency of BFU-E and their size was moderately decreased. Addition of hydroxyurea to these progenitor-derived erythroid cells had no effect on fetal hemoglobin content per cell. Neither did transient exposure of progenitors to hydroxyurea prior to culture in nontoxic concentrations (0 to 500 mumol/L) result in a significant increase in fetal hemoglobin content in progenitor-derived erythroblasts. These data suggest that hydroxyurea does not directly alter the HbF program expressed by progenitor-derived erythroid cells. Instead, it enhances hemoglobin F content secondarily, possibly by inducing alterations in erythropoiesis.     

Risk indicators for the presence and extent of root caries among caries-active adults enrolled in the Xylitol for Adult Caries Trial (X-ACT)

Objective

This paper uses baseline data from a randomized clinical trial to evaluate cross-sectional indicators of root caries in caries-active adults.

Materials and methods

Adults (21–80?years) having at least 12 erupted teeth and between one and ten caries lesions were enrolled. Participants (n?=?437) received caries exams by trained, calibrated examiners and responded to baseline demographic and medical–dental questionnaires. We examined associations between baseline characteristics and (1) the presence of any root caries using Mantel–Haenszel hypothesis tests and odds ratio (OR) estimators and (2) the number of root surfaces with caries among study participants with exposed root surfaces (n?=?349) using Mantel–Haenszel mean score tests and Mann–Whitney estimators.

Results/conclusions

Adjusting for study site and age, male gender [OR, 1.72; 95% confidence interval (CI), 1.08, 2.78], white race (OR, 2.39; 95% CI, 1.43, 3.98), recent dental visit (OR, 1.98; 95% CI, 1.07, 3.66), poor self-described oral health (OR, 2.65; 95% CI, 1.10, 6.39), and recent professional fluoride treatment (OR, 1.85; 95% CI, 1.06, 3.25) were significantly associated with increased odds to have any root caries, and study participants with exposed root surfaces characterized by male gender [Mann–Whitney probability estimate (MW)?=?0.57; 95% CI, 0.51, 0.63), white race (MW, 0.61; 0.55, 0.68), recent dental visit (MW, 0.58; 0.50, 0.67), poor self-described oral health (MW, 0.61; 0.53, 0.69), and flossing at least once per day (MW, 0.57; 95% CI, 0.51, 0.62) were significantly more likely to have a greater number of root surfaces with caries than a randomly selected study participant from their respective complementary subgroups (female gender, non-white, etc.).

Clinical relevance

Our findings may help identify individuals at higher root caries risk.     

Intratumoral inflammation is associated with more aggressive prostate cancer

Purpose

Inflammation may play a role in the development and progression of many cancers, including prostate cancer. We sought to test whether histological inflammation within prostate cancer was associated with more aggressive disease.

Methods

The slides of prostatectomy specimens were reviewed by a board-certified pathologist on 287 men from a Veterans Affairs Medical Center treated with radical prostatectomy from 1992 to 2004. The area with the greatest tumor burden was scored in a blinded manner for the degree of inflammation: absent, mild, or marked. We used logistic and Cox proportional hazards regression analysis to examine whether categorically coded inflammation score was associated with adverse pathology and biochemical progression, respectively.

Results

No inflammation was found in 49 men (17 %), while 153 (53 %) and 85 (30 %) had mild and marked inflammation. During a median follow-up of 77 months, biochemical recurrence occurred among 126 (44 %) men. On multivariate analysis, more inflammation was associated with greater risk of positive margins, capsular penetration, and seminal vesicle invasion (all p < 0.05). Marked inflammation was associated with increased PSA recurrence risk when adjusting for preoperative features only (HR 2.08, 95 % CI 1.02–4.24), but not after adjusting for pathologic features.

Conclusions

Inflammation within prostate cancer was associated with more advanced disease, although it is unclear whether aggressive disease caused increased inflammation or inflammation caused aggressive disease.     

Efficacy of chlorhexidine varnish for the prevention of adult caries: a randomized trial

The Prevention of Adult Caries Study, an NIDCR-funded multicenter, double-blind, randomized clinical trial, enrolled 983 adults (aged 18-80 yrs) at high risk for developing caries (20 or more intact teeth and 2 or more lesions at screening) to test the efficacy of a chlorhexidine diacetate 10% weight per volume (w/v) dental coating (CHX). We excluded participants for whom the study treatment was contraindicated or whose health might affect outcomes or ability to complete the study. Participants were randomly assigned to receive either the CHX coating (n = 490) or a placebo control (n = 493). Coatings were applied weekly for 4 weeks and a fifth time 6 months later. The primary outcome (total net D(1-2)FS increment) was the sum of weighted counts of changes in tooth surface status over 13 months. We observed no significant difference between the two treatment arms in either the intention-to-treat or per-protocol analyses. Analysis of 3 protocol-specified secondary outcomes produced similar findings. This trial failed to find that 10% (w/v) chlorhexidine diacetate coating was superior to placebo coating for the prevention of new caries (Clinicaltrials.gov registration number NCT00357877).     

Does Physical Activity in Adolescence Have Site‐Specific and Sex‐Specific Benefits on Young Adult Bone Size,Content, and Estimated Strength?

The long‐term benefits of habitual physical activity during adolescence on adult bone structure and strength are poorly understood. We investigated whether physically active adolescents had greater bone size, density, content, and estimated bone strength in young adulthood when compared to their peers who were inactive during adolescence. Peripheral quantitative computed tomography (pQCT) was used to measure the tibia and radius of 122 (73 females) participants (age mean ± SD, 29.3 ± 2.3 years) of the Saskatchewan Pediatric Bone Mineral Accrual Study (PBMAS). Total bone area (ToA), cortical density (CoD), cortical area (CoA), cortical content (CoC), and estimated bone strength in torsion (SSI_p) and muscle area (MuA) were measured at the diaphyses (66% tibia and 65% radius). Total density (ToD), trabecular density (TrD), trabecular content (TrC), and estimated bone strength in compression (BSI_c) were measured at the distal ends (4%). Participants were grouped by their adolescent physical activity (PA) levels (inactive, average, and active) based on mean PA Z‐scores obtained from serial questionnaire assessments completed during adolescence. We compared adult bone outcomes across adolescent PA groups in each sex using analysis of covariance followed by post hoc pairwise comparisons with Bonferroni adjustments. When adjusted for adult height, MuA, and PA, adult males who were more physically active than their peers in adolescence had 13% greater adjusted torsional bone strength (SSI_p, p < 0.05) and 10% greater adjusted ToA (p < 0.05) at the tibia diaphysis. Females who were more active in adolescence had 10% larger adjusted CoA (p < 0.05), 12% greater adjusted CoC (p < 0.05) at the tibia diaphysis, and 3% greater adjusted TrC (p < 0.05) at the distal tibia when compared to their inactive peers. Benefits to tibia bone size, content, and strength in those who were more active during adolescence seemed to persist into young adulthood, with greater ToA and SSI_p in males, and greater CoA, CoC, and TrC in females. © 2014 American Society for Bone and Mineral Research.