Evidence That the RNA Methylation and Poly(A) Polymerase Stimulatory Activities of Vaccinia Virus Protein VP39 Do Not Impinge upon One Another

AbstractVaccinia protein VP39 has two RNA modifying activities. In monomeric form, it acts as an mRNA cap-specific 2′-O-methyltransferase, specifically modifying the ribose moiety of the first transcribed nucleotide of m7G-capped mRNA. In association with VP55, the catalytic subunit of the vaccinia poly(A) polymerase, VP39 facilitates the rapid elongation of poly(A) tails that are already greater than ∼35 nt in length. Introducing new assays, we provide evidence that substrates for each of VP39's two activities do not detectably modulate the converse reaction and that VP39's 2′-O-methyltransferase activity is not significantly affected by its association with VP55. In an electrophoretic mobility shift assay, VP39 interacted with a short (5 nucleotide) RNA only when the latter was m7G-capped. Complexes with longer (22 nucleotide) RNAs were more stable (i.e., cap-independent) but were further stabilized by the presence of an m7G cap. An additional complex was observed at elevated RNA:protein molar ratios, indicating the presence of two RNA binding sites per VP39 molecule. Interaction at one of these sites was stabilized by the cap structure. Additional experiments indicated that RNA molecules undergoing poly(A) tail elongation by the VP55-VP39 heterodimer are not favored as cap-methylation substrates

A microarray-based system for the simultaneous analysis of single nucleotide polymorphisms in human genes involved in the metabolism of anti-malarial drugs

Background: In order to provide a cost-effective tool to analyse pharmacogenetic markers in malaria treatment, DNA microarray technology was compared with sequencing of polymerase chain reaction (PCR) fragments to detect single nucleotide polymorphisms (SNPs) in a larger number of samples. Methods: The microarray was developed to affordably generate SNP data of genes encoding the human cytochrome P450 enzyme family (CYP) and N-acetyltransferase-2 (NAT2) involved in antimalarial drug metabolisms and with known polymorphisms, i.e. CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5, and NAT2. Results: For some SNPs, i.e. CYP2A6*2, CYP2B6*5, CYP2C8*3, CYP2C9*3/*5, CYP2C19*3, CYP2D6*4 and NAT2*6/*7/*14, agreement between both techniques ranged from substantial to almost perfect (kappa index between 0.61 and 1.00), whilst for other SNPs a large variability from slight to substantial agreement (kappa index between 0.39 and 1.00) was found, e. g. CYP2D6*17 (2850C>T), CYP3A4*1B and CYP3A5*3. Conclusion: The major limit of the microarray technology for this purpose was lack of robustness and with a large number of missing data or with incorrect specificity

Articular degeneration after subchondral cementation for giant cell tumors at the knee

PURPOSE To quantify joint degeneration and the clinical outcome after curettage and cementation in subchondral giant cell tumors of the bone (GCTB) at the knee. METHODS We conducted a retrospective analysis of 14 consecutive patients (seven female, seven male) with a mean age of 34 years (range 19-51) who underwent curettage and subchondral cementation for a biopsy-confirmed GCTB at the distal femur or the proximal tibia between August 2001 and August 2017, with a mean follow-up period of 54.6 months (range 16.1-156 months). The Whole-Organ Magnetic Resonance Imaging Score (WORMS), Kellgren-Lawrence (KL) classification, and Musculo-Skeletal Tumor Society (MSTS) score were assessed. RESULTS Radiological degeneration progressed from preoperative to the latest follow-up, with a median WORMS from 2.0 to 4.0 (p = 0.006); meanwhile, the median KL score remained at 0 (p = 0.102). Progressive degeneration (WORMS) tended to be associated with the proximity of the tumor to the articular cartilage (mean 1.57 mm; range 0-12 mm) (p = 0.085). The most common degenerative findings were cartilage lesions (n = 11), synovitis (n = 5), and osteophytes (n = 4). Mean MSTS score increased from 23.1 (preoperatively) to 28.3 at the latest follow-up (p < 0.01). Seven patients (50%) were treated for a local recurrence, with six revision surgeries performed. Removal of the cement spacer and filling of the cavity with a cancellous autograft was performed in seven patients. Conversion to a total knee arthroplasty was performed in one patient for local tumor control. CONCLUSIONS Cementation following the curettage of GCTB around the knee is associated with slight degeneration at medium-term follow-up and leads to a significant reduction in pain. Removal of the cement and reconstruction with an autograft may be beneficial in the long term

Diffusion-Weighted Imaging Hyperintensities in Subtypes of Acute Intracerebral Hemorrhage: Meta-Analysis

BACKGROUND AND PURPOSE: Diffusion-weighted imaging (DWI) hyperintensities in intracerebral hemorrhage (ICH) are associated with increased risk of recurrent ICH, cognitive impairment, and death, but whether these lesions are specific to a subtype of ICH remains uncertain. We investigated the association between DWI lesions and ICH subtype and explored the risk factors for DWI lesions. METHODS: In a systematic review of ICH studies, we identified those reporting prevalence of DWI lesions. Two reviewers independently assessed study eligibility and risk of bias and collected data. We determined the pooled prevalence of DWI lesions within 90 days after ICH onset for cerebral amyloid angiopathy- and hypertensive angiopathy-related ICH using random-effects meta-analysis. We calculated odds ratios to compare prevalence of DWI lesions by ICH subtype and to assess risk factors for DWI lesions. RESULTS: Eleven studies (1910 patients) were included. The pooled prevalence of DWI lesions was 18.9% (95% CI, 11.1–26.7) in cerebral amyloid angiopathy- and 21.0% (95% CI, 15.3–26.6) in hypertensive angiopathy-related ICH. There was no difference in the prevalence of DWI lesions between cerebral amyloid angiopathy- (64/292 [21.9%]) and hypertensive angiopathy-related ICH (79/370 [21.4%]; odds ratio, 1.25; 95% CI, 0.73–2.15) in the 5 studies reporting data on both ICH pathogeneses. In all ICH, presence of DWI lesions was associated with neuroimaging features of microangiopathy (leukoaraiosis extension, previous ICH, and presence, and number of microbleeds) but not with vascular risk factors or the use of antithrombotic therapies. CONCLUSIONS: Prevalence of DWI lesions in acute ICH averages 20%, with no difference between cerebral amyloid angiopathy- and hypertensive angiopathy-related ICH. Detection of DWI lesions may add valuable information to assess the progression of the underlying microangiopathy

Improving methods for analysing anti-malarial drug efficacy trials: molecular correction based on length-polymorphic markers msp-1, msp-2 and glurp.

BACKGROUND:Drug efficacy trials monitor the continued efficacy of front-line drugs against falciparum malaria. Over-estimates of efficacy result in a country retaining a failing drug as first-line treatment with associated increases in morbidity and mortality, while under-estimating drug effectiveness leads to removal of an effective treatment with substantial practical and economic implications. Trials are challenging: they require long durations of follow-up to detect drug failures, and patients are frequently re-infected during that period. Molecular correction based on parasite genotypes distinguishes reinfections from drug failures to ensure the accuracy of failure rate estimates. Several molecular correction "algorithms" are proposed, but which is most accurate and/or robust remains unknown. METHODS:We used pharmacological modelling to simulate parasite dynamics and genetic signals that occur in patients enrolled in malaria drug clinical trials. We compared estimates of treatment failure obtained from a selection of proposed molecular correction algorithms against the known "true" failure rate in the model. FINDINGS:(i) Molecular correction is essential to avoid substantial over-estimates of drug failure rates. (ii) The current WHO-recommended algorithm consistently under-estimates the true failure rate. (iii) Newly-proposed algorithms produce more accurate failure rate estimates; the most accurate algorithm depends on the choice of drug, trial follow-up length, and transmission intensity. (iv) Long durations of patient follow-up may be counterproductive; large numbers of new infections accumulate and may be misclassified, over-estimating drug failure rate. (v) Our model was highly consistent with existing in vivo data. INTERPRETATION:The current WHO-recommended method for molecular correction and analysis of clinical trials should be re-evaluated and updated

Rescue and homogenization of 140 years of glacier mass balance data in Switzerland

Glacier monitoring in Switzerland has resulted in some of the longest and most complete data series globally. Mass balance observations at individual locations, starting in the 19th century, are the backbone of the monitoring as they represent the raw and original glaciological data demonstrating the response of snow accumulation and snow/ice melt to changes in climate forcing. So far, however, the variety of sources of historic measurements has not been systematically processed and documented. Here, we present a new complete and extensive point glacier mass balance dataset for the Swiss Alps that provides attributes for data quality and corresponding uncertainties. Original sources were digitized or re-assessed to validate or to correct existing entries and to identify metadata. The sources of data are highly diverse and stem from almost 140 years of records, originating from handwritten field notes, unpublished project documents, various digital sources, published reports, and meta-knowledge of the observers. The project resulted in data series with metadata for 63 individual Swiss glaciers, including more than 60 000 point observations of mass balance. Data were systematically analysed and homogenized, e.g. by supplementing partly missing information based on correlations inferred from direct measurements. A system to estimate uncertainty in all individual observations was developed indicating that annual point balance is measured with a typical error of 0.07 m water equivalent (w.e.), while the average error in winter snow measurements is 0.20 m w.e. Our dataset permits further investigating the climate change impacts on Swiss glaciers. Results show an absence of long-term trends in snow accumulation over glaciers while melt rates have substantially increased over the last 3 decades. The complete dataset is available at DOI https://doi.org/10.18750/massbalance.point.2021.r2021 (GLAMOS, 2021).</p

Predicting the Occurrence of Variants in RAG1 and RAG2

Abstract: While widespread genome sequencing ushers in a new era of preventive medicine, the tools for predictive genomics are still lacking. Time and resource limitations mean that human diseases remain uncharacterized because of an inability to predict clinically relevant genetic variants. A strategy of targeting highly conserved protein regions is used commonly in functional studies. However, this benefit is lost for rare diseases where the attributable genes are mostly conserved. An immunological disorder exemplifying this challenge occurs through damaging mutations in RAG1 and RAG2 which presents at an early age with a distinct phenotype of life-threatening immunodeficiency or autoimmunity. Many tools exist for variant pathogenicity prediction, but these cannot account for the probability of variant occurrence. Here, we present a method that predicts the likelihood of mutation for every amino acid residue in the RAG1 and RAG2 proteins. Population genetics data from approximately 146,000 individuals was used for rare variant analysis. Forty-four known pathogenic variants reported in patients and recombination activity measurements from 110 RAG1/2 mutants were used to validate calculated scores. Probabilities were compared with 98 currently known human cases of disease. A genome sequence dataset of 558 patients who have primary immunodeficiency but that are negative for RAG deficiency were also used as validation controls. We compared the difference between mutation likelihood and pathogenicity prediction. Our method builds a map of most probable mutations allowing pre-emptive functional analysis. This method may be applied to other diseases with hopes of improving preparedness for clinical diagnosis