Quantitative assay for the detection of the V617F variant in the Janus kinase 2 (JAK2) gene using the Luminex xMAP technology

<p>Abstract</p> <p>Background</p> <p>The availability of clinically valid biomarkers contribute to improve the diagnosis and clinical management of diseases. A valine-to-phenylalanine substitution at position 617 (V617F) in the Janus kinase 2 (JAK2) gene has been recently associated with key signaling abnormalities in the transduction of haemopoietic growth-factor receptors and is now considered as a useful clinical marker of myeloproliferative neoplasms. Several methods have recently been reported to detect the JAK2 V617F point mutation and show variable sensitivity.</p> <p>Methods</p> <p>Using the Luminex xMAP technology, we developed a quantitative assay to detect the JAK2V617F variant. The method was based on polymerase chain reaction (PCR) followed by hybridization to specific probes coupled with internally dyed microspheres. The assay comprises 3 steps: genomic DNA extraction, end point PCR reaction, direct hybridization of PCR fragments and quantification. It has been tested with different sources of nucleic acid.</p> <p>Results</p> <p>Applied to whole blood samples, this quantitative assay showed a limit of detection of 2%. A highly sensitive allele-specific primer extension reaction performed in parallel allowed to validate the results and to identify the specimens with values below 2%.</p> <p>Conclusion</p> <p>Direct hybridization assay using the Luminex xMAP technology allows sensitive quantification of JAK2V617F from blood spots. It is simple and can be easily performed in a clinical setting.</p

Spitzer survey of the Large Magellanic Cloud, surveying the agents of a galaxy's evolution (SAGE). IV. Dust properties in the interstellar medium

The goal of this paper is to present the results of a preliminary analysis of the extended infrared (IR) emission by dust in the interstellar medium (ISM) of the Large Magellanic Cloud (LMC). We combine Spitzer Surveying the Agents of Galaxy Evolution (SAGE) and Infrared Astronomical Satellite (IRAS) data and correlate the infrared emission with gas tracers of H I, CO, and Hα. We present a global analysis of the infrared emission as well as detailed modeling of the spectral energy distribution (SED) of a few selected regions. Extended emission by dust associated with the neutral, molecular, and diffuse ionized phases of the ISM is detected at all IR bands from 3.6 μm to 160 μm. The relative abundance of the various dust species appears quite similar to that in the Milky Way (MW) in all the regions we have modeled. We construct maps of the temperature of large dust grains. The temperature map shows variations in the range 12.1-34.7 K, with a systematic gradient from the inner to outer regions, tracing the general distribution of massive stars and individual H II regions as well as showing warmer dust in the stellar bar. This map is used to derive the far-infrared (FIR) optical depth of large dust grains. We find two main departures in the LMC with respect to expectations based on the MW: (1) excess mid-infrared (MIR) emission near 70 μm, referred to as the 70 μm excess, and (2) departures from linear correlation between the FIR optical depth and the gas column density, which we refer to as FIR excess emission. The 70 μm excess increases gradually from the MW to the LMC to the Small Magellanic Cloud (SMC), suggesting evolution with decreasing metallicity. The excess is associated with the neutral and diffuse ionized gas, with the strongest excess region located in a loop structure next to 30 Dor. We show that the 70 μm excess can be explained by a modification of the size distribution of very small grains with respect to that in the MW, and a corresponding mass increase of ≃13% of the total dust mass in selected regions. The most likely explanation is that the 70 μm excess is due to the production of large very small grains (VSG) through erosion of larger grains in the diffuse medium. This FIR excess could be due to intrinsic variations of the dust/gas ratio, which would then vary from 4.6 to 2.3 times lower than the MW values across the LMC, but X_(CO) values derived from the IR emission would then be about three times lower than those derived from the Virial analysis of the CO data. We also investigate the possibility that the FIR excess is associated with an additional gas component undetected in the available gas tracers. Assuming a constant dust abundance in all ISM phases, the additional gas component would have twice the known H I mass. We show that it is plausible that the FIR excess is due to cold atomic gas that is optically thick in the 21 cm line, while the contribution by a pure H_2 phase with no CO emission remains a possible explanation

A Timescale for Evolution, Population Expansion, and Spatial Spread of an Emerging Clone of Methicillin-Resistant Staphylococcus aureus

Due to the lack of fossil evidence, the timescales of bacterial evolution are largely unknown. The speed with which genetic change accumulates in populations of pathogenic bacteria, however, is a key parameter that is crucial for understanding the emergence of traits such as increased virulence or antibiotic resistance, together with the forces driving pathogen spread. Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of hospital-acquired infections. We have investigated an MRSA strain (ST225) that is highly prevalent in hospitals in Central Europe. By using mutation discovery at 269 genetic loci (118,804 basepairs) within an international isolate collection, we ascertained extremely low diversity among European ST225 isolates, indicating that a recent population bottleneck had preceded the expansion of this clone. In contrast, US isolates were more divergent, suggesting they represent the ancestral population. While diversity was low, however, our results demonstrate that the short-term evolutionary rate in this natural population of MRSA resulted in the accumulation of measurable DNA sequence variation within two decades, which we could exploit to reconstruct its recent demographic history and the spatiotemporal dynamics of spread. By applying Bayesian coalescent methods on DNA sequences serially sampled through time, we estimated that ST225 had diverged since approximately 1990 (1987 to 1994), and that expansion of the European clade began in 1995 (1991 to 1999), several years before the new clone was recognized. Demographic analysis based on DNA sequence variation indicated a sharp increase of bacterial population size from 2001 to 2004, which is concordant with the reported prevalence of this strain in several European countries. A detailed ancestry-based reconstruction of the spatiotemporal dispersal dynamics suggested a pattern of frequent transmission of the ST225 clone among hospitals within Central Europe. In addition, comparative genomics indicated complex bacteriophage dynamics

Performance of non-invasive tests and histology for the prediction of clinical outcomes in patients with non-alcoholic fatty liver disease: an individual participant data meta-analysis

BackgroundHistologically assessed liver fibrosis stage has prognostic significance in patients with non-alcoholic fatty liver disease (NAFLD) and is accepted as a surrogate endpoint in clinical trials for non-cirrhotic NAFLD. Our aim was to compare the prognostic performance of non-invasive tests with liver histology in patients with NAFLD.MethodsThis was an individual participant data meta-analysis of the prognostic performance of histologically assessed fibrosis stage (F0–4), liver stiffness measured by vibration-controlled transient elastography (LSM-VCTE), fibrosis-4 index (FIB-4), and NAFLD fibrosis score (NFS) in patients with NAFLD. The literature was searched for a previously published systematic review on the diagnostic accuracy of imaging and simple non-invasive tests and updated to Jan 12, 2022 for this study. Studies were identified through PubMed/MEDLINE, EMBASE, and CENTRAL, and authors were contacted for individual participant data, including outcome data, with a minimum of 12 months of follow-up. The primary outcome was a composite endpoint of all-cause mortality, hepatocellular carcinoma, liver transplantation, or cirrhosis complications (ie, ascites, variceal bleeding, hepatic encephalopathy, or progression to a MELD score ≥15). We calculated aggregated survival curves for trichotomised groups and compared them using stratified log-rank tests (histology: F0–2 vs F3 vs F4; LSM: 2·67; NFS: 0·676), calculated areas under the time-dependent receiver operating characteristic curves (tAUC), and performed Cox proportional-hazards regression to adjust for confounding. This study was registered with PROSPERO, CRD42022312226.FindingsOf 65 eligible studies, we included data on 2518 patients with biopsy-proven NAFLD from 25 studies (1126 [44·7%] were female, median age was 54 years [IQR 44–63), and 1161 [46·1%] had type 2 diabetes). After a median follow-up of 57 months [IQR 33–91], the composite endpoint was observed in 145 (5·8%) patients. Stratified log-rank tests showed significant differences between the trichotomised patient groups (p<0·0001 for all comparisons). The tAUC at 5 years were 0·72 (95% CI 0·62–0·81) for histology, 0·76 (0·70–0·83) for LSM-VCTE, 0·74 (0·64–0·82) for FIB-4, and 0·70 (0·63–0·80) for NFS. All index tests were significant predictors of the primary outcome after adjustment for confounders in the Cox regression.InterpretationSimple non-invasive tests performed as well as histologically assessed fibrosis in predicting clinical outcomes in patients with NAFLD and could be considered as alternatives to liver biopsy in some cases

Data from: The virtues of gaps: xenarthran (edentate) monophyly supported by a unique deletion in αA-crystallin

Shared insertions or deletions (indels) in protein-coding DNA can be strong indicators of the monophyly of a taxon. A three-amino acid deletion had previously been noted in the eye lens protein αA-crystallin of two species of sloths and two species of anteaters, which represent the Pilosa, one of the two infraorders of Xenarthra (Edentata). This deletion has not been observed in 55 species from 16 other eutherian orders, nor in two species of marsupials and 34 non-mammalian vertebrates, from birds to shark. At the genomic level, we have now detected this deletion in two species of armadillos of the second xenarthran infraorder, Cingulata, as well as in an additional species of anteater. Phylogenetic trees were constructed from a 145-bp sequence of the αA-crystallin gene of 39 tetrapod species, supporting xenarthran monophyly with values from 86 to 90%. To quantify the additional support for xenarthran monophyly, as given by the three-residue deletion, we computed the probabilities for the occurrence of this deletion per evolutionary time unit for alternative hypothetical tree topologies. In the obtained estimates, the six trees in which the xenarthran subgroups are unresolved or paraphyletic give an increasingly lower likelihood than the two trees which assume xenarthran monophyly. For the monophyletic trees, the probability that the deletion observed in the xenarthrans is due to a single event is >0.99. Thus, this deletion in αA-crystallin gives strong molecular support for the monophyly of this old and diverse order

Data from: The virtues of gaps: xenarthran (edentate) monophyly supported by a unique deletion in αA-crystallin

Shared insertions or deletions (indels) in protein-coding DNA can be strong indicators of the monophyly of a taxon. A three-amino acid deletion had previously been noted in the eye lens protein αA-crystallin of two species of sloths and two species of anteaters, which represent the Pilosa, one of the two infraorders of Xenarthra (Edentata). This deletion has not been observed in 55 species from 16 other eutherian orders, nor in two species of marsupials and 34 non-mammalian vertebrates, from birds to shark. At the genomic level, we have now detected this deletion in two species of armadillos of the second xenarthran infraorder, Cingulata, as well as in an additional species of anteater. Phylogenetic trees were constructed from a 145-bp sequence of the αA-crystallin gene of 39 tetrapod species, supporting xenarthran monophyly with values from 86 to 90%. To quantify the additional support for xenarthran monophyly, as given by the three-residue deletion, we computed the probabilities for the occurrence of this deletion per evolutionary time unit for alternative hypothetical tree topologies. In the obtained estimates, the six trees in which the xenarthran subgroups are unresolved or paraphyletic give an increasingly lower likelihood than the two trees which assume xenarthran monophyly. For the monophyletic trees, the probability that the deletion observed in the xenarthrans is due to a single event is >0.99. Thus, this deletion in αA-crystallin gives strong molecular support for the monophyly of this old and diverse order