BRAF V600E mutations in urine and plasma cell-free DNA from patients with Erdheim-Chester disease.

Erdheim-Chester disease (ECD) is a rare histiocytosis with a high prevalence of BRAF V600E mutation (>50% of patients). Patients with BRAF-mutant ECD can respond to BRAF inhibitors. Unfortunately, the lack of adequate archival tissue often precludes BRAF testing. We hypothesized that cell-free DNA (cfDNA) from plasma or urine can offer an alternative source of biologic material for testing. We tested for BRAF V600E mutation in cfDNA from the plasma and urine of 6 ECD patients. In patients with available archival tissue, the result of BRAF mutation analysis was concordant with plasma and urine cfDNA results in all 3 patients (100% agreement, kappa 1.00). In all 6 patients, BRAF mutation analysis of plasma and urine cfDNA was concordant in 5 of 6 patients (83% agreement, kappa 0.67). Testing for BRAF V600E mutation in plasma and urine cfDNA should be further investigated as an alternative to archival tissue mutation analysis

A multi-organ transcriptome resource for the Burmese Python (Python molurus bivittatus)

<p>Abstract</p> <p>Background</p> <p>Snakes provide a unique vertebrate system for studying a diversity of extreme adaptations, including those related to development, metabolism, physiology, and venom. Despite their importance as research models, genomic resources for snakes are few. Among snakes, the Burmese python is the premier model for studying extremes of metabolic fluctuation and physiological remodelling. In this species, the consumption of large infrequent meals can induce a 40-fold increase in metabolic rate and more than a doubling in size of some organs. To provide a foundation for research utilizing the python, our aim was to assemble and annotate a transcriptome reference from the heart and liver. To accomplish this aim, we used the 454-FLX sequencing platform to collect sequence data from multiple cDNA libraries.</p> <p>Results</p> <p>We collected nearly 1 million 454 sequence reads, and assembled these into 37,245 contigs with a combined length of 13,409,006 bp. To identify known genes, these contigs were compared to chicken and lizard gene sets, and to all Genbank sequences. A total of 13,286 of these contigs were annotated based on similarity to known genes or Genbank sequences. We used gene ontology (GO) assignments to characterize the types of genes in this transcriptome resource. The raw data, transcript contig assembly, and transcript annotations are made available online for use by the broader research community.</p> <p>Conclusion</p> <p>These data should facilitate future studies using pythons and snakes in general, helping to further contribute to the utilization of snakes as a model evolutionary and physiological system. This sequence collection represents a major genomic resource for the Burmese python, and the large number of transcript sequences characterized should contribute to future research in this and other snake species.</p

The transcriptional repressor protein NsrR senses nitric oxide directly via a [2Fe-2S] cluster

The regulatory protein NsrR, a member of the Rrf2 family of transcription repressors, is specifically dedicated to sensing nitric oxide (NO) in a variety of pathogenic and non-pathogenic bacteria. It has been proposed that NO directly modulates NsrR activity by interacting with a predicted [Fe-S] cluster in the NsrR protein, but no experimental evidence has been published to support this hypothesis. Here we report the purification of NsrR from the obligate aerobe Streptomyces coelicolor. We demonstrate using UV-visible, near UV CD and EPR spectroscopy that the protein contains an NO-sensitive [2Fe-2S] cluster when purified from E. coli. Upon exposure of NsrR to NO, the cluster is nitrosylated, which results in the loss of DNA binding activity as detected by bandshift assays. Removal of the [2Fe-2S] cluster to generate apo-NsrR also resulted in loss of DNA binding activity. This is the first demonstration that NsrR contains an NO-sensitive [2Fe-2S] cluster that is required for DNA binding activity

Cerebrospinal fluid in the differential diagnosis of Alzheimer's disease: clinical utility of an extended panel of biomarkers in a specialist cognitive clinic.

BACKGROUND: Cerebrospinal fluid (CSF) biomarkers are increasingly being used to support a diagnosis of Alzheimer's disease (AD). Their clinical utility for differentiating AD from non-AD neurodegenerative dementias, such as dementia with Lewy bodies (DLB) or frontotemporal dementia (FTD), is less well established. We aimed to determine the diagnostic utility of an extended panel of CSF biomarkers to differentiate AD from a range of other neurodegenerative dementias. METHODS: We used immunoassays to measure conventional CSF markers of amyloid and tau pathology (amyloid beta (Aβ)1-42, total tau (T-tau), and phosphorylated tau (P-tau)) as well as amyloid processing (AβX-38, AβX-40, AβX-42, soluble amyloid precursor protein (sAPP)α, and sAPPβ), large fibre axonal degeneration (neurofilament light chain (NFL)), and neuroinflammation (YKL-40) in 245 patients with a variety of dementias and 30 controls. Patients fulfilled consensus criteria for AD (n = 156), DLB (n = 20), behavioural variant frontotemporal dementia (bvFTD; n = 45), progressive non-fluent aphasia (PNFA; n = 17), and semantic dementia (SD; n = 7); approximately 10% were pathology/genetically confirmed (n = 26). Global tests based on generalised least squares regression were used to determine differences between groups. Non-parametric receiver operating characteristic (ROC) curves and area under the curve (AUC) analyses were used to quantify how well each biomarker discriminated AD from each of the other diagnostic groups (or combinations of groups). CSF cut-points for the major biomarkers found to have diagnostic utility were validated using an independent cohort which included causes of AD (n = 104), DLB (n = 5), bvFTD (n = 12), PNFA (n = 3), SD (n = 9), and controls (n = 10). RESULTS: There were significant global differences in Aβ1-42, T-tau, T-tau/Aβ1-42 ratio, P-tau-181, NFL, AβX-42, AβX-42/X-40 ratio, APPα, and APPβ between groups. At a fixed sensitivity of 85%, AβX-42/X-40 could differentiate AD from controls, bvFTD, and SD with specificities of 93%, 85%, and 100%, respectively; for T-tau/Aβ1-42 these specificities were 83%, 70%, and 86%. AβX-42/X-40 had similar or higher specificity than Aβ1-42. No biomarker or ratio could differentiate AD from DLB or PNFA with specificity > 50%. Similar sensitivities and specificities were found in the independent validation cohort for differentiating AD and other dementias and in a pathology/genetically confirmed sub-cohort. CONCLUSIONS: CSF AβX-42/X-40 and T-tau/Aβ1-42 ratios have utility in distinguishing AD from controls, bvFTD, and SD. None of the biomarkers tested had good specificity at distinguishing AD from DLB or PNFA

Thermal counterflow in a periodic channel with solid boundaries

We perform numerical simulations of finite temperature quantum turbulence produced through thermal counterflow in superfluid 4He, using the vortex filament model. We investigate the effects of solid boundaries along one of the Cartesian directions, assuming a laminar normal fluid with a Poiseuille velocity profile, whilst varying the temperature and the normal fluid velocity. We analyze the distribution of the quantized vortices, reconnection rates, and quantized vorticity production as a function of the wall-normal direction. We find that the quantized vortex lines tend to concentrate close to the solid boundaries with their position depending only on temperature and not on the counterflow velocity. We offer an explanation of this phenomenon by considering the balance of two competing effects, namely the rate of turbulent diffusion of an isotropic tangle near the boundaries and the rate of quantized vorticity production at the center. Moreover, this yields the observed scaling of the position of the peak vortex line density with the mutual friction parameter. Finally, we provide evidence that upon the transition from laminar to turbulent normal fluid flow, there is a dramatic increase in the homogeneity of the tangle, which could be used as an indirect measure of the transition to turbulence in the normal fluid component for experiments

Managing patients with ICD shocks and programming tachycardia therapies during acute heart failure syndromes

We review the pharmacologic, interventional and device programming treatment options for patients with implantable cardioverter-defibrillators who present with acute heart failure and implantable cardioverter-defibrillator shocks

Ancient mtDNA Genetic Variants Modulate mtDNA Transcription and Replication

Although the functional consequences of mitochondrial DNA (mtDNA) genetic backgrounds (haplotypes, haplogroups) have been demonstrated by both disease association studies and cell culture experiments, it is not clear which of the mutations within the haplogroup carry functional implications and which are “evolutionary silent hitchhikers”. We set forth to study the functionality of haplogroup-defining mutations within the mtDNA transcription/replication regulatory region by in vitro transcription, hypothesizing that haplogroup-defining mutations occurring within regulatory motifs of mtDNA could affect these processes. We thus screened >2500 complete human mtDNAs representing all major populations worldwide for natural variation in experimentally established protein binding sites and regulatory regions comprising a total of 241 bp in each mtDNA. Our screen revealed 77/241 sites showing point mutations that could be divided into non-fixed (57/77, 74%) and haplogroup/sub-haplogroup-defining changes (i.e., population fixed changes, 20/77, 26%). The variant defining Caucasian haplogroup J (C295T) increased the binding of TFAM (Electro Mobility Shift Assay) and the capacity of in vitro L-strand transcription, especially of a shorter transcript that maps immediately upstream of conserved sequence block 1 (CSB1), a region associated with RNA priming of mtDNA replication. Consistent with this finding, cybrids (i.e., cells sharing the same nuclear genetic background but differing in their mtDNA backgrounds) harboring haplogroup J mtDNA had a >2 fold increase in mtDNA copy number, as compared to cybrids containing haplogroup H, with no apparent differences in steady state levels of mtDNA-encoded transcripts. Hence, a haplogroup J regulatory region mutation affects mtDNA replication or stability, which may partially account for the phenotypic impact of this haplogroup. Our analysis thus demonstrates, for the first time, the functional impact of particular mtDNA haplogroup-defining control region mutations, paving the path towards assessing the functionality of both fixed and un-fixed genetic variants in the mitochondrial genome

Genetic and Computational Identification of a Conserved Bacterial Metabolic Module

We have experimentally and computationally defined a set of genes that form a conserved metabolic module in the α-proteobacterium Caulobacter crescentus and used this module to illustrate a schema for the propagation of pathway-level annotation across bacterial genera. Applying comprehensive forward and reverse genetic methods and genome-wide transcriptional analysis, we (1) confirmed the presence of genes involved in catabolism of the abundant environmental sugar myo-inositol, (2) defined an operon encoding an ABC-family myo-inositol transmembrane transporter, and (3) identified a novel myo-inositol regulator protein and cis-acting regulatory motif that control expression of genes in this metabolic module. Despite being encoded from non-contiguous loci on the C. crescentus chromosome, these myo-inositol catabolic enzymes and transporter proteins form a tightly linked functional group in a computationally inferred network of protein associations. Primary sequence comparison was not sufficient to confidently extend annotation of all components of this novel metabolic module to related bacterial genera. Consequently, we implemented the Graemlin multiple-network alignment algorithm to generate cross-species predictions of genes involved in myo-inositol transport and catabolism in other α-proteobacteria. Although the chromosomal organization of genes in this functional module varied between species, the upstream regions of genes in this aligned network were enriched for the same palindromic cis-regulatory motif identified experimentally in C. crescentus. Transposon disruption of the operon encoding the computationally predicted ABC myo-inositol transporter of Sinorhizobium meliloti abolished growth on myo-inositol as the sole carbon source, confirming our cross-genera functional prediction. Thus, we have defined regulatory, transport, and catabolic genes and a cis-acting regulatory sequence that form a conserved module required for myo-inositol metabolism in select α-proteobacteria. Moreover, this study describes a forward validation of gene-network alignment, and illustrates a strategy for reliably transferring pathway-level annotation across bacterial species

Sequential induction of three recombination directionality factors directs assembly of tripartite integrative and conjugative elements

Tripartite integrative and conjugative elements (ICE3) are a novel form of ICE that exist as three separate DNA regions integrated within the genomes of Mesorhizobium spp. Prior to conjugative transfer the three ICE3 regions of M. ciceri WSM1271 ICEMcSym1271 combine and excise to form a single circular element. This assembly requires three coordinated recombination events involving three site-specific recombinases IntS, IntG and IntM. Here, we demonstrate that three excisionases–or recombination directionality factors—RdfS, RdfG and RdfM are required for ICE3 excision. Transcriptome sequencing revealed that expression of ICE3 transfer and conjugation genes was induced by quorum sensing. Quorum sensing activated expression of rdfS, and in turn RdfS stimulated transcription of both rdfG and rdfM. Therefore, RdfS acts as a “master controller” of ICE3 assembly and excision. The dependence of all three excisive reactions on RdfS ensures that ICE3 excision occurs via a stepwise sequence of recombination events that avoids splitting the chromosome into a non-viable configuration. These discoveries expose a surprisingly simple control system guiding molecular assembly of these novel and complex mobile genetic elements and highlight the diverse and critical functions of excisionase proteins in control of horizontal gene transfer