A novel epilepsy mutation in the sodium channel SCN1A identifies a cytoplasmic domain for {beta} subunit interaction

A mutation in the sodium channel SCN1A was identified in a small Italian family with dominantly inherited generalized epilepsy with febrile seizures plus (GEFS+). The mutation, D1866Y, alters an evolutionarily conserved aspartate residue in the C-terminal cytoplasmic domain of the sodium channel {alpha} subunit. The mutation decreased modulation of the {alpha} subunit by {beta}1, which normally causes a negative shift in the voltage dependence of inactivation in oocytes. There was less of a shift with the mutant channel, resulting in a 10 mV difference between the wild-type and mutant channels in the presence of {beta}1. This shift increased the magnitude of the window current, which resulted in more persistent current during a voltage ramp. Computational analysis suggests that neurons expressing the mutant channels will fire an action potential with a shorter onset delay in response to a threshold current injection, and that they will fire multiple action potentials with a shorter interspike interval at a higher input stimulus. These results suggest a causal relationship between a positive shift in the voltage dependence of sodium channel inactivation and spontaneous seizure activity. Direct interaction between the cytoplasmic C-terminal domain of the wild-type{alpha} subunit with the {beta}1or {beta}3 subunit was first demonstrated by yeast two-hybrid analysis. The SCN1A peptide K1846-R1886 is sufficient for {beta} subunit interaction. Coimmunoprecipitation from transfected mammalian cells confirmed the interaction between the C-terminal domains of the {alpha} and {beta}1 subunits. The D1866Y mutation weakens this interaction, demonstrating a novel molecular mechanism leading to seizure susceptibility

The analysis of 2-amino-2-thiazoline-4-carboxylic acid in the plasma of smokers and non-smokers

ATCA (2-amino-2-thiazoline-4-carboxylic acid) is a promising marker to assess cyanide exposure because of several advantages of ATCA analysis over direct determination of cyanide and alternative cyanide biomarkers (i.e. stability in biological matrices, consistent recovery, and relatively small endogenous concentrations). Concentrations of ATCA in the plasma of smoking and non-smoking human volunteers were analyzed using gas-chromatography mass-spectrometry to establish the feasibility of using ATCA as a marker for cyanide exposure. The levels of ATCA in plasma of smoking volunteers, 17.2 ng/ml, were found to be significantly (p < 0.001) higher than that of non-smoking volunteers, 11.8 ng/ml. Comparison of ATCA concentrations of smokers relative to non-smokers in both urine and plasma yielded relatively similar results. The concentration ratio of ATCA for smokers versus non-smokers in plasma and urine was compared to similar literature studies of cyanide and thiocyanate, and correlations are discussed. This study supports previous evidence that ATCA can be used to determine past cyanide exposure and indicates that further studies should be pursued to validate the use of ATCA as a marker of cyanide exposure

Sequencing technologies and genome sequencing

The high-throughput - next generation sequencing (HT-NGS) technologies are currently the hottest topic in the field of human and animals genomics researches, which can produce over 100 times more data compared to the most sophisticated capillary sequencers based on the Sanger method. With the ongoing developments of high throughput sequencing machines and advancement of modern bioinformatics tools at unprecedented pace, the target goal of sequencing individual genomes of living organism at a cost of $1,000 each is seemed to be realistically feasible in the near future. In the relatively short time frame since 2005, the HT-NGS technologies are revolutionizing the human and animal genome researches by analysis of chromatin immunoprecipitation coupled to DNA microarray (ChIP-chip) or sequencing (ChIP-seq), RNA sequencing (RNA-seq), whole genome genotyping, genome wide structural variation, de novo assembling and re-assembling of genome, mutation detection and carrier screening, detection of inherited disorders and complex human diseases, DNA library preparation, paired ends and genomic captures, sequencing of mitochondrial genome and personal genomics. In this review, we addressed the important features of HT-NGS like, first generation DNA sequencers, birth of HT-NGS, second generation HT-NGS platforms, third generation HT-NGS platforms: including single molecule Heliscope™, SMRT™ and RNAP sequencers, Nanopore, Archon Genomics X PRIZE foundation, comparison of second and third HT-NGS platforms, applications, advances and future perspectives of sequencing technologies on human and animal genome research

Predictable cholesterol binding sites in GPCRs lack consensus motifs

A rich diversity of transmembrane G protein-coupled receptors (GPCRs) are used by eukaryotes to sense physical and chemical signals. In humans alone, 800 GPCRs comprise the largest and most therapeutically targeted receptor class. Recent advances in GPCR structural biology have produced hundreds of GPCR structures solved by X-ray diffraction and increasingly, cryo-electron microscopy (cryo-EM). Many of these structures are stabilized by site-specific cholesterol binding, but it is unclear whether these interactions are a product of recurring cholesterol-binding motifs and if observed patterns of cholesterol binding differ by experimental technique. Here, we comprehensively analyze the location and composition of cholesterol binding sites in the current set of 473 human GPCR structural chains. Our findings establish that cholesterol binds similarly in cryo-EM and X-ray structures and show that 92% of cholesterol molecules on GPCR surfaces reside in predictable locations that lack discernable cholesterol-binding motifs. [Display omitted] •A rapidly growing number of GPCR structures have been solved by cryo-EM•Cholesterol-binding patterns are similar in cryo-EM and X-ray GPCR structures•92% of cholesterol in GPCR structures map to 12 Cholesterol Network Clusters (CNCs)•There are no broadly recurring cholesterol-binding motifs in GPCRs Cholesterol (CLR) regulates G protein-coupled receptor (GPCR) function, but it is unclear if this is due to binding conserved CLR motifs. Taghon et al. collect, validate, and cluster all GPCR-bound CLR in the PDB and show that 92% reside in 12 distinct GPCR regions that lack consensus CLR-binding sites