The purification and characterisation of a Z-pro-prolinal insensitive Z-gly-pro-MCA degrading peptidase from bovine serum

The study of a novel prolme-specific peptidase from bovine serum is presented. The enzyme readily cleaves Z-Gly-Pro-MCA liberating the fluorophore MCA thus allowing quantification of enzyme activity Unlike prolyl ohgopeptidase (PO) which also hydrolyses this fluorogemc substrate, this peptidase is completely insensitive to Z-Pro- Prolmal and has been designated Z-Pro-Prolmal Insensitive Z-Gly-Pro-MCA degrading Peptidase (ZIP). The two peptidases were successfully separated from each other by phenyl sepharose hydrophobic interaction chromatography and the subsequent purification focused on the isolation of ZIP from bovine serum. In addition to phenyl sepharose, calcium phosphate cellulose and DEAE anion exchange chromatography were employed in the purification, with an overall enzyme yield of 33% and a purification factor of 4023 SDS PAGE and size-exclusion chromatography indicated a heterodimeric structure with a relative molecular mass of 180kDa. The enzyme remained stable at temperatures less than 50X1 for up to an hour, while optimal activity was observed at 37°C ZIP was surprisingly stable over the pH range 2 5-JO 0 Optimal activity was detected in the range pH 7 4-8 0 Isoelectric focusing revealed a pi value of5 68 and the enzyme appears to be 33% glycosylated. Inhibition by AEBSF suggests the peptidase may be a serine protease and ZIP possibly contains a cysteine residue near the active site a2Mfailed to inhibit activity suggesting an ohgopeptidase specificity HPLC analysis revealed a broad substrate specificity for proline-containmg peptides Kinetic analysis indicated that ZIP had a greater affinity for Z-Gly-Pro-MCA than PO with a Km of 54pM deduced. The ohgopeptidase showed complete insensitivity to a number of PO-specific inhibitors, namely JTP-4819 and S- 17092-1. ZIP exhibits similar biophysical characteristics to PO isolated from a number of sources. However the peptidases do appear to be distinct and ZIP may represent a novel prohnespecific serum peptidase, which may play a role in the degradation of oligopeptides

Identification, Replication, and Functional Fine-Mapping of Expression Quantitative Trait Loci in Primary Human Liver Tissue

The discovery of expression quantitative trait loci (“eQTLs”) can help to unravel genetic contributions to complex traits. We identified genetic determinants of human liver gene expression variation using two independent collections of primary tissue profiled with Agilent (n = 206) and Illumina (n = 60) expression arrays and Illumina SNP genotyping (550K), and we also incorporated data from a published study (n = 266). We found that ∼30% of SNP-expression correlations in one study failed to replicate in either of the others, even at thresholds yielding high reproducibility in simulations, and we quantified numerous factors affecting reproducibility. Our data suggest that drug exposure, clinical descriptors, and unknown factors associated with tissue ascertainment and analysis have substantial effects on gene expression and that controlling for hidden confounding variables significantly increases replication rate. Furthermore, we found that reproducible eQTL SNPs were heavily enriched near gene starts and ends, and subsequently resequenced the promoters and 3′UTRs for 14 genes and tested the identified haplotypes using luciferase assays. For three genes, significant haplotype-specific in vitro functional differences correlated directly with expression levels, suggesting that many bona fide eQTLs result from functional variants that can be mechanistically isolated in a high-throughput fashion. Finally, given our study design, we were able to discover and validate hundreds of liver eQTLs. Many of these relate directly to complex traits for which liver-specific analyses are likely to be relevant, and we identified dozens of potential connections with disease-associated loci. These included previously characterized eQTL contributors to diabetes, drug response, and lipid levels, and they suggest novel candidates such as a role for NOD2 expression in leprosy risk and C2orf43 in prostate cancer. In general, the work presented here will be valuable for future efforts to precisely identify and functionally characterize genetic contributions to a variety of complex traits

Genetic fine mapping and genomic annotation defines causal mechanisms at type 2 diabetes susceptibility loci.

We performed fine mapping of 39 established type 2 diabetes (T2D) loci in 27,206 cases and 57,574 controls of European ancestry. We identified 49 distinct association signals at these loci, including five mapping in or near KCNQ1. 'Credible sets' of the variants most likely to drive each distinct signal mapped predominantly to noncoding sequence, implying that association with T2D is mediated through gene regulation. Credible set variants were enriched for overlap with FOXA2 chromatin immunoprecipitation binding sites in human islet and liver cells, including at MTNR1B, where fine mapping implicated rs10830963 as driving T2D association. We confirmed that the T2D risk allele for this SNP increases FOXA2-bound enhancer activity in islet- and liver-derived cells. We observed allele-specific differences in NEUROD1 binding in islet-derived cells, consistent with evidence that the T2D risk allele increases islet MTNR1B expression. Our study demonstrates how integration of genetic and genomic information can define molecular mechanisms through which variants underlying association signals exert their effects on disease

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Bi-allelic Loss-of-Function CACNA1B Mutations in Progressive Epilepsy-Dyskinesia.

The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment.MAK is funded by an NIHR Research Professorship and receives funding from the Wellcome Trust, Great Ormond Street Children's Hospital Charity, and Rosetrees Trust. E.M. received funding from the Rosetrees Trust (CD-A53) and Great Ormond Street Hospital Children's Charity. K.G. received funding from Temple Street Foundation. A.M. is funded by Great Ormond Street Hospital, the National Institute for Health Research (NIHR), and Biomedical Research Centre. F.L.R. and D.G. are funded by Cambridge Biomedical Research Centre. K.C. and A.S.J. are funded by NIHR Bioresource for Rare Diseases. The DDD Study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the Department of Health, and the Wellcome Trust Sanger Institute (grant number WT098051). We acknowledge support from the UK Department of Health via the NIHR comprehensive Biomedical Research Centre award to Guy's and St. Thomas' National Health Service (NHS) Foundation Trust in partnership with King's College London. This research was also supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre. J.H.C. is in receipt of an NIHR Senior Investigator Award. The research team acknowledges the support of the NIHR through the Comprehensive Clinical Research Network. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, Department of Health, or Wellcome Trust. E.R.M. acknowledges support from NIHR Cambridge Biomedical Research Centre, an NIHR Senior Investigator Award, and the University of Cambridge has received salary support in respect of E.R.M. from the NHS in the East of England through the Clinical Academic Reserve. I.E.S. is supported by the National Health and Medical Research Council of Australia (Program Grant and Practitioner Fellowship)

Influence of basolateral condition on the regulation of brain microvascular endothelial tight junction properties and barrier function

Basolateral condition of the brain microvascular endothelium is believed to influence blood–brain barrier (BBB) phenotype, although the precise transcriptional and post-translational mechanisms involved are poorly defined. In vivo, the basolateral surface of the blood–brain endothelium is bathed in serum-free interstitial fluid and encompassed by astrocytic end-feet. We hypothesized that these conditions impact on BBB function by directly modulating expression and biochemical properties of tight junctions. To investigate this, an in vitro transwell culture model was employed to selectively modify the basolateral environment of bovine brain microvascular endothelial cells (BBMvECs). In the absence of basolateral (but not apical) serum, we observed higher levels of expression, association and plasma membrane localization for the tight junction proteins, occludin and zonula occludens-1 (ZO-1), in parallel with elevated transendothelial electrical resistance (TEER) and reduced 14[C]-sucrose permeability of BBMvEC monolayers. We further examined the effects of non-contact co-culture with basolateral astrocytes (C6 glioma) on indices of BBMvEC barrier function in both the presence and absence of serum. Astrocyte co-culture with serum led to enhanced occludin protein expression, occludin/ZO-1 association, and ZO-1 membrane localization, in parallel with increased TEER of BBMvEC monolayers. Astrocyte co-culture in the absence of serum (i.e. basolateral conditions most consistent with in vivo BBB physiology) however, gave the highest increases in BBMvEC barrier indices. Thus, we can conclude that factors influencing condition of the basolateral environment of the brain microvasculature can directly, and independently, modify BBB properties by regulating the expression and biochemical properties of the tight junction proteins, occludin and ZO-1