Molecular electronics sensors on a scalable semiconductor chip: A platform for single-molecule measurement of binding kinetics and enzyme activity.

For nearly 50 years, the vision of using single molecules in circuits has been seen as providing the ultimate miniaturization of electronic chips. An advanced example of such a molecular electronics chip is presented here, with the important distinction that the molecular circuit elements play the role of general-purpose single-molecule sensors. The device consists of a semiconductor chip with a scalable array architecture. Each array element contains a synthetic molecular wire assembled to span nanoelectrodes in a current monitoring circuit. A central conjugation site is used to attach a single probe molecule that defines the target of the sensor. The chip digitizes the resulting picoamp-scale current-versus-time readout from each sensor element of the array at a rate of 1,000 frames per second. This provides detailed electrical signatures of the single-molecule interactions between the probe and targets present in a solution-phase test sample. This platform is used to measure the interaction kinetics of single molecules, without the use of labels, in a massively parallel fashion. To demonstrate broad applicability, examples are shown for probe molecule binding, including DNA oligos, aptamers, antibodies, and antigens, and the activity of enzymes relevant to diagnostics and sequencing, including a CRISPR/Cas enzyme binding a target DNA, and a DNA polymerase enzyme incorporating nucleotides as it copies a DNA template. All of these applications are accomplished with high sensitivity and resolution, on a manufacturable, scalable, all-electronic semiconductor chip device, thereby bringing the power of modern chips to these diverse areas of biosensing.

The Qatar Biobank: background and methods.

BACKGROUND: The Qatar Biobank aims to collect extensive lifestyle, clinical, and biological information from up to 60,000 men and women Qatari nationals and long-term residents (individuals living in the country for ≥15 years) aged ≥18 years (approximately one-fifth of all Qatari citizens), to follow up these same individuals over the long term to record any subsequent disease, and hence to study the causes and progression of disease, and disease burden, in the Qatari population. METHODS: Between the 11(th)-December-2012 and 20(th)-February-2014, 1209 participants were recruited into the pilot study of the Qatar Biobank. At recruitment, extensive phenotype information was collected from each participant, including information/measurements of socio-demographic factors, prevalent health conditions, diet, lifestyle, anthropometry, body composition, bone health, cognitive function, grip strength, retinal imaging, total body dual energy X-ray absorptiometry, and measurements of cardiovascular and respiratory function. Blood, urine, and saliva were collected and stored for future research use. A panel of 66 clinical biomarkers was routinely measured on fresh blood samples in all participants. Rates of recruitment are to be progressively increased in the coming period and the recruitment base widened to achieve a cohort of consented individuals broadly representative of the eligible Qatari population. In addition, it is planned to add additional measures in sub-samples of the cohort, including Magnetic Resonance Imaging (MRI) of the brain, heart and abdomen. RESULTS: The mean time for collection of the extensive phenotypic information and biological samples from each participant at the baseline recruitment visit was 179 min. The 1209 pilot study participants (506 men and 703 women) were aged between 28-80 years (median 39 years); 899 (74.4%) were Qatari nationals and 310 (25.6%) were long-term residents. Approximately two-thirds of pilot participants were educated to graduate level or above. CONCLUSIONS: The pilot has proven that recruitment of volunteers into the Qatar Biobank project with intensive baseline measurements of behavioural, physical, and clinical characteristics is well accepted and logistically feasible. Qatar Biobank will provide a powerful resource to investigate the major determinants of ill-health and well-being in Qatar, providing valuable insights into the current and future public health burden that faces the country.

A genome-wide association study in progressive multiple sclerosis.

BACKGROUND: The role played by genetic factors in influencing the clinical course of multiple sclerosis (MS) is not yet well established. OBJECTIVE: We aimed to identify genetic variants associated with progressive MS (PrMS). METHODS: We conducted a genome-wide association study (GWAS) in 197 patients with PrMS and 234 controls of Italian origin. We tested the top 20 single nucleotide polymorphisms (SNPs) with suggestive evidence of association (p-value<10(-4)) in two independent sets of primary progressive MS cases and controls. RESULTS: We identified a risk-associated SNP in the HLA region in linkage disequilibrium (LD) with DRB1*1501 and DQB*0602 loci, with genome-wide significance (rs3129934(T), p (combined)=6.7×10(-16), OR=2.34, 95% CI=1.90-2.87), and a novel locus on chromosome 7q35 with suggestive evidence of association (rs996343(G), p (combined)=2.4×10(-5), OR=0.70, 95% CI=0.59-0.83) which maps within a human endogenous retroviral (HERV) element. The new locus did not have a 'cis' effect on RNA expression in lymphoblastic cell lines, but pathway analyses of 'trans' effects point to an expression regulation of genes involved in neurodegeneration, including glutamate metabolism (p<0.01) and axonal guidance signalling (p<0.02). CONCLUSIONS: We have confirmed the established association with the HLA region and, despite the low statistical power of the study, we found suggestive evidence for association with a novel locus on chromosome 7, with a putative regulatory role.

CD161(high)CD8+T cells bear pathogenetic potential in multiple sclerosis.

To identify differentially expressed genes in multiple sclerosis, microarrays were used in a stringent experimental setting-leukapheresis from disease-discordant monozygotic twins and gene expression profiling in CD4(+) and CD8(+) T-cell subsets. Disease-related differences emerged only in the CD8(+) T-cell subset. The five differentially expressed genes identified included killer cell lectin-like receptor subfamily B, member 1, also known as natural killer receptor protein 1a/CD161, presented by the International Multiple Sclerosis Genetics Consortium as one of the non-MHC candidate loci. Flow cytometric analysis on peripheral blood of healthy donors and patients with multiple sclerosis and rheumatoid arthritis confirmed an upregulation of CD161 at the protein level, showing also a significant excess of CD161(high)CD8(+) T cells in multiple sclerosis. This subset prevalently included chemokine (C-C motif) receptor 6(+), cytokine-producing, effector-memory T cells with proinflammatory profiles. It also included all circulating interleukin-17(+)CD8(+) T cells. In the CD161(high)CD8(+) subset, interleukin-12 facilitated proliferation and interferon-γ production, with CD161 acting as a co-stimulatory receptor. CD161(+)CD8(+)CD3(+) T cells producing interferon-γ were part of intralesional immune infiltrates and ectopic B cell follicles in autopsy multiple sclerosis brains. Variations of CD161 expression on CD8(+) T cells identify a subset of lymphocytes with proinflammatory characteristics that have not been previously reported in multiple sclerosis and are likely to contribute to disease immunopathology.

Functional whole-genome analysis identifies Polo-like kinase 2 and poliovirus receptor as essential for neuronal differentiation upstream of the negative regulator alphaB-crystallin.

This study aimed at identifying transcriptional changes associated to neuronal differentiation induced by six distinct stimuli using whole-genome microarray hybridization analysis. Bioinformatics analyses revealed the clustering of these six stimuli into two categories, suggesting separate gene/pathway dependence. Treatment with specific inhibitors demonstrated the requirement of both Janus kinase and microtubule-associated protein kinase activation to trigger differentiation with nerve growth factor (NGF) and dibutyryl cAMP. Conversely, activation of protein kinase A, phosphatidylinositol-3-kinase alpha, and mammalian target of rapamycin, although required for dibutyryl cAMP-induced differentiation, exerted a negative feedback on NGF-induced differentiation. We identified Polo-like kinase 2 (Plk2) and poliovirus receptor (PVR) as indispensable for NGF-driven neuronal differentiation and alphaB-crystallin (Cryab) as an inhibitor of this process. Silencing of Plk2 or PVR blocked NGF-triggered differentiation and Cryab down-regulation, while silencing of Cryab enhanced NGF-induced differentiation. Our results position both Plk2 and PVR upstream of the negative regulator Cryab in the pathway(s) leading to neuronal differentiation triggered by NGF.

MGAT5 and disease severity in progressive multiple sclerosis.

MGAT5 was demonstrated to be associated to multiple sclerosis (MS) severity. We evaluated its role in progressive MS. We studied 11 SNPs mapping to MGAT5 in an Italian cohort of primary progressive or progressive-relapsing patients. The rs1257169(G) allele is associated with lower disease severity (p-value = 0.02). Adding the age of onset as covariate, another SNP, rs539588, is nominally significant. None of the SNPs survived after multiple testing correction. These results, even if suggestive of MGAT5 involvement also in progressive MS severity, require a bigger sample size to confirm and better explore the role of this locus in this rare disease course.

Convergent functional genomics of oligodendrocyte differentiation identifies multiple autoinhibitory signaling circuits.

Inadequate remyelination of brain white matter lesions has been associated with a failure of oligodendrocyte precursors to differentiate into mature, myelin-producing cells. In order to better understand which genes play a critical role in oligodendrocyte differentiation, we performed time-dependent, genome-wide gene expression studies of mouse Oli-neu cells as they differentiate into process-forming and myelin basic protein-producing cells, following treatment with three different agents. Our data indicate that different inducers activate distinct pathways that ultimately converge into the completely differentiated state, where regulated gene sets overlap maximally. In order to also gain insight into the functional role of genes that are regulated in this process, we silenced 88 of these genes using small interfering RNA and identified multiple repressors of spontaneous differentiation of Oli-neu, most of which were confirmed in rat primary oligodendrocyte precursors cells. Among these repressors were CNP, a well-known myelin constituent, and three phosphatases, each known to negatively control mitogen-activated protein kinase cascades. We show that a novel inhibitor for one of the identified genes, dual-specificity phosphatase DUSP10/MKP5, was also capable of inducing oligodendrocyte differentiation in primary oligodendrocyte precursors. Oligodendrocytic differentiation feedback loops may therefore yield pharmacological targets to treat disease related to dysfunctional myelin deposition.

Functional variants in the B-cell gene BANK1 are associated with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by production of autoantibodies and complex genetic inheritance. In a genome-wide scan using 85,042 SNPs, we identified an association between SLE and a nonsynonymous substitution (rs10516487, R61H) in the B-cell scaffold protein with ankyrin repeats gene, BANK1. We replicated the association in four independent case-control sets (combined P = 3.7 x 10(-10); OR = 1.38). We analyzed BANK1 cDNA and found two isoforms, one full-length and the other alternatively spliced and lacking exon 2 (Delta2), encoding a protein without a putative IP3R-binding domain. The transcripts were differentially expressed depending on a branch point-site SNP, rs17266594, in strong linkage disequilibrium (LD) with rs10516487. A third associated variant was found in the ankyrin domain (rs3733197, A383T). Our findings implicate BANK1 as a susceptibility gene for SLE, with variants affecting regulatory sites and key functional domains. The disease-associated variants could contribute to sustained B cell-receptor signaling and B-cell hyperactivity characteristic of this disease.

Genetic and physiological data implicating the new human gene G72 and the gene for D-amino acid oxidase in schizophrenia.

A map of 191 single-nucleotide polymorphism (SNPs) was built across a 5-Mb segment from chromosome 13q34 that has been genetically linked to schizophrenia. DNA from 213 schizophrenic patients and 241 normal individuals from Canada were genotyped with this marker set. Two 1,400- and 65-kb regions contained markers associated with the disease. Two markers from the 65-kb region were also found to be associated to schizophrenia in a Russian sample. Two overlapping genes G72 and G30 transcribed in brain were experimentally annotated in this 65-kb region. Transfection experiments point to the existence of a 153-aa protein coded by the G72 gene. This protein is rapidly evolving in primates, is localized to endoplasmic reticulum/Golgi in transfected cells, is able to form multimers and specifically binds to carbohydrates. Yeast two-hybrid experiments with the G72 protein identified the enzyme d-amino acid oxidase (DAAO) as an interacting partner. DAAO is expressed in human brain where it oxidizes d-serine, a potent activator of N-methyl-D-aspartate type glutamate receptor. The interaction between G72 and DAAO was confirmed in vitro and resulted in activation of DAAO. Four SNP markers from DAAO were found to be associated with schizophrenia in the Canadian samples. Logistic regression revealed genetic interaction between associated SNPs in vicinity of two genes. The association of both DAAO and a new gene G72 from 13q34 with schizophrenia together with activation of DAAO activity by a G72 protein product points to the involvement of this N-methyl-d-aspartate receptor regulation pathway in schizophrenia.