Quality control and integration of genotypes from two calling pipelines for whole genome sequence data in the Alzheimer's disease sequencing project

The Alzheimer's Disease Sequencing Project (ADSP) performed whole genome sequencing (WGS) of 584 subjects from 111 multiplex families at three sequencing centers. Genotype calling of single nucleotide variants (SNVs) and insertion-deletion variants (indels) was performed centrally using GATK-HaplotypeCaller and Atlas V2. The ADSP Quality Control (QC) Working Group applied QC protocols to project-level variant call format files (VCFs) from each pipeline, and developed and implemented a novel protocol, termed “consensus calling,” to combine genotype calls from both pipelines into a single high-quality set. QC was applied to autosomal bi-allelic SNVs and indels, and included pipeline-recommended QC filters, variant-level QC, and sample-level QC. Low-quality variants or genotypes were excluded, and sample outliers were noted. Quality was assessed by examining Mendelian inconsistencies (MIs) among 67 parent-offspring pairs, and MIs were used to establish additional genotype-specific filters for GATK calls. After QC, 578 subjects remained. Pipeline-specific QC excluded ~12.0% of GATK and 14.5% of Atlas SNVs. Between pipelines, ~91% of SNV genotypes across all QCed variants were concordant; 4.23% and 4.56% of genotypes were exclusive to Atlas or GATK, respectively; the remaining ~0.01% of discordant genotypes were excluded. For indels, variant-level QC excluded ~36.8% of GATK and 35.3% of Atlas indels. Between pipelines, ~55.6% of indel genotypes were concordant; while 10.3% and 28.3% were exclusive to Atlas or GATK, respectively; and ~0.29% of discordant genotypes were. The final WGS consensus dataset contains 27,896,774 SNVs and 3,133,926 indels and is publicly available.     

A Cys-loop mutation in the Caenorhabditis elegans nicotinic receptor subunit UNC-63 impairs but does not abolish channel function

The nematode Caenorhabditis elegans is an established model organism for studying neurobiology. UNC-63 is a C. elegans nicotinic acetylcholine receptor (nAChR) α-subunit. It is an essential component of the levamisole-sensitive muscle nAChR (L-nAChR) and therefore plays an important role in cholinergic transmission at the nematode neuromuscular junction. Here, we show that worms with the unc-63(x26) allele, with its αC151Y mutation disrupting the Cys-loop, have deficient muscle function reflected by impaired swimming (thrashing). Single-channel recordings from cultured muscle cells from the mutant strain showed a 100-fold reduced frequency of opening events and shorter channel openings of L-nAChRs compared with those of wild-type worms. Anti-UNC-63 antibody staining in both cultured adult muscle and embryonic cells showed that L-nAChRs were expressed at similar levels in the mutant and wild-type cells, suggesting that the functional changes in the receptor, rather than changes in expression, are the predominant effect of the mutation. The kinetic changes mimic those reported in patients with fast-channel congenital myasthenic syndromes. We show that pyridostigmine bromide and 3,4-diaminopyridine, which are drugs used to treat fast-channel congenital myasthenic syndromes, partially rescued the motility defect seen in unc-63(x26). The C. elegans unc-63(x26) mutant may therefore offer a useful model to assist in the development of therapies for syndromes produced by altered function of human nAChRs.     

Low-dose spironolactone reduces reactive oxygen species generation and improves insulin-stimulated glucose transport in skeletal muscle in the TG(mRen2)27 rat

Renin-angiotensin-aldosterone system (RAAS) activation mediates increases in reactive oxygen species (ROS) and impaired insulin signaling. The transgenic Ren2 rat manifests increased tissue renin-angiotensin system activity, elevated serum aldosterone, hypertension, and insulin resistance. To explore the role of aldosterone in the pathogenesis of insulin resistance, we investigated the impact of in vivo treatment with a mineralocorticoid receptor (MR) antagonist on insulin sensitivity in Ren2 and aged-matched Sprague-Dawley (SD) control rats. Both groups (age 6-8 wk) were implanted with subcutaneous time-release pellets containing spironolactone (0.24 mg/day) or placebo over 21 days. Systolic blood pressure (SBP) and intraperitoneal glucose tolerance test were determined. Soleus muscle insulin receptor substrate-1 (IRS-1), tyrosine phosphorylated IRS-1, protein kinase B (Akt) phosphorylation, GLUT4 levels, and insulin-stimulated 2-deoxyglucose uptake were evaluated in relation to NADPH subunit expression/oxidase activity and ROS production (chemiluminescence and 4-hydroxy-2-nonenal immunostaining). Along with increased soleus muscle NADPH oxidase activity and ROS, there was systemic insulin resistance and reduced muscle IRS-1 tyrosine phosphorylation, Akt phosphorylation/activation, and GLUT4 expression in the Ren2 group (each P < 0.05). Despite not decreasing blood pressure, low-dose spironolactone treatment improved soleus muscle insulin signaling parameters and systemic insulin sensitivity in concert with reductions in NADPH oxidase subunit expression/activity and ROS production (each P < 0.05). Our findings suggest that aldosterone contributes to insulin resistance in the transgenic Ren2, in part, by increasing NADPH oxidase activity in skeletal muscle tissue.     

Automated,Quantitative Cognitive/Behavioral Screening of Mice: For Genetics,Pharmacology, Animal Cognition and Undergraduate Instruction

We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and pharmacological manipulations on basic mechanisms of cognition and learning in mice. A standard polypropylene mouse housing tub is connected through an acrylic tube to a standard commercial mouse test box. The test box has 3 hoppers, 2 of which are connected to pellet feeders. All are internally illuminable with an LED and monitored for head entries by infrared (IR) beams. Mice live in the environment, which eliminates handling during screening. They obtain their food during two or more daily feeding periods by performing in operant (instrumental) and Pavlovian (classical) protocols, for which we have written protocol-control software and quasi-real-time data analysis and graphing software. The data analysis and graphing routines are written in a MATLAB-based language created to simplify greatly the analysis of large time-stamped behavioral and physiological event records and to preserve a full data trail from raw data through all intermediate analyses to the published graphs and statistics within a single data structure. The data-analysis code harvests the data several times a day and subjects it to statistical and graphical analyses, which are automatically stored in the "cloud" and on in-lab computers. Thus, the progress of individual mice is visualized and quantified daily. The data-analysis code talks to the protocol-control code, permitting the automated advance from protocol to protocol of individual subjects. The behavioral protocols implemented are matching, autoshaping, timed hopper-switching, risk assessment in timed hopper-switching, impulsivity measurement, and the circadian anticipation of food availability. Open-source protocol-control and data-analysis code makes the addition of new protocols simple. Eight test environments fit in a 48 in x 24 in x 78 in cabinet; two such cabinets (16 environments) may be controlled by one computer.     

Response of seaward-migrating European eel (Anguilla anguilla) to manipulated flow fields

Anthropogenic structures (e.g. weirs and dams) fragment river networks and restrict the movement of migratory fish. Poor understanding of behavioural response to hydrodynamic cues at structures currently limits the development of effective barrier mitigation measures. This study aimed to assess the effect of flow constriction and associated flow patterns on eel behaviour during downstream migration. In a field experiment, we tracked the movements of 40 tagged adult European eels (Anguilla anguilla) through the forebay of a redundant hydropower intake under two manipulated hydrodynamic treatments. Interrogation of fish trajectories in relation to measured and modelled water velocities provided new insights into behaviour, fundamental for developing passage technologies for this endangered species. Eels rarely followed direct routes through the site. Initially, fish aligned with streamlines near the channel banks and approached the intake semi-passively. A switch to more energetically costly avoidance behaviours occurred on encountering constricted flow, prior to physical contact with structures. Under high water velocity gradients, fish then tended to escape rapidly back upstream, whereas exploratory ‘search’ behaviour was common when acceleration was low. This study highlights the importance of hydrodynamics in informing eel behaviour. This offers potential to develop behavioural guidance, improve fish passage solutions and enhance traditional physical screening.