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.     

Structural and Functional Analyses of PAS Domain Interactions of the Clock Proteins Drosophila PERIOD and Mouse PERIOD2

PERIOD proteins are central components of the Drosophila and mammalian circadian clocks. The crystal structure of a Drosophila PERIOD (dPER) fragment comprising two PER-ARNT-SIM (PAS) domains (PAS-A and PAS-B) and two additional C-terminal α-helices (αE and αF) has revealed a homodimer mediated by intermolecular interactions of PAS-A with tryptophane 482 in PAS-B and helix αF. Here we present the crystal structure of a monomeric PAS domain fragment of dPER lacking the αF helix. Moreover, we have solved the crystal structure of a PAS domain fragment of the mouse PERIOD homologue mPER2. The mPER2 structure shows a different dimer interface than dPER, which is stabilized by interactions of the PAS-B β-sheet surface including tryptophane 419 (equivalent to Trp482_dPER). We have validated and quantitatively analysed the homodimer interactions of dPER and mPER2 by site-directed mutagenesis using analytical gel filtration, analytical ultracentrifugation, and co-immunoprecipitation experiments. Furthermore we show, by yeast-two-hybrid experiments, that the PAS-B β-sheet surface of dPER mediates interactions with TIMELESS (dTIM). Our study reveals quantitative and qualitative differences between the homodimeric PAS domain interactions of dPER and its mammalian homologue mPER2. In addition, we identify the PAS-B β-sheet surface as a versatile interaction site mediating mPER2 homodimerization in the mammalian system and dPER-dTIM heterodimer formation in the Drosophila system.     

Isothermal titration calorimetry and surface plasmon resonance allow quantifying substrate binding to different binding sites of Bacillus subtilis xylanase

Isothermal titration calorimetry and surface plasmon resonance were tested for their ability to study substrate binding to the active site (AS) and to the secondary binding site (SBS) of Bacillus subtilis xylanase A separately. To this end, three enzyme variants were compared. The first was a catalytically incompetent enzyme that allows substrate binding to both the AS and SBS. In the second enzyme, binding to the SBS was impaired by site-directed mutagenesis, whereas in the third enzyme, the AS was blocked using a covalent inhibitor. Both techniques were able to show that AS and SBS have a similar binding affinity.     

A β-Lactam Antibiotic Dampens Excitotoxic Inflammatory CNS Damage in a Mouse Model of Multiple Sclerosis

In multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE), impairment of glial “Excitatory Amino Acid Transporters” (EAATs) together with an excess glutamate-release by invading immune cells causes excitotoxic damage of the central nervous system (CNS). In order to identify pathways to dampen excitotoxic inflammatory CNS damage, we assessed the effects of a β-lactam antibiotic, ceftriaxone, reported to enhance expression of glial EAAT2, in “Myelin Oligodendrocyte Glycoprotein” (MOG)-induced EAE. Ceftriaxone profoundly ameliorated the clinical course of murine MOG-induced EAE both under preventive and therapeutic regimens. However, ceftriaxone had impact neither on EAAT2 protein expression levels in several brain areas, nor on the radioactive glutamate uptake capacity in a mixed primary glial cell-culture and the glutamate-induced uptake currents in a mammalian cell line mediated by EAAT2. Moreover, the clinical effect of ceftriaxone was preserved in the presence of the EAAT2-specific transport inhibitor, dihydrokainate, while dihydrokainate alone caused an aggravated EAE course. This demonstrates the need for sufficient glial glutamate uptake upon an excitotoxic autoimmune inflammatory challenge of the CNS and a molecular target of ceftriaxone other than the glutamate transporter. Ceftriaxone treatment indirectly hampered T cell proliferation and proinflammatory INFγ and IL17 secretion through modulation of myelin-antigen presentation by antigen-presenting cells (APCs) e.g. dendritic cells (DCs) and reduced T cell migration into the CNS in vivo. Taken together, we demonstrate, that a β-lactam antibiotic attenuates disease course and severity in a model of autoimmune CNS inflammation. The mechanisms are reduction of T cell activation by modulation of cellular antigen-presentation and impairment of antigen-specific T cell migration into the CNS rather than or modulation of central glutamate homeostasis.     

Constitutive production of lymphocyte activating factors by normal tissues in the adult rat

Lymphocyte activating factors (LAFs), e.g., interleukin-1 (IL-1) and IL-1-like factors, have previously been demonstrated outside the immune system in the skin, thymus epithelium, and the human and rat testis. We have studied the presence of LAFs in normal tissues of the adult rat, utilizing a highly IL-1 sensitive murine thymocyte proliferation assay. We have demonstrated high amounts of LAF activity in the tongue, esophagus, proventricular part of the stomach, and the liver. Some activity was also demonstrated in the duodenum, placenta, spleen, Peyer's patches, glandular stomach, and jejunum, but no bioactivity was present in other gastrointestinal, endocrine, lymphoid, or haematopoeitic tissues. We were also unable to detect any LAF activity in the reproductive organs (except for the testis), urinary tract, skeletal and muscular tissues, brain, eyes, salivary glands, or lung. In the esophagus the activity was mainly localized to the mucosa. The LAF activity in the skin was partly inhibited by treatment with a mixture of antibodies against human IL-1 alpha and IL-1 beta. Dose response curves and gel filtration on a Sephacryl S-200 column suggested the presence of a high molecular weight (90,000-100,000 Da) LAF inhibitory factor in the liver. In all positive tissues, the demonstrated LAFs had a molecular weight of 15,000-25,000 Da, as determined by Sephacryl S-200 gel filtration. Of the positive tissues, the skin, tongue, esophagus, and the proventricular part of the stomach all contain stratified squamous epithelium. It is tempting to suggest that the detected LAFs have a similar function in these barrier tissues, e.g., to serve as host defence factors, or, alternatively or additionally, as tissue growth factors.     

Effects of phosphorus-mobilizing bacteria on tomato growth and soil microbial activity

Aims

The aim of our study was to clarify whether inoculating a soil with Pseudomonas sp. RU47 (RU47) bacteria would stimulate the enzymatic cleavage of organic P compounds in the rhizosphere and bulk soil, promoting plant growth. Adding either viable or heat treated RU47 cells made it possible to separate direct from indirect effects of the inoculum on P cycling in soil and plants.

Methods

We performed a rhizobox experiment in the greenhouse with tomato plants (Solanum lycopersicum) under low P soil conditions. Three inoculation treatments were conducted, using unselectively grown soil bacteria (bacterial mix), heat treated (HT-RU47) and viable RU47 (RU47) cells, and one not inoculated, optimally P-fertilized treatment. We verified plant growth, nutrient availability, enzyme activities and microbial community structure in soil.

Results

A plant growth promotion effect with improved P uptake was observed in both RU47 treatments. Inoculations of RU47 cells increased microbial phosphatase activity (PA) in the rhizosphere.

Conclusions

Plant growth promotion by RU47 cells is primarily associated with increased microbial PA in soil, while promotion of indigenous Pseudomonads as well as phytohormonal effects appear to be the dominant mechanisms when adding HT-RU47 cells. Thus, using RU47 offers a promising approach for more efficient P fertilization in agriculture.

     

Science and craftsmanship: the art of experiment and instrument making

In his two-volume monograph Untersuchungen über thierische Elektricit?t, the Berlin physiologist Emil du Bois-Reymond described the relation between nervous electricity and muscle mechanics by way of a long series of experiments. This work is a key text in the history of the experimental life sciences. But it not only contains new findings about the functioning of muscles and its nerves. Du Bois-Reymond practiced an art of experimentation in which aesthetics of mechanical craftsmanship allied itself with the science of physiology. Experimentation, as du Bois-Reymond understood it, was simultaneously an epistemic and an aesthetic practice. The goal of his science was thus producing both knowledge and aesthetic success.