Clustered Gene Expression Changes Flank Targeted Gene Loci in Knockout Mice

Gene expression profiling using microarrays is a powerful technology widely used to study regulatory networks. Profiling of mRNA levels in mutant organisms has the potential to identify genes regulated by the mutated protein.Using tissues from multiple lines of knockout mice we have examined genome-wide changes in gene expression. We report that a significant proportion of changed genes were found near the targeted gene.The apparent clustering of these genes was explained by the presence of flanking DNA from the parental ES cell. We provide recommendations for the analysis and reporting of microarray data from knockout mice

Proteomic analysis of NMDA receptor–adhesion protein signaling complexes

N-methyl-D-aspartate receptors (NMDAR) mediate long-lasting changes in synapse strength via downstream signaling pathways. We report proteomic characterization with mass spectrometry and immunoblotting of NMDAR multiprotein complexes (NRC) isolated from mouse brain. The NRC comprised 77 proteins organized into receptor, adaptor, signaling, cytoskeletal and novel proteins, of which 30 are implicated from binding studies and another 19 participate in NMDAR signaling. NMDAR and metabotropic glutamate receptor subtypes were linked to cadherins and L1 cell-adhesion molecules in complexes lacking AMPA receptors. These neurotransmitter– adhesion receptor complexes were bound to kinases, phosphatases, GTPase-activating proteins and Ras with effectors including MAPK pathway components. Several proteins were encoded by activity-dependent genes. Genetic or pharmacological interference with 15 NRC proteins impairs learning and with 22 proteins alters synaptic plasticity in rodents. Mutations in three human genes (NF1, Rsk-2, L1) are associated with learning impairments, indicating the NRC also participates in human cognition

G2Cdb: the Genes to Cognition database

Neuroscience databases linking genes, proteins, (patho)physiology, anatomy and behaviour across species will be valuable in a broad range of studies of the nervous system. G2Cdb is such a neuroscience database aiming to present a global view of the role of synapse proteins in physiology and disease. G2Cdb warehouses sets of genes and proteins experimentally elucidated by proteomic mass spectroscopy of signalling complexes and proteins biochemically isolated from mammalian synapse preparations, giving an experimentally validated definition of the constituents of the mammalian synapse. Using automated text-mining and expert (human) curation we have systematically extracted information from published neurobiological studies in the fields of synaptic signalling electrophysiology and behaviour in knockout and other transgenic mice. We have also surveyed the human genetics literature for associations to disease caused by mutations in synaptic genes. The synapse proteome datasets that G2Cdb provides offer a basis for future work in synapse biology and provide useful information on brain diseases. They have been integrated in a such way that investigators can rapidly query whether a gene or protein is found in brain-signalling complex(es), has a phenotype in rodent models or whether mutations are associated with a human disease. G2Cdb can be freely accessed at http://www.genes2cognition.org

Frequency of tuberculous and non-tuberculous mycobacteria in HIV infected patients from Bogota, Colombia

BACKGROUND: The prevalence of infections by Mycobacterium tuberculosis and non-tuberculous Mycobacterium species in the HIV-infected patient population in Colombia was uncertain despite some pilot studies. We determined the frequency of isolation of Mycobacterium tuberculosis and of non-tuberculous Mycobacterium species in diverse body fluids of HIV-infected patients in Bogota, Colombia. METHODS: Patients who attended the three major HIV/AIDS healthcare centres in Bogota were prospectively studied over a six month period. A total of 286 patients were enrolled, 20% of them were hospitalized at some point during the study. Sixty four percent (64%) were classified as stage C, 25% as stage B, and 11% as stage A (CDC staging system, 1993). A total of 1,622 clinical samples (mostly paired samples of blood, sputum, stool, and urine) were processed for acid-fast bacilli (AFB) stain and culture. RESULTS: Overall 43 of 1,622 cultures (2.6%) were positive for mycobacteria. Twenty-two sputum samples were positive. Four patients were diagnosed with M. tuberculosis (1.4%). All isolates of M. tuberculosis were sensitive to common anti-tuberculous drugs. M. avium was isolated in thirteen patients (4.5%), but only in three of them the cultures originated from blood. The other isolates were obtained from stool, urine or sputum samples. In three cases, direct AFB smears of blood were positive. Two patients presented simultaneously with M. tuberculosis and M. avium. CONCLUSIONS: Non-tuberculous Mycobacterium infections are frequent in HIV infected patients in Bogota. The diagnostic sensitivity for infection with tuberculous and non-tuberculous mycobacteria can be increased when diverse body fluids are processed from each patient

Prototype ATLAS IBL Modules using the FE-I4A Front-End Readout Chip

The ATLAS Collaboration will upgrade its semiconductor pixel tracking detector with a new Insertable B-layer (IBL) between the existing pixel detector and the vacuum pipe of the Large Hadron Collider. The extreme operating conditions at this location have necessitated the development of new radiation hard pixel sensor technologies and a new front-end readout chip, called the FE-I4. Planar pixel sensors and 3D pixel sensors have been investigated to equip this new pixel layer, and prototype modules using the FE-I4A have been fabricated and characterized using 120 GeV pions at the CERN SPS and 4 GeV positrons at DESY, before and after module irradiation. Beam test results are presented, including charge collection efficiency, tracking efficiency and charge sharing.Comment: 45 pages, 30 figures, submitted to JINS

Interaction proteomics of synapse protein complexes

The brain integrates complex types of information, and executes a wide range of physiological and behavioral processes. Trillions of tiny organelles, the synapses, are central to neuronal communication and information processing in the brain. Synaptic transmission involves an intricate network of synaptic proteins that forms the molecular machinery underlying transmitter release, activation, and modulation of transmitter receptors and signal transduction cascades. These processes are dynamically regulated and underlie neuroplasticity, crucial to learning and memory formation. In recent years, interaction proteomics has increasingly been used to elucidate the constituents of synaptic protein complexes. Unlike classic hypothesis-based assays, interaction proteomics detects both known and novel interactors without bias. In this trend article, we focus on the technical aspects of recent proteomics to identify synapse protein complexes, and the complementary methods used to verify the protein–protein interaction. Moreover, we discuss the experimental feasibility of performing global analysis of the synapse protein interactome

A proteomic approach based on peptide affinity chromatography, 2-dimensional electrophoresis and mass spectrometry to identify multiprotein complexes interacting with membrane-bound receptors

There is accumulating evidence that membrane-bound receptors interact with many intracellular proteins. Multiprotein complexes associated with ionotropic receptors have been extensively characterized, but the identification of proteins interacting with G protein-coupled receptors (GPCRs) has so far only been achieved in a piecemeal fashion, focusing on one or two protein species. We describe a method based on peptide affinity chromatography, two-dimensional electrophoresis, mass spectrometry and immunoblotting to identify the components of multiprotein complexes interacting directly or indirectly with intracellular domains of GPCRs or, more generally, any other membrane-bound receptor. Using this global approach, we have characterized multiprotein complexes that bind to the carboxy-terminal tail of the 5-hydroxytryptamine type 2C receptor and are important for its subcellular localization in CNS cells (Bécamel et al., EMBO J., 21(10): 2332, 2002)

Dlgap1 knockout mice exhibit alterations of the postsynaptic density and selective reductions in sociability

Abstract The scaffold protein DLGAP1 is localized at the post-synaptic density (PSD) of glutamatergic neurons and is a component of supramolecular protein complexes organized by PSD95. Gain-of-function variants of DLGAP1 have been associated with obsessive-compulsive disorder (OCD), while haploinsufficient variants have been linked to autism spectrum disorder (ASD) and schizophrenia in human genetic studies. We tested male and female Dlgap1 wild type (WT), heterozygous (HT), and knockout (KO) mice in a battery of behavioral tests: open field, dig, splash, prepulse inhibition, forced swim, nest building, social approach, and sucrose preference. We also used biochemical approaches to examine the role of DLGAP1 in the organization of PSD protein complexes. Dlgap1 KO mice were most notable for disruption of protein interactions in the PSD, and deficits in sociability. Other behavioral measures were largely unaffected. Our data suggest that Dlgap1 knockout leads to PSD disruption and reduced sociability, consistent with reports of DLGAP1 haploinsufficient variants in schizophrenia and ASD

Estimation of the number of synapses in the hippocampus and brain-wide by volume electron microscopy and genetic labeling

Determining the number of synapses that are present in different brain regions is crucial to understand brain connectivity as a whole. Membrane-associated guanylate kinases (MAGUKs) are a family of scaffolding proteins that are expressed in excitatory glutamatergic synapses. We used genetic labeling of two of these proteins (PSD95 and SAP102), and Spinning Disc confocal Microscopy (SDM), to estimate the number of fluorescent puncta in the CA1 area of the hippocampus. We also used FIB-SEM, a three-dimensional electron microscopy technique, to calculate the actual numbers of synapses in the same area. We then estimated the ratio between the three-dimensional densities obtained with FIB-SEM (synapses/µm) and the bi-dimensional densities obtained with SDM (puncta/100 µm). Given that it is impractical to use FIB-SEM brain-wide, we used previously available SDM data from other brain regions and we applied this ratio as a conversion factor to estimate the minimum density of synapses in those regions. We found the highest densities of synapses in the isocortex, olfactory areas, hippocampal formation and cortical subplate. Low densities were found in the pallidum, hypothalamus, brainstem and cerebellum. Finally, the striatum and thalamus showed a wide range of synapse densities.This work was supported by grants from the following entities: the Spanish “Ministerio de Ciencia, Innovación y Universidades” (Grant PGC2018-094307-B-I00 and the Cajal Blue Brain Project [C080020-09; the Spanish partner of the Blue Brain Project initiative from EPFL, Switzerland]; the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No. 785907 (Human Brain Project, SGA2); the Wellcome Trust (Technology Development Grant 202932); and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (695568 SYNNOVATE). L.T.-R. is a recipient of grants from the EMBO Long-term fellowship 2016–2018 and the IBRO-PERC InEurope grants programme