Variants of Rab GTPase–Effector Binding Protein-2 Cause Variation in the Collateral Circulation and Severity of Stroke

The extent (number and diameter) of collateral vessels varies widely and is a major determinant, along with arteriogenesis (collateral remodeling), of variation in severity of tissue injury following large artery occlusion. Differences in genetic background underlie the majority of the variation in collateral extent in mice, through alterations in collaterogenesis (embryonic collateral formation). In brain and other tissues, ~80% of the variation in collateral extent among different mouse strains has been linked to a region on chromosome 7. We recently used congenic (CNG) fine-mapping of C57BL/6 (B6, high extent) and BALB/cBy (BC, low extent) mice to narrow the region to a 737 Kb locus, Dce1. Herein, we report the causal gene

Genetic Architecture Underlying Variation in Extent and Remodeling of the Collateral Circulation

Collaterals are arteriole-to-arteriole anastomoses that connect adjacent arterial trees. They lessen ischemic tissue injury by serving as endogenous bypass vessels when the trunk of one tree becomes narrowed by vascular disease. The number and diameter (“extent”) of native (pre-existing) collaterals, plus their amount of lumen enlargement (growth/remodeling) in occlusive disease, show remarkably wide variation among inbred mouse strains (eg, C57BL/6 and BALB/c), resulting in large differences in tissue injury in models of occlusive disease. Evidence suggests similar large differences exist among healthy humans

Association of Utrophin and Multiple Dystrophin Short Forms with the Mammalian Mr 58,000 Dystrophin-associated Protein (Syntrophin)

Electric tissue syntrophin, originally described as an M(r) 58,000 postsynaptic protein having homologs in mammalian muscle, was previously shown to associate with dystrophin in Triton extracts of Torpedo postsynaptic membranes. It also associates with the Torpedo M(r) 87,000 postsynaptic protein (87K), the core of which is a superdomain homologous to the cysteine-rich (CR) and COOH-terminal (CT) domains of human dystrophin. Using immunoaffinity purifications from various rat tissues and immunoblotting, we find that syntrophin associates with dystrophin, utrophin (the chromosome 6-encoded dystrophin homolog formerly known as dystrophin-related protein), multiple proteins which are cross-reactive with 87K, and two subfamilies of 71K-like proteins (CRCT-containing proteins encoded by the dystrophin gene under the control of an alternative promoter in intron 62). One 71K subfamily retains the dystrophin COOH-terminal sequence; the other has an alternative COOH-terminal sequence caused by deletion of the penultimate exon by alternative splicing. The relative masses of the members of the subfamilies suggest they arise by alternative splicing at other previously described sites within CT. These results establish that syntrophin is a general ligand for the CRCT domain in mammalian dystrophin and its homologs. They also reveal a greater diversity in 71K proteins than has previously been apparent

Congenic Fine-Mapping Identifies a Major Causal Locus for Variation in the Native Collateral Circulation and Ischemic Injury in Brain and Lower Extremity

Severity of tissue injury in occlusive disease is dependent on the extent (number and diameter) of collateral vessels, which varies widely among healthy mice and humans. However, the causative genetic elements are unknown. Recently, much of the variation among different mouse strains, including C57Bl/6J (B6, high extent) and BALB/cByJ (Bc, low), was linked to a QTL on chromosome 7 (Candq1)

Yotiao, a Novel Protein of Neuromuscular Junction and Brain That Interacts with Specific Splice Variants of NMDA Receptor Subunit NR1

The molecular machinery underlying neurotransmitter receptor immobilization at postsynaptic sites is poorly understood. The NMDA receptor subunit NR1 can form clusters in heterologous cells via a mechanism dependent on the alternatively spliced C1 exon cassette in its intracellular C-terminal tail, suggesting a functional interaction between NR1 and the cytoskeleton. The yeast two-hybrid screen was used here to identify yotiao, a novel coiled coil protein that interacts with NR1 in a C1 exon-dependent manner. Yotiao mRNA (11 kb) is present modestly in brain and abundantly in skeletal muscle and pancreas. On Western blots, yotiao appears as an approximately 230 kDa band that is present in cerebral cortex, hippocampus, and cerebellum. Biochemical studies reveal that yotiao fractionates with cytoskeleton-associated proteins and with the postsynaptic density. With regard to immunohistochemistry, two anti-yotiao antibodies display a somatodendritic staining pattern similar to each other and to the staining pattern of NR1. Yotiao was colocalized by double-label immunocytochemistry with NR1 in rat brain and could be coimmunoprecipitated with NR1 from heterologous cells. Thus yotiao is an NR1-binding protein potentially involved in cytoskeletal attachment of NMDA receptors. Consistent with a general involvement in postsynaptic structure, yotiao was also found to be specifically concentrated at the neuromuscular junction in skeletal muscle

Structural Abnormalities at Neuromuscular Synapses Lacking Multiple Syntrophin Isoforms

The syntrophins are modular adapter proteins that function by recruiting signaling molecules to the cytoskeleton via their direct association with proteins of the dystrophin protein family. We investigated the physiological function of beta2-syntrophin by generating a line of mice lacking this syntrophin isoform. The beta2-syntrophin null mice show no overt phenotype, or muscular dystrophy, and form structurally normal neuromuscular junctions (NMJs). To determine whether physiological consequences caused by the lack of beta2-syntrophin were masked by compensation from the alpha-syntrophin isoform, we crossed these mice with our previously described alpha-syntrophin null mice to produce mice lacking both isoforms. The alpha/beta2-syntrophin null mice have NMJs that are structurally more aberrant than those lacking only alpha-syntrophin. The NMJs of the alpha/beta2-syntrophin null mice have fewer junctional folds than either parent strain, and the remaining folds are abnormally shaped with few openings to the synaptic space. The levels of acetylcholine receptors are reduced to 23% of wild type in mice lacking both syntrophin isoforms. Furthermore, the alpha/beta2-syntrophin null mice ran significantly shorter distances on voluntary exercise wheels despite having normal neuromuscular junction transmission as determined by micro-electrode recording of endplate potentials. We conclude that both alpha-syntrophin and beta2-syntrophin play distinct roles in forming and maintaining NMJ structure and that each syntrophin can partially compensate for the loss of the other

Evaluating the Revised American Society for Gastrointestinal Endoscopy Guidelines for Common Bile Duct Stone Diagnosis

Background/Aims The American Society for Gastrointestinal Endoscopy (ASGE) revised its guidelines for risk stratification of patients with suspected choledocholithiasis. This study aimed to assess the diagnostic performance of the revision and to compare it to the previous guidelines. Methods We conducted a retrospective cohort study of 267 patients with suspected choledocholithiasis. We identified high-risk patients according to the original and revised guidelines and examined the diagnostic accuracy of both guidelines. We measured the association between individual criteria and choledocholithiasis. Results Under the original guidelines, 165 (62%) patients met the criteria for high risk, of whom 79% had confirmed choledocholithiasis. The categorization had a sensitivity and specificity of 68% and 55%, respectively, for the detection of choledocholithiasis. Under the revised guidelines, 86 (32%) patients met the criteria for high risk, of whom 83% had choledocholithiasis. The revised categorization had a lower sensitivity and higher specificity of 37% and 80%, respectively. The positive predictive value of the high-risk categorization increased with the revision, reflecting a potential decrease in diagnostic endoscopic retrograde cholangiopancreatograpies (ERCPs). Stone visualized on imaging had the greatest specificity for choledocholithiasis. Gallstone pancreatitis was not associated with the risk for choledocholithiasis. Conclusions The 2019 revision of the ASGE guidelines decreases the utilization of ERCP as a diagnostic modality and offers an improved risk stratification tool

Interaction of Muscle and Brain Sodium Channels with Multiple Members of the Syntrophin Family of Dystrophin-Associated Proteins

Syntrophins are cytoplasmic peripheral membrane proteins of the dystrophin-associated protein complex (DAPC). Three syntrophin isoforms, alpha1, beta1, and beta2, are encoded by distinct genes. Each contains two pleckstrin homology (PH) domains, a syntrophin-unique (SU) domain, and a PDZ domain. The name PDZ comes from the first three proteins found to contain repeats of this domain (PSD-95, Drosophila discs large protein, and the zona occludens protein 1). PDZ domains in other proteins bind to the C termini of ion channels and neurotransmitter receptors containing the consensus sequence (S/T)XV-COOH and mediate the clustering or synaptic localization of these proteins. Two voltage-gated sodium channels (NaChs), SkM1 and SkM2, of skeletal and cardiac muscle, respectively, have this consensus sequence. Because NaChs are sarcolemmal components like syntrophins, we have investigated possible interactions between these proteins. NaChs copurify with syntrophin and dystrophin from extracts of skeletal and cardiac muscle. Peptides corresponding to the C-terminal 10 amino acids of SkM1 and SkM2 are sufficient to bind detergent-solubilized muscle syntrophins, to inhibit the binding of native NaChs to syntrophin PDZ domain fusion proteins, and to bind specifically to PDZ domains from alpha1-, beta1-, and beta2-syntrophin. These peptides also inhibit binding of the syntrophin PDZ domain to the PDZ domain of neuronal nitric oxide synthase, an interaction that is not mediated by C-terminal sequences. Brain NaChs, which lack the (S/T)XV consensus sequence, also copurify with syntrophin and dystrophin, an interaction that does not appear to be mediated by the PDZ domain of syntrophin. Collectively, our data suggest that syntrophins link NaChs to the actin cytoskeleton and the extracellular matrix via dystrophin and the DAPC

Genetic Dissection of the Canq1 Locus Governing Variation in Extent of the Collateral Circulation

<div><h3>Background</h3><p>Native (pre-existing) collaterals are arteriole-to-arteriole anastomoses that interconnect adjacent arterial trees and serve as endogenous bypass vessels that limit tissue injury in ischemic stroke, myocardial infarction, coronary and peripheral artery disease. Their extent (number and diameter) varies widely among mouse strains and healthy humans. We previously identified a major quantitative trait locus on chromosome 7 (<em>Canq1</em>, LOD = 29) responsible for 37% of the heritable variation in collateral extent between C57BL/6 and BALB/c mice. We sought to identify candidate genes in <em>Canq1</em> responsible for collateral variation in the cerebral pial circulation, a tissue whose strain-dependent variation is shared by similar variation in other tissues.</p> <h3>Methods and Findings</h3><p>Collateral extent was intermediate in a recombinant inbred line that splits <em>Canq1</em> between the C57BL/6 and BALB/c strains. Phenotyping and SNP-mapping of an expanded panel of twenty-one informative inbred strains narrowed the <em>Canq1</em> locus, and genome-wide linkage analysis of a SWRxSJL-F2 cross confirmed its haplotype structure. Collateral extent, infarct volume after cerebral artery occlusion, bleeding time, and re-bleeding time did not differ in knockout mice for two vascular-related genes located in <em>Canq1</em>, <em>IL4ra</em> and <em>Itgal</em>. Transcript abundance of 6 out of 116 genes within the 95% confidence interval of <em>Canq1</em> were differentially expressed >2-fold (p-value<0.05÷150) in the cortical <em>pia mater</em> from C57BL/6 and BALB/c embryos at E14.5, E16.5 and E18.5 time-points that span the period of collateral formation.</p> <h3>Conclusions</h3><p>These findings refine the <em>Canq1</em> locus and identify several genes as high-priority candidates important in specifying native collateral formation and its wide variation.</p> </div

Consensus guidelines for the use and interpretation of angiogenesis assays

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference