Vascular development is a complex but orderly process that is tightly regulated. A number of secreted factors produced by surrounding cells regulate endothelial cell (EC) differentiation, proliferation, migration and coalescence into cord-like structures. Vascular cords then undergo tubulogenesis to form vessels with a central lumen. But little is known about how tubulogenesis is regulated in vivo. Here we report the identification and characterization of a new EC-derived secreted factor, EGF-like domain 7 (Egfl7). Egfl7 is expressed at high levels in the vasculature associated with tissue proliferation, and is downregulated in most of the mature vessels in normal adult tissues. Loss of Egfl7 function in zebrafish embryos specifically blocks vascular tubulogenesis. We uncover a dynamic process during which gradual separation and proper spatial arrangement of the angioblasts allow subsequent assembly of vascular tubes. This process fails to take place in Egfl7 knockdown embryos, leading to the failure of vascular tube formation. Our study defines a regulator that controls a specific and important step in vasculogenesis.

Large collections of knockout organisms facilitate the elucidation of gene functions. Here we used retroviral insertion or homologous recombination to disrupt 472 genes encoding secreted and membrane proteins in mice, providing a resource for studying a large fraction of this important class of drug target. The knockout mice were subjected to a systematic phenotypic screen designed to uncover alterations in embryonic development, metabolism, the immune system, the nervous system and the cardiovascular system. The majority of knockout lines exhibited altered phenotypes in at least one of these therapeutic areas. To our knowledge, a comprehensive phenotypic assessment of a large number of mouse mutants generated by a gene-specific approach has not been described previously.

Amino acid sequence comparisons of human topoisomerase I (Topo I) with seven other cellular Topo I enzymes reveal that the enzyme can be divided into four major domains: the unconserved NH 2 -terminal domain (24 kDa), the conserved core domain (54 kDa), a poorly conserved linker region (5 kDa), and the highly conserved COOH-terminal domain (8 kDa), which contains the active site tyrosine. To investigate this predicted domain organization, recombinant baculoviruses were engineered to express the 91-kDa full-length enzyme, a 70-kDa NH 2 -terminally truncated enzyme that is missing the first 174 residues, and a 58-kDa NH 2 -and COOHterminally truncated core fragment encompassing residues 175-659. The specific activity of the full-length and , as well as the polycation spermidine, have been shown to stimulate activity (2-5). Phosphodiester bond energy is preserved during the nicking-closing cycle by the formation of a phosphotyrosine bond between the active-site tyrosine and the 3Ј-end of the broken strand (6 -8). This covalent intermediate can be trapped by denaturing the enzyme during catalysis with either SDS or alkali (9 -11). Sequence analyses of a large number of SDS-induced breakage sites indicated that the cellular Topo I enzymes will cleave at specific sequences (9, 12, 13), but there is only limited sequence similarity between such sites (9, 12-17). The SDS-induced cleavage at many breakage sites is enhanced by camptothecin, a plant alkaloid that inhibits the cellular enzymes by reversibly binding to the covalent Topo I-DNA intermediate in a manner that slows the the religation step of catalysis (17-22).The human Topo I is composed of 765 residues with a predicted molecular mass of 91 kDa. Sequence comparisons of cellular eukaryotic Topo I proteins demonstrate that the human Topo I can be divided into four domains (Fig. 1).2 Residues Met 1 -Lys 197 (24 kDa) comprise the unconserved NH 2 -terminal domain, which is highly charged (Asp ϩ Glu ϭ 27%; His ϩ Lys ϩ Arg ϭ 68%) and contains four putative nuclear localization signals (24). Residues Glu 198 -Ile 651 (54 kDa) form the conserved core domain, which is followed by a short positively charged linker domain of unconserved residues Asp 652 -Glu 696 (5 kDa). Finally, residues Gln 697 -Phe 765 (8 kDa) make up the highly conserved COOH-terminal domain, which contains the active site tyrosine at position 723 (25, 26).2 Previous reports have demonstrated that the NH 2 -terminal domain is sensitive to proteolyis (3) and that residues 1-230 can be removed with little if any consequence for Topo I activity (25, 27). In contrast, a 5-amino acid deletion from the COOH terminus abolishes activity. 3 We have used the baculovirus-infected insect cell system to overproduce full-length, as well as NH 2 -and COOH-terminal deletions of human topoisomerase I. The purified recombinant Topo I is by all biochemical criteria tested identical to the native enzyme purified from human cells. Furthermore, we find the activities of the full-length protein and an aminoterminally deleted...

• RASIP1 is required for stabilizing nascent patent blood vessels in both mice and zebrafish.• RASIP1 is a dynamic effector of EPAC1-RAP1 signaling that controls actin bundling and restricts junction remodeling in vitro and in vivo.Establishment and stabilization of endothelial tubes with patent lumens is vital during vertebrate development. Ras-interacting protein 1 (RASIP1) has been described as an essential regulator of de novo lumenogenesis through modulation of endothelial cell (EC) adhesion to the extracellular matrix (ECM). Here, we show that in mouse and zebrafish embryos, Rasip1-deficient vessels transition from an angioblast cord to a hollow tube, permit circulation of primitive erythrocytes, but ultimately collapse, leading to hemorrhage and embryonic lethality. Knockdown of RASIP1 does not alter EC-ECM adhesion, but causes cell-cell detachment and increases permeability of EC monolayers in vitro. We also found that endogenous RASIP1 in ECs binds Ras-related protein 1 (RAP1), but not Ras homolog gene family member A or cell division control protein 42 homolog. Using an exchange protein directly activated by cyclic adenosine monophosphate 1 (EPAC1)-RAP1-dependent model of nascent junction formation, we demonstrate that a fraction of the RASIP1 protein pool localizes to cell-cell contacts. Loss of RASIP1 phenocopies loss of RAP1 or EPAC1 in ECs by altering junctional actin organization, localization of the actin-bundling protein nonmuscle myosin heavy chain IIB, and junction remodeling. Our data show that RASIP1 regulates the integrity of newly formed blood vessels as an effector of EPAC1-RAP1 signaling. (Blood. 2013;122(22):3678-3690)

Disruption or improper activation of the Hedgehog (Hh) pathway is associated with developmental abnormalities and cancer. Although characterized in Drosophila, the mechanisms that mediate the Hh signal downstream of the Smoothened (Smo) seven-transmembrane protein in vertebrates remain poorly understood. In particular, the Fused (Fu) kinase, which mediates Hh signaling in flies, is dispensable in mammals. To identify kinases that positively regulate the Hh pathway in mammals, we screened a mouse kinome small interfering RNA library and validated nine candidates that modulate Hh signaling. Among these candidates, Nek1 and Prkra did not directly function in the Hh pathway but exerted their effects on Hh signaling indirectly through a primary role in ciliogenesis. In contrast, another kinase, Cdc2l1, directly participated in the Hh pathway. Cdc2l1 was necessary and sufficient for activation of the Hh pathway, functioning downstream of Smo and upstream of the Glioma-associated (Gli) transcription factors. More specifically, Cdc2l1 interacted with the negative regulator Suppressor of Fused (Sufu) and relieved its inhibition on Gli, thus providing a mechanism for how Cdc2l1 might play a role in Hh signaling. Finally, with zebrafish as model organism, we showed that Cdc2l1 activated the Hh pathway in vivo. We propose that Cdc2l1 is a previously unrecognized member of the Hh signal transduction cascade.

ImportanceTo date, few multisite investigations have evaluated early interventions for injured patients with posttraumatic stress disorder (PTSD) symptoms.ObjectiveTo simultaneously assess the effectiveness and implementation of a brief stepped collaborative care intervention targeting PTSD and comorbidity.Design, Setting, and ParticipantsA stepped-wedge cluster randomized clinical trial was conducted at 25 US level I trauma centers. Participants included hospitalized survivors of physical injury who underwent a 2-step evaluation for PTSD symptoms. Patients reporting high levels of distress on the PTSD Checklist (PCL-C) were randomized (N = 635) per the stepped-wedge protocol to enhanced usual care control (n = 370) or intervention (n = 265) conditions. The study was conducted from January 4, 2016, through November 2019. Data analysis was performed from November 4, 2019, to December 8, 2020.InterventionsThe Trauma Survivors Outcomes and Support collaborative care intervention included proactive injury case management that assisted patients transitioning from hospital inpatient to outpatient and community settings. The intervention also integrated evidence-based pharmacotherapy and psychotherapeutic elements targeting PTSD symptoms and comorbidity.Main Outcomes and MeasuresThe primary study outcome was PTSD symptoms assessed with the PCL-C at baseline in the surgical ward and at 3, 6, and 12 months postinjury. Secondary outcomes included depressive symptoms, alcohol use, and physical function. Subgroup analyses examined the effect of baseline risk factors for enduring PTSD and quality of protocol implementation on study outcomes. Primary statistical analyses were conducted using the intent-to-treat sample.ResultsA total of 327 men (51.5%) were included in analysis; mean (SD) age was 39.0 (14.2) years. The investigation attained follow-up of 75% to 80% of the participants at 3 to 12 months. The intervention lasted a mean (SD) of 122 (132) minutes. Mixed model regression analyses revealed statistically significant changes in PCL-C scores for intervention patients compared with control patients at 6 months (difference, −2.57; 95% CI, −5.12 to −0.03; effect size, 0.18; P < .05) but not 12 months (difference, −1.27; 95% CI, −4.26 to 1.73; effect size, 0.08; P = .35). Subgroup analyses revealed larger PTSD treatment effects for patients with 3 or more baseline risk factors for enduring PTSD and for patients, including firearm injury survivors, treated at trauma centers with good or excellent protocol implementation. Intervention effects for secondary outcomes did not attain statistical significance.Conclusions and RelevanceA brief stepped collaborative care intervention was associated with significant 6-month but not 12-month PTSD symptom reductions. Greater baseline PTSD risk and good or excellent trauma center protocol implementation were associated with larger PTSD treatment effects. Orchestrated efforts targeting policy and funding should systematically incorporate the study findings into national trauma center requirements and verification criteria.Trial RegistrationClinicalTrials.gov Identifier: NCT02655354

c High-throughput transcriptomics was used to identify Fibroporia radiculosa genes that were differentially regulated during colonization of wood treated with a copper-based preservative. The transcriptome was profiled at two time points while the fungus was growing on wood treated with micronized copper quat (MCQ). A total of 917 transcripts were differentially expressed. Fiftyeight of these genes were more highly expressed when the MCQ was protecting the wood from strength loss and had putative functions related to oxalate production/degradation, laccase activity, quinone biosynthesis, pectin degradation, ATP production, cytochrome P450 activity, signal transduction, and transcriptional regulation. Sixty-one genes were more highly expressed when the MCQ lost its effectiveness (>50% strength loss) and had functions related to oxalate degradation; cytochrome P450 activity; H 2 O 2 production and degradation; degradation of cellulose, hemicellulose, and pectin; hexose transport; membrane glycerophospholipid metabolism; and cell wall chemistry. Ten of these differentially regulated genes were quantified by reverse transcriptase PCR for a more in-depth study (4 time points on wood with or without MCQ treatment). Our results showed that MCQ induced higher than normal levels of expression for four genes (putative annotations for isocitrate lyase, glyoxylate dehydrogenase, laccase, and oxalate decarboxylase 1), while four other genes (putative annotations for oxalate decarboxylase 2, aryl alcohol oxidase, glycoside hydrolase 5, and glycoside hydrolase 10) were repressed. The significance of these results is that we have identified several genes that appear to be coregulated, with putative functions related to copper tolerance and/or wood decay.

Orchestrated investigative and policy efforts should continue to evaluate patient-centered care transition interventions to inform American College of Surgeons' clinical guidelines for U.S. trauma care systems.