Intronic microRNAs have been proposed to complicate the design and interpretation of mouse knockout studies. The endothelialexpressed Egfl7/miR-126 locus contains miR-126 within Egfl7 intron 7, and angiogenesis deficits have been previously ascribed to Egfl7 gene-trap and lacZ knock-in mice. Surprisingly, selectively floxed Egfl7 ⌬ and miR-126 ⌬ alleles revealed that Egfl7 ⌬/⌬ mice were phenotypically normal, whereas miR-126 ⌬/⌬ mice bearing a 289-nt microdeletion recapitulated previously described Egfl7 embryonic and postnatal retinal vascular phenotypes. Regulation of angiogenesis by miR-126 was confirmed by endothelial-specific deletion and in the adult cornea micropocket assay. Furthermore, miR-126 deletion inhibited VEGF-dependent Akt and Erk signaling by derepression of the p85β subunit of PI3 kinase and of Spred1, respectively. These studies demonstrate the regulation of angiogenesis by an endothelial miRNA, attribute previously described Egfl7 vascular phenotypes to miR-126, and document inadvertent miRNA dysregulation as a complication of mouse knockout strategies.KEY WORDS: Angiogenesis, miRNA, miR-126 (Mirn126), Egfl7, p85β (Pik3r2)

Background & Aims In most patients with Parkinson's disease, gastrointestinal (GI) dysfunctions, such as gastroparesis and constipation, are prodromal to the cardinal motor symptoms of the disease. Sporadic Parkinson's disease has been proposed to develop following ingestion of neurotoxicants that affect the brain–gut axis via the vagus nerve, and then travel to higher centers compromising the substantia nigra pars compacta (SNpc), and, later, the cerebral cortex. We aimed to identify the pathway that connects the brainstem vagal nuclei and the SNpc, and to determine whether this pathway is compromised in a rat model of Parkinsonism. Methods To study this neural pathway in rats, we placed tracers in the dorsal vagal complex (DVC) or SNpc; brainstem and midbrain were examined for tracer distribution and neuronal neurochemical phenotype. Rats were given injections of paraquat once weekly for 3 weeks to induce features of Parkinsonism, or vehicle (control). Gastric tone and motility were recorded following NMDA microinjection in the SNpc and/or optogenetic stimulation of nigro-vagal terminals in the DVC. Results Stimulation of the SNpc increased gastric tone and motility via activation of dopamine 1 receptors in the DVC. In the paraquat-induced model of Parkinsonism, this nigro-vagal pathway was compromised during the early stages of motor deficit development. Conclusions We identified and characterized a nigro-vagal monosynaptic pathway in rats that controls gastric tone and motility. This pathway might be involved in the prodromal gastric dysmotility observed in patients with early-stage Parkinson's disease.

Purpose: Plasmalemmal vesicle associated protein-1 (PV-1) is up-regulated in the endothelium of human glioblastoma. We sought to further characterize the expression pattern of PV-1 in human brain tumors and interrogate its role in brain tumor angiogenesis. Experimental Design: Quantitative reverse transcription-PCR and in situ hybridization were used to measure PV-1 expression in a panel of 46 human brain tumors and related pathologic states. Matrigel tubulogenesis assays and cell migration assays were used to show function of PV-1 in primary human endothelial cells (HMVEC) under gene knockdown conditions. Results: PV-1is selectively up-regulated in a variety of high-grade human brain tumors, including glioblastoma and metastatic carcinoma, as well as other cerebral disorders associated with bloodbrain barrier disruption, such as acute ischemia. Expression levels were reduced in low-grade neoplasia; however, tumors associated with the ependyma and choroid plexus, known sites of PV-1 expression, also exhibited robust expression. Cerebral expression of PV-1 mRNA was confined to endothelial cells in all cases. PV-1 expression was induced in HMVEC cells in vitro by exposure to medium conditioned by U87MG and U251MG human brain tumor cell lines and by medium supplemented with exogenous vascular endothelial growth factor or scatter factor/hepatocyte growth factor. RNA interference^mediated inhibition of PV-1 induction in HMVEC cells blocked Matrigel-induced tubulogenesis and inhibited cell migration induced by conditioned medium or angiogenic growth factors. Conclusions: Our results confirm that PV-1 is preferentially induced in the endothelium of highgrade human brain tumors. Inhibition of PV-1 expression is associated with failure of endothelial differentiation in vitro. PV-1 represents a novel marker of brain tumor angiogenesis and integrity of the blood-brain barrier and is a potential therapeutic target.

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As CRISPR-based therapies enter the clinic, evaluation of safety remains a critical and active area of study. Here, we employ a clinical next generation sequencing (NGS) workflow to achieve high sequencing depth and detect ultra-low frequency variants across exons of genes associated with cancer, all exons, and genome wide. In three separate primary human hematopoietic stem and progenitor cell (HSPC) donors assessed in technical triplicates, we electroporated high-fidelity Cas9 protein targeted to three loci (AAVS1, HBB, and ZFPM2) and harvested genomic DNA at days 4 and 10. Our results demonstrate that clinically relevant delivery of high-fidelity Cas9 to primary HSPCs and ex vivo culture up to 10 days does not introduce or enrich for tumorigenic variants and that even a single SNP in a gRNA spacer sequence is sufficient to eliminate Cas9 off-target activity in primary, repair-competent human HSPCs.