Abstract-Vascular endothelial growth factor (VEGF) induces angiogenesis by stimulating endothelial cell proliferation and migration, primarily through the receptor tyrosine kinase VEGF receptor2 (Flk1/KDR). Reactive oxygen species (ROS) derived from NAD(P)H oxidase are critically important in many aspects of vascular cell regulation, and both the small GTPase Rac1 and gp91 phox are critical components of the endothelial NAD(P)H oxidase complex. A role of NAD(P)H oxidase in VEGF-induced angiogenesis, however, has not been defined. In the present study, electron spin resonance spectroscopy is utilized to demonstrate that VEGF stimulates O 2 ·Ϫ production, which is inhibited by the NAD(P)H oxidase inhibitor, diphenylene iodonium, as well as by overexpression of dominant-negative Rac1 (N17Rac1) and transfection of gp91 phox antisense oligonucleotides in human umbilical vein endothelial cells (ECs). Antioxidants, including N-acetylcysteine (NAC), various NAD(P)H oxidase inhibitors, and N17Rac1 significantly attenuate not only VEGF-induced KDR tyrosine phosphorylation but also proliferation and migration of ECs. Importantly, these effects of VEGF are dramatically inhibited in cells transfected with gp91 phox antisense oligonucleotides. By contrast, ROS are not involved in mediating these effects of sphingosine 1-phosphate (S1P) on ECs. Sponge implant assays demonstrate that VEGF-, but not S1P-, induced angiogenesis is significantly reduced in wild-type mice treated with NAC and in gp91 phoxϪ/Ϫ mice, suggesting that ROS derived from gp91 phox -containing NAD(P)H oxidase play an important role in angiogenesis in vivo. These studies indicate that VEGF-induced endothelial cell signaling and angiogenesis is tightly controlled by the reduction/oxidation environment at the level of VEGF receptor and provide novel insights into the NAD(P)H oxidase as a potential therapeutic target for angiogenesis-dependent diseases.

To determine whether reduced striatal D2 receptor binding reported in patients with idiopathic torsion dystonia is associated with the genotype, the authors used PET and [11C]-raclopride to assess non-manifesting carriers of the DYT1 mutation. D2 receptor binding was reduced by approximately 15% in caudate and putamen (p < 0.005). These results suggest that striatal D2 binding reductions are a trait feature of the DYT1 genotype.

The striatum and the subthalamic nucleus are the main entry points for cortical information to the basal ganglia. Parkinson's disease affects not only the function, but also the morphological integrity of some of these inputs and their synaptic targets in the basal ganglia. Significant morphological changes in the cortico-striatal system have already been recognized in patients with Parkinson's disease and in animal models of the disease. To find out whether the primate cortico-subthalamic system is also subject to functionally relevant morphological alterations in parkinsonism, we used a combination of light and electron microscopy anatomical approaches and in vivo electrophysiological methods in monkeys rendered parkinsonian following chronic exposure to low doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). At the light microscopic level, the density of vesicular glutamate transporter 1-positive (i.e. cortico-subthalamic) profiles in the dorsolateral part of the subthalamic nucleus (i.e. its sensorimotor territory) was 26.1% lower in MPTP-treated parkinsonian monkeys than in controls. These results were confirmed by electron microscopy studies showing that the number of vesicular glutamate transporter 1-positive terminals and of axon terminals forming asymmetric synapses in the dorsolateral subthalamic nucleus was reduced by 55.1% and 27.9%, respectively, compared with controls. These anatomical findings were in line with in vivo electrophysiology data showing a 60% reduction in the proportion of pallidal neurons that responded to electrical stimulation of the cortico-subthalamic system in parkinsonian monkeys. These findings provide strong evidence for a partial loss of the hyperdirect cortico-subthalamic projection in MPTP-treated parkinsonian monkeys.

a b s t r a c tSince its outbreak in December 2019, a series of clinical trials on Coronavirus Disease 2019 have been registered or carried out. However, the significant heterogeneity and less critical outcomes of such trials may be leading to a waste of research resources. This study aimed to develop a core outcome set (COS) for clinical trials on COVID-19 in order to tackle the outcome issues. The study was conducted according to the Core Outcome Measures in Effectiveness Trials (COMET) handbook (version 1.0), a guideline for COS development. A research group was set up that included experts in respiratory and critical medicine, traditional Chinese medicine, evidence-based medicine, clinical pharmacology, and statistics, in addition to medical journal editors. Clinical trial registry websites (chictr.org.cn and clinicaltrials.gov) were searched to retrieve clinical trial protocols and outcomes in order to form an outcome pool. A total of 78 clinical trial protocols on COVID-19 were included and 259 outcomes were collected. After standardization, 132 outcomes were identified within seven different categories, of which 58 were selected to develop a preliminary outcome list for further consensus. After two rounds of Delphi survey and one consensus meeting, the most important outcomes for the different clinical classifications of COVID-19 were identified and determined to constitute the COS for clinical trials on COVID-19 (COS-COVID). The COS-COVID includes one outcome for the mild type (time to 2019-nCoV reverse transcriptionpolymerase chain reaction (RT-PCR) negativity), four outcomes for the ordinary type (length of hospital

Fractionation of bean (Phaseolus vulgaris L.) cotyledon storage proteins using an improved separation procedure revealed a protein profile in which globulin‐I was the major fraction (about 40% of the total cotyledon protein), followed in importance by an alkali‐soluble fraction (about 25%) containing glutelin and albumin, free amino acids, ginbulin‐2, prolamine, and residue fractions. SDS‐acrylamideg el electrophoresis was performed to determine banding patterns of total cotyledon polypeptides and the purity of the fractions. Amino acid analyses using an autoanalyzer were performed on total protein and protein fractions. The alkali‐soluble fraction had the highest concentration of methionine (2% of the protein by weight), while the methionine percentage of globulin‐1 (0.88%) and albumin (1.0%) was less than that of the respective cotyledons (1.10%). The alkali‐soluble and globulin‐1 fractions comprised three‐fourths of the total cotyledon methionine. Total cotyledon protein was positively correlated with globulin‐1, free amino acids, alkali‐soluble, and globulin‐2 fractions. Significant positive correlations between amount of methionine in the cotyledons and that in the alkalisoluble fraction were also found. This finding suggests that selection for high protein genotypes in which the alkali‐soluble fraction is increased should result in improved methionine content. Since quantitative estimation of this fracion is time‐consuminga, simplified procedure for determining the amount of protein insoluble in ascorbate‐NaCl solution is suitable for identifying strains with large amountos f the alkali‐soluble fraction.

Objective: To determine whether changes in D 2 receptor availability are present in carriers of genetic mutations for primary dystonia. Methods:Manifesting and nonmanifesting carriers of the DYT1 and DYT6 dystonia mutations were scanned with [11 C] raclopride (RAC) and PET. Measures of D 2 receptor availability in the caudate nucleus and putamen were determined using an automated region-of-interest approach.Values from mutation carriers and healthy controls were compared using analysis of variance to assess the effects of genotype and phenotype. Additionally, voxel-based whole brain searches were conducted to detect group differences in extrastriatal regions.Results: Significant reductions in caudate and putamen D 2 receptor availability were evident in both groups of mutation carriers relative to healthy controls (p Ͻ 0.001). The changes were greater in DYT6 relative to DYT1 carriers (Ϫ38.0 Ϯ 3.0% vs Ϫ15.0 Ϯ 3.0%, p Ͻ 0.001). By contrast, there was no significant difference between manifesting and nonmanifesting carriers of either genotype. Voxel-based analysis confirmed these findings and additionally revealed reduced RAC binding in the ventrolateral thalamus of both groups of mutation carriers. As in the striatum, the thalamic binding reductions were more pronounced in DYT6 carriers and were not influenced by the presence of clinical manifestations. Conclusions:Reduced D 2 receptor availability in carriers of dystonia genes is compatible with dysfunction or loss of D 2 -bearing neurons, increased synaptic dopamine levels, or both. These changes, which may be present to different degrees in the DYT1 and DYT6 genotypes, are likely to represent susceptibility factors for the development of clinical manifestations in mutation carriers. Neurology GLOSSARY ANOVA ϭ analysis of variance; BFM ϭ Burke-Fahn-Marsden; CN ϭ caudate nucleus; FWE ϭ family-wise error rate; GPe ϭ external pallidum; GPi ϭ interal pallidum; RAC ϭ raclopride; ROI ϭ region of interest; SOR ϭ striato-occipital ratio; THX ϭ trihexyphenidyl; VL ϭ ventrolateral tier nuclei.Multiple lines of evidence support the role of altered striatal DA neurotransmission in primary dystonia. 1,2 Nonetheless, imaging studies have revealed only minimal reductions in striatal D 2 receptor binding in patients with idiopathic dystonia 3,4 and in nonmanifesting carriers of the DYT1 dystonia mutation.5 Indeed, similar reductions have been described in DYT1 dystonia patients at postmortem.1 It is not known whether similar abnormalities are also present in primary dystonia associated with other genetic mutations.Experimental models of dystonia have yielded varied results with regard to the striatal DA dynamics. Increased striatal DA turnover was found in a transgenic murine DYT1 model regardless of behavioral phenotype. 6,7 However, in this model, striatal DA levels were reduced

Key points• Acid extrusion from ventricular myocytes typically occurs via Na + /H + exchange (NHE1) and Na + -HCO 3 − co-transporters (NBC). This maintains intracellular pH at ∼7.2: The membrane distribution of these transporters is uncertain.• Immunofluorescence indicates that: NBC isoforms are located in lateral sarcolemma, intercalated discs and transverse tubules, whereas NHE1 is densely expressed at intercalated discs.• Functional experiments with detubulated myocytes indicate reduced acid extrusion on NBC but no effect on NHE1 activity, confirming exclusion of NHE1 function from transverse tubules.• Stimulating NHE1 activity induces sub-sarcolemmal [H + ] i depletion (forming local pH i microdomains), particularly at intercalated discs, while stimulating NBC activity induces no pH i microdomains.• Our results provide the first demonstration that pH i in ventricular myocytes is locally controlled through selective trafficking of membrane ion transporters. NHE1 preferentially controls pH i at intercalated discs, where cell-to-cell gap-junctional channels are located, while NBC influences pH i adjacent to transverse tubules, where key proteins for excitation-contraction coupling are located.Abstract Membrane acid extrusion by Na + /H + exchange (NHE1) and Na + -HCO 3 − co-transport (NBC) is essential for maintaining a low cytoplasmic [H + ] (∼60 nM, equivalent to an intracellular pH (pH i ) of 7.2). This protects myocardial function from the high chemical reactivity of H + ions, universal end-products of metabolism. We show here that, in rat ventricular myocytes, fluorescent antibodies map the NBC isoforms NBCe1 and NBCn1 to lateral sarcolemma, intercalated discs and transverse tubules (t-tubules), while NHE1 is absent from t-tubules. This unexpected difference matches functional measurements of pH i regulation (using AM-loaded SNARF-1, a pH fluorophore). Thus, myocyte detubulation (by transient exposure to 1.5 M formamide) reduces global acid extrusion on NBC by 40%, without affecting NHE1. Similarly, confocal pH i imaging reveals that NBC stimulation induces spatially uniform pH i recovery from acidosis, whereas NHE1 stimulation induces pH i non-uniformity during recovery (of ∼0.1 units, for 2-3 min), particularly at the ends of the cell where intercalated discs are commonly located, and where NHE1 immunostaining is prominent. Mathematical modelling shows that this induction of local pH i microdomains is favoured by low cytoplasmic H + mobility and long H + diffusion distances, particularly to surface NHE1 transporters mediating high membrane flux. Our results provide the first evidence for a spatial localisation of [H + ] i regulation in ventricular myocytes,

Angiotensin II (Ang II) induces transactivation of the epidermal growth factor (EGF) receptor (EGF-R), which serves as a scaffold for various signaling molecules in vascular smooth muscle cells (VSMCs). Cholesterol and sphingomyelin-enriched lipid rafts are plasma membrane microdomains that concentrate various signaling molecules. Caveolae are specialized lipid rafts that are organized by the cholesterol-binding protein, caveolin, and have been shown to be associated with EGF-Rs. Angiotensin II stimulation promotes a rapid movement of AT 1 receptors to caveolae; however, their functional role in angiotensin II signaling has not been elucidated. Here we show that cholesterol depletion by ␤-cyclodextrin disrupts caveolae structure and concomitantly inhibits tyrosine phosphorylation of the EGF-R and subsequent activation of protein kinase B (PKB)/Akt induced by angiotensin II. Similar inhibitory effects were obtained with other cholesterol-binding agents, filipin and nystatin. In contrast, EGF-R autophosphorylation and activation of Akt/PKB in response to EGF are not affected by cholesterol depletion. The early Ang II-induced upstream signaling events responsible for transactivation of the EGF-R, such as the intracellular Ca 2؉ increase and c-Src activation, also remain intact. The EGF-R initially binds caveolin, but these two proteins rapidly dissociate following angiotensin II stimulation during the time when EGF-R transactivation is observed. The activated EGF-R is localized in focal adhesions together with tyrosinephosphorylated caveolin. These findings suggest that 1) a scaffolding role of caveolin is essential for EGF-R transactivation by angiotensin II and 2) cholesterol-rich microdomains as well as focal adhesions are important signal-organizing compartments required for the spatial and temporal organization of angiotensin II signaling in VSMCs.Angiotensin II (Ang II) 1 is a highly pluripotential hormone in vascular smooth muscle cells (VSMCs) and stimulates multiple signaling pathways, including Src family kinases, as well as mitogen-activated protein kinases (MAPKs) and Akt/protein kinase B (PKB), that mediate VSMC hypertrophy and growth via AT 1 receptors (AT 1 Rs). Increasing evidence suggests that transmodulation of the epidermal growth factor receptor (EGF-R), which serves as a scaffold for various signaling molecules, plays an essential role in organizing Ang II-mediated tyrosine kinase signaling pathways. We and others (1, 2) have demonstrated that EGF-R transactivation by Ang II is mediated through Ca 2ϩ , c-Src, and NADPH oxidase-derived reactive oxygen species, leading to activation of downstream signaling such as extracellular signal regulated kinase (ERK) and Akt/ PKB in VSMCs.Relatively little is known of the mechanisms controlling the spatial and temporal organization of AT 1 R signaling in VSMCs or of how specificity is achieved. We showed originally that Ang II signaling in VSMC is biphasic and that internalization or sequestration of the agonist-occupied receptor into a "signaling domain" ...