不実の企業情報開示と民事責任 : 日米比較研究に基づく考察

学位の種別: 課程博士審査委員会委員 : （主査）東京大学教授 神作 裕之, 東京大学教授 沖野 眞巳, 東京大学教授 藤田 友敬, 東京大学教授 中川 淳司, 東京大学教授 山川 隆一University of Tokyo(東京大学

mTORC1 controls lysosomal Ca²⁺ release through the two-pore channel TPC2

The ion channel TPC2 is required for Ca 2+ mobilization from lysosomes in response to mTORC1 inhibition and to NAADP. </jats:p

Chromogranin B (CHGB) is dimorphic and responsible for dominant anion channels delivered to cell surface via regulated secretion

Regulated secretion is conserved in all eukaryotes. In vertebrates granin family proteins function in all key steps of regulated secretion. Phase separation and amyloid-based storage of proteins and small molecules in secretory granules require ion homeostasis to maintain their steady states, and thus need ion conductances in granule membranes. But granular ion channels are still elusive. Here we show that granule exocytosis in neuroendocrine cells delivers to cell surface dominant anion channels, to which chromogranin B (CHGB) is critical. Biochemical fractionation shows that native CHGB distributes nearly equally in soluble and membrane-bound forms, and both reconstitute highly selective anion channels in membrane. Confocal imaging resolves granular membrane components including proton pumps and CHGB in puncta on the cell surface after stimulated exocytosis. High pressure freezing immuno-EM reveals a major fraction of CHGB at granule membranes in rat pancreatic β-cells. A cryo-EM structure of bCHGB dimer of a nominal 3.5 Å resolution delineates a central pore with end openings, physically sufficient for membrane-spanning and large single channel conductance. Together our data support that CHGB-containing (CHGB+) channels are characteristic of regulated secretion, and function in granule ion homeostasis near the plasma membrane or possibly in other intracellular processes

Risk factors associated with amyotrophic lateral sclerosis based on the observational study: a systematic review and meta-analysis

ObjectiveAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder affecting the upper and lower motor neurons. Though the pathogenesis of ALS is still unclear, exploring the associations between risk factors and ALS can provide reliable evidence to find the pathogenesis. This meta-analysis aims to synthesize all related risk factors of ALS to understand this disease comprehensively.MethodsWe searched the following databases: PubMed, EMBASE, Cochrane library, Web of Science, and Scopus. Moreover, observational studies, including cohort studies, and case-control studies, were included in this meta-analysis.ResultsA total of 36 eligible observational studies were included, and 10 of them were cohort studies and the rest were case-control studies. We found six factors exacerbated the progression of disease: head trauma (OR = 1.26, 95% CI = 1.13, 1.40), physical activity (OR = 1.06, 95% CI = 1.04, 1.09), electric shock (OR = 2.72, 95% CI = 1.62, 4.56), military service (OR = 1.34, 95% CI = 1.11, 1.61), pesticides (OR = 1.96, 95% CI = 1.7, 2.26), and lead exposure (OR = 2.31, 95% CI = 1.44, 3.71). Of note, type 2 diabetes mellitus was a protective factor for ALS. However, cerebrovascular disease (OR = 0.99, 95% CI = 0.75, 1.29), agriculture (OR = 1.22, 95% CI = 0.74, 1.99), industry (OR = 1.24, 95% CI = 0.81, 1.91), service (OR = 0.47, 95% CI = 0.19, 1.17), smoking (OR = 1.25, 95% CI = 0.5, 3.09), chemicals (OR = 2.45, 95% CI = 0.89, 6.77), and heavy metal (OR = 1.5, 95% CI = 0.47, 4.84) were not risk factors for ALS based on meta-analyses.ConclusionsHead trauma, physical activity, electric shock, military service, pesticides, and lead were risk factors for ALS onset and progression. But DM was a protective factor. This finding provides a better understanding of ALS risk factors with strong evidence for clinicians to rationalize clinical intervention strategies.INPLSY registration numberhttps://inplasy.com/inplasy-2022-9-0118/, INPLASY202290118

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Fluorescence imaging for nanoscale clustering and lateral diffusion of receptor for advanced glycation endproducts

Receptors on the cell membrane initiate cell signaling upon stimulation with extracellular molecules; this results in signal transduction pathways. Receptors are redistributed as clusters upon extracellular stimuli with the change of receptor dynamics. The lateral diffusion of receptors plays a role in how receptors interface with other membrane proteins, extracellular ligands, intracellular proteins, and how receptors function. In this dissertation, the lateral diffusion and clusters of receptors for advanced glycation end-products (RAGE) at both a single molecule and the ensemble level are discussed. The effects of extracellular ligands, cholesterol levels, and actin polymerization on RAGE diffusion are considered. RAGE is a member of the immunoglobulin superfamily of membrane proteins that are involved in numerous pathological conditions. Ligand binding to the extracellular domain of RAGE drives RAGE clusters and initiates the downstream signal transduction. The signal transduction also consists of the interaction between diaphanous-1 (Diaph1) with the cytoplasmic tail of RAGE. Diaph1 affects the nanoscale clustering and diffusion of RAGE as measured by super-resolution stochastic optical reconstruction microscopy (STORM) and single particle tracking (SPT). A reduced expression level of Diaph1 or a disrupted interaction between Diaph1 and RAGE results in a decreased size and number of RAGE clusters. RAGE diffusion is increased after reduced Diaph1 expression. In contrast, when the interaction site between RAGE and Diaph1 is mutated, RAGE diffusion is slowed. The effect of the ligand on the lateral diffusion of the receptor for advanced glycation endproducts involved in numerous pathological conditions. A methylglyoxal-modified bovine serum albumin (MGO-BSA) RAGE ligand is prepared and characterized. The effect of MGO-BSA on the lateral diffusion of RAGE is measured by SPT. Ligand incubation affects RAGE diffusion and the phosphorylation of extracellular signal-regulated kinases. However, there is no correlation between MGO-BSA ligand binding affinity and the change in RAGE diffusion. Moreover, the mechanism for the ligand-induced change in RAGE diffusion is dependent on cholesterol. The actin cytoskeleton plays a crucial role in RAGE functions. The effect of the actin cytoskeleton on RAGE diffusion is measured by fluorescence recovery after photobleaching at the ensemble-level. When depolymerization of the actin cytoskeleton is inhibited, RAGE diffusion and mobile fraction are decreased. Also, when the actin cytoskeleton is disrupted, the phosphorylation of extracellular signal-regulated kinase is reduced.</p

Diaphanous-1 affects the nanoscale clustering and lateral diffusion of receptor for advanced glycation endproducts (RAGE)

The interactions between the cytoplasmic protein diaphanous-1 (Diaph1) and the receptor for advanced glycation endproducts (RAGE) drive the negative consequences of RAGE signaling in several disease processes. Reported in this work is how Diaph1 affects the nanoscale clustering and diffusion of RAGE measured using super-resolution stochastic optical reconstruction microscopy (STORM) and single particle tracking (SPT). Altering the Diaph1 binding site has a different impact on RAGE diffusion compared to when Diaph1 expression is reduced in HEK293 cells. In cells with reduced Diaph1 expression (RAGE-Diaph1−/−), the average RAGE diffusion coefficient is increased by 35%. RAGE diffusion is known to be influenced by the dynamics of the actin cytoskeleton. Actin labeling shows that a reduced Diaph1 expression leads to cells with reduced filopodia density and length. In contrast, when two RAGE amino acids that interact with Diaph1 are mutated (RAGERQ/AA), the average RAGE diffusion coefficient is decreased by 16%. Since RAGE diffusion is slowed when the interaction between Diaph1 and RAGE is disrupted, the interaction of the two proteins results in faster RAGE diffusion. In both RAGERQ/AA and RAGE-Diaph1−/− cells the number and size of RAGE clusters are decreased compared to cells expressing RAGE and native concentrations of Diaph1. This work shows that Diaph1 has a role in affecting RAGE clusters and diffusion.This is a manuscript of an article published as Zhu, Qiaochu, and Emily A. Smith. "Diaphanous-1 affects the nanoscale clustering and lateral diffusion of receptor for advanced glycation endproducts (RAGE)." Biochimica et Biophysica Acta (BBA)-Biomembranes 1861, no. 1 (2019): 43-49. DOI: 10.1016/j.bbamem.2018.10.015. Posted with permission.</p