各国の海難調査機関に関する比較研究

東京海洋大学修士学位論文 2020年度(2020年3月) 海運ロジスティクス 修士 第3405号指導教員: 竹本孝弘全文公表年月日: 2020-11-18東京海洋大学202

Proton-coupled sugar transport in the prototypical major facilitator superfamily protein XylE.

The major facilitator superfamily (MFS) is the largest collection of structurally related membrane proteins that transport a wide array of substrates. The proton-coupled sugar transporter XylE is the first member of the MFS that has been structurally characterized in multiple transporting conformations, including both the outward and inward-facing states. Here we report the crystal structure of XylE in a new inward-facing open conformation, allowing us to visualize the rocker-switch movement of the N-domain against the C-domain during the transport cycle. Using molecular dynamics simulation, and functional transport assays, we describe the movement of XylE that facilitates sugar translocation across a lipid membrane and identify the likely candidate proton-coupling residues as the conserved Asp27 and Arg133. This study addresses the structural basis for proton-coupled substrate transport and release mechanism for the sugar porter family of proteins

Grounding Language Model with Chunking-Free In-Context Retrieval

This paper presents a novel Chunking-Free In-Context (CFIC) retrieval approach, specifically tailored for Retrieval-Augmented Generation (RAG) systems. Traditional RAG systems often struggle with grounding responses using precise evidence text due to the challenges of processing lengthy documents and filtering out irrelevant content. Commonly employed solutions, such as document chunking and adapting language models to handle longer contexts, have their limitations. These methods either disrupt the semantic coherence of the text or fail to effectively address the issues of noise and inaccuracy in evidence retrieval. CFIC addresses these challenges by circumventing the conventional chunking process. It utilizes the encoded hidden states of documents for in-context retrieval, employing auto-aggressive decoding to accurately identify the specific evidence text required for user queries, eliminating the need for chunking. CFIC is further enhanced by incorporating two decoding strategies, namely Constrained Sentence Prefix Decoding and Skip Decoding. These strategies not only improve the efficiency of the retrieval process but also ensure that the fidelity of the generated grounding text evidence is maintained. Our evaluations of CFIC on a range of open QA datasets demonstrate its superiority in retrieving relevant and accurate evidence, offering a significant improvement over traditional methods. By doing away with the need for document chunking, CFIC presents a more streamlined, effective, and efficient retrieval solution, making it a valuable advancement in the field of RAG systems

A substrate-induced gating mechanism is conserved among Gram-positive IgA1 metalloproteases

The mucosal adaptive immune response is dependent on the production of IgA antibodies and particularly IgA1, yet opportunistic bacteria have evolved mechanisms to specifically block this response by producing IgA1 proteases (IgA1Ps). Our lab was the first to describe the structures of a metal-dependent IgA1P (metallo-IgA1P) produced from Gram-positive Streptococcus pneumoniae both in the absence and presence of its IgA1 substrate through cryo-EM single particle reconstructions. This prior study revealed an active-site gating mechanism reliant on substrate-induced conformational changes to the enzyme that begged the question of whether such a mechanism is conserved among the wider Gram-positive metallo-IgA1P subfamily of virulence factors. Here, we used cryo-EM to characterize the metallo-IgA1P of a more distantly related family member from Gemella haemolysans, an emerging opportunistic pathogen implicated in meningitis, endocarditis, and more recently bacteremia in the elderly. While the substrate-free structures of these two metallo-IgA1Ps exhibit differences in the relative starting positions of the domain responsible for gating substrate, the enzymes have similar domain orientations when bound to IgA1. Together with biochemical studies that indicate these metallo-IgA1Ps have similar binding affinities and activities, these data indicate that metallo-IgA1P binding requires the specific IgA1 substrate to open the enzymes for access to their active site and thus, largely conform to an "induced fit" model.We thank the CU Cryo-EM Structural Biology Shared Resource Facility for screening. Data collection for single particular reconstructions were collected at the Pacific Northwester Cryo-EM Center (PNCC) at Oregon Health and Science University (OHSU), supported by NIH grant U24GM129547 and accessed through EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research. H.Z. was supported by NIH R01 GM126626. E.Z.E. was supported by NIH R21 AI146295 and R01 GM139892.S

Effect of different binders on the electrochemical performance of metal oxide anode for lithium-ion batteries

When testing the electrochemical performance of metal oxide anode for lithium-ion batteries (LIBs), binder played important role on the electrochemical performance. Which binder was more suitable for preparing transition metal oxides anodes of LIBs has not been systematically researched. Herein, five different binders such as polyvinylidene fluoride (PVDF) HSV900, PVDF 301F, PVDF Solvay5130, the mixture of styrene butadiene rubber and sodium carboxymethyl cellulose (SBR+CMC), and polyacrylonitrile (LA133) were studied to make anode electrodes (compared to the full battery). The electrochemical tests show that using SBR+CMC and LA133 binder which use water as solution were significantly better than PVDF. The SBR+CMC binder remarkably improve the bonding capacity, cycle stability, and rate performance of battery anode, and the capacity retention was about 87% after 50th cycle relative to the second cycle. SBR+CMC binder was more suitable for making transition metal oxides anodes of LIBs

Mechanistic Insights Revealed by YbtPQ in the Occluded State

Recently, several ATP-binding cassette (ABC) importers have been found to adopt the typical fold of type IV ABC exporters. Presumably, these importers would function under the transport scheme of “alternating access” like those exporters, cycling through inward-open, occluded, and outward-open conformations. Understanding how the exporter-like importers move substrates in the opposite direction requires structural studies on all the major conformations. To shed light on this, here we report the structure of yersiniabactin importer YbtPQ from uropathogenic Escherichia coli in the occluded conformation trapped by ADP-vanadate (ADP-Vi) at a 3.1 Å resolution determined by cryo-electron microscopy. The structure shows unusual local rearrangements in multiple helices and loops in its transmembrane domains (TMDs). In addition, the dimerization of the nucleotide-binding domains (NBDs) promoted by the vanadate trapping is highlighted by the “screwdriver” action at one of the two hinge points. These structural observations are rare and thus provide valuable information to understand the structural plasticity of the exporter-like ABC importers

Crystal structure of a nitrate/nitrite exchanger.

Mineral nitrogen in nature is often found in the form of nitrate (NO3(-)). Numerous microorganisms evolved to assimilate nitrate and use it as a major source of mineral nitrogen uptake. Nitrate, which is central in nitrogen metabolism, is first reduced to nitrite (NO2(-)) through a two-electron reduction reaction. The accumulation of cellular nitrite can be harmful because nitrite can be reduced to the cytotoxic nitric oxide. Instead, nitrite is rapidly removed from the cell by channels and transporters, or reduced to ammonium or dinitrogen through the action of assimilatory enzymes. Despite decades of effort no structure is currently available for any nitrate transport protein and the mechanism by which nitrate is transported remains largely unknown. Here we report the structure of a bacterial nitrate/nitrite transport protein, NarK, from Escherichia coli, with and without substrate. The structures reveal a positively charged substrate-translocation pathway lacking protonatable residues, suggesting that NarK functions as a nitrate/nitrite exchanger and that protons are unlikely to be co-transported. Conserved arginine residues comprise the substrate-binding pocket, which is formed by association of helices from the two halves of NarK. Key residues that are important for substrate recognition and transport are identified and related to extensive mutagenesis and functional studies. We propose that NarK exchanges nitrate for nitrite by a rocker switch mechanism facilitated by inter-domain hydrogen bond networks