Biology subjects

Yamashita, A.

Publications and source records attributed to Yamashita, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Computational Mechanisms of Neuroimaging Biomarkers Uncovered by Multicenter Resting-State fMRI Connectivity Variation Profile

Resting-state functional connectivity (rsFC) is increasingly used to develop biomarkers for psychiatric disorders. Despite progress, development of the reliable and practical FC biomarker remains an unmet goal, particularly one that is clinically predictive at the individual level with generalizability, robustness, and accuracy. In this study, we propose a new approach to profile each connectivity from diverse perspective, encompassing not only disorder-related differences but also disorder-unrelated variations attributed to individual difference, within-subject across-runs, imaging protocol, and scanner factors. By leveraging over 1500 runs of 10-minute resting-state data from 84 traveling-subjects across 29 sites and 900 participants of the case-control study with three psychiatric disorders, the disorder-related and disorder-unrelated FC variations were estimated for each individual FC. Using the FC profile information, we evaluated the effects of the disorder-related and disorder-unrelated variations on the output of the multi-connectivity biomarker trained with ensemble sparse classifiers and generalizable to the multicenter data. Our analysis revealed hierarchical variations in individual functional connectivity, ranging from within-subject across-run variations, individual differences, disease effects, inter-scanner discrepancies, and protocol differences, which were drastically inverted by the sparse machine-learning algorithm. We found this inversion mainly attributed to suppression of both individual difference and within-subject across-runs variations relative to the disorder-related difference by weighted-averaging of the selected FCs and ensemble computing. This comprehensive approach will provide an analytical tool to delineate future directions for developing reliable individual-level biomarkers.

neuroscience↗

bioRxiv2024

Comprehensive evaluation of pipelines for diagnostic biomarkers of major depressive disorder using multi-site resting-state fMRI datasets

The objective diagnostic and stratification biomarkers developed with resting-state functional magnetic resonance imaging (rs-fMRI) data are expected to contribute to more effective treatment for mental disorders. Unfortunately, there are currently no widely accepted biomarkers, partially due to the large variety of analysis pipelines for developing them. In this study we comprehensively evaluated analysis pipelines using a large-scale, multi-site fMRI dataset for major depressive disorder (MDD) (1162 participants from eight imaging sites). We explored the combinations of options in four subprocesses of analysis pipelines: six types of brain parcellation, four types of estimations of functional connectivity (FC), three types of site difference harmonization, and five types of machine learning methods. 360 different MDD diagnostic biomarkers were constructed using the SRPBS dataset acquired with unified protocols (713 participants from four imaging sites) as a discovery dataset and evaluated with datasets from other projects acquired with heterogeneous protocols (449 participants from four imaging sites) for independent validation. To identify the optimal options regardless of the discovery dataset, we repeated the same procedure after swapping the roles of the two datasets. We found pipelines that included Glassers parcellation, tangent-covariance, no harmonization, and non-sparse machine learning methods tended to result in high classification performance. The diagnosis results of the top 10 biomarkers showed high similarity, and weight similarity was also observed between eight of the biomarkers, except two that used both data-driven parcellation and FC computation. We applied the top 10 pipelines to the datasets of other mental disorders (autism spectral disorder: ASD and schizophrenia: SCZ) and eight of the ten biomarkers showed sufficient classification performances for both disorders, except two pipelines that included Pearson correlation, ComBat harmonization and random forest classifier combination. HighlightsO_LIWe evaluated the analysis pipelines of rsFC biomarker development. C_LIO_LIFour subprocesses in them were investigated with two multi-site datasets. C_LIO_LIGlassers parcellation, tangent covariance, and non-sparse methods were preferred. C_LIO_LIThe weight patterns of eight of the top 10 biomarkers showed high commonality. C_LIO_LIEight of the top 10 pipelines were successful for developing SCZ/ASD biomarkers. C_LI

neuroscience↗

bioRxiv2024

Expression of fibroblast activation protein-α in human deep venous thrombus

BackgroundFibroblast activation protein- (FAP), a type-II transmembrane serine protease, is expressed during wound healing, in cancer-associated fibroblasts, and in chronic fibrosing diseases. However, its expression in deep vein thrombus (DVT) remains unclear. Therefore, in this study, we investigated FAP expression and localization in DVT. MethodsFirst, we pathologically accessed aspirated thrombi of patients with DVT (n=14), classifying thrombotic areas as follows; fresh, cellular lysis, endothelialization, fibroblastic reaction. Endothelialization and fibroblastic reaction were defined as organizing reactions. We immunohistochemically examined FAP-expressed areas and expressed cells. Second, we analyzed FAP expression in cultured dermal fibroblasts. ResultsAll the aspirated thrombi showed a mixture of at least three of the four thrombotic areas. Specifically, 83 % of aspirated thrombi showed fresh and organizing reactions within each thrombus. Immunohistochemical expression of FAP was restricted in organizing area. Further, FAP expression in the thrombi was mainly found in vimentin-positive or -smooth muscle actin-positive fibroblasts in double immunofluorescence. Some CD163-positive macrophages also showed FAP expression. FAP mRNA and protein levels significantly increased in cultured fibroblasts with low- proliferative activity under 0.1% fetal bovine serum (FBS) than that in fibroblasts under 10% FBS. Fibroblasts cultured in 10% FBS showed a significant decrease in FAP mRNA levels following supplementation with hemin, but not with thrombin. ConclusionsThe heterogeneous composition of the venous thrombi suggests the existence of a multistep thrombus formation process in human DVT. Further, fibroblasts or myofibroblasts may express FAP in organizing process in DVT. FAP expression may increase in fibroblasts with low-proliferative activity.

pathology↗

bioRxiv2023

Accelerated Molecular Dynamics and AlphaFold Uncover a Missing Conformational State of Transporter Protein OxlT

Transporter proteins change their conformation to carry their substrate across the cell membrane. The conformational dynamics are vital to understanding the transport function. We have studied the oxalate transporter (OxlT), an oxalate:formate antiporter from Oxalobacter formigenes, significant in avoiding kidney stone formation. The atomic structure of OxlT has been recently solved in the outward-open and occluded states. However, the inward-open conformation is still missing, hindering a complete understanding of the transporter. Here, we performed an accelerated molecular dynamics simulation to sample the extensive conformational space of OxlT and successfully obtained the inward-open conformation where cytoplasmic substrate formate binding was preferred over oxalate binding. We also identified critical interactions for the inward- open conformation. The results were complemented by the highly accurate structure prediction by AlphaFold2. Although AlphaFold2 solely predicted OxlT in the outward-open conformation, mutation of the identified critical residues made it partly predict the inward-open conformation, identifying possible state-shifting mutations. TOC GRAPHICS O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/564285v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@194bb47org.highwire.dtl.DTLVardef@19371b3org.highwire.dtl.DTLVardef@10dd0d1org.highwire.dtl.DTLVardef@8552a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Factor XI localization in human deep venous thrombus and function of activated factor XI on venous thrombus formation and hemostasis in rabbit

BackgroundNovel anticoagulants targeting coagulation factor XI (FXI)/activated FXI (FXIa) are under development. However, whether FXI is present in human deep vein thrombosis (DVT) and whether FXIa and activated factor X (FXa) play different roles in venous thrombus formation and hemostasis remain unclear. This study aimed to determine the presence of FXI in DVT and the effects of direct oral FXIa and FXa inhibitors on venous thrombus formation and hemostasis in rabbits and mural thrombus formation in flow chamber system. MethodsWe immunohistochemically assessed FXI localization in human aspirated DVT (n=15). Additionally, we compared thrombus formation induced by endothelial denudation and stenosis in jugular vein, and skin bleeding time and volume between rabbits treated with direct FXIa inhibitors (ONO-1600586) and FXa inhibitors (rivaroxaban). Ex vivo rabbit and human blood were perfused on a flow chamber under low shear rates (70/s). ResultFXI was localized in all DVT, predominantly in fibrin-rich areas. The FXI-immunopositive area in the non-organizing area was greater than that in the organizing area. Although FXIa and FXa inhibitors comparably inhibited venous thrombus formation, FXIa inhibitors did not affect bleeding time or volume in rabbits. FXIa or FXa inhibitors mildly or strongly inhibited fibrin formation at low shear rates respectively. Furthermore, the FXIa inhibitor suppressed human FXIa activity, thrombin generation, and fibrin formation during perfusion. ConclusionThe pathological findings of human DVT suggest FXIs role in human DVT. FXIa inhibitors may inhibit less fibrin formation than FXa inhibitors, and may explain the minor role of FXIa in hemostasis. EssentialO_LIPresence of factor XI (FXI) in venous thrombus and less bleeding in its inhibition are unclear. C_LIO_LIWe assessed FXI localization in deep vein thrombosis (DVT) and function of FXIa in rabbit. C_LIO_LIFXI localized in human DVT that provide a rationale for FXI inhibition in human DVT. C_LIO_LIFXIa inhibitor inhibited less fibrin formation than factor Xa inhibitor under low-shear rate. C_LI

pathology↗

bioRxiv2023

Structure-based engineering of Tor complexes uncovers different roles of two types of yeast TORC1s

Certain proteins assemble into diverse complex states, each having a distinctive and unique function in the cell. The target of rapamycin complex 1 (TORC1) plays a central role in signaling pathways for cells to respond to their environment, such as nutritional status. TORC1 is widely recognised for its association with various diseases. The budding yeast Saccharomyces cerevisiae has two types of TORC1s comprising different constituent proteins, Tor1- and Tor2-containing TORC1s but are considered to have the same function. Here, we rationally redesigned the complex states by structure-based engineering and constructed a Tor2 mutant to form TORC2 but not TORC1. Functional analysis of the mutant revealed that the two types of TORC1s induced different phenotypes-rapamycin, caffeine and pH dependences of cell growth and replicative and chronological lifespans. These findings are expected to provide further insights into various fields such as molecular evolution and lifespan.

molecular biology↗

bioRxiv2023

Inhibitors of cannabinoid receptor 1 suppress the cellular entry of Lujo virus

Lujo virus (LUJV), which belongs to Mammarenavirus, family arenaviridae, has emerged as pathogen causing severe hemorrhage fever with high mortality. Currently, there are no effective treatments for arenaviruses, including LUJV. Here, we screened chemical compound libraries of Food and Drug Administration (FDA)-approved drug and G protein-coupled receptor-associated drugs to identify effective antivirals against LUJV targeting cell entry using a vesicular stomatitis virus-based pseudotyped virus bearing the LUJV envelope glycoprotein (GP). Cannabinoid receptor 1 (CB1) antagonists, such as rimonabant, AM251 and AM281, have been identified as robust inhibitors of LUJV entry. The IC50 of rimonabant was 0.26 and 0.53 M in Vero and Huh7 cells, respectively. Analysis of the cell fusion activity of the LUJV GP in the presence of CB1 inhibitors revealed that these inhibitors suppressed the fusion activity of the LUJV GP. Moreover, rimonabant, AM251 and AM281 reduced the infectivity of authentic LUJV in vitro, suggesting that the antiviral activity of CB1 antagonists against LUJV is mediated, at least in part, by inhibition of the viral entry, especially, membrane fusion. These findings suggest promising candidates for developing new therapies against LUJV infections. ImportanceTo investigate antiviral drugs against Lujo virus (LUJV), we screened chemical compound libraries to identify effective antivirals against LUJV entry. CB1 antagonists were identified as robust inhibitors of LUJV entry. The cell fusion activity of LUJV GP was suppressed by CB1 inhibitors. Furthermore, CB1 antagonists reduced the infectivity of authentic LUJV. These findings suggest promising candidates for developing new therapies against LUJV infections.

microbiology↗

bioRxiv2023

Generalizable neuromarker for autism spectrum disorder across imaging sites and developmental stages: A multi-site study

Autism spectrum disorder (ASD) is a lifelong condition, and its underlying biological mechanisms remain elusive. The complexity of various factors, including inter-site and development-related differences, makes it challenging to develop generalizable neuroimaging-based biomarkers for ASD. This study used a large-scale, multi-site dataset of 730 Japanese adults to develop a generalizable neuromarker for ASD across independent sites (U.S., Belgium, and Japan) and different developmental stages (children and adolescents). Our adult ASD neuromarker achieved successful generalization for the US and Belgium adults (area under the curve [AUC] = 0.70) and Japanese adults (AUC = 0.81). The neuromarker demonstrated significant generalization for children (AUC = 0.66) and adolescents (AUC = 0.71; all P<0.05, family-wise-error corrected). We identified 141 functional connections (FCs) important for discriminating individuals with ASD from TDCs. These FCs largely centered on social brain regions such as the amygdala, hippocampus, dorsomedial and ventromedial prefrontal cortices, and temporal cortices. Finally, we mapped schizophrenia (SCZ) and major depressive disorder (MDD) onto the biological axis defined by the neuromarker and explored the biological continuity of ASD with SCZ and MDD. We observed that SCZ, but not MDD, was located proximate to ASD on the biological dimension defined by the ASD neuromarker. The successful generalization in multifarious datasets and the observed relations of ASD with SCZ on the biological dimensions provide new insights for a deeper understanding of ASD.

neuroscience↗

bioRxiv2023

Depletion of LONP2 unmasks differential requirements for peroxisomal function between cell types and in cholesterol metabolism.

Peroxisomes play a central role in tuning metabolic and signaling programs in a tissue- and cell type-specific manner. However, the mechanisms by which the status of peroxisomes is communicated and integrated into cellular signaling pathways is not yet understood. Herein, we report the cellular responses to acute peroxisomal proteotoxic stress upon silencing the peroxisomal protease/chaperone LONP2. Depletion of LONP2 triggered accumulation of its substrates, alterations in peroxisome size and numbers, and luminal protein import failure. Gene expression changes and lipidomic analysis revealed striking cell specific differences in the response to siLONP2. Specific to COS-7 cells was a strong activation of the integrated stress response (ISR) and upregulation of ribosomal biogenesis gene expression levels. Common changes between COS-7 and U2OS cell lines included repression of the retinoic acid signaling pathway, and upregulation of sphingolipids. Cholesterol accumulated in the endomembrane compartments in both cell lines, consistent with evidence that peroxisomes are required for cholesterol flux out of late endosomes. These unexpected consequences of peroxisomal stress provide an important insight for our understanding of the tissue-specific responses seen in peroxisomal disorders.

cell biology↗

bioRxiv2023

Simultaneous measurement of nascent transcriptome and translatome using 4-thiouridine metabolic RNA labeling and translating ribosome affinity purification

Regulation of gene expression in response to various biological processes, including extracellular stimulation and environmental adaptation requires de novo RNA synthesis and translation. Analysis of the coordinated and circular regulation of dynamic RNA synthesis and translation is required to determine functional protein production. However, reliable methods for the simultaneous measurement of nascent RNA synthesis and translation at the gene level are limited. Here, we developed a novel method for the simultaneous assessment of nascent RNA synthesis and translation by combining 4-thiouridine (4sU) metabolic RNA labeling and translating ribosome affinity purification (TRAP) using a monoclonal antibody against evolutionarily conserved ribosomal P-stalk proteins. The P-stalk-mediated TRAP (P-TRAP) technique recovered endogenous translating ribosomes, allowing easy translatome analysis of various eukaryotes. We validated this method in mammalian cells by demonstrating that acute unfolded protein response (UPR) in the endoplasmic reticulum (ER) induces dynamic reprograming of nascent RNA synthesis and translation. Our method can serve as a simple and powerful tool for analyzing the coordinated regulation of transcription and translation of individual genes in various eukaryotes.

molecular biology↗

bioRxiv2022

Peripheral vs. Core Body Temperature as Hypocretin/Orexin Neurons Degenerate: Exercise Mitigates Increased Heat Loss

Hypocretins/Orexins (Hcrt/Ox) are hypothalamic neuropeptides implicated in diverse functions, including body temperature regulation through modulation of sympathetic vasoconstrictor tone. In the current study, we measured subcutaneous (Tsc) and core (Tb) body temperature as well as activity in a conditional transgenic mouse strain that allows the inducible ablation of Hcrt/Ox-containing neurons by removal of doxycycline (DOX) from their diet (orexin-DTA mice). Measurements were made during a baseline, when mice were being maintained on food containing DOX, and over 42 days while the mice were fed normal chow which resulted in Hcrt/Ox neuron degeneration. The home cages of the orexin-DTA mice were equipped with running wheels that were either locked or unlocked. In the presence of a locked running wheel, Tsc progressively decreased on days 28 and 42 in the DOX(-) condition, primarily during the dark phase (the major active period for rodents). This nocturnal reduction in Tsc was mitigated when mice had access to unlocked running wheels. In contrast to Tsc, Tb was largely maintained until day 42 in the DOX(-) condition even when the running wheel was locked. Acute changes in both Tsc and Tb were observed preceding, during, and following cataplexy. Our results suggest that ablation of Hcrt/Ox-containing neurons results in elevated heat loss, likely through reduced sympathetic vasoconstrictor tone, and that exercise may have some therapeutic benefit to patients with narcolepsy, a disorder caused by Hcrt/Ox deficiency. Acute changes in body temperature may facilitate prediction of cataplexy onset and lead to interventions to mitigate its occurrence. HighlightsO_LIHypocretin/Orexin (Hcrt/Ox) neuron degeneration results in the sleep disorder Narcolepsy and reduced subcutaneous body temperature (Tsc) during the dark phase of the 24-h light/dark cycle. C_LIO_LIThis reduction in dark phase Tsc is mitigated by access to an exercise opportunity. C_LIO_LIIn contrast to Tsc, core body temperature (Tb) is largely maintained as the Hcrt/Ox neurons degenerate. C_LIO_LIReduced Tsc while Tb is maintained suggests increased heat loss, possibly through modulation of sympathetic vasoconstrictor tone. C_LIO_LIHcrt/Ox neuron loss in Narcolepsy results in cataplexy, whose occurrence is associated with acute changes in both Tsc and Tb. C_LIO_LIExercise may represent an effective intervention for mitigating heat loss resulting from Hcrt/Ox neuron loss in Narcolepsy. C_LI

neuroscience↗

bioRxiv2022

Inflammatory response induced in pulmonary embolic lung: Evaluation usinga reproducible murine pulmonary embolism model.

BackgroundSince previously established models of pulmonary embolism showed a large variability in the degree of ischemia, it is difficult to assess the pathophysiological response in the lung after embolization. Here, we established a model of pulmonary embolism by certain amount of relatively small thrombi, in which the degree of ischemia was reproducible. MethodsThrombi with a maximum diameter of 100 m or 500 m were administered intravenously under anesthesia, and the survival ratio at 4 hours was evaluated. The location of thrombi in the lung was visualized by administration of fluorescent-labeled thrombus, and the hemodynamics of the lung after administration of thrombi was evaluated. CT angiography was also performed to evaluate the ratio of the embolized vessels. In addition, cytokine mRNAs was quantified 4 hours in embolized lung. Immunohistochemical analysis for interleukin (IL)-6 and CD68 as a marker of macrophages were also performed. ResultsIt was found that mice with 100 m clots, but not with 500 m clots, showed a dose-dependence of survival between 2.3 L/g and 3.0 L/g at 4 hours from embolization induction. In mice treated with 2.5 L/g of 100 m thrombus, thrombi were located in the peripheral region of the lung, which was consistent with the disruption of blood circulation the peripheral region. In addition, about 60% of the vessels with a diameter of less than 100 m were occluded in these mice. In the lungs after 4 hours of embolization, IL-6 mRNA and tumor necrosis factor (TNF)- mRNA were significantly higher and lower than control lungs. IL-6 was expressed in CD68-positive macrophages in both embolized and control lungs after 4 hours of embolization, and the number of each positive cells were comparable in both embolized and control lungs. ConclusionsThese results show that the pulmonary embolization model induced by a certain amount of small thrombus is useful for evaluating the pathological responses in the embolized lung. Furthermore, it was found that IL-6 expression was increased in macrophages in the embolized lung, indicating that inflammatory responses may contribute to the pathogenesis of pulmonary embolism.

pathology↗

bioRxiv2022

Hidden Markov Modeling of Molecular Orientations and Structures from High-Speed Atomic Force Microscopy Time-Series Images

High-speed atomic force microscopy (HS-AFM) is a powerful technique for capturing the time-resolved behavior of biomolecules. However, structural information in HS-AFM images is limited to the surface geometry of a sample molecule. Inferring latent three-dimensional structures from the surface geometry is thus important for getting more insights into conformational dynamics of a target biomolecule. Existing methods for estimating the structures are based on the rigid-body fitting of candidate structures to each frame of HS-AFM images. Here, we extend the existing frame-by-frame rigid-body fitting analysis to multiple frames to exploit orientational correlations of a sample molecule between adjacent frames in HS-AFM data due to the interaction with the stage. In the method, we treat HS-AFM data as time-series data, and they are analyzed with the hidden Markov modeling. Using simulated HS-AFM images of the taste receptor type 1 as a test case, the proposed method shows a more robust estimation of molecular orientations than the frame-by-frame analysis. The method is widely applicable in integrative modeling of conformational dynamics using real experimental HS-AFM data. Author summaryBiomolecules dynamically change their structures in cells and perform various functions that are important for life. It is difficult to observe such dynamic structural changes using conventional experimental techniques, such as X-ray crystallography, that mainly observe static atomic structures. Alternative approach is the atomic force microscopy (AFM), a technique to measure the surface geometry of a sample molecule by scanning with an acute tip. Recently, by increasing the imaging rate of the AFM, it has become possible to observe the dynamics of biomolecules at work. However, the information measured with AFM is limited to the surface geometry of a molecule, so it is important to extract three-dimensional structural information from those data. In this study, we develop a method to estimate structural information with higher accuracy.

biophysics↗

bioRxiv2022

A global atlas of substrate specificities for the human serine/threonine kinome

Protein phosphorylation is one of the most widespread post-translational modifications in biology. With the advent of mass spectrometry-based phosphoproteomics, more than 200,000 sites of serine and threonine phosphorylation have been reported, of which several thousand have been associated with human diseases and biological processes. For the vast majority of phosphorylation events, it is not yet known which of the more than 300 protein Ser/Thr kinases encoded in the human genome is responsible. Here, we utilize synthetic peptide libraries to profile the substrate sequence specificity of nearly every functional human Ser/Thr kinase. Viewed in its entirety, the substrate specificity of the kinome was substantially more diverse than expected and was driven extensively by negative selectivity. Our kinome-wide dataset was used to computationally annotate and identify the most likely protein kinases for every reported phosphorylation site in the human Ser/Thr phosphoproteome. For the small minority of phosphosites where the protein kinases involved have been previously identified, our predictions were in excellent agreement. When this approach was applied to examine the signaling response of tissues and cell lines to hormones, growth factors, targeted inhibitors, and environmental or genetic perturbations, it revealed unexpected insights into pathway complexity and compensation. Overall, these studies reveal the full extent of substrate specificity of the human Ser/Thr kinome, illuminate cellular signaling responses, and provide a rich resource to link unannotated phosphorylation events to biological pathways.

systems biology↗

bioRxiv2022

Chloride ion evokes taste sensation by binding to the extracellular ligand-binding domain of sweet/umami taste receptors

Salt taste sensation is multifaceted: NaCl at low or high concentrations is preferably or aversively perceived through distinct pathways. Cl- is thought to participate in taste sensation through an unknown mechanism. Here we describe Cl- ion binding and the response of taste receptor type 1 (T1r), a receptor family composing sweet/umami receptors. The T1r2a/T1r3 heterodimer from the medaka fish, currently the sole T1r amenable to structural analyses, exhibited a specific Cl- binding in the vicinity of the amino-acid-binding site in the ligand-binding domain (LBD) of T1r3, which is likely conserved across species, including human T1r3. The Cl- binding induced a conformational change in T1r2a/T1r3LBD at sub-to low-mM concentrations similar to canonical taste substances. Furthermore, oral Cl- application to mice increased impulse frequencies of taste nerves connected to T1r-expressing taste cells and promoted their behavioral preferences attenuated by a T1r-specific blocker or T1r3 knock-out. These results suggest that the Cl- evokes taste sensations by binding to T1r, thereby serving as another preferred salt taste pathway at a low concentration.

biophysics↗

bioRxiv2021

Structure and mechanism of oxalate transporter OxlT in an oxalate-degrading bacterium in the gut microbiota

Oxalobacter formigenes is an oxalate-degrading bacterium in the gut microbiota that absorbs food-derived oxalate to use this as a carbon and energy source and thereby helps reduce the risk of kidney stone formation of the host animals 1-4. The bacterial oxalate transporter OxlT uptakes oxalate from the gut to bacterial cells and excrete formate as a degradation product, with a strict discrimination from other carboxylates that serve as nutrients 5-7. Nevertheless, the underlying mechanism remains unclear. Here, we present crystal structures of oxalate-bound and ligand-free OxlT in two different conformations, occluded and outward-facing states. The oxalate binding site contains two basic residues that form salt bridges with a dicarboxylate substrate while preventing the conformational switch to the occluded state without an acidic substrate, a disallowed state for an antiporter 8, 9. The occluded ligand-binding pocket can accommodate oxalate but not larger dicarboxylates, such as metabolic intermediates. The permeation pathways from the binding pocket are completely blocked by extensive interdomain hydrophobic and ionic interactions. Nevertheless, a molecular dynamics simulation showed that a flip of a single side chain neighbouring the substrate is sufficient to trigger the gate opening. The OxlT structure indicates the underlying metabolic interactions enabling favourable symbiosis at a molecular level.

biochemistry↗

bioRxiv2021

Effects of upgrading acquisition-techniques and harmonization methods: A multi-modal MRI study with implications for longitudinal designs

A downside of upgrading MRI acquisition sequences is the discontinuity of technological homogeneity of the MRI data. It hampers combining new and old datasets, especially in a longitudinal design. Characterizing upgrading effects on multiple brain parameters and examining the efficacy of harmonization methods are essential. This study investigated the upgrading effects on three structural parameters, including cortical thickness (CT), surface area (SA), cortical volume (CV), and resting-state functional connectivity (rs-FC) collected from 64 healthy volunteers. We used two evaluation metrics, Cohens d and classification accuracy, to quantify the effects. In classification analyses, we built classifiers for differentiating the protocols from brain parameters. We investigated the efficacy of three harmonization methods, including traveling subject (TS), TS-ComBat, and ComBat methods, and the sufficient number of participants for eliminating the effects on the evaluation metrics. Finally, we performed age prediction as an example to confirm that harmonization methods retained biological information. The results without harmonization methods revealed small to large mean Cohens d values on brain parameters (CT:0.85, SA:0.66, CV:0.68, and rs-FC:0.24) with better classification accuracy (>92% accuracy). With harmonization methods, Cohens d values approached zero. Classification performance reached the chance level with TS-based techniques when data from less than 26 participants were used for estimating the effects, while the Combat method required more participants. Furthermore, harmonization methods improved age prediction performance, except for the ComBat method. These results suggest that acquiring TS data is essential to preserve the continuity of MRI data.

neuroscience↗

bioRxiv2021

Unidirectional ion transport mechanism of a light-driven chloride pump revealed using X-ray free electron lasers

Light-driven chloride-pumping rhodopsins actively transport anions, including various halide ions, across cell membranes. Recent studies using time-resolved serial femtosecond crystallography (TR-SFX) have uncovered the structural changes and ion transfer mechanisms in light-driven cation-pumping rhodopsins. However, the mechanism by which the conformational changes pump an anion to achieve unidirectional ion transport, from the extracellular side to the cytoplasmic side, in anion-pumping rhodopsins remains enigmatic. We have collected TR-SFX data of Nonlabens marinus rhodopsin-3 (NM-R3), derived from a marine flavobacterium, at 10 s and 1 ms time-points after photoexcitation. Our structural analysis reveals the conformational alterations during ion transfer and after ion release. Movements of the retinal chromophore initially displace a conserved tryptophan to the cytoplasmic side of NM-R3, accompanied with a slight shift of the halide ion bound to the retinal. After ion release, the inward movements of helix C and helix G and the lateral displacements of the retinal block access to the extracellular side of NM-R3. Anomalous signal data have also been obtained from NM-R3 crystals containing iodide ions. The anomalous density maps provide insight into the halide binding site for ion transfer in NM-R3. SignificanceLight-driven chloride pumps have been identified in various species, including archaea and marine flavobacteria. The function of ion transportation controllable by light is utilized for optogenetics tools in neuroscience. Chloride pumps differ among species, in terms of amino acid homology and structural similarity. Our time-resolved crystallographic studies using X-ray free electron lasers reveal the molecular mechanism of halide ion transfer in a light-driven chloride pump from a marine flavobacterium. Our data indicate a common mechanism in chloride pumping rhodopsins, as compared to previous low temperature trapping studies of chloride pumps. These findings are significant not only for further improvements of optogenetic tools but also for a general understanding of the ion pumping mechanisms of microbial rhodopsins.

molecular biology↗