Biology subjects

Hattori, M.

Publications and source records attributed to Hattori, M..

36 records · Page 2Linked to original sources

bioRxiv2023

Antithetic effects of agonists and antagonists on the structural fluctuations of TRPV1 channel

Transient receptor potential vanilloid member 1 (TRPV1) is a heat and capsaicin receptor that allows cations to permeate and cause pain. As the molecular basis for temperature sensing, the heat capacity ({Delta}Cp) model (D. E. Clapham, C. Miller, Proc. Natl. Acad. Sci. U. S. A. 108, 19492-19497 (2011).) has been proposed and experimentally supported. Theoretically, heat capacity is proportional to a variance in enthalpy, presumably related to structural fluctuation; however, the fluctuation of TRPV1 has not been directly visualized. In this study, we directly visualized single-molecule structural fluctuations of the TRPV1 channels in a lipid bilayer with the ligands resiniferatoxin (RTX: agonist, 1000 times hotter than capsaicin) and capsazepine (CPZ: antagonist) by high-speed atomic force microscopy (HS-AFM). We observed the structural fluctuations of TRPV1 in an apo state and found that RTX binding enhances structural fluctuations, while CPZ binding suppresses fluctuations. These ligand-dependent differences in structural fluctuation would play a key role in the gating of TRPV1.

physiology↗

bioRxiv2022

Lifelong temporal dynamics of the gut microbiome associated with longevity in mice

The temporal changes of the gut microbiome are thought to be critical for understanding its interactions with host aging, but lifelong dynamics within the same individual remain largely unknown. Here we firstly report the high temporal resolution dynamics of gut microbiomes in mice sharing the same genetic background and environment from their birth to natural death, spanning >1,000 days. The 16S rRNA sequencing analysis revealed 9 patterns of OTU temporal dynamics and 38 common "life-core" bacterial species/operational taxonomic units (OTUs) in [≥]80% of all samples across the lifespan of individual mice. The life-core OTUs are largely represented by the phylum Bacteroidota, whereas the transient bacterial group predominantly includes the phylum Firmicutes (Bacillota). Despite the shared genetic background and dietary habits, the gut microbiome structure significantly diversified with age and among individuals. A positive correlation existed between longevity and the microbiome -diversity in middle age (200-500 days) followed by a negative correlation in old age (>700 days), likely influenced by the increase in diversity during the last days of life. The abundance of several "life-core" species also exhibited non-static correlation trends with lifespan. Overall, this research characterized the gut microbiomes based on its persistence over hosts lifetime and suggested a non-static host-microbiome relationship within individual mice.

microbiology↗

bioRxiv2022

ADAMTS2 regulates radial neuronal migration by activating TGF-β signaling at the subplate layer of the developing neocortex

During the development of the mammalian brain, neocortical structures are formed by the sequential radial migration of newborn excitatory neurons. The early migrating neurons exhibit a multipolar shape, but they undergo a multipolar-to-bipolar transition at the subplate (SP) layer, where extracellular matrix (ECM) components are abundantly expressed. In this study, we revealed that the TGF-{beta} signaling-related ECM proteins, such as latent TGF-{beta}-binding protein 1 (LTBP1) and fibrillin 2, and TGF-{beta} receptor II (TGF-{beta}RII) and its downstream effector, p-smad2/3, are selectively expressed at the SP layer, suggesting that TGF-{beta} is sequestered in a latent form by forming complexes with these ECM components and then its signaling is activated by ECM remodeling. We found that the migrating multipolar neurons transiently express a disintegrin and metalloproteinase with thrombospondin motif 2 (ADAMTS2), an ECM metalloproteinase, just below the SP layer. Knockdown and knockout of Adamts2 suppressed the multipolar-to-bipolar transition of migrating neurons, and therefore, disturbed radial migration. Similar phenotypes were observed by the perturbation of TGF-{beta} signaling in the migrating neurons. Time-lapse luminescence imaging of TGF-{beta} signaling indicated that ADAMTS2 activates this signaling pathway in the migrating neurons during the multipolar-to-bipolar transition at the SP layer. These results suggest that the ADAMTS2 secreted by the migrating multipolar neurons activates TGF-{beta} signaling by ECM remodeling of the SP layer, leading to the multipolar-to-bipolar transition. We propose that the SP layer plays an essential role in the radial neuronal migration as a signaling center of the developing neocortex. SIGNIFICANCEThe neocortex is formed by the sequential radial migration of newborn neurons, which undergo a multipolar-to-bipolar transition at the subplate (SP) layer. The extracellular matrix (ECM) is abundantly expressed in the SP layer. However, the roles of the ECM in the SP layer have been unclear. We found that migrating neurons transiently express a disintegrin and a metalloproteinase with thrombospondin motif 2 (ADAMTS2), an ECM metalloproteinase, just below the SP layer. We show that ADAMTS2 secreted by multipolar migrating neurons activates TGF-{beta} signaling through remodeling of the ECM in the SP layer, leading to the multipolar-to-bipolar transition. Thus, the SP layer plays an essential role in radial migration as a signaling center of the developing neocortex

neuroscience↗

bioRxiv2022

Complex host/symbiont integration of a multi-partner symbiotic system in the eusocial aphid Ceratovacuna japonica

Some hemipteran insects rely on multiple endosymbionts for essential nutrients. However, the evolution of multi-partner symbiotic systems is not well-established. Here, we report a co-obligate symbiosis in the eusocial aphid, Ceratovacuna japonica. 16S rRNA amplicon sequencing unveiled co-infection with a novel Arsenophonus sp. symbiont and Buchnera aphidicola, a common obligate endosymbiont in aphids. Both symbionts were housed within distinct bacteriocytes and were maternally transmitted. The Buchnera and Arsenophonus symbionts had streamlined genomes of 432,286 bp and 853,149 bp, respectively, and exhibited metabolic complementarity in riboflavin and peptidoglycan synthesis pathways. These anatomical and genomic properties were similar to those of independently evolved multi-partner symbiotic systems, such as Buchnera-Serratia in Lachninae and Periphyllus aphids, representing remarkable parallelism. Furthermore, symbiont populations and bacteriome morphology differed between reproductive and soldier castes. Our study provides the first example of co-obligate symbiosis in Hormaphidinae and gives insight into the evolutionary genetics of this complex system.

evolutionary biology↗

bioRxiv2022

Extensive gut virome variation and its associations with host and environmental factors in a population-level cohort

Indigenous bacteriophage communities (virome) in the human gut have a huge impact on the gut bacterial communities (bacteriome), but virome variation at a population scale is not fully investigated yet. Here, we analyse the gut virome in a population-level cohort of 4,198 deeply phenotyped individuals. We discovered thousands of high-quality phage genomes including previously uncharacterized ones with different bacterial hosts than known major phage clades. The distribution of host bacteria was a strong determinant for the distribution of phages, and virome diversity was highly correlated with anti-viral defence mechanisms of the bacteriome, such as CRISPR-Cas and restriction-modification systems. We identified 97 various intrinsic/extrinsic factors that significantly affect the virome, including age, sex, lifestyle, and diet, most of which showed consistent associations with both phages and their predicted bacterial hosts. Among the metadata categories, disease and medication had the strongest effects on the virome structure. Overall, these results present a basis to understand the symbiotic communities of bacteria and their viruses in the human gut, which will facilitate the medical and industrial applications of indigenous viruses.

microbiology↗

bioRxiv2021

Structural insights into the ion selectivity of the MgtE channel for Mg2+ over Ca2+

MgtE is a Mg2+-selective ion channel whose orthologs are widely distributed from prokaryotes to eukaryotes, including humans, and play an important role in the maintenance of cellular Mg2+ homeostasis. Previous functional analyses showed that MgtE transports divalent cations with high selectivity for Mg2+ over Ca2+. Whereas the high-resolution structure determination of the MgtE transmembrane (TM) domain in complex with Mg2+ ions revealed a Mg2+ recognition mechanism of MgtE, the previous Ca2+-bound structure of the MgtE TM domain was determined only at moderate resolution (3.2 [A] resolution), which was insufficient to visualize the water molecules coordinated to Ca2+ ions. Thus, the structural basis of the ion selectivity of MgtE for Mg2+ over Ca2+ has remained unclear. Here, we showed that the metal-binding site of the MgtE TM domain binds to Mg2+ [~]500-fold more strongly than Ca2+. We then determined the crystal structure of the MgtE TM domain in complex with Ca2+ ions at a higher resolution (2.5 [A] resolution), allowing us to reveal hexahydrated Ca2+, which is similarly observed in the previously determined Mg2+-bound structure but with extended metal-oxygen bond lengths. Our structural, biochemical, and computational analyses provide mechanistic insights into the ion selectivity of MgtE for Mg2+ over Ca2+.

biophysics↗

bioRxiv2021

Multiple nutritional and gut microbial factors associated with allergic rhinitis: the Hitachi Health Study

Several studies suggest the involvement of dietary habits and gut microbiome in allergic diseases. However, little is known about the nutritional and gut microbial factors associated with the risk of allergic rhinitis (AR). We recruited 186 apparently healthy workers with symptoms of AR and 106 controls at the Hitachi Health Care Center, Japan. The habitual consumption of 42 selected nutrients were examined using the brief-type self-administered diet history questionnaire. Faecal samples were collected and subjected to amplicon sequencing of the 16S ribosomal RNA gene hypervariable regions. Association analysis revealed that four nutrients (retinol, vitamin A, cryptoxanthin, and copper) were negatively associated with AR. Among 40 genera examined, relative abundance of Prevotella and Escherichia were associated with AR. Furthermore, significant statistical interactions were observed between retinol and Prevotella. The age- and sex-adjusted odds of AR were 25-fold lower in subjects with high retinol intake and high Prevotella abundance compared to subjects with low retinol intake and low Prevotella abundance. Our data provide insights into complex interplay between dietary nutrients, gut microbiome, and the development of AR.

microbiology↗

bioRxiv2021

C9ORF72 dipeptide repeat proteins disrupt formation of GEM bodies and induce aberrant accumulation of survival of motor neuron protein

A GGGGCC repeat expansion in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS), a devastating motor neuron disease. In the neurons of ALS patients, dipeptide repeat proteins (DPRs) are produced from repeat-containing RNAs by an unconventional form of translation, and some of these proteins, especially those containing poly(glycine-arginine) and poly(proline-arginine), are toxic to neurons. Gemini of coiled bodies (GEMs) are nuclear structures that harbor survival of motor neuron (SMN) protein, and SMN is essential for the assembly of U-rich small nuclear ribonucleoproteins (snRNPs) that are central for splicing. We previously reported that GEMs are lost and that snRNP biogenesis is misregulated in the motor neurons of ALS patients. Here we show that DPRs interfere with GEM formation and proper SMN localization in HeLa cells and iPSC-derived motor neurons from an ALS patient with the C9ORF72 mutation. The accumulation of poly(glycine-arginine) markedly reduced the number of GEMs and caused the formation of aberrant cytoplasmic RNA granules that sequestered SMN. These findings indicate the functional impairment of SMN in motor neurons expressing DPRs and may provide a mechanism to explain the vulnerability of motor neurons of C9ORF72-ALS patients.

neuroscience↗

bioRxiv2021

Identification and mechanistic analysis of an inhibitor of the CorC Mg2+ transporter

The CorC/CNNM family of Na+-dependent Mg2+ transporters is ubiquitously conserved from bacteria to humans. CorC, the bacterial member of the CorC/CNNM family of proteins, is involved in resistance to antibiotic exposure and in the survival of pathogenic microorganisms in their host environment. The CorC/CNNM family proteins possess a cytoplasmic region containing the regulatory ATP-binding site. While CorC and CNNM have attracted interest as therapeutic targets, inhibitors targeting the ir regulatory ATP-binding site have not yet been identified. Here, we performed a virtual screening of CorC by targeting its regulatory ATP-binding site, identified a chemical compound named IGN95a with inhibitory effects on both ATP binding and Mg2+ export, and determined the cytoplasmic domain structure in complex with IGN95a. Furthermore, a chemical cross-linking experiment indicated that with ATP bound to the cytoplasmic domain, the conformational equilibrium of CorC was shifted more towards the inward-facing state of the transmembrane domain. In contrast, IGN95a did not induce such a shift. Our results provide a structural basis for the further design and optimization of chemical compounds targeting the regulatory ATP-binding site of CorC as well as mechanistic insights into how ATP and chemical compounds modulate the transport activity of CorC.

biophysics↗

bioRxiv2021

Systematic analysis of brain lactate and pH levels in 65 animal models related to neuropsychiatric conditions

Increased levels of lactate, an end-product of glycolysis, have been proposed as a potential surrogate marker for metabolic changes during neuronal excitation. These changes in lactate levels can result in decreased brain pH, which has been implicated in patients with various neuropsychiatric disorders. We previously demonstrated that such alterations are commonly observed in five mouse models of schizophrenia, bipolar disorder, and autism, suggesting a shared endophenotype among these disorders rather than mere artifacts due to medications or agonal state. However, there is still limited research on this phenomenon in animal models, leaving its generality across other disease animal models uncertain. Moreover, the association between changes in brain lactate levels and specific behavioral abnormalities remains unclear. To address these gaps, the International Brain pH Project Consortium investigated brain pH and lactate levels in 109 strains/conditions of 2,294 animals with genetic and other experimental manipulations relevant to neuropsychiatric disorders. Systematic analysis revealed that decreased brain pH and increased lactate levels were common features observed in multiple models of depression, epilepsy, Alzheimers disease, and some additional schizophrenia models. While certain autism models also exhibited decreased pH and increased lactate levels, others showed the opposite pattern, potentially reflecting subpopulations within the autism spectrum. Furthermore, utilizing large-scale behavioral test battery, a multivariate cross-validated prediction analysis demonstrated that poor working memory performance was predominantly associated with increased brain lactate levels. Importantly, this association was confirmed in an independent cohort of animal models. Collectively, these findings suggest that altered brain pH and lactate levels, which could be attributed to dysregulated excitation/inhibition balance, may serve as transdiagnostic endophenotypes of debilitating neuropsychiatric disorders characterized by cognitive impairment, irrespective of their beneficial or detrimental nature.

neuroscience↗

bioRxiv2020

Fluorescence-detection size-exclusion chromatography utilizing nanobody technology for expression screening of membrane proteins

Membrane proteins play numerous physiological roles and are thus of tremendous interest in pharmacology. Nevertheless, stable and homogeneous sample preparation is one of the bottlenecks in biophysical and pharmacological studies of membrane proteins because membrane proteins are typically unstable and poorly expressed. To overcome such obstacles, GFP fusion-based Fluorescence-detection Size-Exclusion Chromatography (FSEC) has been widely employed for membrane protein expression screening for over a decade. However, fused GFP itself may occasionally affect the expression and/or stability of the targeted membrane protein, leading to both false-positive and false-negative results in expression screening. Furthermore, GFP fusion technology is not well suited for some membrane proteins depending on their membrane topology. Here, we developed an FSEC assay utilizing nanobody (Nb) technology, named FSEC-Nb, in which targeted membrane proteins are fused to a small peptide tag and recombinantly expressed. The whole-cell extracts are solubilized, mixed with anti-peptide Nb fused to GFP and applied to a size-exclusion chromatography column attached to a fluorescence detector for FSEC analysis. FSEC-Nb enables one to evaluate the expression, monodispersity and thermostability of membrane proteins without the need of purification by utilizing the benefits of the GFP fusion-based FSEC method, but does not require direct GFP fusion to targeted proteins. We applied FSEC-Nb to screen zinc-activated ion channel (ZAC) family proteins in the Cys-loop superfamily and membrane proteins from SARS-CoV-2 as examples of the practical application of FSEC-Nb. We successfully identified a ZAC ortholog with high monodispersity but moderate expression levels that could not be identified with the previously developed GFP fusion-free FSEC method. Consistent with the results of FSEC-Nb screening, the purified ZAC ortholog showed monodispersed particles by both negative staining EM and cryo-EM. Furthermore, we identified two membrane proteins from SARS-CoV-2 with high monodispersity and expression level by FSEC-Nb, which may facilitate structural and functional studies of SARS-CoV-2. Overall, our results show FSEC-Nb as a powerful tool for membrane protein expression screening that can provide further opportunity to prepare well-behaved membrane proteins for structural and functional studies.

biophysics↗

bioRxiv2020

Cryo-EM structure of the MgtE Mg2+ channel pore domain in Mg2+-free conditions reveals cytoplasmic pore opening

MgtE is a Mg2+ channel conserved in organisms ranging from prokaryotes to eukaryotes, including humans, and plays an important role in Mg2+ homeostasis. The previously determined MgtE structures in the Mg2+-bound, closed state and structure-based functional analyses of MgtE revealed that the binding of Mg2+ ions to the MgtE cytoplasmic domain induces channel inactivation to maintain Mg2+ homeostasis. However, due to the lack of a structure of the MgtE channel, including its transmembrane domain in Mg2+-free conditions, the pore-opening mechanism of MgtE has remained unclear. Here, we determined the cryoelectron microscopy (cryo-EM) structure of the MgtE-Fab complex in the absence of Mg2+ ions. The Mg2+-free MgtE transmembrane domain structure and its comparison with the Mg2+-bound, closed-state structure, together with functional analyses, showed the Mg2+-dependent pore opening of MgtE on the cytoplasmic side and revealed the kink motions of the TM2 and TM5 helices at the glycine residues, which are important for channel activity. Overall, our work provides structure-based mechanistic insights into the channel gating of MgtE.

biophysics↗

bioRxiv2020

Physiological significance of proteolytic processing of Reelin revealed by cleavage-resistant Reelin knock-in mice

Reelin is a secreted protein that plays versatile roles in neuronal development and function, and hypoactivity of Reelin is implicated in many neuropsychiatric disorders. The strength of Reelin signaling is regulated by proteolytic processing, but its importance in vivo is not yet fully understood. Here, we generated Reelin knock-in (PA-DV KI) mice in which the key cleavage site of Reelin was abolished by mutation. As expected, the cleavage of Reelin was severely abrogated in the cerebral cortex and hippocampus of PA-DV KI mice. The amount of Dab1, whose degradation is induced by Reelin signaling, decreased in these tissues, indicating that the signaling strength of Reelin was augmented. The brains of PA-DV KI mice were largely structurally normal, but unexpectedly, the hippocampal layer was disturbed. This phenotype was ameliorated in hemizygote PA-DV KI mice, indicating that excess Reelin signaling is detrimental to hippocampal layer formation. The neuronal dendrites of PA-DV KI mice had more branches and were elongated compared to wild-type mice. These results present the first direct evidence of the physiological importance of Reelin cleavage and suggest that inhibition of Reelin cleavage would counteract neuropsychiatric disorders without causing severe systemic side effects.

neuroscience↗

bioRxiv2019

Deglycosylation of isoflavone C-glucoside puerarin by combination of two recombinant bacterial enzymes and 3-oxo-glucose.

C-Glucosides are resistant to glycoside hydrolase activity because the anomeric carbon of glucose is directly connected to aglycone via carbon-carbon bonding. A human intestinal bacterium strain PUE related to Dorea species can metabolize the isoflavone C-glucoside puerarin (daidzein 8-C-glucoside) to daidzein and glucose by more than three bacterial enzymes which have not been well-characterized. We previously reported that 3\"-oxo-puerarin is an essential reaction intermediate in enzymatic puerarin degradation and characterized a bacterial enzyme of DgpB-C complex which cleaved the C-glycosidic bond in 3\"-oxo-puerarin. However, the exact enzyme catalyzing the oxidation of C-3\" hydroxyl in puerarin has not been identified, and the other metabolite corresponding to the precursor of D-glucose, derived from the sugar moiety in 3\"-oxo-puerarin in the cleaving reaction catalyzed by the DgpB-C complex, remains unknown.\n\nIn this study, we demonstrated that recombinant DgpA, a Gfo/Idh/MocA family oxidoreductase, catalyzed puerarin oxidation in the presence of 3-oxo-glucose as the hydride accepter. In addition, enzymatic C-deglycosylation of puerarin was achieved by a combination of recombinant DgpA, DgpB-C complex, and 3-oxo-glucose. Furthermore, the metabolite derived from the sugar moiety in 3\"-oxo-puerarin cleaving reaction catalyzed by DgpB-C complex was characterized as 1,5-anhydro-D-erythro -hex-1-en-3-ulose, suggesting that the C-glycosidic linkage is cleaved through a {beta}-elimination like mechanism.\n\nImportanceOne important role of the gut microbiota is to metabolize dietary nutrients and supplements such as flavonoid glycosides. Ingested glycosides are metabolized by intestinal bacteria to more absorbable aglycones and further degradation products which show beneficial effects in humans. Although numerous glycoside hydrolases that catalyze O-deglycosylation have been reported, enzymes responsible for C-deglycosylation are still limited. In this study, we characterized enzymes involved in C-deglycosylation of puerarin from a human intestinal bacterium PUE. To our knowledge, this is the first report of the expression, purification and characterization of an oxidoreductase involved in C-glucoside degradation. This study provides new insights for the elucidation of mechanisms of enzymatic C-deglycosylation.

biochemistry↗

bioRxiv2019

Revealing microbial assemblage structure in the human gut microbiome using latent Dirichlet allocation

Recent research has revealed that there are various microbial species in the human gut microbiome. To clarify the structure of the human gut microbiome, many data mining methods have been applied to microbial composition data. Cluster analysis, one of the key data mining methods that have been used in human gut microbiome research, can classify the human gut microbiome into three clusters, called enterotypes. The human gut microbiome has been suggested to be composed of the microbial assemblages or groups of co-occurring microbes, and one human gut microbiome can contain several microbial assemblages. However, cluster analysis can cluster samples into groups without capturing minor assemblages. In addition, a reliable method of assemblage detection has not been established, and little is known about the distributions of microbial assemblages at a population-level scale. Accordingly, the purpose of this study was to clarify the microbial assemblages in the human gut microbiome. In this study, we detected gut microbiome assemblages using a latent Dirichlet allocation (LDA) method, which was first proposed for the classification of documents in natural language processing. We applied LDA to a large-scale human gut metagenome dataset and found that a four-assemblage LDA model can represent relationships between enterotypes and assemblages with high interpretability. This model indicates that each individual tends to have several assemblages, and each of three assemblages corresponded to each enterotype. However, the C-assemblage can exist in all enterotypes. Interestingly, the dominant genera of the C-assemblage (Clostridium, Eubacterium, Faecalibacterium, Roseburia, Coprococcus, and Butyrivibrio) included butyrate-producing species such as Faecalibacterium prausnitzii. Finally, we revealed that genera mainly appearing in the same assemblage were correlated to each other. We conducted an assemblage analysis on a large-scale human gut metagenome dataset using LDA, a powerful method for detection of microbial assemblages. This approach has the potential to reveal the structure of the human gut microbiome.

microbiology↗

bioRxiv2019

Mucin O-glycans facilitate symbiosynthesis to maintain gut immune homeostasis

The dysbiosis of gut microbiota has been implicated in the pathogenesis of inflammatory bowel diseases (IBDs); however, the underlying mechanisms have not yet been elucidated. Heavily glycosylated mucin not only establishes a first-line barrier against pathogens, but also serves as a niche for microbial growth. We hypothesized that dysbiosis may cause abnormal mucin utilization and microbial metabolic dysfunction. To test this hypothesis, we analyzed short-chain fatty acids (SCFAs) and mucin components in the stool samples of 40 healthy subjects, 49 ulcerative colitis (UC) patients, and 44 Crohns disease (CD) patients from Japan. The levels of n-butyrate were significantly lower in the stools of both the CD and UC patients than in those of the healthy subjects. Correlation analysis identified 7 bacterial species positively correlated with n-butyrate levels, among which the major n-butyrate producer, Faecalibacterium prausnitzii, was particularly underrepresented in CD patients, but not in UC patients. In UC patients, there were inverse correlations between mucin O-glycan levels and the production of SCFAs, such as n-butyrate, suggesting that mucin O-glycans act as an endogenous fermentation substrate for n-butyrate production. Indeed, mucin-fed rodents exhibited enhanced n-butyrate production, leading to the expansion of RORgt+Treg cells and IgA-producing cells in the colonic lamina propria. Importantly, the availability of mucin-associated O-glycans to the microbiota was significantly reduced in n-butyrate-deficient UC patients. Taken together, our findings highlight the biological significance of the symbiosynthesis pathway in the production of n-butyrate, which maintains gut immune homeostasis.

microbiology↗

bioRxiv2018

Cryo-EM structure of the volume-regulated anion channel LRRC8

Maintenance of cell volume against osmotic change is crucial for proper cell functions, such as cell proliferation and migration. The leucine-rich repeat-containing 8 (LRRC8) proteins are anion selective channels, and were recently identified as pore components of the volume-regulated anion channels (VRACs), which extrude anions to decrease the cell volume upon cell-swelling. Here, we present the human LRRC8A structure, determined by a single-particle cryo-electron microscopy analysis. The sea anemone-like structure represents a trimer of dimers assembly, rather than a symmetrical hexameric assembly. The four-spanning transmembrane region has a gap junction channel-like membrane topology, while the LRR region containing 15 leucine-rich repeats forms a long twisted arc. The channel pore is along the central axis and constricted on the extracellular side, where the highly conserved polar and charged residues at the tip of the extracellular helix contribute to the anion and other osmolyte permeability. Comparing the two structural populations facilitated the identification of both compact and relaxed conformations, suggesting that the LRR region is flexible and mobile with rigid-body motions, which might be implicated in structural transitions upon pore opening. Overall, our structure provides a framework for understanding the molecular mechanisms of this unique class of ion channels.

molecular biology↗

bioRxiv2018

Ancient DNA analysis of food remains in human dental calculus from the Edo period, Japan

Objectives: Although there are many methods for reconstructing diets of the past, detailed taxon identification is still challenging, and most plants hardly remain at a site. In this study, we applied DNA metabarcoding to dental calculus of Edo people for the taxonomic identification of food species.\n\nMaterials and Methods: DNA was extracted from 13 human dental calculi from the Unko-in site (18th-19th century) of the Edo period, Japan. Polymerase chain reaction (PCR) and sequencing were performed using a primer set specific to the genus Oryza, because rice (Oryza sativa) was a staple food and this was the only member of this genus present in Japan at that time. DNA metabarcoding targeting plants, animals (meat and fish), and fungi was also carried out to investigate dietary diversity.\n\nResults: We detected amplified products of the genus Oryza from more than half of the samples using PCR and Sanger sequencing. DNA metabarcoding enabled us to identify taxa of plants and fungi, although taxa of animals were not detected, except human.\n\nDiscussion: Most of the plant taxonomic groups are present in Japan and include species consumed as food at that time, as confirmed by historical literature. The other groups featured in the lifestyle of Edo people, such as for medicinal purposes and tobacco. The results indicate that plant DNA analysis from calculus provides information about food diversity and lifestyle habits from the past and can complement other analytical methods such as starch analysis and stable isotope analysis.

molecular biology↗