Strain dropouts reveal interactions that govern the metabolic output of the gut microbiome.

The gut microbiome is complex, raising questions about the role of individual strains in the community. Here, we address this question by constructing variants of a complex defined community in which we eliminate strains that occupy the bile acid 7α-dehydroxylation niche. Omitting Clostridium scindens (Cs) and Clostridium hylemonae (Ch) eliminates secondary bile acid production and reshapes the community in a highly specific manner: eight strains change in relative abundance by >100-fold. In single-strain dropout communities, Cs and Ch reach the same relative abundance and dehydroxylate bile acids to a similar extent. However, Clostridium sporogenes increases >1,000-fold in the ΔCs but not ΔCh dropout, reshaping the pool of microbiome-derived phenylalanine metabolites. Thus, strains that are functionally redundant within a niche can have widely varying impacts outside the niche, and a strain swap can ripple through the community in an unpredictable manner, resulting in a large impact on an unrelated community-level phenotype.

Design, construction, and in vivo augmentation of a complex gut microbiome.

Efforts to model the human gut microbiome in mice have led to important insights into the mechanisms of host-microbe interactions. However, the model communities studied to date have been defined or complex, but not both, limiting their utility. Here, we construct and characterize in vitro a defined community of 104 bacterial species composed of the most common taxa from the human gut microbiota (hCom1). We then used an iterative experimental process to fill open niches: germ-free mice were colonized with hCom1 and then challenged with a human fecal sample. We identified new species that engrafted following fecal challenge and added them to hCom1, yielding hCom2. In gnotobiotic mice, hCom2 exhibited increased stability to fecal challenge and robust colonization resistance against pathogenic Escherichia coli. Mice colonized by either hCom2 or a human fecal community are phenotypically similar, suggesting that this consortium will enable a mechanistic interrogation of species and genes on microbiome-associated phenotypes.

Identification of subepithelial mesenchymal cells that induce IgA and diversify gut microbiota.

Immunoglobulin A (IgA) maintains a symbiotic equilibrium with intestinal microbes. IgA induction in the gut-associated lymphoid tissues (GALTs) is dependent on microbial sampling and cellular interaction in the subepithelial dome (SED). However it is unclear how IgA induction is predominantly initiated in the SED. Here we show that previously unrecognized mesenchymal cells in the SED of GALTs regulate bacteria-specific IgA production and diversify the gut microbiota. Mesenchymal cells expressing the cytokine RANKL directly interact with the gut epithelium to control CCL20 expression and microfold (M) cell differentiation. The deletion of mesenchymal RANKL impairs M cell-dependent antigen sampling and B cell-dendritic cell interaction in the SED, which results in a reduction in IgA production and a decrease in microbial diversity. Thus, the subepithelial mesenchymal cells that serve as M cell inducers have a fundamental role in the maintenance of intestinal immune homeostasis.

Mapping the T cell repertoire to a complex gut bacterial community.

Certain bacterial strains from the microbiome induce a potent, antigen-specific T cell response1-5. However, the specificity of microbiome-induced T cells has not been explored at the strain level across the gut community. Here, we colonize germ-free mice with complex defined communities (roughly 100 bacterial strains) and profile T cell responses to each strain. The pattern of responses suggests that many T cells in the gut repertoire recognize several bacterial strains from the community. We constructed T cell hybridomas from 92 T cell receptor (TCR) clonotypes; by screening every strain in the community against each hybridoma, we find that nearly all the bacteria-specific TCRs show a one-to-many TCR-to-strain relationship, including 13 abundant TCR clonotypes that each recognize 18 Firmicutes. By screening three pooled bacterial genomic libraries, we discover that these 13 clonotypes share a single target: a conserved substrate-binding protein from an ATP-binding cassette transport system. Peripheral regulatory T cells and T helper 17 cells specific for an epitope from this protein are abundant in community-colonized and specific pathogen-free mice. Our work reveals that T cell recognition of commensals is focused on widely conserved, highly expressed cell-surface antigens, opening the door to new therapeutic strategies in which colonist-specific immune responses are rationally altered or redirected.

Identification of Factors Necessary for Gatekeeper of Overdose.

Overdose has become a global social problem. The Japanese government requires gatekeeper training to detect and prevent indicators of overdose and suicide. However, knowledge of necessary factors for the gatekeeper of overdose (patient intervention) is limited. This study aimed to investigate the characteristics of individuals who experienced intervening persons expected to overdose, and to identify the factors required of gatekeepers. A Google form was used to survey 298 pharmacists and registered sellers in Japan. We searched for factors by logistic analysis. Knowledge of prescription drugs used for overdose was higher among pharmacists than among registered sellers. Conversely, pharmacists and registered sellers had similar knowledge about OTC drugs. Overall multivariate logistic regression analysis revealed countermeasures against overdose at their workplace (odds ratio (OR): 4.01, 95% confidence interval (CI): 2.25-7.15, p < 0.01) and knowledge that overdose is on the rise (OR: 1.93, 95% CI: 1.04-3.69, p < 0.05) to be significantly associated with intervention experience as a gatekeeper. Countermeasures against overdose at their workplace (OR: 2.40, 95% CI: 1.10-5.25, p < 0.05) in pharmacists and years of work experience (OR: 1.13, 95% CI: 1.04-1.24, p < 0.05), countermeasure against overdose at their workplace (OR: 3.43, 95% CI: 1.18-10.0, p < 0.05), and willingness to participate in study sessions and workshops on overdose (OR: 3.50, 95% CI: 1.51-8.10, p < 0.05) in registered seller were significantly associated with intervention experience as a gatekeeper. These results are useful evidences for countermeasures and gatekeeper training for overdose at pharmacies and drugstores in the community.

Creating and evaluating the score to assess overdose: the OD score.

BACKGROUND: During disasters (including epidemics such as coronavirus disease 2019), the capacity of emergency departments is exceeded, thereby hindering the administration of appropriate lifesaving measures. Furthermore, the number of overdose patients increases because of the stress overload during emergency situation. The fact that overdose patients are forced to be transported to medical facilities that do not typically treat them is becoming worrisome. Moreover, there is no definitive score for overdose. This study aimed to create a patient-specific scoring system to assess overdose. METHODS: This was a retrospective single-center study. The evidence-based OD score was evaluated on a scale of 0-15. Further, logistic analysis and receiver operating characteristic (ROC) curve analysis were performed to evaluate the score. RESULTS: Overall, 262 patients (including 118 overdose patients) receiving care at the intensive care unit of Japan's Teikyo University Hospital in 2021 were targeted. Regarding the total OD score, ROC analysis revealed a cutoff of 8 (area under the curve [AUC]: 0.99, 95% confidence interval [CI]: 0.980-0.997, sensitivity: 0.95, specificity: 0.95, p < 0.05), which was considered to indicate an overdose. Of the items evaluated in the OD score, the scenario at the location of the patient's discovery (adjusted odds ratio [AOR]: 16.8, 95% CI: 5.0-255.9, p = 0.002) and recent experience of mental anxiety (AOR: 55.7, 95% CI: 2.8-5399.5, p = 0.03) significantly predicted an overdose in multivariable logistic regression analysis. External validation revealed that the OD score could also identify overdose in patients treated in a cohort from 2022 (average cutoff: 8.6, average AUC: 1.0, p < 0.0001). CONCLUSIONS: The OD score could accurately assess overdose patients. Medical facilities that do not frequently address overdose patients will benefit from the use of this score.

Peptides Derived from Soybean ß-Conglycinin Induce the Migration of Human Peripheral Polymorphonuclear Leukocytes.

Food-derived peptides have various biological activities. When food proteins are ingested orally, they are digested into peptides by endogenous digestive enzymes and absorbed by the immune cell-rich intestinal tract. However, little is known about the effects of food-derived peptides on the motility of human immune cells. In this study, we aimed to understand the effects of peptides derived from a soybean protein ß-conglycinin on the motility of human peripheral polymorphonuclear leukocytes. We illustrated that MITL and MITLAIPVNKPGR, produced by digestion using in-vivo enzymes (trypsin and pancreatic elastase) of ß-conglycinin, induces the migration of dibutyryl cAMP (Bt2 cAMP)-differentiated human promyelocytic leukemia 60 (HL-60) cells and human polymorphonuclear leukocytes in a dose- and time-dependent manner. This migration was more pronounced in Bt2 cAMP-differentiated HL-60 cells; mRNA expression of formyl peptide receptor (FPR) 1 increased significantly than in all-trans-retinoic acid (ATRA)-differentiated HL-60 cells. This migration was inhibited by tert-butoxycarbonyl (Boc)-MLP, an inhibitor of FPR, and by pretreatment with pertussis toxin (PTX). However, the effect was weak when treated with WRW4, a selective inhibitor of the FPR2. We then demonstrated that MITLAIPVNKPGR induced intracellular calcium responses in human polymorphonuclear leukocytes and Bt2 cAMP-HL60 cells. Furthermore, pre-treatment by fMLP desensitized the calcium response of MITLAIPVNKPGR in these cells. From the above, MITLAIPVNKPGR and MITL derived from soybean ß-conglycinin induced polymorphonuclear leukocyte migration via the FPR1-dependent mechanism. We found chemotactic peptides to human polymorphonuclear leukocytes, which are the endogenous enzyme digests of soybean protein.

Edge-Topological Regulation for in Situ Fabrication of Bridging Nanosensors.

In situ fabrication of well-defined bridging nanostructures is an interesting and unique approach to three-dimensionally design nanosensor structures, which are hardly attainable by other methods. Here, we demonstrate the significant effect of edge-topological regulation on in situ fabrication of ZnO bridging nanosensors. When employing seed layers with a sharp edge, which is a well-defined structure in conventional lithography, the bridging angles and electrical resistances between two opposing electrodes were randomly distributed. The stochastic nature of bridging growth direction at the sharp edges inherently causes such unintentional variation of structural and electrical properties. We propose an edgeless seed layer structure using a two-layers resist method to solve the above uncontrollability of bridging nanosensors. Such bridging nanosensors not only substantially improved the uniformity of structural and electrical properties between two opposing electrodes but also significantly enhanced the sensing responses for NO2 with the smaller variance and the lower limit of detection via in situ controlled electrical contacts.

Image Processing and Machine Learning for Automated Identification of Chemo-/Biomarkers in Chromatography-Mass Spectrometry.

We present a method named NPFimg, which automatically identifies multivariate chemo-/biomarker features of analytes in chromatography-mass spectrometry (MS) data by combining image processing and machine learning. NPFimg processes a two-dimensional MS map (m/z vs retention time) to discriminate analytes and identify and visualize the marker features. Our approach allows us to comprehensively characterize the signals in MS data without the conventional peak picking process, which suffers from false peak detections. The feasibility of marker identification is successfully demonstrated in case studies of aroma odor and human breath on gas chromatography-mass spectrometry (GC-MS) even at the parts per billion level. Comparison with the widely used XCMS shows the excellent reliability of NPFimg, in that it has lower error rates of signal acquisition and marker identification. In addition, we show the potential applicability of NPFimg to the untargeted metabolomics of human breath. While this study shows the limited applications, NPFimg is potentially applicable to data processing in diverse metabolomics/chemometrics using GC-MS and liquid chromatography-MS. NPFimg is available as open source on GitHub (http://github.com/poomcj/NPFimg) under the MIT license.

Self-Anti-Stacking 2D Metal Phosphide Loop-Sheet Heterostructures by Edge-Topological Regulation for Highly Efficient Water Oxidation.

2D metal phosphide loop-sheet heterostructures are controllably synthesized by edge-topological regulation, where Ni2 P nanosheets are edge-confined by the N-doped carbon loop, containing ultrafine NiFeP nanocrystals (denoted as NiFeP@NC/Ni2 P). This loop-sheet feature with lifted-edges prevents the stacking of nanosheets and induces accessible open channels for catalytic site exposure and gas bubble release. Importantly, these NiFeP@NC/Ni2 P hybrids exhibit a remarkable oxygen evolution activity with an overpotential of 223 mV at 20 mA cm-2 and a Tafel slope of 46.1 mV dec-1 , constituting the record-high performance among reported metal phosphide electrocatalysts. The NiFeP@NC/Ni2 P hybrids are also employed as both anode and cathode to achieve an alkaline electrolyzer for overall water splitting, delivering a current density of 10 mA cm-2 with a voltage of 1.57 V, comparable to that of the commercial Pt/C||RuO2 couple (1.56 V). Moreover, a photovoltaic-electrolysis coupling system can as well be effectively established for robust overall water splitting. Evidently, this ingenious protocol would expand the toolbox for designing efficient 2D nanomaterials for practical applications.

Maximizing Conversion of Surface Click Reactions for Versatile Molecular Modification on Metal Oxide Nanowires.

Click reactions (e.g., Huisgen cycloaddition) on metal oxide nanostructures offer a versatile and robust surface molecular modification for various applications because they form strong covalent bonds in a wide range of molecular substrates. This study reports a rational strategy to maximize the conversion rate of surface click reactions on single-crystalline ZnO nanowires by monitoring the reaction progress. p-Polarized multiple-angle incidence resolution spectrometry (pMAIRS) and Fourier-transformed infrared (FT-IR) spectroscopy were employed to monitor the reaction progress of an azide-terminated self-assembled monolayer (SAM) on single-crystalline ZnO nanowires. Although various reaction parameters including the concentration of Cu(I) catalysts, triazolyl ligands, solvents, and target alkynes were systematically examined for the surface click reactions, 10-30% of terminal azide on the nanowire surface remained unreacted. Temperature-dependent FT-IR measurements revealed that such unreacted residual azides deteriorate the thermal stability of the nanowire molecular layer. To overcome this observed conversion limitation of click reactions on nanostructure surfaces, we considered the steric hindrance around the closely packed SAM reaction points, then experimented with dispersing the azide moiety into a methyl-terminated SAM. The mixed-SAM method significantly improved the azide conversion rate to almost 100%. This reaction method enables the construction of spatially patterned molecular surface modifications on metal oxide nanowire arrays without detrimental unreacted azide groups.

Nanowire-based sensor electronics for chemical and biological applications.

Detection and recognition of chemical and biological species via sensor electronics are important not only for various sensing applications but also for fundamental scientific understanding. In the past two decades, sensor devices using one-dimensional (1D) nanowires have emerged as promising and powerful platforms for electrical detection of chemical species and biologically relevant molecules due to their superior sensing performance, long-term stability, and ultra-low power consumption. This paper presents a comprehensive overview of the recent progress and achievements in 1D nanowire synthesis, working principles of nanowire-based sensors, and the applications of nanowire-based sensor electronics in chemical and biological analytes detection and recognition. In addition, some critical issues that hinder the practical applications of 1D nanowire-based sensor electronics, including device reproducibility and selectivity, stability, and power consumption, will be highlighted. Finally, challenges, perspectives, and opportunities for developing advanced and innovative nanowire-based sensor electronics in chemical and biological applications are featured.

Synthesis of Monodispersedly Sized ZnO Nanowires from Randomly Sized Seeds.

We demonstrate the facile, rational synthesis of monodispersedly sized zinc oxide (ZnO) nanowires from randomly sized seeds by hydrothermal growth. Uniformly shaped nanowire tips constructed in ammonia-dominated alkaline conditions serve as a foundation for the subsequent formation of the monodisperse nanowires. By precisely controlling the sharp tip formation and the nucleation, our method substantially narrows the distribution of ZnO nanowire diameters from σ = 13.5 nm down to σ = 1.3 nm and controls their diameter by a completely bottom-up method, even initiating from randomly sized seeds. The proposed concept of sharp tip based monodisperse nanowires growth can be applied to the growth of diverse metal oxide nanowires and thus paves the way for bottom-up grown metal oxide nanowires-integrated nanodevices with a reliable performance.

Controlling Bi-Provoked Nanostructure Formation in GaAs/GaAsBi Core-Shell Nanowires.

We control the formation of Bi-induced nanostructures on the growth of GaAs/GaAsBi core-shell nanowires (NWs). Bi serves as not only a constituent but also a surfactant and nanowire growth catalyst. Thus, we paved a way to achieve unexplored III-V nanostructures employing the characteristic supersaturation of catalyst droplets, structural modifications induced by strain, and incorporation into the host GaAs matrix correlated with crystalline defects and orientations. When Ga is deficient during growth, Bi accumulates on the vertex of core GaAs NWs and serves as a nanowire growth catalyst for the branched structures to azimuthal <112>. We find a strong correlation between Bi accumulation and stacking faults. Furthermore, Bi is preferentially incorporated on the GaAs (112)B surface, leading to spatially selective Bi incorporation into a confined area that has a Bi concentration of over 7%. The obtained GaAs/GaAsBi/GaAs heterostructure with an interface defined by the crystalline twin defects in a zinc-blende structure can be potentially applied to a quantum confined structure. Our finding provides a rational design concept for the creation of GaAsBi based nanostructures and the control of Bi incorporation beyond the fundamental limit.

Unusual Oxygen Partial Pressure Dependence of Electrical Transport of Single-Crystalline Metal Oxide Nanowires Grown by the Vapor-Liquid-Solid Process.

In general, the electrical conductivities of n-type semiconducting metal oxide nanostructures increase with the decrease in the oxygen partial pressure during crystal growth owing to the increased number of crystal imperfections including oxygen vacancies. In this paper, we report an unusual oxygen partial pressure dependence of the electrical conductivity of single-crystalline SnO2 nanowires grown by a vapor-liquid-solid (VLS) process. The electrical conductivity of a single SnO2 nanowire, measured using the four-probe method, substantially decreases by 2 orders of magnitude when the oxygen partial pressure for the crystal growth is reduced from 10-3 to 10-4 Pa. This contradicts the conventional trend of n-type SnO2 semiconductors. Spatially resolved single-nanowire electrical transport measurements, microstructure analysis, plane-view electron energy-loss spectroscopy, and molecular dynamics simulations reveal that the observed unusual oxygen partial pressure dependence of the electrical transport is attributed to the intrinsic differences between the two crystal growth interfaces (LS and VS interfaces) in the critical nucleation of the crystal growth and impurity incorporation probability as a function of the oxygen partial pressure. The impurity incorporation probability at the LS interface is always lower than that at the VS interface, even under reduced oxygen partial pressures.

Rational Method of Monitoring Molecular Transformations on Metal-Oxide Nanowire Surfaces.

Metal-oxide nanowires have demonstrated excellent capability in the electrical detection of various molecules based on their material robustness in liquid and air environments. Although the surface structure of the nanowires essentially determines their interaction with adsorbed molecules, understanding the correlation between an oxide nanowire surface and an adsorbed molecule is still a major challenge. Herein, we propose a rational methodology to obtain this information for low-density molecules adsorbed on metal oxide nanowire surfaces by employing infrared p-polarized multiple-angle incidence resolution spectroscopy and temperature-programmed desorption/gas chromatography-mass spectrometry. As a model system, we studied the surface chemical transformation of an aldehyde (nonanal, a cancer biomarker in breath) on single-crystalline ZnO nanowires. We found that a slight surface reconstruction, induced by the thermal pretreatment, determines the surface chemical reactivity of nonanal. The present results show that the observed surface reaction trend can be interpreted in terms of the density of Zn ions exposed on the nanowire surface and of their corresponding spatial arrangement on the surface, which promotes the reaction between neighboring adsorbed molecules. The proposed methodology will support a better understanding of complex molecular transformations on various nanostructured metal-oxide surfaces.

True Vapor-Liquid-Solid Process Suppresses Unintentional Carrier Doping of Single Crystalline Metal Oxide Nanowires.

Single crystalline nanowires composed of semiconducting metal oxides formed via a vapor-liquid-solid (VLS) process exhibit an electrical conductivity even without an intentional carrier doping, although these stoichiometric metal oxides are ideally insulators. Suppressing this unintentional doping effect has been a challenging issue not only for metal oxide nanowires but also for various nanostructured metal oxides toward their semiconductor applications. Here we demonstrate that a pure VLS crystal growth, which occurs only at liquid-solid (LS) interface, substantially suppresses an unintentional doping of single crystalline SnO2 nanowires. By strictly tailoring the crystal growth interface of VLS process, we found the gigantic difference of electrical conduction (up to 7 orders of magnitude) between nanowires formed only at LS interface and those formed at both LS and vapor-solid (VS) interfaces. On the basis of investigations with spatially resolved single nanowire electrical measurements, plane-view electron energy-loss spectroscopy, and molecular dynamics simulations, we reveal the gigantic suppression of unintentional carrier doping only for the crystal grown at LS interface due to the higher annealing effect at LS interface compared with that grown at VS interface. These implications will be a foundation to design the semiconducting properties of various nanostructured metal oxides.

Substantial Expansion of Detectable Size Range in Ionic Current Sensing through Pores by Using a Microfluidic Bridge Circuit.

Measuring ionic currents passing through nano- or micropores has shown great promise for the electrical discrimination of various biomolecules, cells, bacteria, and viruses. However, conventional measurements have shown there is an inherent limitation to the detectable particle volume (1% of the pore volume), which critically hinders applications to real mixtures of biomolecule samples with a wide size range of suspended particles. Here we propose a rational methodology that can detect samples with the detectable particle volume of 0.01% of the pore volume by measuring a transient current generated from the potential differences in a microfluidic bridge circuit. Our method substantially suppresses the background ionic current from the µA level to the pA level, which essentially lowers the detectable particle volume limit even for relatively large pore structures. Indeed, utilizing a microscale long pore structure (volume of 5.6 × 104 aL; height and width of 2.0 × 2.0 µm; length of 14 µm), we successfully detected various samples including polystyrene nanoparticles (volume: 4 aL), bacteria, cancer cells, and DNA molecules. Our method will expand the applicability of ionic current sensing systems for various mixed biomolecule samples with a wide size range, which have been difficult to measure by previously existing pore technologies.

Targeted deletion of RANKL in M cell inducer cells by the Col6a1-Cre driver.

The gut-associated lymphoid tissues (GALTs), including Peyer's patches (PPs), cryptopatches (CPs) and isolated lymphoid follicles (ILFs), establish a host-microbe symbiosis by the promotion of immune reactions against gut microbes. Microfold cell inducer (MCi) cells in GALTs are the recently identified mesenchymal cells that express the cytokine RANKL and initiate bacteria-specific immunoglobulin A (IgA) production via induction of microfold (M) cell differentiation. In the previous study, the Twist2-Cre driver was utilized for gene deletion in mesenchymal cells including MCi cells. In order to investigate MCi cells more extensively, it will be necessary to develop experimental tools in addition to the Twist2-Cre driver mice and characterize such drivers in specificity and efficiency. Here we show that M cell differentiation and IgA production are impaired in the targeted deletion of RANKL by the Col6a1-Cre driver. We compared Col6a1-Cre with Twist2-Cre in terms of the specificity for mesenchymal cells in GALTs. Col6a1-Cre CAG-CAT-EGFP mice exhibited EGFP expression in podoplanin+CD31- cells including MCi cells, while Twist2-Cre mice were shown to target endothelial cells and podoplanin+CD31- cells. Tnfsf11fl/ΔCol6a1-Cre mice exhibited the absence of M cells and severe IgA reduction together with an alteration in gut microbial composition. Moreover, we analyzed germ free mice to test whether changes in the microbiota are the cause of M cell deficiency. M cell differentiation was normal in the CPs/ILFs of germ free mice, indicating that MCi cells induce M cells independently of microbial colonization. This study demonstrates that Col6a1-Cre driver mice are as useful as Twist2-Cre driver mice for functional analyses of GALT-resident mesenchymal cells, including MCi cells.

Rational Concept for Reducing Growth Temperature in Vapor-Liquid-Solid Process of Metal Oxide Nanowires.

Vapor-liquid-solid (VLS) growth process of single crystalline metal oxide nanowires has proven the excellent ability to tailor the nanostructures. However, the VLS process of metal oxides in general requires relatively high growth temperatures, which essentially limits the application range. Here we propose a rational concept to reduce the growth temperature in VLS growth process of various metal oxide nanowires. Molecular dynamics (MD) simulation theoretically predicts that it is possible to reduce the growth temperature in VLS process of metal oxide nanowires by precisely controlling the vapor flux. This concept is based on the temperature dependent "material flux window" that the appropriate vapor flux for VLS process of nanowire growth decreases with decreasing the growth temperature. Experimentally, we found the applicability of this concept for reducing the growth temperature of VLS processes for various metal oxides including MgO, SnO2, and ZnO. In addition, we show the successful applications of this concept to VLS nanowire growths of metal oxides onto tin-doped indium oxide (ITO) glass and polyimide (PI) substrates, which require relatively low growth temperatures.