Biology subjects

Deguchi, T.

Publications and source records attributed to Deguchi, T..

10 recordsLinked to original sources

bioRxiv2025

Functional Unknomics of the SAR11 clade using bioinformatics approaches

A substantial fraction of the genes in marine bacteria lack detectable sequence similarity to genes with known functions. These functionally uncharacterized genes--collectively referred to as the "unknome"--represent a largely unexplored genetic repertoire harboring insights into marine bacterial ecology. In this study, we explored the function of the unknome of SAR11 clade, the most abundant bacterial lineage in the ocean, with a particular focus on genes that provide insight into their ecology. Based on the COG classification, approximately 58% of SAR11 ortholog groups were classified as unknome. Among the SAR11 unknome, we successfully inferred the functions of 69 ortholog groups that are conserved in SAR11 clade by protein structure similarity searches and genomic context analyses. These ortholog groups include putative transporter components, supporting the current ecological understanding that SAR11 clade is specialized in substrate uptake to adapt to oligotrophic marine environments. Furthermore, structural analysis indicated that the DUF2237-containing protein, enriched in marine environments, has potential interactions with purine nucleotide-containing compounds. This may suggest the existence of unique nucleotide utilization mechanisms in marine bacteria. In addition, we found candidates of virus defense systems within the unknome, which demonstrates that diverse defense systems are present at least in one-third of the cultured SAR11 strains. The conservation of these viral defense systems, even within streamlined SAR11 genomes, suggests that they confer significant ecological advantages. Our exploration provided insights into the genetic basis of bottom-up processes (adaptation to oligotrophic environments) and top-down processes (antiviral defense strategy) contributing to ecological success of SAR11.

microbiology↗

bioRxiv2025

Data-efficient protein mutational effect prediction with weak supervision by molecular simulation and protein language models

Machine learning-based protein mutational effect prediction is widely used in protein engineering and pathogenicity prediction, but training data scarcity remains a major challenge due to high costs of experimental measurements. A previous study proposed data augmentation using computational estimates by molecular simulation. However, this approach has been limited to predicting mutational effects on thermostability. Here, we present a new data augmentation method that combines molecular simulation with zero-shot prediction computed by protein language models. These computational estimates serve as "weak" training data to supplement experimental training data. Our method dynamically adjusts the weight and inclusion of weak training data based on available experimental training data. This reduces potential negative impacts of weak training data while extending applicability to diverse protein properties such as binding affinity and enzymatic activity. Benchmark tests demonstrate that our method improves prediction accuracy particularly when experimental training data are scarce. These results indicate the capability of our approach to advance protein engineering and pathogenicity prediction in small data regimes.

bioinformatics↗

bioRxiv2023

Fast and robust 3D MINFLUX excitation with a variable phase plate

MINFLUX has achieved record resolution in superresolution imaging and single fluorophore tracking. It is based on localizing single fluorophores by rapid probing with a patterned beam that features a local intensity minimum. Current implementations, however, are complex and expensive and are limited in speed and robustness. Here, we show that a combination of an electro-optical modulator with a segmented birefringent element such as a spatial light modulator produces a variable phase plate for which the phase can be scanned on the MHz timescale. Bisected or top-hat phase patterns generate high-contrast compact excitation point-spread functions for MINFLUX localization in the x,y, and z-direction, respectively, which can be scanned around a fluorophore within a microsecond and alternated among different excitation wavelengths. We discuss how to compensate for non-optimal performance of the components and present a robust 3D and multi-color MINFLUX excitation module with record speed, which we envision as an integral component of a high-performance and cost-effective open-source MINFLUX.

biophysics↗

bioRxiv2023

The anti-obesity effect of fish oil in diet-induced obese mice occurs via the induction of heat production in brown but not white adipose tissue

Aims/IntroductionThe {omega}3 polyunsaturated fatty acids in fish oil enhance heat production in adipocytes and exert anti-obesity effects, but the effects of fish oil on heat production in diet-induced obese (DIO) mice are unclear. In this study, we examined whether diets containing fish oil increased the expression of heat-producing genes in adipose tissue and increased body temperature in DIO mice, resulting in weight loss. We also examined fibroblast growth factor 21 (FGF21) levels in blood and the expression of the FGF21 gene in adipose tissue of DIO mice fed fish oil. Materials and MethodsC57BL6/J mice were fed a lard-based high-fat diet for 8 weeks starting at 5 weeks of age and then divided into two groups: one group was fed a fish oil-based high-fat diet, and the other group was fed a lard-based high-fat diet continuously for another 8 weeks. Mice fed a control diet for 16 weeks from the age of 5 weeks served as the control group. Mice were dissected at 21 weeks and used for analysis. ResultsMice fed a fish oil-based high-fat diet lost body weight gain, adipose tissue weight gain, and reduced insulin/leptin resistance. In addition, the rectal temperatures of mice fed a fish oil-based high-fat diet remained higher. The administration of fish oil increased the expression of heat-producing genes in brown adipose tissue (BAT) but did not alter heat-producing genes in inguinal white adipose tissue (WAT). In DIO mice fed a fish oil-based high-fat diet, the FGF21 expression in BAT increased. Furthermore, {beta}klotho expression in BAT increased and the blood FGF21 concentration was decreased compared to mice fed a lard-based high-fat diet. ConclusionsIn DIO mice, fish oil was shown to increase rectal temperature and ameliorate obesity. Furthermore, fish oil enhanced heat production in BAT, but not WAT, in DIO mice.

animal behavior and cognition↗

bioRxiv2022

Direct observation of motor protein stepping in living cells using MINFLUX

Dynamic measurements of molecular machines can provide invaluable insights into their mechanism, but have been challenging in living cells. Here, we developed live-cell tracking of single fluorophores with nanometer spatial and millisecond temporal resolution in 2D and 3D using the recently introduced super-resolution technique MINFLUX. This allowed us to resolve the precise stepping motion of the motor protein kinesin-1 as it walks on microtubules in living cells. In addition, nanoscopic tracking of motors on microtubule of fixed cells enabled us to resolve their spatial organization with protofilament resolution. Our approach will enable future in vivo studies of motor protein kinetics in complex environments and super-resolution mapping of dense microtubule arrays, and pave the way towards monitoring functional conformational changes of protein machines at high spatiotemporal resolution in living systems.

biophysics↗

bioRxiv2022

MicroFPGA: an affordable FPGA platform for microscope control

Modern microscopy relies increasingly on microscope automation to improve throughput, ensure reproducibility or observe rare events. Automation requires in particular computer control of the important elements of the microscope. Furthermore, optical elements that are usually fixed or manually movable can be placed on electronically-controllable elements. In most cases, a central electronics board is necessary to generate the control signals they require and to communicate with the computer. Fur such tasks, Arduino microcontrollers are widely used due to their low cost and programming entry barrier. However, they are limiting in their performance for applications that require high-speed or multiple parallel processes. Field programmable gate arrays (FPGA) are the perfect technology for high-speed microscope control, as they are capable of processing signals in parallel and with high temporal precision. While plummeting price made the technology available to consumers, a major hurdle remains the complex languages used to configure them. In this work, we used an affordable FPGA, delivered with an open-source and friendly-to-use programming language, to create a versatile microscope control platform called MicroFPGA. It is capable of synchronously triggering cameras and multiple lasers following complex patterns, as well as generating various signals used to control microscope elements such as filter wheels, servomotor stages, flip-mirrors, laser power or acoustooptic modulators. MicroFPGA is open-source and we provide online Micro-Manager, Java, Python and LabVIEW libraries, together with blueprints and tutorials.

biophysics↗

bioRxiv2020

Emergence and evolution of antimicrobial resistance genes and mutations in Neisseria gonorrhoeae

Antimicrobial resistance in Neisseria gonorrhoeae is a global health concern. Strains from two internationally circulating sequence types, ST-7363 and ST-1901, have acquired resistance to treatment with third-generation cephalosporins mainly due to the emergence of mosaic penA alleles. These two STs were first detected in Japan; however, when and how the mosaic penA alleles emerged and spread to other countries remains unknown. Here, we addressed the evolution of penA alleles by obtaining complete genomes from three Japanese ST-1901 clinical isolates harboring mosaic penA allele 34 (penA-34) dating from 2005 and generating a phylogenetic representation of 1,075 strains sampled from 37 countries. We also sequenced the genomes of 103 Japanese ST-7363 N. gonorrhoeae isolates from 1996-2005 and reconstructed a phylogeny including 88 previously sequenced genomes. Based on an estimate of the time of emergence of ST-1901 harboring mosaic penA-34 and ST-7363 harboring mosaic penA-10, and >300 additional genome sequences of Japanese strains representing multiple STs isolated in 1996-2015, we suggest that penA-34 in ST-1901 was generated from penA-10 via recombination with another Neisseria species, followed by a second recombination event with a gonococcal strain harboring wildtype penA-1. Following the acquisition of penA-10 in ST-7363, a dominant sub-lineage rapidly acquired fluoroquinolone resistance mutations at GyrA 95 and ParC 87-88, possibly due to independent mutations rather than horizontal gene transfer. Literature data suggest the emergence of these resistance determinants may reflect selection from the standard treatment regimens in Japan at that time. Our findings highlight how recombination and antibiotic use across and within Neisseria species intersect in driving the emergence and spread of drug-resistant gonorrhea. Author summaryAntimicrobial resistance is recognized as one of the greatest threats to human health, and Neisseria gonorrhoeae resistance is classified as one of the most urgent. The two major internationally spreading lineages resistant. to first line drugs likely originated in Japan, but when and how their genetic resistance determinants emerged remain unknown. In this study, we conducted an evolutionary analysis using clinical N. gonorrhoeae isolates from 37 countries, including a historical collection of Japanese isolates, to investigate the emergence of resistance in each of the two major lineages. We showed that the penA allele responsible for resistance to cephalosporins, the first-line treatment for gonorrhea, was possibly generated by two recombination events, one from another Neisseria species and one from another N. gonorrhoeae lineage. We also showed that mutations responsible for resistance to a previously widely used antibiotic treatment occurred twice independently in one of the two major lineages. The emergence of the genetic resistance determinants potentially reflects selection from the standard treatment regimen at that time. Our findings highlight how recombination (horizontal gene transfer) and antibiotic use across and within a bacterial species intersect in driving the emergence and spread of antimicrobial resistance genes and mutations.

microbiology↗

bioRxiv2019

Volumetric Lissajous Confocal Microscopy

Dynamic biological systems present challenges to existing three-dimensional (3D) optical microscopes because of their continuous temporal and spatial changes. Most techniques are based on rigid architectures, as in confocal microscopy, where a laser beam is sequentially scanned at a predefined spatial sampling rate and pixel dwell time. Here, we developed volumetric Lissajous confocal microscopy to achieve unsurpassed 3D scanning speed with a tunable sampling rate. The system combines an acoustic liquid lens for continuous axial focus translation with a resonant scanning mirror. Accordingly, the excitation beam follows a dynamic Lissajous trajectory enabling sub-millisecond acquisitions of image series containing 3D information at a sub-Nyquist sampling rate. By temporal accumulation and/or advanced interpolation algorithms, volumetric imaging rate is selectable using a post-processing step at the desired spatiotemporal resolution for events of interest. We demonstrate multicolor and calcium imaging over volumes of tens of cubic microns with acquisition speeds up to 5 kHz.

biophysics↗

bioRxiv2019

Fourier ring correlation simplifies image restoration in fluorescence microscopy

Fourier ring correlation (FRC) has recently gained some popularity among (super-resolution) fluorescence microscopists as a straightforward and objective method to measure the effective resolution of a microscopy image. While the knowledge of the numeric resolution value is helpful in e.g. interpreting imaging results, much more practical use can be made of FRC analysis - in this article we propose novel blind image restoration methods enabled by it. We apply FRC to perform image de-noising by frequency domain filtering. We propose novel blind linear and non-linear image deconvolution methods that use FRC to estimate the effective point-spread-function, directly from the images, with no need for prior knowledge of the instrument or sample characteristics. The deconvolution is shown to work exquisitely with both two- and three-dimensional images. We also show how FRC can be used as a powerful metric to observe the progress of iterative deconvolution. While developing the image restoration methods, we also addressed two important limitations in FRC that are of more general interest: how to make FRC work with single images and with three-dimensional images with anisotropic resolution.

bioinformatics↗

bioRxiv2018

Image Scanning Microscopy with Single-Photon Detector Array

Image scanning microscopy (ISM) improves the spatial resolution of conventional confocal laser-scanning microscopy (CLSM), but current implementations reduce versatility and restrict its combination with fluorescence spectroscopy techniques, such as fluorescence lifetime. Here, we describe a natural design of ISM based on a fast single-photon detector array, which allows straightforward upgrade of an existing confocal microscope, without compromising any of its functionalities. In contrast to all-optical ISM implementations, our approach provides access to the raw scanned images, opening the way to adaptive reconstruction methods, capable of considering different imaging conditions and distortions. We demonstrate its utility in the context of fluorescence lifetime, deep, multicolor and live-cell imaging. This implementation will pave the way for a transparent and massive transition from conventional CLSM to ISM.\n\nconfocal microscopy | time-resolved spectroscopy | image scanning microscopy | single-photon detector array

bioengineering↗