Biology subjects

Kurokawa, M.

Publications and source records attributed to Kurokawa, M..

7 recordsLinked to original sources

bioRxiv2026

Integrating Segmental Deuteration iCM-SANS with SAXS and MD for Dynamical Analysis of Multi-domain Proteins

Multi-domain proteins (MDPs) adopt diverse conformations arising from cooperative inter-domain motions, and such dynamics are intimately coupled to their biological functions. Quantitative characterization of these motions is crucial for elucidating their functional mechanisms. Although small-angle X-ray scattering (SAXS) provides information on overall domain arrangement, the limited experimental constraints hinder reliable discrimination of conformational ensembles derived from molecular dynamics (MD) simulations. To address this limitation, complementary experimental constraints that enable to observe domain-selective structural information are required. Inverse contrast-matching small-angle neutron scattering (iCM-SANS), combined with segmental deuteration, enables selective visualization of individual domains and thus provides such complementary information. However, practical strategies for preparing segmentally deuterated MDPs with arbitrary domain labelling have yet to be established. Here, we develop an experimental protocol that integrates controlled protein deuteration with high-efficiency multi-step protein ligation to generate a segmentally deuterated MDP in high yield. The combined use of SAXS and iCM-SANS yields complementary structural constraints that enhance discrimination of MD-derived conformational ensembles. This protocol expands the applicability of segment-selective visualization and also provides an opportunity for high-precision analysis of dynamics in complex MDPs. SynopsisSegmental deuteration enabled by high-efficiency multi-step protein ligation, combined with inverse contrast-matching SANS and SAXS, provides structural constraints that improve discrimination of molecular dynamics ensembles of multi-domain proteins. IMPORTANTthis document contains embedded data - to preserve data integrity, please ensure where possible that the IUCr Word tools (available from http://journals.iucr.org/services/docxtemplate/) are installed when editing this document.

biophysics↗

bioRxiv2022

Metagenomic thermometer

Various microorganisms exist in environments, and each of which has an optimal growth temperature (OGT). The relationship between genomic information and OGT of each species has long been studied, and one such study revealed that OGT of prokaryotes can be accurately predicted based on the fraction of seven amino acids (IVYWREL) among all encoded amino-acid sequences in its genome. Extending this discovery, the fraction of IVYWREL among all amino acids encoded in the metagenomic DNA in an environment may reflect the environmental temperature to which the microorganisms have adapted. In the present study, we developed a Metagenomic Thermometer as a means of predicting environmental temperature based on metagenomic sequences. Analysis of 41 metagenomic samples obtained from hot springs of varying temperature revealed that the Metagenomic Thermometer could indeed predict environmental temperature with high accuracy (R2 = 0.75). Although the taxonomic composition of microbial community was substantially affected by pH, the performance of the Metagenomic Thermometer was refractory to changes in community composition. The Metagenomic Thermometer was also applied to 616 human gut metagenomic samples, yielding a reasonably accurate value (i.e., 36.22 {+/-} 1.98{degrees}C) for human body temperature. Our results suggest that environmental temperature can be predicted based on metagenomic sequence data.

bioinformatics↗

bioRxiv2021

Local adaptation mediated niche expansion in correlation with genetic richness

As a central issue in evolution and ecology, the quantitative relationship among the genome, adaptation and the niche was investigated. Local adaptation of five Escherichia coli strains carrying either the wild-type genome or reduced genomes was achieved by experimental evolution. A high-throughput fitness assay of the ancestor and evolved populations across an environmental gradient of eight niches resulted in a total of 80 fitness curves generated from 2,220 growth curves. Further analyses showed that the increases in both local adaptiveness and niche broadness were negatively correlated with genetic richness. Local adaptation caused common niche expansion, whereas niche expansion for generality or speciality was decided by genetic richness. The order of the mutations accumulated stepwise was correlated with the magnitude of the fitness increase attributed to mutation accumulation. Pre-adaptation probably participated in coordination among genetic richness, local adaptation and niche expansion.

evolutionary biology↗

bioRxiv2020

MECOM permits pancreatic acinar cell dedifferentiation avoiding cell death under stress conditions

Maintenance of the pancreatic acinar cell phenotype suppresses tumor formation. Hence, repetitive acute or chronic pancreatitis, stress conditions in which the acinar cells dedifferentiate, predispose for cancer formation in the pancreas. Dedifferentiated acinar cells acquire a large panel of duct cell-specific markers. However, it remains unclear to what extent dedifferentiated acini differ from native duct cells and which genes are uniquely regulating acinar cell dedifferentiation. Moreover, most studies have been performed in mice since the availability of human cells is scarce. Here, we applied a non-genetic lineage tracing method of human pancreatic exocrine acinar and duct cells that allowed cell-type-specific gene expression profiling by RNA sequencing. Subsequent to this discovery analysis, one transcription factor that was unique for dedifferentiated acinar cells was functionally characterized. RNA sequencing analysis showed that human dedifferentiated acinar cells expressed genes in Pathways of cancer with prominence of MECOM (EVI-1), a transcription factor that was not expressed by duct cells. During mouse embryonic development, pre-acinar cells also transiently expressed MECOM and in the adult mouse pancreas, MECOM was re-expressed when mice were subjected to acute and chronic pancreatitis, conditions in which acinar cells dedifferentiate. In human cells and in mice, MECOM expression correlated with and was directly regulated by SOX9. Mouse acinar cells that, by genetic manipulation, lose the ability to upregulate MECOM showed impaired cell adhesion, more prominent acinar cell death and suppressed acinar cell dedifferentiation by limited ERK signaling. In conclusion, we transcriptionally profiled the two major human pancreatic exocrine cell types, acinar and duct cells, during experimental stress conditions. We provide insights that in dedifferentiated acinar cells, cancer pathways are upregulated in which MECOM is a critical regulator that suppresses acinar cell death by permitting cellular dedifferentiation.

cancer biology↗

bioRxiv2020

The common chromosomal periodicity of transcriptomes is correlated with the bacterial growth rate

The growth rate, representing the fitness of a bacterial population, is determined by the whole transcriptome. Chromosomal periodicity is a representative overall feature of the whole transcriptome; however, whether and how it is associated with the bacterial growth rate are unknown. To address these questions, we analyzed a total of 213 transcriptomes of genetically differentiated Escherichia coli strains growing in an assortment of culture conditions varying in terms of temperature, nutrition level and osmotic pressure. Intriguingly, the Fourier transform identified a common chromosomal periodicity of transcriptomes, which was independent of the variation in genomes and environments. In addition, fitting of the theoretical model found that the amplitudes of the periodic transcriptomes were significantly correlated with the growth rates. This novel finding successfully identified a single parameter representing the global pattern of the whole transcriptome for the first time and indicated that bacterial growth was correlated with the magnitude of chromosomal differentiation in gene expression. These results provided an alternative global parameter for evaluating the adaptiveness of a growing bacterial population and provided a quantitative rule that makes it possible to predict the growth dynamics according to the gene expression pattern.

genomics↗

bioRxiv2019

PRDM3 attenuates pancreatitis and pancreatic tumorigenesis by regulating inflammatory response

Pancreatic ductal adenocarcinoma (PDAC) is associated with metaplastic changes in the pancreas but the transcriptional program underlying these changes is incompletely understood. The zinc finger transcription factor, PRDM3, is lowly expressed in normal pancreatic acini and its expression increases during tumorigenesis. Although PRDM3 promotes proliferation and migration of PDAC cell lines, the role of PRDM3 during tumor initiation from pancreatic acinar cells in vivo is unclear. In this study, we showed that high levels of PRDM3 expression in human pancreas was associated with pancreatitis, and well-differentiated but not poorly differentiated carcinoma. We examined PRDM3 function in pancreatic acinar cells during tumor formation and pancreatitis by inactivating Prdm3 using a conditional allele (Ptf1aCreER;Prdm3flox/floxmice) in the context of oncogenic Kras expression and supraphysiological cerulein injections, respectively. In Prdm3-deficient mice, KrasG12D-driven preneoplastic lesions were more abundant and progressed to high-grade precancerous lesions more rapidly. This is consistent with our observations that low levels of PRDM3 in human PDAC was correlated significantly with poorer survival in patient. Moreover, loss of Prdm3 in acinar cells elevated exocrine injury, enhanced immune cell activation and infiltration, and greatly increased acinar-to-ductal cell reprogramming upon cerulein-induced pancreatitis. Whole transcriptome analyses of Prdm3 knockout acini revealed that pathways involved in inflammatory response and Hif-1 signaling were significantly upregulated in Prdm3-depleted acinar cells. Taken together, our results suggest that Prdm3 favors the maintenance of acinar cell homeostasis through modulation of their response to inflammation and oncogenic Kras activation, and thus plays a previously unexpected suppressive role during PDAC initiation.

cancer biology↗

bioRxiv2018

A decay effect of bacterial growth rate associated with genome reduction

Bacterial growth is an important topic in microbiology and of crucial importance to better understand living cells. Bacterial growth dynamics are quantitatively examined using various methods to determine the physical, chemical or biological features of growing populations. Due to methodological differences, the exponential growth rate, which is a parameter that is representative of growth dynamics, should be differentiated. This study experimentally verified the differentiation in growth rates attributed to different methodologies, and demonstrated that the most popular method, optical turbidity, led to the determination of a lower growth rate in comparison to the methods based on colony formation and ATP abundance, due to a decay effect of reading OD600 during a population increase. Accordingly, the logistic model, which is often applied to growth data reading the OD600, was revised by introducing a new parameter: the decay rate, to compensate for the lowered estimation in growth rates. The modified logistic model not only presented an improved goodness of fit in comparison to the original model but also led to an intriguing finding of a correlation between genome reduction and the decay rate. The decay effect seemed to be partially attributed to the decrease in cell size accompanied by a population increase and was medium dependent. In summary, the present study provides not only a better theoretical tool for the high-throughput studies on bacterial growth dynamics linking with experimental data using optical turbidity to the theoretical analysis with biological importance, but also a valuable insight for understanding the genome evolution and fitness increase in microbial life.

microbiology↗