Biology subjects

Sakata, M.

Publications and source records attributed to Sakata, M..

7 recordsLinked to original sources

bioRxiv2025

Transgenic Bax gene efficiently induces lethality in mouse early embryos

Apoptosis is an essential physiological process involved in embryonic development, immune responses, and tissue homeostasis. Despite many studies on pro-apoptotic genes, few reports have directly compared the lethality-inducing potential between them under identical conditions. In this study, we evaluated the lethality-inducing potential of three representative pro-apoptotic genes, Bax, Casp3, and Casp9, in mouse early embryos under defined conditions using the doxycycline (Dox)-inducible tetracycline-regulated gene expression system (Tet-On system) in combination with the PiggyBac transposon system. All genes were transcriptionally induced by Dox, and Bax showed the strongest lethal effect, followed by Casp9, while Casp3 did not show any effect. Notably, Bax expression severely impaired blastocyst formation and led to the intense accumulation of the DNA damage marker {gamma}H2AX. These findings suggest that introducing upstream apoptotic regulators leads to the more efficient and widespread activation of the apoptotic cascade. Additionally, an unexpected Dox-dependent increase in the expression of reverse tetracycline-controlled transactivator, which is typically driven by a constitutive promoter, was observed, raising the possibility of unanticipated regulatory mechanisms within the Tet-On system. Overall, this study is expected to contribute to a deeper understanding of apoptotic mechanisms and future advancements in regenerative medicine, reproductive engineering, and cancer research.

bioengineering↗

bioRxiv2024

Microthrombi-growth in ADAMTS13 deficiency exacerbated ulcerative colitis via mucosal and endothelial dysfunction

BackgroundUlcerative colitis (UC) is a chronic inflammatory bowel disease characterized by mucosal inflammation and ulceration, with systemic immune dysregulation exacerbating disease progression. While the accumulation of von Willebrand factor (VWF), normally digested by a disintegrin-like and metalloproteinase with thrombospondin type 1 motif 13 (ADAMTS13) from ultra-large multimers to small molecules, has been implicated in UC pathogenesis, the association between aberrant microthrombus formation and colitis remains unclear. MethodsPlasma and inflamed colonic tissues from UC patients were analyzed. In the experimental colitis model, we employed an intravital imaging technique to reveal real-time structural dynamics of the mucus layer and the mucosal vasculature. Dextran sulfate sodium (DSS)-induced colitis in wild-type and ADAMTS13-deficient mice was evaluated for disease progression, mucus layer disruption, leukocyte recruitment, and thrombus formation in mucosal vessels using intravital multiphoton excitation microscopy within single-cell spatial resolution. ResultsUC patients exhibited significantly reduced plasma ADAMTS13 activity correlating with disease severity and excessive VWF deposition in inflamed colonic tissues. In DSS-induced colitis mice, ADAMTS13 deficiency showed heightened disease activity and increased mucosal erosion in histochemical analysis compared to wild-type mice. A novel methodology established in this study using intravital microscopy successfully appraised colonic mucus barrier integrity by visualizing fluorescent dextran penetration from the colonic lumen to the crypts. ADAMTS13 deficiency accelerated mucus layer disruption, leukocyte adhesion, and microthrombi formation, particularly close to crypt epithelium regions. Vessel-specific analyses demonstrated that obstructive microthrombi were most prominent in the mucosal layer, contributing to local ischemia and mucosal erosion. Therapeutical usage of recombinant human ADAMTS13 alleviated microthrombus formation, improved mucosal integrity, and mitigated colitis severity in wild-type and ADAMTS13-deficient mice. ConclusionsObstructive thrombi formed in mucosal vessels due to impaired ADAMTS13 activity appeared essential in the disease progression of UC. Advanced intravital imaging provided novel insights into single-cell resolution UC pathogenesis.

physiology↗

bioRxiv2024

Spatial Clustering Analysis with Spectral Imaging-based Single-Step Multiplex Immunofluorescence (SISS-mIF)

Precision medicine, based on spatial biology, is crucial for accurately diagnosing cancer and predicting drug responses. Here, we introduce the Spectral Imaging-based Single-Step Multiplex Immunofluorescence (SISS-mIF) technique, utilizing hyperspectral imaging to capture fluorescence spectra simultaneously. This approach optimizes tissue autofluorescence spectra for each image automatically, allowing the use of fluorescent direct-labeled antibodies for multicolor staining in a single step. Unlike conventional methods, the images are generated as standardized intensity independent of capture conditions, enabling consistent comparisons under different imaging conditions. This technique allows the detection of CD3, CD5, and CD7 in T-cell lymphoma on a single slide. The use of fluorescent direct-labeled antibodies enables triple staining of CD3, CD5, and CD7 without cross-reactivity, maintaining the same intensity as single stains. Furthermore, we developed a joint Non-Negative Matrix Factorization-based Spatial Clustering Analysis (jNMF-SCA) with a modified spectral unmixing system, highlighting its potential as a supportive diagnostic tool for T-cell lymphoma.

pathology↗

bioRxiv2024

Influence of Monosporascus root rot and vine decline disease on the amino acid metabolism of watermelon fruit

Vine decline (VD) is a severe disease of watermelon, melon, and other cucurbits, caused by soil-borne pathogens such as Monosporascus cannonballus and leads to root necrosis and sudden wilting at the later stage of fruit maturation. The present study examined the effects of VD on the metabolism of watermelon fruits. The VD-affected watermelon fruits had significantly lower lycopene and total solid contents. Still, polyphenols content and total antioxidant activities were comparable with the controls, suggesting that VD inhibited the ripening processes but maintained defensive processes in the fruits. The VD fruits showed a lower calcium level than the controls, while the contents of other major nutrition minerals were not significantly altered. The VD fruits had a lower content of total amino acids, and their composition was characterized by an increase in the percentage fractions for several amino acids, including citrulline, which may reflect the physiological response to the VD-related water deficit condition. The PCA plot clearly distinguished amino acid profiles between the VD and control fruits, demonstrating that VD exerted major influences on their amino acid metabolisms. Overall, the present study revealed that VD imposed characteristic impacts on the biochemical behaviors in the watermelon fruits.

plant biology↗

bioRxiv2023

Discovery of Novel Replication Proteins for Large Plasmids in Cyanobacteria and Their Potential Applications in Genetic Engineering

Numerous cyanobacteria capable of oxygenic photosynthesis possess multiple large plasmids exceeding 100 kbp in size. These plasmids are believed to have distinct replication and distribution mechanisms, as they coexist within cells without causing incompatibilities between plasmids. However, information on Rep proteins necessary for plasmid replication initiation in cyanobacteria is limited. Synechocystis sp. PCC 6803 hosts four large plasmids, pSYSM, pSYSX, pSYSA, and pSYSG, but Rep proteins for these plasmids, except for CyRepA1 on pSYSA, are unknown. Using Autonomous Replication sequencing (AR-seq), we identified two potential Rep genes in Synechocystis 6803, slr6031 and slr6090, both located on pSYSX. The corresponding Rep candidates, Slr6031 and Slr6090, share structural similarities with Rep-associated proteins of other bacteria and homologs were also identified in various cyanobacteria. We observed autonomous replication activity for Slr6031 and Slr6090 in Synechococcus elongatus PCC 7942 by fusing their genes with a construct expressing GFP and introducing them via transformation. The slr6031/slr6090-containing plasmids exhibited lower copy numbers and instability in Synechococcus 7942 cells compared to the expression vector pYS. While recombination occurred in the case of slr6090, the engineered plasmid with slr6031 coexisted with plasmids encoding CyRepA1 or Slr6090 in Synechococcus 7942 cells, indicating the compatibility of Slr6031 and Slr6090 with CyRepA1. Based on these results, we designated Slr6031 and Slr6090 as CyRepX1 (Cyanobacterial Rep-related protein encoded on pSYSX) and CyRepX2, respectively, demonstrating that pSYSX is a plasmid with "two Reps in one plasmid". Furthermore, we determined the copy number and stability of plasmids with cyanobacterial Reps in Synechococcus 7942 and Synechocystis 6803 to elucidate their potential applications. The novel properties of CyRepX1 and 2, as revealed by this study, hold promise for the development of innovative genetic engineering tools in cyanobacteria.

microbiology↗

bioRxiv2023

Effectiveness of heat tolerance rice cultivars in preserving grain appearance quality under high temperatures - A meta-analysis

BackgroundClimate change, particularly rising temperatures, negatively affects rice grain quality, increasing chalky grain percentage (CG) and hampering rice grade and price. Heat-tolerant cultivars have been bred and released since the 2000s, but the effectiveness of heat tolerance in reducing the occurrence of CG has yet to be quantified. ObjectivesThis study aimed to measure the effectiveness of breeding for better heat tolerance in reducing the negative impact of high temperatures on rice quality. MethodsThrough a systematic literature search, we developed a dataset including 1297 field observations covering 48 cultivars from five different heat tolerant ranks (HTRs) at 44 sites across Japan. A linear mixed-effect model (LME) and a random forest model (RF) were fitted to the data to analyze the effect of HTR and climatic factors such as the cumulative mean air temperature above 26 {degrees}C (TaHD), mean solar radiation, and mean relative humidity for 20 days after heading on CG. ResultsThe LME model explained 63 % of the variation with a 14% RMSE. The RF partial dependence plot revealed that the logit-transformed CG response to climate factors was linear, supporting the assumption of LME. The statistical analysis showed that CG increased as a function of TaHD (P < 0.001), with significant differences among HTRs (P < 0.001). The strongest effect of TaHD was obtained for the lowest HTR and was found to decrease with increasing HTR. CG also increased with higher relative humidity (P < 0.001) and solar radiation (P < 0.01). Based on our modeling, we estimated that as TaHD increased from 20 to 80 {degrees}Cd (equivalent to a mean temperature increase from 27 {degrees}C to 30 {degrees}C), CG increased by 66 % points (difference in CG) for cultivars with the lowest HTR, 45 % points for cultivars with an intermediate HTR, and 19 % points for cultivars with the highest HTR. Raising HTR by just one step (from intermediate to moderately tolerant) is projected to increase the proportion of first-grade rice at a grain-filling temperature of 27 {degrees}C, but tolerance levels need to be improved further in case of stronger warming. ConclusionsThe effect of high temperatures on CG was highly dependent on the cultivars HTR. Improvements in HTR effectively reduce the negative impacts of high temperatures on rice grain quality. SignificanceHeat-tolerant cultivars are projected to suppress the prevalence of CG more than threefold compared with heat-sensitive cultivars when grain-filling temperature increases from 27 to 30 {degrees}C.

plant biology↗

bioRxiv2022

Regulation of pSYSA defense plasmid copy number in Synechocystis through RNase E and a highly transcribed asRNA

Synthetic biology approaches toward the development of cyanobacterial producer strains require the availability of appropriate sets of plasmid vectors. A factor for the industrial usefulness of such strains is their robustness against pathogens, such as bacteriophages infecting cyanobacteria. Therefore, it is of great interest to understand the native plasmid replication systems and the CRISPR-Cas based defense mechanisms already present in cyanobacteria. In the model cyanobacterium Synechocystis sp. PCC 6803, four large and three smaller plasmids exist. The [~]100 kb plasmid pSYSA is specialized in defense functions by encoding all three CRISPR-Cas systems and several toxin-antitoxin systems. The expression of genes located on pSYSA depends on the plasmid copy number in the cell. The pSYSA copy number is positively correlated with the expression level of the endoribonuclease E. As molecular basis for this correlation we identified the RNase E-mediated cleavage within the pSYSA-encoded ssr7036 transcript. Together with a cis-located abundant antisense RNA (asRNA1), this mechanism resembles the control of ColE1-type plasmid replication by two overlapping RNAs, RNA I and II. In the ColE1 mechanism, two non-coding RNAs interact, supported by the small protein Rop, which is encoded separately. In contrast, in pSYSA the similar-sized protein Ssr7036 is encoded within one of the interacting RNAs and it is this mRNA that likely primes pSYSA replication. Essential for plasmid replication is furthermore the downstream encoded protein Slr7037 featuring primase and helicase domains. Deletion of slr7037 led to the integration of pSYSA into the chromosome or the other large plasmid pSYSX. Moreover, the presence of slr7037 was required for successful replication of a pSYSA-derived vector in another model cyanobacterium, Synechococcus elongatus PCC 7942. Therefore, we annotated the protein encoded by slr7037 as Cyanobacterial Rep protein A1 (CyRepA1). Our findings open new perspectives on the development of shuttle vectors for genetic engineering of cyanobacteria and of modulating the activity of the entire CRISPR-Cas apparatus in Synechocystis sp. PCC 6803.

microbiology↗