{"title":"Enhancing specimen preparation for transmission electron microscopy: Trypan Blue staining and low-melting-point agar embedding for ultra-thin cell sections","authors":"Noriyuki Ishii , Yoshito Koja , Keiichi Noguchi , Masafumi Yohda , Shin Takeda","doi":"10.1016/j.jbiosc.2025.02.003","DOIUrl":"10.1016/j.jbiosc.2025.02.003","url":null,"abstract":"<div><div>The observation of ultrathin sections of cells and other specimens using transmission electron microscopy (TEM) is a standard technique in many biological research laboratories. Cells are typically collected, dehydrated through an ascending series of alcohols, and embedded in resin blocks before being sectioned with an ultramicrotome. This multi-step process can be time consuming and error prone. To address these challenges, we introduced modifications to improve sample visualization while avoiding hazardous substances like osmium tetroxide and epoxy resins (e.g., Araldite), which are increasingly regulated internationally. Specifically, we stained cells with Trypan Blue and used low melting point agar, facilitating visual identification of target areas and enabling precise embedding. As a result, visual tracking of samples prior to embedding for TEM was improved, preventing the cutting of empty blocks and ensuring efficient preparation of ultrathin sections.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"139 5","pages":"Pages 392-398"},"PeriodicalIF":2.3,"publicationDate":"2025-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143585684","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Antibiofilm action of phytochemicals on Enterobacteriaceae","authors":"Anuj Rohatgi , Pratima Gupta","doi":"10.1016/j.jbiosc.2025.01.007","DOIUrl":"10.1016/j.jbiosc.2025.01.007","url":null,"abstract":"<div><div>The biofilm-associated infections pose a great threat to human health. The available drugs are not effective due to the formation of biofilm and limited access to underlying pathogens. The initiation of biofilm formation occurs through adhesion, facilitated by the adhesin protein MrkD1P in the fimbriae tip. This study targeted the MrkD1P protein and employed plant phenols to inhibit biofilm formation in <em>Escherichia coli</em>, <em>Salmonella typhi</em>, and <em>Klebsiella pneumoniae</em>, as major <em>Enterobacteriaceae</em> species. A homology model was constructed for the MrkD1P protein, and 44 phenolic derivatives were assessed for their interaction with this protein. Caffeic acid and 3-hydroxybenzoic acid exhibited the best binding-free energies of 29.61 kcal/mol and 24.24 kcal/mol, respectively. Using a microtiter plates-based minimum biofilm inhibitory concentration assay, it was found that doses of these compounds ranging from 2 to 256 mg/mL effectively reduced biofilm formation. The biofilm inhibition assay demonstrated over 80 % reduction of biofilms in all tested species at inhibitory doses. Further analysis through field emission gun scanning electron micrographs revealed that the compounds disintegrated fimbriae on cell surfaces. Additionally, the re-formation assay demonstrated the inability of biofilm-associated cells to re-form the biofilm on fresh surfaces due to fimbriae inhibition. This study highlights the antibiofilm capabilities of caffeic acid and 3-hydroxybenzoic acid, indicating their potential as effective treatments for illnesses caused by <em>Enterobacteriaceae</em> biofilms.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"139 5","pages":"Pages 362-368"},"PeriodicalIF":2.3,"publicationDate":"2025-02-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143537184","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Controlling the carbon flux between glycolysis and the pentose phosphate pathway via targeted protein degradation in Corynebacterium glutamicum","authors":"Norihiko Takemoto , Wenhui Hao , Hiroki Matsuzawa , Jun Sakurai , Yota Tsuge","doi":"10.1016/j.jbiosc.2025.02.002","DOIUrl":"10.1016/j.jbiosc.2025.02.002","url":null,"abstract":"<div><div>Controlling the carbon flux is important for efficient production of value-added chemicals using microbial cell factories. In this study, we developed a system to control the carbon flux in <em>Corynebacterium glutamicum</em> via targeted protein degradation. We employed an SspB-dependent protein degradation system targeting the SsrA tag and applied it to control the carbon flux. First, we selected a degradation tag efficiently recognized by the ClpXP protease in <em>C. glutamicum</em> using green fluorescent protein (GFP) as a model protein. Among the four tags examined in this study, a mutant SsrA tag with DAS residues from <em>Escherichia coli</em> resulted in specific GFP degradation only when the adaptor SspB was induced in <em>C. glutamicum</em>. Next, we applied this system to control the carbon flux. We selected phosphoglucoisomerase (PGI) encoded by <em>pgi</em>, as a target protein, to control the carbon flux between glycolysis and pentose phosphate pathway (PPP) and 1,5-diaminopentane as a model product to evaluate this control system. Compared with the parental strain, the specific growth rate of the engineered strain decreased by 36 %, whereas the yield and production rate of 1,5-diaminopentane increased by 193 % and 70 %, respectively. This is the first report on the application of a protein degradation system to control carbon flux in <em>C. glutamicum</em>. The system developed in this study can be widely applied for designing <em>C. glutamicum</em> cell factories for efficient production of varied value-added chemicals.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"139 5","pages":"Pages 377-383"},"PeriodicalIF":2.3,"publicationDate":"2025-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143515818","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Mika Nishimura , Kazuki Takahashi , Masahito Hosokawa
{"title":"Recent advances in single-cell RNA sequencing of bacteria: Techniques, challenges, and applications","authors":"Mika Nishimura , Kazuki Takahashi , Masahito Hosokawa","doi":"10.1016/j.jbiosc.2025.01.008","DOIUrl":"10.1016/j.jbiosc.2025.01.008","url":null,"abstract":"<div><div>Single-cell RNA sequencing (scRNA-seq) has revolutionized our understanding of cellular heterogeneity in complex biological systems. While this technology has been widely applied to eukaryotic cells, its adaptation to bacterial systems has been challenging due to the unique characteristics of bacterial transcripts. This review surveys the recent developments in bacterial scRNA-seq techniques, highlighting the technical challenges, methodological innovations, and emerging applications in microbiology. We discuss the key differences between eukaryotic and bacterial RNA-seq approaches, focusing on the strategies to overcome limitations such as the lack of poly-A tails in bacterial mRNAs and the low RNA content in individual bacterial cells. The review covers various bacterial scRNA-seq methods, including plate-based, split-pool barcoding, and droplet-based techniques, comparing their strengths and limitations in terms of sensitivity, throughput, and applicability to different bacterial species. Furthermore, we explore the biological insights gained from these techniques, such as identifying rare cell states, characterization of antibiotic responses, and analysis of bacterial communities. Finally, we discuss future perspectives and potential applications of bacterial scRNA-seq in understanding microbial physiology, host-pathogen interactions, and complex microbial ecosystems. This comprehensive overview aims to provide researchers with a clear understanding of the current state and future directions of single-cell transcriptomics in bacteria.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"139 5","pages":"Pages 341-346"},"PeriodicalIF":2.3,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143472473","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Application of deep learning for evaluation of the growth rate of Daphnia magna","authors":"Shinsuke Inagaki , Yohei Kondo , Pijar Religia , Nikko Adhitama , Yasuhiko Kato , Eiji Watanabe , Hajime Watanabe","doi":"10.1016/j.jbiosc.2025.01.006","DOIUrl":"10.1016/j.jbiosc.2025.01.006","url":null,"abstract":"<div><div>For the safe use of chemicals widely used in human activities, it is crucial to assess their ecological impacts when released into the environment. <em>Daphnia</em>, a well-established environmental indicator species, is commonly used to evaluate the biological effects of chemicals and testing methods have been established. Among various indicators, the growth rate is one of the important parameters, but it requires significant time and effort to measure. In this study, we applied deep learning-based image recognition techniques to extract images of <em>Daphnia</em> from live imaging and assess their size. The estimated size of <em>Daphnia</em>, derived from images processed through deep learning, showed a high correlation with measured values, demonstrating the capability to measure <em>Daphnia</em> size from the images while they are swimming. This approach enables non-invasive measurements of <em>Daphnia</em> size without complicated procedures, which not only streamlines ecological impact assessments but also presents a valuable technique for ecological studies, such as analyzing the size distribution of zooplankton.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"139 5","pages":"Pages 384-391"},"PeriodicalIF":2.3,"publicationDate":"2025-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425452","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Importance of dataset design in developing robust U-Net models for label-free cell morphology evaluation","authors":"Takeru Shiina , Kazue Kimura , Yuto Takemoto , Kenjiro Tanaka , Ryuji Kato","doi":"10.1016/j.jbiosc.2025.01.004","DOIUrl":"10.1016/j.jbiosc.2025.01.004","url":null,"abstract":"<div><div>Advances in regenerative medicine highlighted the need for label-free cell image analysis to replace conventional microscopic observation for non-invasive cell quality evaluation. Image-based evaluation provides an efficient, quantitative, and automated approach to cell analysis, but segmentation remains a critical and challenging step. In this study, we investigated how training dataset design influenced the robustness of U-Net models for cell segmentation, focusing on challenges posed by limited data availability in cell culture. Using 2592 image pairs from four cell types representing key morphological categories, we constructed 42 investigation patterns to evaluate the effects of dataset size, dataset content, and morphological diversity on model performance. Our results showed that robust segmentation models could be developed with approximately 10 raw images captured using a 4× objective lens, a much smaller dataset than typically assumed. The dataset content was found to be crucial: training dataset images that captured commonly observed cell patterns yielded more robust models compared to those capturing rare or irregular cell patterns, which often impaired model performance with large deviations. Additionally, including both spindle and round cell morphologies in the training datasets improved model robustness when tested across all four cell types, while datasets restricted to a single morphology type could not achieve robust models. These findings highlight the importance of curating datasets that capture representative yet diverse cell morphologies. By addressing critical questions about dataset design, this study provides actionable guidance for the effective use of deep learning-based cell segmentation models in manufacturing and research applications.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"139 4","pages":"Pages 329-339"},"PeriodicalIF":2.3,"publicationDate":"2025-02-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143399168","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Cindy Novianti , Lutfi Dewi Nirmala Sari , Husna Nugrahapraja , Sony Suhandono , Fenny Martha Dwivany , Sastia Prama Putri , Eiichiro Fukusaki
{"title":"Metabolic profiling reveals distinctive ripening dynamics in ethylene-treated Musa balbisiana cv. ‘Pisang Klutuk Wulung’ compared to commercial Cavendish banana","authors":"Cindy Novianti , Lutfi Dewi Nirmala Sari , Husna Nugrahapraja , Sony Suhandono , Fenny Martha Dwivany , Sastia Prama Putri , Eiichiro Fukusaki","doi":"10.1016/j.jbiosc.2025.01.001","DOIUrl":"10.1016/j.jbiosc.2025.01.001","url":null,"abstract":"<div><div>As an important crop, bananas still encounter fruit quality and shelf-life problems that are affected by the ripening process. Improving postharvest technologies may effectively address these challenges, such as by studying the ripening mechanism of banana cultivars with a slow ripening process. A banana cultivar that exhibits this characteristic is <em>Musa balbisiana</em> cv. ‘Pisang Klutuk Wulung’ (BB Group) or Pisang Klutuk Wulung (PKW), which has a ripening duration of 14–28 days. However, the metabolomics study on the ripening mechanism of this banana is still limited. This study aimed to analyze metabolite changes in ethylene-treated Pisang Klutuk Wulung during ripening in comparison to commercial bananas (Cavendish). Both bananas were subjected to exogenous ethylene treatment and analyzed using gas chromatography-mass spectrometry to perform metabolite profiling throughout the ripening process. The principal component analysis showed sample separation based on the ripening stages and banana species in pulp and peel. Orthogonal projection to latent structure analysis suggested that metabolite changes accompanied the ripening stages. Potential metabolite markers that distinguished the ripening of PKW and Cavendish were found, such as quinic acid, inositol, and 2-aminoethanol. This study shows differences in metabolite profiles between these bananas, especially the metabolites involved in sugar metabolism, cell wall metabolism, stress response, and biosynthesis of aromatic compounds. This study provides novel insights into the metabolic changes occurring during PKW ripening, contributing to the improvement of banana postharvest strategies.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"139 4","pages":"Pages 302-310"},"PeriodicalIF":2.3,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143374145","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Identification and functional characterization of ammonium transporters in Penicillium purpurogenum","authors":"Ryo Kojima , Taisuke Watanabe , Takafumi Kasumi , Hiroshi Mitsuzawa","doi":"10.1016/j.jbiosc.2025.01.005","DOIUrl":"10.1016/j.jbiosc.2025.01.005","url":null,"abstract":"<div><div>The filamentous fungus <em>Penicillium purpurogenum</em> IAM15392 produces a nitrogen-containing azaphilone pigment, (10<em>Z</em>)-12-carboxyl-monascorubramine (PP-V), which is a potentially valuable natural food colorant. Because ammonium is used as a nitrogen source, and because ammonium uptake is the first step in the synthesis of PP-V, ammonium transporters of <em>P. purpurogenum</em> were identified and characterized. The <em>P. purpurogenum</em> genome was found to contain four putative ammonium transporter genes, designated <em>amtA</em>, <em>amtB</em>, <em>amtC</em>, and <em>amtD</em>, which encode 11 transmembrane proteins of 479, 567, 452, and 475 amino acid residues, respectively. These genes were tested for their ability to complement mutations in the ammonium transporter genes of the fission yeast <em>Schizosaccharomyces pombe</em>. The phenotypes of mutants included defects in growth on low ammonium medium, methylammonium sensitivity, ammonium uptake from the culture medium, and ammonium limitation-induced invasive growth. Furthermore, the transcription of the <em>amt</em> genes was examined in <em>P. purpurogenum</em> grown under different ammonium concentrations. The results suggest that AmtB plays a major role in growth using ammonium as a nitrogen source, whereas AmtA and possibly AmtD function at low ammonium concentrations. Because a medium used for the production of PP-V contains a high concentration of ammonium, our functional characterization of the <em>P. purpurogenum</em> ammonium transporters suggests that AmtB is a potential target of bioengineering for increased PP-V production.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"139 4","pages":"Pages 257-262"},"PeriodicalIF":2.3,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143374144","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"New glycerol glycosides in sake formed by Aspergillus oryzae α-glucosidase A","authors":"Yui Akiyama , Kou Yoshizawa , Asuka Yamada , Izumi Kobayashi , Masafumi Tokuoka , Shigenori Kumazawa , Chihiro Honda","doi":"10.1016/j.jbiosc.2025.01.002","DOIUrl":"10.1016/j.jbiosc.2025.01.002","url":null,"abstract":"<div><div>Sake contains unique glycosides produced by the transglycosylation action of α-glucosidase from <em>Aspergillus oryzae</em>. Besides influencing the taste of sake, some transglycosylation products in sake exhibit beneficial biological activities. In this study, we searched for new transglycosylation products in sake. Liquid chromatography–mass spectrometry (LC–MS) revealed that peaks with <em>m/z</em> values corresponding to the glycosides, diglucopyranosylglycerol, triglucopyranosylglycerol, and tetraglucopyranosylglycerol, are present in sake. The presence of glycosides containing up to four polymerized glucose units is the first observation in sake. The peaks of the compounds were not observed in the sake that was brewed with a rice-koji made by α-glucosidase A (AgdA) gene disruption <em>A</em>. <em>oryzae</em> strain. The <em>in vitro</em> transglycosylation experiment using maltose, glycerol and a recombinant AgdA suggested that the compounds in sake were transglycosylation products composed of glycerol and one to four units of glucose. Nuclear magnetic resonance (NMR) analysis revealed that one of the major products of <em>in vitro</em> synthesis was α-isomaltosylglycerol (α-iMG). α-iMG was detected in commercial sake as a common component at an average of 1095 ppm (mg/L). This is the first study to report the presence of α-iMG in foods and beverages.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"139 4","pages":"Pages 296-301"},"PeriodicalIF":2.3,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143255584","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Yeast diversity during the spontaneous fermentation of wine in a winery and in a laboratory using sterilized equipment","authors":"Hideaki Shimizu , Aya Kamada , Takeshi Akao , Yoshiya Kanno , Kazuya Koyama , Kazuhiro Iwashita , Nami Goto-Yamamoto","doi":"10.1016/j.jbiosc.2024.11.001","DOIUrl":"10.1016/j.jbiosc.2024.11.001","url":null,"abstract":"<div><div>A recent trend in some wineries is the return to using spontaneous fermentation, but it is not clear whether winery flora or vineyard microorganisms drive fermentation. We compared fungal communities during the spontaneous fermentation of wine produced in a winery and in a laboratory with sterilized equipment using three grape cultivars (Chardonnay, Merlot, and Muscat Bailey A) obtained from the same harvest. High-throughput sequencing analysis based on the ITS1 region showed that <em>Saccharomyces cerevisiae</em> was the dominant species in winery batches at the end of fermentation, but it was not always dominant in laboratory batches. The number of laboratory batches where <em>S. cerevisiae</em> reached more than 50% at the end of fermentation was only 10 of 26. Consistent with this, in the grape juice/must before fermentation, <em>S. cerevisiae</em> accounted for 1.71% of fungal species identified in winery batches and 0.04% in laboratory batches. In addition, in laboratory-based winemaking, juice clarification of Chardonnay and cold maceration of Merlot influenced the microbial communities observed during fermentation. Our findings suggest that <em>S. cerevisiae</em> present in the winery environment participates at an early stage of fermentation, leading to its dominance at the end in wine produced by spontaneous fermentation in a winery.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"139 2","pages":"Pages 106-111"},"PeriodicalIF":2.3,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142769289","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}