Biology subjects

Kondo, A.

Publications and source records attributed to Kondo, A..

16 recordsLinked to original sources

bioRxiv2025

Proteinase 3 is involved in presepsin production through neutrophil extracellular trap phagocytosis by macrophages

Presepsin (P-SEP), a soluble subtype of CD14, has been widely reported as a useful biomarker for sepsis in clinical studies. However, the cytological mechanism underlying its production remains poorly understood. Previous research has demonstrated that P-SEP is generated when M1 macrophages (M1 M{Phi}s) phagocytose neutrophil extracellular traps (NETs) and degrade internalized CD14. In this study, we demonstrated that M1 M{Phi}s phagocytose NETs and that proteinase 3 (PR3) within M1 M{Phi}s degrades NET-derived CD14, resulting in the production of P-SEP. Furthermore, we observed a strong correlation between the amount of NETs and P-SEP levels, indicating that NET phagocytosis is a key process in P-SEP generation. Our findings also suggest that PR3 released extracellularly along with NETs may degrade CD14 in plasma, contributing to elevated P-SEP levels. This mechanism may contribute to increased P-SEP concentrations observed in autoimmune diseases associated with NET accumulation, proposing P-SEP as a potential biomarker of disease activity beyond sepsis.

cell biology↗

bioRxiv2024

Engineering Saccharomyces cerevisiae for growth on xylose using an oxidative pathway

The fermentative production of valuable chemicals from lignocellulosic feedstocks has attracted considerable attention. Although Saccharomyces cerevisiae is a promising microbial host, it lacks the ability to efficiently metabolize xylose, a major component of lignocellulosic feedstocks. The xylose oxidative pathway offers advantages such as simplified metabolic regulation and fewer enzymatic steps. Specifically, the pathway involves the conversion of xylose into 2-keto-3-deoxy-xylonate, which can be channeled into two distinct pathways, the Dahms pathway and the Weimberg pathway. However, the growth of yeast on xylose as the sole carbon source through the xylose oxidative pathway has not been achieved, limiting its utilization. We successfully engineered S. cerevisiae to metabolize xylose as its sole carbon source via the xylose oxidative pathways, achieved by enhancing enzyme activities through iron metabolism engineering and rational enzyme selection. We found that increasing the supply of the iron-sulfur cluster to activate the bottleneck enzyme XylD by BOL2 disruption and tTYW1 overexpression facilitated the growth on xylose and the production of ethylene glycol at 1.5 g/L via the Dahms pathway. Furthermore, phylogenetic analysis of xylonate dehydratases led to the identification of a highly active homologous enzyme. A strain possessing the Dahms pathway with this highly active enzyme exhibited reduced xylonate accumulation. Furthermore, the introduction of enzymes based on phylogenetic tree analysis allowed for the utilization of xylose as the sole carbon source through the Weimberg pathway. This study highlights the potential of iron metabolism engineering and phylogenetic enzyme selection for the development of non-native metabolic pathways in yeast. Key pointsO_LI1.5 g/L ethylene glycol was produced via Dahms pathway in S. cerevisiae. C_LIO_LIEnzyme activation enabled growth on xylose via both the Dahms and Weimberg pathways. C_LIO_LITested enzymes in this study may expand application of xylose oxidative pathway. C_LI

bioengineering↗

bioRxiv2024

Genome-scale prediction of gene ontology from mass fingerprints reveals new metabolic gene functions

Mass-based fingerprinting can characterize unknown strains, however expansion of these methods to predict specific gene functions is lacking. Therefore, rapid mass fingerprinting was developed to functionally profile a comprehensive yeast knockout library. Matrix assisted laser desorption ionization (MALDI)-time of flight (TOF) mass fingerprints of 3,238 Saccharomyces cerevisiae knockouts were digitized for correlation with gene ontology (GO) annotations. Random forests and support vector machine (SVM) algorithms precisely assigned GO accessions with AUC scores all above 0.83. SVM was the best predictor with average true positive and true negative rates of 0.975 and 0.991, respectively. The SVM model suggested new functions for 28 uncharacterized yeast genes. Metabolomics analysis of two knockouts (YDR215C and YLR122C) of uncharacterized genes predicted to be involved in methylation-related metabolism, showed altered intracellular contents of methionine-related metabolites. Increased S-adenosylmethionine in YDR215C highlights potential for enhancement of methylation pathways. These results demonstrate that MALDI-TOF fingerprints can be rapidly digitized, resulting in datasets that enable prediction of microbial genotypes and even the function of specific genes. This fingerprinting method can inform optimal bioproduction chassis selection.

systems biology↗

bioRxiv2024

TNFSF13 insufficiency disrupts human colonic epithelial cell-mediated B cell differentiation

Cytokines mediating epithelial and immune cell interactions modulate mucosal healing-a process that goes awry with chronic inflammation as in inflammatory bowel disease. TNFSF13 is a cytokine important for B cell maturation and function, but roles for epithelial TNFSF13 and putative contribution to inflammatory bowel disease are poorly understood. We evaluated functional consequences of a novel monoallelic TNFSF13 variant using biopsies, tissue-derived colonoids and induced pluripotent stem cell (iPSC)-derived colon organoids. TNFSF13 variant colonoids exhibited a >50% reduction in secreted TNFSF13, increased epithelial proliferation, and reduced apoptosis, which was confirmed in iPSC-derived colon organoids. Single cell RNA-sequencing, flow cytometry, and co-immunoprecipitation identified FAS as the predominant colonic epithelial receptor for TNFSF13. Imaging mass cytometry revealed an increase in epithelial-associated B cells in TNFSF13 variant colon tissue sections. Finally, TNFSF13 variant colonoids co-cultured with memory B cells demonstrated a reduction in the production of IgA+ plasma cells compared to control colonoid co-cultures. Our findings support a role for epithelial TNFSF13 as a regulator of colonic epithelial growth and epithelial crosstalk with B cells. SUMMARYEpithelial TNFSF13 regulates colonic epithelial growth and epithelial-B cell interactions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/614260v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1e5b892org.highwire.dtl.DTLVardef@b8857forg.highwire.dtl.DTLVardef@448b95org.highwire.dtl.DTLVardef@2820e2_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Development of a highly efficient base editing system for Lactobacilli to improve probiotics and dissect essential functions

Lactobacilli play essential roles in the food industry and are increasingly explored for their potential as probiotics and therapeutic agents. Beneficial strains are primarily isolated from various natural sources including healthy human bodies, and undergo rigorous characterization and safety evaluations. Genomic and genetic information has increasingly accumulated and been linked to their various functions, to which transgenic approaches are being performed to verify crucial genes. In order to reasonably develop more useful strains, beneficial traits need to be introduced into any given strains and enhanced or combined. However, for practical use as probiotics or foods, organisms with transgene are hardly acceptable. Here, we have introduced the base editing Target-AID system specifically for Lactobacilli, enabling precise installation of point mutations without donor DNA and at multiple genomic loci simultaneously. Lactiplantibacillus plantarum has been successfully engineered to reduce production of imidazole propionate, which has been reported to be associated with type 2 diabetes. Additionally, this system enabled transient knock-out of an essential gene, such as one involved in cell division showing severe filamentous cell phenotype, providing a unique approach for dissecting essential gene function. ImportanceThis work provides highly efficient and multiplexable base editing system that installs precise point mutations in the genomes of the two major Lactobacilli strains. As the advanced CRISPR technology so-called non-cleaving genome editing, base editing is less toxic and does not integrate any foreign DNA into the genomes. Our approaches pave the way for dissecting and improving probiotics and food-grade microbes, ultimately creating better human health.

microbiology↗

bioRxiv2023

Minimized dark cataplerosis of the Calvin cycle ensures prompt photosynthetic initiation in cyanobacteria

As primary contributors to oxygenic photosynthesis, cyanobacteria intricately regulate their metabolic pathways during the diurnal cycle to ensure survival and growth. Under dark conditions, breakdown of stored energy reserves of glycogen replenishes the intermediates, especially the downstream glycolytic metabolites necessary for photosynthetic initiation upon light irradiation. The intracellular level of the intermediates is maintained throughout the dark period. However, it remains unclear how their accumulation is maintained in the dark despite the limited availability of glycogen. Here, we showed that the metabolite accumulation stability is ensured by the low activities of phosphoenolpyruvate (PEP) converting enzymes, namely PEP carboxylase and pyruvate kinase, during the dark period. Overexpression of these enzymes significantly decreased the accumulation of glycolytic intermediates after dark incubation. The oxygen evolution ability simultaneously decreased in the overexpressing strains, indicating that the dark limitation of the PEP-consuming pathway facilitates photosynthetic initiation through the maintenance of glycolytic intermediates. This finding shed light on the importance of controlling cataplerotic flux during the dark for maintaining stable operation of the Calvin cycle.

plant biology↗

bioRxiv2023

Characterizing tissue structures from spatial omics with spatial cellular graph partition

Spatial transcriptomic and proteomic measurements enable high-dimensional characterization of tissues. However, understanding organizations of cells at different spatial scales and extracting tissue structures of interest remain challenging tasks that require extensive human annotations. To address this need for consistent identification of tissue structures, in this work, we present a novel annotation method Spatial Cellular Graph Partitioning (SCGP) that allows unsupervised identification of tissue structures that reflect the anatomical and functional units of human tissues. We further present a reference-query extension pipeline SCGP-Extension that enables the generalization of existing reference tissue structures to previously unseen samples. Our experiments demonstrate reliable and robust partitionings of both spatial transcriptomics and proteomics datasets encompassing different tissue types and profiling techniques. Downstream analysis on SCGP-identified tissue structures reveals disease-relevant insights regarding diabetic kidney disease and skin disorder, underscoring its potential in facilitating spatial analysis and driving new discoveries.

bioinformatics↗

bioRxiv2023

Spatial proteomics of human diabetic kidney disease, from health to class III

Aims/HypothesisDiabetic kidney disease (DKD) remains a significant cause of morbidity and mortality in people with diabetes. Though animal models have taught us much about the molecular mechanisms of DKD, translating these findings to human disease requires greater knowledge of the molecular changes caused by diabetes in human kidneys. Establishing this knowledge base requires building carefully curated, reliable, and complete repositories of human kidney tissue, as well as tissue proteomics platforms capable of simultaneous, spatially resolved examination of multiple proteins. MethodsWe used the multiplexed immunofluorescence platform CO-Detection by indexing (CODEX) to image and analyze the expression of 21 proteins in 23 tissue sections from 12 individuals with diabetes and healthy kidneys (DM, 5 individuals), DKD classes IIA, and IIB (2 individuals per class), IIA-B intermediate (2 individuals), and III (one individual). ResultsAnalysis of the 21-plex immunofluorescence images revealed 18 cellular clusters, corresponding to 10 known kidney compartments and cell types, including proximal tubules, distal nephron, podocytes, glomerular endothelial and peritubular capillaries, blood vessels, including endothelial cells and vascular smooth muscle cells, macrophages, cells of the myeloid lineage, broad CD45+ inflammatory cells and the basement membrane. DKD progression was associated with co-localized increase in collagen IV deposition and infiltration of inflammatory cells, as well as loss of native proteins of each nephron segment at variable rates. Compartment-specific cellular changes corroborated this general theme, with compartment-specific variations. Cell type frequency and cell-to-cell adjacency highlighted (statistically) significant increase in inflammatory cells and their adjacency to tubular and SMA+ cells in DKD kidneys. Finally, DKD progression was marked by substantial regional variability within single tissue sections, as well as variability across patients within the same DKD class. The sizable intra-personal variability in DKD severity impacts pathologic classifications, and the attendant clinical decisions, which are usually based on small tissue biopsies. Conclusions/InterpretationsHigh-plex immunofluorescence images revealed changes in protein expression corresponding to differences in cellular phenotypic composition and microenvironment structure with DKD progression. This initial dataset demonstrates the combined power of curated human kidney tissues, multiplexed immunofluorescence and powerful analysis tools in revealing pathophysiology of human DKD.

molecular biology↗

bioRxiv2023

A multi-kingdom genetic barcoding system for precise target clone isolation

Clonal heterogeneity underlies diverse biological processes, including cancer progression, cell differentiation, and microbial evolution. Cell tagging strategies with DNA barcodes have recently enabled analysis of clone size dynamics and clone-restricted transcriptomic landscapes of heterogeneous populations. However, isolating a target clone that displays a specific phenotype from a complex population remains challenging. Here, we present a new multi-kingdom genetic barcoding system, CloneSelect, in which a target cell clone can be triggered to express a reporter gene for isolation through barcode-specific CRISPR base editing. In CloneSelect, cells are first barcoded and propagated so their subpopulation can be subjected to a given experiment. A clone that shows a phenotype or genotype of interest at a given time can then be isolated from the initial or subsequent cell pools stored throughout the experimental timecourse. This novel CRISPR-barcode genetics platform provides many new ways of analyzing and manipulating mammalian, yeast, and bacterial systems. TeaserA multi-kingdom CRISPR-activatable barcoding system enables the precise isolation of target barcode-labeled clones from a complex cell population.

synthetic biology↗

bioRxiv2022

Accumulated 3-phosphoglycerate in darkness is a primary starting substrate for stepwise photosynthetic induction process in cyanobacteria

Photosynthesis must maintain stability and robustness throughout fluctuating natural environments. In cyanobacteria, dark-to-light transition leads to drastic metabolic changes from dark respiratory metabolism to CO2 fixation through the Calvin-Benson-Bassham (CBB) cycle using energy and redox equivalents provided by photosynthetic electron transfer. Previous studies showed that catabolic metabolism supports the smooth transition into CBB cycle metabolism. However, metabolic mechanisms for robust initiation of photosynthesis are poorly understood due to lack of dynamic metabolic characterizations of dark-to-light transitions. Here, we show rapid (on a time scale of seconds) dynamic changes in absolute metabolite concentrations and 13C tracer incorporation after strong or weak light irradiation in the cyanobacterium Synechocystis sp. PCC 6803. Integration of this data enables estimation of time-resolved nonstationary metabolic flux underlying CBB cycle activation. This dynamic metabolic analysis indicates that downstream glycolytic intermediates including phosphoglycerate and phosphoenolpyruvate accumulate under dark conditions as major substrates for initial CO2 fixation. Compared with wild-type Synechocystis, significant delays in the initiation of oxygen evolution are observed in 12 h dark preincubated mutants deficient in glycogen degradation or oxidative pentose phosphate pathway ({Delta}zwf, {Delta}gnd, and{Delta} glgP). Accordingly, the degree of delay in the oxygen evolution initiation is proportional to the accumulated pool size of the glycolytic intermediates. These observations indicate that the accumulation of glycolytic intermediates is essential for efficient metabolism switching under fluctuating light environments.

plant biology↗

bioRxiv2021

Multiomics single-cell analysis of human pancreatic islets reveals novel cellular states in health and type 1 diabetes

Type 1 diabetes (T1D) is an autoimmune disease of only partially defined etiology in which immune cells destroy insulin-producing beta cells. Using single-cell transcriptomics and an advanced analytical strategy to assess pancreatic islets of T1D, autoantibody-positive, and non-diabetic organ donors, we identified both canonical cell types and rare insulin-expressing cells with a hybrid mixture of endocrine and exocrine gene signatures within all donors. We further found elevated expression of MHC Class II pathway genes in exocrine ductal cells of T1D donors, which we confirmed through CyTOF, in situ imaging mass cytometry, and immunofluorescence analysis. Taken together, our multimodal analyses identify novel cell types and processes that may contribute to T1D immunopathogenesis and provide new cellular and molecular insights into human pancreas function.

genomics↗

bioRxiv2020

Consolidated bioprocessing of corn cob-derived hemicellulose: engineered industrial Saccharomyces cerevisiae as efficient whole cell biocatalysts

Consolidated bioprocessing, which combines saccharolytic and fermentative abilities in a single microorganism, is receiving increased attention to decrease environmental and economic costs in lignocellulosic biorefineries. Nevertheless, the economic viability of lignocellulosic ethanol is also dependent of an efficient utilization of the hemicellulosic fraction, which is mainly composed of xylose and may comprise up to 40 % of the total biomass. This major bottleneck is mainly due to the necessity of chemical/enzymatic treatments to hydrolyze hemicellulose into fermentable sugars and to the fact that xylose is not readily consumed by Saccharomyces cerevisiae – the most used organism for large-scale ethanol production. In this work, industrial S. cerevisiae strains, presenting robust traits such as thermotolerance and improved resistance to inhibitors, were evaluated as hosts for the cell-surface display of hemicellulolytic enzymes and optimized xylose assimilation, aiming at the development of whole-cell biocatalysts for consolidated bioprocessing of corn cob-derived hemicellulose. These modifications allowed the direct production of ethanol from non-detoxified hemicellulosic liquor obtained by hydrothermal pretreatment of corn cob, reaching an ethanol titer of 11.1 g/L corresponding to a yield of 0.328 gram per gram of potential xylose and glucose, without the need for external hydrolytic catalysts. Also, consolidated bioprocessing of pretreated corn cob was found to be more efficient for hemicellulosic ethanol production than simultaneous saccharification and fermentation with addition of commercial hemicellulases. These results show the potential of industrial S. cerevisiae strains for the design of whole-cell biocatalysts and paves the way for the development of more efficient consolidated bioprocesses for lignocellulosic biomass valorization, further decreasing environmental and economic costs.Competing Interest StatementThe authors have declared no competing interest.View Full Text

bioengineering↗

bioRxiv2020

Single cell resolution regulatory landscape of the mouse kidney highlights cellular differentiation programs and renal disease targets

Determining the epigenetic program that generates unique cell types in the kidney is critical for understanding cell-type heterogeneity during tissue homeostasis and injury response. Here, we profiled open chromatin and gene expression in developing and adult mouse kidneys at single cell resolution. We show critical reliance of gene expression on distal regulatory elements (enhancers). We define key cell type-specific transcription factors and major gene-regulatory circuits for kidney cells. Dynamic chromatin and expression changes during nephron progenitor differentiation demonstrated that podocyte commitment occurs early and is associated with sustained Foxl1 expression. Renal tubule cells followed a more complex differentiation, where Hfn4a was associated with proximal and Tfap2b with distal fate. Mapping single nucleotide variants associated with human kidney disease identified critical cell types, developmental stages, genes, and regulatory mechanisms. We provide a global single cell resolution view of chromatin accessibility of kidney development. The dataset is available via interactive public websites.

developmental biology↗

bioRxiv2019

A single CRISPR base editor to induce simultaneous C-to-T and A-to-G mutations

While several Cas9-derived base editors have been developed to induce either C-to-T or A-to-G mutation at target genomic sites, the possible genome editing space when using the current base editors remains limited. Here, we present a novel base editor, Target-ACE, which integrates the abilities of both of the previously developed C-to-T and A-to-G base editors by fusing an activation-induced cytidine deaminase (AID) and an engineered tRNA adenosine deaminase (TadA) to a catalytically impaired Streptococcus pyogenes Cas9. In mammalian cells, Target-ACE enabled heterologous editing of multiple bases in a small sequence window of target sites with increased efficiency compared with a mixture of two relevant base editor enzymes, each of which may block the same target DNA molecule from the other. Furthermore, by modeling editing patterns using deep sequencing data, the editing spectra of Target-ACE and other base editors were simulated across the human genome, demonstrating the highest potency of Target-ACE to edit amino acid coding patterns. Taking these findings together, Target-ACE is a new tool that broadens the capabilities for base editing for various applications.

bioengineering↗

bioRxiv2017

Comprehensive single cell RNAseq analysis of the kidney reveals novel cell types and unexpected cell plasticity

A key limitations to understand kidney function and disease development has been that specific cell types responsible for specific homeostatic kidney function or disease phenotypes have not been defined at the molecular level.\n\nTo fill this gap, we characterized 57,979 cells from healthy mouse kidneys using unbiased single-cell RNA sequencing. We show that genetic mutations that present with similar phenotypes mostly affect genes that are expressed in a single unique differentiated cell type. On the other hand, we found unexpected cell plasticity of epithelial cells in the final segment of the kidney (collecting duct) that is responsible for final composition of the urine. Using computational cell trajectory analysis and in vivo linage tracing, we found that, intercalated cells (that secrete protons) and principal cells (that maintain salt, water and potassium balance) undergo a Notch mediated interconversion via a newly identified transitional cell type. In disease states this transition is shifted towards the principal cell fate. Loss of intercalated cells likely contributes to metabolic acidosis observed in kidney disease.\n\nIn summary, single cell analysis advanced a mechanistic description of kidney diseases by identifying a defective homeostatic cell lineage.\n\nOne Sentence SummaryA comprehensive single cell atlas of the kidney reveals a transitional cell type and cell plasticity determined by Notch signaling which is defective in chronic kidney disease.

cell biology↗

bioRxiv2016

Metabolomic and proteomic analyses of a quiescent Escherichia coli cell factory reveal the mechanisms behind its production efficiency

1Quiescent (Q-Cell) Escherichia coli cultures can be created by using the signalling molecule indole to halt cell division of an hns mutant strain. This uncouples metabolism from cell growth and allows for more efficient use of carbon feedstocks. However, the reason for the increased productivity of cells in this state was previously unknown. We show here that Q-cells can maintain metabolic activity in the absence of growth for up to 24 h, leading to four times greater per-cell productivity of a model metabolite, 3-hydroxybutyrate (3HB), than a control. Metabolomic data show that by disrupting the proton-motive force, indole interrupts the tricarboxylic acid cycle, leading to the accumulation of metabolites in the glycolysis pathway that are excellent starting points for high-value chemical production. By comparing protein expression patterns between wild-type and Q-cell cultures we show that Q-cells overexpress stress response proteins, which prime them to tolerate the metabolic imbalances incurred through indole addition. Quiescent cultures produced half the cell biomass of control cultures lacking indole, but were still able to produce 39.4 g.L-1 of 3HB compared to 18.6 g.L-1 in the control. Therefore, Q-cells have high potential as a platform technology for the efficient production of a wide range of commodity and high value chemicals.

microbiology↗