Biology subjects

Ohara, Y.

Publications and source records attributed to Ohara, Y..

7 recordsLinked to original sources

bioRxiv2025

Heterologous expression of carbonic anhydrase in Acinetobacter sp. Tol 5 for whole-cell biocatalysis

Carbonic anhydrase accelerates the hydration of carbon dioxide (CO2) and is an attractive biocatalyst for carbon capture and utilization. Acinetobacter sp. Tol 5 shows high adhesiveness via its cell-surface protein AtaA. We previously demonstrated its application to bacterial immobilization and gas-phase bioproduction. Here, we developed Tol 5 cells expressing carbonic anhydrase and evaluated CO2 conversion ability as whole-cell biocatalysts. A codon-optimized carbonic anhydrase from Sulfurihydrogenibium yellowstonense (SyCA) was produced in the cytoplasm, but the cells showed little activity as a whole-cell biocatalyst. To enhance activity, we fused six signal peptides (SPs) to SyCA for periplasmic expression. The Omp38-SP fusion of SyCA was properly processed to the mature size, yielding higher whole-cell activity. By contrast, the other constructs were either undetectable or remained unprocessed, resulting in lower activities. These results show that periplasmic expression of SyCA is important for efficient CO2 hydration in Tol 5 cells as whole-cell biocatalysts.

bioengineering↗

bioRxiv2025

Grid partitioning image analysis for bacterial cell aggregates

Bacterial cell aggregation plays a fundamental role in surface colonization, stress tolerance, and interspecies metabolite exchange; yet quantitative analysis remains challenging. Here, we introduce grid partitioning image analysis (GPIA), a simple workflow that quantifies the compositional heterogeneity of bacterial aggregates. Confocal laser scanning microscopy (CLSM) images of fluorescently labeled Acinetobacter sp. Tol 5, which exhibits a self-aggregative nature through its cell surface protein AtaA, were partitioned into 2-{micro}m square grids. Grids containing one or no cells were classified as dispersed, whereas those containing multiple cells were classified as aggregates, and the proportion of EGFP-labeled cells within each grid was recorded. Reference images representing dispersed cells, homo-aggregates, and hetero-aggregates produced characteristic EGFP-ratio histograms that matched binomial predictions. When AtaA production in one cell type was decreased, the histogram changed from a symmetric unimodal histogram with the peak at 40-60 % EGFP-ratio to a skewed distribution, indicating that GPIA can detect differences in cell-to-cell affinity. Using the same procedure, we examined six in-frame deletion variants of AtaA. The deletion of the N-terminal head domain alone prevented co-aggregation with full-length AtaA, suggesting that homophilic recognition by this domain mediated self-aggregation, whereas deletions in all other regions had no measurable effect. GPIA, therefore, offers a simple and rapid approach for quantitative studies on bacterial cell aggregation, bridging the gap between qualitative microscopy and quantitative but technically demanding single-cell analysis. GPIA will accelerate research on cell-cell interactions, which are the foundational processes that drive biofilm formation and the assembly of microbial consortia.

microbiology↗

bioRxiv2025

Identification and functional characterization of toluene degradation genes in Acinetobacter sp. Tol 5

Microbial degradation of aromatic compounds provides sustainable solutions for environmental remediation and bioconversion. Acinetobacter sp. Tol 5 is notable for its strong adhesiveness and potential as a biocatalyst for toluene degradation; however, its toluene metabolic pathway has not been fully elucidated. In this study, genomic analysis identified a cluster of genes in Tol 5 highly similar to the well-known tod operon of Pseudomonas putida, encoding enzymes responsible for toluene metabolism. Phylogenetic analyses indicated that these tod genes, unusual among Acinetobacter species, were likely acquired through horizontal gene transfer. Transcriptomic analyses revealed that todF and todC1 are co-transcribed, while the adjacent fadL2 gene, encoding a putative outer membrane transporter corresponding to P. putida todX, is independently transcribed. Functional characterization using gene-knockout mutants revealed that TodC1, the large subunit of dioxygenase, is essential for growth on toluene, whereas FadL2 is not essential. Growth experiments further showed that the todC1 knockout mutant could metabolize benzoate, but not toluene or benzene, confirming that the TOD pathway is the primary route for toluene and benzene degradation in Tol 5. The identification of the functional TOD pathway, which is unique within Acinetobacter, provides genetic and biochemical insights for the development of Tol 5 as an efficient immobilized biocatalyst for the bioremediation and bioconversion of aromatic compounds.

microbiology↗

bioRxiv2024

ADRA2A promotes the classical/progenitor subtype and reduces disease aggressiveness of pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) manifests diverse molecular subtypes, including the classical/progenitor and basal-like/squamous subtypes, with the latter known for its aggressiveness. We employed integrative transcriptome and metabolome analyses to identify potential genes contributing to the molecular subtype differentiation and its metabolic features. Transcriptome analysis in PDAC patient cohorts revealed downregulation of adrenoceptor alpha 2A (ADRA2A) in the basal-like/squamous subtype, suggesting its potential role as a candidate suppressor of this subtype. Reduced ADRA2A expression was significantly associated with a high frequency of lymph node metastasis, higher pathological grade, advanced disease stage, and decreased survival among PDAC patients. In vitro experiments demonstrated that ADRA2A transgene expression and ADRA2A agonist inhibited PDAC cell invasion. Additionally, ADRA2A-high condition downregulated the basal-like/squamous gene expression signature, while upregulating the classical/progenitor gene expression signature in our PDAC patient cohort and PDAC cell lines. Metabolome analysis conducted on the PDAC cohort and cell lines revealed that elevated ADRA2A levels were associated with suppressed amino acid and carnitine/acylcarnitine metabolism, which are characteristic metabolic profiles of the classical/progenitor subtype. Collectively, our findings suggest that heightened ADRA2A expression induces transcriptome and metabolome characteristics indicative of classical/progenitor subtype with decreased disease aggressiveness in PDAC patients. These observations introduce ADRA2A as a candidate for diagnostic and therapeutic targeting in PDAC. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC="FIGDIR/small/584316v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@edc5ceorg.highwire.dtl.DTLVardef@5579b6org.highwire.dtl.DTLVardef@1a9a000org.highwire.dtl.DTLVardef@147724a_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG HighlightsO_LIADRA2A is downregulated in the basal-like/squamous PDAC while its expression is maintained in the classical/progenitor PDAC subtype C_LIO_LIUpregulated ADRA2A expression correlates with improved PDAC survival and reduced invasion in PDAC cells C_LIO_LIUpregulated ADRA2A downregulates the MYC signaling pathway and promotes the classical/progenitor gene expression profile C_LIO_LIUpregulated ADRA2A induces a unique metabolic signature characterized by diminished amino acid and carnitine/acylcarnitine metabolism, resembling the classical/progenitor PDAC subtype C_LI

cancer biology↗

bioRxiv2023

LMO3 is a suppressor of the basal-like/squamous PDAC subtype and reduces disease aggressiveness of pancreatic cancer through glycerol 3-phosphate metabolism

Pancreatic ductal adenocarcinoma (PDAC) encompasses diverse molecular subtypes, including the classical/progenitor and basal-like/squamous subtypes, each exhibiting distinct characteristics, with the latter known for its aggressiveness. We employed an integrative approach combining transcriptomic and metabolomic analyses to pinpoint potential genes contributing to the basal-like/squamous subtype differentiation. Applying this approach to our NCI-UMD-German and a validation cohort, we identified LIM Domain Only 3 (LMO3), a transcription co-factor, as a candidate suppressor of the basal-like/squamous subtype. Reduced LMO3 expression was significantly associated with higher pathological grade, advanced disease stage, induction of the basal-like/squamous subtype, and decreased survival among PDAC patients. In vitro experiments demonstrated that LMO3 transgene expression inhibited PDAC cell proliferation and migration/invasion, concurrently downregulating the basal-like/squamous gene signature. Metabolomic analysis of patient tumors and PDAC cells revealed a metabolic program linked to elevated LMO3 expression and the classical/progenitor subtype, characterized by enhanced lipogenesis and suppressed amino acid metabolism. Notably, glycerol 3-phosphate (G3P) levels positively correlated with LMO3 expression and associated with improved patient survival. Furthermore, glycerol-3-phosphate dehydrogenase 1 (GPD1), a crucial enzyme in G3P synthesis, showed upregulation in LMO3-high and classical/progenitor PDAC, suggesting its potential role in mitigating disease aggressiveness. Collectively, our findings suggest that heightened LMO3 expression reduces transcriptomic and metabolomic characteristics indicative of basal-like/squamous tumors with decreased disease aggressiveness in PDAC patients. The observations describe LMO3 as a candidate for diagnostic and therapeutic targeting in PDAC. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/564448v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@15ab2f1org.highwire.dtl.DTLVardef@199f209org.highwire.dtl.DTLVardef@1e1661corg.highwire.dtl.DTLVardef@f6eb7e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LILMO3 is downregulated in basal-like/squamous PDAC while its expression is maintained in the classical/progenitor PDAC subtype C_LIO_LIUpregulated LMO3 expression correlates with improved PDAC survival and reduced proliferation and migration/invasion in PDAC cells C_LIO_LIUpregulated LMO3 suppresses basal-like/squamous differentiation and induces a unique metabolic signature characterized by elevated lipogenesis and diminished amino acid metabolism, resembling the classical/progenitor PDAC subtype C_LIO_LIEnhanced LMO3 expression associates with elevated glycerol 3-phosphate levels in PDAC, correlating with improved patient survival in PDAC C_LI

cancer biology↗

bioRxiv2023

ELAPOR1 induces the classical/progenitor subtype and contributes to reduced disease aggressiveness through metabolic reprogramming in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a heterogeneous disease with distinct molecular subtypes classified as classical/progenitor and basal-like/squamous. We hypothesized that integrative transcriptomic and metabolomic approaches can identify candidate genes whose inactivation contributes to the development of the aggressive basal-like/squamous subtype. Using our integrated approach, we identified endosome-lysosome associated apoptosis and autophagy regulator 1 (ELAPOR1/KIAA1324) as a candidate tumor suppressor in both our NCI-UMD-German cohort and validation cohorts. We found that decreased ELAPOR1 expression was significantly associated with high pathological grade, advanced disease stage, the basal-like/squamous subtype, and decreased survival in PDAC patients. In vitro experiments showed that ELAPOR1 transgene expression inhibited migration and invasion of PDAC cells. Metabolomic analysis of patient tumors and PDAC cells revealed a metabolic program associated with both upregulated ELAPOR1 and the classical/progenitor subtype, encompassing upregulated lipogenesis and downregulated amino acid metabolism. 1-methylnicotinamide, an oncometabolite derived from S-adenosylmethionine, was inversely associated with ELAPOR1 expression and promoted migration and invasion of PDAC cells in vitro. Taken together, our data suggest that enhanced ELAPOR1 expression promotes transcriptomic and metabolomic characteristics that are indicative of the classical/progenitor subtype, whereas its reduction associates with basal-like/squamous tumors with increased disease aggressiveness in PDAC patients. This positions ELAPOR1 as a promising candidate for diagnostic and therapeutic targeting in PDAC. Novelty and ImpactPancreatic ductal adenocarcinoma (PDAC) exhibits heterogeneous molecular subtypes: classical/progenitor and basal-like/squamous. Comprehensive transcriptome and metabolome analyses in the PDAC patient cohorts and PDAC cell lines revealed that elevated ELAPOR1 correlates with enhanced survival, reduced PDAC cell invasion, and a distinct metabolic signature resembling the classical/progenitor subtype. Additionally, 1-methylnicotinamide has been identified as an oncometabolite, showing an inverse correlation with ELAPOR1. These findings emphasize ELAPOR1s potential as a diagnostic and therapeutic target in PDAC. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/558894v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@f95c81org.highwire.dtl.DTLVardef@128334corg.highwire.dtl.DTLVardef@3fcb60org.highwire.dtl.DTLVardef@414011_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIELAPOR1 is downregulated in basal-like/squamous PDAC C_LIO_LIUpregulation of ELAPOR1 associates with improved PDAC survival and reduced migration and invasion in PDAC cells C_LIO_LIELAPOR1 expression induces a distinct metabolic signature as characterized by upregulation of lipogenesis and downregulation of amino acid metabolism, commonly observed in the classical/progenitor PDAC subtype C_LIO_LIThe oncometabolite, 1-methylnicotinamide (MNA) is decreased when ELAPOR1 is upregulated, and promotes the migration and invasion of PDAC cells C_LI

cancer biology↗

bioRxiv2023

SERPINB3 induces the basal-like/squamous subtype and enhances disease progression in pancreatic cancer

Pancreatic cancer is a heterogeneous disease with distinct subtypes. Here, we investigated candidate driver genes of the highly aggressive basal-like/squamous molecular subtype of pancreatic ductal adenocarcinoma (PDAC). Integrative transcriptomic analyses identified the upregulated serine/cysteine protease inhibitor, SERPINB3 (squamous cell carcinoma antigen 1, SCCA1) in basal-like/squamous PDAC using discovery and validation approaches. Upregulation of SERPINB3 associated with decreased patient survival and a transcriptome profile indicative of the basal-like/squamous subtype. In human PDAC cell lines, SERPINB3 transgene expression enhanced their invasion capability. Moreover, upregulated expression of SERPINB3 in AsPC-1 cells resulted in enhanced lung metastasis in an orthotopic xenograft model. Molecular analysis of the primary tumor xenografts indicated activation of pathways related to metastasis, increased oxidative damage, and angiogenesis when SERPINB3 was upregulated. Furthermore, metabolomic analysis, using patient cohorts and PDAC cell lines showed a distinct metabolic pattern closely associated with both SERPINB3 and the basal-like/squamous subtype, which included upregulation of carnitine/acylcarnitine, amino acid, glutathione, and purine metabolic pathways, and glycolysis. Further RNA-seq and metabolomic analyses indicated that SERPINB3 may potentially induce the basal-like/squamous subtype and metabolic reprogramming through MYC activation. Taken together, our findings identified SERPINB3 as a candidate marker gene for the basal-like/squamous subtype, which may contribute to the disease aggressiveness in this subtype of PDAC. Abbreviations8-Hydroxy-2-deoxyguanosine (8-OHdG), Diaminobenzene (DAB), Gene Set Enrichment Analysis (GSEA), Human Metabolome Technologies, Inc. (HMT), Immunohistochemistry (IHC), Ingenuity pathway analysis (IPA), Pancreatic ductal adenocarcinoma (PDAC), Serine/Cysteine Proteinase Inhibitor Family B Member 3 (SERPINB3) Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/534766v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@8209f5org.highwire.dtl.DTLVardef@15be96dorg.highwire.dtl.DTLVardef@13a0c6corg.highwire.dtl.DTLVardef@58b2f_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LISERPINB3 is upregulated in basal-like/squamous PDAC and associates with decreased patient survival C_LIO_LISERPINB3 promotes differentiation into the basal-like/squamous subtype and enhances invasion and metastasis of PDAC C_LIO_LISERPINB3 induces metabolic reprogramming and MYC activation and a metabolic signature indicative of basal-like/squamous PDAC C_LI

cancer biology↗