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Cloning, Expression, Characterization and in silico studies of L-asparaginase from Vibrio sp. (GBPx3).
Pub Date : 2025-03-10 DOI: 10.1016/j.biochi.2025.03.003
Sareh Sadat Mousavi Natanzi, Sedigheh Asad, Hossein Mahboudi, Solat Eslami

L-asparaginase is a critical therapeutic enzyme for treating acute lymphoblastic leukemia (ALL), a common childhood malignancy. In this study, the L-asparaginase coding sequence from halophilic Vibrio sp. (GBPx3) was cloned, expressed in Escherichia coli, and characterized. The enzyme exhibited a molecular weight of 39.2 kDa and demonstrated a Km of 4.517 mM, kcat of 2.88 1/s, and Vmax of 0.1055 μmol/min, reflecting high specificity for L-asparagine and minimal activity (0.4%) toward L-glutamine. Optimal activity was observed at physiological conditions (37°C, pH 7.5 and 125-150 mM NaCl), consistent with human serum osmolality. The half-life of the enzyme was 2.64 hours in human serum at 37°C that is longer than the half-life reported for E. coli L-asparaginase. Additionally, the enzyme had no toxic impact on human umbilical vein endothelial cells (HUVEC) and human erythrocytes. The recombinant L-asparaginase was predicted to be 29.3% helix, 35.6% turns, and 35.1% random by circular dichroism spectroscopy. AlphaFold predicted a 3D structure with promising validation scores. The molecular docking study showed that Thr14, Ser60, Thr91, and Asp92 are putative active site residues, with a negative binding energy of -4.5 kJ/mol for the substrate-enzyme interaction. The enzyme's low immunogenicity, high serum stability, and reduced glutaminase activity highlight its potential as a safer therapeutic alternative. Future experiments and protein engineering studies are needed to explore enzyme's in vivo efficacy and improve its clinical effectiveness.

{"title":"Cloning, Expression, Characterization and in silico studies of L-asparaginase from Vibrio sp. (GBPx3).","authors":"Sareh Sadat Mousavi Natanzi, Sedigheh Asad, Hossein Mahboudi, Solat Eslami","doi":"10.1016/j.biochi.2025.03.003","DOIUrl":"https://doi.org/10.1016/j.biochi.2025.03.003","url":null,"abstract":"<p><p>L-asparaginase is a critical therapeutic enzyme for treating acute lymphoblastic leukemia (ALL), a common childhood malignancy. In this study, the L-asparaginase coding sequence from halophilic Vibrio sp. (GBPx3) was cloned, expressed in Escherichia coli, and characterized. The enzyme exhibited a molecular weight of 39.2 kDa and demonstrated a K<sub>m</sub> of 4.517 mM, k<sub>cat</sub> of 2.88 1/s, and V<sub>max</sub> of 0.1055 μmol/min, reflecting high specificity for L-asparagine and minimal activity (0.4%) toward L-glutamine. Optimal activity was observed at physiological conditions (37°C, pH 7.5 and 125-150 mM NaCl), consistent with human serum osmolality. The half-life of the enzyme was 2.64 hours in human serum at 37°C that is longer than the half-life reported for E. coli L-asparaginase. Additionally, the enzyme had no toxic impact on human umbilical vein endothelial cells (HUVEC) and human erythrocytes. The recombinant L-asparaginase was predicted to be 29.3% helix, 35.6% turns, and 35.1% random by circular dichroism spectroscopy. AlphaFold predicted a 3D structure with promising validation scores. The molecular docking study showed that Thr14, Ser60, Thr91, and Asp92 are putative active site residues, with a negative binding energy of -4.5 kJ/mol for the substrate-enzyme interaction. The enzyme's low immunogenicity, high serum stability, and reduced glutaminase activity highlight its potential as a safer therapeutic alternative. Future experiments and protein engineering studies are needed to explore enzyme's in vivo efficacy and improve its clinical effectiveness.</p>","PeriodicalId":93898,"journal":{"name":"Biochimie","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143617969","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Metformin's Anticancer Odyssey: Revealing Multifaceted Mechanisms Across Diverse Neoplastic Terrains- A Critical Review.
Pub Date : 2025-03-07 DOI: 10.1016/j.biochi.2025.03.002
Rashmi Saxena Pal, Talha Jawaid, M A Rahman, Rakesh Verma, Pratap Kumar Patra, Sharma Vedika Vijaypal, Yogendra Pal, Rohit Upadhyay

Metformin, initially prescribed as an oral hypoglycemic medication for type 2 diabetes, has recently gained attention for its potential anticancer effects. Its history dates to 1918, when guanidine, a component of the traditional European herb Galega officinalis, was found to reduce glycemia. This review precisely examines the mechanisms underlying Metformin's anticancer effects across various neoplastic conditions. This investigation explores the complex interactions between metformin and major signaling pathways associated with carcinogenesis, including AMP-activated protein kinase (AMPK), mTOR, and insulin-like growth factor (IGF) pathways. The review emphasizes Metformin's diverse effects on angiogenesis, inflammation, apoptosis, and cellular metabolism in cancer cells. Additionally, new data on metformin's capacity to alter the tumor microenvironment and enhance immune surveillance systems against cancer are examined. The review underscores Metformin's potential for repurposing in oncology, emphasizing its clinical relevance as an adjuvant therapy for various cancers. The review provides insightful information about the complex anticancer mechanisms of metformin by combining data from preclinical and clinical studies. These findings not only broaden our knowledge of the effects of metformin but also open new avenues for oncology research and treatment developments.

{"title":"Metformin's Anticancer Odyssey: Revealing Multifaceted Mechanisms Across Diverse Neoplastic Terrains- A Critical Review.","authors":"Rashmi Saxena Pal, Talha Jawaid, M A Rahman, Rakesh Verma, Pratap Kumar Patra, Sharma Vedika Vijaypal, Yogendra Pal, Rohit Upadhyay","doi":"10.1016/j.biochi.2025.03.002","DOIUrl":"https://doi.org/10.1016/j.biochi.2025.03.002","url":null,"abstract":"<p><p>Metformin, initially prescribed as an oral hypoglycemic medication for type 2 diabetes, has recently gained attention for its potential anticancer effects. Its history dates to 1918, when guanidine, a component of the traditional European herb Galega officinalis, was found to reduce glycemia. This review precisely examines the mechanisms underlying Metformin's anticancer effects across various neoplastic conditions. This investigation explores the complex interactions between metformin and major signaling pathways associated with carcinogenesis, including AMP-activated protein kinase (AMPK), mTOR, and insulin-like growth factor (IGF) pathways. The review emphasizes Metformin's diverse effects on angiogenesis, inflammation, apoptosis, and cellular metabolism in cancer cells. Additionally, new data on metformin's capacity to alter the tumor microenvironment and enhance immune surveillance systems against cancer are examined. The review underscores Metformin's potential for repurposing in oncology, emphasizing its clinical relevance as an adjuvant therapy for various cancers. The review provides insightful information about the complex anticancer mechanisms of metformin by combining data from preclinical and clinical studies. These findings not only broaden our knowledge of the effects of metformin but also open new avenues for oncology research and treatment developments.</p>","PeriodicalId":93898,"journal":{"name":"Biochimie","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143588767","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Over-expression of AeWRKY2 promotes oleanolic acid and hederagenin accumulation in Aralia elata.
Pub Date : 2025-03-05 DOI: 10.1016/j.biochi.2025.03.001
Honghao Xu, Hongfei Liu, Ye Liu, Wenhua Guo, Yue Zhao, Haoze Ying, Yaru Liu, Zhe Li, Yi Zhang, Lei Tao, Tuya Siqin, Wa Gao, Xiangling You

Oleanolic acid (OA) and hederagenin from Aralia elata have important medicinal value due to their anti-cancer and anti-inflammatory activities. Transcription factors (TFs) can affect the production of active substances by regulating the expression of genes in metabolic pathways. In this study, a transcription factor AeWRKY2 was cloned and overexpressed in A. elata. The result of gene expression analysis and high-performance liquid chromatography (HPLC) showed that the key enzyme genes of all transgenic strains analyzed were up-regulated to varying degrees. The contents of oleanolic acid and hederagenin were higher than those of wild type, up to 2.773 mg·g-1 DW and 0.325 mg·g-1 DW, respectively. After one-day MeJA treatment, the contents of oleanolic acid and hederagenin were further increased to 3.31 mg·g-1 and 0.37 mg·g-1, respectively.

{"title":"Over-expression of AeWRKY2 promotes oleanolic acid and hederagenin accumulation in Aralia elata.","authors":"Honghao Xu, Hongfei Liu, Ye Liu, Wenhua Guo, Yue Zhao, Haoze Ying, Yaru Liu, Zhe Li, Yi Zhang, Lei Tao, Tuya Siqin, Wa Gao, Xiangling You","doi":"10.1016/j.biochi.2025.03.001","DOIUrl":"https://doi.org/10.1016/j.biochi.2025.03.001","url":null,"abstract":"<p><p>Oleanolic acid (OA) and hederagenin from Aralia elata have important medicinal value due to their anti-cancer and anti-inflammatory activities. Transcription factors (TFs) can affect the production of active substances by regulating the expression of genes in metabolic pathways. In this study, a transcription factor AeWRKY2 was cloned and overexpressed in A. elata. The result of gene expression analysis and high-performance liquid chromatography (HPLC) showed that the key enzyme genes of all transgenic strains analyzed were up-regulated to varying degrees. The contents of oleanolic acid and hederagenin were higher than those of wild type, up to 2.773 mg·g<sup>-1</sup> DW and 0.325 mg·g<sup>-1</sup> DW, respectively. After one-day MeJA treatment, the contents of oleanolic acid and hederagenin were further increased to 3.31 mg·g<sup>-1</sup> and 0.37 mg·g<sup>-1</sup>, respectively.</p>","PeriodicalId":93898,"journal":{"name":"Biochimie","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143588791","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
The characterization and comparison of femoral bone-derived skeletal stem cells.
Pub Date : 2025-02-27 DOI: 10.1016/j.biochi.2025.02.010
Kayla Howard, William Frank Ferris, Mari van de Vyver

Skeletal stem cells (SSCs) reside in various niche locations within long bones to maintain bone homeostasis and facilitate fracture repair. Bone fragility, associated with ageing, increases the susceptibility of the femoral head to fractures due to an increase in bone adipocytes and concomitant loss of structural integrity. However, the specific contribution of epiphyseal SSCs to fragility is unknown. To explore this, a comparative analysis was performed on the transcriptional profiles and lineage commitment of Wistar rat femoral SSCs derived from the bone marrow (BM-), diaphyseal cortical bone (CB-) and proximal epiphyseal trabecular bone (PF-SSCs) isolated from the same long bones. SSCs were characterized based on morphology, immunophenotype (CD90/CD45), growth rate (population doubling time), gene expression profiles and differentiation capacity (Oil Red O, Alizarin Red S). qRT-PCR micro-arrays were performed on SSCs to evaluate the expression of stemness, SSC and lineage-specific markers in both undifferentiated and differentiated states. Our findings support the hypothesis that SSCs from different bone regions exhibit distinct transcriptional profiles, reflecting their specific niche environments. CB-SSCs displayed superior osteogenic potential as evidenced by the expression of key osteogenic genes and higher levels of mineralization. In contrast, PF-SSCs had a reduced osteogenic capacity with a higher adipogenic potential. Overall, the study revealed the importance of niche-specific stem cell properties for use in regenerative medicine applications and provides insight into the potential role of PF-SSCs in bone fragility and fracture risk.

{"title":"The characterization and comparison of femoral bone-derived skeletal stem cells.","authors":"Kayla Howard, William Frank Ferris, Mari van de Vyver","doi":"10.1016/j.biochi.2025.02.010","DOIUrl":"https://doi.org/10.1016/j.biochi.2025.02.010","url":null,"abstract":"<p><p>Skeletal stem cells (SSCs) reside in various niche locations within long bones to maintain bone homeostasis and facilitate fracture repair. Bone fragility, associated with ageing, increases the susceptibility of the femoral head to fractures due to an increase in bone adipocytes and concomitant loss of structural integrity. However, the specific contribution of epiphyseal SSCs to fragility is unknown. To explore this, a comparative analysis was performed on the transcriptional profiles and lineage commitment of Wistar rat femoral SSCs derived from the bone marrow (BM-), diaphyseal cortical bone (CB-) and proximal epiphyseal trabecular bone (PF-SSCs) isolated from the same long bones. SSCs were characterized based on morphology, immunophenotype (CD90/CD45), growth rate (population doubling time), gene expression profiles and differentiation capacity (Oil Red O, Alizarin Red S). qRT-PCR micro-arrays were performed on SSCs to evaluate the expression of stemness, SSC and lineage-specific markers in both undifferentiated and differentiated states. Our findings support the hypothesis that SSCs from different bone regions exhibit distinct transcriptional profiles, reflecting their specific niche environments. CB-SSCs displayed superior osteogenic potential as evidenced by the expression of key osteogenic genes and higher levels of mineralization. In contrast, PF-SSCs had a reduced osteogenic capacity with a higher adipogenic potential. Overall, the study revealed the importance of niche-specific stem cell properties for use in regenerative medicine applications and provides insight into the potential role of PF-SSCs in bone fragility and fracture risk.</p>","PeriodicalId":93898,"journal":{"name":"Biochimie","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143538235","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Doxorubicin-induced senescence is modulated by the eukaryotic release factor 3a and its polyglycine expansion in HCT116 cells. 多柔比星诱导的衰老受 HCT116 细胞中真核释放因子 3a 及其多聚甘氨酸扩增的调节。
Pub Date : 2025-02-25 DOI: 10.1016/j.biochi.2025.02.009
Béatrice Jolles, Vérène Stierlé

In humans, the release factor eRF3a exists in several forms that differ in the length of the polyglycine tract (7, 10, 11 or 12 glycines) in its N-terminal domain. For the 12-Gly eRF3a, an association with cancer risk and a decreased affinity for the cytoplasmic poly (A) binding protein have already been established. In this work, HCT116 colon cancer cells were treated with low doses of doxorubicin, which is known to induce senescence in these cells with high efficiency. The expression of p21 and p53 (senescence marker proteins) as well as lysosomal β-galactosidase activity were reduced when 12-Gly-eRF3a was overexpressed or eRF3a was depleted in cells. If low activity of mTORC1 pathway might be responsible for reduced senescence onset after eRF3a depletion, its activity is maintained in cells overexpressing 12-Gly-eRF3a. In both cases, a defect in termination efficiency could be involved.

{"title":"Doxorubicin-induced senescence is modulated by the eukaryotic release factor 3a and its polyglycine expansion in HCT116 cells.","authors":"Béatrice Jolles, Vérène Stierlé","doi":"10.1016/j.biochi.2025.02.009","DOIUrl":"10.1016/j.biochi.2025.02.009","url":null,"abstract":"<p><p>In humans, the release factor eRF3a exists in several forms that differ in the length of the polyglycine tract (7, 10, 11 or 12 glycines) in its N-terminal domain. For the 12-Gly eRF3a, an association with cancer risk and a decreased affinity for the cytoplasmic poly (A) binding protein have already been established. In this work, HCT116 colon cancer cells were treated with low doses of doxorubicin, which is known to induce senescence in these cells with high efficiency. The expression of p21 and p53 (senescence marker proteins) as well as lysosomal β-galactosidase activity were reduced when 12-Gly-eRF3a was overexpressed or eRF3a was depleted in cells. If low activity of mTORC1 pathway might be responsible for reduced senescence onset after eRF3a depletion, its activity is maintained in cells overexpressing 12-Gly-eRF3a. In both cases, a defect in termination efficiency could be involved.</p>","PeriodicalId":93898,"journal":{"name":"Biochimie","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143525502","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Difference in the rearrangement of quail histone H1 allelic variants during divergent selection for reduction of body mass coupled to the food withdraw.
Pub Date : 2025-02-21 DOI: 10.1016/j.biochi.2025.02.008
Andrzej Kowalski, Sebastian Knaga

This study was undertaken to show whether chromatin phenotype(s) related to histone H1 polymorphism(s) exhibit connection with breeding practice (selection for reduction of body mass) and/or physiological trait (withdraw of the food) of the organism. For this purpose, a genetic diversity between quail lines selected for high and low reduction of body mass after transient food withdrawal was examined using variation in the histone H1 allelic expression. Symmetric and asymmetric expression of the isoforms of histone H1.b and histone H1.d was identified in 2-D PAGE due to various molecular weight and different intensities, respectively. Low (locus H1.d) to moderate (locus H1.b) differentiated populations conform to Hardy-Weinberg equilibrium but distribution of the phenotypes significantly differ between them. Whereas frequency of the same phenotype of histone H1.b is different between selected lines, the frequency of the same phenotypes of histone H1.d is similar in both selected lines. Thus, line-specific promotion of the phenotypes of histone H1.b suggest a response to conducted selection. Unlike this, a line-nonspecific arrangement of the phenotypes of histone H1.d appear due to the stress evoked by food withdrawal ongoing during the selection. Such a result indicate that histone H1 allelic variants possess individual impact on chromatin states and/or processes associated with breeding practices and physiological conditions of the organism. According to this, functional individualization is a characteristic feature of histone H1 polymorphic variants.

{"title":"Difference in the rearrangement of quail histone H1 allelic variants during divergent selection for reduction of body mass coupled to the food withdraw.","authors":"Andrzej Kowalski, Sebastian Knaga","doi":"10.1016/j.biochi.2025.02.008","DOIUrl":"10.1016/j.biochi.2025.02.008","url":null,"abstract":"<p><p>This study was undertaken to show whether chromatin phenotype(s) related to histone H1 polymorphism(s) exhibit connection with breeding practice (selection for reduction of body mass) and/or physiological trait (withdraw of the food) of the organism. For this purpose, a genetic diversity between quail lines selected for high and low reduction of body mass after transient food withdrawal was examined using variation in the histone H1 allelic expression. Symmetric and asymmetric expression of the isoforms of histone H1.b and histone H1.d was identified in 2-D PAGE due to various molecular weight and different intensities, respectively. Low (locus H1.d) to moderate (locus H1.b) differentiated populations conform to Hardy-Weinberg equilibrium but distribution of the phenotypes significantly differ between them. Whereas frequency of the same phenotype of histone H1.b is different between selected lines, the frequency of the same phenotypes of histone H1.d is similar in both selected lines. Thus, line-specific promotion of the phenotypes of histone H1.b suggest a response to conducted selection. Unlike this, a line-nonspecific arrangement of the phenotypes of histone H1.d appear due to the stress evoked by food withdrawal ongoing during the selection. Such a result indicate that histone H1 allelic variants possess individual impact on chromatin states and/or processes associated with breeding practices and physiological conditions of the organism. According to this, functional individualization is a characteristic feature of histone H1 polymorphic variants.</p>","PeriodicalId":93898,"journal":{"name":"Biochimie","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143485008","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Molecular mechanism of selenite reduction by Bacillus amyloliquefaciens BB61 based on transcriptome analysis.
Pub Date : 2025-02-21 DOI: 10.1016/j.biochi.2025.02.005
Yujie Wang, Fan Wan, Huiqin Xue, Yiqiong Hang, Caixia Pei, Yang Lu

The microbial conversion of selenite represents an effective detoxification and assimilation process, although the underlying mechanisms remain incompletely understood. In this study, strain BB61 was a probiotic isolated from piglet feces and identified as Bacillus amyloliquefaciens, which could almost completely reduce 0.1 g/L Na2SeO3 to SeNPs within 48h. We investigated the potential mechanisms of selenite reduction in this strain through transcriptome sequencing and qPCR. The transcriptome analysis revealed the up-regulation of 829 genes and the down-regulation of 892 genes in response to 1 g/L Se treatment (padj <0.05) in Bacillus amyloliquefaciens BB61. GO (Gene Ontology) enrichment analysis indicated that DEGs (Differentially expressed genes) were predominantly associated with transmembrane transporters, ion transmembrane transport, cytoplasmic and cell membrane composition, cell movement and localization, and carbon metabolism. Additionally, the KEGG (Encyclopedia of Genes and Genomes) pathway annotation analysis revealed that the DEGs were primarily involved in the pentose phosphate pathway, pyruvate metabolism, pyrimidine metabolism, cofactor biosynthesis, and other pathways (P < 0.05). Among the highly expressed reductases, thioredoxin reductase (TrxA/B), nitrite reductase (NfsA), and selenite reductase (NamA) were all found to be up-regulated. Consequently, this study established a reduction pathway model for Se (IV), offering new insights into the molecular mechanisms underlying the bioreduction of selenite to form SeNPs.

亚硒酸盐的微生物转化是一种有效的解毒和同化过程,但其基本机制仍不完全清楚。在本研究中,菌株 BB61 是一种从仔猪粪便中分离出来的益生菌,经鉴定为淀粉芽孢杆菌,它能在 48 小时内将 0.1 g/L Na2SeO3 几乎完全还原为 SeNPs。我们通过转录组测序和 qPCR 研究了该菌株还原亚硒酸盐的潜在机制。转录组分析表明,淀粉芽孢杆菌 BB61 对 1 g/L Se 处理有 829 个基因上调,892 个基因下调(padj < 0.05)。GO(基因本体)富集分析表明,DEGs(差异表达基因)主要与跨膜转运体、离子跨膜转运、细胞质和细胞膜组成、细胞运动和定位以及碳代谢有关。此外,KEGG(基因与基因组百科全书)通路注释分析显示,DEGs 主要参与磷酸戊糖通路、丙酮酸代谢、嘧啶代谢、辅助因子生物合成和其他通路(P < 0.05)。在高表达的还原酶中,硫氧还蛋白还原酶(TrxA/B)、亚硝酸盐还原酶(NfsA)和亚硒酸盐还原酶(NamA)均被上调。因此,本研究建立了 Se (IV) 的还原途径模型,为了解亚硒酸盐生物还原形成 SeNPs 的分子机制提供了新的视角。
{"title":"Molecular mechanism of selenite reduction by Bacillus amyloliquefaciens BB61 based on transcriptome analysis.","authors":"Yujie Wang, Fan Wan, Huiqin Xue, Yiqiong Hang, Caixia Pei, Yang Lu","doi":"10.1016/j.biochi.2025.02.005","DOIUrl":"10.1016/j.biochi.2025.02.005","url":null,"abstract":"<p><p>The microbial conversion of selenite represents an effective detoxification and assimilation process, although the underlying mechanisms remain incompletely understood. In this study, strain BB61 was a probiotic isolated from piglet feces and identified as Bacillus amyloliquefaciens, which could almost completely reduce 0.1 g/L Na<sub>2</sub>SeO<sub>3</sub> to SeNPs within 48h. We investigated the potential mechanisms of selenite reduction in this strain through transcriptome sequencing and qPCR. The transcriptome analysis revealed the up-regulation of 829 genes and the down-regulation of 892 genes in response to 1 g/L Se treatment (padj <0.05) in Bacillus amyloliquefaciens BB61. GO (Gene Ontology) enrichment analysis indicated that DEGs (Differentially expressed genes) were predominantly associated with transmembrane transporters, ion transmembrane transport, cytoplasmic and cell membrane composition, cell movement and localization, and carbon metabolism. Additionally, the KEGG (Encyclopedia of Genes and Genomes) pathway annotation analysis revealed that the DEGs were primarily involved in the pentose phosphate pathway, pyruvate metabolism, pyrimidine metabolism, cofactor biosynthesis, and other pathways (P < 0.05). Among the highly expressed reductases, thioredoxin reductase (TrxA/B), nitrite reductase (NfsA), and selenite reductase (NamA) were all found to be up-regulated. Consequently, this study established a reduction pathway model for Se (IV), offering new insights into the molecular mechanisms underlying the bioreduction of selenite to form SeNPs.</p>","PeriodicalId":93898,"journal":{"name":"Biochimie","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143485013","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Identification and characterization of a laterally transferred alternative oxidase (AOX) in a terrestrial insect, the dipteran Pseudolycoriella hygida.
Pub Date : 2025-02-21 DOI: 10.1016/j.biochi.2025.02.007
Nadia Monesi, Guilherme Magre Fernandes, Felipe Berti Valer, João Vítor Cardoso Uliana, Vitor Trinca, Ana Elisa Caleiro Seixas Azzolini, Eduardo Gorab, Luciane Carla Alberici

Alternative oxidase (AOX) (EC 1.10.3.11) is a terminal oxidase in the mitochondrial inner membrane that branches the canonical electron transport system (ETS). AOX is ubiquitous in plants, frequently found in fungi and protists and presents a more sporadic distribution in metazoans. More recently, AOX has gained attention due to its potential application in gene therapy for treatment of mitochondrial diseases. Here we characterized the AOX in the basal Dipteran, Pseudolycoriella hygida using a combination of genomic analyses, molecular, functional and in vivo survival assays. AOX is a single copy gene that encodes three developmental stage specific protein isoforms. AOX localizes to the mitochondria in adult thoracic muscles, which present cyanide-resistant respiration that is sensitive to the AOX inhibitor salicylhydroxamic acid (SHAM). Both the cyanide-resistant respiration and AOX levels gradually increase during aging, but are not influenced by thermal stress. Thoracic mitochondria respire using substrates derived from several metabolic routes, such as pyruvate, proline, acylcarnitine, NADH and glycerol-3P, and present values of oxidative phosphorylation capacity ((P-L)/E = 0.70) and coupling (P/L = 4.35; L/E = 0.21). Adult flies exhibit a high survival resistance for SHAM-sensitive complex III inhibition. Together, our results demonstrate the presence of a functional AOX in a terrestrial arthropod and provide insights regarding AOX function in animals and evolution of respiratory systems in metazoans. Psl. hygida emerges as a natural and valuable model for comprehensive AOX research at the whole-organism level which complements models expressing the heterologous enzyme.

替代氧化酶(AOX)(EC 1.10.3.11)是线粒体内膜上的一种末端氧化酶,它是典型电子传递系统(ETS)的分支。AOX 在植物中无处不在,在真菌和原生动物中也经常发现,在后生动物中分布较为零散。最近,AOX 因其在治疗线粒体疾病的基因疗法中的潜在应用而备受关注。在这里,我们采用基因组分析、分子、功能和体内生存试验相结合的方法,描述了基底双翅目伪翅虫(Pseudolycoriella hygida)中 AOX 的特征。AOX 是一个单拷贝基因,可编码三种发育阶段特异的蛋白质异构体。AOX 定位于成体胸肌的线粒体中,它具有抗氰化物呼吸作用,对 AOX 抑制剂水杨羟肟酸(SHAM)敏感。抗氰化物呼吸和 AOX 水平在衰老过程中逐渐增加,但不受热应力的影响。胸线粒体呼吸使用的底物来自几种代谢途径,如丙酮酸、脯氨酸、酰基肉碱、NADH 和甘油-3P,并呈现氧化磷酸化能力值((P-L)/E = 0.70)和耦合值(P/L = 4.35;L/E = 0.21)。成蝇对 SHAM 敏感的复合体 III 抑制表现出较高的存活抵抗力。总之,我们的研究结果证明了陆生节肢动物体内存在功能性 AOX,并为动物体内的 AOX 功能以及后生动物呼吸系统的进化提供了启示。Psl. hygida 是在整个生物体水平上对 AOX 进行全面研究的一个天然而有价值的模型,它与表达异源酶的模型相辅相成。
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引用次数: 0
Hijacking a real time detection thermocycler for enzymology: Improvement of a fluorescent bulk assay monitoring helicase activity.
Pub Date : 2025-02-20 DOI: 10.1016/j.biochi.2025.02.002
Jean-Philippe Robin, Vincent Mocquet

Helicases are enzymes involved in all aspects of nucleic acid synthesis, regulation and degradation. As a consequence, several methods were developed to monitor their enzymatic activity. In this report, we described an improvement of bulk fluorescent helicase assays to overcome their specific limitations (cost, health and safety regulations, etc.). Using a real time detection thermocycler to monitor the fluorescence in real-time, we managed to precisely control the initiation of the helicase reaction through temperature tuning. Therefore, we were able to demonstrate that this setup could provide a qualitative and a quantitative evaluation of the helicase domain of the UPF1 helicase (UPF1-HD) and that several fluorophores could be used in parallel during the same run.

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引用次数: 0
Nitazoxanide inhibits pili assembly by targeting BamB to synergize with polymyxin B against drug-resistant Escherichia coli.
Pub Date : 2025-02-19 DOI: 10.1016/j.biochi.2025.02.006
Wenwen Li, Bingjie Ji, Boyu Li, Minghui Du, Linwei Wang, Jiale Tuo, Hongmei Zhou, Jian Gong, Yongshan Zhao

Gram-negative bacteria rely on pili assembly for pathogenicity, with the chaperone-usher (CU) pathway regulating pilus biogenesis. Nitazoxanide (NTZ) inhibits CU pathway-mediated P pilus biogenesis by specifically interfering with the proper folding of the outer membrane protein (OMP) usher, primarily mediated by the β-barrel assembly machinery (BAM) complex. In this study, we identified the BAM complex components BamB and the BamA POTRA2 domain as key binding targets for NTZ. Molecular dynamics simulations and Bio-Layer Interferometry revealed that BamB residues S61 and R195 are critical for NTZ binding. NTZ activated the Cpx two-component system and induced inner membrane perturbations, which resulted from the accumulation of misfolded P pilus subunits. Upregulation of the ibpAB gene, which protects the bacteria against NTZ-induced oxidative stress, was also observed. Importantly, NTZ combined with polymyxin B enhanced the latter's antibacterial activity against both susceptible and MCR-positive E. coli strains. This enhancement was achieved through NTZ-induced increases in inner membrane permeability, oxidative stress, and inhibition of efflux pump activity and biofilm formation. This study provides new insights into the antimicrobial mechanism of NTZ and highlights its potential as an antibiotic adjuvant by targeting BamB to inhibit the CU pathway, restoring the efficacy of polymyxin B against multidrug-resistant bacteria.

{"title":"Nitazoxanide inhibits pili assembly by targeting BamB to synergize with polymyxin B against drug-resistant Escherichia coli.","authors":"Wenwen Li, Bingjie Ji, Boyu Li, Minghui Du, Linwei Wang, Jiale Tuo, Hongmei Zhou, Jian Gong, Yongshan Zhao","doi":"10.1016/j.biochi.2025.02.006","DOIUrl":"10.1016/j.biochi.2025.02.006","url":null,"abstract":"<p><p>Gram-negative bacteria rely on pili assembly for pathogenicity, with the chaperone-usher (CU) pathway regulating pilus biogenesis. Nitazoxanide (NTZ) inhibits CU pathway-mediated P pilus biogenesis by specifically interfering with the proper folding of the outer membrane protein (OMP) usher, primarily mediated by the β-barrel assembly machinery (BAM) complex. In this study, we identified the BAM complex components BamB and the BamA POTRA2 domain as key binding targets for NTZ. Molecular dynamics simulations and Bio-Layer Interferometry revealed that BamB residues S61 and R195 are critical for NTZ binding. NTZ activated the Cpx two-component system and induced inner membrane perturbations, which resulted from the accumulation of misfolded P pilus subunits. Upregulation of the ibpAB gene, which protects the bacteria against NTZ-induced oxidative stress, was also observed. Importantly, NTZ combined with polymyxin B enhanced the latter's antibacterial activity against both susceptible and MCR-positive E. coli strains. This enhancement was achieved through NTZ-induced increases in inner membrane permeability, oxidative stress, and inhibition of efflux pump activity and biofilm formation. This study provides new insights into the antimicrobial mechanism of NTZ and highlights its potential as an antibiotic adjuvant by targeting BamB to inhibit the CU pathway, restoring the efficacy of polymyxin B against multidrug-resistant bacteria.</p>","PeriodicalId":93898,"journal":{"name":"Biochimie","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143473204","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
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Biochimie
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