Pub Date : 2024-02-13DOI: 10.1186/s13568-024-01680-w
Huan Wang, Shuting Zhao, Zhiyang Han, Zexin Qi, Lei Han, Yu Li
Blue light promotes primordium differentiation and fruiting body formation of mushroom. However, the blue light response mechanism of mushroom remains unclear. In this study, mycelium of Flammulina filiformis was exposed to blue light, red light and dark conditions, and then the comparative metabolome and transcriptome analysis was applied to explore metabolic regulation mechanism of F. filiformis under blue light and red light conditions. The yield of the fruiting body of F. filiformis under blue light condition was much higher than that under dark and red light conditions. Metabolome analysis showed that blue light treatment reduced the concentrations of many low molecular weight carbohydrates in the pilei, but it promoted the accumulation of some low molecular weight carbohydrates in the stipes. Blue light also decreased the accumulation of organic acids in the stipes. Blue light treatment reduced the levels of tyrosine and tryptophan in the stipes, but it largely promoted the accumulation of lysine in this organ. In the stipes of F. filiformis, blue light shifted metabolite flow to synthesis of lysine and carbohydrates through inhibiting the accumulation of aromatic amino acids and organic acids, thereby enhancing its nutritional and medicinal values. The transcriptome analysis displayed that blue light enhanced accumulation of lysine in fruiting body of F. filiformis through downregulation of lysine methyltransferase gene and L-lysine 6-monooxygenase gene. Additionally, in the stipes, blue light upregulated many hydrolase genes to improve the ability of the stipe to biodegrade the medium and elevated the growth rate of the fruiting body.
{"title":"Integrated transcriptome and metabolome analysis provides insights into blue light response of Flammulina filiformis.","authors":"Huan Wang, Shuting Zhao, Zhiyang Han, Zexin Qi, Lei Han, Yu Li","doi":"10.1186/s13568-024-01680-w","DOIUrl":"10.1186/s13568-024-01680-w","url":null,"abstract":"<p><p>Blue light promotes primordium differentiation and fruiting body formation of mushroom. However, the blue light response mechanism of mushroom remains unclear. In this study, mycelium of Flammulina filiformis was exposed to blue light, red light and dark conditions, and then the comparative metabolome and transcriptome analysis was applied to explore metabolic regulation mechanism of F. filiformis under blue light and red light conditions. The yield of the fruiting body of F. filiformis under blue light condition was much higher than that under dark and red light conditions. Metabolome analysis showed that blue light treatment reduced the concentrations of many low molecular weight carbohydrates in the pilei, but it promoted the accumulation of some low molecular weight carbohydrates in the stipes. Blue light also decreased the accumulation of organic acids in the stipes. Blue light treatment reduced the levels of tyrosine and tryptophan in the stipes, but it largely promoted the accumulation of lysine in this organ. In the stipes of F. filiformis, blue light shifted metabolite flow to synthesis of lysine and carbohydrates through inhibiting the accumulation of aromatic amino acids and organic acids, thereby enhancing its nutritional and medicinal values. The transcriptome analysis displayed that blue light enhanced accumulation of lysine in fruiting body of F. filiformis through downregulation of lysine methyltransferase gene and L-lysine 6-monooxygenase gene. Additionally, in the stipes, blue light upregulated many hydrolase genes to improve the ability of the stipe to biodegrade the medium and elevated the growth rate of the fruiting body.</p>","PeriodicalId":7537,"journal":{"name":"AMB Express","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10864240/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139728742","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Sixteen strains of basidiomycetous yeasts were evaluated for their capability to produce ergothioneine (EGT), an amino acid derivative with strong antioxidant activity. The cells were cultured in either two synthetic media or yeast mold (YM) medium for 72 h, after which cytosolic constituents were extracted from the cells with hot water. After analyzing the extracts via liquid chromatography-mass spectrometry (LC-MS), we found that all strains produced varying amounts of EGT. The EGT-producing strains, including Ustilago siamensis, Anthracocystis floculossa, Tridiomyces crassus, Ustilago shanxiensis, and Moesziomyces antarcticus, were subjected to flask cultivation in YM medium. U. siamensis CBS9960 produced the highest amount of EGT at 49.5 ± 7.0 mg/L after 120 h, followed by T. crassus at 30.9 ± 1.8 mg/L. U. siamensis was also cultured in a jar fermenter and produced slightly higher amounts of EGT than under flask cultivation. The effects of culture conditions, particularly the addition of precursor amino acids, on EGT production by the selected strains were also evaluated. U. siamensis showed a 1.5-fold increase in EGT production with the addition of histidine, while U. shanxiensis experienced a 1.8-fold increase in EGT production with the addition of methionine. These results suggest that basidiomycetous yeasts could serve an abundant source for natural EGT producers.
{"title":"Biosynthetic ability of diverse basidiomycetous yeast strains to produce the natural antioxidant ergothioneine.","authors":"Shun Sato, Azusa Saika, Kazunori Ushimaru, Tatsuyuki Koshiyama, Yukihiro Higashiyama, Tokuma Fukuoka, Tomotake Morita","doi":"10.1186/s13568-024-01672-w","DOIUrl":"10.1186/s13568-024-01672-w","url":null,"abstract":"<p><p>Sixteen strains of basidiomycetous yeasts were evaluated for their capability to produce ergothioneine (EGT), an amino acid derivative with strong antioxidant activity. The cells were cultured in either two synthetic media or yeast mold (YM) medium for 72 h, after which cytosolic constituents were extracted from the cells with hot water. After analyzing the extracts via liquid chromatography-mass spectrometry (LC-MS), we found that all strains produced varying amounts of EGT. The EGT-producing strains, including Ustilago siamensis, Anthracocystis floculossa, Tridiomyces crassus, Ustilago shanxiensis, and Moesziomyces antarcticus, were subjected to flask cultivation in YM medium. U. siamensis CBS9960 produced the highest amount of EGT at 49.5 ± 7.0 mg/L after 120 h, followed by T. crassus at 30.9 ± 1.8 mg/L. U. siamensis was also cultured in a jar fermenter and produced slightly higher amounts of EGT than under flask cultivation. The effects of culture conditions, particularly the addition of precursor amino acids, on EGT production by the selected strains were also evaluated. U. siamensis showed a 1.5-fold increase in EGT production with the addition of histidine, while U. shanxiensis experienced a 1.8-fold increase in EGT production with the addition of methionine. These results suggest that basidiomycetous yeasts could serve an abundant source for natural EGT producers.</p>","PeriodicalId":7537,"journal":{"name":"AMB Express","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-02-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10858013/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139711187","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-02-09DOI: 10.1186/s13568-023-01648-2
Arezoo Beig Parikhani, Rada Dehghan, Yeganeh Talebkhan, Elham Bayat, Alireza Biglari, Mohammad Ali Shokrgozar, Reza Ahangari Cohan, Esmat Mirabzadeh, Soheila Ajdary, Mahdi Behdani
The immunotherapeutic application of interleukin-2 (IL-2) in cancer treatment is limited by its off-target effects on different cell populations and insufficient activation of anti-tumor effector cells at the site of the tumor upon tolerated doses. Targeting IL-2 to the tumor microenvironment by generating antibody-cytokine fusion proteins (immunocytokine) would be a promising approach to increase efficacy without associated toxicity. In this study, a novel nanobody-based immunocytokine is developed by the fusion of a mutant (m) IL-2 with a decreased affinity toward CD25 to an anti-vascular endothelial growth factor receptor-2 (VEGFR2) specific nanobody, denoted as VGRmIL2-IC. The antigen binding, cell proliferation, IFN-γ-secretion, and cytotoxicity of this new immunocytokine are evaluated and compared to mIL-2 alone. Furthermore, the pharmacokinetic properties are analyzed. Flow cytometry analysis shows that the VGRmIL2-IC molecule can selectively target VEGFR2-positive cells. The results reveal that the immunocytokine is comparable to mIL-2 alone in the stimulation of Primary Peripheral Blood Mononuclear Cells (PBMCs) and cytotoxicity in in vitro conditions. In vivo studies demonstrate improved pharmacokinetic properties of VGRmIL2-IC in comparison to the wild or mutant IL-2 proteins. The results presented here suggest VGRmIL2-IC could be considered a candidate for the treatment of VEGFR2-positive tumors.
{"title":"A novel nanobody-based immunocytokine of a mutant interleukin-2 as a potential cancer therapeutic.","authors":"Arezoo Beig Parikhani, Rada Dehghan, Yeganeh Talebkhan, Elham Bayat, Alireza Biglari, Mohammad Ali Shokrgozar, Reza Ahangari Cohan, Esmat Mirabzadeh, Soheila Ajdary, Mahdi Behdani","doi":"10.1186/s13568-023-01648-2","DOIUrl":"10.1186/s13568-023-01648-2","url":null,"abstract":"<p><p>The immunotherapeutic application of interleukin-2 (IL-2) in cancer treatment is limited by its off-target effects on different cell populations and insufficient activation of anti-tumor effector cells at the site of the tumor upon tolerated doses. Targeting IL-2 to the tumor microenvironment by generating antibody-cytokine fusion proteins (immunocytokine) would be a promising approach to increase efficacy without associated toxicity. In this study, a novel nanobody-based immunocytokine is developed by the fusion of a mutant (m) IL-2 with a decreased affinity toward CD25 to an anti-vascular endothelial growth factor receptor-2 (VEGFR2) specific nanobody, denoted as VGRmIL2-IC. The antigen binding, cell proliferation, IFN-γ-secretion, and cytotoxicity of this new immunocytokine are evaluated and compared to mIL-2 alone. Furthermore, the pharmacokinetic properties are analyzed. Flow cytometry analysis shows that the VGRmIL2-IC molecule can selectively target VEGFR2-positive cells. The results reveal that the immunocytokine is comparable to mIL-2 alone in the stimulation of Primary Peripheral Blood Mononuclear Cells (PBMCs) and cytotoxicity in in vitro conditions. In vivo studies demonstrate improved pharmacokinetic properties of VGRmIL2-IC in comparison to the wild or mutant IL-2 proteins. The results presented here suggest VGRmIL2-IC could be considered a candidate for the treatment of VEGFR2-positive tumors.</p>","PeriodicalId":7537,"journal":{"name":"AMB Express","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-02-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10857990/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139711186","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-02-08DOI: 10.1186/s13568-024-01676-6
William Omuketi Emitaro, Fanuel Kawaka, David Mutisia Musyimi, Asenath Adienge
Plants have diverse and vast niches colonized by endophytic microorganisms that promote the wellbeing of host plant. These microbes inhabit internal plant tissues with no signs of ill health. Bacterial endophytes from many plants have been isolated and characterized due to their beneficial roles however their diversity in leguminous plants still remain unexploited. Diversity of bacterial endophytes isolated from Sesbania sesban, Leucaena diversifolia and Calliandra calothyrsus was assessed using morphological and molecular characteristics. A total of 27 pure isolates were recovered from C. Calothyrsus, L. diversifolia and S. sesban constituting 44.4%, 33.3% and 22.2% from the leaves, stems and roots respectively. The isolates differentiated into Gram positive and negative with rods and spherical shapes. Analysis of 16S rRNA gene sequences revealed 8 closely related bacterial genera that consisted of Bacillus (33.3%), Staphylococcus (22.2%), Alcaligens (11.1%), Pantoea (11.1%), Xanthomonas,and Sphingomonas (7.4%) each. Others included Acinetobacter, and Pseudomonas at 3.7% each. Bacterial endophytes of genus bacillus were isolated from all the three plants. These results indicate the presence of high diversity of endophytic bacteria associated with the different parts of L. diversifolia, S. sesban and C. salothyrsus growing in western Kenya.
植物有多种多样的广阔生态位,其中的内生微生物可促进寄主植物的健康。这些微生物栖息在植物内部组织中,没有健康不良的迹象。由于其有益作用,许多植物的细菌内生菌已被分离出来并定性,但豆科植物中的细菌内生菌多样性仍未得到开发。本研究利用形态学和分子特征评估了从芝麻、Leucaena diversifolia 和 Calliandra calothyrsus 分离的细菌内生菌的多样性。从 C. Calothyrsus、L. diversifolia 和 S. sesban 共分离出 27 个纯分离菌,分别占叶、茎和根的 44.4%、33.3% 和 22.2%。分离物分为革兰氏阳性和阴性,呈棒状和球状。16S rRNA 基因序列分析显示,有 8 个密切相关的细菌属,分别是芽孢杆菌属(33.3%)、葡萄球菌属(22.2%)、弧菌属(11.1%)、泛氏菌属(11.1%)、黄单胞菌属(Xanthomonas)和鞘翅目单胞菌属(Sphingomonas)(7.4%)。其他包括醋杆菌和假单胞菌,各占 3.7%。从这三种植物中都分离出了杆菌属的内生细菌。这些结果表明,与生长在肯尼亚西部的 L. diversifolia、S. sesban 和 C. salothyrsus 不同部位相关的内生细菌具有很高的多样性。
{"title":"Diversity of endophytic bacteria isolated from leguminous agroforestry trees in western Kenya.","authors":"William Omuketi Emitaro, Fanuel Kawaka, David Mutisia Musyimi, Asenath Adienge","doi":"10.1186/s13568-024-01676-6","DOIUrl":"10.1186/s13568-024-01676-6","url":null,"abstract":"<p><p>Plants have diverse and vast niches colonized by endophytic microorganisms that promote the wellbeing of host plant. These microbes inhabit internal plant tissues with no signs of ill health. Bacterial endophytes from many plants have been isolated and characterized due to their beneficial roles however their diversity in leguminous plants still remain unexploited. Diversity of bacterial endophytes isolated from Sesbania sesban, Leucaena diversifolia and Calliandra calothyrsus was assessed using morphological and molecular characteristics. A total of 27 pure isolates were recovered from C. Calothyrsus, L. diversifolia and S. sesban constituting 44.4%, 33.3% and 22.2% from the leaves, stems and roots respectively. The isolates differentiated into Gram positive and negative with rods and spherical shapes. Analysis of 16S rRNA gene sequences revealed 8 closely related bacterial genera that consisted of Bacillus (33.3%), Staphylococcus (22.2%), Alcaligens (11.1%), Pantoea (11.1%), Xanthomonas,and Sphingomonas (7.4%) each. Others included Acinetobacter, and Pseudomonas at 3.7% each. Bacterial endophytes of genus bacillus were isolated from all the three plants. These results indicate the presence of high diversity of endophytic bacteria associated with the different parts of L. diversifolia, S. sesban and C. salothyrsus growing in western Kenya.</p>","PeriodicalId":7537,"journal":{"name":"AMB Express","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-02-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10853127/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139701601","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-02-08DOI: 10.1186/s13568-024-01675-7
Ayman Elbehiry, Mansor Al Shoaibi, Hamzah Alzahrani, Mai Ibrahem, Ihab Moussa, Feras Alzaben, Rousa A Alsubki, Hassan A Hemeg, Dakheel Almutairi, Saleh Althobaiti, Fawaz Alanazi, Sultan A Alotaibi, Hamoud Almutairi, Ali Alzahrani, Akram Abu-Okail
The genus Enterobacter belongs to the ESKAPE group, which includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. This group is characterized by the development of resistance to various antibiotics. In recent years, Enterobacter cloacae (E. cloacae) has emerged as a clinically important pathogen responsible for a wide range of healthcare-associated illnesses. Identifying Enterobacter species can be challenging due to their similar phenotypic characteristics. The emergence of multidrug-resistant E. cloacae is also a significant problem in healthcare settings. Therefore, our study aimed to identify and differentiate E. cloacae using Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as a fast and precise proteomic analytical technique. We also tested hospital-acquired E. cloacae isolates that produce Extended-spectrum beta-lactamases (ESBL) against commonly used antibiotics for treating urinary tract infections (UTIs). We used a total of 189 E. cloacae isolates from 2300 urine samples of patients with UTIs in our investigation. We employed culturing techniques, as well as the BD Phoenix™ automated identification system (Becton, Dickinson) and Analytical Profile Index (API) system for the biochemical identification of E. cloacae isolates. We used the MALDI Biotyper (MBT) device for peptide mass fingerprinting analysis of all isolates. We utilized the single peak intensities and Principal Component Analysis (PCA) created by MBT Compass software to discriminate and cluster the E. cloacae isolates. Additionally, we evaluated the sensitivity and resistance of ESBL-E. cloacae isolates using the Kirby Bauer method. Out of the 189 E. cloacae isolates, the BD Phoenix system correctly identified 180 (95.24%) isolates, while the API system correctly identified 165 (87.30%) isolates. However, the MBT accurately identified 185 (98.95%) isolates with a score of 2.00 or higher. PCA positively discriminated the identified E. cloacae isolates into one group, and prominent peaks were noticed between 4230 mass-to-charge ratio (m/z) and 8500 m/z. The ESBL-E. cloacae isolates exhibited a higher degree of resistance to ampicillin, amoxicillin-clavulanate, cephalothin, cefuroxime, and cefoxitin. Several isolates were susceptible to carbapenems (meropenem, imipenem, and ertapenem); however, potential future resistance against carbapenems should be taken into consideration. In conclusion, MALDI-TOF MS is a powerful and precise technology that can be routinely used to recognize and differentiate various pathogens in clinical samples. Additionally, the growing antimicrobial resistance of this bacterium may pose a significant risk to human health.
{"title":"Enterobacter cloacae from urinary tract infections: frequency, protein analysis, and antimicrobial resistance.","authors":"Ayman Elbehiry, Mansor Al Shoaibi, Hamzah Alzahrani, Mai Ibrahem, Ihab Moussa, Feras Alzaben, Rousa A Alsubki, Hassan A Hemeg, Dakheel Almutairi, Saleh Althobaiti, Fawaz Alanazi, Sultan A Alotaibi, Hamoud Almutairi, Ali Alzahrani, Akram Abu-Okail","doi":"10.1186/s13568-024-01675-7","DOIUrl":"10.1186/s13568-024-01675-7","url":null,"abstract":"<p><p>The genus Enterobacter belongs to the ESKAPE group, which includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. This group is characterized by the development of resistance to various antibiotics. In recent years, Enterobacter cloacae (E. cloacae) has emerged as a clinically important pathogen responsible for a wide range of healthcare-associated illnesses. Identifying Enterobacter species can be challenging due to their similar phenotypic characteristics. The emergence of multidrug-resistant E. cloacae is also a significant problem in healthcare settings. Therefore, our study aimed to identify and differentiate E. cloacae using Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as a fast and precise proteomic analytical technique. We also tested hospital-acquired E. cloacae isolates that produce Extended-spectrum beta-lactamases (ESBL) against commonly used antibiotics for treating urinary tract infections (UTIs). We used a total of 189 E. cloacae isolates from 2300 urine samples of patients with UTIs in our investigation. We employed culturing techniques, as well as the BD Phoenix™ automated identification system (Becton, Dickinson) and Analytical Profile Index (API) system for the biochemical identification of E. cloacae isolates. We used the MALDI Biotyper (MBT) device for peptide mass fingerprinting analysis of all isolates. We utilized the single peak intensities and Principal Component Analysis (PCA) created by MBT Compass software to discriminate and cluster the E. cloacae isolates. Additionally, we evaluated the sensitivity and resistance of ESBL-E. cloacae isolates using the Kirby Bauer method. Out of the 189 E. cloacae isolates, the BD Phoenix system correctly identified 180 (95.24%) isolates, while the API system correctly identified 165 (87.30%) isolates. However, the MBT accurately identified 185 (98.95%) isolates with a score of 2.00 or higher. PCA positively discriminated the identified E. cloacae isolates into one group, and prominent peaks were noticed between 4230 mass-to-charge ratio (m/z) and 8500 m/z. The ESBL-E. cloacae isolates exhibited a higher degree of resistance to ampicillin, amoxicillin-clavulanate, cephalothin, cefuroxime, and cefoxitin. Several isolates were susceptible to carbapenems (meropenem, imipenem, and ertapenem); however, potential future resistance against carbapenems should be taken into consideration. In conclusion, MALDI-TOF MS is a powerful and precise technology that can be routinely used to recognize and differentiate various pathogens in clinical samples. Additionally, the growing antimicrobial resistance of this bacterium may pose a significant risk to human health.</p>","PeriodicalId":7537,"journal":{"name":"AMB Express","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-02-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10853136/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139701602","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-02-01DOI: 10.1186/s13568-024-01669-5
Bai-Wen Fu, Lian Xu, Mei-Xia Zheng, Yan Shi, Yu-Jing Zhu
Bacillus thuringiensis Cry2Ab toxin was a widely used bioinsecticide to control lepidopteran pests all over the world. In the present study, engineering of Bacillus thuringiensis Cry2Ab toxin was performed for improved insecticidal activity using site-specific saturation mutation. Variants L183I were screened with lower LC50 (0.129 µg/cm2) against P. xylostella when compared to wild-type Cry2Ab (0.267 µg/cm2). To investigate the molecular mechanism behind the enhanced activity of variant L183I, the activation, oligomerization and pore-formation activities of L183I were evaluated, using wild-type Cry2Ab as a control. The results demonstrated that the proteolytic activation of L183I was the same as that of wild-type Cry2Ab. However, variant L183I displayed higher oligomerization and pore-formation activities, which was consistence with its increased insecticidal activity. The current study demonstrated that the insecticidal activity of Cry2Ab toxin could be assessed using oligomerization and pore-formation activities, and the screened variant L183I with improved activity might contribute to Cry2Ab toxin's future application.
{"title":"Engineering of Bacillus thuringiensis Cry2Ab toxin for improved insecticidal activity.","authors":"Bai-Wen Fu, Lian Xu, Mei-Xia Zheng, Yan Shi, Yu-Jing Zhu","doi":"10.1186/s13568-024-01669-5","DOIUrl":"10.1186/s13568-024-01669-5","url":null,"abstract":"<p><p>Bacillus thuringiensis Cry2Ab toxin was a widely used bioinsecticide to control lepidopteran pests all over the world. In the present study, engineering of Bacillus thuringiensis Cry2Ab toxin was performed for improved insecticidal activity using site-specific saturation mutation. Variants L183I were screened with lower LC<sub>50</sub> (0.129 µg/cm<sup>2</sup>) against P. xylostella when compared to wild-type Cry2Ab (0.267 µg/cm<sup>2</sup>). To investigate the molecular mechanism behind the enhanced activity of variant L183I, the activation, oligomerization and pore-formation activities of L183I were evaluated, using wild-type Cry2Ab as a control. The results demonstrated that the proteolytic activation of L183I was the same as that of wild-type Cry2Ab. However, variant L183I displayed higher oligomerization and pore-formation activities, which was consistence with its increased insecticidal activity. The current study demonstrated that the insecticidal activity of Cry2Ab toxin could be assessed using oligomerization and pore-formation activities, and the screened variant L183I with improved activity might contribute to Cry2Ab toxin's future application.</p>","PeriodicalId":7537,"journal":{"name":"AMB Express","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10834393/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139650039","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Chinese Hamster Ovary (CHO) cells are widely employed as host cells for biopharmaceutical production. The manufacturing of biopharmaceuticals poses several challenges, including restricted growth potential and inadequate productivity of the host cells. MicroRNAs play a crucial role in regulating gene expression and are considered highly promising tools for cell engineering to enhance protein production. Our study aimed to evaluate the effects of miR-107, which is recognized as an onco-miR, on erythropoietin-producing CHO cells (CHO-hEPO). To assess the impact of miR-107 on CHO cells, a DNA plasmid containing miR-107 was introduced to CHO-hEPO cells through transfection. Cell proliferation and viability were assessed using the trypan blue dye exclusion method. Cell cycle analysis was conducted by utilizing propidium iodide (PI) staining. The quantification of EPO was determined using an immunoassay test. Moreover, the impact of miR-107 on the expression of downstream target genes was evaluated using qRT-PCR. Our findings highlight and underscore the substantial impact of transient miR-107 overexpression, which led to a remarkable 2.7-fold increase in EPO titers and a significant 1.6-fold increase in the specific productivity of CHO cells (p < 0.01). Furthermore, this intervention resulted in significant enhancements in cell viability and growth rate (p < 0.05). Intriguingly, the overexpression of miR‑107 was linked to the downregulation of LATS2, PTEN, and TSC1 genes while concurrently driving upregulation in transcript levels of MYC, YAP, mTOR, and S6K genes within transgenic CHO cells. In conclusion, this study collectively underscores the feasibility of utilizing cancer-associated miRNAs as a powerful tool for CHO cell engineering. However, more in-depth exploration is warranted to unravel the precise molecular intricacies of miR-107's effects in the context of CHO cells.
中国仓鼠卵巢(CHO)细胞被广泛用作生物制药生产的宿主细胞。生物制药的生产面临着一些挑战,包括宿主细胞的生长潜力受限和生产率不足。MicroRNA 在调节基因表达方面起着至关重要的作用,被认为是细胞工程中极具潜力的工具,可提高蛋白质的产量。我们的研究旨在评估 miR-107 对促红细胞生成素 CHO 细胞(CHO-hEPO)的影响。为了评估 miR-107 对 CHO 细胞的影响,研究人员通过转染将含有 miR-107 的 DNA 质粒导入 CHO-hEPO 细胞。采用胰蓝染料排除法评估细胞增殖和活力。细胞周期分析采用碘化丙啶(PI)染色法。用免疫测定法测定了 EPO 的定量。此外,还利用 qRT-PCR 评估了 miR-107 对下游靶基因表达的影响。我们的研究结果突显并强调了瞬时 miR-107 过表达的重大影响,它使 EPO 滴度显著增加了 2.7 倍,CHO 细胞的特异性生产力显著增加了 1.6 倍(p <0.01)。此外,这种干预还显著提高了细胞活力和生长速度(p < 0.05)。耐人寻味的是,miR-107 的过表达与 LATS2、PTEN 和 TSC1 基因的下调有关,而同时又推动了转基因 CHO 细胞中 MYC、YAP、mTOR 和 S6K 基因转录水平的上调。总之,这项研究共同强调了利用癌症相关 miRNA 作为 CHO 细胞工程强大工具的可行性。然而,要揭示 miR-107 在 CHO 细胞中的作用的分子奥秘,还需要更深入的探索。
{"title":"Enhancing protein production and growth in chinese hamster ovary cells through miR-107 overexpression","authors":"Maryam Jari, Shahriyar Abdoli, Zahra Bazi, Fatemeh Tash Shamsabadi, Farnaz Roshanmehr, Majid Shahbazi","doi":"10.1186/s13568-024-01670-y","DOIUrl":"https://doi.org/10.1186/s13568-024-01670-y","url":null,"abstract":"<p>Chinese Hamster Ovary (CHO) cells are widely employed as host cells for biopharmaceutical production. The manufacturing of biopharmaceuticals poses several challenges, including restricted growth potential and inadequate productivity of the host cells. MicroRNAs play a crucial role in regulating gene expression and are considered highly promising tools for cell engineering to enhance protein production. Our study aimed to evaluate the effects of miR-107, which is recognized as an onco-miR, on erythropoietin-producing CHO cells (CHO-hEPO). To assess the impact of miR-107 on CHO cells, a DNA plasmid containing miR-107 was introduced to CHO-hEPO cells through transfection. Cell proliferation and viability were assessed using the trypan blue dye exclusion method. Cell cycle analysis was conducted by utilizing propidium iodide (PI) staining. The quantification of EPO was determined using an immunoassay test. Moreover, the impact of miR-107 on the expression of downstream target genes was evaluated using qRT-PCR. Our findings highlight and underscore the substantial impact of transient miR-107 overexpression, which led to a remarkable 2.7-fold increase in EPO titers and a significant 1.6-fold increase in the specific productivity of CHO cells (p < 0.01). Furthermore, this intervention resulted in significant enhancements in cell viability and growth rate (p < 0.05). Intriguingly, the overexpression of miR‑107 was linked to the downregulation of <i>LATS2, PTEN,</i> and <i>TSC1</i> genes while concurrently driving upregulation in transcript levels of <i>MYC, YAP, mTOR,</i> and <i>S6K</i> genes within transgenic CHO cells. In conclusion, this study collectively underscores the feasibility of utilizing cancer-associated miRNAs as a powerful tool for CHO cell engineering. However, more in-depth exploration is warranted to unravel the precise molecular intricacies of miR-107's effects in the context of CHO cells.</p>","PeriodicalId":7537,"journal":{"name":"AMB Express","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139662790","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-01-29DOI: 10.1186/s13568-024-01666-8
Jae Woong Choi, Nho-Eul Song, Sang-Pil Hong, Young Kyoung Rhee, Hee-Do Hong, Chang-Won Cho
Efficient utilization of galactose by microorganisms can lead to the production of valuable bio-products and improved metabolic processes. While Bacillus subtilis has inherent pathways for galactose metabolism, there is potential for enhancement via evolutionary strategies. This study aimed to boost galactose utilization in B. subtilis using adaptive laboratory evolution (ALE) and to elucidate the genetic and metabolic changes underlying the observed enhancements. The strains of B. subtilis underwent multiple rounds of adaptive laboratory evolution (approximately 5000 generations) in an environment that favored the use of galactose. This process resulted in an enhanced specific growth rate of 0.319 ± 0.005 h-1, a significant increase from the 0.03 ± 0.008 h-1 observed in the wild-type strains. Upon selecting the evolved strain BSGA14, a comprehensive whole-genome sequencing revealed the presence of 63 single nucleotide polymorphisms (SNPs). Two of them, located in the coding sequences of the genes araR and glcR, were found to be the advantageous mutations after reverse engineering. The strain with these two accumulated mutations, BSGALE4, exhibited similar specific growth rate on galactose to the evolved strain BSGA14 (0.296 ± 0.01 h-1). Furthermore, evolved strain showed higher productivity of protease and β-galactosidase in mock soybean biomass medium. ALE proved to be a potent tool for enhancing galactose metabolism in B. subtilis. The findings offer valuable insights into the potential of evolutionary strategies in microbial engineering and pave the way for industrial applications harnessing enhanced galactose conversion.
{"title":"Engineering Bacillus subtilis J46 for efficient utilization of galactose through adaptive laboratory evolution.","authors":"Jae Woong Choi, Nho-Eul Song, Sang-Pil Hong, Young Kyoung Rhee, Hee-Do Hong, Chang-Won Cho","doi":"10.1186/s13568-024-01666-8","DOIUrl":"10.1186/s13568-024-01666-8","url":null,"abstract":"<p><p>Efficient utilization of galactose by microorganisms can lead to the production of valuable bio-products and improved metabolic processes. While Bacillus subtilis has inherent pathways for galactose metabolism, there is potential for enhancement via evolutionary strategies. This study aimed to boost galactose utilization in B. subtilis using adaptive laboratory evolution (ALE) and to elucidate the genetic and metabolic changes underlying the observed enhancements. The strains of B. subtilis underwent multiple rounds of adaptive laboratory evolution (approximately 5000 generations) in an environment that favored the use of galactose. This process resulted in an enhanced specific growth rate of 0.319 ± 0.005 h<sup>-1</sup>, a significant increase from the 0.03 ± 0.008 h<sup>-1</sup> observed in the wild-type strains. Upon selecting the evolved strain BSGA14, a comprehensive whole-genome sequencing revealed the presence of 63 single nucleotide polymorphisms (SNPs). Two of them, located in the coding sequences of the genes araR and glcR, were found to be the advantageous mutations after reverse engineering. The strain with these two accumulated mutations, BSGALE4, exhibited similar specific growth rate on galactose to the evolved strain BSGA14 (0.296 ± 0.01 h<sup>-1</sup>). Furthermore, evolved strain showed higher productivity of protease and β-galactosidase in mock soybean biomass medium. ALE proved to be a potent tool for enhancing galactose metabolism in B. subtilis. The findings offer valuable insights into the potential of evolutionary strategies in microbial engineering and pave the way for industrial applications harnessing enhanced galactose conversion.</p>","PeriodicalId":7537,"journal":{"name":"AMB Express","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-01-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10822834/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139569491","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Streptococcus agalactiae has different virulence factors, from which the capsule has the most significant role in the pathogenesis of this organism. We aimed to investigate the distribution of more prevalent capsular genes among different Random Amplified Polymorphic DNA (RAPD) types of S. agalactiae isolated from pregnant women. A total of 106 isolates were collected from 420 vaginal and rectal swabs obtained from pregnant women. The specimens were transferred using Todd Hewitt Broth and were cultured on a blood agar containing antibiotics. The S. agalactiae isolates were identified by the standard microbiological and biochemical tests. The genomic DNAs of S. agalactiae isolates were extracted using an extraction kit. Then, the PCR method was used to detection of the capsular genes. Moreover, The RAPD PCR was used to genotyping of the isolates. The colonization rate of the pregnant women was 25.23%, and there was a statistically significant correlation between the weeks of gestation and the probability of colonization (p-value < 0.05). Also, 31 (29.24%) and 18 (16.98%) pregnant women had a history of abortion and membrane rupture, respectively. In addition, 20 (18.86%), 32 (30.18%), 4 (3.77%), and 6 (5.66%) isolates carried genes encoding capsular types Ia, Ib, III, and V, respectively. None isolates had the type II capsular gene, and other 44 isolates were non-typeable. Nine clones (clusters) of S. agalactiae were observed in the present study with 70% similarity, and 53 different types were identified among the isolates. Except for capsular types III and V that belonged to clones 3, 5, 7, and 9, other capsular types were detected in different RAPD types. We found that the capsular types Ib and Ia were predominant among pregnant women in this area, indicating their significance for vaccine designation. Also, our isolates showed a lower genotypic diversity in RAPD typing. This may be due to the same sources of most isolates.
{"title":"Capsular gene distribution and RAPD typing of Streptococcus agalactiae isolated from pregnant women.","authors":"Mona Zakerifar, Hamid Reza Goli, Hami Kaboosi, Zahra Rahmani, Fatemeh Peyravii Ghadikolaii","doi":"10.1186/s13568-024-01671-x","DOIUrl":"10.1186/s13568-024-01671-x","url":null,"abstract":"<p><p>Streptococcus agalactiae has different virulence factors, from which the capsule has the most significant role in the pathogenesis of this organism. We aimed to investigate the distribution of more prevalent capsular genes among different Random Amplified Polymorphic DNA (RAPD) types of S. agalactiae isolated from pregnant women. A total of 106 isolates were collected from 420 vaginal and rectal swabs obtained from pregnant women. The specimens were transferred using Todd Hewitt Broth and were cultured on a blood agar containing antibiotics. The S. agalactiae isolates were identified by the standard microbiological and biochemical tests. The genomic DNAs of S. agalactiae isolates were extracted using an extraction kit. Then, the PCR method was used to detection of the capsular genes. Moreover, The RAPD PCR was used to genotyping of the isolates. The colonization rate of the pregnant women was 25.23%, and there was a statistically significant correlation between the weeks of gestation and the probability of colonization (p-value < 0.05). Also, 31 (29.24%) and 18 (16.98%) pregnant women had a history of abortion and membrane rupture, respectively. In addition, 20 (18.86%), 32 (30.18%), 4 (3.77%), and 6 (5.66%) isolates carried genes encoding capsular types Ia, Ib, III, and V, respectively. None isolates had the type II capsular gene, and other 44 isolates were non-typeable. Nine clones (clusters) of S. agalactiae were observed in the present study with 70% similarity, and 53 different types were identified among the isolates. Except for capsular types III and V that belonged to clones 3, 5, 7, and 9, other capsular types were detected in different RAPD types. We found that the capsular types Ib and Ia were predominant among pregnant women in this area, indicating their significance for vaccine designation. Also, our isolates showed a lower genotypic diversity in RAPD typing. This may be due to the same sources of most isolates.</p>","PeriodicalId":7537,"journal":{"name":"AMB Express","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-01-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10822826/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139569488","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-01-22DOI: 10.1186/s13568-024-01665-9
Chunfeng Xia, Yuchao Zhao, Chunlan Liu, Yang Gao
Spatholobus suberectus Dunn as a traditional Chinese herbal medicine, which is susceptible to being infected by molds during storage. In order to explore the diversity characteristics of fungal community and the quality evaluation of Spatholobus suberectus Dunn during the process of mildew. The study used high-throughput sequencing technology to detect the diversity characteristics of fungal community, high-performance liquid chromatography (HPLC) and ultraviolet spectrophotometry (UV-spectrophotometry) methods to detect the content of flavonoids, and enzyme-linked immunosorbent assay (ELISA) method to detect the content of Aflatoxins B1 (AFB1). The result showed that the fungi of all samples belonged to 14 phyla, 336 genera, and the dominant fungi at the early stage of mildew was not obvious, while that at middle and late stages of mildew was Aspergillus. The species diversity of fungal community was the highest at the early stage of mildew, while the species richness of fungal community was the highest at the late stage of mildew. The content of AFB1 showed an upward trend, while the content of flavonoids showed a downward trend during the process of mildew. In brief, the diversity of fungal community decreased gradually, and the number of dominant fungi increased gradually, and the quality of Spatholobus suberectus Dunn decreased gradually during the process of mildew.
{"title":"Diversity of fungal community and quality evaluation of Spatholobus Suberectus Dunn during the process of mildew.","authors":"Chunfeng Xia, Yuchao Zhao, Chunlan Liu, Yang Gao","doi":"10.1186/s13568-024-01665-9","DOIUrl":"10.1186/s13568-024-01665-9","url":null,"abstract":"<p><p>Spatholobus suberectus Dunn as a traditional Chinese herbal medicine, which is susceptible to being infected by molds during storage. In order to explore the diversity characteristics of fungal community and the quality evaluation of Spatholobus suberectus Dunn during the process of mildew. The study used high-throughput sequencing technology to detect the diversity characteristics of fungal community, high-performance liquid chromatography (HPLC) and ultraviolet spectrophotometry (UV-spectrophotometry) methods to detect the content of flavonoids, and enzyme-linked immunosorbent assay (ELISA) method to detect the content of Aflatoxins B<sub>1</sub> (AFB<sub>1</sub>). The result showed that the fungi of all samples belonged to 14 phyla, 336 genera, and the dominant fungi at the early stage of mildew was not obvious, while that at middle and late stages of mildew was Aspergillus. The species diversity of fungal community was the highest at the early stage of mildew, while the species richness of fungal community was the highest at the late stage of mildew. The content of AFB<sub>1</sub> showed an upward trend, while the content of flavonoids showed a downward trend during the process of mildew. In brief, the diversity of fungal community decreased gradually, and the number of dominant fungi increased gradually, and the quality of Spatholobus suberectus Dunn decreased gradually during the process of mildew.</p>","PeriodicalId":7537,"journal":{"name":"AMB Express","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10803692/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139511583","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}