Preeclampsia is a potentially life-threatening condition for both mother and baby, characterized by hypertension and potential organ damage. Early diagnosis is crucial to mitigate its adverse health effects. Traditional diagnostic methods, which focus on late-manifesting symptoms like hypertension and proteinuria, underscore the need for molecular diagnostic approaches for timely detection. This study successfully designs and evaluates novel aptamers with high specificity and affinity for Vascular Endothelial Growth Factor (VEGF) and Placental Growth Factor (PlGF), biomarkers closely associated with preeclampsia. Using molecular docking, molecular dynamics simulations, and BioLayer Interferometry (BLI), we identified aptamers that demonstrated strong binding affinities, comparable or superior to traditional antibodies. Our findings suggest that these aptamers have the potential to be integrated into cost-effective, point-of-care diagnostic tools, significantly improving early detection and intervention strategies for preeclampsia. The robust performance of these aptamers marks a pivotal step toward the development of more reliable and accessible diagnostic solutions, with implications for better maternal and fetal health outcomes.
{"title":"Systematic design and evaluation of aptamers for VEGF and PlGF biomarkers of Preeclampsia.","authors":"Samavath Mallawarachchi, Rümeysa E Cebecioglu, Majed Althumayri, Levent Beker, Sandun Fernando, Hatice Ceylan Koydemir","doi":"10.1186/s12896-024-00891-0","DOIUrl":"https://doi.org/10.1186/s12896-024-00891-0","url":null,"abstract":"<p><p>Preeclampsia is a potentially life-threatening condition for both mother and baby, characterized by hypertension and potential organ damage. Early diagnosis is crucial to mitigate its adverse health effects. Traditional diagnostic methods, which focus on late-manifesting symptoms like hypertension and proteinuria, underscore the need for molecular diagnostic approaches for timely detection. This study successfully designs and evaluates novel aptamers with high specificity and affinity for Vascular Endothelial Growth Factor (VEGF) and Placental Growth Factor (PlGF), biomarkers closely associated with preeclampsia. Using molecular docking, molecular dynamics simulations, and BioLayer Interferometry (BLI), we identified aptamers that demonstrated strong binding affinities, comparable or superior to traditional antibodies. Our findings suggest that these aptamers have the potential to be integrated into cost-effective, point-of-care diagnostic tools, significantly improving early detection and intervention strategies for preeclampsia. The robust performance of these aptamers marks a pivotal step toward the development of more reliable and accessible diagnostic solutions, with implications for better maternal and fetal health outcomes.</p>","PeriodicalId":8905,"journal":{"name":"BMC Biotechnology","volume":"24 1","pages":"64"},"PeriodicalIF":3.5,"publicationDate":"2024-09-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11428563/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142340571","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 : 2024-09-23DOI: 10.1186/s12896-024-00897-8
Eva Forsten, Steffen Gerdes, René Petri, Jochen Büchs, Jørgen Magnus
Background: Vibrio natriegens, a halophilic marine γ-proteobacterium, holds immense biotechnological potential due to its remarkably short generation time of under ten minutes. However, the highest growth rates have been primarily observed on complex media, which often suffer from batch-to-batch variability affecting process stability and performance. Consistent bioprocesses necessitate the use of chemically defined media, which are usually optimized for fermenters with pH and dissolved oxygen tension (DOT) regulation, both of which are not applied during early-stage cultivations in shake flasks or microtiter plates. Existing studies on V. natriegens' growth on mineral media report partially conflicting results, and a comprehensive study examining the combined effects of pH buffering, sodium concentration, and medium osmolality is lacking.
Results: This study evaluates the influence of sodium concentration, pH buffering, and medium osmolality on the growth of V. natriegens under unregulated small-scale conditions. The maximum growth rate, time of glucose depletion, as well as the onset of stationary phase were observed through online-monitoring the oxygen transfer rate. The results revealed optimal growth conditions at an initial pH of 8.0 with a minimum of 300 mM MOPS buffer for media containing 20 g/L glucose or 180 mM MOPS for media with 10 g/L glucose. Optimal sodium chloride supplementation was found to be between 7.5 and 15 g/L, lower than previously reported ranges. This is advantageous for reducing industrial corrosion issues. Additionally, an osmolality range of 1 to 1.6 Osmol/kg was determined to be optimal for growth. Under these optimized conditions, V. natriegens achieved a growth rate of 1.97 ± 0.13 1/h over a period of 1 h at 37 °C, the highest reported rate for this organism on a mineral medium.
Conclusion: This study provides guidelines for cultivating V. natriegens in early-stage laboratory settings without pH and DOT regulation. The findings suggest a lower optimal sodium chloride range than previously reported and establish an osmolality window for optimal growth, thereby advancing the understanding of V. natriegens' physiology. In addition, this study offers a foundation for future research into the effects of different ions and carbon sources on V. natriegens.
{"title":"Unraveling the impact of pH, sodium concentration, and medium osmolality on Vibrio natriegens in batch processes.","authors":"Eva Forsten, Steffen Gerdes, René Petri, Jochen Büchs, Jørgen Magnus","doi":"10.1186/s12896-024-00897-8","DOIUrl":"10.1186/s12896-024-00897-8","url":null,"abstract":"<p><strong>Background: </strong>Vibrio natriegens, a halophilic marine γ-proteobacterium, holds immense biotechnological potential due to its remarkably short generation time of under ten minutes. However, the highest growth rates have been primarily observed on complex media, which often suffer from batch-to-batch variability affecting process stability and performance. Consistent bioprocesses necessitate the use of chemically defined media, which are usually optimized for fermenters with pH and dissolved oxygen tension (DOT) regulation, both of which are not applied during early-stage cultivations in shake flasks or microtiter plates. Existing studies on V. natriegens' growth on mineral media report partially conflicting results, and a comprehensive study examining the combined effects of pH buffering, sodium concentration, and medium osmolality is lacking.</p><p><strong>Results: </strong>This study evaluates the influence of sodium concentration, pH buffering, and medium osmolality on the growth of V. natriegens under unregulated small-scale conditions. The maximum growth rate, time of glucose depletion, as well as the onset of stationary phase were observed through online-monitoring the oxygen transfer rate. The results revealed optimal growth conditions at an initial pH of 8.0 with a minimum of 300 mM MOPS buffer for media containing 20 g/L glucose or 180 mM MOPS for media with 10 g/L glucose. Optimal sodium chloride supplementation was found to be between 7.5 and 15 g/L, lower than previously reported ranges. This is advantageous for reducing industrial corrosion issues. Additionally, an osmolality range of 1 to 1.6 Osmol/kg was determined to be optimal for growth. Under these optimized conditions, V. natriegens achieved a growth rate of 1.97 ± 0.13 1/h over a period of 1 h at 37 °C, the highest reported rate for this organism on a mineral medium.</p><p><strong>Conclusion: </strong>This study provides guidelines for cultivating V. natriegens in early-stage laboratory settings without pH and DOT regulation. The findings suggest a lower optimal sodium chloride range than previously reported and establish an osmolality window for optimal growth, thereby advancing the understanding of V. natriegens' physiology. In addition, this study offers a foundation for future research into the effects of different ions and carbon sources on V. natriegens.</p>","PeriodicalId":8905,"journal":{"name":"BMC Biotechnology","volume":"24 1","pages":"63"},"PeriodicalIF":3.5,"publicationDate":"2024-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11421182/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142307070","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 : 2024-09-18DOI: 10.1186/s12896-024-00890-1
Soad Nasr, Abdelhameed S. Dawood, Amal Mosad Ibrahim, Mohamed S. Abdel-Aziz, Walid Fayad, Anwar Abdelnaser, Faten K. Abd EL-Hady
Inflammation serves as an intricate defense mechanism for tissue repair. However, overactivation of TLR4-mediated inflammation by lipopolysaccharide (LPS) can lead to detrimental outcomes such as sepsis, acute lung injury, and chronic inflammation, often associated with cancer and autoimmune diseases. This study delves into the anti-inflammatory properties of “Aspergillus unguis isolate SP51-EGY” on LPS-stimulated RAW 264.7 macrophages. Through real-time qPCR, we assessed the expression levels of pivotal inflammatory genes, including iNOS, COX-2, TNF-α, and IL-6. Remarkably, our fungal extracts significantly diminished NO production and showed noteworthy reductions in the mRNA expression levels of the aforementioned genes. Furthermore, while Nrf2 is typically associated with modulating inflammatory responses, our findings indicate that the anti-inflammatory effects of our extracts are not Nrf2-dependent. Moreover, the chemical diversity of the potent extract (B Sh F) was elucidated using Q-TOF LC-HRMS, identifying 54 compounds, some of which played vital roles in suppressing inflammation. Most notably, compounds like granisetron, fenofibrate, and umbelliprenin were found to downregulate TNF-α, IL-1β, and IL-6 through the NF-κB signaling pathway. In conclusion, “Aspergillus unguis isolate SP51-EGY”, isolated from the Red Sea, Egypt, has been unveiled as a promising TLR4 inhibitor with significant anti-inflammatory potentials, presenting novel insights for their potential therapeutic use in inflammation.
{"title":"Anti-inflammatory potential of aspergillus unguis SP51-EGY: TLR4-dependent effects & chemical diversity via Q-TOF LC-HRMS","authors":"Soad Nasr, Abdelhameed S. Dawood, Amal Mosad Ibrahim, Mohamed S. Abdel-Aziz, Walid Fayad, Anwar Abdelnaser, Faten K. Abd EL-Hady","doi":"10.1186/s12896-024-00890-1","DOIUrl":"https://doi.org/10.1186/s12896-024-00890-1","url":null,"abstract":"Inflammation serves as an intricate defense mechanism for tissue repair. However, overactivation of TLR4-mediated inflammation by lipopolysaccharide (LPS) can lead to detrimental outcomes such as sepsis, acute lung injury, and chronic inflammation, often associated with cancer and autoimmune diseases. This study delves into the anti-inflammatory properties of “Aspergillus unguis isolate SP51-EGY” on LPS-stimulated RAW 264.7 macrophages. Through real-time qPCR, we assessed the expression levels of pivotal inflammatory genes, including iNOS, COX-2, TNF-α, and IL-6. Remarkably, our fungal extracts significantly diminished NO production and showed noteworthy reductions in the mRNA expression levels of the aforementioned genes. Furthermore, while Nrf2 is typically associated with modulating inflammatory responses, our findings indicate that the anti-inflammatory effects of our extracts are not Nrf2-dependent. Moreover, the chemical diversity of the potent extract (B Sh F) was elucidated using Q-TOF LC-HRMS, identifying 54 compounds, some of which played vital roles in suppressing inflammation. Most notably, compounds like granisetron, fenofibrate, and umbelliprenin were found to downregulate TNF-α, IL-1β, and IL-6 through the NF-κB signaling pathway. In conclusion, “Aspergillus unguis isolate SP51-EGY”, isolated from the Red Sea, Egypt, has been unveiled as a promising TLR4 inhibitor with significant anti-inflammatory potentials, presenting novel insights for their potential therapeutic use in inflammation. ","PeriodicalId":8905,"journal":{"name":"BMC Biotechnology","volume":"35 1","pages":""},"PeriodicalIF":3.5,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142261040","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 : 2024-09-15DOI: 10.1186/s12896-024-00889-8
Rabab M. Thabit, Fatma El-Zahraa A. Abd El-Aziz, A. Abu El-Fadl, A. A. Abu-Sehly, Ahmed M. Sayed
Nanoporous aluminum metal–organic framework (Al-MOF) was synthesized via solvothermal methods and employed as a carrier matrix for in vitro drug delivery of Umbelliferon (Um). The encapsulated Um was gradually released over seven days at 37 °C, using simulated body fluid phosphate-buffered saline (PBS) at pH 7.4 as the release medium. The drug release profile suggests the potential of Al-MOF nanoparticles as effective drug delivery carriers. Structural and chemical analyses of Um-loaded Al-MOF nanoparticles (Um-Al MOF) were conducted using Fourier-transform infrared (FTIR) spectroscopy, X-ray diffractometry (XRD), and ultraviolet–visible (UV–Vis) spectroscopy. Thermal gravimetric analysis (TGA) was employed to investigate the thermal stability of the Al-MOF nanoparticles, while Transmission Electron Microscopy (TEM) was utilized to assess their morphological features. Um-Al MOF nanoparticles demonstrated notable antioxidant and anti-inflammatory properties compared to Um and Al-MOF nanoparticles individually. Moreover, they exhibited significant enhancement in wound healing in an earthworm model. These findings underscore the potential of Al-MOF nanoparticles as a promising drug delivery system, necessitating further investigations to explore their clinical applicability.
{"title":"Synthesis and evaluation of nanosized aluminum MOF encapsulating Umbelliferon: assessing antioxidant, anti-inflammatory, and wound healing potential in an earthworm model","authors":"Rabab M. Thabit, Fatma El-Zahraa A. Abd El-Aziz, A. Abu El-Fadl, A. A. Abu-Sehly, Ahmed M. Sayed","doi":"10.1186/s12896-024-00889-8","DOIUrl":"https://doi.org/10.1186/s12896-024-00889-8","url":null,"abstract":"Nanoporous aluminum metal–organic framework (Al-MOF) was synthesized via solvothermal methods and employed as a carrier matrix for in vitro drug delivery of Umbelliferon (Um). The encapsulated Um was gradually released over seven days at 37 °C, using simulated body fluid phosphate-buffered saline (PBS) at pH 7.4 as the release medium. The drug release profile suggests the potential of Al-MOF nanoparticles as effective drug delivery carriers. Structural and chemical analyses of Um-loaded Al-MOF nanoparticles (Um-Al MOF) were conducted using Fourier-transform infrared (FTIR) spectroscopy, X-ray diffractometry (XRD), and ultraviolet–visible (UV–Vis) spectroscopy. Thermal gravimetric analysis (TGA) was employed to investigate the thermal stability of the Al-MOF nanoparticles, while Transmission Electron Microscopy (TEM) was utilized to assess their morphological features. Um-Al MOF nanoparticles demonstrated notable antioxidant and anti-inflammatory properties compared to Um and Al-MOF nanoparticles individually. Moreover, they exhibited significant enhancement in wound healing in an earthworm model. These findings underscore the potential of Al-MOF nanoparticles as a promising drug delivery system, necessitating further investigations to explore their clinical applicability.\u0000","PeriodicalId":8905,"journal":{"name":"BMC Biotechnology","volume":"90 1","pages":""},"PeriodicalIF":3.5,"publicationDate":"2024-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142261036","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}
Background: Glaesserella parasuis (G. parasuis) is the causative agent of Glässer's disease, which causes significant economic losses in the swine industry. However, research on the pathogenesis of G. parasuis has been hampered by the lack of a simple and efficient marker-free knockout system.
Results: In this study, a marker-free knockout system was developed for G. parasuis using a temperature-sensitive vector. By alternating the incubation of transformants at 30°C and 37°C, we optimized the screening process for this system. The system was successfully applied to knockout the KanR cassette from JS0135ΔnanH::KanR, achieving a knockout efficiency of 90% in the final round of screening. To confirm that temperature variation was a key factor, we proceeded with knocking out the nanH and apd genes in the CF7066 strain. The knockout efficiency reached up to 100%, with the shortest screening time being only four days. The knockout of the nanH gene resulted in a significant reduction in the growth vitality of the strains, while the knockout of the apd gene led to an approximate 56% improvement in the adhesion rate. Additionally, we observed that the expression of recombinant genes in transformants was higher at 30℃ than at 37℃, with the recC gene being upregulated approximately 7-fold. In contrast, there was almost no difference in the expression of recombinant genes between 30℃ and 37℃ in the wild-type strains. This discrepancy was likely due to an elevated copy number of target plasmids at 30℃, which may have resulted in the enhanced expression of recombinant genes.
Conclusions: In conclusion, this newly developed gene knockout system for G. parasuis presents a valuable tool for advancing research on this organism.
{"title":"A marker-free genetic manipulation method for Glaesserella parasuis strains developed by alternately culturing transformants at 37°C and 30°C.","authors":"Jing Xiao, Yuxin Wang, Dongfang Wu, Yuping Song, Xuwang Cai, Huanchun Chen, Hongbo Zhou, Xiaojuan Xu","doi":"10.1186/s12896-024-00887-w","DOIUrl":"10.1186/s12896-024-00887-w","url":null,"abstract":"<p><strong>Background: </strong>Glaesserella parasuis (G. parasuis) is the causative agent of Glässer's disease, which causes significant economic losses in the swine industry. However, research on the pathogenesis of G. parasuis has been hampered by the lack of a simple and efficient marker-free knockout system.</p><p><strong>Results: </strong>In this study, a marker-free knockout system was developed for G. parasuis using a temperature-sensitive vector. By alternating the incubation of transformants at 30°C and 37°C, we optimized the screening process for this system. The system was successfully applied to knockout the Kan<sup>R</sup> cassette from JS0135ΔnanH::Kan<sup>R</sup>, achieving a knockout efficiency of 90% in the final round of screening. To confirm that temperature variation was a key factor, we proceeded with knocking out the nanH and apd genes in the CF7066 strain. The knockout efficiency reached up to 100%, with the shortest screening time being only four days. The knockout of the nanH gene resulted in a significant reduction in the growth vitality of the strains, while the knockout of the apd gene led to an approximate 56% improvement in the adhesion rate. Additionally, we observed that the expression of recombinant genes in transformants was higher at 30℃ than at 37℃, with the recC gene being upregulated approximately 7-fold. In contrast, there was almost no difference in the expression of recombinant genes between 30℃ and 37℃ in the wild-type strains. This discrepancy was likely due to an elevated copy number of target plasmids at 30℃, which may have resulted in the enhanced expression of recombinant genes.</p><p><strong>Conclusions: </strong>In conclusion, this newly developed gene knockout system for G. parasuis presents a valuable tool for advancing research on this organism.</p>","PeriodicalId":8905,"journal":{"name":"BMC Biotechnology","volume":"24 1","pages":"60"},"PeriodicalIF":3.5,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11373133/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142124721","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 : 2024-08-27DOI: 10.1186/s12896-024-00885-y
Nigel Aminake Makoah, Matefo Millicent Litabe, Fredy Brice Nemg Simo, Katlego Keith Maboho, Felicity Jane Burt
Background: Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonotic disease that presents with severe hemorrhagic manifestations and is associated with significant fatality rates. The causative agent, Crimean-Congo Hemorrhagic Fever Virus (CCHFV), is a high-priority pathogen identified by the World Health Organization with no approved vaccine or specific treatment available. In addition, there is a critical need for enhanced diagnostic tools to improve public health awareness, prevention measures, and disease control strategies.
Methods: We designed plasmids to enable the purification of soluble CCHFV glycoprotein Gc expressed in mammalian 293 F cells, followed by purification using affinity and size exclusion chromatography. The purified antigen was analyzed by SDS-PAGE and Western blotting to confirm its reactivity to antibodies from CCHF survivors. Additionally, an in-house indirect ELISA was developed using the purified Gc as a coating antigen.
Results: The optimized expression system successfully produced soluble and pure Gc antigen after affinity chromatography. The protein showed specific reactivity with CCHFV-positive serum antibodies in Western blot analysis. The indirect ELISA assay demonstrated high efficacy in distinguishing between CCHFV-positive and -negative serum samples, indicating its potential as a valuable diagnostic tool. Size exclusion chromatography further confirmed the presence of aggregates in our protein preparation.
Conclusions: The purified Gc antigen shows promise for developing direct diagnostic assays for CCHFV. The antigen's suitability for subunit vaccine development and its application as bait for monoclonal antibody isolation from survivors could be investigated further. This work lays the foundation for future research into the development of rapid diagnostic tests for field deployment.
背景:克里米亚-刚果出血热(CCHF)是一种由蜱虫传播的人畜共患病,表现为严重的出血性症状,致死率很高。病原体克里米亚-刚果出血热病毒(CCHFV)是世界卫生组织确定的高度优先病原体,目前还没有获得批准的疫苗或特效治疗方法。此外,亟需加强诊断工具,以提高公共卫生意识、改进预防措施和疾病控制策略:方法:我们设计了质粒来纯化在哺乳动物 293 F 细胞中表达的可溶性 CCHFV 糖蛋白 Gc,然后使用亲和层析和尺寸排阻层析进行纯化。纯化后的抗原通过 SDS-PAGE 和 Western 印迹法进行分析,以确认其与 CCHF 存活者的抗体的反应性。此外,还使用纯化的 Gc 作为包被抗原开发了一种内部间接 ELISA:结果:经过优化的表达系统在亲和层析后成功产生了可溶性纯Gc抗原。在 Western 印迹分析中,该蛋白与 CCHFV 阳性血清抗体呈特异性反应。间接酶联免疫吸附试验在区分 CCHFV 阳性和阴性血清样本方面表现出很高的效率,表明它有潜力成为一种有价值的诊断工具。尺寸排阻色谱法进一步证实了我们制备的蛋白质中存在聚集体:纯化的 Gc 抗原有望用于开发 CCHFV 的直接诊断测定。结论:纯化的 Gc 抗原有望用于开发 CCHFV 的直接诊断检测,该抗原是否适合亚单位疫苗的开发,以及是否可用作从存活者中分离单克隆抗体的诱饵,还有待于进一步研究。这项工作为今后研究开发用于野外部署的快速诊断测试奠定了基础。
{"title":"Purification and characterization of soluble recombinant Crimean-Congo hemorrhagic fever virus glycoprotein Gc expressed in mammalian 293F cells.","authors":"Nigel Aminake Makoah, Matefo Millicent Litabe, Fredy Brice Nemg Simo, Katlego Keith Maboho, Felicity Jane Burt","doi":"10.1186/s12896-024-00885-y","DOIUrl":"10.1186/s12896-024-00885-y","url":null,"abstract":"<p><strong>Background: </strong>Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonotic disease that presents with severe hemorrhagic manifestations and is associated with significant fatality rates. The causative agent, Crimean-Congo Hemorrhagic Fever Virus (CCHFV), is a high-priority pathogen identified by the World Health Organization with no approved vaccine or specific treatment available. In addition, there is a critical need for enhanced diagnostic tools to improve public health awareness, prevention measures, and disease control strategies.</p><p><strong>Methods: </strong>We designed plasmids to enable the purification of soluble CCHFV glycoprotein Gc expressed in mammalian 293 F cells, followed by purification using affinity and size exclusion chromatography. The purified antigen was analyzed by SDS-PAGE and Western blotting to confirm its reactivity to antibodies from CCHF survivors. Additionally, an in-house indirect ELISA was developed using the purified Gc as a coating antigen.</p><p><strong>Results: </strong>The optimized expression system successfully produced soluble and pure Gc antigen after affinity chromatography. The protein showed specific reactivity with CCHFV-positive serum antibodies in Western blot analysis. The indirect ELISA assay demonstrated high efficacy in distinguishing between CCHFV-positive and -negative serum samples, indicating its potential as a valuable diagnostic tool. Size exclusion chromatography further confirmed the presence of aggregates in our protein preparation.</p><p><strong>Conclusions: </strong>The purified Gc antigen shows promise for developing direct diagnostic assays for CCHFV. The antigen's suitability for subunit vaccine development and its application as bait for monoclonal antibody isolation from survivors could be investigated further. This work lays the foundation for future research into the development of rapid diagnostic tests for field deployment.</p>","PeriodicalId":8905,"journal":{"name":"BMC Biotechnology","volume":"24 1","pages":"59"},"PeriodicalIF":3.5,"publicationDate":"2024-08-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11348531/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142078967","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 : 2024-08-22DOI: 10.1186/s12896-024-00884-z
Meng-Yuan Li, Yan-Ru Li, Cheng-Feng Han, Jie Zhang, Rui-Ying Zhu, Yan Zhang, Jian Li, Shi-Ru Jia, Pei-Pei Han
Based on our previous findings that salicylic acid and jasmonic acid increased Nostoc flagelliforme polysaccharide yield by regulating intracellular nitric oxide (NO) levels, the mechanism through which NO affects polysaccharide biosynthesis in Nostoc flagelliforme was explored from the perspective of S-nitrosylation (SNO). The addition of NO donor and scavenger showed that intracellular NO had a significant positive effect on the polysaccharide yield of N. flagelliforme. To explore the mechanism, we investigated the relationship between NO levels and the activity of several key enzymes involved in polysaccharide biosynthesis, including fructose 1,6-bisphosphate aldolase (FBA), glucokinase (GK), glucose 6-phosphate dehydrogenase (G6PDH), mitochondrial isocitrate dehydrogenase (ICDH), and UDP-glucose dehydrogenase (UGDH). The enzymatic activities of G6PDH, ICDH, and UGDH were shown to be significantly correlated with the shifts in intracellular NO levels. For further validation, G6PDH, ICDH, and UGDH were heterologously expressed in Escherichia coli and purified via Ni+-NAT affinity chromatography, and subjected to a biotin switch assay and western blot analysis, which revealed that UGDH and G6PDH were susceptible to SNO. Furthermore, mass spectrometry analysis of proteins treated with S-nitrosoglutathione (GSNO) identified the SNO modification sites for UGDH and G6PDH as cysteine 423 and cysteine 249, respectively. These findings suggest that NO modulates polysaccharide biosynthesis in N. flagelliforme through SNO of UGDH and G6PDH. This reveals a potential mechanism through which NO promotes polysaccharide synthesis in N. flagelliforme, while also providing a new strategy for improving the industrial production of polysaccharides.
基于水杨酸和茉莉酸通过调节细胞内一氧化氮(NO)水平增加鞭毛藻多糖产量的研究结果,我们从S-亚硝基化(SNO)的角度探讨了NO影响鞭毛藻多糖生物合成的机制。加入 NO 供体和清除剂后发现,细胞内 NO 对鞭毛藻多糖产量有显著的正向影响。为了探索其机制,我们研究了 NO 水平与参与多糖生物合成的几种关键酶活性之间的关系,包括 1,6-二磷酸果糖醛缩酶 (FBA)、葡萄糖激酶 (GK)、6-磷酸葡萄糖脱氢酶 (G6PDH)、线粒体异柠檬酸脱氢酶 (ICDH) 和 UDP-葡萄糖脱氢酶 (UGDH)。研究表明,G6PDH、ICDH 和 UGDH 的酶活性与细胞内 NO 水平的变化显著相关。为了进一步验证,在大肠杆菌中异源表达了 G6PDH、ICDH 和 UGDH,并通过 Ni+-NAT 亲和层析进行纯化,然后进行生物素转换测定和 Western 印迹分析,结果显示 UGDH 和 G6PDH 易受 SNO 影响。此外,经 S-亚硝基谷胱甘肽(GSNO)处理的蛋白质的质谱分析表明,UGDH 和 G6PDH 的 SNO 修饰位点分别为半胱氨酸 423 和半胱氨酸 249。这些发现表明,氮氧化物通过对 UGDH 和 G6PDH 的 SNO 来调节鞭毛虫多糖的生物合成。这揭示了 NO 促进鞭毛菜多糖合成的潜在机制,同时也为改善多糖的工业生产提供了新策略。
{"title":"Nitric oxide mediates positive regulation of Nostoc flagelliforme polysaccharide yield via potential S-nitrosylation of G6PDH and UGDH.","authors":"Meng-Yuan Li, Yan-Ru Li, Cheng-Feng Han, Jie Zhang, Rui-Ying Zhu, Yan Zhang, Jian Li, Shi-Ru Jia, Pei-Pei Han","doi":"10.1186/s12896-024-00884-z","DOIUrl":"10.1186/s12896-024-00884-z","url":null,"abstract":"<p><p>Based on our previous findings that salicylic acid and jasmonic acid increased Nostoc flagelliforme polysaccharide yield by regulating intracellular nitric oxide (NO) levels, the mechanism through which NO affects polysaccharide biosynthesis in Nostoc flagelliforme was explored from the perspective of S-nitrosylation (SNO). The addition of NO donor and scavenger showed that intracellular NO had a significant positive effect on the polysaccharide yield of N. flagelliforme. To explore the mechanism, we investigated the relationship between NO levels and the activity of several key enzymes involved in polysaccharide biosynthesis, including fructose 1,6-bisphosphate aldolase (FBA), glucokinase (GK), glucose 6-phosphate dehydrogenase (G6PDH), mitochondrial isocitrate dehydrogenase (ICDH), and UDP-glucose dehydrogenase (UGDH). The enzymatic activities of G6PDH, ICDH, and UGDH were shown to be significantly correlated with the shifts in intracellular NO levels. For further validation, G6PDH, ICDH, and UGDH were heterologously expressed in Escherichia coli and purified via Ni<sup>+</sup>-NAT affinity chromatography, and subjected to a biotin switch assay and western blot analysis, which revealed that UGDH and G6PDH were susceptible to SNO. Furthermore, mass spectrometry analysis of proteins treated with S-nitrosoglutathione (GSNO) identified the SNO modification sites for UGDH and G6PDH as cysteine 423 and cysteine 249, respectively. These findings suggest that NO modulates polysaccharide biosynthesis in N. flagelliforme through SNO of UGDH and G6PDH. This reveals a potential mechanism through which NO promotes polysaccharide synthesis in N. flagelliforme, while also providing a new strategy for improving the industrial production of polysaccharides.</p>","PeriodicalId":8905,"journal":{"name":"BMC Biotechnology","volume":"24 1","pages":"58"},"PeriodicalIF":3.5,"publicationDate":"2024-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11342573/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142035140","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 : 2024-08-12DOI: 10.1186/s12896-024-00879-w
Qiang Pei, Zihui Li, Jingjing Zhao, Haixi Zhang, Tao Qin, Juan Zhao
Diffuse large B-cell lymphoma (DLBCL) is a malignant tumour. Although some standard therapies have been established to improve the cure rate, they remain ineffective for specific individuals. Therefore, it is meaningful to find more novel therapeutic approaches. Macrophage polarisation is extensively involved in the process of tumour development. Recombinant hirudin (rH) affects macrophages and has been researched frequently in clinical trials lately. Our article validated the regulatory role of rH in macrophage polarisation and the mechanism of PAR-1 by collecting clinical samples and subsequently establishing a cellular model to provide a scientifically supported perspective for discovering new therapeutic approaches. We assessed the expression of macrophage polarisation markers, cytokines and PAR-1 in clinical samples. We established a cell model by co-culture with THP-1 and OCI-Ly10 cell. We determined the degree of cell polarisation and expression of validation cytokines by flow cytometry, ELISA, and RT-qPCR to confirm the success of the cell model. Subsequently, different doses of rH were added to discover the function of rH on cell polarisation. We confirmed the mechanism of PAR-1 in macrophage polarisation by transfecting si-PAR-1 and pcDNA3.1-PAR-1. We found higher expression of M2 macrophage markers (CD163 + CMAF+) and PAR-1 in 32 DLBCL samples. After inducing monocyte differentiation into M0 macrophages and co-culturing with OCI-Ly10 lymphoma cells, we found a trend of these expressions in the cell model consistent with the clinical samples. Subsequently, we discovered that rH promotes the polarisation of M1 macrophages but inhibits the polarisation of M2 macrophages. We also found that PAR-1 regulates macrophage polarisation, inhibiting cell proliferation, migration, invasion and angiogenic capacity. rH inhibits macrophage polarisation towards the M2 type and PAR-1 regulates polarisation, proliferation, migration, invasion, and angiogenesis of DLBCL-associated macrophages.
{"title":"Recombinant hirudin and PAR-1 regulate macrophage polarisation status in diffuse large B-cell lymphoma","authors":"Qiang Pei, Zihui Li, Jingjing Zhao, Haixi Zhang, Tao Qin, Juan Zhao","doi":"10.1186/s12896-024-00879-w","DOIUrl":"https://doi.org/10.1186/s12896-024-00879-w","url":null,"abstract":"Diffuse large B-cell lymphoma (DLBCL) is a malignant tumour. Although some standard therapies have been established to improve the cure rate, they remain ineffective for specific individuals. Therefore, it is meaningful to find more novel therapeutic approaches. Macrophage polarisation is extensively involved in the process of tumour development. Recombinant hirudin (rH) affects macrophages and has been researched frequently in clinical trials lately. Our article validated the regulatory role of rH in macrophage polarisation and the mechanism of PAR-1 by collecting clinical samples and subsequently establishing a cellular model to provide a scientifically supported perspective for discovering new therapeutic approaches. We assessed the expression of macrophage polarisation markers, cytokines and PAR-1 in clinical samples. We established a cell model by co-culture with THP-1 and OCI-Ly10 cell. We determined the degree of cell polarisation and expression of validation cytokines by flow cytometry, ELISA, and RT-qPCR to confirm the success of the cell model. Subsequently, different doses of rH were added to discover the function of rH on cell polarisation. We confirmed the mechanism of PAR-1 in macrophage polarisation by transfecting si-PAR-1 and pcDNA3.1-PAR-1. We found higher expression of M2 macrophage markers (CD163 + CMAF+) and PAR-1 in 32 DLBCL samples. After inducing monocyte differentiation into M0 macrophages and co-culturing with OCI-Ly10 lymphoma cells, we found a trend of these expressions in the cell model consistent with the clinical samples. Subsequently, we discovered that rH promotes the polarisation of M1 macrophages but inhibits the polarisation of M2 macrophages. We also found that PAR-1 regulates macrophage polarisation, inhibiting cell proliferation, migration, invasion and angiogenic capacity. rH inhibits macrophage polarisation towards the M2 type and PAR-1 regulates polarisation, proliferation, migration, invasion, and angiogenesis of DLBCL-associated macrophages.","PeriodicalId":8905,"journal":{"name":"BMC Biotechnology","volume":"23 1","pages":""},"PeriodicalIF":3.5,"publicationDate":"2024-08-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141941949","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}
This study evaluated the effects of supplementing the diet of lactating cows with Acremonium terrestris culture (ATC) on milk production, serum antioxidant capacity, inflammatory indices, and serum lipid metabolomics. Over 90 days, 24 multiparous Chinese Holstein cows in mid-lactation (108 ± 10.4 days in milk, 637 ± 25 kg body weight, 30.23 ± 3.7 kg/d milk yield) were divided into either a control diet (CON) or a diet supplemented with 30 g of ATC daily. All the data were analyzed using Student’s t test with SPSS 20.0 software. The results showed that compared with CON feeding, ATC feeding significantly increased milk yield, antioxidant capacity, and immune function. Lipidome screening identified 143 lipid metabolites that differed between the two groups. Further analysis using “random forest” machine learning revealed three glycerophospholipid serum metabolites that could serve as lipid markers with a predictive accuracy of 91.67%. This study suggests that ATC can be a useful dietary supplement for improving lactational performance in dairy cows and provides valuable insights into developing nutritional strategies to maintain metabolic homeostasis in ruminants.
{"title":"Comparison of lipidome profiles in serum from lactating dairy cows supplemented with Acremonium terrestris culture based on UPLC-QTRAP-MS/MS","authors":"Chenmiao Zhang, Yiran Zhao, Shijiao Guo, Feifei Li, Xu Gong, Jiarui Gao, Linshu Jiang, Jinjin Tong","doi":"10.1186/s12896-024-00881-2","DOIUrl":"https://doi.org/10.1186/s12896-024-00881-2","url":null,"abstract":"This study evaluated the effects of supplementing the diet of lactating cows with Acremonium terrestris culture (ATC) on milk production, serum antioxidant capacity, inflammatory indices, and serum lipid metabolomics. Over 90 days, 24 multiparous Chinese Holstein cows in mid-lactation (108 ± 10.4 days in milk, 637 ± 25 kg body weight, 30.23 ± 3.7 kg/d milk yield) were divided into either a control diet (CON) or a diet supplemented with 30 g of ATC daily. All the data were analyzed using Student’s t test with SPSS 20.0 software. The results showed that compared with CON feeding, ATC feeding significantly increased milk yield, antioxidant capacity, and immune function. Lipidome screening identified 143 lipid metabolites that differed between the two groups. Further analysis using “random forest” machine learning revealed three glycerophospholipid serum metabolites that could serve as lipid markers with a predictive accuracy of 91.67%. This study suggests that ATC can be a useful dietary supplement for improving lactational performance in dairy cows and provides valuable insights into developing nutritional strategies to maintain metabolic homeostasis in ruminants.","PeriodicalId":8905,"journal":{"name":"BMC Biotechnology","volume":"2012 1","pages":""},"PeriodicalIF":3.5,"publicationDate":"2024-08-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141941948","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}