Pub Date : 2024-02-06DOI: 10.1093/biomethods/bpae008
Yeong Yeop Jeong, Yoo-Sun Noh, Suk Weon Kim, P. Seo
Protoplast regeneration has become a key platform for genetic and genome engineering. However, we lack reliable and reproducible methods for efficient protoplast regeneration for tomato (Solanum lycopersicum) cultivars. Here, we optimized cell and tissue culture methods for protoplast isolation, microcallus proliferation, shoot regeneration, and plantlet establishment of the tomato cultivar Micro-Tom. A thin layer of alginate was applied to protoplasts isolated from 3rd and 4th true leaves and cultured at an optimal density of 1 × 105 protoplasts/mL. We determined the optimal culture media for protoplast proliferation, callus formation, de novo shoot regeneration, and root regeneration. Regenerated plantlets exhibited morphologically normal growth and sexual reproduction. The entire regeneration process, from protoplasts to flowering plants, was accomplished within 5 months. The optimized protoplast regeneration platform enables biotechnological applications such as genome engineering as well as basic research on plant regeneration in Solanaceae species.
{"title":"Efficient regeneration of protoplasts from solanum lycopersicum cultivar Micro-Tom","authors":"Yeong Yeop Jeong, Yoo-Sun Noh, Suk Weon Kim, P. Seo","doi":"10.1093/biomethods/bpae008","DOIUrl":"https://doi.org/10.1093/biomethods/bpae008","url":null,"abstract":"\u0000 Protoplast regeneration has become a key platform for genetic and genome engineering. However, we lack reliable and reproducible methods for efficient protoplast regeneration for tomato (Solanum lycopersicum) cultivars. Here, we optimized cell and tissue culture methods for protoplast isolation, microcallus proliferation, shoot regeneration, and plantlet establishment of the tomato cultivar Micro-Tom. A thin layer of alginate was applied to protoplasts isolated from 3rd and 4th true leaves and cultured at an optimal density of 1 × 105 protoplasts/mL. We determined the optimal culture media for protoplast proliferation, callus formation, de novo shoot regeneration, and root regeneration. Regenerated plantlets exhibited morphologically normal growth and sexual reproduction. The entire regeneration process, from protoplasts to flowering plants, was accomplished within 5 months. The optimized protoplast regeneration platform enables biotechnological applications such as genome engineering as well as basic research on plant regeneration in Solanaceae species.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-02-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139801570","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}
Pub Date : 2024-02-05DOI: 10.1093/biomethods/bpae005
Xi Duan, Zhibin Chen, Zhean Zhan, Langhui Li, Xianying Lei, Yang Long, Xie Xiang, Huan Chen
Transurethral catheterization in mice is multifaceted, serving essential functions such as perfusion and drug delivery, and is critical in the development of various urological animal disease models. The complex anatomy of the male mouse urethra presents significant challenges in transurethral catheterization, leading to a predominance of research focused on female specimens. This bias limits the utilization of male mice in lower urinary tract disease studies. Our research aims to develop new reliable methods for transurethral catheterization in adult male mice, thereby expanding their use in relevant disease research. Experiments were conducted on adult male C57BL/6J mice. Utilizing a PE10 catheter measuring 4.5 to 5 cm in length, the catheter was inserted into the bladder via the mouse's urethra under anesthesia. The intubation technique entailed regulating the insertion force, ensuring the catheter's lubrication, using a trocar catheter, modifying the catheter's trajectory, and accommodating the curvature of the bladder neck. Post-catheter insertion, ultrasound imaging was employed to confirm the catheter's accurate positioning within the bladder. Subsequent to catheterization, the bladder was perfused using trypan blue. This method was further validated through its successful application in establishing an acute urinary retention (AUR) model, where the mouse bladder was infused with saline to a pressure of 50 cm or 80 cm H2O, maintained steadily for 30 minutes. A thorough morphological assessment of the mouse bladder was conducted after the infusion. Our study successfully pioneered methods for transurethral catheterization in male mice. This technique not only facilitates precise transurethral catheterization but also proves applicable to male mouse models for lower urinary tract diseases, such as AUR.
{"title":"Establishment of new transurethral catheterization methods for male mice","authors":"Xi Duan, Zhibin Chen, Zhean Zhan, Langhui Li, Xianying Lei, Yang Long, Xie Xiang, Huan Chen","doi":"10.1093/biomethods/bpae005","DOIUrl":"https://doi.org/10.1093/biomethods/bpae005","url":null,"abstract":"\u0000 Transurethral catheterization in mice is multifaceted, serving essential functions such as perfusion and drug delivery, and is critical in the development of various urological animal disease models. The complex anatomy of the male mouse urethra presents significant challenges in transurethral catheterization, leading to a predominance of research focused on female specimens. This bias limits the utilization of male mice in lower urinary tract disease studies. Our research aims to develop new reliable methods for transurethral catheterization in adult male mice, thereby expanding their use in relevant disease research. Experiments were conducted on adult male C57BL/6J mice. Utilizing a PE10 catheter measuring 4.5 to 5 cm in length, the catheter was inserted into the bladder via the mouse's urethra under anesthesia. The intubation technique entailed regulating the insertion force, ensuring the catheter's lubrication, using a trocar catheter, modifying the catheter's trajectory, and accommodating the curvature of the bladder neck. Post-catheter insertion, ultrasound imaging was employed to confirm the catheter's accurate positioning within the bladder. Subsequent to catheterization, the bladder was perfused using trypan blue. This method was further validated through its successful application in establishing an acute urinary retention (AUR) model, where the mouse bladder was infused with saline to a pressure of 50 cm or 80 cm H2O, maintained steadily for 30 minutes. A thorough morphological assessment of the mouse bladder was conducted after the infusion. Our study successfully pioneered methods for transurethral catheterization in male mice. This technique not only facilitates precise transurethral catheterization but also proves applicable to male mouse models for lower urinary tract diseases, such as AUR.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139804370","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}
Pub Date : 2024-02-05DOI: 10.1093/biomethods/bpae005
Xi Duan, Zhibin Chen, Zhean Zhan, Langhui Li, Xianying Lei, Yang Long, Xie Xiang, Huan Chen
Transurethral catheterization in mice is multifaceted, serving essential functions such as perfusion and drug delivery, and is critical in the development of various urological animal disease models. The complex anatomy of the male mouse urethra presents significant challenges in transurethral catheterization, leading to a predominance of research focused on female specimens. This bias limits the utilization of male mice in lower urinary tract disease studies. Our research aims to develop new reliable methods for transurethral catheterization in adult male mice, thereby expanding their use in relevant disease research. Experiments were conducted on adult male C57BL/6J mice. Utilizing a PE10 catheter measuring 4.5 to 5 cm in length, the catheter was inserted into the bladder via the mouse's urethra under anesthesia. The intubation technique entailed regulating the insertion force, ensuring the catheter's lubrication, using a trocar catheter, modifying the catheter's trajectory, and accommodating the curvature of the bladder neck. Post-catheter insertion, ultrasound imaging was employed to confirm the catheter's accurate positioning within the bladder. Subsequent to catheterization, the bladder was perfused using trypan blue. This method was further validated through its successful application in establishing an acute urinary retention (AUR) model, where the mouse bladder was infused with saline to a pressure of 50 cm or 80 cm H2O, maintained steadily for 30 minutes. A thorough morphological assessment of the mouse bladder was conducted after the infusion. Our study successfully pioneered methods for transurethral catheterization in male mice. This technique not only facilitates precise transurethral catheterization but also proves applicable to male mouse models for lower urinary tract diseases, such as AUR.
{"title":"Establishment of new transurethral catheterization methods for male mice","authors":"Xi Duan, Zhibin Chen, Zhean Zhan, Langhui Li, Xianying Lei, Yang Long, Xie Xiang, Huan Chen","doi":"10.1093/biomethods/bpae005","DOIUrl":"https://doi.org/10.1093/biomethods/bpae005","url":null,"abstract":"\u0000 Transurethral catheterization in mice is multifaceted, serving essential functions such as perfusion and drug delivery, and is critical in the development of various urological animal disease models. The complex anatomy of the male mouse urethra presents significant challenges in transurethral catheterization, leading to a predominance of research focused on female specimens. This bias limits the utilization of male mice in lower urinary tract disease studies. Our research aims to develop new reliable methods for transurethral catheterization in adult male mice, thereby expanding their use in relevant disease research. Experiments were conducted on adult male C57BL/6J mice. Utilizing a PE10 catheter measuring 4.5 to 5 cm in length, the catheter was inserted into the bladder via the mouse's urethra under anesthesia. The intubation technique entailed regulating the insertion force, ensuring the catheter's lubrication, using a trocar catheter, modifying the catheter's trajectory, and accommodating the curvature of the bladder neck. Post-catheter insertion, ultrasound imaging was employed to confirm the catheter's accurate positioning within the bladder. Subsequent to catheterization, the bladder was perfused using trypan blue. This method was further validated through its successful application in establishing an acute urinary retention (AUR) model, where the mouse bladder was infused with saline to a pressure of 50 cm or 80 cm H2O, maintained steadily for 30 minutes. A thorough morphological assessment of the mouse bladder was conducted after the infusion. Our study successfully pioneered methods for transurethral catheterization in male mice. This technique not only facilitates precise transurethral catheterization but also proves applicable to male mouse models for lower urinary tract diseases, such as AUR.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139864275","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}
Pub Date : 2024-02-03DOI: 10.1093/biomethods/bpae004
Johnny Sena, L. Karwal, Callum Bell, Nicholas Devitt, Faye Schilkey, Claire Huang, Jill Livengood, Subash Das, Hansi J Dean
The goal of the study was to identify and characterize RNA virus variants containing mutations spread over genomic distances greater than 5 kb. As proof of concept, high quality viral RNA of the Dengue 2 component of Takeda’s tetravalent dengue vaccine candidate (TDV-2) was used to develop an RT-PCR protocol to amplify a ∼5.3 kb cDNA segment that contains the three genetic determinants of TDV-2 attenuation. Unique molecular identifiers were incorporated into each viral cDNA molecule for PacBio library preparation to improve the quantitative precision of the observed variants at the attenuation loci. Following assay optimization, PacBio long read sequencing was validated with multiple clone-derived TDV-2 revertant variants and 4 complex revertant mixtures containing various compositions of TDV-2 and revertant viruses. : PacBio sequencing analysis correctly identified and quantified variant composition in all tested samples, demonstrating that TDV-2 revertants could be identified and characterized and supporting the use of this method in differentiation and quantification of complex variants of other RNA viruses. Long-read sequencing can identify complex RNA virus variants containing multiple mutations on a single genome molecule which is useful for in-depth genetic stability and revertant detection of live-attenuated viral vaccines, as well as research in virus evolution to reveal mechanisms of immune evasion and host cell adaption.
{"title":"Identification and quantitation of multiple variants in RNA virus genomes","authors":"Johnny Sena, L. Karwal, Callum Bell, Nicholas Devitt, Faye Schilkey, Claire Huang, Jill Livengood, Subash Das, Hansi J Dean","doi":"10.1093/biomethods/bpae004","DOIUrl":"https://doi.org/10.1093/biomethods/bpae004","url":null,"abstract":"\u0000 \u0000 \u0000 The goal of the study was to identify and characterize RNA virus variants containing mutations spread over genomic distances greater than 5 kb.\u0000 \u0000 \u0000 \u0000 As proof of concept, high quality viral RNA of the Dengue 2 component of Takeda’s tetravalent dengue vaccine candidate (TDV-2) was used to develop an RT-PCR protocol to amplify a ∼5.3 kb cDNA segment that contains the three genetic determinants of TDV-2 attenuation. Unique molecular identifiers were incorporated into each viral cDNA molecule for PacBio library preparation to improve the quantitative precision of the observed variants at the attenuation loci. Following assay optimization, PacBio long read sequencing was validated with multiple clone-derived TDV-2 revertant variants and 4 complex revertant mixtures containing various compositions of TDV-2 and revertant viruses.\u0000 \u0000 \u0000 \u0000 : PacBio sequencing analysis correctly identified and quantified variant composition in all tested samples, demonstrating that TDV-2 revertants could be identified and characterized and supporting the use of this method in differentiation and quantification of complex variants of other RNA viruses.\u0000 \u0000 \u0000 \u0000 Long-read sequencing can identify complex RNA virus variants containing multiple mutations on a single genome molecule which is useful for in-depth genetic stability and revertant detection of live-attenuated viral vaccines, as well as research in virus evolution to reveal mechanisms of immune evasion and host cell adaption.\u0000","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-02-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139807709","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}
Pub Date : 2024-02-03DOI: 10.1093/biomethods/bpae004
Johnny Sena, L. Karwal, Callum Bell, Nicholas Devitt, Faye Schilkey, Claire Huang, Jill Livengood, Subash Das, Hansi J Dean
The goal of the study was to identify and characterize RNA virus variants containing mutations spread over genomic distances greater than 5 kb. As proof of concept, high quality viral RNA of the Dengue 2 component of Takeda’s tetravalent dengue vaccine candidate (TDV-2) was used to develop an RT-PCR protocol to amplify a ∼5.3 kb cDNA segment that contains the three genetic determinants of TDV-2 attenuation. Unique molecular identifiers were incorporated into each viral cDNA molecule for PacBio library preparation to improve the quantitative precision of the observed variants at the attenuation loci. Following assay optimization, PacBio long read sequencing was validated with multiple clone-derived TDV-2 revertant variants and 4 complex revertant mixtures containing various compositions of TDV-2 and revertant viruses. : PacBio sequencing analysis correctly identified and quantified variant composition in all tested samples, demonstrating that TDV-2 revertants could be identified and characterized and supporting the use of this method in differentiation and quantification of complex variants of other RNA viruses. Long-read sequencing can identify complex RNA virus variants containing multiple mutations on a single genome molecule which is useful for in-depth genetic stability and revertant detection of live-attenuated viral vaccines, as well as research in virus evolution to reveal mechanisms of immune evasion and host cell adaption.
{"title":"Identification and quantitation of multiple variants in RNA virus genomes","authors":"Johnny Sena, L. Karwal, Callum Bell, Nicholas Devitt, Faye Schilkey, Claire Huang, Jill Livengood, Subash Das, Hansi J Dean","doi":"10.1093/biomethods/bpae004","DOIUrl":"https://doi.org/10.1093/biomethods/bpae004","url":null,"abstract":"\u0000 \u0000 \u0000 The goal of the study was to identify and characterize RNA virus variants containing mutations spread over genomic distances greater than 5 kb.\u0000 \u0000 \u0000 \u0000 As proof of concept, high quality viral RNA of the Dengue 2 component of Takeda’s tetravalent dengue vaccine candidate (TDV-2) was used to develop an RT-PCR protocol to amplify a ∼5.3 kb cDNA segment that contains the three genetic determinants of TDV-2 attenuation. Unique molecular identifiers were incorporated into each viral cDNA molecule for PacBio library preparation to improve the quantitative precision of the observed variants at the attenuation loci. Following assay optimization, PacBio long read sequencing was validated with multiple clone-derived TDV-2 revertant variants and 4 complex revertant mixtures containing various compositions of TDV-2 and revertant viruses.\u0000 \u0000 \u0000 \u0000 : PacBio sequencing analysis correctly identified and quantified variant composition in all tested samples, demonstrating that TDV-2 revertants could be identified and characterized and supporting the use of this method in differentiation and quantification of complex variants of other RNA viruses.\u0000 \u0000 \u0000 \u0000 Long-read sequencing can identify complex RNA virus variants containing multiple mutations on a single genome molecule which is useful for in-depth genetic stability and revertant detection of live-attenuated viral vaccines, as well as research in virus evolution to reveal mechanisms of immune evasion and host cell adaption.\u0000","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-02-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139867642","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}
Pub Date : 2024-01-25DOI: 10.1093/biomethods/bpae003
Juliana SanchesTrevizol, A. Dionizio, A. Q. Delgado, T. M. Ventura, Caroline Fernanda da Silva Ribeiro, Nathalia Rabelo Buzalaf, J. Bosqueiro, M. Buzalaf
Pancreatic islets are crucial in diabetes research. Consequently, this protocol aims at optimizing both the protein-extraction process and the proteomic analysis via shotgun methods for pancreatic islets. Six protocols were tested, combining three types of chemical extraction with two mechanical-extraction methods. Furthermore, two protocols incorporated a surfactant to enhance enzymatic cleavage. The steps involved extraction and concentration of protein, protein quantification, reduction, alkylation, digestion, purification and desalination, sample concentration to ∼ 1 µL and proteomic analysis using the mass spectrometer. The most effective protocol involves either a milder chemical extraction paired with a more intensive mechanical process, or a more robust chemical extraction paired with a gentle mechanical process, tailored to the samplés characteristics. Additionally, it was observed that the use of a surfactant proved ineffective for these types of samples. Protocol 5 was recently used with success to examine metabolic changes in pancreatic islets of NOD mice exposed to low doses of fluoride ions (F-) and the primary pathways altered by the treatment.
{"title":"Optimized protocol for shotgun label-free proteomic analysis of pancreatic islets","authors":"Juliana SanchesTrevizol, A. Dionizio, A. Q. Delgado, T. M. Ventura, Caroline Fernanda da Silva Ribeiro, Nathalia Rabelo Buzalaf, J. Bosqueiro, M. Buzalaf","doi":"10.1093/biomethods/bpae003","DOIUrl":"https://doi.org/10.1093/biomethods/bpae003","url":null,"abstract":"\u0000 Pancreatic islets are crucial in diabetes research. Consequently, this protocol aims at optimizing both the protein-extraction process and the proteomic analysis via shotgun methods for pancreatic islets. Six protocols were tested, combining three types of chemical extraction with two mechanical-extraction methods. Furthermore, two protocols incorporated a surfactant to enhance enzymatic cleavage. The steps involved extraction and concentration of protein, protein quantification, reduction, alkylation, digestion, purification and desalination, sample concentration to ∼ 1 µL and proteomic analysis using the mass spectrometer. The most effective protocol involves either a milder chemical extraction paired with a more intensive mechanical process, or a more robust chemical extraction paired with a gentle mechanical process, tailored to the samplés characteristics. Additionally, it was observed that the use of a surfactant proved ineffective for these types of samples. Protocol 5 was recently used with success to examine metabolic changes in pancreatic islets of NOD mice exposed to low doses of fluoride ions (F-) and the primary pathways altered by the treatment.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-01-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139595934","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}
Pub Date : 2024-01-23DOI: 10.1093/biomethods/bpae002
Sivananthan Manoharan, Lee Ying Ying
Due to high heterogeneity and risk of bias (RoB) found in previously published meta-analysis, a concrete conclusion on the efficacy of baricitinib in reducing mortality in COVID-19 patients was unable to form. Hence, this systematic-review and meta-analysis was conducted to analyse whether RoB, heterogeneity and optimal sample size from placebo controlled randomised controlled trials (RCTs) are still the problems to derive a concrete conclusion. Search engines PubMed/MEDLINE, ScienceDirect and other sources like preprints and reference lists were searched with appropriate keywords. The RoB and meta-analysis were conducted using RevMan 5.4. The grading of the articles was conducted using the GRADEPro Guideline Development Tool. Ten (10) RCTs were included in the current systematic-review. Only 5 low RoB articles are Phase III placebo controlled RCTs with high certainty level based on the GRADE grading system. For meta-analysis, based on 5 low RoB articles, baricitinib statistically significantly reduced mortality where the risk ratio (RR) = 0.68 [95% CI: 0.56 to 0.82; p < 0.0001; I2 = 0%; p = 0.85]. The absolute mortality effect (95% CI) based on the grading system was 35 fewer mortalities per 1000 COVID-19 patients where in the baricitinib and control groups the mortality was 7.4% and 10.9%, respectively. With the presence of optimal sample size of 3944 from 5 low RoB-placebo controlled RCTs which represent a minimum of 300 million population of people and with the presence of 0% of heterogeneity from meta-analysis, the effectiveness of baricitinib in reducing the mortality in COVID-19 patients is concretely proven.
由于之前发表的荟萃分析中发现的高度异质性和偏倚风险（RoB），巴利昔替尼在降低COVID-19患者死亡率方面的疗效无法形成具体结论。因此，本系统回顾和荟萃分析旨在分析安慰剂对照随机对照试验（RCT）中的RoB、异质性和最佳样本量是否仍是得出具体结论的问题所在。 研究人员使用适当的关键词搜索了 PubMed/MEDLINE、ScienceDirect 等搜索引擎以及预印本和参考文献目录等其他来源。使用RevMan 5.4进行RoB和荟萃分析。使用 GRADEPro 指南开发工具对文章进行了分级。 本次系统综述共纳入十（10）项 RCT。根据 GRADE 分级系统，只有 5 篇低 RoB 文章是具有高确定性的 III 期安慰剂对照 RCT。在荟萃分析中，基于 5 篇低 RoB 文章，巴利昔尼显著降低了死亡率，风险比 (RR) = 0.68 [95% CI: 0.56 to 0.82; p < 0.0001; I2 = 0%; p = 0.85]。根据分级系统得出的绝对死亡率效应（95% CI）为每1000例COVID-19患者减少35例死亡，其中巴利昔尼组和对照组的死亡率分别为7.4%和10.9%。 5项低RoB-安慰剂对照RCT的最佳样本量为3944个，代表了至少3亿人口，荟萃分析的异质性为0%，因此巴利昔尼在降低COVID-19患者死亡率方面的有效性得到了具体证明。
{"title":"Baricitinib statistically significantly reduced COVID-19 related mortality: a systematic review and Meta-Analysis of five phase III randomised, blinded and placebo controlled clinical trials","authors":"Sivananthan Manoharan, Lee Ying Ying","doi":"10.1093/biomethods/bpae002","DOIUrl":"https://doi.org/10.1093/biomethods/bpae002","url":null,"abstract":"\u0000 \u0000 \u0000 Due to high heterogeneity and risk of bias (RoB) found in previously published meta-analysis, a concrete conclusion on the efficacy of baricitinib in reducing mortality in COVID-19 patients was unable to form. Hence, this systematic-review and meta-analysis was conducted to analyse whether RoB, heterogeneity and optimal sample size from placebo controlled randomised controlled trials (RCTs) are still the problems to derive a concrete conclusion.\u0000 \u0000 \u0000 \u0000 Search engines PubMed/MEDLINE, ScienceDirect and other sources like preprints and reference lists were searched with appropriate keywords. The RoB and meta-analysis were conducted using RevMan 5.4. The grading of the articles was conducted using the GRADEPro Guideline Development Tool.\u0000 \u0000 \u0000 \u0000 Ten (10) RCTs were included in the current systematic-review. Only 5 low RoB articles are Phase III placebo controlled RCTs with high certainty level based on the GRADE grading system. For meta-analysis, based on 5 low RoB articles, baricitinib statistically significantly reduced mortality where the risk ratio (RR) = 0.68 [95% CI: 0.56 to 0.82; p < 0.0001; I2 = 0%; p = 0.85]. The absolute mortality effect (95% CI) based on the grading system was 35 fewer mortalities per 1000 COVID-19 patients where in the baricitinib and control groups the mortality was 7.4% and 10.9%, respectively.\u0000 \u0000 \u0000 \u0000 With the presence of optimal sample size of 3944 from 5 low RoB-placebo controlled RCTs which represent a minimum of 300 million population of people and with the presence of 0% of heterogeneity from meta-analysis, the effectiveness of baricitinib in reducing the mortality in COVID-19 patients is concretely proven.\u0000","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139602770","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}
Pub Date : 2024-01-19DOI: 10.1093/biomethods/bpae001
Hubert Bernauer, Josef Maier, Norbert Bannert, Daniel Ivanusic
Enzyme-linked immunosorbent assay (ELISA) systems use plates coated with peptides or expressed and purified proteins to monitor immunoglobulins derived from patient serum. However, there is currently no easy, flexible, and fast adaptive ELISA-based system for testing antibodies directed against new SARS-CoV-2 variants. In this study, we utilized the tANCHOR protein display system that provides a cell surface decorated with the receptor binding domain (RBD) to monitor specific antibodies derived from SARS-CoV-2 convalescent and vaccinated individuals directed against it. To test sera from vaccinees or convalescent individuals, only the RBD coding sequence needs to be cloned in the tANCHOR vector system and transfected into HeLa cells. Time-consuming protein expression, isolation, and purification followed by coating assay plates is not necessary. With this technique, the immune evasion of new SARS-CoV-2 variants from current vaccination regimes can be examined quickly and reliably.
{"title":"tANCHOR cell-based ELISA approach as a surrogate for antigen-coated plates to monitor specific IgG directed to the SARS-CoV-2 receptor binding domain","authors":"Hubert Bernauer, Josef Maier, Norbert Bannert, Daniel Ivanusic","doi":"10.1093/biomethods/bpae001","DOIUrl":"https://doi.org/10.1093/biomethods/bpae001","url":null,"abstract":"\u0000 Enzyme-linked immunosorbent assay (ELISA) systems use plates coated with peptides or expressed and purified proteins to monitor immunoglobulins derived from patient serum. However, there is currently no easy, flexible, and fast adaptive ELISA-based system for testing antibodies directed against new SARS-CoV-2 variants. In this study, we utilized the tANCHOR protein display system that provides a cell surface decorated with the receptor binding domain (RBD) to monitor specific antibodies derived from SARS-CoV-2 convalescent and vaccinated individuals directed against it. To test sera from vaccinees or convalescent individuals, only the RBD coding sequence needs to be cloned in the tANCHOR vector system and transfected into HeLa cells. Time-consuming protein expression, isolation, and purification followed by coating assay plates is not necessary. With this technique, the immune evasion of new SARS-CoV-2 variants from current vaccination regimes can be examined quickly and reliably.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-01-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139612452","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}
Pub Date : 2024-01-05DOI: 10.1093/biomethods/bpad041
Paula E Adams, Enya Granados, Abby E. Beatty, C. Ballen
Understanding the relationship between science and society is an objective of science education and included as a core competency in the AAAS Vision and Change guidelines for biology education. However, traditional undergraduate biology instruction emphasizes scientific practice and generally avoids potentially controversial issues at the intersection of biology and society. By including these topics in biology coursework, instructors can challenge damaging ideologies and systemic inequalities that have influenced science, such as biological essentialism and health disparities. Specifically, an ideologically aware curriculum highlights how ideologies and paradigms shape our biological knowledge base and the application of that knowledge. Ideologically aware lessons emphasize the relationship between science and society with an aim to create more transparent, scientifically accurate, and inclusive postsecondary biology classrooms. Here we expand upon our ideologically aware curriculum with a new activity that challenges undergraduate biology students to consider the impacts of healthcare disparities. This lesson allows instructors to directly address systemic inequalities and allows students to connect biomedical sciences to real-world issues. Implementing an ideologically aware curriculum enables students to challenge prevailing worldviews and better address societal problems that lead to exclusion and oppression.
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Botrytis cinerea is a well-known plant pathogen responsible for grey mould disease infecting more than 500 plant species. It is listed as the second most important plant pathogen scientifically and economically. Its impact is particularly severe in grapes since it affects both the yield of grape berries and the quality of wines. While various methods for detecting B. cinerea have been investigated, the application of Oxford Nanopore Technology (ONT) for complete ribosomal operon sequencing, which has proven effective in human and animal fungal research and diagnostics, has not yet been explored in grapevine (Vitis vinifera) disease research. In this study, we sequenced complete ribosomal operons (∼5.5 kb amplicons), which encompass the 18S, ITS1, 5.8S, ITS2, and 28S regions, from both pure cultures of B. cinerea and infected grapevine leaf samples. Minimap2, a sequence alignment tool integrated into the EPI2ME software, served as a taxonomy classifier, utilising the custom reference database FRODO. The results demonstrate that B. cinerea was detectable when this pathogen was not the dominant fungal species in leaf samples. Additionally, the method facilitates host DNA-free sequencing and might have a good potential to distinguish other pathogenic and non-pathogenic fungal species hosted within grapevine’s infected leaves, such as Alternaria alternata, Saccharomyces cerevisiae, Saccharomyces boulardii, Mucor racemosus, Ascochyta rabie, etc The sequences were uploaded to the NCBI database. Long Amplicon sequencing method has the capacity to be broadened to other susceptible crops and pathogens, as a valuable tool for early grey rot detection and mycobiome research. Future large-scale studies are needed to overcome challenges, such as comprehensive reference databases for complete fungal ribosomal operons for grape mycobiome studies.
灰霉病是一种著名的植物病原体，感染 500 多种植物。在科学和经济方面，它被列为第二重要的植物病原体。它对葡萄的影响尤为严重，因为它会影响葡萄果实的产量和葡萄酒的质量。虽然已经研究了多种检测葡萄孢病的方法，但牛津纳米孔技术（ONT）在人类和动物真菌研究和诊断中被证明是有效的，但在葡萄（葡萄属）疾病研究中尚未得到应用。在本研究中，我们对纯培养物和受感染葡萄叶片样本中的完整核糖体操作子（∼5.5 kb 扩增子）进行了测序，其中包括 18S、ITS1、5.8S、ITS2 和 28S 区域。EPI2ME 软件中集成的序列比对工具 Minimap2 利用定制的参考数据库 FRODO 作为分类器。结果表明，当病原体不是叶片样本中的主要真菌种类时，也能检测到 B. cinerea。此外，该方法有利于无宿主 DNA 测序，并有可能区分葡萄树感染叶片中寄生的其他病原真菌和非病原真菌，如交替丝核菌、酿酒酵母菌、布拉氏酵母菌、粘孢子菌、雷公藤酵母菌等。长扩增子测序方法可扩展到其他易感作物和病原体，是早期灰腐病检测和真菌生物群研究的重要工具。未来的大规模研究需要克服各种挑战，例如为葡萄真菌生物群研究建立完整的真菌核糖体操作数综合参考数据库。
{"title":"Long amplicon Nanopore sequencing of Botrytis cinerea and other fungal species present in infected grapevine leaf samples","authors":"Vladimer Baramidze, Luca Sella, Tamar Japaridze, Nino Abashidze, Daviti Lamazoshvili, Nino Dzotsenidze, Giorgi Tomashvili","doi":"10.1093/biomethods/bpad042","DOIUrl":"https://doi.org/10.1093/biomethods/bpad042","url":null,"abstract":"\u0000 Botrytis cinerea is a well-known plant pathogen responsible for grey mould disease infecting more than 500 plant species. It is listed as the second most important plant pathogen scientifically and economically. Its impact is particularly severe in grapes since it affects both the yield of grape berries and the quality of wines. While various methods for detecting B. cinerea have been investigated, the application of Oxford Nanopore Technology (ONT) for complete ribosomal operon sequencing, which has proven effective in human and animal fungal research and diagnostics, has not yet been explored in grapevine (Vitis vinifera) disease research. In this study, we sequenced complete ribosomal operons (∼5.5 kb amplicons), which encompass the 18S, ITS1, 5.8S, ITS2, and 28S regions, from both pure cultures of B. cinerea and infected grapevine leaf samples. Minimap2, a sequence alignment tool integrated into the EPI2ME software, served as a taxonomy classifier, utilising the custom reference database FRODO. The results demonstrate that B. cinerea was detectable when this pathogen was not the dominant fungal species in leaf samples. Additionally, the method facilitates host DNA-free sequencing and might have a good potential to distinguish other pathogenic and non-pathogenic fungal species hosted within grapevine’s infected leaves, such as Alternaria alternata, Saccharomyces cerevisiae, Saccharomyces boulardii, Mucor racemosus, Ascochyta rabie, etc The sequences were uploaded to the NCBI database. Long Amplicon sequencing method has the capacity to be broadened to other susceptible crops and pathogens, as a valuable tool for early grey rot detection and mycobiome research. Future large-scale studies are needed to overcome challenges, such as comprehensive reference databases for complete fungal ribosomal operons for grape mycobiome studies.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139383598","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}
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