Johanna Alm, Benoit Fischer, Alexandra Emanuela Burger, Francesca Moretti
Perturbation of angiogenesis is associated with a variety of diseases and pro- as well as antiangiogenic therapies are being actively explored. Additionally, unintended adverse drug effects on angiogenesis might lead to promotion of tumor progression and cardiovascular complications. Several tri-dimensional microfluidic vessel-on-chip systems have been described that allow a more accurate investigation of vascular physiology and pathology, compared to the two-dimensional static culture of endothelial cells. The OrganoPlate® angiogenesis-on-chip system has been demonstrated to be amenable to high-throughput screening for the antiangiogenic properties of molecules. We set out to adapt this system for high-throughput screening of molecules with proangiogenic properties. Our technical advancement of the OrganoPlate® angiogenesis-on-chip assay expands its applicability in the early screening of both anti- as well as proangiogenic properties of compounds for therapeutic modulation of angiogenesis as well as the identification of angiogenesis-associated drug-induced vascular toxicities.
{"title":"Development of a 3D Perfused In Vitro System to Assess Proangiogenic Properties of Compounds.","authors":"Johanna Alm, Benoit Fischer, Alexandra Emanuela Burger, Francesca Moretti","doi":"10.3390/mps6060119","DOIUrl":"10.3390/mps6060119","url":null,"abstract":"<p><p>Perturbation of angiogenesis is associated with a variety of diseases and pro- as well as antiangiogenic therapies are being actively explored. Additionally, unintended adverse drug effects on angiogenesis might lead to promotion of tumor progression and cardiovascular complications. Several tri-dimensional microfluidic vessel-on-chip systems have been described that allow a more accurate investigation of vascular physiology and pathology, compared to the two-dimensional static culture of endothelial cells. The OrganoPlate<sup>®</sup> angiogenesis-on-chip system has been demonstrated to be amenable to high-throughput screening for the antiangiogenic properties of molecules. We set out to adapt this system for high-throughput screening of molecules with proangiogenic properties. Our technical advancement of the OrganoPlate<sup>®</sup> angiogenesis-on-chip assay expands its applicability in the early screening of both anti- as well as proangiogenic properties of compounds for therapeutic modulation of angiogenesis as well as the identification of angiogenesis-associated drug-induced vascular toxicities.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"6 6","pages":""},"PeriodicalIF":2.4,"publicationDate":"2023-12-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10745376/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138830498","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Junchi Huang, Mikael Montelius, Jan-Erik Damber, K. Welén
Bone metastases cause morbidity and mortality in several human cancer forms. Experimental models are used to unravel the mechanisms and identify possible treatment targets. The location inside the skeleton complicates accurate assessment. This study evaluates the performance of magnetic resonance imaging (MRI) of prostate cancer tumors growing intratibially in mice. MRI detected intratibial tumor lesions with a sensitivity and specificity of 100% and 89%, respectively, compared to histological evaluation. Location and some phenotypical features could also be readily detected with MRI. Regarding volume estimation, the correlation between MRI and histological assessment was high (p < 0.001, r = 0.936). In conclusion, this study finds MRI to be a reliable tool for in vivo, non-invasive, non-ionizing, real-time monitoring of intratibial tumor growth.
骨转移在几种人类癌症中引起发病率和死亡率。实验模型用于揭示机制和确定可能的治疗靶点。骨骼内部的位置使准确评估变得复杂。本研究评价了磁共振成像(MRI)对小鼠腹腔内生长的前列腺癌肿瘤的表现。与组织学评估相比，MRI检测胫骨内肿瘤病变的敏感性和特异性分别为100%和89%。位置和一些表型特征也可以很容易地通过MRI检测到。在体积估计方面，MRI与组织学评估的相关性较高(p < 0.001, r = 0.936)。总之，本研究发现MRI是一种可靠的工具，可以在体内、无创、非电离、实时监测胫骨内肿瘤的生长。
{"title":"Magnetic Resonance Imaging as a Tool for Monitoring Intratibial Growth of Experimental Prostate Cancer Metastases in Mice","authors":"Junchi Huang, Mikael Montelius, Jan-Erik Damber, K. Welén","doi":"10.3390/mps6060118","DOIUrl":"https://doi.org/10.3390/mps6060118","url":null,"abstract":"Bone metastases cause morbidity and mortality in several human cancer forms. Experimental models are used to unravel the mechanisms and identify possible treatment targets. The location inside the skeleton complicates accurate assessment. This study evaluates the performance of magnetic resonance imaging (MRI) of prostate cancer tumors growing intratibially in mice. MRI detected intratibial tumor lesions with a sensitivity and specificity of 100% and 89%, respectively, compared to histological evaluation. Location and some phenotypical features could also be readily detected with MRI. Regarding volume estimation, the correlation between MRI and histological assessment was high (p < 0.001, r = 0.936). In conclusion, this study finds MRI to be a reliable tool for in vivo, non-invasive, non-ionizing, real-time monitoring of intratibial tumor growth.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"138 51","pages":""},"PeriodicalIF":2.4,"publicationDate":"2023-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138598703","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}
A. van Duinkerken, M. Bosmans, C. Baliatsas, N. Tak, A. Meerdink, N. Jansen, M. de Vetten-Mc Mahon, E. Marra, M. Dückers
Background: The global COVID-19 pandemic has profoundly affected public health. Directly, the pandemic resulted in over 6.6 million deaths, numerous hospitalizations, and widespread illness. The pandemic has also affected health indirectly through government-imposed protective measures, causing decline in mental well-being and increasing social isolation. Unlike previous disasters or crises, the pandemic’s worldwide and enduring impact necessitates a unique research approach. The Network for Health Research in Disasters in the Netherlands responded by initiating a longitudinal, extensive research project called the Integrated Health Monitor COVID-19. The Integrated Health Monitor COVID-19 explores both the direct and indirect health effects of the pandemic at the population level. Methods: The Integrated Health Monitor COVID-19 employs a dual-pronged monitoring strategy alongside an annual literature review. This strategy comprises short-cycle monitoring (conducted quarterly) and long-cycle monitoring (conducted once every one or two years). This comprehensive approach enables the evaluation of health trends during the pandemic, facilitating comparisons with pre-pandemic levels and identification of risk and protective factors. Both monitoring methods incorporate data from surveys and general practice registries. The integration of annual literature reviews with these measurements enables iterative research, while dialogues on policy and practice improvements enhance the knowledge-to-action process. Discussion: Much of the existing knowledge about the potential impact of the COVID-19 pandemic is derived from research on sudden-onset disasters limited to specific geographical areas. This study is anticipated to provide valuable fresh insights into the evolving dynamics of population health and specific vulnerabilities within the ongoing pandemic context.
{"title":"The Integrated Health Monitor COVID-19: A Protocol for a Comprehensive Assessment of the Short- and Long-Term Health Impact of the Pandemic in the Netherlands","authors":"A. van Duinkerken, M. Bosmans, C. Baliatsas, N. Tak, A. Meerdink, N. Jansen, M. de Vetten-Mc Mahon, E. Marra, M. Dückers","doi":"10.3390/mps6060117","DOIUrl":"https://doi.org/10.3390/mps6060117","url":null,"abstract":"Background: The global COVID-19 pandemic has profoundly affected public health. Directly, the pandemic resulted in over 6.6 million deaths, numerous hospitalizations, and widespread illness. The pandemic has also affected health indirectly through government-imposed protective measures, causing decline in mental well-being and increasing social isolation. Unlike previous disasters or crises, the pandemic’s worldwide and enduring impact necessitates a unique research approach. The Network for Health Research in Disasters in the Netherlands responded by initiating a longitudinal, extensive research project called the Integrated Health Monitor COVID-19. The Integrated Health Monitor COVID-19 explores both the direct and indirect health effects of the pandemic at the population level. Methods: The Integrated Health Monitor COVID-19 employs a dual-pronged monitoring strategy alongside an annual literature review. This strategy comprises short-cycle monitoring (conducted quarterly) and long-cycle monitoring (conducted once every one or two years). This comprehensive approach enables the evaluation of health trends during the pandemic, facilitating comparisons with pre-pandemic levels and identification of risk and protective factors. Both monitoring methods incorporate data from surveys and general practice registries. The integration of annual literature reviews with these measurements enables iterative research, while dialogues on policy and practice improvements enhance the knowledge-to-action process. Discussion: Much of the existing knowledge about the potential impact of the COVID-19 pandemic is derived from research on sudden-onset disasters limited to specific geographical areas. This study is anticipated to provide valuable fresh insights into the evolving dynamics of population health and specific vulnerabilities within the ongoing pandemic context.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"118 2","pages":""},"PeriodicalIF":2.4,"publicationDate":"2023-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138607327","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}
Laurie Nemoz-Billet, Jacques Brocard, Florence Ruggiero, Sandrine Bretaud
Quantifying axonal branching is crucial for understanding neural circuit function, developmental and regeneration processes and disease mechanisms. Factors that regulate patterns of axonal arborization and tune neuronal circuits are investigated for their implication in various disorders in brain connectivity. The lack of a reliable and user-friendly method makes the quantitative analysis of axon morphology difficult. Specifically, methods to visualize and quantify the complex axon arborization are challenging to implement and apply practically. Our study was aimed at developing a robust but simple method of quantification that used ImageJ 2D analysis and compared it with Imaris visualization and analysis of 3D images. We used zebrafish fluorescent transgenic lines to perform in vivo imaging of developing motor neuron axons that adequately reflected the complexity of axonal networks. Our new method, developed on ImageJ, is easy and fast, giving access to new information such as collateral distribution along the axonal shaft. This study describes step-by-step procedures that can be easily applied to a variety of organisms and in vitro systems. Our study provides a basis for further exploration of neural circuits to gain new insights into neuronal disorders and potential therapeutic interventions.
{"title":"Quantitative Image Analysis of Axonal Morphology in In Vivo Model","authors":"Laurie Nemoz-Billet, Jacques Brocard, Florence Ruggiero, Sandrine Bretaud","doi":"10.3390/mps6060116","DOIUrl":"https://doi.org/10.3390/mps6060116","url":null,"abstract":"Quantifying axonal branching is crucial for understanding neural circuit function, developmental and regeneration processes and disease mechanisms. Factors that regulate patterns of axonal arborization and tune neuronal circuits are investigated for their implication in various disorders in brain connectivity. The lack of a reliable and user-friendly method makes the quantitative analysis of axon morphology difficult. Specifically, methods to visualize and quantify the complex axon arborization are challenging to implement and apply practically. Our study was aimed at developing a robust but simple method of quantification that used ImageJ 2D analysis and compared it with Imaris visualization and analysis of 3D images. We used zebrafish fluorescent transgenic lines to perform in vivo imaging of developing motor neuron axons that adequately reflected the complexity of axonal networks. Our new method, developed on ImageJ, is easy and fast, giving access to new information such as collateral distribution along the axonal shaft. This study describes step-by-step procedures that can be easily applied to a variety of organisms and in vitro systems. Our study provides a basis for further exploration of neural circuits to gain new insights into neuronal disorders and potential therapeutic interventions.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":" 863","pages":""},"PeriodicalIF":2.4,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138610525","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}
Christian Wong, Christina Ystrøm Bjerge, Ales Jurca, M. M. Petersen, Soren Boedtker, Andreas Balslev-Clausen, Steen Harsted
Background: The health of children’s lower extremities and feet is a focus area for caregivers and healthcare professionals such as doctors, school nurses, and podiatrists. Our study aims to investigate the general health status of Danish children’s lower extremities and feet to identify anthropometric parameters that might be preconditions for pain and evaluate for foot diseases and whether they are associated with pain intensity and location, three-dimensional foot dimensions and foot pressure mapping, shoe dimensions, types and intensity of sports activity, quality of life, and foot health. The aim is that we will be able to identify parameters pre-dispositioning for pain, thus providing recommendations for sports activities in relation to the anthropometric conditions of a child as a potential preventive measure for pain. This analysis will be stratified by socioeconomic status on a group level, and this perspective will be able to provide preventative recommendations to prevent pain. Methods: This study is a cross-sectional examination of a thousand children in the first, fifth, and ninth grades in randomized selected Danish primary schools. We will perform a clinical examination of the lower extremities and feet for misalignments, deformities, and diseases as well as rotational status and range of motion. Moreover, we will evaluate their pain levels, sports activities, three-dimensional foot dimensions, plantar pressure, footwear, and patient-related outcome measures (PROMs) for foot health and quality of life. Results: We aim to provide an anthropometrical overview of the lower extremities and feet in children. The obtained basic understanding of healthy normal material in children will be analyzed for its relationships with pain level, sports activities, and socioeconomic status on a group level. This could potentially provide us with an understanding of the factors that impact lower extremity and foot diseases in children. In conclusion, examining children’s lower extremities and feet in Danish primary schools is a step toward identifying areas of improvement in self-care and shoe fitting, mapping podiatry-related needs of care in children’s feet, and providing parental recommendations for preventive actions on shoe fitting and the choice and intensity of sports activity concerning pain. Conclusions: The tenet of this study is a long-term follow-up to evaluate the long-term socioeconomic course on a group level, foot status, and sports activity, using patient-related outcome measures evaluating quality of life and other lifestyle factors such as emotional functioning, social functioning and interaction, and school functioning. Potentially, this will improve children’s quality of life and prevent future diseases.
{"title":"Protocol Article: A Cross-Sectional Evaluation of Children’s Feet and Lower Extremities","authors":"Christian Wong, Christina Ystrøm Bjerge, Ales Jurca, M. M. Petersen, Soren Boedtker, Andreas Balslev-Clausen, Steen Harsted","doi":"10.3390/mps6060115","DOIUrl":"https://doi.org/10.3390/mps6060115","url":null,"abstract":"Background: The health of children’s lower extremities and feet is a focus area for caregivers and healthcare professionals such as doctors, school nurses, and podiatrists. Our study aims to investigate the general health status of Danish children’s lower extremities and feet to identify anthropometric parameters that might be preconditions for pain and evaluate for foot diseases and whether they are associated with pain intensity and location, three-dimensional foot dimensions and foot pressure mapping, shoe dimensions, types and intensity of sports activity, quality of life, and foot health. The aim is that we will be able to identify parameters pre-dispositioning for pain, thus providing recommendations for sports activities in relation to the anthropometric conditions of a child as a potential preventive measure for pain. This analysis will be stratified by socioeconomic status on a group level, and this perspective will be able to provide preventative recommendations to prevent pain. Methods: This study is a cross-sectional examination of a thousand children in the first, fifth, and ninth grades in randomized selected Danish primary schools. We will perform a clinical examination of the lower extremities and feet for misalignments, deformities, and diseases as well as rotational status and range of motion. Moreover, we will evaluate their pain levels, sports activities, three-dimensional foot dimensions, plantar pressure, footwear, and patient-related outcome measures (PROMs) for foot health and quality of life. Results: We aim to provide an anthropometrical overview of the lower extremities and feet in children. The obtained basic understanding of healthy normal material in children will be analyzed for its relationships with pain level, sports activities, and socioeconomic status on a group level. This could potentially provide us with an understanding of the factors that impact lower extremity and foot diseases in children. In conclusion, examining children’s lower extremities and feet in Danish primary schools is a step toward identifying areas of improvement in self-care and shoe fitting, mapping podiatry-related needs of care in children’s feet, and providing parental recommendations for preventive actions on shoe fitting and the choice and intensity of sports activity concerning pain. Conclusions: The tenet of this study is a long-term follow-up to evaluate the long-term socioeconomic course on a group level, foot status, and sports activity, using patient-related outcome measures evaluating quality of life and other lifestyle factors such as emotional functioning, social functioning and interaction, and school functioning. Potentially, this will improve children’s quality of life and prevent future diseases.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"170 2","pages":""},"PeriodicalIF":2.4,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138621553","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}
Hongmin Chu, Weonjin Kim, Seongsu Joo, Eunsik Park, Yeong Won Kim, Cheol-Hyun Kim, Sangkwan Lee
Background: The aim of this study was to compare shoulder movement measurements between a Kinect-based markerless ROM assessment device (POM-Checker) and a 3D motion capture analysis system (BTS SMART DX-400).
Methods: This was a single-visit clinical trial designed to evaluate the validity and reliability of the POM-Checker. The primary outcome was to assess the equivalence between two measurement devices within the same set of participants, aiming to evaluate the validity of the POM-Checker compared to the gold standard device (3D Motion Analysis System). As this was a pilot study, six participants were included.
Results: The intraclass correlation coefficient (ICC) and the corresponding 95% confidence intervals (CIs) were used to assess the reproducibility of the measurements. Among the 18 movements analyzed, 16 exhibited ICC values of >0.75, indicating excellent reproducibility.
Conclusion: The results showed that the POM-checker is reliable and validated to measure the range of motion of the shoulder joint.
{"title":"Validity and Reliability of POM-Checker for Measuring Shoulder Range of Motion in Healthy Participants: A Pilot Single-Center Comparative Study.","authors":"Hongmin Chu, Weonjin Kim, Seongsu Joo, Eunsik Park, Yeong Won Kim, Cheol-Hyun Kim, Sangkwan Lee","doi":"10.3390/mps6060114","DOIUrl":"10.3390/mps6060114","url":null,"abstract":"<p><strong>Background: </strong>The aim of this study was to compare shoulder movement measurements between a Kinect-based markerless ROM assessment device (POM-Checker) and a 3D motion capture analysis system (BTS SMART DX-400).</p><p><strong>Methods: </strong>This was a single-visit clinical trial designed to evaluate the validity and reliability of the POM-Checker. The primary outcome was to assess the equivalence between two measurement devices within the same set of participants, aiming to evaluate the validity of the POM-Checker compared to the gold standard device (3D Motion Analysis System). As this was a pilot study, six participants were included.</p><p><strong>Results: </strong>The intraclass correlation coefficient (ICC) and the corresponding 95% confidence intervals (CIs) were used to assess the reproducibility of the measurements. Among the 18 movements analyzed, 16 exhibited ICC values of >0.75, indicating excellent reproducibility.</p><p><strong>Conclusion: </strong>The results showed that the POM-checker is reliable and validated to measure the range of motion of the shoulder joint.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"6 6","pages":""},"PeriodicalIF":2.4,"publicationDate":"2023-11-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10745328/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138830499","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pragya Yadav, V V Santosh Kumar, Jyoti Priya, Shashank Kumar Yadav, Shivani Nagar, Meenu Singh, Viswanathan Chinnusamy
Rice is one of the apex food crops in terms of meeting the daily calorific and dietary requirement of the majority of the world population. However, rice productivity is severely limited by various biotic and abiotic attributes, causing a severe threat to global food security. In the use of functional genomics and genome editing for the generation of trait-enhanced genotypes, it is necessary to have an efficient genetic transformation and regeneration protocol. The recalcitrant nature and paucity of efficient and versatile genetic transformation and regeneration protocols for indica cultivars remains a constraint. In the present study, we have optimized a tissue culture method for MTU1010, a mega indica rice variety. We conducted a combinatorial analysis of different plant growth regulators on embryogenic callus induction efficiency, and it was observed that MSB5 medium supplemented with 2.5 mg/L 2-4D and 0.25 mg/L 6-BAP results in maximum embryogenic callus induction, i.e., 92%. The regeneration efficiency of a transformed callus can be enhanced by up to 50% with the supplementation of 1 mg/L kinetin alongside 2.5 mg/L BAP and 0.5 mg/L NAA in the shooting medium. Furthermore, our results unveiled that the pre-activation of Agrobacterium culture for 30 min with 150 µM acetosyringone significantly increased the transformation efficiency of calli. Additionally, descaling the salt concentration to half strength in resuspension and co-cultivation increased the efficiency of transformation up to 33%. Thus, the protocol developed in this study will be instrumental for the genome editing and genetic engineering of indica rice cultivars for functional genomics studies and crop improvement.
{"title":"A Versatile Protocol for Efficient Transformation and Regeneration in Mega <i>Indica</i> Rice Cultivar MTU1010: Optimization through Hormonal Variables.","authors":"Pragya Yadav, V V Santosh Kumar, Jyoti Priya, Shashank Kumar Yadav, Shivani Nagar, Meenu Singh, Viswanathan Chinnusamy","doi":"10.3390/mps6060113","DOIUrl":"10.3390/mps6060113","url":null,"abstract":"<p><p>Rice is one of the apex food crops in terms of meeting the daily calorific and dietary requirement of the majority of the world population. However, rice productivity is severely limited by various biotic and abiotic attributes, causing a severe threat to global food security. In the use of functional genomics and genome editing for the generation of trait-enhanced genotypes, it is necessary to have an efficient genetic transformation and regeneration protocol. The recalcitrant nature and paucity of efficient and versatile genetic transformation and regeneration protocols for <i>indica</i> cultivars remains a constraint. In the present study, we have optimized a tissue culture method for MTU1010, a mega <i>indica</i> rice variety. We conducted a combinatorial analysis of different plant growth regulators on embryogenic callus induction efficiency, and it was observed that MSB5 medium supplemented with 2.5 mg/L 2-4D and 0.25 mg/L 6-BAP results in maximum embryogenic callus induction, i.e., 92%. The regeneration efficiency of a transformed callus can be enhanced by up to 50% with the supplementation of 1 mg/L kinetin alongside 2.5 mg/L BAP and 0.5 mg/L NAA in the shooting medium. Furthermore, our results unveiled that the pre-activation of <i>Agrobacterium</i> culture for 30 min with 150 µM acetosyringone significantly increased the transformation efficiency of calli. Additionally, descaling the salt concentration to half strength in resuspension and co-cultivation increased the efficiency of transformation up to 33%. Thus, the protocol developed in this study will be instrumental for the genome editing and genetic engineering of <i>indica</i> rice cultivars for functional genomics studies and crop improvement.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"6 6","pages":""},"PeriodicalIF":2.4,"publicationDate":"2023-11-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10745540/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138830497","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Rownock Afruza, Nicole Minerva, Justin B Lack, Moumita Chakraborty, James A Haddad, Rabab O Ali, Christopher Koh, Elliot B Levy, Ohad Etzion, Theo Heller
Cell-free RNAs (cfRNAs) are promising analytes as non-invasive biomarkers and have even greater potential if tied in with metabolomics. Plasma is an optimal source for cfRNAs but is often derived from a variety of anticoagulants. Plasma obtained in heparin is suitable for metabolomics but is difficult to utilize for qPCR-based downstream analysis. In the present study, we aimed to develop a simple, time-efficient, and cost-effective heparinase protocol, followed by library preparation and sequencing of human plasma cfRNAs drawn and stored in heparin at -80 °C for several years. Blood was collected in CPT™ sodium heparin tubes from patients with chronic HCV infection (NCT02400216) at the National Institutes of Health (NIH) Clinical Center. Plasma cfRNAs were treated with heparinase I and used for library preparation and next-generation sequencing (NGS). Heparinase treatment maintained RNA integrity and allowed for successful library preparation for all the study subjects even with 7 ng of cfRNAs as starting material. The classification report derived from Pavian R package v1.2.0 showed no artificial reads. The abundance of chordate over microbial reads suggests no addition of experimental error through heparinase I treatment. We report a novel and practical approach to heparinase treatment for human plasma collected and frozen in sodium heparin for several years. This is an effective demonstration of utilizing heparin plasma for NGS and downstream transcriptomic research, which could then be integrated with metabolomics from the same samples, maximizing efficiency and minimizing blood draws.
{"title":"A Simple, Rapid, and Effective Heparinase Protocol to Enable Nucleic Acid Study from Frozen Heparinized Plasma.","authors":"Rownock Afruza, Nicole Minerva, Justin B Lack, Moumita Chakraborty, James A Haddad, Rabab O Ali, Christopher Koh, Elliot B Levy, Ohad Etzion, Theo Heller","doi":"10.3390/mps6060112","DOIUrl":"10.3390/mps6060112","url":null,"abstract":"<p><p>Cell-free RNAs (cfRNAs) are promising analytes as non-invasive biomarkers and have even greater potential if tied in with metabolomics. Plasma is an optimal source for cfRNAs but is often derived from a variety of anticoagulants. Plasma obtained in heparin is suitable for metabolomics but is difficult to utilize for qPCR-based downstream analysis. In the present study, we aimed to develop a simple, time-efficient, and cost-effective heparinase protocol, followed by library preparation and sequencing of human plasma cfRNAs drawn and stored in heparin at -80 °C for several years. Blood was collected in CPT™ sodium heparin tubes from patients with chronic HCV infection (NCT02400216) at the National Institutes of Health (NIH) Clinical Center. Plasma cfRNAs were treated with heparinase I and used for library preparation and next-generation sequencing (NGS). Heparinase treatment maintained RNA integrity and allowed for successful library preparation for all the study subjects even with 7 ng of cfRNAs as starting material. The classification report derived from Pavian R package v1.2.0 showed no artificial reads. The abundance of chordate over microbial reads suggests no addition of experimental error through heparinase I treatment. We report a novel and practical approach to heparinase treatment for human plasma collected and frozen in sodium heparin for several years. This is an effective demonstration of utilizing heparin plasma for NGS and downstream transcriptomic research, which could then be integrated with metabolomics from the same samples, maximizing efficiency and minimizing blood draws.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"6 6","pages":""},"PeriodicalIF":2.4,"publicationDate":"2023-11-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10660533/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138176753","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
As our readers know, Methods and Protocols is a multidisciplinary peer-reviewed scientific journal that provides a forum to the publication of novel approaches in the fields of Life Sciences, Chemistry, and Biomedical Sciences and their intersection with other related scientific fields such as Physics, Earth Sciences, and Environmental Research [...].
{"title":"<i>Methods and Protocols</i>-Aims and Scope Update.","authors":"Fernando Albericio, Philip Hublitz","doi":"10.3390/mps6060111","DOIUrl":"10.3390/mps6060111","url":null,"abstract":"<p><p>As our readers know, <i>Methods and Protocols</i> is a multidisciplinary peer-reviewed scientific journal that provides a forum to the publication of novel approaches in the fields of Life Sciences, Chemistry, and Biomedical Sciences and their intersection with other related scientific fields such as Physics, Earth Sciences, and Environmental Research [...].</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"6 6","pages":""},"PeriodicalIF":2.4,"publicationDate":"2023-11-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10660513/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138176754","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Tanya Román, Gerardo Acosta, Constanza Cárdenas, Beatriz G. de la Torre, Fanny Guzmán, Fernando Albericio
One approach to enhance the bioavailability and half-life of peptides in vivo is through N-methylation of one or more of the amino acids within the peptide sequence. However, commercially available Fmoc-N-Me-AA-OHs are limited and often expensive. In this study, a solid-phase synthesis method for Fmoc-N-Me-AA-OH was developed using a 2-chlorotrityl chloride (2-CTC) resin as a temporary protective group for the carboxylic acid strategy. Two strategies for the alkylation step were compared, employing either dimethyl sulfate or methyl iodide in the Biron−Kessler method. In this work we tested the protocol with two amino acids: Fmoc-Thr(tBu)-OH and Fmoc-βAla-OH. The first one is an alpha amino acid, very hindered and with the amine group directly influenced by the electronic effects of the carboxy group, whereas in Fmoc-βAla-OH, the presence of a methylene group weakens this influence due to the intervening carbon atoms. The desired amino acids, Fmoc-N-Me-Thr(tBu)-OH and Fmoc-N-Me-βAla-OH, were synthesized by both strategies with high yield and purity.
提高多肽体内生物利用度和半衰期的一种方法是对多肽序列中的一个或多个氨基酸进行n -甲基化。然而，市售的fmoc - n - me - aa - oh数量有限，而且往往价格昂贵。本研究采用2-氯三酰氯(2-CTC)树脂作为羧酸策略的临时保护基，建立了固相合成Fmoc-N-Me-AA-OH的方法。比较了在Biron - Kessler法中采用硫酸二甲酯或碘化甲酯的两种烷基化步骤。在这项工作中，我们测试了两种氨基酸:Fmoc- thr (tBu)-OH和Fmoc-βAla-OH。第一个是α氨基酸，非常受阻，胺基直接受到羧基电子效应的影响，而在Fmoc-βAla-OH中，亚甲基的存在由于中间的碳原子而减弱了这种影响。两种方法合成的氨基酸Fmoc-N-Me- thr (tBu)-OH和Fmoc-N-Me-β - ala -OH收率高，纯度高。
{"title":"Protocol for Facile Synthesis of Fmoc-N-Me-AA-OH Using 2-CTC Resin as Temporary and Reusable Protecting Group","authors":"Tanya Román, Gerardo Acosta, Constanza Cárdenas, Beatriz G. de la Torre, Fanny Guzmán, Fernando Albericio","doi":"10.3390/mps6060110","DOIUrl":"https://doi.org/10.3390/mps6060110","url":null,"abstract":"One approach to enhance the bioavailability and half-life of peptides in vivo is through N-methylation of one or more of the amino acids within the peptide sequence. However, commercially available Fmoc-N-Me-AA-OHs are limited and often expensive. In this study, a solid-phase synthesis method for Fmoc-N-Me-AA-OH was developed using a 2-chlorotrityl chloride (2-CTC) resin as a temporary protective group for the carboxylic acid strategy. Two strategies for the alkylation step were compared, employing either dimethyl sulfate or methyl iodide in the Biron−Kessler method. In this work we tested the protocol with two amino acids: Fmoc-Thr(tBu)-OH and Fmoc-βAla-OH. The first one is an alpha amino acid, very hindered and with the amine group directly influenced by the electronic effects of the carboxy group, whereas in Fmoc-βAla-OH, the presence of a methylene group weakens this influence due to the intervening carbon atoms. The desired amino acids, Fmoc-N-Me-Thr(tBu)-OH and Fmoc-N-Me-βAla-OH, were synthesized by both strategies with high yield and purity.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"4 8","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"136283819","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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