Embryo implantation is the first step in the establishment of a successful pregnancy. An in vitro model for embryo implantation is critical for basic biological research, drug development, and screening. This paper presents a simple, rapid, and highly efficient in vitro model for embryo implantation. In this protocol, we first introduce mouse blastocyst acquisition and human endometrial adenocarcinoma cells (Ishikawa) preparation for implantation, followed by the co-culture method for mouse embryos and Ishikawa cells. Finally, we conducted a study to assess the impact of varying concentrations of 17-β-estradiol (E2) and progesterone (P4) on embryo adhesion rates based on this model. Our findings revealed that high concentrations of E2 significantly reduced embryo adhesion, whereas the addition of progesterone could restore the adhesion rate. This model offers a simple and fast platform for evaluating and screening molecules involved in the adhesion process, such as cytokines, drugs, and transcription factors controlling implantation and endometrial receptivity.
{"title":"Establishment of an Embryo Implantation Model In Vitro.","authors":"Xuemei Wang, Yisi Sun, Huijuan Shi, Aijie Xin","doi":"10.3791/66873","DOIUrl":"https://doi.org/10.3791/66873","url":null,"abstract":"<p><p>Embryo implantation is the first step in the establishment of a successful pregnancy. An in vitro model for embryo implantation is critical for basic biological research, drug development, and screening. This paper presents a simple, rapid, and highly efficient in vitro model for embryo implantation. In this protocol, we first introduce mouse blastocyst acquisition and human endometrial adenocarcinoma cells (Ishikawa) preparation for implantation, followed by the co-culture method for mouse embryos and Ishikawa cells. Finally, we conducted a study to assess the impact of varying concentrations of 17-β-estradiol (E2) and progesterone (P4) on embryo adhesion rates based on this model. Our findings revealed that high concentrations of E2 significantly reduced embryo adhesion, whereas the addition of progesterone could restore the adhesion rate. This model offers a simple and fast platform for evaluating and screening molecules involved in the adhesion process, such as cytokines, drugs, and transcription factors controlling implantation and endometrial receptivity.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141555772","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Chemotherapy-induced nausea and vomiting (CINV) refers to the nausea and vomiting experienced by patients after the application of chemotherapy drugs, significantly affecting their quality of life and physical recovery, as well as increasing the pain of the patients. Basic medicine primarily focuses on acid suppression, gastric protection, and vomiting suppression, but there are still many patients with nausea and vomiting symptoms that cannot be alleviated. Traditional Chinese medicine (TCM) can effectively alleviate nausea and vomiting through acupoint stimulation and pressure, while also offering advantages such as simplicity, affordability, and fewer side effects. The aim of this article is to introduce the method of using acupoint application combined with acupressure as an adjunctive therapy for CINV, using the Multinational Association of Supportive Care in Cancer (MASCC) Antiemesis Tool (MAT) tablet scale as a questionnaire. The article details aspects such as acupoint selection, production, and the use of acupoint application, massage techniques, and operating procedures, all with the goal of ensuring the safety and efficacy of acupoint application combined with acupressure as an adjuvant therapy, thereby improving patients' clinical symptoms and quality of life.
{"title":"Acupoint Application Combined with Acupressure as an Adjunctive Therapy for Chemotherapy-induced Nausea and Vomiting.","authors":"Qiuyu Chen, Ma HongMei, Liu Yi","doi":"10.3791/66865","DOIUrl":"https://doi.org/10.3791/66865","url":null,"abstract":"<p><p>Chemotherapy-induced nausea and vomiting (CINV) refers to the nausea and vomiting experienced by patients after the application of chemotherapy drugs, significantly affecting their quality of life and physical recovery, as well as increasing the pain of the patients. Basic medicine primarily focuses on acid suppression, gastric protection, and vomiting suppression, but there are still many patients with nausea and vomiting symptoms that cannot be alleviated. Traditional Chinese medicine (TCM) can effectively alleviate nausea and vomiting through acupoint stimulation and pressure, while also offering advantages such as simplicity, affordability, and fewer side effects. The aim of this article is to introduce the method of using acupoint application combined with acupressure as an adjunctive therapy for CINV, using the Multinational Association of Supportive Care in Cancer (MASCC) Antiemesis Tool (MAT) tablet scale as a questionnaire. The article details aspects such as acupoint selection, production, and the use of acupoint application, massage techniques, and operating procedures, all with the goal of ensuring the safety and efficacy of acupoint application combined with acupressure as an adjuvant therapy, thereby improving patients' clinical symptoms and quality of life.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141555768","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Filip Klimeš, Andreas Voskrebenzev, Frank Wacker, Jens Vogel-Claussen
Pulmonary magnetic resonance imaging (MRI) offers a variety of radiation-free techniques tailored to assess regional lung ventilation or its surrogates. These techniques encompass direct measurements, exemplified by hyperpolarized gas MRI and fluorinated gas MRI, as well as indirect measurements facilitated by oxygen-enhanced MRI and proton-based Fourier decomposition (FD) MRI. In recent times, there has been substantial progress in the field of FD MRI, which involved improving spatial/temporal resolution, refining sequence design and postprocessing, and developing a comprehensive whole-lung approach. The two-dimensional (2D) phase-resolved functional lung (PREFUL) MRI stands out as an FD-based approach developed for the comprehensive assessment of regional ventilation and perfusion dynamics, all within a single MR acquisition. Recently, a new advancement has been made with the development of 3D PREFUL to assess dynamic ventilation of the entire lung using 8 min exam with a self-gated sequence. The 3D PREFUL acquisition involves employing a stack-of-stars spoiled-gradient-echo sequence with a golden angle increment. Following the compressed sensing image reconstruction of approximately 40 breathing phases, all the reconstructed respiratory-resolved images undergo registration onto a fixed breathing phase. Subsequently, the ventilation parameters are extracted from the registered images. In a study cohort comprising healthy volunteers and patients with chronic obstructive pulmonary disease, the 3D PREFUL ventilation parameters demonstrated strong correlations with measurements obtained from pulmonary function tests. Additionally, the interscan repeatability of the 3D PREFUL technique was deemed to be acceptable, indicating its reliability for repeated assessments of the same individuals. In summary, 3D PREFUL ventilation MRI provides a whole lung coverage and captures ventilation dynamics with enhanced spatial resolution compared to 2D PREFUL. 3D PREFUL technique offers a cost-effective alternative to hyperpolarized 129Xe MRI, making it an attractive option for patient-friendly evaluation of pulmonary ventilation.
{"title":"Three-Dimensional Phase Resolved Functional Lung Magnetic Resonance Imaging.","authors":"Filip Klimeš, Andreas Voskrebenzev, Frank Wacker, Jens Vogel-Claussen","doi":"10.3791/66385","DOIUrl":"https://doi.org/10.3791/66385","url":null,"abstract":"<p><p>Pulmonary magnetic resonance imaging (MRI) offers a variety of radiation-free techniques tailored to assess regional lung ventilation or its surrogates. These techniques encompass direct measurements, exemplified by hyperpolarized gas MRI and fluorinated gas MRI, as well as indirect measurements facilitated by oxygen-enhanced MRI and proton-based Fourier decomposition (FD) MRI. In recent times, there has been substantial progress in the field of FD MRI, which involved improving spatial/temporal resolution, refining sequence design and postprocessing, and developing a comprehensive whole-lung approach. The two-dimensional (2D) phase-resolved functional lung (PREFUL) MRI stands out as an FD-based approach developed for the comprehensive assessment of regional ventilation and perfusion dynamics, all within a single MR acquisition. Recently, a new advancement has been made with the development of 3D PREFUL to assess dynamic ventilation of the entire lung using 8 min exam with a self-gated sequence. The 3D PREFUL acquisition involves employing a stack-of-stars spoiled-gradient-echo sequence with a golden angle increment. Following the compressed sensing image reconstruction of approximately 40 breathing phases, all the reconstructed respiratory-resolved images undergo registration onto a fixed breathing phase. Subsequently, the ventilation parameters are extracted from the registered images. In a study cohort comprising healthy volunteers and patients with chronic obstructive pulmonary disease, the 3D PREFUL ventilation parameters demonstrated strong correlations with measurements obtained from pulmonary function tests. Additionally, the interscan repeatability of the 3D PREFUL technique was deemed to be acceptable, indicating its reliability for repeated assessments of the same individuals. In summary, 3D PREFUL ventilation MRI provides a whole lung coverage and captures ventilation dynamics with enhanced spatial resolution compared to 2D PREFUL. 3D PREFUL technique offers a cost-effective alternative to hyperpolarized <sup>129</sup>Xe MRI, making it an attractive option for patient-friendly evaluation of pulmonary ventilation.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141555778","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Elizabeth Abraham, Mikel Zubillaga, Thomas Roule, Eleonora Stronati, Naiara Akizu, Conchi Estaras
Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, a robust pipeline was designed to perform single-cell and nuclei analysis on mouse embryos from embryonic day E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to wild-type perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, a methodology is presented designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with a same-day genotyping protocol (3 h) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. This method further includes guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate that this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula.
{"title":"Single-Cell RNA Sequencing of Mutant Whole Mouse Embryos: From the Epiblast to the End of Gastrulation.","authors":"Elizabeth Abraham, Mikel Zubillaga, Thomas Roule, Eleonora Stronati, Naiara Akizu, Conchi Estaras","doi":"10.3791/66866","DOIUrl":"10.3791/66866","url":null,"abstract":"<p><p>Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, a robust pipeline was designed to perform single-cell and nuclei analysis on mouse embryos from embryonic day E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to wild-type perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, a methodology is presented designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with a same-day genotyping protocol (3 h) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. This method further includes guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate that this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141471786","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Wanjing Cen, Siegmar Reinert, Meltem Avci-Adali, Dorothea Alexander, Felix Umrath
This corrects the article 10.3791/64016.
本文对文章 10.3791/64016 进行了更正。
{"title":"Erratum: A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro.","authors":"Wanjing Cen, Siegmar Reinert, Meltem Avci-Adali, Dorothea Alexander, Felix Umrath","doi":"10.3791/6601","DOIUrl":"10.3791/6601","url":null,"abstract":"<p><p>This corrects the article 10.3791/64016.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-06-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141301898","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Erratum: Multi-Gene Single Nucleotide Polymorphism Detection in Gastric Cancer Based on Ion Semiconductor Sequencing Platform.","authors":"Kaihua Huang, Xiao Yan, Zhicheng Huang, Huayuan Liang, Zhiwei Li, Minghao Wang, Yong Wan, Suihai Wang, Liying Zhao","doi":"10.3791/6602","DOIUrl":"10.3791/6602","url":null,"abstract":"<p><p>This corrects the article 10.3791/66058.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141248835","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The middle cerebral artery occlusion reperfusion (MCAO/R) model is crucial for understanding the pathological mechanisms of stroke and for drug development.However, among the commonly used modeling methods, the Koizumi method often faces scrutiny due to its ligation of the common carotid artery (CCA) and its inability to achieve adequate reperfusion. Similarly, the Longa method has been criticized for disconnecting and ligating the external carotid artery (ECA). This study aims to introduce a modified model preparation method that preserves the integrity of the ECA, involves inserting a monofilament nylon suture through the CCA, repairing the ligated CCA incision, and maintaining reperfusion from the CCA. Reperfusion of blood flow was confirmed using laser speckle flow imaging. Evaluation methods such as the Longa scale, Modified Neurological Severity Score, triphenyltetrazolium chloride (TTC) staining, and immunofluorescence labeling of neurons demonstrated that this approach could induce stable ischemic nerve damage. This modified MCAO/R model protocol is simple and stable, providing valuable guidance for practitioners in the field of cerebral ischemia.
大脑中动脉闭塞再灌注(MCAO/R)模型对于了解中风的病理机制和药物开发至关重要。然而,在常用的建模方法中,小泉法因结扎颈总动脉(CCA)和无法实现充分再灌注而经常受到质疑。同样,Longa 方法也因断开和结扎颈外动脉(ECA)而受到批评。本研究旨在介绍一种改良的模型制备方法,该方法保留了 ECA 的完整性,包括通过 CCA 插入单丝尼龙缝线,修复结扎的 CCA 切口,并保持 CCA 的再灌注。通过激光斑点血流成像确认血流再灌注。Longa 量表、改良神经严重程度评分、三苯基氯化四氮唑(TTC)染色和神经元免疫荧光标记等评估方法表明,这种方法可诱导稳定的缺血性神经损伤。这种改良的 MCAO/R 模型方案简单而稳定,为脑缺血领域的从业人员提供了宝贵的指导。
{"title":"A Modified Model Preparation for Middle Cerebral Artery Occlusion Reperfusion.","authors":"Rong Ma, Hongyan Yang, Jianlong Liang, Danni Lu, Qian Xie, Xuxin Zeng, Jialiang Guo","doi":"10.3791/67060","DOIUrl":"https://doi.org/10.3791/67060","url":null,"abstract":"<p><p>The middle cerebral artery occlusion reperfusion (MCAO/R) model is crucial for understanding the pathological mechanisms of stroke and for drug development.However, among the commonly used modeling methods, the Koizumi method often faces scrutiny due to its ligation of the common carotid artery (CCA) and its inability to achieve adequate reperfusion. Similarly, the Longa method has been criticized for disconnecting and ligating the external carotid artery (ECA). This study aims to introduce a modified model preparation method that preserves the integrity of the ECA, involves inserting a monofilament nylon suture through the CCA, repairing the ligated CCA incision, and maintaining reperfusion from the CCA. Reperfusion of blood flow was confirmed using laser speckle flow imaging. Evaluation methods such as the Longa scale, Modified Neurological Severity Score, triphenyltetrazolium chloride (TTC) staining, and immunofluorescence labeling of neurons demonstrated that this approach could induce stable ischemic nerve damage. This modified MCAO/R model protocol is simple and stable, providing valuable guidance for practitioners in the field of cerebral ischemia.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141332253","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
He Chang, Zhi Chen, Li Gao, Hong Cao, Yongqiang Wang, Shijun J Zheng
Pyroptosis is an inflammatory type of programmed cell death predominantly driven by the formation of plasma membrane pores by the N-terminus generated from the cleaved Gasdermin (GSDM) family proteins. Examination of membrane-attached GSDM-NT by Western Blot is the most commonly used method for evaluating pyroptosis. However, it is difficult to differentiate cells with pyroptosis from other forms of cell death using this method. In this study, Infectious Bursal Disease Virus (IBDV)-infected DF-1 cells were employed as a model to quantify the proportion of cells undergoing pyroptosis by flow cytometry, utilizing specific antibodies against the N-terminal fragment of chicken GSDME (chGSDME-NT) and propidium iodide (PI) staining. The chGSDME-NT-positive cells were readily detectable by flow cytometry using Alexa Fluor 647-labeled anti-chGSDME-NT antibodies. Moreover, the proportion of chGSDME-NT/PI double-positive cells in IBDV-infected cells (around 33%) was significantly greater than in mock-infected controls (P < 0.001). These findings indicate that examination of membrane-bound chGSDME-NT by flow cytometry is an effective approach for determining pyroptotic cells among cells undergoing cell death.
{"title":"Examination of Pyroptosis by Flow Cytometry.","authors":"He Chang, Zhi Chen, Li Gao, Hong Cao, Yongqiang Wang, Shijun J Zheng","doi":"10.3791/66912","DOIUrl":"https://doi.org/10.3791/66912","url":null,"abstract":"<p><p>Pyroptosis is an inflammatory type of programmed cell death predominantly driven by the formation of plasma membrane pores by the N-terminus generated from the cleaved Gasdermin (GSDM) family proteins. Examination of membrane-attached GSDM-<sup>NT</sup> by Western Blot is the most commonly used method for evaluating pyroptosis. However, it is difficult to differentiate cells with pyroptosis from other forms of cell death using this method. In this study, Infectious Bursal Disease Virus (IBDV)-infected DF-1 cells were employed as a model to quantify the proportion of cells undergoing pyroptosis by flow cytometry, utilizing specific antibodies against the N-terminal fragment of chicken GSDME (chGSDME-<sup>NT</sup>) and propidium iodide (PI) staining. The chGSDME-<sup>NT</sup>-positive cells were readily detectable by flow cytometry using Alexa Fluor 647-labeled anti-chGSDME-<sup>NT</sup> antibodies. Moreover, the proportion of chGSDME-<sup>NT</sup>/PI double-positive cells in IBDV-infected cells (around 33%) was significantly greater than in mock-infected controls (P < 0.001). These findings indicate that examination of membrane-bound chGSDME-<sup>NT</sup> by flow cytometry is an effective approach for determining pyroptotic cells among cells undergoing cell death.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141332299","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Laura C Brown, Jay Chopra, Rachel E Horness, Julia C van Kessel
Bacteria detect local population numbers using quorum sensing, a method of cell-cell communication broadly utilized to control bacterial behaviors. In Vibrio species, the master quorum sensing regulators LuxR/HapR control hundreds of quorum sensing genes, many of which influence virulence, metabolism, motility, and more. Thiophenesulfonamides are potent inhibitors of LuxR/HapR that bind the ligand pocket in these transcription factors and block downstream quorum sensing gene expression. This class of compounds served as the basis for the development of a set of simple, robust, and educational procedures for college students to assimilate their chemistry and biology skills using a CURE model: course-based undergraduate research experience. Optimized protocols are described that comprise three learning stages in an iterative and multi-disciplinary platform to engage students in a year-long CURE: (1) design and synthesize new small molecule inhibitors based on the thiophenesulfonamide core, (2) use structural modeling to predict binding affinity to the target, and (3) assay the compounds for efficacy in microbiological assays against specific Vibrio LuxR/HapR proteins. The described reporter assay performed in E. coli successfully predicts the efficacy of the compounds against target proteins in the native Vibrio species.
{"title":"Synthesis and Assay of Vibrio Quorum Sensing Inhibitors.","authors":"Laura C Brown, Jay Chopra, Rachel E Horness, Julia C van Kessel","doi":"10.3791/66582","DOIUrl":"https://doi.org/10.3791/66582","url":null,"abstract":"<p><p>Bacteria detect local population numbers using quorum sensing, a method of cell-cell communication broadly utilized to control bacterial behaviors. In Vibrio species, the master quorum sensing regulators LuxR/HapR control hundreds of quorum sensing genes, many of which influence virulence, metabolism, motility, and more. Thiophenesulfonamides are potent inhibitors of LuxR/HapR that bind the ligand pocket in these transcription factors and block downstream quorum sensing gene expression. This class of compounds served as the basis for the development of a set of simple, robust, and educational procedures for college students to assimilate their chemistry and biology skills using a CURE model: course-based undergraduate research experience. Optimized protocols are described that comprise three learning stages in an iterative and multi-disciplinary platform to engage students in a year-long CURE: (1) design and synthesize new small molecule inhibitors based on the thiophenesulfonamide core, (2) use structural modeling to predict binding affinity to the target, and (3) assay the compounds for efficacy in microbiological assays against specific Vibrio LuxR/HapR proteins. The described reporter assay performed in E. coli successfully predicts the efficacy of the compounds against target proteins in the native Vibrio species.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141332309","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from appendicular bones, BMMSCs derived from the jaw have greater proliferative and osteogenic differentiation ability, gradually becoming important seed cells for jaw defect repair. However, the mandible has a complex bony structure and less cancellous content than appendicular bones. It is difficult to acquire a large number of high-quality jaw-derived marrow mesenchymal stem cells using traditional methods. This study presents a 'niche-based approach on stemness' for isolating and culturing rat jaw bone marrow mesenchymal stem cells (JBMMSCs). Primary rat JBMMSCs were isolated and cultured using the whole bone marrow adherent method combined with the bone slice digestion method. The isolated cells were identified as JBMMSCs through cell morphology observation, detection of cell surface markers, and multi-directional differentiation induction. The cells extracted by this method exhibit a 'fibroblast-like' spindle shape. The cells are long, spindle-shaped and fibroblast-like. The flow cytometry analysis shows these cells are positive for CD29, CD44, and CD90 but negative for CD11b/c, CD34, and CD45, which is congruent with BMMSCs characteristics. The cells show strong proliferation capacity and can undergo osteogenic, adipogenic, and chondrogenic differentiation. This study provides an effective and stable method for obtaining enough high-quality JBMMSCs with strong differentiation ability in a short time, which could facilitate further studies of the exploration of biological function, regenerative medicine, and related clinical applications.
{"title":"Technique for Isolation and Culture of Rat Jaw Bone Marrow Mesenchymal Stem Cells.","authors":"Tianqi Li, Xiangbo Meng, Shuaichen Li, Sunxin Zhou, Hongkun Li, Shi Quan, Tong Zhang","doi":"10.3791/66765","DOIUrl":"https://doi.org/10.3791/66765","url":null,"abstract":"<p><p>Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from appendicular bones, BMMSCs derived from the jaw have greater proliferative and osteogenic differentiation ability, gradually becoming important seed cells for jaw defect repair. However, the mandible has a complex bony structure and less cancellous content than appendicular bones. It is difficult to acquire a large number of high-quality jaw-derived marrow mesenchymal stem cells using traditional methods. This study presents a 'niche-based approach on stemness' for isolating and culturing rat jaw bone marrow mesenchymal stem cells (JBMMSCs). Primary rat JBMMSCs were isolated and cultured using the whole bone marrow adherent method combined with the bone slice digestion method. The isolated cells were identified as JBMMSCs through cell morphology observation, detection of cell surface markers, and multi-directional differentiation induction. The cells extracted by this method exhibit a 'fibroblast-like' spindle shape. The cells are long, spindle-shaped and fibroblast-like. The flow cytometry analysis shows these cells are positive for CD29, CD44, and CD90 but negative for CD11b/c, CD34, and CD45, which is congruent with BMMSCs characteristics. The cells show strong proliferation capacity and can undergo osteogenic, adipogenic, and chondrogenic differentiation. This study provides an effective and stable method for obtaining enough high-quality JBMMSCs with strong differentiation ability in a short time, which could facilitate further studies of the exploration of biological function, regenerative medicine, and related clinical applications.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141332325","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}