Bio-protocol最新文献_第10页

easyPACId, a Simple Method for Induced Production, Isolation, Identification, and Testing of Natural Products from Proteobacteria. easyacid:一种诱导生产、分离、鉴定和检测变形杆菌天然产物的简单方法。

Bio-protocol Pub Date : 2023-07-05 DOI: 10.21769/BioProtoc.4709

Edna Bode, Daniela Assmann, Petra Happel, Elmar Meyer, Karin Münch, Nicole Rössel, Helge B Bode

{"title":"easyPACId, a Simple Method for Induced Production, Isolation, Identification, and Testing of Natural Products from Proteobacteria.","authors":"Edna Bode, Daniela Assmann, Petra Happel, Elmar Meyer, Karin Münch, Nicole Rössel, Helge B Bode","doi":"10.21769/BioProtoc.4709","DOIUrl":"https://doi.org/10.21769/BioProtoc.4709","url":null,"abstract":"The easyPACId (easy Promoter Activation and Compound Identification) approach is focused on the targeted activation of natural product biosynthetic gene clusters (BGCs) encoding non-ribosomal peptide synthetases (NRPS), polyketide synthases (PKS), NRPS-PKS hybrids, or other BGC classes. It was applied to entomopathogenic bacteria of the genera Xenorhabdus and Photorhabdus by exchanging the natural promoter of desired BGCs against the L-arabinose inducible PBAD promoter in ∆hfq mutants of the respective strains. The crude (culture) extracts of the cultivated easyPACId mutants are enriched with the single compound or compound class and can be tested directly against various target organisms without further purification of the produced natural products. Furthermore, isolation and identification of compounds from these mutants is simplified due to the reduced background in the ∆hfq strains. The approach avoids problems often encountered in heterologous expression hosts, chemical synthesis, or tedious extraction of desired compounds from wild-type crude extracts. This protocol describes easyPACId for Xenorhabdus and Photorhabdus, but it was also successfully adapted to Pseudomonas entomophila and might be suitable for other proteobacteria that carry hfq.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 13","pages":"e4709"},"PeriodicalIF":0.0,"publicationDate":"2023-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/36/51/BioProtoc-13-13-4709.PMC10336570.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9823769","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}

引用次数: 0

Primary Mouse Invariant Natural Killer T (iNKT) Cell Purification and Transduction. 原代小鼠不变性自然杀伤T (iNKT)细胞的纯化和转导。

Bio-protocol Pub Date : 2023-07-05 DOI: 10.21769/BioProtoc.4707

Gloria Delfanti, Paolo Dellabona, Giulia Casorati

{"title":"Primary Mouse Invariant Natural Killer T (iNKT) Cell Purification and Transduction.","authors":"Gloria Delfanti, Paolo Dellabona, Giulia Casorati","doi":"10.21769/BioProtoc.4707","DOIUrl":"https://doi.org/10.21769/BioProtoc.4707","url":null,"abstract":"Invariant natural killer T (iNKT) cells are a non-conventional T-cell population expressing a conserved semi-invariant T-cell receptor (TCR) that reacts to lipid antigens, such as α-galactosyl ceramide (α-GalCer), presented by the monomorphic molecule CD1d. iNKT cells play a central role in tumor immunosurveillance and represent a powerful tool for anti-cancer treatment, notably because they can be efficiently redirected against hematological or solid malignancies by engineering with tumor-specific chimeric antigen receptors (CARs) or TCRs. However, iNKT cells are rare and require specific ex vivo pre-selection and substantial in vitro expansion to be exploited for adoptive cell therapy (ACT). This protocol describes a robust method to obtain a large number of mouse iNKT cells that can be effectually engineered by retroviral (RV) transduction. A major advantage of this protocol is that it requires neither particular instrumentation nor a high number of mice. iNKT cells are enriched from the spleens of iVα14-Jα18 transgenic mice; the rapid purification protocol yields a highly enriched iNKT cell population that is activated by anti-CD3/CD28 beads, which is more reproducible and less time consuming than using bone marrow-derived dendritic cells loaded with α-GalCer, without risks of expanding contaminant T cells. Forty-eight hours after activation, iNKT cells are transduced with the selected RV by spin inoculation. This protocol allows to obtain, in 15 days, millions of ready-to-use, highly pure, and stably transduced iNKT cells that might be exploited for in vitro assays and ACT experiments in preclinical studies.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 13","pages":"e4707"},"PeriodicalIF":0.0,"publicationDate":"2023-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/66/42/BioProtoc-13-13-4707.PMC10336563.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9826326","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}

引用次数: 0

mRNA Delivery Platform Based on Bacterial Outer Membrane Vesicles for Tumor Vaccine. 基于细菌外膜囊泡的肿瘤疫苗 mRNA 运送平台。

Bio-protocol Pub Date : 2023-07-05 DOI: 10.21769/BioProtoc.4774

Xiaoyu Gao, Yao Li, Guangjun Nie, Xiao Zhao

引用次数: 0

Human-rabbit Hybrid Translation System to Explore the Function of Modified Ribosomes. 探索修饰核糖体功能的人兔杂交翻译系统。

Bio-protocol Pub Date : 2023-07-05 DOI: 10.21769/BioProtoc.4714

Eriko Matsuura-Suzuki, Hirotaka Toh, Shintaro Iwasaki

引用次数: 0

In vitro Selection and in vivo Testing of Riboswitch-inspired Aptamers. 核糖体开关诱导核酸适体的体外选择和体内检测。

Bio-protocol Pub Date : 2023-07-05 DOI: 10.21769/BioProtoc.4775

Michael G Mohsen, Ronald R Breaker

{"title":"In vitro Selection and in vivo Testing of Riboswitch-inspired Aptamers.","authors":"Michael G Mohsen, Ronald R Breaker","doi":"10.21769/BioProtoc.4775","DOIUrl":"https://doi.org/10.21769/BioProtoc.4775","url":null,"abstract":"Engineered aptamers for new compounds are typically produced by using in vitro selection methods. However, aptamers that are developed in vitro might not function as expected when introduced into complex cellular environments. One approach that addresses this concern is the design of initial RNA pools for selection that contain structural scaffolds from naturally occurring riboswitch aptamers. Here, we provide guidance on design and experimental principles for developing riboswitch-inspired aptamers for new ligands. The in vitro selection protocol (based on Capture-SELEX) is generalizable to diverse RNA scaffold types and amenable to multiplexing of ligand candidates. We discuss strategies to avoid propagation of selfish sequences that can easily dominate the selection. We also detail the identification of aptamer candidates using next-generation sequencing and bioinformatics, and subsequent biochemical validation of aptamer candidates. Finally, we describe functional testing of aptamer candidates in bacterial cell culture. Key features Develop riboswitch-inspired aptamers for new ligands using in vitro selection. Ligand candidates can be multiplexed to conserve time and resources. Test aptamer candidates in bacterial cells by grafting the aptamer back onto its expression platform.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 13","pages":"e4775"},"PeriodicalIF":0.0,"publicationDate":"2023-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10338711/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9825898","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}

引用次数: 0

3D Ultrastructural Visualization of Mitosis Fidelity in Human Cells Using Serial Block Face Scanning Electron Microscopy (SBF-SEM). 利用连续块面扫描电子显微镜(SBF-SEM)实现人类细胞有丝分裂保真度的三维超微结构可视化。

Bio-protocol Pub Date : 2023-07-05 DOI: 10.21769/BioProtoc.4708

Nuria Ferrandiz, Stephen J Royle

引用次数: 0

In vivo Electroporation of Skeletal Muscle Fibers in Mice. 小鼠骨骼肌纤维的体内电穿孔。

Bio-protocol Pub Date : 2023-07-05 DOI: 10.21769/BioProtoc.4759

Steven J Foltz, Criss H Hartzell, Hyojung J Choo

{"title":"In vivo Electroporation of Skeletal Muscle Fibers in Mice.","authors":"Steven J Foltz, Criss H Hartzell, Hyojung J Choo","doi":"10.21769/BioProtoc.4759","DOIUrl":"10.21769/BioProtoc.4759","url":null,"abstract":"In vitro models are essential for investigating the molecular, biochemical, and cell-biological aspects of skeletal muscle. Still, models that utilize cell lines or embryonic cells do not fully recapitulate mature muscle fibers in vivo. Protein function is best studied in mature differentiated tissue, where biological context is maintained, but this is often difficult when reliable detection reagents, such as antibodies, are not commercially available. Exogenous expression of tagged proteins in vivo solves some of these problems, but this approach can be technically challenging because either a mouse must be engineered for each protein of interest or viral vectors are required for adequate levels of expression. While viral vectors can infect target cells following local administration, they carry the risk of genome integration that may interfere with downstream analyses. Plasmids are another accessible expression system, but they require ancillary means of cell penetration; electroporation is a simple physical method for this purpose that requires minimal training or specialized equipment. Here, we describe a method for in vivo plasmid expression in a foot muscle following electroporation.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 13","pages":"e4759"},"PeriodicalIF":0.0,"publicationDate":"2023-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/d9/c4/BioProtoc-13-13-4759.PMC10339231.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10183526","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}

引用次数: 0

Measuring Myeloperoxidase Activity as a Marker of Inflammation in Gut Tissue Samples of Mice and Rat. 测定小鼠和大鼠肠道组织样本中髓过氧化物酶活性作为炎症标志物。

Bio-protocol Pub Date : 2023-07-05 DOI: 10.21769/BioProtoc.4758

Nikita Hanning, Joris G De Man, Benedicte Y De Winter

{"title":"Measuring Myeloperoxidase Activity as a Marker of Inflammation in Gut Tissue Samples of Mice and Rat.","authors":"Nikita Hanning, Joris G De Man, Benedicte Y De Winter","doi":"10.21769/BioProtoc.4758","DOIUrl":"https://doi.org/10.21769/BioProtoc.4758","url":null,"abstract":"Myeloperoxidase (MPO) is an enzyme contained in lysosomal azurophilic granules of neutrophils. MPO activity has been shown to correlate with the number of neutrophils in histological sections of the gastrointestinal tract and is therefore accepted as a biomarker of neutrophil invasion in the gut. This protocol describes an easy, cost-effective kinetic colorimetric assay to quantify myeloperoxidase activity in intestinal tissue samples. It is explained using tissue collected in mice but can also be used for other laboratory animals. In a first step, tissue specimens are homogenized using a phosphate buffer containing 0.5% hexadecyltrimethylammonium bromide (HTAB), which extracts MPO from neutrophils. The obtained supernatant is added to a reagent solution containing o-dianisidine dihydrochloride, which is a peroxidase substrate. Finally, the change in absorption is measured via spectrophotometry and converted to a standardized unit of enzyme activity. The assay is illustrated and compared to a commercially available enzyme-linked immunoassay (ELISA), demonstrating that MPO activity does not necessarily correlate with MPO protein expression in tissue samples. Key features Optimized for use in mice and rats but can also be used for samples of other species. Measures enzymatic activity instead of mRNA or protein expression. Requires a spectrophotometer. Can be performed in duplo using 10 mg of (dry-blotted) gut tissue or more. Graphical overview.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 13","pages":"e4758"},"PeriodicalIF":0.0,"publicationDate":"2023-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/d4/f9/BioProtoc-13-13-4758.PMC10338346.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10183529","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}

引用次数: 0

Systematic Analysis of Smooth Muscle and Cartilage Ring Formation during Mouse Tracheal Tubulogenesis. 小鼠气管小管形成过程中平滑肌和软骨环形成的系统分析。

Bio-protocol Pub Date : 2023-07-05 DOI: 10.21769/BioProtoc.4711

Haoyu Wu, Ping Wang, Ziying Liu, Chunyan Lu, Wenguang Yin

{"title":"Systematic Analysis of Smooth Muscle and Cartilage Ring Formation during Mouse Tracheal Tubulogenesis.","authors":"Haoyu Wu, Ping Wang, Ziying Liu, Chunyan Lu, Wenguang Yin","doi":"10.21769/BioProtoc.4711","DOIUrl":"https://doi.org/10.21769/BioProtoc.4711","url":null,"abstract":"The trachea tube is the exclusive route to allow gas exchange between the external environment and the lungs. Recent studies have shown the critical role of mesenchymal cells in tracheal tubulogenesis. Improved methods for studying the dynamics of the tracheal mesenchyme development are needed to investigate the cellular and molecular mechanisms during tracheal tubulogenesis. Here, we describe a detailed protocol for a systematic analysis of tracheal tube development to enable observing tracheal smooth muscle (SM) and cartilage ring formation. We describe immunostaining, confocal and stereomicroscopy imaging, and quantitative methods to study the process of tracheal SM and cartilage ring development, including SM cell alignment, polarization, and changes in cell shape as well as mesenchymal condensation. The technologies and approaches described here not only improve analysis of the patterning of the developing trachea but also help uncover the mechanisms underlying airway disease. This protocol also provides a useful technique to analyze cell organization, polarity, and nuclear shape in other organ systems.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 13","pages":"e4711"},"PeriodicalIF":0.0,"publicationDate":"2023-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/92/e0/BioProtoc-13-13-4711.PMC10336568.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9823767","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}

引用次数: 0

Autolysin Production from Chlamydomonas reinhardtii. 莱茵衣藻产自溶素。

Bio-protocol Pub Date : 2023-07-05 DOI: 10.21769/BioProtoc.4705

Justin Findinier

{"title":"Autolysin Production from Chlamydomonas reinhardtii.","authors":"Justin Findinier","doi":"10.21769/BioProtoc.4705","DOIUrl":"https://doi.org/10.21769/BioProtoc.4705","url":null,"abstract":"Chlamydomonas reinhardtii is a model organism for various processes, from photosynthesis to cilia biogenesis, and a great chassis to learn more about biofuel production. This is due to the width of molecular tools available, which have recently expanded with the development of a modular cloning system but, most importantly, with CRISPR/Cas9 editing now being possible. This technique has proven to be more efficient in the absence of a cell wall by using specific mutants or by digesting Chlamydomonas cell wall using the mating-specific metalloprotease autolysin (also called gametolysin). Multiple protocols have been used and shared for autolysin production from Chlamydomonas cells; however, they provide very inconsistent results, which hinders the capacity to routinely perform CRISPR mutagenesis. Here, we propose a simple protocol for autolysin production requiring transfer of cells from plates into a dense liquid suspension, gametogenesis by overnight incubation before mixing of gametes, and enzyme harvesting after 2 h. This protocol has shown to be highly efficient for autolysin production regardless of precise control over cell density at any step. Requiring a minimal amount of labor, it will provide a simple, ready-to-go approach to produce an enzyme critical for the generation of targeted mutants.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 13","pages":"e4705"},"PeriodicalIF":0.0,"publicationDate":"2023-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/d6/ed/BioProtoc-13-13-4705.PMC10336564.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9823771","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}

引用次数: 0