Biology Methods and Protocols最新文献_第10页

Mapping adipocyte interactome networks by HaloTag-enrichment-mass spectrometry. 利用 HaloTag 富集质谱法绘制脂肪细胞相互作用组网络图。

IF 3.6

Biology Methods and Protocols Pub Date : 2024-05-29 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae039

Junshi Yazaki, Takashi Yamanashi, Shino Nemoto, Atsuo Kobayashi, Yong-Woon Han, Tomoko Hasegawa, Akira Iwase, Masaki Ishikawa, Ryo Konno, Koshi Imami, Yusuke Kawashima, Jun Seita

{"title":"Mapping adipocyte interactome networks by HaloTag-enrichment-mass spectrometry.","authors":"Junshi Yazaki, Takashi Yamanashi, Shino Nemoto, Atsuo Kobayashi, Yong-Woon Han, Tomoko Hasegawa, Akira Iwase, Masaki Ishikawa, Ryo Konno, Koshi Imami, Yusuke Kawashima, Jun Seita","doi":"10.1093/biomethods/bpae039","DOIUrl":"10.1093/biomethods/bpae039","url":null,"abstract":"Mapping protein interaction complexes in their natural state in vivo is arguably the Holy Grail of protein network analysis. Detection of protein interaction stoichiometry has been an important technical challenge, as few studies have focused on this. This may, however, be solved by artificial intelligence (AI) and proteomics. Here, we describe the development of HaloTag-based affinity purification mass spectrometry (HaloMS), a high-throughput HaloMS assay for protein interaction discovery. The approach enables the rapid capture of newly expressed proteins, eliminating tedious conventional one-by-one assays. As a proof-of-principle, we used HaloMS to evaluate the protein complex interactions of 17 regulatory proteins in human adipocytes. The adipocyte interactome network was validated using an in vitro pull-down assay and AI-based prediction tools. Applying HaloMS to probe adipocyte differentiation facilitated the identification of previously unknown transcription factor (TF)-protein complexes, revealing proteome-wide human adipocyte TF networks and shedding light on how different pathways are integrated.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"9 1","pages":"bpae039"},"PeriodicalIF":3.6,"publicationDate":"2024-05-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11180226/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141332028","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

Northern blotting of endogenous full-length human-specific LINE-1 RNA. 内源性全长人类特异性 LINE-1 RNA 的 Northern 印迹。

IF 2.5

Biology Methods and Protocols Pub Date : 2024-05-28 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae036

Maisa I Alkailani

引用次数: 0

An optimized ligation-mediated PCR method for chromosome walking and fusion gene chromosomal breakpoints identification. 用于染色体走行和融合基因染色体断点鉴定的优化连接介导 PCR 方法。

IF 3.6

Biology Methods and Protocols Pub Date : 2024-05-25 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae037

Jrhau Lung, Ming-Szu Hung, Chao-Yu Chen, Tsung-Ming Yang, Chin-Kuo Lin, Yu-Hung Fang, Yuan-Yuan Jiang, Hui-Fen Liao, Yu-Ching Lin

引用次数: 0

Macro-based collagen quantification and segmentation in picrosirius red-stained heart sections using light microscopy. 使用光学显微镜对皮色红染色的心脏切片进行基于宏观的胶原蛋白定量和分割。

IF 3.6

Biology Methods and Protocols Pub Date : 2024-04-27 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae027

Julian Kammerer, Alexandra Cirnu, Tatjana Williams, Melanie Hasselmeier, Mike Nörpel, Ruping Chen, Brenda Gerull

{"title":"Macro-based collagen quantification and segmentation in picrosirius red-stained heart sections using light microscopy.","authors":"Julian Kammerer, Alexandra Cirnu, Tatjana Williams, Melanie Hasselmeier, Mike Nörpel, Ruping Chen, Brenda Gerull","doi":"10.1093/biomethods/bpae027","DOIUrl":"10.1093/biomethods/bpae027","url":null,"abstract":"Picrosirius red staining constitutes an important and broadly used tool to visualize collagen and fibrosis in various tissues. Although multiple qualitative and quantitative analysis methods to evaluate fibrosis are available, many require specialized devices and software or lack objectivity and scalability. Here, we aimed to develop a versatile and powerful \"QuantSeg\" macro in the FIJI image processing software capable of automated, robust, and quick collagen quantification in cardiac tissue from light micrographs. To examine different patterns of fibrosis, an optional segmentation algorithm was implemented. To ensure the method's validity, we quantified the collagen content in a set of wild-type versus plakoglobin-knockout murine hearts exhibiting extensive fibrosis using both the macro and an established, fluorescence microscopy-based method, and compared results. To demonstrate the capabilities of the segmentation feature, rat hearts were examined post-myocardial infarction. We found the QuantSeg macro to robustly detect the differences in fibrosis between knockout and control hearts. In sections with low collagen content, the macro yielded more consistent results than using the fluorescence microscopy-based technique. With its wide range of output parameters, ease of use, cost effectiveness, and objectivity, the QuantSeg macro has the potential to become an established method for analysis of PSR-stained tissue. The novel segmentation feature allows for automated evaluation of different patterns of cardiac fibrosis for the first time.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"9 1","pages":"bpae027"},"PeriodicalIF":3.6,"publicationDate":"2024-04-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11116823/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141155716","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

Correction to: An improved method for measuring catalase activity in biological samples. 更正：测量生物样本中过氧化氢酶活性的改进方法。

IF 3.6

Biology Methods and Protocols Pub Date : 2024-04-26 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae025

引用次数: 0

Intra-arterial delivery of neurospheres into isolated perfused porcine colons: a proof of concept. 向离体灌注猪结肠动脉内输送神经球：概念验证。

IF 3.6

Biology Methods and Protocols Pub Date : 2024-04-02 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae022

Richard D Martel, Nicolas A Hoyos, María Ángeles Tapia-Laliena, Irmgard Herrmann, Martin Herrmann, Rasul Khasanov, Karl-Herbert Schäfer

{"title":"Intra-arterial delivery of neurospheres into isolated perfused porcine colons: a proof of concept.","authors":"Richard D Martel, Nicolas A Hoyos, María Ángeles Tapia-Laliena, Irmgard Herrmann, Martin Herrmann, Rasul Khasanov, Karl-Herbert Schäfer","doi":"10.1093/biomethods/bpae022","DOIUrl":"https://doi.org/10.1093/biomethods/bpae022","url":null,"abstract":"Cell replacement in aganglionic intestines is a promising, yet merely experimental tool for the therapy of congenital dysganglionosis of the enteric nervous system like Hirschsprung disease. While the injection of single cells or neurospheres to a defined and very restricted location is trivial, the translation to the clinical application, where large aganglionic or hypoganglionic areas need to be colonized (hundreds of square centimetres), afford a homogeneous distribution of multiple neurospheres all over the affected tissue areas. Reaching the entire aganglionic area in vivo is critical for the restoration of peristaltic function. The latter mainly depends on an intact nervous system that extends throughout the organ. Intra-arterial injection is a common method in cell therapy and may be the key to delivering cells or neurospheres into the capillary bed of the colon with area-wide distribution. We describe an experimental method for monitoring the distribution of a defined number of neurospheres into porcine recta ex vivo, immediately after intra-arterial injection. We designed this method to localize grafting sites of single neurospheres in precise biopsies which can further be examined in explant cultures. The isolated perfused porcine rectum allowed us to continuously monitor the perfusion pressure. A blockage of too many capillaries would lead to an ischaemic situation and an increase of perfusion pressure. Since we could demonstrate that the area-wide delivery of neurospheres did not alter the overall vascular resistance, we showed that the delivery does not significantly impair the local circulation.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"9 1","pages":"bpae022"},"PeriodicalIF":3.6,"publicationDate":"2024-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11018533/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140862191","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

An optimized protocol for generating appendage-bearing skin organoids from human-induced pluripotent stem cells. 利用人体诱导多能干细胞生成附肢皮肤器官组织的优化方案。

IF 3.6

Biology Methods and Protocols Pub Date : 2024-03-22 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae019

Imaan Ahmed, Jane Sun, Jason Brown, Kiarash Khosrotehrani, Abbas Shafiee

引用次数: 0

Alternative community-led intervention to improve uptake of cataract surgery services in rural Tanzania-The Dodoma Community Cataract Acceptance Trial (DoCCAT): a protocol for intervention co-designing and implementation in a cluster-randomized controlled trial. 提高坦桑尼亚农村地区白内障手术服务接受率的替代性社区主导干预措施--多多马社区白内障接受试验（DoCCAT）：群组随机对照试验中干预措施的共同设计和实施方案。

IF 3.6

Biology Methods and Protocols Pub Date : 2024-03-08 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae016

Frank Sandi, Gareth Mercer, Robert Geneau, Kenneth Bassett, Deogratius Bintabara, Albino Kalolo

{"title":"Alternative community-led intervention to improve uptake of cataract surgery services in rural Tanzania-The Dodoma Community Cataract Acceptance Trial (DoCCAT): a protocol for intervention co-designing and implementation in a cluster-randomized controlled trial.","authors":"Frank Sandi, Gareth Mercer, Robert Geneau, Kenneth Bassett, Deogratius Bintabara, Albino Kalolo","doi":"10.1093/biomethods/bpae016","DOIUrl":"https://doi.org/10.1093/biomethods/bpae016","url":null,"abstract":"Age-related lens opacification (cataract) remains the leading cause of visual impairment and blindness worldwide. In low- and middle-income countries, utilization of cataract surgical services is often limited despite community-based outreach programmes. Community-led research, whereby researchers and community members collaboratively co-design intervention is an approach that ensures the interventions are locally relevant and that their implementation is feasible and socially accepted in the targeted contexts. Community-led interventions have the potential to increase cataract surgery uptake if done appropriately. In this study, once the intervention is co-designed it will be implemented through a cluster-randomized controlled trial (cRCT) with ward as a unit of randomization. This study will utilise both the qualitative methods for co-designing the intervention and the quantitative methods for effective assessment of the developed community-led intervention through a cRCT in 80 rural wards of Dodoma region, Tanzania (40 Intervention). The 'intervention package' will be developed through participatory community meetings and ongoing evaluation and modification of the intervention based on its impact on service utilization. Leask's four stages of intervention co-creation will guide the development within Rifkin's CHOICE framework. The primary outcomes are two: the number of patients attending eye disease screening camps, and the number of patients accepting cataract surgery. NVivo version 12 will be used for qualitative data analysis and Stata version 12 for quantitative data. Independent and paired t-tests will be performed to make comparisons between and within groups. P-values less than 0.05 will be considered statistically significant.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"9 1","pages":"bpae016"},"PeriodicalIF":3.6,"publicationDate":"2024-03-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10987207/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140865230","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

A rapid and accurate method for evaluating the degradation of pan-Akt in cells by PROTACs using NanoLuc luciferase. 利用 NanoLuc 荧光素酶快速准确地评估 PROTACs 在细胞中降解 pan-Akt 的方法。

IF 3.6

Biology Methods and Protocols Pub Date : 2024-03-05 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae014

Xiaojun Ji, Lei Miao, Yebin Wu, Tingli Zhao, Yaxuan Si, Xiaoyun Tan, Qiuhua Zhou, Rui Zuo, Junjie Pei, Jian Wu, Changyou Ma, Zhongjun Ma, Dan Xu

{"title":"A rapid and accurate method for evaluating the degradation of pan-Akt in cells by PROTACs using NanoLuc luciferase.","authors":"Xiaojun Ji, Lei Miao, Yebin Wu, Tingli Zhao, Yaxuan Si, Xiaoyun Tan, Qiuhua Zhou, Rui Zuo, Junjie Pei, Jian Wu, Changyou Ma, Zhongjun Ma, Dan Xu","doi":"10.1093/biomethods/bpae014","DOIUrl":"10.1093/biomethods/bpae014","url":null,"abstract":"Proteolysis targeting chimera (PROTAC) is a protein degradation technique that has been increasingly used in the development of new drugs in recent years. Akt is a classical serine/threonine kinase, and its role outside of the kinase has gradually gained attention in recent years, making it one of the proteins targeted by PROTACs. Currently, there are many methods used for the evaluation of intracellular protein degradation, but each has its own advantages or disadvantages. This study aimed to investigate the feasibility of evaluating the degradation of pan-Akt proteins in cells by PROTACs (MS21 and MS170) using the NanoLuc luciferase method. After conducting a thorough comparison between this method and the classical western blot assay in various cells, as well as testing the stability of the experiments between multiple batches, we found that NanoLuc luciferase is a highly accurate, stable, low-cost and easy-to-operate method for the evaluation of intracellular pan-Akt degradation by PROTACs with a short cycle time and high cellular expandability. Given the numerous advantages of this method, it is hypothesized that it could be extended to evaluate the degradation of more target proteins of PROTACs. In summary, the NanoLuc luciferase is a suitable method for early protein degradation screening of PROTAC compounds.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"9 1","pages":"bpae014"},"PeriodicalIF":3.6,"publicationDate":"2024-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10965420/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140307242","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

An improved method for measuring catalase activity in biological samples. 测量生物样本中过氧化氢酶活性的改进方法。

IF 3.6

Biology Methods and Protocols Pub Date : 2024-03-05 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae015

Mahmoud Hussein Hadwan, Marwah Jaber Hussein, Rawa M Mohammed, Asad M Hadwan, Hawraa Saad Al-Kawaz, Saba S M Al-Obaidy, Zainab Abbas Al Talebi

{"title":"An improved method for measuring catalase activity in biological samples.","authors":"Mahmoud Hussein Hadwan, Marwah Jaber Hussein, Rawa M Mohammed, Asad M Hadwan, Hawraa Saad Al-Kawaz, Saba S M Al-Obaidy, Zainab Abbas Al Talebi","doi":"10.1093/biomethods/bpae015","DOIUrl":"10.1093/biomethods/bpae015","url":null,"abstract":"Catalase (CAT) is an important enzyme that protects biomolecules against oxidative damage by breaking down hydrogen peroxide (H2O2) into water and oxygen. CAT is present in all aerobic microbes, animals, and plants. It is, however, absent from normal human urine but can be detected in pathological urine. CAT testing can thus help to detect such urine. This study presents a novel spectrophotometric method for determining CAT activity characterized by its simplicity, sensitivity, specificity, and rapidity. The method involves incubating enzyme-containing samples with a carefully chosen concentration of H2O2 for a specified incubation period. Subsequently, a solution containing ferrous ammonium sulfate (FAS) and sulfosalicylic acid (SSA) is added to terminate the enzyme activity. A distinctive maroon-colored ferrisulfosalicylate complex is formed. The formation of this complex is a direct result of the reaction between FAS and any residual peroxide present. This leads to the generation of ferric ions when coordinated with SSA. The complex has a maximum absorbance of 490 nm. This advanced method eliminates the need for concentrated acids to stop CAT activity, making it safer and easier to handle. A comparative analysis against the standard ferrithiocyanate method showed a correlation coefficient of 0.99, demonstrating the new method's comparable effectiveness and reliability. In conclusion, a simple and reliable protocol for assessing CAT activity, which utilizes a cuvette or microplate, has been demonstrated in this study. This interference-free protocol can easily be used in research and clinical analysis with considerable accuracy and precision.","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"9 1","pages":"bpae015"},"PeriodicalIF":3.6,"publicationDate":"2024-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10957919/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140207689","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