Methods and Protocols最新文献_第3页

An Optimized Protocol for SBEM-Based Ultrastructural Analysis of Cultured Human Cells. 基于sbem的培养人细胞超微结构分析优化方案

IF 2

Methods and Protocols Pub Date : 2025-08-06 DOI: 10.3390/mps8040090

Natalia Diak, Łukasz Chajec, Agnieszka Fus-Kujawa, Karolina Bajdak-Rusinek

{"title":"An Optimized Protocol for SBEM-Based Ultrastructural Analysis of Cultured Human Cells.","authors":"Natalia Diak, Łukasz Chajec, Agnieszka Fus-Kujawa, Karolina Bajdak-Rusinek","doi":"10.3390/mps8040090","DOIUrl":"10.3390/mps8040090","url":null,"abstract":"Serial block-face scanning electron microscopy (SBEM) is a powerful technique for three-dimensional ultrastructural analysis of biological samples, though its application to in vitro cultured human cells remains underutilized. In this study, we present an optimized SBEM sample preparation protocol using human dermal fibroblasts and induced pluripotent stem cells (iPSCs). The method includes key modifications to the original protocol, such as using only glutaraldehyde for fixation and substituting the toxic cacodylate buffer with a less hazardous phosphate buffer. These adaptations result in excellent preservation of cellular ultrastructure, with high contrast and clarity, as validated by transmission electron microscopy (TEM). The loss of natural cell morphology resulted from fixation during passage, when cells formed a precipitate, rather than from fixation directly within the culture medium. The protocol is time-efficient, safe, and broadly applicable to both stem cells and differentiated cells cultured under 2D conditions, providing a valuable tool for ultrastructural analysis in diverse biomedical research settings.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 4","pages":""},"PeriodicalIF":2.0,"publicationDate":"2025-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12388483/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144961775","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

Integrating Cartilage Explant Culture with Simulated Digestion and Hepatic Biotransformation Refines In Vitro Screening of Joint Care Nutraceuticals. 软骨外植体培养与模拟消化和肝脏生物转化相结合，改进关节保健营养品的体外筛选。

IF 2

Methods and Protocols Pub Date : 2025-08-06 DOI: 10.3390/mps8040091

Michelina Crosbie, Kailey Vanderboom, Jamie Souccar-Young, Wendy Pearson

{"title":"Integrating Cartilage Explant Culture with Simulated Digestion and Hepatic Biotransformation Refines In Vitro Screening of Joint Care Nutraceuticals.","authors":"Michelina Crosbie, Kailey Vanderboom, Jamie Souccar-Young, Wendy Pearson","doi":"10.3390/mps8040091","DOIUrl":"10.3390/mps8040091","url":null,"abstract":"In vitro cartilage explant culture has been used to assess nutraceuticals on cartilage responses to inflammatory stimuli. However, applying extracts of nutraceuticals directly to cartilage explants does not account for effects of digestion and hepatic biotransformation, or selective exclusion of product metabolites from joint fluid by the synovial membrane. The current study produced a simulated biological extract of a common nutraceutical (glucosamine; Gsim) by exposing it to a simulated upper gastrointestinal tract digestion, hepatic biotransformation by liver microsomes, and purification to a molecular weight cut-off of 50 kDa. This extract was then used to condition cartilage explants cultured for 120 h in the presence or absence of an inflammatory stimulus (lipopolysaccharide). Media samples were analyzed for prostaglandin E2 (PGE2), glycosaminoglycan (GAG), and nitric oxide (NO). Tissue was digested and analyzed for GAG content and stained for viability. Conditioning of explants with Gsim significantly reduced media GAG in stimulated and unstimulated explants and reduced nitric oxide production in unstimulated explants. These data provide evidence for the value of glucosamine in protecting cartilage from deterioration following an inflammatory challenge, and the model improves applicability of these in vitro data to the in vivo setting.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 4","pages":""},"PeriodicalIF":2.0,"publicationDate":"2025-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12388657/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144961342","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

Expression of Recombinant Hirudin in Bacteria and Yeast: A Comparative Approach. 重组水蛭素在细菌和酵母中的表达比较。

IF 2

Methods and Protocols Pub Date : 2025-08-03 DOI: 10.3390/mps8040089

Zhongjie Wang, Dominique Böttcher, Uwe T Bornscheuer, Christian Müller

{"title":"Expression of Recombinant Hirudin in Bacteria and Yeast: A Comparative Approach.","authors":"Zhongjie Wang, Dominique Böttcher, Uwe T Bornscheuer, Christian Müller","doi":"10.3390/mps8040089","DOIUrl":"10.3390/mps8040089","url":null,"abstract":"The expression of recombinant proteins in heterologous hosts is a common strategy to obtain larger quantities of the \"protein of interest\" (POI) for scientific, therapeutic or commercial purposes. However, the experimental success of such an approach critically depends on the choice of an appropriate host system to obtain biologically active forms of the POI. The correct folding of the molecule, mediated by disulfide bond formation, is one of the most critical steps in that process. Here we describe the recombinant expression of hirudin, a leech-derived anticoagulant and thrombin inhibitor, in the yeast Komagataella phaffii (formerly known and mentioned throughout this publication as Pichia pastoris) and in two different strains of Escherichia coli, one of them being especially designed for improved disulfide bond formation through expression of a protein disulfide isomerase. Cultivation of the heterologous hosts and expression of hirudin were performed at different temperatures, ranging from 22 to 42 °C for the bacterial strains and from 20 to 30 °C for the yeast strain, respectively. The thrombin-inhibitory potencies of all hirudin preparations were determined using the thrombin time coagulation assay. To our surprise, the hirudin preparations of P. pastoris were considerably less potent as thrombin inhibitors than the respective preparations of both E. coli strains, indicating that a eukaryotic background is not per se a better choice for the expression of a biologically active eukaryotic protein. The hirudin preparations of both E. coli strains exhibited comparable high thrombin-inhibitory potencies when the strains were cultivated at their respective optimal temperatures, whereas lower or higher cultivation temperatures reduced the inhibitory potencies.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 4","pages":""},"PeriodicalIF":2.0,"publicationDate":"2025-08-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12388516/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144961795","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

Protocol for the Systematic Quantitative Ultrastructural Analysis of Mitochondria in Cardiac Tissue. 心脏组织线粒体系统定量超微结构分析方案。

IF 2

Methods and Protocols Pub Date : 2025-08-02 DOI: 10.3390/mps8040087

Rebecca Schönmehl, Lina Winter, Daniel H Mendelsohn, Wing-Hoi Cheung, Ronald Man Yeung Wong, Steffen Pabel, Samuel Sossalla, Christoph Brochhausen

引用次数: 0

The Allium cepa Model: A Review of Its Application as a Cytogenetic Tool for Evaluating the Biosafety Potential of Plant Extracts. 葱模型：作为植物提取物生物安全性评价细胞遗传学工具的应用综述。

IF 2

Methods and Protocols Pub Date : 2025-08-02 DOI: 10.3390/mps8040088

Daniela Nicuță, Luminița Grosu, Oana-Irina Patriciu, Roxana-Elena Voicu, Irina-Claudia Alexa

引用次数: 0

Comparative Analysis of a Rapid Quantitative Immunoassay to the Reference Methodology for the Measurement of Blood Vitamin D Levels. 快速定量免疫分析法与参考方法测定血液维生素D水平的比较分析。

IF 2

Methods and Protocols Pub Date : 2025-08-01 DOI: 10.3390/mps8040085

Gary R McLean, Samson Soyemi, Oluwafunmito P Ajayi, Sandra Fernando, Wiktor Sowinski-Mydlarz, Duncan Stewart, Sarah Illingworth, Matthew Atkins, Dee Bhakta

{"title":"Comparative Analysis of a Rapid Quantitative Immunoassay to the Reference Methodology for the Measurement of Blood Vitamin D Levels.","authors":"Gary R McLean, Samson Soyemi, Oluwafunmito P Ajayi, Sandra Fernando, Wiktor Sowinski-Mydlarz, Duncan Stewart, Sarah Illingworth, Matthew Atkins, Dee Bhakta","doi":"10.3390/mps8040085","DOIUrl":"10.3390/mps8040085","url":null,"abstract":"Vitamin D is the only vitamin that is conditionally essential, as it is synthesized from precursors after UV light exposure, whilst also being obtained from the diet. It has numerous health benefits, with deficiency becoming a major concern globally, such that dietary supplementation has more recently achieved vital importance to maintain satisfactory levels. In recent years, measurements made from blood have, therefore, become critical to determine the status of vitamin D levels in individuals and the larger population. Tests for vitamin D have routinely relied on laboratory analysis with sophisticated equipment, often being slow and costly, whilst rapid immunoassays have suffered from poor specificity and sensitivity. Here, we have evaluated a new rapid immunoassay test on the market (Rapi-D & IgLoo) to quickly and accurately measure vitamin D levels in small capillary blood specimens and compared this to measurements made using the standard laboratory method of liquid chromatography and mass spectrometry. Our results show that vitamin D can be measured very quickly and over a broad range using the new method, as well as correlate relatively well with standard laboratory testing; however, it cannot be fully relied upon currently to accurately diagnose deficiency or sufficiency in individuals. Our statistical and comparative analyses find that the rapid immunoassay with digital quantification significantly overestimates vitamin D levels, leading to diminished diagnosis of vitamin D deficiency. The speed and simplicity of the rapid method will likely provide advantages in various healthcare settings; however, further calibration of this rapid method and testing parameters for improving quantification of vitamin D from capillary blood specimens is required before integration of it into clinical decision-making pathways.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 4","pages":""},"PeriodicalIF":2.0,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12388426/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144961783","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

The Effect of Faecal Microbiota Transplantation on Cognitive Function in Cognitively Healthy Adults with Irritable Bowel Syndrome: Protocol for a Randomised, Placebo-Controlled, Double-Blinded Pilot Study. 粪便微生物群移植对认知健康肠易激综合征成人认知功能的影响：一项随机、安慰剂对照、双盲先导研究方案

IF 2

Methods and Protocols Pub Date : 2025-08-01 DOI: 10.3390/mps8040083

Sara Alaeddin, Yanna Ko, Genevieve Z Steiner-Lim, Slade O Jensen, Tara L Roberts, Vincent Ho

{"title":"The Effect of Faecal Microbiota Transplantation on Cognitive Function in Cognitively Healthy Adults with Irritable Bowel Syndrome: Protocol for a Randomised, Placebo-Controlled, Double-Blinded Pilot Study.","authors":"Sara Alaeddin, Yanna Ko, Genevieve Z Steiner-Lim, Slade O Jensen, Tara L Roberts, Vincent Ho","doi":"10.3390/mps8040083","DOIUrl":"10.3390/mps8040083","url":null,"abstract":"Faecal microbiota transplantation (FMT) is an emerging therapy for gastrointestinal and neurological disorders, acting via the microbiota-gut-brain axis. Altering gut microbial composition may influence cognitive function, but this has not been tested in cognitively healthy adults. This randomised, double-blinded, placebo-controlled pilot trial investigates whether FMT is feasible and improves cognition in adults with irritable bowel syndrome (IBS). Participants receive a single dose of FMT or placebo via rectal retention enema. Cognitive performance is the primary outcome, assessed using the Cambridge Neuropsychological Test Automated Battery (CANTAB). Secondary outcomes include IBS symptom severity and mood. Tertiary outcomes include microbiome composition and plasma biomarkers related to inflammation, short-chain fatty acids, and tryptophan metabolism. Outcomes are assessed at baseline and at one, three, six, and twelve months following treatment. We hypothesise that FMT will lead to greater improvements in cognitive performance than placebo, with benefits extending beyond practice effects, emerging at one month and persisting in the long term. The findings will contribute to evaluating the safety and efficacy of FMT and enhance our understanding of gut-brain interactions.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 4","pages":""},"PeriodicalIF":2.0,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12388800/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144961445","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 Study Protocol to Assess the Association Between Ambient Air Pollution and Asthma and Other Respiratory Health Outcomes Amongst Children Below 5 Years of Age in Alexandra Township's Early Childhood Development Centers, Johannesburg. 评估环境空气污染与约翰内斯堡亚历山德拉镇儿童早期发展中心5岁以下儿童哮喘和其他呼吸系统健康结果之间关系的研究方案

IF 2

Methods and Protocols Pub Date : 2025-08-01 DOI: 10.3390/mps8040084

Velisha Thompson, Joyce Shirinde, Masilu D Masekameni, Thokozani P Mbonane

{"title":"A Study Protocol to Assess the Association Between Ambient Air Pollution and Asthma and Other Respiratory Health Outcomes Amongst Children Below 5 Years of Age in Alexandra Township's Early Childhood Development Centers, Johannesburg.","authors":"Velisha Thompson, Joyce Shirinde, Masilu D Masekameni, Thokozani P Mbonane","doi":"10.3390/mps8040084","DOIUrl":"10.3390/mps8040084","url":null,"abstract":"Air pollution is linked to childhood mortality and morbidity in low- and middle-income countries globally. There is growing evidence linking air pollution to asthma and other respiratory diseases in children. Studies have shown that children are likely to experience asthma due to their narrow airways and their heightened sensitivity to environmental irritants. This study aims to investigate the relationship between ambient air pollution and respiratory diseases in children under the age of 5. The study will be conducted in the informal township of Alexandra, north of Johannesburg, South Africa. A quantitative approach will be used in this cross-sectional analytical study. Data will be collected using different tools that include a questionnaire to determine the prevalence of asthma and respiratory disease and potential risk factors. While environmental air pollution will be measured using Radiello passive samplers and Gillian pumps. Data will be analyzed using the latest version of the STATANow/MP 19.5 software. Furthermore, health risk assessment will be conducted for lifetime non-carcinogenic and carcinogenic risk estimation following the USEPA framework. The study will identify environmental triggers that exacerbate asthma and other respiratory conditions in other similar community settings and will contribute to the body of knowledge in public health. Ethical approval was obtained from the Research Ethics Committee, Faculty of Health Sciences at the University of Johannesburg.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 4","pages":""},"PeriodicalIF":2.0,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12388445/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144961798","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

Comparing Nanomechanical Properties and Membrane Roughness Along the Aging of Human Erythrocytes. 红细胞衰老过程中纳米力学性能和膜粗糙度的比较。

IF 2

Methods and Protocols Pub Date : 2025-08-01 DOI: 10.3390/mps8040086

Giovanni Longo, Simone Dinarelli, Federica Collacchi, Marco Girasole

{"title":"Comparing Nanomechanical Properties and Membrane Roughness Along the Aging of Human Erythrocytes.","authors":"Giovanni Longo, Simone Dinarelli, Federica Collacchi, Marco Girasole","doi":"10.3390/mps8040086","DOIUrl":"10.3390/mps8040086","url":null,"abstract":"Erythrocyte (RBC) aging involves significant structural and nanomechanical alterations crucial to their function. This study aims to bridge the gap between analyses based on statistical morphometric parameters, e.g., membrane roughness, and those based on point-dependent nanomechanical properties, e.g., stiffness or Young's modulus. Using Atomic Force Microscopy, we investigated morphology, membrane roughness, and nanomechanical properties on the very same RBCs under dehydrated (air) and hydrated (physiological buffer) conditions. The cells were studied at different stages of in vitro aging: one, seven, and 12 days. Our results quantitatively show that across dehydration, as well as along the aging pathway, RBCs become progressively more rigid while their membrane roughness decreases, a trend observed in both environments. Notably, the differences between the hydrated and dehydrated states were large in young cells but diminished when erythrocytes aged. Despite these parallel trends, high-resolution mapping on the nanoscale revealed that roughness and Young's modulus do not correlate, indicating that these parameters are linked to different properties. In conclusion, this work provides a comprehensive protocol for a biophysical description of RBC aging and establishes that the simultaneous measurement of membrane roughness and nanomechanical properties offers a complementary approach, yielding a more complete characterization of cellular properties.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 4","pages":""},"PeriodicalIF":2.0,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12388609/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144961718","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

Green Chemistry and Multivariate Optimization in the Extraction of Phenolic Compounds: The Potential of NaDES in Alternative Raw Materials for Expanded Extrudates. 酚类化合物提取的绿色化学和多元优化：NaDES在膨化挤出物替代原料中的潜力。

IF 2

Methods and Protocols Pub Date : 2025-07-23 DOI: 10.3390/mps8040082

Mateus Alves Araújo, Bianca Rodrigues Morais, João Pedro da Silva Santos, Larissa Karla de Jesus, Kaliston Aurélio Lomba, Gustavo Costa do Nascimento, Marcus Alvarenga Soares, Nathalia de Andrade Neves, Irene Andressa, Maria Teresa Pedrosa Silva Clerici, Marcio Schmiele

{"title":"Green Chemistry and Multivariate Optimization in the Extraction of Phenolic Compounds: The Potential of NaDES in Alternative Raw Materials for Expanded Extrudates.","authors":"Mateus Alves Araújo, Bianca Rodrigues Morais, João Pedro da Silva Santos, Larissa Karla de Jesus, Kaliston Aurélio Lomba, Gustavo Costa do Nascimento, Marcus Alvarenga Soares, Nathalia de Andrade Neves, Irene Andressa, Maria Teresa Pedrosa Silva Clerici, Marcio Schmiele","doi":"10.3390/mps8040082","DOIUrl":"10.3390/mps8040082","url":null,"abstract":"Phenolic compounds are secondary metabolites widely distributed among plants, with bioactive properties, especially antioxidant activity. The search for sustainable extraction methods has driven the use of natural deep eutectic solvents (NaDESs), formed by combinations of natural compounds, such as organic acids, sugars, alcohols, and amino acids. This study optimized NaDES (sorbitol, citric acid, and glycine) efficiency and compared it to that of 70% methanol solution in extracting total soluble phenolic compounds (TSPCs) from six flours matrices-corn, buckwheat, biofortified orange sweet potato, red lentil, Sudan grass, and chickpea-before and after thermoplastic extrusion cooking. Quantification was performed using the Folin-Ciocalteu method, with statistical analysis at the 10% significance level. In general, the methanolic extracts showed higher TSPC levels in the raw materials, whereas the levels were higher in NaDESs for legumes. After extrusion, a reduction in the TSPC levels was observed, except in the sweet potato. Multivariate analysis (PLS-DA and heatmap) distinguished the raw and extruded samples, revealing structural and chemical changes from thermal processing. The AGREE scores were 0.7 (NaDES) and 0.54 (methanol), favoring NaDES. The BAGI score (75.0) confirmed the method's robustness and suitability for sustainable analytical applications.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 4","pages":""},"PeriodicalIF":2.0,"publicationDate":"2025-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12388042/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144961405","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