Methods and Protocols最新文献_第8页

Assessment of the Integrity and Function of Human Term Placental Explants in Short-Term Culture 评估短期培养的人类末期胎盘外胚层的完整性和功能

IF 2.4

Methods and Protocols Pub Date : 2024-02-15 DOI: 10.3390/mps7010016

Carolina López-Guzmán, Ana María García, Paula Marín, Ana María Vásquez

{"title":"Assessment of the Integrity and Function of Human Term Placental Explants in Short-Term Culture","authors":"Carolina López-Guzmán, Ana María García, Paula Marín, Ana María Vásquez","doi":"10.3390/mps7010016","DOIUrl":"https://doi.org/10.3390/mps7010016","url":null,"abstract":"Human placental explants (HPEs) culture has generated significant interest as a valuable in vitro model for studying tissue functions in response to adverse conditions, such as fluctuations in oxygen levels, nutrient availability, exposure to pathogenic microorganisms, and toxic compounds. HPEs offers the advantage of replicating the intricate microenvironment and cell-to-cell communication involved in this critical and transient organ. Although HPEs culture conditions have been extensively discussed, a protocol for assessing the viability and function of HPEs during short-term culture has not been previously outlined. In this study, we have developed a short-term HPEs culture protocol, specifically up to 72 h, and have employed quantitative, semi-quantitative, and qualitative analyses to evaluate tissue viability and function over time. Under our standardized conditions, placental villi explants began to regain their structural properties (the integrity of the trophoblast and villous stroma) and the functionality of the HPEs (production of angiogenic, endocrine, and immunological factors) starting from 48 h of culture. This restoration ensures a suitable environment for several applications. The data presented here can be highly valuable for laboratories aiming to implement an HPEs model, whether in the process of standardization or seeking to enhance and optimize working conditions and timing with placental tissue.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2024-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139775235","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Pilot Study: Safety and Performance Validation of an Ingestible Medical Device for Collecting Small Intestinal Liquid in Healthy Volunteers. 试点研究：用于收集健康志愿者小肠液的可摄入医疗设备的安全性和性能验证。

IF 2.4

Methods and Protocols Pub Date : 2024-02-04 DOI: 10.3390/mps7010015

Alexandre Tronel, Anne-Sophie Silvent, Elena Buelow, Joris Giai, Corentin Leroy, Marion Proust, Donald Martin, Audrey Le Gouellec, Thomas Soranzo, Nicolas Mathieu

{"title":"Pilot Study: Safety and Performance Validation of an Ingestible Medical Device for Collecting Small Intestinal Liquid in Healthy Volunteers.","authors":"Alexandre Tronel, Anne-Sophie Silvent, Elena Buelow, Joris Giai, Corentin Leroy, Marion Proust, Donald Martin, Audrey Le Gouellec, Thomas Soranzo, Nicolas Mathieu","doi":"10.3390/mps7010015","DOIUrl":"10.3390/mps7010015","url":null,"abstract":"The connection between imbalances in the human gut microbiota, known as dysbiosis, and various diseases has been well established. Current techniques for sampling the small intestine are both invasive for patients and costly for healthcare facilities. Most studies on human gut microbiome are conducted using faecal samples, which do not accurately represent the microbiome in the upper intestinal tract. A pilot clinical investigation, registered as NCT05477069 and sponsored by the Grenoble Alpes University Hospital, is currently underway to evaluate a novel ingestible medical device (MD) designed for collecting small intestinal liquids by Pelican Health. This study is interventional and monocentric, involving 15 healthy volunteers. The primary objective of the study is to establish the safety and the performance of the MD when used on healthy volunteers. Secondary objectives include assessing the device's performance and demonstrating the difference between the retrieved sample from the MD and the corresponding faecal sample. Multi-omics analysis will be performed, including metagenomics, metabolomics, and culturomics. We anticipate that the MD will prove to be safe without any reported adverse effects, and we collected samples suitable for the proposed omics analyses in order to demonstrate the functionality of the MD and the clinical potential of the intestinal content.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2024-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10892249/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139931971","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

Correlation between Tooth Position Parameters and Apical Fenestration: A Cone-Beam Computed Tomography Study 牙齿位置参数与根尖窝洞之间的相关性：锥形束计算机断层扫描研究

IF 2.4

Methods and Protocols Pub Date : 2024-02-02 DOI: 10.3390/mps7010014

Carlos Henrique Ferrari, Lara Steffany de Carvalho, Caroline Trefiglio Rocha, Amjad Abu Hasna

{"title":"Correlation between Tooth Position Parameters and Apical Fenestration: A Cone-Beam Computed Tomography Study","authors":"Carlos Henrique Ferrari, Lara Steffany de Carvalho, Caroline Trefiglio Rocha, Amjad Abu Hasna","doi":"10.3390/mps7010014","DOIUrl":"https://doi.org/10.3390/mps7010014","url":null,"abstract":"This study aimed to assess the relationship between apical fenestration—a defect in the alveolar bone involving the root apex—and tooth position in all tooth groups, excluding the third molars, utilizing cone-beam computed tomography (CBCT) images. A total of 800 CBCT scans (400 maxillary and 400 mandibular) from patients undergoing various treatments were examined by a single professional (radiologist and endodontist). Statistical analyses, including the chi-square test or Fisher’s exact test, were conducted using R software 2.7.3 (R Foundation, Vienna, Austria). Results indicated a significant association (p ≤ 0.05) between apical fenestration and tooth position. In the upper teeth, apical fenestrations were notably present in the mesio-buccal (17.17%) and disto-buccal (11.07%) roots of the first molars. Conversely, apical fenestrations in the lower teeth were relatively less frequent. The study revealed a negative correlation between apical fenestration and mesial inclination, rotation, and extrusion in the upper teeth. However, a positive correlation was observed between apical fenestration and lingual inclination in the upper teeth. In conclusion, this study illuminates the distribution of apical fenestration and its correlation with tooth positions, offering insights into factors influencing this defect in dental anatomy. The findings enhance our understanding of nuanced relationships between tooth position and apical fenestration in the upper and lower dental arches.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2024-02-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139870400","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Correlation between Tooth Position Parameters and Apical Fenestration: A Cone-Beam Computed Tomography Study 牙齿位置参数与根尖窝洞之间的相关性：锥形束计算机断层扫描研究

IF 2.4

Methods and Protocols Pub Date : 2024-02-02 DOI: 10.3390/mps7010014

Carlos Henrique Ferrari, Lara Steffany de Carvalho, Caroline Trefiglio Rocha, Amjad Abu Hasna

{"title":"Correlation between Tooth Position Parameters and Apical Fenestration: A Cone-Beam Computed Tomography Study","authors":"Carlos Henrique Ferrari, Lara Steffany de Carvalho, Caroline Trefiglio Rocha, Amjad Abu Hasna","doi":"10.3390/mps7010014","DOIUrl":"https://doi.org/10.3390/mps7010014","url":null,"abstract":"This study aimed to assess the relationship between apical fenestration—a defect in the alveolar bone involving the root apex—and tooth position in all tooth groups, excluding the third molars, utilizing cone-beam computed tomography (CBCT) images. A total of 800 CBCT scans (400 maxillary and 400 mandibular) from patients undergoing various treatments were examined by a single professional (radiologist and endodontist). Statistical analyses, including the chi-square test or Fisher’s exact test, were conducted using R software 2.7.3 (R Foundation, Vienna, Austria). Results indicated a significant association (p ≤ 0.05) between apical fenestration and tooth position. In the upper teeth, apical fenestrations were notably present in the mesio-buccal (17.17%) and disto-buccal (11.07%) roots of the first molars. Conversely, apical fenestrations in the lower teeth were relatively less frequent. The study revealed a negative correlation between apical fenestration and mesial inclination, rotation, and extrusion in the upper teeth. However, a positive correlation was observed between apical fenestration and lingual inclination in the upper teeth. In conclusion, this study illuminates the distribution of apical fenestration and its correlation with tooth positions, offering insights into factors influencing this defect in dental anatomy. The findings enhance our understanding of nuanced relationships between tooth position and apical fenestration in the upper and lower dental arches.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2024-02-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139810420","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A Detailed Protocol for Constructing a Human Single-Chain Variable Fragment (scFv) Library and Downstream Screening via Phage Display 构建人类单链可变片段 (scFv) 文库并通过噬菌体展示进行下游筛选的详细方案

IF 2.4

Methods and Protocols Pub Date : 2024-02-01 DOI: 10.3390/mps7010013

Ziyi Liu, Dokyun Kim, Seokmin Kang, Jae U. Jung

{"title":"A Detailed Protocol for Constructing a Human Single-Chain Variable Fragment (scFv) Library and Downstream Screening via Phage Display","authors":"Ziyi Liu, Dokyun Kim, Seokmin Kang, Jae U. Jung","doi":"10.3390/mps7010013","DOIUrl":"https://doi.org/10.3390/mps7010013","url":null,"abstract":"The development of monoclonal antibodies (mAbs) represents a significant milestone in both basic research and clinical applications due to their target specificity and versatility in therapeutic and diagnostic applications. The innovative strategy of mAb screening, utilizing phage display, facilitates the in vitro screening of antibodies with high affinity to target antigens. The single-chain variable fragment (scFv) is a subset of mAb derivatives, known for its high binding affinity and smaller size—just one-third of that of human IgG. This report outlines a detailed and comprehensive procedure for constructing a scFv phagemid library derived from human patients, followed by screening via phage display affinity selection. The protocol utilizes 348 primer combinations spanning the entire human antibody repertoire to minimize sequence bias and maintain library diversity during polymerase chain reaction (PCR) for scFv generation, resulting in a library size greater than 1 × 108. Furthermore, we describe a high-throughput phage display screening protocol using enzyme-linked immunosorbent assay (ELISA) to evaluate more than 1200 scFv candidates. The generation of a highly diverse scFv library, coupled with the implementation of a phage display screening methodology, is expected to provide a valuable resource for researchers in pursuit of scFvs with high affinity for target antigens, thus advancing both research and clinical endeavors.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2024-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139687385","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Simplified Method for Agrobacterium-Mediated Genetic Transformation of Populus x berolinensis K. Koch 农杆菌介导的杨树 x berolinensis K. Koch 遗传转化简化方法

IF 2.4

Methods and Protocols Pub Date : 2024-01-26 DOI: 10.3390/mps7010012

V. Pavlichenko, M. Protopopova

{"title":"Simplified Method for Agrobacterium-Mediated Genetic Transformation of Populus x berolinensis K. Koch","authors":"V. Pavlichenko, M. Protopopova","doi":"10.3390/mps7010012","DOIUrl":"https://doi.org/10.3390/mps7010012","url":null,"abstract":"The rapid advancement of genetic technologies has made it possible to modify various plants through both genetic transformation and gene editing techniques. Poplar, with its rapid in vitro growth and regeneration enabling high rates of micropropagation, has emerged as a model system for the genetic transformation of woody plants. In this study, Populus × berolinensis K. Koch. (Berlin poplar) was chosen as the model organism due to its narrow leaves and spindle-shaped crown, which make it highly suitable for in vitro manipulations. Various protocols for the Agrobacterium-mediated transformation of poplar species have been developed to date. However, the genetic transformation procedures are often constrained by the complexity of the nutrient media used for plant regeneration and growth, which could potentially be simplified. Our study presents a cheaper, simplified, and relatively fast protocol for the Agrobacterium-mediated transformation of Berlin poplar. The protocol involved using internode sections without axillary buds as explants, which were co-cultivated in 10 µL droplets of bacterial suspension directly on the surface of a solid agar-based medium without rinsing and sterile paper drying after inoculation. We used only one regeneration Murashige and Skoogbased medium supplemented with BA (0.2 mg·L−1), TDZ (0.02 mg·L−1), and NAA (0.01 mg·L−1). Acetosyringone was not used as an induction agent for vir genes during the genetic transformation. Applying our protocol and using the binary plasmid pBI121 carrying the nptII selective and uidA reporter genes, we obtained the six transgenic lines of poplar. Transgenesis was confirmed through a PCR-based screening of kanamycin-selected regenerants for the presence of both mentioned genes, Sanger sequencing, and tests for detecting the maintained activity of both genes. The transformation efficiency, considering the 100 explants taken originally, was 6%.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2024-01-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139594778","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A Simplified Method for Calculating Surface Area of Mammalian Erythrocytes 计算哺乳动物红细胞表面积的简化方法

IF 2.4

Methods and Protocols Pub Date : 2024-01-25 DOI: 10.3390/mps7010011

I. Udroiu

引用次数: 0

An Ultra-High-Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry Method with Online Solid-Phase Extraction Sample Preparation for the High-Throughput and Sensitive Determination of Ostarine in Human Urine 一种在线固相萃取样品制备的超高效液相色谱-串联质谱法，用于高通量、灵敏地测定人尿液中的奥司他林

IF 2.4

Methods and Protocols Pub Date : 2024-01-23 DOI: 10.3390/mps7010010

Kristián Slíž, J. Piešťanský, P. Mikuš

{"title":"An Ultra-High-Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry Method with Online Solid-Phase Extraction Sample Preparation for the High-Throughput and Sensitive Determination of Ostarine in Human Urine","authors":"Kristián Slíž, J. Piešťanský, P. Mikuš","doi":"10.3390/mps7010010","DOIUrl":"https://doi.org/10.3390/mps7010010","url":null,"abstract":"Ostarine is frequently misused as a selective androgen receptor modulator (SARM) in sports. Consequently, there is a pressing need for reliable and simple approaches to monitor its presence in biological systems. In this work, we developed a two-dimensional analytical method utilizing online solid-phase extraction (online-SPE) in conjunction with ultra-high-performance liquid chromatography and tandem mass spectrometry (triple quadrupole). This automated 2D separation approach is characterized by minimum manual steps in the sample preparation (only dilute-and-shoot), reflecting high sample throughput and the reliability of analytical data. It provides favorable performance parameters, including a limit of detection of 0.5 pg/mL, high accuracy (relative error = 1.6–7.5%), precision (relative standard deviation = 0.8–4.5%), and sensitivity. Additionally, it demonstrates excellent linearity (r2 = 0.9999) in the calibration range of 0.05 to 25 ng/mL and robustness, with no carryover effects observed. This comparative study revealed a two-decadic-order-lower LOD of the SPE-UHPLC-MS/MS method to the corresponding UHPLC-MS/MS method and the lowest one in the group of currently published LC-MS methods. The World Anti-Doping Agency screening and confirmation criteria were met through the analysis of spiked urine samples from ten healthy volunteers. Accordingly, the proposed method is suitable for routine use in antidoping laboratories.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2024-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139604572","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Assessment of Nuclear Gem Quantity for Evaluating the Efficacy of Antisense Oligonucleotides in Spinal Muscular Atrophy Cells 评估反义寡核苷酸对脊髓肌肉萎缩症细胞疗效的核宝石数量

IF 2.4

Methods and Protocols Pub Date : 2024-01-19 DOI: 10.3390/mps7010009

Haya Al-Hilal, M. Maretina, A. Egorova, A. Glotov, A. Kiselev

{"title":"Assessment of Nuclear Gem Quantity for Evaluating the Efficacy of Antisense Oligonucleotides in Spinal Muscular Atrophy Cells","authors":"Haya Al-Hilal, M. Maretina, A. Egorova, A. Glotov, A. Kiselev","doi":"10.3390/mps7010009","DOIUrl":"https://doi.org/10.3390/mps7010009","url":null,"abstract":"Spinal muscular atrophy is a neuromuscular disorder caused by mutations in both copies of the survival motor neuron gene 1 (SMN1), which lead to reduction in the production of the SMN protein. Currently, there are several therapies that have been approved for SMA, with many more undergoing active research. While various biomarkers have been proposed for assessing the effectiveness of SMA treatment, a universally accepted one still has not been identified. This study aimed to describe a fast and reliable method using the number of gems in cell nuclei as a potential tool for assessment of splicing correction of oligonucleotide efficacy in SMA cells. To gain insight into whether the number of gems in cell nuclei varies based on their SMN genotype and whether the increase in gem number is associated with therapeutic response, we utilized fibroblast cell cultures obtained from a patient with SMA type II and from a healthy individual. We discovered a remarkable difference in the number of gems found in the nuclei of these cells, specifically when counting gems per 100 nuclei. The SMA fibroblasts treated with antisense oligonucleotide showed beneficial effects in correcting the abnormal splicing of SMN2 exon 7. It was observed that there was a significant increase in the number of gems in the treated cells compared to the intact SMA cells. The results obtained significantly correlate with an increase of full-length SMN transcript sharing. Based on our findings, we propose using the quantity of gems as a reliable biomarker for SMA drug development.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2024-01-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139612429","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Making the Most of Lateral Flow Immunochromatographic Tests: An Efficient Protocol to Recover DNA. 充分利用侧流免疫层析测试：回收 DNA 的高效方案。

IF 2.3

Methods and Protocols Pub Date : 2024-01-15 DOI: 10.3390/mps7010008

Sara C Zapico, Gabriela Roca

{"title":"Making the Most of Lateral Flow Immunochromatographic Tests: An Efficient Protocol to Recover DNA.","authors":"Sara C Zapico, Gabriela Roca","doi":"10.3390/mps7010008","DOIUrl":"10.3390/mps7010008","url":null,"abstract":"Lateral flow immunochromatographic (LFI) tests are widely used in both biomedical and forensic sciences for different applications. In forensic sciences, their main use is to detect body fluids at crime scenes. However, there are situations in which the amount of potential biological evidence is so low that DNA extraction is favored with respect to the identification of body fluids. Here, an efficient and quick protocol is presented to integrate the detection of body fluids through LFI with DNA extraction from a sample swab and buffer, providing a complete characterization of the biological evidence. This protocol is a modification of a general DNA extraction silica-based kit, whose main application is for blood and tissues. Thus, it could be carried out in different settings (forensic labs, hospitals, other testing labs) without the necessity of buying a specific kit for swabs. The validation of this protocol is supported by the results presented here and previous publications from our group, obtaining DNA in good quantity and with good quality. This proves the potential application of the protocol in both forensic scenarios, to fully characterize biological evidence, and biomedical settings, to molecularly confirm the results of LFI tests.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2024-01-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10801598/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139512995","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