Biology Methods and Protocols最新文献

{"title":"Development of an optimized cell-based selection system for phage display libraries.","authors":"Malgorzata Czarnecka, Nicole Findik, Anja Schlör, Katja Hanack","doi":"10.1093/biomethods/bpaf009","DOIUrl":"10.1093/biomethods/bpaf009","url":null,"abstract":"<p><p>The discovery of antibodies through phage display is significantly influenced by antigen presentation during panning, particularly for membrane-anchored proteins, which pose challenges due to their complex structures. Traditional approaches, such as whole cells expressing the target protein, often result in low antigen density and high background signals. In this study, we describe an alternative method using stably transfected cell lines that express the target antigen on their surface, regulated by an intracellular enhanced green fluorescent protein (EGFP) signal. This system enables high-throughput flow cytometry-based screening of phage display libraries to isolate human antibodies that recognize the native conformation of membrane proteins. Using human epithelial cell adhesion molecule (EpCAM) and human neuroplastin 65 (NP65) as model antigens, we established an optimized screening workflow with polyclonal phage pools. Selected EpCAM-specific single-chain variable fragments (scFvs) from a naïve library were recombinantly expressed with an IgG4 scaffold and characterized for specific binding. This approach provides an effective platform for the identification of antibodies against membrane proteins in their native state.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"10 1","pages":"bpaf009"},"PeriodicalIF":2.5,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11835232/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143450503","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}

{"title":"RUNCOV: a one-pot triplex real-time RT-LAMP as a point-of-care diagnostic tool for detecting SARS-CoV-2.","authors":"Isabelle Robène, Emmanuel Jouen, Véronique Maillot-Lebon, Babbitha T Fenelon, Jérémy Hascoat, Yann Pecrix, Damien Richard, Marie-Christine Jaffar-Bandjee, Patrick Mavingui, Frédéric Chiroleu, Nathalie Becker, Patrice Poubeau, Mahery Ramiandrisoa, Michel Sin, Laurent Costet, Annie Laurent, Philippe Laurent, Aude Chabirand, Aurélie Moreau, Bernard Reynaud, Eric Jeuffrault, Catherine Cêtre-Sossah","doi":"10.1093/biomethods/bpaf010","DOIUrl":"10.1093/biomethods/bpaf010","url":null,"abstract":"<p><p>Given the risk of zoonotic disease emergence, including new SARS-CoV-2 variants of COVID-19, rapid diagnostic tools are urgently needed to improve the control of the spread of infectious diseases. A one-pot triplex real-time RT-LAMP (reverse-transcription-loop-mediated isothermal amplification) assay, based on two regions of the genome SARS-CoV-2-specifically the Orf1ab and N genes-along with one internal control, the human RNase P gene, was developed. The multiplexing relies on the distinct melting peaks produced during an annealing step. This tool, named RUNCOV, was compared to the gold-standard reverse-transcription real-time quantitative PCR (RT-qPCR) assay. A simple sample preparation step was designed alongside the assay, making it ready for use on site, as a point-of-care diagnostic tool. RUNCOV is rapid (typically less than 40 minutes), highly sensitive and specific. When tested on clinical samples with known SARS-CoV-2 status, its limit of detection (LOD) ranges between 5 and 20 copies per reaction and its diagnostic sensitivity (97.44%) and specificity (100%) values are high compared to the RT-qPCR gold standard. These results were supported with an extensive <i>in silico</i> analysis of over 14 million genomes, demonstrating this tool was capable of detecting all known SARS-CoV-2 variants, including the most recent ones KP.3.1.1 and BA2.86.1. This molecular assay is portable, as demonstrated when it was used successfully in La Réunion in different contexts outside the laboratory.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"10 1","pages":"bpaf010"},"PeriodicalIF":2.5,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11882304/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143568466","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}

{"title":"Correction to: Real time-PCR a diagnostic tool for reporting copy number variation and relative gene-expression changes in pediatric B-cell acute lymphoblastic leukemia-a pilot study.","authors":"","doi":"10.1093/biomethods/bpaf005","DOIUrl":"https://doi.org/10.1093/biomethods/bpaf005","url":null,"abstract":"<p><p>[This corrects the article DOI: 10.1093/biomethods/bpae098.].</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"10 1","pages":"bpaf005"},"PeriodicalIF":2.5,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11796088/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143256906","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}

{"title":"Development of a highly sensitive method to detect translational infidelity.","authors":"Max Hartmann, Lisa Neher, Benjamin Grupp, Zhouli Cao, Chloe Chiew, Sebastian Iben","doi":"10.1093/biomethods/bpaf008","DOIUrl":"10.1093/biomethods/bpaf008","url":null,"abstract":"<p><p>Protein homeostasis (proteostasis) is the balance of protein synthesis, protein maintenance, and degradation. Loss of proteostasis contributes to the aging process and characterizes neurodegenerative diseases. It is well established that the processes of protein maintenance and degradation are declining with aging; however, the contribution of a declining quality of protein synthesis to the loss of proteostasis is less well understood. In fact, protein synthesis at the ribosome is an error-prone process and challenges the cell with misfolded proteins. Here, we present the development of a highly sensitive and reproducible reporter assay for the detection of translational errors and the measurement of translational fidelity. Using Nano-luciferase, an enzyme 3 times smaller and 50 times more sensitive than the hitherto used Firefly-luciferase, we introduced stop-codon and amino-acid exchanges that inactivate the enzyme. Erroneous re-activation of luciferase activity indicates ribosomal inaccuracy and translational infidelity. This highly sensitive and reproducible method has broad applications for studying the molecular mechanisms underlying diseases associated with defective protein synthesis and can be used for drug screening to modulate translational fidelity.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"10 1","pages":"bpaf008"},"PeriodicalIF":2.5,"publicationDate":"2025-01-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11805343/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143383845","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}

{"title":"Association of cognitive deficits with sociodemographic characteristics among adults with post-COVID conditions: Findings from the United States household pulse survey.","authors":"Daniel J Wu, Nianjun Liu","doi":"10.1093/biomethods/bpaf006","DOIUrl":"10.1093/biomethods/bpaf006","url":null,"abstract":"<p><p>People infected with coronavirus disease-19 (COVID-19) may continue to experience symptoms for several weeks or even months after acute infection, a condition known as long COVID. Cognitive problems such as memory loss are among the most commonly reported symptoms of long COVID. However, a comprehensive evaluation of the risks of cognitive decline following COVID-19 infection among different sociodemographic groups has not been undertaken at the national level in the USA. We conducted a secondary analysis on the datasets from the U.S. Census Bureau Household Pulse Survey, encompassing data collected from 1 June 2022 to 19 December 2022. Based on a cohort of 385 370 individuals aged 18 years or older, we employed logistic regression analysis to examine the association between self-reported cognitive deficits and different sociodemographic factors among individuals with long COVID conditions. We have demonstrated that individuals with long COVID had a significantly higher risk of cognitive deficits compared to those with no history of COVID infection. Cognitive deficits vary across sociodemographic groups. In individuals without long COVID, men, older adults, and those with higher education reported fewer cognitive deficits, while Hispanics and residents of the South reported more. Long COVID had similar impacts across genders and regions but appeared to have the smallest impact on Hispanics compared to other racial groups. Conversely, the effects of long COVID were most significant in older adults and individuals with higher education. The state-level analysis further suggests potential variation in long COVID's effects across different states. The risks of cognitive deficits among adults with post-COVID conditions are substantial. Various sociodemographic groups can have different risks of developing cognitive deficits after experiencing long COVID. The findings of this large-scale study can help identify sociodemographic groups at higher risk of cognitive deficits, facilitate medical interventions, and guide resource allocation to target populations at risk and prioritize areas with a high rate of cognitive decline.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"10 1","pages":"bpaf006"},"PeriodicalIF":2.5,"publicationDate":"2025-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11785365/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143081613","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}

{"title":"An improved cell nuclear isolation method.","authors":"Pengfei Li, Jingyao Zhang, Xiaojuan Liu, Zhijuan Wu, Y James Kang, Wenjing Zhang","doi":"10.1093/biomethods/bpaf007","DOIUrl":"10.1093/biomethods/bpaf007","url":null,"abstract":"<p><p>Nuclear isolation is crucial for studying gene expression and regulatory mechanisms in eukaryotic cells. This study aimed to improve nuclear isolation and compare the yield, purity, and efficiency of several methods. Human umbilical vein endothelial cells were used to evaluate four different techniques: sucrose centrifugation, a simplified method, homogenization, and the NE-PER kit. For sucrose centrifugation, cells were scraped in Tween buffer, washed with sucrose buffer, and homogenized in a Dounce homogenizer. The pellet was washed with glycerol buffer to isolate the nuclei. In the simplified method, cells were scraped in scraping buffer, washed with sucrose buffer, and the pellet was washed with glycerol buffer to isolate the nuclei. For homogenization, cells were washed with phosphate buffered saline, followed by two washes in extract buffer and lysed with 10 strokes in a Kontes Dounce homogenizer. The NE-PER kit was used according to the manufacturer's protocol. Nuclei isolated by each method were tested by immunoblotting, co-immunoprecipitation (Co-IP), and chromatin immunoprecipitation (Ch-IP) assays. The simplified method produced nuclei with fewer organelles and less cytoplasm than those isolated by homogenization or the NE-PER kit. It was similarly effective as sucrose centrifugation but faster. Co-IP and Ch-IP assays confirmed that the simplified method enriched target proteins and DNA fragments. Overall, the simplified method provides a highly pure nuclear sample optimal for downstream applications requiring purified nuclei.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"10 1","pages":"bpaf007"},"PeriodicalIF":2.5,"publicationDate":"2025-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11805344/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143383844","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}

{"title":"PUPMCR: an R package for image-based identification of color based on Rayner's (1970) terminology and known fungal pigments.","authors":"Niña Rose Zapanta, Rhenz Hannah Santos, Jericho Ivan Pineda, Jireh Sealtiel Pedrosa, Kristine Joyce Rabelas, Charina Samontan, Lourdes Alvarez, Chester Deocaris","doi":"10.1093/biomethods/bpaf004","DOIUrl":"10.1093/biomethods/bpaf004","url":null,"abstract":"<p><p>Fungi are eukaryotic organisms grouped based on different traits of their morphology. In 1970, R. W. Rayner published <i>A Mycological Colour Chart</i> to provide a standardized system for identifying color in fungi. While its terminologies have contributed a standard way of color matching for taxonomic diagnoses, this method using the personal color perception of the observer does not guarantee accuracy. Considering the diversity of fungi, visual color matching is expected to be challenging without a standard assisting instrument. In this study, the R package PUPMCR is developed to approximate the color name and associated pigments of fungal species based on the pixel coordinates of its uploaded image. This software utilizes CIELAB and RGB color spaces as well as Euclidean and Chi-square distance metric systems. The package is tested and validated using 300 fungal images as a dataset for conducting interrater reliability tests. Results showed the highest agreement for parameters utilizing the RGB color space (Cohen's kappa values: 0.655 ± 0.013 for RGB and Euclidean; 0.658 ± 0.004 for RGB and Chi-square), attributed to its computational efficiency, which facilitates more uniform binning and universally scaled distance metrics. The produced color-identifying tool is also available as a Shiny web application (https://pupmcr.shinyapps.io/PUPMCR/) to allow better accessibility for users on the World Wide Web. The development of PUPMCR not only benefits a variety of users from its free accessibility but also provides a more reliable color identification system in the field of mycology.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"10 1","pages":"bpaf004"},"PeriodicalIF":2.5,"publicationDate":"2025-01-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11825390/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143433676","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}

{"title":"A cost-effective and scalable approach for DNA extraction from FFPE tissues.","authors":"Christoph Geisenberger, Edgar Chimal, Philipp Jurmeister, Frederick Klauschen","doi":"10.1093/biomethods/bpaf003","DOIUrl":"10.1093/biomethods/bpaf003","url":null,"abstract":"<p><p>Genomic profiling of cancer plays an increasingly vital role for diagnosis and therapy planning. In addition, research of novel diagnostic applications such as DNA methylation profiling requires large training and validation cohorts. Currently, most diagnostic cases processed in pathology departments are stored as formalin-fixed and paraffin embedded tissue blocks (FFPE). Consequently, there is a growing demand for high-throughput extraction of nucleic acids from FFPE tissue samples. While proprietary kits are available, they are expensive and offer little flexibility. Here, we present ht-HiTE, a high-throughput implementation of a recently published and highly efficient DNA extraction protocol. This approach enables manual and automated processing of 96-well plates with a liquid handler, offers two options for purification and utilizes off-the-shelf reagents. Finally, we show that NGS and DNA methylation microarray data obtained from DNA processed with ht-HiTE are of equivalent quality as compared to a manual, kit-based approach.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"10 1","pages":"bpaf003"},"PeriodicalIF":2.5,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11849955/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143493904","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}

{"title":"Quantification of variegated <i>Drosophila</i> ommatidia with high-resolution image analysis and machine learning.","authors":"Hunter J Hill, William Sullivan, Brandon S Cooper","doi":"10.1093/biomethods/bpaf002","DOIUrl":"10.1093/biomethods/bpaf002","url":null,"abstract":"<p><p>A longstanding challenge in biology is accurately analyzing images acquired using microscopy. Recently, machine learning (ML) approaches have facilitated detailed quantification of images that were refractile to traditional computation methods. Here, we detail a method for measuring pigments in the complex-mosaic adult <i>Drosophila</i> eye using high-resolution photographs and the pixel classifier <i>ilastik</i> [1]. We compare our results to analyses focused on pigment biochemistry and subjective interpretation, demonstrating general overlap, while highlighting the inverse relationship between accuracy and high-throughput capability of each approach. Notably, no coding experience is necessary for image analysis and pigment quantification. When considering time, resolution, and accuracy, our view is that ML-based image analysis is the preferred method.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"10 1","pages":"bpaf002"},"PeriodicalIF":2.5,"publicationDate":"2025-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11739462/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143013382","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}

{"title":"Novel method for detection of Aβ and Iso-D7-Aβ N-terminus-specific B cells and Iso-D7-Aβ-specific antibodies.","authors":"Elizaveta A Kolobova, Irina Yu Petrushanko, Vladimir A Mitkevich, Alexander A Makarov, Irina L Grigorova","doi":"10.1093/biomethods/bpaf001","DOIUrl":"https://doi.org/10.1093/biomethods/bpaf001","url":null,"abstract":"<p><p>Alzheimer's disease (AD) is a multifactorial systemic disease that is triggered, at least in part, by the accumulation of β-amyloid (Aβ) peptides in the brain, but it also depends on immune system-mediated regulation. Recent studies suggest that B cells may play a role in AD development and point to the accumulation of clonally expanded B cells in AD patients. However, the specificity of the clonally expanded B cells is unknown, and the contribution of Aβ-specific B cells to AD pathology development is unclear. In this study, we have developed a novel method to identify Aβ-specific B cells by flow cytometry using fluorescent tetramers. The suggested method also enables the identification of B-cell clones specific to a more pathology-provoking form of Aβ with an isomerized Asp7 residue (Iso-D7-Aβ) that accumulates in elderly people and in AD patients. The method has been verified using mice immunized with antigens containing the isomerized or non-isomerized Aβ N-terminus peptides. In addition, we describe a new method for the detection of Iso-D7-Aβ-specific antibodies, which was tested on mouse serum. These methods are of potential importance in research aimed at studying AD and may be also utilized for diagnostic and therapeutic purposes.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"10 1","pages":"bpaf001"},"PeriodicalIF":2.5,"publicationDate":"2025-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11739456/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143013368","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}