Methods and Protocols最新文献

{"title":"Comparative Analysis of Methods for Assessing On-Target Gene Editing Efficiencies.","authors":"Bing Yao, Qiangbing Yang, Manuel A F V Gonçalves, Raymond Schiffelers, Joost P G Sluijter, Zhiyong Lei","doi":"10.3390/mps8020023","DOIUrl":"10.3390/mps8020023","url":null,"abstract":"<p><p>Genome editing based on CRISPR-derived technologies has become a cornerstone in both fundamental research and clinical applications. Accurately measuring editing efficiency is crucial for developing and applying genome editing strategies. This study offers a detailed comparison of widely used techniques for evaluating on-target gene editing efficiency, including T7 Endonuclease I (T7EI), Tracking of Indels by Decomposition (TIDE), Inference of CRISPR Edits (ICE), droplet digital PCR (ddPCR), and live-cell assays involving engineered fluorescent reporter cells. Through a comparative analysis, this study highlights the unique strengths and limitations of each method, aiding researchers in choosing the most appropriate method for their specific needs, ensuring more tailored monitoring of genome editing outcomes in a precise and reliable manner.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 2","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11932317/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692474","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 Randomized, Double-Blind, Controlled Trial Protocol for Therapeutic Neuroscience Education in Chronic Migraine Patients: A Clinical-Neurophysiological Combined Study Design.","authors":"Matteo Castaldo, Tiziana Atzori, Angela Comanducci, Giacomo Querzola, Chiara-Camilla Derchi, Daniele Lovattini, Carlo Manzoni, Carlo Lovati, Francesca Baglio, Paola Tiberio, Rita De Sanctis, Simone Sarasso, Alessandro Viganò","doi":"10.3390/mps8020022","DOIUrl":"10.3390/mps8020022","url":null,"abstract":"<p><p>Chronic migraine (CM) is a highly disabling condition, affecting about 2% of the global population. Non-pharmacological treatments can be optimal for their non-invasive nature. This prospective, randomized, double-blind, controlled trial aimed to test the efficacy of therapeutic neuroscience education (TNE) in CM. Early response biomarkers were also evaluated. A total of 80 CM patients were consecutively enrolled and randomly allocated to TNE or a general education program. Treatment effectiveness was evaluated at baseline (T1) and 2 months after the end of treatment (T4). We collected the responses to disability and comorbidity questionnaires at the start (T1) and end of treatment (T3, 10 weeks after start). Early response biomarkers were evaluated at screening (T0) and mid-way through the process (T2, 5 weeks after start). We expected that TNE would provide a greater benefit than the general education program, which served as the primary outcome of this study. We also expected that a change in clinical and neurophysiological measures could potentially occur, reflecting plasticity-induced reorganization and predicting clinical response. This is the first study selectively exploring the effect of TNE as a standalone treatment for CM. A new, effective treatment regime without interactions with other medication could be of great interest as an addition to migraine therapeutic strategies.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 2","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11932240/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692377","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":"Optimized SDS-Based Protocol for High-Quality RNA Extraction from <i>Musa</i> spp.","authors":"Kishan Saha, Onyinye C Ihearahu, L H Stevenson Naitchede, Supriyo Ray, George Ude","doi":"10.3390/mps8010021","DOIUrl":"10.3390/mps8010021","url":null,"abstract":"<p><p>The high quantity of polyphenols and polysaccharides present in the tissues of <i>Musa</i> spp. often leads to the degradation of nucleic acids, which is why all previously established methods resulted in lesser yield and poor quality of RNA. In this study, we present a modified SDS-based RNA extraction method to improve the quality and yield of RNA from different tissues of <i>Musa</i> spp. for downstream applications. The modification of RNA extraction buffer, SDS, heat incubation, and use of LiCl resulted in high-intensity RNA bands and increased RNA yield. The clear ribosomal RNA bands ensured the high quality of intact RNA without genomic DNA contamination, along with A260/A280 and A260/A230 ratios ranging from 1.83 to 2.25, which indicated the high quality of RNA across different banana varieties and tissue types. This method was found to be very effective when RNA was extracted from drought-stressed plants yielding 2.92 to 6.30 µg/100 mg fresh weight with high RNA integrity and quality (RNA IQ) 7.8-9.9 from the different groups of <i>Musa</i> tissues. Additionally, the RNA was successfully applied in PCR and quantitative real-time PCR (qRT-PCR), confirming downstream application in gene expression analysis. This method is a reliable and efficient technique for RNA extraction methods like Trizol, NucleoSpin, RNeasy, and CTAB procedures reported so far.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 1","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11858800/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143492966","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":"Real-Time Polymerase Chain Reaction Systems for Detection and Differentiation of Unclassified Viruses of the <i>Phenuiviridae</i> Family.","authors":"Alena V Dereventsova, Alexander S Klimentov, Ivan S Kholodilov, Oxana A Belova, Alexander M Butenko, Galina G Karganova","doi":"10.3390/mps8010020","DOIUrl":"10.3390/mps8010020","url":null,"abstract":"<p><p>The family <i>Phenuiviridae</i>, part of the order <i>Hareavirales</i>, includes arboviruses and arthropod-associated viruses, with sandflies, mosquitoes, and ticks as primary vectors. Historically, only sandfly/mosquito-borne phenuiviruses were associated with human diseases, but the emergence of severe fever with thrombocytopenia syndrome (SFTS) has highlighted the potential of tick-borne phenuiviruses as human pathogens. Recent discoveries of new arthropod-associated viruses, some of which remain unclassified, underscore the need for sensitive detection and differentiation methods, particularly in regions where these viruses may co-circulate. This study aimed to develop real-time PCR test systems for identifying and differentiating unclassified viruses within the <i>Phenuiviridae</i> family. In this study, tick suspensions containing phenuiviruses, previously obtained during the screening of ticks from various regions of Russia using pan-phenuivirus primers, were used. Specific primers and probes were designed to differentiate five <i>Phenuiviridae</i> viruses of genera <i>Uukuvirus</i>, <i>Ixovirus</i>, <i>Phlebovirus</i> and one unclassified phenuivirus, and their analytical sensitivity and specificity were evaluated. These PCR-based tools provide a robust method for detecting and classifying uncharacterized phenuiviruses, contributing to improved surveillance and understanding their potential epidemiological and epizootological impacts.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 1","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11857896/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143492968","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":"The Least Squares Method as a Tool for Assessment of the Stroke Parameters and Velocity in Monofin Swimming.","authors":"Marek Rejman, Paweł Szkudlarek","doi":"10.3390/mps8010019","DOIUrl":"10.3390/mps8010019","url":null,"abstract":"<p><p>This study explores the application of the Least Squares Method to analyze and model the kinematic parameters in monofin swimming, focusing on stroke rate, stroke length, and the amplitudes of joint displacements at the hip, knee, and ankle. The primary aim is to evaluate whether this method provides an objective and diagnostic tool for assessing monofin swimming techniques. Three elite monofin swimmers were evaluated under a progressive fatigue test. Results indicated that the stroke rate increases velocity by 0.95, 0.23, and 0.96 units (for the estimated models respectively). Optimized stroke length (0.01-0.12 units) also significantly correlates with velocity improvements. Joint amplitude reductions, particularly at the hip and ankle, enhanced propulsion by minimizing drag. This study highlights the Least Squares Method as a diagnostic tool for optimizing swimming techniques, with potential applications in performance training.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 1","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11858236/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143492970","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":"Assessment of Endocyn on Dental Pulp Stem Cells (DPSCs): A Pilot Study of Endodontic Irrigant Effects.","authors":"Brennan Truman, Linda Ma, Samuel Stewart, Karl Kingsley, Victoria Sullivan","doi":"10.3390/mps8010018","DOIUrl":"10.3390/mps8010018","url":null,"abstract":"<p><p>Many endodontic procedures within the pediatric population are performed with patients aged 12 years and older, using intracanal irrigants to complement mechanical debridement for the removal of debris and to disinfect the root canal system. The use of antimicrobial irrigants that limit damage to the dental pulp are the goals of endodontic biomaterials research. Using an existing biorepository of dental pulp stem cells (DPSCs), Endocyn was evaluated in varying concentrations in proliferation and viability assays, and compared with positive (sodium hypochlorite or bleach) and negative (phosphate-buffered saline) controls. The DPSC viability was reduced in the range of -8.3% to -15.8%, <i>p</i> = 0.22 to <i>p</i> = 0.042, while the growth inhibition varied between -29.7% and -63%, <i>p</i> = 0.041 to <i>p</i> = 0.022. However, the RNA analysis revealed that no significant changes in biomarker mRNA expression (Nestin, NANOG, Sox2, Oct4, CD73, CD90, and CD105) were observed. These data demonstrated that all of the concentrations of Endocyn inhibited the DPSC viability and growth, although only high concentrations were statistically significant. Moreover, the administration of Endocyn did not alter the DPSC biomarker expression, which are novel and important findings not previously observed or reported that may assist with the development of clinical decision protocols and methods for the treatment of vital pulp tissue.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 1","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11858511/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143492953","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":"Wild Birds' Genetic Resources Bank: Feather Follicle Cell Culture as a Possible Source of Stem Cells.","authors":"Yasmin Godoi Dos Reis, Maria Eduarda Pralon Guerra, Meline de Paula Coutinho, Sarah Ingrid Pinto Santos, Bruna Dias Mota, Lauriene Luiza de Souza Munhoz, Diogo Pascoal Rossetti, Daniele Dos Santos Martins","doi":"10.3390/mps8010017","DOIUrl":"10.3390/mps8010017","url":null,"abstract":"<p><p>Follicular cells represent a valuable resource for genetic research, biotechnology and cryopreservation in biobanks, particularly for the conservation of endangered species. They offer a more practical alternative to gametes, embryos and fibroblasts. Collection of these cells can be achieved through feather plucking. Feather samples were opened with a scalpel and the feather pulp was washed with PBS, cut into cubes and digested in collagenase type IV. Cultivation was carried out in DMEM culture medium with 15% fetal bovine serum, 1% penicillin/streptomycin and 0.5% amphotericin, under incubation conditions of 39.5 °C and 5% CO₂. Passages were carried out with 5% EDTA for 5 min. The culture was successful, with great cell proliferation, adherence to plastic and aggregation into cell colonies. This method was effective in obtaining feather follicle cells from wild birds, especially when collected up to 6 h after their death, and can serve as a base protocol for research with feather follicle cells aiming to create biobanks.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 1","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-02-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11858370/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143492914","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":"Simplified Protocol for the Purification of Native Cas Nucleases for DNA-Free Genome Editing.","authors":"Margherita D'Amico, Flavia Angela Maria Maggiolini, Lucia Rosaria Forleo, Maria Francesca Cardone, Riccardo Velasco, Teodora Basile, Carlo Bergamini","doi":"10.3390/mps8010016","DOIUrl":"10.3390/mps8010016","url":null,"abstract":"<p><p>DNA-free genome editing by the direct delivery of CRISPR-associated nucleases has emerged as a promising technology due to its precision and reduced risk of off-target effects. However, existing purification protocols for native Cas proteins require the use of complex instrumentation, which limits their application. Here, we present a simplified protocol for the purification of native Cas9, Cas12RR and dCas9-VP64 nucleases optimized for DNA-free genome editing. Our approach leverages a streamlined affinity and ion exchange chromatography coupled with minimal downstream processing, ensuring a good yield and activity of the purified proteins. The in vitro analysis of the purified ribonucleoprotein complex demonstrated a good efficiency of DNA target cleavage. This simplified protocol increases the opportunity to adopt CRISPR technology, and enables broader access to DNA-free genome editing tools also for laboratories that are not specifically equipped for protein purification.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 1","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11857876/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143492969","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":"Exploratory Ultrasound Analysis of the Diaphragm and Respiratory Capacity in Women with Primary Dysmenorrhea: A Cross-Sectional Observational Study.","authors":"Rebeca Del Prado-Álvarez, María García-Arrabé, Ángel González-de-la-Flor, Marta de la Plaza San Frutos, Jaime Almazán-Polo, Cecilia Estrada-Barranco","doi":"10.3390/mps8010015","DOIUrl":"10.3390/mps8010015","url":null,"abstract":"<p><p>Primary dysmenorrhea (PD) is a common gynecological condition characterized by menstrual pain without underlying pelvic pathology. It has been linked to functional and structural changes in the core musculature, but limited evidence exists regarding its association with diaphragmatic and respiratory mechanics. This study aimed to elaborate on these potential associations by assessing the diaphragmatic structure and respiratory function in women with PD compared to healthy controls, utilizing ultrasound imaging, spirometry and respiratory pressure measurements.</p><p><strong>Methods: </strong>An observational, cross-sectional study was conducted with 44 female participants (22 with PD and 22 healthy controls). Diaphragmatic structure was evaluated through ultrasound, measuring the intercostal distance, diaphragmatic thickness, and diaphragmatic excursion at rest and during maximum voluntary contraction. Spirometric assessments included forced vital capacity (FVC), forced expiratory volume in the first second (FEV1), and the FVC/FEV1 ratio, along with measurements of maximum inspiratory pressure (MIP) and maximum expiratory pressure (MEP). Group differences were analyzed using Student's <i>t</i>-test and effect sizes were reported with Cohen's d.</p><p><strong>Results: </strong>No significant differences were observed between the groups in diaphragmatic thickness, diaphragmatic excursion, or global respiratory capacity (<i>p</i> > 0.05). However, women with PD presented a significant reduction in the left intercostal distance both at rest (<i>p</i> = 0.035, d = 0.56) and during contraction (<i>p</i> = 0.039, d = 0.54). No other significant group differences were detected.</p><p><strong>Conclusions: </strong>While primary dysmenorrhea does not appear to affect overall diaphragmatic function or respiratory capacity, it is associated with subtle localized changes in the left intercostal dynamics. These findings suggest a potential compensatory mechanical adaptation rather than global respiratory dysfunction. Further longitudinal studies with larger sample sizes are needed to explore the clinical significance of these findings.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 1","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11858394/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143492963","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":"Imaging Flow Cytometry in HIV Infection Research: Advantages and Opportunities.","authors":"Kirill A Elfimov, Dmitriy A Baboshko, Natalya M Gashnikova","doi":"10.3390/mps8010014","DOIUrl":"10.3390/mps8010014","url":null,"abstract":"<p><p>The human immunodeficiency virus (HIV) is a type of retrovirus that infects humans and belongs to the Lentivirus group. Despite the availability of effective treatments, HIV infections are still increasing in some parts of the world, according to the World Health Organization (WHO). Another major challenge is the growing problem of HIV becoming resistant to drugs. This highlights the importance of ongoing research to better understand HIV and find new ways to stop the virus from spreading in the body. Scientists use a variety of methods to study HIV, including techniques from molecular and cellular biology. Many of these methods rely on fluorescent dyes to help visualize specific parts of the virus or infected cells. This article focuses on a technique called imaging flow cytometry, which is particularly useful for studying HIV. Imaging flow cytometry is unique because it not only measures fluorescence (light emitted by the dyes) but also captures images of each cell being analyzed. This allows researchers to see where the fluorescence is located within the cell and to study the cell's shape and structure in detail. Additionally, this method can be combined with machine learning to analyze large amounts of data more efficiently.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 1","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-02-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11858172/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143492964","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}