Methods and Protocols最新文献_第4页

A Double Fluorescent Staining Method Increases the Sensitivity of the Cytokinesis-Block Micronucleus Assay. 双荧光染色法提高细胞分裂阻滞微核试验的灵敏度。

IF 2.3

Methods and Protocols Pub Date : 2025-01-04 DOI: 10.3390/mps8010003

Ion Udroiu

引用次数: 0

In Vivo Imaging of Cobalt-Induced Ocular Toxicity in a Mouse Model. 钴致小鼠眼毒性模型的体内成像。

IF 2.3

Methods and Protocols Pub Date : 2025-01-02 DOI: 10.3390/mps8010001

Basel Obied, Galit Saar, Stephen Richard, Ygal Rotenstreich, Ifat Sher, Alon Zahavi, Nitza Goldenberg-Cohen

{"title":"In Vivo Imaging of Cobalt-Induced Ocular Toxicity in a Mouse Model.","authors":"Basel Obied, Galit Saar, Stephen Richard, Ygal Rotenstreich, Ifat Sher, Alon Zahavi, Nitza Goldenberg-Cohen","doi":"10.3390/mps8010001","DOIUrl":"10.3390/mps8010001","url":null,"abstract":"Cobalt is a trace element, crucial for red blood cell formation and neurological function. Cobalt toxicity is often only diagnosed after severe manifestations, including visual impairment. We aimed to investigate whether optical coherence tomography (OCT) and magnetic resonance imaging (MRI) can effectively detect cobalt-induced ocular toxicity in a murine model. Five wild-type mice (WT, C57Bl6) received daily intraperitoneal cobalt chloride injections for 28 days with a dosage of 12.5 mg/kg. Another 5 WT mice served as controls. After 28 days, all mice underwent manganese contrast-enhanced MRI and OCT examinations. Macroscopic and histological analysis of the enucleated eyes were performed. MRI revealed an increased signal in the optic nerves of injected mice. Anterion OCT provided in vivo visualization of the entire eye, demonstrating incipient cataract formation in the cobalt-injected mice. Both Spectralis domain OCT and Anterion, followed by histological analyses, confirmed preserved retinal structure with decreased thickness in the cobalt-injected group, with only minor neuronal damage and cell loss. Optic nerve analysis demonstrated myelin loss and increased inflammation with high levels of reactive gliosis. This study demonstrates optic neuropathy induced by cobalt toxicity, as shown by increased optic nerve signal on MRI without significant retinopathy. Anterion OCT showed incipient cataracts in the anterior segment.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 1","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11755644/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143024017","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

Differential DNA Extraction from Lateral Flow Immunochromatographic Tests via the EZ1^® Advanced XL System. 通过EZ1®高级XL系统从横向流动免疫层析测试中提取差异DNA。

IF 2.3

Methods and Protocols Pub Date : 2025-01-02 DOI: 10.3390/mps8010002

Scarlet Neilson, Leah Nangeroni, Mirna Ghemrawi

{"title":"Differential DNA Extraction from Lateral Flow Immunochromatographic Tests via the EZ1® Advanced XL System.","authors":"Scarlet Neilson, Leah Nangeroni, Mirna Ghemrawi","doi":"10.3390/mps8010002","DOIUrl":"10.3390/mps8010002","url":null,"abstract":"This differential extraction protocol details the steps for isolating DNA from sample pads used in lateral flow immunochromatographic (LFI) tests, particularly for cases involving mixed biological samples such as semen and menstrual blood, or other evidence related to sexual assault. This procedure utilizes a differential extraction technique applied to sample pads from immunochromatographic tests, where the sample pads serve as the substrate. The method involves two sequential lysis steps to effectively separate non-sperm and sperm fractions, enabling the targeted isolation of distinct cell types for downstream DNA analysis. The efficiency of this procedure is demonstrated by the results within this paper, which highlights the successful recovery of both male autosomal and Y-STR profiles, even in mixed samples with a high female presence. Overall, this protocol demonstrates the effective recovery of DNA from sample pads, which is beneficial for forensic practitioners dealing with limited sample quantities, underscoring the value of using these pads in forensic analysis.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 1","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11755612/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143023962","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

Evaluation of Protocols for DNA Extraction from Individual Culex pipiens to Assess Pyrethroid Resistance Using Genotyping Real-Time Polymerase Chain Reaction. 实时聚合酶链反应基因分型法检测库蚊对拟除虫菊酯类杀虫剂抗性的方法研究

IF 2.3

Methods and Protocols Pub Date : 2024-12-23 DOI: 10.3390/mps7060106

Ilaria Congiu, Elisa Cugini, Daniele Smedile, Federico Romiti, Manuela Iurescia, Valentina Donati, Claudio De Liberato, Antonio Battisti

{"title":"Evaluation of Protocols for DNA Extraction from Individual Culex pipiens to Assess Pyrethroid Resistance Using Genotyping Real-Time Polymerase Chain Reaction.","authors":"Ilaria Congiu, Elisa Cugini, Daniele Smedile, Federico Romiti, Manuela Iurescia, Valentina Donati, Claudio De Liberato, Antonio Battisti","doi":"10.3390/mps7060106","DOIUrl":"10.3390/mps7060106","url":null,"abstract":"Culex pipiens is a major vector of pathogens, including West Nile and Usutu viruses, that poses a significant public health risk. Monitoring pyrethroid resistance in mosquito populations is essential for effective vector control. This study aims to evaluate four DNA extraction protocols-QIAsymphony, DNAzol® Direct reagent, PrepMan® Ultra Sample Preparation Reagent (USPR), and Chelex® 100-to identify an optimal method to extract DNA from individual Culex pipiens, as part of a high-throughput surveillance of pyrethroid resistance using Real-Time Genotyping PCR. The target is the L1014F mutation in the voltage-sensitive sodium channel (VSSC) gene, which confers knockdown (kdr) resistance to pyrethroids. Mosquitoes were collected from wintering and summer habitats in Lazio and Tuscany, Italy, and DNA was extracted using the four methods. The quality, quantity, extraction time, and cost of the DNA were compared among the various methods. The PrepMan® USPR protocol was the most efficient, providing high-quality DNA with a 260/280 purity ratio within the optimal range at the lowest cost and in a short time. This method also demonstrated the highest amplification success rate (77%) in subsequent real-time PCR assays, making it the preferred protocol for large-scale genotyping studies.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 6","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11677584/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142896143","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

Formalin and 2.5% Glutaraldehyde/2% Paraformaldehyde in 0.1 M Cacodylate Buffer Inactivation Protocols to Ensure the Proper Fixation of Positive Sense RNA Viruses and Genomic Material Prior to Removal from Containment. 福尔马林和2.5%戊二醛/2%多聚甲醛在0.1 M草酸缓冲液中灭活方案，以确保在从容器中移除之前正确固定阳性RNA病毒和基因组物质。

IF 2.3

Methods and Protocols Pub Date : 2024-12-21 DOI: 10.3390/mps7060105

Lauren E Panny, Ashley E Piper, Christina L Gardner, Crystal W Burke

{"title":"Formalin and 2.5% Glutaraldehyde/2% Paraformaldehyde in 0.1 M Cacodylate Buffer Inactivation Protocols to Ensure the Proper Fixation of Positive Sense RNA Viruses and Genomic Material Prior to Removal from Containment.","authors":"Lauren E Panny, Ashley E Piper, Christina L Gardner, Crystal W Burke","doi":"10.3390/mps7060105","DOIUrl":"10.3390/mps7060105","url":null,"abstract":"Recommendations released by the CDC in 2023 address the need to demonstrate that the RNA genome of positive-strand RNA viruses is inactivated in addition to viral particles. This recommendation is in response to the similarities between host mRNA and the viral genome that allow the viral RNA to be used as a template by host replication mechanisms to produce infectious viruses; therefore, there is concern that through artificial introduction into host cells, active positive-strand RNA genomes can be utilized to produce infectious viruses out of a containment facility. Utilizing 10% formalin for 7 days or 2.5% glutaraldehyde/2% paraformaldehyde in 0.1 M cacodylate buffer (glut/PFA) for 2 days to fix eastern equine encephalitis virus (EEEV)-infected non-human primate (NHP) brain tissue was found to effectively inactivate EEEV particles and genomic RNA. The methods assessed in this paper outline an effective means to validate both genomic RNA and viral particle inactivation.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 6","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-12-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11676695/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142896144","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 Measurement of Spatiotemporal Parameters in Running at Different Velocities: A Comparison Between a GPS Unit and an Infrared Mat. 不同运动速度下时空参数的测量：GPS单元与红外垫的比较。

IF 2.3

Methods and Protocols Pub Date : 2024-12-20 DOI: 10.3390/mps7060103

Thomas Provot, Benjamin Millot, Eline Hazotte, Thomas Rousseau, Jean Slawinski

{"title":"The Measurement of Spatiotemporal Parameters in Running at Different Velocities: A Comparison Between a GPS Unit and an Infrared Mat.","authors":"Thomas Provot, Benjamin Millot, Eline Hazotte, Thomas Rousseau, Jean Slawinski","doi":"10.3390/mps7060103","DOIUrl":"10.3390/mps7060103","url":null,"abstract":"The accurate measurement of spatiotemporal parameters, such as step length and step frequency, is crucial for analyzing running and sprinting performance. Traditional methods like video analysis and force platforms are either time consuming or limited in scope, prompting the need for more efficient technologies. This study evaluates the effectiveness of a commercial Global Positioning System (GPS) unit integrated with an Inertial Measurement Unit (IMU) in capturing these parameters during sprints at varying velocities. Five experienced male runners performed six 40 m sprints at three velocity conditions (S: Slow, M: Medium, F: Fast) while equipped with a GPS-IMU system and an optical system as the gold standard reference. A total of 398 steps were analyzed for this study. Step frequency, step length and step velocity were extracted and compared using statistical methods, including the coefficient of determination (r2) and root mean square error (RMSE). Results indicated a very large agreement between the embedded system and the reference system, for the step frequency (r2 = 0.92, RMSE = 0.14 Hz), for the step length (r2 = 0.91, RMSE = 0.07 m) and the step velocity (r2 = 0.99, RMSE = 0.17 m/s). The GPS-IMU system accurately measured spatiotemporal parameters across different running velocities, demonstrating low relative errors and high precision. This study demonstrates that GPS-IMU systems can provide comprehensive spatiotemporal data, making them valuable for both training and competition. The integration of these technologies offers practical benefits, helping coaches better understand and enhance running performance. Future improvements in sample rate acquisition GPS-IMU technology could further increase measurement accuracy and expand its application in elite sports.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 6","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11676369/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142896149","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

Establishment of Stable Knockdown of MACC1 Oncogene in Patient-Derived Ovarian Cancer Organoids. 在患者源性卵巢癌类器官中稳定敲低MACC1癌基因的建立。

IF 2.3

Methods and Protocols Pub Date : 2024-12-20 DOI: 10.3390/mps7060104

Sophia Hierlmayer, Liliia Hladchenko, Juliane Reichenbach, Christoph Klein, Sven Mahner, Fabian Trillsch, Mirjana Kessler, Anca Chelariu-Raicu

{"title":"Establishment of Stable Knockdown of MACC1 Oncogene in Patient-Derived Ovarian Cancer Organoids.","authors":"Sophia Hierlmayer, Liliia Hladchenko, Juliane Reichenbach, Christoph Klein, Sven Mahner, Fabian Trillsch, Mirjana Kessler, Anca Chelariu-Raicu","doi":"10.3390/mps7060104","DOIUrl":"10.3390/mps7060104","url":null,"abstract":"High-grade serous ovarian cancer (HGSOC) remains the most lethal gynecological malignancy, and there is still an unmet medical need to deepen basic research on its origins and mechanisms of progression. Patient-derived organoids of high-grade serous ovarian cancer (HGSOC-PDO) are a powerful model to study the complexity of ovarian cancer as they maintain, in vitro, the mutational profile and cellular architecture of the cancer tissue. Genetic modifications by lentiviral transduction allow novel insights into signaling pathways and the potential identification of biomarkers regarding the evolution of drug resistance. Here, we provide an in-depth and detailed protocol to successfully modify the gene expression of HGSOC-PDOs by lentiviral transduction. As an example, we validate our protocol and create a stable knockdown of the MACC1 oncogene with an efficacy of ≥72% in two HGSOC-PDO lines, which remained stable for >3 months in culture. Moreover, we explain step-by-step the sample preparation for the validation procedures on transcriptional (qPCR) and protein (Western Blot) levels. Sustained downregulation of specific genes by lentiviral transduction enables the analysis of the resulting phenotypic and morphological changes. It serves as a valuable in-vitro model to study the mechanisms of ovarian cancer pathogenesis and allows for the evaluation of therapeutic approaches.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 6","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11678392/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142896142","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

Studies on Square Wave and Cyclic Voltammetric Behavior of 1,2- and 1,4-Dihydroxybenzenes and Their Derivatives in Acetic Acid, Ethyl Acetate and Mixtures of the Two. 1,2-和1,4-二羟基苯及其衍生物在乙酸、乙酸乙酯和两者混合物中的方波和循环伏安行为研究

IF 2.3

Methods and Protocols Pub Date : 2024-12-20 DOI: 10.3390/mps7060102

László Kiss

{"title":"Studies on Square Wave and Cyclic Voltammetric Behavior of 1,2- and 1,4-Dihydroxybenzenes and Their Derivatives in Acetic Acid, Ethyl Acetate and Mixtures of the Two.","authors":"László Kiss","doi":"10.3390/mps7060102","DOIUrl":"10.3390/mps7060102","url":null,"abstract":"An electrochemical investigation of 1,2- and 1,4-dihydroxybenzenes was carried out with platinum macro- and microelectrodes using square wave and cyclic voltammetry techniques. Furthermore, the effect of the two solvents-acetic acid and ethyl acetate-was compared. When using square wave voltammetry, signals only appeared at lower frequencies and only when the supporting electrolyte was in excess, as expected due to the relatively low permittivity of the used solvents. The behavior of hydroquinone and catechol did not differ significantly from that of their derivatives (dihydroxybenzaldehydes, dihydroxybenzoic acids and 2',5'-dihydroxyacetophenone). When the cyclic voltammetric experiments using a microelectrode were extended to higher anodic potentials, electrode fouling was very significant in ethyl acetate after the potential region where steady-state oxidation to the corresponding quinone occurs. The substituent effect was not significant here either, which was proven by using different functional groups in different positions. In contrast, the position had a dramatic influence on the susceptibility to electropolymerization, as 1,2-dihydroxybenzenes-independent of the nature of the substituent on the benzene ring-deactivated the electrode, while 1,4-dihydroxybenzenes did not, possibly due to the different solubilities of the polymers formed from the primary oxidation product (quinones). A user-friendly analytical procedure is also proposed that uses an electropolymerization reaction and does not require frequent cleaning of the electrode via polishing, which is required usually especially with a microelectrode.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 6","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11679714/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142896148","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

Proof of Concept Study: Comparison of Semi-Automated RNA Isolation Methods from Archived Formalin-Fixed, Paraffin-Embedded Tissues with Clinical Routine RNA Isolation Methods. 概念验证研究：从存档的福尔马林固定石蜡包埋组织中半自动RNA分离方法与临床常规RNA分离方法的比较。

IF 2.3

Methods and Protocols Pub Date : 2024-12-19 DOI: 10.3390/mps7060101

Patrick Hannibal Dalsbo Petersen, Jaslin Pallikkunnath James, Lene Buhl Riis, Claus Kim Høgdall, Estrid Vilma Høgdall

{"title":"Proof of Concept Study: Comparison of Semi-Automated RNA Isolation Methods from Archived Formalin-Fixed, Paraffin-Embedded Tissues with Clinical Routine RNA Isolation Methods.","authors":"Patrick Hannibal Dalsbo Petersen, Jaslin Pallikkunnath James, Lene Buhl Riis, Claus Kim Høgdall, Estrid Vilma Høgdall","doi":"10.3390/mps7060101","DOIUrl":"10.3390/mps7060101","url":null,"abstract":"High-quality RNA is crucial in clinical diagnostics and precision medicine. Formalin-fixed and paraffin-embedded (FFPE) tissues pose a challenge due to nucleic acid fragmentation and crosslinking. In this pilot study, various commercially available techniques for extracting RNA from small FFPE samples were compared. We evaluated the KingFisher Duo automated system or the manual MagMAX FFPE DNA/RNA Ultra Kit as an RNA extraction method combined with either a xylene, d-limonene, or AutoLys M tubes deparaffinization method. Additionally, the automated Maxwell RSC RNA FFPE kit and the High Pure FFPET RNA Isolation Kit were examined using FFPE samples from inflammatory bowel disease (IBD) patients, as well as samples from ovarian, kidney, and breast cancer and the skin. The KingFisher Duo system gave a higher yield and more consistent RNA quantities, especially from small volumes of IBD samples, compared to manual extraction. The deparaffinization method also impacted results, with AutoLys M tubes proving effective in combination with the KingFisher Duo system. Conversely, the High Pure kit exhibited higher yields for larger FFPE samples. While RNA integrity is a critical factor, particularly for messenger RNA (mRNA) expression studies, its role is less prominent in microRNA (miRNA) analyses. Recognizing this, our study focused on RNA yield and purity (A260/A230) to evaluate RNA extraction methods for various sample types. These findings emphasize the importance of selecting appropriate RNA extraction methods based on sample characteristics and research goals, highlighting the performance of automated methods and the impact of deparaffinization choices. The findings contribute to refining RNA extraction for molecular biology analyses, suggesting avenues for further exploration, including cost-effectiveness under specific experimental conditions.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 6","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11678837/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142896147","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

Liver Cancer Neuroscience: Regulating Liver Tumors via Selective Hepatic Vagotomy. 肝癌神经科学：通过选择性肝迷走神经切开术调节肝脏肿瘤。

IF 2.3

Methods and Protocols Pub Date : 2024-12-11 DOI: 10.3390/mps7060099

Kylynda C Bauer, Shadin Ghabra, Chi Ma, Lee Chedester, Tim F Greten

引用次数: 0