Methods and Protocols最新文献_第10页

A Novel and Quantitative Detection Assay (effluxR) for Identifying Efflux-Associated Resistance Genes Using Multiplex Digital PCR in Clinical Isolates of Pseudomonas aeruginosa. 一种新的定量检测方法（effluxR），用于在铜绿假单胞菌临床分离株中使用多重数字PCR鉴定与流出相关的耐药性基因。

IF 2.4

Methods and Protocols Pub Date : 2023-10-08 DOI: 10.3390/mps6050096

Nontaporn Rattanachak, Sattaporn Weawsiangsang, Robert A Baldock, Theerasak Jaifoo, Touchkanin Jongjitvimol, Jirapas Jongjitwimol

{"title":"A Novel and Quantitative Detection Assay (effluxR) for Identifying Efflux-Associated Resistance Genes Using Multiplex Digital PCR in Clinical Isolates of Pseudomonas aeruginosa.","authors":"Nontaporn Rattanachak, Sattaporn Weawsiangsang, Robert A Baldock, Theerasak Jaifoo, Touchkanin Jongjitvimol, Jirapas Jongjitwimol","doi":"10.3390/mps6050096","DOIUrl":"10.3390/mps6050096","url":null,"abstract":"The rise of multidrug resistance of Pseudomonas aeruginosa highlights an increased need for selective and precise antimicrobial treatment. Drug efflux pumps are one of the major mechanisms of antimicrobial resistance found in many bacteria, including P. aeruginosa. Detection of efflux genes using a polymerase chain reaction (PCR)-based system would enable resistance detection and aid clinical decision making. Therefore, we aimed to develop and optimize a novel method herein referred to as \"effluxR detection assay\" using multiplex digital PCR (mdPCR) for detection of mex efflux pump genes in P. aeruginosa strains. The annealing/extension temperatures and gDNA concentrations were optimized to amplify mexB, mexD, and mexY using the multiplex quantitative PCR (mqPCR) system. We established the optimal mqPCR conditions for the assay (Ta of 59 °C with gDNA concentrations at or above 0.5 ng/µL). Using these conditions, we were able to successfully detect the presence of these genes in a quantity-dependent manner. The limit of detection for mex genes using the effluxR detection assay with mdPCR was 0.001 ng/µL (7.04-34.81 copies/µL). Moreover, using blind sample testing, we show that effluxR detection assay had 100% sensitivity and specificity for detecting mex genes in P. aeruginosa. In conclusion, the effluxR detection assay, using mdPCR, is able to identify the presence of multiple mex genes in P. aeruginosa that may aid clinical laboratory decisions and further epidemiological studies.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2023-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10608825/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54230070","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

Allergens and Other Harmful Substances in Hydroalcoholic Gels: Compliance with Current Regulation. 水酒精凝胶中的过敏原和其他有害物质：符合现行法规。

IF 2.4

Methods and Protocols Pub Date : 2023-10-07 DOI: 10.3390/mps6050095

Ana Castiñeira-Landeira, Lua Vazquez, Thierry Dagnac, Maria Celeiro, María Llompart

{"title":"Allergens and Other Harmful Substances in Hydroalcoholic Gels: Compliance with Current Regulation.","authors":"Ana Castiñeira-Landeira, Lua Vazquez, Thierry Dagnac, Maria Celeiro, María Llompart","doi":"10.3390/mps6050095","DOIUrl":"10.3390/mps6050095","url":null,"abstract":"Hydroalcoholic gels or hand sanitisers have become essential products to prevent and mitigate the transmission of COVID-19. Depending on their use, they can be classified as cosmetics (cleaning the skin) or biocides (with antimicrobial effects). The aim of this work was to determine sixty personal care products frequently found in cosmetic formulations, including fragrance allergens, synthetic musks, preservatives and plasticisers, in hydroalcoholic gels and evaluate their compliance with the current regulation. A simple and fast analytical methodology based on solid-phase microextraction followed by gas chromatography-tandem mass spectrometry (SPME-GC-MS/MS) was validated and applied to 67 real samples. Among the 60 target compounds, 47 of them were found in the analysed hand sanitisers, highlighting the high number of fragrance allergens (up to 23) at concentrations of up to 32,458 μg g-1. Most of the samples did not comply with the labelling requirements of the EU Regulation No 1223/2009, and some of them even contained compounds banned in cosmetic products such as plasticisers. Method sustainability was also evaluated using the metric tool AGREEPrep, demonstrating its greenness.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2023-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10609030/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54230075","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A Simple and Fast Method for the Formation and Downstream Processing of Cancer-Cell-Derived 3D Spheroids: An Example Using Nicotine-Treated A549 Lung Cancer 3D Spheres. 用于癌细胞衍生的3D球体的形成和下游处理的简单且快速的方法：使用尼古丁处理的A549癌症3D球体的实例。

IF 2.4

Methods and Protocols Pub Date : 2023-10-04 DOI: 10.3390/mps6050094

Irida Papapostolou, Florian Bochen, Christine Peinelt, Maria Constanza Maldifassi

{"title":"A Simple and Fast Method for the Formation and Downstream Processing of Cancer-Cell-Derived 3D Spheroids: An Example Using Nicotine-Treated A549 Lung Cancer 3D Spheres.","authors":"Irida Papapostolou, Florian Bochen, Christine Peinelt, Maria Constanza Maldifassi","doi":"10.3390/mps6050094","DOIUrl":"10.3390/mps6050094","url":null,"abstract":"Although 2D in vitro cancer cell cultures have been used for decades as a first line-of-research tool to investigate antitumoral drugs and treatments, their use presents many drawbacks, including the poor resemblance of such cultures to the characteristics of in vivo tumors. To mitigate these drawbacks, 3D culture models have emerged as a more representative alternative. Cancer cells cultured as 3D structures have the advantage of resembling solid tumors in their architecture and in their resistance to chemotherapeutic drugs, in part because of restrained drug penetration. Additionally, these 3D structures create a more physiological environment for the study of immune cell invasion and migration, comparable to solid tumors. In this paper, we describe a fast and cost-effective step-by-step protocol for the generation of 3D spheres using ultra-low-attachment (ULA) multiwell plates, which can be incorporated into the normal workflow of any laboratory. Using this protocol, spheroids of different human cancer cell lines can be obtained and can then be characterized on the basis of their morphology, viability, and expression of specific markers.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2023-10-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10609300/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54230071","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A Simplified Microscopy Technique to Rapidly Characterize Individual Fiber Traits in Cotton. 一种快速表征棉花单个纤维性状的简化显微镜技术。

IF 2.4

Methods and Protocols Pub Date : 2023-10-03 DOI: 10.3390/mps6050092

Quinn LaFave, Shalini P Etukuri, Chaney L Courtney, Neha Kothari, Trevor W Rife, Christopher A Saski

{"title":"A Simplified Microscopy Technique to Rapidly Characterize Individual Fiber Traits in Cotton.","authors":"Quinn LaFave, Shalini P Etukuri, Chaney L Courtney, Neha Kothari, Trevor W Rife, Christopher A Saski","doi":"10.3390/mps6050092","DOIUrl":"10.3390/mps6050092","url":null,"abstract":"Recent advances in phenotyping techniques have substantially improved the ability to mitigate type-II errors typically associated with high variance in phenotyping data sets. In particular, the implementation of automated techniques such as the High-Volume Instrument (HVI) and the Advanced Fiber Information System (AFIS) have significantly enhanced the reproducibility and standardization of various fiber quality measurements in cotton. However, micronaire is not a direct measure of either maturity or fineness, lending to limitations. AFIS only provides a calculated form of fiber diameter, not a direct measure, justifying the need for a visual-based reference method. Obtaining direct measurements of individual fibers through cross-sectional analysis and electron microscopy is a widely accepted standard but is time-consuming and requires the use of hazardous chemicals and specialized equipment. In this study, we present a simplified fiber histology and image acquisition technique that is both rapid and reproducible. We also introduce an automated image analysis program that utilizes machine learning to differentiate good fibers from bad and to subsequently collect critical phenotypic measurements. These methods have the potential to improve the efficiency of cotton fiber phenotyping, allowing for greater precision in unravelling the genetic architecture of critical traits such as fiber diameter, shape, areas of the secondary cell wall/lumen, and others, ultimately leading to larger genetic gains in fiber quality and improvements in cotton.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2023-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10609321/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54230073","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

pH and NaCl Optimisation to Improve the Stability of Gold and Silver Nanoparticles' Anti-Zearalenone Antibody Conjugates for Immunochromatographic Assay. pH和NaCl优化以提高金和银纳米粒子的抗玉米赤霉烯酮抗体缀合物的稳定性，用于免疫色谱测定。

IF 2.4

Methods and Protocols Pub Date : 2023-10-03 DOI: 10.3390/mps6050093

Thasmin Shahjahan, Bilal Javed, Vinayak Sharma, Furong Tian

{"title":"pH and NaCl Optimisation to Improve the Stability of Gold and Silver Nanoparticles' Anti-Zearalenone Antibody Conjugates for Immunochromatographic Assay.","authors":"Thasmin Shahjahan, Bilal Javed, Vinayak Sharma, Furong Tian","doi":"10.3390/mps6050093","DOIUrl":"10.3390/mps6050093","url":null,"abstract":"The aim of this research is to define optimal conditions to improve the stability of gold and silver nanoparticles' anti-zearalenone antibody conjugates for their utilisation in lateral flow immunochromatographic assay (LFIA). The Turkevich-Frens method was used to synthesise gold nanoparticles (AuNPs), which were between 10 and 110 nm in diameter. Silver nanoparticles (AgNPs) with a size distribution of 2.5 to 100 nm were synthesised using sodium borohydride as a reducing agent. The onset of AuNP and AgNP aggregation occurred at 150 mM and 80 mM NaCl concentrations, respectively. Stable Au and Ag nanoparticle-antibody conjugates were achieved at 1.2 mM of K2CO3 concentration, which corresponds to the pH value of ≈7. Lastly, the highest degree of conjugation between Au and Ag nanoparticles and anti-zearalenone antibodies was at 4 and 6 µg/mL of antibody concentrations. The optimisation of the conjugation conditions can contribute to better stability of nanoparticles and their antibody conjugate and can improve the reproducibility of results of bioreporter molecules in biosensing lateral flow devices.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2023-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10609120/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54230082","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

Diet and Depression during Peri- and Post-Menopause: A Scoping Review Protocol. 更年期前后饮食与抑郁：范围界定审查方案。

IF 2.4

Methods and Protocols Pub Date : 2023-10-02 DOI: 10.3390/mps6050091

Alexandra M Bodnaruc, Miryam Duquet, Denis Prud'homme, Isabelle Giroux

{"title":"Diet and Depression during Peri- and Post-Menopause: A Scoping Review Protocol.","authors":"Alexandra M Bodnaruc, Miryam Duquet, Denis Prud'homme, Isabelle Giroux","doi":"10.3390/mps6050091","DOIUrl":"10.3390/mps6050091","url":null,"abstract":"The aim of the proposed scoping review is to describe and summarize studies assessing the associations between diet-related variables and depression in peri- and post-menopausal women. Studies examining the associations between diet-related variables and mental health indicators in women undergoing menopausal transition or in the post-menopausal period will be systematically retrieved via Medline, EMBASE, PsycINFO, Web of Science, and Scopus databases. All articles identified through the database searches will be imported into Covidence. Following the removal of duplicates, two authors will independently perform title and abstract screening, as well as full-text assessment against eligibility criteria. Data will be extracted using tables developed for observational and experimental studies. The methodological quality of randomized trials, cohort and cross-sectional studies, and case–control studies, will be assessed using the Cochrane risk-of-bias (RoB-2) tool, the NHLBI Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies, and the NHLBI Quality Assessment Tool for Case–Control studies, respectively. Data extraction tables will be used to produce two tables summarizing the main characteristics and findings of the studies included in the review. In the proposed review, we will systematically identify and summarize the currently available evidence on the association between diet-related variables and depression in peri- and post-menopausal women. To our knowledge, this is the first review focusing on this subgroup of the population. Protocol registration: osf.io/b89r6.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2023-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10609501/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54230077","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

Generation of a Recombinant scFv against Deoxycholic Acid and Its Conversion to a Quenchbody for One-Step Immunoassay. 抗脱氧胆酸的重组单链抗体的产生及其转化为用于一步免疫测定的淬灭体。

IF 2.4

Methods and Protocols Pub Date : 2023-09-30 DOI: 10.3390/mps6050090

Hiroshi Ueda, Hee-Jin Jeong

引用次数: 0

High-Efficiency Transformation and Expression of Genomic Libraries in Yeast. 基因组文库在酵母中的高效转化和表达。

IF 2.4

Methods and Protocols Pub Date : 2023-09-21 DOI: 10.3390/mps6050089

Mira Loock, Luiza Berenguer Antunes, Rhiannon T Heslop, Antonio Alfonso De Lauri, Andressa Brito Lira, Igor Cestari

{"title":"High-Efficiency Transformation and Expression of Genomic Libraries in Yeast.","authors":"Mira Loock, Luiza Berenguer Antunes, Rhiannon T Heslop, Antonio Alfonso De Lauri, Andressa Brito Lira, Igor Cestari","doi":"10.3390/mps6050089","DOIUrl":"https://doi.org/10.3390/mps6050089","url":null,"abstract":"Saccharomyces cerevisiae is a powerful system for the expression of genome-wide or combinatorial libraries for diverse types of screening. However, expressing large libraries in yeast requires high-efficiency transformation and controlled expression. Transformation of yeast using electroporation methods is more efficient than chemical methods; however, protocols described for electroporation require large amounts of linearized plasmid DNA and often yield approximately 106 cfu/µg of plasmid DNA. We optimized the electroporation of yeast cells for the expression of whole-genome libraries to yield up to 108 cfu/µg plasmid DNA. The protocol generates sufficient transformants for 10-100× coverage of diverse genome libraries with small amounts of genomic libraries (0.1 µg of DNA per reaction) and provides guidance on calculations to estimate library size coverage and transformation efficiency. It describes the preparation of electrocompetent yeast cells with lithium acetate and dithiothreitol conditioning step and the transformation of cells by electroporation with carrier DNA. We validated the protocol using three yeast surface display libraries and demonstrated using nanopore sequencing that libraries' size and diversity are preserved. Moreover, expression analysis confirmed library functionality and the method's efficacy. Hence, this protocol yields a sufficient representation of the genome of interest for downstream screening purposes while limiting the amount of the genomic library required.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2023-09-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10514863/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41141128","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A Robust and Standardized Approach to Quantify Wound Closure Using the Scratch Assay. 一种稳健和标准化的方法来量化伤口闭合使用划痕测定。

IF 2.4

Methods and Protocols Pub Date : 2023-09-17 DOI: 10.3390/mps6050087

Stefan Balko, Evan Kerr, Edward Buchel, Sarvesh Logsetty, Afshin Raouf

引用次数: 0

Epidemiology of Maternal Nutritional Status and Risk of Adverse Birth Outcomes in Undernourished Mothers with Sickle Cell Disease: A Systematic Review and Meta-Analysis Protocol. 镰状细胞病营养不良母亲营养状况和不良出生结局风险的流行病学：系统综述和荟萃分析方案。

IF 2.4

Methods and Protocols Pub Date : 2023-09-17 DOI: 10.3390/mps6050088

Lauren J Klein, John Benaiah Ayete-Nyampong, Annette M Williams, Lori A Harding, Samuel A Oppong, Sari Acra, Michael R DeBaun, Aamer Imdad

{"title":"Epidemiology of Maternal Nutritional Status and Risk of Adverse Birth Outcomes in Undernourished Mothers with Sickle Cell Disease: A Systematic Review and Meta-Analysis Protocol.","authors":"Lauren J Klein, John Benaiah Ayete-Nyampong, Annette M Williams, Lori A Harding, Samuel A Oppong, Sari Acra, Michael R DeBaun, Aamer Imdad","doi":"10.3390/mps6050088","DOIUrl":"10.3390/mps6050088","url":null,"abstract":"In pregnancies complicated by sickle cell disease (SCD), the maternal-fetal dyad is at high risk for mortality and morbidity. In healthy pregnancies, maternal nutritional status is a critical factor for the healthy growth and development of the fetus. However, there are no reviews of the current research on the nutritional status of pregnant women with SCD and pregnancy outcomes. First, we aim to assess the burden of malnutrition in pregnant women with SCD. Next, we aim to systematically evaluate if pregnant women with SCD who have poor nutritional status are at increased risk for adverse birth outcomes compared to pregnant women with sickle cell disease and normal nutritional status. We will systematically search multiple electronic databases. Our exposure is pregnant women with SCD and poor nutritional status. The primary outcomes of interest include low birth weight (categorical) and birth weight z-scores (continuous). We will also evaluate maternal and perinatal outcomes as secondary outcomes. We will evaluate the risk of bias and overall certainty of evidence with Risk of Bias in Non-randomized Studies-of Interventions (ROBINS-I), and the overall evidence will be assessed using Grading of Recommendation Assessment, Development, and Evaluation (GRADE) criteria. We will pool findings with a meta-analysis if sufficient homogeneity exists among studies. Findings will be published in a peer-reviewed journal and disseminated to SCD advocacy groups. PROSPERO registration number: 429412.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2023-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10514847/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41141678","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