Methods and Protocols最新文献

{"title":"Development of a Quantitative PCR Method for Detecting Enterococcus faecalis Cytolysin in Human Stool Samples","authors":"Noemí Cabré, Yongqiang Yang, Yanhan Wang, Bernd Schnabl","doi":"10.3390/mps6060107","DOIUrl":"https://doi.org/10.3390/mps6060107","url":null,"abstract":"Alcohol-associated liver disease (ALD) is a major global health issue, contributing significantly to morbidity and mortality worldwide. Among the ALD subtypes, alcohol-associated hepatitis poses a severe and urgent medical challenge with high short-term mortality rates. Despite extensive research, the current therapeutic approaches for alcohol-associated hepatitis have limited efficacy, necessitating novel interventions. Recent studies have highlighted the crucial role of the gut microbiota in ALD pathogenesis, particularly Enterococcus faecalis (E. faecalis) and its cytolysin exotoxin. This study presents the development of a standardized real-time quantitative polymerase chain reaction (RT-qPCR) assay to detect and quantify cytolysin in fecal samples from patients with alcohol-associated hepatitis. The diagnostic assay allows for an association analysis between cytolysin-positive E. faecalis and disease severity as well as mortality. This assay was developed to standardize the identification of cytolysin-positive patients who can be selected for clinical trials.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"347 5","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135392586","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}

{"title":"Improved Separation in Horizontal Protein SDS-PAGE with Double-Deck Flat Electrodes and a Field Inversion Gel Electrophoresis Module","authors":"Dong Woo Lim, Tae-Sung Yoon, Kyung Ho Han, Saba Sajjad, Heung-Seon Shin, Sunghyun Kang","doi":"10.3390/mps6060106","DOIUrl":"https://doi.org/10.3390/mps6060106","url":null,"abstract":"The horizontal flatbed electrophoresis method is employed to separate protein samples, providing greater flexibility for various electrophoretic applications and easier sample loading compared to its vertical counterpart. In the currently available equipment setup, cathode and anode electrodes are positioned on top of a gel at each end. Since an electric field enters the gel from the top, its strength gradually weakens from the top to the bottom of the gel. When examining the interior of gels following electrophoretic separation, the uneven electric field causes the protein bands to lie down forward in the direction of migration, leading to an increase in bandwidth. This issue has remained unaddressed for several decades. To address this problem, new clamp-shaped and double-deck electrodes were developed to apply an electric field simultaneously from both the top and bottom of the gel. Both of these new electrodes facilitated the formation of perpendicular protein band shapes and enhanced resolution at a comparable level. Due to their ease of use, double-deck electrodes are recommended. By combining these new electrodes with the field inversion gel electrophoresis (FIGE) technique, the protein bands could be focused and aligned nearly vertically, resulting in the highest level of electrophoretic resolution. Our electrodes are compatible with polyacrylamide gels of varying sizes, buffer systems, and sample well formats. They can be easily manufactured and seamlessly integrated into existing laboratory instruments for practical use.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"11 20","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135818214","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}

{"title":"Method for Extraction and Evaluation of Heliocarpus popayanensis and Triumfetta bogotensis as Natural Coagulants for the Treatment of Wastewater","authors":"Yeison Alberto Garcés-Gómez, Sebastián Isaac Pacheco-Gonzalez","doi":"10.3390/mps6060105","DOIUrl":"https://doi.org/10.3390/mps6060105","url":null,"abstract":"This research evaluates extracts from the bark of Heliocarpus popayanensis and Triumfetta bogotensis as coagulating agents for removing turbidity in domestic wastewater, considering the coagulant dosage and pH of the wastewater. ANOVA was conducted to assess differences between the coagulants, dosages, and pH, with three pH levels (5, 8, and 9) and six dosages (7, 9, 11, 13, 15, and 17 mL per 1000 mL of wastewater) at a significance level of α = 0.05, and both the p-value and effect size were evaluated. This study found that the mucilaginous compound from the bark of Triumfetta bogotensis performed better in reducing turbidity levels, with an average reduction of 30.2 NTU (Nephelometric Turbidity Unit) (CI [25.9 NTU; 34.5 NTU], α = 0.05) at a pH of 5, and an average initial NTU of 102.2. This represents an average reduction of 70.45%. The dosage factor did not show significant effects on turbidity reduction, which opens the possibility for further study to determine the optimal dosage of the best coagulant.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"70 7","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135933005","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}

{"title":"Development of a HPLC Method for Analysis of a Combination of Clofazimine, Isoniazid, Pyrazinamide, and Rifampicin Incorporated into a Dermal Self-Double-Emulsifying Drug Delivery System","authors":"Daniélle van Staden, Richard K. Haynes, Frank Van der Kooy, Joe M. Viljoen","doi":"10.3390/mps6060104","DOIUrl":"https://doi.org/10.3390/mps6060104","url":null,"abstract":"We describe the development and validation of a new high performance liquid chromatography (HPLC) method for analysis of a combination of the first-line anti-tubercular drugs isoniazid, pyrazinamide, and rifampicin together with clofazimine. This is a unique challenge since clofazimine and rifampicin are relatively highly lipophilic drugs, whereas isoniazid and pyrazinamide are considerably more hydrophilic. Thus, clear separation of peaks and quantification of four individual drugs can present difficulties during the development of an analytical method. Detection was established at two wavelengths—254 nm for isoniazid and pyrazinamide and 320 nm for clofazimine and rifampicin. Gradient elution was employed using 0.1% aqueous formic acid (A) and acetonitrile (B); clear separation of the four drugs was achieved within 10 min. A linear relationship was indicated by a correlation coefficient (r2) of 0.9999 for each anti-tubercular drug, respectively. The limit of detection (LOD) for the individual drugs was 0.70 µg/mL (isoniazid), 0.30 µg/mL (pyrazinamide), 0.20 µg/mL (rifampicin) and 0.20 µg/mL (clofazimine). Precision experiments rendered a mean recovery percentage of 101.25% (isoniazid), 98.70% (pyrazinamide), 99.68% (rifampicin) and 97.14% (clofazimine). This HPLC method was validated and is reliable, repeatable, and accurate for the purpose of conducting simultaneous HPLC analyses of the four anti-tubercular drugs.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"119 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135325948","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}

{"title":"Safeguarding against Dementia in Aboriginal and Torres Strait Islander Communities through the Optimisation of Primary Health Care: A Project Protocol.","authors":"Yvonne C Hornby-Turner,&nbsp;Sarah G Russell,&nbsp;Rachel Quigley,&nbsp;Veronica Matthews,&nbsp;Sarah Larkins,&nbsp;Noel Hayman,&nbsp;Prabha Lakhan,&nbsp;Leon Flicker,&nbsp;Kate Smith,&nbsp;Dallas McKeown,&nbsp;Diane Cadet-James,&nbsp;Alan Cass,&nbsp;Gail Garvey,&nbsp;Dina LoGiudice,&nbsp;Gavin Miller,&nbsp;Edward Strivens","doi":"10.3390/mps6050103","DOIUrl":"10.3390/mps6050103","url":null,"abstract":"<p><p>This protocol describes the methodology and methods for a collaborative project with eight Aboriginal and Torres Strait Islander primary health care (PHC) organisations, across three Australian states and one territory, to increase clinical service performance and access to preventive health and health promotion services for preventing, identifying, treating, and managing dementia risk in Aboriginal and Torres Strait Islander communities. Aboriginal participatory action research (APAR) methodology will be the framework for this project, incorporating continuous quality improvement (CQI), informed by research yarning with stakeholder groups, comprising community members and PHC staff and service providers and data collected from the auditing of client health records and the mapping of existing clinical processes and health services at each partnering PHC organisation. The qualitative and quantitative data will be summarised and discussed with stakeholder groups. Priorities will be identified and broken down into tangible PHC organisation deliverable strategies and programs, which will be co-developed with stakeholder groups and implemented cyclically over 24 months using the Plan, Do, Study, Act model of change. Key project outcome measures include increased clinical service performance and availability of preventive health and health promotion services for safeguarding against dementia. Project implementation will be evaluated for quality and transparency from an Indigenous perspective using an appropriate appraisal tool. The project processes, impact, and sustainability will be evaluated using the RE-AIM framework. A dementia safeguarding framework and accompanying tool kit will be developed from this work to support Aboriginal and Torres Strait Islander PHC organisations to identify, implement, and evaluate dementia safeguarding practice and service improvements on a broader scale.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"6 5","pages":""},"PeriodicalIF":2.4,"publicationDate":"2023-10-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10609630/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54230083","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":"Isolation of the Sarcoplasmic Reticulum Ca²⁺-ATPase from Rabbit Fast-Twitch Muscle.","authors":"Miguel A Rivera-Morán, José G Sampedro","doi":"10.3390/mps6050102","DOIUrl":"10.3390/mps6050102","url":null,"abstract":"<p><p>The sarcoendoplasmic reticulum Ca²⁺-ATPase (SERCA) is a membrane protein that is destabilized during purification in the absence of calcium ions. The disaccharide trehalose is a protein stabilizer that accumulates in the yeast cytoplasm when under stress. In the present work, SERCA was purified by including trehalose in the purification protocol. The purified SERCA showed high protein purity (~95%) and ATPase activity. ATP hydrolysis was dependent on the presence of Ca²⁺ and the enzyme kinetics showed a hyperbolic dependence on ATP (<i>K</i>_m = 12.16 ± 2.25 μM ATP). FITC labeling showed the integrity of the ATP-binding site and the identity of the isolated enzyme as a P-type ATPase. Circular dichroism (CD) spectral changes at a wavelength of 225 nm were observed upon titration with ATP, indicating α-helical rearrangements in the nucleotide-binding domain (N-domain), which correlated with ATP affinity (<i>K</i>_m). The presence of Ca²⁺ did not affect FITC labeling or the ATP-mediated structural changes at the N-domain. The use of trehalose in the SERCA purification protocol stabilized the enzyme. The isolated SERCA appears to be suitable for structural and ligand binding studies, e.g., for testing newly designed or natural inhibitors. The use of trehalose is recommended for the isolation of unstable enzymes.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"6 5","pages":""},"PeriodicalIF":2.3,"publicationDate":"2023-10-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10608927/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54230080","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 Systematic Review to Inform the Development of a Reporting Guideline for Concept Mapping Research.","authors":"Sandesh Pantha, Martin Jones, Pragya Gartoulla, Richard Gray","doi":"10.3390/mps6050101","DOIUrl":"10.3390/mps6050101","url":null,"abstract":"<p><p>Concept mapping is a phased, mixed-method approach that is increasingly used in health research to develop an understanding of complex phenomena. The six phases of concept mapping are preparation, idea generation, structuring (clustering and prioritization), data analysis, interpretation, and utilization of the map. The reporting of concept mapping research requires the development of a specific reporting guideline. We conducted a systematic review to identify candidate reporting items for inclusion in a reporting guideline. Three databases (MEDLINE, CINAHL, and PsycInfo) were searched to identify studies that used concept mapping methodology. We included 75 concept mapping studies published since 2019 from which we extracted information about the quality of reporting. A third of the studies focused on public health. We identified 71 candidate items that relate to the quality of reporting concept mapping research. The rationale for the study, the focus prompt, procedures for brainstorming, and structuring statements were consistently reported across the included studies. The process for developing the focus prompt, the rationale for the size of the stakeholder groups, and the process for determining the final concept map were generally not reported. The findings from the review will be used to inform the development of our reporting guideline for concept mapping research.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"6 5","pages":""},"PeriodicalIF":2.3,"publicationDate":"2023-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10609252/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54230074","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 Simple Protocol for Isolating Hemolymph from Single <i>Drosophila melanogaster</i> Adult Flies.","authors":"Kirah Jones,&nbsp;Ioannis Eleftherianos","doi":"10.3390/mps6050100","DOIUrl":"10.3390/mps6050100","url":null,"abstract":"<p><p><i>Drosophila melanogaster</i> is an excellent model for dissecting innate immune signaling and functions. Humoral and cellular immune mechanisms in the fly take place in the hemolymph, where host defense components are secreted and act in response to microbial invaders. Studying hemolymph factors is critical for understanding the regulation of the host's antimicrobial immune system. Therefore, methods for extracting the fly hemolymph efficiently and in sufficient quantities are essential for isolating and characterizing immune proteins and peptides. Here, we describe a novel and simple hemolymph isolation protocol for single <i>D. melanogaster</i> male and female adults. This procedure substantially improves the already used technique and allows fly immunologists to explore innate immune hemolymph activity in <i>D. melanogaster</i> individuals.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"6 5","pages":""},"PeriodicalIF":2.4,"publicationDate":"2023-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10609455/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54230072","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":"Efficacy of Sonic-Powered and Manual Toothbrushes on Plaque-Induced Gingivitis in Pregnant Women: A Randomized Controlled Trial.","authors":"Céline Clément,&nbsp;Denis Bourgeois,&nbsp;Flavia Vitiello,&nbsp;Herve Perrier,&nbsp;Ariane Tabary,&nbsp;Florence Carrouel","doi":"10.3390/mps6050099","DOIUrl":"10.3390/mps6050099","url":null,"abstract":"<p><p>Hormonal changes and physiological alterations in pregnancy increase the susceptibility of the woman to oral diseases such as plaque-induced gingivitis. In individual oral prophylaxis, effective tooth brushing can reduce gingival inflammation. Therefore, it is necessary to update the scientific evidence to identify which type of toothbrush, manual or sonic-powered, is most effective in reducing the incidence of gingivitis in pregnant women. The aim of this clinical trial is to compare the biofilm control effectiveness of two manual and two sonic toothbrushes in pregnant women. This study is designed as a four-arm, parallel, randomized controlled trial with an allocation ratio of 1:1:1:1. The pregnant woman will be included at 15-18 weeks of amenorrhea and followed for 3 months. The primary outcome will be the change in the incidence of gingival bleeding from a baseline and various follow-up periods of the study. Secondary outcomes measures will be to compare clinical effects of the toothbrushes tested on (i) gingival inflammation, (ii) dental plaque, (iii) gingival attachment and (iv) periodontal pocket; and to assess toothbrush acceptability. Thus, identifying the best device for effective tooth brushing in pregnancy could be helpful in reducing and improving the incidence of gingival inflammation.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"6 5","pages":""},"PeriodicalIF":2.4,"publicationDate":"2023-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10608920/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54230078","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":"Maternal Antibiotic Exposure and the Risk of Developing Antenatal or Postpartum Depressive Symptoms: The Maternal Experience Study Protocol.","authors":"Mahsa Pouranayatihosseinabad,&nbsp;Maggie Taylor,&nbsp;Jason Hawrelak,&nbsp;Gregory M Peterson,&nbsp;Felicity Veal,&nbsp;Tristan Ling,&nbsp;Mackenzie Williams,&nbsp;Megan Whatley,&nbsp;Kyan Ahdieh,&nbsp;Corinne Mirkazemi","doi":"10.3390/mps6050098","DOIUrl":"10.3390/mps6050098","url":null,"abstract":"<p><p>Limited epidemiological evidence suggests a link between antibiotic use and developing depression. This study seeks to investigate this association in depth, using a cohort of pregnant individuals. The primary aim is to explore any association between the use of antibiotics during pregnancy and the development of antenatal depressive symptoms up to the third trimester, as well as the use of antibiotics during pregnancy and within 12 months postpartum and the development of postpartum depressive symptoms. A national prospective, observational, longitudinal cohort study has been designed to examine these relationships. A sample size of 1500 pregnant individuals has been sought for this study, assuming 10 potential predictor variables (including antibiotic use) in the final multiple logistic regression model and allowing for a 30% drop-out rate. The development of depressive symptoms is considered either a diagnosis by a medical doctor and/or a scoring 13 or higher on the Edinburgh Postnatal Depression Scale. Data will be collected during the third trimester and at 6 weeks, 6 months, and 12 months postpartum. These surveys include variables previously identified as associated with antenatal and postpartum depression (e.g., level of social support, experience of intimate partner abuse, and obstetric complications), as well as antibiotic and probiotic use. This study will provide an update on the prevalence of the symptoms of depression during pregnancy and postpartum and its associated risk factors. It will also, for the first time, comprehensively explore the potential association between antibiotic use during pregnancy and up to 12 months postpartum and the development of depressive symptoms.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"6 5","pages":""},"PeriodicalIF":2.4,"publicationDate":"2023-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10609134/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54230081","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}