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Standardized Protocol for Comprehensive, Non-Invasive Phenotyping of Atrial Myopathy in Sprague-Dawley Rat Models of Metabolic Syndrome Using Clinical-Grade Echocardiography and Electrophysiology Systems. 使用临床级超声心动图和电生理系统对Sprague-Dawley代谢综合征大鼠模型心房肌病进行全面、无创表型分析的标准化方案。
IF 2.5
Methods and Protocols Pub Date : 2026-07-02 DOI: 10.3390/mps9040103
Ardian Rizal, Mohammad Saifur Rohman, Fatchiyah Fatchiyah, Hidayat Sujuti, Anna Fuji Rahimah, Wella Karolina, Victor Alvianoes Guterez Hose, Mokhammad Afifudin
{"title":"Standardized Protocol for Comprehensive, Non-Invasive Phenotyping of Atrial Myopathy in Sprague-Dawley Rat Models of Metabolic Syndrome Using Clinical-Grade Echocardiography and Electrophysiology Systems.","authors":"Ardian Rizal, Mohammad Saifur Rohman, Fatchiyah Fatchiyah, Hidayat Sujuti, Anna Fuji Rahimah, Wella Karolina, Victor Alvianoes Guterez Hose, Mokhammad Afifudin","doi":"10.3390/mps9040103","DOIUrl":"10.3390/mps9040103","url":null,"abstract":"<p><p><b>Background:</b> Small animal models are essential for atrial fibrillation (AF) research. Researchers in AF use an electrocardiogram (ECG), echocardiography and invasive electrophysiology study (EPS) to assess atrial structural and electrical remodeling. In relatively smaller cardiac structures and rapid heart rates, the examination can be challenging without special tools designed for animal study. Moreover, conventional invasive EPSs often cause significant trauma, alter autonomic tone, and limit longitudinal evaluations. This study aimed to evaluate the feasibility of repurposing hospital-grade medical devices for the non-invasive, multi-modality assessment of atrial myopathy in a rat model of metabolic syndrome (MetS). <b>Methods:</b> A total of 12 male Sprague-Dawley rats underwent the multi-modality assessment. Structural remodeling was evaluated using hospital-grade echocardiography (8-12 MHz) to measure left atrial (LA) dimensions and volume. Surface ECG was used to determine P-wave duration. Electrical remodeling and AF inducibility were assessed using transesophageal pacing (TEP)-based EPS, evaluating the atrial effective refractory period (AERP), sinus node recovery time (SNRT), and response to rapid atrial burst pacing. <b>Results:</b> The protocols showed high procedural safety (survival rate 91.67%) and successfully characterized atrial myopathy. Surface ECG showed marked intra-atrial conduction delay with prolonged P-wave duration in the MetS group (30.17 ± 4.62 vs. 22.33 ± 1.86 ms, <i>p</i> < 0.05). Echocardiography revealed signs of structural remodeling in the MetS group, evidenced by marked prolonged Isovolumic Relaxation Time (IVRT: 35.602 ± 3.043 vs. 19.187 ± 3.631 ms; <i>p</i> < 0.001) and increased Left Atrial Area (0.223 ± 0.0556 vs. 0.134 ± 0.033; <i>p</i> = 0.007). Furthermore, TEP-based EPS quantified electrical remodeling. The MetS group had shorter AERP (73.33 ± 10.33 ms vs. 120.00 ± 34.06 ms; <i>p</i> = 0.010) and Corrected SNRT (100.67 ± 53.98 ms) versus controls (208.33 ± 76.97 ms; <i>p</i> = 0.018). The MetS group exhibited a higher absolute AF inducibility rate (50%, three out of six rats) compared to the SH group (33.3%, two out of six rats). <b>Conclusions:</b> The integration of surface ECG, echocardiography, and TEP-based EPS provides a safe, highly reproducible, and comprehensive method for evaluating both structural and electrical components of atrial myopathy in small animal models, allowing for robust longitudinal studies.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13398038/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148579434","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
Evaluating Effectiveness of the FiTeens Intervention for Health Behavior Change in Students: A Study Protocol. 青少年干预对学生健康行为改变的有效性评估:一项研究方案。
IF 2.5
Methods and Protocols Pub Date : 2026-07-01 DOI: 10.3390/mps9040101
Taavi Rand, Henri Tilga, Ángel Abós, Luis García-González, Sergio Diloy Peña, Rafael Burgueño-Mengibar, Andre Koka
{"title":"Evaluating Effectiveness of the FiTeens Intervention for Health Behavior Change in Students: A Study Protocol.","authors":"Taavi Rand, Henri Tilga, Ángel Abós, Luis García-González, Sergio Diloy Peña, Rafael Burgueño-Mengibar, Andre Koka","doi":"10.3390/mps9040101","DOIUrl":"10.3390/mps9040101","url":null,"abstract":"<p><p>(1) Background: Previous school-based interventions have addressed adolescent health behaviors such as physical activity, screen time, and sleep, but have predominantly targeted these behaviors independently rather than simultaneously. The Erasmus+ project FiTeens developed an integrated intervention combining theoretical content, videos, infographics, and interactive tasks to promote multiple health behaviors concurrently. The objective of the current article is to present the protocol for a school-based intervention study designed to examine the effects of the FiTeens program on adolescents' physical activity, screen time, and sleep behaviors. We hypothesize that students receiving the FiTeens intervention will demonstrate increased physical activity, reduced screen time, and improved sleep outcomes compared with students in the control group. (2) Methods: Teachers will be introduced to the FiTeens tools prior to delivering the intervention to students in grades 5-9. Students will participate in an eight-week intervention program combining structured lessons and behavior-change challenges. Primary outcomes include changes in physical activity, screen time, and sleep duration and quality. Secondary outcomes include psychological determinants such as motivation and behavioral intentions. Data will be collected at baseline and at 1-, 3-, and 6-month follow-ups and analyzed using repeated measures ANOVA. (3) Expected results: The study will evaluate whether the intervention may contribute to improvements in health-related behaviors among adolescents, including increased physical activity, reduced screen time, and improved sleep outcomes. (4) Conclusions: The intervention based on FiTeens tools could have the potential to promote healthier lifestyle behaviors among students by increasing physical activity during leisure time, supporting the effective limitation of screen time and enhancing bedtime routines to improve sleep quality.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13397910/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148579421","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 Miniaturised Protocol for Feeding Measurements in Daphnids. 水蚤摄食测量的小型化方案。
IF 2.5
Methods and Protocols Pub Date : 2026-07-01 DOI: 10.3390/mps9040102
Izabela Antepowicz, Antonia Despotidi, Emma Rowan, Mbuyiselwa Shadrack Moloi, Silke Aulhorn, Harry Esmonde, Konstantinos Grintzalis, Eberhard Küster
{"title":"A Miniaturised Protocol for Feeding Measurements in Daphnids.","authors":"Izabela Antepowicz, Antonia Despotidi, Emma Rowan, Mbuyiselwa Shadrack Moloi, Silke Aulhorn, Harry Esmonde, Konstantinos Grintzalis, Eberhard Küster","doi":"10.3390/mps9040102","DOIUrl":"10.3390/mps9040102","url":null,"abstract":"<p><p>Daphnids, commonly known as water fleas, are freshwater planktonic microcrustacean species used as model organisms in ecotoxicology, particularly in regulatory frameworks that adhere to OECD and ISO standards. Mortality is the most common endpoint in toxicity testing; however, more sensitive indicators are required to assess sublethal acute effects of pollutants. The use of feeding impairment as a toxicity phenotypic endpoint in daphnids is considered a cost-effective approach that aligns with the 3Rs principle (Replace, Reduce, Refine) and is more physiologically and environmentally relevant. Current feeding methods are inefficient due to the large test volumes and extended incubation periods required. In this paper, we present a miniaturised protocol to assess feeding behaviour following exposure to chemicals in daphnids. The method is based on the consumption of algae, which is measured with chlorophyll fluorescence. The optimised protocol is more robust and rapid, and results can be obtained in 30 min and in a 96-well plate. Responses in feeding rate were investigated using this miniaturised protocol following exposure to a range of prevalent pollutants, which include two metals and, as a more realistic sample, a leachate from smoked cigarette filters. All three pollutants were tested at sublethal concentrations. This method provides an efficient approach to assess the toxicity of chemicals and water quality.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13398296/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148579387","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
An Efficient TetR/TetO-Integrated Packaging System for Fowl Adenovirus 4 Vector Carrying Toxic Transgene. 禽腺病毒4携带毒性转基因载体的高效TetR/ teto集成包装系统
IF 2.5
Methods and Protocols Pub Date : 2026-06-22 DOI: 10.3390/mps9030100
Qian-Wen Ma, Zhi Li, Zhi-Chao Zhang, Xiao-Juan Guo, Xiao-Hui Zou, Tao Hung, Zhuo-Zhuang Lu
{"title":"An Efficient TetR/TetO-Integrated Packaging System for Fowl Adenovirus 4 Vector Carrying Toxic Transgene.","authors":"Qian-Wen Ma, Zhi Li, Zhi-Chao Zhang, Xiao-Juan Guo, Xiao-Hui Zou, Tao Hung, Zhuo-Zhuang Lu","doi":"10.3390/mps9030100","DOIUrl":"10.3390/mps9030100","url":null,"abstract":"<p><p>Adenoviral vectors are widely used for gene therapy and vaccine development. To circumvent pre-existing immunity against commonly used human adenovirus type 5, vectors based on rare human serotype or animal adenoviruses have attracted increasing interest. Previously, we constructed vectors based on fowl adenovirus 4 (FAdV-4) and replaced the knob of FAdV-4 fiber2 with that of FAdV-1 fiber1 to generate FAdV4-CF1K vectors with enhanced transduction efficiency in human cells. In this study, we aimed to modify the packaging system to efficiently produce FAdV-4 vectors carrying transgenes toxic to viral replication. Chicken LMH cells failed to form colonies at low seeding densities. We collected used medium from LMH cell cultures and used it as a supplement to adapt LMH cells, generating the colony-competent subclone LMH-C3532. A lentiviral vector encoding a codon-optimized tetracycline repressor (tetR) was transduced into LMH-C3532 to establish a tetR-integrated cell line, LMH-tetR24. An adenoviral plasmid, pKFAV4-CF1K-CtG, was constructed in which a tetracycline operator (tetO)-bearing CMV promoter controlled GFP expression. The SwaI-flanked GFP in this plasmid was replaced with the HA gene from an H5N1 influenza virus to generate pKFAV4-CF1K-CtHA. Linearized adenoviral plasmids were transfected into LMH-tetR24 cells, and recombinant FAdV4-CF1K-CtG and FAdV4-CF1K-CtHA viruses were successfully rescued, amplified, and purified. When infected with FAdV4-CF1K-CtG at various multiplicities of infection (MOI), the progeny virus yield from LMH-tetR24 cells was 4-10 times higher than that from LMH-C3532 cells. For FAdV4-CF1K-CtHA, the yield difference between the two cell lines was even more pronounced, reaching 3-4 orders of magnitude. Overexpression of HA in LMH-C3532 cells negatively affected FAdV4-CF1K-CtHA replication, resulting in smaller and fewer plaques. In conclusion, by separately integrating tetR into packaging cells and TetO into the adenoviral plasmid, we established a system that can be routinely used to package FAdV-4 vectors. Notably, this system facilitates the propagation of FAdV-4 vectors carrying toxic transgenes.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 3","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-06-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13306192/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148318069","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
Exploring Barriers and Facilitators to COVID-19 Vaccination Uptake Among Individuals with Mental Illness in the Australian Healthcare System: A Qualitative Study Protocol. 探索澳大利亚医疗保健系统中精神疾病患者接种COVID-19疫苗的障碍和促进因素:一项定性研究方案。
IF 2.5
Methods and Protocols Pub Date : 2026-06-16 DOI: 10.3390/mps9030099
Soumitra Das, James Killian, Mahesh Jayaram, Naveen Thomas, Chi Jonasi
{"title":"Exploring Barriers and Facilitators to COVID-19 Vaccination Uptake Among Individuals with Mental Illness in the Australian Healthcare System: A Qualitative Study Protocol.","authors":"Soumitra Das, James Killian, Mahesh Jayaram, Naveen Thomas, Chi Jonasi","doi":"10.3390/mps9030099","DOIUrl":"10.3390/mps9030099","url":null,"abstract":"<p><p>Individuals living with mental illness face disproportionately higher COVID-19 morbidity and mortality than the general population. Despite their prioritisation in Australia's national vaccination rollout, vaccination rates among this population are significantly lower than those without mental illness. No previous study has employed a qualitative research paradigm to explore the barriers and facilitators to COVID-19 vaccination among people with mental illness in the Australian context. This qualitative study will employ Braun and Clarke's reflexive thematic analysis framework. Participants will be recruited through Western Health Mental Health and Wellbeing Services in Victoria, Australia. Semi-structured individual interviews will be conducted with approximately 17-20 participants (aged 18-65) who have a DSM-5 diagnosed mental illness and any experience (vaccinated or unvaccinated) with COVID-19 vaccination. Interviews will be audio-recorded, transcribed, and then analysed and collated into themes using NVivo software. Code saturation will guide the final sample size. This study aims to produce a rich thematic map that captures the experience of individual, illness, and system-level barriers and facilitators to COVID-19 vaccination in this cohort. Findings are anticipated to inform targeted public health interventions to improve equitable vaccine uptake and contribute to closing the mortality gap for those with mental illness. This protocol has been approved by the Western Health Low Risk Ethics Panel (ERM ID: 113351).</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 3","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-06-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13305169/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148318176","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
In Vitro Capacitation in Boar Sperm: Evaluation of Selected Detection Techniques. 猪精子体外获能:选定检测技术的评价。
IF 2.5
Methods and Protocols Pub Date : 2026-06-15 DOI: 10.3390/mps9030098
Barbora Klusackova, Zuzana Pilsova, Katerina Nemeckova, Aneta Pilsova, Pavla Postlerova
{"title":"In Vitro Capacitation in Boar Sperm: Evaluation of Selected Detection Techniques.","authors":"Barbora Klusackova, Zuzana Pilsova, Katerina Nemeckova, Aneta Pilsova, Pavla Postlerova","doi":"10.3390/mps9030098","DOIUrl":"10.3390/mps9030098","url":null,"abstract":"<p><p>Sperm capacitation is essential for fertilization and involves coordinated changes in membrane organization, ion fluxes, and intracellular signaling. However, commonly used detection methods may reflect different biological events, which can be strongly influenced by experimental methodology. This study critically evaluated fluorescence-based approaches for assessing capacitation in boar spermatozoa, focusing on their specificity, interpretative limits, and methodological sensitivity. Ejaculated boar spermatozoa were incubated under in vitro capacitating conditions in TALP medium. Selected samples were subsequently treated with calcium ionophore to induce the acrosome reaction (AR). Phosphotyrosine (PTyr) immunofluorescence was assessed using five fixation and labeling protocols, acrosin redistribution was evaluated with the ACR.2 antibody, calcium ion redistribution was assessed using chlortetracycline (CTC) fluorescence, and acrosomal responsiveness was monitored by peanut agglutinin (PNA) lectin labeling. PTyr immunofluorescence was highly dependent on fixation protocol, indicating marked methodological sensitivity. Acrosin immunodetection revealed a clear capacitation-associated redistribution from weak or diffuse staining to a well-defined acrosomal pattern, whereas ionophore treatment caused a pronounced signal loss consistent with acrosomal exocytosis. PNA labeling confirmed that capacitation alone did not increase spontaneous acrosome loss, whereas ionophore treatment induced a robust AR. CTC staining showed a significant shift from whole-head pattern to acrosome in TALP-treated spermatozoa, indicating capacitation-associated Ca<sup>2+</sup> redistribution. Together with CTC and Western blot data, these findings show that sperm capacitation status should be evaluated using multiple complementary markers rather than a single gold-standard assay.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 3","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13304760/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148318173","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
Multiparametric Flow Cytometry Panel for Characterization of Mouse T Cell Differentiation and NK Cell Maturation Following Inflammatory Challenge. 炎症刺激后小鼠T细胞分化和NK细胞成熟的多参数流式细胞仪。
IF 2.5
Methods and Protocols Pub Date : 2026-06-12 DOI: 10.3390/mps9030097
Tim Bozic, Bostjan Markelc, Simona Kranjc Brezar, Ziva Pisljar, Tanja Jesenko, Maja Cemazar
{"title":"Multiparametric Flow Cytometry Panel for Characterization of Mouse T Cell Differentiation and NK Cell Maturation Following Inflammatory Challenge.","authors":"Tim Bozic, Bostjan Markelc, Simona Kranjc Brezar, Ziva Pisljar, Tanja Jesenko, Maja Cemazar","doi":"10.3390/mps9030097","DOIUrl":"10.3390/mps9030097","url":null,"abstract":"<p><p>Lymph nodes are central hubs of immune regulation and coordination, serving as primary sites for antigen presentation, lymphocyte activation, and the orchestration of adaptive immune responses. The composition and activation state of lymph node-resident immune cells critically shape both local and systemic immunity. Comprehensive immunophenotyping of these populations is therefore essential for understanding immune organization and functional heterogeneity. Here, we present an optimized protocol for the characterization of mouse lymph node-associated immune populations using 14-color multiparametric flow cytometry. The method combines lymph node isolation based on anatomical landmarks with mechanical dissociation and enzymatic digestion to generate high-quality single-cell suspensions suitable for downstream analysis. Furthermore, the described flow cytometry panel and gating strategy enable reliable identification and quantification of major lymphoid subsets, including helper CD4<sup>+</sup> and cytotoxic CD8<sup>+</sup> T cells with their differentiation states, as well as natural killer (NK) cells across distinct maturation stages. Although optimized for assessing lymphocyte maturation after lipopolysaccharide (LPS) challenge, the protocol serves as a reproducible platform for broad immunophenotyping of T and NK cell subsets in mouse lymphoid tissues under experimental conditions.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 3","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-06-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13305854/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148318209","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
Design, Preparation and Characterization of Nationally Representative Synthetic Food Waste for Reproducible Waste Valorization Research. 具有全国代表性的可再生垃圾合成物的设计、制备及表征。
IF 2.5
Methods and Protocols Pub Date : 2026-06-10 DOI: 10.3390/mps9030093
Ryan Scott Anderson, Sybil Sharvelle, Susan K De Long
{"title":"Design, Preparation and Characterization of Nationally Representative Synthetic Food Waste for Reproducible Waste Valorization Research.","authors":"Ryan Scott Anderson, Sybil Sharvelle, Susan K De Long","doi":"10.3390/mps9030093","DOIUrl":"10.3390/mps9030093","url":null,"abstract":"<p><p>Food waste is a readily digestible and fermentable feedstock for waste to energy bioprocesses. Approximately one third of food is wasted, thus making improvements in food waste valorization is essential for a circular economy. Laboratory results must be reproducible and as representative of scaled performance as possible to facilitate knowledge sharing between research groups. Food waste used in laboratory studies is often collected in situ or overly simplistic synthetic mixtures are used. Food waste collected in situ from any one local source at a single time point (e.g., grab samples from a cafeteria or restaurant) are not reproducible or nationally representative; additionally, overly simple synthetic mixtures are reproducible, but lack the complexity of real food waste and are not nationally representative. Thus, an adequately complex, reproducible, and nationally representative food waste recipe is needed to standardize the feedstocks used in laboratory scale food waste digestion and fermentation studies. In this work, we developed a food waste recipe made from widely and commercially available ingredients which is based on national-scale food wastage data in the United States. The nationally representative food waste mixture was 45.4% carbohydrates, 32.5% lipids, and 13.4% proteins. The biomethane potential was 495 ± 44 mL CH<sub>4</sub>/g VS and the food waste mixture was suitable for use in low-pH bench-scale arrested anaerobic digesters. This design approach can be adapted for other regions and countries where food loss data are available.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 3","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-06-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13304604/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148318194","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
Cumulative Frameworks as a Pragmatic Alternative to Multivariable Modeling in Rare-Event Clinical Settings: A Coronary Care Unit Case Study. 在罕见事件临床环境中,累积框架作为多变量模型的实用替代方案:冠状动脉护理单位案例研究。
IF 2.5
Methods and Protocols Pub Date : 2026-06-10 DOI: 10.3390/mps9030092
Daniela Mirela Vîrtosu, Simina Crișan, Oana Pătru, Angela Dragomir, Silvia Luca, Ruxandra-Maria Băghină, Mihai-Andrei Lazăr, Alina-Ramona Cozlac, Stela Iurciuc, Constantin Tudor Luca
{"title":"Cumulative Frameworks as a Pragmatic Alternative to Multivariable Modeling in Rare-Event Clinical Settings: A Coronary Care Unit Case Study.","authors":"Daniela Mirela Vîrtosu, Simina Crișan, Oana Pătru, Angela Dragomir, Silvia Luca, Ruxandra-Maria Băghină, Mihai-Andrei Lazăr, Alina-Ramona Cozlac, Stela Iurciuc, Constantin Tudor Luca","doi":"10.3390/mps9030092","DOIUrl":"10.3390/mps9030092","url":null,"abstract":"<p><p><b>Background</b>: Risk stratification models are widely used in clinical research; however, their development becomes methodologically challenging in settings characterized by low outcome incidence. In coronary care unit (CCU) populations, healthcare-associated infections (HAIs) occur relatively infrequently, limiting the feasibility of conventional multivariable predictive modeling. <b>Methods</b>: A retrospectively assembled CCU cohort comprising 870 patients with 16 HAI events (1.8%) was used as an illustrative example to examine methodological constraints associated with low events-per-variable (EPV) ratios. The implications of limited event frequency for multivariable logistic regression were evaluated, including risks of overfitting, coefficient instability, and reduced reproducibility. As an alternative strategy, a prespecified cumulative additive framework integrating baseline vulnerability and exposure-related variables was conceptually and analytically explored. <b>Results</b>: With four candidate predictors and 16 outcome events, the resulting EPV was approximately four, indicating a high risk of instability for conventional multivariable modeling. The cumulative framework allowed structured cumulative stratification without coefficient optimization. Infection occurrence increased progressively across cumulative framework levels, illustrating a graded pattern of increasing HAI occurrence with accumulating vulnerability and exposure-related burden. <b>Conclusions</b>: In clinical datasets with limited outcome events, modeling strategies should be aligned with the informational capacity of the data. Cumulative additive frameworks may represent a pragmatic structural alternative to coefficient-based modeling approaches in rare-event clinical settings. The present work provides a structured methodological framework for risk stratification under low-events-per-variable conditions rather than proposing a novel clinical scoring system.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 3","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-06-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13306005/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148318182","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
Contact Lens-Associated Ocular Surface and Corneal Disorders. 隐形眼镜相关的眼表和角膜疾病。
IF 2.5
Methods and Protocols Pub Date : 2026-06-10 DOI: 10.3390/mps9030095
Omar Abdelaziz, Seyyedehfatemeh Ghalibafan, Raul E Ruiz-Lozano, Jeffrey C Peterson, Ryan A Gallo, Ali R Djalilian
{"title":"Contact Lens-Associated Ocular Surface and Corneal Disorders.","authors":"Omar Abdelaziz, Seyyedehfatemeh Ghalibafan, Raul E Ruiz-Lozano, Jeffrey C Peterson, Ryan A Gallo, Ali R Djalilian","doi":"10.3390/mps9030095","DOIUrl":"10.3390/mps9030095","url":null,"abstract":"<p><p>Contact lens wear is widely used for vision correction by millions of individuals worldwide; however, it remains associated with a spectrum of ocular complications ranging from mild inflammatory conditions to vision-threatening infections. Common contact lens-related complications are predominantly noninfectious, including contact lens discomfort, dry eye syndromes, and papillary conjunctivitis. These conditions are typically mild and manageable with conservative measures. In contrast, corneal inflammatory conditions, such as contact lens-induced acute red eye and peripheral ulcers, represent an intermediate spectrum and may clinically overlap with early infection, creating diagnostic uncertainty. The most serious complication is microbial keratitis, a vision-threatening infection that remains challenging to recognize in its early stages due to its variable and often subtle presentation. Delayed identification may lead to rapid progression and significant visual morbidity. Patients with contact lens-related complaints often present to frontline settings, where early recognition is essential. Distinguishing benign from infectious conditions can be challenging; a risk-based approach with prompt triage and referral, along with proper lens hygiene and patient education, is key.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 3","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-06-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13304652/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148318152","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}
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