Methods and Protocols最新文献

{"title":"The PROMISE of Precision Medicine in Myocardial Infarction with Non-Obstructive Coronary Arteries.","authors":"Giulia La Vecchia, Vincenzo Scarica, Ludovica Leo, Rocco A Montone","doi":"10.3390/mps8030044","DOIUrl":"10.3390/mps8030044","url":null,"abstract":"<p><p>Myocardial infarction with non-obstructive coronary arteries (MINOCA) is a working diagnosis encompassing several pathophysiological mechanisms with specific treatments and different prognoses. Despite the absence of obstructive coronary artery disease, MINOCA has proven to be associated with a significant risk of mortality, angina burden, and socioeconomic costs. However, due to the heterogeneous nature of this clinical condition and the absence of randomized clinical trials, evidence supporting a standardized diagnostic algorithm and the clinical management of these patients is lacking. The PROMISE trial is the first randomized clinical trial evaluating the effectiveness of a precision medicine approach strategy in improving the outcomes and quality of life of patients with MINOCA, offering new insights into personalized treatment strategies. This review article discusses the promise of a precision medicine approach in patients with MINOCA, highlighting the potential innovations and challenges of a personalized medicine strategy in MINOCA.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 3","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-04-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12101212/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144128239","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The Validation of Antibodies Suitable for Flow Cytometric Analysis and Immunopeptidomics of Peptide-MHC Complexes in the Outbred Swiss Albino Mouse Strain.","authors":"Shanzou Chung, Isambard G Knox-Johnson, Sarah E Gazzard, Runqiu Song, Ngoc H Le, Luise A Cullen-McEwen, John F Bertram, Anthony W Purcell, Asolina Braun","doi":"10.3390/mps8030043","DOIUrl":"10.3390/mps8030043","url":null,"abstract":"<p><p>Antigen presentation on major histocompatibility complex (MHC) molecules is central to the initiation of immune responses, and a lot of our understanding about the antigen processing and presentation pathway has been gained through studies in mice. MHC molecules are the most genetically diverse genes; consequently, mouse strains differ substantially in their MHC make up and resulting antigen presentation. Swiss mice are commonly used in pharmacological research, yet our understanding of antigen presentation in this strain is surprisingly limited. Here, we have tested a range of anti-MHC antibodies and present a range of clones suitable to analyse MHC class I and class II molecules in Swiss mice who have the H2-q MHC haplotype. Moreover, we demonstrate using immunopeptidomics that clones 28-12-8, 34-1-2, MKD6, and N22 are also suited to isolate MHC class I and class II ligands in this mouse strain. Thus, this work also establishes a first experimental account of the H2-q-derived thymus and spleen immunopeptidome in Swiss mice which bears strong resemblance with ligands isolated from the H2-d MHC haplotype of Balb/C mice. The analysis of source proteins shows common but also organ- and function-specific antigen presentation in line with the involvement of the thymus in tolerance induction and the function of the spleen as a site of immune responses.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 3","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12101307/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144128337","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":"Quantification of Circulating Cell-Free DNA as a NETosis Marker in Trauma Patients with Type 2 Diabetes Mellitus.","authors":"Filiz Sahin, Regina Breinbauer, Caren Linnemann, Melike Tombaz, Andreas K Nussler, Sabrina Ehnert","doi":"10.3390/mps8020042","DOIUrl":"https://doi.org/10.3390/mps8020042","url":null,"abstract":"<p><p>Type 2 diabetes mellitus (T2DM) significantly impairs fracture healing, with neutrophils playing a crucial role in this process. In T2DM, these immune cells are over-activated, leading to the excessive release of neutrophil extracellular traps (NETs), increasing inflammation and hindering recovery. Thus, a need for markers to assess patients in the risk group arises. This study demonstrates that circulating cell-free DNA (cfDNA) can be efficiently quantified from serum samples by a single-step qPCR and be used as a marker for NETosis. Our results revealed that trauma patients with T2DM have the highest cfDNA levels, followed by trauma patients, and the healthy group has the lowest. The method shows strong correlations between cfDNA and neutrophil-specific markers such as MPO, citH3, AZU1, and α-defensin, highlighting its potential as a rapid indicator of NETosis. This approach could allow the timely interference for high-risk patients, ultimately improving healing outcomes and reducing complications such as chronic inflammation, non-union fractures, and diabetic foot ulcers.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 2","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-04-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12029683/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144036068","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":"Concurrent Validity of Three Photogrammetric Methods for Assessing Knee Alignment in Sagittal Plane.","authors":"Bruna Nichele da Rosa, Paula Andryelly Gomes Giendruczak, Marina Ziegler Frantz, Matias Noll, Cláudia Tarragô Candotti","doi":"10.3390/mps8020041","DOIUrl":"https://doi.org/10.3390/mps8020041","url":null,"abstract":"<p><p><i>Background</i>: Evidence supporting the validity of photogrammetry for assessing body segment alignment remains limited, with most studies focusing on spinal evaluation. Thus, there is a lack of robust research examining its use for other body segments such as the lower limbs. <i>Objective</i>: This study aimed to evaluate the concurrent validity of three photogrammetric methods for measuring knee alignment in the sagittal plane with and without corrections for potential rotational deviations in the participant's thigh and leg. <i>Methods</i>: A total of 21 adults underwent sequential evaluations involving panoramic radiography of the lower limbs and photogrammetry at a private radiology clinic. Photogrammetric analysis involved identifying the following anatomical landmarks: the greater trochanter of the femur (GTF), the lateral condyle of the femur (LCF), the head of the fibula (HF), and lateral malleolus (LM). Three photogrammetric methods were employed: (1) the condylar angle (CA) defined by the GTF, LCF, and LM points; (2) the fibula head angle (FHA) defined by the GTF, HF, and LM points; and (3) the four-point angle (4PA) incorporating the GTF, LCF, HF, and LM. Concurrent validity was assessed using correlation analysis, agreement with radiographic measurements, and the root mean square error (RMSE). Each photogrammetric method was tested using raw (CA, FHA, and 4PA) and corrected (CAcorr, FHAcorr, and 4PAcorr) values, accounting for thigh and/or leg rotational deviations. <i>Results</i>: Correcting for thigh and leg rotations significantly improved the validity metrics for all methods. The best performance was observed with the corrected condylar angle (CAcorr: r = 0.746; adjusted r² = 0.533; RMSE = 2.9°) and the corrected four-point angle (4PAcorr: r = 0.733; adjusted r² = 0.513; RMSE = 3.0°); however, the measurements presented proportional errors, possible due the method of assessment of rotations. <i>Conclusions</i>: The findings validate the evaluated photogrammetric methods for assessing sagittal knee alignment. Accounting for thigh and leg rotational deviations is critical for achieving accurate measurements, raising the need of accurate tools for measuring rotational changes in the lower limbs to avoid errors.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 2","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-04-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12029935/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143971952","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":"Physical Exercise and Psychophysical Learnings on Basic Strength Development.","authors":"Gaetano Raiola, Giovanni Esposito, Sara Aliberti, Francesca D'Elia, Tiziana D'Isanto","doi":"10.3390/mps8020040","DOIUrl":"https://doi.org/10.3390/mps8020040","url":null,"abstract":"<p><p>Strength development through physical exercise enhances neuromodulator production, neural connectivity, and motor unit efficiency. Beyond physical benefits, understanding individuals' perceptions, opinions, and knowledge can optimize engagement in exercise. However, existing literature lacks studies examining these factors alongside strength development. This study aimed to investigate whether the effectiveness of strength training protocols is associated with individuals' perceptions, opinions, and knowledge, thereby establishing a link between performance enhancement and awareness of the physiological demands of exercise. The findings seek to highlight the educational potential of physical exercise in promoting psychophysical well-being. A total of 24 participants (14 males, 10 females), aged 35-55 years with varying occupational backgrounds and sedentary levels, were recruited. A strength development protocol was administered, and the participants completed perception-based questionnaires at three time points. Statistical analyses, including repeated-measures ANOVA, Friedman's test, and post hoc comparisons, were conducted. Significant strength improvements were observed, specifically in the Hand Grip Test (<i>p</i> < 0.01). An increase, but non-significant, emerged in the Isometric Mid-Thigh Pull from 1850 ± 210 N to 2270 ± 190 N. The participants also reported a 35% increase in motivation to engage in exercise and a 42% reduction in sedentary behavior. Additionally, 78% of the participants demonstrated greater awareness of exercise benefits, correlating positively with physical improvements. The findings indicate that strength development is associated with increased awareness of the benefits of physical exercise, supporting its use as an educational tool to enhance engagement and adherence to exercise protocols.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 2","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12029894/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144008309","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":"Experimental Investigation of the Effect of Solvent Type and Its Concentration on the Performance of ES-SAGD.","authors":"Sajjad Esmaeili, Brij Maini, Zain Ul Abidin, Apostolos Kantzas","doi":"10.3390/mps8020039","DOIUrl":"https://doi.org/10.3390/mps8020039","url":null,"abstract":"<p><p>Steam-assisted gravity drainage (SAGD) is a widely used thermal enhanced oil recovery (EOR) technique in North America, particularly in high-permeability oil sand reservoirs. While effective, its economic viability has declined due to low oil prices and high greenhouse gas (GHG) emissions from the steam generation. To improve cost-effectiveness and reduce emissions, solvent-assisted SAGD techniques have been explored. Expanding Solvent-SAGD (ES-SAGD) involves co-injecting light hydrocarbons like propane or butane with steam to enhance oil viscosity reduction. This approach lowers the steam-oil ratio by combining solvent dissolution effects with thermal effects. However, the high cost of solvents, particularly butane, challenges its commercial feasibility. Propane is cheaper but less effective, while butane improves performance but remains expensive. This research aims to optimize ES-SAGD by using a propane-butane mixture to achieve efficient performance at a lower cost than pure butane. A linear sand pack is used to evaluate different propane/butane compositions, maintaining constant operational conditions and a solvent concentration of 15 vol.%. Temperature monitoring provides insights into steam chamber growth. Results show that solvent injection significantly enhances ES-SAGD performance compared to conventional SAGD. Performance improves with increasing butane concentration, up to 80% butane in the C₃-C₄ mixture at the test pressure and ambient temperature. Propane alone results in the lowest system temperature, while conventional SAGD reaches the highest temperature. These findings highlight the potential of optimized solvent mixtures to improve ES-SAGD efficiency while reducing costs and GHG emissions.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 2","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12029696/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143972307","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":"Enzyme-Linked Immunosorbent Assay (ELISA) Development for Equine Serum Amyloid A (SAA) Determination Using Recombinant Proteins.","authors":"Pollyanna C Souto, Marcus R Santos, Andrés M Ortega Orozco, Lucas D Bento, Camilo J Ramirez-Lopez, Fabrícia M Girardi, Júlia C Assis Machado, Leandro L de Oliveira, Leandro A da Fonseca","doi":"10.3390/mps8020037","DOIUrl":"https://doi.org/10.3390/mps8020037","url":null,"abstract":"<p><p>We aimed to develop a species-specific ELISA for qualitatively and quantitatively determining serum amyloid A (SAA) in horses. Current methods for measuring SAA in horses utilize ELISA or immunoturbidimetric tests designed for human SAA, which are not specific to horses. Mice and rabbits were used to generate polyclonal antibodies against equine SAA. The study examined serum samples from 32 horses with acute inflammatory disease (SG) and 25 clinically healthy horses. Furthermore, the SAAeq kinetics were observed in three horses from the SG group at three different timepoints. The SAA-ELISA established a cut-off at 0.06 OD₄₉₂nm, where values equal to or higher than this were deemed positive, while values below it was considered negative. The test exhibited a sensitivity of 94% and specificity of 92%, resulting in an overall accuracy of 93%. The positive and negative predictive values were 94% and 92%, respectively. Coefficients of variation for inter- and intra-assay were 6.1% and 7.46% for SG and 9.6% and 9.63% for the control group (CG). The detection limit was determined to be 0.067. The SAA-ELISA proved its worth by demonstrating satisfactory performance, paving the way for the development of automated quantitative tests and species-specific semi-quantitative tests. This paves the way for their application in practical field settings.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 2","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12029847/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144027237","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":"Analysis of Processing, Post-Maturation, and By-Products of shRNA in Gene and Cell Therapy Applications.","authors":"Zhenyi Hong, Nikola Tesic, Xavier Bofill-De Ros","doi":"10.3390/mps8020038","DOIUrl":"https://doi.org/10.3390/mps8020038","url":null,"abstract":"<p><p>Short hairpin RNAs (shRNAs) are potent tools for gene silencing, offering therapeutic potential for gene and cell therapy applications. However, their efficacy and safety depend on precise processing by the RNA interference machinery and the generation of minimal by-products. In this protocol, we describe how to systematically analyze the processing of therapeutic small RNAs by DROSHA and DICER1 and their incorporation into functional AGO complexes. Using standard small RNA sequencing and tailored bioinformatic analysis (QuagmiR), we evaluate the different steps of shRNA maturation that influence processing efficiency and specificity. We provide guidelines for troubleshooting common design pitfalls and off-target effects in transcriptome-wide profiling to identify unintended mRNA targeting via the miRNA-like effect. We provide examples of the bioinformatic analysis that can be performed to characterize therapeutic shRNA. Finally, we provide guidelines for troubleshooting shRNA designs that result in suboptimal processing or undesired off-target effects. This protocol underscores the importance of rational shRNA design to enhance specificity and reduce biogenesis by-products that can lead to off-target effects, providing a framework for optimizing the use of small RNAs in gene and cell therapies.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 2","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12029666/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144012886","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":"Specific Aspects of SELEX Protocol: Different Approaches for ssDNA Generation.","authors":"Alexandr Garanin, Andrey Shalaev, Lidia Zabegina, Ekaterina Kadantseva, Tatiana Sharonova, Anastasia Malek","doi":"10.3390/mps8020036","DOIUrl":"https://doi.org/10.3390/mps8020036","url":null,"abstract":"<p><strong>Background: </strong>Synthetic DNA aptamers are a class of molecules with potential applications in medicine, serving as molecular sensors or ligands for targeted drug delivery. Systematic evolution of ligands by exponential enrichment (SELEX) is a technology for selecting functional aptamers that was first reported three decades ago and has been actively developed since. SELEX involves multiple iterations of two fundamental steps: (i) target affinity-based partitioning of aptamers from a random library and (ii) amplification of selected aptamers by PCR, followed by isolation of single-stranded DNA (ssDNA). SELEX protocols have diversified considerably, with numerous variations possible for each step. This heterogeneity makes it challenging to identify optimal methods. Comparative analysis of different approaches for the major stages of SELEX is therefore of considerable practical importance.</p><p><strong>Methods: </strong>Four widely used methods for ssDNA generation were performed in parallel: (a) PCR followed by digestion of the antisense strand with exonuclease lambda, (b) PCR with an extended primer followed by size-dependent strand separation using denaturing PAGE, (c) asymmetric PCR, and (d) asymmetric PCR with a primer-blocker.</p><p><strong>Results: </strong>The specificity, efficiency, reproducibility, and duration of each method were compared.</p><p><strong>Conclusions: </strong>Asymmetric PCR with a primer-blocker yielded the most favorable results.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 2","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-04-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12029403/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144035375","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The Handling and Sampling of Radical Cystectomy Specimens: A Standardized Approach for Pathological Evaluation.","authors":"Francesca Sanguedolce, Angelo Cormio, Magda Zanelli, Maurizio Zizzo, Andrea Palicelli, Alessandra Filosa, Ugo Giovanni Falagario, Andrea Benedetto Galosi, Luigi Cormio, Giuseppe Carrieri, Roberta Mazzucchelli","doi":"10.3390/mps8020035","DOIUrl":"https://doi.org/10.3390/mps8020035","url":null,"abstract":"<p><p>An accurate histopathological evaluation of radical cystectomy (RC) specimens is crucial for optimal tumor staging, prognosis, and therapeutic decision making. The increasing demand for precision medicine and multidisciplinary oncological management emphasizes the necessity for standardized protocols in the handling and sampling of bladder cancer specimens. The effective processing of RC specimens begins with the integration of clinical and anamnestic data, along with appropriate formalin fixation methods to meet diagnostic needs. The pathologist must meticulously document the macroscopic characteristics and dimensions of the surgical specimen, especially in post-neoadjuvant chemotherapy (post-NAC) cases where the primary tumor may not be macroscopically visible. Sampling strategies should ensure a comprehensive assessment of the primary tumor and any extra-organ or metastatic involvement. Despite international guidelines, variability in pathology practices persists, particularly concerning prostate sampling in RC and the use of frozen sections for margin assessment. Addressing these challenges necessitates a consensus-driven, standardized approach to improve the reproducibility and quality of histopathological data. By addressing gaps in current pathology practices, this review advocates for uniform protocols that enhance diagnostic accuracy, ultimately improving patient care and clinical decision making.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 2","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12029177/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144035603","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}