Methods and Protocols最新文献

{"title":"Clinical Wound Healing After Lower Third Molar Surgery with Envelope and Bayonet Flaps: A Randomized Clinical Trial.","authors":"Roberto Pippi, Chiara Mazzei, Alessandra Pietrantoni","doi":"10.3390/mps8050101","DOIUrl":"10.3390/mps8050101","url":null,"abstract":"<p><strong>Objectives: </strong>The present study mainly aimed to identify whether the envelope and triangular flaps affected wound healing and patient quality of life differently. Secondarily, the study aimed to investigate whether some anatomical and operational variables may also affect healing.</p><p><strong>Study design: </strong>A prospective randomized study was conducted with 56 fully impacted lower third molars, randomly divided into two groups, one treated with the envelope flap and the other with the bayonet flap. Qualitative variables were transformed into quantitative ones and then analyzed using independent samples <i>t</i>-tests or analysis of variance. An analysis of bivariate correlations with Pearson's coefficient was also used. The chi-square test was used to verify the association between each flap and the categorical variables considered.</p><p><strong>Results: </strong>No statistically significant associations were found between flap types and dehiscence, although the mean dehiscence diameter was consistently greater in the envelope flap group. The maximum diameter of the dehiscence at 14 days was found to be significantly and negatively related to the 14-day wound healing indices. Analyses relating to the quality of life did not show significant associations.</p><p><strong>Conclusions: </strong>Despite some significant healing differences between the two considered flaps exist, they do not have relevant effects on the patient's post-operative quality of life.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 5","pages":""},"PeriodicalIF":2.0,"publicationDate":"2025-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12452354/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145113544","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":"Development and Validation of an HPLC-UV/PDA Method for the Determination of Cannflavins in Different <i>Cannabis sativa</i> Chemovars.","authors":"Mostafa A Elhendawy, Mohamed M Radwan, Elsayed A Ibrahim, Amira S Wanas, Adel A Marzouk, Suman Chandra, Murelle Godfrey, Mahmoud A ElSohly","doi":"10.3390/mps8050100","DOIUrl":"10.3390/mps8050100","url":null,"abstract":"<p><p><i>Cannabis sativa</i> (<i>C. sativa</i>) is a psychoactive plant that has been used for millennia for medicinal, recreational, and industrial purposes. The main constituents of cannabis are the cannabinoids, with other constituents including terpenes and flavonoids that contribute to its bioactivity. Among the flavonoid class, there is a subclass, specific to cannabis, namely the cannflavins (A, B, and C), which are biologically active. This study is directed to the analysis of these constituents in various cannabis chemovars. In this study, an HPLC-PDA method was validated and applied to determine the content of cannflavins, namely, cannflavin A (CF-A), cannflavin B (CF-B), and cannflavin C (CF-C), in six different cannabis chemovars. The HPLC separation was achieved using a Luna^® C18 (150 × 4.6 mm × 3 μm) with isocratic elution using a mobile phase consisting of acetonitrile and water (65:35, <i>v</i>/<i>v</i>), both containing 0.1% formic acid at a flow rate of 1 mL/min, with the detector set at 342.4 nm. The method was validated according to the ICH guidelines and exhibited a linear relationship in the 5-500 ppm range with R² > 0.99. The method showed good recovery, ranging from 82% to 98%. The intra-day and inter-day relative standard deviations (% RSDs) were ≤5.29%. Consequently, the method was applied for the determination of all these cannflavins in the different cannabis chemovars. CF-A was the most abundant cannflavin in the examined samples (15.2-478.38 ppm). The method was shown to be simple, accurate, and selective.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 5","pages":""},"PeriodicalIF":2.0,"publicationDate":"2025-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12452342/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145113678","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":"Usefulness of Chromogenic Media in the Identification of <i>Candida</i> spp. Yeasts Compared to Mass Spectrometry.","authors":"Agata Bloch, Tomasz Bogiel, Małgorzata Prażyńska, Eugenia Gospodarek-Komkowska","doi":"10.3390/mps8050098","DOIUrl":"10.3390/mps8050098","url":null,"abstract":"<p><p>Yeasts of the <i>Candida</i> genus are part of the normal human microbiota but can cause infections (candidiasis) under certain conditions. While <i>Candida albicans</i> remains the most common etiological agent, the prevalence of non-albicans <i>Candida</i> species-such as <i>C. glabrata</i>, <i>C. tropicalis</i>, <i>C. krusei</i>, <i>C. parapsilosis</i>, <i>C. kefyr</i>, <i>C. lusitaniae</i>, and the emerging multidrug-resistant <i>C. auris</i>-has been increasing. Effective treatment of candidiasis requires rapid and accurate identification of the causative species, particularly due to species-specific antifungal agent resistance patterns. The aim of this study was to evaluate the usefulness of five chromogenic media for the differentiation of <i>Candida</i> species: BD CHROMagar Candida (Becton Dickinson), CHROM ID Candida (<i>bio</i>Mérieux), CHROMAgar Candida Plus (CHROMAgar France, Biomaxima), CHROMAgar Candida Plus (GRASO Biotech), and Brilliance Candida Agar (OXOID). A total of 175 strains from the following species were tested: <i>C. albicans</i>, <i>C. parapsilosis</i>, <i>C. dubliniensis</i>, <i>C. lusitaniae</i>, <i>C. tropicalis</i>, <i>C. glabrata</i>, <i>C. kefyr</i>, <i>C. krusei</i>, and <i>C. auris</i>. Species identification was confirmed by MALDI-TOF mass spectrometry using the MALDI Biotyper system (Bruker). Colony morphology, especially color characteristics, was assessed on each medium. The morphological features of most <i>Candida</i> species were consistent with the manufacturer's descriptions and allowed for presumptive species-level identification. However, some species showed reproducible but previously undescribed morphological traits, including variations in colony shade. Notably, <i>C. auris</i> could not be reliably identified using BD, <i>bio</i>Mérieux, or OXOID media. In conclusion, while chromogenic media are a helpful preliminary diagnostic tool, subtle differences in colony coloration can complicate interpretation. Diagnostic caution is recommended, and confirmatory methods such as MALDI-TOF remain essential for reliable identification, especially for emerging or less common <i>Candida</i> species.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 5","pages":""},"PeriodicalIF":2.0,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12452300/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145113636","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 the Effect of the Tablet Matrix on the Polymorphism of Ibuprofen, Naproxen, and Naproxen Sodium in Commercially Available Pharmaceutical Formulations.","authors":"Edyta Leyk, Marcin Środa, Gracjan Maślanka, Patrycja Nowaczyk, Amelia Orzołek, Hanna Grodzka, Aleksandra Kurek, Olaf Knut, Julia Michalak, Jonatan Płachciak, Alina Plenis","doi":"10.3390/mps8050099","DOIUrl":"10.3390/mps8050099","url":null,"abstract":"<p><p>Pharmaceutical formulations, in addition to the medicinal substance(s), contain added excipients that make it possible to create a pharmaceutical product that exhibits required properties in terms of mechanical, physical, chemical, and microbiological stability. Additionally, these substances can act as release modifiers or improve bioavailability parameters. Literature data indicate that excipients, especially polymeric ones, can also affect the polymorphism of the active substance, resulting in drug bioavailability enhancement or reduction. This influence can be evaluated using thermal and spectroscopic methods. In the study, differential scanning calorimetry (DSC), vibrational spectroscopic studies (Fourier transform infrared spectroscopy, FTIR), Raman spectroscopy, and X-ray diffraction (XRD) assay of ibuprofen, naproxen, and naproxen sodium standards and pharmaceutical preparations containing these medicinal substances in their compositions were carried out. DSC results indicated that a sharp melting peak was observed on the DSC curves of the standards, confirming their crystalline form. DSC results obtained for pharmaceutical formulations also indicated that the enthalpy of melting is sometimes lower than calculated from the percentage of active ingredients in the formulations. In addition, the melting peak is often broadened and shifted toward lower temperatures, suggesting the influence of excipients on the polymorphism of drug substances. The FTIR and Raman spectra of pharmaceutical formulations contained all characteristics of the active substances. XRD analysis was also performed. Therefore, possible chemical interactions between the components of the preparations have been excluded. At the same time, FTIR and Raman spectroscopy results as well as XRD assay showed a reduction in the height of signals corresponding to the crystalline API form, confirming the possibility of reducing API crystallinity in pharmaceutical formulations.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 5","pages":""},"PeriodicalIF":2.0,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12452562/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145113551","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 Protocol for Modeling Human Bone Inflammation: Co-Culture of Osteoblasts and Osteoclasts Exposed to Different Inflammatory Microenvironments.","authors":"Araceli Valverde, Afsar Raza Naqvi","doi":"10.3390/mps8050097","DOIUrl":"10.3390/mps8050097","url":null,"abstract":"<p><p>Bone remodeling relies on the coordinated activity of osteoblasts (OBs) and osteoclasts (OCs). Disruptions in OB-OC balance can lead to diseases such as periodontitis, a chronic microbial-induced inflammatory disease. To investigate how inflammation affects OB-OC interactions, we standardized an in vitro 2D indirect co-culture system using primary human OB and OC precursors from peripheral blood mononuclear cells in a transwell setup, which allows paracrine signaling and separate analysis of each cell type. When exposed to bacterial lipopolysaccharides (<i>Aa LPS</i> and <i>E. coli LPS</i>) and proinflammatory cytokines (IL-6 and TNF-α), we observed that inflammatory stimuli significantly increased OC differentiation, particularly TNF-α, while <i>E. coli LPS</i> specifically suppressed OB activity as observed by the expression of key markers and cellular staining. These results demonstrate that microbial and host-derived inflammatory factors can differentially modulate bone cell behavior. This approach offers a physiologically relevant and ethically advantageous alternative to animal models to screen dual-targeted bone therapies to restore perturbed metabolism.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 5","pages":""},"PeriodicalIF":2.0,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12452566/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145113510","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":"Development of an Ex Vivo Platform to Model Urethral Healing.","authors":"Christopher Foster, Ryan Tran, Khushi Grover, Abdullah Salama, Courtney K Rowe","doi":"10.3390/mps8040096","DOIUrl":"10.3390/mps8040096","url":null,"abstract":"<p><strong>Background: </strong>Urethral strictures impact millions, causing significant morbidity and millions in healthcare costs. Testing new interventions is limited by the lack of inexpensive urethral healing models. We developed an ex vivo model of early urethral wound healing using explanted rabbit urethral tissue. This was used to test the impact of six growth factors (GFs).</p><p><strong>Methods: </strong>The rabbit urethra was detubularized by cutting it between the corpora cavernosa, and then it was stitched flat using a custom 3D-printed platform. The tissue was carefully scratched to produce a visible wound, and the specimens were placed in media containing growth factors at 100 ng/mL and 10 ng/mL. Images were taken at 0, 24, 48, 72, and 96 h, and the wound area was measured by blinded reviewers to determine the rate of wound contraction.</p><p><strong>Results: </strong>Specimens with IGF at 100 ng/mL showed a statistically significant difference in wound contraction when compared to those with GF-free control medium, showing that IGF-1 supports early urethral epithelization and may improve healing.</p><p><strong>Conclusions: </strong>The developed protocol provides a simple explant platform that can be used to investigate methods of enhancing early phases of urethral healing or used to investigate other areas of urethral health, including drug delivery, infection, and mechanical properties.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 4","pages":""},"PeriodicalIF":2.0,"publicationDate":"2025-08-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12388648/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144961737","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":"Functional Methods for Studying Sperm-Zona Pellucida Interaction in Mammals.","authors":"Natalie Zelenkova, Veronika Kraus, Alexandra Maresova, Zuzana Pilsova, Aneta Pilsova, Barbora Klusackova, Eva Chmelikova, Katerina Komrskova, Pavla Postlerova","doi":"10.3390/mps8040095","DOIUrl":"10.3390/mps8040095","url":null,"abstract":"<p><p>The initial molecular events mediating mammalian sperm binding to the zona pellucida (ZP) of the oocyte are highly complex and still not fully elucidated. Recent advances have identified multiple candidate sperm surface proteins, often functioning as part of high-molecular-weight complexes that mediate this critical fertilization event in a species-specific and coordinated manner. To address a significant gap in the literature, we provide an in-depth overview of the functional assays employed to investigate sperm-ZP interactions, emphasizing their underlying principles, potential applications, and key methodological strengths and limitations. The techniques discussed range from classical in vitro sperm-oocyte and hemizona binding assays, including antibody-blocking and competitive strategies, to cutting-edge in vivo genetic models, each contributing unique insights into the physiological relevance of the proposed ZP receptors. Robust experimental design, including the use of appropriate controls and validation strategies, is essential for accurately interpreting the role of candidate sperm receptors. This review provides a structured overview of current methodologies to support researchers in critically evaluating and applying functional assays in future studies.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 4","pages":""},"PeriodicalIF":2.0,"publicationDate":"2025-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12388836/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144961709","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":"Exploring the Relationship of Cognitive Disengagement Syndrome and Attention Deficit/Hyperactivity Disorder with Emotional Dysregulation: A Twin Study in Childhood and Adolescence.","authors":"Simona Scaini, Stefano De Francesco, Ludovica Giani, Marco Battaglia, Emanuela Medda, Corrado Fagnani","doi":"10.3390/mps8040094","DOIUrl":"10.3390/mps8040094","url":null,"abstract":"<p><p>Data on the genetic and environmental factors underlying the co-occurrence of Cognitive Disengagement Syndrome (CDS), Attention Deficit Hyperactivity Disorder (ADHD), and Emotional Dysregulation (ED) are limited. This study aimed to explore the nature of the associations between CDS, ADHD with ED, and to assess the role of shared etiological factors in explaining their comorbidity. We analyzed a sample of 400 Italian twin pairs aged 8-18, from Northern Italy and enrolled in the Italian Twin Registry. Bivariate genetic analyses were conducted using parent-rated CBCL scores for CDS, ADHD, and ED. For both CDS-ED and ADHD-ED associations, the best-fitting models were Cholesky AE models (-2LL = -849.167 and -339.030, respectively; <i>p</i> > 0.05), suggesting that the covariation was mainly due to additive genetic factors (CDS-ED-A = 0.81, 95% CI [0.66-0.95]; ADHD-ED-A = 0.86, 95% CI [0.75-0.95]). More than half of the genes were shown to be shared among the phenotypes. Non-shared environmental contributions were smaller (CDS-ED-E = 0.19, 95% CI [0.05-0.34]; ADHD-ED-E = 0.14, 95% CI [0.05-0.25]), indicating interrelated but distinct constructs. Despite some limitations, particularly the exclusive use of the CBCL, findings highlight the importance of monitoring ED symptoms in individuals with CDS or ADHD, and vice versa.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 4","pages":""},"PeriodicalIF":2.0,"publicationDate":"2025-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12388083/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144961764","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":"Optimizing Cell Density and Unveiling Cytotoxic Profiles of DMSO and Ethanol in Six Cancer Cell Lines: Experimental and In Silico Insights.","authors":"Abutaleb Asiri, Munazzah Tasleem, Muwadah Al Said, Abdulaziz Asiri, Ali Ahmed Al Qarni, Ahmed Bakillah","doi":"10.3390/mps8040093","DOIUrl":"10.3390/mps8040093","url":null,"abstract":"<p><p><b>Background:</b> Accurate assessment of drug cytotoxicity in vitro is essential for preclinical evaluation of anticancer agents. Methodological parameters such as cell density and solvent concentrations can significantly influence the reproducibility and reliability of cell-based assay results. <b>Objective:</b> This study aims to optimize cell seeding density and evaluate the cytotoxic effects of common solvents (DMSO and ethanol) on different cancer cell lines, complemented by in silico analysis to elucidate underlying mechanisms. <b>Materials and Methods:</b> Six cancer cell lines (HepG2, Huh7, HT29, SW480, MCF-7, and MDA-MB-231) were seeded at different densities to determine the optimal cell seeding number ideal for cell viability assay at 24, 48, and 72 h. The cytotoxicity of DMSO and ethanol was assessed in these cell lines using an MTT assay at multiple time points. In silico docking studies were conducted to investigate the interactions between solvents and key proteins involved in apoptosis, membrane function, and metabolism. <b>Results:</b> A cell density of 2000 cells per well yielded consistent linear viability across cell lines and time points. DMSO at 0.3125% showed minimal cytotoxicity across all cell lines (except MCF-7) and time points; the cytotoxic effect at higher concentrations is variable depending on cell type and exposure duration. Ethanol exhibited rapid and concentration-dependent cytotoxicity, reducing viability by more than 30% at as low as 0.3125% concentration after 24 h. Docking analyses revealed that DMSO binds specifically to apoptotic and membrane proteins, suggesting a role in inducing apoptosis. In contrast, ethanol primarily interacts with metabolic proteins, consistent with its effect on membrane disruption and rapid cell death. <b>Conclusion:</b> DMSO at 0.3125% is a good choice as a solvent since it has low toxicity in most tested cell lines; however, the safe concentration limit is dependent on cell type and exposure duration. Ethanol exhibited higher cytotoxicity, necessitating careful concentration management. The in silico analysis supports these findings, indicating that DMSO interacts with apoptosis-related proteins, whereas ethanol primarily affects metabolic processes. These results highlight the importance of precise cell density optimization and solvents for reliable cytotoxicity assessment in cell-based assays.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 4","pages":""},"PeriodicalIF":2.0,"publicationDate":"2025-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12388702/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144961366","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":"¹H Nuclear Magnetic Resonance (NMR) Metabolomics in Rodent Plasma: A Reproducible Framework for Preclinical Biomarker Discovery.","authors":"Mohd Naeem Mohd Nawi, Ranina Radzi, Azizan Ali, Siti Zubaidah Che Lem, Azlina Zulkapli, Ezarul Faradianna Lokman, Mansor Fazliana, Sreelakshmi Sankara Narayanan, Karuthan Chinna, Mohd Fairulnizal Md Noh, Zulfitri Azuan Mat Daud, Tilakavati Karupaiah","doi":"10.3390/mps8040092","DOIUrl":"10.3390/mps8040092","url":null,"abstract":"<p><p>This protocol paper outlines a robust and reproducible framework for a ¹H nuclear magnetic resonance (NMR) metabolomics analysis of rodent plasma, designed to facilitate preclinical biomarker discovery. The protocol details optimised steps for plasma collection in a preclinical rodent model, sample preparation, and NMR data acquisition using presaturation Carr-Purcell-Meiboom-Gill (PRESAT-CPMG) pulse sequences, ensuring high-quality spectral data and effective suppression of macromolecule signals. Comprehensive spectral processing and metabolite assignment are described, with guidance on multivariate and univariate statistical analyses to identify metabolic changes and potential biomarkers. The framework emphasises methodological rigour and reproducibility, enabling accurate quantification and interpretation of metabolites relevant to disease mechanisms or therapeutic interventions. By providing a standardised approach, this protocol supports longitudinal and translational studies, bridging findings from rodent models to clinical applications and advancing the reliability of metabolomics-based biomarker discovery in preclinical research.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 4","pages":""},"PeriodicalIF":2.0,"publicationDate":"2025-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12388194/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144961706","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}