Methods and Protocols

19 pages, 3668 KB

Open AccessProtocol

Optimized Protocol for Primary Rat Hepatocyte Isolation and a Model for Investigating Experimental Steatosis

by Amani A. Harb, Mohammad AlSalem and Shtaywy Abdalla

Methods Protoc. 2025, 8(5), 111; https://doi.org/10.3390/mps8050111 - 19 Sep 2025

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Background: Primary hepatocytes are excellent models for studying liver functions and liver diseases. However, obtaining high yields of viable hepatocytes remains technically challenging, limiting their broader applications. Most conventional methods rely on a two-step collagenase perfusion technique. Despite its widespread use, this approach [...] Read more.

Background: Primary hepatocytes are excellent models for studying liver functions and liver diseases. However, obtaining high yields of viable hepatocytes remains technically challenging, limiting their broader applications. Most conventional methods rely on a two-step collagenase perfusion technique. Despite its widespread use, this approach has several limitations that reduce the success rate of hepatocyte isolation and culture. The procedure involves multiple parameters that are continually being optimized in order to obtain hepatocytes in high yield and quality that can be used to provide insights into their physiology and pathophysiology. Aim: We aimed to enhance the success rate and reproducibility of hepatocyte isolation with high yield, enabling analysis of diverse physiological and pathophysiological aspects of lipid metabolism. It also establishes an in vitro steatosis model for evaluating therapeutic drugs and molecular interventions. Methods: Rat liver was perfused in situ with EDTA buffer followed by collagenase IV. Liver was then isolated, and hepatocytes were mechanically liberated, filtered, and purified through density-gradient centrifugation. Viable cells were cultured at 700,000 or 1 million cells/well for 24 h. The monolayer was incubated in lipogenic media for an additional 24 or 48 h. Hepatocytes were fixed, neutral lipids were stained using Oil Red O, and the stained area was quantified using Image J software version 1.54. Results: Yield of hepatocytes was ~75–90 million cells/liver, with viability of 86–93%. Cells seeded at 700,000 and 1 million cells/well reached confluences of 60% and 80%, respectively, after 24 h. Steatosis was then induced with lipid accumulation reaching 21% of image area after 24 h and 25% after 48 h. Conclusions: The current protocol presents an efficient and highly reproducible method for isolating primary rat hepatocytes in high yield with high viability. Additionally, the protocol provides a foundation for studying the pathophysiology of fatty liver disease. Full article

(This article belongs to the Section Molecular and Cellular Biology)

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19 pages, 2541 KB

Open AccessArticle

A Comparative Bioinformatic Analysis of Optic Nerve Axon Regeneration Lipidomes Using the Xenopus laevis as a Model System

by Vernon S. Volante, Fiona L. Watson and Sanjoy K. Bhattacharya

Methods Protoc. 2025, 8(5), 110; https://doi.org/10.3390/mps8050110 - 15 Sep 2025

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Abstract

Lipidomics is a rapidly growing branch of metabolomics that identifies lipid compositions of samples to learn more about disease and identify potential novel therapeutic targets. In the context of ophthalmology, lipidomic research has increased our understanding of optic nerve regeneration. The diversity of [...] Read more.

Lipidomics is a rapidly growing branch of metabolomics that identifies lipid compositions of samples to learn more about disease and identify potential novel therapeutic targets. In the context of ophthalmology, lipidomic research has increased our understanding of optic nerve regeneration. The diversity of experimental designs for lipidomic research and the large datasets generated are two obstacles that must be addressed by bioinformatic tools to perform statistical analysis on lipidomics data. Our study provides an objective comparison of the features in two freely accessible web-based bioinformatics tools, MetaboAnalyst 6.0 and LipidOne 2.3, for analyzing an optic nerve regeneration model lipidome. A publicly available lipidomic dataset of the optic nerve axon regeneration model, Xenopus laevis, was used to compare the analytic capabilities of both tools. Though both tools offered univariate and multivariate analysis methods, MetaboAnalyst 6.0 had advantages in customizable data processing, normalization, analysis, and image generation. It also offered consistent multiple-comparison testing correction and comprehensive results/dataset export. Meanwhile LipidOne 2.3 uniquely allowed for univariate and multivariate analysis of lipid classes and lipid building blocks. Full article

(This article belongs to the Section Molecular and Cellular Biology)

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17 pages, 591 KB

Open AccessProtocol

Comprehensive Protocols for Detecting Xenotransplantation-Relevant Viruses

by Hina Jhelum, Benedikt B. Kaufer and Joachim Denner

Methods Protoc. 2025, 8(5), 109; https://doi.org/10.3390/mps8050109 - 12 Sep 2025

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Abstract

Xenotransplantation using pig cells, tissues, or organs is advancing toward clinical application to address the shortage of human donor organs for treating organ failure. However, this emerging technology carries the risk of transmitting pathogenic porcine microorganisms, particularly viruses. The recent transmission of a [...] Read more.

Xenotransplantation using pig cells, tissues, or organs is advancing toward clinical application to address the shortage of human donor organs for treating organ failure. However, this emerging technology carries the risk of transmitting pathogenic porcine microorganisms, particularly viruses. The recent transmission of a porcine herpesvirus to the first human recipient of a pig heart highlights the urgent need for more rigorous screening of donor pigs. To identify potentially pathogenic porcine viruses, highly sensitive and specific detection methods are required. PCR-based techniques able to detect porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV), hepatitis E virus (HEV), porcine circoviruses (PCV1-4), porcine lymphotropic herpesviruses (PLHV-1-3), porcine endogenous retroviruses (PERVs), porcine parvovirus (PPV), Torque teno sus viruses (TTSuV1,2), atypical porcine pestivirus (APPV) and SARS-CoV-2 were established. Immunological assays that detect antibodies as indirect indicators of infection were established for PCMV/PRV, HEV, PLHVs and PERVs. Since most veterinary laboratories focus on detecting viruses that are pathogenic to pigs and cause economic losses to the swine industry, screening for viruses relevant to xenotransplantation should be conducted in specialized virological diagnostic units. In this context, we present a complete collection of the newest and detailed protocols for comprehensive viral screening, along with guidance on how to implement these methods effectively. Full article

(This article belongs to the Section Public Health Research)

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43 pages, 1075 KB

Open AccessReview

Mechanisms of Resistance to CAR T-Cells and How to Overcome Them

by Luca Legato, Matteo Bisio, Filippo Fasano, Corrado Benevolo Savelli, Carolina Secreto, Chiara Maria Dellacasa, Barbara Botto, Alessandro Busca, Marco Cerrano, Roberto Freilone and Mattia Novo

Methods Protoc. 2025, 8(5), 108; https://doi.org/10.3390/mps8050108 - 11 Sep 2025

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Abstract

In the last few decades, chimeric antigen receptor (CAR) T-cell therapy has led to a paradigm shift in the treatment of hematological malignancies, including various subtypes of B-cell non-Hodgkin’s lymphoma, B-cell acute lymphoblastic leukemia, and multiple myeloma. However, most patients experience refractoriness to [...] Read more.

In the last few decades, chimeric antigen receptor (CAR) T-cell therapy has led to a paradigm shift in the treatment of hematological malignancies, including various subtypes of B-cell non-Hodgkin’s lymphoma, B-cell acute lymphoblastic leukemia, and multiple myeloma. However, most patients experience refractoriness to CAR T-cells or relapse after treatment. Many efforts are underway to understand the mechanisms behind CAR T-cell failure, which are mainly related to CAR T-cell dysfunction, tumor-intrinsic resistance, an immunosuppressive tumor microenvironment, manufacturing issues, or patient-related factors. Several strategies are being developed to overcome these resistance mechanisms, including the engineering of more functional allogeneic CAR T-cell products, the targeting of alternative tumor antigens, and combination therapies with other drugs such as checkpoint inhibitors or small molecules to enhance CAR T-cell efficacy. In this review, we will provide an updated overview of the mechanisms of CAR T-cell failure and the therapeutic advances currently under development to address them. Full article

(This article belongs to the Special Issue Current Methodology Advances in Cell Therapy Applications)

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21 pages, 4438 KB

Open AccessProtocol

Compendium of Agrobacterium-Mediated Tissue Culture Transformation Methods of Various Solanaceae Species

by Caterina Brancato, Najmeh Heusch and Kenneth Wayne Berendzen

Methods Protoc. 2025, 8(5), 107; https://doi.org/10.3390/mps8050107 - 11 Sep 2025

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Abstract

The routine transformation and genetic modification of many plant species other than Arabidopsis thaliana is still an arduous task. In many cases, it is necessary to use special tissues or conditions performed under sterile tissue culture conditions. Nevertheless, this approach is often the [...] Read more.

The routine transformation and genetic modification of many plant species other than Arabidopsis thaliana is still an arduous task. In many cases, it is necessary to use special tissues or conditions performed under sterile tissue culture conditions. Nevertheless, this approach is often the most expedient one, and streamlining protocols to maximize efficiency and minimize effort without sacrificing quality is paramount to today’s research agendas. The Solanaceae family tends to be amicable to tissue culture and relatively easy to transform. Here, we present our optimized, routine tissue culture protocols for the transformation of Nicotiana benthamiana, Nicotiana tabacum, Solanum tuberosum (potato), and Solanum lycopersicum (tomato). We highlight their commonalities and their differences, thus giving the researcher a framework for optimizing their own protocols in their laboratory if needed. Tissue culture transformation is still an important and dynamic field for the advancement of plant research in molecular genetics, physiology, and plant pathology, and will continue to be a viable and important resource into the future. Full article

(This article belongs to the Section Tissue Engineering and Organoids)

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10 pages, 1163 KB

Open AccessArticle

A Rapid LC–MS/MS Method for Quantification of Biogenic Amines in Meat: Validation and Application for Food Safety Monitoring

by Giulia Rampazzo, Giacomo Depau, Giampiero Pagliuca, Elisa Zironi, Andrea Serraino, Federica Savini and Teresa Gazzotti

Methods Protoc. 2025, 8(5), 106; https://doi.org/10.3390/mps8050106 - 10 Sep 2025

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Abstract

Biogenic amines (BAs) are nitrogenous compounds naturally present in protein-rich foods, whose accumulation may indicate spoilage and pose health risks. This study presents the development and validation of a rapid LC–MS/MS method for the simultaneous quantification of six BAs—putrescine (PUT), cadaverine (CAD), histamine [...] Read more.

Biogenic amines (BAs) are nitrogenous compounds naturally present in protein-rich foods, whose accumulation may indicate spoilage and pose health risks. This study presents the development and validation of a rapid LC–MS/MS method for the simultaneous quantification of six BAs—putrescine (PUT), cadaverine (CAD), histamine (HIS), tyramine (TYR), spermidine (SPD), and spermine (SPM)—in meat products, without requiring derivatisation. Sample preparation was optimized to enhance extraction efficiency and reproducibility, using 0.5 M HCl and a double-centrifugation protocol to avoid matrix interference. Chromatographic separation was optimized using a C18 column and acidified ammonium formate/acetonitrile mobile phases. The method showed good linearity (R² > 0.99), trueness between −20% and +20%, and acceptable precision (RSD_r and RSD_R ≤ 25%). Limits of quantification were established at 10 µg/g for all analytes. The method was applied to ten commercial meat samples, where PUT, TYR, and SPD were the most frequently detected amines. Although HIS and TYR levels were below toxicological thresholds for healthy individuals, one sample showed TYR levels potentially concerning for monoamine oxidase inhibitors -treated consumers. The Biogenic Amine Index (BAI) further supported product quality assessment, identifying early spoilage in selected cases. This method offers a rapid, robust and efficient tool for routine monitoring of BAs in meat products, supporting food safety and quality control initiatives. Full article

(This article belongs to the Special Issue Analytical Methods in Natural Sciences and Archaeometry)

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20 pages, 5563 KB

Open AccessArticle

Creation of a Novel Coding Program to Identify Genes Controlled by miRNAs During Human Rhinovirus Infection

by Pax Bosner, Emily Smith, Victoria Cappleman, Alka Tomicic, Ahmed Alrefaey, Ibemusu Michael Otele, Aref Kyyaly and Jamil Jubrail

Methods Protoc. 2025, 8(5), 105; https://doi.org/10.3390/mps8050105 - 9 Sep 2025

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Abstract

Human rhinovirus (RV) is the most frequent cause of the common cold, as well as severe exacerbations of chronic obstructive pulmonary disease (COPD) and asthma. Currently, there are no effective and accurate diagnostic tools or antiviral therapies. MicroRNAs (miRNAs) are small, non-coding sections [...] Read more.

Human rhinovirus (RV) is the most frequent cause of the common cold, as well as severe exacerbations of chronic obstructive pulmonary disease (COPD) and asthma. Currently, there are no effective and accurate diagnostic tools or antiviral therapies. MicroRNAs (miRNAs) are small, non-coding sections of RNA involved in the regulation of gene expression and have been shown to be associated with different pathologies. However, the precise role of miRNAs in RV infection is not yet well established. Also, no unified computational framework exists to specifically link miRNA expression with functional gene targets during RV infection. This study aimed to first analyse the impact of RV16 on miRNA expression across the viral life cycle to identify a small panel with altered expression. We then developed a novel bioinformatics pipeline that integrated time-resolved miRNA profiling with multi-database gene-phenotype mapping to identify diagnostic biomarkers and their regulatory networks. Our in-house Python-based tool, combining mirDIP, miRDB and VarElect APIs, predicted seven genes (EZH2, RARG, PTPN13, OLFML3, STAG2, SMARCA2 and CD40LG) implicated in antiviral responses and specifically targeted by RV16 and regulated by our miRNAs. This method therefore offers a scalable approach to interrogate miRNA-gene interactions for viral infections, with potential applications in rapid diagnostics and therapeutic target discovery. Full article

(This article belongs to the Section Molecular and Cellular Biology)

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17 pages, 2350 KB

Open AccessProtocol

A Safe and Accessible Cell-Based Spike–ACE2 Binding Assay for Evaluating SARS-CoV-2 Neutralization Activity in Biological Samples Using Flow Cytometry

by Martin A. Rossotti, Shannon Ryan, Greg Hussack, Jamshid Tanha, Bassel Akache and Tyler M. Renner

Methods Protoc. 2025, 8(5), 104; https://doi.org/10.3390/mps8050104 - 8 Sep 2025

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Abstract

SARS-CoV-2, the agent responsible for coronavirus disease in 2019 (COVID-19), has caused extensive global health and socioeconomic impact due to its transmissibility and pathology. As a result, it was classified as a Risk Group 3 human pathogen, and handling samples containing live virus [...] Read more.

SARS-CoV-2, the agent responsible for coronavirus disease in 2019 (COVID-19), has caused extensive global health and socioeconomic impact due to its transmissibility and pathology. As a result, it was classified as a Risk Group 3 human pathogen, and handling samples containing live virus requires enhanced biological containment facilities (i.e., CL3) to reduce the potential of laboratory infection to personnel and the spread of the virus into the community. While the use of an authentic live virus remains the gold standard for biological assays, alternative methods have been developed to effectively evaluate neutralization activity in the absence of a replicating viral agent. Here, we describe a cell-based spike–ACE2 binding assay as a surrogate for neutralization of SARS-CoV-2 spike to identify potential neutralizing antibodies. A main advantage of this approach is the exclusion of infectious viral particles, increasing biosafety for laboratory personnel. The interaction of recombinant SARS-CoV-2 trimeric spike protein with ACE2 is monitored and quantified by flow cytometry. Notably, our previous studies have demonstrated the utility of this assay for other viruses, beyond SARS-CoV-2. The methodology presented here has exhibited a strong correlation to other widely accepted methods, such as pseudotyped lentiviral and live virus neutralization assays, in identifying neutralizing antibodies. Full article

(This article belongs to the Section Molecular and Cellular Biology)

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11 pages, 1696 KB

Open AccessArticle

Recombinant Expression and Purification of the Cyanobacterial Chaperone HtpG from Synechococcus elongatus PCC 7942

by Liqun Jiang, Ibrahim D. Boyenle, Nicolas Delaeter and Yanxin Liu

Methods Protoc. 2025, 8(5), 103; https://doi.org/10.3390/mps8050103 - 6 Sep 2025

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Abstract

The 90 kDa Heat Shock Protein (Hsp90) is an essential and highly conserved molecular chaperone that supports the folding and maturation of a diverse array of client proteins across prokaryotic and eukaryotic organisms. In bacteria, HtpG, the Hsp90 homolog, plays a central role [...] Read more.

The 90 kDa Heat Shock Protein (Hsp90) is an essential and highly conserved molecular chaperone that supports the folding and maturation of a diverse array of client proteins across prokaryotic and eukaryotic organisms. In bacteria, HtpG, the Hsp90 homolog, plays a central role in stress response and protein homeostasis, particularly under high-temperature and other stress conditions. Despite extensive studies on HtpG from E. coli, the biochemical properties and functional roles of cyanobacterial HtpG remain poorly characterized. Here, we focus on HtpG from the cyanobacterium Synechococcus elongatus PCC 7942 (seHtpG), a model organism for photosynthesis and circadian rhythm research. We developed a method for the overexpression and purification of seHtpG in E. coli, achieving high purity and yield suitable for biochemical and structural studies. Biophysical and biochemical assays show that seHtpG forms dimers and hydrolyzes ATP at a rate of 1.9 ATP/min, 4-fold faster than that of E. coli HtpG. This work establishes seHtpG as a model for studying the roles of HtpG in cyanobacterial protein homeostasis, photosynthesis, and stress response, enabling further exploration of cyanobacterial Hsp90 in ecosystem dynamics and biotechnological applications. Full article

(This article belongs to the Section Molecular and Cellular Biology)

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15 pages, 2311 KB

Open AccessProtocol

A Scalable Protocol for Ex Vivo Production of CAR-Engineered Human NK Cells

by Supreet Khanal and Nirjal Bhattarai

Methods Protoc. 2025, 8(5), 102; https://doi.org/10.3390/mps8050102 - 5 Sep 2025

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Abstract

Chimeric antigen receptor-expressing NK (CAR-NK) cells represent an advancing frontier in cancer immunotherapy, building upon decades of natural killer cell research and recent breakthroughs in CAR technology. While early CAR-NK manufacturing protocols have demonstrated feasibility, existing manufacturing methods, whether utilizing cord blood or [...] Read more.

Chimeric antigen receptor-expressing NK (CAR-NK) cells represent an advancing frontier in cancer immunotherapy, building upon decades of natural killer cell research and recent breakthroughs in CAR technology. While early CAR-NK manufacturing protocols have demonstrated feasibility, existing manufacturing methods, whether utilizing cord blood or peripheral blood sources, often require extended culture periods and intensive labor, creating bottlenecks for widespread therapeutic application. To address these manufacturing hurdles, we have developed an optimized protocol for ex vivo CAR-NK cell production from human peripheral blood that incorporates lessons learned from previous methodologies while introducing novel efficiency improvements. This protocol offers a practical solution for scalable CAR-NK cell manufacturing that can be readily adapted across different production facilities, potentially accelerating the clinical development of CAR-NK therapies. Full article

(This article belongs to the Special Issue Current Methodology Advances in Cell Therapy Applications)

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15 pages, 1461 KB

Open AccessArticle

Clinical Wound Healing After Lower Third Molar Surgery with Envelope and Bayonet Flaps: A Randomized Clinical Trial

by Roberto Pippi, Chiara Mazzei and Alessandra Pietrantoni

Methods Protoc. 2025, 8(5), 101; https://doi.org/10.3390/mps8050101 - 4 Sep 2025

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Abstract

Objectives: 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. Study design: A prospective [...] Read more.

Objectives: 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. Study design: 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 t-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. Results: 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. Conclusions: 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. Full article

(This article belongs to the Section Public Health Research)

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18 pages, 1484 KB

Open AccessArticle

Development and Validation of an HPLC–UV/PDA Method for the Determination of Cannflavins in Different Cannabis sativa Chemovars

by Mostafa A. Elhendawy, Mohamed M. Radwan, Elsayed A. Ibrahim, Amira S. Wanas, Adel A. Marzouk, Suman Chandra, Murelle Godfrey and Mahmoud A. ElSohly

Methods Protoc. 2025, 8(5), 100; https://doi.org/10.3390/mps8050100 - 3 Sep 2025

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Abstract

Cannabis sativa (C. sativa) 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 [...] Read more.

Cannabis sativa (C. sativa) 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, v/v), 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. Full article

(This article belongs to the Special Issue Spectroscopic Methods of Analysis)

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22 pages, 6643 KB

Open AccessArticle

Analysis of the Effect of the Tablet Matrix on the Polymorphism of Ibuprofen, Naproxen, and Naproxen Sodium in Commercially Available Pharmaceutical Formulations

by Edyta Leyk, Marcin Środa, Gracjan Maślanka, Patrycja Nowaczyk, Amelia Orzołek, Hanna Grodzka, Aleksandra Kurek, Olaf Knut, Julia Michalak, Jonatan Płachciak and Alina Plenis

Methods Protoc. 2025, 8(5), 99; https://doi.org/10.3390/mps8050099 - 1 Sep 2025

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Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Analytical Methods in Natural Sciences and Archaeometry)

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12 pages, 250 KB

Open AccessArticle

Usefulness of Chromogenic Media in the Identification of Candida spp. Yeasts Compared to Mass Spectrometry

by Agata Bloch, Tomasz Bogiel, Małgorzata Prażyńska and Eugenia Gospodarek-Komkowska

Methods Protoc. 2025, 8(5), 98; https://doi.org/10.3390/mps8050098 - 1 Sep 2025

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Abstract

Yeasts of the Candida genus are part of the normal human microbiota but can cause infections (candidiasis) under certain conditions. While Candida albicans remains the most common etiological agent, the prevalence of non-albicans Candida species—such as C. glabrata, C. tropicalis, C. [...] Read more.

Yeasts of the Candida genus are part of the normal human microbiota but can cause infections (candidiasis) under certain conditions. While Candida albicans remains the most common etiological agent, the prevalence of non-albicans Candida species—such as C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, C. kefyr, C. lusitaniae, and the emerging multidrug-resistant C. auris—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 Candida species: BD CHROMagar Candida (Becton Dickinson), CHROM ID Candida (bioMérieux), 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: C. albicans, C. parapsilosis, C. dubliniensis, C. lusitaniae, C. tropicalis, C. glabrata, C. kefyr, C. krusei, and C. auris. 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 Candida 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, C. auris could not be reliably identified using BD, bioMérieux, 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 Candida species. Full article

(This article belongs to the Section Public Health Research)

15 pages, 2089 KB

Open AccessProtocol

A Protocol for Modeling Human Bone Inflammation: Co-Culture of Osteoblasts and Osteoclasts Exposed to Different Inflammatory Microenvironments

by Araceli Valverde and Afsar Raza Naqvi

Methods Protoc. 2025, 8(5), 97; https://doi.org/10.3390/mps8050097 - 1 Sep 2025

Viewed by 440

Abstract

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 [...] Read more.

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 (Aa LPS and E. coli LPS) and proinflammatory cytokines (IL-6 and TNF-α), we observed that inflammatory stimuli significantly increased OC differentiation, particularly TNF-α, while E. coli LPS 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. Full article

(This article belongs to the Section Molecular and Cellular Biology)

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