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Methods Protoc., Volume 8, Issue 1 (February 2025) – 15 articles

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11 pages, 2060 KiB  
Article
Exploratory Ultrasound Analysis of the Diaphragm and Respiratory Capacity in Women with Primary Dysmenorrhea: A Cross-Sectional Observational Study
by Rebeca del Prado-Álvarez, María García-Arrabé, Ángel González-de-la-Flor, Marta de la Plaza San Frutos, Jaime Almazán-Polo and Cecilia Estrada-Barranco
Methods Protoc. 2025, 8(1), 15; https://doi.org/10.3390/mps8010015 - 4 Feb 2025
Viewed by 302
Abstract
Primary dysmenorrhea (PD) is a common gynecological condition characterized by menstrual pain without underlying pelvic pathology. It has been linked to functional and structural changes in the core musculature, but limited evidence exists regarding its association with diaphragmatic and respiratory mechanics. This study [...] Read more.
Primary dysmenorrhea (PD) is a common gynecological condition characterized by menstrual pain without underlying pelvic pathology. It has been linked to functional and structural changes in the core musculature, but limited evidence exists regarding its association with diaphragmatic and respiratory mechanics. This study aimed to elaborate on these potential associations by assessing the diaphragmatic structure and respiratory function in women with PD compared to healthy controls, utilizing ultrasound imaging, spirometry and respiratory pressure measurements. Methods: An observational, cross-sectional study was conducted with 44 female participants (22 with PD and 22 healthy controls). Diaphragmatic structure was evaluated through ultrasound, measuring the intercostal distance, diaphragmatic thickness, and diaphragmatic excursion at rest and during maximum voluntary contraction. Spirometric assessments included forced vital capacity (FVC), forced expiratory volume in the first second (FEV1), and the FVC/FEV1 ratio, along with measurements of maximum inspiratory pressure (MIP) and maximum expiratory pressure (MEP). Group differences were analyzed using Student’s t-test and effect sizes were reported with Cohen’s d. Results: No significant differences were observed between the groups in diaphragmatic thickness, diaphragmatic excursion, or global respiratory capacity (p > 0.05). However, women with PD presented a significant reduction in the left intercostal distance both at rest (p = 0.035, d = 0.56) and during contraction (p = 0.039, d = 0.54). No other significant group differences were detected. Conclusions: While primary dysmenorrhea does not appear to affect overall diaphragmatic function or respiratory capacity, it is associated with subtle localized changes in the left intercostal dynamics. These findings suggest a potential compensatory mechanical adaptation rather than global respiratory dysfunction. Further longitudinal studies with larger sample sizes are needed to explore the clinical significance of these findings. Full article
(This article belongs to the Section Public Health Research)
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18 pages, 5152 KiB  
Review
Imaging Flow Cytometry in HIV Infection Research: Advantages and Opportunities
by Kirill. A. Elfimov, Dmitriy. A. Baboshko and Natalya. M. Gashnikova
Methods Protoc. 2025, 8(1), 14; https://doi.org/10.3390/mps8010014 - 3 Feb 2025
Viewed by 393
Abstract
The human immunodeficiency virus (HIV) is a type of retrovirus that infects humans and belongs to the Lentivirus group. Despite the availability of effective treatments, HIV infections are still increasing in some parts of the world, according to the World Health Organization (WHO). [...] Read more.
The human immunodeficiency virus (HIV) is a type of retrovirus that infects humans and belongs to the Lentivirus group. Despite the availability of effective treatments, HIV infections are still increasing in some parts of the world, according to the World Health Organization (WHO). Another major challenge is the growing problem of HIV becoming resistant to drugs. This highlights the importance of ongoing research to better understand HIV and find new ways to stop the virus from spreading in the body. Scientists use a variety of methods to study HIV, including techniques from molecular and cellular biology. Many of these methods rely on fluorescent dyes to help visualize specific parts of the virus or infected cells. This article focuses on a technique called imaging flow cytometry, which is particularly useful for studying HIV. Imaging flow cytometry is unique because it not only measures fluorescence (light emitted by the dyes) but also captures images of each cell being analyzed. This allows researchers to see where the fluorescence is located within the cell and to study the cell’s shape and structure in detail. Additionally, this method can be combined with machine learning to analyze large amounts of data more efficiently. Full article
(This article belongs to the Section Molecular and Cellular Biology)
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10 pages, 1347 KiB  
Communication
The Reconstructed Individual Patient Data from Kaplan–Meier (IPDfromKM) Method for Non-Inferiority Analyses: A New Potential Application
by Eugenia Piragine, Sabrina Trippoli, Sara Veneziano, Andrea Messori and Vincenzo Calderone
Methods Protoc. 2025, 8(1), 13; https://doi.org/10.3390/mps8010013 - 2 Feb 2025
Viewed by 344
Abstract
The IPDfromKM method, or Shiny method, is an artificial intelligence tool that enables indirect comparisons between studies by reconstructing individual patient data (IPD) from Kaplan–Meier (KM) curves. The IPDfromKM method is generally used for superiority analyses, but a further application could be represented [...] Read more.
The IPDfromKM method, or Shiny method, is an artificial intelligence tool that enables indirect comparisons between studies by reconstructing individual patient data (IPD) from Kaplan–Meier (KM) curves. The IPDfromKM method is generally used for superiority analyses, but a further application could be represented by non-inferiority analyses. However, there are no studies supporting this methodological hypothesis. The aim of this work was to validate the IPDfromKM method for non-inferiority analyses by “exploiting” the well-described non-inferiority of implantable devices occluding the left atrial appendage compared to oral anticoagulants in patients with atrial fibrillation. We performed a systematic review searching for randomized controlled trials (RCTs) in the PubMed database and found five studies. The R software (version 4.3.3) was used to perform a standard survival analysis comparing Watchman and Amlet devices with warfarin. The hazard ratio (HR), with 95% confidence interval (CI), was the main parameter of our analysis. The results confirmed the non-inferiority of Amlet and Watchman compared to warfarin (HR of Watchman vs. warfarin: 1.23, 95% CI 0.80 to 1.9; HR of Amlet vs. warfarin: 1.05, 95% CI 0.61 to 1.80). Therefore, we proposed a new application of the IPDfromKM method that could be potentially relevant in decision-making for the management of this common cardiac arrhythmia and a wide range of other pathological conditions. Full article
(This article belongs to the Special Issue Feature Papers in Methods and Protocols 2024)
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16 pages, 2428 KiB  
Article
Procurement, Processing, and Storage of Human Amniotic Membranes for Implantation Purposes in Non-Healing Pressure Ulcers
by Lina A. Gómez, Carlos Domínguez-Paz, Juan F. Ospina and Elga J. Vargas
Methods Protoc. 2025, 8(1), 12; https://doi.org/10.3390/mps8010012 - 1 Feb 2025
Viewed by 345
Abstract
The human amniotic membrane (hAM) has been used as an implant to enhance the regenerative process and control inflammation in different diseases, given their structure, biocompatibility, presence of stem cells and multiple growth factors. The objective of this study was to generate a [...] Read more.
The human amniotic membrane (hAM) has been used as an implant to enhance the regenerative process and control inflammation in different diseases, given their structure, biocompatibility, presence of stem cells and multiple growth factors. The objective of this study was to generate a standardized protocol for obtaining, processing, and storing hAMs that guarantee the conservation of their structural and cellular characteristics as well as their mechanical properties, ensuring their ease of handling, sterility, and quality that allows their implementation for therapeutic purposes in the field of regenerative medicine. The hAMs were obtained from mothers with healthy, full-term, controlled pregnancies and by cesarean section. The hAMs were processed under sterile conditions, manually separated from the placenta and, subsequently, they were frozen in a solution of culture medium plus 50% v/v glycerol. The protocol allows obtaining sterile hAMs composed of both epithelium and stroma with adequate preservation of the amniotic cells. The glycerol’s impact on the mechanical properties may enhance the membrane’s adaptability and conformability to diverse wound surfaces, potentially improving the healing process. It is necessary to repeat microbiological, cell viability and mechanical studies at 6 and 12 months to ensure that long-term frozen conditions do not affect the quality of the hAMs. Full article
(This article belongs to the Section Tissue Engineering and Organoids)
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11 pages, 1928 KiB  
Article
Comparison of Commercially Available Thermostable DNA Polymerases with Reverse Transcriptase Activity in Coupled Reverse Transcription Polymerase Chain Reaction Assays
by Evgeniya V. Smirnova, Konstantin A. Blagodatskikh, Ekaterina V. Barsova, Dmitriy A. Varlamov, Vladimir M. Kramarov and Konstantin B. Ignatov
Methods Protoc. 2025, 8(1), 11; https://doi.org/10.3390/mps8010011 - 26 Jan 2025
Viewed by 417
Abstract
Reverse transcription polymerase chain reaction (RT-PCR) is an important tool for the detection of target RNA molecules and the assay of RNA pathogens. Coupled RT-PCR is performed with an enzyme mixture containing a reverse transcriptase and a thermostable DNA polymerase. To date, several [...] Read more.
Reverse transcription polymerase chain reaction (RT-PCR) is an important tool for the detection of target RNA molecules and the assay of RNA pathogens. Coupled RT-PCR is performed with an enzyme mixture containing a reverse transcriptase and a thermostable DNA polymerase. To date, several biotechnological companies offer artificial thermostable DNA polymerases with a built-in reverse transcriptase activity for use in the coupled RT-PCR instead of the enzyme mixtures. Here, we compared the artificial DNA polymerases and conventional enzyme mixtures for the RT-PCR by performing end-point and real-time RT-PCR assays using severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV2) RNA and endogenous mRNA molecules as templates. We found that the artificial enzymes were suitable for different RT-PCR applications, including SARS-CoV2 RNA detection but not for long-fragment RT-PCR amplification. Full article
(This article belongs to the Section Molecular and Cellular Biology)
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19 pages, 4194 KiB  
Article
Optimized Methods to Quantify Tumor Treating Fields (TTFields)-Induced Permeabilization of Glioblastoma Cell Membranes
by Melisa Martinez-Paniagua, Sabbir Khan, Nikita W. Henning, Sri Vaishnavi Konagalla and Chirag B. Patel
Methods Protoc. 2025, 8(1), 10; https://doi.org/10.3390/mps8010010 - 22 Jan 2025
Viewed by 659
Abstract
Glioblastoma (GBM) is a lethal primary brain cancer with a 5.6% five-year survival rate. Tumor treating fields (TTFields) are alternating low-intensity electric fields that have demonstrated a GBM patient survival benefit. We previously reported that 0.5–24 h of TTFields exposure resulted in an [...] Read more.
Glioblastoma (GBM) is a lethal primary brain cancer with a 5.6% five-year survival rate. Tumor treating fields (TTFields) are alternating low-intensity electric fields that have demonstrated a GBM patient survival benefit. We previously reported that 0.5–24 h of TTFields exposure resulted in an increased uptake of FITC-dextran fluorescent probes (4–20 kDa) in human GBM cells. However, this approach, in which a fluorescence plate-based detector is used to evaluate cells attached to glass coverslips, cannot distinguish FITC-dextran uptake in live vs. dead cells. The goal of the study was to report the optimization and validation of two independent methods to quantify human GBM cell membrane permeabilization induced by TTFields exposure. First, we optimized flow cytometry by measuring mean fluorescence intensity at 72 h for 4 kDa (TTFields 6726 ± 958.0 vs. no-TTFields 5093 ± 239.7, p = 0.016) and 20 kDa (7087 ± 1137 vs. 5055 ± 897.8, p = 0.031) probes. Second, we measured the ratio of lactate dehydrogenase (LDH) to cell viability (measured using the CellTiter-Glo [CTG] viability assay); the LDH/CTG ratio was higher under TTFields (1.47 ± 0.15) than no-TTFields (1.08 ± 0.08) conditions, p < 0.0001. The findings using these two independent methods reproducibly demonstrated their utility for time-dependent evaluations. We also showed that these methods can be used to relate the cell membrane-permeabilizing effects of the non-ionizing radiation of TTFields to that of an established cell membrane permeabilizer, the non-ionic detergent Triton-X-100. Evaluating carboplatin ± TTFields, the LDH/CTG ratio was significantly higher in the TTFields vs. no-TTFields condition at each carboplatin concentration (0–30 µM), p = 0.014. We successfully optimized and validated two cost-effective methods to reproducibly quantify TTFields-induced human GBM cancer cell membrane permeabilization. Full article
(This article belongs to the Section Molecular and Cellular Biology)
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9 pages, 890 KiB  
Communication
Validity of a New Portable Sensor to Measure Velocity-Based Resistance Training
by Alejandro Justo-Álvarez, Juan García-López, Rafael Sabido and Adrián García-Valverde
Methods Protoc. 2025, 8(1), 9; https://doi.org/10.3390/mps8010009 - 20 Jan 2025
Viewed by 440
Abstract
This study evaluated the concurrent validity of the Vitruve linear encoder compared to the T-Force device for measuring mean propulsive velocity (MPV) and peak velocity (PV) during the free-weight bench press exercise. Thirteen resistance-trained men participated in three sessions, during which MPV and [...] Read more.
This study evaluated the concurrent validity of the Vitruve linear encoder compared to the T-Force device for measuring mean propulsive velocity (MPV) and peak velocity (PV) during the free-weight bench press exercise. Thirteen resistance-trained men participated in three sessions, during which MPV and PV were recorded simultaneously by both devices. The data were analysed using one-way ANOVA, Pearson’s correlation, Bland–Altman analysis, and effect size calculations, with statistical significance set at p ≤ 0.05. The results showed discrepancies between the Vitruve and T-Force devices across different intensity levels. Specifically, the Vitruve device generally reported higher MPV and lower PV values, particularly at moderate and low intensities. Vitruve was deemed useful for MPV measurements, especially at velocities below 0.65 m/s during free-weight bench press exercises. In conclusion, the Vitruve device overestimated MPV and underestimated PV at moderate and low loads (>0.65 m·s−1), with the discrepancies increasing as velocity rose. It can provide valuable data for monitoring and assessing resistance training programs focused on MPV at heavier loads (<0.65 m·s−1). Researchers and practitioners should take these findings into account when incorporating the Vitruve into velocity-based strength training protocols. Full article
(This article belongs to the Special Issue Methods on Sport Biomechanics)
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20 pages, 42067 KiB  
Article
Finite Element Analysis of Functionally Loaded Subperiosteal Implants Evaluated on a Realistic Model Reproducing Severe Atrophic Jaws
by Gerardo Pellegrino, Maryia Karaban, Veronica Scalchi, Marco Urbani, Amerigo Giudice, Carlo Barausse and Pietro Felice
Methods Protoc. 2025, 8(1), 8; https://doi.org/10.3390/mps8010008 - 18 Jan 2025
Viewed by 397
Abstract
Implant-supported prosthetic rehabilitation for patients with severely atrophic jaws is challenging due to complex anatomical considerations and the limitations of conventional augmentation techniques. This study explores the potential of subperiosteal (juxta-osseous) implants as an alternative solution, using finite element analysis (FEA) to evaluate [...] Read more.
Implant-supported prosthetic rehabilitation for patients with severely atrophic jaws is challenging due to complex anatomical considerations and the limitations of conventional augmentation techniques. This study explores the potential of subperiosteal (juxta-osseous) implants as an alternative solution, using finite element analysis (FEA) to evaluate mechanical performance. Realistic jaw models, developed from radiographic data, are utilized to simulate various implant configurations and load scenarios. Results indicate that different screw placements, implant designs, and structural modifications can significantly influence stress distribution and biomechanical behavior. Upper and lower jaw models were assessed under multiple load conditions to determine optimal configurations. Findings suggest that strategic adjustments, such as adding posterior screws or altering implant connections, can enhance load distribution and reduce stress concentration, particularly in critical areas. Tensile loads in critical bone areas near cortical fixing screws exceeded 50 MPa under anterior loading, while configurations with larger load distributions reduced stress on both implant and bone. The study provides evidence-based insights into optimizing subperiosteal implant design to improve stability, longevity, and patient outcomes. Full article
(This article belongs to the Section Biomedical Sciences and Physiology)
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12 pages, 453 KiB  
Article
Development and Validation of a High-Throughput Quantification Method of Crown Procyanidins in Different Wines by UHPLC-Q-TOF
by Manon Ferreira, Pierre-Louis Teissedre and Michaël Jourdes
Methods Protoc. 2025, 8(1), 7; https://doi.org/10.3390/mps8010007 - 11 Jan 2025
Viewed by 373
Abstract
Procyanidins are widely distributed in plant-derived foods, and consist of flavanol oligomers and polymers. Recently, the crown procyanidin sub-family, characterised by a unique macrocyclic structure, has been identified in grapes and wine. This study reports the development and validation of a rapid and [...] Read more.
Procyanidins are widely distributed in plant-derived foods, and consist of flavanol oligomers and polymers. Recently, the crown procyanidin sub-family, characterised by a unique macrocyclic structure, has been identified in grapes and wine. This study reports the development and validation of a rapid and quantitative analytical method measuring crown procyanidin concentration in red and white wines using ultra-high-performance liquid chromatography (UHPLC) coupled with a Q-TOF mass spectrometer. Validation followed international standards, demonstrating high sensitivity (LOQ = 0.033 mg/L), accuracy (recovery = 88.21% to 107.64%), repeatability (RSD = 1.99% to 11.03%), and intermediate reproducibility (RSD = 2.51% to 19.05%). Minimal matrix effects were observed, ensuring reliable and precise quantification across both wine types. The applicability of the method was confirmed through the successful analysis of wine samples, leading to the first quantification of crown procyanidins in white wine. Concentrations ranged from 0.81 mg/L to 15.88 mg/L in the different analysed wines. This validated method provides a valuable tool for the study of crown procyanidin profiles in various wine matrices and establishes a foundation for future research into the role of crown procyanidins in wines and other food and beverage matrices where these compounds may be present. Full article
(This article belongs to the Special Issue Spectroscopic Methods of Analysis)
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8 pages, 187 KiB  
Study Protocol
Heidelberg Adult and Pediatric Airway Registry (HAPA-Registry)
by Davut D. Uzun, Kim Bittlinger, Emily Wibbe, Stefan Mohr, Markus A. Weigand and Felix C. F. Schmitt
Methods Protoc. 2025, 8(1), 6; https://doi.org/10.3390/mps8010006 - 7 Jan 2025
Viewed by 536
Abstract
Background: Advanced airway management is of fundamental importance in almost all areas of anesthesiology, emergency medicine, and critical care. Securing the airway is of the utmost importance, as this is a prerequisite for the oxygenation of the human organism. The clinical relevance of [...] Read more.
Background: Advanced airway management is of fundamental importance in almost all areas of anesthesiology, emergency medicine, and critical care. Securing the airway is of the utmost importance, as this is a prerequisite for the oxygenation of the human organism. The clinical relevance of airway management is particularly evident in the fact that the primary cause of significant anesthesia-related complications can be attributed to this field. The need for the systematic recording of these procedures and their complications, as well as structured training in airway management and the evaluation of outcome parameters, is, therefore, evident. Methods: The HAPA-registry is a prospective and monocentric observational trial at the Department of Anesthesiology, Medical Faculty Heidelberg, University of Heidelberg, Germany. All patients requiring general anesthesia with consecutive advanced airway management during a surgical procedure were included. We, therefore, planned to include approximately 9000 patients in the first period. Following successful airway management, the anesthetist completed a case report form (CRF) in person. The intention was to record airway management cases on an annual basis for a period of several months, thus ensuring that the register remains up-to-date and that airway management procedures are continuously recorded. Discussion: The aim of this study was to establish an airway registry that enables the systematic recording and evaluation of different methods of airway management. The registry can be used to monitor and evaluate the implementation of current guidelines and recommendations for action, as well as the rates of success and complications. Full article
(This article belongs to the Section Biomedical Sciences and Physiology)
12 pages, 1930 KiB  
Article
Fluorescence Analysis of Quinine in Commercial Tonic Waters
by Artturi Harcher, Connor Ricard, Danielle Connolly, Isabel Gibbs, Jarve Shaw, Jillian Butler, Julia Perschbacher, Lindsay Replogle, Michaela Eide, Morgan Grissom, Oliver O’Neal, Quan Nguyen, Van Hac Nguyen, Michael Hunnicutt, Roaa Mahmoud and Soma Dhakal
Methods Protoc. 2025, 8(1), 5; https://doi.org/10.3390/mps8010005 - 6 Jan 2025
Viewed by 719
Abstract
Quinine is known for treating malaria, muscle cramps, and, more recently, has been used as an additive in tonic water due to its bitter taste. However, it was shown that excessive consumption of quinine can have severe side effects on health. In this [...] Read more.
Quinine is known for treating malaria, muscle cramps, and, more recently, has been used as an additive in tonic water due to its bitter taste. However, it was shown that excessive consumption of quinine can have severe side effects on health. In this work, we utilized fluorescence spectroscopy to measure the concentration of quinine in commercial tonic water samples. An external standard method was used to calculate the concentrations of quinine in two commercially available tonic water brands, namely Canada Dry and Schweppes, and compare them to the maximum allowable concentration of quinine in beverages. Upon analysis of the data collected by five different groups, the levels of quinine were found to be above the average concentration in most commercial tonic water samples, but below the maximum permitted concentration. Moreover, the five replicate sets of data demonstrated high reproducibility of the method employed in this study. The simple yet instructive protocol that we developed can be adapted to determine the concentration of other fluorescent compounds in foods and beverages. Further, the presented method and detailed protocol can be easily adopted for undergraduate labs and in chemical education. Full article
(This article belongs to the Special Issue Feature Papers in Methods and Protocols 2024)
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16 pages, 5027 KiB  
Article
Evaluation of Drug–Polymer and Drug–Drug Interaction in Cellulosic Multi-Drug Delivery Matrices
by Abdullah Isreb, Mohamed A. Alhnan, Abdulrahman Mkia, Khaled Al-Jammal, Abdallah Yaghi, Enoche Florence Oga, Peter Timmins, Michael Bonner and Robert T. Forbes
Methods Protoc. 2025, 8(1), 4; https://doi.org/10.3390/mps8010004 - 6 Jan 2025
Viewed by 701
Abstract
Multi-drug delivery systems have gained increasing interest from the pharmaceutical industry. Alongside this is the interest in amorphous solid dispersions as an approach to achieve effective oral delivery of compounds with solubility-limited bioavailability. Despite this, there is limited information regarding predicting the behavior [...] Read more.
Multi-drug delivery systems have gained increasing interest from the pharmaceutical industry. Alongside this is the interest in amorphous solid dispersions as an approach to achieve effective oral delivery of compounds with solubility-limited bioavailability. Despite this, there is limited information regarding predicting the behavior of two or more drugs (in amorphous forms) in a polymeric carrier and whether molecular interactions between the compounds, between each compound, and if the polymer have any effect on the physical properties of the system. This work studies the interaction between model drug combinations (two of ibuprofen, malonic acid, flurbiprofen, or naproxen) dispersed in a polymeric matrix of hypromellose acetate succinate (HPMCAS) using a solvent evaporation technique. Hildebrand and Hansen calculations were used to predict the miscibility of compounds as long as the difference in their solubility parameter values was not greater than 7 MPa1/2. It was observed that the selected APIs (malonic acid, ibuprofen, naproxen, and flurbiprofen) were miscible within the formed polymeric matrix. Adding the API caused depression in the Tg of the polymer to certain concentrations (17%, 23%, 13%) for polymeric matrices loaded with malonic acid, ibuprofen, and naproxen, respectively. Above this, large crystals started to form, and phase separation was seen. Adding two APIs to the same matrix resulted in reducing the saturation concentration of one of the APIs. A trend was observed and linked to Hildebrand and Hansen solubility parameters (HSP). Full article
(This article belongs to the Special Issue Feature Papers in Methods and Protocols 2024)
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6 pages, 775 KiB  
Technical Note
A Double Fluorescent Staining Method Increases the Sensitivity of the Cytokinesis-Block Micronucleus Assay
by Ion Udroiu
Methods Protoc. 2025, 8(1), 3; https://doi.org/10.3390/mps8010003 - 4 Jan 2025
Viewed by 574
Abstract
The micronucleus test is one of the most popular genotoxicity assays. In order to avoid underestimation of micronuclei frequencies by counting non-replicating cells, the cytokinesis-blocked micronucleus test has been developed. In this technique, only binucleated cells are scored. One underestimated problem is the [...] Read more.
The micronucleus test is one of the most popular genotoxicity assays. In order to avoid underestimation of micronuclei frequencies by counting non-replicating cells, the cytokinesis-blocked micronucleus test has been developed. In this technique, only binucleated cells are scored. One underestimated problem is the potential difficulty in discriminating binucleated from mononucleated cells when using DAPI staining, i.e., the possibility that two neighboring mononucleated cells could be mistaken for a binucleated one. The new protocol presented here comprises the addition of acridine orange in order to stain the cytoplasm (in addition to DAPI to stain nuclei and micronuclei). This new technique can increase the sensitivity of the cytokinesis-blocked micronucleus test and avoid underestimation of micronuclei frequencies, an important issue when high doses are employed. Full article
(This article belongs to the Section Molecular and Cellular Biology)
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13 pages, 1064 KiB  
Protocol
Differential DNA Extraction from Lateral Flow Immunochromatographic Tests via the EZ1® Advanced XL System
by Scarlet Neilson, Leah Nangeroni and Mirna Ghemrawi
Methods Protoc. 2025, 8(1), 2; https://doi.org/10.3390/mps8010002 - 2 Jan 2025
Viewed by 684
Abstract
This differential extraction protocol details the steps for isolating DNA from sample pads used in lateral flow immunochromatographic (LFI) tests, particularly for cases involving mixed biological samples such as semen and menstrual blood, or other evidence related to sexual assault. This procedure utilizes [...] Read more.
This differential extraction protocol details the steps for isolating DNA from sample pads used in lateral flow immunochromatographic (LFI) tests, particularly for cases involving mixed biological samples such as semen and menstrual blood, or other evidence related to sexual assault. This procedure utilizes a differential extraction technique applied to sample pads from immunochromatographic tests, where the sample pads serve as the substrate. The method involves two sequential lysis steps to effectively separate non-sperm and sperm fractions, enabling the targeted isolation of distinct cell types for downstream DNA analysis. The efficiency of this procedure is demonstrated by the results within this paper, which highlights the successful recovery of both male autosomal and Y-STR profiles, even in mixed samples with a high female presence. Overall, this protocol demonstrates the effective recovery of DNA from sample pads, which is beneficial for forensic practitioners dealing with limited sample quantities, underscoring the value of using these pads in forensic analysis. Full article
(This article belongs to the Section Molecular and Cellular Biology)
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12 pages, 3302 KiB  
Article
In Vivo Imaging of Cobalt-Induced Ocular Toxicity in a Mouse Model
by Basel Obied, Galit Saar, Stephen Richard, Ygal Rotenstreich, Ifat Sher, Alon Zahavi and Nitza Goldenberg-Cohen
Methods Protoc. 2025, 8(1), 1; https://doi.org/10.3390/mps8010001 - 2 Jan 2025
Viewed by 725
Abstract
Cobalt is a trace element, crucial for red blood cell formation and neurological function. Cobalt toxicity is often only diagnosed after severe manifestations, including visual impairment. We aimed to investigate whether optical coherence tomography (OCT) and magnetic resonance imaging (MRI) can effectively detect [...] Read more.
Cobalt is a trace element, crucial for red blood cell formation and neurological function. Cobalt toxicity is often only diagnosed after severe manifestations, including visual impairment. We aimed to investigate whether optical coherence tomography (OCT) and magnetic resonance imaging (MRI) can effectively detect cobalt-induced ocular toxicity in a murine model. Five wild-type mice (WT, C57Bl6) received daily intraperitoneal cobalt chloride injections for 28 days with a dosage of 12.5 mg/kg. Another 5 WT mice served as controls. After 28 days, all mice underwent manganese contrast-enhanced MRI and OCT examinations. Macroscopic and histological analysis of the enucleated eyes were performed. MRI revealed an increased signal in the optic nerves of injected mice. Anterion OCT provided in vivo visualization of the entire eye, demonstrating incipient cataract formation in the cobalt-injected mice. Both Spectralis domain OCT and Anterion, followed by histological analyses, confirmed preserved retinal structure with decreased thickness in the cobalt-injected group, with only minor neuronal damage and cell loss. Optic nerve analysis demonstrated myelin loss and increased inflammation with high levels of reactive gliosis. This study demonstrates optic neuropathy induced by cobalt toxicity, as shown by increased optic nerve signal on MRI without significant retinopathy. Anterion OCT showed incipient cataracts in the anterior segment. Full article
(This article belongs to the Section Biomedical Sciences and Physiology)
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