Journal Description
Methods and Protocols
Methods and Protocols
is an international, peer-reviewed, open access journal aiming to establish and describe new experimental techniques in the fields of Life Sciences, Chemistry, and Biomedical Sciences, published bimonthly online by MDPI.
- Open Access— free for readers, with article processing charges (APC) paid by authors or their institutions.
- High Visibility: indexed within Scopus, ESCI (Web of Science), PubMed, PMC, CAPlus / SciFinder, and other databases.
- Journal Rank: CiteScore - Q2 (Biochemistry, Genetics and Molecular Biology (miscellaneous))
- Rapid Publication: manuscripts are peer-reviewed and a first decision is provided to authors approximately 24.9 days after submission; acceptance to publication is undertaken in 3.7 days (median values for papers published in this journal in the first half of 2024).
- Recognition of Reviewers: reviewers who provide timely, thorough peer-review reports receive vouchers entitling them to a discount on the APC of their next publication in any MDPI journal, in appreciation of the work done.
Impact Factor:
2.3 (2023);
5-Year Impact Factor:
2.2 (2023)
Latest Articles
Protocol for the Development and Initial Validation of the COG-IMPACT Tool: A Purpose-Built Unmet Needs Assessment for Cancer-Related Cognitive Impairment
Methods Protoc. 2024, 7(4), 54; https://doi.org/10.3390/mps7040054 - 10 Jul 2024
Abstract
(1) Background: A significant proportion of cancer survivors report experiencing a cognitive ‘fog’ that affects their ability to think coherently and quickly, and reason with clarity. This has been referred to as cancer-related cognitive impairment (CRCI). CRCI has extensive impacts on the
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(1) Background: A significant proportion of cancer survivors report experiencing a cognitive ‘fog’ that affects their ability to think coherently and quickly, and reason with clarity. This has been referred to as cancer-related cognitive impairment (CRCI). CRCI has extensive impacts on the daily lives of people living with or beyond cancer, including occupational, social, and psychological functioning. Oncology health professionals report feeling under-resourced to effectively assess the needs of an individual with CRCI and then provide optimal care and referral. (2) Methods: The objective of this project is to develop and provide an initial validation of the first purpose-built unmet needs assessment for CRCI: the Unmet Needs Assessment of Cancer-Related Cognitive Impairment Impact (COG-IMPACT). We will use a multiple-stage, co-design, mixed-methods approach to develop and provide an initial validation of the COG-IMPACT. (3) Results: The primary anticipated result of this research is the production of the COG-IMPACT, the first purpose-built unmet needs assessment for CRCI. The assessment could be used by health professionals to understand the unmet needs and facilitate optimal care and referral for cancer survivors, by survivors to elucidate their supportive needs and advocate for their care, and by researchers to examine the correlates of unmet needs relating to CRCI, as well as how best to support people with CRCI.
Full article
(This article belongs to the Section Public Health Research)
Open AccessCommunication
Suitability of XRF for Routine Analysis of Multi-Elemental Composition: A Multi-Standard Verification
by
Riccardo Fedeli, Luigi Antonello Di Lella and Stefano Loppi
Methods Protoc. 2024, 7(4), 53; https://doi.org/10.3390/mps7040053 - 5 Jul 2024
Abstract
This study investigated the suitability of X-ray fluorescence (XRF) analysis for routine multi-elemental composition analysis, checking its analytical capabilities by measuring a wide array of certified reference materials of soil and plant origin. A portable XRF analyzer was used to evaluate 32 soil
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This study investigated the suitability of X-ray fluorescence (XRF) analysis for routine multi-elemental composition analysis, checking its analytical capabilities by measuring a wide array of certified reference materials of soil and plant origin. A portable XRF analyzer was used to evaluate 32 soil and 12 plant standard materials, using both the Soil and Geochem mode, with sequential beams, allowing the detection of a wide range of elements. Recovery rates were calculated by comparing XRF measurements with certified values, and their correlations were verified through the Spearman coefficient. The results demonstrated the reliability of XRF measurements for soil samples, with a large number of elements showing a good or very good recovery and strong correlations with certified values. For plant samples, XRF largely overestimated the certified values, but the strong statistically significant correlations for almost all tested elements allowed us to correct this systematic bias, using the reported median value for dividing the value obtained via XRF. The Geochem mode emerged as more reliable for a larger number of elements. It was concluded that XRF may be a suitable alternative to ICP-MS in routine multi-elemental composition analysis.
Full article
(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
Open AccessReview
Methods to Investigate the Secretome of Senescent Cells
by
Afshin Samiminemati, Domenico Aprile, Dario Siniscalco and Giovanni Di Bernardo
Methods Protoc. 2024, 7(4), 52; https://doi.org/10.3390/mps7040052 - 2 Jul 2024
Abstract
The word “secretome” was first used to describe the proteins that cells secrete under different circumstances; however, recent studies have proven the existence of other molecules such as RNA and chemical compounds in the secretome. The study of secretome has significance for the
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The word “secretome” was first used to describe the proteins that cells secrete under different circumstances; however, recent studies have proven the existence of other molecules such as RNA and chemical compounds in the secretome. The study of secretome has significance for the diagnosis and treatment of disease as it provides insight into cellular functions, including immune responses, development, and homeostasis. By halting cell division, cellular senescence plays a role in both cancer defense and aging by secreting substances known as senescence-associated secretory phenotypes (SASP). A variety of techniques could be used to analyze the secretome: protein-based approaches like mass spectrometry and protein microarrays, nucleic acid-based methods like RNA sequencing, microarrays, and in silico prediction. Each method offers unique advantages and limitations in characterizing secreted molecules. Top-down and bottom-up strategies for thorough secretome analysis are became possible by mass spectrometry. Understanding cellular function, disease causes, and proper treatment targets is aided by these methodologies. Their approaches, benefits, and drawbacks will all be discussed in this review.
Full article
(This article belongs to the Section Molecular and Cellular Biology)
Open AccessReview
A Review of Histological Techniques for Differentiating Human Bone from Animal Bone
by
Emanuela Stan, Camelia-Oana Muresan, Ecaterina Daescu, Raluca Dumache, Veronica Ciocan, Stefania Ungureanu, Dan Costachescu and Alexandra Enache
Methods Protoc. 2024, 7(4), 51; https://doi.org/10.3390/mps7040051 - 30 Jun 2024
Abstract
The first step in anthropological study is the positive identification of human remains, which can be a challenging undertaking when bones are broken. When bone pieces from different species are mixed together, it can be crucial to distinguish between them in forensic and
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The first step in anthropological study is the positive identification of human remains, which can be a challenging undertaking when bones are broken. When bone pieces from different species are mixed together, it can be crucial to distinguish between them in forensic and archaeological contexts. For years, anthropology and archaeology have employed the histomorphological analysis of bones to evaluate species-specific variations. Based on variations in the dimensions and configuration of Haversian systems between the two groups, these techniques have been devised to distinguish between non-human and human bones. All of those techniques concentrate on a very particular kind of bone, zone, and segment. Histomorphometric techniques make the assumption that there are size, form, and quantity variations between non-humans and humans. The structural components of Haversian bones are significant enough to use discriminant function analysis to separate one from the other. This review proposes a comprehensive literature analysis of the various strategies or techniques available for distinguishing human from non-human bones to demonstrate that histomorphological analysis is the most effective method to be used in the case of inadequate or compromised samples.
Full article
(This article belongs to the Section Tissue Engineering and Organoids)
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Open AccessProtocol
In Vitro Methods for Assessing the Antibacterial and Antibiofilm Properties of Essential Oils as Potential Root Canal Irrigants—A Simplified Description of the Technical Steps
by
Jihad Diouchi, Jelena Marinković, Milica Nemoda, Lhoussaine El Rhaffari, Babacar Toure and Sonia Ghoul
Methods Protoc. 2024, 7(4), 50; https://doi.org/10.3390/mps7040050 - 25 Jun 2024
Abstract
Background: Essential oils have gained in significance due to their various biological activities, and there is a growing demand for them in many industries. The present article focuses on the technical steps for an in vitro evaluation of the antibacterial and antibiofilm activities
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Background: Essential oils have gained in significance due to their various biological activities, and there is a growing demand for them in many industries. The present article focuses on the technical steps for an in vitro evaluation of the antibacterial and antibiofilm activities of essential oils for potential use as root canal irrigant in dentistry. Methods: The bioactivities of the essential oil were investigated through in vitro assays. The gram-positive bacterium Enterococcus faecalis was used as a micro-organism model. The antibacterial activity of the essential oil was assessed using the microdilution method, and resazurin staining to determine the minimal inhibition concentrations (MICs) and the minimal bactericidal concentrations (MBCs). The antibiofilm effect was evaluated spectrophotometrically at 570 nm using the microplate cultivation technique and crystal violet staining. Conclusions: This article features a detailed in vitro protocol to facilitate the preparation of the essential oil samples, the bacterial suspension, and the methods used for assessment of the antibiofilm and antibacterial activities of the essential oil. The advantages of these approaches are presented in relation to the limits linked to the choice of the bacteria and the essential oil.
Full article
(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
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Open AccessProtocol
Simple Determination of Affinity Constants of Antibodies by Competitive Immunoassays
by
Janina Fischer, Jan Ole Kaufmann and Michael G. Weller
Methods Protoc. 2024, 7(3), 49; https://doi.org/10.3390/mps7030049 - 13 Jun 2024
Abstract
The affinity constant, also known as the equilibrium constant, binding constant, equilibrium association constant, or the reciprocal value, the equilibrium dissociation constant (Kd), can be considered as one of the most important characteristics for any antibody–antigen pair. Many methods based on
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The affinity constant, also known as the equilibrium constant, binding constant, equilibrium association constant, or the reciprocal value, the equilibrium dissociation constant (Kd), can be considered as one of the most important characteristics for any antibody–antigen pair. Many methods based on different technologies have been proposed and used to determine this value. However, since a very large number of publications and commercial datasheets do not include this information, significant obstacles in performing such measurements seem to exist. In other cases where such data are reported, the results have often proved to be unreliable. This situation may indicate that most of the technologies available today require a high level of expertise and effort that does not seem to be available in many laboratories. In this paper, we present a simple approach based on standard immunoassay technology that is easy and quick to perform. It relies on the effect that the molar IC50 approaches the Kd value in the case of infinitely small concentrations of the reagent concentrations. A two-dimensional dilution of the reagents leads to an asymptotic convergence to Kd. The approach has some similarity to the well-known checkerboard titration used for the optimization of immunoassays. A well-known antibody against the FLAG peptide, clone M2, was used as a model system and the results were compared with other methods. This approach could be used in any case where a competitive assay is available or can be developed. The determination of an affinity constant should belong to the crucial parameters in any quality control of antibody-related products and assays and should be mandatory in papers using immunochemical protocols.
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(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
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Open AccessProtocol
A Comprehensive Methodology for Microbial Strain Typing Using Fourier-Transform Infrared Spectroscopy
by
Francis Muchaamba and Roger Stephan
Methods Protoc. 2024, 7(3), 48; https://doi.org/10.3390/mps7030048 - 11 Jun 2024
Abstract
Timely and accurate detection and characterization of microbial threats is crucial for effective infection and outbreak management. Additionally, in food production, rapid microbe identification is indispensable for maintaining quality control and hygiene standards. Current methods for typing microbial strains often rely on labor-intensive,
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Timely and accurate detection and characterization of microbial threats is crucial for effective infection and outbreak management. Additionally, in food production, rapid microbe identification is indispensable for maintaining quality control and hygiene standards. Current methods for typing microbial strains often rely on labor-intensive, time-consuming, and expensive DNA- and sera-serotyping techniques, limiting their applicability in rapid-response scenarios. In this context, the IR Biotyper®, utilizing Fourier-transform infrared (FTIR) spectroscopy, offers a novel approach, providing specific spectra for fast strain typing within 3 h. This methodology article serves as a comprehensive resource for researchers and technicians aiming to utilize FTIR spectroscopy for microbial strain typing. It encompasses detailed guidelines on sample preparation, data acquisition, and analysis techniques, ensuring the generation of reliable and reproducible results. We highlight the IR Biotyper®’s rapid and accurate discrimination capabilities, showcasing its potential for real-time pathogen monitoring and source-tracking to enhance public health and food safety. We propose its integration as an early screening method, followed by more detailed analysis with whole-genome sequencing, to optimize detection accuracy and response efficiency in microbial surveillance systems.
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(This article belongs to the Section Molecular and Cellular Biology)
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Open AccessStudy Protocol
Evaluation of the Sexually Transmitted Infections Programme among Key and Priority Populations in Primary Healthcare Facilities to Inform a Targeted Response: A Protocol Paper
by
Mohlago Ablonia Seloka, Edith Phalane and Refilwe Nancy Phaswana-Mafuya
Methods Protoc. 2024, 7(3), 47; https://doi.org/10.3390/mps7030047 - 5 Jun 2024
Abstract
Primary healthcare facilities lack routine diagnostic screening due to resource limitations and dependence on syndromic management, resulting in an unprecedented prevalence and incidence of sexually transmitted infections (STIs), particularly among key and priority populations. Specific focuses are essential to strengthen current STI control
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Primary healthcare facilities lack routine diagnostic screening due to resource limitations and dependence on syndromic management, resulting in an unprecedented prevalence and incidence of sexually transmitted infections (STIs), particularly among key and priority populations. Specific focuses are essential to strengthen current STI control measures. Therefore, this article describes the protocol for evaluating STI programme among key and priority populations in selected primary healthcare facilities in South Africa. We will employ an exploratory, descriptive research design to assess the STI programme in terms of its facility operations, functions, scope, gaps, delivery services, STI surveillance methods, and indicators in the selected primary healthcare facilities. A purposive sample of 15–20 STI programme stakeholders will be selected from five primary healthcare facilities in Limpopo Province, South Africa. The programme evaluation will use the World Health Organization assessment checklist tool, a globally recognised and validated instrument comprising open- and closed-ended questions to assess the STI programme. This tool, known for its credibility and reliability, ensures the study’s validity. Quantitative data will be captured on STATA software (College Station, TX, USA) version 18 for descriptive analysis and presented as the mean and standard deviation for continuous variables, proportions and percentages for categorical variables. A p ≤ 0.05 will demonstrate a statistically significant level. Thematic content analysis will be conducted for the qualitative data using Atlas. ti software (Technical University, Berlin, Germany) version 23.1. The study’s results will inform new approaches to strengthen STI coverage, service delivery, and linkage to care.
Full article
(This article belongs to the Section Public Health Research)
Open AccessReview
Advances in 3D Inner Ear Reconstruction Software for Cochlear Implants: A Comprehensive Review
by
Michail Athanasopoulos, Pinelopi Samara and Ioannis Athanasopoulos
Methods Protoc. 2024, 7(3), 46; https://doi.org/10.3390/mps7030046 - 23 May 2024
Abstract
Auditory impairment stands as a pervasive global issue, exerting significant effects on individuals’ daily functioning and interpersonal engagements. Cochlear implants (CIs) have risen as a cutting-edge solution for severe to profound hearing loss, directly stimulating the auditory nerve with electrical signals. The success
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Auditory impairment stands as a pervasive global issue, exerting significant effects on individuals’ daily functioning and interpersonal engagements. Cochlear implants (CIs) have risen as a cutting-edge solution for severe to profound hearing loss, directly stimulating the auditory nerve with electrical signals. The success of CI procedures hinges on precise pre-operative planning and post-operative evaluation, highlighting the significance of advanced three-dimensional (3D) inner ear reconstruction software. Accurate pre-operative imaging is vital for identifying anatomical landmarks and assessing cochlear deformities. Tools like 3D Slicer, Amira and OTOPLAN provide detailed depictions of cochlear anatomy, aiding surgeons in simulating implantation scenarios and refining surgical approaches. Post-operative scans play a crucial role in detecting complications and ensuring CI longevity. Despite technological advancements, challenges such as standardization and optimization persist. This review explores the role of 3D inner ear reconstruction software in patient selection, surgical planning, and post-operative assessment, tracing its evolution and emphasizing features like image segmentation and virtual simulation. It addresses software limitations and proposes solutions, advocating for their integration into clinical practice. Ultimately, this review underscores the impact of 3D inner ear reconstruction software on cochlear implantation, connecting innovation with precision medicine.
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(This article belongs to the Section Biomedical Sciences and Physiology)
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Open AccessStudy Protocol
MUltiparametric Score for Ventilation Discontinuation in Intensive Care Patients: A Protocol for an Observational Study
by
Iacopo Cappellini, Andrea Cardoni, Lorenzo Campagnola and Guglielmo Consales
Methods Protoc. 2024, 7(3), 45; https://doi.org/10.3390/mps7030045 - 20 May 2024
Abstract
Background: Mechanical ventilation significantly improves patient survival but is associated with complications, increasing healthcare costs and morbidity. Identifying optimal weaning times is paramount to minimize these risks, yet current methods rely heavily on clinical judgment, lacking specificity. Methods: This study introduces a novel
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Background: Mechanical ventilation significantly improves patient survival but is associated with complications, increasing healthcare costs and morbidity. Identifying optimal weaning times is paramount to minimize these risks, yet current methods rely heavily on clinical judgment, lacking specificity. Methods: This study introduces a novel multiparametric predictive score, the MUSVIP (MUltiparametric Score for Ventilation discontinuation in Intensive care Patients), aimed at accurately predicting successful extubation. Conducted at Santo Stefano Hospital’s ICU, this single-center, observational, prospective cohort study will span over 12 months, enrolling adult patients undergoing invasive mechanical ventilation. The MUSVIP integrates variables measured before and during a spontaneous breathing trial (SBT) to formulate a predictive score. Results: Preliminary analyses suggest an Area Under the Curve (AUC) of 0.815 for the MUSVIP, indicating high predictive capacity. By systematically applying this score, we anticipate identifying patients likely to succeed in weaning earlier, potentially reducing ICU length of stay and associated healthcare costs. Conclusion: This study’s findings could significantly influence clinical practices, offering a robust, easy-to-use tool for optimizing weaning processes in ICUs.
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(This article belongs to the Section Biomedical Sciences and Physiology)
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Open AccessArticle
Pole Dancing-Specific Muscle Strength: Development and Reliability of a Novel Assessment Protocol
by
Despoina Ignatoglou, Achilleas Paliouras, Eleftherios Paraskevopoulos, Nikolaos Strimpakos, Paraskevi Bilika, Maria Papandreou and Eleni Kapreli
Methods Protoc. 2024, 7(3), 44; https://doi.org/10.3390/mps7030044 - 18 May 2024
Abstract
Background: Pole dancing is a physically demanding sport that combines dance and acrobatic movements on a vertical pole. Despite its highly growing popularity, there is currently limited research in the field. The aim of this study was to create and evaluate a strength
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Background: Pole dancing is a physically demanding sport that combines dance and acrobatic movements on a vertical pole. Despite its highly growing popularity, there is currently limited research in the field. The aim of this study was to create and evaluate a strength assessment protocol for athletes in pole dancing, with a specific focus on functional positions on the pole. Methods: Thirty-two female pole dancing athletes participated in this study. Maximal voluntary isometric contractions (MVIC) were measured at three different sport-specific positions on the pole (shoulder abduction and adduction, and hip adduction), on two separate days (test and re-test) with a five to seven day interval between them. A hand-held dynamometer (Activ5- Activbody) stabilized on the pole was used for this study. Results: The intra-session reliability was good to excellent for all sports-specific positions and for both sides of the body, across all different movements (ICC = 0.837–0.960, SEM = 5.02 Kg–2.24 Kg, and SDD = 27.46%–14.92%). Slightly better results were found regarding inter-session reliability (ICC = 0.927–0.970, SEM = 3.72 Kg–1.97 Kg, and SDD = 22.86%–15.19%). There was not a statistically significant difference between the MVICs between the left and right or dominant and non-dominant side in shoulder abduction (p = 0.105) and hip adduction (p = 0.282), in contrast to shoulder adduction (p = 0.00). Conclusion: The strength assessment protocol developed in the current study has proven to be a reliable and functional tool, with the potential for utilization in clinical practice as part of objective strength testing. Further studies are needed in order to expand the protocol to other muscle groups and positions and to generalize the results in all pole dancing populations such as male athletes.
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(This article belongs to the Special Issue Methods on Sport Biomechanics)
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Open AccessProtocol
Isolation of Intestinal Macrophage Subpopulations for High-Quality Total RNA Purification in Zebrafish
by
Yalén Del Río-Jay, Audrey Barthelaix, Cristian Reyes-Martínez, Christophe Duperray, Camila J. Solis-Cascante, Yessia Hidalgo, Patricia Luz-Crawford, Farida Djouad and Carmen G. Feijoo
Methods Protoc. 2024, 7(3), 43; https://doi.org/10.3390/mps7030043 - 17 May 2024
Abstract
Intestinal macrophages have been poorly studied in fish, mainly due to the lack of specific molecular markers for their identification and isolation. To address this gap, using the zebrafish Tg(mpeg1:EGFP) transgenic line, we developed a fluorescence-activated cell sorting strategy (FACS)
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Intestinal macrophages have been poorly studied in fish, mainly due to the lack of specific molecular markers for their identification and isolation. To address this gap, using the zebrafish Tg(mpeg1:EGFP) transgenic line, we developed a fluorescence-activated cell sorting strategy (FACS) that allows us to isolate different intestinal macrophage subpopulations, based on GFP expression and morphological differences. Also, we achieved the purification of high-quality total RNA from each population to perform transcriptomic analysis. The complete strategy comprises three steps, including intestine dissection and tissue dissociation, the isolation of each intestinal macrophage population via FACS, and the extraction of total RNA. To be able to characterize molecularly different macrophage subpopulations and link them to their functional properties will allow us to unravel intestinal macrophage biology.
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(This article belongs to the Section Molecular and Cellular Biology)
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Open AccessArticle
Syrian Hamsters Model Does Not Reflect Human-like Disease after Aerosol Exposure to Encephalitic Alphaviruses
by
Christina L. Gardner, Rebecca A. Erwin-Cohen, Bridget S. Lewis, Russell R. Bakken, Shelley P. Honnold, Pamela J. Glass and Crystal W. Burke
Methods Protoc. 2024, 7(3), 42; https://doi.org/10.3390/mps7030042 - 15 May 2024
Abstract
Venezuelan (VEE), eastern (EEE), and western (WEE) equine encephalitis viruses are encephalitic New World alphaviruses that cause periodic epizootic and epidemic outbreaks in horses and humans that may cause severe morbidity and mortality. Currently there are no FDA-licensed vaccines or effective antiviral therapies.
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Venezuelan (VEE), eastern (EEE), and western (WEE) equine encephalitis viruses are encephalitic New World alphaviruses that cause periodic epizootic and epidemic outbreaks in horses and humans that may cause severe morbidity and mortality. Currently there are no FDA-licensed vaccines or effective antiviral therapies. Each year, there are a limited number of human cases of encephalitic alphaviruses; thus, licensure of a vaccine or therapeutic would require approval under the FDA animal rule. Approval under the FDA animal rule requires the disease observed in the animal model to recapitulate what is observed in humans. Currently, initial testing of vaccines and therapeutics is performed in the mouse model. Unfortunately, alphavirus disease manifestations in a mouse do not faithfully recapitulate human disease; the VEEV mouse model is lethal whereas in humans VEEV is rarely lethal. In an effort to identify a more appropriate small animal model, we evaluated hamsters in an aerosol exposure model of encephalitic alphavirus infection. The pathology, lethality, and viremia observed in the infected hamsters was inconsistent with what is observed in NHP models and humans. These data suggest that hamsters are not an appropriate model for encephalitic alphaviruses to test vaccines or potential antiviral therapies.
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(This article belongs to the Special Issue Feature Papers in Methods and Protocols 2024)
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Open AccessCommentary
Methodological Considerations Regarding the Quantification of DNA Impurities in the COVID-19 mRNA Vaccine Comirnaty®
by
Brigitte König and Jürgen O. Kirchner
Methods Protoc. 2024, 7(3), 41; https://doi.org/10.3390/mps7030041 - 8 May 2024
Abstract
DNA impurities can impact the safety of genetically engineered pharmaceuticals; thus, a specific limit value must be set for them during marketing authorisation. This particularly applies to mRNA vaccines, as large quantities of DNA templates are used for their production. Furthermore, when quantifying
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DNA impurities can impact the safety of genetically engineered pharmaceuticals; thus, a specific limit value must be set for them during marketing authorisation. This particularly applies to mRNA vaccines, as large quantities of DNA templates are used for their production. Furthermore, when quantifying the total DNA content in the final product, we must observe that, in addition to the mRNA active ingredient, DNA impurities are also encased in lipid nanoparticles and are therefore difficult to quantify. In fact, the manufacturer of the mRNA vaccine Comirnaty (BioNTech/Pfizer) only measures DNA impurities in the active substance by means of a quantitative polymerase chain reaction (qPCR), whose DNA target sequence is less than just 1% of the originally added DNA template. This means that no direct DNA quantification takes place, and compliance with the limit value for DNA contamination is only estimated from the qPCR data using mathematical extrapolation methods. However, it is also possible to dissolve the lipid nanoparticles with a detergent to directly measure DNA contamination in the final product by using fluorescence spectroscopic methods. Experimental testing of this approach confirms that reliable values can be obtained in this way.
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(This article belongs to the Section Biomedical Sciences and Physiology)
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Open AccessArticle
Quantifying mRNA in Highly Degraded Fixed Tissues by Nanostring Technology: A Comparative Study
by
Eros Azzalini, Barbara Di Stefano, Vincenzo Canzonieri, Tiziana Venesio, Umberto Miglio, Caterina Marchiò, Anna Sapino, Carlo Previderè, Paolo Fattorini and Serena Bonin
Methods Protoc. 2024, 7(3), 40; https://doi.org/10.3390/mps7030040 - 7 May 2024
Abstract
Archive tissues are the most available source of human tissues useful for molecular analysis in translational research. The main issues for those specimens are the modification and degradation of biomolecules, namely proteins, DNA, and RNA. In the last decade, several high-throughput analytical methods
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Archive tissues are the most available source of human tissues useful for molecular analysis in translational research. The main issues for those specimens are the modification and degradation of biomolecules, namely proteins, DNA, and RNA. In the last decade, several high-throughput analytical methods have been applied to archive tissues. Although histological tissues are fixed in neutral-buffered formalin nowadays, in the recent past, Bouin’s solution was also used in tissue processing. The present study aims to investigate the feasibility of nCounter Nanostring hybridization in quantifying mRNA in highly degraded samples, such as Bouin’s fixed and paraffin-embedded (BFPE) tissues, in comparison to the standard formalin-fixed and paraffin-embedded (FFPE) tissues as a source of RNA. A total of 16 paraffin-embedded tissue blocks from eight patients were analyzed (8 were FFPE and 8 were BEPE). Nanostring technology was applied to 300 ng of each RNA sample, whereas 360 ng of the same templates were retrotranscribed and submitted to qPCR and ddPCR. Our results show that the Nanostring technology outperforms the reference methods (ddPCR and qPCR) in detecting target mRNA in FFPE and BFPE samples. However, even Nanostring technology does not escape the limitation imposed by the degradation of the RNA templates, which could lead to misleading conclusions on the gene expression level.
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(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
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Open AccessProtocol
A Protocol for Comprehensive Analysis of Gait in Individuals with Incomplete Spinal Cord Injury
by
Emelie Butler Forslund, Minh Tat Nhat Truong, Ruoli Wang, Åke Seiger and Elena M. Gutierrez-Farewik
Methods Protoc. 2024, 7(3), 39; https://doi.org/10.3390/mps7030039 - 4 May 2024
Abstract
This is a protocol for comprehensive analysis of gait and affecting factors in individuals with incomplete paraplegia due to spinal cord injury (SCI). A SCI is a devastating event affecting both sensory and motor functions. Due to better care, the SCI population is
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This is a protocol for comprehensive analysis of gait and affecting factors in individuals with incomplete paraplegia due to spinal cord injury (SCI). A SCI is a devastating event affecting both sensory and motor functions. Due to better care, the SCI population is changing, with a greater proportion retaining impaired ambulatory function. Optimizing ambulatory function after SCI remains challenging. To investigate factors influencing optimal ambulation, a multi-professional research project was grounded with expertise from clinical rehabilitation, neurophysiology, and biomechanical engineering from Karolinska Institutet, the Spinalis Unit at Aleris Rehab Station (Sweden’s largest center for specialized neurorehabilitation), and the Promobilia MoveAbility Lab at KTH Royal Institute of Technology. Ambulatory adults with paraplegia will be consecutively invited to participate. Muscle strength, sensitivity, and spasticity will be assessed, and energy expenditure, 3D movements, and muscle function (EMG) during gait and submaximal contractions will be analyzed. Innovative computational modeling and data-driven analyses will be performed, including the identification of clusters of similar movement patterns among the heterogeneous population and analyses that study the link between complex sensorimotor function and movement performance. These results may help optimize ambulatory function for persons with SCI and decrease the risk of secondary conditions during gait with a life-long perspective.
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(This article belongs to the Section Biomedical Sciences and Physiology)
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Open AccessReview
Chromatographic Methods for the Determination of Glyphosate in Cereals Together with a Discussion of Its Occurrence, Accumulation, Fate, Degradation, and Regulatory Status
by
Maurizio Masci, Roberto Caproni and Teresina Nevigato
Methods Protoc. 2024, 7(3), 38; https://doi.org/10.3390/mps7030038 - 2 May 2024
Abstract
The European Union’s recent decision to renew the authorization for the use of glyphosate until 15 December 2033 has stimulated scientific discussion all around the world regarding its toxicity or otherwise for humans. Glyphosate is a chemical of which millions of tons have
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The European Union’s recent decision to renew the authorization for the use of glyphosate until 15 December 2033 has stimulated scientific discussion all around the world regarding its toxicity or otherwise for humans. Glyphosate is a chemical of which millions of tons have been used in the last 50 years worldwide to dry out weeds in cultivated fields and greenhouses and on roadsides. Concern has been raised in many areas about its possible presence in the food chain and its consequent adverse effects on health. Both aspects that argue in favor of toxicity and those that instead may indicate limited toxicity of glyphosate are discussed here. The widespread debate that has been generated requires further investigations and field measurements to understand glyphosate’s fate once dispersed in the environment and its concentration in the food chain. Hence, there is a need for validated analytical methods that are available to analysts in the field. In the present review, methods for the analytical determination of glyphosate and its main metabolite, AMPA, are discussed, with a specific focus on chromatographic techniques applied to cereal products. The experimental procedures are explained in detail, including the cleanup, derivatization, and instrumental conditions, to give the laboratories involved enough information to proceed with the implementation of this line of analysis. The prevalent chromatographic methods used are LC-MS/MS, GC-MS/SIM, and GC-MS/MS, but sufficient indications are also given to those laboratories that wish to use the better performing high-resolution MS or the simpler HPLC-FLD, HPLC-UV, GC-NPD, and GC-FPD techniques for screening purposes. The concentrations of glyphosate from the literature measured in wheat, corn, barley, rye, oats, soybean, and cereal-based foods are reported, together with its regulatory status in various parts of the world and its accumulation mechanism. As for its accumulation in cereals, the available data show that glyphosate tends to accumulate more in wholemeal flours than in refined ones, that its concentration in the product strictly depends on the treatment period (the closer it is to the time of harvesting, the higher the concentration), and that in cold climates, the herbicide tends to persist in the soil for a long time.
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(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
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Open AccessProtocol
Dedicated Protocol for Ultrastructural Analysis of Farmed Rainbow Trout (Oncorhynchus mykiss) Tissues with Red Mark Syndrome: The Skin—Part One
by
Diana Torge, Sara Bernardi, Giulia Ciciarelli, Guido Macchiarelli and Serena Bianchi
Methods Protoc. 2024, 7(3), 37; https://doi.org/10.3390/mps7030037 - 24 Apr 2024
Abstract
The present study aims to provide a specific protocol for transmission electron microscopy of a sample of skin of rainbow trout affected by red mark syndrome (RMS). The red mark syndrome is a skin disease that affects the rainbow trout (Oncorhynchus mykiss
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The present study aims to provide a specific protocol for transmission electron microscopy of a sample of skin of rainbow trout affected by red mark syndrome (RMS). The red mark syndrome is a skin disease that affects the rainbow trout (Oncorhynchus mykiss). The disease, probably due to the Midichloria-like organism infection, is not lethal, but morbidity can reach up to 60%, leading to significant economic impact associated with the downgrading of the commercial product, increased labor, and susceptibility to secondary infections. The ultrastructure analyses allowed an earlier study to identify the presence of scattered microorganisms characterized by an oval shape, mainly in the cytoplasm of the cells. The protocol developed in this study will be instrumental in visualizing the ultrastructure of the microorganism, which is probably responsible for red mark syndrome infection.
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(This article belongs to the Section Tissue Engineering and Organoids)
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Open AccessArticle
Simplifying Data Analysis in Biomedical Research: An Automated, User-Friendly Tool
by
Rúben Araújo, Luís Ramalhete, Ana Viegas, Cristiana P. Von Rekowski, Tiago A. H. Fonseca, Cecília R. C. Calado and Luís Bento
Methods Protoc. 2024, 7(3), 36; https://doi.org/10.3390/mps7030036 - 24 Apr 2024
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Robust data normalization and analysis are pivotal in biomedical research to ensure that observed differences in populations are directly attributable to the target variable, rather than disparities between control and study groups. ArsHive addresses this challenge using advanced algorithms to normalize populations (e.g.,
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Robust data normalization and analysis are pivotal in biomedical research to ensure that observed differences in populations are directly attributable to the target variable, rather than disparities between control and study groups. ArsHive addresses this challenge using advanced algorithms to normalize populations (e.g., control and study groups) and perform statistical evaluations between demographic, clinical, and other variables within biomedical datasets, resulting in more balanced and unbiased analyses. The tool’s functionality extends to comprehensive data reporting, which elucidates the effects of data processing, while maintaining dataset integrity. Additionally, ArsHive is complemented by A.D.A. (Autonomous Digital Assistant), which employs OpenAI’s GPT-4 model to assist researchers with inquiries, enhancing the decision-making process. In this proof-of-concept study, we tested ArsHive on three different datasets derived from proprietary data, demonstrating its effectiveness in managing complex clinical and therapeutic information and highlighting its versatility for diverse research fields.
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Open AccessStudy Protocol
Effects of a WhatsApp-Assisted Health Educational Intervention for Cardiac Rehabilitation: A Randomized Controlled Clinical Trial Protocol
by
Adriana Marcela Jacome-Hortua, Zully Rocio Rincon-Rueda, Diana C. Sanchez-Ramirez and Adriana Angarita-Fonseca
Methods Protoc. 2024, 7(2), 35; https://doi.org/10.3390/mps7020035 - 19 Apr 2024
Abstract
Although the effectiveness of cardiac rehabilitation (CR) programs in secondary prevention is well-recognized, there is a lack of studies exploring the potential of mobile health to enhance educational interventions within CR. The objective is to assess the impact of a structured WhatsApp-assisted health
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Although the effectiveness of cardiac rehabilitation (CR) programs in secondary prevention is well-recognized, there is a lack of studies exploring the potential of mobile health to enhance educational interventions within CR. The objective is to assess the impact of a structured WhatsApp-assisted health educational intervention, in conjunction with the usual care, compared to the usual care alone among participants enrolled in a CR program. The trial will recruit 32 participants enrolled in a CR program, who will be randomly assigned to a structured WhatsApp-assisted health educational intervention plus usual care or usual care alone group. The intervention will span 4 weeks, with assessments at baseline, 4 weeks, and 3, 6, and 12 months. The primary outcome measure is the cardiovascular risk factors knowledge score. Secondary outcomes include physical activity levels, anxiety and depression, and quality of life. Expected results include improved knowledge of cardiovascular risk factors, increased physical activity levels, and better mental health outcomes in the intervention group. Additionally, an enhancement in the overall quality of life is anticipated. These findings are expected to underscore the value of integrating mHealth with traditional CR methods, potentially shaping future approaches in chronic disease management and prevention.
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(This article belongs to the Section Public Health Research)
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