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Methods Protoc., Volume 7, Issue 3 (June 2024) – 10 articles

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12 pages, 437 KiB  
Study 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
Viewed by 156
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 [...] Read more.
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. Full article
(This article belongs to the Section Biomedical Sciences and Physiology)
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15 pages, 943 KiB  
Article
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
Viewed by 358
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 [...] Read more.
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 = 5.02Kg-2.24Kg, and SDD = 27.46%-14.92%). Slightly better results were found regarding inter-session reliability (ICC = 0.927–0.970, SEM = 3.72Kg-1.97Kg, 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. Full article
(This article belongs to the Special Issue Methods on Sport Biomechanics)
10 pages, 2239 KiB  
Protocol
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
Viewed by 215
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) [...] Read more.
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. Full article
(This article belongs to the Section Molecular and Cellular Biology)
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21 pages, 1609 KiB  
Article
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
Viewed by 215
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. [...] Read more.
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. Full article
(This article belongs to the Special Issue Feature Papers in Methods and Protocols 2024)
8 pages, 1187 KiB  
Commentary
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
Viewed by 23853
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 [...] Read more.
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. Full article
(This article belongs to the Section Biomedical Sciences and Physiology)
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14 pages, 3728 KiB  
Article
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
Viewed by 293
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 [...] Read more.
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. Full article
(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
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11 pages, 1014 KiB  
Protocol
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
Viewed by 504
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 [...] Read more.
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. Full article
(This article belongs to the Section Biomedical Sciences and Physiology)
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23 pages, 3119 KiB  
Review
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
Viewed by 590
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 [...] Read more.
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. Full article
(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
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13 pages, 8114 KiB  
Protocol
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
Viewed by 700
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 [...] Read more.
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. Full article
(This article belongs to the Section Tissue Engineering and Organoids)
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20 pages, 3689 KiB  
Article
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
Viewed by 697
Abstract
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., [...] Read more.
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. Full article
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