Next Issue
Volume 7, June
Previous Issue
Volume 7, February
 
 

Methods Protoc., Volume 7, Issue 2 (April 2024) – 18 articles

Cover Story (view full-size image): In immunological research, bone marrow-derived dendritic cells (BMDCs) are a valuable tool in the study of antigen processing and presentation, as well as T-cell responses. However, the isolation of murine bone marrow for large-scale experiments can be time-consuming. In this protocol, we evaluated a faster isolation method that uses centrifugation prior to the differentiation of FMS-like tyrosine kinase 3 ligand-driven BMDCs. Compared to the conventional flush-out method, we found that the new approach resulted in a similar initial bone marrow cell count as well as viability and an increased yield of BMDCs upon eight days of differentiation, while preserving the proportions of individual dendritic cell subsets (cDC1, cDC2 and pDC). View this paper
  • Issues are regarded as officially published after their release is announced to the table of contents alert mailing list.
  • You may sign up for e-mail alerts to receive table of contents of newly released issues.
  • PDF is the official format for papers published in both, html and pdf forms. To view the papers in pdf format, click on the "PDF Full-text" link, and use the free Adobe Reader to open them.
Order results
Result details
Section
Select all
Export citation of selected articles as:
10 pages, 686 KiB  
Study 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://0-doi-org.brum.beds.ac.uk/10.3390/mps7020035 - 19 Apr 2024
Viewed by 1009
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 [...] Read more.
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. Full article
(This article belongs to the Section Public Health Research)
Show Figures

Figure 1

11 pages, 404 KiB  
Study Protocol
Financial Toxicity in Renal Patients (FINTORE) Study: A Cross-Sectional Italian Study on Financial Burden in Kidney Disease—A Project Protocol
by Rossella Siligato, Guido Gembillo, Emanuele Di Simone, Alessio Di Maria, Simone Nicoletti, Laura Maria Scichilone, Matteo Capone, Francesca Maria Vinci, Marta Bondanelli, Cristina Malaventura, Alda Storari, Domenico Santoro, Marco Di Muzio, Sara Dionisi and Fabio Fabbian
Methods Protoc. 2024, 7(2), 34; https://0-doi-org.brum.beds.ac.uk/10.3390/mps7020034 - 14 Apr 2024
Viewed by 958
Abstract
Financial toxicity (FT) refers to the negative impact of health-care costs on clinical conditions. In general, social determinants of health, especially poverty, socioenvironmental stressors, and psychological factors, are increasingly recognized as important determinants of non-communicable diseases, such as chronic kidney disease (CKD), and [...] Read more.
Financial toxicity (FT) refers to the negative impact of health-care costs on clinical conditions. In general, social determinants of health, especially poverty, socioenvironmental stressors, and psychological factors, are increasingly recognized as important determinants of non-communicable diseases, such as chronic kidney disease (CKD), and their consequences. We aim to investigate the prevalence of FT in patients at different stages of CKD treated in our universal health-care system and from pediatric nephrology, hemodialysis, peritoneal dialysis and renal transplantation clinics. FT will be assessed with the Patient-Reported Outcome for Fighting Financial Toxicity (PROFFIT) score, which was first developed by Italian oncologists. Our local ethics committee has approved the study. Our population sample will answer the sixteen questions of the PROFFIT questionnaire, seven of which are related to the outcome and nine the determinants of FT. Data will be analyzed in the pediatric and adult populations and by group stratification. We are confident that this study will raise awareness among health-care professionals of the high risk of adverse health outcomes in patients who have both kidney disease and high levels of FT. Strategies to reduce FT should be implemented to improve the standard of care for people with kidney disease and lead to truly patient-centered care. Full article
(This article belongs to the Section Public Health Research)
Show Figures

Figure 1

20 pages, 14205 KiB  
Article
Research and Simulation on the Development of a Hydraulic Prop Support System of Powered Roof Support to Increase Work Safety
by Beata Borska and Dawid Szurgacz
Methods Protoc. 2024, 7(2), 33; https://0-doi-org.brum.beds.ac.uk/10.3390/mps7020033 - 11 Apr 2024
Viewed by 1027
Abstract
The underground mining environment is currently based on technology that uses mainly analogue sensors in machine and equipment control systems. The primary machine performing the most important functions in a mining system is the powered roof support. In order for it to work [...] Read more.
The underground mining environment is currently based on technology that uses mainly analogue sensors in machine and equipment control systems. The primary machine performing the most important functions in a mining system is the powered roof support. In order for it to work properly, it is important that it achieves the required power. To ensure this, it is necessary to continuously and precisely monitor the pressure in the under-piston space of the prop. Due to the extreme environmental conditions, pressure sensors should have high sensitivity, large transmission capacity, small size and light weight. To achieve these requirements, the authors of the article propose to implement a monitoring system based on photonics technology. To achieve this goal, several studies were carried out. The range of these studies included simulations, bench tests and tests under real conditions. The obtained test results showed the possibility of developing the control system for the powered roof support, the additional function to supercharge power. Based on the analysis of the obtained test results, assumptions were developed for the development of a power charging system with monitoring sensors. Based on the guidelines obtained from the research results, thedevelopment of the above prototype based on photonics technology is proposed. Full article
Show Figures

Figure 1

8 pages, 2411 KiB  
Communication
Optimization of the Amplification of Equine Muscle-Derived Mesenchymal Stromal Cells in a Hollow-Fiber Bioreactor
by Julien Duysens, Hélène Graide, Ariane Niesten, Ange Mouithys-Mickalad, Justine Ceusters and Didier Serteyn
Methods Protoc. 2024, 7(2), 32; https://0-doi-org.brum.beds.ac.uk/10.3390/mps7020032 - 2 Apr 2024
Cited by 1 | Viewed by 1216
Abstract
The main causes of mortality in horses are the gastrointestinal pathologies associated with septic shock. Stem cells have shown, through systemic injection, a capacity to decrease inflammation and to regenerate injured tissue faster. Nevertheless, to achieve this rapid and total regeneration, systemic injections [...] Read more.
The main causes of mortality in horses are the gastrointestinal pathologies associated with septic shock. Stem cells have shown, through systemic injection, a capacity to decrease inflammation and to regenerate injured tissue faster. Nevertheless, to achieve this rapid and total regeneration, systemic injections of 1 to 2 million cells per kilogram of body weight must be considered. Here, we demonstrate for the first time the feasibility and expansion capacity of equine muscle-derived mesenchymal stromal cells (mdMSCs) in a functionally closed, automated, perfusion-based, hollow-fiber bioreactor (HFBR) called the Quantum™ Cell Expansion System (Terumo Blood and Cell Technologies). This feature greatly increases the number of generated cells with a surface area of 1.7 m2. The expansion of mdMSCs is very efficient in this bioreactor. The maximum expansion generated twenty times more cells than the initial seeding in nine days. The best returns were observed with an optimal seeding between 10 and 25 million mdMSCs, using the Bull’s eye loading method and with a run duration between 7 and 10 days. Moreover, all the generated cells kept their stem properties: the ability to adhere to plastic and to differentiate into chondroblasts, osteoblasts and adipocytes. They also showed the expression of CD-44 and CD-90 markers, with a positive rate above 93%, while CD-45 and MHCII were non-expressed, with a positive rate below 0.5%. By capitalizing on the scalability, automation and 3D culture capabilities of the Quantum™, it is possible to generate large quantities of high-quality equine mdMSCs for gastrointestinal disorders and other clinical applications. Full article
(This article belongs to the Special Issue Feature Papers in Methods and Protocols 2024)
Show Figures

Figure 1

12 pages, 2158 KiB  
Study Protocol
Assessing Stress Induced by Fluid Shifts and Reduced Cerebral Clearance during Robotic-Assisted Laparoscopic Radical Prostatectomy under Trendelenburg Positioning (UroTreND Study)
by Tobias Peschke, Matthias Feuerecker, Daniel Siegl, Nathalie Schicktanz, Christian Stief, Peter Zu Eulenburg, Alexander Choukér and Judith-Irina Buchheim
Methods Protoc. 2024, 7(2), 31; https://0-doi-org.brum.beds.ac.uk/10.3390/mps7020031 - 1 Apr 2024
Viewed by 1108
Abstract
In addition to general anesthesia and mechanical ventilation, robotic-assisted laparoscopic radical prostatectomy (RALP) necessitates maintaining a capnoperitoneum and placing the patient in a pronounced downward tilt (Trendelenburg position). While the effects of the resulting fluid shift on the cardiovascular system seem to be [...] Read more.
In addition to general anesthesia and mechanical ventilation, robotic-assisted laparoscopic radical prostatectomy (RALP) necessitates maintaining a capnoperitoneum and placing the patient in a pronounced downward tilt (Trendelenburg position). While the effects of the resulting fluid shift on the cardiovascular system seem to be modest and well tolerated, the effects on the brain and the blood–brain barrier have not been thoroughly investigated. Previous studies indicated that select patients showed an increase in the optic nerve sheath diameter (ONSD), detected by ultrasound during RALP, which suggests an elevation in intracranial pressure. We hypothesize that the intraoperative fluid shift results in endothelial dysfunction and reduced cerebral clearance, potentially leading to transient neuronal damage. This prospective, monocentric, non-randomized, controlled clinical trial will compare RALP to conventional open radical prostatectomy (control group) in a total of 50 subjects. The primary endpoint will be the perioperative concentration of neurofilament light chain (NfL) in blood using single-molecule array (SiMoA) as a measure for neuronal damage. As secondary endpoints, various other markers for endothelial function, inflammation, and neuronal damage as well as the ONSD will be assessed. Perioperative stress will be evaluated by questionnaires and stress hormone levels in saliva samples. Furthermore, the subjects will participate in functional tests to evaluate neurocognitive function. Each subject will be followed up until discharge. Conclusion: This trial aims to expand current knowledge as well as to develop strategies for improved monitoring and higher safety of patients undergoing RALP. The trial was registered with the German Clinical Trials Register DRKS00031041 on 11 January 2023. Full article
(This article belongs to the Section Biomedical Sciences and Physiology)
Show Figures

Figure 1

11 pages, 2990 KiB  
Protocol
Diverting the Use of Hand-Operated Tablet Press Machines to Bioassays: A Novel Protocol to Test ‘Waste’ Insoluble Shell Matrices
by Camille Lutet-Toti, Marie Da Silva Feliciano, Nelly Debrosse, Jérôme Thomas, Laurent Plasseraud and Frédéric Marin
Methods Protoc. 2024, 7(2), 30; https://0-doi-org.brum.beds.ac.uk/10.3390/mps7020030 - 1 Apr 2024
Viewed by 1046
Abstract
To mineralize their shells, molluscs secrete a complex cocktail of proteins—collectively defined as the calcifying shell matrix—that remains occluded in the exoskeleton. Nowadays, protein extracts from shells are recognized as a potential source of bioactive substances, among which signalling molecules, bactericides or protease [...] Read more.
To mineralize their shells, molluscs secrete a complex cocktail of proteins—collectively defined as the calcifying shell matrix—that remains occluded in the exoskeleton. Nowadays, protein extracts from shells are recognized as a potential source of bioactive substances, among which signalling molecules, bactericides or protease inhibitors offer the most tangible perspectives in applied sciences, health, and aquaculture. However, one technical obstacle in testing the activity of shell extracts lies in their high insolubility. In this paper, we present a protocol that circumvents this impediment. After an adapted shell protein extraction and the production of two organic fractions—one soluble, one insoluble—we employ a hand-operated tablet press machine to generate well-calibrated tablets composed of 100% insoluble shell matrix. FT-IR monitoring of the quality of the tablets shows that the pressure used in the press machine does not impair the molecular properties of the insoluble extracts. The produced tablets can be directly tested in different biological assays, such as the bactericidal inhibition zone assay in Petri dish, as illustrated here. Diverting the use of the hand-operated tablet press opens new perspectives in the analysis of insoluble shell matrices, for discovering novel bioactive components. Full article
(This article belongs to the Section Synthetic and Systems Biology)
Show Figures

Figure 1

1 pages, 167 KiB  
Correction
Correction: Kharb et al. A Genotype-Independent, Simple, Effective and Efficient in Planta Agrobacterium-Mediated Genetic Transformation Protocol. Methods Protoc. 2022, 5, 69
by Pushpa Kharb, Rinku Chaudhary, Narendra Tuteja and Prashant Kaushik
Methods Protoc. 2024, 7(2), 29; https://0-doi-org.brum.beds.ac.uk/10.3390/mps7020029 - 27 Mar 2024
Viewed by 840
Abstract
Additional Affiliation(s): Updated Affiliation [...] Full article
14 pages, 557 KiB  
Review
Imaging Flow Cytometry: Development, Present Applications, and Future Challenges
by Savvas Dimitriadis, Lefkothea Dova, Ioannis Kotsianidis, Eleftheria Hatzimichael, Eleni Kapsali and Georgios S. Markopoulos
Methods Protoc. 2024, 7(2), 28; https://0-doi-org.brum.beds.ac.uk/10.3390/mps7020028 - 23 Mar 2024
Viewed by 1721
Abstract
Imaging flow cytometry (ImFC) represents a significant technological advancement in the field of cytometry, effectively merging the high-throughput capabilities of flow analysis with the detailed imaging characteristics of microscopy. In our comprehensive review, we adopt a historical perspective to chart the development of [...] Read more.
Imaging flow cytometry (ImFC) represents a significant technological advancement in the field of cytometry, effectively merging the high-throughput capabilities of flow analysis with the detailed imaging characteristics of microscopy. In our comprehensive review, we adopt a historical perspective to chart the development of ImFC, highlighting its origins and current state of the art and forecasting potential future advancements. The genesis of ImFC stemmed from merging the hydraulic system of a flow cytometer with advanced camera technology. This synergistic coupling facilitates the morphological analysis of cell populations at a high-throughput scale, effectively evolving the landscape of cytometry. Nevertheless, ImFC’s implementation has encountered hurdles, particularly in developing software capable of managing its sophisticated data acquisition and analysis needs. The scale and complexity of the data generated by ImFC necessitate the creation of novel analytical tools that can effectively manage and interpret these data, thus allowing us to unlock the full potential of ImFC. Notably, artificial intelligence (AI) algorithms have begun to be applied to ImFC, offering promise for enhancing its analytical capabilities. The adaptability and learning capacity of AI may prove to be essential in knowledge mining from the high-dimensional data produced by ImFC, potentially enabling more accurate analyses. Looking forward, we project that ImFC may become an indispensable tool, not only in research laboratories, but also in clinical settings. Given the unique combination of high-throughput cytometry and detailed imaging offered by ImFC, we foresee a critical role for this technology in the next generation of scientific research and diagnostics. As such, we encourage both current and future scientists to consider the integration of ImFC as an addition to their research toolkit and clinical diagnostic routine. Full article
(This article belongs to the Special Issue Feature Papers in Methods and Protocols 2024)
Show Figures

Figure 1

15 pages, 2082 KiB  
Article
Indirect Measurement of Variables in a Heterogeneous Reaction for Biodiesel Production
by Ana Paloma González-García, Lourdes Díaz-Jiménez, Padmasree K. Padmadas and Salvador Carlos-Hernández
Methods Protoc. 2024, 7(2), 27; https://0-doi-org.brum.beds.ac.uk/10.3390/mps7020027 - 23 Mar 2024
Viewed by 1551
Abstract
This research focuses on the development of a state observer for performing indirect measurements of the main variables involved in the soybean oil transesterification reaction with a guishe biochar-based heterogeneous catalyst; the studied reaction takes place in a batch reactor. The mathematical model [...] Read more.
This research focuses on the development of a state observer for performing indirect measurements of the main variables involved in the soybean oil transesterification reaction with a guishe biochar-based heterogeneous catalyst; the studied reaction takes place in a batch reactor. The mathematical model required for the observer design includes the triglycerides’ conversion rate, and the reaction temperature. Since these variables are represented by nonlinear differential equations, the model is linearized around an operation point; after that, the pole placement and linear quadratic regulator (LQR) methods are considered for calculating the observer gain vector L(x). Then, the estimation of the conversion rate and the reaction temperature provided by the observer are used to indirectly measure other variables such as esters, alcohol, and byproducts. The observer performance is evaluated with three error indexes considering initial condition variations up to 30%. With both methods, a fast convergence (less than 3 h in the worst case) of the observer is remarked. Full article
(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
Show Figures

Figure 1

15 pages, 1016 KiB  
Study Protocol
The Frailty Reduction via Implementation of Exercise, Nutrition, and Deprescribing (FRIEND) Trial: Study Protocol and Recruitment Results
by Michael Inskip, Carolina Almendrales Rangel, Chidiamara Maria Njoku, Fiona Barnett, Isabel Shih, Leonie O’Neill, Maria A. Fiatarone Singh and Trinidad Valenzuela
Methods Protoc. 2024, 7(2), 26; https://0-doi-org.brum.beds.ac.uk/10.3390/mps7020026 - 22 Mar 2024
Viewed by 2032
Abstract
Introduction: Virtually all adults in aged care facilities are frail, a condition which contributes to falls, cognitive decline, hospitalisation, and mortality. Polypharmacy, malnutrition, sedentariness, and sarcopenia are risk factors amenable to intervention. The Asia–Pacific Frailty Management Guidelines recommend anabolic exercise and the optimisation [...] Read more.
Introduction: Virtually all adults in aged care facilities are frail, a condition which contributes to falls, cognitive decline, hospitalisation, and mortality. Polypharmacy, malnutrition, sedentariness, and sarcopenia are risk factors amenable to intervention. The Asia–Pacific Frailty Management Guidelines recommend anabolic exercise and the optimisation of medications and nutrition. However, no study has evaluated this best practice intervention triad in aged care. Methods: The Frailty Reduction via the Implementation of Exercise, Nutrition, and Deprescribing (FRIEND) Trial (ANZCTR No.ACTRN12622000926730p) is a staged 6-month translational trial evaluating resident outcomes, staff/caregiver knowledge, and institutional implementation in a Townsville aged care facility. Residents received high-intensity resistance exercise and balance training and medication and nutrition optimisation co-implemented by investigators (exercise physiologist, geriatrician, pharmacist, and nutritionist) and facility staff. Staff and caregivers completed comprehensive education modules and training. We report the trial protocol and recruitment results. Results: 29 residents (21 female, age: 88.6 ± 6.3 years) were recruited. At baseline, the residents were frail (frailty scale nursing home (FRAIL-NH); 6.3 ± 2.4/14), cognitively impaired (Montreal Cognitive Assessment; 13.8 ± 6.8/30), functionally impaired (Short Physical Performance Battery; 4.9 ± 3.1/12, 6 min walk distance; 222.2 ± 104.4 m), and were prescribed numerous medications (15.5 ± 5.9). Two residents died and one withdrew before the intervention’s commencement. Thirty family members and 19 staff (carers, allied health assistants, nurse managers, registered nurses, lifestyle–leisure officers, kitchen/hospitality staff, and senior leadership) were recruited to receive frailty education modules. Conclusions: The FRIEND trial is currently being implemented with results expected in mid-2024. This is the first trial to evaluate the implementation of the best practice frailty guidelines including anabolic exercise and medication/nutritional optimisation in residential aged care. Full article
(This article belongs to the Section Public Health Research)
Show Figures

Figure 1

11 pages, 1388 KiB  
Protocol
Development and Validation of Micro-Azocasein Assay for Quantifying Bromelain
by Krishna Pillai, Javed Akhter, Ahmed H. Mekkawy, Sarah J. Valle and David L. Morris
Methods Protoc. 2024, 7(2), 25; https://0-doi-org.brum.beds.ac.uk/10.3390/mps7020025 - 15 Mar 2024
Viewed by 1286
Abstract
The proteolytic activity of enzymes may be evaluated by a colorimetric method with azocasein. Hence, we developed a micro-assay to quantify bromelain using azocasein. A total of 250 µL of 1.0% azocasein in dH2O was added to 250 µL of test [...] Read more.
The proteolytic activity of enzymes may be evaluated by a colorimetric method with azocasein. Hence, we developed a micro-assay to quantify bromelain using azocasein. A total of 250 µL of 1.0% azocasein in dH2O was added to 250 µL of test solution, vortexed and incubated at ambient room temperature/30 min. The reaction was terminated with 1500 µL of 5% trichloroacetic acid, vortexed and centrifuged. A total of 150 µL of 0.5M NaOH was added to 150 µL of supernatant in triplicates, and absorbance was recorded at 410 nm. The linearity of the calibration curve was tested with 200–800 µg/mL serial dilutions. The detection limit, precision, accuracy, and robustness were tested along with the substrate enzyme reaction time and solvent matrix effect. Good linearity was seen with serially diluted 200 µg/mL bromelain. The limit of quantification and limit of detection were 5.412 and 16.4 µg/mL, respectively. Intra-day and inter-day analyses showed a relative standard deviation below 2.0%. The assay was robust when tested over 400–450 nm wavelengths. The assays performed using dH2O or PBS diluents indicated a higher sensitivity in dH2O. The proteolytic activity of bromelain was enhanced with L-cysteine or N-acetylcysteine. Hence, this micro-azocasein assay is reliable for quantifying bromelain. Full article
(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
Show Figures

Figure 1

10 pages, 942 KiB  
Protocol
Effects on Quality of Life of a Telemonitoring Platform amongst Patients with Cancer (EQUALITE): A Randomized Trial Protocol
by Felipe Martínez, Carla Taramasco, Manuel Espinoza, Johanna Acevedo, Carolina Goic and Bruno Nervi
Methods Protoc. 2024, 7(2), 24; https://0-doi-org.brum.beds.ac.uk/10.3390/mps7020024 - 15 Mar 2024
Viewed by 1408
Abstract
Cancer, a pervasive global health challenge, necessitates chemotherapy or radiotherapy treatments for many prevalent forms. However, traditional follow-up approaches encounter limitations, exacerbated by the recent COVID-19 pandemic. Consequently, telemonitoring has emerged as a promising solution, although its clinical implementation lacks comprehensive evidence. This [...] Read more.
Cancer, a pervasive global health challenge, necessitates chemotherapy or radiotherapy treatments for many prevalent forms. However, traditional follow-up approaches encounter limitations, exacerbated by the recent COVID-19 pandemic. Consequently, telemonitoring has emerged as a promising solution, although its clinical implementation lacks comprehensive evidence. This report depicts the methodology of a randomized trial which aims to investigate whether leveraging a smartphone app called Contigo for disease monitoring enhances self-reported quality of life among patients with various solid cancers compared to standard care. Secondary objectives encompass evaluating the app’s impact on depressive symptoms and assessing adherence to in-person appointments. Randomization will be performed independently using an allocation sequence that will be kept concealed from clinical investigators. Contigo offers two primary functions: monitoring cancer patients’ progress and providing educational content to assist patients in managing common clinical situations related to their disease. The study will assess outcomes such as quality of life changes and depressive symptom development using validated scales, and adherence to in-person appointments. Specific scales include the EuroQol Group’s EQ-5D questionnaire and the Patient Health Questionnaire (PHQ-9). We hypothesize that the use of Contigo will assist and empower patients receiving cancer treatment, which will translate to better quality of life scores and a reduced incidence of depressive symptoms. All analyses will be undertaken with the intention-to-treat principle by a statistician unaware of treatment allocation. This trial is registered in ClinicalTrials under the registration number NCT06086990. Full article
(This article belongs to the Section Public Health Research)
Show Figures

Figure 1

12 pages, 1763 KiB  
Study Protocol
A Virtual Case Presentation Platform: Protocol Study
by Imad Alex Awada, Adina Magda Florea and Alexandru Scafa-Udriște
Methods Protoc. 2024, 7(2), 23; https://0-doi-org.brum.beds.ac.uk/10.3390/mps7020023 - 8 Mar 2024
Viewed by 1261
Abstract
Gaining practical experience is indispensable for medical students. Therefore, when medical students were prevented access to hospitals during the COVID-19 pandemic in Romania, there was an urgent need to find a solution that would allow medical students to develop the skills they would [...] Read more.
Gaining practical experience is indispensable for medical students. Therefore, when medical students were prevented access to hospitals during the COVID-19 pandemic in Romania, there was an urgent need to find a solution that would allow medical students to develop the skills they would usually develop in hospitals but without the need to be physically present in a hospital. This was the reason behind the idea of developing a Virtual Case Presentation Platform. The platform offers the possibility for medical students to reproduce virtually, in clinically valid scenarios, the diagnostic process and treatment recommendation, as well as the interactions with patients that usually take place in hospitals using natural language through speech and text. On the platform, the students receive valuable feedback from the professors about their performance. In order to reproduce the whole targeted experience for students, without missing anything, before starting the development of the platform, it was mandatory to identify and understand all the aspects that should be covered by the platform. The proposed platform covers the different aspects that have been identified for the diagnostic process and treatment recommendation. It enables medical students to develop essential skills for their future careers as doctors. Full article
Show Figures

Figure 1

11 pages, 4167 KiB  
Protocol
Isolation and Culture of Primary Human Dental Pulp Cells—A Description of Technical and Methodological Steps to Maximise Predictability and Yield
by Michaela Kearney, David E. McReynolds and Henry F. Duncan
Methods Protoc. 2024, 7(2), 22; https://0-doi-org.brum.beds.ac.uk/10.3390/mps7020022 - 1 Mar 2024
Viewed by 1663
Abstract
The dental pulp has critical functions in tooth development as well as an ongoing role in promoting and maintaining the vitality of teeth. In particular, its regenerative ability allows dental tissues to be restored following damage caused by traumatic injury or caries. Regenerative [...] Read more.
The dental pulp has critical functions in tooth development as well as an ongoing role in promoting and maintaining the vitality of teeth. In particular, its regenerative ability allows dental tissues to be restored following damage caused by traumatic injury or caries. Regenerative endodontic procedures aim to utilise these processes to stimulate dental pulp repair in a minimally invasive manner and reduce the need for more invasive procedures such as root canal treatment. Dental pulp is a source of dental pulp cells (DPCs), which has a subpopulation of dental pulp stem cells (DPSCs), which are attractive for use in regenerative medicine due to their high proliferation rate, ability to differentiate into multiple cell types, and their preserved vitality following cryopreservation. The development of next-generation clinical therapeutics that maximise the potential of dental pulp relies on strong empirical evidence arising from in vitro experimentation. Here, we describe a modified method for the efficient isolation of primary human DPCs from sound third molar teeth for culture using an explant outgrowth method on basement membrane-coated flasks, as well as using high-resolution macro-photography to illustrate the methods. Critically, steps are taken to minimise potential physical and mechanical trauma to the cells and maximise yield. Human DPCs cultured using this method can be further expanded in cell culture flasks to facilitate their use in various in vitro experimental procedures. Full article
(This article belongs to the Section Tissue Engineering and Organoids)
Show Figures

Figure 1

13 pages, 629 KiB  
Study Protocol
Acceptability, Feasibility, and Effectiveness of a Worksite Intervention to Lower Cardiometabolic Risk in South Africa: Protocol
by Evonne Shanita Singh, Ashika Naicker and Shivneta Singh
Methods Protoc. 2024, 7(2), 21; https://0-doi-org.brum.beds.ac.uk/10.3390/mps7020021 - 1 Mar 2024
Viewed by 1352
Abstract
As an important way to translate cardiovascular disease prevention efforts, worksite intervention programs can be used to effectively facilitate healthy food choices, health education, and social support among employees, in a targeted approach to improve health outcomes and physical activity levels of employees. [...] Read more.
As an important way to translate cardiovascular disease prevention efforts, worksite intervention programs can be used to effectively facilitate healthy food choices, health education, and social support among employees, in a targeted approach to improve health outcomes and physical activity levels of employees. In this study, the effectiveness of a canteen and a behavioral intervention on cardiometabolic risk among prediabetic and prehypertensive employees at two multinational worksites in South Africa will be measured. This two-arm randomized controlled trial (RCT) will be structured to provide a six-week intervention at two multinational companies spread across eight worksites and will include a canteen and behavioral arm (CB) and a canteen only (CO) arm. Participants who are either prediabetic or prehypertensive will complete the baseline assessments, which will include anthropometry, a demographic and lifestyle survey, the global physical activity questionnaire (GPAQ) and the 24 h food recall. Participants will be randomized into the CO and the canteen and CB intervention groups. The CO group will receive six weeks of canteen intervention [changes to enable a healthy food environment], while the CB group will receive six weeks of canteen intervention along with a behavioral intervention. The behavioral intervention will include an intense six-week lifestyle program aligned to the Diabetes Prevention Program (DPP). This study will assess the added benefit of environmental-level changes aimed at lowering cardiometabolic risk in a low–middle-income country (LMIC) and has the potential for scale-up to other worksites in South Africa and globally. Full article
Show Figures

Figure 1

10 pages, 1943 KiB  
Protocol
A Simple and Rapid Protocol for the Isolation of Murine Bone Marrow Suitable for the Differentiation of Dendritic Cells
by Runqiu Song, Mariam Bafit, Kirsteen M. Tullett, Peck Szee Tan, Mireille H. Lahoud, Meredith O’Keeffe, Anthony W. Purcell and Asolina Braun
Methods Protoc. 2024, 7(2), 20; https://0-doi-org.brum.beds.ac.uk/10.3390/mps7020020 - 27 Feb 2024
Viewed by 1827
Abstract
The generation of bone-marrow-derived dendritic cells is a widely used approach in immunological research to study antigen processing and presentation, as well as T-cell activation responses. However, the initial step of isolating the bone marrow can be time-consuming, especially when larger numbers of [...] Read more.
The generation of bone-marrow-derived dendritic cells is a widely used approach in immunological research to study antigen processing and presentation, as well as T-cell activation responses. However, the initial step of isolating the bone marrow can be time-consuming, especially when larger numbers of precursor cells are required. Here, we assessed whether an accelerated bone marrow isolation method using centrifugation is suitable for the differentiation of FMS-like tyrosine kinase 3 ligand-driven dendritic cells. Compared to the conventional flushing method, the centrifugation-based isolation method resulted in a similar bone marrow cell yield on Day 0, increased cell numbers by Day 8, similar proportions of dendritic cell subsets, and consequently a higher number of type 1 conventional dendritic cells (cDC1) from the culture. Although the primary purpose of this method of optimization was to improve experimental efficiency and increase the output of cDC1s, the protocol is also compatible with the differentiation of other dendritic cell subsets such as cDC2 and plasmacytoid dendritic cells, with an improved output cell count and a consistent phenotype. Full article
(This article belongs to the Section Biomedical Sciences and Physiology)
Show Figures

Figure 1

18 pages, 6303 KiB  
Article
Scale-Up of the Fermentation Process for the Production and Purification of Serratiopeptidase Using Silkworm Pupae as a Substrate
by Jhon Jairo Melchor-Moncada, Alejandra García-Barco, Augusto Zuluaga-Vélez, Luz Angela Veloza and Juan Carlos Sepúlveda-Arias
Methods Protoc. 2024, 7(2), 19; https://0-doi-org.brum.beds.ac.uk/10.3390/mps7020019 - 25 Feb 2024
Cited by 1 | Viewed by 1684
Abstract
Serratiopeptidase, a bacterial metalloprotease known for its pain-relieving and anti-inflammatory properties, can be produced through fermentation with S. marcescens. This study aimed to identify key factors related to nutrient composition and physicochemical conditions for production in Erlenmeyer flasks and to scale up [...] Read more.
Serratiopeptidase, a bacterial metalloprotease known for its pain-relieving and anti-inflammatory properties, can be produced through fermentation with S. marcescens. This study aimed to identify key factors related to nutrient composition and physicochemical conditions for production in Erlenmeyer flasks and to scale up the mixture to a bioreactor to obtain the maximum proteolytic activity. A Plackett–Burman design was used to determine whether the presence of silkworm pupae (at 1.5%) was a significant parameter for serratiopeptidase production. Along with the variables pH, temperature, and time, they were optimized using a Taguchi experimental design, resulting in values of 7, 25 °C, and 36 h, respectively. Scaling up with a kLa of 25.45 ± 3.12 h−1 showed the highest serratiopeptidase production at 24 h. A factorial design was used for ultrafiltration, resulting in an LMH (liters per square meter per hour) of 960 L/m2h, a TMP (transmembrane pressure) of 15 psi, and a concentration factor of five, with a specific activity of 24,325.81 ± 1515.69 U/mg. Afterward, the retentate was purified using strong anion exchange chromatography and ultrafiltration, yielding a 19.94 ± 3.07% recovery and a purification factor of 1.59 ± 0.31. In conclusion, waste from the sericulture industry can be used for serratiopeptidase production. Full article
(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
Show Figures

Figure 1

12 pages, 1278 KiB  
Article
Housing European Ground Squirrels (Spermophilus citellus) for an Ex Situ Conservation Program
by Boróka Bárdos, Vilmos Altbacker, Henrietta Kinga Török and István Nagy
Methods Protoc. 2024, 7(2), 18; https://0-doi-org.brum.beds.ac.uk/10.3390/mps7020018 - 20 Feb 2024
Viewed by 1428
Abstract
European ground squirrel (Spermophilus citellus) populations have declined precipitously over the last 70 years. Its protection cannot be ensured solely by protecting its habitat; it is also necessary to protect the animals ex situ. In our study, within a European ground [...] Read more.
European ground squirrel (Spermophilus citellus) populations have declined precipitously over the last 70 years. Its protection cannot be ensured solely by protecting its habitat; it is also necessary to protect the animals ex situ. In our study, within a European ground squirrel species protection program, we examined two elements of indoor housing technology. Knowledge of the animals’ needs is essential for captive housing and breeding success, so in our tests, the animals could freely choose both nest-building materials and feed. In the nest material preference test, the animals could choose from three materials with different structures: paper, Lignocel and hay. In the feed preference test, the animals could also choose from three types of feed: commercial rabbit feed, complete rabbit feed and a natural feed mixture. The first two feeds were in granulated format, and the third was a grain feed mix. Among the nesting materials, they preferred hay, which allowed them to build better-quality nests. Among the feeds, they preferred the grain feed mix, the composition closest to their natural feed, and it was the only one that contained animal protein. Our results contribute to the successful maintenance and breeding the European ground squirrel in captivity. Full article
Show Figures

Figure 1

Previous Issue
Next Issue
Back to TopTop