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 27.9 days after submission; acceptance to publication is undertaken in 3.9 days (median values for papers published in this journal in the second half of 2023).
- 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:
2.4 (2022);
5-Year Impact Factor:
2.4 (2022)
Latest Articles
Development and Validation of Micro-Azocasein Assay for Quantifying Bromelain
Methods Protoc. 2024, 7(2), 25; https://doi.org/10.3390/mps7020025 - 15 Mar 2024
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
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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.
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(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
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Open AccessProtocol
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://doi.org/10.3390/mps7020024 - 15 Mar 2024
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
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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.
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(This article belongs to the Section Public Health Research)
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Open AccessStudy 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://doi.org/10.3390/mps7020023 - 08 Mar 2024
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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
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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.
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Open AccessProtocol
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://doi.org/10.3390/mps7020022 - 01 Mar 2024
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
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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.
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(This article belongs to the Section Tissue Engineering and Organoids)
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Open AccessStudy 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://doi.org/10.3390/mps7020021 - 01 Mar 2024
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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.
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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.
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Open AccessProtocol
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://doi.org/10.3390/mps7020020 - 27 Feb 2024
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
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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.
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(This article belongs to the Section Biomedical Sciences and Physiology)
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Open AccessArticle
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://doi.org/10.3390/mps7020019 - 25 Feb 2024
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
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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)
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Open AccessArticle
Housing European Ground Squirrels (Spermophilus citellus) for an Ex Situ Conservation Program
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Boróka Bárdos, Vilmos Altbacker, Henrietta Kinga Török and István Nagy
Methods Protoc. 2024, 7(2), 18; https://doi.org/10.3390/mps7020018 - 20 Feb 2024
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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
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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.
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Open AccessProtocol
Crafting a Rigorous, Clinically Relevant Large Animal Model of Chronic Myocardial Ischemia: What Have We Learned in 20 Years?
by
Christopher R. Stone, Dwight D. Harris, Mark Broadwin, Meghamsh Kanuparthy, Sharif A. Sabe, Cynthia Xu, Jun Feng, M. Ruhul Abid and Frank W. Sellke
Methods Protoc. 2024, 7(1), 17; https://doi.org/10.3390/mps7010017 - 19 Feb 2024
Abstract
The past several decades have borne witness to several breakthroughs and paradigm shifts within the field of cardiovascular medicine, but one component that has remained constant throughout this time is the need for accurate animal models for the refinement and elaboration of the
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The past several decades have borne witness to several breakthroughs and paradigm shifts within the field of cardiovascular medicine, but one component that has remained constant throughout this time is the need for accurate animal models for the refinement and elaboration of the hypotheses and therapies crucial to our capacity to combat human disease. Numerous sophisticated and high-throughput molecular strategies have emerged, including rational drug design and the multi-omics approaches that allow extensive characterization of the host response to disease states and their prospective resolutions, but these technologies all require grounding within a faithful representation of their clinical context. Over this period, our lab has exhaustively tested, progressively refined, and extensively contributed to cardiovascular discovery on the basis of one such faithful representation. It is the purpose of this paper to review our porcine model of chronic myocardial ischemia using ameroid constriction and the subsequent myriad of physiological and molecular–biological insights it has allowed our lab to attain and describe. We hope that, by depicting our methods and the insight they have yielded clearly and completely—drawing for this purpose on comprehensive videographic illustration—other research teams will be empowered to carry our work forward, drawing on our experience to refine their own investigations into the pathogenesis and eradication of cardiovascular disease.
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(This article belongs to the Special Issue Feature Papers in Methods and Protocols 2023)
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Open AccessArticle
Assessment of the Integrity and Function of Human Term Placental Explants in Short-Term Culture
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Carolina López-Guzmán, Ana María García, Paula Marín and Ana María Vásquez
Methods Protoc. 2024, 7(1), 16; https://doi.org/10.3390/mps7010016 - 15 Feb 2024
Abstract
Human placental explants (HPEs) culture has generated significant interest as a valuable in vitro model for studying tissue functions in response to adverse conditions, such as fluctuations in oxygen levels, nutrient availability, exposure to pathogenic microorganisms, and toxic compounds. HPEs offers the advantage
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Human placental explants (HPEs) culture has generated significant interest as a valuable in vitro model for studying tissue functions in response to adverse conditions, such as fluctuations in oxygen levels, nutrient availability, exposure to pathogenic microorganisms, and toxic compounds. HPEs offers the advantage of replicating the intricate microenvironment and cell-to-cell communication involved in this critical and transient organ. Although HPEs culture conditions have been extensively discussed, a protocol for assessing the viability and function of HPEs during short-term culture has not been previously outlined. In this study, we have developed a short-term HPEs culture protocol, specifically up to 72 h, and have employed quantitative, semi-quantitative, and qualitative analyses to evaluate tissue viability and function over time. Under our standardized conditions, placental villi explants began to regain their structural properties (the integrity of the trophoblast and villous stroma) and the functionality of the HPEs (production of angiogenic, endocrine, and immunological factors) starting from 48 h of culture. This restoration ensures a suitable environment for several applications. The data presented here can be highly valuable for laboratories aiming to implement an HPEs model, whether in the process of standardization or seeking to enhance and optimize working conditions and timing with placental tissue.
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(This article belongs to the Section Biomedical Sciences and Physiology)
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Open AccessStudy Protocol
Pilot Study: Safety and Performance Validation of an Ingestible Medical Device for Collecting Small Intestinal Liquid in Healthy Volunteers
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Alexandre Tronel, Anne-Sophie Silvent, Elena Buelow, Joris Giai, Corentin Leroy, Marion Proust, Donald Martin, Audrey Le Gouellec, Thomas Soranzo and Nicolas Mathieu
Methods Protoc. 2024, 7(1), 15; https://doi.org/10.3390/mps7010015 - 04 Feb 2024
Abstract
The connection between imbalances in the human gut microbiota, known as dysbiosis, and various diseases has been well established. Current techniques for sampling the small intestine are both invasive for patients and costly for healthcare facilities. Most studies on human gut microbiome are
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The connection between imbalances in the human gut microbiota, known as dysbiosis, and various diseases has been well established. Current techniques for sampling the small intestine are both invasive for patients and costly for healthcare facilities. Most studies on human gut microbiome are conducted using faecal samples, which do not accurately represent the microbiome in the upper intestinal tract. A pilot clinical investigation, registered as NCT05477069 and sponsored by the Grenoble Alpes University Hospital, is currently underway to evaluate a novel ingestible medical device (MD) designed for collecting small intestinal liquids by Pelican Health. This study is interventional and monocentric, involving 15 healthy volunteers. The primary objective of the study is to establish the safety and the performance of the MD when used on healthy volunteers. Secondary objectives include assessing the device’s performance and demonstrating the difference between the retrieved sample from the MD and the corresponding faecal sample. Multi-omics analysis will be performed, including metagenomics, metabolomics, and culturomics. We anticipate that the MD will prove to be safe without any reported adverse effects, and we collected samples suitable for the proposed omics analyses in order to demonstrate the functionality of the MD and the clinical potential of the intestinal content.
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(This article belongs to the Section Biomedical Sciences and Physiology)
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Open AccessArticle
Correlation between Tooth Position Parameters and Apical Fenestration: A Cone-Beam Computed Tomography Study
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Carlos Henrique Ferrari, Lara Steffany de Carvalho, Caroline Trefiglio Rocha and Amjad Abu Hasna
Methods Protoc. 2024, 7(1), 14; https://doi.org/10.3390/mps7010014 - 02 Feb 2024
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This study aimed to assess the relationship between apical fenestration—a defect in the alveolar bone involving the root apex—and tooth position in all tooth groups, excluding the third molars, utilizing cone-beam computed tomography (CBCT) images. A total of 800 CBCT scans (400 maxillary
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This study aimed to assess the relationship between apical fenestration—a defect in the alveolar bone involving the root apex—and tooth position in all tooth groups, excluding the third molars, utilizing cone-beam computed tomography (CBCT) images. A total of 800 CBCT scans (400 maxillary and 400 mandibular) from patients undergoing various treatments were examined by a single professional (radiologist and endodontist). Statistical analyses, including the chi-square test or Fisher’s exact test, were conducted using R software 2.7.3 (R Foundation, Vienna, Austria). Results indicated a significant association (p ≤ 0.05) between apical fenestration and tooth position. In the upper teeth, apical fenestrations were notably present in the mesio-buccal (17.17%) and disto-buccal (11.07%) roots of the first molars. Conversely, apical fenestrations in the lower teeth were relatively less frequent. The study revealed a negative correlation between apical fenestration and mesial inclination, rotation, and extrusion in the upper teeth. However, a positive correlation was observed between apical fenestration and lingual inclination in the upper teeth. In conclusion, this study illuminates the distribution of apical fenestration and its correlation with tooth positions, offering insights into factors influencing this defect in dental anatomy. The findings enhance our understanding of nuanced relationships between tooth position and apical fenestration in the upper and lower dental arches.
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Open AccessProtocol
A Detailed Protocol for Constructing a Human Single-Chain Variable Fragment (scFv) Library and Downstream Screening via Phage Display
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Ziyi Liu, Dokyun Kim, Seokmin Kang and Jae U. Jung
Methods Protoc. 2024, 7(1), 13; https://doi.org/10.3390/mps7010013 - 01 Feb 2024
Abstract
The development of monoclonal antibodies (mAbs) represents a significant milestone in both basic research and clinical applications due to their target specificity and versatility in therapeutic and diagnostic applications. The innovative strategy of mAb screening, utilizing phage display, facilitates the in vitro screening
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The development of monoclonal antibodies (mAbs) represents a significant milestone in both basic research and clinical applications due to their target specificity and versatility in therapeutic and diagnostic applications. The innovative strategy of mAb screening, utilizing phage display, facilitates the in vitro screening of antibodies with high affinity to target antigens. The single-chain variable fragment (scFv) is a subset of mAb derivatives, known for its high binding affinity and smaller size—just one-third of that of human IgG. This report outlines a detailed and comprehensive procedure for constructing a scFv phagemid library derived from human patients, followed by screening via phage display affinity selection. The protocol utilizes 348 primer combinations spanning the entire human antibody repertoire to minimize sequence bias and maintain library diversity during polymerase chain reaction (PCR) for scFv generation, resulting in a library size greater than 1 × 108. Furthermore, we describe a high-throughput phage display screening protocol using enzyme-linked immunosorbent assay (ELISA) to evaluate more than 1200 scFv candidates. The generation of a highly diverse scFv library, coupled with the implementation of a phage display screening methodology, is expected to provide a valuable resource for researchers in pursuit of scFvs with high affinity for target antigens, thus advancing both research and clinical endeavors.
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(This article belongs to the Section Molecular and Cellular Biology)
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Open AccessArticle
Simplified Method for Agrobacterium-Mediated Genetic Transformation of Populus x berolinensis K. Koch
by
Vasiliy V. Pavlichenko and Marina V. Protopopova
Methods Protoc. 2024, 7(1), 12; https://doi.org/10.3390/mps7010012 - 26 Jan 2024
Abstract
The rapid advancement of genetic technologies has made it possible to modify various plants through both genetic transformation and gene editing techniques. Poplar, with its rapid in vitro growth and regeneration enabling high rates of micropropagation, has emerged as a model system for
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The rapid advancement of genetic technologies has made it possible to modify various plants through both genetic transformation and gene editing techniques. Poplar, with its rapid in vitro growth and regeneration enabling high rates of micropropagation, has emerged as a model system for the genetic transformation of woody plants. In this study, Populus × berolinensis K. Koch. (Berlin poplar) was chosen as the model organism due to its narrow leaves and spindle-shaped crown, which make it highly suitable for in vitro manipulations. Various protocols for the Agrobacterium-mediated transformation of poplar species have been developed to date. However, the genetic transformation procedures are often constrained by the complexity of the nutrient media used for plant regeneration and growth, which could potentially be simplified. Our study presents a cheaper, simplified, and relatively fast protocol for the Agrobacterium-mediated transformation of Berlin poplar. The protocol involved using internode sections without axillary buds as explants, which were co-cultivated in 10 µL droplets of bacterial suspension directly on the surface of a solid agar-based medium without rinsing and sterile paper drying after inoculation. We used only one regeneration Murashige and Skoogbased medium supplemented with BA (0.2 mg·L−1), TDZ (0.02 mg·L−1), and NAA (0.01 mg·L−1). Acetosyringone was not used as an induction agent for vir genes during the genetic transformation. Applying our protocol and using the binary plasmid pBI121 carrying the nptII selective and uidA reporter genes, we obtained the six transgenic lines of poplar. Transgenesis was confirmed through a PCR-based screening of kanamycin-selected regenerants for the presence of both mentioned genes, Sanger sequencing, and tests for detecting the maintained activity of both genes. The transformation efficiency, considering the 100 explants taken originally, was 6%.
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(This article belongs to the Collection Gene Editing)
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Open AccessTechnical Note
A Simplified Method for Calculating Surface Area of Mammalian Erythrocytes
by
Ion Udroiu
Methods Protoc. 2024, 7(1), 11; https://doi.org/10.3390/mps7010011 - 25 Jan 2024
Abstract
Knowledge of the geometric quantities of the erythrocyte is useful in several physiological studies, both for zoologists and veterinarians. While the diameter and volume (MCV) are easily obtained from observations of blood smears and complete blood count, respectively, the thickness and surface area
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Knowledge of the geometric quantities of the erythrocyte is useful in several physiological studies, both for zoologists and veterinarians. While the diameter and volume (MCV) are easily obtained from observations of blood smears and complete blood count, respectively, the thickness and surface area are instead much more difficult to measure. The precise description of the erythrocyte geometry is given by the equation of the oval of Cassini, but the formulas deriving from it are very complex, comprising elliptic integrals. In this article, three solids are proposed as models approximating the erythrocyte: sphere, cylinder and a spheroid with concave caps. The volumes and surface areas obtained with these models are compared to those effectively measured. The spheroid with concave caps gives the best approximation and can be used as a simple model to determine the erythrocyte surface area. With this model, a simple method that allows one to estimate the surface area by knowing only the diameter and MCV is proposed.
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(This article belongs to the Section Biomedical Sciences and Physiology)
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Open AccessArticle
An Ultra-High-Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry Method with Online Solid-Phase Extraction Sample Preparation for the High-Throughput and Sensitive Determination of Ostarine in Human Urine
by
Kristián Slíž, Juraj Piešťanský and Peter Mikuš
Methods Protoc. 2024, 7(1), 10; https://doi.org/10.3390/mps7010010 - 23 Jan 2024
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Ostarine is frequently misused as a selective androgen receptor modulator (SARM) in sports. Consequently, there is a pressing need for reliable and simple approaches to monitor its presence in biological systems. In this work, we developed a two-dimensional analytical method utilizing online solid-phase
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Ostarine is frequently misused as a selective androgen receptor modulator (SARM) in sports. Consequently, there is a pressing need for reliable and simple approaches to monitor its presence in biological systems. In this work, we developed a two-dimensional analytical method utilizing online solid-phase extraction (online-SPE) in conjunction with ultra-high-performance liquid chromatography and tandem mass spectrometry (triple quadrupole). This automated 2D separation approach is characterized by minimum manual steps in the sample preparation (only dilute-and-shoot), reflecting high sample throughput and the reliability of analytical data. It provides favorable performance parameters, including a limit of detection of 0.5 pg/mL, high accuracy (relative error = 1.6–7.5%), precision (relative standard deviation = 0.8–4.5%), and sensitivity. Additionally, it demonstrates excellent linearity (r2 = 0.9999) in the calibration range of 0.05 to 25 ng/mL and robustness, with no carryover effects observed. This comparative study revealed a two-decadic-order-lower LOD of the SPE-UHPLC-MS/MS method to the corresponding UHPLC-MS/MS method and the lowest one in the group of currently published LC-MS methods. The World Anti-Doping Agency screening and confirmation criteria were met through the analysis of spiked urine samples from ten healthy volunteers. Accordingly, the proposed method is suitable for routine use in antidoping laboratories.
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Open AccessProtocol
Assessment of Nuclear Gem Quantity for Evaluating the Efficacy of Antisense Oligonucleotides in Spinal Muscular Atrophy Cells
by
Haya Al-Hilal, Marianna Maretina, Anna Egorova, Andrey Glotov and Anton Kiselev
Methods Protoc. 2024, 7(1), 9; https://doi.org/10.3390/mps7010009 - 19 Jan 2024
Abstract
Spinal muscular atrophy is a neuromuscular disorder caused by mutations in both copies of the survival motor neuron gene 1 (SMN1), which lead to reduction in the production of the SMN protein. Currently, there are several therapies that have been approved
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Spinal muscular atrophy is a neuromuscular disorder caused by mutations in both copies of the survival motor neuron gene 1 (SMN1), which lead to reduction in the production of the SMN protein. Currently, there are several therapies that have been approved for SMA, with many more undergoing active research. While various biomarkers have been proposed for assessing the effectiveness of SMA treatment, a universally accepted one still has not been identified. This study aimed to describe a fast and reliable method using the number of gems in cell nuclei as a potential tool for assessment of splicing correction of oligonucleotide efficacy in SMA cells. To gain insight into whether the number of gems in cell nuclei varies based on their SMN genotype and whether the increase in gem number is associated with therapeutic response, we utilized fibroblast cell cultures obtained from a patient with SMA type II and from a healthy individual. We discovered a remarkable difference in the number of gems found in the nuclei of these cells, specifically when counting gems per 100 nuclei. The SMA fibroblasts treated with antisense oligonucleotide showed beneficial effects in correcting the abnormal splicing of SMN2 exon 7. It was observed that there was a significant increase in the number of gems in the treated cells compared to the intact SMA cells. The results obtained significantly correlate with an increase of full-length SMN transcript sharing. Based on our findings, we propose using the quantity of gems as a reliable biomarker for SMA drug development.
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(This article belongs to the Section Molecular and Cellular Biology)
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Open AccessProtocol
Making the Most of Lateral Flow Immunochromatographic Tests: An Efficient Protocol to Recover DNA
by
Sara C. Zapico and Gabriela Roca
Methods Protoc. 2024, 7(1), 8; https://doi.org/10.3390/mps7010008 - 15 Jan 2024
Abstract
Lateral flow immunochromatographic (LFI) tests are widely used in both biomedical and forensic sciences for different applications. In forensic sciences, their main use is to detect body fluids at crime scenes. However, there are situations in which the amount of potential biological evidence
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Lateral flow immunochromatographic (LFI) tests are widely used in both biomedical and forensic sciences for different applications. In forensic sciences, their main use is to detect body fluids at crime scenes. However, there are situations in which the amount of potential biological evidence is so low that DNA extraction is favored with respect to the identification of body fluids. Here, an efficient and quick protocol is presented to integrate the detection of body fluids through LFI with DNA extraction from a sample swab and buffer, providing a complete characterization of the biological evidence. This protocol is a modification of a general DNA extraction silica-based kit, whose main application is for blood and tissues. Thus, it could be carried out in different settings (forensic labs, hospitals, other testing labs) without the necessity of buying a specific kit for swabs. The validation of this protocol is supported by the results presented here and previous publications from our group, obtaining DNA in good quantity and with good quality. This proves the potential application of the protocol in both forensic scenarios, to fully characterize biological evidence, and biomedical settings, to molecularly confirm the results of LFI tests.
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(This article belongs to the Section Molecular and Cellular Biology)
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Open AccessArticle
Mapping m6A Sites on HIV-1 RNA Using Oligonucleotide LC-MS/MS
by
Alice Baek, Asif Rayhan, Ga-Eun Lee, Sarah Golconda, Hannah Yu, Shihyoung Kim, Patrick A. Limbach, Balasubrahmanyam Addepalli and Sanggu Kim
Methods Protoc. 2024, 7(1), 7; https://doi.org/10.3390/mps7010007 - 10 Jan 2024
Abstract
The biological significance of chemical modifications to the ribonucleic acid (RNA) of human immunodeficiency virus type-1 (HIV-1) has been recognized. However, our understanding of the site-specific and context-dependent roles of these chemical modifications remains limited, primarily due to the absence of nucleotide-resolution mapping
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The biological significance of chemical modifications to the ribonucleic acid (RNA) of human immunodeficiency virus type-1 (HIV-1) has been recognized. However, our understanding of the site-specific and context-dependent roles of these chemical modifications remains limited, primarily due to the absence of nucleotide-resolution mapping of modification sites. In this study, we present a method for achieving nucleotide-resolution mapping of chemical modification sites on HIV-1 RNA using liquid chromatography and tandem mass spectrometry (LC–MS/MS). LC–MS/MS, a powerful tool capable of directly analyzing native RNAs, has proven effective for mapping RNA modifications in small RNA molecules, including ribosomal RNA and transfer RNA. However, longer RNAs have posed challenges, such as the 9 Kb HIV-1 virion RNA, due to the complexity of and ambiguity in mass differences among RNase T1-cleaved RNA fragments in LC-MS/MS data. Here, we introduce a new target RNA enrichment method to isolate small local RNA fragments of HIV-1 RNA that potentially harbor site-specific N6-methyladenosine (m6A) modifications. In our initial trial, we used target-specific DNA probes only and encountered insufficient RNA fragmentation due to inefficient S1 digestion near the target site. Recognizing that inefficient S1 digestion by HIV-1 RNA is likely due to the formation of secondary structures in proximity to the target site, we designed multiple DNA probes annealing to various sites of HIV-1 RNA to better control the structures of RNA substrates for S1 digestion. The use of these non-target DNA probes significantly improved the isolation of more homogeneous target RNA fragments of approximately 50 bases in length. Oligonucleotide LC-MS/MS analysis of these isolated target RNA fragments successfully separated and detected both m6A-methylated and non-methylated oligomers at the two m6A-predicted sites. The principle of this new target enrichment strategy holds promise and should be broadly applicable to the analysis of any lengthy RNA that was previously deemed infeasible for investigation using oligonucleotide LC-MS/MS.
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(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
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Optimal Selection of Sampling Points within Sewer Networks for Wastewater-Based Epidemiology Applications
by
Yao Yao, Yibo Zhu, Regina Nogueira, Frank Klawonn and Markus Wallner
Methods Protoc. 2024, 7(1), 6; https://doi.org/10.3390/mps7010006 - 05 Jan 2024
Abstract
Wastewater-based epidemiology (WBE) has great potential to monitor community public health, especially during pandemics. However, it faces substantial hurdles in pathogen surveillance through WBE, encompassing data representativeness, spatiotemporal variability, population estimates, pathogen decay, and environmental factors. This paper aims to enhance the reliability
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Wastewater-based epidemiology (WBE) has great potential to monitor community public health, especially during pandemics. However, it faces substantial hurdles in pathogen surveillance through WBE, encompassing data representativeness, spatiotemporal variability, population estimates, pathogen decay, and environmental factors. This paper aims to enhance the reliability of WBE data, especially for early outbreak detection and improved sampling strategies within sewer networks. The tool implemented in this paper combines a monitoring model and an optimization model to facilitate the optimal selection of sampling points within sewer networks. The monitoring model utilizes parameters such as feces density and average water consumption to define the detectability of the virus that needs to be monitored. This allows for standardization and simplicity in the process of moving from the analysis of wastewater samples to the identification of infection in the source area. The entropy-based model can select optimal sampling points in a sewer network to obtain the most specific information at a minimum cost. The practicality of our tool is validated using data from Hildesheim, Germany, employing SARS-CoV-2 as a pilot pathogen. It is important to note that the tool’s versatility empowers its extension to monitor other pathogens in the future.
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(This article belongs to the Section Public Health Research)
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