Journal Description
Methods and Protocols
Methods and Protocols
is an international, peer-reviewed, open access journal aiming to establish and describe new experimental techniques in the fields of Life Sciences, Chemistry, and Biomedical Sciences, published bimonthly online by MDPI.
- Open Access— free for readers, with article processing charges (APC) paid by authors or their institutions.
- High Visibility: indexed within Scopus, ESCI (Web of Science), PubMed, PMC, CAPlus / SciFinder, and other databases.
- Journal Rank: CiteScore - Q2 (Biochemistry, Genetics and Molecular Biology (miscellaneous))
- Rapid Publication: manuscripts are peer-reviewed and a first decision is provided to authors approximately 24.9 days after submission; acceptance to publication is undertaken in 3.7 days (median values for papers published in this journal in the first half of 2024).
- Recognition of Reviewers: reviewers who provide timely, thorough peer-review reports receive vouchers entitling them to a discount on the APC of their next publication in any MDPI journal, in appreciation of the work done.
Impact Factor:
2.3 (2023);
5-Year Impact Factor:
2.2 (2023)
Latest Articles
A Scalable Method to Fabricate 2D Hydrogel Substrates for Mechanobiology Studies with Independent Tuning of Adhesiveness and Stiffness
Methods Protoc. 2024, 7(5), 75; https://doi.org/10.3390/mps7050075 (registering DOI) - 26 Sep 2024
Abstract
Mechanical signals from the extracellular matrix are crucial in guiding cellular behavior. Two-dimensional hydrogel substrates for cell cultures serve as exceptional tools for mechanobiology studies because they mimic the biomechanical and adhesive characteristics of natural environments. However, the interdisciplinary knowledge required to synthetize
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Mechanical signals from the extracellular matrix are crucial in guiding cellular behavior. Two-dimensional hydrogel substrates for cell cultures serve as exceptional tools for mechanobiology studies because they mimic the biomechanical and adhesive characteristics of natural environments. However, the interdisciplinary knowledge required to synthetize and manipulate these biomaterials typically restricts their widespread use in biological laboratories, which may not have the material science expertise or specialized instrumentation. To address this, we propose a scalable method that requires minimal setup to produce 2D hydrogel substrates with independent modulation of the rigidity and adhesiveness within the range typical of natural tissues. In this method, norbornene-terminated 8-arm polyethylene glycol is stoichiometrically functionalized with RGD peptides and crosslinked with a di-cysteine terminated peptide via a thiol–ene click reaction. Since the synthesis process significantly influences the final properties of the hydrogels, we provide a detailed description of the chemical procedure to ensure reproducibility and high throughput results. We demonstrate examples of cell mechanosignaling by monitoring the activation state of the mechanoeffector proteins YAP/TAZ. This method effectively dissects the influence of biophysical and adhesive cues on cell behavior. We believe that our procedure will be easily adopted by other cell biology laboratories, improving its accessibility and practical application.
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(This article belongs to the Section Molecular and Cellular Biology)
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Open AccessProtocol
Co-Culture of Gut Bacteria and Metabolite Extraction Using Fast Vacuum Filtration and Centrifugation
by
Asha Guraka, Richard Duff, Joe Waldron, Gyanendra Tripathi and Ali Kermanizadeh
Methods Protoc. 2024, 7(5), 74; https://doi.org/10.3390/mps7050074 - 19 Sep 2024
Abstract
This protocol describes a robust method for the extraction of intra and extracellular metabolites of gut bacterial mono and co-cultures. In recent years, the co-culture techniques employed in the field of microbiology have demonstrated significant importance in regard to understanding cell–cell interactions, cross-feeding,
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This protocol describes a robust method for the extraction of intra and extracellular metabolites of gut bacterial mono and co-cultures. In recent years, the co-culture techniques employed in the field of microbiology have demonstrated significant importance in regard to understanding cell–cell interactions, cross-feeding, and the metabolic interactions between different bacteria, fungi, and microbial consortia which enable the mimicking of complex co-habitant conditions. This protocol highlights a robust reproducible physiologically relevant culture and extraction protocol for the co-culture of gut bacterium. The novel extraction steps are conducted without using quenching and cell disruption through bead-cell methods, freeze–thaw cycles, and sonication, which tend to affect the physical and biochemical properties of intracellular metabolites and secretome. The extraction procedure of inoculated bacterial co-cultures and monocultures use fast vacuum filtration and centrifugation. The extraction methodology is fast, effective, and robust, requiring 4 h to complete.
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(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
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Open AccessProtocol
Microneedle System Coated with Hydrogels Containing Protoporphyrin IX for Potential Application in Pharmaceutical Technology
by
Beata Czarczynska-Goslinska, Tomasz Goslinski, Agata Roszak, Anna Froelich, Piotr Szyk, Dariusz T. Mlynarczyk, Lukasz Sobotta, Irena Budnik, Oliwia Kordyl and Tomasz Osmałek
Methods Protoc. 2024, 7(5), 73; https://doi.org/10.3390/mps7050073 - 13 Sep 2024
Abstract
The article aims to outline the potential of treating malignant skin cancer with microneedles covered with polymer layers containing a photosensitizer—protoporphyrin IX disodium salt (PPIX). The usefulness of stereolithography (SLA), which is a form of 3D-printing technology, for the preparation of a microneedle
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The article aims to outline the potential of treating malignant skin cancer with microneedles covered with polymer layers containing a photosensitizer—protoporphyrin IX disodium salt (PPIX). The usefulness of stereolithography (SLA), which is a form of 3D-printing technology, for the preparation of a microneedle system with protoporphyrin IX was demonstrated. The SLA method allowed for pyramid-shaped microneedles to be printed that were covered with three different 0.1% PPIX hydrogels based on sodium alginate, xanthan, and poloxamer. Rheological tests and microscopic analysis of the hydrogels were performed. Microneedles coated with two layers of poloxamer-based hydrogel containing 0.1% PPIX were subjected to release tests in Franz diffusion cells. The release profile of PPIX initially increased and then remained relatively constant. The amount of substance released after a four-hour test in three Franz cells was 0.2569 ± 0.0683 mg/cm2. Moreover, the acute toxicity of this type of microneedle was assessed using the Microtox system. The obtained results show the usefulness of further development studies on microneedles as carriers of photosensitizing agents.
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(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
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Generating Cytokines and Growth Factors for Functional Activity: Feasibility of Method Using MIF Protein
by
Hiba Osmani, Ishrya Sharma and Shannon Moonah
Methods Protoc. 2024, 7(5), 72; https://doi.org/10.3390/mps7050072 - 12 Sep 2024
Abstract
Cytokines and growth factors are signaling molecules that regulate a variety of biological processes. Understanding their role is essential for basic research and clinical utilization. Thus, cytokines and growth factors are widely used throughout research labs in a significant number of applications. Additionally,
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Cytokines and growth factors are signaling molecules that regulate a variety of biological processes. Understanding their role is essential for basic research and clinical utilization. Thus, cytokines and growth factors are widely used throughout research labs in a significant number of applications. Additionally, genetic polymorphisms result in variant forms of cytokines and growth factors, which can alter their function. Becoming more common, researchers will need to generate these important proteins and their variants themselves in functional forms for activity studies. The expression systems used to generate these proteins can have a major impact on their function. In some instances, post-translational modifications are needed to produce a functionally active protein, which can only be conducted using eukaryotic expression systems. Ideally, for functional relevance, a human expression system should be used for human-related research and applications. Most human cell-based expression systems primarily use HEK (Human Embryonic Kidney) cells; however, relying on just one cell type can lead to several issues, considering the variety of proteins derived from various cell sources. Here, we provide a protocol to effectively and efficiently generate functional recombinant proteins, taking into consideration the diverse range of proteins from different cell types throughout the human body.
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(This article belongs to the Section Molecular and Cellular Biology)
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Open AccessProtocol
FAMS—A Targeted Fatty Acid Mass Spectrometry Method for Monitoring Free Fatty Acids from Polysorbate Hydrolysis
by
Anja Bathke, Sina Hoelterhoff, Jan Wendler, Inn H. Yuk and Christian H. Bell
Methods Protoc. 2024, 7(5), 71; https://doi.org/10.3390/mps7050071 - 7 Sep 2024
Abstract
Polysorbates are the predominant surfactants used to stabilize protein formulations. Unfortunately, polysorbates can undergo hydrolytic degradation, which releases fatty acids that can accumulate to form visible particles. The detection and quantitation of these fatty acid degradation products are critical for assessing the extent
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Polysorbates are the predominant surfactants used to stabilize protein formulations. Unfortunately, polysorbates can undergo hydrolytic degradation, which releases fatty acids that can accumulate to form visible particles. The detection and quantitation of these fatty acid degradation products are critical for assessing the extent of polysorbate degradation and the associated risks of particle formation. We previously developed a user-friendly mass spectrometric method called Fatty Acids by Mass Spectrometry (FAMS) to quantify the free fatty acids. The FAMS method was validated according to ICH Q2 (R1) guidelines and is suitable for a wide range of products, buffers and protein concentrations. The end-to-end workflow can be automated from sample preparation to data analysis. To broaden method accessibility, the QDa detector selected for fatty acid measurement does not require specific mass spectrometry experience. We provide here a detailed procedure for both manual and automated sample preparation for high-throughput analysis. In addition, we highlight in this protocol the critical operational details, procedural watchouts and troubleshooting tips to support the successful execution of this method in another laboratory.
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(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
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Open AccessStudy Protocol
The Burden of Sleep/Wake Disorders: Excessive Daytime Sleepiness and Insomnia Project
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Marina Tüzün, Ulf Kallweit, Stefan Seidel, Olga Endrich, Sven Trelle, Maurizio A. Leone, Oliviero Bruni, Richard Dodel, Maria Konti, Maria Lolich, Elisabetta Pupillo, Dauren Ramankulov, Luca Vignatelli, Carla Meyer-Massetti, Markus Schmidt and Claudio L. A. Bassetti
Methods Protoc. 2024, 7(5), 70; https://doi.org/10.3390/mps7050070 - 6 Sep 2024
Abstract
Excessive daytime sleepiness (EDS) and insomnia (IN) complaints represent the most common sleep/wake disorders. Currently, the specific needs of these patients and their relatives, as well as the overall socio-economic burden of IN and EDS remains widely unexplored. This pilot study to be
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Excessive daytime sleepiness (EDS) and insomnia (IN) complaints represent the most common sleep/wake disorders. Currently, the specific needs of these patients and their relatives, as well as the overall socio-economic burden of IN and EDS remains widely unexplored. This pilot study to be carried out in Switzerland is a retro- and prospective, national, one-center cohort observational study for the systematic evaluation of the burden of EDS and IN and its evolution 12 months after the first assessment. Patient recruitment will be organized through 7–8 primary care providers (primary/general care practitioners and pharmacies). Primary outcomes are the prevalence of EDS/IN in the primary care setting and the association between EDS/IN with health-related quality of life (QOL) as assessed with the established instruments. Secondary outcomes are the association between EDS/IN with the presence of comorbidities, number of injuries/accidents, and number of sick/leave days for the subgroup of working subjects. Calculation of direct per-patient costs will be undertaken to analyze the economic implications of sleep/wake disorders, providing valuable insights into the financial burden experienced by affected individuals within the healthcare system. This research will provide information on the feasibility of such a study and inform on aspects of the QOL most associated with EDS/IN. Based on this pilot project, a European multicenter study on the burden of sleep/wake disorders will be conducted by the European Academy of Neurology.
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(This article belongs to the Section Public Health Research)
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Open AccessArticle
CO2-Free On-Stage Incubator for Live Cell Imaging of Cholangiocarcinoma Cell Migration on Microfluidic Device
by
Shahab Ud Din, Puey Ounjai, Arthit Chairoungdua and Werasak Surareungchai
Methods Protoc. 2024, 7(5), 69; https://doi.org/10.3390/mps7050069 - 4 Sep 2024
Abstract
Long-term live cell imaging requires sophisticated and fully automated commercial-stage incubators equipped with specified inverted microscopes to regulate temperature, CO2 content, and humidity. In this study, we present a CO2-free on-stage incubator specifically designed for use across various cell culture
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Long-term live cell imaging requires sophisticated and fully automated commercial-stage incubators equipped with specified inverted microscopes to regulate temperature, CO2 content, and humidity. In this study, we present a CO2-free on-stage incubator specifically designed for use across various cell culture platforms, enabling live cell imaging applications. A simple and transparent incubator was fabricated from acrylic sheets to be easily placed on the stages of most inverted microscopes. We successfully performed live-cell imaging of cholangiocarcinoma (CCA) cells and HeLa cell dynamics in both 2D and 3D microenvironments over three days. We also analyzed directed cell migration under high serum induction within a microfluidic device. Interesting phenomena such as “whole-colony migration”, “novel type of collective cell migration” and “colony formation during cell and colony migration” are reported here for the first time, to the best of our knowledge. These phenomena may improve our understanding of the nature of cell migration and cancer metastasis.
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(This article belongs to the Section Biomedical Sciences and Physiology)
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Open AccessArticle
Optimization of Methodologies to Study Freeze/Thaw Processes in Drug Substance Bottles
by
Sarah S. Peláez, Hanns-Christian Mahler, Jörg Huwyler and Andrea Allmendinger
Methods Protoc. 2024, 7(5), 68; https://doi.org/10.3390/mps7050068 - 4 Sep 2024
Cited by 1
Abstract
Biological drug substance (DS) is often frozen to enhance storage stability, prolong shelf life, and increase flexibility during manufacturing. However, the freezing and thawing (F/T) of bulk DS at the manufacturing scale can impact product quality as a result of various critical conditions,
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Biological drug substance (DS) is often frozen to enhance storage stability, prolong shelf life, and increase flexibility during manufacturing. However, the freezing and thawing (F/T) of bulk DS at the manufacturing scale can impact product quality as a result of various critical conditions, including cryo-concentration during freezing, which are influenced, among other things, by product-independent process parameters (e.g., container type, fill level, F/T equipment, and protocols). In this article, we report the optimization of two major methodologies to study product-independent process parameters in DS bottles at the manufacturing scale, namely the recording of temperature profiles and liquid sampling after thawing to quantify the concentration gradients in the solution. We report experimentally justified measuring positions for temperature recordings, especially for the selection of the last point to freeze position, and highlight the implementation of camera-assisted inspection to determine the last point to thaw and the actual thawing time. In particular, we provide, for the first time, a detailed description of the technical implementation of these two measuring set-ups. Based on the reported case studies, we recommend choosing relevant measuring positions as a result of initial equipment characterization, resulting in a resource-conscious study set-up.
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(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
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Comparison of Antigen Retrieval Methods for Immunohistochemical Analysis of Cartilage Matrix Glycoproteins Using Cartilage Intermediate Layer Protein 2 (CILP-2) as an Example
by
Taavi Torga, Siim Suutre, Kalle Kisand, Marina Aunapuu and Andres Arend
Methods Protoc. 2024, 7(5), 67; https://doi.org/10.3390/mps7050067 - 24 Aug 2024
Abstract
The aim of this study was to compare different antigen retrieval methods to improve the outcome of immunohistochemistry (IHC) performed on osteoarthritic (OA) cartilage obtained from total knee replacement operation. A voluminous and dense extracellular matrix of articular cartilage inhibits antibody penetration, and
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The aim of this study was to compare different antigen retrieval methods to improve the outcome of immunohistochemistry (IHC) performed on osteoarthritic (OA) cartilage obtained from total knee replacement operation. A voluminous and dense extracellular matrix of articular cartilage inhibits antibody penetration, and therefore, proteins present at low concentrations and masked during fixation may need antigen retrieval to enhance an IHC outcome. We focused on the IHC detection of a minor but diagnostically promising cartilage glycoprotein, CILP-2 (cartilage intermediate layer protein 2), to demonstrate the effect of four different protocols: (1) heat-induced epitope retrieval (HIER), (2) proteolytic-induced epitope retrieval applying proteinase K and hyaluronidase (PIER), (3) HIER combined with PIER, and (4) no antigen retrieval (control). A semi-quantitative staining assessment based on the CILP-2 staining extent was applied. Out of the tested antigen retrieval protocols, the best CILP-2 IHC staining results were achieved by PIER. Combining PIER with HIER did not improve CILP-2 staining in the given experimental setting. Rather the opposite, the application of heat reduced the positive effect of PIER on CILP-2 staining and resulted in the frequent detachment of sections from the slides. Our findings emphasize the need for proper adaptation of antigen retrieval protocols for IHC to maximize the quantitative evaluation of minor matrix proteins in OA articular cartilage samples.
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(This article belongs to the Section Biomedical Sciences and Physiology)
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Open AccessTechnical Note
Nanoparticle Lysis of Cryptosporidium Oocysts
by
Ameya Vaidya, Claire Bankier, Helinor Johnston and Helen Bridle
Methods Protoc. 2024, 7(5), 66; https://doi.org/10.3390/mps7050066 - 23 Aug 2024
Abstract
The extraction of DNA from Cryptosporidium oocysts is challenging due to the robust oocyst wall. Nanoparticles have been applied to disinfect Cryptosporidium oocysts; here, we demonstrate the utilisation of nanoparticles to disrupt the oocyst wall to enable sporozoite lysis and detection via PCR.
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The extraction of DNA from Cryptosporidium oocysts is challenging due to the robust oocyst wall. Nanoparticles have been applied to disinfect Cryptosporidium oocysts; here, we demonstrate the utilisation of nanoparticles to disrupt the oocyst wall to enable sporozoite lysis and detection via PCR. Both silver and zinc oxide nanoparticles are investigated under different conditions and compared to existing techniques. Zinc oxide nanoparticles are shown to be as effective as freeze–thaw methods, suggesting that a nanoparticle lysis approach offers a viable alternative to existing methods.
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(This article belongs to the Section Molecular and Cellular Biology)
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Open AccessProtocol
Parasitological Examination of the Digestive System of Wild Boar from a Practical Point of View—Endoparasitological Sampling under Field Conditions
by
Csaba Farkas, Alexandra Juhász, Balázs Fekete and Borisz Egri
Methods Protoc. 2024, 7(4), 65; https://doi.org/10.3390/mps7040065 - 21 Aug 2024
Abstract
From 2015 to 2023, we conducted a comprehensive study in the 11,893-hectare hunting area managed by the Marcal-Bitvaközi Hunting Company, characterised by its substantial wild boar population. The research was carried out across various settings, including a free-range wild boar garden during large-scale
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From 2015 to 2023, we conducted a comprehensive study in the 11,893-hectare hunting area managed by the Marcal-Bitvaközi Hunting Company, characterised by its substantial wild boar population. The research was carried out across various settings, including a free-range wild boar garden during large-scale hunts and free-living areas during individual hunts. We examined 216 wild boars in total, with 173 individuals from free-living areas and 43 from free-range areas. Throughout the sample collection process, we encountered numerous technical challenges that are infrequently detailed in the professional literature, often mentioned only tangentially. This oversight in existing publications neglects the significance of addressing field sampling difficulties, which are crucial for ensuring the precision and accuracy of research. This paper details the equipment requirements, sampling methodologies, and practical solutions to streamline fieldwork. While our primary focus was on endoparasitic infections of the stomach and small intestine, the described methodologies and findings are broadly applicable to research involving all internal organs.
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(This article belongs to the Special Issue Feature Papers in Methods and Protocols 2024)
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Full Validation and Application to Clinical Research of a High-Performance Liquid Chromatography Method for the Assessment of Urinary 3-Indoxyl Sulfate in Pediatric Patients with Hematopoietic Stem Cell Transplant
by
Christian Ezequiel Olivetti, María Florencia Fernández, Jana Stojanova, Silvina Ruvinsky, Andrea Mangano and Paula Schaiquevich
Methods Protoc. 2024, 7(4), 64; https://doi.org/10.3390/mps7040064 - 19 Aug 2024
Abstract
3-indoxyl sulfate (3-IS) results from a hepatic transformation of indole, a tryptophan degradation product produced by commensal gut bacteria. The metabolite has shown promise as a biomarker of dysbiosis and clinical outcomes following hematopoietic stem cell transplant (HSCT) in adults. Nonetheless, there is
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3-indoxyl sulfate (3-IS) results from a hepatic transformation of indole, a tryptophan degradation product produced by commensal gut bacteria. The metabolite has shown promise as a biomarker of dysbiosis and clinical outcomes following hematopoietic stem cell transplant (HSCT) in adults. Nonetheless, there is a paucity of data regarding microbiome health and outcomes in the pediatric HSCT setting. We developed and thoroughly validated an affordable high-performance liquid chromatography/fluorescence detector (HPLC-FLD) method to quantify 3-IS in urine for use in the pediatric setting. Chromatographic separation was achieved on a C18 column (250 × 4.6 mm × 5 μm) with a mobile phase consisting of pH 4.0 acetic acid-triethylamine buffer and acetonitrile (88:12, v/v), eluted isocratically at 1 mL/min. 3-IS fluorescence detection was set at excitation/emission of 280 and 375, respectively. The method was fully validated according to FDA-specified limits including selectivity, linearity (0.10 to 10.00 mg/L, r2 > 0.997), intra- and inter-day accuracy, and precision. 3-IS stability was confirmed after three freeze–thaw cycles, for short- and medium-term on a benchtop and at 4 °C and for long-term up to 60 days at −20 °C. The validated method was used to quantify 3-IS in urine samples from HSCT pediatric patients.
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(This article belongs to the Special Issue Feature Papers in Methods and Protocols 2024)
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Open AccessProtocol
Evaluation of an Italian Population-Based Programme for Risk Assessment and Genetic Counselling and Testing for BRCA1/2-Related Hereditary Breast and Ovarian Cancer after 10 Years of Operation: An Observational Study Protocol
by
Stefano Ferretti, Priscilla Sassoli de Bianchi, Debora Canuti, Cinzia Campari, Laura Cortesi, Valentina Arcangeli, Elena Barbieri, Cecilia D’Aloia, Rita Danesi, Pierandrea De Iaco, Margherita De Lillo, Laura Lombardo, Gabriella Moretti, Antonino Musolino, Dante Palli, Caterina Palmonari, Mila Ravegnani, Alfredo Tafà, Alessandra Tononi, Daniela Turchetti, Claudio Zamagni, Valentina Zampiga, Lauro Bucchi and the HBOC Study Groupadd
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Methods Protoc. 2024, 7(4), 63; https://doi.org/10.3390/mps7040063 - 13 Aug 2024
Abstract
Hereditary breast/ovarian cancer (HBOC) syndrome is caused by the inheritance of monoallelic germline BRCA1/2 gene mutations. If BRCA1/2 mutation carriers are identified before the disease develops, effective actions against HBOC can be taken, including intensive screening, risk-reducing mastectomy and salpingo-oophorectomy, and risk-reducing medications.
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Hereditary breast/ovarian cancer (HBOC) syndrome is caused by the inheritance of monoallelic germline BRCA1/2 gene mutations. If BRCA1/2 mutation carriers are identified before the disease develops, effective actions against HBOC can be taken, including intensive screening, risk-reducing mastectomy and salpingo-oophorectomy, and risk-reducing medications. The Italian National Prevention Plan mandates the creation of regional BRCA genetic testing programmes. So far, however, only informal data have been reported on their implementation. We have designed a study aimed at evaluating the results of a population-based programme for risk assessment and genetic counselling and testing for BRCA1/2-related HBOC that is underway in the Emilia-Romagna region (northern Italy). The programme—which is entirely free—includes basic screening with an estimate of the likelihood of carrying a BRCA1/2 mutation using a familial risk assessment tool, a closer examination of women with suspected risk increase, an assessment of the need for further genetic counselling and, if needed, genetic testing and risk-reducing interventions. In this paper, the design of the programme and the protocol of the study are presented. The study has an observational, historical cohort design. Eligible are the women found to be at an increased risk of HBOC (profile 3 women). The main objectives are (i) to determine the precision of the programme in measuring the level of risk of HBOC for profile 3 women; (ii) to determine the characteristics of profile 3 women and their association with the risk management strategy chosen; (iii) to compare the age at onset, histologic type, tumour stage, molecular subtype, and prognosis of breast/ovarian cancers observed in the cohort of profile 3 women with the features of sporadic cancers observed in the general female population; (iv) to determine the level and the determinants of adherence to recommendations; and (v) to determine the appropriateness and timing of risk-reducing surgery and medications. Investigating the quality and results of the programme is necessary because the best practices in risk assessment and genetic counselling and testing for BRCA1/2-related cancer and the challenges they encounter should be identified and shared. The study has the potential to provide sound empirical evidence for the factors affecting the effectiveness of this type of service.
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(This article belongs to the Section Biomedical Sciences and Physiology)
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Open AccessArticle
A Mathematical Model of Pressure Ulcer Formation to Facilitate Prevention and Management
by
Ioannis G. Violaris, Konstantinos Kalafatakis, Nikolaos Giannakeas, Alexandros T. Tzallas and Markos Tsipouras
Methods Protoc. 2024, 7(4), 62; https://doi.org/10.3390/mps7040062 - 13 Aug 2024
Abstract
Pressure ulcers are a frequent issue involving localized damage to the skin and underlying tissues, commonly arising from prolonged hospitalization and immobilization. This paper introduces a mathematical model designed to elucidate the mechanics behind pressure ulcer formation, aiming to predict its occurrence and
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Pressure ulcers are a frequent issue involving localized damage to the skin and underlying tissues, commonly arising from prolonged hospitalization and immobilization. This paper introduces a mathematical model designed to elucidate the mechanics behind pressure ulcer formation, aiming to predict its occurrence and assist in its prevention. Utilizing differential geometry and elasticity theory, the model represents human skin and simulates its deformation under pressure. Additionally, a system of ordinary differential equations is employed to predict the outcomes of these deformations, estimating the cellular death rate in skin tissues and underlying layers. The model also incorporates changes in blood flow resulting from alterations in skin geometry. This comprehensive approach provides new insights into the optimal bed surfaces required to prevent pressure ulcers and offers a general predictive method to aid healthcare personnel in making informed decisions for at-risk patients. Compared to existing models in the literature, our model delivers a more thorough prediction method that aligns well with current data. It can forecast the time required for an immobilized individual to develop an ulcer in various body parts, considering different initial health conditions and treatment strategies.
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(This article belongs to the Section Biomedical Sciences and Physiology)
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The Sri Lanka Mother and Newborn Growth (S-MaNGro) Cohort: Protocol of a Nationwide Prospective Study
by
Malshani Lakshika Pathirathna, Megumi Haruna, Satoshi Sasaki, Kaori Yonezawa, Yuriko Usui and Yasuhiro Hagiwara
Methods Protoc. 2024, 7(4), 61; https://doi.org/10.3390/mps7040061 - 6 Aug 2024
Abstract
Perinatal cohort studies with a prospective longitudinal design are critical for determining the effects of early-life exposures on offspring’s health outcomes. The Sri Lanka Mother and Newborn Growth cohort study aims to investigate the impact of maternal nutritional and psychosocial factors on newborns’
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Perinatal cohort studies with a prospective longitudinal design are critical for determining the effects of early-life exposures on offspring’s health outcomes. The Sri Lanka Mother and Newborn Growth cohort study aims to investigate the impact of maternal nutritional and psychosocial factors on newborns’ birth weight in the Sri Lankan context. This paper presents the methodology of participant recruitment, follow-ups, an overview of measurements, and planned data analyses. This study included a nationally representative sample of Sri Lankan pregnant women recruited in their first trimester of pregnancy. Follow-up assessments were conducted once during the second and third trimesters of pregnancy and after the baby’s birth, prospectively tracking the women’s dietary intake, mental health, hemoglobin concentrations, and gestational weight gain data. Once the participants delivered their babies, the data on gestational age, sex of the newborn, birth weight, length and occipitofrontal circumference at birth, and mode of delivery were collected. Between August 2022 and August 2023, we recruited 2000 first-trimester pregnant women to the cohort and continued to follow up with them until the baby’s birth. The response rates were 90.4%, 81.4%, and 75.2% in the first, second, and third follow-ups. We plan to analyze the data in July 2024. We expect this study to provide valuable insights into various early-life exposures affecting neonatal birth weight. The study’s findings will serve as a valuable information resource for a broader scientific community, enabling the development of effective policies to prevent low-birth-weight deliveries in low-resource settings.
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(This article belongs to the Section Public Health Research)
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Open AccessArticle
Phytochemical Composition and Antimicrobial and Antibiofilm Effect of Myrciaria cauliflora Hydroethanolic Extract against Staphylococcus aureus and Acinetobacter baumannii
by
Luciane Dias de Oliveira, Ana Luisa Monteiro Ribeiro, Sthéfani de Oliveira Dias, Geovani Moreira da Cruz, Raquel Teles de Menezes, Lara Steffany de Carvalho, Mariana Gadelho Gimenez Diamantino, Thaís Cristine Pereira, Maria Cristina Marcucci and Amjad Abu Hasna
Methods Protoc. 2024, 7(4), 60; https://doi.org/10.3390/mps7040060 - 4 Aug 2024
Abstract
Staphylococcus aureus and Acinetobacter baumannii are opportunistic pathogens, and both are involved in different oral infections. This work aimed to analyze the phytochemical composition of Myrciaria cauliflora hydroethanolic extract and to evaluate its antimicrobial and antibiofilm action against Staphylococcus aureus (ATCC 6538) and
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Staphylococcus aureus and Acinetobacter baumannii are opportunistic pathogens, and both are involved in different oral infections. This work aimed to analyze the phytochemical composition of Myrciaria cauliflora hydroethanolic extract and to evaluate its antimicrobial and antibiofilm action against Staphylococcus aureus (ATCC 6538) and Acinetobacter baumannii (ATCC 19606; multi-resistant clinical strains 58004, 50098, 566006, and H557). Myrciaria cauliflora hydroethanolic extract was prepared, and the content of soluble solids, flavonoids, and phenols was quantified. High-performance liquid chromatography (HPLC) was performed later. The minimum inhibitory concentration was determined using the broth microdilution method according to the Clinical and Laboratory Standards Institute, standard M7-A6, and subsequently, its minimum bactericidal concentration was determined. Then, the most effective concentrations were analyzed against biofilms. Statistical analysis was performed using the ANOVA method with Tukey’s test. The soluble solids content in the prepared hydroethanolic extract of M. cauliflora was 2.22%. Additionally, the total flavonoid content, measured using the quercetin standard curve, was 0.040 mg/mL. Furthermore, the total phenol content, determined using the gallic acid standard curve, was 0.729 mg/mL. HPLC analysis presented peaks of gallic acid (11.80 m), p-coumaric acid (12.09 m), cinnamic acid derivative (19.02 m), and ellagic acid (29.83 m). The extract demonstrated antimicrobial and antibiofilm action against all tested strains. However, the most effective antibacterial concentration against all the tested bacteria was 5.55 mg/mL. Therefore, these chemical components justify that M. cauliflora hydroethanolic extract is effective in reducing biofilm formation in S. aureus (standard strain) and A. baumannii (standard and clinical strains).
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(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
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Open AccessProtocol
Research on the Design and Performance of Plant Volatile Organic Compounds Water Removal Device Based on Optimized Filler Ratio
by
Yali Yuan, Huasen Wang, Zhihong Sun and Chao Yu
Methods Protoc. 2024, 7(4), 59; https://doi.org/10.3390/mps7040059 - 31 Jul 2024
Abstract
This study focuses on the development and optimization of a water removal device for biogenic volatile organic compounds (BVOCs) from plant emissions. BVOCs play a crucial role in various ecological processes and have potential therapeutic effects on human health. However, it is challenging
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This study focuses on the development and optimization of a water removal device for biogenic volatile organic compounds (BVOCs) from plant emissions. BVOCs play a crucial role in various ecological processes and have potential therapeutic effects on human health. However, it is challenging to accurately detect and analyze BVOCs due to their very low concentrations and interference by water vapor. This study systematically evaluates different filler materials and ratios to alleviate water vapor interference while maintaining BVOCs’ integrity. The experimental results demonstrate that the combination of MgSO4 + Na2SO4 mixed filling and CuSO4 layered filling in a 3:3:1 ratio can effectively improve the collection efficiency and detection accuracy of BVOCs. Meanwhile, the effectiveness of the device in improving the detection of volatile compounds in plant samples is also confirmed by the VOC verification experiments on Michelia maudiae and Cinnamomum camphora tree species after mechanical damage. The experimental results show that the device is effective in improving the detection of volatile compounds in plant samples. The findings provide a powerful technical means for exploring the role of BVOCs in environmental monitoring and scientific research.
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(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
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Open AccessStudy Protocol
A Multicenter, Single-Arm, Prospective Trial to Evaluate Efficacy and Safety of Dose-Dense Methotrexate, Vinblastine, Doxorubicin, and Carboplatin (DD-MVACarbo) Chemotherapy for Cisplatin-Ineligible Patients with Advanced Urothelial Cancer: Study Protocol of the CARBUNCLE Trial
by
Makito Miyake, Satoshi Anai, Yusuke Iemura, Kazuki Ichikawa, Tatsuki Miyamoto, Atsushi Tomioka, Masaomi Kuwada, Yoshitaka Itami, Yukinari Hosokawa, Yoshiaki Matsumura, Eijiro Okajima, Kazumasa Torimoto, Nobutaka Nishimura, Mitsuru Tomizawa, Takuto Shimizu, Shunta Hori, Yosuke Morizawa, Daisuke Gotoh, Yasushi Nakai and Kiyohide Fujimoto
Methods Protoc. 2024, 7(4), 58; https://doi.org/10.3390/mps7040058 - 29 Jul 2024
Abstract
Unresectable, metastatic, advanced urothelial carcinoma (aUC) is an aggressive disease and is treated with platinum-containing first-line chemotherapy, followed by immune checkpoint inhibitors and antibody–drug conjugates. Response to first-line chemotherapy is a vital priority in sequential treatment strategies because a better response to first-line
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Unresectable, metastatic, advanced urothelial carcinoma (aUC) is an aggressive disease and is treated with platinum-containing first-line chemotherapy, followed by immune checkpoint inhibitors and antibody–drug conjugates. Response to first-line chemotherapy is a vital priority in sequential treatment strategies because a better response to first-line chemotherapy is associated with a better response to subsequent therapies. Gemcitabine plus carboplatin chemotherapy is conventionally recommended for cisplatin-ineligible patients. This multicenter, single-arm prospective trial will investigate whether dose-dense methotrexate, vinblastine, doxorubicin, and carboplatin (DD-MVACarbo) chemotherapy is superior to gemcitabine plus carboplatin chemotherapy in terms of efficacy in platinum-naïve, cisplatin-ineligible patients with aUC. After screening and registration, a total of 46 patients will be treated with this novel chemotherapy regimen. The primary endpoint is the objective response rate. The secondary endpoints include disease control rate, patient-reported outcomes, and adverse events. No evidence of this novel intervention is available as of July 2024. The results are expected to change the standard of care and improve the management of patients with aUC.
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(This article belongs to the Section Public Health Research)
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Open AccessProtocol
A Protocol to Extract a Specific Genomic Region from a Public Whole-Genome Database and Modify Analytical Bin Length for Population Genetic Studies
by
Muhammad Shoaib Akhtar and Shoji Kawamura
Methods Protoc. 2024, 7(4), 57; https://doi.org/10.3390/mps7040057 - 27 Jul 2024
Abstract
With the advent of “next-generation” sequencing and the continuous reduction in sequencing costs, an increasing amount of genomic data has emerged, such as whole-genome, whole-exome, and targeted sequencing data. These applications are popular not only in mega sequencing projects, such as the 1000
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With the advent of “next-generation” sequencing and the continuous reduction in sequencing costs, an increasing amount of genomic data has emerged, such as whole-genome, whole-exome, and targeted sequencing data. These applications are popular not only in mega sequencing projects, such as the 1000 Genomes Project and UK BioBank, but also among individual researchers. Evolutionary genetic analyses, such as the dN/dS ratio and Tajima’s D, are demanded more and more for whole-genome-level population data. These analyses are often carried out under a uniform custom bin size across the genome. However, these analyses require subdivision of a genomic region into functional units, such as protein-coding regions, introns, and untranslated regions, and computing these genetic measures for large-scale data remains challenging. In a recent investigation, we successfully devised a method to address this issue. This method requires a multi-sample VCF file containing population data, a reference genome, target regions in the BED file, and a list of samples to be included in the analysis. Given that the targeted regions are extracted in a new VCF file, targeted population genetic analysis can be performed. We conducted Tajima’s D analysis using this approach on intact and pseudogenes, as well as non-coding regions.
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(This article belongs to the Section Omics and High Throughput)
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Open AccessArticle
Image Processing Analysis of Plant Morphometry with Examples from the Genus Sedum (Crassulaceae)
by
Mădălina Borcă, Alexandru Borcă, Alin Ciobica, Gabriela Halitchi and Andrei Stoie
Methods Protoc. 2024, 7(4), 56; https://doi.org/10.3390/mps7040056 - 24 Jul 2024
Abstract
The complex systematics of the genus Sedum, the difficulties of its classification and the ambiguity of the concrete identification of the taxa brought about the need to implement a measurement system adaptable to field conditions, so as to facilitate the accuracy of
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The complex systematics of the genus Sedum, the difficulties of its classification and the ambiguity of the concrete identification of the taxa brought about the need to implement a measurement system adaptable to field conditions, so as to facilitate the accuracy of data collection, avoiding the etiolation of samples and, therefore, the deterioration of the morphological structures subject to analysis. Thus, our study describes a digitization of the classic method of making measurements using millimeter paper, thus facilitating the subsequent statistical processing of quantifiable values. Depending on the number of pixels in the photos taken and the pixel/millimeter ratio, a variable measurement scale can be created depending on the size of the analyzed taxomes. The method used adds to the classic taxonomy, which is based on the analysis of morphological characteristics to determine the species of these succulent plants. The applicability of our method is shown by means of the example of an analysis performed on the flowers of the native species of the genus Sedum in the territory of Romania.
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(This article belongs to the Special Issue Plant Tissue Culture for Crop Improvement)
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