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Volume 7
Issue 5
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Methods Protoc., Volume 7, Issue 5 (October 2024) – 12 articles

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16 pages, 1666 KiB  
Study Protocol
Oral Pre-Exposure Prophylaxis Innovative Interventions among Adolescent Girls and Young Women in South Africa: A Protocol Paper
by Lerato Lucia Olifant, Edith Phalane and Refilwe Nancy Phaswana-Mafuya
Methods Protoc. 2024, 7(5), 77; https://doi.org/10.3390/mps7050077 (registering DOI) - 29 Sep 2024
Abstract
Although South Africa was the first country to register and roll out oral pre-exposure prophylaxis (PrEP) biomedical human immunodeficiency virus (HIV) prevention intervention in sub-Saharan Africa (SSA), its uptake remains low, particularly among adolescent girls and young women (AGYW). The uptake of PrEP [...] Read more.
Although South Africa was the first country to register and roll out oral pre-exposure prophylaxis (PrEP) biomedical human immunodeficiency virus (HIV) prevention intervention in sub-Saharan Africa (SSA), its uptake remains low, particularly among adolescent girls and young women (AGYW). The uptake of PrEP may have worsened during the Coronavirus disease 2019 (COVID-19) pandemic. Some innovative interventions to improve PrEP uptake among AGYW have been implemented. This study aims to evaluate the effectiveness of PrEP innovative interventions implemented during COVID-19 towards reducing the risk of HIV infection among AGYW in South Africa. An exploratory, descriptive design will be conducted to carry out four study objectives. Firstly, to carry out a systematic review of innovative PrEP interventions implemented during COVID-19 in SSA countries. Secondly, to conduct a stakeholder analysis to identify PrEP stakeholders and interview them on their views on the implemented interventions. Thirdly, to assess the implementation outcomes of the innovative interventions using document reviews and Consolidated Framework for Implementation Research. Fourthly, to develop a framework for an improved PrEP service delivery among AGYW. Qualitative data will be captured in ATLAS.ti software (Technical University, Berlin, Germany) version 23 and analysed via thematic analysis. A statistical software package (STATA) version 18 (College Station, TX, USA) will be used to capture quantitative data and analyse them via descriptive analysis. The generated evidence will be used towards the development of framework, guidelines, and policies to strengthen the uptake of, scale-up, and adherence to PrEP among AGYW. Full article
(This article belongs to the Section Public Health Research)
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11 pages, 2947 KiB  
Protocol
Use of Decellularized Bio-Scaffolds for the Generation of a Porcine Artificial Intestine
by Sharon Arcuri, Georgia Pennarossa, Madhusha Prasadani, Fulvio Gandolfi and Tiziana A. L. Brevini
Methods Protoc. 2024, 7(5), 76; https://doi.org/10.3390/mps7050076 - 27 Sep 2024
Viewed by 375
Abstract
In recent years, great interest has been focused on the development of highly reproducible 3D in vitro models that are able to mimic the physiological architecture and functionality of native tissues. To date, a wide range of techniques have been proposed to recreate [...] Read more.
In recent years, great interest has been focused on the development of highly reproducible 3D in vitro models that are able to mimic the physiological architecture and functionality of native tissues. To date, a wide range of techniques have been proposed to recreate an intestinal barrier in vitro, including synthetic scaffolds and hydrogels, as well as complex on-a-chip systems and organoids. Here, we describe a novel protocol for the generation of an artificial intestine based on the creation of decellularized bio-scaffolds and their repopulation with intestinal stromal and epithelial cells. Organs collected at the local slaughterhouse are subjected to a decellularization protocol that includes a freezing/thawing step, followed by sequential incubation in 1% SDS for 12 h, 1% Triton X-100 for 12 h, and 2% deoxycholate for 12 h. At the end of the procedure, the generated bio-scaffolds are repopulated with intestinal fibroblasts and then with epithelial cells. The protocol described here represents a promising and novel strategy to generate an in vitro bioengineered intestine platform able to mimic some of the complex functions of the intestinal barrier, thus constituting a promising 3D strategy for nutritional, pharmaceutical, and toxicological studies. Full article
(This article belongs to the Section Molecular and Cellular Biology)
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13 pages, 10019 KiB  
Protocol
A Scalable Method to Fabricate 2D Hydrogel Substrates for Mechanobiology Studies with Independent Tuning of Adhesiveness and Stiffness
by Alessandro Gandin, Veronica Torresan, Tito Panciera and Giovanna Brusatin
Methods Protoc. 2024, 7(5), 75; https://doi.org/10.3390/mps7050075 - 26 Sep 2024
Viewed by 205
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 [...] Read more.
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. Full article
(This article belongs to the Section Molecular and Cellular Biology)
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13 pages, 2324 KiB  
Protocol
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
Viewed by 392
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, [...] Read more.
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. Full article
(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
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19 pages, 3313 KiB  
Protocol
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
Viewed by 427
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 [...] Read more.
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. Full article
(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
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7 pages, 837 KiB  
Protocol
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
Viewed by 457
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, [...] Read more.
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. Full article
(This article belongs to the Section Molecular and Cellular Biology)
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13 pages, 1725 KiB  
Protocol
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
Viewed by 417
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 [...] Read more.
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. Full article
(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
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9 pages, 363 KiB  
Study Protocol
The Burden of Sleep/Wake Disorders: Excessive Daytime Sleepiness and Insomnia Project
by 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
Viewed by 437
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 [...] Read more.
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. Full article
(This article belongs to the Section Public Health Research)
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14 pages, 5354 KiB  
Article
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
Viewed by 419
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 [...] Read more.
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. Full article
(This article belongs to the Section Biomedical Sciences and Physiology)
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10 pages, 2377 KiB  
Article
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 | Viewed by 417
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, [...] Read more.
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. Full article
(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
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10 pages, 1453 KiB  
Article
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
Viewed by 480
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 [...] Read more.
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. Full article
(This article belongs to the Section Biomedical Sciences and Physiology)
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7 pages, 807 KiB  
Technical 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
Viewed by 384
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. [...] Read more.
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. Full article
(This article belongs to the Section Molecular and Cellular Biology)
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