Methods and Protocols 2023

A special issue of Methods and Protocols (ISSN 2409-9279).

Deadline for manuscript submissions: closed (31 March 2023) | Viewed by 18448

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Guest Editor
1. School of Chemistry and Physics, University of KwaZulu-Natal, Durban, South Africa
2. Department of Organic Chemistry, University of Barcelona, Barcelona, Spain
Interests: antimicrobial peptides; solid-phase chemistry; combinatorial chemistry; drug delivery systems; peptide drug conjugates; orthogonal chemistry; drug discovery; biomaterials
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Special Issue Information

Dear Colleagues,

We are pleased to announce this Special Issue, entitled “Methods and Protocols in 2023”, which is part of the MDPI journal New Year Special Issue Series. This Special Issue will be a collection of high-quality reviews and original research articles from Advisory Board Members, Editor-in-Chief, Associate Editor, Editorial Board Members, Guest Editors, Topical Advisory Panel Members, Reviewer Board Members, Authors, and Reviewers from Methods and Protocols, in addition to excellent editorials from high-profile scholars in the fields of biological, medical, chemical, life sciences and public health et al. Submissions on new protocols based on established techniques as well as significant improvements and developments of cutting-edge methods are welcome.

Prof. Dr. Fernando Albericio
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

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Published Papers (8 papers)

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Research

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15 pages, 622 KiB  
Article
Creating Implicit Measure Stimulus Sets Using a Multi-Step Piloting Method
by Daniel J. Phipps and Kyra Hamilton
Methods Protoc. 2023, 6(3), 47; https://doi.org/10.3390/mps6030047 - 03 May 2023
Viewed by 1828
Abstract
The effect of arbitrary stimulus selection is a persistent concern when employing implicit measures. The current study tests a data-driven multi-step procedure to create stimulus items using a combination of free-recall and survey data. Six sets of stimulus items were created, representing healthy [...] Read more.
The effect of arbitrary stimulus selection is a persistent concern when employing implicit measures. The current study tests a data-driven multi-step procedure to create stimulus items using a combination of free-recall and survey data. Six sets of stimulus items were created, representing healthy food and high sugar items in children, adolescents, and adults. Selected items were highly representative of the target concepts, in frequent use, and of near equal length. Tests of the piloted items in two samples showed slightly higher implicit measure–behavior relations compared to a previously used measure, providing preliminary support for the value in empirically based stimulus selection. Further, the items reported as being the most associated with their target concepts differed notably from what one may expect from the guidelines or population consumption patterns, highlighting the importance of informed stimulus selection. Full article
(This article belongs to the Special Issue Methods and Protocols 2023)
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8 pages, 824 KiB  
Article
Intrarater Reliability and Analysis of Learning Effects in the Y Balance Test
by Olli Kattilakoski, Noora Kauranen, Mari Leppänen, Pekka Kannus, Kati Pasanen, Tommi Vasankari and Jari Parkkari
Methods Protoc. 2023, 6(2), 41; https://doi.org/10.3390/mps6020041 - 07 Apr 2023
Viewed by 1936
Abstract
While the general reliability of the Y balance test has been previously found to be excellent, earlier reviews highlighted a need for a more consistent methodology between studies. The purpose of this test–retest intrarater reliability study was to assess the intrarater reliability of [...] Read more.
While the general reliability of the Y balance test has been previously found to be excellent, earlier reviews highlighted a need for a more consistent methodology between studies. The purpose of this test–retest intrarater reliability study was to assess the intrarater reliability of the YBT using different methodologies regarding normalisation for leg length, number of repetitions, and score calculation. Sixteen healthy adult novice recreational runners aged 18–55 years, both women and men, were reviewed in a laboratory environment. Mean calculated scores, intraclass correlation coefficient, standard error of measurement, and minimal detectable change were calculated and analysed between different leg length normalisation and score calculation methods. The number of repetitions needed to reach a plateauing of results was analysed from the mean proportion of maximal reach per successful repetition. The intrarater reliability of the YBT was found to be good to excellent, and it was not affected by the method of score calculation or leg length measurement. The test results plateaued after the sixth successful repetition. Based on this study, it is suggested to use anterior superior iliac spine–medial malleolus length for leg length normalisation because this method was proposed in the original YBT protocol. At least seven successful repetitions should be performed to reach a result plateau. The average of the best three repetitions should be used to mitigate possible outliers and account for the learning effects seen in this study. Full article
(This article belongs to the Special Issue Methods and Protocols 2023)
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Review

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13 pages, 1464 KiB  
Review
Established and Emerging Methods for Protecting Linear DNA in Cell-Free Expression Systems
by Trevor J. Fochtman and Javin P. Oza
Methods Protoc. 2023, 6(2), 36; https://doi.org/10.3390/mps6020036 - 30 Mar 2023
Cited by 2 | Viewed by 3986
Abstract
Cell-free protein synthesis (CFPS) is a method utilized for producing proteins without the limits of cell viability. The plug-and-play utility of CFPS is a key advantage over traditional plasmid-based expression systems and is foundational to the potential of this biotechnology. A key limitation [...] Read more.
Cell-free protein synthesis (CFPS) is a method utilized for producing proteins without the limits of cell viability. The plug-and-play utility of CFPS is a key advantage over traditional plasmid-based expression systems and is foundational to the potential of this biotechnology. A key limitation of CFPS is the varying stability of DNA types, limiting the effectiveness of cell-free protein synthesis reactions. Researchers generally rely on plasmid DNA for its ability to support robust protein expression in vitro. However, the overhead required to clone, propagate, and purify plasmids reduces the potential of CFPS for rapid prototyping. While linear templates overcome the limits of plasmid DNA preparation, linear expression templates (LETs) were under-utilized due to their rapid degradation in extract based CFPS systems, limiting protein synthesis. To reach the potential of CFPS using LETs, researchers have made notable progress toward protection and stabilization of linear templates throughout the reaction. The current advancements range from modular solutions, such as supplementing nuclease inhibitors and genome engineering to produce strains lacking nuclease activity. Effective application of LET protection techniques improves expression yields of target proteins to match that of plasmid-based expression. The outcome of LET utilization in CFPS is rapid design–build–test–learn cycles to support synthetic biology applications. This review describes the various protection mechanisms for linear expression templates, methodological insights for implementation, and proposals for continued efforts that may further advance the field. Full article
(This article belongs to the Special Issue Methods and Protocols 2023)
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Other

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9 pages, 1145 KiB  
Protocol
An Improved Technique for Genotyping the ABCB1 Gene Variant of Exon 21
by Johanna Romina Zuccoli, Priscila Ayelén Pagnotta, Viviana Alicia Melito, Jimena Verónica Lavandera, Victoria Estela Parera and Ana María Buzaleh
Methods Protoc. 2023, 6(3), 53; https://doi.org/10.3390/mps6030053 - 26 May 2023
Viewed by 1548
Abstract
The Multidrug Resistance protein (ABCB1, MDR1) is involved in the transport of xenobiotics and antiretroviral drugs. Some variants of the ABCB1 gene are of clinical importance; among them, exon 12 (c.1236C>T, rs1128503), 21 (c.2677G>T/A, rs2032582), and 26 (c.3435C>T, rs1045642) have [...] Read more.
The Multidrug Resistance protein (ABCB1, MDR1) is involved in the transport of xenobiotics and antiretroviral drugs. Some variants of the ABCB1 gene are of clinical importance; among them, exon 12 (c.1236C>T, rs1128503), 21 (c.2677G>T/A, rs2032582), and 26 (c.3435C>T, rs1045642) have a high incidence in Caucasians. Several protocols have been used for genotyping the exon 21 variants, such as allele-specific PCR-RFLP using adapted primer to generate a digestion site for several enzymes and automatic sequencing to detect the SNVs, TaqMan Allele Discrimination assay and High-Resolution Melter analysis (HRMA). The aim was to describe a new approach to genotype the three variants c.2677G>T/A for the exon 21 doing only one PCR with the corresponding primers and the digestion of the PCR product with two restriction enzymes: BrsI to identify A allele and BseYI to differentiate between G or T. An improvement of this methodology was also described. The proposal technique here described is demonstrated to be very efficient, easy, fast, reproducible, and cost-effective. Full article
(This article belongs to the Special Issue Methods and Protocols 2023)
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10 pages, 470 KiB  
Protocol
The Impact of Introducing Midwives and also Mentoring on the Quality of Sexual, Reproductive, Maternal, Newborn, and Adolescent Health Services in Low- and Middle-Income Countries: An Integrative Review Protocol
by Rondi Anderson, Sojib Bin Zaman and Mark Limmer
Methods Protoc. 2023, 6(3), 48; https://doi.org/10.3390/mps6030048 - 05 May 2023
Cited by 1 | Viewed by 1376
Abstract
Introduction: Midwives have the potential to significantly contribute to health-delivery systems by providing sexual, reproductive, maternal, newborn, and adolescent health (SRMNAH) care. However, scant research finds barriers to understanding what midwives need to realize their full potential. There are gaps in the definition [...] Read more.
Introduction: Midwives have the potential to significantly contribute to health-delivery systems by providing sexual, reproductive, maternal, newborn, and adolescent health (SRMNAH) care. However, scant research finds barriers to understanding what midwives need to realize their full potential. There are gaps in the definition of a midwife and an understanding of effective means to support the implementation of midwifery care. Mentorship has been found to support systems and healthcare providers to improve care availability and quality. Objectives: We describe the methodology of an integrative review that aims to generate evidence of the impact of introducing midwives and also on-site facility mentoring to better understand facilitators and barriers to implementation of the quality and availability of SRMNAH services in low- and middle-income countries (LMICs). Methods: The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines will be used to carry out the integrative review. Four electronic bibliographic databases, PubMed MEDLINE, EMBASE, Scopus, and CINAHL, will be used to identify eligible studies. All types of qualitative or quantitative studies will be considered. Eligible studies will be screened according to Population, Intervention, Comparison, and Outcome (PICO) inclusion criteria, and data will be extracted against a predetermined format. The aspects of health system strengthening in providing improved SRMNCH care will be examined in this review to generate evidence on how midwives and mentorship can improve routine care and health outcomes using the World Health Organization’s Six Building Blocks approach. The quality of the articles will be thematically analyzed in four areas: coherence and integrity, appropriateness for answering the question, relevance and focus, and overall assessment using the Gough weight-of-evidence framework. Expected results: The literature review will consider assessing both upstream health systems regulators and downstream effectors for implementing midwifery interventions. Within this building block framework, this research will report on the outcomes and experiences of introducing midwives and the effectiveness of mentoring midwives and other staff in midwives’ roles in improving care quality and health outcomes. Full article
(This article belongs to the Special Issue Methods and Protocols 2023)
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13 pages, 2832 KiB  
Protocol
A Multiparametric Protocol for the Detailed Phytochemical and Antioxidant Characterisation of Plant Extracts
by Anna Michalaki and Konstantinos Grintzalis
Methods Protoc. 2023, 6(2), 40; https://doi.org/10.3390/mps6020040 - 05 Apr 2023
Viewed by 2009
Abstract
Medicinal and herbal plants are abundant sources of phytochemicals, which are biologically active compounds with potential health benefits. The characterisation of phytochemicals has been the subject of many studies, but there is a lack of comprehensive assays to accurately assess the main phytochemical [...] Read more.
Medicinal and herbal plants are abundant sources of phytochemicals, which are biologically active compounds with potential health benefits. The characterisation of phytochemicals has been the subject of many studies, but there is a lack of comprehensive assays to accurately assess the main phytochemical categories and their antioxidant properties. To address this, the present study has developed a multiparametric protocol comprising eight biochemical assays, which quantify the major categories of phytochemicals, including polyphenols, tannins and flavonoids, as well as their antioxidant and scavenging potential. The presented protocol offers several advantages over other methods, including higher sensitivity and significantly lower cost, making it a simpler and more affordable approach compared to commercial kits. The protocol was tested on two datasets with seventeen distinct herbal and medicinal plants, and the results demonstrated its effectiveness in accurately characterising the phytochemical composition of plant samples. The modular design of the protocol allows its adaptation to any spectrophotometric instrumentation, while all assays are simple to follow and require a minimum number of analytical steps. Full article
(This article belongs to the Special Issue Methods and Protocols 2023)
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8 pages, 584 KiB  
Protocol
Interventions to Increase Physical Activity in Community-Dwelling Older Adults in Regional and Rural Areas: A Realist Synthesis Review Protocol
by Stephen Cousins, Rebecca McKechnie, Patricia Jackman, Geoff Middleton, Tshepo Rasekaba and Irene Blackberry
Methods Protoc. 2023, 6(2), 29; https://doi.org/10.3390/mps6020029 - 12 Mar 2023
Viewed by 2354
Abstract
The importance of physical activity (PA) for the health and wellbeing of older adults is well documented, yet many older adults are insufficiently active. This issue is more salient in regional and rural areas, where evidence of the most critical components of interventions [...] Read more.
The importance of physical activity (PA) for the health and wellbeing of older adults is well documented, yet many older adults are insufficiently active. This issue is more salient in regional and rural areas, where evidence of the most critical components of interventions that explain PA participation and maintenance in older populations is sparse. This realist review will (1) systematically identify and synthesise literature on PA interventions in community-dwelling older adults in regional and rural areas, and (2) explore how and why those interventions increase PA in that population. Using a realist synthesis framework and the behaviour change wheel (BCW), context–mechanism–outcome (C-M-O) patterns of PA interventions for older adults in regional and rural areas will be synthesised. Thematic analysis will be employed to compare, contrast, and refine emerging C-M-O patterns to understand how contextual factors trigger mechanisms that influence regional and rural community-dwelling older adults’ participation in PA interventions. This realist review will be the first to adopt a BCW analysis and a realist synthesis framework to explore PA interventions in community-dwelling older adults in regional and rural areas. This review will provide recommendations for evidence-based interventions to improve PA participation and adherence by revealing the important mechanisms apparent in this context. Systematic review registration: (PROSPERO CRD42023402499). Full article
(This article belongs to the Special Issue Methods and Protocols 2023)
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15 pages, 2909 KiB  
Technical Note
Sustainable Methodologies for Efficient Gel Electrophoresis and Streamlined Screening of Difficult Plasmids
by Nadeem Asad, Emily Smith, Sudeep Shakya, Sutton Stegman and Lisa Timmons
Methods Protoc. 2023, 6(2), 25; https://doi.org/10.3390/mps6020025 - 06 Mar 2023
Viewed by 2274
Abstract
We describe a workflow for efficient, environmentally attentive, and sustainable practices related to routine agarose gel electrophoresis. The methods reduce plastic waste and improve efficiency, especially for the exhaustive screening of difficult-to-obtain plasmids. Sustainability is increased when agarose is used ten times over [...] Read more.
We describe a workflow for efficient, environmentally attentive, and sustainable practices related to routine agarose gel electrophoresis. The methods reduce plastic waste and improve efficiency, especially for the exhaustive screening of difficult-to-obtain plasmids. Sustainability is increased when agarose is used ten times over by virtue of a thorough recycling regimen. The workflow optimizes workspaces and standardizes lab practices for handling potentially hazardous waste, minimizing environmental harm. Safety, efficiency, and sustainability improve laboratory productivity, help minimize environmental contamination, and increase cost-effectiveness. Full article
(This article belongs to the Special Issue Methods and Protocols 2023)
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