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Feature Papers in Methods and Protocols 2023
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Feature Papers in Methods and Protocols 2023

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

Deadline for manuscript submissions: closed (31 December 2023) | Viewed by 9235

Special Issue Editors

Prof. Dr. Fernando Albericio

E-Mail Website
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
Special Issues, Collections and Topics in MDPI journals
Dr. Kejin Hu

E-Mail Website
Guest Editor
Department of Microbiology, Immunology and Genetics, Health Science Center, University of North Texas, Fort Worth, TX 76107, USA
Interests: cellular reprogramming; induced pluripotent stem cells; iPSCs; bromodomain extra terminal (BET) protein; epigenetics; molecular biology; cell biology
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

We are glad to announce this Special Issue on “Feature Papers in Methods and Protocols”. In this Special Issue, we will collect articles from top researchers describing new approaches or new cutting-edge developments to tackle questions in the fields of chemistry, biological and biomedical sciences. In particular, we encourage submissions in the emerging fields of “Omics and High Throughput”, “Public Health Research”, “Synthetic and Systems Biology” and “Tissue Engineering and Organoids”. Our aim is to encourage scientists to publish new protocols based on established techniques, as well as significant improvements and developments of cutting-edge methods. For all articles, background information necessary to understand the underlying principles, full experimental details, and comparison with available protocols/methods must be provided. Scientists are encouraged to provide accompanying video demonstrations, which will be published online, for ease of reproducibility.

You are welcome to send short proposals for the submission of feature papers to our Managing Editor Ms. Suzie Li (suzie.li@mdpi.com) or Editorial Office (mps@mdpi.com)  before submission. All papers will be subjected to thorough and rigorous peer review.

We look forward to receiving your excellent work.

Prof. Dr. Fernando Albericio
Dr. Kejin Hu
Guest Editors

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.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Methods and Protocols is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 1800 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Published Papers (5 papers)

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Research

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21 pages, 5228 KiB  
Article
Development of Solid Lipid Nanoparticles for Controlled Amiodarone Delivery
by Andreea Creteanu, Gabriela Lisa, Cornelia Vasile, Maria-Cristina Popescu, Adrian Florin Spac and Gladiola Tantaru
Methods Protoc. 2023, 6(5), 97; https://doi.org/10.3390/mps6050097 - 09 Oct 2023
Cited by 1 | Viewed by 1530
Abstract
In various drug delivery systems, solid lipid nanoparticles are dominantly lipid-based nanocarriers. Amiodarone hydrochloride is an antiarrhythmic agent used to treat severe rhythm disturbances. It has variable and hard-to-predict absorption in the gastrointestinal tract because of its low solubility and high permeability. The [...] Read more.
In various drug delivery systems, solid lipid nanoparticles are dominantly lipid-based nanocarriers. Amiodarone hydrochloride is an antiarrhythmic agent used to treat severe rhythm disturbances. It has variable and hard-to-predict absorption in the gastrointestinal tract because of its low solubility and high permeability. The aims of this study were to improve its solubility by encapsulating amiodarone into solid lipid nanoparticles using two excipients—Compritol® 888 ATO (pellets) (C888) as a lipid matrix and Transcutol® (T) as a surfactant. Six types of amiodarone-loaded solid lipid nanoparticles (AMD-SLNs) were obtained using a hot homogenization technique followed by ultrasonication with varying sonication parameters. AMD-SLNs were characterized by their size distribution, polydispersity index, zeta potential, entrapment efficiency, and drug loading. Based on the initial evaluation of the entrapment efficiency, only three solid lipid nanoparticle formulations (P1, P3, and P5) were further tested. They were evaluated through scanning electron microscopy, Fourier-transform infrared spectrometry, near-infrared spectrometry, thermogravimetry, differential scanning calorimetry, and in vitro dissolution tests. The P5 formulation showed optimum pharmaco-technical properties, and it had the greatest potential to be used in oral pharmaceutical products for the controlled delivery of amiodarone. Full article
(This article belongs to the Special Issue Feature Papers in Methods and Protocols 2023)
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17 pages, 4687 KiB  
Article
A Novel and Quantitative Detection Assay (effluxR) for Identifying Efflux-Associated Resistance Genes Using Multiplex Digital PCR in Clinical Isolates of Pseudomonas aeruginosa
by Nontaporn Rattanachak, Sattaporn Weawsiangsang, Robert A. Baldock, Theerasak Jaifoo, Touchkanin Jongjitvimol and Jirapas Jongjitwimol
Methods Protoc. 2023, 6(5), 96; https://doi.org/10.3390/mps6050096 - 08 Oct 2023
Viewed by 1743
Abstract
The rise of multidrug resistance of Pseudomonas aeruginosa highlights an increased need for selective and precise antimicrobial treatment. Drug efflux pumps are one of the major mechanisms of antimicrobial resistance found in many bacteria, including P. aeruginosa. Detection of efflux genes [...] Read more.
The rise of multidrug resistance of Pseudomonas aeruginosa highlights an increased need for selective and precise antimicrobial treatment. Drug efflux pumps are one of the major mechanisms of antimicrobial resistance found in many bacteria, including P. aeruginosa. Detection of efflux genes using a polymerase chain reaction (PCR)-based system would enable resistance detection and aid clinical decision making. Therefore, we aimed to develop and optimize a novel method herein referred to as “effluxR detection assay” using multiplex digital PCR (mdPCR) for detection of mex efflux pump genes in P. aeruginosa strains. The annealing/extension temperatures and gDNA concentrations were optimized to amplify mexB, mexD, and mexY using the multiplex quantitative PCR (mqPCR) system. We established the optimal mqPCR conditions for the assay (Ta of 59 °C with gDNA concentrations at or above 0.5 ng/µL). Using these conditions, we were able to successfully detect the presence of these genes in a quantity-dependent manner. The limit of detection for mex genes using the effluxR detection assay with mdPCR was 0.001 ng/µL (7.04–34.81 copies/µL). Moreover, using blind sample testing, we show that effluxR detection assay had 100% sensitivity and specificity for detecting mex genes in P. aeruginosa. In conclusion, the effluxR detection assay, using mdPCR, is able to identify the presence of multiple mex genes in P. aeruginosa that may aid clinical laboratory decisions and further epidemiological studies. Full article
(This article belongs to the Special Issue Feature Papers in Methods and Protocols 2023)
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21 pages, 11785 KiB  
Protocol
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
Viewed by 1367
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Feature Papers in Methods and Protocols 2023)
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16 pages, 6348 KiB  
Protocol
Protocol for Facile Synthesis of Fmoc-N-Me-AA-OH Using 2-CTC Resin as Temporary and Reusable Protecting Group
by Tanya Román, Gerardo Acosta, Constanza Cárdenas, Beatriz G. de la Torre, Fanny Guzmán and Fernando Albericio
Methods Protoc. 2023, 6(6), 110; https://doi.org/10.3390/mps6060110 - 13 Nov 2023
Viewed by 1816
Abstract
One approach to enhance the bioavailability and half-life of peptides in vivo is through N-methylation of one or more of the amino acids within the peptide sequence. However, commercially available Fmoc-N-Me-AA-OHs are limited and often expensive. In this study, a solid-phase synthesis method [...] Read more.
One approach to enhance the bioavailability and half-life of peptides in vivo is through N-methylation of one or more of the amino acids within the peptide sequence. However, commercially available Fmoc-N-Me-AA-OHs are limited and often expensive. In this study, a solid-phase synthesis method for Fmoc-N-Me-AA-OH was developed using a 2-chlorotrityl chloride (2-CTC) resin as a temporary protective group for the carboxylic acid strategy. Two strategies for the alkylation step were compared, employing either dimethyl sulfate or methyl iodide in the Biron−Kessler method. In this work we tested the protocol with two amino acids: Fmoc-Thr(tBu)-OH and Fmoc-βAla-OH. The first one is an alpha amino acid, very hindered and with the amine group directly influenced by the electronic effects of the carboxy group, whereas in Fmoc-βAla-OH, the presence of a methylene group weakens this influence due to the intervening carbon atoms. The desired amino acids, Fmoc-N-Me-Thr(tBu)-OH and Fmoc-N-Me-βAla-OH, were synthesized by both strategies with high yield and purity. Full article
(This article belongs to the Special Issue Feature Papers in Methods and Protocols 2023)
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10 pages, 1294 KiB  
Protocol
Improving 2-Chlorotrityl Chloride (2-CTC) Resin Activation
by Tanya Román, Gerardo Acosta, Beatriz G. de la Torre, Constanza Cárdenas, Fanny Guzmán and Fernando Albericio
Methods Protoc. 2023, 6(5), 82; https://doi.org/10.3390/mps6050082 - 08 Sep 2023
Cited by 1 | Viewed by 2255
Abstract
Used in solid-phase peptide synthesis (SPPS) for peptides with an acid termination, the 2-chlorotrityl chloride (2-CTC) resin is highly susceptible to moisture, leading to reduced resin loading and lower synthetic yields. It is therefore recommended that the resin be activated with thionyl chloride [...] Read more.
Used in solid-phase peptide synthesis (SPPS) for peptides with an acid termination, the 2-chlorotrityl chloride (2-CTC) resin is highly susceptible to moisture, leading to reduced resin loading and lower synthetic yields. It is therefore recommended that the resin be activated with thionyl chloride (SOCl2) before peptide assembly. Here we present an optimized procedure for resin activation that minimizes the use of SOCl2 as the activation reagent and reduces the activation time. Additionally, we demonstrate the feasibility of reusing the 2-CTC resin when following the activation protocol, achieving comparable results to the first usage of the resin. Moreover, we achieved different degrees of resin activation by varying the amount of SOCl2. For instance, the use of 2% SOCl2 in anhydrous dichloromethane (DCM) allowed up to 44% activation of the resin, thereby making it suitable for the synthesis of longer peptides. Alternatively, employing 25% SOCl2 in anhydrous DCM resulted in up to 80% activation with a reaction time of only 5 min in both cases. Full article
(This article belongs to the Special Issue Feature Papers in Methods and Protocols 2023)
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