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Feature Papers in Methods and Protocols 2024
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Feature Papers in Methods and Protocols 2024

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

Deadline for manuscript submissions: closed (31 December 2024) | Viewed by 15917

Special Issue Editor

Prof. Dr. Fernando Albericio

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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
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 life sciences, chemistry, 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 the 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. Eunice Fu ([email protected]) or Editorial Office ([email protected]) 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
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.

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.

Keywords

  • cell biology
  • biochemistry
  • chemistry
  • nanotechnology
  • biotechnology
  • imaging
  • applied computation and bioinformatics
  • genomics
  • proteomics
  • developmental biology
  • model organisms
  • pharmacology
  • structural biology/chemistry
  • biophysics
  • tissue engineering
  • public health

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Published Papers (9 papers)

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Research

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10 pages, 1347 KiB  
Communication
The Reconstructed Individual Patient Data from Kaplan–Meier (IPDfromKM) Method for Non-Inferiority Analyses: A New Potential Application
by Eugenia Piragine, Sabrina Trippoli, Sara Veneziano, Andrea Messori and Vincenzo Calderone
Methods Protoc. 2025, 8(1), 13; https://doi.org/10.3390/mps8010013 - 2 Feb 2025
Viewed by 255
Abstract
The IPDfromKM method, or Shiny method, is an artificial intelligence tool that enables indirect comparisons between studies by reconstructing individual patient data (IPD) from Kaplan–Meier (KM) curves. The IPDfromKM method is generally used for superiority analyses, but a further application could be represented [...] Read more.
The IPDfromKM method, or Shiny method, is an artificial intelligence tool that enables indirect comparisons between studies by reconstructing individual patient data (IPD) from Kaplan–Meier (KM) curves. The IPDfromKM method is generally used for superiority analyses, but a further application could be represented by non-inferiority analyses. However, there are no studies supporting this methodological hypothesis. The aim of this work was to validate the IPDfromKM method for non-inferiority analyses by “exploiting” the well-described non-inferiority of implantable devices occluding the left atrial appendage compared to oral anticoagulants in patients with atrial fibrillation. We performed a systematic review searching for randomized controlled trials (RCTs) in the PubMed database and found five studies. The R software (version 4.3.3) was used to perform a standard survival analysis comparing Watchman and Amlet devices with warfarin. The hazard ratio (HR), with 95% confidence interval (CI), was the main parameter of our analysis. The results confirmed the non-inferiority of Amlet and Watchman compared to warfarin (HR of Watchman vs. warfarin: 1.23, 95% CI 0.80 to 1.9; HR of Amlet vs. warfarin: 1.05, 95% CI 0.61 to 1.80). Therefore, we proposed a new application of the IPDfromKM method that could be potentially relevant in decision-making for the management of this common cardiac arrhythmia and a wide range of other pathological conditions. Full article
(This article belongs to the Special Issue Feature Papers in Methods and Protocols 2024)
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12 pages, 1930 KiB  
Article
Fluorescence Analysis of Quinine in Commercial Tonic Waters
by Artturi Harcher, Connor Ricard, Danielle Connolly, Isabel Gibbs, Jarve Shaw, Jillian Butler, Julia Perschbacher, Lindsay Replogle, Michaela Eide, Morgan Grissom, Oliver O’Neal, Quan Nguyen, Van Hac Nguyen, Michael Hunnicutt, Roaa Mahmoud and Soma Dhakal
Methods Protoc. 2025, 8(1), 5; https://doi.org/10.3390/mps8010005 - 6 Jan 2025
Viewed by 650
Abstract
Quinine is known for treating malaria, muscle cramps, and, more recently, has been used as an additive in tonic water due to its bitter taste. However, it was shown that excessive consumption of quinine can have severe side effects on health. In this [...] Read more.
Quinine is known for treating malaria, muscle cramps, and, more recently, has been used as an additive in tonic water due to its bitter taste. However, it was shown that excessive consumption of quinine can have severe side effects on health. In this work, we utilized fluorescence spectroscopy to measure the concentration of quinine in commercial tonic water samples. An external standard method was used to calculate the concentrations of quinine in two commercially available tonic water brands, namely Canada Dry and Schweppes, and compare them to the maximum allowable concentration of quinine in beverages. Upon analysis of the data collected by five different groups, the levels of quinine were found to be above the average concentration in most commercial tonic water samples, but below the maximum permitted concentration. Moreover, the five replicate sets of data demonstrated high reproducibility of the method employed in this study. The simple yet instructive protocol that we developed can be adapted to determine the concentration of other fluorescent compounds in foods and beverages. Further, the presented method and detailed protocol can be easily adopted for undergraduate labs and in chemical education. Full article
(This article belongs to the Special Issue Feature Papers in Methods and Protocols 2024)
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16 pages, 5027 KiB  
Article
Evaluation of Drug–Polymer and Drug–Drug Interaction in Cellulosic Multi-Drug Delivery Matrices
by Abdullah Isreb, Mohamed A. Alhnan, Abdulrahman Mkia, Khaled Al-Jammal, Abdallah Yaghi, Enoche Florence Oga, Peter Timmins, Michael Bonner and Robert T. Forbes
Methods Protoc. 2025, 8(1), 4; https://doi.org/10.3390/mps8010004 - 6 Jan 2025
Viewed by 648
Abstract
Multi-drug delivery systems have gained increasing interest from the pharmaceutical industry. Alongside this is the interest in amorphous solid dispersions as an approach to achieve effective oral delivery of compounds with solubility-limited bioavailability. Despite this, there is limited information regarding predicting the behavior [...] Read more.
Multi-drug delivery systems have gained increasing interest from the pharmaceutical industry. Alongside this is the interest in amorphous solid dispersions as an approach to achieve effective oral delivery of compounds with solubility-limited bioavailability. Despite this, there is limited information regarding predicting the behavior of two or more drugs (in amorphous forms) in a polymeric carrier and whether molecular interactions between the compounds, between each compound, and if the polymer have any effect on the physical properties of the system. This work studies the interaction between model drug combinations (two of ibuprofen, malonic acid, flurbiprofen, or naproxen) dispersed in a polymeric matrix of hypromellose acetate succinate (HPMCAS) using a solvent evaporation technique. Hildebrand and Hansen calculations were used to predict the miscibility of compounds as long as the difference in their solubility parameter values was not greater than 7 MPa1/2. It was observed that the selected APIs (malonic acid, ibuprofen, naproxen, and flurbiprofen) were miscible within the formed polymeric matrix. Adding the API caused depression in the Tg of the polymer to certain concentrations (17%, 23%, 13%) for polymeric matrices loaded with malonic acid, ibuprofen, and naproxen, respectively. Above this, large crystals started to form, and phase separation was seen. Adding two APIs to the same matrix resulted in reducing the saturation concentration of one of the APIs. A trend was observed and linked to Hildebrand and Hansen solubility parameters (HSP). Full article
(This article belongs to the Special Issue Feature Papers in Methods and Protocols 2024)
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15 pages, 1931 KiB  
Article
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
Viewed by 1359
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Feature Papers in Methods and Protocols 2024)
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10 pages, 917 KiB  
Article
Odours in Asphalt: Analysis of the Release of H2S from Bitumen by a Mass Spectrometric Residual Gas Analyser
by Marcello Monteleone, Giuseppe Di Luca, Marcello Filomia, Alessio Fuoco, Alberto Figoli and Johannes Carolus Jansen
Methods Protoc. 2024, 7(4), 55; https://doi.org/10.3390/mps7040055 - 15 Jul 2024
Cited by 1 | Viewed by 1950
Abstract
During the production and laying phases of hot-mixing asphalt (HMA), various volatile organic compounds (VOCs) and noxious gases such as H2S are released into the atmosphere. These emissions are a serious environmental problem, a risk to human health, and expose workers [...] Read more.
During the production and laying phases of hot-mixing asphalt (HMA), various volatile organic compounds (VOCs) and noxious gases such as H2S are released into the atmosphere. These emissions are a serious environmental problem, a risk to human health, and expose workers and residents to unfriendly odours. The aim of this study was the development of a fast and sensitive analytical method to detect the H2S emitted from hot bituminous binder that is generally used in the various stages of asphalt production, processing, handling and during road construction. The method consisted in the analysis of evolved H2S from a flask with molten bitumen, using nitrogen as a carrier gas to lead the volatile compounds into a residual gas analyser equipped with a quadrupole mass spectrometer. The analysis was performed following the H2S-specific signals at m/z 33 (HS+) and at m/z 34 (H2S+) in real time, directly on the sample without laborious and expensive pre-treatments and with short response times (<6 s). Calibration with a standard mixture of 1000 ppm of H2S in nitrogen allows semi-quantitative H2S detection. The sensitivity and rapidity of the method were evaluated by quenching the release of sulphur compounds with commercial odour-suppressing agents. Upon addition of 0.1% of additive in two minutes, the H2S signal drops about 80% in two minutes, confirming the good response of the method, even with a very complex matrix. Full article
(This article belongs to the Special Issue Feature Papers in Methods and Protocols 2024)
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21 pages, 15036 KiB  
Article
Syrian Hamsters Model Does Not Reflect Human-like Disease after Aerosol Exposure to Encephalitic Alphaviruses
by Christina L. Gardner, Rebecca A. Erwin-Cohen, Bridget S. Lewis, Russell R. Bakken, Shelley P. Honnold, Pamela J. Glass and Crystal W. Burke
Methods Protoc. 2024, 7(3), 42; https://doi.org/10.3390/mps7030042 - 15 May 2024
Cited by 1 | Viewed by 1361
Abstract
Venezuelan (VEE), eastern (EEE), and western (WEE) equine encephalitis viruses are encephalitic New World alphaviruses that cause periodic epizootic and epidemic outbreaks in horses and humans that may cause severe morbidity and mortality. Currently there are no FDA-licensed vaccines or effective antiviral therapies. [...] Read more.
Venezuelan (VEE), eastern (EEE), and western (WEE) equine encephalitis viruses are encephalitic New World alphaviruses that cause periodic epizootic and epidemic outbreaks in horses and humans that may cause severe morbidity and mortality. Currently there are no FDA-licensed vaccines or effective antiviral therapies. Each year, there are a limited number of human cases of encephalitic alphaviruses; thus, licensure of a vaccine or therapeutic would require approval under the FDA animal rule. Approval under the FDA animal rule requires the disease observed in the animal model to recapitulate what is observed in humans. Currently, initial testing of vaccines and therapeutics is performed in the mouse model. Unfortunately, alphavirus disease manifestations in a mouse do not faithfully recapitulate human disease; the VEEV mouse model is lethal whereas in humans VEEV is rarely lethal. In an effort to identify a more appropriate small animal model, we evaluated hamsters in an aerosol exposure model of encephalitic alphavirus infection. The pathology, lethality, and viremia observed in the infected hamsters was inconsistent with what is observed in NHP models and humans. These data suggest that hamsters are not an appropriate model for encephalitic alphaviruses to test vaccines or potential antiviral therapies. Full article
(This article belongs to the Special Issue Feature Papers in Methods and Protocols 2024)
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8 pages, 2411 KiB  
Communication
Optimization of the Amplification of Equine Muscle-Derived Mesenchymal Stromal Cells in a Hollow-Fiber Bioreactor
by Julien Duysens, Hélène Graide, Ariane Niesten, Ange Mouithys-Mickalad, Justine Ceusters and Didier Serteyn
Methods Protoc. 2024, 7(2), 32; https://doi.org/10.3390/mps7020032 - 2 Apr 2024
Cited by 1 | Viewed by 1802
Abstract
The main causes of mortality in horses are the gastrointestinal pathologies associated with septic shock. Stem cells have shown, through systemic injection, a capacity to decrease inflammation and to regenerate injured tissue faster. Nevertheless, to achieve this rapid and total regeneration, systemic injections [...] Read more.
The main causes of mortality in horses are the gastrointestinal pathologies associated with septic shock. Stem cells have shown, through systemic injection, a capacity to decrease inflammation and to regenerate injured tissue faster. Nevertheless, to achieve this rapid and total regeneration, systemic injections of 1 to 2 million cells per kilogram of body weight must be considered. Here, we demonstrate for the first time the feasibility and expansion capacity of equine muscle-derived mesenchymal stromal cells (mdMSCs) in a functionally closed, automated, perfusion-based, hollow-fiber bioreactor (HFBR) called the Quantum™ Cell Expansion System (Terumo Blood and Cell Technologies). This feature greatly increases the number of generated cells with a surface area of 1.7 m2. The expansion of mdMSCs is very efficient in this bioreactor. The maximum expansion generated twenty times more cells than the initial seeding in nine days. The best returns were observed with an optimal seeding between 10 and 25 million mdMSCs, using the Bull’s eye loading method and with a run duration between 7 and 10 days. Moreover, all the generated cells kept their stem properties: the ability to adhere to plastic and to differentiate into chondroblasts, osteoblasts and adipocytes. They also showed the expression of CD-44 and CD-90 markers, with a positive rate above 93%, while CD-45 and MHCII were non-expressed, with a positive rate below 0.5%. By capitalizing on the scalability, automation and 3D culture capabilities of the Quantum™, it is possible to generate large quantities of high-quality equine mdMSCs for gastrointestinal disorders and other clinical applications. Full article
(This article belongs to the Special Issue Feature Papers in Methods and Protocols 2024)
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Review

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14 pages, 557 KiB  
Review
Imaging Flow Cytometry: Development, Present Applications, and Future Challenges
by Savvas Dimitriadis, Lefkothea Dova, Ioannis Kotsianidis, Eleftheria Hatzimichael, Eleni Kapsali and Georgios S. Markopoulos
Methods Protoc. 2024, 7(2), 28; https://doi.org/10.3390/mps7020028 - 23 Mar 2024
Cited by 2 | Viewed by 5039
Abstract
Imaging flow cytometry (ImFC) represents a significant technological advancement in the field of cytometry, effectively merging the high-throughput capabilities of flow analysis with the detailed imaging characteristics of microscopy. In our comprehensive review, we adopt a historical perspective to chart the development of [...] Read more.
Imaging flow cytometry (ImFC) represents a significant technological advancement in the field of cytometry, effectively merging the high-throughput capabilities of flow analysis with the detailed imaging characteristics of microscopy. In our comprehensive review, we adopt a historical perspective to chart the development of ImFC, highlighting its origins and current state of the art and forecasting potential future advancements. The genesis of ImFC stemmed from merging the hydraulic system of a flow cytometer with advanced camera technology. This synergistic coupling facilitates the morphological analysis of cell populations at a high-throughput scale, effectively evolving the landscape of cytometry. Nevertheless, ImFC’s implementation has encountered hurdles, particularly in developing software capable of managing its sophisticated data acquisition and analysis needs. The scale and complexity of the data generated by ImFC necessitate the creation of novel analytical tools that can effectively manage and interpret these data, thus allowing us to unlock the full potential of ImFC. Notably, artificial intelligence (AI) algorithms have begun to be applied to ImFC, offering promise for enhancing its analytical capabilities. The adaptability and learning capacity of AI may prove to be essential in knowledge mining from the high-dimensional data produced by ImFC, potentially enabling more accurate analyses. Looking forward, we project that ImFC may become an indispensable tool, not only in research laboratories, but also in clinical settings. Given the unique combination of high-throughput cytometry and detailed imaging offered by ImFC, we foresee a critical role for this technology in the next generation of scientific research and diagnostics. As such, we encourage both current and future scientists to consider the integration of ImFC as an addition to their research toolkit and clinical diagnostic routine. Full article
(This article belongs to the Special Issue Feature Papers in Methods and Protocols 2024)
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Other

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11 pages, 2482 KiB  
Protocol
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
Viewed by 848
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Feature Papers in Methods and Protocols 2024)
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