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Methods Protoc., Volume 4, Issue 1 (March 2021) – 17 articles

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Open AccessProtocol
Concentration and Quantification of SARS-CoV-2 RNA in Wastewater Using Polyethylene Glycol-Based Concentration and qRT-PCR
Methods Protoc. 2021, 4(1), 17; https://doi.org/10.3390/mps4010017 - 23 Feb 2021
Viewed by 189
Abstract
Wastewater-based epidemiology has become an important tool for the surveillance of SARS-CoV-2 outbreaks. However, the detection of viruses in sewage is challenging and to date there is no standard method available which has been validated for the sensitive detection of SARS-CoV-2. In this [...] Read more.
Wastewater-based epidemiology has become an important tool for the surveillance of SARS-CoV-2 outbreaks. However, the detection of viruses in sewage is challenging and to date there is no standard method available which has been validated for the sensitive detection of SARS-CoV-2. In this paper, we describe a simple concentration method based on polyethylene glycol (PEG) precipitation, followed by RNA extraction and a one-step quantitative reverse transcription PCR (qRT-PCR) for viral detection in wastewater. PEG-based concentration of viruses is a simple procedure which is not limited by the availability of expensive equipment and has reduced risk of disruption to consumable supply chains. The concentration and RNA extraction steps enable 900–1500× concentration of wastewater samples and sufficiently eliminates the majority of organic matter, which could inhibit the subsequent qRT-PCR assay. Due to the high variation in the physico-chemical properties of wastewater samples, we recommend the use of process control viruses to determine the efficiency of each step. This procedure enables the concentration and the extraction the DNA/RNA of different viruses and hence can be used for the surveillance of different viral targets for the comprehensive assessment of viral diseases in a community. Full article
(This article belongs to the Section Public Health Research)
Open AccessStudy Protocol
A Modified Limiting Dilution Method for Monoclonal Stable Cell Line Selection Using a Real-Time Fluorescence Imaging System: A Practical Workflow and Advanced Applications
Methods Protoc. 2021, 4(1), 16; https://doi.org/10.3390/mps4010016 - 20 Feb 2021
Viewed by 183
Abstract
Stable cell lines are widely used in laboratory research and pharmaceutical industry. They are mainly applied in recombinant protein and antibody productions, gene function studies, drug screens, toxicity assessments, and for cancer therapy investigation. There are two types of cell lines, polyclonal and [...] Read more.
Stable cell lines are widely used in laboratory research and pharmaceutical industry. They are mainly applied in recombinant protein and antibody productions, gene function studies, drug screens, toxicity assessments, and for cancer therapy investigation. There are two types of cell lines, polyclonal and monoclonal origin, that differ regarding their homogeneity and heterogeneity. Generating a high-quality stable cell line, which can grow continuously and carry a stable genetic modification without alteration is very important for most studies, because polyclonal cell lines of multicellular origin can be highly variable and unstable and lead to inconclusive experimental results. The most commonly used technologies of single cell originate monoclonal stable cell isolation in laboratory are fluorescence-activated cell sorting (FACS) sorting and limiting dilution cloning. Here, we describe a modified limiting dilution method of monoclonal stable cell line selection using the real-time fluorescence imaging system IncuCyte®S3. Full article
(This article belongs to the Section Molecular and Cellular Biology)
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Open AccessArticle
Total Face Approach (TFA): A Novel 3D Approach to Describe the Main Cephalometric Craniomaxillofacial Parameters
Methods Protoc. 2021, 4(1), 15; https://doi.org/10.3390/mps4010015 - 20 Feb 2021
Viewed by 142
Abstract
The aim of this study is to propose a 3D skeletal classification and relative normal values of reference. Method: from a pool of 271 cone-beam computerized tomography images 108 chin-summit examinations of the skull were selected and divided into 3 traditional skeletal [...] Read more.
The aim of this study is to propose a 3D skeletal classification and relative normal values of reference. Method: from a pool of 271 cone-beam computerized tomography images 108 chin-summit examinations of the skull were selected and divided into 3 traditional skeletal classes. The same Cone-beam Computerized Tomography (CBCT) images were then assessed using the cephalometric multiplanar analysis following the total face approach protocol. Results: the results of this study indicate standard 3D cephalometric norms for the vertical and sagittal evaluation of the skull. Conclusion: data obtained from our measurements allowed the creation of intervals supplying nosological classification that could be used in orthodontics, orthognatic surgery and implant surgery in fully edentulous patients. Full article
Open AccessProtocol
The Enzyme-Modified Neutral Comet (EMNC) Assay for Complex DNA Damage Detection
Methods Protoc. 2021, 4(1), 14; https://doi.org/10.3390/mps4010014 - 16 Feb 2021
Viewed by 183
Abstract
The comet assay is a versatile, simple, and sensitive gel electrophoresis–based method that can be used to measure and accurately quantify DNA damage, particularly single and double DNA strand breaks, in single cells. While generally this is used to measure variation in DNA [...] Read more.
The comet assay is a versatile, simple, and sensitive gel electrophoresis–based method that can be used to measure and accurately quantify DNA damage, particularly single and double DNA strand breaks, in single cells. While generally this is used to measure variation in DNA strand break levels and repair capacity within a population of cells, the technique has more recently been adapted and evolved into more complex analysis and detection of specific DNA lesions, such as oxidized purines and pyrimidines, achieved through the utilization of damage-specific DNA repair enzymes following cell lysis. Here, we detail a version of the enzyme-modified neutral comet (EMNC) assay for the specific detection of complex DNA damage (CDD), defined as two or more DNA damage lesions within 1–2 helical turns of the DNA. CDD induction is specifically relevant to ionizing radiation (IR), particularly of increasing linear energy transfer (LET), and is known to contribute to the cell-killing effects of IR due to the difficult nature of its repair. Consequently, the EMNC assay reveals important details regarding the extent and complexity of DNA damage induced by IR, but also has potential for the study of other genotoxic agents that may induce CDD. Full article
(This article belongs to the Section Molecular and Cellular Biology)
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Open AccessCommunication
Specific Assay of Negative Strand Template to Quantify Intracellular Levels of Rhinovirus Double-Stranded RNA
Methods Protoc. 2021, 4(1), 13; https://doi.org/10.3390/mps4010013 - 11 Feb 2021
Viewed by 184
Abstract
Human rhinovirus infections are a major trigger for acute exacerbations of lower airway diseases, including asthma and chronic obstructive pulmonary disease. Disease exacerbation is thought to be regulated via double-stranded RNA (dsRNA)-mediated signaling of proinflammatory and host defense responses in airway epithelial cells. [...] Read more.
Human rhinovirus infections are a major trigger for acute exacerbations of lower airway diseases, including asthma and chronic obstructive pulmonary disease. Disease exacerbation is thought to be regulated via double-stranded RNA (dsRNA)-mediated signaling of proinflammatory and host defense responses in airway epithelial cells. Despite the central role of dsRNA in regulating host cell responses, no method for the quantitative assessment of dsRNA levels during HRV infections has been developed. Conventional RT-PCR for the negative strand template is not effective as self-priming results in apparent signals, even in the absence of primer during reverse transcription. To avoid these issues, we developed a selective assay for the negative strand template that uses a chimeric primer containing a 5′ non-viral sequence for reverse transcription and a primer using the non-viral sequence during subsequent PCR. We established that this assay avoided issues of self-priming and is strand specific, as it is unaffected even in the presence of a 1000-fold excess of positive strand. Assays in primary human airway epithelial cells showed that negative strand was detectable within 6 h of virus exposure and peaked at 18 h after virus exposure. The temporal pattern of negative strand induction mirrored that of genomic RNA but was always 1000-fold lower than positive strand, indicating that the negative strand levels regulate levels of dsRNA formation. This assay will permit relative quantification of dsRNA during studies of HRV regulation of epithelial cell function. Full article
Open AccessCommentary
The Protocol Gap
Methods Protoc. 2021, 4(1), 12; https://doi.org/10.3390/mps4010012 - 03 Feb 2021
Viewed by 426
Abstract
Although peer review is considered one of the main pillars of modern science, experimental methods and protocols seem to be not a rigorous subject of this process in many papers. Commercial equipment, test kits, labeling kits, previously published concepts, and standard protocols are [...] Read more.
Although peer review is considered one of the main pillars of modern science, experimental methods and protocols seem to be not a rigorous subject of this process in many papers. Commercial equipment, test kits, labeling kits, previously published concepts, and standard protocols are often considered to be not worth a detailed description or validation. Even more disturbing is the extremely biased citation behavior in this context, which sometimes leads to surrogate citations to avoid low-impact journals, preprints, or to indicate traditional practices. This article describes some of these surprising habits and suggests some measures to avoid the most unpleasant effects, which in the long term may undermine the credibility of science as a whole. Full article
Open AccessReview
The Past, Present and Future of Flow Cytometry in Central Nervous System Malignancies
Methods Protoc. 2021, 4(1), 11; https://doi.org/10.3390/mps4010011 - 26 Jan 2021
Viewed by 374
Abstract
Central nervous system malignancies (CNSMs) are categorized among the most aggressive and deadly types of cancer. The low median survival in patients with CNSMs is partly explained by the objective difficulties of brain surgeries as well as by the acquired chemoresistance of CNSM [...] Read more.
Central nervous system malignancies (CNSMs) are categorized among the most aggressive and deadly types of cancer. The low median survival in patients with CNSMs is partly explained by the objective difficulties of brain surgeries as well as by the acquired chemoresistance of CNSM cells. Flow Cytometry is an analytical technique with the ability to quantify cell phenotype and to categorize cell populations on the basis of their characteristics. In the current review, we summarize the Flow Cytometry methodologies that have been used to study different phenotypic aspects of CNSMs. These include DNA content analysis for the determination of malignancy status and phenotypic characterization, as well as the methodologies used during the development of novel therapeutic agents. We conclude with the historical and current utility of Flow Cytometry in the field, and we propose how we can exploit current and possible future methodologies in the battle against this dreadful type of malignancy. Full article
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Open AccessPerspective
Recent Advances in miRNA Delivery Systems
Methods Protoc. 2021, 4(1), 10; https://doi.org/10.3390/mps4010010 - 20 Jan 2021
Viewed by 414
Abstract
MicroRNAs (miRNAs) represent a family of short non-coding regulatory RNA molecules that are produced in a tissue and time-specific manner to orchestrate gene expression post-transcription. MiRNAs hybridize to target mRNA(s) to induce translation repression or mRNA degradation. Functional studies have demonstrated that miRNAs [...] Read more.
MicroRNAs (miRNAs) represent a family of short non-coding regulatory RNA molecules that are produced in a tissue and time-specific manner to orchestrate gene expression post-transcription. MiRNAs hybridize to target mRNA(s) to induce translation repression or mRNA degradation. Functional studies have demonstrated that miRNAs are engaged in virtually every physiological process and, consequently, miRNA dysregulations have been linked to multiple human pathologies. Thus, miRNA mimics and anti-miRNAs that restore miRNA expression or downregulate aberrantly expressed miRNAs, respectively, are highly sought-after therapeutic strategies for effective manipulation of miRNA levels. In this regard, carrier vehicles that facilitate proficient and safe delivery of miRNA-based therapeutics are fundamental to the clinical success of these pharmaceuticals. Here, we highlight the strengths and weaknesses of current state-of-the-art viral and non-viral miRNA delivery systems and provide perspective on how these tools can be exploited to improve the outcomes of miRNA-based therapeutics. Full article
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Open AccessArticle
Validity and Cost-Effectiveness of Pediatric Home Respiratory Polygraphy for the Diagnosis of Obstructive Sleep Apnea in Children: Rationale, Study Design, and Methodology
Methods Protoc. 2021, 4(1), 9; https://doi.org/10.3390/mps4010009 - 19 Jan 2021
Viewed by 341
Abstract
Obstructive sleep apnea (OSA) in children is a prevalent, albeit largely undiagnosed disease associated with a large spectrum of morbidities. Overnight in-lab polysomnography remains the gold standard diagnostic approach, but is time-consuming, inconvenient, and expensive, and not readily available in many places. Simplified [...] Read more.
Obstructive sleep apnea (OSA) in children is a prevalent, albeit largely undiagnosed disease associated with a large spectrum of morbidities. Overnight in-lab polysomnography remains the gold standard diagnostic approach, but is time-consuming, inconvenient, and expensive, and not readily available in many places. Simplified Home Respiratory Polygraphy (HRP) approaches have been proposed to reduce costs and facilitate the diagnostic process. However, evidence supporting the validity of HRP is still scarce, hampering its implementation in routine clinical use. The objectives were: Primary; to establish the diagnostic and therapeutic decision validity of a simplified HRP approach compared to PSG among children at risk of OSA. Secondary: (a) Analyze the cost-effectiveness of the HRP versus in-lab PSG in evaluation and treatment of pediatric OSA; (b) Evaluate the impact of therapeutic interventions based on HRP versus PSG findings six months after treatment using sleep and health parameters and quality of life instruments; (c) Discovery and validity of the urine biomarkers to establish the diagnosis of OSA and changes after treatment. Full article
(This article belongs to the Section Public Health Research)
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Open AccessProtocol
The Impact of Diversified Farming Practices on Terrestrial Biodiversity Outcomes and Agricultural Yield Worldwide: A Systematic Review Protocol
Methods Protoc. 2021, 4(1), 8; https://doi.org/10.3390/mps4010008 - 14 Jan 2021
Viewed by 445
Abstract
The expansion and intensification of agriculture have led to global declines in biodiversity. This paper presents a systematic review protocol to clarify under what management and landscape contexts diversified farming practices are effective at improving outcomes for terrestrial biodiversity, and potential trade-offs or [...] Read more.
The expansion and intensification of agriculture have led to global declines in biodiversity. This paper presents a systematic review protocol to clarify under what management and landscape contexts diversified farming practices are effective at improving outcomes for terrestrial biodiversity, and potential trade-offs or synergies with agricultural yields. The systematic review will be developed following the Reporting Standards for Systematic Evidence Syntheses (ROSES). The review will include articles that compare levels of diversity (e.g., abundance, richness, Shannon’s diversity index) of any terrestrial taxon (e.g., arthropods, mammals) in diversified farming systems to levels in simplified farming systems and/or natural habitats, prioritising articles that also report agricultural yields. We will search for relevant peer-reviewed primary studies in two global repositories: Scopus and Web of Science, and among primary studies included in previous meta-analyses that are retrieved from the search. Full-texts of identified articles will be screened using a clear inclusion/exclusion eligibility criteria. All included articles will be assessed to determine their internal validity. A narrative synthesis will be performed to summarize, describe and present the results, and where the articles provide sufficient and appropriate data, we will conduct a quantitative meta-analysis. Full article
(This article belongs to the Section Synthetic and Systems Biology)
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Open AccessLetter
Inactivation of Material from SARS-CoV-2-Infected Primary Airway Epithelial Cell Cultures
Methods Protoc. 2021, 4(1), 7; https://doi.org/10.3390/mps4010007 - 07 Jan 2021
Viewed by 528
Abstract
Given that the airway epithelium is the initial site of infection, study of primary human airway epithelial cells (AEC) infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) will be crucial to improved understanding of viral entry factors and innate immune responses to [...] Read more.
Given that the airway epithelium is the initial site of infection, study of primary human airway epithelial cells (AEC) infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) will be crucial to improved understanding of viral entry factors and innate immune responses to the virus. Centers for Disease Control and Prevention (CDC) guidance recommends work with live SARS-CoV-2 in cell culture be conducted in a Biosafety Level 3 (BSL-3) laboratory. To facilitate downstream assays of materials from experiments there is a need for validated protocols for SARS-CoV-2 inactivation to facilitate safe transfer of material out of a BSL-3 laboratory. We propagated stocks of SARS-CoV-2, then evaluated the effectiveness of heat (65 °C) or ultraviolet (UV) light inactivation. We infected differentiated human primary AECs with SARS-CoV-2, then tested protocols designed to inactivate SARS-CoV-2 in supernatant, protein isolate, RNA, and cells fixed for immunohistochemistry by exposing Vero E6 cells to materials isolated/treated using these protocols. Heating to 65 °C for 10 min or exposing to UV light fully inactivated SARS-CoV-2. Furthermore, we found in SARS-CoV-2-infected primary AEC cultures that treatment of supernatant with UV light, isolation of RNA with Trizol®, isolation of protein using a protocol including sodium dodecyl sulfate (SDS) 0.1% and Triton X100 1%, and fixation of AECs using 10% formalin and Triton X100 1%, each fully inactivated SARS-CoV-2. Full article
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Open AccessStudy Protocol
Novel In Vivo Mouse Cryoablation Model to Explore Unique Therapeutic Approaches for Premalignant Columnar Lesions
Methods Protoc. 2021, 4(1), 6; https://doi.org/10.3390/mps4010006 - 05 Jan 2021
Viewed by 324
Abstract
Patients with epithelial metaplasias have an increased risk of developing malignancies. In Barrett’s esophagus, neo-columnar epithelium develops proximal to the squamous-columnar junction (SCJ) in the esophagus as the result of prolonged exposure to bile and acid reflux. Patients require lifetime periodic surveillance, due [...] Read more.
Patients with epithelial metaplasias have an increased risk of developing malignancies. In Barrett’s esophagus, neo-columnar epithelium develops proximal to the squamous-columnar junction (SCJ) in the esophagus as the result of prolonged exposure to bile and acid reflux. Patients require lifetime periodic surveillance, due to lack of effective eradication therapies. The shortage of innovative treatment options is mostly attributable to the paucity of adequate in vivo models of neo-columnar epithelium regeneration. This protocol describes the generation of a cryoablation model to study regeneration of neo-epithelia at the SCJ. Cryoablation of the columnar and squamous mucosa at the SCJ was achieved through local application of liquid N2O in wild-type and reporter mice in combination with acid suppression. Acid suppression alone, showed restoration of the SCJ with normal histological features of both the neo-columnar and neo-squamous epithelium within 14 days. As a proof of principle, mice were treated with mNoggin, an inhibitor of bone morphogenetic proteins (BMPs), which are involved in the development of columnar epithelia. Local application of mNoggin to the ablated area at the SCJ significantly reduced the development of the neo-columnar mucosa. Although this model does not faithfully recapitulate the exact characteristics of Barrett’s esophagus, it is a well-suited tool to study the mechanisms of therapeutic inhibition of neo-columnar regeneration. It therefore represents an efficient and easy platform to test novel pharmacological therapies for treatment of neo-epithelial lesions at the SCJ. Full article
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Open AccessArticle
Application of microRNA Database Mining in Biomarker Discovery and Identification of Therapeutic Targets for Complex Disease
Methods Protoc. 2021, 4(1), 5; https://doi.org/10.3390/mps4010005 - 30 Dec 2020
Viewed by 468
Abstract
Over the past two decades, it has become increasingly evident that microRNAs (miRNA) play a major role in human diseases such as cancer and cardiovascular diseases. Moreover, their easy detection in circulation has made them a tantalizing target for biomarkers of disease. This [...] Read more.
Over the past two decades, it has become increasingly evident that microRNAs (miRNA) play a major role in human diseases such as cancer and cardiovascular diseases. Moreover, their easy detection in circulation has made them a tantalizing target for biomarkers of disease. This surge in interest has led to the accumulation of a vast amount of miRNA expression data, prediction tools, and repositories. We used the Human microRNA Disease Database (HMDD) to discover miRNAs which shared expression patterns in the related diseases of ischemia/reperfusion injury, coronary artery disease, stroke, and obesity as a model to identify miRNA candidates for biomarker and/or therapeutic intervention in complex human diseases. Our analysis identified a single miRNA, hsa-miR-21, which was casually linked to all four pathologies, and numerous others which have been detected in the circulation in more than one of the diseases. Target analysis revealed that hsa-miR-21 can regulate a number of genes related to inflammation and cell growth/death which are major underlying mechanisms of these related diseases. Our study demonstrates a model for researchers to use HMDD in combination with gene analysis tools to identify miRNAs which could serve as biomarkers and/or therapeutic targets of complex human diseases. Full article
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Open AccessProtocol
An Efficient Workflow for Screening and Stabilizing CRISPR/Cas9-Mediated Mutant Lines in Bombyx mori
Methods Protoc. 2021, 4(1), 4; https://doi.org/10.3390/mps4010004 - 29 Dec 2020
Viewed by 550
Abstract
The domestic silkworm Bombyx mori is extensively studied as a model organism for lepidopteran genetics and has an economic value in silk production. Silkworms also have applications in biomedical and cosmetic industries, and the production of mutant B. mori strains significantly enhances basic [...] Read more.
The domestic silkworm Bombyx mori is extensively studied as a model organism for lepidopteran genetics and has an economic value in silk production. Silkworms also have applications in biomedical and cosmetic industries, and the production of mutant B. mori strains significantly enhances basic and applied silkworm research. In recent years, CRISPR/Cas9 technology is being rapidly adopted as the most efficient molecular tool for generating silkworm lines carrying mutations in target genes. Here we illustrate a complete and efficient workflow to screen, characterize rapidly and follow mutations through generations, allowing the generation of B. mori lines, stably inheriting single CRISPR/Cas9-induced mutations. This approach relies on the use of different molecular methods, the heteroduplex assay, cloning followed by Sanger sequencing, and the amplification refractory mutation system PCR. The use of these methodologies in a sequential combination allows the identification of CRISPR/Cas9-induced mutations in genes mapping on both autosomes and sex chromosomes, and the selection of appropriate individuals to found stable mutant B. mori lines. This protocol could be further applied to screen CRISPR/Cas9 mutations in haploid insects. Full article
(This article belongs to the Special Issue Advances of CRISPR-Cas Systems for Genome Engineering)
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Open AccessProtocol
Purification and Characterization of Recombinant Expressed Apple Allergen Mal d 1
Methods Protoc. 2021, 4(1), 3; https://doi.org/10.3390/mps4010003 - 27 Dec 2020
Viewed by 497
Abstract
Mal d 1 is the primary apple allergen in northern Europe. To explain the differences in the allergenicity of apple varieties, it is essential to study its properties and interaction with other phytochemicals, which might modulate the allergenic potential. Therefore, an optimized production [...] Read more.
Mal d 1 is the primary apple allergen in northern Europe. To explain the differences in the allergenicity of apple varieties, it is essential to study its properties and interaction with other phytochemicals, which might modulate the allergenic potential. Therefore, an optimized production route followed by an unsophisticated purification step for Mal d 1 and respective mutants is desired to produce sufficient amounts. We describe a procedure for the transformation of the plasmid in competent E. coli cells, protein expression and rapid one-step purification. r-Mal d 1 with and without a polyhistidine-tag are purified by immobilized metal ion affinity chromatography (IMAC) and fast-protein liquid chromatography (FPLC) using a high-resolution anion-exchange column, respectively. Purity is estimated by SDS-PAGE using an image-processing program (Fiji). For both mutants an appropriate yield of r-Mal d 1 with purity higher than 85% is achieved. The allergen is characterized after tryptic in gel digestion by peptide analyses using HPLC-MS/MS. Secondary structure elements are calculated based on CD-spectroscopy and the negligible impact of the polyhistidine-tag on the folding is confirmed. The formation of dimers is proved by mass spectrometry and reduction by DTT prior to SDS-PAGE. Furthermore, the impact of the freeze and thawing process, freeze drying and storage on dimer formation is investigated. Full article
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Open AccessArticle
Predictive Quantitative Structure–Activity Relationship Modeling of the Antifungal and Antibiotic Properties of Triazolothiadiazine Compounds
Methods Protoc. 2021, 4(1), 2; https://doi.org/10.3390/mps4010002 - 27 Dec 2020
Viewed by 490
Abstract
Predictive models were developed using two-dimensional quantitative structure activity relationship (QSAR) methods coupled with B3LYP/6-311+G** density functional theory modeling that describe the antimicrobial properties of twenty-four triazolothiadiazine compounds against Aspergillus niger, Aspergillus flavus and Penicillium sp., as well as the bacteria Staphylococcus [...] Read more.
Predictive models were developed using two-dimensional quantitative structure activity relationship (QSAR) methods coupled with B3LYP/6-311+G** density functional theory modeling that describe the antimicrobial properties of twenty-four triazolothiadiazine compounds against Aspergillus niger, Aspergillus flavus and Penicillium sp., as well as the bacteria Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa. B3LYP/6-311+G** density functional theory calculations indicated the triazolothiadiazine derivatives possess only modest variation between the frontier orbital properties. Genetic function approximation (GFA) analysis identified the topological and density functional theory derived descriptors for antimicrobial models using a population of 200 models with one to three descriptors that were crossed for 10,000 generations. Two or three descriptor models provided validated predictive models for antifungal and antibiotic properties with R2 values between 0.725 and 0.768 and no outliers. The best models to describe antimicrobial activities include descriptors related to connectivity, electronegativity, polarizability, and van der Waals properties. The reported method provided robust two-dimensional QSAR models with topological and density functional theory descriptors that explain a variety of antifungal and antibiotic activities for structurally related heterocyclic compounds. Full article
(This article belongs to the Special Issue Methods for Antifungal Discovery and Development)
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Open AccessReview
miRNA Targets: From Prediction Tools to Experimental Validation
Methods Protoc. 2021, 4(1), 1; https://doi.org/10.3390/mps4010001 - 24 Dec 2020
Viewed by 586
Abstract
MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression in both animals and plants. By pairing to microRNA responsive elements (mREs) on target mRNAs, miRNAs play gene-regulatory roles, producing remarkable changes in several physiological and pathological processes. Thus, the identification of miRNA-mRNA target interactions [...] Read more.
MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression in both animals and plants. By pairing to microRNA responsive elements (mREs) on target mRNAs, miRNAs play gene-regulatory roles, producing remarkable changes in several physiological and pathological processes. Thus, the identification of miRNA-mRNA target interactions is fundamental for discovering the regulatory network governed by miRNAs. The best way to achieve this goal is usually by computational prediction followed by experimental validation of these miRNA-mRNA interactions. This review summarizes the key strategies for miRNA target identification. Several tools for computational analysis exist, each with different approaches to predict miRNA targets, and their number is constantly increasing. The major algorithms available for this aim, including Machine Learning methods, are discussed, to provide practical tips for familiarizing with their assumptions and understanding how to interpret the results. Then, all the experimental procedures for verifying the authenticity of the identified miRNA-mRNA target pairs are described, including High-Throughput technologies, in order to find the best approach for miRNA validation. For each strategy, strengths and weaknesses are discussed, to enable users to evaluate and select the right approach for their interests. Full article
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