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Optimized Lambda Exonuclease Digestion or Purification Using Streptavidin-Coated Beads: Which One Is Best for Successful DNA Aptamer Selection?
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Physical Movement Habit Formation in Sedentary Office Workers: Protocol Paper
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Studying miRNA–mRNA Interactions: An Optimized CLIP-Protocol for Endogenous Ago2-Protein
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Inexpensive, Accurate, and Stable Method to Quantitate Blood Alanine Aminotransferase (ALT) Levels
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The Design and Implementation of the ECOVIR Project: A Primary Health Care Surveillance System to Strengthen Co-Detection of Respiratory Viruses in Normandy
Journal Description
Methods and Protocols
Methods and Protocols
is an international, peer-reviewed, open access journal aiming to establish and describe new experimental techniques in the biological and medical sciences, published bimonthly online by MDPI.
- Open Access— free for readers, with article processing charges (APC) paid by authors or their institutions.
- High Visibility: indexed within Scopus, ESCI (Web of Science), PubMed, PMC, CAPlus / SciFinder, and other databases.
- Journal Rank: CiteScore - Q2 (Biochemistry, Genetics and Molecular Biology (miscellaneous))
- Rapid Publication: manuscripts are peer-reviewed and a first decision is provided to authors approximately 26.3 days after submission; acceptance to publication is undertaken in 3.7 days (median values for papers published in this journal in the second half of 2022).
- Recognition of Reviewers: reviewers who provide timely, thorough peer-review reports receive vouchers entitling them to a discount on the APC of their next publication in any MDPI journal, in appreciation of the work done.
Latest Articles
Search and Identification of Amyloid Proteins
Methods Protoc. 2023, 6(1), 16; https://doi.org/10.3390/mps6010016 - 04 Feb 2023
Abstract
Amyloids are fibrillar proteins with a cross-β structure. Pathological amyloids are associated with the development of a number of incurable diseases, while functional amyloids regulate vital processes. The detection of unknown amyloids in living objects is a difficult task, and therefore the question
[...] Read more.
Amyloids are fibrillar proteins with a cross-β structure. Pathological amyloids are associated with the development of a number of incurable diseases, while functional amyloids regulate vital processes. The detection of unknown amyloids in living objects is a difficult task, and therefore the question of the prevalence and biological significance of amyloids remains open. We present a description of two methods, the combination of which makes it possible to find and identify amyloid proteins in the proteome of various organisms. The method of proteomic screening for amyloids allows the detection of the proteins that form SDS-resistant aggregates. SDS resistance is a general feature of amyloid fibrils. Protein aggregates resistant to SDS treatment can be collected by ultracentrifugation and further identified by mass spectrometry. However, in addition to amyloids, SDS-resistant aggregates contain some non-amyloid proteins. To test the amyloid properties of proteins identified by proteomic screening, we developed the method of fibril immunoprecipitation followed by Congo red staining and birefringence analysis. The methods of proteomic screening and immunoprecipitation of fibrillar proteins have been successfully tested and applied for the identification of amyloid proteins in yeast and vertebrates.
Full article
(This article belongs to the Section Molecular and Cellular Biology)
Open AccessProtocol
A Production and Fractionation Protocol for Polyvinyl Chloride Microplastics
Methods Protoc. 2023, 6(1), 15; https://doi.org/10.3390/mps6010015 - 31 Jan 2023
Abstract
Concerns about the presence of microplastics in the environment has increased in recent years, prompting more attention from scientists. Thorough exposure studies using artificially produced microplastics containing additives are required to assess their potentially hazardous effects. Therefore, an efficient microplastic production and fractionation
[...] Read more.
Concerns about the presence of microplastics in the environment has increased in recent years, prompting more attention from scientists. Thorough exposure studies using artificially produced microplastics containing additives are required to assess their potentially hazardous effects. Therefore, an efficient microplastic production and fractionation protocol was established using a cryogenic grinding and wet-sieving approach. The developed cryogenic grinding method was able to produce (20–40 g/h) polyvinyl chloride (PVC) microplastics having a volume-weighted mean particle size of 391 µm and a span of 2.12. Performing a second grinding cycle on the same particles resulted in microplastics which were smaller (volume-weighted mean size = 219 μm) and had a narrower particle size distribution (span = 1.59). In addition, the microplastics were also fractionated into different particle size ranges using a vibrating wet-sieving tower. The latter technique allowed separating 10 g of PVC microplastics into seven different fractions using six sieves (Ø 200 mm) for 30 min while shaking. By using the developed method, PVC microplastics could easily be made and fractionated into desired particle-size ranges. The proposed protocol could also be adjusted to produce and fractionate microplastics of other plastics.
Full article
(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
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Open AccessProtocol
Optimized Protocol for Isolation and Culture of Murine Neonatal Primary Lung Fibroblasts
Methods Protoc. 2023, 6(1), 14; https://doi.org/10.3390/mps6010014 - 24 Jan 2023
Abstract
During all the stages of lung development, the lung mesoderm (or mesenchyme) is closely related to the endoderm, and their cross-regulation promotes, controls, and drives all lung developmental processes. Generation of 3D organoids in vitro, RNA assays, and mitochondrial respiration studies are used
[...] Read more.
During all the stages of lung development, the lung mesoderm (or mesenchyme) is closely related to the endoderm, and their cross-regulation promotes, controls, and drives all lung developmental processes. Generation of 3D organoids in vitro, RNA assays, and mitochondrial respiration studies are used to analyze lung development and regeneration to better understand the interactions between epithelium and mesenchyme, as well as for the study of redox alterations and the metabolic status of the cells. Moreover, to avoid using immortalized cell lines in these in vitro approaches, standardized murine neonatal primary lung fibroblast isolation techniques are essential. Here, we present an optimized method to isolate, culture, and freeze primary lung fibroblasts from neonatal lungs. Our current method includes step-by-step instructions accompanied by graphical explanations and critical steps.
Full article
(This article belongs to the Section Molecular and Cellular Biology)
Open AccessStudy Protocol
The Changes in Red Blood Cell Indices That Occur in Pre-Diabetic Patients of all Ethnicities from the 25–45 Years of Age: A Protocol for a Systematic Review and Meta-Analysis
by
, , and
Methods Protoc. 2023, 6(1), 13; https://doi.org/10.3390/mps6010013 - 24 Jan 2023
Abstract
Introduction: Pre-diabetes is an intermediate, asymptomatic state between normoglycaemia and the onset of type 2 diabetes mellitus (T2D). Recent reports indicate that there are sub-clinical changes observed in red blood cells during pre-diabetes. This systematic review protocol will provide an outline of
[...] Read more.
Introduction: Pre-diabetes is an intermediate, asymptomatic state between normoglycaemia and the onset of type 2 diabetes mellitus (T2D). Recent reports indicate that there are sub-clinical changes observed in red blood cells during pre-diabetes. This systematic review protocol will provide an outline of all procedures in the synthesis of the available data on the changes in red blood cell indices. Methods and Analysis: This protocol was prepared by adhering to the PRISMA 2015 guidelines for reporting protocols. Published clinical studies that involve observation, whether it is cross-sectional, comparative cross-sectional, case-control or cohort study designs that involve normal/non-diabetic and pre-diabetes reports were used. Additionally, this was accomplished by using clinical MeSH headings to search on MEDLINE, COCHRANE library and African Journal Online. Three reviewers (NCM, AMS & AK) screened all the results for eligibility criteria. Then, Downs and Black checklist was used to check the risk of bias. Review Manager v5.4 Forrest plot was used for meta-analysis and sensitivity analysis. Strength of evidence was then assessed using the Grading of Recommendations Assessment, Development, and Evaluation approach (GRADE). Results and Conclusion: This protocol will give direction on the exploration of articles that report on changes in red blood cell indices in the pre-diabetic state. The results obtained from this protocol will further give direction on the research to be done at in the eThekwini district of South Africa. Ethics and Dissemination: The data that will be analyzed will be data that has already been published thus there will be no data collection from subjects. Therefore, no ethical clearance is required. Registration Details: This protocol has been registered with the International Prospective Registry of Systematic Reviews (PROSPERO) registration number “CRD42020189080” dated 05-07-2020.
Full article
(This article belongs to the Section Biomedical Sciences and Physiology)
Open AccessProtocol
Protocol for a Randomized Control Trial for Tungiasis Treatment in Homa Bay County, Kenya: Dimeticone versus Sodium Carbonate
Methods Protoc. 2023, 6(1), 12; https://doi.org/10.3390/mps6010012 - 23 Jan 2023
Abstract
Tungiasis, a World Health Organization neglected tropical disease, is caused by the female sand flea. Most clinical trials for tungiasis use expensive or impractical drugs, which are difficult for residents to use. However, in western Kenya, communities successfully treat tungiasis with sodium carbonate.
[...] Read more.
Tungiasis, a World Health Organization neglected tropical disease, is caused by the female sand flea. Most clinical trials for tungiasis use expensive or impractical drugs, which are difficult for residents to use. However, in western Kenya, communities successfully treat tungiasis with sodium carbonate. We hypothesise that the topical risk-difference of 5% sodium carbonate is no more than 10% non-inferior to dimeticone (NYDA®) for tungiasis treatment. This is a protocol for a non-inferiority study, which will be randomised and with an observer-blinded control. The study will have two arms: 5% sodium carbonate and NYDA®, one on each foot, and will take place at state primary schools in Homa Bay County, Kenya. Fleas identified among school children aged 8–14 years with sand-flea lesions will be enrolled in the study. For each participant, the viability of the embedded fleas, clinical signs including inflammation, and symptoms will be monitored for seven days after treatment. The proportion of dead fleas will be compared in the primary analysis. All adverse events will be monitored throughout the study period. We expect to identify the most effective treatment between sodium carbonate and NYDA® for tungiasis, which can be adopted in the community.
Full article
(This article belongs to the Section Public Health Research)
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Open AccessArticle
Quantitative 1H Nuclear Magnetic Resonance (qNMR) of Aromatic Amino Acids for Protein Quantification
Methods Protoc. 2023, 6(1), 11; https://doi.org/10.3390/mps6010011 - 23 Jan 2023
Abstract
Hydrolysis of protein samples into amino acids facilitates the use of NMR spectroscopy for protein and peptide quantification. Different conditions have been tested for quantifying aromatic amino acids and proteins. The pH-dependent signal shifts in the aromatic region of amino acid samples were
[...] Read more.
Hydrolysis of protein samples into amino acids facilitates the use of NMR spectroscopy for protein and peptide quantification. Different conditions have been tested for quantifying aromatic amino acids and proteins. The pH-dependent signal shifts in the aromatic region of amino acid samples were examined. A pH of 12 was found to minimize signal overlap of the four aromatic amino acids. Several aromatic compounds, such as terephthalic acid, sulfoisophthalic acid, and benzene tricarboxylic acid, were applied as internal standards. The quantification of amino acids from an amino acid standard was performed. Using the first two suggested internal standards, recovery was ~97% for histidine, phenylalanine, and tyrosine at a concentration of approximately 1 mM in solution. Acidic hydrolysis of a certified reference material (CRM) of bovine serum albumin (BSA) and subsequent quantification of Phe and Tyr yielded recoveries of 98% ± 2% and 88% ± 4%, respectively, at a protein concentration of 16 g/L or 250 µM.
Full article
(This article belongs to the Special Issue Methods and Protocols 2022)
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Open AccessProtocol
Ion Selective Electrode (ISE) Method for Determination of Total Fluorine and Total Organic Fluorine in Packaging Substrates
by
, , , and
Methods Protoc. 2023, 6(1), 10; https://doi.org/10.3390/mps6010010 - 18 Jan 2023
Abstract
Various testing methods and techniques have been used to identify and quantify per- and polyfluoroalkyl substances (PFAS) in food packaging. A common indirect measurement of PFAS is total fluorine (TF) and total organic fluorine (TOF). These methods are critical in rapidly screening food
[...] Read more.
Various testing methods and techniques have been used to identify and quantify per- and polyfluoroalkyl substances (PFAS) in food packaging. A common indirect measurement of PFAS is total fluorine (TF) and total organic fluorine (TOF). These methods are critical in rapidly screening food packaging materials for the >9000 PFAS and are often globally used for regulatory limits. However, this destructive approach requires careful sample preparation, combustion, and the analysis of the solution by a fluoride-specific electrode. The method described herein is a cost-effective, rapid, quantitative, and externally validated initial screening of packaging materials for fluoro-chemistry. This study presents validated protocols for measuring TF and TOF in packaging substrates using oxygen combustion sample preparation coupled with fluoride ion-selective electrode (F-ISE); the materials and required equipment are provided, and the step-by-step procedure from sample preparation to the analysis are described, including critical steps to minimize contamination and interferences during sample preparation.
Full article
(This article belongs to the Special Issue Analytical Methodologies in Food Authenticity and Traceability)
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Open AccessEditorial
Acknowledgment to the Reviewers of Methods and Protocols in 2022
Methods Protoc. 2023, 6(1), 9; https://doi.org/10.3390/mps6010009 - 16 Jan 2023
Abstract
High-quality academic publishing is built on rigorous peer review [...]
Full article
Open AccessProtocol
Advanced Flow Cytometry Using the SYTO-13 Dye for the Assessment of Platelet Reactivity and Maturity in Whole Blood
by
, , , , and
Methods Protoc. 2023, 6(1), 8; https://doi.org/10.3390/mps6010008 - 13 Jan 2023
Abstract
Newly produced immature platelets are larger, contain higher amounts of residual RNA, and are more reactive than mature platelets. Flow cytometry using the SYTO-13 dye is a method for the subdivision of immature platelets from mature platelets based on the labelling of intracellular
[...] Read more.
Newly produced immature platelets are larger, contain higher amounts of residual RNA, and are more reactive than mature platelets. Flow cytometry using the SYTO-13 dye is a method for the subdivision of immature platelets from mature platelets based on the labelling of intracellular platelet RNA, enabling the simultaneous investigation of the reactivity of each platelet population. This method provides detailed information on several aspects of platelet physiology using a combination of platelet surface markers and agonists. Currently, no standardized protocol exists across laboratories. Here, we describe a flow cytometry protocol in detail to investigate platelet reactivity and its relation to platelet maturity. We analyzed 20 healthy individuals with the protocol and compared the platelet subpopulation with the highest SYTO-13 labelling (in the first quintile, “SYTO-high”) corresponding to the most immature platelets (highest RNA content) with the platelet subpopulation with the lowest SYTO-13 labelling (in the fifth quintile, “SYTO-low”) corresponding to the mature platelets with the lowest RNA content. SYTO-high platelets had overall significantly increased platelet reactivity compared with that of SYTO-low platelets. The presented method may be a valuable research tool for the analysis of platelet reactivity and its relation to platelet maturity.
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(This article belongs to the Special Issue Methods and Protocols 2022)
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Open AccessProtocol
Efficacy and Long-Term Safety of Ibuprofen Gargle for Oral Lichen Planus: A Study Protocol of Randomized Crossover and Long-Term Extension Trials
by
, , , , , , , , and
Methods Protoc. 2023, 6(1), 7; https://doi.org/10.3390/mps6010007 - 10 Jan 2023
Abstract
Oral lichen planus (OLP) is a type of chronic and refractory stomatitis characterized by abnormal keratinization, which is often painful. There is no consensus regarding treatment options for OLP, particularly in the presence of pain. The current study protocol focuses on the short-term
[...] Read more.
Oral lichen planus (OLP) is a type of chronic and refractory stomatitis characterized by abnormal keratinization, which is often painful. There is no consensus regarding treatment options for OLP, particularly in the presence of pain. The current study protocol focuses on the short-term efficacy and long-term safety of an ibuprofen gargle for pain management in patients with OLP. Patients (n = 24) with painful OLP will be enrolled. During a crossover study period, patients in the ibuprofen–placebo (IP) group will receive an ibuprofen gargle (0.6%) on day 1, a placebo gargle on day 2, and an ibuprofen gargle on days 3–5 at least once daily. Patients in the placebo–ibuprofen (PI) group will receive a placebo gargle on day 1, an ibuprofen gargle on day 2, and an ibuprofen gargle on days 3–5 at least once daily. The primary endpoint of the crossover study period is the change in pain level as measured by a visual analogue scale score from before gargle administration to 5 min after gargle administration on days 1 and 2. The primary endpoint of the long-term extension study is assessment of long-term safety. The results of this study may support existing evidence regarding the effectiveness of ibuprofen rinses in treating OLP.
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(This article belongs to the Section Public Health Research)
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Open AccessArticle
Adsorption Kinetic Model Predicts and Improves Reliability of Electrochemical Serotonin Detection
Methods Protoc. 2023, 6(1), 6; https://doi.org/10.3390/mps6010006 - 09 Jan 2023
Abstract
Serotonin (5-HT) is a neurotransmitter involved in many biophysiological processes in the brain and in the gastrointestinal tract. Electrochemical methods are commonly used to quantify 5-HT, but their reliability may suffer due to the time-dependent nature of adsorption-limited 5-HT detection, as well as
[...] Read more.
Serotonin (5-HT) is a neurotransmitter involved in many biophysiological processes in the brain and in the gastrointestinal tract. Electrochemical methods are commonly used to quantify 5-HT, but their reliability may suffer due to the time-dependent nature of adsorption-limited 5-HT detection, as well as electrode fouling over repeated measurements. Mathematical characterization and modeling of adsorption-based electrochemical signal generation would improve reliability of 5-HT measurement. Here, a model was developed to track 5-HT electrode adsorption and resulting current output by combining Langmuir adsorption kinetic equations and adsorption-limited electrochemical equations. 5-HT adsorption binding parameters were experimentally determined at a carbon-nanotube coated Au electrode: KD = 7 × 10−7 M, kon = 130 M−1 s−1, koff = 9.1 × 10−5 s−1. A computational model of 5-HT adsorption was then constructed, which could effectively predict 5-HT fouling over 50 measurements (R2 = 0.9947), as well as predict electrode responses over varying concentrations and measurement times. The model aided in optimizing the measurement of 5-HT secreted from a model enterochromaffin cell line—RIN14B—minimizing measurement time. The presented model simplified and improved the characterization of 5-HT detection at the selected electrode. This could be applied to many other adsorption-limited electrochemical analytes and electrode types, contributing to the improvement of application-specific modeling and optimization processes.
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(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
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Open AccessProtocol
COVID-19 Related Protocol Considerations and Modifications within a Rural, Community-Engaged Health Promotion Randomized Trial
by
, , , , and
Methods Protoc. 2023, 6(1), 5; https://doi.org/10.3390/mps6010005 - 05 Jan 2023
Abstract
Rural communities are at higher risk for physical inactivity, poor dietary behaviors, and related chronic diseases and obesity. These disparities are largely driven by built environment, socioeconomic, and social factors. A community-based cluster randomized controlled trial of an intervention, the Change Club, aims
[...] Read more.
Rural communities are at higher risk for physical inactivity, poor dietary behaviors, and related chronic diseases and obesity. These disparities are largely driven by built environment, socioeconomic, and social factors. A community-based cluster randomized controlled trial of an intervention, the Change Club, aims to address some of these disparities via civic engagement for built environment change. Baseline data collection began in February 2020, only to be paused by the COVID-19 pandemic. In this context, the investigators evaluated multiple approaches for collecting data when the study resumed, focusing on Life’s Simple 7, and additional anthropometric, physiologic, and behavioral outcomes in rural and micropolitan (<50,000 population) communities in Texas and New York. Life’s Simple 7 includes fasting blood glucose, total cholesterol, blood pressure, weight, physical activity, diet, and smoking. Rigor and feasibility were considered across a variety of in-person versus at-home measurement options. After a comprehensive input from participants, partners, staff, researchers, and the funding liaison, the study team chose self-measurement and use of validated questionnaires/surveys to measure the Life’s Simple 7 components. This case provides an example of how a study team might adjust data collection protocol during unexpected and acute events while giving consideration to rigor, feasibility, stakeholder views, and participants’ health and safety.
Full article
(This article belongs to the Section Public Health Research)
Open AccessProtocol
Analytical Validation of SOD2 Genotyping
Methods Protoc. 2023, 6(1), 4; https://doi.org/10.3390/mps6010004 - 31 Dec 2022
Abstract
Manganese superoxide dismutase-2 (SOD2) plays a crucial role in cells’ protection against mitochondrial oxidative damage. A genetic polymorphism in the mitochondrial targeting sequence of the SOD2 gene has been implicated in various diseases, including prostate cancer. Paller et al. have shown
[...] Read more.
Manganese superoxide dismutase-2 (SOD2) plays a crucial role in cells’ protection against mitochondrial oxidative damage. A genetic polymorphism in the mitochondrial targeting sequence of the SOD2 gene has been implicated in various diseases, including prostate cancer. Paller et al. have shown an increase in prostate-specific antigen (PSA) doubling time in patients with the Ala/Ala (wildtype) genotype when treated with pomegranate/grape extract antioxidants. We developed and validated a pyrosequencing assay that detects the common germline SOD2 SNP (rs_4880) with the aim of identifying men with castrate-resistant prostate cancer eligible for an antioxidant therapy clinical trial. We first selected 37 samples from the 1000 genomes study with known genotypes determined using Illumina-based sequencing and confirmed them by Sanger sequencing. In a blinded design, we then performed the new pyrosequencing assay on these samples and assigned genotypes. Genotypes for all 37 samples (13 homozygous Ala, 12 heterozygous Ala/Val, and 12 homozygous Val) were all concordant by pyrosequencing. The pyrosequencing assay has been live since May 2018 and has proven to be robust and accurate.
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(This article belongs to the Section Biomedical Sciences and Physiology)
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Open AccessProtocol
Isolation and Cultivation of Adipose-Derived Mesenchymal Stem Cells Originating from the Infrapatellar Fat Pad Differentiated with Blood Products: Method and Protocol
by
, , , , , and
Methods Protoc. 2023, 6(1), 3; https://doi.org/10.3390/mps6010003 - 25 Dec 2022
Abstract
Adipose-derived mesenchymal stem cells (ASCs) are a promising source for clinical application in regenerative orthopedics. ASCs derived from the infra-patellar fat pad (IFP)—a distinct adipose structure in the knee—show superior regenerative potential compared to subcutaneous-fat-derived cells. Furthermore, it has been shown that blood
[...] Read more.
Adipose-derived mesenchymal stem cells (ASCs) are a promising source for clinical application in regenerative orthopedics. ASCs derived from the infra-patellar fat pad (IFP)—a distinct adipose structure in the knee—show superior regenerative potential compared to subcutaneous-fat-derived cells. Furthermore, it has been shown that blood products enhance ASCs’ viability. A major challenge for clinical translation of both ASCs and blood products is the low comparability of obtained data due to non-standardized harvesting, isolation and preparation methods. The aim of this method-paper is to provide reproducible protocols to help standardize basic research in the field to build a sound basis for clinical translation with an emphasize on practicability. The presented protocols include (i) ASC isolation from the IFP, (ii) blood product preparation and (iii) ASC incubation with blood products.
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(This article belongs to the Section Molecular and Cellular Biology)
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Open AccessProtocol
Identifying Gut Microbiota Conditions Associated with Disease in the African Continent: A Scoping Review Protocol
Methods Protoc. 2023, 6(1), 2; https://doi.org/10.3390/mps6010002 - 24 Dec 2022
Abstract
The gut microbiota has been immensely studied over the past years because of its involvement in the pathogenesis of numerous diseases. However, gut microbiota data in Africa are limited. Therefore, it is crucial to have studies that reflect various populations in order to
[...] Read more.
The gut microbiota has been immensely studied over the past years because of its involvement in the pathogenesis of numerous diseases. However, gut microbiota data in Africa are limited. Therefore, it is crucial to have studies that reflect various populations in order to fully capture global microbial diversity. In the proposed scoping review, we will describe the gut microbiota’s appearance in terms of gut microbiota markers, in both health and disease in African populations. Relevant publications will be searched for in the PubMed, Scopus, Web of Science, Academic Search Premier, Africa-Wide Information, African journals online, CINAHL, and EBSCOhost and Embase databases. We will focus on articles published between January 2005 and March 2023. We will also determine if the studies to be included in the review would provide enough data to identify quantifiable gut microbiome traits that could be used as health or disease markers, identify the types of diseases that were mostly focused on in relation to gut microbiota research in Africa, as well as to discover and analyze knowledge gaps in the gut microbiota research field in the continent. We will include studies involving African countries regardless of race, gender, age, health status, disease type, study design, or care setting. Two reviewers will conduct a literature search and screen the titles/abstracts against the eligibility criteria. The reviewers will subsequently screen full-text articles and identify studies that meet the inclusion criteria. This will be followed by charting the data using a charting tool and analysis of the evidence. The proposed scoping review will follow a qualitative approach such that a narrative summary will accompany the tabulated/graphical results which will describe how the results relate to the review objectives and questions. As a result, this review may play a significant role in the identification of microbiota-related adjunctive therapies in the African region where multiple comorbidities coexist. Scoping review registration: Open Science Framework.
Full article
(This article belongs to the Section Public Health Research)
Open AccessArticle
Engineering Ag43 Signal Peptides with Bacterial Display and Selection
Methods Protoc. 2023, 6(1), 1; https://doi.org/10.3390/mps6010001 - 23 Dec 2022
Abstract
Protein display, secretion, and export in prokaryotes are essential for utilizing microbial systems as engineered living materials, medicines, biocatalysts, and protein factories. To select for improved signal peptides for Escherichia coli protein display, we utilized error-prone polymerase chain reaction (epPCR) coupled with single-cell
[...] Read more.
Protein display, secretion, and export in prokaryotes are essential for utilizing microbial systems as engineered living materials, medicines, biocatalysts, and protein factories. To select for improved signal peptides for Escherichia coli protein display, we utilized error-prone polymerase chain reaction (epPCR) coupled with single-cell sorting and microplate titer to generate, select, and detect improved Ag43 signal peptides. Through just three rounds of mutagenesis and selection using green fluorescence from the 56 kDa sfGFP-beta-lactamase, we isolated clones that modestly increased surface display from 1.4- to 3-fold as detected by the microplate plate-reader and native SDS-PAGE assays. To establish that the functional protein was displayed extracellularly, we trypsinized the bacterial cells to release the surface displayed proteins for analysis. This workflow demonstrated a fast and high-throughput method leveraging epPCR and single-cell sorting to augment bacterial surface display rapidly that could be applied to other bacterial proteins.
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(This article belongs to the Special Issue Methods and Protocols 2022)
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Open AccessStudy Protocol
Association of Ultrasonography and Health Education during Antenatal Visits among Pregnant Women to Reduce Unnecessary Caesarean Section in Bangladesh: Study Protocol for a Cluster Randomized Control Trial
by
, , , , , , and
Methods Protoc. 2022, 5(6), 101; https://doi.org/10.3390/mps5060101 - 17 Dec 2022
Abstract
The rate of caesarean section (C/S) has been increasing globally. The proposed study aims to evaluate the effects of ultrasonography (USG) and health education in reducing unnecessary C/S among pregnant women. The secondary objectives are to increase antenatal care (ANC) and post-natal care
[...] Read more.
The rate of caesarean section (C/S) has been increasing globally. The proposed study aims to evaluate the effects of ultrasonography (USG) and health education in reducing unnecessary C/S among pregnant women. The secondary objectives are to increase antenatal care (ANC) and post-natal care (PNC) visit rates, increase USG use, increase institutional (hospitals and clinics) delivery, reduce delivery related complications and reduce the number of still births. This is a prospective, open-label, two-arm cluster randomized controlled trial (RCT). A total of 288 pregnant women are enrolled from two urban and two rural hospitals in Bangladesh. Women were screened during their first ANC visits, then written informed consent was taken from the participants. Women in intervention hospitals receive eight ANC visits, two additional USG, and health education eight times during their ANC visits. In contrast, participants at control hospitals receive usual care consisting of four ANC visits and two USG during their ANC visits. The primary outcome is to compare the rate of unnecessary C/S during PNC visits. This study obtained approval from the Institutional Review Board of North South University (2021/OR-NSU/IRB/0804), Bangladesh in November 2021 and was registered in clinicalTrails.gov (#NCT05135026).
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(This article belongs to the Section Public Health Research)
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Open AccessArticle
Development and Characterization of a Luminescence-Based High-Throughput Serum Bactericidal Assay (L-SBA) to Assess Bactericidal Activity of Human Sera against Nontyphoidal Salmonella
Methods Protoc. 2022, 5(6), 100; https://doi.org/10.3390/mps5060100 - 16 Dec 2022
Abstract
Salmonella Typhimurium and Salmonella Enteritidis are leading causative agents of invasive nontyphoidal Salmonella (iNTS) disease, which represents one of the major causes of death and morbidity in sub-Saharan Africa, still partially underestimated. Large sero-epidemiological studies are necessary to unravel the burden of disease
[...] Read more.
Salmonella Typhimurium and Salmonella Enteritidis are leading causative agents of invasive nontyphoidal Salmonella (iNTS) disease, which represents one of the major causes of death and morbidity in sub-Saharan Africa, still partially underestimated. Large sero-epidemiological studies are necessary to unravel the burden of disease and guide the introduction of vaccines that are not yet available. Even if no correlate of protection has been determined so far for iNTS, the evaluation of complement-mediated functionality of antibodies generated towards natural infection or elicited upon vaccination may represent a big step towards this achievement. Here we present the setup and the intra-laboratory characterization in terms of repeatability, intermediate precision, linearity, and specificity of a high-throughput luminescence-based serum bactericidal assay (L-SBA). This method could be useful to perform sero-epidemiological studies across iNTS endemic countries and for evaluation of antibodies raised against iNTS vaccine candidates in upcoming clinical trials.
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(This article belongs to the Section Omics and High Throughput)
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Open AccessProtocol
RIAM: A Universal Accessible Protocol for the Isolation of High Purity DNA from Various Soils and Other Humic Substances
by
, , , , and
Methods Protoc. 2022, 5(6), 99; https://doi.org/10.3390/mps5060099 - 16 Dec 2022
Abstract
A single universal open protocol RIAM (named after Research Institute for Agricultural Microbiology) for the isolation of high purity DNA from different types of soils and other substrates (high and low in humic, clay content, organic fertilizer, etc.) is proposed. The main features
[...] Read more.
A single universal open protocol RIAM (named after Research Institute for Agricultural Microbiology) for the isolation of high purity DNA from different types of soils and other substrates (high and low in humic, clay content, organic fertilizer, etc.) is proposed. The main features of the RIAM protocol are the absence of the sorption–desorption stage on silica columns, the use of high concentrations of phosphate in buffers, which prevents DNA sorption on minerals, and DNA precipitation using CTAB. The performance of RIAM was compared with a reference commercial kit and showed very good results in relation to the purity and quantity of DNA, as well as the absence of inhibitory activity on PCR. In all cases, the RIAM ensured the isolation of DNA in quantities much greater than the commercial kit without the effect of PCR inhibition up to 50 ng DNA per reaction in a volume of 15 µL. The latter circumstance along with the ability of the protocol to extract low molecular weight DNA fractions makes the method especially suitable for those cases where quantitative assessments, detection of minor components of soil microbiota, and completeness of isolation of all DNA fractions are required.
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(This article belongs to the Section Molecular and Cellular Biology)
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Open AccessProtocol
The Design and Implementation of the ECOVIR Project: A Primary Health Care Surveillance System to Strengthen Co-Detection of Respiratory Viruses in Normandy
by
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Methods Protoc. 2022, 5(6), 98; https://doi.org/10.3390/mps5060098 - 13 Dec 2022
Abstract
Acute respiratory infections (ARIs) need to be better understood and treated, as they are critical to public health, especially during crises such as the SARS-CoV2 pandemic. These are the most abundant infections in the general population and are seen primarily in primary care
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Acute respiratory infections (ARIs) need to be better understood and treated, as they are critical to public health, especially during crises such as the SARS-CoV2 pandemic. These are the most abundant infections in the general population and are seen primarily in primary care by general practitioners (GPs). Many different viruses are involved, according to epidemic variations. Viral co-detections account for a significant proportion of ARIs in hospital cohorts. The objective of the ECOVIR cohort was to study viral co-detections by setting up a biobank of respiratory tract samples from patients consulting their general practitioner for ARI symptoms. We report here on the course of the study: the design, the conduct, and the difficulties encountered. ECOVIR (Etude des CO-detections VIrales dans les prélèvements Respiratoires) was a prospective, multicenter cohort conducted in France during two epidemic seasons (2018–2019 and 2019–2020). We recruited GPs. Each GP investigator (GPI) saw patients weekly for examination, clinical data collection, and nasopharyngeal swabbing. Each sample was sent to the virology unit for biobanking and molecular analysis. Clinical and sociodemographic data were collected 7 days after inclusion. ECOVIR involved 36 GPIs. Patients with symptoms of an ARI were included (n = 685). The median number of inclusions was 16 patients per GPI over both seasons (IC25–75% [4.75; 27]). Patients aged 18 to 64 years were the most numerous (57%), followed by children (30%), and the elderly (13% over 65 years). This age distribution emphasizes the young adult and middle-aged population. Residents participated in the project and called patients on day 7 to obtain clinical and sociodemographic data. Our study triggered the creation of an original network, which plans to establish a functional link between research and primary health care. Primary care is unfortunately poorly represented in research protocols, particularly in respiratory infections, even though it is a cornerstone of our French health care system, as demonstrated every day in this period of crisis.
Full article
(This article belongs to the Section Public Health Research)
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