Methods Protoc., Volume 6, Issue 1 (February 2023) – 20 articles

The potential of antioxidants in preventing several diseases has attracted great attention in recent years. Indeed, these products are part of a multi-billion industry. However, there is a lack of scientific information about safety, quality, doses, and changes over time. In the present work, a simple multisample methodology based on chemiluminiscent imaging to determine chlorogenic acid (CHLA) in green coffee samples has been proposed. The multi-chemiluminiscent response was obtained after a luminol-persulfate reaction at pH 10.8 in a multiplate followed by image capture with a charge-coupled device (CCD) camera as a readout system. The chemiluminiscent image was used as an analytical response by measuring the luminescent intensity at 0 °C with the CCD camera. Under the optimal conditions, the detection limit was 20 µM and precision was also adequate with RSD < 12%. The accuracy of the proposed system was evaluated by studying the matrix effect, using a standard addition method. Recoveries of chlorogenic acid ranged from 93–94%. The use of the CCD camera demonstrated advantages such as analysis by image inspection, portability, and easy-handling which is of particular relevance in the application for quality control in industries. Furthermore, multisample analysis was allowed by one single image saving time, energy, and cost. The proposed methodology is a promising sustainable analytical tool for quality control to ensure green coffee safety through dosage control and proper labelling preventing potential frauds. Full article

Patients feel more vulnerable when accessing community mental health programs for the first time or after being discharged from psychiatric inpatient units. Long wait times for follow-up appointments, shortage of mental health professionals, lack of service integration, and scarcity of tailored support can weaken their connection to the health care system. As a result, patients can present low adherence, dissatisfaction with treatment, and recurrent hospitalizations. Finding solutions to avoid unnecessary high-cost services and providing tailored and cost-effective mental health interventions may reduce the health system burden and augment patient support. We propose implementing an add-on, supportive text messaging service (Text4Support), developed using cognitive–behavioural therapy (CBT) principles to augment mental health support for patients attending to or being discharged from psychiatric care in Nova Scotia, Canada. This randomized controlled trial aims to investigate the effectiveness of Text4Support in improving mental health outcomes and overall mental well-being compared with usual care. We also will examine the intervention’s impact on health services utilization and patient satisfaction. The results from this study will provide evidence on stepped and technology-based mental health care, which will contribute to generating new knowledge about mental health innovations in various clinical contexts, which is not only helpful for the local context but to other jurisdictions in Canada and abroad that are seeking to improve their health care. Full article

Flow cytometry gives a unique opportunity to analyze thousands of individual cells for multiple parameters in a course of minutes. The most commonly used flow cytometry application in plant biology is estimation of nuclear DNA content. This becomes an indispensable tool in different areas of plant research, including breeding, taxonomy, plant development, evolutionary biology, populational studies and others. DNA content analysis can provide an insight into natural ploidy changes that reflect evolutionary processes, such as interspecific hybridization and polyploidization. It is also widely used for processing samples with biotechnologically induced ploidy changes, for instance, plants produced by doubled haploid technology. Absolute genome size data produced by cytometric analysis serve as useful taxon-specific markers since genome size vary between different taxa. It often allows the distinguishing of species within a genus or even different subspecies. Introducing flow cytometry method in the lab is extremely appealing, but new users face a significant challenge of learning instrument management, quality sample preparation and data processing. Not only is flow cytometry a complex method, but plant samples have unique features that make plants a demanding research subject. Without proper training, researchers risk damaging the expensive instrument or publishing poor quality data, artifacts or unreproducible results. We bring together information from our experience, key papers and online resources to provide step by step protocols and give a starting point for exploring the abundant cytometry literature. Full article

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous organic compounds coming from natural or anthropogenic activities. Tree organs such as leaves and barks have been used to monitor urban air quality and have achieved remarkable ecological importance. However, the potential of many tree species as biomonitors is still unknown and efforts should be focused on conducting studies that analyze their capabilities with a viable analytical method. In this work, an analytical method for quantification of the 16 EPA priority PAHs from the leaves and bark of Sambucus nigra was validated. In general, the method showed good linearity, detection limits, precision, and recoveries, demonstrating that it is suitable for analyzing PAHs in both the leaves and bark of the Sambucus nigra species for which no analytical method for PAHs is yet available. The high prevalence of fluoranthene in the samples, which is a PAH related to coal combustion and biomass burning, and benzo[a]pyrene, which has a carcinogenic effect, was identified. Full article

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

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

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

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

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

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

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

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. Full article

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. Full article

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: K_D = 7 × 10⁻⁷ M, k_on = 130 M⁻¹ s⁻¹, k_off = 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. Full article

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

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. Full article

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. Full article

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

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. Full article