Methods Protoc., Volume 5, Issue 2 (April 2022) – 15 articles

Lipedema is a multifaceted chronic fat disorder characterized by the bilateral and disproportionate accumulation of fat predominantly in the lower body regions of females. Research strongly supports that estrogen factors likely contribute to the pathophysiology of this disease. We aim to help demonstrate this link by quantifying estrogen factor differences between women with and without lipedema. For time and lipedema adipose tissue conservation, the Protein Simple WES machine will be utilized in place of traditional western blotting. Here, we are interested in evaluating estrogen related factors, such as, but not limited to, estrogen receptors and enzymes involved in the successive conversions of cholesterol and androgens to estrogens in human subcutaneous adipose. Evaluation of these factors within adipose tissue, however, is novel for this instrument. Thus, we optimized tissue lysis and protein extraction for 11 proteins of interest. Antibodies and their working concentrations were determined based upon specific and distinguishable (signal-to-noise) peaks from electropherogram outputs across different tissue lysate concentrations. We found that overnight acetone precipitation proved to be the best procedure for extracting protein from lipid rich adipose tissue samples. Six of the eleven proteins were found to migrate to their expected molecular weights, however, five did not. For proteins that did not migrate as expected, overexpression lysates and empty vector controls were used to validate detection antibodies. Protein extract from subcutaneous adipose tissue and overexpression lysates were then combined to understand if migration was specifically altered by adipose tissue. From these results, we concluded that the lipid rich nature of adipose tissue in combination with the separation matrix designated for use with the WES were preventing the appropriate migration of some proteins rather than non-specific antibody binding or inappropriate preparation methods. Full article

Background: Local food environments are recognized by experts as a determinant of healthy eating. Food cooperatives (coop) can promote the accessibility to healthier foods and thus improve the health of the population, particularly in remote rural communities. Objective: To measure the effects of implementing a food coop in a disadvantaged community with poor access to food. We have two main research questions: (1). Does the establishment of a food coop in rural areas described as food deserts have an impact on accessibility, frequency of use, food consumption, food quality, and ultimately the health of individuals? (2). Does the establishment of a food coop in rural areas described as food deserts have an impact on food security and community vitality? Design: A natural experiment with a mixed pre/post method will be used. The sample is composed of households that came from geographically isolated communities (population: 215 to 885 inhabitants) which qualified as food deserts and located in rural areas of Quebec (Canada). All communities plan to open a food coop (in the years 2022–2023), and as their opening will be staggered over time, participants from communities with a new food coop (intervention) will be compared to communities awaiting the opening of their food coop (control). Data collection was carried out at three time points: (1) before; (2) 1 to 5 months after; and (3) 13 to 17 months after the opening of the coop. Questionnaires were used to measure sociodemographic variables, dietary intake, residents’ health, and community vitality. Semi-structured interviews were conducted with community stakeholders. Results: Few natural experiments have been conducted regarding the impact of implementing food coops. Gathering concrete data on the effectiveness and processes surrounding these interventions through natural experiments will help to quantify their impact and guide knowledge users and policymakers to make more informed decisions. Full article

Medication administration is recognized as a risk-prone activity where errors and near misses have multiple opportunities to occur along the route from manufacturing, through transportation, storage, prescription, dispensing, point-of-care administration, and post-administration documentation. While substantial research, education, and tools have been invested in the detection of medication errors on either side of point-of-care administration, less attention has been placed on this finite phase, leaving a gap in the error detection process. This protocol proposes to undertake a scoping review of the literature related to the detection of medication errors at the point-of-care to understand the potential size, nature, and extent of available literature. The aim is to identify research evidence to guide clinical practice and future research at the medication and patient point-of-care intersection. The search strategy will review literature from PubMed, CINAHL, Cochrane Collaboration, Embase, Scopus, PsychInfo, Web of Science, TRIP, TROVE, JBI Systematic Reviews, Health Collection (Informit), Health Source Nursing Academic, Prospero, Google Scholar, and graylit.org dated 1 January 2000–31 December 2021. Two independent reviewers will screen the literature for relevancy to the review objective, and critically appraise the citations for quality, validity, and reliability using the Joanna Briggs scoping review methodology and System for Unified Management, Assessment and Review of Information (SUMARI) tool. The data will be systematically synthesized to identify and compare the medication error administration detection method findings. A descriptive narrative discussion will accompany the findings. Full article

Silver diamine fluoride (SDF) has been useful in clinical dentistry for the purpose of caries arrest and prevention. Although methods for the application of SDF are well-known among dental professionals, such as microbrush applications, few studies have explored the effect of light curing, which accelerates precipitation onto dentin, and whether this has any effect on the antimicrobial properties of SDF. To assess this technique, single (Streptococcus gordonii) and polymicrobial (mixed salivary) colonies were grown and plated using SDF applied to hydroxyapatite discs with and without treatment with curing light. Kirby–Bauer Zone of Inhibition assay results revealed no significant differences in the areas between the two treatment groups (SDF: 1.27 mm, SDF plus curing light: 1.25 mm), p = 0.887 in the single culture (S. gordonii) experiments. In addition, no significant differences were found between the two treatment groups (SDF: 1.26 mm, SDF plus curing light: 1.24 mm), p = 0.771 in the polymicrobial culture experiments. Although there may be specific properties associated with SDF induced following light curing, these differences do not appear to be associated with the antimicrobial properties affecting gram-positive or polymicrobial films. Full article

The complete dissolution of silicate-containing materials, often necessary for elemental determination, is generally performed by microwave-assisted digestion involving the forced use of hydrofluoric acid (HF). Although highly efficient in dissolving silicates, this acid exhibits many detrimental effects (e.g., formation of precipitates, corrosiveness to glassware) that make its removal after digestion essential. The displacement of HF is normally achieved by evaporation in open-vessel systems: atmospheric contamination or loss of analytes can occur when fuming-off HF owing to the non-ultraclean conditions necessarily adopted for safety reasons. This aspect strongly hinders determination at the ultra-trace level. To overcome this issue, we propose a clean and safe microwave-assisted procedure to induce the evaporative migration of HF inside a sealed “vessel-inside-vessel” system: up to 99.9% of HF can be removed by performing two additional microwave cycles after sample dissolution. HF migrates from the digestion solution to a scavenger (ultrapure H₂O) via a simple physical mechanism, and then, it can be safely dismissed/recycled. The procedure was validated by a soil reference material (NIST 2710), and no external or cross-contamination was observed for the 27 trace elements studied. The results demonstrate the suitability of this protocol for ultra-trace analysis when the utilization of HF is mandatory. Full article

Plankton, at the bottom of the food web, play a central role in the entry of mercury into the aquatic biota. To investigate their role in mercury uptake, reliable analytical procedures for Hg analysis are highly sought. Wet digestion procedures for determining total mercury in different biological matrices have been established since years, however only few studies focused on planktonic samples. In the present work, a simple and cost-effective wet digestion method was developed for the determination of total mercury in samples of small plankton material using a cold vapor atomic fluorescence spectroscopy (CVAFS). The optimization of the digestion method was achieved by using glass vessels with Teflon caps, low amount of acids (3 mL w/w 65% HNO₃ or 3 mL 50% v/v HNO₃), a constant temperature of 85 °C, the presence and absence of pre-ultrasound treatment, and a continuous digestion period (12 h). Certified reference materials IAEA-450 (unicellular alga Scenedesmus obliquus) and BRC-414 (plankton matrix) were used to optimize and validate the digestion method. The recovery efficiency of the proposed method for IAEA-450 and BCR-414 (3.1 mg and 21.5 mg) ranged between 94.1 ± 7.6% and 97.2 ± 4.6%. The method displayed a good recovery efficiency and precision for plankton matrices of low size. Thus, allowing better digestion of planktonic samples for mercury analysis using CVAFS techniques. Full article

The reprogramming of somatic cells to obtain induced pluripotent stem cells (iPSCs) is an important biological and medical breakthrough, providing important applications for fields such as regenerative medicine and disease modeling. However, this promising technology is damped due to its low efficiency and slow kinetics. Therefore, we generated a practical workflow to rapidly and efficiently induce iPSCs from mouse embryonic fibroblasts (MEFs) using iCD1 (iPS chemically-defined medium 1). This protocol can easily be implemented in a standard cell culture laboratory and be applied to cell fate research. Full article

The rise of modern gene expression profiling techniques, such as RNA-Seq, has generated a wealth of high-quality datasets spanning all fields of current biological research. The large data sets and the continually expanding applications for which they can be mined, such as the investigation of alternative splicing and others, have created novel challenges for data management, exploration, analysis, and visualization. Although a large variety of RNA-Seq data analysis software packages has emerged, both open-source and commercial, most fail to simultaneously address the above challenges, while they lack obvious functionalities, such as estimating RNA abundance over non-annotated genomic regions of interest in real time. We have developed SeqCVIBE, an R Shiny web application for the interactive exploration, analysis, visualization, and genome browsing of large RNA-Seq datasets. SeqCVIBE allows for multiple on-the-fly visualizations and calculations, such as differential expression analysis, averaging genomic signals over specific regions of the genome, and calculating RNA abundances over custom, potentially non-annotated regions, such as novel long non-coding RNAs. In addition, SeqCVIBE comprises a database for pre-analyzed data, where users can navigate and explore results, as well as perform a variety of basic on-the-fly analyses and export the outcomes. Finally, we demonstrate the value of SeqCVIBE in the elucidation of the interplay of a novel lincRNA, WiNTRLINC1, and Wnt signaling in colon cancer. Full article

Molecular evolution analyses, such as detection of adaptive/purifying selection or ancestral protein reconstruction, typically require three inputs for a target gene (or gene family) in a particular group of organisms: sequence alignment, model of evolution, and phylogenetic tree. While modern advances in high-throughput sequencing techniques have led to rapid accumulation of genomic-scale data in public repositories and databases, mining such vast amount of information often remains a challenging enterprise. Here, we describe a comprehensive, versatile workflow aimed at the preparation of genome-extracted datasets readily available for molecular evolution research. The workflow involves: (1) fishing (searching and capturing) specific gene sequences of interest from taxonomically diverse genomic data available in databases at variable levels of annotation, (2) processing and depuration of retrieved sequences, (3) production of a multiple sequence alignment, (4) selection of best-fit model of evolution, and (5) solid reconstruction of a phylogenetic tree. Full article

Since mid-2020 there have been complexities and difficulties in the standardisation and administration of nasopharyngeal swabs. Coupled with the variable and/or poor accuracy of lateral flow devices, this has led to increased societal ‘testing fatigue’ and reduced confidence in test results. Consequently, asymptomatic individuals have developed reluctance towards repeat testing, which remains the best way to monitor COVID-19 cases in the wider population. On the other hand, saliva-based PCR, a non-invasive, highly sensitive, and accurate test suitable for everyone, is gaining momentum as a straightforward and reliable means of detecting SARS-CoV-2 in symptomatic and asymptomatic individuals. Here, we provide an itemised list of the equipment and reagents involved in the process of sample submission, inactivation and analysis, as well as a detailed description of how each of these steps is performed. Full article

Background: In various cancer types, the first step towards extended metastatic disease is the presence of lymph node metastases. Imaging methods with sufficient diagnostic accuracy are required to personalize treatment. Lymph node metastases can be detected with ultrasmall superparamagnetic iron oxide (USPIO)-enhanced magnetic resonance imaging (MRI), but this method needs validation. Here, a workflow is presented, which is designed to compare MRI-visible lymph nodes on a node-to-node basis with histopathology. Methods: In patients with prostate, rectal, periampullary, esophageal, and head-and-neck cancer, in vivo USPIO-enhanced MRI was performed to detect lymph nodes suspicious of harboring metastases. After lymphadenectomy, but before histopathological assessment, a 7 Tesla preclinical ex vivo MRI of the surgical specimen was performed, and in vivo MR images were radiologically matched to ex vivo MR images. Lymph nodes were annotated on the ex vivo MRI for an MR-guided pathological examination of the specimens. Results: Matching lymph nodes of ex vivo MRI to pathology was feasible in all cancer types. The annotated ex vivo MR images enabled a comparison between USPIO-enhanced in vivo MRI and histopathology, which allowed for analyses on a nodal, or at least on a nodal station, basis. Conclusions: A workflow was developed to validate in vivo USPIO-enhanced MRI with histopathology. Guiding the pathologist towards lymph nodes in the resection specimens during histopathological work-up allowed for the analysis at a nodal basis, or at least nodal station basis, of in vivo suspicious lymph nodes with corresponding histopathology, providing direct information for validation of in vivo USPIO-enhanced, MRI-detected lymph nodes. Full article

In the context of increased rates of frailty and chronic disease among older people, there is a need to develop age-friendly, integrated primary care models that place the older person at the centre of their care. However, there is little evidence about how age-friendly integrated care frameworks that are sensitive to the challenges of rural regions can be developed. This protocol paper outlines a study that will examine how the use of an age-friendly care framework (the Indigo 4Ms Framework) within a co-design process can facilitate the development of models of integrated care for rural older people within the Upper Hume region (Victoria, Australia). A co-design team will be assembled, which will include older people and individuals from local health, aged care, and community organisations. Process and outcome evaluation of the co-design activities will be undertaken to determine (1) the processes, activities and outputs that facilitate or hinder the co-design of a 4Ms integrated approach, and (2) how the use of the Indigo 4Ms Framework within a co-design process contributes to more integrated working practices. This protocol contributes to the development of a field of study examining how rural health and aged care services can become more age-friendly, with an emphasis on the role of co-design in developing integrated approaches to health care for older adults. Full article

Risk factors that predispose individuals towards major non-communicable diseases (NCDs) and periodontal disease (PD) often co-occur in the same individual. The common risk factor approach (CRFA) for controlling the risk factors associated with NCDs and PD ensures that modifying a few risk factors has an incredible impact on regulating many chronic conditions. To apply CRFA to NCDs and PD, it is essential to quantify the common risk factors of these conditions. The proposed hospital-based analytical cross-sectional study aims to assess the proportion overlap of risk factors that are common or shared between NCDs (cardiovascular diseases (CVD) and diabetes mellitus (DM) type 2) and PD. The risk factors for PD and NCDs will be estimated in subjects aged 18 years and above, diagnosed with NCDs (DM type 2, CVD) or PD. This will be a non-directional study. The dependent variables analyzed will be PD and NCDs (DM type 2, CVD). The explanatory variables that are assessed will be: age, gender, address, occupation, access and affordability of care, familial pattern, family size, insurance, socioeconomic status, obesity, tobacco usage, physical activity, alcohol consumption, food frequency, stress, and oral hygiene. The research is expected to provide data which will aid in the development of advocacy initiatives to implement CRFA for PD and NCDs. Full article

Retinoblastoma (RB) is a primary intraocular malignancy in childhood. Relapses may develop and cause secondary cancers during later development. This study was set up to identify optimal cell culture conditions for RB cell growth in vitro and to optimize tumor growth in an in vivo model. RB cell lines (Y79 and WERI-Rb1) were cultivated under three different in vitro conditions and apoptosis, proliferation and cell growth, as well as expression profiles of two epithelial-mesenchymal transition (EMT) markers, were analyzed. EMT gene expression profiles were not generally changed, whereas apoptosis levels, tumor cell proliferation, and in vitro growth were significantly influenced by different cell culture conditions. In order to optimize the time-limited chick chorioallantoic membrane (CAM) assay, we investigated two different time points of tumor cell inoculation (embryonic development day EDD8 and EDD10) as well as three different cell concentrations. We showed that inoculation at EDD8 led to decreased tumor formation and chicken viability, whereas different cell concentrations did not change size and weight of developing tumors. Our findings demonstrate that medium conditions in vitro as well as the starting point for CAM inoculation in ovo significantly influence the experimental outcome of investigations using RB cell lines. Full article

Managing medical procedures for children with problematic disorders is a challenging approach, especially in the case of blood withdrawal for autism spectrum disorder-affected children. Peripheral blood mononuclear cells (PBMC) represent an important cellular model to study immune responses and drug toxicity. The monocytic cells, a fraction of PBMC, are strongly involved in some pathophysiological processes, such as inflammation and immune system changes. Here, we propose a simple, reliable protocol for obtaining peripheral blood-derived mononuclear cells from small volumes of blood samples. Full article