Methods Protoc., Volume 4, Issue 2 (June 2021) – 20 articles

Polystyrene (PS) is one of the major plastics contributing to environmental pollution with its durability and resistance to natural biodegradation. Recent research showed that mealworms (Tenebrio molitor) and superworms (Zophobas morio) are naturally able to consume PS as a carbon food source and degrade them without observable toxic effects. In this study, we explored the effects of possible food additives and use of worm frass as potential plant fertilizers. We found that small amounts of sucrose and bran increased PS consumption and that the worm frass alone could support dragon fruit cacti (Hylocereus undatus) growth, with superworm frass in particular, supporting better growth and rooting than mealworm frass and control media over a fortnight. As known fish and poultry feed, these findings present worms as a natural solution to simultaneously tackle both the global plastic problem and urban farming issue in a zero-waste sustainable bioremediation cycle. Full article

(1) Background: Genomic precision medicine (PM) utilises people’s genomic data to inform the delivery of preventive and therapeutic health care. PM has not been well-established for use with people of Aboriginal and Torres Strait Islander ancestry due to the paucity of genomic data from these communities. We report the development of a new protocol using co-design methods to enhance the potential use of PM for Aboriginal Australians. (2) Methods: This iterative qualitative study consists of five main phases. Phase-I will ensure appropriate governance of the project and establishment of a Project Advisory Committee. Following an initial consultation with the Aboriginal community, Phase-II will invite community members to participate in co-design workshops. In Phase-III, the Chief Investigators will participate in co-design workshops and document generated ideas. The notes shall be analysed thematically in Phase-IV with Aboriginal community representatives, and the summary will be disseminated to the communities. In Phase-V, we will evaluate the co-design process and adapt our protocol for the use in partnership with other communities. (3) Discussion: This study protocol represents a crucial first step to ensure that PM research is relevant and acceptable to Aboriginal Australians. Without fair access to PM, the gap in health outcome between Aboriginal and non-Aboriginal Australians will continue to widen. Full article

Despite the public availability, finding experts in any field when relying on academic publications can be challenging, especially with the use of jargons. Even after overcoming these issues, the discernment of expertise by authorship positions is often also absent in the many publication-based search platforms. Given that it is common in many academic fields for the research group lead or lab head to take the position of the last author, some of the existing authorship scoring systems that assign a decreasing weightage from the first author would not reflect the last author correctly. To address these problems, we incorporated natural language processing (Common Crawl using fastText) to retrieve related keywords when using jargons as well as a modified authorship positional scoring that allows the assignment of greater weightage to the last author. The resulting output is a ranked scoring system of researchers upon every search that we implemented as a webserver for internal use called the APD lab Capability & Expertise Search (ACES). Full article

Advances in the field of CRISPR/Cas systems are expanding our ability to modulate cellular genomes and transcriptomes precisely and efficiently. Here, we assessed the Cas13a-mediated targeted disruption of RNA in chicken fibroblast DF1 cells. First, we developed a Tol2 transposon vector carrying the Cas13a-msGFP-NLS (pT-Cas13a) transgene, followed by a stable insertion of the Cas13a transgene into the genome of DF1 cells to generate stable DF1-Cas13a cells. To assess the Cas13a-mediated functional knockdown, DF1-Cas13a cells were transfected with the combination of a plasmid encoding DsRed coding sequence (pDsRed) and DsRed-specific crRNA (crRNA-DsRed) or non-specific crRNA (crRNA-NS). Fluorescence-activated cell sorting (FACS) and a microscopy analysis showed reduced levels of DsRed expression in cells transfected with crRNA-DsRed but not in crRNA-NS, confirming a sequence-specific Cas13a mediated mRNA knockdown. Next, we designed four crRNAs (crRNA-IAV) against the PB1, NP and M genes of influenza A virus (IAV) and cloned in tandem to express from a single vector. DF1-Cas13a cells were transfected with plasmids encoding the crRNA-IAV or crRNA-NS, followed by infection with WSN or PR8 IAV. DF1 cells transfected with crRNA-IAV showed reduced levels of viral titers compared to cells transfected with crRNA-NS. These results demonstrate the potential of Cas13a as an antiviral strategy against highly pathogenic strains of IAV in chickens. Full article

Organic solvent dibenzyl ether (DBE)-based protocols have been widely used in adipose tissue clearing. However, benzyl alcohol/benzyl benzoate (BABB)-based clearing has been shown to offer better transparency in other tissues. The addition of diphenyl ether (DPE) to BABB (BABB-D4) is often included to preserve fluorescent signals, but its effects on adipose tissue transparency and shrinkage have not been explored. Distinct adipocyte subpopulations contribute to its cellular composition and biological activity. Here, we compared clearing solvents to create an optimized clearing methodology for the study of adipocyte subpopulations. Adipose tissues were cleared with BABB, BABB-D4, and DBE, and post-clearing transparency and tissue shrinkage were measured. An optimized protocol, including BABB-D4 clearing, delipidation, and extensive immunofluorescence blocking steps, was created to examine the spatial distribution of Wt-1 positive progenitor-derived (Type-1) adipocytes in intact mesenteric fat. Both BABB and BABB-D4 lead to significantly increased tissue transparency with reduced tissue shrinkage compared to DBE-cleared adipose tissue. Type-1 adipocytes are found in a clustered distribution with predominant residence in fat associated with the ileum and colon. This paper details an optimized clearing methodology for adipose tissue with increased tissue transparency and reduced shrinkage, and therefore will be a useful tool for investigating adipose tissue biology. Full article

DNA origami has garnered great attention due to its excellent programmability and precision. It offers a powerful means to create complex nanostructures which may not be possible by other methods. The macromolecular structures may be used as static templates for arranging proteins and other molecules. They are also capable of undergoing structural transformation in response to external signals, which may be exploited for sensing and actuation at the nanoscale. Such on-demand reconfigurations are executed mostly by DNA oligomers through base-pairing and/or strand displacement, demonstrating drastic shape changes between two different states, for example, open and close. Recent studies have developed new mechanisms to modulate the origami conformation in a controllable, progressive manner. Here we present several methods for conformational control of DNA origami nanostructures including chemical adducts and UV light as well as widely applied DNA oligomers. The detailed methods should be useful for beginners in the field of DNA nanotechnology. Full article

Injury-related disability burden extends well beyond two years post-injury, especially for Māori (Indigenous) New Zealanders. Māori also experience greater difficulty accessing health services. This prospective cohort study extension uses mixed-methods and aims to understand and identify factors contributing to long-term experiences and outcomes (positive and negative) at 12 years post-injury for injured Māori and their whānau (families), and explore the barriers and facilitators to whānau flourishing, and access to health and rehabilitation services. Five hundred and sixty-six Māori, who were injured between 2007–2009, participated in the Prospective Outcomes of Injury Study (POIS). Of these, 544 consented to long-term follow up, and will be invited to participate in a POIS-10 Māori interview at 12 years post-injury. We anticipate a 65% follow-up rate (~n = 350). Aligned with the Meihana Model, interviews will collect information about multiple inter-related dimensions. Administrative injury and hospitalisation data up to 12 years post-injury will also be collected. Regression models will be developed to examine predictors of long-term health and disability outcomes, after adjusting for a range of confounders. POIS-10 Māori will identify key points in the injury and rehabilitation pathway to inform future interventions to improve post-injury outcomes for Māori and whānau, and will highlight the support required for Māori flourishing post-injury. Full article

Background: Cephalometry is fundamental in diagnosis, analysis, and planning of orthodontic-surgical treatment as it reveals skeletal relationship between the upper and lower jaw as well as facial aesthetic parameters. Nevertheless, 3D cephalometry has still not become the exam of choice in orthognathic treatment even though today CBCT (Cone Beam Computed Tomography) is routinely used in other branches of dentistry. Methods: In a sample of 13 patients undergoing bimaxillary orthognathic surgery a chin-vertex CBCT exam was prescribed prior to orthodontic treatment (OT) and 12 months after surgery (T1). The DICOM files uploaded to MaterialiseSimplant Ortho software pro 2.1 (Materialise Co., Leuven, Belgium) were analyzed following the multiplane 3D Total Face cephalometry protocol (TFA). Results: Results comparing pre-op and post-op TFA 3D cephalometry, were then evaluated considering reference values reported in literature. The CBCT, carried out pre- and post-surgery, were subsequently analyzed employing the superimposition method using cranial base as reference. The aim of this study is to evaluate the advantages and disadvantages of the two methods in orthognathic surgery. Conclusions: Multiplane 3D TFA allows the clinician to locate where major or minor skeletal discrepancies are found with respect to ideal parameters and is also useful in classifying skeletal intermaxillary relation. The superimposition method is highly intuitive but does not provide information on the quantity and location of osteotomic movement. Full article

Injury is a leading cause of disability and is costly. This prospective cohort study extension aims to improve disability, health, and wellbeing outcomes for injured New Zealanders, including for Māori. We will identify predictors and modifiable risk factors of long-term outcomes (positive and negative), and develop an Injury Early Care Tool (INJECT) to inform the implementation of effective interventions to improve outcomes. In the Prospective Outcomes of Injury Study (POIS), 2856 people participated following an injury (occurring between 2007 and 2009) registered with New Zealand’s no-fault accident compensation scheme (ACC). POIS-10 will invite 2121 people (including 358 Māori) who completed a 24-month POIS interview and agreed to follow-up, anticipating 75% participation (n = 1591). Interviews will collect sociodemographic characteristics, life events, comorbidities, and new injuries since participants’ 24-month interview, as well as key disability, health, and wellbeing outcomes 12 years post-injury. Injury-related data will be collected from ACC and hospitalisation records 12 years post-injury. Regression models for the main outcomes will examine the direct effects of predictor variables after adjustment for a wide range of confounders. POIS-10 is enhanced by our partnership with ACC, and expert advisors and will benefit injured people, including Māori, through increased understanding of mechanisms and interventions to improve long-term post-injury outcomes. Full article

A 56-year-old female patient with vertical atrophy of the right posterior mandible was treated adopting an interpositional bone block approach using a cancellous heterologous bone block. Osteotomies of the patient’s mandible were performed with the help of dynamic computer-assisted surgery using virtual anatomical patient information obtained from a cone beam computed tomography (CBCT). The use of the dynamic computer-assisted surgery allowed authors to perform the horizontal osteotomy line as planned preoperatively on the CBCT virtual reconstruction, trying to minimize the risks of the inlay technique. No neurological complications were observed after surgery. The inlay technique could benefit from the aid of dynamic navigation technologies in posterior atrophic mandibles, increasing the reproducibility of the technique. A likely safer method for performing osteotomies with the “sandwich” technique in the posterior atrophic mandible is reported. Full article

Self-reported fruit and vegetable (FV) consumption in children has limitations that could be overcome with objective measures that are easy to implement. The Veggie Meter (VM) is a non-invasive portable device that measures skin carotenoid levels, a concentration biomarker of usual FV intake. While VM has been used to measure FV consumption in adults, few studies have explored its use in elementary school settings. Designing research studies using the VM with elementary school-age children requires an understanding of how well this device can be used in a school setting and of the distribution of VM scores in this population. We used VM to measure skin carotenoids in a diverse sample of 143 elementary school children who also answered commonly asked questions about consumption frequency of FV the previous day. Multivariable regression was used to assess the independent association of demographic variables with VM scores. VM scores were also compared with student-reported FV intake. There was a weak but statistically significant correlation between reported frequency of total vegetable consumption the previous day and observed VM scores (r = 0.174, p = 0.042). This study provides an example of the successful use of the VM in a school setting to collect an objective measure of FV intake and provides important description of data that can inform future studies. Full article

The COVID-19 pandemic is a global crisis and continues to impact communities as the disease spreads. Clinical testing alone provides a snapshot of infected individuals but is costly and difficult to perform logistically across whole populations. The virus which causes COVID-19, SARS-CoV-2, is shed in human feces and urine and can be detected in human waste. SARS-CoV-2 can be shed in high concentrations (>10⁷ genomic copies/mL) due to its ability to replicate in the gastrointestinal tract of humans through attachment to the angiotensin-converting enzyme 2 (ACE-2) receptors there. Monitoring wastewater for SARS-CoV-2, alongside clinical testing, can more accurately represent the spread of disease within a community. This protocol describes a reliable and efficacious method to recover SARS-CoV-2 in wastewater, quantify genomic RNA levels, and evaluate concentration fluctuations over time. Using this protocol, viral levels as low as 10 genomic copies/mL were successfully detected from 30 mL of wastewater in more than seven-hundred samples collected between August 2020 and March 2021. Through the adaptation of traditional enteric virus methods used in food safety research, targets have been reliably detected with no inhibition of detection (RT-qPCR) observed in any sample processed. This protocol is currently used for surveillance of wastewater systems across New Castle County, Delaware. Full article

Extracellular vesicles (EVs) are cell-secreted, lipid membrane-enclosed nanoparticles without functional nucleus. EV is a general term that includes various subtypes of particles named microvesicles, microparticles, ectosomes or exosomes. EVs transfer RNA, DNA and protein cargo between proximal and distant cells and tissues, thus constituting an organism-wide signal transduction network. Pathological tissues secrete EVs that differ in their cargo composition compared to their healthy counterparts. The detection of biomarkers in EVs from biological fluids may aid the diagnosis of disease and/or monitor its progression in a minimally invasive manner. Among biological fluids, pleural effusions (PEs) are integrated to clinical practice, as they accompany a wide variety of lung disorders. Due to the proximity with the pleura and the lungs, PEs are expected to be especially enriched in EVs that originate from diseased tissues. However, PEs are among the least studied biofluids regarding EV-specialized isolation methods and related biomarkers. Herein, we describe a practical EV isolation method from PEs for the screening of EV RNA biomarkers in clinical routine. It is based on a Proteinase K treatment step to digest contaminants prior to standard polyethylene-glycol precipitation. The efficiency of the method was confirmed by transmission electron microscopy, nanoparticle tracking analysis and Western blot. The reliability and sensitivity of the method towards the detection of EV-enriched RNA biomarkers from multiple PEs was also demonstrated. Full article

Introduction: Silver Diammine Fluoride (SDF) is an emerging caries preventive treatment option that is inexpensive, safe, and easily accessible. The evidence is clear that the use of SDF at concentrations of 38% is effective for arresting caries in primary teeth. However, the determination of an optimal SDF application frequency for a cavitated lesion in pragmatic settings is warranted especially among high dental caries risk groups. Hence, the primary objective of this clinical trial is to compare the effectiveness of annual, bi-annual, and four times a year application of 38% SDF application in arresting active coronal dentinal carious lesions on primary teeth among tribal preschool children aged 2–6 years. Methods and Analysis: This study is designed as a randomized, controlled trial consisting of three parallel arms with an allocation ratio of 1:1:1. The trial will enroll 480 preschool tribal children with a cavitated carious lesion (2–6 years) attending a primary health care Centre in Wayanad district, India. Each arm will receive 38% SDF application on an annual (baseline), bi-annual (baseline and 6 months), and four times a year (baseline, 2nd, 4th, and 8th week), respectively. The analysis will be performed both at the tooth- and person-level. Ethics and Dissemination: This trial will be conducted following the principles of the Declaration of Helsinki and local guidelines (Indian Council of Medical Research). The protocol has been approved by Institutional Review Committee (IRB). This trial has been registered prospectively with the Clinical Trial Registry of India [Registration No: CTRI/2020/03/024265]. Full article

Exercise therapy in the intermediate stages of peripheral artery disease (PAD) represents an effective solution to improve mobility and quality of life (QoL). Home-based programs, although less effective than supervised programs, have been found to be successful when conducted at high intensity by walking near maximal pain. In this randomized trial, we aim to compare a low-intensity, pain-free structured home-based exercise (SHB) program to an active control group that will be advised to walk according to guidelines. Sixty PAD patients aged > 60 years with claudication will be randomized with a 1:1 ratio to SHB or Control. Patients in the training group will be prescribed an interval walking program at controlled speed to be performed at home; the speed will be increased weekly. At baseline and after 6 months, the following outcomes will be collected: pain-free walking distance and 6-min walking distance (primary outcome), ankle-brachial index, QoL by the VascuQoL-6 questionnaire, foot temperature by thermal camera, 5-time sit-to-stand test, and long-term clinical outcomes including revascularization rate and mortality. The home-based pain-free exercise program may represent a sustainable and cost effective option for patients and health services. The trial has been approved by the CE-AVEC Ethics Committee (898/20). Registration details: Clinicaltrials.gov NCT04751890 [Registered: 12 February 2021]. Full article

Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) is a recently established multimodal single cell analysis technique combining the immunophenotyping capabilities of antibody labeling and cell sorting with the resolution of single-cell RNA sequencing (scRNA-seq). By simply adding a 12-bp nucleotide barcode to antibodies (cell hashing), CITE-seq can be used to sequence antibody-bound tags alongside the cellular mRNA, thus reducing costs of scRNA-seq by performing it at the same time on multiple barcoded samples in a single run. Here, we illustrate an ideal CITE-seq data analysis workflow by characterizing the transcriptome of SH-SY5Y neuroblastoma cell line, a widely used model to study neuronal function and differentiation. We obtained transcriptomes from a total of 2879 single cells, measuring an average of 1600 genes/cell. Along with standard scRNA-seq data handling procedures, such as quality checks and cell filtering procedures, we performed exploratory analyses to identify most stable genes to be possibly used as reference housekeeping genes in qPCR experiments. We also illustrate how to use some popular R packages to investigate cell heterogeneity in scRNA-seq data, namely Seurat, Monocle, and slalom. Both the CITE-seq dataset and the code used to analyze it are freely shared and fully reusable for future research. Full article

Efficient decision-making is crucial to ensure adequate rehabilitation with optimal use of healthcare resources. Establishing the factors associated with making decisions concerning rehabilitation provision is important to guide clinical staff towards person-centred decisions for rehabilitation after severe stroke. In this study we conduct a best–worst scaling (BWS) experiment to identify the most important factors and their relative weight of importance for deciding the type of ongoing rehabilitation services a person with severe stroke might receive post hospital discharge. Fractional, efficient designs are applied regarding the survey design. Key multidisciplinary staff regularly involved in making decisions for rehabilitation in a stroke unit will be recruited to participate in an online BWS survey. Hierarchical Bayes estimation will be used as the main analysis method, with the best–worst count analysis as a secondary analysis. The survey is currently being piloted prior to commencing the process of data collection. Results are expected by the end of September 2021. The research will add to the current literature on clinical decision-making in stroke rehabilitation. Findings will quantify the preferences of factors among key multi-disciplinary clinicians working in stroke units in the UK, involved in decision-making concerning rehabilitation after stroke. Full article

Uremic toxins play a pathological role in atherosclerosis and represent an important risk factor in dialysis patients. Online hemodiafiltration (HDF) has been introduced to improve the clearance of middle- and large-molecular-weight solutes (>500 Da) and has been associated with reduced cardiovascular mortality compared to standard hemodialysis. This non-randomized, open-label observational study will explore the efficacy of two dialyzers currently used for online HDF, a polysulfone-based high-flux membrane, and a cellulose triacetate membrane, in hemodialysis patients with signs of middle-molecule intoxication or intradialytic hypotension. In particular, the two filters will be evaluated for their ability in uremic toxin removal and modulation of inflammatory status. Sixteen subjects in standard chronic bicarbonate hemodialysis requiring a switch to online HDF in view of their clinical status will be enrolled and divided into two treatment arms, according to the previous history of hypersensitivity to polysulfone/polyethersulfone dialysis filters and hypersensitivity to drugs or other allergens. Group A will consist of 16 patients without a previous history of hypersensitivity and will be treated with a polysulfone filter (Helixone FX100), and group B, also consisting of 16 patients, with a previous history of hypersensitivity and will be treated with asymmetric triacetate (ATA; SOLACEA 21-H) dialyzer. Each patient will be followed for a period of 24 months, with monthly assessments of circulating middle-weight toxins and protein-bound toxins, markers of inflammation and oxidative stress, lymphocyte subsets, activated lymphocytes, and monocytes, cell apoptosis, the accumulation of advanced glycation end-products (AGEs), variations in arterial stiffens measured by pulse wave velocity (PWV), and mortality rate. The in vitro effect on endothelial cells of uremic serum collected from patients treated with the two different dialyzers will also be investigated to examine the changes in angiogenesis, cell migration, differentiation, apoptosis and proliferative potential, and gene and protein expression profile. The expected results will be a better awareness of the different effects of polysulfone gold-standard membrane for online HDF and the new ATA membrane on the removal of uremic toxins removal and inflammation due to blood–membrane interaction. Full article

We present a new and straightforward method by which standard cell culture plates can be sealed off from ambient air and be placed under controlled hypoxic cell culture conditions without costly or highly specialized materials. The method was established on a murine cell culture system using the dendritic cell line JAWS II but can be readily adapted to other cell cultures. The procedure was designed to be easy to implement in cell culture laboratories with standard incubators and requires only readily available materials, resources, and consumables, such as six-well plates, degassed culture medium, CoCl₂, a vacuum sealer, etc., and no further complicated laboratory equipment. The simple hypoxic cell culture method presented here is technically reliable and experimentally safe. As it can be performed in any standard incubator, it is suitable for use at both low and higher biosafety levels. Full article

Measured variability of product within Cell and Gene Therapy (CGT) manufacturing arises from numerous sources across pre-analytical to post-analytical phases of testing. Operators are a function of the manufacturing process and are an important source of variability as a result of personal differences impacted by numerous factors. This research uses measurement uncertainty in comparison to Coefficient of Variation to quantify variation of participants when they complete Flow Cytometry data analysis through a 5-step gating sequence. Two study stages captured participants applying gates using their own judgement, and then following a diagrammatical protocol, respectively. Measurement uncertainty was quantified for each participant (and analysis phase) by following Guide to the Expression of Uncertainty in Measurement protocols, combining their standard deviations in quadrature from each gating step in the respective protocols. When participants followed a diagrammatical protocol, variation between participants reduced by 57%, increasing confidence in a more uniform reported cell count percentage. Measurement uncertainty provided greater resolution to the analysis processes, identifying that most variability contributed in the Flow Cytometry gating process is from the very first gate, where isolating target cells from dead or dying cells is required. This work has demonstrated the potential for greater usage of measurement uncertainty within CGT manufacturing scenarios, due to the resolution it provides for root cause analysis and continuous improvement. Full article