Methods Protoc., Volume 4, Issue 4 (December 2021) – 23 articles

Empathy is positively related to healthcare workers and patients’ wellbeing. There is, however, limited research on the effects of empathy education delivered in acute clinical settings and its impact on healthcare consumers. This research tests the feasibility and the potential efficacy outcomes of an immersive education programme developed by the research team in collaboration with clinical partners and a multidisciplinary advisory group. Healthcare worker participants in the intervention ward will receive an 8-week immersive empathy education. The primary outcome (feasibility) will be assessed by evaluating the acceptability of the intervention and the estimated resources. The secondary outcome (efficacy) will be assessed using a quasi-experimental study design. Non-parametric tests will be used to test healthcare worker participants’ empathy, burnout, and organisational satisfaction (within-group and across groups), and healthcare consumer participants’ satisfaction (between-group) over time. Despite growing interest in the importance of empathy in professional relationships, to our knowledge, the present pilot study is the first to explore the feasibility and efficacy of an immersive empathy education in New Zealand. Our findings will provide critical evidence to support the development of a randomised cluster trial and potentially provide preliminary evidence for the effectiveness of this type of empathy education. Full article

To sustain energy-demanding developmental processes, oocytes must accumulate adequate stores of metabolic substrates and mitochondrial numbers prior to the initiation of maturation. In the past, researchers have utilized pooled samples to study oocyte metabolism, and studies that related multiple metabolic outcomes in single oocytes, such as ATP concentration and mitochondrial DNA copy number, were not possible. Such scenarios decreased sensitivity to intraoocyte metabolic relationships and made it difficult to obtain adequate sample numbers during studies with limited oocyte availability. Therefore, we developed and validated procedures to measure both mitochondrial DNA (mtDNA) copy number and ATP quantity in single oocytes. Validation of our procedures revealed that we could successfully divide oocyte lysates into quarters and measure consistent results from each of the aliquots for both ATP and mtDNA copy number. Coefficient of variation between the values retrieved for mtDNA copy number and ATP quantity quadruplicates were 4.72 ± 0.98 and 1.61 ± 1.19, respectively. We then utilized our methodology to concurrently measure mtDNA copy number and ATP quantity in germinal vesicle (GV) and metaphase two (MII) stage oocytes. Our methods revealed a significant increase in ATP levels (GV = 628.02 ± 199.53 pg, MII = 1326.24 ± 199.86 pg, p < 0.001) and mtDNA copy number (GV = 490,799.4 ± 544,745.9 copies, MII = 1,087,126.9 ± 902,202.8 copies, p = 0.035) in MII compared to GV stage oocytes. This finding is consistent with published literature and provides further validation of the accuracy of our methods. The ability to produce consistent readings and expected results from aliquots of the lysate from a single oocyte reveals the sensitivity and feasibility of using this method. Full article

Ichthyoplankton is the cluster of planktonic organisms that consists of fish eggs and larvae. These planktonic stages belong to the temporary zooplankton, representing future exploitable stocks. The study of the early ontogenesis of fish plays a key role in the understanding and evaluation of these populations through the study of their abundance and their spatio-temporal distribution. To better understand and protect these fisheries resources, it is essential to identify the different stages of fish embryonic development. This identification is usually performed using the classical method, based on morphological criteria under a binocular magnifying glass; however, this methodology is not always sufficient and is time consuming and, therefore, it is necessary to rely increasingly on molecular tools. The major problem with these tools is the yield and quality of the nucleic acids extracted from ichthyoplankton, especially in the case of eggs, which are small. Several methods have been used for DNA extraction from ichthyoplankton, either automated or manual, but very often from larvae or adults. In the present work, five fish egg DNA extraction protocols were compared based on their DNA yield and extraction quality, verified by agarose gel electrophoresis and quantitative PCR amplification. The results showed that extraction by our heat-protocol for direct PCR (Hp-dPCR) presents the simplest and cheapest protocol of all the kits used in this study, providing a sufficient quantity and quality of nucleic acids to be used for PCR amplification, and being within the reach of third world laboratories that often do not have sufficiently large budgets to obtain automated kits. Full article

Microglia, the resident brain immune effectors cells, show dynamic activation level changes for most neuropsychiatric diseases, reflecting their complex regulatory function and potential as a therapeutic target. Emerging single-cell molecular biology studies are used to investigate the genetic modification of individual cells to better understand complex gene regulatory pathways. Although multiple protocols for microglia isolation from adult mice are available, it is always challenging to get sufficient purified microglia from a single brain for simultaneous DNA and RNA extraction for subsequent downstream analysis. Moreover, for data comparison between treated and untreated groups, standardized cell isolation techniques are essential to decrease variability. Here, we present a combined method of microglia isolation from a single adult mouse brain, using a magnetic bead-based column separation technique, and a column-based extraction of purified DNA-RNA from the isolated microglia for downstream application. Our current method provides step-by-step instructions accompanied by visual explanations of important steps for isolating DNA-RNA simultaneously from a highly purified microglia population. Full article

Drug counterfeits have been an international issue for almost two decades, and the latest statistics show that fake medications will continue to penetrate legitimate pharmaceutical supply chains (PSCs). Therefore, identifying the issues faced by PSCs is essential to combat the counterfeit drug problem, which will require the implementation of technologies in various phases of the PSC to gain better visibility. In this regard, a literature review was conducted to fulfill the following objectives: (i) review the application of traceability technologies in various PSC phases to detect counterfeits; (ii) analyze the various barriers affecting the establishment of a safe PSC and the critical success factors used to overcome those barriers; and (iii) develop a conceptual framework and guidelines to demonstrate the influence of traceability technologies and success factors on overcoming the various barriers in different phases of the PSC. The major finding of this review was that traceability technologies and the critical success factors have a significant influence on overcoming the barriers to establishing a safe PSC. Full article

Plant leaves present an intricate array of layers providing a robust barrier against pathogens and abiotic stressors. However, these layers may also constitute an obstacle for the assessment of intracellular processes, especially when using fluorescence microscopy approaches. Current methods for leaf mitochondrial membrane potential determinations have been traditionally performed in thin mesophyll sections, in isolated protoplasts or in fluorescent protein-expressing transgenic plants. This may limit the amount of information obtained about overall mitochondrial morphology in intact leaves. Here, we detail a fast and straightforward protocol to assess changes in leaf mitochondrial membrane potential associated with mitochondrial dysfunction in the model plant Arabidopsis thaliana. This protocol also permits mitochondrial shape, dynamics and polarity assessment in leaves subjected to diverse stress conditions. Full article

To explore the biological and immunological basis of human rheumatoid arthritis and human atherosclerosis, we planned and reported a detailed design and rationale for Orencia Atherosclerosis and Rheumatoid Arthritis Study (ORACLE Arthritis Study) using highly sensitive, high-throughput, human autoantibody measurement methods with cell-free protein synthesis technologies. Our previous study revealed that subjects with atherosclerosis had various autoantibodies in their sera, and the titers of anti-Th2 cytokine antibodies were correlated with the severity of atherosclerosis. Because rheumatoid arthritis is a representative autoimmune disease, we hypothesized that both rheumatoid arthritis and atherosclerosis are commonly developed by autoantibody-mediated autoimmune processes, leading to incessant inflammatory changes in both articular joint tissues and vessel walls. We planned a detailed examination involving carotid artery ultrasonography, measurements of adhesion molecules, such as ICAM-1 (intercellular adhesion molecule 1) and VCAM-1 (vascular cell adhesion molecule 1) for the evaluation of atherosclerosis progression, and high-throughput, high-sensitivity, autoantibody analyses using cell-free technologies, with detailed examinations of the disease activity of rheumatoid arthritis. Analyses of correlations and associations between biological markers and degrees of carotid atherosclerosis over time under consistent conditions may enable us to understand the biological and humoral immunity background of human atherosclerosis and autoimmune diseases. Full article

Internal derangement (ID) in the temporomandibular joint (TMJ) is defined as a mechanical problem of the joint that interferes with its function. It is attributed to an abnormal interaction among the articular disc, condyle, and joint eminence. The aim of this study is to evaluate diagnostic efficacy of non-invasive hand-carried ultrasonography instrumentation (US) to provide high-level images for a correct diagnosis of ID. Twenty-eight ID patients, 15 female and 13 males, were examined both clinically and by MRI images in order to achieve a diagnosis of ID (using Helkimo index). Then, they were submitted to US examination with a 12 MHz transducer by using hand-carried instrumentation by a clinician that was blind to their diagnosis and clinical data. TMJ US examination was performed with the mouth closed and mouth open, with proper technique. Each position was then evaluated with two different orientations of the transducer. US showed acceptable results in identifying bone structures. Lower values of diagnostic efficacy were obtained for disc position during joint movements with respect to MRI images. MRI still represents the gold standard for the identification of joint structures. If not corroborated by clinical and anamnestic data, the diagnostic efficacy of US in identifying the position of the disc during opening and closing jaw movements appears limited than compared to MRI. Full article

Stratum corneum (SC) represents the outermost layer of the skin, being an effective barrier against the entry of molecules and pathogens. Skin research has given particular focus to SC as it hampers effective drug delivery for cosmetical and therapeutical purposes. Following recommendations to develop alternative models to animals, the SC isolated from skin obtained from medical procedures or from pigs has gained extensive attention. Yet, there is still missing a standard and simple procedure accepted within the scientific community to avoid application of different isolated SC methodologies, a fact that may hamper progress in skin research. Considering this challenge, the present study evaluated different experimental conditions aiming to establish a useful and sustainable solvent-free procedure for the obtention of a realistic SC model. The studied trypsin digestion parameters included concentration, incubation period and temperature. Isolated SC was characterized using histological analysis and calcein’s permeability, after the procedure and during a 6-week storage period. Data recommend trypsin digestion at 4 °C for 20 h as the most effective procedure to isolate SC from pig ear skin. This work contributes to standardize the SC isolation procedure, and to obtain a valuable and reliable SC mimetic model for skin drug development. Full article

The rat model is a common model for intervertebral disc (IVD) and spinal research. However, complications remain challenging. Standard Operating Procedures (SOPs) are validated methods to minimize complications and improve safety and quality of studies. However, a SOP for rat spinal fusion surgery has been missing until now. Therefore, the aim of the study was to develop a SOP for spinal tail disc surgery in elderly Wistar rats (419.04 ± 54.84 g). An initial preoperative, intraoperative, and postoperative surgical setup, including specific anaesthesia and pain management protocols, was developed. Anaesthesia was induced by subcutaneous injection of a pre-mixture of fentanyl, midazolam, and medetomidin with the addition of 0.5% isoflurane in oxygen and caudal epidural analgesia. The surgery itself consisted of the fixation of a customized external ring fixator with ⌀ 0.8 mm Kirschner wires at the proximal rat tail and a discectomy and replacement with bone morphogenetic protein coated beta-tricalcium-phosphate carrier. The postoperative setup included heating, analgesia with buprenorphine, and meloxicam, as well as special supplementary food. Anaesthesia, surgery, and pain management were sufficient. In the presented optimized SOP, no animals developed any complications. A SOP for spinal surgery in elderly rats in an in vivo spinal fusion model was developed successfully. This novel protocol can improve transparency, reproducibility, and external validity in experimental rat spinal surgery experiments. Full article

The nutritional components of brown rice have been reported to be effective against diabetes mellitus. Recent animal studies have suggested that it is also effective in maintaining cognitive function. Therefore, in this study, we examined the effect of a brown rice diet on cognitive function in individuals aged over 60 years. The study participants were recruited from a pool of individuals aged ≥60 years who were using elderly care facilities. The participants were provided with four servings of brown or white rice per week for 6 months, and their cognitive function was measured before and after the intervention period. Prior to the intervention, participants tasted the white and brown rice to determine which type they would like to be offered over the 6-month period. Since rice is the staple food of the participants in this study, they were allowed to decide whether they wanted to eat white or brown rice. Full article

Damage to the hyaline layer of the articular surface is an urgent problem for millions of people around the world. At present, a large number of experimental methods are being developed to address this problem, including the transplantation of a cell-engineered construct (CEC) composed of a biodegradable scaffold with a premixed cell culture into the damaged area of the articular surface. However, current methods for analyzing the effectiveness of such CECs have significant limitations. This study aimed to compare the SEM technique, classical histology, and cryosectioning for the analysis of CECs transplanted to hyaline cartilage. Full article

Depression, anxiety, and insomnia are common in cancer patients. Mind-body therapies (MBTs) are promising forms of treatment for cancer patients living with depression, anxiety, and insomnia. The objective of this study is to assess the effectiveness and acceptability of MBTs in cancer patients living with depression, anxiety, or insomnia. EMBase, PubMed, Cinahl, PsychINFO, IndMED, CSI-NISCAIR, CNKI, Clinicaltrial.gov, ChiCTR, and CTRI will be searched until October 2020 for relevant studies. Randomized controlled studies in which MBTs were tested in a cancer population will be selected. The authors of the selected studies will be contacted to obtain individual participant data. The participants who reached a defined clinical threshold for depression, anxiety, or insomnia will be selected for the three sub-studies on depression, anxiety, and insomnia, respectively. Pairwise and network meta-analyses will be used to assess the changes in depression, anxiety, sleep quality, and completion rate. We will assess the effect of the treatment dose (number and frequency of interventions) on effectiveness. The results of this study will inform clinical decision-making for the treatment of psychological disturbances in cancer patients. If MBTs are found effective, they will potentially be recommended as treatments for cancer patients with psychological symptoms. Full article

In inflammatory myopathies, the self-reactive immune cells involved in muscle aggression have been studied mostly using histological assessment of muscle biopsy sections; this methodology provides the advantage of visualizing and identifying cells within the tissue, but it does not allow further investigation. To gain access to live and isolated cells, many studies utilized blood samples; however, in the absence of biological tools to discriminate the leukocytes associated with the autoimmune process from those that emerged from responses against pathogens, the information observed on circulating immune cells often lacks in specificity, and thus result interpretation may prove difficult. In order to selectively retrieve self-reactive immune cells, we developed a protocol to isolate live leukocytes from human muscle biopsies, which allows for further analysis using a large range of methodologies. The protocol uses enzymatic digestion to release live leukocytes from freshly collected skeletal muscle samples, followed by filtration and separation of the leukocytes from the myocytes by density gradient centrifugation. The isolated cells can be submitted immediately to various analysis strategies to characterize ex vivo the specific cellular and molecular mechanisms responsible for self-directed immune muscle aggression or may be placed in culture for expansion. Full article

Homeostasis of vascular tone is intricately and delicately maintained systemically and locally, by autonomic nerves and hormones in the blood and by intimal vasoactive substances, respectively. The balance can be acutely or chronically interrupted secondary to many alterations, especially under pathological conditions. Excessive matricellular glycoprotein thrombospondin 1 (TSP1) levels in circulation have been found to play an important role in ischemia-reperfusion injuries of different organs, by acutely suppressing vasorelaxation and chronically remodeling vascular bed. Our laboratory has been interested in identifying new drug moieties, which can selectively and effectively counteract TSP1-induced vascular dysfunction, in order to address associated clinical complications. Preliminary studies using computational docking and molecular models revealed potential drug candidates for further evaluation via vascular functional bioassay to prove the antagonism using an ex vivo vascular model. Herein, we described an efficient screening method for the identification of active drug candidates, by adapting a multiwire myograph system to perform a protocol with different treatments, in the presence of pathological levels of TSP1. We discussed the promising pharmacological evaluation results and suggested suitable modification for versatile applications. We also described the necessity of pre-determination of optimal resting tension to obtain the maximal response, if the experimental test model is different from those with determined optimal resting tension. Full article

Natural reduced water is natural water that contains active hydrogen and reduces oxidation. It is rare in the world, and in Japan, it is produced in the Hita area of Oita Prefecture (Hita Tenryosui water). Previous studies in humans have examined the effects of natural reduced water on diabetes, which is one of the known risks for dementia. Animal studies of natural reduced water have revealed anti-obesity and anti-anxiety effects. However, the effects of natural reduced water on cognitive function, body composition, and psychological function in humans are unknown. Therefore, we investigated the relationship between these items in elderly people who continuously consume natural reduced water. In this study, we recruited participants aged between 65 and 74 years. The participants were randomly and blindly assigned to a natural reduced water (Hita Tenryosui water) group or a control (tap water) group and drank 1 L of water daily for 6 months. Cognitive function, body composition, and psychological function were measured before and after the 6-month intervention period. Full article

Serology tests are extremely useful for assessing whether a person has been infected with a pathogen. Since the onset of the COVID-19 pandemic, measurement of anti-SARS-CoV-2-specific antibodies has been considered an essential tool in identifying seropositive individuals and thereby understanding the extent of transmission in communities. The Luminex system is a bead-based technology that has the capacity to assess multiple antigens simultaneously using very low sample volumes and is ideal for high-throughput studies. We have adapted this technology to develop a COVID-19 multi-antigen serological assay. This protocol described here carefully outlines recommended steps to optimize and establish this method for COVID-19-specific antibody measurement in plasma and in saliva. However, the protocol can easily be customized and thus the assay is broadly applicable to measure antibodies to other pathogens. Full article

A large number of bacterial toxin–antitoxin (TA) systems have been identified so far and different experimental approaches have been explored to investigate their activity and regulation both in vivo and in vitro. Nonetheless, a common feature of these methods is represented by the difficulty in cell transformation, culturing, and stability of the transformants, due to the expression of highly toxic proteins. Recently, in dealing with the type I Lpt/RNAII and the type II YafQ/DinJ TA systems, we encountered several of these problems that urged us to optimize methodological strategies to study the phenotype of recombinant Escherichia coli host cells. In particular, we have found conditions to tightly repress toxin expression by combining the pET expression system with the E. coli C41(DE3) pLysS strain. To monitor the RNase activity of the YafQ toxin, we developed a fluorescence approach based on Thioflavin-T which fluoresces brightly when complexed with bacterial RNA. Fluorescence microscopy was also applied to reveal loss of membrane integrity associated with the activity of the type I toxin Lpt, by using DAPI and ethidium bromide to selectively stain cells with impaired membrane permeability. We further found that atomic force microscopy can readily be employed to characterize toxin-induced membrane damages. Full article

Background: Community-dwelling older clients are becoming increasingly complex. Detecting this complexity in clinical practice is limited, with greater reliance on community nurses’ clinical judgment and skills. The lack of a consistent approach to complexity impacts the level of care and support for older clients to remain in their homes for longer. Objective: To examine the effectiveness of the Patient Complexity Instrument (PCI) in addition to nurses’ clinical judgment to enhance detection of complexity, and subsequent older clients’ resource allocation compared to usual nursing assessment. Design: A pragmatic randomized controlled trial will be conducted within a community nursing service in regional Victoria, Australia. Clients 65 years and over referred to the service who are eligible for Commonwealth Home Support Programme (CHSP) funding will be randomized into Control group: usual nursing assessment or Intervention group: usual nursing assessment plus the PCI. Nurse participants are Registered Nurses currently employed in the community nursing service. Results: This study will explore whether introducing the PCI in a community nursing service enhances detection of complexity and client care resource allocation compared to nurses’ clinical judgment based on usual nursing assessment. Conclusion: This protocol outlines the study to enhance the detection of complexity by nurses delivering care for community-dwelling older people in the regional Australian context. The findings will inform the use of a standardized tool to detect complexity among community-dwelling older Australians. Full article

Gout is an inflammatory arthritis, which causes intense, acute pain due to the buildup of uric acid crystals in synovial fluid. The gold standard for gout diagnosis consists of synovial fluid analysis by polarized light microscopy, which is costly, time-intensive, and technique-dependent, therefore meriting a more efficient, inexpensive, and accessible method for diagnosis. We previously developed and validated a novel colorimetric gout detection method and device based on the reduction of silver nitrate by uric acid; here, we clinically validated our method and device using arthroscopically obtained synovial fluid samples from gout patients. We successfully identified uric acid crystals in clinical samples via our colorimetric method, visualized uric acid crystals in synovial fluid via handheld microscopy, and determined that silver nitrate stain did not interfere with the microscopic visualization of uric acid crystals necessary for diagnosis. We also developed and validated a method of processing turbid clinical samples for use in our device to prevent the obscuration of uric acid crystals by suspended material. Our method and device will clinically facilitate the immediate colorimetric diagnosis of gout and the subsequent bedside visualization of uric acid crystals in both ideal and turbid synovial fluid samples, allowing for a point-of-care diagnosis of gout. Full article

RNA sequencing has become the standard technique for high resolution genome-wide monitoring of gene expression. As such, it often comprises the first step towards understanding complex molecular mechanisms driving various phenotypes, spanning organ development to disease genesis, monitoring and progression. An advantage of RNA sequencing is its ability to capture complex transcriptomic events such as alternative splicing which results in alternate isoform abundance. At the same time, this advantage remains algorithmically and computationally challenging, especially with the emergence of even higher resolution technologies such as single-cell RNA sequencing. Although several algorithms have been proposed for the effective detection of differential isoform expression from RNA-Seq data, no widely accepted golden standards have been established. This fact is further compounded by the significant differences in the output of different algorithms when applied on the same data. In addition, many of the proposed algorithms remain scarce and poorly maintained. Driven by these challenges, we developed a novel integrative approach that effectively combines the most widely used algorithms for differential transcript and isoform analysis using state-of-the-art machine learning techniques. We demonstrate its usability by applying it on simulated data based on several organisms, and using several performance metrics; we conclude that our strategy outperforms the application of the individual algorithms. Finally, our approach is implemented as an R Shiny application, with the underlying data analysis pipelines also available as docker containers. Full article

Sperm binding within the oviductal sperm reservoir plays an important role for reproductive success by enabling sperm survival and maintaining fertilizing capacity. To date, numerous in vitro technologies have been established to measure sperm binding capacity to cultured oviductal cells or oviductal explants. However, these methods do not accurately represent the microenvironment and complex multi-molecular nature of the oviduct. In this paper, we describe a novel protocol for assessing sperm binding capacity in the tubal sperm reservoir using an ex vivo oviduct culture in the bovine model. This protocol includes the staining of frozen-thawed bovine spermatozoa with the DNA-binding dye Hoechst 33342, the co-incubation of stained sperm in closed segments of the oviduct and the visualization and quantification of bound spermatozoa by fluorescence microscopy. By generating overlays of multiple Z-stacks of randomly selected regions of interest (ROIs), spermatozoa bound in the sperm reservoir can be visualized and quantified within the 3D arrangement of the oviductal folds. This method, which is applicable to multiple species, can be used to assess individual sperm binding capacity in males for prognostic purposes as well as to assess the impact of diseases and medications on the formation of the sperm reservoir in the oviduct in humans and animals. Full article