Methods Protoc., Volume 3, Issue 4 (December 2020) – 21 articles

A full-arch rehabilitation of the edentulous upper jaw without grafting procedures exploits the residual alveolar or the basal bone, with the necessity of long implants placed with a particular orientation. The precision in planning and placing the fixtures is fundamental to avoid clinical problems and to allow an acceptable connection with the prosthesis. The computer-aided implantology resulted in more accuracy than the traditional one, with a high standard of correspondence between the virtual project and the real outcome. This paper reports about the two different digital protocols, static and dynamic, as support to implant-borne prosthetic rehabilitation of edentulous maxillae. Two pterygoid and two/four anterior standard implants were seated in both cases by two different operators, without flap raising, and immediately loaded. This approach avoided the posterior cantilever by-passing the maxillary sinus and was adequately planned and realized without any surgical or prosthetic error. The two digital flow-charts were described step by step, underlining each other’s advantages and drawbacks compared to a free-hand approach. Full article

Sedentariness of patients affected by end-stage kidney disease (ESKD) expose them to high risk of unfavorable clinical outcomes. Exercise training is effective in improving physical function, quality of life (QoL) and long-term outcomes. However, the existing barriers related to patients, programs and dialysis staff limit patient participation and call for new strategies. This pragmatic nonrandomized trial will test the impact on ESKD population of an intervention proposed by an exercise facilitator regularly present in a dialysis center. The patient will be free to choose among three-month walking and/or resistance low-intensity training programs: (a) guided physical activity increase; (b) home-based exercise; (c) in-hospital (pre/post dialysis) supervised exercise; (d) performance assessment only. The first phase will define feasibility and the characteristics and preference of responders. The second phase will evaluate safety and patients’ adherence. Outcome measures will be collected at baseline, after three-month and at six-month follow-up. They will include: aerobic capacity, QoL, gait speed, strength, depression and long-term clinical outcomes (hospitalization and mortality). The trial was approved by the Area-Vasta Emilia-Romagna Centro Ethics Committee with approval number 48/2019. Written informed consent will be obtained from all participants. The results of the study will be presented in international congresses, published in peer-reviewed journals and communicated to the patient community. Registration details: Clinicaltrials.gov NCT04282616 [Registered:24/02/2020]. Full article

The limited amount of fusion protein transported into cytosol milieu has made it challenging to obtain a sufficient amount for further applications. To avoid the laborious and expensive task, T7 promoter-driving pET-30a(+) coding for chimeric gene of thymidine phosphorylase and core streptavidin as a model system was constructed and transformed into a variety of E. coli strains with T7 expression system. Our results demonstrated that the pET-30a(+)-TP-coreSA/Lemo21(DE3) system is able to provide efficient expression of soluble TP-coreSA fusion protein for purification. Moreover, the eluted TP-coreSA fusion protein tethered on biotinylated A549 carcinoma cells could effectively eliminate these malignant cells after administrating prodrug 5′-DFUR. Full article

Amylin is a pancreatic peptide hormone that regulates glucose homeostasis but also aggregates to form islet amyloid in type-2 diabetes. Given its role in both health and disease, there is renewed interest in proteolytic degradation of amylin by insulin-degrading enzyme (IDE) and other proteases. Here, we describe the development and detailed characterization of three novel assays for amylin degradation, two based on a fluoresceinated and biotinylated form of rodent amylin (fluorescein-rodent amylin-biotin, FrAB), which can be used for any amylin protease, and another based on an internally quenched fluorogenic substrate (FRET-based amylin, FRAM), which is more specific for IDE. The FrAB-based substrate can be used in a readily implemented fluorescence-based protocol or in a fluorescence polarization (FP)-based protocol that is more amenable to high-throughput screening (HTS), whereas the FRAM substrate has the advantage of permitting continuous monitoring of proteolytic activity. All three assays yield highly quantitative data and are resistant to DMSO, and the FRAM and FP-based FrAB assay are ideally suited to HTS applications. Full article

The chick embryo chorioallantoic membrane (CAM) represents a powerful in vivo model to study several physiological and pathological processes including inflammation and tumor progression. Nevertheless, the possibility of deepening the molecular processes in the CAM system is biased by the absence/scarcity of chemical and biological reagents, designed explicitly for avian species. This is particularly true for transcriptional factors, proteinaceous molecules that regulate various cellular responses, including proliferation, survival, and differentiation. Here, we propose a detailed antibody-independent protocol to visualize the activation and nuclear translocation of transcriptional factors in cells or in tissues of different animal species. As a proof of concept, DNA/cAMP response element-binding protein (CREB) interaction was characterized on the CAM tissue using oligonucleotides containing the palindromic binding sequence of CREB. Scrambled oligonucleotides were used as controls. In situ DNA/protein interaction protocol is a versatile method that is useful for the study of transcription factors in the cell and tissue of different origins. Full article

While clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing techniques have been widely adapted for use in immortalised immune cells, efficient manipulation of primary T cells has proved to be more challenging. Nonetheless, the rapid expansion of the CRISPR toolbox accompanied by the development of techniques for delivery of CRISPR components into primary T cells now affords the possibility to genetically manipulate primary T cells both with precision and at scale. Here, we review the key features of the techniques for primary T cell editing and discuss how the new generation of CRISPR-based tools may advance genetic engineering of these immune cells. This improved ability to genetically manipulate primary T cells will further enhance our fundamental understanding of cellular signalling and transcriptional networks in T cells and more importantly has the potential to revolutionise T cell-based therapies. Full article

The precise knowledge of intracellular polarity, a physiological parameter that involves complex and intertwined intracellular mechanisms, may be relevant in the study of important diseases like cancer or Alzheimer’s. In this technical note, we illustrate our recently developed, accurate method for obtaining intracellular polarity maps employing potent fluorescence microscopy techniques. Our method is based on the selection of appropriate luminescent probes, in which several emission properties vary with microenvironment polarity, specifically spectral shifts and luminescence lifetime. A multilinear calibration is performed, correlating polarity vs. spectral shift vs. luminescence lifetime, to generate a powerful and error-free 3D space for reliable interpolation of microscopy data. Multidimensional luminescence microscopy is then used to obtain simultaneously spectral shift and luminescence lifetime images, which are then interpolated in the 3D calibration space, resulting in accurate, quantitative polarity maps. Full article

Epigenetic aging has been found to be associated with a number of phenotypes and diseases. A few studies have investigated its effect on lung function in relatively older people. However, this effect has not been explored in the younger population. This study examines whether lung function in adolescence can be predicted with epigenetic age accelerations (AAs) using machine learning techniques. DNA methylation based AAs were estimated in 326 matched samples at two time points (at 10 years and 18 years) from the Isle of Wight Birth Cohort. Five machine learning regression models (linear, lasso, ridge, elastic net, and Bayesian ridge) were used to predict FEV₁ (forced expiratory volume in one second) and FVC (forced vital capacity) at 18 years from feature selected predictor variables (based on mutual information) and AA changes between the two time points. The best models were ridge regression (R² = 75.21% ± 7.42%; RMSE = 0.3768 ± 0.0653) and elastic net regression (R² = 75.38% ± 6.98%; RMSE = 0.445 ± 0.069) for FEV₁ and FVC, respectively. This study suggests that the application of machine learning in conjunction with tracking changes in AA over the life span can be beneficial to assess the lung health in adolescence. Full article

Daily human activities and vast green areas produce substantial amounts of organic wastes that end up in landfills with minimal treatment. In addition to the problems associated with landfills, disposal through this method is unsustainable in the long run and does not allow recovering materials from the waste. This paper focuses on the co-composting of different organic wastes produced in The Emirate of Sharjah, United Arab Emirates (UAE), to optimize mixing proportions of three different kinds of wastes—sewage sludge (SS), green waste (GW), and food waste (FW). All three organic wastes were analyzed to determine their chemical composition and the mixing proportions. Ten different mixing proportions as a function of carbon:nitrogen (C:N ratios) were determined and mixed in a NatureMill composter. Compost samples were tested for pH, salinity, conductivity, moisture content, organic matter, organic carbon, phosphorus, total nitrogen, and final C:N ratio after 6 weeks. Results indicate that a period of 5–6 weeks is sufficient for the compost to stabilize. The varying mixing proportions produced a good-quality compost (C:N up to 20:1) with high nutrient content. The study reaffirms that co-composting can be a potential sustainable organic waste management option for the United Arab Emirates. Full article

Dynamic Navigation is a computer-aided technology that allows the surgeon to track the grip instruments while preparing the implant site in real time based on radiological anatomy and accurate pre-operative planning. The support of this technology to the zygoma implant placement aims to reduce the risks and the errors associated with this complex surgical and prosthetic treatment. Various navigation systems are available to clinicians currently, distinguished by handling, reliability, and the associated economic and biological benefits and disadvantages. The present paper reports on the different protocols of dynamic navigations following a standard workflow in correlation with zygomatic implant supported rehabilitations and describes a case of maxillary atrophy successfully resolved with this technology. An innovative and minimally invasive dynamic navigation system, with the use of an intraoral anchored trust marker plate and a patient reference tool, has been adopted to support the accurate insertion of four zygomatic implants, which rapidly resolved maxillary atrophy from a 75-year-old male system. This approach provided an optimal implant placement accuracy reducing surgical invasiveness. Full article

The human eye is a specialized organ with a complex anatomy and physiology, because it is characterized by different cell types with specific physiological functions. Given the complexity of the eye, ocular tissues are finely organized and orchestrated. In the last few years, many in vitro models have been developed in order to meet the 3Rs principle (Replacement, Reduction and Refinement) for eye toxicity testing. This procedure is highly necessary to ensure that the risks associated with ophthalmic products meet appropriate safety criteria. In vitro preclinical testing is now a well-established practice of significant importance for evaluating the efficacy and safety of cosmetic, pharmaceutical, and nutraceutical products. Along with in vitro testing, also computational procedures, herein described, for evaluating the pharmacological profile of potential ocular drug candidates including their toxicity, are in rapid expansion. In this review, the ocular cell types and functionality are described, providing an overview about the scientific challenge for the development of three-dimensional (3D) in vitro models. Full article

Background: Although mortality rates have declined in Uganda over the last decade, maternal mortality is still high at 336 deaths per 100,000 live births, as is infant mortality at 43 deaths per 1000 live births. One in every 19 babies born in Uganda does not live to celebrate their first birthday. Many of these deaths occur within the first 28 days of life, forming the single largest category of death. Promising effects for preventing death are expected from timed and targeted counselling (ttC), an intervention package of key messages and actions that address integrated health and nutrition needs of the mothers and children, barriers and negotiation agreement, to cause sustainable behavioural change at specific timelines in the first 1000 days. Methods: The study has a quasi-experimental design in order to evaluate the implementation and effectiveness of the ttC intervention. Participants are pregnant women who have been registered by village health team (VHT) members and who live in Hoima (intervention region) or Masindi (control region) districts, who will be monitored throughout their pregnancy up to at least six weeks after delivery. A multi-stage sampling technique will be employed to select participants, the study sites being purposively chosen. Sample size is determined using the pregnancy rate from the population estimates, resulting in a total required sample of 1218 (609 each in the intervention and control group). Study instruments that will be used include the Ugandan VHT household register (in which all mothers to be studied will be registered), the ttC register (an additional tool for the study area), and a study questionnaire, to collect data at outcome level. Univariate, bivariate and multivariate analyses will be performed using SPSS to evaluate intervention effects on outcomes (e.g., relationship between pregnancy outcomes and antenatal attendance). In addition, quantitative findings will be triangulated with qualitative data, and collected through interviews and focus group discussions with participants and implementers. Discussion: The proposed study will examine the effectiveness of implementing ttC to improve maternal and child outcomes in Uganda. If ttC is effective, broader implementation of appropriate antenatal services can be advised as essential newborn care improvements. Trial registration: PACTR, PACTR202002812123868. Registered on 25 February 2020. Full article

Butterflies are well known for their beautiful wings and have been great systems to understand the ecology, evolution, genetics, and development of patterning and coloration. These color patterns are mosaics on the wing created by the tiling of individual units called scales, which develop from single cells. Traditionally, bulk RNA sequencing (RNA-seq) has been used extensively to identify the loci involved in wing color development and pattern formation. RNA-seq provides an averaged gene expression landscape of the entire wing tissue or of small dissected wing regions under consideration. However, to understand the gene expression patterns of the units of color, which are the scales, and to identify different scale cell types within a wing that produce different colors and scale structures, it is necessary to study single cells. This has recently been facilitated by the advent of single-cell sequencing. Here, we provide a detailed protocol for the dissociation of cells from Bicyclus anynana pupal wings to obtain a viable single-cell suspension for downstream single-cell sequencing. We outline our experimental design and the use of fluorescence-activated cell sorting (FACS) to obtain putative scale-building and socket cells based on size. Finally, we discuss some of the current challenges of this technique in studying single-cell scale development and suggest future avenues to address these challenges. Full article

Worldwide, injuries due to falls in the home impose a substantial burden and merit considerable effort to find effective prevention measures. The current study is one of very few randomized controlled trials that assess the effectiveness of home modification for preventing falls. It is the first carried out with a minority or indigenous community and focused on reducing inequities. Just over 250 households in Aotearoa, New Zealand, with Māori occupants were recruited in two strata, 150 from the Wellington region and 100 from the Taranaki region. These were randomly allocated to equally sized treatment and control groups within the respective regions, the treatment group receiving a package of home modifications designed to prevent falls at the start of the study, and the control group receiving the package at the end of the study. Injury data came from the Accident Compensation Corporation, a state-owned no-fault injury insurer. This provided coverage of virtually all unintentional injuries requiring medical treatment. Matched injury claims were made available for analysis once all identifying fields had been removed. These data will be pooled with data for Māori households from the already-conducted Home Injury Prevention Intervention (HIPI) study, which tested an identical intervention on the general population. In the analysis, the primary outcome measure will be fall injury rates over time, comparing treatment and control households, adjusting for the stratum and prior falls in the household. A secondary measure will be the rates of specific injuries, which are most likely to be prevented by the package of modifications tested. We anticipate that the findings will provide robust evidence for effective injury prevention measures that can reduce an important contributor to health inequities for indigenous populations such as the Māori. Full article

Dynamic navigation (DN) is a computer-guided technique employed in different surgical fields and recently adopted in dental implantology to improve the accuracy of dental implant insertion. Medication-related osteonecrosis of the jaws (MRONJ) often requires the surgical removal of the impaired, hard tissue, trying at the same time to spare the healthy tissue and the noble anatomical structures. A case of extensive bilateral medication-related osteonecrosis, with the symptomatic involvement of the right mandibular canal, was successfully resolved with the use of ultrasonic surgery associated with a dynamic navigation, in order to limit the invasiveness of the surgical approach improving its reliability and accuracy. The usefulness of this technology in the management of MRONJ can be considered in future clinical trials to confirm the advantages and standardize the technique. Full article

In this article, we present several protocols that describe the steps from cloning and obtaining a large amount of pure plasmid DNA to generation of lentiviruses based on these constructs. The protocols have been worked out on human cell culture HEK293T but can be adapted for other cell cultures. This protocol was designed to be simple to execute and cheap since it requires only materials and consumables widely available in molecular laboratories, such as salts, alcohols, etc., and no complicated laboratory equipment. These protocols are highly effective and can be performed in any standard molecular biology laboratory. Full article

Immunosuppressant agents are essential in every transplant recipient’s care yet walking the fine line of over- or under-immunosuppression is a constant struggle for both patients and transplant providers alike. Optimization and personalization of immunosuppression has been limited by the need for non-invasive graft surveillance methods that are specific enough to identify organ injury in real time. With this in mind, we propose a pilot study protocol utilizing both donor derived cell free DNA (dd-cfDNA, gene expression profiling (GEP), and machine learning (iBox), called KidneyCare, to assess the feasibility and safety in reducing immunosuppressant exposure without increasing the risk of clinical rejection, graft injury, or allograft loss. Patients randomized to the immunominimization arm will be enrolled in one of two protocols designed to eliminate one immunosuppressant and optimize the dose of the Calcineurin Inhibitors (CNIs) using the KidneyCare platform. All patients will be maintained on dual therapy of either steroids and a low dose CNI, or mycophenolate mofetil (MMF) and low dose CNI. Their outcomes will be compared to patients who have their immunosuppressants managed using standard clinical assessment and treatment protocols to determine the impact of immuno-optimization on graft function, complications, and patient reported outcomes. Full article

Disturbance in the balance between fibrin formation and fibrinolysis can lead to either bleeding or thrombosis; however, our current routine coagulation assays are not sensitive to altered fibrinolysis. The clot formation and lysis assay is a dynamic plasma-based analysis that assesses the patient’s capacity for fibrin formation and fibrinolysis by adding an activator of coagulation as well as fibrinolysis to plasma and measuring ex vivo fibrin clot formation and breakdown over time. This assay provides detailed information on the fibrinolytic activity but is currently used for research only, as the assay is prone to inter-laboratory variation and as it demands experienced laboratory technicians as well as specialized personnel to validate and interpret the results. Here, we describe a protocol for the clot formation and lysis assay used at our research laboratory. Full article

Oxidative stress is prominent in many neurodegenerative diseases. Along with mitochondrial dysfunction and pathological protein aggregation, increased levels of reactive oxygen and nitrogen species, together with impaired antioxidant defense mechanisms, are frequently observed in Alzheimer’s, Parkinson’s, Huntington’s disease and amyotrophic lateral sclerosis. The presence of oxidative stress markers in patients’ plasma and cerebrospinal fluid may aid early disease diagnoses, as well as provide clues regarding the efficacy of experimental disease-modifying therapies in clinical trials. In preclinical animal models, the detection and localization of oxidatively damaged lipids, proteins and nucleic acids helps to identify most vulnerable neuronal populations and brain areas, and elucidate the molecular pathways and the timeline of pathology progression. Here, we describe the protocol for the detection of oxidative stress markers using immunohistochemistry on formaldehyde-fixed, paraffin-embedded tissue sections, applicable to the analysis of postmortem samples and tissues from animal models. In addition, we provide a simple method for the detection of malondialdehyde in tissue lysates and body fluids, which is useful for screening and the identification of tissues and structures in the nervous system which are most affected by oxidative stress. Full article

Parallel plate flow chambers are widely used to expose cultured cells to physiological flows for the investigation of a variety of diseases. These applications usually involve the generation of continuous and steady fluid flow over cell monolayers for extended durations, usually a few days. Another technique is to generate a fast high-stress wave over the cells to see the immediate effect of flow-induced stresses. This can be achieved by propagating an air/liquid interface, in other words, a bubble, over cell monolayers. The approach is relevant to the reopening event of fluid-filled lung bronchioles and alveoli during mechanical ventilation therapy of Acute Respiratory Distress Syndrome. This article explains how we generate a stress wave using a parallel plate flow chamber and presents representative results of this wave on cultured lung epithelial cells. Full article

The boxplot is a powerful visualization tool of sampled continuous data sets because of its rich information delivered, compact size, and effective visual expression. The advantage of boxplots is not widely appreciated. Many top journals suggest that boxplots should be used in place of bar charts, but have been wrongly replaced by bar charts. One technical barrier to the usage of boxplots in reporting quantitative data is that bench scientists are not competent in generating boxplots, and are afraid of R, a programming tool. This tutorial provides an effective training material in that even a novice without prior R experience can become competent, within one day, in generating professional boxplots. The available R scripts for boxplots are very limited in scope and are aimed at specialists, and the bench scientists have difficulty in following these scripts. This tutorial provides extensive step-by-step R scripts and instructions, as well as 29 illustrations for customizing every detail of the boxplot structures. Basic R commands and concepts are introduced for users without prior R experiences, which can be skipped by audiences with R knowledge. Violin plots are the enhanced version of boxplots, and therefore, this tutorial also provides a brief introduction and usage of the R package vioplot with one additional illustration. While the protocol is prepared for the newbies and trainees it will be a handy tool for infrequent users, and may benefit the experienced users as well since it provides scripts for customizing every detail of boxplots. Full article