Methods and Protocols

Given that the airway epithelium is the initial site of infection, study of primary human airway epithelial cells (AEC) infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) will be crucial to improved understanding of viral entry factors and innate immune responses to the virus. Centers for Disease Control and Prevention (CDC) guidance recommends work with live SARS-CoV-2 in cell culture be conducted in a Biosafety Level 3 (BSL-3) laboratory. To facilitate downstream assays of materials from experiments there is a need for validated protocols for SARS-CoV-2 inactivation to facilitate safe transfer of material out of a BSL-3 laboratory. We propagated stocks of SARS-CoV-2, then evaluated the effectiveness of heat (65 °C) or ultraviolet (UV) light inactivation. We infected differentiated human primary AECs with SARS-CoV-2, then tested protocols designed to inactivate SARS-CoV-2 in supernatant, protein isolate, RNA, and cells fixed for immunohistochemistry by exposing Vero E6 cells to materials isolated/treated using these protocols. Heating to 65 °C for 10 min or exposing to UV light fully inactivated SARS-CoV-2. Furthermore, we found in SARS-CoV-2-infected primary AEC cultures that treatment of supernatant with UV light, isolation of RNA with Trizol^®, isolation of protein using a protocol including sodium dodecyl sulfate (SDS) 0.1% and Triton X100 1%, and fixation of AECs using 10% formalin and Triton X100 1%, each fully inactivated SARS-CoV-2. Full article

Patients with epithelial metaplasias have an increased risk of developing malignancies. In Barrett’s esophagus, neo-columnar epithelium develops proximal to the squamous-columnar junction (SCJ) in the esophagus as the result of prolonged exposure to bile and acid reflux. Patients require lifetime periodic surveillance, due to lack of effective eradication therapies. The shortage of innovative treatment options is mostly attributable to the paucity of adequate in vivo models of neo-columnar epithelium regeneration. This protocol describes the generation of a cryoablation model to study regeneration of neo-epithelia at the SCJ. Cryoablation of the columnar and squamous mucosa at the SCJ was achieved through local application of liquid N₂O in wild-type and reporter mice in combination with acid suppression. Acid suppression alone, showed restoration of the SCJ with normal histological features of both the neo-columnar and neo-squamous epithelium within 14 days. As a proof of principle, mice were treated with mNoggin, an inhibitor of bone morphogenetic proteins (BMPs), which are involved in the development of columnar epithelia. Local application of mNoggin to the ablated area at the SCJ significantly reduced the development of the neo-columnar mucosa. Although this model does not faithfully recapitulate the exact characteristics of Barrett’s esophagus, it is a well-suited tool to study the mechanisms of therapeutic inhibition of neo-columnar regeneration. It therefore represents an efficient and easy platform to test novel pharmacological therapies for treatment of neo-epithelial lesions at the SCJ. Full article

Over the past two decades, it has become increasingly evident that microRNAs (miRNA) play a major role in human diseases such as cancer and cardiovascular diseases. Moreover, their easy detection in circulation has made them a tantalizing target for biomarkers of disease. This surge in interest has led to the accumulation of a vast amount of miRNA expression data, prediction tools, and repositories. We used the Human microRNA Disease Database (HMDD) to discover miRNAs which shared expression patterns in the related diseases of ischemia/reperfusion injury, coronary artery disease, stroke, and obesity as a model to identify miRNA candidates for biomarker and/or therapeutic intervention in complex human diseases. Our analysis identified a single miRNA, hsa-miR-21, which was casually linked to all four pathologies, and numerous others which have been detected in the circulation in more than one of the diseases. Target analysis revealed that hsa-miR-21 can regulate a number of genes related to inflammation and cell growth/death which are major underlying mechanisms of these related diseases. Our study demonstrates a model for researchers to use HMDD in combination with gene analysis tools to identify miRNAs which could serve as biomarkers and/or therapeutic targets of complex human diseases. Full article

The domestic silkworm Bombyx mori is extensively studied as a model organism for lepidopteran genetics and has an economic value in silk production. Silkworms also have applications in biomedical and cosmetic industries, and the production of mutant B. mori strains significantly enhances basic and applied silkworm research. In recent years, CRISPR/Cas9 technology is being rapidly adopted as the most efficient molecular tool for generating silkworm lines carrying mutations in target genes. Here we illustrate a complete and efficient workflow to screen, characterize rapidly and follow mutations through generations, allowing the generation of B. mori lines, stably inheriting single CRISPR/Cas9-induced mutations. This approach relies on the use of different molecular methods, the heteroduplex assay, cloning followed by Sanger sequencing, and the amplification refractory mutation system PCR. The use of these methodologies in a sequential combination allows the identification of CRISPR/Cas9-induced mutations in genes mapping on both autosomes and sex chromosomes, and the selection of appropriate individuals to found stable mutant B. mori lines. This protocol could be further applied to screen CRISPR/Cas9 mutations in haploid insects. Full article

Mal d 1 is the primary apple allergen in northern Europe. To explain the differences in the allergenicity of apple varieties, it is essential to study its properties and interaction with other phytochemicals, which might modulate the allergenic potential. Therefore, an optimized production route followed by an unsophisticated purification step for Mal d 1 and respective mutants is desired to produce sufficient amounts. We describe a procedure for the transformation of the plasmid in competent E. coli cells, protein expression and rapid one-step purification. r-Mal d 1 with and without a polyhistidine-tag are purified by immobilized metal ion affinity chromatography (IMAC) and fast-protein liquid chromatography (FPLC) using a high-resolution anion-exchange column, respectively. Purity is estimated by SDS-PAGE using an image-processing program (Fiji). For both mutants an appropriate yield of r-Mal d 1 with purity higher than 85% is achieved. The allergen is characterized after tryptic in gel digestion by peptide analyses using HPLC-MS/MS. Secondary structure elements are calculated based on CD-spectroscopy and the negligible impact of the polyhistidine-tag on the folding is confirmed. The formation of dimers is proved by mass spectrometry and reduction by DTT prior to SDS-PAGE. Furthermore, the impact of the freeze and thawing process, freeze drying and storage on dimer formation is investigated. Full article

Predictive models were developed using two-dimensional quantitative structure activity relationship (QSAR) methods coupled with B3LYP/6-311+G** density functional theory modeling that describe the antimicrobial properties of twenty-four triazolothiadiazine compounds against Aspergillus niger, Aspergillus flavus and Penicillium sp., as well as the bacteria Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa. B3LYP/6-311+G** density functional theory calculations indicated the triazolothiadiazine derivatives possess only modest variation between the frontier orbital properties. Genetic function approximation (GFA) analysis identified the topological and density functional theory derived descriptors for antimicrobial models using a population of 200 models with one to three descriptors that were crossed for 10,000 generations. Two or three descriptor models provided validated predictive models for antifungal and antibiotic properties with R² values between 0.725 and 0.768 and no outliers. The best models to describe antimicrobial activities include descriptors related to connectivity, electronegativity, polarizability, and van der Waals properties. The reported method provided robust two-dimensional QSAR models with topological and density functional theory descriptors that explain a variety of antifungal and antibiotic activities for structurally related heterocyclic compounds. Full article

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression in both animals and plants. By pairing to microRNA responsive elements (mREs) on target mRNAs, miRNAs play gene-regulatory roles, producing remarkable changes in several physiological and pathological processes. Thus, the identification of miRNA-mRNA target interactions is fundamental for discovering the regulatory network governed by miRNAs. The best way to achieve this goal is usually by computational prediction followed by experimental validation of these miRNA-mRNA interactions. This review summarizes the key strategies for miRNA target identification. Several tools for computational analysis exist, each with different approaches to predict miRNA targets, and their number is constantly increasing. The major algorithms available for this aim, including Machine Learning methods, are discussed, to provide practical tips for familiarizing with their assumptions and understanding how to interpret the results. Then, all the experimental procedures for verifying the authenticity of the identified miRNA-mRNA target pairs are described, including High-Throughput technologies, in order to find the best approach for miRNA validation. For each strategy, strengths and weaknesses are discussed, to enable users to evaluate and select the right approach for their interests. Full article

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