Methods Protoc., Volume 5, Issue 3 (June 2022) – 18 articles

In this study, the investigation of the oil painting on canvas I Tesori del Mare made by Plinio Nomellini in 1901 is presented. The aim of the research was threefold: the examination of the state of conservation in view of the restoration treatment, together with the identification of the causes of degradation and the study of the artistic technique. During the years, the artwork underwent several cleaning and fixing interventions, resulting in a patchy appearance of the surface. Nevertheless, the presence of consistent liftings persists, while the protective coating shows uneven chromatic alteration, both requiring further analysis. Multispectral imaging allowed for better visualization of the figuration’s structure and the restored areas. The combined use of Raman spectroscopy, Fourier Transform Infrared spectroscopy in the Attenuated Total Reflection mode (FT-IR ATR), and Scanning Electron Microscopy coupled with an Energy Dispersive Spectroscopy (SEM/EDS) enabled better understanding of the stratigraphy through the identification of some pigments, the binder, and the aged varnish layer on the top. SEM/EDS highlighted the presence of zinc in both the ground layer and the paint layers. Furthermore, FT-IR ATR spectroscopy showed peaks related to metal soaps such as zinc stearate, which are known to cause severe delamination of the paint layers, explaining the recurring lifting issues. Eventually, the varnish layer was found to be acrylic resin, presumably mixed with varnishes applied in past restoration treatments. Full article

Organoids are a type of three-dimensional (3D) cell culture that more closely mimic the in vivo environment and can be maintained in the long term. To date, oviductal organoids have only been reported in laboratory mice, women, and cattle. Equine oviductal organoids were generated and cultured for 42 days (including 3 passages and freeze–thawing at passage 1). Consistent with the reports in mouse and human oviductal organoids, the equine oviductal organoids revealed round cell clusters with a central lumen. Developing a 3D model of the mare oviduct may allow for an increased understanding of their normal physiology, including hormonal regulation. These organoids may provide an environment that mimics the in vivo equine oviduct and facilitate improved in vitro embryo production in equids. Full article

Congenital heart defects (CHD) are the most common type of birth defects. Several human case studies and genetically altered animal models have identified abnormalities in the development of ventricular conduction system (VCS) in the heart. While cell-based therapies hold promise for treating CHDs, translational efforts are limited by the lack of suitable in vitro models for feasibility and safety studies. A better understanding of cell differentiation pathways can lead to development of cell-based therapies for individuals living with CHD/VCS disorders. Here, we describe a new and reproducible 3-D cell culture method for studying cardiac cell lineage differentiation in vitro. We used primary ventricular cells isolated from embryonic day 11.5 (E11.5) mouse embryos, which can differentiate into multiple cardiac cell types including VCS cells. We compared 3-D cultures with three types of basement membrane extracts (BME) for their abilities to support E11.5 ventricular cell differentiation. In addition, the effects of atrial natriuretic peptide (ANP) and an inhibitor for its high affinity receptor were tested on cell differentiation in 3-D cultures. Following the cell culture, protocols for immunofluorescence imaging, cell extraction and protein isolation from the 3-D culture matrix and in-cell western methods are described. Further, these approaches can be used to study the effects of various ligands and genetic interventions on VCS cell development. We propose that these methodologies may also be extended for differentiation studies using other sources of stem cells such as induced pluripotent stem cells. Full article

Various methods for detecting malaria have been developed in recent years, each with its own set of advantages. These methods include microscopic, antigen-based, and molecular-based analysis of blood samples. This study aimed to develop a new, alternative procedure for clinical use by using a large data set of surface-enhanced Raman spectra to distinguish normal and infected red blood cells. PCA-LDA algorithms were used to produce models for separating P. falciparum (3D7)-infected red blood cells and normal red blood cells based on their Raman spectra. Both average normalized spectra and spectral imaging were considered. However, these initial spectra could hardly differentiate normal cells from the infected cells. Then, discrimination analysis was applied to assist in the classification and visualization of the different spectral data sets. The results showed a clear separation in the PCA-LDA coordinate. A blind test was also carried out to evaluate the efficiency of the PCA-LDA separation model and achieved a prediction accuracy of up to 80%. Considering that the PCA-LDA separation accuracy will improve when a larger set of training data is incorporated into the existing database, the proposed method could be highly effective for the identification of malaria-infected red blood cells. Full article

Hospitalised patients with extreme obesity have poorer healthcare outcomes compared to normal weight patients. How hospital services are coordinated and delivered to meet the care needs of patients with extreme obesity is not well understood. The aim of the proposed evidence gap map (EGM) is to identify and assess the available evidence on healthcare interventions to improve healthcare outcomes for hospitalised patients with extreme obesity. This research will use standardised evidence gap map methods to undertake a five-stage process to develop an intervention–outcome framework; identify the current evidence; critically appraise the quality of the evidence, extract, code, and summarise the data in relation to the EGM objectives; and create a visualisation map to present findings. This EGM will provide a means of determining the nature and quality of health service initiatives available, identify the components of the services delivered and the outcome measures used for evaluation, and will identify areas where there is a lack of research that validates the funding of new research studies. Full article

Spine surgery patients with a history of organ transplantation are a complex population due to their unique anesthetic considerations, immunologic profiles, drug interactions, and potential organ dysfunction. It is common for these patients to develop neck/back pain and pathology that warrants surgical intervention. However, there is a relative dearth of literature examining their outcomes and clinical considerations. The purpose of this protocol is to investigate their clinical outcomes following spine surgery and medical management. We perform a systematic literature search using PRISMA-ScR guidelines to include case reports, cohort studies, and retrospective analyses. The search terms include kidney, liver, heart, pancreas, lung, and bone marrow for transplants of interest and contain an extensive list of terms covering spine surgery. The search is conducted using Medline, Embase, and the Cochrane Central Register of Controlled Trials. A thorough examination of titles and abstracts is performed followed by data extraction. The data points include patient demographics, past medical history, spine procedural information, and clinical outcomes. This systematic review will aid clinicians in identifying demographics, medical management, and clinical outcomes for spine surgery patients with a previous organ transplant. These findings will highlight the gaps in the knowledge of this complex population and stimulate further research. Full article

Interstitial fluid (ISF) bathes the cells and tissues and is in constant exchange with blood. As an exchange medium for waste, nutrients, exosomes, and signaling molecules, ISF is recognized as a plentiful source of biomolecules. Many basic and pre-clinical small animal studies could benefit from an inexpensive and efficient technique that allows for the in vivo extraction of ISF for the subsequent quantification of molecules in the interstitial space. We have previously reported on a minimally invasive technique for the extraction of ISF using a 3D-printed microneedle array (MA) platform for comprehensive biomedical applications. Previously, hairless animal models were utilized, and euthanasia was performed immediately following the procedure. Here, we demonstrate the technique in Sprague Dawley rats, without the need for hair removal, over multiple extractions and weeks. As an example of this technique, we report simultaneous quantification of the heavy metals Copper (Cu), Lead (Pb), Lithium (Li), and Nickel (Ni) within the ISF, compared with whole blood. These results demonstrate the MA technique applicability to a broader range of species and studies and the reuse of animals, leading to a reduction in number of animals needed to successfully complete ISF extraction experiments. Full article

Human Activity Recognition (HAR) is increasingly used in a variety of applications, including health care, fitness tracking, and rehabilitation. To reduce the impact on the user’s daily activities, wearable technologies have been advanced throughout the years. In this study, an improved smart insole-based HAR system is proposed. The impact of data segmentation, sensors used, and feature selection on HAR was fully investigated. The Support Vector Machine (SVM), a supervised learning algorithm, has been used to recognise six ambulation activities: downstairs, sit to stand, sitting, standing, upstairs, and walking. Considering the impact that data segmentation can have on the classification, the sliding window size was optimised, identifying the length of 10 s with 50% of overlap as the best performing. The inertial sensors and pressure sensors embedded into the smart insoles have been assessed to determine the importance that each one has in the classification. A feature selection technique has been applied to reduce the number of features from 272 to 227 to improve the robustness of the proposed system and to investigate the importance of features in the dataset. According to the findings, the inertial sensors are reliable for the recognition of dynamic activities, while pressure sensors are reliable for stationary activities; however, the highest accuracy ($94.66\%$) was achieved by combining both types of sensors. Full article

Genome editing using the CRISPR/Cas9 system is one of the trendiest methodologies in the scientific community. Many genome editing approaches require recombinant Streptococcus pyogenes Cas9 (SpCas9) at some point during their application, for instance, for in vitro validation of single guide RNAs (SgRNAs) or for the DNA-free editing of genes of interest. Hereby, we provide a simple and detailed expression and purification protocol for SpCas9 as a protein fused to GFP and MBP. This protocol improves protein yield and simplifies the purification process by overcoming the frequently occurring obstacles such as plasmid loss, inconsistent protein expression levels, or inadequate protein binding to affinity resins. On average, this protocol yields 10 to 30 mg of purified, active, His6−MBP−SpCas9 NLS−GFP protein. The purity addressed through SDS-PAGE is > 80%. Full article

In order to improve a recently established cell-based assay to assess the potency of botulinum neurotoxin, neuroblastoma-derived SiMa cells and induced pluripotent stem-cells (iPSC) were modified to incorporate the coding sequence of a reporter luciferase into a genetic safe harbor utilizing CRISPR/Cas9. A novel method, the double-control quantitative copy number PCR (dc-qcnPCR), was developed to detect off-target integrations of donor DNA. The donor DNA insertion success rate and targeted insertion success rate were analyzed in clones of each cell type. The dc-qcnPCR reliably quantified the copy number in both cell lines. The probability of incorrect donor DNA integration was significantly increased in SiMa cells in comparison to the iPSCs. This can possibly be explained by the lower bundled relative gene expression of a number of double-strand repair genes (BRCA1, DNA2, EXO1, MCPH1, MRE11, and RAD51) in SiMa clones than in iPSC clones. The dc-qcnPCR offers an efficient and cost-effective method to detect off-target CRISPR/Cas9-induced donor DNA integrations. Full article

The relationship between the host and the microbiome, or the assemblage of microorganisms (including bacteria, archaea, fungi, and viruses), has been proven crucial for its health and disease development. The high dimensionality of microbiome datasets has often been addressed as a major difficulty for data analysis, such as the use of machine-learning (ML) and deep-learning (DL) models. Here, we present BiGAMi, a bi-objective genetic algorithm fitness function for feature selection in microbial datasets to train high-performing phenotype classifiers. The proposed fitness function allowed us to build classifiers that outperformed the baseline performance estimated by the original studies by using as few as 0.04% to 2.32% features of the original dataset. In 35 out of 42 performance comparisons between BiGAMi and other feature selection methods evaluated here (sequential forward selection, SelectKBest, and GARS), BiGAMi achieved its results by selecting 6–93% fewer features. This study showed that the application of a bi-objective GA fitness function against microbiome datasets succeeded in selecting small subsets of bacteria whose contribution to understood diseases and the host state was already experimentally proven. Applying this feature selection approach to novel diseases is expected to quickly reveal the microbes most relevant to a specific condition. Full article

Raman spectroscopy has recently been used for quantitative analyses of cortical bone tissue and related materials, such as dentin and enamel. While those analyses have proven useful as potential diagnostic tools, the Raman spectrum of bone encrypts a wealth of additional molecular scale details about structure and crystal arrangement, which are yet to be unfolded. Such details directly link to both bone physiology and pathology. In this work, a triple monochromator spectrometer with high spectral resolution, employed in polarized light configurations, was used to extract quantitative details about the preferential crystallographic orientation of apatite and collagen components in a human proximal femoral cortical bone sample. This body of information was then used to model the bone structure at the nanometric scale through a methodology that could be key in assessments of bone structure in health and disease. Full article

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic condition characterized by pelvic pain coupled with urinary frequency and urgency. The underlying cause of IC/BPS is unknown; there is no cure. Dietary components exacerbate symptoms. The Anti-Inflammatory Diet for Interstitial Cystitis (AID-IC) employs a randomized, crossover design to evaluate the effect of a plant-based, low saturated fat diet on the quality of life of women with IC/BPS. Insights on the implementation of the protocol and reflections on the facilitators and barriers experienced during the pilot study follow. The logistics of the protocol proved time-consuming; however, the barriers were surmountable. Quantitative and qualitative findings suggest that the AID-IC therapeutic diet may have lessened symptoms and improved the quality of life for many of the women in the study. Full article

Magnetic resonance neurography (MRN), the MR imaging of peripheral nerves, is clinically used for assessing and monitoring peripheral neuropathies based on qualitative, weighted MR imaging. Recently, quantitative MRN has been increasingly reported with various MR parameters as potential biomarkers. An evidence synthesis mapping the available methodologies and normative values of quantitative MRN of human peripheral nerves, independent of the anatomical location and type of neuropathy, is currently unavailable and would likely benefit this young field of research. Therefore, the proposed scoping review will include peer-reviewed literature describing methodologies and normative values of quantitative MRN of human peripheral nerves. The literature search will include the databases MEDLINE (PubMed), EMBASE (Ovid), Web of Science, and Scopus. At least two independent reviewers will screen the titles and abstracts against the inclusion criteria. Potential studies will then be screened in full against the inclusion criteria by two or more independent reviewers. From all eligible studies, data will be extracted by two or more independent reviewers and presented in a diagrammatic or tabular form, separated by MR parameter and accompanied by a narrative summary. The reporting will follow the guidelines of the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR). Upon completion, the scoping review will provide a map of the available literature, identify possible gaps, and inform on possible future research. SCOPING REVIEW REGISTRATION: Open Science Framework 9P3ZM. Full article

Aquaculture, a mass supplier of seafood, relies on plastic materials that may contain the endocrine disruptors bisphenol-A (BPA) and tert-octylphenol (t-OCT). These pollutants present toxicity to Artemia, the live aquaculture feed, and are transferred through it to the larval stages of the cultured organisms. The purpose of this work is the development and validation of an analytical method to determine BPA and t-OCT in Artemia and their culture medium, using n-octylphenol as the internal standard. Extraction of the samples was performed with H₂O/TFA (0.08%)–methanol (3:1), followed by SPE. Analysis was performed in a Nucleosil column with mobile phases A (95:5, v/v, 0.1% TFA in H₂O:CH₃CN) and B (5:95, v/v, 0.08% TFA in H₂O:CH₃CN). Calibration curves were constructed in the range of concentrations expected following a 24 h administration of BPA (10 μg/mL) or t-OCT (0.5 μg/mL), below their respective LC₅₀. At the end of exposure to the pollutants, their total levels appeared reduced by about 32% for BPA and 35% for t-OCT, and this reduction could not be accounted for by photodegradation (9–19%). The developed method was validated in terms of linearity, accuracy, and precision, demonstrating the uptake of BPA and t-OCT in Artemia. Full article

Bio-colonization is a dynamic and multiphasic process headed by microorganisms. Conventional treatments to process affected stone materials include chemical biocides, whose formulations are mainly composed of quaternary ammonium salts(QAs), reported to be toxic for human health, dangerous for the environment, and not biodegradable. Accordingly, novel green and eco-friendly products are a promising alternative to treat stone materials deteriorated by microorganism colonization. In this study, the efficacy of pure essential oils (EOs) and a mix of EOs was assessed in situ and compared to a conventional biocide based on QAs, and two commercially green products based on EOs, which were taken as references, through application on a mosaic located at the Archaeological Park of Ostia Antica (Rome). The EO biocide efficacy was analyzed by ultraviolet induced luminescence, spectro-colorimetry and bio-luminometry analyses while the possibility of their permanence on simulated substrate was studied by FTIR spectroscopy. It was observed by FTIR analysis, that EOs considered volatile can leave a residue after the application; typical fingerprint bands at about 2926, 1510, and 1455 cm⁻¹ were recorded in the EO spectra. Every tested oil was confirmed to have a biocide action although minimal in relation to the most conventional products based on QAs. The synergy of the essential oils revealed positive results, showing a stronger biocide efficacy. Further investigation should be carried out to develop the method of application and study of essential oils on cultural heritage. Full article

The brown algae are an important but understudied group of multicellular marine organisms. A number of genetic and genomic tools have been developed for the model brown alga Ectocarpus; this includes, most recently, chromatin immunoprecipitation methodology, which allows genome-wide detection and analysis of histone post-translational modifications. Post-translational modifications of histone molecules have been shown to play an important role in gene regulation in organisms from other major eukaryotic lineages, and this methodology will therefore be a very useful tool to investigate genome function in the brown algae. This article provides a detailed, step-by-step description of the Ectocarpus ChIP protocol, which effectively addresses the difficult problem of efficiently extracting chromatin from cells protected by a highly resistant cell wall. The protocol described here will be an essential tool for the future application of chromatin analysis methodologies in brown algal research. Full article

Obesity is known to impair the efficacy of glucocorticoid medications for asthma control. Glucocorticoid-induced gene expression studies may be useful to discriminate those obese asthmatic patients who present a poor response to glucocorticoids. The expression of genes of interest is normalized with respect to reference genes (RGs). Ideally, RGs have a stable expression in different samples and are not affected by experimental conditions. The objective of this work was to analyze suitable RGs to study the role of glucocorticoid-induced genes in obese asthmatic patients in further research. The gene expression of eight potential RGs (GUSB, B2M, POLR2A, PPIA, ACTB, GAPDH, HPRT1, and TBP) was assessed with reverse transcription–quantitative polymerase chain reaction in peripheral blood mononuclear cells (PBMCs) from asthmatic, obese asthmatic, and healthy individuals. Their stability was analyzed using four different algorithms—BestKeeper, ΔCt, geNorm, and NormFinder. geNorm analysis recommended the use of a minimum of three genes for normalization. Moreover, intergroup variation due to the treatment was calculated by NormFinder, which found that B2M was the gene that was least affected by different treatments. Comprehensive rankings indicated GUSB and HPRT1 as the best RGs for qPCR in PBMCs from healthy and asthmatic subjects, while B2M and PPIA were the best for obese asthmatic subjects. Finally, our results demonstrated that B2M and HPRT1 were the most stable RGs among all groups, whereas ACTB, TBP, and GAPDH were the worst shared ones. Full article