Methods Protoc., Volume 3, Issue 2 (June 2020) – 21 articles

Several microwave-assisted digestion methods were tested at the Centro de Estudios Aplicados en Química laboratory in Quito, Ecuador, to determine the accuracy and performance efficiency of the mineralization process for the determination of total mercury in fish tissue by cold vapor atomic fluorescence spectrophotometry. The use of MARSEasyPrep high-pressure vessels, low amounts of reagents (1 cm³ HNO₃, 1 cm³ H₂O₂, and 1 cm³ HClO₄), an irradiation temperature of 210 °C, and 35 min of mineralization time resulted in accurate performance, with recoveries of certified reference material DORM-4 between 90.1% and 105.8%. This is better than the Association of Official Analytical Chemists 2015.01 method, which has a reported accuracy of 81%. The repeatability precision and intermediate precision were established at three concentration levels (0.167, 0.500, and 0.833 mg·kg⁻¹) and expressed as the percentage of the relative standard deviation ranging from 1.5% to 3.0% and 1.7% to 4.2%, respectively. Further, the method was satisfactorily applied to analyze fortified samples of tilapia (Oreochromis niloticus), with recoveries ranging from 98.3% to 104.3%. The instrumental limits of detection and quantification were 0.118 µg·dm⁻³ and 0.394 µg·dm⁻³, respectively. Full article

Precise creation, maintenance, and monitoring of neuronal circuits would facilitate the investigation of subjects such as neuronal development or synaptic plasticity, or assist in the development of neuronal prosthetics. Here we present a method to precisely control the placement of multiple types of neuronal retinal cells onto a commercially available multiple electrode array (MEA), using custom-built optical tweezers. We prepared the MEAs by coating a portion of the MEA with a non-adhesive substrate (Poly (2-hydroxyethyl methacrylate)), and the electrodes with an adhesive cell growth substrate. We then dissociated the retina of adult tiger salamanders, plated them onto prepared MEAs, and utilized the optical tweezers to create retinal circuitry mimicking in vivo connections. In our hands, the optical tweezers moved ~75% of photoreceptors, bipolar cells, and multipolar cells, an average of ~2000 micrometers, at a speed of ~16 micrometers/second. These retinal circuits were maintained in vitro for seven days. We confirmed electrophysiological activity by stimulating the photoreceptors with the MEA and measuring their response with calcium imaging. In conclusion, we have developed a method of utilizing optical tweezers in conjunction with MEAs that allows for the design and maintenance of custom neural circuits for functional analysis. Full article

The problem of illicit drug use and addiction is an escalating issue worldwide. As such, fast and precise detection methods are needed to help combat the problem. Herein, the synthesis method for an anti-methamphetamine Quenchbody (Q-body), a promising sensor for use in simple and convenient assays, has been described. The fluorescence intensity of the Q-body generated by two-site labeling of Escherichia coli produced anti-methamphetamine antigen-binding fragment (Fab) with TAMRA-C2-maleimide dyes increased 5.1-fold over background in the presence of a hydroxyl methamphetamine derivative, 3-[(2S)-2-(methylamino)propyl]phenol. This derivative has the closest structure to methamphetamine of the chemicals available for use in a laboratory. Our results indicate the potential use of this Q-body as a novel sensor for the on-site detection of methamphetamine, in such occasions as drug screening at workplace, suspicious substance identification, and monitoring patients during drug rehabilitation. Full article

Characterized by their covalently closed structure and thus an elevated stability compared to linear RNA molecules, circular RNAs (circRNAs) form a novel class of mainly non-coding RNAs. Although the biological functions of naturally occurring circRNAs are largely unknown, they were reported to act as molecular sponges, sequestering microRNAs (miRNAs), resulting in a de-repression of target mRNAs. Taking these characteristics of naturally occurring circRNAs into account, artificial circRNAs could be a potential tool in molecular biology and medicine. Using the Hepatitis C virus (HCV) as a model system, this application of artificial circular RNAs was demonstrated. The virus requires cellular miRNA miR-122 for its life cycle, and circRNAs specifically engineered to efficiently sequester this miRNA impacted viral propagation. Since in this context the production of engineered circRNA remains the limiting factor, we present a method to produce and efficiently purify artificial circRNA sponges (ciRS) in vitro. In this protocol we provide insights into a small-scale and large-scale production technique of artificial circular RNA sponges relying on in vitro transcription and RNA ligation. Full article

A simple and fast manual centrifuge was developed to spin down solutions in 96-well polymerase chain reaction (PCR) plates. A commercially available salad spinner was utilized for this purpose. Acceleration and deceleration of the centrifuge were faster than those of a conventional electric centrifuge using 96-well PCR plates. Solutions in a 96-well PCR plate settled quickly after centrifuging for only 3 s. This lightweight centrifuge can be stored under a laboratory bench or on a shelf and can be put on the bench only when required, whereas the electric centrifuge is immobile due to its weight and the requirement of electric cables. This simple centrifuge is inexpensive, requires minimal effort for making, and can be used anywhere. Full article

In transfection experiments with mammalian cells aiming to overexpress a specific protein, it is often necessary to correctly quantify the level of the recombinant and the corresponding endogenous mRNA. In our case, mouse calvarial osteoblasts were transfected with a vector containing the complete Pex11β cDNA (plasmid DNA). The Pex11β mRNA level, as calculated using the RT-qPCR product, was unrealistically higher (>1000-fold) in transfected compared to non-transfected cells, and we assumed that there were large amounts of contaminating plasmid DNA in the RNA sample. Thus, we searched for a simple way to distinguish between plasmid-derived mRNA, endogenous genome-derived mRNA and plasmid DNA, with minimal changes to standard RT-PCR techniques. We succeeded by performing a plasmid mRNA-specific reverse transcription, and the plasmid cDNA was additionally tagged with a nonsense tail. A subsequent standard qPCR was conducted using appropriate PCR primers annealing to the plasmid cDNA and to the nonsense tail. Using this method, we were able to determine the specific amount of mRNA derived from the transfected plasmid DNA in comparison to the endogenous genome-derived mRNA, and thus the transfection and transcription efficiency. Full article

Culture-independent molecular techniques have advanced the characterization of environmental and human samples including the human milk (HM) bacteriome. However, extraction of high-quality genomic DNA that is representative of the bacterial population in samples is crucial. Lipids removal from HM prior to DNA extraction is common practice, but this may influence the bacterial population detected. The objective of this study was to compare four commercial DNA extraction kits and lipid removal in relation to HM bacterial profiles. Four commercial DNA extraction kits, QIAamp^® DNA Microbiome Kit, ZR Fungal/Bacterial DNA MiniPrep™, QIAsymphony DSP DNA Kit and ZymoBIOMICS™ DNA Miniprep Kit, were assessed using milk collected from ten healthy lactating women. The kits were evaluated based on their ability to extract high quantities of pure DNA from HM and how well they extracted DNA from bacterial communities present in a commercial mock microbial community standard spiked into HM. Finally, the kits were evaluated by assessing their extraction repeatability. Bacterial profiles were assessed using Illumina MiSeq sequencing targeting the V4 region of the 16S rRNA gene. The ZR Fungal/Bacterial DNA MiniPrep™ and ZymoBIOMICS™ DNA Miniprep (Zymo Research Corp., Irvine, CA, USA) kits extracted the highest DNA yields with the best purity. DNA extracted using ZR Fungal/Bacterial DNA MiniPrep™ best represented the bacteria in the mock community spiked into HM. In un-spiked HM samples, DNA extracted using the QIAsymphony DSP DNA kit showed statistically significant differences in taxa prevalence from DNA extracted using ZR Fungal/Bacterial DNA MiniPrep™ and ZymoBIOMICS™ DNA Miniprep kits. The only difference between skim and whole milk is observed in bacterial profiles with differing relative abundances of Enhydrobacter and Acinetobacter. DNA extraction, but not lipids removal, substantially influences bacterial profiles detected in HM samples, emphasizing the need for careful selection of a DNA extraction kit to improve DNA recovery from a range of bacterial taxa. Full article

Due to their high bioreactivity, the in-vitro analysis of bioactive glasses (BGs) can be challenging when it comes to maintaining a physiological pH. To improve BG biocompatibility, a heterogenic spectrum of preconditioning approaches, such as “passivation” of the BGs by incubation in cell culture medium, are used but have never been directly compared. In this study, the effect of passivation periods of up to 72 h on pH alkalization and viability of human bone marrow-derived mesenchymal stromal cells was evaluated to determine a time-efficient passivation protocol using granules based on the 45S5-BG composition (in wt%: 45.0 SiO₂, 24.5 Na₂O, 24.5 CaO, 6.0 P₂O₅) in different concentrations. pH alkalization was most reduced after passivation of 24 h. Cell viability continuously improved with increasing passivation time being significantly higher after passivation of at least 24 h compared to non-passivated 45S5-BG and the necessary passivation time increased with increasing BG concentrations. In this setting, a passivation period of 24 h presented as an effective approach to provide a biocompatible cell culture setting. In conclusion, before introduction of BGs in cell culture, different passivation periods should be evaluated in order to meet the respective experimental settings, e.g., by following the experimental protocols used in this study. Full article

A method for the positive selection of specific antibodies for target proteins expressed as fusion proteins for the production of antiserum is presented. As proof of concept, the fusion protein FLAG::His::GFP::His::FLAG was expressed in Escherichia coli, purified, and used for the immunization of rabbits. The obtained serum was precleared via protein A affinity. A CusF::FLAG fusion protein was expressed in the periplasm of E. coli and purified. GFP without tags was also expressed in E. coli and purified via organic extraction. These proteins were then coupled to NHS-activated sepharose and used for the positive selection of Anti-GFP and Anti-FLAG antibodies. The obtained sera were tested for their specificity against different protein samples and fusion proteins in Western blots. A high specificity of the antibodies could be achieved by a single affinity chromatography step. In general, we advise to express the target protein with different tags and in different E. coli compartments for antibody production and affinity chromatography. Full article

Fatty acids (FAs) represent an important class of metabolites, impacting on membrane building blocks and signaling compounds in cellular regulatory networks. In nature, prokaryotes are characterized with the most impressing FA structural diversity and the highest relative content of free fatty acids (FFAs). In this context, nitrogen-fixing bacteria (order Rhizobiales), the symbionts of legumes, are particularly interesting. Indeed, the FA profiles influence the structure of rhizobial nodulation factors, required for successful infection of plant root. Although FA patterns can be assessed by gas chromatography—(GC-) and liquid chromatography—mass spectrometry (LC-MS), sample preparation for these methods is time-consuming and quantification suffers from compromised sensitivity, low stability of derivatives and artifacts. In contrast, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) represents an excellent platform for high-efficient metabolite fingerprinting, also applicable to FFAs. Therefore, here we propose a simple and straightforward protocol for high-throughput relative quantification of FFAs in rhizobia by combination of Langmuir technology and MALDI-TOF-MS featuring a high sensitivity, accuracy and precision of quantification. We describe a step-by-step procedure comprising rhizobia culturing, pre-cleaning, extraction, sample preparation, mass spectrometric analysis, data processing and post-processing. As a case study, a comparison of the FFA metabolomes of two rhizobia species—Rhizobium leguminosarum and Sinorhizobium meliloti, demonstrates the analytical potential of the protocol. Full article

Bisphenol-A (BPA), bisphenol A glycerolate dimethacrylate (Bis-GMA), triethylene glycol dimethacrylate (TEGDMA), and urethane dimethacrylate (UDMA) are organic monomers that can be released from dental composites into the oral cavity. Over specific concentrations, they can act as endocrine disruptors or cause toxic effects. The purpose of this work is to develop and validate an analytical method to determine BPA, Bis-GMA, TEGDMA, and UDMA monomers released from synthetic dental resins in artificial saliva. The method was validated before its application to new hybrid ceramic materials used in computer-aided design and computer-aided manufacturing (CAD/CAM) restorations to determine the release of monomers in various time intervals (e.g., 24 h, and 7, 14, 30, and 60 days), both in methanolic solutions, as well as in artificial saliva. Chromatographic analysis was performed isocratically on a Perfect Sil Target ODS-3 analytical column (250 mm × 4.6 mm, 5 µm) with CH₃CN/H₂O, 58/42% v/v as mobile phase within 23 min. The developed method was validated in terms of selectivity, linearity, accuracy, and precision. Full article

Modern vibrational spectroscopy techniques enable the rapid collection of thousands of spectra in a single hyperspectral image, allowing researchers to study spatially heterogeneous samples at micrometer resolution. A number of algorithms have been developed to correct for effects such as atmospheric absorption, light scattering by cellular structures and varying baseline levels. After preprocessing, spectra are commonly decomposed and clustered to reveal informative patterns and subtle spectral changes. Several of these steps are slow, labor-intensive and require programming skills to make use of published algorithms and code. We here present a free and platform-independent graphical toolbox that allows rapid preprocessing of large sets of spectroscopic images, including atmospheric correction and a new algorithm for resonant Mie scattering with improved speed. The software also includes modules for decomposition into constituent spectra using the popular Multivariate Curve Resolution–Alternating Least Squares (MCR-ALS) algorithm, augmented by region-of-interest selection, as well as clustering and cluster annotation. Full article

The analysis of glycosylphosphatidylinositol (GPI)-anchored receptor distribution and dynamics in live cells is challenging, because their clusters exhibit subdiffraction-limited sizes and are highly dynamic. However, the cellular response depends on the GPI-anchored receptor clusters’ distribution and dynamics. Here, we compare three approaches to GPI-anchored receptor labeling (with antibodies, fluorescent proteins, and enzymatically modified small peptide tags) and use several variants of Förster resonance energy transfer (FRET) detection by confocal microscopy and flow cytometry in order to obtain insight into the distribution and the ligand-induced dynamics of GPI-anchored receptors. We found that the enzyme-mediated site-specific fluorescence labeling of T-cadherin modified with a short peptide tag (12 residues in length) have several advantages over labeling by fluorescent proteins or antibodies, including (i) the minimized distortion of the protein’s properties, (ii) the possibility to use a cell-impermeable fluorescent substrate that allows for selective labeling of surface-exposed proteins in live cells, and (iii) superior control of the donor to acceptor molar ratio. We successfully detected the FRET of GPI-anchored receptors, T-cadherin, and ephrin-A1, without ligands, and showed in real time that adiponectin induces stable T-cadherin cluster formation. In this paper (which is complementary to our recent research (Balatskaya et al., 2019)), we present the practical aspects of labeling and the heteroFRET measurements of GPI-anchored receptors to study their dynamics on a plasma membrane in live cells. Full article

In this protocol, we introduced a method of measuring mitochondrial dysfunction to confirm the epithelial–mesenchymal transition (EMT) in pancreatic cancer cells under a hypoxic environment. There are many expertized and complicated methods to verify EMT. However, our methods have indicated that EMT can be identified by examining changes in reactive oxygen species (ROS) generation and membrane potential in mitochondria. To demonstrate whether the changes in the indicators of mitochondrial dysfunction are correlative to EMT, cell morphology, and expression of E-cadherin and N-cadherin were additionally observed. The results verified that a decrease in membrane potential and an increase in ROS in mitochondria were associated with EMT of pancreatic cancer cells. This protocol would be useful as a basis for providing an additional indicator for changes in the tumor microenvironment of pancreatic cancer cells relating to EMT under a hypoxic environment. Full article

The complex color patterns on the wings and body of Drosophila guttifera (D. guttifera) are emerging as model systems for studying evolutionary and developmental processes. Studies regarding these processes depend on overexpression and downregulation of developmental genes, which ultimately rely upon an effective transgenic system. Methods describing transgenesis in Drosophila melanogaster (D. melanogaster) have been reported in several studies, but they cannot be applied to D. guttifera due to the low egg production rate and the delicacy of the eggs. In this protocol, we describe extensively a comprehensive method used for generating transgenic D. guttifera. Using the protocol described here, we are able to establish transgenic lines, identifiable by the expression of enhanced green fluorescent protein (EGFP) in the eye disks of D. guttifera larvae. The entire procedure, from injection to screening for transgenic larvae, can be completed in approximately 30 days and should be relatively easy to adapt to other non-model Drosophila species, for which no white-eyed mutants exist. Full article

Novel bone substitute materials need to be evaluated in terms of their osteogenic differentiation capacity and possible unwanted cytotoxic effects in order to identify promising candidates for the therapy of bone defects. The activity of alkaline phosphatase (ALP) is frequently quantified as an osteogenic marker, while various colorimetric assays, like MTT assay, are used to monitor cell viability. In addition, the DNA or protein content of the samples needs to be quantified for normalization purposes. As this approach is time consuming and often requires the analysis of multiple samples, we aimed to simplify this process and established a protocol for the combined fluorescence-based quantification of ALP activity and cell viability within one single measurement. We demonstrate that the fluorogenic substrate 4-methylumbelliferone-phosphate (4-MUP) and the commonly used para-nitrophenylphosphate (p-NPP) produce comparable and highly correlating results. We further show that fluorescein–diacetate (FDA) can be used to quantify both cell viability and cell number without interfering with the quantification of ALP activity. The measurement of additional normalization parameters is, therefore, unnecessary. Therefore, the presented assay allows for a time-efficient, simple and reliable analysis of both ALP activity and cell viability from one sample and might facilitate experiments evaluating the osteogenic differentiation of osteoblast precursor cells. Full article

Hemocompatibility is a critical consideration when designing cardiovascular devices. Methods of assessing hemocompatibility range from in vitro protein adsorption and static platelet attachment to in vivo implantation. A standard preclinical assessment of biomaterial hemocompatibility is ex vivo quantification of thrombosis in a chronic arteriovenous shunt. This technique utilizes flowing blood and quantifies platelet accumulation and fibrin deposition. However, the physical parameters of the thrombus have remained unknown. This study presents the development of a novel method to quantify the 3D physical properties of the thrombus on different biomaterials: expanded polytetrafluoroethylene and a preclinical hydrogel, poly(vinyl alcohol). Tubes of 4–5 mm inner diameter were exposed to non-anticoagulated blood flow for 1 hour and fixed. Due to differences in biomaterial water absorption properties, unique methods, requiring either the thrombus or the lumen to be radiopaque, were developed to quantify average thrombus volume within a graft. The samples were imaged using X-ray microcomputed tomography (microCT). The methodologies were strongly and significantly correlated to caliper-measured graft dimensions (R² = 0.994, p < 0.0001). The physical characteristics of the thrombi were well correlated to platelet and fibrin deposition. MicroCT scanning and advanced image analyses were successfully applied to quantitatively measure 3D physical parameters of thrombi on cardiovascular biomaterials under flow. Full article

Quantitative trait loci (QTL) are genomic regions associated with phenotype variation of quantitative traits. To date, a total of 313 QTL for 31 quantitative traits have been reported in 14 studies on flax. Of these, 200 QTL from 12 studies were identified based on genetic maps, the scaffold sequences, or the pre-released chromosome-scale pseudomolecules. Molecular markers for QTL identification differed across studies but the most used ones were simple sequence repeats (SSRs) or single nucleotide polymorphisms (SNPs). To uniquely map the SSR and SNP markers from different references onto the recently released chromosome-scale pseudomolecules, methods with several scripts and database files were developed to locate PCR- and SNP-based markers onto the same reference, co-locate QTL, and scan genome-wide candidate genes. Using these methods, 195 out of 200 QTL were successfully sorted onto the 15 flax chromosomes and grouped into 133 co-located QTL clusters; the candidate genes that co-located with these QTL clusters were also predicted. The methods and tools presented in this article facilitate marker re-mapping to a new reference, genome-wide QTL analysis, candidate gene scanning, and breeding applications in flax and other crops. Full article

Telomeres represent the nucleotide repeat sequences at the ends of chromosomes and are essential for chromosome stability. They can shorten at each round of DNA replication mainly because of incomplete DNA synthesis of the lagging strand. Reduced relative telomere length is associated with aging and a range of disease states. Different methods such as terminal restriction fragment analysis, real-time quantitative PCR (qPCR) and fluorescence in situ hybridization are available to measure telomere length; however, the qPCR-based method is commonly used for large population-based studies. There are multiple variations across qPCR-based methods, including the choice of the single-copy gene, primer sequences, reagents, and data analysis methods in the different reported studies so far. Here, we provide a detailed step-by-step protocol that we have optimized and successfully tested in the hands of other users. This protocol will help researchers interested in measuring relative telomere lengths in cells or across larger clinical cohort/study samples to determine associations of telomere length with health and disease. Full article

BACKGROUND: The introduction of multiparametric magnetic resonance imaging (mpMRI) has improved the diagnosis of suspected prostate cancer, accurately risk-stratifying men before a biopsy. However, pre-biopsy mpMRI represents a significant deviation from the traditional approach of prostate specific antigen testing with subsequent systematic transrectal ultrasound-guided prostate biopsy and we have not yet explored the views of men who experience this new pathway. The purpose of the PACT study (PAtient views and aCceptance of mulTiparametric MRI) is to explore men’s perceptions of mpMRI. METHODS: PACT will be conducted at teaching hospitals in which mpMRI is central to the prostate cancer diagnostic pathway using a two-phase, mixed-methods, quantitative and qualitative approach. In phase I, men referred with suspected prostate cancer will complete detailed surveys to explore their views on the mpMRI-directed pathway compared to the traditional pathway and on what constitutes ‘significant’ prostate cancer. In phase II, these themes will be expanded upon with in-depth, semi-structured interviews. Qualitative data will be transcribed and thematically analysed, and quantitative questionnaire responses will be analysed statistically. DISCUSSION: PACT will provide the first detailed insight into patient perceptions on the use and acceptability of mpMRI. Furthermore, results from PACT will help contribute to the resolution of outstanding controversies that surround this technology. Full article

Recently there is a great social expectation that scientists should produce more sustainable and environmentally friendly chemical processes. Within this necessity, biocatalysis presents many attractive features because reactions are often performed in water, under mild conditions, the catalyst is biodegradable and can be obtained from renewable raw materials. In this work, we propose a simple, rapid and low-cost method for the preparation and application of an enzymatic extract from turnip root. The protocol described includes (1) the preparation of the enzymatic extract, (2) the procedure for the assessment of the more favorable working parameters (temperature, pH) and (3) the methodology for the application of the extract as the catalyst for biotransformation reactions. We anticipate that the protocol in this research will provide a simple way for obtaining an enzymatic extract which can operate efficiently under mild conditions and can effectively catalyze the biotransformation of simple phenols. Full article