Methods Protoc., Volume 5, Issue 4 (August 2022) – 15 articles

The assignment of specific patterns of gene expression to specific cells in a complex tissue facilitates the connection between genotype and phenotype. Single-cell sequencing of whole tissues produces single-cell transcript resolution but lacks the spatial information of the derivation of each cell, whereas techniques such as multiplex FISH localize transcripts to specific cells in a tissue but require a priori information of the target transcripts to examine. Laser dissection of tissues followed by transcriptome analysis is an efficient and cost-effective technique that provides both unbiased gene expression discovery together with spatial information. Here, we detail a laser dissection protocol for total RNA extraction from butterfly larval and pupal wing tissues, without the need of paraffin embedding or the use of a microtome, that could be useful to researchers interested in the transcriptome of specific areas of the wing during development. This protocol can bypass difficulties in extracting high quality RNA from thick fixed tissues for sequencing applications. Full article

A co-culture of neurons and Schwann cells has frequently been used to investigate myelin sheath formation. However, this approach is restricted to myelin-related diseases of the peripheral nervous system. This study introduces and compares an ex vivo model of adult-mouse-derived dorsal root ganglia (DRG) explant, with an in vitro co-culture of dissociated neurons from mouse embryo DRG and Schwann cells from a mouse sciatic nerve. The 2D co-culture has disadvantages of different mouse isolation for neurons and Schwann cells, animal number, culture duration, and the identification of disease model. However, 3D DRG explant neurons and myelination cells in Matrigel-coated culture are obtained from the same mouse, the culture period is shorter than that of 2D co-culture, and fewer animals are needed. In addition, it has simpler and shorter experimental steps than 2D co-culture. This culture system may prove advantageous in studies of biological functions and pathophysiological mechanisms of disease models, since it can reflect disease characteristics as traditional co-culture does. Therefore, it is suggested that a DRG explant culture is a scientifically, ethically, and economically more practical option than a co-culture system for studying myelin dynamics, myelin sheath formation, and demyelinating disease. Full article

With the plethora of commercially available UV-C devices exhibiting different intensity and lifespans, it is critical to consumer safety that companies verify and clearly communicate the efficacy of their devices as per the intended use. The purpose of this study was to define a low-cost protocol for investigating the antimicrobial efficacy of commercial UV devices for industry use. The tested devices included: a wall-mounted unit (Device A), a troffer unit (Device B), and an induction lamp unit (Device C). The devices were installed within an enclosed tower to prevent the transmission of UV-C radiation outside of the testing area. The procedure details determining the devices′ antimicrobial efficacy using plastic coupons inoculated with Escherichia coli or Staphylococcus aureus. The protocol includes suggested time–distance treatments according to the potential application of each device type and reports the results as log CFU/mL reduction or percent reduction. Full article

The combination of histological and biomolecular analyses provides deep understanding of different biological processes and is of high interest for basic and applied research. However, the available analytical methods are still limited, especially when considering bone samples. This study compared different fixation media to identify a sufficient analytical method for the combination of histological, immuno-histological and biomolecular analyses of the same fixed, processed and paraffin embedded bone sample. Bone core biopsies of rats’ femurs were fixed in different media (RNAlater + formaldehyde (R + FFPE), methacarn (MFPE) or formaldehyde (FFPE)) for 1 week prior to decalcification by EDTA and further histological processing and paraffin embedding. Snap freezing (unfixed frozen tissue, UFT) and incubation in RNAlater were used as additional controls. After gaining the paraffin sections for histological and immunohistological analysis, the samples were deparaffined and RNA was isolated by a modified TRIZOL protocol. Subsequently, gene expression was evaluated using RT-qPCR. Comparable histo-morphological and immuno-histological results were evident in all paraffin embedded samples of MFPE, FFPE and R + FFPE. The isolated RNA in the group of MFPE showed a high concentration and high purity, which was comparable to the UFT and RNAlater groups. However, in the groups of FFPE and R + FFPE, the RNA quality and quantity were statistically significantly lower when compared to MFPE, UFT and RNAlater. RT-qPCR results showed a comparable outcome in the group of MFPE and UFT, whereas the groups of FFPE and R + FFPE did not result in a correctly amplified gene product. Sample fixation by means of methacarn is of high interest for clinical samples to allow a combination of histological, immunohistological and biomolecular analysis. The implementation of such evaluation method in clinical research may allow a deeper understanding of the processes of bone formation and regeneration. Full article

For-profit donor human milk organizations have DNA-based proprietary methodology for testing incoming milk for adulteration with other species’ milk. However, there is currently no standardized methodology for extracting DNA from human milk. Microbiome research has shown that DNA purity and quantity can vary depending on the extraction methodology and storage conditions. This study assessed the purity and quantity of DNA extracted from four commercially available DNA extraction kits—including one kit that was developed for human milk. This study was for method validation only. One donor provided a 90 mL human milk sample. The sample was aliquoted into 70 × 1 mL microcentrifuge tubes. Aliquots were randomized into one of three categories: fresh extraction, extraction after freezing, and extraction after purification and storage at room temperature. DNA was analyzed for purity and quantity using a NanoDrop Spectrophotometer. Results confirmed differences in DNA purity and quantity between extraction kits. The Plasma/Serum Circulating DNA Purification Mini Kit (Norgen Biotek, ON, Canada) provided significantly more DNA, and consistent purity as measured by 260/280 and 260/230 ratios. DNA quantity and purity were similar between fresh and frozen human milk samples. These results suggest that DNA purity and quantity is highest and most consistent when extracted from human milk using the Plasma/Serum Circulating DNA Purification Mini Kit amongst the kits tested in this study. Standardized methodology for extracting DNA from human milk is necessary for improvement of research in the field of human milk. To do this, future studies are recommended for optimization of DNA extraction from human milk using larger sample sizes and multiple donor parents. Full article

Monitoring electrical signals in plants allows the examination of their acute and chronic physiological changes and responses to stimuli. Understanding how plant roots/rhizoids respond to chemical cues in their environment will provide insight into how these structures acquire resources. Chronic exposure to L-glutamate alters root growth and is known to alter Ca²⁺ flux inside roots. The ionic flux can be detected by electrical changes. A rapid and relatively easy approach is presented to screen the electrical sensitivity of roots/rhizoids to compounds such as amino acids and known agonists/antagonists to receptors and ion channels. The approach uses a background-flow system of basal salt or water; then, the administered compounds are added to the roots/rhizoids while monitoring their electrical responses. As a proof of concept, the response to flow-through of glutamate (1 mM) was targeted at the root/rhizoids of three plants (Arabidopsis thaliana, Pisum sativum and Marchantia inflexa). Both Arabidopsis thaliana and Pisum sativum produced rapid depolarization upon exposure to glutamate, while M. inflexa did not show an electrical response. In some experiments, simultaneous recordings with impedance measures for acute changes and glass electrodes for chronic electrical potential changes were used. The effect of potassium chloride (300 mM) as a depolarizing stimulus produced responses in both P. sativum and M. inflexa. The protocol presented can be used to screen various compounds in a relatively rapid manner for responsiveness by the roots/rhizoids of plants. Full article

Nanomaterials are materials in which at least one of the three dimensions ranges from 1 to 100 nm, according to the International Organization for Standardization (ISO). Nanomaterials can be categorized according to various parameters, such as their source, their shape, and their origin. Their increasing use in industrial settings, everyday items, electronic devices, etc. poses an environmental and biological risk that needs to be assessed and appropriately addressed. The development of reliable analytical methods for both characterization and quantification of nanomaterials in various matrices is essential. This review summarized the recent trends in analytical methodologies for the characterization and determination of nanoparticles in biological matrices. Full article

Machine learning (ML) in healthcare data analytics is attracting much attention because of the unprecedented power of ML to extract knowledge that improves the decision-making process. At the same time, laws and ethics codes drafted by countries to govern healthcare data are becoming stringent. Although healthcare practitioners are struggling with an enforced governance framework, we see the emergence of distributed learning-based frameworks disrupting traditional-ML-model development. Splitfed learning (SFL) is one of the recent developments in distributed machine learning that empowers healthcare practitioners to preserve the privacy of input data and enables them to train ML models. However, SFL has some extra communication and computation overheads at the client side due to the requirement of client-side model synchronization. For a resource-constrained client side (hospitals with limited computational powers), removing such conditions is required to gain efficiency in the learning. In this regard, this paper studies SFL without client-side model synchronization. The resulting architecture is known as multi-head split learning (MHSL). At the same time, it is important to investigate information leakage, which indicates how much information is gained by the server related to the raw data directly out of the smashed data—the output of the client-side model portion—passed to it by the client. Our empirical studies examine the Resnet-18 and Conv1-D architecture model on the ECG and HAM-10000 datasets under IID data distribution. The results find that SFL provides 1.81% and 2.36% better accuracy than MHSL on the ECG and HAM-10000 datasets, respectively (for cut-layer value set to 1). Analysis of experimentation with various client-side model portions demonstrates that it has an impact on the overall performance. With an increase in layers in the client-side model portion, SFL performance improves while MHSL performance degrades. Experiment results also demonstrate that information leakage provided by mutual information score values in SFL is more than MHSL for ECG and HAM-10000 datasets by $2\times {10}^{-5}$ and $4\times {10}^{-3}$, respectively. Full article

Digestibility trials need a viable rumen fluid as inoculum to degrade feeds. The variability of rumen fluid depends on the animal’s diet, while its viability is greatly influenced by the sampling and handling procedures. In this article, we present a replicable protocol for sampling the rumen fluid from slaughtered animals for in vitro digestibility trials. A detailed list of the tools and a step-by-step standardized procedure for the collection, storage and the transportation of the rumen fluid from the slaughterhouse to the laboratory is presented. We also describe a digestibility trial for establishing the maximum storage time of rumen fluid from sampling to its use. The results show that the rumen fluid, collected and maintained according to the proposed protocol, can be stored and used from 30 to 300 min from sampling without significantly compromising the fermentative activity of the microbial population. Full article

The neuroblastoma cell line SH-SY5Y has been a well-established and very popular in vitro model in neuroscience for decades, especially focusing on neurodevelopmental disorders, such as Parkinson’s disease. The ability of this cell type to differentiate compared with other models in neurobiology makes it one of the few suitable models without having to rely on a primary culture of neuronal cells. Over the years, various, partly contradictory, methods of cultivation have been reported. This study is intended to provide a comprehensive guide to the in vitro cultivation of undifferentiated SH-SY5Y cells. For this purpose, the morphology of the cell line and the differentiation of the individual subtypes are described, and instructions for cell culture practice and long-term cryoconservation are provided. We describe the key growth characteristics of this cell line, including proliferation and confluency data, optimal initial seeding cell numbers, and a comparison of different culture media and cell viability during cultivation. Furthermore, applying an optimized protocol in a long-term cultivation over 60 days, we show that cumulative population doubling (CPD) is constant over time and does not decrease with incremental passage, enabling stable cultivation, for example, for recurrent differentiation to achieve the highest possible reproducibility in subsequent analyses. Therefore, we provide a solid guidance for future research that employs the neuroblastoma cell line SH-SY5Y. Full article

The molecular analysis of small or rare patient tissue samples is challenging and often limited by available technologies and resources, such as reliable antibodies against a protein of interest. Although targeted approaches provide some insight, here, we describe the workflow of two complementary mass spectrometry approaches, which provide a more comprehensive and non-biased analysis of the molecular features of the tissue of interest. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) generates spatial intensity maps of molecular features, which can be easily correlated with histology. Additionally, liquid chromatography tandem mass spectrometry (LC-MS/MS) can identify and quantify proteins of interest from a consecutive section of the same tissue. Here, we present data from concurrent precancerous lesions from the endometrium and fallopian tube of a single patient. Using this complementary approach, we monitored the abundance of hundreds of proteins within the precancerous and neighboring healthy regions. The method described here represents a useful tool to maximize the number of molecular data acquired from small sample sizes or even from a single case. Our initial data are indicative of a migratory phenotype in these lesions and warrant further research into their malignant capabilities. Full article

Electrical activity is widely used for assessing a plant’s response to an injury or environmental stimulus. Commonly, a differential electrode recording between silver wire leads with the reference wire connected to the soil, or a part of the plant, is used. One method uses KCl-filled glass electrodes placed into the plant, similar to recording membrane/cell potentials in animal tissues. This method is more susceptible to artifacts of equipment noise and photoelectric effects than an impedance measure. An impedance measure using stainless steel wires is not as susceptible to electrically induced noises. Impedance measurements are able to detect injury in plants as well as exposure of the roots to environmental compounds (glutamate). The impedance measures were performed in 5 different plants (tomato, eggplant, pepper, liverwort, and Coleus scutellarioides), and responses to mechanical movement of the plant, as well as injury, were recorded. Monitoring electrical activity in a plant that arises in a distant plant was also demonstrated using the impedance method. The purpose of this report is to illustrate the ease in using impedance measures for monitoring electrical signals from individual plants or aggregates of plants for potentially scaling for high throughput and monitoring controlled culturing and outdoor field environments. Full article

The measurement of antibodies to vaccine antigens is crucial for research towards a safe and effective vaccine for Streptococcus pyogenes (Strep A). We describe the establishment and detailed characterisation of a four-plex assay to measure IgG to the Strep A vaccine antigens SpyCEP, Slo, SpyAD and GAC using the Luminex multiplex platform. A standard curve was established and characterized to allow the quantification of antigen-specific IgG. Assay specificity, precision, linearity, reproducibility and repeatability were determined via the measurement of antigen-specific IgG from pooled human serum. The assay is highly specific, reproducible and performs well across a large range of antibody concentrations against all four antigens. It is, therefore, suitable for future clinical trials in humans with a four-component vaccine, as well as for seroepidemiological studies to gain insights into naturally occurring immunity. Full article

The existing methods of callose quantification include epifluorescence microscopy and fluorescence spectrophotometry of aniline blue-stained callose particles, immuno-fluorescence microscopy and indirect assessment of both callose synthase and β-(1,3)-glucanase enzyme activities. Some of these methods are laborious, time consuming, not callose-specific, biased and require high technical skills. Here, we describe a method of callose quantification based on Sandwich Enzyme-Linked Immunosorbent Assay (S-ELISA). Tissue culture-derived banana plantlets were inoculated with Xanthomonas campestris pv. musacearum (Xcm) bacteria as a biotic stress factor inducing callose production. Banana leaf, pseudostem and corm tissue samples were collected at 14 days post-inoculation (dpi) for callose quantification. Callose levels were significantly different in banana tissues of Xcm-inoculated and control groups except in the pseudostems of both banana genotypes. The method described here could be applied for the quantification of callose in different plant species with satisfactory level of specificity to callose, and reproducibility. Additionally, the use of 96-well plate makes this method suitable for high throughput callose quantification studies with minimal sampling and analysis biases. We provide step-by-step detailed descriptions of the method. Full article

Intron evolution may be readily imaged through the combined use of the “dot plot” function of the NCBI BLAST, aligning two sequences at a time, and the Vertebrate “Multiz” alignment and conservation tool of the UCSC Genome Browser. With the NCBI BLAST, an ideal alignment of two highly conserved sequences generates a diagonal straight line in the plot from the lower left corner to the upper right corner. Gaps in this line correspond to non-conserved sections. In addition, the dot plot of the alignment of a sequence with the same sequence after the removal of the Transposable Elements (TEs) can be observed along the diagonal gaps that correspond to the sites of TE insertion. The UCSC Genome Browser can graph, along the entire sequence of a single gene, the level of overall conservation in vertebrates. This level can be compared with the conservation level of the gene in one or more selected vertebrate species. As an example, we show the graphic analysis of the intron conservation in two genes: the mitochondrial solute carrier 21 (SLC25A21) and the growth hormone receptor (GHR), whose coding sequences are conserved through vertebrates, while their introns show dramatic changes in nucleotide composition and even length. In the SLC25A21, a few short but significant nucleotide sequences are conserved in zebrafish, Xenopus and humans, and the rate of conservation steadily increases from chicken/human to mouse/human alignments. In the GHR, a less conserved gene, the earlier indication of intron conservation is a small signal in chicken/human alignment. The UCSC tool may simultaneously display the conservation level of a gene in different vertebrates, with reference to the level of overall conservation in Vertebrates. It is shown that, at least in SLC25A21, the sites of higher conservation are not always coincident in chicken and zebrafish nor are the sites of higher vertebrate conservation. Full article