Sensitive visualization of SARS-CoV-2 RNA with CoronaFISH

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The positive-sense single-stranded RNA virus contains a single linear RNA segment that serves as a template for transcription and replication, leading to the synthesis of positive and negative-stranded viral RNA (vRNA) in infected cells. Tools to visualize vRNA directly in infected cells are critical to analyze the viral replication cycle, screen for therapeutic molecules, or study infections in human tissue. Here, we report the design, validation, and initial application of FISH probes to visualize positive or negative RNA of SARS-CoV-2 (CoronaFISH). We demonstrate sensitive visualization of vRNA in African green monkey and several human cell lines, in patient samples and human tissue. We further demonstrate the adaptation of CoronaFISH probes to electron microscopy. We provide all required oligonucleotide sequences, source code to design the probes, and a detailed protocol. We hope that CoronaFISH will complement existing techniques for research on SARS-CoV-2 biology and COVID-19 pathophysiology, drug screening, and diagnostics.

Extracting abundance information from DNA-based data

The accurate extraction of species-abundance information from DNA-based data (metabarcoding, metagenomics) could contribute usefully to diet reconstruction and quantitative food webs, the inference of species interactions, the modelling of population dynamics and species distributions, the biomonitoring of environmental state and change, and the inference of false positives and negatives. However, capture bias, capture noise, species pipeline biases, and pipeline noise all combine to inject error into DNA-based datasets. We focus on methods for correcting the latter two error sources, as the first two are addressed extensively in the ecological literature. To extract abundance information, it is useful to distinguish two concepts. (1) Across-species quantification describes relative species abundances within one sample. (2) In contrast, within-species quantification describes how the abundance of each individual species varies from sample to sample, as in a time series, an environmental gradient, or different experimental treatments. Firstly, we review methods to remove species pipeline biases and pipeline noise. Secondly, we demonstrate experimentally (with a detailed protocol) how to use a 'DNA spike-in' to remove pipeline noise and recover within-species abundance information. We also introduce a statistical estimator that can partially remove pipeline noise from datasets that lack a physical DNA spike-in.

Defining Blood Plasma and Serum Metabolome by GC-MS

Metabolomics uses advanced analytical chemistry methods to analyze metabolites in biological samples. The most intensively studied samples are blood and its liquid components: plasma and serum. Armed with advanced equipment and progressive software solutions, the scientific community has shown that small molecules’ roles in living systems are not limited to traditional “building blocks” or “just fuel” for cellular energy. As a result, the conclusions based on studying the metabolome are finding practical reflection in molecular medicine and a better understanding of fundamental biochemical processes in living systems. This review is not a detailed protocol of metabolomic analysis. However, it should support the reader with information about the achievements in the whole process of metabolic exploration of human plasma and serum using mass spectrometry combined with gas chromatography.

Improvement of the selection method for potato breeding for resistance to the Colorado potato beetle

Создание сортов картофеля, устойчивых к повреждению колорадским жуком, – основное направление стабилизации картофельных агроценозов в долгосрочной перспективе. Цель настоящих исследований – совершенствование методики отбора генотипов с относительно высокой устойчивостью к повреждению колорадским жуком. Предложена методика отбора по фенотипическому признаку интенсивности проявления реакции сверхчувствительности (СВЧ) листовой пластинки листьев картофеля к яйцекладкам колорадского жука. Чтобы исключить зависимость селекционного отбора от наличия кладки насекомого на листовой пластинке из состава химических соединений, находящихся на поверхности хориона откладываемых яиц, был выделен эффектор, запускающий реакцию сверхчувствительности (СВЧ-реакцию) к появлению на поверхности листовой пластинки яиц колорадского жука. На основе результатов двухлетних полевых опытов на большом количестве сортообразцов установлено наличие положительной корреляционной зависимости средней степени (коэффициент 0,451) между интенсивностью образования некроза в ответ на обработку листовой пластинки эффектором СВЧ-реакции и уровнем устойчивости растений картофеля к фитофагу. Использование эффектора, запускающего СВЧ-реакцию на кладки яиц колорадского жука, позволяет оценивать гибриды в младших питомниках в отсутствии фитофага. В статье приводится подробное описание усовершенствованной методики оценки и отбора устойчивых к насекомому генотипов с использованием эффектора, запускающего СВЧ-реакцию на кладки яиц колорадского жука. Воспроизводимость СВЧ-реакции листьев картофеля на кладки яиц колорадского жука в полевых условиях при использовании биохимического эффектора позволяет провести визуализацию наличия устойчивости растений к вредителю, значительно облегчить оценку и отбор устойчивых генотипов, что в конечном счете существенно повышает результативность селекционного процесса. Creation of resistant potato varieties to damage by the Colorado potato beetle is the main direction of stabilization of potato fields in the long term. The aim of these studies is to improve the methodology for the selection of genotypes with a relatively high resistance to damage by the Colorado potato beetle. A method is proposed for the selection of genotypes with a relatively high resistance to damage by the Colorado potato beetle according to the phenotypic sign of the intensity of the hypersensitivity reaction (HR) of the leaf blade of potato leaves to the clutches of eggs of the Colorado potato beetle. In order to exclude the dependence of the selection screening on the presence of an insect clutch on the leaf blade, an effector that triggers a HR – response to clutches was isolated from the composition of chemical compounds on the chorion surface of the laid eggs. On the basis of two-year field experiments on a large number of cultivars, the presence of a positive correlation dependence of the average degree (coefficient 0.451) between the intensity of necrosis formation in response to the treatment of the leaf blade by the effector that triggers a HR – response and the level of resistance of potato plants to phytophagous was established. The use of the triggering effector HR – the reaction to the egg clutches of the Colorado potato beetle makes it possible to evaluate hybrids in younger nurseries in the absence of a phytophages. The article provides a detailed protocol of an improved methodology for assessing and selecting insect-resistant genotypes using the triggers effector HR – response to egg clutches of the Colorado potato beetle. Stable repeatability of the HR – response of potato leaves to clutches of Colorado potato beetle eggs in the field using the microwave response effector allows visualizing the presence of plant resistance to the pest, greatly facilitating the assessment and selection of resistant genotypes, which ultimately significantly increases the effectiveness of the breeding process.

A drone-based survey for large, basking freshwater turtle species

Conservation concerns are increasing for numerous freshwater turtle species, including Pseudemys gorzugi, which has led to a call for more research. However, traditional sampling methodologies are often time consuming, labor intensive, and invasive, restricting the amount of data that can be collected. Biases of traditional sampling methods can further impair the quality of the data collected, and these shortfalls may discourage their use. The use of unmanned aerial vehicles (UAVs, drones) for conducting wildlife surveys has recently demonstrated the potential to bridge gaps in data collection by offering a less labor intensive, minimally invasive, and more efficient process. Photographs and video can be obtained by camera attachments during a drone flight and analyzed to determine population counts, abundance, and other types of data. In this study we developed a detailed protocol to survey for large, freshwater turtle species in an arid, riverine landscape. This protocol was implemented with a DJI Matrice 600 Pro drone and a SONY ILCE α6000 digital camera to determine P. gorzugi and sympatric turtle species occurrence across 42 sites in southwestern Texas, USA. The use of a large drone and high-resolution camera resulted in high identification percentages, demonstrating the potential of drones to survey for large, freshwater turtle species. Numerous advantages to drone-based surveys were identified as well as some challenges, which were addressed with additional refinement of the protocol. Our data highlight the utility of drones for conducting freshwater turtle surveys and provide a guideline to those considering implementing drone-mounted high-resolution cameras as a survey tool.

Skin swabbing protocol to collect DNA samples from small-bodied fish species

Fish species are commonly used as experimental models in the laboratory. DNA is routinely collected from these animals to permit identification of their genotype. The current standard procedure to sample DNA is fin clipping, which involves anaesthetising individuals and removing a portion of the caudal fin. While fin clipping reliably generates good quality DNA samples for downstream applications, there is evidence that it can alter health and welfare, leading to infection and impacting on the fish’s behaviour. This in turn can result in greater variation in the data collected. In a recent study we adapted a skin swabbing protocol to collect DNA from small-bodied fish, including sticklebacks and zebrafish, without the use of anaesthetics or sharp instruments. A rayon-tipped swab was used to collect mucus from the flank of the fish, which was then used for DNA extraction. We subsequently demonstrated that compared to fin clipping, skin swabbing triggered fewer changes in stress axis activation and behaviour. We also found that data collected from fish that had been swabbed were less variable than data from fish that had been fin clipped, potentially allowing smaller sample sizes in experimental groups after using this technique, and thereby reducing animal use. Here we provide a detailed protocol explaining how to collect DNA samples from small laboratory fish using skin swabs.

Tutorial: Guidelines for Single-Cell RT-qPCR

Reverse transcription quantitative PCR (RT-qPCR) has delivered significant insights in understanding the gene expression landscape. Thanks to its precision, sensitivity, flexibility, and cost effectiveness, RT-qPCR has also found utility in advanced single-cell analysis. Single-cell RT-qPCR now represents a well-established method, suitable for an efficient screening prior to single-cell RNA sequencing (scRNA-Seq) experiments, or, oppositely, for validation of hypotheses formulated from high-throughput approaches. Here, we aim to provide a comprehensive summary of the scRT-qPCR method by discussing the limitations of single-cell collection methods, describing the importance of reverse transcription, providing recommendations for the preamplification and primer design, and summarizing essential data processing steps. With the detailed protocol attached in the appendix, this tutorial provides a set of guidelines that allow any researcher to perform scRT-qPCR measurements of the highest standard.

Sperm Accumulation Induced by the Female Reproductive Fluid: Putative Evidence of Chemoattraction Using a New Tool

There is considerable evidence that female reproductive fluid (FRF) interacts intimately with sperm, affecting several sperm traits, including sperm motility and longevity, and ultimately fertilization success. One of the first documented interactions between FRF and sperm is the ability of FRF to attract and guide sperm towards the eggs. However, most of the evidence of FRF’s chemoattraction proprieties comes from a limited number of taxa, specifically mammals and invertebrate broadcasting spawners. In other species, small FRF volumes and/or short sperm longevity often impose methodological difficulties resulting in this gap in chemoattraction studies in non-model species. One of the outcomes of sperm chemotaxis is sperm accumulation towards high chemoattractant concentrations, which can be easily quantified by measuring sperm concentration. Here, we tested sperm accumulation towards FRF in the zebrafish, Danio rerio, using an ad hoc developed, 3D printed, device (‘sperm selection chamber’). This easy-to-use tool allows to select and collect the sperm that swim towards a chemical gradient, and accumulate in a chemoattractant-filled well thus providing putative evidence for chemoattraction. We found that sperm accumulate in FRF in zebrafish. We also found that none of the sperm quality traits we measured (sperm swimming velocity and trajectory, sperm motility, and longevity) were correlated with this response. Together with the 3D printable project, we provide a detailed protocol for using the selection chamber. The chamber is optimized for the zebrafish, but it can be easily adapted for other species. Our device lays the foundation for a standardized way to measure sperm accumulation and in general chemoattraction, stimulating future research aimed at understanding the role and the mechanisms of sperm chemoattraction by FRF.

Development, validation, and application of the ribosome separation and reconstitution system for protein translation in vitro

Stress-induced molecular damage to ribosomes can impact protein synthesis in cells, but cell-based assays do not provide a clear way to distinguish the effects of ribosome damage from stress responses and damage to other parts of the translation machinery. Here we describe a detailed protocol for the separation of yeast ribosomes from other translational machinery constituents, followed by reconstitution of the translation mixture in vitro. This technique, which we refer to as Ribosome Separation and Reconstitution (RSR), allows chemical modifications of yeast ribosomes without compromising other key translational components. In addition to the characterization of stress-induced ribosome damage, RSR can be applied to a broad range of experimental problems in studies of yeast translation.