Environmental science

Ecological and evolutionary studies often require high quality biodiversity data. This information is readily available through the many online databases that have compiled biodiversity data from herbaria, museums, and human observations. However, the process of preparing this information for analysis is complex and time consuming. In this study, we have developed a protocol in R language to process spatial data (download, merge, clean, and correct) and extract climatic data, using some genera of the ginseng family (Araliaceae) as an example. The protocol provides an automated way to process spatial and climatic data for numerous taxa independently and from multiple online databases. The script uses GBIF, BIEN, and WorldClim as the online data sources, but can be easily adapted to include other online databases. The script also uses genera as the sampling unit but provides a way to use species as the target. The cleaning process includes a filter to remove occurrences outside the natural range of the taxa, gardens, and other human environments, as well as erroneous locations and aspatial correction for misplaced occurrences (i.e., occurrences within a distance buffer from the coastal boundary). Additionally, each step of the protocol can be run independently. Thus, the protocol can begin with data cleaning, if the database has already been compiled, or with climatic data extraction, if the database has already been parsed. Each line of the R script is commented so that it can also be run by users with little knowledge of R.

Mixed communities of fungi and bacteria have been shown to be more efficient in degrading wood than fungi alone. Some standardised protocols for quantification of the wood decay ability of fungi have been developed (e.g., DIN V ENV 12038:2002 as the legal standard to test for the resistance of wood against wood-destroying basidiomycetes in Germany). Here, we describe a step-by-step protocol developed from the official standard DIN V ENV12038 to test combinations of bacteria and fungi for their combined wood degradation ability. Equally sized wood blocks are inoculated with wood decay fungi and bacterial strains. Axenic controls allow the analysis of varying degradation rates via comparison of the wood dry weights at the end of the experiments. This protocol provides new opportunities in exploration of inter- and intra-kingdom interactions in the wood-related environment and forms the basis for microcosm experiments.

Key features

• Quantification of wood decay ability of mixed cultures.

• Allows testing if fungi are more efficient in degrading wood when bacteria are present.

Mandelonitrile is a nitrogen-containing compound, considered an essential secondary metabolite. Chemically, it is a cyanohydrin derivative of benzaldehyde, with relevant functions in different physiological processes including defense against phytophagous arthropods. So far, procedures for detecting mandelonitrile have been effectively applied in cyanogenic plant species such as Prunus spp. Nevertheless, its presence in Arabidopsis thaliana, considered a non-cyanogenic species, has never been determined. Here, we report the development of an accurate protocol for mandelonitrile quantification in A. thaliana within the context of A. thaliana–spider mite interaction. First, mandelonitrile was isolated from Arabidopsis rosettes using methanol; then, it was derivatized by silylation to enhance detection and, finally, it was quantified using gas chromatography–mass spectrometry. The selectivity and sensitivity of this method make it possible to detect low levels of mandelonitrile (LOD 3 ppm) in a plant species considered non-cyanogenic that, therefore, will have little to no cyanogenic compounds, using a small quantity of starting material (≥100 mg).

Paraquat is a cost-effective herbicide, widely used in many countries, that can induce severe oxidative stress in photosynthetic tissues. Studying plant herbicide resistance or antioxidant stress mechanisms requires determining the cellular paraquat level when plants are treated by paraquat. The traditional isotopic labeling method has the potential risk to cause problems to both human health and the environment. For radioisotope manipulation, special operation spaces and strict environmental inspection are also required. In addition, the radiolabeled paraquat is increasingly hard to buy due to the extended production cycle. Here, we describe a nonradioactive method to determine the paraquat level in a small number of Arabidopsis tissues or protoplasts, using a high resolution ultra-high-performance liquid chromatography (UHPLC)-mass spectrometry (MS)/MS method. This method is highly selective and sensitive, and more environmentally compatible and technically feasible than the isotope detection method.

The study of haloarchaea provides an opportunity to expand understanding of the mechanisms used by extremophiles to thrive in and respond to harsh environments, including hypersaline and oxidative stress conditions. A common strategy used to investigate molecular mechanisms of stress response involves the deletion and/or site-directed mutagenesis of genes identified through omics studies followed by a comparison of the mutant and wild-type strains for phenotypic differences. The experimental methods used to monitor these differences must be controlled and reproducible. Current methods to examine recovery of halophilic archaea from extreme stress are complicated by extended incubation times, nutrients not typically encountered in the environment, and other related limitations. Here we describe a method for assessing the function of genes during hypochlorite stress in the halophilic archaeon Haloferax volcanii that overcomes these types of limitations. The method was found reproducible and informative in identifying genes needed for H. volcanii to recover from hypochlorite stress.

Acute respiratory distress syndrome (ARDS) is a life-threatening, high mortality pulmonary condition characterized by acute lung injury (ALI) resulting in diffuse alveolar damage. Despite progress regarding the understanding of ARDS pathophysiology, there are presently no effective pharmacotherapies. Due to the complexity and multiorgan involvement typically associated with ARDS, animal models remain the most commonly used research tool for investigating potential new therapies. Experimental models of ALI/ARDS use different methods of injury to acutely induce lung damage in both small and large animals. These models have historically played an important role in the development of new clinical interventions, such as fluid therapy and the use of supportive mechanical ventilation (MV). However, failures in recent clinical trials have highlighted the potential inadequacy of small animal models due to major anatomical and physiological differences, as well as technical challenges associated with the use of clinical co-interventions [e.g., MV and extracorporeal membrane oxygenation (ECMO)]. Thus, there is a need for larger animal models of ALI/ARDS, to allow the incorporation of clinically relevant measurements and co-interventions, hopefully leading to improved rates of clinical translation. However, one of the main challenges in using large animal models of preclinical research is that fewer species-specific experimental tools and metrics are available for evaluating the extent of lung injury, as compared to rodent models. One of the most relevant indicators of ALI in all animal models is evidence of histological tissue damage, and while histological scoring systems exist for small animal models, these cannot frequently be readily applied to large animal models. Histological injury in these models differs due to the type and severity of the injury being modeled. Additionally, the incorporation of other clinical support devices such as MV and ECMO in large animal models can lead to further lung damage and appearance of features absent in the small animal models. Therefore, semi-quantitative histological scoring systems designed to evaluate tissue-level injury in large animal models of ALI/ARDS are needed. Here we describe a semi-quantitative scoring system to evaluate histological injury using a previously established porcine model of ALI via intratracheal and intravascular lipopolysaccharide (LPS) administration. Additionally, and owing to the higher number of samples generated from large animal models, we worked to implement a more sustainable and greener histopathological workflow throughout the entire process.

Microorganisms have evolved adaptive strategies to respond to the autonomous degradation of their environment. Indeed, a growing culture progressively exhausts nutrients from its media and modifies its composition. Yet, how single cells react to these modifications remains difficult to study since it requires population-scale growth experiments to allow cell proliferation to have a collective impact on the environment, while monitoring the same individuals exposed to this environment for days. For this purpose, we have previously described an integrated microfluidic pipeline, based on continuous separation of the cells from the media and subsequent perfusion of the filtered media in an observation chamber containing isolated single cells. Here, we provide a detailed protocol to implement this methodology, including the setting up of the microfluidic system and the processing of timelapse images.

Iron (Fe) is an indispensable micronutrient for plant growth and development. Since both deficiency, as well as a surplus of Fe, can be detrimental to plant health, plants need to constantly tune uptake rates to maintain an optimum level of Fe. Quantification of Fe serves as an important parameter for analyzing the fitness of plants from different accessions, or mutants and transgenic lines with altered expression of specific genes. To quantify metals in plant samples, methods based on inductively coupled plasma-optical emission spectrometry (ICP-OES) or inductively coupled plasma-mass spectrometry (ICP-MS) have been widely employed. Although these methods are highly accurate, these methodologies rely on sophisticated equipment which is not always available. Moreover, ICP-OES and ICP-MS allow for surveying several metals in the same sample, which may not be necessary if only the Fe status is to be determined. Here, we outline a simple and cost-efficient protocol to quantify Fe concentrations in roots and shoots of Arabidopsis seedlings, by using a spectroscopy-based assay to quantify Fe²⁺-BPDS₃ complexes against a set of standards. This protocol provides a fast and reproducible method to determine Fe levels in plant samples with high precision and low costs, which does not depend on expensive equipment and expertise to operate such equipment.

We describe a method to test the preference of insects in response to (3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT). We use a device that includes a horizontal glass tube, two grooves (with activated carbon), air flow, rubber stoppers/tubes, transparent glass containers (optional), and a holder for the glass tube (optional). Equal amounts of activated carbon in the groove (removable) are placed at both ends to avoid air contamination. The air flow is generated by an air pump. In the closed device, different samples are placed at each end of the glass tube. The air pump at the top of the glass tube forms an air flow that converges to the middle site of the glass tube. In each test, insect larvae are located in the middle of the glass test tube. If the test samples release DMNT that can be sensed by insects, the insects will selectively move to one specific end of the glass tube. The number of insects that move to each end will be recorded for further studies. This method can also be used to test the preference of insects in response to other volatile compounds.

The ongoing COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As this virus is classified as a biosafety level-3 (BSL-3) agent, the development of countermeasures and basic research methods is logistically difficult. Recently, using reverse genetics, we developed a BSL-2 cell culture system for production of transcription- and replication-component virus-like-particles (trVLPs) by genetic transcomplementation. The system consists of two parts: SARS-CoV-2 GFP/ΔN genomic RNA, in which the nucleocapsid (N) gene, a critical gene for virion packaging, is replaced by a GFP reporter gene; and a packaging cell line for ectopic expression of N (Caco-2-N). The complete viral life cycle can be recapitulated and confined to Caco-2-N cells, with GFP positivity serving as a surrogate readout for viral infection. In addition, we utilized an intein-mediated protein splicing technique to split the N gene into two independent vectors and generated the Caco-2-N^intein cells as a packaging cell line to further enhance the security of this cell culture model. Altogether, this system provides for a safe and convenient method to produce trVLPs in BSL-2 laboratories. These trVLPs can be modified to incorporate desired mutations, permitting high-throughput screening of antiviral compounds and evaluation of neutralizing antibodies. This protocol describes the details of the trVLP cell culture model to make SARS-CoV-2 research more readily accessible.