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Burris, D. M., Gillespie, S. W., Campbell, E. J., Ice, S. N., Yadav, V., Picking, W. D., Lorson, C. L., & Singh, K. (2024). Applications of surface plasmon resonance (SPR) to the study of diverse protein-ligand interactions. Current Protocols, 4, e1030. doi: 10.1002/cpz1.1030

In the above-referenced article:

Figure 6 has been updated to include a key to the symbols used in the figure.

The current version online now includes these corrections and may be considered the authoritative version of record.

Orsay virus infection in the nematode Caenorhabditis elegans presents an opportunity to study host-virus interactions in an easily culturable, whole-animal host. Previously, a major limitation of C. elegans as a model for studying antiviral immunity was the lack of viruses known to naturally infect the worm. With the 2011 discovery of the Orsay virus, a naturally occurring viral pathogen, C. elegans has emerged as a compelling model for research on antiviral defense. From the perspective of the host, the genetic tractability of C. elegans enables mechanistic studies of antiviral immunity while the transparency of this animal allows for the observation of subcellular processes in vivo. Preparing infective virus filtrate and performing infections can be achieved with relative ease in a laboratory setting. Moreover, several tools are available to measure the outcome of infection. Here, we describe workflows for generating infective virus filtrate, achieving reproducible infection of C. elegans, and assessing the outcome of viral infection using molecular biology approaches and immunofluorescence. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Preparation of Orsay virus filtrate

Support Protocol: Synchronize C. elegans development by bleaching

Basic Protocol 2: Orsay virus infection

Basic Protocol 3: Quantification of Orsay virus RNA1/RNA2 transcript levels by qRT-PCR

Basic Protocol 4: Quantification of infection rate and fluorescence in situ hybridization (FISH) fluorescence intensity

Basic Protocol 5: Immunofluorescent labeling of dsRNA in virus-infected intestinal tissue

Traditional skin sampling methods include punch or shave biopsies to produce a solid tissue sample for analysis. These biopsy procedures are painful, require anesthesia, and leave permanent scars. This unit describes a suction blister skin biopsy method that can be used in place of traditional biopsy methodologies as a minimally invasive, non-scarring skin sampling technique. The induction of suction blisters uses an instrument with a chamber that applies negative pressure and gentle heat to the skin. Blister formation occurs within 1 hr, producing up to five blisters, each 10 mm in diameter per biopsy site. Blister fluid can be extracted and centrifuged to retrieve cells from the epidermis and upper dermis for flow cytometry, single-cell RNA sequencing, cell culture, and more without the need for digestion protocols. In addition, the blister fluid can be used to measure soluble proteins and metabolites. This unit describes the preparation of supplies and subjects, the suction blister biopsy procedure and blister formation, fluid extraction, and post-blistering care. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Preparation of supplies and subject

Basic Protocol 2: Suction blister biopsy procedure and formation

Basic Protocol 3: Blister fluid extraction

Basic Protocol 4: Post-blister care and clean up

The Affective Bias Test (ABT) quantifies acute changes in affective state based on the affective biases they generate in an associative reward learning task. The Reward Learning Assay (RLA) provides a control assay for the ABT and reward-induced biases generated in this model are sensitive to changes in core affective state. Both tasks involve training animals to associate a specific digging substrate with a food reward. Animals learn to discriminate between two digging substrates placed in ceramic bowls, one rewarded and one unrewarded. In the ABT, the animal learns two independent substrate-reward associations with a fixed reward value following either an affective state or drug manipulation, or under control conditions. Affective biases generated are quantified in a choice test where the animals exhibit a bias (make more choices) for one of the substrates which is specifically related to affective state at the time of learning. The ABT is used to investigate biases generated during learning as well as modulation of biases associated with past experiences. The RLA follows a similar protocol, but the animal remains in the same affective state throughout and a reward-induced bias is generated by pairing one substrate with a higher value reward. The RLA provides a control to determine if drug treatments affect memory retrieval more generally. Studies in depression models and following environmental enrichment suggest that reward-induced biases are sensitive to core changes in affective state. Each task offers different insights into affective processing mechanisms and may help improve the translational validity of animal studies and benefit pre-clinical drug development. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Bowl digging and discrimination training

Basic Protocol 2: The reward learning assay

Basic Protocol 3: The affective bias test - new learning

Basic Protocol 4: The affective bias test - modulation of affective biases associated with past experiences

Functional characterization of enzymes/proteins requires determination of the binding affinity of small molecules or other biomolecules with the target proteins. Several available techniques, such as proteomics and drug discovery strategies, require a precise and high-throughput assay for rapid and reliable screening of potential candidates for further testing. Surface plasmon resonance (SPR), a well-established label-free technique, directly measures biomolecular affinities. SPR assays require immobilization of one interacting component (ligand) on a conductive metal (mostly gold or silver) and a continuous flow of solution containing potential binding partner (analyte) across the surface. The SPR phenomenon occurs when polarized light excites the electrons at the interface of the metal and the dielectric medium to generate electromagnetic waves that propagate parallel to the surface. Changes in the refractive index due to interaction between the ligand and analyte are measured by detecting the reflected light, providing real-time data on kinetics and specificity. A prominent use of SPR is identifying compounds in crude plant extracts that bind to specific molecules. Procedures that utilize SPR are becoming increasingly applicable outside the laboratory setting, and SPR imaging and localized SPR (LSPR) are cheaper and more portable alternative for in situ detection of plant or mammalian pathogens and drug discovery studies. LSPR, in particular, has the advantage of direct attachment to test tissues in live-plant studies. Here, we describe three protocols utilizing SPR-based assays for precise analysis of protein-ligand interactions. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: SPR comparison of binding affinities of viral reverse transcriptase polymorphisms

Basic Protocol 2: SPR screening of crude plant extract for protein-binding agents

Basic Protocol 3: Localized SPR–based antigen detection using antibody-conjugated gold nanoparticles

Ascariasis, caused by both Ascaris lumbricoides and Ascaris suum, is the most prevalent parasitic disease worldwide, affecting both human and porcine populations. However, due to the difficulties of assessing the early events of infection in humans, most studies of human ascariasis have been restricted to the chronic intestinal phase. Therefore, the Ascaris mouse model has become a fundamental tool for investigating the immunobiology and pathogenesis of the early infection stage referred to as larval ascariasis because of the model's practicality and ability to replicate the natural processes involved. The Ascaris mouse model has been widely used to explore factors such as infection resistance/susceptibility, liver inflammation, lung immune-mediated pathology, and co-infections and, notably, as a pivotal element in preclinical vaccine trials. Exploring the immunobiology of larval ascariasis may offer new insights into disease development and provide a substantial understanding of key components that trigger a protective immune response. This article focuses on creating a comprehensive guide for conducting Ascaris experimental infections in the laboratory as a foundation for future research efforts. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Acquisition and embryonation of Ascaris suum eggs from adult females

Alternate Protocol: Cleaning and purification of Ascaris suum from female A. suum uteri

Basic Protocol 2: Preparation of Ascaris suum eggs and murine infection

Basic Protocol 3: Measurement of larval burden and Ascaris-larva-induced pathogenesis

Basic Protocol 4: In vitro hatching and purification of Ascaris L3 larvae

Support Protocol: Preparation of crude antigen from Ascaris infectious stages

Basic Protocol 5: Ultrastructure-expansion microscopy (U-ExM) of Ascaris suum larval stages

This article describes a step-by-step process of lumbar intrathecal injection of Evans blue dye and AAV9-EGFP in adult (2-month-old) and neonatal (postnatal day 10) mice. Intrathecal injection is a clinically translatable technique that has already been extensively applied in humans. In mice, intrathecal injection is considered a challenging procedure that requires a trained and experienced researcher. For both adult and neonatal mice, lumbar intrathecal injection is directed into the L5-L6 intervertebral space. Intrathecally injected material enters the cerebrospinal fluid (CSF) within the intrathecal space from where it can directly access the central nervous system (CNS) parenchyma. Simultaneously, intrathecally injected material exits the CSF with pressure gradient and enters the endoneurial fluid and ultimately the peripheral nerves. While in the CSF, the injectable material also enters the bloodstream and systemic circulation through the arachnoid villi. A successful lumbar intrathecal injection results in adequate biodistribution of the injectable material in the CNS, PNS, and peripheral organs. When correctly applied, this technique is considered as minimally invasive and non-disruptive and can be used for the lumbar delivery of any solute. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: C57BL/6 adult and P10 mice lumbar intrathecal injection

Basic Protocol 2: Tissue collection and preparation for evaluating Evans blue dye diffusion

Basic Protocol 3: Tissue collection and preparation for immunohistochemistry staining

Basic Protocol 4: Tissue collection and vector genome copy number analysis

In the event of a sunlight-blocking, temperature-lowering global catastrophe, such as a global nuclear war, super-volcano eruption or large asteroid strike, normal agricultural practices would be severely disrupted with a devastating impact on the global food supply. Despite the improbability of such an occurrence, it is prudent to consider how to sustain the surviving population following a global catastrophe until normal weather and climate patterns resume. Additionally, the ongoing challenges posed by climate change, droughts, flooding, soil salinization, and famine highlight the importance of developing food systems with resilient inputs such as lignocellulosic biomass. With its high proportion of cellulose, the abundant lignocellulosic biomass found across the Earth's land surfaces could be a source of energy and nutrition, but it would first need to be converted into foods. To understand the potential of lignocellulosic biomass to provide energy and nutrition to humans in post-catastrophic and other food crisis scenarios, compositional analyses should be completed to gauge the amount of energy (soluble sugars) and other macronutrients (protein and lipids) that might be available and the level of difficulty in extracting them. Suitable preparation of the lignocellulosic biomass is critical to achieve consistent and comparable results from these analyses. Here we describe a compilation of protocols to prepare lignocellulosic biomass and analyze its composition to understand its potential as a precursor to produce post-catastrophic foods which are those that could be foraged, grown, or produced under the new climate conditions to supplement reduced availability of traditional foods. These foods have sometimes been referred to in the literature as emergency, alternate, or resilient foods. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Convection oven drying (1 to 2 days)

Alternate Protocol 1: Air-drying (2 to 3 days)

Alternate Protocol 2: Lyophilization (1 to 4 days)

Support Protocol 1: Milling plant biomass

Support Protocol 2: Measuring moisture content

Basic Protocol 2: Cellulose determination

Basic Protocol 3: Lignin determination

Basic Protocol 4: Crude protein content by total nitrogen

Basic Protocol 5: Crude fat determination via soxtec extraction system

Basic Protocol 6: Sugars by HPLC

Basic Protocol 7: Ash content

Fluorescence in situ hybridization (FISH) is a cytogenetic assay that is widely used in both clinical and research settings to validate genetic aberrations. Simple in principle, it is based on denaturation and hybridization of a DNA probe and its complementary sequence; however, it is subject to continuous optimization. Here we share how in-house FISH can be optimized using different control tissues to visualize and ultimately validate common and novel genetic abnormalities unearthed by next-generation sequencing (NGS). Seven specific FISH probes were designed and labeled, and conditions for eight tissue types and one patient-derived tumor organoid were optimized. Formalin-fixed paraffin-embedded (FFPE) tissue slides were used for each experiment. Slides were first deparaffinized, then placed in a pretreatment solution followed by a digestion step. In-house FISH probes were then added to the tissue to be denatured and hybridized, and then washed twice. To obtain optimal results, probe concentration, pepsin incubation time, denaturation, and the two post-hybridization washes were optimized for each sample. By modifying the above conditions, all FISH experiments were optimized in separate tissue types to investigate specific genomic alterations in tumors arising in those tissues. Signals were clear and distinct, allowing for visualization of the selected probes. Following this protocol, our lab has quickly optimized 11 directly labeled in-house FISH probes to support genetic aberrations nominated by NGS, including most recent discoveries through whole-genome sequencing analyses. We describe a robust approach of how to advance in-house labeled FISH probes. By following these guidelines, reliable and reproducible FISH results can be obtained to interrogate FFPE slides from benign, tumor tissues, and patient-derived tumor organoid specimens. This is of most relevance in the era of NGS and precision oncology. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Metaphase FISH optimization

Support Protocol 1: In-house probe labeling and preparation

Support Protocol 2: Metaphase spread preparation

Basic Protocol 2: Optimization of FISH on formalin-fixed paraffin-embedded tissue

The middle (MID) domain of eukaryotic Argonaute (Ago) proteins and archaeal and bacterial homologues mediates the interaction with the 5′-terminal nucleotide of miRNA and siRNA guide strands. The MID domain of human Ago2 (hAgo2) is comprised of 139 amino acids with a molecular weight of 15.56 kDa. MID adopts a Rossman-like beta1-alpha1-beta2-alpha2-beta3-alpha3-beta4-alpha4 fold with a nucleotide specificity loop between beta3 and alpha3. Multiple crystal structures of nucleotides bound to hAgo2 MID have been reported, whereby complexes were obtained by soaking ligands into crystals of MID domain alone. This protocol describes a simplified one-step approach to grow well-diffracting crystals of hAgo2 MID-nucleotide complexes by mixing purified His₆-SUMO-MID fusion protein, Ulp1 protease, and excess nucleotide in the presence of buffer and precipitant. The crystal structures of MID complexes with UMP, UTP and 2′-3′ linked α-L-threofuranosyl thymidine-3′-triphosphate (tTTP) are presented. This article also describes fluorescence-based assays to measure dissociation constants (K_d) of MID-nucleotide interactions for nucleoside 5′-monophosphates and nucleoside 3′,5′-bisphosphates. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Crystallization of Ago2 MID-nucleotide complexes

Basic Protocol 2: Measurement of dissociation constant K_d between Ago2 MID and nucleotides