Current protocols最新文献

Mucin-domain glycoproteins are characterized by their high density of glycosylated serine and threonine residues, which complicates their analysis by mass spectrometry. The dense glycosylation renders the protein backbone inaccessible to workhorse proteases like trypsin, the vast heterogeneity of glycosylation often results in ion suppression from unmodified peptides, and search algorithms struggle to confidently analyze and site-localize O-glycosites. We have made a number of advances to address these challenges, rendering mucinomics possible for the first time. Here, we summarize these contributions and provide a detailed protocol for mass spectrometric analysis of mucin-domain glycoproteins. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Enrichment of mucin-domain glycoproteins

Basic Protocol 2: Enzymatic digestion of mucin-domain glycoprotein(s)

Basic Protocol 3: Mass spectrometry data collection for O-glycopeptides

Basic Protocol 4: Mass spectrometry data analysis of O-glycopeptides

Cardiovascular diseases have emerged as one of the leading causes of human mortality, but the discovery of new drugs has been hindered by the absence of suitable in vitro platforms. In recent decades, continuously refined protocols for differentiating human induced pluripotent stem cells (hiPSCs) into hiPSC-derived cardiomyocytes (hiPSC-CMs) have significantly advanced disease modeling and drug screening; however, this has led to an increasing need to monitor the function of hiPSC-CMs. The precise regulation of action potentials (APs) and intracellular calcium (Ca²⁺) transients is critical for proper excitation-contraction coupling and cardiomyocyte function. These important parameters are usually adversely affected in cardiovascular diseases or under cardiotoxic conditions and can be measured using optical imaging–based techniques. However, this procedure is complex and technologically challenging. We have adapted the IonOptix system to simultaneously measure APs and Ca²⁺ transients in hiPSC-CMs loaded with the fluorescent dyes FluoVolt and Rhod 2, respectively. This system serves as a powerful high-throughput platform to facilitate the discovery of new compounds to treat cardiovascular diseases with the cellular phenotypes of abnormal APs and Ca²⁺ handling. Here, we present a comprehensive protocol for hiPSC-CM preparation, device setup, optical imaging, and data analysis. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Maintenance and seeding of hiPSC-CMs

Basic Protocol 2: Simultaneous detection of action potentials and Ca²⁺ transients in hiPSC-CMs

A variety of metals, e.g., lead (Pb), cadmium (Cd), and lithium (Li), are in the environment and are toxic to humans. Hematopoietic stem cells (HSCs) reside at the apex of hematopoiesis and are capable of generating all kinds of blood cells and self-renew to maintain the HSC pool. HSCs are sensitive to environmental stimuli. Metals may influence the function of HSCs by directly acting on HSCs or indirectly by affecting the surrounding microenvironment for HSCs in the bone marrow (BM) or niche, including cellular and extracellular components. Investigating the impact of direct and/or indirect actions of metals on HSCs contributes to the understanding of immunological and hematopoietic toxicology of metals. Treatment of HSCs with metals ex vivo, and the ensuing HSC transplantation assays, are useful for evaluating the impacts of the direct actions of metals on the function of HSCs. Investigating the mechanisms involved, given the rarity of HSCs, methods that require large numbers of cells are not suitable for signal screening; however, flow cytometry is a useful tool for signal screening HSCs. After targeting signaling pathways, interventions ex vivo and HSCs transplantation are required to confirm the roles of the signaling pathways in regulating the function of HSCs exposed to metals. Here, we describe protocols to evaluate the mechanisms of direct and indirect action of metals on HSCs. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Identify the impact of a metal on the competence of HSCs

Basic Protocol 2: Identify the impact of a metal on the lineage bias of HSC differentiation

Basic Protocol 3: Screen the potential signaling molecules in HSCs during metal exposure

Alternate Protocol 1: Ex vivo treatment with a metal on purified HSCs

Alternate Protocol 2: Ex vivo intervention of the signaling pathway regulating the function of HSCs during metal exposure

Current Protocols is issuing corrections for the following protocol article.

Bryant, J. D., Lei, Y., VanPortfliet, J. J., Winters, A. D., & West, A. P. (2022). Assessing mitochondrial DNA release into the cytosol and subsequent activation of innate immune-related pathways in mammalian cells. Current Protocols, 2, e372. doi: 10.1002/cpz1.372

In the above-referenced article:

In Basic Protocol 2, step 13, the tube label has been changed from “B-2” to “A-2”.

In Basic Protocol 2, the following instruction has been added to step 16: “Save the pellet to use in step 21.”

In Basic Protocol 2, step 21, the step number has been changed from “step 18” to “step 16”.

In Basic Protocol 2, the following instruction has been added to step 23: “Save the pellet to use in step 26.”

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

Short tandem repeat (STR) expansions are associated with more than 60 genetic disorders. The size and stability of these expansions correlate with the severity and age of onset of the disease. Therefore, being able to accurately detect the absolute length of STRs is important. Current diagnostic assays include laborious lab experiments, including repeat-primed PCR and Southern blotting, that still cannot precisely determine the exact length of very long repeat expansions. Optical genome mapping (OGM) is a cost-effective and easy-to-use alternative to traditional cytogenetic techniques and allows the comprehensive detection of chromosomal aberrations and structural variants >500 bp in length, including insertions, deletions, duplications, inversions, translocations, and copy number variants. Here, we provide methodological guidance for preparing samples and performing OGM as well as running the analysis pipelines and using the specific repeat expansion workflows to determine the exact repeat length of repeat expansions expanded beyond 500 bp. Together these protocols provide all details needed to analyze the length and stability of any repeat expansion with an expected repeat size difference from the expected wild-type allele of >500 bp. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Genomic ultra-high-molecular-weight DNA isolation, labeling, and staining

Basic Protocol 2: Data generation and genome mapping using the Bionano Saphyr® System

Basic Protocol 3: Manual De Novo Assembly workflow

Basic Protocol 4: Local guided assembly workflow

Basic Protocol 5: EnFocus Fragile X workflow

Basic Protocol 6: Molecule distance script workflow

Current Protocols is issuing corrections for the following protocol article.

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