Current protocols最新文献

In this article, we describe the synthesis of N³-methyluridine (m³U) and 2′-O-alkyl/2′-fluoro-N³-methyluridine (2′-O-alkyl/2′-F-m³U) phosphoramidites as well as their incorporation into a 14-mer DNA and RNA oligonucleotide sequence. Synthesis of the 2′-O-alkyl-m³U phosphoramidite starts with commercially available uridine to achieve a tritylated m³U intermediate, followed by 2′-O-alkylation and finally phosphitylation. Synthesis of the 2′-F-m³U phosphoramidite is obtained from a commercially available 2′-F-uridine nucleoside. These phosphoramidite monomers are compatible with DNA and RNA oligonucleotide synthesis using conventional phosphoramidite chemistry. This strategy offers efficient synthetic access to various modifications at the 2′-position of m³U that can be employed in numerous nucleic acid–based therapeutic applications, including antisense technologies, small interfering RNAs, CRISPR-Cas9, and aptamers. The data presented in this article are based on our previously published reports. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Synthesis of 2′-O-alkyl-N³-methyluridine analogs and their corresponding phosphoramidites

Alternate Protocol 1: Synthesis of 2′-O-TBDMS-N³-methyluridine and its phosphoramidite

Alternate Protocol 2: Synthesis of 2′-fluoro-N³-methyluridine and its phosphoramidite

Basic Protocol 2: Solid-phase synthesis of N³-methyluridine-modified DNA and RNA oligonucleotides

Understanding the genetic basis of antimicrobial resistance is crucial for developing effective mitigation strategies. One necessary step is to identify the antimicrobial resistance genes (ARGs) within a microbial population, referred to as the resistome, as well as the mobile genetic elements (MGEs) harboring ARGs. Although shotgun metagenomics has been successful in detecting ARGs and MGEs within a microbiome, it is limited by low sensitivity. Enrichment using cRNA biotinylated probes has been applied to address this limitation, enhancing the detection of rare ARGs and MGEs, especially when combined with long-read sequencing. Here, we present the TELCoMB protocol, a Snakemake workflow that elucidates resistome and mobilome composition and diversity and uncovers ARG-MGE colocalizations. The protocol supports both short- and long-read sequencing and does not require enrichment, making it versatile for various genomic data types. TELCoMB generates publication-ready figures and CSV files for comprehensive analysis, improving our understanding of antimicrobial resistance mechanisms and spread. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Installing TELCOMB Locally

Alternate Protocol: Installing TELCOMB on a SLURM Cluster

Basic Protocol 2: Data Preprocessing

Basic Protocol 3: Calculation of Resistome Distribution and Composition

Basic Protocol 4: Identification of ARG-MGE Colocalizations

Calcium plays a pivotal role in the excitation-contraction coupling process in cardiomyocytes, a critical multi-parametric event leading to rhythmic contraction. Over the past few decades, calcium signaling in cardiomyocytes has been extensively studied in cardiovascular sciences. However, a standard methodology is needed not only to trace the calcium within cells but also to remove signal processing biases and to accurately interpret the features of calcium transient signals in relation to cardiomyocyte electrophysiology. This article outlines the use of genetically encoded calcium indicator (GCaMP) human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to record calcium transients. These cells express a green fluorescent signal when calcium binds to intracellular calmodulin, a key regulator of calcium signaling. The extraction and processing of calcium transient waveforms are performed using ImageJ and MATLAB software. Key features of these waveforms are then identified and categorized based on their physiological relevance to cardiomyocyte function. Additionally, this work includes a Support Protocol for the successful replating of cardiomyocytes onto non-traditional culture platforms, such as metallic sensors and polymer-based substrates, to facilitate data multiplexing. The three Basic Protocols outlined here provide a comprehensive approach for maintaining, expanding, and differentiating the GCaMP hiPSCs, video recording of calcium transients, and the subsequent signal extraction, preprocessing, analysis, and data visualization. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Maintenance, expansion, and differentiation of genetically encoded calcium indicator hiPSCs

Support Protocol: Replating GCaMP hiPSC-CMs for stimulation and multielectrode array studies

Basic Protocol 2: Video recording from calcium transients of GCaMP hiPSC-CMs

Basic Protocol 3: Signal extraction, preprocessing, analysis, and data visualization

Current Protocols is issuing corrections for the following protocol article:

Andreou, I., Storbeck, M., Hahn, P., Rulli, S., & Lader, E. (2024). Exome sequencing starting from single cells. Current Protocols, 4, e70017. doi: 10.1002/cpz1.70017

In the above-referenced article:

A new Table 10 has been added and the existing Table 10 has been renumbered as Table 12.

The existing Tables 12 to 14 have been renumbered as Tables 13 to 15.

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

Kindling models are widely used animal models to study the pathobiology of epilepsy and epileptogenesis. These models exhibit distinctive features whereby sub-threshold stimuli instigate the initial induction of brief focal seizures. Over time, the severity and duration of these seizures progressively increase, leading to a fully epileptic state, which is marked by consistent development of generalized tonic-clonic seizures. Kindling involves focal stimulation via implanted depth electrodes or repeated administration of chemoconvulsants such as pentylenetetrazol. Comparative analysis of preclinical and clinical findings has confirmed a high predictive validity of fully kindled animals for testing novel antiseizure medications. Thus, kindling models remain an essential component of anticonvulsant drug development programs. This article provides a comprehensive guide to working protocols, testing of therapeutic drugs, outcome parameters, troubleshooting, and data analysis for various electrical and chemical kindling epileptogenesis models for new therapeutic development and optimization. The use of pharmacological agents or genetically modified mice in kindling experiments is valuable, offering insights into the impact of a specific target on various aspects of seizures, including thresholds, initiation, spread, termination, and the generation of a hyperexcitable network. These kindling epileptogenesis paradigms are helpful in identifying mechanisms and disease-modifying interventions for epilepsy. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Hippocampal kindling

Basic Protocol 2: Amygdala kindling

Basic Protocol 3: Rapid hippocampal kindling

Basic Protocol 4: Chemical kindling

The laboratory mouse has been described as a “miracle” model organism, providing a window by which we may gain an understanding of ourselves. Since the first recorded mouse experiment in 1664, the mouse has become the most used animal model in biomedical research. Mice are ideally suited as a model organism because of their small size, short gestation period, large litter size, and genetic similarity to humans. This article provides a broad overview of the laboratory mouse as a model organism and is intended for undergraduates and those new to working with mice. We delve into the history of the laboratory mouse and outline important terminology to accurately describe research mice. The types of laboratory mice available to researchers are reviewed, including outbred stocks, inbred strains, immunocompromised mice, and genetically engineered mice. The critical role mice have played in advancing knowledge in the areas of oncology, immunology, and pharmacology is highlighted by examining the significant contribution of mice to Nobel Prize winning research. International mouse mutagenesis programs and accurate phenotyping of mouse models are outlined. We also explain important considerations for working with mice, including animal ethics; the welfare principles of replacement, refinement, and reduction; and the choice of mouse model in experimental design. Finally, we present practical advice for maintaining a mouse colony, which involves adequate training of staff, the logistics of mouse housing, monitoring colony health, and breeding strategies. Useful resources for working with mice are also listed. The aim of this overview is to equip the reader with a broad appreciation of the enormous potential and some of the complexities of working with the laboratory mouse in a quest to improve human health. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Cancer cell lines are important tools to investigate the biology of cancer and test hypotheses to improve cancer treatments. A major challenge in establishing epithelial cancer cell lines is the removal of cancer-associated fibroblasts (CAFs). CAFs are abundant within the tumor microenvironment. CAFs generally proliferate faster than epithelial cancer cells in culture. CAFs can be mistakenly identified as cancer cells, especially when cancer cells display spindle-shaped morphology. Among all cancer types, pancreatic ductal adenocarcinoma (PDAC) is characterized by an abundant desmoplastic stroma. Here, we describe protocols for establishing epithelial cancer cell lines from mouse models of PDAC and verifying that they are not CAFs. The approach is cost-effective and can be used for other types of cancer. If needed, CAF cell lines can also be established and preserved using this protocol. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Isolation of cells from tumors

Basic Protocol 2: Isolation and cryopreservation of cancer cell clones

Basic Protocol 3: Assessment of the identity of cancer cell lines and CAFs by western blotting

Mouse models remain at the forefront of immuno-oncology research, providing invaluable insights into the complex interactions between the immune system and developing tumors. While several flow cytometry panels have been developed to study cancer immunity in mice, most are limited in their capacity to address the complexity of anti-cancer immune responses. For example, many of the panels developed to date focus on a restricted number of leukocyte populations (T cells or antigen-presenting cells), failing to include the multitude of other subsets that participate in anti-cancer immunity. In addition, these panels were developed using blood or splenic leukocytes. While the immune composition of the blood or spleen can provide information on systemic immune responses to cancer, it is in the tumor microenvironment (TME) that local immunity takes place. Therefore, we optimized this spectral flow cytometry panel to identify the chief cell types that take part in cancer immunity using immune cells from cancer tissue. We used pancreatic tumors implanted both orthotopically and subcutaneously to demonstrate the panel's flexibility and suitability in diverse mouse models. The panel was also validated in peripheral immune districts (the blood, spleen, and liver of tumor-bearing mice) to allow comparisons between local and systemic anti-tumor immunity. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Tumor induction—Orthotopic

Alternate Protocol: Tumor induction—Subcutaneous

Basic Protocol 2: Preparation of single-cell suspensions from the tumor, spleen, liver, and blood of tumor-bearing mice

Basic Protocol 3: Staining single-cell suspensions from the tumor, spleen, liver, and blood of tumor-bearing mice

This protocol describes the synthesis of long oligonucleotides (up to 401-mer), their isolation from complex mixtures using the catching-by-polymerization (CBP) method, and the selection of error-free sequence via cloning followed by Sanger sequencing. Oligo synthesis is achieved under standard automated solid-phase synthesis conditions with only minor yet critical adjustments using readily available reagents. The CBP method involves tagging the full-length sequence with a polymerizable tagging phosphoramidite (PTP), co-polymerizing the sequence into a polymer, washing away failure sequences, and cleaving the full-length sequence from the polymer. Cloning and sequencing guided selection of error-free sequence overcome the problems of substitution, deletion, and addition errors that cannot be addressed using any other methods, including CBP. Long oligos are needed in many areas such as protein engineering and synthetic biology. The methods described here are particularly important for projects requiring long oligos containing long repeats or stable higher-order structures, which are difficult or impossible to produce using any other existing technologies. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Long oligo synthesis

Support Protocol 1: Synthesis of polymerizable tagging phosphoramidite (PTP)

Support Protocol 2: Synthesis of 5′-O-Bz phosphoramidite

Basic Protocol 2: Catching-by-polymerization (CBP) purification

Basic Protocol 3: Error-free sequence selection via cloning and sequencing

Neurological deficits, psychiatric disorders, and cognitive impairments often accompany stroke, brain injury, epilepsy, and many neurological disorders, which present intricate comorbidities that challenge recognition and management. There are many tools and paradigms for evaluating learning, memory, anxiety, and depression-like behaviors in lab animal models of brain disorders. However, there is a significant gap between clinical observations and experimental models, which limit understanding of the complex interplay between chronic brain conditions and their impact on cognitive dysfunction and psychiatric impairments. This article describes an overview of experimental rationale, methods, protocols, and strategies for evaluating sensorimotor, affective and cognitive-associated comorbid behaviors in epilepsy, traumatic brain injury (TBI), stroke, spinal cord injury (SCI), and many other neurological disorders. First, we delve into clinical evidence elucidating the profound impact of comorbidities, e.g., psychiatric disorders and cognitive deficits, in individuals with epilepsy. Then, we discuss diverse approaches to assess these comorbidities in experimental models of brain diseases. Finally, we explore the methodologies for assessing motor function, sensorimotor, behavior, and psychiatric health. We cover strategies and protocols enabling these assays, including implementing behavioral paradigms to assess learning and memory, anxiety, and depression-like behaviors in rodents in health and disease conditions. It is essential to consider a comprehensive battery of tests to investigate various behavioral deficits, considering environment, age, and sex differences relevant to the disease, such as TBI, SCI, epilepsy, stroke, and other complex neurological conditions. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.