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Journal Methods Mol. Biol.

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Measurement of Antigen-Specific IgG Titers by Direct ELISA.

Bassel Akache, Felicity C Stark, Michael J McCluskie,

Direct ELISA allows for the measurement of antibody levels to a particular antigen. Serum or plasma from the vaccinated subject are incubated on high-binding capacity microplates precoated with the antigen of interest and detected utilizing an enzyme-linked secondary antibody. Herein, using influenza hemagglutinin as model antigen, we describe the quantification ... Read more >>

Methods Mol. Biol. (Methods in molecular biology (Clifton, N.J.))
[2021, 2183:537-547]

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Flavivirus DNA Vaccine Design and Adjuvant Selection.

Lei Li, Yoshikazu Honda-Okubo, Nikolai Petrovsky,

A DNA vaccine is a plasmid encoding a vaccine antigen together with an efficient eukaryotic promoter to drive protein expression. The chief problem of DNA vaccines has been their suboptimal immunogenicity in humans. Many different flaviviruses infect and cause serious illness and even death in humans, but human vaccines are ... Read more >>

Methods Mol. Biol. (Methods in molecular biology (Clifton, N.J.))
[2021, 2183:405-422]

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Vaccine Delivery and Immune Response Basics.

Andrew Hill, Marie Beitelshees, Blaine A Pfeifer,

In this opening chapter, we outline the basics of vaccine delivery and subsequent immune reactivity. Vaccine delivery is an augmentation to immunization more generally in that a delivery reagent is harnessed to improve administration of the key ingredient (i.e., the antigen) needed to provoke an immune response. In this chapter, ... Read more >>

Methods Mol. Biol. (Methods in molecular biology (Clifton, N.J.))
[2021, 2183:1-8]

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Studying the Metabolism of Epithelial-Mesenchymal Plasticity Using the Seahorse XFe96 Extracellular Flux Analyzer.

Sugandha Bhatia, Erik W Thompson, Jennifer H Gunter,

The critical role of metabolism in facilitating cancer cell growth and survival has been demonstrated by a combination of methods including, but not limited to, genomic sequencing, transcriptomic and proteomic analyses, measurements of radio-labelled substrate flux and the high throughput measurement of oxidative metabolism in unlabelled live cells using the ... Read more >>

Methods Mol. Biol. (Methods in molecular biology (Clifton, N.J.))
[2021, 2179:327-340]

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Using Xenopus Neural Crest Explants to Study Epithelial-Mesenchymal Transition.

Nadège Gouignard, Christian Rouvière, Eric Theveneau,

The epithelial-mesenchymal transition (EMT) converts coherent epithelial structures into single cells. EMT is a dynamic cellular process that is not systematically completed (not all EMTs lead to single cells) and reversible (cells can re-epithelialize). EMT is orchestrated at multiple levels from transcription, to posttranslational modifications, to protein turnover. It involves ... Read more >>

Methods Mol. Biol. (Methods in molecular biology (Clifton, N.J.))
[2021, 2179:257-274]

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4D Live Imaging and Analysis of Chick Embryo Somites.

Gi Fay Mok, James McColl, Andrea Münsterberg,

Avian (chick) embryos are an established and accessible model organism making them ideal for studying developmental processes. Chick embryos can be harvested from the egg and cultured allowing real-time observations and imaging. Here, we describe ex vivo culture and preparation of somite tissue followed by time-lapse multi-photon microscopy, image capture ... Read more >>

Methods Mol. Biol. (Methods in molecular biology (Clifton, N.J.))
[2021, 2179:173-181]

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Live Imaging of the Neural Crest Cell Epithelial-to-Mesenchymal Transition in the Chick Embryo.

Mary Cathleen McKinney, Paul M Kulesa,

Live embryo imaging may provide a wealth of information on intact cell and tissue dynamics, but can be technically challenging to sustain embryo orientation and health for long periods under a microscope. In this protocol, we describe an in vivo method to mount and image cell movements during the epithelial-to-mesenchymal ... Read more >>

Methods Mol. Biol. (Methods in molecular biology (Clifton, N.J.))
[2021, 2179:107-114]

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Partial EMT/MET: An Army of One.

Sofiane Hamidi, Hiroki Nagai, Guojun Sheng,

As our understanding of Epithelial Mesenchymal Transition (EMT) increases, the original binary concept of E versus M no longer fits with experimental evidence. Re-definition of the EMT paradigm as spectral transitions between a full epithelium and a full mesenchyme suggests the existence of a virtual infinity of intermediate cellular states. ... Read more >>

Methods Mol. Biol. (Methods in molecular biology (Clifton, N.J.))
[2021, 2179:29-33]

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Controlling the Activity of CRISPR Transcriptional Regulators with Inducible sgRNAs.

Quentin R V Ferry, Tudor A Fulga,

The type-II CRISPR-Cas9 system has been repurposed to create synthetic programmable transcriptional regulators (CRISPR-TRs). Subsequent modifications of the system now allow for spatiotemporal control of CRISPR-mediated gene activation and repression. Among these solutions, the development of inducible spacer-blocking hairpin guide RNAs (iSBH-sgRNAs) provide an easy to implement and versatile way ... Read more >>

Methods Mol. Biol. (Methods in molecular biology (Clifton, N.J.))
[2021, 2162:153-184]

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Gene Disruption Using Chemically Modified CRISPR-Cpf1 RNA.

Moira A McMahon, Meghdad Rahdar,

CRISPR-based gene editing in mammalian cells is a powerful research tool which has demonstrated efficient site-specific gene modifications and is showing promise as a therapeutic for patients with genetic diseases. Multiple different CRISPR systems have been identified, each with its own target DNA recognition sequence, expanding the editable mammalian genome. ... Read more >>

Methods Mol. Biol. (Methods in molecular biology (Clifton, N.J.))
[2021, 2162:49-60]

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Quantitative Analysis of Bile Acid with UHPLC-MS/MS.

Yuan Tian, Jingwei Cai, Erik L Allman, Philip B Smith, Andrew D Patterson,

Bile acids are important end products of cholesterol metabolism, having been shown to serve as signaling molecules and intermediates between the host and the gut microbiota. Here we describe a robust and accurate method using ultrahigh-pressure liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) for the quantification of bile acids ... Read more >>

Methods Mol. Biol. (Methods in molecular biology (Clifton, N.J.))
[2021, 2194:291-300]

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Investigating Inter- and Intrasample Diversity of Single-Cell RNA Sequencing Datasets.

Meghan C Ferrall-Fairbanks, Philipp M Altrock,

Tumor heterogeneity can arise from a variety of extrinsic and intrinsic sources and drives unfavorable outcomes. With recent technological advances, single-cell RNA sequencing has become a way for researchers to easily assay tumor heterogeneity at the transcriptomic level with high resolution. However, ongoing research focuses on different ways to analyze ... Read more >>

Methods Mol. Biol. (Methods in molecular biology (Clifton, N.J.))
[2021, 2194:177-186]

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Variable Selection for Time-to-Event Data.

Ai Ni, Chi Song,

With the increasing availability of large scale biomedical and -omics data, researchers are offered with unprecedented opportunities to discover novel biomarkers for clinical outcomes. At the same time, they are also faced with great challenges to accurately identify important biomarkers from numerous candidates. Many novel statistical methodologies have been developed ... Read more >>

Methods Mol. Biol. (Methods in molecular biology (Clifton, N.J.))
[2021, 2194:61-76]

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Determination of Antimicrobial Resistance of Salmonella in Pork.

Jing Wang, Dan Gu,

Antimicrobial susceptibilities testing is used for evaluating and monitoring the resistance of bacteria to antimicrobial agents. Here we describe three commonly used methods for testing susceptibility to antimicrobial agents in Salmonella, including the disk diffusion method, the broth microdilution method, and the agar dilution method. ... Read more >>

Methods Mol. Biol. (Methods in molecular biology (Clifton, N.J.))
[2021, 2182:179-186]

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Production of Murine Macrophages from Hoxb8-Immortalized Myeloblasts: Utility and Use in the Context of Salmonella Infection.

Ziyan Fang, Margaux Lagier, Stéphane Méresse,

Salmonella enterica is a Gram-negative intracellular pathogen that causes a range of life-threatening diseases in humans and animals worldwide. In a systemic infection, the ability of Salmonella to survive/replicate in macrophages, particularly in the liver and spleen, is crucial for virulence. Transformed macrophage cell lines and primary macrophages prepared from ... Read more >>

Methods Mol. Biol. (Methods in molecular biology (Clifton, N.J.))
[2021, 2182:117-126]

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CRISPR Typing of Salmonella Isolates from Different Resources.

Qiuchun Li,

CRISPR typing is a newly developed method used to reveal the genetic relationship of bacterial isolates from different resources. For Salmonella, CRISPR typing can not only reveal the phylogenic difference among isolates belonging to the identical serotype, but also show good correspondence with Salmonella serotypes. Here we describe the protocol ... Read more >>

Methods Mol. Biol. (Methods in molecular biology (Clifton, N.J.))
[2021, 2182:45-50]

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Next-Day Salmonella spp. Detection Method Based on Real-Time PCR for Foods.

David Rodríguez-Lázaro, Marta Hernández,

Molecular techniques such as real-time polymerase chain reaction (qPCR) have become a very effective alternative in food microbiology diagnostic for rapid and specific detection of foodborne pathogens such as Salmonella in foods and food-related environments. qPCR is a simple, sensitive, specific, and reproducible assay. Here, we describe the application of ... Read more >>

Methods Mol. Biol. (Methods in molecular biology (Clifton, N.J.))
[2021, 2182:1-6]

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Optical Manipulation of Perfused Mouse Heart Expressing Channelrhodopsin-2 in Rhythm Control.

Xi Wang, Yue Cheng,

Optogenetics is a new approach using light intensity to modulate the electrical activity of excitable cells by the interaction of light-sensitive proteins. This method has been widely and enthusiastically utilized in some fields over the last decade. Localizing a photosensitive protein to a specific place in the membrane of cardiomyocytes ... Read more >>

Methods Mol. Biol. (Methods in molecular biology (Clifton, N.J.))
[2021, 2191:377-390]

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Application of Targeting-Optimized Chronos for Stimulation of the Auditory Pathway.

Antoine Tarquin Huet, Vladan Rankovic,

In the last 15 years, optogenetics has revolutionized the life sciences and enabled studies of complex biological systems such as the brain. Applying optogenetics also has great potential for restorative medicine, such as hearing restoration, by stimulating genetically modified spiral ganglion neurons of the cochlea with light. To this end, opsins ... Read more >>

Methods Mol. Biol. (Methods in molecular biology (Clifton, N.J.))
[2021, 2191:261-285]

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Patterned Optogenetic Stimulation Using a DMD Projector.

Aanchal Bhatia, Sahil Moza, Upinder S Bhalla,

Optical manipulation is a powerful way to control neural activity in vitro and in vivo with millisecond precision. Patterning of light provides the remarkable ability to simultaneously target spatially segregated neurons from a population. Commercially available projectors provide one of the simplest and most economical ways of achieving spatial light ... Read more >>

Methods Mol. Biol. (Methods in molecular biology (Clifton, N.J.))
[2021, 2191:173-188]

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Probing Channelrhodopsin Electrical Activity in Algal Cell Populations.

Oleg A Sineshchekov, Elena G Govorunova, John L Spudich,

Photoelectric recording from populations of phototactic flagellate algae provides a means to study channelrhodopsin functions in vivo. Technical simplicity, versatility, high sensitivity, and reproducibility are the advantages of this assay over recording from individual algal cells by the suction pipette technique. Here we describe the principles and procedures of this ... Read more >>

Methods Mol. Biol. (Methods in molecular biology (Clifton, N.J.))
[2021, 2191:85-96]

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In Vitro Synthesis of Modified RNA for Cardiac Gene Therapy.

Nishat Sultana, Mohammad Tofael Kabir Sharkar, Yoav Hadas, Elena Chepurko, Lior Zangi,

Modified mRNA (modRNA) is a promising new gene therapy approach that has safely and effectively delivered genes into different tissues, including the heart. Current efforts to use DNA-based or viral gene therapy to induce cardiac regeneration postmyocardial infarction (MI) or in heart failure (HF) have encountered key challenges, e.g., genome ... Read more >>

Methods Mol. Biol. (Methods in molecular biology (Clifton, N.J.))
[2021, 2158:281-294]

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Cardiac Resection Injury in Zebrafish.

Delicia Z Sheng, Dawei Zheng, Kazu Kikuchi,

The zebrafish (Danio rerio) possesses a spectacular capacity for cardiac regeneration. Zebrafish have been used in cardiac regeneration research for nearly two decades, contributing to the identification of signals and cellular mechanisms as potential targets for human heart repair. Investigations into cardiac regeneration in zebrafish have been facilitated by multiple ... Read more >>

Methods Mol. Biol. (Methods in molecular biology (Clifton, N.J.))
[2021, 2158:63-69]

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Myocardial Infarction Techniques in Adult Mice.

Elad Bassat, Dahlia E Perez, Eldad Tzahor,

The discovery of endogenous regenerative potential of the heart in zebrafish and neonatal mice has shifted the cardiac regenerative field towards the utilization of intrinsic regenerative mechanisms in the mammalian heart. The goal of these studies is to understand, and eventually apply, the neonatal regenerative mechanisms into adulthood. To facilitate these ... Read more >>

Methods Mol. Biol. (Methods in molecular biology (Clifton, N.J.))
[2021, 2158:3-21]

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Cultivation of Testicular Germ Cell Cancer Cell Lines and Establishment of Gene-Edited Subclones Using CRISPR/Cas9.

Sina Jostes, Daniel Nettersheim, Simon Schneider, Hubert Schorle,

Type II testicular germ cell tumors (GCTs) can be classified as seminoma or embryonal carcinoma. Both subtypes present distinct cellular morphologies and characteristics. Seminomas closely resemble primordial germ cells (PGCs) with respect to their transcriptome and epigenetic signature (DNA hypomethylation). They express the pluripotency markers LIN28, NANOG, and OCT3/4 and ... Read more >>

Methods Mol. Biol. (Methods in molecular biology (Clifton, N.J.))
[2021, 2195:85-97]

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