Showing papers in "Methods in Cell Biology in 2022"

Gel-electrophoresis based method for biomolecular interaction.

Abstract: Electrophoresis is one of the most important analytical technologies for characterization of macromolecules and their interactions. Among them, native gel electrophoresis is used to analyze the macromolecules in the native structure. It differs in principle and information from those obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) or blue native polyacrylamide gel electrophoresis (BN-PAGE). SDS-PAGE is carried out in the presence of strong denaturant, SDS, while BN-PAGE is done in the presence of negatively charged dye, e.g., Coomassie brilliant blue, G-250. Here, we describe native gel electrophoresis using agarose gel and a buffer at pH 6.1 composed of histidine and 2-(N-morpholino) ethanesulfonic acid. First, a protocol for vertical and horizontal formats of agarose native gel electrophoresis is described followed by different staining procedures. Then, various examples obtained using the developed procedure will be shown to demonstrate how the technology can be applied to specific cases and the advantages or caveats of the present technology.

An intrabody sensor to monitor conformational activation of β-arrestins.

Abstract: Agonist-induced interaction of β-arrestins with GPCRs is critically involved in downstream signaling and regulation. This interaction is associated with activation and major conformational changes in β-arrestins. Although there are some assays available to monitor the conformational changes in β-arrestins in cellular context, additional sensors to report β-arrestin activation, preferably with high-throughput capability, are likely to be useful considering the structural and functional diversity in GPCR-β-arrestin complexes. We have recently developed an intrabody-based sensor as an integrated approach to monitor GPCR-β-arrestin interaction and conformational change, and generated a luminescence-based reporter using NanoBiT complementation technology. This sensor is derived from a synthetic antibody fragment referred to as Fab30 that selectively recognizes activated and receptor-bound conformation of β-arrestin1. Here, we present a step-by-step protocol to employ this intrabody sensor to measure the interaction and conformational activation of β-arrestin1 upon agonist-stimulation of a prototypical GPCR, the complement C5a receptor (C5aR1). This protocol is potentially applicable to other GPCRs and may also be leveraged to deduce qualitative differences in β-arrestin1 conformations induced by different ligands and receptor mutants.

Interaction of alpha-synuclein with lipids.

Abstract: Alpha-synuclein (α-syn) is a natively unfolded protein that is abundantly expressed in the central nervous system. Although it has been shown to be involved in neurotransmission and cognition, its exact functions remain elusive. The misfolding of this protein into β-sheet-rich amyloid structures and subsequent aggregation has been associated with several neurodegenerative diseases, including Parkinson's disease. The interaction of α-syn with lipid membranes has been implicated in the formation of these pathological aggregates. At the same time, some physiological functions of α-syn also seem to require membrane interactions. A majority of the disease-associated mutations of α-syn occur in the lipid binding domain, further indicating the importance of membrane interactions in health and disease. A comprehensive understanding of the factors that modulate these interactions will help delineate the physiological and pathological states of this protein.

Nanoluciferase-based complementation assay for systematic profiling of GPCR-GRK interactions.

Abstract: G protein-coupled receptor kinases (GRKs) are a family of seven soluble receptor-modifying enzymes which are essential regulators of GPCR activity. Following agonist-induced receptor activation and G protein dissociation, GRKs prime the receptor for desensitization through phosphorylation of its C terminus, which subsequently allows arrestins to bind and initiate the receptor internalization process. While GRKs constitute key GPCR-interacting proteins, to date, no method has been put forward to readily and systematically determine the preference of a specific GPCR towards the seven different GRKs (GRK1-7). This chapter describes a simple and standardized approach for systematic profiling of GRK1-7–GPCR interactions relying on the complementation of the split Nanoluciferase (NanoBiT). When applied to a set of GPCRs (MOR, 5-HT1A, B2AR, CXCR3, AVPR2, CGRPR), including two intrinsically β-arrestin-biased receptors (ACKR2 and ACKR3), this methodology yields highly reproducible results highlighting different GRK recruitment profiles. Using this assay, further characterization of MOR, a crucial target in the development of analgesics, reveals not only its GRK fingerprint but also related kinetics and activity of various ligands for a single GRK.

Proximity labeling for investigating protein-protein interactions.

Abstract: The study of protein complexes and protein-protein interactions is of great importance due to their fundamental roles in cellular function. Proximity labeling, often coupled with mass spectrometry, has become a powerful and versatile tool for studying protein-protein interactions by enriching and identifying proteins in the vicinity of a specified protein-of-interest. Here, we describe and compare traditional approaches to investigate protein-protein interactions to current day state-of-the-art proximity labeling methods. We focus on the wide array of proximity labeling strategies and underscore studies using diverse model systems to address numerous biological questions. In addition, we highlight current advances in mass spectrometry-based technology that exhibit promise in improving the depth and breadth of the data acquired in proximity labeling experiments. In all, we show the diversity of proximity labeling strategies and emphasize the broad range of applications and biological inquiries that can be addressed using this technology.

Clonal analysis of human clonogenic keratinocytes.

Abstract: Regenerative medicine has its roots in harnessing stem cells for permanent restoration of damaged or diseased tissues. The first procedure for the transplantation of epidermal cultures in massive full-thickness burns was established in the 1980s. Since then, epithelial stem cell-based therapies have been further developed in cell and gene therapy protocols aimed at restoring visual acuity in severe ocular burns and treating patients affected by genetic skin diseases, as Epidermolysis Bullosa. The clinical success of these Advanced Therapy Medicinal Products (ATMPs) requires the presence of a defined number of epithelial stem cells in the grafts, detected as holoclone-forming cells. To date, the most trustworthy method to identify and measure holoclones in a culture is the clonal analysis of clonogenic keratinocytes. Here we describe in detail how to perform such a clonal analysis and identify each epidermal clonal type.

Nanoluciferase-based methods to monitor activation, modulation and trafficking of atypical chemokine receptors.

Abstract: Chemokines regulate directed cell migration, proliferation and survival and are key components in various physiological and pathological processes. They exert their functions by interacting with seven-transmembrane domain receptors that signal through G proteins (GPCRs). Atypical chemokine receptors (ACKRs) play important roles in the chemokine–receptor network by regulating chemokine bioavailability for the classical receptors through chemokine sequestration, scavenging or transport. Currently, this subfamily of receptors comprises four members: ACKR1, ACKR2, ACKR3 and ACKR4. They differ notably from the classical chemokine receptors by their inability to elicit G protein-mediated signaling, which precludes the use of classical assays relying on the activation of G proteins and related downstream secondary messengers to investigate ACKRs. There is therefore a need for alternative approaches to monitor ACKR activation, modulation and trafficking. This chapter details sensitive and versatile methods based on Nanoluciferase Binary Technology (NanoBiT) and Nanoluciferase Bioluminescence Resonance Energy Transfer (NanoBRET) to monitor ACKR2 and ACKR3 activity through the measurement of β-arrestin and GRK recruitment, and receptor trafficking, including internalization and delivery to early endosomes.

Evaluation of CAR-T cell cytotoxicity: Real-time impedance-based analysis.

Abstract: This chapter describes the most common method for evaluating cytotoxicity of chimeric antigen receptor (CAR) T cells, the xCELLigence real-time cell analysis (RTCA) platform (Agilent Technologies, Inc., Santa Clara, CA). Though there are a variety of assays used to evaluate conventional and engineered T cell cytotoxicity, the benefit of the xCELLigence platform is the depth of real-time data collected. This chapter begins by providing information on the conceptual basis underlying the xCELLigence assay, followed by a detailed protocol for the application of this assay to evaluate your own CAR-T cells, as well as specific insight and helpful tips for assay design, usage, and data analysis. Application of the information and methods discussed within this chapter will provide a greater understanding for evaluating cytotoxicity of CAR-T cells using this in vitro model system.

Assessment of hypoxia by pimonidazole staining following radiotherapy.

Abstract: The rapid proliferation of cancer cells and the aberrant vasculature present in most solid tumors frequently result in the lack of oxygen generating a hypoxic tumor microenvironment. Low levels of oxygen not only affect the tumor cell biology and tumorigenesis, but also the other components of the tumor microenvironment such as the tumor stroma and the immune infiltrate, promoting a more suppressive environment. In addition, tumor hypoxia has been associated with reduced sensitivity to chemotherapy (CH) and radiotherapy (RT), leading to poor outcomes in cancer patients. Therefore, the evaluation of tumor oxygen status has become clinically relevant. Tumor hypoxia can be assessed by different methods that include the analysis of the oxygen concentration or the expression of endogenous markers directly related to hypoxia. In this paper, we focus on the use of the hypoxia-specific marker pimonidazole as a straightforward way to measure tumor hypoxia following radiotherapy in a preclinical melanoma model.

Generation of CAR-T cells using lentiviral vectors.

Abstract: Cancer immunotherapy is nowadays largely focused on the development of therapeutic antibodies and chimeric antigen receptors (CARs). Two CARs targeting CD19 have been approved recently for the treatment of some hematological malignancies. This demonstrates the capability of engineered CAR T cells in generating effective tumor responses. Furthermore, several hundred ongoing clinical trials are exploring the feasibility of CAR-based approaches to target tumor-associated antigens in solid tumors. However, there still remain significant challenges and limitations in the design and production of CAR-modified T cells that need to be addressed, such as more effective transduction methods, expression and exhaustion issues, reliable in vitro and in vivo characterization methods, etc. Here we describe current techniques for generating CAR T cells using lentiviral vectors as well as detailed protocols for their functional characterization.

Discovery of Virus-Host interactions using bioinformatic tools.

Abstract: Viruses are a diverse biological group capable of infecting several hosts such as bacteria, plants, and animals, including humans. Viral infections constitute a threat to the human population as they may cause high mortality rates, decrease food production, and generate large economical losses. Viruses co-evolve with their hosts and this constant evolution must be clarified to better predict possible viral outbreaks, and to develop improved diagnostic methods and therapeutical approaches. In this review, we summarize several viral databases that store key information retrieved from a variety of omics approaches. Furthermore, we explore the use of such databases to predict Virus-Host interactions through artificial intelligence algorithms, focusing on the latest methodologies to characterize biological networks.

Induction of pancreatitis in mice with susceptibility to pancreatic cancer.

Abstract: Chronic inflammation is known to be associated with pancreatic cancer, however a complete picture regarding how these pathologies intersect is still being characterized. In vivo model systems are critical for the study of mechanisms underlying how inflammation accelerates neoplasia. Repeat injection of cerulein, a cholecystokinin (CCK) analog, is widely used to experimentally induce acute and chronic pancreatitis in vivo. Chronic cerulein administration into genetically engineered mouse models (GEMMs) with predisposition to pancreatic cancer can induce a pro-inflammatory immune response, pancreatic acinar cell damage, pancreatic stellate cell activation, and accelerate the onset of neoplasia. Here we provide a detailed protocol and insights into using cerulein to induce pancreatitis in GEMMs, and methods to experimentally assess inflammation and pancreatic neoplasia.

In vitro preparation of human Dental Pulp Stem Cell grafts with biodegradable polymer scaffolds for nerve tissue engineering.

Abstract: Human Dental Pulp Stem Cells (hDPSCs) are one of the most promising stem cell sources for tissue engineering and regeneration, due to their extraordinary multi-lineage differentiation ability, ease of extraction from biological waste in dental clinics, safe non-tumorigenic phenotype, immune-tolerance upon in vivo transplantation, and great possibilities of application in autologous tissue reconstruction. The in vitro manipulation of hDPSCs paves the way for drug screening and tailor-made regeneration of damaged tissues, in the context of personalized medicine. The neural crest phenotype of these stem cells gives them the capacity to differentiate to a large variety of cell types, including neural-lineage cells. In this chapter, we describe various culture methods to generate different cellular phenotypes from hDPSCs, which can not only grow as mesenchymal-like plastic adherent cells, but also as non-adherent neurogenic dentospheres in serum-free medium. Floating dentospheres can be used to generate large populations of mature neuronal and glial marker expressing cells, which may be cultured over a substrate of nanopatterned scaffold based on biodegradable poly(lactide-co-caprolactone) (PLCL) to induce a controlled alignment of neurites and cell migration, to generate in vivo biocompatible constructs for nerve tissue bioengineering.

In-cellulo chemical cross-linking to visualize protein-protein interactions.

Abstract: Reversible protein-protein interaction in cells is an integral and central aspect of intracellular signaling mechanisms. This allows distinct signaling cascades to become active upon stimulation with external signal resulting in cellular and physiological responses. Several distinct methods are currently available and utilized routinely to monitor protein-protein interactions including co-immunoprecipitation (co-IP). An inherent limitation associated with co-IP assay however is the inability to efficiently capture transient and short-lived interactions in cells. Chemical cross-linking of such transient interactions in cellular context using cell permeable reagents followed by co-IP overcomes this limitation, and allows a simplified approach without requiring any sophisticated instrumentation. In this chapter, we present a step-by-step protocol for monitoring protein-protein interaction by combining chemical cross-linking and co-immunoprecipitation using GPCR-β-arrestin complex as a case example. This protocol is based on previously validated method that can potentially be adapted to capture and visualize transient protein-protein interactions in general.

Immunofluorescence staining of colorectal cancer patient-derived organoids.

Abstract: Organoids recapitulate the cellular heterogeneity, functionality, architecture and molecular signature of the organ or diseased tissue from which they are derived. They thus provide a bridge between traditional 2D culture systems and animal models and have profoundly enhanced our ability to study organ development and disease in vitro . Fluorescence microscopy has been an essential method in characterizing the cellular and morphological composition of organoids and demonstrating that they faithfully recapitulate the in vivo tissue of origin. Here we provide a straightforward method for immunofluorescence staining and confocal microscopy imaging of colorectal cancer (CRC) patient-derived organoids (PDOs) in basement matrix. The method is applicable to other types of human organoids, and we have also successfully used it on organoids derived from the mouse mammary gland.

Flow cytometry detection and quantification of CAR T cells into solid tumors.

Abstract: Adoptive T cell therapy (ACT) is a therapeutic approach which employs genetically manipulated autologous T cells to target and eliminate a patient's malignancy. This novel therapeutic approach, when employing a chimeric antigen receptor (CAR) targeting CD19-expressing B cells, has shown remarkable success in treating acute B-cell lymphocytic leukemia. However, blood born malignancies represent only a fraction of cancers which affect patients. Unfortunately, the utilization of ACT to target solid malignancies has only shown marginal success rates. There are many known obstacles which hinder CAR T cell therapy in patients suffering from solid cancer, one notable obstacle is the effective trafficking of CAR T cells to the tumor site. With the rapid advancement of novel approaches and targets which may enhance CAR T cell infiltration into solid tumors, a standardized approach to assess and measure CAR T cell infiltration becomes imperative in order to compare these different approaches across platforms. Here we describe a flow cytometry method which enables the rapid detection and quantification of CAR T cells which have reached and entered the tumor mass following intravenous injection. Competence with single cell preparation and flow cytometry is required for optimal results.

Small animal models of thermal injury.

Abstract: Burns are a severe form of trauma that account for 1.1 million cases necessitating medical attention and 4500 mortalities annually in the United States alone. Importantly, the initial trauma is succeeded by extensive, prolonged physiological alterations that detrimentally impact multiple organ systems. Given the complexity of post-burn pathophysiology, in vitro experiments are insufficient to model thermal injuries. Therefore, compatible animal burn models are essential for studying burn-related phenomena. In this chapter, we discuss commonly employed small animal burn models and their comparability and applicability to human studies. In particular, we compare post-burn wound healing between the species as well as relevant hypermetabolic and inflammatory characteristics, providing a better understanding of the pros and cons of utilizing a small animal surrogate for human burns. We further provide an overview of the rodent scald burn model methodology as well as a comparison between elderly, aged and young animals, providing a guide for tailoring animal model choice based on the relevant research question.

Methods to isolate adipose tissue-derived stem cells.

Abstract: The use of adipose tissue has seen increasing interest in recent years for treating plastic surgery defects and for regenerative medicine applications. Adipose tissue is considered an optimal source of stem cells, as it contains more multipotent cells than bone marrow for the same volume. The adipose tissue-derived stem cells (ADSCs), isolated from the heterogeneous stromal vascular fraction (SVF), possess self-renewal properties and multilineage differentiation potential. In addition, adipose tissue can be obtained with less invasive procedures and patient morbidity than other tissue. For these reasons, numerous enzymatic, and non-enzymatic isolation methods have been developed over the years. The traditional method for isolation and culture of primary ADSCs from adipose tissue relies on enzymatic digestion with collagenase, followed by multiple steps of centrifugation. Alternative non-enzymatic isolation methods are based closed, sterile, and safe isolation processes that differ from each other for parameters such as the centrifugation force, pressure, filtration, and washing. Despite the existence of this multitude of systems, the best isolation method has not been identified to date. Therefore, the great challenge remains the achievement of the standardization of cellular products to allow the comparability between clinical studies and trials.

Label-free impedance measurements to unravel biomolecular interactions involved in G protein-coupled receptor signaling.

Abstract: G protein-coupled receptors (GPCRs) are among the most heavily addressed drug targets in medicinal chemistry and pharmacology. The screening for new agonists or antagonists has been largely based on genetically engineered cells overexpressing the receptor to study binding of ligands directly or via intracellular signaling events downstream of receptor activation. These approaches are often invasive in nature, need to be conducted as endpoint assays, require isotope- or fluorophore-labeling and significant genetic manipulation. In contrast to that, non-invasive and label-free impedance measurements are capable of monitoring ligand-receptor interactions in target cells with endogenous receptor expression in real time. The cells expressing the receptor are grown on planar gold-film electrodes that are integrated into regular cell culture dishes. This article will highlight several impedance-based assay formats to characterize biomolecular interactions between ligands and their GPCRs in vitro, comprising agonist and antagonist characterization, dose-response relationships, receptor desensitization, and signal transduction profiling.

Mouse models of graft-versus-host disease.

Abstract: Transplantation of allogeneic hematopoietic stem and progenitor cells (allo-HCT) allows for cure of life-limiting malignant and non-malignant hematologic diseases. Crossing the human leukocyte antigen (HLA) barrier, however, comes at the cost of graft-versus-host disease (GVHD), a life-threatening syndrome mediated in part by the same donor T-lymphocytes that eliminate malignant cells. Acute GVHD occurs in the skin, gut, and/or liver in 25-55% of patients with a mortality rate of 15-40%, while chronic GVHD develops in 30-65% of patients who survive at least 3 months following allo-HCT and is highly debilitating in its extensive form, with a 30-50% 5year mortality rate stemming in part from immune dysregulation and opportunistic infections. Knowledge gaps remain in understanding the pathogenesis and in developing novel and effective treatments for the acute and chronic GVHD, which have distinct biology and yet are both treated with front line systemic corticosteroids. Novel and informative mouse models remain the primary means by which these diseases are studied and drugs initially developed prior to testing in humans. In this chapter, we describe allo-HCT mouse models and protocols using these mouse models by which to study acute and chronic GVHD with the goal of improving prevention and therapy.

In vivo experimental mouse model to test CD19CAR T cells generated with different methods.

Abstract: Pre-clinical evaluation of CAR T cells includes procedures testing T-cell efficacy and safety in as close to real world conditions as possible. An important step in efficacy testing is the in vivo study, most often using immunodeficient murine models into which both the poison and the cure are injected; namely a human cancer cell line and genetically modified human T cells. The capacity of the T cells to control the cancer progression will provide information about the CAR T-cell efficacy. We here provide a protocol to test CAR T cells in vivo using the validated anti-B-cell marker CD19CAR and an aggressive lymphoma model.

Assessment of type I interferon responses as a feature of immunogenic cell death.

Abstract: The radiochemotherapy- or chemotherapy-induced stimulation of immunogenic cell death (ICD) affecting malignant cells ignites antitumor immune responses that are clinically relevant as they allow to achieve durable responses beyond treatment discontinuation. The mechanistic exploration of ICD and the discovery of agents and interventions that are endowed with the capacity to elicit ICD is of the utmost importance. Here, we describe an assay for the assessment of type I interferon (IFN) production, which is one of the salient features of ICD. Biosensor cells that express GFP under the control of the IFN-inducible MX dynamin like GTPase 1 (MX1) gene promoter are employed, and the fluorescent signal is assessed by automated microscopy. The described workflow is automation-friendly, rendering it compatible with high-throughput screening (HTS) for drug discovery.

Orthotopic brain tumor models derived from glioblastoma stem-like cells.

Abstract: There is an urgency for identifying effective therapies for glioblastoma (GBM), an incurable and lethal primary malignant brain tumor. Patient-derived xenograft mouse models, in which glioma stem cells, which retain the characteristics of the original tumor, are implanted into the brain of immunocompromised mice, represent a well-suited model for studying GBM. Such models are essential for studies involving the tumor microenvironment and for testing experimental therapeutics for brain tumors. In this chapter, we detail various steps for generating an orthotopic brain tumor model in mice. We provide step-by-step guidance for enrichment and expansion of glioma stem cells for surgical specimens, surgical injection of these cells into the brain of immunocompromised mice, as well as monitoring of tumor growth.

Modeling acute graft-versus-host disease (aGVHD) in murine bone marrow transplantation (BMT) models with MHC disparity.

Abstract: For more than 50years, hematopoietic stem cell transplantation (HSCT) has been the major curative therapy for hematological malignancies and genetic disorders, but its success is limited by the development of graft-versus-host disease (GVHD). GVHD represents a post-transplantation disorder representing the immune-mediated attack of transplant-derived T cells against recipient tissue finally leading to increased morbidity and mortality of the recipient. GVHD develops if donor and recipient are disparate in major or minor histocompatibility antigens (MHC, miHA). Most of the initial knowledge about the biology of GVHD is derived from murine bone marrow transplantation (BMT) models. Of course, GVHD mouse models do not reflect one to one the human situation, but they contribute significantly to our understanding how conditioning and danger signals activate the immune system, enlighten the role of individual molecules, e.g., cytokines, chemokines, death-inducing ligands, define the function of lymphocytes subpopulations for GVHD development and have significant impact on establishing new treatment and prevention strategies used in clinical HSCT. This chapter describes in detail the procedure of allogeneic BMT and the development of GVHD in two commonly used allogeneic murine BMT models (B6→B6.bm1, B6→B6D2F1) with different MHC disparities, which can be used as a basis for advanced studies of GVHD pathology or the development of new treatment strategies.

ELISA-based quantification of type I IFN secretion by irradiated cancer cells.

Abstract: Cancer cell-intrinsic type I interferon (IFN-I) activation is required to initiate early innate immune responses and the subsequent radiation-induced anti-tumor immunity. Investigating the secretion of IFN-I cytokines in response to radiation therapy (RT) is therefore a critical readout for selecting the best immunogenic radiation dose-fractionation regimen. In this chapter, we present different ELISA-based quantification techniques that can be utilized to assess the secretion of tumor-derived IFN-I cytokines, namely IFN-α and IFN-β.

Methods for induction and assessment of intestinal permeability in rodent models of radiation injury.

Abstract: Ionizing radiation (IR) is a significant contributor to the contemporary market of energy production and an important diagnostic and treatment modality. Besides having numerous useful applications, it is also a ubiquitous environmental stressor and a potent genotoxic and epigenotoxic agent, capable of causing substantial damage to organs and tissues of living organisms. The gastrointestinal (GI) tract is highly sensitive to IR. This problem is further compounded by the fact that there is no FDA-approved medication to mitigate acute radiation-induced GI syndrome. Therefore, establishing the animal model for studying IR-induced GI-injury is crucially important to understand the harmful consequences of intestinal radiation damage. Here, we discuss two different animal models of IR-induced acute gastrointestinal syndrome and two separate methods for measuring the magnitude of intestinal radiation damage.

Assessment of transcription inhibition as a characteristic of immunogenic cell death.

Abstract: Anticancer drugs that suppress DNA-to-RNA transcription are particularly efficient in stimulating immunogenic cell death and hence eradicate malignant cells in a way that they will ignite an antitumor immune response. This is therapeutically relevant as it allows treatment response to last beyond drug discontinuation. For this reason, it is important to measure transcription inhibition in a precise fashion. Here, we detail two complementary assays for the assessment of transcription inhibition, one that detects the physical separation of fibrillarin and nucleolin by two-color immunofluorescence and another that measures the diminution of incorporated 5-ethynyl uridine (EU) into RNA, as revealed by click chemistry and the per-cell-intensity of a fluorescent signal.

The use of foodborne infection to evaluate bacterial pathogenesis and host response.

Abstract: Foodborne bacterial infections are a major cause of gastrointestinal illness. Murine models have been widely used to interrogate bacterial pathogenesis and host response to better understand the pathogens that cause gastrointestinal disease. Humans are usually exposed to these pathogens through consumption of contaminated food products. However, most murine models of foodborne infection rely on oral gavage to deliver pathogens directly into the stomach. While expedient, the gavage procedure may lead to microabrasions in the esophagus that allow direct access of the pathogen to the blood, which can alter bacterial pathogenesis and the host response under study. In this chapter, the alternative approach of foodborne infection through the consumption of inoculated food is described using the human pathogen Listeria monocytogenes (Lm). A detailed protocol of this methodology is provided with details of assessing bacterial burden and the host immune response. Translation of these methods to other foodborne pathogens will allow a more accurate assessment of bacterial pathogenesis and host immunity in more physiologic murine models.

Preparation of a stable CCL5·CCR5·Gi signaling complex for Cryo-EM analysis.

Abstract: The numerous chemokines and their cognate G protein-coupled chemokine receptors on the surface of leukocytes form a complex signaling network, which regulates the immune response and also other key physiological processes. Currently only a very limited number of structures of chemokine•chemokine receptor complexes have been solved. More structures are needed for the understanding of their mechanism of action and the rational design of drugs against these highly relevant therapeutic targets. Recently, we have determined the cryo-EM structure of the human wild-type CCR5 chemokine receptor, which is also the HIV-1 coreceptor, in its active conformation bound to the chemokine super-agonist [6P4]CCL5 and the heterotrimeric Gi protein. The structure provides the rationale for the sequence-activity relation of agonist and antagonist CCR5 chemokine ligands. In this chapter, we present a detailed protocol for the preparation of the active agonist chemokine•CCR5•Gi complex for cryo-EM studies including quality controls and caveats. As such the protocol may serve as starting point for structural and biophysical studies of other chemokine•chemokine receptor complexes.

Small animal models of localized heart irradiation.

Abstract: A subset of cancer patients treated with radiation therapy may experience radiation-induced heart disease (RIHD) that develops within weeks to several years after cancer treatment. Rodent models are most commonly used to examine the biological effects of local X-rays in the heart and test potential strategies to reduce RIHD. While developments in technology over the last decades have changed the procedures for local heart irradiation in animal models, the X-ray settings and radiation doses have remained quite consistent in time and between different research laboratories. This chapter provides a protocol for whole heart irradiation in rodent models, using an X-ray machine with cone beam computed tomography (CBCT) capabilities. Some methods for the quantification of common histological changes after whole heart irradiation in the rodent are also described.