Preparation of Barley Roots for Histological, Structural, and Immunolocalization Studies Using Light and Electron Microscopy.
Abstract Microscopic investigations of biological objects are an integral part in plant research and most fields of life sciences. They allow the description of morphological, histological, and structural aspects of individual cells or tissues. Based on various cell biological tools and methods it is possible to characterize different plant genotypes or study their adaptation to changing environmental conditions. In combination with antibodies raised against specific antigens and epitopes light and electron microscopy enable investigation of the function of single genes/proteins in plant growth and development or ...
Source: Mol Biol Cell - November 23, 2018 Category: Molecular Biology Authors: Marzec M, Melzer M Tags: Methods Mol Biol Source Type: research

Preparation of Barley Pollen Mother Cells for Confocal and Super Resolution Microscopy.
Abstract Recombination (crossover) drives the release of genetic diversity in plant breeding programs. However, in barley, recombination is skewed toward the telomeric ends of its seven chromosomes, restricting the re-assortment of about 30% of the genes located in the centromeric regions of its large 5.1 Gb genome. A better understanding of meiosis and recombination could provide ways of modulating crossover distribution and frequency in barley as well as in other grasses, including wheat. While most research on recombination has been carried out in the model plant Arabidopsis thaliana, recent studies in barley (...
Source: Mol Biol Cell - November 23, 2018 Category: Molecular Biology Authors: Mittmann S, Arrieta M, Ramsay L, Waugh R, Colas I Tags: Methods Mol Biol Source Type: research

Microarrays for High-Throughput Gene Expression Analysis of Barley.
Abstract Microarrays remain an extremely robust and cost-efficient technology for transcriptome analysis. A custom Agilent microarray has been designed from the predicted gene transcripts of the barley genome reference. Here, we describe total RNA extraction, fluorescent labeling, hybridization, and image analysis of barley arrays. Quality control measures are detailed, along with recommendations for downstream data analysis. PMID: 30460566 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - November 23, 2018 Category: Molecular Biology Authors: Morris JA, Hedley PE Tags: Methods Mol Biol Source Type: research

High-Resolution RT-PCR Analysis of Alternative Barley Transcripts.
Abstract Assembly of the barley genome and extensive use of RNA-seq has resulted in an abundance of gene expression data and the recognition of wide-scale production of alternatively spliced transcripts. Here, we describe in detail a high-resolution reverse transcription-PCR based panel (HR RT-PCR) that confirms the accuracy of alternatively spliced transcripts from RNA-seq and allows quantification of changes in the proportion of splice isoforms between different experimental conditions, time points, tissues, genotypes, ecotypes, and treatments. By validating a selection of barley genes, use of the panel gives co...
Source: Mol Biol Cell - November 23, 2018 Category: Molecular Biology Authors: Simpson CG, Fuller J, Rapazote-Flores P, Mayer CD, Calixto CPG, Milne L, Hedley PE, Booth C, Waugh R, Brown JWS Tags: Methods Mol Biol Source Type: research

Usage of Droplet Digital PCR (ddPCR) Assays for T Cell Quantification in Cancer.
Abstract T cells fulfill a central role in cell-mediated immunity and can be found in the circulation and lymphoid organs upon maturation. For clinical applications, it can be important to quantify (infiltrated) T cells accurately in a variety of body fluids and tissues of benign, inflammatory, or malignant origin. For decades, flow cytometry and immunohistochemistry have been the accustomed methods to quantify T cells. Although these methods are widely used, they depend on the accessibility of T-cell epitopes and therefore require fresh, frozen, or fixated material of a certain quality. Whenever samples are low i...
Source: Mol Biol Cell - November 23, 2018 Category: Molecular Biology Authors: Zoutman WH, Nell RJ, van der Velden PA Tags: Methods Mol Biol Source Type: research

An Economical, Quantitative, and Robust Protocol for High-Throughput T Cell Receptor Sequencing from Tumor or Blood.
We describe a protocol for amplifying, sequencing, and analyzing T cell receptors which is economical, robust, sensitive, and versatile. The key experimental step is the ligation of a single-stranded oligonucleotide to the 3' end of the T cell receptor cDNA, which allows easy amplification of all possible rearrangements using only a single set of primers per locus, while simultaneously introducing a unique molecular identifier to label each starting cDNA molecule. After sequencing, this molecular identifier can be used to correct both sequence errors and the effects of differential PCR amplification efficiency, thus produc...
Source: Mol Biol Cell - November 23, 2018 Category: Molecular Biology Authors: Uddin I, Joshi K, Oakes T, Heather JM, Swanton C, TRACERx consortium, Chain B Tags: Methods Mol Biol Source Type: research

Generation of Tumor Antigen-Specific Cytotoxic T Lymphocytes from Pluripotent Stem Cells.
Abstract Immunotherapy is a developing but very promising arsenal to treat cancer. Acquiring a more potent and effective approach in cancer immunotherapy is always the ultimate pursuance. CTL-based therapies are highly acclaimed recently due to its direct killing property. However, difficulty in obtaining adequate number of CTLs is still a major obstacle. In previous studies, it is shown that pluripotent stem cell-derived cytotoxic T lymphocytes (CTL)-especially the genetically engineered tumor antigen-specific CTLs-may serve as a good candidate for this goal. Here we introduce a novel approach in generating tumor...
Source: Mol Biol Cell - November 23, 2018 Category: Molecular Biology Authors: Chen X, Lei F, Wang L, Xiong X, Song J Tags: Methods Mol Biol Source Type: research

Expansion and Adoptive Transfer of Human V δ2+ T Cells to Assess Antitumor Effects In Vivo.
Expansion and Adoptive Transfer of Human Vδ2+ T Cells to Assess Antitumor Effects In Vivo. Methods Mol Biol. 2019;1884:57-72 Authors: Sharma A, Zumwalde NA, Gumperz JE Abstract Recent clinical trials have yielded promising results suggesting that γδ T cell62-based immunotherapies can be effective against hematological malignancies. Human T cells expressing Vγ9Vδ2+ receptors are particularly attractive candidates for this application, since they can be readily expanded in vitro in large quantities for adoptive transfer and do not require HLA-matching of donors and recipien...
Source: Mol Biol Cell - November 23, 2018 Category: Molecular Biology Authors: Sharma A, Zumwalde NA, Gumperz JE Tags: Methods Mol Biol Source Type: research

Usage of Single-Cell RNA Sequencing to Unveil Immune Lymphoid Cell Precursors.
Abstract Single-cell RNA sequencing (scRNA-seq) is a powerful tool to study immune cells, which enables an unbiased way to discover novel cell populations, biological meaningful cellular heterogeneity, and cell lineage development trajectories. Advances in scRNA-seq technologies and computational data analysis have driven a revolution in our understanding of the immune system in health and disease. Technically, the key step for scRNA-seq analysis is making a high-quality cDNA library for sequencing. Here, we describe a plate-based protocol to prepare single-cell cDNA library of bone marrow innate lymphoid precurso...
Source: Mol Biol Cell - November 23, 2018 Category: Molecular Biology Authors: Yu Y, Liu P Tags: Methods Mol Biol Source Type: research

Isolation, Expansion, and Characterization of Natural Killer Cells and Their Precursors as a Tool to Study Cancer Immunosurveillance.
Abstract This chapter will describe the current methodologies to isolate and expand NK cells from Peripheral Blood (PB) or tissues for "in vitro" studies, including NK cell antitumor immune function. In addition, methods to induce NK cell maturation, differentiation, and expansion from CD34+ precursors will also be described. Finally, it will also be treated the topical issue of the characterization of new functionally and phenotypically defined NK cell subsets. PMID: 30465197 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - November 23, 2018 Category: Molecular Biology Authors: Parodi M, Meazza R, Vitale C, Pietra G, Carrega P, Vitale M Tags: Methods Mol Biol Source Type: research

CD107a Degranulation Assay to Evaluate Immune Cell Antitumor Activity.
oto A Abstract Cancer development is under surveillance by the immune system of the host. Tumor cells can be recognized and killed by cytotoxic lymphocytes- such as CD8+ T lymphocytes and natural killer (NK) cells-mainly through the immune secretion of lytic granules that kill target cells. This process involves the fusion of the granule membrane with the cytoplasmic membrane of the immune effector cell, resulting in surface exposure of lysosomal-associated proteins that are typically present on the lipid bilayer surrounding lytic granules, such as CD107a. Therefore, membrane expression of CD107a constitutes a mar...
Source: Mol Biol Cell - November 23, 2018 Category: Molecular Biology Authors: Lorenzo-Herrero S, Sordo-Bahamonde C, Gonzalez S, López-Soto A Tags: Methods Mol Biol Source Type: research

A Flow Cytometric NK Cell-Mediated Cytotoxicity Assay to Evaluate Anticancer Immune Responses In Vitro.
;pez-Soto A Abstract The immune system is able to detect and eliminate nascent and developing tumors. Thus, T lymphocytes of the adaptive immunity recognize cancer cells by detecting tumor-associated antigens, whereas certain innate immune cells scan for molecules that are mainly overexpressed on malignant and infected cells. The best example of the latter is natural killer (NK) cells. The activity of these immune cells is strictly governed by a balance between positive and negative signals provided by stress-regulated molecules that bind NK cell membrane receptors, such as the activatory receptor NKG2D. Given the...
Source: Mol Biol Cell - November 23, 2018 Category: Molecular Biology Authors: Lorenzo-Herrero S, Sordo-Bahamonde C, González S, López-Soto A Tags: Methods Mol Biol Source Type: research

Mouse Models to Study Natural Killer Cell-Mediated Immunosurveillance and Metastatic Latency.
Abstract Metastatic latency is a major concern in the clinic, yet how these disseminated cancer cells survive and initiate metastases is unknown (Massagué and Obenauf, Nature 529:298-306, 2016). Here, we describe an approach to isolate latency competent cancer (LCC) cells from early stage human lung and breast carcinoma cell lines using mouse xenograft models (Malladi, Cell 165:45-60, 2016). Cancer cell lines labeled with GFP-luciferase and antibiotic selection markers were injected intracardially into athymic mice. Three months, post-injection, LCC cells were identified in situ and isolated. Upon reinjecti...
Source: Mol Biol Cell - November 23, 2018 Category: Molecular Biology Authors: Nair VR, Malladi S Tags: Methods Mol Biol Source Type: research

Tumor-Associated Macrophage Isolation and In Vivo Analysis of Their Tumor-Promoting Activity.
Abstract Characterization of individual cell populations from the tumor microenvironment is critical to understand their functional contribution to tumor progression. Magnetic bead enrichment and fluorescence-activated cell sorting (FACS) allow for the isolation of specific cell types that can be used in downstream applications, including in vitro and in vivo functional studies and molecular profiling. In this chapter, we describe the process of isolation of tumor-associated macrophages (TAMs) from primary murine breast tumors subsequent to therapeutic or experimental intervention. Additionally, we further detail ...
Source: Mol Biol Cell - November 23, 2018 Category: Molecular Biology Authors: Clark NM, Bos PD Tags: Methods Mol Biol Source Type: research

Conditional Genetic Ablation Mouse Models as a Tool to Study Cancer Immunosurveillance In Vivo.
Abstract Over the last decades, it has been established that the immune system is crucial for the impediment of cancer development by recognizing and destroying transformed cells. This process has been termed cancer immunosurveillance. Small animal models have significantly facilitated our understanding of it. Dissecting the contribution of any specific immune cell type participating in this process requires the ability to specifically target it while leaving the other immune components as well as the cancer model system unperturbed in vivo. Here, we provide a simple and rapid protocol for the generation of transg...
Source: Mol Biol Cell - November 23, 2018 Category: Molecular Biology Authors: Dupont M, Vosshenrich CAJ Tags: Methods Mol Biol Source Type: research

Determination and Isolation of Immune Populations from Brain Tumor Microenvironments.
Abstract Flow cytometry analysis and fluorescence-activated cell sorting (FACS) allow the determination and isolation of different cell types from a given tumor sample. Here we describe and comment a method consisting of the preparation of a single cell suspension from a freshly dissected mouse brain tumor mass, staining with a combination of fluorescently labeled antibodies and analysis by flow cytometry to determine, characterize, and isolate different immune populations. PMID: 30465203 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - November 23, 2018 Category: Molecular Biology Authors: Godino J, Schuhmacher AJ Tags: Methods Mol Biol Source Type: research

Mouse Model of Colitis-Associated Colorectal Cancer (CAC): Isolation and Characterization of Mucosal-Associated Lymphoid Cells.
Abstract Colorectal cancer (CRC) is the third most common malignancy worldwide presenting high mortality due to low treatment efficacy. Existing evidence indicates that inflammatory bowel disease (IBD) is associated with a higher risk of developing CRC. Many murine models of inflammation-related colon carcinogenesis (CAC) have been developed to study colon carcinogenesis and novel treatments. A commonly used model involves the combination of a single dose of azoxymethane (AOM), together with three cycles of the inflammatory agent dextran sodium sulfate (DSS) (5 days in drinking water followed by a two-week rest). ...
Source: Mol Biol Cell - November 23, 2018 Category: Molecular Biology Authors: Santiago L, Castro M, Pardo J, Arias M Tags: Methods Mol Biol Source Type: research

Cancer Exome-Based Identification of Tumor Neo-Antigens Using Mass Spectrometry.
Abstract Neo-antigens expressed on tumors are targets for development of cancer immunotherapy strategies. Use of prediction algorithms to identify neo-antigens yields a significant number of peptides that must be validated in laborious and time-consuming methods; many prove to be false-positive identifications. The use of HLA peptidomics allows the isolation of the HLA-peptide complexes directly from cells and can be done on fresh tumor, patient-derived xerographs, or cell lines when the tissue sample is limited. This method can be used to identify both HLA class I and HLA class II or any different MHC from differ...
Source: Mol Biol Cell - November 23, 2018 Category: Molecular Biology Authors: Kalaora S, Samuels Y Tags: Methods Mol Biol Source Type: research

Single-Cell Mass Cytometry of Archived Human Epithelial Tissue for Decoding Cancer Signaling Pathways.
We present example data for using DISSECT on a cryopreserved specimen of the human colon to profile its immune and epithelial composition. These techniques can be used for high-resolution studies for monitoring disease-related alternations in different cellular compartments using specimens stored in cryopreserved or FFPE tissue banks. PMID: 30465206 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - November 23, 2018 Category: Molecular Biology Authors: Scurrah CR, Simmons AJ, Lau KS Tags: Methods Mol Biol Source Type: research

Purification of Leukemia-Derived Exosomes to Study Microenvironment Modulation.
Abstract Exosomes are membrane-enclosed vesicles released by different cell types into the extracellular space. As mediators of intercellular communication, they are involved in multiple physiological processes, but they are also associated with the pathogenesis of human malignancies including leukemia. Isolation of exosomes enables the characterization of their role in microenvironment modulation as well as their participation in disease pathology. A variety of strategies and techniques exists to purify exosomes from many biological fluids (e.g., blood, urine, and saliva). Here, we describe the efficient producti...
Source: Mol Biol Cell - November 23, 2018 Category: Molecular Biology Authors: Wierz M, Pierson S, Gargiulo E, Guerin C, Moussay E, Paggetti J Tags: Methods Mol Biol Source Type: research

Quantitative Identification of Senescent Cells in Cancer.
Abstract Cellular senescence, a state of permanent growth arrest, is an important mechanism preventing the propagation of damaged cells. It suppresses cancer development in premalignant lesions in response to activated oncogenes and in tumors following therapy. The presence of senescent cells in premalignant lesions and tumors is controlled by the immune system. The ability to identify and quantify senescent cells more efficiently in vivo is necessary in order to evaluate the effect of these cells on tumorigenesis and cancer therapy. Through combining senescent-associated beta-galactosidase staining with ImageStre...
Source: Mol Biol Cell - November 23, 2018 Category: Molecular Biology Authors: Biran A, Porat Z, Krizhanovsky V Tags: Methods Mol Biol Source Type: research

Generation and Harnessing of Heterotypic Tumor-Stroma Spheroids to Study Cancer Immunosurveillance.
g A Abstract Clinically apparent tumors have often established an immunosuppressive tumor microenvironment which renders them "cold," meaning that there are low numbers of immune cells within the tumor. Consequently, novel immunotherapy approaches such as checkpoint inhibitors fail to reactivate the tumor-targeted immune cells. Here we describe the generation of heterotypic tumor-stroma spheroids to study various approaches aiming at the reactivation of cancer immunosurveillance. These spheroids allow to investigate whether a certain immunotherapy or a combination treatment is able to stimulate antitumor...
Source: Mol Biol Cell - November 23, 2018 Category: Molecular Biology Authors: Zboralski D, Frömming A Tags: Methods Mol Biol Source Type: research

Establishment of Slice Cultures as a Tool to Study the Cancer Immune Microenvironment.
Abstract Although immunotherapy is currently being widely applied to treat a variety of cancers, there is great heterogeneity in the response to these treatments. Many in the field hypothesize that this may be attributable to the characteristics of each individual tumor immune microenvironment, in addition to systemic immune factors. Therefore, understanding the immune cell microenvironment in a variety of tumors is critically important. Specifically, the interactions among immune, stromal, and cancer cells, along with other factors in tumors, may hold the key to developing rational personalized combinations of im...
Source: Mol Biol Cell - November 23, 2018 Category: Molecular Biology Authors: Jiang X, Seo YD, Sullivan KM, Pillarisetty VG Tags: Methods Mol Biol Source Type: research

Gold Standard Assessment of Immunogenic Cell Death in Oncological Mouse Models.
ol JG Abstract The efficacy of cancer therapies strongly relies on their ability to reinstate cancer immunosurveillance. Numerous biomedical approaches with immunotherapeutic activity have been developed to reeducate the host immune system to detect and clear tumor cells. Cytotoxicants have been primarily designed to slow down malignant cell proliferation and to induce programmed cell death. Some cytotoxic stimuli are able to activate a particular type of apoptosis, which is referred to as immunogenic cell death (ICD), that de facto convert cancer cells into their own vaccine. This effect ultimately facilitates th...
Source: Mol Biol Cell - November 23, 2018 Category: Molecular Biology Authors: Humeau J, Lévesque S, Kroemer G, Pol JG Tags: Methods Mol Biol Source Type: research

A Novel Dendritic Cell-Based Vaccination Protocol to Stimulate Immunosurveillance of Aggressive Cancers.
Abstract A major challenge in the development of a successful tumor vaccination is to break immune tolerance and to sensitize efficiently the immune system toward relevant tumor antigens, thus enabling T-cell-mediated antitumor responses in vivo. Dendritic cell (DC)-based immunotherapy shows the advantage to induce an adaptive immune response against the tumor, with the potential to generate a long-lasting immunological memory able to prevent further relapses and hopefully metastasis. Recently different preclinical studies highlighted the golden opportunity to exploit the features of immunogenic cell death (ICD) t...
Source: Mol Biol Cell - November 23, 2018 Category: Molecular Biology Authors: Nigro A, Montico B, Casolaro V, Dal Col J Tags: Methods Mol Biol Source Type: research

Development of Bispecific Antibody Derivatives for Cancer Immunotherapy.
Abstract Development of antibody-based immunotherapeutics has progressed from direct tumor-targeting, with antibodies such as rituximab, to blocking of immune checkpoints to reactivate antitumor immunity. In addition, bispecific antibodies/antibody fragments are also of great interest in cancer therapy, as these constructs have the ability to redirect immune effector cells to cancer targets and, thereby, enhance therapeutic efficacy. A number of bispecific antibody formats have been reported, with the first FDA-approved bispecific antibody being blinatumomab, a so-called bispecific T cell engager (BiTE), which red...
Source: Mol Biol Cell - November 23, 2018 Category: Molecular Biology Authors: He Y, Helfrich W, Bremer E Tags: Methods Mol Biol Source Type: research

Generation of CAR-T Cells for Cancer Immunotherapy.
Abstract T cells engineered with chimeric antigen receptors (CARs) are emerging as powerful cancer immunotherapies. Remarkable efficacies have been demonstrated in treating B-cell malignancies with CAR-T cells, leading to the FDA's first approval of gene therapy. Currently, numerous clinical trials for hematological malignancies and solid tumors are underway worldwide. Production of CAR-T cells with proper qualities is essential for CAR-T success in vivo. Here we detail optimized protocols for the generation of CAR-T cells for preclinical studies using lentiviral gene transfer, expansion of CAR-T cells in culture,...
Source: Mol Biol Cell - November 23, 2018 Category: Molecular Biology Authors: Xu Q, Harto H, Berahovich R, Xu S, Zhou H, Golubovskaya V, Wu L Tags: Methods Mol Biol Source Type: research

Thermogenic fat.
PMID: 30449311 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - November 20, 2018 Category: Molecular Biology Authors: Marx SJ, Cohen P Tags: Biochim Biophys Acta Mol Cell Biol Lipids Source Type: research

Isoelectric Focusing on Non-Denaturing Flatbed Gels.
Abstract Isoelectric focusing (IEF) serves as a very useful procedure for cell protein separation and characterization. We have used this method to study antibody clonotype changes. Here we discuss the use of a sensitive native flatbed isoelectric focusing method to analyze specific antibody clonotype changes in a patient with systemic lupus erythematosus, who developed autoantibodies to the Ro 60 autoantigen under observation. Patient sera samples collected over several years were used for analysis using flatbed IEF. Following electrofocusing, the gel is analyzed by affinity immunoblotting utilizing Ro 60-coated ...
Source: Mol Biol Cell - November 16, 2018 Category: Molecular Biology Authors: Kurien BT, Scofield RH Tags: Methods Mol Biol Source Type: research

Identification of Proteins Interacting with Single Nucleotide Polymorphisms (SNPs) by DNA Pull-Down Assay.
Abstract Single nucleotide polymorphisms (SNPs) are the most abundantly found common form of DNA variation in the human genome. Many genetic association studies have proved that some of these SNPs are involved in regulating several cellular/physiological processes ranging from gene regulation to disease development. Analysis of the protein complex that binds to these SNPs is a crucial step in studying the mechanisms by which gene expressions are regulated in cis- or trans-acting manner. Commonly used techniques to determine DNA-protein interaction, such as electrophoretic mobility shift assay (EMSA), have limited ...
Source: Mol Biol Cell - November 16, 2018 Category: Molecular Biology Authors: Singh B, Nath SK Tags: Methods Mol Biol Source Type: research

A Combined Free-Flow Electrophoresis and DIGE Approach to Compare Proteins in Complex Biological Samples.
Abstract Free-flow electrophoresis has been applied in numerous studies as a protein separation technique due to its multiple advantages such as fast and efficient sample recovery, high resolving power, high reproducibility and wide applicability to protein classes. As a stand-alone platform, however, its utility in comparative proteomic analysis is limited as protein samples must be run sequentially rather than simultaneously which introduces inherent variability when attempting to perform quantitative analysis. Here we describe an approach combining fluorescent CyDye technology (DIGE) with free-flow electrophore...
Source: Mol Biol Cell - November 16, 2018 Category: Molecular Biology Authors: Fung KYC, Cursaro C, Lewanowitsch T, Cosgrove L, Hoffmann P Tags: Methods Mol Biol Source Type: research

Cell Surface Protein Biotinylation for SDS-PAGE Analysis.
Abstract Proteins expressed at the cell surface play important roles in physiology and represent valuable targets for new therapeutic agents. Indeed, the so-called druggable proteome consists, for about two thirds, of proteins that are either integral to or associated with the cell membrane. In spite of its importance, however, a complete characterization of the cell surface proteome has remained elusive because of the difficulty to efficiently purify these proteins from other contaminants. Methods exploiting the strong interaction between biotin and streptavidin have paved the way for the most significant ad...
Source: Mol Biol Cell - November 16, 2018 Category: Molecular Biology Authors: Elia G Tags: Methods Mol Biol Source Type: research

Single-Cell High-Resolution Detection and Quantification of Protein Isoforms Differing by a Single Charge Unit.
Abstract Isoelectric focusing (IEF) is an electrophoretic technique that enables the separation of proteins based on their isoelectric points. Until recently, this valuable method was not feasible for single-cell applications, which are necessary to interrogate heterogeneous cell populations. Herein we highlight a recently published method enabling the analysis of single-cell proteomics, which utilizes microfluidics coupled with IEF, photocapture, and immunoprobing of the protein in the same micro-gel, which can be stripped and reprobed multiple times. PMID: 30426445 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - November 16, 2018 Category: Molecular Biology Authors: Koelsch KA Tags: Methods Mol Biol Source Type: research

Artifacts and Common Errors in Protein Gel Electrophoresis.
Abstract In spite of taking precautions, some common mistakes creep into well-planned gel electrophoresis experiments. This occurs commonly in relation to calculating the cross-linking factor of a gel, polymerization temperature and time for a polyacrylamide gel, inducing aggregates in samples for electrophoresis, titrating the running buffer in electrophoresis, proper sample preparation, amount of protein to be loaded on a gel, sample buffer-to-protein ratios, incompletely removing phosphate-buffered saline from cells prior to cell lysis, and over-focusing of IPG strip in two-dimensional gel electrophoresis. In a...
Source: Mol Biol Cell - November 16, 2018 Category: Molecular Biology Authors: Kurien BT, Scofield RH Tags: Methods Mol Biol Source Type: research

Mass Spectrometry-Based Microbial Metabolomics: Techniques, Analysis, and Applications.
Abstract The demand for understanding the roles genes play in biological systems has steered the biosciences into the direction the metabolome, as it closely reflects the metabolic activities within a cell. The importance of the metabolome is further highlighted by its ability to influence the genome, transcriptome, and proteome. Consequently, metabolomic information is being used to understand microbial metabolic networks. At the forefront of this work is mass spectrometry, the most popular metabolomics measurement technique. Mass spectrometry-based metabolomic analyses have made significant contributions to micr...
Source: Mol Biol Cell - November 15, 2018 Category: Molecular Biology Authors: Baidoo EEK, Teixeira Benites V Tags: Methods Mol Biol Source Type: research

Fatty Acid Metabolome Extraction from Mycobacterial Cells for GC-MS Metabolomics Analysis.
Abstract Metabolomics is becoming an increasingly popular research tool for identifying new biomarkers, which can, among other applications, be applied to elucidate various microbial growth and virulence mechanisms. Since the lipid composition of numerous microorganisms are unique and characteristic of the particular species, and in many instances also associated with several of their growth and virulence features, we developed a method for extracting the total free fatty acid metabolome from mycobacterial cells, in order to better characterize these using a gas chromatography-mass spectrometry (GC-MS) metabolomic...
Source: Mol Biol Cell - November 15, 2018 Category: Molecular Biology Authors: du Preez I, Beukes D, Loots DT Tags: Methods Mol Biol Source Type: research

Total Metabolome Extraction from Mycobacterial Cells for GC-MS Metabolomics Analysis.
Abstract Over the past 10 years, the number of metabolomics based publications in the available scientific literature has exponentially grown, a large portion of which describing new biomarkers better elucidating microbial disease mechanisms and improved diagnostics and treatment thereof. Here, we describe a metabolomics method for extracting the total metabolome (all compounds present in the microbial cell irrespective of the compound class), for analysis in a single analytical run using only one analytical instrument. This method includes disruption of robust microbial cell walls, and the precipitation of p...
Source: Mol Biol Cell - November 15, 2018 Category: Molecular Biology Authors: Beukes D, du Preez I, Loots DT Tags: Methods Mol Biol Source Type: research

Exploiting High-Resolution Mass Spectrometry for Targeted Metabolite Quantification and 13C-Labeling Metabolism Analysis.
Abstract Quantification of targeted metabolites, especially trace metabolites and structural isomers, in complex biological materials is an ongoing challenge for metabolomics. In this chapter, we summarize high-resolution mass spectrometry-based approaches mainly used for targeted metabolite and metabolomics analysis, and then introduce an MS1/MS2-combined PRM workflow for quantification of central carbon metabolism intermediates, amino acids, and shikimate pathway-related metabolites. Major steps in the workflow, including cell culture, metabolite extraction, LC-MS analysis and data processing, are described. Fur...
Source: Mol Biol Cell - November 15, 2018 Category: Molecular Biology Authors: Li Z, Li Y, Tang YJ, Shui W Tags: Methods Mol Biol Source Type: research

Genome-Scale 13C Fluxomics Modeling for Metabolic Engineering of Saccharomyces cerevisiae.
ín H Abstract Synthetic biology is a rapidly developing field that pursues the application of engineering principles and development approaches to biological engineering. Synthetic biology is poised to change the way biology is practiced, and has important practical applications: for example, building genetically engineered organisms to produce biofuels, medicines, and other chemicals. Traditionally, synthetic biology has focused on manipulating a few genes (e.g., in a single pathway or genetic circuit), but its combination with systems biology holds the promise of creating new cellular architectures and co...
Source: Mol Biol Cell - November 15, 2018 Category: Molecular Biology Authors: Ando D, García Martín H Tags: Methods Mol Biol Source Type: research

Repurposing Macromolecule Delivery Tools for Plant Genetic Modification in the Era of Precision Genome Engineering.
This article reviews the major progress in the development of tools for large biomolecule delivery into plant cells in the new era of precision genome engineering. PMID: 30415325 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - November 13, 2018 Category: Molecular Biology Authors: Que Q, Chilton MM, Elumalai S, Zhong H, Dong S, Shi L Tags: Methods Mol Biol Source Type: research

Mesophyll Protoplasts and PEG-Mediated Transfections: Transient Assays and Generation of Stable Transgenic Canola Plants.
Abstract Plant protoplasts are derived by controlled enzymatic digestion that removes the plant cell wall without damaging the cell membrane. Protoplasts represent a true single-cell system and are useful for various biochemical and physiological studies. Protoplasts from several agriculturally important crop species can be regenerated into a fertile whole plant, extending the utility of protoplasts from transient expression assays to the generation of stable transformation events. Here we describe procedures for transient and stable transformation of leaf mesophyll protoplasts obtained from axenic shoot cultures ...
Source: Mol Biol Cell - November 13, 2018 Category: Molecular Biology Authors: Sahab S, Hayden MJ, Mason J, Spangenberg G Tags: Methods Mol Biol Source Type: research

Genetic Modification of Grapevine Embryogenic Cultures.
Abstract Precision breeding is an approach to grapevine genetic improvement that transfers only specific traits among sexually compatible species via the relatively stable mitotic cell division pathway in order to avoid the significant disruption imposed upon conventional breeding by meiosis. Factors enabling precision breeding include the availability of the Vitis genome sequence combined with highly optimized gene insertion and plant regeneration protocols. A protocol for the production of grapevine embryogenic cultures and their genetic transformation is described. Embryogenic cultures are produced from either ...
Source: Mol Biol Cell - November 13, 2018 Category: Molecular Biology Authors: Dhekney SA, Sessions SK, Brungart-Rosenberg M, Claflin C, Li ZT, Gray DJ Tags: Methods Mol Biol Source Type: research

CRISPR/Cas9 for Mutagenesis in Rice.
Abstract CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) provides a workhorse for genome editing biotechnology. CRISPR/Cas9 tailored for enabling genome editing has been extensively interrogated and widely utilized for precise genomic alterations in eukaryotic organisms including in plant species. The technology holds the great promise to better understand gene functions, elucidate networks, and improve the performance of crop plants such as increasing grain yields, improving nutritional content, and better combating the biotic and abiotic stresses. Various method...
Source: Mol Biol Cell - November 13, 2018 Category: Molecular Biology Authors: Char SN, Li R, Yang B Tags: Methods Mol Biol Source Type: research

Genome Size Estimation and Quantitative Cytogenetics in Insects.
Abstract With care, it is possible using flow cytometry to create a precise and accurate estimate of the genome size of an insect that is useful for genomics, genetics, molecular/cell biology, or systematics. Genome size estimation is a useful first step in a complete genome sequencing project. The number of sequencing reads required to produce a given level of coverage depends directly upon the 1C amount of DNA per cell, while an even more critical need is an accurate 1C genome size estimate to compare against the final assembly. Here we present a detailed protocol to estimate genome size using flow cytometry. Pu...
Source: Mol Biol Cell - November 12, 2018 Category: Molecular Biology Authors: Johnston JS, Bernardini A, Hjelmen CE Tags: Methods Mol Biol Source Type: research

Using Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) to Identify Functional Regulatory DNA in Insect Genomes.
Abstract Differential regulation of gene expression determines cell-type-specific function, making identification of the cis-regulatory elements that control gene expression a central goal of developmental biology. In addition, changes in the sequence of cis-regulatory elements are thought to drive changes in gene expression patterns between species, making comparisons of cis-regulatory element usage important for evolutionary biology as well. Due to the number of extant species and the incredible morphological diversity that they exhibit, insects are favorite model organisms for both developmental and evolutionar...
Source: Mol Biol Cell - November 12, 2018 Category: Molecular Biology Authors: McKay DJ Tags: Methods Mol Biol Source Type: research

Common Cell Lines Used to Study Bone Morphogenetic Proteins (BMPs).
Abstract Many research methods exist to elucidate the functions of BMPs during osteogenesis. This chapter briefly reviews common immortalized mesenchymal cell types used to measure the efficacy of osteogenic factors like BMP-2. Detailed information regarding media and culture conditions are provided. Parameters relevant to experimental reproducibility and cell line authentication are discussed. PMID: 30414121 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - November 12, 2018 Category: Molecular Biology Authors: Cottrell JA, Burgess D, Michaels S, Rogers MB Tags: Methods Mol Biol Source Type: research

In Vitro and In Vivo Osteogenic Differentiation of Human Adipose-Derived Stromal Cells.
Abstract Adipose-derived stromal cells (ASCs) are a promising population of cells that may be useful for the regeneration of human tissue defects. ASCs are capable of forming bone tissue in vitro and in vivo. Further work is required to determine the optimal conditions that will allow human ASCs to regenerate tissue in clinically significant tissue defects. Here we present three experimental protocols that are indispensable for the study of ASC osteogenic activity. PMID: 30414122 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - November 12, 2018 Category: Molecular Biology Authors: Marshall CD, Brett EA, Moore AL, Wan DC, Longaker MT Tags: Methods Mol Biol Source Type: research

Cell-Based Gene Therapy System for Delivering BMPs.
Abstract The use of an adenoviral vector to transduce cells allows for certain secreted proteins or growth factors to be generated in vivo in eukaryotic cells with accurate posttranslational processing. The use of transduced cells eliminates viral toxicity, allows for targeted expression of the secreted factor at a specific site, and ensures that the therapy will be turned off when the cells are cleared by the organism. Here we describe the delivery system which utilizes cells transduced with a non-replicating adenovirus containing bone morphogenetic protein 2 (BMP-2) in the E1 region of the cassette. With this me...
Source: Mol Biol Cell - November 12, 2018 Category: Molecular Biology Authors: Dickerson A, Davis EL, Sonnet C, Davis AR, Olmsted-Davis EA Tags: Methods Mol Biol Source Type: research

Generation of Endogenous BMP Transcriptional Reporter Cells Through CRISPR/Cas9 Genome Editing.
Abstract Transcriptional reporter systems allow researchers to investigate the function and regulation of transcription factors. Conventional systems employ artificial cDNA overexpression vectors containing either a promoter fragment or specific nucleotide sequence repeats upstream of firefly luciferase or fluorescent reporters, such as green fluorescence protein (GFP) cDNA. These systems suffer mainly from the lack of chromatin context. Here, we describe the rapid generation of endogenous transcriptional reporter cells for the bone morphogenetic protein (BMP) pathway using CRISPR/Cas9 genome editing. In principle...
Source: Mol Biol Cell - November 12, 2018 Category: Molecular Biology Authors: Hutchinson LD, Bozatzi P, Macartney T, Sapkota GP Tags: Methods Mol Biol Source Type: research

High-Throughput, Biosensor-Based Approach to Examine Bone Morphogenetic Protein (BMP)-Receptor Interactions.
Abstract Binding of a BMP to its cognate cell surface receptors is the initiating step in the BMP signaling cascade. Thus, knowing which BMP-receptor complexes form is vital for understanding the physiological activities of a particular BMP. Here, we describe a surface plasmon resonance (SPR)-based, high-throughput approach that allows fast identification and evaluation of BMP-receptor complexes. Briefly, the extracellular, BMP-binding domains of receptors are produced as human IgG1-Fc-fusion proteins. The Fc moiety enables simple capture of the Fc-receptor-fusion protein on the sensor chip, supports a highly repr...
Source: Mol Biol Cell - November 12, 2018 Category: Molecular Biology Authors: Aykul S, Martinez-Hackert E Tags: Methods Mol Biol Source Type: research