Droplet Digital PCR for Measuring Absolute Copies of Gene Transcripts in Human Islet-Derived Progenitor Cells.
Abstract Transcript analysis is a routinely used method to assess the expression profile of progenitor cells at different stages starting from their isolation to differentiation into specific lineages. It is a powerful way to understand similarities and differences between different cell types as well to estimate successful differentiation process. Transcript measurement is most commonly done using polymerase chain reaction (PCR) but other methods such as in situ hybridization, RNA sequencing are available. The quantitative PCR using TaqMan chemistry is a highly sensitive and reproducible method that measures gene...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Maynard CL, Wong WKM, Hardikar AA, Joglekar MV Tags: Methods Mol Biol Source Type: research
Isolation and In Vitro Culture of Human Gut Progenitor Cells.
Abstract The gastrointestinal epithelium is a highly regenerative organ, where each cell is replaced in a cycle of 4-6 days, depending on the mammalian species. This highly proliferative state is driven by gastrointestinal stem and progenitor cells, located at the base of crypts. These cells give rise to at least six types of differentiated epithelial cells, each with a distinct function in maintaining homeostasis between the intestinal interface and the luminal environment. The isolation and culture of these cells from mammalian gastrointestinal tissue is a novel technique, which allows for the generation an...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Bruce J, Kaiko GE, Keely S Tags: Methods Mol Biol Source Type: research
Isolation and Characterization of Colony-Forming Progenitor Cells from Adult Pancreas.
Abstract Obtaining, growing, and analysis of pancreatic progenitor cells. Adult stem and progenitor cells have been successfully used for cell-based therapies such as transplantation of hematopoietic stem cells for various diseases. Whether stem and progenitor cells in the adult pancreas can be identified and used for replacement therapy has been a highly controversial topic. To address this controversy, our laboratory has developed in vitro colony assays to detect and characterize individual pancreatic stem and progenitor-like cells. We found that a subpopulation of ductal cells in the adult murine pancreas has t...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Quijano JC, Tremblay JR, Rawson J, Ku HT Tags: Methods Mol Biol Source Type: research
A Novel Gene Delivery Approach Using Metal Organic Frameworks in Human Islet-Derived Progenitor Cells.
Abstract The ability to regenerate insulin-producing β cells is the ultimate goal for treatment of type 1 diabetes. Several sources of stem cells have been investigated by studying their differential potential to form insulin-producing β cells that can be used for replacement therapy. Progenitor cells derived from human islets that are lineage committed have been shown to be better alternatives with regard to their differentiation capabilities for the generation of insulin-producing β-like cells. Controlling the differentiation of progenitor cells is a vital approach in exploiting cellular expansion...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Poddar A, Joglekar MV, Hardikar AA, Shukla R Tags: Methods Mol Biol Source Type: research
Encapsulation and Transplantation of Pancreatic Progenitor Cells.
Abstract Type 1 diabetes, characterized by autoimmune destruction of pancreatic beta cells, affects 41 million people worldwide. Beta cell replacement therapies have immense potential as a treatment option because pancreatic progenitors derived from human pluripotent stem cells can provide a near limitless supply of transplantable tissue. The key limitation of this approach is the need for lifelong use of immunosuppressive drugs that have undesirable side effects. Microencapsulation is an option for providing protection for transplanted cells from mechanical stress and immune attack. Traditionally, pluripotent cel...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Carroll L, Mridha AR, Tuch BE Tags: Methods Mol Biol Source Type: research
Differentiation of Urothelium from Mouse Embryonic Stem Cells in Chemically Defined Conditions.
Abstract The urothelium of the bladder and urethra are derived from the definitive endoderm during development. Cellular signaling molecules important to the developmental specification of the urothelium are also implicated in the dysregulation of the tissue repair mechanism characteristic of bladder disease. Hence, a complete understanding of the regulation of urothelium development is central to understanding the processes of bladder disease, and in development of simple chemically defined methods for use in regenerative medicine. Key to this is a suitable in vitro model that readily allows for the prosecution o...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Boumelhem BB, Fraser ST, Assinder SJ Tags: Methods Mol Biol Source Type: research
Identification and Analysis of Mouse Erythroid Progenitor Cells.
Abstract The most common cell type in the human body, the red blood cell or erythrocyte, has a life span of approximately 3 months. To compensate for this massive cellular requirement and short life span, the major blood producing tissues contain vast numbers of erythroid progenitor cells. Erythroid progenitors differentiate progressively from hematopoietic stem cells to committed erythroid progenitors to reticulocytes lacking a nucleus and finally to functionally mature erythrocytes in the circulation. Different erythroid progenitor activity, representative of distinct stages of erythropoiesis, can be observ...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Colonne CK, Yeo JH, McKenzie CV, Fraser ST Tags: Methods Mol Biol Source Type: research
Single-Cell Assays Using Hematopoietic Stem and Progenitor Cells.
Abstract Hematopoietic stem cells (HSCs) undergo division, making two daughter cells with unique fate decision choices, that is, whether to self-renew to maintain stemness or differentiate to committed progenitors. Since HSCs are heterogeneous in nature understanding this phenomenon at the single cell level is important. In vitro single-cell assays like the paired-daughter cell and myeloid multilineage differentiation are useful to understand this unique stem cell process. Both assays are performed using cytokine combination which allows four-lineage myeloid differentiation-neutrophil, erythroid, macrophage/monocy...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Hinge AS, Filippi MD Tags: Methods Mol Biol Source Type: research
Mitochondrial Assays Using Cardiac Stem Cells.
Abstract Ischemic heart disease is the leading cause of death worldwide. Stem cell therapy to repair and regenerate the infarcted myocardium is a promising approach to address this unmet medical need. However, the poor survival of transplanted cells in the hostile ischemic myocardium has been a major hurdle in achieving an effective cell therapy against myocardial infarction. As such, novel strategies to promote the survival of transplanted cells are highly sought after. Mitochondria are intimately involved in cell survival and have been the main organelles being targeted for cytoprotection. Mitochondrial morpholo...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Rosdah AA, Delbridge LMD, Lim SY Tags: Methods Mol Biol Source Type: research
High-Efficiency Lentiviral Gene Modification of Primary Murine Bone-Marrow Mesenchymal Stem Cells.
Abstract Lentiviral vectors are the method of choice for stable gene modification of a variety of cell types. However, the efficiency with which they transduce target cells varies significantly, in particular their typically poor capacity to transduce primary stem cells. Here we describe the isolation and enrichment of murine bone-marrow mesenchymal stem cells (MSCs) via fluorescence-activated cell sorting (FACS); the cloning, production, and concentration of high-titer second generation lentiviral vectors via combined tangential flow filtration (TFF) and ultracentrifugation; and the subsequent high-efficiency gen...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Gerace D, Ren B, Martiniello-Wilks R, Simpson AM Tags: Methods Mol Biol Source Type: research
Bone Marrow-Derived Progenitor Cells Mediate Immune Cell Regulation.
Abstract Human bone marrow (BM) derived mesenchymal stem cells (MSC) have high capacity to propagate ex vivo with superior reparative, immunosuppressive, and anti-inflammatory properties. Here we describe standardized protocols and culture conditions that enable the isolation, expansion and maintenance of a highly purified and homogenous population of human MSC. These third party-derived off-the-shelf MSC from healthy human bone marrow donors can potently inhibit mitogenically or allogeneically activated human T cells in proliferation assays. The standard operating procedures described in this chapter can be appli...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Sivanathan KN, Coates PT Tags: Methods Mol Biol Source Type: research
Flow Cytometry and Cell Sorting Using Hematopoietic Progenitor Cells.
Abstract Flow cytometry is a widely used laser-based technology for rapid analysis of the expression of cell surface antigens and intracellular molecules in various cell types including hematopoietic stem/progenitor cells (HSPCs). Multiparametric analysis of individual cells within a short time frame makes this tool attractive and indispensable in the field of stem cell research. This is accomplished by harnessing the specific light scattering ability of the cell type, which determines its size and internal complexity. In addition, use of fluorescently conjugated antibodies allows the detection of a specific surfa...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Vadakke-Madathil S, Limaye LS, Kale VP, Chaudhry HW Tags: Methods Mol Biol Source Type: research
Isolation of Epidermal Progenitor Cells from Rat Tympanic Membrane.
Abstract The eardrum is an important structural component for hearing, but it is delicate and subject to traumatic injury and disease. Healing mechanisms are activated after injury but sometimes healing fails and chronic perforations develop, requiring surgical intervention. To model the wound healing responses we established a simple method for isolating keratinocytes and progenitors from individual eardrums. The central region of the eardrum contains epidermal proliferative centers that produce keratinocytes which migrate to cover the eardrum surface. We dissected out the central region and explanted it to the p...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Liew LJ, Wang AY, Dilley RJ Tags: Methods Mol Biol Source Type: research
Measurement of Store-Operated Calcium Entry in Human Neural Cells: From Precursors to Differentiated Neurons.
Abstract Calcium imaging in an ex-vivo setup is used to understand the calcium status of isolated cells or tissue. In this chapter we explain the use of the ratiometric chemical indicator Fura-2 which can be loaded into isolated cells in the form of lipophilic acetomethyl (AM) esters. Fura-2 is a combination of calcium chelator and fluorophore, and can be used with dual wavelength excitation (340/380 nm) for quantitative calcium concentrations. The cells can then be viewed using a fluorescence microscope and captured by a CCD camera. We specifically discuss the technique involved in understanding the end...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Gopurappilly R, Deb BK, Chakraborty P, Hasan G Tags: Methods Mol Biol Source Type: research
Embryoid Body Differentiation of Mouse Embryonic Stem Cells into Neurectoderm and Neural Progenitors.
Abstract Mouse embryonic stem cells (mESCs) are pluripotent cells capable of differentiating in vitro to form the ~200 types of cells of the developing embryo and adult, including cells of the nervous system. This makes mESCs a useful tool for studying the molecular mechanisms of mammalian embryonic development. Many protocols involving the use of growth factors and small molecules to differentiate mESCs into neural progenitors and neurons currently exist. However, there is a paucity of protocols available that recapitulate the developmental process. Our laboratory has developed a protocol to recapitulate mammalia...
Source: Mol Biol Cell - July 6, 2019 Category: Molecular Biology Authors: Shparberg RA, Glover HJ, Morris MB Tags: Methods Mol Biol Source Type: research
G1 Premature Chromosome Condensation (PCC) Assay.
Abstract Premature chromosome condensation (PCC) is a sensitive and unique way to detect interphase chromosome damage and its recovery in mammalian cells irradiated with ionizing radiation. In this chapter, we describe G1 PCC assay with which one can measure immediate chromosome breaks in G1 type chromosomes and their repair/rejoining. In order to induce G1 PCC, one needs to fuse mitotic cells with G1 cells to be tested. There are two methods to fuse cells; one is to use Sendai virus or its equivalent, and another method needs polyethylene glycol (PEG) as a fusing agent. The date obtained with PCC assay can bridge...
Source: Mol Biol Cell - July 5, 2019 Category: Molecular Biology Authors: Okayasu R, Liu C Tags: Methods Mol Biol Source Type: research
G2 Chromosomal Radiosensitivity Assay for Testing Individual Radiation Sensitivity.
Abstract The G2 chromosomal radiosensitivity assay or, simply G2 assay, measures the number of chromatid type aberrations induced by radiation in G2 phase. Typically, asynchronous growing cells are irradiated with less than 1 Gy and allowed 0.5-1 h for cells in mitosis, at the time of irradiation, to transit into G1. Later, the G2 phase cells, at the time irradiation, are blocked by colcemid for 1-4 h at metaphase. Cells are collected by standard hypotonic solution and Carnoy solution fixation or directly fixed onto the culture vessels. The G2 assay can detect severe radiosensitivity in ATM homozygo...
Source: Mol Biol Cell - July 5, 2019 Category: Molecular Biology Authors: Haskins JS, Kato TA Tags: Methods Mol Biol Source Type: research
G2 Premature Chromosome Condensation/Chromosome Aberration Assay: Drug-Induced Premature Chromosome Condensation (PCC) Protocols and Cytogenetic Approaches in Mitotic Chromosome and Interphase Chromatin for Radiation Biology.
Abstract Chromosome analysis is a fundamental technique for a wide range of cytogenetic studies. Chromosome aberrations are easily introduced by many kinds of clastogenic agents such as ionizing irradiation, UV, or alkylating agents, and damaged chromosomes may be prone to cancer. Chromosomes are conventionally prepared from mitotic cells arrested by the colcemid block method. However, obtaining of mitotic chromosomes is sometimes hampered under several circumstances, for example after high-dose (over several Gys of γ-rays) ionizing irradiation exposure accident. As a result, cytogenetic analysis will be oft...
Source: Mol Biol Cell - July 5, 2019 Category: Molecular Biology Authors: Gotoh E Tags: Methods Mol Biol Source Type: research
Sister Chromatid Exchange as a Genotoxic Stress Marker.
Abstract Sister chromatid exchange (SCE) is the phenomenon of partial DNA exchange during DNA replication. SCE detection has been developed through eliciting DNA's semiconservative replicative nature. Thymidine analogues such as 5'-bromodeoxyuridine (BrdU) and ethynyldeoxyuridine (EdU) are incorporated into the newly synthesized DNA for two cell cycles. The addition of Colcemid to the culture blocks and synchronizes cells at mitosis, and conventional cytogenetic preparations are made. Differential staining methods with Hoechst dye and Giemsa (Fluorescence Plus Giemsa staining), antibody detection against...
Source: Mol Biol Cell - July 5, 2019 Category: Molecular Biology Authors: Sunada S, Haskins JS, Kato TA Tags: Methods Mol Biol Source Type: research
DNA Damage Focus Formation Assay.
Abstract Advanced techniques allow investigating cellular DNA damage measurements. Ionizing radiation produces multiple DNA damages. Among them, DNA double strand breaks are most toxic to cells. DSBs can form mutations, chromosome aberrations, and cell killing. Although DSBs in cells can be detected directly by neutral elution, pulse field gel electrophoresis, and premature chromosome condensation, recent technologies like cellular immunocytochemistry-based fluorescence detection allow us to visualize the DSBs in cells. Here, we describe gamma-H2AX and Rad51 focus formation assay, which play an important...
Source: Mol Biol Cell - July 5, 2019 Category: Molecular Biology Authors: Fujii Y Tags: Methods Mol Biol Source Type: research
Nuclear Foci Assays in Live Cells.
Abstract DNA double strand breaks (DSBs) are a serious threat to genome stability and cell viability. Accurate detection of DSBs is critical for the basic understanding of cellular response to ionizing radiation. Recruitment and retention of DNA repair and response proteins at DSBs can be conveniently visualized by fluorescence imaging (often called ionizing radiation-induced foci) both in live and fixed cells. In this chapter, we describe a live cell imaging methodology that directly monitors induction and repair of single DSB, recruitment kinetics of DSB repair/sensor factors to DSB sites, and dynamic interactio...
Source: Mol Biol Cell - July 5, 2019 Category: Molecular Biology Authors: Mori E, Asaithamby A Tags: Methods Mol Biol Source Type: research
Semiautomated Small-Scale Purification Method for High-Throughput Expression Analysis of Recombinant Proteins.
chroeder K, Tam C, Ti S, Win YN, Bowman K Abstract The expression analysis of recombinant proteins is a challenging step in any high-throughput protein production pipeline. Often multiple expression systems and a variety of expression construct designs are considered for the production of a protein of interest. There is a strong need to triage constructs rapidly and systematically. This chapter describes a semiautomated method for the simultaneous purification and characterization of proteins expressed from multiple samples of expression cultures from the E. coli, baculovirus expression vector system, and mammalia...
Source: Mol Biol Cell - July 5, 2019 Category: Molecular Biology Authors: Kraft E, Franke Y, Heeringa K, Shriver S, Zilberleyb I, Kugel C, Dela Vega T, Wong A, Brillantes B, Ciferri C, Dutina G, Lee G, Lehoux I, Li ZR, Lior-Hoffmann L, Hwang J, Lonergan C, Martin L, Mortara K, Nguyen L, Payandeh J, Perez A, Sampang J, Singh L, Tags: Methods Mol Biol Source Type: research
High-Throughput Protein Production in Yeast.
S, Vega MC Abstract Yeasts are versatile single-celled fungi that grow to high cell densities on inexpensive media. With well-studied genetics and metabolism and a wealth of knowledge available about their propagation and growth in academic as well as industrial settings, yeasts have long been used for recombinant protein production of isolated proteins and multisubunit complexes. They can be easily adapted to high-throughput protein expression pipelines. Importantly, the outcome from small-scale expression evaluations in high-throughput mode is scalable to laboratory and industrial scales using well-established ...
Source: Mol Biol Cell - July 5, 2019 Category: Molecular Biology Authors: Fernández FJ, Gómez S, Vega MC Tags: Methods Mol Biol Source Type: research
A High-Throughput System for Transient and Stable Protein Production in Mammalian Cells.
Abstract Recombinant protein expression and purification is an essential component of biomedical research and drug discovery. Advances in automation and laboratory robotics have enabled the development of highly parallel and rapid processes for cell culture and protein expression, purification, and analysis. Human embryonic kidney (HEK) cells and Chinese hamster ovary (CHO) cells have emerged as the standard host cell workhorses for producing recombinant secreted mammalian proteins by using both transient and stable production strategies. In this chapter we describe a fully automated custom platform, Protein Expre...
Source: Mol Biol Cell - July 5, 2019 Category: Molecular Biology Authors: Rue SM, Anderson PW, Gaylord MR, Miller JJ, Glaser SM, Lesley SA Tags: Methods Mol Biol Source Type: research
High-Throughput Production of Influenza Virus-Like Particle (VLP) Array by Using VLP-factory ™, a MultiBac Baculoviral Genome Customized for Enveloped VLP Expression.
High-Throughput Production of Influenza Virus-Like Particle (VLP) Array by Using VLP-factory™, a MultiBac Baculoviral Genome Customized for Enveloped VLP Expression. Methods Mol Biol. 2019;2025:213-226 Authors: Sari-Ak D, Bahrami S, Laska MJ, Drncova P, Fitzgerald DJ, Schaffitzel C, Garzoni F, Berger I Abstract Baculovirus-based expression of proteins in insect cell cultures has emerged as a powerful technology to produce complex protein biologics for many applications ranging from multiprotein complex structural biology to manufacturing of therapeutic proteins including virus-like particles (VL...
Source: Mol Biol Cell - July 5, 2019 Category: Molecular Biology Authors: Sari-Ak D, Bahrami S, Laska MJ, Drncova P, Fitzgerald DJ, Schaffitzel C, Garzoni F, Berger I Tags: Methods Mol Biol Source Type: research
High-Throughput E. coli Cell-Free Expression: From PCR Product Design to Functional Validation of GPCR.
ine S Abstract This chapter outlines a protocol to express GPCRs libraries for screening of targets. High-throughput screening of GPCR expression raised a big interest in the development of proteomic drug candidates, protein engineering, and microarrays. However, GPCRs represent a large family of difficult-to-express proteins which can be successfully produced by cell-free systems in the presence of liposomes. The open and flexible nature of this in vitro expression system allows the manipulation of transcription and translation as well as the modulation of the cell-free reaction environment by the addition of any...
Source: Mol Biol Cell - July 5, 2019 Category: Molecular Biology Authors: Cortès S, Hibti FE, Chiraz F, Ezzine S Tags: Methods Mol Biol Source Type: research
High-Throughput Isolation of Soluble Protein Domains Using a Bipartite Split-GFP Complementation System.
Abstract The identification of soluble, folded domains of proteins is a recurring task in modern molecular biology. We detail a protocol for identifying compact soluble protein domains using a self-assembling two-part split-GFP comprised of a detector fragment (GFP β-strands 1 through 10, or GFP1-10) and a tagging fragment (GFP β-strand 11, or GFP11). The assay is performed in E. coli cells and in cell extracts. A selection step insures the protein fragments are in frame and contain no stop codons, while an inverse PCR is used to enrich protein fragment libraries containing a specific target sequence. ...
Source: Mol Biol Cell - July 5, 2019 Category: Molecular Biology Authors: Massemin A, Cabantous S, Waldo GS, Pedelacq JD Tags: Methods Mol Biol Source Type: research
High-Throughput Analytical Light Scattering for Protein Quality Control and Characterization.
We present a review of high-throughput techniques for the characterization and quality control of proteins in the course of purification, evaluation, and formulation, based on static and dynamic light scattering. Multi-angle static light scattering (MALS) in combination with rapid, low-volume UHPLC size exclusion chromatography is effective in characterizing key biophysical properties, while dynamic light scattering (DLS) in high-throughput microwell-plate format provides large quantities of data in a short time to screen many conditions, excipients, cell lines, or candidate biotherapeutics. PMID: 31267461 [PubMed - i...
Source: Mol Biol Cell - July 5, 2019 Category: Molecular Biology Authors: Some D, Razinkov V Tags: Methods Mol Biol Source Type: research
Adaption of the Leishmania Cell-Free Expression System to High-Throughput Analysis of Protein Interactions.
Abstract In this chapter, we present methods for adapting the eukaryotic cell-free expression system based on Leishmania tarentolae to high-throughput analysis of protein interactions. Specifically, we present a lysate optimization technique that minimizes the amount of unwanted premature termination products while balancing protein expression level and protein aggregation. Finally, we present methods for adapting the Leishmania cell-free system to the AlphaLISA-based protein interaction assay. PMID: 31267464 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 5, 2019 Category: Molecular Biology Authors: Johnston WA, Moradi SV, Alexandrov K Tags: Methods Mol Biol Source Type: research
High-Throughput Protein-Protein Interaction Assays Using Tripartite Split-GFP Complementation.
Abstract Most cellular processes are driven by complex protein-protein interaction networks. Identifying key players and characterizing their interactions at the cellular and molecular level is of key importance to understand biochemical mechanisms that control cellular responses. Here, we detail a protocol for monitoring protein-protein interactions in E. coli cells or in cell extracts using a tripartite split-GFP system comprised of a protein interaction detector fragment (GFP ß-strands 1 through 9 or GFP1-9) and small tagging fragments of GFP ß-strands 10 (GFP10) and 11 (GFP11). Interaction of bait ...
Source: Mol Biol Cell - July 5, 2019 Category: Molecular Biology Authors: Pedelacq JD, Waldo GS, Cabantous S Tags: Methods Mol Biol Source Type: research
An Update on Artemisinin Resistance.
ard D Abstract While the precise mode of action of artemisinin (ART) derivatives remains obscure, it is nonetheless commonly accepted that ART generates reactive oxygen intermediates that contribute to cell death. Also, numerous studies confirm that point mutations in the propeller domain of K13 protein play a key role in resistance to ART derivatives. Because of its homology with the KEAP1 protein, it is thought that this protein may have a role in the polyubiquitination of proteins and that its alteration may cause resistance of young parasite stages to the drug. In this chapter, we present our current knowledge...
Source: Mol Biol Cell - July 5, 2019 Category: Molecular Biology Authors: Ariey F, Ménard D Tags: Methods Mol Biol Source Type: research
Mol Biol Cell; +117 new citations
117 new pubmed citations were retrieved for your search. Click on the search hyperlink below to display the complete search results: Mol Biol Cell These pubmed results were generated on 2019/07/03PubMed comprises more than millions of citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites. (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 3, 2019 Category: Molecular Biology Tags: Report Source Type: research
Enzymatic Release of Glycoprotein N-Glycans and Fluorescent Labeling.
We present here two robust protocols to achieve N-glycan release. The first one uses trypsin to disrupt protein structure whereas the other involves the use of detergents prior to PNGase F digestion. Thereafter, N-glycans are isolated from peptides using reverse-phase cartridges and are desalted with carbograph cartridges before finally being derivatized with the fluorescent label 2AB. PMID: 31256372 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 2, 2019 Category: Molecular Biology Authors: Grunow D, Blanchard V Tags: Methods Mol Biol Source Type: research
Glycosylation of Type I Collagen.
Abstract Fibrillar type I collagen is the most abundant structural protein in most tissues and organs. One of the unique and functionally important characteristics of collagen is sequential posttranslational modifications of lysine (Lys) residues. In the endoplasmic reticulum, hydroxylation of specific Lys occurs producing 5-hydroxylysine (Hyl). Then, to the 5-hydroxyl group of Hyl, a single galactose unit can be attached to form galactosyl-Hyl (Gal-Hyl) and further glucose can be added to Gal-Hyl to form glucosylgalactosyl-Hyl (GlcGal-Hyl). These are the only two O-linked glycosides found in mature type I collage...
Source: Mol Biol Cell - July 2, 2019 Category: Molecular Biology Authors: Yamauchi M, Sricholpech M, Terajima M, Tomer KB, Perdivara I Tags: Methods Mol Biol Source Type: research
Metabolic Labeling and Structural Analysis of Glycosylphosphatidylinositols from Parasitic Protozoa.
Abstract Glycosylphosphatidylinositol (GPI) is a complex glycolipid structure that acts as a membrane anchor for many cell-surface proteins of eukaryotes. GPI-anchored proteins are particularly abundant in protozoa and represent the major carbohydrate modification of many cell-surface parasite proteins. A minimal GPI-anchor precursor consists of core glycan (ethanolamine-PO4-Manα1-2Manα1-6Manα1-4GlcNH2) linked to the 6-position of the D-myo-inositol ring of phosphatidylinositol. Although the GPI core glycan is conserved in all organisms, many differences in additional modifications to GPI structu...
Source: Mol Biol Cell - July 2, 2019 Category: Molecular Biology Authors: Azzouz N, Gerold P, Schwarz RT Tags: Methods Mol Biol Source Type: research
Analysis of Sumoylation.
Abstract Protein regulation by reversible attachment of SUMO (small ubiquitin-related modifier) plays an important role in several cellular processes such as transcriptional regulation, nucleo-cytoplasmic transport, cell-cycle progression, meiosis, and DNA repair. However, most sumoylated proteins are of marginal abundance at steady state levels, which is due to strict regulation and/or rapid turnover of modification and de-modification. Consequently, analysis of protein sumoylation in vivo is very challenging. Nonetheless, a novel method was established that allows detection of sumoylated proteins at endogenous l...
Source: Mol Biol Cell - July 2, 2019 Category: Molecular Biology Authors: Breucker J, Pichler A Tags: Methods Mol Biol Source Type: research
S-Acylation of Proteins.
Abstract Palmitoylation or S-acylation is the posttranslational attachment of fatty acids to cysteine residues and is common among integral and peripheral membrane proteins. Palmitoylated proteins have been found in every eukaryotic cell type examined (yeast, insect, and vertebrate cells), as well as in viruses grown in these cells. The exact functions of protein palmitoylation are not well understood. Intrinsically hydrophilic proteins, especially signaling molecules, are anchored by long-chain fatty acids to the cytoplasmic face of the plasma membrane. Palmitoylation may also promote targeting to membrane subdom...
Source: Mol Biol Cell - July 2, 2019 Category: Molecular Biology Authors: Kordyukova L, Krabben L, Serebryakova M, Veit M Tags: Methods Mol Biol Source Type: research
Harnessing epithelial homeostatic mechanisms to fight cancer.
Abstract Cancer treatments have, in general, targeted the cancer cell itself. This approach has often been unsuccessful in the long term, especially for solid tumors. Even targeted therapies based on sequencing cancer genomes can be thwarted by genetic heterogeneity within tumors. Furthermore, genomic instability in cancer cells accelerates the generation of variants that are resistant to the treatment. Immunotherapies and anti-angiogenic treatments, which target the tumor-interacting and tumor-adjacent cells, have overcome some of these challenges, suggesting that other methods that target wild-type cells could b...
Source: Mol Biol Cell - June 29, 2019 Category: Molecular Biology Authors: Lahvic JL, Hariharan IK Tags: Mol Biol Cell Source Type: research
Rockets, gauges, and pendulums: applying engineering principles to cell biology.
Abstract From flight to radar to Velcro, biological form and function have inspired engineers for centuries. It is equally valuable to consider whether concepts in engineering might provide insights into core biological processes. To explore this idea, cell cycle checkpoints, biological clocks, and signaling pathways are viewed here from an engineering perspective. Engineering concepts covered include gauge error, the distinction between precision and accuracy, and the Taguchi method of robust design. Also discussed is the Pareto principle, which describes the observation that, in complex systems, a minority of th...
Source: Mol Biol Cell - June 29, 2019 Category: Molecular Biology Authors: Sullivan W Tags: Mol Biol Cell Source Type: research
Measurement of Phototropism of the Sporangiophore of Phycomyces blakesleeanus.
We describe the basic methods for characterizing phototropic bending and the correlated elongation and rotation responses of the sporangiophore. PMID: 30694468 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - June 22, 2019 Category: Molecular Biology Authors: Corrochano LM, Galland P Tags: Methods Mol Biol Source Type: research
Quantitative Analysis of Tip Growth, Phototropic Responses, and Other Blue Light-Dependent Photoresponses of Vaucheria.
Abstract The coenocytic tip-growing alga Vaucheria exhibits positive and negative phototropism, apical expansion, polarotropism, and branch induction from the illuminated region of the cell, all of which are caused by blue light. The bending response of Vaucheria is a blue light-mediated growth response. Differently from diffuse-growing cells or organs, the apical hemispherical dome of the Vaucheria cell is the site of not only maximum growth activity but also the site of blue light perception. Thence the phototropic response is initiated by the bulging mechanism: that is, a quick shift of the growth center to the...
Source: Mol Biol Cell - June 22, 2019 Category: Molecular Biology Authors: Kataoka H Tags: Methods Mol Biol Source Type: research
Immunolocalization of IAA Using an Anti-IAA-C-Antibody Raised Against Carboxyl-Linked IAA.
Abstract Plant hormone indole-3-acetic acid (IAA) plays a crucial role in plant physiological events such as plant development, differentiation, and environmental responses. IAA is synthesized in specific focal cells and/or tissues such as the coleoptile tip in maize and the root tip and young leaf primordia in Arabidopsis thaliana. Recent studies have shown that formation of an IAA maxima or concentration gradient, created by the changing expression and cellular localization of IAA transport proteins, crucially controls plant physiological events. For this reason, visualization of IAA molecules at the cell and ti...
Source: Mol Biol Cell - June 22, 2019 Category: Molecular Biology Authors: Nishimura T, Koshiba T Tags: Methods Mol Biol Source Type: research
The Pathometabolism of Legionella Studied by Isotopologue Profiling.
Abstract Metabolic pathways and fluxes can be analyzed under in vivo conditions by incorporation experiments using general 13C-labelled precursors. On the basis of the isotopologue compositions in amino acids or other metabolites, the incorporation rates of the supplied precursors and the pathways of their utilization can be studied in considerable detail. In this chapter, the method of isotopologue profiling is illustrated with recent work on the metabolism of intracellular living Legionella pneumophila. PMID: 30694483 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - June 22, 2019 Category: Molecular Biology Authors: Heuner K, Kunze M, Chen F, Eisenreich W Tags: Methods Mol Biol Source Type: research
Quantification of Viable but Non-Culturable Cells of Legionella pneumophila.
Abstract Legionella pneumophila, among other bacteria, may enter a viable but non-culturable state as a means for survival in stressful conditions. Bacterial cells in the viable but non-culturable state cannot grow on standard medium; however, they continue to exhibit characteristics that are associated with live cells, such as respiration, transcription, and cell wall integrity. The present paper outlines a detailed protocol for the detection of viable but non-culturable L. pneumophila cells via Syto® 9 and propidium iodide staining coupled with flow cytometry. PMID: 30694484 [PubMed - indexed for MEDLIN...
Source: Mol Biol Cell - June 22, 2019 Category: Molecular Biology Authors: Braun RS, Mendis N, Li L, Faucher SP Tags: Methods Mol Biol Source Type: research
Migration of Acanthamoeba castellanii Through Legionella Biofilms.
Abstract The amoeba-resistant bacterium Legionella pneumophila infects humans through aerosols and thereby can cause a life-threatening pneumonia termed Legionnaires' disease. In the environment L. pneumophila forms and colonizes biofilms, which usually comprise complex multispecies communities. In these biofilms L. pneumophila persists and replicates intracellularly in protozoa, such as the amoeba Acanthamoeba castellanii. The interactions between sessile L. pneumophila in biofilms and their natural protozoan hosts are not understood on a molecular level. Here, we describe a method to visualize by confocal m...
Source: Mol Biol Cell - June 22, 2019 Category: Molecular Biology Authors: Hochstrasser R, Hilbi H Tags: Methods Mol Biol Source Type: research
Sorting of Phagocytic Cells Infected with Legionella pneumophila.
Abstract The ability of Legionella pneumophila to colonize host cells and to form a replicative vacuole depends on its ability to counteract the host cell response by secreting more than 300 effectors. The host cell responds to this bacterial invasion with extensive intracellular signaling to counteract the replication of the pathogen. When studying L. pneumophila infection in vitro, only a small proportion of the cell lines or primary cells used to analyze the host response are infected; the study of such a mixed cell population leads to unprecise results. In order to study the multitude of pathogen-ind...
Source: Mol Biol Cell - June 22, 2019 Category: Molecular Biology Authors: Rolando M, Buchrieser C Tags: Methods Mol Biol Source Type: research
Single Cell Analysis of Legionella and Legionella-Infected Acanthamoeba by Agarose Embedment.
Abstract Legionella pneumophila resides in multispecies biofilms, where it infects and replicates in environmental protozoa such as Acanthamoeba castellanii. Studies on L. pneumophila physiology and host-pathogen interactions are frequently conducted using clonal bacterial populations and population level analysis, overlooking the remarkable differences in single cell behavior. The fastidious nutrient requirements of extracellular L. pneumophila and the extraordinary motility of Acanthamoeba castellanii hamper an analysis at single cell resolution. In this chapter, we describe a method to study L. pneumophila and ...
Source: Mol Biol Cell - June 22, 2019 Category: Molecular Biology Authors: Personnic N, Striednig B, Hilbi H Tags: Methods Mol Biol Source Type: research
Mitochondrial Dynamics and Activity in Legionella-Infected Cells.
Abstract The study of Legionella pneumophila interactions with host mitochondria during infection has been historically limited by the techniques available to analyze and quantify mitochondrial dynamics and activity in living cells. Recently, new, powerful techniques such as high-content microscopy or mitochondrial respiration assays (Seahorse) have been developed to quantitatively analyze mitochondrial parameters. Here we present state-of-the-art methods adapted to analyze mitochondrial dynamics and activity during Legionella infection of living human primary macrophages. PMID: 30694494 [PubMed - indexed for...
Source: Mol Biol Cell - June 22, 2019 Category: Molecular Biology Authors: Song OR, Brodin P, Buchrieser C, Escoll P Tags: Methods Mol Biol Source Type: research
Perturbation of Legionella Cell Infection by RNA Interference.
Abstract Legionella pneumophila is a facultative intracellular bacterium, which grows in amoebae as well as in macrophages and epithelial cells. Depletion of genes of interest by RNA interference (RNAi) has proven to be a robust and economic technique to study L. pneumophila-host cell interactions. Predesigned and often validated double-stranded (ds) RNA oligonucleotides that silence specific genes are commercially available. RNAi results in a reduced level of distinct proteins, which allows studying the specific role of host cell components involved in L. pneumophila infection. Here, we describe how to assess RNA...
Source: Mol Biol Cell - June 22, 2019 Category: Molecular Biology Authors: Steiner B, Swart AL, Hilbi H Tags: Methods Mol Biol Source Type: research
Methods for Noncanonical Ubiquitination and Deubiquitination Catalyzed by Legionella pneumophila Effector Proteins.
Abstract Protein ubiquitination is one of the most prevalent posttranslational modifications; it regulates a wide range of critical cellular processes in eukaryotes. This modification occurs by covalent attachment of the ubiquitin molecule to other proteins via an isopeptide bond in reactions typically catalyzed by sequential actions of three enzymes, including ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3). Ubiquitination is a reversible process catalyzed by a group of proteins known as deubiquitinase (DUB), which specifically cleaves the isopeptide bond between ubi...
Source: Mol Biol Cell - June 22, 2019 Category: Molecular Biology Authors: Qiu J, Luo ZQ Tags: Methods Mol Biol Source Type: research