Measurement of Inner Bark and Leaf Osmolality.
We describe how to implement this procedure for gymnosperm as well as angiosperm trees. This method uses mechanical sap extraction with a centrifuge from inner bark and leaf samples, which have gone through a deep freeze treatment and thawing. The osmotic potential of these samples is then analyzed with a freezing point or vapor pressure osmometer. The aim of these measurements is to study the spatial and temporal dynamics of phloem function. PMID: 31197792 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Paljakka T, Lintunen A, Salmon Y, Hölttä T Tags: Methods Mol Biol Source Type: research

Quantification of Symplasmic Phloem Loading Capacity with Live-Cell Microscopy.
Abstract Sugars produced by photosynthesis in leaves get transported to other organs in the phloem vascular tissue. Three general mechanisms have been proposed for the loading of sugars into the phloem. These differ in the involvement of active transport across the phloem cell's membrane and their capacity for passive intercellular transport through plasmodesmata. This capacity for diffusion from the mesophyll into the phloem cells can be quantified by live-cell microscopy. Instead of sugar molecules, the movement of fluorescent tracers of similar size can be observed. In this chapter, a simple method is desc...
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Martens HJ, Gao C, Liesche J Tags: Methods Mol Biol Source Type: research

Measurement of Subcellular Metabolite Concentrations in Relation to Phloem Loading.
Abstract The key step of carbon export from green leaves is the loading of sugars into the phloem. To fully understand and quantify this process, it is essential to know the concentration of sugars in the different compartments of the cells along the phloem loading pathway. However, determining subcellular metabolite concentrations has been technically challenging. This paper describes a technique to measure metabolite levels in the chloroplast, the cytosol, and the vacuole of mesophyll cells with high accuracy. The nonaqueous fractionation (NAF) technique is arguably the method of choice to analyze the subcellula...
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Lohaus G Tags: Methods Mol Biol Source Type: research

The AtSUC2 Promoter: A Powerful Tool to Study Phloem Physiology and Development.
Abstract The sucrose carrier AtSUC2 of Arabidopsis thaliana is localized in the phloem, where it catalyzes the uptake of sucrose from the apoplast into companion cells. Imported sucrose moves passively via plasmodesmata from the companion cells into the neighboring sieve elements that distribute this disaccharide to the different sink organs. Phloem loading of sucrose by the AtSUC2 protein is an essential process, and mutants lacking this protein stay tiny, develop no or only few flowers, and have a strongly reduced root system. The promoter of the AtSUC2 gene is active exclusively in companion cells of the p...
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Stadler R, Sauer N Tags: Methods Mol Biol Source Type: research

Methods for Assessing the Role of Phloem Transport in Plant Stress Responses.
Abstract Delivery of carbohydrates to tissues that need them under stress is important for plant defenses and survival. Yet, little is known on how phloem function is altered under stress, and how that influences plant responses to stress. This is because phloem is a challenging tissue to study. It consists of cells of various types with soft cell walls, and the cells show strong wounding reactions to protect their integrity, making both imaging and functional studies challenging. This chapter summarizes theories on how phloem transport is affected by stress and presents methods that have been used to gain the cur...
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Sevanto S Tags: Methods Mol Biol Source Type: research

Analyzing and Predicting Phloem Mobility of Macromolecules with an Online Database.
Abstract Phloem, a specialized plant tissue, serves as a superhighway for macromolecular exchanges between different organs or tissues in plants. These mobile macromolecules may function as signaling molecules to sense intrinsic developmental cues or environmental inputs. Among these mobile molecules, RNAs generally need non-cell-autonomous pathway proteins (NCAPPs) to bind to and help them move along the symplasmic passage (through plasmodesmata) into the phloem stream. Grafting experiments combined with next-generation sequencing discovered that around 11.4% of identified Arabidopsis mobile mRNAs have a tRNA-lik...
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Guan D, Xia Y, Zhang S Tags: Methods Mol Biol Source Type: research

What Can Cell Culture Systems Reveal About Sieve Element Differentiation?
Abstract Tissue culture systems can be powerful tools for studying the process of cell differentiation in detail. Although a large number of cultures for xylem differentiation have been developed and utilized, there are only few reports on culture systems for ectopic phloem differentiation. Recently, a novel tissue culture system named Vascular cell Induction culture System Using Arabidopsis Leaves (VISUAL) was established, in which both xylem and phloem cells can be rapidly and efficiently induced in the model plant Arabidopsis thaliana. This chapter discusses the principle of VISUAL and how it can be used to inv...
Source: Mol Biol Cell - June 16, 2019 Category: Molecular Biology Authors: Saito M, Kondo Y Tags: Methods Mol Biol Source Type: research

Mol Biol Cell; +177 new citations
177 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 2019/06/15PubMed 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 - June 15, 2019 Category: Molecular Biology Tags: Report Source Type: research

Transcriptome profiling revealed multiple genes and ECM-receptor interaction pathways that may be associated with breast cancer.
Conclusion: Several potential breast cancer-related genes and pathways were captured, including 7 novel upregulated genes and 76 novel downregulated genes that were not found in other studies. These genes are related to cell proliferation, movement and adhesion. They may be important for research into breast cancer mechanisms, particularly CST2 and CA4. A key signaling pathway, the ECM-receptor interaction signal pathway, was also identified as possibly involved in the development of breast cancer. PMID: 31182966 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - June 13, 2019 Category: Molecular Biology Authors: Bao Y, Wang L, Shi L, Yun F, Liu X, Chen Y, Chen C, Ren Y, Jia Y Tags: Cell Mol Biol Lett Source Type: research

Mechanisms of plant protection against two oxalate-producing fungal pathogens by oxalotrophic strains of Stenotrophomonas spp.
in FL Abstract KEY MESSAGE: Oxalotrophic Stenotrophomonas isolated from tomato rhizosphere are able to protect plants against oxalate-producing pathogens by a combination of actions including induction of plant defence signalling callose deposition and the strengthening of plant cell walls and probably the degradation of oxalic acid. Oxalic acid plays a pivotal role in the virulence of the necrotrophic fungi Botrytis cinerea and Sclerotinia sclerotiorum. In this work, we isolated two oxalotrophic strains (OxA and OxB) belonging to the bacterial genus Stenotrophomonas from the rhizosphere of tomato plants. Both str...
Source: Mol Biol Cell - June 12, 2019 Category: Molecular Biology Authors: Marina M, Romero FM, Villarreal NM, Medina AJ, Gárriz A, Rossi FR, Martinez GA, Pieckenstain FL Tags: Plant Mol Biol Source Type: research

The adhesion modulation domain of C. elegans α-catenin regulates actin binding during morphogenesis.
The adhesion modulation domain of C. elegans α-catenin regulates actin binding during morphogenesis. Mol Biol Cell. 2019 Jun 12;:mbcE19010018 Authors: Shao X, Lucas B, Strauch J, Hardin J Abstract Maintaining tissue integrity during epidermal morphogenesis depends on α-catenin, which connects the cadherin complex to F-actin. We show that the adhesion modulation domain (AMD) of C. elegans HMP-1/α-catenin regulates its F-actin binding activity and organization of junctional proximal actin in vivo. Deleting the AMD increases F-actin binding in vitro and leads to excess actin recruitment...
Source: Mol Biol Cell - June 12, 2019 Category: Molecular Biology Authors: Shao X, Lucas B, Strauch J, Hardin J Tags: Mol Biol Cell Source Type: research

A reversible autophagy inhibitor blocks autophagosome-lysosome fusion by preventing Stx17 loading onto autophagosomes.
Abstract Autophagy is an evolutionarily conserved intracellular lysosomal degradation pathway. It is a multi-step process involving de novo formation of double membrane autophagosomes that capture cytosolic constituents (cargo) and eventually fuse with lysosomes wherein the cargo gets degraded and resulting simpler biomolecules get recycled. In addition to their autophagy function, several of the autophagy related proteins work at the interface of other vesicular trafficking pathways. Hence, development of specific autophagy modulators that do not perturb general endo-lysosomal traffic possesses unique challenges....
Source: Mol Biol Cell - June 12, 2019 Category: Molecular Biology Authors: Vats S, Manjithaya R Tags: Mol Biol Cell Source Type: research

Specialized eRpL22 paralogue-specific ribosomes regulate specific mRNA translation in spermatogenesis in Drosophila melanogaster.
This study reveals specialized roles in translation for eRpL22 and eRpL22-like ribosomes in germline differentiation. PMID: 31188709 [PubMed - as supplied by publisher] (Source: Mol Biol Cell)
Source: Mol Biol Cell - June 12, 2019 Category: Molecular Biology Authors: Mageeney CM, War VC Tags: Mol Biol Cell Source Type: research

Constricted migration modulates stem cell differentiation.
Abstract Tissue regeneration at an injured site depends on proliferation, migration, and differentiation of resident stem or progenitor cells, but solid tissues are often sufficiently dense and constricting that nuclei are stressed by migration. Here, constricted migration of myoblastic cell types and mesenchymal stem cells (MSCs) increases nuclear rupture, increases DNA damage, and modulates differentiation. Fewer myoblasts fuse into regenerating muscle in vivo after constricted migration in vitro, and myo-differentiation in vitro is likewise suppressed. Myosin-II inhibition rescues rupture and DNA damage, implic...
Source: Mol Biol Cell - June 12, 2019 Category: Molecular Biology Authors: Smith LR, Irianto J, Xia Y, Pfeifer CR, Discher DE Tags: Mol Biol Cell Source Type: research

Identification of a Mitofusin specificity region that confers unique activities to Mfn1 and Mfn2.
Abstract Mitochondrial structure can be maintained at steady state or modified in response to changes in cellular physiology. This is achieved by the coordinated regulation of dynamic properties including mitochondrial fusion, division and transport. Disease states, including neurodegeneration, are associated with defects in these processes. In vertebrates, two Mitofusin paralogs, Mfn1 and Mfn2, are required for efficient mitochondrial fusion. The Mitofusins share a high degree of homology and have very similar domain architecture, including an amino terminal GTPase domain and two extended helical bundles that are...
Source: Mol Biol Cell - June 12, 2019 Category: Molecular Biology Authors: Sloat SR, Whitley BN, Engelhart EA, Hoppins S Tags: Mol Biol Cell Source Type: research

The Drosophila Afadin and ZO-1 homologs Canoe and Polychaetoid act in parallel to maintain epithelial integrity when challenged by adherens junction remodeling.
Abstract During morphogenesis cells must change shape and move without disrupting tissue integrity. This requires cell-cell junctions to allow dynamic remodeling while resisting force generated by the actomyosin cytoskeleton. Multiple proteins play roles in junctional-cytoskeletal linkage, but the mechanisms by which they act remain unclear. Drosophila Canoe maintains adherens junction-cytoskeletal linkage during gastrulation. Canoe's mammalian homolog Afadin plays similar roles in cultured cells, working in parallel with ZO-1 proteins, particularly at multicellular junctions. We took these insights back into the ...
Source: Mol Biol Cell - June 12, 2019 Category: Molecular Biology Authors: Manning LA, Perez-Vale KZ, Schaefer KN, Sewell MT, Peifer M Tags: Mol Biol Cell Source Type: research

Augmentation of CFTR function in human bronchial epithelial cells via SLC6A14-dependent amino acid uptake: Implications for treatment of Cystic Fibrosis.
CONCLUSION: SLC6A14-mediated L-arginine transport augments residual F508del-CFTR channel function via a non-canonical, NO pathway. This effect is enhanced with increasing pharmacological rescue of F508del-CFTR to the membrane. The current study demonstrates how endogenous pathways can be utilized for the development of companion therapy in CF. PMID: 31189070 [PubMed - as supplied by publisher] (Source: Mol Biol Cell)
Source: Mol Biol Cell - June 12, 2019 Category: Molecular Biology Authors: Ahmadi S, Wu YS, Li M, Ip W, Lloyd-Kuzik A, Di Paola M, Du K, Xia S, Lew A, Bozoky Z, Forman-Kay J, Bear CE, Gonska T Tags: Am J Respir Cell Mol Biol Source Type: research

Rapid evolution of gained essential developmental functions of a young gene via interactions with other essential genes.
Abstract New genes are of recent origin and only present in a subset of species in a phylogeny. Accumulated evidence suggests that new genes, like old genes that are conserved across species, can also take on important functions and be essential for the survival and reproductive success of organisms. While there are detailed analyses of the mechanisms underlying new genes' gaining fertility functions, how new genes rapidly become essential for viability remains unclear. We focused on a young retro-duplicated gene (CG7804, which we named Cocoon) in Drosophila that originated between 4-10 million years ago. We found...
Source: Mol Biol Cell - June 11, 2019 Category: Molecular Biology Authors: Lee YCG, Ventura IM, Rice GR, Chen DY, Colmenares SU, Long M Tags: Mol Biol Evol Source Type: research

Frontiers of Pluripotency.
Abstract Humans develop from a unique group of pluripotent cells in early embryos that can produce all cells of the human body. While pluripotency is only transiently manifest in the embryo, scientists have identified conditions that sustain pluripotency indefinitely in the laboratory. Pluripotency is not a monolithic entity, however, but rather comprises a spectrum of different cellular states. Questions regarding the scientific value of examining the continuum of pluripotent stem (PS) cell states have gained increased significance in light of attempts to generate interspecies chimeras between humans and animals....
Source: Mol Biol Cell - June 10, 2019 Category: Molecular Biology Authors: De Los Angeles A Tags: Methods Mol Biol Source Type: research

Highly Efficient Derivation of Pluripotent Stem Cells from Mouse Preimplantation and Postimplantation Embryos in Serum-Free Conditions.
We describe generation of mouse naïve PS cells using LIF and inhibitors of MEK and GSK3 kinases and of mouse primed PS cells using FGF2 and IWR1 compound which induces the stabilization of Axin proteins. PMID: 31175643 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - June 10, 2019 Category: Molecular Biology Authors: De Los Angeles A, Okamura D, Wu J Tags: Methods Mol Biol Source Type: research

Derivation of Hematopoietic Stem and Progenitor Cells from Human Pluripotent Stem Cells.
Abstract Hematopoietic stem cell (HSC) transplantation is a curative treatment for hematologic malignancies and innate immunodeficiency, but its applications are limited to matched donors, and the availability of umbilical cord blood of immune types. Therefore, derivation of HSCs from patient-specific human induced pluripotent stem cells (hiPSCs) is a holy grail in regenerative medicine. However, derivation of HSCs from iPSCs has proven elusive. Here, the authors developed a method to derive hematopoietic stem and progenitor cells (HSPCs) by combining two established methods. The first method mimics embryonic deve...
Source: Mol Biol Cell - June 10, 2019 Category: Molecular Biology Authors: Sugimura R Tags: Methods Mol Biol Source Type: research

Cancer Stem Cells: Concepts, Challenges, and Opportunities for Cancer Therapy.
Abstract Cancer stem cells (CSCs) are a subpopulation of cancer cells with self-renewal capacity, that fuel tumor growth and contribute to the heterogeneous nature of tumors. First identified in hematological malignancies, CSC populations have to date been proposed in solid tumors in various organs. In vitro and in vivo assays, mouse genetic models, and more recently single-cell sequencing technologies and other '-omics' methodologies have not only facilitated the identification of novel CSC populations but also revealed and clarified novel properties of CSCs. Increasingly, both cell-autonomous and CSC niche ...
Source: Mol Biol Cell - June 10, 2019 Category: Molecular Biology Authors: Lee MY, Giraddi RR, Tam WL Tags: Methods Mol Biol Source Type: research

Informed Consent Issues for Cell Donors.
Abstract Stem cell-based chimera research depends on the free and voluntary provision of human biomaterials necessary for the derivation of pluripotent stem cell lines. Informed consent requirements for the procurement of human embryos, gametes, and somatic cells must take into account unique features of biomedical research involving the use of immortal cell lines that carry their donors' genetic information. The extent and basis for donors' rights, including the right to withdraw from research, are explored here in detail. PMID: 31175646 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - June 10, 2019 Category: Molecular Biology Authors: Hyun I Tags: Methods Mol Biol Source Type: research

Chick Models and Human-Chick Organizer Grafts.
Abstract The combination of affordability, large size, and ease of access at almost every stage of development renders the chick an excellent model organism for studying vertebrate development. Not only is it a great system in and of itself, but these qualities make it a great host for interspecies chimera experiments. In this chapter we highlight some notable examples of mammalian-chick chimeras, and show how one can for instance use the chick to push mammalian stem cell experiments further to learn about the behavior and capabilities of these cells in vivo. In particular, here we present the methodology necessar...
Source: Mol Biol Cell - June 10, 2019 Category: Molecular Biology Authors: Martyn I, Kanno TY, Brivanlou AH Tags: Methods Mol Biol Source Type: research

The Engraftment of Lentiviral Vector-Transduced Human CD34+ Cells into Humanized Mice.
Abstract Humanized mouse models have been developed to study human hematopoiesis and therapeutic application of hematopoietic stem cell transplantation. To evaluate the safety and efficacy of lentiviral vectors for gene therapy, human CD34+ cells have been transduced with lentiviral vectors and transplanted into the humanized mice. Recipient mice are monitored over time and sacrificed for bone marrow analyses with regard to human cell engraftment, lineage distribution, and vector transduction. This chapter details the procedure for lentiviral transduction and transplantation of human hematopoietic stem/progenitor ...
Source: Mol Biol Cell - June 10, 2019 Category: Molecular Biology Authors: Kim YS, Wielgosz M, Ryu B Tags: Methods Mol Biol Source Type: research

Pig Chimeric Model with Human Pluripotent Stem Cells.
Abstract Interspecies chimera formation provides a unique platform for studying donor cell developmental potential, modeling disease in vivo, as well as in vivo production of tissues and organs. The derivation of human pluripotent stem cells (hPSC) from either human embryos or somatic cell reprogramming facilitates our understanding of human development, as well as accelerates our exploration of regenerative medicine for human health. Due to similar organ size, close anatomy, and physiology between pig and human, human-Pig interspecies chimeric model in which pig serves as the host species may open new avenues for...
Source: Mol Biol Cell - June 10, 2019 Category: Molecular Biology Authors: Zhong C, Wu J, Izpisua Belmonte JC Tags: Methods Mol Biol Source Type: research

Embryonic Chimeras with Human Pluripotent Stem Cells.
We describe how to generate interspecies primate-mouse chimeras at the blastocyst and postimplantation embryo stages. Systematic evaluation of the chimeric competency of human and primate PS cells will aid in efforts to overcome species barriers and achieve higher grade chimerism in postimplantation conceptuses that could enable organ-specific enrichment of human xenogeneic PS cell derivatives in large animals such as pigs and sheep. PMID: 31175650 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - June 10, 2019 Category: Molecular Biology Authors: De Los Angeles A, Sakurai M, Wu J Tags: Methods Mol Biol Source Type: research

Neural Stem Cell Transplantation into a Mouse Model of Stroke.
Abstract Stroke is the fifth leading cause of death among Americans each year. Current standard-of-care treatment for stroke deploys intravenous tissue-type plasminogen activator (tPA), mechanical thrombolysis, or delivery of fibrinolytics. Although these therapies have reduced stroke-induced damage, therapeutic options still remain limited. Transplantation of patient-specific neural stem (NS) cells represents a promising strategy for the treatment of stroke. Basic science research has shown that transplanted NS cells can differentiate in the brain of rodent models of stroke and promote behavioral recovery. Clinic...
Source: Mol Biol Cell - June 10, 2019 Category: Molecular Biology Authors: De Los Angeles A Tags: Methods Mol Biol Source Type: research

Ethical Standards for Chimera Research Oversight.
Abstract Ethical standards for stem cell-based chimera research should build on current ethical standards for all animal research, adding stem cell-specific guidance only when necessary to address potential negative effects on animal welfare resulting from heavy biological modification. PMID: 31175652 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - June 10, 2019 Category: Molecular Biology Authors: Hyun I Tags: Methods Mol Biol Source Type: research

Isolation, Cryopreservation, and Transplantation of Spermatogonial Stem Cells.
Abstract Spermatogonial stem cell (SSC) culture and transplantation pave the way for clinical restoration of fertility in male prepubertal cancer survivors. In this chapter we detail the steps for isolating and freezing testicular tissue along with protocols for the subsequent recovery from cryopreservation and transplantation of cells into a recipient testis. Transplantation of cultured or thawed SSCs provides not only a functional assay for identification of stem cells, a critical tool for the study of the germline stem cell niche in model organisms, but also a framework for reconstitution of spermatogenesis in ...
Source: Mol Biol Cell - June 10, 2019 Category: Molecular Biology Authors: Sinha N, Whelan EC, Brinster RL Tags: Methods Mol Biol Source Type: research

Human-Monkey Chimeras for Modeling Human Disease: Opportunities and Challenges.
Abstract The search for a better animal model to simulate human disease has been a "holy grail" of biomedical research for decades. Recent identification of different types of pluripotent stem cells (PS cells) and advances in chimera research might soon permit the generation of interspecies chimeras from closely related species, such as those between humans and other primates. Here, we suggest that the creation of human-primate chimeras-specifically, the transfer of human stem cells into (non-ape) primate hosts-could surpass the limitations of current monkey models of neurological and psychiatric disease...
Source: Mol Biol Cell - June 10, 2019 Category: Molecular Biology Authors: De Los Angeles A, Hyun I, Latham SR, Elsworth JD, Redmond DE Tags: Methods Mol Biol Source Type: research

Mouse Models of Yersiniosis.
Abstract Yersiniosis is common foodborne gastrointestinal disease caused by the enteric pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis. The mouse model of oral infection serves as a useful tool to study enteropathogenic Yersinia infection in mammals. The following protocol describes two distinct oral infection methods: the commonly used oral gavage method in which the bacterial inoculum is instilled directly into the mouse stomach using a feeding needle, and an alternative method in which mice are fed bread soaked with Yersinia culture. PMID: 31177430 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - June 10, 2019 Category: Molecular Biology Authors: Hooker-Romero D, Schwiesow L, Wei Y, Auerbuch V Tags: Methods Mol Biol Source Type: research

Intravital Confocal Microscopy of Dermal Innate Immune Responses to Flea-Transmitted Yersinia pestis.
Abstract The technique known as intravital microscopy (IVM), when used in conjunction with transgenic mice expressing fluorescent proteins in various cell populations, is a powerful tool with the potential to provide new insights into host-pathogen interactions in infectious disease pathogenesis in vivo. Yersinia pestis, the causative agent of plague, is typically deposited in a host's skin during feeding of an infected flea. IVM has been used to characterize the innate immune response to Y. pestis in the skin and identify differences between the responses to needle-inoculated and flea-transmitted bacteria that wo...
Source: Mol Biol Cell - June 10, 2019 Category: Molecular Biology Authors: Shannon JG, Hinnebusch BJ Tags: Methods Mol Biol Source Type: research

Detection of Cells Translocated with Yersinia Yops in Infected Tissues Using β-Lactamase Fusions.
Detection of Cells Translocated with Yersinia Yops in Infected Tissues Using β-Lactamase Fusions. Methods Mol Biol. 2019;2010:117-139 Authors: Nguyen GT, McCabe AL, Fasciano AC, Mecsas J Abstract Development of the TEM-CCF2/4-AM FRET-based system has enabled investigators to track translocation of effector proteins into mammalian cells during infection. This allows for separation of translocated and non-translocated cell populations for further study. Yersinia strains expressing translational Yop-TEM fusions, containing the secretion and translocation signals of a Yop with the TEM-1 portion of &b...
Source: Mol Biol Cell - June 10, 2019 Category: Molecular Biology Authors: Nguyen GT, McCabe AL, Fasciano AC, Mecsas J Tags: Methods Mol Biol Source Type: research

Drosophila as a Model for Understanding the Insect Host of Yersinia pestis.
Abstract With the limited availability of genomic sequence information and no established methods for genetic knockdowns or the creation of transgenic fleas and flea cell lines, we have adopted Drosophila melanogaster as a model for the study of the insect life cycle of Yersinia pestis. Infection of Drosophila larvae can be used to model early colonization of fleas, while the established embryonic cell lines can be used to model insect-pathogen interactions that underlie the unique capacity of Y. pestis to colonize the gut of its flea host. In this chapter, we present the methods we developed for infection of Dros...
Source: Mol Biol Cell - June 10, 2019 Category: Molecular Biology Authors: Ludlow AB, Pauling CD, Marketon MM, Anderson DM Tags: Methods Mol Biol Source Type: research

Intracellular Assays to Monitor Survival and Growth of Yersinia pestis Within Macrophages.
Abstract Yersinia pestis is able to survive and replicate within macrophages, while also being able to live in the extracellular milieu of the host. Assays that facilitate better understanding of how Y. pestis survives intracellularly and subverts normal host antimicrobial defenses require the ability to monitor intracellular Y. pestis survival and replication. In this chapter three different assays for monitoring intracellular survival and replication will be described, along with the formulas and methods to quantify and present the acquired data. These assays are fundamental to answering a multitude of questions...
Source: Mol Biol Cell - June 10, 2019 Category: Molecular Biology Authors: Pulsifer AR, VanCleave TT, Lawrenz MB Tags: Methods Mol Biol Source Type: research

Human Neutrophil Isolation and Degranulation Responses to Yersinia pestis Infection.
We describe a cell culture model for the infection of human neutrophils with Y. pestis. Neutrophils are isolated from human peripheral blood at high purity and subsequently infected with Y. pestis. We specifically focus on the application of this in vitro infection assay to the analysis of neutrophil degranulation responses. PMID: 31177440 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - June 10, 2019 Category: Molecular Biology Authors: Eichelberger KR, Goldman WE Tags: Methods Mol Biol Source Type: research

Measurement of Yersinia Translocon Pore Formation in Erythrocytes.
Abstract Many Gram-negative pathogens produce a type III secretion system capable of intoxicating eukaryotic cells with immune-modulating effector proteins. Fundamental to this injection process is the prior secretion of two translocator proteins destined for injectisome translocon pore assembly within the host cell plasma membrane. It is through this pore that effectors are believed to travel to gain access to the host cell interior. Yersinia species especially pathogenic to humans and animals assemble this translocon pore utilizing two hydrophobic translocator proteins-YopB and YopD. Although a full molecular un...
Source: Mol Biol Cell - June 10, 2019 Category: Molecular Biology Authors: Costa TRD, Francis MK, Farag SI, Edgren T, Francis MS Tags: Methods Mol Biol Source Type: research

Analysis of Inflammasome Activation in Response to Yersinia Infection by Fluorescence Microscopy Detection of Active Caspase-1 Puncta.
Abstract The type of cell death triggered by a particular environmental stimulus influences the outcome of infection or inflammatory disease processes. The ability to identify the cell death pathway that is activated in response to infection is essential for understanding the pathogenesis and host response to infection. Activation of the cysteine protease caspase-1 in various inflammasome complexes indicates that cells are undergoing pyroptosis, a regulated, proinflammatory cell death. Inflammasome assembly and caspase activation can be measured by various methods ranging from detection of inflammasome-dependent c...
Source: Mol Biol Cell - June 10, 2019 Category: Molecular Biology Authors: Zwack EE, Brodsky IE Tags: Methods Mol Biol Source Type: research

Methods for Detection of Pyrin Inflammasome Assembly in Macrophages Infected with Yersinia spp.
Abstract The Yersinia effector proteins YopE and YopT are important bacterial virulence factors that are secreted into infected host cells and can inactivate Rho GTPases, like RhoA, Rac1, and Cdc42. In order to compensate for the consequences of this effect, the host cell can sense RhoA modifications and trigger a proinflammatory reaction to control the infection. This host response, known as pyrin inflammasome assembly, is normally prevented by another important effector, YopM, allowing Yersinia to counteract this conserved innate immune response. Once assembled, the pyrin inflammasome can activate caspase-1 via ...
Source: Mol Biol Cell - June 10, 2019 Category: Molecular Biology Authors: Medici NP, Bliska JB Tags: Methods Mol Biol Source Type: research

T Cell: Primary Culture from Peripheral Blood.
Abstract Peripheral blood mononuclear cells (PBMCs) can be used to assess cell-mediated immunity in general or via antigen-specific stimulation to detect previous exposure to a variety of antigens/allergens and to monitor the response to immunotherapies. Peripheral blood is the most common source of mononuclear cells for in vitro cultures, although mononuclear cells can be obtained from other sources involved in the allergic reaction. PBMCs from individuals previously exposed to an antigen proliferate in vitro when stimulated with the specific antigen. Proliferation is measured by the incorporation of (3H)-thymidi...
Source: Mol Biol Cell - June 10, 2019 Category: Molecular Biology Authors: Raulf M Tags: Methods Mol Biol Source Type: research

Measurement of Allergen-Specific Inhibitory Antibody Activity.
Abstract Specific allergen immunotherapy (AIT) is an effective treatment for IgE-mediated allergic diseases and involves T- and B-cell-mediated events. IgE receptors on the surface of antigen-presenting cells facilitate the presentation of allergens in the presence of specific IgE antibody resulting in T-cell activation. Interference with these IgE-dependent mechanisms by "blocking" IgG antibodies suppresses pro-inflammatory Th2 cell responses and manifests as a reduction in allergic responses in vivo.In vitro assays used to measure the inhibition of binding of allergen-IgE complexes have previously util...
Source: Mol Biol Cell - June 10, 2019 Category: Molecular Biology Authors: Shamji MH, Francis JN Tags: Methods Mol Biol Source Type: research

Monocyte-Derived Dendritic Cells as Antigen-Presenting Cells in T-Cell Proliferation and Cytokine Production.
Abstract Dendritic cells (DC) are the most potent antigen-presenting cells that link innate with adaptive immunity. They circulate the body and sample the microenvironments for maintaining homeostasis and for mounting T-cell responses against invading pathogens, foreign antigens, and aberrant self-proteins. In humans, DC derived from blood monocytes (MDC) by cytokine treatment provide the most abundant and versatile source for studying DC and T-cell biology, and for use as adjuvants in cancer therapy. In asthma patients, T-cell functions are studied by using autologous MDC as accessory cells for allergen presentat...
Source: Mol Biol Cell - June 10, 2019 Category: Molecular Biology Authors: Sung SJ Tags: Methods Mol Biol Source Type: research

The Use of Dual-Cell-Tracker Dye Staining for the Identification and Characterization of Peanut-Specific T-Cell Subsets.
Abstract Cell-tracker fluorescent dye labeling is widely used for investigating antigen-specific immune responses in vitro and in vivo. Here we describe a development of this technique-the use of dual-cell-tracker dye staining for the identification and characterization of the responses of different T-cell subsets to peanut proteins in vitro. PMID: 31177497 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - June 10, 2019 Category: Molecular Biology Authors: Dunsterville C, Stephens AC, Lack G, Turcanu V Tags: Methods Mol Biol Source Type: research

The Use of pMHCII Tetramer Technology for the Identification and Characterization of CD4+ T Cells.
Abstract Understanding the mechanism underlying allergic disease is dependent upon definition of the heterogeneity and complexity of the cellular immune response toward allergens both in the context of disease and clinical intervention. Among all components of the immune system, CD4+ T cells play a key role in the orchestration of immune response toward allergen and have become a dynamic area of research. Because of their unique ability to identify antigen-specific CD4+ T cells irrespective of functional outputs, fluorescently labeled peptide-MHC class II (pMHCII) tetramers in combination with multiparameter flow ...
Source: Mol Biol Cell - June 10, 2019 Category: Molecular Biology Authors: Wambre E Tags: Methods Mol Biol Source Type: research

Isolation and Characterization of Lymphocytes from Human Mucosal Biopsies.
Abstract The important role of the local mucosal environment in both the initiation and progression of allergic disease is well established. Analysis of tissue-resident lymphocyte subsets by flow cytometry requires isolation of viable cells from mucosal samples.Here we describe an advanced method to dissociate lymphocytes from human mucosal (e.g., nasal, bronchial) biopsies. Single-cell suspensions are obtained through a combination of gentle mechanical disruption and incubation of tissue with proteolytic enzymes. This method fully utilizes limited clinical samples and is amenable to a variety of downstream applic...
Source: Mol Biol Cell - June 10, 2019 Category: Molecular Biology Authors: Upton NEG, Hayday TS, James LK Tags: Methods Mol Biol Source Type: research

Isolation of Competitive Phage Display-Modified Bacteriophage T4 with Affinity Chromatography.
Abstract Phage recovery from various solutions, including physiological samples, as well as phage purification from crude lysates often requires a specific isolation method. Here, we demonstrate that T4-like phages can be efficiently isolated by affinity chromatography. This approach employs specific affinity tags (GST (glutathione S-transferase) or His-tag) that allow for the isolation of the phage. These affinity tags are exposed on the phage head using phage display. By combining competitive phage display and affinity chromatography, wild-type phages can be specifically recovered from mixtures with other phage/...
Source: Mol Biol Cell - June 9, 2019 Category: Molecular Biology Authors: Dąbrowska K Tags: Methods Mol Biol Source Type: research

Mol Biol Cell; +174 new citations
174 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 2019/06/07PubMed 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 - June 8, 2019 Category: Molecular Biology Tags: Report Source Type: research

Mol Biol Cell; +136 new citations
136 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 2019/06/06PubMed 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 - June 6, 2019 Category: Molecular Biology Tags: Report Source Type: research

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114 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 2019/06/05PubMed 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 - June 5, 2019 Category: Molecular Biology Tags: Report Source Type: research