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Methods in Molecular Biology

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https://www.readbyqxmd.com/read/27787775/the-three-dimensional-culture-of-epithelial-organoids-derived-from-embryonic-chicken-intestine
#1
Malgorzata Pierzchalska, Malgorzata Panek, Malgorzata Czyrnek, Maja Grabacka
The intestinal epithelium isolated from chicken embryos in last 3 days of development can be used to establish the 3D culture of intestinal organoids. When fragments of epithelial tissue released by incubation with EGTA (2.5 mM, 2 h) are embedded in Matrigel matrix on cell culture inserts the formation of empty spheres covered by epithelial cells is observed in first 24 h of culture. The growth and survival of organoids are supported by the addition of R-spondin 1, Noggin, and prostaglandin E2 to the culture medium...
October 28, 2016: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27730537/construction-of-thymus-organoids-from-decellularized-thymus-scaffolds
#2
Asako Tajima, Isha Pradhan, Xuehui Geng, Massimo Trucco, Yong Fan
One of the hallmarks of modern medicine is the development of therapeutics that can modulate immune responses, especially the adaptive arm of immunity, for disease intervention and prevention. While tremendous progress has been made in the past decades, manipulating the thymus, the primary lymphoid organ responsible for the development and education of T lymphocytes, remains a challenge. One of the major obstacles is the difficulty to reproduce its unique extracellular matrix (ECM) microenvironment that is essential for maintaining the function and survival of thymic epithelial cells (TECs), the predominant population of cells in the thymic stroma...
October 12, 2016: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27914091/toward-clinical-use-of-the-igg-specific-enzymes-ides-and-endos-against-antibody-mediated-diseases
#3
Mattias Collin, Lars Björck
The endoglycosidase EndoS and the protease IdeS from the human pathogen Streptococcus pyogenes are immunomodulating enzymes hydrolyzing human IgG. IdeS cleaves IgG in the lower hinge region, while EndoS hydrolyzes the conserved N-linked glycan in the Fc region. Both enzymes are remarkably specific for human IgG that after hydrolysis loses most of its effector functions, such as binding to leukocytes and complement activation, all contributing to bacterial evasion of adaptive immunity. However, taken out of their infectious context, we and others have shown that IdeS and EndoS can alleviate autoimmune disease in a number of animal models of antibody-mediated disorders...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27914090/measuring-antibody-orientation-at-the-bacterial-surface
#4
Oonagh Shannon, Pontus Nordenfelt
Many bacteria have the ability to interact with antibodies as a means to circumvent the immune response. This includes binding to the Fc portion of antibodies, effectively reversing the antibody orientation and thus decreasing the Fc-mediated immune signaling. Since antibody orientation at the bacterial surface has been shown to be important in human disease, it is valuable to be able to assess how antibodies are interacting with bacterial pathogens. Here, we describe a method to measure the proportion of human IgG that are bound via their Fc or Fabs to a bacterial surface...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27914089/generating-and-purifying-fab-fragments-from-human-and-mouse-igg-using-the-bacterial-enzymes-ides-speb-and-kgp
#5
Jonathan Sjögren, Linda Andersson, Malin Mejàre, Fredrik Olsson
Fab fragments are valuable research tools in various areas of science including applications in imaging, binding studies, removal of Fc-mediated effector functions, mass spectrometry, infection biology, and many others. The enzymatic tools for the generation of Fab fragments have been discovered through basic research within the field of molecular bacterial pathogenesis. Today, these enzymes are widely applied as research tools and in this chapter, we describe methodologies based on bacterial enzymes to generate Fab fragments from both human and mouse IgG...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27914088/murine-mycobacterium-marinum-infection-as-a-model-for-tuberculosis
#6
Julia Lienard, Fredric Carlsson
Mycobacteria are a major human health problem globally. Regarding tuberculosis the situation is worsened by the poor efficacy of current vaccine regimens and by emergence of drug-resistant strains (Manjelievskaia J et al, Trans R Soc Trop Med Hyg 110: 110, 2016; Pereira et al., Lancet Infect Dis 12:300-306, 2012; http://www.who.int/tb/publications/global_report/en/) undermining both disease-prevention and available treatments. Thus, increased basic understanding of mycobacterial-and particularly Mycobacterium tuberculosis-virulence strategies and pathogenesis is of great importance...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27914087/in-vitro-and-in-vivo-biofilm-formation-by-pathogenic-streptococci
#7
Yashuan Chao, Caroline Bergenfelz, Anders P Håkansson
This manuscript presents novel approaches to grow and evaluate Streptococcal biofilm formation using the human respiratory pathogen Streptococcus pneumoniae (the pneumococcus) as the main model organism on biological surfaces in vitro and in vivo. Most biofilm models are based on growth on abiotic surfaces, which is relevant for many pathogens whose growth on surfaces or medical devices is a major cause of disease transmission and infections, especially in hospital environments. However, most infections with commensal organisms require biofilm formation on biological surfaces in the host at the site of colonization or infection...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27914086/killing-bacteria-with-cytotoxic-effector-proteins-of-human-killer-immune-cells-granzymes-granulysin-and-perforin
#8
Diego López León, Isabelle Fellay, Pierre-Yves Mantel, Michael Walch
Bacterial pathogens represent a constant threat to human health that was exacerbated in recent years by a dramatic increase of strains resistant to last resort antibiotics. The immune system of higher vertebrates generally evolved several efficient innate and adaptive mechanisms to fight ubiquitous bacterial pathogens. Among those mechanisms, immune proteases were recognized to contribute essentially to antibacterial immune defense. The effector serine proteases of the adaptive immune system, the granzymes, exert potent antimicrobial activity when they are delivered into the bacterial cytosol by prokaryotic membrane disrupting proteins, such as granulysin...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27914085/determining-platelet-activation-and-aggregation-in-response-to-bacteria
#9
Oonagh Shannon
Many pathogenic bacteria have been reported to interact with human platelets to mediate platelet activation and aggregation. The importance of these interactions to the immune response or pathogenesis of bacterial infection has not been clarified. It may therefore be valuable to assess platelet responses mediated by diverse strains of bacteria. Here, I describe a method to study platelet integrin activation and granule release using flow cytometry, and a complementary method to study platelet aggregation using a dedicated platelet aggregometer...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27914084/the-zebrafish-as-a-model-for-human-bacterial-infections
#10
Melody N Neely
The development of the zebrafish (Danio rerio) infectious disease model has provided new insights and information into pathogenesis. Many of these new discoveries would not have been possible using a typical mammalian model. The advantages of using this model are many and in the last 15 years the model has been exploited for the analysis of many different pathogens. Here, we describe in detail how to perform a bacterial infection using either the adult zebrafish or zebrafish larvae using microinjection. Multiple methods of analysis are described that can be used to address specific questions pertaining to disease progression and the interactions with the immune system...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27914083/antibody-guided-molecular-imaging-of-infective-endocarditis
#11
Kenneth L Pinkston, Peng Gao, Kavindra V Singh, Ali Azhdarinia, Barbara E Murray, Eva M Sevick-Muraca, Barrett R Harvey
In this protocol, we describe the application of using a high affinity monoclonal antibody generated against the major pilin protein component of the pilin structure of Enterococcus faecalis as a PET imaging agent for enterococcal endocarditis detection. The anti-pilin -mAb 64Cu conjugate was able to specifically label enterococcal endocarditis vegetation in vivo in a rodent endocarditis model. By targeting pili, a covalently linked surface antigen extending from the bacterial surface, we provided evidence that gram-positive pilin represent a logical surface antigen to define or target an infectious agent for molecularly guided imaging...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27914082/detection-of-intracellular-proteins-by-high-resolution-immunofluorescence-microscopy-in-streptococcus-pyogenes
#12
Assaf Raz
Immunofluorescence microscopy is an invaluable tool for the study of biological processes at the cellular level. While the localization of surface-exposed antigens can easily be determined using fluorescent antibodies, localization of intracellular antigens requires permeabilization of the bacterial cell wall and membrane. Here, we describe an immunofluorescence protocol tailored specifically for Streptococcus pyogenes, applying the phage lysin PlyC for cell wall permeabilization. This protocol allows a high level of morphological preservation, suitable for high-resolution microscopy...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27914081/negative-staining-and-transmission-electron-microscopy-of-bacterial-surface-structures
#13
Matthias Mörgelin
Negative staining is an essential and versatile staining technique in transmission electron microscopy that can be employed for visualizing bacterial cell morphology, size, and surface architecture at high resolution. Bacteria are usually transferred by passive electrostatic adsorption from suspensions in physiological saline onto suitable hydrophilic support films on electron microscopic grids. There they are contrasted, or "stained," by heavy metal ions in solution such as tungsten, uranyl, molybdate, or vanadate compounds...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27914080/immobilization-techniques-of-bacteria-for-live-super-resolution-imaging-using-structured-illumination-microscopy
#14
Amy L Bottomley, Lynne Turnbull, Cynthia B Whitchurch, Elizabeth J Harry
Advancements in optical microscopy technology have allowed huge progression in the ability to understand protein structure and dynamics in live bacterial cells using fluorescence microscopy. Paramount to high-quality microscopy is good sample preparation to avoid bacterial cell movement that can result in motion blur during image acquisition. Here, we describe two techniques of sample preparation that reduce unwanted cell movement and are suitable for application to a number of bacterial species and imaging methods...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27914079/assessing-vacuolar-escape-of-listeria-monocytogenes
#15
Juan J Quereda, Martin Sachse, Damien Balestrino, Théodore Grenier, Jennifer Fredlund, Anne Danckaert, Nathalie Aulner, Spencer Shorte, Jost Enninga, Pascale Cossart, Javier Pizarro-Cerdá
Listeria monocytogenes is a bacterial pathogen which invades and multiplies within non-professional phagocytes. Signaling cascades involved in cellular entry have been extensively analyzed, but the events leading to vacuolar escape remain less clear. In this chapter, we detail a microscopy FRET-based assay which allows quantitatively measuring L. monocytogenes infection and escape from its internalization vacuole, as well as a correlative light/electron microscopy method to investigate the morphological features of the vacuolar compartments containing L...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27914078/computer-vision-based-image-analysis-of-bacteria
#16
Jonas Danielsen, Pontus Nordenfelt
Microscopy is an essential tool for studying bacteria, but is today mostly used in a qualitative or possibly semi-quantitative manner often involving time-consuming manual analysis. It also makes it difficult to assess the importance of individual bacterial phenotypes, especially when there are only subtle differences in features such as shape, size, or signal intensity, which is typically very difficult for the human eye to discern. With computer vision-based image analysis - where computer algorithms interpret image data - it is possible to achieve an objective and reproducible quantification of images in an automated fashion...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27914077/making-fluorescent-streptococci-and-enterococci-for-live-imaging
#17
Sarah Shabayek, Barbara Spellerberg
Since the discovery of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria, outstanding fluorescent labeling tools with numerous applications in vastly different areas of life sciences have been developed. To optimize GFP for diverse life science applications, a large variety of GFP derivatives with different environmental characteristics have been generated by mutagenesis. The enhanced green fluorescent protein (EGFP) is a well-known GFP derivative with highly increased fluorescence intensity compared to the GFP wild-type molecule...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27914076/reconstructing-the-ancestral-relationships-between-bacterial-pathogen-genomes
#18
Caitlin Collins, Xavier Didelot
Following recent developments in DNA sequencing technology, it is now possible to sequence hundreds of whole genomes from bacterial isolates at relatively low cost. Analyzing this growing wealth of genomic data in terms of ancestral relationships can reveal many interesting aspects of the evolution, ecology, and epidemiology of bacterial pathogens. However, reconstructing the ancestry of a sample of bacteria remains challenging, especially for the majority of species where recombination is frequent. Here, we review and describe the computational techniques currently available to infer ancestral relationships, including phylogenetic methods that either ignore or account for the effect of recombination, as well as model-based and model-free phylogeny-independent approaches...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27914075/development-of-a-single-locus-sequence-typing-slst-scheme-for-typing-bacterial-species-directly-from-complex-communities
#19
Christian F P Scholz, Anders Jensen
The protocol describes a computational method to develop a Single Locus Sequence Typing (SLST) scheme for typing bacterial species. The resulting scheme can be used to type bacterial isolates as well as bacterial species directly from complex communities using next-generation sequencing technologies.
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27914074/common-challenges-in-studying-the-structure-and-function-of-bacterial-proteins-case-studies-from-helicobacter-pylori
#20
Daniel A Bonsor, Eric J Sundberg
Employing biophysical and structural methods is a powerful way to elucidate mechanisms of molecular recognition in bacterial pathogenesis. Such studies invariably depend on the production of pure, folded and stable proteins. Many proteins that can be expressed recombinantly ultimately fail to meet one or more of these criteria. The cag proteins from Helicobacter pylori form a secretion system that delivers the oncoprotein, CagA, into human gastric epithelial cells through an interaction between CagL and host cell integrins, where it can cause gastric adenocarcinoma...
2017: Methods in Molecular Biology
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