Development of multiplex PCR systems for expression profiling of human cardiomyocytes induced to proliferate by lentivirus transduction of upcyte genes

George, Sandra; Rödiger, Stefan; Schröder, Christian; Knaut, Michael; Küpper, Jan-Heiner

doi:10.3233/jcb-15025

Cited by 4 publications

(3 citation statements)

References 52 publications

(55 reference statements)

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“…Next, the reverse transcriptase reaction was performed as in the Section “Sequencing, Sequence Alignment, and Comparison for FimH Genes of Salmonella .” PCR was performed using standard conditions and primers. RPLP0 was used as a reference gene ( George et al, 2016 ). Primer sequences used in this section for cloning and RT PCR are shown in Supplementary Table 2 .…”

Section: Methodsmentioning

confidence: 99%

Adhesion of Salmonella to Pancreatic Secretory Granule Membrane Major Glycoprotein GP2 of Human and Porcine Origin Depends on FimH Sequence Variation

et al. 2018

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Bacterial host tropism is a primary determinant of the range of host organisms they can infect. Salmonella serotypes are differentiated into host-restricted and host-adapted specialists, and host-unrestricted generalists. In order to elucidate the underlying molecular mechanisms of host specificity in Salmonella infection, we investigated the role of the intestinal host cell receptor zymogen granule membrane glycoprotein 2 (GP2), which is recognized by FimH adhesin of type 1 fimbriae found in Enterobacteriaceae. We compared four human and two porcine GP2 isoforms. Isoforms were expressed in Sf9 cells as well as in one human (HEp-2) and one porcine (IPEC-J2) cell line. FimH genes of 128 Salmonella isolates were sequenced and the 10 identified FimH variants were compared regarding adhesion (static adhesion assay) and infection (cell line assay) using an isogenic model. We expressed and characterized two functional porcine GP2 isoforms differing in their amino acid sequence to human isoforms by approximately 25%. By comparing all isoforms in the static adhesion assay, FimH variants were assigned to high, low or no-binding phenotypes. This FimH variant-dependent binding was neither specific for one GP2 isoform nor for GP2 in general. However, cell line infection assays revealed fundamental differences: using HEp-2 cells, infection was also FimH variant-specific but mainly independent of human GP2. In contrast, this FimH variant dependency was not obvious using IPEC-J2 cells. Here, we propose an alternative GP2 adhesion/infection mechanism whereby porcine GP2 is not a receptor that determined host-specificity of Salmonella. Salmonella specialists as well as generalists demonstrated similar binding to GP2. Future studies should focus on spatial distribution of GP2 isoforms in the human and porcine intestine, especially comparing health and disease.

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Section: Methodsmentioning

confidence: 99%

Adhesion of Salmonella to Pancreatic Secretory Granule Membrane Major Glycoprotein GP2 of Human and Porcine Origin Depends on FimH Sequence Variation

et al. 2018

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“…Finally, the qPCR was performed with a CFX96 touch real-time PCR detection system (Bio-Rad, Hercules, USA) and the following conditions: 95°C for 3 min; and then 40 cycles of 95°C for 30 s, 57°C for 30 s, and 72°C for 1 min. Additionally, RPLP0 was used as a reference gene for the quantification of GP2 mRNA ( 72 ). The primer sequences are shown in Table 3 .…”

Section: Methodsmentioning

confidence: 99%

Adhesion of Enteropathogenic, Enterotoxigenic, and Commensal Escherichia coli to the Major Zymogen Granule Membrane Glycoprotein 2

Bartlitz

Kolenda

Chilimoniuk

et al. 2022

Appl Environ Microbiol

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Pathogenic bacteria, such as enteropathogenic (EPEC) and enterotoxigenic Escherichia coli (ETEC), cause diarrhea in mammals. In particular, E. coli colonize and infect the gastrointestinal tract via type 1 fimbriae (T1F). Here the major zymogen granule membrane glycoprotein 2 (GP2) acts as host cell receptor. GP2 is also secreted by the pancreas and various mucous glands, interacting with luminal type 1 fimbriae-positive E. coli . It is unknown whether GP2 isoforms demonstrate specific E. coli pathotype binding. In this study, we investigated interactions of human, porcine and bovine EPEC, ETEC as well as commensal E. coli isolates with human, porcine and bovine GP2. We first defined pathotype- and host-associated FimH variants. Secondly, we could prove that GP2 isoforms bound to FimH variants to varying degrees. However, the GP2-FimH interactions did not seem to be influenced by the host specificity of E. coli . In contrast, soluble GP2 affected ETEC infection and phagocytosis rates of macrophages. Pre-incubation of ETEC pathotype with GP2 reduced infection of cell lines. Furthermore, pre-incubation of E. coli with GP2 improved the phagocytosis rate of macrophages. Our findings suggest that GP2 plays a role in the defense against E. coli infection and in the corresponding host immune response. IMPORTANCE Infection by pathogenic bacteria such as certain Escherichia coli pathotypes results in diarrhea in mammals. Pathogens, including zoonotic agents, can infect different hosts or show host-specificity. There are Escherichia coli strains which are frequently transmitted between humans and animals, whereas other Escherichia coli strains tend to colonize only one host. This host-specificity is still not fully understood. We show that glycoprotein 2 is a selective receptor for particular Escherichia coli strains or variants of the adhesin FimH but not a selector for a species-specific Escherichia coli group. We demonstrate that GP2 is involved in the regulation of colonization and infection and thus represents a molecule of interest for the prevention or treatment of disease.

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“…DCM has a mortality rate of 50 % within five years and is the most common cause of heart transplantation. Characteristics for DCM are the strong volume increase (dilatation) of the left and right ventricles and a homogeneous contractile dysfunction of the myocardium with the consequence of a restricted systolic function [6][7][8][9].…”

Section: Introductionmentioning

confidence: 99%

A multiplex microchamber diffusion assay for the antibody-based detection of microRNAs on randomly ordered microbeads

Geithe

Zeng

Schmidt

et al. 2021

Preprint

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Background: MicroRNAs (miRNAs) are small, conserved, noncoding RNAs regulating gene expression that functions in RNA silencing and post-transcriptional regulation of gene expression. Altered miRNA profiles have been implicated in many human diseases, and due to their circulating abilities, they have excited great interest in their use as clinical biomarkers. The development of innovative methods for miRNA detection has become of high scientific and clinical interest. Methods: We developed a diffusion-driven microbead assay and combined it with an antibody-based miRNA detection. The diffusion process was carried out in two different approaches a) co-diffusion of miRNA and antibodies (termed diffusion approach I, DAI) and b) diffusion of miRNA in an antibody-saturated environment (DAII). In both approaches, neutravidin-coated microbeads were loaded with specific biotinylated DNA capture probes, which targets either miR-21-5p, miR-30a-3p or miR-93-5p. The miRNAs were time- and dose-dependently detected in a diffusion microchamber by primary anti-DNA:RNA hybrid and fluorescence-labeled secondary antibodies using our in-house developed inverse fluorescence microscope imaging platform VideoScan. Results: Our assay offers the advantage that several target molecules can be detected simultaneously and in real-time in one reaction environment (multiplex), without any amplification steps. We recorded the diffusion process over a period of 24 h and found that the reaction was almost completed after 2 h. The specificity of the assay was 96.7 % for DAI and 92.3 % for DAII. The detection limits were in a concentration range of 0.03-0.43 nM for DAI and 0.14-1.09 nM for DAII, depending on the miRNA. Conclusion: The miRNAs are successively exposed to the capture probe-loaded randomly ordered microbeads (p value of CSR 0.23-0.96), which leads to microbeads that become saturated with the target molecules first in front rows. Non-bonded miRNAs continue to diffuse further and can therefore subsequently bind to the microbeads with free binding sites. Our detection principle differs from other microbead assays, in which all microbeads are simultaneously mixed with the sample solution, so that all target molecules bind equally distributed to the microbeads, resulting in an averaged signal intensity.

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Development of multiplex PCR systems for expression profiling of human cardiomyocytes induced to proliferate by lentivirus transduction of upcyte genes

Cited by 4 publications

References 52 publications

Adhesion of Salmonella to Pancreatic Secretory Granule Membrane Major Glycoprotein GP2 of Human and Porcine Origin Depends on FimH Sequence Variation

Adhesion of Salmonella to Pancreatic Secretory Granule Membrane Major Glycoprotein GP2 of Human and Porcine Origin Depends on FimH Sequence Variation

Adhesion of Enteropathogenic, Enterotoxigenic, and Commensal Escherichia coli to the Major Zymogen Granule Membrane Glycoprotein 2

A multiplex microchamber diffusion assay for the antibody-based detection of microRNAs on randomly ordered microbeads

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