scholarly journals Co-Cultures ofPseudomonas aeruginosaandRoseobacter denitrificansReveal Shifts in Gene Expression Levels Compared to Solo Cultures

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
Crystal A. Conway ◽  
Nwadiuto Esiobu ◽  
Jose V. Lopez
Keyword(s):  
Gene Expression ◽  
Target Gene ◽  
Beta Lactamase ◽  
Interspecies Interactions ◽  
Quantitative Real Time Pcr ◽  
Bacterial Strains ◽  
Microbial Cultures ◽  
Culturing Conditions ◽  
Gene Expression Levels

Consistent biosynthesis of desired secondary metabolites (SMs) from pure microbial cultures is often unreliable. In a proof-of-principle study to induce SM gene expression and production, we describe mixed “co-culturing” conditions and monitoring of messages via quantitative real-time PCR (qPCR). Gene expression of model bacterial strains (Pseudomonas aeruginosaPAO1 andRoseobacter denitrificansOch114) was analyzed in pure solo and mixed cocultures to infer the effects of interspecies interactions on gene expressionin vitro, TwoP. aeruginosagenes (PhzHcoding for portions of the phenazine antibiotic pathway leading to pyocyanin (PCN) and theRhdAgene for thiosulfate: cyanide sulfurtransferase (Rhodanese)) and twoR. denitrificansgenes (BetaLactfor metallo-beta-lactamase and theDMSPgene for dimethylpropiothetin dethiomethylase) were assessed for differential expression. Results showed thatR. denitrificansDMSPandBetaLactgene expression became elevated in a mixed culture. In contrast,P. aeruginosaco-cultures withR. denitrificansor a third species did not increase target gene expression above control levels. This paper provides insight for better control of target SM gene expressionin vitroand bypass complex genetic engineering manipulations.

scholarly journals SAT-726 Estrogen Receptor Alpha as a Potential Target for Bisphenol A-Mediated Epigenetic Reprogramming: An in Vitro Analysis

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Morgan Gallo ◽  
Lindsey S Treviño ◽  
Tiffany A Katz
Keyword(s):  
Gene Expression ◽  
Target Gene ◽  
Target Genes ◽  
Hepatic Cell ◽  
Time Points ◽  
Target Gene Expression ◽  
Er Alpha ◽  
Hepatic Cell Lines ◽  
Gene Expression Levels

Abstract Perinatal exposure to bisphenol A (BPA) has been shown to reprogram the hepatic epigenome of rodents and may promote the development of various metabolic diseases later in life, such as nonalcoholic fatty liver disease (NAFLD). This developmental reprogramming is characterized by the creation of “super promoters” at target genes implicated in metabolic pathways. While it is unclear how these “super promoters” are created, their creation is potentially mediated through BPA and estrogen receptor (ER) interaction. In order to test this potential mechanism, in vitro methods were used to examine ER target gene expression via RT-qPCR in 2 human hepatic cell lines transiently transfected with the ER isoform, ER alpha, prior to BPA exposure for various lengths of time. Within individual time points, there were no significant differences in target gene expression levels between cells that had been transfected with ER alpha and the vector control. Gene expression levels in the target genes were visibly increased at the 24-hour exposure mark in both transfection groups in comparison to the 0- and 6-hour time points, however only a fraction of these increases were found to be statistically significant. These gene expression patterns are not only consistent with previous studies examining target gene expression in BPA-treated hepatic cell lines, but more importantly, suggest BPA does not act via ER alpha to orchestrate the epigenetic changes seen in vitro. BPA may interact with a different ER isoform or an unknown target to create the observed “super promoters” at target genes, reinforcing the promiscuity of BPA and other xenoestrogens in facilitating epigenetic modifications, and ultimately, disease phenotypes.

scholarly journals Gene Transfer for Enhancing Plant Disease Resistance to Bacterial Pathogens

HortScience ◽  
1995 ◽  
Vol 30 (4) ◽  
pp. 788A-788
Author(s):  
Zhanyuan Zhang ◽  
D.P. Coyne ◽  
A. Mitra
Keyword(s):  
Gene Expression ◽  
Antibacterial Activity ◽  
Disease Resistance ◽  
Gene Transfer ◽  
Plant Disease Resistance ◽  
Bacterial Strains ◽  
Gene Encoding ◽  
High Antibacterial Activity ◽  
Gene Expression Levels

Gene transfer can provide plants with a novel source of disease resistance. Two different antibacterial peptides, Shiva-1 and lactoferrin, were tested in vitro for antibacterial activity. The former is from cecropin B in insects, and the latter from human or mammal fluids such as milk. Both peptides exhibited high antibacterial activity against all tested gram-negative phytopathogenic bacterial strains. Lactoferrin was more lethal than Shiva-1. A particular lactoferrin domain showed a much higher activity against bacterial strains. A gene encoding lactoferrin was then transferred to Nicotinia tabacum L. xanthi-nc to evaluate the gene expression using Agrobacterium. Stable transformation was confirmed by Southern, Northern, and Western blot analysis. Delayed wilting of the transgenic plants inoculated with Pseudomonas solanacearum was observed. A significant positive relationship between the gene expression levels and resistance was also found by either Northern or Western blotting. Biolistic transformation using a gene gun is currently underway to transfer this novel gene to common beans.

2049-P: The Chd4 Helicase of the Nucleosome Remodeling and Deacetylase Complex Modulates Pdx1 Target Gene Expression In Vitro

Diabetes ◽  
2020 ◽  
Vol 69 (Supplement 1) ◽  
pp. 2049-P
Author(s):  
REBECCA K. DAVIDSON ◽  
NOLAN CASEY ◽  
JASON SPAETH
Keyword(s):  
Gene Expression ◽  
Target Gene ◽  
Nucleosome Remodeling ◽  
Target Gene Expression

scholarly journals 1564. Activity of bacteriophage against multidrug resistant Klebsiella pneumoniae

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S782-S782
Author(s):  
Sailaja Puttagunta ◽  
Maya Kahan-Haanum ◽  
Sharon Kredo-Russo ◽  
Eyal Weinstock ◽  
Efrat Khabra ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Unmet Need ◽  
Multidrug Resistant ◽  
Beta Lactamase ◽  
Bacterial Strains ◽  
Antibiotic Resistant ◽  
Extended Spectrum Beta Lactamase ◽  
Novel Approach ◽  
Infection Types

Abstract Background The prevalence of extended-spectrum beta-lactamase (ESBL) producing and carbapenem resistant (CR) Klebsiella pneumoniae (KP) has significantly risen in all geographic regions. Infections due to these bacteria are associated with high mortality across different infection types. Even with newer options, there remains an unmet need for safe and effective therapeutic options to treat infections caused by ESBL and CR KP. Phage therapy offers a novel approach with an unprecedented and orthogonal mechanism of action for treatment of diseases caused by pathogenic bacterial strains that are insufficiently addressed by available antibiotics. Phage-based therapies confer a high strain-level specificity and have a strong intrinsic safety profile. Here we describe the identification of novel phages that can effectively target antibiotic resistant KP strains. Host range of the 21 phages on 33 strain KP panel via solid culture infectivity assays. Red marks resistance to infection while sensitivity to phage is marked in green Methods KP clinical strains were isolated from human stool specimens preserved in glycerol. Selective culturing was carried, followed by testing of individual colonies for motility, indole and urease production, sequenced and analyzed by Kleborate tool to determine antibiotic resistant genes. Natural phages were isolated from plaques that developed on susceptible bacterial targets, sequenced and characterized. Results Antibiotic-resistant KP strains encoding beta lactamase genes or a carbapenemase (n=33) were isolated from healthy individuals (n=3), and patients with inflammatory bowel disease (n=26) or primary sclerosing cholangitis (n=3). Isolates sequencing revealed bla CTX-M15 and/or bla SHV encoding strains and carbapenamase KPC-2. A panel of 21 phages targeting the beta-lactamase- and carbapenemase-producing KP strains were identified. Phage sequencing revealed that all phages belong to the Caudovirales order and include 6 Siphoviridae, 14 Myoviridae, and 1 Podoviridae. In vitro lytic activity of the phages was tested on the isolated bacteria and revealed a coverage of 70% of the 33 isolated antibiotic resistant strains, >50% of which were targeted by multiple phages. Conclusion Collectively, these results demonstrate the feasibility of identifying phage with potent activity against antibiotic resistant KP strains, and may provide a novel therapeutic approach for treatment of ESBL and CR KP infections. Disclosures All Authors: No reported disclosures

scholarly journals Assessment of brain reference genes for RT-qPCR studies in neurodegenerative diseases

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Rasmus Rydbirk ◽  
Jonas Folke ◽  
Kristian Winge ◽  
Susana Aznar ◽  
Bente Pakkenberg ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Reference Genes ◽  
Target Gene ◽  
Structural Changes ◽  
A Priori ◽  
Brain Regions ◽  
Quantitative Real Time Pcr ◽  
Human Brain Tissue ◽  
Web Based ◽  
Gene Expression Levels

Abstract Evaluation of gene expression levels by reverse transcription quantitative real-time PCR (RT-qPCR) has for many years been the favourite approach for discovering disease-associated alterations. Normalization of results to stably expressed reference genes (RGs) is pivotal to obtain reliable results. This is especially important in relation to neurodegenerative diseases where disease-related structural changes may affect the most commonly used RGs. We analysed 15 candidate RGs in 98 brain samples from two brain regions from Alzheimer’s disease (AD), Parkinson’s disease (PD), Multiple System Atrophy, and Progressive Supranuclear Palsy patients. Using RefFinder, a web-based tool for evaluating RG stability, we identified the most stable RGs to be UBE2D2, CYC1, and RPL13 which we recommend for future RT-qPCR studies on human brain tissue from these patients. None of the investigated genes were affected by experimental variables such as RIN, PMI, or age. Findings were further validated by expression analyses of a target gene GSK3B, known to be affected by AD and PD. We obtained high variations in GSK3B levels when contrasting the results using different sets of common RG underlining the importance of a priori validation of RGs for RT-qPCR studies.

In vitro chemosensitivity of freshly explanted tumor cells to pemetrexed is correlated with target gene expression

2007 ◽  
Vol 25 (5) ◽  
pp. 417-423 ◽  
Author(s):  
Axel-Rainer Hanauske ◽  
Ulrike Eismann ◽  
Olaf Oberschmidt ◽  
Heike Pospisil ◽  
Steve Hoffmann ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tumor Cells ◽  
Target Gene ◽  
Target Gene Expression ◽  
In Vitro Chemosensitivity

Mutants of cubitus interruptus that are independent of PKA regulation are independent of hedgehog signaling

Development ◽  
1999 ◽  
Vol 126 (16) ◽  
pp. 3607-3616 ◽  
Author(s):  
Y. Chen ◽  
J.R. Cardinaux ◽  
R.H. Goodman ◽  
S.M. Smolik
Keyword(s):  
Gene Expression ◽  
Target Gene ◽  
Ectopic Expression ◽  
Proteolytic Processing ◽  
Dominant Negative ◽  
Cubitus Interruptus ◽  
Target Gene Expression ◽  
Exogenous Expression

Hedgehog (HH) is an important morphogen involved in pattern formation during Drosophila embryogenesis and disc development. cubitus interruptus (ci) encodes a transcription factor responsible for transducing the hh signal in the nucleus and activating hh target gene expression. Previous studies have shown that CI exists in two forms: a 75 kDa proteolytic repressor form and a 155 kDa activator form. The ratio of these forms, which is regulated positively by hh signaling and negatively by PKA activity, determines the on/off status of hh target gene expression. In this paper, we demonstrate that the exogenous expression of CI that is mutant for four consensus PKA sites [CI(m1-4)], causes ectopic expression of wingless (wg) in vivo and a phenotype consistent with wg overexpression. Expression of CI(m1-4), but not CI(wt), can rescue the hh mutant phenotype and restore wg expression in hh mutant embryos. When PKA activity is suppressed by expressing a dominant negative PKA mutant, the exogenous expression of CI(wt) results in overexpression of wg and lethality in embryogenesis, defects that are similar to those caused by the exogenous expression of CI(m1-4). In addition, we demonstrate that, in cell culture, the mutation of any one of the three serine-containing PKA sites abolishes the proteolytic processing of CI. We also show that PKA directly phosphorylates the four consensus phosphorylation sites in vitro. Taken together, our results suggest that positive hh and negative PKA regulation of wg gene expression converge on the regulation of CI phosphorylation.

Effects of the HDAC inhibitor scriptaid on the in vitro development of bovine embryos and on imprinting gene expression levels

2018 ◽  
Vol 110 ◽  
pp. 79-85 ◽  
Author(s):  
R. Laguna-Barraza ◽  
M.J. Sánchez-Calabuig ◽  
A. Gutiérrez-Adán ◽  
D. Rizos ◽  
S. Pérez-Cerezales
Keyword(s):  
Gene Expression ◽  
Hdac Inhibitor ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
Expression Levels ◽  
Imprinting Gene ◽  
Vitro Development ◽  
Gene Expression Levels

scholarly journals Reversible Gene Expression Control inYersinia pestisby Using an Optimized CRISPR Interference System

2019 ◽  
Vol 85 (12) ◽  
Author(s):  
Tong Wang ◽  
Min Wang ◽  
Qingwen Zhang ◽  
Shiyang Cao ◽  
Xiang Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
High Throughput ◽  
Biofilm Formation ◽  
Prevention And Control ◽  
Target Gene ◽  
Gene Knockdown ◽  
Content Type ◽  
Short Palindromic Repeat ◽  
And Control

ABSTRACTMany genes in the bacterial pathogenYersinia pestis, the causative agent of three plague pandemics, remain uncharacterized, greatly hampering the development of measures for plague prevention and control. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been shown to be an effective tool for gene knockdown in model bacteria. In this system, a catalytically dead Cas9 (dCas9) and a small guide RNA (sgRNA) form a complex, binding to the specific DNA target through base pairing, thereby impeding RNA polymerase binding and causing target gene repression. Here, we introduce an optimized CRISPRi system usingStreptococcus pyogenesCas9-derived dCas9 for gene knockdown inY. pestis. Multiple genes harbored on either the chromosome or plasmids ofY. pestiswere efficiently knocked down (up to 380-fold) in a strictly anhydrotetracycline-inducible manner using this CRISPRi approach. Knockdown ofhmsH(responsible for biofilm formation) orcspB(encoding a cold shock protein) resulted in greatly decreased biofilm formation or impaired cold tolerance inin vitrophenotypic assays. Furthermore, silencing of the virulence-associated genesyscBorailusing this CRISPRi system resulted in attenuation of virulence in HeLa cells and mice similar to that previously reported foryscBandailnull mutants. Taken together, our results confirm that this optimized CRISPRi system can reversibly and efficiently repress the expression of target genes inY. pestis, providing an alternative to conventional gene knockdown techniques, as well as a strategy for high-throughput phenotypic screening ofY. pestisgenes with unknown functions.IMPORTANCEYersiniapestisis a lethal pathogen responsible for millions of human deaths in history. It has also attracted much attention for potential uses as a bioweapon or bioterrorism agent, against which new vaccines are desperately needed. However, manyY. pestisgenes remain uncharacterized, greatly hampering the development of measures for plague prevention and control. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been successfully used in a variety of bacteria in functional genomic studies, but no such genetic tool has been reported inY. pestis. Here, we systematically optimized the CRISPRi approach for use inY. pestis, which ultimately repressed target gene expression with high efficiency in a reversible manner. Knockdown of functional genes using this method produced phenotypes that were readily detected byin vitroassays, cell infection assays, and mouse infection experiments. This is a report of a CRISPRi approach inY. pestisand highlights the potential use of this approach in high-throughput functional genomics studies of this pathogen.

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