scholarly journals Tissue Distribution of Doxycycline in Animal Models of Tuberculosis

2020 ◽  
Vol 64 (5) ◽  
Author(s):  
Martin Gengenbacher ◽  
Matthew D. Zimmerman ◽  
Jansy P. Sarathy ◽  
Firat Kaya ◽  
Han Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Control ◽  
Target Genes ◽  
Calcium Content ◽  
Tissue Penetration ◽  
Lung Lesions ◽  
Content Type ◽  
Clearance Kinetics

ABSTRACT Doxycycline, an FDA-approved tetracycline, is used in tuberculosis in vivo models for the temporal control of mycobacterial gene expression. In these models, animals are infected with recombinant Mycobacterium tuberculosis carrying genes of interest under transcriptional control of the doxycycline-responsive TetR-tetO unit. To minimize fluctuations of plasma levels, doxycycline is usually administered in the diet. However, tissue penetration studies to identify the minimum doxycycline content in food achieving complete repression of TetR-controlled genes in tuberculosis (TB)-infected organs and lesions have not been conducted. Here, we first determined the tetracycline concentrations required to achieve silencing of M. tuberculosis target genes in vitro. Next, we measured doxycycline concentrations in plasma, major organs, and lung lesions in TB-infected mice and rabbits and compared these values to silencing concentrations measured in vitro. We found that 2,000 ppm doxycycline supplemented in mouse and rabbit feed is sufficient to reach target concentrations in TB lesions. In rabbit chow, the calcium content had to be reduced 5-fold to minimize chelation of doxycycline and deliver adequate oral bioavailability. Clearance kinetics from major organs and lung lesions revealed that doxycycline levels fall below concentrations that repress tet promoters within 7 to 14 days after doxycycline is removed from the diet. In summary, we have shown that 2,000 ppm doxycycline supplemented in standard mouse diet and in low-calcium rabbit diet delivers concentrations adequate to achieve full repression of tet promoters in infected tissues of mice and rabbits.

scholarly journals Epigenetic regulation of neural cell differentiation plasticity in the adult mammalian brain

2008 ◽  
Vol 105 (46) ◽  
pp. 18012-18017 ◽  
Author(s):  
Jun Kohyama ◽  
Takuro Kojima ◽  
Eriko Takatsuka ◽  
Toru Yamashita ◽  
Jun Namiki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Target Genes ◽  
Epigenetic Modification ◽  
Mammalian Brain ◽  
Cytokine Signaling ◽  
Specific Gene ◽  
Neural Cells ◽  
Specific Gene Expression

Neural stem/progenitor cells (NSCs/NPCs) give rise to neurons, astrocytes, and oligodendrocytes. It has become apparent that intracellular epigenetic modification including DNA methylation, in concert with extracellular cues such as cytokine signaling, is deeply involved in fate specification of NSCs/NPCs by defining cell-type specific gene expression. However, it is still unclear how differentiated neural cells retain their specific attributes by repressing cellular properties characteristic of other lineages. In previous work we have shown that methyl-CpG binding protein transcriptional repressors (MBDs), which are expressed predominantly in neurons in the central nervous system, inhibit astrocyte-specific gene expression by binding to highly methylated regions of their target genes. Here we report that oligodendrocytes, which do not express MBDs, can transdifferentiate into astrocytes both in vitro (cytokine stimulation) and in vivo (ischemic injury) through the activation of the JAK/STAT signaling pathway. These findings suggest that differentiation plasticity in neural cells is regulated by cell-intrinsic epigenetic mechanisms in collaboration with ambient cell-extrinsic cues.

scholarly journals 18β-Glycyrrhetinic Acid Inhibits Methicillin-Resistant Staphylococcus aureus Survival and Attenuates Virulence Gene Expression

2012 ◽  
Vol 57 (1) ◽  
pp. 241-247 ◽  
Author(s):  
Danyelle R. Long ◽  
Julia Mead ◽  
Jay M. Hendricks ◽  
Michele E. Hardy ◽  
Jovanka M. Voyich
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Virulence Gene ◽  
Glycyrrhetinic Acid ◽  
Licorice Root ◽  
Methicillin Resistant ◽  
Content Type ◽  
Virulence Gene Expression

ABSTRACTMethicillin-resistantStaphylococcus aureus(MRSA) has become a major source of infection in hospitals and in the community. Increasing antibiotic resistance inS. aureusstrains has created a need for alternative therapies to treat disease. A component of the licorice rootGlycyrrhizaspp., 18β-glycyrrhetinic acid (GRA), has been shown to have antiviral, antitumor, and antibacterial activity. This investigation explores thein vitroandin vivoeffects of GRA on MRSA pulsed-field gel electrophoresis (PFGE) type USA300. GRA exhibited bactericidal activity at concentrations exceeding 0.223 μM. Upon exposure ofS. aureusto sublytic concentrations of GRA, we observed a reduction in expression of key virulence genes, includingsaeRandhla. In murine models of skin and soft tissue infection, topical GRA treatment significantly reduced skin lesion size and decreased the expression ofsaeRandhlagenes. Our investigation demonstrates that at high concentrations GRA is bactericidal to MRSA and at sublethal doses it reduces virulence gene expression inS. aureusbothin vitroandin vivo.

scholarly journals New Shuttle Vectors for Real-Time Gene Expression Analysis in Multidrug-Resistant Acinetobacter Species: In Vitro and In Vivo Responses to Environmental Stressors

2019 ◽  
Vol 85 (18) ◽  
Author(s):  
Massimiliano Lucidi ◽  
Daniela Visaggio ◽  
Elisa Prencipe ◽  
Francesco Imperi ◽  
Giordano Rampioni ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Multidrug Resistant ◽  
Antibiotic Selection ◽  
Content Type ◽  
Shuttle Vectors ◽  
Reporter Systems

ABSTRACT The Acinetobacter genus includes species of opportunistic pathogens and harmless saprophytes. The type species, Acinetobacter baumannii, is a nosocomial pathogen renowned for being multidrug resistant (MDR). Despite the clinical relevance of infections caused by MDR A. baumannii and a few other Acinetobacter spp., the regulation of their pathogenicity remains elusive due to the scarcity of adequate genetic tools, including vectors for gene expression analysis. Here, we report the generation and testing of a series of Escherichia coli-Acinetobacter promoter-probe vectors suitable for gene expression analysis in Acinetobacter spp. These vectors, named pLPV1Z, pLPV2Z, and pLPV3Z, carry both gentamicin and zeocin resistance markers and contain lux, lacZ, and green fluorescent protein (GFP) reporter systems downstream of an extended polylinker, respectively. The presence of a toxin-antitoxin gene system and the high copy number allow pLPV plasmids to be stably maintained even without antibiotic selection. The pLPV plasmids can easily be introduced by electroporation into MDR A. baumannii belonging to the major international lineages as well as into species of the Acinetobacter calcoaceticus-A. baumannii complex. The pLPV vectors have successfully been employed to study the regulation of stress-responsive A. baumannii promoters, including the DNA damage-inducible uvrABC promoter, the ethanol-inducible adhP and yahK promoters, and the iron-repressible promoter of the acinetobactin siderophore biosynthesis gene basA. A lux-tagged A. baumannii ATCC 19606T strain, carrying the iron-responsive pLPV1Z::PbasA promoter fusion, allowed in vivo and ex vivo monitoring of the bacterial burden in the Galleria mellonella infection model. IMPORTANCE The short-term adaptive response to environmental cues greatly contributes to the ecological success of bacteria, and profound alterations in bacterial gene expression occur in response to physical, chemical, and nutritional stresses. Bacteria belonging to the Acinetobacter genus are ubiquitous inhabitants of soil and water though some species, such as Acinetobacter baumannii, are pathogenic and cause serious concern due to antibiotic resistance. Understanding A. baumannii pathobiology requires adequate genetic tools for gene expression analysis, and to this end we developed user-friendly shuttle vectors to probe the transcriptional responses to different environmental stresses. Vectors were constructed to overcome the problem of antibiotic selection in multidrug-resistant strains and were equipped with suitable reporter systems to facilitate signal detection. By means of these vectors, the transcriptional response of A. baumannii to DNA damage, ethanol exposure, and iron starvation was investigated both in vitro and in vivo, providing insights into A. baumannii adaptation during stress and infection.

scholarly journals Analysis of theIn VivoTranscriptome ofBordetella pertussisduring Infection of Mice

mSphere ◽  
2019 ◽  
Vol 4 (2) ◽  
Author(s):  
Ting Y. Wong ◽  
Jesse M. Hall ◽  
Evan S. Nowak ◽  
Dylan T. Boehm ◽  
Laura A. Gonyar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Iron Acquisition ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Growth Conditions ◽  
Rna Seq ◽  
Content Type ◽  
Murine Lung

ABSTRACTBordetella pertussiscauses the disease whooping cough through coordinated control of virulence factors by theBordetellavirulence gene system. Microarrays and, more recently, RNA sequencing (RNA-seq) have been used to describein vitrogene expression profiles ofB. pertussisand other pathogens. In previous studies, we have analyzed thein vitrogene expression profiles ofB. pertussis, and we hypothesize that the infection transcriptome profilein vivois significantly different from that under laboratory growth conditions. To study the infection transcriptome ofB. pertussis, we developed a simple filtration technique for isolation of bacteria from infected lungs. The work flow involves filtering the bacteria out of the lung homogenate using a 5-μm-pore-size syringe filter. The captured bacteria are then lysed to isolate RNA for Illumina library preparation and RNA-seq analysis. Upon comparing thein vitroandin vivogene expression profiles, we identified 351 and 255 genes as activated and repressed, respectively, during murine lung infection. As expected, numerous genes associated with virulent-phase growth were activated in the murine host, including pertussis toxin (PT), the PT secretion apparatus, and the type III secretion system. A significant number of genes encoding iron acquisition and heme uptake proteins were highly expressed during infection, supporting iron acquisition as critical forB. pertussissurvivalin vivo. Numerous metabolic genes were repressed during infection. Overall, these data shed light on the gene expression profile ofB. pertussisduring infection, and this method will facilitate efforts to understand how this pathogen causes infection.IMPORTANCEIn vitrogrowth conditions for bacteria do not fully recapitulate the host environment. RNA sequencing transcriptome analysis allows for the characterization of the infection gene expression profiles of pathogens in complex environments. Isolation of the pathogen from infected tissues is critical because of the large amounts of host RNA present in crude lysates of infected organs. A filtration method was developed that enabled enrichment of the pathogen RNA for RNA-seq analysis. The resulting data describe the “infection transcriptome” ofB. pertussisin the murine lung. This strategy can be utilized for pathogens in other hosts and, thus, expand our knowledge of what bacteria express during infection.

scholarly journals CodY Deletion EnhancesIn VivoVirulence of Community-Associated Methicillin-Resistant Staphylococcus aureus Clone USA300

2012 ◽  
Vol 80 (7) ◽  
pp. 2382-2389 ◽  
Author(s):  
Christopher P. Montgomery ◽  
Susan Boyle-Vavra ◽  
Agnès Roux ◽  
Kazumi Ebine ◽  
Abraham L. Sonenshein ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Virulence Genes ◽  
Target Genes ◽  
Gene Encoding ◽  
Methicillin Resistant ◽  
Content Type ◽  
Global Regulators ◽  
Alpha Hemolysin

ABSTRACTTheStaphylococcus aureusglobal regulator CodY responds to nutrient availability by controlling the expression of target genes.In vitro, CodY represses the transcription of virulence genes, but it is not known if CodY also represses virulencein vivo. The dominant community-associated methicillin-resistantS. aureus(CA-MRSA) clone, USA300, is hypervirulent and has increased transcription of global regulators and virulence genes; these features are reminiscent of a strain defective in CodY. Sequence analysis revealed, however, that thecodYgenes of USA300 and other sequencedS. aureusisolates are not significantly different from thecodYgenes in strains known to have active CodY.codYwas expressed in USA300, as well as in other pulsotypes assessed. Deletion ofcodYfrom a USA300 clinical isolate resulted in modestly increased expression of the global regulatorsagrandsaeRS, as well as the gene encoding the toxin alpha-hemolysin (hla). A substantial increase (>30-fold) in expression of thelukF-PVgene, encoding part of the Panton-Valentine leukocidin (PVL), was observed in thecodYmutant. All of these expression differences were reversed by complementation with a functionalcodYgene. Moreover, purified CodY protein bound upstream of thelukSF-PVoperon, indicating that CodY directly represses expression oflukSF-PV. Deletion ofcodYincreased the virulence of USA300 in necrotizing pneumonia and skin infection. Interestingly, deletion oflukSF-PVfrom thecodYmutant did not attenuate virulence, indicating that the hypervirulence of thecodYmutant was not explained by overexpression of PVL. These results demonstrate that CodY is active in USA300 and that CodY-mediated repression restrains the virulence of USA300.

scholarly journals Use of a Multiplex Transcript Method for Analysis of Pseudomonas aeruginosa Gene Expression Profiles in the Cystic Fibrosis Lung

2016 ◽  
Vol 84 (10) ◽  
pp. 2995-3006 ◽  
Author(s):  
Alex H. Gifford ◽  
Sven D. Willger ◽  
Emily L. Dolben ◽  
Lisa A. Moulton ◽  
Dana B. Dorman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Expression Profiles ◽  
Clinical Isolates ◽  
Probe Design ◽  
Content Type ◽  
Lung Infections

The discovery of therapies that modulatePseudomonas aeruginosavirulence or that can eradicate chronicP. aeruginosalung infections associated with cystic fibrosis (CF) will be advanced by an improved understanding ofP. aeruginosabehaviorin vivo. We demonstrate the use of multiplexed Nanostring technology to monitor relative abundances ofP. aeruginosatranscripts across clinical isolates, in serial samples, and for the purposes of comparing microbial physiologyin vitroandin vivo. The expression of 75 transcripts encoded by genes implicated in CF lung disease was measured in a variety ofP. aeruginosastrains as well as RNA serial sputum samples from fourP. aeruginosa-colonized subjects with CF collected over 6 months. We present data on reproducibility, the results from different methods of normalization, and demonstrate high concordance between transcript relative abundance data obtained by Nanostring or transcriptome sequencing (RNA-Seq) analysis. Furthermore, we address considerations regarding sequence variation between strains during probe design. Analysis ofP. aeruginosagrownin vitroidentified transcripts that correlated with the different phenotypes commonly observed in CF clinical isolates.P. aeruginosatranscript profiles in RNA from CF sputum indicated alginate productionin vivo, and transcripts involved in quorum-sensing regulation were less abundant in sputum than strains grown in the laboratory.P. aeruginosagene expression patterns from sputum clustered closely together relative to patterns for laboratory-grown cultures; in contrast, laboratory-grownP. aeruginosashowed much greater transcriptional variation with only loose clustering of strains with different phenotypes. The clustering within and between subjects was surprising in light of differences in inhaled antibiotic and respiratory symptoms, suggesting that the pathways represented by these 75 transcripts are stable in chronic CFP. aeruginosalung infections.

Proto-oncogene abnormalities and their relationship to tumorigenicity in some human glioblastomas

1989 ◽  
Vol 71 (1) ◽  
pp. 83-90 ◽  
Author(s):  
Hoi Sang U ◽  
Patricia Y. Kelley ◽  
James D. Hatton ◽  
Jin Y. Shew
Keyword(s):  
Gene Expression ◽  
Cell Growth ◽  
Growth Factor Receptor ◽  
Growth Stimulation ◽  
Soft Agar ◽  
R Gene ◽  
Primary Tumors ◽  
Content Type

✓ Human glioblastomas are highly malignant intracranial tumors, some of which demonstrate amplification of the epidermal growth factor-receptor (EGF-R) gene. Overexpression of this gene is seen in the majority of primary tumors; however, the role of the EGF-R gene in glial tumorigenesis is unknown. The authors explored the relationship between EGF-R gene expression and glioblastoma cell growth in vitro and in vivo and found that the level of EGF-R gene expression did not correlate with tumor cell growth either in soft agar or in the nude mouse. This suggests that the EGF-R gene is not involved in effecting direct growth stimulation in glial oncogenesis. Tumorigenesis involves differentiation arrest; therefore, the expression of several proto-oncogenes in neuroectodermal tumors was investigated to evaluate the potential involvement of the EGF-R gene in glial differentiation. A nonoverlapping expression of the N-myc and EGF-R genes was found in neuronal-derived and glial-derived tumors, respectively. This suggests that the EGF-R gene may be involved in differentiation or its arrest in glia.

scholarly journals EPCR-dependent PAR2 activation by the blood coagulation initiation complex regulates LPS-triggered interferon responses in mice

Blood ◽  
2015 ◽  
Vol 125 (18) ◽  
pp. 2845-2854 ◽  
Author(s):  
Hai Po H. Liang ◽  
Edward J. Kerschen ◽  
Irene Hernandez ◽  
Sreemanti Basu ◽  
Mark Zogg ◽  
...  
Keyword(s):  
Gene Expression ◽  
Blood Coagulation ◽  
Target Genes ◽  
Adaptor Protein ◽  
Messenger Rnas ◽  
Initiation Complex ◽  
Induced Expression ◽  
Regulated Gene Expression

Abstract Infection and inflammation are invariably associated with activation of the blood coagulation mechanism, secondary to the inflammation-induced expression of the coagulation initiator tissue factor (TF) on innate immune cells. By investigating the role of cell-surface receptors for coagulation factors in mouse endotoxemia, we found that the protein C receptor (ProcR; EPCR) was required for the normal in vivo and in vitro induction of lipopolysaccharide (LPS)-regulated gene expression. In cultured bone marrow–derived myeloid cells and in monocytic RAW264.7 cells, the LPS-induced expression of functionally active TF, assembly of the ternary TF-VIIa-Xa initiation complex of blood coagulation, and the EPCR-dependent activation of protease-activated receptor 2 (PAR2) by the ternary TF-VIIa-Xa complex were required for the normal LPS induction of messenger RNAs encoding the TLR3/4 signaling adaptor protein Pellino-1 and the transcription factor interferon regulatory factor 8. In response to in vivo challenge with LPS, mice lacking EPCR or PAR2 failed to fully initiate an interferon-regulated gene expression program that included the Irf8 target genes Lif, Iigp1, Gbp2, Gbp3, and Gbp6. The inflammation-induced expression of TF and crosstalk with EPCR, PAR2, and TLR4 therefore appear necessary for the normal evolution of interferon-regulated host responses.

scholarly journals Cryptococcus neoformans Host Adaptation: Toward Biological Evidence of Dormancy

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Alexandre Alanio ◽  
Frédérique Vernel-Pauillac ◽  
Aude Sturny-Leclère ◽  
Françoise Dromer
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
Cryptococcus Neoformans ◽  
Immune Cells ◽  
Gene Expression Analysis ◽  
Yeast Cells ◽  
Content Type ◽  
Dormant Cells

ABSTRACTCryptococcosis is an opportunistic infection due to the ubiquitous yeastCryptococcus neoformans. This yeast interacts closely with innate immune cells, leading to various fates, including fungal persistence within cells, making possible the dissemination of the yeast cells with monocytes via a Trojan horse strategy. In humans, the natural history of the infection begins with primoinfection during childhood, which is followed by dormancy and, in some individuals, reactivation upon immunosuppression. To address the question of dormancy, we studiedC. neoformansinfection at the macrophage level (in vitroH99-macrophage interaction) and at the organ level in a murine model of cryptococcosis. We analyzed the diversity of yeast adaptation to the host by characterizing severalC. neoformanspopulations with new assays based on flow cytometry (quantitative flow cytometry, multispectral imaging flow cytometry, sorting), microscopy (dynamic imaging), and gene expression analysis. On the basis of parameters of multiplication and stress response, various populations of yeast cells were observed over timein vivoandin vitro. Cell sorting allowed the identification of a subpopulation that was less prone to grow under standard conditions than the other populations, with growth enhanced by the addition of serum. Gene expression analysis revealed that this population had specific metabolic characteristics that could reflect dormancy. Our data suggest that dormant yeast cells could existin vitroandin vivo.C. neoformansexhibits a huge plasticity and adaptation to hosts that deserves further study.In vitrogeneration of dormant cells is now the main challenge to overcome the limited number of yeast cells recovered in our models.IMPORTANCECryptococcus neoformansis a sugar-coated unicellular fungus that interacts closely with various cells and organisms, including amoebas, nematodes, and immune cells of mammals. This yeast is able to proliferate and survive in the intracellular environment.C. neoformanscauses cryptococcosis, and yeast dormancy in humans has been suggested on the basis of epidemiological evidence obtained years ago. By studying anin vitromodel of yeast-macrophage interaction and murine models of cryptococcosis, we observed that yeast cells evolve in heterogeneous populations during infection on the basis of global metabolic activity. We compared the growth ability and gene expression of yeast cells belonging to various populations in those two models. We eventually found a population of yeast cells with low metabolism that fit some of the criteria for dormant cells. This paves the way for further characterization of dormancy inC. neoformans.

scholarly journals Bile acids inhibit duodenal secretin expression via orphan nuclear receptor small heterodimer partner (SHP)

2009 ◽  
Vol 297 (1) ◽  
pp. G90-G97 ◽  
Author(s):  
Ian P. Y. Lam ◽  
Leo T. O. Lee ◽  
Hueng-Sik Choi ◽  
Gianfranco Alpini ◽  
Billy K. C. Chow
Keyword(s):  
Gene Expression ◽  
Bile Acids ◽  
Nuclear Receptor ◽  
Target Genes ◽  
In Vivo Studies ◽  
Control Group ◽  
Orphan Nuclear Receptor ◽  
Small Heterodimer Partner

Small heterodimer partner (SHP) is an orphan nuclear receptor in which gene expression can be upregulated by bile acids. It regulates its target genes by repressing the transcriptional activities of other nuclear receptors including NeuroD, which has been shown to regulate secretin gene expression. Here, we evaluated the regulation on duodenal secretin gene expression by SHP and selected bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA). In vitro treatment of CDCA or fexaramine elevated the SHP transcript level and occupancy on secretin promoter. The increase in the SHP level, induced by bile acid treatment or overexpression, reduced secretin gene expression, whereas this gene inhibitory effect was reversed by silencing of endogenous SHP. In in vivo studies, double-immunofluorescence staining demonstrated the coexpression of secretin and SHP in mouse duodenum. Feeding mice with 1% CA-enriched rodent chow resulted in upregulation of SHP and a concomitant decrease in secretin transcript and protein levels in duodenum compared with the control group fed with normal chow. A diet enriched with 5% cholestyramine led to a decrease in SHP level and a corresponding increase in secretin expression. Overall, this study showed that bile acids via SHP inhibit duodenal secretin gene expression. Because secretin is a key hormone that stimulates bile flow in cholangiocytes, this pathway thus provides a novel means to modulate secretin-stimulated choleresis in response to intraduodenal bile acids.

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