[The effect of DNA hydroxymethylase Tet2 on γ globin activation in the treatment of β-thalassemia].

PubMed

Li, W X; Ma, Q W; Zeng, F Y

2018-03-01

Objective: To study the function of ten-eleven translocation 2 (Tet2) in γ globin gene expression in patients with β- thalassemia. Methods: Gamma globin expression was induced by 5-azacytidine and Tet2 gene expression was knocked down by short hairpin RNA (shRNA) in a human immortalized myelogenous leukemia K562 cell line. The global 5-hydroxymethylcytosine (5hmC) level was measured by an ELISA kit. 5hmC level of γ globin gene was quantified by sulfite sequencing. The mRNA level of Tet2, γ globin, and related transcription factors Nfe4 and Klf1 were quantified by real-time PCR. Results: Tet2 knockdown resulted in a decreased global 5hmC level from 0.14% to 0.03% as of the control group in K562 cells. The expression of γ globin was enhanced after 5-azacytidine treatment in vitro. However, γ globin mRNA level in Tet2 knockdown cells was only 55% as that in control group. The CG sites on γ globin gene were unmethylated. As Tet2 was down-regulated, the expression levels of Nfe4 and Klf1 decreased by about 80% and increased to 3.5 folds, respectively. Conclusions: Tet2 appears to maintain 5hmC level and facilitates γ globin gene activation. Moreover, Tet2 more likely regulates γ globin expression via affecting transcription factors rather than the gene itself. Thus, Tet2 could be a potential therapeutic target for β thalassemias.

Characterization of the product of a nonribosomal peptide synthetase-like (NRPS-like) gene using the doxycycline dependent Tet-on system in Aspergillus terreus.

PubMed

Sun, Wei-Wen; Guo, Chun-Jun; Wang, Clay C C

2016-04-01

Genome sequencing of the fungus Aspergillus terreus uncovered a number of silent core structural biosynthetic genes encoding enzymes presumed to be involved in the production of cryptic secondary metabolites. There are five nonribosomal peptide synthetase (NRPS)-like genes with the predicted A-T-TE domain architecture within the A. terreus genome. Among the five genes, only the product of pgnA remains unknown. The Tet-on system is an inducible, tunable and metabolism-independent expression system originally developed for Aspergillus niger. Here we report the adoption of the Tet-on system as an effective gene activation tool in A. terreus. Application of this system in A. terreus allowed us to uncover the product of the cryptic NRPS-like gene, pgnA. Furthermore expression of pgnA in the heterologous Aspergillus nidulans host suggested that the pgnA gene alone is necessary for phenguignardic acid (1) biosynthesis. Copyright © 2016 Elsevier Inc. All rights reserved.

Mechanism of action of tetrandrine, a natural inhibitor of Candida albicans drug efflux pumps.

PubMed

Zhang, Hong; Gao, Aili; Li, Fengxia; Zhang, Gehua; Ho, Hon In; Liao, Wanqing

2009-05-01

Synergistic effects have previously been observed for a natural compound, tetrandrine (TET), with fluconazole (FLC) in vitro and in the treatment of Candida albicans-infected mice. To investigate the mechanisms of these synergistic effects, 16 strains of C. albicans from the same parent but with different FLC sensitivities were examined using flow cytometry and fluorescent spectrophotometry. Rhodamine 123 (Rh123)-positive cells and intracellular Rh123 fluorescence intensity were determined in accumulation/efflux experiments involving no or a noncytotoxic dose of TET. Total RNA extracted from each strain was used to compare the expressions of drug efflux pump genes in FLC-susceptible, -susceptible dose-dependent, and -resistant strains before and 24 h after TET administration. Accumulation experiments determined that mean percentages of Rh123-positive cells were 26.65% (TET-free) and 70.99% (TET 30 microg/ml), and mean respective intracellular Rh123 fluorescence intensities were 11.34 and 18.00. Efflux experiments showed that percentages of Rh123-positive cells were 1.79% (TET free) and 42.57% (TET 30 microg/ml), respectively, and respective mean intracellular Rh123 fluorescence intensities were 0.74 and 2.19. Differences in MDR1, FLU1, CDR1, and CDR2 expression levels in the absence of TET were statistically significant (p<0.05) between FLC-susceptible, -susceptible dose-dependent, and -resistant strains. Compared with TET-free conditions, 24 h TET-treated strains showed statistically different (p<0.05) expression of MDR1 (FLC-resistant strain), FLU1 (FLC-susceptible dose-dependent and -resistant strains), and CDR1 and CDR2 (FLC-susceptible, -susceptible dose-dependent, and -resistant strains). Thus TET can inhibit the C. albicans drug efflux system and reduce drug efflux. Its mechanism of action is related to the inhibition of expression of the drug efflux pump genes MDR1, FLU1, CDR1, and CDR2.

Influence of TRAIL gene on biomechanical properties of the human leukemic cell line Jurkat.

PubMed

Yao, Weijuan; Chen, Kai; Wang, Xinjuan; Xie, Lide; Wen, Zongyao; Yan, Zongyi; Chien, Shu

2002-12-01

We cloned the cDNA fragment of human TNF-related apoptosis inducing ligand (TRAIL) into RevTet-On, a Tet-regulated and high-level gene expression system. Making use of the TRAIL gene expression system in Jurkat as a cell model, we studied the influence of TRAIL gene on the biomechanics properties of Jurkat through measuring changes of cellular biomechanics properties before and after the TRAIL gene expression, which was induced by adding tetracycline derivative doxycycline (Dox). The results indicated that the TRAIL gene expression led to significant changes in cellular biomechanics properties. The osmotic fragility increased and the cell stiffness increased after the expression of TRAIL gene. Thus, the apoptosis-inducing TRAIL gene caused significant changes in the biomechanics properties of Jurkat cells.

TetR-dependent gene regulation in intracellular Listeria monocytogenes demonstrates the spatiotemporal surface distribution of ActA.

PubMed

Schmitter, Sibylle; Fieseler, Lars; Klumpp, Jochen; Bertram, Ralph; Loessner, Martin J

2017-08-01

To enable specific and tightly controlled gene expression both in vitro and during the intracellular lifecycle of the pathogen Listeria monocytogenes, a TetR-dependent genetic induction system was developed. Highest concentration of cytoplasmic TetR and best repression of tetO-controlled genes was obtained by tetR expression from the synthetic promoter Pt 17 . Anhydrotetracycline (ATc) as inducer permitted concentration-dependent, fine-tuned expression of genes under control of the tetO operator and a suitable promoter. The actin-polymerizing ActA protein represents a major virulence factor of L. monocytogenes, required for actin-based motility and cell-to-cell spread in infected host cells. To be able to observe its spatial and temporal distribution on intracellular L. monocytogenes cells, conditional mutants featuring actA placed under TetR control were used to infect PtK2 epithelial cells. Following induction at different time intervals, the subsequent recruitment of actin by L. monocytogenes could be monitored. We found that cells displayed functional ActA after approximately 15 min, while formation of polarized actin tail was complete after 90-120 min. At this point, intracellular motility of the induced mutants was indistinguishable from wild-type bacteria. Interestingly, de novo ActA synthesis in intracellular Listeria also demonstrated the temporal, asymmetric redistribution of the membrane-anchored proteins from the lateral walls toward the cell poles. © 2017 John Wiley & Sons Ltd.

Tetracycline-inducible system for regulation of skeletal muscle-specific gene expression in transgenic mice

NASA Technical Reports Server (NTRS)

Grill, Mischala A.; Bales, Mark A.; Fought, Amber N.; Rosburg, Kristopher C.; Munger, Stephanie J.; Antin, Parker B.

2003-01-01

Tightly regulated control of over-expression is often necessary to study one aspect or time point of gene function and, in transgenesis, may help to avoid lethal effects and complications caused by ubiquitous over-expression. We have utilized the benefits of an optimized tet-on system and a modified muscle creatine kinase (MCK) promoter to generate a skeletal muscle-specific, doxycycline (Dox) controlled over-expression system in transgenic mice. A DNA construct was generated in which the codon optimized reverse tetracycline transactivator (rtTA) was placed under control of a skeletal muscle-specific version of the mouse MCK promoter. Transgenic mice containing this construct expressed rtTA almost exclusively in skeletal muscles. These mice were crossed to a second transgenic line containing a bi-directional promoter centered on a tet responder element driving both a luciferase reporter gene and a tagged gene of interest; in this case the calpain inhibitor calpastatin. Compound hemizygous mice showed high level, Dox dependent muscle-specific luciferase activity often exceeding 10,000-fold over non-muscle tissues of the same mouse. Western and immunocytochemical analysis demonstrated similar Dox dependent muscle-specific induction of the tagged calpastatin protein. These findings demonstrate the effectiveness and flexibility of the tet-on system to provide a tightly regulated over-expression system in adult skeletal muscle. The MCKrtTA transgenic lines can be combined with other transgenic responder lines for skeletal muscle-specific over-expression of any target gene of interest.

A novel isoform of TET1 that lacks a CXXC domain is overexpressed in cancer

PubMed Central

Good, Charly R.; Madzo, Jozef; Patel, Bela; Maegawa, Shinji; Engel, Nora; Jelinek, Jaroslav

2017-01-01

Abstract TET1 oxidizes methylated cytosine into 5-hydroxymethylcytosine (5hmC), resulting in regulation of DNA methylation and gene expression. Full length TET1 (TET1FL) has a CXXC domain that binds to unmethylated CpG islands (CGIs). This CXXC domain allows TET1 to protect CGIs from aberrant methylation, but it also limits its ability to regulate genes outside of CGIs. Here, we report a novel isoform of TET1 (TET1ALT) that has a unique transcription start site from an alternate promoter in intron 2, yielding a protein with a unique translation start site. Importantly, TET1ALT lacks the CXXC domain but retains the catalytic domain. TET1ALT is repressed in embryonic stem cells (ESCs) but becomes activated in embryonic and adult tissues while TET1FL is expressed in ESCs, but repressed in adult tissues. Overexpression of TET1ALT shows production of 5hmC with distinct (and weaker) effects on DNA methylation or gene expression when compared to TET1FL. TET1ALT is aberrantly activated in multiple cancer types including breast, uterine and glioblastoma, and TET1 activation is associated with a worse overall survival in breast, uterine and ovarian cancers. Our data suggest that the predominantly activated isoform of TET1 in cancer cells does not protect from CGI methylation and likely mediates dynamic site-specific demethylation outside of CGIs. PMID:28531272

Drug-loaded nanoparticles induce gene expression in human pluripotent stem cell derivatives

NASA Astrophysics Data System (ADS)

Gajbhiye, Virendra; Escalante, Leah; Chen, Guojun; Laperle, Alex; Zheng, Qifeng; Steyer, Benjamin; Gong, Shaoqin; Saha, Krishanu

2013-12-01

Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained higher fibroblast proliferation levels and MMP activity. The results demonstrate that the PEG-H40-DXC nanoparticle system provides an effective tool to controlling gene expression in human stem cell derivatives.Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained higher fibroblast proliferation levels and MMP activity. The results demonstrate that the PEG-H40-DXC nanoparticle system provides an effective tool to controlling gene expression in human stem cell derivatives. Electronic supplementary information (ESI) available: ESI containing 1H NMR spectra and additional fibroblast characterization data. See DOI: 10.1039/c3nr04794f

TET1 promotes cisplatin-resistance via demethylating the vimentin promoter in ovarian cancer.

PubMed

Han, Xi; Zhou, Yuanyuan; You, Yuanyi; Lu, Jiaojiao; Wang, Lijie; Hou, Huilian; Li, Jing; Chen, Wei; Zhao, Le; Li, Xu

2017-04-01

The development of chemo-resistance impairs the outcome of the first line platinum-based chemotherapies for ovarian cancer. Deregulation of DNA methylation/demethylation provides a critical mechanism for the occurrence of chemo-resistance. The ten-eleven translocation (TET) family of dioxygenases including TET1/2/3 plays an important part in DNA demethylation, but their roles in cisplatin resistance have not been elucidated. Using cisplatin-sensitive and cisplatin-resistant ovarian cancer cell models, we found that TET1 was significantly upregulated in cisplatin-resistant CP70 cells compared with that in cisplatin-sensitive A2780 cells. Ectopic expression of TET1 in A2780 cells promoted cisplatin resistance and decreased cytotoxicity induced by cisplatin, while inhibition of TET1 by siRNA transfection in CP70 cells attenuated cisplatin resistance and enhanced cytotoxicity of cisplatin. Increased TET1 induced re-expression of vimentin through active DNA demethylation, and cause partial epithelial-to-mesenchymal (EMT) in A2780 cells. Contrarily, knocking down of TET1 in CP70 cells reduced vimentin expression and reversed EMT process. Immunohistochemical analysis of TET1 in human ovarian cancer tissues revealed that TET1 existed in nucleus and cytoplasm in ovarian cancer tissues. And the expression of nuclear TET1 was positively correlated with residual tumor and chemotherapeutic response. Thus, TET1 expression causes resistance to cisplatin and one of the targets of TET1 action is vimentin in ovarian cancer. © 2017 International Federation for Cell Biology.

Airway-Specific Inducible Transgene Expression Using Aerosolized Doxycycline

PubMed Central

Tata, Purushothama Rao; Pardo-Saganta, Ana; Prabhu, Mythili; Vinarsky, Vladimir; Law, Brandon M.; Fontaine, Benjamin A.; Tager, Andrew M.

2013-01-01

Tissue-specific transgene expression using tetracycline (tet)-regulated promoter/operator elements has been used to revolutionize our understanding of cellular and molecular processes. However, because most tet-regulated mouse strains use promoters of genes expressed in multiple tissues, to achieve exclusive expression in an organ of interest is often impossible. Indeed, in the extreme case, unwanted transgene expression in other organ systems causes lethality and precludes the study of the transgene in the actual organ of interest. Here, we describe a novel approach to activating tet-inducible transgene expression solely in the airway by administering aerosolized doxycycline. By optimizing the dose and duration of aerosolized doxycycline exposure in mice possessing a ubiquitously expressed Rosa26 promoter–driven reverse tet-controlled transcriptional activator (rtTA) element, we induce transgene expression exclusively in the airways. We detect no changes in the cellular composition or proliferative behavior of airway cells. We used this newly developed method to achieve airway basal stem cell–specific transgene expression using a cytokeratin 5 (also known as keratin 5)–driven rtTA driver line to induce Notch pathway activation. We observed a more robust mucous metaplasia phenotype than in mice receiving doxycycline systemically. In addition, unwanted phenotypes outside of the lung that were evident when doxycycline was received systemically were now absent. Thus, our approach allows for rapid and efficient airway-specific transgene expression. After the careful strain by strain titration of the dose and timing of doxycycline inhalation, a suite of preexisting transgenic mice can now be used to study airway biology specifically in cases where transient transgene expression is sufficient to induce a phenotype. PMID:23848320

A novel pair of inducible expression vectors for use in Methylobacterium extorquens.

PubMed

Chubiz, Lon M; Purswani, Jessica; Carroll, Sean Michael; Marx, Chistopher J

2013-05-06

Due to the ever increasing use of diverse microbial taxa in basic research and industrial settings, there is a growing need for genetic tools to alter the physiology of these organisms. In particular, there is a dearth of inducible expression systems available for bacteria outside commonly used γ-proteobacteria, such as Escherichia coli or Pseudomonas species. To this end, we have sought to develop a pair of inducible expression vectors for use in the α-proteobacterium Methylobacterium extorquens, a model methylotroph. We found that the P(R) promoter from rhizobial phage 16-3 was active in M. extorquens and engineered the promoter to be inducible by either p-isopropyl benzoate (cumate) or anhydrotetracycline. These hybrid promoters, P(R/cmtO) and P(R/tetO), were found to have high levels of expression in M. extorquens with a regulatory range of 10-fold and 30-fold, respectively. Compared to an existing cumate-inducible (10-fold range), high-level expression system for M. extorquens, P(R/cmtO) and P(R/tetO) have 33% of the maximal activity but were able to repress gene expression 3 and 8-fold greater, respectively. Both promoters were observed to exhibit homogeneous, titratable activation dynamics rather than on-off, switch-like behavior. The utility of these promoters was further demonstrated by complementing loss of function of ftfL--essential for growth on methanol--where we show P(R/tetO) is capable of not only fully complementing function but also producing a conditional null phenotype. These promoters have been incorporated into a broad-host-range backbone allowing for potential use in a variety of bacterial hosts. We have developed two novel expression systems for use in M. extorquens. The expression range of these vectors should allow for increased ability to explore cellular physiology in M. extorquens. Further, the P(R/tetO) promoter is capable of producing conditional null phenotypes, previously unattainable in M. extorquens. As both expression systems rely on the use of membrane permeable inducers, we suspect these expression vectors will be useful for ectopic gene expression in numerous proteobacteria.

An Efficient and Versatile System for Visualization and Genetic Modification of Dopaminergic Neurons in Transgenic Mice

PubMed Central

Kramer, Edgar R.

2015-01-01

Background & Aims The brain dopaminergic (DA) system is involved in fine tuning many behaviors and several human diseases are associated with pathological alterations of the DA system such as Parkinson’s disease (PD) and drug addiction. Because of its complex network integration, detailed analyses of physiological and pathophysiological conditions are only possible in a whole organism with a sophisticated tool box for visualization and functional modification. Methods & Results Here, we have generated transgenic mice expressing the tetracycline-regulated transactivator (tTA) or the reverse tetracycline-regulated transactivator (rtTA) under control of the tyrosine hydroxylase (TH) promoter, TH-tTA (tet-OFF) and TH-rtTA (tet-ON) mice, to visualize and genetically modify DA neurons. We show their tight regulation and efficient use to overexpress proteins under the control of tet-responsive elements or to delete genes of interest with tet-responsive Cre. In combination with mice encoding tet-responsive luciferase, we visualized the DA system in living mice progressively over time. Conclusion These experiments establish TH-tTA and TH-rtTA mice as a powerful tool to generate and monitor mouse models for DA system diseases. PMID:26291828

A Tet-on and Cre-loxP Based Genetic Engineering System for Convenient Recycling of Selection Markers in Penicillium oxalicum

PubMed Central

Jiang, Baojie; Zhang, Ruiqin; Feng, Dan; Wang, Fangzhong; Liu, Kuimei; Jiang, Yi; Niu, Kangle; Yuan, Quanquan; Wang, Mingyu; Wang, Hailong; Zhang, Youming; Fang, Xu

2016-01-01

The lack of selective markers has been a key problem preventing multistep genetic engineering in filamentous fungi, particularly for industrial species such as the lignocellulose degrading Penicillium oxalicum JUA10-1(formerly named as Penicillium decumbens). To resolve this problem, we constructed a genetic manipulation system taking advantage of two established genetic systems: the Cre-loxP system and Tet-on system in P. oxalicum JUA10-1. This system is efficient and convenient. The expression of Cre recombinase was activated by doxycycline since it was controlled by Tet-on system. Using this system, two genes, ligD and bglI, were sequentially disrupted by loxP flanked ptrA. The successful application of this procedure will provide a useful tool for genetic engineering in filamentous fungi. This system will also play an important role in improving the productivity of interesting products and minimizing by-product when fermented by filamentous fungi. PMID:27148179

The antimicrobial resistance patterns and associated determinants in Streptococcus suis isolated from humans in southern Vietnam, 1997-2008

PubMed Central

2011-01-01

Background Streptococcus suis is an emerging zoonotic pathogen and is the leading cause of bacterial meningitis in adults in Vietnam. Systematic data on the antimicrobial susceptibility profiles of S. suis strains isolated from human cases are lacking. We studied antimicrobial resistance and associated resistance determinants in S. suis isolated from patients with meningitis in southern Vietnam. Methods S. suis strains isolated between 1997 and 2008 were investigated for their susceptibility to six antimicrobial agents. Strains were screened for the presence and expression of tetracycline and erythromycin resistance determinants and the association of tet(M) genes with Tn916- like transposons. The localization of tetracycline resistance gene tet(L) was determined by pulse field gel electrophoresis and Southern blotting. Results We observed a significant increase in resistance to tetracycline and chloramphenicol, which was concurrent with an increase in multi-drug resistance. In tetracycline resistance strains, we identified tet(M), tet(O), tet(W) and tet(L) and confirmed their expression. All tet(M) genes were associated with a Tn916-like transposon. The co-expression of tet(L) and other tetracycline resistance gene(s) encoding for ribosomal protection protein(s) was only detected in strains with a minimum inhibitory concentration (MIC) of tetracycline of ≥ 64 mg/L Conclusions We demonstrated that multi-drug resistance in S. suis causing disease in humans in southern Vietnam has increased over the 11-year period studied. We report the presence and expression of tet(L) in S. suis strains and our data suggest that co-expression of multiple genes encoding distinct mechanism is required for an MIC ≥ 64 mg/L to tetracycline. PMID:21208459

The antimicrobial resistance patterns and associated determinants in Streptococcus suis isolated from humans in southern Vietnam, 1997-2008.

PubMed

Hoa, Ngo T; Chieu, Tran T B; Nghia, Ho D T; Mai, Nguyen T H; Anh, Pham H; Wolbers, Marcel; Baker, Stephen; Campbell, James I; Chau, Nguyen V V; Hien, Tran T; Farrar, Jeremy; Schultsz, Constance

2011-01-06

Streptococcus suis is an emerging zoonotic pathogen and is the leading cause of bacterial meningitis in adults in Vietnam. Systematic data on the antimicrobial susceptibility profiles of S. suis strains isolated from human cases are lacking. We studied antimicrobial resistance and associated resistance determinants in S. suis isolated from patients with meningitis in southern Vietnam. S. suis strains isolated between 1997 and 2008 were investigated for their susceptibility to six antimicrobial agents. Strains were screened for the presence and expression of tetracycline and erythromycin resistance determinants and the association of tet(M) genes with Tn916- like transposons. The localization of tetracycline resistance gene tet(L) was determined by pulse field gel electrophoresis and Southern blotting. We observed a significant increase in resistance to tetracycline and chloramphenicol, which was concurrent with an increase in multi-drug resistance. In tetracycline resistance strains, we identified tet(M), tet(O), tet(W) and tet(L) and confirmed their expression. All tet(M) genes were associated with a Tn916-like transposon. The co-expression of tet(L) and other tetracycline resistance gene(s) encoding for ribosomal protection protein(s) was only detected in strains with a minimum inhibitory concentration (MIC) of tetracycline of ≥ 64 mg/L. We demonstrated that multi-drug resistance in S. suis causing disease in humans in southern Vietnam has increased over the 11-year period studied. We report the presence and expression of tet(L) in S. suis strains and our data suggest that co-expression of multiple genes encoding distinct mechanism is required for an MIC ≥ 64 mg/L to tetracycline.

Increased 5-hydroxymethylcytosine and Ten-eleven Translocation Protein Expression in Ultraviolet B-irradiated HaCaT Cells

PubMed Central

Wang, Dan; Huang, Jin-Hua; Zeng, Qing-Hai; Gu, Can; Ding, Shu; Lu, Jian-Yun; Chen, Jing; Yang, Sheng-Bo

2017-01-01

Background: DNA hydroxymethylation refers to a chemical modification process in which 5-methylcytosine (5mC) is catalyzed to 5- hydroxymethylcytosine (5hmC) by ten-eleven translocation (TET) family proteins. Recent studies have revealed that aberrant TETs expression or 5hmC level may play important roles in the occurrence and development of various pathological and physiological processes including cancer and aging. This study aimed to explore the relation between aberrant DNA hydroxymethylation with skin photoaging and to investigate the levels of TETs, 5mC, and 5hmC expression 24 h after 40 mJ/cm2 and 80 mJ/cm2 doses of ultraviolet B (UVB) irradiation to HaCaT cells. Methods: To explore whether aberrant DNA hydroxymethylation is also related to skin photoaging, 40 mJ/cm2 and 80 mJ/cm2 doses of UVB were chosen to treat keratinocytes (HaCaT cells). After 24 h of UVB irradiation, 5mC and 5hmC levels were determined by immunohistochemistry (IHC) and immunofluorescence (IF), and at the same time, the expression levels of matrix metalloproteinase 1 (MMP-1) and TETs were assessed by reverse transcription-polymerase chain reaction or Western blot analysis. Results: After 40 mJ/cm2 and 80 mJ/cm2 doses of UVB exposure, both IHC and IF results showed that 5hmC levels increased significantly, while the 5mC levels did not exhibit significant changes in HaCaT cells, compared with HaCat cells without UVB exposure. Moreover, compared with HaCat cells without UVB exposure, the levels of TET1, TET2, and TET3 mRNA and protein expression were significantly upregulated (mRNA: P = 0.0022 and 0.0043 for TET1; all P < 0.0001 for TET2; all P = 0.0006 for TET3; protein: P = 0.0012 and 0.0006 for TET1; all P = 0.0022 for TET2; and all P = 0.0002 for TET3), and the levels of MMP-1 mRNA expression increased dose dependently in 40 mJ/cm2 and 80 mJ/cm2 UVB-irradiated groups. Conclusion: UVB radiation could cause increased 5hmC and TET expression, which might become a novel biomarker in UVB-related skin aging. PMID:28229992

Functional Analysis of the Arabidopsis TETRASPANIN Gene Family in Plant Growth and Development.

PubMed

Wang, Feng; Muto, Antonella; Van de Velde, Jan; Neyt, Pia; Himanen, Kristiina; Vandepoele, Klaas; Van Lijsebettens, Mieke

2015-11-01

TETRASPANIN (TET) genes encode conserved integral membrane proteins that are known in animals to function in cellular communication during gamete fusion, immunity reaction, and pathogen recognition. In plants, functional information is limited to one of the 17 members of the Arabidopsis (Arabidopsis thaliana) TET gene family and to expression data in reproductive stages. Here, the promoter activity of all 17 Arabidopsis TET genes was investigated by pAtTET::NUCLEAR LOCALIZATION SIGNAL-GREEN FLUORESCENT PROTEIN/β-GLUCURONIDASE reporter lines throughout the life cycle, which predicted functional divergence in the paralogous genes per clade. However, partial overlap was observed for many TET genes across the clades, correlating with few phenotypes in single mutants and, therefore, requiring double mutant combinations for functional investigation. Mutational analysis showed a role for TET13 in primary root growth and lateral root development and redundant roles for TET5 and TET6 in leaf and root growth through negative regulation of cell proliferation. Strikingly, a number of TET genes were expressed in embryonic and seedling progenitor cells and remained expressed until the differentiation state in the mature plant, suggesting a dynamic function over developmental stages. The cis-regulatory elements together with transcription factor-binding data provided molecular insight into the sites, conditions, and perturbations that affect TET gene expression and positioned the TET genes in different molecular pathways; the data represent a hypothesis-generating resource for further functional analyses. © 2015 American Society of Plant Biologists. All Rights Reserved.

Functional Analysis of the Arabidopsis TETRASPANIN Gene Family in Plant Growth and Development1[OPEN

PubMed Central

Wang, Feng; Muto, Antonella; Van de Velde, Jan; Neyt, Pia; Himanen, Kristiina; Vandepoele, Klaas; Van Lijsebettens, Mieke

2015-01-01

TETRASPANIN (TET) genes encode conserved integral membrane proteins that are known in animals to function in cellular communication during gamete fusion, immunity reaction, and pathogen recognition. In plants, functional information is limited to one of the 17 members of the Arabidopsis (Arabidopsis thaliana) TET gene family and to expression data in reproductive stages. Here, the promoter activity of all 17 Arabidopsis TET genes was investigated by pAtTET::NUCLEAR LOCALIZATION SIGNAL-GREEN FLUORESCENT PROTEIN/β-GLUCURONIDASE reporter lines throughout the life cycle, which predicted functional divergence in the paralogous genes per clade. However, partial overlap was observed for many TET genes across the clades, correlating with few phenotypes in single mutants and, therefore, requiring double mutant combinations for functional investigation. Mutational analysis showed a role for TET13 in primary root growth and lateral root development and redundant roles for TET5 and TET6 in leaf and root growth through negative regulation of cell proliferation. Strikingly, a number of TET genes were expressed in embryonic and seedling progenitor cells and remained expressed until the differentiation state in the mature plant, suggesting a dynamic function over developmental stages. The cis-regulatory elements together with transcription factor-binding data provided molecular insight into the sites, conditions, and perturbations that affect TET gene expression and positioned the TET genes in different molecular pathways; the data represent a hypothesis-generating resource for further functional analyses. PMID:26417009

TET2 binds the androgen receptor and loss is associated with prostate cancer

PubMed Central

Nickerson, ML; Das, S; Im, KM; Turan, S; Berndt, SI; Li, H; Lou, H; Brodie, SA; Billaud, JN; Zhang, T; Bouk, AJ; Butcher, D; Wang, Z; Sun, L; Misner, K; Tan, W; Esnakula, A; Esposito, D; Huang, WY; Hoover, RN; Tucker, MA; Keller, JR; Boland, J; Brown, K; Anderson, SK; Moore, LE; Isaacs, WB; Chanock, SJ; Yeager, M; Dean, M; Andresson, T

2016-01-01

Genetic alterations associated with prostate cancer (PCa) may be identified by sequencing metastatic tumor genomes to identify molecular markers at this lethal stage of disease. Previously, we characterized somatic alterations in metastatic tumors in the methylcytosine dioxygenase ten-eleven translocation 2 (TET2), which is altered in 5–15% of myeloid, kidney, colon and prostate cancers. Genome-wide association studies previously identified non-coding risk variants associated with PCa and melanoma. We performed fine-mapping of PCa risk across TET2 using genotypes from the PEGASUS case-control cohort and identified six new risk variants in introns 1 and 2. Oligonucleotides containing two risk variants were bound by the transcription factor octamer-binding protein 1 (Oct1/POU2F1) and TET2 and Oct1 expression were positively correlated in prostate tumors. TET2 is expressed in normal prostate tissue and reduced in a subset of tumors from the Cancer Genome Atlas (TCGA). Small interfering RNA (siRNA)-mediated TET2 knockdown (KD) increases LNCaP cell proliferation, migration, and wound healing, verifying loss drives a cancer phenotype. Endogenous TET2 bound the androgen receptor (AR) and AR-coactivator proteins in LNCaP cell extracts, and TET2 KD increases prostate-specific antigen (KLK3/PSA) expression. Published data reveal TET2 binding sites and hydroxymethylcytosine (hmC) proximal to KLK3. A gene co-expression network identified using TCGA prostate tumor RNA-sequencing identifies co-regulated cancer genes associated with 2-oxoglutarate (2-OG) and succinate metabolism, including TET2, lysine demethylase (KDM) KDM6A, BRCA1-associated BAP1, and citric acid cycle enzymes IDH1/2, SDHA/B, and FH. The co-expression signature is conserved across 31 TCGA cancers suggesting a putative role for TET2 as an energy sensor (of 2-OG) that modifies aspects of androgen-AR signaling. Decreased TET2 mRNA expression in TCGA PCa tumors is strongly associated with reduced patient survival indicating reduced expression in tumors maybe an informative biomarker of disease progression and perhaps metastatic disease. PMID:27819678

Immunization-induced perturbation of human blood plasma cell pool: progressive maturation, IL-6 responsiveness, and high PRDI-BF1/BLIMP1 expression are critical distinctions between antigen-specific and nonspecific plasma cells.

PubMed

González-García, Inés; Ocaña, Esther; Jiménez-Gómez, Gema; Campos-Caro, Antonio; Brieva, José A

2006-04-01

The present study shows that reimmunization with tetanus toxoid (tet) caused a transient increase of the human blood plasma cell (PC) pool, detectable from 6th to 15th day postboost, as well as the temporal alteration of several PC features. Labeling of specific PC with FITC-tet C fragment (tetC) allowed kinetics analysis of the tetC(+) and tetC(-) PC, and revealed remarkable differences between them: 1) the kinetics of tetC(+) PC occurrence was exponential, and most of them appeared in a narrow time frame (5th to 8th day postboost), whereas the tetC(-) PC increase was lower (three to five times) and more prolonged (4th to 15th day postboost). 2) The tetC(+) PC subset contained a fraction of cycling cells, expressed high levels of DR, CD138, and CD126, and responded to IL-6 by improving their survival and Ig secretion; in contrast, the tetC(-) PC showed higher CXCR4 and lower DR and CD138, did not respond to IL-6, and contained a fraction of apoptotic cells. 3) Sequential phenotypic analysis revealed maturational changes within the tetC(+), but not tetC(-), PC subset; sequencing of tetC(+) PC IgVH genes showed clear features of Ag selection. 4) The tetC(+) PC expressed several times more positive regulatory domain I- binding factor 1/B lymphocyte-induced maturation protein 1 transcription factor than the tetC(-) PC. 5) The tetC(-) PC and bone marrow resident PC similarly expressed low DR and high CXCR4, but differed in that the latter exhibited higher levels of CD31, CD138, and positive regulatory domain I- binding factor 1/B lymphocyte-induced maturation protein 1. These findings support the view that tetC(+) PC contain bone marrow PC precursors, and tetC(-) PC probably belong to a removable compartment of aged PC.

Expression and characterization of an M cell-specific ligand-fused dengue virus tetravalent epitope using Saccharomyces cerevisiae.

PubMed

Nguyen, Ngoc-Luong; So, Kum-Kang; Kim, Jung-Mi; Kim, Sae-Hae; Jang, Yong-Suk; Yang, Moon-Sik; Kim, Dae-Hyuk

2015-01-01

A fusion construct (Tet-EDIII-Co1) consisting of an M cell-specific peptide ligand (Co1) at the C-terminus of a recombinant tetravalent gene encoding the amino acid sequences of dengue envelope domain III (Tet-EDIII) from four serotypes was expressed and tested for binding activity to the mucosal immune inductive site M cells for the development of an oral vaccine. The yeast episomal expression vector, pYEGPD-TER, which was designed to direct gene expression using the glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, a functional signal peptide of the amylase 1A protein from rice, and the GAL7 terminator, was used to clone the Tet-EDIII-Co1 gene and resultant plasmids were then used to transform Saccharomyces cerevisiae. PCR and back-transformation into Escherichia coli confirmed the presence of the Tet-EDIII-Co1 gene-containing plasmid in transformants. Northern blot analysis of transformed S. cerevisiae identified the presence of the Tet-EDIII-Co1-specific transcript. Western blot analysis indicated that the produced Tet-EDIII-Co1 protein with the expected molecular weight was successfully secreted into the culture medium. Quantitative Western blot analysis and ELISA revealed that the recombinant Tet-EDIII-Co1 protein comprised approximately 0.1-0.2% of cell-free extracts (CFEs). In addition, 0.1-0.2 mg of Tet-EDIII-Co1 protein per liter of culture filtrate was detected on day 1, and this quantity peaked on day 3 after cultivation. In vivo binding assays showed that the Tet-EDIII-Co1 protein was delivered specifically to M cells in Peyer's patches (PPs) while the Tet-EDIII protein lacking the Co1 ligand did not, which demonstrated the efficient targeting of this antigenic protein through the mucosal-specific ligand. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

[DNA hydroxymethylase 10-11 translocation 2 (TET2) inhibits mouse macrophage activation and polarization].

PubMed

Li, Bingyi; Huo, Yi; Lin, Zhifeng; Wang, Tao

2017-09-01

Objective To study the role of DNA hydroxymethylase 10-11 translocation 2 (TET2) in macrophage activation and polarization. Methods RAW264.7 macrophages were cultured in vitro and stimulated with 100 ng/mL LPS for 0, 1, 2, 4, 6 hours. Real-time quantitative PCR was used to detect TET2 mRNA expression. TET2 expression was knocked down with siRNA and the knock-down efficiency was evaluated by real-time quantitative PCR and Western blotting. Following siRNA transfection for 48 hours, RAW264.7 cells were stimulated by LPS for 4 hours, and then real-time quantitative PCR and ELISA were performed to detect the expressions of interleukin 6 (IL-6), tumor necrosis factor α (TNF-α) and IL-12. The M1 polarizing markers TNF-α, inducible nitric oxide synthase (iNOS) and IL-12, and M2 polarizing markers mannose receptor (MR), arginase 1 (Arg-1) and chitinase 3-like molecule 1 (Ym1) were tested after M1 or M2 induction by LPS/IFN-γ or IL-4. Results TET2 expression increased after LPS treatment in RAW264.7 cells and reached the peak at 2 hours later. The siRNA effectively reduced the expression of TET2. The expressions of IL-6, TNF-α and IL-12 mRNAs increased after TET2 knock-down and LPS stimulation. The expressions of M1 polarization markers and M2 markers were up-regulated by the corresponding stimulations after TET2 knock-down. Conclusion TET2 has the effect of inhibiting LPS-induced macrophage activation and plays an inhibitory role in macrophage M1 and M2 polarization.

HSP27 and 70 expression in thymic epithelial tumors and benign thymic alterations: diagnostic, prognostic and physiologic implications.

PubMed

Janik, S; Schiefer, A I; Bekos, C; Hacker, P; Haider, T; Moser, J; Klepetko, W; Müllauer, L; Ankersmit, H J; Moser, B

2016-04-21

Thymic Epithelial Tumors (TETs), the most common tumors in the anterior mediastinum in adults, show a unique association with autoimmune Myasthenia Gravis (MG) and represent a multidisciplinary diagnostic and therapeutic challenge. Neither risk factors nor established biomarkers for TETs exist. Predictive and diagnostic markers are urgently needed. Heat shock proteins (HSPs) are upregulated in several malignancies promoting tumor cell survival and metastases. We performed immunohistochemical staining of HSP27 and 70 in patients with TETs (n = 101) and patients with benign thymic alterations (n = 24). Further, serum HSP27 and 70 concentrations were determined in patients with TETs (n = 46), patients with benign thymic alterations (n = 33) and volunteers (n = 49) by using ELISA. HSPs were differentially expressed in histologic types and pathological tumor stages of TETs. Weak HSP tumor expression correlated with worse freedom from recurrence. Serum HSP concentrations were elevated in TETs and MG, correlated with clinical tumor stage and histologic subtype and decreased significantly after complete tumor resection. To conclude, we found HSP expression in the vast majority of TETs, in physiologic thymus and staining intensities in patients with TETs have been associated with prognosis. However, although interesting and promising the role of HSPs in TETs as diagnostic and prognostic or even therapeutic markers need to be further evaluated.

HSP27 and 70 expression in thymic epithelial tumors and benign thymic alterations: diagnostic, prognostic and physiologic implications

PubMed Central

Janik, S.; Schiefer, A. I.; Bekos, C.; Hacker, P.; Haider, T.; Moser, J.; Klepetko, W.; Müllauer, L.; Ankersmit, H. J.; Moser, B.

2016-01-01

Thymic Epithelial Tumors (TETs), the most common tumors in the anterior mediastinum in adults, show a unique association with autoimmune Myasthenia Gravis (MG) and represent a multidisciplinary diagnostic and therapeutic challenge. Neither risk factors nor established biomarkers for TETs exist. Predictive and diagnostic markers are urgently needed. Heat shock proteins (HSPs) are upregulated in several malignancies promoting tumor cell survival and metastases. We performed immunohistochemical staining of HSP27 and 70 in patients with TETs (n = 101) and patients with benign thymic alterations (n = 24). Further, serum HSP27 and 70 concentrations were determined in patients with TETs (n = 46), patients with benign thymic alterations (n = 33) and volunteers (n = 49) by using ELISA. HSPs were differentially expressed in histologic types and pathological tumor stages of TETs. Weak HSP tumor expression correlated with worse freedom from recurrence. Serum HSP concentrations were elevated in TETs and MG, correlated with clinical tumor stage and histologic subtype and decreased significantly after complete tumor resection. To conclude, we found HSP expression in the vast majority of TETs, in physiologic thymus and staining intensities in patients with TETs have been associated with prognosis. However, although interesting and promising the role of HSPs in TETs as diagnostic and prognostic or even therapeutic markers need to be further evaluated. PMID:27097982

DNA methylcytosine dioxygenase ten-eleven translocation 2 enhances lipopolysaccharide-induced cytokine expression in human dental pulp cells by regulating MyD88 hydroxymethylation.

PubMed

Wang, Xinxuan; Feng, Zhihui; Li, Qimeng; Yi, Baicheng; Xu, Qiong

2018-04-13

Dental pulp inflammation is a bacterially driven inflammation process characterized by the local accumulation of cytokines/chemokines that participate in destructive processes in the pulp. Multiple mechanisms are involved in dental pulp inflammation, including epigenetic events, such as DNA methylation/demethylation. Ten-eleven translocation 2 (TET2) is a recently discovered DNA methylcytosine dioxygenase that plays important roles in inflammatory disease. However, its role in the inflammatory response of dental pulp is unknown. We observed elevated mRNA and protein levels of TET2 after lipopolysaccharide (LPS) stimulation in human dental pulp cells (hDPCs). To identify the effects of TET2 on cytokine expression, TET2 was knocked down and cytokines were detected using a cytokine antibody array after LPS stimulation. The protein expression of GM-CSF, IL-6, IL-8 and RANTES decreased in the LPS-induced hDPCs following TET2 knockdown. The downregulated expression levels of IL-6 and IL-8 were further confirmed by real-time quantitative polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). Additionally, the phosphorylation levels of IKK-α/β, p65 and IκBα of the NF-κB signaling pathway were decreased in the TET2-silenced group. Furthermore, the global 5-hydroxymethylcytosine (5hmC) level was significantly decreased and the genomic 5-methylcytosine (5mC) level was increased in the TET2-deficient hDPCs; TET2 depletion resulted in a decrease in the 5hmC level of the MyD88 promoter following LPS stimulation. These findings indicate that TET2 knockdown inhibits LPS-induced inflammatory response in hDPCs by downregulating MyD88 hydroxymethylation. Thus, TET2-dependent DNA demethylation might play an important role in dental pulp inflammation as an epigenetic regulator.

Contribution of Efflux Pumps, Porins, and β-Lactamases to Multidrug Resistance in Clinical Isolates of Acinetobacter baumannii

PubMed Central

Rumbo, C.; Gato, E.; López, M.; Ruiz de Alegría, C.; Fernández-Cuenca, F.; Martínez-Martínez, L.; Vila, J.; Pachón, J.; Cisneros, J. M.; Rodríguez-Baño, J.; Pascual, A.

2013-01-01

We investigated the mechanisms of resistance to carbapenems, aminoglycosides, glycylcyclines, tetracyclines, and quinolones in 90 multiresistant clinical strains of Acinetobacter baumannii isolated from two genetically unrelated A. baumannii clones: clone PFGE-ROC-1 (53 strains producing the OXA-58 β-lactamase enzyme and 18 strains with the OXA-24 β-lactamase) and clone PFGE-HUI-1 (19 strains susceptible to carbapenems). We used real-time reverse transcriptase PCR to correlate antimicrobial resistance (MICs) with expression of genes encoding chromosomal β-lactamases (AmpC and OXA-51), porins (OmpA, CarO, Omp33, Dcap-like, OprB, Omp25, OprC, OprD, and OmpW), and proteins integral to six efflux systems (AdeABC, AdeIJK, AdeFGH, CraA, AbeM, and AmvA). Overexpression of the AdeABC system (level of expression relative to that by A. baumannii ATCC 17978, 30- to 45-fold) was significantly associated with resistance to tigecycline, minocycline, and gentamicin and other biological functions. However, hyperexpression of the AdeIJK efflux pump (level of expression relative to that by A. baumannii ATCC 17978, 8- to 10-fold) was significantly associated only with resistance to tigecycline and minocycline (to which the TetB efflux system also contributed). TetB and TetA(39) efflux pumps were detected in clinical strains and were associated with resistance to tetracyclines and doxycycline. The absence of the AdeABC system and the lack of expression of other mechanisms suggest that tigecycline-resistant strains of the PFGE-HUI-1 clone may be associated with a novel resistance-nodulation-cell efflux pump (decreased MICs in the presence of the inhibitor Phe-Arg β-naphthylamide dihydrochloride) and the TetA(39) system. PMID:23939894

Tet2 and Tet3 cooperate with B-lineage transcription factors to regulate DNA modification and chromatin accessibility.

PubMed

Lio, Chan-Wang; Zhang, Jiayuan; González-Avalos, Edahí; Hogan, Patrick G; Chang, Xing; Rao, Anjana

2016-11-21

Ten-eleven translocation (TET) enzymes oxidize 5-methylcytosine, facilitating DNA demethylation and generating new epigenetic marks. Here we show that concomitant loss of Tet2 and Tet3 in mice at early B cell stage blocked the pro- to pre-B cell transition in the bone marrow, decreased Irf4 expression and impaired the germline transcription and rearrangement of the Igκ locus. Tet2/3-deficient pro-B cells showed increased CpG methylation at the Igκ 3' and distal enhancers that was mimicked by depletion of E2A or PU.1, as well as a global decrease in chromatin accessibility at enhancers. Importantly, re-expression of the Tet2 catalytic domain in Tet2/3-deficient B cells resulted in demethylation of the Igκ enhancers and restored their chromatin accessibility. Our data suggest that TET proteins and lineage-specific transcription factors cooperate to influence chromatin accessibility and Igκ enhancer function by modulating the modification status of DNA.

The Success of Thread-embedding Therapy in Generating Hair Re-growth in Mice Points to Its Possibly Having a Similar Effect in Humans

PubMed Central

Shin, Hyun Jong; Lee, Dong-Jin; Kwon, Kang; Seo, Hyung-Sik; Jeong, Han-Sol; Lee, Ji-Yeon; Ha, Ki-Tae; Lee, Chang-Hyun; Jang, Yong-Suk; Lee, Byung-Wook; Kim, Byung Joo; Jung, Myeong-Ho

2015-01-01

Objectives: Recently, thread-embedding therapy (TET) has been widely applied in Korean medicine for cosmetic purposes such as reducing skin wrinkles. An inserted thread was reported to have induced continuous stimulation, followed by support for connective tissue regeneration. However, the potential role of TET in hairgrowth has not yet been reported. Methods: We designed this study to evaluate whether TET has a hair-growth-promoting effect. C57 black 6 (C57BL/6) mice were divided into three groups: normal saline-treated, minoxidil-treated, and thread-embedded groups. Normal saline or 5% minoxidil was topically sprayed on the dorsal skin of the mice once a day for 16 days. Medical threads were embedded into the dorsal skin of the mice in a single application. Hair growth activity was evaluated by using dermoscopic and microscopic observations. Sections of the dorsal skin were stained with hematoxylin and eosin. Expressions of bromodeoxyuridine (BrdU), proliferating cell nuclear antigen (PCNA), fibroblast growth factor-7 (FGF-7), and fibroblast growth factor-5 (FGF-5) were detected by using immunohistochemical staining. A reverse transcription-polymerase chain reaction (RT-PCR) analysis was adopted to measure the messenger RNA (mRNA) expressions of FGF-7 and FGF-5. Results: TET enhanced anagen development in the hair follicles of C57BL/6 mice. The expressions of BrdU and PCNA, both of which imply active cellular proliferation, were increased by using TET. Moreover, TET increased the expression of FGF-7, an anagen-inducing growth factor, while decreasing the expression of FGF-5, an anagen-cessation growth factor, both at the protein and the mRNA levels. Conclusion: TET enhanced hair re-growth in C57BL/6 mice. TET regulated the expressions of anagen-associated growth factors and activated the proliferation of hair follicular cells in depilated skin lesions. Considering its long-lasting effect, TET may be a good alternative therapeutic for the treatment of alopecia. PMID:26998386

[Effects of Tetrandrine Prenatal Intervention on Alveolar Epithelial Cells Type I Differentiation in Rat Model of Nitrofen-induced Congenital Diaphragmatic Hernia].

PubMed

Xiao, Bin; Xu, Chang; Liu, Min; Ji, Yi; Yang Li-xun; Li, Tai-ming; Jiang, Jun; He, Tao-zhen

2016-03-01

To investigate the effects of Tetrandrine (TET) prenatal intervention on the differentiation of alveolar epithelial cells type I (AEC I) in rat model of Nitrofen-induced congenital diaphragmatic hernia (CDH). Timed-pregnant Sprague-Dawley rats were divided into three groups, namely control, CDH and TET group on day 9.5 of gestation. The rats in TET group and CDH group were given 125 mg of Nitrofen by gavage one time, while the rats in control group were given the same dose of seed fat. After that, the rats in TET group was given 30 mg/kg of TET by gavage once a day for three days from day 18.5 of gestation, while the rats in CDH and control group were given the same dose of normal saline. On day 21.5 of gestation, all fetuses were delivered by cesarean, the lungs of fetuses were histologically evaluated by microscope and electron microscope. The expressions of type I cell-specific protein (RT140) and thyroid transcription factor 1 (TTF1) in alveolar fluid content were analyzed by RT-PCR and immunohistochemistry staining. To detect the number of AEC I and AEC II of each group by transmission electron microscopy and calculate the percentage of AEC I and AEC II (I/II%). The microscope and electron microscope study found the lungs of fetuses in CDH group showed marked hypoplasia, in contrast to the improvement of hypoplasia in TET fetuses. The pulmonary alveolar area had significant difference statistically (P < 0.01) in each group, which present as control > TET > CDH. I/II% had significant difference statistically (P < 0.01) in each group, which present as control > TET > CDH. The expression level of TTF1 was up-regulated in both CDH and TET groups, and it was higher in CDH group (P < 0.01). The expression level of RT140 were down-regulated in CDH and TET groups, which was lower in CDH group (P < 0.01). The development of AEC I was interfered in CDH rat model, TET prenatal treatment could improve the lung development of CDH.

Tet2 Regulates Osteoclast Differentiation by Interacting with Runx1 and Maintaining Genomic 5-Hydroxymethylcytosine (5hmC).

PubMed

Chu, Yajing; Zhao, Zhigang; Wayne Sant, David; Zhu, Ganqian; Greenblatt, Sarah M; Liu, Lin; Wang, Jinhuan; Cao, Zeng; Cheng Tho, Jeanette; Chen, Shi; Liu, Xiaochen; Zhang, Peng; Maciejewski, Jaroslaw P; Nimer, Stephen; Wang, Gaofeng; Yuan, Weiping; Yang, Feng-Chun; Xu, Mingjiang

2018-06-13

As a dioxygenase, Ten-eleven translocation 2 (TET2) catalyzes subsequent steps of 5-methylcytosine (5mC) oxidation. Tet2 plays a critical role in the self-renewal, proliferation, and differentiation of hematopoietic stem cells, but its impact on mature hematopoietic cells is not well-characterized. Here we show that Tet2 plays an essential role in osteoclastogenesis. Deletion of Tet2 impairs the differentiation of osteoclast precursor cells (macrophages) and their maturation into bone-resorbing osteoclasts in vitro. Furthermore, Tet2 -/- mice exhibit mild osteopetrosis, accompanied by decreased number of osteoclasts in vivo. Tet2 loss in macrophages results in the altered expression of a set of genes implicated in osteoclast differentiation, such as Cebpa, Mafb, and Nfkbiz. Tet2 deletion also leads to a genome-wide alteration in the level of 5-hydroxymethylcytosine (5hmC) and altered expression of a specific subset of macrophage genes associated with osteoclast differentiation. Furthermore, Tet2 interacts with Runx1 and negatively modulates its transcriptional activity. Our studies demonstrate a novel molecular mechanism controlling osteoclast differentiation and function by Tet2, that is, through interactions with Runx1 and the maintenance of genomic 5hmC. Targeting Tet2 and its pathway could be a potential therapeutic strategy for the prevention and treatment of abnormal bone mass caused by the deregulation of osteoclast activities. Copyright © 2018. Production and hosting by Elsevier B.V.

A tetO Toolkit To Alter Expression of Genes in Saccharomyces cerevisiae.

PubMed

Cuperus, Josh T; Lo, Russell S; Shumaker, Lucia; Proctor, Julia; Fields, Stanley

2015-07-17

Strategies to optimize a metabolic pathway often involve building a large collection of strains, each containing different versions of sequences that regulate the expression of pathway genes. Here, we develop reagents and methods to carry out this process at high efficiency in the yeast Saccharomyces cerevisiae. We identify variants of the Escherichia coli tet operator (tetO) sequence that bind a TetR-VP16 activator with differential affinity and therefore result in different TetR-VP16 activator-driven expression. By recombining these variants upstream of the genes of a pathway, we generate unique combinations of expression levels. Here, we built a tetO toolkit, which includes the I-OnuI homing endonuclease to create double-strand breaks, which increases homologous recombination by 10(5); a plasmid carrying six variant tetO sequences flanked by I-OnuI sites, uncoupling transformation and recombination steps; an S. cerevisiae-optimized TetR-VP16 activator; and a vector to integrate constructs into the yeast genome. We introduce into the S. cerevisiae genome the three crt genes from Erwinia herbicola required for yeast to synthesize lycopene and carry out the recombination process to produce a population of cells with permutations of tetO variants regulating the three genes. We identify 0.7% of this population as making detectable lycopene, of which the vast majority have undergone recombination at all three crt genes. We estimate a rate of ∼20% recombination per targeted site, much higher than that obtained in other studies. Application of this toolkit to medically or industrially important end products could reduce the time and labor required to optimize the expression of a set of metabolic genes.

Safety profile, efficacy, and biodistribution of a bicistronic high-capacity adenovirus vector encoding a combined immunostimulation and cytotoxic gene therapy as a prelude to a phase I clinical trial for glioblastoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Puntel, Mariana; Department of Cell and Developmental Biology, The University of Michigan School of Medicine, MSRB II, RM 4570C, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5689; Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048

2013-05-01

Adenoviral vectors (Ads) are promising gene delivery vehicles due to their high transduction efficiency; however, their clinical usefulness has been hampered by their immunogenicity and the presence of anti-Ad immunity in humans. We reported the efficacy of a gene therapy approach for glioma consisting of intratumoral injection of Ads encoding conditionally cytotoxic herpes simplex type 1 thymidine kinase (Ad-TK) and the immunostimulatory cytokine fms-like tyrosine kinase ligand 3 (Ad-Flt3L). Herein, we report the biodistribution, efficacy, and neurological and systemic effects of a bicistronic high-capacity Ad, i.e., HC-Ad-TK/TetOn-Flt3L. HC-Ads elicit sustained transgene expression, even in the presence of anti-Ad immunity, andmore » can encode large therapeutic cassettes, including regulatory elements to enable turning gene expression “on” or “off” according to clinical need. The inclusion of two therapeutic transgenes within a single vector enables a reduction of the total vector load without adversely impacting efficacy. Because clinically the vectors will be delivered into the surgical cavity, normal regions of the brain parenchyma are likely to be transduced. Thus, we assessed any potential toxicities elicited by escalating doses of HC-Ad-TK/TetOn-Flt3L (1 × 10{sup 8}, 1 × 10{sup 9}, or 1 × 10{sup 10} viral particles [vp]) delivered into the rat brain parenchyma. We assessed neuropathology, biodistribution, transgene expression, systemic toxicity, and behavioral impact at acute and chronic time points. The results indicate that doses up to 1 × 10{sup 9} vp of HC-Ad-TK/TetOn-Flt3L can be safely delivered into the normal rat brain and underpin further developments for its implementation in a phase I clinical trial for glioma. - Highlights: ► High capacity Ad vectors elicit sustained therapeutic gene expression in the brain. ► HC-Ad-TK/TetOn-Flt3L encodes two therapeutic genes and a transcriptional switch. ► We performed a dose escalation study at acute and chronic time points. ► Doses up to 1 × 10{sup 9} vp of HC-Ad-TK/TetOn-Flt3L can be safely delivered in the brain. ► Efficacy and safety of HC-Ad-TK/TetOn-Flt3L merits its use in a GBM Phase I trial.« less

The Modification of Tet1 in Male Germline Stem Cells and Interact with PCNA, HDAC1 to promote their Self-renewal and Proliferation

PubMed Central

Zheng, Liming; Zhai, Yuanxin; Li, Na; Ma, Fanglin; Zhu, Haijing; Du, Xiaomin; Li, Guangpeng; Hua, Jinlian

2016-01-01

Epigenetic modification plays key roles in spermatogenesis, especially DNA methylation dynamic is important in sustaining normal spermatogenesis. Ten-eleven translocation 1 (Tet1) is not only a key demethylase, which works in specific gene regions, but also crosstalks with partners to regulate epigenetic progress as protein complexes. Dairy goat is an important livestock in China, while the unstable culture system in vitro inhibits optimization of new dairy goat species. The study of epigenetic modification in male germline stem cells (mGSCs) is beneficial to the optimization of adult stem cell culture system in vitro, and the improvement of sperm quality and breeding of selected livestock. In our study, we not only analyzed the morphology, gene expression, DNA methylation and histone methylation dynamic in mouse Tet1 (mTet1) modified mGSCs, we also analyzed the stemness ability by in vivo transplantation and explored the functional mechanism of Tet1 in dairy goat mGSCs. The results showed mTet1 modified mGSCs had better self-renewal and proliferation ability than wild-type mGSCs, mTet1 could also up-regulate JMJD3 to decrease H3K27me3, which also showed to suppress the MEK-ERK pathway. Furthermore, Co-IP analysis demonstrated that TET1 interact with PCNA and HDAC1 by forming protein complexes to comprehensively regulate dairy goat mGSCs and spermatogenesis. PMID:27857213

5-Hydroxymethylcytosine-mediated alteration of transposon activity associated with the exposure to adverse in utero environments in human

PubMed Central

Sun, Miao; Song, Mingxi M.; Wei, Bin; Gao, Qinqin; Li, Lingjun; Yao, Bing; Chen, Li; Lin, Li; Dai, Qing; Zhou, Xiuwen; Tao, Jianying; Chen, Jie; He, Chuan; Jin, Peng; Xu, Zhice

2016-01-01

Preeclampsia and gestational diabetes mellitus (GDM) are the most common clinical conditions in pregnancy that could result in adverse in utero environments. Fetal exposure to poor environments may raise the long-term risk of postnatal disorders, while epigenetic modifications could be involved. Recent research has implicated involvement of 5-hydroxymethylcytosine (5hmC), a DNA base derived from 5-methylcytosine, via oxidation by ten–eleven translocation (TET) enzymes, in DNA methylation-related plasticity. Here, we show that the TET2 expression and 5hmC abundance are significantly altered in the umbilical veins of GDM and preeclampsia. Genome-wide profiling of 5hmC revealed its specific reduction on intragenic regions from both GDM and preeclampsia compared to healthy controls. Gene Ontology analysis using loci bearing unique GDM- and preeclampsia-specific loss-of-5hmC indicated its impact on several critical biological pathways. Interestingly, the substantial alteration of 5hmC on several transposons and repetitive elements led to their differential expression. The alteration of TET expression, 5hmC levels and 5hmC-mediated transposon activity was further confirmed using established hypoxia cell culture model, which could be rescued by vitamin C, a known activator of TET proteins. Together, these results suggest that adverse pregnancy environments could influence 5hmC-mediated epigenetic profile and contribute to abnormal development of fetal vascular systems that may lead to postnatal diseases. PMID:27005421

Controlled insertional mutagenesis using a LINE-1 (ORFeus) gene-trap mouse model.

PubMed

O'Donnell, Kathryn A; An, Wenfeng; Schrum, Christina T; Wheelan, Sarah J; Boeke, Jef D

2013-07-16

A codon-optimized mouse LINE-1 element, ORFeus, exhibits dramatically higher retrotransposition frequencies compared with its native long interspersed element 1 counterpart. To establish a retrotransposon-mediated mouse model with regulatable and potent mutagenic capabilities, we generated a tetracycline (tet)-regulated ORFeus element harboring a gene-trap cassette. Here, we show that mice expressing tet-ORFeus broadly exhibit robust retrotransposition in somatic tissues when treated with doxycycline. Consistent with a significant mutagenic burden, we observed a reduced number of double transgenic animals when treated with high-level doxycycline during embryogenesis. Transgene induction in skin resulted in a white spotting phenotype due to somatic ORFeus-mediated mutations that likely disrupt melanocyte development. The data suggest a high level of transposition in melanocyte precursors and consequent mutation of genes important for melanoblast proliferation, differentiation, or migration. These findings reveal the utility of a retrotransposon-based mutagenesis system as an alternative to existing DNA transposon systems. Moreover, breeding these mice to different tet-transactivator/reversible tet-transactivator lines supports broad functionality of tet-ORFeus because of the potential for dose-dependent, tissue-specific, and temporal-specific mutagenesis.

5-Hydroxymethylcytosine Promotes Proliferation of Human Uterine Leiomyoma: A Biological Link to a New Epigenetic Modification in Benign Tumors

PubMed Central

Navarro, Antonia; Yin, Ping; Ono, Masanori; Monsivais, Diana; Moravek, Molly B.; Coon, John S.; Dyson, Matthew T.; Wei, Jian-Jun

2014-01-01

Context: Uterine leiomyoma, or fibroids, represent the most common benign tumors of the female reproductive tract. A newly discovered epigenetic modification, 5-hydroxymethylation (5-hmC), and its regulators, the TET (Ten Eleven Translocation) enzymes, were implicated in the pathology of malignant tumors; however, their roles in benign tumors, including uterine fibroids, remain unknown. Objective: To determine the role of 5-hmC and TET proteins in the pathogenesis of leiomyoma using human uterine leiomyoma and normal matched myometrial tissues and primary cells. Design: 5-hmC levels were determined by ELISA and immunofluorescent staining in matched myometrial and leiomyoma tissues. TET expression was analyzed by quantitative RT-PCR and immunoblotting. TET1 or TET3 were silenced or inhibited by small interfering RNA or 2-hydroxyglutarate to study their effects on 5-hmC content and cell proliferation. Results: We demonstrated significantly higher 5-hmC levels in the genomic DNA of leiomyoma tissue compared to normal myometrial tissue. The increase in 5-hmC levels was associated with the up-regulation of TET1 or TET3 mRNA and protein expression in leiomyoma tissue. TET1 or TET3 knockdown significantly reduced 5-hmC levels in leiomyoma cells and decreased cell proliferation. Treatment with 2-hydroxyglutarate, a competitive TET enzyme inhibitor, significantly decreased both 5-hmC content and cell proliferation of leiomyoma cells. Conclusion: An epigenetic imbalance in the 5-hmC content of leiomyoma tissue, caused by up-regulation of the TET1 and TET3 enzymes, might lead to discovery of new therapeutic targets in leiomyoma. PMID:25057885

TET1-mediated hypomethylation activates oncogenic signaling in triple-negative breast cancer.

PubMed

Good, Charly Ryan; Panjarian, Shoghag; Kelly, Andrew D; Madzo, Jozef; Patel, Bela; Jelinek, Jaroslav; Issa, Jean-Pierre J

2018-06-11

Both gains and losses of DNA methylation are common in cancer, but the factors controlling this balance of methylation remain unclear. Triple-negative breast cancer (TNBC), a subtype that does not overexpress hormone receptors or HER2/NEU, is one of the most hypomethylated cancers observed. Here we discovered that the TET1 DNA demethylase is specifically overexpressed in about 40% of patients with TNBC, where it is associated with hypomethylation of up to 10% of queried CpG sites and a worse overall survival. Through bioinformatic analyses in both breast and ovarian cancer cell line panels, we uncovered an intricate network connecting TET1 to hypomethylation and activation of cancer-specific oncogenic pathways including PI3K, EGFR, and PDGF. TET1 expression correlated with sensitivity to drugs targeting the PI3K-mTOR pathway, and CRISPR-mediated deletion of TET1 in two independent TNBC cell lines resulted in reduced expression of PI3K pathway genes, upregulation of immune response genes, and substantially reduced cellular proliferation, suggesting dependence of oncogenic pathways on TET1 overexpression. Our work establishes TET1 as a potential oncogene that contributes to aberrant hypomethylation in cancer and suggests that TET1 could serve as a druggable target for therapeutic intervention. Copyright ©2018, American Association for Cancer Research.

Detection and Diversity Evaluation of Tetracycline Resistance Genes in Grassland-Based Production Systems in Colombia, South America

PubMed Central

Santamaría, Johanna; López, Liliana; Soto, Carlos Yesid

2011-01-01

Grassland-based production systems use ∼26% of land surface on earth. However, there are no evaluations of these systems as a source of antibiotic pollution. This study was conducted to evaluate the presence, diversity, and distribution of tetracycline resistance genes in the grasslands of the Colombian Andes, where administration of antibiotics to animals is limited to treat disease and growth promoters are not included in animals’ diet. Animal (ruminal fluid and feces) and environmental (soil and water) samples were collected from different dairy cattle farms and evaluated by PCR for the genes tet(M), tet(O), tetB(P), tet(Q), tet(W), tet(S), tet(T), otr(A), which encode ribosomal protection proteins (RPPs), and the genes tet(A), tet(B), tet(D), tet(H), tet(J), and tet(Z), encoding efflux pumps. A wide distribution and high frequency for genes tet(W) and tet(Q) were found in both sample types. Genes tet(O) and tetB(P), detected in high frequencies in feces, were detected in low frequencies or not detected at all in the environment. Other genes encoding RPPs, such as tet(M), tet(S), and tet(T), were detected at very low frequencies and restricted distributions. Genes encoding efflux pumps were not common in this region, and only two of them, tet(B) and tet(Z), were detected. DGGE–PCR followed by comparative sequence analysis of tet(W) and tet(Q) showed that the sequences detected in animals did not differ from those coming from soil and water. Finally, the farms sampled in this study showed more than 50% similarity in relation to the tet genes detected. In conclusion, there was a remarkable presence of tet genes in these production systems and, although not all genes detected in animal reservoirs were detected in the environment, there is a predominant distribution of tet(W) and tet(Q) in both animal and environmental reservoirs. Sequence similarity analysis suggests the transmission of these genes from animals to the environment. PMID:22174707

Regulated Expression of Adenoviral Vectors-Based Gene Therapies

PubMed Central

Curtin, James F.; Candolfi, Marianela; Puntel, Mariana; Xiong, Weidong; Muhammad, A. K. M.; Kroeger, Kurt; Mondkar, Sonali; Liu, Chunyan; Bondale, Niyati; Lowenstein, Pedro R.; Castro, Maria G.

2008-01-01

Summary Regulatable promoter systems allow gene expression to be tightly controlled in vivo. This is highly desirable for the development of safe, efficacious adenoviral vectors that can be used to treat human diseases in the clinic. Ideally, regulatable cassettes should have minimal gene expression in the “OFF” state, and expression should quickly reach therapeutic levels in the “ON” state. In addition, the components of regulatable cassettes should be non-toxic at physiological concentrations and should not be immunogenic, especially when treating chronic illness that requires long-lasting gene expression. In this chapter, we will describe in detail protocols to develop and validate first generation (Ad) and high-capacity adenoviral (HC-Ad) vectors that express therapeutic genes under the control of the TetON regulatable system. Our laboratory has successfully used these protocols to regulate the expression of marker genes, immune stimulatory genes, and toxins for cancer gene therapeutics, i.e., glioma that is a deadly form of brain cancer. We have shown that this third generation TetON regulatable system, incorporating a doxycycline (DOX)-sensitive rtTA2S-M2 inducer and tTSKid silencer, is non-toxic, relatively non-immunogenic, and can tightly regulate reporter transgene expression downstream of a TRE promoter from adenoviral vectors in vitro and also in vivo. PMID:18470649

TET1-GPER-PI3K/AKT pathway is involved in insulin-driven endometrial cancer cell proliferation.

PubMed

Xie, Bing-Ying; Lv, Qiao-Ying; Ning, Cheng-Cheng; Yang, Bing-Yi; Shan, Wei-Wei; Cheng, Ya-Li; Gu, Chao; Luo, Xue-Zhen; Zhang, Zhen-Bo; Chen, Xiao-Jun; Xi, Xiao-Wei; Feng, You-Ji

2017-01-22

Large amount of clinical evidence has demonstrated that insulin resistance is closely related to oncogenesis of endometrial cancer (EC). Despite recent studies showed the up-regulatory role of insulin in G protein-coupled estrogen receptor (GPER/GPR30) expression, GPER expression was not decreased compared to control when insulin receptor was blocked even in insulin treatment. The purpose of this study was to explore the possible mechanism by which insulin up-regulates GPER that drives EC cell proliferation. For this purpose, we first investigated the GPER expression in tissues of endometrial lesions, further explored the effect of GPER on EC cell proliferation in insulin resistance context. Then we analyzed the role of Ten-Eleven Translocation 1 (TET1) in insulin-induced GEPR expression and EC cell proliferation. The results showed that GPER was highly expressed in endometrial atypical hyperplasia and EC tissues. Mechanistically, insulin up-regulated TET1 expression and the latter played an important role in up-regulating GPER expression and activating PI3K/AKT signaling pathway. TET1 mediated GPER up-regulation was another mechanism that insulin promotes EC cell proliferation. Copyright © 2016 Elsevier Inc. All rights reserved.

Induced DNA demethylation by targeting Ten-Eleven Translocation 2 to the human ICAM-1 promoter

PubMed Central

Chen, Hui; Kazemier, Hinke G; de Groote, Marloes L.; Ruiters, Marcel H. J.; Xu, Guo-Liang; Rots, Marianne G.

2014-01-01

Increasing evidence indicates that active DNA demethylation is involved in several processes in mammals, resulting in developmental stage-specificity and cell lineage-specificity. The recently discovered Ten-Eleven Translocation (TET) dioxygenases are accepted to be involved in DNA demethylation by initiating 5-mC oxidation. Aberrant DNA methylation profiles are associated with many diseases. For example in cancer, hypermethylation results in silencing of tumor suppressor genes. Such silenced genes can be re-expressed by epigenetic drugs, but this approach has genome-wide effects. In this study, fusions of designer DNA binding domains to TET dioxygenase family members (TET1, -2 or -3) were engineered to target epigenetically silenced genes (ICAM-1, EpCAM). The effects on targeted CpGs’ methylation and on expression levels of the target genes were assessed. The results indicated demethylation of targeted CpG sites in both promoters for targeted TET2 and to a lesser extent for TET1, but not for TET3. Interestingly, we observed re-activation of transcription of ICAM-1. Thus, our work suggests that we provided a mechanism to induce targeted DNA demethylation, which facilitates re-activation of expression of the target genes. Furthermore, this Epigenetic Editing approach is a powerful tool to investigate functions of epigenetic writers and erasers and to elucidate consequences of epigenetic marks. PMID:24194590

Generation of iPS-derived model cells for analyses of hair shaft differentiation.

PubMed

Kido, Takumi; Horigome, Tomoatsu; Uda, Minori; Adachi, Naoki; Hirai, Yohei

2017-09-01

Biological evaluation of hair growth/differentiation activity in vitro has been a formidable challenge, primarily due to the lack of relevant model cell systems. To solve this problem, we generated a stable model cell line in which successive differentiation via epidermal progenitors to hair components is easily inducible and traceable. Mouse induced pluripotent stem (iPS) cell-derived cells were selected to stably express a tetracycline (Tet)-inducible bone morphogenic protein-4 (BMP4) expression cassette and a luciferase reporter driven by a hair-specific keratin 31 gene (krt31) promoter (Tet-BMP4-KRT31-Luc iPS). While Tet- BMP4-KRT31-Luc iPS cells could be maintained as stable iPS cells, the cells differentiated to produce luciferase luminescence in the presence of all-trans retinoic acid (RA) and doxycycline (Dox), and addition of a hair differentiation factor significantly increased luciferase fluorescence. Thus, this cell line may provide a reliable cell-based screening system to evaluate drug candidates for hair differentiation activity.

TET proteins regulate the lineage specification and TCR-mediated expansion of iNKT cells.

PubMed

Tsagaratou, Ageliki; González-Avalos, Edahí; Rautio, Sini; Scott-Browne, James P; Togher, Susan; Pastor, William A; Rothenberg, Ellen V; Chavez, Lukas; Lähdesmäki, Harri; Rao, Anjana

2017-01-01

TET proteins oxidize 5-methylcytosine in DNA to 5-hydroxymethylcytosine and other oxidation products. We found that simultaneous deletion of Tet2 and Tet3 in mouse CD4 + CD8 + double-positive thymocytes resulted in dysregulated development and proliferation of invariant natural killer T cells (iNKT cells). Tet2-Tet3 double-knockout (DKO) iNKT cells displayed pronounced skewing toward the NKT17 lineage, with increased DNA methylation and impaired expression of genes encoding the key lineage-specifying factors T-bet and ThPOK. Transfer of purified Tet2-Tet3 DKO iNKT cells into immunocompetent recipient mice resulted in an uncontrolled expansion that was dependent on the nonclassical major histocompatibility complex (MHC) protein CD1d, which presents lipid antigens to iNKT cells. Our data indicate that TET proteins regulate iNKT cell fate by ensuring their proper development and maturation and by suppressing aberrant proliferation mediated by the T cell antigen receptor (TCR).

ATNT: an enhanced system for expression of polycistronic secondary metabolite gene clusters in Aspergillus niger.

PubMed

Geib, Elena; Brock, Matthias

2017-01-01

Fungi are treasure chests for yet unexplored natural products. However, exploitation of their real potential remains difficult as a significant proportion of biosynthetic gene clusters appears silent under standard laboratory conditions. Therefore, elucidation of novel products requires gene activation or heterologous expression. For heterologous gene expression, we previously developed an expression platform in Aspergillus niger that is based on the transcriptional regulator TerR and its target promoter P terA . In this study, we extended this system by regulating expression of terR  by the doxycycline inducible Tet-on system. Reporter genes cloned under the control of the target promoter P terA remained silent in the absence of doxycycline, but were strongly expressed when doxycycline was added. Reporter quantification revealed that the coupled system results in about five times higher expression rates compared to gene expression under direct control of the Tet-on system. As production of secondary metabolites generally requires the expression of several biosynthetic genes, the suitability of the self-cleaving viral peptide sequence P2A was tested in this optimised expression system. P2A allowed polycistronic expression of genes required for Asp-melanin formation in combination with the gene coding for the red fluorescent protein tdTomato. Gene expression and Asp-melanin formation was prevented in the absence of doxycycline and strongly induced by addition of doxycycline. Fluorescence studies confirmed the correct subcellular localisation of the respective enzymes. This tightly regulated but strongly inducible expression system enables high level production of secondary metabolites most likely even those with toxic potential. Furthermore, this system is compatible with polycistronic gene expression and, thus, suitable for the discovery of novel natural products.

TET1-mediated DNA hypomethylation regulates the expression of MUC4 in lung cancer

PubMed Central

Yokoyama, Seiya; Higashi, Michiyo; Tsutsumida, Hideaki; Wakimoto, Jouji; Hamada, Tomofumi; Wiest, Edwin; Matsuo, Kei; Kitazono, Ikumi; Goto, Yuko; Guo, Xin; Hamada, Taiji; Yamada, Sohsuke; Hiraki, Tsubasa; Yonezawa, Suguru; Batra, Surinder K.; Hollingsworth, Michael A.; Tanimoto, Akihide

2017-01-01

Lung cancer remains a disease of high mortality, despite advanced diagnostic techniques. Mucins (MUC) play crucial roles in carcinogenesis and tumor invasion in lung neoplasms. Our immunohistochemistry (IHC) studies have shown that high MUC4 expression correlates with a poor outcome. We have also shown that the expression of several mucin genes in cancer cell lines is regulated by DNA methylation. We evaluated the expression level of MUC4, mRNA and several DNA hypomethylation factors in lung tissue samples from 33 patients with various lung lesions. The results indicated that the DNA methylation status of MUC4 matched the expression level of mRNA. In addition, the TET1 (Ten-Eleven Translocation) mRNA showed a significant correlation with the status of DNA methylation of MUC4. Furthermore, the treatment of a lung cancer cell line with TET1 siRNA caused a reduction in MUC4 mRNA expression. Thus, we suggest that TET1 mediated DNA hypomethylation plays a key role in the expression of MUC4. This is the first report that TET1 mediated DNA hypomethylation regulates the expression of MUC4 in lung cancer. The analysis of these epigenetic changes may be useful for diagnosing carcinogenic risk. PMID:28680536

TET1-mediated DNA hypomethylation regulates the expression of MUC4 in lung cancer.

PubMed

Yokoyama, Seiya; Higashi, Michiyo; Tsutsumida, Hideaki; Wakimoto, Jouji; Hamada, Tomofumi; Wiest, Edwin; Matsuo, Kei; Kitazono, Ikumi; Goto, Yuko; Guo, Xin; Hamada, Taiji; Yamada, Sohsuke; Hiraki, Tsubasa; Yonezawa, Suguru; Batra, Surinder K; Hollingsworth, Michael A; Tanimoto, Akihide

2017-03-01

Lung cancer remains a disease of high mortality, despite advanced diagnostic techniques. Mucins (MUC) play crucial roles in carcinogenesis and tumor invasion in lung neoplasms. Our immunohistochemistry (IHC) studies have shown that high MUC4 expression correlates with a poor outcome. We have also shown that the expression of several mucin genes in cancer cell lines is regulated by DNA methylation. We evaluated the expression level of MUC4, mRNA and several DNA hypomethylation factors in lung tissue samples from 33 patients with various lung lesions. The results indicated that the DNA methylation status of MUC4 matched the expression level of mRNA. In addition, the TET1 (Ten-Eleven Translocation) mRNA showed a significant correlation with the status of DNA methylation of MUC4 . Furthermore, the treatment of a lung cancer cell line with TET1 siRNA caused a reduction in MUC4 mRNA expression. Thus, we suggest that TET1 mediated DNA hypomethylation plays a key role in the expression of MUC4. This is the first report that TET1 mediated DNA hypomethylation regulates the expression of MUC4 in lung cancer. The analysis of these epigenetic changes may be useful for diagnosing carcinogenic risk.

Fungal Gene Expression on Demand: an Inducible, Tunable, and Metabolism-Independent Expression System for Aspergillus niger▿†

PubMed Central

Meyer, Vera; Wanka, Franziska; van Gent, Janneke; Arentshorst, Mark; van den Hondel, Cees A. M. J. J.; Ram, Arthur F. J.

2011-01-01

Filamentous fungi are the cause of serious human and plant diseases but are also exploited in biotechnology as production platforms. Comparative genomics has documented their genetic diversity, and functional genomics and systems biology approaches are under way to understand the functions and interaction of fungal genes and proteins. In these approaches, gene functions are usually inferred from deletion or overexpression mutants. However, studies at these extreme points give only limited information. Moreover, many overexpression studies use metabolism-dependent promoters, often causing pleiotropic effects and thus limitations in their significance. We therefore established and systematically evaluated a tunable expression system for Aspergillus niger that is independent of carbon and nitrogen metabolism and silent under noninduced conditions. The system consists of two expression modules jointly targeted to a defined genomic locus. One module ensures constitutive expression of the tetracycline-dependent transactivator rtTA2S-M2, and one module harbors the rtTA2S-M2-dependent promoter that controls expression of the gene of interest (the Tet-on system). We show here that the system is tight, responds within minutes after inducer addition, and allows fine-tuning based on the inducer concentration or gene copy number up to expression levels higher than the expression levels of the gpdA promoter. We also validate the Tet-on system for the generation of conditional overexpression mutants and demonstrate its power when combined with a gene deletion approach. Finally, we show that the system is especially suitable when the functions of essential genes must be examined. PMID:21378046

Overexpression of Tet3 in donor cells enhances goat somatic cell nuclear transfer efficiency.

PubMed

Han, Chengquan; Deng, Ruizhi; Mao, Tingchao; Luo, Yan; Wei, Biao; Meng, Peng; Zhao, Lu; Zhang, Qing; Quan, Fusheng; Liu, Jun; Zhang, Yong

2018-05-23

Ten-eleven translocation 3 (TET3) mediates active DNA demethylation of paternal genomes during mouse embryonic development. However, the mechanism of DNA demethylation in goat embryos remains unknown. In addition, aberrant DNA methylation reprogramming prevalently occurs in embryos cloned by somatic cell nuclear transfer (SCNT). In this study, we reported that TET3 is a key factor in DNA demethylation in goat pre-implantation embryos. Knockdown of Tet3 hindered DNA demethylation at the two- to four-cell stage in goat embryos and decreased Nanog expression in blastocysts. Overexpression of Tet3 in somatic cells can initiate DNA demethylation, reduce 5-methylcytosine level, increase 5-hydroxymethylcytosine level and promote the expression of key pluripotency genes. After SCNT, overexpression of Tet3 in donor cells corrected abnormal DNA hypermethylation of cloned embryos and significantly enhanced in vitro and in vivo developmental rate (P < 0.05). We conclude that overexpression of Tet3 in donor cells significantly improves goat SCNT efficiency. © 2018 Federation of European Biochemical Societies.

A new tetracycline efflux gene, tet(40), is located in tandem with tet(O/32/O) in a human gut firmicute bacterium and in metagenomic library clones.

PubMed

Kazimierczak, Katarzyna A; Rincon, Marco T; Patterson, Andrea J; Martin, Jennifer C; Young, Pauline; Flint, Harry J; Scott, Karen P

2008-11-01

The bacterium Clostridium saccharolyticum K10, isolated from a fecal sample obtained from a healthy donor who had received long-term tetracycline therapy, was found to carry three tetracycline resistance genes: tet(W) and the mosaic tet(O/32/O), both conferring ribosome protection-type resistance, and a novel, closely linked efflux-type resistance gene designated tet(40). tet(40) encodes a predicted membrane-associated protein with 42% amino acid identity to tetA(P). Tetracycline did not accumulate in Escherichia coli cells expressing the Tet(40) efflux protein, and resistance to tetracycline was reduced when cells were incubated with an efflux pump inhibitor. E. coli cells carrying tet(40) had a 50% inhibitory concentration of tetracycline of 60 microg/ml. Analysis of a transconjugant from a mating between donor strain C. saccharolyticum K10 and the recipient human gut commensal bacterium Roseburia inulinivorans suggested that tet(O/32/O) and tet(40) were cotransferred on a mobile element. Sequence analysis of a 37-kb insert identified on the basis of tetracycline resistance from a metagenomic fosmid library again revealed a tandem arrangement of tet(O/32/O) and tet(40), flanked by regions with homology to parts of the VanG operon previously identified in Enterococcus faecalis. At least 10 of the metagenomic inserts that carried tet(O/32/O) also carried tet(40), suggesting that tet(40), although previously undetected, may be an abundant efflux gene.

Novel Epigenetic Controlling of Hypoxia Pathway Related to Overexpression and Promoter Hypomethylation of TET1 and TET2 in RPE Cells.

PubMed

Alivand, Mohammad Reza; Soheili, Zahra-Soheila; Pornour, Majid; Solali, Saeed; Sabouni, Farzaneh

2017-10-01

CpG methylation of DNA takes part in a specific epigenetic memory that plays crucial roles in the differentiation and abnormality of the cells. The methylation pattern aberration of genomes is affected in three ways, namely DNA methyltransferase (DNMT), ten-eleven translocation (TET), and methyl-binding domain (MBD) proteins. Of these, TET enzymes have recently been demonstrated to be master modifier enzymes in the DNA methylation process. Additionally, recent studies emphasize that not only epigenetic phenomena play a role in controlling hypoxia pathway, but the hypoxia condition also triggers hypomethylation of genomes that may help with the expression of hypoxia pathway genes. In this study, we suggested that TET1 and TET2 could play a role in the demethylation of genomes under chemical hypoxia conditions. Herein, the evaluating methylation status and mRNA expression of mentioned genes were utilized through real-time PCR and methylation-specific PCR (MSP), respectively. Our results showed that TET1 and TET2 genes were overexpressed (P < 0.05) under chemical hypoxia conditions in Retinal Pigment Epithelial (RPE) cells, whereas the promoter methylation status of them were hypomethylated in the same condition. Therefore, chemical hypoxia not only causes overexpression of TET1 and TET2 but also could gradually do promoter demethylation of same genes. This is the first study to show the relationship between epigenetics and the expression of mentioned genes related to hypoxia pathways. Furthermore, it seems that these associations in RPE cells are subjected to chemical hypoxia as a mechanism that could play a crucial role in methylation pattern changes of hypoxia-related diseases such as cancer and ischemia. J. Cell. Biochem. 118: 3193-3204, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

5-Hydroxymethylcytosine-mediated alteration of transposon activity associated with the exposure to adverse in utero environments in human.

PubMed

Sun, Miao; Song, Mingxi M; Wei, Bin; Gao, Qinqin; Li, Lingjun; Yao, Bing; Chen, Li; Lin, Li; Dai, Qing; Zhou, Xiuwen; Tao, Jianying; Chen, Jie; He, Chuan; Jin, Peng; Xu, Zhice

2016-06-01

Preeclampsia and gestational diabetes mellitus (GDM) are the most common clinical conditions in pregnancy that could result in adverse in utero environments. Fetal exposure to poor environments may raise the long-term risk of postnatal disorders, while epigenetic modifications could be involved. Recent research has implicated involvement of 5-hydroxymethylcytosine (5hmC), a DNA base derived from 5-methylcytosine, via oxidation by ten-eleven translocation (TET) enzymes, in DNA methylation-related plasticity. Here, we show that the TET2 expression and 5hmC abundance are significantly altered in the umbilical veins of GDM and preeclampsia. Genome-wide profiling of 5hmC revealed its specific reduction on intragenic regions from both GDM and preeclampsia compared to healthy controls. Gene Ontology analysis using loci bearing unique GDM- and preeclampsia-specific loss-of-5hmC indicated its impact on several critical biological pathways. Interestingly, the substantial alteration of 5hmC on several transposons and repetitive elements led to their differential expression. The alteration of TET expression, 5hmC levels and 5hmC-mediated transposon activity was further confirmed using established hypoxia cell culture model, which could be rescued by vitamin C, a known activator of TET proteins. Together, these results suggest that adverse pregnancy environments could influence 5hmC-mediated epigenetic profile and contribute to abnormal development of fetal vascular systems that may lead to postnatal diseases. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Topical Tetracycline Improves MC903-induced Atopic Dermatitis in Mice through Inhibition of Inflammatory Cytokines and Thymic Stromal Lymphopoietin Expression.

PubMed

Liu, Xiao-Jing; Mu, Zhang-Lei; Zhao, Yan; Zhang, Jian-Zhong

2016-06-20

Tetracycline (TET) has been found to have both antibiotic and anti-inflammatory properties. The anti-inflammatory effect of topical TET on atopic dermatitis (AD) has not been reported. The purpose of this study was to explore the potential role of topical TET and its anti-inflammatory effects in a mouse model of AD. The 2% TET was applied topically to ears of MC903-induced AD-like BALB/c mice once a day. AD-like symptoms and severity were evaluated by assessing skin scoring of dermatitis, ear thickness, and frequency of scratching. Serum IgE and thymic stromal lymphopoietin (TSLP) levels were measured by enzyme-linked immunosorbent assay. Western blot was used for analyzing the expressions of TSLP, protease-activated receptor 2 (PAR2), and nuclear factor-kappa B (NF-κB) in skin lesions. Real-time polymerase chain reaction was performed to assess the mRNA levels of TSLP and inflammatory cytokines including interleukin (IL)-4, IL-13, tumor necrosis factor (TNF)-α, and IL-1β in skin lesions. Scoring of dermatitis (9.00 ± 0.63 vs. 6.67 ± 1.03, P = 0.001), ear thickness (0.44 ± 0.02 mm vs. 0.40 ± 0.03 mm, P = 0.018), and serum IgE level (421.06 ± 212.13 pg/ml vs. 244.15 ± 121.39 pg/ml, P = 0.047) were all improved in the 2% TET treatment group compared with AD group. Topical TET significantly reduced the serum level of TSLP (119.04 ± 38.92 pg/ml vs. 65.95 ± 54.61 pg/ml, P = 0.011) and both mRNA and protein expressions of TSLP in skin lesions compared with AD group (P = 0.003 and 0.011, respectively), and NF-κB and PAR2 expression in skin lesions were also suppressed (P = 0.016 and 0.040, respectively). Furthermore, expressions of inflammatory cytokines IL-4, IL-13, and TNF-α in skin lesions were down-regulated in 2% TET group compared with AD group (P = 0.035, 0.008, and 0.044, respectively). Topical TET exerted anti-inflammatory effects through suppression of TSLP and inflammatory cytokines in AD mouse model, suggesting TET as a potential agent for the topical treatment of AD in the future.

A Comprehensive Analysis on Spread and Distribution Characteristic of Antibiotic Resistance Genes in Livestock Farms of Southeastern China.

PubMed

Wang, Na; Guo, Xinyan; Yan, Zheng; Wang, Wei; Chen, Biao; Ge, Feng; Ye, Boping

2016-01-01

The pollution of antibiotic resistance genes (ARGs) in livestock farms is a problem which need to be paid more attention to, due to the severe resistance dissemination and the further human health risk. In this study, all the relevant exposure matrices (manure, soil and water) of sixteen animal farms in Southeastern China were sampled to determine twenty-two ARGs conferring resistance to five major classes of antibiotics including tetracyclines, sulfonamides, quinolones, aminoglycosides, and macrolides. The results showed that the spread property of sul genes was most extensive and strong, followed by tet and erm genes. The abundance of tet genes expressing ribosomal protection proteins (tetM, tetO, tetQ, tetT and tetW) was higher than that expressing efflux pump proteins (tetA, tetC, tetE and tetG) in each type of samples. The high abundance and frequency of ermB gene in the matrices should be paid more attention, because macrolides is a major medicine for human use. For manures, it was found that the similar ARGs distribution rules were existing in poultry manure or porcine manure samples, despite of the different origins of these two types of livestock farms. Meanwhile, it was interesting that the distribution rule of tet genes in animal manure was nearly the same as all the ARGs. For soils, the result of nonmetric multi-dimensional scaling (NMDS) analysis showed that the pollution of ARGs in the soils fertilized by poultry and cattle manures were more substantial in northern Jiangsu, but no significant ARGs diversity was observed among porcine manured soils of five different regions. Furthermore, most ARGs showed significant positive relationships with environmental variables such as concentration of sulfonamides, tetracyclines, Cu, Zn and total organic carbon (TOC). The pollution profile and characteristics of so many ARGs in livestock farms can provide significative foundation for the regulation and legislation of antibiotics in China.

Functions of TET Proteins in Hematopoietic Transformation.

PubMed

Han, Jae-A; An, Jungeun; Ko, Myunggon

2015-11-01

DNA methylation is a well-characterized epigenetic modification that plays central roles in mammalian development, genomic imprinting, X-chromosome inactivation and silencing of retrotransposon elements. Aberrant DNA methylation pattern is a characteristic feature of cancers and associated with abnormal expression of oncogenes, tumor suppressor genes or repair genes. Ten-eleven-translocation (TET) proteins are recently characterized dioxygenases that catalyze progressive oxidation of 5-methylcytosine to produce 5-hydroxymethylcytosine and further oxidized derivatives. These oxidized methylcytosines not only potentiate DNA demethylation but also behave as independent epigenetic modifications per se. The expression or activity of TET proteins and DNA hydroxymethylation are highly dysregulated in a wide range of cancers including hematologic and non-hematologic malignancies, and accumulating evidence points TET proteins as a novel tumor suppressor in cancers. Here we review DNA demethylation-dependent and -independent functions of TET proteins. We also describe diverse TET loss-of-function mutations that are recurrently found in myeloid and lymphoid malignancies and their potential roles in hematopoietic transformation. We discuss consequences of the deficiency of individual Tet genes and potential compensation between different Tet members in mice. Possible mechanisms underlying facilitated oncogenic transformation of TET-deficient hematopoietic cells are also described. Lastly, we address non-mutational mechanisms that lead to suppression or inactivation of TET proteins in cancers. Strategies to restore normal 5mC oxidation status in cancers by targeting TET proteins may provide new avenues to expedite the development of promising anti-cancer agents.

Eicosapentaenoic acid induces DNA demethylation in carcinoma cells through a TET1-dependent mechanism.

PubMed

Ceccarelli, Veronica; Valentini, Virginia; Ronchetti, Simona; Cannarile, Lorenza; Billi, Monia; Riccardi, Carlo; Ottini, Laura; Talesa, Vincenzo Nicola; Grignani, Francesco; Vecchini, Alba

2018-05-14

In cancer cells, global genomic hypomethylation is found together with localized hypermethylation of CpG islands within the promoters and regulatory regions of silenced tumor suppressor genes. Demethylating agents may reverse hypermethylation, thus promoting gene re-expression. Unfortunately, demethylating strategies are not efficient in solid tumor cells. DNA demethylation is mediated by ten-eleven translocation enzymes (TETs). They sequentially convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which is associated with active transcription; 5-formylcytosine; and finally, 5-carboxylcytosine. Although α-linolenic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid, the major n-3 polyunsaturated fatty acids, have anti-cancer effects, their action, as DNA-demethylating agents, has never been investigated in solid tumor cells. Here, we report that EPA demethylates DNA in hepatocarcinoma cells. EPA rapidly increases 5hmC on DNA, inducing p21 Waf1/Cip1 gene expression, which slows cancer cell-cycle progression. We show that the underlying molecular mechanism involves TET1. EPA simultaneously binds peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor α (RXRα), thus promoting their heterodimer and inducing a PPARγ-TET1 interaction. They generate a TET1-PPARγ-RXRα protein complex, which binds to a hypermethylated CpG island on the p21 gene, where TET1 converts 5mC to 5hmC. In an apparent shuttling motion, PPARγ and RXRα leave the DNA, whereas TET1 associates stably. Overall, EPA directly regulates DNA methylation levels, permitting TET1 to exert its anti-tumoral function.-Ceccarelli, V., Valentini, V., Ronchetti, S., Cannarile, L., Billi, M., Riccardi, C., Ottini, L., Talesa, V. N., Grignani, F., Vecchini, A., Eicosapentaenoic acid induces DNA demethylation in carcinoma cells through a TET1-dependent mechanism.

Osteopontin regulates adhesion of calcium oxalate crystals to renal epithelial cells.

PubMed

Yasui, Takahiro; Fujita, Keiji; Asai, Kiyofumi; Kohri, Kenjiro

2002-02-01

The association of calcium crystals with renal tubular cells is an important factor during the formation of urinary stones. We previously reported the strong expression of osteopontin (OPN) on renal tubular cells in the stone-forming kidney, suggesting that OPN plays a role in the crystal-cell interaction. In the present study, we examined the biological consequences of inhibiting OPN expression at the translational level on the formation and adhesion of crystals. We synthesized antisense OPN expression vector (pTet-OPNas) using the tetracycline-regulated expression system. The pTet-OPNas was constructed using a mouse OPN cDNA sequence in an inverted (antisense) orientation. Two clones (NRK-52E/ASs) were identified by transfection of pTet-OPNas into NRK-52E cells and they showed a marked reduction of OPN synthesis in the absence of tetracycline. Calcium oxalate (CaOx) crystal suspension was spread homogeneously on top of the NRK-52E cells. After incubation, the association of CaOx crystals and cells was visualized by scanning electron microscopy. Intact NRK-52E cells, NRK-52E cells transfected with empty vector and tetracycline-treated antisense clones (NRK-52E/ASs), under identical conditions, were associated with CaOx crystals. In contrast, the expression of antisense OPN prevented the association of CaOx crystals with NRK-52E cells. Osteopontin plays a crucial role in the adhesion process of CaOx crystals to renal tubular cells in stone formation.

A gene expression system offering multiple levels of regulation: the Dual Drug Control (DDC) system.

PubMed

Sudomoina, Marina; Latypova, Ekaterina; Favorova, Olga O; Golemis, Erica A; Serebriiskii, Ilya G

2004-04-29

Whether for cell culture studies of protein function, construction of mouse models to enable in vivo analysis of disease epidemiology, or ultimately gene therapy of human diseases, a critical enabling step is the ability to achieve finely controlled regulation of gene expression. Previous efforts to achieve this goal have explored inducible drug regulation of gene expression, and construction of synthetic promoters based on two-hybrid paradigms, among others. In this report, we describe the combination of dimerizer-regulated two-hybrid and tetracycline regulatory elements in an ordered cascade, placing expression of endpoint reporters under the control of two distinct drugs. In this Dual Drug Control (DDC) system, a first plasmid expresses fusion proteins to DBD and AD, which interact only in the presence of a small molecule dimerizer; a second plasmid encodes a cassette transcriptionally responsive to the first DBD, directing expression of the Tet-OFF protein; and a third plasmid encodes a reporter gene transcriptionally responsive to binding by Tet-OFF. We evaluate the dynamic range and specificity of this system in comparison to other available systems. This study demonstrates the feasibility of combining two discrete drug-regulated expression systems in a temporally sequential cascade, without loss of dynamic range of signal induction. The efficient layering of control levels allowed by this combination of elements provides the potential for the generation of complex control circuitry that may advance ability to regulate gene expression in vivo.

Elevated CRP levels predict poor outcome and tumor recurrence in patients with thymic epithelial tumors: A pro- and retrospective analysis

PubMed Central

Janik, Stefan; Bekos, Christine; Hacker, Philipp; Raunegger, Thomas; Ghanim, Bahil; Einwallner, Elisa; Beer, Lucian; Klepetko, Walter; Müllauer, Leonhard; Ankersmit, Hendrik J.; Moser, Bernhard

2017-01-01

Objective Scarce information exists on the pathogenesis of thymic epithelial tumors (TETs), comprising thymomas, thymic carcinomas (TCs) and neuroendocrine tumors. C-reactive protein (CRP) increases during certain malignancies. We aimed to investigate the clinical relevance of CRP in patients with TETs. Results Pretreatment CRP serum concentrations were significantly elevated in patients with TETs, particularly TCs and metastatic TETs. After complete tumor resection CRP serum concentrations were decreased (p = 0.135) but increased significantly in case of tumor recurrence (p = 0.001). High pretreatment CRP was associated with significantly worse 5- and 10-year freedom-from recurrence (FFR) (p = 0.010) and was a negative prognostic factor for FFR (HR 3.30; p = 0.015). IL-6 (not IL-1β) serum concentrations were significantly elevated in patients with TETs but we did not detect CRP tissue expression in TETs. Materials and Methods Pretreatment CRP serum concentrations were retrospectively analyzed from 128 surgical patients (1990–2015). In a subset of 68 patients longitudinal analysis of CRP was performed. Additionally, immunohistochemical tumor CRP expression and serum concentrations of interleukin (IL)-6 and IL-1β were measured. Conclusions Hence, diagnostic measurement of serum CRP might be useful to indicate highly aggressive TETs and to make doctors consider tumor recurrences during oncological follow-up. PMID:28514756

Transcript levels of ten-eleven translocation type 1–3 in cervical cancer and non-cancerous cervical tissues

PubMed Central

Bronowicka-Kłys, Dorota Ewa; Roszak, Andrzej; Pawlik, Piotr; Sajdak, Stefan; Sowińska, Anna; Jagodziński, Paweł Piotr

2017-01-01

Decreased expression of ten-eleven translocation (TET1, TET2 and TET3) proteins has been reported in various types of cancer. However, the expression levels of TET proteins in cervical cancer (CC) remain to be elucidated. The present study determined the levels of TET1, TET2 and TET3 transcripts in cancerous (n=80) and non-cancerous cervical tissues (n=41). The results revealed a significant reduction in TET1 transcripts (P=0.0000001) in cervical tissue samples from patients with primary CC compared with samples from control patients. Significantly decreased TET1 transcript levels, as compared to non-cancerous cervical tissues, were also observed in tissue samples with the following characteristics: Stage I (P=0.016), II (P<0.0001), III (P=0.00007) and grade of differentiation G1 (P=0.026), G2 (P=0.00006), G3 (P=0.0007) and Gx (P=0.0004) and squamous histological type (P<0.00001). TET1 transcript levels were significantly lower in patients aged 45–60 years (P=0.0002) and patients age >60 years (P=0.003), as compared with non-cancerous cervical tissues. TET2 transcript levels were lower in cervical cancer tissues classified as stage II (P=0.043) and TET3 transcript levels were lower in stage III samples (P=0.010), tissue samples with a grade of differentiation of G3 (P=0.025) and tissue with squamous type histology (P=0.047), all compared with non-cancerous cervical tissues. The present study demonstrated a significantly reduced level of TET1 transcripts in cancerous cervical tissues, as compared with non-cancerous tissues. Furthermore, decreased TET1-3 transcript levels were identified when patients with CC were stratified by clinicopathological variables, as compared with non-cancerous cervical tissues. PMID:28521490

Methylation and expression profiles of MGMT gene in thymic epithelial tumors.

PubMed

Mokhtar, Mohamed; Kondo, Kazuya; Namura, Toshiaki; Ali, Abdellah H K; Fujita, Yui; Takai, Chikako; Takizawa, Hiromitsu; Nakagawa, Yasushi; Toba, Hiroaki; Kajiura, Koichiro; Yoshida, Mitsuteru; Kawakami, Gyokei; Sakiyama, Shoji; Tangoku, Akira

2014-02-01

A key challenge in diagnosis and treatment of thymic epithelial tumors (TET) is in improving our understanding of the genetic and epigenetic changes of these relatively rare tumors. Methylation specific PCR (MSP) and immunohistochemistry were applied to 66 TET to profile the methylation status of DNA repair gene O6-methylguanine DNA methyltransferase (MGMT) and its protein expression in TET to clarify the association between MGMT status and clinicopathological features, response to chemotherapy and overall survival. MGMT methylation was significantly more frequent in thymic carcinoma than in thymoma (17/23, 74% versus 13/44, 29%; P<0.001). Loss of expression of MGMT protein was significantly more frequent in thymic carcinoma than in thymoma (20/23, 87% versus 10/44, 23%; P<0.0001). There is a significant correlation between of MGMT methylation and loss of its protein expression (P<0.0003). MGMT methylation and loss of expression were significantly more frequent in advanced thymic epithelial tumors (III/IV) than in early tumors (I/II). MGMT methylation plays a soul role in development of TET, especially in thymic carcinoma. Therefore, translation of our results from basic molecular research to clinical practice may have important implication for considering MGMT methylation as a marker and a target of future therapies in TET. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes for Methanosarcina species

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guss, Adam M.; Rother, Michael; Zhang, Jun Kai

A highly efficient method for chromosomal integration of cloned DNA into Methanosarcina spp. was developed utilizing the site-specific recombination system from the Streptomyces phage φC31. Host strains expressing the φC31 integrase gene and carrying an appropriate recombination site can be transformed with non-replicating plasmids carrying the complementary recombination site at efficiencies similar to those obtained with self-replicating vectors. We have also constructed a series of hybrid promoters that combine the highly expressed M. barkeri P mcrB promoter with binding sites for the tetracycline-responsive, bacterial TetR protein. These promoters are tightly regulated by the presence or absence of tetracycline in strainsmore » that express the tetR gene. The hybrid promoters can be used in genetic experiments to test gene essentiality by placing a gene of interest under their control. Thus, growth of strains with tetR -regulated essential genes becomes tetracycline-dependent. A series of plasmid vectors that utilize the site-specific recombination system for construction of reporter gene fusions and for tetracycline regulated expression of cloned genes are reported. These vectors were used to test the efficiency of translation at a variety of start codons. Fusions using an ATG start site were the most active, whereas those using GTG and TTG were approximately one half or one fourth as active, respectively. The CTG fusion was 95% less active than the ATG fusion.« less

New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes for Methanosarcina species

DOE PAGES

Guss, Adam M.; Rother, Michael; Zhang, Jun Kai; ...

2008-01-01

A highly efficient method for chromosomal integration of cloned DNA into Methanosarcina spp. was developed utilizing the site-specific recombination system from the Streptomyces phage φC31. Host strains expressing the φC31 integrase gene and carrying an appropriate recombination site can be transformed with non-replicating plasmids carrying the complementary recombination site at efficiencies similar to those obtained with self-replicating vectors. We have also constructed a series of hybrid promoters that combine the highly expressed M. barkeri P mcrB promoter with binding sites for the tetracycline-responsive, bacterial TetR protein. These promoters are tightly regulated by the presence or absence of tetracycline in strainsmore » that express the tetR gene. The hybrid promoters can be used in genetic experiments to test gene essentiality by placing a gene of interest under their control. Thus, growth of strains with tetR -regulated essential genes becomes tetracycline-dependent. A series of plasmid vectors that utilize the site-specific recombination system for construction of reporter gene fusions and for tetracycline regulated expression of cloned genes are reported. These vectors were used to test the efficiency of translation at a variety of start codons. Fusions using an ATG start site were the most active, whereas those using GTG and TTG were approximately one half or one fourth as active, respectively. The CTG fusion was 95% less active than the ATG fusion.« less

Aberrant TET1 Methylation Closely Associated with CpG Island Methylator Phenotype in Colorectal Cancer.

PubMed

Ichimura, Norihisa; Shinjo, Keiko; An, Byonggu; Shimizu, Yasuhiro; Yamao, Kenji; Ohka, Fumiharu; Katsushima, Keisuke; Hatanaka, Akira; Tojo, Masayuki; Yamamoto, Eiichiro; Suzuki, Hiromu; Ueda, Minoru; Kondo, Yutaka

2015-08-01

Inactivation of methylcytosine dioxygenase, ten-eleven translocation (TET) is known to be associated with aberrant DNA methylation in cancers. Tumors with a CpG island methylator phenotype (CIMP), a distinct subgroup with extensive DNA methylation, show characteristic features in the case of colorectal cancer. The relationship between TET inactivation and CIMP in colorectal cancers is not well understood. The expression level of TET family genes was compared between CIMP-positive (CIMP-P) and CIMP-negative (CIMP-N) colorectal cancers. Furthermore, DNA methylation profiling, including assessment of the TET1 gene, was assessed in colorectal cancers, as well as colon polyps. The TET1 was silenced by DNA methylation in a subset of colorectal cancers as well as cell lines, expression of which was reactivated by demethylating agent. TET1 methylation was more frequent in CIMP-P (23/55, 42%) than CIMP-N (2/113, 2%, P < 0.0001) colorectal cancers. This trend was also observed in colon polyps (CIMP-P, 16/40, 40%; CIMP-N, 2/24, 8%; P = 0.002), suggesting that TET1 methylation is an early event in CIMP tumorigenesis. TET1 methylation was significantly associated with BRAF mutation but not with hMLH1 methylation in the CIMP-P colorectal cancers. Colorectal cancers with TET1 methylation have a significantly greater number of DNA methylated genes and less pathological metastasis compared to those without TET1 methylation (P = 0.007 and 0.045, respectively). Our data suggest that TET1 methylation may contribute to the establishment of a unique pathway in respect to CIMP-mediated tumorigenesis, which may be incidental to hMLH1 methylation. In addition, our findings provide evidence that TET1 methylation may be a good biomarker for the prediction of metastasis in colorectal cancer. ©2015 American Association for Cancer Research.

Microbial phylogeny determines transcriptional response of resistome to dynamic composting processes.

PubMed

Wang, Cheng; Dong, Da; Strong, P J; Zhu, Weijing; Ma, Zhuang; Qin, Yong; Wu, Weixiang

2017-08-16

Animal manure is a reservoir of antibiotic resistance genes (ARGs) that pose a potential health risk globally, especially for resistance to the antibiotics commonly used in livestock production (such as tetracycline, sulfonamide, and fluoroquinolone). Currently, the effects of biological treatment (composting) on the transcriptional response of manure ARGs and their microbial hosts are not well characterized. Composting is a dynamic process that consists of four distinct phases that are distinguished by the temperature resulting from microbial activity, namely the mesophilic, thermophilic, cooling, and maturing phases. In this study, changes of resistome expression were determined and related to active microbiome profiles during the dynamic composting process. This was achieved by integrating metagenomic and time series metatranscriptomic data for the evolving microbial community during composting. Composting noticeably reduced the aggregated expression level of the manure resistome, which primarily consisted of genes encoding for tetracycline, vancomycin, fluoroquinolone, beta-lactam, and aminoglycoside resistance, as well as efflux pumps. Furthermore, a varied transcriptional response of resistome to composting at the ARG levels was highlighted. The expression of tetracycline resistance genes (tetM-tetW-tetO-tetS) decreased during composting, where distinctive shifts in the four phases of composting were related to variations in antibiotic concentration. Composting had no effect on the expression of sulfonamide and fluoroquinolone resistance genes, which increased slightly during the thermophilic phase and then decreased to initial levels. As indigenous populations switched greatly throughout the dynamic composting, the core resistome persisted and their reservoir hosts' composition was significantly correlated with dynamic active microbial phylogenetic structure. Hosts for sulfonamide and fuoroquinolone resistance genes changed notably in phylognetic structure and underwent an initial increase and then a decrease in abundance. By contrast, hosts for tetracycline resistance genes (tetM-tetW-tetO-tetS) exhibited a constant decline through time. The transcriptional patterns of a core resistome over the course of composting were identified, and microbial phylogeny was the key determinant in defining the varied transcriptional response of resistome to this dynamic biological process. This research demonstrated the benefits of composting for manure treatment. It reduced the risk of emerging environmental contaminants such as tetracyclines, tetracycline resistance genes, and clinically relevant pathogens carrying ARGs, as well as RNA viruses and bacteriophages.

Selective inhibition of CTCF binding by iAs directs TET-mediated reprogramming of 5-hydroxymethylation patterns in iAs-transformed cells

PubMed Central

Rea, Matthew; Gripshover, Tyler; Fondufe-Mittendorf, Yvonne

2017-01-01

Methylation at cytosine (5mC) is a fundamental epigenetic DNA modification recently associated with iAs-mediated carcinogenesis. In contrast, the role of 5-hydroxymethylcytosine (5hmC), the oxidation product of 5mC in iAs-mediated carcinogenesis is unknown. Here we assess the hydroxymethylome in iAs-transformed cells, showing that dynamic modulation of hydroxymethylated DNA is associated with specific transcriptional networks. Moreover, this pathologic iAs-mediated carcinogenesis is characterized by a shift toward a higher hydroxymethylation pattern genome-wide. At specific promoters, hydroxymethylation correlated with increased gene expression. Furthermore, this increase in hydroxymethylation occurs concurrently with an upregulation of ten-eleven translocation (TET) enzymes that oxidize 5-methylcytosine (5mC) in DNA. To gain an understanding into how iAs might impact TET expression, we found that iAs inhibits the binding of CTCF at the proximal, weak CTCF binding sites of the TET1 and TET2 gene promoters and enhances CTCF binding at the stronger distal binding site. Further analyses suggest that this distal site acts as an enhancer, thus high CTCF occupancy at the enhancer region of TET1 and TET2 possibly drives their high expression in iAs-transformed cells. These results have major implications in understanding the impact of differential CTCF binding, genome architecture and its consequences in iAs-mediated pathogenesis. PMID:29175454

Automatic Control of Gene Expression in Mammalian Cells.

PubMed

Fracassi, Chiara; Postiglione, Lorena; Fiore, Gianfranco; di Bernardo, Diego

2016-04-15

Automatic control of gene expression in living cells is paramount importance to characterize both endogenous gene regulatory networks and synthetic circuits. In addition, such a technology can be used to maintain the expression of synthetic circuit components in an optimal range in order to ensure reliable performance. Here we present a microfluidics-based method to automatically control gene expression from the tetracycline-inducible promoter in mammalian cells in real time. Our approach is based on the negative-feedback control engineering paradigm. We validated our method in a monoclonal population of cells constitutively expressing a fluorescent reporter protein (d2EYFP) downstream of a minimal CMV promoter with seven tet-responsive operator motifs (CMV-TET). These cells also constitutively express the tetracycline transactivator protein (tTA). In cells grown in standard growth medium, tTA is able to bind the CMV-TET promoter, causing d2EYFP to be maximally expressed. Upon addition of tetracycline to the culture medium, tTA detaches from the CMV-TET promoter, thus preventing d2EYFP expression. We tested two different model-independent control algorithms (relay and proportional-integral (PI)) to force a monoclonal population of cells to express an intermediate level of d2EYFP equal to 50% of its maximum expression level for up to 3500 min. The control input is either tetracycline-rich or standard growth medium. We demonstrated that both the relay and PI controllers can regulate gene expression at the desired level, despite oscillations (dampened in the case of the PI controller) around the chosen set point.

Myelin basic protein-messenger RNA (MBP-mRNA) expression during triethyltin-induced myelin edema.

PubMed

Veronesi, B; Jones, K; Gupta, S; Pringle, J; Mezei, C

1991-01-01

Triethyltin (TET) is a neurotoxicant that produces severe but transient cerebral edema, characterized ultrastructurally by vacuolation of the intraperiod line of central nervous system (CNS) myelin. TET has been reported to depress levels of myelin basic protein (MBP), a protein thought to play a critical role in myelin compaction. In the present study, the genomic expression (i.e., mRNA) of MBP was monitored throughout the pathogenesis of TET-induced myelin edema and recovery in Sprague-Dawley rats given a single injection of a neuropathic (8.0 mg/kg) or non-neuropathic (0.8 mg/kg) dose of TET-bromide. Levels of MBP-mRNA from the anterior and posterior brain were collected 1 hr, 3 hr, 2d, and 7d, postexposure. The optic nerve and caudal brainstem, representing anterior and posterior brain sites, respectively, were examined at the same time-points for ultrastructural evidence of edema and recovery. Our data indicate that neuropathic doses (8.0 mg/kg) of TET significantly stimulated MBP transcript throughout the brain at all exposure time-points. The magnitude and time-course of this stimulation differed in the anterior and posterior brain, with the latter region showing higher levels of MBP-mRNA. In the posterior brain, the highest levels of mRNA correlated with the appearance of edema in the caudal brainstem. In the anterior brain, MBP-mRNA levels were only marginally increased over controls. Ultrastructural evidence of myelin edema was confined to the brainstem in rats treated with neuropathic dose of TET. Intralamellar vacuolation appeared at 3 hr and 2d postexposure and could be correlated with peak levels of MBP transcript, whereas, recompacted myelin, which appeared by 7d postexposure, was associated with declining levels of the mRNA. Ultrastructural changes in the oligodendroglia were suggestive of metabolic stimulation and correlated with high MBP-mRNA levels. In summary, these data indicate that an initial genomic event in TET-induced myelin edema is stimulation of MBP transcript.

Tet1 is required for Rb phosphorylation during G1/S phase transition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Shengsong; Zhu, Ziqi; Wang, Yiqin

2013-05-03

Highlights: •Tet1 was required for NIT3T3 proliferation. •Tet1 depletion inhibited G1-S entry. •Cyclin D1 accumulation and Rb phosphorylation was blocked by Tet1 knockdown. -- Abstract: DNA methylation plays an important role in many biological processes, including regulation of gene expression, maintenance of chromatin conformation and genomic stability. TET-family proteins convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which indicates that these enzymes may participate in DNA demethylation. The function of TET1 has not yet been well characterized in somatic cells. Here, we show that depletion of Tet1 in NIH3T3 cells inhibits cell growth. Furthermore, Tet1 knockdown blocks cyclin D1 accumulation in G1more » phase, inhibits Rb phosphorylation and consequently delays entrance to G1/S phase. Taken together, this study demonstrates that Tet1 is required for cell proliferation and that this process is mediated through the Rb pathway.« less

Decrease of 5-hydroxymethylcytosine in rat liver with subchronic exposure to genotoxic carcinogens riddelliine and aristolochic acid.

PubMed

Lian, Christine Guo; Xu, Shuyun; Guo, Weimin; Yan, Jian; Frank, Maximilian Y M; Liu, Robert; Liu, Cynthia; Chen, Ying; Murphy, George F; Chen, Tao

2015-11-01

The level of 5-hydroxymethylcytosine (5-hmC) converted by ten-eleven translocation (TET) family is decreased in cancers. However, whether 5-hmC level is perturbed in early stages of carcinogenesis caused by genotoxic carcinogens is not defined. 5-hmC levels and TET2 expression were measured in liver of rats treated with genotoxic carcinogens, riddelliine, or aristolochic acid. Levels of 5-hmC and TET2 expression decreased in the liver of the carcinogens-treated rats. Loss of 5-hmC correlates well with documented induction of genetic mutations by the carcinogens, suggesting that TET2-mediated 5-hydroxymethylation plays an epigenetic role in early state of carcinogenesis. © 2014 Wiley Periodicals, Inc.

Decrease of 5-Hydroxymethylcytosine in Rat Liver with Subchronic Exposure to Genotoxic Carcinogens Riddelliine and Aristolochic Acid

PubMed Central

Lian, Christine Guo; Xu, Shuyun; Guo, Weimin; Yan, Jian; Frank, Maximilian Y M; Liu, Robert; Liu, Cynthia; Chen, Ying; Murphy, George F.; Chen, Tao

2018-01-01

The level of 5-hydroxymethylcytosine (5-hmC) converted by ten-eleven translocation (TET) family is decreased in cancers. However, whether 5-hmC level is perturbed in early stages of carcinogenesis caused by genotoxic carcinogens is not defined. 5-hmC levels and TET2 expression were measured in liver of rats treated with genotoxic carcinogens, riddelliine, or aristolochic acid. Levels of 5-hmC and TET2 expression decreased in the liver of the carcinogens-treated rats. Loss of 5-hmC correlates well with documented induction of genetic mutations by the carcinogens, suggesting that TET2-mediated 5-hydroxymethylation plays an epigenetic role in early state of carcinogenesis. PMID:25154389

Retrieval-Induced Upregulation of Tet3 in Pyramidal Neurons of the Dorsal Hippocampus Mediates Cocaine-Associated Memory Reconsolidation.

PubMed

Liu, Cao; Sun, Xue; Wang, Zhilin; Le, Qiumin; Liu, Peipei; Jiang, Changyou; Wang, Feifei; Ma, Lan

2018-03-01

Memory retrieval refers to reexposure to information previously encoded and stored in the brain. Following retrieval, a once-consolidated memory destabilizes and undergoes reconsolidation, during which gene expression changes to restabilize memory. Investigating epigenetic regulation during reconsolidation could provide insights into normal memory formation and pathological memory associated with psychiatric disorders. We used cocaine-induced conditioned place preference to assess the cocaine-associated memory of mice and used chemogenetic methods to manipulate the activity of the pyramidal neurons in the dorsal hippocampus. We isolated the ribosome-associated transcripts from the excitatory neurons in the dorsal hippocampus by RiboTag purification to identify the potential epigenetic regulators, and we specifically knocked down gene expression in pyramidal neurons with a Cre-dependent lentivirus. Chemogenetically silencing the activity of the pyramidal neurons in the dorsal hippocampus immediately after memory retrieval markedly impaired memory reconsolidation, and the ribosome-associated mRNA level of the ten-eleven translocation (Tet) family methylcytosine dioxygenase Tet3, but not Tet1 or Tet2, was dramatically upregulated 10 minutes after memory retrieval. The protein level of Tet3 in the dorsal hippocampus but not in the anterior cingulate cortex was dramatically increased 1 hour after memory retrieval. Specifically, knockdown of Tet3 in pyramidal neurons in the dorsal hippocampus decreased the activation of pyramidal neurons and impaired the reconsolidation of cocaine-associated memory. Our findings highlight the new function of the DNA demethylation regulator Tet3 in pyramidal neurons of the dorsal hippocampus in regulating the reconsolidation of cocaine-associated memory. © The Author 2017. Published by Oxford University Press on behalf of CINP.

Regulated expression of the rat recombinant P2X(3) receptor in stably transfected CHO-K1 tTA cells.

PubMed

Lachnit, W G; Oglesby, I B; Gever, J R; Gever, M; Huang, C; Li, X C; Jin, H; McGivern, J G; Ford, A P

2000-07-03

In this report, the regulatable expression by tetracycline of the rat recombinant P2X(3) receptor in stably transfected Chinese hamster ovary (CHO-K1) expressing the tetracycline-controlled transactivator (tTA) is described. cDNA encoding the rat P2X(3)-receptor was subcloned into pTRE (a tetracycline-repressible expression vector) which was used to transfect stably CHO-K1 tTA cells. Using whole cell patch clamp techniques, 100 microM ATP evoked inward currents of 2.9+/-1.6 nA in transfected cells grown in the absence of tetracycline (tet-). The P2X(3) receptor protein was detectable by immunoblot as early as 24 h and protein expression levels continued to increase as much as 192 h following activation of tTA by the removal of the antibiotic. Saturation binding isotherms using [35S]ATP gamma S yielded a pK(d) of 8.2+/-0.1 and a B(max) of 31.9+/-3.5 pmol/mg protein in tet- cell membranes and a pK(d) of 8.1+/-0.1 and a B(max) of 5.8+/-0.8 pmol/mg protein in tet+ cell membranes. The agonist ligands 2MeSATP and alpha beta MeATP displaced the binding of [35S]ATP gamma S in tet- cell membranes with very high affinity, yielding pIC(50) values of 9.4+/-0.2 and 7.5+/-0. 2, respectively. In tet+ cell membrane, displacement of [35S]ATP gamma S by 2MeSATP and alpha beta MeATP was of much lower affinity (pIC(50) values of 7.8 and 6.2, respectively). ATP, ADP and UTP showed similar displacement of [35S]ATP gamma S binding in tet- and tet+ cell membranes. In other experiments, cytosolic Ca(2+) was monitored using the fluorescent indicator, fluo-3. Increases in cytosolic Ca(2+) were elicited by 100 nM alpha beta MeATP in tet- cells while no increases in cytosolic Ca(2+) were detected below 100 microM alpha beta MeATP in either tet+ cells or untransfected cells. These calcium responses to alpha beta MeATP had a pEC(50) of 6.7 and were transient, returning to baseline within 120 s. Suramin produced concentration-dependent, parallel, dextral shifts of E/[A] curves to alpha beta MeATP yielding a pK(B) of 5.6. PPADS produced non-parallel, dextral shifts of E/[A] curves to alpha beta MeATP which were insurmountable. These results show for the first time, expression of a functional, homomeric recombinant rat P2X(3) receptor which is under regulated expression in a stably transfected mammalian cell line.

A downstream box fusion allows stable accumulation of a bacterial cellulase in Chlamydomonas reinhardtii chloroplasts.

PubMed

Richter, Lubna V; Yang, Huijun; Yazdani, Mohammad; Hanson, Maureen R; Ahner, Beth A

2018-01-01

We investigated strategies to improve foreign protein accumulation in the chloroplasts of the model algae Chlamydomonas reinhardtii and tested the outcome in both standard culture conditions as well as one pertinent to algal biofuel production. The downstream box (DB) of the TetC or NPTII genes, the first 15 codons following the start codon, was N -terminally fused to the coding region of cel6A , an endoglucanase from Thermobifida fusca . We also employed a chimeric regulatory element, consisting of the 16S rRNA promoter and the atpA 5'UTR, previously reported to enhance protein expression, to regulate the expression of the TetC- cel6A gene. We further investigated the accumulation of TetC-Cel6A under N -deplete growth conditions. Both of the DB fusions improved intracellular accumulation of Cel6A in transplastomic C. reinhardtii strains though the TetC DB was much more effective than the NPTII DB. Furthermore, using the chimeric regulatory element, the TetC-Cel6A protein accumulation displayed a significant increase to 0.3% total soluble protein (TSP), whereas NPTII-Cel6A remained too low to quantify. Comparable levels of TetC- and NPTII- cel6A transcripts were observed, which suggests that factors other than transcript abundance mediate the greater TetC-Cel6A accumulation. The TetC-Cel6A accumulation was stable regardless of the growth stage, and the transplastomic strain growth rate was not altered. When transplastomic cells were suspended in N -deplete medium, cellular levels of TetC-Cel6A increased over time along with TSP, and were greater than those in cells suspended in N -replete medium. The DB fusion holds great value as a tool to enhance foreign protein accumulation in C. reinhardtii chloroplasts and its influence is related to translation or other post-transcriptional processes. Our results also suggest that transplastomic protein production can be compatible with algal biofuel production strategies. Cells displayed a consistent accumulation of recombinant protein throughout the growth phase and nitrogen starvation, a strategy used to induce lipid production in algae, led to higher cellular heterologous protein content. The latter result is contrary to what might have been expected a priori and is an important result for the development of future algal biofuel systems, which will likely require co-products for economic sustainability.

Synergistic Effects of Tetrandrine with Posaconazole Against Aspergillus fumigatus.

PubMed

Li, Shui-Xiu; Song, Yan-Jun; Jiang, Ling; Zhao, Ya-Jing; Guo, Hui; Li, Dong-Mei; Zhu, Kun-Ju; Zhang, Hong

2017-09-01

In our earlier in vitro and in vivo studies, synergistic effects were observed when itraconazole or voriconazole were combined with tetrandrine (TET) against Aspergillus fumigatus, and the synergistic mechanism was related to inhibition of the drug efflux pump. Posaconazole (PCZ) is a broad-spectrum triazole antifungal agent used for the treatment of diverse fungal infections, including aspergillosis and candidiasis. Herein, the antifungal effects of TET are further investigated in vitro and in vivo alone or combined with PCZ against 20 clinical isolates of A. fumigatus. We found that the minimal inhibitory concentrations (MICs) of PCZ were decreased one- to twofold and three- to fivefold across a series of concentration gradients in vitro in presence of TET. Time-killing curves revealed that the synergy was dependent on TET and PCZ concentrations as well as incubation time. The combination could further downregulate the expression of MDR2, MDR3, MDR4, and ATRF in PCZ-resistant strain, however, it has subtle effects on TET-synergized mechanism. In addition, TET in combination with PCZ significantly prolonged mice survival time and reduced kidney and brain tissue burdens in vivo. Our data in vitro and in vivo demonstrate that TET is an effective synergist with azoles against A. fumigates.

Regulatable Transgene Expression for Prevention of Chemotherapy-Induced Peripheral Neuropathy.

PubMed

Kawata, Daisuke; Wu, Zetang

2017-09-15

Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating complication associated with drug treatment of cancer for which there are no effective strategies of prevention or treatment. In this study, we examined the effect of intermittent expression of neurotophin-3 (NT-3) or interleukin-10 (IL-10) from replication-defective herpes simplex virus (HSV)-based regulatable vectors delivered by subcutaneous inoculation to the dorsal root ganglion (DRG) on the development of paclitaxel-induced peripheral neuropathy. We constructed two different tetracycline (tet)-on-based regulatable HSV vectors, one expressing NT-3 and the other expressing IL-10, in which the transactivator expression in the tet-on system was under the control of HSV latency-associated promoter 2 (LAP-2), and expression of the transgene was controlled by doxycycline (DOX). We examined the therapeutic effect of intermittent expression of the transgene in animals with paclitaxel-induced peripheral neuropathy modeled by intraperitoneal injection of paclitaxel (16 mg/kg) once a week for 5 weeks. Intermittent expression of either NT-3 or IL-10 3 days before and 1 day after paclitaxel administration protected animals against paclitaxel-induced peripheral neuropathy over the course of 5 weeks. These results suggest the potential of regulatable vectors for prevention of chemotherapy-induced peripheral neuropathy.

Prevalence of tetracycline resistance genes among multi-drug resistant bacteria from selected water distribution systems in southwestern Nigeria.

PubMed

Adesoji, Ayodele T; Ogunjobi, Adeniyi A; Olatoye, Isaac O; Call, Douglas R; Douglas, Douglas R

2015-06-25

Antibiotic resistance genes [ARGs] in aquatic systems have drawn increasing attention they could be transferred horizontally to pathogenic bacteria. Water treatment plants (WTPs) are intended to provide quality and widely available water to the local populace they serve. However, WTPs in developing countries may not be dependable for clean water and they could serve as points of dissemination for antibiotic resistant bacteria. Only a few studies have investigated the occurrence of ARGs among these bacteria including tetracycline resistance genes in water distribution systems in Nigeria. Multi-drug resistant (MDR) bacteria, including resistance to tetracycline, were isolated from treated and untreated water distribution systems in southwest Nigeria. MDR bacteria were resistant to >3 classes of antibiotics based on break-point assays. Isolates were characterized using partial 16S rDNA sequencing and PCR assays for six tetracycline-resistance genes. Plasmid conjugation was evaluated using E. coli strain DH5α as the recipient strain. Out of the 105 bacteria, 85 (81 %) and 20 (19 %) were Gram- negative or Gram- positive, respectively. Twenty-nine isolates carried at least one of the targeted tetracycline resistance genes including strains of Aeromonas, Alcaligenes, Bacillus, Klebsiella, Leucobacter, Morganella, Proteus and a sequence matching a previously uncultured bacteria. Tet(A) was the most prevalent (16/29) followed by tet(E) (4/29) and tet30 (2/29). Tet(O) was not detected in any of the isolates. Tet(A) was mostly found with Alcaligenes strains (9/10) and a combination of more than one resistance gene was observed only amongst Alcaligenes strains [tet(A) + tet30 (2/10), tet(A) + tet(E) (3/10), tet(E) + tet(M) (1/10), tet(E) + tet30 (1/10)]. Tet(A) was transferred by conjugation for five Alcaligenes and two E. coli isolates. This study found a high prevalence of plasmid-encoded tet(A) among Alcaligenes isolates, raising the possibility that this strain could shuttle resistance plasmids to pathogenic bacteria.

Characteristic profiles of DNA epigenetic modifications in colon cancer and its predisposing conditions-benign adenomas and inflammatory bowel disease.

PubMed

Dziaman, Tomasz; Gackowski, Daniel; Guz, Jolanta; Linowiecka, Kinga; Bodnar, Magdalena; Starczak, Marta; Zarakowska, Ewelina; Modrzejewska, Martyna; Szpila, Anna; Szpotan, Justyna; Gawronski, Maciej; Labejszo, Anna; Liebert, Ariel; Banaszkiewicz, Zbigniew; Klopocka, Maria; Foksinski, Marek; Marszalek, Andrzej; Olinski, Ryszard

2018-01-01

Active demethylation of 5-methyl-2'-deoxycytidine (5-mdC) in DNA occurs by oxidation to 5-(hydroxymethyl)-2'-deoxycytidine (5-hmdC) and further oxidation to 5-formyl-2'-deoxycytidine (5-fdC) and 5-carboxy-2'-deoxycytidine (5-cadC), and is carried out by enzymes of the ten-eleven translocation family (TETs 1, 2, 3). Decreased level of epigenetic DNA modifications in cancer tissue may be a consequence of reduced activity/expression of TET proteins. To determine the role of epigenetic DNA modifications in colon cancer development, we analyzed their levels in normal colon and various colonic pathologies. Moreover, we determined the expressions of TETs at mRNA and protein level.The study included material from patients with inflammatory bowel disease (IBD), benign polyps (AD), and colorectal cancer (CRC). The levels of epigenetic DNA modifications and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in examined tissues were determined by means of isotope-dilution automated online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS). The expressions of TET mRNA were measured with RT-qPCR, and the expressions of TET proteins were determined immunohistochemically. IBD was characterized by the highest level of 8-oxodG among all analyzed tissues, as well as by a decrease in 5-hmdC and 5-mdC levels (at a midrange between normal colon and CRC). AD had the lowest levels of 5-hmdC and 5-mdC of all examined tissues and showed an increase in 8-oxodG and 5-(hydroxymethyl)-2'-deoxyuridine (5-hmdU) levels. CRC was characterized by lower levels of 5-hmdC and 5-mdC, the lowest level of 5-fdC among all analyzed tissues, and relatively high content of 5-cadC. The expression of TET1 mRNA in CRC and AD was significantly weaker than in IBD and normal colon. Furthermore, CRC and AD showed significantly lower levels of TET2 and AID mRNA than normal colonic tissue. Our findings suggest that a complex relationship between aberrant pattern of DNA epigenetic modification and cancer development does not depend solely on the transcriptional status of TET proteins, but also on the characteristics of premalignant/malignant cells. This study showed for the first time that the examined colonic pathologies had their unique epigenetic marks, distinguishing them from each other, as well as from normal colonic tissue. A decrease in 5-fdC level may be a characteristic feature of largely undifferentiated cancer cells.

Multifunctional magnetic Fe3O4 nanoparticles combined with chemotherapy and hyperthermia to overcome multidrug resistance

PubMed Central

Ren, Yanyan; Zhang, Haijun; Chen, Baoan; Cheng, Jian; Cai, Xiaohui; Liu, Ran; Xia, Guohua; Wu, Weiwei; Wang, Shuai; Ding, Jiahua; Gao, Chong; Wang, Jun; Bao, Wen; Wang, Lei; Tian, Liang; Song, Huihui; Wang, Xuemei

2012-01-01

Background Multidrug resistance in cancer is a major obstacle for clinical therapeutics, and is the reason for 90% of treatment failures. This study investigated the efficiency of novel multifunctional Fe3O4 magnetic nanoparticles (Fe3O4-MNP) combined with chemotherapy and hyperthermia for overcoming multidrug resistance in an in vivo model of leukemia. Methods Nude mice with tumor xenografts were randomly divided into a control group, and the treatment groups were allocated to receive daunorubicin, 5-bromotetrandrine (5-BrTet) and daunorubicin, Fe3O4-MNP, and Fe3O4-MNP coloaded with daunorubicin and 5-bromotetrandrine (Fe3O4-MNP-DNR-5-BrTet), with hyperthermia in an alternating magnetic field. We investigated tumor volume and pathology, as well as P-glycoprotein, Bcl-2, Bax, and caspase-3 protein expression to elucidate the effect of multimodal treatment on overcoming multidrug resistance. Results Fe3O4-MNP played a role in increasing tumor temperature during hyperthermia. Tumors became significantly smaller, and apoptosis of cells was observed in both the Fe3O4-MNP and Fe3O4-MNP-DNR-5-BrTet groups, especially in the Fe3O4-MNP-DNR-5-BrTet group, while tumor volumes in the other groups had increased after treatment for 12 days. Furthermore, Fe3O4-MNP-DNR-5-BrTet with hyperthermia noticeably decreased P-glycoprotein and Bcl-2 expression, and markedly increased Bax and caspase-3 expression. Conclusion Fe3O4-MNP-DNR-5-BrTet with hyperthermia may be a potential approach for reversal of multidrug resistance in the treatment of leukemia. PMID:22619560

Multifunctional magnetic Fe3O4 nanoparticles combined with chemotherapy and hyperthermia to overcome multidrug resistance.

PubMed

Ren, Yanyan; Zhang, Haijun; Chen, Baoan; Cheng, Jian; Cai, Xiaohui; Liu, Ran; Xia, Guohua; Wu, Weiwei; Wang, Shuai; Ding, Jiahua; Gao, Chong; Wang, Jun; Bao, Wen; Wang, Lei; Tian, Liang; Song, Huihui; Wang, Xuemei

2012-01-01

Multidrug resistance in cancer is a major obstacle for clinical therapeutics, and is the reason for 90% of treatment failures. This study investigated the efficiency of novel multifunctional Fe(3)O(4) magnetic nanoparticles (Fe(3)O(4)-MNP) combined with chemotherapy and hyperthermia for overcoming multidrug resistance in an in vivo model of leukemia. Nude mice with tumor xenografts were randomly divided into a control group, and the treatment groups were allocated to receive daunorubicin, 5-bromotetrandrine (5-BrTet) and daunorubicin, Fe(3)O(4)-MNP, and Fe(3)O(4)-MNP coloaded with daunorubicin and 5-bromotetrandrine (Fe(3)O(4)-MNP-DNR-5-BrTet), with hyperthermia in an alternating magnetic field. We investigated tumor volume and pathology, as well as P-glycoprotein, Bcl-2, Bax, and caspase-3 protein expression to elucidate the effect of multimodal treatment on overcoming multidrug resistance. Fe(3)O(4)-MNP played a role in increasing tumor temperature during hyperthermia. Tumors became significantly smaller, and apoptosis of cells was observed in both the Fe(3)O(4)-MNP and Fe(3)O(4)-MNP-DNR-5-BrTet groups, especially in the Fe(3)O(4)-MNP-DNR-5-BrTet group, while tumor volumes in the other groups had increased after treatment for 12 days. Furthermore, Fe(3)O(4)-MNP-DNR-5-BrTet with hyperthermia noticeably decreased P-glycoprotein and Bcl-2 expression, and markedly increased Bax and caspase-3 expression. Fe(3)O(4)-MNP-DNR-5-BrTet with hyperthermia may be a potential approach for reversal of multidrug resistance in the treatment of leukemia.

In Vivo magnetic resonance imaging of xenografted tumors using FTH1 reporter gene expression controlled by a tet-on switch.

PubMed

He, Xiaoya; Cai, Jinhua; Li, Hao; Liu, Bo; Qin, Yong; Zhong, Yi; Wang, Longlun; Liao, Yifan

2016-11-29

As a promising magnetic resonance imaging (MRI) reporter, ferritin has been used to track cells in vivo; however, its continuous overexpression can be cytotoxic, which restricts its application. In this study, we aimed to develop a switch to turn this genetic reporter "on" or "off" while monitoring cell grafts via MRI. To accomplish this, we genetically modified the ferritin heavy chain (FTH1) with a Tet-On switch and assessed the expression of FTH1 in transduced neuroblastoma cells (SK-N-SH) in vitro and in xenografted tumors in vivo. We found that FTH1 expression induced by doxycycline (Dox) in SK-N-SH-FTH1 cells depended on treatment dose and duration. We successfully detected T2-weighted MRI contrast in cell grafts after switching "on" the reporter gene using Dox, and this contrast disappeared when we switched it "off". The genetic reporter FTH1 can thus be switched "on" or "off" throughout longitudinal monitoring of cell grafts, limiting expression to when MRI contrast is needed. The controllable imaging system we have developed minimizes risks from constitutive reporter gene overexpression and facilitates tumor cell monitoring in vitro and in vivo.

DNA hydroxymethylation profiling reveals that WT1 mutations result in loss of TET2 function in acute myeloid leukemia

PubMed Central

Rampal, Raajit; Alkalin, Altuna; Madzo, Jozef; Vasanthakumar, Aparna; Pronier, Elodie; Patel, Jay; Li, Yushan; Ahn, Jihae; Abdel-Wahab, Omar; Shih, Alan; Lu, Chao; Ward, Patrick S.; Tsai, Jennifer J.; Hricik, Todd; Tosello, Valeria; Tallman, Jacob E.; Zhao, Xinyang; Daniels, Danette; Dai, Qing; Ciminio, Luisa; Aifantis, Iannis; He, Chuan; Fuks, Francois; Tallman, Martin S.; Ferrando, Adolfo; Nimer, Stephen; Paietta, Elisabeth; Thompson, Craig B.; Licht, Jonathan D.; Mason, Chris; Godley, Lucy A.; Melnick, Ari; Figueroa, Maria E.; Levine, Ross L.

2014-01-01

Summary Somatic mutations in IDH1/2 and TET2 result in impaired TET2 mediated conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC). The observation that WT1 inactivating mutations anti-correlate with TET2/IDH1/2 mutations in AML led us to hypothesize that WT1 mutations may impact TET2 function. WT1 mutant acute myeloid leukemia (AML) patients have reduced 5-hmC levels similar to TET2/IDH1/2-mutant AML. These mutations are characterized by convergent, site-specific alterations in DNA hydroxymethylation, which drive differential gene expression more than alterations in DNA promoter methylation. WT1 overexpression increases global levels of 5-hmC, and WT1 silencing reduced 5-hmC levels. WT1 physically interacts with TET2 and TET3, and WT1 loss of function results in a similar hematopoietic differentiation phenotype as observed with TET2 deficiency. These data provide a novel role for WT1 in regulating DNA hydroxymethylation and suggest that TET2 IDH1/2, and WT1 mutations define a novel AML subtype defined by dysregulated DNA hydroxymethylation. PMID:25482556

Pluripotency transcription factors and Tet1/2 maintain Brd4-independent stem cell identity.

PubMed

Finley, Lydia W S; Vardhana, Santosha A; Carey, Bryce W; Alonso-Curbelo, Direna; Koche, Richard; Chen, Yanyang; Wen, Duancheng; King, Bryan; Radler, Megan R; Rafii, Shahin; Lowe, Scott W; Allis, C David; Thompson, Craig B

2018-05-01

A robust network of transcription factors and an open chromatin landscape are hallmarks of the naive pluripotent state. Recently, the acetyllysine reader Brd4 has been implicated in stem cell maintenance, but the relative contribution of Brd4 to pluripotency remains unclear. Here, we show that Brd4 is dispensable for self-renewal and pluripotency of embryonic stem cells (ESCs). When maintained in their ground state, ESCs retain transcription factor binding and chromatin accessibility independent of Brd4 function or expression. In metastable ESCs, Brd4 independence can be achieved by increased expression of pluripotency transcription factors, including STAT3, Nanog or Klf4, so long as the DNA methylcytosine oxidases Tet1 and Tet2 are present. These data reveal that Brd4 is not essential for ESC self-renewal. Rather, the levels of pluripotency transcription factor abundance and Tet1/2 function determine the extent to which bromodomain recognition of protein acetylation contributes to the maintenance of gene expression and cell identity.

Prevalence and characterization of antimicrobial-resistant Escherichia coli isolated from conventional and organic vegetables.

PubMed

Kim, Sara; Woo, Gun-Jo

2014-10-01

To compare the characteristics and to identify the epidemiological relationships of Escherichia coli isolated from organic and conventional vegetables, the antimicrobial resistance and genetic properties of E. coli were investigated from 2010 to 2011. E. coli was isolated from 1 of 111 (0.9%) organic vegetables and from 20 of 225 (8.9%) conventional vegetables. The majority of strains were isolated from the surrounding farming environment (n=27/150 vs. 49/97 in organic vs. conventional samples). The majority of the vegetable strains were isolated from the surrounding farming environments. E. coli isolated from organic vegetables showed very low antimicrobial resistance rates except for cephalothin, ranging from 0% to 17.9%, while the resistance rates to cephalothin (71%) were extremely high in both groups. E. coli isolates expressed various resistance genes, which most commonly included blaTEM, tet(A), strA, strB, and qnrS. However, none of the isolates harbored tet(D), tet(E), tet(K), tet(L), tet(M), or qnrA. The transferability of tet gene, tet(A), and tet(B) was identified in tetracycline-resistant E. coli, and the genetic relationship was confirmed in a few cases from different sources. With regard to the lower antimicrobial resistance found in organic produce, this production mode seems able to considerably reduce the selection of antimicrobial-resistant bacteria on vegetables.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Iyer, Sukanya; Karig, David K; Norred, Sarah E

Engineered gene circuits offer an opportunity to harness biological systems for biotechnological and biomedical applications. However, reliance on host E. coli promoters for the construction of circuit elements, such as logic gates, makes implementation of predictable, independently functioning circuits difficult. In contrast, T7 promoters offer a simple orthogonal expression system for use in a variety of cellular backgrounds and even in cell free systems. Here we develop a T7 promoter system that can be regulated by two different transcriptional repressors for the construction of a logic gate that functions in cells and in cell free systems. We first present LacImore » repressible T7lacO promoters that are regulated from a distal lac operator site for repression. We next explore the positioning of a tet operator site within the T7lacO framework to create T7 promoters that respond to tet and lac repressors and realize an IMPLIES gate. Finally, we demonstrate that these dual input sensitive promoters function in a commercially available E. coli cell-free protein expression system. Together, our results contribute to the first demonstration of multi-input regulation of T7 promoters and expand the utility of T7 promoters in cell based as well as cell-free gene circuits.« less

Tetracycline Resistance and Presence of Tetracycline Resistance Determinants tet(V) and tap in Rapidly Growing Mycobacteria from Agricultural Soils and Clinical Isolates

PubMed Central

Kyselková, Martina; Chron̂áková, Alica; Volná, Lucie; Nêmec, Jan; Ulmann, Vít; Scharfen, Josef; Elhottová, Dana

2012-01-01

Rapidly growing mycobacteria (RGM) inhabit soil and water but certain strains represent a health risk for human and animals. Both clinical and soil RGM may be under selection pressure for resistance to tetracycline (TET) antibiotics, since tetracyclines are administrated to humans and farm animals, and TET residues enter soil through manuring; however, resistance to TET and the presence of TET-resistance genes have been assessed only in clinical isolates. We were therefore interested in comparing soil and clinical RGM in terms of TET resistance and the presence of TET-resistance genes. We used 44 RGM from grasslands with different exposure to animal manure, and 38 clinical RGM from Czech hospitals. There was no difference between the clinical and soil isolates in TET resistance, with >50% resistant isolates in both groups. otr(A), otr(B), tet(K), tet(L) or tet(M) were not detected in any soil or clinical isolate. In contrast, most isolates harbored tet(V) and tap, both encoding mycobacterial efflux pumps, including species where these genes have never been evidenced before. The phylogeny of tet(V) correlated with isolates’ BOX-PCR profiles, suggesting that this gene evolved along with mycobacterial genomes as a part of the intrinsic resistome. In certain cases, tet(V) and/or tap were found in TET-sensitive isolates, or inversely, were not found in resistant strains. Concluding, intrinsic efflux pumps may be more important for TET resistance than horizontally transferred genes in both soil and clinical RGM. Their simple presence, however, does not attest to resistance, and therefore their diversity, function and expression merit further research. PMID:22673307

Downregulation of TLX induces TET3 expression and inhibits glioblastoma stem cell self-renewal and tumorigenesis.

PubMed

Cui, Qi; Yang, Su; Ye, Peng; Tian, E; Sun, Guoqiang; Zhou, Jiehua; Sun, Guihua; Liu, Xiaoxuan; Chen, Chao; Murai, Kiyohito; Zhao, Chunnian; Azizian, Krist T; Yang, Lu; Warden, Charles; Wu, Xiwei; D'Apuzzo, Massimo; Brown, Christine; Badie, Behnam; Peng, Ling; Riggs, Arthur D; Rossi, John J; Shi, Yanhong

2016-02-03

Glioblastomas have been proposed to be maintained by highly tumorigenic glioblastoma stem cells (GSCs) that are resistant to current therapy. Therefore, targeting GSCs is critical for developing effective therapies for glioblastoma. In this study, we identify the regulatory cascade of the nuclear receptor TLX and the DNA hydroxylase Ten eleven translocation 3 (TET3) as a target for human GSCs. We show that knockdown of TLX expression inhibits human GSC tumorigenicity in mice. Treatment of human GSC-grafted mice with viral vector-delivered TLX shRNA or nanovector-delivered TLX siRNA inhibits tumour development and prolongs survival. Moreover, we identify TET3 as a potent tumour suppressor downstream of TLX to regulate the growth and self-renewal in GSCs. This study identifies the TLX-TET3 axis as a potential therapeutic target for glioblastoma.

Inducible expression of photoacoustic reporter gene tyrosinase in cells using a single plasmid

NASA Astrophysics Data System (ADS)

Paproski, Robert J.; Zemp, Roger J.

2012-02-01

We have previously demonstrated that tyrosinase is a reporter gene for photoacoustic imaging since tyrosinase is the rate-limiting step in the synthesis of melanin, a pigment capable of producing strong photoacoustic signals. We previously created a cell line capable of inducible tyrosinase expression (important due to toxicity of melanin) by stably transfecting tyrosinase in MCF-7 Tet-OnR cell line (Clontech) which expresses a doxycycline-controlled transactivator. Unfortunately, Clontech provides few Tet-On Advanced cell lines making it difficult to have inducible tyrosinase expression in cell lines not provided by Clontech. In order to simplify the creation of cell lines with inducible expression of tyrosinase, we created a single plasmid that encodes both the transactivator as well as tyrosinase. PCR was used to amplify both the transactivator and tyrosinase from the Tet-OnR Advanced and pTRE-Tight-TYR plasmids, respectively. Both PCR products were cloned into the pEGFP-N1 plasmid and the newly created plasmid was transfected into ZR-75-1, MCF-7, and MIA PaCa-1 cells using lipofectamine. After several days, brown melanin was only observed in cells incubated with doxycycline, suggesting that the newly created single plasmid allowed inducible tyrosinase expression in many different cells lines.

Optimal Design of Litz Wire Coils With Sandwich Structure Wirelessly Powering an Artificial Anal Sphincter System.

PubMed

Ke, Lei; Yan, Guozheng; Yan, Sheng; Wang, Zhiwu; Li, Xiaoyang

2015-07-01

Transcutaneous energy transfer system (TETS) is widely used to energize implantable biomedical devices. As a key part of the TETS, a pair of applicable coils with low losses, high unloaded Q factor, and strong coupling is required to realize an efficient TETS. This article presents an optimal design methodology of planar litz wire coils sandwiched between two ferrite substrates wirelessly powering a novel mechanical artificial anal sphincter system for treating severe fecal incontinence, with focus on the main parameters of the coils such as the wire diameter, number of turns, geometry, and the properties of the ferrite substrate. The theoretical basis of optimal power transfer efficiency in an inductive link was analyzed. A set of analytical expressions are outlined to calculate the winding resistance of a litz wire coil on ferrite substrate, taking into account eddy-current losses, including conduction losses and induction losses. Expressions that describe the geometrical dimension dependence of self- and mutual inductance are derived. The influence of ferrite substrate relative permeability and dimensions is also considered. We have used this foundation to devise an applicable coil design method that starts with a set of realistic constraints and ends with the optimal coil pair geometries. All theoretical predictions are verified with measurements using different types of fabricated coils. The results indicate that the analysis is useful for optimizing the geometry design of windings and the ferrite substrate in a sandwich structure as part of which, in addition to providing design insight, allows speeding up the system efficiency-optimizing design process. Copyright © 2015 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Retrieval-Induced Upregulation of Tet3 in Pyramidal Neurons of the Dorsal Hippocampus Mediates Cocaine-Associated Memory Reconsolidation

PubMed Central

Liu, Cao; Sun, Xue; Wang, Zhilin; Le, Qiumin; Liu, Peipei; Jiang, Changyou; Wang, Feifei; Ma, Lan

2018-01-01

Abstract Background Memory retrieval refers to reexposure to information previously encoded and stored in the brain. Following retrieval, a once-consolidated memory destabilizes and undergoes reconsolidation, during which gene expression changes to restabilize memory. Investigating epigenetic regulation during reconsolidation could provide insights into normal memory formation and pathological memory associated with psychiatric disorders. Methods We used cocaine-induced conditioned place preference to assess the cocaine-associated memory of mice and used chemogenetic methods to manipulate the activity of the pyramidal neurons in the dorsal hippocampus. We isolated the ribosome-associated transcripts from the excitatory neurons in the dorsal hippocampus by RiboTag purification to identify the potential epigenetic regulators, and we specifically knocked down gene expression in pyramidal neurons with a Cre-dependent lentivirus. Results Chemogenetically silencing the activity of the pyramidal neurons in the dorsal hippocampus immediately after memory retrieval markedly impaired memory reconsolidation, and the ribosome-associated mRNA level of the ten-eleven translocation (Tet) family methylcytosine dioxygenase Tet3, but not Tet1 or Tet2, was dramatically upregulated 10 minutes after memory retrieval. The protein level of Tet3 in the dorsal hippocampus but not in the anterior cingulate cortex was dramatically increased 1 hour after memory retrieval. Specifically, knockdown of Tet3 in pyramidal neurons in the dorsal hippocampus decreased the activation of pyramidal neurons and impaired the reconsolidation of cocaine-associated memory. Conclusions Our findings highlight the new function of the DNA demethylation regulator Tet3 in pyramidal neurons of the dorsal hippocampus in regulating the reconsolidation of cocaine-associated memory. PMID:29106571

α7 nAChR mediated Fas demethylation contributes to prenatal nicotine exposure-induced programmed thymocyte apoptosis in mice.

PubMed

Liu, Han-Xiao; Liu, Sha; Qu, Wen; Yan, Hui-Yi; Wen, Xiao; Chen, Ting; Hou, Li-Fang; Ping, Jie

2017-11-07

This study aimed to investigate the effects of prenatal nicotine exposure (PNE) on thymocyte apoptosis and postnatal immune impairments in vivo and further explore the epigenetic mechanisms of the pro-apoptotic effect of nicotine in vitro . The results showed that PNE caused immune impairments in offspring on postnatal day 49, manifested as increased IL-4 production and an increased IgG1/IgG2a ratio in serum. Enhanced apoptosis of total and CD4+SP thymocytes was observed both in fetus and in offspring. Further, by exposing thymocytes to 0-100 μM of nicotine in vitro for 48 h, we found that nicotine increased α7 nicotinic acetylcholine receptor (nAChR) expression, activated the Fas apoptotic pathway, and promoted thymocyte apoptosis in concentration-dependent manners. In addition, nicotine could induce Tet methylcytosine dioxygenase (TET) 2 expression and Fas promoter demethylation, which can be abolished by TET2 siRNA transfection. Moreover, the α7 nAChR specific antagonist α-bungarotoxin can abrogate nicotine-induced TET2 increase, and the following Fas demethylation and Fas-mediated apoptosis. In conclusion, our findings showed, for the first time, that α7 nAChR activation could induce TET2-mediated Fas demethylation in thymocytes and results in the upregulation of Fas apoptotic pathway, which provide evidence for elucidating the PNE-induced programmed thymocyte apoptosis.

Individual retrotransposon integrants are differentially controlled by KZFP/KAP1-dependent histone methylation, DNA methylation and TET-mediated hydroxymethylation in naïve embryonic stem cells.

PubMed

Coluccio, Andrea; Ecco, Gabriela; Duc, Julien; Offner, Sandra; Turelli, Priscilla; Trono, Didier

2018-02-26

The KZFP/KAP1 (KRAB zinc finger proteins/KRAB-associated protein 1) system plays a central role in repressing transposable elements (TEs) and maintaining parent-of-origin DNA methylation at imprinting control regions (ICRs) during the wave of genome-wide reprogramming that precedes implantation. In naïve murine embryonic stem cells (mESCs), the genome is maintained highly hypomethylated by a combination of TET-mediated active demethylation and lack of de novo methylation, yet KAP1 is tethered by sequence-specific KZFPs to ICRs and TEs where it recruits histone and DNA methyltransferases to impose heterochromatin formation and DNA methylation. Here, upon removing either KAP1 or the cognate KZFP, we observed rapid TET2-dependent accumulation of 5hmC at both ICRs and TEs. In the absence of the KZFP/KAP1 complex, ICRs lost heterochromatic histone marks and underwent both active and passive DNA demethylation. For KAP1-bound TEs, 5mC hydroxylation correlated with transcriptional reactivation. Using RNA-seq, we further compared the expression profiles of TEs upon Kap1 removal in wild-type, Dnmt and Tet triple knockout mESCs. While we found that KAP1 represents the main effector of TEs repression in all three settings, we could additionally identify specific groups of TEs further controlled by DNA methylation. Furthermore, we observed that in the absence of TET proteins, activation upon Kap1 depletion was blunted for some TE integrants and increased for others. Our results indicate that the KZFP/KAP1 complex maintains heterochromatin and DNA methylation at ICRs and TEs in naïve embryonic stem cells partly by protecting these loci from TET-mediated demethylation. Our study further unveils an unsuspected level of complexity in the transcriptional control of the endovirome by demonstrating often integrant-specific differential influences of histone-based heterochromatin modifications, DNA methylation and 5mC oxidation in regulating TEs expression.

Design and implementation of a synthetic pre-miR switch for controlling miRNA biogenesis in mammals

PubMed Central

Atanasov, Janina; Groher, Florian

2017-01-01

Abstract Synthetic RNA-based systems have increasingly been used for the regulation of eukaryotic gene expression. Due to their structural properties, riboregulators provide a convenient basis for the development of ligand-dependent controllable systems. Here, we demonstrate reversible conditional control of miRNA biogenesis with an aptamer domain as a sensing unit connected to a natural miRNA precursor for the first time. For the design of the pre-miR switch, we replaced the natural terminal loop with the TetR aptamer. Thus, the TetR aptamer was positioned close to the Dicer cleavage sites, which allowed sterical control over pre-miR processing by Dicer. Our design proved to be highly versatile, allowing us to regulate the biogenesis of three structurally different miRNAs: miR-126, -34a and -199a. Dicer cleavage was inhibited up to 143-fold via co-expression of the TetR protein, yet could be completely restored upon addition of doxycycline. Moreover, we showed the functionality of the pre-miR switches for gene regulation through the interaction of the respective miRNA with its specific target sequence. Our designed device is capable of robust and reversible control of miRNA abundance. Thus, we offer a novel investigational tool for functional miRNA analysis. PMID:29036355

Identification and characterization of tetracycline resistance in Lactococcus lactis isolated from Polish raw milk and fermented artisanal products.

PubMed

Zycka-Krzesinska, Joanna; Boguslawska, Joanna; Aleksandrzak-Piekarczyk, Tamara; Jopek, Jakub; Bardowski, Jacek K

2015-10-15

To assess the occurrence of antibiotic-resistant Lactic Acid Bacteria (LAB) in Polish raw milk and fermented artisanal products, a collection comprising 500 isolates from these products was screened. Among these isolates, six strains (IBB28, IBB160, IBB161, IBB224, IBB477 and IBB487) resistant to tetracycline were identified. The strains showing atypical tetracycline resistance were classified as Lactococcus lactis: three of them were identified as L. lactis subsp. cremoris (IBB224, IBB477 and IBB487) and the other three (IBB28, IBB160, IBB161) were identified as L. lactis subsp. lactis. The mechanism involving Ribosomal Protection Proteins (RPP) was identified as responsible for tetracycline resistance. Three of the tested strains (IBB28, IBB160 and IBB224) had genes encoding the TetS protein, whereas the remaining three (IBB161, IBB477 and IBB487) expressed TetM. The results also demonstrated that the genes encoding these proteins were located on genetic mobile elements. The tet(S) gene was found to be located on plasmids, whereas tet(M) was found within the Tn916 transposon. Copyright © 2015 Elsevier B.V. All rights reserved.

Downregulation of TLX induces TET3 expression and inhibits glioblastoma stem cell self-renewal and tumorigenesis

PubMed Central

Cui, Qi; Yang, Su; Ye, Peng; Tian, E.; Sun, Guoqiang; Zhou, Jiehua; Sun, Guihua; Liu, Xiaoxuan; Chen, Chao; Murai, Kiyohito; Zhao, Chunnian; Azizian, Krist T.; Yang, Lu; Warden, Charles; Wu, Xiwei; D'Apuzzo, Massimo; Brown, Christine; Badie, Behnam; Peng, Ling; Riggs, Arthur D.; Rossi, John J.; Shi, Yanhong

2016-01-01

Glioblastomas have been proposed to be maintained by highly tumorigenic glioblastoma stem cells (GSCs) that are resistant to current therapy. Therefore, targeting GSCs is critical for developing effective therapies for glioblastoma. In this study, we identify the regulatory cascade of the nuclear receptor TLX and the DNA hydroxylase Ten eleven translocation 3 (TET3) as a target for human GSCs. We show that knockdown of TLX expression inhibits human GSC tumorigenicity in mice. Treatment of human GSC-grafted mice with viral vector-delivered TLX shRNA or nanovector-delivered TLX siRNA inhibits tumour development and prolongs survival. Moreover, we identify TET3 as a potent tumour suppressor downstream of TLX to regulate the growth and self-renewal in GSCs. This study identifies the TLX-TET3 axis as a potential therapeutic target for glioblastoma. PMID:26838672

Cytoskeleton structure and total methylation of mouse cardiac and lung tissue during space flight.

PubMed

Ogneva, Irina V; Loktev, Sergey S; Sychev, Vladimir N

2018-01-01

The purpose of this work was to evaluate the protein and mRNA expression levels of multiple cytoskeletal proteins in the cardiac and lung tissue of mice that were euthanized onboard the United States Orbital Segment of the International Space Station 37 days after the start of the SpaceX-4 mission (September 2014, USA). The results showed no changes in the cytoskeletal protein content in the cardiac and lung tissue of the mice, but there were significant changes in the mRNA expression levels of the associated genes, which may be due to an increase in total genome methylation. The mRNA expression levels of DNA methylases, the cytosine demethylases Tet1 and Tet3, histone acetylase and histone deacetylase did not change, and the mRNA expression level of cytosine demethylase Tet2 was significantly decreased.

Cytoskeleton structure and total methylation of mouse cardiac and lung tissue during space flight

PubMed Central

Loktev, Sergey S.; Sychev, Vladimir N.

2018-01-01

The purpose of this work was to evaluate the protein and mRNA expression levels of multiple cytoskeletal proteins in the cardiac and lung tissue of mice that were euthanized onboard the United States Orbital Segment of the International Space Station 37 days after the start of the SpaceX-4 mission (September 2014, USA). The results showed no changes in the cytoskeletal protein content in the cardiac and lung tissue of the mice, but there were significant changes in the mRNA expression levels of the associated genes, which may be due to an increase in total genome methylation. The mRNA expression levels of DNA methylases, the cytosine demethylases Tet1 and Tet3, histone acetylase and histone deacetylase did not change, and the mRNA expression level of cytosine demethylase Tet2 was significantly decreased. PMID:29768411

Tet methylcytosine dioxygenase 2 inhibits atherosclerosis via upregulation of autophagy in ApoE−/− mice

PubMed Central

Peng, Juan; Yang, Qin; Li, A-Fang; Li, Rong-Qing; Wang, Zuo; Liu, Lu-Shan; Ren, Zhong; Zheng, Xi-Long; Tang, Xiao-Qing; Li, Guo-Hua; Tang, Zhi-Han; Jiang, Zhi-Sheng; Wei, Dang-Heng

2016-01-01

Tet methylcytosine dioxygenase 2 (TET2) mediates the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). The loss of TET2 is associated with advanced atherosclerotic lesions. Our previous study showed that TET2 improves endothelial cell function by enhancing endothelial cell autophagy. Accordingly, this study determined the role of TET2 in atherosclerosis and potential mechanisms. In ApoE−/− mice fed high-fat diet, TET2 overexpression markedly decreased atherosclerotic lesions with uniformly increased level of 5hmC and decreased level of 5mC in genomic DNA. TET2 overexpression also promoted autophagy and downregulated inflammation factors, such as vascular cell adhesion molecule 1, intercellular adhesion molecule 1, monocyte chemotactic protein 1, and interleukin-1. Consistently, TET2 knockdown with small hairpin RNA (shRNA) in ApoE−/− mice decreased 5hmC and increased 5mC levels in atherosclerotic lesions. Meanwhile, autophagy was inhibited and atherosclerotic lesions progressed with an unstable lesion phenotype characterized by large lipid core, macrophage accumulation, and upregulated inflammation factor expression. Experiments with the cultured endothelial cells revealed that oxidized low-density lipoprotein (ox-LDL) inhibited endothelial cell autophagy. TET2 shRNA strengthened impaired autophagy and autophagic flux in the ox-LDL-treated endothelial cells. TET2 overexpression reversed these effects by decreasing the methylation level of the Beclin 1 promoter, which contributed to the downregulation of inflammation factors. Overall, we identified that TET2 was downregulated during the pathogenesis of atherosclerosis. The downregulation of TET2 promotes the methylation of the Beclin 1 promoter, leading to endothelial cell autophagy, impaired autophagic flux, and inflammatory factor upregulation. Upregulation of TET2 may be a novel therapeutic strategy for treating atherosclerosis. PMID:27821816

Behavior of antibiotics and antibiotic resistance genes in eco-agricultural system: A case study.

PubMed

Cheng, Weixiao; Li, Jianan; Wu, Ying; Xu, Like; Su, Chao; Qian, Yanyun; Zhu, Yong-Guan; Chen, Hong

2016-03-05

This study aims to determine abundance and persistence of antibiotics and antibiotic resistance genes (ARGs) in eco-agricultural system (EAS), which starts from swine feces to anaerobic digestion products, then application of anaerobic digestion solid residue (ADSR) and anaerobic digestion liquid residue (ADLR) to the soil to grow ryegrass, one of swine feed. Oxytetracycline had the highest concentration in manure reaching up to 138.7 mg/kg. Most of antibiotics could be effectively eliminated by anaerobic digestion and removal rates ranged from 11% to 86%. ARGs abundance fluctuated within EAS. TetQ had the highest relative abundance and the relative abundance of tetG had the least variation within the system, which indicates that tetG is persistent in the agricultural environment and requires more attention. Compared to the relative abundance in manure, tetC and tetM increased in biogas residue while three ribosomal protection proteins genes (tetO, tetQ, tetW) decreased (p<0.05), with other genes showing no significant change after anaerobic fermentation (p>0.05). Most ARGs in downstream components (soils and fishpond) of EAS showed significantly higher relative abundance than the control agricultural system (p<0.05), except for tetG and sulI. Copyright © 2015 Elsevier B.V. All rights reserved.

Detection of tetQ and ermF antibiotic resistance genes in Prevotella and Porphyromonas isolates from clinical specimens and resident microbiota of humans.

PubMed

Arzese, A R; Tomasetig, L; Botta, G A

2000-05-01

Gram-negative anaerobes belonging to the genera Fusobacterium, Prevotella and Porphyromonas were investigated for the presence of tetQ and ermF, which have been shown to be spread by conjugal elements. One hundred isolates from either sites of infection or various body sites in healthy subjects were studied. PCR was used to detect tetQ, and DNA-DNA hybridization studies on EcoRI chromosomal digests were undertaken to detect the presence of tetQ and ermF. Antibiotic sensitivity assays were performed on selected isolates to detect tetracycline, erythromycin and penicillin resistance. Twenty Fusobacterium isolates lacked tetQ, and were tetracycline sensitive. Twenty per cent of Prevotella spp. isolates both from clinical specimens and from healthy subjects were found to possess tetQ. Of 20 Porphyromonas isolates tested, one (Porphyromonas levii) from a case of bacterial vaginosis was shown to possess tetQ in the chromosome. The presence of tetQ was always associated with tetracycline resistance. Four isolates of Prevotella melaninogenica and one isolate of Prevotella were ermF-positive, although expression of erythromycin resistance was not consistently associated with detection of this gene. Antibiotic resistance phenotypes of Prevotella isolates were shown to be related to specific chromosomal restriction patterns by hybridization studies: tetracycline resistance and tetracycline/erythromycin resistance are conferred by Bacteroides tetracycline-resistant ERL elements, whereas the tetracycline/penicillin resistance phenotype could be due to spread of elements identified in Prevotella only. Tetracycline/erythromycin-resistant and tetracycline/erythromycin/penicillin-resistant P. melaninogenica isolates were found in this study. It appeared that the presence of tetQ and ermF in Bacteroides and Prevotella contributed to the persistence of antibiotic resistance isolates within the host and to potential spread to other organisms through conjugal elements.

Synthetic dual-input mammalian genetic circuits enable tunable and stringent transcription control by chemical and light.

PubMed

Chen, Xianjun; Li, Ting; Wang, Xue; Du, Zengmin; Liu, Renmei; Yang, Yi

2016-04-07

Programmable transcription factors can enable precise control of gene expression triggered by a chemical inducer or light. To obtain versatile transgene system with combined benefits of a chemical inducer and light inducer, we created various chimeric promoters through the assembly of different copies of the tet operator and Gal4 operator module, which simultaneously responded to a tetracycline-responsive transcription factor and a light-switchable transactivator. The activities of these chimeric promoters can be regulated by tetracycline and blue light synergistically or antagonistically. Further studies of the antagonistic genetic circuit exhibited high spatiotemporal resolution and extremely low leaky expression, which therefore could be used to spatially and stringently control the expression of highly toxic protein Diphtheria toxin A for light regulated gene therapy. When transferring plasmids engineered for the gene switch-driven expression of a firefly luciferase (Fluc) into mice, the Fluc expression levels of the treated animals directly correlated with the tetracycline and light input program. We suggest that dual-input genetic circuits using TET and light that serve as triggers to achieve expression profiles may enable the design of robust therapeutic gene circuits for gene- and cell-based therapies. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Efficient Power-Transfer Capability Analysis of the TET System Using the Equivalent Small Parameter Method.

PubMed

Yanzhen Wu; Hu, A P; Budgett, D; Malpas, S C; Dissanayake, T

2011-06-01

Transcutaneous energy transfer (TET) enables the transfer of power across the skin without direct electrical connection. It is a mechanism for powering implantable devices for the lifetime of a patient. For maximum power transfer, it is essential that TET systems be resonant on both the primary and secondary sides, which requires considerable design effort. Consequently, a strong need exists for an efficient method to aid the design process. This paper presents an analytical technique appropriate to analyze complex TET systems. The system's steady-state solution in closed form with sufficient accuracy is obtained by employing the proposed equivalent small parameter method. It is shown that power-transfer capability can be correctly predicted without tedious iterative simulations or practical measurements. Furthermore, for TET systems utilizing a current-fed push-pull soft switching resonant converter, it is found that the maximum energy transfer does not occur when the primary and secondary resonant tanks are "tuned" to the nominal resonant frequency. An optimal turning point exists, corresponding to the system's maximum power-transfer capability when optimal tuning capacitors are applied.

Chiral Antioxidant-based Gold Nanoclusters Reprogram DNA Epigenetic Patterns

PubMed Central

Ma, Yue; Fu, Hualin; Zhang, Chunlei; Cheng, Shangli; Gao, Jie; Wang, Zhen; Jin, Weilin; Conde, João; Cui, Daxiang

2016-01-01

Epigenetic modifications sit ‘on top of’ the genome and influence DNA transcription, which can force a significant impact on cellular behavior and phenotype and, consequently human development and disease. Conventional methods for evaluating epigenetic modifications have inherent limitations and, hence, new methods based on nanoscale devices are needed. Here, we found that antioxidant (glutathione) chiral gold nanoclusters induce a decrease of 5-hydroxymethylcytosine (5hmC), which is an important epigenetic marker that associates with gene transcription regulation. This epigenetic change was triggered partially through ROS activation and oxidation generated by the treatment with glutathione chiral gold nanoclusters, which may inhibit the activity of TET proteins catalyzing the conversion of 5-methylcytosine (5mC) to 5hmC. In addition, these chiral gold nanoclusters can downregulate TET1 and TET2 mRNA expression. Alteration of TET-5hmC signaling will then affect several downstream targets and be involved in many aspects of cell behavior. We demonstrate for the first time that antioxidant-based chiral gold nanomaterials have a direct effect on epigenetic process of TET-5hmC pathways and reveal critical DNA demethylation patterns. PMID:27633378

Time-controllable Nkcc1 knockdown replicates reversible hearing loss in postnatal mice.

PubMed

Watabe, Takahisa; Xu, Ming; Watanabe, Miho; Nabekura, Junichi; Higuchi, Taiga; Hori, Karin; Sato, Mitsuo P; Nin, Fumiaki; Hibino, Hiroshi; Ogawa, Kaoru; Masuda, Masatsugu; Tanaka, Kenji F

2017-10-19

Identification of the causal effects of specific proteins on recurrent and partially reversible hearing loss has been difficult because of the lack of an animal model that provides reversible gene knockdown. We have developed the transgenic mouse line Actin-tTS::Nkcc1 tetO/tetO for manipulatable expression of the cochlear K + circulation protein, NKCC1. Nkcc1 transcription was blocked by the binding of a tetracycline-dependent transcriptional silencer to the tetracycline operator sequences inserted upstream of the Nkcc1 translation initiation site. Administration of the tetracycline derivative doxycycline reversibly regulated Nkcc1 knockdown. Progeny from pregnant/lactating mothers fed doxycycline-free chow from embryonic day 0 showed strong suppression of Nkcc1 expression (~90% downregulation) and Nkcc1 null phenotypes at postnatal day 35 (P35). P35 transgenic mice from mothers fed doxycycline-free chow starting at P0 (delivery) showed weaker suppression of Nkcc1 expression (~70% downregulation) and less hearing loss with mild cochlear structural changes. Treatment of these mice at P35 with doxycycline for 2 weeks reactivated Nkcc1 transcription to control levels and improved hearing level at high frequency; i.e., these doxycycline-treated mice exhibited partially reversible hearing loss. Thus, development of the Actin-tTS::Nkcc1 tetO/tetO transgenic mouse line provides a mouse model for the study of variable hearing loss through reversible knockdown of Nkcc1.

Tetracycline Susceptibility in Chlamydia suis Pig Isolates.

PubMed

Donati, Manuela; Balboni, Andrea; Laroucau, Karine; Aaziz, Rachid; Vorimore, Fabien; Borel, Nicole; Morandi, Federico; Vecchio Nepita, Edoardo; Di Francesco, Antonietta

2016-01-01

The aims of the present study were to assess the prevalence of Chlamydia suis in an Italian pig herd, determine the tetracycline susceptibility of C. suis isolates, and evaluate tet(C) and tetR(C) gene expression. Conjunctival swabs from 20 pigs were tested for C. suis by real-time polymerase chain reaction, and 55% (11) were positive. C. suis was then isolated from 11 conjunctival swabs resampled from the same herd. All positive samples and isolates were positive for the tet(C) resistance gene. The in vitro susceptibility to tetracycline of the C. suis isolates showed MIC values ranging from 0.5 to 4 μg/mL. Tet(C) and tetR(C) transcripts were found in all the isolates, cultured both in the absence and presence of tetracycline. This contrasts with other Gram-negative bacteria in which both genes are repressed in the absence of the drug. Further investigation into tet gene regulation in C. suis is needed.

Aid is a key regulator of myeloid/erythroid differentiation and DNA methylation in hematopoietic stem/progenitor cells

PubMed Central

Kunimoto, Hiroyoshi; McKenney, Anna Sophia; Meydan, Cem; Shank, Kaitlyn; Nazir, Abbas; Rapaport, Franck; Durham, Benjamin; Garrett-Bakelman, Francine E.; Pronier, Elodie; Shih, Alan H.; Melnick, Ari; Chaudhuri, Jayanta

2017-01-01

Recent studies have reported that activation-induced cytidine deaminase (AID) and ten-eleven-translocation (TET) family members regulate active DNA demethylation. Genetic alterations of TET2 occur in myeloid malignancies, and hematopoietic-specific loss of Tet2 induces aberrant hematopoietic stem cell (HSC) self-renewal/differentiation, implicating TET2 as a master regulator of normal and malignant hematopoiesis. Despite the functional link between AID and TET in epigenetic gene regulation, the role of AID loss in hematopoiesis and myeloid transformation remains to be investigated. Here, we show that Aid loss in mice leads to expansion of myeloid cells and reduced erythroid progenitors resulting in anemia, with dysregulated expression of Cebpa and Gata1, myeloid/erythroid lineage-specific transcription factors. Consistent with data in the murine context, silencing of AID in human bone marrow cells skews differentiation toward myelomonocytic lineage. However, in contrast to Tet2 loss, Aid loss does not contribute to enhanced HSC self-renewal or cooperate with Flt3-ITD to induce myeloid transformation. Genome-wide transcription and differential methylation analysis uncover the critical role of Aid as a key epigenetic regulator. These results indicate that AID and TET2 share common effects on myeloid and erythroid lineage differentiation, however, their role is nonredundant in regulating HSC self-renewal and in myeloid transformation. PMID:28077417

Rescue of TET2 Haploinsufficiency in Myelodysplastic Syndrome Patients Using Turbo Cosubstrate

DTIC Science & Technology

2017-07-01

Annual PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 DISTRIBUTION STATEMENT: Approved for...2446 9. SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S) U.S. Army Medical Research and Materiel Command Fort...enhance the activity of TET2 enzymes from MDS patient. Further, we published a manuscript on convenient expression, purification and quantitative liquid

Reversal of multidrug resistance by magnetic Fe3O4 nanoparticle copolymerizating daunorubicin and 5-bromotetrandrine in xenograft nude-mice

PubMed Central

Chen, Baoan; Cheng, Jian; Wu, Yanan; Gao, Feng; Xu, Wenlin; Shen, Huilin; Ding, Jiahua; Gao, Chong; Sun, Qian; Sun, Xinchen; Cheng, Hongyan; Li, Guohong; Chen, Wenji; Chen, Ningna; Liu, Lijie; Li, Xiaomao; Wang, Xuemei

2009-01-01

In this paper we establish the xenograft leukemia model with stable multidrug resistance in nude mice and to investigate the reversal effect of 5-bromotetrandrine (5-BrTet) and magnetic nanoparticle of Fe3O4 (MNP-Fe3O4) combined with daunorubicin (DNR) in vivo. Two subclones of K562 and K562/A02 cells were inoculated subcutaneously into the back of athymic nude mice (1 × 107 cells/each) respectively to establish leukemia xenograft models. Drug-resistant and sensitive tumor-bearing nude mice were assigned randomly into five groups which were treated with normal saline; DNR; NP-Fe3O4 combined with DNR; 5-BrTet combined with DNR; 5-BrTet and MNP-Fe3O4 combined with DNR, respectively. The incidence of formation, growth characteristics, weight, and volume of tumors were observed. The histopathologic examination of tumors and organs were detected. For resistant tumors, the protein levels of Bcl-2, and BAX were detected by Western blot. Bcl-2, BAX, and caspase-3 genes were also detected. For K562/A02 cells xenograft tumors, 5-BrTet and MNP-Fe3O4 combined with DNR significantly suppressed growth of tumor. A histopathologic examination of tumors clearly showed necrosis of the tumors. Application of 5-BrTet and MNP-Fe3O4 inhibited the expression of Bcl-2 protein and upregulated the expression of BAX and caspase-3 proteins in K562/A02 cells xenograft tumor. It is concluded that 5-BrTet and MNP-Fe3O4 combined with DNR had a significant tumor-suppressing effect on a MDR leukemia cells xenograft model. PMID:19421372

Reversal of multidrug resistance by magnetic Fe3O4 nanoparticle copolymerizating daunorubicin and 5-bromotetrandrine in xenograft nude-mice.

PubMed

Chen, Baoan; Cheng, Jian; Wu, Yanan; Gao, Feng; Xu, Wenlin; Shen, Huilin; Ding, Jiahua; Gao, Chong; Sun, Qian; Sun, Xinchen; Cheng, Hongyan; Li, Guohong; Chen, Wenji; Chen, Ningna; Liu, Lijie; Li, Xiaomao; Wang, Xuemei

2009-01-01

In this paper we establish the xenograft leukemia model with stable multidrug resistance in nude mice and to investigate the reversal effect of 5-bromotetrandrine (5-BrTet) and magnetic nanoparticle of Fe(3)O(4) (MNP-Fe(3)O(4)) combined with daunorubicin (DNR) in vivo. Two subclones of K562 and K562/A02 cells were inoculated subcutaneously into the back of athymic nude mice (1 x 10(7) cells/each) respectively to establish leukemia xenograft models. Drug-resistant and sensitive tumor-bearing nude mice were assigned randomly into five groups which were treated with normal saline; DNR; NP-Fe(3)O(4) combined with DNR; 5-BrTet combined with DNR; 5-BrTet and MNP-Fe(3)O(4) combined with DNR, respectively. The incidence of formation, growth characteristics, weight, and volume of tumors were observed. The histopathologic examination of tumors and organs were detected. For resistant tumors, the protein levels of Bcl-2, and BAX were detected by Western blot. Bcl-2, BAX, and caspase-3 genes were also detected. For K562/A02 cells xenograft tumors, 5-BrTet and MNP-Fe(3)O(4) combined with DNR significantly suppressed growth of tumor. A histopathologic examination of tumors clearly showed necrosis of the tumors. Application of 5-BrTet and MNP-Fe(3)O(4) inhibited the expression of Bcl-2 protein and upregulated the expression of BAX and caspase-3 proteins in K562/A02 cells xenograft tumor. It is concluded that 5-BrTet and MNP-Fe(3)O(4) combined with DNR had a significant tumor-suppressing effect on a MDR leukemia cells xenograft model.

Distribution of tetracycline resistance genes and AmpC β-lactamase genes in representative non-urban sewage plants and correlations with treatment processes and heavy metals.

PubMed

Xu, Yan-Bin; Hou, Mao-Yu; Li, Ya-Fei; Huang, Lu; Ruan, Jing-Jing; Zheng, Li; Qiao, Qing-Xia; Du, Qing-Ping

2017-03-01

The mixed development of livestock breeding and industry in non-urban zones is a very general phenomenon in China. Distribution of antibiotic resistance genes (ARGs) in non-urban sewage treatment systems has not been paid enough attentions. In this study, eleven tetracycline resistance genes (tetA, tetB, tetC, tetE, tetG, tetL, tetM, tetO, tetQ, tetS and tetX), four AmpC β-lactamase genes (EBC, MOX, FOX and CIT) and four heavy metals (Cu, Zn, Cd and Pb) were detected and analyzed in four non-urban sewage plants with different sewage sources and different treatment processes in Guangzhou. The results showed that tetA and tetC were the most prevalent tetracycline resistance genes with the same detection frequency of 85% and EBC was the most prevalent AmpC β-lactamase gene with a detection frequency of 75%. The relative abundance of tetracycline resistance genes was approximately 1.6 orders of magnitudes higher than that of AmpC β-lactamase genes in all samples. A/O was the most effective process for the non-urban sewage plant receiving industrial or agricultural wastewater. Sedimentation was the most key process to eliminate ARGs from liquid phase. Most ARGs were carried in excess sludge rather than effluent. Significant correlation was found between the tet gene and Zn (r = 0.881, p < 0.01), followed by the AmpC gene and Cu (r = 0.847, p < 0.01), the tet gene and Cu (r = 0.714, p < 0.05). Therefore, the pollution of ARGs in the sewage treatment systems of non-urban zones co-polluted by heavy metals should be paid more attentions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Preparation, characterization, pharmacokinetics and tissue distribution of solid lipid nanoparticles loaded with tetrandrine.

PubMed

Li, Su; Ji, Zhaoshuai; Zou, Meijuan; Nie, Xin; Shi, Yijie; Cheng, Gang

2011-09-01

Tetrandrine (TET) is a poorly water-soluble bisbenzylisoquinoline alkaloid. In this study, TET solid lipid nanoparticles (SLNs) were prepared by a melt-emulsification and ultrasonication technique. Precirol(®) ATO 5, glyceryl monostearate, and stearic acid were used as the lipid matrix for the SLNs, while Lipoid E80, Pluronic F68, and sodium deoxycholate were used as emulsifying and stabilizing agents. The physicochemical characteristics of the TET-SLNs were investigated when it was found that the mean particle size and zeta potential of the TET-SLNs were 134 ± 1.3 nm and -53.8 ± 1.7 mV, respectively, and the entrapment efficiency (EE) was 89.57% ± 0.39%. Differential scanning calorimetry indicated that TET was in an amorphous state in SLNs. TET-SLNs exhibited a higher release rate at a lower pH and a lower release rate at a higher pH. The release pattern of the TET-SLNs followed the Weibull model. The pharmacokinetics of TET-SLNs after intravenous administration to male rats was studied. TET-SLN resulted in a higher plasma concentration and lower clearance. The biodistribution study indicated that TET-SLN showed a high uptake in reticuloendothelial system organs. In conclusion, TET-SLNs with a small particle size, and high EE, can be produced by the method described in this study. The SLN system is a promising approach for the intravenous delivery of tetrandrine.

Magnetic-Fe/Fe(3)O(4)-nanoparticle-bound SN38 as carboxylesterase-cleavable prodrug for the delivery to tumors within monocytes/macrophages.

PubMed

Wang, Hongwang; Shrestha, Tej B; Basel, Matthew T; Dani, Raj Kumar; Seo, Gwi-Moon; Balivada, Sivasai; Pyle, Marla M; Prock, Heidy; Koper, Olga B; Thapa, Prem S; Moore, David; Li, Ping; Chikan, Viktor; Troyer, Deryl L; Bossmann, Stefan H

2012-01-01

The targeted delivery of therapeutics to the tumor site is highly desirable in cancer treatment, because it is capable of minimizing collateral damage. Herein, we report the synthesis of a nanoplatform, which is composed of a 15 ± 1 nm diameter core/shell Fe/Fe(3)O(4) magnetic nanoparticles (MNPs) and the topoisomerase I blocker SN38 bound to the surface of the MNPs via a carboxylesterase cleavable linker. This nanoplatform demonstrated high heating ability (SAR = 522 ± 40 W/g) in an AC-magnetic field. For the purpose of targeted delivery, this nanoplatform was loaded into tumor-homing double-stable RAW264.7 cells (mouse monocyte/macrophage-like cells (Mo/Ma)), which have been engineered to express intracellular carboxylesterase (InCE) upon addition of doxycycline by a Tet-On Advanced system. The nanoplatform was taken up efficiently by these tumor-homing cells. They showed low toxicity even at high nanoplatform concentration. SN38 was released successfully by switching on the Tet-On Advanced system. We have demonstrated that this nanoplatform can be potentially used for thermochemotherapy. We will be able to achieve the following goals: (1) Specifically deliver the SN38 prodrug and magnetic nanoparticles to the cancer site as the payload of tumor-homing double-stable RAW264.7 cells; (2) Release of chemotherapeutic SN38 at the cancer site by means of the self-containing Tet-On Advanced system; (3) Provide localized magnetic hyperthermia to enhance the cancer treatment, both by killing cancer cells through magnetic heating and by activating the immune system.

Helicobacter pylori gene silencing in vivo demonstrates urease is essential for chronic infection

PubMed Central

Walton, Senta M.; Liao, Tingting; Stubbs, Keith A.; Marshall, Barry J.; Fulurija, Alma; Benghezal, Mohammed

2017-01-01

Helicobacter pylori infection causes chronic active gastritis that after many years of infection can develop into peptic ulceration or gastric adenocarcinoma. The bacterium is highly adapted to surviving in the gastric environment and a key adaptation is the virulence factor urease. Although widely postulated, the requirement of urease expression for persistent infection has not been elucidated experimentally as conventional urease knockout mutants are incapable of colonization. To overcome this constraint, conditional H. pylori urease mutants were constructed by adapting the tetracycline inducible expression system that enabled changing the urease phenotype of the bacteria during established infection. Through tight regulation we demonstrate that urease expression is not only required for establishing initial colonization but also for maintaining chronic infection. Furthermore, successful isolation of tet-escape mutants from a late infection time point revealed the strong selective pressure on this gastric pathogen to continuously express urease in order to maintain chronic infection. In addition to mutations in the conditional gene expression system, escape mutants were found to harbor changes in other genes including the alternative RNA polymerase sigma factor, fliA, highlighting the genetic plasticity of H. pylori to adapt to a changing niche. The tet-system described here opens up opportunities to studying genes involved in the chronic stage of H. pylori infection to gain insight into bacterial mechanisms promoting immune escape and life-long infection. Furthermore, this genetic tool also allows for a new avenue of inquiry into understanding the importance of various virulence determinants in a changing biological environment when the bacterium is put under duress. PMID:28644872

Helicobacter pylori gene silencing in vivo demonstrates urease is essential for chronic infection.

PubMed

Debowski, Aleksandra W; Walton, Senta M; Chua, Eng-Guan; Tay, Alfred Chin-Yen; Liao, Tingting; Lamichhane, Binit; Himbeck, Robyn; Stubbs, Keith A; Marshall, Barry J; Fulurija, Alma; Benghezal, Mohammed

2017-06-01

Helicobacter pylori infection causes chronic active gastritis that after many years of infection can develop into peptic ulceration or gastric adenocarcinoma. The bacterium is highly adapted to surviving in the gastric environment and a key adaptation is the virulence factor urease. Although widely postulated, the requirement of urease expression for persistent infection has not been elucidated experimentally as conventional urease knockout mutants are incapable of colonization. To overcome this constraint, conditional H. pylori urease mutants were constructed by adapting the tetracycline inducible expression system that enabled changing the urease phenotype of the bacteria during established infection. Through tight regulation we demonstrate that urease expression is not only required for establishing initial colonization but also for maintaining chronic infection. Furthermore, successful isolation of tet-escape mutants from a late infection time point revealed the strong selective pressure on this gastric pathogen to continuously express urease in order to maintain chronic infection. In addition to mutations in the conditional gene expression system, escape mutants were found to harbor changes in other genes including the alternative RNA polymerase sigma factor, fliA, highlighting the genetic plasticity of H. pylori to adapt to a changing niche. The tet-system described here opens up opportunities to studying genes involved in the chronic stage of H. pylori infection to gain insight into bacterial mechanisms promoting immune escape and life-long infection. Furthermore, this genetic tool also allows for a new avenue of inquiry into understanding the importance of various virulence determinants in a changing biological environment when the bacterium is put under duress.

Seasonal variation and removal efficiency of antibiotic resistance genes during wastewater treatment of swine farms.

PubMed

Sui, Qianwen; Zhang, Junya; Tong, Juan; Chen, Meixue; Wei, Yuansong

2017-04-01

The seasonal variation and removal efficiency of antibiotic resistance genes (ARGs), including tetracycline resistance genes (tetG, tetM, and tetX) and macrolide (ermB, ermF, ereA, and mefA), were investigated in two typical swine wastewater treatment systems in both winter and summer. ARGs, class 1 integron gene, and 16S rRNA gene were quantified using real-time polymerase chain reaction assays. There was a 0.31-3.52 log variation in ARGs in raw swine wastewater, and the abundance of ARGs in winter was higher than in summer. tetM, tetX, ermB, ermF, and mefA were highly abundant. The abundance of ARGs was effectively reduced by most individual treatment process and the removal efficiencies of ARGs were higher in winter than in summer. However, when examining relative abundance, the fate of ARGs was quite variable. Anaerobic digestion reduced the relative abundance of tetX, ermB, ermF, and mefA, while lagoon treatment decreased tetM, ermB, ermF, and mefA. Sequencing batch reactor (SBR) decreased tetM, ermB, and ermF, but biofilters and wetlands did not display consistent removal efficiency on ARGs in two sampling seasons. As far as the entire treatment system is concerned, ermB and mefA were effectively reduced in both winter and summer in both total and relative abundance. The relative abundances of tetG and ereA were significantly correlated with intI1 (p < 0.01), and both tetG and ereA increased after wastewater treatment. This may pose a great threat to public health.

Hypoxia-Mediated Epigenetic Regulation of Stemness in Brain Tumor Cells.

PubMed

Prasad, Pankaj; Mittal, Shivani Arora; Chongtham, Jonita; Mohanty, Sujata; Srivastava, Tapasya

2017-06-01

Activation of pluripotency regulatory circuit is an important event in solid tumor progression and the hypoxic microenvironment is known to enhance the stemness feature of some cells. The distinct population of cancer stem cells (CSCs)/tumor initiating cells exist in a niche and augment invasion, metastasis, and drug resistance. Previously, studies have reported global hypomethylation and site-specific aberrant methylation in gliomas along with other epigenetic modifications as important contributors to genomic instability during glioma progression. Here, we have demonstrated the role of hypoxia-mediated epigenetic modifications in regulating expression of core pluripotency factors, OCT4 and NANOG, in glioma cells. We observe hypoxia-mediated induction of demethylases, ten-eleven-translocation (TET) 1 and 3, but not TET2 in our cell-line model. Immunoprecipitation studies reveal active demethylation and direct binding of TET1 and 3 at the Oct4 and Nanog regulatory regions. Tet1 and 3 silencing assays further confirmed induction of the pluripotency pathway involving Oct4, Nanog, and Stat3, by these paralogues, although with varying degrees. Knockdown of Tet1 and Tet3 inhibited the formation of neurospheres in hypoxic conditions. We observed independent roles of TET1 and TET3 in differentially regulating pluripotency and differentiation associated genes in hypoxia. Overall, this study demonstrates an active demethylation in hypoxia by TET1 and 3 as a mechanism of Oct4 and Nanog overexpression thus contributing to the formation of CSCs in gliomas. Stem Cells 2017;35:1468-1478. © 2017 AlphaMed Press.

Live-cell imaging combined with immunofluorescence, RNA, or DNA FISH to study the nuclear dynamics and expression of the X-inactivation center.

PubMed

Pollex, Tim; Piolot, Tristan; Heard, Edith

2013-01-01

Differentiation of embryonic stem cells is accompanied by changes of gene expression and chromatin and chromosome dynamics. One of the most impressive examples for these changes is inactivation of one of the two X chromosomes occurring upon differentiation of mouse female embryonic stem cells. With a few exceptions, these events have been mainly studied in fixed cells. In order to better understand the dynamics, kinetics, and order of events during differentiation, one needs to employ live-cell imaging techniques. Here, we describe a combination of live-cell imaging with techniques that can be used in fixed cells (e.g., RNA FISH) to correlate locus dynamics or subnuclear localization with, e.g., gene expression. To study locus dynamics in female ES cells, we generated cell lines containing TetO arrays in the X-inactivation center, the locus on the X chromosome regulating X-inactivation, which can be visualized upon expression of TetR fused to fluorescent proteins. We will use this system to elaborate on how to generate ES cell lines for live-cell imaging of locus dynamics, how to culture ES cells prior to live-cell imaging, and to describe typical live-cell imaging conditions for ES cells using different microscopes. Furthermore, we will explain how RNA, DNA FISH, or immunofluorescence can be applied following live-cell imaging to correlate gene expression with locus dynamics.

Synthetic Promoters Functional in Francisella novicida and Escherichia coli

PubMed Central

McWhinnie, Ralph L.

2014-01-01

In this work, we describe the identification of synthetic, controllable promoters that function in the bacterial pathogen Francisella novicida, a model facultative intracellular pathogen. Synthetic DNA fragments consisting of the tetracycline operator (tetO) flanked by a random nucleotide sequence were inserted into a Francisella novicida shuttle plasmid upstream of a promoterless artificial operon containing the reporter genes cat and lacZ. Fragments able to promote transcription were selected for based on their ability to drive expression of the cat gene, conferring chloramphenicol resistance. Promoters of various strengths were found, many of which were repressed in the presence of the tetracycline repressor (TetR) and promoted transcription only in the presence of the TetR inducer anhydrotetracycline. A subset of both constitutive and inducible synthetic promoters were characterized to find their induction ratios and to identify their transcription start sites. In cases where tetO was located between or downstream of the −10 and −35 regions of the promoter, control by TetR was observed. If the tetO region was upstream of the −35 region by more than 9 bp, it did not confer TetR control. We found that three of three promoters isolated in F. novicida functioned at a comparable level in E. coli; however, none of the 10 promoters isolated in E. coli functioned at a significant level in F. novicida. Our results allowed us to isolate minimal F. novicida promoters of 47 and 48 bp in length. PMID:24141126

WP1: transgenic opto-animals

NASA Astrophysics Data System (ADS)

UŻarowska, E.; Czajkowski, Rafał; Konopka, W.

2014-11-01

We aim to create a set of genetic tools where permanent opsin expression (ChR or NpHR) is precisely limited to the population of neurons that express immediate early gene c-fos during a specific temporal window of behavioral training. Since the c-fos gene is only expressed in neurons that form experience-dependent ensemble, this approach will result in specific labeling of a small subset of cells that create memory trace for the learned behavior. To this end we employ two alternative inducible gene expression systems: Tet Expression System and Cre/lox System. In both cases, the temporal window for opsin induction is controlled pharmacologically, by doxycycline or tamoxifen, respectively. Both systems will be used for creating lines of transgenic animals.

A mutation in the tuft mouse disrupts TET1 activity and alters the expression of genes that are crucial for neural tube closure.

PubMed

Fong, Keith S K; Hufnagel, Robert B; Khadka, Vedbar S; Corley, Michael J; Maunakea, Alika K; Fogelgren, Ben; Ahmed, Zubair M; Lozanoff, Scott

2016-05-01

Genetic variations affecting neural tube closure along the head result in malformations of the face and brain. Neural tube defects (NTDs) are among the most common birth defects in humans. We previously reported a mouse mutant called tuft that arose spontaneously in our wild-type 3H1 colony. Adult tuft mice present midline craniofacial malformations with or without an anterior cephalocele. In addition, affected embryos presented neural tube closure defects resulting in insufficient closure of the anterior neuropore or exencephaly. Here, through whole-genome sequencing, we identified a nonsense mutation in the Tet1 gene, which encodes a methylcytosine dioxygenase (TET1), co-segregating with the tuft phenotype. This mutation resulted in premature termination that disrupts the catalytic domain that is involved in the demethylation of cytosine. We detected a significant loss of TET enzyme activity in the heads of tuft embryos that were homozygous for the mutation and had NTDs. RNA-Seq transcriptome analysis indicated that multiple gene pathways associated with neural tube closure were dysregulated in tuft embryo heads. Among them, the expressions of Cecr2, Epha7 and Grhl2 were significantly reduced in some embryos presenting neural tube closure defects, whereas one or more components of the non-canonical WNT signaling pathway mediating planar cell polarity and convergent extension were affected in others. We further show that the recombinant mutant TET1 protein was capable of entering the nucleus and affected the expression of endogenous Grhl2 in IMCD-3 (inner medullary collecting duct) cells. These results indicate that TET1 is an epigenetic determinant for regulating genes that are crucial to closure of the anterior neural tube and its mutation has implications to craniofacial development, as presented by the tuft mouse. © 2016. Published by The Company of Biologists Ltd.

Transcriptional activation of transposable elements in mouse zygotes is independent of Tet3-mediated 5-methylcytosine oxidation.

PubMed

Inoue, Azusa; Matoba, Shogo; Zhang, Yi

2012-12-01

The methylation state of the paternal genome is rapidly reprogrammed shortly after fertilization. Recent studies have revealed that loss of 5-methylcytosine (5mC) in zygotes correlates with appearance of 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). This process is mediated by Tet3 and the 5mC oxidation products generated in zygotes are gradually lost during preimplantation development through a replication-dependent dilution process. Despite these findings, the biological significance of Tet3-mediated oxidation of 5mC to 5hmC/5fC/5caC in zygotes is unknown. DNA methylation plays an important role in silencing gene expression including the repression of transposable elements (TEs). Given that the activation of TEs during preimplantation development correlates with loss of DNA methylation, it is believed that paternal DNA demethylation may have an important role in TE activation. Here we examined this hypothesis and found that Tet3-mediated 5mC oxidation does not have a significant contribution to TE activation. We show that the expression of LINE-1 (long interspersed nucleotide element 1) and ERVL (endogenous retroviruses class III) are activated from both paternal and maternal genomes in zygotes. Inhibition of 5mC oxidation by siRNA-mediated depletion of Tet3 affected neither TE activation, nor global transcription in zygotes. Thus, our study provides the first evidence demonstrating that activation of both TEs and global transcription in zygotes are independent of Tet3-mediated 5mC oxidation.

A transcutaneous energy transmission system for artificial heart adapting to changing impedance.

PubMed

Fu, Yang; Hu, Liang; Ruan, Xiaodong; Fu, Xin

2015-04-01

This article presents a coil-coupling-based transcutaneous energy transmission system (TETS) for wirelessly powering an implanted artificial heart. Keeping high efficiency is especially important for TETS, which is usually difficult due to transmission impedance changes in practice, which are commonly caused by power requirement variation for different body movements and coil-couple malposition accompanying skin peristalsis. The TETS introduced in this article is designed based on a class-E power amplifier (E-PA), of which efficiency is over 95% when its load is kept in a certain range. A resonance matching and impedance compressing functions coupled network based on parallel-series capacitors is proposed in the design, to enhance the energy transmission efficiency and capacity of the coil-couple through resonating, and meanwhile compress the changing range of the transmission impedance to meet the load requirements of the E-PA and thus keep the high efficiency of TETS. An analytical model of the designed TETS is built to analyze the effect of the network and also provide bases for following parameters determination. Then, according algorithms are provided to determine the optimal parameters required in the TETS for good performance both in resonance matching and impedance compressing. The design is tested by a series of experiments, which validate that the TETS can transmit a wide range of power with a total efficiency of at least 70% and commonly beyond 80%, even when the coil-couple is seriously malpositioned. The design methodology proposed in this article can be applied to any existing TETS based on E-PA to improve their performance in actual applications. Copyright © 2014 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Abundance and persistence of antibiotic resistance genes in livestock farms: a comprehensive investigation in eastern China.

PubMed

Cheng, Weixiao; Chen, Hong; Su, Chao; Yan, Shuhai

2013-11-01

Increases of antibiotic resistance genes in the environment may pose a threat to public health. The purpose of this study was to investigate the abundance and diversity of tetracycline (tet) and sulfonamide (sul) resistance genes in eight livestock farms in Hangzhou, eastern China. Ten tet genes (tetA, tetB, tetC, tetG, tetL, tetM, tetO, tetQ, tetW, and tetX), two sul genes (sulI and sulII), and one genetic element associated with mobile antibiotic resistance genes [class 1 integron (intI1)] were quantified by real-time polymerase chain reaction. No significant difference was found in the abundance of the tet and sul genes in various scales of pig, chicken, and duck farms (P>0.05). The average abundance of ribosomal protection protein genes (tetQ, tetM, tetW, and tetO) in the manure and wastewater samples was higher than most of the efflux pump genes (tetA, tetB, tetC, and tetL) and enzymatic modification gene (tetX) (P<0.05), except for efflux pump gene tetG, which was abundant and showed no difference from tetM. Most ARGs had higher relative abundance in the wastewater lagoon than in manures even after treatment. Although the three ribosomal protection protein genes (tetQ, tetW, and tetO) had higher relative abundance, numbers were reduced during the complete wastewater treatment process in pig farms (P<0.05). The relative abundance of tetG, sulI, and sulII increased after the wastewater treatment and the removal of these three genes exhibited significant positive correlations with the intI1 gene (tetG: R(2)=0.60, P<0.05; sulI: R(2)=0.72, P<0.05; sulII: R(2)=0.62, P<0.05), suggesting that intI1 may be involved in their proliferation. As for tetM and sulII genes, a highly significant difference was found in manure samples between pig farms and duck farms (P<0.001). Phylogenetic analysis showed that tetM was more diverse in duck farms than in pig farms. Additionally, sulII sequence was conserved both in pig and duck farms. This is the first comprehensive study to detail the relative abundance of specific ARGs in animal manures and agricultural wastewater treatment systems, potentially providing knowledge for managing antibiotic resistance emanating from agricultural activities. © 2013.

Plasmid-Encoded Tetracycline Efflux Pump Protein Alters Bacterial Stress Responses and Ecological Fitness of Acinetobacter oleivorans

PubMed Central

Hong, Hyerim; Jung, Jaejoon; Park, Woojun

2014-01-01

Acquisition of the extracellular tetracycline (TC) resistance plasmid pAST2 affected host gene expression and phenotype in the oil-degrading soil bacterium, Acinetobacter oleivorans DR1. Whole-transcriptome profiling of DR1 cells harboring pAST2 revealed that all the plasmid genes were highly expressed under TC conditions, and the expression levels of many host chromosomal genes were modulated by the presence of pAST2. The host energy burden imposed by replication of pAST2 led to (i) lowered ATP concentrations, (ii) downregulated expression of many genes involved in cellular growth, and (iii) reduced growth rate. Interestingly, some phenotypes were restored by deleting the plasmid-encoded efflux pump gene tetH, suggesting that the membrane integrity changes resulting from the incorporation of efflux pump proteins also resulted in altered host response under the tested conditions. Alteration of membrane integrity by tetH deletion was shown by measuring permeability of fluorescent probe and membrane hydrophobicity. The presence of the plasmid conferred peroxide and superoxide resistance to cells, but only peroxide resistance was diminished by tetH gene deletion, suggesting that the plasmid-encoded membrane-bound efflux pump protein provided peroxide resistance. The downregulation of fimbriae-related genes presumably led to reduced swimming motility, but this phenotype was recovered by tetH gene deletion. Our data suggest that not only the plasmid replication burden, but also its encoded efflux pump protein altered host chromosomal gene expression and phenotype, which also alters the ecological fitness of the host in the environment. PMID:25229538

Plasmid-encoded tetracycline efflux pump protein alters bacterial stress responses and ecological fitness of Acinetobacter oleivorans.

PubMed

Hong, Hyerim; Jung, Jaejoon; Park, Woojun

2014-01-01

Acquisition of the extracellular tetracycline (TC) resistance plasmid pAST2 affected host gene expression and phenotype in the oil-degrading soil bacterium, Acinetobacter oleivorans DR1. Whole-transcriptome profiling of DR1 cells harboring pAST2 revealed that all the plasmid genes were highly expressed under TC conditions, and the expression levels of many host chromosomal genes were modulated by the presence of pAST2. The host energy burden imposed by replication of pAST2 led to (i) lowered ATP concentrations, (ii) downregulated expression of many genes involved in cellular growth, and (iii) reduced growth rate. Interestingly, some phenotypes were restored by deleting the plasmid-encoded efflux pump gene tetH, suggesting that the membrane integrity changes resulting from the incorporation of efflux pump proteins also resulted in altered host response under the tested conditions. Alteration of membrane integrity by tetH deletion was shown by measuring permeability of fluorescent probe and membrane hydrophobicity. The presence of the plasmid conferred peroxide and superoxide resistance to cells, but only peroxide resistance was diminished by tetH gene deletion, suggesting that the plasmid-encoded membrane-bound efflux pump protein provided peroxide resistance. The downregulation of fimbriae-related genes presumably led to reduced swimming motility, but this phenotype was recovered by tetH gene deletion. Our data suggest that not only the plasmid replication burden, but also its encoded efflux pump protein altered host chromosomal gene expression and phenotype, which also alters the ecological fitness of the host in the environment.

The Development of a Viral Mediated CRISPR/Cas9 System with Doxycycline Dependent gRNA Expression for Inducible In vitro and In vivo Genome Editing

PubMed Central

de Solis, Christopher A.; Ho, Anthony; Holehonnur, Roopashri; Ploski, Jonathan E.

2016-01-01

The RNA-guided Cas9 nuclease, from the type II prokaryotic Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) adaptive immune system, has been adapted and utilized by scientists to edit the genomes of eukaryotic cells. Here, we report the development of a viral mediated CRISPR/Cas9 system that can be rendered inducible utilizing doxycycline (Dox) and can be delivered to cells in vitro and in vivo utilizing adeno-associated virus (AAV). Specifically, we developed an inducible gRNA (gRNAi) AAV vector that is designed to express the gRNA from a H1/TO promoter. This AAV vector is also designed to express the Tet repressor (TetR) to regulate the expression of the gRNAi in a Dox dependent manner. We show that H1/TO promoters of varying length and a U6/TO promoter can edit DNA with similar efficiency in vitro, in a Dox dependent manner. We also demonstrate that our inducible gRNAi vector can be used to edit the genomes of neurons in vivo within the mouse brain in a Dox dependent manner. Genome editing can be induced in vivo with this system by supplying animals Dox containing food for as little as 1 day. This system might be cross compatible with many existing S. pyogenes Cas9 systems (i.e., Cas9 mouse, CRISPRi, etc.), and therefore it likely can be used to render these systems inducible as well. PMID:27587996

Aldolase B inhibits metastasis through Ten-Eleven Translocation 1 and serves as a prognostic biomarker in hepatocellular carcinoma.

PubMed

Tao, Qi-Fei; Yuan, Sheng-Xian; Yang, Fu; Yang, Sen; Yang, Yuan; Yuan, Ji-Hang; Wang, Zhen-Guang; Xu, Qing-Guo; Lin, Kong-Ying; Cai, Jie; Yu, Jian; Huang, Wei-Long; Teng, Xiao-Lei; Zhou, Chuan-Chuan; Wang, Fang; Sun, Shu-Han; Zhou, Wei-Ping

2015-09-17

Downregulation of Aldolase B (ALDOB) has been reported in hepatocellular carcinoma. However, its clinical significance and its role in pathogenesis of HCC remain largely unknown. We analyzed the expression of ALDOB and its clinical features in a large cohort of 313 HCC patients using tissue microarray and immunohistochemistry. Moreover, the function of stably overexpressed ALDOB in HCC cells was explored in vitro and in vivo. Gene expression microarray analysis was performed on ALDOB-overexpressing SMMC7721 cells to elucidate its mechanism of action. ALDOB downregulation in HCC was significantly correlated with aggressive characteristics including absence of encapsulation, increased tumor size (>5 cm) and early recurrence. ALDOB downregulation was indicative of a shorter recurrence-free survival (RFS) and overall survival (OS) for all HCC patients and early-stage HCC patients (BCLC 0-A and TNM I stage patients). Multiple analyses revealed that ALDOB downregulation was an independent risk factor of RFS and OS. Stable expression of ALDOB in HCC cell lines reduced cell migration in vitro and inhibited lung metastasis, intrahepatic metastasis, and reduced circulating tumor cells in vivo. Mechanistically, we found that cells stably expressing ALDOB show elevated Ten-Eleven Translocation 1 (TET1) expression. Moreover, ALDOB expressing cells have higher levels of methylglyoxal than do control cells, which can upregulate TET1 expression. The downregulation of ALDOB could indicate a poor prognosis for HCC patients, and therefore, ALDOB might be considered a prognostic biomarker for HCC, especially at the early stage. In addition, ALDOB inhibits the invasive features of cell lines partly through TET1 expression.

The organotin compounds trimethyltin (TMT) and triethyltin (TET) but not tributyltin (TBT) induce activation of microglia co-cultivated with astrocytes.

PubMed

Röhl, C; Grell, M; Maser, E

2009-12-01

The organotin compounds trimethyltin (TMT), triethyltin (TET) and tributyltin (TBT) show different organotoxicities in vivo. While TMT and TET induce a strong neurotoxicity accompanied by microglial and astroglial activation, TBT rather effects the immune system. Previously, we have shown in an in vitro co-culture model that microglial cells can be activated by TMT in the presence of astrocytes. In this study, we wanted to investigate (a) if the neurotoxic organotin compound TET can also activate microglial cells in vitro similar to TMT and (b) if differences between the neurotoxicants TMT and TET on the one side and TBT on the other exist concerning microglial activation. Therefore, purified microglial and astroglial cell cultures from neonatal rat brains were treated either alone or in co-cultures for 24h with different concentrations of TMT, TET or TBT and the basal cytotoxicity and nitric oxide formation was determined. Furthermore, morphological changes of astrocytes were examined. Our results show that microglial activation can be increased in subcytolethal concentrations, but only in the presence of astrocytes and not in microglial cell cultures alone. This increase was induced by the neurotoxicants TMT and TET but not by TBT. Taken together, the differing microglia activating effect of the organotin compounds may contribute to the differing neurotoxic potential of this group of chemicals in vivo. In addition, our results emphasize the need for co-culture systems when studying interactions between different cell types for toxicity assessment.

Magnetic-Fe/Fe3O4-nanoparticle-bound SN38 as carboxylesterase-cleavable prodrug for the delivery to tumors within monocytes/macrophages

PubMed Central

Shrestha, Tej B; Basel, Matthew T; Dani, Raj Kumar; Seo, Gwi-Moon; Balivada, Sivasai; Pyle, Marla M; Prock, Heidy; Koper, Olga B; Thapa, Prem S; Moore, David; Li, Ping; Chikan, Viktor

2012-01-01

Summary The targeted delivery of therapeutics to the tumor site is highly desirable in cancer treatment, because it is capable of minimizing collateral damage. Herein, we report the synthesis of a nanoplatform, which is composed of a 15 ± 1 nm diameter core/shell Fe/Fe3O4 magnetic nanoparticles (MNPs) and the topoisomerase I blocker SN38 bound to the surface of the MNPs via a carboxylesterase cleavable linker. This nanoplatform demonstrated high heating ability (SAR = 522 ± 40 W/g) in an AC-magnetic field. For the purpose of targeted delivery, this nanoplatform was loaded into tumor-homing double-stable RAW264.7 cells (mouse monocyte/macrophage-like cells (Mo/Ma)), which have been engineered to express intracellular carboxylesterase (InCE) upon addition of doxycycline by a Tet-On Advanced system. The nanoplatform was taken up efficiently by these tumor-homing cells. They showed low toxicity even at high nanoplatform concentration. SN38 was released successfully by switching on the Tet-On Advanced system. We have demonstrated that this nanoplatform can be potentially used for thermochemotherapy. We will be able to achieve the following goals: (1) Specifically deliver the SN38 prodrug and magnetic nanoparticles to the cancer site as the payload of tumor-homing double-stable RAW264.7 cells; (2) Release of chemotherapeutic SN38 at the cancer site by means of the self-containing Tet-On Advanced system; (3) Provide localized magnetic hyperthermia to enhance the cancer treatment, both by killing cancer cells through magnetic heating and by activating the immune system. PMID:23016149

A review of the influence of treatment strategies on antibiotic resistant bacteria and antibiotic resistance genes.

PubMed

Sharma, Virender K; Johnson, Natalie; Cizmas, Leslie; McDonald, Thomas J; Kim, Hyunook

2016-05-01

Antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARG) in the aquatic environment have become an emerging contaminant issue, which has implications for human and ecological health. This review begins with an introduction to the occurrence of ARB and ARG in different environmental systems such as natural environments and drinking water resources. For example, ARG or ARB with resistance to ciprofloxacin, sulfamethoxazole, trimethoprim, quinolone, vancomycin, or tetracycline (e.g., tet(A), tet(B), tet(C), tet(G), tet(O), tet(M), tet(W), sul I, and sul II) have been detected in the environment. The development of resistance may be intrinsic, may be acquired through spontaneous mutations (de novo), or may occur due to horizontal gene transfer from donor bacteria, phages, or free DNA to recipient bacteria. An overview is also provided of the current knowledge regarding inactivation of ARB and ARG, and the mechanism of the effects of different disinfection processes in water and wastewater (chlorination, UV irradiation, Fenton reaction, ozonation, and photocatalytic oxidation). The effects of constructed wetlands and nanotechnology on ARB and ARG are also summarized. Copyright © 2015 Elsevier Ltd. All rights reserved.

Tet1 and Tet2 maintain mesenchymal stem cell homeostasis via demethylation of the P2rX7 promoter.

PubMed

Yang, Ruili; Yu, Tingting; Kou, Xiaoxing; Gao, Xiang; Chen, Chider; Liu, Dawei; Zhou, Yanheng; Shi, Songtao

2018-06-01

Ten-eleven translocation (Tet) family-mediated DNA oxidation represents an epigenetic modification capable of converting 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), which regulates various biological processes. However, it is unknown whether Tet family affects mesenchymal stem cells (MSCs) or the skeletal system. Here we show that depletion of Tet1 and Tet2 results in impaired self-renewal and differentiation of bone marrow MSCs (BMMSCs) and a significant osteopenia phenotype. Tet1 and Tet2 deficiency reduces demethylation of the P2rX7 promoter and downregulates exosome release, leading to intracellular accumulation of miR-297a-5p, miR-297b-5p, and miR-297c-5p. These miRNAs inhibit Runx2 signaling to impair BMMSC function. We show that overexpression of P2rX7 rescues the impaired BMMSCs and osteoporotic phenotype in Tet1 and Tet2 double knockout mice. These results indicate that Tet1 and Tet2 play a critical role in maintaining BMMSC and bone homeostasis through demethylation of P2rX7 to control exosome and miRNA release. This Tet/P2rX7/Runx2 cascade may serve as a target for the development of novel therapies for osteopenia disorders.

Polycistronic gene expression in Aspergillus niger.

PubMed

Schuetze, Tabea; Meyer, Vera

2017-09-25

Genome mining approaches predict dozens of biosynthetic gene clusters in each of the filamentous fungal genomes sequenced so far. However, the majority of these gene clusters still remain cryptic because they are not expressed in their natural host. Simultaneous expression of all genes belonging to a biosynthetic pathway in a heterologous host is one approach to activate biosynthetic gene clusters and to screen the metabolites produced for bioactivities. Polycistronic expression of all pathway genes under control of a single and tunable promoter would be the method of choice, as this does not only simplify cloning procedures, but also offers control on timing and strength of expression. However, polycistronic gene expression is a feature not commonly found in eukaryotic host systems, such as Aspergillus niger. In this study, we tested the suitability of the viral P2A peptide for co-expression of three genes in A. niger. Two genes descend from Fusarium oxysporum and are essential to produce the secondary metabolite enniatin (esyn1, ekivR). The third gene (luc) encodes the reporter luciferase which was included to study position effects. Expression of the polycistronic gene cassette was put under control of the Tet-On system to ensure tunable gene expression in A. niger. In total, three polycistronic expression cassettes which differed in the position of luc were constructed and targeted to the pyrG locus in A. niger. This allowed direct comparison of the luciferase activity based on the position of the luciferase gene. Doxycycline-mediated induction of the Tet-On expression cassettes resulted in the production of one long polycistronic mRNA as proven by Northern analyses, and ensured comparable production of enniatin in all three strains. Notably, gene position within the polycistronic expression cassette matters, as, luciferase activity was lowest at position one and had a comparable activity at positions two and three. The P2A peptide can be used to express at least three genes polycistronically in A. niger. This approach can now be applied to heterologously express entire secondary metabolite gene clusters polycistronically or to co-express any genes of interest in equimolar amounts.

Diabetes Mellitus in Pregnancy Leads to Growth Restriction and Epigenetic Modification of the Srebf2 Gene in Rat Fetuses.

PubMed

Golic, Michaela; Stojanovska, Violeta; Bendix, Ivo; Wehner, Anika; Herse, Florian; Haase, Nadine; Kräker, Kristin; Fischer, Caroline; Alenina, Natalia; Bader, Michael; Schütte, Till; Schuchardt, Mirjam; van der Giet, Markus; Henrich, Wolfgang; Muller, Dominik N; Felderhoff-Müser, Ursula; Scherjon, Sicco; Plösch, Torsten; Dechend, Ralf

2018-05-01

Diabetic pregnancy is correlated with increased risk of metabolic and neurological disorders in the offspring putatively mediated epigenetically. Little is known about epigenetic changes already present in fetuses of diabetic pregnancies. We aimed at characterizing the perinatal environment after preexisting maternal diabetes mellitus and at identifying relevant epigenetic changes in the fetus. We focused on the transcription factor Srebf2 (sterol regulatory element binding transcription factor 2), a master gene in regulation of cholesterol metabolism. We tested whether diabetic pregnancy induces epigenetic changes in the Srebf2 promoter and if they become manifest in altered Srebf2 gene expression. We worked with a transgenic rat model of type 2 diabetes mellitus (Tet29) in which the insulin receptor is knocked down by doxycycline-induced RNA interference. Doxycycline was administered preconceptionally to Tet29 and wild-type control rats. Only Tet29 doxycycline dams were hyperglycemic, hyperinsulinemic, and hyperlipidemic. Gene expression was analyzed with quantitative real-time reverse transcriptase polymerase chain reaction and CpG promoter methylation with pyrosequencing. Immunohistochemistry was performed on fetal brains. Fetuses from diabetic Tet29 dams were hyperglycemic and growth restricted at the end of pregnancy. They further displayed decreased liver and brain weight with concomitant decreased microglial activation in the hippocampus in comparison to fetuses of normoglycemic mothers. Importantly, diabetic pregnancy induced CpG hypermethylation of the Srebf2 promoter in the fetal liver and brain, which was associated with decreased Srebf2 gene expression. In conclusion, diabetic and hyperlipidemic pregnancy induces neurological, metabolic, and epigenetic alterations in the rat fetus. Srebf2 is a potential candidate mediating intrauterine environment-driven epigenetic changes and later diabetic offspring health. © 2018 American Heart Association, Inc.

Expression of BCL-2 in liver grafts after adenoviral transfer improves survival following prolonged ischemia and reperfusion in rat liver transplantation.

PubMed

Kienle, K; Rentsch, M; Müller, T; Engelhard, N; Vogel, M; Jauch, K W; Beham, A

2005-01-01

Apoptosis represents a crucial mechanism of ischemia-reperfusion injury after liver transplantation. Bcl-2 may inhibit apoptosis. This study investigates the effect on ischemia/reperfusion injury and survival after rat liver transplantation of adenoviral bcl-2 transfer into donor livers. A nonreplicative adenovirus, expressing bcl-2 under control of a tetracyclin-inducible promoter (adv TetOn bcl-2) was used to treat male Lewis rats in combination with a second adenovirus transferring the TetOn repressor protein under control of a cytomegalovirus promoter (advCMVRep). Virus induction was achieved by addition of doxycyclin to the drinking water. Controls were pretreated with a control adenovirus (advCMV GFP) or with doxycycline. Liver transplantations were performed after 16-hour graft storage. Bcl-2 expression was evaluated by Western blot and immunohistology. Survival was monitored for 7 days, and tissue specimens were collected at 24 hours and 7 days post reperfusion. After pretreatment with advTetOn bcl-2/adv CMVRep, intrahepatic bcl-2 expression was evident at 24 hours and 7 days but was absent among controls. Bcl-2 expression was detected in hepatocytes and, to a high degree, in sinusoidal lining cells. TUNEL-positive sinusoidal lining cells were strikingly reduced after bcl-2 transfer (0.1 +/- 0.3 cells/hpf, mean +/- SD) compared to control virus (4.8 +/- 2.3) or doxycyclin-treated grafts (1.3 +/- 0.2); P < .05. After bcl-2 treatment, survival after transplantation was 100%, whereas it was 50% in both control groups (P = .035). The study shows the feasibility of transient, doxycyclin-controlled adenoviral gene transfer in a transplantation model. Bcl-2 expression increased survival after ischemia/reperfusion in rat liver transplantation, potentially through protection of sinusoidal lining cells.

Genetic modification of embryonic stem cells with VEGF enhances cell survival and improves cardiac function.

PubMed

Xie, Xiaoyan; Cao, Feng; Sheikh, Ahmad Y; Li, Zongjin; Connolly, Andrew J; Pei, Xuetao; Li, Ren-Ke; Robbins, Robert C; Wu, Joseph C

2007-01-01

Cardiac stem cell therapy remains hampered by acute donor cell death posttransplantation and the lack of reliable methods for tracking cell survival in vivo. We hypothesize that cells transfected with inducible vascular endothelial growth factor 165 (VEGF(165)) can improve their survival as monitored by novel molecular imaging techniques. Mouse embryonic stem (ES) cells were transfected with an inducible, bidirectional tetracycline (Bi-Tet) promoter driving VEGF(165) and renilla luciferase (Rluc). Addition of doxycycline induced Bi-Tet expression of VEGF(165) and Rluc significantly compared to baseline (p<0.05). Expression of VEGF(165) enhanced ES cell proliferation and inhibited apoptosis as determined by Annexin-V staining. For noninvasive imaging, ES cells were transduced with a double fusion (DF) reporter gene consisting of firefly luciferase and enhanced green fluorescence protein (Fluc-eGFP). There was a robust correlation between cell number and Fluc activity (R(2)=0.99). Analysis by immunostaining, histology, and RT-PCR confirmed that expression of Bi-Tet and DF systems did not affect ES cell self-renewal or pluripotency. ES cells were differentiated into beating embryoid bodies expressing cardiac markers such as troponin, Nkx2.5, and beta-MHC. Afterward, 5 x 10(5) cells obtained from these beating embryoid bodies or saline were injected into the myocardium of SV129 mice (n=36) following ligation of the left anterior descending (LAD) artery. Bioluminescence imaging (BLI) and echocardiography showed that VEGF(165) induction led to significant improvements in both transplanted cell survival and cardiac function (p<0.05). This is the first study to demonstrate imaging of embryonic stem cell-mediated gene therapy targeting cardiovascular disease. With further validation, this platform may have broad applications for current basic research and further clinical studies.

Hypoxia, Epithelial-Mesenchymal Transition, and TET-Mediated Epigenetic Changes

PubMed Central

Kao, Shih-Han; Wu, Kou-Juey; Lee, Wen-Hwa

2016-01-01

Tumor hypoxia is a pathophysiologic outcome of disrupted microcirculation with inadequate supply of oxygen, leading to enhanced proliferation, epithelial-mesenchymal transition (EMT), metastasis, and chemo-resistance. Epigenetic changes induced by hypoxia are well documented, and they lead to tumor progression. Recent advances show that DNA demethylation mediated by the Ten-eleven translocation (TET) proteins induces major epigenetic changes and controls key steps of cancer development. TET enzymes serve as 5mC (5-methylcytosine)-specific dioxygenases and cause DNA demethylation. Hypoxia activates the expression of TET1, which also serves as a co-activator of HIF-1α transcriptional regulation to modulate HIF-1α downstream target genes and promote epithelial-mesenchymal transition. As HIF is a negative prognostic factor for tumor progression, hypoxia-activated prodrugs (HAPs) may provide a favorable therapeutic approach to lessen hypoxia-induced malignancy. PMID:26861406

Streptomyces venezuelae TX-TL - a next generation cell-free synthetic biology tool.

PubMed

Moore, Simon J; Lai, Hung-En; Needham, Hannah; Polizzi, Karen M; Freemont, Paul S

2017-04-01

Streptomyces venezuelae is a promising chassis in synthetic biology for fine chemical and secondary metabolite pathway engineering. The potential of S. venezuelae could be further realized by expanding its capability with the introduction of its own in vitro transcription-translation (TX-TL) system. TX-TL is a fast and expanding technology for bottom-up design of complex gene expression tools, biosensors and protein manufacturing. Herein, we introduce a S. venezuelae TX-TL platform by reporting a streamlined protocol for cell-extract preparation, demonstrating high-yield synthesis of a codon-optimized sfGFP reporter and the prototyping of a synthetic tetracycline-inducible promoter in S. venezuelae TX-TL based on the tetO-TetR repressor system. The aim of this system is to provide a host for the homologous production of exotic enzymes from Actinobacteria secondary metabolism in vitro. As an example, the authors demonstrate the soluble synthesis of a selection of enzymes (12-70 kDa) from the Streptomyces rimosus oxytetracycline pathway. © 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Decreased expression of ten-eleven translocation 1 protein is associated with some clinicopathological features in gastric cancer.

PubMed

Frycz, Bartosz Adam; Murawa, Dawid; Borejsza-Wysocki, Maciej; Marciniak, Ryszard; Murawa, Paweł; Drews, Michał; Kołodziejczak, Anna; Tomela, Katarzyna; Jagodziński, Paweł Piotr

2014-03-01

A decrease in ten-eleven translocation 1 (TET1) transcript and 5-Hydroxymethylcytosine (5hmC) levels has recently been demonstrated in primary gastric cancer (GC). However, little is known about TET1 protein levels in gastric tumoral and nontumoral tissue. Therefore, using reverse transcription, real-time quantitative polymerase chain reaction and western blotting analysis, we determined the TET1 transcript and protein levels in tumoral and nontumoral tissue from 38 patients with GC. We also assessed the association between the decrease in TET1 transcript and protein levels and some clinicopathological features in primary GC. We found significantly decreased levels of TET1 transcript (P=0.0023) and protein (P=0.00024) in primary tumoral tissues as compared to nontumoral tissues in patients with GC. Moreover, we also observed significantly lower amounts of TET1 transcript (P=0.03) and protein (P=0.00018) in tumoral tissues in patients aged>60. We also found significant lowered TET1 protein levels in male patients (P=0.0014), stomach (P=0.044) and cardia (P=0.013) tumor localization, T3 depth of invasion (P=0.019), N1 (P=0.012) and N3 lymph node metastasis (P=0.013) and G3 histological grade (P=0.0012). There were also significant decreases in TET1 transcript levels in female patients (P=0.042), intestinal histological types (P=0.0079) and T4 depth of invasion (P=0.037). Our results demonstrated that a decrease in TET1 transcript and protein levels is associated with some clinicopathological features in GC. Copyright © 2014. Published by Elsevier Masson SAS.

Effect from low-level exposure of oxytetracycline on abundance of tetracycline resistance genes in arable soils.

PubMed

Shentu, Jia-Li; Zhang, Kun; Shen, Dong-Sheng; Wang, Mei-Zhen; Feng, Hua-Jun

2015-09-01

To evaluate the effect from low-level exposure of antibiotics on the abundance of antibiotic resistance genes, unpolluted arable soils were treated with oxytetracycline (OTC)-containing manure, with OTC (0, 2, 20, or 70 μg kg(-1)) added every 2 weeks on 10 occasions. Six tetracycline resistance genes [TRGs-tet(A), tet(L), tet(M), tet(Q), tet(O), and tet(W)] and the 16S ribosomal RNA (rRNA) gene were monitored using real-time quantitative polymerase chain reaction. The relative abundance of tet(A), tet(L), tet(M), and tet(Q) genes in soil increased 10-1000 times after application of OTC-containing manure. Tet(A) abundance per unit of residual OTC on day 140 was 1.53-4.42 times higher than that on day 28, while tet(L) abundance was 1.04-1.74 times higher. Treatment with >40 μg kg(-1) OTC significantly increased abundance of tet(A) and tet(L), while tet(M) and tet(Q) abundance was positively correlated (R (2) = 0.965 and 0.932, p < 0.01) with residual OTC concentrations. There was a significant accumulation of TRGs associated with low-level OTC exposure in arable soils. Besides OTC residual, the effects from exposure time and application frequencies should also be considered to limit the increase in abundance of tet(A) and tet(L).

Analysis of the machinery and intermediates of the 5hmC-mediated DNA demethylation pathway in aging on samples from the MARK-AGE Study.

PubMed

Valentini, Elisabetta; Zampieri, Michele; Malavolta, Marco; Bacalini, Maria Giulia; Calabrese, Roberta; Guastafierro, Tiziana; Reale, Anna; Franceschi, Claudio; Hervonen, Antti; Koller, Bernhard; Bernhardt, Jürgen; Slagboom, P Eline; Toussaint, Olivier; Sikora, Ewa; Gonos, Efstathios S; Breusing, Nicolle; Grune, Tilman; Jansen, Eugène; Dollé, Martijn E T; Moreno-Villanueva, María; Sindlinger, Thilo; Bürkle, Alexander; Ciccarone, Fabio; Caiafa, Paola

2016-08-29

Gradual changes in the DNA methylation landscape occur throughout aging virtually in all human tissues. A widespread reduction of 5-methylcytosine (5mC), associated with highly reproducible site-specific hypermethylation, characterizes the genome in aging. Therefore, an equilibrium seems to exist between general and directional deregulating events concerning DNA methylation controllers, which may underpin the age-related epigenetic changes. In this context, 5mC-hydroxylases (TET enzymes) are new potential players. In fact, TETs catalyze the stepwise oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), driving the DNA demethylation process based on thymine DNA glycosylase (TDG)-mediated DNA repair pathway. The present paper reports the expression of DNA hydroxymethylation components, the levels of 5hmC and of its derivatives in peripheral blood mononuclear cells of age-stratified donors recruited in several European countries in the context of the EU Project 'MARK-AGE'. The results provide evidence for an age-related decline of TET1 , TET3 and TDG gene expression along with a decrease of 5hmC and an accumulation of 5caC. These associations were independent of confounding variables, including recruitment center, gender and leukocyte composition. The observed impairment of 5hmC-mediated DNA demethylation pathway in blood cells may lead to aberrant transcriptional programs in the elderly.

Analysis of the machinery and intermediates of the 5hmC-mediated DNA demethylation pathway in aging on samples from the MARK-AGE Study

PubMed Central

Valentini, Elisabetta; Zampieri, Michele; Malavolta, Marco; Bacalini, Maria Giulia; Calabrese, Roberta; Guastafierro, Tiziana; Reale, Anna; Franceschi, Claudio; Hervonen, Antti; Koller, Bernhard; Bernhardt, Jürgen; Slagboom, P. Eline; Toussaint, Olivier; Sikora, Ewa; Gonos, Efstathios S.; Breusing, Nicolle; Grune, Tilman; Jansen, Eugène; Dollé, Martijn E.T.; Moreno-Villanueva, María; Sindlinger, Thilo; Bürkle, Alexander; Ciccarone, Fabio; Caiafa, Paola

2016-01-01

Gradual changes in the DNA methylation landscape occur throughout aging virtually in all human tissues. A widespread reduction of 5-methylcytosine (5mC), associated with highly reproducible site-specific hypermethylation, characterizes the genome in aging. Therefore, an equilibrium seems to exist between general and directional deregulating events concerning DNA methylation controllers, which may underpin the age-related epigenetic changes. In this context, 5mC-hydroxylases (TET enzymes) are new potential players. In fact, TETs catalyze the stepwise oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), driving the DNA demethylation process based on thymine DNA glycosylase (TDG)-mediated DNA repair pathway. The present paper reports the expression of DNA hydroxymethylation components, the levels of 5hmC and of its derivatives in peripheral blood mononuclear cells of age-stratified donors recruited in several European countries in the context of the EU Project ‘MARK-AGE’. The results provide evidence for an age-related decline of TET1, TET3 and TDG gene expression along with a decrease of 5hmC and an accumulation of 5caC. These associations were independent of confounding variables, including recruitment center, gender and leukocyte composition. The observed impairment of 5hmC-mediated DNA demethylation pathway in blood cells may lead to aberrant transcriptional programs in the elderly. PMID:27587280

Modification of Tet1 and histone methylation dynamics in dairy goat male germline stem cells.

PubMed

Zheng, Liming; Zhai, Yuanxin; Li, Na; Wu, Chongyang; Zhu, Haijing; Wei, Zhuying; Bai, Chunling; Li, Guangpeng; Hua, Jinlian

2016-04-01

Tet (ten-eleven translocation) protein 1 is a key enzyme for DNA demethylation, which modulates DNA methylation and gene transcription. DNA methylation and histone methylation are critical elements in self-renewal of male germline stem cells (mGSCs) and spermatogenesis. mGSCs are the only type of adult stem cells able to achieve intergenerational transfer of genetic information, which is accomplished through differentiated sperm cells. However, numerous epigenetic obstacles including incomplete DNA methylation and histone methylation dynamics make establishment of stable livestock mGSC cell lines difficult. The present study was conducted to detect effects of DNA methylation and histone methylation dynamics in dairy goat mGSCs self-renewal and proliferation, through overexpression of Tet1. An immortalized dairy goat mGSC cell line bearing mouse Tet1 (mTet1) gene was screened and characteristics of the cells were assayed by quantitative real-time PCR (qRT-PCR), immunofluorescence assay, western blotting, fluorescence activated cell sorting (FACS) and use of the cell counting kit (CCK8) assay. The screened immortalized dairy goat mGSC cell line bearing mTet1, called mGSC-mTet1 cells was treated with optimal doxycycline (Dox) concentration to maintain Tet1 gene expression. mGSC-mTet1 cells proliferated at a significantly greater rate than wild-type mGSCs, and mGSCs-specific markers such as proliferating cell nuclear antigen (PCNA), cyclinD1 (CCND1), GDNF family receptor alpha 1 (Gfra1) and endogenic Tet1, Tet2 were upregulated. The cells exhibited not only reduction in level of histone methylation but also changes in nuclear location of that methylation marker. While H3K9me3 was uniformly distributed throughout the nucleus of mGSC-mTet1 cells, it was present in only particular locations in mGSCs. H3K27me3 was distributed surrounding the edges of nuclei of mGSC-mTet1 cells, while it was uniformly distributed throughout nuclei of mGSCs. Our results conclusively demonstrate that modification of mGSCs with mTet1 affected mGSC maintenance and seemed to promote establishment of stable goat mGSC cell lines. Taken together, our data suggest that Tet1 had novel and dynamic roles for regulating maintenance of pluripotency and proliferation of mGSCs by forming complexes with PCNA and histone methylation dynamics. This may provide new solutions for mGSCs stability and livestock mGSC cell line establishment. © 2016 John Wiley & Sons Ltd.

Abundance of antibiotic resistance genes in environmental bacteriophages.

PubMed

Anand, Taruna; Bera, Bidhan Ch; Vaid, Rajesh K; Barua, Sanjay; Riyesh, Thachamvally; Virmani, Nitin; Hussain, Mubarik; Singh, Raj K; Tripathi, Bhupendra N

2016-12-01

The ecosystem is continuously exposed to a wide variety of antimicrobials through waste effluents, agricultural run-offs and animal-related and anthropogenic activities, which contribute to the spread of antibiotic resistance genes (ARGs). The contamination of ecosystems with ARGs may create increased opportunities for their transfer to naive microbes and eventually lead to entry into the human food chain. Transduction is a significant mechanism of horizontal gene transfer in natural environments, which has traditionally been underestimated as compared to transformation. We explored the presence of ARGs in environmental bacteriophages in order to recognize their contribution in the spread of ARGs in environmental settings. Bacteriophages were isolated against environmental bacterial isolates, purified and bulk cultured. They were characterized, and detection of ARG and intI genes including blaTEM, blaOXA-2, intI1, intI2, intI3, tetA and tetW was carried out by PCR. This study revealed the presence of various genes [tetA (12.7 %), intI1 (10.9 %), intI2 (10.9 %), intI3 (9.1 %), tetW (9.1 %) and blaOXA-2 (3.6 %)] and blaTEM in a significantly higher proportion (30.9 %). blaSHV, blaOXA-1, tetO, tetB, tetG, tetM and tetS were not detected in any of the phages. Soil phages were the most versatile in terms of ARG carriage. Also, the relative abundance of tetA differed significantly vis-à-vis source. The phages from organized farms showed varied ARGs as compared to the unorganized sector, although blaTEM ARG incidences did not differ significantly. The study reflects on the role of phages in dissemination of ARGs in environmental reservoirs, which may provide an early warning system for future clinically relevant resistance mechanisms.

Tetracycline-regulated expression of OLIG2 gene in human dental pulp stem cells lead to mouse sciatic nerve regeneration upon transplantation.

PubMed

Askari, N; Yaghoobi, M M; Shamsara, M; Esmaeili-Mahani, S

2015-10-01

Numerous studies have indicated dental pulp stem cells (DPSCs) potency to differentiate into several types of cell lineages. Oligodendrocyte lineage transcription factor 2 (OLIG2) plays an important role in the oligodendrogenic pathway. In this study, a tetracycline (Tet)-inducible system expressing OLIG2 gene was transfected into human DPSCs to direct their differentiation toward oligodendrocyte progenitor cells (OPCs). Following induction, the expression of stage-specific markers was studied by Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR), immunocytochemistry and western blotting. In the following, the cells were transplanted into the mouse model of local sciatic demyelination damage by lysolecithin. Recovery of lysolecithin-induced lesions in sciatic nerve was studied by treadmill exercise, von Frey filament test and hind paw withdrawal in response to a thermal stimulus. Improvement of behavioral symptoms was efficiently observed from the second week to the sixth week post-transplantation. Our findings showed that exogenous expression of the OLIG2 gene by a Tet-regulated system could be used as an efficient way to induce the differentiation of DPSCs into functional oligodendrocytes. Meanwhile, the DPSC-derived OPCs have relevant therapeutic potential in the animal model of sciatic nerve injury and therefore might represent a valuable tool for stem cell-based therapy in inflammatory and degenerative diseases of the peripheral and central nervous systems (CNSs). Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Safety considerations for wireless delivery of continuous power to implanted medical devices.

PubMed

Lucke, Lori; Bluvshtein, Vlad

2014-01-01

Wireless power systems for use with implants are referred to as transcutaneous energy transmission systems (TETS) and consist of an implanted secondary coil and an external primary coil along with supporting electronics. A TETS system could be used to power ventricular assist systems and eliminate driveline infections. There are both direct and indirect safety concerns that must be addressed when continuously transferring power through the skin. Direct safety concerns include thermal tissue damage caused by exposure to the electromagnetic fields, coil heating effects, and potential unwanted nerve stimulation. Indirect concerns are those caused by potential interference of the TETS system with other implanted devices. Wireless power systems are trending towards higher frequency operation. Understanding the limits for safe operation of a TETS system across a range of frequencies is important. A low frequency and a high frequency implementation are simulated to demonstrate the impact of this trend for a VAD application.

Combination of antibiotics suppressed the increase of a part of ARGs in fecal microorganism of weaned pigs.

PubMed

Li, Huizhi; Chu, Qingpo; Xu, Feilong; Fu, Lingling; Liang, Tingting; Li, Yuan; Zhou, Bo

2016-09-01

The presence of antibiotic resistance genes (ARGs) is one of the most important public health concerns. Six tetracycline resistance genes (TRGs-tetA, tetC, tetL, tetO, tetW, and tetX) were quantified using real-time quantitative polymerase chain reaction (qPCR) in the fecal microorganisms of weaned pigs. Two hundred 35-day-old weaned pigs were fed different dietary antibiotics for 28 days: (1) no antibiotic as the control treatment (CT); (2) chlortetracycline, bacitracin zinc and colistin sulfate (CBC); (3) bacitracin zinc and colistin sulfate (BC); and (4) chlortetracycline (CTC). The detection frequencies (DFs) of tetC, tetL, and tetW were 100 %; and the DFs of tetA, tetD, tetM, tetO, and tetX were 65 %. The relative abundances (tet/16S rRNA gene copy numbers) of six tet genes (tetA, tetC, tetL, tetO, tetW and tetX) were between 1.5 × 10(-4) and 2.0 × 10(-1). In the group CTC, the relative abundances of tetC (P < 0.01), tetL (P < 0.01), tetO (P < 0.05), tetW (P < 0.01), and tetX (P < 0.01) were greater than those of the group CT. Compared with the group CTC, the relative abundances of tetC (P < 0.01), tetL (P < 0.01), and tetW (P < 0.01) were decreased in the CBC and BC groups. These results indicate that a combination of different antibiotics suppressed the abundance increase of a part of tet genes, which suggests that a combination of antibiotics produces multiple selection pressures on fecal microorganism of pigs.

Co-delivery of paclitaxel and tetrandrine via iRGD peptide conjugated lipid-polymer hybrid nanoparticles overcome multidrug resistance in cancer cells

PubMed Central

Zhang, Jinming; Wang, Lu; Fai Chan, Hon; Xie, Wei; Chen, Sheng; He, Chengwei; Wang, Yitao; Chen, Meiwan

2017-01-01

One of the promising strategies to overcome tumor multidrug resistance (MDR) is to deliver anticancer drug along with P-glycoprotein (P-gp) inhibitor simultaneously. To enhance the cancer cellular internalization and implement the controlled drug release, herein an iRGD peptide-modified lipid-polymer hybrid nanosystem (LPN) was fabricated to coload paclitaxel (PTX) and tetrandrine (TET) at a precise combination ratio. In this co-delivery system, PTX was covalently conjugated to poly (D,L-lactide-co-glycolide) polymeric core by redox-sensitive disulfide bond, while TET was physically capsulated spontaneously for the aim to suppress P-gp in advance by the earlier released TET in cancer cells. As a result, the PTX+TET/iRGD LPNs with a core-shell structure possessed high drug loading efficiency, stability and redox-sensitive drug release profiles. Owing to the enhanced cellular uptake and P-gp suppression mediated by TET, significantly more PTX accumulated in A2780/PTX cells treated with PTX+TET/iRGD LPNs than either free drugs or non-iRGD modified LPNs. As expected, PTX+TET/iRGD LPNs presented the highest cytotoxicity against A2780/PTX cells and effectively promoted ROS production, enhanced apoptosis and cell cycle arrests particularly. Taken together, the co-delivery system demonstrated great promise as potential treatment for MDR-related tumors based on the synergistic effects of P-gp inhibition, enhanced endocytosis and intracellular sequentially drug release. PMID:28470171

Elimination of veterinary antibiotics and antibiotic resistance genes from swine wastewater in the vertical flow constructed wetlands.

PubMed

Liu, Lin; Liu, Chaoxiang; Zheng, Jiayu; Huang, Xu; Wang, Zhen; Liu, Yuhong; Zhu, Gefu

2013-05-01

This paper investigated the efficiency of two vertical flow constructed wetlands characterized by volcanic (CW1) and zeolite (CW2) respectively, at removing three common antibiotics (ciprofloxacin HCl, oxytetracycline HCl, and sulfamethazine) and tetracycline resistance (tet) genes (tetM, tetO, and tetW) from swine wastewater. The result indicated that the two systems could significantly reduce the wastewater antibiotics content, and elimination rates were in the following sequence: oxytetracycline HCl>ciprofloxacin HCl>sulfamethazine. The zeolite-medium system was superior to that of the volcanic-medium system vis-à-vis removal, perhaps because of the differing pH values and average pore sizes of the respective media. A higher concentration of antibiotics accumulated in the soil than in the media and vegetation, indicating that soil plays the main role in antibiotics removal from wastewater in vertical flow constructed wetlands. The characteristics of the wetland medium may also affect the antibiotic resistance gene removal capability of the system; the total absolute abundances of three tet genes and of 16S rRNA were reduced by 50% in CW1, and by almost one order of magnitude in CW2. However, the relative abundances of target tet genes tended to increase following CW1 treatment. Copyright © 2013 Elsevier Ltd. All rights reserved.

ERF is a Potential ERK-Modulated Tumor Suppressor in Prostate Cancer

DTIC Science & Technology

2017-10-01

15 20 P = 0 .0 17 P < 0 .0 00 1 CRISPR –Cas9 sgERF CRISPR –Cas9 sgNT –dox (TetOn_Flag–ERF) +dox (TetOn_Flag–ERF) shNT, sgNT shERF_2, sgNT shERF_2...Pten−/−;R26ERG/ERG organoids infected with CRISPR –Cas9 targeting ERF (sgERF). Data are median ± interquartile range; n = 10 tumours per condition, P...cancer21. We next asked whether ERF could modulate oncogenicity mediated by ERG expression. First, we used CRISPR –Cas9 (clustered regularly interspaced

Assessment of global DNA methylation in peripheral blood cell subpopulations of early rheumatoid arthritis before and after methotrexate.

PubMed

de Andres, María C; Perez-Pampin, Eva; Calaza, Manuel; Santaclara, Francisco J; Ortea, Ignacio; Gomez-Reino, Juan J; Gonzalez, Antonio

2015-08-29

DNA methylation is an epigenetic mechanism regulating gene expression that has been insufficiently studied in the blood of rheumatoid arthritis (RA) patients, as only T cells and total peripheral blood mononuclear cells (PBMCs) from patients with established RA have been studied and with conflicting results. Five major blood cell subpopulations: T, B and NK cells, monocytes, and polymorphonuclear leukocytes, were isolated from 19 early RA patients and 17 healthy controls. Patient samples were taken before and 1 month after the start of treatment with methotrexate (MTX). Analysis included DNA methylation with high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring (HPLC-ESI-MS/MS-SRM) and expression levels of seven methylation-specific enzymes by quantitative polymerase chain reaction (qPCR). Disease-modifying anti-rheumatic drug (DMARD)-naïve early RA patients showed global DNA hypomethylation in T cells and monocytes, together with a lower expression of DNA methyltrasnferase 1 (DNMT1), the maintenance DNA methyltransferase, which was also decreased in B cells. Furthermore, significantly increased expression of ten-eleven translocation1 (TET1), TET2 and TET3, enzymes involved in demethylation, was found in monocytes and of TET2 in T cells. There was also modest decreased expression of DNMT3A in B cells and of growth arrest and DNA-damage-inducible protein 45A (GADD45A) in T and B cells. Treatment with MTX reverted hypomethylation in T cells and monocytes, which were no longer different from controls, and increased global methylation in B cells. In addition, DNMT1 and DNMT3A showed a trend to reversion of their decreased expression. Our results confirm global DNA hypomethylation in patients with RA with specificity for some blood cell subpopulations and their reversal with methotrexate treatment. These changes are accompanied by parallel changes in the levels of enzymes involved in methylation, suggesting the possibility of regulation at this level.

Mutations in TET2 and DNMT3A genes are associated with changes in global and gene-specific methylation in acute myeloid leukemia.

PubMed

Ponciano-Gómez, Alberto; Martínez-Tovar, Adolfo; Vela-Ojeda, Jorge; Olarte-Carrillo, Irma; Centeno-Cruz, Federico; Garrido, Efraín

2017-10-01

Acute myeloid leukemia is characterized by its high biological and clinical heterogeneity, which represents an important barrier for a precise disease classification and accurate therapy. While epigenetic aberrations play a pivotal role in acute myeloid leukemia pathophysiology, molecular signatures such as change in the DNA methylation patterns and genetic mutations in enzymes needed to the methylation process can also be helpful for classifying acute myeloid leukemia. Our study aims to unveil the relevance of DNMT3A and TET2 genes in global and specific methylation patterns in acute myeloid leukemia. Peripheral blood samples from 110 untreated patients with acute myeloid leukemia and 15 healthy control individuals were collected. Global 5-methylcytosine and 5-hydroxymethylcytosine in genomic DNA from peripheral blood leukocytes were measured by using the MethylFlashTM Quantification kits. DNMT3A and TET2 expression levels were evaluated by real-time quantitative polymerase chain reaction. The R882A hotspot of DNMT3A and exons 6-10 of TET2 were amplified by polymerase chain reaction and sequenced using the Sanger method. Methylation patterns of 16 gene promoters were evaluated by pyrosequencing after treating DNA with sodium bisulfite, and their transcriptional products were measured by real-time quantitative polymerase chain reaction.Here, we demonstrate altered levels of 5-methylcytosine and 5-hydroxymethylcytosine and highly variable transcript levels of DNMT3A and TET2 in peripheral blood leukocytes from acute myeloid leukemia patients. We found a mutation prevalence of 2.7% for DNMT3A and 11.8% for TET2 in the Mexican population with this disease. The average overall survival of acute myeloid leukemia patients with DNMT3A mutations was only 4 months. In addition, we showed that mutations in DNMT3A and TET2 may cause irregular DNA methylation patterns and transcriptional expression levels in 16 genes known to be involved in acute myeloid leukemia pathogenesis. Our findings suggest that alterations in DNMT3A and TET2 may be associated with acute myeloid leukemia prognosis. Furthermore, alterations in these enzymes affect normal methylation patterns in acute myeloid leukemia- specific genes, which in turn, may influence patient survival.

The TetO rat as a new translational model for type 2 diabetic retinopathy by inducible insulin receptor knockdown.

PubMed

Reichhart, Nadine; Crespo-Garcia, Sergio; Haase, Nadine; Golic, Michaela; Skosyrski, Sergej; Rübsam, Anne; Herrspiegel, Christina; Kociok, Norbert; Alenina, Natalia; Bader, Michael; Dechend, Ralf; Strauss, Olaf; Joussen, Antonia M

2017-01-01

Although the renin-angiotensin system plays an important role in the progression of diabetic retinopathy, its influence therein has not been systematically evaluated. Here we test the suitability of a new translational model of diabetic retinopathy, the TetO rat, for addressing the role of angiotensin-II receptor 1 (AT1) blockade in experimental diabetic retinopathy. Diabetes was induced by tetracycline-inducible small hairpin RNA (shRNA) knockdown of the insulin receptor in rats, generating TetO rats. Systemic treatment consisted of an AT1 blocker (ARB) at the onset of diabetes, following which, 4-5 weeks later the retina was analysed in vivo and ex vivo. Retinal function was assessed by Ganzfeld electroretinography (ERG). Retinal vessels in TetO rats showed differences in vessel calibre, together with gliosis. The total number and the proportion of activated mononuclear phagocytes was increased. TetO rats presented with loss of retinal ganglion cells (RGC) and ERG indicated photoreceptor malfunction. Both the inner and outer blood-retina barriers were affected. The ARB treated group showed reduced gliosis and an overall amelioration of retinal function, alongside RGC recovery, whilst no statistically significant differences in vascular and inflammatory features were detected. The TetO rat represents a promising translational model for the early neurovascular changes associated with type 2 diabetic retinopathy. ARB treatment had an effect on the neuronal component of the retina but not on the vasculature.

Enterococcus faecium ST17 from Coastal Marine Sediment Carrying Transferable Multidrug Resistance Plasmids.

PubMed

Morroni, Gianluca; Di Cesare, Andrea; Di Sante, Laura; Brenciani, Andrea; Vignaroli, Carla; Pasquaroli, Sonia; Giovanetti, Eleonora; Sabatino, Raffaella; Rossi, Luigia; Magnani, Mauro; Biavasco, Francesca

2016-10-01

The multidrug-resistant Enterococcus faecium 17i48, sequence type 17, from marine sediment, carrying erm(B), tet(M), and tet(L) genes, was analyzed for the presence of antibiotic resistance plasmids and for the ability to transfer resistance genes. The strain was found to harbor the replicon type (repA) of pRE25, pRUM, pHTβ, and the axe-txe toxin-antitoxin (TA) system. In mating experiments, tet(M) and tet(L) were cotransferred with the repA pRE25 , whereas erm(B) was consistently cotransferred with the axe-txe and repA pRUM , suggesting that tetracycline and erythromycin resistance genes were carried on different elements both transferable by conjugation, likely via pHTβ-mediated mobilization. Hybridization and PCR mapping demonstrated that tet(M) and tet(L) were located in tandem on a pDO1-like plasmid that also carried the repA pRE25 , whereas erm(B) was carried by a pRUM-like plasmid. Sequencing of the latter plasmid showed a high nucleotide identity with pRUM and the presence of cat, aadE, sat4, and a complete aphA resistance genes. These findings show that the genetic features of E. faecium 17i48 are consistent with a hospital-adapted clone and suggest that antibiotic resistance may spread in the environment, also in the absence of antibiotic pressure, due to TA system plasmid maintenance.

Autism-associated R451C mutation in neuroligin3 leads to activation of the unfolded protein response in a PC12 Tet-On inducible system

PubMed Central

Ulbrich, Lisa; Favaloro, Flores Lietta; Trobiani, Laura; Marchetti, Valentina; Patel, Vruti; Pascucci, Tiziana; Comoletti, Davide; Marciniak, Stefan J.; De Jaco, Antonella

2015-01-01

Several forms of monogenic heritable autism spectrum disorders are associated with mutations in the neuroligin genes. The autism-linked substitution R451C in neuroligin3 induces local misfolding of its extracellular domain, causing partial retention in the ER (endoplasmic reticulum) of expressing cells. We have generated a PC12 Tet-On cell model system with inducible expression of wild-type or R451C neuroligin3 to investigate whether there is activation of the UPR (unfolded protein response) as a result of misfolded protein retention. As a positive control for protein misfolding, we also expressed the mutant G221R neuroligin3, which is known to be completely retained within the ER. Our data show that overexpression of either R451C or G221R mutant proteins leads to the activation of all three signalling branches of the UPR downstream of the stress sensors ATF6 (activating transcription factor 6), IRE1 (inositol-requiring enzyme 1) and PERK [PKR (dsRNA-dependent protein kinase)-like endoplasmic reticulum kinase]. Each branch displayed different activation profiles that partially correlated with the degree of misfolding caused by each mutation. We also show that up-regulation of BiP (immunoglobulin heavy-chain-binding protein) and CHOP [C/EBP (CCAAT/enhancer-binding protein)-homologous protein] was induced by both mutant proteins but not by wild-type neuroligin3, both in proliferative cells and cells differentiated to a neuron-like phenotype. Collectively, our data show that mutant R451C neuroligin3 activates the UPR in a novel cell model system, suggesting that this cellular response may have a role in monogenic forms of autism characterized by misfolding mutations. PMID:26621873

Application of manure containing tetracyclines slowed down the dissipation of tet resistance genes and caused changes in the composition of soil bacteria.

PubMed

Xiong, Wenguang; Wang, Mei; Dai, Jinjun; Sun, Yongxue; Zeng, Zhenling

2018-01-01

Manure application contributes to the increased environmental burden of antibiotic resistance genes (ARGs). We investigated the response of tetracycline (tet) resistance genes and bacterial taxa to manure application amended with tetracyclines over two months. Representative tetracyclines (oxytetracycline, chlorotetracycline and doxycycline), tet resistance genes (tet(M), tet(O), tet(W), tet(S), tet(Q) and tet(X)) and bacterial taxa in the untreated soil, +manure, and +manure+tetracyclines groups were analyzed. The abundances of all tet resistance genes in the +manure group were significantly higher than those in the untreated soil group on day 1. The abundances of all tet resistance genes (except tet(Q) and tet(X)) were significantly lower in the +manure group than those in the +manure+tetracyclines group on day 30 and 60. The dissipation rates were higher in the +manure group than those in the +manure+tetracyclines group. Disturbance of soil bacterial community composition imposed by tetracyclines was also observed. The results indicated that tetracyclines slowed down the dissipation of tet resistance genes in arable soil after manure application. Application of manure amended with tetracyclines may provide a significant selective advantage for species affiliated to the taxonomical families of Micromonosporaceae, Propionibacteriaceae, Streptomycetaceae, Nitrospiraceae and Clostridiaceae. Copyright © 2017 Elsevier Inc. All rights reserved.

A complex mechanism involving LysR and TetR/AcrR that regulates iron scavenger biosynthesis in Pseudomonas donghuensis HYS.

PubMed

Chen, Min; Wang, Panning; Xie, Zhixiong

2018-04-23

7-Hydroxytropolone (7-HT) is a symmetrical, seven-membered heteroatomic ring with a carboxyl group and two hydroxyl groups and was recently reported to be an iron scavenger of Pseudomonas donghuensis HYS. Cluster 1 encodes 12 genes related to the synthesis of 7-HT; among these genes, two regulators, ORF1 and ORF12, were predicted to regulate 7-HT biosynthesis and to be LysR-type transcriptional regulators (LTTRs) and TetR/AcrR family transcriptional regulators, respectively. Data from real-time quantitative PCR, β-galactosidase and classical siderophore assays indicated that the transcription levels of orf1 and orf12, as well as those of crucial genes orf6-orf9, were repressed under high-iron conditions. The deletion of orf1 and orf12 led to an absence of 7-HT and a decrease in orf6-orf9 expression. ORF1 and ORF12 were essential for the production of 7-HT through orf6-orf9 These two regulators are regulated by the Gac/Rsm system; ORF1 facilitates the expression of ORF12, and ORF12 concomitantly stimulates the expression of orf6-orf9 to synthesize 7-HT. Overexpression of ORF12 decreased 7-HT yields possibly through decreased orf6-orf9 expression. This work thus outlines a complex mechanism regulating the biosynthesis of the iron scavenger 7-HT in P. donghuensis HYS. The synergy between ORF1 and ORF12 ensures that 7-HT acts as an iron chelator despite being toxic to bacteria and provides new ideas for the novel regulation of dual-functional secondary metabolism and research on 7-HT and its derivates in other bacteria. IMPORTANCE A complex regulation mechanism including two regulators, LysR and TetR/AcrR, of the biosynthesis of the novel iron scavenger 7-HT was verified in Pseudomonas donghuensis HYS. The coaction of LysR ORF1 and TetR/AcrR ORF12 may balance the toxicity and iron chelation of 7-HT in P. donghuensis HYS to overcome iron deficiency, as well as improve the bacterial competitiveness in iron-scarce conditions because of the toxicity of 7-HT toward other bacteria, making the accurate regulation of 7-HT biosynthesis indispensable. This regulation mechanism may be ubiquitous in the Pseudomonas putida group but may better explain the group's strong adaptability. Copyright © 2018 American Society for Microbiology.

Occurrence and diversity of tetracycline resistance genes in the agricultural soils of South Korea.

PubMed

Kim, Song Yeob; Kuppusamy, Saranya; Kim, Jang Hwan; Yoon, Young-Eun; Kim, Kwon-Rae; Lee, Yong Bok

2016-11-01

Reports on the occurrence and diversity of antibiotic-resistant bacteria and genes, which are considered to be emerging pollutants worldwide, have, to date, not been published on South Korean agricultural soils. This is the first study to investigate the persistence of tetracycline (oxytetracycline, tetracycline, and chlortetracycline)-resistant bacterial community and genes in natural and long-term fertilized (NPK, pig, and cattle manure composts) agricultural soils in South Korea. The results showed that oxytetracycline and chlortetracycline could be the dominant residues in animal manures; regular fertilization of manures, particularly pig manures, may be the prime cause for the spread and abundance of tetracycline resistance in South Korean agricultural soils. Both the country's natural and agricultural soils are reservoirs of antibiotic-resistant species. Of the 113 tetracycline-resistant isolates identified (19 typical bacterial genera and 36 distinct species), approximately 40 to 99 % belonged to Gram-positive bacteria and Bacillus constituted the predominant genera. Of the 24 tet genes targeted, tetG, tetH, tetK, tetY, tetO, tetS, tetW, and tetQ were detected in all soil samples, highlighting their predominance and robust adaptability in soils. Meanwhile, it is suggested that tetC, tetE, tetZ, tetM, tetT, and tetP(B) are the common residues in pig manures, and furthermore, the treatment of soils with pig manures may wield a different impact on the tet gene resistome in agricultural soils. This study thus highlights the necessity for regulating the usage of tetracyclines in South Korean animal farming. This must be followed by proper monitoring of the subsequent usage of animal manures especially that derived from pig farms located in agricultural soils.

Performance of vertical up-flow constructed wetlands on swine wastewater containing tetracyclines and tet genes.

PubMed

Huang, Xu; Liu, Chaoxiang; Li, Ke; Su, Jianqiang; Zhu, Gefu; Liu, Lin

2015-03-01

Antibiotics and antibiotic resistance genes (ARGs) pollution in animal feeding farms received more public attention recently. Livestock wastewater contains large quantities of antibiotics and ARGs even after traditional lagoon treatment. In this study, the performance of vertical up-flow constructed wetlands (VUF-CWs) on swine wastewater containing tetracycline compounds (TCs) and tet genes was evaluated based on three aspects, TCs and tet genes removal efficiencies, residual TCs and tet genes in soils and plants, and the effect of TCs accumulation on nutrients removal and tet genes development. High removal efficiencies (69.0-99.9%) were achieved for oxytetracycline (OTC), tetracycline (TC) and chlortetracycline (CTC) with or without OTC spiked in the influent additionally. TCs concentrations in surface soils increased at first two sampling periods and then decreased after plants were harvested. Satisfactory nutrients removal efficiencies were also obtained, but TN and NH4-N removal efficiencies were significantly negative correlated with total concentration of TCs (∑TCs) in the soils (p < 0.01). The absolute abundances of all the target genes (tetO, tetM, tetW, tetA, tetX and intI1) were greatly reduced with their log units ranging from 0.26 to 3.3. However, the relative abundances of tetO, tetM and tetX in some effluent samples were significantly higher than those in the influent (p < 0.05). The relative abundances of tet genes except for tetO were significantly correlated with ∑TCs in the soils (p < 0.05). In summary, the proposed VUF-CWs are effective alternative for the removal of TCs and tet genes. But it is of great importance to prevent large accumulation of TCs in the soils. Copyright © 2014 Elsevier Ltd. All rights reserved.

[Effect of DOT1L gene silence on proliferation of acute monocytic leukemia cell line THP-1].

PubMed

Zhang, Yu-Juan; Li, Hua-Wen; Chang, Guo-Qiang; Zhang, Hong-Ju; Wang, Jian; Lin, Ya-Ni; Zhou, Jia-Xi; Li, Qing-Hua; Pang, Tian-Xiang

2013-08-01

This study was aimed to investigate the influence of short hairpin RNA (shRNA) on proliferation of human leukemia cell line THP-1. The shRNA targeting the site 732-752 of DOT1L mRNA was designed and chemically synthesized, then a single-vector lentiviral, tet-inducible shRNA-DOT1L system (Plko-Tet-On) was generated. Thereafter, the THP-1 cells with lentivirus were infected to create stable cell line with regulatable shRNA expression. The expression of DOT1L in the THP-1 cell line was assayed by RT-PCR. Effect of shRNA-DOT1L on the proliferation of THP-1 cells was detected with MTT method,and the change of colony forming potential of THP-1 cells was analyzed by colony forming unit test. Cell cycle distribution was tested by flow cytometry. The results indicated that the expression of DOT1L was statistically lower than that in the control groups. The proliferation and colony forming capacity of THP-1 cells were significantly inhibited. The percentage of cells at G0/G1 phase increased in THP-1/shRNA cells treated with Dox while the percentage of cells at S phase significantly decreased as compared with that in the control group. It is concluded that the shRNA targeting DOT1L can effectively inhibit the proliferation of acute monocytic leukemia cell line THP-1.

Preeclampsia is associated with hypermethylation of IGF-1 promoter mediated by DNMT1.

PubMed

Ma, Min; Zhou, Qiong-Jie; Xiong, Yu; Li, Bin; Li, Xiao-Tian

2018-01-01

Previous studies have demonstrated a dynamic epigenetic regulation of genes expression in placenta trophoblasts and a dynamic imbalance of DNA methylation and hydroxymethylation. Reduced IGF-1 has been observed in preeclampsia. This study was to investigate the interactive roles between IGF-1 and the global DNA methylation/hydroxymethylation, and the status of DNA methylation/hydroxymethylation and associated enzymes such as DNMTs and TETs in peeeclamptic placentas and hypoxic trophoblasts. It was found that IGF-1 was decreased in preeclamptic placentas and hypoxic trophoblasts when compared to the control group using immunohistochemisty, western blot, qRT-PCR and ELISA. Pyrophosphate sequencing showed IGF-1 promoter was significantly hypermethylated in preeclamptic placentas, which was responsible for reduced IGF-1 expression. Preeclamptic placentas and hypoxic trophoblasts were hypermethylated and hypohydroxymethylated accompanied by remarkably higher 5mC, DNMT1 and DNMT3b, and lower DNMT3a, 5hmC, TET1, TET2 and TET3 detected by immunohistochemisty, western blot, qRT-PCR and ELISA. Pearson's correlation confirmed a statistically significant negative correlation between IGF-1 and DNMT1. Furthermore, both treatment with 5-Aza-dc and DNMT1-siRNA significantly increased the expression of IGF-1 in HTR8 cells, indicating the potential mechanism of DNMT1-mediated DNA methylation in IGF-1 regulation. However, IGF-1 didn't change DNA methylation or hydroxymethylation. These findings suggest that preeclampsia is associated with hypermethylation of IGF-1 promoter mediated by DNMT1 and provide new insights into the diagnosis and treatment of preeclampsia.

Preeclampsia is associated with hypermethylation of IGF-1 promoter mediated by DNMT1

PubMed Central

Ma, Min; Zhou, Qiong-Jie; Xiong, Yu; Li, Bin; Li, Xiao-Tian

2018-01-01

Previous studies have demonstrated a dynamic epigenetic regulation of genes expression in placenta trophoblasts and a dynamic imbalance of DNA methylation and hydroxymethylation. Reduced IGF-1 has been observed in preeclampsia. This study was to investigate the interactive roles between IGF-1 and the global DNA methylation/hydroxymethylation, and the status of DNA methylation/hydroxymethylation and associated enzymes such as DNMTs and TETs in peeeclamptic placentas and hypoxic trophoblasts. It was found that IGF-1 was decreased in preeclamptic placentas and hypoxic trophoblasts when compared to the control group using immunohistochemisty, western blot, qRT-PCR and ELISA. Pyrophosphate sequencing showed IGF-1 promoter was significantly hypermethylated in preeclamptic placentas, which was responsible for reduced IGF-1 expression. Preeclamptic placentas and hypoxic trophoblasts were hypermethylated and hypohydroxymethylated accompanied by remarkably higher 5mC, DNMT1 and DNMT3b, and lower DNMT3a, 5hmC, TET1, TET2 and TET3 detected by immunohistochemisty, western blot, qRT-PCR and ELISA. Pearson’s correlation confirmed a statistically significant negative correlation between IGF-1 and DNMT1. Furthermore, both treatment with 5-Aza-dc and DNMT1-siRNA significantly increased the expression of IGF-1 in HTR8 cells, indicating the potential mechanism of DNMT1-mediated DNA methylation in IGF-1 regulation. However, IGF-1 didn’t change DNA methylation or hydroxymethylation. These findings suggest that preeclampsia is associated with hypermethylation of IGF-1 promoter mediated by DNMT1 and provide new insights into the diagnosis and treatment of preeclampsia. PMID:29422991

Immunohistochemical loss of 5-hydroxymethylcytosine expression in acute myeloid leukaemia: relationship to somatic gene mutations affecting epigenetic pathways.

PubMed

Magotra, Minoti; Sakhdari, Ali; Lee, Paul J; Tomaszewicz, Keith; Dresser, Karen; Hutchinson, Lloyd M; Woda, Bruce A; Chen, Benjamin J

2016-12-01

Genes affecting epigenetic pathways are frequently mutated in myeloid malignancies, including acute myeloid leukaemia (AML). The genes encoding TET2, IDH1 and IDH2 are among the most commonly mutated genes, and cause defective conversion of 5-methylcytosine into 5-hydroxymethylcytosine (5hmC), impairing demethylation of DNA, and presumably serving as driver mutations in leukaemogenesis. The aim of this study was to correlate 5hmC immunohistochemical loss with the mutation status of genes involved in epigenetic pathways in AML. Immunohistochemical staining with an anti-5hmC antibody was performed on 41 decalcified, formalin-fixed paraffin-embedded (FFPE) bone marrow biopsies from patients with AML. Archived DNA was subjected to next-generation sequencing for analysis of a panel of genes, including TET2, IDH1, IDH2, WT1 and DNMT3A. TET2, IDH1, IDH2, WT1 and DNMT3A mutations were found in 46% (19/41) of the cases. Ten of 15 cases (67%) with TET2, IDH1, IDH2 or WT1 mutations showed deficient 5hmC staining, whereas nine of 26 cases (35%) without a mutation in these genes showed loss of 5hmC. It is of note that all four cases with TET2 mutations showed deficient 5hmC staining. Overall, somatic mutations in TET2, IDH1, IDH2, WT1 and DNMT3A were common in our cohort of AML cases. Immunohistochemical staining for 5hmC was lost in the majority of cases harbouring mutations in these genes, reflecting the proposed relationship between dysfunctional epigenetic pathways and leukaemogenesis. © 2016 John Wiley & Sons Ltd.

Antimicrobial resistance and virulence profile of enterococci isolated from poultry and cattle sources in Nigeria.

PubMed

Ngbede, Emmanuel Ochefije; Raji, Mashood Abiola; Kwanashie, Clara Nna; Kwaga, Jacob Kwada Paghi

2017-03-01

This study investigated the occurrence, antimicrobial resistance and virulence of Enterococcus from poultry and cattle farms. Three hundred and ninety samples: cloacal/rectal swabs (n = 260) and manure (n = 130] were processed for recovery of Enterococcus species. Standard bacteriological methods were used to isolate, identify and characterize Enterococcus species for antimicrobial susceptibility and expression of virulence traits. Detection of antibiotic resistance and virulence genes was carried out by polymerase chain reaction. Enterococcus was recovered from 167 (42.8%) of the 390 samples tested with a predominance of Enterococcus faecium (27.7%). Other species detected were Enterococcus gallinarum, Enterococcus faecalis, Enterococcus hirae, Enterococcus raffinosus, Enterococcus avium, Enterococcus casseliflavus, Enterococcus mundtii and Enterococcus durans. All the isolates tested were susceptible to vancomycin, but resistance to tetracycline, erythromycin, ampicillin and gentamicin was also observed among 61.0, 61.0, 45.1 and 32.7% of the isolates, respectively. Sixty (53.1%) of the isolates were multidrug resistant presenting as 24 different resistance patterns with resistance to gentamicin-erythromycin-streptomycin-tetracycline (CN-ERY-STR-TET) being the most common (n = 11) pattern. In addition to expression of virulence traits (haemolysin, gelatinase, biofilm production), antibiotic resistance (tetK, tetL, tetM, tetO and ermB) and virulence (asa1, gelE, cylA) genes were detected among the isolates. Also, in vitro transfer of resistance determinants was observed among 75% of the isolates tested. Our data revealed poultry, cattle and manure in this area are hosts to varying Enterococcus species harbouring virulence and resistance determinants that can be transferred to other organisms and also are important for causing nosocomial infection.

Enhanced Neuroprotective Effects of Coadministration of Tetrandrine with Glutathione in Preclinical Model of Parkinson's Disease

PubMed Central

Li, Xiang-Yun; Mei, Guang-Hai; Dong, Qiang; Zhang, Yu; Guo, Zhuang-Li; Su, Jing-Jing; Tang, Yu-Ping; Jin, Xue-Hong; Zhou, Hou-Guang; Huang, Yan-Yan

2015-01-01

Aim. In this study we examined the influence of tetrandrine (Tet) on the neuroprotective effects of glutathione (GSH) in the 6-hydroxydopamine- (6-OHDA-) lesioned rat model of Parkinson's disease (PD). Methods. Levels in the redox system, dopamine (DA) metabolism, dopaminergic neuronal survival, and apoptosis of the substantia nigra (SN) and striatum, as well as the rotational behavior of animals were examined after a 50-day administration of GSH + Tet (or GSH) and/or L-3,4-dihydroxyphenylalanine (L-dopa) to PD rats. Ethics Committee of Huashan Hospital, Fudan University approved the protocol (number SYXK2009-0082). Results. Administration of GSH or Tet alone did not show any significant effects on the factors evaluated in the PD rats. However, in the GSH + Tet group, we observed markedly decreased oxidative damage, inhibition of DA metabolism and enhanced DA synthesis, increased tyrosine hydroxylase- (TH-) immunopositive neuronal survival, and delayed apoptosis of dopaminergic neurons in the SN. Animal rotational behavior was improved in the GSH + Tet group. Additionally, coadministration of GSH + Tet appeared to offset the possible oxidative neurotoxicity induced by L-dopa. Conclusion. In this study, we demonstrated that tetrandrine allowed occurrence of the neuroprotective effect of glutathione probably due to inhibition of P-glycoprotein on 6-hydroxydopamine-lesioned rat models of Parkinson's disease, including rats undergoing long-term L-dopa treatment. PMID:26664824

Stabilization of Foxp3 expression by CRISPR-dCas9-based epigenome editing in mouse primary T cells.

PubMed

Okada, Masahiro; Kanamori, Mitsuhiro; Someya, Kazue; Nakatsukasa, Hiroko; Yoshimura, Akihiko

2017-01-01

Epigenome editing is expected to manipulate transcription and cell fates and to elucidate the gene expression mechanisms in various cell types. For functional epigenome editing, assessing the chromatin context-dependent activity of artificial epigenetic modifier is required. In this study, we applied clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9-based epigenome editing to mouse primary T cells, focusing on the Forkhead box P3 (Foxp3) gene locus, a master transcription factor of regulatory T cells (Tregs). The Foxp3 gene locus is regulated by combinatorial epigenetic modifications, which determine the Foxp3 expression. Foxp3 expression is unstable in transforming growth factor beta (TGF-β)-induced Tregs (iTregs), while stable in thymus-derived Tregs (tTregs). To stabilize Foxp3 expression in iTregs, we introduced dCas9-TET1CD (dCas9 fused to the catalytic domain (CD) of ten-eleven translocation dioxygenase 1 (TET1), methylcytosine dioxygenase) and dCas9-p300CD (dCas9 fused to the CD of p300, histone acetyltransferase) with guide RNAs (gRNAs) targeted to the Foxp3 gene locus. Although dCas9-TET1CD induced partial demethylation in enhancer region called conserved non-coding DNA sequences 2 (CNS2), robust Foxp3 stabilization was not observed. In contrast, dCas9-p300CD targeted to the promoter locus partly maintained Foxp3 transcription in cultured and primary T cells even under inflammatory conditions in vitro. Furthermore, dCas9-p300CD promoted expression of Treg signature genes and enhanced suppression activity in vitro. Our results showed that artificial epigenome editing modified the epigenetic status and gene expression of the targeted loci, and engineered cellular functions in conjunction with endogenous epigenetic modification, suggesting effective usage of these technologies, which help elucidate the relationship between chromatin states and gene expression.

Reversible immortalization of Nestin-positive precursor cells from pancreas and differentiation into insulin-secreting cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wei, Pei; Li, Li; Qi, Hui

2012-02-10

Highlights: Black-Right-Pointing-Pointer The NPPCs from mouse pancreas were isolated. Black-Right-Pointing-Pointer Tet-on system for SV40 large in NPPCs was used to get RINPPCs. Black-Right-Pointing-Pointer The RINPPCs can undergo at least 80 population doublings without senescence. Black-Right-Pointing-Pointer The RINPPCs can be induced to differentiate into insulin-producing cells. Black-Right-Pointing-Pointer The combination of GLP-1 and sodium butyrate promoted the differentiation process. -- Abstract: Pancreatic stem cells or progenitor cells posses the ability of directed differentiation into pancreatic {beta} cells. However, these cells usually have limited proliferative capacity and finite lifespan in vitro. In the present study, Nestin-positive progenitor cells (NPPCs) from mouse pancreas thatmore » expressed the pancreatic stem cells or progenitor cell marker Nestin were isolated to obtain a sufficient number of differentiated pancreatic {beta} cells. Tet-on system for SV40 large T-antigen expression in NPPCs was used to achieve reversible immortalization. The reversible immortal Nestin-positive progenitor cells (RINPPCs) can undergo at least 80 population doublings without senescence in vitro while maintaining their biological and genetic characteristics. RINPPCs can be efficiently induced to differentiate into insulin-producing cells that contain a combination of glucagon-like peptide-1 (GLP-1) and sodium butyrate. The results of the present study can be used to explore transplantation therapy of type I diabetes mellitus.« less

Response of ginger growth to a tetracycline-contaminated environment and residues of antibiotic and antibiotic resistance genes.

PubMed

Liu, Xiaohui; Lv, Yao; Xu, Kun; Xiao, Xinxin; Xi, Beidou; Lu, Shaoyong

2018-06-01

The presence of antibiotic residues in vegetables has been highlighted as a risk to human health; antibiotics not only cause toxic effects to plants but can also induce antibiotic resistance gene (ARG) expression. Using a soil-free approach, this study aimed to explore the response of ginger growth to tetracycline (TC) pollution and to assess the levels of antibiotic residues in different plant organs and the presence of ARGs in the rhizome. Ginger growth in a highly TC-contaminated environment was remarkably inhibited. Photosynthetic parameters, fluorescence parameters, and some physiological indicators (oxidative substances, photosynthetic pigments, enzyme activity, etc.) were negatively influenced by TC contamination. Although the superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activity levels significantly increased, their effects appear to be limited. The accumulation of TC in the rhizome (28.1 mg kg -1 ) was greater than that in the roots, stem, or leaves. All tested antibiotic resistance genes except for tetL were detectable in the rhizome, and their relative abundance was in the order integron1>tetG > tetA > tetC > tetB > tetM. The level of TC in ginger rhizomes was much higher than the maximum residue limits. The potential dose of TC acquired from the consumption of ginger grown in a highly TC-contaminated environment poses no obvious risk to adults but may be a threat to children. Copyright © 2018 Elsevier Ltd. All rights reserved.

Precise integration of inducible transcriptional elements (PrIITE) enables absolute control of gene expression.

PubMed

Pinto, Rita; Hansen, Lars; Hintze, John; Almeida, Raquel; Larsen, Sylvester; Coskun, Mehmet; Davidsen, Johanne; Mitchelmore, Cathy; David, Leonor; Troelsen, Jesper Thorvald; Bennett, Eric Paul

2017-07-27

Tetracycline-based inducible systems provide powerful methods for functional studies where gene expression can be controlled. However, the lack of tight control of the inducible system, leading to leakiness and adverse effects caused by undesirable tetracycline dosage requirements, has proven to be a limitation. Here, we report that the combined use of genome editing tools and last generation Tet-On systems can resolve these issues. Our principle is based on precise integration of inducible transcriptional elements (coined PrIITE) targeted to: (i) exons of an endogenous gene of interest (GOI) and (ii) a safe harbor locus. Using PrIITE cells harboring a GFP reporter or CDX2 transcription factor, we demonstrate discrete inducibility of gene expression with complete abrogation of leakiness. CDX2 PrIITE cells generated by this approach uncovered novel CDX2 downstream effector genes. Our results provide a strategy for characterization of dose-dependent effector functions of essential genes that require absence of endogenous gene expression. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Prevalence and proliferation of antibiotic resistance genes in two municipal wastewater treatment plants.

PubMed

Mao, Daqing; Yu, Shuai; Rysz, Michal; Luo, Yi; Yang, Fengxia; Li, Fengxiang; Hou, Jie; Mu, Quanhua; Alvarez, P J J

2015-11-15

The propagation of antibiotic resistance genes (ARGs) is an emerging health concern worldwide. Thus, it is important to understand and mitigate their occurrence in different systems. In this study, 30 ARGs that confer resistance to tetracyclines, sulfonamides, quinolones or macrolides were detected in two activated sludge wastewater treatment plants (WWTPs) in northern China. Bacteria harboring ARGs persisted through all treatment units, and survived disinfection by chlorination in greater percentages than total Bacteria (assessed by 16S rRNA genes). Although the absolute abundances of ARGs were reduced from the raw influent to the effluent by 89.0%-99.8%, considerable ARG levels [(1.0 ± 0.2) × 10(3) to (9.5 ± 1.8) × 10(5) copies/mL)] were found in WWTP effluent samples. ARGs were concentrated in the waste sludge (through settling of bacteria and sludge dewatering) at (1.5 ± 2.3) × 10(9) to (2.2 ± 2.8) × 10(11) copies/g dry weight. Twelve ARGs (tetA, tetB, tetE, tetG, tetH, tetS, tetT, tetX, sul1, sul2, qnrB, ermC) were discharged through the dewatered sludge and plant effluent at higher rates than influent values, indicating overall proliferation of resistant bacteria. Significant antibiotic concentrations (2%-50% of raw influent concentrations) remained throughout all treatment units. This apparently contributed selective pressure for ARG replication since the relative abundance of resistant bacteria (assessed by ARG/16S rRNA gene ratios) was significantly correlated to the corresponding effluent antibiotic concentrations. Similarly, the concentrations of various heavy metals (which induce a similar bacterial resistance mechanism as antibiotics - efflux pumps) were also correlated to the enrichment of some ARGs. Thus, curtailing the release of antibiotics and heavy metals to sewage systems (or enhancing their removal in pre-treatment units) may alleviate their selective pressure and mitigate ARG proliferation in WWTPs. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cardiac overexpression of Mammalian enabled (Mena) exacerbates heart failure in mice

PubMed Central

Belmonte, Stephen L.; Ram, Rashmi; Mickelsen, Deanne M.; Gertler, Frank B.

2013-01-01

Mammalian enabled (Mena) is a key regulator of cytoskeletal actin dynamics, which has been implicated in heart failure (HF). We have previously demonstrated that cardiac Mena deletion produced cardiac dysfunction with conduction abnormalities and hypertrophy. Moreover, elevated Mena expression correlates with HF in human and animal models, yet the precise role of Mena in cardiac pathophysiology is unclear. In these studies, we evaluated mice with cardiac myocyte-specific Mena overexpression (TTA/TgTetMena) comparable to that observed in cardiac pathology. We found that the hearts of TTA/TgTetMena mice were functionally and morphologically comparable to wild-type littermates, except for mildly increased heart mass in the transgenic mice. Interestingly, TTA/TgTetMena mice were particularly susceptible to cardiac injury, as these animals experienced pronounced decreases in ejection fraction and fractional shortening as well as heart dilatation and hypertrophy after transverse aortic constriction (TAC). By “turning off” Mena overexpression in TTA/TgTetMena mice either immediately prior to or immediately after TAC surgery, we discovered that normalizing Mena levels eliminated cardiac hypertrophy in TTA/TgTetMena animals but did not preclude post-TAC cardiac functional deterioration. These findings indicate that hearts with increased levels of Mena fare worse when subjected to cardiac injury and suggest that Mena contributes to HF pathophysiology. PMID:23832697

Cardiac overexpression of Mammalian enabled (Mena) exacerbates heart failure in mice.

PubMed

Belmonte, Stephen L; Ram, Rashmi; Mickelsen, Deanne M; Gertler, Frank B; Blaxall, Burns C

2013-09-15

Mammalian enabled (Mena) is a key regulator of cytoskeletal actin dynamics, which has been implicated in heart failure (HF). We have previously demonstrated that cardiac Mena deletion produced cardiac dysfunction with conduction abnormalities and hypertrophy. Moreover, elevated Mena expression correlates with HF in human and animal models, yet the precise role of Mena in cardiac pathophysiology is unclear. In these studies, we evaluated mice with cardiac myocyte-specific Mena overexpression (TTA/TgTetMena) comparable to that observed in cardiac pathology. We found that the hearts of TTA/TgTetMena mice were functionally and morphologically comparable to wild-type littermates, except for mildly increased heart mass in the transgenic mice. Interestingly, TTA/TgTetMena mice were particularly susceptible to cardiac injury, as these animals experienced pronounced decreases in ejection fraction and fractional shortening as well as heart dilatation and hypertrophy after transverse aortic constriction (TAC). By "turning off" Mena overexpression in TTA/TgTetMena mice either immediately prior to or immediately after TAC surgery, we discovered that normalizing Mena levels eliminated cardiac hypertrophy in TTA/TgTetMena animals but did not preclude post-TAC cardiac functional deterioration. These findings indicate that hearts with increased levels of Mena fare worse when subjected to cardiac injury and suggest that Mena contributes to HF pathophysiology.

Genetic Manipulation of Lactococcus lactis by Using Targeted Group II Introns: Generation of Stable Insertions without Selection

PubMed Central

Frazier, Courtney L.; San Filippo, Joseph; Lambowitz, Alan M.; Mills, David A.

2003-01-01

Despite their commercial importance, there are relatively few facile methods for genomic manipulation of the lactic acid bacteria. Here, the lactococcal group II intron, Ll.ltrB, was targeted to insert efficiently into genes encoding malate decarboxylase (mleS) and tetracycline resistance (tetM) within the Lactococcus lactis genome. Integrants were readily identified and maintained in the absence of a selectable marker. Since splicing of the Ll.ltrB intron depends on the intron-encoded protein, targeted invasion with an intron lacking the intron open reading frame disrupted TetM and MleS function, and MleS activity could be partially restored by expressing the intron-encoded protein in trans. Restoration of splicing from intron variants lacking the intron-encoded protein illustrates how targeted group II introns could be used for conditional expression of any gene. Furthermore, the modified Ll.ltrB intron was used to separately deliver a phage resistance gene (abiD) and a tetracycline resistance marker (tetM) into mleS, without the need for selection to drive the integration or to maintain the integrant. Our findings demonstrate the utility of targeted group II introns as a potential food-grade mechanism for delivery of industrially important traits into the genomes of lactococci. PMID:12571038

Small-angle neutron scattering reveals the assembly mode and oligomeric architecture of TET, a large, dodecameric aminopeptidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Appolaire, Alexandre; Girard, Eric; Colombo, Matteo

2014-11-01

The present work illustrates that small-angle neutron scattering, deuteration and contrast variation, combined with in vitro particle reconstruction, constitutes a very efficient approach to determine subunit architectures in large, symmetric protein complexes. In the case of the 468 kDa heterododecameric TET peptidase machine, it was demonstrated that the assembly of the 12 subunits is a highly controlled process and represents a way to optimize the catalytic efficiency of the enzyme. The specific self-association of proteins into oligomeric complexes is a common phenomenon in biological systems to optimize and regulate their function. However, de novo structure determination of these important complexesmore » is often very challenging for atomic-resolution techniques. Furthermore, in the case of homo-oligomeric complexes, or complexes with very similar building blocks, the respective positions of subunits and their assembly pathways are difficult to determine using many structural biology techniques. Here, an elegant and powerful approach based on small-angle neutron scattering is applied, in combination with deuterium labelling and contrast variation, to elucidate the oligomeric organization of the quaternary structure and the assembly pathways of 468 kDa, hetero-oligomeric and symmetric Pyrococcus horikoshii TET2–TET3 aminopeptidase complexes. The results reveal that the topology of the PhTET2 and PhTET3 dimeric building blocks within the complexes is not casual but rather suggests that their quaternary arrangement optimizes the catalytic efficiency towards peptide substrates. This approach bears important potential for the determination of quaternary structures and assembly pathways of large oligomeric and symmetric complexes in biological systems.« less

Evolution of corresponding resistance genes in the water of fish tanks with multiple stresses of antibiotics and heavy metals.

PubMed

He, Xiaolin; Xu, Yanbin; Chen, Jinliang; Ling, Jiayin; Li, Yafei; Huang, Lu; Zhou, Xiao; Zheng, Li; Xie, Guangyan

2017-11-01

Abuse of antibiotics and heavy metals in aquaculture has been widely concerned and might aggravate the spread of resistance genes in environment. To investigate the occurrence and proliferation of antibiotic resistance genes (ARGs) and heavy metal resistance genes (HMRGs), three commonly used antibiotics (tetracycline, sulfanilamide, cefotaxime) and two heavy metals (Zn and Cu) were designed to add individually or jointly in nine fish tanks including five individual exposure tanks of tetracycline (tet), sulfanilamide (sul), cefotaxime (cef), Cu, Zn and four combination exposure tanks of tetracycline + sulfanilamide (tet + sul), tetracycline + sulfanilamide + cefotaxime (tet + sul + cef), tetracycline + sulfanilamide + Cu (tet + sul + Cu), tetracycline + sulfanilamide + Zn (tet + sul + Zn) as well as the control during the experiment period of 180 days. Nineteen ARGs (tetA, tetB, tetC, tetD, tetE, tetG, tetM, tetO, tetQ, tetS, tetW, tetX, tetY, sul1, sul2, sul3, bla DHA , bla MOX , bla FOX ), two HMRGs (copA, czcA) and the class 1 integron gene (intI 1) in fish tanks water were investigated. The results showed that the residual rate of antibiotics and heavy metals ranged from 0.03% to 2.46% and 9.25%-52.97%, respectively, positively related to their original concentration and types. Tetracycline resistance genes were more sensitive to antibiotics and easier to be induced and developed than sulfanilamide resistance genes and AmpC β-lactamase resistance genes. The total relative abundances of ARGs in combined stresses exposure tanks (tet + sul, tet + sul + cef, tet + sul + Cu, tet + sul + Zn) were about 1.01-1.55 times more than the sum of their individual ones. The co-selective effects of cefotaxime on the abundance and diversity of tetracycline resistance genes were stronger than Zn and Cu. Besides, multivariate correlation analysis revealed that tetO, tetQ, tetW and sul3 were in significant correlation with the concentrations of Cu and Zn (r = 0.882-0.992, p < 0.05 or p < 0.01). The significant correlations between tetO and intI1 (p < 0.01), tetW and intI1 (p < 0.05), and sul3 and intI1 (p < 0.05) hinted a potentially serious and undesirable dissemination risk of ribosomal protection proteins gene of ARGs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Liquid Crystalline Nanoparticles as an Ophthalmic Delivery System for Tetrandrine: Development, Characterization, and In Vitro and In Vivo Evaluation

NASA Astrophysics Data System (ADS)

Liu, Rui; Wang, Shuangshuang; Fang, Shiming; Wang, Jialu; Chen, Jingjing; Huang, Xingguo; He, Xin; Liu, Changxiao

2016-05-01

The purpose of this study was to develop novel liquid crystalline nanoparticles (LCNPs) that display improved pre-ocular residence time and ocular bioavailability and that can be used as an ophthalmic delivery system for tetrandrine (TET). The delivery system consisted of three primary components, including glyceryl monoolein, poloxamer 407, and water, and two secondary components, including Gelucire 44/14 and amphipathic octadecyl-quaternized carboxymethyl chitosan. The amount of TET, the amount of glyceryl monoolein, and the ratio of poloxamer 407 to glyceryl monoolein were selected as the factors that were used to optimize the dependent variables, which included encapsulation efficiency and drug loading. A three-factor, five-level central composite design was constructed to optimize the formulation. TET-loaded LCNPs (TET-LCNPs) were characterized to determine their particle size, zeta potential, entrapment efficiency, drug loading capacity, particle morphology, inner crystalline structure, and in vitro drug release profile. Corneal permeation in excised rabbit corneas was evaluated. Pre-ocular retention was determined using a noninvasive fluorescence imaging system. Finally, pharmacokinetic study in the aqueous humor was performed by microdialysis technique. The optimal formulation had a mean particle size of 170.0 ± 13.34 nm, a homogeneous distribution with polydispersity index of 0.166 ± 0.02, a positive surface charge with a zeta potential of 29.3 ± 1.25 mV, a high entrapment efficiency of 95.46 ± 4.13 %, and a drug loading rate of 1.63 ± 0.07 %. Transmission electron microscopy showed spherical particles that had smooth surfaces. Small-angle X-ray scattering profiles revealed an inverted hexagonal phase. The in vitro release assays showed a sustained drug release profile. A corneal permeation study showed that the apparent permeability coefficient of the optimal formulation was 2.03-fold higher than that of the TET solution. Pre-ocular retention capacity study indicated that the retention of LCNPs was significantly longer than that of the solution ( p < 0.01). In addition, a pharmacokinetic study of rabbit aqueous humors demonstrated that the TET-LCNPs showed 2.65-fold higher ocular bioavailability than that of TET solution. In conclusion, a LCNP system could be a promising method for increasing the ocular bioavailability of TET by enhancing its retention time and permeation into the cornea.

Characterization of antimicrobial susceptibility and virulence genes of Salmonella serovars collected at a commercial turkey processing plant.

PubMed

Nde, C W; Logue, C M

2008-01-01

To determine the antimicrobial susceptibility profiles, distribution of class 1 integrons, virulence genes and genes encoding resistance to tetracycline (tetA, tetC, tetD and tetE) and streptomycin (strA, strB and aadA1) in Salmonella recovered from turkeys. The antimicrobial susceptibility of 80 isolates was determined using National Antimicrobial Resistance Monitoring System. The distribution of resistance genes, class 1 integrons and virulence genes was determined using PCR. Resistances to tetracycline (76 x 3%) and streptomycin (40%) were common. Sixty-two (77 x 5%) isolates displayed resistance against one or more antimicrobials and 33 were multi-drug resistant. tetA was detected in 72 x 5% of the isolates, while tetC, tetD and tetE were not detected. The strA and strB genes were detected in 73 x 8% of the isolates. Two isolates possessed class 1 integrons of 1 kb in size, containing the aadA1 gene conferring resistance to streptomycin and spectinomycin. Fourteen of the virulence genes were detected in over 80% of the isolates. This study shows that continuous use of tetracycline and streptomycin in poultry production selects for resistant strains. The Salmonella isolates recovered possess significant ability to cause human illness. Information from this study can be employed in guiding future strategies for the use of antimicrobials in poultry production.

Genetic algorithm optimization of transcutaneous energy transmission systems for implantable ventricular assist devices.

PubMed

Byron, Kelly; Bluvshtein, Vlad; Lucke, Lori

2013-01-01

Transcutaneous energy transmission systems (TETS) wirelessly transmit power through the skin. TETS is particularly desirable for ventricular assist devices (VAD), which currently require cables through the skin to power the implanted pump. Optimizing the inductive link of the TET system is a multi-parameter problem. Most current techniques to optimize the design simplify the problem by combining parameters leading to sub-optimal solutions. In this paper we present an optimization method using a genetic algorithm to handle a larger set of parameters, which leads to a more optimal design. Using this approach, we were able to increase efficiency while also reducing power variability in a prototype, compared to a traditional manual design method.

Cow excrements enhance the occurrence of tetracycline resistance genes in soil regardless of their oxytetracycline content.

PubMed

Kyselková, Martina; Jirout, Jiří; Chroňáková, Alica; Vrchotová, Naděžda; Bradley, Robert; Schmitt, Heike; Elhottová, Dana

2013-11-01

Fertilizing soils with animal excrements from farms with common antibiotic use represents a risk of disseminating antibiotic resistance genes into the environment. In the case of tetracycline antibiotics, it is not clear, however, whether the presence of antibiotic residues further enhances the gene occurrence in manured soils. We established a microcosm experiment in which 3 farm soils that had no recent history of fertilization with animal excrements were amended on a weekly basis (9 times) with excrements from either an oxytetracycline-treated or an untreated cow. Throughout the study, the concentration of oxytetracycline in excrements from the treated cow was above 500 μg g(-1)dw, whereas no oxytetracycline was detected in excrements from the healthy cow. Both excrements contained tetracycline resistance (TC-r) genes tet(L), tet(M), tet(V), tet(Z), tet(Q) and tet(W). The excrements from the treated cow also contained the tet(B) gene, and a higher abundance of tet(Z), tet(Q) and tet(W). Three weeks after the last excrement addition, the individual TC-r genes differed in their persistence in soil: tet(Q) and tet(B) were not detectable while tet(L), tet(M), tet(Z) and tet(W) were found in all 3 soils. There were, however, no significant differences in the total number, nor in the abundance, of TC-r genes between soil samples amended with each excrement type. The oxytetracycline-rich and the oxytetracycline-free excrement therefore contributed equally to the increase of tetracycline resistome in soil. Our results indicate that other mechanisms than OTC-selection pressure may be involved in the maintenance of TC-r genes in manured soils. Copyright © 2013. Published by Elsevier Ltd.

The dynamics of DNA methylation and hydroxymethylation during amelogenesis.

PubMed

Yoshioka, Hirotaka; Minamizaki, Tomoko; Yoshiko, Yuji

2015-11-01

Amelogenesis is a multistep process that relies on specific temporal and spatial signaling networks between the dental epithelium and mesenchymal tissues. Epigenetic modifications of key developmental genes in this process may be closely linked to a network of molecular events. However, the role of epigenetic regulation in amelogenesis remains unclear. Here, we have uncovered the spatial distributions of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) to determine epigenetic events in the mandibular incisors of mice. Immunohistochemistry and dot blotting showed that 5-hmC in ameloblasts increased from the secretory stage to the later maturation stage. We also demonstrated the distribution of 5-mC-positive ameloblasts with punctate nuclear labeling from sometime after the initiation of the secretory stage to the later maturation stage; however, dot blotting failed to detect this change. No obvious alteration of 5-mC/5-hmC staining in odontoblasts and dental pulp cells was observed. Concomitant with quantitative expression data, immunohistochemistry showed that maintenance DNA methyltransferase DNMT1 was highly expressed in immature dental epithelial cells and subsequently decreased at later stages of development. Meanwhile, de novo DNA methyltransferase Dnmt3a and Dnmt3b and DNA demethylase Tet family genes were universally expressed, except Tet1 that was highly expressed in immature dental epithelial cells. Thus, DNMT1 may sustain the undifferentiated status of dental epithelial cells through the maintenance of DNA methylation, while the hydroxylation of 5-mC may occur through the whole differentiation process by TET activity. Taken together, these data indicate that the dynamic changes of 5-mC and 5-hmC may be critical for the regulation of amelogenesis.

Prevalence of antibiotic resistance genes from effluent of coastal aquaculture, South Korea.

PubMed

Jang, Hyun Min; Kim, Young Beom; Choi, Sangki; Lee, Yunho; Shin, Seung Gu; Unno, Tatsuya; Kim, Young Mo

2018-02-01

The wide use of antibiotics in aquaculture for prophylactic and therapeutic purposes can potentially lead to the prevalence of antibiotic resistance genes (ARGs). This study reports for the first time the profile of ARGs from effluents of coastal aquaculture located in South Jeolla province and Jeju Island, South Korea. Using quantitative PCR (qPCR), twenty-two ARGs encoding tetracycline resistance (tetA, tetB, tetD, tetE, tetG, tetH, tetM, tetQ, tetX, tetZ, tetBP), sulfonamide resistance (sul1, sul2), quinolone resistance (qnrD, qnrS, aac(6')-Ib-cr), β-lactams resistance (bla TEM , bla CTX , bla SHV ), macrolide resistance (ermC), florfenicol resistance (floR) and multidrug resistance (oqxA) and a class 1 integrons-integrase gene (intI1) were quantified. In addition, Illumina Miseq sequencing was applied to investigate microbial community differences across fish farm effluents. Results from qPCR showed that the total number of detected ARGs ranged from 4.24 × 10 -3 to 1.46 × 10 -2 copies/16S rRNA gene. Among them, tetB and tetD were predominant, accounting for 74.8%-98.0% of the total ARGs. Furthermore, intI1 gene showed positive correlation with tetB, tetD, tetE, tetH, tetX, tetZ tetQ and sul1. Microbial community analysis revealed potential host bacteria for ARGs and intI1. Two genera, Vibrio and Marinomonas belonging to Gammaproteobacteria, showed significant correlation with tetB and tetD, the most dominant ARGs in all samples. Also, operational taxonomic units (OTUs)-based network analysis revealed that ten OTUs, classified into the phyla Proteobacteria, Cyanobacteria/Chloroplast, Bacteroidetes, Verrucomicrobia and an unclassified phylum, were potential hosts of tetracycline resistance genes (i.e., tetA, tetG, tetH, tetM, tetQ and tetZ). Further systematic monitoring of ARGs is warranted for risk assessment and management of antibacterial resistance from fish farm effluents. Copyright © 2017 Elsevier Ltd. All rights reserved.

Quantitative Proteomic and Microarray Analysis of the Archaeon Methanosarcina Acetivorans Grown with Acetate Versus Methanol*

PubMed Central

Li, Lingyun; Li, Qingbo; Rohlin, Lars; Kim, UnMi; Salmon, Kirsty; Rejtar, Tomas; Gunsalus, Robert P.; Karger, Barry L.; Ferry, James G.

2008-01-01

Summary Methanosarcina acetivorans strain C2A is an acetate- and methanol-utilizing methane-producing organism for which the genome, the largest yet sequenced among the Archaea, reveals extensive physiological diversity. LC linear ion trap-FTICR mass spectrometry was employed to analyze acetate- vs. methanol-grown cells metabolically labeled with 14N vs. 15N, respectively, to obtain quantitative protein abundance ratios. DNA microarray analyses of acetate- vs. methanol-grown cells was also performed to determine gene expression ratios. The combined approaches were highly complementary, extending the physiological understanding of growth and methanogenesis. Of the 1081 proteins detected, 255 were ≥ 3-fold differentially abundant. DNA microarray analysis revealed 410 genes that were ≥ 2.5-fold differentially expressed of 1972 genes with detected expression. The ratios of differentially abundant proteins were in good agreement with expression ratios of the encoding genes. Taken together, the results suggest several novel roles for electron transport components specific to acetate-grown cells, including two flavodoxins each specific for growth on acetate or methanol. Protein abundance ratios indicated that duplicate CO dehydrogenase/acetyl-CoA complexes function in the conversion of acetate to methane. Surprisingly, the protein abundance and gene expression ratios indicated a general stress response in acetate- vs. methanol-grown cells that included enzymes specific for polyphosphate accumulation and oxidative stress. The microarray analysis identified transcripts of several genes encoding regulatory proteins with identity to the PhoU, MarR, GlnK, and TetR families commonly found in the Bacteria domain. An analysis of neighboring genes suggested roles in controlling phosphate metabolism (PhoU), ammonia assimilation (GlnK), and molybdopterin cofactor biosynthesis (TetR). Finally, the proteomic and microarray results suggested roles for two-component regulatory systems specific for each growth substrate. PMID:17269732

Loss of heterozygosity 4q24 and TET2 mutations associated with myelodysplastic/myeloproliferative neoplasms

PubMed Central

Jankowska, Anna M.; Szpurka, Hadrian; Tiu, Ramon V.; Makishima, Hideki; Afable, Manuel; Huh, Jungwon; O'Keefe, Christine L.; Ganetzky, Rebecca; McDevitt, Michael A.

2009-01-01

Chromosomal abnormalities are frequent in myeloid malignancies, but in most cases of myelodysplasia (MDS) and myeloproliferative neoplasms (MPN), underlying pathogenic molecular lesions are unknown. We identified recurrent areas of somatic copy number–neutral loss of heterozygosity (LOH) and deletions of chromosome 4q24 in a large cohort of patients with myeloid malignancies including MDS and related mixed MDS/MPN syndromes using single nucleotide polymorphism arrays. We then investigated genes in the commonly affected area for mutations. When we sequenced TET2, we found homozygous and hemizygous mutations. Heterozygous and compound heterozygous mutations were found in patients with similar clinical phenotypes without LOH4q24. Clinical analysis showed most TET2 mutations were present in patients with MDS/MPN (58%), including CMML (6/17) or sAML (32%) evolved from MDS/MPN and typical MDS (10%), suggesting they may play a ubiquitous role in malignant evolution. TET2 mutations affected conserved domains and the N terminus. TET2 is widely expressed in hematopoietic cells but its function is unknown, and it lacks homology to other known genes. The frequency of mutations in this candidate myeloid regulatory gene suggests an important role in the pathogenesis of poor prognosis MDS/MPN and sAML and may act as a disease gene marker for these often cytogenetically normal disorders. PMID:19372255

Genomic and epigenomic analysis of high-risk prostate cancer reveals changes in hydroxymethylation and TET1.

PubMed

Spans, Lien; Van den Broeck, Thomas; Smeets, Elien; Prekovic, Stefan; Thienpont, Bernard; Lambrechts, Diether; Karnes, R Jeffrey; Erho, Nicholas; Alshalalfa, Mohammed; Davicioni, Elai; Helsen, Christine; Gevaert, Thomas; Tosco, Lorenzo; Haustermans, Karin; Lerut, Evelyne; Joniau, Steven; Claessens, Frank

2016-04-26

The clinical heterogeneity of prostate cancer (PCa) makes it difficult to identify those patients that could benefit from more aggressive treatments. As a contribution to a better understanding of the genomic changes in the primary tumor that are associated with the development of high-risk disease, we performed exome sequencing and copy number determination of a clinically homogeneous cohort of 47 high-risk PCas. We confirmed recurrent mutations in SPOP, PTEN and TP53 among the 850 point mutations we detected. In seven cases, we discovered genomic aberrations in the TET1 (Ten-Eleven Translocation 1) gene which encodes a DNA hydroxymethylase than can modify methylated cytosines in genomic DNA and thus is linked with gene expression changes. TET1 protein levels were reduced in tumor versus non-tumor prostate tissue in 39 of 40 cases. The clinical relevance of changes in TET1 levels was demonstrated in an independent PCa cohort, in which low TET1 mRNA levels were significantly associated with worse metastases-free survival. We also demonstrate a strong reduction in hydroxymethylated DNA in tumor tissue in 27 of 41 cases. Furthermore, we report the first exploratory (h)MeDIP-Seq analyses of eight high-risk PCa samples. This reveals a large heterogeneity in hydroxymethylation changes in tumor versus non-tumor genomes which can be linked with cell polarity.

The role of zero valent iron on the fate of tetracycline resistance genes and class 1 integrons during thermophilic anaerobic co-digestion of waste sludge and kitchen waste.

PubMed

Gao, Pin; Gu, Chaochao; Wei, Xin; Li, Xiang; Chen, Hong; Jia, Hanzhong; Liu, Zhenhong; Xue, Gang; Ma, Chunyan

2017-03-15

Activated sludge has been identified as a potential significant source of antibiotic resistance genes (ARGs) to the environment. Anaerobic digestion is extensively used for sludge stabilization and resource recovery, and represents a crucial process for controlling the dissemination of ARGs prior to land application of digested sludge. The objective of this study is to investigate the effect of zero valent iron (Fe 0 ) on the attenuation of seven representative tetracycline resistance genes (tet, tet(A), tet(C), tet(G), tet(M), tet(O), tet(W), and tet(X)), and the integrase gene intI1 during thermophilic anaerobic co-digestion of waste sludge and kitchen waste. Significant decrease (P < 0.05) in the quantities of tet (except tet(W)) and intI1 genes was observed at Fe 0 dosage of 5 g/L, whereas no significant differences (P > 0.05) were found for all gene targets between digesters with Fe 0 dosages of 5 and 60 g/L. A first-order kinetic model favorably described the trends in concentrations of tet and intI1 gene targets during thermophilic anaerobic digestion with or without Fe 0 . Notably, tet genes encoding different resistance mechanisms behaved distinctly in anaerobic digesters, although addition of Fe 0 could enhance their reduction. The overall results of this research suggest that thermophilic anaerobic digestion with Fe 0 can be a potential alternative technology for the attenuation of tet and intI1 genes in waste sludge. Copyright © 2016 Elsevier Ltd. All rights reserved.

The New Payload Handling System for the G erman On- Orbit Verification Satellite TET with the Sensor Bus as Example for Payloads

NASA Astrophysics Data System (ADS)

Heyer, H.-V.; Föckersperger, S.; Lattner, K.; Moldenhauer, W.; Schmolke, J.; Turk, M.; Willemsen, P.; Schlicker, M.; Westerdorff, K.

2008-08-01

The technology verification satellite TET (Technologie ErprobungsTräger) is the core element of the German On-Orbit-Verification (OOV) program of new technologies and techniques. The goal of this program is the support of the German space industry and research facilities for on-orbit verification of satellite technologies. The TET satellite is a small satellite developed and built in Germany under leadership of Kayser-Threde. The satellite bus is based on the successfully operated satellite BIRD and the newly developed payload platform with the new payload handling system called NVS (Nutzlastversorgungs-system). The NVS can be detailed in three major parts: the power supply the processor boards and the I/O-interfaces. The NVS is realized via several PCBs in Europe format which are connected to each other via an integrated backplane. The payloads are connected by front connectors to the NVS. This paper describes the concept, architecture, and the hard-/software of the NVS. Phase B of this project was successfully finished last year.

A quantitative real-time PCR assay for the detection of tetR of Tn10 in Escherichia coli using SYBR Green and the Opticon.

PubMed

Morsczeck, Christian; Langendörfer, Daniel; Schierholz, Jörg Michael

2004-06-30

Bacteria of implant infections are extremely resistant to antibiotics. One reason for this antibiotic resistance are transposons; the well-known transposon Tn10, for example, mediates tetracycline resistance to Escherichia coli. Two genes of Tn10, tetA and tetR, are essential for the mechanism of resistance. These genes encode a drug-specific efflux protein and a tetracycline repressor protein, respectively. Tn10 is also widely used in molecular biology. For example, tTA, a recombinant derivate of tetR, has been utilised for a highly efficient gene regulation system in mammalian cells. We have examined E. coli isolates from implant infections for tetracycline resistance and for the presence of tetR. A real-time PCR assay was developed for detection of tetR with SybrGreen using the Opticon PCR machine of MJ Research. This method offers a quick, sensitive, efficient, and reliable approach to the detection and quantification of genes. Clinical isolates of E. coli were examined successfully for tetracycline resistance and for the presence of tetR. The real-time PCR is effective using a variety of templates including isolated E. coli DNA, pure colonies, or liquid culture sources. Using quantified standard DNA, this assay can accurately detect as few as 15 copies. Moreover, this assay has the ability to quantify the number of tetR genes in the presence of contaminating mammalian DNA. In conclusion, the tetR real-time PCR offers new methods for detection and quantification of tetracycline-resistant bacteria and tTA in transfected cell-lines or transgenic animals.

Stringent and reproducible tetracycline-regulated transgene expression by site-specific insertion at chromosomal loci with pre-characterised induction characteristics

PubMed Central

Brough, Rachel; Papanastasiou, Antigoni M; Porter, Andrew CG

2007-01-01

Background The ability to regulate transgene expression has many applications, mostly concerning the analysis of gene function. Desirable induction characteristics, such as low un-induced expression, high induced expression and limited cellular heterogeneity, can be seriously impaired by chromosomal position effects at the site of transgene integration. Many clones may therefore need to be screened before one with optimal induction characteristics is identified. Furthermore, such screens must be repeated for each new transgene investigated, and comparisons between clones with different transgenes is complicated by their different integration sites. Results To circumvent these problems we have developed a "screen and insert" strategy in which clones carrying a transgene for a fluorescent reporter are first screened for those with optimal induction characteristics. Site-specific recombination (SSR) is then be used repeatedly to insert any new transgene at the reporter transgene locus of such clones so that optimal induction characteristics are conferred upon it. Here we have tested in a human fibrosarcoma cell line (HT1080) two of many possible implementations of this approach. Clones (e.g. Rht14-10) in which a GFP reporter gene is very stringently regulated by the tetracycline (tet) transactivator (tTA) protein were first identified flow-cytometrically. Transgenes encoding luciferase, I-SceI endonuclease or Rad52 were then inserted by SSR at a LoxP site adjacent to the GFP gene resulting stringent tet-regulated transgene expression. In clone Rht14-10, increases in expression from essentially background levels (+tet) to more than 104-fold above background (-tet) were reproducibly detected after Cre-mediated insertion of either the luciferase or the I-SceI transgenes. Conclusion Although previous methods have made use of SSR to integrate transgenes at defined sites, none has effectively combined this with a pre-selection step to identify integration sites that support optimal regulatory characteristics. Rht14-10 and similar HT1080-derived clones can now be used in conjunction with a convenient delivery vector (pIN2-neoMCS), in a simple 3-step protocol leading to stringent and reproducible transgene regulation. This approach will be particularly useful for transgenes whose products are very active at low concentrations and/or for comparisons of multiple related transgenes. PMID:17493262

Aging and alcohol interact to alter hepatic DNA hydroxymethylation

PubMed Central

Tammen, Stephanie A.; Dolnikowski, Gregory G.; Ausman, Lynne M.; Liu, Zhenhua; Sauer, Julia; SimonettaFriso; Choi, Sang-Woon

2014-01-01

Background Aging and chronic alcohol consumption are both modifiers of DNA methylation but it is not yet known whether chronic alcohol consumption also alters DNA hydroxymethylation, a newly discovered epigenetic mark produced by oxidation of methylcytosine. Furthermore, it has not been tested whether aging and alcohol interact to modify this epigenetic phenomenon, thereby having an independent effect on gene expression. Methods Old (18 months) and young (4 months) male C57BL/6 mice were pair-fed either a Lieber-DeCarli liquid diet with alcohol (18% of energy) or an isocaloricLieber-DeCarli control diet for 5 weeks. Global DNA hydroxymethylation and DNA methylation were analyzed from hepatic DNA using a new LC/MS-MS method. Hepatic mRNA expression of the Tet enzymes and Cyp2e1 were measured via qRTPCR. Results In young mice, mild chronic alcohol exposure significantly reduced global DNA hydroxymethylation compared with control mice (0.22%±0.01% vs 0.29±0.06%, p = 0.004). Alcohol did not significantly alter hydroxymethylcytosine levels in old mice. Old mice fed the control diet showed decreased global DNA hydroxymethylation compared with young mice fed the control diet (0.24±0.02% vs 0.29±0.06%, p = 0.04). This model suggests an interaction between aging and alcohol in determining DNA hydroxymethylation (pinteraction = 0.009). Expression of Tet2 and Tet3 enzymes was decreased in the old mice relative to the young (p < 0.005). Conclusions The observation that alcohol alters DNA hydroxymethylation indicates a new epigenetic effect of alcohol. This is the first study demonstrating the interactive effects of chronic alcohol consumption and aging on DNA hydroxymethylation. PMID:25070523

The study of membrane formation via phase inversion method by cloud point and light scattering experiment

NASA Astrophysics Data System (ADS)

Arahman, Nasrul; Maimun, Teuku; Mukramah, Syawaliah

2017-01-01

The composition of polymer solution and the methods of membrane preparation determine the solidification process of membrane. The formation of membrane structure prepared via non-solvent induced phase separation (NIPS) method is mostly determined by phase separation process between polymer, solvent, and non-solvent. This paper discusses the phase separation process of polymer solution containing Polyethersulfone (PES), N-methylpirrolidone (NMP), and surfactant Tetronic 1307 (Tet). Cloud point experiment is conducted to determine the amount of non-solvent needed on induced phase separation. Amount of water required as a non-solvent decreases by the addition of surfactant Tet. Kinetics of phase separation for such system is studied by the light scattering measurement. With the addition of Tet., the delayed phase separation is observed and the structure growth rate decreases. Moreover, the morphology of fabricated membrane from those polymer systems is analyzed by scanning electron microscopy (SEM). The images of both systems show the formation of finger-like macrovoids through the cross-section.

Tet1 facilitates hypoxia tolerance by stabilizing the HIF-α proteins independent of its methylcytosine dioxygenase activity.

PubMed

Wang, Jing; Zhang, Dawei; Du, Juan; Zhou, Chi; Li, Zhi; Liu, Xing; Ouyang, Gang; Xiao, Wuhan

2017-12-15

Because of the requirement of oxygen (O2) to produce energy, aerobic organisms developed mechanisms to protect themselves against a shortage of oxygen in both acute status and chronic status. To date, how organisms tolerate acute hypoxia and the underlying mechanisms remain largely unknown. Here, we identify that Tet1, one member of the ten-eleven translocation (TET) family of methylcytosine dioxygenases, is required for hypoxia tolerance in zebrafish and mice. Tet1-null zebrafish and mice are more sensitive to hypoxic conditions compared with their wild-type siblings. We demonstrate that Tet1 stabilizes hypoxia-inducible factor α (HIF-α) and enhances HIF-α transcription activity independent of its enzymatic activity. In addition, we show that Tet1 modulates HIF-2α and HIF-1α through different mechanisms. Tet1 competes with prolyl hydroxylase protein 2 (PHD2) to bind to HIF-2α, resulting in a reduction of HIF-2α hydroxylation by PHD2. For HIF-1α, however, Tet1 has no effect on HIF-1α hydroxylation, but rather it appears to stabilize the C-terminus of HIF-1α by affecting lysine site modification. Furthermore, we found that Tet1 enhances rather than prevents poly-ubiquitination on HIF-α. Our results reveal a previously unrecognized function of Tet1 independent of its methylcytosine dioxygenase activity in hypoxia signaling. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Cytogenetic correlates of TET2 mutations in 199 patients with myeloproliferative neoplasms

PubMed Central

Hussein, Kebede; Abdel-Wahab, Omar; Lasho, Terra L.; Van Dyke, Daniel L.; Levine, Ross L.; Hanson, Curtis A.; Pardanani, Animesh; Tefferi, Ayalew

2015-01-01

TET2 is a putative tumor suppressor gene located at chromosome 4q24. TET2 mutations were recently described in several myeloid neoplasms but correlations with cytogenetic findings have not been studied. Among a recently described cohort of patients with myeloproliferative neoplasms (MPN) who underwent TET2 mutation analysis, 199 had information on karyotype at diagnosis or time of TET2 testing: 71 polycythemia vera (PV), 55 primary myelofibrosis (PMF), 43 essential thrombocythemia (ET), 13 post-PV MF, 7 post-ET MF, and 10 blast phase MPN. Forty eight patients (24%) exhibited abnormal karyotype: 15 favorable (sole 20q-, 13q-, or +9), 8 unfavorable (complex karyotype or sole +8), and 25 “other” cytogenetic abnormalities. We found no significant difference either in the incidence or type of cytogenetic abnormalities between TET2 mutated (n = 25) and unmutated (n = 174) cases. Seventy nine patients, including 14 with TET2 mutations, underwent follow-up cytogenetic testing and the findings were again not affected by TET2 mutational status. We conclude that TET2 mutated MPN patients are not cytogenetically different than their TET2 unmutated counterparts. PMID:19957346

Thoratec transcutaneous energy transformer system: a review and update.

PubMed

Rintoul, Thomas C; Dolgin, Alexander

2004-01-01

A transcutaneous energy transformer system (TETS) can provide power to an implanted ventricular assist device (VAD) across an unbroken layer of skin. A TETS includes a subcutaneous secondary coil, which traditionally connects to remote power conditioning circuitry located to avoid eddy current losses and heating that occur in metal near operating TETS coils. Litz wire, used to construct the coil and connect it to that circuitry, efficiently conducts the high frequency alternating current but is bulky and stiff. A novel concept (US Patent No. 6,327,504 B1) packages the secondary coil's output power conditioning circuitry within the unused aperture of the coil while minimizing eddy current losses. The concept allows use of a more flexible cable for its direct current power output. The result is improved reliability, functionality, and efficiency along with decreased implant volume and a thinner, more flexible lead system to interconnect to the VAD. This in turn enhances system versatility by expanding sites available for module implantation. A TETS using this concept has demonstrated efficiency exceeding 80% and peak power outputs of 45 W with good tissue compatibility in the bovine model after a 30 day implant.

Detection of tet(M) and tet(O) using the polymerase chain reaction in bacteria isolated from patients with periodontal disease.

PubMed

Olsvik, B; Olsen, I; Tenover, F C

1995-04-01

The polymerase chain reaction was used to examine 114 tetracycline-resistant anaerobic and facultative anaerobic bacterial isolates from patients with periodontal disease for the tet(M) and tet(O) genes. A 740-base-pair fragment of the tet(M) gene was amplified from 84 of 114 isolates, and a 519-base-pair fragment of the tet(O) gene was amplified from 13 streptococcal isolates. Six of 7 tetracycline-resistant isolates of Veillonella spp. and tetracycline-resistant isolates of Eubacterium spp. (n = 3), Eubacterium saburreum (n = 1), Streptococcus intermedius (n = 5) and Gemella morbillorum (n = 2) all harbored the tet(M) gene. The tet(M) and tet(O) negative as well as selected positive isolates were tested for the tet(K) and tet(L) genes using DNA probes. All isolates of Staphylococcus spp. (n = 11) hybridized with the tet(K) probe. None of the isolates tested hybridized with the probe for tet(L). This is the first report of the tet(M) gene in the facultative bacterium G. morbillorum and in E. saburreum.

[Identification of lactic acid bacteria in commercial yogurt and their antibiotic resistance].

PubMed

Qin, Yuxuan; Li, Jing; Wang, Qiuya; Gao, Kexin; Zhu, Baoli; Lv, Na

2013-08-04

To identify lactic acid bacteria (LAB) in commercial yogurts and investigate their antibiotic resistance. LABs were cultured from 5 yogurt brands and the isolates were identified at the species level by 16S rRNA sequence. Genotyping was performed by repetitive extragenic palindromic PCR (rep-PCR). The sensitivity to 7 antibiotics was tested for all LAB isolates by Kirby-Bauer paper diffusion (K-B method). Meanwhile, 9 antibiotic resistance genes (ARGs), including erythromycin resistance genes (ermA and ermB) and tetracycline resistance genes (tetM, tetK, tetS, tetQ, tetO, tetL and tetW), were detected by PCR amplification in the identified LAB isolates. The PCR products were confirmed by sequencing. Total 100 LABs were isolated, including 23 Lactobacillus delbrueckii ssp. bulgaricus, 26 Lactobacillus casei, 30 Streptococcus thermophilus, 5 Lactobacillus acidophilus, 6 Lactobacillus plantarum, and 10 Lactobacillus paracasei. The drug susceptibility test shows that all 100 isolates were resistant to gentamicin and streptomycin, 42 isolates were resistant to vancomycin, and on the contrary all were sensitive to cefalexin, erythromycin, tetracycline and oxytetracycline. Moreover, 5 ARGs were found in the 28 sequencing confirmed isolates, ermB gene was detected in 8 isolates, tet K in 4 isolates, tetL in 2 isolates, tetM in 4 isolates, tetO in 2 isolates. erm A, tet S, tet Q and tet W genes were not detected in the isolates. Antibiotic resistance genes were found in 53.57% (15/28) sequenced isolates, 2 -3 antibiotic resistance genes were detected in 4 isolates of L. delbrueckii ssp. bulgaricus. Some LABs were not labeled in commercial yogurt products. Antibiotic resistance genes tend to be found in the starter culture of L. delbrueckii ssp. Bulgaricus and S. thermophilus. All the LAB isolates were sensitive to erythromycin and tetracycline, even though some carried erythromycin and/or tetracycline resistance genes. We proved again that LAB could carry antibiotic resistance gene(s) though it is sensitive to antibiotics.

Elevated catalase expression in a fungal pathogen is a double-edged sword of iron

PubMed Central

Belmonte, Rodrigo; Budge, Susan; Lopez Garcia, Angela; Lee, Keunsook K.; Bebes, Attila; Quinn, Janet

2017-01-01

Most fungal pathogens of humans display robust protective oxidative stress responses that contribute to their pathogenicity. The induction of enzymes that detoxify reactive oxygen species (ROS) is an essential component of these responses. We showed previously that ectopic expression of the heme-containing catalase enzyme in Candida albicans enhances resistance to oxidative stress, combinatorial oxidative plus cationic stress, and phagocytic killing. Clearly ectopic catalase expression confers fitness advantages in the presence of stress, and therefore in this study we tested whether it enhances fitness in the absence of stress. We addressed this using a set of congenic barcoded C. albicans strains that include doxycycline-conditional tetON-CAT1 expressors. We show that high basal catalase levels, rather than CAT1 induction following stress imposition, reduce ROS accumulation and cell death, thereby promoting resistance to acute peroxide or combinatorial stress. This conclusion is reinforced by our analyses of phenotypically diverse clinical isolates and the impact of stochastic variation in catalase expression upon stress resistance in genetically homogeneous C. albicans populations. Accordingly, cat1Δ cells are more sensitive to neutrophil killing. However, we find that catalase inactivation does not attenuate C. albicans virulence in mouse or invertebrate models of systemic candidiasis. Furthermore, our direct comparisons of fitness in vitro using isogenic barcoded CAT1, cat1Δ and tetON-CAT1 strains show that, while ectopic catalase expression confers a fitness advantage during peroxide stress, it confers a fitness defect in the absence of stress. This fitness defect is suppressed by iron supplementation. Also high basal catalase levels induce key iron assimilatory functions (CFL5, FET3, FRP1, FTR1). We conclude that while high basal catalase levels enhance peroxide stress resistance, they place pressure on iron homeostasis through an elevated cellular demand for iron, thereby reducing the fitness of C. albicans in iron-limiting tissues within the host. PMID:28542620

Elevated catalase expression in a fungal pathogen is a double-edged sword of iron.

PubMed

Pradhan, Arnab; Herrero-de-Dios, Carmen; Belmonte, Rodrigo; Budge, Susan; Lopez Garcia, Angela; Kolmogorova, Aljona; Lee, Keunsook K; Martin, Brennan D; Ribeiro, Antonio; Bebes, Attila; Yuecel, Raif; Gow, Neil A R; Munro, Carol A; MacCallum, Donna M; Quinn, Janet; Brown, Alistair J P

2017-05-01

Most fungal pathogens of humans display robust protective oxidative stress responses that contribute to their pathogenicity. The induction of enzymes that detoxify reactive oxygen species (ROS) is an essential component of these responses. We showed previously that ectopic expression of the heme-containing catalase enzyme in Candida albicans enhances resistance to oxidative stress, combinatorial oxidative plus cationic stress, and phagocytic killing. Clearly ectopic catalase expression confers fitness advantages in the presence of stress, and therefore in this study we tested whether it enhances fitness in the absence of stress. We addressed this using a set of congenic barcoded C. albicans strains that include doxycycline-conditional tetON-CAT1 expressors. We show that high basal catalase levels, rather than CAT1 induction following stress imposition, reduce ROS accumulation and cell death, thereby promoting resistance to acute peroxide or combinatorial stress. This conclusion is reinforced by our analyses of phenotypically diverse clinical isolates and the impact of stochastic variation in catalase expression upon stress resistance in genetically homogeneous C. albicans populations. Accordingly, cat1Δ cells are more sensitive to neutrophil killing. However, we find that catalase inactivation does not attenuate C. albicans virulence in mouse or invertebrate models of systemic candidiasis. Furthermore, our direct comparisons of fitness in vitro using isogenic barcoded CAT1, cat1Δ and tetON-CAT1 strains show that, while ectopic catalase expression confers a fitness advantage during peroxide stress, it confers a fitness defect in the absence of stress. This fitness defect is suppressed by iron supplementation. Also high basal catalase levels induce key iron assimilatory functions (CFL5, FET3, FRP1, FTR1). We conclude that while high basal catalase levels enhance peroxide stress resistance, they place pressure on iron homeostasis through an elevated cellular demand for iron, thereby reducing the fitness of C. albicans in iron-limiting tissues within the host.

Occurrence of tetracycline resistance genes in aquaculture facilities with varying use of oxytetracycline

PubMed Central

Seyfried, Erin E.; Newton, Ryan J.; Rubert, Kennedy F.; Pedersen, Joel A.; McMahon, Katherine D.

2014-01-01

The contribution of human activities to environmental reservoirs of antibiotic resistance is poorly understood. The purpose of this study was to determine if oxytetracycline (OTC) use in aquaculture facilities increased the detection frequency (i.e. prevalence) of tetracycline resistance genes relative to facilities with no recent OTC treatment. We used PCR to screen water and sediment from four non-commercial fish farms in northwestern Wisconsin for the presence of ten tetracycline resistance determinants (tetR): tet(A), tet(B), tet(D), tet(E), tet(G), tet(M), tet(O), tet(Q), tet(S) and tet(W). Water from farms with recent OTC use had significantly higher tetR detection frequencies than did water from farms without recent OTC use, with prevalence in raceways and rearing ponds of farms with recent OTC use exceeded by more than two-fold that of farms not using OTC. Effluent from all farms, regardless of treatment regime, had higher tetR detection frequencies than their corresponding influent for all genes, but the specific combinations of tetR genes detected in a sample were not different from their corresponding influent. Although OTC use was associated with the increased occurrence and diversity of tetR genes in water samples, it was not found to relate to tetR gene occurrence in sediment samples. Sediment samples from facilities with no recent OTC use had significantly higher frequencies of tetR gene detection than did samples from facilities with recent OTC use. All of the tetR genes were detected in both the medicated and non-medicated feed samples analyzed in this study. These findings suggest that both OTC treatment in aquaculture facilities, and the farms themselves, may be sources of tetR gene introduction to the environment. To our knowledge, this is the first study to use genotypic and cultivation-independent methods to examine tetR gene occurrence associated with OTC use in aquaculture. PMID:20217406

The re-design at the transformer portion of transcutaneous energy transmission system for all implantable devices.

PubMed

Watada, Masaya; Saisho, Ryohei; Kim, Yong-Jae; Ohuchi, Katsuhiro; Takatani, Setsuo; Um, Yong-Su

2007-01-01

All implantable devices, such as an artificial heart, an artificial lung, a pacemaker, a defibrillator, need electric power. So the electric power supply through the skin is requested. Then, it is transcutaneous energy transmission system (TETS) that has been studied and used a lot. TETS is the system which performs an electric power supply by non-contact transcutaneously using the electromagnetic induction phenomenon of an external primary side coil and a secondary side coil in human body. In this research, we are developing the core type TETS which applied for the implantable devices. In this paper, corresponding to various conditions, such as a difference in required electric power and transmission distance change, the core type transformer which can hold high transmission efficiency is designed.

Triplet–triplet energy transfer in artificial and natural photosynthetic antennas

DOE PAGES

Ho, Junming; Kish, Elizabeth; Méndez-Hernandez, Dalvin D.; ...

2017-06-26

In photosynthetic organisms, protection against photo-oxidative stress due to singlet oxygen is provided by carotenoid molecules, which quench chlorophyll triplet species before they can sensitize singlet oxygen formation. In anoxygenic photosynthetic organisms, in which exposure to oxygen is low, chlorophyll to carotenoid triplet-triplet energy transfer (T-TET) is slow, in the tens of nanoseconds range, while it is ultrafast in the oxygen-rich chloroplasts of oxygen evolving photosynthetic organisms. In order to better understand the structural features and resulting electronic coupling that leads to T-TET dynamics adapted to ambient oxygen activity, we have carried out experimental and theoretical studies of two isomericmore » carotenoporphyrin molecular dyads having different conformations and therefore different interchromophore electronic interactions. This pair of dyads reproduces the characteristics of fast and slow T-TET including a resonance Raman based spectroscopic marker of strong electronic coupling and fast T-TET that has been observed in photosynthesis. As identified by DFT calculations, the spectroscopic marker associated with fast T-TET is due primarily to a geometrical perturbation of the carotenoid backbone in the triplet state induced by the interchromophore interaction. This is also the case for the natural systems, as demonstrated by the hybrid quantum mechanics/molecular mechanics (QM/MM) simulations of light harvesting proteins from oxygenic (LHCII) and anoxygenic organisms (LH2). In conclusion, both DFT and EPR analysis further indicates that upon T-TET, the triplet wave function is localized on the carotenoid in both dyads.« less

Triplet–triplet energy transfer in artificial and natural photosynthetic antennas

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ho, Junming; Kish, Elizabeth; Méndez-Hernandez, Dalvin D.

In photosynthetic organisms, protection against photo-oxidative stress due to singlet oxygen is provided by carotenoid molecules, which quench chlorophyll triplet species before they can sensitize singlet oxygen formation. In anoxygenic photosynthetic organisms, in which exposure to oxygen is low, chlorophyll to carotenoid triplet-triplet energy transfer (T-TET) is slow, in the tens of nanoseconds range, while it is ultrafast in the oxygen-rich chloroplasts of oxygen evolving photosynthetic organisms. In order to better understand the structural features and resulting electronic coupling that leads to T-TET dynamics adapted to ambient oxygen activity, we have carried out experimental and theoretical studies of two isomericmore » carotenoporphyrin molecular dyads having different conformations and therefore different interchromophore electronic interactions. This pair of dyads reproduces the characteristics of fast and slow T-TET including a resonance Raman based spectroscopic marker of strong electronic coupling and fast T-TET that has been observed in photosynthesis. As identified by DFT calculations, the spectroscopic marker associated with fast T-TET is due primarily to a geometrical perturbation of the carotenoid backbone in the triplet state induced by the interchromophore interaction. This is also the case for the natural systems, as demonstrated by the hybrid quantum mechanics/molecular mechanics (QM/MM) simulations of light harvesting proteins from oxygenic (LHCII) and anoxygenic organisms (LH2). In conclusion, both DFT and EPR analysis further indicates that upon T-TET, the triplet wave function is localized on the carotenoid in both dyads.« less

Triplet–triplet energy transfer in artificial and natural photosynthetic antennas

PubMed Central

Ho, Junming; Kish, Elizabeth; Méndez-Hernández, Dalvin D.; WongCarter, Katherine; Pillai, Smitha; Kodis, Gerdenis; Niklas, Jens; Poluektov, Oleg G.; Gust, Devens; Moore, Thomas A.; Moore, Ana L.; Batista, Victor S.

2017-01-01

In photosynthetic organisms, protection against photooxidative stress due to singlet oxygen is provided by carotenoid molecules, which quench chlorophyll triplet species before they can sensitize singlet oxygen formation. In anoxygenic photosynthetic organisms, in which exposure to oxygen is low, chlorophyll-to-carotenoid triplet–triplet energy transfer (T-TET) is slow, in the tens of nanoseconds range, whereas it is ultrafast in the oxygen-rich chloroplasts of oxygen-evolving photosynthetic organisms. To better understand the structural features and resulting electronic coupling that leads to T-TET dynamics adapted to ambient oxygen activity, we have carried out experimental and theoretical studies of two isomeric carotenoporphyrin molecular dyads having different conformations and therefore different interchromophore electronic interactions. This pair of dyads reproduces the characteristics of fast and slow T-TET, including a resonance Raman-based spectroscopic marker of strong electronic coupling and fast T-TET that has been observed in photosynthesis. As identified by density functional theory (DFT) calculations, the spectroscopic marker associated with fast T-TET is due primarily to a geometrical perturbation of the carotenoid backbone in the triplet state induced by the interchromophore interaction. This is also the case for the natural systems, as demonstrated by the hybrid quantum mechanics/molecular mechanics (QM/MM) simulations of light-harvesting proteins from oxygenic (LHCII) and anoxygenic organisms (LH2). Both DFT and electron paramagnetic resonance (EPR) analyses further indicate that, upon T-TET, the triplet wave function is localized on the carotenoid in both dyads. PMID:28652359

Nickel(ii) inhibits the oxidation of DNA 5-methylcytosine in mammalian somatic cells and embryonic stem cells.

PubMed

Yin, Ruichuan; Mo, Jiezhen; Dai, Jiayin; Wang, Hailin

2018-03-01

Nickel is found widely in the environment. It is an essential microelement but also toxic. However, nickel displays only weak genotoxicity and mutagenicity. Exploration of the epigenetic toxicity of nickel is extremely interesting. Iron(ii)- and 2-oxoglutarate-dependent Tet dioxygenases are a class of epigenetic enzymes that catalyze the oxidation of DNA 5-methylcytosine (5mC). Thus, they are critical for DNA demethylation and, importantly, are involved with nuclear reprogramming, embryonic development, and regulation of gene expression. Here, we demonstrated that nickel(ii) dramatically inhibits Tet proteins-mediated oxidation of DNA 5mC in cells ranging from somatic cell lines to embryonic stem cells, as manifested by the consistent observation of a significant decrease in 5-hydroxymethylcytosine, a critical intermediate resulting from the oxidation of 5mC. The inhibitory effects of nickel(ii) were concentration- and time-dependent. Using HEK293T cells overexpressing Tet proteins and ascorbic acid-stimulated Tet-proficient ES cells, we observed that nickel(ii) significantly reduced DNA demethylation at the global level. Interestingly, we also showed that nickel(ii) might affect the naïve or ground state of pluripotent embryonic stem cells. Here we show, for the first time, that nickel(ii) represses the oxidation of DNA 5mC and potentially alters the Tet proteins-regulated DNA methylation landscape in human cells. These findings provide new insights into the epigenetic toxicology of nickel.

Effects of advanced treatment systems on the removal of antibiotic resistance genes in wastewater treatment plants from Hangzhou, China.

PubMed

Chen, Hong; Zhang, Mingmei

2013-08-06

This study aimed at quantifying the concentration and removal of antibiotic resistance genes (ARGs) in three municipal wastewater treatment plants (WWTPs) employing different advanced treatment systems [biological aerated filter, constructed wetland, and ultraviolet (UV) disinfection]. The concentrations of tetM, tetO, tetQ, tetW, sulI, sulII, intI1, and 16S rDNA genes were examined in wastewater and biosolid samples. In municipal WWTPs, ARG reductions of 1-3 orders of magnitude were observed, and no difference was found among the three municipal WWTPs with different treatment processes (p > 0.05). In advanced treatment systems, 1-3 orders of magnitude of reductions in ARGs were observed in constructed wetlands, 0.6-1.2 orders of magnitude of reductions in ARGs were observed in the biological aerated filter, but no apparent decrease by UV disinfection was observed. A significant difference was found between constructed wetlands and biological filter (p < 0.05) and between constructed wetlands and UV disinfection (p < 0.05). In the constructed wetlands, significant correlations were observed in the removal of ARGs and 16S rDNA genes (R(2) = 0.391-0.866; p < 0.05). Constructed wetlands not only have the comparable ARG removal values with WWTP (p > 0.05) but also have the advantage in ARG relative abundance removal, and it should be given priority to be an advanced treatment system for further ARG attenuation from WWTP.

Cilostazol Upregulates Autophagy via SIRT1 Activation: Reducing Amyloid-β Peptide and APP-CTFβ Levels in Neuronal Cells.

PubMed

Lee, Hye Rin; Shin, Hwa Kyoung; Park, So Youn; Kim, Hye Young; Bae, Sun Sik; Lee, Won Suk; Rhim, Byung Yong; Hong, Ki Whan; Kim, Chi Dae

2015-01-01

Autophagy is a vital pathway for the removal of β-amyloid peptide (Aβ) and the aggregated proteins that cause Alzheimer's disease (AD). We previously found that cilostazol induced SIRT1 expression and its activity in neuronal cells, and thus, we hypothesized that cilostazol might stimulate clearances of Aβ and C-terminal APP fragment β subunit (APP-CTFβ) by up-regulating autophagy.When N2a cells were exposed to soluble Aβ1-42, protein levels of beclin-1, autophagy-related protein5 (Atg5), and SIRT1 decreased significantly. Pretreatment with cilostazol (10-30 μM) or resveratrol (20 μM) prevented these Aβ1-42 evoked suppressions. LC3-II (a marker of mammalian autophagy) levels were significantly increased by cilostazol, and this increase was reduced by 3-methyladenine. To evoke endogenous Aβ overproduction, N2aSwe cells (N2a cells stably expressing human APP containing the Swedish mutation) were cultured in medium with or without tetracycline (Tet+ for 48 h and then placed in Tet- condition). Aβ and APP-CTFβ expressions were increased after 12~24 h in Tet- condition, and these increased expressions were significantly reduced by pretreating cilostazol. Cilostazol-induced reductions in the expressions of Aβ and APP-CTFβ were blocked by bafilomycin A1 (a blocker of autophagosome to lysosome fusion). After knockdown of the SIRT1 gene (to ~40% in SIRT1 protein), cilostazol failed to elevate the expressions of beclin-1, Atg5, and LC3-II, indicating that cilostazol increases these expressions by up-regulating SIRT1. Further, decreased cell viability induced by Aβ was prevented by cilostazol, and this inhibition was reversed by 3-methyladenine, indicating that the protective effect of cilostazol against Aβ induced neurotoxicity is, in part, ascribable to the induction of autophagy. In conclusion, cilostazol modulates autophagy by increasing the activation of SIRT1, and thereby enhances Aβ clearance and increases cell viability.

Antibiotic resistant salmonella and Escherichia coli isolated from indigenous Gallus domesticus in Nairobi, Kenya.

PubMed

Wesonga, S M; Muluvi, G M; Okemo, P O; Kariuki, S

2010-05-01

To characterise and investigate antimicrobial resistance of Esherichia coli and salmonella strains isolated from indigenous Gallus gallus in a leading slaughterhouse/market outlet in Nairobi-Kenya. A repeated cross sectional study and based on random sampling was used. The study was carried out in a leading market outlet in Nairobi, Kenya. A hundred and four indigenous chicken rectal swabs were analysed, of which 67.3% were contaminated with Escherichia coli and 12.5% with Salmonella typhimurium. Seventy Escherichia coli isolates showed resistance phenotypes to one, two or more antibiotics. The most common antimicrobial resistance pattern was the single resistance to Tet (21.43%), followed by Amp Cot Tet (14%), Aug Amp Cot Tet (4.29%), Aug Amp Cot Tet Kan Chl (2.86%), Amp Cot Tet Chl, Cot Tet (2.86%) and Crx Amp Cot Tet Chl, Crx Amp Cot Chi, Amp Cot, Aug Amp, (1.43%) respectively. The highest rate of resistance was against Tet (55.7%), followed by Cot (40%). Third in line of resistance was Amp 32.86%, followed by Aug (11.43%), low or moderate resistance was against Chl (8.57%), Kan (4.29%), and Crx (2.86%) (P<0.0002). Salmonella typhimurium recovered displayed single resistance pattern to Tet (16.67%), Gen Cot Tet (8.33%), Amp Cot Tet (8.33%), Aug Amp Cot Tet (8.33%) and Amp Cot Tet Chl (16.67%). The highest resistance was against Tet (58.3%), Cot (41.7%), Amp (33.3%), Chl (16.7%), Aug and Gen (8.3%) respectively (P<0.0001). 3.0kb and 5.6kb plasmids isolated were not transferable by conjugation. Routine surveillance at slaughter/market outlets of Escherichia coli and Salmonella enterica should be done to identify infected flocks as a regulatory procedure for food safety and security programme.

Electrospun matrices for localised controlled drug delivery: release of tetracycline hydrochloride from layers of polycaprolactone and poly(ethylene-co-vinyl acetate).

PubMed

Alhusein, Nour; Blagbrough, Ian S; De Bank, Paul A

2012-12-01

We report the controlled release of tetracycline (Tet) HCl from a three-layered electrospun matrix for the first time. Five formulations of electrospun poly-ε-caprolactone (PCL) and poly(ethylene-co-vinyl acetate) (PEVA) have been designed, prepared as micro/nanofibre layers, and assayed for the controlled release of the clinically useful antibiotic Tet HCl with potential applications in wound healing and especially in complicated skin and skin-structure infections. Tet HCl was also chosen as a model drug possessing a good ultraviolet (UV) chromophore and capable of fluorescence together with limited stability. Tet HCl was successfully incorporated (essentially quantitatively at 3 %, w/w) and provided controlled release from multilayered electrospun matrices. The Tet HCl release test was carried out by a total immersion method on 2 × 2 cm(2) electrospun fibrous mats in Tris or phosphate-buffered saline heated to 37 °C. The formulation PCL/PEVA/PCL with Tet HCl in each layer gave a large initial (burst) release followed by a sustained release. Adding a third layer to the two-layered formulations led to release being sustained from 6 days to more than 15 days. There was no detectable loss of Tet chemical stability (as shown by UV and NMR) or bioactivity (as shown by a modified Kirby-Bauer disc assay). Using Tet HCl-sensitive bacteria, Staphylococcus aureus (ATCC 25923), the Tet HCl-loaded three-layered matrix formulations were still showing significantly higher antibacterial effects on days 4 and 5 than commercially available Antimicrobial Susceptibility Test Discs of Tet HCl. Electrospinning provides good encapsulation efficiency of Tet HCl within PCL/PEVA/PCL polymers in micro/nanofibre layers which display sustained antibiotic release.

Reversal of multidrug resistance in xenograft nude-mice by magnetic Fe(3)O(4) nanoparticles combined with daunorubicin and 5-bromotetrandrine.

PubMed

Wu, Ya-Nan; Chen, Bao-An; Cheng, Jian; Gao, Feng; Xu, Wen-Lin; Ding, Jia-Hua; Gao, Chong; Sun, Xin-Chen; Li, Guo-Hong; Chen, Wen-Ji; Liu, Li-Jie; Li, Xiao-Mao; Wang, Xue-Mei

2009-02-01

This study was aimed to investigate the reversal effect of 5-bromotetrandrine (5-BrTet) and magnetic nanoparticle of Fe(3)O(4) (Fe(3)O(4)-MNPs) combined with DNR in vivo. The xenograft leukemia model with stable multiple drug resistance in nude mice was established. The two sub-clones of K562 and K562/A02 cells were respectively inoculated subcutaneously into back of athymic nude mice (1 x 10(7) cells/each) to establish the leukemia xenograft models. Drug resistant and the sensitive tumor-bearing nude mice were both assigned randomly into 5 groups: group A was treated with NS; group B was treated with DNR; group C was treated with nanoparticle of Fe(3)O(4) combined with DNR; group D was treated with 5-BrTet combined with DNR; group E was treated with 5-bromotetrandrine and magnetic nanoparticle of Fe(3)O(4) combined with DNR. The incidence of tumor formation, growth characteristics, weight and volume of tumor were observed. The histopathologic examination of tumors and organs were carried out. The protein levels of BCL-2, BAX, and Caspase-3 in resistant tumors were detected by Western blot. The results indicated that 5-BrTet and magnetic nanoparticle of Fe(3)O(4) combined with DNR significantly suppressed growth of K562/A02 cell xenograft tumor, histopathologic examination of tumors showed the tumors necrosis obviously. Application of 5-BrTet and magnetic nanoparticle of Fe(3)O(4) inhibited the expression of BCL-2 protein and up-regulated the expression of BAX, and Caspase-3 protein in K562/A02 cell xenograft tumor. It is concluded that 5-bromotetrandrine and magnetic nanoparticle of Fe(3)O(4) combined with DNR have significant tumor-suppressing effect on MDR leukemia cell xenograft model.

Assessment of phenotypic and genotypic antibiotic susceptibility of vaginal Lactobacillus sp.

PubMed

Štšepetova, J; Taelma, H; Smidt, I; Hütt, P; Lapp, E; Aotäht, E; Mändar, R

2017-08-01

To assess antibiotic susceptibility of vaginal lactobacilli strains and provide the data required for assessing the potential of antibiotic resistance risk of new strains selected as probiotic. Potential probiotic vaginal lactobacilli used in the study included 31 vaginal strains of Lactobacillus crispatus (n = 27), Lactobacillus gasseri (n = 3) and Lactobacillus jensenii (n = 1) obtained from the collection of Competence Centre on Health Technologies. Two commercial probiotic strains were used as controls (Lactobacillus rhamnosus GR-1 and Lactobacillus fermentum RC-14). The phenotypic and genotypic antibiotic resistances of the strains were determined by E-test and PCR methods. The location (chromosomal DNA or plasmid) of antibiotic resistance genes was also detected. All lactobacilli strains expressed high level of resistance to kanamycin, metronidazole, norfloxacin and trimethoprim/sulphamethoxazole. Some of the strains also expressed resistance to other antibiotics (chloramphenicol, vancomycin) indicating acquired resistance. I class integrons were found in 20% (6/31) of the strains. The RPP (ribosomal protection protein) gene was found to be positive in 30% (9/31) of the strains. Only one L. jensenii strain was determined with tet(M) gene. The tet(K) gene was positive in 26·7% (8/31) and erm(B) gene in 43·3% (13/31) of strains. Three RPP and both four tet(K) and erm(B) genes were located in plasmids. High antibiotic resistance to clinically important antibiotics was demonstrated, including metronidazole, sulphonamides, aminoglycoside and quinolones. In addition, acquired tetracycline and erythromycin resistance genes were detected in either plasmid or chromosomal DNA of certain isolates, in some of the cases for the first time in the literature. It appears that antibiotic resistance genes erm(B) and tet(K) are widely spread in vaginal lactobacilli. This study provides new data about antimicrobial resistance and genotypic diversity of vaginal Lactobacillus isolates. In addition, it provides data assessing the potential of antibiotic resistance risk of new strains selected as probiotic. © 2017 The Society for Applied Microbiology.

Oxidative Stress Induced Inflammation Initiates Functional Decline of Tear Production

PubMed Central

Uchino, Yuichi; Kawakita, Tetsuya; Miyazawa, Masaki; Ishii, Takamasa; Onouchi, Hiromi; Yasuda, Kayo; Ogawa, Yoko; Shimmura, Shigeto; Ishii, Naoaki; Tsubota, Kazuo

2012-01-01

Oxidative damage and inflammation are proposed to be involved in an age-related functional decline of exocrine glands. However, the molecular mechanism of how oxidative stress affects the secretory function of exocrine glands is unclear. We developed a novel mev-1 conditional transgenic mouse model (Tet-mev-1) using a modified tetracycline system (Tet-On/Off system). This mouse model demonstrated decreased tear production with morphological changes including leukocytic infiltration and fibrosis. We found that the mev-1 gene encodes Cyt-1, which is the cytochrome b560 large subunit of succinate-ubiquinone oxidoreductase in complex II of mitochondria (homologous to succinate dehydrogenase C subunit (SDHC) in humans). The mev-1 gene induced excessive oxidative stress associated with ocular surface epithelial damage and a decrease in protein and aqueous secretory function. This new model provides evidence that mitochondrial oxidative damage in the lacrimal gland induces lacrimal dysfunction resulting in dry eye disease. Tear volume in Tet-mev-1 mice was lower than in wild type mice and histopathological analyses showed the hallmarks of lacrimal gland inflammation by intense mononuclear leukocytic infiltration and fibrosis in the lacrimal gland of Tet-mev-1 mice. These findings strongly suggest that oxidative stress can be a causative factor for the development of dry eye disease. PMID:23071526

Muddying the Waters: A New Area of Concern for Drinking Water Contamination in Cameroon

PubMed Central

Healy Profitós, Jessica M.; Mouhaman, Arabi; Lee, Seungjun; Garabed, Rebecca; Moritz, Mark; Piperata, Barbara; Tien, Joe; Bisesi, Michael; Lee, Jiyoung

2014-01-01

In urban Maroua, Cameroon, improved drinking water sources are available to a large majority of the population, yet this water is frequently distributed through informal distribution systems and stored in home containers (canaries), leaving it vulnerable to contamination. We assessed where contamination occurs within the distribution system, determined potential sources of environmental contamination, and investigated potential pathogens. Gastrointestinal health status (785 individuals) was collected via health surveys. Drinking water samples were collected from drinking water sources and canaries. Escherichia coli and total coliform levels were evaluated and molecular detection was performed to measure human-associated faecal marker, HF183; tetracycline-resistance gene, tetQ; Campylobacter spp.; and Staphylococcus aureus. Statistical analyses were performed to evaluate the relationship between microbial contamination and gastrointestinal illness. Canari samples had higher levels of contamination than source samples. HF183 and tetQ were detected in home and source samples. An inverse relationship was found between tetQ and E. coli. Presence of tetQ with lower E. coli levels increased the odds of reported diarrhoeal illness than E. coli levels alone. Further work is warranted to better assess the relationship between antimicrobial-resistant bacteria and other pathogens in micro-ecosystems within canaries and this relationship’s impact on drinking water quality. PMID:25464137

Muddying the waters: a new area of concern for drinking water contamination in Cameroon.

PubMed

Profitós, Jessica M Healy; Mouhaman, Arabi; Lee, Seungjun; Garabed, Rebecca; Moritz, Mark; Piperata, Barbara; Tien, Joe; Bisesi, Michael; Lee, Jiyoung

2014-11-28

In urban Maroua, Cameroon, improved drinking water sources are available to a large majority of the population, yet this water is frequently distributed through informal distribution systems and stored in home containers (canaries), leaving it vulnerable to contamination. We assessed where contamination occurs within the distribution system, determined potential sources of environmental contamination, and investigated potential pathogens. Gastrointestinal health status (785 individuals) was collected via health surveys. Drinking water samples were collected from drinking water sources and canaries. Escherichia coli and total coliform levels were evaluated and molecular detection was performed to measure human-associated faecal marker, HF183; tetracycline-resistance gene, tetQ; Campylobacter spp.; and Staphylococcus aureus. Statistical analyses were performed to evaluate the relationship between microbial contamination and gastrointestinal illness. Canari samples had higher levels of contamination than source samples. HF183 and tetQ were detected in home and source samples. An inverse relationship was found between tetQ and E. coli. Presence of tetQ with lower E. coli levels increased the odds of reported diarrhoeal illness than E. coli levels alone. Further work is warranted to better assess the relationship between antimicrobial-resistant bacteria and other pathogens in micro-ecosystems within canaries and this relationship's impact on drinking water quality.

Consecutive epigenetically-active agent combinations act in ID1-RUNX3-TET2 and HOXA pathways for Flt3ITD+ve AML.

PubMed

Sayar, Hamid; Liu, Yan; Gao, Rui; Zaid, Mohammad Abu; Cripe, Larry D; Weisenbach, Jill; Sargent, Katie J; Nassiri, Mehdi; Li, Lang; Konig, Heiko; Suvannasankha, Attaya; Pan, Feng; Shanmugam, Rajasubramaniam; Goswami, Chirayu; Kapur, Reuben; Xu, Mingjiang; Boswell, H Scott

2018-01-19

Co-occurrence of Flt3ITD and TET2 mutations provoke an animal model of AML by epigenetic repression of Wnt pathway antagonists, including RUNX3, and by hyperexpression of ID1, encoding Wnt agonist. These affect HOXA over-expression and treatment resistance. A comparable epigenetic phenotype was identified among adult AML patients needing novel intervention. We chose combinations of targeted agents acting on distinct effectors, at the levels of both signal transduction and chromatin remodeling, in relapsed/refractory AML's, including Flt3ITD+ve, described with a signature of repressed tumor suppressor genes, involving Wnt antagonist RUNX3 , occurring along with ID1 and HOXA over-expressions. We tracked patient response to combination of Flt3/Raf inhibitor, Sorafenib, and Vorinostat, pan-histone deacetylase inhibitor, without or with added Bortezomib, in consecutive phase I trials. A striking association of rapid objective remissions (near-complete, complete responses) was noted to accompany induced early pharmacodynamic changes within patient blasts in situ, involving these effectors, significantly linking RUNX3 /Wnt antagonist de-repression (80%) and ID1 downregulation (85%), to a response, also preceded by profound HOXA9 repression. Response occurred in context of concurrent TET2 mutation/hypomorphy and Flt3ITD+ve mutation (83% of complete responses). Addition of Bortezomib to the combination was vital to attainment of complete response in Flt3ITD+ve cases exhibiting such Wnt pathway dysregulation.

Establishment of the Inducible Tet-On System for the Activation of the Silent Trichosetin Gene Cluster in Fusarium fujikuroi

PubMed Central

Janevska, Slavica; Arndt, Birgit; Baumann, Leonie; Apken, Lisa Helene; Mauriz Marques, Lucas Maciel; Humpf, Hans-Ulrich; Tudzynski, Bettina

2017-01-01

The PKS-NRPS-derived tetramic acid equisetin and its N-desmethyl derivative trichosetin exhibit remarkable biological activities against a variety of organisms, including plants and bacteria, e.g., Staphylococcus aureus. The equisetin biosynthetic gene cluster was first described in Fusarium heterosporum, a species distantly related to the notorious rice pathogen Fusarium fujikuroi. Here we present the activation and characterization of a homologous, but silent, gene cluster in F. fujikuroi. Bioinformatic analysis revealed that this cluster does not contain the equisetin N-methyltransferase gene eqxD and consequently, trichosetin was isolated as final product. The adaption of the inducible, tetracycline-dependent Tet-on promoter system from Aspergillus niger achieved a controlled overproduction of this toxic metabolite and a functional characterization of each cluster gene in F. fujikuroi. Overexpression of one of the two cluster-specific transcription factor (TF) genes, TF22, led to an activation of the three biosynthetic cluster genes, including the PKS-NRPS key gene. In contrast, overexpression of TF23, encoding a second Zn(II)2Cys6 TF, did not activate adjacent cluster genes. Instead, TF23 was induced by the final product trichosetin and was required for expression of the transporter-encoding gene MFS-T. TF23 and MFS-T likely act in consort and contribute to detoxification of trichosetin and therefore, self-protection of the producing fungus. PMID:28379186

Establishment of the Inducible Tet-On System for the Activation of the Silent Trichosetin Gene Cluster in Fusarium fujikuroi.

PubMed

Janevska, Slavica; Arndt, Birgit; Baumann, Leonie; Apken, Lisa Helene; Mauriz Marques, Lucas Maciel; Humpf, Hans-Ulrich; Tudzynski, Bettina

2017-04-05

The PKS-NRPS-derived tetramic acid equisetin and its N -desmethyl derivative trichosetin exhibit remarkable biological activities against a variety of organisms, including plants and bacteria, e.g., Staphylococcus aureus . The equisetin biosynthetic gene cluster was first described in Fusarium heterosporum , a species distantly related to the notorious rice pathogen Fusarium fujikuroi . Here we present the activation and characterization of a homologous, but silent, gene cluster in F. fujikuroi . Bioinformatic analysis revealed that this cluster does not contain the equisetin N -methyltransferase gene eqxD and consequently, trichosetin was isolated as final product. The adaption of the inducible, tetracycline-dependent Tet-on promoter system from Aspergillus niger achieved a controlled overproduction of this toxic metabolite and a functional characterization of each cluster gene in F. fujikuroi . Overexpression of one of the two cluster-specific transcription factor (TF) genes, TF22 , led to an activation of the three biosynthetic cluster genes, including the PKS-NRPS key gene. In contrast, overexpression of TF23 , encoding a second Zn(II)₂Cys₆ TF, did not activate adjacent cluster genes. Instead, TF23 was induced by the final product trichosetin and was required for expression of the transporter-encoding gene MFS-T . TF23 and MFS-T likely act in consort and contribute to detoxification of trichosetin and therefore, self-protection of the producing fungus.

Effects of chlortetracycline and copper supplementation on the prevalence, distribution, and quantity of antimicrobial resistance genes in the fecal metagenome of weaned pigs.

PubMed

Agga, Getahun E; Scott, H Morgan; Vinasco, Javier; Nagaraja, T G; Amachawadi, Raghavendra G; Bai, Jianfa; Norby, Bo; Renter, David G; Dritz, Steve S; Nelssen, Jim L; Tokach, Mike D

2015-05-01

Use of in-feed antibiotics such as chlortetracycline (CTC) in food animals is fiercely debated as a cause of antimicrobial resistance in human pathogens; as a result, alternatives to antibiotics such as heavy metals have been proposed. We used a total community DNA approach to experimentally investigate the effects of CTC and copper supplementation on the presence and quantity of antimicrobial resistance elements in the gut microbial ecology of pigs. Total community DNA was extracted from 569 fecal samples collected weekly over a 6-week period from groups of 5 pigs housed in 32 pens that were randomized to receive either control, CTC, copper, or copper plus CTC regimens. Qualitative and quantitative PCR were used to detect the presence of 14 tetracycline resistance (tet) genes and to quantify gene copies of tetA, tetB, blaCMY-2 (a 3rd generation cephalosporin resistance gene), and pcoD (a copper resistance gene), respectively. The detection of tetA and tetB decreased over the subsequent sampling periods, whereas the prevalence of tetC and tetP increased. CTC and copper plus CTC supplementation increased both the prevalence and gene copy numbers of tetA, while decreasing both the prevalence and gene copies of tetB. In summary, tet gene presence was initially very diverse in the gut bacterial community of weaned pigs; thereafter, copper and CTC supplementation differentially impacted the prevalence and quantity of the various tetracycline, ceftiofur and copper resistance genes resulting in a less diverse gene population. Published by Elsevier B.V.

Overexpression of neurofilament H disrupts normal cell structure and function

NASA Technical Reports Server (NTRS)

Szebenyi, Gyorgyi; Smith, George M.; Li, Ping; Brady, Scott T.

2002-01-01

Studying exogenously expressed tagged proteins in live cells has become a standard technique for evaluating protein distribution and function. Typically, expression levels of experimentally introduced proteins are not regulated, and high levels are often preferred to facilitate detection. However, overexpression of many proteins leads to mislocalization and pathologies. Therefore, for normative studies, moderate levels of expression may be more suitable. To understand better the dynamics of intermediate filament formation, transport, and stability in a healthy, living cell, we inserted neurofilament heavy chain (NFH)-green fluorescent protein (GFP) fusion constructs in adenoviral vectors with tetracycline (tet)-regulated promoters. This system allows for turning on or off the synthesis of NFH-GFP at a selected time, for a defined period, in a dose-dependent manner. We used this inducible system for live cell imaging of changes in filament structure and cell shape, motility, and transport associated with increasing NFH-GFP expression. Cells with low to intermediate levels of NFH-GFP were structurally and functionally similar to neighboring, nonexpressing cells. In contrast, overexpression led to pathological alterations in both filament organization and cell function. Copyright 2002 Wiley-Liss, Inc.

The nucleolar protein SURF-6 is essential for viability in mouse NIH/3T3 cells.

PubMed

Polzikov, Mikhail; Magoulas, Charalambos; Zatsepina, Olga

2007-09-01

SURF-6 is a bona fide nucleolar protein comprising an evolutionary conserved family that extends from human to yeast. The expression of the mammalian SURF-6 has been recently found to be regulated during the cell cycle. In order to determine the importance of SURF-6 in mammalian cells, we applied the Tet-On system to regulate conditionally, in response to tetracycline, the expression of an antisense RNA (asRNA) that targets Surf-6 mRNA in mouse NIH/3T3 cells. Induced Surf-6 asRNA caused an effective depletion of SURF-6 protein resulted in cell death and in an apparent arrest in the G1 phase of the cell cycle. These results provide for the first time evidence that expression of SURF-6 is essential for mammalian cell viability, and suggest that SURF-6 might participate in the progression of cell cycle.

CD52, CD22, CD26, EG5 and IGF-1R expression in thymic malignancies.

PubMed

Remon, J; Abedallaa, N; Taranchon-Clermont, E; Bluthgen, V; Lindsay, C R; Besse, B; Thomas de Montpréville, V

2017-06-01

Thymic epithelial tumours are rare cancers for which new treatment options are required. Identification of putative predictive markers is important for developing clinical trials. We studied the expression of five putative predictive biomarkers, potentially actionable by approved experimental drugs. CD52, CD22, CD26, EG5, and IGF-1R expression were investigated by immunohistochemistry in formalin-fixed surgical samples of thymic epithelial tumour patients. All samples containing 10% positive epithelial tumour cells, independent of tumour cell intensity, were considered as positive. Correlation with histological subtype was performed. 106 surgical samples (89 thymomas, 12 thymic carcinoma, and 5 thymic neuroendocrine tumours) were evaluated. Overall, CD52, CD22, CD26, EG5 and IGF-1R expression was observed in 7%, 42%, 25%, 42% and 77% of samples, respectively. CD52 expression was more frequent in B2 and B3 thymoma. All TET subtypes stained for CD22, mainly AB thymoma (68%). CD26 expression also correlated with AB thymoma (68%), and A thymoma (50%) subtype, while IGFR1 was the most common marker expressed by thymic carcinoma samples (92%), followed by EG5 (60%). Only EG5 expression was significantly higher in thymic carcinomas than in thymomas (75% vs. 38%, p=0.026). Our data were consistent with a previous study of IGF-1R expression. Based on their expression, activity of agents targeting CD52, CD 22, CD26 and EG5 could be further explored in TET patients. Copyright © 2017 Elsevier B.V. All rights reserved.

Endothelial cells actively concentrate insulin during its transendothelial transport

PubMed Central

Genders, Amanda J.; Frison, Vera; Abramson, Sarah R.; Barrett, Eugene J.

2013-01-01

We examined insulin's uptake and transendothelial transport (TET) by cultured bovine aortic endothelial cells (BAECs) in order to: a) ascertain whether insulin accumulates within the cells to concentrations greater than in the media; b) compare the TET of insulin to that of inulin (using the latter as a tracer for passive transport or leak); and c) determine whether insulin's TET depended on insulin action. Using 125I-insulin at physiologic concentrations, we found that BAECs accumulate insulin >5-fold above media concentrations and that the TET of insulin, but not inulin, is saturable and requires intact PI-3-kinase and MEK-kinase signaling. We conclude that the insulin receptor and downstream signaling from the receptor regulate endothelial insulin transport. Based on comparison of the kinetics of BAEC insulin uptake with insulin TET, we suggest that insulin uptake is rate limiting for insulin TET. PMID:23350546

Dependence of regenerated sensory axons on continuous neurotrophin-3 delivery.

PubMed

Hou, Shaoping; Nicholson, LaShae; van Niekerk, Erna; Motsch, Melanie; Blesch, Armin

2012-09-19

Previous studies have shown that injured dorsal column sensory axons extend across a spinal cord lesion site if axons are guided by a gradient of neurotrophin-3 (NT-3) rostral to the lesion. Here we examined whether continuous NT-3 delivery is necessary to sustain regenerated axons in the injured spinal cord. Using tetracycline-regulated (tet-off) lentiviral gene delivery, NT-3 expression was tightly controlled by doxycycline administration. To examine axon growth responses to regulated NT-3 expression, adult rats underwent a C3 dorsal funiculus lesion. The lesion site was filled with bone marrow stromal cells, tet-off-NT-3 virus was injected rostral to the lesion site, and the intrinsic growth capacity of sensory neurons was activated by a conditioning lesion. When NT-3 gene expression was turned on, cholera toxin β-subunit-labeled sensory axons regenerated into and beyond the lesion/graft site. Surprisingly, the number of regenerated axons significantly declined when NT-3 expression was turned off, whereas continued NT-3 expression sustained regenerated axons. Quantification of axon numbers beyond the lesion demonstrated a significant decline of axon growth in animals with transient NT-3 expression, only some axons that had regenerated over longer distance were sustained. Regenerated axons were located in white matter and did not form axodendritic synapses but expressed presynaptic markers when closely associated with NG2-labeled cells. A decline in axon density was also observed within cellular grafts after NT-3 expression was turned off possibly via reduction in L1 and laminin expression in Schwann cells. Thus, multiple mechanisms underlie the inability of transient NT-3 expression to fully sustain regenerated sensory axons.

Influence of tetracycline on tetracycline-resistant heterotrophs and tet genes in activated sludge process.

PubMed

Yu, Jie; Liu, Dongfang; Li, Kexun

2015-03-01

The concentrations of tetracycline-intermediate resistant, tetracycline-resistant heterotrophic bacteria, and total heterotrophic bacteria were examined to assess the influence of tetracycline on tetracycline-resistant heterotrophs by the R2A agar cultivation method in the tetracycline fortified activated sludge process and in the natural background. Results showed that the percentages of both tetracycline-intermediate resistant and tetracycline-resistant heterotrophic bacteria in total heterotrophic bacteria were significantly increased, after tetracycline was fed to activated sludge for a 3 months period under four different operating conditions, as compared with the background. In order to investigate the mechanism of activated sludge resistance to tetracycline, polymerase chain reaction experiments were carried out to analyze the existence and evolution of tet genes in the presence of tetracycline. Results revealed that only tet A and tet B genes out of the 11 target tet genes were observed in tetracycline treated activated sludge while no tet gene was detected in background. This indicated that tet A gene could accumulate in activated sludge with slower and continuous influent, while the accumulation of tet B gene could be attributed to shorter hydraulic retention time. Therefore, it was proposed in this study that tetracycline-resistant genes created by efflux pumps spread earlier and quicker to encode resistance to tetracycline, which facilitated the increase in tetracycline-resistance.

Effects of oxytetracycline on archaeal community, and tetracycline resistance genes in anaerobic co-digestion of pig manure and wheat straw.

PubMed

Wang, Xiaojuan; Pan, Hongjia; Gu, Jie; Qian, Xun; Gao, Hua; Qin, Qingjun

2016-12-01

In this study, the effects of different concentrations of oxytetracycline (OTC) on biogas production, archaeal community structure, and the levels of tetracycline resistance genes (TRGs) were investigated in the anaerobic co-digestion products of pig manure and wheat straw. PCR denaturing gradient gel electrophoresis analysis and real-time quantitative polymerase chain reaction (RT-qPCR) (PCR) were used to detect the archaeal community structure and the levels of four TRGs: tet(M), tet(Q), tet(W), and tet(C). The results showed that anaerobic co-digestion with OTC at concentrations of 60, 100, and 140 mg/kg (dry weight of pig manure) reduced the cumulative biogas production levels by 9.9%, 10.4%, and 14.1%, respectively, compared with that produced by the control, which lacked the antibiotic. The addition of OTC substantially modified the structure of the archaeal community. Two orders were identified by phylogenetic analysis, that is, Pseudomonadales and Methanomicrobiales, and the methanogen present during anaerobic co-digestion with OTC may have been resistant to OTC. The abundances of tet(Q) and tet(W) genes increased as the OTC concentration increased, whereas the abundances of tet(M) and tet(C) genes decreased as the OTC concentration increased.

TET-1- A German Microsatellite for Technology On -Orbit Verification

NASA Astrophysics Data System (ADS)

Föckersperger, S.; Lattner, K.; Kaiser, C.; Eckert, S.; Bärwald, W.; Ritzmann, S.; Mühlbauer, P.; Turk, M.; Willemsen, P.

2008-08-01

Due to the high safety standards in the space industry every new product must go through a verification process before qualifying for operation in a space system. Within the verification process the payload undergoes a series of tests which prove that it is in accordance with mission requirements in terms of function, reliability and safety. Important verification components are the qualification for use on the ground as well as the On-Orbit Verification (OOV), i.e. proof that the product is suitable for use under virtual space conditions (on-orbit). Here it is demonstrated that the product functions under conditions which cannot or can only be partially simulated on the ground. The OOV-Program of the DLR serves to bridge the gap between the product tested and qualified on the ground and the utilization of the product in space. Due to regular and short-term availability of flight opportunities industry and research facilities can verify their latest products under space conditions and demonstrate their reliability and marketability. The Technologie-Erprobungs-Tr&äger TET (Technology Experiments Carrier) comprises the core elements of the OOV Program. A programmatic requirement of the OOV Program is that a satellite bus already verified in orbit be used in the first segment of the program. An analysis of suitable satellite buses showed that a realization of the TET satellite bus based on the BIRD satellite bus fulfilled the programmatic requirements best. Kayser-Threde was selected by DLR as Prime Contractor to perform the project together with its major subcontractors Astro- und Feinwerktechnik, Berlin for the platform development and DLR-GSOC for the ground segment development. TET is now designed to be a modular and flexible micro-satellite for any orbit between 450 and 850 km altitude and inclination between 53° and SSO. With an overall mass of 120 kg TET is able to accommodate experiments of up to 50 kg. A multipurpose payload supply systemThere is significant confusion in the space industry today over the terms used to describe satellite bus architectures. Terms such as "standard bus" (or "common bus"), "modular bus" and "plug-and-play bus" are often used with little understanding of what the terms actually mean, and even less understanding of what the differences in these space architectures mean. It may seem that these terms are subtle differentiators, but in reality these terms describe radically different ways to design, build, test, and operate satellites. Furthermore, these terms imply very different business models for the acquisition, operation, and sustainment of space systems. This paper will define and describe the difference between "standard buses", "modular buses" and "plug-and-play buses"; giving examples of each kind with a cost/benefit discussion of each type. under Kayser-Threde responsibility provides the necessary interfaces to the experiments. The first TET mission is scheduled for mid of 2010. TET will be launched as piggy-back payload on any available launcher worldwide to reduce launch cost and provide maximum flexibility. Finally, TET will provide all services required by the experimenters for a one year mission operation to perform a successful OOV-mission with its technology experiments leading to an efficient access to space for German industry and institutions.

High yield cell-free production of integral membrane proteins without refolding or detergents.

PubMed

Wuu, Jessica J; Swartz, James R

2008-05-01

Integral membrane proteins act as critical cellular components and are important drug targets. However, difficulties in producing membrane proteins have hampered investigations of structure and function. In vivo production systems are often limited by cell toxicity, and previous in vitro approaches have required unnatural folding pathways using detergents or lipid solutions. To overcome these limitations, we present an improved cell-free expression system which produces high yields of integral membrane proteins without the use of detergents or refolding steps. Our cell-free reaction activates an Escherichia coli-derived cell extract for transcription and translation. Purified E. coli inner membrane vesicles supply membrane-bound components and the lipid environment required for insertion and folding. Using this system, we demonstrated successful synthesis of two complex integral membrane transporters, the tetracycline pump (TetA) and mannitol permease (MtlA), in yields of 570+/-50 microg/mL and 130+/-30 microg/mL of vesicle-associated protein, respectively. These yields are up to 400 times typical in vivo concentrations. Insertion and folding of these proteins are verified by sucrose flotation, protease digestion, and activity assays. Whereas TetA incorporates efficiently into vesicle membranes with over two-thirds of the synthesized protein being inserted, MtlA yields appear to be limited by insufficient concentrations of a membrane-associated chaperone.

Prevalence of sulfonamide and tetracycline resistance genes in drinking water treatment plants in the Yangtze River Delta, China.

PubMed

Guo, Xueping; Li, Jing; Yang, Fan; Yang, Jie; Yin, Daqiang

2014-09-15

The occurrence and distribution of antibiotic resistance genes (ARGs) in drinking water treatment plants (DWTPs) and finished water are not well understood, and even less is known about the contribution of each treatment process to resistance gene reduction. The prevalence of ten commonly detected sulfonamide and tetracycline resistance genes, namely, sul I, sul II, tet(C), tet(G), tet(X), tet(A), tet(B), tet(O), tet(M) and tet(W) as well as 16S-rRNA genes, were surveyed in seven DWTPs in the Yangtze River Delta, China, with SYBR Green I-based real-time quantitative polymerase chain reaction. All of the investigated ARGs were detected in the source waters of the seven DWTPs, and sul I, sul II, tet(C) and tet(G) were the four most abundant ARGs. Total concentrations of ARGs belonging to either the sulfonamide or tetracycline resistance gene class were above 10(5) copies/mL. The effects of a treatment process on ARG removal varied depending on the overall treatment scheme of the DWTP. With combinations of the treatment procedures, however, the copy numbers of resistance genes were reduced effectively, but the proportions of ARGs to bacteria numbers increased in several cases. Among the treatment processes, the biological treatment tanks might serve as reservoirs of ARGs. ARGs were found in finished water of two plants, imposing a potential risk to human health. The results presented in this study not only provide information for the management of antibiotics and ARGs but also facilitate improvement of drinking water quality. Copyright © 2014 Elsevier B.V. All rights reserved.

How Different Are Our Perceptions of Equal-Tempered and Microtonal Intervals? A Behavioural and EEG Survey.

PubMed

Bailes, Freya; Dean, Roger T; Broughton, Mary C

2015-01-01

For listeners familiar with Western twelve-tone equal-tempered (12-TET) music, a novel microtonal tuning system is expected to present additional processing challenges. We aimed to determine whether this was the case, focusing on the extent to which our perceptions can be considered bottom-up (psychoacoustic and primarily perceptual) and top-down (dependent on familiarity and cognitive processing). We elicited both overt response ratings, and covert event-related potentials (ERPs), so as to compare subjective impressions of sounds with the neurophysiological processing of the acoustic signal. We hypothesised that microtonal intervals are perceived differently from 12-TET intervals, and that the responses of musicians (n = 10) and non-musicians (n = 10) are distinct. Two-note chords were presented comprising 12-TET intervals (consonant and dissonant) or microtonal (quarter tone) intervals, and ERP, subjective roughness ratings, and liking ratings were recorded successively. Musical experience mediated the perception of differences between dissonant and microtone intervals, with non-musicians giving similar ratings for each, and musicians preferring dissonant over the less commonly used microtonal intervals, rating them as less rough. ERP response amplitude was greater for consonant intervals than other intervals. Musical experience interacted with interval type, suggesting that musical expertise facilitates the sensory and perceptual discrimination of microtonal intervals from 12-TET intervals, and an increased ability to categorize such intervals. Non-musicians appear to have perceived microtonal intervals as instances of neighbouring 12-TET intervals.

Thermal Destruction of TETS: Experiments and Modeling ...

EPA Pesticide Factsheets

Symposium Paper In the event of a contamination event involving chemical warfare agents (CWAs) or toxic industrial chemicals (TICs), large quantities of potentially contaminated materials, both indoor and outdoor, may be treated with thermal incineration during the site remediation process. Even if the CWAs or TICs of interest are not particularly thermally stable and might be expected to decompose readily in a high temperature combustion environment, the refractory nature of many materials found inside and outside buildings may present heat transfer challenges in an incineration system depending on how the materials are packaged and fed into the incinerator. This paper reports on a study to examine the thermal decomposition of a banned rodenticide, tetramethylene disulfotetramine (TETS) in a laboratory reactor, analysis of the results using classical reactor design theory, and subsequent scale-up of the results to a computer-simulation of a full-scale commercial hazardous waste incinerator processing ceiling tile contaminated with residual TETS.

MeCP2 regulates Tet1-catalyzed demethylation, CTCF binding, and learning-dependent alternative splicing of the BDNF gene in Turtle

PubMed Central

Zheng, Zhaoqing; Ambigapathy, Ganesh; Keifer, Joyce

2017-01-01

MECP2 mutations underlying Rett syndrome cause widespread misregulation of gene expression. Functions for MeCP2 other than transcriptional are not well understood. In an ex vivo brain preparation from the pond turtle Trachemys scripta elegans, an intraexonic splicing event in the brain-derived neurotrophic factor (BDNF) gene generates a truncated mRNA transcript in naïve brain that is suppressed upon classical conditioning. MeCP2 and its partners, splicing factor Y-box binding protein 1 (YB-1) and methylcytosine dioxygenase 1 (Tet1), bind to BDNF chromatin in naïve but dissociate during conditioning; the dissociation correlating with decreased DNA methylation. Surprisingly, conditioning results in new occupancy of BDNF chromatin by DNA insulator protein CCCTC-binding factor (CTCF), which is associated with suppression of splicing in conditioning. Knockdown of MeCP2 shows it is instrumental for splicing and inhibits Tet1 and CTCF binding thereby negatively impacting DNA methylation and conditioning-dependent splicing regulation. Thus, mutations in MECP2 can have secondary effects on DNA methylation and alternative splicing. DOI: http://dx.doi.org/10.7554/eLife.25384.001 PMID:28594324

Molecular Characterization of tet(M) Genes in Lactobacillus Isolates from Different Types of Fermented Dry Sausage

PubMed Central

Gevers, Dirk; Danielsen, Morten; Huys, Geert; Swings, Jean

2003-01-01

The likelihood that products prepared from raw meat and milk may act as vehicles for antibiotic-resistant bacteria is currently of great concern in food safety issues. In this study, a collection of 94 tetracycline-resistant (Tcr) lactic acid bacteria recovered from nine different fermented dry sausage types were subjected to a polyphasic molecular study with the aim of characterizing the host organisms and the tet genes, conferring tetracycline resistance, that they carry. With the (GTG)5-PCR DNA fingerprinting technique, the Tcr lactic acid bacterial isolates were identified as Lactobacillus plantarum, L. sakei subsp. carnosus, L. sakei subsp. sakei, L. curvatus, and L. alimentarius and typed to the intraspecies level. For a selection of 24 Tcr lactic acid bacterial isolates displaying unique (GTG)5-PCR fingerprints, tet genes were determined by means of PCR, and only tet(M) was detected. Restriction enzyme analysis with AccI and ScaI revealed two different tet(M) allele types. This grouping was confirmed by partial sequencing of the tet(M) open reading frame, which indicated that the two allele types displayed high sequence similarities (>99.6%) with tet(M) genes previously reported in Staphylococcus aureus MRSA 101 and in Neisseria meningitidis, respectively. Southern hybridization with plasmid profiles revealed that the isolates contained tet(M)-carrying plasmids. In addition to the tet(M) gene, one isolate also contained an erm(B) gene on a different plasmid from the one encoding the tetracycline resistance. Furthermore, it was also shown by PCR that the tet(M) genes were not located on transposons of the Tn916/Tn1545 family. To our knowledge, this is the first detailed molecular study demonstrating that taxonomically and genotypically diverse Lactobacillus strains from different types of fermented meat products can be a host for plasmid-borne tet genes. PMID:12571056

Effects of TET2 mutations on DNA methylation in chronic myelomonocytic leukemia

USDA-ARS?s Scientific Manuscript database

TET2 enzymatically converts 5-methyl-cytosine to 5-hydroxymethyl-cytosine, possibly leading to loss of DNA methylation. TET2 mutations are common in myeloid leukemia and were proposed to contribute to leukemogenesis through DNA methylation. To expand on this concept, we studied chronic myelomonocyti...

High-level tetracycline resistance mediated by efflux pumps Tet(A) and Tet(A)-1 with two start codons.

PubMed

Wang, Weixia; Guo, Qinglan; Xu, Xiaogang; Sheng, Zi-ke; Ye, Xinyu; Wang, Minggui

2014-11-01

Efflux is the most common mechanism of tetracycline resistance. Class A tetracycline efflux pumps, which often have high prevalence in Enterobacteriaceae, are encoded by tet(A) and tet(A)-1 genes. These genes have two potential start codons, GTG and ATG, located upstream of the genes. The purpose of this study was to determine the start codon(s) of the class A tetracycline resistance (tet) determinants tet(A) and tet(A)-1, and the tetracycline resistance level they mediated. Conjugation, transformation and cloning experiments were performed and the genetic environment of tet(A)-1 was analysed. The start codons in class A tet determinants were investigated by site-directed mutagenesis of ATG and GTG, the putative translation initiation codons. High-level tetracycline resistance was transferred from the clinical strain of Klebsiella pneumoniae 10-148 containing tet(A)-1 plasmid pHS27 to Escherichia coli J53 by conjugation. The transformants harbouring recombinant plasmids that carried tet(A) or tet(A)-1 exhibited tetracycline MICs of 256-512 µg ml(-1), with or without tetR(A). Once the ATG was mutated to a non-start codon, the tetracycline MICs were not changed, while the tetracycline MICs decreased from 512 to 64 µg ml(-1) following GTG mutation, and to ≤4 µg ml(-1) following mutation of both GTG and ATG. It was presumed that class A tet determinants had two start codons, which are the primary start codon GTG and secondary start codon ATG. Accordingly, two putative promoters were predicted. In conclusion, class A tet determinants can confer high-level tetracycline resistance and have two start codons. © 2014 The Authors.

Tet and sul antibiotic resistance genes in livestock lagoons of various operation type, configuration, and antibiotic occurrence

USGS Publications Warehouse

McKinney, C.W.; Loftin, K.A.; Meyer, M.T.; Davis, J.G.; Pruden, A.

2010-01-01

Although livestock operations are known to harbor elevated levels of antibiotic resistant bacteria, few studies have examined the potential of livestock waste lagoons to reduce antibiotic resistance genes (ARGs). The purpose of this study was to determine the prevalence and examine the behavior of tetracycline [tet(O) and tet(W)] and sulfonamide [sul(I) and su/(II)] ARGsin a broad cross-section of livestock lagoons within the same semiarid western watershed. ARGs were monitored for one year in the water and the settled solids of eight lagoon systems by quantitative polymerase chain reaction. In addition, antibiotic residues and various bulk water quality constituents were analyzed. It was found that the lagoons of the chicken layer operation had the lowest concentrations of both tet and sul ARGs and low total antibiotic concentrations, whereas su ARGs were highest in the swine lagoons, which generally corresponded to the highest total antibiotic concentrations. A marginal benefit of organic and small dairy operations also was observed compared to conventional and large dairies, respectively. In all lagoons, su ARGs were observed to be generally more recalcitrant than tet ARGs. Also, positive correlations of various bulk water quality constituents were identified with tet ARGs but not sul ARGs. Significant positive correlations were identified between several metals and tet ARGs, but Pearson's correlation coefficients were mostly lower than those determined between antibiotic residues and ARGs. This study represents a quantitative characterization of ARGs in lagoons across a variety of livestock operations and provides insight into potential options for managing antibiotic resistance emanating from agricultural activities. ?? 2010 American Chemical Society.

Variable effects of oxytetracycline on antibiotic resistance gene abundance and the bacterial community during aerobic composting of cow manure.

PubMed

Qian, Xun; Sun, Wei; Gu, Jie; Wang, Xiao-Juan; Sun, Jia-Jun; Yin, Ya-Nan; Duan, Man-Li

2016-09-05

Livestock manure is often subjected to aerobic composting but little is known about the variation in antibiotic resistance genes (ARGs) during the composting process under different concentrations of antibiotics. This study compared the effects of three concentrations of oxytetracycline (OTC; 10, 60, and 200mg/kg) on ARGs and the succession of the bacterial community during composting. Very similar trends were observed in the relative abundances (RAs) of each ARG among the OTC treatments and the control during composting. After composting, the RAs of tetC, tetX, sul1, sul2, and intI1 increased 2-43 times, whereas those of tetQ, tetM, and tetW declined by 44-99%. OTC addition significantly increased the absolute abundances and RAs of tetC and intI1, while 200mg/kg OTC also enhanced those of tetM, tetQ, and drfA7. The bacterial community could be grouped according to the composting time under different treatments. The highest concentration of OTC had a more persistent effect on the bacterial community. In the present study, the succession of the bacterial community appeared to have a greater influence on the variation of ARGs during composting than the presence of antibiotics. Aerobic composting was not effective in reducing most of the ARGs, and thus the compost product should be considered as an important reservoir for ARGs. Copyright © 2016 Elsevier B.V. All rights reserved.

Comparison of two fabricated aptasensors based on modified carbon paste/oleic acid and magnetic bar carbon paste/Fe3O4@oleic acid nanoparticle electrodes for tetracycline detection.

PubMed

Jahanbani, Shahriar; Benvidi, Ali

2016-11-15

In this research, we have improved two aptasensors based on a modified carbon paste electrode (CPE) with oleic acid (OA), and a magnetic bar carbon paste electrode (MBCPE) with Fe3O4 magnetic nanoparticles and oleic acid (OA). After the immobilization process of anti-TET at the electrode surfaces, the aptasensors were named CPE/OA/anti-TET and MBCPE/Fe3O4NPs/OA/anti-TET respectively. In this paper, the detection of tetracycline is compared using CPE/OA/anti-TET and MBCPE/Fe3O4NPs/OA/anti-TET aptasensors. These modified electrodes were characterized by infrared spectroscopy (IR), scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS), UV-vis spectroscopy, and voltammetric methods. The linear range and the detection limit for TET with the CPE/OA/anti-TET aptasensor were found to be 1.0×10(-12)-1.0×10(-7)M and 3.0×10(-13)M respectively by EIS method. The linear range and the detection limit for TET with the CPE/OA/anti-TET aptasensor were found to be 1.0×10(-10)-1.0×10(-7)M with a limit of detection of 2.9×10(-11)M using differential pulse voltammetry (DPV) technique. The MBCPE/Fe3O4NPs/OA/anti-TET aptasensor was used for determination of TET, and a liner range of 1.0×10(-14)-1.0×10(-6)M with a detection limit of 3.8×10(-15)M was obtained by EIS method. Also, the linear range and detection limit of 1.0×10(-12)-1.0×10(-6)M and 3.1×10(-13)M respectively, were obtained for MBCPE/Fe3O4NPs/OA/anti-TET aptasensor using DPV. The proposed aptasensors were applied for determination of tetracycline in some real samples such as drug, milk, honey and blood serum samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Electrostatic immobilization of antimicrobial peptides on polyethylenimine and their antibacterial effect against Staphylococcus epidermidis.

PubMed

Hernandez-Montelongo, J; Corrales Ureña, Y R; Machado, D; Lancelloti, M; Pinheiro, M P; Rischka, K; Lisboa-Filho, P N; Cotta, M A

2018-04-01

Staphylococcus epidermidis is a gram-positive bacterium, and one of the most prevalent causes of nosocomial infections due to its strong ability to form biofilms on catheters and surgical implants. Here we explore the antimicrobial properties of Tet-124 peptides, which are part of the innate defense against different multicellular organisms in nature. Two different Tet-124 peptides were immobilized on a polyethylenimine (PEI) film to determine their impact on the antimicrobial properties: KLWWMIRRW (Tet-124), which contains only natural amino acids, and KLWWMIRRWG-(F-Br)-G (F-Br = 4-Bromophenylalanine), a modified Tet-124 sequence with the addition of an unnatural amino acid. The immobilization was obtained as a result of the electrostatic interaction between PEI amino groups and the C-terminal carboxylic groups of tryptophan and glycine amino acids of Tet-124 and Tet-124-Br peptides, respectively. The process was monitored and studied by water contact angle, Atomic Force Microscopy (AFM), X-ray Photoelectron Spectroscopy (XPS) and Quartz Crystal Microbalance with Dissipation (QCM-D) measurements. The antibacterial effect of our samples against S. epidermis was evaluated by the spread plate counting method, and cytotoxicity was tested using fibroblast cultures. Our results indicate the feasibility to immobilize electrostatically both Tet-124 peptides for biomedical applications. Copyright © 2018 Elsevier B.V. All rights reserved.

CPT1{alpha} over-expression increases long-chain fatty acid oxidation and reduces cell viability with incremental palmitic acid concentration in 293T cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jambor de Sousa, Ulrike L.; Koss, Michael D.; Fillies, Marion

2005-12-16

To test the cellular response to an increased fatty acid oxidation, we generated a vector for an inducible expression of the rate-limiting enzyme carnitine palmitoyl-transferase 1{alpha} (CPT1{alpha}). Human embryonic 293T kidney cells were transiently transfected and expression of the CPT1{alpha} transgene in the tet-on vector was activated with doxycycline. Fatty acid oxidation was measured by determining the conversion of supplemented, synthetic cis-10-heptadecenoic acid (C17:1n-7) to C15:ln-7. CPT1{alpha} over-expression increased mitochondrial long-chain fatty acid oxidation about 6-fold. Addition of palmitic acid (PA) decreased viability of CPT1{alpha} over-expressing cells in a concentration-dependent manner. Both, PA and CPT1{alpha} over-expression increased cell death. Interestingly,more » PA reduced total cell number only in cells over-expressing CPT1{alpha}, suggesting an effect on cell proliferation that requires PA translocation across the mitochondrial inner membrane. This inducible expression system should be well suited to study the roles of CPT1 and fatty acid oxidation in lipotoxicity and metabolism in vivo.« less

Tet(L) and Tet(K) Tetracycline-Divalent Metal/H+ Antiporters: Characterization of Multiple Catalytic Modes and a Mutagenesis Approach to Differences in Their Efflux Substrate and Coupling Ion Preferences

PubMed Central

Jin, Jie; Guffanti, Arthur A.; Bechhofer, David H.; Krulwich, Terry A.

2002-01-01

The Tet(L) protein encoded in the Bacillus subtilis chromosome and the closely related Tet(K) protein from Staphylococcus aureus plasmids are multifunctional antiporters that have three cytoplasmic efflux substrates: a tetracycline-divalent metal (TC-Me2+) complex that bears a net single positive charge, Na+, and K+. Tet(L) and Tet(K) had been shown to couple efflux of each of these substrates to influx of H+ as the coupling ion. In this study, competitive cross-inhibition between K+ and other cytoplasmic efflux substrates was demonstrated. Tet(L) and Tet(K) had also been shown to use K+ as an alternate coupling ion in support of Na+ or K+ efflux. Here they were shown to couple TC-Me2+ efflux to K+ uptake as well, exhibiting greater use of K+ as a coupling ion as the external pH increased. The substrate and coupling ion preferences of the two Tet proteins differed, especially in the higher preference of Tet(K) than Tet(L) for K+, both as a cytoplasmic efflux substrate and as an external coupling ion. Site-directed mutagenesis was employed to test the hypothesis that some feature of the putative “antiporter motif,” motif C, of Tet proteins would be involved in these characteristic preferences. Mutation of the A157 in Tet(L) to a hydroxyamino acid resulted in a more Tet(K)-like K+ preference both as coupling ion and efflux substrate. A reciprocal S157A mutant of Tet(K) exhibited reduced K+ preference. Competitive inhibition among substrates and the parallel effects of the single mutation upon K+ preference, as both an efflux substrate and coupling ion, are compatible with a model in which a single translocation pathway through the Tet(L) and Tet(K) transporters is used both for the cytoplasmic efflux substrates and for the coupling ions, in an alternating fashion. However, the effects of the A157 and other mutations of Tet(L) indicate that even if there are a shared binding site and translocation pathway, some elements of that pathway are used by all substrates and others are important only for particular substrates. PMID:12169596

Detection of a Common and Persistent tet(L)-Carrying Plasmid in Chicken-Waste-Impacted Farm Soil

PubMed Central

Hilpert, Markus; Ward, Mandy J.

2012-01-01

The connection between farm-generated animal waste and the dissemination of antibiotic resistance in soil microbial communities, via mobile genetic elements, remains obscure. In this study, electromagnetic induction (EMI) surveying of a broiler chicken farm assisted soil sampling from a chicken-waste-impacted site and a marginally affected site. Consistent with the EMI survey, a disparity existed between the two sites with regard to soil pH, tetracycline resistance (Tcr) levels among culturable soil bacteria, and the incidence and prevalence of several tet and erm genes in the soils. No significant difference was observed in these aspects between the marginally affected site and several sites in a relatively pristine regional forest. When the farm was in operation, tet(L), tet(M), tet(O), erm(A), erm(B), and erm(C) genes were detected in the waste-affected soil. Two years after all waste was removed from the farm, tet(L), tet(M), tet(O), and erm(C) genes were still detected. The abundances of tet(L), tet(O), and erm(B) were measured using quantitative PCR, and the copy numbers of each were normalized to eubacterial 16S rRNA gene copy numbers. tet(L) was the most prevalent gene, whereas tet(O) was the most persistent, although all declined over the 2-year period. A mobilizable plasmid carrying tet(L) was identified in seven of 14 Tcr soil isolates. The plasmid's hosts were identified as species of Bhargavaea, Sporosarcina, and Bacillus. The plasmid's mobilization (mob) gene was quantified to estimate its prevalence in the soil, and the ratio of tet(L) to mob was shown to have changed from 34:1 to 1:1 over the 2-year sampling period. PMID:22389375

Constrained variability of modeled T:ET ratio across biomes

NASA Astrophysics Data System (ADS)

Fatichi, Simone; Pappas, Christoforos

2017-07-01

A large variability (35-90%) in the ratio of transpiration to total evapotranspiration (referred here as T:ET) across biomes or even at the global scale has been documented by a number of studies carried out with different methodologies. Previous empirical results also suggest that T:ET does not covary with mean precipitation and has a positive dependence on leaf area index (LAI). Here we use a mechanistic ecohydrological model, with a refined process-based description of evaporation from the soil surface, to investigate the variability of T:ET across biomes. Numerical results reveal a more constrained range and higher mean of T:ET (70 ± 9%, mean ± standard deviation) when compared to observation-based estimates. T:ET is confirmed to be independent from mean precipitation, while it is found to be correlated with LAI seasonally but uncorrelated across multiple sites. Larger LAI increases evaporation from interception but diminishes ground evaporation with the two effects largely compensating each other. These results offer mechanistic model-based evidence to the ongoing research about the patterns of T:ET and the factors influencing its magnitude across biomes.

Occurrence and Diversity of Tetracycline Resistance Genes in Lagoons and Groundwater Underlying Two Swine Production Facilities

USGS Publications Warehouse

Chee-Sanford, J. C.; Aminov, R.I.; Krapac, I.J.; Garrigues-Jeanjean, N.; Mackie, R.I.

2001-01-01

In this study, we used PCR typing methods to assess the presence of tetracycline resistance determinants conferring ribosomal protection in waste lagoons and in groundwater underlying two swine farms. All eight classes of genes encoding this mechanism of resistance [tet(O), tet(Q), tet(W), tet(M), tetB(P), tet(S), tet(T), and otrA] were found in total DNA extracted from water of two lagoons. These determinants were found to be seeping into the underlying groundwater and could be detected as far as 250 m downstream from the lagoons. The identities and origin of these genes in groundwater were confirmed by PCR-denaturing gradient gel electrophoresis and sequence analyses. Tetracycline-resistant bacterial isolates from groundwater harbored the tet(M) gene, which was not predominant in the environmental samples and was identical to tet(M) from the lagoons. The presence of this gene in some typical soil inhabitants suggests that the vector of antibiotic resistance gene dissemination is not limited to strains of gastrointestinal origin carrying the gene but can be mobilized into the indigenous soil microbiota. This study demonstrated that tet genes occur in the environment as a direct result of agriculture and suggested that groundwater may be a potential source of antibiotic resistance in the food chain.

Global gene expression analyses of hematopoietic stem cell-like cell lines with inducible Lhx2 expression

PubMed Central

Richter, Karin; Wirta, Valtteri; Dahl, Lina; Bruce, Sara; Lundeberg, Joakim; Carlsson, Leif; Williams, Cecilia

2006-01-01

Background Expression of the LIM-homeobox gene Lhx2 in murine hematopoietic cells allows for the generation of hematopoietic stem cell (HSC)-like cell lines. To address the molecular basis of Lhx2 function, we generated HSC-like cell lines where Lhx2 expression is regulated by a tet-on system and hence dependent on the presence of doxycyclin (dox). These cell lines efficiently down-regulate Lhx2 expression upon dox withdrawal leading to a rapid differentiation into various myeloid cell types. Results Global gene expression of these cell lines cultured in dox was compared to different time points after dox withdrawal using microarray technology. We identified 267 differentially expressed genes. The majority of the genes overlapping with HSC-specific databases were those down-regulated after turning off Lhx2 expression and a majority of the genes overlapping with those defined as late progenitor-specific genes were the up-regulated genes, suggesting that these cell lines represent a relevant model system for normal HSCs also at the level of global gene expression. Moreover, in situ hybridisations of several genes down-regulated after dox withdrawal showed overlapping expression patterns with Lhx2 in various tissues during embryonic development. Conclusion Global gene expression analysis of HSC-like cell lines with inducible Lhx2 expression has identified genes putatively linked to self-renewal / differentiation of HSCs, and function of Lhx2 in organ development and stem / progenitor cells of non-hematopoietic origin. PMID:16600034

Aggregates assembled from overexpression of wild-type alpha-synuclein are not toxic to human neuronal cells.

PubMed

Ko, Li-Wen; Ko, Hwai-Hwa C; Lin, Wen-Lang; Kulathingal, Jayanranyan G; Yen, Shu-Hui C

2008-11-01

Filamentous alpha-synuclein (alpha-syn) aggregates form Lewy bodies (LBs), the neuropathologic hallmarks of Parkinson disease and related alpha-synucleinopathies. To model Lewy body-associated neurodegeneration, we generated transfectant 3D5 of human neuronal-type in which expression of human wild-type alpha-syn is regulated by the tetracycline off (TetOff)-inducible mechanism. Retinoic acid-elicited differentiation promoted assembly of alpha-syn aggregates after TetOff induction in 3D5 cells. The aggregates accumulated 14 days after TetOff induction were primarily soluble and showed augmented thioflavin affinity with concomitant phosphorylation and nitration of alpha-syn. Extension of the induction led to the formation of sarkosyl-insoluble aggregates that appeared concurrently with thioflavin-positive inclusions. Immunoelectron microscopy revealed that the inclusions consist of dense bundles of 8- to 12-nm alpha-syn fibrils that congregate in the perikarya and resemble Lewy bodies. Most importantly, accumulation of soluble and insoluble aggregates after TetOff induction for 14 and 28 days was reversible and did not compromise the viability of the cells or their subsequent survival. Thus, this chemically defined culture paradigm provides a useful means to elucidate how oxidative injuries and other insults that are associated with aging promote alpha-syn to self-assemble or interact with other molecules leading to neuronal degeneration in alpha-synucleinopathies.

Epigenetic modification of the PD-1 (Pdcd1) promoter in effector CD4+ T cells tolerized by peptide immunotherapy

PubMed Central

McPherson, Rhoanne C; Konkel, Joanne E; Prendergast, Catriona T; Thomson, John P; Ottaviano, Raffaele; Leech, Melanie D; Kay, Oliver; Zandee, Stephanie E J; Sweenie, Claire H; Wraith, David C; Meehan, Richard R; Drake, Amanda J; Anderton, Stephen M

2014-01-01

Clinically effective antigen-based immunotherapy must silence antigen-experienced effector T cells (Teff) driving ongoing immune pathology. Using CD4+ autoimmune Teff cells, we demonstrate that peptide immunotherapy (PIT) is strictly dependent upon sustained T cell expression of the co-inhibitory molecule PD-1. We found high levels of 5-hydroxymethylcytosine (5hmC) at the PD-1 (Pdcd1) promoter of non-tolerant T cells. 5hmC was lost in response to PIT, with DNA hypomethylation of the promoter. We identified dynamic changes in expression of the genes encoding the Ten-Eleven-Translocation (TET) proteins that are associated with the oxidative conversion 5-methylcytosine and 5hmC, during cytosine demethylation. We describe a model whereby promoter demethylation requires the co-incident expression of permissive histone modifications at the Pdcd1 promoter together with TET availability. This combination was only seen in tolerant Teff cells following PIT, but not in Teff that transiently express PD-1. Epigenetic changes at the Pdcd1 locus therefore determine the tolerizing potential of TCR-ligation. DOI: http://dx.doi.org/10.7554/eLife.03416.001 PMID:25546306

The fragment spin difference scheme for triplet-triplet energy transfer coupling

NASA Astrophysics Data System (ADS)

You, Zhi-Qiang; Hsu, Chao-Ping

2010-08-01

To calculate the electronic couplings in both inter- and intramolecular triplet energy transfer (TET), we have developed the "fragment spin difference" (FSD) scheme. The FSD was a generalization from the "fragment charge difference" (FCD) method of Voityuk et al. [J. Chem. Phys. 117, 5607 (2002)] for electron transfer (ET) coupling. In FSD, the spin population difference was used in place of the charge difference in FCD. FSD is derived from the eigenstate energies and populations, and therefore the FSD couplings contain all contributions in the Hamiltonian as well as the potential overlap effect. In the present work, two series of molecules, all-trans-polyene oligomers and polycyclic aromatic hydrocarbons, were tested for intermolecular TET study. The TET coupling results are largely similar to those from the previously developed direct coupling scheme, with FSD being easier and more flexible in use. On the other hand, the Dexter's exchange integral value, a quantity that is often used as an approximate for the TET coupling, varies in a large range as compared to the corresponding TET coupling. To test the FSD for intramolecular TET, we have calculated the TET couplings between zinc(II)-porphyrin and free-base porphyrin separated by different numbers of p-phenyleneethynylene bridge units. Our estimated rate constants are consistent with experimentally measured TET rates. The FSD method can be used for both intermolecular and intramolecular TET, regardless of their symmetry. This general applicability is an improvement over most existing methodologies.

Identification of Group G Streptococcal Isolates from Companion Animals in Japan and Their Antimicrobial Resistance Patterns.

PubMed

Tsuyuki, Yuzo; Kurita, Goro; Murata, Yoshiteru; Goto, Mieko; Takahashi, Takashi

2017-07-24

In this study, we conducted a species-level identification of group G streptococcal (GGS) isolates from companion animals in Japan and analyzed antimicrobial resistance (AMR) patterns. Strains were isolated from sterile and non-sterile specimens collected from 72 animals with clinical signs or symptoms in April-May, 2015. We identified the strain by 16S rRNA sequencing, mass spectrometry (MS), and an automated method based on their biochemical properties. Antimicrobial susceptibility was determined using the broth microdilution method and E-test. AMR determinants (erm(A), erm(B), mef(A), tet(M), tet(O), tet(K), tet(L), and tet(S)) in corresponding resistant isolates were amplified by PCR. The 16S rRNA sequencing identified the GGS species as Streptococcus canis (n = 68), Streptococcus dysgalactiae subsp. equisimilis (n = 3), and S. dysgalactiae subsp. dysgalactiae (n = 1). However, there were discrepancies between the sequencing data and both the MS and automated identification data. MS and the automated biochemical technique identified 18 and 37 of the 68 sequencing-identified S. canis strains, respectively. The AMR rates were 20.8% for tetracycline and 5.6% for clarithromycin, with minimum inhibitory concentrations (MIC) 50 -MIC 90 of 2-64 and ≤ 0.12-0.25μg/mL, respectively. AMR genotyping showed single or combined genotypes: erm(B) or tet(M)-tet(O)-tet(S). Our findings show the unique characteristics of GGS isolates from companion animals in Japan in terms of species-level identification and AMR patterns.

Dissemination of antibiotic resistance genes and their potential removal by on-farm treatment processes in nine swine feedlots in Shandong Province, China.

PubMed

Ben, Weiwei; Wang, Jian; Pan, Xun; Qiang, Zhimin

2017-01-01

This work investigated the dissemination of antibiotic resistance genes (ARGs) encoding resistance to sulfonamide and tetracycline antibiotics in nine swine feedlots located in Shandong Province of China, and examined their potential removal by various on-farm treatment processes. Results indicate that the target ARGs were widely distributed in swine wastes, with mean relative abundances ranging from 3.3 × 10 -5 (tetC) to 5.2 × 10 -1 (tetO) in swine manure and from 7.3 × 10 -3 (tetC) to 1.7 × 10 -1 (tetO) in swine wastewater. The mean relative ARG abundances ranged from 9.9 × 10 -5 (tetW) to 1.1 × 10 -2 (tetO) in soils and from 3.1 × 10 -4 (tetW) to 1.1 × 10 -2 (sul2) in receiving river sediments, indicating that the farmland application of swine manure compost and the discharge of swine wastewater promoted the dissemination of ARGs into adjacent environments. Microbial fermentation bed (MFB) could reduce the relative ARG abundances by 0-1.18 logs. However, septic tank, biogas digester and natural drying methods were relatively ineffective for ARG removal, and the relative abundances of some ARGs (i.e., tetC, tetG, sul1, and sul2) even increased by 0.74-3.90 logs in treated wastes. Bacterial diversity analysis indicates that the evolution of bacterial communities in the MFB played a crucial role in eliminating the ARGs. This study helps the effective assessment and management of ecological risks arising from ARGs in swine feedlots. Copyright © 2016 Elsevier Ltd. All rights reserved.

Post-exposure administration of diazepam combined with soluble epoxide hydrolase inhibition stops seizures and modulates neuroinflammation in a murine model of acute TETS intoxication

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vito, Stephen T., E-mail: stvito@ucdavis.edu; Austin, Adam T., E-mail: aaustin@ucdavis.edu; Banks, Christopher N., E-mail: Christopher.Banks@oehha.ca.gov

Tetramethylenedisulfotetramine (TETS) is a potent convulsant poison for which there is currently no approved antidote. The convulsant action of TETS is thought to be mediated by inhibition of type A gamma-aminobutyric acid receptor (GABA{sub A}R) function. We, therefore, investigated the effects of post-exposure administration of diazepam, a GABA{sub A}R positive allosteric modulator, on seizure activity, death and neuroinflammation in adult male Swiss mice injected with a lethal dose of TETS (0.15 mg/kg, ip). Administration of a high dose of diazepam (5 mg/kg, ip) immediately following the second clonic seizure (approximately 20 min post-TETS injection) effectively prevented progression to tonic seizuresmore » and death. However, this treatment did not prevent persistent reactive astrogliosis and microglial activation, as determined by GFAP and Iba-1 immunoreactivity and microglial cell morphology. Inhibition of soluble epoxide hydrolase (sEH) has been shown to exert potent anti-inflammatory effects and to increase survival in mice intoxicated with other GABA{sub A}R antagonists. The sEH inhibitor TUPS (1 mg/kg, ip) administered immediately after the second clonic seizure did not protect TETS-intoxicated animals from tonic seizures or death. Combined administration of diazepam (5 mg/kg, ip) and TUPS (1 mg/kg, ip, starting 1 h after diazepam and repeated every 24 h) prevented TETS-induced lethality and influenced signs of neuroinflammation in some brain regions. Significantly decreased microglial activation and enhanced reactive astrogliosis were observed in the hippocampus, with no changes in the cortex. Combining an agent that targets specific anti-inflammatory mechanisms with a traditional antiseizure drug may enhance treatment outcome in TETS intoxication. - Highlights: • Acute TETS intoxication causes delayed and persistent neuroinflammation. • Diazepam given post-TETS prevents lethal tonic seizures but not neuroinflammation. • A soluble epoxide hydrolase inhibitor alters TETS-induced neuroinflammation. • Acute TETS intoxication may be more effectively treated by a combinatorial therapy.« less

Food isolate Listeria monocytogenes harboring tetM gene plasmid-mediated exchangeable to Enterococcus faecalis on the surface of processed cheese.

PubMed

Haubert, Louise; Cunha, Carlos Eduardo Pouey da; Lopes, Graciela Völz; Silva, Wladimir Padilha da

2018-05-01

The genetic basis of tetracycline resistance in a food isolate Listeria monocytogenes (Lm16) was evaluated. Resistance to tetracycline was associated with the presence of the tetM gene in plasmid DNA. The sequence of tetM showed 100% of similarity with the Enterococcus faecalis sequences found in the EMBL database, suggesting that Lm16 received this gene from E. faecalis. Various size bands were detected in the DNA plasmid analysis, the largest being approximately 54.38 kb. Transferability of the tetM gene was achieved in vitro by agar matings between Lm16 and E. faecalis JH2-2, proving the potential for the spread of tetM by horizontal gene transfer. Furthermore, the conjugation experiments were performed on the surface of processed cheese, confirming the transferability in a food matrix. PCR assays were used to confirm the identity of E. faecalis and to detect the tetM gene in transconjugant bacteria. Additionally, the minimal inhibitory concentration for tetracycline and rifampicin and plasmid profiling were performed. This is the first report of a food isolate L. monocytogenes carrying the tetM gene in plasmid DNA, and it highlights the potential risk of spreading antimicrobial resistance genes between different bacteria. Copyright © 2018 Elsevier Ltd. All rights reserved.

Epigenetic alterations mediate iPSC normalization of DNA-repair expression and TNR stability in Huntington's disease.

PubMed

Mollica, Peter A; Zamponi, Martina; Reid, John A; Sharma, Deepak K; White, Alyson E; Ogle, Roy C; Bruno, Robert D; Sachs, Patrick C

2018-06-13

Huntington's disease (HD) is a rare autosomal dominant neurodegenerative disorder caused by a cytosine-adenine-guanine (CAG) trinucleotide repeat (TNR) expansion within the HTT gene. The mechanisms underlying HD-associated cellular dysfunction during pluripotency and neurodevelopment, are poorly understood. Here we tested the hypothesis that hypomethylation during cellular reprogramming leads to up-regulation of DNA repair genes and stabilization of TNRs in HD cells. We sought to determine how the HD TNR region is affected by global epigenetic changes through cellular reprogramming and early neurodifferentiation. We find that early-stage HD-affected neural stem cells (NSCs) contain increased levels of global 5-hydroxymethylation (5-hmC) and normalized DNA repair gene expression. We confirm TNR stability is induced during pluripotency, and maintained in HD-NSCs. We also identify up-regulation of 5-hmC catalyzing ten-eleven translocation (TET1/2) proteins, and show their knockdown leads to a corresponding decrease in select DNA repair gene expression. We further confirm decreased expression of TET regulating miR-29 family members in HD-NSCs. Our findings demonstrate that mechanisms involved in pluripotency recover the selected DNA repair gene expression and stabilizes pathogenic TNRs in HD. © 2018. Published by The Company of Biologists Ltd.

Behavioral Assessment of NIH Swiss Mice Acutely Intoxicated with Tetramethylenedisulfotetramine

PubMed Central

Flannery, Brenna M.; Silverman, Jill L.; Bruun, Donald A.; Puhger, Kyle R.; McCoy, Mark R.; Hammock, Bruce D.; Crawley, Jacqueline N.; Lein, Pamela J.

2014-01-01

Tetramethylenedisulfotetramine (TETS) is a potent convulsant poison that is thought to trigger seizures by inhibiting the function of the type A gamma-aminobutyric acid receptor (GABAAR). Acute intoxication with TETS can cause vomiting, convulsions, status epilepticus (SE) and even death. Clinical case reports indicate that individuals who survive poisoning may exhibit long-term neuropsychological issues and cognitive deficits. Therefore, the objective of this research was to determine whether a recently described mouse model of acute TETS intoxication exhibits persistent behavioral deficits. Young adult male NIH Swiss mice received a seizure-inducing dose of TETS (0.15 mg/kg, ip) and then were rescued from lethality by administration of diazepam (5 mg/kg, ip) approximately 20 min post-TETS-exposure. TETS-intoxicated mice typically exhibited 2 clonic seizures prior to administration of diazepam with no subsequent seizures post-diazepam injection as assessed using behavioral criteria. Seizures lasted an average of 72 seconds. Locomotor activity, anxiety-like and depression-relevant behaviors and cognition were assessed at 1 week, 1 month and 2 months post-TETS exposure using open field, elevated-plus maze, light↔dark transitions, tail suspension, forced swim and novel object recognition tasks. Interestingly, preliminary validation tests indicated that NIH Swiss mice do not respond to the shock in fear conditioning tasks. Subsequent evaluation of hot plate and tail flick nociception tasks revealed that this strain exhibits significantly decreased pain sensitivity relative to age- and sex-matched C57BL/6J mice, which displayed normal contextual fear conditioning. NIH Swiss mice acutely intoxicated with TETS exhibited no significant anxiety-related, depression-relevant, learning or memory deficits relative to vehicle controls at any of the time points assessed with the exception of significantly increased locomotor activity at 2 months post-TETS intoxication. The general absence of long-term behavioral deficits in TETS-intoxicated mice on these six assays suggests that the neurobehavioral consequences of TETS exposure described in human survivors of acute TETS intoxication are likely due to sustained seizure activity, rather than a direct effect of the chemical itself. Future research efforts are directed towards developing an animal model that better recapitulates the SE and seizure duration reported in humans acutely intoxicated with TETS. PMID:25446016

Spread of tetracycline resistance genes at a conventional dairy farm

PubMed Central

Kyselková, Martina; Jirout, Jiří; Vrchotová, Naděžda; Schmitt, Heike; Elhottová, Dana

2015-01-01

The use of antibiotics in animal husbandry contributes to the worldwide problem of increasing antibiotic resistance in animal and human pathogens. Intensive animal production is considered an important source of antibiotic resistance genes released to the environment, while the contribution of smaller farms remains to be evaluated. Here we monitor the spread of tetracycline resistance (TC-r) genes at a middle-size conventional dairy farm, where chlortetracycline (CTC, as intrauterine suppository) is prophylactically used after each calving. Our study has shown that animals at the farm acquired the TC-r genes in their early age (1–2 weeks), likely due to colonization with TC-resistant bacteria from their mothers and/or the farm environment. The relative abundance of the TC-r genes tet(W), tet(Q), and tet(M) in fresh excrements of calves was about 1–2 orders of magnitude higher compared to heifers and dairy cows, possibly due to the presence of antibiotic residues in milk fed to calves. The occurrence and abundance of TC-r genes in fresh excrements of heifers and adult cows remained unaffected by intrauterine CTC applications, with tet(O), tet(Q), and tet(W) representing a “core TC-resistome” of the farm, and tet(A), tet(M), tet(Y), and tet(X) occurring occasionally. The genes tet(A), tet(M), tet(Y), and tet(X) were shown to be respectively harbored by Shigella, Lactobacillus and Clostridium, Acinetobacter, and Wautersiella. Soil in the farm proximity, as well as field soil to which manure from the farm was applied, was contaminated with TC-r genes occurring in the farm, and some of the TC-r genes persisted in the field over 3 months following the manure application. Concluding, our study shows that antibiotic resistance genes may be a stable part of the intestinal metagenome of cattle even if antibiotics are not used for growth stimulation, and that smaller dairy farms may also contribute to environmental pollution with antibiotic resistance genes. PMID:26074912

Phenotypic and genotypic bacterial antimicrobial resistance in liquid pig manure is variously associated with contents of tetracyclines and sulfonamides.

PubMed

Hölzel, C S; Harms, K S; Küchenhoff, H; Kunz, A; Müller, C; Meyer, K; Schwaiger, K; Bauer, J

2010-05-01

Antibiotic residues as well as antibiotic-resistant bacteria in environmental samples might pose a risk to human health. This study aimed to investigate the association between antibiotic residues and bacterial antimicrobial resistance in liquid pig manure used as fertilizer. Concentrations of tetracyclines (TETs) and sulfonamides (SULs) were determined by liquid chromatography-mass spectrometry in 305 pig manure samples; antibiotic contents were correlated to the phenotypic resistance of Escherichia coli (n = 613) and enterococci (n = 564) towards up to 24 antibiotics. In 121 samples, the concentration of the TET resistance genes tet(M), tet(O) and tet(B) was quantified by real-time-PCR. TETs were found in 54% of the samples. The median sum concentration of all investigated TETs in the positive samples was 0.73 mg kg(-1). SULs were found with a similar frequency (51%) and a median sum concentration of 0.15 mg kg(-1) in the positive samples. Associated with the detection of TETs and/or SULs, resistance rates were significantly elevated for several substances - some of them not used in farm animals, e.g. chloramphenicol and synercid. In addition, multiresistant isolates were found more often in samples containing antibiotics. Analysis of the resistance genes tet(M) and tet(O) already showed a significant increase in their concentrations - but not in tet(B) - in the lowest range of total TET concentration. Mean tet(M) concentrations increased by the factor of 4.5 in the TET concentration range of 0.1-1 mg kg(-1), compared to negative manure samples. Antibiotic contamination of manure seems to be associated with a variety of changes in bacterial resistance, calling for a prudent use of antibiotics in farm animals. This study provides an interdisciplinary approach to assess antimicrobial resistance by combining the microbiological analysis of bacterial resistance with high quality chemical analysis of antibiotic residues in a representative number of environmental samples.

Occurrence of tetracycline-resistant fecal coliforms and their resistance genes in an urban river impacted by municipal wastewater treatment plant discharges.

PubMed

Zhang, Chong-Miao; Du, Cong; Xu, Huan; Miao, Yan-Hui; Cheng, Yan-Yan; Tang, Hao; Zhou, Jin-Hong; Wang, Xiao-Chang

2015-01-01

Antibiotic resistance of fecal coliforms in an urban river poses great threats to both human health and the environment. To investigate the occurrence and distribution of antibiotic resistant bacteria in an urban river, water samples were collected from the Chanhe River in Xi'an, China. After membrane filtration of water samples, the tetracycline resistance rate of fecal coliforms and their resistance genes were detected by plating and polymerase chain reaction (PCR), respectively. We found that fecal coliforms were generally resistant to tetracycline and saw average resistance rates of 44.7%. The genes tetA and tetB were widely detected, and their positive rate was 60%-100% and 40%-90%, respectively. We found few strains containing tetC, tetK, tetQ and tetX, and we did not identify any strains containing tetG, tetM or tetO. The prevalence of tetA and tetB over other genes indicated that the main mechanism for resistance to tetracycline is by changes to the efflux pump. Our analysis of the types and proportion of tetracycline resistance genes in the Chanhe River at locations upstream and downstream of the urban center suggests that the increased number of tetracycline-resistant fecal coliforms and spatial variation of tetracycline resistance genes diversity were related to municipal wastewater treatment plant discharge.

Identification of Sequence Specificity of 5-Methylcytosine Oxidation by Tet1 Protein with High-Throughput Sequencing.

PubMed

Kizaki, Seiichiro; Chandran, Anandhakumar; Sugiyama, Hiroshi

2016-03-02

Tet (ten-eleven translocation) family proteins have the ability to oxidize 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxycytosine (caC). However, the oxidation reaction of Tet is not understood completely. Evaluation of genomic-level epigenetic changes by Tet protein requires unbiased identification of the highly selective oxidation sites. In this study, we used high-throughput sequencing to investigate the sequence specificity of mC oxidation by Tet1. A 6.6×10(4) -member mC-containing random DNA-sequence library was constructed. The library was subjected to Tet-reactive pulldown followed by high-throughput sequencing. Analysis of the obtained sequence data identified the Tet1-reactive sequences. We identified mCpG as a highly reactive sequence of Tet1 protein. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Dynamic changes in DNA demethylation in the tree shrew (Tupaia belangeri chinensis) brain during postnatal development and aging.

PubMed

Wei, Shu; Hua, Hai-Rong; Chen, Qian-Quan; Zhang, Ying; Chen, Fei; Li, Shu-Qing; Li, Fan; Li, Jia-Li

2017-03-18

Brain development and aging are associated with alterations in multiple epigenetic systems, including DNA methylation and demethylation patterns. Here, we observed that the levels of the 5-hydroxymethylcytosine (5hmC) ten-eleven translocation (TET) enzyme-mediated active DNA demethylation products were dynamically changed and involved in postnatal brain development and aging in tree shrews ( Tupaia belangeri chinensis ). The levels of 5hmC in multiple anatomic structures showed a gradual increase throughout postnatal development, whereas a significant decrease in 5hmC was found in several brain regions in aged tree shrews, including in the prefrontal cortex and hippocampus, but not the cerebellum. Active changes in Tet mRNA levels indicated that TET2 and TET3 predominantly contributed to the changes in 5hmC levels. Our findings provide new insight into the dynamic changes in 5hmC levels in tree shrew brains during postnatal development and aging processes.

Characterization of resistance to tetracyclines and aminoglycosides of sheep mastitis pathogens: study of the effect of gene content on resistance.

PubMed

Lollai, S A; Ziccheddu, M; Duprè, I; Piras, D

2016-10-01

Mastitis causes economic losses and antimicrobials are frequently used for mastitis treatment. Antimicrobial resistance surveys are still rare in the ovine field and characterization of strains is important in order to acquire information about resistance and for optimization of therapy. Bacterial pathogens recovered in milk samples from mastitis-affected ewes were characterized for resistance to tetracyclines and aminoglycosides, members of which are frequently used antimicrobials in small ruminants. A total of 185 strains of staphylococci, streptococci, and enterococci, common mastitis pathogens, were tested for minimal inhibitory concentration (MIC) to tetracycline, doxycycline, minocycline, gentamicin, kanamycin, streptomycin, and for resistance genes by PCR. Effects of different tet genes arrangements on MICs were also investigated. Staphylococci expressed the lowest MIC for tetracycline and tet(K) was the most common gene recovered; tet(M) and tet(O) were also found. Gene content was shown to influence the tetracycline MIC values. Enterococci and streptococci showed higher MICs to tetracyclines and nonsusceptible strains always harboured at least one ribosomal protection gene (MIC above 8 μg ml(-1) ). Streptococci often harboured two or more tet determinants. As regards the resistance to aminoglycosides, staphylococci showed the lowest gentamicin and kanamycin median MIC along with streptomycin high level resistant (HLR) strains (MIC >1024 μg ml(-1) ) all harbouring str gene. The resistance determinant aac(6')-Ie-aph(2″)-Ia was present in few strains. Streptococci were basically nonsusceptible to aminoglycosides but neither HLR isolates nor resistance genes were detected. Enterococci revealed the highest MICs for gentamicin; two str harbouring isolates were shown to be HLR to streptomycin. Evidence was obtained for the circulation of antimicrobial-resistant strains and genes in sheep dairy farming. Tetracycline MIC of 64 μg ml(-1) and high-level resistance were detected for streptomycin (MIC >1024 μg ml(-1) ), so that effectiveness of common treatments may be at risk. © 2016 The Society for Applied Microbiology.

Electromagnetic Compatibility of Transcutaneous Energy Transmission Systemfor Totally Implantable Artificial Heart

NASA Astrophysics Data System (ADS)

Shiba, Kenji; Koshiji, Kohji

Transcutaneous Energy Transmission (TET) is one way of providing the energy needed to power a totally implantable artificial heart (TIAH). In the present study, an externally coupled TET system was implanted in a prototype human phantom to evaluate emission and immunity. In the emission evaluation, measurements were conducted based on CISPR Pub.11 and VDE 0871 standards, while immunity tests were based on the standards of the IEC 61000-4 series. The magnetic field of the radiated emission was measured using a loop antenna. At 0.1[MHz], we found the greatest magnetic field of 47.8 [dBμA/m], somewhat less than CISPR’s upper limit of 54 [dBμA/m]. For the conducted emission, by installing a noise filter and ferrite beads in the input section of the DC-power supply, conducted emission could be kept within the allowable limits of CISPR Pub.11 and VDE 0871. Finally, the immunity tests against radiated and conducted emission, electrostatic discharge and voltage fluctuation proved that the prototype could withstand the maximum level of disturbance. These results confirmed that the TET system implanted in a human phantom could, through modification, meet the emission and immunity standards.

A novel cell model to study the function of the adrenoleukodystrophy-related protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gueugnon, Fabien; Volodina, Natalia; Taouil, Jaoued Et

2006-03-03

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder due to mutations in the ABCD1 (ALD) gene. ALDRP, the closest homolog of ALDP, has been shown to have partial functional redundancy with ALDP and, when overexpressed, can compensate for the loss-of-function of ALDP. In order to characterize the function of ALDRP and to understand the phenomenon of gene redundancy, we have developed a novel system that allows the controlled expression of the ALDRP-EGFP fusion protein (normal or non-functional mutated ALDRP) using the Tet-On system in H4IIEC3 rat hepatoma cells. The generated stable cell lines express negligible levels of endogenous ALDRP and doxycyclinemore » dosage-dependent levels of normal or mutated ALDRP. Importantly, the ALDRP-EGFP protein is targeted correctly to peroxisome and is functional. The obtained cell lines will be an indispensable tool in our further studies aimed at the resolution of the function of ALDRP to characterize its potential substrates in a natural context.« less

Utilization of NASA Lewis mobile terminals for the Hermes satellite

NASA Technical Reports Server (NTRS)

Edelman, E. A.; Fiala, J. L.; Rizzolla, L.

1977-01-01

The paper describes the portable earth terminal (PET) and the transportable earth terminal (TET) which enable two-way television and voice communication. Both terminals were developed by NASA and utilize the high power of the Hermes satellite. PET is a bus-type vehicle which has receiving equipment for full duplex color television and which can transmit programs originating in either the on-board PET studio or in nearby buildings. PET has a collapsible 2.4-m diameter parabolic antenna interfacing with a 500-watt 14-GHz wideband TV transmitter and a 12-GHz wideband TV receiver system. TET uses two parabolic reflector antennas, 3 m and 1.2 m in diameter, mounted on a flat trailer towed by a truck. TET can receive and relay color TV signals, and its narrowband transmitter can serve as a return audio link permitting a question-and-answer format. Also described are uplink and downlink performance characteristics, operation procedures, and field demonstrations which enabled personnel at several hospitals to participate in a distant medical conference.

Monitoring tetracycline through a solid-state nanopore sensor

NASA Astrophysics Data System (ADS)

Zhang, Yuechuan; Chen, Yanling; Fu, Yongqi; Ying, Cuifeng; Feng, Yanxiao; Huang, Qimeng; Wang, Chao; Pei, De-Sheng; Wang, Deqiang

2016-06-01

Antibiotics as emerging environmental contaminants, are widely used in both human and veterinary medicines. A solid-state nanopore sensing method is reported in this article to detect Tetracycline, which is based on Tet-off and Tet-on systems. rtTA (reverse tetracycline-controlled trans-activator) and TRE (Tetracycline Responsive Element) could bind each other under the action of Tetracycline to form one complex. When the complex passes through nanopores with 8 ~ 9 nanometers in diameter, we could detect the concentrations of Tet from 2 ng/mL to 2000 ng/mL. According to the Logistic model, we could define three growth zones of Tetracycline for rtTA and TRE. The slow growth zone is 0-39.5 ng/mL. The rapid growth zone is 39.5-529.7 ng/mL. The saturated zone is > 529.7 ng/mL. Compared to the previous methods, the nanopore sensor could detect and quantify these different kinds of molecule at the single-molecule level.

Tet38 Efflux Pump Affects Staphylococcus aureus Internalization by Epithelial Cells through Interaction with CD36 and Contributes to Bacterial Escape from Acidic and Nonacidic Phagolysosomes

PubMed Central

Truong-Bolduc, Q. C.; Khan, N. S.

2016-01-01

ABSTRACT We previously reported that the Tet38 efflux pump is involved in internalization of Staphylococcus aureus by A549 lung epithelial cells. A lack of tet38 reduced bacterial uptake by A549 cells to 36% of that of the parental strain RN6390. Using invasion assays coupled with confocal microscopy imaging, we studied the host cell receptor(s) responsible for bacterial uptake via interaction with Tet38. We also assessed the ability of S. aureus to survive following alkalinization of the phagolysosomes by chloroquine. Antibody to the scavenger receptor CD36 reduced the internalization of S. aureus RN6390 by A549 cells, but the dependence on CD36 was reduced in QT7 tet38, suggesting that an interaction between Tet38 and CD36 contributed to S. aureus internalization. Following fusion of the S. aureus-associated endosomes with lysosomes, alkalinization of the acidic environment with chloroquine led to a rapid increase in the number of S. aureus RN6390 bacteria in the cytosol, followed by a decrease shortly thereafter. This effect of chloroquine was not seen in the absence of intact Tet38 in mutant QT7. These data taken together suggest that Tet38 plays a role both in bacterial internalization via interaction with CD36 and in bacterial escape from the phagolysosomes. PMID:27956597

A novel quick transendoscopic enteral tubing in mid-gut: technique and training with video.

PubMed

Long, Chuyan; Yu, Yan; Cui, Bota; Jagessar, Sabreen Abdul Rahman; Zhang, Jie; Ji, Guozhong; Huang, Guangming; Zhang, Faming

2018-03-13

This study aimed to evaluate the feasibility, safety, and value of a quick technique for transendoscopic enteral tubing (TET) through mid-gut. A prospective interventional study was performed in a single center. A TET tube was inserted into mid-gut through the nasal orifice and fixed on the pylorus wall by one tiny titanium endoscopic clip under anesthesia. The feasibility, safety, success rate, and satisfaction with TET placement were evaluated for enteral nutrition or fecal microbiota transplantation. A total of 86 patients underwent mid-gut TET. The success rate of the TET procedure was 98.8% (85/86). Mean tubing time of the TET procedure was 4.2 ± 1.9 min. 10 cases of procedure was enough for training of general endoscopist to shorten the procedure time (7.0 min vs 4.0 min, p < 0.05). 97.7% (84/86) of patients were satisfied with the TET placement. Procedure-related and tube-related adverse events were observed in 8.1% (7/86) and 7.0% (6/86) of patients respectively. There were no moderate to severe adverse events during tube extubation. TET through mid-gut is a novel, convenient, reliable and safe procedure for mid-gut administration with a high degree of patient satisfaction. This research was retrospectively registered with clinicaltrials.gov. Trial registration date: 29th November 2017. NCT03335982 .

Monitoring and source tracking of tetracycline resistance genes in lagoons and groundwater adjacent to swine production facilities over a 3-year period

USGS Publications Warehouse

Koike, S.; Krapac, I.G.; Oliver, H.D.; Yannarell, A.C.; Chee-Sanford, J. C.; Aminov, R.I.; Mackie, R.I.

2007-01-01

To monitor the dissemination of resistance genes into the environment, we determined the occurrence of tetracycline resistance (Tcr) genes in groundwater underlying two swine confinement operations. Monitoring well networks (16 wells at site A and 6 wells at site C) were established around the lagoons at each facility. Groundwater (n = 124) and lagoon (n = 12) samples were collected from the two sites at six sampling times from 2000 through 2003. Total DNA was extracted, and PCR was used to detect seven Tcr genes [tet(M), tet(O), tet(Q), tet(W), tet(C), tet(H), and tet(Z)]. The concentration of Tcr genes was quantified by real-time quantitative PCR. To confirm the Tcr gene source in groundwater, comparative analysis of tet(W) gene sequences was performed on groundwater and lagoon samples. All seven Tcr genes were continually detected in groundwater during the 3-year monitoring period at both sites. At site A, elevated detection frequency and concentration of Tcr genes were observed in the wells located down-gradient of the lagoon. Comparative analysis of tet(W) sequences revealed that the impacted groundwater contained gene sequences almost identical (99.8% identity) to those in the lagoon, but these genes were not found in background libraries. Novel sequence clusters and unique indigenous resistance gene pools were also found in the groundwater. Thus, antibiotic resistance genes in groundwater are affected by swine manure, but they are also part of the indigenous gene pool. Copyright ?? 2007, American Society for Microbiology. All Rights Reserved.

Monitoring and Source Tracking of Tetracycline Resistance Genes in Lagoons and Groundwater Adjacent to Swine Production Facilities over a 3-Year Period▿

PubMed Central

Koike, S.; Krapac, I. G.; Oliver, H. D.; Yannarell, A. C.; Chee-Sanford, J. C.; Aminov, R. I.; Mackie, R. I.

2007-01-01

To monitor the dissemination of resistance genes into the environment, we determined the occurrence of tetracycline resistance (Tcr) genes in groundwater underlying two swine confinement operations. Monitoring well networks (16 wells at site A and 6 wells at site C) were established around the lagoons at each facility. Groundwater (n = 124) and lagoon (n = 12) samples were collected from the two sites at six sampling times from 2000 through 2003. Total DNA was extracted, and PCR was used to detect seven Tcr genes [tet(M), tet(O), tet(Q), tet(W), tet(C), tet(H), and tet(Z)]. The concentration of Tcr genes was quantified by real-time quantitative PCR. To confirm the Tcr gene source in groundwater, comparative analysis of tet(W) gene sequences was performed on groundwater and lagoon samples. All seven Tcr genes were continually detected in groundwater during the 3-year monitoring period at both sites. At site A, elevated detection frequency and concentration of Tcr genes were observed in the wells located down-gradient of the lagoon. Comparative analysis of tet(W) sequences revealed that the impacted groundwater contained gene sequences almost identical (99.8% identity) to those in the lagoon, but these genes were not found in background libraries. Novel sequence clusters and unique indigenous resistance gene pools were also found in the groundwater. Thus, antibiotic resistance genes in groundwater are affected by swine manure, but they are also part of the indigenous gene pool. PMID:17545324

Modelling non-alcoholic fatty liver disease in human hepatocyte-like cells.

PubMed

Lyall, Marcus J; Cartier, Jessy; Thomson, John P; Cameron, Kate; Meseguer-Ripolles, Jose; O'Duibhir, Eoghan; Szkolnicka, Dagmara; Villarin, Baltasar Lucendo; Wang, Yu; Blanco, Giovanny Rodriguez; Dunn, Warwick B; Meehan, Richard R; Hay, David C; Drake, Amanda J

2018-07-05

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of liver disease in developed countries. An in vitro NAFLD model would permit mechanistic studies and enable high-throughput therapeutic screening. While hepatic cancer-derived cell lines are a convenient, renewable resource, their genomic, epigenomic and functional alterations mean their utility in NAFLD modelling is unclear. Additionally, the epigenetic mark 5-hydroxymethylcytosine (5hmC), a cell lineage identifier, is rapidly lost during cell culture, alongside expression of the Ten-eleven-translocation ( TET ) methylcytosine dioxygenase enzymes, restricting meaningful epigenetic analysis. Hepatocyte-like cells (HLCs) derived from human embryonic stem cells can provide a non-neoplastic, renewable model for liver research. Here, we have developed a model of NAFLD using HLCs exposed to lactate, pyruvate and octanoic acid (LPO) that bear all the hallmarks, including 5hmC profiles, of liver functionality. We exposed HLCs to LPO for 48 h to induce lipid accumulation. We characterized the transcriptome using RNA-seq, the metabolome using ultra-performance liquid chromatography-mass spectrometry and the epigenome using 5-hydroxymethylation DNA immunoprecipitation (hmeDIP) sequencing. LPO exposure induced an NAFLD phenotype in HLCs with transcriptional and metabolomic dysregulation consistent with those present in human NAFLD. HLCs maintain expression of the TET enzymes and have a liver-like epigenome. LPO exposure-induced 5hmC enrichment at lipid synthesis and transport genes. HLCs treated with LPO recapitulate the transcriptional and metabolic dysregulation seen in NAFLD and additionally retain TET expression and 5hmC. This in vitro model of NAFLD will be useful for future mechanistic and therapeutic studies.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'. © 2018 The Authors.

Characterisation of penicillin and tetracycline resistance in Staphylococcus aureus isolated from bovine milk samples in Minas Gerais, Brazil.

PubMed

Martini, Caroline L; Lange, Carla C; Brito, Maria Avp; Ribeiro, João B; Mendonça, Letícia C; Vaz, Eliana K

2017-05-01

This Regional Research Communication describes the characterisation of ampicillin, penicillin and tetracycline resistance in Staphylococcus aureus isolated from bovine subclinical mastitis in Minas Gerais State, Brazil. Ninety S. aureus isolates from bovine mastitis exhibiting phenotypic resistance to ampicillin, penicillin and/or tetracycline were selected for this study. The minimum inhibitory concentration (MIC) of each antibiotic was determined using the E-Test® and the production of beta-lactamase was determined by cefinase disks. The resistance genes blaZ, tet(K), tet(L), tet(M), and tet(O) were investigated by PCR in all of the isolates. The MIC results classified 77, 83 and 71% of the isolates as resistant to ampicillin, penicillin and tetracycline, respectively. The MIC50 and MIC90 were, respectively, 1 and 2 µg/ml for ampicillin, 0·5 and 1 µg/ml for penicillin and 32 and 64 µg/ml for tetracycline. Eighty-six per cent of beta-lactamase producing isolates were detected. Of the 90 isolates investigated, 97% amplified blaZ, 84% amplified tet(K), 9% amplified tet(L), 2% amplified tet(M) and 1% amplified tet(O). Seventy-nine isolates (88%) showed blaZ together with at least one tet gene. S. aureus isolates showed high MIC50 and MIC90 values for the three antimicrobials. The blaZ and tet(K) genes were widespread in the herds studied, and most of the isolates harboured blaZ and tet(K) concomitantly.

TET2 functions as a resistance factor against DNA methylation acquisition during Epstein-Barr virus infection.

PubMed

Namba-Fukuyo, Hiroe; Funata, Sayaka; Matsusaka, Keisuke; Fukuyo, Masaki; Rahmutulla, Bahityar; Mano, Yasunobu; Fukayama, Masashi; Aburatani, Hiroyuki; Kaneda, Atsushi

2016-12-06

Extensive DNA methylation is observed in gastric cancer with Epstein-Barr virus (EBV) infection, and EBV infection is the cause to induce this extensive hypermethylaton phenotype in gastric epithelial cells. However, some 5' regions of genes do not undergo de novo methylation, despite the induction of methylation in surrounding regions, suggesting the existence of a resistance factor against DNA methylation acquisition. We conducted an RNA-seq analysis of gastric epithelial cells with and without EBV infection and found that TET family genes, especially TET2, were repressed by EBV infection at both mRNA and protein levels. TET2 was found to be downregulated by EBV transcripts, e.g. BARF0 and LMP2A, and also by seven human miRNAs targeting TET2, e.g., miR-93 and miR-29a, which were upregulated by EBV infection, and transfection of which into gastric cells repressed TET2. Hydroxymethylation target genes by TET2 were detected by hydroxymethylated DNA immunoprecipitation sequencing (hMeDIP-seq) with and without TET2 overexpression, and overlapped significantly with methylation target genes in EBV-infected cells. When TET2 was knocked down by shRNA, EBV infection induced de novo methylation more severely, including even higher methylation in methylation-acquired promoters or de novo methylation acquisition in methylation-protected promoters, leading to gene repression. TET2 knockdown alone without EBV infection did not induce de novo DNA methylation. These data suggested that TET2 functions as a resistance factor against DNA methylation in gastric epithelial cells and repression of TET2 contributes to DNA methylation acquisition during EBV infection.

Quorum Sensing Gene Regulation by LuxR/HapR Master Regulators in Vibrios

PubMed Central

Ball, Alyssa S.; Chaparian, Ryan R.

2017-01-01

ABSTRACT The coordination of group behaviors in bacteria is accomplished via the cell-cell signaling process called quorum sensing. Vibrios have historically been models for studying bacterial communication due to the diverse and remarkable behaviors controlled by quorum sensing in these bacteria, including bioluminescence, type III and type VI secretion, biofilm formation, and motility. Here, we discuss the Vibrio LuxR/HapR family of proteins, the master global transcription factors that direct downstream gene expression in response to changes in cell density. These proteins are structurally similar to TetR transcription factors but exhibit distinct biochemical and genetic features from TetR that determine their regulatory influence on the quorum sensing gene network. We review here the gene groups regulated by LuxR/HapR and quorum sensing and explore the targets that are common and unique among Vibrio species. PMID:28484045

Antibiotics and Antibiotic Resistance Genes in Sediment of Honghu Lake and East Dongting Lake, China.

PubMed

Yang, Yuyi; Cao, Xinhua; Lin, Hui; Wang, Jun

2016-11-01

Sediment is an ideal medium for the aggregation and dissemination of antibiotics and antibiotic resistance genes (ARGs). The levels of antibiotics and ARGs in Honghu Lake and East Dongting Lake of central China were investigated in this study. The concentrations of eight antibiotics (four sulfonamides and four tetracyclines) in Honghu Lake were in the range 90.00-437.43 μg kg -1 (dry weight (dw)) with mean value of 278.21 μg kg -1 dw, which was significantly higher than those in East Dongting Lake (60.02-321.04 μg kg -1 dw, mean value of 195.70 μg kg -1 dw). Among the tested three sulfonamide resistance genes (sul) and eight tetracycline resistance genes (tet), sul1, sul2, tetA, tetC, and tetM had 100 % detection frequency in sediment samples of East Dongting Lake, while only sul1, sul2, and tetC were observed in all samples of Honghu Lake. The relative abundance of sul2 was higher than that of sul1 at p < 0.05 level in both lakes. The relative abundance of tet genes in East Dongting Lake was in the following order: tetM > tetB > tetC > tetA. The relative abundance of sul1, sul2, and tetC in East Dongting Lake was significantly higher than those in Honghu Lake. The abundance of background bacteria may play an important role in the horizontal spread of sul2 and tetC genes in Honghu Lake and sul1 in East Dongting Lake, respectively. Redundancy analysis indicated that tetracyclines may play a more important role than sulfonamides in the abundance of sul1, sul2, and tetC gens in Honghu Lake and East Dongting Lake.

Getting insight into the prevalence of antibiotic resistance genes in specimens of marketed edible insects.

PubMed

Milanović, Vesna; Osimani, Andrea; Pasquini, Marina; Aquilanti, Lucia; Garofalo, Cristiana; Taccari, Manuela; Cardinali, Federica; Riolo, Paola; Clementi, Francesca

2016-06-16

This study was aimed at investigating the occurrence of 11 transferable antibiotic resistance (AR) genes [erm(A), erm(B), erm(C), vanA, vanB, tet(M), tet(O), tet(S), tet(K), mecA, blaZ] in 11 species of marketed edible insects (small crickets powder, small crickets, locusts, mealworm larvae, giant waterbugs, black ants, winged termite alates, rhino beetles, mole crickets, silkworm pupae, and black scorpions) in order to provide a first baseline for risk assessment. Among the AR genes under study, tet(K) occurred with the highest frequency, followed by erm(B), tet(S) and blaZ. A high variability was seen among the samples, in terms of occurrence of different AR determinants. Cluster Analysis and Principal Coordinates Analysis allowed the 11 samples to be grouped in two main clusters, one including all but one samples produced in Thailand and the other including those produced in the Netherlands. Copyright © 2016 Elsevier B.V. All rights reserved.

Early de novo DNA methylation and prolonged demethylation in the muscle lineage.

PubMed

Tsumagari, Koji; Baribault, Carl; Terragni, Jolyon; Varley, Katherine E; Gertz, Jason; Pradhan, Sirharsa; Badoo, Melody; Crain, Charlene M; Song, Lingyun; Crawford, Gregory E; Myers, Richard M; Lacey, Michelle; Ehrlich, Melanie

2013-03-01

Myogenic cell cultures derived from muscle biopsies are excellent models for human cell differentiation. We report the first comprehensive analysis of myogenesis-specific DNA hyper- and hypo-methylation throughout the genome for human muscle progenitor cells (both myoblasts and myotubes) and skeletal muscle tissue vs. 30 non-muscle samples using reduced representation bisulfite sequencing. We also focused on four genes with extensive hyper- or hypo-methylation in the muscle lineage (PAX3, TBX1, MYH7B/MIR499 and OBSCN) to compare DNA methylation, DNaseI hypersensitivity, histone modification, and CTCF binding profiles. We found that myogenic hypermethylation was strongly associated with homeobox or T-box genes and muscle hypomethylation with contractile fiber genes. Nonetheless, there was no simple relationship between differential gene expression and myogenic differential methylation, rather only for subsets of these genes, such as contractile fiber genes. Skeletal muscle retained ~30% of the hypomethylated sites but only ~3% of hypermethylated sites seen in myogenic progenitor cells. By enzymatic assays, skeletal muscle was 2-fold enriched globally in genomic 5-hydroxymethylcytosine (5-hmC) vs. myoblasts or myotubes and was the only sample type enriched in 5-hmC at tested myogenic hypermethylated sites in PAX3/CCDC140 andTBX1. TET1 and TET2 RNAs, which are involved in generation of 5-hmC and DNA demethylation, were strongly upregulated in myoblasts and myotubes. Our findings implicate de novo methylation predominantly before the myoblast stage and demethylation before and after the myotube stage in control of transcription and co-transcriptional RNA processing. They also suggest that, in muscle, TET1 or TET2 are involved in active demethylation and in formation of stable 5-hmC residues.

Early de novo DNA methylation and prolonged demethylation in the muscle lineage

PubMed Central

Tsumagari, Koji; Baribault, Carl; Terragni, Jolyon; Varley, Katherine E.; Gertz, Jason; Pradhan, Sirharsa; Badoo, Melody; Crain, Charlene M.; Song, Lingyun; Crawford, Gregory E.; Myers, Richard M.; Lacey, Michelle; Ehrlich, Melanie

2013-01-01

Myogenic cell cultures derived from muscle biopsies are excellent models for human cell differentiation. We report the first comprehensive analysis of myogenesis-specific DNA hyper- and hypo-methylation throughout the genome for human muscle progenitor cells (both myoblasts and myotubes) and skeletal muscle tissue vs. 30 non-muscle samples using reduced representation bisulfite sequencing. We also focused on four genes with extensive hyper- or hypo-methylation in the muscle lineage (PAX3, TBX1, MYH7B/MIR499 and OBSCN) to compare DNA methylation, DNaseI hypersensitivity, histone modification, and CTCF binding profiles. We found that myogenic hypermethylation was strongly associated with homeobox or T-box genes and muscle hypomethylation with contractile fiber genes. Nonetheless, there was no simple relationship between differential gene expression and myogenic differential methylation, rather only for subsets of these genes, such as contractile fiber genes. Skeletal muscle retained ~30% of the hypomethylated sites but only ~3% of hypermethylated sites seen in myogenic progenitor cells. By enzymatic assays, skeletal muscle was 2-fold enriched globally in genomic 5-hydroxymethylcytosine (5-hmC) vs. myoblasts or myotubes and was the only sample type enriched in 5-hmC at tested myogenic hypermethylated sites in PAX3/CCDC140 andTBX1. TET1 and TET2 RNAs, which are involved in generation of 5-hmC and DNA demethylation, were strongly upregulated in myoblasts and myotubes. Our findings implicate de novo methylation predominantly before the myoblast stage and demethylation before and after the myotube stage in control of transcription and co-transcriptional RNA processing. They also suggest that, in muscle, TET1 or TET2 are involved in active demethylation and in formation of stable 5-hmC residues. PMID:23417056

Anabolic and Antiresorptive Modulation of Bone Homeostasis by the Epigenetic Modulator Sulforaphane, a Naturally Occurring Isothiocyanate*

PubMed Central

Thaler, Roman; Maurizi, Antonio; Roschger, Paul; Sturmlechner, Ines; Khani, Farzaneh; Spitzer, Silvia; Rumpler, Monika; Zwerina, Jochen; Karlic, Heidrun; Dudakovic, Amel; Klaushofer, Klaus; Teti, Anna; Rucci, Nadia; Varga, Franz; van Wijnen, Andre J.

2016-01-01

Bone degenerative pathologies like osteoporosis may be initiated by age-related shifts in anabolic and catabolic responses that control bone homeostasis. Here we show that sulforaphane (SFN), a naturally occurring isothiocyanate, promotes osteoblast differentiation by epigenetic mechanisms. SFN enhances active DNA demethylation via Tet1 and Tet2 and promotes preosteoblast differentiation by enhancing extracellular matrix mineralization and the expression of osteoblastic markers (Runx2, Col1a1, Bglap2, Sp7, Atf4, and Alpl). SFN decreases the expression of the osteoclast activator receptor activator of nuclear factor-κB ligand (RANKL) in osteocytes and mouse calvarial explants and preferentially induces apoptosis in preosteoclastic cells via up-regulation of the Tet1/Fas/Caspase 8 and Caspase 3/7 pathway. These mechanistic effects correlate with higher bone volume (∼20%) in both normal and ovariectomized mice treated with SFN for 5 weeks compared with untreated mice as determined by microcomputed tomography. This effect is due to a higher trabecular number in these mice. Importantly, no shifts in mineral density distribution are observed upon SFN treatment as measured by quantitative backscattered electron imaging. Our data indicate that the food-derived compound SFN epigenetically stimulates osteoblast activity and diminishes osteoclast bone resorption, shifting the balance of bone homeostasis and favoring bone acquisition and/or mitigation of bone resorption in vivo. Thus, SFN is a member of a new class of epigenetic compounds that could be considered for novel strategies to counteract osteoporosis. PMID:26757819

Antibiotic-resistant genes and antibiotic-resistant bacteria in the effluent of urban residential areas, hospitals, and a municipal wastewater treatment plant system.

PubMed

Li, Jianan; Cheng, Weixiao; Xu, Like; Strong, P J; Chen, Hong

2015-03-01

In this study, we determined the abundance of 8 antibiotics (3 tetracyclines, 4 sulfonamides, and 1 trimethoprim), 12 antibiotic-resistant genes (10 tet, 2 sul), 4 antibiotic-resistant bacteria (tetracycline, sulfamethoxazole, and combined resistance), and class 1 integron integrase gene (intI1) in the effluent of residential areas, hospitals, and municipal wastewater treatment plant (WWTP) systems. The concentrations of total/individual targets (antibiotics, genes, and bacteria) varied remarkably among different samples, but the hospital samples generally had a lower abundance than the residential area samples. The WWTP demonstrated removal efficiencies of 50.8% tetracyclines, 66.8% sulfonamides, 0.5 logs to 2.5 logs tet genes, and less than 1 log of sul and intI1 genes, as well as 0.5 log to 1 log removal for target bacteria. Except for the total tetracycline concentration and the proportion of tetracycline-resistant bacteria (R (2) = 0.330, P < 0.05), there was no significant correlation between antibiotics and the corresponding resistant bacteria (P > 0.05). In contrast, various relationships were identified between antibiotics and antibiotic resistance genes (P < 0.05). Tet (A) and tet (B) displayed noticeable relationships with both tetracycline and combined antibiotic-resistant bacteria (P < 0.01).

CBL, CBLB, TET2, ASXL1, and IDH1/2 mutations and additional chromosomal aberrations constitute molecular events in chronic myelogenous leukemia

PubMed Central

Makishima, Hideki; Jankowska, Anna M.; McDevitt, Michael A.; O'Keefe, Christine; Dujardin, Simon; Cazzolli, Heather; Przychodzen, Bartlomiej; Prince, Courtney; Nicoll, John; Siddaiah, Harish; Shaik, Mohammed; Szpurka, Hadrian; Hsi, Eric; Advani, Anjali; Paquette, Ronald

2011-01-01

Progression of chronic myelogenous leukemia (CML) to accelerated (AP) and blast phase (BP) is because of secondary molecular events, as well as additional cytogenetic abnormalities. On the basis of the detection of JAK2, CBL, CBLB, TET2, ASXL1, and IDH1/2 mutations in myelodysplastic/myeloproliferative neoplasms, we hypothesized that they may also contribute to progression in CML. We screened these genes for mutations in 54 cases with CML (14 with chronic phase, 14 with AP, 20 with myeloid, and 6 with nonmyeloid BP). We identified 1 CBLB and 2 TET2 mutations in AP, and 1 CBL, 1 CBLB, 4 TET2, 2 ASXL1, and 2 IDH family mutations in myeloid BP. However, none of these mutations were found in chronic phase. No cases with JAK2V617F mutations were found. In 2 cases, TET2 mutations were found concomitant with CBLB mutations. By single nucleotide polymorphism arrays, uniparental disomy on chromosome 5q, 8q, 11p, and 17p was found in AP and BP but not involving 4q24 (TET2) or 11q23 (CBL). Microdeletions on chromosomes 17q11.2 and 21q22.12 involved tumor associated genes NF1 and RUNX1, respectively. Our results indicate that CBL family, TET2, ASXL1, and IDH family mutations and additional cryptic karyotypic abnormalities can occur in advanced phase CML. PMID:21346257

Contamination profiles of antibiotic resistance genes in the sediments at a catchment scale.

PubMed

Su, Hao-Chang; Pan, Chang-Gui; Ying, Guang-Guo; Zhao, Jian-Liang; Zhou, Li-Jun; Liu, You-Sheng; Tao, Ran; Zhang, Rui-Quan; He, Liang-Ying

2014-08-15

The aim of this study was to investigate the contamination profiles of tetracycline, sulfonamide, and macrolide resistance genes, as well as integrons in sediments of Dongjiang River basin of South China by real time quantitative polymerase chain reaction. sul2 was the most abundant resistance gene, with the average concentration of 6.97×10(8) copies/g and 1.00×10(8) copies/g in the dry and wet seasons, respectively, followed by ermF, sul3, sul1, intI1, tetA, ermB, tetX, tetM, tetQ, tetO, tetW, tetS, ermC, and tetB. The abundance of intI2 gene was the lowest in the sediment samples. Significant correlations existed between the ARGs and sediment properties as well as metals (Cu and Zn) and corresponding antibiotic classes, suggesting that the contamination of ARGs is related to chemical pollution of the sediments in the river basin. Principal component analysis showed distinct groupings of the sampling sites, reflecting that human activities are the key player in the dissemination of ARGs in the catchment environment. Copyright © 2014 Elsevier B.V. All rights reserved.

Parp inhibition prevents ten-eleven translocase enzyme activation and hyperglycemia-induced DNA demethylation.

PubMed

Dhliwayo, Nyembezi; Sarras, Michael P; Luczkowski, Ernest; Mason, Samantha M; Intine, Robert V

2014-09-01

Studies from human cells, rats, and zebrafish have documented that hyperglycemia (HG) induces the demethylation of specific cytosines throughout the genome. We previously documented that a subset of these changes become permanent and may provide, in part, a mechanism for the persistence of complications referred to as the metabolic memory phenomenon. In this report, we present studies aimed at elucidating the molecular machinery that is responsible for the HG-induced DNA demethylation observed. To this end, RNA expression and enzymatic activity assays indicate that the ten-eleven translocation (Tet) family of enzymes are activated by HG. Furthermore, through the detection of intermediates generated via conversion of 5-methyl-cytosine back to the unmethylated form, the data were consistent with the use of the Tet-dependent iterative oxidation pathway. In addition, evidence is provided that the activity of the poly(ADP-ribose) polymerase (Parp) enzyme is required for activation of Tet activity because the use of a Parp inhibitor prevented demethylation of specific loci and the accumulation of Tet-induced intermediates. Remarkably, this inhibition was accompanied by a complete restoration of the tissue regeneration deficit that is also induced by HG. The ultimate goal of this work is to provide potential new avenues for therapeutic discovery. © 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

CRISPR/Cas9-mediated knock-in of an optimized TetO repeat for live cell imaging of endogenous loci.

PubMed

Tasan, Ipek; Sustackova, Gabriela; Zhang, Liguo; Kim, Jiah; Sivaguru, Mayandi; HamediRad, Mohammad; Wang, Yuchuan; Genova, Justin; Ma, Jian; Belmont, Andrew S; Zhao, Huimin

2018-06-15

Nuclear organization has an important role in determining genome function; however, it is not clear how spatiotemporal organization of the genome relates to functionality. To elucidate this relationship, a method for tracking any locus of interest is desirable. Recently clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) or transcription activator-like effectors were adapted for imaging endogenous loci; however, they are mostly limited to visualization of repetitive regions. Here, we report an efficient and scalable method named SHACKTeR (Short Homology and CRISPR/Cas9-mediated Knock-in of a TetO Repeat) for live cell imaging of specific chromosomal regions without the need for a pre-existing repetitive sequence. SHACKTeR requires only two modifications to the genome: CRISPR/Cas9-mediated knock-in of an optimized TetO repeat and its visualization by TetR-EGFP expression. Our simplified knock-in protocol, utilizing short homology arms integrated by polymerase chain reaction, was successful at labeling 10 different loci in HCT116 cells. We also showed the feasibility of knock-in into lamina-associated, heterochromatin regions, demonstrating that these regions prefer non-homologous end joining for knock-in. Using SHACKTeR, we were able to observe DNA replication at a specific locus by long-term live cell imaging. We anticipate the general applicability and scalability of our method will enhance causative analyses between gene function and compartmentalization in a high-throughput manner.

Quantification of Tetramethylenedisulfotetramine (TETS) in Various Food Matrices by Solid Phase Extraction Liquid ChromatographyIon Trap Mass Spectrometry

DTIC Science & Technology

2017-04-01

used to deliberately contaminate food or water. TETS is not absorbed through the skin; the most common route of exposure is ingestion of... contaminated foods . Thus, the development of a reliable extraction and detection technique for TETS in different foods is essential: when accidental and...TETS in various complex food matrices. TETS is a relatively persistent environmental contaminant due to its high stability in water. This extraction

An exclusive α/β code directs allostery in TetR-peptide complexes.

PubMed

Sevvana, Madhumati; Goetz, Christoph; Goeke, Dagmar; Wimmer, Cornelius; Berens, Christian; Hillen, Wolfgang; Muller, Yves A

2012-02-10

The allosteric mechanism of one of the best characterized bacterial transcription regulators, tetracycline repressor (TetR), has recently been questioned. Tetracycline binding induces cooperative folding of TetR, as suggested by recent unfolding studies, rather than switching between two defined conformational states, namely a DNA-binding-competent conformation and a non-DNA-binding conformation. Upon ligand binding, a host of near-native multiconformational structures collapse into a single, highly stabilized protein conformation that is no longer able to bind DNA. Here, structure-function studies performed with four synthetic peptides that bind to TetR and mimic the function of low-molecular-weight effectors, such as tetracyclines, provide new means to discriminate between different allosteric models. Whereas two inducing peptides bind in an extended β-like conformation, two anti-inducing peptides form an α-helix in the effector binding site of TetR. This exclusive bimodal interaction mode coincides with two distinct overall conformations of TetR, namely one that is identical with induced TetR and one that mirrors the DNA-bound state of TetR. Urea-induced unfolding studies show no increase in thermodynamic stability for any of the peptide complexes, although fluorescence measurements demonstrate peptide binding to TetR. This strongly suggests that, at least for these peptide effectors, a classical two-state allosteric model best describes TetR function. Copyright © 2011 Elsevier Ltd. All rights reserved.

Comparison of ozone and thermal hydrolysis combined with anaerobic digestion for municipal and pharmaceutical waste sludge with tetracycline resistance genes.

PubMed

Pei, Jin; Yao, Hong; Wang, Hui; Ren, Jia; Yu, Xiaohua

2016-08-01

Biosolids from wastewater treatment plant (WWTP) are environmental reservoirs of antibiotic resistance genes, which attract great concerns on their efficient treatments. Anaerobic digestion (AD) is widely used for sewage sludge treatment but its effectiveness is limited due to the slow hydrolysis. Ozone and thermal hydrolysis pre-treatment were employed to improve AD efficiency and reduce antibiotic-resistant genes in municipal and pharmaceutical waste sludge (MWS and PWS, respectively) in this study. Sludge solubilization achieved 15.75-25.09% and 14.85-33.92% after ozone and thermal hydrolysis, respectively. Both pre-treatments improved cumulative methane production and the enhancements were greater on PWS than MWS. Five tetracycline-resistant genes (tet(A), tet(G), tet(Q), tet(W), tet(X)) and one mobile element (intI1) were qPCR to assess pre-treatments. AD of pre-treated sludge reduced more tet genes than raw sludge for both ozonation and thermal hydrolysis in PWS and MWS. Thermal hydrolysis pre-treatment was more efficient than ozone for reduction after AD. Results of this study help support management options for reducing the spread of antibiotic resistance from biosolids. Copyright © 2016. Published by Elsevier Ltd.

Tet38 Efflux Pump Affects Staphylococcus aureus Internalization by Epithelial Cells through Interaction with CD36 and Contributes to Bacterial Escape from Acidic and Nonacidic Phagolysosomes.

PubMed

Truong-Bolduc, Q C; Khan, N S; Vyas, J M; Hooper, D C

2017-02-01

We previously reported that the Tet38 efflux pump is involved in internalization of Staphylococcus aureus by A549 lung epithelial cells. A lack of tet38 reduced bacterial uptake by A549 cells to 36% of that of the parental strain RN6390. Using invasion assays coupled with confocal microscopy imaging, we studied the host cell receptor(s) responsible for bacterial uptake via interaction with Tet38. We also assessed the ability of S. aureus to survive following alkalinization of the phagolysosomes by chloroquine. Antibody to the scavenger receptor CD36 reduced the internalization of S. aureus RN6390 by A549 cells, but the dependence on CD36 was reduced in QT7 tet38, suggesting that an interaction between Tet38 and CD36 contributed to S. aureus internalization. Following fusion of the S. aureus-associated endosomes with lysosomes, alkalinization of the acidic environment with chloroquine led to a rapid increase in the number of S. aureus RN6390 bacteria in the cytosol, followed by a decrease shortly thereafter. This effect of chloroquine was not seen in the absence of intact Tet38 in mutant QT7. These data taken together suggest that Tet38 plays a role both in bacterial internalization via interaction with CD36 and in bacterial escape from the phagolysosomes. Copyright © 2017 American Society for Microbiology.

Characterization of tet(Y)-carrying LowGC plasmids exogenously captured from cow manure at a conventional dairy farm.

PubMed

Kyselková, Martina; Chrudimský, Tomáš; Husník, Filip; Chroňáková, Alica; Heuer, Holger; Smalla, Kornelia; Elhottová, Dana

2016-06-01

Manure from dairy farms has been shown to contain diverse tetracycline resistance genes that are transferable to soil. Here, we focus on conjugative plasmids that may spread tetracycline resistance at a conventional dairy farm. We performed exogenous plasmid isolation from cattle feces using chlortetracycline for transconjugant selection. The transconjugants obtained harbored LowGC-type plasmids and tet(Y). A representative plasmid (pFK2-7) was fully sequenced and this was compared with previously described LowGC plasmids from piggery manure-treated soil and a GenBank record from Acinetobacter nosocomialis that we also identified as a LowGC plasmid. The pFK2-7 plasmid had the conservative backbone typical of LowGC plasmids, though this region was interrupted with an insert containing the tet(Y)-tet(R) tetracycline resistance genes and the strA-strB streptomycin resistance genes. Despite Acinetobacter populations being considered natural hosts of LowGC plasmids, these plasmids were not found in three Acinetobacter isolates from the study farm. The isolates harbored tet(Y)-tet(R) genes in identical genetic surroundings as pFK2-7, however, suggesting genetic exchange between Acinetobacter and LowGC plasmids. Abundance of LowGC plasmids and tet(Y) was correlated in manure and soil samples from the farm, indicating that LowGC plasmids may be involved in the spread of tet(Y) in the environment. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Tetracycline-Containing MCM-41 Mesoporous Silica Nanoparticles for the Treatment of Escherichia coli.

PubMed

Koneru, Bhuvaneswari; Shi, Yi; Wang, Yu-Chieh; Chavala, Sai H; Miller, Michael L; Holbert, Brittany; Conson, Maricar; Ni, Aiguo; Di Pasqua, Anthony J

2015-10-30

Tetracycline (TC) is a well-known broad spectrum antibiotic, which is effective against many Gram positive and Gram negative bacteria. Controlled release nanoparticle formulations of TC have been reported, and could be beneficial for application in the treatment of periodontitis and dental bone infections. Furthermore, TC-controlled transcriptional regulation systems (Tet-on and Tet-off) are useful for controlling transgene expression in vitro and in vivo for biomedical research purposes; controlled TC release systems could be useful here, as well. Mesoporous silica nanomaterials (MSNs) are widely studied for drug delivery applications; Mobile crystalline material 41 (MCM-41), a type of MSN, has a mesoporous structure with pores forming channels in a hexagonal fashion. We prepared 41 ± 4 and 406 ± 55 nm MCM-41 mesoporous silica nanoparticles and loaded TC for controlled dug release; TC content in the TC-MCM-41 nanoparticles was 18.7% and 17.7% w/w, respectively. Release of TC from TC-MCM-41 nanoparticles was then measured in phosphate-buffered saline (PBS), pH 7.2, at 37 °C over a period of 5 h. Most antibiotic was released from both over this observation period; however, the majority of TC was released over the first hour. Efficacy of the TC-MCM-41 nanoparticles was then shown to be superior to free TC against Escherichia coli (E. coli) in culture over a 24 h period, while blank nanoparticles had no effect.

Improvement in magnetic field immunity of externally-coupled transcutaneous energy transmission system for a totally implantable artificial heart.

PubMed

Yamamoto, Takahiko; Koshiji, Kohji; Homma, Akihiko; Tatsumi, Eisuke; Taenaka, Yoshiyuki

2008-01-01

Transcutaneous energy transmission (TET) that uses electromagnetic induction between the external and internal coils of a transformer is the most promising method to supply driving energy to a totally implantable artificial heart without invasion. Induction-heating (IH) cookers generate magnetic flux, and if a cooker is operated near a transcutaneous transformer, the magnetic flux generated will link with the external and internal coils of the transcutaneous transformer. This will affect the performance of the TET and the artificial heart system. Hence, it is necessary to improve the magnetic field immunity of the TET system. During operation of the system, if the transcutaneous transformer is in close proximity to an IH cooker, the electric power generated by the cooker and coupled to the transformer can drive the artificial heart system. To prevent this coupling, the external coil was shielded with a conductive shield that had a slit in it. This reduces the coupling between the transformer and the magnetic field generated by the induction cooker. However, the temperature of the shield increased due to heating by eddy currents. The temperature of the shield can be reduced by separating the IH cooker and the shield.

Behavior of tetracycline and sulfamethazine with corresponding resistance genes from swine wastewater in pilot-scale constructed wetlands.

PubMed

Liu, Lin; Liu, Yu-Hong; Wang, Zhen; Liu, Chao-Xiang; Huang, Xu; Zhu, Ge-Fu

2014-08-15

Four pilot-scale constructed wetlands (free water surface, SF; horizontal subsurface flow, HSF; vertical subsurface flows with different water level, VSF-L and VSF-H) were operated to assess their ability to remove sulfamethazine (SMZ) and tetracycline (TC) from wastewaters, and to investigate the abundance level of corresponding resistance genes (sulI, sulII, tetM, tetW and tetO) in the CWs. The results indicated that CWs could significantly reduce the concentration of antibiotics in wastewater, and the mass removal rate range of SMZ and TC were respectively 11%-95% and 85%-95% in the four systems on the basis of hydraulic equilibrium; further relatively high removal rate was observed in VSF with low water level. Seasonal condition had a significant effect on SMZ removal in the CWs (especially SMZ in SF), but TC removal in VSFs were not considered to have statistically significant differences in winter and summer. At the end period, the relative abundances of target genes in the CWs showed obvious increases compared to initial levels, ranging from 2.98 × 10(-5) to 1.27 × 10(-1) for sul genes and 4.68 × 10(-6) to 1.54 × 10(-1) for tet genes after treatment, and those abundances showed close relation to both characteristic of wastewater and configuration of CWs. Copyright © 2014 Elsevier B.V. All rights reserved.

An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells.

PubMed

Guo, Jianying; Ma, Dacheng; Huang, Rujin; Ming, Jia; Ye, Min; Kee, Kehkooi; Xie, Zhen; Na, Jie

2017-05-01

Human pluripotent stem cells (hPSCs) are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR-ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline (Dox) inducible dCas9-VP64-p65-Rta (dCas9-VPR) transcription activator and a reverse Tet transactivator (rtTA) expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by the addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG promoter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level promoted naïve pluripotent gene expression, enhanced cell survival and clonogenicity, and enabled hESCs to integrate with the inner cell mass (ICM) of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs.

Cortex and hippocampus DNA epigenetic response to a long-term arsenic exposure via drinking water.

PubMed

Du, Xiaoyan; Tian, Meiping; Wang, Xiaoxue; Zhang, Jie; Huang, Qingyu; Liu, Liangpo; Shen, Heqing

2018-03-01

The neurotoxicity of arsenic is a serious health problem, especially for children. DNA epigenetic change may be an important pathogenic mechanism, but the molecular pathway remains obscure. In this study, the weaned male Sprague-Dawly (SD) rats were treated with arsenic trioxide via drinking water for 6 months, simulating real developmental exposure situation of children. Arsenic exposure impaired the cognitive abilities, and altered the expression of neuronal activity-regulated genes. Total arsenic concentrations of cortex and hippocampus tissues were significantly increased in a dose-dependent manner. The reduction in 5-methylcytosine (5 mC) and 5-hydroxymethylcytosine (5hmC) levels as well as the down-regulation of DNA methyltransferases (DNMTs) and ten-eleven translocations (TETs) expression suggested that DNA methylation/demethylation processes were significantly suppressed in brain tissues. S-adenosylmethionine (SAM) level wasn't changed, but the expression of the important indicators of oxidative/anti-oxidative balance and tricarboxylic acid (TCA) cycle was significantly deregulated. Overall, arsenic can disrupt oxidative/anti-oxidative balance, further inhibit TETs expression through TCA cycle and alpha-ketoglutarate (α-KG) pathway, and consequently cause DNA methylation/demethylation disruption. The present study implies oxidative stress but not SAM depletion may lead to DNA epigenetic alteration and arsenic neurotoxicity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Frequency of heterozygous TET2 deletions in myeloproliferative neoplasms

PubMed Central

Tripodi, Joseph; Hoffman, Ronald; Najfeld, Vesna; Weinberg, Rona

2010-01-01

The Philadelphia chromosome (Ph)-negative myeloproliferative neoplasms (MPNs), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are a group of clonal hematopoietic stem cell disorders with overlapping clinical and cytogenetic features and a variable tendency to evolve into acute leukemia. These diseases not only share overlapping chromosomal abnormalities but also a number of acquired somatic mutations. Recently, mutations in a putative tumor suppressor gene, ten-eleven translocation 2 (TET2) on chromosome 4q24 have been identified in 12% of patients with MPN. Additionally 4q24 chromosomal rearrangements in MPN, including TET2 deletions, have also been observed using conventional cytogenetics. The goal of this study was to investigate the frequency of genomic TET2 rearrangements in MPN using fluorescence in situ hybridization as a more sensitive method for screening and identifying genomic deletions. Among 146 MPN patients, we identified two patients (1.4%) who showed a common 4q24 deletion, including TET2. Our observations also indicated that the frequency of TET2 deletion is increased in patients with an abnormal karyotype (5%). PMID:21188113

Modelling insights on the partition of evapotranspiration components across biomes

NASA Astrophysics Data System (ADS)

Fatichi, Simone; Pappas, Christoforos

2017-04-01

Recent studies using various methodologies have found a large variability (from 35 to 90%) in the ratio of transpiration to total evapotranspiration (denoted as T:ET) across biomes or even at the global scale. Concurrently, previous results suggest that T:ET is independent of mean precipitation and has a positive correlation with Leaf Area Index (LAI). We used the mechanistic ecohydrological model, T&C, with a refined process-based description of soil resistance and a detailed treatment of canopy biophysics and ecophysiology, to investigate T:ET across multiple biomes. Contrary to observation-based estimates, simulation results highlight a well-constrained range of mean T:ET across biomes that is also robust to perturbations of the most sensitive parameters. Simulated T:ET was confirmed to be independent of average precipitation, while it was found to be uncorrelated with LAI across biomes. Higher values of LAI increase evaporation from interception but suppress ground evaporation with the two effects largely cancelling each other in many sites. These results offer mechanistic, model-based, evidence to the ongoing research about the range of T:ET and the factors affecting its magnitude across biomes.

Acute loss of TET function results in aggressive myeloid cancer in mice

PubMed Central

An, Jungeun; González-Avalos, Edahí; Chawla, Ashu; Jeong, Mira; López-Moyado, Isaac F.; Li, Wei; Goodell, Margaret A.; Chavez, Lukas; Ko, Myunggon; Rao, Anjana

2015-01-01

TET-family dioxygenases oxidize 5-methylcytosine (5mC) in DNA, and exert tumour suppressor activity in many types of cancers. Even in the absence of TET coding region mutations, TET loss-of-function is strongly associated with cancer. Here we show that acute elimination of TET function induces the rapid development of an aggressive, fully-penetrant and cell-autonomous myeloid leukaemia in mice, pointing to a causative role for TET loss-of-function in this myeloid malignancy. Phenotypic and transcriptional profiling shows aberrant differentiation of haematopoietic stem/progenitor cells, impaired erythroid and lymphoid differentiation and strong skewing to the myeloid lineage, with only a mild relation to changes in DNA modification. We also observe progressive accumulation of phospho-H2AX and strong impairment of DNA damage repair pathways, suggesting a key role for TET proteins in maintaining genome integrity. PMID:26607761

Mosaic tetracycline resistance genes and their flanking regions in Bifidobacterium thermophilum and Lactobacillus johnsonii.

PubMed

van Hoek, Angela H A M; Mayrhofer, Sigrid; Domig, Konrad J; Flórez, Ana B; Ammor, Mohammed S; Mayo, Baltasar; Aarts, Henk J M

2008-01-01

For the first time, mosaic tetracycline resistance genes were identified in Lactobacillus johnsonii and in Bifidobacterium thermophilum strains. The L. johnsonii strain investigated contains a complex hybrid gene, tet(O/W/32/O/W/O), whereas the five bifidobacterial strains possess two different mosaic tet genes: i.e., tet(W/32/O) and tet(O/W). As reported by others, the crossover points of the mosaic tet gene segments were found at similar positions within the genes, suggesting a hot spot for recombination. Analysis of the sequences flanking these genes revealed that the upstream part corresponds to the 5' end of the mosaic open reading frame. In contrast, the downstream region was shown to be more variable. Surprisingly, in one of the B. thermophilum strains a third tet determinant was identified, coding for the efflux pump Tet(L).

Factors influencing the removal of antibiotic-resistant bacteria and antibiotic resistance genes by the electrokinetic treatment.

PubMed

Li, Hongna; Li, Binxu; Zhang, Zhiguo; Tian, Yunlong; Ye, Jing; Lv, Xiwu; Zhu, Changxiong

2018-09-30

The performance of the electrokinetic remediation process on the removal of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) was evaluated with different influencing factors. With chlortetracycline (CTC), oxytetracycline (OTC), and tetracycline (TC) as template chemicals, the removal of both ARB and ARGs was enhanced with the increase of voltage gradient (0.4-1.2 V cm -1 ) and prolonged reaction time (3-14 d). The greatest removal (26.01-31.48% for ARB, 37.93-83.10% for ARGs) was obtained applying a voltage of 1.2 V cm -1 , leading to the highest electrical consumption. The effect of polarity reversal intervals on the inactivation ratio of ARB followed the order of 0 h (66.06-80.00%) > 12 h (17.07-24.75%) > 24 h (10.44-13.93%). Lower pH, higher current density, and more evenly-distributed voltage drop was observed with a polarity reversal interval of 12 h compared with that of 24 h, leading to more efficient electrochemical reactions in soil. Compared with sul genes, tet genes were more vulnerable to be attacked in an electric field. It was mainly attributed to the lower abundance of tet genes (except tetM) and the varied effects of electrokinetic remediation process on different ARGs. Moreover, a relatively less removal ratio of tetC and tetG was obtained mainly due to the mechanism of the efflux pump upregulation. Both tet and sul genes were positively correlated with TC-resistant bacteria. The efflux pump genes like tetG and the cellular protection genes like tetM showed different correlations with ARB. This study enhances the current understanding on the removal strategies of ARB and ARGs, and it provides important parameters for their destruction by the electrokinetic treatment. Copyright © 2018 Elsevier Inc. All rights reserved.

Immunogenicity and safety of the first indigenously developed Indian tetravalent influenza vaccine (split virion) in healthy adults ≥ 18 years of age: A randomized, multicenter, phase II / III clinical trial.

PubMed

Sharma, Shrikant; Singh, Veer Bahadur; Kumar, Sanjay; Prajapati, Vipul; Patel, Jitendra; Vukkala, Rajesh; Jangid, Sanjay Kumar; Sanmukhani, Jayesh; Gupta, Gaurav; Patel, Pradip; Mittal, Ravindra; Glueck, Reinhard

2018-02-20

This phase II / III clinical trial was conducted to evaluate the immunogenicity and safety of the Tetravalent Influenza vaccine (Split virion) I.P. (TetIV) developed indigenously in the country for the first time by M/s Cadila Healthcare Limited, India containing two influenza A and two influenza B strains, one of each, Yamagata (B/Phuket) and Victoria (B/Brisbane) lineage and also compare it to that of an licensed seasonal Trivalent Influenza vaccine (TriIV) of Sanofi Pasteur India Private Limited, containing the two influenza A and only the Yamagata lineage (B/Phuket) strain. Three hundred and fifty subjects of either sex, aged more than 18 years of age, were randomized in a 1:1 ratio to receive either the TetIV or TriIV. Immunogenicity assessments (antibody against A/H1N1, A/H3N2, B/Phuket and B/Brisbane) were done by Haemagglutination Inhibition assay at baseline and 21 d after vaccination. Solicited (local and systemic) and unsolicited adverse events were recorded for up to 42 d following vaccination. The TetIV was found to fulfill the criteria set by the European and the US regulatory authorities and WHO guidance on the requirements of clinical data for licensure of seasonal inactivated influenza vaccines. The seroconversion rates with TetIV were 93.5% for A/H1N1, 90.0% for A/H3N2, 70.0% for B/Phuket and 82.9% for B/Brisbane strain. There was no significant difference in the seroconversion and seroprotection rates at day 21 for A/H1N1, A/H3N2 and B/Phuket in the two groups while the TetIV was superior to the TrivIV for the seroconversion and the seroprotection rate for the B/Brisbane strain (Victoria lineage). Both the vaccines were well tolerated by all the study participants; addition of the fourth strain in the TetIV did not compromise the safety as compared to TriIV. The most common systemic adverse event reported in both the groups was headache followed by fever.

Conjugative transfer of resistance determinants among human and bovine Streptococcus agalactiae

PubMed Central

Pinto, Tatiana Castro Abreu; Costa, Natália Silva; Corrêa, Ana Beatriz de Almeida; de Oliveira, Ivi Cristina Menezes; de Mattos, Marcos Correa; Rosado, Alexandre Soares; Benchetrit, Leslie Claude

2014-01-01

Streptococcus agalactiae (GBS) is a major source of human perinatal diseases and bovine mastitis. Erythromycin (Ery) and tetracycline (Tet) are usually employed for preventing human and bovine infections although resistance to such agents has become common among GBS strains. Ery and Tet resistance genes are usually carried by conjugative transposons (CTns) belonging to the Tn916 family, but their presence and transferability among GBS strains have not been totally explored. Here we evaluated the presence of Tet resistance genes (tetM and tetO) and CTns among Ery-resistant (Ery-R) and Ery-susceptible (Ery-S) GBS strains isolated from human and bovine sources; and analyzed the ability for transferring resistance determinants between strains from both origins. Tet resistance and int-Tn genes were more common among Ery-R when compared to Ery-S isolates. Conjugative transfer of all resistance genes detected among the GBS strains included in this study (ermA, ermB, mef, tetM and tetO), in frequencies between 1.10−7 and 9.10−7, was possible from bovine donor strains to human recipient strain, but not the other way around. This is, to our knowledge, the first report of in vitro conjugation of Ery and Tet resistance genes among GBS strains recovered from different hosts. PMID:25477908

The expression of dominant negative TCF7L2 in pancreatic beta cells during the embryonic stage causes impaired glucose homeostasis.

PubMed

Shao, Weijuan; Xiong, Xiaoquan; Ip, Wilfred; Xu, Fenghao; Song, Zhuolun; Zeng, Kejing; Hernandez, Marcela; Liang, Tao; Weng, Jianping; Gaisano, Herbert; Nostro, M Cristina; Jin, Tianru

2015-04-01

Disruption of TCF7L2 in mouse pancreatic β-cells has generated different outcomes in several investigations. Here we aim to clarify role of β-cell TCF7L2 and Wnt signaling using a functional-knockdown approach. Adenovirus-mediated dominant negative TCF7L2 (TCF7L2DN) expression was conducted in Ins-1 cells. The fusion gene in which TCF7L2DN expression is driven by P TRE3G was utilized to generate the transgenic mouse line TCF7L2DN Tet . The double transgenic line was created by mating TCF7L2DN Tet with Ins2-rtTA, designated as βTCFDN. β-cell specific TCF7L2DN expression was induced in βTCFDN by doxycycline feeding. TCF7L2DN expression in Ins-1 cells reduced GSIS, cell proliferation and expression of a battery of genes including incretin receptors and β-cell transcription factors. Inducing TCF7L2DN expression in βTCFDN during adulthood or immediately after weaning generated no or very modest metabolic defect, while its expression during embryonic development by doxycycline feeding in pregnant mothers resulted in significant glucose intolerance associated with altered β-cell gene expression and reduced β-cell mass. Our observations support a cell autonomous role for TCF7L2 in pancreatic β-cells suggested by most, though not all, investigations. βTCFDN is a novel model for further exploring the role of TCF7L2 in β-cell genesis and metabolic homeostasis.

Computational identification of novel biochemical systems involved in oxidation, glycosylation and other complex modifications of bases in DNA.

PubMed

Iyer, Lakshminarayan M; Zhang, Dapeng; Burroughs, A Maxwell; Aravind, L

2013-09-01

Discovery of the TET/JBP family of dioxygenases that modify bases in DNA has sparked considerable interest in novel DNA base modifications and their biological roles. Using sensitive sequence and structure analyses combined with contextual information from comparative genomics, we computationally characterize over 12 novel biochemical systems for DNA modifications. We predict previously unidentified enzymes, such as the kinetoplastid J-base generating glycosyltransferase (and its homolog GREB1), the catalytic specificity of bacteriophage TET/JBP proteins and their role in complex DNA base modifications. We also predict the enzymes involved in synthesis of hypermodified bases such as alpha-glutamylthymine and alpha-putrescinylthymine that have remained enigmatic for several decades. Moreover, the current analysis suggests that bacteriophages and certain nucleo-cytoplasmic large DNA viruses contain an unexpectedly diverse range of DNA modification systems, in addition to those using previously characterized enzymes such as Dam, Dcm, TET/JBP, pyrimidine hydroxymethylases, Mom and glycosyltransferases. These include enzymes generating modified bases such as deazaguanines related to queuine and archaeosine, pyrimidines comparable with lysidine, those derived using modified S-adenosyl methionine derivatives and those using TET/JBP-generated hydroxymethyl pyrimidines as biosynthetic starting points. We present evidence that some of these modification systems are also widely dispersed across prokaryotes and certain eukaryotes such as basidiomycetes, chlorophyte and stramenopile alga, where they could serve as novel epigenetic marks for regulation or discrimination of self from non-self DNA. Our study extends the role of the PUA-like fold domains in recognition of modified nucleic acids and predicts versions of the ASCH and EVE domains to be novel 'readers' of modified bases in DNA. These results open opportunities for the investigation of the biology of these systems and their use in biotechnology.

Computational identification of novel biochemical systems involved in oxidation, glycosylation and other complex modifications of bases in DNA

PubMed Central

Iyer, Lakshminarayan M.; Zhang, Dapeng; Maxwell Burroughs, A.; Aravind, L.

2013-01-01

Discovery of the TET/JBP family of dioxygenases that modify bases in DNA has sparked considerable interest in novel DNA base modifications and their biological roles. Using sensitive sequence and structure analyses combined with contextual information from comparative genomics, we computationally characterize over 12 novel biochemical systems for DNA modifications. We predict previously unidentified enzymes, such as the kinetoplastid J-base generating glycosyltransferase (and its homolog GREB1), the catalytic specificity of bacteriophage TET/JBP proteins and their role in complex DNA base modifications. We also predict the enzymes involved in synthesis of hypermodified bases such as alpha-glutamylthymine and alpha-putrescinylthymine that have remained enigmatic for several decades. Moreover, the current analysis suggests that bacteriophages and certain nucleo-cytoplasmic large DNA viruses contain an unexpectedly diverse range of DNA modification systems, in addition to those using previously characterized enzymes such as Dam, Dcm, TET/JBP, pyrimidine hydroxymethylases, Mom and glycosyltransferases. These include enzymes generating modified bases such as deazaguanines related to queuine and archaeosine, pyrimidines comparable with lysidine, those derived using modified S-adenosyl methionine derivatives and those using TET/JBP-generated hydroxymethyl pyrimidines as biosynthetic starting points. We present evidence that some of these modification systems are also widely dispersed across prokaryotes and certain eukaryotes such as basidiomycetes, chlorophyte and stramenopile alga, where they could serve as novel epigenetic marks for regulation or discrimination of self from non-self DNA. Our study extends the role of the PUA-like fold domains in recognition of modified nucleic acids and predicts versions of the ASCH and EVE domains to be novel ‘readers’ of modified bases in DNA. These results open opportunities for the investigation of the biology of these systems and their use in biotechnology. PMID:23814188

Interaction centres of pyrimidine nucleotides: cytidine-5'-diphosphate (CDP) and cytidine-5'-triphosphate (CTP) in their reactions with tetramines and Cu(II) ions.

PubMed

Gasowska, A

2005-08-01

The interactions between pyrimidine nucleotides: cytidine-5'-diphosphate (CDP) and cytidine-5'-triphosphate (CTP) and Cu(II) ions, spermine (Spm) and 1,11-diamino-4,8-diazaundecane (3,3,3-tet) have been studied. The composition and stability constants of the complexes formed have been determined by means of the potentiometric method, while the centres of interactions in the ligands have been identified by the spectral methods (UV-Vis, Ultraviolet and Visible spectroscopy; EPR, electron spin resonance; NMR). In the systems without metal, formation of the molecular complexes nucleotide-polyamine with the interaction centres at the endocyclic nitrogen atom of purine ring N3, the oxygen atoms of the phosphate group from the nucleotide and protonated nitrogen atoms of the polyamine have been detected. Significant differences have been found in the metallation between the systems with Spm and with 3,3,3-tet. In the systems with spermine, mainly protonated species are formed with the phosphate group of the nucleotide and deprotonated nitrogen atoms of the polyamine making the coordination centres, while the donor nitrogen atom of the nucleotide N3 is involved in the intramolecular interligand interactions, additionally stabilising the complex. In the systems with 3,3,3-tet, the MLL' type species are formed in which the oxygen atoms of the phosphate group and nitrogen atoms of the polyamine are involved in metallation, whereas the N3 atom from the pyrimidine ring of the nucleotide is located outside the inner coordination sphere of copper ion. The main centre of Cu(II) interaction in the nucleotide, both in the system with Spm and 3,3,3-tet is the phosphate group of the nucleotide.

Effects of horizontal grid resolution on evapotranspiration partitioning using TerrSysMP

NASA Astrophysics Data System (ADS)

Shrestha, P.; Sulis, M.; Simmer, C.; Kollet, S.

2018-02-01

Biotic leaf transpiration (T) and abiotic evaporation (E) are the two major pathways by which water is transferred from land surfaces to the atmosphere. Earth system models simulating the terrestrial water, carbon and energy cycle are required to reliably embed the role of soil and vegetation processes in order to realistically reproduce both fluxes including their relative contributions to total evapotranspiration (ET). Earth system models are also being used with increasing spatial resolutions to better simulate the effects of surface heterogeneity on the regional water and energy cycle and to realistically include effects of subsurface lateral flow paths, which are expected to feed back on the exchange fluxes and their partitioning in the model. Using the hydrological component of the Terrestrial Systems Modeling Platform (TerrSysMP), we examine the uncertainty in the estimates of T/ET ratio due to horizontal model grid resolution for a dry and wet year in the Inde catchment (western Germany). The aggregation of topography results in smoothing of slope magnitudes and the filtering of small-scale convergence and divergence zones, which directly impacts the surface-subsurface flow. Coarsening of the grid resolution from 120 m to 960 m increased the available soil moisture for ground evaporation, and decreased T/ET ratio by about 5% and 8% for dry and wet year respectively. The change in T/ET ratio was more pronounced for agricultural crops compared to forested areas, indicating a strong local control of vegetation on the ground evaporation, affecting the domain average statistics.

Spectrum of mutations in RARS-T patients includes TET2 and ASXL1 mutations.

PubMed

Szpurka, Hadrian; Jankowska, Anna M; Makishima, Hideki; Bodo, Juraj; Bejanyan, Nelli; Hsi, Eric D; Sekeres, Mikkael A; Maciejewski, Jaroslaw P

2010-08-01

While a majority of patients with refractory anemia with ring sideroblasts and thrombocytosis harbor JAK2V617F and rarely MPLW515L, JAK2/MPL-negative cases constitute a diagnostic problem. 23 RARS-T cases were investigated applying immunohistochemical phospho-STAT5, sequencing and SNP-A-based karyotyping. Based on the association of TET2/ASXL1 mutations with MDS/MPN we studied molecular pattern of these genes. Two patients harbored ASXL1 and another 2 TET2 mutations. Phospho-STAT5 activation was present in one mutated TET2 and ASXL1 case. JAK2V617F/MPLW515L mutations were absent in TET2/ASXL1 mutants, indicating that similar clinical phenotype can be produced by various MPN-associated mutations and that additional unifying lesions may be present in RARS-T. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Nucleotide sequences of the tet(M) genes from the American and Dutch type tetracycline resistance plasmids of Neisseria gonorrhoeae.

PubMed

Gascoyne-Binzi, D M; Heritage, J; Hawkey, P M

1993-11-01

High-level tetracycline-resistant Neisseria gonorrhoeae (TRNG) has been associated with the presence of a plasmid approximately 25.2 MDa in size which carries a Tet M tetracycline resistance determinant. Two different plasmid types, American and Dutch, have previously been described, based on the restriction endonuclease digestion pattern. In this study, the tet(M) genes from the two plasmid types have been amplified by the polymerase chain reaction (PCR) and then sequenced. The gene sequences from the two plasmids shared 96.8% identity, and showed similarities with different segments of the tet(M) gene sequences from Tn1545, Tn916 and Ureaplasma urealyticum. The data suggest that it is highly likely that the Tet M determinant found in the American type plasmid has a different origin from that present in the Dutch plasmid.

Efficient antitumor effect of co-drug-loaded nanoparticles with gelatin hydrogel by local implantation

PubMed Central

Zhang, Hao; Tian, Yong; Zhu, Zhenshu; Xu, Huae; Li, Xiaolin; Zheng, Donghui; Sun, Weihao

2016-01-01

Tetrandrine (Tet) could enhance the antitumor effect of Paclitaxel (Ptx) by increasing intracellular Reactive Oxygen Species (ROS) levels, which leads to the possibility of co-delivery of both drugs for synergistic antitumor effect. In the current study, we reported an efficient, local therapeutic strategy employing effective Tet and Ptx delivery with a nanoparticle-loaded gelatin system. Tet- and Ptx co-loaded mPEG-PCL nanoparticles (P/T-NPs) were encapsulated into the physically cross-linked gelatin hydrogel and then implanted on the tumor site for continuous drug release. The drug-loaded gelatin hydrogel underwent a phase change when the temperature slowly increased. In vitro study showed that Tet/Ptx-loaded PEG-b-PCL nanoparticles encapsulated within a gelatin hydrogel (P/T-NPs-Gelatin) inhibited the growth and invasive ability of BGC-823 cells more effectively than the combination of free drugs or P/T-NPs. In vivo study validated the therapeutic potential of P/T-NPs-Gelatin. P/T-NPs-Gelatin significantly inhibited the activation of p-Akt and the downstream anti-apoptotic Bcl-2 protein and also inducing the activation of pro-apoptotic Bax protein. Moreover, the molecular-modulating effect of P/T-NPs-Gelatin on related proteins varied slightly under the influence of NAC, which was supported by the observations of the tumor volumes and weights. Based on these findings, local implantation of P/T-NPs-Gelatin may be a promising therapeutic strategy for the treatment of gastric cancer. PMID:27226240

Structure and Function of TET Enzymes.

PubMed

Yin, Xiaotong; Xu, Yanhui

2016-01-01

Mammalian DNA methylation mainly occurs at the carbon-C5 position of cytosine (5mC). TET enzymes were discovered to successively oxidize 5mC to 5-hydromethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). TET enzymes and oxidized 5mC derivatives play important roles in various biological and pathological processes, including regulation of DNA demethylation, gene transcription, embryonic development, and oncogenesis. In this chapter, we will discuss the discovery of TET-mediated 5mC oxidation and the structure, function, and regulation of TET enzymes.

Transgenic APP expression during postnatal development causes persistent locomotor hyperactivity in the adult.

PubMed

Rodgers, Shaefali P; Born, Heather A; Das, Pritam; Jankowsky, Joanna L

2012-06-18

Transgenic mice expressing disease-associated proteins have become standard tools for studying human neurological disorders. Transgenes are often expressed using promoters chosen to drive continuous high-level expression throughout life rather than temporal and spatial fidelity to the endogenous gene. This approach has allowed us to recapitulate diseases of aging within the two-year lifespan of the laboratory mouse, but has the potential for creating aberrant phenotypes by mechanisms unrelated to the human disorder. We show that overexpression of the Alzheimer's-related amyloid precursor protein (APP) during early postnatal development leads to severe locomotor hyperactivity that can be significantly attenuated by delaying transgene onset until adulthood. Our data suggest that exposure to transgenic APP during maturation influences the development of neuronal circuits controlling motor activity. Both when matched for total duration of APP overexpression and when matched for cortical amyloid burden, animals exposed to transgenic APP as juveniles are more active in locomotor assays than animals in which APP overexpression was delayed until adulthood. In contrast to motor activity, the age of APP onset had no effect on thigmotaxis in the open field as a rough measure of anxiety, suggesting that the interaction between APP overexpression and brain development is not unilateral. Our findings indicate that locomotor hyperactivity displayed by the tet-off APP transgenic mice and several other transgenic models of Alzheimer's disease may result from overexpression of mutant APP during postnatal brain development. Our results serve as a reminder of the potential for unexpected interactions between foreign transgenes and brain development to cause long-lasting effects on neuronal function in the adult. The tet-off APP model provides an easy means of avoiding developmental confounds by allowing transgene expression to be delayed until the mice reach adulthood.

Chronic impacts of oxytetracycline on mesophilic anaerobic digestion of excess sludge: Inhibition of hydrolytic acidification and enrichment of antibiotic resistome.

PubMed

Tian, Zhe; Zhang, Yu; Yang, Min

2018-07-01

We evaluated the chronic impact of oxytetracycline (OTC) on performance and antibiotic resistance development during the mesophilic anaerobic digestion (AD) of antibiotic-containing biomass. Mesophilic AD was conducted in a completely stirred tank reactor by constantly feeding municipal excess sludge spiked with increasing concentrations of OTC (0-1000 mg L -1 ) under a solid retention time of 20 days over a period of 265 days. Results showed that methane generation of mesophilic AD was inhibited when the OTC concentration in digested sludge was increased to around 18,000 mg kg -1 (OTC dose, 1000 mg L -1 ), due to the inhibition of fermenting and acidogenic bacteria. Metagenomic sequencing and high-throughput quantitative PCR analysis demonstrated that tetracycline resistance genes were the most dominant type (38.47-43.76%) in the resistome, with tetG, tetX, tetM, tetR, tetQ, tetO, and tetL as the dominant resistant subtypes throughout the whole experimental period. The relative abundance of these tet genes increased from 2.10 × 10 -1 before spiking OTC (OTC concentration in digested sludge, 8.97 mg kg -1 ) to 2.83 × 10 -1 (p < 0.05) after spiking OTC at a dose of 40 mg L -1 (OTC concentration in digested sludge, 528.52 mg kg -1 ). Furthermore, mobile genetic elements, including integrons, transposons, and plasmids, were also enriched with the increase in OTC dose. Based on partial canonical correspondence analysis, the contributions of horizontal (mobile element alteration) and vertical (bacterial community shift) gene transfer to antibiotic resistome variation were 29.35% and 21.51%, respectively. Thus, considering the inhibition of hydrolytic acidification and enrichment of antibiotic resistome, mesophilic AD is not suggested to directly treat the biomass containing OTC concentration higher than 200 mg L -1 . Copyright © 2018 Elsevier Ltd. All rights reserved.

Construction and modelling of an inducible positive feedback loop stably integrated in a mammalian cell-line.

PubMed

Siciliano, Velia; Menolascina, Filippo; Marucci, Lucia; Fracassi, Chiara; Garzilli, Immacolata; Moretti, Maria Nicoletta; di Bernardo, Diego

2011-06-01

Understanding the relationship between topology and dynamics of transcriptional regulatory networks in mammalian cells is essential to elucidate the biology of complex regulatory and signaling pathways. Here, we characterised, via a synthetic biology approach, a transcriptional positive feedback loop (PFL) by generating a clonal population of mammalian cells (CHO) carrying a stable integration of the construct. The PFL network consists of the Tetracycline-controlled transactivator (tTA), whose expression is regulated by a tTA responsive promoter (CMV-TET), thus giving rise to a positive feedback. The same CMV-TET promoter drives also the expression of a destabilised yellow fluorescent protein (d2EYFP), thus the dynamic behaviour can be followed by time-lapse microscopy. The PFL network was compared to an engineered version of the network lacking the positive feedback loop (NOPFL), by expressing the tTA mRNA from a constitutive promoter. Doxycycline was used to repress tTA activation (switch off), and the resulting changes in fluorescence intensity for both the PFL and NOPFL networks were followed for up to 43 h. We observed a striking difference in the dynamics of the PFL and NOPFL networks. Using non-linear dynamical models, able to recapitulate experimental observations, we demonstrated a link between network topology and network dynamics. Namely, transcriptional positive autoregulation can significantly slow down the "switch off" times, as compared to the non-autoregulated system. Doxycycline concentration can modulate the response times of the PFL, whereas the NOPFL always switches off with the same dynamics. Moreover, the PFL can exhibit bistability for a range of Doxycycline concentrations. Since the PFL motif is often found in naturally occurring transcriptional and signaling pathways, we believe our work can be instrumental to characterise their behaviour.

TET2 mutations predict response to hypomethylating agents in myelodysplastic syndrome patients

PubMed Central

Lord, Allegra; Stevenson, Kristen; Bar-Natan, Michal; Pérez-Ladaga, Albert; Zaneveld, Jacques; Wang, Hui; Caughey, Bennett; Stojanov, Petar; Getz, Gad; Garcia-Manero, Guillermo; Kantarjian, Hagop; Chen, Rui; Stone, Richard M.; Neuberg, Donna; Steensma, David P.; Ebert, Benjamin L.

2014-01-01

Only a minority of myelodysplastic syndrome (MDS) patients respond to hypomethylating agents (HMAs), but strong predictors of response are unknown. We sequenced 40 recurrently mutated myeloid malignancy genes in tumor DNA from 213 MDS patients collected before treatment with azacitidine (AZA) or decitabine (DEC). Mutations were examined for association with response and overall survival. The overall response rate of 47% was not different between agents. Clonal TET2 mutations predicted response (odds ratio [OR] 1.99, P = .036) when subclones unlikely to be detected by Sanger sequencing (allele fraction <10%) were treated as wild-type (WT). Response rates were highest in the subset of TET2 mutant patients without clonal ASXL1 mutations (OR 3.65, P = .009). Mutations of TP53 (hazard ratio [HR] 2.01, P = .002) and PTPN11 (HR 3.26, P = .006) were associated with shorter overall survival but not drug response. Murine-competitive bone marrow transplantation followed by treatment with AZA demonstrated that Tet2-null cells have an engraftment advantage over Tet2-WT cells. AZA significantly decreased this advantage for Tet2-null cells (P = .002) but not Tet2-WT cells (P = .212). Overall, Tet2 loss appears to sensitize cells to treatment with AZA in vivo, and TET2 mutations can identify patients more likely to respond to HMAs. PMID:25224413

TET2 mutations predict response to hypomethylating agents in myelodysplastic syndrome patients.

PubMed

Bejar, Rafael; Lord, Allegra; Stevenson, Kristen; Bar-Natan, Michal; Pérez-Ladaga, Albert; Zaneveld, Jacques; Wang, Hui; Caughey, Bennett; Stojanov, Petar; Getz, Gad; Garcia-Manero, Guillermo; Kantarjian, Hagop; Chen, Rui; Stone, Richard M; Neuberg, Donna; Steensma, David P; Ebert, Benjamin L

2014-10-23

Only a minority of myelodysplastic syndrome (MDS) patients respond to hypomethylating agents (HMAs), but strong predictors of response are unknown. We sequenced 40 recurrently mutated myeloid malignancy genes in tumor DNA from 213 MDS patients collected before treatment with azacitidine (AZA) or decitabine (DEC). Mutations were examined for association with response and overall survival. The overall response rate of 47% was not different between agents. Clonal TET2 mutations predicted response (odds ratio [OR] 1.99, P = .036) when subclones unlikely to be detected by Sanger sequencing (allele fraction <10%) were treated as wild-type (WT). Response rates were highest in the subset of TET2 mutant patients without clonal ASXL1 mutations (OR 3.65, P = .009). Mutations of TP53 (hazard ratio [HR] 2.01, P = .002) and PTPN11 (HR 3.26, P = .006) were associated with shorter overall survival but not drug response. Murine-competitive bone marrow transplantation followed by treatment with AZA demonstrated that Tet2-null cells have an engraftment advantage over Tet2-WT cells. AZA significantly decreased this advantage for Tet2-null cells (P = .002) but not Tet2-WT cells (P = .212). Overall, Tet2 loss appears to sensitize cells to treatment with AZA in vivo, and TET2 mutations can identify patients more likely to respond to HMAs. © 2014 by The American Society of Hematology.

Persistence of bacterial pathogens, antibiotic resistance genes, and enterococci in tidal creek tributaries.

PubMed

Jones, Chance E; Maddox, Anthony; Hurley, Dorset; Barkovskii, Andrei L

2018-05-19

Intertidal creeks form the primary hydrologic link between estuaries and land-based activities on barrier islands. Fecal indicators Enterococcus spp. (Entero1), pathogens Shigella spp. (ipaH), Salmonella spp. (invA), E. coli of EHEC/EPEC groups (eaeA), E. coli of EAEC, EIEC, and UPEC groups (set1B), E. coli of STEC group (stx1); and tetracycline resistance genes (tet(B), tet(C), tet(D), tet(E), tet(K), tet(Q), tet(W), and tet(X); TRG) were detected in the headwater of Oakdale Creek (Sapelo Island, GA) receiving runoffs from Hog Hammock village. Excavation of drainage ditches around the village caused a high increase in the incidence of the above determinants. Water samples were collected from the headwater, transferred to diffusion chambers, submersed in the headwater, saltmarsh, and mouth of the creek; and the determinants were monitored for 3 winter months. With some exceptions, their persistence decreased in order headwater > saltmarsh > mouth. Genes associated with Enterococcus spp. were the most persistent at all the sites, following in the headwater with determinants for Salmonella spp. and E. coli of EAEC, EIEC, and UPEC groups. In the mouth, the most persistent gene was eaeA indicating EHEC, EPEC, and STEC. Tet(B) and tet(C) persisted the longest in headwater and saltmarsh. No TRG persisted after 11 days in the mouth. Most determinants revealed correlations with temperature and pH, and inverse correlations with dissolved oxygen. Decay rates of the above determinants varied in the range of -0.02 to -0.81/day, and were up to 40 folds higher in the saltmarsh and mouth than in the headwater. Our data demonstrated that water parameters could to some extent predict a general trend in the fate of virulence and antibiotic resistance determinants in tidal creek tributaries but strongly suggested that their persistence in these tributaries cannot be predicted from that of enterococci, or extrapolated from one biological contaminant to another. Copyright © 2018 Elsevier Ltd. All rights reserved.

Nickel(II) Inhibits Tet-Mediated 5-Methylcytosine Oxidation by High Affinity Displacement of the Cofactor Iron(II).

PubMed

Yin, Ruichuan; Mo, Jiezhen; Dai, Jiayin; Wang, Hailin

2017-06-16

Ten-eleven translocation (Tet) family proteins are Fe(II)- and 2-oxoglutarate-dependent dioxygenases that regulate the dynamics of DNA methylation by catalyzing the oxidation of DNA 5-methylcytosine (5mC). To exert physiologically important functions, redox-active iron chelated in the catalytic center of Tet proteins directly involves the oxidation of the multiple substrates. To understand the function and interaction network of Tet dioxygenases, it is interesting to obtain high affinity and a specific inhibitor. Surprisingly, here we found that natural Ni(II) ion can bind to the Fe(II)-chelating motif (HXD) with an affinity of 7.5-fold as high as Fe(II). Consistently, we further found that Ni(II) ion can displace the cofactor Fe(II) of Tet dioxygenases and inhibit Tet-mediated 5mC oxidation activity with an estimated IC 50 of 1.2 μM. Essentially, Ni(II) can be used as a high affinity and selective inhibitor to explore the function and dynamics of Tet proteins.

Clonal hematopoiesis associated with TET2 deficiency accelerates atherosclerosis development in mice.

PubMed

Fuster, José J; MacLauchlan, Susan; Zuriaga, María A; Polackal, Maya N; Ostriker, Allison C; Chakraborty, Raja; Wu, Chia-Ling; Sano, Soichi; Muralidharan, Sujatha; Rius, Cristina; Vuong, Jacqueline; Jacob, Sophia; Muralidhar, Varsha; Robertson, Avril A B; Cooper, Matthew A; Andrés, Vicente; Hirschi, Karen K; Martin, Kathleen A; Walsh, Kenneth

2017-02-24

Human aging is associated with an increased frequency of somatic mutations in hematopoietic cells. Several of these recurrent mutations, including those in the gene encoding the epigenetic modifier enzyme TET2, promote expansion of the mutant blood cells. This clonal hematopoiesis correlates with an increased risk of atherosclerotic cardiovascular disease. We studied the effects of the expansion of Tet2 -mutant cells in atherosclerosis-prone, low-density lipoprotein receptor-deficient ( Ldlr -/- ) mice. We found that partial bone marrow reconstitution with TET2-deficient cells was sufficient for their clonal expansion and led to a marked increase in atherosclerotic plaque size. TET2-deficient macrophages exhibited an increase in NLRP3 inflammasome-mediated interleukin-1β secretion. An NLRP3 inhibitor showed greater atheroprotective activity in chimeric mice reconstituted with TET2-deficient cells than in nonchimeric mice. These results support the hypothesis that somatic TET2 mutations in blood cells play a causal role in atherosclerosis. Copyright © 2017, American Association for the Advancement of Science.

Clonal hematopoiesis associated with TET2 deficiency accelerates atherosclerosis development in mice

PubMed Central

Fuster, José J.; MacLauchlan, Susan; Zuriaga, María A.; Polackal, Maya N.; Ostriker, Allison C.; Chakraborty, Raja; Wu, Chia-Ling; Sano, Soichi; Muralidharan, Sujatha; Rius, Cristina; Vuong, Jacqueline; Jacob, Sophia; Muralidhar, Varsha; Robertson, Avril A. B.; Cooper, Matthew A.; Andrés, Vicente; Hirschi, Karen K.; Martin, Kathleen A.; Walsh, Kenneth

2017-01-01

Human aging is associated with an increased frequency of somatic mutations in hematopoietic cells. Several of these recurrent mutations, including those in the gene encoding the epigenetic modifier enzyme TET2, promote expansion of the mutant blood cells. This clonal hematopoiesis correlates with an increased risk of atherosclerotic cardiovascular disease. We studied the effects of the expansion of Tet2-mutant cells in atherosclerosis-prone, low-density lipoprotein receptor–deficient (Ldlr−/−) mice. We found that partial bone marrow reconstitution with TET2-deficient cells was sufficient for their clonal expansion and led to a marked increase in atherosclerotic plaque size. TET2-deficient macrophages exhibited an increase in NLRP3 inflammasome–mediated interleukin-1β secretion. An NLRP3 inhibitor showed greater atheroprotective activity in chimeric mice reconstituted with TET2-deficient cells than in nonchimeric mice. These results support the hypothesis that somatic TET2 mutations in blood cells play a causal role in atherosclerosis. PMID:28104796

The pINDUCER lentiviral toolkit for inducible RNA interference in vitro and in vivo

PubMed Central

Meerbrey, Kristen L.; Hu, Guang; Kessler, Jessica D.; Roarty, Kevin; Fang, Justin E.; Herschkowitz, Jason I.; Burrows, Anna E.; Ciccia, Alberto; Sun, Tingting; Schmitt, Earlene M.; Bernardi, Ronald J.; Fu, Xiaoyong; Bland, Christopher S.; Cooper, Thomas A.; Schiff, Rachel; Rosen, Jeffrey M.; Westbrook, Thomas F.; Elledge, Stephen J.

2011-01-01

The discovery of RNAi has revolutionized loss-of-function genetic studies in mammalian systems. However, significant challenges still remain to fully exploit RNAi for mammalian genetics. For instance, genetic screens and in vivo studies could be broadly improved by methods that allow inducible and uniform gene expression control. To achieve this, we built the lentiviral pINDUCER series of expression vehicles for inducible RNAi in vivo. Using a multicistronic design, pINDUCER vehicles enable tracking of viral transduction and shRNA or cDNA induction in a broad spectrum of mammalian cell types in vivo. They achieve this uniform temporal, dose-dependent, and reversible control of gene expression across heterogenous cell populations via fluorescence-based quantification of reverse tet-transactivator expression. This feature allows isolation of cell populations that exhibit a potent, inducible target knockdown in vitro and in vivo that can be used in human xenotransplantation models to examine cancer drug targets. PMID:21307310

Inhibition of the cancer-associated TASK 3 channels by magnetically induced thermal release of Tetrandrine from a polymeric drug carrier.

PubMed

Shi, Chen; Thum, Carolin; Zhang, Qian; Tu, Wei; Pelaz, Beatriz; Parak, Wolfgang J; Zhang, Yu; Schneider, Marc

2016-09-10

Two-pore domain (K2P) potassium channels have recently attracted growing interest in the field of cancer research. These channels play an important role in cancer biology specifically for cancer progression, including proliferation, migration, and apoptosis, which makes them an attractive target for novel cancer therapies. Here, we examined the effect of Tetrandrine (Tet), a natural compound known as a channel modulator, which is associated with anticancer activities, as potential drug in this regard. Xenopus oocyte with overexpression of K2P 9.1 (TASK 3) channels has been chosen as model system for this purpose. In order to release Tet and trigger the channels we developed a polymeric magnetic delivery system: Tetrandrine-Magnetite co-loaded poly (lactic-co-glycolic) acid particles. The embedded iron oxide magnetite (Fe3O4) nanoparticles (NPs) allow to inductively heat the particles by applying a high frequency alternating magnetic field, and thus trigger the release of the co-encapsulated Tet. As a proof of concept the nanoparticulate drug delivery system was heated by raising the suspension's temperature proving the temperature dependent release behaviour. Both heating approaches were then successfully applied for measuring the TASK 3 channels current in response to the released drug. It was found that the released Tet amount is sufficient to inhibit the TASK 3 channels in a dose dependent manner. Thus, such a stimulus responsive drug delivery system holds great promise as a novel approach for the treatment of various cancer types such as for the interaction with the two-pore domain potassium channels K2P 9.1. Copyright © 2016 Elsevier B.V. All rights reserved.

Antibiotic Resistance in Animal-waste-impacted Farm Soil: From Molecular Mechanisms to Microbial Evolution and Ecology

NASA Astrophysics Data System (ADS)

You, Y.; Ward, M. J.; Hilpert, M.

2012-12-01

Antibiotic resistance is a growing public health problem worldwide and the routine use of antibiotics in industrial animal production has sparked debate on whether this practice might constitute an environmental and public health concern. At a broiler farm, electromagnetic induction (EMI) surveying assisted soil sampling from a chicken-waste-impacted site and a marginally affected site. Consistent with the EMI survey, disparity existed between the two sites with regard to soil pH, tetracycline resistance (TcR) levels among heterotrophic culturable soil bacteria, and the incidence/prevalence of a number of tet and erm genes in the soils. No significant difference was observed in these aspects between the marginally affected site and several sites in a regional state forest that has not been in agricultural use for decades. Shortly after our sampling, the farm closed down and all the waste was removed. This unique change in situation offered us an unusual opportunity to examine the reversibility of any impact of the chicken waste on the soil microbial community. Two years after the event, several antibiotic resistance genes (ARGs) were still detected in the waste-impacted soil, and quantitative real-time PCR (qPCR) data showed that their relative abundance remained at substantial levels. A mobilizable tet(L)-carrying plasmid, pSU1, was identified in several chicken-waste-exposed soil bacteria of three different genera. Quantification of the plasmid's mobilization gene suggested that pSU1 had contributed to the prevalence and persistence of tet(L) in the waste-impacted soil. A second mobilizable tet(L)-carrying plasmid, pBSDMV9, isolated from the same soil, contained a region with 98.8% nucleotide identity to pSU1. The mosaic structure of the plasmids and the highly conserved nature of the tet(L) genes suggested that plasmid rearrangement favoring the acquisition of tet(L) may have occurred in the soil relatively recently. Additionally, in one chicken-waste-exposed soil isolate, Bhargavaea cecembensis DMV42A, cloning and sequencing identified a new TcR gene, tet(45), adjacent to putative arsenic resistance genes. Tet45 was shown to function as an efflux pump in Escherichia coli. A CTn3-like mobile genetic element (MGE) harboring tet(M) was also identified in DMV42A. After filter matings with DMV42A, both tet(45) and a CTn3 gene were identified in newly TcR E. coli transconjugants, indicating that both genes are transmissible. In the waste-impacted soil, tet(M) and CTn3-like elements (as well as other Tn916-family MGEs) were found by qPCR to be more abundant than tet(45). By the end of the 2-year sampling period, these tet genes and MGEs were still present at similar levels. None of them were found at the marginally affected farm site, nor at any state forest site. While none of the ARGs/MGEs identified in this study were shown to be present in the chicken waste generated on the farm (no chicken waste was analyzed), several characteristics of the ARGs (including their prevalence, persistence, transmissibility, and association with MGEs) indicate that they are perhaps not associated with the natural soil resistome, but are instead part of the potentially worrisome expanded resistome associated with agricultural practices at concentrated animal feeding operations.

Structure of Naegleria Tet-like dioxygenase (NgTet1) in complexes with a reaction intermediate 5-hydroxymethylcytosine DNA

DOE PAGES

Hashimoto, Hideharu; Pais, June E.; Dai, Nan; ...

2015-08-31

The family of ten-eleven translocation (Tet) dioxygenases is widely distributed across the eukaryotic tree of life, from mammals to the amoeboflagellate Naegleria gruberi. Like mammalian Tet proteins, the Naegleria Tet-like protein, NgTet1, acts on 5-methylcytosine (5mC) and generates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) in three consecutive, Fe(II)- and α-ketoglutarate-dependent oxidation reactions. The two intermediates, 5hmC and 5fC, could be considered either as the reaction product of the previous enzymatic cycle or the substrate for the next cycle. Here we present a new crystal structure of NgTet1 in complex with DNA containing a 5hmC. Along with the previously solvedmore » NgTet1–5mC structure, the two complexes offer a detailed picture of the active site at individual stages of the reaction cycle. In the crystal, the hydroxymethyl (OH-CH 2-) moiety of 5hmC points to the metal center, representing the reaction product of 5mC hydroxylation. The hydroxyl oxygen atom could be rotated away from the metal center, to a hydrophobic pocket formed by Ala212, Val293 and Phe295. Such rotation turns the hydroxyl oxygen atom away from the product conformation, and exposes the target CH 2 towards the metal-ligand water molecule, where a dioxygen O 2 molecule would occupy to initiate the next round of reaction by abstracting a hydrogen atom from the substrate. The Ala212-to-Val (A212V) mutant profoundly limits the product to 5hmC, probably due to the reduced hydrophobic pocket size restricts the binding of 5hmC as a substrate.« less

Nuclear accumulation of epidermal growth factor receptor and acceleration of G1/S stage by Epstein-Barr-encoded oncoprotein latent membrane protein 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tao Yongguang; Song Xing; Deng Xiyun

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is considered to be the major oncogenic protein of EBV-encoded proteins and has always been the core of the oncogenic mechanism of EBV. Advanced studies on nuclear translocation of the epidermal growth factor receptor (EGFR) family have greatly improved our knowledge of the biological function of cell surface receptors. In this study, we used the Tet-on LMP1 HNE2 cell line as a cell model, which is a dual-stable LMP1-integrated nasopharyngeal carcinoma (NPC) cell line and the expression of LMP1 which could be regulated by the Tet system. We found that LMP1 couldmore » regulate the nuclear accumulation of EGFR in a dose-dependent manner quantitatively and qualitatively. We also demonstrated that the nuclear localization sequence of EGFR played some roles in the location of the protein within the nucleus under LMP1 regulation and EGFR in the nucleus could bind to the promoters of cyclinD1 and cyclinE, respectively. We further demonstrated that EGFR is involved in the acceleration of the G1/S phase transition by LMP1 through binding to cyclinD1 and cyclinE directly. These findings provided a novel view that the acceleration of LMP1 on the G1/S transition via the nuclear accumulation of EGFR was critical in the process of nasopharyngeal carcinoma.« less

Influence of zeolite and superphosphate as additives on antibiotic resistance genes and bacterial communities during factory-scale chicken manure composting.

PubMed

Peng, Shuang; Li, Huijie; Song, Dan; Lin, Xiangui; Wang, Yiming

2018-04-30

Factory-scale chicken manure composting added with zeolite (F), superphosphate (G), or zeolite and ferrous sulfate (FL) simultaneously, were evaluate for their effects on the behaviors of antibiotic resistance genes (ARGs) and bacterial communities. After composting, ARGs in manure decreased by 67.3% in the control, whereas the reductions were 86.5%, 68.6% and 72.2% in F, G and FL, respectively. ARGs encoding ribosomal protection proteins (tetO, tetB(P), and tetM) were reduced to a greater extent than tetG, tetL, sul1 and sul2. Bacteria pathogens were also effectively removed by composting. Network analysis showed that Firmicutes were the important potential host bacteria for ARGs. The bacterial communities and environmental factors, as well as the intI gene, contributed significantly to the variation of ARGs. The ARGs and integrons were reduced more when zeolite was added than when superphosphate was added; thus, it may be useful for reducing the risks of ARGs in chicken manure. Copyright © 2018 Elsevier Ltd. All rights reserved.

Genetic diversity of Streptococcus suis clinical isolates from pigs and humans in Italy (2003-2007).

PubMed

Princivalli, M S; Palmieri, C; Magi, G; Vignaroli, C; Manzin, A; Camporese, A; Barocci, S; Magistrali, C; Facinelli, B

2009-08-20

Streptococcus suis, a major porcine pathogen, is emerging as a zoonotic agent capable of causing severe invasive disease in humans exposed to pigs or pork products. S. suis infection is rare in industrialised countries and usually arises as sporadic cases, with meningitis the most common clinical presentation in humans. Recent reports of two cases of meningitis in Sardinia and northeastern Italy prompted this first characterisation of Italian S. suis isolates. Fifty-nine S. suis strains, the two recent human strains and 57 swine clinical isolates collected between 2003 and 2007 from different Italian herds and regions, were tested for antimicrobial susceptibility, PCR-screened for virulence and antibiotic resistance genes, and subjected to molecular typing. Phenotypic and genotypic analysis demonstrated an overall high genetic diversity among isolates, the majority of which were resistant to macrolides (78%) and tetracyclines (90%). The erm(B), tet(O), mosaic tet(O/W/32/O), tet(W), and tet(M) genes were detected. The tet(O/W/32/O) gene, the most frequent tet gene after tet(O), had never been described in the genus Streptococcus before. In addition, a virulent cps2, erm(B) tet(O) clone, belonging to sequence type 1 (ST1) of the ST1 complex, was found to be prevalent and persistent in Italian swine herds. Finally, the two human isolates (both ST1) carrying cps2, erm(B) and tet(W) were seen to be closely related to each other.

Antimicrobial susceptibility, tetracycline and erythromycin resistance genes, and multilocus sequence typing of Streptococcus suis isolates from diseased pigs in China.

PubMed

Chen, Lei; Song, Yajing; Wei, Zigong; He, Hongkui; Zhang, Anding; Jin, Meilin

2013-01-01

Streptococcus suis (S. suis) is an emerging zoonotic pathogen causing significant economic losses in the swine industry. Here, we investigated the antimicrobial susceptibility, associated antibiotic-resistant determinants and sequence type (ST) of S. suis isolates from diseased pigs in China from 2008 to 2010. Serotype 2 was the most frequently observed strain (n=95) among the 106 S. suis strains collected, followed by serotypes 3 (n=3), 5 (n=3), 4 (n=2), 7 (n=1), 11 (n=1) and 28 (n=1). Multilocus sequence typing analysis revealed that ST1 (n=21) and ST7 (n=74) were the predominant STs, and serotype 2 was found to be significantly correlated with ST7 (P=0.017, Fisher's exact test) and CC1 (P=0.024, Fisher's exact test). The antimicrobial susceptibility results indicated that the antibiotic resistance rate was highest for tetracycline (99.1%), followed by azithromycin (68.9%), erythromycin (67.9%), clindamycin (67.9%), trimethoprim/sulfamethoxazole (16%), levofloxacin (2.8%), chloramphenicol (1.9%), cefaclor (0.9%) and ceftriaxone (0.9%). Antibiotic-resistant genes tet(M), tet(O), tet(O/W/32/O), tet(O/32/O), tet(S), tet(W), tet(L), tet(40), erm(B), mef(A/E) and msr(D) could be detected, and several tandem organizations of antibiotic resistance genes were also found in this study. In conclusion, S. suis strains isolated from diseased pigs in China were less diverse and multi-drug resistant.

Enrichment of the Antibiotic Resistance Gene tet(L) in an Alkaline Soil Fertilized With Plant Derived Organic Manure.

PubMed

Peng, Shuang; Dolfing, Jan; Feng, Youzhi; Wang, Yiming; Lin, Xiangui

2018-01-01

Fifteen antibiotic resistance genes (ARGs) and intI1 , a gene involved in horizontal gene transfer (HGT) of ARGs, were quantified in three different soil samples from a 22 year old field experiment that had received inorganic fertilizer (NPK), organic manure (OM; a mixture of wheat straw, soybean oil cake and cotton cake), and control fields that had received no fertilizer and manure (CK). Tet (L) was the most abundant ARG in OM, which also contained considerable levels of intI1 . Molecular analysis of yearly collected archived soils over the past 22 years showed that tet (L) and intI1 were higher in OM soils than in NPK soils. The relative abundance of tet (L) was essentially constant during these years, while the level of intI1 in OM soils decreased over time. The main genotype of tet (L) was the same in archived and in fresh soil, OM, and irrigation water. Phylogenetic analysis of the 16S rRNA genes of tetracycline-resistant bacteria (TRB) isolates indicated that the Firmucutes carrying tet (L) in OM were similar to those in the OM soil, suggesting that OM transferred TRB into the OM soils where they survived. Almost all of the TRB isolated from OM carried tet (L) and belonged to the Firmicutes . Survival of bacteria from the organic manure that carried tet (L) may be the cause of the increased level of tet (L) in OM soil.

Tet-mediated imprinting erasure in H19 locus following reprogramming of spermatogonial stem cells to induced pluripotent stem cells

USDA-ARS?s Scientific Manuscript database

Selective methylation of CpG islands at imprinting control regions (ICR) determines the monoparental expression of a subset of genes. The imprinting marks are protected from global demethylation taking place during pre-implantation development before being reset in primordial germ cells. However, it...

Detection of Endotoxin Contamination of Graphene Based Materials Using the TNF-α Expression Test and Guidelines for Endotoxin-Free Graphene Oxide Production

PubMed Central

Del Rio-Castillo, Antonio E.; Newman, Leon; Vázquez, Ester; Kostarelos, Kostas; Wick, Peter; Fadeel, Bengt

2016-01-01

Nanomaterials may be contaminated with bacterial endotoxin during production and handling, which may confound toxicological testing of these materials, not least when assessing for immunotoxicity. In the present study, we evaluated the conventional Limulus amebocyte lysate (LAL) assay for endotoxin detection in graphene based material (GBM) samples, including graphene oxide (GO) and few-layered graphene (FLG). Our results showed that some GO samples interfered with various formats of the LAL assay. To overcome this problem, we developed a TNF-α expression test (TET) using primary human monocyte-derived macrophages incubated in the presence or absence of the endotoxin inhibitor, polymyxin B sulfate, and found that this assay, performed with non-cytotoxic doses of the GBM samples, enabled unequivocal detection of endotoxin with a sensitivity that is comparable to the LAL assay. FLG also triggered TNF-α production in the presence of the LPS inhibitor, pointing to an intrinsic pro-inflammatory effect. Finally, we present guidelines for the preparation of endotoxin-free GO, validated by using the TET. PMID:27880838

Detection of Endotoxin Contamination of Graphene Based Materials Using the TNF-α Expression Test and Guidelines for Endotoxin-Free Graphene Oxide Production.

PubMed

Mukherjee, Sourav P; Lozano, Neus; Kucki, Melanie; Del Rio-Castillo, Antonio E; Newman, Leon; Vázquez, Ester; Kostarelos, Kostas; Wick, Peter; Fadeel, Bengt

2016-01-01

Nanomaterials may be contaminated with bacterial endotoxin during production and handling, which may confound toxicological testing of these materials, not least when assessing for immunotoxicity. In the present study, we evaluated the conventional Limulus amebocyte lysate (LAL) assay for endotoxin detection in graphene based material (GBM) samples, including graphene oxide (GO) and few-layered graphene (FLG). Our results showed that some GO samples interfered with various formats of the LAL assay. To overcome this problem, we developed a TNF-α expression test (TET) using primary human monocyte-derived macrophages incubated in the presence or absence of the endotoxin inhibitor, polymyxin B sulfate, and found that this assay, performed with non-cytotoxic doses of the GBM samples, enabled unequivocal detection of endotoxin with a sensitivity that is comparable to the LAL assay. FLG also triggered TNF-α production in the presence of the LPS inhibitor, pointing to an intrinsic pro-inflammatory effect. Finally, we present guidelines for the preparation of endotoxin-free GO, validated by using the TET.

Excretion of Antibiotic Resistance Genes by Dairy Calves Fed Milk Replacers with Varying Doses of Antibiotics

PubMed Central

Thames, Callie H.; Pruden, Amy; James, Robert E.; Ray, Partha P.; Knowlton, Katharine F.

2012-01-01

Elevated levels of antibiotic resistance genes (ARGs) in soil and water have been linked to livestock farms and in some cases feed antibiotics may select for antibiotic resistant gut microbiota. The purpose of this study was to examine the establishment of ARGs in the feces of calves receiving milk replacer containing no antibiotics versus subtherapeutic or therapeutic doses of tetracycline and neomycin. The effect of antibiotics on calf health was also of interest. Twenty-eight male and female dairy calves were assigned to one of the three antibiotic treatment groups at birth and fecal samples were collected at weeks 6, 7 (prior to weaning), and 12 (5 weeks after weaning). ARGs corresponding to the tetracycline (tetC, tetG, tetO, tetW, and tetX), macrolide (ermB, ermF), and sulfonamide (sul1, sul2) classes of antibiotics along with the class I integron gene, intI1, were monitored by quantitative polymerase chain reaction as potential indicators of direct selection, co-selection, or horizontal gene transfer of ARGs. Surprisingly, there was no significant effect of antibiotic treatment on the absolute abundance (gene copies per gram wet manure) of any of the ARGs except ermF, which was lower in the antibiotic-treated calf manure, presumably because a significant portion of host bacterial cells carrying ermF were not resistant to tetracycline or neomycin. However, relative abundance (gene copies normalized to 16S rRNA genes) of tetO was higher in calves fed the highest dose of antibiotic than in the other treatments. All genes, except tetC and intI1, were detectable in feces from 6 weeks onward, and tetW and tetG significantly increased (P < 0.10), even in control calves. Overall, the results provide new insight into the colonization of calf gut flora with ARGs in the early weeks. Although feed antibiotics exerted little effect on the ARGs monitored in this study, the fact that they also provided no health benefit suggests that the greater than conventional nutritional intake applied in this study overrides previously reported health benefits of antibiotics. The results suggest potential benefit of broader management strategies, and that cost and risk may be avoided by minimizing incorporation of antibiotics in milk replacer. PMID:22514550

High levels of BRC4 induced by a Tet-On 3G system suppress DNA repair and impair cell proliferation in vertebrate cells

PubMed Central

Abe, Takuya; Branzei, Dana

2014-01-01

Transient induction or suppression of target genes is useful to study the function of toxic or essential genes in cells. Here we apply a Tet-On 3G system to DT40 lymphoma B cell lines, validating it for three different genes. Using this tool, we then show that overexpression of the chicken BRC4 repeat of the tumor suppressor BRCA2 impairs cell proliferation and induces chromosomal breaks. Mechanistically, high levels of BRC4 suppress double strand break-induced homologous recombination, inhibit the formation of RAD51 recombination repair foci, reduce cellular resistance to DNA damaging agents and induce a G2 damage checkpoint-mediated cell-cycle arrest. The above phenotypes are mediated by BRC4 capability to bind and inhibit RAD51. The toxicity associated with BRC4 overexpression is exacerbated by chemotherapeutic agents and reversed by RAD51 overexpression, but it is neither aggravated nor suppressed by a deficit in the non-homologous end-joining pathway of double strand break repair. We further find that the endogenous BRCA2 mediates the cytotoxicity associated with BRC4 induction, thus underscoring the possibility that BRC4 or other domains of BRCA2 cooperate with ectopic BRC4 in regulating repair activities or mitotic cell division. In all, the results demonstrate the utility of the Tet-On 3G system in DT40 research and underpin a model in which BRC4 role on cell proliferation and chromosome repair arises primarily from its suppressive role on RAD51 functions. PMID:25218467

Differential regulation of genomic imprinting by TET proteins in embryonic stem cells.

PubMed

Liu, Lizhi; Mao, Shi-Qing; Ray, Chelsea; Zhang, Yu; Bell, Fong T; Ng, Sheau-Fang; Xu, Guo-Liang; Li, Xiajun

2015-09-01

TET proteins have been found to play an important role in active demethylation at CpG sites in mammals. There are some reports implicating their functions in removal of DNA methylation imprint at the imprinted regions in the germline. However, it is not well established whether TET proteins can also be involved in demethylation of DNA methylation imprint in embryonic stem (ES) cells. Here we report that loss of TET proteins caused a significant increase in DNA methylation at the Igf2-H19 imprinted region in ES cells. We also observed a variable increase in DNA methylation at the Peg1 imprinted region in the ES clones devoid of TET proteins, in particular in the differentiated ES cells. By contrast, we did not observe a significant increase of DNA methylation imprint at the Peg3, Snrpn and Dlk1-Dio3 imprinted regions in ES cells lacking TET proteins. Interestingly, loss of TET proteins did not result in a significant increase of DNA methylation imprint at the Igf2-H19 and Peg1 imprinted regions in the embryoid bodies (EB). Therefore, TET proteins seem to be differentially involved in maintaining DNA methylation imprint at a subset of imprinted regions in ES cells and EBs. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Molecular serotyping and antimicrobial resistance profiles of Actinobacillus pleuropneumoniae isolated from pigs in South Korea.

PubMed

Kim, Boram; Hur, Jin; Lee, Ji Yeong; Choi, Yoonyoung; Lee, John Hwa

2016-09-01

Actinobacillus pleuropneumoniae (APP) causes porcine pleuropneumonia (PP). Serotypes and antimicrobial resistance patterns in APP isolates from pigs in Korea were examined. Sixty-five APP isolates were genetically serotyped using standard and multiplex PCR (polymerase chain reaction). Antimicrobial susceptibilities were tested using the standardized disk-agar method. PCR was used to detect β-lactam, gentamicin and tetracycline-resistance genes. The random amplified polymorphic DNA (RAPD) patterns were determined by PCR. Korean pigs predominantly carried APP serotypes 1 and 5. Among 65 isolates, one isolate was sensitive to all 12 antimicrobials tested in this study. Sixty-two isolates was resistant to tetracycline and 53 isolates carried one or five genes including tet(B), tet(A), tet(H), tet(M)/tet(O), tet(C), tet(G) and/or tet(L)-1 markers. Among 64 strains, 9% and 26.6% were resistance to 10 and three or more antimicrobials, respectively. Thirteen different antimicrobial resistance patterns were observed and RAPD analysis revealed a separation of the isolates into two clusters: cluster II (6 strains resistant to 10 antimicrobials) and cluster I (the other 59 strains). Results show that APP serotypes 1 and 5 are the most common in Korea, and multi-drug resistant strains are prevalent. RAPD analysis demonstrated that six isolates resistant to 10 antimicrobials belonged to the same cluster.

TET2 mutations in B cells of patients affected by angioimmunoblastic T-cell lymphoma.

PubMed

Schwartz, Friederike H; Cai, Qian; Fellmann, Eva; Hartmann, Sylvia; Mäyränpää, Mikko I; Karjalainen-Lindsberg, Marja-Liisa; Sundström, Christer; Scholtysik, René; Hansmann, Martin-Leo; Küppers, Ralf

2017-06-01

Angioimmunoblastic T-cell lymphomas (AITLs) frequently carry mutations in the TET2 and IDH2 genes. TET2 mutations represent early genetic lesions as they had already been detected in haematopoietic precursor cells of AITL patients. We show by analysis of whole-tissue sections and microdissected PD1 + cells that the frequency of TET2-mutated AITL is presumably even higher than reported (12/13 cases in our collection; 92%). In two-thirds of informative AITLs (6/9), a fraction of B cells was also TET2-mutated. Investigation of four AITLs by TET2 and IGHV gene sequencing of single microdissected B cells showed that between 10% and 60% of polyclonal B cells in AITL lymph nodes harboured the identical TET2 mutations of the respective T-cell lymphoma clone. Thus, TET2-mutated haematopoietic precursor cells in AITL patients not only give rise to the T-cell lymphoma but also generate a large population of mutated mature B cells. Future studies will show whether this is a reason why AITL patients frequently also develop B-cell lymphomas. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Epigallocatechin-3-gallate (EGCG)-stabilized selenium nanoparticles coated with Tet-1 peptide to reduce amyloid-β aggregation and cytotoxicity.

PubMed

Zhang, Jingnan; Zhou, Xianbo; Yu, Qianqian; Yang, Licong; Sun, Dongdong; Zhou, Yanhui; Liu, Jie

2014-06-11

Alzheimer's disease (AD), the most common neurodegenerative disease, is caused by an accumulation of amyloid-β (Aβ) plaque deposits in the brains. Evidence is increasingly showing that epigallocatechin-3-gallate (EGCG) can partly protect cells from Aβ-mediated neurotoxicity by inhibiting Aβ aggregation. In order to better understand the process of Aβ aggregation and amyloid fibril disaggregation and reduce the cytotoxicity of EGCG at high doses, we attached EGCG onto the surface of selenium nanoparticles (EGCG@Se). Given the low delivery efficiency of EGCG@Se to the targeted cells and the involvement of selenoprotein in antioxidation and neuroprotection, which are the key factors for preventing the onset and progression of AD, we synthesized EGCG-stabilized selenium nanoparticles coated with Tet-1 peptide (Tet-1-EGCG@Se, a synthetic selenoprotein analogue), considering the affinity of Tet-1 peptide to neurons. We revealed that Tet-1-EGCG@Se can effectively inhibit Aβ fibrillation and disaggregate preformed Aβ fibrils into nontoxic aggregates. In addition, we found that both EGCG@Se and Tet-1-EGCG@Se can label Aβ fibrils with a high affinity, and Tet-1 peptides can significantly enhance the cellular uptake of Tet-1-EGCG@Se in PC12 cells rather than in NIH/3T3 cells.

Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T cells.

PubMed

Fraietta, Joseph A; Nobles, Christopher L; Sammons, Morgan A; Lundh, Stefan; Carty, Shannon A; Reich, Tyler J; Cogdill, Alexandria P; Morrissette, Jennifer J D; DeNizio, Jamie E; Reddy, Shantan; Hwang, Young; Gohil, Mercy; Kulikovskaya, Irina; Nazimuddin, Farzana; Gupta, Minnal; Chen, Fang; Everett, John K; Alexander, Katherine A; Lin-Shiao, Enrique; Gee, Marvin H; Liu, Xiaojun; Young, Regina M; Ambrose, David; Wang, Yan; Xu, Jun; Jordan, Martha S; Marcucci, Katherine T; Levine, Bruce L; Garcia, K Christopher; Zhao, Yangbing; Kalos, Michael; Porter, David L; Kohli, Rahul M; Lacey, Simon F; Berger, Shelley L; Bushman, Frederic D; June, Carl H; Melenhorst, J Joseph

2018-06-01

Cancer immunotherapy based on genetically redirecting T cells has been used successfully to treat B cell malignancies 1-3 . In this strategy, the T cell genome is modified by integration of viral vectors or transposons encoding chimaeric antigen receptors (CARs) that direct tumour cell killing. However, this approach is often limited by the extent of expansion and persistence of CAR T cells 4,5 . Here we report mechanistic insights from studies of a patient with chronic lymphocytic leukaemia treated with CAR T cells targeting the CD19 protein. Following infusion of CAR T cells, anti-tumour activity was evident in the peripheral blood, lymph nodes and bone marrow; this activity was accompanied by complete remission. Unexpectedly, at the peak of the response, 94% of CAR T cells originated from a single clone in which lentiviral vector-mediated insertion of the CAR transgene disrupted the methylcytosine dioxygenase TET2 gene. Further analysis revealed a hypomorphic mutation in this patient's second TET2 allele. TET2-disrupted CAR T cells exhibited an epigenetic profile consistent with altered T cell differentiation and, at the peak of expansion, displayed a central memory phenotype. Experimental knockdown of TET2 recapitulated the potency-enhancing effect of TET2 dysfunction in this patient's CAR T cells. These findings suggest that the progeny of a single CAR T cell induced leukaemia remission and that TET2 modification may be useful for improving immunotherapies.

Distributive Processing by the Iron(II)/α-Ketoglutarate-Dependent Catalytic Domains of the TET Enzymes Is Consistent with Epigenetic Roles for Oxidized 5-Methylcytosine Bases.

PubMed

Tamanaha, Esta; Guan, Shengxi; Marks, Katherine; Saleh, Lana

2016-08-03

The ten-eleven translocation (TET) proteins catalyze oxidation of 5-methylcytosine ((5m)C) residues in nucleic acids to 5-hydroxymethylcytosine ((5hm)C), 5-formylcytosine ((5f)C), and 5-carboxycytosine ((5ca)C). These nucleotide bases have been implicated as intermediates on the path to active demethylation, but recent reports have suggested that they might have specific regulatory roles in their own right. In this study, we present kinetic evidence showing that the catalytic domains (CDs) of TET2 and TET1 from mouse and their homologue from Naegleria gruberi, the full-length protein NgTET1, are distributive in both chemical and physical senses, as they carry out successive oxidations of a single (5m)C and multiple (5m)C residues along a polymethylated DNA substrate. We present data showing that the enzyme neither retains (5hm)C/(5f)C intermediates of preceding oxidations nor slides along a DNA substrate (without releasing it) to process an adjacent (5m)C residue. These findings contradict a recent report by Crawford et al. ( J. Am. Chem. Soc. 2016 , 138 , 730 ) claiming that oxidation of (5m)C by CD of mouse TET2 is chemically processive (iterative). We further elaborate that this distributive mechanism is maintained for TETs in two evolutionarily distant homologues and posit that this mode of function allows the introduction of (5m)C forms as epigenetic markers along the DNA.

MicroRNA-133 modulates the β1-adrenergic receptor transduction cascade.

PubMed

Castaldi, Alessandra; Zaglia, Tania; Di Mauro, Vittoria; Carullo, Pierluigi; Viggiani, Giacomo; Borile, Giulia; Di Stefano, Barbara; Schiattarella, Gabriele Giacomo; Gualazzi, Maria Giovanna; Elia, Leonardo; Stirparo, Giuliano Giuseppe; Colorito, Maria Luisa; Pironti, Gianluigi; Kunderfranco, Paolo; Esposito, Giovanni; Bang, Marie-Louise; Mongillo, Marco; Condorelli, Gianluigi; Catalucci, Daniele

2014-07-07

The sympathetic nervous system plays a fundamental role in the regulation of myocardial function. During chronic pressure overload, overactivation of the sympathetic nervous system induces the release of catecholamines, which activate β-adrenergic receptors in cardiomyocytes and lead to increased heart rate and cardiac contractility. However, chronic stimulation of β-adrenergic receptors leads to impaired cardiac function, and β-blockers are widely used as therapeutic agents for the treatment of cardiac disease. MicroRNA-133 (miR-133) is highly expressed in the myocardium and is involved in controlling cardiac function through regulation of messenger RNA translation/stability. To determine whether miR-133 affects β-adrenergic receptor signaling during progression to heart failure. Based on bioinformatic analysis, β1-adrenergic receptor (β1AR) and other components of the β1AR signal transduction cascade, including adenylate cyclase VI and the catalytic subunit of the cAMP-dependent protein kinase A, were predicted as direct targets of miR-133 and subsequently validated by experimental studies. Consistently, cAMP accumulation and activation of downstream targets were repressed by miR-133 overexpression in both neonatal and adult cardiomyocytes following selective β1AR stimulation. Furthermore, gain-of-function and loss-of-function studies of miR-133 revealed its role in counteracting the deleterious apoptotic effects caused by chronic β1AR stimulation. This was confirmed in vivo using a novel cardiac-specific TetON-miR-133 inducible transgenic mouse model. When subjected to transaortic constriction, TetON-miR-133 inducible transgenic mice maintained cardiac performance and showed attenuated apoptosis and reduced fibrosis compared with control mice. miR-133 controls multiple components of the β1AR transduction cascade and is cardioprotective during heart failure. © 2014 American Heart Association, Inc.

Homology among tet determinants in conjugative elements of streptococci.

PubMed Central

Smith, M D; Hazum, S; Guild, W R

1981-01-01

A mutation to tetracycline sensitivity in a resistant strain of Streptococcus pneumoniae was shown by several criteria to be due to a point mutation in the conjugative omega (cat-tet) element found in the chromosomes of strains derived from BM6001, a clinical strain resistant to tetracycline and chloramphenicol. Strains carrying the mutation were transformed back to tetracycline resistance with the high efficiency of a point marker by donor deoxyribonucleic acids from its ancestral strain and from nine other clinical isolates of pneumococcus and by deoxyribonucleic acids from group D Streptococcus faecalis and group B Streptococcus agalactiae strains that also carry conjugative tet elements in their chromosomes. It was not transformed to resistance by tet plasmid deoxyribonucleic acids from either gram-negative or gram-positive species, except for one that carried transposon Tn916, the conjugative tet element present in the chromosomes of some S. faecalis strains. The results showed that the tet determinants in conjugative elements of several streptococcal species share a high degree of deoxyribonucleic acid sequence homology and suggested that they differ from other tet genes. PMID:6270063

Quantitative Analysis and Stability of the Rodenticide TETS ...

EPA Pesticide Factsheets

Journal Article The determination of the rodenticide tetramethylenedisulfotetramine (TETS) in drinking water is reportable through the use of automated sample preparation via solid phase extraction and detection using isotope dilution gas chromatography-mass spectrometry. The method was characterized over twenty-two analytical batches with quality control samples. Accuracies for low and high concentration quality control pools were 100 and 101%, respectively. The minimum reporting level (MRL) for TETS in this method is 0.50 ug/L. Five drinking waters representing a range of water quality parameters and disinfection practices were fortified with TETS at ten times the MRL and analyzed over a 28 day period to determine the stability of TETS in these waters. The amount of TETS measured in these samples averaged 100 ± 6% of the amount fortified suggesting that tap water samples may be held for up to 28 days prior to analysis.

Antibiotic Susceptibility Profiles of Dairy Leuconostoc, Analysis of the Genetic Basis of Atypical Resistances and Transfer of Genes In Vitro and in a Food Matrix

PubMed Central

Delgado, Susana; Alegría, Ángel; Salvetti, Elisa; Felis, Giovanna E.; Mayo, Baltasar; Torriani, Sandra

2016-01-01

In spite of a global concern on the transfer of antibiotic resistances (AR) via the food chain, limited information exists on this issue in species of Leuconostoc and Weissella, adjunct cultures used as aroma producers in fermented foods. In this work, the minimum inhibitory concentration was determined for 16 antibiotics in 34 strains of dairy origin, belonging to Leuconostoc mesenteroides (18), Leuconostoc citreum (11), Leuconostoc lactis (2), Weissella hellenica (2), and Leuconostoc carnosum (1). Atypical resistances were found for kanamycin (17 strains), tetracycline and chloramphenicol (two strains each), and erythromycin, clindamycin, virginiamycin, ciprofloxacin, and rifampicin (one strain each). Surprisingly, L. mesenteroides subsp. mesenteroides LbE16, showed resistance to four antibiotics, kanamycin, streptomycin, tetracycline and virginiamycin. PCR analysis identified tet(S) as responsible for tetracycline resistance in LbE16, but no gene was detected in a second tetracycline-resistant strain, L. mesenteroides subsp. cremoris LbT16. In Leuconostoc mesenteroides subsp. dextranicum LbE15, erythromycin and clindamycin resistant, an erm(B) gene was amplified. Hybridization experiments proved erm(B) and tet(S) to be associated to a plasmid of ≈35 kbp and to the chromosome of LbE15 and LbE16, respectively. The complete genome sequence of LbE15 and LbE16 was used to get further insights on the makeup and genetic organization of AR genes. Genome analysis confirmed the presence and location of erm(B) and tet(S), but genes providing tetracycline resistance in LbT16 were again not identified. In the genome of the multi-resistant strain LbE16, genes that might be involved in aminoglycoside (aadE, aphA-3, sat4) and virginiamycin [vat(E)] resistance were further found. The erm(B) gene but not tet(S) was transferred from Leuconostoc to Enterococcus faecalis both under laboratory conditions and in cheese. This study contributes to the characterization of AR in the Leuconostoc-Weissella group, provides evidence of the genetic basis of atypical resistances, and demonstrates the inter-species transfer of erythromycin resistance. PMID:26726815

Characteristics of Escherichia coli from raw vegetables at a retail market in the Czech Republic.

PubMed

Skočková, Alena; Karpíšková, Renáta; Koláčková, Ivana; Cupáková, Šárka

2013-10-15

A large epidemic caused by shigatoxigenic Escherichia coli (E. coli) in spring 2011 in Germany resulted in reduction of trust in the health safety of raw vegetables and sprouted seeds. This study focused on the detection and characterization of E. coli in raw vegetables and sprouted seeds sold in the Czech Republic. Out of 91 samples, 24 (26.4%) were positive for the presence of E. coli. Resistance to antimicrobial agents was determined by the disk diffusion method and E-test. Polymerase chain reaction was used for the detection of selected genes encoding virulence--eaeA, hly, stx1, and stx2 and genes encoding resistance to tetracycline--tet(A), tet(B), tet(C), and tet(G) and to β-lactams--blaTEM, blaSHV, and blaCTX. The blaTEM gene was detected in two isolates, the tet(B) gene in three and tet(A) in one isolate. No hly, stx1, or stx2 genes were present, but the eaeA gene was found in three (11.1%) isolates from imported vegetables. These isolates can be considered as potentially enteropathogenic. Results of this study show that raw vegetables and sprouted seeds sold in the retail market can represent a potential risk for consumers. © 2013.

Antimicrobial-Resistant Fecal Bacteria from Ceftiofur-Treated and Nonantimicrobial-Treated Comingled Beef Cows at a Cow-Calf Operation.

PubMed

Agga, Getahun E; Schmidt, John W; Arthur, Terrance M

2016-10-01

We compared the occurrences of 3rd-generation cephalosporin-resistant (3GC r ), tetracycline-resistant (TET r ), and trimethoprim-sulfamethoxazole-resistant (COT r ) Escherichia coli, 3GC r and nalidixic acid-resistant (NAL r ) Salmonella enterica, and erythromycin-resistant (ERY r ) enterococci from the fecal samples of ceftiofur-treated (n = 162) and nonantimicrobial-treated (n = 207) comingled beef cows ≥8 years old, for which complete antimicrobial treatment records were available. The prevalence of 3GC r (17%; n = 369), TET r (88%), COT r E. coli (22%), and ERY r enterococci (69%) was not significantly (p > 0.05) associated with ceftiofur treatment, prior history of other antimicrobial treatments, or duration of time between last antimicrobial treatment and sampling. 3GC r and NAL r S. enterica were not detected. The prevalence of tetB was significantly (p < 0.05) higher compared with tetA among TET r E. coli. However, the prevalence of tetA was significantly (p < 0.05) higher than tetB among 3GC r and COT r E. coli. There was a significant (p < 0.05) association between tetM and ermB among ERY r enterococci. In conclusion, occurrences of 3GC r , TET r , and COT r E. coli and ERY r enterococci in comingled antimicrobial-treated and nonantimicrobial-treated beef cows were not associated with ceftiofur or other antimicrobial use, indicating that other factors influenced the observed levels of antimicrobial-resistant bacteria in feces of beef cows.

Frequency and Genetic Determinants of Tigecycline Resistance in Clinically Isolated Stenotrophomonas maltophilia in Beijing, China.

PubMed

Zhao, Jin; Liu, Yunxi; Liu, Yi; Wang, Dong; Ni, Wentao; Wang, Rui; Liu, Youning; Zhang, Bo

2018-01-01

Stenotrophomonas maltophilia is an emerging nosocomial pathogen with high resistance to most clinically used antimicrobials. Tigecycline is a potential alternative antimicrobial for S. maltophilia infection treatment, but its resistance mechanism in clinical isolates is not fully elucidated. We investigated the antimicrobial susceptibility of 450 S. maltophilia isolated during 2012-2015 from three university hospitals in Beijing, China. These strains exhibited high susceptibility to minocycline (98.44%), sulfamethoxazole/trimethoprim (87.56%), tigecycline (77.78 %), doxycycline (81.33%), levofloxacin (67.56%), and ticarcillin/clavulanate (73.00%). The susceptibility of tigecycline-nonsusceptible strains (TNS) to doxycycline and levofloxacin was much lower than that of tigecycline-susceptible strains (TSS) (25.00% vs. 97.71% for doxycycline, P < 0.001; 17.00% vs. 82.00% for levofloxacin, P < 0.001). We further selected 48 TNS and TSS and compared the detection rate of eight tetracycline-specific genes by PCR and the expression level of six intrinsic multidrug resistance efflux pumps by real-time PCR. Only one tetB and two tetH genes in TNS and three tetH genes in TSS were detected, and the detection rate had no difference. The average expression level of smeD in TNS was higher than that in TSS [20.59 (11.53, 112.54) vs. 2.07 (0.80, 4.96), P < 0.001], while the average expression levels of smeA , smeI , smeO , smeV , and smrA were not significantly different, indicating that smeDEF was the predominant resistance genetic determinant in clinical S. maltophilia . Higher smeD expression was also observed in levofloxacin- and doxycycline-nonsusceptible isolates than in their corresponding susceptible isolates [16.46 (5.83, 102.24) vs. 2.72 (0.80, 6.25) for doxycycline, P < 0.001; 19.69 (8.07, 115.10) vs. 3.01(1.00, 6.03), P < 0.001], indicating that smeDEF was also the resistance genetic determinant to levofloxacin and doxycycline. The consistent resistance profile and common resistance genetic determinant highlight the importance of rational use of tigecycline for preventing the occurrence and spread of multidrug resistance.

Detection of the tetM resistance determinant among phenotypically sensitive Ureaplasma species by a novel real-time PCR method.

PubMed

Kotrotsiou, Tzimoula; Tzimoula, Kotrotsiou; Exindari, Maria; Maria, Exindari; Diza, Eudoxia; Eudoxia, Diza; Gioula, Georgia; Georgia, Gioula; Melidou, Angeliki; Angeliki, Melidou; Malisiovas, Nikolaos; Nikolaos, Malisiovas

2015-02-01

The study aimed to identify the proportion of tetM-positive Ureaplasma spp. isolates phenotypically susceptible to tetracycline by real-time PCR. Ureaplasma spp. strains of urogenital origin were isolated from 100 female or male adults on A7 agar plates. The presence of Ureaplasma was confirmed by the presence of urease gene by a novel real-time PCR method. Genotyping and sensitivity to tetracyclines were examined using commercial methods. The tetM gene was detected by a novel real-time PCR method especially designed for this study. Ureaplasma parvum was isolated from 87 of the specimens; Ureaplasma urealyticum, from 12; and both species were isolated from a single specimen. All isolates were phenotypically susceptible to tetracyclines. Thirty-five strains were tetM carriers; 29 (82.9%), U. parvum; 5 (14.3%), U. urealyticum; and 1 (2.9%), U. parvum/U. urealyticum. No statistically significant difference was observed between the 3 groups. Four (40%) tetM carriers were isolated from 10 symptomatic men; 11 (32.4%), from 34 symptomatic women; and 20 (35.7%), from 56 asymptomatic women. No statistically significant difference was observed between the 3 groups. The tetM determinant is detected in 35% of phenotypically susceptible to tetracycline Ureaplasma spp. Greek isolates. The use of a real-time PCR technique is particularly helpful, as it makes its detection easy; cost-effective; rapid; and, therefore, more convenient for the surveillance of the dissemination of the tetM resistance gene. Copyright © 2015 Elsevier Inc. All rights reserved.

Determining the efficiency of subjecting finely dispersed emulsions to physical coagulation in a packed layer under turbulent conditions

NASA Astrophysics Data System (ADS)

Laptev, A. G.; Basharov, M. M.; Farakhova, A. I.

2013-09-01

The process through which small droplets contained in emulsions are physically coagulated on the surface of random packing elements is considered. The theory of turbulent migration of a finely dispersed phase is used for determining the coagulation efficiency. Expressions for calculating coagulation efficiency and turbulent transfer rate are obtained by applying models of a turbulent boundary layer. An example of calculating the enlargement of water droplets in hydrocarbon medium represented by a wide fraction of light hydrocarbons (also known as natural gas liquid) is given. The process flowchart of a system for removing petroleum products from effluent waters discharged from the Kazan TETs-1 cogeneration station is considered. Replacement of the mechanical filter by a thin-layer settler with a coagulator is proposed.

Occurrence of antibiotics and antibiotic resistance genes in a sewage treatment plant and its effluent-receiving river.

PubMed

Xu, Jian; Xu, Yan; Wang, Hongmei; Guo, Changsheng; Qiu, Huiyun; He, Yan; Zhang, Yuan; Li, Xiaochen; Meng, Wei

2015-01-01

The extensive use of antibiotics has caused the contamination of both antibiotics and antibiotic resistance genes (ARGs) in the environment. In this study, the abundance and distribution of antibiotics and ARGs from a sewage treatment plant (STP) and its effluent-receiving river in Beijing China were characterized. Three classes of antibiotics including tetracycline, sulfonamide and quinolone were quantified by LC-MS/MS. In the secondary effluent they were detected at 195, 2001 and 3866 ng L(-1), respectively, which were higher than in the receiving river water. A total of 13 ARGs (6 tet genes: tetA, tetB, tetE, tetW, tetM and tetZ, 3 sulfonamide genes: sul1, sul2 and sul3, and 4 quinolone genes: gryA, parC, qnrC and qnrD) were determined by quantitative PCR. For all ARGs, sulfonamide resistance genes were present at relatively high concentrations in all samples, with the highest ARG concentration above 10(-1). ARGs remained relatively stable along each sewage treatment process. The abundances of detected ARGs from the STP were also higher than its receiving river. Bivariate correlation analysis showed that relative tet gene copies (tetB/16S-rRNA and tetW/16S-rRNA) were strongly correlated with the concentrations of tetracycline residues (r(2)>0.8, p<0.05), while no significant correlations occurred between sulfonamides and sul genes. A negative correlation between the relative abundance of quinolone resistance gene (qnrC/16S-rRNA) and the concentrations of enrofloxacin (ENR) was also determined. The difference of ARGs levels in the raw influent and secondary effluent suggested that the STP treatment process may induce to increase the abundance of resistance genes. The results showed that the sewage was an important repository of the resistance genes, which need to be effectively treated before discharge into the natural water body. Copyright © 2014 Elsevier Ltd. All rights reserved.

Feasibility of controlling CD38-CAR T cell activity with a Tet-on inducible CAR design

PubMed Central

Poels, Renée; Mulders, Manon J.; van de Donk, Niels W. C. J.; Themeli, Maria; Lokhorst, Henk M.; Mutis, Tuna

2018-01-01

Recent clinical advances with chimeric antigen receptor (CAR) T cells have led to the accelerated clinical approval of CD19-CARs to treat acute lymphoblastic leukemia. The CAR T cell therapy is nevertheless associated with toxicities, especially if the CARs are not entirely tumor-specific. Therefore, strategies for controlling the CAR T cell activity are required to improve their safety profile. Here, by using the multiple myeloma (MM)-associated CD38 molecule as target molecule, we tested the feasibility and utility of a doxycycline (DOX) inducible Tet-on CD38-CAR design to control the off-target toxicities of CAR T cells. Using CARs with high affinity to CD38, we demonstrate that this strategy allows the proper induction of CD38-CARs and CAR-mediated T cell cytotoxicity in a DOX-dose dependent manner. Especially when the DOX dose was limited to 10ng/ml, its removal resulted in a relatively rapid decay of CAR- related off-tumor effects within 24 hours, indicating the active controllability of undesired CAR activity. This Tet-on CAR design also allowed us to induce the maximal anti-MM cytotoxic activity of affinity-optimized CD38-CAR T cells, which already display a low toxicity profile, hereby adding a second level of safety to these cells. Collectively, these results indicate the possibility to utilize this DOX inducible CAR-design to actively regulate the CAR-mediated activities of therapeutic T cells. We therefore conclude that the Tet-on system may be more advantageous above suicide-genes to control the potential toxicities of CAR T cells without the need to destroy them permanently. PMID:29847570

Antimicrobial resistance genes in Actinobacillus pleuropneumoniae, Haemophilus parasuis and Pasteurella multocida isolated from Australian pigs.

PubMed

Dayao, Dae; Gibson, J S; Blackall, P J; Turni, C

2016-07-01

To identify genes associated with the observed antimicrobial resistance in Actinobacillus pleuropneumoniae, Haemophilus parasuis and Pasteurella multocida isolated from Australian pigs. Isolates with known phenotypic resistance to β-lactams, macrolides and tetracycline were screened for the presence of antimicrobial resistance genes. A total of 68 A. pleuropneumoniae, 62 H. parasuis and 20 P. multocida isolates exhibiting phenotypic antimicrobial resistance (A. pleuropneumoniae and P. multocida) or elevated minimal inhibitory concentrations (MICs) (H. parasuis) to any of the following antimicrobial agents - ampicillin, erythromycin, penicillin, tetracycline, tilmicosin and tulathromycin - were screened for a total of 19 associated antimicrobial resistance genes (ARGs) by PCR. The gene bla ROB-1 was found in all ampicillin- and penicillin-resistant isolates, but none harboured the bla TEM-1 gene. The tetB gene was found in 76% (74/97) of tetracycline-resistant isolates, 49/53 A. pleuropneumoniae, 17/30 H. parasuis and 8/14 P. multocida. One A. pleuropneumoniae isolate harboured the tetH gene, but none of the 97 isolates had tetA, tetC, tetD, tetE, tetL, tetM or tetO. A total of 92 isolates were screened for the presence of macrolide resistance genes. None was found to have ermA, ermB, ermC, erm42, mphE, mefA, msrA or msrE. The current study has provided a genetic explanation for the resistance or elevated MIC of the majority of isolates of Australian porcine respiratory pathogens to ampicillin, penicillin and tetracycline. However, the macrolide resistance observed by phenotypic testing remains genetically unexplained and further studies are required. © 2016 Australian Veterinary Association.

Genetic analysis of leukemic transformation of chronic myeloproliferative neoplasms

PubMed Central

Abdel-Wahab, Omar; Manshouri, Taghi; Patel, Jay; Harris, Kelly; Yao, JinJuan; Hedvat, Cyrus; Heguy, Adriana; Bueso-Ramos, Carlos; Kantarjian, Hagop; Levine, Ross L.; Verstovsek, Srdan

2009-01-01

The genetic events which contribute to transformation of myeloproliferative neoplasms (MPN) to acute myeloid leukemia (AML) are not well characterized. We investigated the role of JAK2, TET2, ASXL1, and IDH1 mutations in leukemic transformation of MPNs through mutational analysis of 63 patients with AML secondary to a preexisting MPN (sAML). We identified frequent TET2 (26.3%), ASXL1 (19.3%), IDH1 (9.5%), and JAK2 (36.8%) mutations in sAML; all possible mutational combinations of these genes were observed. Analysis of 14 patients for which paired samples from MPN and sAML were available demonstrated TET2 mutations were frequently acquired at leukemic transformation (6/14=43%). In contrast, ASXL1 mutations were almost always detected in both the MPN and AML clones from individual patients. A case was also observed where TET2 and ASXL1 mutations were found before the patient acquired a JAK2 mutation or developed clinical evidence of MPN. We conclude that mutations in TET2, ASXL1, and IDH1 are common in sAML derived from a pre-existing MPN. Although TET2/ASXL1 mutations may precede acquisition of JAK2 mutations by the MPN clone, mutations in TET2, but not ASXL1, are commonly acquired at the time of leukemic transformation. These data suggest the mutational order of events in MPN and sAML varies in different patients, and that TET2 and ASXL1 mutations have distinct roles in MPN pathogenesis and leukemic transformation. The presence of sAML with no pre-existing JAK2/TET2/ASXL1/IDH1 mutations indicates the existence of other mutations necessary for leukemic transformation. PMID:20068184

Partitioning Evapotranspiration for Three Typical Ecosystems in the Heihe River Basin, Northwestern China

NASA Astrophysics Data System (ADS)

Zhou, S.; Yu, B.; Zhang, Y.; Huang, Y.; Wang, G.

2017-12-01

It is crucial to improve water use efficiency (WUE) and the transpiration fraction of evapotranspiration (T/ET) for water conservation in arid regions. As a link between carbon and water cycling, WUE is defined as the ratio of gross primary productivity (GPP) and ET at the ecosystem scale. By incorporating the effect of vapor pressure deficit (VPD), two underlying WUE (uWUE) formulations, i.e. a potential uWUE (uWUEp=GPP·VPD0.5/T) and an apparent uWUE (uWUEa=GPP·VPD0.5/ET), were proposed. uWUEp is nearly constant for a given vegetation type, while uWUEa varies with T/ET. The ratio of uWUEa and uWUEp was then used to estimate T/ET. This new method for ET partitioning was applied to three typical ecosystems in the Heihe River Basin. Growing season T/ET at the Daman site (0.63) was higher than that at the Arou and Huyanglin sites (0.55) due to the application of plastic film mulching. The effect of leaf area index (LAI) on seasonal variations in T/ET was strong for Arou (R2=0.74) and Daman (R2=0.76) sites, but weak for Huyanglin (R2=0.44) site. Daily T/ET derived using the uWUE method agreed with that using the isotope and lysimeter/eddy covariance methods during the peak growth season at the Daman site. The estimated T using the uWUE method showed consistent seasonal and diurnal patterns and magnitudes with that using the sap flow method at the Huyanglin site. In addition, the uWUE method is scale-independent, and can effectively capture T/ET variations in relation to LAI changes and the abrupt T/ET changes in response to individual irrigation events. These advantages make the uWUE method more effective for ET partitioning at the ecosystem scale, and can be used for water resources management by predicting seasonal pattern of irrigation water requirements in arid regions.

Reactions of rhenium(V) and molybdenum(V) oxypentahalides with 5,7,7,12,14,14-hexamethyl-1,4,8,11-tetraazacyclotetradecane

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ashurova, N.Kh.; Yakubov, K.G.

1993-11-01

The author studied complexation reactions of rhenium(V) and molybdenum(V) with a macrocyclic ligand 5,7,7,12,14,14-hexamethyl-1,4,8,11-tetraazacyclotetradecane (Tet a). The authors found that in hydrohalic acid medium, the onium complexes [H{sub 2}(Tet a)][EOX{sub 5}] are formed, with E = Re, Mo; X = Cl, Br. Reactions of [EOX{sub 5}]{sup 2{minus}} with (Tet a) in nitrometane yield complexes of molecular and cation types [EO(Tet a)Cl{sub 3}] and [EO(Tet a)Br]Br{sub 2}. Composition and structure of the obtained compounds are established by elemental analysis, conductometry, and mid- and far-IR spectroscopy.

Atomic layer deposition of tin oxide and zinc tin oxide using tetraethyltin and ozone

DOE Office of Scientific and Technical Information (OSTI.GOV)

Warner, Ellis J.; Gladfelter, Wayne L., E-mail: wlg@umn.edu; Johnson, Forrest

Silicon or glass substrates exposed to sequential pulses of tetraethyltin (TET) and ozone (O{sub 3}) were coated with thin films of SnO{sub 2}. Self-limiting deposition was found using 8 s pulse times, and a uniform thickness per cycle (TPC) of 0.2 nm/cycle was observed in a small, yet reproducible, temperature window from 290 to 320 °C. The as-deposited, stoichiometric SnO{sub 2} films were amorphous and transparent above 400 nm. Interspersing pulses of diethylzinc and O{sub 3} among the TET:O{sub 3} pulses resulted in deposition of zinc tin oxide films, where the fraction of tin, defined as [at. % Sn/(at. % Sn + at. %more » Zn)], was controlled by the ratio of TET pulses, specifically n{sub TET}:(n{sub TET} + n{sub DEZ}) where n{sub TET} and n{sub DEZ} are the number of precursor/O{sub 3} subcycles within each atomic layer deposition (ALD) supercycle. Based on film thickness and composition measurements, the TET pulse time required to reach saturation in the TPC of SnO{sub 2} on ZnO surfaces was increased to >30 s. Under these conditions, film stoichiometry as a function of the TET pulse ratio was consistent with the model devised by Elliott and Nilsen. The as-deposited zinc tin oxide (ZTO) films were amorphous and remained so even after annealing at 450 °C in air for 1 h. The optical bandgap of the transparent ZTO films increased as the tin concentration increased. Hall measurements established that the n-type ZTO carrier concentration was 3 × 10{sup 17} and 4 × 10{sup 18} cm{sup −3} for fractional tin concentrations of 0.28 and 0.63, respectively. The carrier mobility decreased as the concentration of tin increased. A broken gap pn junction was fabricated using ALD-deposited ZTO and a sputtered layer of cuprous oxide. The junction demonstrated ohmic behavior and low resistance consistent with similar junctions prepared using sputter-deposited ZTO.« less

High levels of BRC4 induced by a Tet-On 3G system suppress DNA repair and impair cell proliferation in vertebrate cells.

PubMed

Abe, Takuya; Branzei, Dana

2014-10-01

Transient induction or suppression of target genes is useful to study the function of toxic or essential genes in cells. Here we apply a Tet-On 3G system to DT40 lymphoma B cell lines, validating it for three different genes. Using this tool, we then show that overexpression of the chicken BRC4 repeat of the tumor suppressor BRCA2 impairs cell proliferation and induces chromosomal breaks. Mechanistically, high levels of BRC4 suppress double strand break-induced homologous recombination, inhibit the formation of RAD51 recombination repair foci, reduce cellular resistance to DNA damaging agents and induce a G2 damage checkpoint-mediated cell-cycle arrest. The above phenotypes are mediated by BRC4 capability to bind and inhibit RAD51. The toxicity associated with BRC4 overexpression is exacerbated by chemotherapeutic agents and reversed by RAD51 overexpression, but it is neither aggravated nor suppressed by a deficit in the non-homologous end-joining pathway of double strand break repair. We further find that the endogenous BRCA2 mediates the cytotoxicity associated with BRC4 induction, thus underscoring the possibility that BRC4 or other domains of BRCA2 cooperate with ectopic BRC4 in regulating repair activities or mitotic cell division. In all, the results demonstrate the utility of the Tet-On 3G system in DT40 research and underpin a model in which BRC4 role on cell proliferation and chromosome repair arises primarily from its suppressive role on RAD51 functions. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Dual genetic selection of synthetic riboswitches in Escherichia coli.

PubMed

Nomura, Yoko; Yokobayashi, Yohei

2014-01-01

This chapter describes a genetic selection strategy to engineer synthetic riboswitches that can chemically regulate gene expression in Escherichia coli. Riboswitch libraries are constructed by randomizing the nucleotides that potentially comprise an expression platform and fused to the hybrid selection/screening marker tetA-gfpuv. Iterative ON and OFF selections are performed under appropriate conditions that favor the survival or the growth of the cells harboring the desired riboswitches. After the selection, rapid screening of individual riboswitch clones is performed by measuring GFPuv fluorescence without subcloning. This optimized dual genetic selection strategy can be used to rapidly develop synthetic riboswitches without detailed computational design or structural knowledge.

Cellular immune reaction in the pancreas is induced by constitutively active IκB kinase‐2

PubMed Central

Aleksic, Tamara; Baumann, Bernd; Wagner, Martin; Adler, Guido; Wirth, Thomas

2007-01-01

Background Activation of the nuclear factor κB (NF‐κB) system is a major event in acute and chronic inflammatory processes. NF‐κB cascades are comprised of IκB kinases, IκBs and NF‐κB dimers. Little is known of the individual roles of these proteins in organ specific inflammation. The aim of the present study was to analyse the consequences of ectopic IκB kinase‐2 (IKK2) activation in the pancreas of mice. Methods Transgenic mice were generated using an inducible genetic system (tet system) to conditionally overexpress a gain of function mutant of IKK2 (tetO‐IKK2‐EE) in the pancreas. To achieve transgene expression in the pancreas, these animals were crossed with CMV‐rtTA mice that are known to express the rtTA protein in the pancreas. Results In these double transgenic animals, doxycycline treatment induced expression of IKK2‐EE (IKK2CA) in pancreatic acinar cells resulting in moderate activation of the IκB kinase complex, as measured by the immune complex kinase assay, and up to 200‐fold activation of the transgene expression cassette, as detected by luciferase assay. IKK2CA expression in the pancreas had a mosaic appearance. Ectopic IKK2CA mostly activated the classical NF‐κB pathway. The activation level of the NF‐κB cascade induced by IKK2CA was considerably lower compared with that observed after supramaximal caerulein stimulation but still led to the formation of leucocyte infiltrates first observed after 4 weeks of doxycycline stimulation with a maximum after 8–12 weeks. The infiltrates were mainly composed of B lymphocytes and macrophages. Increased mRNA levels of tumour necrosis factor α and RANTES were detected in pancreatic acinar cells. However, only minor damage to pancreatic tissue was observed. A combination of supramaximal caerulein stimulation with induction of IKK2CA caused increased tissue damage compared with either IKK2CA or caerulein alone. Conclusions Our observations suggest that the role of IKK2 activation in pancreatic acini is to induce leucocyte infiltration, but at a moderate level of activation it is not sufficient to induce pancreatic damage in mice. The IKK2CA induced infiltrations resemble those observed in autoimmune pancreatitis, indicating a role for IKK2/NF‐κB in this disease. IKK2CA in pancreatic acinar cells increases tissue damage of secretagogue induced experimental pancreatitis underlining the proinflammatory role of the IKK/NF‐κB pathway in this disease. PMID:16870717

Focal gene misexpression in zebrafish embryos induced by local heat shock using a modified soldering iron.

PubMed

Hardy, Melissa E; Ross, Louis V; Chien, Chi-Bin

2007-11-01

Misexpression of genes in a temporally and spatially controlled fashion is an important tool for assessing gene function during development. Because few tissue-specific promoters have been identified in zebrafish, inducible systems such as the Cre/LoxP and Tet repressor systems are of limited utility. Here we describe a new method of misexpression: local heat shock using a modified soldering iron. Zebrafish carrying transgenes under the control of a heat shock promoter (hsp70) are focally heated with the soldering iron to induce gene expression in a small area of the embryo. We have validated this method in three stable transgenic lines and at three developmental timepoints. Local heat shock is a fast, easy, and inexpensive method for gene misexpression. Copyright 2007 Wiley-Liss, Inc.

Dissemination of antibiotic resistance genes in representative broiler feedlots environments: identification of indicator ARGs and correlations with environmental variables.

PubMed

He, Liang-Ying; Liu, You-Sheng; Su, Hao-Chang; Zhao, Jian-Liang; Liu, Shuang-Shuang; Chen, Jun; Liu, Wang-Rong; Ying, Guang-Guo

2014-11-18

Livestock operations are known to harbor elevated levels of antibiotic resistance genes (ARGs) that may pose a threat to public health. Broiler feedlots may represent an important source of ARGs in the environment. However, the prevalence and dissemination mechanisms of various types of ARGs in the environment of broiler feedlots have not previously been identified. We examined the occurrence, abundance and variation of ARGs conferring resistance to chloramphenicols, sulfonamides and tetracyclines in the environments of two representative types of broiler feedlots (free range and indoor) by quantitative PCR, and assessed their dissemination mechanisms. The results showed the prevalence of various types of ARGs in the environmental samples of the broiler feedlots including manure/litter, soil, sediment, and water samples, with the first report of five chloramphenicol resistance genes (cmlA, floR, fexA, cfr, and fexB) in broiler feedlots. Overall, chloramphenicol resistance genes and sulfonamides sul genes were more abundant than tetracyclines tet genes. The ARG abundances in the samples from indoor boiler feedlots were generally different to the free range feedlots, suggesting the importance of feeding operations in ARG dissemination. Pearson correlation analysis showed significant correlations between ARGs and mobile genetic element genes (int1 and int2), and between the different classes of ARGs themselves, revealing the roles of horizontal gene transfer and coselection for ARG dissemination in the environment. Further regression analysis revealed that fexA, sul1 and tetW could be reliable indicator genes to surrogate anthropogenic sources of ARGs in boiler feedlots (correlations of fexA, sul1 and tetW to all ARGs: R = 0.95, 0.96 and 0.86, p < 0.01). Meanwhile, significant correlations were also identified between indicator ARGs and their corresponding antibiotics. In addition, some ARGs were significantly correlated with typical metals (e.g., Cu, Zn, and As with fexA, fexB, cfr, sul1, tetW, tetO, tetS: R = 0.52-0.71) and some environmental parameters (e.g., TOC, TN, TP, NH3-N with fexA, fexB, cfr, sul1, tetW, tetO, tetQ, tetS: R = 0.53-0.87) (p < 0.01). Further redundancy analysis demonstrated that the distribution and transportation of ARGs from the boiler feedlots to the receiving environments were correlated with environmental variables. The findings highlight the contribution of some chemicals such as antibiotics and metals to the development of ARGs in broiler feedlots environments; and the observed ARG dissemination mechanism in the broiler feedlots facilitates the development of effective mitigation measures.

Practical use of advanced mouse models for lung cancer.

PubMed

Safari, Roghaiyeh; Meuwissen, Ralph

2015-01-01

To date a variety of non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) mouse models have been developed that mimic human lung cancer. Chemically induced or spontaneous lung cancer in susceptible inbred strains has been widely used, but the more recent genetically engineered somatic mouse models recapitulate much better the genotype-phenotype correlations found in human lung cancer. Additionally, improved orthotopic transplantation of primary human cancer tissue fragments or cells into lungs of immune-compromised mice can be valuable tools for preclinical research such as antitumor drug tests. Here we give a short overview of most somatic mouse models for lung cancer that are currently in use. We accompany each different model with a description of its practical use and application for all major lung tumor types, as well as the intratracheal injection or direct injection of fresh or freeze-thawed tumor cells or tumor cell lines into lung parenchyma of recipient mice. All here presented somatic mouse models are based on the ability to (in) activate specific alleles at a time, and in a tissue-specific cell type, of choice. This spatial-temporal controlled induction of genetic lesions allows the selective introduction of main genetic lesions in an adult mouse lung as found in human lung cancer. The resulting conditional somatic mouse models can be used as versatile powerful tools in basic lung cancer research and preclinical translational studies alike. These distinctively advanced lung cancer models permit us to investigate initiation (cell of origin) and progression of lung cancer, along with response and resistance to drug therapy. Cre/lox or FLP/frt recombinase-mediated methods are now well-used techniques to develop tissue-restricted lung cancer in mice with tumor-suppressor gene and/or oncogene (in)activation. Intranasal or intratracheal administration of engineered adenovirus-Cre or lentivirus-Cre has been optimized for introducing Cre recombinase activity into pulmonary tissues, and we discuss here the different techniques underlying these applications. Concomitant with Cre/Flp recombinase-based models are the tetracycline (Tet)-inducible bitransgenic systems in which presence or absence of doxycycline can turn the expression of a specific oncogene on or off. The use of several Tet-inducible lung cancer models for NSCLC is presented here in which the reversal of oncogene expression led to complete tumor regression and provided us with important insight of how oncogene dependence influence lung cancer survival and growth. As alternative to Tet-inducible models, we discuss the application of reversible expressed, transgenic mutant estrogen receptor (ER) fusion proteins, which are regulated via systemic tamoxifen administration. Most of the various lung cancer models can be combined through the generation of transgenic compound mice so that the use of these somatic mouse models can be even more enhanced for the study of specific molecular pathways that facilitate growth and maintenance of lung cancer. Finally, this description of the practical application and methodology of mouse models for lung cancer should be helpful in assisting researchers to make the best choices and optimal use of (existing) somatic models that suits the specific experimental needs in their study of lung cancer.

Loss of Function of TET2 Cooperates with Constitutively Active KIT in Murine and Human Models of Mastocytosis

PubMed Central

De Vita, Serena; Schneider, Rebekka K.; Garcia, Michael; Wood, Jenna; Gavillet, Mathilde; Ebert, Benjamin L.; Gerbaulet, Alexander; Roers, Axel; Levine, Ross L.; Mullally, Ann; Williams, David A.

2014-01-01

Systemic Mastocytosis (SM) is a clonal disease characterized by abnormal accumulation of mast cells in multiple organs. Clinical presentations of the disease vary widely from indolent to aggressive forms, and to the exceedingly rare mast cell leukemia. Current treatment of aggressive SM and mast cell leukemia is unsatisfactory. An imatinib-resistant activating mutation of the receptor tyrosine kinase KIT (KIT D816V) is most frequently present in transformed mast cells and is associated with all clinical forms of the disease. Thus the etiology of the variable clinical aggressiveness of abnormal mast cells in SM is unclear. TET2 appears to be mutated in primary human samples in aggressive types of SM, suggesting a possible role in disease modification. In this report, we demonstrate the cooperation between KIT D816V and loss of function of TET2 in mast cell transformation and demonstrate a more aggressive phenotype in a murine model of SM when both mutations are present in progenitor cells. We exploit these findings to validate a combination treatment strategy targeting the epigenetic deregulation caused by loss of TET2 and the constitutively active KIT receptor for the treatment of patients with aggressive SM. PMID:24788138

Epigenetic Modifications in the Biology of Nonalcoholic Fatty Liver Disease

PubMed Central

Pirola, Carlos J.; Scian, Romina; Gianotti, Tomas Fernández; Dopazo, Hernán; Rohr, Cristian; Martino, Julio San; Castaño, Gustavo O.; Sookoian, Silvia

2015-01-01

Abstract The 5-Hydroxymethylcytosine (5-hmC) is an epigenetic modification whose role in the pathogenesis of metabolic-related complex diseases remains unexplored; 5-hmC appears to be prevalent in the mitochondrial genome. The Ten-Eleven-Translocation (TET) family of proteins is responsible for catalyzing the conversion of 5-methylcytosine to 5-hmC. We hypothesized that epigenetic editing by 5-hmC might be a novel mechanism through which nonalcoholic fatty liver disease (NAFLD)-associated molecular traits could be explained. Hence, we performed an observational study to explore global levels of 5-hmC in fresh liver samples of patients with NAFLD and controls (n = 90) using an enzyme-linked-immunosorbent serologic assay and immunohistochemistry. We also screened for genetic variation in TET 1–3 loci by next generation sequencing to explore its contribution to the disease biology. The study was conducted in 2 stages (discovery and replication) and included 476 participants. We observed that the amount of 5-hmC in the liver of both NAFLD patients and controls was relatively low (up to 0.1%); a significant association was found with liver mitochondrial DNA copy number (R = 0.50, P = 0.000382) and PPARGC1A-mRNA levels (R = −0.57, P = 0.04). We did not observe any significant difference in the 5-hmC nuclear immunostaining score between NAFLD patients and controls; nevertheless, we found that patients with NAFLD (0.4 ± 0.5) had significantly lower nonnuclear-5-hmC staining compared with controls (1.8 ± 0.8), means ± standard deviation, P = 0.028. The missense p.Ile1123Met variant (TET1-rs3998860) was significantly associated with serum levels of caspase-generated CK-18 fragment-cell death biomarker in the discovery and replication stage, and the disease severity (odds ratio: 1.47, 95% confidence interval: 1.10–1.97; P = 0.005). The p.Ile1762Val substitution (TET2-rs2454206) was associated with liver PPARGC1A-methylation and transcriptional levels, and Type 2 diabetes. Our results suggest that 5-hmC might be involved in the pathogenesis of NAFLD by regulating liver mitochondrial biogenesis and PPARGC1A expression. Genetic diversity at TET loci suggests an “epigenetic” regulation of programmed liver-cell death and a TET-mediated fine-tuning of the liver PPARGC1A-transcriptional program. PMID:26356709

Entity Recognition Via Multimodal Sensor Fusion With Smart Phones

DTIC Science & Technology

2015-03-26

Xs ,t|Et = 1] P[ Xs ,t|Et = 0] ≥ τ However, an event such as an earthquake, due to its’ rarity, does not have suffi- cient data to obtain good...Faulkner et al. develop a methodology to estimate the distribution of normal observations over time L̂0( Xs ,t) = P̂[ Xs ,t|Et = 0] for non-events. This is done...by using a parametric 37 approach: P[ Xs ,t|Et0] = φ( Xs ,t, θ) This model improves when the time span of sensing increases and thus the availability of

TET2 Mutations Are Associated with Specific 5-Methylcytosine and 5-Hydroxymethylcytosine Profiles in Patients with Chronic Myelomonocytic Leukemia

PubMed Central

Pérez, Cristina; Martínez-Calle, Nicolas; Martín-Subero, José Ignacio; Segura, Victor; Delabesse, Eric; Fernandez-Mercado, Marta; Garate, Leire; Alvarez, Sara; Rifon, José; Varea, Sara; Boultwood, Jacqueline; Wainscoat, James S.; Cigudosa, Juan Cruz; Calasanz, María José; Cross, Nicholas C. P.

2012-01-01

Chronic myelomonocytic leukemia (CMML) has recently been associated with a high incidence of diverse mutations in genes such as TET2 or EZH2 that are implicated in epigenetic mechanisms. We have performed genome-wide DNA methylation arrays and mutational analysis of TET2, IDH1, IDH2, EZH2 and JAK2 in a group of 24 patients with CMML. 249 genes were differentially methylated between CMML patients and controls. Using Ingenuity pathway analysis, we identified enrichment in a gene network centered around PLC, JNK and ERK suggesting that these pathways, whose deregulation has beenrecently described in CMML, are affected by epigenetic mechanisms. Mutations of TET2, JAK2 and EZH2 were found in 15 patients (65%), 4 patients (17%) and 1 patient (4%) respectively while no mutations in the IDH1 and IDH2 genes were identified. Interestingly, patients with wild type TET2 clustered separately from patients with TET2 mutations, showed a higher degree of hypermethylation and were associated with higher risk karyotypes. Our results demonstrate the presence of aberrant DNA methylation in CMML and identifies TET2 mutant CMML as a biologically distinct disease subtype with a different epigenetic profile. PMID:22328940

Inducible Expression of spo0A as a Universal Tool for Studying Sporulation in Clostridium difficile.

PubMed

Dembek, Marcin; Willing, Stephanie E; Hong, Huynh A; Hosseini, Siamand; Salgado, Paula S; Cutting, Simon M

2017-01-01

Clostridium difficile remains a leading nosocomial pathogen, putting considerable strain on the healthcare system. The ability to form endospores, highly resistant to environmental insults, is key to its persistence and transmission. However, important differences exist between the sporulation pathways of C. difficile and the model Gram-positive organism Bacillus subtilis . Amongst the challenges in studying sporulation in C. difficile is the relatively poor levels of sporulation and high heterogeneity in the sporulation process. To overcome these limitations we placed P tet regulatory elements upstream of the master regulator of sporulation, spo0A , generating a new strain that can be artificially induced to sporulate by addition of anhydrotetracycline (ATc). We demonstrate that this strain is asporogenous in the absence of ATc, and that ATc can be used to drive faster and more efficient sporulation. Induction of Spo0A is titratable and this can be used in the study of the spo0A regulon both in vitro and in vivo , as demonstrated using a mouse model of C. difficile infection (CDI). Insights into differences between the sporulation pathways in B. subtilis and C. difficile gained by study of the inducible strain are discussed, further highlighting the universal interest of this tool. The P tet -spo0A strain provides a useful background in which to generate mutations in genes involved in sporulation, therefore providing an exciting new tool to unravel key aspects of sporulation in C. difficile.

Synergistic anti-candidal activity of tetrandrine on ketoconazole: an experimental study.

PubMed

Zhang, Hong; Wang, Kaili; Zhang, Gehua; Ho, Hon In; Gao, Aili

2010-01-01

With the widespread use of azoles, drug resistant Candida albicans strains are increasing. The study examined the synergism of tetrandrine (TET) on ketoconazole (KCZ) candidacidal activity. The protocol M27-A2 of the Clinical and Laboratory Standards Institute (CLSI) was adopted and the minimum inhibitory concentrations (MICs) were determined for KCZ alone and in combination with a TET level that was noncytotoxic for C. albicans strains CA-1 through CA-17, with no CA-10. Colony counting techniques were used to construct time-kill curves. CA-15 was used to build the mouse candidal vaginitis model. After randomization, drugs were administered vaginally once daily from days 3-10 (both KCZ and TET were 26 mg/kg/day and 13 mg/kg/day, respectively, administered in different combinations). Mouse vaginal lavage fluid was obtained at days 2, 6, and 11 after inoculation for fungal load analysis, and vaginal tissue was obtained for pathological examination. MICs of KCZ alone and combined with 30 microg/mL TET for the C. albicans strains were 1-32 microg/mL and 0.0038-0.2500 microg/mL, respectively ( T = 24.624, p = 0.000). Time-kill curves showed that at 48 h the viable cell counts of strains treated with KCZ + TET were at least 2 log(10) CFU/mL lower compared to strains treated with corresponding doses of KCZ (p = 0.000). At day 6, the fungal load in the KCZ 26 mg/kg/day + TET 26 mg/kg/day mice was significantly lower than the KCZ 26 mg/kg/day mice (1.17 +/- 1.17 x 10(4) CFU/mL and 9.33 +/- 3.08 x 10(4) CFU/mL, respectively, p = 0.000). Mucosal and submucosal fungal clearances were complete and vaginal mucosal edema was slight with minimal inflammatory cell infiltration. We conclude that noncytotoxic doses of TET synergistically enhance KCZ candidacidal activity in vitro and in vivo. Copyright Georg Thieme Verlag KG Stuttgart . New York.

Horizontal Transfer of Tetracycline Resistance Genes in the Subsurface of a Poultry Farm

NASA Astrophysics Data System (ADS)

You, Y.; Ward, M.; Hilpert, M.

2008-12-01

Concentrated animal feeding operations (CAFOs) are considered to be important man-made reservoirs of antibiotic resistant bacteria and antibiotic resistance genes. At a poultry farm, we, together with Mr.~James Doolittle from USDA, measured the apparent subsurface electrical conductivity (ECa) using a EM38 meter. The resulting ECaR) associated with the poultry farm due to the fact that tetracycline (Tc) is one of the most frequently used antibiotics in food animal production and therefore is probably used at this farm. Soil and aquifer samples were taken from the farm. TcR bacteria were detected, with higher concentrations in the top layer of soil than in the aquifer. TcR bacteria were then enriched from a soil sample, and two classes of TcR genes were detected: tet(M) genes encoding ribosomal protection proteins and tet(L) genes encoding tet efflux pumps. Sequences of the PCR products were compared to known tet(M) and tet(L) genes in GenBank using BLASTN. Phylogenetic trees were also built based on the sequence information. The tet(M) genes found in our soil sample were highly similar to those located on transposons. In a soil microcosm experiment, we used the aforementioned soil sample as incubation medium as well as genetic donor (TcR soil bacteria), and a green fluorescent strain of E. coli as a model genetic recipient to study horizontal transfer of TcR genes from soil bacteria to naïve bacteria. Concentrations of inoculated E. coli were continuously monitored for 15 days, TcR E. coli isolated, and colony PCR performed. The tet(M) genes were found to be transferred to naïve E. coli. The highest horizontal transfer ratio, 0.62 transconjugant per recipient, was observed when Tc was supplemented to a soil microcosm at a concentration of 140 μg/kg soil. Modeling is also ongoing to obtain a better understanding of this complex phenomenon.

Effect of Mutation Order on Myeloproliferative Neoplasms

PubMed Central

Nangalia, Jyoti; Silber, Yvonne; Wedge, David C.; Grinfeld, Jacob; Baxter, E. Joanna; Massie, Charles E.; Papaemmanuil, Elli; Menon, Suraj; Godfrey, Anna L.; Dimitropoulou, Danai; Guglielmelli, Paola; Bellosillo, Beatriz; Besses, Carles; Döhner, Konstanze; Harrison, Claire N.; Vassiliou, George S.; Vannucchi, Alessandro; Campbell, Peter J.; Green, Anthony R.

2015-01-01

BACKGROUND Cancers result from the accumulation of somatic mutations, and their properties are thought to reflect the sum of these mutations. However, little is known about the effect of the order in which mutations are acquired. METHODS We determined mutation order in patients with myeloproliferative neoplasms by genotyping hematopoietic colonies or by means of next-generation sequencing. Stem cells and progenitor cells were isolated to study the effect of mutation order on mature and immature hematopoietic cells. RESULTS The age at which a patient presented with a myeloproliferative neoplasm, acquisition of JAK2 V617F homozygosity, and the balance of immature progenitors were all influenced by mutation order. As compared with patients in whom the TET2 mutation was acquired first (hereafter referred to as “TET2-first patients”), patients in whom the Janus kinase 2 (JAK2) mutation was acquired first (“JAK2-first patients”) had a greater likelihood of presenting with polycythemia vera than with essential thrombocythemia, an increased risk of thrombosis, and an increased sensitivity of JAK2-mutant progenitors to ruxolitinib in vitro. Mutation order influenced the proliferative response to JAK2 V617F and the capacity of double-mutant hematopoietic cells and progenitor cells to generate colony-forming cells. Moreover, the hematopoietic stem-and-progenitor-cell compartment was dominated by TET2 single-mutant cells in TET2-first patients but by JAK2–TET2 double-mutant cells in JAK2-first patients. Prior mutation of TET2 altered the transcriptional consequences of JAK2 V617F in a cell-intrinsic manner and prevented JAK2 V617F from up-regulating genes associated with proliferation. CONCLUSIONS The order in which JAK2 and TET2 mutations were acquired influenced clinical features, the response to targeted therapy, the biology of stem and progenitor cells, and clonal evolution in patients with myeloproliferative neoplasms. (Funded by Leukemia and Lymphoma Research and others.) PMID:25671252

Maternal TET3 is dispensable for embryonic development but is required for neonatal growth.

PubMed

Tsukada, Yu-Ichi; Akiyama, Tomohiko; Nakayama, Keiichi I

2015-10-28

The development of multicellular organisms is accompanied by reprogramming of the epigenome in specific cells, with the epigenome of most cell types becoming fixed after differentiation. Genome-wide reprogramming of DNA methylation occurs in primordial germ cells and in fertilized eggs during mammalian embryogenesis. The 5-methylcytosine (5mC) content of DNA thus undergoes a marked decrease in the paternal pronucleus of mammalian zygotes. This loss of DNA methylation has been thought to be mediated by an active demethylation mechanism independent of replication and to be required for development. TET3-mediated sequential oxidation of 5mC has recently been shown to contribute to the genome-wide loss of 5mC in the paternal pronucleus of mouse zygotes. We now show that TET3 localizes not only to the paternal pronucleus but also to the maternal pronucleus and oxidizes both paternal and maternal DNA in mouse zygotes, although these phenomena are less pronounced in the female pronucleus. Genetic ablation of TET3 in oocytes had no significant effect on oocyte development, maturation, or fertilization or on pregnancy, but it resulted in neonatal sublethality. Our results thus indicate that zygotic 5mC oxidation mediated by maternal TET3 is required for neonatal growth but is not essential for development.

Antibiotic resistance genes in surface water of eutrophic urban lakes are related to heavy metals, antibiotics, lake morphology and anthropic impact.

PubMed

Yang, Yuyi; Xu, Chen; Cao, Xinhua; Lin, Hui; Wang, Jun

2017-08-01

Urban lakes are impacted by heavy human activities and represent potential reservoirs for antibiotic resistance genes. In this study, six urban lakes in Wuhan, central China were selected to analyze the distribution of sulfonamide resistance (sul) genes, tetracycline resistance (tet) genes and quinolone resistance (qnr) genes and their relationship with heavy metals, antibiotics, lake morphology and anthropic impact. sul1 and sul2 were detected in all six lakes and dominated the types of antibiotic resistance genes, which accounted for 86.28-97.79% of the total antibiotic resistance gene abundance. For eight tested tet genes, antibiotic efflux pumps (tetA, tetB, tetC, and tetG) genes were all observed in six lakes and had higher relative abundance than ribosomal protection protein genes (tetM and tetQ). For 4 plasmid mediated quinolone resistance genes, only qnrD is found in all six lakes. The class I integron (intI1) is also found to be a very important media for antibiotic resistance gene propagation in urban lakes. The results of redundancy analysis and variation partitioning analysis showed that antibiotic and co-selection with heavy metals were the major factors driving the propagation of antibiotic resistance genes in six urban lakes. The heavily eutrophic Nanhu Lake and Shahu Lake which located in a high density building area with heavy human activities had the higher relative abundance of total antibiotic resistance genes. Our study could provide a useful reference for antibiotic resistance gene abundance in urban lakes with high anthropic impact.

Effect of different oral oxytetracycline treatment regimes on selection of antimicrobial resistant coliforms in nursery pigs.

PubMed

Herrero-Fresno, Ana; Zachariasen, Camilla; Nørholm, Nanna; Holm, Anders; Christiansen, Lasse Engbo; Olsen, John Elmerdahl

2017-09-01

A major concern derived from using antimicrobials in pig production is the development of resistance. This study aimed to assess the impact of selected combinations of oral dose and duration of treatment with oxytetracycline (OTC) on selection of tetracycline resistant (TET-R) coliforms recovered from swine feces. The work encompassed two studies: 1) OTC 5mg/kg and 20mg/kg were administered to nursery pigs for 3 and 10days, respectively, under controlled experimental conditions, and 2) 10mg/kg, 20mg/kg and 30mg/kg OTC were given to a higher number of pigs for 6, 3 and 2days, respectively, under field conditions. Statistical modeling was applied to analyze trends in the proportion of TET-R coliforms. In the experimental study, no statistical difference in proportion of TET-R coliforms was observed between treatments at the end of the trial (day 18) and compared to day 0. In the field study, treatment had a significant effect on the proportion of TET-R bacteria two days after the end of treatment (2dAT) with the regimes "low dose-six days" and "medium dose-three days" yielding the highest and lowest proportions of TET-R strains, respectively. No indication of co-selection for ampicillin- and sulphonamide -R bacteria was observed for any treatment at 2dAT. By the end of the nursery period, the proportion of TET-R bacteria was not significantly different between treatments and compared to day 0. Our results suggest that similar resistance levels might be obtained by using different treatment regimes regardless of the combinations of oral dose-duration of treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

Acute and Chronic Electroconvulsive Seizures (ECS) Differentially Regulate the Expression of Epigenetic Machinery in the Adult Rat Hippocampus.

PubMed

Pusalkar, Madhavi; Ghosh, Shreya; Jaggar, Minal; Husain, Basma Fatima Anwar; Galande, Sanjeev; Vaidya, Vidita A

2016-09-01

Electroconvulsive seizure treatment is a fast-acting antidepressant therapy that evokes rapid transcriptional, neurogenic, and behavioral changes. Epigenetic mechanisms contribute to altered gene regulation, which underlies the neurogenic and behavioral effects of electroconvulsive seizure. We hypothesized that electroconvulsive seizure may modulate the expression of epigenetic machinery, thus establishing potential alterations in the epigenetic landscape. We examined the influence of acute and chronic electroconvulsive seizure on the gene expression of histone modifiers, namely histone acetyltransferases, histone deacetylases, histone methyltransferases, and histone (lysine) demethylases as well as DNA modifying enzymes, including DNA methyltransferases, DNA demethylases, and methyl-CpG-binding proteins in the hippocampi of adult male Wistar rats using quantitative real time-PCR analysis. Further, we examined the influence of acute and chronic electroconvulsive seizure on global and residue-specific histone acetylation and methylation levels within the hippocampus, a brain region implicated in the cellular and behavioral effects of electroconvulsive seizure. Acute and chronic electroconvulsive seizure induced a primarily unique, and in certain cases bidirectional, regulation of histone and DNA modifiers, and methyl-CpG-binding proteins, with an overlapping pattern of gene regulation restricted to Sirt4, Mll3, Jmjd3, Gadd45b, Tet2, and Tet3. Global histone acetylation and methylation levels were predominantly unchanged, with the exception of a significant decline in H3K9 acetylation in the hippocampus following chronic electroconvulsive seizure. Electroconvulsive seizure treatment evokes the transcriptional regulation of several histone and DNA modifiers, and methyl-CpG-binding proteins within the hippocampus, with a predominantly distinct pattern of regulation induced by acute and chronic electroconvulsive seizure. © The Author 2016. Published by Oxford University Press on behalf of CINP.

Adenoviral bcl-2 transfer improves survival and early graft function after ischemia and reperfusion in rat liver transplantation.

PubMed

Rentsch, Markus; Kienle, Klaus; Mueller, Thomas; Vogel, Mandy; Jauch, Karl Walter; Püllmann, Kerstin; Obed, Aiman; Schlitt, Hans J; Beham, Alexander

2005-11-27

Primary graft dysfunction due to ischemia and reperfusion injury represents a major problem in liver transplantation. The related cell stress may induce apoptosis, which can be suppressed by bcl-2. The purpose of the study was to investigate the effect of adenoviral bcl-2 gene transfer on early graft function and survival in rat liver transplantation. An adenoviral construct that transfers bcl-2 under the control of a tetracycline inducible promoter was generated (advTetOn bcl-2) and used with a second adenovirus that transfers the repressor protein (advCMV Rep). Forty-eight hours before explantation, donor rats were treated with advTetOn bcl-2/ advCMV Rep (n=7) and doxycyclin, with the control adenoviral construct advCMV GFP (n=8) or with doxycyclin alone (n=8). Liver transplantation was performed following 16 hours of cold storage (UW). Bcl-2 expression and intrahepatic apoptosis was assessed. Bile flow was monitored 90 min posttransplantation. The endpoint for survival was 7 days. Bcl-2 was expressed in hepatocytes and sinusoidal lining cells. This was associated with a significant reduction of apoptotic sinusoidal lining cells and hepatocytes after 24 hours and 7 days. Bile production was significantly higher following bcl-2 pretreatment. Furthermore, bcl-2 transfer resulted in significantly improved survival (100% vs. 50% both control groups). Adenoviral bcl-2 transfer results in protein expression in hepatocytes and sinusoidal lining cells resulting in early graft function and survival enhancement after prolonged ischemia and reperfusion injury. The inhibition of apoptosis in the context of liver transplantation might be a reasonable approach in the treatment of graft dysfunction.

Rapid Detection of Bacterial Antibiotic Resistance: Preliminary Evaluation of PCR Assays Targeting Tetracycline Resistance Genes

DTIC Science & Technology

2007-08-01

gonorrheae strain 2309 plasmid pOZ101; AF440277, Lactobacillus plantarum plasmid pMD5057; X75073, Neisseria meningitidis plasmid DNA for tet(M...tetracycline resistance tet(M) gene; AY057892, Staphylococcus aureus strain 1802 tetracycline resistance protein tet(M) gene; AY149596, Lactobacillus sakei

Antibiotic Resistance in Czech Urban Wastewater Treatment Plants: Microbial and Molecular Genetic Characterization.

PubMed

Svobodová, Kateřina; Semerád, Jaroslav; Petráčková, Denisa; Novotný, Čeněk

2018-05-30

Quantitative changes in antibiotic resistance genes (ARGs) were investigated in six urban wastewater treatment plants (WWTPs) treating municipal and industrial wastewaters. In a selected WWTP, the fate of ARGs was studied in a 1-year time interval and in two phases of wastewater treatment process. Nine ARGs (tetW, tetO, tetA, tetB, tetM, bla TEM , ermB, sul1, and intl1) were quantified in total and their relative abundance assessed by ARG copies/16SrRNA copies. From the tetracycline resistance genes, tetW was the only one detected in all sampled WWTPs. Its relative abundance in the nitrification tank of WWTP5 was found stable during the 1-year period, but was lowered by secondary sedimentation processes in the wastewater treatment down to 24% compared to the nitrification tank. Bacterial isolates showing high tetracycline resistance (minimal inhibition concentrations >100 μg/mL) were identified as members of Acinetobacter, Klebsiella, Citrobacter, Bacillus, and Enterobacter genera. Dynamic shifts in the relative abundance of ermB and sul1 were also demonstrated in wastewater samples from WWTP5.

Direct quantification and distribution of tetracycline-resistant genes in meat samples by real-time polymerase chain reaction.

PubMed

Guarddon, Mónica; Miranda, Jose M; Vázquez, Beatriz I; Cepeda, Alberto; Franco, Carlos M

2012-07-01

The evolution of antimicrobial-resistant bacteria has become a threat to food safety and methods to control them are necessary. Counts of tetracycline-resistant (TR) bacteria by microbiological methods were compared with those obtained by quantitative PCR (qPCR) in 80 meat samples. TR Enterobacteriaceae counts were similar between the count plate method and qPCR (P= 0.24), whereas TR aerobic mesophilic bacteria counts were significantly higher by the microbiological method (P < 0.001). The distribution of tetA and tetB genes was investigated in different types of meat. tetA was detected in chicken meat (40%), turkey meat (100%), pork (20%), and beef (40%) samples, whereas tetB was detected in chicken meat (45%), turkey meat (70%), pork (30%), and beef (35%) samples. The presence of tetracycline residues was also investigated by a receptor assay. This study offers an alternative and rapid method for monitoring the presence of TR bacteria in meat and furthers the understanding of the distribution of tetA and tetB genes. © 2012 Institute of Food Technologists®

Occurrence and distribution of antibiotics, antibiotic resistance genes in the urban rivers in Beijing, China.

PubMed

Xu, Yan; Guo, Changsheng; Luo, Yi; Lv, Jiapei; Zhang, Yuan; Lin, Haixia; Wang, Li; Xu, Jian

2016-06-01

The occurrence and distribution of sulfonamide and tetracycline, corresponding bacterial resistant rate and resistance genes (ARGs) and two integrase genes were investigated in seven urban rivers in Beijing, China. The total concentration of sulfonamide and tetracycline ranged from 1.3 × 10(1)-1.5 × 10(3) ng/L and 3.9 × 10(1)-5.4 × 10(4) ng/L for water, and 1.0 × 10(0)-2.7 × 10(2) and 3.1 × 10(1)-1.6 × 10(2) ng/g for sediment, respectively. The sul resistant rate was 2-3 times higher than tet resistant rate in both surface water and sediment. The average rate of sul resistance and tet resistance were up to 81.3% and 38.6% in surface water, 89.1% and 69.4% in the sediment, respectively. The sul1, tetA and tetE genes were predominant in term of the absolute abundance. The absolute abundance of ARGs in Wenyu River and Qinghe River, which were close to the direct discharging sites, were 5-50 times higher than those in the other investigated urban rivers, suggesting that the source release played an important role in the distribution of ARGs. The sul1 and sul2 genes had positive correlation (p < 0.05) with sulfonamides, and the tet resistance genes was significantly correlated with tetracyclines (p < 0.05), indicating that some ARGs and antibiotics in the urban rivers had identical sources of pollution. Considering principal component analysis, sampling sites (QH5, QH6, B1, B2, B3 and BX2) intimated that a complex interplay of processes govern fate and transport of ARGs in the junction of rivers. These results are significant to understand the fate, and the contribution of ARGs from the source release. In view of the large-scale investigation of urban rivers system in Beijing, it reflected the bacterial resistance in sewage drainage system. Such investigation highlights the management on controlling the pollutant release which was seemed as a major driving force for the maintenance and propagation of many ARGs during the development of urbanization in the future. Copyright © 2016 Elsevier Ltd. All rights reserved.

Determination of Chlortetracycline Residues, Antimicrobial Activity and Presence of Resistance Genes in Droppings of Experimentally Treated Broiler Chickens.

PubMed

Cornejo, Javiera; Yevenes, Karina; Avello, Constanza; Pokrant, Ekaterina; Maddaleno, Aldo; Martin, Betty San; Lapierre, Lisette

2018-05-25

Tetracyclines are important antimicrobial drugs for poultry farming that are actively excreted via feces and urine. Droppings are one of the main components in broiler bedding, which is commonly used as an organic fertilizer. Therefore, bedding becomes an unintended carrier of antimicrobial residues into the environment and may pose a highly significant threat to public health. For this depletion study, 60 broiler chickens were treated with 20% chlortetracycline (CTC) under therapeutic conditions. Concentrations of CTC and 4-epi-CTC were then determined in their droppings. Additionally, this work also aimed to detect the antimicrobial activity of these droppings and the phenotypic susceptibility to tetracycline in E. coli isolates, as well as the presence of tet(A) , tet(B) , and tet(G) resistance genes. CTC and 4-epi-CTC concentrations that were found ranged from 179.5 to 665.8 µg/kg. Based on these data, the depletion time for chicken droppings was calculated and set at 69 days. All samples presented antimicrobial activity, and a resistance to tetracyclines was found in bacterial strains that were isolated from these samples. Resistance genes tet(A) and tet(B) were also found in these samples.

Antimicrobial Resistance and Resistance Genes in Aerobic Bacteria Isolated from Pork at Slaughter.

PubMed

Li, Lili; Heidemann Olsen, Rikke; Ye, Lei; Yan, He; Nie, Qing; Meng, Hecheng; Shi, Lei

2016-04-01

The aim of this study was to investigate the phenotypic and genotypic antimicrobial resistance, integrons, and transferability of resistance markers in 243 aerobic bacteria recovered from pork at slaughter in the People's Republic of China. The organisms belonged to 22 genera of gram-negative bacteria (92.2%) and gram-positive bacteria (7.8%). High levels of resistance were detected to tetracycline, trimethoprim-sulfamethoxazole, and ampicillin (36.2 to 54.3%), and lower levels were detected to nitrofurantoin, cefotaxime, gentamicin, ciprofloxacin, and chloramphenicol (7.8 to 29.2%). Across species, genes conferring antimicrobial resistance were observed with the following frequencies: blaTEM, 40.7%; blaCMY-2, 15.2%; blaCTX-M, 11.5%; sul2, 27.2%; sul1, 14.4%; tet(A), 5.4%; tet(L), 5.4%; tet(M), 5.0%; tet(E), 3.7%; tet(C), 3.3%; tet(S), 2.5%; and tet(K), 0.8%. Various antimicrobial resistance genes were found in new carriers: blaTEM in Lactococcus garvieae, Myroides odoratimimus, Aeromonas hydrophila, Staphylococcus sciuri, Raoultella terrigena, Macrococcus caseolyticus, Acinetobacter ursingii, Sphingobacterium sp., and Oceanobacillus sp.; blaCMY-2 in Lactococcus lactis, Klebsiella oxytoca, Serratia marcescens, Acinetobacter baumannii, and Myroides phaeus; tet(L) in M. caseolyticus; sul1 in Vibrio cincinnatiensis; sul2 in Acinetobacter bereziniae, Acinetobacter johnsonii, and V. cincinnatiensis; and the class 1 integron and gene cassette aadA2 in V. cincinnatiensis. Approximately 6.6% of isolates contained class 1 integrons, and one isolate harbored class 2 integrons. Plasmid associated intI1 and androgen receptor- encoding genes were transferred into Escherichia coli J53 and E. coli DH5α by conjugation and transformation experiments, respectively. Our study highlights the importance of aerobic bacteria from pork as reservoirs for antimicrobial resistance genes and mobile genetic elements that can readily be transferred intra- and interspecies.

Occurrence of antibiotic resistance genes in the fecal DNA of healthy omnivores, ovo-lacto vegetarians and vegans.

PubMed

Milanović, Vesna; Osimani, Andrea; Aquilanti, Lucia; Tavoletti, Stefano; Garofalo, Cristiana; Polverigiani, Serena; Litta-Mulondo, Alice; Cocolin, Luca; Ferrocino, Ilario; Di Cagno, Raffaella; Turroni, Silvia; Lazzi, Camilla; Pellegrini, Nicoletta; Clementi, Francesca

2017-09-01

The effects of long-term omnivore, ovo-lacto vegetarian and vegan diets on the occurrence of 12 antibiotic resistance (AR) genes in the human gut were studied. The feces of 144 healthy volunteers recruited from Turin, Bari, Bologna, and Parma were screened for the occurrence of genes conferring resistance to tetracyclines, macrolide-lincosamide-streptogramin B, vancomycin, and β-lactams. Overall, erm(B), tet(W) and tet(M) were detected at the highest frequency. A low effect from the diet on the AR gene distribution emerged, with tet(K) and vanB occurring at a lower and higher frequency in vegans and omnivores, respectively. A correlation of the intake of eggs, milk from animal sources and cheese with an increased occurrence of tet(K) was observed, together with a higher incidence of vanB in consumers of eggs, poultry meat, fish and seafood. When the detection frequencies of AR genes in volunteers from Bari and the other sites were comparatively evaluated, a north-to-south gradient was observed, whereas no effect of sex or age was highlighted. Except for tet(K), a negligible three-factor interaction was seen. A high impact of the geographical location on AR gene distribution was seen in the cohort of subjects analyzed, irrespective of their dietary habits. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A fatal case of streptococcal toxic shock syndrome caused by Streptococcus suis carrying tet (40) and tet (O/W/32/O), Italy.

PubMed

Mancini, Fabiola; Adamo, Francesco; Creti, Roberta; Monaco, Monica; Alfarone, Giovanna; Pantosti, Annalisa; Ciervo, Alessandra

2016-11-01

We report the first human fatal case of streptococcal toxic shock syndrome (STSS) caused by Streptococcus suis serotype 2 carrying the tetracycline efflux tet (40) gene and the tetracycline ribosomal protection tet (O/W/32/O) gene. The patient was splenectomized. The case was characterized by multi-organ dysfunction and disseminated intravascular coagulopathy, in accordance with the clinical parameters of STSS. More investigations are needed to improve the epidemiology and the pathogenesis of S. suis in human infection. Published by Elsevier Ltd.

Industrial water supply for the Kursk TETs-1 heating and electric power plant with the ecological safety of the River Seim taken into account

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kyakk, V. A.

2009-09-15

The Kursk TETs-1 heating and electric power plant is discussed as an illustration of preventing thermal contamination of its water supply (River Seim) by circulating water. An effective water supply system is taken to be one for which the overheating of the water at a monitoring site (near the outlet location) does not exceed an approved level for the water use conditions. The required cooling capacity of a spray pond for circulating and flow-through water supplies is determined.

Piano-stool lutetium amido and imido compounds supported by a constrained bis(oxazoline)cyclopentadienyl ligand

DOE PAGES

Lampland, Nicole L.; Zhu, Jing; Hovey, Megan; ...

2015-06-25

{Bo MCp tet}Lu(CH 2Ph) 2 ( 1; Bo MCp tet = MeC(Ox Me2 2C 5Me 4; Ox Me2 = 4,4-dimethyl-2-oxazoline) was prepared in 95% yield from the reaction of Bo MCp tetH and Lu(CH 2Ph) 3THF 3. Compound 1 reacts with 1 or 2 equiv of H 2NCH 2R (R = C 6H 5, 1-C 10H 7) to give the corresponding imido complexes [{Bo MCp tet}LuNCH 2R] 2 (R = C 6H 5 ( 2a), 1-C 10H 7 ( 2b)) or amido complexes {Bo MCp tet}Lu(NHCH 2R) 2 (R = C 6H 5 ( 3a), 1-C 10H 7 ( 3b)).more » When isolated, the imido species are insoluble in nonprotic organic solvents. Crystallographic characterization reveals dimeric [{Bo MCp tet}LuNCH 2(1-C 10H 7)] 2 in the solid state. The reaction of 1 and NH3B(C6F5)3 affords crystallographically characterized {Bo MCp tet}Lu{NHB(C 6F 5) 2}C 6F 5. This species is proposed to form via a transient lutetium imido, which undergoes C6F5 migration to the lutetium center.« less

Sensitive detection of proteasomal activation using the Deg-On mammalian synthetic gene circuit.

PubMed

Zhao, Wenting; Bonem, Matthew; McWhite, Claire; Silberg, Jonathan J; Segatori, Laura

2014-04-08

The ubiquitin proteasome system (UPS) has emerged as a drug target for diverse diseases characterized by altered proteostasis, but pharmacological agents that enhance UPS activity have been challenging to establish. Here we report the Deg-On system, a genetic inverter that translates proteasomal degradation of the transcriptional regulator TetR into a fluorescent signal, thereby linking UPS activity to an easily detectable output, which can be tuned using tetracycline. We demonstrate that this circuit responds to modulation of UPS activity in cell culture arising from the inhibitor MG-132 and activator PA28γ. Guided by predictive modelling, we enhanced the circuit's signal sensitivity and dynamic range by introducing a feedback loop that enables self-amplification of TetR. By linking UPS activity to a simple and tunable fluorescence output, these genetic inverters will enable a variety of applications, including screening for UPS activating molecules and selecting for mammalian cells with different levels of proteasome activity.

Implantable control, telemetry, and solar energy system in the moving actuator type total artificial heart.

PubMed

Ahn, J M; Lee, J H; Choi, S W; Kim, W E; Omn, K S; Park, S K; Kim, W G; Roh, J R; Min, B G

1998-03-01

The moving actuator type total artificial heart (TAH) developed in the Seoul National University has numerous design improvements based upon the digital signal processor (DSP). These improvements include the implantability of all electronics, an automatic control algorithm, and extension of the battery run-time in connection with an amorphous silicon solar system (SS). The implantable electronics consist of the motor drive, main processor, intelligent Li ion battery management (LIBM) based upon the DSP, telemetry system, and transcutaneous energy transmission (TET) system. Major changes in the implantable electronics include decreasing the temperature rise by over 21 degrees C on the motor drive, volume reduction (40 x 55 x 33 mm, 7 cell assembly) of the battery pack using a Li ion (3.6 V/cell, 900 mA.h), and improvement of the battery run-time (over 40 min) while providing the cardiac output (CO) of 5 L/min at 100 mm Hg afterload when the external battery for testing is connected with the SS (2.5 W, 192.192, 1 kg) for the external battery recharge or the partial TAH drive. The phase locked loop (PLL) based telemetry system was implemented to improve stability and the error correction DSP algorithm programmed to achieve high accuracy. A field focused light emitting diode (LED) was used to obtain low light scattering along the propagation path, similar to the optical property of the laser and miniature sized, mounted on the pancake type TET coils. The TET operating resonance frequency was self tuned in a range of 360 to 410 kHz to provide enough power even at high afterloads. An automatic cardiac output regulation algorithm was developed based on interventricular pressure analysis and carried out in several animal experiments successfully. All electronics have been evaluated in vitro and in vivo and prepared for implantation of the TAH. Substantial progress has been made in designing a completely implantable TAH at the preclinical stage.

Teaching Evaluation Tools as Robust Ethical Codes

ERIC Educational Resources Information Center

Talanker, Sergei

2018-01-01

I argue that teaching evaluation tools (TETs) may function as ethical codes (ECs), and answer certain demands that ECs cannot sufficiently fulfill. In order to be viable, an EC related to the teaching profession must assume a different form, and such a form is already present in several of the contemporary TETs. The TET matrix form allows for…

Molecular epidemiology of tetM genes in Neisseria gonorrhoeae

PubMed Central

Turner, A.; Gough, K. R.; Leeming, J. P.

1999-01-01

OBJECTIVE: To examine the epidemiology of the tetM gene in Neisseria gonorrhoeae strains with high level resistance to tetracycline (TRNG) using a polymerase chain reaction (PCR) assay. METHODS: A single tube PCR was developed which distinguishes between the American and Dutch variants of the tetM gene. Between 1988 and 1995, 518 strains of TRNG (tetracycline MIC > 8.mg/l) were referred to the Gonococcus Reference Unit by other laboratories or isolated from routine swabs taken at local clinics. The strains were analysed for plasmid content, auxotype, serovar, and the tetM gene type. Travel details of the patients were determined by a questionnaire. RESULTS: A PCR product was obtained from all TRNG examined. 387 TRNG strains produced a 778 bp PCR product (American type tetM) and 131 produced a 443 by PCR product (Dutch type tetM). Infections acquired in the United Kingdom contributed 57% of the TRNG strains included in this study; 82% of these carried the American type of tetM. The number of UK acquired TRNG received by the GRU increased each year except 1993--from four strains received in 1990 to 92 in 1995. After the United Kingdom, Caribbean and African countries contributed most strains, with 56 and 60 TRNG acquired in each area respectively. All strains originating in Africa, except one from South Africa, contained the American type tetM. Infections caught in Nigeria and Kenya contributed most strains (15 and 14 respectively). The TRNG originating from Caribbean countries comprised 36% Dutch tetM type. Infections caught in Jamaica accounted for 82% of the Caribbean strains. All 35 TRNG strains originating in the Far East contained the Dutch type tetM. 25 of the Far East strains were also penicillinase producing (PPNG). Infections originating in Indonesia accounted for 49% of the Far East strains but these belonged to 12 different auxotype/serovar combinations. A geographical variation in the type of penicillinase coding plasmids found in PPNG/TRNG was also detected. CONCLUSIONS: These data suggest that the Dutch type tetM may have originated in the Far East and the American type in the African continent. Subsequent spread has resulted in a heterogeneous distribution of TRNG types in other parts of the world. At completion of the survey the numbers of TRNG imported each year from the major overseas sources had reached a plateau while UK contracted TRNG continued to rise providing evidence for the establishment of endemic TRNG strains in the United Kingdom. 


 PMID:10448346

Characterization of a Streptococcus suis tet(O/W/32/O)-carrying element transferable to major streptococcal pathogens.

PubMed

Palmieri, Claudio; Magi, Gloria; Mingoia, Marina; Bagnarelli, Patrizia; Ripa, Sandro; Varaldo, Pietro E; Facinelli, Bruna

2012-09-01

Mosaic tetracycline resistance determinants are a recently discovered class of hybrids of ribosomal protection tet genes. They may show different patterns of mosaicism, but their final size has remained unaltered. Initially thought to be confined to a small group of anaerobic bacteria, mosaic tet genes were then found to be widespread. In the genus Streptococcus, a mosaic tet gene [tet(O/W/32/O)] was first discovered in Streptococcus suis, an emerging drug-resistant pig and human pathogen. In this study, we report the molecular characterization of a tet(O/W/32/O) gene-carrying mobile element from an S. suis isolate. tet(O/W/32/O) was detected, in tandem with tet(40), in a circular 14,741-bp genetic element (39.1% G+C; 17 open reading frames [ORFs] identified). The novel element, which we designated 15K, also carried the macrolide resistance determinant erm(B) and an aminoglycoside resistance four-gene cluster including aadE (streptomycin) and aphA (kanamycin). 15K appeared to be an unstable genetic element that, in the absence of recombinases, is capable of undergoing spontaneous excision under standard growth conditions. In the integrated form, 15K was found inside a 54,879-bp integrative and conjugative element (ICE) (50.5% G+C; 55 ORFs), which we designated ICESsu32457. An ∼1.3-kb segment that apparently served as the att site for excision of the unstable 15K element was identified. The novel ICE was transferable at high frequency to recipients from pathogenic Streptococcus species (S. suis, Streptococcus pyogenes, Streptococcus pneumoniae, and Streptococcus agalactiae), suggesting that the multiresistance 15K element can successfully spread within streptococcal populations.

A Fluorescence Polarization Biophysical Assay for the Naegleria DNA Hydroxylase Tet1.

PubMed

Marholz, Laura J; Wang, Wei; Zheng, Yu; Wang, Xiang

2016-02-11

The discovery of the 5-methylcytosine (5mC) oxidation by the ten-eleven translocation (Tet) protein family was an important advancement in our understanding of DNA-modified epigenetics. Potent inhibitors of these proteins are greatly desired for both the understanding of the functions of these enzymes and to serve as eventual therapeutic leads. So far, the discovery of such small molecules with high affinity has been quite limited. Original tools to screen for activity are greatly needed in order to accelerate this process. Here we present a novel fluorescent probe, and the results of a fluorescence polarization-based binding assay for Naegleria Tet1, a homologue to mammalian Tet. A fluorescence polarization-based competition assay was also established and applied to the rapid and quantitative measurement of the binding affinity of the cofactor αKG and several known Tet1 inhibitors.

Expression of CYP2E1 in human nasopharynx and its metabolic effect in vitro.

PubMed

Hou, De-Fu; Wang, Shui-Liang; He, Zhi-Min; Yang, Fang; Chen, Zhu-Chu

2007-04-01

It was evident that nitrosamines can act directly on target tissue and result in carcinogenesis. As has been shown, the carcinogenic activity of nitrosamines relied on its bioactivation by Cytochrome P450 2E1 (CYP2E1). In this study, we investigated the expression of CYP2E1 in Nasopharyngeal carcinoma (NPC) cells, embryonic nasopharyngeal epithelial tissue (ENET) specimens, and NPC biopsies by RT-PCR analysis. CYP2E1 was expressed in all NPC cell lines (6/6, including 7429) and ENET (6/6), and 80% of NPC biopsie (8/10). The fact that Human nasopharynx expresses CYP2E1 suggests that CYP2E1 may play an important role in the course of NPC by indirect carcinogens nitrosamines. To further evaluate the function of CYP2E1, the CYP2E1 was stably expressed in the cell line NIH 3T3/rtTA under a tetracycline-controlled transactivator. The expression of CYP2E1 was tightly regulated in a dose-dependent manner by Doxycycline (Dox) When the catalytic activity of CYP2E1 was assayed, the result showed that the generation of 6-hydroxychlorzoxazone (6-OH-CZ) from chlorzoxazone (CZ) was dose- and time-dependent on Dox addition to the medium. In the presence of 1 microg/ml Dox, the CZ 6-hydroxylase activity of the cell line was found to be 0.986 +/- 0.034 nmol/10(6) cells/h. The metabolic activation of Tet/3T3/2E1-6 cells was also assayed by N,N'-dinitrosopiperazine (DNP) cytotoxicity, and the viability of Tet/3T3/2E1-6 cells treated with Dox was lower than that of untreated cells with a significant difference between them in 80 and 160 microg/ml DNP (P ( 0.05, t test. This cell line will be useful not only to assess the metabolic characteristics of CYP2E1, but also will be useful to investigate the role of CYP2E1 in metabolic activation of carcinogenic nitrosamines in vitro.

The Chlamydia suis Genome Exhibits High Levels of Diversity, Plasticity, and Mobile Antibiotic Resistance: Comparative Genomics of a Recent Livestock Cohort Shows Influence of Treatment Regimes.

PubMed

Seth-Smith, Helena M B; Wanninger, Sabrina; Bachmann, Nathan; Marti, Hanna; Qi, Weihong; Donati, Manuela; di Francesco, Antonietta; Polkinghorne, Adam; Borel, Nicole

2017-03-01

Chlamydia suis is an endemic pig pathogen, belonging to a fascinating genus of obligate intracellular pathogens. Of particular interest, this is the only chlamydial species to have naturally acquired genes encoding for tetracycline resistance. To date, the distribution and mobility of the Tet-island are not well understood. Our study focused on whole genome sequencing of 29 C. suis isolates from a recent porcine cohort within Switzerland, combined with data from USA tetracycline-resistant isolates. Our findings show that the genome of C. suis is very plastic, with unprecedented diversity, highly affected by recombination and plasmid exchange. A large diversity of isolates circulates within Europe, even within individual Swiss farms, suggesting that C. suis originated around Europe. New World isolates have more restricted diversity and appear to derive from European isolates, indicating that historical strain transfers to the United States have occurred. The architecture of the Tet-island is variable, but the tetA(C) gene is always intact, and recombination has been a major factor in its transmission within C. suis. Selective pressure from tetracycline use within pigs leads to a higher number of Tet-island carrying isolates, which appear to be lost in the absence of such pressure, whereas the loss or gain of the Tet-island from individual strains is not observed. The Tet-island appears to be a recent import into the genome of C. suis, with a possible American origin. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

A novel method to partition evapotranspiration based on the concept of underlying water use efficiency

NASA Astrophysics Data System (ADS)

Zhou, Sha; Yu, Bofu; Zhang, Yao; Huang, Yuefei; Wang, Guangqian

2017-04-01

Evapotranspiration (ET) is dominated by transpiration (T) in the terrestrial water cycle. However, continuous measurement of transpiration is still difficult, and the effect of vegetation on ET partitioning is unclear. The concept of underlying water use efficiency (uWUE) was used to develop a new method for ET partitioning by assuming that the maximum, or the potential uWUE is related to T while the averaged or apparent uWUE is related to ET. T/ET was thus estimated as the ratio of the apparent over the potential uWUE using half-hourly flux data from 17 AmeriFlux sites. The estimated potential uWUE was shown to be essentially constant for the 14 sites with a single vegetation type, and was broadly consistent with the uWUE evaluated at the leaf scale. The annual T/ET was the highest for croplands, i.e., 0.69 for corn and 0.62 for soybean, followed by grasslands (0.60) and evergreen needle leaf forests (0.56), and was the lowest for deciduous broadleaf forests (0.52). The enhanced vegetation index (EVI) was shown to be significantly correlated with T/ET and could explain about 75% of the variation in T/ET among the 71 site-years. The coefficients of determination between EVI and T/ET were 0.84 and 0.82 for corn and soybean, respectively, and 0.77 for deciduous broadleaf forests and grasslands, but only 0.37 for evergreen needle leaf forests. This ET partitioning method is sound in principle and simple to apply in practice, and would enhance the value and role of global FLUXNET in estimating T/ET variations and monitoring ecosystem dynamics.

Acute and Chronic Electroconvulsive Seizures (ECS) Differentially Regulate the Expression of Epigenetic Machinery in the Adult Rat Hippocampus

PubMed Central

Pusalkar, Madhavi; Ghosh, Shreya; Jaggar, Minal; Husain, Basma Fatima Anwar; Galande, Sanjeev

2016-01-01

Background: Electroconvulsive seizure treatment is a fast-acting antidepressant therapy that evokes rapid transcriptional, neurogenic, and behavioral changes. Epigenetic mechanisms contribute to altered gene regulation, which underlies the neurogenic and behavioral effects of electroconvulsive seizure. We hypothesized that electroconvulsive seizure may modulate the expression of epigenetic machinery, thus establishing potential alterations in the epigenetic landscape. Methods: We examined the influence of acute and chronic electroconvulsive seizure on the gene expression of histone modifiers, namely histone acetyltransferases, histone deacetylases, histone methyltransferases, and histone (lysine) demethylases as well as DNA modifying enzymes, including DNA methyltransferases, DNA demethylases, and methyl-CpG-binding proteins in the hippocampi of adult male Wistar rats using quantitative real time-PCR analysis. Further, we examined the influence of acute and chronic electroconvulsive seizure on global and residue-specific histone acetylation and methylation levels within the hippocampus, a brain region implicated in the cellular and behavioral effects of electroconvulsive seizure. Results: Acute and chronic electroconvulsive seizure induced a primarily unique, and in certain cases bidirectional, regulation of histone and DNA modifiers, and methyl-CpG-binding proteins, with an overlapping pattern of gene regulation restricted to Sirt4, Mll3, Jmjd3, Gadd45b, Tet2, and Tet3. Global histone acetylation and methylation levels were predominantly unchanged, with the exception of a significant decline in H3K9 acetylation in the hippocampus following chronic electroconvulsive seizure. Conclusions: Electroconvulsive seizure treatment evokes the transcriptional regulation of several histone and DNA modifiers, and methyl-CpG-binding proteins within the hippocampus, with a predominantly distinct pattern of regulation induced by acute and chronic electroconvulsive seizure. PMID:27207907

Effect of the selective pressure of sub-lethal level of heavy metals on the fate and distribution of ARGs in the catchment scale.

PubMed

Xu, Yan; Xu, Jian; Mao, Daqing; Luo, Yi

2017-01-01

Our previous study demonstrated that high levels of antibiotic resistance genes (ARGs) in the Haihe River were directly attributed to the excessive use of antibiotics in animal agriculture. The antibiotic residues of the Xiangjiang River determined in this study were much lower than those of the Haihe River, but the relative abundance of 16 detected ARGs (sul1, sul2 and sul3, qepA, qnrA, qnrB, qnrD and qnrS, tetA, tetB, tetW, tetM, tetQ and tetO, ermB and ermC), were as high as the Haihe River particularly in the downstream of the Xiangjiang River which is close to the extensive metal mining. The ARGs discharged from the pharmaceutical wastewater treatment plant (PWWTP) are a major source of ARGs in the upstream of the Xiangjiang River. In the downstream, selective stress of heavy metals rather than source release had a significant influence on the distinct distribution pattern of ARGs. Some heavy metals showed a positive correlation with certain ARG subtypes. Additionally, there is a positive correlation between individual ARG subtypes and heavy metal resistance genes, suggesting that heavy metals may co select the ARGs on the same plasmid of antibiotic resistant bacteria. The co-selection mechanism between specific metal and antibiotic resistance was further confirmed by these isolations encoding the resistance genotypes to antibiotics and metals. To our knowledge, this is the first study on the fate and distribution of ARGs under the selective pressure exerted by heavy metals in the catchment scale. These results are beneficial to understand the fate, and to discern the contributors of ARGs from either the source release or the selective pressure by sub-lethal levels of environmental stressors during their transport on a river catchment scale. Copyright © 2016 Elsevier Ltd. All rights reserved.

Synthesis and Characterization of Photoactivatable Doxycycline Analogues Bearing Two-Photon-Sensitive Photoremovable Groups Suitable for Light-Induced Gene Expression.

PubMed

Goegan, Bastien; Terzi, Firat; Bolze, Frédéric; Cambridge, Sidney; Specht, Alexandre

2018-06-18

We report the synthesis and photolytic properties of caged 9-aminodoxycycline derivatives modified with 2-{4'-bis-[2-(2methoxyethoxy)ethyl]-4-nitrobiphenyl-3-yl}prop-1-oxy (EANBP) and PEG7-ylated (7-diethylamino-2-oxo-2H-chromen-4-yl)methyl (PEG7-DEACM) groups. 9-Aminodoxycycline is a tetracycline analogue capable of activating transcription through the inducible TetOn transgene expression system and can be regioselectively coupled to two-photon-sensitive photo-removable protecting groups by carbamoylation. The EANBP-based caged 9-aminodoxycycline showed complex photochemical reactions but did release 10 % of 9-aminodoxycycline. However, 9-(PEG7-DEACMamino)doxycycline exhibited excellent photolysis efficiency at 405 nm with quantitative release of 9-aminodoxycycline and a 0.21 uncaging quantum yield. Thanks to the good two-photon sensitivity of the DEACM chromophore, 9-aminodoxycycline release by two-photon photolysis is possible, with calculated action cross-sections of up to 4.0 GM at 740 nm. Therefore, 9-(PEG7-DEACMamino)doxycycline represents a very attractive tool for the development of a light-induced gene expression method in living cells. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Re-engineering adenovirus vector systems to enable high-throughput analyses of gene function.

PubMed

Stanton, Richard J; McSharry, Brian P; Armstrong, Melanie; Tomasec, Peter; Wilkinson, Gavin W G

2008-12-01

With the enhanced capacity of bioinformatics to interrogate extensive banks of sequence data, more efficient technologies are needed to test gene function predictions. Replication-deficient recombinant adenovirus (Ad) vectors are widely used in expression analysis since they provide for extremely efficient expression of transgenes in a wide range of cell types. To facilitate rapid, high-throughput generation of recombinant viruses, we have re-engineered an adenovirus vector (designated AdZ) to allow single-step, directional gene insertion using recombineering technology. Recombineering allows for direct insertion into the Ad vector of PCR products, synthesized sequences, or oligonucleotides encoding shRNAs without requirement for a transfer vector Vectors were optimized for high-throughput applications by making them "self-excising" through incorporating the I-SceI homing endonuclease into the vector removing the need to linearize vectors prior to transfection into packaging cells. AdZ vectors allow genes to be expressed in their native form or with strep, V5, or GFP tags. Insertion of tetracycline operators downstream of the human cytomegalovirus major immediate early (HCMV MIE) promoter permits silencing of transgenes in helper cells expressing the tet repressor thus making the vector compatible with the cloning of toxic gene products. The AdZ vector system is robust, straightforward, and suited to both sporadic and high-throughput applications.

Enhanced expression of VEGF-A in β cells increases endothelial cell number but impairs islet morphogenesis and β cell proliferation

PubMed Central

Cai, Qing; Brissova, Marcela; Reinert, Rachel B.; Pan, Fong Cheng; Brahmachary, Priyanka; Jeansson, Marie; Shostak, Alena; Radhika, Aramandla; Poffenberger, Greg; Quaggin, Susan E.; Jerome, W. Gray; Dumont, Daniel J.; Powers, Alvin C.

2012-01-01

There is a reciprocal interaction between pancreatic islet cells and vascular endothelial cells (EC) in which EC-derived signals promote islet cell differentiation and islet development while islet cell-derived angiogenic factors promote EC recruitment and extensive islet vascularization. To examine the role of angiogenic factors in the coordinated development of islets and their associated vessels, we used a “tet-on” inducible system (mice expressing rat insulin promoter-reverse tetracycline activator transgene and a tet-operon-angiogenic factor transgene) to increase the β cell production of vascular endothelial growth factor-A (VEGF-A), angiopoietin-1 (Ang1), or angiopoietin-2 (Ang2) during islet cell differentiation and islet development. In VEGF-A overexpressing embryos, ECs began to accumulate around epithelial tubes residing in the central region of the developing pancreas (associated with endocrine cells) as early as embryonic day 12.5 (E12.5) and increased dramatically by E16.5. While α and β cells formed islet cell clusters in control embryos at E16.5, the increased EC population perturbed endocrine cell differentiation and islet cell clustering in VEGF-A overexpressing embryos. With continued overexpression of VEGF-A, α and β cells became scattered, remained adjacent to ductal structures, and never coalesced into islets, resulting in a reduction in β cell proliferation and β cell mass at postnatal day 1. A similar impact on islet morphology was observed when VEGF-A was overexpressed in β cells during the postnatal period. In contrast, increased expression of Ang1 or Ang2 in β cells in developing or adult islets did not alter islet differentiation, development, or morphology, but altered islet EC ultrastructure. These data indicate that 1) increased EC number does not promote, but actually impairs β cell proliferation and islet formation; 2) the level of VEGF-A production by islet endocrine cells is critical for islet vascularization during development and postnatally; 3) Angiopoietin-Tie2 signaling in endothelial cells does not have a crucial role in the development or maintenance of islet vascularization. PMID:22546694

Fate of tetracycline, sulfonamide and fluoroquinolone resistance genes and the changes in bacterial diversity during composting of swine manure.

PubMed

Selvam, Ammaiyappan; Xu, Delin; Zhao, Zhenyong; Wong, Jonathan W C

2012-12-01

This study monitored the abundance of antibiotic resistant genes (ARGs) and the bacterial diversity during composting of swine manure spiked with chlortetracycline, sulfadiazine and ciprofloxacin at two different levels and a control without antibiotics. Resistance genes of tetracycline (tetQ, tetW, tetC, tetG, tetZ and tetY), sulfonamide (sul1, sul2, dfrA1 and dfrA7) and fluoroquinolone (gyrA and parC) represented 0.02-1.91%, 0.67-10.28% and 0.00005-0.0002%, respectively, of the total 16S rDNA copies in the initial composting mass. After 28-42 days of composting, these ARGs, except parC, were undetectable in the composting mass indicating that composting is a potential method of manure management. Polymerase chain reaction-denaturing gradient gel electrophoresis analysis of bacterial 16S rDNA of the composting mass indicated that the addition of antibiotics up to 100, 20 and 20mg/kg of chlortetracycline, sulfadiazine and ciprofloxacin, respectively, elicited only a transient perturbation and the bacterial diversity was restored in due course of composting. Copyright © 2012 Elsevier Ltd. All rights reserved.

SACE_0012, a TetR-family transcriptional regulator, affects the morphogenesis of Saccharopolyspora erythraea.

PubMed

Yin, Xiaojuan; Xu, Xinqiang; Wu, Hang; Yuan, Li; Huang, Xunduan; Zhang, Buchang

2013-12-01

Saccharopolyspora erythraea, a mycelium-forming actinomycete, produces a clinically important antibiotic erythromycin. Extensive investigations have provided insights into erythromycin biosynthesis in S. erythraea, but knowledge of its morphogenesis remains limited. By gene inactivation and complementation strategies, the TetR-family transcriptional regulator SACE_0012 was identified to be a negative regulator of mycelium formation of S. erythraea A226. Detected by quantitative real-time PCR, the relative transcription of SACE_7115, the amfC homolog for an aerial mycelium formation protein, was dramatically increased in SACE_0012 mutant, whereas erythromycin biosynthetic gene eryA, a pleiotropic regulatory gene bldD, and the genes SACE_2141, SACE_6464, SACE_6040, that are the homologs to the sporulation regulators WhiA, WhiB, WhiG, were not differentially expressed. SACE_0012 disruption could not restore its defect of aerial development in bldD mutant, and also did not further accelerate the mycelium formation in the mutant of SACE_7040 gene, that was previously identified to be a morphogenesis repressor. Furthermore, the transcriptional level of SACE_0012 had not markedly changed in bldD and SACE_7040 mutant over A226. Taken together, these results suggest that SACE_0012 is a negative regulator of S. erythraea morphogenesis by mainly increasing the transcription of amfC gene, independently of the BldD regulatory system.

A Computational Approach to Estimating Nondisjunction Frequency in Saccharomyces cerevisiae

PubMed Central

Chu, Daniel B.; Burgess, Sean M.

2016-01-01

Errors segregating homologous chromosomes during meiosis result in aneuploid gametes and are the largest contributing factor to birth defects and spontaneous abortions in humans. Saccharomyces cerevisiae has long served as a model organism for studying the gene network supporting normal chromosome segregation. Measuring homolog nondisjunction frequencies is laborious, and involves dissecting thousands of tetrads to detect missegregation of individually marked chromosomes. Here we describe a computational method (TetFit) to estimate the relative contributions of meiosis I nondisjunction and random-spore death to spore inviability in wild type and mutant strains. These values are based on finding the best-fit distribution of 4, 3, 2, 1, and 0 viable-spore tetrads to an observed distribution. Using TetFit, we found that meiosis I nondisjunction is an intrinsic component of spore inviability in wild-type strains. We show proof-of-principle that the calculated average meiosis I nondisjunction frequency determined by TetFit closely matches empirically determined values in mutant strains. Using these published data sets, TetFit uncovered two classes of mutants: Class A mutants skew toward increased nondisjunction death, and include those with known defects in establishing pairing, recombination, and/or synapsis of homologous chromosomes. Class B mutants skew toward random spore death, and include those with defects in sister-chromatid cohesion and centromere function. Epistasis analysis using TetFit is facilitated by the low numbers of tetrads (as few as 200) required to compare the contributions to spore death in different mutant backgrounds. TetFit analysis does not require any special strain construction, and can be applied to previously observed tetrad distributions. PMID:26747203

Implementation of a novel in vitro model of infection of reconstituted human epithelium for expression of virulence genes in methicillin-resistant Staphylococcus aureus strains isolated from catheter-related infections in Mexico

PubMed Central

2014-01-01

Background Methicillin-resistant Staphylococcus aureus (MRSA) are clinically relevant pathogens that cause severe catheter-related nosocomial infections driven by several virulence factors. Methods We implemented a novel model of infection in vitro of reconstituted human epithelium (RHE) to analyze the expression patterns of virulence genes in 21 MRSA strains isolated from catheter-related infections in Mexican patients undergoing haemodialysis. We also determined the phenotypic and genotypic co-occurrence of antibiotic- and disinfectant-resistance traits in the S. aureus strains, which were also analysed by pulsed-field-gel electrophoresis (PFGE). Results In this study, MRSA strains isolated from haemodialysis catheter-related infections expressed virulence markers that mediate adhesion to, and invasion of, RHE. The most frequent pattern of expression (present in 47.6% of the strains) was as follows: fnbA, fnbB, spa, clfA, clfB, cna, bbp, ebps, eap, sdrC, sdrD, sdrE, efb, icaA, and agr. Seventy-one percent of the strains harboured the antibiotic- and disinfectant-resistance genes ermA, ermB, tet(M), tet(K), blaZ, qacA, qacB, and qacC. PFGE of the isolated MRSA revealed three identical strains and two pairs of identical strains. The strains with identical PFGE patterns showed the same phenotypes and genotypes, including the same spa type (t895), suggesting hospital personnel manipulating the haemodialysis equipment could be the source of catheter contamination. Conclusion These findings help define the prevalence of MRSA virulence factors in catheter-related infections. Some of the products of the expressed genes that we detected in this work may serve as potential antigens for inclusion in a vaccine for the prevention of MRSA-catheter-related infections. PMID:24405688

Probing the target search of DNA-binding proteins in mammalian cells using TetR as model searcher

NASA Astrophysics Data System (ADS)

Normanno, Davide; Boudarène, Lydia; Dugast-Darzacq, Claire; Chen, Jiji; Richter, Christian; Proux, Florence; Bénichou, Olivier; Voituriez, Raphaël; Darzacq, Xavier; Dahan, Maxime

2015-07-01

Many cellular functions rely on DNA-binding proteins finding and associating to specific sites in the genome. Yet the mechanisms underlying the target search remain poorly understood, especially in the case of the highly organized mammalian cell nucleus. Using as a model Tet repressors (TetRs) searching for a multi-array locus, we quantitatively analyse the search process in human cells with single-molecule tracking and single-cell protein-DNA association measurements. We find that TetRs explore the nucleus and reach their target by 3D diffusion interspersed with transient interactions with non-cognate sites, consistent with the facilitated diffusion model. Remarkably, nonspecific binding times are broadly distributed, underlining a lack of clear delimitation between specific and nonspecific interactions. However, the search kinetics is not determined by diffusive transport but by the low association rate to nonspecific sites. Altogether, our results provide a comprehensive view of the recruitment dynamics of proteins at specific loci in mammalian cells.

Response to 5-azacytidine in a patient with TET2-mutated angioimmunoblastic T-cell lymphoma and chronic myelomonocytic leukaemia preceded by an EBV-positive large B-cell lymphoma.

PubMed

Saillard, Colombe; Guermouche, Helene; Derrieux, Coralie; Bruneau, Julie; Frenzel, Laurent; Couronne, Lucile; Asnafi, Vahid; Macintyre, Elizabeth; Trinquand, Amélie; Lhermitte, Ludovic; Molina, Thierry; Suarez, Felipe; Lemonnier, Francois; Kosmider, Olivier; Delarue, Richard; Hermine, Olivier; Cheminant, Morgane

2017-12-01

We report the case of a patient with a history of Epstein-Barr virus-positive large B-cell lymphoma, who relapsed with an angioimmunoblastic T-cell lymphoma (AITL) associated with a chronic myelomonocytic leukaemia (CMML). We performed targeted next-generation sequencing on CMML and AITL DNA, which revealed mutations of TET2, DNMT3A, SRSF2, NRAS and IDH1, thus confirming that the spectrum of AITL mutations share similarities with myeloid disorders. The frequencies of TET2/DNMT3A and SRSF2 variants could support the hypothesis that TET2/DNMT3A mutations occurred in an early progenitor cell, which later progressed to both the AITL and CMML clones. Treatment with 5-azacytidine led to the complete remission of both diseases. Thus, targeting DNA methylation abnormalities in AITL may be an alternative strategy to chemotherapy. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Real-time fluorescence imaging of the DNA damage repair response during mitosis.

PubMed

Miwa, Shinji; Yano, Shuya; Yamamoto, Mako; Matsumoto, Yasunori; Uehara, Fuminari; Hiroshima, Yukihiko; Toneri, Makoto; Murakami, Takashi; Kimura, Hiroaki; Hayashi, Katsuhiro; Yamamoto, Norio; Efimova, Elena V; Tsuchiya, Hiroyuki; Hoffman, Robert M

2015-04-01

The response to DNA damage during mitosis was visualized using real-time fluorescence imaging of focus formation by the DNA-damage repair (DDR) response protein 53BP1 linked to green fluorescent protein (GFP) (53BP1-GFP) in the MiaPaCa-2(Tet-On) pancreatic cancer cell line. To observe 53BP1-GFP foci during mitosis, MiaPaCa-2(Tet-On) 53BP1-GFP cells were imaged every 30 min by confocal microscopy. Time-lapse imaging demonstrated that 11.4 ± 2.1% of the mitotic MiaPaCa-2(Tet-On) 53BP1-GFP cells had increased focus formation over time. Non-mitotic cells did not have an increase in 53BP1-GFP focus formation over time. Some of the mitotic MiaPaCa-2(Tet-On) 53BP1-GFP cells with focus formation became apoptotic. The results of the present report suggest that DNA strand breaks occur during mitosis and undergo repair, which may cause some of the mitotic cells to enter apoptosis in a phenomenon possibly related to mitotic catastrophe. © 2014 Wiley Periodicals, Inc.

Systemic administration of low dosage of tetanus toxin decreases cell proliferation and neuroblast differentiation in the mouse hippocampal dentate gyrus

PubMed Central

Yan, Bing Chun; Kim, In Hye; Park, Joon Ha; Ahn, Ji Hyeon; Cho, Jeong-Hwi; Chen, Bai Hui; Lee, Jae-Chul; Choi, Jung Hoon; Yoo, Ki-Yeon; Lee, Choong Hyun; Cho, Jun Hwi

2013-01-01

In the present study, we investigated the effect of Tetaus toxin (TeT) on cell proliferation and neuroblast differentiation using specific markers: 5-bromo-2-deoxyuridine (BrdU) as an exogenous marker for cell proliferation, Ki-67 as an endogenous marker for cell proliferation and doublecortin (DCX) as a marker for neuroblasts in the mouse hippocampal dentate gyrus (DG) after TeT treatment. Mice were intraperitoneally administered 2.5 and 10 ng/kg TeT and sacrificed 15 days after the treatment. In both the TeT-treated groups, no neuronal death occurred in any layers of the DG using neuronal nuclei (NeuN, a neuron nuclei maker) and Fluoro-Jade B (F-J B, a high-affinity fluorescent marker for the localization of neuronal degeneration). In addition, no significant change in glial activation in both the 2.5 and 10 ng/kg TeT-treated-groups was found by GFAP (a marker for astrocytes) and Iba-1 (a marker for microglia) immunohistochemistry. However, in the 2.5 ng/kg TeT-treated-group, the mean number of BrdU, Ki-67 and DCX immunoreactive cells, respectively, were apparently decreased compared to the control group, and the mean number of each in the 10 ng/kg TeT-treated-group was much more decreased. In addition, processes of DCX-immunoreactive cells, which projected into the molecular layer, were short compared to those in the control group. In brief, our present results show that low dosage (10 ng/kg) TeT treatment apparently decreased cell proliferation and neuroblast differentiation in the mouse hippocampal DG without distinct gliosis as well as any loss of adult neurons. PMID:24106509

A site-specific genetic modification for induction of pluripotency and subsequent isolation of derived lung alveolar epithelial type II cells.

PubMed

Yan, Qing; Quan, Yuan; Sun, Huanhuan; Peng, Xinmiao; Zou, Zhengyun; Alcorn, Joseph L; Wetsel, Rick A; Wang, Dachun

2014-02-01

Human induced pluripotent stem cells (hiPSCs) have great therapeutic potential in repairing defective lung alveoli. However, genetic abnormalities caused by vector integrations and low efficiency in generating hiPSCs, as well as difficulty in obtaining transplantable hiPSC-derived cell types are still major obstacles. Here we report a novel strategy using a single nonviral site-specific targeting vector with a combination of Tet-On inducible gene expression system, Cre/lox P switching gene expression system, and alveolar epithelial type II cell (ATIIC)-specific Neomycin(R) transgene expression system. With this strategy, a single copy of all of the required transgenes can be specifically knocked into a site immediately downstream of β-2-microglobulin (B2M) gene locus at a high frequency, without causing B2M dysfunction. Thus, the expression of reprogramming factors, Oct4, Sox2, cMyc, and Klf4, can be precisely regulated for efficient reprogramming of somatic cells into random integration-free or genetic mutation-free hiPSCs. The exogenous reprogramming factor transgenes can be subsequently removed after reprogramming by transient expression of Cre recombinase, and the resulting random integration-free and exogenous reprogramming factor-free hiPSCs can be selectively differentiated into a homogenous population of ATIICs. In addition, we show that these hiPSC-derived ATIICs exhibit ultrastructural characteristics and biological functions of normal ATIICs. When transplanted into bleomycin-challenged mice lungs, hiPSC-derived ATIICs efficiently remain and re-epithelialize injured alveoli to restore pulmonary function, preventing lung fibrosis and increasing survival without tumorigenic side effect. This strategy allows for the first time efficient generation of patient-specific ATIICs for possible future clinical applications. © 2013 AlphaMed Press.

A site-specific genetic modification for induction of pluripotency and subsequent isolation of derived lung alveolar epithelial type II cells

PubMed Central

Yan, Qing; Quan, Yuan; Sun, Huanhuan; Peng, Xinmiao; Zou, Zhengyun; Alcorn, Joseph L.; Wetsel, Rick A.; Wang, Dachun

2013-01-01

Human induced pluripotent stem cells (hiPSCs) have great therapeutic potential in repairing defective lung alveoli. However, genetic abnormalities caused by vector-integrations and low efficiency in generating hiPSCs, as well as difficulty in obtaining transplantable hiPSC-derived cell types, are still major obstacles. Here we report a novel strategy using a single non-viral site-specific-targeting vector with a combination of Tet-On inducible gene expression system, Cre/lox P switching gene expression system, and alveolar epithelial type II cell (ATIIC)-specific NeomycinR trangene expression system. With this strategy, a single copy of all of the required transgenes can be specifically knocked into a site immediately downstream of beta-2-microglobulin (B2M) gene locus at a high frequency, without causing B2M dysfunction. Thus, the expression of reprogramming factors, Oct4, Sox2, cMyc and Klf4, can be precisely regulated for efficient reprogramming of somatic cells into random-integration-free or genetic mutation-free hiPSCs. The exogenous reprogramming factor transgenes can be subsequently removed after reprogramming by transient expression of Cre recombinase, and the resulting random-integration-free and exogenous reprogramming-factor-free hiPSCs can be selectively differentiated into a homogenous population of ATIICs. In addition, we show that these hiPSC-derived ATIICs exhibit ultra-structural characteristics and biological functions of normal ATIICs. When transplanted into bleomycin-challenged mice lungs, hiPSC-derived ATIICs efficiently remain and re-epithelialize injured alveoli to restore pulmonary function, preventing lung fibrosis and increasing survival without tumorigenic side effect. This strategy allows for the first time efficient generation of patient-specific ATIICs for possible future clinical applications. PMID:24123810

Relating Androgen Receptor Conformation to Function in Prostate Cancer Cells

DTIC Science & Technology

2005-01-01

line development , but have made progress towards resolving these issues and in development of alternate strategies. Task 1. Development of AR and...conformation. Task 2. Development of LNCaP Cells to Express Human AR mutants. We experienced unexpected difficulties in Task 2. We transfected the TET...Coactivators in AR Transactivation Summary Androgens drive sex differentiation, bone and muscle development , and promote growth of hormone dependent cancers

Disconnect between alcohol-induced alterations in chromatin structure and gene transcription in a mouse embryonic stem cell model of exposure

PubMed Central

Veazey, Kylee J.; Wang, Haiqing; Behdi, Yudhishtar S.; Skiles, William M.; Chang, Richard Cheng-An; Golding, Michael C.

2017-01-01

Alterations to chromatin structure induced by environmental insults have become an attractive explanation for the persistence of exposure effects into subsequent life stages. However, a growing body of work examining the epigenetic impact alcohol and other drugs of abuse exert consistently note a disconnect between induced changes in chromatin structure and patterns of gene transcription. Thus, an important question is whether perturbations in the ‘histone code’ induced by prenatal exposures to alcohol implicitly subvert gene expression, or if the hierarchy of cellular signaling networks driving development is such that they retain control over the transcriptional program. To address this question, we examined the impact of ethanol exposure in mouse embryonic stem cells cultured under 2i conditions, where the transcriptional program is rigidly enforced through the use of small molecule inhibitors. We find that ethanol-induced changes in post-translational histone modifications are dose-dependent, unique to the chromatin modification under investigation, and that the extent and direction of the change differ between the period of exposure and the recovery phase. Similar to in vivo models, we find post-translational modifications affecting histone 3 lysine 9 are the most profoundly impacted, with the signature of exposure persisting long after alcohol has been removed. These changes in chromatin structure associate with dose-dependent alterations in the levels of transcripts encoding Dnmt1, Uhrf1, Tet1, Tet2, Tet3, and Polycomb complex members Eed and Ezh2. However, in this model, ethanol-induced changes to the chromatin template do not consistently associate with changes in gene transcription, impede the process of differentiation or impact the acquisition of monoallelic patterns of expression for the imprinted gene Igf2R. These findings question the inferred universal relevance of epigenetic changes induced by drugs of abuse and suggest changes in chromatin structure cannot unequivocally explain dysgenesis in isolation. PMID:28433419

Disconnect between alcohol-induced alterations in chromatin structure and gene transcription in a mouse embryonic stem cell model of exposure.

PubMed

Veazey, Kylee J; Wang, Haiqing; Bedi, Yudhishtar S; Skiles, William M; Chang, Richard Cheng-An; Golding, Michael C

2017-05-01

Alterations to chromatin structure induced by environmental insults have become an attractive explanation for the persistence of exposure effects into subsequent life stages. However, a growing body of work examining the epigenetic impact that alcohol and other drugs of abuse exert consistently notes a disconnection between induced changes in chromatin structure and patterns of gene transcription. Thus, an important question is whether perturbations in the 'histone code' induced by prenatal exposures to alcohol implicitly subvert gene expression, or whether the hierarchy of cellular signaling networks driving development is such that they retain control over the transcriptional program. To address this question, we examined the impact of ethanol exposure in mouse embryonic stem cells cultured under 2i conditions, where the transcriptional program is rigidly enforced through the use of small molecule inhibitors. We find that ethanol-induced changes in post-translational histone modifications are dose-dependent, unique to the chromatin modification under investigation, and that the extent and direction of the change differ between the period of exposure and the recovery phase. Similar to in vivo models, we find post-translational modifications affecting histone 3 lysine 9 are the most profoundly impacted, with the signature of exposure persisting long after alcohol has been removed. These changes in chromatin structure associate with dose-dependent alterations in the levels of transcripts encoding Dnmt1, Uhrf1, Tet1, Tet2, Tet3, and Polycomb complex members Eed and Ezh2. However, in this model, ethanol-induced changes to the chromatin template do not consistently associate with changes in gene transcription, impede the process of differentiation, or affect the acquisition of monoallelic patterns of expression for the imprinted gene Igf2R. These findings question the inferred universal relevance of epigenetic changes induced by drugs of abuse and suggest that changes in chromatin structure cannot unequivocally explain dysgenesis in isolation. Copyright © 2017 Elsevier Inc. All rights reserved.

Mechanisms of reduced susceptibility and genotypic prediction of antibiotic resistance in Prevotella isolated from cystic fibrosis (CF) and non-CF patients

PubMed Central

Sherrard, Laura J.; Schaible, Bettina; Graham, Kathryn A.; McGrath, Stef J.; McIlreavey, Leanne; Hatch, Joseph; Wolfgang, Matthew C.; Muhlebach, Marianne S.; Gilpin, Deirdre F.; Schneiders, Thamarai; Elborn, J. Stuart; Tunney, Michael M.

2014-01-01

Objectives To investigate mechanisms of reduced susceptibility to commonly used antibiotics in Prevotella cultured from patients with cystic fibrosis (CF), patients with invasive infection and healthy control subjects and to determine whether genotype can be used to predict phenotypic resistance. Methods The susceptibility of 157 Prevotella isolates to seven antibiotics was compared, with detection of resistance genes (cfxA-type gene, ermF and tetQ), mutations within the CfxA-type β-lactamase and expression of efflux pumps. Results Prevotella isolates positive for a cfxA-type gene had higher MICs of amoxicillin and ceftazidime compared with isolates negative for this gene (P < 0.001). A mutation within the CfxA-type β-lactamase (Y239D) was associated with ceftazidime resistance (P = 0.011). The UK CF isolates were 5.3-fold, 2.7-fold and 5.7-fold more likely to harbour ermF compared with the US CF, UK invasive and UK healthy control isolates, respectively. Higher concentrations of azithromycin (P < 0.001) and clindamycin (P < 0.001) were also required to inhibit the growth of the ermF-positive isolates compared with ermF-negative isolates. Furthermore, tetQ-positive Prevotella isolates had higher MICs of tetracycline (P = 0.001) and doxycycline (P < 0.001) compared with tetQ-negative isolates. Prevotella spp. were also shown, for the first time, to express resistance nodulation division (RND)-type efflux pumps. Conclusions This study has demonstrated that Prevotella isolated from various sources harbour a common pool of resistance genes and possess RND-type efflux pumps, which may contribute to tetracycline resistance. The findings indicate that antibiotic resistance is common in Prevotella spp., but the genotypic traits investigated do not reflect phenotypic antibiotic resistance in every instance. PMID:24917582

40 CFR 721.6120 - Phosphoric acid, 1,2-eth-a-ne-diyl tet-ra-kis(2-chloro-1-meth-yl-ethyl) ester.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Phosphoric acid, 1,2-eth-a-ne-diyl tet-ra-kis(2-chloro-1-meth-yl-ethyl) ester. 721.6120 Section 721.6120 Protection of Environment...-diyl tet-ra-kis(2-chloro-1-meth-yl-ethyl) ester. (a) Chemical substances and significant new uses...

40 CFR 721.6120 - Phosphoric acid, 1,2-eth-a-ne-diyl tet-ra-kis(2-chloro-1-meth-yl-ethyl) ester.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Phosphoric acid, 1,2-eth-a-ne-diyl tet-ra-kis(2-chloro-1-meth-yl-ethyl) ester. 721.6120 Section 721.6120 Protection of Environment...-diyl tet-ra-kis(2-chloro-1-meth-yl-ethyl) ester. (a) Chemical substances and significant new uses...

40 CFR 721.6120 - Phosphoric acid, 1,2-eth-a-ne-diyl tet-ra-kis(2-chloro-1-meth-yl-ethyl) ester.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Phosphoric acid, 1,2-eth-a-ne-diyl tet-ra-kis(2-chloro-1-meth-yl-ethyl) ester. 721.6120 Section 721.6120 Protection of Environment...-diyl tet-ra-kis(2-chloro-1-meth-yl-ethyl) ester. (a) Chemical substances and significant new uses...

40 CFR 721.6120 - Phosphoric acid, 1,2-eth-a-ne-diyl tet-ra-kis(2-chloro-1-meth-yl-ethyl) ester.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Phosphoric acid, 1,2-eth-a-ne-diyl tet-ra-kis(2-chloro-1-meth-yl-ethyl) ester. 721.6120 Section 721.6120 Protection of Environment...-diyl tet-ra-kis(2-chloro-1-meth-yl-ethyl) ester. (a) Chemical substances and significant new uses...

40 CFR 721.6120 - Phosphoric acid, 1,2-eth-a-ne-diyl tet-ra-kis(2-chloro-1-meth-yl-ethyl) ester.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Phosphoric acid, 1,2-eth-a-ne-diyl tet-ra-kis(2-chloro-1-meth-yl-ethyl) ester. 721.6120 Section 721.6120 Protection of Environment...-diyl tet-ra-kis(2-chloro-1-meth-yl-ethyl) ester. (a) Chemical substances and significant new uses...

A qualified presumption of safety approach for the safety assessment of Grana Padano whey starters.

PubMed

Rossetti, Lia; Carminati, Domenico; Zago, Miriam; Giraffa, Giorgio

2009-03-15

A Qualified Presumption of Safety (QPS) approach was applied to dominant lactic acid bacteria (LAB) associated with Grana Padano cheese whey starters. Thirty-two strains belonging to Lactobacillus helveticus, Lactobacillus delbrueckii subsp. lactis, Streptococcus thermophilus, and Lactobacillus fermentum, and representing the overall genotypic LAB diversity associated with 24 previously collected whey starters [Rossetti, L., Fornasari, M.E., Gatti, M., Lazzi, C., Neviani, E., Giraffa, G., 2008. Grana Padano cheese whey starters: microbial composition and strain distribution. International Journal of Food Microbiology 127, 168-171], were analyzed. All L. helveticus, L. delbrueckii subsp. lactis, and S. thermophilus isolates were susceptible to four (i.e. vancomycin, gentamicin, tetracycline, and erythromycin) of the clinically most relevant antibiotics. One L. fermentum strain displayed phenotypic resistance to tetracycline (Tet(R)), with MIC of 32 microg/ml, and gentamycin (Gm(R)), with MIC of 32 microg/ml. PCR was applied to this strain to test the presence of genes tet(L), tet(M), tet(S), and aac(6')-aph(2')-Ia, which are involved in horizontal transfer of Tet(R) and Gm(R), respectively but no detectable amplification products were observed. According to QPS criteria, we conclude that Grana cheese whey starters do not present particular safety concerns.

Dnmts and Tet target memory-associated genes after appetitive olfactory training in honey bees

PubMed Central

Biergans, Stephanie D.; Giovanni Galizia, C.; Reinhard, Judith; Claudianos, Charles

2015-01-01

DNA methylation and demethylation are epigenetic mechanisms involved in memory formation. In honey bees DNA methyltransferase (Dnmt) function is necessary for long-term memory to be stimulus specific (i.e. to reduce generalization). So far, however, it remains elusive which genes are targeted and what the time-course of DNA methylation is during memory formation. Here, we analyse how DNA methylation affects memory retention, gene expression, and differential methylation in stimulus-specific olfactory long-term memory formation. Out of 30 memory-associated genes investigated here, 9 were upregulated following Dnmt inhibition in trained bees. These included Dnmt3 suggesting a negative feedback loop for DNA methylation. Within these genes also the DNA methylation pattern changed during the first 24 hours after training. Interestingly, this was accompanied by sequential activation of the DNA methylation machinery (i.e. Dnmts and Tet). In sum, memory formation involves a temporally complex epigenetic regulation of memory-associated genes that facilitates stimulus specific long-term memory in the honey bee. PMID:26531238

Characterization of an epoxide hydrolase from the Florida red tide dinoflagellate, Karenia brevis.

PubMed

Sun, Pengfei; Leeson, Cristian; Zhi, Xiaoduo; Leng, Fenfei; Pierce, Richard H; Henry, Michael S; Rein, Kathleen S

2016-02-01

Epoxide hydrolases (EH, EC 3.3.2.3) have been proposed to be key enzymes in the biosynthesis of polyether (PE) ladder compounds such as the brevetoxins which are produced by the dinoflagellate Karenia brevis. These enzymes have the potential to catalyze kinetically disfavored endo-tet cyclization reactions. Data mining of K. brevis transcriptome libraries revealed two classes of epoxide hydrolases: microsomal and leukotriene A4 (LTA4) hydrolases. A microsomal EH was cloned and expressed for characterization. The enzyme is a monomeric protein with molecular weight 44kDa. Kinetic parameters were evaluated using a variety of epoxide substrates to assess substrate selectivity and enantioselectivity, as well as its potential to catalyze the critical endo-tet cyclization of epoxy alcohols. Monitoring of EH activity in high and low toxin producing cultures of K. brevis over a three week period showed consistently higher activity in the high toxin producing culture implicating the involvement of one or more EH in brevetoxin biosynthesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Finite Element Simulation of Articular Contact Mechanics with Quadratic Tetrahedral Elements

PubMed Central

Maas, Steve A.; Ellis, Benjamin J.; Rawlins, David S.; Weiss, Jeffrey A.

2016-01-01

Although it is easier to generate finite element discretizations with tetrahedral elements, trilinear hexahedral (HEX8) elements are more often used in simulations of articular contact mechanics. This is due to numerical shortcomings of linear tetrahedral (TET4) elements, limited availability of quadratic tetrahedron elements in combination with effective contact algorithms, and the perceived increased computational expense of quadratic finite elements. In this study we implemented both ten-node (TET10) and fifteen-node (TET15) quadratic tetrahedral elements in FEBio (www.febio.org) and compared their accuracy, robustness in terms of convergence behavior and computational cost for simulations relevant to articular contact mechanics. Suitable volume integration and surface integration rules were determined by comparing the results of several benchmark contact problems. The results demonstrated that the surface integration rule used to evaluate the contact integrals for quadratic elements affected both convergence behavior and accuracy of predicted stresses. The computational expense and robustness of both quadratic tetrahedral formulations compared favorably to the HEX8 models. Of note, the TET15 element demonstrated superior convergence behavior and lower computational cost than both the TET10 and HEX8 elements for meshes with similar numbers of degrees of freedom in the contact problems that we examined. Finally, the excellent accuracy and relative efficiency of these quadratic tetrahedral elements was illustrated by comparing their predictions with those for a HEX8 mesh for simulation of articular contact in a fully validated model of the hip. These results demonstrate that TET10 and TET15 elements provide viable alternatives to HEX8 elements for simulation of articular contact mechanics. PMID:26900037

Tetracycline resistance phenotypes and genotypes of coagulase-negative staphylococcal isolates from bubaline mastitis in Egypt.

PubMed

El-Razik, K A Abd; Arafa, A A; Hedia, R H; Ibrahim, E S

2017-06-01

This study was devoted to elucidate the tetracycline resistance of coagulase-negative staphylococci (CNS) derived from normal and subclinical mastitic (SCM) buffaloes' milk in Egypt. A total of 81 milk samples from 46 normal buffalo milk samples and 35 SCM buffalo milk samples at private dairy farms of Egypt were used in this study. CNS were identified using phenotypic and molecular methods (polymerase chain reaction [PCR]). CNS isolates were tested for tetracycline resistance using routine methods and multiplex PCR targeting tetracycline ( tet ) resistance genes followed by sequencing of positive PCR products and phylogenetic analysis. Isolation and identification of 28 (34.5%) CNS from normal and SCM buffaloes' milk, namely, Staphylococcus intermedius (39.2%), Staphylococcus xylosus (25.0%), Staphylococcus epidermidis (10.7%), Staphylococcus hominis (10.7%), and 3.5% to each of Staphylococcus sciuri , Staphylococcus hyicus , Staphylococcus lugdunensis , and Staphylococcus simulans . Using nested PCR, all the 28 CNS isolates revealed positive for 16srRNA gene specific for genus staphylococci and negative for thermonuclease ( nuc ) gene specific for Staphylococcus aureus species. The presence of tetracycline resistance-encoding genes ( tet K, tet L, tet M, and tet O) was detected by multiplex PCR. All isolates were negative for tet L, M, and O genes while 14 (50%) CNS isolates were positive for tet K gene, namely, S. lugdunensis (100%), S. hominis (100%), S. epidermidis (66.6%), S. intermedius (45.4%), and S. xylosus (42.8%). Nucleotide sequencing of tet K gene followed by phylogenetic analysis showed the high homology between our CNS isolates genes of tetracycline resistance with S. aureus isolates including Egyptian ones. This proves the transfer of the tetracycline resistance encoding genes between coagulase-negative and coagulase positive Staphylococcus spp. CNS isolates have distinguishingly high resistance to tetracycline. Abundant tetracycline usage for mastitis treatment leads to the spread of genetic resistance mechanisms inside CNS strains and among all Staphylococcus spp. Consequently, tetracycline is not effective anymore.

Tetracycline resistance phenotypes and genotypes of coagulase-negative staphylococcal isolates from bubaline mastitis in Egypt

PubMed Central

El-Razik, K. A. Abd; Arafa, A. A.; Hedia, R. H.; Ibrahim, E. S.

2017-01-01

Aim:: This study was devoted to elucidate the tetracycline resistance of coagulase-negative staphylococci (CNS) derived from normal and subclinical mastitic (SCM) buffaloes’ milk in Egypt. Materials and Methods: :: A total of 81 milk samples from 46 normal buffalo milk samples and 35 SCM buffalo milk samples at private dairy farms of Egypt were used in this study. CNS were identified using phenotypic and molecular methods (polymerase chain reaction [PCR]). CNS isolates were tested for tetracycline resistance using routine methods and multiplex PCR targeting tetracycline (tet) resistance genes followed by sequencing of positive PCR products and phylogenetic analysis. Results:: Isolation and identification of 28 (34.5%) CNS from normal and SCM buffaloes’ milk, namely, Staphylococcus intermedius (39.2%), Staphylococcus xylosus (25.0%), Staphylococcus epidermidis (10.7%), Staphylococcus hominis (10.7%), and 3.5% to each of Staphylococcus sciuri, Staphylococcus hyicus, Staphylococcus lugdunensis, and Staphylococcus simulans. Using nested PCR, all the 28 CNS isolates revealed positive for 16srRNA gene specific for genus staphylococci and negative for thermonuclease (nuc) gene specific for Staphylococcus aureus species. The presence of tetracycline resistance-encoding genes (tetK, tetL, tetM, and tetO) was detected by multiplex PCR. All isolates were negative for tetL, M, and O genes while 14 (50%) CNS isolates were positive for tetK gene, namely, S. lugdunensis (100%), S. hominis (100%), S. epidermidis (66.6%), S. intermedius (45.4%), and S. xylosus (42.8%). Nucleotide sequencing of tetK gene followed by phylogenetic analysis showed the high homology between our CNS isolates genes of tetracycline resistance with S. aureus isolates including Egyptian ones. This proves the transfer of the tetracycline resistance encoding genes between coagulase-negative and coagulase positive Staphylococcus spp. Conclusion:: CNS isolates have distinguishingly high resistance to tetracycline. Abundant tetracycline usage for mastitis treatment leads to the spread of genetic resistance mechanisms inside CNS strains and among all Staphylococcus spp. Consequently, tetracycline is not effective anymore. PMID:28717325

Detection and Characterization of Low Temperature Peat Fires during the 2015 Fire Catastrophe in Indonesia Using a New High-Sensitivity Fire Monitoring Satellite Sensor (FireBird)

PubMed Central

Atwood, Elizabeth C.; Englhart, Sandra; Lorenz, Eckehard; Halle, Winfried; Wiedemann, Werner; Siegert, Florian

2016-01-01

Vast and disastrous fires occurred on Borneo during the 2015 dry season, pushing Indonesia into the top five carbon emitting countries. The region was affected by a very strong El Niño-Southern Oscillation (ENSO) climate phenomenon, on par with the last severe event in 1997/98. Fire dynamics in Central Kalimantan were investigated using an innovative sensor offering higher sensitivity to a wider range of fire intensities at a finer spatial resolution (160 m) than heretofore available. The sensor is onboard the TET-1 satellite, part of the German Aerospace Center (DLR) FireBird mission. TET-1 images (acquired every 2–3 days) from the middle infrared were used to detect fires continuously burning for almost three weeks in the protected peatlands of Sebangau National Park as well as surrounding areas with active logging and oil palm concessions. TET-1 detection capabilities were compared with MODIS active fire detection and Landsat burned area algorithms. Fire dynamics, including fire front propagation speed and area burned, were investigated. We show that TET-1 has improved detection capabilities over MODIS in monitoring low-intensity peatland fire fronts through thick smoke and haze. Analysis of fire dynamics revealed that the largest burned areas resulted from fire front lines started from multiple locations, and the highest propagation speeds were in excess of 500 m/day (all over peat > 2m deep). Fires were found to occur most often in concessions that contained drainage infrastructure but were not cleared prior to the fire season. Benefits of implementing this sensor system to improve current fire management techniques are discussed. Near real-time fire detection together with enhanced fire behavior monitoring capabilities would not only improve firefighting efforts, but also benefit analysis of fire impact on tropical peatlands, greenhouse gas emission estimations as well as mitigation measures to reduce severe fire events in the future. PMID:27486664

Detection and Characterization of Low Temperature Peat Fires during the 2015 Fire Catastrophe in Indonesia Using a New High-Sensitivity Fire Monitoring Satellite Sensor (FireBird).

PubMed

Atwood, Elizabeth C; Englhart, Sandra; Lorenz, Eckehard; Halle, Winfried; Wiedemann, Werner; Siegert, Florian

2016-01-01

Vast and disastrous fires occurred on Borneo during the 2015 dry season, pushing Indonesia into the top five carbon emitting countries. The region was affected by a very strong El Niño-Southern Oscillation (ENSO) climate phenomenon, on par with the last severe event in 1997/98. Fire dynamics in Central Kalimantan were investigated using an innovative sensor offering higher sensitivity to a wider range of fire intensities at a finer spatial resolution (160 m) than heretofore available. The sensor is onboard the TET-1 satellite, part of the German Aerospace Center (DLR) FireBird mission. TET-1 images (acquired every 2-3 days) from the middle infrared were used to detect fires continuously burning for almost three weeks in the protected peatlands of Sebangau National Park as well as surrounding areas with active logging and oil palm concessions. TET-1 detection capabilities were compared with MODIS active fire detection and Landsat burned area algorithms. Fire dynamics, including fire front propagation speed and area burned, were investigated. We show that TET-1 has improved detection capabilities over MODIS in monitoring low-intensity peatland fire fronts through thick smoke and haze. Analysis of fire dynamics revealed that the largest burned areas resulted from fire front lines started from multiple locations, and the highest propagation speeds were in excess of 500 m/day (all over peat > 2m deep). Fires were found to occur most often in concessions that contained drainage infrastructure but were not cleared prior to the fire season. Benefits of implementing this sensor system to improve current fire management techniques are discussed. Near real-time fire detection together with enhanced fire behavior monitoring capabilities would not only improve firefighting efforts, but also benefit analysis of fire impact on tropical peatlands, greenhouse gas emission estimations as well as mitigation measures to reduce severe fire events in the future.

Structures of the TetR-like simocyclinone efflux pump repressor, SimR, and the mechanism of ligand-mediated derepression.

PubMed

Le, Tung B K; Stevenson, Clare E M; Fiedler, Hans-Peter; Maxwell, Anthony; Lawson, David M; Buttner, Mark J

2011-04-22

Simocyclinone D8 (SD8), a potent DNA gyrase inhibitor made by Streptomyces antibioticus, is exported from the producing organism by the SimX efflux pump. The expression of simX is under the control of SimR, a member of the TetR family of transcriptional regulators. SimR represses simX transcription by binding to operators in the intergenic region between simR and simX. Previously, we have shown that the mature antibiotic SD8 or its biosynthetic intermediate, simocyclinone C4, can dissociate SimR from its operators, leading to derepression of simX and export of SD8 from the cell. This provides a mechanism that couples the biosynthesis of the antibiotic to its export. Here, we report the crystal structures of SimR alone and in complex with either SD8 or simocyclinone C4. The ligand-binding pocket is unusual compared to those of other characterized TetR-family transcriptional regulators: the structures show an extensive ligand-binding pocket spanning both monomers in the functional dimeric unit, with the aminocoumarin moiety of SD8 buried in the protein core, while the angucyclic polyketide moiety is partially exposed to bulk solvent. Through comparisons of the structures, we postulate a derepression mechanism for SimR that invokes rigid-body motions of the subunits relative to one another, coupled with a putative locking mechanism to restrict further conformational change. Copyright © 2011 Elsevier Ltd. All rights reserved.

Regulation of BDNF chromatin status and promoter accessibility in a neural correlate of associative learning

PubMed Central

Ambigapathy, Ganesh; Zheng, Zhaoqing; Keifer, Joyce

2015-01-01

Brain-derived neurotrophic factor (BDNF) gene expression critically controls learning and its aberrant regulation is implicated in Alzheimer's disease and a host of neurodevelopmental disorders. The BDNF gene is target of known DNA regulatory mechanisms but details of its activity-dependent regulation are not fully characterized. We performed a comprehensive analysis of the epigenetic regulation of the turtle BDNF gene (tBDNF) during a neural correlate of associative learning using an in vitro model of eye blink classical conditioning. Shortly after conditioning onset, the results from ChIP-qPCR show conditioning-dependent increases in methyl-CpG-binding protein 2 (MeCP2) and repressor basic helix-loop-helix binding protein 2 (BHLHB2) binding to tBDNF promoter II that corresponds with transcriptional repression. In contrast, enhanced binding of ten-eleven translocation protein 1 (Tet1), extracellular signal-regulated kinase 1/2 (ERK1/2), and cAMP response element-binding protein (CREB) to promoter III corresponds with transcriptional activation. These actions are accompanied by rapid modifications in histone methylation and phosphorylation status of RNA polymerase II (RNAP II). Significantly, these remarkably coordinated changes in epigenetic factors for two alternatively regulated tBDNF promoters during conditioning are controlled by Tet1 and ERK1/2. Our findings indicate that Tet1 and ERK1/2 are critical partners that, through complementary functions, control learning-dependent tBDNF promoter accessibility required for rapid transcription and acquisition of classical conditioning. PMID:26336984

Direct regulation of E-cadherin by targeted histone methylation of TALE-SET fusion protein in cancer cells.

PubMed

Cho, Hyun-Soo; Kang, Jeong Gu; Lee, Jae-Hye; Lee, Jeong-Ju; Jeon, Seong Kook; Ko, Jeong-Heon; Kim, Dae-Soo; Park, Kun-Hyang; Kim, Yong-Sam; Kim, Nam-Soon

2015-09-15

TALE-nuclease chimeras (TALENs) can bind to and cleave specific genomic loci and, are used to engineer gene knockouts and additions. Recently, instead of using the FokI domain, epigenetically active domains, such as TET1 and LSD1, have been combined with TAL effector domains to regulate targeted gene expression via DNA and histone demethylation. However, studies of histone methylation in the TALE system have not been performed. Therefore, in this study, we established a novel targeted regulation system with a TAL effector domain and a histone methylation domain. To construct a TALE-methylation fusion protein, we combined a TAL effector domain containing an E-Box region to act as a Snail binding site and the SET domain of EHMT 2 to allow for histone methylation. The constructed TALE-SET module (TSET) repressed the expression of E-cadherin via by increasing H3K9 dimethylation. Moreover, the cells that overexpressed TSET showed increased cell migration and invasion. This is the first phenotype-based study of targeted histone methylation by the TALE module, and this new system can be applied in new cancer therapies to reduce side effects.

An affinity-directed protein missile system for targeted proteolysis

PubMed Central

Fulcher, Luke J.; Macartney, Thomas; Bozatzi, Polyxeni; Hornberger, Annika; Rojas-Fernandez, Alejandro

2016-01-01

The von Hippel–Lindau (VHL) protein serves to recruit the hypoxia-inducible factor alpha (HIF1α) protein under normoxia to the CUL2 E3 ubiquitin ligase for its ubiquitylation and degradation through the proteasome. In this report, we modify VHL to engineer an affinity-directed protein missile (AdPROM) system to direct specific endogenous target proteins for proteolysis in mammalian cells. The proteolytic AdPROM construct harbours a cameloid anti-green fluorescence protein (aGFP) nanobody that is fused to VHL for either constitutive or tetracycline-inducible expression. For target proteins, we exploit CRISPR/Cas9 to rapidly generate human kidney HEK293 and U2OS osteosarcoma homozygous knock-in cells harbouring GFP tags at the VPS34 (vacuolar protein sorting 34) and protein associated with SMAD1 (PAWS1, aka FAM83G) loci, respectively. Using these cells, we demonstrate that the expression of the VHL-aGFP AdPROM system results in near-complete degradation of the endogenous GFP-VPS34 and PAWS1-GFP proteins through the proteasome. Additionally, we show that Tet-inducible destruction of GFP-VPS34 results in the degradation of its associated partner, UVRAG, and reduction in levels of cellular phosphatidylinositol 3-phosphate. PMID:27784791

Genetic Characterization of Circulating African Swine Fever Viruses in Nigeria (2007-2015).

PubMed

Luka, P D; Achenbach, J E; Mwiine, F N; Lamien, C E; Shamaki, D; Unger, H; Erume, J

2017-10-01

Sequencing and analysis of three discrete genome regions of African swine fever viruses (ASFV) from archival samples collected in 2007-2011 and active and passive surveillance between 2012 and 2015 in Nigeria were carried out. Analysis was conducted by genotyping of three single-copy African swine fever (ASF) genes. The E183L and B646L genes that encode structural proteins p54 and p72, respectively, were utilized to delineate genotypes before intragenotypic resolution by characterization of the tetrameric amino acid repeat region within the hypervariable central variable region of the B602L gene. The results showed no variation in the p72 and p54 gene regions sequenced. Phylogeny of p72 sequences revealed that all the Nigerian isolates belonged to genotype I, while that of the p54 recovered the Ia genotype. Analysis of B602L gene revealed the differences in the number of tetrameric repeats. Four new variants (Tet-15, Tet-17a, Tet-17b and Tet-48) were recovered, while a fifth variant (Tet-20) was the most widely distributed in the country displacing Tet-36 reported previously in 2003-2006. The viruses responsible for ASF outbreaks in Nigeria are from very closely related but mutated variants of the virus that have been circulating since 1997. A practical implication of the genetic variability of the Nigerian viral isolates in this study is the need for continuous sampling and analysis of circulating viruses, which will provide epidemiological information on the evolution of ASFV in the field versus new incursion for informed strategic control of the disease in the country. © 2016 Blackwell Verlag GmbH.

PigZ, a TetR/AcrR family repressor, modulates secondary metabolism via the expression of a putative four-component resistance-nodulation-cell-division efflux pump, ZrpADBC, in Serratia sp. ATCC 39006.

PubMed

Gristwood, Tamzin; Fineran, Peter C; Everson, Lee; Salmond, George P C

2008-07-01

The Gram-negative enterobacterium, Serratia sp. ATCC 39006 synthesizes several secondary metabolites, including prodigiosin (Pig) and a carbapenem antibiotic (Car). A complex hierarchical network of regulatory proteins control Pig and Car production. In this study we characterize a TetR family regulator, PigZ, which represses transcription of a divergently transcribed putative resistance-nodulation-cell-division (RND) efflux pump, encoded by zrp (PigZ repressed pump) ADBC, via direct binding to the zrpA-pigZ intergenic region. Unusually, this putative RND pump contains two predicted membrane fusion proteins (MFPs), ZrpA and ZrpD. A mutation in pigZ resulted in multiple phenotypic changes, including exoenzyme production, motility and differential regulation of Pig and Car production. A polar suppressor mutation, within zrpA, restored all tested phenotypes to parental strain levels, indicating that the changes observed are due to the increase in expression of ZrpADBC in the absence of the repressor, PigZ. Genomic deletions of zrpA and zrpD indicate that the MFP ZrpD, but not ZrpA, is essential for activity of the putative pump. Bioinformatic analysis revealed that putative RND efflux pumps encoding two MFP components are not uncommon, particularly among plant-associated, Gram-negative bacteria. In addition, based on phylogenetic analysis, we propose that these pairs of MFPs consist of two distinct subtypes.

Autoclave treatment of pig manure does not reduce the risk of transmission and transfer of tetracycline resistance genes in soil: successive determinations with soil column experiments.

PubMed

Kang, Yijun; Gu, Xian; Hao, Yangyang; Hu, Jian

2016-03-01

The increasing use of antibiotics, especially tetracycline, in livestock feed adversely affects animal health and ecological integrity. Therefore, approaches to decrease this risk are urgently needed. High temperatures facilitate antibiotic degradation; whether this reduces transmission risk and transfer of tetracycline-resistant bacteria (TRBs) and tetracycline resistance genes (TRGs) in soil remains unknown. Successive experiments with soil columns evaluated the effects of autoclaving pig manure (APM) on soil TRB populations and TRGs over time at different soil depths. The data showed sharp increases in TRB populations and TRGs in each subsoil layer of PM (non-APM) and APM treatments within 30 days, indicating that TRBs and TRGs transferred rapidly. The level of TRBs in the upper soil layers was approximately 15-fold higher than in subsoils. TRBs were not dependent on PM and APM levels, especially in the late phase. Nevertheless, higher levels of APM led to rapid expansion of TRBs as compared to PM. Moreover, temporal changes in TRB frequencies in total culturable bacteria (TCBs) were similar to TRBs, indicating that the impact of PM or APM on TRBs was more obvious than for TCBs. TRBs were hypothesized to depend on the numbers of TRGs and indigenous recipient bacteria. In the plough layer, five TRGs (tetB, tetG, tetM, tetW, and tetB/P) existed in each treatment within 150 days. Selective pressure of TC may not be a necessary condition for the transfer and persistence of TRGs in soil. High temperatures might reduce TRBs in PM, which had minimal impact on the transmission and transfer of TRGs in soil. Identifying alternatives to decrease TRG transmission remains a major challenge.

Deletions in the tetracycline resistance determinant reduce the thermosensitivity of a trfA(Ts) derivative of plasmid RP1 in Pseudomonas aeruginosa.

PubMed

Rella, M; Watson, J M; Thomas, C M; Haas, D

1987-01-01

A derivative of the broad-host-range plasmid RP1, pME301, was temperature-sensitive (Ts) at 43 degrees C for maintenance in Pseudomonas aeruginosa, P. mendocina, Klebsiella aerogenes and Escherichia coli. In E. coli, the Ts defect of pME301 could be complemented in trans by the cloned trfA gene, which is known to be essential for RP1 replication in E. coli and P. aeruginosa. Because pME301 expressed a Ts phenotype in P. mendocina and K. aerogenes, we assume that the trfA function is also vital in these organisms. When plasmid-encoded carbenicillin resistance (on transposon Tn801) was selected at non-permissive temperatures in P. aeruginosa strain PAO carrying pME301, we obtained either Tn801 insertions into the chromosome or pME301 derivatives having a deletion (or point mutation) in their tet genes, which determine resistance to tetracycline and are not transposable. From cloning experiments, we infer that the tet gene product(s) destabilize the pME301 replicon in P. aeruginosa at 40-43 degrees C.

Stabilization of the μ-opioid receptor by truncated single transmembrane splice variants through a chaperone-like action.

PubMed

Xu, Jin; Xu, Ming; Brown, Taylor; Rossi, Grace C; Hurd, Yasmin L; Inturrisi, Charles E; Pasternak, Gavril W; Pan, Ying-Xian

2013-07-19

The μ-opioid receptor gene, OPRM1, undergoes extensive alternative pre-mRNA splicing, as illustrated by the identification of an array of splice variants generated by both 5' and 3' alternative splicing. The current study reports the identification of another set of splice variants conserved across species that are generated through exon skipping or insertion that encodes proteins containing only a single transmembrane (TM) domain. Using a Tet-Off system, we demonstrated that the truncated single TM variants can dimerize with the full-length 7-TM μ-opioid receptor (MOR-1) in the endoplasmic reticulum, leading to increased expression of MOR-1 at the protein level by a chaperone-like function that minimizes endoplasmic reticulum-associated degradation. In vivo antisense studies suggested that the single TM variants play an important role in morphine analgesia, presumably through modulation of receptor expression levels. Our studies suggest the functional roles of truncated receptors in other G protein-coupled receptor families.

Novel mutations and their functional and clinical relevance in myeloproliferative neoplasms: JAK2, MPL, TET2, ASXL1, CBL, IDH and IKZF1

PubMed Central

Tefferi, A

2010-01-01

Myeloproliferative neoplasms (MPNs) originate from genetically transformed hematopoietic stem cells that retain the capacity for multilineage differentiation and effective myelopoiesis. Beginning in early 2005, a number of novel mutations involving Janus kinase 2 (JAK2), Myeloproliferative Leukemia Virus (MPL), TET oncogene family member 2 (TET2), Additional Sex Combs-Like 1 (ASXL1), Casitas B-lineage lymphoma proto-oncogene (CBL), Isocitrate dehydrogenase (IDH) and IKAROS family zinc finger 1 (IKZF1) have been described in BCR-ABL1-negative MPNs. However, none of these mutations were MPN specific, displayed mutual exclusivity or could be traced back to a common ancestral clone. JAK2 and MPL mutations appear to exert a phenotype-modifying effect and are distinctly associated with polycythemia vera, essential thrombocythemia and primary myelofibrosis; the corresponding mutational frequencies are ∼99, 55 and 65% for JAK2 and 0, 3 and 10% for MPL mutations. The incidence of TET2, ASXL1, CBL, IDH or IKZF1 mutations in these disorders ranges from 0 to 17% these latter mutations are more common in chronic (TET2, ASXL1, CBL) or juvenile (CBL) myelomonocytic leukemias, mastocytosis (TET2), myelodysplastic syndromes (TET2, ASXL1) and secondary acute myeloid leukemia, including blast-phase MPN (IDH, ASXL1, IKZF1). The functional consequences of MPN-associated mutations include unregulated JAK-STAT (Janus kinase/signal transducer and activator of transcription) signaling, epigenetic modulation of transcription and abnormal accumulation of oncoproteins. However, it is not clear as to whether and how these abnormalities contribute to disease initiation, clonal evolution or blastic transformation. PMID:20428194

Finite element simulation of articular contact mechanics with quadratic tetrahedral elements.

PubMed

Maas, Steve A; Ellis, Benjamin J; Rawlins, David S; Weiss, Jeffrey A

2016-03-21

Although it is easier to generate finite element discretizations with tetrahedral elements, trilinear hexahedral (HEX8) elements are more often used in simulations of articular contact mechanics. This is due to numerical shortcomings of linear tetrahedral (TET4) elements, limited availability of quadratic tetrahedron elements in combination with effective contact algorithms, and the perceived increased computational expense of quadratic finite elements. In this study we implemented both ten-node (TET10) and fifteen-node (TET15) quadratic tetrahedral elements in FEBio (www.febio.org) and compared their accuracy, robustness in terms of convergence behavior and computational cost for simulations relevant to articular contact mechanics. Suitable volume integration and surface integration rules were determined by comparing the results of several benchmark contact problems. The results demonstrated that the surface integration rule used to evaluate the contact integrals for quadratic elements affected both convergence behavior and accuracy of predicted stresses. The computational expense and robustness of both quadratic tetrahedral formulations compared favorably to the HEX8 models. Of note, the TET15 element demonstrated superior convergence behavior and lower computational cost than both the TET10 and HEX8 elements for meshes with similar numbers of degrees of freedom in the contact problems that we examined. Finally, the excellent accuracy and relative efficiency of these quadratic tetrahedral elements was illustrated by comparing their predictions with those for a HEX8 mesh for simulation of articular contact in a fully validated model of the hip. These results demonstrate that TET10 and TET15 elements provide viable alternatives to HEX8 elements for simulation of articular contact mechanics. Copyright © 2016 Elsevier Ltd. All rights reserved.

Detection of the antibiotic resistance genes blaTem-1, cfxA, tetQ, tetM, tetW, and ermC in endodontic infections of a Mexican population.

PubMed

Domínguez-Pérez, Rubén Abraham; De la Torre-Luna, Rocio; Ahumada-Cantillano, Mariana; Vázquez-Garcidueñas, Ma Soledad; Pérez-Serrano, Rosa Martha; Martínez-Martínez, Rita Elizabeth; Guillén-Nepita, Ana Laura

2018-05-22

To identify the prevalence of genes encoding resistance to three groups of antibiotics in root canals with primary infection or post-treatment disease. Sixty four subjects who needed root canal treatment because of primary infection or post-treatment disease were enrolled in the present cross-sectional analytic study. Root canal samples were obtained, and DNA isolated. Specific primers for six antibiotic resistance genes and seven bacterial taxa (five genera and two species) were used. Student t test, chi-square test, and the Fisher's exact test were applied when appropriate to detect statistical differences. blaTEM-1, ermC, and tetM were more frequently found in the post-treatment disease group. While tetQ and cfxA were not found in any case. The occurrence of assessed bacteria were similar in both groups, except for Enterococcus spp. and P. endodontalis, which were found with a significant higher frequency in the post-treatment disease group. It was evident that the post-treatment disease group harboured more antibiotic resistance genes. The most frequent was tetW whereas tetQ and cfxA were not detected. With respect to bacterial taxa, Fusobacterium spp. was present in the 100% while the species Porphyromonas gingivalis was not in any of the samples. In all cases, at least one antibiotic resistance gene was detected, 32.8% were positive to four resistance genes, 54.6% to three, 9.3% to two and only 3.1% to one resistance gene. This indicates a high prevalence and diversity of antibiotic resistance genes in the sample. Copyright © 2018. Published by Elsevier Ltd.

Effects of chlortetracycline and copper on tetracyclines and copper resistance genes and microbial community during swine manure anaerobic digestion.

PubMed

Wang, Rui; Chen, Meixue; Feng, Feng; Zhang, Junya; Sui, Qianwen; Tong, Juan; Wei, Yuansong; Wei, Dongbin

2017-08-01

As antibiotic and heavy metals are over used in the livestock industry, animal manure is a reservoir of antibiotic resistance genes (ARGs). Anaerobic digestion has been reported to have the potential to reduce ARGs. However, few studies investigated whether reduction of ARGs would be affected by different external pressures including antibiotics and heavy metals during anaerobic digestion. The purpose of this study was thus to investigate effects of both chlortetracycline (CTC) and Cu on reduction of ARGs, heavy metal resistance genes (HMRGs) and mobile genetic elements (MGEs) during the swine manure anaerobic digestion. The results showed that the predominant ARGs (tetO, tetW, tetX, tetL) could be effectively reduced (approximately 1.00 log copies/g TS) through mesophilic anaerobic digestion. Microbial community evolution was the main driver. It was interesting that Treponema might indicate the termination of anaerobic digestion and compete with ARGs host bacteria. Addition of CTC, Cu and CTC+Cu affected microbial community change and hindered removal of ARGs, especially, CTC+Cu seriously affected Treponema and ARGs during anaerobic digestion. Copyright © 2017 Elsevier Ltd. All rights reserved.

Analysis of antimicrobial resistance genes detected in multidrug-resistant Salmonella enterica serovar Typhimurium isolated from food animals.

PubMed

Glenn, LaShanda M; Lindsey, Rebecca L; Frank, Joseph F; Meinersmann, Richard J; Englen, Mark D; Fedorka-Cray, Paula J; Frye, Jonathan G

2011-09-01

Multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium is the most prevalent penta-resistant serovar isolated from animals by the U.S. National Antimicrobial Resistance Monitoring System. Penta-resistant isolates are often resistant to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline. To investigate MDR in Salmonella Typhimurium (including variant 5-), one isolate each from cattle, poultry, and swine with at least the ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline phenotype were selected for each year from 1997 to 2007 (n = 33) for microarray analysis of antimicrobial resistance, incompatibility IncA/C, and HI1 plasmid genes. Cluster analysis based on these data separated 31 of the isolates into two groups A and B (15 and 16 isolates, respectively). Isolates in group A were phage type DT104 or U302 and were mostly swine isolates (7/15). Genes detected included intI1, bla(PSE-1), floR, aadA, sulI, tet(G), and tetR, which are often found in Salmonella Genomic Island I. Isolates in group B had numerous IncA/C plasmid genes detected and were mostly cattle isolates (9/16). Genes detected included bla(CMY-2), floR, aac(3), aadA, aphA1, strA, strB, sulI, sulII, dfrA, dhf, tet(A)(B)(C)(D), and tetR, which are often found on MDR-AmpC IncA/C plasmids. The IncA/C replicon was also detected in all group B isolates. The two remaining isolates did not cluster with any others and both had many HI1 plasmid genes detected. Linkage disequilibrium analysis detected significant associations between plasmid replicon type, phage type, and animal source. These data suggest that MDR in Salmonella Typhimurium is associated with DT104/Salmonella Genomic Island I or IncA/C MDR-AmpC encoding plasmids and these genetic elements have persisted throughout the study period.

A protein functional leap: how a single mutation reverses the function of the transcription regulator TetR.

PubMed

Resch, Marcus; Striegl, Harald; Henssler, Eva Maria; Sevvana, Madhumati; Egerer-Sieber, Claudia; Schiltz, Emile; Hillen, Wolfgang; Muller, Yves A

2008-08-01

Today's proteome is the result of innumerous gene duplication, mutagenesis, drift and selection processes. Whereas random mutagenesis introduces predominantly only gradual changes in protein function, a case can be made that an abrupt switch in function caused by single amino acid substitutions will not only considerably further evolution but might constitute a prerequisite for the appearance of novel functionalities for which no promiscuous protein intermediates can be envisaged. Recently, tetracycline repressor (TetR) variants were identified in which binding of tetracycline triggers the repressor to associate with and not to dissociate from the operator DNA as in wild-type TetR. We investigated the origin of this activity reversal by limited proteolysis, CD spectroscopy and X-ray crystallography. We show that the TetR mutant Leu17Gly switches its function via a disorder-order mechanism that differs completely from the allosteric mechanism of wild-type TetR. Our study emphasizes how single point mutations can engender unexpected leaps in protein function thus enabling the appearance of new functionalities in proteins without the need for promiscuous intermediates.

Widespread distribution of a tet W determinant among tetracycline-resistant isolates of the animal pathogen Arcanobacterium pyogenes.

PubMed

Billington, Stephen J; Songer, J Glenn; Jost, B Helen

2002-05-01

Tetracycline resistance is common among isolates of the animal commensal and opportunistic pathogen Arcanobacterium pyogenes. The tetracycline resistance determinant cloned from two bovine isolates of A. pyogenes was highly similar at the DNA level (92% identity) to the tet(W) gene, encoding a ribosomal protection tetracycline resistance protein, from the rumen bacterium Butyrivibrio fibrisolvens. The tet(W) gene was found in all 20 tetracycline-resistant isolates tested, indicating that it is a widely distributed determinant of tetracycline resistance in this organism. In 25% of tetracycline-resistant isolates, the tet(W) gene was associated with a mob gene, encoding a functional mobilization protein, and an origin of transfer, suggesting that the determinant may be transferable to other bacteria. In fact, low-frequency transfer of tet(W) was detected from mob+ A. pyogenes isolates to a tetracycline-sensitive A. pyogenes recipient. The mobile nature of this determinant and the presence of A. pyogenes in the gastrointestinal tract of cattle and pigs suggest that A. pyogenes may have inherited this determinant within the gastrointestinal tracts of these animals.

An HDAC2-TET1 switch at distinct chromatin regions significantly promotes the maturation of pre-iPS to iPS cells

PubMed Central

Wei, Tingyi; Chen, Wen; Wang, Xiukun; Zhang, Man; Chen, Jiayu; Zhu, Songcheng; Chen, Long; Yang, Dandan; Wang, Guiying; Jia, Wenwen; Yu, Yangyang; Duan, Tao; Wu, Minjuan; Liu, Houqi; Gao, Shaorong; Kang, Jiuhong

2015-01-01

The maturation of induced pluripotent stem cells (iPS) is one of the limiting steps of somatic cell reprogramming, but the underlying mechanism is largely unknown. Here, we reported that knockdown of histone deacetylase 2 (HDAC2) specifically promoted the maturation of iPS cells. Further studies showed that HDAC2 knockdown significantly increased histone acetylation, facilitated TET1 binding and DNA demethylation at the promoters of iPS cell maturation-related genes during the transition of pre-iPS cells to a fully reprogrammed state. We also found that HDAC2 competed with TET1 in the binding of the RbAp46 protein at the promoters of maturation genes and knockdown of TET1 markedly prevented the activation of these genes. Collectively, our data not only demonstrated a novel intrinsic mechanism that the HDAC2-TET1 switch critically regulates iPS cell maturation, but also revealed an underlying mechanism of the interplay between histone acetylation and DNA demethylation in gene regulation. PMID:25934799

Tetracycline residues and tetracycline resistance genes in groundwater impacted by swine production facilities

USGS Publications Warehouse

Mackie, R.I.; Koike, S.; Krapac, I.; Chee-Sanford, J.; Maxwell, Susan; Aminov, R.I.

2006-01-01

Antibiotics are used at therapeutic levels to treat disease; at slightly lower levels as prophylactics; and at low, subtherapeutic levels for growth promotion and improvement of feed efficiency. Over 88% of swine producers in the United States gave antimicrobials to grower/finisher pigs in feed as a growth promoter in 2000. It is estimated that ca. 75% of antibiotics are not absorbed by animals and are excreted in urine and feces. The extensive use of antibiotics in swine production has resulted in antibiotic resistance in many intestinal bacteria, which are also excreted in swine feces, resulting in dissemination of resistance genes into the environment.To assess the impact of manure management on groundwater quality, groundwater samples have been collected near two swine confinement facilities that use lagoons for manure storage and treatment. Several key contaminant indicators-including inorganic ions, antibiotics, and antibiotic resistance genes-were analyzed in groundwater collected from the monitoring wells. Chloride, ammonium, potassium, and sodium were predominant inorganic constituents in the manure samples and served as indicators of groundwater contamination. Based on these analyses, shallow groundwater has been impacted by lagoon seepage at both sites. Liquid chromatography-mass spectroscopy (LC-MS) was used to measure the dissolved concentrations of tetracycline, chlortetracycline, and oxytetracycline in groundwater and manure. Although tetracyclines were regularly used at both facilities, they were infrequently detected in manure samples and then at relatively trace concentrations. Concentrations of all tetracyclines and their breakdown products in the groundwater sampled were generally less than 0.5 ??g/L.Bacterial tetracycline resistance genes served as distinct genotypic markers to indicate the dissemination and mobility of antibiotic resistance genes that originated from the lagoons. Applying PCR to genomic DNA extracted from the lagoon and groundwater samples, four commonly occurring tetracycline (tet) resistance genes-tet(M), tet(O), tet(Q), and tet(W)-were detected. The detection frequency of tet genes was much higher in wells located closer to and down-gradient from the lagoons than in wells more distant from the lagoons. These results suggested that in the groundwater underlying both facilities tetracycline resistance genes exist and are somewhat persistent, but that the distribution and potentially the flux for each tet gene varied throughout the study period.

Preferential 5-Methylcytosine Oxidation in the Linker Region of Reconstituted Positioned Nucleosomes by Tet1 Protein.

PubMed

Kizaki, Seiichiro; Zou, Tingting; Li, Yue; Han, Yong-Woon; Suzuki, Yuki; Harada, Yoshie; Sugiyama, Hiroshi

2016-11-07

Tet (ten-eleven translocation) family proteins oxidize 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxycytosine (caC), and are suggested to be involved in the active DNA demethylation pathway. In this study, we reconstituted positioned mononucleosomes using CpG-methylated 382 bp DNA containing the Widom 601 sequence and recombinant histone octamer, and subjected the nucleosome to treatment with Tet1 protein. The sites of oxidized methylcytosine were identified by bisulfite sequencing. We found that, for the oxidation reaction, Tet1 protein prefers mCs located in the linker region of the nucleosome compared with those located in the core region. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Persistently Altered Epigenetic Marks in the Mouse Uterus After Neonatal Estrogen Exposure

PubMed Central

Jefferson, Wendy N.; Chevalier, Dominique M.; Phelps, Jazma Y.; Cantor, Amy M.; Padilla-Banks, Elizabeth; Newbold, Retha R.; Archer, Trevor K.; Kinyamu, H. Karimi

2013-01-01

Neonatal exposure to diethylstilbestrol (DES) causes permanent alterations in female reproductive tract gene expression, infertility, and uterine cancer in mice. To determine whether epigenetic mechanisms could explain these phenotypes, we first tested whether DES altered uterine expression of chromatin-modifying proteins. DES treatment significantly reduced expression of methylcytosine dioxygenase TET oncogene family, member 1 (TET1) on postnatal day 5; this decrease was correlated with a subtle decrease in DNA 5-hydroxymethylcytosine in adults. There were also significant reductions in histone methyltransferase enhancer of zeste homolog 2 (EZH2), histone lysine acetyltransferase 2A (KAT2A), and histone deacetylases HDAC1, HDAC2, and HDAC3. Uterine chromatin immunoprecipitation was used to analyze the locus-specific association of modified histones with 2 genes, lactoferrin (Ltf) and sine oculis homeobox 1 (Six1), which are permanently upregulated in adults after neonatal DES treatment. Three histone modifications associated with active transcription, histone H3 lysine 9 acetylation (H3K9ac), H3 lysine 4 trimethylation (H3K4me3), and H4 lysine 5 acetylation (H4K5ac) were enriched at specific Ltf promoter regions after DES treatment, but this enrichment was not maintained in adults. H3K9ac, H4K5ac, and H3K4me3 were enriched at Six1 exon 1 immediately after neonatal DES treatment. As adults, DES-treated mice had greater differences in H4K5ac and H3K4me3 occupancy at Six1 exon 1 and new differences in these histone marks at an upstream region. These findings indicate that neonatal DES exposure temporarily alters expression of multiple chromatin-modifying proteins and persistently alters epigenetic marks in the adult uterus at the Six1 locus, suggesting a mechanism for developmental exposures leading to altered reproductive function and increased cancer risk. PMID:24002655

Relative contribution of CTR1 and DMT1 in copper transport by the blood–CSF barrier: Implication in manganese-induced neurotoxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Gang; Department of Occupational and Environmental Health and the Ministry of Education Key Lab of Hazard Assessment and Control in Special Operational Environment, School of Public Health, Fourth Military Medical University, Xi'an, Shanxi 710032; Chen, Jingyuan

2012-05-01

The homeostasis of copper (Cu) in the cerebrospinal fluid (CSF) is partially regulated by the Cu transporter-1 (CTR1) and divalent metal transporter-1 (DMT1) at the blood–CSF barrier (BCB) in the choroid plexus. Data from human and animal studies suggest an increased Cu concentration in blood, CSF, and brains following in vivo manganese (Mn) exposure. This study was designed to investigate the relative role of CTR1 and DMT1 in Cu transport under normal or Mn-exposed conditions using an immortalized choroidal Z310 cell line. Mn exposure in vitro resulted in an increased cellular {sup 64}Cu uptake and the up-regulation of both CTR1more » and DMT1. Knocking down CTR1 by siRNA counteracted the Mn-induced increase of {sup 64}Cu uptake, while knocking down DMT1 siRNA resulted in an increased cellular {sup 64}Cu uptake in Mn-exposed cells. To distinguish the roles of CTR1 and DMT1 in Cu transport, the Z310 cell-based tetracycline (Tet)-inducible CTR1 and DMT1 expression cell lines were developed, namely iZCTR1 and iZDMT1 cells, respectively. In iZCTR1 cells, Tet induction led to a robust increase (25 fold) of {sup 64}Cu uptake with the time course corresponding to the increased CTR1. Induction of DMT1 by Tet in iZDMT1 cells, however, resulted in only a slight increase of {sup 64}Cu uptake in contrast to a substantial increase in DMT1 mRNA and protein expression. These data indicate that CTR1, but not DMT1, plays an essential role in transporting Cu by the BCB in the choroid plexus. Mn-induced cellular overload of Cu at the BCB is due, primarily, to Mn-induced over-expression of CTR1. -- Highlights: ► This study compares the relative role of CTR1 and DMT1 in Cu transport by the BCB. ► Two novel tetracycline-inducible CTR1 and DMT1 expression cell lines are created. ► CTR1, but not DMT1, plays an essential role in transporting Cu by the BCB. ► Mn-induced cellular Cu overload is due to its induction of CTR1 rather than DMT1. ► Induction of CTR1 by Mn in the BCB contributes to an elevated Cu level in the CSF.« less

Metagenomic insights into tetracycline effects on microbial community and antibiotic resistance of mouse gut.

PubMed

Yin, Jinbao; Zhang, Xu-Xiang; Wu, Bing; Xian, Qiming

2015-12-01

Antibiotics have been widely used for disease prevention and treatment of the human and animals, and for growth promotion in animal husbandry. Antibiotics can disturb the intestinal microbial community, which play a fundamental role in animals' health. Misuse or overuse of antibiotics can result in increase and spread of microbial antibiotic resistance, threatening human health and ecological safety. In this study, we used Illumina Hiseq sequencing, (1)H nuclear magnetic resonance spectroscopy and metagenomics approaches to investigate intestinal microbial community shift and antibiotic resistance alteration of the mice drinking the water containing tetracycline hydrochloride (TET). Two-week TET administration caused reduction of gut microbial diversity (from 194 to 89 genera), increase in Firmicutes abundance (from 24.9 to 39.8%) and decrease in Bacteroidetes abundance (from 69.8 to 51.2%). Metagenomic analysis showed that TET treatment affected the intestinal microbial functions of carbohydrate, ribosomal, cell wall/membrane/envelope and signal transduction, which is evidenced by the alteration in the metabolites of mouse serum. Meanwhile, in the mouse intestinal microbiota, TET treatment enhanced the abundance of antibiotic resistance genes (ARGs) (from 307.3 to 1492.7 ppm), plasmids (from 425.4 to 3235.1 ppm) and integrons (from 0.8 to 179.6 ppm) in mouse gut. Our results indicated that TET administration can disturb gut microbial community and physiological metabolism of mice, and increase the opportunity of ARGs and mobile genetic elements entering into the environment with feces discharge.

Antimicrobial Resistance Genes in Pigeons from Public Parks in Costa Rica.

PubMed

Blanco-Peña, K; Esperón, F; Torres-Mejía, A M; de la Torre, A; de la Cruz, E; Jiménez-Soto, M

2017-11-01

Antimicrobial resistance is known to be an emerging problem, but the extent of the issue remains incomplete. The aim of this study was to determine the presence or absence of nine resistance genes (bla TEM , catI, mecA, qnrS, sulI, sulII, tet(A), tet(Q), vanA) in the faeces of 141 pigeons from four urban parks in Alajuela, Guadalupe, Tres Ríos and San José in Costa Rica. The genes were identified by real-time PCR directly from enema samples. About 30% of the samples were positive for genes catI and sulI; between 13% and 17% were positive for qnrS, sulII, tet(A) and tet(Q); and 4% were positive for bla TEM . The mecA and vanA genes were not detected. The average of antimicrobial resistance genes detected per pigeon was 2. Eight different patterns of resistance were identified, without differences in the sampling areas, being the most common pattern 2 (sulII positive samples). During rainy season, the genes more frequently found were sulI and tet(A). In conclusion, the urban inhabiting pigeons tested are currently carrying antimicrobial resistance genes, potentially acting as reservoirs of resistant bacteria and vectors to humans. To the authors' knowledge, this is the first study carried out on direct detection of resistance genes in the digestive metagenomes of pigeons. © 2017 The Authors. Zoonoses and Public Health Published by Blackwell Verlag GmbH.

Environmental Dissemination of Multidrug Methicillin-Resistant Staphylococcus sciuri After Application of Manure from Commercial Swine Production Systems.

PubMed

Kumar, Deepak; Pornsukarom, Suchawan; Sivaraman, G K; Thakur, Siddhartha

2018-04-01

The deposition of manure originating from food animal farms in the environment can lead to the dissemination of antimicrobial-resistant (AMR) bacterial foodborne pathogens, thereby potentially impacting human health. The objective of our study was to determine the dissemination of multidrug methicillin-resistant Staphylococcus sciuri (MDR-MRSS) in the environment after land application of manure on commercial swine farms. A total of 400 environmental samples (40 manure and 360 soil) were collected after repeated sampling from four commercial swine farms located in North Carolina (n = 1) and Iowa (n = 3) in the United States. At each farm, we collected 10 manure and 40 soil samples (20 samples before and after 2 h of manure application) from four plots (five soil samples/plot) on day 0. Subsequently, 20 soil samples were collected on day 7, 14, and 21 from the same plots. A total of 67 (16.75%) MRSS were isolated from the 400 samples. The prevalence in soil and manure was 13.33% (48/360) and 47.5% (19/40), respectively. Prevalence was highest in the soil samples collected after 2 h of manure application on day 0 and decreased subsequently on 7, 14, and 21 days. Antimicrobial susceptibility testing was done against a panel of 12 antibiotics. A majority of S. sciuri isolates exhibited resistance against ampicillin (AMP; 95.5%), penicillin (PEN; 95.5%), clindamycin (CLI; 95.5%), cefoxitin (FOX; 92.5%), ceftiofur (XNL; 92.5%), tetracycline (TET; 86.56%), and erythromycin (ERY; 50.74%). The MDR pattern AMP FOX CLI PEN TET XNL (n = 24; 35.8%) was the most commonly observed. We detected multiple AMR genes, including mecA, aac(6'), Ie-aph(2″)Ia, tetM, tetK, mphC, ermA, ermB, and ermC. Pulsed-field gel electrophoresis clustered isolates from different sample collection days from the same farm into one group. Overall, our study identifies swine manure as an important reservoir of MDR-MRSS and highlights its dissemination in the environment upon spreading of manure.

Evaluation of the impacts of cooperative adaptive cruise control on reducing rear-end collision risks on freeways.

PubMed

Li, Ye; Wang, Hao; Wang, Wei; Xing, Lu; Liu, Shanwen; Wei, Xueyan

2017-01-01

Although plenty of studies have been conducted recently about the impacts of cooperative adaptive cruise control (CACC) system on traffic efficiency, there are few researches analyzing the safety effects of this advanced driving-assistant system. Thus, the primary objective of this study is to evaluate the impacts of the CACC system on reducing rear-end collision risks on freeways. The CACC model is firstly developed, which is based on the Intelligent Driver Model (IDM). Then, two surrogated safety measures, derived from the time-to-collision (TTC), denoting time exposed time-to-collision (TET) and time integrated time-to-collision (TIT), are introduced for quantifying the collision risks. And the safety effects are analyzed both theoretically and experimentally, by the linear stability analysis and simulations. The theoretical and simulation results conformably indicate that the CACC system brings dramatic benefits for reducing rear-end collision risks (TET and TIT are reduced more than 90%, respectively), when the desired time headway and time delay are set properly. The sensitivity analysis indicates there are few differences among different values of the threshold of TTC and the length of a CACC platoon. The results also show that the safety improvements weaken with the decrease of the penetration rates of CACC on the market and the increase of time delay between platoons. We also evaluate the traffic efficiency of the CACC system with different desired time headway. Copyright © 2016 Elsevier Ltd. All rights reserved.

Genotypic Characterization of Egypt Enterotoxigenic Escherichia coli Isolates Expressing Coli Surface Antigen 6

DTIC Science & Technology

2013-02-01

47%) of these isolates were resistant to ampicillin, a third (37%) of the isolates were resistant to trimethoprim -sulfamethoxazole, and 24% of the...isolates were tetracycline-resistant. A blaTEM gene was detected in 24 (83%) ampicillin-resistant isolates. Trimethoprim -sulfamethoxazole-resistant...ciprofloxacin (CIP) 5 μg, amikacin (AN) 30 μg, gentamicin (GEN) 10 μg, tetracycline (TET) 30 μg, trimethoprim 1.25 μg / sulfamethoxazole 23.75 μg (SXT

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miyamae, Yusaku, E-mail: ymiyamae@lif.kyoto-u.ac.jp; Nishito, Yukina; Nakai, Naomi

Macroautophagy, or autophagy, is a cellular response in which unnecessary cytoplasmic components, including lipids and organelles, are self-degraded. Recent studies closely related autophagy to activation of hepatic stellate cells (HSCs), a process critical in the pathogenesis of liver fibrosis. During HSC activation, cytoplasmic lipid droplets (LDs) are degraded as autophagic cargo, and then cells express fibrogenic genes. Thus, inhibition of autophagy in HSCs is a potential therapeutic approach for attenuating liver fibrosis. We found that tetrandrine, a bisbenzylisoquinoline alkaloid isolated from Stephania tetrandra, induced lipid accumulation, a phenotype associated with quiescent HSCs, through blockade of autophagy in the rat-derived HSCmore » line HSC-T6. Tetrandrine inhibited autophagic flux without affecting lysosomal function. A phenotypic comparison using siRNA knockdown suggested that tetrandrine may target regulators, involved in fusion between autophagosomes and lysosomes (e.g., syntaxin 17). Moreover, perilipin 1, an LD-coated protein, co-localized specifically with LC3, a marker protein for autophagosomes, in tetrandrine-treated HSC-T6 cells. This suggests a potential role for perilipin 1 in autophagy-mediated LD degradation in HSCs. Our results identified tetrandrine as a potential tool for prevention and treatment of HSC activation. - Highlights: • Autophagy is closely related to lipid degradation in hepatic stellate cells. • Tetrandrine (Tet) causes lipid accumulation via blockade of autophagy in HSC-T6 cells. • Tet blocked autophagy without affecting lysosomal function unlike bafilomycin A{sub 1}. • Perilipin 1 was specifically co-localized with LC3 in Tet-treated cells. • Perilipin 1 may play potential roles in autophagy-mediated lipid degradation.« less

Sodium-dependent Vitamin C transporter 2 deficiency impairs myelination and remyelination after injury: Roles of collagen and demethylation.

PubMed

Röhr, Dominik; Halfter, Hartmut; Schulz, Jörg B; Young, Peter; Gess, Burkhard

2017-07-01

Peripheral nerve myelination involves rapid production of tightly bound lipid layers requiring cholesterol biosynthesis and myelin protein expression, but also a collagen-containing extracellular matrix providing mechanical stability. In previous studies, we showed a function of ascorbic acid in peripheral nerve myelination and extracellular matrix formation in adult mice. Here, we sought the mechanism of action of ascorbic acid in peripheral nerve myelination using different paradigms of myelination in vivo and in vitro. We found impaired myelination and reduced collagen expression in Sodium-dependent Vitamin C Transporter 2 heterozygous mice (SVCT2 +/- ) during peripheral nerve development and after peripheral nerve injury. In dorsal root ganglion (DRG) explant cultures, hypo-myelination could be rescued by precoating with different collagen types. The activity of the ascorbic acid-dependent demethylating Ten-eleven-translocation (Tet) enzymes was reduced in ascorbic acid deprived and SVCT2 +/- DRG cultures. Further, in ascorbic acid-deprived DRG cultures, methylation of a CpG island in the collagen alpha1 (IV) and alpha2 (IV) bidirectional promoter region was increased compared to wild-type and ascorbic acid treated controls. Taken together, these results provide further evidence for the function of ascorbic acid in myelination and extracellular matrix formation in peripheral nerves and suggest a putative molecular mechanism of ascorbic acid function in Tet-dependent demethylation of collagen promoters. © 2017 Wiley Periodicals, Inc.

Deletion of Asxl1 results in myelodysplasia and severe developmental defects in vivo

PubMed Central

Abdel-Wahab, Omar; Gao, Jie; Adli, Mazhar; Dey, Anwesha; Trimarchi, Thomas; Chung, Young Rock; Kuscu, Cem; Hricik, Todd; Ndiaye-Lobry, Delphine; LaFave, Lindsay M.; Koche, Richard; Shih, Alan H.; Guryanova, Olga A.; Kim, Eunhee; Li, Sheng; Pandey, Suveg; Shin, Joseph Y.; Telis, Leon; Liu, Jinfeng; Bhatt, Parva K.; Monette, Sebastien; Zhao, Xinyang; Mason, Christopher E.; Park, Christopher Y.; Bernstein, Bradley E.

2013-01-01

Somatic Addition of Sex Combs Like 1 (ASXL1) mutations occur in 10–30% of patients with myeloid malignancies, most commonly in myelodysplastic syndromes (MDSs), and are associated with adverse outcome. Germline ASXL1 mutations occur in patients with Bohring-Opitz syndrome. Here, we show that constitutive loss of Asxl1 results in developmental abnormalities, including anophthalmia, microcephaly, cleft palates, and mandibular malformations. In contrast, hematopoietic-specific deletion of Asxl1 results in progressive, multilineage cytopenias and dysplasia in the context of increased numbers of hematopoietic stem/progenitor cells, characteristic features of human MDS. Serial transplantation of Asxl1-null hematopoietic cells results in a lethal myeloid disorder at a shorter latency than primary Asxl1 knockout (KO) mice. Asxl1 deletion reduces hematopoietic stem cell self-renewal, which is restored by concomitant deletion of Tet2, a gene commonly co-mutated with ASXL1 in MDS patients. Moreover, compound Asxl1/Tet2 deletion results in an MDS phenotype with hastened death compared with single-gene KO mice. Asxl1 loss results in a global reduction of H3K27 trimethylation and dysregulated expression of known regulators of hematopoiesis. RNA-Seq/ChIP-Seq analyses of Asxl1 in hematopoietic cells identify a subset of differentially expressed genes as direct targets of Asxl1. These findings underscore the importance of Asxl1 in Polycomb group function, development, and hematopoiesis. PMID:24218140

Occurrence and transformation of veterinary antibiotics and antibiotic resistance genes in dairy manure treated by advanced anaerobic digestion and conventional treatment methods.

PubMed

Wallace, Joshua S; Garner, Emily; Pruden, Amy; Aga, Diana S

2018-05-01

Manure treatment technologies are rapidly developing to minimize eutrophication of surrounding environments and potentially decrease the introduction of antibiotics and antibiotic resistant genes (ARGs) into the environment. While laboratory and pilot-scale manure treatment systems boast promising results, antibiotic and ARG removals in full-scale systems receiving continuous manure input have not been evaluated. The effect of treatment on ARGs is similarly lacking. This study examines the occurrence and transformation of sulfonamides, tetracyclines, tetracycline degradation products, and related ARGs throughout a full-scale advanced anaerobic digester (AAD) receiving continuous manure and antibiotic input. Manure samples were collected throughout the AAD system to evaluate baseline antibiotic and ARG input (raw manure), the effect of hygenization (post-pasteurized manure) and anaerobic digestion (post-digestion manure) on antibiotic and ARG levels. Antibiotics were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the ARGs tet(O), tet(W), sul1 and sul2 were analyzed by quantitative polymerase chain reaction (Q-PCR). Significant reductions in the concentrations of chlortetracycline, oxytetracycline, tetracycline and their degradation products were observed in manure liquids following treatment (p < 0.001), concomitant to significant increases in manure solids (p < 0.001). These results suggest sorption is the major removal route for tetracyclines during AAD. Significant decreases in the epimer-to-total residue ratios for chlortetracycline and tetracycline in manure solids further indicate degradation is desorption-limited. Moreover, sul1 and sul2 copies decreased significantly (p < 0.001) following AAD in the absence of sulfonamide antibiotics, while tetracyclines-resistant genes remained unchanged. A cross-sectional study of dairy farms utilizing natural aeration and liquid-solid separation treatments was additionally performed to compare levels of antibiotics and ARGs found in AAD with the levels in common manure management systems. The concentration of antibiotics in raw manure varied greatly between farms while minimal differences in ARGs were observed. However, significant (p < 0.01) differences in the levels of antibiotics and ARGs (except tet(W)) were observed in the effluents from the three different manure management systems. Copyright © 2018 Elsevier Ltd. All rights reserved.

Coagulase-negative staphylococci (CoNS) isolated from ready-to-eat food of animal origin--phenotypic and genotypic antibiotic resistance.

PubMed

Chajęcka-Wierzchowska, Wioleta; Zadernowska, Anna; Nalepa, Beata; Sierpińska, Magda; Łaniewska-Trokenheim, Łucja

2015-04-01

The aim of this work was to study the pheno- and genotypical antimicrobial resistance profile of coagulase negative staphylococci (CoNS) isolated from 146 ready-to-eat food of animal origin (cheeses, cured meats, sausages, smoked fishes). 58 strains were isolated, they were classified as Staphylococcus xylosus (n = 29), Staphylococcus epidermidis (n = 16); Staphylococcus lentus (n = 7); Staphylococcus saprophyticus (n = 4); Staphylococcus hyicus (n = 1) and Staphylococcus simulans (n = 1) by phenotypic and genotypic methods. Isolates were tested for resistance to erythromycin, clindamycin, gentamicin, cefoxitin, norfloxacin, ciprofloxacin, tetracycline, tigecycline, rifampicin, nitrofurantoin, linezolid, trimetoprim, sulphamethoxazole/trimethoprim, chloramphenicol, quinupristin/dalfopristin by the disk diffusion method. PCR was used for the detection of antibiotic resistance genes encoding: methicillin resistance--mecA; macrolide resistance--erm(A), erm(B), erm(C), mrs(A/B); efflux proteins tet(K) and tet(L) and ribosomal protection proteins tet(M). For all the tet(M)-positive isolates the presence of conjugative transposons of the Tn916-Tn1545 family was determined. Most of the isolates were resistant to cefoxitin (41.3%) followed by clindamycin (36.2%), tigecycline (24.1%), rifampicin (17.2%) and erythromycin (13.8%). 32.2% staphylococcal isolates were multidrug resistant (MDR). All methicillin resistant staphylococci harboured mecA gene. Isolates, phenotypic resistant to tetracycline, harboured at least one tetracycline resistance determinant on which tet(M) was most frequent. All of the isolates positive for tet(M) genes were positive for the Tn916-Tn1545 -like integrase family gene. In the erythromycin-resistant isolates, the macrolide resistance genes erm(C) or msr(A/B) were present. Although coagulase-negative staphylococci are not classical food poisoning bacteria, its presence in food could be of public health significance due to the possible spread of antibiotic resistance. Copyright © 2014 Elsevier Ltd. All rights reserved.

Quantitative detection of antibiotic resistance genes using magnetic/luminescent core-shell nanoparticles

NASA Astrophysics Data System (ADS)

Son, Ahjeong; Hristova, Krassimira R.; Dosev, Dosi; Kennedy, Ian M.

2008-02-01

Nanoscale magnetic/luminescent core-shell particles were used for DNA quantification in a hybridization-in-solution format. We demonstrated a simple, high-throughput, and non-PCR based DNA assay for quantifying antibiotic resistance gene tetQ. Fe 3O 4/Eu:Gd IIO 3 nanoparticles (NPs) synthesized by spray pyrolysis were biofunctionalized by passive adsorption of NeutrAvidin. Following immobilization of biotinylated probe DNA on the particles' surfaces, target dsDNA and signaling probe DNA labeled with Cy3 were hybridized with NPs-probe DNA. Hybridized DNA complexes were separated from solution by a magnet, while non-hybridized DNA remained in solution. A linear quantification (R2 = 0.99) of a target tetQ gene was achieved based on the normalized fluorescence (Cy3/NPs) of DNANP hybrids. A real-time qPCR assay was used for evaluation of the NPs assay sensitivity and range of quantification. The quantity of antibiotic resistance tetQ genes in activated sludge microcosms, with and without addition of tetracycline or triclosan has been determined, indicating the potential of the optimized assay for monitoring the level of antibiotic resistance in environmental samples. In addition, the tetQ gene copy numbers in microcosms determined by NPhybridization were well correlated with the numbers measured by real-time qPCR assay (R2 = 0.92).

Metagenomic insights into chlorination effects on microbial antibiotic resistance in drinking water.

PubMed

Shi, Peng; Jia, Shuyu; Zhang, Xu-Xiang; Zhang, Tong; Cheng, Shupei; Li, Aimin

2013-01-01

This study aimed to investigate the chlorination effects on microbial antibiotic resistance in a drinking water treatment plant. Biochemical identification, 16S rRNA gene cloning and metagenomic analysis consistently indicated that Proteobacteria were the main antibiotic resistant bacteria (ARB) dominating in the drinking water and chlorine disinfection greatly affected microbial community structure. After chlorination, higher proportion of the surviving bacteria was resistant to chloramphenicol, trimethoprim and cephalothin. Quantitative real-time PCRs revealed that sulI had the highest abundance among the antibiotic resistance genes (ARGs) detected in the drinking water, followed by tetA and tetG. Chlorination caused enrichment of ampC, aphA2, bla(TEM-1), tetA, tetG, ermA and ermB, but sulI was considerably removed (p < 0.05). Metagenomic analysis confirmed that drinking water chlorination could concentrate various ARGs, as well as of plasmids, insertion sequences and integrons involved in horizontal transfer of the ARGs. Water pipeline transportation tended to reduce the abundance of most ARGs, but various ARB and ARGs were still present in the tap water, which deserves more public health concerns. The results highlighted prevalence of ARB and ARGs in chlorinated drinking water and this study might be technologically useful for detecting the ARGs in water environments. Copyright © 2012 Elsevier Ltd. All rights reserved.

miR-125b promotes MLL-AF9–driven murine acute myeloid leukemia involving a VEGFA-mediated non–cell-intrinsic mechanism

PubMed Central

Liu, Jun; Guo, Bo; Chen, Zhuo; Wang, Nayi; Iacovino, Michelina; Cheng, Jijun; Roden, Christine; Pan, Wen; Khan, Sajid; Chen, Suning; Kyba, Michael; Fan, Rong; Guo, Shangqin

2017-01-01

The hematopoietic stem cell–enriched miR-125 family microRNAs (miRNAs) are critical regulators of hematopoiesis. Overexpression of miR-125a or miR-125b is frequent in human acute myeloid leukemia (AML), and the overexpression of these miRNAs in mice leads to expansion of hematopoietic stem cells accompanied by perturbed hematopoiesis with mostly myeloproliferative phenotypes. However, whether and how miR-125 family miRNAs cooperate with known AML oncogenes in vivo, and how the resultant leukemia is dependent on miR-125 overexpression, are not well understood. We modeled the frequent co-occurrence of miR-125b overexpression and MLL translocations by examining functional cooperation between miR-125b and MLL-AF9. By generating a knock-in mouse model in which miR-125b overexpression is controlled by doxycycline induction, we demonstrated that miR-125b significantly enhances MLL-AF9–driven AML in vivo, and the resultant leukemia is partially dependent on continued overexpression of miR-125b. Surprisingly, miR-125b promotes AML cell expansion and suppresses apoptosis involving a non–cell-intrinsic mechanism. MiR-125b expression enhances VEGFA expression and production from leukemia cells, in part by suppressing TET2. Recombinant VEGFA recapitulates the leukemia-promoting effects of miR-125b, whereas knockdown of VEGFA or inhibition of VEGF receptor 2 abolishes the effects of miR-125b. In addition, significant correlation between miR-125b and VEGFA expression is observed in human AMLs. Our data reveal cooperative and dependent relationships between miR-125b and the MLL oncogene in AML leukemogenesis, and demonstrate a miR-125b-TET2-VEGFA pathway in mediating non–cell-intrinsic leukemia-promoting effects by an oncogenic miRNA. PMID:28053194

Frequent silencing of RASSF1A by DNA methylation in thymic neuroendocrine tumours.

PubMed

Kajiura, Koichiro; Takizawa, Hiromitsu; Morimoto, Yuki; Masuda, Kiyoshi; Tsuboi, Mitsuhiro; Kishibuchi, Reina; Wusiman, Nuliamina; Sawada, Toru; Kawakita, Naoya; Toba, Hiroaki; Yoshida, Mitsuteru; Kawakami, Yukikiyo; Naruto, Takuya; Imoto, Issei; Tangoku, Akira; Kondo, Kazuya

2017-09-01

Aberrant methylation of promoter CpG islands (CGIs) of tumour suppressor genes is a common epigenetic mechanism underlying cancer pathogenesis. The methylation patterns of thymic tumours have not been studied in detail since such tumours are rare. Herein, we sought to identify genes that could serve as epigenetic targets for thymic neuroendocrine tumour (NET) therapy. Genome-wide screening for aberrantly methylated CGIs was performed in three NET samples, seven thymic carcinoma (TC) samples, and eight type-B3 thymoma samples. The methylation status of thymic epithelial tumours (TETs) samples was validated by pyrosequencing in a larger cohort. The expression status was analysed by quantitative polymerase chain reaction (PCR) and immunohistochemistry. We identified a CGI on a novel gene, RASSF1A, which was strongly hypermethylated in NET, but not in thymic carcinoma or B3 thymoma. RASSF1A was identified as a candidate gene statistically and bibliographically, as it showed frequent CGI hypermethylation in NET by genome-wide screening. Pyrosequencing confirmed significant hypermethylation of a RASSF1A CGI in NET. Low-grade NET tissue was more strongly methylated than high-grade NET. Quantitative PCR and immunohistochemical staining revealed that RASSF1A mRNA and protein expression levels were negatively regulated by DNA methylation. RASSF1A is a tumour suppressor gene epigenetically dysregulated in NET. Aberrant methylation of RASSF1A has been reported in various tumours, but this is the first report of RASSF1A hypermethylation in TETs. RASSF1A may represent an epigenetic therapeutic target in thymic NET. Copyright © 2017 Elsevier B.V. All rights reserved.

First study on characterization of virulence and antibiotic resistance genes in verotoxigenic and enterotoxigenic E. coli isolated from raw milk and unpasteurized traditional cheeses in Romania.

PubMed

Tabaran, Alexandra; Mihaiu, Marian; Tăbăran, Flaviu; Colobatiu, Liora; Reget, Oana; Borzan, Mihai Marian; Dan, Sorin Daniel

2017-03-01

The study focused on the incidence of enterotoxigenic Escherichia coli (ETEC) and verotoxigenic E. coli (VTEC) in raw milk and traditional dairy cheeses marketed in Romania, characterizing the virulence and antibiotic resistance genes of these isolates. One hundred and twenty samples of raw milk and 80 samples of unpasteurized telemy cheese were collected and cultured according to the international standard protocol. All the characteristic E. coli cultures were analyzed for the presence of STa, STb, LT, stx1, and stx2 toxicity genes. The ETEC/VTEC strains were tested for the presence of antibiotic resistance genes, such as aadA1, tetA, tetB, tetC, tetG, dfrA1, qnrA, aaC, sul1, bla SHV , bla CMY , bla TEM , and ere(A), using PCR. The results showed that 27 samples (18.62%) were positive for one of the virulence genes investigated. 48.1% (n = 13) tested positive at the genes encoding for tetracycline resistance, tetA being the most prevalent one (61.5%; n = 8). A high percent (33.3%; n = 9) revealed the beta-lactamase (bla TEM ) resistance gene, and none of the samples tested positive for bla CMY and bla SHV genes. The genes responsible for resistance to sulfonamides (sul1) and trimethoprim (dfrA1) were detected in rates of 14.8% (n = 4) and 7.4% (n = 2), respectively. E. coli is highly prevalent in raw milk and unpasteurized cheeses marketed in Romania. These strains might represent an important reservoir of resistance genes which can easily spread into other European countries, given the unique market.

[Serotype identification and antibiotic susceptibility of Shiga toxin-producing Escherichia coli in the Weishan area in Shandong Province, China].

PubMed

Shao, C C; Hu, B; Bi, Z W; Kou, Z Q; Fang, M; Chen, B L; Bi, Z Q

2017-01-06

Objective: To determine the serotypes and drug resistance profiles of Shiga toxin-producing Escherichia coli (STEC) in animal stools from the Weishan area in Shandong Province, China. To provide the basis for further study. Methods: Five hundred animal stool samples (from pigs, cattle, sheep, dogs and birds) were collected from the Weishan area and STEC strains were isolated from these samples. Strains were serotyped by a serum agglutination test, and their drug resistance profiles were determined through antimicrobial sensitivity experiments. In this study, PCR was used to detect tetracycline resistance genes ( tetA , tetB , tetC , tetD ) and beta-lactam resistance genes ( blaSHV -1, blaCTX - M , blaTEM ). Results: Sixteen strains of STEC were isolated from animal stool samples. Thirteen strains were isolated from pig stool samples, two from bovine stool samples and one from a sheep stool sample. Two of the strains were identified as E. coli O157:H7, and other 14 strains were non-O157 STEC of different serotypes. Antimicrobial sensitivity experiments showed that 15 of the strains were multidrug resistant. The rates of resistance were as follows: nalidixic acid (12/16 strains), sulfisoxazole (11/16), trimethoprim and sulphame-thoxazole (11/16), doxycycline (9/16), azithromycin (9/16), tetracycline (9/16), chloramphenicol (8/16) and streptomycin (8/16). Therefore, nalidixic acid showed the highest rate of resistance among the strains, followed by trimethoprim and sulphame-thoxazole, and sulfisoxazole. Resistance to cefepime or imipenem was not detected. In total, three types of drug resistance genes ( tetA , tetB and tetC ) were detected among the 16 strains. Conclusion: The results showed that STEC strains isolated from animals in the Weishan area were of a range of serotypes. The 16 strains of STEC isolated from animal stools in this area were resistant to a number of antibiotics, with many strains displaying multidrug resistance.

Transposon Tn10 contains two structural genes with opposite polarity between tetA and IS10R.

PubMed Central

Schollmeier, K; Hillen, W

1984-01-01

The nucleotide sequence of the central part of Tn10 has been determined from the rightmost HindIII site to IS10R. This sequence contains two open reading frames with opposite polarity. The in vivo transcription start points in this sequence have been determined by S1 mapping. These results define one minor and two major promoters. The transcription starts of the two major promoters are only 18 base pairs apart, and the transcripts show different polarity and overlap by 18 base pairs. The nucleotide sequence reveals two regions with palindromic symmetry which may serve as operators. Their possible involvement in the regulation of transcription of both genes is discussed. Taken together these results allow for a maximal coding capacity of 138 amino acids directed toward IS10R and 197 amino acids directed toward tetA. The possible function of these gene products is discussed. The accompanying article (Braus et al., J. Bacteriol. 160:504-509, 1984) presents evidence that these genes are expressed. Images PMID:6094471

Genetic diversity of Streptococcus suis isolated from three pig farms of China obtained by acquiring antibiotic resistance genes.

PubMed

Huang, Jinhu; Shang, Kexin; Kashif, Jam; Wang, Liping

2015-05-01

Acquiring antibiotic resistance genes may change an organism's genetic characteristics and the effect of antibiotics, resulting in a rapid transmission of microbial pathogens. The objectives of this experiment were to identify the features of Streptococcus suis (S. suis) isolated from three pig farms in China which are geographically isolated. Among the isolates, 56.52% were sequence type 7 (ST7), followed by ST1 (26.09%), indicating that ST7 prevails in China, as revealed by multi-locus sequence typing (MLST). Statistical analysis indicated an association between geography, sequence types and antibiotic resistance genotypes. 66.67% of the isolates in Sichuan province presented a (ermB(-) + mefA(-) + tetO(-) + tetM(-)) + ST7 type. The tetM(+) +ST7 type was the most prevalent in Jiangsu province, whereas the strains from Hebei province had a phenotype ermB(+) +tetO(+) +ST1 (63.64%). Pulsed-field gel electrophoresis (PGFE) pattern A2 with 100% similarity reflected the clonal dissemination between Sichuan and Jiangsu provinces. Strains carrying or not carrying antibiotic resistance genes presented different PFGE patterns in Hebei province. ST7 is widespread in many regions of China and a clonal dissemination occurred between Sichuan and Jiangsu provinces in diseased pigs. However, ST1 strains with macrolide and tetracycline resistance (ermB(+) +tetO(+) +ST1) isolated from a farm in Hebei province demonstrated that the genetic diversity was contributed by horizontal acquiring of ermB and tetO carrying elements. © 2014 Society of Chemical Industry.

An affinity-directed protein missile system for targeted proteolysis.

PubMed

Fulcher, Luke J; Macartney, Thomas; Bozatzi, Polyxeni; Hornberger, Annika; Rojas-Fernandez, Alejandro; Sapkota, Gopal P

2016-10-01

The von Hippel-Lindau (VHL) protein serves to recruit the hypoxia-inducible factor alpha (HIF1α) protein under normoxia to the CUL2 E3 ubiquitin ligase for its ubiquitylation and degradation through the proteasome. In this report, we modify VHL to engineer an affinity-directed protein missile (AdPROM) system to direct specific endogenous target proteins for proteolysis in mammalian cells. The proteolytic AdPROM construct harbours a cameloid anti-green fluorescence protein (aGFP) nanobody that is fused to VHL for either constitutive or tetracycline-inducible expression. For target proteins, we exploit CRISPR/Cas9 to rapidly generate human kidney HEK293 and U2OS osteosarcoma homozygous knock-in cells harbouring GFP tags at the VPS34 (vacuolar protein sorting 34) and protein associated with SMAD1 (PAWS1, aka FAM83G) loci, respectively. Using these cells, we demonstrate that the expression of the VHL-aGFP AdPROM system results in near-complete degradation of the endogenous GFP-VPS34 and PAWS1-GFP proteins through the proteasome. Additionally, we show that Tet-inducible destruction of GFP-VPS34 results in the degradation of its associated partner, UVRAG, and reduction in levels of cellular phosphatidylinositol 3-phosphate. © 2016 The Authors.

Development of a targeted transgenesis strategy in highly differentiated cells: a powerful tool for functional genomic analysis.

PubMed

Puttini, Stefania; Ouvrard-Pascaud, Antoine; Palais, Gael; Beggah, Ahmed T; Gascard, Philippe; Cohen-Tannoudji, Michel; Babinet, Charles; Blot-Chabaud, Marcel; Jaisser, Frederic

2005-03-16

Functional genomic analysis is a challenging step in the so-called post-genomic field. Identification of potential targets using large-scale gene expression analysis requires functional validation to identify those that are physiologically relevant. Genetically modified cell models are often used for this purpose allowing up- or down-expression of selected targets in a well-defined and if possible highly differentiated cell type. However, the generation of such models remains time-consuming and expensive. In order to alleviate this step, we developed a strategy aimed at the rapid and efficient generation of genetically modified cell lines with conditional, inducible expression of various target genes. Efficient knock-in of various constructs, called targeted transgenesis, in a locus selected for its permissibility to the tet inducible system, was obtained through the stimulation of site-specific homologous recombination by the meganuclease I-SceI. Our results demonstrate that targeted transgenesis in a reference inducible locus greatly facilitated the functional analysis of the selected recombinant cells. The efficient screening strategy we have designed makes possible automation of the transfection and selection steps. Furthermore, this strategy could be applied to a variety of highly differentiated cells.

Abundances of Tetracycline, Sulphonamide and Beta-Lactam Antibiotic Resistance Genes in Conventional Wastewater Treatment Plants (WWTPs) with Different Waste Load

PubMed Central

Voolaid, Veiko; Ritz, Christian; Tenson, Tanel; Virta, Marko; Kisand, Veljo

2014-01-01

Antibiotics and antibiotic resistant bacteria enter wastewater treatment plants (WWTPs), an environment where resistance genes can potentially spread and exchange between microbes. Several antibiotic resistance genes (ARGs) were quantified using qPCR in three WWTPs of decreasing capacity located in Helsinki, Tallinn, and Tartu, respectively: sulphonamide resistance genes (sul1 and sul2), tetracycline resistance genes (tetM and tetC), and resistance genes for extended spectrum beta-lactams (blaoxa-58, blashv-34, and blactx-m-32). To avoid inconsistencies among qPCR assays we normalised the ARG abundances with 16S rRNA gene abundances while assessing if the respective genes increased or decreased during treatment. ARGs were detected in most samples; sul1, sul2, and tetM were detected in all samples. Statistically significant differences (adjusted p<0.01) between the inflow and effluent were detected in only four cases. Effluent values for blaoxa-58 and tetC decreased in the two larger plants while tetM decreased in the medium-sized plant. Only blashv-34 increased in the effluent from the medium-sized plant. In all other cases the purification process caused no significant change in the relative abundance of resistance genes, while the raw abundances fell by several orders of magnitude. Standard water quality variables (biological oxygen demand, total phosphorus and nitrogen, etc.) were weakly related or unrelated to the relative abundance of resistance genes. Based on our results we conclude that there is neither considerable enrichment nor purification of antibiotic resistance genes in studied conventional WWTPs. PMID:25084517

Soil texture-depending effects of doxycycline and streptomycin applied with manure on the bacterial community composition and resistome.

PubMed

Blau, Khald; Casadevall, Laia; Wolters, Birgit; Van den Meersche, Tina; Kreuzig, Robert; Smalla, Kornelia; Jechalke, Sven

2018-02-01

Veterinary antibiotics, bacteria carrying antibiotic resistance determinants located on mobile genetic elements and nutrients are spread on agricultural soil using manure as fertilizer. However, systematic quantitative studies linking antibiotic concentrations and antimicrobial resistance genes (ARGs) in manure and the environment are scarce but needed to assess environmental risks. In this microcosm study, a sandy and a loamy soil were mixed with manure spiked with streptomycin or doxycycline at five concentrations. Total-community DNA was extracted on days 28 and 92, and the abundances of ARGs (aadA, strA, tet(A), tet(M), tet(W), tet(Q), sul1, qacE/qacEΔ1) and class 1 and 2 integron integrase genes (intI1 and intI2) were determined by qPCR relative to 16S rRNA genes. Effects on the bacterial community composition were evaluated by denaturing gradient gel electrophoresis of 16S rRNA gene amplicons. Manure application to the soils strongly increased the relative abundance of most tested genes. Antibiotics caused further enrichments which decreased over time and were mostly seen at high concentrations. Strikingly, the effects on relative gene abundances and soil bacterial community composition were more pronounced in sandy soil. The concept of defining antibiotic threshold concentrations for environmental risk assessments remains challenging due to the various influencing factors. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Design and simulation of proportional biological operational Mu-circuit.

PubMed

Xu, Dechang; Cai, Zhipeng; Liu, Ke; Zeng, Xiangmiao; Ouyang, Yujing; Dai, Cuihong; Hou, Aiju; Cheng, Dayou; Li, Jianzhong

2015-03-01

It is challenging yet desirable to quantitatively control the expression of a target gene in practice. We design a device-Proportional Biological Operational Mu-circuit (P-BOM) incorporating AND/OR gate and operational amplifier into one circuit and explore its behaviors through simulation. The results imply that will be possible to regulate input-output proportionally by manipulating the RBS of hrpR, hrpS, tetR and output gene and used in the sensing of environmental weak signals such as dioxins.

Nuclear hormone retinoid X receptor (RXR) negatively regulates the glucose-stimulated insulin secretion of pancreatic ß-cells.

PubMed

Miyazaki, Satsuki; Taniguchi, Hidenori; Moritoh, Yusuke; Tashiro, Fumi; Yamamoto, Tsunehiko; Yamato, Eiji; Ikegami, Hiroshi; Ozato, Keiko; Miyazaki, Jun-ichi

2010-11-01

Retinoid X receptors (RXRs) are members of the nuclear hormone receptor superfamily and are thought to be key regulators in differentiation, cellular growth, and gene expression. Although several experiments using pancreatic β-cell lines have shown that the ligands of nuclear hormone receptors modulate insulin secretion, it is not clear whether RXRs have any role in insulin secretion. To elucidate the function of RXRs in pancreatic β-cells, we generated a double-transgenic mouse in which a dominant-negative form of RXRβ was inducibly expressed in pancreatic β-cells using the Tet-On system. We also established a pancreatic β-cell line from an insulinoma caused by the β-cell-specific expression of simian virus 40 T antigen in the above transgenic mouse. In the transgenic mouse, expression of the dominant-negative RXR enhanced the insulin secretion with high glucose stimulation. In the pancreatic β-cell line, the suppression of RXRs also enhanced glucose-stimulated insulin secretion at a high glucose concentration, while 9-cis-retinoic acid, an RXR agonist, repressed it. High-density oligonucleotide microarray analysis showed that expression of the dominant-negative RXR affected the expression levels of a number of genes, some of which have been implicated in the function and/or differentiation of β-cells. These results suggest that endogenous RXR negatively regulates the glucose-stimulated insulin secretion. Given these findings, we propose that the modulation of endogenous RXR in β-cells may be a new therapeutic approach for improving impaired insulin secretion in type 2 diabetes.

Selective eradication of cancer cells by delivery of adenovirus-based toxins

PubMed Central

Shapira, Shiran; Shapira, Assaf; Kazanov, Diana; Hevroni, Gil; Kraus, Sarah; Arber, Nadir

2017-01-01

Background and objective KRAS mutation is an early event in colorectal cancer carcinogenesis. We previously reported that a recombinant adenovirus, carrying a pro-apoptotic gene (PUMA) under the regulation of Ets/AP1 (RAS-responsive elements) suppressed the growth of cancer cells harboring hyperactive KRAS. We propose to exploit the hyperactive RAS pathway, rather than to inhibit it as was previously tried and failed repeatedly. We aim to improve efficacy by substituting PUMA with a more potent toxin, the bacterial MazF-MazE toxin-antitoxin system, under a very tight regulation. Results A massive cell death, in a dose-dependent manner, reaching 73% at MOI 10 was seen in KRAS cells as compared to 22% in WT cells. Increase expression of MazE (the anti-toxin) protected normal cells from any possible internal or external leakage of the system and confirmed the selectivity, specificity and safety of the targeting system. Considerable tumor shrinkage (61%) was demonstrated in vivo following MazEF-encoding adenovirus treatment without any side effects. Design Efficient vectors for cancer-directed gene delivery were constructed; “pAdEasy-Py4-SV40mP-mCherry-MazF”“pAdEasy-Py4-SV40mP-mCherry-MazF-IRES-TetR-CMVmp-MazE-IRES-EGFP“,“pAdEasy-ΔPy4-SV40mP-mCherry-MazF-IRES-TetR-CMVmp-MazE-IRES-EGFP “and “pAdEasy-mCherry”. Virus particles were produced and their potency was tested. Cell death was measured qualitatively by using the fluorescent microscopy and colony formation assay, and was quantified by MTT. FACS analysis using annexin V and RedDot2 dyes was performed for measuring apoptotic and dead cells, respectively. In vivo tumor formation was measured in a xenograft model. Conclusions A proof of concept for a novel cancer safe and effective gene therapy exploiting an aberrant hyperactive pathway is achievable. PMID:28445136

Epigenetic determinants of space radiation-induced cognitive dysfunction

PubMed Central

Acharya, Munjal M.; Baddour, Al Anoud D.; Kawashita, Takumi; Allen, Barrett D.; Syage, Amber R.; Nguyen, Thuan H.; Yoon, Nicole; Giedzinski, Erich; Yu, Liping; Parihar, Vipan K.; Baulch, Janet E.

2017-01-01

Among the dangers to astronauts engaging in deep space missions such as a Mars expedition is exposure to radiations that put them at risk for severe cognitive dysfunction. These radiation-induced cognitive impairments are accompanied by functional and structural changes including oxidative stress, neuroinflammation, and degradation of neuronal architecture. The molecular mechanisms that dictate CNS function are multifaceted and it is unclear how irradiation induces persistent alterations in the brain. Among those determinants of cognitive function are neuroepigenetic mechanisms that translate radiation responses into altered gene expression and cellular phenotype. In this study, we have demonstrated a correlation between epigenetic aberrations and adverse effects of space relevant irradiation on cognition. In cognitively impaired irradiated mice we observed increased 5-methylcytosine and 5-hydroxymethylcytosine levels in the hippocampus that coincided with increased levels of the DNA methylating enzymes DNMT3a, TET1 and TET3. By inhibiting methylation using 5-iodotubercidin, we demonstrated amelioration of the epigenetic effects of irradiation. In addition to protecting against those molecular effects of irradiation, 5-iodotubercidin restored behavioral performance to that of unirradiated animals. The findings of this study establish the possibility that neuroepigenetic mechanisms significantly contribute to the functional and structural changes that affect the irradiated brain and cognition. PMID:28220892

A dual brain-targeting curcumin-loaded polymersomes ameliorated cognitive dysfunction in intrahippocampal amyloid-β1-42-injected mice.

PubMed

Jia, Tingting; Sun, Zhiguo; Lu, Ying; Gao, Jie; Zou, Hao; Xie, Fangyuan; Zhang, Guoqing; Xu, Hao; Sun, Duxin; Yu, Yuan; Zhong, Yanqiang

2016-01-01

Due to the impermeability of the blood-brain barrier and the nonselective distribution of drugs in the brain, the therapeutic access to intractable neurological disorders is challenging. In this study, dual brain-targeting polymersomes (POs) functionalized by transferrin and Tet-1 peptide (Tf/Tet-1-POs) promoted the transportation of curcumin into the brain and provided neuroprotection. The modification of the ligands that bind to the surface of POs was revealed by X-ray photoelectron spectroscopy analysis. The cell uptake of a coculture model of mouse brain capillary endothelial cells with neurons showed that the Tf/Tet-1-POs had significant transportation properties and possessed affinity for neurons. The pharmacokinetic analysis showed that the blood-brain barrier permeability-surface efficiency of the Tf/Tet-1-POs was 0.28 mL/h/g and that the brain tissue uptake rate (% ID/g) was 0.08, which were significant compared with the controls (P<0.05). The curcumin-encapsulated Tf/Tet-1-POs provided neuroprotection and ameliorated cognitive dysfunction in intrahippocampal amyloid-β1-42-injected mice. These results suggest that the dual brain-targeting POs are more capable of drug delivery to the brain that can be exploited as a multiple noninvasive vehicle for targeting therapeutics.

Conditional lethality strains for the biological control of Anastrepha species

USDA-ARS?s Scientific Manuscript database

Pro-apoptotic cell death genes are promising candidates for biologically-based autocidal control of pest insects as demonstrated by tetracycline (tet)-suppressible systems for conditional embryonic lethality in Drosophila melanogaster (Dm) and the medfly, Ceratitis capitata (Cc). However, for medfly...

Msx1-modulated muscle satellite cells retain a primitive state and exhibit an enhanced capacity for osteogenic differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ding, Ke, E-mail: dingke@med.uestc.edu.cn; Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital, Chengdu 610072; Department of Orthopaedics, Southwest Hospital, Third Military Medical University, Chongqing 400038

Multipotent muscle satellite cells (MuSCs) have been identified as potential seed cells for bone tissue engineering. However, MuSCs exhibit a rapid loss of stemness after in vitro culturing, thereby compromising their therapeutic efficiency. Muscle segment homeobox gene 1 (msx1) has been found to induce the dedifferentiation of committed progenitor cells, as well as terminally differentiated myotubes. In this study, a Tet-off retroviral gene delivery system was used to modulate msx1 expression. After ten passages, MuSCs that did not express msx-1 (e.g., the non-msx1 group) were compared with MuSCs with induced msx-1 expression (e.g., the msx1 group). The latter group exhibitedmore » a more juvenile morphology, it contained a significantly lower percentage of senescent cells characterized by positive β-galactosidase staining, and it exhibited increased proliferation and a higher proliferation index. Immunocytochemical stainings further detected a more primitive gene expression profile for the msx1 group, while osteogenic differentiation assays and ectopic bone formation assays demonstrated an improved capacity for the msx1 group to undergo osteogenic differentiation. These results suggest that transient expression of msx1 in MuSCs can retain a primitive state, thereby enhancing their capacity for osteogenic differentiation and restoring the potential for MuSCs to serve as seed cells for bone tissue engineering.« less

Msx1-modulated muscle satellite cells retain a primitive state and exhibit an enhanced capacity for osteogenic differentiation.

PubMed

Ding, Ke; Liu, Wen-Ying; Zeng, Qiang; Hou, Fang; Xu, Jian-Zhong; Yang, Zhong

2017-03-01

Multipotent muscle satellite cells (MuSCs) have been identified as potential seed cells for bone tissue engineering. However, MuSCs exhibit a rapid loss of stemness after in vitro culturing, thereby compromising their therapeutic efficiency. Muscle segment homeobox gene 1 (msx1) has been found to induce the dedifferentiation of committed progenitor cells, as well as terminally differentiated myotubes. In this study, a Tet-off retroviral gene delivery system was used to modulate msx1 expression. After ten passages, MuSCs that did not express msx-1 (e.g., the non-msx1 group) were compared with MuSCs with induced msx-1 expression (e.g., the msx1 group). The latter group exhibited a more juvenile morphology, it contained a significantly lower percentage of senescent cells characterized by positive β-galactosidase staining, and it exhibited increased proliferation and a higher proliferation index. Immunocytochemical stainings further detected a more primitive gene expression profile for the msx1 group, while osteogenic differentiation assays and ectopic bone formation assays demonstrated an improved capacity for the msx1 group to undergo osteogenic differentiation. These results suggest that transient expression of msx1 in MuSCs can retain a primitive state, thereby enhancing their capacity for osteogenic differentiation and restoring the potential for MuSCs to serve as seed cells for bone tissue engineering. Copyright © 2017 Elsevier Inc. All rights reserved.

Two-fluid (plasma-neutral) Extended-MHD simulations of spheromak configurations in the HIT-SI experiment with PSI-Tet

NASA Astrophysics Data System (ADS)

Sutherland, D. A.; Hansen, C. J.; Jarboe, T. R.

2017-10-01

A self-consistent, two-fluid (plasma-neutral) dynamic neutral model has been implemented into the 3-D, Extended-MHD code PSI-Tet. A monatomic, hydrogenic neutral fluid reacts with a plasma fluid through elastic scattering collisions and three inelastic collision reactions: electron-impact ionization, radiative recombination, and resonant charge-exchange. Density, momentum, and energy are evolved for both the plasma and neutral species. The implemented plasma-neutral model in PSI-Tet is being used to simulate decaying spheromak configurations in the HIT-SI experimental geometry, which is being compare to two-photon absorption laser induced fluorescence measurements (TALIF) made on the HIT-SI3 experiment. TALIF is used to measure the absolute density and temperature of monatomic deuterium atoms. Neutral densities on the order of 1015 m-3 and neutral temperatures between 0.6-1.7 eV were measured towards the end of decay of spheromak configurations with initial toroidal currents between 10-12 kA. Validation results between TALIF measurements and PSI-Tet simulations with the implemented dynamic neutral model will be presented. Additionally, preliminary dynamic neutral simulations of the HIT-SI/HIT-SI3 spheromak plasmas sustained with inductive helicity injection will be presented. Lastly, potential benefits of an expansion of the two-fluid model into a multi-fluid model that includes multiple neutral species and tracking of charge states will be discussed.

An inducible tool for random mutagenesis in Aspergillus niger based on the transposon Vader.

PubMed

Paun, Linda; Nitsche, Benjamin; Homan, Tim; Ram, Arthur F; Kempken, Frank

2016-07-01

The ascomycete Aspergillus niger is widely used in the biotechnology, for instance in producing most of the world's citric acid. It is also known as a major food and feed contaminant. While generation of gene knockouts for functional genomics has become feasible in ku70 mutants, analyzing gene functions or metabolic pathways remains a laborious task. An unbiased transposon-based mutagenesis approach may aid this process of analyzing gene functions by providing mutant libraries in a short time. The Vader transposon is a non-autonomous DNA-transposon, which is activated by the homologous tan1-transposase. However, in the most commonly used lab strain of A. niger (N400 strain and derivatives), we found that the transposase, encoded by the tan1 gene, is mutated and inactive. To establish a Vader transposon-based mutagenesis system in the N400 background, we expressed the functional transposase of A. niger strain CBS 513.88 under the control of an inducible promoter based on the Tet-on system, which is activated in the presence of the antibiotic doxycycline (DOX). Increasing amounts of doxycycline lead to higher Vader excision frequencies, whereas little to none activity of Vader was observed without addition of doxycycline. Hence, this system appears to be suitable for producing stable mutants in the A. niger N400 background.

DNA demethylation of inflammasome-associated genes is enhanced in patients with cryopyrin-associated periodic syndromes.

PubMed

Vento-Tormo, Roser; Álvarez-Errico, Damiana; Garcia-Gomez, Antonio; Hernández-Rodríguez, José; Buján, Segundo; Basagaña, Maria; Méndez, Maria; Yagüe, Jordi; Juan, Manel; Aróstegui, Juan I; Ballestar, Esteban

2017-01-01

Inflammasomes are cytosolic multiprotein complexes in macrophages. They assemble after infection- or stress-associated stimuli, activating both caspase-1-mediated inflammatory cytokine secretion and pyroptosis. Increased inflammasome activity resulting from gene mutations is related to monogenic autoinflammatory syndromes. However, variable penetrance among patients with the same gene mutations suggests involvement of additional mechanisms associated with inflammasome gene regulation. We sought to investigate the role of DNA demethylation in activating inflammasome genes during macrophage differentiation and monocyte activation in healthy control subjects and patients with autoinflammatory syndrome. Inflammasome-related genes were tested for DNA methylation and mRNA levels by using bisulfite pyrosequencing and quantitative RT-PCR in monocytes in vitro differentiated to macrophages and exposed to inflammatory conditions. The contribution of Tet methylcytosine dioxygenase 2 (TET2) and nuclear factor κB to DNA demethylation was tested by using chromatin immunoprecipitation, small interfering RNA-mediated downregulation, and pharmacologic inhibition. We observed that inflammasome-related genes are rapidly demethylated in both monocyte-to-macrophage differentiation and on monocyte activation. Demethylation associates with increased gene expression, and both mechanisms are impaired when TET2 and nuclear factor κB are downregulated. We analyzed DNA methylation levels of inflammasome-related genes in patients with cryopyrin-associated periodic syndromes (CAPS) and familial Mediterranean fever, 2 archetypical monogenic autoinflammatory syndromes. Under the above conditions, monocytes from untreated patients with CAPS undergo more efficient DNA demethylation than those of healthy subjects. Interestingly, patients with CAPS treated with anti-IL-1 drugs display methylation levels similar to those of healthy control subjects. Our study is the first to demonstrate the involvement of DNA methylation-associated alterations in patients with monogenic autoinflammatory disease and opens up possibilities for novel clinical markers. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Detection of antibiotic resistance genes in samples from acute and chronic endodontic infections and after treatment.

PubMed

Rôças, Isabela N; Siqueira, José F

2013-09-01

The purpose of this study was twofold: survey samples from acute and chronic endodontic infections for the presence of genes encoding resistance to beta-lactams, tetracycline and erythromycin, and evaluate the ability of treatment to eliminate these genes from root canals. DNA extracts from samples of abscess aspirates (n=25) and root canals of teeth with asymptomatic apical periodontitis (n=24) were used as template for direct detection of the genes blaTEM, cfxA, tetM, tetQ, tetW, and ermC using real-time polymerase chain reaction (PCR). Bacterial presence was determined using PCR with universal bacterial primers. Root canals of the asymptomatic cases were also sampled and evaluated after chemomechanical procedures using NiTi instruments with 2.5% NaOCl irrigation. All abscess and initial root canal samples were positive for bacteria. At least one of the target resistance genes was found in 36% of the abscess samples and 67% of the asymptomatic cases. The most prevalent genes in abscesses were blaTEM (24%) and ermC (24%), while tetM (42%) and tetW (29%) prevailed in asymptomatic cases. The blaTEM gene was significantly associated with acute cases (p=0.02). Conversely, tetM was significantly more prevalent in asymptomatic cases (p=0.008). Treatment eliminated resistance genes from most cases. Acute and chronic endodontic infections harboured resistance genes for 3 classes of widely used antibiotics. In most cases, treatment was effective in eliminating these genes, but there were a few cases in which they persisted. The implications of persistence are unknown. Direct detection of resistance genes in abscesses may be a potential method for rapid diagnosis and establishment of proactive antimicrobial therapy. Copyright © 2013 Elsevier Ltd. All rights reserved.

Observations of volcanic hotspots with TET-1

NASA Astrophysics Data System (ADS)

Zakšek, Klemen; Hort, Matthias; Lorenz, Eckehard

2016-04-01

The most important source of uncertainties in thermal monitoring of active volcanoes from space stems from the lack of dedicated satellite instruments. Considering the currently available technology, we usually have to make a compromises between spatial and temporal resolution - if the data is available at high temporal resolution (from geostationary instruments), it is impossible to provide high spatial resolution data. The most promising solution seems to be a constellation of small satellites, for they can provide data at high spatial resolution and provide a short revisit time as there is a high number of satellites in the constellation. It is also difficult to provide narrow spectral channels at high radiometric accuracy for monitoring high and low temperatures at the same time. Instruments designed for meteorological applications are usually used in remote sensing of volcanic thermal anomalies. These instruments contain a mid-infrared channel, which provides crucial data for monitoring active volcanoes. However, the settings of meteorological instruments are optimized for monitoring low temperatures, which results in often saturated data over active volcanoes. The volcanological community can partially overcome the gap between the available meteorological satellites and its requirements with the small satellite TET-1 German abbreviation for "Technologie-Erprobungsträger 1" meaning Technology Experiment Carrier). TET-1 is the first satellite within the FireBird constellation. This consists of two small satellites which are predominantly dedicated to investigating high temperature events. They were built and are operated by the German Aerospace Center. TET-1 was launched in June 2012. Here we present the first results obtained from TET-1 data. The data were retrieved over several volcanoes: Etna, Stromboli, Bárdarbunga, etc. We show that using TET-1 data, it is possible to better constrain the time averaged lava discharge from other satellite data sources.

Different Genetic Elements Carrying the tet(W) Gene in Two Human Clinical Isolates of Streptococcus suis▿ †

PubMed Central

Palmieri, Claudio; Princivalli, Maria Stella; Brenciani, Andrea; Varaldo, Pietro E.; Facinelli, Bruna

2011-01-01

The genetic support for tet(W), an emerging tetracycline resistance determinant, was studied in two strains of Streptococcus suis, SsCA and SsUD, both isolated in Italy from patients with meningitis. Two completely different tet(W)-carrying genetic elements, sharing only a tet(W)-containing segment barely larger than the gene, were found in the two strains. The one from strain SsCA was nontransferable, and aside from an erm(B)-containing insertion, it closely resembled a genomic island recently described in an S. suis Chinese human isolate in sequence, organization, and chromosomal location. The tet(W)-carrying genetic element from strain SsUD was transferable (at a low frequency) and, though apparently noninducible following mitomycin C treatment, displayed a typical phage organization and was named ΦSsUD.1. Its full sequence was determined (60,711 bp), the highest BLASTN score being Streptococcus pyogenes Φm46.1. ΦSsUD.1 exhibited a unique combination of antibiotic and heavy metal resistance genes. Besides tet(W), it contained a MAS (macrolide-aminoglycoside-streptothricin) fragment with an erm(B) gene having a deleted leader peptide and a cadC/cadA cadmium efflux cassette. The MAS fragment closely resembled the one recently described in pneumococcal transposons Tn6003 and Tn1545. These resistance genes found in the ΦSsUD.1 phage scaffold differed from, but were in the same position as, cargo genes carried by other streptococcal phages. The chromosome integration site of ΦSsUD.1 was at the 3′ end of a conserved tRNA uracil methyltransferase (rum) gene. This site, known to be an insertional hot spot for mobile elements in S. pyogenes, might play a similar role in S. suis. PMID:21115784

Different genetic elements carrying the tet(W) gene in two human clinical isolates of Streptococcus suis.

PubMed

Palmieri, Claudio; Princivalli, Maria Stella; Brenciani, Andrea; Varaldo, Pietro E; Facinelli, Bruna

2011-02-01

The genetic support for tet(W), an emerging tetracycline resistance determinant, was studied in two strains of Streptococcus suis, SsCA and SsUD, both isolated in Italy from patients with meningitis. Two completely different tet(W)-carrying genetic elements, sharing only a tet(W)-containing segment barely larger than the gene, were found in the two strains. The one from strain SsCA was nontransferable, and aside from an erm(B)-containing insertion, it closely resembled a genomic island recently described in an S. suis Chinese human isolate in sequence, organization, and chromosomal location. The tet(W)-carrying genetic element from strain SsUD was transferable (at a low frequency) and, though apparently noninducible following mitomycin C treatment, displayed a typical phage organization and was named ΦSsUD.1. Its full sequence was determined (60,711 bp), the highest BLASTN score being Streptococcus pyogenes Φm46.1. ΦSsUD.1 exhibited a unique combination of antibiotic and heavy metal resistance genes. Besides tet(W), it contained a MAS (macrolide-aminoglycoside-streptothricin) fragment with an erm(B) gene having a deleted leader peptide and a cadC/cadA cadmium efflux cassette. The MAS fragment closely resembled the one recently described in pneumococcal transposons Tn6003 and Tn1545. These resistance genes found in the ΦSsUD.1 phage scaffold differed from, but were in the same position as, cargo genes carried by other streptococcal phages. The chromosome integration site of ΦSsUD.1 was at the 3' end of a conserved tRNA uracil methyltransferase (rum) gene. This site, known to be an insertional hot spot for mobile elements in S. pyogenes, might play a similar role in S. suis.

Dissipation of antimicrobial resistance genes in compost originating from cattle manure after direct oral administration or post-excretion fortification of antimicrobials.

PubMed

Xu, Shanwei; Amarakoon, Inoka D; Zaheer, Rahat; Smith, Alanna; Sura, Srinivas; Wang, George; Reuter, Tim; Zvomuya, Francis; Cessna, Allan J; Larney, Francis J; McAllister, Tim A

2018-03-21

Dissipation of antimicrobial resistance genes (ARG) during composting of cattle manure generated through fortification versus administration of antimicrobials in feed was compared. Manure was collected from cattle fed diets containing (kg -1 ) dry matter (DM): (1) 44 mg chlortetracycline (CTC), (2) a mixture of 44 mg each of chlortetracycline and sulfamethazine (CTCSMZ), (3) 11 mg tylosin (TYL) or (4) Control, no antimicrobials. Manures were composted for 30 d with a single mixing after 16 d to generate the second heating cycle. Quantitative PCR (qPCR) was used to measure 16S rDNA and tetracycline (tet), erythromycin (erm) and sulfamethazine (sul) genes. Temperature peaks ranged from 48 to 68°C across treatments in the first composting cycle, but except for the control, did not exceed 55°C in the second cycle. Copy numbers of 16S rDNA decreased (P < 0.05) during composting, but were not altered by antimcrobials. Except tet(L), all ARG decreased by 0.1-1.6 log 10 g DM -1 in the first cycle, but some genes (tet[B], tet[L], erm[F], erm[X]) increased (P < 0.05) by 1.0-3.1 log 10 g DM -1 in the second. During composting, levels of tet(M) and tet(W) in CTC, erm(A), erm(B) and erm(X) in TYL, and sul(1) in CTCSMZ remained higher (P < 0.05) in fed than fortified treatments. The dissipation of ARG during composting of manure fortified with antimicrobials differs from manure generated by cattle that are administered antimicrobials in feed, and does not always align with the dissipation of antimicrobial residues.

Cyclopentadienyl-bis(oxazoline) magnesium and zirconium complexes in aminoalkene hydroaminations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eedugurala, Naresh; Hovey, Megan; Ho, Hung -An

Here, a new class of cyclopentadiene-bis(oxazoline) compounds and their piano-stool-type organometallic complexes have been prepared as catalysts for hydroamination of aminoalkenes. The two compounds MeC(Ox Me2) 2C 5H 5 (Bo MCpH; Ox Me2 = 4,4-dimethyl-2-oxazoline) and MeC(Ox Me2) 2C 5Me 4H (Bo MCp tetH) are synthesized from C 5R 4HI (R = H, Me) and MeC(Ox Me2) 2Li. These cyclopentadiene-bis(oxazolines) are converted into ligands that support a variety of metal centers in piano-stool-type geometries, and here we report the preparation of Mg, Tl, Ti, and Zr compounds. Bo MCpH and Bo MCp tetH react with MgMe 2(O 2C 4H 8)more » 2 to give the magnesium methyl complexes {Bo MCp}MgMe and {Bo MCp tet}MgMe. Bo MCpH and Bo MCp tetH are converted to Bo MCpTl and Bo MCp tetTl by reaction with TlOEt. The thallium derivatives react with TiCl 3(THF) 3 to provide [{Bo MCp}TiCl(μ-Cl)] 2 and [{Bo MCptet}TiCl(μ-Cl)] 2, the former of which is crystallographically characterized as a dimeric species. Bo MCpH and Zr(NMe 2) 4 react to eliminate dimethylamine and afford {Bo MCp}Zr(NMe 2) 3, which is crystallographically characterized as a monomeric four-legged piano-stool compound. {Bo MCp}Zr(NMe 2) 3, {Bo MCp}MgMe, and {Bo MCp tet}MgMe are efficient catalysts for the hydroamination/cyclization of aminoalkenes under mild conditions.« less

Cyclopentadienyl-bis(oxazoline) magnesium and zirconium complexes in aminoalkene hydroaminations

DOE PAGES

Eedugurala, Naresh; Hovey, Megan; Ho, Hung -An; ...

2015-11-25

Here, a new class of cyclopentadiene-bis(oxazoline) compounds and their piano-stool-type organometallic complexes have been prepared as catalysts for hydroamination of aminoalkenes. The two compounds MeC(Ox Me2) 2C 5H 5 (Bo MCpH; Ox Me2 = 4,4-dimethyl-2-oxazoline) and MeC(Ox Me2) 2C 5Me 4H (Bo MCp tetH) are synthesized from C 5R 4HI (R = H, Me) and MeC(Ox Me2) 2Li. These cyclopentadiene-bis(oxazolines) are converted into ligands that support a variety of metal centers in piano-stool-type geometries, and here we report the preparation of Mg, Tl, Ti, and Zr compounds. Bo MCpH and Bo MCp tetH react with MgMe 2(O 2C 4H 8)more » 2 to give the magnesium methyl complexes {Bo MCp}MgMe and {Bo MCp tet}MgMe. Bo MCpH and Bo MCp tetH are converted to Bo MCpTl and Bo MCp tetTl by reaction with TlOEt. The thallium derivatives react with TiCl 3(THF) 3 to provide [{Bo MCp}TiCl(μ-Cl)] 2 and [{Bo MCptet}TiCl(μ-Cl)] 2, the former of which is crystallographically characterized as a dimeric species. Bo MCpH and Zr(NMe 2) 4 react to eliminate dimethylamine and afford {Bo MCp}Zr(NMe 2) 3, which is crystallographically characterized as a monomeric four-legged piano-stool compound. {Bo MCp}Zr(NMe 2) 3, {Bo MCp}MgMe, and {Bo MCp tet}MgMe are efficient catalysts for the hydroamination/cyclization of aminoalkenes under mild conditions.« less

Fine-scale mapping of the 4q24 locus identifies two independent loci associated with breast cancer risk.

PubMed

Guo, Xingyi; Long, Jirong; Zeng, Chenjie; Michailidou, Kyriaki; Ghoussaini, Maya; Bolla, Manjeet K; Wang, Qin; Milne, Roger L; Shu, Xiao-Ou; Cai, Qiuyin; Beesley, Jonathan; Kar, Siddhartha P; Andrulis, Irene L; Anton-Culver, Hoda; Arndt, Volker; Beckmann, Matthias W; Beeghly-Fadiel, Alicia; Benitez, Javier; Blot, William; Bogdanova, Natalia; Bojesen, Stig E; Brauch, Hiltrud; Brenner, Hermann; Brinton, Louise; Broeks, Annegien; Brüning, Thomas; Burwinkel, Barbara; Cai, Hui; Canisius, Sander; Chang-Claude, Jenny; Choi, Ji-Yeob; Couch, Fergus J; Cox, Angela; Cross, Simon S; Czene, Kamila; Darabi, Hatef; Devilee, Peter; Droit, Arnaud; Dörk, Thilo; Fasching, Peter A; Fletcher, Olivia; Flyger, Henrik; Fostira, Florentia; Gaborieau, Valerie; García-Closas, Montserrat; Giles, Graham G; Grip, Mervi; Guénel, Pascal; Haiman, Christopher A; Hamann, Ute; Hartman, Mikael; Hollestelle, Antoinette; Hopper, John L; Hsiung, Chia-Ni; Ito, Hidemi; Jakubowska, Anna; Johnson, Nichola; Kabisch, Maria; Kang, Daehee; Khan, Sofia; Knight, Julia A; Kosma, Veli-Matti; Lambrechts, Diether; Le Marchand, Loic; Li, Jingmei; Lindblom, Annika; Lophatananon, Artitaya; Lubinski, Jan; Mannermaa, Arto; Manoukian, Siranoush; Margolin, Sara; Marme, Frederik; Matsuo, Keitaro; McLean, Catriona A; Meindl, Alfons; Muir, Kenneth; Neuhausen, Susan L; Nevanlinna, Heli; Nord, Silje; Olson, Janet E; Orr, Nick; Peterlongo, Paolo; Putti, Thomas Choudary; Rudolph, Anja; Sangrajrang, Suleeporn; Sawyer, Elinor J; Schmidt, Marjanka K; Schmutzler, Rita K; Shen, Chen-Yang; Shi, Jiajun; Shrubsole, Martha J; Southey, Melissa C; Swerdlow, Anthony; Teo, Soo Hwang; Thienpont, Bernard; Toland, Amanda Ewart; Tollenaar, Robert A E M; Tomlinson, Ian P M; Truong, Thérèse; Tseng, Chiu-Chen; van den Ouweland, Ans; Wen, Wanqing; Winqvist, Robert; Wu, Anna; Yip, Cheng Har; Zamora, M Pilar; Zheng, Ying; Hall, Per; Pharoah, Paul D P; Simard, Jacques; Chenevix-Trench, Georgia; Dunning, Alison M; Easton, Douglas F; Zheng, Wei

2015-11-01

A recent association study identified a common variant (rs9790517) at 4q24 to be associated with breast cancer risk. Independent association signals and potential functional variants in this locus have not been explored. We conducted a fine-mapping analysis in 55,540 breast cancer cases and 51,168 controls from the Breast Cancer Association Consortium. Conditional analyses identified two independent association signals among women of European ancestry, represented by rs9790517 [conditional P = 2.51 × 10(-4); OR, 1.04; 95% confidence interval (CI), 1.02-1.07] and rs77928427 (P = 1.86 × 10(-4); OR, 1.04; 95% CI, 1.02-1.07). Functional annotation using data from the Encyclopedia of DNA Elements (ENCODE) project revealed two putative functional variants, rs62331150 and rs73838678 in linkage disequilibrium (LD) with rs9790517 (r(2) ≥ 0.90) residing in the active promoter or enhancer, respectively, of the nearest gene, TET2. Both variants are located in DNase I hypersensitivity and transcription factor-binding sites. Using data from both The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC), we showed that rs62331150 was associated with level of expression of TET2 in breast normal and tumor tissue. Our study identified two independent association signals at 4q24 in relation to breast cancer risk and suggested that observed association in this locus may be mediated through the regulation of TET2. Fine-mapping study with large sample size warranted for identification of independent loci for breast cancer risk. ©2015 American Association for Cancer Research.