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https://www.readbyqxmd.com/read/28335006/a-platform-for-functional-assessment-of-large-variant-libraries-in-mammalian-cells
#1
Kenneth A Matreyek, Jason J Stephany, Douglas M Fowler
Sequencing-based, massively parallel genetic assays have revolutionized our ability to quantify the relationship between many genotypes and a phenotype of interest. Unfortunately, variant library expression platforms in mammalian cells are far from ideal, hindering the study of human gene variants in their physiologically relevant cellular contexts. Here, we describe a platform for phenotyping variant libraries in transfectable mammalian cell lines in two steps. First, a landing pad cell line with a genomically integrated, Tet-inducible cassette containing a Bxb1 recombination site is created...
March 15, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28333198/a-tetracycline-inducible-integrative-expression-system-for-streptococcus-pneumoniae
#2
Marina Meiers, Anke Laux, Daniela Eichinger, Anne Sexauer, Patrick Marx, Ralph Bertram, Reinhold Brückner
Inducible gene expression systems are very useful to analyze cellular processes. The ability to switch the expression state of genes of interest may even be crucial if essential traits or genetic instability are involved. An integrative plasmid, pTEX2, was designed using the (anhydro)tetracycline-inducible promoter Pxyl/tet from staphylococcal plasmid pRAB11 to control gene expression in Streptococcus pneumoniae. The system was evaluated by expressing genes of the two-component regulatory system ciaRH of S...
February 21, 2017: FEMS Microbiology Letters
https://www.readbyqxmd.com/read/28321439/overexpression-of-parkinson-s-disease-associated-mutation-lrrk2-g2019s-in-mouse-forebrain-induces-behavioral-deficits-and-%C3%AE-synuclein-pathology
#3
Yulan Xiong, Stewart Neifert, Senthilkumar S Karuppagounder, Jeannette N Stankowski, Byoung Dae Lee, Jonathan C Grima, Guanxing Chen, Han Seok Ko, Yunjong Lee, Debbie Swing, Lino Tessarollo, Ted M Dawson, Valina L Dawson
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified as an unambiguous cause of late-onset, autosomal-dominant familial Parkinson's disease (PD) and LRRK2 mutations are the strongest genetic risk factor for sporadic PD known to date. A number of transgenic mice expressing wild-type or mutant LRRK2 have been described with varying degrees of LRRK2-related abnormalities and modest pathologies. None of these studies directly addressed the role of the kinase domain in the changes observed and none of the mice present with robust features of the human disease...
March 2017: ENeuro
https://www.readbyqxmd.com/read/28288190/tetrandrine-inhibits-migration-and-invasion-of-human-renal-cell-carcinoma-by-regulating-akt-nf-%C3%AE%C2%BAb-mmp-9-signaling
#4
Shurui Chen, Wei Liu, Ke Wang, Yizeng Fan, Jiaqi Chen, Jianbin Ma, Xinyang Wang, Dalin He, Jin Zeng, Lei Li
Renal cell carcinoma (RCC) is known as one of the most lethal malignancies in the urological system because of its high incidence of metastasis. Tetrandrine (Tet), a traditional Chinese herbal medicine, exerts a potent anti-cancer effect in a variety of cancer cells. However, the anti-metastatic effect of Tet and its possible mechanism in RCC is still unclear. The present study revealed that Tet significantly suppressed the migration and invasion of RCC 786-O and 769-P cells in vitro. Mechanistically, the protein levels of matrix metalloproteinases 9 (MMP-9), phosphorylated PI3K, PDK1, Akt and NF-κB were markedly reduced after Tet treatment...
2017: PloS One
https://www.readbyqxmd.com/read/28277978/targeted-dna-demethylation-in-human-cells-by-fusion-of-a-plant-5-methylcytosine-dna-glycosylase-to-a-sequence-specific-dna-binding-domain
#5
Jara Teresa Parrilla-Doblas, Rafael R Ariza, Teresa Roldán-Arjona
DNA methylation is a crucial epigenetic mark associated to gene silencing, and its targeted removal is a major goal of epigenetic editing. In animal cells, DNA demethylation involves iterative 5mC oxidation by TET enzymes followed by replication-dependent dilution and/or replication-independent DNA repair of its oxidized derivatives. In contrast, plants use specific DNA glycosylases that directly excise 5mC and initiate its substitution for unmethylated C in a base excision repair process. In this work, we have fused the catalytic domain of Arabidopsis ROS1 5mC DNA glycosylase (ROS1_CD) to the DNA binding domain of yeast GAL4 (GBD)...
February 23, 2017: Epigenetics: Official Journal of the DNA Methylation Society
https://www.readbyqxmd.com/read/28274739/dual-role-of-act1-in-keratinocyte-differentiation-and-host-defense-traf3ip2-silencing-alters-keratinocyte-differentiation-while-inhibiting-il-17-responses
#6
Sylviane Lambert, William R Swindell, Lam C Tsoi, Stefan W Stoll, James T Elder
TRAF3IP2 is a candidate psoriasis susceptibility gene encoding Act1, an adaptor protein with ubiquitin ligase activity that couples the IL-17 receptor to downstream signaling pathways. We investigated the role of Act1 in keratinocyte responses to IL-17 using a tetracycline inducible shRNA targeting TRAF3IP2. Tet exposure for seven days effectively silenced TRAF3IP2 mRNA and Act1 protein, resulting in 761 genes with significant changes in expression (495 down, 266 up, >1.5-fold, p<0.05). Gene Ontology analysis revealed that genes affected by TRAF3IP2 silencing are involved in epidermal differentiation, with early differentiation genes (KRT1, KRT10, DSC1, DSG1) being downregulated and late differentiation genes (SPRR2, SPRR3, LCE3) being upregulated...
March 5, 2017: Journal of Investigative Dermatology
https://www.readbyqxmd.com/read/28267381/dynamics-of-5-carboxylcytosine-during-hepatic-differentiation-potential-general-role-for-active-demethylation-by-dna-repair-in-lineage-specification
#7
Lara C Lewis, Peggy Cho Kiu Lo, Jeremy M Foster, Nan Dai, Ivan R Corrêa, Paulina M Durczak, Gary Duncan, Ashley Ramsawhook, Guruprasad Padur Aithal, Chris Denning, Nicholas R F Hannan, Alexey Ruzov
Patterns of DNA methylation (5-methylcytosine, 5mC) are rearranged during differentiation contributing to the regulation of cell type-specific gene expression. TET proteins oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Both 5fC and 5caC can be recognized and excised from DNA by thymine-DNA glycosylase (TDG) followed by the subsequent incorporation of unmodified cytosine into the abasic site via the base excision repair (BER) pathway. We previously demonstrated that 5caC accumulates during lineage specification of neural stem cells (NSCs) suggesting that such active demethylation pathway is operative in this system; however, it is still unknown if TDG/BER-dependent demethylation is utilized during other types of cellular differentiation...
March 7, 2017: Epigenetics: Official Journal of the DNA Methylation Society
https://www.readbyqxmd.com/read/28252217/novel-epigenetic-controlling-of-hypoxia-pathway-related-to-overexpression-and-promoter-hypomethylation-of-tet1-and-tet2-in-rpe-cells
#8
Mohammad Reza Alivand, Zahra-Soheila Soheili, Majid Pornour, Saeed Solali, Farzaneh Sabouni
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 do 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...
March 2, 2017: Journal of Cellular Biochemistry
https://www.readbyqxmd.com/read/28251343/methyl-donor-depletion-of-head-and-neck-cancer-cells-in-vitro-establishes-a-less-aggressive-tumour-cell-phenotype
#9
Vanessa Hearnden, Hilary J Powers, Abeir Elmogassabi, Rosanna Lowe, Craig Murdoch
PURPOSE: DNA methylation plays a fundamental role in the epigenetic control of carcinogenesis and is, in part, influenced by the availability of methyl donors obtained from the diet. In this study, we developed an in-vitro model to investigate whether methyl donor depletion affects the phenotype and gene expression in head and neck squamous cell carcinoma (HNSCC) cells. METHODS: HNSCC cell lines (UD-SCC2 and UPCI-SCC72) were cultured in medium deficient in methionine, folate, and choline or methyl donor complete medium...
March 1, 2017: European Journal of Nutrition
https://www.readbyqxmd.com/read/28250235/methylation-dynamics-during-folliculogenesis-and-early-embryo-development-in-sheep
#10
Laura Masala, Giovanni Pietro Burrai, Emanuela Bellu, Federica Ariu, Luisa Bogliolo, Sergio Ledda, Daniela Bebbere
Genome-wide DNA methylation reprogramming occurs during mammalian gametogenesis and early embryogenesis. Post-fertilization demethylation of paternal and maternal genomes is considered to occur by an active and passive mechanism, respectively, in most mammals but sheep; in this species no loss of methylation was observed in either pronucleus. Post-fertilization reprogramming relies on methylating and de-methylating enzymes and co-factors that are stored during oocyte growth, concurrently with the re-methylation of the oocyte itself...
March 1, 2017: Reproduction: the Official Journal of the Society for the Study of Fertility
https://www.readbyqxmd.com/read/28243323/doxycycline-regulated-p16-mts1-expression-suppresses-the-anchorage-independence-and-tumorigenicity-of-breast-cancer-cell-lines-that-lack-endogenous-p16
#11
Maria C Todd, Thomas A Langan, Robert A Sclafani
The RB pathway controls the critical transition from G1 into S phase of the mammalian cell cycle. Deregulation of the RB pathway by means of RB or p16 inactivation has been implicated in the development of virtually all human cancers. Such findings have led to the view that the loss of RB-mediated regulation at the G1/S checkpoint is a precondition for human malignancy. Our analysis of the RB-positive MCF-7 and ZR75.1 breast cancer cell lines revealed a lack of endogenous p16 protein expression as a result of the homozygous deletion and methylation of the p16 gene at the CDKN2A locus, respectively...
2017: Journal of Cancer
https://www.readbyqxmd.com/read/28242787/tet2-in-normal-and-malignant-hematopoiesis
#12
Robert L Bowman, Ross L Levine
The ten-eleven translocation (TET) family of enzymes were originally cloned from the translocation breakpoint of t(10;11) in infant acute myeloid leukemia (AML) with subsequent genomic analyses revealing somatic mutations and suppressed expression of TET family members across a range of malignancies, particularly enriched in hematological neoplasms. The TET family of enzymes is responsible for the hydroxylation of 5-methylcytosines (5-mC) to 5-hydroxymethylcytosine (5-hmC), followed by active and passive mechanisms leading to DNA demethylation...
February 27, 2017: Cold Spring Harbor Perspectives in Medicine
https://www.readbyqxmd.com/read/28237036/mycobacterium-tuberculosis-rv1474c-is-a-tetr-like-transcriptional-repressor-that-regulates-aconitase-an-essential-enzyme-and-rna-binding-protein-in-an-iron-responsive-manner
#13
Kannan Balakrishnan, Krishnaveni Mohareer, Sharmistha Banerjee
Mycobacterium tuberculosis (M.tb), tuberculosis (TB) causing bacteria, employs several mechanisms to maintain iron homeostasis which is critical for its survival and pathogenesis. M.tb aconitase (Acn), a [4Fe-4S] cluster-containing essential protein, apart from participating in energy cycle, also binds to predicted iron-responsive RNA elements. In this study, we identified Rv1474c as a regulator of its operonic partner acn and carried out its biochemical and functional characterization. The binding motif for Rv1474c in the upstream region of acn (Rv1475c)-Rv1474c operon was verified by gel-shift assays...
March 2017: Tuberculosis
https://www.readbyqxmd.com/read/28230192/an-endoparasitoid-wasp-influences-host-dna-methylation
#14
Sunil Kumar, Yonggyun Kim
Parasitism by endoparasitoid wasps changes the expression of various host genes, and alters host immune and developmental processes. However, it is not clearly understood how parasitism changes host gene expression in a whole genome scale. This study focused on an epigenetic control of Cotesia plutellae, an endoparasitoid wasp, against its host, Plutella xylostella. Two DNA methyltransferases (DNMT-1 and DNMT-2) are encoded in the genome of P. xylostella. In addition, methyl-binding domain proteins (MBDs) and DNA demethylation factor, ten-eleven translation protein (TET) are encoded...
February 23, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28229992/increased-5-hydroxymethylcytosine-and-ten-eleven-translocation-protein-expression-in-ultraviolet-b-irradiated-hacat-cells
#15
Dan Wang, Jin-Hua Huang, Qing-Hai Zeng, Can Gu, Shu Ding, Jian-Yun Lu, Jing Chen, Sheng-Bo Yang
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...
March 5, 2017: Chinese Medical Journal
https://www.readbyqxmd.com/read/28229923/epigenetic-regulation-of-reln-and-gad1-in-the-frontal-cortex-fc-of-autism-spectrum-disorder-asd-subjects
#16
Adrian Zhubi, Ying Chen, Alessandro Guidotti, Dennis R Grayson
Both Reelin (RELN) and glutamate decarboxylase 67 (GAD1) have been implicated in the pathophysiology of Autism Spectrum Disorders (ASD). We have previously shown that both mRNAs are reduced in the cerebella (CB) of ASD subjects through a mechanism that involves increases in the amounts of MECP2 binding to the corresponding promoters. In the current study, we examined the expression of RELN, GAD1, GAD2, and several other mRNAs implicated in this disorder in the frontal cortices (FC) of ASD and CON subjects. We also focused on the role that epigenetic processes play in the regulation of these genes in ASD brain...
February 13, 2017: International Journal of Developmental Neuroscience
https://www.readbyqxmd.com/read/28228863/medulloblastoma-and-ependymoma-cells-display-increased-levels-of-5-carboxylcytosine-and-elevated-tet1-expression
#17
Ashley Ramsawhook, Lara Lewis, Beth Coyle, Alexey Ruzov
BACKGROUND: Alteration of DNA methylation (5-methylcytosine, 5mC) patterns represents one of the causes of tumorigenesis and cancer progression. Tet proteins can oxidise 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine and 5-carboxylcytosine (5caC). Although the roles of these oxidised forms of 5mC (oxi-mCs) in cancer pathogenesis are still largely unknown, there are indications that they may be involved in the mechanisms of malignant transformation. Thus, reduction of 5hmC content represents an epigenetic hallmark of human tumours, and according to our recent report, 5caC is enriched in a proportion of breast cancers and gliomas...
2017: Clinical Epigenetics
https://www.readbyqxmd.com/read/28212413/scarless-deletion-of-up-to-seven-methyl-accepting-chemotaxis-genes-with-an-optimized-method-highlights-key-function-of-chem-in-salmonella-typhimurium
#18
Stefanie Hoffmann, Christiane Schmidt, Steffi Walter, Jennifer K Bender, Roman G Gerlach
Site-directed scarless mutagenesis is an essential tool of modern pathogenesis research. We describe an optimized two-step protocol for genome editing in Salmonella enterica serovar Typhimurium to enable multiple sequential mutagenesis steps in a single strain. The system is based on the λ Red recombinase-catalyzed integration of a selectable antibiotics resistance marker followed by replacement of this cassette. Markerless mutants are selected by expressing the meganuclease I-SceI which induces double-strand breaks in bacteria still harboring the resistance locus...
2017: PloS One
https://www.readbyqxmd.com/read/28197642/tgf-%C3%AE-1-pten-pi3k-signaling-plays-a-critical-role-in-the-anti-proliferation-effect-of-tetrandrine-in-human-colon-cancer-cells
#19
Qian-Zhao Chen, Yang Li, Ying Shao, Yu-Hua Zeng, Wen-Yan Ren, Rong-Xing Liu, Lin-Yun Zhou, Xue-Lian Hu, Ming Huang, Fang He, Wen-Juan Sun, Ke Wu, Bai-Cheng He
The diagnosis and treatment for colon cancer have been greatly developed, but the prognosis remains unsatisfactory. There is still a great clinical need to explore new efficacious drugs for colon cancer treatment. Tetrandrine (Tet) is a bis-benzylisoquinoline alkaloid. It has been shown that Tet may be a potential candidate for cancer treatment, but the explicit mechanism underlying this activity remains unclear. In this study, we investigated the anticancer activity of Tet in human colon cancer cells and dissected the possible mechanism...
March 2017: International Journal of Oncology
https://www.readbyqxmd.com/read/28193047/accurate-direct-and-high-throughput-analyses-of-a-broad-spectrum-of-endogenously-generated-dna-base-modifications-with-isotope-dilution-two-dimensional-ultraperformance-liquid-chromatography-with-tandem-mass-spectrometry-possible-clinical-implication
#20
Daniel Gackowski, Marta Starczak, Ewelina Zarakowska, Martyna Modrzejewska, Anna Szpila, Zbigniew Banaszkiewicz, Ryszard Olinski
Our hereby presented methodology is suitable for reliable assessment of the most common unavoidable DNA modifications which arise as a product of fundamental metabolic processes. 8-Oxoguanine, one of the oxidatively modified DNA bases, is a typical biomarker of oxidative stress. A noncanonical base, uracil, may be also present in small quantities in DNA. A set of ten-eleven translocation (TET) proteins are involved in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine which can be further oxidized to 5-formylcytosine and 5-carboxycytosine...
December 20, 2016: Analytical Chemistry
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