Bases methylated in vitro by cell-free extracts of adenovirus-18-induced tumours.
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ABSTRACT:
SUBMITTER: McFarlane ES
PROVIDER: S-EPMC1174154 | biostudies-other | 1972 Sep
REPOSITORIES: biostudies-other
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Project description:Raw reads of RNA products generated upon incubation of circular and linear pLac-Tet plasmid in human cell-free extract.
2017-12-16 | GSE75512 | GEO
Project description:BACKGROUND:The bacterial genus Exiguobacterium includes several species that inhabit environments with a wide range of temperature, salinity, and pH. This is why the microorganisms from this genus are known generically as polyextremophiles. Several environmental isolates have been explored and characterized for enzyme production as well as for bioremediation purposes. In this line, toxic metal(loid) reduction by these microorganisms represents an approach to decontaminate soluble metal ions via their transformation into less toxic, insoluble derivatives. Microbial-mediated metal(loid) reduction frequently results in the synthesis of nanoscale structures-nanostructures (NS) -. Thus, microorganisms could be used as an ecofriendly way to get NS. RESULTS:We analyzed the tolerance of Exiguobacterium acetylicum MF03, E. aurantiacum MF06, and E. profundum MF08 to Silver (I), gold (III), and tellurium (IV) compounds. Specifically, we explored the ability of cell-free extracts from these bacteria to reduce these toxicants and synthesize NS in vitro, both in the presence or absence of oxygen. All isolates exhibited higher tolerance to these toxicants in anaerobiosis. While in the absence of oxygen they showed high tellurite- and silver-reducing activity at pH?9.0, whereas AuCl4- which was reduced at pH?7.0 in both conditions. Given these results, cell-free extracts were used to synthesize NS containing silver, gold or tellurium, characterizing their size, morphology and chemical composition. Silver and tellurium NS exhibited smaller size under anaerobiosis and their morphology was circular (silver NS), starred (tellurium NS) or amorphous (gold NS). CONCLUSIONS:This nanostructure-synthesizing ability makes these isolates interesting candidates to get NS with biotechnological potential.
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Project description:The tRNA methylase activity in vitro of adenovirus-12-induced tumour and normal hamster tissues was studied. Enzyme extracts from adenovirus-12-induced tumour, hamster muscle-connective tissue and hamster liver methylated both Escherichia coli tRNA and yeast tRNA. The source of the enzyme and the pH of the incubation mixture influenced the bases methylated. However, the methylated bases that were isolated were not affected by the presence of NH(4) (+) ions in the incubation mixture.
| S-EPMC1179629 | biostudies-other
Project description:Cell-free systems using crude cell extracts present appealing opportunities for designing biosynthetic pathways and enabling sustainable chemical synthesis. However, the lack of tools to effectively manipulate the underlying host metabolism in vitro limits the potential of these systems. Here, we create an integrated framework to address this gap that leverages cell extracts from host strains genetically rewired by multiplexed CRISPR-dCas9 modulation and other metabolic engineering techniques. As a model, we explore conversion of glucose to 2,3-butanediol in extracts from flux-enhanced Saccharomyces cerevisiae strains. We show that cellular flux rewiring in several strains of S. cerevisiae combined with systematic optimization of the cell-free reaction environment significantly increases 2,3-butanediol titers and volumetric productivities, reaching productivities greater than 0.9 g/L-h. We then show the generalizability of the framework by improving cell-free itaconic acid and glycerol biosynthesis. Our coupled in vivo/in vitro metabolic engineering approach opens opportunities for synthetic biology prototyping efforts and cell-free biomanufacturing.
| S-EPMC8390474 | biostudies-literature
Project description:We have explored the mechanism of adenovirus type 12 (Ad12) DNA integration because of its importance for viral oncogenesis and as an example of insertional recombination. We have used a fractionated cell-free system from nuclear extracts of hamster cells and have partly purified nuclear proteins that could catalyze in vitro recombination. As recombination partners, the 20,880- to 24,049-nucleotide Pst I D fragment of Ad12 DNA and the hamster preinsertion sequence p7 from the Ad12-induced tumor CLAC1 have proven to recombine at higher frequencies than randomly selected adenoviral or cellular DNA sequences. A preinsertion sequence might carry elements essential in eliciting recombination. Patch homologies between the recombination partners seem to play a role in the selection of sites for recombination in vivo and in the cell-free system. Nuclear extracts from BHK21 cells were prepared by incubating the nuclei in 0.42 M (NH4)2SO4 and fractionated by Sephacryl S-300 gel filtration, followed by chromatography on Mono S and Mono Q columns. The purified products active in recombination contained a limited number of different protein bands, as determined by polyacrylamide gel electrophoresis and silver staining. The most highly purified fraction IV had helicase and topoisomerase I activities. We used two different methods to assess the in vitro generation of hamster DNA-Ad12 DNA recombinants upon incubation with the purified protein fractions: (i) transfection of the recombination products into recA- strains of Escherichia coli and (ii) the polymerase chain reaction by using amplification primers unique for each of the two recombination partners. In p7 hamster DNA, the nucleotide sequence 5'-CCTCTCCG-3' or similar sequences served repeatedly as a preferred recombination target for Ad12 DNA in the tumor CLAC1 and in five independent cell-free recombination experiments.
| S-EPMC47136 | biostudies-other
Project description:Mammalian cells defective in DNA end-joining are highly sensitive to ionizing radiation and are immunodeficient because of a failure to complete V(D)J recombination. By using cell-free extracts prepared from human lymphoblastoid cell lines, an in vitro system for end-joining has been developed. Intermolecular ligation was found to be accurate and to depend on DNA ligase IV/Xrcc4 and requires Ku70, Ku86, and DNA-PKcs, the three subunits of the DNA-activated protein kinase DNA-PK. Because these activities are involved in the cellular resistance to x-irradiation and V(D)J recombination, the development of this in vitro system provides an important advance in the study of the mechanism of DNA end-joining in human cells.
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Project description:Trinucleotide repeat expansions cause 17 heritable human neurological disorders. In some diseases, somatic expansions occur in non-proliferating tissues such as brain where DNA replication is limited. This finding stimulated significant interest in replication-independent expansion mechanisms. Aberrant DNA repair is a likely source, based in part on mouse studies showing that somatic expansions are provoked by the DNA repair protein MutS? (Msh2-Msh3 complex). Biochemical studies to date used cell-free extracts or purified DNA repair proteins to yield partial reactions at triplet repeats. The findings included expansions on one strand but not the other, or processing of DNA hairpin structures thought to be important intermediates in the expansion process. However, it has been difficult to recapitulate complete expansions in vitro, and the biochemical role of MutS? remains controversial. Here, we use a novel in vitro assay to show that human cell-free extracts catalyze expansions and contractions of trinucleotide repeats without the requirement for DNA replication. The extract promotes a size range of expansions that is similar to certain diseases, and triplet repeat length and sequence govern expansions in vitro as in vivo. MutS? stimulates expansions in the extract, consistent with aberrant repair of endogenous DNA damage as a source of expansions. Overall, this biochemical system retains the key characteristics of somatic expansions in humans and mice, suggesting that this important mutagenic process can be restored in the test tube.
| S-EPMC3616437 | biostudies-literature
Project description:Detection of cellular changes in tissue biopsies has been the basis for cancer diagnostics. However, tissue biopsies are invasive and limited by inaccuracies due to sampling locations, restricted sampling frequency, and poor representation of tissue heterogeneity. Liquid biopsies are emerging as a complementary approach to traditional tissue biopsies to detect dynamic changes in specific cell populations. Cell-free DNA (cfDNA) fragments released into the circulation from dying cells can be traced back to the tissues and cell types they originated from using DNA methylation, an epigenetic regulatory mechanism that is highly cell-type specific. Decoding changes in the cellular origins of cfDNA over time can reveal altered host tissue homeostasis due to local cancer invasion and metastatic spread to distant organs as well as treatment responses. In addition to host-derived cfDNA, changes in cancer cells can be detected from cell-free, circulating tumor DNA (ctDNA) by monitoring DNA mutations carried by cancer cells. Here, we will discuss computational approaches to identify and validate robust biomarkers of changed tissue homeostasis using cell-free, methylated DNA in the circulation. We highlight studies performing genome-wide profiling of cfDNA methylation and those that combine genetic and epigenetic markers to further identify cell-type specific signatures. Finally, we discuss opportunities and current limitations of these approaches for implementation in clinical oncology.
| S-EPMC8353442 | biostudies-literature
Project description:Aim:This study aimed to evaluate and compare the antimicrobial efficacy of root canal sealers of different bases when mixed with herbal extracts. Materials and methods:An evaluation of three sealers (Endomethasone, AH plus, Apexit plus) mixed with three herbal extracts [Emblica officinalis (Amla), Myristica fragrans (Nutmeg) and Salvadora persica (Miswak)] was done against nine strains of bacteria at various time intervals using the agar diffusion test. Moreover, measurement of the mean zones of inhibition was done. Results:The largest zones of bacterial growth inhibition were observed with Endomethasone mixed with Myristica fragrans (Nutmeg) followed in the descending order by AH plus mixed with Salvadora persica (Miswak) and Apexit plus mixed with Salvadora persica (Miswak) respectively. The differences between zones of inhibition among different groups were found to be statistically significant. Conclusion:Zinc-oxide-eugenol-based sealer (Endomethasone) mixed with herbal extracts produced the largest inhibitory zones followed in the descending order by resin-based sealer (AH plus) and calcium-hydroxide-based sealer (Apexit plus), respectively. Clinical significance:Herbal plants such as [Emblica officinalis (Amla), Myristica fragrans (Nutmeg) and Salvadora persica (Miswak)] are rich sources of bioactive compounds that possess antimicrobial properties. Mixing their extracts with endodontic sealers can produce additive antimicrobial effect against microbes found in inflamed pulp. How to cite this article:Devi MT, Saha S, Tripathi AM, et al. Evaluation of the Antimicrobial Efficacy of Herbal Extracts Added to Root Canal Sealers of Different Bases: An In Vitro Study. Int J Clin Pediatr Dent 2019;12(5):398-404.
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Project description:AID/APOBEC family enzymes are best known for deaminating cytosine bases to uracil in single-stranded DNA, with characteristic sequence preferences that can produce mutational signatures in targets such as retroviral and cancer cell genomes. These deaminases have also been proposed to function in DNA demethylation via deamination of either 5-methylcytosine (mC) or TET-oxidized mC bases (ox-mCs), which include 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine. One specific family member, APOBEC3A (A3A), has been shown to readily deaminate mC, raising the prospect of broader activity on ox-mCs. To investigate this claim, we developed a novel assay that allows for parallel profiling of activity on all modified cytosines. Our steady-state kinetic analysis reveals that A3A discriminates against all ox-mCs by >3700-fold, arguing that ox-mC deamination does not contribute substantially to demethylation. A3A is, by contrast, highly proficient at C/mC deamination. Under conditions of excess enzyme, C/mC bases can be deaminated to completion in long DNA segments, regardless of sequence context. Interestingly, under limiting A3A, the sequence preferences observed with targeting unmodified cytosine are further exaggerated when deaminating mC. Our study informs how methylation, oxidation, and deamination can interplay in the genome and suggests A3A's potential utility as a biotechnological tool to discriminate between cytosine modification states.
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