TET-mediated DNA hydroxymethylation is negatively influenced by the PARP-dependent PARylation

Background Poly(ADP-ribosyl)ation (PARylation), a posttranslational modification introduced by PARP-1 and PARP-2, has first been implicated in DNA demethylation due to its role in base excision repair. Recent evidence indicates a direct influence of PARP-dependent PARylation on TET enzymes which catalyse hydroxymethylation of DNA—the first step in DNA demethylation. However, the exact nature of influence that PARylation exerts on TET activity is still ambiguous. In our recent study, we have observed a negative influence of PARP-1 on local TET-mediated DNA demethylation of a single gene and in this study, we further explore PARP–TET interplay. Results Expanding on our previous work, we show that both TET1 and TET2 can be in vitro PARylated by PARP-1 and PARP-2 enzymes and that TET1 PARylation negatively affects the TET1 catalytic activity in vitro. Furthermore, we show that PARylation inhibits TET-mediated DNA demethylation at the global genome level in cellulo. Conclusions According to our findings, PARP inhibition can positively influence TET activity and therefore affect global levels of DNA methylation and hydroxymethylation. This gives a strong rationale for future examination of PARP inhibitors' potential use in the therapy of cancers characterised by loss of 5-hydroxymethylcytosine

Genotoxic potential of Cotinus coggygria Scop. (Anacardiaceae) stem extract in vivo

The intention was to evaluate the possible in vivo genotoxic potential in different cell-types, of a methanol extract obtained from the plant stem of Cotinus coggygria Scop., using the sex-linked recessive lethal (or SLRL) test and alkaline comet assay. The SLRL test, revealed the genotoxic effect of this extract in postmeiotic and premeiotic germ-cell lines. The comet assay was carried out on rat liver and bone marrow at 24 and 72 h after intraperitoneal administration. For genotoxic evaluation, three concentrations of the extract were tested, viz., 500, 1000 and 2000 mg/kg body weight (bw), based on the solubility limit of the extract in saline. Comet tail moment and total scores in the group treated with 500 mg/kg bw, 24 and 72 h after treatment, were not significantly different from the control group, whereas in the groups of animals, under the same conditions, but with 1000 and 2000 mg/kg bw of the extract, scores were statistically so. A slight decrease in the comet score and tail moment observed in all the doses in the 72 h treatment, gave to understand that DNA damage induced by Cotinus coggygria extract decreased with time. The results of both tests revealed the genotoxic effect of Cotinus coggygria under our experimental conditions

EpiCRISPR targeted methylation of Arx gene initiates transient switch of mouse pancreatic alpha to insulin-producing cells

Introduction: Beta cell dysfunction by loss of beta cell identity, dedifferentiation, and the presence of polyhormonal cells are main characteristics of diabetes. The straightforward strategy for curing diabetes implies reestablishment of pancreatic beta cell function by beta cell replacement therapy. Aristaless-related homeobox (Arx) gene encodes protein which plays an important role in the development of pancreatic alpha cells and is a main target for changing alpha cell identity. Results: In this study we used CRISPR/dCas9-based epigenetic tools for targeted hypermethylation of Arx gene promoter and its subsequent suppression in mouse pancreatic αTC1-6 cell line. Bisulfite sequencing and methylation profiling revealed that the dCas9-Dnmt3a3L-KRAB single chain fusion constructs (EpiCRISPR) was the most efficient. Epigenetic silencing of Arx expression was accompanied by an increase in transcription of the insulin gene (Ins2) mRNA on 5th and 7th post-transfection day, quantified by both RT-qPCR and RNA-seq. Insulin production and secretion was determined by immunocytochemistry and ELISA assay, respectively. Eventually, we were able to induce switch of approximately 1% of transiently transfected cells which were able to produce 35% more insulin than Mock transfected alpha cells. Conclusion: In conclusion, we successfully triggered a direct, transient switch of pancreatic alpha to insulin-producing cells opening a future research on promising therapeutic avenue for diabetes management. 1 Introductio

The moss flora in the central urban area of Belgrade

In floristic research on mosses in the central urban area of Belgrade, the presence of 58 species classified into 23 genera and 12 families was verified. The mosses occur in different specific and extreme microhabitats. Diversity of the bryoflora in urban ecosystems can be explained in terms of the large number of different habitats found on large greens, old fortress ruins, and roofs, as well as in gardens in the center of the Old Town

New and interesting bryophyte records for the flora of Serbia

During a revision of the bryophyte collections in the Herbarium of the Balkan Peninsula, Natural History Museum of Belgrade (BEO) and the Herbarium of the University of Belgrade (BEOU), as well as historical literature data, four bryophytes were identified as new to the flora of Serbia, namely, the mosses Fontinalis hypnoides, Leucobryum juniperoideum, Dicranum spurium and the hornwort Phaeoceros laevis. Fontinalis hypnoides is relatively recently recorded in SW Serbia, while Leucobryum juniperoideum and Dicranum spurium are known from historical collections. The hornwort Phaeoceros laevis was cited for SE Serbia in 1907, but up to now not found again

Effects of feeding buffering mineral mixture on subacute rumen acidosis and some production traits in dairy cows

This trial was designed in order to evaluate the incidence of subacute rumen acidosis (SARA) during early lactation and to investigate the possibilities for its prevention by use of a buffering mineral mixture. On the beginning of the trial it was found that the pH value of rumen fluid in 4 animals was lower than normal (pH < 6.0) and that 20% of animals have had SARA. The control and the experimental group of cows were fed the same meal with exception of concentrated feed which in the experimental group contained the mineral mix with buffering activity in amount of 1%. Continuous addition of buffering mineral mixture in the amount of 1% in concentrated feed for early lactation cows successfully prevents SARA formation and leads to increased milk production, as well as increased milk fat and protein content

Natural and anthropogenic radioactivity of feedstuffs, mosses and soil in the Belgrade environment, Serbia

By gamma spectroscopic measurement a content of natural radio-nuclides ((40)K, (238)U, (226)Ra, (232)Th) and (137)Cs was determined in samples of soil, alfalfa, maize and moss on six sites in the surroundings of Belgrade. Natural radio-nuclides in the soil were at the level characteristic for Serbia, whereas a relatively high level of activity of (137)Cs (around 30 Bq kg(-1)) was determined. On the other hand, in plant samples mostly used as feed (such as alfalfa and maize) the concentration of natural radio-nuclide activity and (137)Cs was relatively low, i.e. below the range of detection. The content of natural radio-nuclides in moss was within the standard range of values specific for Serbia. However, the activity level of (137)Cs in moss gathered from the wider area around Belgrade, was high, the highest measured level being in the Avala-Zuce area (158-221 Bq kg(-1)). Our results show that this radio-nuclide is still present in the living environment of Belgrade even 20 years after the Chernobyl disaster, and that moss is a good indicator of living environment (137)Cs contamination