Single Cell Analysis of Human RAD18-Dependent DNA Post-Replication Repair by Alkaline Bromodeoxyuridine Comet Assay

Damage to DNA can block replication progression resulting in gaps in the newly synthesized DNA. Cells utilize a number of post-replication repair (PRR) mechanisms such as the RAD18 controlled translesion synthesis or template switching to overcome the discontinuities formed opposite the DNA lesions and to complete DNA replication. Gaining more insights into the role of PRR genes promotes better understanding of DNA damage tolerance and of how their malfunction can lead to increased genome instability and cancer. However, a simple and efficient method to characterise gene specific PRR deficiencies at a single cell level has not been developed. Here we describe the so named BrdU comet PRR assay to test the contribution of human RAD18 to PRR at a single cell level, by which we kinetically characterized the consequences of the deletion of human RAD18 on the replication of UV-damaged DNA. Moreover, we demonstrate the capability of our method to evaluate PRR at a single cell level in unsynchronized cell population. © 2013 Mórocz et al

Immun tényezők, parvalbumin és PARP aktiváció jelentősége neurodegeneratív kórképekben = The role of immune factors, parvalbumin and activation of PARP, neurodegenerative diseases

A neurodegeneratív betegségekben a károsodás közös útja az intraneuronális Ca emelkedése. Amyotrophiás laterálsclerosisban (ALS), /humán motoneuron (MN) betegség/, Parkinson kórban (PK) és állatkísérletes modelljeikben azok az idegsejtek, amelyek képesek kompenzatórikusan megemelni a Ca-kötő parvalbumin szintjüket, rezisztensek a károsodásra. A sérülékeny sejtek NMDA receptor antagonistákkal, Ca-csatorna gátlókkal és a parvalbumin gén beültetéssel megóvhatók a pusztulástól a kísérletes MN betegségben. DNS károsodás és az intraneuronális Ca emelkedés aktiválja a poly(ADP-ribose) polymerase (PARP) DNS javító enzymet ALS-ben a corticális MN-okban, PK-ban a dopaminerg sejtekben. A túlfokozott aktiváció elhasználva a sejt energia forrásait sejthalált okoz. A PARP aktiválódik a microgliában is, mely szabályozza a helyi immun-gyulladásos reakciót. A PARP gátlók adásának kedvező hatását tapasztaltuk a MN betegség immun-mediált kísérletes modelljében, s potenciálisan használhatónak tűnnek ALS-ben és PK-ban. Az autoimmun IgG intraneuronális felvételéből, a microglia aktiválódásából és antigén prezentáló dendritikus sejtekké alakulásából, T lymphocyták odavándorlásából álló immun-gyulladásos reakció figyelhető meg ALS-ből, PK-ból származó boncolási anyagban az érintett régiókban. A microglia aktiváció gátlók csökkentik a neuronokra káros anyagok elválasztását, akadályozzák a lokális antigén prezentációt és így a neuronokat károsító másodlagos autoimmun reakciót. | The rise in intraneuronal Ca is the common pathway of cell injury in neurodegenerative diseases. The neurons which are able to upregulate a calcium-buffering protein, parvalbumin are resistant to the damage in amyotrophic lateral sclerosis (ALS), in Parkinson disease (PD) and in their animal models. NMDA receptor antagonists, Ca-channel blockers and parvalbumin gene transfer were successfully used for ameliorating the MN damage in animal models. The rise in intraneuronal Ca upregulates the DNA repair enzyme poly(ADP-ribose) polymerase in the cortical MNs in ALS and in the dopaminergic neurons in PD. The overactivation of PARP exhausts the energy sources of the cells leading to death. The PARP is also upregulated in microglia, which dictates the local immune-inflammatory reaction. The beneficial effect of the use of PARP inhibitors is predicted in the treatment of the diseases and was experienced in an immune-mediated model of MN disease. An immune-inflammatory reaction consisting of autoimmune IgG uptake by the neurons, microglia activation and conversion to antigen presenting dendritic cells, and the recruitment of T lymphocytes was noted in autopsy materials from ALS, PD and from Alzheimer disease in the affected areas. The inhibitors of the activation of microglia may be useful to diminish the harmful effect of the materials secreted by it to damage neurons and can prevent the local antigen presentation and the secondary autoimmune attack targeting neurons

Y-chromosome haplogroups from Hun, Avar and conquering Hungarian period nomadic people of the Carpathian Basin.

Hun, Avar and conquering Hungarian nomadic groups arrived to the Carpathian Basin from the Eurasian Steppes and significantly influenced its political and ethnical landscape, however their origin remains largely unknown. In order to shed light on the genetic affinity of above groups we have determined Y chromosomal haplogroups and autosomal loci, suitable to predict biogeographic ancestry, from 49 individuals, supposed to represent the power/military elit. Haplogroups from the Hun-age are consistent with Xiongnu ancestry of European Huns. Most of the Avar-age individuals carry east Eurasian Y haplogroups typical for modern north-eastern Siberian and Buryat populations and their autosomal loci indicate mostly un-admixed Asian characteristics. In contrast the conquering Hungarians seem to be a recently assembled population incorporating un-admixed European, Asian as well as admixed components. Their heterogeneous paternal and maternal lineages indicate similar supposed phylogeographic origin of males and females, derived from Central-Inner Asian and European Pontic Steppe sources