Interpretation of bafilomycin, pH neutralizing or protease inhibitor treatments in autophagic flux experiments: novel considerations.

Recent publications showed that the kinase MTOR localizes to lysosomes and its activation depends on amino acids inside the lysosomal lumen, implying that autophagic protein degradation is a positive regulator of MTOR in this setting. Since decreased MTOR activity results in autophagy induction, drug treatments that block autolysosomal degradation (a commonly used technique to estimate autophagic flux) may actually interfere not only with lysosomal breakdown, but also increase autophagosome generation through impaired MTOR signaling

Az autofágiában szereplő kinázok és foszfatázok azonosítása in vivo RNSi teszteléssel ecetmuslicában = An in vivo RNAi screen of Drosophila kinases and phosphatases involved in autophagy

Az autofágia során a sejt saját citoplazmája kerül lizoszómális lebontásra és újrahasznosításra. Az éhezés a folyamat nagymérvű indukciójához vezet. Mivel az autofágia megváltozása különféle emberi betegségeket okoz, a folyamat megértése terápiás szempontból is igen fontos. Munkánkban jól táplált és éhező muslica lárvák génexpressziós mintázatát hasonlítottuk össze, kontroll és autofág mutáns állatokban. A genomszintű transzkripciós változások és egy kis léptékű RNSi vizsgálatsorozat alapján két gént választottunk ki részletesebb jellemzésre. A lipid foszfatáz EDTP autofágiában betöltött szerepét jelenleg is vizsgáljuk. A Rack1 (az aktivált protein kináz C kötőpartnere) expressziója megnőtt az éhezés során, és a gén hiánya az indukált autofágia gátlását okozta. Az endogén Rack1 fehérje jelenlétét a korai autofág struktúrákban kimutattuk fény- és elektronmikroszkópos szinten is, ami arra utal, hogy ezek kialakulásában játszik szerepet. Érdekes módon a Rack1 fehérje a glikogén szemcséken is jelen volt, a glikogén szintáz kinázzal együtt. Ezzel összhangban a Rack1 hiánya sejt-autonóm módon a glikogén raktárak erőteljes csökkenéséhez vezetett az emberi májhoz és zsírszövethez hasonlóan működő Drosophila lárvális zsírtestben. Megfigyeléseink alapján a Rack1 állványfehérje alapvető szerepet játszik az autofágia korai lépéseiben és a glikogén szintézis során. | Autophagy delivers cytoplasmic material for lysosomal degradation in eukaryotic cells. Starvation induces high levels of autophagy to promote survival in the lack of nutrients. As alterations in autophagy strongly impact human health and disease, complete understanding of this process is necessary for potential future therapies. In this work, we compared the transcriptional profiles of fed and starved control and autophagy-deficient mutant Drosophila larvae to find novel regulators of autophagy. Based on these transcriptional changes and a small-scale RNAi screen, we chose two genes for detailed studies. We are still investigating how the lipid phosphatase EDTP functions in autophagy. Rack1 (Receptor of activated protein kinase C 1) expression increased during nutrient deprivation, and loss of Rack1 function strongly decreased the autophagic response to starvation. Endogenous Rack1 partially colocalized with Atg8a reporters and early autophagic structures on the ultrastructural level, suggesting its involvement in autophagosome formation. Interestingly, endogenous Rack1 also colocalized with glycogen particles in the larval fat body, and with Shaggy, the Drosophila homolog of Glycogen synthase kinase 3B (GSK-3B). In line with these observations, glycogen stores were decreased in Rack1 mutant cells. These results, for the first time, demonstrate the fundamental role of the scaffold protein Rack1 in autophagosome generation, and also in the formation of glycogen particles

A Bayesian Approach to Identify Bitcoin Users

Bitcoin is a digital currency and electronic payment system operating over a peer-to-peer network on the Internet. One of its most important properties is the high level of anonymity it provides for its users. The users are identified by their Bitcoin addresses, which are random strings in the public records of transactions, the blockchain. When a user initiates a Bitcoin-transaction, his Bitcoin client program relays messages to other clients through the Bitcoin network. Monitoring the propagation of these messages and analyzing them carefully reveal hidden relations. In this paper, we develop a mathematical model using a probabilistic approach to link Bitcoin addresses and transactions to the originator IP address. To utilize our model, we carried out experiments by installing more than a hundred modified Bitcoin clients distributed in the network to observe as many messages as possible. During a two month observation period we were able to identify several thousand Bitcoin clients and bind their transactions to geographical locations

Genetic variability of anther donor versus spontaneous doubled haploid descendents and colchicine induced doubled haploid sweet pepper (Capsicum annuum L.) lines

Haploid (n) and doubled haploid (DH) plants were developed in anther culture of sweet pepper (Capsicum annuum L.). Regenerants were analyzed by flow cytometry for haploid (n = 12) and spontaneous doubled haploid (2n = 24) genomes. Haploid plants were forwarded to colchicine-treatment for induced doubled haploid (2n·) plant production. Molecular polymorphism of anther donor plants (2n), the haploid regenerants (n), the spontaneous (2n) and induced (2n·)-DH plants were analysed by RAPD-, SSR- and ISSR-PCR. The analysis of anther-donor plants compared to DH-descendents showed an unexpectedly wide range of molecular polymorphism. Our results suggest that genetic changes occurring during meiotic recombination is higher than those of occurring during colchicine-induced genomic duplication

Synthetic and steel fibers in prestressed, precast long span beams

Four large-scale, prismatic, T-shaped beams with 19 m span were produced without stirrups, but 2 with synthetic fibres and 2 with steel fibres. Four point bending tests were made to simulate the load bearing process of built-in beams. Three point shear tests were made in the uncracked ends of the beams to prove the shear resistance of FRC. The behaviour of beams made of synthetic and steel fibre reinforced concrete was compared. Finite element calculation was made with the fibre reinforced concrete and plain concrete as well. The material model was according to Eurocode and the effect of the fibre was according to RILEM guideline. The numerical and real test load-deflection results showed close correlation