Gene up-regulation by DNA demethylation in 35S-gshI-transgenic poplars (Populus x canescens)

Gene expression levels of transgene 35S-gshI (γ-glutamylcysteine synthetase) cloned from E. coli, and the endogenous gene gsh1 of poplar (Populus x canescens) were upregulated by the DNA demethylating agent DHAC (5,6-dihydro-5'-azacytidine hydrochloride) (10-4 M for 7 days) in aseptic leaf discs cultures. Two 35S-gshI-transgenic (6lgl and 11ggs) and wild type (WT) poplar clones were used. The efficiency of gene upregulation was also analyzed under herbicide paraquat stress (4 x 10-7 M). Levels of gshI-mRNA and gsh1-mRNA were determined by RT-qPCR (reverse transcriptase quantitative PCR) after cDNA synthesis. For internal control, the constitutively expressed housekeeping poplar genes α-tubulin and actin were used, and the 2−HHCt method was applied for data analysis. In long term DHAC treatment (21 days), a morphogenetic response of de novo root development was observed on leaf discs in a wide concentration range of DHAC (10-8 to 10-6 M). Adventitious shoots (11ggs clone) also emerged from leaf discs after a combined treatment with DHAC (10-4 M) and paraquat (10-7 M). Shoots were dissected, rooted and transplanted in glass houses for further analyses for phytoremediation capacity. Since DNA methylation patterns are inherited (epigenetic memory), these poplar plants with increased gene expression levels of both transgene 35S-gshI and endogenous gene gsh1 provide novel plant sources for in situ application

Comparison of the efficiency of Na+/Ca2+ exchanger or Na+/H+ exchanger inhibition and their combination in reducing coronary reperfusion-induced arrhythmias

During ischaemia/reperfusion, the rise in [Na+]i, induced by simultaneous depression of the Na+/K+-ATPase and activation of the Na+/H+ exchanger (NHE), shifts the Na+/Ca2+ exchanger (NCX) into reverse transport mode, resulting in Ca2+ i overload, which is a critical factor in enhancing the liability to cardiac arrhythmias. The inhibition of NHE, and recently NCX has been suggested to effectively protect the heart from reperfusion-induced arrhythmias. In this study, we investigated and compared the efficacy of individual or the simultaneous inhibition of the NHE and NCX against reperfusion-induced arrhythmias in Langendorff-perfused rat hearts by applying a commonly used regional ischaemiareperfusion protocol. The NHE and NCX were inhibited by cariporide and SEA0400 or the novel, more selective ORM10103, respectively. Arrhythmia diagrams calculated for the reperfusion period were analysed for the incidence and duration of extrasystoles (ESs), ventricular tachycardia (VT) and ventricular fibrillation (VF). NHE inhibition by cariporide was highly efficient in reducing the recorded reperfusion-induced arrhythmias. Following the application of SEA0400 or ORM-10103, the number and duration of arrhythmic periods were efficiently or moderately decreased. While both NCX inhibitors effectively reduced ESs, the most frequently triggered arrhythmias, they exerted limited or no effect on VTs and VFs. Of the NCX inhibitors, ORM-10103 was more effective. Surprisingly, the simultaneous inhibition of the NCX and NHE failed to significantly improve the antiarrhythmic efficacy reached by NCX blockade alone. In conclusion, although principal simultaneous NHE+NCX inhibition should be highly effective against all types of the recorded reperfusion-induced arrhythmias, NCX inhibitors, alone or in combination with cariporide, seem to be moderately suitable to provide satisfactory cardioprotection - at least in the present arrhythmia model. Since ORM10103 and SEA0400 are known to effectively inhibit after-depolarisations, it is suggested that their efficacy and that of other NCX inhibitors may be higher and more pronounced in the predominantly Ca2+ i-dependent triggered arrhythmias. © 2015, Polish Physiological Society. All rights reserved

A gst gén DNS-demetilált overexpressziója a szürkenyár (Populus x canescens) fitoremediációs kapacitásának növelésére

A szürkenyár (Populus x canescens) gst (glutation S-transzferáz) génexpresszióját növeltük meg DHAC-indukált (5,6-dihidro-5'-azacitidin hidroklorid) DNSdemetilációval. Három klónt vizsgáltunk, a természetes (WT) és két glutationtúltermelő 35S-gshI nyárfaklónt (11ggs, 6lgl) RT-qPCR elemzésben. A gst gén megemelt expressziós szintje a DHAC-kezelt kontroll növényekben 4,9-szeresre emelkedett, amely tovább nőtt paraquat stresszben (11,2-szeres), amely eredmény azt bizonyítja, hogy a DNS demetilációjával az endogén gének expressziós szintje nagyságrendekkel emelhető meg. Ismert, hogy a DNS demetiláció a vegetativ klónokban öröklődik (epigenetikus memória), ezért a demetilációs eljárás (gén upreguláció) új lehetőséget ad stressztűrő nyárfaklónok előállítására. | In the study presented gst (glutathione S-transferase) gene expression levels of poplar (Populus x canescens) were analyzed in response to the DNA demethylating agent DHAC (5,6-dihydro-5'-azacytidine hydrochloride). Aseptic leaf discs cultures of wild type (WT) and two gshI-transgenic clones (6lgl and 11ggs) clones were analyzed by RT-qPCR. High expression levels of DHAC treated control plants (4.9-fold increment, and a further 11.2-fold increment after paraquat treatment) proves that DNA demethylation provides powerful tools in gene-upregulation. As DNA methylation patterns are inherited (‘epigenetic memory’) novel poplar plant sources are provided with increased gst gene expression levels

Metilviologén (paraquat) toleráns nyárfaklónok (Populus x canescens) szelekciója és alkalmazása fitoremediációban

Paraquat (syn.: metilviologén) toleráns nyárfa (Populus x canescens) klónok szelekcióját végeztük el in vitro kultúrában. A szintetikus talaj összetétele: WPM (Woody Plant Media) tápsók, 1% szacharóz, 0,8 % agar 1 mg/l benziladenin, 0,2 mg/l naftilecetsav és paraquat koncentráció sor (2×10(-6) M, 1,47×10(-6) M, 9,3×10(-7) M, 4×10(-7) M) volt. A regeneránsokat szelekciós táptalajon történő tesztelés után felszaporítottuk (mikroszaporítás), gyökeresítettük, majd üvegházban felneveltük. A molekuláris (RTqPCR) és biokémiai elemzések (aszkorbát peroxidáz, glutation peroxidáz, glutation Stranszferáz, lipoxigenáz) igazolták egy rendkívül stabil paraquat toleráns klón sikeres szelekcióját. A klónokat az in vitro vizsgálatokat követően in situ (Fűzfőgyártelep) teszteljük gyomirtószer maradványok toleranciájára. | Paraquat (syn.: methylviologen) tolerant poplar (P. x canescens) clones (PQT) were selected in in vitro cultures at concentration series of paraquat (2×10(-6) M, 1.47×10(-6) M, 9.3×10(-7) M, 4×10(-7) M). After testing on tissue culture media, regenerants were micropropagated. After rooting, PQT-clones were transplanted to a greenhouse. PQT clones showed significantly higher gst gene expression then wild type (WT) analyzed by RT-qPCR (quantitative reverse transcriptase PCR). For functional analysis enzyme activities of GST (glutathione S-transferase), APOX (ascorbate peroxides), GR (glutathione reductase), and LOX (lipoxygenase, pH 8.0) were determined. After rooting, PQT-clones were transplanted in glass houses, followed by field performance analyses for phytoremediation (environmental clean up using plants) capacity in heavily contaminated area at Balatonfűzfő, Hungar

An Online Toolkit for Applications Featuring Collaborative Robots Across Different Domains

Collaborative robots (cobots) are being applied in areas such as healthcare, rehabilitation, agriculture and logistics, beyond the typical manufacturing setting. This is leading to a marked increase in the number of cobot stakeholders with little or no experience in traditional safety engineering. Considering the importance of human safety in collaborative robotic applications, this is currently proving to be a barrier to more widespread cobot usage. A web-based Toolkit that targets cobot end-users and manufacturers with varying levels of safety expertise was developed, helping them to understand how to consider the safety of their cobot applications. In this work, we will provide an overview of the state of the art for ensuring cobot safety, highlight the support provided by the “COVR Toolkit” and introduce three examples where third parties applied the Toolkit for their collaborative robotics application