181. Insertion of a specific fungal 3'-phosphoadenosine-5'-phosphatase motif into a plant homologue improves halotoleranceand drought tolerance of plantsMeti Buh Gašparič, Metka Lenassi, Cene Gostinčar, Ana Rotter, Ana Plemenitaš, Nina Gunde-Cimerman, Kristina Gruden, Jana Žel, 2013, izvirni znanstveni članek Povzetek: Soil salinity and drought are among the most serious agricultural and environmental problems of today. Therefore, investigations of plant resistance to abiotic stress have received a lot of attention in recent years. In this study, we identified the complete coding sequence of a 3′-phosphoadenosine-5′-phosphatase protein, ApHal2, from the halotolerant yeast Aureobasidium pullulans. Expression of the ApHAL2 gene in a Saccharomyces cerevisiae hal2 mutant complemented the mutant auxotrophy for methionine, and rescued the growth of the hal2 mutant in media with high NaCl concentrations. A 21-amino-acids-long region of the ApHal2 enzyme was inserted into the Arabidopsis thaliana homologue of Hal2, the SAL1 phosphatase. The inserted sequence included the META motif, which has previously been implicated in increased sodium tolerance of the Hal2 homologue from a related fungal species. Transgenic Arabidopsis plants overexpressing this modified SAL1 (mSAL1) showed improved halotolerance and drought tolerance. In a medium with an elevated salt concentration, mSAL1-expressing plants were twice as likely to have roots in a higher length category in comparison with the wild-type Arabidopsis and with plants overexpressing the native SAL1, and had 5% to 10% larger leaf surface area under moderate and severe salt stress, respectively. Similarly, after moderate drought exposure, the mSAL1-expressing plants showed 14% increased dry weight after revitalisation, with no increase in dry weight of the wild-type plants. With severe drought, plants overexpressing native SAL1 had the worst rehydration success, consistent with the recently proposed role of SAL1 in severe drought. This was not observed for plants expressing mSAL1. Therefore, the presence of this fungal META motif sequence is beneficial under conditions of increased salinity and moderate drought, and shows no drawbacks for plant survival under severe drought. This demonstrates that adaptations of extremotolerant fungi should be considered as a valuable resource for improving stress-tolerance in plant breeding in the future. Ključne besede: soil salinity and drought, plant resistance, abiotic stress Objavljeno v DiRROS: 02.08.2024; Ogledov: 180; Prenosov: 174
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182. Increased mitochondrial activity in a novel IDH1-R132H mutant human oligodendroglioma xenograft model : in situ detection of 2-HG and [alpha]-KGAnna C. Navis, Simone P. Niclou, Fred Fack, Daniel Stieber, Sanne A. M. van Lith, Kiek Verrijp, Alan F. Wright, Jonathan Stauber, Bastiaan Tops, Irene Otte-Holler, Ron A. Wevers, Arno van Rooij, Stefan Pusch, Andreas von Deimling, Wikky Tigchelaar, Cornelis J. F. van Noorden, Pieter Wesseling, William P. J. Leenders, 2013, izvirni znanstveni članek Povzetek: Background
Point mutations in genes encoding NADP+-dependent isocitrate dehydrogenases (especially IDH1) are common in lower grade diffuse gliomas and secondary glioblastomas and occur early during tumor development. The contribution of these mutations to gliomagenesis is not completely understood and research is hampered by the lack of relevant tumor models. We previously described the development of the patient-derived high-grade oligodendroglioma xenograft model E478 that carries the commonly occurring IDH1-R132H mutation. We here report on the analyses of E478 xenografts at the genetic, histologic and metabolic level.
Results
LC-MS and in situ mass spectrometric imaging by LESA-nano ESI-FTICR revealed high levels of the proposed oncometabolite D-2-hydroxyglutarate (D-2HG), the product of enzymatic conversion of α-ketoglutarate (α-KG) by IDH1-R132H, in the tumor but not in surrounding brain parenchyma. α-KG levels and total NADP+-dependent IDH activity were similar in IDH1-mutant and -wildtype xenografts, demonstrating that IDH1-mutated cancer cells maintain α-KG levels. Interestingly, IDH1-mutant tumor cells in vivo present with high densities of mitochondria and increased levels of mitochondrial activity as compared to IDH1-wildtype xenografts. It is not yet clear whether this altered mitochondrial activity is a driver or a consequence of tumorigenesis.
Conclusions
The oligodendroglioma model presented here is a valuable model for further functional elucidation of the effects of IDH1 mutations on tumor metabolism and may aid in the rational development of novel therapeutic strategies for the large subgroup of gliomas carrying IDH1 mutations. Ključne besede: gliomaI, IDH mutations, xenograft, D-2-hydroxyglutarate, [alpha]-ketoglutarate, mitochondria: LESA-nano ESI-FTIC Objavljeno v DiRROS: 02.08.2024; Ogledov: 171; Prenosov: 170
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183. Endothelial tip cells in ocular angiogenesis : potential target for anti-angiogenesis therapyMartin J. Siemerink, Ingeborg Klaassen, Cornelis J. F. van Noorden, Reinier O. Schlingemann, 2013, izvirni znanstveni članek Povzetek: Endothelial tip cells are leading cells at the tips of vascular sprouts coordinating multiple processes during angiogenesis. In the developing retina, tip cells play a tightly controlled, timely role in angiogenesis. In contrast, excessive numbers of tip cells are a characteristic of the chaotic pathological blood vessels in proliferative retinopathies. Tip cells control adjacent endothelial cells in a hierarchical manner to form the stalk of the sprouting vessel, using, among others, the VEGF-DLL-Notch signaling pathway, and recruit pericytes. Tip cells are guided toward avascular areas by signals from the local extracellular matrix that are released by cells from the neuroretina such as astrocytes. Recently, tip cells were identified in endothelial cell cultures, enabling identification of novel molecular markers and mechanisms involved in tip cell biology. These mechanisms are relevant for understanding proliferative retinopathies. Agents that primarily target tip cells can block pathological angiogenesis in the retina efficiently and safely without adverse effects. A striking example is platelet-derived growth factor, which was recently shown to be an efficacious additional target in the treatment of retinal neovascularization. Here we discuss these and other tip cell-based strategies with respect to their potential to treat patients with ocular diseases dominated by neovascularization.
Ključne besede: angiogenesis, endothelial tip cell, proliferative retinopathy, anti-angiogenesis therapy, retinal neovascularization, vascular sprouts, endothelial stalk cell, molecular mediators of angiogenesis, pericytes Objavljeno v DiRROS: 02.08.2024; Ogledov: 168; Prenosov: 108
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184. The efficient method for simultaneous monitoring of the culturable as well as nonculturable airborne microorganismsBarbara Hubad, Aleš Lapanje, 2013, izvirni znanstveni članek Povzetek: Cultivation-based microbiological methods are a gold standard for monitoring of airborne micro-organisms to determine the occupational exposure levels or transmission paths of a particular infectious agent. Some highly contagious microorganisms are not easily culturable but it is becoming evident that cultivation and molecular methods are complementary and in these cases highly relevant. We report a simple and efficient method for sampling and analyzing airborne bacteria with an impactor-type high-flow-rate portable air sampler, currently used for monitoring culturable bacteria or fungi. A method is reported for extraction of nucleic acids from impacted cells without prior cultivation and using agarose as a sampling matrix. The DNA extraction efficiency was determined in spiked samples and, samples taken from a wastewater treatment plant and an alpine area. The abundance, diversity and quantity of total bacteria were analysed by a quantitative polymerase chain reaction, and by construction and analysis of clone libraries. The method does not interfere with downstream PCR analysis and can cover the gap between traditional culture and molecular techniques of bioaerosol monitoring. Objavljeno v DiRROS: 02.08.2024; Ogledov: 158; Prenosov: 87
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185. SACE_5599, a putative regulatory protein, is involved in morphological differentiation and erythromycin production in Saccharopolyspora erythraeaBenjamin Kirm, Vasilka Magdevska, Miha Tome, Marinka Horvat, Katarina Karničar, Marko Petek, Robert Vidmar, Špela Baebler, Polona Jamnik, Štefan Fujs, Jaka Horvat, Marko Fonović, Boris Turk, Kristina Gruden, Hrvoje Petković, Gregor Kosec, 2013, izvirni znanstveni članek Povzetek: Background
Erythromycin is a medically important antibiotic, biosynthesized by the actinomycete Saccharopolyspora erythraea. Genes encoding erythromycin biosynthesis are organized in a gene cluster, spanning over 60 kbp of DNA. Most often, gene clusters encoding biosynthesis of secondary metabolites contain regulatory genes. In contrast, the erythromycin gene cluster does not contain regulatory genes and regulation of its biosynthesis has therefore remained poorly understood, which has for a long time limited genetic engineering approaches for erythromycin yield improvement.
Results
We used a comparative proteomic approach to screen for potential regulatory proteins involved in erythromycin biosynthesis. We have identified a putative regulatory protein SACE_5599 which shows significantly higher levels of expression in an erythromycin high-producing strain, compared to the wild type S. erythraea strain. SACE_5599 is a member of an uncharacterized family of putative regulatory genes, located in several actinomycete biosynthetic gene clusters. Importantly, increased expression of SACE_5599 was observed in the complex fermentation medium and at controlled bioprocess conditions, simulating a high-yield industrial fermentation process in the bioreactor. Inactivation of SACE_5599 in the high-producing strain significantly reduced erythromycin yield, in addition to drastically decreasing sporulation intensity of the SACE_5599-inactivated strains when cultivated on ABSM4 agar medium. In contrast, constitutive overexpression of SACE_5599 in the wild type NRRL23338 strain resulted in an increase of erythromycin yield by 32%. Similar yield increase was also observed when we overexpressed the bldD gene, a previously identified regulator of erythromycin biosynthesis, thereby for the first time revealing its potential for improving erythromycin biosynthesis.
Conclusions
SACE_5599 is the second putative regulatory gene to be identified in S. erythraea which has positive influence on erythromycin yield. Like bldD, SACE_5599 is involved in morphological development of S. erythraea, suggesting a very close relationship between secondary metabolite biosynthesis and morphological differentiation in this organism. While the mode of action of SACE_5599 remains to be elucidated, the manipulation of this gene clearly shows potential for improvement of erythromycin production in S. erythraea in industrial setting. We have also demonstrated the applicability of the comparative proteomics approach for identifying new regulatory elements involved in biosynthesis of secondary metabolites in industrial conditions. Ključne besede: erythromycin, polyketide, regulator, SACE_5599, lmbU, differentiation (biology), sporulation, strain improvement, metabolic engineering Objavljeno v DiRROS: 02.08.2024; Ogledov: 136; Prenosov: 162
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187. The GMOseek matrix : a decision support tool for optimizing the detection of genetically modified plantsAnnette Block, Frédéric Debode, Lutz Grohmann, Julie Hulin, Isabel Taverniers, Linda Kluga, Elodie Barbau-Piednoir, Sylvia Broeders, Ingrid Huber, Marc Bulcke, Petra Heinze, Gilbert Berben, Ulrich Busch, Nancy Roosens, Erik Janssen, Jana Žel, Kristina Gruden, Dany Morisset, 2013, izvirni znanstveni članek Povzetek: Background
Since their first commercialization, the diversity of taxa and the genetic composition of transgene sequences in genetically modified plants (GMOs) are constantly increasing. To date, the detection of GMOs and derived products is commonly performed by PCR-based methods targeting specific DNA sequences introduced into the host genome. Information available regarding the GMOs’ molecular characterization is dispersed and not appropriately organized. For this reason, GMO testing is very challenging and requires more complex screening strategies and decision making schemes, demanding in return the use of efficient bioinformatics tools relying on reliable information.
Description
The GMOseek matrix was built as a comprehensive, online open-access tabulated database which provides a reliable, comprehensive and user-friendly overview of 328 GMO events and 247 different genetic elements (status: 18/07/2013). The GMOseek matrix is aiming to facilitate GMO detection from plant origin at different phases of the analysis. It assists in selecting the targets for a screening analysis, interpreting the screening results, checking the occurrence of a screening element in a group of selected GMOs, identifying gaps in the available pool of GMO detection methods, and designing a decision tree. The GMOseek matrix is an independent database with effective functionalities in a format facilitating transferability to other platforms. Data were collected from all available sources and experimentally tested where detection methods and certified reference materials (CRMs) were available.
Conclusions
The GMOseek matrix is currently a unique and very valuable tool with reliable information on GMOs from plant origin and their present genetic elements that enables further development of appropriate strategies for GMO detection. It is flexible enough to be further updated with new information and integrated in different applications and platforms. Ključne besede: genetically modified organism, GMO, GMO screening, matrix approach, genetically modified plant Objavljeno v DiRROS: 02.08.2024; Ogledov: 163; Prenosov: 140
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188. Chemical composition of natural sea salt from the Sečovlje salina (Gulf of Trieste, northern Adriatic)Nives Kovač, Neli Glavaš, Matej Dolenc, Nastja Rogan Šmuc, Zdenka Šlejkovec, 2013, izvirni znanstveni članek Povzetek: In our research the concentrations of major and minor elements were determined in natural sea salts from the Sečovlje salina (Piran salts, Slovenia) and compared to those of selected samples of commercially available unrefined salts with different geographical origins (Croatia, Austria, Italy, Portugal, India, and Pakistan). In the case of major element contents such as sodium (Na), iron (Fe), aluminum (Al), manganese (Mn), and titanium (Ti) many similarities were observed among the analysed salt samples. On the other hand, Piran salts are characterized by lower silicon (Si) values. Among the salts from the Sečovlje salina, the salt with the trade name Piran salt has a higher Mg content while Flower of salt has a lower concentration of calcium (Ca). In Slovenian samples the majority of trace element values were lower than 0.5 μg g–1, which was comparable to the results from commercially available unrefined salts. The salt composition differences observed indicate area-specific signatures related to geographic origin and diverse salt production processes. The quality of the studied salt samples is in accordance with standards established by the Codex Alimentarius Commission and the Piran salts are also suitable regarding issues of national food control. Ključne besede: microbial mat-petola, natural salt, elemental composition Objavljeno v DiRROS: 02.08.2024; Ogledov: 156; Prenosov: 87
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189. Double strand breaks and cell-cycle arrest induced by the cyanobacterial toxin cylindrospermopsin in HepG2 cellsAlja Štern, Metka Filipič, Matjaž Novak, Bojana Žegura, 2013, izvirni znanstveni članek Povzetek: The newly emerging cyanobacterial cytotoxin cylindrospermopsin (CYN) is increasingly found in surface freshwaters, worldwide. It poses a potential threat to humans after chronic exposure as it was shown to be genotoxic in a range of test systems and is potentially carcinogenic. However, the mechanisms of CYN toxicity and genotoxicity are not well understood. In the present study CYN induced formation of DNA double strand breaks (DSBs), after prolonged exposure (72 h), in human hepatoma cells, HepG2. CYN (0.1–0.5 µg/mL, 24–96 h) induced morphological changes and reduced cell viability in a dose and time dependent manner. No significant increase in lactate dehydrogenase (LDH) leakage could be observed after CYN exposure, indicating that the reduction in cell number was due to decreased cell proliferation and not due to cytotoxicity. This was confirmed by imunocytochemical analysis of the cell-proliferation marker Ki67. Analysis of the cell-cycle using flow-cytometry showed that CYN has an impact on the cell cycle, indicating G0/G1 arrest after 24 h and S-phase arrest after longer exposure (72 and 96 h). Our results provide new evidence that CYN is a direct acting genotoxin, causing DSBs, and these facts need to be considered in the human health risk assessment. Ključne besede: cylindrospermopsin, cell-cycle, cell-proliferation, double-strand breaks, HepG2 cells Objavljeno v DiRROS: 02.08.2024; Ogledov: 159; Prenosov: 128
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190. APS8, a polymeric alkylpyridinium salt blocks [alpha]7 nAChR and induces apoptosis in non-small cell lung carcinomaAna Zovko, Kristina Viktorsson, Rolf Lewensohn, Katja Kološa, Metka Filipič, Hong Xing, William R. Kem, Laura Paleari, Tom Turk, 2013, izvirni znanstveni članek Povzetek: Naturally occurring 3-alkylpyridinium polymers (poly-APS) from the marine sponge Reniera sarai, consisting of monomers containing polar pyridinium and nonpolar alkyl chain moieties, have been demonstrated to exert a wide range of biological activities, including a selective cytotoxicity against non-small cell lung cancer (NSCLC) cells. APS8, an analog of poly-APS with defined alkyl chain length and molecular size, non-competitively inhibits α7 nicotinic acetylcholine receptors (nAChRs) at nanomolar concentrations that are too low to be acetylcholinesterase (AChE) inhibitory or generally cytotoxic. In the present study we show that APS8 inhibits NSCLC tumor cell growth and activates apoptotic pathways. APS8 was not toxic for normal lung fibroblasts. Furthermore, in NSCLC cells, APS8 reduced the adverse anti-apoptotic, proliferative effects of nicotine. Our results suggest that APS8 or similar compounds might be considered as lead compounds to develop antitumor therapeutic agents for at least certain types of lung cancer. Ključne besede: 3-alkylpyridinium polymers, apoptosis, nicotine, nicotinic acetylcholine receptor, non-small cell lung carcinoma Objavljeno v DiRROS: 02.08.2024; Ogledov: 136; Prenosov: 121
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