161. Gene expression profiling of recombinant protein producing E. coli at suboptimal growth temperatureMitja Mahnič, Špela Baebler, Andrej Blejec, Špela Jalen, Kristina Gruden, Viktor Menart, Simona Jevševar, 2012, original scientific article Abstract: Recent studies have revealed that at lower cultivation temperatures (25 °C) much higher percentage of correctly folded recombinant hG-CSF protein can be extracted from inclusion bodies. Hence, the goal of our research was to investigate mechanisms determining characteristics of non-classical inclusion bodies production using gene expression profiling, focusing on proteases and chaperones gene expression. Statistical analysis of microarray data showed prominent changes in energy metabolism, in metabolism of amino acids and nucleotides, as well as in biosynthesis of cofactors and secondary metabolites if the culture was grown below its optimal temperature. Moreover, 24 differentially expressed up to now known genes classified among proteases, chaperones and other heat or stress related genes. Among chaperones UspE and among proteases YaeL and YeaZ might play an important role in accumulation of correctly folded recombinant proteins. Membrane localized protease yaeL gene was found to have higher activity at 25 °C and is thus potentially functionally related to the more efficient recombinant protein production at lower temperatures. The results of this study represent advance in the understanding of recombinant protein production in E. coli. Genes potentially influencing production of recombinant protein at lower growth temperature represent basis for further research towards improvement of E. coli production strains as well as fermentation process. Keywords: recombinant protein production, non-classical inclusion bodies, expression microarrays, YaeL protease, GroEL chaperone Published in DiRROS: 05.08.2024; Views: 222; Downloads: 208
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162. Comparison and transfer testing of multiplex ligation detection methods for GM plantsGabriella Ujhelyi, Jeroen P. van Dijk, Theo W. Prins, Marleen Voorhuijzen, Angeline Van Hoef, Henriek G. Beenen, Dany Morisset, Kristina Gruden, Esther Kok, 2012, original scientific article Abstract: Background
With the increasing number of GMOs on the global market the maintenance of European GMO regulations is becoming more complex. For the analysis of a single food or feed sample it is necessary to assess the sample for the presence of many GMO-targets simultaneously at a sensitive level. Several methods have been published regarding DNA-based multidetection. Multiplex ligation detection methods have been described that use the same basic approach: i) hybridisation and ligation of specific probes, ii) amplification of the ligated probes and iii) detection and identification of the amplified products. Despite they all have this same basis, the published ligation methods differ radically. The present study investigated with real-time PCR whether these different ligation methods have any influence on the performance of the probes. Sensitivity and the specificity of the padlock probes (PLPs) with the ligation protocol with the best performance were also tested and the selected method was initially validated in a laboratory exchange study.
Results
Of the ligation protocols tested in this study, the best results were obtained with the PPLMD I and PPLMD II protocols and no consistent differences between these two protocols were observed. Both protocols are based on padlock probe ligation combined with microarray detection. Twenty PLPs were tested for specificity and the best probes were subjected to further evaluation. Up to 13 targets were detected specifically and simultaneously. During the interlaboratory exchange study similar results were achieved by the two participating institutes (NIB, Slovenia, and RIKILT, the Netherlands).
Conclusions
From the comparison of ligation protocols it can be concluded that two protocols perform equally well on the basis of the selected set of PLPs. Using the most ideal parameters the multiplicity of one of the methods was tested and 13 targets were successfully and specifically detected. In the interlaboratory exchange study it was shown that the selected method meets the 0.1% sensitivity criterion. The present study thus shows that specific and sensitive multidetection of GMO targets is now feasible. Keywords: geneticallz modified organisms, GMO Published in DiRROS: 05.08.2024; Views: 208; Downloads: 172
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163. Vodne ptice in ujede Cerkniškega polja (Južna Slovenija) v letih 2007 in 2008, s pregledom zanimivejših opazovanj do konca leta 2010Dejan Bordjan, 2012, original scientific article Abstract: Od konca februarja 2007 do sredine februarja 2008 je bilo na Cerkniškem polju (južna Slovenija) v desetdnevnih obdobjih (dekadah) opravljenih 73 sistematičnih popisov vodnih ptic in ujed. Cilji raziskave so bili: ugotoviti številčnost in časovno dinamiko pojavljanja vrst, opredeliti njihov status, napraviti primerjavo s preteklim obdobjem sistematičnega zbiranja podatkov v letih 1991–1992, podati ocene gnezdečih, selitvenih in prezimujočih populacij ter na njihovi osnovi naravovarstveno ovrednotiti to območje. Zbrani so bili tudi drugi ornitološki podatki, pridobljeni do konca leta 2010. Med začetkom maja in začetkom septembra leta 2007 je voda na območju raziskave presahnila, tako da se je zadržala le v strugah potokov, vodna površina pa je bila deloma ali v celoti zaledenela od sredine novembra 2007 do začetka februarja 2008. Do konca leta 2010 je bilo na Cerkniškem polju skupaj zabeleženih 129 vrst vodnih ptic in ujed, v obdobju 2007/2008 pa 83. Dinamika pojavljanja vrst z več kot devetimi opazovanji je podrobneje predstavljena v pregledu vrst. Status gnezdilke ima 27 vrst, še nadaljnjih sedem pa je prehranskih gostov, ki gnezdijo v okolici območja raziskave. Največ vrst (118) ima status preletnega gosta, medtem ko je vrst s statusom poletnega (34) oziroma zimskega gosta (40) manj. Med zabeleženimi vrstami je 34 izjemnih in 21 redkih gostov. Prezimovalcev je 16, letovalcev sedem, vse leto pa se je tukaj zadrževalo šest vrst. V vseh dekadah obdobja raziskave so bile opažene štiri vrste: mlakarica Anas plathyrhynchos, siva čaplja Ardea cinerea, kanja Buteo buteo in postovka Falco tinnunculus. V več kot 90 % dekad je bila zabeležena še velika bela čaplja Ardea alba. Dve vrsti sta bili v obdobju 2007/2008 evdominantni (mlakarica 27,2 %, kanja 10,1 %), še dve pa dominantni (reglja Anas querquedula 7,4 %, liska Fulica atra 6,4 %). Največ osebkov je bilo zabeleženih konec marca in v začetku aprila (do 1978 os.), največ vrst (48) pa sredi aprila. Število vrst in osebkov je bilo med majem in avgustom zaradi presahlega jezera majhno. Najmanjše območje pojavljanja so imeli kormoran Phalacrocorax carbo ter skupina rac iz rodov Aythya, Bucephala in Mergellus. Galebi, čaplje, pobrežniki, lunji Circus sp. in rdečenoga postovka Falco vespertinus so se pojavljali na večjem delu območja raziskave. 10 gnezdilk izpolnjuje kriterije za oznako varstveno najpomembnejše vrste, od katerih sta dve (kostanjevka Aythya nyroca, veliki škurh Numenius arquata) vrsti globalne varstvene pozornosti, sedem pa vrste evropske varstvene pozornosti. Med negnezdilkami se 14 vrst vodnih ptic in ujed pojavlja v pomembnem številu (> 0,1 % biogeografske populacije), od tega se jih pet vsaj občasno pojavlja z več kot 1 % biogeografske populacije. Dve vrsti (rjavovrati ponirek Podiceps grisegena, kozica Gallinago gallinago) v Sloveniji gnezdita samo na Cerkniškem polju, še šest vrst pa ima tukaj vsaj 40 % slovenske gnezdeče populacije. Keywords: vodne ptice, ujede, številčnost (biologija), dinamika pojavljanja (biologija), naravovarstveno vrednotenje Published in DiRROS: 02.08.2024; Views: 144; Downloads: 102
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164. In vitro corrosion-fatigue behaviour of rare-earth containing magnesium WE43 in sterile complex cell culture mediumJulia Nachtsheim, Songyun Ma, Jaka Burja, Alexander Köppl, Jan-Marten Seitz, Bernd Markert, 2024, original scientific article Abstract: Rare-earth containing magnesium alloys are promising biomedical materials for a new generation of biodegradable orthopaedic implant systems due to their excellent biocompatibility, mechanical and biodegradation properties. However, chemo-mechanical interactions in aggressive physiological corrosion environments result in rapid degradation and early loss of mechanical integrity, limiting its broader application for orthopaedic implants. To date, only few studies have assessed the corrosion-fatigue behaviour of medical-grade magnesium alloys in an organic physiological corrosion environment, especially under sterile test conditions. In the present work, the corrosion-fatigue behaviour of fine-grained medical-grade magnesium alloy WE43MEO was systematically analysed under in vitro conditions using an organic physiological fluid DMEM. The experimental results showed that the fatigue strength of the alloy is nearly unaffected by a 1-day precorrosion, while a 7-day precorrosion resulted in a significant deterioration of mechanical integrity. In corrosion-fatigue experiments, the fatigue life was considerably reduced by interactions between corrosion and fatigue damages. The SEM analysis revealed that the mixed mode of intergranular and transgranular fracture in the crack propagation zone transits to intergranular cracking dominant mode under the corrosion-fatigue conditions due to hydrogen embrittlement. Keywords: corrosion-fatigue behaviour, stress corrosion mechanisms, biodegradable magnesium alloys, WE43, DMEM Published in DiRROS: 02.08.2024; Views: 161; Downloads: 113
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165. VEGF levels in plasma in relation to platelet activation, glycemic control, and microvascular complications in type 1 diabetesReinier O. Schlingemann, Cornelis J. F. van Noorden, Mattheus J.M. Diekman, Anna Tiller, Joost C.M. Meijers, Pieter Koolwijk, Wilmar M. Wiersinga, 2013, original scientific article Abstract: OBJECTIVE
Increased levels of vascular endothelial growth factor (VEGF) in human plasma samples have suggested that circulating VEGF is a cause of endothelial dysfunction in diabetes mellitus. However, artificial release of VEGF from platelets as a source of VEGF in plasma samples, as also occurs in serum samples, has not been ruled out in these studies.
RESEARCH DESIGN AND METHODS
We determined VEGF levels in plasma collected in both citrate and PECT, a medium that inactivates platelets, in a cross-sectional cohort of 21 healthy subjects and 64 patients with type 1 diabetes. In addition, we evaluated whether VEGF levels in both types of plasma correlated with the presence of diabetes, glycemic control, markers of in vivo or ex vivo platelet activation, and degree of diabetic retinopathy and nephropathy.
RESULTS
VEGF levels were invariably low in PECT plasma of both nondiabetic and diabetic subjects and were unrelated to any other diabetes-related variable studied. In contrast, VEGF levels in citrate plasma were 150% higher in diabetic patients than in control subjects and correlated with diabetes-related variables. Multiple linear regression analysis showed that levels of platelet factor 4, a marker for ex vivo platelet activation, and HbA1c were the independent predictors of VEGF levels in citrate plasma. Platelet activation, in vivo and ex vivo, was similar in diabetic persons and control subjects.
CONCLUSIONS
Like serum, citrate plasma is not suitable for reliable measurements of circulating VEGF. The low levels of VEGF in vivo, as represented by measurements in PECT plasma in our study, do not support a role of circulating VEGF in endothelial dysfunction in type 1 diabetes. Higher levels of VEGF in citrate plasma samples of diabetic persons do not represent the in vivo situation, but mainly originate from higher artificial ex vivo release from platelets correlating with the degree of glycemic control. Keywords: vascular endothelial growth factor, VEGF, diabetes mellitus Published in DiRROS: 02.08.2024; Views: 140; Downloads: 76
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167. Rocky macrozoobenthos mediolittoral community in the Gulf of Trieste (North Adriatic) along a gradient of hydromorphological modificationsValentina Pitacco, Borut Mavrič, Martina Orlando-Bonaca, Lovrenc Lipej, 2013, original scientific article Abstract: Despite the increasing urban and industrial development in coastal areas our knowledge on direct consequences of coastal modifications on benthic communities is still limited. The aim of the present study was to assess the response of rocky macrozoobenthos mediolittoral communities to human-induced hydromorphological pressures. Sampling was carried out by SCUBA diving and snorkeling in June 2008. Ten sites were selected along a gradient of hydromorphological alterations in the southern part of the Gulf of Trieste. Variables used to describe the stressor gradient were: water retention (from normal hydrology in unprotected coast to closed areas with only one opening), substrate composition, texture and rugosity. Despite natural differences between upper and lower mediolittoral subbelts, the present work showed that human-induced alterations of the coastal zone impact biological assemblages. There was a marked difference in biodiversity among sites with pristine conditions and stressed zones, mainly due to evenness of species distribution. Structural complexity of the substrate resulted to be the main factor influencing benthic diversity in the upper mediolittoral subbelt, while in the lower subbelt also the human-induced water retention seemed to play a key role. Anyhow, this response was complex, and the major human-induced alterations considered had different level of pressure within the two subbelts. The current study has a good potential to contribute to existing coastal assessment methods, since the impact of hydromorphological pressures on mediolittoral communities was almost neglected in the past. However, further work is needed to fully explain the impact of main human-induced threats on benthic communities. Keywords: sea, Zoobenthos, artificial substrata, intertidal environment, hydromorphological modifications, ecological distribution, coastal waters, biodiversity, bio-indicators, macrozoobenthos, rocky bottom, biocoenoses, ecological aspects, Gulf of Trieste Published in DiRROS: 02.08.2024; Views: 126; Downloads: 85
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168. Roost-site characteristics of the Mediterranean shag Phalacrocorax aristotelis desmarestii along the Slovenian coastDejan Bordjan, Matej Gamser, Aleksander Kozina, Jure Novak, Mitja Denac, 2013, original scientific article Abstract: Several bird species utilize artificial structures for communal roosting. Between 26 May and 28 Jun 2012, the selection of buoys and times of departure by Mediterranean Shags Phalacrocorax aristotelis desmarestii were studied at three communal roosts within shellfish farms in the Bays of Sv. Jernej (Debeli rtič), Strunjan and Piran (Sečovlje). A total of 3,110 buoys were counted and categorized into two groups according to their shape (horizontal and vertical) and colours. The black horizontally floating buoys were of two types (barrelshaped and oval). The Shags chose to utilize the horizontally floating buoys
only, most often black and white in colour. Owing to their poorer stability and smaller standing surface, the vertically floating buoys are clearly unsuitable for them. The highest share of Shags with regard to the number of buoys of separate types was registered on black barrel-shaped buoys. As the percentage of occupied buoys was similar at all roost sites (36–39%), it was deduced that Shags distribute evenly among roosts, regardless of the number of individuals present in the Slovenian sea. At the larger roost at Debeli rtič, the percentage of adult individuals (73.5%) was greater than at Strunjan (42.5%). This could be due to the competition for better places, given that competitively stronger individuals select safer larger roosts. Between 5.42 and 9.00 hrs, 53.3% and
69.1% of Shags departed from the roost sites at Debeli rtič and Strunjan, respectively, with the majority of departures recorded between 7.50 and 8.30 hrs. They left their roosts mostly individually (48.7%) or in pairs (23.3%), at Debeli rtič predominantly in the SW (58.9%) and W (16.9%) directions, and at Strunjan in the NE (42.3%) and N (38.5%) directions. Keywords: Mediterranean Shag, Phalacrocorax aristotelis desmarestii, roost-site, buoys, Slovenian coast Published in DiRROS: 02.08.2024; Views: 125; Downloads: 67
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169. 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, original scientific article Abstract: 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. Keywords: soil salinity and drought, plant resistance, abiotic stress Published in DiRROS: 02.08.2024; Views: 151; Downloads: 138
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170. 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, original scientific article Abstract: 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. Keywords: gliomaI, IDH mutations, xenograft, D-2-hydroxyglutarate, [alpha]-ketoglutarate, mitochondria: LESA-nano ESI-FTIC Published in DiRROS: 02.08.2024; Views: 135; Downloads: 138
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