171. 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: 157; Downloads: 92
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173. 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: 153; Downloads: 111
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174. 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: 159; Downloads: 81
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175. 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: 179; Downloads: 174
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176. 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: 169; Downloads: 170
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177. 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, original scientific article Abstract: 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.
Keywords: angiogenesis, endothelial tip cell, proliferative retinopathy, anti-angiogenesis therapy, retinal neovascularization, vascular sprouts, endothelial stalk cell, molecular mediators of angiogenesis, pericytes Published in DiRROS: 02.08.2024; Views: 167; Downloads: 106
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178. The efficient method for simultaneous monitoring of the culturable as well as nonculturable airborne microorganismsBarbara Hubad, Aleš Lapanje, 2013, original scientific article Abstract: 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. Published in DiRROS: 02.08.2024; Views: 158; Downloads: 86
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179. 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, original scientific article Abstract: 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. Keywords: erythromycin, polyketide, regulator, SACE_5599, lmbU, differentiation (biology), sporulation, strain improvement, metabolic engineering Published in DiRROS: 02.08.2024; Views: 136; Downloads: 162
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