71. IGF2 and IGF1R identified as novel tip cell genes in primary microvascular endothelial cell monolayersMarchien G. Dallinga, Bahar Yetkin-Arik, Richelle P. Kayser, Ilse M.C. Vogels, Patrycja Nowak-Sliwinska, Arjan W. Griffioen, Cornelis J. F. van Noorden, Ingeborg Klaassen, Reinier O. Schlingemann, 2018, original scientific article Abstract: Tip cells, the leading cells of angiogenic sprouts, were identified in cultures of human umbilical vein endothelial cells (HUVECs) by using CD34 as a marker. Here, we show that tip cells are also present in primary human microvascular endothelial cells (hMVECs), a more relevant endothelial cell type for angiogenesis. By means of flow cytometry, immunocytochemistry, and qPCR, it is shown that endothelial cell cultures contain a dynamic population of CD34+ cells with many hallmarks of tip cells, including filopodia-like extensions, elevated mRNA levels of known tip cell genes, and responsiveness to stimulation with VEGF and inhibition by DLL4. Furthermore, we demonstrate that our in vitro tip cell model can be exploited to investigate cellular and molecular mechanisms in tip cells and to discover novel targets for anti-angiogenesis therapy in patients. Small interfering RNA (siRNA) was used to knockdown gene expression of the known tip cell genes angiopoietin 2 (ANGPT2) and tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE1), which resulted in similar effects on tip cells and sprouting as compared to inhibition of tip cells in vivo. Finally, we identified two novel tip cell-specific genes in CD34+ tip cells in vitro: insulin-like growth factor 2 (IGF2) and IGF-1-receptor (IGF1R). Knockdown of these genes resulted in a significant decrease in the fraction of tip cells and in the extent of sprouting in vitro and in vivo. In conclusion, this study shows that by using our in vitro tip cell model, two novel essential tip cells genes are identified.
Keywords: Angiogenesis, tip cells, CD34, IGF2, endothelial cells, cultured cells, endothelial growth factors
Published in DiRROS: 24.07.2024; Views: 21; Downloads: 5
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72. Fungi between extremotolerance and opportunistic pathogenicity on humansCene Gostinčar, Janja Zajc, Metka Lenassi, Ana Plemenitaš, Sybren de Hoog, Abdullah M. S. Al-Hatmi, Nina Gunde-Cimerman, 2018, original scientific article Abstract: Numerous agents of infections in humans and other mammals are found among fungi that are able to survive extreme environmental conditions and to quickly adapt to novel habitats. Nevertheless, the relationship between opportunistic potential and polyextremotolerance was not yet studied systematically in fungi. Here, the link between polyextremotol- erance and opportunistic pathogenicity is shown in a kingdom-wide phylogenetic analysis as a statistically significant co- occurrence of extremotolerance (e.g. osmotolerance and psychrotolerance) and opportunism at the level of fungal orders. In addition to extremotolerance, fungal opportunists share another characteristic%an apparent lack of specialised virulence traits. This is illustrated by a comparative genomic analysis of 20 dothideomycetous and eurotiomycetous black fungi. While the genomes of specialised fungal plant pathogens were significantly enriched in known virulence-associated genes that encode secreted proteases, carbohydrate active enzyme families, polyketide synthases, and non-ribosomal peptide synthetases, no such signatures were observed in human opportunists. Together the presented results have several implications. If infection of human hosts is a side effect of fungal stress tolerance and adaptability, the human body is most likely neither the preferred habitat of such species, nor important for their evolutionary success. This defines opportunism as opposed to pathogenicity, where infection is advantageous for the species% fitness. Since opportunists are generally incapable of the host-to-host transmission, any host-specific adaptations are likely to be lost with the resolution of the infection, explaining the observed lack of specialised virulence traits. In this scenario opportunistic infections should be seen as an evolutionary dead end and unlikely to lead to true pathogenicity.
Keywords: fungi, pathogenicity, infections in humans
Published in DiRROS: 24.07.2024; Views: 17; Downloads: 5
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73. Effects of oils and solid fats on blood lipids : a systematic review and network meta-analysisLukas Schwingshackl, Berit Bogensberger, Aleksander Benčič, Sven Knüppel, Heiner Boeing, Georg Hofmann, 2018, original scientific article Abstract: The aim of this network meta-analysis (NMA) is to compare the effects of different oils/solid fats on blood lipids. Literature searches were performed until March 2018. Inclusion criteria were as follows: i) randomized trial (≥3 weeks study length) comparing at least two of the following oils/solid fats: safflower, sunflower, rapeseed, hempseed, flaxseed, corn, olive, soybean, palm, and coconut oil, and lard, beef-fat, and butter; ii) outcomes LDL-cholesterol (LDL-C), total cholesterol (TC), HDL-cholesterol (HDL-C), and triacylglycerols (TGs). A random dose-response (per 10% isocaloric exchange) NMA was performed and surface under the cumulative ranking curve (SUCRA) was estimated. Fifty-four trials were included in the NMA. Safflower oil had the highest SUCRA value for LDL-C (82%) and TC (90%), followed by rapeseed oil (76% for LDL-C, 85% for TC); whereas, palm oil (74%) had the highest SUCRA value for TG, and coconut oil (88%) for HDL-C. Safflower, sunflower, rapeseed, flaxseed, corn, olive, soybean, palm, and coconut oil as well beef fat were more effective in reducing LDL-C (−0.42 to −0.23 mmol/l) as compared with butter. Despite limitations in these data, our NMA findings are in line with existing evidence on the metabolic effects of fat and support current recommendations to replace high saturated-fat food with unsaturated oils.
Keywords: cardiovascular disease, evidence synthesis, fatty acids
Published in DiRROS: 24.07.2024; Views: 20; Downloads: 4
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74. Diversity and flight activity of aphid species as potential vectors of oilseed pumpkin viruses in SerbiaAna Vučurović, Olivera Petrović-Obradović, Anđa Radonjić, Dušan Nikolić, Katarina Zečević, Ivana Stanković, Branka Krstić, 2018, original scientific article Abstract: The fauna of aphids appearing on cucurbits, including oilseed pumpkin have been poorly investigated in Serbia. Yellow water traps were used to determine the diversity of aphid species visiting cucurbits in Serbia and to monitor their flight activity. During the years 2009 to 2011, a total of 1,447 specimens were collected and a total of 57 different aphid taxa were identified. In 2009, the highest total number of aphids were caught (755), followed by 203 and 489 aphids in 2010 and 2011, respectively. The most abundant species were Aphis fabae (15%), Myizus persicae (13.4%) and Acyrthosiphon pisum (11.7%), followed by Anoecia corni (6.4%)and Aphis gossypii(5.6%). All of them, except A. corni,are reported as efficient vectors of cucurbit viruses. These five species represent 52.1% of all aphid species collected within this study. Out of 57 taxa found in oilseed pumpkin crops, 17 are known as vectors of ZYMV, 18 of WMV and 15 of CMV. Generally, 25 out of a total of 57 taxa are known as vectors of at least one of three viruses present in Serbia. From total of 1,447 specimens collected during three years of investigation, 917 (63.4%) are vectors of at least one of three viruses. Individual oilseed pumpkin plants were frequently colonized by A. gossypiiduring all three years of investigation. Results of this study showed that Serbian oilseed pumpkin growing localities are grouped among medium to reach in aphidofauna, according to total Shannon-Weaver index values which varied from 1.8 to 2.8. The highest value of Shannon-Weaver (2.8) was recorded in the Bački Petrovac I locality in 2009, while the lowest Shannon-Weaver (1.8) was recorded in Kisač locality in 2010. The maximum values of Shanon-Weaver diversity index in almost all crops were recorded in the second week of investigation or in early stages of oilseed pumpkin growth when plants had 5-6 true leaves or were in flowering stage, when potential virus infection could lead to significant yield losses.
Published in DiRROS: 24.07.2024; Views: 13; Downloads: 3
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76. Comparative ultrastructure of cells and cuticle in the anterior chamber and papillate region of Porcellio scaber (Crustacea, Isopoda) hindgutUrban Bogataj, Monika Praznik, Polona Mrak, Jasna Štrus, Magda Tušek-Žnidarič, Nada Žnidaršič, 2018, original scientific article Abstract: Isopod hindgut consists of two anatomical and functional parts, the anterior chamber, and the papillate region. This study provides a detailed ultrastructural comparison of epithelial cells in the anterior chamber and the papillate region with focus on cuticle ultrastructure, apical and basal plasma membrane labyrinths, and cell junctions. Na+/K+-ATPase activity in the hindgut epithelial cells was demonstrated by cytochemical localisation. The main difference in cuticle ultrastructure is in the thickness of epicuticle which is almost as thick as the procuticle in the papillate region and only about one sixth of the thickness of procuticle in the anterior chamber. The apical plasma membrane in both hindgut regions forms an apical plasma membrane labyrinth of cytoplasmic strands and extracellular spaces. In the papillate region the membranous infoldings are deeper and the extracellular spaces are wider. The basal plasma membrane is extensively infolded and associated with numerous mitochondria in the papillate region, while it forms relatively scarce basal infoldings in the anterior chamber. The junctional complex in both hindgut regions consists of adherens and septate junctions. Septate junctions are more extensive in the papillate region. Na+/K+-ATPase was located mostly in the apical plasma membranes in both hindgut regions. The ultrastructural features of hindgut cuticle are discussed in comparison to exoskeletal cuticle and to cuticles of other arthropod transporting epithelia from the perspective of their mechanical properties and permeability. The morphology of apical and basal plasma membranes and localisation of Na+/K+-ATPase are compared with other arthropod-transporting epithelia according to different functions of the anterior chamber and the papillate region.
Keywords: cell junctions, digestive system, extracellular matrix, ion transporting epithelium, plasma membrane labyrinth
Published in DiRROS: 24.07.2024; Views: 23; Downloads: 5
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77. A European multi lake survey dataset of environmental variables, phytoplankton pigments and cyanotoxinsEvanthia Mantzouki, Špela Remec-Rekar, Tina Eleršek, 2018, original scientific article Abstract: Under ongoing climate change and increasing anthropogenic activity, which continuously challenge ecosystem resilience, an in-depth understanding of ecological processes is urgently needed. Lakes, as providers of numerous ecosystem services, face multiple stressors that threaten their functioning. Harmful cyanobacterial blooms are a persistent problem resulting from nutrient pollution and climate-change induced stressors, like poor transparency, increased water temperature and enhanced stratification. Consistency in data collection and analysis methods is necessary to achieve fully comparable datasets and for statistical validity, avoiding issues linked to disparate data sources. The European Multi Lake Survey (EMLS) in summer 2015 was an initiative among scientists from 27 countries to collect and analyse lake physical, chemical and biological variables in a fully standardized manner. This database includes in-situ lake variables along with nutrient, pigment and cyanotoxin data of 369 lakes in Europe, which were centrally analysed in dedicated laboratories. Publishing the EMLS methods and dataset might inspire similar initiatives to study across large geographic areas that will contribute to better understanding lake responses in a changing environment.
Keywords: biological pigments, cyanotoxins, ecology, hydrobiology, data, datasets
Published in DiRROS: 24.07.2024; Views: 10; Downloads: 4
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78. Loop-mediated isothermal amplification : rapid molecular detection of virulence genes associated with avian pathogenic Escherichia coli in poultryPolona Kogovšek, Jerneja Ambrožič, Alenka Dovč, Tanja Dreo, Hristo Hristov, Uroš Krapež, Maja Ravnikar, Brigita Slavec, Marjetka Lotrič, Jana Žel, Olga Zorman-Rojs, 2018, original scientific article Abstract: Infections with pathogenic Escherichia colican lead to different animal- and human-associated diseases. E. coliinfections are common in intensive poultry farming, and important economic losses can be expected during infections with avian pathogenic E. coli(APEC) strains followed by colibacillosis. Loop-mediated isothermal amplification (LAMP) assays were developed for rapid detection of 3 APEC-associated virulence genes: sitA, traT, and ompT. All 3 LAMP assays are shown to be specific, repeatable, and reproducible. High sensitivities of the assays are shown, where as few as 1,000 bacterial cells/mL can be detected in different matrices. On-site applicability of this LAMP method is demonstrated through testing of different sample types, from animal swabs and tissues, and from environmental samples collected from 6 commercial poultry farms. All 3 virulence genes were detected at high rates (above 85%) in samples from layer and broiler chickens with clinical signs and, interestingly, high prevalence of those genes was detected also in samples collected from clinically healthy broiler flock (above 75%) while lower prevalence was observed in remaining 3 clinically healthy chicken flocks (less than 75%). Importantly, these virulence genes were detected in almost all of the air samples from 11 randomly selected poultry houses, indicating air as an important route of E. colispread. Three LAMP assays that target APEC-associated virulence genes are shown to be sensitive and robust and are therefore applicable for rapid on-site testing of various sample types, from animal swabs to air. This on-site LAMP testing protocol offers rapid diagnostics, with results obtained in <35 min, and it can be applied to other important microorganisms to allow the required prompt measures to be taken.
Keywords: APEC virulence genes, isothermal amplification LAMP, on-site detection, colibacillosis
Published in DiRROS: 24.07.2024; Views: 26; Downloads: 6
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79. High-throughput sequencing facilitates characterisation of a ʺforgottenʺ plant virus : the case of a henbane mosaic virus infecting tomatoAnja Pecman, Denis Kutnjak, Nataša Mehle, Magda Tušek-Žnidarič, Ion Gutiérrez-Aguirre, Patricija Pirnat, Ian Adams, Neil Boonham, Maja Ravnikar, 2018, original scientific article Abstract: High-throughput sequencing has dramatically broadened the possibilities for plant virus research and diagnostics, enabling discovery of new or obscure viruses, and virus strains and rapid sequencing of their genomes. In this research, we employed high-throughput sequencing to discover a new virus infecting tomato, Henbane mosaic virus (Potyvirus, Potyviridae), which was first discovered at the beginning of 20th century in the United Kingdom in cultivated henbane. A field tomato plant with severe necrotic symptoms of unknown etiology was sampled in Slovenia and high-throughput sequencing analysis using small RNA and ribosomal RNA depleted total RNA approaches revealed a mixed infection with Potato virus M (Carlavirus, Betaflexiviridae), Southern tomato virus (Amalgavirus, Amalgamaviridae) and henbane mosaic virus in the sample. The complete genomic sequence of henbane mosaic virus was assembled from the sequencing reads. By re-inoculation of the infected material on selected test plants, henbane mosaic virus was isolated and a host range analysis was performed, demonstrating the virus was pathogenic on several plant species. Due to limited metadata in public repositories, the taxonomic identification of the virus isolate was initially putative. Thus, in the next step, we used small RNA sequencing to determine genomic sequences of four historic isolates of the virus, obtained from different virus collections. Phylogenetic analyses performed using this new sequence information enabled us to taxonomically position Henbane mosaic virus as a member of the Potyvirus genus within the chili veinal mottle virus phylogenetic cluster and define the relationship of the new tomato isolate with the historic ones, indicating the existence of at least four putative strains of the virus. The first detection of henbane mosaic virus in tomato and demonstration of its pathogenicity on this host is important for plant protection and commercial tomato production. Since the virus was initially present in a mixed infection, and its whole genome was not sequenced, it has probably been overlooked in routine diagnostics. This study confirms the applicability of a combination of high-throughput sequencing and classic plant virus characterization methods for identification and phylogenetic classification of obscure viruses and historical viral isolates, for which no or limited genome sequence data is available.
Keywords: henbane mosaic virus, Tomato, high-throughput sequencing, host range analysis, phylogeny, Potyvirus
Published in DiRROS: 24.07.2024; Views: 16; Downloads: 7
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80. Localization patterns of cathepsins K and X and their predictive value in glioblastomaBarbara Breznik, Clara Limbaeck Stanic, Andrej Porčnik, Andrej Blejec, Miha Koprivnikar Krajnc, Roman Bošnjak, Janko Kos, Cornelis J. F. van Noorden, Tamara Lah Turnšek, 2018, original scientific article Abstract: Background
Glioblastoma is a highly aggressive central nervous system neoplasm characterized by extensive infiltration of malignant cells into brain parenchyma, thus preventing complete tumor eradication. Cysteine cathepsins B, S, L and K are involved in cancer progression and are overexpressed in glioblastoma. We report here for the first time that cathepsin X mRNA and protein are also abundantly present in malignant glioma.
Materials and methods
Gene expression of cathepsins K and X was analyzed using publically-available tran-scriptomic datasets and correlated with glioma grade and glioblastoma subtype. Kaplan-Maier survival analysis was performed to evaluate the predictive value of cathepsin K and X mRNA expression. Cathepsin protein expression was localized and semi-quantified in tumor tissues by immunohistochemistry.
Results
Highest gene expression of cathepsins K and X was found in glioblastoma, in particular in the mesenchymal subtype. Overall, high mRNA expression of cathepsin X, but not that of cathepsin K, correlated with poor patients’ survival. Cathepsin K and X proteins were abundantly and heterogeneously expressed in glioblastoma tissue. Immuno-labeling of cathepsins K and X was observed in areas of CD133-positive glioblastoma stem cells, localized around arterioles in their niches that also expressed SDF-1α and CD68. mRNA levels of both cathepsins K and X correlated with mRNA levels of markers of glioblastoma stem cells and their niches.
Conclusions
The presence of both cathepsins in glioblastoma stem cell niche regions indicates their possible role in regulation of glioblastoma stem cell homing in their niches. The clinical relevance of this data needs to be elaborated in further prospective studies.
Keywords: cathepsins, glioblastoma, immunohistochemistry, patient survival, cancer stem cell niches
Published in DiRROS: 24.07.2024; Views: 20; Downloads: 8
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