711. CellTracker Green labelling vs. Rose Bengal staining: CTG wins by points in distinguishing living from dead anoxia-impacted copepods and nematodsMateja Grego, Michael Stachowitsch, Marleen De Troch, Bettina Riedel, 2013, original scientific article Abstract: Hypoxia and anoxia have become a key threat to shallow coastal seas. Much is known about their impact on macrofauna, less on meiofauna. In an attempt to shed more light on the latter group, in particular from a process-oriented view, we experimentally induced short-term anoxia (1 week) in the northern Adriatic Sea (Mediterranean) and examined the two most abundant meiofauna taxa – harpacticoid copepods and nematodes. Both taxa also represent different ends of the tolerance spectrum, with copepods being the most sensitive and nematodes among the most tolerant. We compared two methods: CellTracker Green (CTG) – new labelling approach for meiofauna – with the traditional rose bengal (RB) staining method. CTG binds to active enzymes and therefore colours live organisms only. The two methods show considerable differences in the number of living and dead individuals of both meiofauna taxa. Generally, RB will stain dead but not yet decomposed copepods and nematodes equally as it does live ones. Specifically, RB significantly overestimated the number of living copepods in all sediment layers in anoxic samples, but not in any normoxic samples. In contrast, for nematodes, the methods did not show such a clear difference between anoxia and normoxia. RB overestimated the number of living nematodes in the top sediment layer of normoxic samples, which implies an overestimation of the overall live nematofauna. For monitoring and biodiversity studies, the RB method might be sufficient, but for more precise quantification of community degradation, especially after an oxygen depletion event, CTG labelling is a better tool. Moreover, it clearly highlights the surviving species within the copepod or nematode community. As already accepted for foraminiferal research, we demonstrate that the CTG labelling is also valid for other meiofauna groups.
Keywords: morje, plankton, kopepodi, živalski plankton, trofični status, morski plankton, CTG, CellTracker Green
Published in DiRROS: 02.08.2024; Views: 278; Downloads: 203
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712. Vloga matičnih celic pri napredovanju in zdravljenju gliomaUrška Verbovšek, Tamara Lah Turnšek, Helena Motaln, Mateja Robič, 2013, original scientific article Abstract: Izvor tumorjev in stohastično naravo procesa karcinogeneze najbolje opisuje hierarhični model, ki predvideva obstoj tumorskih matičnih celic (TMC). Slednje predstavljajo populacijo celic z neomejenim samoobnovitvenim potencialom, ki so se sposobne diferencirati v vrste celic vseh treh zarodnih linij in so manj občutljive na večino protirakavih učinkovin. Zato predstavljajo glavni vir za razvoj in rast tumorja, zaradi svoje odpornosti na kemoterapijo pa so vzrok za ponovitev bolezni. Za uspešno zdravljenje možganskega tumorja glioma in njegove najbolj maligne oblike, glioblastoma multiformae (GBM), bi zato bilo potrebno odstraniti prav vse TMC. Žal slednjega zaradi prehitre infiltrativne vrasti subpopulacije GBM celic z visoko izraženimi geni za gibljivost (migratom) v okolno zdravo možgansko tkivo, ni možno doseči s trenutno uporabljanimi načini zdravljenja (npr. kirurškim izrezom)
Poleg TMC, ki so ključne za razvoj in razrast tumorja, tkivo tumorja vsebuje še hematopoetske matične celice, endotelne predniške celice in mezenhimske matične celice (MMC). Delovanje teh drugih vrst matičnih celic, kjer je bila celicam MMC že dokazana protitumorska aktivnost v GBM, pa je odvisno od tumorskega mikrookolja. Žal mehanizmi in delovanje MMC med modulacijo rasti tumorja preko parakrinih in neposrednih interakcij z GBM (matičnimi) celicami še niso znani. Kljub temu pa matične celice, s poudarkom na MMC, predstavljajo nove nosilce npr. za ciljni vnos terapevtske učinkovine v tumor, ki bi lahko izboljšali učinkovitost trenutnih protitumorskih terapij. Razvoj celičnih zdravil veliko obeta, saj so MMC, poleg svojih imunomodulacijskih lastnosti, sposobne tudi usmerjenega gibanja v GBM in tam učinkovati, o čemer razpravlja ta prispevek.
Keywords: tumorske matične celice, mezenhimske matične celice, mikrookolje tumorja, celična terapija
Published in DiRROS: 02.08.2024; Views: 451; Downloads: 181
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713. Identification of plasma biomarker candidates in glioblastoma using an antibody-array-based proteomic approachKlemen Zupančič, Marjan Koršič, Urška Verbovšek, Primož Rožman, Tamara Lah Turnšek, Andrej Blejec, Kristina Gruden, Helena Motaln, Miomir Knežević, Matija Veber, Ana Herman, 2014, original scientific article Abstract: Background. Glioblastoma multiforme (GBM) is a brain tumour with a very high patient mortality rate, with a median survival of 47 weeks. This might be improved by the identification of novel diagnostic, prognostic and predictive
therapy-response biomarkers, preferentially through the monitoring of the patient blood. The aim of this study was to define the impact of GBM in terms of alterations of the plasma protein levels in these patients.
Materials and methods. We used a commercially available antibody array that includes 656 antibodies to analyse
blood plasma samples from 17 healthy volunteers in comparison with 17 blood plasma samples from patients with
GBM.
Results. We identified 11 plasma proteins that are statistically most strongly associated with the presence of GBM.
These proteins belong to three functional signalling pathways: T-cell signalling and immune responses; cell adhesion and migration; and cell-cycle control and apoptosis. Thus, we can consider this identified set of proteins as potential diagnostic biomarker candidates for GBM. In addition, a set of 16 plasma proteins were significantly associated with the overall survival of these patients with GBM. Guanine nucleotide binding protein alpha (GNAO1) was associated with both GBM presence and survival of patients with GBM.
Conclusions. Antibody array analysis represents a useful tool for the screening of plasma samples for potential cancer biomarker candidates in small-scale exploratory experiments; however, clinical validation of these candidates requires their further evaluation in a larger study on an independent cohort of patients.
Keywords: glioblastoma, proteomics, biomarker
Published in DiRROS: 02.08.2024; Views: 293; Downloads: 222
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714. New records of opistobranch gastropods in the waters off Slovenia (Gulf of Trieste, Northern Adriatic Sea)Lovrenc Lipej, Borut Mavrič, Jan Simič, Domen Trkov, 2014, original scientific article Abstract: The paper deals with four opisthobranch molluscs, which were found in the Slovenian marine waters as new records. The pleurobranchomorph Pleurobranchea meckeli was found on two occasions on muddy detritic bottom in the Gulf of Piran in June of 2013 and 2014. The nudibranch Favorinus branchialis was found in May and June 2014 on turf vegetation in a very shallow area off the pier in Koper harbour. Its spawn with white eggs was also found nearby. The second nudibranch Facelina rubrovittata was found in March 2010 crawling in the intertidal zone of the Nature reserve Strunjan. The third nudibranch Dondice banyulensis was found in waters of the Nature Monument Debeli rtic on sedimentary bottom. With the fi nding of these four species, the total number of opisthobranchs recorded to date in the Slovenian part of the Adriatic Sea increased to 75 species.
Keywords: opistobranch fauna, sea, Slovenia, Mediterranean Sea, Gulf of Trieste
Published in DiRROS: 02.08.2024; Views: 300; Downloads: 137
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715. The lesser-known medusa Drymonema dalmatinum Haeckel 1880 (Scyphozoa, Discomedusae) in the Adriatic SeaAlenka Malej, Martin Vodopivec, Davor Lučić, Ivona Onofri, Branka Pestorić, 2014, original scientific article Abstract: Authors report historical and recent records of the little-known medusa Drymonema dalmatinum in the Adriatic
Sea. This large scyphomedusa, which may develop a bell diameter of more than 1 m, was fi rst described in 1880 by
Haeckel based on four specimens collected near the Dalmatian island Hvar. The paucity of this species records since
its description confi rms its rarity, however, in the last 15 years sightings of D. dalmatinum have been more frequent.
Keywords: scyphomedusae, historical occurrence, recent observations, Adriatic Sea
Published in DiRROS: 02.08.2024; Views: 288; Downloads: 117
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716. Recurrence of Sargassum vulgare C. Agardh in Slovenian coastal waters (Adriatic Sea)Martina Orlando-Bonaca, Borut Mavrič, 2014, original scientific article Abstract: Perennial species from the genus Sargassum are considered to be indicators of high environmental quality and are therefore used in the assessment of the Ecological Status of Mediterranean coastal waters according to the European Water Framework Directive (WFD, 2000/60/EC). Over the past three decades a signifi cant decline in Sargassum populations has been reported in the Gulf of Trieste, as well as in other Adriatic and Mediterranean areas. In Slovenian coastal waters the presence of Sargassum spp. had not been confi rmed since 1980, after a severe decline due to overgrazing by the sea urchin Paracentrotus lividus. Recently, however, some thalli of S. vulgare were found in Piran Bay. The recurrence of this species is discussed in the paper, as well as the possible causes that led to the non-recovery in its populations in last decades in Slovenian coastal waters.
Keywords: macroalgae, Gulf of Trieste, Sargassum, Mediterranean Sea, non-recovery of populations
Published in DiRROS: 02.08.2024; Views: 273; Downloads: 146
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717. Reverse transcriptase droplet digital PCR shows high resilience to PCR inhibitors from plant, soil and water samplesNejc Rački, Tanja Dreo, Ion Gutiérrez-Aguirre, Andrej Blejec, Maja Ravnikar, 2014, original scientific article Abstract: Background
Detection and quantification of plant pathogens in the presence of inhibitory substances can be a challenge especially with plant and environmental samples. Real-time quantitative PCR has enabled high-throughput detection and quantification of pathogens; however, its quantitative use is linked to standardized reference materials, and its sensitivity to inhibitors can lead to lower quantification accuracy. Droplet digital PCR has been proposed as a method to overcome these drawbacks. Its absolute quantification does not rely on standards and its tolerance to inhibitors has been demonstrated mostly in clinical samples. Such features would be of great use in agricultural and environmental fields, therefore our study compared the performance of droplet digital PCR method when challenged with inhibitors common to plant and environmental samples and compared it with quantitative PCR.
Results
Transfer of an existing Pepper mild mottle virus assay from reverse transcription real-time quantitative PCR to reverse transcription droplet digital PCR was straight forward. When challenged with complex matrices (seeds, plants, soil, wastewater) and selected purified inhibitors droplet digital PCR showed higher resilience to inhibition for the quantification of an RNA virus (Pepper mild mottle virus), compared to reverse transcription real-time quantitative PCR.
Conclusions
This study confirms the improved detection and quantification of the PMMoV RT-ddPCR in the presence of inhibitors that are commonly found in samples of seeds, plant material, soil, and wastewater. Together with absolute quantification, independent of standard reference materials, this makes droplet digital PCR a valuable tool for detection and quantification of pathogens in inhibition prone samples.
Keywords: PCR amplification, inhibition, qPCR, droplet digital PCR, environmental samples
Published in DiRROS: 02.08.2024; Views: 264; Downloads: 154
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718. TRIM28 and [beta]-actin identified via nanobody-based reverse proteomics approach as possible human glioblastoma biomarkersIvana Jovchevska, Neja Šamec, Nina Kočevar Britovšek, Daniela Cesselli, Neža Podergajs, Clara Limbaeck Stanic, Michael P. Myers, Serge Muyldermans, Gholamreza Hassanzadeh Ghassabeh, Helena Motaln, Maria Elisabetta Ruaro, Evgenia Bourkoula, Tamara Lah Turnšek, Radovan Komel, 2014, original scientific article Abstract: Malignant gliomas are among the rarest brain tumours, and they have the worst prognosis. Grade IV astrocytoma, known as glioblastoma multiforme (GBM), is a highly lethal disease where the standard therapies of surgery, followed by radiation and chemotherapy, cannot significantly prolong the life expectancy of the patients. Tumour recurrence shows more aggressive form compared to the primary tumour, and results in patient survival from 12 to 15 months only. Although still controversial, the cancer stem cell hypothesis postulates that cancer stem cells are responsible for early relapse of the disease after surgical intervention due to their high resistance to therapy. Alternative strategies for GBM therapy are thus urgently needed. Nanobodies are single-domain antigen-binding fragments of heavy-chain antibodies, and together with classical antibodies, they are part of the camelid immune system. Nanobodies are small and stable, and they share a high degree of sequence identity to the human heavy chain variable domain, and these characteristics offer them advantages over classical antibodies or antibody fragments. We first immunised an alpaca with a human GBM stem-like cell line prepared from primary GBM cultures. Next, a nanobody library was constructed in a phage-display vector. Using nanobody phage-display technology, we selected specific GBM stem-like cell binders through a number of affinity selections, using whole cell protein extracts and membrane protein-enriched extracts from eight different GBM patients, and membrane protein-enriched extracts from two established GBM stem-like cell lines (NCH644 and NCH421K cells). After the enrichment, periplasmic extract ELISA was used to screen for specific clones. These nanobody clones were recloned into the pHEN6 vector, expressed in Escherichia coli WK6, and purified using immobilised metal affinity chromatography and size-exclusion chromatography. Specific nanobody:antigen pairs were obtained and mass spectrometry analysis revealed two proteins, TRIM28 and β-actin, that were up-regulated in the GBM stem-like cells compared to the controls.
Keywords: malignant gliomas, cancer stem cells, nanobodies
Published in DiRROS: 02.08.2024; Views: 349; Downloads: 248
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719. Seasonal variation in marine-snow-associated and ambient-water prokaryotic communities in the northern Adriatic SeaJana Vojvoda, Dominique Lamy, Eva Sintes, Juan A.L. Garcia, Valentina Turk, Gerhard J. Herndl, 2014, original scientific article Abstract: The structure and activity of prokaryotic communities were determined in marine snow and in the ambient water of the northern Adriatic Sea in different seasons (autumn, spring and summer). The seasonal variation in the composition of marine-snow-associated and ambient-water bacterial communities was assessed by T-RFLP (Terminal Restriction Fragment Length Polymorphism) on the 16S rRNA gene (16S rDNA) and 16S rRNA transcript (16S rRNA) level. On the 16S rDNA level, the bacterial community composition of the marine snow and ambient water was similar in summer and autumn, but not in spring. In contrast, on the 16S rRNA level, indicative of the active bacterial community, the marine-snow-associated bacterial community was different from that of the ambient-water, and different from the bacterial community on the 16S rDNA level, except in autumn. To phylogenetically characterize the bacterial and archaeal community composition associated with marine snow and the ambient water, clone libraries of 16S rDNA and 16S rRNA were constructed from 2 contrasting seasons. Phylogenetic profiling revealed a higher similarity among bacterial communities in summer compared to late autumn. Certain bacterial and archaeal groups were exclusively associated with summer or autumn marine snow, suggesting that marine-snow-associated prokaryotic communities are subjected to successional changes similar to ambient-water communities. Moreover, the presence of bacterial groups enriched in marine snow including Vibrionales and sulphate-reducing bacteria is consistent with niche partitioning and metabolic adaptations of the particle-associated microbiota.
Keywords: marine snow, free-living-bacteria, Northern Adriatic sea
Published in DiRROS: 02.08.2024; Views: 307; Downloads: 158
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720. Expression analysis of all protease genes reveals cathepsin K to be overexpressed in glioblastomaUrška Verbovšek, Helena Motaln, Ana Rotter, Nadia A. Atai, Kristina Gruden, Cornelis J. F. van Noorden, Tamara Lah Turnšek, 2014, original scientific article Abstract: Background
Cancer genome and transcriptome analyses advanced our understanding of cancer biology. We performed transcriptome analysis of all known genes of peptidases also called proteases and their endogenous inhibitors in glioblastoma multiforme (GBM), which is one of the most aggressive and deadly types of brain cancers, where unbalanced proteolysis is associated with tumor progression.
Methods
Comparisons were performed between the transcriptomics of primary GBM tumors and unmatched non-malignant brain tissue, and between GBM cell lines (U87-MG and U373) and a control human astrocyte cell line (NHA). Publicly-available data sets and our own datasets were integrated and normalized using bioinformatics tools to reveal protease and protease inhibitor genes with deregulated expression in both malignant versus non-malignant tissues and cells.
Results
Of the 311 protease genes identified to be differentially expressed in both GBM tissues and cells, 5 genes were highly overexpressed, 2 genes coding for non-peptidase homologues transferrin receptor (TFRC) and G protein-coupled receptor 56 (GPR56), as well as 3 genes coding for the proteases endoplasmic reticulum aminopeptidase 2 (ERAP2), glutamine-fructose-6-phosphate transaminase 2 (GFPT2) and cathepsin K (CTSK), whereas one gene, that of the serine protease carboxypeptidase E (CPE) was strongly reduced in expression. Seventy five protease inhibitor genes were differentially expressed, of which 3 genes were highly overexpressed, the genes coding for stefin B (CSTB), peptidase inhibitor 3 (PI3 also named elafin) and CD74. Seven out of 8 genes (except CSTB) were validated using RT-qPCR in GBM cell lines. CTSK overexpression was validated using RT-qPCR in GBM tissues as well. Cathepsin K immunohistochemical staining and western blotting showed that only proteolytically inactive proforms of cathepsin K were overexpressed in GBM tissues and cells.
Conclusions
The presence of high levels of inactive proforms of cathepsin K in GBM tissues and cells indicate that in GBM the proteolytic/collagenolytic role is not its primary function but it plays rather a different yet unknown role.
Keywords: glioblastoma multiforme, genes
Published in DiRROS: 02.08.2024; Views: 360; Downloads: 210
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