1311. HepG2 spheroids as a biosensor-like cell-based system for (geno)toxicity assessmentMartina Štampar, Sonja Žabkar, Metka Filipič, Bojana Žegura, 2022, original scientific article Abstract: 3D spheroids developed from HepG2 cells were used as a biosensor-like system for the detection of (geno)toxic effects induced by chemicals. Benzo(a)pyrene (B(a)P) and amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) with well-known mechanisms of action were used for system validation. HepG2 spheroids grown for 3 days were exposed to BaP and PhIP for 24 and 72 h. The growth and viability of spheroids were monitored by planimetry and Live/Dead staining of cells. Multi-parametric flow cytometric analysis was applied for simultaneous detection of specific end-effects including cell cycle analysis (Hoechst staining), cell proliferation (KI67 marker), and DNA double-strand breaks (ℽH2AX) induced by genotoxic compounds. Depending on the exposure concentration/time, BaP reduced spheroid growth, affected cell proliferation by arresting cells in S and G2 phase and induced DNA double-strand breaks (DSB). Simultaneous staining of ℽH2AX formation and cell cycle analysis revealed that after BaP (10 μM; 24 h) exposure 60% of cells in G0/G1 phase had DNA DSB, while after 72 h only 20% of cells contained DSB indicating efficient repair of DNA lesions. PhIP did not influence the spheroid size whereas accumulation of cells in the G2 phase occurred after both treatment times. The evaluation of DNA damage revealed that at 200 μM PhIP 50% of cells in G0/G1 phase had DNA DSB, which after 72-h exposure dropped to 40%, showing lower repair capacity of PhIP-induced DSB compared to BaP-induced. The developed approach using simultaneous detection of several parameters provides mechanistic data and thus contributes to more reliable genotoxicity assessment of chemicals as a high-content screening tool.
Keywords: in vitro 3D cell model, HepG2, flow cytometry, cell cycle, proliferation, DNA strand, breaks
Published in DiRROS: 16.07.2024; Views: 312; Downloads: 180
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1312. An assessment of the reproducibility of reverse transcription digital PCR quantification of HIV-1Samreen Falak, Rainer Macdonald, Eloise J. Busby, Denise M. O'Sullivan, Mojca Milavec, Annabell Plauth, Martin Kammel, Heinz Zeichhardt, Hans-Peter Grunert, Andreas Kummrow, Jim F. Huggett, 2022, original scientific article Abstract: Viral load monitoring in human immunodeficiency virus type 1 (HIV-1) infection is often performed using reverse transcription quantitative PCR (RT-qPCR) to observe response to treatment and identify the development of resistance. Traceability is achieved using a calibration hierarchy traceable to the International Unit (IU). IU values are determined using consensus agreement derived from estimations by different laboratories. Such a consensus approach is necessary due to the fact that there are currently no reference measurement procedures available that can independently assign a reference value to viral reference materials for molecular in vitro diagnostic tests. Digital PCR (dPCR) is a technique that has the potential to be used for this purpose. In this paper, we investigate the ability of reverse transcriptase dPCR (RT-dPCR) to quantify HIV-1 genomic RNA without calibration. Criteria investigated included the performance of HIV-1 RNA extraction steps, choice of reverse transcription approach and selection of target gene with assays performed in both single and duplex format. We developed a protocol which was subsequently applied by two independent laboratories as part of an external quality assurance (EQA) scheme for HIV-1 genome detection. Our findings suggest that RT-dPCR could be used as reference measurement procedure to aid the value assignment of HIV-1 reference materials to support routine calibration of HIV-1 viral load testing by RT-qPCR.
Published in DiRROS: 16.07.2024; Views: 266; Downloads: 145
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1313. The performance of human cytomegalovirus digital PCR reference measurement procedure in seven external quality assessment schemes over four yearsMojca Milavec, Jernej Pavšič, Alexandra Bogožalec Košir, Gerwyn M. Jones, Denise M. O'Sullivan, Alison S. Devonshire, Fran Van Heuverswyn, Maria Karczmarczyk, Jannika Neeb, Annabell Plauth, Philippe Corbisier, Heinz Schimmel, Andreas Kummrow, Jörg Neukammer, Carole A. Foy, Martin Kammel, Hans-Peter Grunert, Heinz Zeichhardt, Jim F. Huggett, 2022, original scientific article Published in DiRROS: 15.07.2024; Views: 371; Downloads: 148
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1315. Diferential roles of eNOS in late efects ofVEGF‑A on hyperpermeability in diferent types of endothelial cellsEsmeralda K. Bosma, Shahan Darwesh, Yasmin I. Habani, Maxime Cammeraat, Paola Serrano Martinez, Mathilda E. van Breest Smallenburg, JiaY. Zheng, Ilse M.C. Vogels, Cornelis J. F. van Noorden, Reinier O. Schlingemann, Ingeborg Klaassen, 2023, original scientific article Abstract: Vascular endothelial growth factor (VEGF)-A induces endothelial hyperpermeability, but the molecular
pathways remain incompletely understood. Endothelial nitric oxide synthase (eNOS) regulates acute
efects of VEGF-A on permeability of endothelial cells (ECs), but it remains unknown whether and how
eNOS regulates late efects of VEGF-A-induced hyperpermeability. Here we show that VEGF-A induces
hyperpermeability via eNOS-dependent and eNOS-independent mechanisms at 2 days after VEGF-A
stimulation. Silencing of expression of the eNOS gene (NOS3) reduced VEGF-A-induced permeability
for dextran (70 kDa) and 766 Da-tracer in human dermal microvascular ECs (HDMVECs), but not in
human retinal microvascular ECs (HRECs) and human umbilical vein ECs (HUVECs). However, silencing
of NOS3 expression in HRECs increased permeability to dextran, BSA and 766 Da-tracer in the absence
of VEGF-A stimulation, suggesting a barrier-protective function of eNOS. We also investigated how
silencing of NOS3 expression regulates the expression of permeability-related transcripts, and
found that NOS3 silencing downregulates the expression of PLVAP, a molecule associated with
trans-endothelial transport via caveolae, in HDMVECs and HUVECs, but not in HRECs. Our fndings
underscore the complexity of VEGF-A-induced permeability pathways in ECs and the role of eNOS
therein, and demonstrate that diferent pathways are activated depending on the EC phenotype.
Keywords: endocytosis, RNAi, hyperpermeability
Published in DiRROS: 15.07.2024; Views: 319; Downloads: 210
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1316. The function of mating plugs in the spider Neriene emphana: mating strategy or sperm protection?Shuang Tian, He Jiang, Yongjia Zhan, Qingqing Wu, Matjaž Kuntner, Lihong Tu, 2023, original scientific article Abstract: Introduction: It is generally thought that mating plugs, where present, impede or reduce the possibilities of female subsequent mating. Behavioral studies on numerous spiders, where mating plugs are common, have generally supported this function. However, mating plugs in spiders could plausibly serve other functions as well. Namely, the structure of entelegyne spermathecae—the morphology of most spiders—could require a mechanism that would prevent sperm from leakage, desiccation, and backflow. Although the form and function of mating plugs in several spider species imply their potential adaptation for sperm protection, this function has never been empirically tested. Methods: Here, we test whether mating plugs in the sheet-web spider Neriene emphana serve as a sperm protective device by investigating its genital morphology, its copulation process, and the precise formation of its amorphous mating plugs. Results: This species constructs secretion plugs through male-female cooperation. Additionally, we found sperm plugs to be formed as a side product of sperm transfer, as well as an intermediate type of secretion plugs. These plug materials are transferred in different mating stages as documented by variations in the rhythm of male palpal application during copulation. We showed that complete copulations always resulted in formation of secretion plugs at spermathecal entrances via laborious deposition of male materials. Discussion: While our findings do not reject that secretion plugs in N. emphana prevent females from subsequent mating, we suggest that they must have evolved to provide sperm protection.
Keywords: entelegyne spermatheca, mating behavior, mating strategy, sperm protection mechanism, mating plug, secretion plug, sperm plug
Published in DiRROS: 15.07.2024; Views: 261; Downloads: 168
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1317. Transcriptional and epigenetic changes during tomato yellow leaf curl virus infection in tomatoBeatriz Romero-Rodríguez, Marko Petek, Chen Jiao, Maja Križnik, Maja Zagorščak, Zhangjun Fei, Eduardo Rodríguez Bejarano, Kristina Gruden, Araceli G. Castillo, 2023, original scientific article Abstract: Background Geminiviruses are DNA plant viruses that cause highly damaging diseases affecting crops worldwide. During the infection, geminiviruses hijack cellular processes, suppress plant defenses, and cause a massive reprogramming of the infected cells leading to major changes in the whole plant homeostasis. The advances in sequencing technologies allow the simultaneous analysis of multiple aspects of viral infection at a large scale, generating new insights into the molecular mechanisms underlying plant-virus interactions. However, an integrative study of the changes in the host transcriptome, small RNA profile and methylome during a geminivirus infection has not been performed yet. Using a time-scale approach, we aim to decipher the gene regulation in tomato in response to the infection with the geminivirus, tomato yellow leaf curl virus (TYLCV). Results We showed that tomato undergoes substantial transcriptional and post-transcriptional changes upon TYLCV infection and identified the main altered regulatory pathways. Interestingly, although the principal plant defense-related processes, gene silencing and the immune response were induced, this cannot prevent the establishment of the infection. Moreover, we identified extra- and intracellular immune receptors as targets for the deregulated microRNAs (miRNAs) and established a network for those that also produced phased secondary small interfering RNAs (phasiRNAs). On the other hand, there were no significant genome-wide changes in tomato methylome at 14 days post infection, the time point at which the symptoms were general, and the amount of viral DNA had reached its maximum level, but we were able to identify differentially methylated regions that could be involved in the transcriptional regulation of some of the differentially expressed genes. Conclusion We have conducted a comprehensive and reliable study on the changes at transcriptional, post-transcriptional and epigenetic levels in tomato throughout TYLCV infection. The generated genomic information is substantial for understanding the genetic, molecular and physiological changes caused by TYLCV infection in tomato.
Keywords: geminivirus, TYLCV, tomato, transcriptome, miRNA
Published in DiRROS: 15.07.2024; Views: 288; Downloads: 187
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1318. Biodiversity patterns of cyanobacterial oligotypes in lakes and rivers : results of a large-scale metabarcoding survey in the Alpine regionNico Salmaso, Serena Bernabei, Adriano Boscaini, Camilla Capelli, Leonardo Cerasino, Isabelle Domaizon, Tina Eleršek, Claudia Greco, Aleksandra Krivograd-Klemenčič, Paolo Tomassetti, Rainer Kurmayer, 2023, original scientific article Abstract: In this work, we characterised the cyanobacterial communities in the plankton and littoral biofilm of 38 lakes and in the biofilm of 21 rivers in the Alps and surrounding subalpine regions by 16S rRNA gene metabarcoding. We found little overlap in the distribution of amplicon sequence variants (ASVs) between the three habitats and between water bodies. The differences were caused by environmental filtering acting on the selection of the most abundant ASVs and a high contribution of rare oligotypes. The differentiation of community and genotype composition from specific water bodies was explained to a significant extent by environmental variables and morphometry. The taxonomic consistency of ASVs classified under the same genus name was assessed by phylogenetic analyses performed on three representative dominant genera, namely Cyanobium, Tychonema and Planktothrix. The analyses revealed eco-evolutionary adaptations in lakes and rivers, including some evidence for a polyphyletic nature. Monitoring individual genotypes in relation to environmental conditions will be useful to define the ecological amplitude of these taxa. However, the persistence or ephemeral nature of some of the rarest and most unusual ASVs has remained unknown.
Keywords: plankton, littoral, bioflm, European Alps, phylogenetic analysis, toxigenic cyanobacteria
Published in DiRROS: 15.07.2024; Views: 302; Downloads: 210
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1319. Cell-type proteomic and metabolomic resolution of early and late grain filling stages of wheat endospermShuang Zhang, Arindam Ghatak, Mitra Mohammadi Bazargani, Hannes Kramml, Fujuan Zang, Shuang Gao, Živa Ramšak, Kristina Gruden, Rajeev K. Varshney, Dong Jiang, Palak Chaturvedi, Wolfram Weckwerth, 2023, original scientific article Abstract: The nutritional value of wheat grains, particularly their protein and metabolite composition, is a result of the grain-filling process, especially in the endosperm. Here, we employ laser microdissection (LMD) combined with shotgun proteomics and metabolomics to generate a cell type-specific proteome and metabolome inventory of developing wheat endosperm at the early (15 DAA) and late (26 DAA) grain-filling stages. We identified 1803 proteins and 41 metabolites from four different cell types (aleurone (AL), sub-aleurone (SA), starchy endosperm (SE) and endosperm transfer cells (ETCs). Differentially expressed proteins were detected, 67 in the AL, 31 in the SA, 27 in the SE and 50 in the ETCs between these two-time points. Cell-type accumulation of specific SUT and GLUT transporters, sucrose converting and starch biosynthesis enzymes correlate well with the respective sugar metabolites, suggesting sugar upload and starch accumulation via nucellar projection and ETC at 15 DAA in contrast to the later stage at 26 DAA. Changes in various protein levels between AL, SA and ETC support this metabolic switch from 15 to 26 DAA. The distinct spatial and temporal abundances of proteins and metabolites revealed a contrasting activity of nitrogen assimilation pathways, e.g. for GOGAT, GDH and glutamic acid, in the different cell types from 15 to 26 DAA, which can be correlated with specific protein accumulation in the endosperm. The integration of cell-type specific proteome and metabolome data revealed a complex metabolic interplay of the different cell types and a functional switch during grain development and grain-filling processes.
Keywords: wheat, proteomics and metabolomics, aleurone, sub-aleurone, starchy endosperm, endosperm transfer cells
Published in DiRROS: 15.07.2024; Views: 654; Downloads: 194
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1320. Adrenal vein sampling for primary aldosteronism : a 15-year national referral center experienceTomaž Kocjan, Mojca Jensterle Sever, Gaj Vidmar, Rok Vrčkovnik, Pavel Berden, Milenko Stankovič, 2020, original scientific article Abstract: Background Adrenal vein sampling (AVS) is essential for diagnostics of primary aldosteronism, distinguishing unilateral from bilateral disease and determining treatment options. We reviewed the performance of AVS for primary aldosteronism at our center during first 15 years, comparing the initial period to the period after the introduction of a dedicated radiologist. Additionally, AVS outcomes were checked against CT findings and the proportion of operated patients with proven unilateral disease was estimated. Patients and methods A retrospective cross-sectional study conducted at the national endocrine referral center included all patients with primary aldosteronism who underwent AVS after its introduction in 2004 until the end of 2018. AVS was performed sequentially during Synacthen infusion. When the ratio of cortisol concentrations from adrenal vein and inferior vena cava was at least 5, AVS was considered successful. Results Data from 235 patients were examined (168 men; age 32%73, median 56 years; BMI 18%48, median 30.4 kg/ m2). Average number of annual AVS procedures increased from 7 in the 2004%2011 period to 29 in the 2012%2018 period (p < 0.001). AVS had to be repeated in 10% of procedures; it was successful in 77% of procedures and 86% of patients. The proportion of patients with successful AVS (92% in 2012%2018 vs. 66% in 2004%2011, p < 0.001) and of successful AVS procedures (82% vs. 61%, p < 0.001) was statistically significantly higher in the recent period. Conclusions Number of AVS procedures and success rate at our center increased over time. Introduction of a dedicated radiologist and technical advance expanded and improved the AVS practice.
Keywords: angiography, adrenal gland, endocrine disorders
Published in DiRROS: 15.07.2024; Views: 341; Downloads: 166
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