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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: 433; Downloads: 288
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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: 443; Downloads: 243
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Abstract: In the field, plants are challenged by more than one biotic stressor at the same time. In this study, the molecular interactions between potato (Solanum tuberosum L.), Colorado potato beetle (Leptinotarsa decemlineata Say; CPB) and Potato virus YNTN (PVYNTN) were investigated through analyses of gene expression in the potato leaves and the gut of the CPB larvae, and of the release of potato volatile compounds. CPB larval growth was enhanced when reared on secondary PVYNTN-infected plants, which was linked to decreased accumulation of transcripts associated with the antinutritional properties of potato. In PVYNTN-infected plants, ethylene signalling pathway induction and induction of auxin response transcription factors were attenuated, while no differences were observed in jasmonic acid (JA) signalling pathway. Similarly to rearing on virus-infected plants, CPB larvae gained more weight when reared on plants silenced in JA receptor gene (coi1). Although herbivore-induced defence mechanism is regulated predominantly by JA, response in coi1-silenced plants only partially corresponded to the one observed in PVYNTN-infected plants, confirming the role of other plant hormones in modulating this response. The release of β-barbatene and benzyl alcohol was different in healthy and PVYNTN-infected plants before CPB larvae infestation, implicating the importance of PVYNTN infection in plant communication with its environment. This was reflected in gene expression profiles of neighbouring plants showing different degree of defence response. This study thus contributes to our understanding of plant responses in agro-ecosystems.
Keywords: insect midgut transcriptional response, gene expression, plant defence, volatile organic compounds, potato
Published in DiRROS: 02.08.2024; Views: 405; Downloads: 208
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Abstract: The ability to identify and locate conspecifics depends on reliable transfer of information between emitter and receiver. For a majority of plant-dwelling insects communicating with substrate-borne vibrations, localization of a potential partner may be a difficult task due to their small body size and complex transmission properties of plants. In the present study, we used the leafhopper Scaphoideus titanus as a model to investigate duetting and mate searching associated with pair formation. Studying these insects on a natural substrate, we showed that the spatio-temporal structure of a vibrational duet and the perceived intensity of partner's signals influence the mating behaviour. Identification, localization and courtship stages were each characterized by a specific duet structure. In particular, the duet structure differed in synchronization between male and female pulses, which enables identification of the partner, while the switch between behavioural stages was associated with the male-perceived intensity of vibrational signals. This suggests that males obtain the information about their distance from the female and optimize their strategy accordingly. More broadly, our results show that even in insects smaller than 1 cm, vibrational signals provide reliable information needed to find a mating partner.
Keywords: mating behaviour, identification, localization, courtship, vibrational communication
Published in DiRROS: 02.08.2024; Views: 371; Downloads: 211
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Abstract: The risk posed by complex chemical mixtures in the environment to wildlife and humans is increasingly debated, but has been rarely tested under environmentally relevant scenarios. To address this issue, two mixtures of 14 or 19 substances of concern (pesticides, pharmaceuticals, heavy metals, polyaromatic hydrocarbons, a surfactant, and a plasticizer), each present at its safety limit concentration imposed by the European legislation, were prepared and tested for their toxic effects. The effects of the mixtures were assessed in 35 bioassays, based on 11 organisms representing different trophic levels. A consortium of 16 laboratories was involved in performing the bioassays. The mixtures elicited quantifiable toxic effects on some of the test systems employed, including i) changes in marine microbial composition, ii) microalgae toxicity, iii) immobilization in the crustacean Daphnia magna, iv) fish embryo toxicity, v) impaired frog embryo development, and vi) increased expression on oxidative stress-linked reporter genes. Estrogenic activity close to regulatory safety limit concentrations was uncovered by receptor-binding assays. The results highlight the need of precautionary actions on the assessment of chemical mixtures even in cases where individual toxicants are present at seemingly harmless concentrations.
Keywords: morje, analizna kemija, biologija, evtrofikacija, hranilne snovi, fitoplankton, onesnaževanje, Piranski zaliv
Published in DiRROS: 02.08.2024; Views: 389; Downloads: 302
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