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Abstract: Nanosized spinel-type ferrites have gained recognition as a unique class of engineered nanomaterials with promising applications, but their safety profiles remain insufficiently explored. Although iron (Fe), zinc (Zn), and manganese (Mn) are biologically relevant elements, the use of Zn- and Mn-containing ferrite nanoparticles in biomedical contexts demands careful (geno)toxicity evaluation. In this study, three ferrite nanoparticles – γFe2O3 (FeNPs), Zn0.7Fe2.3O4 (ZnNPs), and Mn0.4Fe2.6O4 (MnNPs) – synthesised through a microwave-assisted polyol route, functionalized with citric acid to improve colloidal stability, were evaluated for their potential (geno)toxic effects in an advanced in vitro 3D cell model, HepG2 spheroids. Cellular stress responses upon exposure to the particle were assessed using toxicogenomic analysis.This approach allows the identification of early molecular events that may precede overt toxicity, supporting a mechanistic understanding of adverse outcomes and facilitating the development of predictive biomarkers for hazard assessment. In the present study, the expression of selected DNA damage-responsive genes (TP53, MDM2, GADD45a, CDKN1A, OGG1, and JUNB), apoptosis-related genes (BCL2 and BAX) and oxidative stress response genes (SOD1, CAT, GPX1, GCLC, and GSR) was evaluated. The expression of the selected genes after exposure to the tested nanoparticles was analysed by qPCR primer assays (Applied Biosystems, USA) and One 48.48 Dynamic Array IFC for Gene Expression (Fluidigm, USA). After 24 and 96 hours of exposure, the spheroids were collected, and total RNA was isolated using the RNeasy Mini Kit from Qiagen (Qiagen, Germany) according to the manufacturer's instructions. 10 µg/mL etoposide served as athe positive control for the toxicogenomic analysis. RNA concentration and purity were assessed using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific) by measuring absorbance at 260/280 nm and gele efectrophoresis (Figure 1). Reverse transcription of 1 µg total RNA per sample was performed with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, MA, USA) on a BIO-RAD T100 thermal cycler under conditions listed in Table 3. For preamplification, 4 µL of each of the 24 selected TaqMan assays (SM2) were pooled into a primer mix. The reaction mixture was prepared using TATAA PreAmp GrandMasterMix (Tataa Biocenter, Sweden), the primer pool, and nuclease-free water, following manufacturer instructions. Negative controls (NTC for preamplification and NTCq for qPCR) were included. Each reaction contained 8 µL of mix and 2 µL of 5× diluted cDNA, processed in a 96-deep well plate, sealed, vortexed, and centrifuged (1000 g, 1 min). Preamplification was carried out on a BIO-RAD T100 thermal cycler under conditions in Table 4. Gene expression analysis used TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assays (Table 6). Preamplified samples were diluted 10× with nuclease-free water. Assays were prepared by mixing equal volumes (6 µL) of each assay with Fluidigm Assay Loading Reagent Kit – 10IFCS. The reaction premix combined DNA Sample Loading Reagent and Fast Probe Master Mix (Biotium/Roche) and was added to each diluted cDNA sample. qPCR was performed on 48.48 Dynamic Array™ IFC chips using the Fluidigm BioMark™ HD System under conditions in Table 5. Data were analysed with Fluidigm Gene Expression Analysis Software and quantGenious. Fold changes >1.5 or <0.66 were considered biologically relevant. Statistical significance between NP-exposed cells and solvent controls was assessed using ANOVA and Dunnett’s test in GraphPad Prism v9 (GraphPad Software, CA, USA).
Keywords: ferrite-based nanoparticles, HepG2 spheroids, toxicogenomics, changes in gene expression
Published in DiRROS: 24.03.2026; Views: 147; Downloads: 142
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Abstract: The main objective of this study was to investigate transcriptomic differences in subcutaneous adipose tissue between the local Krškopolje pig and modern hybrids, and to assess the effects of a reduced-protein diet in both genotypes. Comparative analysis between Krškopolje pigs and modern crossbreeds revealed 375 differentially expressed genes, with 189 upregulated and 186 downregulated in Krškopolje pigs. The upregulated genes were enriched in processes related to adipogenesis (SLC7A10, ADIRF, INHBB, SFRP2), extracellular matrix remodeling (COL6A5, COL4A5, COL2A1), calcium signaling (TRPV4, CAMK2A, CALML5), pro-inflammatory cytokines (IL1A, TNFSF9, CXCL8, PTGS2), and cholesterol metabolism (CYP1A1, CYP2A19, CES1). In contrast, the reduced-protein diet induced only minor transcriptional changes at the individual gene level in both Krškopolje pigs and modern crossbreeds.
Keywords: RNA sequences, pigs, adipose tissue, high throughput sequencing, RNA, transcriptome profiles, gene expression, locel breeds, hybrid breeds, data, data set
Published in DiRROS: 20.03.2026; Views: 220; Downloads: 37
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Abstract: Drought stress induces pronounced metabolic and transcriptional reprogramming of glucosinolate (GLS) biosynthesis in Brassica oleracea. An integrative approach combining HPLC-based quantification of individual GLSs, quantitative real-time PCR of core biosynthetic and regulatory genes, correlation-based network analysis, and in silico promoter characterization was applied to evaluate drought responses across genetically diverse accessions. Drought triggered strong, accession-specific shifts in GLS composition, with sinigrin content increasing from 35.9% to 55.1% in BR1 and glucoerucin reaching up to 80.2% in CCP1, while indolic GLSs such as glucobrassicin and neoglucobrassicin accounted for >75% of total GLSs in CV2 and CCP3. Hierarchical clustering separated accessions into four distinct drought response clusters independent of morphotype. Correlation analysis revealed drought-induced rewiring of GLS interdependencies, characterized by strengthened positive associations among aliphatic GLSs (r > 0.75). Gene expression profiling identified conserved MYB-centered regulatory modules (MYB28, MYB29, MYB34, MYB122) alongside strong accession-specific induction of CYP79F1 (up to 6.3-fold), FMOGS-OX5 (up to 4.8-fold), and ST5a (up to 5.1-fold). Promoter analysis revealed enrichment of ABA- and stress-responsive cis-regulatory elements. These findings delineate a genotype-dependent regulatory framework underlying GLS plasticity and identify quantitative metabolic and transcriptional markers relevant for breeding drought-resilient Brassica cultivars.
Keywords: glucosinolate, gene expression, qRT-PCR, abiotic stress response, drought stress
Published in DiRROS: 10.02.2026; Views: 519; Downloads: 180
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Abstract: Bisphenol AF (BPAF), Bisphenol AP (BPAP), and Bisphenol PH (BPPH) are being introduced into consumer products to replace BPA and are subsequently detected in ecosystems. This study investigates the genotoxic and endocrine-disruptive potential of these emerging bisphenols using a 3D in vitro liver spheroid model derived from Danio rerio (ZFL cell line), on the transcriptional level. The selected genes targeted DNA damage response pathways (TP53, NER, BER) and endocrine-related signalling (Table 1). ZFL spheroids were prepared by a force floating method as described by Štampar et al. (2019)1. Four-day-old ZFL spheroids were exposed to BPA (50 and 200 µM), BPAF (25 and 100 µM), BPAP (25 and 100 µM), and BPPH (10 and 50 µM) for 24 (Table 2) and 96 (Table 3) hours. Following exposure, total RNA was extracted using the RNeasy® Mini Kit (Qiagen, Germany). RNA quality and quantity were assessed prior to reverse transcription (Applied Biosystems, USA), followed by gene-specific preamplification (TATAA PreAmp GrandMasterMix, Tataa Biocenter, Sweden). Gene expression analysis was performed using TaqMan Gene Expression Assays (Applied Biosystems, USA) on the Fluidigm One 48.48 Dynamic Array IFC microfluidic system as described by Štern et al. (2024)2. The generated data was analysed using the Fluidigm Gene Expression Analysis Software and with a free-accessible web program, quantGenious3. The difference in gene expression greater than 1.5-fold was considered a biologically important up/downregulation (relative expression >1.5 or <0.66, respectively). Statistically significant differences were analysed using ANOVA and Dunnett’s multiple comparison test in GraphPad Prism v9 (GraphPad Software, San Diego, CA, USA).
Keywords: bisphenols, genotoxicity, endocrine disruption, ZFL spheroids, gene expression, data
Published in DiRROS: 30.09.2025; Views: 582; Downloads: 419
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Abstract: Measurement of RNA can be used to study and monitor a range of infectious and non-communicable diseases, with profiling of multiple gene expression mRNA transcripts being increasingly applied to cancer stratification and prognosis. An international comparison study (Consultative Committee for Amount of Substance (CCQM)-P103.1) was performed in order to evaluate the comparability of measurements of RNA copy number ratio for multiple gene targets between two samples. Six exogenous synthetic targets comprising of External RNA Control Consortium (ERCC) standards were measured alongside transcripts for three endogenous gene targets present in the background of human cell line RNA. The study was carried out under the auspices of the Nucleic Acids (formerly Bioanalysis) Working Group of the CCQM. It was coordinated by LGC (United Kingdom) with the support of National Institute of Standards and Technology (USA) and results were submitted from thirteen National Metrology Institutes and Designated Institutes. The majority of laboratories performed RNA measurements using RT-qPCR, with datasets also being submitted by two laboratories based on reverse transcription digital polymerase chain reaction and one laboratory using a next-generation sequencing method. In RT-qPCR analysis, the RNA copy number ratios between the two samples were quantified using either a standard curve or a relative quantification approach. In general, good agreement was observed between the reported results of ERCC RNA copy number ratio measurements. Measurements of the RNA copy number ratios for endogenous genes between the two samples were also consistent between the majority of laboratories. Some differences in the reported values and confidence intervals (‘measurement uncertainties’) were noted which may be attributable to choice of measurement method or quantification approach. This highlights the need for standardised practices for the calculation of fold change ratios and uncertainties in the area of gene expression profiling.
Keywords: RNA copy number ratio, RT-qPCR, gene expression, normalisation, standardisation, molecular diagnostic, transcriptomics, cancer, diagnostics, biomarker identification and validation
Published in DiRROS: 04.03.2025; Views: 914; Downloads: 484
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