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Directed DNA Methylation of the Arx gene Regulatory Region via CRISPR/dCas9 System in αTC1-6 Cells
No full textIntroduction and Rationale: Cell transdifferentiation is the process of directly converting one terminally differentiated cell type into another, without passing through a pluripotent state. DNA methylation pattern plays a key regulatory role in cell transdifferentiation by influencing the activation or repression of genes. The aim of this study was to investigate whether a CRISPR/dCas9 construct containing gRNAs targeting the regulatory region of the Arx gene which represent key factor in maintaining the identity of alpha pancreatic cells (2 kb upstream of the transcription start site) could induce targeted DNA methylation at this locus.
Methods: The experiment was conducted on a mouse pancreatic alpha cell line (αTC1-6). The CRISPR/dCas9 construct consisted of a catalytically inactive dCas9 protein, the DNMT3a effector domain, four gRNAs specific to the Arx regulatory region, and a mClover selection marker. This construct, herein referred to as EpiC, was delivered into the experimental samples, while control samples were transfected with the same construct lacking the specific gRNAs, using nucleofection. At fifth day post-transfection, cells were sorted using a FACSIII/Aria cell sorter based on mClover expression. Genomic DNA was isolated from sorted cells, bisulfide-converted, and amplified using primers specific to the targeted region. Sequencing was performed using a SeqStudio Genetic Analyser to assess whether DNA methylation was established.
Results: Seven CpG sites within the targeted region were analysed. Comparison between control samples and in EpiC transfected samples showed that the construct significantly increased cytosine methylation in all CpG sites. In control samples, thymine peaks were observed indicating unmethylated cytosines. In contrast, experimental samples retained cytosine signals, consistent with successful methylation at the targeted loci.
Conclusions: These results demonstrate that the designed gRNAs successfully directed the EpiC construct to the targeted region, leading to targeted DNA methylation. This supports the potential of this system for precise epigenetic editing and highlights its utility in modulating cell identity.Abstract book: FEBS-IUBMB-enable conference: 4th International Molecular Biosciences PhD and Postdoc Conference: Bridging Minds, interdisciplinary research for the future of the life sciences; 2025 Sep 10-12; Glasgow, United Kingdom. Federation of European Biochemical Societies; 2025. Poster no. 14
Impact of yogurt consumption on bone health markers in adults with or without osteoporosis: a systematic review and meta-analysis
No full textYogurt is a fermented dairy product widely recognized for its nutritional
value and potential health benefits, particularly due to its high content of
calcium, phosphorus, vitamins, and other bioactive compounds. Its purported
role in promoting bone health has attracted increasing attention, especially
among adults at risk of osteoporosis. However, the specific impact of yogurt
consumption on bone health remains controversial, as current evidence is
limited and often inconclusive. This systematic review and meta-analysis sought
to evaluate the exclusive effects of yogurt consumption on bone health in adults
with and without osteoporosis. Employing rigorous inclusion and exclusion
criteria and standardized methodologies, the study reviewed data from both
observational and clinical studies to assess the effect of yogurt consumption
on bone mineral density (BMD), fracture risk, and other bone health markers.
In addition, yogurt characteristics were documented in accordance with
EFSA guidelines, the possible bioactive components listed, and their potential
mechanism of action in relation to bone health discussed. Studies fitting the
inclusion and exclusion criteria (n = 12) were all observational, limiting the ability
to infer causality. The meta-analysis of cohort studies (n = 6) found no significant
association between yogurt consumption and hip fracture risk (HR=1.01,95% CI: 0.96–1.07), while the observed positive effect on BMD was minimal and clinically irrelevant (SMD = 0.009). Although some studies reported modest improvements in BMD and bone biomarkers, the overall evidence remains inconclusive, hindered by the heterogeneity in study designs and inconsistent yogurt intake. In conclusion, current evidence does not support a significant role of yogurt consumption in preventing fractures or improving BMD in adults. Well-designed randomized controlled studies are needed to clarify its effects,
particularly in adults at risk of or with osteoporosis or osteopenia. In conclusion, the effectiveness of yogurt as a primary strategy for enhancing bone health is not fully substantiated by current data, though its inclusion within a balanced diet may still offer benefits for bone health
Health benefits of ethnic fermented foods
No full textFood science and technology have evolved to enhance food availability, nutrition,
safety, and marketability, addressing global challenges such as hunger and malnutrition.
However, projections indicate that by 2030, over 670 million people may face
hunger, emphasizing the need for innovative and sustainable solutions. Ethnic
fermented foods (EFF) originate from the heritage and culture of various ethnic
groups who utilize their ethnomicrobiological knowledge of fermentation with
local plant- or animal-based ingredients. EFF provide a promising approach to
promote a healthier, sustainable, and more inclusive global food system. They are
also traditionally produced by combining local ingredients with artisanal practices.
However, urbanization and changes in eating habits threaten the preservation of EFF
and the associated native microorganisms that are essential for their production.
This research systematically examines health-related endpoints reported in human
clinical studies of EFF. While some EFF such as natto, kimchi, and tempeh, originating
from Asia have gained global recognition and consumption, multiple others remain
unknown and understudied. This comprehensive narrative review aims to identify
the potential health benefits associated with these foods. A systematic search
in PubMed, Scopus, and the Cochrane Library Central initially resulted in 3,488
entries, which were narrowed down to 125 reports fulfilling the selection criteria
defined to identify human studies investigating the functionality of EFF. CADIMA
software was used to manage and select references. Data synthesis focused on
clinical indications, offering valuable insights into health benefits of EFF, such as gut
health, immune modulation, metabolic benefit, cognitive function, cardiovascular,
bone, oral, eye, skin health and antimicrobial effect. This systematic narrative
review highlights the importance of EFF in improving health, emphasizing their
role in sustainable food systems, and underscoring the importance of preserving
traditional practices
Connecting brain and gut inflammation in the murine model of Alzheimer’s disease
No full textIn the mouse model of Alzheimer’s disease, App NL-G-F NL-G-F (Alz) mice that carry the Arctic, Swedish
and Beyreuther/Iberian mutations, aggressive Aβ amyloidosis with cortical and subcortical deposition
were documented. We investigated the composition and function of immune cells in the gut associated
lymphoid tissue in Alz mice and their littermate controls (LC) at the age of 6 months, when they display
microgliosis, astrocytosis and memory impairment. The small intestine (SI) lamina propria (LP) of Alz
mice harbour fewer ILC3 cells and IL-22-producing ILC3, important for the maintenance of intestinal homeostasis. SI homogenates from Alz mice show lower IL-10 and IL-22, but higher IL-17 levels. In line with
these, the frequency of Treg cells in the SI LP is lower. Simultaneously, the cortex and hippocampus of
these animals have higher concentrations of TNF and IFN-γ. Alz mice differ from the LC in microbiota
composition and the abundance of short chain fatty acids in the SI. Data from SI’s TEM shows that there
is no difference in the morphological measurements of intestinal barrier integrity. Also, the motility and
peristaltic of SI is the same. However, the expression of mRNA for tight junction proteins claudin 4 and
cadherin 1 is significantly downregulated in Alz mice, compared to LC.Robajac D, editor. Book of abstracts: FEBS3+ meeting: Advances in molecular biosciences: from genes to personalized therapies; 2025 Sep 24-27; Belgrade, Serbia. Belgrade: Serbian Biochemical Society; Faculty of Chemistry, University; 2025. p. 50-1
Polystyrene-nickel interactions in soil: Implications for metal mobility, plant uptake, and human health
No full textThe coexistence of microplastics (MPs) and metals is ubiquitous in terrestrial ecosystems. However, their coupled effects on soil biota and human health remain unknown. The present study investigated the interactive impacts of polystyrene (PS)-nickel (Ni) contamination on Ni mobility in the soil-medicinal plant system and the associated health risks. A soil pot experiment was conducted with Capsella bursa-pastoris under six treatments: control (CK), PS alone (1 % w/w, PS), Ni at 50 and 500 mg kg−1 (Ni50 and Ni500), and their combinations (Ni50 +PS, Ni500 +PS). The introduction of PS into soil promoted Ni transformation to the reducible soil fraction (F2) and increased its bioavailability by up to 81.2 % compared to treatments without MPs. The mechanism underlying Ni redistribution involved PS–soil associations that mask mineral binding sites and induce a “dilution effect,” whereby Ni loosely associated with PS surfaces became more mobile and bioavailable in the soil matrix. The most prominent increase was recorded in control soil (CK) and soil with 50 mg kg−1 Ni (Ni50). PS addition to CK and Ni50 also stimulated Ni uptake by C. bursa-pastoris and increased the carcinogenic risk by 62.5 % compared to CK and by 28.6 % in Ni50 +PS compared to Ni50. Structural Equation Modeling (SEM) and Principal Component Analysis (PCA) confirmed that Ni bioavailability and transfer to and within the plant were strongly influenced by the PS presence. The findings of this study provided valuable insights into the toxic effects of PS-Ni exposure on the food safety of medicinal plants
Micropropagation of Micromeria graeca (L.) Benth. ex Rchb. (Lamiaceae)
No full textMicromeria graeca (L.) Benth. ex Rchb. subsp. graeca (Lamiaceae), a perennial subshrub widely distributed in the Mediterranean area, is a promising medicinal plant with significant phenolic content, traditionally used in folk medicine and as a condiment for its aromatic properties. This study aimed to optimize in vitro propagation of M. graeca to enhance biomass accumulation and secondary metabolite production for potential pharmacological and commercial applications. Shoot multiplication was investigated using nodal segments cultured on Murashige and Skoog (MS) medium supplemented with various plant growth regulators (PGRs): N6-benzyladenine (BA), kinetin (Kin), indole-3-acetic acid (IAA), BA or Kin in combination with IAA. Basal MS medium devoid of PGRs was used as a control. Axillary shoots developed on all media, with the highest mean shoot length observed on PGR-free medium. Shoot multiplication was achieved across all treatments, with comparable multiplication rates. The highest number of shoots per explant was recorded on medium supplemented with 10 µM BA and 0.57 µM IAA, indicating a synergistic effect of auxin and cytokinin. However, maximum biomass accumulation occurred on cytokinin-free media, suggesting that high cytokinin levels may prioritize shoot number over biomass. These results establish an effective, scalable protocol for M. graeca in vitro propagation, enhancing biomass production to support potential pharmacological and commercial applications.Golub D, editor. Book of asbtracts: V Symposium of biologists and ecologists of Republic of Srpska SBERS2025; 2025 Nov 13-25; Banja Luka, Bosnia and Herzegovina. Banja Luka: Faculty of Natural Sciences and Mathematics, University of Banja Luka; 2025. p. 36
Eriodictyol and Diosmetin Protective Potential in Skin Infection: Antimicrobial Action, Gene and Molecular Targets, and Keratinocyte Protection Against Bacteria-Induced Damage
No full textEriodictyol and diosmetin are bioactive flavonoids. This study explored their antimicrobial activities and antibiofilm potential along with the effect on pyocyanin and protease production and virulence-linked gene expression, followed by in silico molecular target predictions. Moreover, keratinocytes were used for the evaluation of cytotoxicity and protective antioxidant and anti-inflammatory effects in the infected cells. Both compounds have shown significant antibacterial capacity towards skin pathogens (minimal inhibitory concentrations 0.025–0.2 mg/mL). Their ability to prevent biofilm formation of Pseudomonas aeruginosa was drastic, as well as the impact on other virulence factors, proteases, and pyocyanin production. RT-qPCR determined downregulation of almost all genes examined (lasI, lasR, lasB, rhlI, rhlR, rhlC, pqsH, pqsR, pvdS, pvdF, phzM, and algK), while molecular docking predicted strong binding affinities to the LasI, LasR, PqsR, and QscR quorum-sensing proteins. Moreover, both compounds were not toxic to HaCaT and were able to reduce damage induced by P. aeruginosa in this cell line. Precisely, eriodictyol reduced levels of secreted IL-6 (from 335.32 to 261.76 pg), while both compounds reduced the formation of superoxide. Both eriodictyol and diosmetin displayed remarkable antimicrobial potential while employing a wide array of antimicrobial mechanisms, making them attractive candidates for further assessment and eventual incorporation into novel therapeutic strategies
Artificial Light at Night (ALAN) and Activity Patterns in Hyla Arborea Tadpoles: a Preliminary Study
No full textArtificial light at night (ALAN) is human-made illumination that disrupts the natural day–night
cycle. In amphibians, ALAN can suppress nocturnal melatonin and alter corticosterone regulation,
affecting activity and risk-related behaviours. Studies from the past decade indicate that ALAN can
modify activity timing, foraging, anti-predator responses, swimming performance, and crypsis in anuran
tadpoles. However, these effects vary considerably among species and depend on light intensity,
spectrum, and developmental stage. In this preliminary study, we investigated how exposure to cold LED
light affects the behaviour of tree frog (Hyla arborea) tadpoles. Two night-time treatments were applied:
a natural dark control (ND, <0.3 lx) and a 90-lx LED illumination (LED). We analysed only night activity
patterns under natural and artificial light. Activity and rest were recorded in seconds over a 300-second
observation period in tadpoles (stage Gosner 38). Our results show that exposure to 90 lx LED light
increases activity regardless of treatment. Although differences between treatments and the interaction
were not statistically significant (p = 0.08), clear tendencies are apparent. Activity levels under natural
light were similar across individuals, regardless of their treatment origin. In contrast, individuals exposed
to LED light showed nearly double the activity, originating from the natural night–day regime. These
preliminary patterns suggest that ALAN may differentially shape behavioural adaptation and activity
rhythms in H. arborea, and that prior exposure history could modulate sensitivity to artificial night
lighting. Further analyses with the full dataset and longer behavioural recordings are needed to confirm
these trends.Book of abstracts : The International Conference on Light Pollution and Dark Sky Protection ENLIGHT; 2025 Nov 28-30; Novi Sad, Serbia. Belgrade: Institute for Biological Research "Siniša Stanković" – National Institute of Republic of Serbia, University of Belgrade; 2025. p. 21
Antioxidant defense in resurrection fern Asplenium ceterach: role of enzymes and phenolic compounds
No full textDrought stress disrupts critical physiological processes in plants leading to oxidative stress through the excessive accumulation of reactive oxygen species (ROS). An extensive antioxidant defense system, consisting of enzymes and metabolites, enables plants to prevent oxidative damage and maintain the redox balance under normal or unfavorable environmental conditions. The present study was conducted to evaluate the defensive response of the desiccation-tolerant rustyback fern (Asplenium ceterach L.) to dehydration and subsequent rehydration, with a particular focus on selected enzymatic and non-enzymatic components of the antioxidant system.
All desiccation-tolerant plants increase antioxidant enzyme activity upon dehydration. Similarly, when rustyback fern sporophytes were exposed to water scarcity, they showed a 2.6-fold increase in activity of peroxidase compared to control plants after 21 days of dehydration. After rehydration, enzyme activity returned to control values within a few hours. On the other hand, polyphenol oxidase (PPO) activity was significantly higher during the rehydration process. As this enzyme contributes to the oxidation of phenolic compounds, it can be assumed that a decrease in PPO activity would lead to the accumulation of phenolics in A. ceterach sporophytes, thus mitigating oxidative stress in desiccated plants and further strengthening their endogenous antioxidant defense. Metabolic analysis of the rustyback fern sporophyte confirmed that the content of the dominant phenolic compound, chlorogenic acid (CGA), was higher in desiccated plants compared to the control and increased further in the first hours of rehydration.Book of abstracts: International Conference on Natural Sciences and Biotechnology: Kliment’s Days 2025: with satellite scientific conference: Reintroduction of Conservation: Reliant species; 2025 Nov 6-7; Sofia, Bulgaria. Sofia: The Faculty of Biology at Sofia University “St. Kliment Ohridski”; 2025. p. 193
The establishment of an apoptosis-induced compensatory proliferation model: challenges, optimization, and variabilities between different mouse cancer models
No full textBackground: Although apoptosis of cancer cells is typically considered a desirable therapeutic effect, numerous studies have emerged describing the paradoxical relationship between chemotherapy-induced apoptosis and an increased cell proliferation rate in the tumor microenvironment. Notably, apoptosis triggers the activation of protective mechanisms that tend to compensate for cell loss in tumor tissue, unveiling the phenomenon described as compensatory proliferation. To better understand the strategy of cell death-induced proliferation in tumors, we developed a suitable advanced mouse model that appropriately mimics the communication between dying and living cells within the tumor mass. We have also established mouse-derived organoids from isolated tumors and used transcriptional analysis to assess the gene expression similarity and evaluate the potential of the formed organoids as 3D models to replace animal studies in the future.
Material and Methods: The compensatory proliferation model was developed by subcutaneous inoculation of a mixture of live and dead cancer cells of different origins into mice (MC38 or B16 cells into C57BL/6 mice; CT26 cells into BALB/c mice). The dead cells were collected after in vitro treatment with 5-FU (MC38 and CT26 cells) or paclitaxel (B16 cells). The number of live cells and the live:dead cells ratio in the mixture were varied. Both tumors and organoids from MC38 tumor model were further analysed by RNA-seq.
Results: While the optimization of MC38 compensatory proliferation model in C57BL/6 mice was successful, and live:dead cells mixture led to the earlier tumor onset, faster growth, and higher incidence rate compared to the group that received only live cells, CT26 model in BALB/c mice failed due to low incidence rate in all groups. Conversely, the mixture of live and dead B16 cells induced smaller tumors compared to the group that received only live cells, indicating a strong immune response of animals to dead melanoma cells. The transcriptional analysis of isolated tumors and their matching organoids from the MC38 compensatory proliferation model showed over 80% homology in gene expression.
Conclusions: The obtained data revealed that the establishment of a compensatory proliferation model depends on multiple factors, such as the origin, growth rate, invasiveness, and immunogenicity of the cell lines used.Čavić M, editor. Proceedings book: The 1st Regional SDIR-HDIR-MOKAD Congress; 2025 Oct 8-10; Belgrade, Serbia. Belgrade : Serbian Association for Cancer Research; 2025. p. 168. (Oncology Insights; No. 3)