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Development of a Clinically Relevant Temozolomide-Resistant Human Glioblastoma A-172 Cell Line
No full textBackground: Glioblastoma is the most aggressive and deadly form of brain cancer, with a median survival of
approximately 14 months. A major factor contributing to an unfavorable prognosis is the development of resistance
to temozolomide (TMZ), the current first-line chemotherapeutic drug for glioblastoma. Thus, developing reliable
preclinical models of TMZ resistance is essential for evaluating clinically relevant therapeutic strategies.
Material and Methods: To generate an in vitro resistance model that mirrors the cyclical TMZ administration used in
the Stupp regimen (5 days of treatment followed by a 3-week break), we implemented an adapted treatment schedule
compatible with cell culture conditions. The TMZ-sensitive human glioblastoma cell line A-172 was treated with 10 µM
TMZ for two consecutive cycles of 3-day exposure, each followed by a 3-week recovery phase. Sensitivity to TMZ was
evaluated in both parental cells and TMZ-exposed cells via MTT assay. The MTT assay was performed on cells treated
with TMZ once, twice, and three times (i.e., single, double and triple TMZ treatment). To further characterize the TMZexposed cells, we assessed changes in cell migration (wound healing assay), EGFR, CD44, SOX2 and CD133 expression
(immunostaining), and cellular senescence (beta-galactosidase activity assay after exposure to 10 and 50 µM TMZ
during three days).
Results: Relative to the parental cells, the TMZ-exposed population showed progressively higher IC50 values, in
single, double and triple TMZ treatment, suggesting a gradual development of resistance. In the wound healing assay,
resistant cells showed reduced motility, closing the wound at approximately half the rate of the parental line after 72
h. Immunofluorescence assays showed a minor increase in EGFR expression and a slight reduction in CD44 expression,
while SOX2 and CD133 levels remained unchanged. In the beta-galactosidase activity assay after exposure to both 10
and 50 µM TMZ, the resistant cell line showed a reduced proportion of senescent cells relative to the parental line.
Conclusions: The cyclic TMZ regimen adapted for in vitro use successfully induced resistance in A-172 cells, generating a
cell line with reduced migratory capacity, slightly increased EGFR expression, decreased CD44 expression, and reduced
senescence.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. 171. (Oncology Insights; No. 3)
Cell dynamics investigation of the Mucoromycetes fungus, Phycomyces blakesleeanus, via nonlinear laser scanning microscopy
No full textMultiphoton nonlinear laser microscopy (NLM) is a powerful, non-invasive optical technique. Its unique advantages make it a valuable tool for a wide range of applications, from basic research to clinical diagnostics. These advantages include in vivo and label-free imaging, high-contrast images, high-resolution 3D imaging and reduced sample damage and photo-bleaching due to the use of infrared ultrafast pulsed lasers (1). Different NLM modalities provide the structural and functional imaging as well as selective manipulation of cells and intracellular structures. NLM have rarely been used to study filamentous fungi. Here we present a comprehensive application of NLM modalities to investigate the lipid droplets (LDs) and mitochondria dynamics under various environmental conditions. Exposure to 1 mM selenite and temperature shift of 3 degrees outside the optimal range had a clear effect on organelle dynamics, significantly increasing LD number and inducing the appearance of smaller LDs and predominantly tubular mitochondrial morphology. Nitrogen starvation also significantly increased the LD number after 4.5 hours, but induced the appearance of a new morphological type with semicircular tubules. Due to refractive index mismatch, between LDs and cytoplasm, third harmonic generation microscopy enables the visualization and quantified changes of quite small fungal LDs (2). In synergy with two-photon excitation fluorescence, NLM provides fundamental insights into organelle dynamics and physiological adaptation of hyphal metabolism. We also present femtosecond (fs) laser nanosurgery of the cell wall of filamentous fungi, which enables patch-clamp measurements on viable protoplasts released from different regions of hyphae (3). The high precision of fs laser ablation allowed us to cut a small portion of the cell wall without damaging the cell membrane (only a few micrometers away), while the ability to select an area for surgery allowed us to obtain information on the distribution of different ion channels along the hyphae.International Fungal Biology Conference: from Molecules to Communities: IFBC-2025; 2025 Sep 27-30; Chania, Crete, Greece. International Fungal Biology Conference; 2025. p. 46
Structural characterisation of iridoid glycosides in the aerial parts of Nepeta nuda L.
No full textWithin the family Lamiaceae, the genus Nepeta contains approximately 280 species,
with the greatest diversity found in southwestern Asia and the western Himalayan region
[1]. Belonging to the cyclopenta[c]pyran monoterpenoid family, iridoids form a large
and continually increasing group of compounds present in many folk medicinal plants,
where they serve as bitter tonics, fever reducers, sedatives, blood pressure regulators,
and agents for healing wounds and skin-related disorders, due to the wide spectrum of
bioactivity of both aglyconic and glycosidic forms [2]. In this study, iridoid glycoside
was isolated from air-dried aerial parts of Nepeta nuda L. The plant material underwent
maceration in methanol over a period of three days, which was then evaporated. The
obtained extract was suspended in water and extracted with chloroform. The remaining
aqueous fraction was then further extracted with n-butanol, and this butanol extract was
evaporated to yield the extract used for iridoid isolation. The purification procedures
included chromatography using a Sephadex LH-20 column, implementation of dry flash
chromatography, and semi-preparative high-performance liquid chromatography
(HPLC). Isolated compound, identified as one of the predominant iridoid glycosides
beside 1,5,9-epi-deoxyloganic acid, was comprehensively analyzed by detailed NMR
spectroscopic analysis, employing both 1D (1H and 13C) and 2D (COSY, HSQC, HMBC,
and NOESY) experiments. This compound, to the best of our knowledge, has not been
fully investigated so far either in the plant species N. nuda L.or in the genus Nepeta.Book of abstracts: 11th Conference of Young Chemists of Serbia; 2025 Oct 25; Kragujevac; Serbia. Belgrade: Serbian Chemical Society; 2025. p. 103
Sweeteners affect biofilm formation and virulence gene expression in Pseudomonas aeruginosa PAO1
No full textPseudomonas aeruginosa is an opportunistic pathogen able to form biofilms, contributing to its
virulence. With the increasing use of sweeteners in various foods, understanding their influence
on bacterial behavior is critical. This study investigated the virulence of P. aeruginosa PAO1
exposed to sweeteners (erythritol, stevia, fructose, coconut sugar, cane sugar, demerara).
Sweeteners didn’t affect growth rates. Erythritol stimulated biofilm (100 mg/mL, 159.98% formation),
while 10 mg/mL of coconut sugar, cane sugar, and demerara promoted lower levels (�70%
formation). Erythritol stimulated exopolysaccharides production but reduced biofilm eDNA.
Stevia, fructose, and coconut sugar increased the expression of lasI, lasR, rhlI, rhlR, pqsA, mvfR,
and pvdF. HPLC analysis confirmed sucrose as the major sugar in demerara, coconut and cane
sugar. Erythritol stimulated biofilm and some virulence genes expression, while other sweeteners’
effects varied. Cane sugar was a biofilm inhibitor with a limited gene expression effect.
The sweeteners’ impact on microorganisms is diverse and should be further investigated
Perifithic diatom community of the Danube river (Belgrade area) in the summer season
No full textNa reci Dunav u letnjem periodu 2024. godine prikupljeni su uzorci perifitona sa četiri lokaliteta na području Beograda, primenom standardne metodologije. Cilj rada je utvrđivanje sastava zajednice, kao i ekološkog stanja ispitivanog sektora Dunava na osnovu ekoloških gildi i ocenom preliminarnog ekološkog statusa/potencijala prema nacionalnim propisima zasnovanih na EU Direktivi o vodama. Identifikovano je ukupno 76 taksona silikatnih algi i opisane su ekološke gilde, od kojih je pokretna gilda najbrojnija, potom gilda niskih formi i planktonska. Preliminarni ekološki status/potencijal na osnovu dijatomnih indeksa IPS i CEE bio je ,,zadovoljavajuć“ i ,,umeren“ na istraživanim lokalitetima.In the summer period of the year 2024, periphyton samples were collected from four localities in the Belgrade area of the Danube River using standard methodology. The goal of this study was to determine diatom community composition, as well as the ecological state according to ecological guilds and to assess preliminary ecological status/potential by the national regulations established on the EU Water Framework Directive. A total of 76 taxa were identified and ecological guilds were described, of which motile is the most numerous, followed by low profile and planktonic. Preliminarly ecological status/potential at the investigated localities, based on diatom indices IPS and CEE was a "satisfactory" and "moderate".Đukić A, editor. Conference proceedings: 54th Annual Conference of the Serbian Water Pollution Control Society: Water 2025 ; 2025 Jun 16-18; Kopaonik, Serbia. Belgrade: Serbian Water Pollution Control Society; 2025. p. 61-70
Goat’s Skim Milk Enriched with Agrocybe aegerita (V. Brig.) Vizzini Mushroom Extract: Optimization, Physico-Chemical Characterization, and Evaluation of Techno-Functional, Biological and Antimicrobial Properties
No full textThe aim of this study was the optimization of skimmed goat milk ingredient enriched with Agrocybe aegerita (V. Brig.) Vizzini mushroom extract (ME/M), using centrale composite design. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) and Scanning electron microscopy (SEM) were employed for analysing optimized ME/M ingredient. Optimized ME/M ingredient contained glucans derived from mush-room, as well as new polypeptide obtained by selective cleavage of goat milk proteins. Optimized ME/M is a multicompound ingredient that exhibited a polymodal particle size distribution. The 0.1% solution of ME/M exhibited good emulsifying and foaming properties. In addition, ME/M showed a good anti-proliferative effect on tested human cancer cell lines, especially against Caco-2 colorectal and MCF7 breast cancer cell lines. The ME/M ingredient promoted HaCaT cell growth, without cytotoxicity, suggesting their safe use, as well as promising wound closure activity. The addition of ME/M in-gredient showed reduced IL-6 levels compared to Staphilococus aureus alone, indicating a dose-dependent effect on inflammatory response and good anti-inflammatory activ-ity. Optimized ME/M ingredient showed antibacterial, antifungal, anticandidial and antibiofilm (¼ MIC) activity. Finally, this study can be a promising model for formu-lating optimal ingredients that have the best techno-functional and functional proper-ties
Antiferroptotic effects of H2S donors against diabetic injury of β-cells in vivo and in vitro
No full textFerroptosis is a regulated non-apoptotic cell deathcharacterized by the increased lipid peroxidationtriggered by the accumulation of labile iron andreactive oxygen species. The major antiferroptoticsignalling pathway is the GSH/GPX4/xCT axis, which iscontrolled by the transcription factor Nrf2.• The involvement of ferroptosis in diabetes-induced β-cell injury was recently described by our team,suggesting that it represents a potential target fordiabetes prevention and therapy.• Originally considered a toxic gas, H₂S is nowrecognised as a gasotransmitter and with its reactivemetabolites (persulfides, polysulfides and otherreactive sulphur species (RSS)) as important redoxsignalling molecules.• Recently, the antiferroptotic effect of H₂S andrelated RSS has been demonstrated in variouspathologies. The antidiabetic role of H₂S has alsobeen demonstrated, although it remains unclear whetherit also reduces ferroptosis of β-cells.• Here, we aimed to investigate the potential of H₂S/RSSdonors in preventing the development of diabetes,focussing on their antiferroptotic effects.22nd SFRRI Biennial Meeting 2025: The New Era of Redox Biology: from Basic Biochemistry to Redox Omics; 2025 Jun 3-6; Galway, Ireland. Elsevier; 2025. p. 100-1. (Free Radical Biology and Medicine; Vol. 233; Suppl. 1)
Exploring health beneficial effects of poisonous mushroom Paxillus involutus Batsch Fr.
No full textIn the present study, phenolic and flavonoid composition and biological properties of methanolic extract of wild growing Paxillus involutus collected in Serbia have been investigated. Ellagic acid was the most abundant phenolic compound (34.92 µg g-1), followed by 5-O-caffeoylquinic acid (4.51 µg g-1), whereas isoorientin was the most abundant flavonoid (3.42 µg g-1). P. involutus turned out to be a rich source of phenolic compounds (74.67 mg GAE g-1 d.w.), whereas total flavonoid content was significantly lower (4.05 mg QE g-1 d.w.). As for the various investigated biological activities, methanolic extract exerted high level of antioxidant, antimicrobial and antibiofilm activities. The highest antioxidative potential was measured by TAC (350 TE mg g-1 d.w.), whereas evaluation of antimicrobial properties showed selective antimicrobial potential toward tested pathogenic microorganisms, with resistant strain of E. coli being the most susceptible to the activity of the extract (MIC 0.08 mg mL-1, MBC 0.16 mg mL-1). Furthermore, methanolic extract of P. involutus demonstrated genotoxicity, severe hemolysis effects and selective cytotoxicity against colon cancer cells. From the obtained data, it may be concluded that investigated mushroom albeit being toxic for human consumption, may be considered as a source of highly bioactive components with potential application in drug development
Integration of In Vitro and In Silico Results From Chemical and Biological Assays of Rheum turkestanicum and Calendula officinalis Flower Extracts
No full textIn this study, we conducted a thorough analysis of Rheum turkestanicum (RT) and Calendula officinalis flowers (COF) extracts with varying polarities using LC–MS chemical profiling and biological tests (antioxidant, antimicrobial, enzyme inhibition, and cytotoxic effects). The highest level of total phenolic content in the ethanol extract of RT with 75.82 mg GAE/g, followed by the infusions of RT (65.00 mg GAE/g) and COF (40.99 mg GAE/g). A total of 20 bioactive compounds were identified and quantified. The ethanol extract of COF was rich in terms of 5-O-caffeoylquinic acid (2780.56 μg/g), isorhamnetin-O-rutinoside (1653.59 μg/g), and rutin (1356.97 μg/g). However, RF extracts were rich in catechin gallate (21.66–80.01 μg/g) and 5-O-caffeoylquinic acid. Except for metal chelating ability, the ethanol extract of RT exhibited the strongest ability (DPPH: 171.5 mg TE/g; ABTS: 387.35 mg TE/g; CUPRAC: 449.80 mg TE/g; FRAP: 195.60 mg TE/g; and PBD: 1.52 mmol TE/g). In the enzyme inhibition tests, the tested ethanol extracts for both species were more active than the infusion. The highest values for tyrosinase were recorded as 72.47 mg KAE/g (in RT extracts) and 71.74 mg KAE/g (in COF extracts). Furthermore, all extracts underwent assessment for their antibacterial and antifungal properties, targeting both Gram-positive and Gram-negative bacteria, as well as clinical yeast and fungal microorganisms. In silico studies yielded valuable insights into the potential therapeutic applications of the bioactive compounds identified in COF and RT extracts. Stable interactions were observed between key compounds, such as isorhamnetin 3-O-glucoside and 3-O-caffeoylquinic acid, with crucial target proteins (AChE, BChE, and MurE). These compounds formed stable hydrogen bonds with minimal root mean square deviation (RMSD) fluctuations, particularly in the isorhamnetin 3-O-glucoside-Staphylococcus aureus MurE and 3-O-caffeoylquinic acid-MurE of S. aureus complexes. These findings further underscore the potential of these compounds as promising candidates for therapeutic development
TFT fertility in different nuclear background
No full textData sets for fertility of female and male seed beetles (Acanthoscelides obtectus) harbouring one of the three naturally occurring mitochondrial haplotypes (MG1a, MG1d and MG3b), expressed in three nuclear backgrounds originating from different natural populations in Serbia (Bela Palanka (BP - southern Serbia), Srpska Crnja (SC - northern Serbia) and Ravnje (Ra - western Serbia))