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Optimizacija biosinteze iridoida kod vrsta roda Nepeta primenom metaboličkog inženjeringa
No full textRod Nepeta obuhvata oko 300 vrsta, poznatih po svojim aromatičnim i lekovitim svojstvima,
od kojih se mnoga mogu pripisati iridoidnim jedinjenjima. Iridoidi predstavljaju biciklične
monoterpene koji se kod vrsta roda Nepeta mogu naći kao aglikoni (IA) i u formi glikozida
(IG), i predstavljaju produkt jedinstvenog i evolutivno-specifičnog iridoidnog biosintetskog
puta. Na osnovu grupe iridoidnih jedinjenja koje produkuju, vrste roda Nepeta možemo
grupisati u tri hemotipa – hemotip čiji predstavnici produkuju IA i IG (Hemotip A), hemotip
čiji predstavnici produkuju samo IG (Hemotip B) i hemotip čiji predstavnici ne produkuju
iridoide (Hemotip C). Glavnu prepreku ka njihovoj široj komercijalnoj upotrebi predstavlja
ograničena proizvodnja ovih jedinjenja in planta, te je osnovni cilj naših istraživanja
identifikacija ključnih gena iridoidnog biosintetskog puta kod odabranih vrsta roda Nepeta, i
modulacija njihove aktivnosti primenom biotehnoloških postupaka, a u cilju optimizacije
iridoidne produkcije. Tako je pretragom transkriptoma N. grandiflora, vrste koja pripada
hemotipu B, identifikovano odsustvo transkripata NEPS1 i NEPS5, ključnih za oksidaciju
nepetalaktola, čime se može objasniti odsustvo IA kod ove vrste. Tranzijentna ekspresija
NEPS1 poreklom iz vrste N. rtanjensis (hemotip A), u listovima N. grandiflora dovela je do
produkcije IA – cis,trans-nepetalaktona, što je potvrđeno UHPLC/DAD/(±)HESI-MS2 i
GC/MS analizom. U slučaju vrste N. nuda, predstavnika hemotipa A, tranzijentno povećanje
ekspresije gena za rani enzim iridoidnog biosintetskog puta geraniol-sintazu (GES) dovela je
do porasta, dok je njegovo utišavanje dovelo do sniženja produkcije analiziranih iridoidnih
jedinjenja, što je potvrđeno UHPLC/DAD/(±)HESI-MS2 analizom. Rezultati istraživanja
ukazuju na potencijal ispitivanih gena kao vrednih biotehnoloških alata za optimizaciju
iridoidne produkcije kod vrsta roda Nepeta.The genus Nepeta comprises of about 300 species known for their aromatic and medicinal
properties which can be attributed to iridoid compounds. Iridoids are bicyclic monoterpenes
that can be present as aglycones (IAs) and in the form of glycosides (IGs), representing the
products of a evolutionarily specific iridoid biosynthetic pathway. Based on the iridoids they
produce, Nepeta species can be classified into three chemotypes: chemotype A, producing both
IAs and IGs; chemotype B, containing only IGs; and chemotype C, lacking iridoids. The main
obstacle for their commercial application is limited iridoid production in planta. The aim of
this research was to identify key limiting genes of the iridoid biosynthetic pathway in selected
Nepeta species, as well as to modulate their activity towards optimizing iridoid production.
Transcriptome analysis of N. grandiflora, a species belonging to chemotype B, revealed the
absence of NEPS1 and NEPS5 transcripts, which are crucial for nepetalactol oxidation.
Transient expression of NEPS1 from N. rtanjensis (chemotype A) through agroinfiltration of
N. grandiflora leaves resulted in the production of the IA – cis,trans-nepetalactone, as
confirmed by UHPLC/DAD/(±)HESI-MS² and GC/MS analysis. In the case of N. nuda, a
representative of the chemotype A, transient overexpression of the gene encoding the early
iridoid pathway enzyme geraniol synthase (GES) led to an increase, while its silencing led to
a decrease in the production of the analyzed iridoid compounds, which was confirmed by
UHPLC/DAD/(±)HESI-MS² analysis. Obtained results are marking the investigated genes as
valuable biotechnological tools for optimizing iridoid production in Nepeta species.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. 81-
Multi-biomarker assessment of metal-oxide nanoparticle toxicity in Chironomus riparius
No full textThe increasing production and utilization of metal-oxide nanoparticles (NPs) such as titanium dioxide (TiO2), cerium dioxide (CeO2), and magnetite (Fe3O4) have raised concerns regarding their environmental impact. This study aimed to assess the sub-organismal biomarkers of Chironomus riparius larvae exposed to these NPs, focusing on target organs, potential DNA damage, oxidative stress parameters, and hemoglobin levels. Chronic exposure experiments were conducted using OECD guidelines, with histological analyses revealing shortened microvilli in the midgut of larvae treated with CeO2 and Fe3O4 NPs. DNA damage, as measured by the comet assay, was significantly higher in larvae treated with CeO2 and Fe3O4 NPs. Oxidative stress parameters, including thiobarbituric acid reactive substances (TBARS) and advanced oxidation protein products (AOPP), were elevated in groups treated with CeO2 and Fe3O4 NPs, with significantly higher activity of catalase (CAT) was also noticed in the latter group. Group treated with TiO2 NPs had increased activity of superoxide dismutase (SOD). Hemoglobin levels were significantly affected, with increased concentrations in TiO2 and Fe3O4 treated groups, and decreased levels in the CeO2 treated group. The results of the study support the multi-biomarker approach as highly beneficial in understanding the mechanisms of NPs toxicity in aquatic ecotoxicology by detecting histopathological biomarkers along with sub-organismal biomarkers such as DNA damage, oxidative stress parameters, and hemoglobin concentration
Food-derived bioactive pigment phycocyanobilin binds to SARS-CoV-2 spike protein both covalently and noncovalently affecting its conformation and functionality
No full textPhycocyanobilin (PCB), tetrapyrrole chromophore of Spirulina phycocyanin, is bilirubin analog and weak thiolmodifying agent. SARS-CoV-2 spike protein (SP) has bilirubin binding pocket, lacks free sulfhydryl, but it has two pairs of functionally important semi-stable disulfides reactive towards thiol-modifying agents. We investigated covalent and noncovalent binding of PCB to SP and its receptor-binding domain (RBD) and impact of covalent PCB conjugation to RBD on structure and binding to human angiotensin-converting enzyme 2 (ACE-2). PCB shows high-affinity for SP (Ka = 2.1 x 107 M-1), moderate-affinity for RBD (Ka = 8.4 x 104 M-1) and binds covalently to SP and RBD in reaction involving thiols. PCB binding alters RBD conformation. Molecular docking identified two binding sites of PCB to SP, bilirubin/biliverdin binding site and hydrophobic pocket of RBD in vicinity of Cys432, preferential target for covalent binding in in silico covalent docking of PCB to RBD. Redox proteomics mapped reactive Cys432, Cys391 and Cys525 in RBD. PCB-modified RBD exhibited reduced ability to bind to ACE-2. This is the first study demonstrating PCB reactivity towards semi-stable disulfides of proteins lacking free sulfhydryl groups. PCB may affect functionality and structure of SP and its RBD by noncovalent and covalent binding
Comparative Study of Natamycin Encapsulation in Liposomes: Thin-Film vs. Proliposome Methods for Enhanced Stability, Controlled Release, and Efficacy Against Milk Spoilage and Pathogenic Microorganisms
No full textThe aim of this study was to evaluate liposomal particles as a potential delivery system for
natamycin, a widely known antimicrobial agent used in the food industry. The goal was to
prolong its diffusion into the surrounding medium. Natamycin-loaded liposomes were
prepared using two methods (proliposome and thin-film) and two different phospholipid
mixtures. The characterization of natamycin-loaded liposomes was performed in terms of
their chemical composition (FT-IR analysis), encapsulation efficiency (EE), and antimicrobial
potential against spoilage and pathogenic microorganisms that can be found in milk and
milk products. During the 60-day storage period, their size, polydispersity index (PDI), and
zeta potential were measured. The in vitro release kinetics of natamycin from liposomes
were also assessed, and the results showed a significantly lower release rate of the drug
when it was encapsulated. EE showed a high level of natamycin encapsulation (>80%),
which was confirmed with FT-IR analysis. The stability study indicated that these systems
were stable over a 60-day storage period, as the zeta potential of all formulations was
~−25 mV. Satisfactory antimicrobial performance of the developed liposomes against
Listeria monocytogenes, Yersinia enterocolitica, Candida tropicalis, Candida parapsilosis, and
Aspergillus flavus (MIC values from 0.00625 to 4 mg/mL) indicates that loading of natamycin
into liposomal carriers was an adequate method for their encapsulation and delivery in the
milk industry
Assessment of Hepatic Enzyme Biomarkers in Northern Pike (Esox lucius) from Lotic and Lentic Freshwater Habitats: Implications for Monitoring Metal Pollution and Ecological Stress in Aquatic Ecosystems
No full textRapid urbanization and increased anthropogenic activities have led to the release of an increasing number of pollutants, including metals, into freshwater ecosystems, posing a significant threat to aquatic life. In this study, the bioaccumulation of metals and hepatic enzyme activities in northern pike (Esox lucius) from two contrasting freshwater ecosystems in Serbia, the lotic Tisza River and the lentic Bela Crkva Reservoirs, were investigated. A total of 22 specimens (11 per site) were sampled in autumn 2024. The liver tissue was analyzed for the concentrations of 11 metals (As, Cd, Cr, Co, Cu, Pb, Li, Fe, Zn, Mn, Ni) and the activities of the liver enzymes (ALT, AST, AP, GGT). The results showed ecosystem-specific patterns of metal accumulation, with the northern pike in the rivers showing significantly higher Cu and Fe levels, while the fish in the reservoirs showed increased Zn concentrations. Enzymatic biomarkers showed different responses between ecosystems, with river fish showing increased ALT and AST activities, indicating hepatocellular stress, while reservoir fish showed increased GGT, indicating enhanced detoxification processes. The body condition factor was negatively correlated with liver Mn and Zn concentrations, emphasizing its utility as an integrative bioindicator of metal-induced stress. No significant sex-specific differences in metal or enzyme levels were found. These results suggest the suitability of northern pike as a sentinel species for environmental monitoring in freshwaters and highlight the different physiological adaptations to local metal stress in lentic and lotic habitats
Biophysical profile of the inward inactivating anion current recorded in the native membrane of the sporangiophore and mycelium of Phycomyces blakesleeanus
No full textFunctional characterization of novel ion channels in the native membrane of filamentous fungi is a challenging field of research, mostly due to inaccessibility of the membrane for patch clamp recordings. Cytoplasmic droplets (CD) of Phycomyces blakesleeanus sporangiophores are an alternative model system for patch clamp measurements (1), that has yielded characterization of several ion currents so far (1,2). With ambition to exceed our research beyond the CD membrane, we have recently developed an advanced protocol for precise laser microsurgery of the single hyphae of P. blakesleeanus mycelium (3). A distinct difference was noticed in the profile of dominant currents from sporangiophore and mycelium cell membrane. Here we present the preliminary biophysical profile of the inward inactivated current from both cell surgery-derived protoplasts and CD, so far shown to have a role in export of chloride and nitrate from the cell. It is active in hyperpolarizing range of membrane potential and has a voltage-dependent inactivating pattern starting from -110 mV, with the time constant of inactivation decreasing with the increase of hyperpolarization. It was present in every recording of the cell- surgery derived protoplast membrane. In CD it was most often recorded in large outside-out patches with 1 mM of Ca2+ present in the recording pipette, but was seldom dominant in the recordings of the entire droplet membrane. Interestingly, this current profile is relatively similar to the recorded activity of bestrophin homologue of Aspergillus nidulans that was investigated after heterologous expression in yeast (4), while P. blakesleeanus genome holds sequences of bestrophin-like channel domains (5). The further interplay between genetical approach and direct recording of ion channels in their natural surrounding would significantly advance the field of filamentous fungi ion channel studies.
1. Zivić et al. (2005). Ann. N. Y. Acad. Sci 1048 (June): 491–95 2. Stevanović et al. (2023) J Fungi (Basel) May 31;9(6):637. 3. Pajić, et al Microsyst Nanoeng (2024) 10, 47. 4. Roberts et al (2011). FG & B 48 (9): 928–38. 5. UniProt Consortium. (2019) 47(D1), D506–D515. [Accessed entry: A0A167R601]International Fungal Biology Conference: from Molecules to Communities: IFBC-2025; 2025 Sep 27-30; Chania, Crete, Greece. International Fungal Biology Conference; 2025. p. 90
Nonlinear Imaging and Laser Nano Surgery of Hyphae to Enable Electrophysiological Measurements
No full textElectrophysiology studies of ion channels, in live filamentous fungi by patch clamp method are not possible due to presence of rigid chitinous cell wall that prevents patch clamp pipette to access the plasma membrane. We present laser nano-surgery of the fungal cell wall that enables patch clamp electrophysiology studies. Similar approaches as one-time reports utilizing nanosecond laser pulses long time ago were not pursued further [1, 2]. Here, we demonstrate reproducible method using femtosecond lasers accompanied by two-photon excitation fluorescence (TPEF) imaging of hyphae [3]. A wild-type strain of filamentous fungus Phycomyces blakesleeanus (Burgeff) [NRRL 1555(-)] were grown on glass coverslips with hand-etched grid, coated with a thin layer of 50% collagen type I as an immobilizer. Home built nonlinear laser scanning microscope [4] utilizing Ti:Sa tunable fs laser was used for TPEF imaging of hyphae and the cell surgery. The latter is enabled with the custom made add-on in software. The dwell time (1s) and the laser power (4-15mW) were set with fixed repetition rate (76MHz), pulse duration (160fs) and laser wavelength (730nm). Coverslip with hyphae is transferred to another microscope setup for patch clamp, consisting of micromanipulators and precise electronics for pA current measurements. Obtained current recordings provide valuable information on fungal cell membrane ionic channelsBook of Abstracts: 1st Carpathian Biophotonics Meeting (CBM 2025); 2025 Sep 8-12; Sinaia; Romania. Carpathian Biophotonics Meeting; 2025. p. 43
Incorporating Alkylthio Groups Provides Potent In Vitro Anticancer Activity to Water Soluble and Aqueous Stable Diiron(I) Bis-Cyclopentadienyl Complexes
No full textWe synthesized five novel diiron(I) complexes with a varying S-functionalized vinyliminium ligand, 3a-e, which were structurally characterized by IR and NMR spectroscopy, and single crystal X-ray diffraction in one representative case. Based on NMR and UV-Vis methods, these complexes show adequate water solubility (3 - 19 mM), balanced amphiphilicity (Log Pow values ranging from −0.46 to 0.16), and substantial stability in physiological-like solutions (> 90% unchanged after 24 h in DMEM cell culture medium). The cytotoxicity of the N-cyclohexyl complexes 3a-d was determined on human (A2780, A375, MCF-7) and murine cancer (B16F10, 4T1) cell lines. The leading compound 3d, featuring methylthio-group, N-cyclohexyl and 4-methoxyphenyl substituents, exhibited IC50 values in the low micromolar range and demonstrated remarkable selectivity for malignant phenotypes. Several in-depth experiments were conducted to elucidate the biochemistry of 3d, including evaluation of cell death induction, proliferation rate and cellular redox status. Spectroelectrochemical and binding studies with DNA and bovine serum albumin (BSA) were also conducted. Despite its similarity to cisplatin in inducing apoptosis, the redox signature of 3d is distinct, characterized by a strong scavenging potential of reactive oxygen and nitrogen species (ROS and RNS)
Structural characterization of iridoid glycosides in leaves of Nepeta nuda l.
No full textPlant species of the Nepeta genus are distributed across Central and
Southern Europe, the Middle East, Africa, and Central and Southern Asia.
Nepeta spp. are a rich source of iridoids, a class of monoterpenes known
for their pronounced pharmacological and biopesticidal properties and
behavioral effects on cats [1]. Iridoids from Nepeta spp. have unique stereochemistry,
and some of these compounds are exclusively present in this
group of plants, with nepetalactone being the most recognized. Iridoid
glycosides of Nepeta spp., in comparison to the aglycone forms, remain underexplored
and underutilized, which, along with their biological potential,
highlights the need for further research. Nepeta iridoid glycosides are acetal
derivatives of iridodial, which likely arises from nepetalactol, a common
precursor of all iridoids in these plants [2]. In this study, isolation of iridoid
glycosides was conducted using air-dried aerial plant parts. The extraction
process was conducted through maceration using methanol over a period
of three days. Subsequently, the methanolic extract was suspended in
aqueous solution and subjected to extraction with chloroform. The remaining
aqueous layer underwent a further extraction with n-butanol, which
was then evaporated and used for the isolation of iridoids. Purification
was performed on a Sephadex LH-20 column and semi-preparative HPLC.
Structural characterization of the major iridoid glycosides was done using
1D (1H and 13C) and 2D (COSY, NOESY, HSQC, and HMBC) NMR experiments. This was accompanied by UHPLC/Orbitrap MS profiling of extracts, fractions,
and pure compounds.Book of abstracts: The 6th International conference on natural product utilization: from plants to pharmacy shelf; 2025 May 27-30; Bansko, Bulgaria. 2025; p. 221-2