Optimizacija biosinteze iridoida kod vrsta roda Nepeta primenom metaboličkog inženjeringa

Rod 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-

Polystyrene-nickel interactions in soil: Implications for metal mobility, plant uptake, and human health

The 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

Strain-Dependent Differences in Inflammatory/Immune Activity in Cutaneous Wound Tissue Repair in Rats: The Significance of Body Mass/Proneness to Obesity

Inflammatory/immune cells and mediators are substantial for wound healing because they orchestrate biological activities in this complex process. Among factors that affect wound healing, obesity, and metabolic diseases are among the most significant, particularly because of a relationship between obesity and a prediabetic state with immune reactivity. Using Dark Agouti (DA) and Albino Oxford (AO) rats, which differ in immune responses as well as in proneness to obesity, we examined the impact of these intrinsic factors on cutaneous wound healing. Dynamics of the process were monitored at days 3, 5, and 7 post-wounding parallel in both rat strains by analysis of selected basic aspects of the wound repair process (cytokine and growth factor responses) in granulation tissue. Strain-related differences in the extent of reduction of the wound area were shown, which coincided with differential proinflammatory and immune-regulatory cytokines, as well as growth factors response in these rats. Some of these differences seem related to their dissimilarities in the proneness to obesity. Results in this study extended so far known differences in inflammatory/immune responses to a variety of stimuli between AO and DA rats and showed, for the first time, immune-based differences in wound healing between rats that differ in body mass (BM) and obesity proneness (under ad libitum feeding conditions with normal rodent chow)

Via Air or Rhizosphere: The Phytotoxicity of Nepeta Essential Oils and Malus Dihydrochalcones

Many specialized metabolites found in plants have significant potential for developing environmentally friendly weed management solutions. This review focuses on the phytotoxic effects of volatile terpenes and phenolic compounds, particularly nepetalactone, an iridoid monoterpenoid from Nepeta species, and phloretin, a dihydrochalcone predominantly found in the genus Malus. We highlight current findings on their herbicidal effects, including morphological, physiological, and biochemical responses in target plants. These results underscore their potential for developing sustainable herbicides that could control weeds with minimal environmental impact. We also discuss their soil persistence and methods to enhance their solubility, chemical stability, and bioavailability. Additionally, the possible effects on non-target organisms, such as pollinators, non-pollinating insects, and soil microbiota, are considered. However, further research and a deeper understanding of their long-term ecological impact, along with a resistance development risk assessment, is essential for the potential development of bioherbicides that could be applied in sustainable weed management practices

Alterations in non-REM sleep and EEG spectra precede REM-sleep deficits in a model of synucleinopathy

Background Sleep disturbances often precede motor symptoms in neurodegenerative diseases like Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Neuroinflammation is implicated in PD pathophysiology and may contribute to non-motor symptoms such as sleep disturbances. The Thy1-αSyn mouse model, overexpressing human alpha-synuclein (αSyn), mimics key aspects of PD and DLB, making it valuable for studying related sleep disturbances and neuroinflammatory changes. Objective To investigate early-stage alterations in sleep architecture, electroencephalographic (EEG) patterns, and neuroinflammation in Thy1-αSyn mice. Methods We used telemetric EEG/electromyography (EMG) with video surveillance to compare sleep patterns and EEG spectral power between 2.5- and 4.5-month-old male Thy1-αSyn transgenic mice and wild-type littermates. Neuroinflammation was assessed by examining microglial (Iba1) and astrocytic (GFAP) activation in key sleep-regulating brain regions. Results Thy1-αSyn mice showed decreased resting wake time and increased non-REM sleep, with altered sleep bout frequency and length, indicating significant sleep architecture changes. Spectral analysis revealed a shift from higher to lower frequency bands, suggesting early neural circuitry disruptions due to αSyn overexpression. Significant microglial activation was observed at 3 months, with astrogliosis progressing by 5 months in key sleep-regulating regions, indicating that neuroinflammation may contribute to the observed sleep disturbances. Conclusions Early-stage Thy1-αSyn mice exhibit significant sleep architecture changes, EEG spectral shifts, and neuroinflammatory alterations. These findings suggest that neuroinflammation may play a role in the initial pathophysiological changes in PD and related synucleinopathies. Sleep, EEG, and neuroinflammatory changes could serve as early biomarkers for these diseases

Associating Patient Responses with Drug Sensitivity in Non-Small Cell Lung Carcinoma Using an Immunoassay on Patient-Derived Cell Cultures

Abstract Background/Objectives: Non-small cell lung carcinoma (NSCLC) is characterized by its diverse molecular profiles and varying responses to treatment, highlighting the importance of precision medicine in optimizing therapeutic outcomes. A promising approach involves using patient-derived cellular models, which provide insights into the unique biology of individual tumors and their responsiveness to treatment. Methods: We established short-term primary cell cultures from thirteen patients with NSCLC of different subtypes and stages, including both cancer and stromal cells. To evaluate the ex vivo cytotoxicity and selectivity of eight chemotherapeutics and erlotinib, we employed an immunoassay, and the results were analyzed using an automated imaging system. Scoring of the obtained results was also performed. The ex vivo responses to cisplatin, etoposide, and paclitaxel were correlated with the patients’ responses to therapy. We used Kaplan–Meier analysis to assess progression-free survival (PFS) differences among patient groups. Results: NSCLC cells exhibited significant variability in their responses to drugs, with stromal cells demonstrating greater sensitivity. Tumors at stages I-III responded to multiple treatments, whereas stage IV cells showed considerable resistance. Erlotinib effectively reduced cancer cell growth at lower doses but plateaued at higher concentrations. The immunoassay indicated 67% sensitivity and 100% specificity in predicting patient responses to chemotherapy. Sensitivity to etoposide and paclitaxel correlated with progression-free survival (PFS). Conclusions: A personalized treatment strategy, such as our immunoassay based on the ex vivo responses of cancer patients’ cells, can guide treatment decisions and, in some cases, serve as surrogate biomarkers for tumor types that lack actionable biomarkers

Metal bioaccumulation in muscle tissue of the northern pike Esox lucius Linnaeus, 1758 from Tisza River

Fish inhabiting contaminated aquatic environment tend to accumulate essential, but also toxic metals in their tissues. The extent of metal accumulation is influenced by several factors, including metal concentration in the environment, duration and frequency of exposure, mechanisms of metal uptake and intrinsic factors such as fish age, sex, or feeding habits. Various metals, depending on their type and role, have a different affinity to fish tissues. Fish muscles usually contain the lowest amounts of metals compared to other tissues, but are often used in studies because they are a part of the fish body used in the human nutrition [1, 2]. The northern pike is the top piscivore and a phytophilic species in a trophic pyramid of freshwater ecosystems that is highly dependent on backwaters and vegetated riverine areas for spawning and recruitment. In the autumn season 2024, 11 samples of northern pike were collected from the Tisza River using electrofishing (HONDA 1.2кW, 6 А). The content of metals (Ag, Al, As, Cd, Cr, Co, Cu, Fe, Li, Mn, Ni, Pb, Sr, Zn) was determined in muscle samples of fish of different ages using inductively coupled plasma optical spectrometry (ICP-OES). The age determination indicated that the sampled fish ranged from age classes 1+ to 5+. This study aimed to evaluate the potential human health risk associated with the consumption of fish muscle by calculating the estimated daily intake (EDI) of metals, following the methodology described by Javed and Usmani [3]. The risk assessment of metal exposure determined by parameters such as the EDI, involves not only the amount of contaminant ingested, but also factors such as the frequency and duration of exposure, the average body weight of the fish, and the oral reference dose. Notably, nickel (Ni) and cobalt (Co) were below detection limits in the analyzed fish muscle samples. The concentrations of other detected metals were below the maximum concentrations set by the EU [4] and the Republic of Serbia [5]. The observed values for EDI were significantly lower than the corresponding reference doses, therefore all confirming safe human consumption.Zarić N, Aničić Urošević M, editors. Book of Abstracts: The 3rd Conference of the International Association for Biomonitoring of Environmental Pollution: IABEP2025; 2025 Oct 13-15; Belgrade, Serbia. Belgrade: Institute of Physics; 2025. p. 109

The Influence of Adolescent Dietary Restriction on Memory Performance at 18 and 24 Months in Aged Wistar Rats

Dietary restriction (DR), defined as a 20–40% reduction in food intake without malnutrition, impacts the organism depending on the age of onset and duration. The developmental window from postnatal day (PND) 28 to PND 60 in rats— spanning early through late adolescence—is a critical and highly sensitive period for dietary interventions, as it coincides with ongoing maturation of prefrontal, hippocampal and limbic systems that underlie adult social, emotional, and cognitive behaviors. This study investigated the effects of 30% DR applied to female Wistar rats during different adolescent phases: early (EADR, PND 28–35), middle (MADR, PND 35–42), and early plus middle adolescence (EMADR, PND 28– 42), compared to ad libitum (AL) controls. Animals were tested at 18 and 24 months using the novel object recognition (NOR) task for recognition memory and the Y maze spontaneous alternation test for spatial working memory. The NOR test results showed that at 18 months old animals short-term memory was preserved in the MADR and EMADR groups, while long-term memory was preserved in the EADR and MADR groups. On the other hand, Y maze results showed that EMADR preserved spatial working memory at both 18 and 24 months of age, as these females spent more time in a new arm of Y maze, compared to AL females of the same age. The effects of adolescent dietary restriction on memory preservation at 18 and 24 months were assessed, demonstrating that early-life caloric limitation preserves recognition and spatial memory in aged rats.Dragić M, editor. Book of abstracts: 9th Congress of Serbian Neuroscience Society; 2025 Oct 6-8; Belgrade, Serbia. Belgrade: Serbian Neuroscience Society; 2025

Chemical profile and enzyme-inhibitory activity from different parts of blue poppy (Papaver somniferum)

Due to the significant side effects associated with synthetic drugs, there has been a growing interest in natural agents in recent years. In an effort to prevent numerous diseases, plant-based natural therapeutics are receiving increasing scientific attention. Poppy (Papaver somniferum) is one of the oldest plants used in traditional and alternative medicine, primarily due to its rich chemical composition. In this study, different parts of the poppy were examined, namely: stems, leaves, root, capsule, pressedcake (PC) and defatted pressed-cake (DPC). The main focus of this research was to determine the chemical profile and potential enzyme-inhibitory activity of extracts obtained from different parts of poppy. Chemical composition of obtained extracts was determined by liquid chromatography/mass spectroscopy (LC-MS). Enzyme inhibitory effect was investigated against α-amylase, α-glucosidase, tyrosinase and acetylcholinesterase. To obtain extracts from different parts of poppy, conventional extraction (CE) was used, where different percentages of ethanol (0, 20, 40, 60, 80, and 96% w/v) were used. The extraction lasted for 24 hours, at room temperature, and the shaking speed was 180 rpm. One portion of PC was defatted using Soxhlet extraction with methylene-chloride as a solvent, and then extracted by CE. According to LC-MS analysis, 62 compounds were identified in different parts of the poppy. Some of the compounds which are present in all parts of the plant were: glutaric acid, malic acid, p-coumaric acid, narcein. As a result, for α-amylase and α-glucosidase, the extracts from PC and leaves showed the greatest inhibitory power on these enzymes. Tyrosinase was most effectively inhibited by the extract from the DPC, while the capsule exhibited the best activity in the case of acetylcholinesterase. It can be concluded that poppy represents a plant rich in bioactive compounds, and as such can be used as an alternative to synthetic drugs.Popović-Đorđević J, Kostić A, editors. Book of Abstracts: 3rd European Symposium on Phytochemicals in Medicine and Food (3-EuSPMF); 2025 Jul 1-4; Belgrade, Serbia. Belgrade: University of Belgrade, Faculty of Agriculture; 2025. p. 92

Biocontrol Potential of Native Trichoderma Strains Toward Soil-Borne Phytopathogenic and Saprotrophic Fungi

The potential of Trichoderma fungi as biocontrol agents has not yet been fully explored, as there is a large repertoire of inter- and intra-species variation in their phytopathogenic antagonistic effects due to different adaptations of individual Trichoderma strains. In the present study, we investigated the biocontrol efficacy of eight native isolates of Trichoderma spp. against the soilborne phytopathogens Sclerotinia sclerotiorum and Rhizoctonia solani and a representative of the Mucoromycota, Phycomyces blakesleeanus. An in vitro dual culture test showed a complete (100%) inhibition of S. sclerotiorum and P. blakesleeanus by each tested Trichoderma strain and a high (80–100%) inhibition of R. solani. The crude chloroform extracts, whose peptide contents were confirmed by thin-layer chromatography, caused a concentration-dependent reduction in the growth of the target fungi, with inhibition comparable to the effect of the peptaibol standard alamethicin. Despite the differences between fungi from the phyla Basidiomycota, Ascomycota, and Mucoromycota, their inhibition by alamethicin followed the same dose–response dependence. The growth inhibition of P. blakesleeanus induced by Trichoderma extracts was characterized by a significantly increased activity of antioxidative defense enzymes. Both variants of biocontrol agents, the native strains of Trichoderma spp. and their extracts, are efficient in controlling fungal growth and should be considered for the development of new potent bioformulations applicable in agriculture