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Prostate cancer is one of the most common types of cancer and is still difficult to cure with currently available antitumor agents. New compounds showing more efficient antitumor properties are being intensively developed. One of these compounds are imidazolium oximes, since these heterocyclic aromatic structures have numerous pharmacological properties, achieved through various modes of action and interactions with many cellular targets. In view of the therapeutic importance of oximes in suppressing tumor growth, the present study focused on newly synthesized imidazolium oximes ; hydroxyimino-methyl imidazolium bromides (compounds IV, VI, VII and X) in PC-3 prostate cancer cells. Cells were exposed to oximes in a concentration range of 6.25–800 μM for 1 h, 4 h, and 24 h. The effect on cell viability was investigated by monitoring mitochondrial succinate dehydrogenase activity in metabolically active cells by MTS assay. It was then examined whether the tested oximes impaired cell membrane integrity by measuring the activity of the enzyme lactate dehydrogenase (LDH). Finally, flow cytometry was used to elucidate if the tested oximes could induce programmed cell death, apoptosis. The results indicate that tested hydroxyimino-methyl imidazolium bromides caused a significant cytotoxic effect on PC-3 cells, in a time- and dose-dependent manner. Compounds VII and X, having in their structure aromatic side branch, exhibited the highest inhibitory effect on cell viability and induced a significant release of LDH into the medium, which points to the induction of necrosis. Conversely, none of the compounds induced apoptosis, which implies the need for further modification of these oximes in order for them to be suitable in future studies as potential antitumor drugs.

Mechanisms of aging, and cancer as one of main associated pathology are only on verge of understanding. Even in healthy aging gradually loss of function in excretion organs kidneys and liver appears. Previously, we did not find expected elevated tissue concentrations of reactive oxidative species (ROS), that are assumed to be present in old humans and experimental animals and that they can be reversed by antioxidants (AO) treatment. The new hypothesis was that the expression of ROS inducible, potent antioxidant and metal scavenging proteins, metallothioneins Mt1 and Mt2 (MTs), among others, increases in old age to maintain ROS levels and that they can be further modulated with AO. To that aim we studied male and female Wistar rats regularly aged together with these treated with melatonin and resveratrol. Starting from 3 months of age, for the next 9 or 21 months treated rats were drinking AO in water (~1 mg/kg b.w./day), whereas the control animals were drinking water or AO vehicle (0.01% ethanol). Trace elements (TE) were analysed by ICP-MS and the expression of MTs mRNA and proteins in kidneys and liver were determinate by end-point RT-PCR and immunochemical methods, respectively. AO did not change TE accumulation and expression of MTs in both sexes, but known iron accumulation in aged animals was followed by copper and toxic metal cadmium with known female domination. After 1 year, aging increased MTs expression in both sexes that did only slightly further increased after 2 years. We conclude that Fenton reactive metals accumulated in aged animals may influence MTs mRNA and protein expression to help retain physiological ROS level in old rats. (Croatian Science Foundation project IP-2013-11-1481).

EDAM is a well-established ontology of operations, topics, types of data, and data formats that are used in bioinformatics and its neighbouring fields [1, 2] . EDAM-bioimaging is an extension of EDAM dedicated to bioimage analysis, bioimage informatics, and bioimaging. It is being developed in collaboration between the ELIXIR research infrastructure and the NEUBIAS and COMULIS COST Actions, in close contact with the Euro-BioImaging research infrastructure and the Global BioImaging network.

Povodom Sveučilišnog tjedna karijera, 14.05.2019., Osijek, održano je prevadavanje o Genetičkoj i molekularnoj toksikologiji na Institutu za medicinska istraživanja i medicinu rada te aktualnom projektu kojim se istražuju protuotrovi i potencijalni lijekovi na staničnoj razini.

Introduction: The ferritin complex consists of two protein chains structured in a 24 subunit nanocage for iron storage. Very early research on liver in experimental animals found sex differences in favor of females in the amount of iron, which was later associated with sex differences in the ferritin amount in the liver as an iron storage organ. Materials and Methods: We used specific antibodies for two ferritin subunits followed by immunochemical methods for sex and age differences in the expression of light and heavy subunits of ferritin in the liver of three-month and two-year old Wistar rats together with gonadectomized three-month animals for steroid hormone influence determination. Distribution of ferritin subunits inside the organ was obtained by immunohistochemistry, while differences in the expression of individual subunits were determined by western analysis. Results: The results of immunochemical analyses confirmed sex differences in both ferritin subunits and these remained with age, while the amount of ferritin in liver increased with age. Gonadectomy showed a positive effect of estrogen on the expression of both chains and a negative of testosterone. Both ferritin subunits displayed an overlapping distribution in liver cells, which was most pronounced in hepatocytes around the central veins. Conclusion: We can conclude that both ferritin subunits were expressed in the liver and showed sex differences influenced by steroid hormones and age differences which may be correlated with an enlarged amount of stored iron and thus with a great threat of increased oxidative stress crucial to the pathophysiological mechanisms of the aging process.

Introduction: Aging-related impaired body structure and functions are assumed to be, at least partially, caused by elevated oxidative stress (OS). A red wine polyphenol resveratrol may act as an antioxidant and anti-aging medium, but its actions in vivo are controversial (1). Here we report on sex- related effects of resveratrol treatment (RT) on blood/plasma parameters of DNA damage, oxidative status, and concentrations of sex hormones (SH) in a rat model of aging (2). Material & Methods: Starting from their age of 3-mo, for the next 9-mo or 21-mo male and female Wistar rats were drinking resveratrol in water (10 mg/L ; predicted intake: ~1 mg/kg b.m./day) or only water (controls). In their blood/plasma we performed the alkaline comet assay (3) and determined glutathione (4) and malondialdehyde (5) by the established methods. SH were determined by commercial kits. Results: Compared with controls, the 9-mo RT did not change any parameter in both sexes. After the 21-mo RT, the OS-related parameters remained unchanged, but testosterone levels in males were ⁓45% higher (P

A better selection of a lead candidate for preclinical drug development has become an unconditional requirement in recent years. For that reason, early, rapid and robust results that unambiguously rank compounds according to their desirable and undesirable effects placed cell-based in vitro toxicology in major focus, overcoming the criticised use of animals in toxicological studies. The focus of our research has been set on the early safety evaluation of potential antidotes for organophosphorus compounds (OPs), which still attract great attention due to their continuous use in modern industrial society (as in agriculture and chemical industry), but unfortunately also due to their use as chemical agents (soman, satin, tabun, VX) in the battlefield or in terrorist attacks. The therapeutically approved antidotes (i.e. oximes 2-PAM or HI-6) have several major limitations, and the search for new, more efficient ones is still ongoing. Until now, hundreds of new oximes have been tested but none passed to the final step of approval even if they acted efficiently on their main target in the organism. Their crucial drawback is always linked to the observed side effects like neuropathy or myopathy or toxicity in general. Therefore, by a simple addition of a cell-based assay identification of specific molecular targets of the tested compounds’ unwanted action at the beginning of research could help reduce the risk of missing this critical information, which often proves costly in later stages of development. We selected two cell types as the two main targets of OP poisoning and antidote therapy in vivo: skeletal muscle cells (myoblasts and myotubes) and neuronal cells SH-SY5Y. We tested several defined sets of structurally related compounds, marked as potent OP antidotes in Current research. The selected compounds showed a different cell toxicity profile and the myoblasts were more sensitive to exposure. However, compounds that were toxic to muscle cells were toxic to neuronal cells as well, and the obtained results could be linked to the structure of the compounds itself. Thus, defining the structural features probably responsible for a negative effect enabled us to give efficient feedback to the designers of these compounds who could then modify them in the early lead compound structure refinement process without performing any in vivo studies. Moreover, the results obtained here could be useful and applied to any of the drug design and developmental studies, as the determined cell effects of specific compound structural features are general.

Compounds known as oximes are investigated today as antidotes in highly toxic organophosphorus pesticide and nerve warfare agent (OPs) poisonings. Their major advantage is the ability to act fast by reactivating the essential enzyme acetylcholinesterase (AChE, EC 3.1.1.7), the main OP target in neural synapses. However, in the last 70 years, only two oximes have been introduced to medical practice, unfortunately, both with a narrow scope of action. The search for more efficient oxime antidotes is still ongoing and diverse chemical structures are being tested with groups added to the core structure to enhance properties such as lipophilicity and blood brain barrier penetration. The major issue for oximes in the early stages of drug development is the high cut-off rate due to observed side- effects and a possible negative influence on organs. For that reason, we wanted to investigate what drives this toxicity and whether it could be related to oxime’s specific structural motives. We tested the influence of four diverse sets of potential oxime antidotes on the viability of several different cell types representing nerves, muscles, liver and kidneys. The toxic effect was monitored for each compound in a time- and dose-dependent manner, and the type of cell death was assessed. As the results indicated, the observed negative effects were triggered by the presence of specific moieties within the oxime structure or by the presence of specific structural feature combinations. The most toxic oximes had the 6, 7-dimethoxy-isoquinoline group or chlorine atoms present in their structure. With respect to this finding, studies designing these drugs will have to consider other options for oxime structure modifications to aid the challenging development of a more effective treatment for OP poisoning. Furthermore, we designed a pharmacophore model for the tested set of oximes and ran it through the database carrying information about associated cell targets and mechanisms of action responsible for the observed biological phenotype. The provided information will subsequently be used to narrow down the choice of possible cell targets of the tested oximes as well as the experimental method used to ascertain the assumed interaction. Acknowledgment: This work was supported in part by the Croatian Science Foundation under project UIP-2017-05-7260 and by the Slovenian Research Agency research programme P3-0043.

The objective of our study was to investigate neuropathy target esteraserelated enzyme (NRE; PNPLA7) expression and its functional characteristics in human muscle cells as a model organism for insulintargeted tissues. More specifically, our previous preliminary studies confirmed the presence of this unexplored enzyme in both myoblasts and myotubes and indicated a role for NRE in the energy metabolism. The human NRE enzyme is predicted to be around 146 kDa. Though the crystal structure of NRE has not yet been defined, gene sequence analysis and homology modelling predict the presence of Nterminal singlepass transmembrane domain, three cyclic nucleotide binding sites, possible glycosylation sites and Cterminal patatinlike catalytic domain. Furthermore, analysis also predicts the existence of several isomers some of which do not have a catalytic domain or in other words, possibly lack esterase/lipase functions. Sequence alignment reveals that NRE is conserved through rat, mice and human species. High NTE homology with more investigated enzyme NTE or PNPLA6 suggests that NRE might also be a target of organophosphorus compounds (OP) which implies NRE involvement in OP caused pathological conditions including poorly defined intermediatemyopathy syndrome. Therefore, by following changes in NRE mRNA, the protein and activity level in cells exposed to different stimuli mimicking real life conditions, we tried to define NRE’s physiological role and its potential to be used as a new therapeutic target in OP poisoning or in wider research. The enzyme was cloned, expressed and studied as the esterase in kinetic experiments in vitro following interactions with potential substrate and inhibitor. In this way, we also evaluated its enzymatic properties. Since little is known about this enzyme's physiological role and biological relevance, any findings would most certainly contribute to the understanding of the importance of NRE, which still calls for a detailed clarification.

Apigenin is a dietary flavonoid found in several types of vegetables and fruits. Its potential anticancer properties were investigated in two types of human breast cancer cells: ER-positive MCF-7 and triple-negative MDA MB-231 cells. Human peripheral blood lymphocytes were used to elucidate the apigenin’s toxicological safety regarding the normal cells. MTT, comet and lipid peroxidation assays were used to evaluate cyto- and genotoxicity of apigenin towards cancer cells. Furthermore, the type of apigenin-induced cell death was analysed using several biomarkers. Our results revealed that the treatment with apigenin caused changes in cell morphology, in a dose- and time-dependent manner. This was followed with apoptosis as a dominant type of cell death in both cell lines. Moreover, apigenin exhibited genotoxicity towards cancer cells by inducing oxidative damage. Importantly, cell viability and comet assays showed that apigenin was not cytogenotoxic to normal cells. The observed anticancer activities of apigenin accompanied by its low toxicity towards normal cells indicate the possibility of using this dietary flavonoid as an anticancer agent. Even though the beneficial effects of apigenin are promising, further in vitro and in vivo studies are needed to enable its translation from bench to bedside.

Water is the most abundant molecule in living cells. Aquaporin protein channels (AQPs) facilitate the passive movement of water across cell membranes. In mammalian kidneys, AQP1 and AQP2 facilitate water reabsorption from primary urine across the apical membrane of proximal and distal nephron tubules. Studies indicate that renal functional/structural changes in humans and experimental animals develop with age. Aging promotes fluid loss in tissues which was suggested to reflect changes in the expression/activity of AQPs. Here we investigated the potential age-ameliorating effects of two potential geroprotector substances, melatonin (endogenous hormone) and resveratrol (plant flavonoid), on AQP1 and AQP2 expressions in the rat model of experimental aging. During 21 months, rats of both sexes were given melatonin or resveratrol via drinking water (~1 mg/kg b.w./day), whereas control animals drank vehicle (0.01% ethanol in drinking water). The expression of both AQPs in the kidneys of control and treated animals was determined using immunofluorescence cytochemistry in tissue cryosections and Western blot analysis in isolated cell membranes. Results showed that melatonin and resveratrol treatment had no effect on renal AQP1 expression in both sexes. AQP2 expression in male and female kidneys of resveratrol-treated rats also did not change compared to controls. However, only in female rats, AQP2 expression was upregulated by melatonin in the inner stripe and papilla kidney regions. Therefore, in 24-month old rat kidneys, chronic oral intake of melatonin and resveratrol had no effect on AQP1, whereas melatonin exerted sex-dependent upregulation of AQP2. (Supported by Croatian Science Foundation project IP-2013-11-1481).

Svaka ljudska stanica u našem tijelu djeluje poput mini-me: stvara energiju, raste, reproducira se, razboli, pa čak i umire - baš kao i mi. Osim građevne uloge u tijelu, stanice kontroliraju i svaku funkciju ljudskog tijela te svako obilježje, poput visine i boje kose, kože, očiju, a imaju i zaštitnu ulogu od bolesti. Kako postoje različiti ljudi, tako postoje i različite vrste stanica, a svaka od njih ima svoju posebnu zadaću. Kao što vam je sigurno poznato, stare i istrošene stanice odumiru i zamjenjuju se novima. No, je li to stvarno tako? I kako to možemo iskoristiti? Sve ćete saznati tijekom ovog slikovitog predavanja o stanicama. Poslušajte „njihovu stranu priče“.

Telomeres are the main guardians of genome stability. During DNA replication, progressive shortening of telomeres takes place with each cell division. Critically short telomeres induce cell cycle arrest or apoptosis. Oxidative stress also plays an important role in telomere dynamics. Recent evidence highlights direct molecular connection between telomere attrition and mitochondrial dysfunction. Although reactive oxygen species (ROS) are normal products in intermediary metabolism, and are crucial for different intracellular signalling pathways, a disbalance resulting in ROS excess induces oxidative stress and accelerates telomere shortening. It is assumed that with ageing, production of ROS may be increased causing faster telomere shortening, and that both may be ameliorated by antioxidants. To test this hypothesis, we treated male and female Wistar rats for 9 or 21 months with melatonin and resveratrol and investigated their effect on telomere attrition rate in liver and kidney (various zones) tissues. Telomere length was assessed by Southern blotting of genomic DNA. The results in 1- and 2- year-old male and female rats showed an absence of significant differences in telomere length in the tissues from both organs after treatment with antioxidants compared to control (vehicle-treated) animals (N=4 per group). Based on obtained results, we did not observe beneficial properties of tested antioxidants towards age-dependent telomere shortening.

Many industrialized countries are facing demographic changes that manifest themselves in population aging. It is estimated that roughly one third of Europe’s population is 50+ with this share likely to increase. Therefore, the investigation of aging molecular mechanisms is a priority to facilitate healthy aging and improve the productivity/longevity of the aged population in both men and women. Within the frame of project Aging-related Expression of Membrane Transporters in Rats (AGEMETAR), aging is investigated in the Wistar rat model over 24 months, which corresponds to the elder age of humans. The aim of the current study was to investigate baseline DNA damage using the comet assay in kidneys, liver and blood mononuclear cells (BMCs) of adult 3-month-old rats of both sexes. In order to establish the influence of sex-hormones on baseline DNA damage, gonadectomized rats of both sexes were compared to sex-matched sham-operated animals (N=10/group). The sex-related differences of baseline DNA damage were not observed for any tissue studied in 3-month-old rats ; the percentage of comet DNA was in: BMCs 0.62±0.13% (male) compared to 0.63±0.20% (female) ; kidneys 1.00±0.24% (male) compared to 1.28±0.49% (female) ; and liver 0.86±0.21% (male) compared to 0.84±0.26% (female). Similarly, castration in males or ovariectomy in females had no effect on baseline DNA damage in: BMCs 0.55±0.13% (castration) and 0.53±0.14% (ovariectomy) ; kidneys 0.92±0.25% (castration) and 1.00±0.39% (ovariectomy) ; and liver 0.83±0.20% (castration) and 0.93±0.15% (ovariectomy) when compared to sex-matched sham-operated animals. Altogether, these results indicate that sex hormones have no effect on baseline DNA damage in the BMCs, kidneys and liver of 3-month-old rats. Furthermore, they provide useful information for further investigation of sex- and age-related differences of baseline DNA damage in the Wistar rat animal model of aging. (Supported by the Croatian Science Foundation Grant No. IP-2013-11-1481 ; AGEMETAR).

U ovom popularno-znanstvenom radu ukratko su sažeta povijesna saznanja o pojavi medicinskih pristupa liječenju kamenaca u mokraćnom sustavu čovjeka. Počinje spominjanjem drevnih zapisa o bolesti kamenaca u mokraćnom sustavu, nastavlja o napretku kirurških i nekirurških metoda liječenja kroz povijest i završava sa opisom novih pristupa u modernoj medicini.

For last couple of decades, natural products have served us well in combating different types of cancer. The main sources of these useful compounds are from both animal and plant origin. Here we will present anticancer ability of bee venom (BV) and apigenin (API) towards different types of cancer cells in vitro. BV from honey bees is a complex mixture of a variety of different active peptides while API is a natural flavonoid found in several dietary plant foods. Anticancer effect of whole BV was tested in human cervical carcinoma HeLa cells and their drug- resistant HeLa CK subline while anticancer effect of API was tested in human breast cancer MCF-7 and MDA MB-231 cells. Cytotoxicity of both compounds towards cancer cells was evaluated by MTT assay whereas type of cell death was analysed by differential staining using acridine orange/ethidium bromide and was further verified by Western blot analysis. BV displayed dose- dependent cytotoxicity against both cell lines tested with drug-resistant HeLa CK cells being more sensitive to BV than their parental cell lines. Similarly, API inhibited the growth of both cell lines in a dose-dependent manner with MCF- 7 cells being more sensitive. Treatment with BV induced a necrotic type of cell death, as shown by characteristic morphological features, fast staining with ethidium bromide and a lack of cleavage of apoptotic marker poly (ADP-ribose) polymerase (PARP) on Western blot. On the contrary, cell treated with API showed apoptosis as a dominant type of cell death in both cell lines which was further verified by Western blot analysis detecting cleaved PARP. In view of accumulating evidence on anti- proliferative and pro-cell death activity, both tested compounds could be used in the development of future anticancer drugs. Undoubtedly, therapeutic applications of BV and API are promising, however further in vitro and in vivo studies are warranted to resolve precise mechanisms responsible for their anticancer properties

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Cellular uptake and metabolism of glucose (G) serves as an important energy source. In mammals, SGLT1 (humans)/Sglt1 (rodents) mediates an almost complete absorption of G in small intestine (SI) and a limited (3-15%) reabsorption of G in kidneys (K). Novel antidiabetic drugs were recently developed aiming to lower blood G by inhibiting SGLT1 in SI and K. However, if SGLT1 is expressed in insufficiently explored other organs, these drugs could affect their function and induce health disturbances in diabetic patients. To reveal SGLT1/Sglt1 expression in other organs, we performed mRNA and immunochemical studies in various human, mouse and rat organs. In humans, SGLT1 mRNA was detected in SI, K, liver, lung, and heart, but not in brain, whereas the SGLT1 protein was localized in the luminal membrane (LM) of SI enterocytes, K nephron and hepatic duct cells, in lung alveolar type 2 and bronchiolar Clara cells, and in heart capillaries. In mice, Sglt1 mRNA expression was highest in SI, high in seminal vesicles, K and salivary glands, medium in prostate, tongue, eyes and uterus, and very low in pancreas, lungs, liver, and brain. The Sglt1 protein in wild-type, but not in Sglt1 knockout mice, was immunolocalized in the LM of SI enterocytes, K proximal tubule, liver bile ducts, pancreatic ducts, salivary glands (acinal and duct cells), prostate epithelium, bulbourethral gland ducts, uterine endometrium, tongue taste cells and eyes (optical nerve), while brain, lungs and seminal vesicles were Sglt1-negative. In rats, we localized the Sglt1 protein in SI, K, and submandibular glands (similar to the findings in mice), but also in lungs, brain, and heart capillaries (different from the findings in mice). The novel extrarenal localizations implicate so far unrecognized (patho)physiological roles of SGLT1/Sglt1 in the mammalian organs, indicate species differences in this protein expression in some of them, and represent potential targets for the novel inhibitors. Since the Sglt1 expression in rodents does not fully reflect that in humans, it is thus important to carefully select an animal model for each organ of interest to study the role of SGLT1 in human health and disease.

Apigenin, one of the most common flavonoids found in a variety of fruits, leafy vegetables as well as tea and wine, has been shown to possess anti- oxidant, anti-inflammatory and anti-cancer properties. In this study, hormone responsive, ER-positive MCF-7 and triple-negative MDA MB-231 breast tumour cells were used to investigate potential cytogenotoxic effect of apigenin as well as the type of cell death in vitro. Based on our results, apigenin treatment induced changes in cell morphology in dose- (10-100 µM) and time- dependent manner. Moreover, apigenin caused cell death in both cell lines (with MCF-7 cells being more sensitive) leading to significant toxicity, which was evaluated by MTT and XTT assays. Additionally, apigenin induced DNA damage in both cell lines in dose-dependent manner with MDA MB- 231 cells being more sensitive compared to MCF-7 cells based on the comet assay results. Differential staining using acridine orange/ethidium bromide indicates apoptosis as a dominant type of cell death in both cells. This was also confirmed by Western blot analysis which detected cleaved PARP (poly(ADP-ribose) polymerase), as a marker of apoptotic cell death, after treatment with apigenin in concentrations of IC50 and above. Cytogenotoxic effect of apigenin was also evaluated on non-tumour circulating blood cells, human lymphocytes. According to our results, apigenin showed no effect on lymphocyte viability and there was no significant DNA damaging effect observed as determined by the comet assay, indicating its safety from the aspect of cytogenotoxicity towards normal human cells. Observed cytogenotoxic and pro-cell death activities of apigenin coupled with lack of toxicity (concentrations up to 100 µM) towards non-tumour cells, indicates that this natural product could be a useful candidate in the future breast cancer therapy.

Introduction. In diabetics, major organs that contribute to glucose handling are small intestine (SI ; SGLT1-mediated glucose absorption), kidneys (SGLT1- and SGLT2-mediated glucose reabsorption), and liver (glucose transport supposed to be SGLTs-independent). Recently, novel phlorizin-derived antidiabetic drugs (inhibitors of SGLT2 or both SGLT2+SGLT1) were developed aiming to lower blood glucose by inhibiting the SGLTs-mediated transport in SI and kidneys. However, SGLTs are insufficiently explored in other organs ; where present, these drugs could affect their function and patient's health. Material and methods. SGLT1 and SGLT2 mRNA and protein expression were explored, respectively, with quantitative RT-PCR and immunocytochemistry in various organs from humans, rats, and wild type (WT) and SGLT1 knockout (KO) mice. Results. In all three species, SGLT2 mRNA and protein were detected only in kidneys, while SGLT1 was expressed in various organs. In WT, but not in KO mice, variable expression of SGLT1 mRNA and protein was found in SI, kidneys, salivary glands, prostate, tongue, optical nerve, uterus, pancreas, liver, and periurethral gland ; lungs, heart, seminal vesicles, and brain were variably positive for mRNA and negative for protein. In rats, the SGLT1 protein was located in SI, kidneys, submandibular glands, lungs, heart, and brain. In humans, the SGLT1 mRNA and protein were detected in SI, kidneys, liver, lungs, and heart, Conclusions. In contrast to selective SGLT2 inhibitors that will inhibit only the transporter in kidneys, the numerous extrarenal SGLT1 localizations represent potential targets for the novel, dual (SGLT2+SGLT1) inhibitors with unpredictable health consequences. (Supported by Croatian Science Foundation project #1481).

The chloride/formate exchanger Slc26a6/Cfex transports chloride, bicarbonate, oxalate, formate and hydroxyl ions in mammalian organs. In contrast to mice, Cfex expression in rat organs has been poorly studied. So, we investigated the cell localization and possible sex-related expression of Cfex in various rat organs at mRNA and protein level by quantitative RT-PCR (qRT-PCR), immunofluorescence analysis (IFC) and western blotting (WB), respectively. Specificity of the anti-CFEX antibody (CFEX-Ab) was verified by IFC in HEK293 cells transiently transfected with rCfex cDNA ; the CFEX-Ab strongly stained the plasma membrane of rCfex-transfected HEK293 cells, whereas this staining was not observed in mock-transfected cells. By IFC, the Cfex protein was localized in the brush-border membrane of renal proximal tubules (S1S2S3) and intestinal enterocytes (duodenumjejunum, ileum was negative), canalicular membrane of hepatocytes and apical domain of pancreatic ducts. WB analysis of total cell membranes isolated from respective organs detected a single protein band of 120 kDa whose density matched the IFC findings. The Cfex mRNA expression roughly followed protein expression. Sex-dependent expression of Cfex at mRNA (qRT-PCR) and protein (WB/IFC) level was exclusively detected in kidneys of adult rats (malesfemales), whereas in pre-pubertal rats its expression was low and sex-independent. Furthermore, renal Cfex protein expression was downregulated by castration and unaffected by ovariectomy. Finally, testosterone upregulated, while estrogen and progesterone had no effect on renal Cfex protein expression in castrated males. Thus, Cfex is expressed in various rat organs, with male-dominant expression in kidneys that appears after puberty due to androgen stimulation.Funded by Croatian Science Foundation-project#1481.

Novel antidiabetic drugs have been developed aimed to lower blood glucose by inhibiting sodium-D-glucose cotransporter 1 (Sglt1) in intestine and kidneys. In mice, the intestinal Sglt1, localized in the enterocyte brush-border membrane (BBM), is responsible for bulk (>80%) glucose absorption, whereas the transporter in the BBM of renal proximal tubules (PT) contributes to ~3% glucose reabsorption. The presence of Sglt1 in other mammalian organs, which could represent possible targets of novel inhibitors, is poorly known. Here we compared the expression of Sglt1 mRNA and its protein in various organs of wild type (WT) and Sglt1 knockout (KO) mice by quantitative RT-PCR and immunocytochemistry (IC). In WT, but not in KO mice, the Sglt1 mRNA expression was highest in small intestine ; high in seminal vesicles, kidneys and salivary glands ; medium in prostate, tongue, eyes and uterus, and small in pancreas, lungs and liver. By IC in WT mice, Sglt1 protein was detected in small intestine (enterocytes, BBM), kidneys (PT, BBM ; thick ascending limbs, luminal membrane (LM)), eyes (optical nerves), liver (bile ducts, LM), pancreas (ducts, LM), salivary glands (serous acini and initial ducts, LM), tongue (taste epithelium, plasma membrane), prostate (myoepithelial cells), bulbourethral gland (duct cells, LM), seminal vesicles (myoepithelial cells), and uterus (endometrial cells, LM). In kidneys, we observed sex-dependent expression of Sglt1 mRNA (females>males) and its protein (males>females) indicating different transcriptional and post-translational regulations. Diverse localizations of Sglt1 in various organs may represent targets of novel inhibitors with unpredictable health consequences in diabetic patients. Funded by Croatian Science Foundation-project#1481.

Aging is a physiological process characterized by a reduced ability to respond to environmental stressors. Despite many aging theories, this process is still poorly understood. As evidenced in recent studies, aging may be associated with increased oxidative stress (OS), which may affect an organ’s functional and morphological characteristics. Reactive oxygen species can change the structure and function of membrane lipids, inhibit enzyme activities and oxidative phosphorylation, and oxidize nucleic acids and proteins. This study used gonadectomised rats (N=5) as an experimental model of aging and sham-operated rats as the controls to investigate if sex hormones affect OS parameters in the major internal organs. Experiments were performed on three-month old Wistar rats of both sexes six weeks after the surgical procedures. Glutathione (GSH) was measured in the kidneys, liver, brain and plasma. Significant difference in GSH concentration was observed only in plasma of sham-operated females (3.62±0.34 nmol g-1 tissue) compared to sham-operated males (4.93±0.68 nmol g-1 tissue, mean±SD ; P

Breast cancer is one of the most prevalent malignant diseases in women and its occurrence is rapidly increasing due to modern life stress. Targeted therapy includes treatment with selective estrogen receptor modulators (SERM), such as tamoxifen, in cells that express estrogen receptors (ER-positive cells) or antibodies like trastuzumab in HER2/Neu expressing breast cancer cells. However, not all breast cancer cells express ER, progesterone or HER2/Neu receptor. Such triple-negative breast cancer cells responds poorly to therapy and is often associated with negative prognosis. Several studies have shown that consumption of cruciferous vegetables suppresses the progression of malignant tumors including breast cancer. One of such natural phytochemicals is apigenin, a non-mutagenic and low toxicity flavone existing in fruits and vegetables. The aim of our study was to investigate possible anticancer effect of apigenin on the morphology and viability of breast cancer cells. Our model cells were hormone responsive, ER-positive MCF-7 and triple-negative MDA MB-231. After the treatment with apigenin we observed changes in cell morphology in dose- (10- 100 μM) and time-dependent manner. Moreover, apigenin caused cell death in both cell lines (with MCF-7 cells being more sensitive towards apigenin treatment) leading to significant toxicity, which was evaluated by MTT and XTT tetrazolium colorimetric assay. Differential staining using acridine orange/ethidium bromide on fluorescent microscope showed apoptosis as dominant type of cell death in both cell lines after 24 and 72 h treatment. This was further proved by Western blot analysis which detected cleaved PARP (poly (ADP-ribose) polymerase) in samples treated for 72 h with apigenin concentrations of IC50 and above. Observed cytotoxic and pro-cell death activities of apigenin coupled with its low toxicity towards normal cells, indicate that this natural product could be used as a future anticancer agent.

Testicular seminoma (SE) represents the most common type of testicular germ cell tumours, which are affecting young men. Thus, it is important to find novel biomarkers for early detection which could improve the life and reproductive health of the patients after the diagnosis and treatment. Copy number variation (CNV) has already been associated with various cancers as well as with SE. In this study, we selected two genes, NANOG and KITLG for CNV analysis which are located on chromosomes susceptible to gains, and whose aberrant expression was already detected in SE. CNV analysis was performed on genomic DNA (gDNA) and cell-free DNA (cfDNA) from seminal plasma as liquid biopsy. The aim of the study was to investigate the CNV of selected genes and determine their potential as a possible SE biomarker. Twenty-four SE patients and thirty-five healthy volunteers (HV) were recruited for this study. CNV analysis was performed by droplet digital polymerase chain reaction (ddPCR) on gDNA from SE and nonmalignant testicular tissue. Seminal plasma cfDNA from SE patients before and after surgery was analysed, as well as from healthy volunteers. Statistically significant gains were detected in gDNA for both analysed genes. Comparison between CNVs detected in cfDNA from seminal plasma of HV, preoperative, and postoperative samples disclosed that CNV of NANOG and KITLG was increased in preoperative samples, indicating the reflection of increased NANOG and KITLG CNV from gDNA SE tissue in cfDNA from seminal plasma. Although statistically not significant, a decrease in CNV trend in analysed genes in postoperative compared to preoperative seminal plasma samples indicates that operational treatment induces at least slight normalization in CNV to an HV level. Although clinical value is yet to be determined, presented data emphasize a potential use of CNV as a potential SE biomarker from a liquid biopsy.

During the co-evolution of microbial pathogens and their hosts, pathogens developed a wide array of effectors – powerful molecular weapons increasing their virulence by disturbing hosts’ developmental processes and immune system. The term effector usually denotes a small secreted protein interacting with host cell ; however, effectors can include toxins, elicitors, analogues of (phyto)hormones and different enzymes. Phytoplasmas (genus ‘Candidatus Phytoplasma’) are endocellular wall-less bacteria with small genomes affecting numerous plant species and causing significant damage in agriculture worldwide. They have a dual parasitic cycle colonizing plant phloem and insect cells. Due to the inability of obtaining a pure culture in vitro, studies related to their pathogenicity mechanisms are still hindered. However, the ascent of metagenomic era and new technologies delivered new tools and possibilities for finding potential effectors. ‘Ca. P. solani’ is a cosmopolitan pathogen with a broad host range transmitted by many insect species and one of the most important phytoplasmas in Croatia. The aim this study was to sequence two ‘Ca. P. solani’ strains in order to get insight into their pathogenic potential and the repertoire of putative effectors. Whole plant samples of periwinkle infected with ‘Ca. P. solani’ were used for generation of libraries sequenced on Illumina MiSeq and MinION nanopore sequencing platforms. Raw reads were mapped to the reference genome and reads originating from periwinkle were filtered out. De novo assembly was performed as well as whole genome alignment and comparative analyses. Two draft genomes were generated with total size of 647, 316 bp (ST19) and 668, 749 bp (STOL) and longest contigs of 140, 634 bp and 158, 078 bp, respectively. Whole genome alignment with reference SA-1 revealed synteny and conserved gene order. Prediction and search for putative effectors showed the presence of homologues of SAP11 and SAP54 effectors as well as some putative effectors that were strain specific. Nonetheless, sequencing and comparative genome analyses of ‘Ca. P. solani’ genomes confirmed their highly repetitive nature and the presence of diverse putative effectors as a mechanism for successful adaptation and pathogenicity of this versatile pathogen.

Protein-protein interactions are crucial for many cellular processes and their research allows us to understand these processes from a biochemical point of view. Since changes in the regulation of signaling pathways in the cell often lead to the development of various diseases, studying protein- protein interactions can help us better understand their origin, development and impact on human health. SH2D3C acts as an adapter protein in signaling pathways involved in cell adhesion and migration, tissue organization, and regulation of the immune response. The protein contains a Ras GEF-like C-terminal domain that has no significant GEF activity, but may interact with other proteins. Analysis of the cellular proteome by SILAC-MS revealed a potential interaction of SH2D3C with dipeptidyl peptidase III (DPP III) involved in the regulation of oxidative stress by competitively binding to the KEAP1 protein which is a key participant in the Nrf2/KEAP1 signaling pathway. This would mean that their interaction represents a possible link between the Nrf2/KEAP1 signaling pathway and cell migration regulation. To confirm the interaction by other methods such as micro-scale thermophoresis (MST), the C- terminal domain of SH2D3C was overexpressed and purified in E. coli with two different tags, GST and MBP.

4-Aminoquinolines are a class of compounds known mostly as antimalarials, among which chloroquine, hydroxychloroquine and amodiaquine are the most used drugs. These compounds also have antiinfective and anti-inflammatory activity and have been successfully used for the treatment of certain autoimmune diseases. Due to their ability to cross the blood-brain barrier, they have attracted attention as potentially CNS active agents. In our pilot study, we determined the ability of eight 4- aminoquinolines differing in the substituents attached to the C(4)-amino group and C(7) to inhibit the activity of human acetyl- and butyrylcholinesterases, the main targets in developing drugs for treating Alzheimer’s disease (AD). In continuation of our studies, we pointed out two major goals: the first to explore the impact of length, conformational flexibility, steric demands, basicity and electronic density of the linker between aminoquinoline moiety and terminal amino group, and the second to explore the impact of different N- terminus substituents, as are adamantane or substituted benzyl, on the inhibition of human acetyland butyrylcholinesterase. Starting from the corresponding 4- chloroquinoline and using different alkyl- (normal or branched) or aryldiamines, in various solvents (net diamine, phenol, NMP or EtOH) and heating in the inert atmosphere, corresponding 4- aminoquinolines were obtained. Further modifications of the terminal amino group by reductive amination yielded two series of derivatives that have N-benzyl or Nadamantyl moieties. Alternatively, previously synthesized side chains were directly coupled with the corresponding 4- chloroquinoline. Structure-activity analysis revealed that n- octenyl as a linker is favourable for the inhibition of both acetyl- and butyrylcholinesterase, without pronounced selectivity toward either. Docking studies rationalized our kinetic results and provided us insight into potential ligand-enzyme interactions. An in silico evaluation of ligand’s ability to cross the blood-brain barrier and their drug- likeness was also evaluated.

The aim of this study was to test cytotoxicity, genotoxicity, and oxidative stress as a mechanism of toxicity of bisphenol A (BPA) using Allium test. Allium test is considered as a good model for detecting environmental mutagens and good correlation in cytogenetic studies of Allium test with other test organisms is confirmed. Onion bulbs (Allium cepa L.) were germinated for 24 h, and afterwards, rooted bulbs (n = 10) were exposed to BPA in concentration range 1-50 mg/L or to distilled water (negative control) for 72 h. Following 72 h exposure, morphometry (the root length and the fresh weight) was assessed. In root meristem cells mitotic index (MI) and frequency of micronuclei (MNi) were determined using a light microscope analysing minimum 500 cells per slide. In root homogenate as oxidative stress parameters the level of glutathione (GSH) and protein carbonyls (PC) was assessed spectrophotometrically. The results were analysed by use of one-way ANOVA followed by Dunnett’s test. Statistical significance level was set at p < 0.05. After 72 h exposure to BPA (1-50 mg/L) a concentration-dependent decrease in the root length, root fresh weight as well as decrease in the percentage of MI of meristem cells was observed. Even the lowest concentration of BPA (1 mg/L) inhibited the root growth while the root fresh weight was reduced after exposure to BPA at 5 mg/L compared to negative control. The lowest BPA concentration (1 mg/L) decreased the percentage of MI as well as the number of cells in each of the cell cycle phase. With the increase of BPA concentration, an increase in MNi formation was observed. A decrease in GSH level was observed already after exposure to the lowest BPA concentration (1 mg/L) while an increase of PC was observed after exposure to BPA in concentration 10 mg/L. Based on the obtained results, it can be concluded that BPA induces cyto/genotoxic effects in root meristem cells and that oxidative stress is involved in its toxicity.

The object of our study was the kinetic characterization of the neuropathy target esterase-related enzyme (NRE, PNPLA7) in different cell lines. NRE enzyme is a member of the PNPLA (patatin-like phospholipase domain containing proteins) family and a membrane protein associated with endoplasmic reticulum or lipids droplets and is highly expressed in insulin-targeted tissues. Its crystal structure has not been resolved, yet, but according to the sequence analysis and homology modelling it consists of an N-terminal domain with a transmembrane segment and three cyclic nucleotide binding sites, and a C-terminal domain where the active site with catalytic dyad Ser-Asp is located. NRE has been identified as a lysophospholipase that hydrolyses sn-1 esters in lysophosphatidylcholine and lysophosphatidic acid, but its physiological roles are not fully understood. NRE enzyme shares high homology with neuropathy target esterase (NTE, PNPLA6), which suggests that NRE might also be a target of highly toxic organophosphorus compounds (OP) implying involvement of NRE in OP caused pathological conditions such as poorly defined intermediate myopathy syndrome. In our research, we used two commercially available donors of human skeletal muscle cells and the liver hepatocellular carcinoma cell line HepG2 to kinetically characterize NRE activity using known substrate p-nitrophenyl valerate (p-NPV). Firstly, we confirmed presence of NRE in myotubes, differentiated human skeletal muscle cells, and HepG2 by Western blot. Afterwards, esterase activity measurement was optimised for cell lysates. In order to confirm esterase activity, we incubated cell lysates with different OP compounds and measured esterase activity with p-NPV. Our results showed a difference in the NRE activity in different donors of human skeletal muscle cells, probably due to difference in the protein expression. Also, we have concluded that optimal p-NPV concentration for activity measurement is 1 mM with the Km value of 0.4827 mM. Since little is known about this enzyme's physiological role and biological relevance, any findings would most certainly contribute to the understanding of the importance of NRE, which still calls for a detailed clarification. This research was supported by the Croatian Science Foundation, grant number HrZZ-UIP-2017-05- 7260, Slovenian Research Agency (J3-9263 and J3- 2523, P3-0043 and J7-8276) and Croatian-Slovenian Bilateral grant 2020-2022 (BI-HR/20-21-041).

Psoriasis vulgaris (PV) is a common, chronic inflammatory skin disease driven by aberrant activation of T cells. In addition to autoreactive conventional T cell lines, innate-like lymphocytes, such as MAIT (mucosal associated invariant T cells) and γ δT cells, have been recently implicated in the initiation and progression of PV. Both MAIT and γ δT lymphocytes seed multiple epithelial tissues and demonstrate various functional capacities in host tissue repair, pathogen clearance, tumor surveillance, and inflammation. In case of injury or antigen encounter in the skin, these innate-like players exhibit rapid effector responses and directly contribute to local IL-17 production, a key pro- inflammatory mediator of epithelial, stromal, and vascular remodeling in psoriasis. Emerging evidence suggest an important role for MAIT and γ δT cells in PV immunopathology, but their diversity, distribution and trafficking in psoriatic skin plaques remain less well understood. To determine the number and distribution of IL-17-producing MAIT and γδT cells in psoriatic and healthy skin, a multiplex immunofluorescence analysis was performed with MR1 5-OP-RU PEconjugated tetramer in combination with primary monoclonal antibodies against CD3, IL-17A and γ δTCR. Psoriatic (n=6) and healthy (n=3) skin biopsies were initially fixed in 4% paraformaldehyde, cryoprotected in sucrose gradient and snap frozen, before being cut on the cryostat. Primary antibodies were detected with fluorescently labeled secondary antibodies, and nuclei stained with DAPI. All slides were covered with a VectaShield covering media and imaged under a fluorescent microscope. Compared to healthy skin, psoriatic skin sections were significantly enriched in CD3+ and IL-17+ T cells, of which only a minor fraction were either γ δTCR+ or MR1- restricted lymphocytes. Although rarely evident in examined skin sections, both MR1-tet+IL-17+ and γ δTCR+IL17+ T cells were more frequently observed in psoriatic sections, predominantly in dermal area, between elongated rete ridges of the epidermis. In conclusion, our results provide novel insights on MAIT and γδT cell distribution in healthy and psoriatic skin, and warrant further research on their phenotype and function in PV.

Ball shaped cellular structures called spheroids are the basis of 3D cell culture. Shift from 2D to 3D cell culture was necessary in various fields since 3D structures mimic live tissue better. There are many different ways to form spheroids from single cells. One of the methods is magnetic levitation where cells are grown under the influence of a magnetic field after taking up ferrous nanoparticles. Spheroids formed this way are well defined and compact. Our work set out to establish the baseline composition of these spheroids to better characterize them for further use in toxicity assays. Spheroids in question were formed from glioblastoma cells (namely D54 cell line) by magnetic levitation. They were formed and grown for up to 15 days prior to analysis with the growth media changed as needed. Completely grown spheroids were harvested and dissolved in 0.5% trypsin before staining with Annexin and PI. Samples were analyzed by flow cytometry to determine the ratio of live, apoptotic and necrotic cells in the samples. Additional samples were frozen, cryostat cut and stained to observe changes in morphology and collagen production. Results showed that the ration changes over time with spheroids of different size having different proportions of apoptotic and live cells, but relatively constant number of necrotic cells. The ratio of apoptotic and live cells rises and falls respectively during the growth period. The intent is to determine how spheroids behave in control conditions as to better interpret toxicity results. Longer growth time resulted in bigger, less compact spheroids that show beginnings of ECM synthesis.

Prostate cancer (PCa) represents a malignancy with the highest prevalence and very high incidence among men worldwide. Clinical challenge is to differentiate localized PCa from benign prostate hyperplasia (BPH) due to the lack of specificity of routinely used biomarker PSA. Epigenetic biomarkers in liquid biopsies, especially miRNA, could address this challenge. The absolute expression of miR375-3p, miR-182-5p, miR-21-5p, and miR-148a-3p were quantified in blood plasma and seminal plasma of 65 PCa and 58 BPH patients by digital droplet PCR. The sensitivity and specificity of these microRNAs were determined using ROC curve analysis. The higher expression of miR-182- 5p and miR-375-3p in the blood plasma of PCa patients was statistically significant as compared to BPH (p = 0.0363 and 0.0226, respectively). Their combination achieved a specificity of 90.2 % for predicting positive or negative biopsy results, while PSA cut-off of 4 µg/L performed with only 1.7 % specificity. In seminal plasma, miR-375- 3p, miR-182-5p, and miR- 21-5p showed a statistically significantly higher expression in PCa patients with PSA >10 µg/L compared to ones with PSA >10 µg/L. MiR182-5p and miR-375-3p in blood plasma show higher performance than PSA in differentiating PCa from BPH. Seminal plasma requires further investigation as it represents an obvious source for PCa biomarker identification.

Gangliosides are membrane glycosphingolipids particularly abundant in mammalian nervous tissue. Being harboured in the outer leaflet of the plasma membrane, they are implicated in various physiological processes including brain development, aging and signal transduction pathways by modulating the function and structure of specific membrane proteins. Our previous studies revealed that Na⁺/K⁺-ATPase (NKA), a membrane ion transporter, appears to be one of the proteins affected by ganglioside composition. NKA dysfunction leads to a disbalance in ion homeostasis in nervous tissue reported in numerous neurodegenerative and neurological diseases. Although the effect of gangliosides on NKA had been observed, detailed clarification of their interrelations still awaits elucidation. Hence, we investigated NKA protein expression and localization in brain tissue derived from the St8sia1 null mice - a transgenic mouse model with impaired synthesis of gangliosides, characterized by lack of the b- and c- series and overexpression of the a-series gangliosides. Protein expression of NKA was determined in cortical and cerebellar tissue homogenates from the null and wild-type (WT) mice by Western blotting using the NKA-specific antibody. Immunohistochemical staining was performed on frozen brain tissue slices from the null and WT mice using the same NKA- specific antibody. Results revealed a statistically lower amount of NKA expressed in the cortices of null mice compared to WTs, with no disparity in the cerebella. Immunohistochemical staining revealed the lower intensity of NKA staining in the cortical tissue and cerebellar molecular and granular layers of null mice compared to WT mice. These results clearly demonstrate and strengthen our previously reported findings that NKA is affected by ganglioside environment in a way that gangliosides provide the proper lipid milieu needed for optimal pump functioning and ion homeostasis upkeep. Considering the vital and ubiquitous role of NKA in maintaining resting potential and regulating cellular volume, the paramount importance of ganglioside surroundings on NKA is even further highlighted and should be considered when conducting studies regarding NKA function.

Inner filter effect (IFE) arises from the absorption of excitation and/or emission light during fluorescence measurements. Therefore, fluorescence intensities are proportional to fluorophore concentration only in a limited, dilute range. Numerous mathematical models and experimental techniques have been used to extend the range of linear response of the fluorescence signal versus concentration, but most are inappropriate for measurements in microplates. A common method for IFE correction requires separate absorbance measurements and can only be used in flat-bottomed transparent microplates. Here we present a Numerical INner Filter Effect (NINFE) method that takes advantage of the variable position of the optical element perpendicular to the sample well (z-position) available in modern microplate readers. By using at least two fluorescence measurements at different z-positions, the linear range can be extended with NINFE. We have successfully applied the presented method to various plate well geometries, including the flat-bottom and round-bottom 96-well microplates (transparent, white, and black) and PCR microplates. NINFE corrections have been performed for protein solutions (BSA and human transferrin) and fluorescent amino acids (tyrosine and tryptophan) to demonstrate that this correction method is applicable to biological systems. Current investigations are aimed at integration with real-time PCR measurements.

Diabetes mellitus and its comorbidities are one of the major health issues worldwide. Diabetes- related complications such as chronic ulcerations are the leading causes of hospitalization of diabetic patients. Dipeptidyl-peptidase IV or CD26 molecule (DPP IV/CD26) is widely known, besides the involvement in a broad range of physiological and pathological processes, for its role in the regulation of glycaemia. DPP IV/CD26 inhibitors are known therapeutics in diabetes. It has been previously shown that DPP IV/CD26 inhibition improves the healing of chronic diabetic ulcers. However, the mechanisms of its actions in the processes and mechanisms of wound healing are unknown. Our hypothesis was that DPP IV/CD26 plays a significant role in the process of cutaneous wound healing in hyperglycaemia through a mechanism of induction enhanced angiogenesis. We aimed to research the process of wound healing in CD26 deficient diabetic mice in order to broaden the knowledge on the role of DPP IV/CD26 in processes of cutaneous reparation and regeneration. A streptozotocin-induced model of diabetes was established in CD26-deficient as well as C57BL/6 (wild-type) mice. Experimental wounds were made on the dorsal region of the mice. Animals were sacrificed in scheduled time periods according to the protocol of wound healing monitoring. Tissue samples were subjected to pathohistological, histomorphometrical, immunohistochemical and immunochemical analyses. Serum samples were analyzed for DPP IV/CD26 activity and concentration of target vascularization factors. The results of this study confirm the hypothesis that DPP IV/CD26 plays an important role in the regulation of glycaemia. Moreover, parameters of wound healing in hyperglycaemia were found to be improved in CD26 deficient mice, indicating that inhibition of DPP IV/CD26 has beneficial effects on the wound healing process in diabetes. Therefore, the significance of DPP IV/CD26 inhibition as a therapeutic strategy for the treatment of diabetes and diabetic wounds is emphasized. DPP IV/CD26 inhibitors are proposed as potential therapeutics in chronic diabetic ulcers.

Grape pomace (GP) is the residue of winemaking, rich in polyphenolic compounds. Polyphenols have antioxidant, anti-inflammatory, anti-allergic and regenerative potential, which makes them a subject of medical and pharmaceutical research. The aim of this study is to investigate the effect of GP extracts on Caco-2 and SW 620 cells, focusing on their antiproliferative and antioxidant properties. The cytotoxic effect of 5 samples of the GP extracts (Merlot 2018 (GP1) ; Cabernet Sauvignon 2018 (GP2) ; Cabernet Sauvignon 2016 (GP3) ; Frankovka 2017(GP4) ; Mix GP 2016: Graševina, Sauvignon Blanc, Muscat Blanc, Incrocio Manzoni (GP5)) at three concentrations (0.02 ; 0.1 ; 0.5 mg/mL) was tested by MTT assay comparing 2D and 3D cell growth. The antioxidant capacity of the GP extracts was determined spectrophotometrically after exposure to UV light for 20 minutes. The absorption rate was evaluated on the Caco-2 cell model after 20 days of cultivation. Polyphenols were quantified by the HPLC method. Data were analysed using Statistica 13.1 (P < 0.05). GP extracts at the highest concentration (0.5 mg/mL) were most effective in suppressing cell growth. Caco-2 was suppressed from 53.7 – 79.2 % (0.5 mg/mL) in the 2D model (P < 0.05), whereas SW-620 was inhibited from 41.3 – 74.5 % at (0.5 mg/mL ; P < 0.05). The efficacy of GP extracts on the 3D model of cell growth was statistically insignificant. Measurement of total cell antioxidant capacity showed sensitivity of Caco-2 to UV treatment and a reduction in cell antioxidant status of 17.0% (GP2) -20.0% (GP4) at 0.5 mg/mL (P < 0.05). The Caco-2 absorption model shows an absorption rate for polyphenols of 30 to 45 minutes, resulting in an overall absorption efficiency of 85 to 95%. HPLC analysis of the GP extracts revealed catechin as the most abundant polyphenol, followed by epicatechin, procyanidin B1 and B2, gallic acid, resveratrol, caffeic acid and p-coumaric acid. GP is a good source of polyphenols that show significant antiproliferative effect on Caco-2 cells depending on the model used (2D vs 3D). The most effective concentration of the studied GP extracts was 0.5 mg/mL.

Proper cell-substratum adhesion is crucial for motility, phagocytosis and cytokinesis of single cells, and is also essential for complex biological processes such as embryonic development, wound healing, and the maintenance of tissue integrity. Amoeba Dictyostelium discoideum is a unicellular model organism that possesses homologs of many proteins involved in cell- substratum adhesion in higher eukaryotes, for instance, talin, paxillin, and small GTPase RapA. IqgC is a RasGAP protein that inactivates small GTPase RasG and negatively regulates phagocytosis and macropinocytosis. We observed that cells lacking IqgC have impaired attachment to the substratum. Indeed, when the detachment of cells from the bottom of petri dishes was assessed using a shaking assay, iqgc-null cells were more easily detached than wild-type cells, and the expression of recombinant IqgC in mutant cells rescued this defect. We also observed the localization of fluorescently tagged IqgC to the punctiform adhesion structures on the ventral part of moving cells using TIRF microscopy. To determine which protein domain is important for the localization and function of IqgC in adhesion, truncated variants of the protein were expressed in mutant cells. IqgC consists of two main functional domains – a GRD (GAP-related domain) and an RGCt (RasGAP C-terminal domain). The GRD domain binds small GTPases in homologous proteins, for example in human IQGAP1, while the RGCt domain is important for binding PtdIns(4, 5)P2, E- cadherin and β-catenin. In order to compare the localization of these two domains with the fulllength protein, the iqgc-null cells were transfected with the fluorescently labeled YFP- IqgC, YFP-GRD and YFP-RGCt constructs. YFP-IqgC and YFP-RGCt localized to the ventral punctiform adhesion structures, while YFP-GRD did not. Furthermore, we tested the ability of these constructs to rescue the adhesion defect of iqgc- null cells. Only YFP-IqgC, and none of the truncated constructs, rescued the impaired adhesion. Therefore, we conclude that the RGCt domain is sufficient for the localization of IqgC to the adhesion structures, but it needs additional parts of the full-length protein (possibly the GRD domain) to fully exert its role in the cell-substratum adhesion.

Glycosylation is present in all living organisms, ranging from bacteria and archaea all the way to humans. Glycan structures attached to lipids and proteins are not coded into the genome but are precisely added by enzymes involved in the glycosylation process. The observed increase in glycan structure complexity through evolution suggests that the glycosylation processes are under continuous evolutionary change. To reconstruct the evolutionary origin of the genes involved in N-glycosylation and their target proteins in H. sapiens we applied the phylostratigraphic approach. Results showed that homologs of most human genes involved in N- glycosylation could be traced back to the last universal common ancestor (LUCA) suggesting that the protein glycosylation is essential to all cellular life. Apart from that, we observed significant enrichment in the last eukaryotic common ancestor (LECA), probably linked to the emergence of the endomembrane system which plays important role in eukaryotic N-glycosylation. Focusing on the enzymes of the N-glycosylation biosynthetic pathway localized on the cytosolic and luminal side of the membrane of the endoplasmic reticulum, it has been observed that these enzymes are grouped into blocks and that evolutionarily older enzymes, originated in LUCA, are oriented toward the cytosolic side, while evolutionarily newer enzymes, originated in LECA, are oriented towards the lumen of the endoplasmic reticulum. Such rearrangement supports suggested theories of eukaryogenesis which theorize the development of the endomembrane system from the cell membrane of the host cell.

Organophosphate compounds (OPs) comprise an important class of pesticides, chemical agents used in agriculture for management of diseases and pests. Due to their widespread and frequent use as insecticides and herbicides during the last 80 years, they are considered as an increasing environmental and public health concern. The uptake of contaminated food and water or exposure through agricultural activities is harmful to humans and other species because of their low target selectivity and severe toxicity. OPs act mainly by inhibiting enzyme acetylcholinesterase (AChE) which hydrolizes essential neurotransmitter acetylcholine (ACh), leading to a paralysis of cholinergic synaptic transmission in the central and peripheral nervous system which may induce convulsions, respiratory failure, loss of consciousness and death. In this study we investigated in vitro and in silico kinetics of inhibition and interactions of human AChE with four OP herbicides (anilofos, bensulide, butamifos and piperophos) and compared them to a potent AChE inhibitor, insecticide ethoprophos. Their overall rate constants of inhibition (ki) were up to 300-fold lower than ki of ethoprophos and they achieved a maximum inhibition of 85% of enzyme activity. Since medical therapy for restoration of AChE activity in case of poisoning includes oxime reactivators which dephosphylate AChE’s active center, we also evaluated potential of standard pyridinium oximes (2-PAM and HI-6) and a novel zwitterionic oxime RS194B to reactivate selected herbicide- and ethoprophos-inhibited AChE. Our results provide insight into neurotoxic effects of pesticides and facilitate further development of antidotes directed against OP pesticide poisoning. This research was supported by the Croatian Science Foundation (HrZZ-IP-2018-01-7683).

Grape pomace is a by-product of wine production and is rich in natural antioxidants, fiber and phenolic components. Therefore, it is used, among other things, in the development of various food products that acquire new functional properties through the addition of grape pomace. In this paper, the influence of using different sample amounts (5 g, 1 g and 0.2 g) on the prediction of the quality of biscuits made from wheat flour with the addition of Cabernet Sauvignon grape pomace was studied. The methods used were Solvent Retention Capacity (SRC) and Alkaline Water Retention Capacity (AWRC). These are methods used to test the quality of wheat flour, measuring the ability of the flour to retain various solutions after centrifugation of the suspension. The test results showed that there is a significant correlation between the retention values of the solutions used, regardless of the sample weight used. The best results for the retention capacity of flour were obtained for samples of 5 g. The obtained results show that we can use with high statistical significance (p < 0.01) the AWRC, SRC- H2O, SRC-Sucrose and SRC-Na2CO3 methods to predict the biscuit volume, the SRC-Sucrose and SRC-Na2CO3 methods to predict the width and the SRC-Sucrose method to predict the spreading factor. The prediction of the height of the biscuit with the addition of Cabernet Sauvignon grape pomace can be performed with high statistical significance (p < 0.01) for 1 g samples using the AWRC method. According to the obtained results, SRC and AWC are methods for determining the quality of wheat flour that can be used to predict the quality of biscuit with grape pomace addition when we need quick results or when we have a small amount of samples.

Thallium is a naturally occurring, highly toxic trace element. Human exposure to thallium is mainly connected to occupational exposure, environmental contamination and food accumulation. The exact mechanism of thallium toxicity is still poorly understood, however its toxic effect is often connected to disruption of potassium-dependent processes and development of oxidative stress. The aim of this study was to access the cytotoxic effect of thallium on several cell culture lines and relate the obtained results with its specific toxicity to a particular organ. In addition, possible protective effect of potassium on toxicity of thallium was also examined. Human keratinocytes (HaCaT), human hepatocellular carcinoma (HepG2), porcine kidney epithelial cells (PK15), human neuroblastoma (SH-SY5Y) and Chinese hamster lung fibroblast cells (V79) were treated with thallium (I) acetate in a wide concentration range (3.9-500 µg/ml) for 24h, 48h and 72h. Based on cytotoxicity results, four concentrations (15.6, 31.25, 62.5, 125 µg/ml) were chosen to be tested in combination with potassium acetate (500 μg/ml) to assess their competitive interaction during 24h treatment. Cell morphological changes were monitored on inverted microscope, and cytotoxic effect was assessed by 3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide (MTT-test). Thallium toxicity for each cell line tested was both, time- and dose-dependent. For each measurement point SH-SY5Y cells had the lowest IC50 values and appeared to be most sensitive, while HepG2 were most resistant to thallium exposure. Simultaneous treatment of cells with thallium and potassium (I) acetate resulted with higher viability compared to treatment with thallium (I) acetate alone. Results of our study indicate that tissue origin contributes to cell’s susceptibility to thallium toxicity. Different toxic effect of thallium could be attributed to cell’s antioxidant capacity as well as transport systems through which thallium and potassium compete for entering the cell. The obtained results point to importance of monitoring thallium concentrations in the environment and call for further research into the mechanism of its toxicity.

Changes in expression of integrins, heterodimeric transmembrane cell adhesion molecules used by cells to bind to the extracellular matrix and control cytoskeletal network organization, are frequently associated with cancer. Integrins foster tumour cell survival, proliferation and migration during invasion and metastasis through bidirectional signalling via multimolecular integrin adhesion complexes (IACs). Our previously published data in melanoma cell line RPMI-7951 showed that knockdown of integrin αV decreased migration and invasion. Mass spectrometry (MS) analysis of biochemically isolated IACs revealed that this cell line preferentially uses integrin αVβ5 for adhesion forming FAs, and we confirmed its exclusive role in regulating cell migration. MS also revealed that αVβ5 IACs contain actin cross-linking proteins α- actinin 1 (ACTN1) and α- actinin 4 (ACTN4). These are ubiquitously expressed cytoskeleton proteins that cross-link actin filaments, and through interaction with a number of IAC proteins and integrins anchor them to focal adhesions at the leading edge of migrating cells. As such, α- actinins can influence actin cytoskeleton organization and dynamics, focal adhesion maturation and cell migration. To investigate their localisation, elucidate their individual role in formation of IACs, actin dynamics and migration, we performed immunofluorescence and western blot analyses of ACTN1 and ACTN4 localisation/ expression, actin and FAs visualisation as well as migration assay upon knockdown of each ACTNs. Both knockdown of ACTN1 and ACTN4 resulted in diminished cell size and decreased expression of integrin αVβ5 FAs. However, the integrin αVβ5 FAs size only changed upon ACTN1 knockdown, indicating its possible role in maturation of αVβ5 FAs. Additional data pointing to the role of ACTN1 in FAs maintenance was the increased expression of ACTN1 upon ACTN4 knockdown. Also, we observed a transition from dorsal actin fibers with the transverse arc to ventral stress fibers upon ACTN4 knockdown. We are currently performing migration assays and live cell imaging upon each ACTN knockdown using fluorescently labelled ACTN4. These data will elucidate the differential role of ACTN isoforms in αVβ5 FAs which contribute to actin organisation and migration in melanoma.