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Cancer is the second leading cause of death worldwide, resulting in around 9 million deaths annually [1]. Among the approved anticancer drugs, around 80% of them are derived from natural compounds. Coumarin derivatives are ubiquitous in nature and can readily interact with a variety of enzymes and receptors such as kinase, telomerase, aromatase, sulfatase, monocarboxylate transporters, and carbonic anhydrase, thereby exhibiting a potential activity against various cancer cell lines including multidrug‐resistant cells [24]. Thus, the coumarin moiety is a useful template for the development of novel anticancer agents. On the other hand, metal-based drugs have gained attention to address the negative aspects of platinum-based drugs, like high toxicity, resistance to treatment, low specificity for target DNA, and poor absorption [5]. Considering the promising anticancer activity of metal complexes, we designed and synthesized coumarin– pyridine hybrids and their metal complexes with the aim to evaluate their antiproliferative activity (Fig. 1). Herein, we report syntheses, spectroscopic characterization and anticancer activity of coumarin ligands and their Ru(II) and Re(I) complexes. References [1] A. Kopacz-Bednarska, T. Król, Oncol. Clin. Pract. 18 (2022) 166176. [2] T. Gazivoda Kraljević, A. Harej, M. Sedić, S. Kraljević Pavelić, Višnja Stepanić, D. Drenjančević, J. Talapko, S. Raić-Malić, Eur. J. Med. Chem. 124 (2016) 794808. [3] I. Sokol, M. Toma, M. Krnić, A. Meščić Macan, D. Drenjančević, S. Liekens, S. Raić- Malić, T. Gazivoda Kraljević, Future Med. Chem. 13 (2021) 18651884. [4] A. Bistrović, N. Stipaničev, T. Opačak- Bernardi, M. Jukić, S. Martinez, Lj. Glavaš- Obrovac, S. Raić-Malić, New J. Chem. 41 (2017) 75317543. [5] S. Jakopec, N. Pantalon Juraj, A. Brozovic, D. Jadreško, B. Perić, S. I. Kirin, S. Raić-Malić, Appl. Organomet. Chem. 36 (2022) e6575.

One of the biggest challenges in modern medicine is the treatment of neuronal diseases and nerve injury repair due to incapability of the neural tissue to self-renew. Regenerative medicine and stem cell technology holds tremendous potential to treat a wide range of such medical problems. Although there is substantial work done with two dimensional cultures used for neural differentiation and proliferation protocols, surrounding environment and three dimensional culturing are important candidates to enhance cell proliferation and differentiation to desired cell lineage. Alginate hydrogel is a polysaccharide biocompatible with human tissue and it has appropriate physicochemical characteristics. Its gel-forming property is achieved through a cross- linking with calcium ions. Neural stem cells from the brain cortex of 14 days old mouse embryos were isolated and encapsulated in soft alginate hydrogel beads in concentration of 2 million cells per ml of 1% alginate. Cells were cultured following established neural proliferation protocols. On 7th day following encapsulation, live/dead test was performed. Confocal microscopy showed that cells were both viable and formed clusters. Alginate hydrogel was a good candidate for encapsulation and culturing of neuronal stem cells improving their proliferation and viability. Obtained results opened opportunities for further research on therapeutic treatment of neural tissue injuries

Interprofessional collaboration is the process in which different professional groups work together to positively impact health care. We aimed to explore physicians' attitudes toward interprofessional collaboration in the context of chronic pain management with the implication that if attitudes are not positive, appropriate interventions could be developed. Design: A quantitative attitudes study. The ethical committee approved the study. A web-based survey about interprofessional treatment of chronic pain was administered to physicians. Outcome measures were as follows: physicians' demographic and workplace information, previous experience of working within an interprofessional team, and attitudes towards interprofessional collaboration in chronic pain management. There were 90 physicians who responded to the survey. Physicians had positive attitudes towards team work in the context of chronic pain, but they were undecided about sharing their role within an interprofessional team. The family physician was singled out as the most important as well as the most common collaborator in chronic pain treatment. Interprofessional educational seminars and workshops were suggested as methods for improving interprofessional collaboration. Interprofessional collaboration may be enhanced with continuing medical education that will bring together different healthcare professionals, enable them to exchange experiences and learn about their potential roles within a team.

Introduction: Red wine is antioxidant and direct, endothelium-dependent vasodilator. Diabetes mellitus (DM) alters vascular responsiveness as a result of oxidative stress and chronic high blood glucose. The present study was designed to examine red wine vasodilatory activity in the rats with streptozotocin-induced type 1 DM. Materials and methods: DM was induced with intaperitoneal injection of streptozotocin (55 mg/kg) to male Sprague-Dawley rats. The DM rats were treated once a week with long-lasting insulin therapy. 14 months after induction of DM, control (C, non- diabetic, 14 months old) and diabetic rats were anesthetized and thoracic aortas excised (N=15 per group). The isolated aortic rings were precontracted with noradrenaline (10-7 M) and randomly exposed to red wine (“Zlatan Plavac” 2008, Plenković), acetylcholine or sodium nitroprusside. Vasodilation was measured, dose- response curves analyzed. Results: Red wine induced vasodilation in control and diabetic aorta. DM did not significantly affect maximal vasodilatory activity (Emax) of red wine (Emax 63, 75±5, 01% and 55, 75±5, 68% for C and DM, respectively). However, 50% effective concentration, EC50 of wine was increased in diabetic aorta (EC50 of 2, 01‰ (95% CI of 1, 68- 2, 41‰)) in comparison to control aorta (EC50 of 1, 06‰ (95% CI of 0, 87-1, 28‰)). Furthermore, the vasodilatory response to acetylcholine was significantly decreased in diabetic compared to the control aorta, as indicated by decreased Emax and EC50. No differences between diabetic and control aortas were observed in the vasodilatory response to sodium nitroprusside. Conclusion: Acetylcholine vasodilation was decreased in diabetic rats. DM also impaired red wine vasodilation potency, but did not affect its effectiveness.

Background: The kidney expresses relatively high levels of glycosphingolipid globotriaosylceramide (Gb3Cer or CD77) compared with other organs. Glycosphingolipids are found in membrane lipid rafts where they influence function of receptors and transporters embedded in these domains. In addition, CD77 serves se E. coli verotoxin receptor. Renal glucose reabsorption in diabetes mellitus (DM) is altered, and glycosuria provides a favorable environment for bacteria to reside and grow in the urinary tract, often leading to urinary tract infection. Therefore, the aim of our study was to determine CD77 expression on renal endothelial and non-endothelial cells, in rat models of DM type 1 (T1DM) and type 2 (T2DM). Methods: Diabetes was induced with streptozotocin (55 mg kg−1 for T1DM and 35 mg kg−1 for T2DM) injection to rats which were fed with normal pellet diet (T1DM) or high-fat diet (T2DM). The endothelial marker CD34 and glycosphingolipid CD77 were determined in renal cell suspensions by flow cytometry. Results: Rats of T2DM model showed twice lower proportion of renal CD77 positive non-endothelial cells in comparison to corresponding control rats (P = 0.016). Conclusions: Our study showed that the CD77 positive non-endothelial renal cells could specifically mirror diabetic nephropathy. The reason of increased susceptibility to bacteria other than E. coli in diabetic patients could lay in changed membrane glycosphingolipid composition.

AIM The aim of this study was to compare expression of total calcium/calmodulin- dependent protein kinase II (tCaMKII) and its α, β γ and δ isoforms in dorsal root ganglia (DRG) in rat models of diabetes mellitus type I (DM1), 6 months and 1 year after diabetes induction. METHODS A total of 45 male Sprague– Dawley rats weighing 160-200 g were assigned into four experimental groups: 6-months DM1 and its control group, 1-year and its control group. For the induction of DM1, after overnight fasting animals were injected intraperitonealy with 55 mg/kg of the streptozotocine (STZ). Rats were sacrificed 6 months and 1 year after the diabetes induction. The L4 and L5 ganglia were removed, fixed, embedded in freezing medium and sectioned on a cryostat. Immunofluorescence analysis was performed for detection of tCaMKII and its α, β, γ and δ isoforms. Image J software was used for analysis of immunofluorescence. RESULTS The diabetes was successfully induced as confirmed by measurement of glucose levels and weight increase. Analysis of tCaMKII expression in DRGs revealed no differences between DM1 and control animals after 6 and 12 months. In diabetic animals, the expression of α and β isoforms decreased significantly after 6 months, compared to the controls, while decrease of γ and δ was observed after one year of diabetes in diabetic animals. CONCLUSIONS The observed changes in the expression of CaMKII isoforms reveal plastic changes of this enzyme during the chronic diabetic state and may be involved in the chronic neuropathic pain development.

The promise of stem cell therapy to achieve tissue regeneration and replace the lost cells is in particular difficult to achieve in the brain. The three dimensional structure of the complicated network of neural cell, where new cells are rarely added in the mammalian brain after birth, represent a major challenge for the incorporation of the transplanted cells. To enhance the abilities of neural stem cells to be incorporated in the brain in particular, the neural stem cells were cultivated using alginate hydrogels. The cells were isolated from the brains of mouse fetuses 14 days old, and cultivated within the alginate beds of standardized 3 mm diameter. Their survival, differentiation and the behavior after the transplantation were followed in different alginate concentrations. The neural stem cells survived and differentiated within alginate beds. The neural markers were present after differentiation. In particular, the alginate concentration suitable for brain transplantation was investigated. The results showed that alginate hydrogels have potential to enhance the abilities of neural stem cells and to serve as a biomaterial supporting their transplantation to the brain.

Sensory changes and increase in calcium/calmodulin-dependent protein kinase II in animal models of diabetes type 1 and 2

Pain catastrophizing changes during menstrual cycle

AIMS: Abnormalities in peripheral nerve and dorsal root ganglion (DRG) have been noted in early stages of experimentally-induced diabetes. The enzyme calcium/calmodulin-dependent protein kinase II (CaMKII) has been linked with neuropathic pain. The aim of this study was to compare expression of total CaMKII (tCaMKII) and its phosphorilated alpha isoform (pCaMKII-alpha) in rat models of diabets mellitus type 1 (DM1) and II (DM2), as well as their pain-related behavior. METHODS: Sprague-Dawley rats were injected with streptozotocin to get DM1, while DM2 was induced with combination of high-fat diet and low-doze streptozotocin. Pain-related behavior was analyzed with thermal and mechanical stimuli. On the 14th post-injection day, rats were sacrificed and DRGs were sectioned. For detection of tCaMKII and pCaMKII-alpha, immunofluorescence was used and measured with Metamorph software. RESULTS: Comparison between DM1 and control rats revealed significant difference following cold stimuli. In DM2 control group we saw increase in pain responsiveness following thermal (cold) and mechanical stimuli, comparing to DM2 group. In DM1 rats we observed significant difference between control and diabetic rats in tCaMKII expression, while this difference was not observed in DM2 model. In both DM models pCaMKII-alpha expression was increased. CONCLUSIONS: Differences in pain-related behavior were observed in rat diabetes models after only two weeks. Experimetnaly-induced diabetes increases expression of tCaMKII (in DM1) and pCaMKII-alpha (DM1 and 2) in sensory neurons. This may indicate involvement of this enzyme in tramsission of nociceptive input early in diabetes. CaMKII may be a suitable pharmacological target for diabetic neuropathy. The experiments are continued in DM1 and DM2 rats that will live for 2 months, 6 months and 12 months.

Globotetraosylceramide (Gb4Cer, GalNAc alpha1-3Gal alpha1-4Gal alpha1-4 Glc alpha1-1Cer) has been identified among the major renal neutral glycosphingolipids. Gb4Cer molecules are found in membrane lipid rafts where they can influence the function of receptors and transporters embedded in these domains. Apical membranes of the kidney proximal tubule epithelial cells contain lipid rafts that support absorptive strategy for nutrient reabsorption. Considering diabetic nephropathy and changed glycosphingolipid metabolism in diabetes, the aim of our study was to determine renal Gb4Cer expression in rat model of diabetes type 1. Diabetes was induced with streptozotocin (55 mg kg−1) injection two weeks before Gb4Cer analysis in the kidney of Sprague-Dawley rats weighing 140-160 g (8 males and 5 females) and compared to respective control animals (3 males and 3 females). Gb4Cer fractions in the tissues of diabetic and control rats were determined by high performance thin-layer chromatography (HPTLC), followed by immunostaining with specific anti-Gb4Cer polyclonal antibody. Diabetic male rats showed increased expression of Gb4Cer. In addition, two bands of Gb4Cer derivative (corresponding to sulphated Gb4Cer), chromatographed bellow Gb4Cer, were also increased in diabetic male rats compared to control. In contrast to control male rats, control female rats had lower Gb4Cer and only one Gb4Cer derivative band. Diabetic female rats showed lower expression of Gb4Cer and Gb4Cer derivative compared to control female rats. Results of this study are significant in the view of the risk to develop end-stage renal disease that is doubled in men compared with women when age at diabetes type 1 onset is ≥15 years. Knowing that estrogen treatment decreases the content of Gb4Cer in rat kidney, our results point at the role of Gb4Cer in nephropathy development particularly in males.

Croatia flora is rich with plant species from Asteraceae familiy. Some of these species are well known medicinal plants, widely used in ethnomedicine. Since the substances used in treatment of some diseases are potentially harmful for human health, it is challenge to find active substances of natural origin like medicinal plants. Alzheimer disease (AD) is an incurable form of dementia which causes severe cognitive dysfunctions. AD is manifested as reduction of neurotransmitter acetylcholine inside the cholinergic system, hence the inhibition of acetylcholinesterase (AChE), enzyme that hydrolyses acetylcholine, has an important role in AD treatment. It is also considered that oxidative stress could be a trigger for AD development ; hence the research of AChE inhibitors is accompanied by assessment of their antioxidative activity. The aim of this work was to test AChE inhibitory activity as well as antioxidative activity of extracts from selected Asteraceae species: Centaurea ragusina L., Inula verbascifolia (Willd.) Hausskn and Pallenis spinosa (Cass.) Cass.