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Anafilaksija je resna, sistemska akutna alergijska reakcija do katere lahko pride zaradi različnih vzrokov in preko različnih mehanizmov. Določanje serumske triptaze je trenutno najpogosteje uporabljen laboratorijski test, ki ima številne pomanjkljivosti in omejitve. Tekom naše študije smo se osredotočili na potencialne nove diagnostične biomarkerje, ki so bolj kot z mastociti povezani z bazofilci. V našo raziskavo smo skupno vključili 204 anafilaktične bolnike, ki so bili na urgentni oddelek Univerzitetne klinike za pljučne bolezni in alergijo Golnik, pripeljani v časovnem okviru od januarja 2011 do decembra 2018. Bolnikom smo odvzeli parne vzorce: v akutni fazi ob anafilaksiji in v bazalnem stanju (približno 1 mesec po reakciji). Najpogostejši vzrok anafilaktičnih reakcij je bil pik / strup kožekrilca. Največ bolnikov je utrpelo najhujšo, IV stopnjo reakcije po Muellerju. Za primerjavo smo skupno vključili 203 zdrave kontrole. Rezultati so pokazali, da pri anafilaksiji, med izbranimi 11-imi kemokini, pride do signifikantnega povišanja samo pri kemokinskem ligandu z motivom C-C tipa 2 (CCL2), njegove koncentracije in odstotek spremembe glede na njegovo bazalno vrednost, pa so povezane tudi s stopnjo reakcije. CCL2 korelira s triptazo, povišanje CCL2 pa je bilo zabeleženo tudi pri 30 % tistih bolnikov, pri katerih se triptaza ni povišala. Med anafilaksijo pride do signifikantnega znižanja absolutnega števila bazofilcev v krvi ter posledično tudi do znižane relativne genske ekspresije vseh treh bazofilnih markerjev ?-podenote visokoafinitetnega IgE receptorja Fc?RI (FCER1A), karboksipeptidaze A3 (CPA3) in L-histidin dekarboksilaze (HDC). Signifikantno znižan je tudi kemokinski receptor z motivom C-C tipa 2 (CCR2) na površini bazofilcev in delež CCR2 pozitivnih celic. Ti rezultati potrjujejo hipotezo o potencialni bazofilni migraciji iz krvi na mesta vnetja med anafilaktično reakcijo. In vitro stimulacija polne krvi je pokazala nižje vrednosti receptorja CCR2 na površini bazofilcev in nespremenjene koncentracije CCL2 v plazmi, kar nakazuje, da aktivirani bazofilci bolnikov s predhodno znanimi akutnimi alergijskimi reakcijami, kemokina CCL2 ne proizvajajo de novo in tudi ne izločajo njegovih povišanih koncentracij kot odgovor na ponovno stimulacijo z istim alergenom. Zaključimo lahko, da bazofilno usmerjeni biomarkerji, poleg mastocitne triptaze, predstavljajo nove potencialne (in dodatne) diagnostične biomarkerje anafilaksije. Anaphylaxis is a serious, systemic acute allergic reaction that occurs due to various triggers and through a variety of mechanisms. Measurement of serum mast cell tryptase is currently the most commonly used laboratory test, but has many limitations. In our study, we focused on potential new diagnostic biomarkers of anaphylaxis that are more basophil- than mast cell-related. We included a total of 204 anaphylactic patients who were presented to the Emergency Department of University Clinic Golnik in the time frame from January 2011 to December 2018. Paired serum samples were collected: in acute phase during the reaction and later in basal state (approximately 1 month after the reaction). The most common trigger of reactions was Hymenoptera venom and most patients suffered the worst, Mueller's IV grade of reaction. A total of 203 healthy controls were included for comparison. The results showed, that during anaphylaxis, among 11 selected chemokines, there is a significant increase in C-C Motif Chemokine Ligand 2 (CCL2) only, and its concentration and the percentage change from baseline values are both related to the reaction severity. CCL2 strongly correlates with tryptase, and an increase in CCL2 was present also in 30 % of those patients in whom tryptase levels were not elevated. During anaphylaxis, there is a significant decrease in the absolute number of basophils and, consequently, a decrease in relative gene expression of all three basophil markers the α-subunit of the high-affinity IgE receptor FcεRI (FCER1A), carboxypeptidase A3 (CPA3) and L-histidine decarboxylase (HDC). Additionally, significant decrease is also observed in C-C Motif Chemokine Receptor 2 (CCR2) on the surface of basophils and in the proportion of CCR2 positive cells. These results support the hypothesis of potential basophil migration during an anaphylactic reaction from blood to the site of inflammation. In vitro whole blood stimulations showed lower CCR2 receptors on basophils surface and unchanged concentrations of CCL2 in plasma, suggesting that activated basophils in patients with previously known acute allergic reactions history, do not produce de novo and secrete elevated levels of CCL2 as a response to re-stimulation with the same allergen. We can conclude that basophil-related biomarkers, in addition to mast cell tryptase, represent new potential (and additional) diagnostic biomarkers of anaphylaxis.

The dynamics of bio-conjugated magnetic nanoparticles suspended in buffer-saline solutions containing target proteins (i.e., analytes) is investigated numerically on a mesoscopic level. To simulate the dispersion of magnetic nanoparticles the dissipative particle dynamics is employed, which allows to study rather large systems, while still retaining important microscopic nanoparticle properties. In addition, the method is coupled to the Landau-Lifshitz-Gilbert equation, describing the dynamics of the magnetic nanocrystals within the macrospin approximation. The binding of multivalent analytes to the recognition ligands of the nanoparticles leads to the formation of clusters of magnetic nanoparticles, which in turn drastically changes the macroscopic magnetic response of the solution. Such colloidal changes are experimentally observable, allowing to explore new approaches to quantify the analyte amount. The ratio of the concentrations between the analytes (biomarkers) and the recognition ligands on the nanoparticles is found to play an important role in the formation and hydrodynamic size of the clusters. The proposed computational framework has great potential to be integrated with experimental efforts to advance the development of nanoparticle-based biosensors for disease diagnostics and other biomedical applications.

Endometrial cancer (EC) is the most frequent gynecological malignancy in developed countries and requires a relatively invasive diagnostic evaluation and operative therapy as the primary therapeutic approach. Angiogenesis is one of the main processes needed for cancer growth and spread. The production of angiogenic factors (AFs) appears early in the process of carcinogenesis. The detection of AFs in plasma and tissue and a better understanding of the angiogenic properties of EC may contribute not only to earlier but also more specific diagnosis and consequently tailored and individual therapeutic approaches. AFs and their receptors also have high potential as binding sites for targeted cancer therapy. In this review, we discuss angiogenesis in EC and the characteristics of the AFs that most contribute to angiogenesis in EC. We also highlight therapeutic strategies that target angiogenesis as potential EC therapy.

Cancer was the leading cause of death in 2020 worldwide, consequently it exerts a huge burden on the health care system and on people’s lives. An early detection and correct treatment are crucial for curing cancer. Biomarkers are an effective tool used by clinicians to predict who will benefit from a certain treatment. One such potential biomarker is the protein TP53INP2, more specifically for the anticancer treatment TRAIL. TP53INP2 is an intrinsically disordered protein with many different roles in the cell. Protein levels of TP53INP2 are increased when kinases such as Checkpoint 1 kinase (Chk1), Casein kinase 2 (CK2), and Cyclin-dependent kinases 4 or 6 (CDK4,6) are inhibited. In this thesis it is shown that in the Ishikawa, SH-SY5Y and MDA-MB-231 cell lines TP53INP2 levels increase when treated with inhibitors Palbociclib (CDK4,6 inhibitor), Silmitasertib (CK2 inhibitor), or Chk1 inhibitor. Moreover, this increase in protein abundance is not due to an increase in gene expression, therefore phosphorylation probably marks TP53INP2 for degradation. TP53INP2 sensitizes cancer cells to synergistic treatment with TRAIL and kinase inhibitors of CDK4,6, CK2, and Chk1. The Ishikawa cells die more often by apoptosis when treated with the combined treatment (TRAIL and kinase inhibition) than when treated with TRAIL alone. However, the loss of TP53INP2 abrogated apoptosis, suggesting that the levels of TP53INP2 could be used as a biomarker for this combined treatment. Our results indicate that TP53INP2 is a potential biomarker for combination anticancer treatment with TRAIL and the above mentioned kinase inhibitors. Further research is needed to better understand the full importance of this protein in the fight against cancer. Rak je bil v letu 2020 najpogostejši vzrok smrti na svetu in tako predstavlja veliko breme za zdravstveni sistem in življenja ljudi. Zgodnje odkrivanje in pravilno zdravljenje sta ključna za ozdravitev teh bolezni. Biomarkerji so učinkovito orodje, ki ga zdravniki uporabljajo za napovedovanje, komu bo določeno zdravljenje koristilo. Eden od takšnih primerov je protein TP53INP2, ki je potencialni biomarker zlasti za protirakavo zdravilo TRAIL. TP53INP2 je intrinzično neurejen protein s številnimi različnimi vlogami v celici. Raven proteina TP53INP2 se poveča, kadar so kinaze, kot so kinaza Chk 1, kazein kinaza 2 (CK2) ter od ciklina odvisni kinazi 4 in 6 (CDK4,6), inhibirane. V tem magistrskem delu smo ugotovili, da se v celicah celičnh linij Ishikawa, SH-SY5Y in MDA-MB-231 raven proteina TP53INP2 poveča, če jih izpostavimo inhibitorjem Palbociclib (zaviralec CDK4,6), Silmitasertib (zaviralec CK2) oziroma inhibitorjem Chk1. Poleg tega to povečanje koncentracije proteina ni posledica povečanja izražanja gena, tako da fosforilacija verjetno označi TP53INP2, da se ta razgradi. TP53INP2 senzibilizira rakave celice tako, da te postanejo bolj občutljive za kombinirano sinergistično zdravljenje s TRAIL in inhibitorji kinaz CDK4,6, CK2 in Chk1. Celice Ishikawa v večji meri umirajo z apoptozo, če jih zdravimo s kombiniranim zdravljenjem (TRAIL in inhibitorji kinaz), kakor če jih zdravimo samo s TRAIL. Izguba TP53INP2 pa je odpravila apoptozo, kar nakazuje, da bi lahko raven proteina TP53INP2 uporabili kot biomarker za to kombinirano zdravljenje. Naši rezultati kažejo, da je TP53INP2 potencialni biomarker za kombinirano protirakavo zdravljenje s TRAIL in prej omenjenimi zaviralci kinaz.

We have recently shown that minor subclinical creatinine dynamic changes enable the excellent detection of acute kidney injury (AKI) within 6–12 h after cardiac surgery. The aim of the present study was to examine a combination of neutrophil gelatinase-associated lipocalin (NGAL), cystatin C (CysC) and creatinine for enhanced AKI detection early after cardiac surgery. Elective patients with normal renal function undergoing cardiac surgery using cardiopulmonary bypass were enrolled. Concentrations of plasma NGAL, serum CysC and serum creatinine were determined after the induction of general anesthesia, at the termination of the cardiopulmonary bypass and 2 h thereafter. Out of 119 enrolled patients, 51 (43%) developed AKI. A model utilizing an NGAL, CysC and creatinine triple biomarker panel including sequential relative changes provides a better prediction of cardiac surgery-associated acute kidney injury than any biomarker alone already 2 h after the termination of the cardiopulmonary bypass. The area under the receiver-operator curve was 0.77, sensitivity 77% and specificity 68%.

The rapid spread of COVID-19 outbreak lead to a global pandemic declared in March 2020. The common features of corona virus family helped to resolve structural characteristics and entry mechanism of SARS-CoV-2. However, rapid mutagenesis leads to the emergence of new strains that may have different reproduction rates or infectivity and may impact the course and severity of the disease. Host related factors may also play a role in the susceptibility for infection as well as the severity and outcomes of the COVID-19. We have performed a literature and database search to summarize potential viral and host-related genomic and epigenomic biomarkers, such as genetic variability, miRNA, and DNA methylation in the molecular pathway of SARS-CoV-2 entry into the host cell, that may be related to COVID-19 susceptibility and severity. Bioinformatics tools may help to predict the effect of mutations in the spike protein on the binding to the ACE2 receptor and the infectivity of the strain. SARS-CoV-2 may also target several transcription factors and tumour suppressor genes, thus influencing the expression of different host genes and affecting cell signalling. In addition, the virus may interfere with RNA expression in host cells by exploiting endogenous miRNA and its viral RNA. Our analysis showed that numerous human miRNA may form duplexes with different coding and non-coding regions of viral RNA. Polymorphisms in human genes responsible for viral entry and replication, as well as in molecular damage response and inflammatory pathways may also contribute to disease prognosis and outcome. Gene ontology analysis shows that proteins encoded by such polymorphic genes are highly interconnected in regulation of defense response. Thus, virus and host related genetic and epigenetic biomarkers may help to predict the course of the disease and the response to treatment.

miRNAs play an important role in neurodegenerative diseases. Many miRNA-target gene interactions (MTI) have been experimentally confirmed and associated with Alzheimer’s disease (AD). miRNAs may also be contained within extracellular vesicles (EVs), mediators of cellular communication and a potential source of circulating biomarkers in body fluids. Therefore, EV-associated miRNAs (EV-miRNAs) in peripheral blood could support earlier and less invasive AD diagnostics. We aimed to prioritize EV-related miRNA with AD-related genes and to identify the most promising candidates for novel AD biomarkers. A list of unique EV-miRNAs from the literature was combined with a known set of AD risk genes and enriched for MTI. Additionally, miRNAs associated with the AD phenotype were combined with all known target genes in MTI enrichment. Expression in different sample types was analyzed to identify AD-associated miRNAs with the greatest potential as AD circulating biomarkers. Four common MTI were observed between EV-miRNAs and AD-associated miRNAs: hsa-miR-375–APH1B, hsa-miR-107–CDC42SE2, hsa-miR-375–CELF2, and hsa-miR-107–IL6. An additional 61 out of 169 unique miRNAs (36.1%) and seven out of 84 unique MTI (8.3%), observed in the body fluids of AD patients, were proposed as very strong AD-circulating biomarker candidates. Our analysis summarized several potential novel AD biomarkers, but further studies are needed to evaluate their potential in clinical practice.

Endometriosis is an estrogen-dependent inflammatory disease affecting women in their reproductive age. Due to non-specific symptoms, women with endometriosis are often misdiagnosed or are accurately diagnosed only after several years. Diagnosis of peritoneal endometriosis is especially challenging and relies only on laparoscopic surgery. To date, different molecules have been proposed as potential non-invasive biomarkers of endometriosis; however, none have been confirmed as clinically useful. Therefore, this study aimed to discover novel plasma biomarker candidates for peritoneal endometriosis using an antibody array platform. This study included patients with endometriosis-like symptoms characterized by the absence (controls) or presence of peritoneal endometriosis (cases) after laparoscopic surgery and histological evaluation. Patients were further divided into secretory and proliferative groups, according to the phase of their menstrual cycle. Their plasma samples were collected and analyzed on an antibody array platform targeting more than 1350 proteins with over 1820 antibodies. In the proliferative group, the analysis revealed three differential proteins between cases and controls: ITB3, ITA2B2, and ACVL-1. In the secretory group, none of the examined proteins reached the log-fold change (logFC) and significance thresholds simultaneously. The potential of the identified differential proteins as plasma biomarker candidates for peritoneal endometriosis should be evaluated on a larger cohort, and their role in endometriosis should be investigated in further studies.

Exposure to actual or simulated microgravity results in alterations of renal function, fluid redistribution, and bone loss, which is coupled to a rise of urinary calcium excretion. We provided evidence that high calcium delivery to the collecting duct reduces local Aquaporin 2 (AQP2)-mediated water reabsorption under vasopressin action, thus limiting the maximal urinary concentration to reduce calcium saturation. To investigate early renal adaptation into simulated microgravity, we investigated the effects of 10 days of strict bedrest in 10 healthy volunteers. We report here that 10 days of inactivity are associated with a transient, significant decrease (day 5) in vasopressin (copeptin) paralleled by a decrease in AQP2 excretion, consistent with an increased central volume to the heart, resulting in reduced water reabsorption. Moreover, bedrest caused a significant increase in calciuria secondary to bone demineralization paralleled by a decrease in PTH. Urinary osteopontin, a glycoprotein exerting a protective effect on stone formation, was significantly reduced during bedrest. Moreover, a significant increase in adrenomedullin (day 5), a peptide with vasodepressor properties, was observed at day 5, which may contribute to the known reduced orthostatic capacity post-bedrest. We conclude that renal function is altered in simulated microgravity and is associated with an early increase in the risk of stone formation and reduced orthostatic capacity post-bedrest within a few days of inactivity.

Preoperative determination of the extent of endometrial cancer (EC) would avoid the complications associated with radical surgery. Screening of patients' plasma biomarkers might enable a more precise diagnosis of EC and a tailored treatment approach. This prospective case-control monocentric pilot study included 76 postmenopausal women (38 endometrioid EC patients and 38 control patients with benign gynecological conditions), and 37 angiogenic factors (AFs) were investigated as potential biomarkers for EC. AF concentrations in preoperative plasma samples were measured using Luminex xMAP$^{TM}$ multiplexing technology. The plasma levels of sTie-2 and G-CSF were significantly lower in EC compared to control patients, whereas the plasma levels of leptin were significantly higher in EC patients. Neuropilin-1 plasma levels were significantly higher in patients with type 2 EC (grade 3) compared to patients with lower grade cancer or controls. Follistatin levels were significantly higher in patients with lymphovascular invasion, and IL-8 plasma levels were significantly higher in patients with metastases. If validated, the plasma concentrations of the indicated AFs could represent an important additional diagnostic tool for the early detection and characterization of EC. This could guide the decision-making on the extent of surgery. Further studies with larger patient numbers are currently ongoing.

Alzheimer’s Disease (AD) is a progressive neurodegenerative disorder and the most common cause of dementia; however, early diagnosis of the disease is challenging. Research suggests that biomarkers found in blood, such as microRNAs (miRNA), may be promising for AD diagnostics. Experimental data on miRNA–target interactions (MTI) associated with AD are scattered across databases and publications, thus making the identification of promising miRNA biomarkers for AD difficult. In response to this, a list of experimentally validated AD-associated MTIs was obtained from miRTarBase. Cytoscape was used to create a visual MTI network. STRING software was used for protein–protein interaction analysis and mirPath was used for pathway enrichment analysis. Several targets regulated by multiple miRNAs were identified, including: BACE1, APP, NCSTN, SP1, SIRT1, and PTEN. The miRNA with the highest numbers of interactions in the network were: miR-9, miR-16, miR-34a, miR-106a, miR-107, miR-125b, miR-146, and miR-181c. The analysis revealed seven subnetworks, representing disease modules which have a potential for further biomarker development. The obtained MTI network is not yet complete, and additional studies are needed for the comprehensive understanding of the AD-associated miRNA targetome.

Preterm birth (before 37 weeks gestation) accounts for ~10% of births worldwide and remains one of the leading causes of death in children under 5 years of age. Preterm born adults have been consistently shown to be at an increased risk for chronic disorders including cardiovascular, endocrine/metabolic, respiratory, renal, neurologic, and psychiatric disorders that result in increased death risk. Oxidative stress was shown to be an important risk factor for hypertension, metabolic syndrome and lung disease (reduced pulmonary function, long-term obstructive pulmonary disease, respiratory infections, and sleep disturbances). The aim of this study was to explore the differences between preterm and full-term male participants’ levels of urine and fecal proton nuclear magnetic resonance (1H-NMR) metabolomes, during rest and exercise in normoxia and hypoxia and to assess general differences in human gut-microbiomes through metagenomics at the level of taxonomy, diversity, functional genes, enzymatic reactions, metabolic pathways and predicted gut metabolites. Significant differences existed between the two groups based on the analysis of 1H-NMR urine and fecal metabolomes and their respective metabolic pathways, enabling the elucidation of a complex set of microbiome related metabolic biomarkers, supporting the idea of distinct host-microbiome interactions between the two groups and enabling the efficient classification of samples; however, this could not be directed to specific taxonomic characteristics.

Glioblastoma (GBM), the most common primary brain tumor, is a complex and extremely aggressive disease. Despite recent advances in molecular biology, there is a lack of biomarkers, which would improve GBM&rsquo, s diagnosis, prognosis, and therapy. Here, we analyzed by qPCR the expression levels of a set of miRNAs in GBM and lower-grade glioma human tissue samples and performed a survival analysis in silico. We then determined the expression of same miRNAs and their selected target mRNAs in small extracellular vesicles (sEVs) of GBM cell lines. We showed that the expression of miR-21-5p was significantly increased in GBM tissue compared to lower-grade glioma and reference brain tissue, while miR-124-3p and miR-138-5p were overexpressed in reference brain tissue compared to GBM. We also demonstrated that miR-9-5p and miR-124-3p were overexpressed in the sEVs of GBM stem cell lines (NCH421k or NCH644, respectively) compared to the sEVs of all other GBM cell lines and astrocytes. VIM mRNA, a target of miR-124-3p and miR-138-5p, was overexpressed in the sEVs of U251 and U87 GBM cell lines compared to the sEVs of GBM stem cell line and also astrocytes. Our results suggest VIM mRNA, miR-9-5p miRNA, and miR-124-3p miRNA could serve as biomarkers of the sEVs of GBM cells.

In the last ten years, clinical oncology has been revolutionized by the introduction of oncological immunotherapy, mainly in the form of immune checkpoint inhibitors (ICIs) that transformed the standard of care of several advanced solid malignancies. Using ICIs for advanced gynecological cancers has yielded good results, especially for endometrial cancer. In ovarian or cervical cancer, combining ICIs with other established agents has shown some promise. Concurrently with the clinical development of ICIs, biomarkers that predict responses to such therapy have been discovered and used in clinical trials. The translation of these biomarkers to clinical practice was somewhat hampered by lacking assay standardization and non-comprehensive reporting of biomarker status in trials often performed on a small number of gynecological cancer patients. We can expect increased use of ICIs combined with other agents in gynecological cancer in the near future. This will create a need for reliable response prediction tools, which we believe will be based on biomarker, clinical, and tumor characteristics. In this article, we review the basic biology of ICIs and response prediction biomarkers, as well as the latest clinical trials that focus on subgroup effectiveness based on biomarker status in gynecological cancer patients.

Na osnovi z bakrom katalizirane azometin imin-alkin cikloadicije smo sintetizirali pet novih, pod UV svetlobo fluorescentnih cilkloaduktov. Kot alkin smo uporabili inone sintetizirane iz anhidrida jantarne kisline ter iz propiolne kisline. Inoni so bili v namen vezave na aminske skupine biomolekul aktivirani ali z N-hidroksisukcinimidom (NHS) ali z benzotriazolom (BtH). Na inone smo ciklizirali različne N,N-ciklične azometin imine, reakcijo pa smo katalizirali z Cu0 v obliki bakrovega prahu ali bakrove žičke. Za začetni preizkus biokonjugacije smo z NHS in BtH aktivirane fluorescentne cikloadukte vezali na (aminometil)polistiren (AMP). Na AMP smo vezali tudi z NHS in BtH aktivirane ter jih v fluorescentne produkte z CuAIAC ciklizirali na (aminometil) polistirenu. Z BtH aktivirane cikloadukte smo vezali še na realno biomolekulo, goveji serumski albumin (BSA), ter merili emisijske spektre tako označenih proteinov. We used copper-catalyzed azomethine imine–alkyne cycloaddition (CuAIAC) to prepare five novel cycloadducts that exhibited fluorescence under UV light. As alkynes in CuAIAC we used two ynones that we synthesized one from succinic anhydride and the other from propiolic acid. The ynones were activated with either N-hydroxysuccinimide (NHS) or benzotriazole (BtH) in order to facilitate a covalent bonding with amine groups of biomolecules, thus making bioconjugation possible. Ynones were cyclized with various N,N-cyclic azomethine imines and the reaction was catalyzed with Cu0 in the form of copper particles or copper wire. NHS and BtH activated fluorescent cycloadducts were first bonded to (aminomethyl)polystyrene (AMP) as a preliminary test of bioconjugation. We also bonded AMP with activated ynones and then performed CuAIAC on the ynone-marked AMP to again yield a fluorescent polystyrene. Lastly, we bonded BtH activated cycloadducts onto amine groups of bovine serum albumin (BSA) and measured their emission spectra.

Gigantocelični arteritis (GCA) je najpogostejši primarni sistemski vaskulitis pri odraslih po 50. letu starosti v Evropi. Prizadene velike in srednje velike arterije in vnetni proces, ki zožuje ali popolnoma zapre svetlino žile, lahko dovede do hudih/trajnih ishemičnih zapletov kot so oslepitev, možganska kap ali miokardni infarkt. V zadnjem desetletju se je z vključitvijo slikovnih preiskav v diagnostični postopek pomembno skrajšal čas do prepoznave bolezni (t.i. zgodnji GCA). Pospešena obravnava bolnikov (ang. “fast track clinic”) je vodila v zmanjšanje pojavnosti najresnejših ishemičnih zapletov bolezni in znižanje stroškov zdravljenja. Vendar pa bolezen praviloma poteka kronično, s poslabšanji, kar skupaj s kroničnim glukokortikoidnem zdravljenjem vodi v kopičenje okvar organov in tkiv. Prav zato se intenzivno preučuje patogeneza bolezni, z možnostjo implementacije izsledkov kot so sodobne molekularno in celično usmerjene tarčne terapije. Glavni cilji našega preglednega članka so bili: a) analiza raziskav z navedenim časom trajanja od začetka simptomov do postavitve diagnoze, b) raziskava obetavnih molekularnih tarč za zdravljenje GCA in c) prepoznava klinično pomembnih celičnih podtipov. Najbolj obetavne tarčne molekule za tarčno zdravljenje so IL-6, IL-12/IL-23 in citototoksični z limfociti T povezan protein 4, medtem ko terapija z zaviralci TNF-α ni bila uspešna. Kliničnih raziskav z učinkovinami, usmerjenimi proti IL-17, še ni. V prispevku pa smo se dotaknili tudi drugih potencialnih terapevtskih tarč, vključno z molekulami, ki sodelujejujo v signalnih poteh.

Glioblastoma (GB) is the most aggressive brain malignancy. Although some potential glioblastoma biomarkers have already been identified, there is a lack of cell membrane-bound biomarkers capable of distinguishing brain tissue from glioblastoma and/or glioblastoma stem cells (GSC), which are responsible for the rapid post-operative tumor reoccurrence. In order to find new GB/GSC marker candidates that would be cell surface proteins (CSP), we have performed meta-analysis of genome-scale mRNA expression data from three data repositories (GEO, ArrayExpress and GLIOMASdb). The search yielded ten appropriate datasets, and three (GSE4290/GDS1962, GSE23806/GDS3885, and GLIOMASdb) were used for selection of new GB/GSC marker candidates, while the other seven (GSE4412/GDS1975, GSE4412/GDS1976, E-GEOD-52009, E-GEOD-68848, E-GEOD-16011, E-GEOD-4536, and E-GEOD-74571) were used for bioinformatic validation. The selection identified four new CSP-encoding candidate genes—CD276, FREM2, SPRY1, and SLC47A1—and the bioinformatic validation confirmed these findings. A review of the literature revealed that CD276 is not a novel candidate, while SLC47A1 had lower validation test scores than the other new candidates and was therefore not considered for experimental validation. This validation revealed that the expression of FREM2—but not SPRY1—is higher in glioblastoma cell lines when compared to non-malignant astrocytes. In addition, FREM2 gene and protein expression levels are higher in GB stem-like cell lines than in conventional glioblastoma cell lines. FREM2 is thus proposed as a novel GB biomarker and a putative biomarker of glioblastoma stem cells. Both FREM2 and SPRY1 are expressed on the surface of the GB cells, while SPRY1 alone was found overexpressed in the cytosol of non-malignant astrocytes.

Hemorrhagic fever with renal syndrome (HFRS) and Crimean-Congo hemorrhagic fever (CCHF) are important viral hemorrhagic fevers (VHF), especially in the Balkan region. Infections with Dobrava or Puumala orthohantavirus and Crimean-Congo hemorrhagic fever orthonairovirus can vary from a mild, nonspecific febrile illness, to a severe disease with a fatal outcome. The pathogenesis of both diseases is poorly understood, but it has been suggested that a host&rsquo, s immune mechanism might influence the pathogenesis of the diseases and survival. The aim of our study is to characterize cytokine response in patients with VHF in association with the disease progression and viral load. Forty soluble mediators of the immune response, coagulation, and endothelial dysfunction were measured in acute serum samples in 100 HFRS patients and 70 CCHF patients. HFRS and CCHF patients had significantly increased levels of IL-6, IL-12p70, IP-10, INF-&gamma, TNF-&alpha, GM-CSF, MCP-3, and MIP-1b in comparison to the control group. Interestingly, HFRS patients had higher concentrations of serum MIP-1&alpha, MIP-1&beta, which promote activation of macrophages and NK cells. HFRS patients had increased concentrations of IFN-&gamma, and TNF-&alpha, while CCHF patients had significantly higher concentrations of IFN-&alpha, and IL-8. In both, CCHF and HFRS patients&rsquo, viral load significantly correlated with IP-10. Patients with fatal outcome had significantly elevated concentrations of IL-6, IFN-&alpha, 2 and MIP-1&alpha, while GRO-&alpha, chemokine related to activation of neutrophils and basophils, was downregulated. Our study provided a comprehensive characterization of biomarkers released in the acute stages of CCHF and HFRS.

Determination of food authenticity is an important issue in quality control and food safety. Recent studies predict a growing demand for natural and more authentic food and beverage products. The quality and authenticity of apple juice is also of a great economic importance since the popularity and demand for apple juice consumption has increased. The growth of the market for organically produced apples and apple juice is due to the increasing demand for healthy food requirements, protection of the environment and the promotion of biotic diversity. Organic foods have a higher nutritional and health value, but they are more expensive, because their production is more difficult and less profitable. In addition to how food is produced, consumers are increasingly placing emphasis on food products of specific region, which are known for their unique natural flavours and taste. The presented thesis is based on four separate but closely interrelated studies, in which a combination of different isotopic ratios of bioelements (2H/1H, 13C/12C, 15N/14N, 18O/16O), multi-element analysis, and major primary and secondary metabolite profiles were exploited to differentiate the geographical origin and agricultural production practice (organic vs integrated/conventional) of Slovenian apples. These parameters were used to establish the first database of authentic Slovenian apple juice, which can be used to verify the authenticity of commercially available apple juice in Slovenia. The first preliminary study was entitled “Organic Cultivation ~ Geographical Origin (OCGO)” and was performed using apples from the 2009 growing season. Its aim was to examine the use of stable isotope and multi-element data for determining the geographical origin and agricultural production practice of fresh apple juices. Fruits of six apple (Malus domestica Borkh) cultivars (Topaz, Idared, Golden Delicious, Goldrush, Gala, Gloster) were collected from four different geographical regions of Slovenia (Alpine, Dinaric, Pannonian and Mediterranean) grown under organic and integrated/conventional orchard management systems. The results revealed that stable isotope parameters in sugar, pulp and water were the most significant variables for differentiating between the regions. Good separation was achieved between the geographical regions in Slovenia based on the δ18O and δ2H values in water and Rb and S levels in the apple fruit juice. The most significant variables that distinguished between organically and integrated/conventionally cultivated apples were the 15N/14N ratio and antioxidant activity of the apple juice. Significant differences were also observed in the ascorbic acid content of the juice. Based on these results the number and types of apples and the minimum number of samples needed from the same region for determining geographical origin were determined. The second study was called “Organic ~ Conventional Apple Cultivation” (OCAC) and was performed in 2010 and 2011 in a Gala apple orchard. The aim was to determine the effect of different fertilizers allowed either in organic or conventional/integrated agricultural regimes on different parameters. Quality parameters, isotopic composition of C in sugars and in pulp together with N and elemental analysis were investigated. The following five fertilizers were applied: Biosol and Plantella organic (organic) and Ca cyanamide, KAN and UREA (mineral) at a rate of 60 and 120 kg of nitrogen per hectare. From the obtained data it was possible to differentiate between organic and integrated/conventional apple production when taking into account the following parameters: mass, skin and flesh firmness (SFF), total soluble solids (TSS), and the content of Cl as well as δ15N and δ13C in the pulp. The “Organic Cultivation ~ Geographical Origin” (OCGO) study, which took place during the 2011 and 2012 growing seasons included a greater number of samples and samples from five different geographical regions: Alpine, Dinaric, Nedavne študije nakazujejo na povečevanje zanimanja za pristnost hrane in pijače s strani potrošnika. Določitev pristnosti pa je pomembna tudi z vidika nadzora nad varnostjo in kakovostjo živil. Povečevanje priljubljenosti in povpraševanje po jabolčnem soku, pa vpliva na to, da sta kakovost in pristnost le-tega tudi velikega gospodarskega pomena. Rast tržišča z ekološkimi pridelki, med katerimi so tudi jabolka in iz njih pridelan jabolčni sok, je pogojena s povečanim povpraševanjem po zdravi hrani, z zahtevami po varstvu okolja in s spodbujanjem biotske raznovrstnosti. Organska hrana ima večjo hranilno vrednost in je bolj zdrava, vendar pa je na drugi strani tudi dražja, ker je njena proizvodnja težja in zaradi tega manj donosna. Poleg pridelave dajejo potrošniki vedno večji poudarek tudi geografskemu izvoru proizvodov, ki so zaradi tega edinstveni po okusu in vonju. Predložena doktorska disertacija obsega 4 ločene, vendar med seboj tesno povezane poskuse, v kateri sem želela z določitvijo izotopskih razmerij različnih bioelementov (2H/1H, 13C/12C, 15N/14N, 18O/16O), elementno sestavo, ter profilom glavnine primarnih in sekundarnih metabolitov najti tiste parametre, ki omogočajo razlikovanje slovenskih jabolk po geografskem poreklu ali glede na način pridelave (organska vs. integrirana/konvencionalna). Ustvarjena je bila tudi prva baza podatkov pristnih slovenskih jabolčnih sokov, ki je bila kasneje uporabljena za testiranje komercialnih jabolčnih sokov dostopnih na slovenskem trgu. Predposkus imenovan OCGO (Ekološka pridelava ~ Geografsko poreklo) 2009, je bil izveden v rastni sezoni 2009. Namen tega poskusa je bil preučiti uporabo stabilnih izotopov in multi-elementno analizo za določanje geografskega porekla in načina pridelave jabolčnih sokov. Zbrala sem vzorce šestih sort jabolk (gala, gloster, gold rush, zlati delišes, topaz in idared) iz štirih slovenskih geografskih regij (panonska, mediteranska, dinarska in alpska). Sedem vzorcev je bilo ekološko, dvanajst pa konvencionalno pridelanih. Rezultati so pokazali, da na ločitev po geografskem poreklu signifikantno vplivajo stabilni izotopi določeni v sladkorjih, pulpi in vodi, ki so sestavni del jabolčnega soka. Jabolka iz omenjenih slovenskih regij so bila ločena na podlagi δ18O in δ2H vrednosti v vodi ter vsebnosti rubidija in žvepla v iztisnjenem jabolčnem soku. Kot statistično najbolj signifikantni parametri za razlikovanje med organsko in integrirano/konvencionalno pridelanimi jabolki pa so se izkazali 15N/14N razmerje, antioksidacijski potencial in vsebnost vitamina C. Na podlagi opisanega predposkusa je bilo določeno potrebno število vzorcev in sort jabolk ter minimalno število vzorcev iz posamezne slovenske geografske regije za razvrstitev le teh glede na geografsko poreklo. Drugi poskus, ki sem ga poimenovala Organska ~ konvencionalna pridelava jabolk (OCAC), je potekal v letih 2010 in 2011, v sadovnjaku jabolk sorte Gala. Cilj je bil ugotoviti vpliv različnih gnojil dovoljenih v ekološki ali v integrirani/konvencionalni pridelavi na različne parametre. Določeni so bili parametri kakovosti, izotopska sestava C v sladkorjih ter C in N v pulpi ter elementna analiza. Jablanam so bila aplicirana naslednja gnojila: Biosol in Plantella organik (organska) ter apneni dušik, KAN in UREA (mineralna) v količini, s katero je bilo zagotovljeno 60 oziroma 120 kg dušika na hektar. Rezultati so pokazali, da je razlikovanje med jabolki, pognojenimi z različnimi gnojili mogoče na podlagi naslednjih parametov: povprečne mase plodov, trdote jabolk (SFF), celokupne suhe snovi (TSS), vsebnosti klora ter δ15N in δ13C vrednosti v pulpi. Poskus “Ekološka pridelava ~ Geografsko poreklo" (OCGO) je potekal v rastnih sezonah 2011 in 2012 in je glede na OCGO 2009 vključeval večje število vzorcev iz petih različnih geografskih regij: alpske, dinarske, panonske, mediteranske in submediteranske. Izbrane so bile tri sorte jabolk: idared in zlati delišes pridelana na integriran/konvencionale

Analiza potencijalnog toksičnog učinka neonikotinoidnog insekticida imidakloprida i njegovih transformacijskih produkata prilikom okolišne degradacije ili metabolizma na različitim nivoima biološke organizacije uz korištenje in vivo i in vitro testnih modela.