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Hot spring biofilms are stable, highly complex microbial structures. They form at dynamic redox and light gradients and are composed of microorganisms adapted to the extreme temperatures and fluctuating geochemical conditions of geothermal environments. In Croatia, a large number of poorly investigated geothermal springs hosts biofilm communities. Here, we investigated the microbial community composition of biofilms collected over several seasons at 12 geothermal springs and wells. We found biofilm microbial communities to be temporally stable and highly dominated by Cyanobacteria in all but one high-temperature sampling site (Bizovac well). Of the physiochemical parameters recorded, temperature was found to have the strongest influence on biofilm microbial community composition. Besides Cyanobacteria, the biofilms were mainly inhabited by Chloroflexi, Gammaproteobacteria and Bacteroides. In a series of incubations with Cyanobacteria-dominated biofilms from Tuhelj spring, and Chloroflexi- and Proteobacteria-dominated biofilms from Bizovac well, we stimulated either chemoorganotrophic or chemolithotrophic community members, to determine the fraction of microorganisms in biofilms dependent on organic carbon (in situ predominantly produced via photosynthesis) versus energy derived from geochemical redox gradients (here simulated by addition of thiosulfate). We found a surprisingly similar level of activity in response to all substrates in these two distinct biofilm communities and observed microbial community composition and hot spring geochemistry to be poor predictors of microbial activity in the studies systems.

In Croatia, a variety of geothermal springs with a wide temperature range and varied hydrochemical conditions exist, and they may harbor different niches for the distribution of microbial communities. In this study, 19 different sites, mainly located in central and eastern Croatia, were selected for primary characterization of spring hydrochemistry and microbial community composition. Using 16S rRNA gene amplicon sequencing, it was found that the bacterial communities that dominated most geothermal waters were related to Proteobacteria and Campylobacteria, while most archaeal sequences were related to Crenarchaeota. At the genus level, the prokaryotic community was highly site-specific and was often dominated by a single genus, including sites dominated by Hydrogenophilus, Sulfuricurvum, Sulfurovum, Thiofaba and Nitrospira, while the most abundant archaeal genera were affiliated to the ammonia- oxidizing archaea, Candidatus Nitrosotenuis and Candidatus Nitrososphaera. Whereas the microbial communities were overall highly location- specific, temperature, pH, ammonia, nitrate, total nitrogen, sulfate and hydrogen sulfide, as well as dissolved organic and inorganic carbon, were the abiotic factors that significantly affected microbial community composition. Furthermore, an aquifer-type effect was observed in the community composition, but there was no pronounced seasonal variability for geothermal spring communities (i.e. the community structure was mainly stable during the three seasons sampled). These results surprisingly pointed to stable and geographically unique microbial communities that were adapted to different geothermal water environments throughout Croatia. Knowing which microbial communities are present in these extreme habitats is essential for future research. They will allow us to explore further the microbial metabolisms prevailing at these geothermal sites that have high potential for biotechnological uses, as well as the establishment of the links between microbial community structure and the physicochemical environment of geothermal waters.

Microbial biofilms in hot springs represent complex, self-sustaining communities with complete biogeochemical cycles and food webs. Due to the harsh environmental conditions and complex interactions of individual microorganisms in the biofilms, it is not trivial to understand the drivers behind the observed community structure and mechanisms of biogeochemical cycling in these environments. While a portion of the biofilm- dwelling microorganisms can be isolated into pure culture, pure culture activities do not necessary reflect the in situ activities and crossfeeding interactions in the biofilm community. Without the need for prior cultivation, bioorthogonal non- canonical amino acid tagging (BONCAT) can be used in combination with fluorescence activated cell sorting (FACS) to identify translationally active taxa in the community and their substrate preferences. The aim of this study was to investigate the community composition and metabolic activity of two hot spring biofilms in a range of enviromentaly relevant substrates. Translational activity was monitored by incorporation of a synthetic amino acid L- homopropargylglycine (HPG), whereupon HPG- containing proteins were detected by a copper- catalysed azide-alkyne click chemistry reaction. FACS was used to sort active members of a biofilm community whose taxonomic identification was performed using 16S rRNA gene amplicon sequencing. Although the sites were differing in geochemistry and the biofilms in overall community composition, biofilms from both sites showed a similar pattern in substrate preference. Metabolic activity was highest when biofilm material was incubated with simple organic substrates (acetate and pyruvate), whereas the preferred inorganic substrate for the chemolitotrophs and chemoorganotrophs was thiosulfate in both cases.

Mikrobni biofilmovi iz geotermalnih izvora su kompleksne, samoodržive zajednice čiji je sastav određen teškim uvjetima u kojima egzistiraju. Osim tlaka i visokih temperatura, prisutnost svjetla i dostupnost različitih elektron donora i akceptora predstavljaju glavne čimbenike koji utječu na metaboličku aktivnost ovih zajednica. Ciljevi istraživanja bili su identifikacija i vizualizacija aktivnih populacija biofilma iz Bizovačkih toplica u različitim, ekološki relevantnim supstratima i uvjetima. Biofilm je inkubiran tijekom 24 i 48 sati na temperaturi bušotine u acetatu, piruvatu, glukozi i tiosulfatu te sa i bez prisustva svjetla. BONCAT (eng. Bioorthogonal Non-Canonical Amino Acid Tagging) metoda omogućila je označavanje translacijski aktivnih mikroorganizama inkorporacijom sintetske aminokiseline HPG (eng. L-homopropargylglycine) u novo sintetizirane proteine fluorescentno obojane azid- alkin KLIK reakcijom. Potom, filogenetski identitet aktivnih stanica i staničnih agregata određen je CARD-FISH (eng. Catalyzed Reporter Deposition rRNA- targeted Fluorescence in Situ Hybridization) metodom ciljajući najzastupljenije skupine, identificirane pomoću 16S rRNA amplikona sekvenci. Uzorci su pregledani na invertnom mikroskopu (Leica DMi8, Njemačka) te je analiza biovolumena provedena u daime software-u (v. 2.2.3.). Utjecaj svjetla doveo je do niže aktivnosti Gammaproteobacteria, Chloroflexia i Campylobacterota u usporedbi s inkubacijom u mraku, dok je najveći postotak aktivnih Thermoanaerobacteriuma bio prilikom dvodnevne inkubacije na svjetlu. Najveća aktivnost Gammaproteobacteria i Chloroflexia uočena je prilikom inkubacija s acetatom i tiosulfatom. Duža inkubacija u piruvatu dovela je do veće aktivnosti svih skupina u usporedbi s aktivnošću tijekom prvih 24 sata. Gammaproteobacterie imale su podjednak postotak aktivnosti (28, 2%) prilikom inkubacije s glukozom kroz oba dana.

This chapter will explore the impact of cognitive skills on education. In the case of OECD (Organisation for Economic Co-operation and Development) countries, it will be examined the relation between the level of education to the economic situation of a state. Case study work is based on a statistical approach of OECD countries ranking, based on The Global Index of Cognitive Skills and Educational Attainment (GICSES) and Ivanovic distance (I-distance). The chapter will be presented to rank these countries based on the value of the global index. Using I-distance method it will be formed a new order, and then it will be carried out a comparative analysis of these two ways of ranking. The aim of the chapter is to present a new approach to the evaluation of a composite indicator based on the multivariate statistical analysis.

ST2 is a member of the IL-1 receptor family and IL-33 was recently identified as its natural ligand. The IL-33/ST2 pathway regulates Th1/Th2 immune responses in autoimmune and inflammatory conditions, but the role of ST2 signaling in tumor growth and metastasis has not been investigated. We aimed to investigate whether ST2 gene deletion affects tumor appearance, growth, and metastasis, and antitumor immunity in an experimental metastatic breast cancer model. Deletion of ST2 in BALB/c mice bearing mammary carcinoma attenuated tumor growth and metastasis, which was accompanied by increased serum levels of IL-17, IFN-γ, and TNF-α and decreased IL-4. Tumor-bearing ST2−/− mice had significantly higher percentages of activated CD27highCD11bhigh NK cells, CD69+ and KLRG− NK cells and higher cytotoxic activity of splenocytes, NK cells, and CD8+ T cells in vitro. A significantly higher number of NK cells expressing IFN-γ were found in ST2−/− mice compared with WT recipients. In vivo depletion of CD8+ or NK cells revealed a key role for NK cells in enhanced antitumor immunity in ST2−/− mice. We report for the first time that suppressed breast cancer progression and metastasis in mice lacking ST2 corresponds mainly with enhanced cytotoxic activity of NK cells, and increased systemic Th1/Th17 cytokines.

Galectin-3, a β galactoside–binding lectin, plays an important role in the processes relevant to tumorigenesis such as malignant cell transformation, invasion and metastasis. We have investigated whether deletion of Galectin-3 in the host affects the metastasis of B16F1 malignant melanoma. Galectin-3-deficient (Gal-3−/−) mice are more resistant to metastatic malignant melanoma as evaluated by number and size of metastatic colonies in the lung. In vitro assays showed lower number of attached malignant cells in the tissue section derived from Gal-3−/− mice. Furthermore, lack of Galectin-3 correlates with higher serum levels of IFN-γ and IL-17 in tumor bearing hosts. Interestingly, spleens of Gal-3−/− mice have lower number of Foxp3+ T cells after injection of B16F1 melanoma cells. Finally, we found that while CD8+ T cell and adherent cell cytotoxicity were similar, there was greater cytotoxic activity of splenic NK cells of Gal-3−/− mice compared with “wild-type” (Gal-3 +/+ ) mice. Despite the reduction in total number of CD3e−NK1.1+, Gal-3−/− mice constitutively have a significantly higher percentage of effective cytotoxic CD27highCD11bhigh NK cells as well as the percentage of immature CD27highCD11blow NK cells. In contrast, CD27lowCD11bhigh less functionally exhausted NK cells and NK cells bearing inhibitory KLRG1 receptor were more numerous in Gal-3 +/+ mice. It appears that lack of Galectin-3 affects tumor metastasis by at least two independent mechanisms: by a decrease in binding of melanoma cells onto target tissue and by enhanced NK-mediated anti-tumor response suggesting that Galectin-3 may be considered as therapeutic target.

Natural killer cell recognition of “missing self” contributes meaningfully to control of mouse cytomegalovirus infection in vivo., Cytomegaloviruses (CMVs) are renowned for interfering with the immune system of their hosts. To sidestep antigen presentation and destruction by CD8+ T cells, these viruses reduce expression of major histocompatibility complex class I (MHC I) molecules. However, this process sensitizes the virus-infected cells to natural killer (NK) cell–mediated killing via the “missing self” axis. Mouse cytomegalovirus (MCMV) uses m152 and m06 encoded proteins to inhibit surface expression of MHC I molecules. In addition, it encodes another protein, m04, which forms complexes with MHC I and escorts them to the cell surface. This mechanism is believed to prevent NK cell activation and killing by restoring the “self” signature and allowing the engagement of inhibitory Ly49 receptors on NK cells. Here we show that MCMV lacking m04 was attenuated in an NK cell– and MHC I–dependent manner. NK cell–mediated control of the infection was dependent on the presence of NK cell subsets expressing different inhibitory Ly49 receptors. In addition to providing evidence for immunoevasion strategies used by CMVs to avoid NK cell control via the missing-self pathway, our study is the first to demonstrate that missing self–dependent NK cell activation is biologically relevant in the protection against viral infection in vivo.

International audience; The immune response against a variety of pathogens can lead to activation of blood formation at ectopic sites, a process termed extramedullary hematopoiesis (EMH). The underlying mechanisms of EMH have been enigmatic. Investigating splenic EMH in mice infected with murine cytomegalovirus (MCMV), we find that, while cells of the adaptive immune system were dispensable for EMH, natural killer (NK) cells were essential. EMH required recognition of infected cells via activating NK cell receptors Ly49H or NKG2D, and correspondingly, viral interference with NK cell recognition abolished EMH. Surprisingly, development of EMH was not induced by NK cell-derived cytokines but was dependent on perforin-mediated cytotoxicity in order to control virus spread. Spreading virus reduced the numbers of F4/80(+) macrophages that were crucial for inflammatory EMH. Hence, whereas MCMV suppresses inflammation-induced EMH, NK cells confine virus spread, thereby protecting extramedullary hematopoietic niches and facilitating EMH.

Natural killer (NK) cells and CD8 + T cells play a prominent role in the clearance of mouse cytomegalovirus (MCMV) infection. The role of NK cells in modulating the CD8 + T-cell response to MCMV infection is still the subject of intensive research. For analyzing the impact of NK cells on mounting of a CD8 + T-cell response and the contribution of these cells to virus control during the first days postinfection (p.i.), we used C57BL/6 mice in which NK cells are specifically activated through the Ly49H receptor engaged by the MCMV-encoded ligand m157. Our results indicate that the requirement for CD8 + T cells in early MCMV control inversely correlates with the engagement of Ly49H. While depletion of CD8 + T cells has only a minor effect on the early control of wild-type MCMV, CD8 + T cells are essential in the control of Δ m157 virus. The frequencies of virus epitope-specific CD8 + T cells and their activation status were higher in mice infected with Δ m157 virus. In addition, these mice showed elevated levels of alpha interferon (IFN-α) and several other proinflammatory cytokines as early as 1.5 days p.i. Although the numbers of conventional dendritic cells (cDCs) were reduced later during infection, particularly in Δ m157 -infected mice, they were not significantly affected at the peak of the cytokine response. Altogether, we concluded that increased antigen load, preservation of early cDCs' function, and higher levels of innate cytokines collectively account for an enhanced CD8 + T-cell response in C57BL/6 mice infected with a virus unable to activate NK cells via the Ly49H–m157 interaction.

Natural killer (NK) cells play a crucial role in early immune response against cytomegalovirus infection. A large and mounting body of data indicate that these cells are involved in the regulation of the adaptive immune response as well. By using mouse cytomegalovirus (MCMV) as a model, several groups provided novel insights into the role of NK cells in the development and kinetics of antiviral CD8(+) T cell response. Depending on infection conditions, virus strain and the genetic background of mice used, NK cells are either positive or negative regulators of the CD8(+) T cell response. At present, there is no unique explanation for the observed differences between various experimental systems used. In this review we discuss the mechanisms involved in the interplay between NK and CD8(+) T cells in the early control of MCMV infection.

Murine cytomegalovirus (MCMV) Smith strain has been cloned as a bacterial artificial chromosome (BAC) named pSM3fr and used for analysis of virus gene functions in vitro and in vivo . When sequencing the complete BAC genome, we identified a frameshift mutation within the open reading frame (ORF) encoding MCMV chemokine homologue MCK-2. This mutation would result in a truncated MCK-2 protein. When mice were infected with pSM3fr-derived virus, we observed reduced virus production in salivary glands, which could be reverted by repair of the frameshift mutation. When looking for the source of the mutation, we consistently found that virus stocks of cell culture-passaged MCMV Smith strain are mixtures of viruses with or without the MCK-2 mutation. We conclude that the MCK-2 mutation in the pSM3fr BAC is the result of clonal selection during the BAC cloning procedure.

Human CMV (HCMV) is a major cause of morbidity and mortality in both congenitally infected and immunocompromised individuals. Development of an effective HCMV vaccine would help protect these vulnerable groups. NK group 2, member D (NKG2D) is a potent activating receptor expressed by cells of the innate and adaptive immune systems. Its importance in HCMV immune surveillance is indicated by the elaborative evasion mechanisms evolved by the virus to avoid NKG2D. In order to study this signaling pathway, we engineered a recombinant mouse CMV expressing the high-affinity NKG2D ligand RAE-1γ (RAE-1γMCMV). Expression of RAE-1γ by MCMV resulted in profound virus attenuation in vivo and lower latent viral DNA loads. RAE-1γMCMV infection was efficiently controlled by immunodeficient hosts, including mice lacking type I interferon receptors or immunosuppressed by sublethal γ-irradiation. Features of MCMV infection in neonates were also diminished. Despite tight innate immune control, RAE-1γMCMV infection elicited strong and long-lasting protective immunity. Maternal RAE-1γMCMV immunization protected neonatal mice from MCMV disease via placental transfer of antiviral Abs. Despite strong selective pressure, the RAE-1γ transgene did not exhibit sequence variation following infection. Together, our results indicate that use of a recombinant virus encoding the ligand for an activating NK cell receptor could be a powerful approach to developing a safe and immunogenic HCMV vaccine.

Svrha ovog rada jest prikazati stvarno stanje mirnog načina rješavanja sporova u Hrvatskoj. Polazište rada su određena pitanja o svrsishodnosti rješavanja sporova mirnim putem u hrvatskom kontekstu i oportunost takvog rješavanja. Na primjeru različitih vrsta sporova za koje postoji normativna podloga rješavanja mirnim putem (trgovački i građanski sporovi, radni sporovi, sporovi u kojima je stranka Republika Hrvatska, pod određenim pretpostavkama obiteljski sporovi te potrošački sporovi) provjerava se opravdanost takvih i sličnih pitanja na način da se predstavlja dosadašnji razvoj mirnog rješavanja te se daju procjene dosega njegova razvoja i postojeća ograničenja. Na temelju provedenog istraživanja dolazi se do nedvojbenog zaključka da se potencijali koje mirno rješavanje nosi u sebi po svojoj prirodi u Hrvatskoj koriste daleko ispod zadovoljavajuće razine. Zaključno, predlaže se način kojim bi se stanje moglo poboljšati kako bi prednosti mirnog rješavanja sporova mogle doći do izražaja., The paper gives an outline of the present situation in mediation for dispute settlement in Croatia. It starts by questioning the purposefulness of certain issues relating to dispute mediation in the Croatian context and considers whether such mediation is opportune. Examples are given of different types of disputes that have a normative base and can be settled through mediation (commercial disputes, civil suits, labour suits, disputes to which the Republic of Croatia is a party, family disputes, under certain circumstances, and consumer disputes; furthermore, the authors question how justifiable these mediations have been so far, estimating the benefits and existing limitations. The paper analyzes court-based and out-of-court mediation, starting from the beginnings of mediation to the present-day situation in mediation services, as well as the institutional framework in which such services were first applied and where mediation services are now provided. The authors present the background of the Croatian model of mediation and the possible and predictable future development of the existing institutional framework of peaceful settlement of disputes. The inevitable conclusion that can be drawn from the research is that the mediation potential in Croatia has not been adequately used. The paper questions why this is so and whether the situation could improve with time under the influence of mediation development and application in the European Union, considering that Croatia strives to become aligned with the EU's cultural and legal environment. In conclusion, the authors propose ways in which the situation could be improved so that the advantages of peaceful resolution of disputes could be fully appreciated., Das Ziel dieser Arbeit ist es, eine realistische Bestandsaufnahme zur gütlichen Beilegung von Streitigkeiten in Kroatien abzuliefern. Ausgangspunkt sind bestimmte Fragen zur Zweckmäßigkeit der gütlichen Beilegung von Streitigkeiten in Anbetracht der Verhältnisse in Kroatien und zur Opportunität einer solchen Beilegung. Anhand verschiedener Arten von Verfahren, für deren gütliche Beilegung eine normative Grundlage besteht (Handels- und Zivilsachen, Arbeitssachen, Verfahren, in denen die Republik Kroatien Prozessbeteiligte ist, unter bestimmten Voraussetzungen Familiensachen sowie Verbrauchersachen) wird die Berechtigung solcher und ähnlicher Fragen geprüft, indem die bisherige Entwicklung der gütlichen Streitbeilegung dargestellt und ihre mögliche Reichweite und bestehenden Beschränkungen beurteilt werden. Die Arbeit analysiert die gerichtliche und außergerichtliche Schlichtung unter Beachtung der Anfänge und des aktuellen Stands sowie den institutionellen Rahmen, in dem die Dienstleistung der Schlichtung erstmals erbracht wurde und auch weiterhin nachzufragen ist. Es werden auch der Hintergrund des kroatischen Schlichtungsmodells und die mögliche und absehbare Weiterentwicklung des bestehenden institutionellen Rahmens der gütlichen Streitbeilegung aufgezeigt. Die Untersuchungsergebnisse führen uns eindeutig zu dem Schluss, dass die Potenziale, die der gütlichen Streitbeilegung von der Sache her immanent sind, in Kroatien weit weniger genutzt werden, als dass dies zufrieden stellend wäre. Es wird versucht, die Gründe eines solchen Zustands zu bestimmen und die Frage zu beantworten, ob er sich mit der Zeit unter dem Einfluss des Stellenwerts der Schlichtung in der Europäischen Union, deren Kultur- und Rechtskreis Kroatien sich verbunden fühlt, verbessern wird. Abschließend wird vorgeschlagen, wie der Zustand verbessert werden kann, damit die Vorteile der gütlichen Streitbeilegung zum Ausdruck kommen können.