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Over the last few years, cancer-associated genes gain more research interest from an evolutionary perspective. Cancer is known as a disease of multicellular animals caused by the errors within the multicellular system, leading to the proliferation of "selfish" cell lines. It had most likely appeared in parallel with multicellularity and the development of true tissues, since comparative genomics studies have shown that many genes linked to human cancer were already present in simple non-bilaterian animals, such as sponges (Porifera). Nevertheless, their function and mechanisms of action still remain unknown. Despite of their simple morphology, with only few cell types and without true tissues and organs, sponges possess complex genomes harbouring many genes highly similar to their vertebrate homologs. Therefore, they provide an excellent model for studying the evolution of different genes that were most possibly present in the genome of animal ancestor. To get a broader insight into the evolutionary history of cancer, we have identified and characterized a number of sponge homologs of human genes associated with cancer development, such as Myc and Ras. Analyses of the primary gene structure and positions of introns have revealed that these genes show similarity in primary, but also in the predicted secondary and tertiary structures, to their homologs in "higher" animals. The phylogenetic analysis implies that sponge homologs of cancer- associated genes are highly conserved when compared to the cancer-associated genes in "higher" animals. Altogether, our work indicates that sponge proteins probably reflect the structures of ancestral proteins present in the last common ancestor of all animals, which have probably had similar properties as their homologs from the extant complex animals. Our next goal is to biochemically characterize the selected sponge homologs of cancer-associated genes and compare their structures and functions to their human homologs. We expect that a deeper insight into the physiological properties of cancer-related homologues in sponges will help to explain their complex interactions in humans and hopefully improve our overall understanding of this disease.

The Nme gene/protein family of nucleoside diphosphate kinases (NDPK), was originally named after its member Nm23-H1/Nme1. It was the first gene identified as metastasis suppressor. To date, ten different Nme genes have been identified in humans, with Nme1 and Nme2 as the most studied representatives. Nme proteins in humans have been divided in Group I (Nme1-Nme4) and Group II (Nme5-Nme9) and all of them possess nucleoside diphosphate kinase domain (NDK). There is a single histidine residue involved in the catalytic mechanism, conserved in all known active NDPK enzymes. Group I proteins display a single type NDK domain, whereas Group II proteins display a single or several NDK domains of different types, associated or not with extra-domains. In contrast to Group I members, the accumulating data strongly suggest that none of the Group II members display measurable NDPK activities, even though some of them are able to autophosphorylate. Multimeric form of any Group II member is not determined yet. Our comprehensive analyses of the Nme family in eukaryotes revealed conservancy of ancestral type of Group II protein in several species from three eukaryotic supergroups. Therefore we analyzed Nme protein from early branching eukaryotic lineage. We chose Chondrus crispus (Irish moss), recently sequenced red alga which is an excellent model organism for understanding Nme evolution. Genomic DNA from C. crispus was isolated and plasmids for overproduction of recombinant protein, localization assay, cell proliferation, apoptosis and soft agar colonization assay were constructed. NDPK activity was measured using a coupled PK-LDH (pyruvate kinase-lactate dehydrogenase) assay and the fast protein liquid chromatography (FPLC) system was employed for determination of protein MW. HeLa and HEK293T cells were used for transfection. Our analyses revealed that ancestral type of protein, in contrast to human homologue, was fully functional multimeric NDP kinase. This three-domain compound protein shows high affinity to various types of DNA and displays a dispersed localization throughout the eukaryotic cell. Overexpression of enzyme inhibits both cell proliferation and anchorage independent growth in soft agar, but does not deregulate the cell apoptosis. We conclude that the structure of ancestral gene has changed during eukaryotic evolution possibly in correlation with the function of the protein.

A huge therapeutic potential of non-coding RNAs (ncRNAs) was recognized early upon their discovery, and has been explored intensively and continuously ever since. The great perspective of ncRNAs in medicine is due to their inherent mechanism of gene regulation that might be applicable in therapy of wide spectrum of diseases. Small nucleolar RNAs (snoRNAs) are believed to be the most ancient ncRNAs, essential for the correct assembly of the ribosome. Many examples of ncRNAs displaying both snoRNA and microRNA (miRNA) characteristics suggest a possible evolution from one type to the other. Numerous studies already provided evidence for the functional importance of snoRNAs in cancerogenesis. The accumulating genomic data strongly confirm the tendency of snoRNAs to colonize ribosomal protein genes (RPGs) and ribosome related genes in eukaryotes. SNORA62 is found in ribosomal protein S30 gene (FAU) in the subset of sponges from the genus Suberites, and is located in the human ribosomal protein gene SA (RPSA). Both host RPGs possess an extraribosomal function involved in the maintenance of cellular viability through the caspase-dependent regulation of apoptosis. Since SNORA62 is the most upregulated gene in diallyl sulfide (DAS)- induced apoptosis in HeLa cells, we tested overexpression of SNORA62 in human cells. Plasmids for overproduction of recombinant proteins RPSA, FAU and SNORA62 were constructed and HeLa and HEK293T cells were used for transfection. Cell proliferation was measured using CellTiter-Glo Luminescent Cell Viability Assay (Promega) and caspase activity was measured using the Caspase-Glo 3/7 Assay Kit (Promega). Our results show that SNORA62 can modify cell growth rate and act like tumor suppressor in HEK293T and HeLa cells, but also interfere and modulate similar functions of its host ribosomal protein genes. Analyses of SNORA62 presented here provide the opportunity to determine direction for further investigation and evaluation of its potential for cancer treatment.

The amazing complexity of metazoan genomes originated early in the evolution of the animal kingdom. Rapid advances of next-generation sequencing (NGS) technologies and bioinformatic data analysis are enabling elucidation of early metazoan gene regulation and development. Sponges have a unique evolutionary position of being the simplest and arguably the earliest branching metazoans, and are therefore critical for understanding the genomic traits of the ancestor of metazoa - the first multicellular animal. However, available genomic data from the phylum Porifera is still very scarce. We sequenced the genome of the freshwater sponge Eunapius subterraneus, Sket & Velikonja, 1984 using a combination of NGS methods (Illumina and Oxford Nanopore) and assembled a draft version of its genome. Eunapius subterraneus is the only cave-adapted sponge species, known from only a small region in Croatia. The sponge possess unique morphological and ecological features. Using the -omics methodologies in combination with experimental approaches we aim to advance our understanding of 1. basic processes involved in multicellular development and differentiation and 2. the process of adaptation to different environmental conditions. Financial support by the Croatian National Science Foundation (Project No. 6400) is gratefully acknowledged.

Sirtuin 3 (Sirt3), a major mitochondrial NAD+ dependent deacetylase, has bifunctional role in cancer tumorigenesis, acting as both oncogene and tumor suppressor, depending on the tissue and cancer-type specific metabolic programs. Changes in its expression are associated with the excessive production of reactive oxygen species (ROS), thus contributing to mitochondrial dysfunction and age-related pathologies. Hyperoxic treatment (i.e. generator of ROS) was shown to support some tumorigenic properties, but finally suppresses growth of certain mammary carcinoma cells. 70% of all breast cancer cases are estrogen receptor (ER) positive and express ER-alpha (ER- α). While ER-α positive cancers are more receptive to hormonal therapy, triple negative breast cancers (TNBC) are characterized by an aggressive behaviour and the lack of targeted therapeutic strategies. Due to strikingly reduced Sirt3 level in many breast cancer cells, we aimed at deciphering the effect of de novo Sirt3 expression in normoxic and hyperoxic conditions in the human breast cancer cells. Although we have recently shown that Sirt3 acts as a tumor suppressor in non-invasive (ER-α positive) breast cancer cells, it remains unclear whether this effect is mediated through ER-α signalling pathway. Therefore, we stably transfected both MCF-7 (ER-α positive) and MDA-MB-231 (TNBC) cells with Flag-tagged Sirt-3 plasmid and characterized Sirt-3 overexpressing clones in normoxic and hyperoxic conditions. To further characterize clones and decipher the cause of the observed differences in their response, we used combination of treatments to alter the expression and activity of different proteins. We monitored the expression of proteins involved in mitochondrial biogenesis, glycolysis, metabolic regulation and antioxidant defense. Furthermore, we compared the growth rate, metabolic activity, mitochondrial ROS production and the cell cycle of the clones. Initial findings showed enhanced susceptibility of MCF-7 cells to hyperoxia and decreased cellular growth upon de novo Sirt3 expression. On the other hand, Sirt-3 markedly promoted growth of highly invasive MDA-MB-231 cells. Collectively, results suggested that Sirt-3 may either have a tumor suppressing or tumor promoting role in breast cancer cells depending on their invasiveness, thus giving us a rationale for further studies on Sirt3 and hyperoxia as an adjuvant tumor therapy in breast cancer malignancies.

Large scale –omics projects have provided valuable data across the animal kingdom and have revealed remarkable complexity and diversity of animal genomes. Given the rapid advances in genomic data collection and bioinformatics analysis methods, unique opportunity opens up towards the elucidation of early metazoan gene regulation and development. With their pivotal evolutionary position of arguably the simplest extant and earliest branching metazoans, sponges are rapidly gaining focus in genomic research, however, more genomic information within the sponge phylum is required. We sequenced the genome of the freshwater sponge Eunapius subterraneus using a combination of next-generation sequencing technologies (Illumina and Oxford Nanopore) and assembled a draft version of its genome. Eunapius subterraneus is the only stygobitic sponge species, an endemic member of the suborder Spongillina (freshwater sponges) known from merely six caves near Ogulin, Croatia. This endemic sponge was shown to possess numerous unique morphological, molecular and ecological features. Using the - omics methodologies in combination with experimental approaches, we aim to dive more deeply into the understanding of some of the basic processes involved in multicellular development and differentiation.

Mitochondria, as the major reactive oxygen species (ROS) producer and the major antioxidant producer exert a crucial role within the cell mediating processes such as apoptosis, detoxification, Ca2+ buffering, etc. Oxidative stress and high levels of ROS cause DNA damage and genomic instability, which lead to changes in cell metabolism and ultimately loss of control of the cell cycle. These conditions support continuous growth and proliferation, the main characteristics of tumor cells. Although the role of pure oxygen (hyperoxia) as a promoter of malignant diseases has long been debated, today hyperoxia is used as a routine therapy to treat such diseases. Sirtuin-3 (Sirt-3) is a major mitochondrial NAD+-dependent deacetylase that plays critical role in activation of mitochondrial proteins involved in energy metabolism, ATP production and mitochondrial biogenesis which effectively keeps the cell in energetic homeostasis. In addition, Sirt-3 has a role as an oncogene, but also as a tumor suppressor in tumor cells, and it is not clear which factors lead to the change in the protein’s function. In this research we investigated the effect of Sirt-3 on the proliferation, survival, growth, metabolic activity, oxidant/antioxidant status, apoptosis and mitochondrial function of the MCF-7 breast cancer tumor cell line in normoxia and after treatment in hyperoxia. We confirmed that in normoxic conditions, Sirt-3 functions as an oncogene, and causes oxidative stress and other factors responsible for tumor growth. We also confirmed that in hyperoxic conditions, Sirt-3 functions as a tumor suppressor because it decreases the tumor cell characteristics usually present in breast cancer MCF-7 cells. With our research we have shown that Sirt-3 regulates numerous cell processes essential for the proliferation and metastasis of tumor cells, and that hyperoxia encourages the tumor suppressor activity of Sirt-3. These characteristics make Sirt-3 a potential therapeutic target in combination with hyperoxia to treat malignant diseases.

INTRODUCTION & OBJECTIVES: Fungal scalp infections caused by Microsporum (M.) canis tend to be noninflammatory, but recently, progressive numbers of cases of tinea capitis characterized with highly inflammatory infection caused by M. canis and M. gypseum, have been registered. We present case of highly inflammatory tinea capitis, also known as kerion celsi with dermatophytide reaction caused by M. canis. MATERIAL & METHODS: A 6-year-old, otherwise healthy girl, with tender, infiltrated, 6x4 cm large plaque in the occipital region of the scalp, of 2.5 months duration has been examined. Almost complete loss of hair, a few satellite plaques with pustules on the surface, and exudation from some of the follicular orifices could be observed. Multiple annular lesions spread over the neck and shoulders. In addition, papular exanthema of the trunk and bilateral cervical lymphadenopathy were present. The patient was in a good condition, and routine investigations were all within the normal limits. Fungal culture on modified Sabouraud dextrose agar medium, revealed flat, white to creamy colonies with cottony surface and golden brownish-yellow reversed pigment. Diagnosis of kerion due to M. canis was established. M. canis has been confirmed in the isolate by the use of the molecular methods (PCR-RFLP), as well. Thus, ITS1 and ITS4 universal primers were used to amplify ITS1-5.8s rDNA-ITS2 region. By the RFLP analysis of the PCR product of the isolated strain and reference strain of M. canis ATCC 36299, using Hinf1 restriction enzyme a unique pattern for M. canis has been revealed. Mycological examination of the papular lesions of the trunk was negative, so these lesions were considered as dermatophytide reaction. The treatment with oral terbinafine 125 mg/day had been prescribed together with topical gyrodalate ointment and triamcinolone acetonide cream followed with miconazole cream. Systemic prednisolone, 10 mg/day for 7 days has been also administered in order to relive symptoms of “ide” reaction and to prevent scarring. After 12 weeks of therapy, complete clinical and mycological regression was observed. CONCLUSIONS: Changes in epidemiology and clinical pattern of fungal infections due to M. canis have become significant in the recent years. M. canis, thus not only Trichophyton spp. as previously reported can also be responsible for kerion and dermatophytide reactions. Greater awareness is needed in order to establish proper diagnosis and successful treatment strategy for those patients.

Molecular studies impose many changes in the taxonomy of certain animal groups whose relations are based solely on morphological characters. Many freshwater and calcarean sponges have been wrongly identified at morphological level, hence concurrent molecular analyses are necessary. The analysis of repetitive elements in the intergenic regions of freshwater sponge mitochondrial genomes suggests that their families are probably evolutionary related. Based on molecular markers, together with morphological traits, 6 new calcarean species are described here.