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Goal: Mitochondrial haplogroup (hg) X was estimated to originate in the Near East cca 30 ka years ago. Its approximate prevalence in the general European population is 2%, with most sublineages belonging to the X2 clade. The X2 clade is far less common in Croatian insular populations according to our previous findings - 6 out of 13 detected X samples belong to a new, local variant based on specific mutations in both the control and coding region of the mitochondrial genome. The aim of this study was to fully explore mentioned 6 mitochondrial haplotypes found in islands of Rab and Cres. Materials and methods: Complete sequencing of one Rab and one Cres mitochondrial genome was performed using both Sanger sequencing and massively parallel sequencing with the Illumina® Human mtDNA Genome assay on MiSeq FGx™ instrument. Results: Sequenced samples indicate a novel lineage within the global mitochondrial X hg phylogeny according to both PhyloTree and Mitomap. The lineage is connected with the hg X3 by a common polymorphism G3531A in the coding region of the mitochondrial genome, but it lacks all other X3 defining mutations. Mutations specific for this lineage are T195C, C338T, A7518G, G7853A, A10113G, C10673T, C10920T, A11380G, A13614G, C13950T, A15903G, G15927A, T16136C and A16289G. Analysis of non-phylogenetic variants exposed a rare, A7518G mutation in the tRNA gene, which is predicted as possibly pathogenic by MitoTIP. Findings of different heteroplasmic mutation patterns between samples indicate a more distant kinship within the same mitochondrial lineage. Conclusion: Our finding indicates a recent local microdifferentiation process within hg X. Described lineage could possibly be marked as a new, island-specific X twig formed within the Croatian population. The extension of present research, including genealogical and clinical data, is needed to confirm further enrichment of existing mtDNA phylogeny and to establish possible functional manifestations of locally specific variants.

With the recent advances in genomic research, long non-coding RNAs (lncRNAs) have been recognized as key players in gene expression regulation [1]. However, research on basal metazoan lncRNAs has been scarce, with a few studies exploring the non-coding transcriptomes in developmental stages of the Amphimedon queenslandica sponge [2]. In this study, we enriched the catalog of sponge lncRNAs by sequencing the transcriptomes of Eunapius subterraneus, an endemic stygobiotic demosponge found in Ogulin, Croatia. Using our lncRNA identification pipeline, we found a comprehensive yet highly confidential set of 1981 poriferan lncRNA transcripts. We discovered that identified lncRNAs exhibit many of the vertebrate lncRNA characteristics, such as relatively low levels of alternative splicing, two-exon bias, shorter length compared to mRNA transcripts and splice sites characteristic for mRNA introns. The majority of identified lncRNAs were found inside the introns of protein-coding genes (59.3%), followed by lncRNAs without overlaps with protein-coding genes (34.1%) and lncRNAs which contain protein-coding genes within their introns (6.6%). We also found an enrichment of transposable element insertions in the identified lncRNAs, highlighting the importance of transposons in poriferan lncRNA origin. Additionally, a surprisingly large number of identified lncRNAs was similar to or syntenic with lncRNAs found in Ephydatia muelleri, a demosponge closely related to Eunapius subterraneus. When analyzing protein-coding genes close to identified lncRNA loci, we found numerous protein-coding genes involved in various biological processes, including DNA binding, DNA integration and protein deubiquitination. Moreover, the majority of lncRNAs proximal to protein-coding genes involved in these processes were conserved or syntenic in multiple sponge species. This finding represents evidence for the potential importance of cis-acting sponge lncRNAs in regulating the expression of protein-coding genes involved in multiple important cellular processes. Taken together, our results demonstrate a surprising similarity between poriferan and bilaterian lncRNAs.

Cancer is a disease caused by errors within the multicellular system and it represents a major health issue in multicellular organisms. Although cancer research has advanced substantially, new approaches focusing on fundamental aspects of cancer origin and mechanisms of spreading are necessary. Comparative genomic studies have shown that most genes linked to human cancer emerged during the early evolution of Metazoa. Thus, basal animals without true tissues and organs, such as sponges, might be an innovative model system for understanding the molecular mechanisms of proteins involved in cancer biology. One of these proteins is Developmentally regulated GTP-binding protein 1 (DRG1), a GTPase stabilized by interaction with DRG family regulatory protein 1 (DFRP1). This study reveals a high evolutionary conservation of DRG1 gene/protein in metazoans. Biochemical analysis and structural predictions demonstrate that recombinant DRG1 from humans and sponge Eunapius subterraneus are predominantly monomers that form complexes with DFRP1 and bind non- specifically to RNA and DNA. Our transfection experiments show the intracellular localization of exogenous sponge and human DRG1 in human tumor cells. We reveal the conservation of sponge and human DRG1 biological features, including DRG1:DFRP1 binding, function of DRG1 in α-tubulin dynamics, and its role in cancer biology demonstrated by increased proliferation, migration and colonization of human cancer cells. These results suggest that the ancestor of all Metazoa already possessed DRG1 that is structurally and functionally similar to the human DRG1, even before the development of real tissues or tumors, indicating an important function of DRG1 in fundamental cellular pathways.

Spužve (Porifera) su jedne od najjednostavnijih životinja koje ne posjeduju pravo tkivo niti organe te su evolucijski očuvane [1]. Jednostavne su morfologije, građene su od nekoliko tipova stanica, ali posjeduju složen genom sa mnoštvom gena koji pokazuju visoku sačuvanost s homolozima kod kralješnjaka, uključujući i gene povezane s patologijom raka [2, 3]. Stoga spužve predstavljaju dobar modelni organizam za istraživanje gena uključenih u nastanak raka u ljudi [4]. Rak je patološko stanje povezano s pojavom višestaničnosti, čiji su uzrok nastanka „greške“ koje dovode do proliferacije „sebičnih“ staničnih linija. Naš cilj je istraživanje evolucijski očuvanih proteina povezanih s nastankom raka, kao što je supresor metastaziranja DRG1 (od engl. Developmentally Regulated GTP Binding Protein 1) [5]. Bioinformatičkim i filogenetskim analizama pokazali smo da je protein DRG1 evolucijski očuvan kod Metazoa. S obzirom da aktivnost proteina DRG1 pojačava protein LEREPO4 (od engl. Zinc Finger CCCH-Type Containing protein 15) [6], oba proteina i njihovi homolozi iz čovjeka su klonirani i prekomjerno eksprimirani u E. coli. Potvrđena je intrinzična GTP-azna aktivnost spužvinog i ljudskog proteina DRG1. Transfekcijom ljudskih tumorskih stanica pokazali smo unutarstaničnu lokalizaciju spužvinih i ljudskih proteina DRG1 i LEREPO4. Daljnje biokemijske i biološke analize su u tijeku. Naše istraživanje pridonijet će boljem razumijevanju unutarstaničnih procesa supresije metastaziranja, kao i same patologije raka i metastaza. LITERATURA [1] Dohrmann, M., et al. Syst. Biol., 2008. 57(3): p. 388-405. [2] Srivastava, M., et al. Nature, 2010. 466(7307): p. 720-6. [3] Harcet, M., et al. Mol. Biol. Evol., 2010. 27(12): p. 2747-56. [4] Cetkovic, H., M. Halasz, and M. Herak Bosnar, Mar. Drugs, 2018. 16(1). [5] Cetkovic, H., et al. Lab. Invest., 2018. 98(5): p. 554-570. [6] Perez-Arellano, I., M. Spinola

Bakterija Borrelia burgdorferi vektorski se prenosi putem krpelja i primarni je uzročnik Lajamske bolesti. U laboratorijskim uvjetima stanice B. burgdorferi mogu postojati u nekoliko različitih pleomorfnih oblika (morfotipova). Dok su funkcionalne i strukturne razlike među nekim morfotipovima već prethodno opisane, ekspresijske razlike na razini transkriptoma do sada nisu istražene. S tim ciljem uzgojili smo laboratorijske kulture različitih morfotipova (spiralni, okrugli, bleb i biofilm), izolirali ukupnu RNA, sekvencirali je te skupili podatke o razlici u ekspresiji na razini transkriptoma. Otkrili smo da unatoč morfološkim razlikama, spiralni i okrugli morfotipovi imaju sličan ekspresijski profil, dok bleb i biofilm morfotipovi pokazuju značajnu razliku u ekspresiji kako međusobno tako i u odnosu na spiralni i okrugli morfotip. Unatoč tome što okrugli morfotip ima transkripciju sličnu spiralnom morfotipu, u okruglom morfotipu su pojačano su eksprimirani geni uključeni u procese transkripcije i translacije. Iako bleb i biofilm morfotipovi imaju veći broj pojačano eksprimiranih gena o odnosu na okrugli oblik, funkcija tih gena je pretežito nepoznata. Geni koji su pojačano eksprimirani u okruglom morfotipu su pretežito locirani na bakterijskom kromosomu, dok se pojačano eksprimirani geni bleb i biofilm morfotipova uglavnom nalaze na plazmidima. Kako bi definirali evolucijsko podrijetlo diferencijalno eksprimiranih gena bakterije B. burgdorferi koristili smo genomsku filostratigrafiju. Otkrili smo da su geni koji su pojačano eksprimirani u okruglom morfotipu obogaćeni evolucijski starim genima karakterističnim za sve stanične organizme, dok su geni pojačano eksprimirani u bleb i biofilm morfotipovima evolucijski mlađi i specifični za porodicu Borreliaceae. Naši rezultati upućuju na to da je tranzicija iz spiralnog u okrugli morfotip regulirana malim brojem evolucijski visoko konzerviranih gena koji sudjeluju u transkripciji i translaciji, dok je prijelaz iz spiralnog morfotipa u bleb ili biofilm morfotip popraćen velikim ekspresijskim promjenama koje uključuju pojačanu ekspresiju evolucijski mlađih gena nepoznate funkcije.

Circulating cell-free DNA (cfDNA) present in the plasma of healthy individuals is believed to mostly be a product of apoptosis of cells of the hematopoietic lineage. Differences in the distribution of tissues of origin of circulating cfDNA between healthy and disease states or specific physiological conditions can be informative of the underlying biological mechanisms contributing to specific states and have the substantial potential of improving the diagnostics of various diseases. Several approaches for identification of the tissue of origin from cfDNA have been developed, including methods based on the analysis of nucleosome patterns or transcription factor binding sites inferred from cfDNA fragmentation patterns, DNA methylation, or ChIP-seq of cfDNA. However, methods that rely only on cfDNA fragmentation patterns can usually differentiate between hematopoietic vs non-hematopoietic tissues of origin but are less sensitive to differences in the changes of proportions of different blood cells. To increase the sensitivity of the method to subtle changes in proportions of cells of origin of cfDNA, whose cfDNA fragmentation patterns are highly similar, we developed a novel approach based on the comparison of average fragmentation patterns of cfDNA in tissue-specific regulatory regions and the cfDNA signal intensity. We tested our approach on a whole genome sequencing dataset of individuals before and after exercising. Vigorous exercise has been shown to increase the concentration of cfDNA in plasma and the cell types that increase after exercise are largely unknown. We analyzed the cfDNA patterns before and after exercise using our approach and identified the cell types that increase after exercise. This method can be used to study other conditions that may be associated with changes in the release of cell-free DNA from specific blood cell types.

Nedavna istraživanja fokusirana na korelaciju filogenije i ontogenije pokazala su kako je ta korelacija na molekularnoj razini prisutna u različitim skupinama složenih višestaničnih eukariotskih organizama [1-3]. Takve korelacije nisu istraživane u skupinama organizama gdje nedostaje jasna definicija razvoja i višestanične složenosti, što je slučaj kod bakterijskih biofilmova. Od ranije je poznato da se bakterijski biofilmovi, najzastupljeniji pojavni oblik bakterija u prirodi, formiraju kao posljedica višestaničnog ponašanja bakterijskih stanica [4], ali nije poznato dijele li biofilmovi osnovne značajke razvoja s kompleksnim eukariotima. U ovom radu pokazali smo na modelu rastućeg biofilma vrste Bacillus subtilis da ontogenija rekapitulira njegovu filogeniju na ekspresijskoj razini. Koristeći transkriptomske i proteomske podatke iz različitih stadija razvoja biofilma, uočili smo da ontogenija biofilma korelira s evolucijskim mjerama, odnosno da mlađi i više divergirani geni pokazuju veću ekspresiju u krajnjim stadijima rasta biofilma. Također, pokazali smo da je na molekularnoj i morfološkoj razini rast biofilma strogo reguliran i organiziran u odvojene ontogenetske stadije, analogno eukariotskim embrijima. To dovodi do zaključka da je formiranje bakterijskog biofilma istinski razvojni proces usporediv s razvojem u životinja, biljaka i gljiva.

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.

Tool for analysis of codon usage in various unannotated or KEGG/COG annotated DNA sequences. Calculates different measures of CU bias and CU-based predictors of gene expressivity, and performs gene set enrichment analysis for annotated sequences. Implements several methods for visualization of CU and enrichment analysis results.

Uvod: Primarne imunodeficijencije (PID) su nezarazne i nasljedne bolesti razvoja i/ili funkcije imunosnog sustava uzorkovane genskim mutacijama. PID predstavljaju rastuću skupinu od preko 320 različitih genetski determiniranih poremećaja za koje se smatra da su pojedinačno i kolektivno rijetki. Stvarna učestalost PID-a kod djece i odraslih ostaje nejasna, ali nedavne epidemiološke studije upućuju na to da su primarne imunodeficijencije u stalnom porastu. Najčešće PID su povezane s poremećajem humoralnog imuniteta. Navedeni poremećaji mogu biti karakterizirani odsutnošću ili prisutnošću B stanica i njihovih produkata - imunoglobulina. U kliničkoj slici kod pedijatrijske populacije koja ima nedostatak protutijela prevladavaju rekurentne upale srednjeg uha, sinusa i pluća, te teške infekcije gornjeg i donjeg dišnog sustava. Isto tako vrijedi i za odrasle, iako je u tom slučaju upala srednjeg uha manje uobičajena. Pojava infekcija oportunističkim uzročnicima dovela je do razumijevanja individualne osjetljivosti na infekcije. Međutim, učinkovito djelovanje terapije antibiotikom može istovremeno zamaskirati prepoznavanje i otkloniti sumnju na PID nakon uspješno riješene infekcije. Mnogi pacijenti imaju dugu povijest ukorijenjenih infekcija prije nego što je PID dijagnosticirana kada su kronične komplikacije već razvijene (npr. bronhiektazije, malapsorpcija). Kompleksnost kliničkog stanja oboljelih od PID-a utječe i na kakvoću života i životnu prognozu samog bolesnika. Stoga je od posebnog značaja rana dijagnostika i rana terapija kako bi se izbjegle nepotrebne komplikacije. Cilj: Procijeniti pojavnost najčešćih prirođenih imunodeficijencija vezanih uz poremećaje produkcije protutijela, te bolesti koje su uz njih vezane. Ispitanici i rezultati: Ispitivanje je provedeno u Dječjoj bolnici Srebrnjak u djece dobi 4-18 godina, (prosječna dob 9.3 godine). Pojavnost poremećaja produkcije protutijela u djece, posebno selektivni deficit sinteze IgA protutijela (teški i parcijalni) najčešći je poremećaj humoralnog imuniteta u naših ispitanika. Poremećaji produkcije protutijela najčešći su tip PID-a, te oni čine oko 70% svih primarnih imunodeficijencija. Učestalost rekurentnih infekcija, astme i alergija u djece sa selektivnim deficitom IgA protutijela (N=95) prema našim istraživanjima bila je statistički značajno viša (p

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.

In a translational medicine concept, we aim to combine clinical, immunological and genetic aspects of the primary immunodeficiency diagnostics in order to improve treatment decisions and prevention strategies of these disorders. In this approach, the assessment of the immune imbalance by flow cytometry combined with the next generation sequencing (NGS) technology represents an unprecedented tool in immunogenetic diagnostics of rare and monogenic diseases in the new era of precision medicine. A 16 year-old male patient has been admitted to our hospital due to recurrent respiratory infections and hypogammaglobulinemia, and was diagnosed with ataxia telangiectasia (A-T)-a rare genetic disorder of neurologic impairment and immune deficiency. We performed the flow cytometry immunophenotyping of peripheral blood using a 6- and 7-colour antibody panels for the analysis of T cells, B cells and NK cells (Table 1, Panel 1 and 2) and the B cell subsets (Table 2), as well as the targeted NGS analysis using the TrueSeq Custom Amplicon panel and the TruSeq Custom Amplicon Low Imput kit followed by sequencing on the MiniSeq system (Illumina, USA) to screen for the variants of the immunodeficiency genes involved in the predominantly antibody deficiency and combined immunodeficiencies with associated or syndromic features (Table 3, Panel 1 and 2). Immunophenotyping revealed a decrease or a complete loss of the CD4+ T lymphocytes, B lymphocytes, transitional B lymphocytes, class- switched memory B lymphocytes and plasmablasts (the latter not shown) ; an increase of the CD3+HLA-DR+ activated T lymphocytes (Figure 1) ; and an appearance of a peculiar CD45dimCD38++CD27+CD56+ population (Figure 2). The targeted NGS analysis identified 3 novel missense variants in i) meiotic recombination 11 (MRE11) gene, ii) ataxia telangiectasia and Rad3-related protein (ATR) gene, and iii) B-cell linker protein (BLNK) gene, with the in silico predictions as possibly deleterious (Figure 3). We here report novel variants detected by NGS in the A-T patient previously not reported in clinical practice. Given their potentially pathogenic nature, i.e. the phenotypic association with leukemia (BLNK gene), the appearance of the CD45dimCD38+CD27+CD56+ population, and an increased alpha-fetoprotein level, a further treatment of the patient requires a close monitoring for the potential development of malignancy - the main cause of premature death of A-T patients, by means of a tight integration of immunogenetic diagnostic tools in a personalized medicine perspective.