Transcription – coupled repair

Popravak DNA udružen s transkripcijom jedan je od putova NER, a javlja se kad RNAP zastane na oštećenju u nekodirajućem lancu. Nekodirajući lanac popravlja se puno brže nego kodirajući lanac istog gena ili neeksprimirana regija genoma. Iako je TCR prvi put uočen kod eukariota taj fenomen najbolje je okarakteriziran kod E. coli. Glavnu ulogu u TCR kod bakterija ima protein Mfd kao ATP-ovisna DNA translokaza, a poznat je i kao faktor povezan s transkripcijskim popravkom (TRCF). Model djelovanja Mfd pretpostavlja da Mfd prepoznaje zaustavljenu RNAP te da reverzno translociranu RNAP pomoću energije ATP translocira naprijed na njenu prvotnu poziciju. Zatim dolazi do otpuštanja RNAP i dovođenja enzima NER. Postoji i alternativni put Mfd-ovisnom TCR, a to je TCR u kojem otpuštanje RNAP i popravak DNA povezuje elongacijski faktor NusA. Puno se toga ulaže u poznavanje mnogo kompleksnijeg mehanizma TCR kod eukariota zbog činjenice da mutacije u genima povezanima s gubitkom TCR dovode do teških bolesti kod ljudi. Mehanizam TCR kod ljudi je uglavnom nepoznat usprkos tome što su poznati neki od glavnih sudionika tog procesa poput proteina CSA, CSB, TFIIH i XPG. Važno je ustvrditi mehanizam TCR ne samo zbog problema koje zaustavljena RNAP stvara u stanici i bolesti koje uzrokuje nefunkcionalni TCR već i zbog toga što je TRCF uključen u povezivanje centralnih staničnih procesa i što bi daljnja saznanja o načinu na koji TRCF pomoću svojih domena utječe na RNAP i NER pomoglo u otkrivanju točnog mehanizma ovog procesa.Transcription-coupled DNA repair (TCR) is one of the subpathways of nucleotide excision repair (NER) that occurs when RNA polymerase is stalled at sites of DNA damage in the template strand. Template strand is repaired more quickly that similar lesions in coding strand or unexpressed parts of genome. Although the TCR was first observed in eukaryotes TCR phenomenon is best characterized in E. coli. The main role of TCR in bacteria has Mfd (ATP-dependent DNA translocase), also known as a transcription repair coupling factor (TRCF). Model for TCR postulate that Mfd recognizes and binds specifically to stalled and reverse translocated RNAP and using energy derived from ATP hydrolysis translocates forward RNAP to the transcription block. That results in RNAP release, transcript termination and recruitment of the NER machinery to the site of lesion. Recent studies show existence of alternative Mfd-independent TCR pathway mediated by elongation factor NusA. Alot is invested in understanding of more complex mechanism of TCR in eukaryotes due to the fact that mutations in genes associated with loss of TCR lead to severe disease in humans. The mechanism of TCR in humans is largely unknown despite having known some of the major participants in this process, such as CSA, CSB, TFIIH and XPG proteins. It is important to establish the mechanism of TCR not only because of the problems that stalled RNAP causes in the cell and diseases caused by dysfunctional TCR but also because the TRCF is involved in linking central cellular processes and knowing how TRCF uses its multiple domains to manipulate RNAP and the NER would help shed the light on the functional mechanism of TCR

Transcription – coupled repair

Popravak DNA udružen s transkripcijom jedan je od putova NER, a javlja se kad RNAP zastane na oštećenju u nekodirajućem lancu. Nekodirajući lanac popravlja se puno brže nego kodirajući lanac istog gena ili neeksprimirana regija genoma. Iako je TCR prvi put uočen kod eukariota taj fenomen najbolje je okarakteriziran kod E. coli. Glavnu ulogu u TCR kod bakterija ima protein Mfd kao ATP-ovisna DNA translokaza, a poznat je i kao faktor povezan s transkripcijskim popravkom (TRCF). Model djelovanja Mfd pretpostavlja da Mfd prepoznaje zaustavljenu RNAP te da reverzno translociranu RNAP pomoću energije ATP translocira naprijed na njenu prvotnu poziciju. Zatim dolazi do otpuštanja RNAP i dovođenja enzima NER. Postoji i alternativni put Mfd-ovisnom TCR, a to je TCR u kojem otpuštanje RNAP i popravak DNA povezuje elongacijski faktor NusA. Puno se toga ulaže u poznavanje mnogo kompleksnijeg mehanizma TCR kod eukariota zbog činjenice da mutacije u genima povezanima s gubitkom TCR dovode do teških bolesti kod ljudi. Mehanizam TCR kod ljudi je uglavnom nepoznat usprkos tome što su poznati neki od glavnih sudionika tog procesa poput proteina CSA, CSB, TFIIH i XPG. Važno je ustvrditi mehanizam TCR ne samo zbog problema koje zaustavljena RNAP stvara u stanici i bolesti koje uzrokuje nefunkcionalni TCR već i zbog toga što je TRCF uključen u povezivanje centralnih staničnih procesa i što bi daljnja saznanja o načinu na koji TRCF pomoću svojih domena utječe na RNAP i NER pomoglo u otkrivanju točnog mehanizma ovog procesa.Transcription-coupled DNA repair (TCR) is one of the subpathways of nucleotide excision repair (NER) that occurs when RNA polymerase is stalled at sites of DNA damage in the template strand. Template strand is repaired more quickly that similar lesions in coding strand or unexpressed parts of genome. Although the TCR was first observed in eukaryotes TCR phenomenon is best characterized in E. coli. The main role of TCR in bacteria has Mfd (ATP-dependent DNA translocase), also known as a transcription repair coupling factor (TRCF). Model for TCR postulate that Mfd recognizes and binds specifically to stalled and reverse translocated RNAP and using energy derived from ATP hydrolysis translocates forward RNAP to the transcription block. That results in RNAP release, transcript termination and recruitment of the NER machinery to the site of lesion. Recent studies show existence of alternative Mfd-independent TCR pathway mediated by elongation factor NusA. Alot is invested in understanding of more complex mechanism of TCR in eukaryotes due to the fact that mutations in genes associated with loss of TCR lead to severe disease in humans. The mechanism of TCR in humans is largely unknown despite having known some of the major participants in this process, such as CSA, CSB, TFIIH and XPG proteins. It is important to establish the mechanism of TCR not only because of the problems that stalled RNAP causes in the cell and diseases caused by dysfunctional TCR but also because the TRCF is involved in linking central cellular processes and knowing how TRCF uses its multiple domains to manipulate RNAP and the NER would help shed the light on the functional mechanism of TCR

Transcription – coupled repair

Popravak DNA udružen s transkripcijom jedan je od putova NER, a javlja se kad RNAP zastane na oštećenju u nekodirajućem lancu. Nekodirajući lanac popravlja se puno brže nego kodirajući lanac istog gena ili neeksprimirana regija genoma. Iako je TCR prvi put uočen kod eukariota taj fenomen najbolje je okarakteriziran kod E. coli. Glavnu ulogu u TCR kod bakterija ima protein Mfd kao ATP-ovisna DNA translokaza, a poznat je i kao faktor povezan s transkripcijskim popravkom (TRCF). Model djelovanja Mfd pretpostavlja da Mfd prepoznaje zaustavljenu RNAP te da reverzno translociranu RNAP pomoću energije ATP translocira naprijed na njenu prvotnu poziciju. Zatim dolazi do otpuštanja RNAP i dovođenja enzima NER. Postoji i alternativni put Mfd-ovisnom TCR, a to je TCR u kojem otpuštanje RNAP i popravak DNA povezuje elongacijski faktor NusA. Puno se toga ulaže u poznavanje mnogo kompleksnijeg mehanizma TCR kod eukariota zbog činjenice da mutacije u genima povezanima s gubitkom TCR dovode do teških bolesti kod ljudi. Mehanizam TCR kod ljudi je uglavnom nepoznat usprkos tome što su poznati neki od glavnih sudionika tog procesa poput proteina CSA, CSB, TFIIH i XPG. Važno je ustvrditi mehanizam TCR ne samo zbog problema koje zaustavljena RNAP stvara u stanici i bolesti koje uzrokuje nefunkcionalni TCR već i zbog toga što je TRCF uključen u povezivanje centralnih staničnih procesa i što bi daljnja saznanja o načinu na koji TRCF pomoću svojih domena utječe na RNAP i NER pomoglo u otkrivanju točnog mehanizma ovog procesa.Transcription-coupled DNA repair (TCR) is one of the subpathways of nucleotide excision repair (NER) that occurs when RNA polymerase is stalled at sites of DNA damage in the template strand. Template strand is repaired more quickly that similar lesions in coding strand or unexpressed parts of genome. Although the TCR was first observed in eukaryotes TCR phenomenon is best characterized in E. coli. The main role of TCR in bacteria has Mfd (ATP-dependent DNA translocase), also known as a transcription repair coupling factor (TRCF). Model for TCR postulate that Mfd recognizes and binds specifically to stalled and reverse translocated RNAP and using energy derived from ATP hydrolysis translocates forward RNAP to the transcription block. That results in RNAP release, transcript termination and recruitment of the NER machinery to the site of lesion. Recent studies show existence of alternative Mfd-independent TCR pathway mediated by elongation factor NusA. Alot is invested in understanding of more complex mechanism of TCR in eukaryotes due to the fact that mutations in genes associated with loss of TCR lead to severe disease in humans. The mechanism of TCR in humans is largely unknown despite having known some of the major participants in this process, such as CSA, CSB, TFIIH and XPG proteins. It is important to establish the mechanism of TCR not only because of the problems that stalled RNAP causes in the cell and diseases caused by dysfunctional TCR but also because the TRCF is involved in linking central cellular processes and knowing how TRCF uses its multiple domains to manipulate RNAP and the NER would help shed the light on the functional mechanism of TCR

Developing bioinformatics pipeline for processing environmental DNA metabarcoding sequencing data

Environmental DNA (eDNA) is DNA present in an environmental sample, originating from any biological material released from organisms living in that environment. This DNA can be isolated, amplified, sequenced, and analyzed in order to examine the taxonomic richness and abundance of different organism groups in the targeted environment. Methods of eDNA metabarcoding thus offer a unique opportunity to systematically streamline and scale-up regular biological assessments across many different environments of interest. Recently, as a part of the project funded by European structural and investment funds, Labena d.o.o. company established a modern laboratory in Zagreb focused on the research and provision of services in the field of eDNA. In collaboration with the Institute Ruđer Bošković we have been working on developing tests for analysis of water quality based on the eDNA and, as part of the standardization and optimization of sample-to-results eDNA analysis process, we developed a custom bioinformatics pipeline to facilitate efficient and effective eDNA sequencing data analysis. The pipeline was was written in Bash and utilizes several different algorithms to filter, trim, merge, denoise and classify targeted eDNA sequences. Python-based scripts which allow automatically download, filter, and format the data available on various online platforms were included in the pipeline to facilitate the curation of custom reference databases needed for taxonomic classification of targeted organism groups. User-friendly and interactive pipeline report generation, comprised of both wet- and dry-lab step-bystep sample statistics and graphical representations or the main results, is supported using Rmarkdown and Plotly and DataTables libraries. The pipeline is containerized in Docker, allowing for easier environment building and pipeline deployment.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202

Ostavština Tihomila Vidošića

Zadatak diplomskog rada bio je srediti građu iz skladateljeve ostavštine, razvrstati, popisati te utvrditi stanje i vrstu materijala kako bi se osigurali preduvjeti za daljnja istraživanja Vidošićevog rada. Cilj rada bio je napraviti popis građe u Vidošićevoj ostavštini, katalogizirati je i razvrstati po vrsti materijala kako bi se iz tako sređenih podataka mogao dobiti uvid u barem dio Visošićevih interesa i usmjerenja na skladateljskom i pedagoškom području

Ostavština Tihomila Vidošića

Zadatak diplomskog rada bio je srediti građu iz skladateljeve ostavštine, razvrstati, popisati te utvrditi stanje i vrstu materijala kako bi se osigurali preduvjeti za daljnja istraživanja Vidošićevog rada. Cilj rada bio je napraviti popis građe u Vidošićevoj ostavštini, katalogizirati je i razvrstati po vrsti materijala kako bi se iz tako sređenih podataka mogao dobiti uvid u barem dio Visošićevih interesa i usmjerenja na skladateljskom i pedagoškom području

Putevi biosinteze lignana u Linaceae : povezivanje pinoresinol-lariciresinol reduktaza s biosintezom pojedinih lignana

Le lin cultivé (Linum usitatissimum L.) est l’une des principales sources de lignanes, faisant de cette plante un modèle d’étude de cette voie du métabolisme spécialisé. Les principaux lignanes de lin dérivent de composés optiquement actifs, en particuliers les stéréoisomères du sécoisolaricirésinol qui sont synthétisés à partir de pinorésinol via laricirésinol. Les principales enzymes impliquées dans la synthèse de ces stéréoisomères sont deux isoformes de pinorésinol-laricirésinol réductases (PLR) déjà caractérisées et possédant des énantiospécificitées opposées. Néanmoins l'action de ces deux réductases bifonctionnelles ne permet pas d'expliquer les profils d'accumulation complexes notamment de laricirésinol et ses dérivés observés dans les graines, tiges et suspensions cellulaires de lin. Afin de mieux comprendre les mécanismes mis en oeuvre menant à ces profils d’accumulation de lignanes chez cette plante, la recherche de nouvelles PLRs a révélé l’existence de deux nouvelles isoformes. L’analyse de l'expression des gènes ainsi que l'activité enzymatique in vitro de ces deux nouvelles PLR putatives, LuPLR3 et LuPLR4 ont été élucidées. LuPLR4, in vitro, présente une activité réductase uniquement du pinorésinol. Ce type d’activité est ici décrit pour la première fois en dehors de la famille Brassicées et permet d’expliquer en partie les profils d’accumulation complexes observés chez le lin. De par leurs propriétés biocides, les lignanes sont suspectés jouer un rôle dans les mécanismes de défense des plantes. Dans le cadre de ce travail, suite à une élicitation fongique à l’aide d’extraits de Fusarium oxysporum spp. linii, un agent pathogène commun du lin, l’analyse de l’expression des différents gènes codant les isoformes de PLRs a révélées une induction globale et coordonnée. En particulier, dans le cas de l’isoforme LuPLR1, des délétions et mutations dans la région promotrice de son gène ont permis de mettre en évidence une région impliquée dans la régulation de la réponse à l’élicitation par F. oxysporum. Cette région contient plusieurs boîtes W, sites de liaison putatifs pour des facteurs de transcription de type WRKY. Les facteurs de transcription WRKY jouent un rôle dans les réponses aux stress biotique et abiotique. Un facteur de transcription candidat LuWRKY36 a été isolé à partir de suspensions cellulaires traitées avec des éliciteurs de F. oxysporum ou de l'acide abscissique. En particulier, les expériences de gel-retard et DPI-ELISA ont montré la capacité de liaison de LuWRKY36 à la boîte W3 présente du promoteur du gène LuPLR1. Cette régulation a ensuite été confirmée in vivo. Nous rapportons également l'impact différentiel de l'élicitation par des extraits de F. oxysporum sur l’expression des gènes LuWRKY36 et LuPLR1 ainsi que la production de sécoisolaricirésinol dans les variétés de lin sensible (Barbara) et résistante (Baïkal) à la fusariose. Enfin, la pleine exploitation des nombreux effets bénéfiques (en santé humaine ou cosmétique notamment) du sécoisolaricirésinol et des autres composés phénoliques accumulés dans les graines de lin nécessitent la mise au point de procédés d’extraction “verts”, efficaces voir sélectifs. Nous rapportons ici que l’utilisation de solvants eutectiques de type NADES (Natural Deep Eutectic Solvent) qui couplée à une extraction assistée par ultrasons, dans le cadre d’un procédé de type cracking, utilisant comme matériel de départ un coproduit d’extraction de l’huile de lin produit de manière innovante, permet d’obtenir des rendements d’extraction élevés et sélectifs de ces différents composés d’intérêt dans le cadre d’une démarche d’éco-extraction.L. usitatissimum is one of the richest sources of lignans. Main flax lignan is optically active secoisolariciresinol that is synthesized from pinoresinol via lariciresinol. Key enzymes involved in the synthesis of this lignans are two isoforms of pinoresinol-lariciresinol reductases with opposite enantiospecificity. The action of bifunctional reductase does not allow for an explanation for the accumulation of lariciresinol and its derivates in seeds, stem and cell suspension. To try and better understand complex lignan profile we report expression and activity of two new putative PLRs, LuPLR3 and LuPLR4. LuPLR4 in vitro acts only as pinoresinol reductase what has only been seen in Brassicaceae family until now. Lignans play a role in plant defense. All PLRs are upregulated following Fusarium oxysporum attack, a common flax pathogen. Promoter deletions and mutation evidenced region involved in regulation of LuPLR1 gene response to Fusarium. The region contains several W boxes, putative binding sites for WRKY transcription factors. WRKY transcription factors play a role in response to biotic and abiotic stress. We have isolated LuWRKY36 from two cell suspension treated with Fusarium oxysporum or abscisic acid. Gel-shift assay and DPI-ELISA showed binding of LuWRKY36 to W box present in the LuPLR1 gene promoter. This regulation was also confirmed in vivo. We also report the differential impact of F. oxysporum elicitation on LuWRKY36 and LuPLR1 gene expression and secoisolariciresinol production in flax cultivars Barbara (Fusarium sensitive) and Baikal (Fusarium tolerant). Many beneficial effects of secoisolariciresinol and other phenolic compounds found in flax require “green” extraction and sometimes targeted purification of a specific compound. We report here that natural deep eutectic solvents using ultrasound assisted extraction can extract phenolic compounds from flax seed coat and that results indicate that by tuning different parameters of extraction we can target purification of desired plant product

Ostavština Tihomila Vidošića

Zadatak diplomskog rada bio je srediti građu iz skladateljeve ostavštine, razvrstati, popisati te utvrditi stanje i vrstu materijala kako bi se osigurali preduvjeti za daljnja istraživanja Vidošićevog rada. Cilj rada bio je napraviti popis građe u Vidošićevoj ostavštini, katalogizirati je i razvrstati po vrsti materijala kako bi se iz tako sređenih podataka mogao dobiti uvid u barem dio Visošićevih interesa i usmjerenja na skladateljskom i pedagoškom području

Production of Antidiabetic Lignans in Flax Cell Cultures.

Lignans are a group of polyphenols that mimic endogenous estrogen in a structural context. Clear evidence was provided by animal studies and preclinical models that phytoestrogens could have antidiabetic activity in both estrogen-dependent and estrogen-independent pathways. A number of studies, including epidemiological studies and clinical studies conducted in different populations, link lignans, diabetes prevention, and management. Several lignans have been reported as potent antidiabetic compounds, in particular, secoisolariciresinol diglucoside (SDG), the major antidiabetic lignan from flax seeds. SDG have to be converted by the human intestinal microbiota into the bioactive mammalian lignans enterodiol and enterolactone. Most of the published research indicate that SDG may have a great potential to reduce type 1 diabetes mellitus incidence and to delay the development of type 2 diabetes mellitus in humans. Flax (Linum usitatissimum L.) cell cultures can provide an attractive and sustainable resource for the production and extraction of antidiabetic lignans. The advantage of using cell cultures as opposed to whole plants is that they can be used independently of climatic or seasonal considerations. This ensures more reproducible production of economically important bioactive extracts. This chapter provides an overview of the biotechnological approaches to the production of antidiabetic lignans in flax cell cultures