Structure of telomere and role of telomeric proteins

Telomere su zaštitne strukture na krajevima linearnih kromosoma koje su nužne na staničnoj razini za stabilnost genoma, dok na razini organizma sudjeluju u procesu starenja koji ima funkciju tumorske supresije. Telomere mogu regulirati duljinu životnog vijeka stanica i štite prirodne krajeve kromosoma od neželjenih aktivnosti popravka DNA kao što su nehomologno spajanje krajeva DNA i homologna rekombinacija. Kromosomi imaju na svojim krajevima telomernu DNA s uzastopnim ponavljanjem heksanukleotidne sekvence koja oblikuje omčastu strukturu uz pomoć specifičnih proteina, tzv. T petlju. Šelterin, zaštitni proteinski kompleks, sastoji se od šest proteina: TRF1, TRF2, TIN2, Rap1, TPP1 i POT1 koji su međusobno povezani i vežu telomernu sekvencu. Inhibicija TRF2 rezultira poremećajem telomernih struktura, aktivacijom p53 i procesima apoptoze ili starenja, odnosno fuzijom krajeva kromosoma, ovisno o tipu stanica. Najnovijim je istraživanjima dokazano da se na telomerama nalazi i velika nekodirajuća RNA koja stvara cjelovitu komponentu telomernog heterokromatina, TERRA. Na temelju njene funkcije, zaključeno je da je takva RNA uključena u regulaciju enzima telomeraze i remodeliranje kromatina tijekom razvoja i diferencijacije stanice.Telomeres are protective structures at the ends of linear chromosomes. They are critical at the cellular level for maintenance of the genome stability. At the organismal level they may contribute to ageing, which is considered to be tumor supressing mechanism. They can regulate the lifespan of cells and protect natural chromosome ends from unwanted DNA repair activities, such as non-homologous end joining and homologous recombination. The chromosomes end in telomeric DNA containing tandem hexanucleotid repeats in specific loop structure. The complex of specific proteins, named Shelterin, takes part in the maintenance of the T loop and telomere function. Shelterin consists of six proteins: TRF1, TRF2, TIN2, Rap1, TPP1 and POT1. The complex has a high affinity toward telomeric sequence. TRF2 inhibition compromises the structure of the telomeres and can cause p53 activation, apoptosis or cell senescence as well as chromosome end fusion, depending on the cell type. The recent investigations had identified telomeric repeat containing RNA, TERRA, a large noncoding RNA forming an integral component of telomeric heterochromatin. TERRA functions suggest important roles in the regulation of telomerase and in orchestrating chromatin remodelling throughout development and cellular differentiation

CpG methylation of MGAT3 and MGAT5 glyco-genes and glycosylation of immunoglobulin G in inflammatory bowel diseases

Upalne bolesti crijeva (engl. inflammatory bowel diseases, IBD) su kronične bolesti probavnog trakta koje nastaju zbog neodgovarajućeg upalnog odgovora imunološkog sustava na crijevnu mikrofloru u osoba s genetičkom predispozicijom. Cjelogenomske studije povezanosti su povezale gliko-gen MGAT3 s glikozilacijom imunoglobulina G (IgG) i IBD-em. Druga istraživanja su s razvojem IBD-a povezala i gliko-gen MGAT5. Bisulfitnim pirosekvenciranjem analizirala sam metilaciju gena MGAT3 i MGAT5 iz krvi pacijenata s IBD-em dviju velikih neovisnih kohorti te pokazala da je metilacija ovih gena značajno promijenjena u ulceroznom kolitisu i Crohnovoj bolesti. Korelacijske analize upućuju na smanjenu ekspresiju gena MGAT3 (kodira za enzim GnT-III) u IBD-u, ako je točna pretpostavka da su analizirana CpG mjesta unutar promotora tog gena uključena u regulaciju ekspresije. U trećoj manjoj kohorti analizirala sam metilaciju gena MGAT3 i MGAT5 u mononuklearnim stanicama krvi, T-limfocitima izoliranim iz krvi i iz tkiva crijeva, te pokazala značajnu promjenu metilacije oba gena u T-limfocitima iz crijeva, te promijenjenu metilaciju i ekspresiju gena MGAT5 u T-limfocitima iz tkiva crijeva pacijenata s aktivnim oblikom ulceroznog kolitisa.Inflammatory bowel diseases (IBD) are a group of intestinal disorders that occur due to inappropriate immune response to intestinal microbiome in genetically susceptible individuals. Genome wide association studies associated MGAT3 gene with IgG glycosylation and IBD. Previous studies associated another glyco-gene, MGAT5 with IBD, too. Methylation of MGAT3 and MGAT5 genes was analysed from whole blood of IBD patients from two large independent cohorts using bisulfite pyrosequencing. The results showed significant change in methylation level between patients with Crohn's disease and ulcerative colitis, and healthy controls. Correlation analysis between MGAT3 methylation and IgG glycans suggests that MGAT3 (coding for GnT-III enzyme) expression is reduced in IBD, if CpG sites encompassed with methylation assays are relevant for transcriptional regulation. In third cohort with smaller number of patients with ulcerative colitis, methylation of MGAT3 and MGAT5 genes was analysed in mononuclear cells from blood, T cells from blood, and T cells from inflamed bowel tissue. Change in methylation level of both genes was observed only in T cells from bowel tissue. The methylation and expression of MGAT5 gene was significantly changed in T cells from bowel tissue in active ulcerative colitis

CRISPR-Cas in Escherichia coli: regulation by H-NS, LeuO and temperature

CRISPR-Cas adaptive immune systems are present in many bacteria and archaea and provide protection against invading DNA such as phages and plasmids. These systems are very versatile and complex in their gene composition and genomic architecture. CRISPR-Cas systems are classified into 2 classes, 6 types and 33 subtypes although this number is not definitive and the research is ongoing. All CRISPR-Cas systems have been thoroughly investigated in order to better understand the mechanism of CRISPR immunity enabling its use as a tool in genome editing and other biotechnological applications. However, regulation of the CRISPR-Cas system is also very complex and still not fully understood; it must provide optimal protection without introducing harmful consequences to the host. In this review we give an overview on the regulation of the CRISPR-Cas system Class 1 Type I-E in Escherichia coli with the emphasis on the role of temperature in regulation of the CRISPR-Cas activity and the interplay of the key regulators H-NS and StpA repressors and LeuO antirepressor in regulation of cas gene expression and HtpG chaperone in maintaining functional levels of Cas3.</p

Genome Editing Tools for Functional Analysis of HNF1A and IL6ST Genes

Genome editing tools, such as TALEN (transcription activator-like effector nuclease) or CRISPR-Cas9 (CRISPR-associated protein-9 nuclease) systems, enable functional studies by targeted gene knockout. They introduce double-stranded breaks (DSBs) into a DNA molecule in a sequence-specific manner, thereby stimulating the error-prone non-homologous end joining repair mechanism, leading to probable gene inactivation when the coding sequence is targeted. Vectors for expression of TALEN and Cas9-based constructs targeting the human IL6ST and HNF1A genes were assembled and tested for their ability to introduce DSBs when transfected into cultured cells using the luciferase assay. The Cas9-based construct targeting the IL6ST gene was shown to be active, while the two TALEN-based constructs did not introduce DSBs above background level. Both the TALEN and the CRISPR-Cas9 constructs targeting the HNF1A gene were found to be active, with the TALEN showing higher activity in a dose-dependent manner. The constructed genome-editing tools can be used for functional analysis of the putative role of HNF1A and IL6ST genes in IgG glycosylation, as shown previously by genome wide association studies. This work is licensed under a Creative Commons Attribution 4.0 International License

A tryptophan ‘gate’ in the CRISPR-Cas3 nuclease controls ssDNA entry into the nuclease site, that when removed results in nuclease hyperactivity

Cas3 is a ssDNA-targeting nuclease-helicase essential for class 1 prokaryotic CRISPR immunity systems, which has been utilized for genome editing in human cells. Cas3-DNA crystal structures show that ssDNA follows a pathway from helicase domains into a HD-nuclease active site, requiring protein conformational flexibility during DNA translocation. In genetic studies, we had noted that the efficacy of Cas3 in CRISPR immunity was drastically reduced when temperature was increased from 30C to 37C, caused by an unknown mechanism. Here, using E. coli Cas3 proteins, we show that reduced nuclease activity at higher temperature corresponds with measurable changes in protein structure. This effect of temperature on Cas3 was alleviated by changing a single highly conserved tryptophan residue (Trp-406) into an alanine. This Cas3W406A protein is a hyperactive nuclease that functions independently from temperature and from the interference effector module Cascade. Trp-406 is situated at the interface of Cas3 HD and RecA1 domains that is important for maneuvering DNA into the nuclease active site. Molecular dynamics simulations based on the experimental data showed temperature-induced changes in positioning of Trp-406 that either blocked or cleared the ssDNA pathway. We propose that Trp- 406 forms a ‘gate’ for controlling Cas3 nuclease activity via access of ssDNA to the nuclease active site. The effect of temperature in these experiments may indicate allosteric control of Cas3 nuclease activity caused by changes in protein conformations. The hyperactive Cas3W406A protein may offer improved Cas3-based genetic editing in human cells

Impact of Traffic Demand on Air Traffic Complexity

Analizom prometnih podataka objavljenih od strane HKZP-a vidljivo je povećanje prometa i prometne potražnje zadnjih nekoliko godina. Prometne prognoze prometne potražnje koje je objavio EUROCONTROL predviđaju rast prometa bez obzira na zadnje dvije godine koje su zabilježile veliki pad prometa zbog pandemije uzrokovane virusom SARS Covid-19. Prometna potražnja povezana je s kompleksnosti koja ovisi o različitim indikatorima. Pomoću programa NEST mogu se izračunati ti indikatori i napraviti konačan rezultat kompleksnosti pomoću formula za izračun kompleksnosti. U programu NEST mogu se napraviti različite simulacije prometa. U ovom radu su napravljene simulacije povećanja prometne potražnje prometa 2018. godine za 5, 10 i 15 % te je uspoređen promet i kompleksnost u odnosu na 2018. i 2019. godinu. Konačni rezultati pokazuju kako je prometna potražnja utjecala na kompleksnost zračnog prometa.An analysis of traffic data published by HKZP shows an increase in traffic and traffic demand over the past few years. Traffic demand forecasts published by EUROCONTROL predict traffic growth regardless of the last two years, which have seen a large drop in traffic due to the pandemic caused by the SARS Covid-19 virus. Traffic demand is associated with complexity that depends on different indicators. Using the NEST program, these indicators can be calculated and a final complexity result can be made using formulas for calculating complexity. Various traffic simulations can be made in the NEST program. In this paper, simulations of the increase in traffic demand in 2018 by 5, 10 and 15% were made, and traffic and complexity were compared to 2018 and 2019. The final results show how traffic demand affected the complexity of air traffic

Impact of Traffic Demand on Air Traffic Complexity

Analizom prometnih podataka objavljenih od strane HKZP-a vidljivo je povećanje prometa i prometne potražnje zadnjih nekoliko godina. Prometne prognoze prometne potražnje koje je objavio EUROCONTROL predviđaju rast prometa bez obzira na zadnje dvije godine koje su zabilježile veliki pad prometa zbog pandemije uzrokovane virusom SARS Covid-19. Prometna potražnja povezana je s kompleksnosti koja ovisi o različitim indikatorima. Pomoću programa NEST mogu se izračunati ti indikatori i napraviti konačan rezultat kompleksnosti pomoću formula za izračun kompleksnosti. U programu NEST mogu se napraviti različite simulacije prometa. U ovom radu su napravljene simulacije povećanja prometne potražnje prometa 2018. godine za 5, 10 i 15 % te je uspoređen promet i kompleksnost u odnosu na 2018. i 2019. godinu. Konačni rezultati pokazuju kako je prometna potražnja utjecala na kompleksnost zračnog prometa.An analysis of traffic data published by HKZP shows an increase in traffic and traffic demand over the past few years. Traffic demand forecasts published by EUROCONTROL predict traffic growth regardless of the last two years, which have seen a large drop in traffic due to the pandemic caused by the SARS Covid-19 virus. Traffic demand is associated with complexity that depends on different indicators. Using the NEST program, these indicators can be calculated and a final complexity result can be made using formulas for calculating complexity. Various traffic simulations can be made in the NEST program. In this paper, simulations of the increase in traffic demand in 2018 by 5, 10 and 15% were made, and traffic and complexity were compared to 2018 and 2019. The final results show how traffic demand affected the complexity of air traffic

Relation of DNA traces from different surfaces with donor age and gender

Ovim se istraživanjem nastojalo otkriti ovisnost količine DNA-traga o različitim čimbenicima. Analizirala se povezanost dobi i spola donora te količine DNA koju su ispitanici ostavili na predmetima različite teksture (papir, plastika, plastificirani metal) prilikom trljanja prstima i dlanom ili držanja predmeta u ruci te ovisnost količine DNA-traga o vremenu kontakta ispitanika s predmetima. Predmeti su prebrisani navlaženim vatiranim štapićem, DNA se izolirala metodom Chelex, a za kvantifikaciju DNA se koristila lančana reakcija polimerazom u stvarnom vremenu (engl. Real Time PCR). Rezultati su pokazali da je količina DNA-traga neovisna o vremenu u kojem je ispitanik bio u dodiru s predmetima. Nasuprot tome, ovisna je o teksturi predmeta pri čemu je najveća količina DNA-traga ostala na plastificiranom metalu, a najmanja na papiru. Osobe između 35 i 44 godine starosti te muškarci ostavili su najveću količinu DNA-traga. Konačno, u ovom smo radu pokazali da je svojstvo ostavljanja određene količine DNA na predmetima individualno.The aim of this research was to discover the dependence of trace DNA quantity on various factors. We analyzed the correlation between handling time, donor age and gender and quantity of DNA trace left by individuals on items of different textures (paper, plastic, plastic coated metal) during rubbing with fingers and palm or holding items in a hand. Items were swabbed with moistened cotton swab, DNA was isolated by Chelex method and quantified by Real Time PCR. The results showed the amount of DNA transferred to an item was independent of the handling time. In contrast, it was dependent of item texture where the greatest amount was left on plastic coated metal and the least on paper. The greatest amount of DNA trace was left by individuals ranging in age from 35 to 44 years and men. Finally, this study showed that the tendency to deposit DNA on an item was an independent characteristic of a person

Impact of Traffic Demand on Air Traffic Complexity

Analizom prometnih podataka objavljenih od strane HKZP-a vidljivo je povećanje prometa i prometne potražnje zadnjih nekoliko godina. Prometne prognoze prometne potražnje koje je objavio EUROCONTROL predviđaju rast prometa bez obzira na zadnje dvije godine koje su zabilježile veliki pad prometa zbog pandemije uzrokovane virusom SARS Covid-19. Prometna potražnja povezana je s kompleksnosti koja ovisi o različitim indikatorima. Pomoću programa NEST mogu se izračunati ti indikatori i napraviti konačan rezultat kompleksnosti pomoću formula za izračun kompleksnosti. U programu NEST mogu se napraviti različite simulacije prometa. U ovom radu su napravljene simulacije povećanja prometne potražnje prometa 2018. godine za 5, 10 i 15 % te je uspoređen promet i kompleksnost u odnosu na 2018. i 2019. godinu. Konačni rezultati pokazuju kako je prometna potražnja utjecala na kompleksnost zračnog prometa.An analysis of traffic data published by HKZP shows an increase in traffic and traffic demand over the past few years. Traffic demand forecasts published by EUROCONTROL predict traffic growth regardless of the last two years, which have seen a large drop in traffic due to the pandemic caused by the SARS Covid-19 virus. Traffic demand is associated with complexity that depends on different indicators. Using the NEST program, these indicators can be calculated and a final complexity result can be made using formulas for calculating complexity. Various traffic simulations can be made in the NEST program. In this paper, simulations of the increase in traffic demand in 2018 by 5, 10 and 15% were made, and traffic and complexity were compared to 2018 and 2019. The final results show how traffic demand affected the complexity of air traffic

Surface Coating-Modulated Phytotoxic Responses of Silver Nanoparticles in Plants and Freshwater Green Algae

Silver nanoparticles (AgNPs) have been implemented in a wide range of commercial products, resulting in their unregulated release into aquatic as well as terrestrial systems. This raises concerns over their impending environmental effects. Once released into the environment, they are prone to various transformation processes that modify their reactivity. In order to increase AgNP stability, different stabilizing coatings are applied during their synthesis. However, coating agents determine particle size and shape and influence their solubility, reactivity, and overall stability as well as their behavior and transformations in the biological medium. In this review, we attempt to give an overview on how the employment of different stabilizing coatings can modulate AgNP-induced phytotoxicity with respect to growth, physiology, and gene and protein expression in terrestrial and aquatic plants and freshwater algae