Automated Slovene crosswords solving with the help of the internet

The goal of the thesis was to create a program, that automatically solves Slovene crosswords with the help of puzzle dictionary and web search. For the purpose of creating the program, we used a database of 367 crosswords and puzzle dictionary with 104.123 clue answers. We also created a list of web answers for all the crosswords in the database. Because of the enormous amount of the web answers searching the whole solution space would not be feasible. That is why the program solves each crossword in two separate phases. In the first phase, a crossword gets partially solved with the help of the puzzle dictionary, which creates a list of most probable solution candidates for each clue. In the second phase, a crossword is solved with puzzle dictionary and web answers combined. For the purpose of building the program, we used a set of 37 training crosswords. The final results were obtained from a set of 330 testing crosswords

Exposure of honey bee larvae to thiamethoxam and its interaction with Nosema ceranae infection in adult honey bees

During their lifetime honey bees (Apis mellifera) rarely experience optimal conditions. Sometimes, a simultaneous action of multiple stressors, natural and chemical, results in even greater effect than of any stressor alone. Therefore, integrative investigations of different factors affecting honey bees have to be carried out. In this study, adult honey bees exposed to thiamethoxam in larval and/or adult stage and infected with Nosema ceranae were examined. Newly emerged bees from colonies, non-treated or treated with thiamethoxam, were organized in six groups and kept in cages. Thiamethoxam treated bees were further exposed to either thiamethoxam or Nosema (groups TT and TN), or simultaneously to both (group TTN). Newly emerged bees from non-treated colonies were exposed to Nosema (group CN). From both, treated and non-treated colonies two groups were organized and further fed only with sugar solution (groups C and TC). Here, we present the expression profile of 19 genes in adult worker honey bees comprising those involved in immune, detoxification, development and apoptosis response. Results showed that gene expression patterns changed with time and depended on the treatment. In group TC at the time of emergence the majority of tested genes were downregulated, among which nine were significantly altered. The same gene pattern was observed on day six, where the only significantly upregulated gene was defensin-1. On day nine most of analyzed genes in all experimental groups showed upregulation compared to control group, where upregulation of antimicrobial peptide genes abaecin, defensin-1 and defensin-2 was significant in groups TT and TTN. On day 15 we observed a similar pattern of expression in groups TC and TT exposed to thiamethoxam only, where most of the detoxification genes were downregulated. Additionally RNA loads of Nosema and honey bee viruses were recorded. We detected a synergistic interaction of thiamethoxam and Nosema, reflected in lowest honey bee survival

implications of glycemic control

Funding Information: The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was supported by the Slovenian Research and Innovation Agency ARIS (P3-0343, and J7-1820). It was partially funded by the ISPAD JDRF Fellowship Grant 2018. JG was supported by the ESPE fellowship grant. Publisher Copyright: Copyright © 2024 Čugalj Kern, Kovač, Šket, Tesovnik, Jenko Bizjan, Galhardo, Battelino, Bratina and Dovč.Background: Prolonged hyperglycemia causes diabetes-related micro- and macrovascular complications, which combined represent a significant burden for individuals living with diabetes. The growing scope of evidence indicates that hyperglycemia affects the development of vascular complications through DNA methylation. Methods: A genome-wide differential DNA methylation analysis was performed on pooled peripheral blood DNA samples from individuals with type 1 diabetes (T1D) with direct DNA sequencing. Strict selection criteria were used to ensure two age- and sex-matched groups with no clinical signs of chronic complications according to persistent mean glycated hemoglobin (HbA1c) values over 5 years: HbA1c8% (N=10). Results: Between the two groups, 8385 differentially methylated CpG sites, annotated to 1802 genes, were identified. Genes annotated to hypomethylated CpG sites were enriched in 48 signaling pathways. Further analysis of key CpG sites revealed four specific regions, two of which were hypermethylated and two hypomethylated, associated with long non-coding RNA and processed pseudogenes. Conclusions: Prolonged hyperglycemia in individuals with T1D, who have no clinical manifestation of diabetes-related complications, is associated with multiple differentially methylated CpG sites in crucial genes and pathways known to be linked to chronic complications in T1D.publishersversionpublishe

Response of adult honey bees treated in larval stage with prochloraz to infection with Nosema ceranae

Among numerous factors that contribute to honey bee colony losses and problems in beekeeping, pesticides and Nosema ceranae have been often reported. In contrast to insecticides, whose effects on bees have been widely studied, fungicides did not attract considerable attention. Prochloraz, an imidazole fungicide widely used in agriculture, was detected in honey and pollen stored inside hives and has been already proven to alter immune gene expression of honey bees at different developmental stages. The aim of this study was to simulate the realistic conditions of migratory beekeeping, where colonies, both uninfected and infected with N. ceranae, are frequently transported to the vicinity of crop fields treated with prochloraz. We investigated the combined effect of prochloraz and N. ceranae on honey bees that faced fungicide during the larval stage through food consumption and microsporidium infection afterwards. The most pronounced changes in gene expression were observed in newly emerged Nosema-free bees originating from colonies previously contaminated with prochloraz. As exclusively upregulation was registered, prochloraz alone most likely acts as a challenge that induces activation of immune pathways in newly emerged bees. The combination of both stressors (prochloraz and Nosema infection) exerted the greatest effect on six-day-old honey bees. Among ten genes with significantly altered expression, half were upregulated and half downregulated. N. ceranae as a sole stressor had the weakest effects on immune gene expression modulation with only three genes significantly dysregulated. In conclusion, food contaminated with prochloraz consumed in larval stage could present a threat to the development of immunity and detoxification mechanisms in honey bees

Exploring early DNA methylation alterations in type 1 diabetes: implications of glycemic control

BackgroundProlonged hyperglycemia causes diabetes-related micro- and macrovascular complications, which combined represent a significant burden for individuals living with diabetes. The growing scope of evidence indicates that hyperglycemia affects the development of vascular complications through DNA methylation.MethodsA genome-wide differential DNA methylation analysis was performed on pooled peripheral blood DNA samples from individuals with type 1 diabetes (T1D) with direct DNA sequencing. Strict selection criteria were used to ensure two age- and sex-matched groups with no clinical signs of chronic complications according to persistent mean glycated hemoglobin (HbA1c) values over 5 years: HbA1c<7% (N=10) and HbA1c>8% (N=10).ResultsBetween the two groups, 8385 differentially methylated CpG sites, annotated to 1802 genes, were identified. Genes annotated to hypomethylated CpG sites were enriched in 48 signaling pathways. Further analysis of key CpG sites revealed four specific regions, two of which were hypermethylated and two hypomethylated, associated with long non-coding RNA and processed pseudogenes.ConclusionsProlonged hyperglycemia in individuals with T1D, who have no clinical manifestation of diabetes-related complications, is associated with multiple differentially methylated CpG sites in crucial genes and pathways known to be linked to chronic complications in T1D

Extracellular vesicles derived small non-coding RNA in type 1 diabetes

UVOD Sladkorna bolezen tipa 1 nastane kot posledica avtoimunskega uničenja celic beta Langerhansovih otočkov trebušne slinavke, ki izločajo glavni anabolni hormon inzulin. Zaradi pomanjkanja lastnega hormona inzulina so osebe s sladkorno boleznijo tipa 1 primorane doživljenjsko prejemati zdravljenje z dodajanjem eksogenih odmerkov inzulina in dosledno izvajati meritve krvnega sladkorja, s čemer lahko preprečijo oziroma odložijo nastanek zapletov sladkorne bolezni. Vzrok nastanka avtoimunosti še vedno ni pojasnjen, med drugim tudi zaradi pomanjkanja bioloških označevalcev za zgodnje odkrivanje bolezni in zaradi tkivne nedostopnosti celic beta Langerhansovih otočkov v človeškem organizmu. Zunajcelični vezikli so majhne sferične strukture, ki jih celice izločajo v zunajcelični prostor z namenom celične komunikacije in usklajenega delovanja organizma. Poleg vloge pri zagotavljanju normalnega delovanja organizma lahko zunajcelični vezikli in miRNA sodelujejo tudi pri patogenezi številnih bolezni. Z doktorskim delom smo poskušali opredeliti zunajcelične vezikle kot potencialne biološke označevalce in posrednike pri nastanku sladkorne bolezni tipa 1. METODE Prisotnost zunajceličnih veziklov v izolirani frakciji krvne plazme z verjetnim izvorom iz celic beta Langerhansovih otočkov smo dokazovali z označevanjem vzorcev s specifičnimi protitelesi in slikanjem s transmisijsko elektronsko mikroskopijo. Z metodo sekvenciranja naslednje generacije smo določali miRNA zunajceličnih veziklov izoliranih iz krvne plazme desetih posameznikov s sladkorno boleznijo tipa 1 ob izbruhu bolezni, desetih posameznikov z več kot deset let trajajočo boleznijo in desetih zdravih preiskovancev. Z namenom opredelitve miRNA zunajceličnih veziklov, ki so prisotni v krvni plazmi ob intenzivnem propadu celic beta, smo kronološko analizirali tudi vzorce zunajceličnih veziklov izoliranih iz krvne plazme dveh transplantirancev Langerhansovih otočkov. Vpliv izbranih različno izraženih miRNA smo določali z in vitro testiranjem na vzorcih polne krvi, pri čemer smo spremljali izražanje označevalca zgodnje aktivacije CD69 in citotoksičnosti CD107a celic imunskega sistema s pretočno citometrijo. S fluorescenčno označeno miRNA smo s pretočno citometrijo in fluorescenčno mikroskopijo opredeljevali kopičenje veziklov v krvnih celicah imunskega sistema. Aktivacijo krvnih celic imunskega sistema ob izpostavitvi z miRNA s sintetičnimi vezikli smo utišali z dodatkom inhibitorja endosomalnih TLR7/8 receptorjev in predpostavili potencialen model učinkovanja miRNA zunajceličnih veziklov na imunski sistem. Pri preiskovancih s sladkorno boleznijo tipa 1 smo dodatno opredelili tudi dolžine telomer, ki so tudi uravnavane z miRNA in veljajo za označevalce staranja organizma ter delovanja oksidativnega stresa. REZULTATI S transmisijsko elektronsko mikroskopijo smo dokazali prisotnost zunajceličnih veziklov v krvni plazmi, ki potencialno izvirajo iz celic beta Langerhansovih otočkov, kar nakazuje na možno komunikacijo celic beta z drugimi tkivi v človeškem telesu. S sekvenciranjem naslednje generacije smo določili različno izražene miRNA pri preiskovancih s sladkorno boleznijo tipa 1 in intenzivnemu propadu celic beta ob transplantaciji Langerhansovih otočkov. Na podlagi rezultatov primerjalnih analiz izražanja miRNA smo izbrali osem zvrsti miRNA (hsa-miR-122-5p, hsa-miR-192-5p, hsa-miR-193b-5p, hsa-miR-185-5p, hsa-miR-195-3p, hsa-miR-455-5p, hsa-miR-375-3p, hsa-miR-129-5p), ki smo jih s sintetičnimi vezikli izpostavili nativnim krvnim celicam oseb s sladkorno boleznijo tipa 1 in zdravih preiskovancev pri in vitro pogojih, kar je povzročilo aktivizacijo celic imunskega sistema. Aktivacija imunskega sistema se je odražala s povečanim izražanjem zgodnjega označevalca aktivacije CD69 in citotoksičnosti CD107a celic T CD4+ in CD8+ in naravnih celic ubijalk CD56+. Pokazali smo, da se vezikli preferenčno kopičijo v fagocitnih krvnih celicah, kjer se nahajajo TLR7 in TLR8 receptorji, ki so najverjetneje odgovorni za prepoznavanje vezikularnih miRNA. Vpletenost TLR7 in TLR8 smo dokazovali s TLR7/8 inhibitorjem klorokinom, ki je ob stimulaciji celic imunskega sistema z miRNA in sintetičnimi vezikli znižal aktivacijski odziv. Prepoznavanje človeških miRNA zunajceličnih veziklov v endosomalni poti fagocitnih celic s TLR7 in TLR8 sproži imunski odziv in aktivacijo efektorskih celic imunskega sistema. Rezultati naših raziskav so pokazali tudi razlike v izražanju miRNA, katerih vloga je že bila vpisana pri regulaciji dolžin telomer. Dolžine telomer pri preiskovancih s sladkorno boleznijo tipa 1 s slabo glikemično urejenostjo so se krajšale hitreje kot pri preiskovancih z dobro glikemično urejenostjo, k čemur so morda pripomogle tudi miRNA zunajceličnih veziklov. ZAKLJUČEK Rezultati naše raziskave nakazujejo na pomembno vlogo človeških miRNA pri regulaciji imunskega sistema in staranja organizma. miRNA zunajceličnih veziklov so lahko udeležene pri aktivaciji imunskega sistema in lahko pripomorejo k nastanku začetnega vnetja Langerhansovih otočkov. Človeške miRNA tako lahko sodelujejo pri razvoju avtoimunosti in nastanku sladkorne bolezni tipa 1. Raziskava dodatno razkriva potencialne tarče za preprečevanje in zdravljenje sladkorne bolezni tipa 1.INTRODUCTION Type 1 diabetes is caused by autoimmune destruction of Langerhans islets\u27 beta-cells, resulting in an absolute lack of one’s own insulin. Hence, individuals with type 1 diabetes require a life long intensive exogenous insulin therapy with glucose monitoring to prevent the development of diabetes-related complications, which can dramatically shorten their lifespan. The cause of type 1 diabetes is still unknown due to a lack of biomarkers and difficulties in gaining the insight into beta-cells functioning during the developmental stages of the disease. With their molecular cargo extracellular vesicles can modulate a target cell response and may affect the pathogenesis of the diseases. The extracellular vesicles containing miRNAs are often studied as disease biomarkers, but rarely as mediators of development of the disease. The role of extracellular vesicles derived miRNAs in type 1 diabetes is currently not well established. Our work aimed to evaluate the extracellular vesicles as biomarkers in type 1 diabetes and evaluate their role in the modulation of the immune system in the development of the disease. METHODS Transmission electron microscopy and vesicle labelling with specific antibodies was used to characterize the extracellular vesicles originating from insulin-producing pancreatic beta cells in blood plasma. In the next step, differentially expressed extracellular vesicles’ miRNAs were evaluated using the next-generation sequencing approach in ten individuals with type 1 diabetes at the disease onset, ten individuals with ten years duration of the disease, and ten healthy controls. To assess the extracellular vesicle miRNAs in intensive beta-cell destruction, Langerhans islet transplantation individuals’ chronological blood plasma samples were sequenced and analysed. Eight differentially expressed miRNAs in type 1 diabetes and Langerhans islets destruction were selected for the in vitro immunomodulation testing with the synthetic vesicle delivery system on the whole blood samples. Flow cytometry was applied to evaluate the immune system activation with the detection of CD107a degranulation and CD69 early activation markers on CD4+ and CD8+ T-cells and CD56+ NK cells. The vesicle miRNA accumulation in the cells of the immune system was evaluated with microscopy and flow cytometry after the whole blood cells exposure to synthetic vesicles and fluorescent labeled miRNA. The endosomal TLR7/8 inhibition in the transfection experiments was performed to evaluate the role of these receptors in the recognition of extracellular vesicle delivered miRNA in the immune system activation. Additionally, miRNAs regulate telomere lengths in the cells of a human organism and these are biomarkers of oxidative stress and organsim aging. Telomere lengths were assessed in the individuals with type 1 diabetes according to their glycemic control. RESULTS Transmission electron microscopy imaging confirmed Langerhans islets beta-cell-released extracellular vesicles in the blood plasma and potential beta-cells’ communication with other tissues. Based on the next-generation sequencing differential expression analysis results, eight significantly differentially expressed miRNA in type 1 diabetes and Langerhans transplantation individuals were selected (hsa-miR-122-5p, hsa-miR-192-5p, hsa-miR-193b-5p, hsa-miR-185-5p, hsa-miR-195-3p, hsa-miR-455-5p, hsa-miR-375-3p and hsa-miR-129-5p). The selected differentially expressed miRNA delivered with synthetic vesicles to the whole blood samples from participants with type 1 diabetes and healthy controls showed the immunomodulation activity with the overexpression of an early activation marker CD69 and a cytotoxicity marker CD107a on CD4+ and CD8+ T-cells and CD 56+ NK cells. The phagocytic cells accumulated vesicles with miRNAs in the endolysosomal pathway, where TLR7 and TLR8 were expressed. The potential involvement of the endolysosomal TLR7/8 in the activation of the immune system was shown with the endosomal TLR7/8 inhibitor chloroquine, which effectively inhibited a vesicle delivered miRNAs activation. The sequencing results also revealed differentially expressed miRNAs, whose role was reported in the telomere length regulation. The telomere length erosion in the individuals with type 1 diabetes with poor glyceamic control was accelerated compared to the good glycemic controlled individuals. SUMMARY Our results illustrate miRNAs derived from human blood plasma extracellular vesicles as modulators of the immune system in type 1 diabetes autoimmunity, providing a potentially new insight into the pathogenesis of the disease, and novel molecular targets for intervention and type 1 diabetes prevention

Natural and anthropogenic impact on Holocene marine sediment sedimentation in Gulf of Trieste

V okviru magistrskega dela smo proučevali vrtino MC-2, ki predstavlja točkovni odvzem zaporedja sedimentov morskega dna na izbrani lokaciji med Ankaranom in Koprom. Vrtina je dolga 636 cm in zajema celotno holocensko sedimentacijo. Na podlagi analize sedimentoloških značilnosti in granulometričnih lastnosti smo ugotovili, da je vrtina sestavljena iz plasti sedimenta različnih debelin (1-86 cm), ki se glede na sestavo klasificirajo kot glinasti melj, peščeni melj in meljasti pesek. V holocenski sekvenci prevladuje glinasti melj (85,88 %), sledita mu peščeni melj (9,24 %) in meljasti pesek (4,87 %). V vseh jedrih smo opazili prisotnost ostankov polžev, školjk in ostalega biogenega drobirja. Mineraloško smo identificirali prisotnost kalcita, dolomita, kremena, pirita, halita, illita, albita, klorita in kaolinita. Opazili smo trend naraščanja deleža kremena in dolomita z globino ter obraten trend padanja deleža kalcita z globino. Na podlagi analize PCA smo merjene geokemične, granulometrične in mineraloške parametre razvrstili glede na njihovo medsebojno korelacijo v štiri glavne klastre. Prvi klaster združuje parametre, kot so silicij (Si), razmerje organskega ogljika/totalnega dušika (TOC/TN), kremen, magnezij (Mg) in dolomit, ki jih pripisujemo terigenemu doprinosu iz okoliških zalednih kamnin. Vrednosti parametrov prvega klastra z globino naraščajo. Drugi klaster združuje aluminij (Al), železo (Fe), kalij (K) ter frakcijo melja in gline, ki jih povezujemo z minerali glin (kaolinit, illit, klorit). Tretji klaster združuje žveplo (S), krom (Cr), mangan (Mn), nikelj (Ni), baker (Cu), cink (Zn), arzen (As), litij (Li), kadmij (Cd), svinec (Pb), kalcit, pirit, skupni dušik (TN), organski ogljik (TOC) ter izotopsko sestavo ogljika (δ13Corg). Izvor tega klastra ni enoznačno določen, vendar verjetno vključuje parametre, povezane z bioprodukcijo okolja (S, TOC), avtigenimi karbonati bioklastov (kalcit, TN, δ13Corg) ter mešanim vplivom organske snovi, antropogenega vpliva in doprinosa okolnega fliša (Cr, Mn, Ni, Cu, Zn, As, Li, Cd, Pb). Vrednosti parametrov drugega klastra generalno z globino padajo. Vsebnosti Cr, Pb, Cu, Ni in Zn so višje kot v okolnem flišu, kar kaže na antropogeno onesnaženje v zgornjem delu. Četrti klaster predstavlja arzen (As) in frakcijo peska, katerih izvor je lahko povezan s piritom, halitom, organsko snovjo, železo/manganovimi oksidi in oksihidroksidi. Z absolutno starostno datacijo s 14C smo opredeli holocensko sekvenco sedimentov, pri čemer je najgloblji vzorec na globini 640 cm star 10.515 ± 84 let. V zgornjem delu vrtine do globine 166 cm smo opazili odstopanje, ki ga pripisujemo antropogenemu mešanju sedimenta kot posledica poglabljanja ladijskega koridorja pristanišča Luke Koper ali vpliva erozijskih tokov. V vrtini MC-2 identificirali dve vrsti prevladujoče sedimentacije. Globlje od 445 cm smo opazili visoke deleže dolomita in kremena, zelo nizke vrednosti izotopskega razmerja δ13Corg ter višje vrednosti atomskega razmerja TOC/TN, kar pripisujemo prevladujoči terestrični sedimentaciji. V vrhnjem delu do globine 445 cm so merjene višje vrednosti δ13Corg in nižja atomska razmerja TOC/TN, ki kažejo na mešano morsko in terestrično sedimentacijo.The seabed sediment borehole MC -2, from area between Ankaran and Koper, was investigated as part of the master\u27s thesis. The borehole is 636 cm long and covers the entire Holocene sedimentary sequence. Based on the analysis of sedimentological and granulometric properties, the borehole consists of sedimentary layers of different thicknesses (1-86 cm), classified as clayey silt, sandy silt, and silty sand depending on their composition. The Holocene sequence is dominated by clayey silt (85.87 %), followed by sandy clay (9,24 %) and silty sand (4.87%). The presence of snail remains, biogenic detritus and bivalves was observed in all cores. Calcite, dolomite, quartz, pyrite, halite, illite, albite, chlorite, and kaolinite were identified. We observed an increasing trend in the proportion of quartz and dolomite with depth and a reverse trend of decreasing proportion of calcite with depth. Based on PCA analysis, the measured geochemical, granulometric, and mineralogical parameters were classified into four main clusters according to their correlation. The first cluster includes parameters such as silicon (Si), organic carbon/total nitrogen ratio (TOC/TN), quartz, magnesium (Mg), and dolomite, which are due to terrigenous inputs from the surrounding bedrock. The values of the parameters of the first cluster increase with depth. The second cluster includes aluminium (Al), iron (Fe), potassium (K), and a proportion of silt and clay associated with clay minerals (kaolinite, illite, chlorite). The third cluster includes sulphur (S), chromium (Cr), manganese (Mn), nickel (Ni), copper (Cu), zinc (Zn), arsenic (As), lithium (Li), cadmium (Cd), lead (Pb), calcite, pyrite, total nitrogen (TN), total organic carbon (TOC) and the isotopic composition of carbon (δ13Corg). The origin of this cluster is not clearly defined, but it probably includes parameters related to environmental bioproduction (S, TOC), authigenic carbonates of bioclasts (calcite, TN, δ13Corg), and the mixed influence of organic material, anthropogenic influence, and the contribution of surrounding flysch rocks (Cr, Mn, Ni, Cu, Zn, As, Li, Cd, Pb). The values of the parameters of the second cluster generally decrease with depth. The contents of Cr, Pb, Cu, Ni and Zn are higher than in the surrounding felsic material, indicating anthropogenic pollution in the upper part. The fourth group is represented by arsenic (As) and the sand fraction, whose origin can be associated with pyrite, halite, organic material, iron/manganese oxides, and oxyhydroxides. Absolute age dating with 14C was used to define a Holocene sedimentary sequence, with the deepest sample at 640 cm depth being 10,515 ± 84 years old. In the upper part of the borehole, to a depth of 166 cm, a deviation was observed, which is attributed to anthropogenic mixing of the sediment due to dredging of the shipping corridor of the Port of Koper or the influence of erosional currents. Two types of dominant sedimentation were identified in the borehole MC -2. At depths greater than 445 cm, high proportions of dolomite and quartz, very low values of the δ13Corg isotope ratio, and higher values of the TOC/TN atomic ratio were observed, which we attribute to dominant terrestrial sedimentation. In the upper part up to a depth of 445 cm, higher δ13Corg values and lower TOC/TN atomic ratios are measured, indicating mixing of sediments