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Cepiva na osnovi nukleinskih kislin, kot je DNA-cepivo, so ena izmed možnosti cepljenja proti bolezni covid-19. Z uporabo metode genskega elektroprenosa izboljšamo slabo imunogenost DNA-cepiv in zagotovimo boljšo učinkovitost. Pripravili smo cepivo na osnovi plazmidne DNA, z zapisoma za antigena virusa SARS-CoV-2, in sicer za protein bodice (S) in protein nukleokaspide (N). Transfekcijo plazmidne DNA v celice mišjih fibroblastov L929 smo izvedli z genskim elektroprenosom. V in vitro študiji smo preverili izražanje antigenov na ravni mRNA in proteinov. Z uporabo metode RT-qPCR smo dokazali povečano izražanje genov za protein S in N v poskusnih skupinah S, N in N+S. Z metodo ELISA smo dokazali protein N v celičnem mediju in celičnem lizatu. V skupini N+S smo v primerjavi s skupino N v celičnem mediju dokazali šestkrat večjo koncentracijo proteina N. V celičnem lizatu nam je uspelo dokazati trikrat večjo koncentracijo proteina N v poskusni skupini N+S v primerjavi s skupino N. Proteina S s testom ELISA nismo uspeli zaznati ter ga tako nismo potrditi na proteinski ravni. Rezultate in povzetke in vitro študije je Oddelek za Eksperimentalno onkologijo, OI v nadaljevanju uporabil za pripravo in vivo študije na mišjih modelih. Nucleic acid-based vaccines, such as DNA vaccine, are one of the options for vaccination against the disease COVID-19. Using the Gene Electro Transfer method, we improve the poor immunogenicity of the DNA vaccine and provide better efficacy. We have produced a vaccine based on plasmid DNA. As antigens were used two plasmids that encode SARS-CoV-2 spike (S) or nucleocapsid (N) protein. Transfection of mouse fibroblast cells L929 was performed by Gene Electro Transfer. In in vitro study, we checked the expression of the antigens at the mRNA and protein levels. Using RT-qPCR, we have shown that in experimental groups S, N, and N+S, gene expression for N and S proteins occurs. Finally, antigens were determined at the protein level. N protein was detected by ELISA in the cell medium and cell lysate. In the N+S group in the cell medium, we detected a 6 times higher concentration of N protein than in the N group. In cell lysate, we were able to demonstrate a 3-fold higher concentration of N protein in the N+S experimental group compared to the N group. We were not able to detect S protein with the ELISA assay, thus, could not confirm it at the protein level. The final outcomes of the in vitro study were used by the Department of Experimental oncology, OI for in vivo studies.

Protein staufen je RNA-vezavni protein, ki skupaj z molekulo RNA tvori ribonukleoproteinske komplekse kot so na primer stresne granule, ki omogočajo preživetje stresnih pogojev. Spremembe RNA-vezavnih proteinov lahko povzročijo številne nevrodegenerativne bolezni, za katere je značilno propadanje nevronov. V naši raziskavi smo z metodo označevanja okolice z biotinom označili proteine, ki interagirajo ali so v bližini proteina staufen. Uporabili smo dve biotin-ligazi: BirA* in TurboID. Ko smo dokazali, da se je fuzijski protein sintetiziral v celicah HEK293 in je bila biotinilacija proteinov uspešna smo izolirane proteine poslali na analizo z masno spektrometrijo. Rezultati so pokazali, da večina proteinov, ki smo jih določili, sodeluje pri vezavi RNA ali pri strukturi ribosoma, kar nekaj proteinov pa se veže z molekulo DNA. Večje razlike med proteini v okolici proteinov staufen 1 in staufen 2 nismo zaznali. Uporaba različnih biotin-ligaz pa nam je dala vpogled v časovno dinamiko interakcij. Pokazali smo, da se protein staufen nahaja v okolici ribosoma in molekule RNA. Staufen protein is an RNA-binding protein that forms ribonucleoprotein complexes with the RNA, e.g., stress granules which help survive stress conditions. Changes of RNA-binding proteins can cause a number of neurodegenerative diseases characterised by neuronal degeneration. In our study, we used a proximity labelling method to label proteins that interact or are in the vicinity of the Staufen protein. Two biotin ligases were used: BirA* and TurboID. After confirming that the fusion protein was correctly expressed and that the biotinylation of the proteins was successful, we sent the isolated proteins for analysis by mass spectrometry. We concluded that most of the proteins we identified are involved in RNA binding or ribosome structure, and few proteins bind to DNA. We did not detect any major difference between the proteins surrounding Staufen 1 and Staufen 2 proteins. Using different biotin-ligases gave us insight into the temporal dynamics of the interactions. We showed that the Staufen protein is located near the the ribosome and the RNA molecule.

Infections with pathogenic Escherichia colican lead to different animal- and human-associated diseases. E. coliinfections are common in intensive poultry farming, and important economic losses can be expected during infections with avian pathogenic E. coli(APEC) strains followed by colibacillosis. Loop-mediated isothermal amplification (LAMP) assays were developed for rapid detection of 3 APEC-associated virulence genes: sitA, traT, and ompT. All 3 LAMP assays are shown to be specific, repeatable, and reproducible. High sensitivities of the assays are shown, where as few as 1,000 bacterial cells/mL can be detected in different matrices. On-site applicability of this LAMP method is demonstrated through testing of different sample types, from animal swabs and tissues, and from environmental samples collected from 6 commercial poultry farms. All 3 virulence genes were detected at high rates (above 85%) in samples from layer and broiler chickens with clinical signs and, interestingly, high prevalence of those genes was detected also in samples collected from clinically healthy broiler flock (above 75%) while lower prevalence was observed in remaining 3 clinically healthy chicken flocks (less than 75%). Importantly, these virulence genes were detected in almost all of the air samples from 11 randomly selected poultry houses, indicating air as an important route of E. colispread. Three LAMP assays that target APEC-associated virulence genes are shown to be sensitive and robust and are therefore applicable for rapid on-site testing of various sample types, from animal swabs to air. This on-site LAMP testing protocol offers rapid diagnostics, with results obtained in

Konec decembra 2019 so iz Kitajskega mesta Vuhan poročali o izbruhu atipične pljučnice neznanega izvora. Analize genoma so pokazale, da gre za novega predstavnika koronavirusov, ki so ga poimenovali virus SARS-CoV-2. V manj kot enem letu od izbruha je virus zaradi visoke stopnje kužnosti povzročil pandemijo. Za njeno obvladovanje je bil razvoj učinkovitega cepiva nujno potreben. Do danes so bila odobrena 4 cepiva proti SARS-CoV-2, ki temeljijo na mRNA in adenovirusnih vektorjih. Veliko pozornosti je bilo usmerjene tudi v razvoj DNA-cepiv proti SARS-CoV-2. Učinkovitost DNA-cepiva je pogojena z metodo vnosa v tarčno tkivo. Ena od metod je elektroporacija, s katero povečamo permeabilnost celičnih membran in olajšamo vstop DNA v citoplazmo celic. Transfekcijo DNA v tarčno celico z elektroporacijo imenujemo genski elektroprenos. Glavni namen magistrske naloge je bil primerjati imunski odziv po genskem elektroprenosu DNA-cepiva proti virusu SARS-CoV-2 v mišico in kožo eksperimentalnih miši. Za genski elektroprenos smo pripravili mešanice plazmidnih DNA z geni, ki kodirajo za N in S proteina virusa SARS-CoV-2 in citokin IL-12. Imunski odziv smo vrednotili v vzorcih odvzetih na 13. in 28. dan po cepljenju. S komercialno dostopnimi testi ELISA smo določali prisotnost in koncentracije IFNγ (nespecifična imunost), ter titer anti-N in anti-S IgG protiteles (specifična humoralna imunost). S testom tetramerov smo določali delež CD8+N+ in CD8+S+ T celic (specifična celična imunost). Dokazali smo, da genski elektroprenos DNA-cepiva izzove tako nespecifično kot specifično imunost proti N in S proteinu. Ugotovili smo, da je aktivacija imunskega odziva intenzivnejša po genskem elektroprenosu v mišico in, da je v našem primeru N protein bolj imunogen od S proteina. Potrdili smo tudi, da je genski elektroprenos ustrezna metoda za vnašanje DNA-cepiva v tarčno tkivo. At the end of December 2019, an outbreak of atypical pneumonia of unknown origin was reported from the Wuhan, China. Genomic analysis has shown that it is a new representative of coronviruses, which has been named SARS-CoV-2. In less than a year after the outbreak, the virus caused a pandemic due to its high infectivity. The development of an effective vaccine was necessary for its control. To date, four SARS-CoV-2 vaccines based on mRNA and adenoviral vectors have been approved. A lot of attention has also been focused on the development of DNA-vaccines against SARS-CoV-2. The effectiveness of the DNA-vaccine depends on the method of introduction into the target tissue. One of the methods is electroporation, which increases the permeability of cell membranes and facilitates the entry od DNA into the cell cytoplasm. The transfection of DNA into a target cell by electroporation is called gene electrotransfer. The aim of the master's thesis was to compare the immune response after gene electrotransfer of the DNA-vaccine against the SARS-CoV-2 virus and cytokine IL-12 in the muscle and skin tissue. The immune response was evaluated in samples taken on the 13th and 28th day after vaccination. Commercially available ELISA tests were used to determine the presence and concentrations of IFNγ (non-specific immunity), as well as the titer of anti-N and anti-S IgG antibodies (specific humoral immunity). The tetramer test was used to determine portion of CD8+N+ and CD8+S+ T cells (specific cellular immunity). We have demonstrated that gene electrotransfer of DNA-vaccine elicits both non-specific and specific immunity against N and S protein. We found that the activation of the immune response is more intense after gene electrotransfer into the muscle and that in our case the N protein is more immunogenic than the S protein. We have also confirmed that gene electrotransfer is suitable method for introducing a DNA-vaccine into the target tissue.

The development of bioinformatic solutions is guided by biological knowledge of the subject. In some cases, we use unambiguous biological models, while in others we rely on assumptions. A commonly used assumption for genomes is that related species have similar genome sequences. This is even more obvious in the case of chloroplast genomes due to their slow evolution. We investigated whether the lengths of complete chloroplast sequences are closely related to the taxonomic proximity of the species. The study was performed using all available RefSeq sequences from the asterid and rosid clades. In general, chloroplast length distributions are narrow at both the family and genus levels. In addition, clear biological explanations have already been reported for families and genera that exhibit particularly wide distributions. The main factors responsible for the length variations are parasitic life forms, IR loss, IR expansions and contractions, and polyphyly. However, the presence of outliers in the distribution at the genus level is a strong indication of possible inaccuracies in sequence assembly. Nasl. z nasl. zaslona. Opis vira z dne 25. 1. 2023. Bibliografija: str. 10-12. Abstract.

Rastline imajo razvite raznolike in kompleksne mehanizme za regulacijo izražanja genov. Nedavno je bil odkrit nov mehanizem, imenovan RNK interferenca (RNKi). Osrednjo vlogo v RNKi imajo male nekodirajoče RNK (sRNK) dolge od 21-24 nukleotidov, ki z vezavo na komplementarna mesta v transkriptih preprečijo njihovo prevajanje v proteine. Ker sRNK definira izvor, strukturne lastnosti prekurzorjev ter sekvenčne lastnosti, jih delimo v več različnih razredov. mikroRNK (miRNK) ter sekundarne male interferenčne RNK (siRNK), med katere prištevamo tasiRNK in phasiRNK imajo pomembno vlogo v regulaciji izražanja genov v številnih bioloških procesih ter odzivu rastlin na biotske ali abiotske dejavnike stresa. Kljub številnim ohranjenim sRNK med rastlinskimi vrstami ter karakterizaciji njihovega delovanja, do danes še ni celovitega razumevanja njihove vloge v obrambnem odzivu rastlin pred fitopatogeni. Ta pregled povzema trenutno razumevanje patogeneze verticilijske uvelosti, obrambnega mehanizma rastlin pred fitopatogeni in biogeneze ter vloge miRNK, tasiRNK ter phasiRNK v obrambnem odzivu rastlin pred glivnimi patogeni. Nadaljnje raziskave rastlinskih sRNK in njihovo izražanje v odzivu rastlin na različne fitopatogene organizme so potrebne za jasno določitev njihove vloge. Novi pristopi sekvenciranja ter bioinformacijske analize in napovedovanja vloge miRNK tarč v času okužb nam lahko pri nemodelnih organizmih omogočijo razvoj novih načinov varstva rastlin. Plants have evolved diverse and complex mechanisms to regulate gene expression. Recently, a new mechanism called RNA interference (RNAi) has been discovered. At the core of RNAi are small non-coding RNAs (sRNAs), 21-24 nucleotides in length, that prevent the translation of transcripts into proteins by binding to complementary sites in transcripts. Because sRNAs are determined by origin, precursor structural properties, and sequence characteristics, they are classified into several classes like microRNAs (miRNAs) and secondary small interfering RNAs (siRNAs), which include tasiRNAs and phasiRNAs. They play important roles in regulating gene expression in a wide range of biological processes and in plant responses to biotic or abiotic stresses. Despite the numerous conserved sRNAs among plant species and the characterization of their function, there is still no comprehensive understanding of their role in plant defense responses against phytopathogens. This review summarizes the current understanding of Verticillium wilt pathogenesis, plant defense mechanisms against phytopathogens, and the biogenesis and roles of miRNAs, tasiRNAs, and phasiRNAs in plant defense responses against fungal pathogens. Further studies on plant sRNAs and their expression in response to various phytopathogens are needed to clearly define their roles. New sequencing approaches, bioinformatic analysis, and prediction of the role of miRNA targets during infection may allow us to develop new forms of plant protection in non-model organisms.

The development of next-generation sequencing technology and the increasing amount of sequencing data have brought the bioinformatic tools used in genome assembly into focus. The final step of the process is genome annotation, which works on assembled genome sequences to identify the location of genome features. In the case of organelle genomes, specialized annotation tools are used to identify organelle genes and structural features. Numerous annotation tools target chloroplast sequences. Most chloroplast DNA genomes have a quadripartite structure caused by two copies of a large inverted repeat. We investigated the strategies of six annotation tools (Chloë, Chloroplot, GeSeq, ORG.Annotate, PGA, Plann) for identifying inverted repeats and analyzed their success using publicly available complete chloroplast sequences of taxa belonging to the asterid and rosid clades. The annotation tools use two different approaches to identify inverted repeats, using existing general search tools or implementing stand-alone solutions. The chloroplast sequences studied show that there are different types of imperfections in the assembled data and that each tool performs better on some sequences than the others.

Probiotični izdelki so sestavljeni iz živih mikroorganizmov, ki ob zastopanju v pravih količinah povzročajo pozitivne učinke na gostitelja. V procesu proizvodnje probiotičnih izdelkov je eden od parametrov zagotavljanja kakovostnih probiotičnih izdelkov količina zastopanih koristnih probiotičnih bakterij v probiotičnih izdelkih. Mikrobiološko testiranje kakovosti probiotičnih izdelkov sloni na tradicionalnih metodah, ki temeljijo na rasti mikroorganizmov v tekočih in trdnih hranilnih gojiščih. Metode so pogosto nezanesljive, z dolgimi časi inkubacije in slabo selekcijo med mikroorganizmi. Ena izmed mikrobioloških tehnik je metoda štetja na ploščah, ki se uporablja za določanje količine bakterij v probiotičnem izdelku. Razvoj gre v smeri uporabe natančnejših in bolj zanesljivih molekularnih metod. Potencialna metoda za ugotavljanje števila točno določenih probiotičnih bakterij je digitalna PCR. Metoda omogoča absolutno določanje števila bakterij v vzorcu probiotičnega izdelka na podlagi flurescence. Tekom razvoja smo tehniko optimizirali do te mere, da lahko določamo celokupno količino probiotičnih bakterij v probiotičnem izdelku. Nova metoda predstavlja potencialno orodje za določanje količine probiotičnih bakterij v probiotičnih izdelkih, vendar jo bo za vsesplošno uporabo potrebno optimizirati do te mere, da bomo lahko z njo razlikovali med živimi in mrtvimi probiotičnimi bakterijami. Probiotic products are composed of living microorganisms that, when present in the right quantity, have beneficial effects on the host. One of the parameters for the quality of probiotic products during their production is the amount of beneficial probiotic bacteria present in them. Microbiological testing of probiotic product quality is based on traditional methods that measure the growth of microorganisms in liquid and solid culture media. Traditional methods are often unreliable, have long incubation times, and provide poor differentiation between microorganisms. Plate counting method is one of the microbiological techniques used to determine the number of bacteria in a probiotic product. Novel microbiological tests are based on the use of more accurate and reliable molecular methods. One possible method for determining specific probiotic bacteria is digital PCR. It allows the determination of the absolute number of bacteria in a sample of a probiotic product based on fluorescence. We have optimized the technique to the point where we can determine the total amount of probiotic bacteria in a probiotic product. The method is a potential tool for determining the number of probiotic bacteria in probiotic products but needs to be optimized for general use so that it can distinguish between live and dead probiotic bacteria.

Sevi Escherichia coli imajo pogosto genske zapise za encime betalaktamaze z razširjenim spektrom delovanja (ESBL) na konjugativnih plazmidih. V raziskavi smo analizirali konjugativni plazmid pES6, povezan z odpornostjo proti cefotaksimu (blaSHV-12) in nizkim koncentracijam fluorokinolonov (qnrS1), iz ESBL-pozitivnih sevov E. coli, vzorčenih v perutninskih rejah v Sloveniji. Ugotovljene frekvence konjugativnega prenosa odpornosti proti cefotaksimu so bile pri poskusih konjugacij z donorskim sevom VF2017-A5 (A5) od 10-7 do 10-5, z donorskim sevom VF2017-A6 (A6) pa od 10-5 do 10-3. S sekvenciranjem plazmidne DNA (pDNA) izbranih transkonjugant (TK) smo ugotovili, da so poleg plazmida pES6, ki je enak kot v literaturi opisani plazmid pEC-243, v navedenih donorskih sevih prisotni še dodatni plazmidi pV5, pV6 in pK6. Pri TK iz donorja A5 je prisoten domnevni plazmid pV5 z zapisom za kolicin B in kolicin M, pri TK iz donorja A6 pa domnevni plazmid pV6 z zapisom za kolicin Ia in mikrocin V ter kriptični plazmid pK6. Njihovo prisotnost smo pri TK potrdili tudi s PCR s plazmidno-specifičnimi začetnimi oligonukleotidi, plazmida pV5 in pV6 pa tudi fenotipsko na podlagi kolicinske aktivnosti. S primerjavo rezultatov, dobljenih z bioinformacijsko analizo nukleotidnih zaporedij plazmidov sevov A5 in A6 s podatki, dobljenih z analizo izolirane nerazrezane in razrezane pDNA po gelski elektroforezi, smo ugotovili, da z elektroforezo pDNA, zgolj vizualno, ne zaznamo in/ali težko opredelimo vse plazmide v izolirani pDNA posameznega seva. Escherichia coli strains have extended-spectrum beta-lactamases (ESBL) genes that are frequently encoded on conjugative plasmids. In our study, we analyzed the conjugative plasmid pES6, isolated from ESBL-positive E. coli strains from selected poultry farms in Slovenia, that confer resistance to low concentrations of fluoroquinolones (qnrS1) and cefotaxime (blaSHV-12). We analyzed the frequency of conjugative transfer of resistance to cefotaxime from source donors VF2017-A5 (A5) and VF2017-A6 (A6). The frequency of A5 conjugation experiments varied from 10-7 to 10-5, and the frequency of A6 conjugation experiments from 10-5 to 10-3. By sequencing the plasmid DNA (pDNA) of selected transconjugants (TK), we found that in addition to plasmid pES6, which is identical to plasmid pEC-243 described in the literature, additional plasmids pV5, pV6, and pK6 are present. Supposed plasmid pV5, present in TK derived from the A5 donor, encodes the colicins B and M. Supposed plasmid pV6, encoding the colicin Ia and microcin V and cryptic plasmid pK6 are present in TK derived from the A6 donor. Their presence in TK was confirmed by PCR with plasmid-specific primers. For plasmids pV5 and pV6, their presence was also confirmed phenotipically by colicin activity. Comparison of the results of bioinformatic analysis with data obtained from analysis of isolated, uncut and cut pDNA after gel electrophoresis, led to the conclusion that pDNA electrophoresis methods are not sufficient to visually detect all plasmids in the isolated pDNA of a single strain.

Zaznavanje z nanoporami (angl. nanopore sensing) je analitska metoda, ki omogoča detekcijo posameznih molekul na podlagi sprememb v ionskem toku, ki teče skozi poro. Zaradi vedno večjega nabora kompleksnejših analitov pa se povečujejo potrebe po novih porah s specifičnimi lastnostmi. V magistrski nalogi smo želeli pripraviti homogene pore perfringolizina O (PFO) za uporabo v zaznavanju z nanoporami. V ta namen smo z uporabo SDS-AGE (angl. sodium dodecyl sulphate agarose gel electrophoresis) analizirali vpliv pogojev priprave na velikost, homogenost in končno koncentracijo PFO oligomerov, ki smo jih pripravili na velikih enoslojnih veziklih in jih s teh sprostili z uporabo različnih detergentov. Nato smo za nadaljnjo analizo na planarnih lipidnih membranah in s krio transmisijskim elektronskim mikroskopom vzorce tako pripravljenih por s pomočjo ionsko-izmenjevalne kromatografije, SDS-AGE in gelske izključitvene kromatografije očistili ostankov priprave por in obenem poskusili ločiti pore po obliki in velikosti. Sestava veziklov se je izkazala kot ključna za optimalno pripravo PFO por in je morala zagotavljati, da je dovoljšna količina holesterola izpostavljena na površini membrane ter tako dostopna za vezavo PFO monomerov. Hkrati pa se je potrebno izogniti kombinacijam lipidov, ki s PFO oligomeri tvorijo agregate, po tem ko se vezikle raztopi z detergentom. Ostali pogoji priprave niso imeli vpliva na pripravo por ali pa so povzročili agregacijo oligomerov z lipidi po dodatku detergenta. S prej navedenimi metodami nam je uspelo očistiti vzorce pripravljenih por, ni pa nam uspelo doseči ločbe por po velikosti in obliki. Ker se očiščene pore niso vstavljale v lipidne dvosloje, jim na planarnih lipidnih membranah nismo uspeli opredeliti lastnosti. Nanopore sensing is an analytical method that enables single molecule detection by identifying changes in ionic current that runs through the pore, as it interacts with the analyte. Due to an ever-increasing assortment of complex analytes the demand for pores with novel characteristics in growing as well. Herein we attempted to prepare homogeneous pores of perfringolysin O (PFO) for use in nanopore sensing. To this end we used sodium dodecyl sulphate agarose gel electrophoresis (SDS-AGE) to analyse the effects of different pore preparation conditions on size, homogeneity and final concentration of PFO oligomers, which we prepared on large unilamellar vesicles and solubilised with different detergents. Using ion exchange chromatography, SDS-AGE and size exclusion chromatography we then purified pore samples of pore preparation residues and simultaneously tried to resolve pores of varying shapes and sizes for further analysis on planar lipid membranes and with cryo transmission electron microscopy. Major requirement for adequate pore preparation was vesicle lipid composition, which had to ensure sufficient cholesterol exposure for binding of PFO to the membrane, while also being incapable of forming aggregates with PFO oligomers after vesicles were solubilised with detergent. Other tested conditions either had no effect on pore preparation or influenced it by affecting aggregation of oligomers with lipids after detergent addition. Using aforementioned methods, we successfully removed residues of pore formation from samples, but failed to resolve pores according to their sizes and shapes. Due to inability of purified pores to insert into lipid bilayers, we did not manage to characterise them on planar lipid membranes.

Bisfenoli (BP) so skupina industrijskih kemikalij, ki se uporabljajo pri proizvodnji plastike in epoksi smol. Iz njih izdelujejo najrazličnejše izdelke kot so obloge za konzerve, stekleničke za dojenčke, igrače, papir za račune in mnoge druge, ki se uporabljajo v našem vsakdanjem življenju in jih posledično zasledimo v okolju. Zaradi kopičenja podatkov o nevarnih lastnostih in škodljivih vplivih najpogostejšega izmed BP-jev, bisfenola A (BPA), predvsem motenj endokrinega sistema, je bila njegova uporaba v Evropski uniji postopoma omejena oziroma celo prepovedana, da bi zaščitili zdravje ljudi in okolje. Prepoved uporabe BPA je spodbudila razvoj domnevno varnejših analogov, za katere pa v literaturi ni veliko toksikoloških podatkov. Tako smo v magistrski nalogi proučevali citotoksično in genotoksično aktivnost BPA in njegovih analogov: bisfenol AF (BPAF), bisfenol AP (BPAP), bisfenol C (BPC), bisfenol FL (BPFL), bisfenol S (BPS) na jetrnem in vitro 3D celičnem modelu. Sferoide smo pripravili s prisilno plavajočo metodo, vpliv na živost celic sferoidov po izpostavitvi BP-jem smo merili s testom MTS. S pomočjo mikroskopije, smo spremljali površino, obliko in kompaktnost sferoidov, s testom komet pa smo preučevali genotoksičnost v časovnih izpostavitvah 24 in 96 ur. Statistično značilen vpliv na živost celic v sferoidu po 24 urah so imeli BPFL > BPAF = BPAP, po 96 urah pa BPFL > BPC = BPAP > BPA = BPAF. Mikroskopske analize so pokazale, da sta imela največji vpliv na rast in spremembo površine ter kompaktnosti sferoidov BPC in BPFL po 96-urni izpostavitvi. S testom komet smo pokazali, da sta bila po 24 urah izpostavitve najbolj genotoksična BPAF in BPS (⡥ 10 μM), sledil je BPAP ( ⡥ 40 μM). Po 96 urah izpostavitve je BPAP povzročil poškodbe DNA pri koncentraciji ⡥ 0,1 μM, pri vseh ostalih BP-jih smo poškodbe DNA zaznali pri višjih koncentracijah. Povzamemo lahko, da so za analoge BPA potrebne dodatne študije mehanizmov delovanja za pripravo ustrezne ocene tveganja, saj je toksična aktivnost za posamezne analoge, kljub podobni kemijski strukturi, različna. Bisphenols (BPs) are a group of industrial chemicals used in the manufacture of plastics and epoxy resin. They are found in various products, including the inner linings of cans, baby bottles, toys, thermal paper, and others used in our daily life. Therefore, BPs are present in the environment. The most widely used BP is bisphenol A (BPA), on which most studies have been conducted showing its adverse effects on human health, especially its endocrine disrupting activity. Therefore, the European Union began to restrict and eventually ban the use of BPA to protect human health and the environment. The ban has led to the development of supposedly safer BPA analogues, for which few toxicological data are available. In the master thesis, we investigated the cytotoxic and genotoxic activity of BPA and its analogues (BPAF, BPAP, BPC, BPFL, BPS) in a liver in vitro 3D cell model. In this study, we prepared spheroids by the forced floating method and measured cell viability after exposure to BPs using the MTS assay. We measured the surface area and observed the shape and compactness of the spheroids with microscopy and studied the genotoxic effects with the comet assay at 24 and 96-hour exposure. After 24-hour exposure to BPs, BPFL > BPAF = BPAP statistically significantly decreased the viability of HepG2 and after 96 hours, we observed a similar effect on cell viability in BPFL > BPC = BPAP > BPA = BPAF. BPC and BPFL had the greatest effect on morphological changes in growth, surface area, compactness, and shape of the spheroids that were evident at 96-hour exposure. Of the six BPs tested by the comet assay, BPAF and BPS (⡥ 10 μM) and BPAP (⡥ 40 μM) exhibited stronger genotoxic potential than BPA, BPFL, and BPC after 24 hours of exposure. After 96 hours of exposure, BPAP was the most genotoxic causing/inducing DNA damage at ⡥1 µM. All others BPs studied statistically significantly increased DNA damage at higher concentrations. To summarize, despite their similar chemical structure, the toxic activity of the analogues is not the same, and therefore further studies on the BPA analogues and their molecular activity are needed to provide/make accurate risk assessment.

Metagenomic approaches used for virus diagnostics allow for rapid and accurate detection of all viral pathogens in the plants. In order to investigate the occurrence of viruses and virus-like organisms infecting grapevine from the Ampelographic collection Kromberk in Slovenia, we used Ion Torrent small RNA sequencing (sRNA-seq) and the VirusDetect pipeline to analyze the sRNA-seq data. The used method revealed the presence of: Grapevine leafroll-associated virus 1 (GLRaV-1), Grapevine leafroll-associated virus 2 (GLRaV-2), Grapevine leafroll-associated virus 3 (GLRaV-3), Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine fanleaf virus (GFLV) and its satellite RNA (satGFLV), Grapevine fleck virus (GFkV), Grapevine rupestris vein feathering virus (GRVFV), Grapevine Pinot gris virus (GPGV), Grapevine satellite virus (GV-Sat), Hop stunt viroid (HSVd), and Grapevine yellow speckle viroid 1 (GYSVd-1). Multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for validation of sRNA-seq predicted infections, including various combinations of viruses or viroids and satellite RNA. mRT-PCR could further be used for rapid and cost-effective routine molecular diagnosis, including widespread, emerging, and seemingly rare viruses, as well as viroids which testing is usually overlooked.

The genus Verticillium is a group of ascomycete fungi that includes several pathogenic plant species. In 2011, a new taxonomic classification, proposed by Inderbitzin and coworkers (2011), re-defined the genus as Verticillium sensu stricto. The objective of our study was the re-classification of the fungal species held in the culture collection in the Slovenian Institute of Hop Research and Brewing in accordance with the newly established taxonomy. With the PCR marker system proposed by Inderbitzin and coworkers in 2011, we re-classified 88 Verticillium isolates out of the 105 samples that are held in the institute’s bank, which were obtained from different geographic locations in Europe, North America, and Japan, and from different host plants, including alfalfa, cotton, hop, olive, potato, and tomato. However, the PCR marker for the V. dahliae identification proved to be less specific, and it resulted in the positive amplification of Gibellulopsis nigrescens, V. isaacii, and V. longisporum. To enable the accurate distinction of the fungi, the SSR and LAMP markers were added to the analyses. The 12 newly identified SSR markers, which were used in simplex PCR reactions or in combination, enabled the accurate identification of all included Verticillium isolates and could potentially be used as biomarkers for rapid and easy species identification. Nasl. z nasl. zaslona. Opis vira z dne 30. 3. 2023. Bibliografija: str. 17-18. Abstract.

Olive is considered one of the oldest and the most important cultivated fruit trees in Albania. In the present study, the genetic diversity and structure of Albanian olive germplasm is represented by a set of 194 olive genotypes collected in-situ in their natural ecosystems and in the ex-situ collection. The study was conducted using 26 microsatellite markers (14 genomic SSR and 12 Expressed Sequence Tag microsatellites). The identity analysis revealed 183 unique genotypes. Genetic distance-based and model-based Bayesian analyses were used to investigate the genetic diversity, relatedness, and the partitioning of the genetic variability among the Albanian olive germplasm. The genetic distance-based analysis grouped olives into 12 clusters, with an average similarity of 50.9%. Albanian native olives clustered in one main group separated from introduced foreign cultivars, which was also supported by Principal Coordinate Analysis (PCoA) and model-based methods. A core collection of 57 genotypes representing all allelic richness found in Albanian germplasm was developed for the first time. Herein, we report the first extended genetic characterization and structure of olive germplasm in Albania. The findings suggest that Albanian olive germplasm is a unique gene pool and provides an interesting genetic basis for breeding programs.

Familial Mediterranean Fever (FMF) is the most prevalent genetic autoinflammatory disorder. In most patients, treatment with colchicine can prevent attacks of fever and inflammation. However, 5%–10% of patients are resistant to colchicine treatment, while a similar percentage cannot tolerate colchicine in doses needed to prevent attacks. For these patients, Canakinumab, a full human antibody against IL-1β, has been approved recently by the FDA and EMA. In this article, we present a systematic review of the long-term efficacy, safety, and tolerability of Canakinumab in FMF patients who cannot tolerate colchicine or who are resistant to colchicine treatment.

Some wild, morphologically diverse taxa of the genus Iris in the broad Alpine-Dinaric area have never been explored molecularly, and/or have ambiguous systematic status. The main aims of our research were to perform a molecular study of critical Iris taxa from that area (especially a narrow endemic accepted species I. adriatica, for which we also analysed genome size) and to explore the contribution of eight microsatellites and highly variable chloroplast DNA (ndhJ, rpoC1) markers to the understanding of the Iris taxa taxonomy and phylogeny. Both the microsatellite-based UPGMA and plastid markers-based maximum likelihood analysis discriminated three main clusters in the set of 32 analysed samples, which correspond well to the lower taxonomic categories of the genus, and support separate status of ambiguous regional taxa (e.g., I. sibirica subsp. erirrhiza, I. x croatica and I. x rotschildii). The first molecular data on I. adriatica revealed its genome size (2C = 12.639 &plusmn, 0.202 pg) and indicated the existence of ecotypes. For future molecular characterisation of the genus we recommend the utilisation of microsatellite markers supplemented with a combination of plastid markers.

Verticillium nonalfalfae (V. nonalfalfae) is one of the most problematic hop (Humulus lupulus L.) pathogens, as the highly virulent fungal pathotypes cause severe annual yield losses due to infections of entire hop fields. In recent years, the RNA interference (RNAi) mechanism has become one of the main areas of focus in plant—fungal pathogen interaction studies and has been implicated as one of the major contributors to fungal pathogenicity. MicroRNA-like RNAs (milRNAs) have been identified in several important plant pathogenic fungi; however, to date, no milRNA has been reported in the V. nonalfalfae species. In the present study, using a high-throughput sequencing approach and extensive bioinformatics analysis, a total of 156 milRNA precursors were identified in the annotated V. nonalfalfae genome, and 27 of these milRNA precursors were selected as true milRNA candidates, with appropriate microRNA hairpin secondary structures. The stem-loop RT-qPCR assay was used for milRNA validation; a total of nine V. nonalfalfae milRNAs were detected, and their expression was confirmed. The milRNA expression patterns, determined by the absolute quantification approach, imply that milRNAs play an important role in the pathogenicity of highly virulent V. nonalfalfae pathotypes. Computational analysis predicted milRNA targets in the V. nonalfalfae genome and in the host hop transcriptome, and the activity of milRNA-mediated RNAi target cleavage was subsequently confirmed for two selected endogenous fungal target gene models using the 5′ RLM-RACE approach.

Viroids are small non-capsidated, single-stranded, covalently-closed circular noncoding RNA replicons of 239&ndash, 401 nucleotides that exploit host factors for their replication, and some cause disease in several economically important crop plants, while others appear to be benign. The proposed mechanisms of viroid pathogenesis include direct interaction of the genomic viroid RNA with host factors and post-transcriptional or transcriptional gene silencing via viroid-derived small RNAs (vd-sRNAs) generated by the host defensive machinery. Humulus lupulus (hop) plants are hosts to several viroids among which Hop latent viroid (HLVd) and Citrus bark cracking viroid (CBCVd) are attractive model systems for the study of viroid-host interactions due to the symptomless infection of the former and severe symptoms induced by the latter in this indicator host. To better understand their interactions with hop plant, a comparative transcriptomic analysis based on RNA sequencing (RNA-seq) was performed to reveal the transcriptional alterations induced as a result of single HLVd and CBCVd infection in hop. Additionally, the effect of HLVd on the aggressiveness of CBCVd that underlies severe stunting in hop in a mixed infection was studied by transcriptomic analysis. Our analysis revealed that CBCVd infection resulted in dynamic changes in the activity of genes as compared to single HLVd infection and their mixed infection. The differentially expressed genes that are involved in defense, phytohormone signaling, photosynthesis and chloroplasts, RNA regulation, processing and binding, protein metabolism and modification, and other mechanisms were more modulated in the CBCVd infection of hop. Nevertheless, Gene Ontology (GO) classification and pathway enrichment analysis showed that the expression of genes involved in the proteolysis mechanism is more active in a mixed infection as compared to a single one, suggesting co-infecting viroids may result in interference with host factors more prominently. Collectively, our results provide a deep transcriptome of hop and insight into complex single HLVd, CBCVd, and their coinfection in hop-plant interactions

The SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) is essential for virus replication, therefore it is a promising drug target. Here we present a surface plasmon resonance approach to study the interaction of RdRp with drugs in real time. We monitored the effect of favipiravir, ribavirin, sofosbuvir triphosphate PSI-7409 and suramin on RdRp binding to RNA immobilized on the chip. Suramin precluded interaction of RdRp with RNA and even displaced RdRp from RNA.

Biallelic mutations in SLC29A3 cause histiocytosis-lymphadenopathy plus syndrome, also known as H syndrome (HS). HS is a complex disorder, with ~ 25% of patients developing autoinflammatory complications consisting of unexplained fevers, persistently elevated inflammatory markers, and unusual lymphadenopathies, with infiltrating CD68+, S100+, and CD1a− histiocytes, resembling the immunophenotype found in Rosai-Dorfman disease (RDD). We investigated the transcriptomic profiles of monocytes, non-activated (M0), classically activated (M1), and alternatively activated macrophages (M2) in two patients with HS, one without autoinflammatory (HS1) and one with autoinflammatory complications (HS2). RNA sequencing revealed a dysregulated transcriptomic profile in both HS patients compared to healthy controls (HC). HS2, when compared to HS1, had several differentially expressed genes, including genes associated with lymphocytic-histiocytic predominance (e.g. NINL) and chronic immune activation (e.g. B2M). The transcriptomic and cytokine profiles of HS patients were comparable to patients with SAID with high levels of TNF. SERPINA1 gene expression was found to be upregulated in all patients studied. Moreover, higher levels of IFNγ were found in the serum of both HS patients when compared to HC. Gene ontology (GO) enrichment analysis of the DEGs in HS patients revealed the terms “type I IFN,” “IFNγ signaling pathway,” and “immune responses” as the top 3 most significant terms for monocytes. Gene expression analysis of lymph node biopsies from sporadic and H syndrome-associated RDD suggests common underlying pathological process. In conclusion, monocytes and macrophages from both HS patients showed transcriptomic profiles similar to SAIDs and also uniquely upregulated IFNγ signature. These findings may help find better therapeutic options for this rare disorder.

Tobacco (Nicotiana tabacum) pollen is a well-suited model for studying many fundamental biological processes owing to its well-defined and distinct development stages. It is also one of the major agents involved in the transmission of infectious viroids, which is the primary mechanism of viroid pathogenicity in plants. However, some viroids are non-transmissible and may be possibly degraded or eliminated during the gradual process of pollen development maturation. The molecular details behind the response of developing pollen against the apple fruit crinkle viroid (AFCVd) infection and viroid eradication is largely unknown. In this study, we performed an integrative analysis of the transcriptome and proteome profiles to disentangle the molecular cascade of events governing the three pollen development stages: early bicellular pollen (stage 3, S3), late bicellular pollen (stage 5, S5), and 6 h-pollen tube (PT6). The integrated analysis delivered the molecular portraits of the developing pollen against AFCVd infection, including mechanistic insights into the viroid eradication during the last steps of pollen development. The isobaric tags for label-free relative quantification (iTRAQ) with digital gene expression (DGE) experiments led us to reliably identify subsets of 5321, 5286, and 6923 proteins and 64,033, 60,597, and 46,640 expressed genes in S3, S5, and PT6, respectively. In these subsets, 2234, 2108 proteins and 9207 and 14,065 mRNAs were differentially expressed in pairwise comparisons of three stages S5 vs. S3 and PT6 vs. S5 of control pollen in tobacco. Correlation analysis between the abundance of differentially expressed mRNAs (DEGs) and differentially expressed proteins (DEPs) in pairwise comparisons of three stages of pollen revealed numerous discordant changes in mRNA/protein pairs. Only a modest correlation was observed, indicative of divergent transcription, and its regulation and importance of post-transcriptional events in the determination of the fate of early and late pollen development in tobacco. The functional and enrichment analysis of correlated DEGs/DEPs revealed the activation in pathways involved in carbohydrate metabolism, amino acid metabolism, lipid metabolism, and cofactor as well as vitamin metabolism, which points to the importance of these metabolic pathways in pollen development. Furthermore, the detailed picture of AFCVd-infected correlated DEGs/DEPs was obtained in pairwise comparisons of three stages of infected pollen. The AFCVd infection caused the modulation of several genes involved in protein degradation, nuclear transport, phytohormone signaling, defense response, and phosphorylation. Intriguingly, we also identified several factors including, DNA-dependent RNA-polymerase, ribosomal protein, Argonaute (AGO) proteins, nucleotide binding proteins, and RNA exonucleases, which may plausibly involve in viroid stabilization and eradication during the last steps of pollen development. The present study provides essential insights into the transcriptional and translational dynamics of tobacco pollen, which further strengthens our understanding of plant-viroid interactions and support for future mechanistic studies directed at delineating the functional role of candidate factors involved in viroid elimination.

(1) Background: Verticillium wilt (VW) of hop is a devastating disease caused by the soil-borne fungi Verticillium nonalfalfae and Verticillium dahliae. As suggested by quantitative trait locus (QTL) mapping and RNA-Seq analyses, the underlying molecular mechanisms of resistance in hop are complex, consisting of preformed and induced defense responses, including the synthesis of various phenolic compounds. (2) Methods: We determined the total polyphenolic content at two phenological stages in roots and stems of 14 hop varieties differing in VW resistance, examined the changes in the total polyphenols of VW resistant variety Wye Target (WT) and susceptible Celeia (CE) on infection with V. nonalfalfae, and assessed the antifungal activity of six commercial phenolic compounds and total polyphenolic extracts from roots and stems of VW resistant WT and susceptible CE on the growth of two different V. nonalfalfae hop pathotypes. (3) Results: Generally, total polyphenols were higher in roots than stems and increased with maturation of the hop. Before flowering, the majority of VW resistant varieties had a significantly higher content of total polyphenols in stems than susceptible varieties. At the symptomatic stage of VW disease, total polyphenols decreased in VW resistant WT and susceptible CE plants in both roots and stems. The antifungal activity of total polyphenolic extracts against V. nonalfalfae was higher in hop extracts from stems than those from roots. Among the tested phenolic compounds, only p-coumaric acid and tyrosol markedly restricted fungal growth. (4) Conclusions: Although the correlation between VW resistance and total polyphenols content is not straightforward, higher levels of total polyphenols in the stems of the majority of VW resistant hop varieties at early phenological stages probably contribute to fast and efficient activation of signaling pathways, leading to successful defense against V. nonalfalfae infection.

Phenolic compounds are involved in plant responses to various biotic and abiotic stress factors, with many studies suggesting their role in defense mechanisms against fungal pathogens. Soilborne vascular pathogen Verticillium nonalfalfae causes severe wilting and consequent dieback in a wide range of economically important crops, including hops (Humulus lupulus L.). In this study, we investigated the differential accumulation of phenolics in the susceptible &ldquo, Celeia&rdquo, and resistant &ldquo, Wye Target&rdquo, hop cultivars during the pathogenesis of Verticillium wilt. Quantitative polymerase chain reaction showed that colonization in the roots of both cultivars was intensive, but decreased continuously throughout the experiment in the resistant cultivar, while the relative fungal amount continuously increased in the stems of the susceptible cultivar. In response to colonization in the roots of the resistant cultivar, a significant increase in total flavanols was detected at three days postinoculation (dpi), suggesting a possible role in preventing fungus spread into the stems. The accumulation of phenolic compounds was less pronounced in the stems of the resistant cultivar since, compared to the latter, significant increases in flavonols at 3 and 15 dpi and hydroxycinnamic acids at 6 dpi were observed in the stems of the susceptible cultivar.

Population-wide tolerance and persisters enable susceptible bacterial cells to endure hostile environments, including antimicrobial exposure. The SOS response can play a significant role in the generation of persister cells, population-wide tolerance, and shielding. The SOS pathway is an inducible DNA damage repair system that is also pivotal for bacterial adaptation, pathogenesis, and diversification. In addition to the two key SOS regulators, LexA and RecA, some other stressors and stress responses can control SOS factors. Bacteria are exposed to DNA-damaging agents and other environmental and intracellular factors, including cigarette smoke, that trigger the SOS response at a number of sites within the host. The Escherichia coli TisB/IstR module is as yet the only known SOS-regulated toxin–antitoxin module involved in persister formation. Nevertheless, the SOS response plays a key role in the formation of biofilms that are highly recalcitrant to antimicrobials and can be abundant in persisters. Furthermore, the dynamic biofilm environment generates DNA-damaging factors that trigger the SOS response within the biofilm, fueling bacterial adaptation and diversification. This review highlights the SOS response in relation to antimicrobial recalcitrance to antimicrobials in four clinically significant species, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Mycobacterium tuberculosis.

The mediator (MED) represents a large, conserved, multi-subunit protein complex that regulates gene expression through interactions with RNA polymerase II and enhancer-bound transcription factors. Expanding research accomplishments suggest the predominant role of plant MED subunits in the regulation of various physiological and developmental processes, including the biotic stress response against bacterial and fungal pathogens. However, the involvement of MED subunits in virus/viroid pathogenesis remains elusive. In this study, we investigated for the first time the gene expression modulation of selected MED subunits in response to five viroid species (Apple fruit crinkle viroid (AFCVd), Citrus bark cracking viroid (CBCVd), Hop latent viroid (HLVd), Hop stunt viroid (HSVd), and Potato spindle tuber viroid (PSTVd)) in two model plant species (Nicotiana tabacum and N. benthamiana) and a commercially important hop (Humulus lupulus) cultivar. Our results showed a differential expression pattern of MED subunits in response to a viroid infection. The individual plant MED subunits displayed a differential and tailored expression pattern in response to different viroid species, suggesting that the MED expression is viroid- and plant species-dependent. The explicit evidence obtained from our results warrants further investigation into the association of the MED subunit with symptom development. Together, we provide a comprehensive portrait of MED subunit expression in response to viroid infection and a plausible involvement of MED subunits in fine-tuning transcriptional reprogramming in response to viroid infection, suggesting them as a potential candidate for rewiring the defense response network in plants against pathogens.

The hop plant (Humulus lupulus L.) produces several valuable secondary metabolites, such as prenylflavonoid, bitter acids, and essential oils. These compounds are biosynthesized in glandular trichomes (lupulin glands) endowed with pharmacological properties and widely implicated in the beer brewing industry. The present study is an attempt to generate exhaustive information of transcriptome dynamics and gene regulatory mechanisms involved in biosynthesis and regulation of these compounds, developmental changes including trichome development at three development stages, namely leaf, bract, and mature lupulin glands. Using high-throughput RNA-Seq technology, a total of 61.13, 50.01, and 20.18 Mb clean reads in the leaf, bract, and lupulin gland libraries, respectively, were obtained and assembled into 43,550 unigenes. The putative functions were assigned to 30,996 transcripts (71.17%) based on basic local alignment search tool similarity searches against public sequence databases, including GO, KEGG, NR, and COG families, which indicated that genes are principally involved in fundamental cellular and molecular functions, and biosynthesis of secondary metabolites. The expression levels of all unigenes were analyzed in leaf, bract, and lupulin glands tissues of hop. The expression profile of transcript encoding enzymes of BCAA metabolism, MEP, and shikimate pathway was most up-regulated in lupulin glands compared with leaves and bracts. Similarly, the expression levels of the transcription factors and structural genes that directly encode enzymes involved in xanthohumol, bitter acids, and terpenoids biosynthesis pathway were found to be significantly enhanced in lupulin glands, suggesting that production of these metabolites increases after the leaf development. In addition, numerous genes involved in primary metabolism, lipid metabolism, photosynthesis, generation of precursor metabolites/energy, protein modification, transporter activity, and cell wall component biogenesis were differentially regulated in three developmental stages, suggesting their involvement in the dynamics of the lupulin gland development. The identification of differentially regulated trichome-related genes provided a new foundation for molecular research on trichome development and differentiation in hop. In conclusion, the reported results provide directions for future functional genomics studies for genetic engineering or molecular breeding for augmentation of secondary metabolite content in hop.

Background: Wallemia ichthyophaga is a highly specialized basidiomycetous fungus. It is one of the most halophilic fungi ever described, only able to grow at low water activity. This specialization is thought to explain why it is only rarely isolated from nature.Results: Genomes of 21 W. ichthyophaga strains were sequenced with PE150 reads on BGISEQ500 platform. The genomes shared high similarity with the reference genome of the species, they were all smaller than 10 Mbp and had a low number of predicted genes. Groups of strains isolated in the same location encompassed clones as well as very divergent strains. There was little concordance between phylogenies of predicted genes. Linkage disequilibrium of pairs of polymorphic loci decayed relatively quickly as a function of distance between the loci (LD decay distance 1270 bp). For the first time a putative mating-type locus was identified in the genomes of W. ichthyophaga.Conclusion: Based on the comparison of W. ichthyophaga genomes it appears that some phylogenetic lineages of the species can persist in the same location over at least several years. Apart from this, the differences between the strains do not reflect the isolation habitat or geographic location. Together with results supporting the existence of (sexual) recombination in W. ichthyophaga, the presented results indicate that strains of W. ichthyophaga can form a single recombining population even between different habitats and over large geographical distances.

Hop (Humulus lupulus L.) is grown mostly as flavouring and bittering ingredient for beer and is also appreciated in the herbal and cosmetic industry, as well as in pharmacology. Among several diseases that damage hop growing, the most devastating in European hop production is verticillium wilt, caused by the soil-borne fungal pathogen Verticillium nonalfalfae. Colonization pattern and differential expression of selected genes after artificial infection of susceptible and resistant hop cultivars with V. nonalfalafae in stems and roots have been analysed recently Švara et al., 2019. Here, we present the dataset related to verification of plant samples infections after artificial inoculation (fungi- and mock-inoculated). After inoculation plant samples were tested for the positive infection by PCR amplification of the V. nonalfalfae ITS DNA region with species specific primers developed and optimised for this purpose. For more insight please see the article “Temporal and spatial assessment of defence responses in resistant and susceptible hop cultivars during infection with Verticillium nonalfalfae”. Keywords: Verticilium nonalfalfae, Hop, Artificial infection rate, Molecular markers, ITS primers

The conserved RNA interference mechanism (RNAi) in the fungal kingdom has become a focus of intense scientific investigation. The three catalytic core components, Dicer-like (DCL), Argonaute (AGO), and RNA-dependent RNA polymerase (RdRP), and their associated small interfering RNA molecules (siRNAs) have been identified and characterised in several fungal species. Recent studies have proposed that RNAi is a major contributor to the virulence of fungal pathogens as a result of so-called trans-kingdom RNA silencing. In the present study, we report on the existence of three core RNAi proteins in the pathogenic plant fungus Verticillium nonalfalfae, which is a soilborne plant pathogen that causes severe wilting disease in hops (Humulus lupulus L.). Two DCL proteins, two AGO proteins, and two RdRP proteins were identified, and their conserved RNAi domains were characterised. Our phylogeny results confirm the existing taxonomic relationships in the Ascomycete fungal phylum and show that the fungi of the Hypocreomycetidae subclass of the Sordariomycetes class have high amino acid sequence similarity. The expression analysis revealed a potential role of RNAi in the pathogenicity of the fungi, since all the RNAi genes were highly upregulated in the highly virulent isolate T2 and were also differentially expressed in the V. nonalfalfae-susceptible Celeia and V. nonalfalfae-resistant Wye Target cultivars.

The complexity of designed bionano-scale architectures is rapidly increasing mainly due to the expanding field of DNA-origami technology and accurate protein design approaches. The major advantage offered by polypeptide nanostructures compared with most other polymers resides in their highly programmable complexity. Proteins allow in vivo formation of well-defined structures with a precise spatial arrangement of functional groups, providing extremely versatile nano-scale scaffolds. Extending beyond existing proteins that perform a wide range of functions in biological systems, it became possible in the last few decades to engineer and predict properties of completely novel protein folds, opening the field of protein nanostructure design. This review offers an overview on rational and computational design approaches focusing on the main achievements of novel protein nanostructure design.

RNA interference is an evolutionary conserved mechanism by which organisms regulate the expression of genes in a sequence-specific manner to modulate defense responses against various abiotic or biotic stresses. Hops are grown for their use in brewing and, in recent years, for the pharmaceutical industry. Hop production is threatened by many phytopathogens, of which Verticillium, the causal agent of Verticillium wilt, is a major contributor to yield losses. In the present study, we performed identification, characterization, phylogenetic, and expression analyses of three Argonaute, two Dicer-like, and two RNA-dependent RNA polymerase genes in the susceptible hop cultivar Celeia and the resistant cultivar Wye Target after infection with Verticillium nonalfalfae. Phylogeny results showed clustering of hop RNAi proteins with their orthologues from the closely related species Cannabis sativa, Morus notabilis and Ziziphus jujuba which form a common cluster with species of the Rosaceae family. Expression analysis revealed downregulation of argonaute 2 in both cultivars on the third day post-inoculation, which may result in reduced AGO2-siRNA-mediated posttranscriptional gene silencing. Both cultivars may also repress ta-siRNA biogenesis at different dpi, as we observed downregulation of argonaute 7 in the susceptible cultivar on day 1 and downregulation of RDR6 in the resistant cultivar on day 3 after inoculation.

The main challenge associated with genotyping based on conventional length polymorphisms is the cross-laboratory standardization of allele sizes. This step requires the inclusion of standards and manual sizing to avoid false results. Capillary electrophoresis (CE) approaches limit the information to the length polymorphism and do not allow the determination of a complete marker sequence. As an alternative, high-throughput sequencing (HTS) offers complete information regarding marker sequences and their flanking regions. In this work, we investigated the suitability of a semi-quantitative sequencing approach for microsatellite genotyping using Illumina paired-end technology. Twelve microsatellite loci that are well established for grapevine CE typing were analysed on 96 grapevine samples from six different countries. We redesigned primers to the length of the amplicon for short sequencing (~100 bp). The primer pair was flanked with a 10 bp overhang for the introduction of barcodes on both sides of the amplicon to enable high multiplexing. The highest data peaks were determined as simple sequence repeat (SSR) alleles and compared with the CE dataset based on 12 reference samples. The comparison showed that HTS SSR genotyping can successfully replace the CE system in further experiments. We believe that, with next-generation sequencing, genotyping can be improved in terms of its speed, accuracy, and price.

Viroids are smallest known pathogen that consist of non-capsidated, single-stranded non-coding RNA replicons and they exploits host factors for their replication and propagation. The severe stunting disease caused by Citrus bark cracking viroid (CBCVd) is a serious threat, which spreads rapidly within hop gardens. In this study, we employed comprehensive transcriptome analyses to dissect host-viroid interactions and identify gene expression changes that are associated with disease development in hop. Our analysis revealed that CBCVd-infection resulted in the massive modulation of activity of over 2000 genes. Expression of genes associated with plant immune responses (protein kinase and mitogen-activated protein kinase), hypersensitive responses, phytohormone signaling pathways, photosynthesis, pigment metabolism, protein metabolism, sugar metabolism, and modification, and others were altered, which could be attributed to systemic symptom development upon CBCVd-infection in hop. In addition, genes encoding RNA-dependent RNA polymerase, pathogenesis-related protein, chitinase, as well as those related to basal defense responses were up-regulated. The expression levels of several genes identified from RNA sequencing analysis were confirmed by qRT-PCR. Our systematic comprehensive CBCVd-responsive transcriptome analysis provides a better understanding and insights into complex viroid-hop plant interaction. This information will assist further in the development of future measures for the prevention of CBCVd spread in hop fields.

Cholesterol is an essential component of mammalian cells, but its role in cancer is unclear. We have determined the total cholesterol content in the healthy and the cancerous lung tissues of the same patient. Tissues of 13 patients (7 women and 6 men, different histological type, different stages of the disease) were obtained during surgical intervention. The samples (0.056–2.004 g) were hydrolyzed in alkaline medium, and the total cholesterol was extracted into n-hexane and simultaneously determined by thin-layer chromatography (TLC) on silica gel high-performance thin-layer chromatography (HPTLC) plates and nonaqueous reversed-phase high-performance liquid chromatography (HPLC), which both proved to be suitable for the determination of cholesterol in lung tissues and gave comparable results. This is the first report about the comparison of the total cholesterol content in the healthy and the cancerous tissue of the same patient. The difference in the results for the total cholesterol from both types of tissues was remarkable. For 13 patients, the mean contents and standard deviations determined by TLC were 4.01 ± 0.75 mg g−1 in the healthy lung tissue and 7.75 ± 1.93 mg g−1 in the cancerous lung tissue. Comparable results were obtained by HPLC analyses of the same samples: 3.91 ± 0.73 mg g−1 in the healthy and 6.95 ± 1.83 mg g−1 in the cancerous lung tissue. The range of total cholesterol content determined by TLC was between 3.20 mg g−1 and 5.83 mg g−1 in healthy lung tissues, and between 4.64 mg g−1 and 12.01 mg g−1 in lung cancer tissues, while the ranges obtained by HPLC were between 2.80 mg g−1 and 5.49 mg g−1 for healthy lung tissue, and between 4.38 mg g−1 and 11.24 mg g−1 for lung cancer tissue. The cancerous lung tissue of each of the thirteen patients contained higher amounts of total cholesterol compared to their healthy lung tissue. In 8 of the patients, the total chole sterol in the cancer tissue was more than 60% higher than in the healthy tissue; furthermore, in 5 patients, it was more than 100% higher.

Background: The SOS response including two main proteins LexA and RecA, maintains the integrity of bacterial genomes after DNA damage due to metabolic or environmental assaults. Additionally, derepression of LexA-regulated genes can result in mutations, genetic exchange and expression of virulence factors. Here we describe the first comprehensive description of the in silico LexA regulon in Clostridium difficile, an important human pathogen. Results: We grouped thirty C. difficile strains from different ribotypes and toxinotypes into three clusters according to lexA gene/protein variability. We applied in silico analysis coupled to surface plasmon resonance spectroscopy (SPR) and determined 16 LexA binding sites in C. difficile. Our data indicate that strains within the cluster, as defined by LexA variability, harbour several specific LexA regulon genes. In addition to core SOS genes: lexA, recA, ruvCA and uvrBA, we identified a LexA binding site on the pathogenicity locus (PaLoc) and in the putative promoter region of several genes involved in housekeeping, sporulation and antibiotic resistance. Conclusions: Results presented here suggest that in C. difficile LexA is not merely a regulator of the DNA damage response genes but also controls the expression of dozen genes involved in various other biological functions. Our in vitro results indicate that in C. difficile inactivation of LexA repressor depends on repressor`s dissociation from the operators. We report that the repressor`s dissociation rates from operators differentiate, thus the determined LexA-DNA dissociation constants imply on the timing of SOS gene expression in C. difficile.

Polypeptides and polynucleotides are natural programmable biopolymers that can self-assemble into complex tertiary structures. We describe a system analogous to designed DNA nanostructures in which protein coiled-coil (CC) dimers serve as building blocks for modular de novo design of polyhedral protein cages that efficiently self-assemble in vitro and in vivo. We produced and characterized gt 20 single-chain protein cages in three shapes-tetrahedron, four-sided pyramid, and triangular prism-with the largest containing gt 700 amino-acid residues and measuring 11 nm in diameter. Their stability and folding kinetics were similar to those of natural proteins. Solution small-angle X-ray scattering (SAXS), electron microscopy (EM), and biophysical analysis confirmed agreement of the expressed structures with the designs. We also demonstrated self-assembly of a tetrahedral structure in bacteria, mammalian cells, and mice without evidence of inflammation. A semi-automated computational design platform and a toolbox of CC building modules are provided to enable the design of protein cages in any polyhedral shape. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3212]

Transcription factors (TFs) play a major role in controlling gene expression by intricately regulating diverse biological processes such as growth and development, the response to external stimuli and the activation of defense responses. The systematic identification and classification of TF genes are essential to gain insight into their evolutionary history, biological roles, and regulatory networks. In this study, we performed a global mining and characterization of hop TFs and their involvement in Citrus bark cracking viroid CBCVd infection by employing a digital gene expression analysis. Our systematic analysis resulted in the identification of a total of 3,818 putative hop TFs that were classified into 99 families based on their conserved domains. A phylogenetic analysis classified the hop TFs into several subgroups based on a phylogenetic comparison with reference TF proteins from Arabidopsis thaliana providing glimpses of their evolutionary history. Members of the same subfamily and subgroup shared conserved motif compositions. The putative functions of the CBCVd-responsive hop TFs were predicted using their orthologous counterparts in A. thaliana. The analysis of the expression profiling of the CBCVd-responsive hop TFs revealed a massive differential modulation, and the expression of the selected TFs was validated using qRT-PCR. Together, the comprehensive integrated analysis in this study provides better insights into the TF regulatory networks associated with CBCVd infections in the hop, and also offers candidate TF genes for improving the resistance in hop against viroids.

Knots are some of the most remarkable topological features in nature. Self-assembly of knotted polymers without breaking or forming covalent bonds is challenging, as the chain needs to be threaded through previously formed loops in an exactly defined order. Here we describe principles to guide the folding of highly knotted single-chain DNA nanostructures as demonstrated on a nano-sized square pyramid. Folding of knots is encoded by the arrangement of modules of different stability based on derived topological and kinetic rules. Among DNA designs composed of the same modules and encoding the same topology, only the one with the folding pathway designed according to the ‘free-end' rule folds efficiently into the target structure. Besides high folding yield on slow annealing, this design also folds rapidly on temperature quenching and dilution from chemical denaturant. This strategy could be used to design folding of other knotted programmable polymers such as RNA or proteins., Driven by complementary base pairing, artificial single-chain DNA is capable of forming complex 3D architectures if an appropriate folding pathway can be realised. Here, the authors describe the design principles for rapidly folding structures, exemplified through fabrication of a nanosized square pyramid.

The aim of the present study was to investigate indigenous fungal communities isolated from extreme environments (hypersaline waters of solar salterns and subglacial ice), for the production of metabolic compounds with selected biological activities: hemolysis, antibacterial, and acetylcholinesterase inhibition. In their natural habitats, the selected fungi are exposed to environmental extremes, and therefore the production of bioactive metabolites was tested under both standard growth conditions for mesophilic microorganisms, and at high NaCl and sugar concentrations and low growth temperatures. The results indicate that selected halotolerant and halophilic species synthesize specific bioactive metabolites under conditions that represent stress for non-adapted species. Furthermore, adaptation at the level of the chemical nature of the solute lowering the water activity of the medium was observed. Increased salt concentrations resulted in higher hemolytic activity, particularly within species dominating the salterns. The appearance of antibacterial potential under stress conditions was seen in the similar pattern of fungal species as for hemolysis. The active extracts exclusively affected the growth of the Gram-positive bacterium tested, Bacillus subtilis. None of the extracts tested showed inhibition of acetylcholinesterase activity.