Monitoring of Genotoxicity in Drinking Water Using in vitro Comet Assay and Ames Test

A screening strategy for evaluation of genotoxic potential of drinking water has been proposed in the present work. Genotoxicity assays with tap water collected at three different sampling points in Ljubljana drinking water region are presented here. In vitro alkaline version of the comet assay was performed with human HepG2 and Caco2 cell lines and protozoa (Tetrahymena thermophila) cells. Parallel genotoxicity evaluation on the same samples was carried out by the Ames test (with/without exogenous metabolic activation) using Salmonella typhimurium TA97a, TA100 and TA1535 strains. Nonconcentrated and concentrated water samples were tested in both bioassays, and chemical analyses were performed to check the contents of pesticides and nitrates. There was no indication of genotoxic activity in any of the drinking water samples according to the Ames test. The results of the comet assays showed differences and possible genotoxic potential among the water samples tested on different cell types, which were, however, statistically not significant, except in two cases. Statistical analyses showed the comet assay was more sensitive than the Ames test for genotoxicity detection in drinking water samples

Potential of Selected Rumen Bacteria for Cellulose and Hemicellulose Degradation

U probavnom se sustavu biljojeda nalaze vrlo učinkoviti mikroorganizmi što razgrađuju celulolozu i hemicelulozu iz sažvakanog biljnog materijala, te ih opskrbljuju hranjivim tvarima. Osim protozoa i gljivica, razgradnji otporne (hemi)celulozne biomase u buragu znatno pridonose i bakterije. U fokusu je ovog preglednog rada opis enzimskog sustava triju predstavnika bakterija iz buraga što proizvode celulazu, i to: Ruminococcus flavefaciens, Prevotella bryantii i Pseudobutyrivibrio xylanivorans. R. flavefaciens je poznat po proizvodnji jedne od najkompleksnijih enzimskih struktura, pa bi se mogao upotrijebiti za dizajn enzimskog kompleksa za razgradnju stanične stijenke biljaka. S druge strane, bakterije Prevotella bryantii i Pseudobutyrivibrio xylanivorans proizvode jednostavne, slobodne i vrlo aktivne ksilanaze. Bakterija P. xylanivorans ima i probiotička svojstva, pa se može upotrijebiti za proizvodnju bioplina i kao dodatak krmivu. Ispitivanje je genoma i proteoma bakterija što razgrađuju celulozu i hemicelulozu usmjereno na identifikaciju novih enzima, koji se nakon kloniranja i ekspresije u odgovarajućim domaćinima mogu upotrijebiti za izradu vrlo aktivnih rekombinantnih hidrolitičkih mikroorganizama, te primijeniti u različitim biotehnološkim procesima.Herbivorous animals harbour potent cellulolytic and hemicellulolytic microorganisms that supply the host with nutrients acquired from degradation of ingested plant material. In addition to protozoa and fungi, rumen bacteria contribute a considerable part in the breakdown of recalcitrant (hemi)cellulosic biomass. The present review is focused on the enzymatic systems of three representative fibrolytic rumen bacteria, namely Ruminococcus flavefaciens, Prevotella bryantii and Pseudobutyrivibrio xylanivorans. R. flavefaciens is known for one of the most elaborated cellulosome architectures and might represent a promising candidate for the construction of designer cellulosomes. On the other hand, Prevotella bryantii and Pseudobutyrivibrio xylanivorans produce multiple free, but highly efficient xylanases. In addition, P. xylanivorans was also shown to have some probiotic traits, which makes it a promising candidate not only for biogas production, but also as an animal feed supplement. Genomic and proteomic analyses of cellulolytic and hemicellulolytic bacterial species aim to identify novel enzymes, which can then be cloned and expressed in adequate hosts to construct highly active recombinant hydrolytic microorganisms applicable for different biotechnological tasks

Vpliv mikrobiote na delovanje imunskega sistema in zaviralcev imunskih kontrolnih točk

Imunski sistem je ključen pri nadzoru nastanka in rasti malignih celic. Z imunoterapijo z zaviralci imunskih kontrolnih točk je imunski sistem postal eden pomembnejših tarč onkološkega zdravljenja. Sorazmerno visok delež onkoloških bolnikov na to zdravljenje še vedno ne odgovori. Črevesna mikrobiota bi glede na zadnja spoznanja lahko postala način premagovanja odpornosti na imunoterapijo. Možni načini modulacije mikrobiote so prehranska strategija, probiotiki in fekalna transplantacija mikrobiote. Ovire pri raziskovanju mikrobiote in njene uporabe pri zdravljenju z zaviralci imunskih kontrolnih točk so zaenkrat nestandardizirani pristopi k analizi mikrobiote, geografske razlike v mikrobioti, vpliv kulture ter načina življenja itd. V teku so številne predklinične in klinične raziskave, nekatere že z obetavnimi rezultati. Z obdelavo mikrobiote bi bila lahko obravnava onkoloških bolnikov še bolj personalizirana in holistična

Ispitivanje estrogene aktivnosti u vodama pomoću kvasaca

The presence of endocrine-disrupting compounds in wastewater, surface water, groundwater and even drinking water has become a major concern worldwide, since they negatively affect wildlife and humans. Therefore, these substances should be effectively removed from effluents before they are discharged into surface water to prevent pollution of groundwater, which can be a source of drinking water. Furthermore, an efficient control of endocrine-disrupting compounds in wastewater based on biological and analytical techniques is required. In this study, a yeast estrogen screen (YES) bioassay has been introduced and optimized with the aim to assess potential estrogenic activity of waters. First, assay duration, concentration of added substrate to the assay medium and wavelength used to measure the absorbance of the substrate were estimated. Several compounds, such as 17-β-estradiol, 17-α-ethinylestradiol, bisphenol A, nonylphenol, genisteine, hydrocortisone, dieldrin, atrazine, methoxychlor, testosterone and progesterone were used to verify its specificity and sensitivity. The optimized YES assay was sensitive and responded specifically to the selected estrogenic and nonestrogenic compounds in aqueous samples. Potential estrogenicity of influent and effluent samples of two wastewater treatment plants was assessed after the samples had been concentrated by solid-phase extraction (SPE) procedure using Oasis® HLB cartridges and methanol as eluting solvent. Up to 90 % of relative estrogenic activity was detected in concentrated samples of influents to wastewater treatment plants and estrogenic activity was still present in the concentrated effluent samples. We found that the introduced YES assay is a suitable screening tool for monitoring the potential estrogenicity of effluents that are discharged into surface water.Prisutnost spojeva koji imaju estrogeni učinak u otpadnim, površinskim, podzemnim, a čak i pitkim vodama sve više zabrinjava stručnjake diljem svijeta zbog negativnog učinka na zdravlje ljudi i životinja. Stoga takve spojeve treba odstraniti iz otpadnih voda prije njihovog ispuštanja u površinske vode, te na taj način spriječiti zagađivanje podzemnih voda, što se koriste kao izvor pitke vode. Kemijskom analizom i biološkim testovima potrebno je učinkovito pratiti prisutnost spojeva s estrogenim učinkom u otpadnim vodama. U ovom smo radu razvili i optimirali metodu ispitivanja estrogenog učinka spojeva pomoću kvasca (test YES) radi procjene potencijalne estrogene aktivnosti u vodama. Optimirali smo trajanje testa, koncentraciju supstrata dodanog podlozi i valnu duljinu za mjerenje apsorbancije razgrađenog supstrata. Specifičnost i osjetljivost testa YES provjerili smo pomoću sljedećih spojeva: 17-β-estradiol, 17-α-etinilestradiol, bisfenol A, nonilfenol, genistein, hidrokortizon, dieldrin, atrazin, metoksiklor, testosteron i progesteron. Optimiranim testom YES mogu se u uzorcima voda detektirati odabrani spojevi s estrogenim učinkom ili bez njega. Moguća estrogenost pritoka i istoka vode iz dva postrojenja za obradu otpadnih voda određena je prethodnom ekstrakcijom uzoraka vode na čvrstoj fazi (SPE ekstrakcija) pomoću kolone Oasis® HLB i metanola kao otapala. U koncentriranim je uzorcima pritoka u postrojenjima određeno 90 % relativne estrogene aktivnosti. Isto tako je estrogena aktivnost pronađena i u koncentriranim uzorcima istoka iz postrojenja. Iz dobivenih se rezultata može zaključiti da je test YES prikladan za ispitivanje estrogene aktivnosti otpadnih voda, što naknadno dospijevaju u površinske vode

Expression of Cellulosome Components and Type IV Pili within the Extracellular Proteome of Ruminococcus flavefaciens 007

Funding: The Rowett Institute receives funding from SG-RESAS (Scottish Government Rural and Environmental Science and Analysis Service). Visit of M.V. was supported by research grants from FEMS and Slovene human resources development and scholarship funds. Parts of this work were funded by grants from the United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel – BSF Energy Research grant to E.A.B. and B.A.W. and Regular BSF Research grants to R.L. and B.A.W. – and by the Israel Science Foundation (grant nos 966/09 and 159/07 291/08). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

How Can We Advance Integrative Biology Research in Animal Science in 21st Century?:Experience at University of Ljubljana from 2002 to 2022

In this perspective analysis, we strive to answer the following question: how can we advance integrative biology research in the 21st century with lessons from animal science? At the University of Ljubljana, Biotechnical Faculty, Department of Animal Science, we share here our three lessons learned in the two decades from 2002 to 2022 that we believe could inform integrative biology, systems science, and animal science scholarship in other countries and geographies. Cultivating multiomics knowledge through a conceptual lens of integrative biology is crucial for life sciences research that can stand the test of diverse biological, clinical, and ecological contexts. Moreover, in an era of the current COVID-19 pandemic, animal nutrition and animal science, and the study of their interactions with human health (and vice versa) through integrative biology approaches hold enormous prospects and significance for systems medicine and ecosystem health

Probiotics in animal nutrition

Probiotiki so živi mikroorganizmi, ki zaužiti v ustreznem številu, ugodno vplivajo na zdravje gostitelja. Njihovi učinki so praviloma povezani z vzpostavitvijo ugodnega mikrobnega ravnovesja v prebavilih gostitelja ter uravnavanjem njegovega imunskega odziva. Pri domačih živalih so ključni učinki probiotikov povezani z izboljšano učinkovitostjo prireje. Poleg ugodnega vpliva na zdravstveno stanje (predvsem mladih) živali, slednje obsega tudi izboljšano konverzijo krme, povečano hitrost rasti in nekatere druge. Probiotični krmni dodatki registrirani v EU vsebujejo predvsem Gram-pozitivne bakterije iz rodov Bacillus, Enterococcus, Lactobacillus, Pediococcus, Streptococcus ter kvasovke Saccharomyces cerevisiae in Kluyveromyces sp. Medtem, ko je večina omenjenih mikroorganiymov načeloma varnih, imajo nekateri lastnosti, ki so lahko škodljive tako za živali, kot tudi za ljudi. Tak primer so enterokoki, pri katerih pogosto opažajo zapise za prenosljive determinante odpornosti proti antibiotikom. Slednji tako predstavljajo potencialno nevarnost za širjenje odpornosti v patogenih mikrobnih populacijah ljudi in živali. Hiter napredek na področju sintetične in sistemske biologije združen s podporo bioinformatike in novimi orodji genskega inženirstva v prihodnosti obeta skoraj neskončne možnosti za pripravo probiotičnih sevov s poljubnimi lastnostmi, vendar pa bodo le-ti lahko prestopili meje laboratorijev šele ob ustrezni spremembi zakonodaje in javnega mnenja.Probiotics are defined as living micro-organisms, that upon ingestion in certain numbers, exert health benefits to the host. Their use is linked to proven efficacy on the gastrointestinal microbial equilibrium as well as immunomodulation. The positive effect in animals exerts not only in an improved health status, especially in young animals, but also in improved animal performance, including growth rate and feed conversion efficiency. Microorganisms that are used in animal feeds in the EU are mainly Gram-positive bacteria belonging to genera Bacillus, Enterococcus, Lactobacillus, Pediococcus and Streptococcus and yeasts, such as Saccharomyces cerevisiae or Kluyveromyces species. While most of the species are apparently safe, certain microorganisms may exert harmful properties for animals as well as humans. Enterococci, for example, might harbour transmissible antibiotic resistance determinants, which have the potential to spread in animal and human-associated pathogenic microbial populations. Recent developments in synthetic and systems biology, coupled with bioinformatics and novel tools for genetic engineering, will soon enable the construction of \u27artificial\u27 probiotic microorganisms with virtually any combination of properties. Whether and when these \u27designer probiotics\u27 will reach out of the labs depends on legislation as well as public opinion

Expression patterns of Ruminococcus flavefaciens 007S cellulases as revealed by zymogram approach

The expression of Ruminococcus flavefaciens 007S cellulases in different incubation time points (growth stages) and their substrate inducibility were analyzed by comparing the zymogram expression profiles of cultures grown on insoluble cellulose (avicel) with celliose-grown cultures. The molecular weights of the enzymes were compared to (putative) cellulases encoded in the R. flavefaciens FD-1 genome