Identifikacija bakterija octenog vrenja izoliranih iz organski i konvencionalno proizvedenog jabučnog octa

Organic apple cider vinegar is produced from apples that go through very restricted treatment in orchard. During the first stage of the process, the sugars from apples are fermented by yeasts to cider. The produced ethanol is used as a substrate by acetic acid bacteria in a second separated bioprocess. In both, the organic and conventional apple cider vinegars the ethanol oxidation to acetic acid is initiated by native microbiota that survived alcohol fermentation. We compared the cultivable acetic acid bacterial microbiota in the production of organic and conventional apple cider vinegars from a smoothly running oxidation cycle of a submerged industrial process. In this way we isolated and characterized 96 bacteria from organic and 72 bacteria from conventional apple cider vinegar. Using the restriction analysis of the PCR-amplifi ed 16S-23S rRNA gene ITS regions, we identified four different HaeIII and five different HpaII restriction profiles for bacterial isolates from organic apple cider vinegar. Each type of restriction profile was further analyzed by sequence analysis of the 16S-23S rRNA gene ITS regions, resulting in identification of the following species: Acetobacter pasteurianus (71.90 %), Acetobacter ghanensis (12.50 %), Komagataeibacter oboediens (9.35 %) and Komagataeibacter saccharivorans (6.25 %). Using the same analytical approach in conventional apple cider vinegar, we identified only two different HaeIII and two different HpaII restriction profiles of the 16S‒23S rRNA gene ITS regions, which belong to the species Acetobacter pasteurianus (66.70 %) and Komagataeibacter oboediens (33.30 %). Yeasts that are able to resist 30 g/L of acetic acid were isolated from the acetic acid production phase and further identified by sequence analysis of the ITS1-5.8S rDNA‒ITS2 region as Candida ethanolica, Pichia membranifaciens and Saccharomycodes ludwigii. This study has shown for the first time that the bacterial microbiota for the industrial production of organic apple cider vinegar is clearly more heterogeneous than the bacterial microbiota for the industrial production of conventional apple cider vinegar. Further chemical analysis should reveal if a difference in microbiota composition influences the quality of different types of apple cider vinegar.„Organski“ jabučni ocat proizvodi se od jabuka uzgojenim prema vrlo strogim ekološkim kriterijima. U prvoj fazi proizvodnje kvasci fermentiraju šećer iz jabuka u jabučno vino. U drugoj fazi bakterije octenog vrenja fermentiraju nastali etanol u jabučni ocat. Oksidacija etanola u octenu kiselinu u oba postupka proizvodnje octa, organskom i konvencionalnom, potaknuta je aktivnošću bakterija koje su preživjele alkoholno vrenje. U radu su ispitane bakterije octenog vrenja izolirane tijekom submerznog postupka organske i konvencionalne proizvodnje jabučnog octa. Izolirano je i okarakterizirano 96 sojeva bakterija iz organskog i 72 soja iz konvencionalno proizvedenog jabučnog octa. Restrikcijskom su analizom intergenskih regija 16S‒23S rDNA bakterija izoliranih iz organskog jabučnog octa, umnoženih lančanom reakcijom polimeraze, identificirana četiri restrikcijska profila HaeIII i pet profila HpaII. Sekvencioniranjem i analizom restrikcijskih profila identificirane su sljedeće vrste bakterija: Acetobacter pasteurianus (71,90 %), Acetobacter ghanensis (12,50 %), Komagataeibacter oboediens (9,35 %) i Komagataeibacter saccharivorans (6,25 %). Na isti su način u konvencionalno proizvedenom jabučnom octu identificirana samo dva HaeIII i dva HpaII restrikcijska profila, koji pripadaju vrstama Acetobacter pasteurianus (66,70 %) i Komagataeibacter oboediens (33,30 %). Tijekom proizvodnje octene kiseline izolirani su kvasci koji mogu rasti pri koncentraciji octene kiseline od 30 g/L, te je sekvencijskom analizom potvrđeno da pripadaju sljedećim vrstama: Candida ethanolica, Pichia membranifaciens and Saccharomycodes ludwigii. Rezultati rada po prvi put dokazuju da su bakterije koje se koriste u proizvodnji organskog jabučnog octa raznovrsnije od onih korištenih u konvencionalnoj industrijskoj proizvodnji octa. Dodatnim bi se kemijskim analizama trebalo ispitati utječe li raznovrsnost mikrobiote na kakvoću različitih tipova jabučnog octa

Chromatographic tools for optimization of IVT reaction and improving mRNA purification process

The recently demonstrated efficacy of mRNA-based Covid-19 vaccines has shown the promise of this therapeutic format, but also highlighted the need for higher efficiency of mRNA production to meet enormous needs for global vaccine supply. Typical mRNA production process involves three key steps: 1) plasmid DNA (pDNA) production in supercoiled (sc) isoform, linearization and purification, 2) in-vitro transcription (IVT) reaction and 3) mRNA purification. Here we present a chromatographic toolbox for integrated mRNA production from pDNA to mRNA purification, including in-process analytics. The pDNA purification approach presented here was designed to fit the specific requirements of mRNA vaccines. It integrates a linearisation step before polishing (removal of unwanted isoforms) of plasmid DNA. The polishing step after enzymatic linearisation, separates linear pDNA from enzyme and other unwanted products. Supporting in-process analytical tools are presented. IVT reaction monitoring with novel HPLC approaches includes CIMac PrimaS analysis of mRNA content as a function of time, with concomitant monitoring of NTP consumption. With information on NTPs, capping reagent and mRNA content, IVT reaction can be rapidly optimized for maximum productivity, in near real-time. Advantage of at-line monitoring is to prevent degradation of mRNA in IVT mixture which would occur after maximum productivity is reached. Purification of mRNA from IVT reaction mixture can be achieved using selective binding to polyA tail (using OligodT chromatography) or multimodal chromatography (PrimaS) which separates the ssRNA product from DNA template and IVT reaction mixture. Polishing approaches for final separation of ssRNA and dsRNA using hydrophobic interaction chromatography (HIC) and reverse-phase (RP) chromatography achieve high purity of final product. Supporting in-process analytical HPLC tools, including multimodal chromatography resin, facilitate a rapid read-out of mRNA concentration and purity profile

Determination of the sensitivity of human tumor cells to electrochemotherapy with platinum (II) compounds in vitro

Elektroporacija (EP) je fizikalna metoda s katero z dovajanjem električnih pulzov v celični membrani za kratek čas ustvarimo pore, kar olajša prehod v celice določenim molekulam. Za zdravljenje rakavih obolenj se vse bolj uporablja EP v kombinaciji s cisplatinom (CDDP) in bleomicinom, čemur rečemo elektrokemoterapija (EKT). Cisplatin je platinova (II) spojina, ki s kovalentnimi povezavami povzroči spremembe v strukturi DNA in vodi v smrt rakave celice. Kljub temu, da je CDDP eden najpogosteje uporabljenih kemoterapevtikov, pa povzroča hude stranske učinke in tudi odpornost rakavih celic. Zaradi tega se raziskave usmerjajo v razvoj novih platinovih (II) analogov s protitumorskim delovanjem, kamor spadata tudi [PtCl (5,7-dibromo-8-hidroksikinolinato)(S-dmso)] (spojina A) in njen analog, [PtCl (5,7-dibromo-8-hidroksikinolinato)(pta)] (spojina B). Citotoksičnost obeh spojin v kombinaciji z EP smo preverili na humani celični liniji raka debelega črevesja (HT29) in raka dojke (MCF7). S testom klonogenosti smo izračunali vrednosti IC50 in jih primerjali z vrednostmi IC50 CDDP, s pripravo citospinov in barvanjem po Giemsi pa smo na podlagi morfoloških značilnosti ugotovili delež apoptotičnih in nekrotičnih celic. Ugotovili smo, da spojini A in B delujeta citotoksično na tumorske celice in povzročata predvsem apoptozo ter deloma nekrozo. Elektroporacija statistično značilno poveča citotoksično delovanje vseh treh platinovih (II) spojin in je učinkovita metoda za izboljšanje vnosa ne samo CDDP, pač pa tudi spojine A in B. Spojina B se je v kombinaciji z in brez EP izkazala za bolj toksično od spojine A in hkrati tudi enako ali celo bolj citotoksično kot CDDP pri obeh celičnih linijah in je zato primerna za nadaljne testiranje, kot potencialni kemoterapevtik.Electroporation (EP) is a physical technique in which an electric field is applied to cells in order to increase the permeability of cell membrane for poorly permeating molecules. Electroporation in combination with cisplatin (CDDP), also called electrochemotherapy (ECT), is used for local treatment of several malignant tumors. Cisplatin is a well-known platinum (II) antitumor drug which antitumor activity has been linked to its ability to form DNA adducts, which subsequently lead to apoptosis of cancer cells. Although CDDP is broadly used, its main limitations are severe side effects and tumor cells’ resistance. Because of this, researchers are focusing on developing new platinum (II) complexes with antitumor activity, including [PtCl (5,7-dibromo-8-hydroxyquinolinato)(S-dmso)] (compound A) and it\u27s analog, [PtCl (5,7-dibromo-8-hydroxyquinolinato)(pta)] (compound B). In the thesis, we analysed the cytotoxicity of compound A and B in combination with EP in human colon cancer (HT29) and human breast cancer cell line (MCF7). We determined IC50 values with clonogenic assay and compared it to IC50 values of CDDP. We used Giemsa staining to determine the percentage of apoptotic and necrotic cells. Results show that all compounds, A, B and CDDP have a cytotoxic effect on both tumor cell lines and cause primarily apoptotic cell death. Furthermore, EP has proved to be a very effective method for drug uptake as it has significantly improved cytotoxicity of all three platinum (II) complexes. Compound B has turned out to be more cytotoxic with and without EP than compound A and showed similar or better cytotoxicity than CDDP in both cell lines. Based on our results, compound B is very promising potential therapeutic agent

Comparison of the impact of the working environment on employees’ satisfaction at work in the selected public and private sector organizations

Zadovoljstvo zaposlenih pri delu je eden izmed tistih ključnih dejavnikov, ki vodi do organizacijskega uspeha in na katerega so dandanes pozorne praktično vse organizacije. Vedno bolj se poudarja pomen zadovoljstva zaposlenih pri delu, saj ta omogoča veliko pozitivnih učinkov tako za organizacijo kot tudi za zaposlene. Večja produktivnost (storilnost), boljši medosebni odnosi, manjša fluktuacija in s tem manjši stroški so le nekateri izmed teh učinkov. Poznavanje dejavnikov, ki vplivajo na zadovoljstvo zaposlenih pri delu pomaga identificirati razloge in področja, s katerimi in na katerih zaposleni niso zadovoljni. Na osnovi tega lahko organizacija uvede določene spremembe in prilagoditve ter tako izboljša raven zadovoljstva zaposlenih pri delu znotraj organizacije. Namen magistrskega dela je teoretično osvetliti področje delovnega okolja in z izvedbo raziskave v izbrani organizaciji državne uprave in izbrani organizaciji zasebnega sektorja ugotoviti, kako dejavniki delovnega okolja vplivajo na zadovoljstvo zaposlenih pri delu ter oblikovati priporočila za uvedbo sprememb v izbranih organizacijah. V magistrskem delu je bila izvedena mednarodna primerjava sekundarnih podatkov štirih držav. Izvedena je bila analiza izbranih dejavnikov kakovosti delovnega okolja, ki se meri tako s subjektivnimi ocenami zaposlenih kot z objektivnimi pokazatelji. Z metodo anketiranja je bila izvedena tudi raziskava na vzorcu dveh izbranih organizacij javnega in zasebnega sektorja. Osredotočilo se je predvsem na oceno dejavnikov zadovoljstva z delom in delovnim mestom ter na zaznavo dejavnikov delovnega okolja. Rezultati mednarodne primerjave kažejo, da med spremljanimi dejavniki delovnega okolja v Sloveniji, Avstriji, Nemčiji in na Danskem ni bistvenih razlik ter da so razmere za delo v vseh državah varne. Rezultati anketne raziskave so pokazali, da so sodelujoči v raziskavi v obeh sektorjih najbolj zadovoljni z ožjimi sodelavci, s katerimi delajo, medtem ko so najmanj zadovoljni z intenzivnostjo nagrajevanja, ki so je deležni. Obstaja kar nekaj statistično značilnih razlik med skupinama zaposlenih iz javnega in zasebnega sektorja, in sicer so povprečno višje ocene zadovoljstva zaposlenih pri delu v skupini iz zasebnega sektorja. Na podlagi rezultatov raziskave se predlaga, da vodje storijo več za dvig motiviranosti zaposlenih in za izboljšanje delovnega okolja, kar posledično vpliva tudi na višje zadovoljstvo zaposlenih pri delu in večjo uspešnost organizacij.Employee satisfaction at work is one of those key factors that enable organizational success and to which virtually all organizations pay attention today. The importance of job satisfaction is increasingly emphasized because it provides many positive effects for both the organization and employees. Higher productivity (performance), better interpersonal relationships, less fluctuation, and, thus, lower costs are just some of these effects. Knowing the factors that affect employee satisfaction at work helps to identify the reasons and areas with which and in which employees are dissatisfied. Based on this, the organization can introduce certain changes and adjustments. Thus, it improves the level of satisfaction within the organization. The purpose of the master\u27s thesis is to highlight the field of work environment theoretically and, by researching a selected state administration organization and a selected private sector organization, determine how work environment factors affect employee satisfaction at work, as well as to make recommendations for changes in the selected organizations. In the master\u27s thesis, an international comparison of secondary data from four countries was performed. An analysis of the selected factors of the quality of the working environment was performed. It is measured both by subjective assessments of employees and by objective indicators. A survey was also conducted on a sample of the two selected public and private sector organizations using the survey method. It focused mainly on the assessment of job satisfaction and satisfaction-with-work factors and the perception of work environment factors. The results of the international comparison show that there are no significant differences between the monitored factors of the work environment in Slovenia, Austria, Germany, and Denmark and that the working conditions in all countries are safe. The results of the survey showed that survey participants in both sectors are most satisfied with the close associates they work with while they are least satisfied with the intensity of rewards they receive. There are quite a few statistically significant differences between the groups of employees from the public and private sectors, i.e. on average, there are higher satisfaction ratings in the group of employees from the private sector. Based on the results of the research, it is proposed that managers do more to increase employee motivation and to improve the work environment, which, in turn, affects also higher employee satisfaction and greater performance of organizations

Vpliv bakterije Klebsiella oxytoca na privzem cinka in svinca pri sončnici (Helianthus annuus)

Ugotoviti vpliv bakterije Klebsiella oxytoca na privzem težkih kovin, predvsem cinka in svinca, pri sončnici (Helianthus annuus). Kalitev sončnice in spremljanje rasti korenin ter poganjkov v treh serijah (kontrola (K), živa bakterija (ŽB), avtoklavirana bakterija (AB)), primerjava sveže in suhe mase poganjkov in korenin, analiza koncentracije kovin v koreninah in poganjkih, merjenje vsebnosti fotosinteznih pigmentov poganjkov, merjenje transpiracije, merjenje učinkovitosti fotosistema II. Statistična obdelava v R studiu, z enosmerno analizo variance (ANOVA), post-hoc s Tukyevim testom ter obdelava podatkov iz rentgenske fluorescenčne spektroskopije s programom Microsoft Office Excel z dodatkom XL Toolbox z metodo Dunnetovega testa. Vidne so nekroze listov pri vseh testiranih serijah, pri seriji z dodatkom živih bakterij opazimo povišane koncentracije izmerjenih elementov (osredotočili smo se na koncentracijo cinka in svinca), v seriji ŽB sta opazni zmanjšana transpiracija in dejanska fotosintezna učinkovitost fotosistema II. Statističnih razlik pri vsebnosti fotosinteznih barvil nismo opazili. Prisotnost bakterije Klebsiella oxytoca poveča privzem snovi v rastlino, kar je v našem primeru predvsem negativno vplivalo na nekatere merljive fiziološke parametre. Za zanesljivejše zaključke bi morali izvesti več ponovitev