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21.
Preliminary experiments into colonization of microorganisms from activated sludge on different types of plastics
Tjaša Matjašič, Tanja Dreo, Zoran Samardžija, Oliver Bajt, Tjaša Kanduč, Tatjana Simčič, Nataša Mori, 2020, original scientific article

Abstract: The presence of plastics in the environment is currently one of the most pressing global environmental problems. Microorganisms start to form biofilms on plastic surfaces when they first come in contact with the biosphere; however, these interactions and processes are little understood, especially in freshwaters. This study aimed to better understand the colonization process of microorganisms from activated sludge on plastic materials exhibiting different surface characteristics. We inoculated synthetic fabric (PET), water bottles (PET), and plastic bags for packing vegetables and fruits (HDPE) with microorganisms from activated sludge. Mixtures of plastics and activated sludge, as well as the control, were incubated at 22-24°C in Bushnell Haas (BH) liquid medium and shaken at 120 rpm for two months. The mixtures were sub-sampled weekly and seeded into fresh BH medium with test plastic materials to avoid feeding microorganisms on dead biomass. The colonization was followed by measuring optical density (OD600) of liquid medium, by measurements of isotopic composition of carbon (δ13C) in untreated and treated plastic materials and, with in-specting the plastics surface with scanning electron microscopy (SEM). Overall, the study confirmed differences between colonizing microorganisms on different plastic material when comparing SEM micrographs of materials from the flasks inoculated with activated sludge. The texture of the HDPE bag changed during the experiment in both, control and inoculated flasks, but it is not clear whether the observed changes were due to abiotic or biotic factors. We concluded that microorganisms from activated sludge are capable of colonizing both PET and HDPE materials, and biofilm formation is most probably influenced by the chemical composition of plastics and their surface characteristics.
Keywords: biofilm, plastics, SEM, isotopic composition of carbon, co-cultivation, UV sterilization
Published in DiRROS: 31.07.2024; Views: 261; Downloads: 193
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22.
Metabolic potential, respiration rate and their relationship in offspring of different sizes of marble trout (Salmo marmoratus Cuvier)
Anton Brancelj, Tatjana Simčič, Dušan Jesenšek, 2015, original scientific article

Abstract: The size and composition of fish eggs are related to female’s characteristics, such as age, size and individual conditions, and they have an impact on the properties of offspring that are important for their fitness. Electron transport system (ETS) activity and respiration rate (R) of early life history stages (i.e. non-fertilized eggs, eggs at eyed stage and larvae with yolk sac) of 13 females of marble trout (Salmo marmoratus) were measured separately in order to determine their metabolic properties in relation to size. The results showed that larger females produced larger eggs in higher numbers. Growth experiments on the survival of offspring of a single female revealed that the survival rate of early embryos was higher for smaller eggs during the earliest stages, but ultimately the percentage of surviving larvae did not correlate with egg size. The ETS activities and respiration rates of non-fertilized eggs, eyed eggs and larvae differed significantly between 13 females. Both parameters increased with increasing dry mass of the early life history stages, but the increase of respiration rate was greater than that of ETS activity. The lower ETS/R ratios in larger individuals therefore indicate that their energy metabolism was less adaptable to environmental changes than that of smaller ones. Larger egg size could be an advantage under favourable conditions, whereas smaller size could be optimal under stressing circumstances in which the higher metabolic potential enables production of the energy required for metabolism. This is first report on the relationship between ETS activity and respiration rate of the early life history stages in salmonids.
Keywords: Salmo marmoratus, fitness, metabolism, ETS activity, ETS/R ratio, fishes
Published in DiRROS: 29.07.2024; Views: 222; Downloads: 127
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23.
The five-year KRAS, NRAS and BRAF analysis results and treatment patterns in daily clinical practice in Slovenia in 1st line treatment of metastatic colorectal (mCRC) patients with RASwild-type tumour (wtRAS) : a real- life data report 2013–2018
Tanja Mesti, Martina Reberšek, Janja Ocvirk, 2023, original scientific article

Abstract: Background. We preformed a Phase IV non-interventional study to assess KRAS, NRAS and BRAF status in metastatic colorectal cancer (mCRC) patients suitable for 1st line treatment and to evaluate the decisions for 1st line treatment considering the treatment goals in the RAS wild type (wt) patients. The aim of our study was also to evaluate the influ-ence of a waiting period for biomarkers analysis on the start of first-line treatment.Patients and methods. Patients with histologically confirmed mCRC adenocarcinoma suitable for first-line treat-ment fulfilling all inclusion criteria were included in the study. The KRAS, NRAS and BRAF analysis was performed from tissue samples of primary tumor site or metastatic site. All included patients have given consent to participate in the study by signing the informed consent form. Results. From April 2013 to March 2018 at the Institute of Oncology Ljubljana 650 patients were included, 637 of them were treated with first- line systemic treatment according to RAS and BRAF status. Remaining 13 patients with mCRC did not receive systemic first-line treatment. The distribution of patients with KRAS mutated and wild-type tumors, was almost equal, 48.8% and 47.9% respectively, 89 % of the patients had wt NRAS tumours and 86.1% had wt BRAF tu-mours. The most frequently prescribed treatment was bevacizumab-based therapy (53.1%), either in combination with doublet chemotherapy or with mono-chemotherapy. EGFR inhibitors cetuximab and panitumumab were prescribed in wt RAS mCRC patients (30.9%). The waiting period for biomarkers analysis was two weeks.Conclusions. Our real-world data, single centre 5-year analysis showed that the distribution between wild type and mutated type tumors of the patients with mCRC was approximately the same, as worldwide, so the Slovenian popula-tion with mCRC has the same ratio distribution of KRAS, NRAS and BRAF wild and mutated genes. We concluded that a two-week waiting period for biomarkers analysis did not influence the first line treatment decision, so it was in the accordance with the worldwide treatment guidelines based on evidence-based medicine.
Keywords: metastatic colorectal cancer, RAS and BRAF biomarkers, systemic treatment
Published in DiRROS: 25.07.2024; Views: 197; Downloads: 84
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24.
Treatment of vulvar cancer recurrences with electrochemotherapy : a detailed analysis of possible causes for unsuccessful treatment
Gregor Vivod, Tanja Jesenko, Gorana Gašljević, Nina Kovačević, Maša Omerzel, Gregor Serša, Sebastjan Merlo, Maja Čemažar, 2023, original scientific article

Abstract: Background. Electrochemotherapy has good local effectiveness in the treatment of vulvar cancer. Most studies have reported the safety and effectiveness of electrochemotherapy for palliative treatment of gynecological cancers and mostly vulvar squamous cell carcinoma. Some tumors, however, fail to respond to electrochemotherapy. The biological features/determinants for the nonresponsiveness are not determined yet. Patient and methods. A recurrence of vulvar squamous cell carcinoma was treated by electrochemotherapy using intravenous administration of bleomycin. The treatment was performed by hexagonal electrodes according to standard operating procedures. We analyzed the factors that could determine nonresponsiveness to electrochemotherapy. Results. Based on the presented case of nonresponsive vulvar recurrence to electrochemotherapy, we hypothesize that the vasculature of the tumors prior to treatment may predict the response to electrochemotherapy. The histological analysis showed minimal presence of blood vessels in the tumor. Thus, low perfusion may reduce drug delivery and lead to a lower response rate because of the minor antitumor effectiveness of vascular disruption. In this case, no immune response in the tumor was elicited by electrochemotherapy. Conclusions. In this case, of nonresponsive vulvar recurrence treated by electrochemotherapy, we analyzed possible factors that could predict treatment failure. Based on histological analysis, low vascularization of the tumor was observed, which hampered drug delivery and distribution and resulted in no vascular disrupting action of electrochemotherapy. All these factors could contribute to ineffective treatment with electrochemotherapy.
Keywords: electrochemotherapy, bleomycin, vulvar cancer
Published in DiRROS: 25.07.2024; Views: 203; Downloads: 121
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25.
Newly isolated bacteriophages from the Podoviridae, Siphoviridae, and Myoviridae families have variable effects on putative novel Dickeya spp.
Špela Alič, Tina Mikuletič, Magda Tušek-Žnidarič, Maja Ravnikar, Nejc Rački, Matjaž Peterka, Tanja Dreo, 2017, original scientific article

Abstract: Soft rot pathogenic bacteria from the genus Dickeya cause severe economic losses in orchid nurseries worldwide, and there is no effective control currently available. In the last decade, the genus Dickeya has undergone multiple changes as multiple new taxa have been described, and just recently a new putative Dickeya species was reported. This study reports the isolation of three bacteriophages active against putative novel Dickeya spp. isolates from commercially produced infected orchids that show variable host-range profiles. Bacteriophages were isolated through enrichment from Dickeya-infected orchid tissue. Convective interaction media monolith chromatography was used to isolate bacteriophages from wastewaters, demonstrating its suitability for the isolation of infective bacteriophages from natural sources. Based on bacteriophage morphology, all isolated bacteriophages were classified as being in the order Caudovirales, belonging to three different families, Podoviridae, Myoviridae, and Siphoviridae. The presence of three different groups of bacteriophages was confirmed by analyzing the bacteriophage specificity of bacterial hosts, restriction fragment length polymorphism and plaque morphology. Bacteriophage BF25/12, the first reported Podoviridae bacteriophage effective against Dickeya spp., was selected for further characterization. Its genome sequence determined by next-generation sequencing showed limited similarity to other characterized Podoviridae bacteriophages. Interactions among the bacteriophages and Dickeya spp. were examined using transmission electron microscopy, which revealed degradation of electron-dense granules in response to bacteriophage infection in some Dickeya strains. The temperature stability of the chosen Podoviridae bacteriophage monitored over 1 year showed a substantial decrease in the survival of bacteriophages stored at -20∘C over longer periods. It showed susceptibility to low pH and UV radiation but was stable in neutral and alkaline pH. Furthermore, the stability of the tested bacteriophage was also connected to the incubation medium and bacteriophage concentration at certain pH values. Finally, the emergence of bacteriophage-resistant bacterial colonies is highly connected to the concentration of bacteriophages in the bacterial environment. This is the first report on bacteriophages against Dickeya from the Podoviridae family to expand on potential bacteriophages to include in bacteriophage cocktails as biocontrol agents. Some of these bacteriophage isolates also showed activity against Dickeya solani, an aggressive strain that causes the soft rot of potatoes, which indicates their broad potential as biocontrol agents.
Keywords: bacteriophages, genome sequencing, resistance development, convective interaction media monolith chromatography
Published in DiRROS: 25.07.2024; Views: 234; Downloads: 177
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26.
Droplet volume variability as a critical factor for accuracy of absolute quantification using droplet digital PCR
Alexandra Bogožalec Košir, Carla Divieto, Jernej Pavšič, Stefano Pavarelli, David Dobnik, Tanja Dreo, Roberto Bellotti, Maria Paola Sassi, Jana Žel, 2017, original scientific article

Abstract: Accurate and precise nucleic-acid quantification is crucial for clinical and diagnostic decisions, as overestimation or underestimation can lead to misguided treatment of a disease or incorrect labelling of the products. Digital PCR is one of the best tools for absolute nucleic-acid copy-number determination. However, digital PCR needs to be well characterised in terms of accuracy and sources of uncertainty. With droplet digital PCR, discrepancies between the droplet volume assigned by the manufacturer and measured by independent laboratories have already been shown in previous studies. In the present study, we report on the results of an inter-laboratory comparison of different methods for droplet volume determination that is based on optical microscopy imaging and is traceable to the International System of Units. This comparison was conducted on the same DNA material, with the examination of the influence of parameters such as droplet generators, supermixes, operators, inter-cartridge and intra-cartridge variability, and droplet measuring protocol. The mean droplet volume was measured using a QX200™ AutoDG™ Droplet Digital™ PCR system and two QX100™ Droplet Digital™ PCR systems. The data show significant volume differences between these two systems, as well as significant differences in volume when different supermixes are used. We also show that both of these droplet generator systems produce droplets with significantly lower droplet volumes (13.1%, 15.9%, respectively) than stated by the manufacturer and previously measured by other laboratories. This indicates that to ensure precise quantification, the droplet volumes should be assessed for each system.
Keywords: droplet digital PCR, droplet volume, DNA quantification, optical microscopy imaging
Published in DiRROS: 25.07.2024; Views: 221; Downloads: 182
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27.
Real-life long-term outcomes of upfront surgery in patients with resectable stage I-IIIA non-small cell lung cancer
Marko Bitenc, Tanja Čufer, Izidor Kern, Martina Miklavčič, Sabrina Petrovič, Vida Groznik, Aleksander Sadikov, 2022, original scientific article

Abstract: Treatment of early-stage non-small cell lung cancer (NSCLC) is rapidly evolving. When introducing novelties, real-life data on effectiveness of currently used treatment strategies are needed. The present study evaluated outcomes of stage I–IIIA NSCLC patients treated with upfront radical surgery in everyday clinical practice, between 2010–2017. Data of 539 consecutive patients were retrieved from a prospective hospital-based registry. All diagnostic, treatment and follow-up procedures were performed at the same thoracic oncology centre according to the valid guidelines. The primary outcome was overall survival (OS) analysed by clinical(c) and pathological(p) TNM (tumour, node, metastases) stage. The impact of clinicopathological characteristics on OS was evaluated using univariable (UVA) and multivariable regression analysis (MVA). With a median follow-up of 53.9 months, median OS and 5-year OS rate in the overall population were 90.4 months and 64.4%. Five-year OS rates by pTNM stage I, II and IIIA were 70.2%, 60.21%, and 49.9%, respectively. Both cTNM and pTNM stages were associated with OS; but only pTNM retained its independent prognostic value (p = 0.003) in MVA. Agreement between cTNM and pTNM was 69.0%. Next to pTNM, age (p = 0.001) and gender (p = 0.004) retained their independent prognostic value for OS. The study showed favourable outcomes of resectable stage I–IIIA NSCLC treated with upfront surgery in real-life. Relatively low agreement between cTNM and pTNM stages and independent prognostic value of only pTNM, observed in real-life data, suggest that surgery remains the most accurate provider of the anatomical stage of disease and important upfront therapy.
Keywords: resectable NSCLC, upfront surgery, real-life data, overall survival, prognostic factors
Published in DiRROS: 25.07.2024; Views: 199; Downloads: 164
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28.
1-aminocyclopropane-1-carboxylate oxidase induction in tomato flower pedicel phloem and abscission related processes are differentially sensitive to ethylene
Marko Chersicola, Aleš Kladnik, Magda Tušek-Žnidarič, Tanja Mrak, Kristina Gruden, Marina Dermastia, 2017, original scientific article

Abstract: Ethylene has impact on several physiological plant processes, including abscission, during which plants shed both their vegetative and reproductive organs. Cell separation and programmed cell death are involved in abscission, and these have also been correlated with ethylene action. However, the detailed spatiotemporal pattern of the molecular events during abscission remains unknown. We examined the expression of two tomato ACO genes, LeACO1, and LeACO4 that encode the last enzyme in ethylene biosynthesis, 1-aminocyclopropane-1-carboxylate oxidase (ACO), together with the expression of other abscission-associated genes involved in cell separation and programmed cell death, during a period of 0–12 h after abscission induction in the tomato flower pedicel abscission zone and nearby tissues. In addition, we determined their localization in specific cell layers of the flower pedicel abscission zone and nearby tissues obtained by laser microdissection before and 8 h after abscission induction. The expression of both ACO genes was localized to the vascular tissues in the pedicel. While LeACO4 was more uniformly expressed in all examined cell layers, the main expression site of LeACO1 was in cell layers just outside the abscission zone in its proximal and distal part. We showed that after abscission induction, ACO1 protein was synthesized in phloem companion cells, in which it was localized mainly in the cytoplasm. Samples were additionally treated with 1-methylcyclopropene (1-MCP), a competitive inhibitor of ethylene actions, and analyzed 8 h after abscission induction. Cell-layer-specific changes in gene expression were observed together with the specific localization and ethylene sensitivity of the hallmarks of cell separation and programmed cell death. While treatment with 1-MCP prevented separation of cells through inhibition of the expression of polygalacturonases, which are the key enzymes involved in degradation of the middle lamella, this had less impact on the occurrence of different kinds of membrane vesicles and abscission-related programmed cell death. In the flower pedicel abscission zone, the physical progressions of cell separation and programmed cell death are perpendicular to each other and start in the vascular tissues.
Keywords: abscission, ACO, cell separation, ethylene, laser microdissection, programmed cell death, tomato, ultrastructure
Published in DiRROS: 24.07.2024; Views: 196; Downloads: 249
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29.
Testing the influence of sediment granulometry on heterotrophic respiration with a new laboratory flow-through system
Nataša Mori, Barbara Debeljak, David Kocman, Tatjana Simčič, 2017, original scientific article

Abstract: Purpose Increased sedimentation due to land use intensification is increasingly affecting carbon processing in streams and rivers around the globe. This study describes the design of a laboratory-scale flow-through incubation system as a tool for the rapid estimation of sediment respiration. The measurements were compared with those obtained using an in situ closed chamber respiration method. The influence of sediment size on respiration rates was also investigated. Materials and methods Measurements were conducted on a pre-alpine gravel-bed river sediment separated into the following grain size fractions: > 60 mm (14.3%), 60–5 mm (60.2%), 5–2 mm (13.7%), 2–0.063 mm (11.1%) and <0.063 mm (0.6%). Concurrently, in situ and laboratory measurements were carried out on a naturally heterogeneous sediment. In situ respiration was determined in closed chambers as O2 consumption over time, while in the laboratory, respiration was determined using flow-through respiration chambers. Oxygen concentrations were measured using a fibre-optic oxygen meter positioned at the inflow and outflow from the chamber. Results and discussion The mean respiration rates within naturally mixed riverbed sediments were 1.27 ± 0.3 mg O2 dm−3 h−1 (n = 4) and 0.77 ± 0.1 mg O2 dm−3 h−1 (n = 3) for the flow-through chamber system and closed chamber system, respectively. Respiration rates were statistically significantly higher in the flow-through chamber system (t test, p < 0.05), indicating that closed chamber measurements underestimated the oxygen consumption within riverbed sediments. Sediment grain size was found to significantly affect respiration rates in both systems (ANOVA, p < 0.001) with the fine sediment fraction (particle size <0.063 mm) having the highest respiration rate (rflow-through = 51 ± 23 mg O2 dm−3 h−1). The smallest fractions (2–0.063 and <0.063 mm), which represent approximately 12% of total sediment volume, contributed 60% of total respiration. Conclusions The study demonstrated that flow-through respiration chambers more accurately estimate the respiration rate within riverbed sediments than in situ closed chambers, since the former experiment imitates the natural conditions where continuous interstitial flow occurs in the sediments. We also demonstrated that fine sediments (<5 mm) substantially contribute to heterotrophic respiration in the studied gravel-bed river.
Keywords: carbon fluxes, freshwaters, geomorphology, hyporheic zone, respiration, sediments
Published in DiRROS: 24.07.2024; Views: 205; Downloads: 147
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30.
Treatment of skin tumors with intratumoral interleukin 12 gene electrotransfer in the head and neck region : a first-in-human clinical trial protocol
Aleš Grošelj, Maša Omerzel, Tanja Jesenko, Maja Čemažar, Boštjan Markelc, Primož Strojan, Gregor Serša, 2022, original scientific article

Abstract: Immune therapies are currently under intensive investigation providing in many cases excellent re-sponses in different tumors. Other possible approach for immunotherapy is a targeted intratumoral delivery of inter-leukin 12 (IL-12), a cytokine with anti-tumor effectiveness. Due to its immunomodulatory action, it can be used as an imunostimulating component to in situ vaccinating effect of local ablative therapies. We have developed a phIL12 plasmid devoid of antibiotic resistance marker with a transgene for human IL-12 p70 protein. The plasmid can be delivered intratumorally by gene electrotransfer (GET). Patients and methods. Here we present a first-in-human clinical trial protocol for phIL12 GET (ISRCTN15479959, ClinicalTrials NCT05077033). The study is aimed at evaluating the safety and tolerability of phIL12 GET in treatment of basal cell carcinomas in patients with operable tumors in the head and neck region. The study is designed as an ex-ploratory, dose escalating study with the aim to determine the safety and tolerability of the treatment and to identify the dose of plasmid phIL12 that is safe and elicits its biological activity. Conclusions. The results of this trail protocol will therefore provide the basis for the use of phIL12 GET as an adjuvant treatment to local ablative therapies, to potentially increase their local and elicit a systemic response.
Keywords: skin tumors, gene electrotransfer, interleukin 12, clinical trial
Published in DiRROS: 24.07.2024; Views: 199; Downloads: 72
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