1. Gene expression profiling of recombinant protein producing E. coli at suboptimal growth temperatureMitja Mahnič, Špela Baebler, Andrej Blejec, Špela Jalen, Kristina Gruden, Viktor Menart, Simona Jevševar, 2012, izvirni znanstveni članek Povzetek: Recent studies have revealed that at lower cultivation temperatures (25 °C) much higher percentage of correctly folded recombinant hG-CSF protein can be extracted from inclusion bodies. Hence, the goal of our research was to investigate mechanisms determining characteristics of non-classical inclusion bodies production using gene expression profiling, focusing on proteases and chaperones gene expression. Statistical analysis of microarray data showed prominent changes in energy metabolism, in metabolism of amino acids and nucleotides, as well as in biosynthesis of cofactors and secondary metabolites if the culture was grown below its optimal temperature. Moreover, 24 differentially expressed up to now known genes classified among proteases, chaperones and other heat or stress related genes. Among chaperones UspE and among proteases YaeL and YeaZ might play an important role in accumulation of correctly folded recombinant proteins. Membrane localized protease yaeL gene was found to have higher activity at 25 °C and is thus potentially functionally related to the more efficient recombinant protein production at lower temperatures. The results of this study represent advance in the understanding of recombinant protein production in E. coli. Genes potentially influencing production of recombinant protein at lower growth temperature represent basis for further research towards improvement of E. coli production strains as well as fermentation process.
Ključne besede: recombinant protein production, non-classical inclusion bodies, expression microarrays, YaeL protease, GroEL chaperone
Objavljeno v DiRROS: 05.08.2024; Ogledov: 221; Prenosov: 207
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2. Identification of plasma biomarker candidates in glioblastoma using an antibody-array-based proteomic approachKlemen Zupančič, Marjan Koršič, Urška Verbovšek, Primož Rožman, Tamara Lah Turnšek, Andrej Blejec, Kristina Gruden, Helena Motaln, Miomir Knežević, Matija Veber, Ana Herman, 2014, izvirni znanstveni članek Povzetek: Background. Glioblastoma multiforme (GBM) is a brain tumour with a very high patient mortality rate, with a median survival of 47 weeks. This might be improved by the identification of novel diagnostic, prognostic and predictive
therapy-response biomarkers, preferentially through the monitoring of the patient blood. The aim of this study was to define the impact of GBM in terms of alterations of the plasma protein levels in these patients.
Materials and methods. We used a commercially available antibody array that includes 656 antibodies to analyse
blood plasma samples from 17 healthy volunteers in comparison with 17 blood plasma samples from patients with
GBM.
Results. We identified 11 plasma proteins that are statistically most strongly associated with the presence of GBM.
These proteins belong to three functional signalling pathways: T-cell signalling and immune responses; cell adhesion and migration; and cell-cycle control and apoptosis. Thus, we can consider this identified set of proteins as potential diagnostic biomarker candidates for GBM. In addition, a set of 16 plasma proteins were significantly associated with the overall survival of these patients with GBM. Guanine nucleotide binding protein alpha (GNAO1) was associated with both GBM presence and survival of patients with GBM.
Conclusions. Antibody array analysis represents a useful tool for the screening of plasma samples for potential cancer biomarker candidates in small-scale exploratory experiments; however, clinical validation of these candidates requires their further evaluation in a larger study on an independent cohort of patients.
Ključne besede: glioblastoma, proteomics, biomarker
Objavljeno v DiRROS: 02.08.2024; Ogledov: 134; Prenosov: 106
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3. Reverse transcriptase droplet digital PCR shows high resilience to PCR inhibitors from plant, soil and water samplesNejc Rački, Tanja Dreo, Ion Gutiérrez-Aguirre, Andrej Blejec, Maja Ravnikar, 2014, izvirni znanstveni članek Povzetek: Background
Detection and quantification of plant pathogens in the presence of inhibitory substances can be a challenge especially with plant and environmental samples. Real-time quantitative PCR has enabled high-throughput detection and quantification of pathogens; however, its quantitative use is linked to standardized reference materials, and its sensitivity to inhibitors can lead to lower quantification accuracy. Droplet digital PCR has been proposed as a method to overcome these drawbacks. Its absolute quantification does not rely on standards and its tolerance to inhibitors has been demonstrated mostly in clinical samples. Such features would be of great use in agricultural and environmental fields, therefore our study compared the performance of droplet digital PCR method when challenged with inhibitors common to plant and environmental samples and compared it with quantitative PCR.
Results
Transfer of an existing Pepper mild mottle virus assay from reverse transcription real-time quantitative PCR to reverse transcription droplet digital PCR was straight forward. When challenged with complex matrices (seeds, plants, soil, wastewater) and selected purified inhibitors droplet digital PCR showed higher resilience to inhibition for the quantification of an RNA virus (Pepper mild mottle virus), compared to reverse transcription real-time quantitative PCR.
Conclusions
This study confirms the improved detection and quantification of the PMMoV RT-ddPCR in the presence of inhibitors that are commonly found in samples of seeds, plant material, soil, and wastewater. Together with absolute quantification, independent of standard reference materials, this makes droplet digital PCR a valuable tool for detection and quantification of pathogens in inhibition prone samples.
Ključne besede: PCR amplification, inhibition, qPCR, droplet digital PCR, environmental samples
Objavljeno v DiRROS: 02.08.2024; Ogledov: 103; Prenosov: 74
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4. Transcriptome study and identification of potential marker genes related to the stable expression of recombinant proteins in CHO clonesAndrej Blejec, Marjanca Blas, Petra Nikolić, Dominik Gaser, Kristina Gruden, Aleš Belič, Špela Baebler, Uroš Jamnikar, Andrej Francky, Holger Laux, 2015, izvirni znanstveni članek Povzetek: Background
Chinese hamster ovary (CHO) cells have become the host of choice for the production of recombinant proteins, due to their capacity for correct protein folding, assembly, and posttranslational modifications. The most widely used system for recombinant proteins is the gene amplification procedure that uses the CHO-Dhfr expression system. However, CHO cells are known to have a very unstable karyotype. This is due to chromosome rearrangements that can arise from translocations and homologous recombination, especially when cells with the CHO-Dhfr expression system are treated with methotrexate hydrate. The present method used in the industry for testing clones for their long-term stability of recombinant protein production is empirical, and it involves their cultivation over extended periods of time prior to the selection of the most suitable clone for further bioprocess development. The aim of the present study was the identification of marker genes that can predict stable expression of recombinant genes in particular clones early in the development stage.
Results
The transcriptome profiles of CHO clones with stable and unstable recombinant protein production were investigated over 10-weeks of cultivation, using a DNA microarray. We identified 14 genes that were differentially expressed between the stable and unstable clones already at 2 weeks from the beginning of the cultivation. Their expression was validated by reverse-transcription quantitative real-time PCR (RT-qPCR). Furthermore, the k-nearest neighbour algorithm approach shows that the combination of the gene expression patterns of only five of these 14 genes is sufficient to predict stable recombinant protein production in clones in the early phases of cell-line development.
Conclusions
The exact molecular mechanisms that cause unstable recombinant protein production are not fully understood. However, the expression profiles of some genes in clones with stable and unstable recombinant protein production allow prediction of such instability early in the cell-line development stage. We have thus developed a proof-of-concept for a novel approach to eliminate unstable clones in the CHO-Dhfr expression system, which saves time and labour-intensive work in cell-line development.
Ključne besede: CHO cell line, stable recombinant protein production, gene expression, RT-qPCR, DNA microarray, mMarker genes
Objavljeno v DiRROS: 29.07.2024; Ogledov: 132; Prenosov: 232
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5. The effect of timing of female vibrational reply on male signalling and searching behaviour in the leafhopper Aphrodes makaroviMeta Virant-Doberlet, Maarten De Groot, Andrej Blejec, Ana Kuhelj, 2015, izvirni znanstveni članek Povzetek: Sexual communication in animals often involves duetting characterized by a coordinated reciprocal exchange of acoustic signals. We used playback experiments to study the role of timing of a female reply in the species-specific duet structure in the leafhopper Aphrodes makarovi (Hemiptera: Cicadellidae). In leafhoppers, mate recognition and location is mediated exclusively by species- and sex-specific substrate-borne vibrational signals and a female signal emitted in reply to male advertisement calls is essential for recognition and successful location of the female. In A. makarovi, males have to initiate each exchange of vibrational signals between partners, and in a duet the beginning of a female reply overlaps the end of the male advertisement call. Results of playback treatments in which female replies were delayed and did not overlap with the male call revealed that in order to trigger an appropriate behavioural response of the male, female reply has to appear in a period less than 400 ms after the end of the initiating male call. Results also suggest that males are not able to detect a female reply while calling, since female reply that did not continue after the end of male call triggered male behaviour similar to behaviour observed in the absence of female reply. Together, our results show that vibrational duets are tightly coordinated and that the species-specific duet structure plays an important role in mate recognition in location processes.
Ključne besede: vibration, insects, bioacoustics, animal signaling and communication
Objavljeno v DiRROS: 29.07.2024; Ogledov: 115; Prenosov: 76
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6. Bimodal dynamics of primary metabolism-related responses in tolerant potato-Potato virus Y interactionNeža Turnšek, Živa Ramšak, Katja Stare, Tjaša Stare, Dominik Vodnik, Andrej Blejec, Kristina Gruden, Wolfram Weckwerth, Stefanie Wienkoop, 2015, izvirni znanstveni članek Povzetek: Background
Potato virus Y (PVY) is a major pathogen that causes substantial economic losses in worldwide potato production. Different potato cultivars differ in resistance to PVY, from severe susceptibility, through tolerance, to complete resistance. The aim of this study was to better define the mechanisms underlying tolerant responses of potato to infection by the particularly aggressive PVYNTN strain. We focused on the dynamics of the primary metabolism-related processes during PVYNTN infection.
Results
A comprehensive analysis of the dynamic changes in primary metabolism was performed, which included whole transcriptome analysis, nontargeted proteomics, and photosynthetic activity measurements in potato cv. Désirée and its transgenic counterpart depleted for accumulation of salicylic acid (NahG-Désirée). Faster multiplication of virus occurred in the NahG-Désirée, with these plants developing strong disease symptoms. We show that while the dynamics of responses at the transcriptional level are extensive and bimodal, this is only partially translated to the protein level, and to the final functional outcome. Photosynthesis-related genes are transiently induced before viral multiplication is detected and it is down-regulated later on. This is reflected as a deficiency of the photosynthetic apparatus at the onset of viral multiplication only. Interestingly, specific and constant up-regulation of some RuBisCO transcripts was detected in Désirée plants, which might be important, as these proteins have been shown to interact with viral proteins.
In SA-deficient and more sensitive NahG-Désirée plants, consistent down-regulation of photosynthesis-related genes was detected. A constant reduction in the photochemical efficiency from the onset of viral multiplication was identified; in nontransgenic plants this decrease was only transient. The transient reduction in net photosynthetic rate occurred in both genotypes with the same timing, and coincided with changes in stomatal conductivity.
Conclusions
Down-regulation of photosynthesis-related gene expression and decreased photosynthetic activity is in line with other studies that have reported the effects of biotic stress on photosynthesis. Here, we additionally detected induction of light-reaction components in the early stages of PVYNTN infection of tolerant interaction. As some of these components have already been shown to interact with viral proteins, their overproduction might contribute to the absence of symptoms in cv. Désirée.
Ključne besede: plant-pathogen interactions, Potato virus Y, potyviridae, salicylic acid, whole transcriptome analysis, shot-gun proteomics, photosynthetic parameters
Objavljeno v DiRROS: 29.07.2024; Ogledov: 121; Prenosov: 149
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7. Analysis of glioblastoma patients' plasma revealed the presence of microRNAs with a prognostic impact on survival and those of viral originKlemen Zupančič, Helena Motaln, Miomir Knežević, Urška Verbovšek, Marjan Koršič, Tamara Lah Turnšek, Primož Rožman, Matjaž Jeras, Matjaž Hren, Kristina Gruden, Andrej Blejec, Matija Veber, Ana Herman, Andrej Porčnik, Vid Podpečan, 2015, izvirni znanstveni članek Povzetek: Background
Glioblastoma multiforme (GBM) is among the most aggressive cancers with a poor prognosis in spite of a plethora of established diagnostic and prognostic biomarkers and treatment modalities. Therefore, the current goal is the detection of novel biomarkers, possibly detectable in the blood of GBM patients that may enable an early diagnosis and are potential therapeutic targets, leading to more efficient interventions.
Experimental Procedures
MicroRNA profiling of 734 human and human-associated viral miRNAs was performed on blood plasma samples from 16 healthy individuals and 16 patients with GBM, using the nCounter miRNA Expression Assay Kits.
Results
We identified 19 miRNAs with significantly different plasma levels in GBM patients, compared to the healthy individuals group with the difference limited by a factor of 2. Additionally, 11 viral miRNAs were found differentially expressed in plasma of GBM patients and 24 miRNA levels significantly correlated with the patients’ survival. Moreover, the overlap between the group of candidate miRNAs for diagnostic biomarkers and the group of miRNAs associated with survival, consisted of ten miRNAs, showing both diagnostic and prognostic potential. Among them, hsa miR 592 and hsa miR 514a 3p have not been previously described in GBM and represent novel candidates for selective biomarkers. The possible signalling, induced by the revealed miRNAs is discussed, including those of viral origin, and in particular those related to the impaired immune response in the progression of GBM.
Conclusion
The GBM burden is reflected in the alteration of the plasma miRNAs pattern, including viral miRNAs, representing the potential for future clinical application. Therefore proposed biomarker candidate miRNAs should be validated in a larger study of an independent cohort of patients
Ključne besede: microRNAs, glioblastoma multiforme, biomarkers, RNA extraction, viral disease diagnosis
Objavljeno v DiRROS: 26.07.2024; Ogledov: 159; Prenosov: 70
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8. Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detectionDavid Dobnik, Dejan Štebih, Andrej Blejec, Dany Morisset, Jana Žel, 2016, izvirni znanstveni članek Povzetek: The advantages of the digital PCR technology are already well documented until now. One way to achieve better cost efficiency of the technique is to use it in a multiplexing strategy. Droplet digital PCR platforms, which include two fluorescence filters, support at least duplex reactions and with some developments and optimization higher multiplexing is possible. The present study not only shows a development of multiplex assays in droplet digital PCR, but also presents a first thorough evaluation of several parameters in such multiplex digital PCR. Two 4-plex assays were developed for quantification of 8 different DNA targets (7 genetically modified maize events and maize endogene). Per assay, two of the targets were labelled with one fluorophore and two with another. As current analysis software does not support analysis of more than duplex, a new R- and Shiny-based web application analysis tool (http://bit.ly/ddPCRmulti) was developed that automates the analysis of 4-plex results. In conclusion, the two developed multiplex assays are suitable for quantification of GMO maize events and the same approach can be used in any other field with a need for accurate and reliable quantification of multiple DNA targets.
Ključne besede: digital PCR, DNA targets, GMO detection
Objavljeno v DiRROS: 25.07.2024; Ogledov: 155; Prenosov: 96
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9. Inter-laboratory assessment of different digital PCR platforms for quantification of human cytomegalovirus DNAJernej Pavšič, Alison S. Devonshire, Andrej Blejec, Carole A. Foy, Fran Van Heuverswyn, Gerwyn M. Jones, Heinz Schimmel, Jana Žel, Jim F. Huggett, Nicholas Redshaw, Maria Karczmarczyk, Erkan Mozioglu, Sema Akyürek, Müslüm Akgöz, Mojca Milavec, 2017, izvirni znanstveni članek Povzetek: Quantitative PCR (qPCR) is an important tool in pathogen detection. However, the use of different qPCR components, calibration materials and DNA extraction methods reduces comparability between laboratories, which can result in false diagnosis and discrepancies in patient care. The wider establishment of a metrological framework for nucleic acid tests could improve the degree of standardisation of pathogen detection and the quantification methods applied in the clinical context. To achieve this, accurate methods need to be developed and implemented as reference measurement procedures, and to facilitate characterisation of suitable certified reference materials. Digital PCR (dPCR) has already been used for pathogen quantification by analysing nucleic acids. Although dPCR has the potential to provide robust and accurate quantification of nucleic acids, further assessment of its actual performance characteristics is needed before it can be implemented in a metrological framework, and to allow adequate estimation of measurement uncertainties. Here, four laboratories demonstrated reproducibility (expanded measurement uncertainties below 15%) of dPCR for quantification of DNA from human cytomegalovirus, with no calibration to a common reference material. Using whole-virus material and extracted DNA, an intermediate precision (coefficients of variation below 25%) between three consecutive experiments was noted. Furthermore, discrepancies in estimated mean DNA copy number concentrations between laboratories were less than twofold, with DNA extraction as the main source of variability. These data demonstrate that dPCR offers a repeatable and reproducible method for quantification of viral DNA, and due to its satisfactory performance should be considered as candidate for reference methods for implementation in a metrological framework.
Ključne besede: digital PCR, DNA quantification, inter-laboratory assessment, human cytomegalovirus, virus reference materials
Objavljeno v DiRROS: 24.07.2024; Ogledov: 120; Prenosov: 100
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10. Localization patterns of cathepsins K and X and their predictive value in glioblastomaBarbara Breznik, Clara Limbaeck Stanic, Andrej Porčnik, Andrej Blejec, Miha Koprivnikar Krajnc, Roman Bošnjak, Janko Kos, Cornelis J. F. van Noorden, Tamara Lah Turnšek, 2018, izvirni znanstveni članek Povzetek: Background
Glioblastoma is a highly aggressive central nervous system neoplasm characterized by extensive infiltration of malignant cells into brain parenchyma, thus preventing complete tumor eradication. Cysteine cathepsins B, S, L and K are involved in cancer progression and are overexpressed in glioblastoma. We report here for the first time that cathepsin X mRNA and protein are also abundantly present in malignant glioma.
Materials and methods
Gene expression of cathepsins K and X was analyzed using publically-available tran-scriptomic datasets and correlated with glioma grade and glioblastoma subtype. Kaplan-Maier survival analysis was performed to evaluate the predictive value of cathepsin K and X mRNA expression. Cathepsin protein expression was localized and semi-quantified in tumor tissues by immunohistochemistry.
Results
Highest gene expression of cathepsins K and X was found in glioblastoma, in particular in the mesenchymal subtype. Overall, high mRNA expression of cathepsin X, but not that of cathepsin K, correlated with poor patients’ survival. Cathepsin K and X proteins were abundantly and heterogeneously expressed in glioblastoma tissue. Immuno-labeling of cathepsins K and X was observed in areas of CD133-positive glioblastoma stem cells, localized around arterioles in their niches that also expressed SDF-1α and CD68. mRNA levels of both cathepsins K and X correlated with mRNA levels of markers of glioblastoma stem cells and their niches.
Conclusions
The presence of both cathepsins in glioblastoma stem cell niche regions indicates their possible role in regulation of glioblastoma stem cell homing in their niches. The clinical relevance of this data needs to be elaborated in further prospective studies.
Ključne besede: cathepsins, glioblastoma, immunohistochemistry, patient survival, cancer stem cell niches
Objavljeno v DiRROS: 24.07.2024; Ogledov: 145; Prenosov: 105
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