1. Stabilization of fish protein‐based adhesive by reduction of its hygroscopicityBranka Mušič, Jaka Gašper Pečnik, Andreja Pondelak, 2024, original scientific article Abstract: Protein-based fish adhesives have historically been used in various bonding applications; however, due to the protein’s high affinity for water absorption, these adhesives become destabilized in high-moisture environments, resulting in reduced bondline strength and early failure. This limitation makes them unsuitable for industrial applications with higher demands. To address this issue, water-insoluble raw powder materials such as iron, copper, or zeolite were incorporated into natural fish adhesives. In this study, the hygroscopicity, dry matter content, thermal analysis (TGA/DSC), FT-IR spectroscopy, surface tension measurements, vapour permeability, and scanning electron microscope (SEM) of the modified adhesives were determined. In addition, the bonding properties of the modified adhesives were evaluated by the tensile shear strength of the lap joints, and mould growth was visually inspected. The resulting modified protein-based adhesives demonstrated improved stability in high humidity environments. Enhancing the hygroscopic properties of protein-based fish adhesives has the potential to unlock new opportunities and applications, providing a healthier and more environmentally sustainable alternative to petroleum-based adhesives. Keywords: protein‐based adhesive, polymer stabilization, hygroscopicity, fish adhesive modification, fish industry waste, circular economy, bonding properties Published in DiRROS: 12.08.2024; Views: 66; Downloads: 215
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2. The role of plasmalemma vesicle-associated protein in pathological breakdown of blood-brain and blood-retinal barriers : potential novel therapeutic target for cerebral edema and diabetic macular edemaEsmeralda K. Bosma, Cornelis J. F. van Noorden, Reinier O. Schlingemann, Ingeborg Klaassen, 2018, review article Abstract: Breakdown of the blood–brain barrier (BBB) or inner blood–retinal barrier (BRB), induced by pathologically elevated levels of vascular endothelial growth factor (VEGF) or other mediators, can lead to vasogenic edema and significant clinical problems such as neuronal morbidity and mortality, or vision loss. Restoration of the barrier function with corticosteroids in the brain, or by blocking VEGF in the eye are currently the predominant treatment options for brain edema and diabetic macular edema, respectively. However, corticosteroids have side effects, and VEGF has important neuroprotective, vascular protective and wound healing functions, implying that long-term anti-VEGF therapy may also induce adverse effects. We postulate that targeting downstream effector proteins of VEGF and other mediators that are directly involved in the regulation of BBB and BRB integrity provide more attractive and safer treatment options for vasogenic cerebral edema and diabetic macular edema. The endothelial cell-specific protein plasmalemma vesicle-associated protein (PLVAP), a protein associated with trans-endothelial transport, emerges as candidate for this approach. PLVAP is expressed in a subset of endothelial cells throughout the body where it forms the diaphragms of caveolae, fenestrae and trans-endothelial channels. However, PLVAP expression in brain and eye barrier endothelia only occurs in pathological conditions associated with a compromised barrier function such as cancer, ischemic stroke and diabetic retinopathy. Here, we discuss the current understanding of PLVAP as a structural component of endothelial cells and regulator of vascular permeability in health and central nervous system disease. Besides providing a perspective on PLVAP identification, structure and function, and the regulatory processes involved, we also explore its potential as a novel therapeutic target for vasogenic cerebral edema and retinal macular edema. Keywords: plasmalemma vesicle-associated protein, blood-brain barrier, blood-retinal barrier, cerebral edema, diabetic macular edema Published in DiRROS: 06.08.2024; Views: 80; Downloads: 57
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3. 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, original scientific article Abstract: 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. Keywords: recombinant protein production, non-classical inclusion bodies, expression microarrays, YaeL protease, GroEL chaperone Published in DiRROS: 05.08.2024; Views: 222; Downloads: 208
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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, original scientific article Abstract: 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. Keywords: CHO cell line, stable recombinant protein production, gene expression, RT-qPCR, DNA microarray, mMarker genes Published in DiRROS: 29.07.2024; Views: 133; Downloads: 234
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5. Learning deep representations of enzyme thermal adaptationGang Li, Filip Buric, Jan Zrimec, Sandra Viknander, Jens Nielsen, Aleksej Zelezniak, Martin K. M. Engqvist, 2022, original scientific article Abstract: Temperature is a fundamental environmental factor that shapes the evolution of organisms. Learning thermal determinants of protein sequences in evolution thus has profound significance for basic biology, drug discovery, and protein engineering. Here, we use a data set of over 3 million BRENDA enzymes labeled with optimal growth temperatures (OGTs) of their source organisms to train a deep neural network model (DeepET). The protein-temperature representations learned by DeepET provide a temperature-related statistical summary of protein sequences and capture structural properties that affect thermal stability. For prediction of enzyme optimal catalytic temperatures and protein melting temperatures via a transfer learning approach, our DeepET model outperforms classical regression models trained on rationally designed features and other deep-learning-based representations. DeepET thus holds promise for understanding enzyme thermal adaptation and guiding the engineering of thermostable enzymes. Keywords: bioinformatics, deep neural networks, enzyme catalytic temperatures, optimal growth temperatures, protein thermostability, transfer learning Published in DiRROS: 17.07.2024; Views: 152; Downloads: 125
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6. Subunits of an E3 ligase complex as degrons for efficient degradation of cytosolic, nuclear, and membrane proteinsAnže Verbič, Tina Lebar, Arne Praznik, Roman Jerala, 2024, original scientific article Keywords: synthetic biology, degrons, control of protein expression, E3 ligase Published in DiRROS: 05.07.2024; Views: 187; Downloads: 148
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7. Rotational dynamics of a protein under shear flow studied by the eckart frame formalismPetra Papež, Franci Merzel, Matej Praprotnik, 2023, original scientific article Keywords: energy, kinetics, molecules, protein structure, rotational dynamics, molecules, protein structure, rotational dynamics Published in DiRROS: 06.09.2023; Views: 495; Downloads: 260
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8. Analysis of putative apoplastic effectors from the nematode, Globodera rostochiensis, and identification of an expansin-like protein that can induce and suppress host defensesBarbara Gerič Stare, Shawkat Ali, Maxime Magne, Shiyan Chen, Olivier Côte, Natasa Obradovic, Lubna Jamshaid, Xiaohong Wang, Guy Bélair, Peter Moffett, 2015, original scientific article Keywords: nematode, expansin-like protein, expansin, potato cyst nematode, potato pest, parasitic nematodes, plant celular function Published in DiRROS: 06.09.2022; Views: 674; Downloads: 250
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9. Fractional heat shock protein 27 urine excretion as a short-term predictor in acute exacerbation of chronic obstructive pulmonary diseaseDenise Traxler, Matthias Zimmermann, Elisabeth Simader, Elisa Einwallner, Dragan Copic, Alexandra Graf, Thomas Mueller, Cecilia Veraar, Mitja Lainščak, Robert Marčun, Mitja Košnik, Matjaž Fležar, Aleš Rozman, Peter Korošec, 2020, original scientific article Abstract: Background: Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality and is characterized by episodes of acute exacerbations. Finding a systemic biomarker that reliably predicts outcome after an acute exacerbation remains a major challenge. Heat shock protein 27 (HSP27) has been previously studied in COPD, however, urine excretion trajectory and prognostic value after an exacerbation is unknown. Methods: In this retrospective post hoc analysis of a prospective study that included 253 COPD patients who were hospitalized for acute exacerbation, 207 patients were analyzed. Urine and serum were sampled at admission, discharge, and 180 days after discharge; urine excretion trajectory was analyzed and correlated with clinicopathological and survival data. Results: HSP27 urine excretion increased after an exacerbation episode [1.8% admission, 1.8% discharge, 2.3% 180 days after discharge (P=0.091)]. In severely ill patients (GOLD IV) this course was even more distinct [1.6% admission, 2.1% discharge, 2.8% 180 days after discharge (P=0.007)]. Furthermore, fractional HSP27 urine excretion at discharge was increased in GOLD IV patients (P=0.031). In Kaplan-Meier and univariable Cox proportional hazard models patients with HSP27 urine excretion below 0.845% showed significantly worse survival at 30, 90 and 180 days after discharge. In a multivariable Cox proportional hazard model including established COPD outcome parameters fractional HSP27 urine excretion remained a significant predictor of survival at 30 and 90 days after discharge. Comparing this model to our already published model that includes HSP27 serum concentration we could show that fractional HSP27 urine excretion performs better in short-term survival. Conclusions: Our findings provide novel information about fractional HSP27 urine excretion trajectory in acute exacerbation of COPD. Fractional HSP27 urine excretion may be significantly reduced during an episode of acute exacerbation in COPD patients and may be used as a predictor of short-term all-cause mortality. Keywords: biomarkers, heat-shock proteins, chronic obstructive pulmonary disease, urine, heat shock protein 27 Published in DiRROS: 25.01.2021; Views: 1643; Downloads: 1134
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10. Design of protein logic gate system operating on lipid membranesNeža Omersa, Saša Rezelj, Matic Kisovec, Marjetka Podobnik, Gregor Anderluh, 2020, original scientific article Keywords: pore-forming toxin, listeriolysin O, protein logic gates, lipid membrane, DARPin, pH Published in DiRROS: 25.11.2020; Views: 1768; Downloads: 975
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