Records export, transfer and ingest recommendations and SIP Creation Tools

This report describes a software deliverable as it delivers a number of E-ARK tools: • ERMS Export Module (a tool for exporting records and their metadata from ERMS in a controlled manner); • Database Preservation Toolkit (a tool for exporting relational databases as SIARD 2.0 or other formats); • ESSArch Tools for Producer (a tool for SIP creation); • ESSArch Tools for Archive (a tool for SIP ingestion); • RODA-in (a tool for SIP creation); • Universal Archiving Module (a tool for SIP creation). In addition, an overview of Pre-Ingest and Ingest processes will be provided by this report which will help to understand the tools and their use

Update of controller for automation of continuous casting system

Diplomsko delo obsega tako strojno nadgradnjo sistema za kontinuirno litje, kot tudi programsko nadgradnjo, narejeno po zahtevah Laboratorija za materiale na Fakulteti za strojništvo. Podrobneje je opisan spremenjen grafični vmesnik in logika, ki se skriva v odsekih kode nadzornega programa. Prav tako je opisan celotni postopek nadgraditve obstoječe strojne opreme do stopnje za namestitev in priklop na peč za kontinuirno litje. Posodobljen sistem je bil priklopljen na priključke starega sistema, testirano je bilo delovanje spremenjenega programa in simulacija kontinuirnega litja.The diploma thesis focuses on the hardware and software upgrade of the system for continuous casting in accordance with the Laboratory of materials of the Faculty of Mechanical Engineering. The thesis includes an in-depth analysis of the modified user interface and the logic behind sections of code of the monitoring program. We described the complete upgrade procedure of the existing hardware as well as the phase of installation and coupling to the casting furnace. At the final phase the upgraded system was coupled with the previous system. Furthermore, we tested the operation of the upgraded program and ran simulations of continuous casting

The Phospholipase Activity of Ammodytoxin, a Prototype Snake Venom β-Neurotoxin, Is Not Obligatory for Cell Internalisation and Translocation to Mitochondria

β-Neurotoxins are secreted phospholipase A2 molecules that inhibit transmission in neuromuscular synapses by poisoning the motor neurons. These toxins specifically and rapidly internalise into the nerve endings of motor neurons. Ammodytoxin (Atx) is a prototype β-neurotoxin from the venom of the nose-horned viper (Vipera ammodytes ammodytes). Here, we studied the relevance of the enzymatic activity of Atx in cell internalisation and subsequent intracellular movement using transmission electron microscopy (TEM). We prepared a recombinant, enzymatically inactive mutant of Atx, Atx(D49S), labelled with gold nanoparticles (GNP), and incubated this with PC12 cells, to analyse its localisation by TEM. Atx(D49S)-GNP internalised into the cells. Inside the cells, Atx(D49S)-GNP was detected in different vesicle-like structures, cytosol, endoplasmic reticulum and mitochondria, where it was spotted in the intermembrane space and matrix. Co-localization of fluorescently labelled Atx(D49S) with mitochondria in PC12 cells by confocal fluorescence microscopy confirmed the reliability of results generated using Atx(D49S)-GNP and TEM and allowed us to conclude that the phospholipase activity of Atx is not obligatory for its cell internalisation and translocation into the mitochondrial intermembrane space and matrix

Anti-vimentin, anti-TUFM, anti-NAP1L1 and anti-DPYSL2 nanobodies display cytotoxic effect and reduce glioblastoma cell migration

Background: Glioblastoma is a particularly common and very aggressive primary brain tumour. One of the main causes of therapy failure is the presence of glioblastoma stem cells that are resistant to chemotherapy and radiotherapy, and that have the potential to form new tumours. This study focuses on validation of eight novel antigens, TRIM28, nucleolin, vimentin, nucleosome assembly protein 1-like 1 (NAP1L1), mitochondrial translation elongation factor (EF-TU) (TUFM), dihydropyrimidinase-related protein 2 (DPYSL2), collapsin response mediator protein 1 (CRMP1) and Aly/REF export factor (ALYREF), as putative glioblastoma targets, using nanobodies. Methods: Expression of these eight antigens was analysed at the cellular level by qPCR, ELISA and immunocytochemistry, and in tissues by immunohistochemistry. The cytotoxic effects of the nanobodies were determined using AlamarBlue and water-soluble tetrazolium tests. Annexin V/propidium iodide tests were used to determine apoptotsis/necrosis of the cells in the presence of the nanobodies. Cell migration assays were performed to determine the effects of the nanobodies on cell migration. Results: NAP1L1 and CRMP1 were significantly overexpressed in glioblastoma stem cells in comparison with astrocytes and glioblastoma cell lines at the mRNA and protein levels. Vimentin, DPYSL2 and ALYREF were overexpressed in glioblastoma cell lines only at the protein level. The functional part of the study examined the cytotoxic effects of the nanobodies on glioblastoma cell lines. Four of the nanobodies were selected in terms of their specificity towards glioblastoma cells and protein overexpression: anti-vimentin (Nb79), anti-NAP1L1 (Nb179), anti-TUFM (Nb225) and anti-DPYSL2 (Nb314). In further experiments to optimise the nanobody treatment schemes, to increase their effects, and to determine their impact on migration of glioblastoma cells, the anti-TUFM nanobody showed large cytotoxic effects on glioblastoma stem cells, while the anti-vimentin, anti-NAP1L1 and anti-DPYSL2 nanobodies were indicated as agents to target mature glioblastoma cells. The anti-vimentin nanobody also had significant effects on migration of mature glioblastoma cells. Conclusion: Nb79 (anti-vimentin), Nb179 (anti-NAP1L1), Nb225 (anti-TUFM) and Nb314 (anti-DPYSL2) nanobodies are indicated for further examination for cell targeting. The anti-TUFM nanobody, Nb225, is particularly potent for inhibition of cell growth after long-term exposure of glioblastoma stem cells, with minor effects seen for astrocytes. The anti-vimentin nanobody represents an agent for inhibition of cell migration

Rat Group IIA Secreted Phospholipase A2 Binds to Cytochrome c Oxidase and Inhibits Its Activity: A Possible Episode in the Development of Alzheimer’s Disease

Alzheimer’s disease (AD), a progressive form of dementia, is characterized by the increased expression of secreted phospholipase A2 group IIA (GIIA) in the affected tissue and the dysfunction of neuronal mitochondria, similar to that induced by an orthologous GIIA from snake venom, β-neurotoxic ammodytoxin (Atx), in the motor neurons. To advance our knowledge about the role of GIIA in AD, we studied the effect of rat GIIA on the neuronal mitochondria and compared it with that of the Atx. We produced recombinant rat GIIA (rGIIA) and its enzymatically inactive mutant, rGIIA(D49S), and demonstrated that they interact with the subunit II of cytochrome c oxidase (CCOX-II) as Atx. rGIIA and rGIIA(D49S) bound to this essential constituent of the respiratory chain complex with an approximately 100-fold lower affinity than Atx; nevertheless, both rGIIA molecules potently inhibited the CCOX activity in the isolated rat mitochondria. Like Atx, rGIIA was able to reach the mitochondria in the PC12 cells from the extracellular space, independent of its enzymatic activity. Consistently, the inhibition of the CCOX activity in the intact PC12 cells and in the rat’s brain tissue sections was clearly demonstrated using rGIIA(D49S). Our results show that the effects of mammalian and snake venom β-neurotoxic GIIA on the neuronal mitochondria have similar molecular backgrounds. They suggest that the elevated extracellular concentration of GIIA in the AD tissue drives the translocation of this enzyme into local neurons and their mitochondria to inhibit the activity of the CCOX in the respiratory chain. Consequently, the process of oxidative phosphorylation in the neurons is attenuated, eventually leading to their degeneration. Atx was thus revealed as a valuable molecular tool for further investigations of the role of GIIA in AD

3D models of complexes between sPLA2s and hPDI.

<p>(A) Structure of hPDI was superimposed on the structure of yPDI in the complex with Atx bound to the bb'-binding site and the 3D model optimised by HADDOCK. The model is presented using the PyMOL program with hPDI in space-filling style and Atx in the ribbon style. (B) By substitution of Atx in the Atx—hPDI model with GIB, GIIA, GV or GX sPLA₂, and HADDOCK molecular docking, the models of mammalian sPLA₂s bound to hPDI were generated. GV sPLA₂ (as also GIB and GIIA) occupies the same area on hPDI as Atx (upper picture), while GX sPLA₂ occupies only part of this area (lower picture). (C) Heterologous competition of sulfo-SBED-Atx binding to hPDI by GV and GX sPLA₂s confirms validity of the proposed sPLA₂–hPDI 3D model. Human PDI was incubated with sulfo-SBED-Atx in the dark in the presence of 26 to 100-fold molar excess of AtxC, hGV or hGX sPLA₂ over the sulfo-SBED-Atx. The photo cross-linking reaction was triggered by irradiation at 312 nm. SDS-PAGE analysis under reducing conditions was followed by Western blotting of the samples and biotin-specific detection on the nitrocellulose membranes. Black arrowhead is pointing at the biotinylated hPDI. White arrows are pointing at positions with biotinylated monomer and dimer of Atx. Experimental details are described in Materials and Methods section.</p

Atx interacts with several domains of yPDI.

<p>(A) yPDI is a multi-domain protein. It consists of domains a, a’, b, b’ and c. Domain b’ is linked to domain a’ by a linker sequence x. Separate domains and their fusions, as schematically presented, were employed in affinity-labelling experiments using photo-reactive sulfo-SBED-Atx. Atx photo-probe was incubated with the wt yPDI (wt-PDI) (B) or its various parts (C) for 30 min, followed by the UV light-initiated reaction of the photo-probe with proteins in close contact, which were thus biotinylated. Samples were analysed on SDS-PAGE under reducing conditions, electro-transferred to a PVDF membrane. Biotin-containing bands on the PVDF membrane were detected using streptavidin-HRP. Labelling specificity in (B) was verified with the addition of 100-fold unlabelled Atx over sulfo-SBED-Atx (wt-PDI+). Black arrowheads in (A) point at those elements of yPDI which bind and react with sulfo-SBED-Atx. In (B) and (C) black arrowheads are pointing at biotinylated yPDI-structures. Positions on the membrane with biotinylated monomer and dimer of Atx are pointed at by white arrowheads. Experimental details are described in section Materials and Methods. Experiment was performed in triplicate.</p

On the Role of Protein Disulfide Isomerase in the Retrograde Cell Transport of Secreted Phospholipases A₂

<div><p>Following the finding that ammodytoxin (Atx), a neurotoxic secreted phospholipase A₂ (sPLA₂) in snake venom, binds specifically to protein disulfide isomerase (PDI) <i>in vitro</i> we show that these proteins also interact in living rat PC12 cells that are able to internalize this group IIA (GIIA) sPLA₂. Atx and PDI co-localize in both differentiated and non-differentiated PC12 cells, as shown by fluorescence microscopy. Based on a model of the complex between Atx and yeast PDI (yPDI), a three-dimensional model of the complex between Atx and human PDI (hPDI) was constructed. The Atx binding site on hPDI is situated between domains b and b’. Atx interacts hPDI with an extensive area on its interfacial binding surface. The mammalian GIB, GIIA, GV and GX sPLA₂s have the same fold as Atx. The first three sPLA₂s have been detected intracellularly but not the last one. The models of their complexes with hPDI were constructed by replacement of Atx with the respective mammalian sPLA₂ in the Atx—hPDI complex and molecular docking of the structures. According to the generated models, mammalian GIB, GIIA and GV sPLA₂s form complexes with hPDI very similar to that with Atx. The contact area between GX sPLA₂ and hPDI is however different from that of the other sPLA₂s. Heterologous competition of Atx binding to hPDI with GV and GX sPLA₂s confirmed the model-based expectation that GV sPLA₂ was a more effective inhibitor than GX sPLA₂, thus validating our model. The results suggest a role of hPDI in the (patho)physiology of some snake venom and mammalian sPLA₂s by assisting the retrograde transport of these molecules from the cell surface. The sPLA₂–hPDI model constitutes a valuable tool to facilitate further insights into this process and into the (patho)physiology of sPLA₂s in relation to their action intracellularly.</p></div

Detailed view of the Atx—yPDI interaction areas.

<p>(A) Details of the interaction between Atx and the a’c-binding site of yPDI. (B) Details of the interaction between Atx and the bb’-binding site of yPDI. Structures are prepared using the PyMOL program. yPDI is presented in space-filling mode while Atx in the ribbon mode, with side chains of the yPDI-interacting amino acids shown. (C) The yPDI-contacting amino acid residues of Atx are marked on its primary structure (arrowheads). Black arrowheads point at residues contacting yPDI at the bb’-binding site, while red at those contacting yPDI at the a’c-binding site. Elements of the secondary structure of Atx, α-helices and β-sheets, are shown behind corresponding parts of the primary structure.</p