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Deliverable 4.4 is part of Task 4.2 that deals with the development of the NanoCommons Knowledge Base and Warehouse that aims to collect raw and processed data generated by different projects and to provide support and processes for preparing datasets for upload to the NanoCommons data warehouse or other specialized databases linked into the infrastructure, as well as templates for data collection (linked to the online notebooks). Additionally, repositories for protocol description directly linked to the relevant datasets are provided in order for complete coverage of the experimental procedure and results to be included into the system. The work in this task is based on the concepts and aligned with developments from previous and ongoing projects (e.g. eNanoMapper, NANoREG, NanoMILE, NanoFASE, SmartNanoTox, ACEnano, etc.) and is extended in order to cover additional areas of nano safety research. It considers requirements for regulatory reporting and Adverse Outcome Pathway (AOP) development, as well as the support of ontology development and semantic annotation. In this way, the warehouse facilitates data transfer to and from other databases as part of a federated data ecosystem.

The scope of Work Package 5 (WP5) is the integration of the state-of-the-art tools for data management, mining, handling, analysis and predictive modelling to facilitate their joint provision as tools and services for Transnational Access (TA). To achieve this, a linked data approach is being utilised to exploit, extract, and integrate knowledge from all available information types (raw and processed experimental and modelling data, and metadata) to be captured in the NanoCommons Knowledge Base (KB). These tools, once linked and interoperable via the NanoCommons platform, will be made available to the nanosafety community either as standalone or through the NanoCommons TA open calls that will offer funded access to the tools and services offered by NanoCommons to develop tailored solutions for individual user or project’s data management needs. The TA calls will allow successful applicants to take advantage of the NanoCommons tools and services free-of-charge, including help to make their existing data FAIR (Findable, Accessible, Interoperable, and Reusable), to implement data management, mining, handling, analysis, visualisation and modelling workflows into their experimental design and data collection processes and to facilitate direct integration of resulting datasets into relevant databases (experimental and in silico) with all of the required metadata and semantic annotation. NanoCommons TA Users are thus becoming part of a wider attempt at cross-field and cross-data harmonisation, leading to increased data quality and thus better predictive models for nanosafety, thereby resulting in ground breaking insights and novel materials. This advance in nanosafety and safe-by-design (SdD) NMs will also be facilitated through the further development of the existing tools and the design and creation of new tools to fill in any identified research gaps or lack of services. This deliverable (D5.1) presents the tools and respective services implemented during Year 1 within NanoCommons that are offered during the first TA call. Additional services will continue to be added to the NanoCommons portfolio and refinements to current services will also be made based on experience gained during the first round of TA provision.

The current deliverable is part of Work Package 5 (WP5) Joint Research Activity 3 (JRA3) - Analysis and Modelling Tools, which aims to integrate the current state of the art tools for data mining and data analysis, utilising a linked data approach that will exploit, extract, and integrate knowledge from all available information (raw experimental and modelling data, and metadata) contained in the NanoCommons data warehouse (DW) and KnowledgeBase (KB). These tools and the expertise to apply them and interpret the outputs in a meaningful manner, once linked and interoperable via the NanoCommons platform, will be made available to the NanoCommons User community via the open calls for Transnational Access. This Deliverable report describes the initial set of tools directed towards analysis of “omics” datasets, including transcriptomics (analysis of changes in gene expression), proteomics (analysis of changes in protein expression), metabolomics and lipidomics (changes in expression of small molecules and lipids, respectively).

The objective of the present study was to describe two non-invasive methods for fat quantification in normal canine liver by using magnetic resonance imaging (MRI) and spectroscopy. Eleven adult beagle dogs were anesthetized and underwent magnetic resonance examination of the cranial abdomen by performing morphologic, modified Dixon (mDixon) dual gradient echo sequence, and proton magnetic resonance spectroscopy (1H MRS) imaging. In addition, ultrasonographic liver examination was performed, fine-needle liver aspirates and liver biopsies were obtained, and hepatic triglyceride content was assayed. Ultrasonographic, cytologic, and histologic examination results were unremarkable in all cases. The median hepatic fat fraction calculated was 2.1% (range, 1.3%–5.5%) using mDixon, 0.3% (range, 0.1%–1.0%) using 1H MRS, and 1.6% (range 1.0%–2.5%) based on triglyceride content. The hepatic fat fractions calculated using mDixon and 1H MRS imaging were highly correlated to that based on triglyceride content. A weak correlation between mDixon and 1H MRS imaging was detected. The results show that hepatic fat content can be estimated using non-invasive techniques (mDixon or 1H MRS) in healthy dogs. Further studies are warranted to evaluate the use of these techniques in dogs with varying hepatic fat content and different hepatic disorders., Journal of Veterinary Science, 19 (4), ISSN:1229-845X, ISSN:1976-555X

Cerebrolysin (EVER Neuro Pharma, Austria) is a peptidergic drug indicated for clinical use in stroke, traumatic brain injury and dementias. The therapeutic effect of Cerebrolysin is thought to ensure from its neurotrophic activity, which shares some properties with naturally occurring neurotrophic factors. However, the exact mechanism(s) of action of Cerebrolysin is yet to be fully deciphered. This study aimed to investigate the neuroprotective effect of Cerebrolysin in a widely used in vitro model of hypoxia-induced neuronal cytotoxicity, namely CoCl2-treatment of PC12 cells. CoCl2 cytotoxicity was indicated by a reduced cell-diameter, cell shrinkage, increased pro-apoptotic caspase-activities and a decreased metabolic activity. Cerebrolysin maintained the cell-diameter of CoCl2-treated naïve PC12 cells, decreased the activation of caspase 3/7 in naïve PC12 cells and restored the cells’ metabolic activity in naïve and differentiated PC12 cells. Cerebrolysin treatment also decreased the levels of superoxides observed after exposure to CoCl2. Investigating the mechanism of action, we could demonstrate that Cerebrolysin application to CoCl2-stressed PC12 cells increased the phosphorylation of GSK3ß, resulting in the inhibition of GSK3ß. This might become clinically relevant for Alzheimer’s disease, since GSK3ß activity has been linked to the production of amyloid beta. Taken together, Cerebrolysin was found to have neuroprotective effects in CoCl2-induced cytotoxicity in PC12 cells.

Thogoto and Dhori viruses are tick-borne orthomyxoviruses infecting humans and livestock in Africa, Asia, and Europe. Here, we show that human MxA protein is an efficient inhibitor of Thogoto virus but is inactive against Dhori virus. When expressed in the cytoplasm of stably transfected cell lines, MxA protein interfered with the accumulation of Thogoto viral RNA and proteins. Likewise, MxA(R645), a mutant MxA protein known to be active against influenza virus but inactive against vesicular stomatitis virus, was equally efficient in blocking Thogoto virus growth. Hence, a common antiviral mechanism that is distinct from the antiviral action against vesicular stomatitis virus may operate against both influenza virus and Thogoto virus. When moved to the nucleus with the help of a foreign nuclear transport signal, MxA(R645) remained active against Thogoto virus, indicating that a nuclear step of virus replication was inhibited. In contrast, Dhori virus was not affected by wild-type or mutant MxA protein, indicating substantial differences between these two tick-transmitted orthomyxoviruses. Human MxB protein had no antiviral activity against either virus.

The genetic determinants of enterobacterial common antigen (ECA) include the rfe and rff genes located between ilv and cya near min 85 on the Escherichia coli chromosome. The rfe-rff gene cluster of E. coli K-12 was cloned in the cosmid pHC79. The cosmid clone complemented mutants defective in the synthesis of ECA due to lesions in the rfe, rffE, rffD, rffA, rffC, rffT, and rffM genes. Restriction endonuclease mapping combined with complementation studies of the original cosmid clone and six subclones revealed the order of genes in this region to be rfe-rffD/rffE-rffA/rffC-rffT-rffM . The rfe gene was localized to a 2.54-kilobase ClaI fragment of DNA, and the complete nucleotide sequence of this fragment was determined. The nucleotide sequencing data revealed two open reading frames, ORF-1 and ORF-2, located on the same strand of DNA. The putative initiation codon of ORF-1 was found to be 570 nucleotides downstream from the termination codon of rho. ORF-1 and ORF-2 specify putative proteins of 257 and 348 amino acids with calculated Mr values of 29,010 and 39,771, respectively. ORF-1 was identified as the rfe gene since ORF-1 alone was able to complement defects in the synthesis of ECA and 08-side chain synthesis in rfe mutants of E. coli. Data are also presented which suggest the possibility that the rfe gene is the structural gene for the tunicamycin sensitive UDP-GlcNAc:undecaprenylphosphate GlcNAc-1-phosphate transferase that catalyzes the synthesis of GlcNAc-pyrophosphorylundecaprenol (lipid I), the first lipid-linked intermediate involved in ECA synthesis.

Twelve independent Tn10 insertion mutants of Escherichia coli K12 were isolated that were defective in the synthesis of enterobacterial common antigen (ECA). The mutants were identified by screening a random pool of Tn10 insertion mutants for their ECA phenotype using a colony-immunoblot assay. All 12 of the Tn10 insertion mutants were found to be located in the chromosomal region of the rff-rfe genes. Four of the Tn10 insertions were in rfe genes while the remaining eight Tn10 insertions were in rff genes. All of the rfe::Tn10 insertion mutants were defective in the synthesis of GlcNAc-pyrophosphorylundecaprenol (C55-PP-GlcNAc, lipid I), the first lipid-linked intermediate involved in ECA synthesis. Biochemical characterization of the rff::Tn10 insertion mutants revealed that they were defective in various steps of ECA synthesis subsequent to the synthesis of lipid I. These defects included: (i) the inability to synthesize UDP-ManNAcA due to Tn10 insertions in the structural genes for UDP-GlcNAc-2-epimerase (rffE) and UDP-ManNAcA (N-acetyl-D-mannosaminuronic acid) dehydrogenase (rffD), (ii) defects in the synthesis of C55-GlcNAc-ManNAcA (lipid II) due to insertion of transposon Tn10 in the structural gene for the UDP-ManNAcA transferase (rffM), (iii) the inability to synthesize TDP-Fuc4NAc (4-acetamido-4,6-dideoxy-D-galactose) due to Tn10 insertions in the structural gene for the transaminase that catalyzes the conversion of TDP-4-keto-6-deoxy-D-glucose to TDP-4-amino-4,6-dideoxy-D-galactose (rffA), and (iv) defects in steps subsequent to the synthesis of C55-GlcNAc-ManNAcA-Fuc4NAc (lipid III). In addition, a re-examination of a mutant possessing the rff-726 lesion revealed that it was defective in the synthesis of lipid III due to a defect in the structural gene for the Fuc4NAc transferase (rffT).