Norvaline is accumulated after a down-shift of oxygen in Escherichia coli W3110

<p>Abstract</p> <p>Background</p> <p>Norvaline is an unusual non-proteinogenic branched-chain amino acid which has been of interest especially during the early enzymological studies on regulatory mutants of the branched-chain amino acid pathway in <it>Serratia marcescens</it>. Only recently norvaline and other modified amino acids of the branched-chain amino acid synthesis pathway got attention again when they were found to be incorporated in minor amounts in heterologous proteins with a high leucine or methionine content. Earlier experiments have convincingly shown that norvaline and norleucine are formed from pyruvate being an alternative substrate of α-isopropylmalate synthase, however so far norvaline accumulation was not shown to occur in non-recombinant strains of <it>E. coli</it>.</p> <p>Results</p> <p>Here we show that oxygen limitation causes norvaline accumulation in <it>E. coli </it>K-12 W3110 during grow in glucose-based mineral salt medium. Norvaline accumulates immediately after a shift to oxygen limitation at high glucose concentration. On the contrary free norvaline is not accumulated in <it>E. coli </it>W3110 in aerobic cultures. The analysis of medium components, supported by transcriptomic studies proposes a purely metabolic overflow mechanism from pyruvate into the branched chain amino acid synthesis pathway, which is further supported by the significant accumulation of pyruvate after the oxygen downshift. The results indicate overflow metabolism from pyruvate as necessary and sufficient, but deregulation of the branched chain amino acid pathway may be an additional modulating parameter.</p> <p>Conclusion</p> <p>Norvaline synthesis has been so far mainly related to an imbalance of the synthesis of the branched chain amino acids under conditions were pyruvate level is high. Here we show that simply a downshift of oxygen is sufficient to cause norvaline accumulation at a high glucose concentration as a consequence of the accumulation of pyruvate and its direct chain elongation over α-ketobutyrate and α-ketovalerate.</p> <p>Although the flux to norvaline is low, millimolar concentrations are accumulated in the cultivation broth, which is far above the level which has been discussed for being relevant for misincorporation of norvaline into recombinant proteins. Therefore we believe that our finding is relevant for recombinant protein production but also may even have implications for the physiology of <it>E. coli </it>under oxygen limitation in general.</p

Composite Finishing for Reuse

Coating processes are emerging for new applications related to remanufactured products from End-of-Life materials. In this perspective, their employment can generate interesting scenarios for the design of products and solutions in circular economy frameworks, especially for composite materials. This chapter would give an overview of coating design and application for recycled glass fiber reinforced polymers on the base of the experimentation made within the FiberEUse project. New cosmetic and functional coatings were developed and tested on different polymer composite substrates filled with mechanically recycled End-of-Life glass fibers. Afterwards, recycled glass fiber reinforced polymer samples from water-solvable 3D printed molds were successfully coated. Finally, new industrial applications for the developed coatings and general guidelines for the coating of recycled glass fiber reinforced polymers were proposed by using the FiberEUse Demo Cases as a theoretical proof-of-concept

Integrity of APS, HVOF and HVAF sprayed NiCr and NiCrBSi coatings based on the tensile stress-strain response

The interlamellar cohesion of thermal spray coatings influences greatly their mechanical properties and ability to use coatings in different loading conditions and wear/erosion resistance. In the present study, micro-tensile testing of free-standing coatings was utilized to evaluate the mechanical response of thermally sprayed coatings. In addition, the longitudinal uniaxial fracture strength of free-standing coatings could be determined by a tensile test. The coating materials studied were NiCr and NiCrBSi coatings sprayed by atmospheric plasma spraying (APS), high velocity oxy-fuel (HVOF), and high velocity air-fuel (HVAF) processes. The different materials used for the coatings sprayed by different methods yield different microstructures, different stress-strain relation in tensile testing. Different tensile test response was found to be related to cohesion strength between lamellas, and thus was affecting the cavitation erosion wear. The effect of other factors such as hardness and residual stresses on cavitation resistance were also discussed. Such results are crucial to understand the suitability of microstructures obtained by TS processes for different wear conditions.publishedVersionPeer reviewe

On the Applicability of Iron-Based Coatings Against Abrasion and Cavitation Erosion Wear

Iron-based coatings are often considered as replacement of hard chromium and WC-Co, as they pose lower health and environmental impact. In many cases, the combination of mechanical and chemical properties of iron-based alloys may be satisfactory and their relatively low cost makes these coatings an interesting candidate for many applications. This study is inspired by opportunities to harden the iron-base materials by strain hardening, solid solution strengthening, and precipitation hardening. Already commercially available Fe-based coating materials with precipitates of mixed carbides and borides in the metastable austenitic matrix achieve a high hardness. In this study, the cavitation erosion and abrasion resistance of various Fe-based coatings produced by HVAF and HVOF processes were investigated. Four experimental precipitates containing materials were prepared, and the sprayed coatings were tested for abrasive wear and cavitation erosion. In addition to precipitates, the contributions of martensite and retained austenite phases were investigated by influencing the microstructure through heat treatments, as the ability of these phases to influence ductility and toughness can be crucial to achieve the desired material properties. The properties of experimental and two commercial Fe-based alloys are compared with WC-Co and Cr3C2-NiCr coatings by property mapping.publishedVersionPeer reviewe

Human Bone Marrow Mesenchymal Stem Cells Induce Collagen Production and Tongue Cancer Invasion

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Human mitochondrial DNA lineages in Iron-Age Fennoscandia suggest incipient admixture and eastern introduction of farming-related maternal ancestry

Human ancient DNA studies have revealed high mobility in Europe's past, and have helped to decode the human history on the Eurasian continent. Northeastern Europe, especially north of the Baltic Sea, however, remains less well understood largely due to the lack of preserved human remains. Finland, with a divergent population history from most of Europe, offers a unique perspective to hunter-gatherer way of life, but thus far genetic information on prehistoric human groups in Finland is nearly absent. Here we report 103 complete ancient mitochondrial genomes from human remains dated to AD 300-1800, and explore mtDNA diversity associated with hunter-gatherers and Neolithic farmers. The results indicate largely unadmixed mtDNA pools of differing ancestries from Iron-Age on, suggesting a rather late genetic shift from hunter-gatherers towards farmers in North-East Europe. Furthermore, the data suggest eastern introduction of farmer-related haplogroups into Finland, contradicting contemporary genetic patterns in Finns.Peer reviewe

Human GNAL, C18orf2, and MPPE1 genes:genomic organization of the human GNAL gene and characterization of two novel genes, C18orf2 and MPPE1, on chromosome 18p11.2, a susceptibility region for schizophrenia and bipolar disorder

Abstract The genomic organization and mRNA expression of the human GNAL gene on chromosome 18p11.2, a region that has been associated with bipolar disorder and schizophrenia, was determined. The GNAL gene was shown to span over 80 kb and consist of twelve exons, and its structure was very similar to adenyl cyclase stimulating G protein GSα. The start site of transcription was revealed by 5'-RACE. Two polyadenylation signals were found, and 3'-RACE assay was used to verify the functional site. The GNAL gene was expressed as approximately 6 kb transcripts in various regions of the human brain, and no alternative splicing was detected. One informative CA-dinucleotide repeat of 11 alleles and 74% heterozygosity was found in intron 5, and two single nucleotide polymorphisms in introns 3 and 10 were detected by SSCP. During characterization of the GNAL gene, two previously unknown genes were found. A novel intronless gene C18orf2 coding for a functionally unknown protein was localized to intron 5 of the GNAL gene. By semiquatitative RT-PCR, C18orf2 mRNA was found to be moderately expressed in all tissues studied here. Another novel gene, metallophosphoesterase MPPE1, was found to reside adjacent to the 3'-end of the GNAL gene in a tail-to-tail orientation. The deduced amino acid sequence revealed a highly conserved metallophosphoesterase motif gDxH..(16-60)..GDxxdr..(13-34)..GNH[DE], which is typical for various phosphate hydrolyzing enzymes, especially serine/threonine protein phosphatases. The MPPE1 gene contained fourteen exons and spanned about 27 kb. MPPE1 was expressed as a single mRNA of 2.2 kb in various regions of the human brain but not in any other tissues. Four different alternatively spliced forms of MPPE1 were detected by RT-PCR, and each transcript was shown to partially overlap with the 3'-untranslated region of the GNAL gene

Suunnittelumanuaalin kehittämistyö : Vesiturbiinin juoksupyörä

Opinnäytetyön aiheena oli kehittää vesiturbiinin juoksupyörän suunnittelumanuaali Andritz Hydro Oy:n tarpeisiin. Edellisen harjoittelun loppukeskustelujen pohjalta tuli ilmi, että on tarve luoda suunnitteluohjeet juoksupyörän suunnittelulle. Projektin tavoitteena oli yhtenäistää suunnittelukäytäntöjä yrityksessä laatimalla ajantasaisia, piirustusteknillisesti oikeaoppisia sekä valmistusteknillisesti ja toiminnallisesti tarkoituksenmukaisia piirustusmalleja. Lisäksi haluttiin lisätä kustannustehokkuutta suunnittelussa tarkennetulla toleranssien asettamisella sekä valmistusmahdollisuuksien tarkastamisella ja tehostamisella. Kustannuslaskennan esimerkkien haluttiin tuoda lisänäkemystä valmistusteknillisestä ja kustannustehokkaasta näkökulmasta suunnittelutyössä. Samalla ohjekirja toimisi perehdytys- ja koulutusmateriaalina uusille työntekijöille. Opinnäytetyön tarkoituksena oli käsitellä ohjekirjan laadintaprojektiin kuuluvan asiasisällön käsittelyä. Pääpaino työssä oli ohjekirjan sisältöön kuuluvien osuuksien esittelyllä yleisellä tasolla ja varsinaisen ohjekirjan laadintaan kuuluvien vaiheiden käsittelyllä. Suunnittelumanuaali koostuu tehtävänannon mukaan laadituista mallipiirustuksista ja niitä tukevasta tekstiosuudesta. Se käsittelee mallipiirustuksien ja ohjesivujen avulla juoksupyörän osissa huomioitavia erityispiirteitä suunnittelussa, kuten erilaisia rakenneratkaisuja, mitoituksen lähtökohtia, toleransseja, materiaaleja ja materiaalivaatimuksia sekä tarkastusvaatimuksia. Lopulliseksi suunnittelumanuaaliksi kehittyi tehtävänannon mukainen ohjekirja, jolla luodaan linjaukset juoksupyörän suunnitteluun. Työstä tuli laaja, kaikki tärkeimmät juoksupyörän osat kattava, yleiset suunnittelun linjaukset tarkentava ohjekirja. Suunnittelumanuaalia tullaan käyttämään yrityksessä suunnittelijoiden ohjeistukseen ja sitä voidaan laajentaa ja kehittää tarpeen vaatiessa. Projektin luottamuksellisuuden vuoksi tässä työssä ei käsitellä yritykselle laadittua suunnittelumanuaalin sisältöä kuin vain yleisellä tasolla ja luottamuksellinen aineisto on poistettu julkaistavasta raportista.The subject of this thesis was to develop a design guide line for water turbine runners for Andritz Hydro Oy. The final discussions after my last internship in the company revealed that there was a need to define design instructions for runners. The purpose of the project was standardize design practices by creating proper drawing models that would serve the needs of manufacturing process and the operation of the components. Cost efficiency would increase with setting up more suitable tolerances for components and rationalizing manufacturing processes. In addition the design guide line is aimed to be a training material for new employees. The purpose of the thesis was to cover the matters that need to be considered in designing a runner guide line. The main focus was in the introduction of the matters that need to be considered in design process and in theory of designing a guide line. The design guide line consists of model drawings and text documents supporting information expressed in the drawings. It covers special perspectives in designing a runner, for example different structure options, tolerances, materials, material requirements and requirements concerning inspections of the components. The final form of the guide line developed into design guide line covering design practices concerning runners. The project was extensive and the guide line covers all main components of the runner and it defines general design instructions. It is intended to be used to instruct the designers. The design guide line that was created in the project is left unpublished because of its confidentiality. The matters that need to be considered in design process are introduced in general level

Developmental &amp; Comparative Immunology Iap2 is required for a sustained response in the Drosophila Imd pathway

Abstract Fruit flies have effective immune response against Gram-negative bacteria. Upon infection, early JNK-signaling pathway mediated response is followed by the action of the Immune deficiency (Imd) signaling cascade, a Drosophila equivalent of mammalian TNF-receptor pathway, leading to the release of antimicrobial peptides. Recently, Tak1-binding protein 2 (Tab2) and Inhibitor of apoptosis 2 (Iap2) were identified as components of the Imd pathway. In this study, we carried out a genome-wide kinetic analysis of the role of Tab2 and Iap2 for immune response in Drosophila S2 cells using oligonucleotide microarrays. Tab2 RNAi abolished the induction of all immune response genes in S2 cells indicating its requirement for signaling both via the Imd and the JNK pathway. The role of Iap2 was more specific. Kinetic analysis indicated that Iap2 is required to sustain antimicrobial peptide gene expression in S2 cells. Furthermore, inactivation of Iap2 by RNAi resulted in impaired microbial resistance in Drosophila in vivo.

Effect of heat treatments on the wear resistance of HVAF and HVOF sprayed tool steel coatings

Iron-based coatings are an attractive solution for many wear applications, considering sustainability requirements such as avoidance of critical raw materials and toxic substances. Cold worked tool steels are one potential, yet very undiscovered, material option for coating applications. As a bulk, their careful heat treatment typically produces a martensitic carbide microstructure with very good wear resistance. Compared to bulk materials, the thermal history of a thermally sprayed coating in the as sprayed state is quite different as the microstructure of the sprayed coating does not form in equilibrium. This study explored the potential of AISI D2 cold worked tool steel as a thermal spray coating under different wear conditions. The study investigated different heat treatments to achieve different microstructures of the powder and coatings and their effect on the wear and cavitation erosion properties of the HVOF and HVAF sprayed thermally sprayed tool steel coating. It is important to understand whether the properties of thermally sprayed coatings, which initially have a high defect density, can be improved by heat treatments, and how modification of the phase structures of iron-based coatings affects their properties, in order to extend the use of these coatings.Peer reviewe