AP-CVD ZrB2 process development for discrete and duplex UHTC coatings

Refractory borides are a class materials with properties ideally suited to extreme operating conditions such as leading edges, spaceplanes, hypersonic vehicles and volumetric solar power receivers. The ability to coat complex geometries and varying materials with refractory borides such as zirconium diboride, (ZrB2), is offered through chemical vapour deposition, (CVD), methods. CVD ZrB2 can offer high purity, uniform and fully dense coatings economically, negating many of the current issues with powder processing methods that dominate commercial availability. The study was conducted to establish ZrB2 coating processes by atmospheric pressure CVD, (AP-CVD), on a variety of substrate materials including graphite, CMCs, SiC coated CMCs, and C and SiC fibre fabric. Single layer coatings were developed, although these are limited in temperature performance, as well as multilayer ZrB2-SiC coatings to offer an innovative and effective solution to operation in the high and ultra-high temperature regimes. Literature studies demonstrated CVD processes could be achieved through direct chlorination of zirconium metal to generate a metal chloride precursor that underwent subsequent reduction with BCl3 and H2 to generate ZrB2 coatings. An iterative process of parameter selection and modification, deposition trials and analysis was undertaken to evaluate optimal deposition conditions. Please click Additional Files below to see the full abstract

Characterization of C-S Lyase from C. diphtheriae

The emergence of antibiotic resistance in microbial pathogens requires the identification of new antibacterial drugs. The biosynthesis of methionine is an attractive target because of its central importance in cellular metabolism. Moreover, most of the steps in methionine biosynthesis pathway are absent in mammals, lowering the probability of unwanted side effects. Herein, detailed biochemical characterization of one enzyme required for methionine biosynthesis, a pyridoxal-5′-phosphate (PLP-) dependent C-S lyase from Corynebacterium diphtheriae, a pathogenic bacterium that causes diphtheria, has been performed. We overexpressed the protein in E. coli and analyzed substrate specificity, pH dependence of steady state kinetic parameters, and ligand-induced spectral transitions of the protein. Structural comparison of the enzyme with cystalysin from Treponema denticola indicates a similarity in overall folding. We used site-directed mutagenesis to highlight the importance of active site residues Tyr55, Tyr114, and Arg351, analyzing the effects of amino acid replacement on catalytic properties of enzyme. Better understanding of the active site of C. diphtheriae C-S lyase and the determinants of substrate and reaction specificity from this work will facilitate the design of novel inhibitors as antibacterial therapeutics

Structural insights into the heme pocket and oligomeric state of non-symbiotic hemoglobins from Arabidopsis thaliana

Non-symbiotic hemoglobins AHb1 and AHb2 from Arabidopsis thaliana are hexacoordinate heme-proteins that likely have different biological roles, in view of diverse tissue localization, expression pattern, and ligand binding properties. Herein, we expand upon previous biophysical studies on these isoforms, focusing on their oligomeric states and circular dichroism (CD) characteristics. We found that AHb1 exists in solution in a concentration-dependent monomer-dimer equilibrium, while AHb2 is present only as a monomer. The quaternary structure of AHb1 affects its degree of hexacoordination with the formation of the dimer that enhances pentacoordination. Accordingly, the mutant of a conserved residue within the dimeric interface, AHb1-T45A, which is mostly monomeric in solution, has an equilibrium that is shifted toward a hexacoordinate form compared to the wild-type protein. CD studies further support differences in the globin's structure and heme moiety. The Soret CD spectra for AHb2 are opposite in sense to those for AHb1, reflecting different patterns of heme-protein side chain contacts in the two proteins. Moreover, the smaller contribution of the heme to the near-UV CD in AHb2 compared to AHb1 suggests a weaker heme-protein association in AHb2. Our data corroborate the structural diversity of AHb1 and AHb2 and confirm the leghemoglobin-like structural properties of AHb2

Residues in the Distal Heme Pocket of Arabidopsis Non-Symbiotic Hemoglobins: Implication for Nitrite Reductase Activity

It is well-established that plant hemoglobins (Hbs) are involved in nitric oxide (NO) metabolism via NO dioxygenase and/or nitrite reductase activity. The ferrous-deoxy Arabidopsis Hb1 and Hb2 (AHb1 and AHb2) have been shown to reduce nitrite to NO under hypoxia. Here, to test the hypothesis that a six- to five-coordinate heme iron transition might mediate the control of the nitrite reduction rate, we examined distal pocket mutants of AHb1 and AHb2 for nitrite reductase activity, NO production and spectroscopic features. Absorption spectra of AHbs distal histidine mutants showed that AHb1 mutant (H69L) is a stable pentacoordinate high-spin species in both ferrous and ferric states, whereas heme iron in AHb2 mutant (H66L) is hexacoordinated low-spin with Lys69 as the sixth ligand. The bimolecular rate constants for nitrite reduction to NO were 13.3 ± 0.40, 7.3 ± 0.5, 10.6 ± 0.8 and 171.90 ± 9.00 M(-1)·s(-1) for AHb1, AHb2, AHb1 H69L and AHb2 H66L, respectively, at pH 7.4 and 25 °C. Consistent with the reductase activity, the amount of NO detected by chemiluminescence was significantly higher in the AHb2 H66L mutant. Our data indicate that nitrite reductase activity is determined not only by heme coordination, but also by a unique distal heme pocket in each AHb

Residues in the Distal Heme Pocket of Arabidopsis Non-Symbiotic Hemoglobins: Implication for Nitrite Reductase Activity

It is well-established that plant hemoglobins (Hbs) are involved in nitric oxide (NO) metabolism via NO dioxygenase and/or nitrite reductase activity. The ferrous-deoxy Arabidopsis Hb1 and Hb2 (AHb1 and AHb2) have been shown to reduce nitrite to NO under hypoxia. Here, to test the hypothesis that a six- to five-coordinate heme iron transition might mediate the control of the nitrite reduction rate, we examined distal pocket mutants of AHb1 and AHb2 for nitrite reductase activity, NO production and spectroscopic features. Absorption spectra of AHbs distal histidine mutants showed that AHb1 mutant (H69L) is a stable pentacoordinate high-spin species in both ferrous and ferric states, whereas heme iron in AHb2 mutant (H66L) is hexacoordinated low-spin with Lys69 as the sixth ligand. The bimolecular rate constants for nitrite reduction to NO were 13.3 \ub1 0.40, 7.3 \ub1 0.5, 10.6 \ub1 0.8 and 171.90 \ub1 9.00 M(-1)\ub7s(-1) for AHb1, AHb2, AHb1 H69L and AHb2 H66L, respectively, at pH 7.4 and 25 \ub0C. Consistent with the reductase activity, the amount of NO detected by chemiluminescence was significantly higher in the AHb2 H66L mutant. Our data indicate that nitrite reductase activity is determined not only by heme coordination, but also by a unique distal heme pocket in each AHb

Analysis of the lipid binding properties of mutant murine Bid proteins

Bid is a BH3-only member of the Bcl-2 family that regulates cell death at the level of mitochondrial membranes. It is generally assumed that the full length Bid protein becomes activated after a proteolytic cleavage catalized by apical caspases, like caspase 8. The cleaved protein then re-locates to mitochondria and promotes membrane permeabilization, presumably by interaction with mitochondrial lipids and other Bcl-2 proteins that facilitate the release of apoptogenic proteins like cytochrome c. The un-cleaved Bid also has proapoptotic potential when ectopically expressed in cells or in vitro. It has been demonstrated that full length Bid can insert specific lysolipids into the membrane surface and this lipid transfer activity participates to the release of apoptogenic factors from mitochondria. The binding properties of Bid are still unknown. In this work we will present new full length Bid mutants that possess altered lipid binding properties and proapoptotic activities in vitro. We have analysed the binding properties of Bid mutants to LPC species and MCL (or LPG) in order to investigate the protein dual specificity for the diverse lysolipids

Lipid exchange in mitochondrial cytochrome c release: pro-apoptotic effect of maize lipid transfer protein

Membrane lipids and protein-lipid interactions are attracting increasing interest in the field of cell death and apoptosis. Some pro-apoptotic proteins, like Bid, appear to have an intrinsic capacity of binding and exchange lipids but it is still unclear whether this function could be relevant for apoptotic signalling cascade. We have studied the ability of a plant lipid transfer protein, not related to animal apoptotic cascade, to induce cytochrome c release from mammalian mitochondria. Non -specific lipid transfer proteins (nsLTPs) are ubiquitous plant proteins that have been shown to bind, in vitro, various amphiphilic molecules including lysolipids and glycolipids and to facilitate in vitro transfer of phospholipids between membranes. The results showed that, in the presence of specific lipid molecules (i.e. lysolipids), ns-LTP from maize is able to induce cytochrome c release from the intermembrane space of mouse liver mitochondria. These data are discussed with respect to the role played by lipids and lipid binding in apoptosis

Role of myristoylation in modulating PCaP1 interaction with calmodulin

Plasma membrane-associated Cation-binding Protein 1 (PCaP1) belongs to the plant-unique DREPP protein family with largely unknown biological functions but ascertained roles in plant development and calcium (Ca2+) signaling. PCaP1 is anchored to the plasma membrane via N-myristoylation and a polybasic cluster, and its N-terminal region can bind Ca2+/calmodulin (CaM). However, the molecular determinants of PCaP1-Ca2+-CaM interaction and the functional impact of myristoylation in the complex formation and Ca2+ sensitivity of CaM remained to be elucidated. Herein, we investigated the direct interaction between Arabidopsis PCaP1 (AtPCaP1) and CaM1 (AtCaM1) using both myristoylated and non-myristoylated peptides corresponding to the N-terminal region of AtPCaP1. ITC analysis showed that AtCaM1 forms a high affinity 1:1 complex with AtPCaP1 peptides and the interaction is strictly Ca2+-dependent. Spectroscopic and kinetic Ca2+ binding studies showed that the myristoylated peptide dramatically increased the Ca2+-binding affinity of AtCaM1 and slowed the Ca2+ dissociation rates from both the C- and N-lobes, thus suggesting that the myristoylation modulates the mechanism of AtPCaP1 recognition by AtCaM1. Furthermore, NMR and CD spectroscopy revealed that the structure of both the N- and C-lobes of Ca2+-AtCaM1 changes markedly in the presence of the myristoylated AtPCaP1 peptide, which assumes a helical structure in the final complex. Overall, our results indicate that AtPCaP1 biological function is strictly related to the presence of multiple ligands, i.e., the myristoyl moiety, Ca2+ ions and AtCaM1 and only a full characterization of their equilibria will allow for a complete molecular understanding of the putative role of PCaP1 as signal protein

Does MtN5 play a double role in root responses to symbiontic and pathogenic microorganisms?

MtN5, a new Lipid Transfer Protein, has been identified in nodulated roots of Medicago truncatula andpreliminarily classified as early nodulin, which is expressed in response to rhizobial symbiosis. Wehave shown that the recombinant MtN5 exerts antifungal and antimicrobial activity in vitro againstFusarium semitectum and Rhizobium leguminosarum, respectively. In vivo, the fungal infection leadsto the expression of MtN5 in the whole root apparatus of M. truncatula plants, whereas the inoculationwith rhizobia induces an early and nodule-specific expression of the protein, that is also maintained inmature nodules. These two different expression patterns suggest a putative double role for MtN5, whichcould be involved both in a general response mechanism against fungi and in sensing or controlling theinfection of the symbiont. This last hypothesis is supported by the observation that M.truncatula rootstransformed with an hairpin construct aiming to silence endogenous MtN5, are impaired in noduleformation respect to control roots. Therefore, MtN5 is hereby proposed as a novel, multifunctionalprotein taking part in the symbiotic process

BIOCHEMICAL AND FUNCTIONAL CHARACTERIZATION OF THE BACTERIAL PsaR1 SENSOR IN THE PSEUDOMONAS SYRINGAE PV. ACTINIDIAE-KIWIFRUIT INTERACTION

Kiwifruit bacterial canker, caused by Pseudomonas syringae pv. actinidiae (Psa), is responsible for important economic losses in all major areas of kiwifruit production worldwide, including Italy. As for many other bacterial diseases, current plant defense strategies against Psa are mainly based on the use of copper-containing products, which raise eco-toxicological problems. Revision and restriction processes regarding the use of high amounts of copper in agriculture impose an urgent study of new solutions, efficient and eco-compatible, avoiding at the same time the occurrence of new resistance to active molecules. Innovative strategies are based for instance on the application of targeted treatments for \u201cweakening\u201d the pathogen, i.e. to reduce its virulence within its host. However, this requires improving our knowledge regarding molecular mechanisms controlling bacterial virulence induction. A key regulator of bacterial virulence is the so-called \u2018quorum-sensing\u2019 (QS), that links bacterial density to gene expression. This mechanism allows bacteria to communicate within the bacterial community and with their environment, via small diffusible molecules. The prototypical QS system of Gram-negative bacteria consists of a LuxI-type synthase that produces the signal molecules acyl homoserine lactones (AHLs) and a cognate LuxR-type receptor/regulator that senses signal specific threshold concentration. An interesting subgroup of LuxR receptors lacks a genetically linked LuxI and has been termed \u201csolos\u201d. These \u201csolos\u201d are assumed to sense AHLs from neighboring bacteria, bacterial molecules other than AHLs or still unknown plant-produced compounds in the case of phytopathogenic bacteria. Interestingly, Psa does not produce AHLs but possesses three LuxR solos, which likely contribute to Psa virulence. As a first candidate for a targeted inhibition strategy against Psa, we are currently investigating the biochemical properties of the sensor PsaR1. To that purpose, several tentative have been made to obtain the soluble recombinant sensor in a heterologous system. Once achieved, we demonstrated that it does not bind AHLs, thus excluding the possibility to sense AHLs from neighboring bacteria, and we are currently setting a chemical screening, based on thermal shift assay, to identify the class(es) of molecules able to bind to the sensor. On the other hand, we aim to identify the pathway(s) regulated by PsaR1 during Psa interaction with kiwifruit, during different phases of the infection. Thus, a microarray analysis is being performed to compare the transcriptomic profiles of wild-type and psaR1 knockout Psa strains at both exponential and stationary growth phase, in conditions mimicking the interaction with the host plant, i.e. minimal medium supplemented with kiwifruit extract