Applicability of heat transfer and solidification simulations in investigating microstructural banding in continuously cast steel

Abstract Microstructural banding is observed as alternating microstructures in cast steel products, parallel to cast/rolling direction. Banding, causing hardenability issues and anisotropy of mechanical properties, is considered to originate from the interdenritic segregation during the solidification of steel. In this study, a three-dimensional heat transfer model (3DHTM) was used to simulate the steady-state local temperatures in a casting strand of 0.34C low alloy carbon steel, taking into account both primary and secondary cooling as well as other casting parameters. The calculated temperature profiles for a set of selected locations along the strand were used as input data for a solidification and microstructure model (SMM) for the continuous casting of steel. To assess the microstructure of the cast bloom, the prior austenite grain size, dendrite arm spacing, and the magnitude of elemental microsegregation between the dendrites were calculated with the selected temperature profiles for the steel grade. For validation purposes, bloom and bar samples were prepared from industrial trials. The calculated results are compared to the microstructural characterization of austenite grain size, and local elemental concentrations obtained with electron probe microanalyzer (EPMA). Based on the results, elemental microsegregation and microstructural banding is assessed, affected by casting parameters and the total composition of steel. Additionally, a brief discussion of the segregation between the bloom and bar samples is presented

Trimeric Form of Intracellular ATP Synthase Subunit β of Aggregatibacter actinomycetemcomitans Binds Human Interleukin-1β.

Bacterial biofilms resist host defenses and antibiotics partly because of their decreased metabolism. Some bacteria use proinflammatory cytokines, such as interleukin (IL)-1β, as cues to promote biofilm formation and to alter virulence. Although one potential bacterial IL-1β receptor has been identified, current knowledge of the bacterial IL-1β sensing mechanism is limited. In chronic biofilm infection, periodontitis, Aggregatibacter actinomycetemcomitans requires tight adherence (tad)-locus to form biofilms, and tissue destroying active lesions contain more IL-1β than inactive ones. The effect of IL-1β on the metabolic activity of A. actinomycetemcomitans biofilm was tested using alamarBlue™. The binding of IL-1β to A. actinomycetemcomitans cells was investigated using transmission electron microscopy and flow cytometry. To identify the proteins which interacted with IL-1β, different protein fractions from A. actinomycetemcomitans were run in native-PAGE and blotted using biotinylated IL-1β and avidin-HRP, and identified using mass spectroscopy. We show that although IL-1β slightly increases the biofilm formation of A. actinomycetemcomitans, it reduces the metabolic activity of the biofilm. A similar reduction was observed with all tad-locus mutants except the secretin mutant, although all tested mutant strains as well as wild type strains bound IL-1β. Our results suggest that IL-1β might be transported into the A. actinomycetemcomitans cells, and the trimeric form of intracellular ATP synthase subunit β interacted with IL-1β, possibly explaining the decreased metabolic activity. Because ATP synthase is highly conserved, it might universally enhance biofilm resistance to host defense by binding IL-1β during inflammation

Binding of IL-1β to intracellular protein fraction of <i>A. actinomycetemcomitans</i>.

<p>The samples were run in native-PAGE, and the proteins were transferred to nitrocellulose membrane. The reactive bands were then detected using biotinylated IL-1β and, HRP-linked streptavidin. Control detection included biotinylated soybean trypsin inhibitor (sti). Reactive protein band 305 (named according to its travelling distance in these gels), which was more pronounced in IL-1β incubated detection than in the control detection, was isolated from identical gel after silver staining. The isolated protein band 305 was digested with trypsin before peptide separation and peptide identification with LC-MS/MS. Panel A shows the blots treated with either IL-1β or sti. Panel B shows the intensities of the three reactive bands in IL-1β-treated membranes compared to sti-treated membranes. Panel C presents the silver stained native-PAGE gel. Panel D shows the seven peptides that were identified from protein band 305 (denoted by the sequences given in bold type). According to the peptide sequence data, the protein was identified as ATP synthase subunit β (<i>Aggregatibacter actinomycetemcomitans</i> D11S-1) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018929#pone.0018929-Chen1" target="_blank">[42]</a>. The Mascot program combined with Sprot-Trembl (uniprot) protein sequence database was used to the protein identification. The gels in Panels A and B contained PageRuler™ Plus Prestained Protein Ladder (denoted by “S”), and some of the proteins in the ladder are denoted (a–f).</p

Effect of IL-1β on the formation of <i>A. actinomycetemcomitans</i> biofilm.

<p>The tested strains D7S (serotype a) and SA1151 (serotype c) were rough-colony forming clinical isolates, which produced fimbriae. Pre-grown (approximately 18 h) biofilms were grown with/without IL-1β (10 ng/ml) in RPMI 1640 medium, and the formation of biofilm was estimated with crystal violet staining after 6 h incubation. The box-plot represents data from four independent experiments.</p

Effect of IL-1β on the metabolic activity of <i>A. actinomycetemcomitans</i>.

<p>The tested strains D7S (serotype a) was rough colony forming isolate, which produced fimbriae. Pre-grown (approximately 18 h) biofilms were grown with human IL-1β (10 ng/ml) or without (control) in RPMI 1640 medium and the metabolic activity was followed with alamarBlue™ (Panel A). Data is shown as percentage of reduced alamarBlue compared to control. The spontaneous smooth-colony variant (D7SS) of D7S and its planktonic single-gene deletion mutants <i>rcpA</i>, <i>rcpB</i>, <i>tadD</i>, <i>tadG</i>, and <i>flp1-flp2</i> were studied (Panel B and C). The pre-grown (18 h) cells were incubated with or without human IL-1β (10 ng/ml) in RPMI 1640 medium, and the metabolic activity was followed using alamarBlue™. The results are shown as percentage of metabolic activities of IL-1β-containing reactions compared to control cultures (Panel B). The metabolic activities of planktonic control cultures (sterile water was substituted for IL-1β) are presented as the fluorescence of the reduced form of alamarBlue™ (Panel C). All results are shown as means ± SD from three independent experiments. Each experiment contained duplications of each reaction.</p

Binding of IL-1β on <i>A. actinomycetemcomitans</i> cells.

<p>The binding on fixed <i>A. actinomycetemcomitans</i> cells was studied using flow cytometry. The cells were first treated with biotinylated IL-1β after which they were stained with avidin-FITC. Panel A presents the percentage of positively stained cells as detected with flow cytometer. Panel B shows the mean fluorescence intensity in positively stained cells. Results are shown as means + SD of three independent experiments. Statistically significant (Two sample paired T-test) differences between the test strain and Δ<i>flp1-flp2</i> mutant (flp1-flp2-) are marked with * and **, indicating p≤0.05 and p≤0.01, respectively. Solid line indicates the level obtained with the Δ<i>flp1-flp2</i> mutant. Biotinylated soybean trypsin inhibitor was used as a negative control protein.</p

IL-1β binding capacity of recombinant ATP synthase subunit β of <i>A. actinomycetemcomitans</i>.

<p>Recombinant ATP synthase subunit β (88 µM) was incubated with or without IL-1β (0.29 µM) for 1 h, after which the samples were run in native-PAGE and immunoblotted with anti-IL-1β (Panel A), or silver stained (Panel B). Three prominent forms could be observed from recombinant ATP synthase subunit β (Panel B). IL-1β bound to the trimeric form of recombinant ATP synthase subunit β. IL-1β was not detectable from immunoblotted native-PAGE without pre-incubation with ATP synthase subunit β (Panel A). However, IL-1β was released from the trimeric form of ATP synthase subunit β under denaturing conditions of SDS-PAGE (Panel C). The sizes of ATP synthase subunit β and IL-1β were 51 kDa and 17 kDa, respectively. The binding of the biotinylated control protein soy bean trypsin inhibitor (STI) to ATP synthase subunit β was was estimated similarly (Panel D and E) by using streptavidin-HRP in detection, except the SDS-page was not run. The control protein bound only to the ATP synthase subunit β monomer.</p

Internalization of IL-1β by <i>A. actinomycetemcomitans</i> co-cultured with organotypic oral mucosa.

<p>Formalin fixed paraffin sections of <i>A. actinomycetemcomitans</i> biofilm containing co-cultures were treated with anti-IL-1β (Panel A), anti-N-terminal-RcpA (Panel B), or control IgG (Panel C) after which the binding antibodies were detected with the NovoLink™ Polymer Detection System (Novocastra™). The sections with the DAB-label were stained with osmium for electron microscopy. Anti-IL-1β stained samples showed structures of <i>A. actinomycetemcomitans</i> cell shape and size (Panel D), with dark precipitate in both extra- and intracellular space (Panel G). The anti-RcpA stained positive control showed intense staining (Panel B) with similar structures (Panel E), although the cell structures are less visible due to the extent of the extracellular precipitate (Panel H). Control IgG antibody showed less staining (Panel C) revealing similar structures (Panel F) without dark precipitates bound to the cell membranes (Panel I).</p

<i>Aggregatibacter actinomycetemcomitans</i> strains used in this study.

a<p>: spontaneous non-fimbriated variant of D7S;</p>b<p>: <i>flp</i> locus coding for Flp pili subunit Flp1 and a pseudogene <i>flp2</i>;</p>c<p>: spectinomycin resistance cassette;</p>d<p>: gene coding for outer membrane rough colony protein A;</p>e<p>: gene coding for outer membrane rough colony protein B;</p>f<p>: gene coding for outer membrane protein TadD;</p>g<p>: gene coding for inner membrane protein TadG.</p

Type 2 Diabetes Whole-Genome Association Study in Four Populations: The DiaGen Consortium

Type 2 diabetes (T2D) is a common, polygenic chronic disease with high heritability. The purpose of this whole-genome association study was to discover novel T2D-associated genes. We genotyped 500 familial cases and 497 controls with >300,000 HapMap-derived tagging single-nucleotide–polymorphism (SNP) markers. When a stringent statistical correction for multiple testing was used, the only significant SNP was at TCF7L2, which has already been discovered and confirmed as a T2D-susceptibility gene. For a replication study, we selected 10 SNPs in six chromosomal regions with the strongest association (singly or as part of a haplotype) for retesting in an independent case-control set including 2,573 T2D cases and 2,776 controls. The most significant replicated result was found at the AHI1-LOC441171 gene region