Primary Initiation Site Research Articles

Effect of weld microstructure on brittle fracture initiation in the thermally-aged boiling water reactor pressure vessel head weld metal

Effects of the weld microstructure and inclusions on brittle fracture initiation are investigated in a thermally aged ferritic high-nickel weld of a reactor pressure vessel head from a decommissioned nuclear power plant. As-welded and reheated regions mainly consist of acicular and polygonal ferrite, respectively. Fractographic examination of Charpy V-notch impact toughness specimens reveals large inclusions (0.5–2.5 µm) at the brittle fracture primary initiation sites. High impact energies were measured for the specimens in which brittle fracture was initiated from a small inclusion or an inclusion away from the V-notch. The density, geometry, and chemical composition of the primary initiation inclusions were investigated. A brittle fracture crack initiates as a microcrack either within the multiphase oxide inclusions or from the de-bonded interfaces between the uncracked inclusions and weld metal matrix. Primary fracture sites can be determined in all the specimens tested in the lower part of the transition curve at and below the 41-J reference impact toughness energy but not above the mentioned value because of the changes in the fracture mechanism and resulting changes in the fracture appearance.

Differential phenotypes and localization of CD8 T cell responses to acute and chronic viral infections

Abstract Norovirus is the most common viral cause of gastroenteritis globally. Most infections are either acute, symptomatic, and rapidly cleared or persist asymptomatically. However, the mechanisms underlying these outcomes are unknown. MNV-CW3 and -CR6 are murine noroviruses which demonstrate immune correlates of acute and chronic infection respectively. This research interrogates the mechanisms underlying strain-specific differential antiviral CD8 T cell responses to acute or chronic MNV. At days 3, 4, 5 and 8 post-infection, the phenotype and quantity of adoptively transferred MNV specific CD8 T cells in the spleen, mesenteric lymph node (MLN), and intestines are analyzed by flow cytometry. Concurrently, fluorescent microscopy is used to determine the biogeographical distribution of antiviral CD8 T cells throughout the intestine. Flow cytometry shows activated MNV-specific CD8 T cells first accumulate in the MLN following oral infection with either virus suggesting this is the site of immune activation though CR6 infection results in a delayed, reduced CD8 T cell response as shown by CFSE dilution. Supporting the MLN as a primary initiation site of the antiviral CD8 T cell response, prevention of T cell egress from activation sites by FTY720 treatment enriches activated CD8 T cells in the MLN in CW3 infections with a concurrent decrease in the spleen. Furthermore, by day 8 post-infection CD8 T cell populations are skewed to memory precursors or short-lived effectors in CW3 and CR6, respectively. These data suggest these highly related viral strains may induce activation of distinct populations of, or pathways in, APC populations with long-lasting effects on CD8 T cell function and the outcome of infection.

Understanding the damage initiation mechanism of precipitation-strengthened Fe-Ni-Cr based austenitic steel

The present study aims at enhancing the understanding of damage evolution mechanism in precipitation-strengthened Fe-Ni-Cr based steel (SUH660). Tensile deformation at room temperature, subsequent damage quantification and microstructural characterization was performed. The primary damage initiation sites were observed at inclusions and spots where dislocation slip impinges grain boundary. Early damage initiation was also observed.

Influence of Rotational Nucleosome Positioning on Transcription Start Site Selection in Animal Promoters

The recruitment of RNA-Pol-II to the transcription start site (TSS) is an important step in gene regulation in all organisms. Core promoter elements (CPE) are conserved sequence motifs that guide Pol-II to the TSS by interacting with specific transcription factors (TFs). However, only a minority of animal promoters contains CPEs. It is still unknown how Pol-II selects the TSS in their absence. Here we present a comparative analysis of promoters’ sequence composition and chromatin architecture in five eukaryotic model organisms, which shows the presence of common and unique DNA-encoded features used to organize chromatin. Analysis of Pol-II initiation patterns uncovers that, in the absence of certain CPEs, there is a strong correlation between the spread of initiation and the intensity of the 10 bp periodic signal in the nearest downstream nucleosome. Moreover, promoters’ primary and secondary initiation sites show a characteristic 10 bp periodicity in the absence of CPEs. We also show that DNA natural variants in the region immediately downstream the TSS are able to affect both the nucleosome-DNA affinity and Pol-II initiation pattern. These findings support the notion that, in addition to CPEs mediated selection, sequence–induced nucleosome positioning could be a common and conserved mechanism of TSS selection in animals.

Influence of Weld Preparation Procedure and Heat Tinting on Sulfide Stress Corrosion Cracking of Duplex Stainless Steel

Corrosion resistant alloys, such as duplex stainless steel (DSS), are increasingly the material of choice for oilfield applications as a result of the trend toward more extreme temperature, higher pressure, and elevated levels of hydrogen sulfide. However, the susceptibility of such materials to localized corrosion and sulfide stress cracking (SSC) is a major consideration, particularly in the vicinity of welds. Here we investigate the effect of surface preparation and oxygen content in the backing gas during welding on the pitting and SSC susceptibility of 22Cr DSS. Four-point bend testing of welded specimens under conditions close to the pass/fail boundary revealed a greater tendency for pitting as a function of oxygen content in the backing gas. Pitting was concentrated in the heat-tinted region, whereas cracking was mainly observed in the adjacent coarse ground parent material, with surface defects from the grinding process acting as primary crack initiation sites. The results demonstrate that control...

Low cycle fatigue behavior of laser melting deposited TC18 titanium alloy

Low cycle fatigue (LCF) behavior of laser melting deposited (LMD) TC18 titanium alloy was studied at room temperature. Microstructure consisting of fine lamella-like primary α phase and transformed β matrix was obtained by double annealed treatment, and inhomogeneous grain boundary α phase was detected. Fatigue fracture surfaces and longitudinal sections of LCF specimens were examined by optical microscopy and scanning electron microscopy. Results indicate that more than one crack initiation site can be detected on the LCF fracture surface. The fracture morphology of the secondary crack initiation site is different from that of the primary crack initiation site. When the crack grows along the grain boundary α phase, continuous grain boundary α phase leads to a straight propagating manner while discontinuous grain boundary α phase gives rise to flexural propagating mode.

SP0021 The Onset of RA: Disease Processes at Extraarticular Sites

Rheumatoid arthritis (RA) is a chronic inflammatory disease resulting from the complex interaction between genes and environment. In a large majority of patients this interaction leads to formation of anti-citrullinated...

P5-S6.21 Acyclovir for managing genital ulcer disease in South Africa: experiences of guideline introduction, implementation and uptake

BackgroundHerpes simplex virus type 2 (HSV-2) is the leading cause of genital ulcer disease (GUD) worldwide. Treatment with acyclovir has been shown to shorten GUD duration, decrease plasma and genital...

Microstructure and mechanical properties in TIG welding of CLAM steel

Tungsten insert gas (TIG) welding on China low activation martensitic (CLAM) steel under identical conditions was performed. Microhardness test, tensile test, Charpy impact test and microstructure measurements were carried out on TIG welded joints after post weld heat-treatment. Hardening at WM and softening in HAZ is detected in the TIG weld joint. Microhardness in WM decreased when the temperature of PWHT increased. The ultimate tensile stress of weld metal is higher than that of HAZ and BM. Absorbed energy increased with PWHT temperature rising, until PWHT was done at 760 °C/30 min, the specimen ductile fractured in local area. The microstructure of the weld metal for every specimen was found to be tempered martensite with a little of delta ferrite. M 23C 6 particles are the predominant type of carbides. Oxide precipitate phases appeared in WM, which are the primary crack initiation sites and it is critically important minimize their formation.

Variability of Pseudo-nitzschia and domoic acid in the Juan de Fuca eddy region and its adjacent shelves

The Juan de Fuca eddy is a toxic “hot spot.” Domoic acid (DA) was detected in the eddy during each of six cruises over a 4-yr study, although Pseudo-nitzschia abundance and toxin concentrations were highly variable. During the September 2004 eddy bloom, Pseudo-nitzschia spp. exceeded 13 × 106 cells L−1, and particulate DA reached 80 nmol L−1. Of the >10 species of Pseudo-nitzschia identified in this region, those coincident with the most toxic blooms are P. cf. pseudodelicatissima, P. cuspidata, P. multiseries, and P. australis. However, the presence of any particular species could not be used as an indicator of toxicity because of the high level of variability in intracellular DA in field assemblages. Pseudo-nitzschia cells were typically associated with blooms of other diatom taxa but also were coincident with blooms of euglenoids and dinoflagellates in the eddy region. Pseudo-nitzschia always comprised <17% of the total carbon biomass, thereby rendering remote sensing an unsuitable means for predicting toxigenic Pseudo-nitzschia blooms in this region. Our results support the hypothesis that the Juan de Fuca eddy region and not the nearshore zone is the primary initiation site for toxic blooms of Pseudo-nitzschia affecting the Washington coast. Although particulate DA was observed near the edges of the Columbia River plume, whether toxin can be produced in situ in plume water is not resolved. No first-order predictive relationships were found for either Pseudo-nitzschia abundance or DA concentration and environmental data from all six cruises.

Translational Regulation of Rat Multidrug Resistance-Associated Protein 2 Expression Is Mediated by Upstream Open Reading Frames in the 5′ Untranslated Region

Multidrug resistance-associated protein 2 (Mrp2/Abcc2), an organic anion transporter present in the apical membrane of hepatocytes, renal epithelial cells, and enterocytes, is postulated to undergo translational regulation. Transcription of rat hepatic Mrp2 mRNA is initiated at multiple sites (-213, -163, -132, and -98 nucleotides relative to the Mrp2 ATG) and contains potential upstream open reading frames (uORFs) in the 5' untranslated region (UTR) starting at -213, -149 and -109 nucleotides. Ribonuclease protection assays demonstrated that transcription of the Mrp2 gene at the various initiation sites was tissue-specific, with the major initiation site in the liver and kidney being -98 and -132 nucleotides, respectively. In the jejunum, the primary and secondary initiation sites were -98 and -132 nucleotides, respectively, with the converse true in the ileum. The relative abundance of these Mrp2 transcripts expressed in tissues varied with age from birth to the adult. HepG2 transient expression assays and in vitro translation assays in which the 5'UTRs were fused with a luciferase reporter showed that the 5'UTR without any uORF (-98 nucleotide) expressed maximal luciferase activity compared with those with one (-132 nucleotides), two (-163 nucleotides), or three (-213 nucleotides) uORFs. Disruption of the uORF by site-directed mutagenesis at nucleotide -109 enhanced luciferase activity 2- to 3-fold, whereas disruption of the uORF at nucleotide -149 had little effect. We conclude that among the uORFs in the Mrp2 5'UTR, the uORF starting at nucleotide -109 probably plays an important role in the regulation of Mrp2 protein expression.

Isolating Apparently Pure Libraries of Replication Origins from Complex Genomes

Because of the complexity of higher eukaryotic genomes and the lack of a reliable autonomously replicating sequence (ARS) assay for isolating potential replicators, the identification of origins has proven to be extremely challenging and time consuming. We have developed a new origin-trapping method based on the partially circular nature of restriction fragments containing replication bubbles and have prepared a library of approximately 1,000 clones from early S phase CHO cells. When 15 randomly selected clones were analyzed by a stringent two-dimensional (2D) gel replicon mapping method, all were shown to correspond to active, early firing origins. Furthermore, most of these appear to derive from broad zones of potential sites, and the five that were analyzed in a time-course study are all inefficient. This bubble-trapping scheme will allow the construction of comprehensive origin libraries from any complex genome so that their natures and distributions vis-a-vis other chromosomal markers can be established.

Dissecting the Molecular Mechanisms of Mutant C/EBPalpha (p30) in Normal and Malignant Human Hematopoiesis.

Acute myelogenous leukemia (AML) is often associated with mutations in transcription factors that are essential for normal hematopoietic development and differentiation. The basic leucine zipper (bZIP) protein C/EBPalpha is mutated in 7–10% of AML. Two different classes of C/EBPalpha mutations are found in AML: 1) mutations that introduce a stop codon between the primary translational initiation site and a downstream ATG, resulting in translation of an N-terminally truncated protein (p30) 2) mutations that disrupt the basic region of the bZIP domain in the C-terminus, abolishing DNA binding. Of note, biallellic mutations are often but not always observed. It has been proposed that p30 is dominant-negative over wt C/EBPalpha (p42), blocking its transcriptional activity. We have previously shown that p30 blocks erythrocytic commitment and inhibits terminal granulocytic differentiation when expressed in human but not murine primary hematopoietic cells. To identify the molecular mechanism by which the p30 inhibits differentiation and to evaluate the role of homo- and heterodimerization between p30 and p42 in leukemogenesis, we created a p30 that carries point mutations in bZIP (p30-L12V) that disrupt dimerization. Expression of p30-L12V had no effect on the differentiation of human CD34+ cells, arguing for the requirement of dimerization. To inhibit potential heterodimerization between p30 and p42 but permit p30 homodimerization, we generated a p30-GZ, in which the leucine zipper was replaced by an artificial dimerization domain. Expression of p30-GZ induced a similar differentiation block as the normal p30, arguing against a dominant-negative function of p30 over p42 mediated by dimerization. In a second approach to understand the mechanism by which p30 inhibits differentiation, we wanted to identify gene targets of p30. For this purpose, p30 was fused to the modified ligand-binding domain of the estrogen receptor (p30-ERtm). Expression of p30-ERtm in CD34+ cells in the presence of tamoxifen showed a phenotype very similar to the one observed after the expression of p30, while in the absence of tamoxifen no effect was observed. We used this system of inducible p30 expression in human CD34+ cells to evaluate gene expression patterns. If p30 is dominant-negative and interferes with the transcriptional activation of p42, reciprocal expression patterns of target genes should be observed for wt C/EBPalpha and p30. Our data show that homodimerization of p30 is necessary and sufficient to block erythrocytic commitment and terminal granulocytic differentiation of human CD34+ cells and to induce known C/EBPalpha target genes. These data argue that the formation of p30/p42 heterodimers is not required for the effect of C/EBPalpha p30, questioning the proposed dominant-negative function.

Primary structure of guinea pig preprodynorphin and preproenkephalin mRNAs: multiple transcription initiation sites for preprodynorphin

Preprodynorphin and preproenkephalin are protein precursors from which are derived two classes of opioid neurotransmitter peptides. Dynorphin A (1–17) is produced by proteolytic processing of prodynorphin, and processing of proenkephalin yields the enkephalin peptides. We report here on the isolation and sequencing of multiple clones for these two mRNAs from a cDNA library. Two cDNA clones of preprodynorphin contained the full-length sequence (2.35 kb) with the primary structure predicted from the guinea pig gene sequence. In contrast, one clone encoded the full-length sequence but also an additional 192 nt at the 5′ end. This sequence has high homology to the 5′ flanking region of the human preprodynorphin gene, and RNase protection assays demonstrated that in addition to a primary initiation site, transcription of this mRNA is initiated at several sites 160–190 nt 5′ with respect to the primary site. This difference may alter translational efficiency or mRNA stability. The sequence of preproenkephalin cDNA clones confirmed the structure predicted from the gene sequence. One clone, however, contained sequences encoded by exons 2 and 3, and initiated within the first intron (intron A) of the gene. We used RNase protection mapping to assess the abundance in the brain and pituitary of preproenkephalin transcripts that initiate within intron A. These studies confirmed that the primary transcription start site is 28 nucleotides downstream from the TATAA site, and that intron A sequences are not present in significant amounts in these tissues.

Phase relationships between segmentally organized oscillators in the leech heartbeat pattern generating network.

Motor pattern generating networks that produce segmentally distributed motor outflow are often portrayed as a series of coupled segmental oscillators that produce a regular progression (constant phase differences) in their rhythmic activity. The leech heartbeat central pattern generator is paced by a core timing network, which consists of two coupled segmental oscillators in segmental ganglia 3 and 4. The segmental oscillators comprise paired mutually inhibitory oscillator interneurons and the processes of intersegmental coordinating interneurons. As a first step in understanding the coordination of segmental motor outflow by this pattern generator, we describe the functional synaptic interactions, and activity and phase relationships of the heart interneurons of the timing network, in isolated nerve cord preparations. In the timing network, most (approximately 75%) of the coordinating interneuron action potentials were generated at a primary spike initiation site located in ganglion 4 (G4). A secondary spike initiation site in ganglion 3 (G3) became active in the absence of activity at the primary site. Generally, the secondary site was characterized by a reluctance to burst and a lower spike frequency, when compared with the primary site. Oscillator interneurons in G3 inhibited spike activity at both initiation sites, whereas oscillator interneurons in G4 inhibited spike activity only at the primary initiation site. This asymmetry in the control of spike activity in the coordinating interneurons may account for the observation that the phase of the coordinating interneurons is more tightly linked to the G3 than G4 oscillator interneurons. The cycle period of the timing network and the phase difference between the ipsilateral G3 and G4 oscillator interneurons were regular within individual preparations, but varied among preparations. This variation in phase differences observed across preparations implies that modulated intrinsic membrane and synaptic properties, rather than the pattern of synaptic connections, are instrumental in determining phase within the timing network.

Comparative Squalene Synthase Gene Expression in Mouse Liver and Testis

RNA from various mouse organs was analyzed by Northern hybridization to determine the response of squalene synthase (SQS) mRNA to dietary cholesterol, or lovastatin and cholestyramine, administration. Two size-classes of highly abundant mouse SQS (mSQS) mRNAs of ∼1.9 and 2.0 kb were found in testis. These transcripts were unresponsive to sterol regulation. A single size-class of liver mSQS mRNA of ∼1.9 kb was sterol-regulated. Studies using primer extension and 5′ rapid amplification of cDNA ends (RACE) indicated that the size differences in liver and testis mSQS transcripts were due to variations in the lengths of the 5′ untranslated regions (UTRs). The longest testis 5′ UTR extended ∼106 nt 5′ of the primary transcription initiation site in liver of mice fed lovastatin and cholestyramine. These results suggest that tissue-specific promoter elements control the transcriptional regulation of the promoters for the mSQS gene in liver and testis.

Murine cysteine dioxygenase gene: structural organization, tissue-specific expression and promoter identification

The murine gene encoding cysteine dioxygenase (CDO; EC 1.13.11.20), a key enzyme of l-cysteine metabolism, was isolated and characterized, and the proximal promoter was identified. A bacterial artificial chromosome mouse library was screened and a single clone containing the entire CDO gene was isolated. The murine CDO gene contains five exons and spans about 15 kb. The open reading frame is encoded within all five exons. All intron/exon splice junctions and all intron sizes are conserved with the rat CDO gene and are very similar to those of the human CDO gene. The primary transcriptional initiation site is located 213 bp upstream of the initiation ATG codon. The nucleotide sequence of the 5′-promoter region is highly conserved between the mouse and rat genes and contains a TATA-box-like sequence and GC boxes. A variety of consensus cis-acting elements were also identified in the 5′-flanking region. These included HNF-3β, HFH-1, HFH-2, HFH-3, C/EBP, and C/EBPβ, all of which are consistent with the tissue-specific expression profiles of the gene. Gene reporter studies of the CDO 5′-region indicated the presence of an active promoter within the first 223 bp upstream of the transcriptional initiation site and the possible presence of repressor elements upstream of bp −223. Northern blot analyses indicated that the CDO gene displays tissue-specific expression, with the highest mRNA level present in liver and with detectable levels found in kidney, lung, brain and small intestine. Western blot analyses indicated that CDO protein levels parallel mRNA levels. These results are consistent with the known function of CDO in whole-body cysteine homeostasis.

Oligonucleotide mapping of the core genomic RNA dimer linkage in human T-cell leukaemia virus type-1

We have previously mapped the sequences required for dimerisation of the 5′ leader of the human T-cell leukaemia virus type-1 (HTLV-1) genome. The smallest sequence necessary and sufficient for dimer formation, in vitro, was ascertained to be a 37 nucleotide (nt) region downstream of the splice donor and just upstream of the primer binding site. Deletion of a 32 base-pair sequence encompassing this region within the provirus was associated with a minor decrease in infectivity of the virus in an in vitro system. To further map and help elucidate the nature of the dimer linkage, we used RNA and DNA oligonucleotide competition assays to define the nucleotides involved. These experiments revealed that a 14 nt sequence containing a potential stem loop structure, formed from a palindromic sequence, is important for dimer formation. This was confirmed by the ability of this RNA sequence to form heterodimers with larger RNA transcripts from the same region, while sequences lacking this motif could not. RNA transcripts containing the reverse sequence, the same nucleotides in a random arrangement, and complementary DNA oligos, all failed to form heterodimers with the 14 nt sequence. The primary dimer initiation site of HTLV-1 has thus been located to a 14 nt palindrome containing sequence, and dimerisation is shown to be dependent on specific sense–sense RNA interactions.

An origin of bidirectional DNA replication is located within a CpG island at the 3" end of the chicken lysozyme gene.

We previously identified a broad initiation zone of DNA replication at the chicken lysozyme gene locus. However, the existence of a highly preferred origin of bidirectional replication (OBR), often found in initiation zones, remained elusive. In order to re-examine this issue we used a competitive PCR assay to determine the abundance of closely spaced genomic segments in a 1 kb size fraction of nascent DNA. A sharp peak of nascent strand abundance occurred at the 3" end of the gene, where initiation events were 17 times more frequent than upstream of the gene. This primary initiation site, active in lysozyme expressing myelomonocytic HD11 cells and non-expressing hepatic DU249 cells, was found to reside within an unusually located CpG island. While most CpG islands are found at the 5" end of genes, the lysozyme gene island extends from the 3" end of the second intron and includes approximately 1.2 kb of 3" flanking DNA. As diagnosed by methylation-sensitive restriction enzymes, the island is largely non-methylated in HD11 cells, DU249 cells and inactive chicken erythrocytes. Furthermore, a DNase I hypersensitive site (HS) that is composed of two subsites separated by approximately 100 bp, was localised very close to the segment with the highest initiation activity. Our results suggest that the non-methylated CpG island and the HS provide an accessible chromatin structure for the lysozyme gene origin of replication.

Occluded solution chemistry control and the role of alloy sulfur on the initiation of crevice corrosion in type 304ss

Because sulfide inclusions are recognized as the primary initiation sites for localized corrosion of austenitic stainless steels, the influence of alloy sulfur on the crevice solution chemistry and nature of the surface in the crevice at initiation were investigated. Through the coordinated use of occluded solution analysis, high resolution TEM⧹EDS, small-spot XPS, and electrochemical measurements, the dominance of the effects of occluded solution chemistry evolution on the initiation of crevice corrosion was shown. The chemical dissolution of MnS inclusions to form aqueous sulfide increases the corrosivity of the low pH, high [Cl −] occluded solution until crevice corrosion occurs at rates on the order of 35 mA cm [2]for an applied potential of +0.05 V SCE. Neither ohmic potential drop, nor changes in local pH and⧹or [Cl −] alone can rationalize the crevice dissolution rates observed without the addition of aqueous sulfide to the solution. No evidence was found for salt film formation or enrichment⧹deposition of elemental sulfur on the corroding surfaces.