Bobbin-Tool Friction-Stir Welding of Thick-Walled Aluminum Alloy Pressure Vessels

It was desired to assemble thick-walled Al alloy 2219 pressure vessels by bobbin-tool friction-stir welding. To develop the welding-process, mechanical-property, and fitness-for-service information to support this effort, extensive friction-stir welding-parameter studies were conducted on 2.5 cm. and 3.8 cm. thick 2219 Al alloy plate. Starting conditions of the plate were the fully-heat-treated (-T62) and in the annealed (-O) conditions. The former condition was chosen with the intent of using the welds in either the 'as welded' condition or after a simple low-temperature aging treatment. Since preliminary stress-analyses showed that stresses in and near the welds would probably exceed the yield-strength of both 'as welded' and welded and aged weld-joints, a post-weld solution-treatment, quenching, and aging treatment was also examined. Once a suitable set of welding and post-weld heat-treatment parameters was established, the project divided into two parts. The first part concentrated on developing the necessary process information to be able to make defect-free friction-stir welds in 3.8 cm. thick Al alloy 2219 in the form of circumferential welds that would join two hemispherical forgings with a 102 cm. inside diameter. This necessitated going to a bobbin-tool welding-technique to simplify the tooling needed to react the large forces generated in friction-stir welding. The bobbin-tool technique was demonstrated on both flat-plates and plates that were bent to the curvature of the actual vessel. An additional issue was termination of the weld, i.e. closing out the hole left at the end of the weld by withdrawal of the friction-stir welding tool. This was accomplished by friction-plug welding a slightly-oversized Al alloy 2219 plug into the termination-hole, followed by machining the plug flush with both the inside and outside surfaces of the vessel. The second part of the project involved demonstrating that the welds were fit for the intended service. This involved determining the room-temperature tensile and elastic-plastic fracture-toughness properties of the bobbin-tool friction-stir welds after a post-weld solution-treatment, quenching, and aging heat-treatment. These mechanical properties were used to conduct fracture-mechanics analyses to determine critical flaw sizes. Phased-array and conventional ultrasonic non-destructive examination was used to demonstrate that no flaws that match or exceed the calculated critical flaw-sizes exist in or near the friction-stir welds

Inactivation of the dnaK gene in Clostridium difficile 630 Δerm yields a temperature-sensitive phenotype and increases biofilm-forming ability

Abstract Clostridium difficile infection is a growing problem in healthcare settings worldwide and results in a considerable socioeconomic impact. New hypervirulent strains and acquisition of antibiotic resistance exacerbates pathogenesis; however, the survival strategy of C. difficile in the challenging gut environment still remains incompletely understood. We previously reported that clinically relevant heat-stress (37–41 °C) resulted in a classical heat-stress response with up-regulation of cellular chaperones. We used ClosTron to construct an insertional mutation in the dnaK gene of C. difficile 630 Δerm. The dnaK mutant exhibited temperature sensitivity, grew more slowly than C. difficile 630 Δerm and was less thermotolerant. Furthermore, the mutant was non-motile, had 4-fold lower expression of the fliC gene and lacked flagella on the cell surface. Mutant cells were some 50% longer than parental strain cells, and at optimal growth temperatures, they exhibited a 4-fold increase in the expression of class I chaperone genes including GroEL and GroES. Increased chaperone expression, in addition to the non-flagellated phenotype of the mutant, may account for the increased biofilm formation observed. Overall, the phenotype resulting from dnaK disruption is more akin to that observed in Escherichia coli dnaK mutants, rather than those in the Gram-positive model organism Bacillus subtilis

Increased sporulation underpins adaptation of Clostridium difficile strain 630 to a biologically–relevant faecal environment, with implications for pathogenicity

Abstract Clostridium difficile virulence is driven primarily by the processes of toxinogenesis and sporulation, however many in vitro experimental systems for studying C. difficile physiology have arguably limited relevance to the human colonic environment. We therefore created a more physiologically–relevant model of the colonic milieu to study gut pathogen biology, incorporating human faecal water (FW) into growth media and assessing the physiological effects of this on C. difficile strain 630. We identified a novel set of C. difficile–derived metabolites in culture supernatants, including hexanoyl– and pentanoyl–amino acid derivatives by LC-MSn. Growth of C. difficile strain 630 in FW media resulted in increased cell length without altering growth rate and RNA sequencing identified 889 transcripts as differentially expressed (p < 0.001). Significantly, up to 300–fold increases in the expression of sporulation–associated genes were observed in FW media–grown cells, along with reductions in motility and toxin genes’ expression. Moreover, the expression of classical stress–response genes did not change, showing that C. difficile is well–adapted to this faecal milieu. Using our novel approach we have shown that interaction with FW causes fundamental changes in C. difficile biology that will lead to increased disease transmissibility

Semiquantitative Analysis of Clinical Heat Stress in Clostridium difficile Strain 630 Using a GeLC/MS Workflow with emPAI Quantitation.

<div><p><i>Clostridium difficile</i> is considered to be the most frequent cause of infectious bacterial diarrhoea in hospitals worldwide yet its adaptive ability remains relatively uncharacterised. Here, we used GeLC/MS and the exponentially modified protein abundance index (emPAI) calculation to determine proteomic changes in response to a clinically relevant heat stress. Reproducibility between both biological and technical replicates was good, and a 37°C proteome of 224 proteins was complemented by a 41°C proteome of 202 proteins at a 1% false discovery rate. Overall, 236 <i>C. difficile</i> proteins were identified and functionally categorised, of which 178 were available for comparative purposes. A total of 65 proteins (37%) were modulated by 1.5-fold or more at 41°C compared to 37°C and we noted changes in the majority of proteins associated with amino acid metabolism, including upregulation of the reductive branch of the leucine fermentation pathway. Motility was reduced at 41°C as evidenced by a 2.7 fold decrease in the flagellar filament protein, FliC, and a global increase in proteins associated with detoxification and adaptation to atypical conditions was observed, concomitant with decreases in proteins mediating transcriptional elongation and the initiation of protein synthesis. Trigger factor was down regulated by almost 5-fold. We propose that under heat stress, titration of the GroESL and dnaJK/grpE chaperones by misfolded proteins will, in the absence of trigger factor, prevent nascent chains from emerging efficiently from the ribosome causing translational stalling and also an increase in secretion. The current work has thus allowed development of a heat stress model for the key cellular processes of protein folding and export.</p></div

Polyphosphates as a source of enhanced P fluxes in marine sediments overlain by anoxic waters: Evidence from (31)P NMR

Sedimentary phosphorus (P) composition was investigated in Effingham Inlet, a fjord located on the west coast of Vancouver Island in Barkley Sound. Solid-state (31)P nuclear magnetic resonance (NMR) spectroscopy was applied to demineralized sediment samples from sites overlain by oxic and anoxic bottom waters. The two sites were similar in terms of key diagenetic parameters, including the mass accumulation rate, integrated sulfate reduction rate, and bulk sediment organic carbon content. In contrast, P benthic fluxes were much higher at the anoxic site. (31)P NMR results show that P esters and phosphonates are the major organic P species present at the surface and at depth in sediments at both sites. Polyphosphates were only found in the surface sediment of the site overlain by oxic waters. The varying stability of polyphosphates in microorganisms under different redox conditions may, in part, explain their distribution as well as differences in P flux between the two sites

Increase of gut protection bioactivity induced by strawberry anthocyanins after in vitro digestion

Berries are one of the most consumed sources of bioactive polyphenols, including anthocyanins, and these compounds exert protective effects against initiation of colorectal cancer (CRC) by reducing DNA damage. Some varieties of freshly collected wild strawberry can reach up to 200mg/100g of anthocyanins. We hypothesised that physiologically relevant levels of Italian wild strawberry metabolites exiting the ileum would be both bioavailable and would exert positive effects on gut health. Five ileostomists completed a wild strawberry feeding study (11/NI/0112), ileal fluid was collected pre (0 h) and post (8 h) consumption of strawberries (225 g) and assessed for phytochemical composition by LCMSn. We simulated the interaction of the ileal fluids with colonic microbiota over a 24 h period (0, 5,10, 24 hr) using in vitro gut fermenter models. Nutri-kinetic analysis using LCMSn demonstrated significant increases in the concentration of gut microbiota-mediated polyphenolic metabolites over time. While changes in the bacterial composition of the gut fermenter models were monitored using fluorescent in situ hybridisation analysis with validated probes for Total bacteria, Bifidobacterium genus, Clostridium histolyticum/perfringens group, Faecalibacterium prausnitzii, Eubacterium rectale group, Bacteroides, Lactobacilli and Enterobacteria; limited changes observed. Bioactivity of the post-berry consumption ileal fermentates was assessed on two colonocyte cell lines (HT29 and CCD841 CON (normal)) using COMET assay. Post-berry ileal fermentate from all five ileostomists significantly (p<0.01) decreased DNA damage in both HT29 cells and CCD841 cells compared to untreated controls. To conclude, strawberry phytochemicals were available for colonic fermentation following ileal digestion and human microbiota-mediated fermentation which subsequently increased overall levels of polyphenolic metabolites, the post berry fermentates were demonstrated to reduce DNA damage in colonocytes