Creep analysis of fuel plates for the Advanced Neutron Source

The reactor for the planned Advanced Neutron Source will use closely spaced arrays of fuel plates. The plates are thin and will have a core containing enriched uranium silicide fuel clad in aluminum. The heat load caused by the nuclear reactions within the fuel plates will be removed by flowing high-velocity heavy water through narrow channels between the plates. However, the plates will still be at elevated temperatures while in service, and the potential for excessive plate deformation because of creep must be considered. An analysis to include creep for deformation and stresses because of temperature over a given time span has been performed and is reported herein

Dynamic pressure approach to analysis of reactor fuel plate stability

The dynamic pressure model can conveniently be used to evaluate the critical stress regions as a function of flow velocity. For some of the preliminary advanced neutron source reactor plate designs this could be very significant since the flow velocity could be limited by peak stresses in the plates more than by deflection or stability. The dynamic pressure results predicts the differential pressure across a plate as a function of flow velocity. The pressure differential can then be used to find the deflection and/or stress of the plate using traditional plate analyses. Instability would occur when plates are touching at mid-channel such that rapid oscillations of pressure can occur. The technique is conservative and gives a design limit for the plate. This model is one of several methods being used in the design of the ANS fuel elements. 10 refs., 4 figs

The AlgZR Two-Component System Recalibrates the RsmAYZ Posttranscriptional Regulatory System To Inhibit Expression of the Pseudomonas aeruginosa Type III Secretion System

Pseudomonas aeruginosa causes chronic airway infections in cystic fibrosis (CF) patients. A classic feature of CF airway isolates is the mucoid phenotype. Mucoidy arises through mutation of the mucA anti-sigma factor and subsequent activation of the AlgU regulon. Inactivation of mucA also results in reduced expression of the Vfr transcription factor. Vfr regulates several important virulence factors, including a type III secretion system (T3SS). In the present study, we report that ExsA expression, the master regulator of T3SS gene expression, is further reduced in mucA mutants through a Vfr-independent mechanism involving the RsmAYZ regulatory system. RsmA is an RNA binding protein required for T3SS gene expression. Genetic experiments suggest that the AlgZR two-component system, part of the AlgU regulon, inhibits ExsA expression by increasing the expression of RsmY and RsmZ, two small noncoding RNAs that sequester RsmA from target mRNAs. Epistasis analyses revealed that increasing the concentration of free RsmA, through either rsmYZ deletion or increased RsmA expression, partially restored T3SS gene expression in the mucA mutant. Furthermore, increasing RsmA availability in combination with Vfr complementation fully restored T3SS expression. Recalibration of the RsmAYZ system by AlgZR, however, did not alter the expression of other selected RsmA-dependent targets. We account for this observation by showing that ExsA expression is more sensitive to changes in free RsmA than other members of the RsmA regulon. Together, these data indicate that recalibration of the RsmAYZ system partially accounts for reduced T3SS gene expression in mucA mutants

Pressure reversal study through tensile tests

This paper is a summary of the results from a study of the variables related to pressure reversal and was sponsored by the US Department of Transportation, Office of Pipeline Safety. The circumferential pipe stress, which is the most significant variable in pressure reversal, was examined by using tensile specimens and then relating the results to pressurized pipe. A model is proposed that gives some insight into how pressure reversal can be minimized when a section of pipe is being hydrotested. Twenty tensile specimens from X-42 electric resistance welded (ERW) pipe and twenty specimens from X-52 ERW pipe were tested. Each specimen had a machined flaw. The flaw regions were monitored using strain gages and photoelasticity. These tensile tests represent the first phase of a research effort to examine and understand the variables related to pressure reversal. The second phase of this effort will be with pipe specimens and presently is in progress

Scotland’s biodiversity progress to 2020 Aichi Targets:Conserving genetic diversity- development of a national approach for addressing Aichi Biodiversity Target 13 that includes wild species

Aichi Target 13 (T13) focuses on the conservation of genetic diversity. •Major challenges in implementing T13 are that the type of genetic diversity to conserve is not clearly defined, and that key issues in genetic conservation vary across different sectors (e.g., forestry vs agriculture vs other species of socio-economic importance). •In Scotland and the UK more widely, baseline mechanisms are well established for assessing and reporting on genetic diversity in species of agricultural importance (e.g., rare livestock breeds, crop wild relatives), and a methodology has been established for ornamental plants. •A new UK Strategy for Forest Genetics Resources was launched in 2019, creating a framework for linking forest trees into T13 reporting. •However, there is no clear strategy to deal with ‘other species of socio-economic importance’ in Scotland, the UK or indeed elsewhere, and addressing this gap is the major focus of this report. •There is a lack of guidance for identifying focal species of socio-economic importance, and no clear mechanism for addressing T13 for these species once they have been identified. •To address this, we have identified a set of criteria for defining terrestrial and freshwater species of socio-economic importance in Scotland, and selected an initial list of 26 species. •The criteria applied were: -National conservation priority wild species. -Species of national cultural importance. -Species providing key ecosystem services. -Species of importance for wild harvesting (food and medicine). -Economically important game species. •We then developed a simple, readily applicable scorecard method for assessing risks to the conservation of genetic diversity in these species. •The scorecard approach is not dependent on prior genetic knowledge, and instead uses structured expert opinion assessments of whether: -Demographic declines are likely to lead to loss of genetic diversity (genetic erosion). -Hybridisation is likely to lead to undesirable replacement of genetic diversity. -Restrictions to regeneration/turnover are likely to impede evolutionary change. •For plant species where seed-banking is a viable mechanism for holding genetic resources ex situ,we also report on the representativeness of these ex situ collections. •Overall, this scorecard provides a mechanism for incorporating ‘other species of socio-economic importance’ into T13 actions and reporting. •Furthermore, its application is not restricted to Aichi T13 as the approach is designed as a generic scorecard for genetic diversity. It is thus relevant to post-2020 CBD targets focusing on genetic diversity. •Future priorities include: -Extension to other species of socio-economic, commercial and cultural importance (with the inclusion of marine species being a particularly high priority). -Harmonising genetic conservation strategies between sectors (drawing on commonalities), whilst minimising disruption of existing well-established methodologies within sectors. -Greater incorporation of genomic data into monitoring genetic diversity (particularly in the agricultural and forestry sectors where data availability is potentially high)

The Pseudomonas aeruginosa Vfr Regulator Controls Global Virulence Factor Expression through Cyclic AMP-Dependent and -Independent Mechanisms

Vfr is a global regulator of virulence factor expression in the human pathogen Pseudomonas aeruginosa. Although indirect evidence suggests that Vfr activity is controlled by cyclic AMP (cAMP), it has been hypothesized that the putative cAMP binding pocket of Vfr may accommodate additional cyclic nucleotides. In this study, we used two different approaches to generate apo-Vfr and examined its ability to bind a representative set of virulence gene promoters in the absence and presence of different allosteric effectors. Of the cyclic nucleotides tested, only cAMP was able to restore DNA binding activity to apo-Vfr. In contrast, cGMP was capable of inhibiting cAMP-Vfr DNA binding. Further, we demonstrate that vfr expression is autoregulated and cAMP dependent and involves Vfr binding to a previously unidentified site within the vfr promoter region. Using a combination of in vitro and in vivo approaches, we show that cAMP is required for Vfr-dependent regulation of a specific subset of virulence genes. In contrast, we discovered that Vfr controls expression of the lasR promoter in a cAMP-independent manner. In summary, our data support a model in which Vfr controls virulence gene expression by distinct (cAMP-dependent and -independent) mechanisms, which may allow P. aeruginosa to fine-tune its virulence program in response to specific host cues or environments

An unusual CsrA family member operates in series with RsmA to amplify posttranscriptional responses in Pseudomonas aeruginosa

Members of the CsrA family of prokaryotic mRNA-binding proteins alter the translation and/or stability of transcripts needed for numerous global physiological processes. The previously described CsrA family member in Pseudomonas aeruginosa (RsmA) plays a central role in determining infection modality by reciprocally regulating processes associated with acute (type III secretion and motility) and chronic (type VI secretion and biofilm formation) infection. Here we describe a second, structurally distinct RsmA homolog in P. aeruginosa (RsmF) that has an overlapping yet unique regulatory role. RsmF deviates from the canonical 5 β-strand and carboxyl-terminal α-helix topology of all other CsrA proteins by having the α-helix internally positioned. Despite striking changes in topology, RsmF adopts a tertiary structure similar to other CsrA family members and binds a subset of RsmA mRNA targets, suggesting that RsmF activity is mediated through a conserved mechanism of RNA recognition. Whereas deletion of rsmF alone had little effect on RsmA-regulated processes, strains lacking both rsmA and rsmF exhibited enhanced RsmA phenotypes for markers of both type III and type VI secretion systems. In addition, simultaneous deletion of rsmA and rsmF resulted in superior biofilm formation relative to the wild-type or rsmA strains. We show that RsmF translation is derepressed in an rsmA mutant and demonstrate that RsmA specifically binds to rsmF mRNA in vitro, creating a global hierarchical regulatory cascade that operates at the posttranscriptional level

Modified Needle-Tip PcrV Proteins Reveal Distinct Phenotypes Relevant to the Control of Type III Secretion and Intoxication by Pseudomonas aeruginosa

The type III secretion system (T3SS) is employed to deliver effector proteins to the cytosol of eukaryotic hosts by multiple species of Gram-negative bacteria, including Pseudomonas aeruginosa. Translocation of effectors is dependent on the proteins encoded by the pcrGVHpopBD operon. These proteins form a T3S translocator complex, composed of a needle-tip complex (PcrV), translocons (PopB and PopD), and chaperones (PcrG and PcrH). PcrV mediates the folding and insertion of PopB/PopD in host plasmic membranes, where assembled translocons form a translocation channel. Assembly of this complex and delivery of effectors through this machinery is tightly controlled by PcrV, yet the multifunctional aspects of this molecule have not been defined. In addition, PcrV is a protective antigen for P. aeruginosa infection as is the ortholog, LcrV, for Yersinia. We constructed PcrV derivatives containing in-frame linker insertions and site-specific mutations. The expression of these derivatives was regulated by a T3S-specific promoter in a pcrV-null mutant of PA103. Nine derivatives disrupted the regulation of effector secretion and constitutively released an effector protein into growth medium. Three of these regulatory mutants, in which the linker was inserted in the N-terminal globular domain, were competent for the translocation of a cytotoxin, ExoU, into eukaryotic host cells. We also isolated strains expressing a delayed-toxicity phenotype, which secrete translocators slowly despite the normal level of effector secretion. Most of the cytotoxic translocation-competent strains retained the protective epitope of PcrV derivatives, and Mab166 was able to protect erythrocytes during infection with these strains. The use of defined PcrV derivatives possessing distinct phenotypes may lead to a better understanding of the functional aspects of T3 needle-tip proteins and the development of therapeutic agents or vaccines targeting T3SS-mediated intoxication

Bacterial Delivery of Nuclear Proteins into Pluripotent and Differentiated Cells

Numerous Gram negative pathogens possess a type III secretion system (T3SS) which allows them to inject virulent proteins directly into the eukaryotic cell cytoplasm. Injection of these proteins is dependent on a variable secretion signal sequence. In this study, we utilized the N-terminal secretion signal sequence of Pseudomonas aeruginosa exotoxin ExoS to translocate Cre recombinase containing a nuclear localization sequence (Cre-NLS). Transient exposure of human sarcoma cell line, containing Cre-dependent lacZ reporter, resulted in efficient recombination in the host chromosome, indicating that the bacterially delivered protein was not only efficiently localized to the nucleus but also retained its biological function. Using this system, we also illustrate the ability of P. aeruginosa to infect mouse embryonic stem cells (mESC) and the susceptibility of these cells to bacterially delivered Cre-NLS. A single two-hour infection caused as high as 30% of the mESC reporter cells to undergo loxP mediated chromosomal DNA recombination. A simple antibiotic treatment completely eliminated the bacterial cells following the delivery, while the use of an engineered mutant strain greatly reduced cytotoxicity. Utility of the system was demonstrated by delivery of the Cre-NLS to induced pluripotent stem cells to excise the floxed oncogenic nuclear reprogramming cassette. These results validate the use of T3SS for the delivery of transcription factors for the purpose of cellular reprogramming

Enzymatic Depilation of Animal Hide: Identification of Elastase (LasB) from Pseudomonas aeruginosa MCM B-327 as a Depilating Protease

Conventional leather processing involving depilation of animal hide by lime and sulphide treatment generates considerable amounts of chemical waste causing severe environmental pollution. Enzymatic depilation is an environmentally friendly process and has been considered to be a viable alternative to the chemical depilation process. We isolated an extracellular protease from Pseudomonas aeruginosa strain MCM B-327 with high depilation activity using buffalo hide as a substrate. This 33 kDa protease generated a peptide mass fingerprint and de novo sequence that matched perfectly with LasB (elastase), of Pseudomonas aeruginosa. In support of this data a lasB mutant of MCM B-327 strain lacked depilatory activity and failed to produce LasB. LasB heterologously over-produced and purified from Escherichia coli also exhibited high depilating activity. Moreover, reintroduction of the lasB gene to the P. aeruginosa lasB mutant via a knock-in strategy also successfully restored depilation activity thus confirming the role of LasB as the depilating enzyme