Assessing the suitability of three proxy sources for the development of detectors of special nuclear materials.

Numerous techniques and equipment have been developed to provide a capability for the detection of special nuclear materials (SNM), but due to the necessary security measures surrounding these materials alternate, or proxy, neutron sources are often utilised in their stead. In this paper we report the neutron and gamma pulse shape discrimination response of plastic scintillator to mixed neutron/gamma beams produced from two radionuclide neutron sources, and also from an SNM source of weapons-grade plutonium. We discuss the suitability of using radionuclide sources, with appropriate shielding configurations as proxy sources for SNM. A 3σnth-γ discrimination level has been achieved for an SNM source at a low-level energy threshold of ∼220 keVee when a shielding configuration of 5 cm of lead was implemented. Varying amounts of lead and high-density polyethylene (HDPE) shielding were also investigated with the 3σ limit being reached by ∼240 keVee. This work shows that an AmBe neutron source serves as an appropriate SNM proxy achieving a comparable value for figure of merit above ∼1 MeVee. For energies below 1 MeVee down to ∼100 keVee a closer approximation of the expected FoM for SNM can be attained when using 252Cf as a proxy source or by utilising an 'enhanced' AmBe source with the addition of a further low energy γ ray source.

Loss of Dynamin 2 GTPase function results in microcytic anaemia.

In a dominant mouse ethylnitrosurea mutagenesis screen for genes regulating erythropoiesis, we identified a pedigree with a novel microcytic hypochromia caused by a V235G missense mutation in Dynamin 2 (Dnm2). Mutations in Dnm2, a GTPase, are highly disease-specific and have been implicated in four forms of human diseases: centronuclear myopathy, Charcot-Marie Tooth neuropathy and, more recently, T-cell leukaemia and Hereditary Spastic Paraplegia, but red cell abnormalities have not been reported to date. The V235G mutation lies within a crucial GTP nucleotide-binding pocket of Dnm2, and resulted in defective GTPase activity and incompatibility with life in the homozygous state. Dnm2 is an essential mediator of clathrin-mediated endocytosis, which is required for the uptake of transferrin (Tf) into red cells for incorporation of haem. Accordingly, we observed significantly reduced Tf uptake by Dnm2+/V235G cells, which led to impaired endosome formation. Despite these deficiencies, surprisingly all iron studies were unchanged, suggesting an unexplained alternative mechanism underlies microcytic anaemia in Dnm2+/V235G mice. This study provides the first in vivo evidence for the requirements of Dnm2 in normal erythropoiesis.

SH3 Domains Differentially Stimulate Distinct Dynamin I Assembly Modes and G Domain Activity.

Dynamin I is a highly regulated GTPase enzyme enriched in nerve terminals which mediates vesicle fission during synaptic vesicle endocytosis. One regulatory mechanism involves its interactions with proteins containing Src homology 3 (SH3) domains. At least 30 SH3 domain-containing proteins bind dynamin at its proline-rich domain (PRD). Those that stimulate dynamin activity act by promoting its oligomerisation. We undertook a systematic parallel screening of 13 glutathione-S-transferase (GST)-tagged endocytosis-related SH3 domains on dynamin binding, GTPase activity and oligomerisation. No correlation was found between dynamin binding and their potency to stimulate GTPase activity. There was limited correlation between the extent of their ability to stimulate dynamin activity and the level of oligomerisation, indicating an as yet uncharacterised allosteric coupling of the PRD and G domain. We examined the two variants, dynamin Iab and Ibb, which differ in the alternately splice middle domain α2 helix. They responded differently to the panel of SH3s, with the extent of stimulation between the splice variants varying greatly between the SH3s. This study reveals that SH3 binding can act as a heterotropic allosteric regulator of the G domain via the middle domain α2 helix, suggesting an involvement of this helix in communicating the PRD-mediated allostery. This indicates that SH3 binding both stabilises multiple conformations of the tetrameric building block of dynamin, and promotes assembly of dynamin-SH3 complexes with distinct rates of GTP hydrolysis.

Altered transcription of murine genes induced in the small bowel by administration of probiotic strain Lactobacillus rhamnosus HN001.

Lactobacillus rhamnosus HN001 is a probiotic strain reported to increase resistance to epithelium-adherent and -invasive intestinal pathogens in experimental animals. To increase understanding of the relationship between strain HN001 and the bowel, transcription of selected genes in the mucosa of the murine small bowel was measured. Mice previously naive to lactobacilli (Lactobacillus-free mice) were examined after daily exposure to HN001 in drinking water. Comparisons were made to results from matched Lactobacillus-free mice. Infant and adult mice were investigated to provide a temporal view of gene expression in response to exposure to HN001. Genes sgk1, angptl4, and hspa1b, associated with the apoptosis pathway, were selected for investigation by reverse transcription-quantitative PCR on the basis of a preliminary duodenal DNA microarray screen. Normalized to gapdh gene transcription, these three genes were upregulated after 6 to 10 days exposure of adult mice to HN001. Angptl4 was shown by immunofluorescence to be upregulated in duodenal epithelial cells of mucosal samples. Epithelial cell migration was faster in HN001-exposed mice than in the Lactobacillus-free controls. Transcriptional responses in infant mice differed according to bowel region and age. For example, sgk1 was upregulated in duodenal, jejunal, and ileal mucosa of mice less than 25 days old, whereas angptl4 and hspa1b were upregulated at 10 days in the duodenum but downregulated in the jejunal mucosa until mice were 25 days old. Overall, the results provide links between a probiotic strain, mucosal gene expression, and host phenotype, which may be useful in delineating mechanisms of probiotic action.

Unique secreted-surface protein complex of Lactobacillus rhamnosus, identified by phage display.

Proteins are the most diverse structures on bacterial surfaces; hence, they are candidates for species- and strain-specific interactions of bacteria with the host, environment, and other microorganisms. Genomics has decoded thousands of bacterial surface and secreted proteins, yet the function of most cannot be predicted because of the enormous variability and a lack of experimental data that would allow deduction of function through homology. Here, we used phage display to identify a pair of interacting extracellular proteins in the probiotic bacterium Lactobacillus rhamnosus HN001. A secreted protein, SpcA, containing two bacterial immunoglobulin-like domains type 3 (Big-3) and a domain distantly related to plant pathogen response domain 1 (PR-1-like) was identified by screening of an L. rhamnosus HN001 library using HN001 cells as bait. The SpcA-"docking" protein, SpcB, was in turn detected by another phage display library screening, using purified SpcA as bait. SpcB is a 3275-residue cell-surface protein that contains general features of large glycosylated Serine-rich adhesins/fibrils from gram-positive bacteria, including the hallmark signal sequence motif KxYKxGKxW. Both proteins are encoded by genes within a L. rhamnosus-unique gene cluster that distinguishes this species from other lactobacilli. To our knowledge, this is the first example of a secreted-docking protein pair identified in lactobacilli.

FRQ-interacting RNA helicase mediates negative and positive feedback in the Neurospora circadian clock.

The Neurospora circadian oscillator comprises FREQUENCY (FRQ) and its transcription activator, the White Collar Complex (WCC). Repression of WCC's transcriptional activity by FRQ via negative feedback is indispensable for clock function. An unbiased genetic screen that targeted mutants with defects in negative feedback regulation yielded a fully viable arrhythmic strain bearing a novel allele of FRQ-interacting RNA helicase (frh), an essential gene that encodes a putative exosome component protein. In the allele, frh(R806H), clock function is completely disturbed, while roles of FRQ-interacting RNA helicase (FRH) essential for viability are left intact. FRH(R806H) still interacts with FRQ, but interaction between the FRQ-FRH(R806H) complex (FFC) and WCC is severely affected. Phosphorylation of WC-1 is reduced in the mutant leading to constantly elevated WCC activity, which breaks the negative feedback loop. WCC levels are considerably reduced in the mutant, especially those of WC-1, consistent both with loss of positive feedback (FRQ-dependent WC-1 stabilization) and with a reduced level of the FRQ-mediated WCC phosphorylation that leads to high WCC activity accompanied by rapid transcription-associated turnover. FRH overexpression promotes WC-1 accumulation, confirming that FRH together with FRQ plays a role in WC-1 stabilization. Identification of a viable allele of frh, displaying virtually complete loss of both negative and positive circadian feedback, positions FRH as a core component of the central oscillator that is permissive for rhythmicity but appears not to modulate periodicity. Moreover, the results suggest that there are clock-specific roles for FRH that are distinct from the predicted essential exosome-associated functions for the protein.

Direct selection and phage display of a Gram-positive secretome.

Surface, secreted and transmembrane protein-encoding open reading frames, collectively the secretome, can be identified in bacterial genome sequences using bioinformatics. However, functional analysis of translated secretomes is possible only if many secretome proteins are expressed and purified individually. We have now developed and applied a phage display system for direct selection, identification, expression and purification of bacterial secretome proteins.

Functionality of probiotics - potential for product development.

It is becoming increasingly accepted by consumers that live lactic acid bacteria do exert health benefits when eaten. In addition, it is also becoming recognised that not all probiotic bacteria are equal. It is now no longer just a question of providing sufficient numbers of viable bacteria in a product; industry must also provide proof of efficacy for each strain. In the early 1990s, Fonterra embarked on a programme to develop proprietary probiotic strains, and as a result, commercialised two strains, Bifidobacterium lactis HN019 and Lactobacillus rhamnosus HN001. Over the past decade, Fonterra has developed a significant body of peerreviewed published reports around these strains, including studies showing safety in animal and human trials, protection against pathogens such as Salmonella typhimurium and Escherichia coli O157:H7, modulation of human and animal immune markers at realistic dose rates, and efficacy in human clinical trials. Based on this work, HN019 and HN001 have been applied to several functional foods both by Fonterra (under the DR10 and DR20 brands, respectively) and by third parties (e.g. under the HOWARU brand by Danisco). While the 'gold standard' of proof of efficacy is a phase III clinical trial, ethical considerations as well as expense preclude the use of clinical trials as screening tools for probiotics. Therefore, biomarkers have to be employed to identify strains with probiotic utility, and to define the different positive health benefits of existing probiotic strains. However, as the mechanisms by which most probiotic bacteria exert their health benefits remain unclear, the question of which biomarkers accurately reflect efficacy in vivo remains unresolved. With recent technological advances, and the shift toward probiotics targeted to specific conditions, researchers are beginning to tease out how probiotic bacteria work, and it is this knowledge that will inform biomarker development and improve the ability to offer the market safe and effective probiotic functional foods.

Light and clock expression of the Neurospora clock gene frequency is differentially driven by but dependent on WHITE COLLAR-2.

Visible light is thought to reset the Neurospora circadian clock by acting through heterodimers of the WHITE COLLAR-1 and WHITE COLLAR-2 proteins to induce transcription of the frequency gene. To characterize this photic entrainment we examined frq expression in constant light, under which condition the mRNA and protein of this clock gene were strongly induced. In continuous illumination FRQ accumulated in a highly phosphorylated state similar to that seen at subjective dusk, the time at which a step from constant light to darkness sets the clock. Examination of frq expression in several wc-2 mutant alleles surprisingly revealed differential regulation when frq expression was compared between constant light, following a light pulse, and darkness (clock-driven expression). Construction of a wc-2 null strain then demonstrated that WC-2 is absolutely required for both light and clock-driven frq expression, in contrast to previous expectations based on presumptive nulls containing altered Zn-finger function. Additionally, we found that frq light signal transduction differs from that of other light-regulated genes. Thus clock and light-driven frq expression is differentially regulated by, but dependent on, WC-2.