Comparative Genomics to Delineate Pathogenic Potential in Non-O157 Shiga Toxin-Producing Escherichia coli (STEC) from Patients with and without Haemolytic Uremic Syndrome (HUS) in Norway

Shiga toxin-producing Escherichia coli (STEC) cause infections in humans ranging from asymptomatic carriage to bloody diarrhoea and haemolytic uremic syndrome (HUS). Here we present whole genome comparison of Norwegian non-O157 STEC strains with the aim to distinguish between strains with the potential to cause HUS and less virulent strains. Whole genome sequencing and comparisons were performed across 95 non-O157 STEC strains. Twenty-three of these were classified as HUS-associated, including strains from patients with HUS (n = 19) and persons with an epidemiological link to a HUS-case (n = 4). Genomic comparison revealed considerable heterogeneity in gene content across the 95 STEC strains. A clear difference in gene profile was observed between strains with and without the Locus of Enterocyte Effacement (LEE) pathogenicity island. Phylogenetic analysis of the core genome showed high degree of diversity among the STEC strains, but all HUS-associated STEC strains were distributed in two distinct clusters within phylogroup B1. However, non-HUS strains were also found in these clusters. A number of accessory genes were found to be significantly overrepresented among HUS-associated STEC, but none of them were unique to this group of strains, suggesting that different sets of genes may contribute to the pathogenic potential in different phylogenetic STEC lineages. In this study we were not able to clearly distinguish between HUS-associated and non-HUS non-O157 STEC by extensive genome comparisons. Our results indicate that STECs from different phylogenetic backgrounds have independently acquired virulence genes that determine pathogenic potential, and that the content of such genes is overlapping between HUS-associated and non-HUS strains.     

Functional Genetic Analysis of the GarML Gene Cluster in Lactococcus garvieae DCC43 Gives New Insights into Circular Bacteriocin Biosynthesis

Garvicin ML (GarML) is a circular bacteriocin produced by Lactococcus garvieae DCC43. The recently published draft genome of this strain allowed determination of the genetic background for bacteriocin production. Bioinformatic analysis identified a gene cluster consisting of nine open reading frames likely involved in the production of and immunity to GarML. The garA gene encodes the bacteriocin precursor, garX a large transmembrane protein, garBCDE a putative immunity protein (garB) followed by an ATPase and two transmembrane proteins, and garFGH a putative ABC transporter complex. Functional genetic analysis revealed that deletion of garFGH had no effect on sensitivity to or production of GarML. In contrast, deletion of garBCDE or inactivation of garX resulted in high-level sensitivity to GarML and completely abolished production of active bacteriocin. Mass spectrometry of culture supernatants revealed that wild-type cultures contained the mature circular form as well as the linear forms of the bacteriocin, both with and without the three-amino-acid leader sequence, while bacteriocin-negative mutants contained only the linear forms. These results indicate that cleavage of the leader peptide precedes circularization and is likely performed by a functional entity separate from the GarML gene cluster. To our knowledge, this is the first conclusive evidence for these processes being separated in time. Loss of immunity and antimicrobial activity in addition to our inability to detect the circular bacteriocin in the ΔgarBCDE and garX::pCG47 mutants demonstrate that both these units are indispensable for GarML biosynthesis as well as immunity. Furthermore, the results indicate that these genes are implicated in the circularization of the bacteriocin and that their functions are probably interlinked.     

Synovial explant inflammatory mediator production corresponds to rheumatoid arthritis imaging hallmarks: a cross-sectional study

Introduction

Despite the widespread use of magnetic resonance imaging (MRI) and Doppler ultrasound for the detection of rheumatoid arthritis (RA) disease activity, little is known regarding the association of imaging-detected activity and synovial pathology. The purpose of this study was to compare site-specific release of inflammatory mediators and evaluate the corresponding anatomical sites by examining colour Doppler ultrasound (CDUS) and MRI scans.

Methods

RA patients were evaluated on the basis of CDUS and 3-T MRI scans and subsequently underwent synovectomy using a needle arthroscopic procedure of the hand joints. The synovial tissue specimens were incubated for 72 hours, and spontaneous release of monocyte chemoattractant protein 1 (MCP-1), interleukin 6 (IL-6), macrophage inflammatory protein 1β (MIP-1β) and IL-8 was measured by performing multiplex immunoassays. Bone marrow oedema (BME), synovitis and erosion scores were estimated on the basis of the rheumatoid arthritis magnetic resonance imaging score (RAMRIS). Mixed models were used for the statistical analyses. Parsimony was achieved by omitting covariates with P > 0.1 from the statistical model.

Results

Tissue samples from 58 synovial sites were obtained from 25 patients. MCP-1 was associated with CDUS activity (P = 0.009, approximate Spearman’s ρ = 0.41), RAMRIS BME score (P = 0.01, approximate Spearman’s ρ = 0.42) and RAMRIS erosion score (P = 0.03, approximate Spearman’s ρ = 0.31). IL-6 was associated with RAMRIS synovitis score (P = 0.04, approximate Spearman’s ρ = 0.50), BME score (P = 0.04, approximate Spearman’s ρ = 0.31) and RAMRIS erosion score (P = 0.03, approximate Spearman’s ρ = 0.35). MIP-1β was associated with CDUS activity (P = 0.02, approximate Spearman’s ρ = 0.38) and RAMRIS synovitis scores (P = 0.02, approximate Spearman’s ρ = 0.63). IL-8 associations with imaging outcome measures did not reach statistical significance.

Conclusions

The association between imaging activity and synovial inflammatory mediators underscores the high sensitivity of CDUS and MRI in the evaluation of RA disease activity. The associations found in our present study have different implications for synovial mediator releases and corresponding imaging signs. For example, MCP-1 and IL-6 were associated with both general inflammation and bone destruction, in contrast to MIP-1β, which was involved solely in general synovitis. The lack of association of IL-8 with synovitis was likely underestimated because of a large proportion of samples above assay detection limits among the patients with the highest synovitis scores.     

Validation of water flux and body composition in glaucous gulls (Larus hyperboreus)

Water influx rates (WIR) measured with tritiated water dilution were compared with direct measures of water and energy intake in glaucous gulls (Larus hyperboreus). Total body water (TBW) measured isotopically was also compared with TBW determined by body composition analysis (BCA) of the same birds. Seventeen wild gulls were captured and studied in outdoor enclosures at Ny-Alesund, Svalbard, in July 2002. Gulls were hand-fed known quantities of Arctic cod (Boreogadus saida) or given water on the basis of one of four experimental treatments: (A) fasting, (B) fish only, (C) water only, or (D) fish and water. Water and energy content of Arctic cod was also determined. WIR of gulls (after subtracting metabolic water production) in treatments A, B, C, and D were 0, 101 +/- 5, 62 +/- 19, and 122 +/- 21 SD g d(-1), respectively. Measured water intake in each group was 0, 111 +/- 2, 64 +/- 3, and 134 +/- 15 SD g d(-1), respectively. On average, WIR underestimated measured water intake in each group. Errors were lowest but most variable for gulls fed water only (-2.2% +/- 32.8%) compared with gulls fed fish only (-9.0% +/- 5.4%) or fish and water (-9.0% +/- 7.0%). Compared with measured water intake, errors in WIR were relatively low overall (-6.9% +/- 17.4%) and comparable to previous validation studies. The difference in TBW determined by BCA versus isotopic dilution ranged between -1.02% and +8.59% of mass. On average, TBW measured isotopically (632 +/- 24 g kg(-1)) overestimated true body water by a factor of 1.033.     

Effect of spaceflight on the subcutaneous venoarteriolar reflex in the human lower leg.

Whenever the legs are lowered in humans, a venoarteriolar reflex is activated by the hydrostatic distension of the venules. Through local axon reflexes, the adjacent arterioles are contracted to decrease blood flow and prevent formation of edema. Because the venoarteriolar reflex is activated by gravity, we tested the hypothesis that long-term weightlessness would attenuate it. The reduction in subcutaneous blood flow was measured by the (133)Xe washout technique just proximal to the ankle joint in dependent lower legs of eight supine astronauts, where the knee joint was passively bent by 90 degrees . The measurements were conducted before spaceflight and 3-6 h on landing following 4-6.5 mo in space. Activation of the venoarteriolar reflex reduced subcutaneous blood flow by 37 +/- 9% (P = 0.016) before flight and by 64 +/- 8% (P < 0.001) following landing with no statistical significant difference between the two reductions (P = 0.062). Therefore, our results show that the venoarteriolar reflex is not attenuated by weightlessness and therefore does not need the everyday stimulus of gravity to maintain efficiency.     

Thyroid Hormones Correlate with Basal Metabolic Rate but Not Field Metabolic Rate in a Wild Bird Species

Thyroid hormones (TH) are known to stimulate in vitro oxygen consumption of tissues in mammals and birds. Hence, in many laboratory studies a positive relationship between TH concentrations and basal metabolic rate (BMR) has been demonstrated whereas evidence from species in the wild is scarce. Even though basal and field metabolic rates (FMR) are often thought to be intrinsically linked it is still unknown whether a relationship between TH and FMR exists. Here we determine the relationship between the primary thyroid hormone triiodothyronine (T3) with both BMR and FMR in a wild bird species, the black-legged kittiwake (Rissa tridactyla). As predicted we found a strong and positive relationship between plasma concentrations of T3 and both BMR and mass-independent BMR with coefficients of determination ranging from 0.36 to 0.60. In contrast there was no association of T3 levels with either whole-body or mass-independent FMR (R² = 0.06 and 0.02, respectively). In accordance with in vitro studies our data suggests that TH play an important role in modulating BMR and may serve as a proxy for basal metabolism in wild birds. However, the lack of a relationship between TH and FMR indicates that levels of physical activity in kittiwakes are largely independent of TH concentrations and support recent studies that cast doubt on a direct linkage between BMR and FMR.     

Genome evolution of a tertiary dinoflagellate plastid

The dinoflagellates have repeatedly replaced their ancestral peridinin-plastid by plastids derived from a variety of algal lineages ranging from green algae to diatoms. Here, we have characterized the genome of a dinoflagellate plastid of tertiary origin in order to understand the evolutionary processes that have shaped the organelle since it was acquired as a symbiont cell. To address this, the genome of the haptophyte-derived plastid in Karlodinium veneficum was analyzed by Sanger sequencing of library clones and 454 pyrosequencing of plastid enriched DNA fractions. The sequences were assembled into a single contig of 143 kb, encoding 70 proteins, 3 rRNAs and a nearly full set of tRNAs. Comparative genomics revealed massive rearrangements and gene losses compared to the haptophyte plastid; only a small fraction of the gene clusters usually found in haptophytes as well as other types of plastids are present in K. veneficum. Despite the reduced number of genes, the K. veneficum plastid genome has retained a large size due to expanded intergenic regions. Some of the plastid genes are highly diverged and may be pseudogenes or subject to RNA editing. Gene losses and rearrangements are also features of the genomes of the peridinin-containing plastids, apicomplexa and Chromera, suggesting that the evolutionary processes that once shaped these plastids have occurred at multiple independent occasions over the history of the Alveolata.     

Structural characterisation of Tpx from Yersinia pseudotuberculosis reveals insights into the binding of salicylidene acylhydrazide compounds

Thiol peroxidase, Tpx, has been shown to be a target protein of the salicylidene acylhydrazide class of antivirulence compounds. In this study we present the crystal structures of Tpx from Y. pseudotuberculosis (ypTpx) in the oxidised and reduced states, together with the structure of the C61S mutant. The structures solved are consistent with previously solved atypical 2-Cys thiol peroxidases, including that for "forced" reduced states using the C61S mutant. In addition, by investigating the solution structure of ypTpx using small angle X-ray scattering (SAXS), we have confirmed that reduced state ypTpx in solution is a homodimer. The solution structure also reveals flexibility around the dimer interface. Notably, the conformational changes observed between the redox states at the catalytic triad and at the dimer interface have implications for substrate and inhibitor binding. The structural data were used to model the binding of two salicylidene acylhydrazide compounds to the oxidised structure of ypTpx. Overall, the study provides insights into the binding of the salicylidene acylhydrazides to ypTpx, aiding our long-term strategy to understand the mode of action of this class of compounds.