The Conserved nhaAR Operon Is Drastically Divergent between B2 and Non-B2 Escherichia coli and Is Involved in Extra-Intestinal Virulence

The Escherichia coli species is divided in phylogenetic groups that differ in their virulence and commensal distribution. Strains belonging to the B2 group are involved in extra-intestinal pathologies but also appear to be more prevalent as commensals among human occidental populations. To investigate the genetic specificities of B2 sub-group, we used 128 sequenced genomes and identified genes of the core genome that showed marked difference between B2 and non-B2 genomes. We focused on the gene and its surrounding region with the strongest divergence between B2 and non-B2, the antiporter gene nhaA. This gene is part of the nhaAR operon, which is in the core genome but flanked by mobile regions, and is involved in growth at high pH and high sodium concentrations. Consistently, we found that a panel of non-B2 strains grew faster than B2 at high pH and high sodium concentrations. However, we could not identify differences in expression of the nhaAR operon using fluorescence reporter plasmids. Furthermore, the operon deletion had no differential impact between B2 and non-B2 strains, and did not result in a fitness modification in a murine model of gut colonization. Nevertheless, sequence analysis and experiments in a murine model of septicemia revealed that recombination in nhaA among B2 strains was observed in strains with low virulence. Finally, nhaA and nhaAR operon deletions drastically decreased virulence in one B2 strain. This effect of nhaAR deletion appeared to be stronger than deletion of all pathogenicity islands. Thus, a population genetic approach allowed us to identify an operon in the core genome without strong effect in commensalism but with an important role in extra-intestinal virulence, a landmark of the B2 strains.     

Correction to: Preliminary insights into the population characteristics and distribution of reef (Mobula alfredi) and oceanic (M. birostris) manta rays in French Polynesia

Coral Reefs - This erratum has been initiated as many corrections which were submitted during proofing were overlooked by vendor.     

SERS‐based biosensor for Alzheimer disease evaluation through the fast analysis of human serum

Alzheimer disease (AD) is the most common form of dementia in the elderly, progressively affecting the cognitive functions with a complex diagnostic procedure that limits the time for a prompt intervention. In this study we optimized a reliable protocol for the analysis of AD patients and healthy subjects' serum using the Surface Enhanced Raman Spectroscopy (SERS), taking into consideration the effect of different variables on the final spectra, analyzed and compared through multivariate analysis and correlated with hippocampus volume. As results, we demonstrated a statistical difference between the spectra collected from the two investigated groups, with an accuracy, precision and specificity of respectively 83%, 86%, and 86%. The correlation of these data with those obtained from MRI, demonstrated a direct correlation between Raman spectra and hippocampus degeneration showing the Raman Spectroscopy (RS) as a potential tool for the monitoring of AD progression and rehabilitation treatments.     

Estimates of forest floor litter frog communities: A comparison of two methods

Estimates of forest leaf litter frog density, mass, richness and diversity given by the widely used 8 m × 8 m large plot method (LPM) were compared with estimates obtained by a newly proposed method (small 2 m × 1 m plots with leaf removal; SPLR). The study site was an undisturbed area of the Atlantic Rainforest of Ilha Grande, an island located in the south of Rio de Janeiro State, Brazil. Twenty‐four LPM (totalling 1536 m² of forest floor) and 90 SPLR (totalling 180 m² of forest floor) were performed. The estimates obtained by the two methods differed markedly, indicating that even using a much smaller sampling area (11.7% of that of LPM), SPLR gave frog density estimates six times higher, and frog mass estimates approximately 2.5 times higher than estimates provided by LPM. The species richness and diversity obtained by the two methods were similar, despite the fact that the total area sampled with SPLR was much smaller. These data suggest that LPM may underestimate the abundance and biomass of leaf litter frogs in a given area.     

Over-expression and refolding of MAP kinase phosphatase 3

MAP kinase phosphatase 3 (MKP3, also known as DUSP6 and PYST1) is involved in extracellular signal receptor kinase (ERK) regulation and functions as a specific phosphatase to the activated (phosphorylated) forms of ERK1 and ERK2. MKP3 displays allosteric activation, which aids in tightly regulating its function to ERK substrates, but not other related MAPKs. Due to MKP3's specificity for the ERK signaling pathway, the development of specific activators or inhibitors to the enzyme have been suggested in order to expressly influence the ERK1 and ERK2 pathways. To produce the high yields of MKP3 protein necessary for physico-chemical characterization of MKP3 and for high throughput screening of its small-molecule activators and inhibitors, we have cloned, purified and, and identified refolding conditions suitable for producing full-length, human MKP3 from Escherichia coli inclusion bodies. Furthermore, we demonstrate the use of a 96-well plate format refolding assay in which the ERK-induced activity of MKP3 is simulated by 33% DMSO. The assay allowed for rapid detection of MKP3's function following a refolding screen in the absence of ERK and thus provides quick and inexpensive testing of MKP3 activity. Following screening, the refolded product was confirmed to be correctly folded by steady-state kinetic analysis and by the CD spectroscopy-determined secondary structure content. CD data were consistent with 36% helix and 14% sheet, which compared to an expected 32.9% helix and 12.4% sheet. These data indicated that MKP3 was properly folded, making it a suitable protein for use in functional studies.     

Identifying Hendra virus diversity in pteropid bats

Hendra virus (HeV) causes a zoonotic disease with high mortality that is transmitted to humans from bats of the genus Pteropus (flying foxes) via an intermediary equine host. Factors promoting spillover from bats to horses are uncertain at this time, but plausibly encompass host and/or agent and/or environmental factors. There is a lack of HeV sequence information derived from the natural bat host, as previously sequences have only been obtained from horses or humans following spillover events. In order to obtain an insight into possible variants of HeV circulating in flying foxes, collection of urine was undertaken in multiple flying fox roosts in Queensland, Australia. HeV was found to be geographically widespread in flying foxes with a number of HeV variants circulating at the one time at multiple locations, while at times the same variant was found circulating at disparate locations. Sequence diversity within variants allowed differentiation on the basis of nucleotide changes, and hypervariable regions in the genome were identified that could be used to differentiate circulating variants. Further, during the study, HeV was isolated from the urine of flying foxes on four occasions from three different locations. The data indicates that spillover events do not correlate with particular HeV isolates, suggesting that host and/or environmental factors are the primary determinants of bat-horse spillover. Thus future spillover events are likely to occur, and there is an on-going need for effective risk management strategies for both human and animal health.     

Remodelling of cortical actin where lytic granules dock at natural killer cell immune synapses revealed by super-resolution microscopy

Natural Killer (NK) cells are innate immune cells that secrete lytic granules to directly kill virus-infected or transformed cells across an immune synapse. However, a major gap in understanding this process is in establishing how lytic granules pass through the mesh of cortical actin known to underlie the NK cell membrane. Research has been hampered by the resolution of conventional light microscopy, which is too low to resolve cortical actin during lytic granule secretion. Here we use two high-resolution imaging techniques to probe the synaptic organisation of NK cell receptors and filamentous (F)-actin. A combination of optical tweezers and live cell confocal microscopy reveals that microclusters of NKG2D assemble into a ring-shaped structure at the centre of intercellular synapses, where Vav1 and Grb2 also accumulate. Within this ring-shaped organisation of NK cell proteins, lytic granules accumulate for secretion. Using 3D-structured illumination microscopy (3D-SIM) to gain super-resolution of ~100 nm, cortical actin was detected in a central region of the NK cell synapse irrespective of whether activating or inhibitory signals dominate. Strikingly, the periodicity of the cortical actin mesh increased in specific domains at the synapse when the NK cell was activated. Two-colour super-resolution imaging revealed that lytic granules docked precisely in these domains which were also proximal to where the microtubule-organising centre (MTOC) polarised. Together, these data demonstrate that remodelling of the cortical actin mesh occurs at the central region of the cytolytic NK cell immune synapse. This is likely to occur for other types of cell secretion and also emphasises the importance of emerging super-resolution imaging technology for revealing new biology.     

Comparing expression level-dependent features in codon usage with protein abundance: an analysis of 'predictive proteomics'

Synonymous codon usage is a commonly used means for estimating gene expression levels of Escherichia coli genes and has also been used for predicting highly expressed genes for a number of prokaryotic genomes. By comparison of expression level-dependent features in codon usage with protein abundance data from two proteome studies of exponentially growing E. coli and Bacillus subtilis cells, we try to evaluate whether the implicit assumption of this approach can be confirmed with experimental data. Log-odds ratio scores are used to model differences in codon usage between highly expressed genes and genomic average. Using these, the strength and significance of expression level-dependent features in codon usage were determined for the genes of the Escherichia coli, Bacillus subtilis and Haemophilus influenzae genomes. The comparison of codon usage features with protein abundance data confirmed a relationship between these to be present, although exceptions to this, possibly related to functional context, were found. For species with expression level-dependent features in their codon usage, the applied methodology could be used to improve in silico simulations of the outcome of two-dimensional gel electrophoretic experiments.     

Proteins containing non-native disulfide bonds generated by oxidative stress can act as signals for the induction of the heat shock response

While oxidative stress can induce a heat shock response, the primary signals that initiate activation have not been identified. To identify such signals, HepG2 and V 79 cells were exposed to menadione, a compound that redox-cycles to generate superoxide. The oxidative stress generated by menadione resulted in oxidation of protein thiols in a dose-dependent manner. This was followed by protein destabilization and denaturation, as determined by differential scanning calorimetry of whole cells. To directly evaluate the effect of non-native disulfides on protein conformation, Ca²⁺-ATPase, isolated from rabbit sarcoplasmic reticulum, was chemically modified to contain non-native intermolecular or glutathione (GHS)-mixed disulfides. Differential scanning calorimetry profiles and 1-anilinonaphthalene-8-sulfonic acid fluorescence indicated that formation of non-native disulfides produced protein destabilization, denaturation, and exposure of hydrophobic domains. Cellular proteins shown to contain oxidized thiols formed detergent-insoluble aggregates. Cells treated with menadione exhibited activation of HSF-1, accumulated Hsp 70 mRNA, and increased synthesis of Hsp 70. This work demonstrates that formation of physiologically relevant, non-native intermolecular and GSH-mixed disulfides causes proteins to destabilize, unfold such that hydrophobic domains are exposed, and initiate a signal for induction of the heat shock response. J. Cell. Physiol. 171:143–151, 1997. © 1997 Wiley-Liss, Inc.