Both ends ofEscherichia coli ribosomal protein S13 are immunochemically accessiblein situ

To investigate the structure ofEscherichia coli ribosomal protein S13 in 30S ribosomal subunits, we have previously generated 22 S13 specific monoclonal antibodies and mapped their specific epitopes to the S13 sequence. The availability of these S13 epitopesin situ has been further examined by incubating these monoclonal antibodies with 30S ribosomal subunits and analyzing formation of monoclonal antibody-linked ribosome dimers by sucrose gradients centrifugation. We have found that none of the 22 monoclonal antibodies makes ribosome dimers individually as do typical antisera. However, one monoclonal antibody, designated AS13-MAb 2, reacts with 30S ribosomal subunits to form immunocomplexes sedimenting faster than subunit monomers. When AS13-MAb 2 is paired with any one of three monoclonal antibodies directed to the S13 C-terminal epitopes, dimer formation is observed. Other pairs of monoclonal antibodies directed to distinct S13 epitopes have been tested similarly for dimer formation. Monoclonal antibody AS13-MAb 22, directed to the N-terminal region of 22 residues, also causes subunits to form typical dimers, but only if paired with one of the three monoclonal antibodies directed to the S13 C-terminal region. The close proximity of the epitopes recognized by AS13-MAbs 2 and 22 has been established by the mutual competition between the antibodies binding to intact 30S subunits. These results corroborate our previous observation, using polyclonal antibodies, that S13 has more than one epitope exposed on 30S subunits. Our finding that sequences on both ends of the S13 molecule are immunochemically accessible provides information about the molecular organization of S13in situ.     

The sensitivity of Afromontane tarns in the Maloti-Drakensberg region of South Africa and Lesotho to acidic deposition

Despite their remoteness from sources of atmospheric pollutant emissions, the Afromontane tarns in the Maloti-Drakensberg region are perfect candidates to study the negative effects of acidifying atmospheric pollution, because mountain lakes are widely recognised as sentinel ecosystems, unimpacted by direct human disturbance within their catchments. Thirty-four tarns were sampled in the Maloti-Drakensberg region and most were found to be extremely sensitive to acidic deposition, as indicated by their low acid neutralising capacity. There are very few studies of freshwater critical loads for any region within South Africa. The steady-state water chemistry model (SSWC) was adapted and used to determine critical loads, whereas exceedance was estimated relative to modelled regional deposition data, in order to understand the risk of harmful effects to aquatic ecosystems. Seventy-six percent of sampled sites across the Maloti-Drakensberg would exceed critical loads even at the lowest modelled deposition levels, but there are no current measured deposition data for the region. The sensitivity of the Maloti-Drakensberg tarns needs to be considered in future policy formulation regarding acceptable levels of acidifying atmospheric pollution from South Africa’s energy sector and indicates the need for assessing aquatic ecosystem impacts in other regions of South Africa.     

OrthoMaM: A database of orthologous genomic markers for placental mammal phylogenetics

Background  

Molecular sequence data have become the standard in modern day phylogenetics. In particular, several long-standing questions of mammalian evolutionary history have been recently resolved thanks to the use of molecular characters. Yet, most studies have focused on only a handful of standard markers. The availability of an ever increasing number of whole genome sequences is a golden mine for modern systematics. Genomic data now provide the opportunity to select new markers that are potentially relevant for further resolving branches of the mammalian phylogenetic tree at various taxonomic levels.     

The Effect of Enterohemorrhagic E. coli Infection on the Cell Mechanics of Host Cells

Enterohaemorrhagic E. coli (EHEC) is a type of human pathogenic bacteria. The main virulence characteristics of EHEC include the formation of attaching and effacing lesions (A/E lesions) and the production of one or more Shiga-like toxins, which may induce human uremic complications. When EHEC infects host cells, it releases translocated intimin receptor (Tir) and effector proteins inside the host cells, inducing the rearrangement and accumulation of the F-actin cytoskeleton, a phenotype leading to the formation of pedestals in the apical cell surface, and the growth of stress fibers at the base of the cells. To examine the effect of EHEC infection on cell mechanics, we carried out a series of experiments to examine HeLa cells with and without EHEC infection to quantify the changes in (1) focal adhesion area, visualized by anti-vinculin staining; (2) the distribution and orientation of stress fibers; and (3) the intracellular viscoelasticity, via directional video particle tracking microrheology. Our results indicated that in EHEC-infected HeLa cells, the focal adhesion area increased and the actin stress fibers became thicker and more aligned. The cytoskeletal reorganization induced by EHEC infection mediated a dramatic increase in the cytoplasmic elastic shear modulus of the infected cells, and a transition in the viscoelastic behavior of the cells from viscous-like to elastic-like. These changes in mechanobiological characteristics might modulate the attachments between EHEC and the host cell to withstand exfoliation, and between the host cell and the extracellular matrix, and might also alter epithelial integrity.     

ARTIFICIAL SELECTION ON RELATIVE BRAIN SIZE REVEALS A POSITIVE GENETIC CORRELATION BETWEEN BRAIN SIZE AND PROACTIVE PERSONALITY IN THE GUPPY

Animal personalities range from individuals that are shy, cautious, and easily stressed (a “reactive” personality type) to individuals that are bold, innovative, and quick to learn novel tasks, but also prone to routine formation (a “proactive” personality type). Although personality differences should have important consequences for fitness, their underlying mechanisms remain poorly understood. Here, we investigated how genetic variation in brain size affects personality. We put selection lines of large‐ and small‐brained guppies (Poecilia reticulata), with known differences in cognitive ability, through three standard personality assays. First, we found that large‐brained animals were faster to habituate to, and more exploratory in, open field tests. Large‐brained females were also bolder. Second, large‐brained animals excreted less cortisol in a stressful situation (confinement). Third, large‐brained animals were slower to feed from a novel food source, which we interpret as being caused by reduced behavioral flexibility rather than lack of innovation in the large‐brained lines. Overall, the results point toward a more proactive personality type in large‐brained animals. Thus, this study provides the first experimental evidence linking brain size and personality, an interaction that may affect important fitness‐related aspects of ecology such as dispersal and niche exploration.     

Comparison of inflammatory markers in induced and spontaneous sputum in a cohort of COPD patients

Background

Sputum induction is a non-invasive method for obtaining measurements of inflammation in the airways. Whether spontaneously sampled sputum can be a valid surrogate is unknown. The aim of this study was to compare levels of six inflammatory markers in sputum pairs consisting of induced and spontaneous sputum sampled on the same consultation either in a stable state or during exacerbations of chronic obstructive pulmonary disease (COPD).

Methods

433 COPD patients aged 40–76, Global initiative for chronic Obstructive Lung Disease (GOLD) stage II-IV were enrolled in 2006/07 and followed every six months for three years. 356 patients were followed for potential exacerbations. Interleukin-6, interleukin-8, interleukin-18, interferon gamma-inducible protein-10, monokine induced by gamma interferon and tumor necrosis factor-alpha (IL-6, IL-8, IL-18, IP-10, MIG and TNF-α) were measured by bead based multiplex immunoassay in 60 paired sputum samples from 45 patients. Albumin was measured by enzyme immunoassay, for concentration correction. Culturing for bacterial growth was performed on 24 samples. Bland-Altman plots were used to assess agreement. The paired non-parametric Wilcoxon signed-rank test, the non-parametric Spearman’s rank correlation test and Kruskal-Wallis test were used for statistical analyses. For all analyses, a p-value < 0.05 was considered significant.

Results

Agreement between the two measurements was generally low for all six markers. TNF-α was significantly higher in spontaneous sputum at exacerbations (p = 0.002) and trending higher at the steady state (p = 0.06). Correlation coefficients between the levels of markers in induced and spontaneous sputum varied between 0.58 (IL-18) to 0.83 (IP-10). In spontaneous sputum IL-18 and MIG were higher in ex-smokers (p < 0.05). The levels of all markers were higher in GOLD stage III & IV except for IL-6 in spontaneous sputum and IL-18 in induced sputum, compared with GOLD stage II, although not statistically significant. In spontaneous sputum the levels of IL-6 were significantly higher if Haemophilus influenzae (HI) was not cultured.

Conclusion

We observed a low agreement and significant differences in inflammatory markers between induced and spontaneous sputum, both at steady state and exacerbations. We recommend considering sampling method when reporting on inflammatory markers in sputum.     

Cytotoxic and aromatic constituents from Salvia miltiorrhiza

As part of an ongoing study of traditional Chinese medicinal plants, the root tissue of Salvia miltiorrhiza was further investigated for its chemical constituents. Five naturally occurring products along with 13 known constituents were isolated from an ethyl acetate-soluble portion of its ethanol extract. Their structures were elucidated by means of spectroscopic methods. Some selected compounds were also evaluated for biological activity.     

Enterohaemorrhagic Escherichia coli O157:H7 Shiga‐like toxin 1 is required for full pathogenicity and activation of the p38 mitogen‐activated protein kinase pathway in Caenorhabditis elegans

Enterohaemorrhagic Escherichia coli (EHEC) causes life‐threatening infections in humans as a consequence of the production of Shiga‐like toxins. Lack of a good animal model system currently hinders in vivo study of EHEC virulence by systematic genetic methods. Here we applied the genetically tractable animal, Caenorhabditis elegans, as a surrogate host to study the virulence of EHEC as well as the host immunity to this human pathogen. Our results show that E. coli O157:H7, a serotype of EHEC, infects and kills C. elegans. Bacterial colonization and induction of the characteristic attaching and effacing (A/E) lesions in the intact intestinal epithelium of C. elegans by E. coli O157:H7 were concomitantly demonstrated in vivo. Genetic analysis indicated that the Shiga‐like toxin 1 (Stx1) of E. coli O157:H7 is a virulence factor in C. elegans and is required for full toxicity. Moreover, the C. elegans p38 mitogen‐activated protein kinase (MAPK) pathway, anevolutionarily conserved innate immune and stress response signalling pathway, is activated in the regulation of host susceptibility to EHEC infection in a Stx1‐dependent manner. Our results validate the EHEC–C. elegans interaction as suitable for future comprehensive genetic screens for both novel bacterial and host factors involved in the pathogenesis of EHEC infection.