Structure and Interactions of the Cytoplasmic Domain of the Yersinia Type III Secretion Protein YscD

The virulence of a large number of Gram-negative bacterial pathogens depends on the type III secretion (T3S) system, which transports select bacterial proteins into host cells. An essential component of the Yersinia T3S system is YscD, a single-pass inner membrane protein. We report here the 2.52-Å resolution structure of the cytoplasmic domain of YscD, called YscDc. The structure confirms that YscDc consists of a forkhead-associated (FHA) fold, which in many but not all cases specifies binding to phosphothreonine. YscDc, however, lacks the structural properties associated with phosphothreonine binding and thus most likely interacts with partners in a phosphorylation-independent manner. Structural comparison highlighted two loop regions, L3 and L4, as potential sites of interactions. Alanine substitutions at L3 and L4 had no deleterious effects on protein structure or stability but abrogated T3S in a dominant negative manner. To gain insight into the function of L3 and L4, we identified proteins associated with YscD by affinity purification coupled to mass spectrometry. The lipoprotein YscJ was found associated with wild-type YscD, as was the effector YopH. Notably, the L3 and L4 substitution mutants interacted with more YopH than did wild-type YscD. These substitution mutants also interacted with SycH (the specific chaperone for YopH), the putative C-ring component YscQ, and the ruler component YscP, whereas wild-type YscD did not. These results suggest that substitutions in the L3 and L4 loops of YscD disrupted the dissociation of SycH from YopH, leading to the accumulation of a large protein complex that stalled the T3S apparatus.     

Morphometric and molecular variation in concert: taxonomy and genetics of the reticulate Pyrenean and Iberian alpine spiny fescues (Festuca eskia complex,Poaceae)

The Iberian mountain spiny fescues are a reticulate group of five diploid grass taxa consisting of three parental species and two putative hybrids: F. × souliei (F. eskia × F. quadriflora) and F. × picoeuropeana (F. eskia × F. gautieri). Phenotypic and molecular studies were conducted with the aim of determining the taxonomic boundaries and genetic relationships of the five taxa and disentangling the origins of the two hybrids. Statistical analyses of 31 selected phenotypic traits were conducted on individuals from 159 populations and on nine type specimens. Molecular analyses of random amplified polymorphic DNA (RAPD) markers were performed on 29 populations. The phenotypic analyses detected significant differences between the five taxa and demonstrated the overall intermediacy of the F. × picoeuropeana and F. × souliei between their respective parents. The RAPD analysis corroborated the genetic differentiation of F. eskia, F. gautieri and F. quadriflora and the intermediate nature of the two hybrids; however, they also detected genetic variation within F. × picoeuropeana. These results suggest distinct origins for F. × picoeuropeana in the Cantabrian and Pyrenean mountains, with the sporadic Pyrenean populations having potentially resulted from recent hybridizations and the stabilized Cantabrian ones from older events followed by potential displacements of the parents. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173 , 676–706.     

前科迪勒拉（阿根廷）寒武─奥陶纪牙形刺和沉积演化

阿根廷前科迪勒拉区寒武─奥陶系地层发育。中晚奥陶世时,前科迪勒拉东部与中部山区为浅水陆棚相,西部山区为斜坡─盆地相。牙形刺的分布主要与平静的暖水大洋有关。寒武纪仅发现晚寒武世Ｐｒｏｃｏｎｏｄｏｎｔｕｓ带,此期间有一次海侵事件。在奥陶纪发现了牙形刺并可识别出四次海侵事件三次海退事件和二次缺氧事件。     

The Fate of Threatened Coastal Dune Habitats in Italy under Climate Change Scenarios

Coastal dunes worldwide harbor threatened habitats characterized by high diversity in terms of plant communities. In Italy, recent assessments have highlighted the insufficient state of conservation of these habitats as defined by the EU Habitats Directive. The effects of predicted climate change could have dramatic consequences for coastal environments in the near future. An assessment of the efficacy of protection measures under climate change is thus a priority. Here, we have developed environmental envelope models for the most widespread dune habitats in Italy, following two complementary approaches: an “indirect” plant-species-based one and a simple “direct” one. We analyzed how habitats distribution will be altered under the effects of two climate change scenarios and evaluated if the current Italian network of protected areas will be effective in the future after distribution shifts. While modeling dune habitats with the “direct” approach was unsatisfactory, “indirect” models had a good predictive performance, highlighting the importance of using species’ responses to climate change for modeling these habitats. The results showed that habitats closer to the sea may even increase their geographical distribution in the near future. The transition dune habitat is projected to remain stable, although mobile and fixed dune habitats are projected to lose most of their actual geographical distribution, the latter being more sensitive to climate change effects. Gap analysis highlighted that the habitats’ distribution is currently adequately covered by protected areas, achieving the conservation target. However, according to predictions, protection level for mobile and fixed dune habitats is predicted to drop drastically under the climate change scenarios which we examined. Our results provide useful insights for setting management priorities and better addressing conservation efforts to preserve these threatened habitats in future.     

Multifunctionalized CMCht/PAMAM dendrimer nanoparticles modulate the cellular uptake by astrocytes and oligodendrocytes in primary cultures of glial cells

The efficiency of the treatments involving CNS disorders is commonly diminished by the toxicity, reduced stability and lack of targeting of the administered neuroactive compounds. In this study, we have successfully multifunctionalized CMCht/PAMAM dendrimer nanoparticles by coupling the CD11b antibody and loading MP into the nanoparticles. The modification of the new antibody-conjugated nanoparticles was confirmed by S-TEM observation and (1) H NMR and FTIR spectroscopy. Cytotoxicity assays revealed that the conjugates did not affect the viability of both primary cultures of glial and microglial cells. Trace analyses of FITC-labelled nanoparticles revealed that the uptake of antibody-conjugated nanoparticles was conserved in microglial cells but significantly decreased in astrocytes and oligodendrocytes. Thus, this study demonstrates that antibody conjugation contributes to a modulation of the internalization of these nanocarriers by different cell types, which might be of relevance for specific targeting of CNS cell populations.     

The assessment of post-vasectomy pain in mice using behaviour and the Mouse Grimace Scale

Background

Current behaviour-based pain assessments for laboratory rodents have significant limitations. Assessment of facial expression changes, as a novel means of pain scoring, may overcome some of these limitations. The Mouse Grimace Scale appears to offer a means of assessing post-operative pain in mice that is as effective as manual behavioural-based scoring, without the limitations of such schemes. Effective assessment of post-operative pain is not only critical for animal welfare, but also the validity of science using animal models.

Methodology/Principal Findings

This study compared changes in behaviour assessed using both an automated system (“HomeCageScan”) and using manual analysis with changes in facial expressions assessed using the Mouse Grimace Scale (MGS). Mice (n = 6/group) were assessed before and after surgery (scrotal approach vasectomy) and either received saline, meloxicam or bupivacaine. Both the MGS and manual scoring of pain behaviours identified clear differences between the pre and post surgery periods and between those animals receiving analgesia (20 mg/kg meloxicam or 5 mg/kg bupivacaine) or saline post-operatively. Both of these assessments were highly correlated with those showing high MGS scores also exhibiting high frequencies of pain behaviours. Automated behavioural analysis in contrast was only able to detect differences between the pre and post surgery periods.

Conclusions

In conclusion, both the Mouse Grimace Scale and manual scoring of pain behaviours are assessing the presence of post-surgical pain, whereas automated behavioural analysis could be detecting surgical stress and/or post-surgical pain. This study suggests that the Mouse Grimace Scale could prove to be a quick and easy means of assessing post-surgical pain, and the efficacy of analgesic treatment in mice that overcomes some of the limitations of behaviour-based assessment schemes.     

Structural determination of the O-specific polysaccharide from Aeromonas hydrophila strain A19 (serogroup O:14) with S-layer

Bacteria belonging to the genus Aeromonas are Gram-negative mesophilic and essentially ubiquitous in the microbial biosphere; moreover they are considered very important pathogens in fish and responsible for a great variety of human infections.The virulence of Gram-negative bacteria is often associated with the structure of lipopolysaccharides, which consist of three regions covalently linked: the glycolipid (lipid A), the oligosaccharide region (core region) and the O-specific polysaccharide (O-chain, O-antigen).The O-chain region seems to play an important role in host-pathogen interaction. In the case of Aeromonas hydrophila the majority of pathogenic strains belongs to serogroups O:11, O:16, O:18 and O:34. In this paper, we report the complete structure of the O-chain of A. hydrophila strain A19 (serogroup O:14), a pathogenic strain isolated from European eels, which showed high virulence when tested in trout or mice. Dried cells were extracted by the PCP (phenol/chloroform/petroleum ether) method obtaining the lipopolysaccharide. After mild acid hydrolysis the lipid A was removed by centrifugation and the obtained polysaccharide was fully characterized by means of chemical analysis and one- and two-dimensional NMR spectroscopy. All the data collected are directed towards the following structure: