Requirement of Signal Peptidase ComC and Thiol-Disulfide Oxidoreductase DsbA for Optimal Cell Surface Display of Pseudopilin ComGC in Staphylococcus aureus

Staphylococcus aureus is an important Gram-positive bacterial pathogen producing many secreted and cell surface-localized virulence factors. Here we report that the staphylococcal thiol-disulfide oxidoreductase DsbA is essential for stable biogenesis of the ComGC pseudopilin. The signal peptidase ComC is indispensable for ComGC maturation and optimal cell surface exposure.     

A new analytical approach to landscape genetic modelling: least-cost transect analysis and linear mixed models

Landscape genetics aims to assess the effect of the landscape on intraspecific genetic structure. To quantify interdeme landscape structure, landscape genetics primarily uses landscape resistance surfaces (RSs) and least-cost paths or straight-line transects. However, both approaches have drawbacks. Parameterization of RSs is a subjective process, and least-cost paths represent a single migration route. A transect-based approach might oversimplify migration patterns by assuming rectilinear migration. To overcome these limitations, we combined these two methods in a new landscape genetic approach: least-cost transect analysis (LCTA). Habitat-matrix RSs were used to create least-cost paths, which were subsequently buffered to form transects in which the abundance of several landscape elements was quantified. To maintain objectivity, this analysis was repeated so that each landscape element was in turn regarded as migration habitat. The relationship between explanatory variables and genetic distances was then assessed following a mixed modelling approach to account for the nonindependence of values in distance matrices. Subsequently, the best fitting model was selected using the statistic. We applied LCTA and the mixed modelling approach to an empirical genetic dataset on the endangered damselfly, Coenagrion mercuriale. We compared the results to those obtained from traditional least-cost, effective and resistance distance analysis. We showed that LCTA is an objective approach that identifies both the most probable migration habitat and landscape elements that either inhibit or facilitate gene flow. Although we believe the statistical approach to be an improvement for the analysis of distance matrices in landscape genetics, more stringent testing is needed.     

Short-term effect of different teaching methods on nasopharyngeal carcinoma for general practitioners in Jakarta, Indonesia

In Indonesia, Nasopharyngeal Carcinoma (NPC) is the most frequent cancer of the head and neck region. At first presentation in the hospital most patients already have advanced NPC. Our previous study showed that general practitioners (GPs) working in Yogyakarta, Indonesia lack the knowledge necessary for early detection of NPC. By providing training on early symptoms of NPC we hope that the diagnosis and referral will occur at an earlier stage. Here we assess the current NPC knowledge levels of GPs in Jakarta, evaluate improvement after training, compare the effectiveness of two training formats, and estimate the loss of recall over a two week period.

Methods

Two Indonesian GPs visited 31 Primary Health Care Centres (PHCCs) and provided a lecture on NPC. The alternative format consisted of a symposium at the Universitas Indonesia, Jakarta, presented by local head and neck surgeons, with all GPs in the region being invited. To evaluate the effect of both formats a questionnaire was conducted before and after.

Results

The lecture in the PHCCs was attended by 130 GPs. Sixty-six GPs attended the training in the university hospital and 40 GPs attended both. Pre training the NPC knowledge level was poor with an average of 1.6 symptoms being correctly identified out of a potential maximum of 12, this was increased to 4.9 post training (p<0.0001). GPs attending the PHCC course recorded a greater increase in correct symptoms than those attending the symposium (3.8 vs. 2.8; p = 0.01). After a two week period the knowledge levels had declined slightly from 5.5 correctly identified symptoms to 4.2 (p = 0.25).

Conclusion

These results confirm our findings regarding GPs insufficient knowledge of NPC. Lectures in the PHCC and a symposium have both been proven to be effective training tools in the education of GPs.     

Toward a global phylogeny of the "living fossil" crustacean order of the Notostraca

Tadpole shrimp (Crustacea, Notostraca) are iconic inhabitants of temporary aquatic habitats worldwide. Often cited as prime examples of evolutionary stasis, surviving representatives closely resemble fossils older than 200 mya, suggestive of an ancient origin. Despite significant interest in the group as 'living fossils' the taxonomy of surviving taxa is still under debate and both the phylogenetic relationships among different lineages and the timing of diversification remain unclear. We constructed a molecular phylogeny of the Notostraca using model based phylogenetic methods. Our analyses supported the monophyly of the two genera Triops and Lepidurus, although for Triops support was weak. Results also revealed high levels of cryptic diversity as well as a peculiar biogeographic link between Australia and North America presumably mediated by historic long distance dispersal. We concluded that, although some present day tadpole shrimp species closely resemble fossil specimens as old as 250 mya, no molecular support was found for an ancient (pre) Mesozoic radiation. Instead, living tadpole shrimp are most likely the result of a relatively recent radiation in the Cenozoic era and close resemblances between recent and fossil taxa are probably the result of the highly conserved general morphology in this group and of homoplasy.     

Batrachochytrium dendrobatidis zoospore secretions rapidly disturb intercellular junctions in frog skin

Global amphibian declines are in part driven by the chytrid fungus Batrachochytrium dendrobatidis, causing superficial dermatomycosis with epidermal hyperplasia and hyperkeratosis in infected amphibians. The susceptibility to chytridiomycosis and the severity of epidermal lesions in amphibians with chytridiomycosis are not consistent across species or even among individuals. Severe infections cause death of the animal most likely through disturbance of ion homeostasis. The mechanism by which this superficial skin infection results in epidermal lesions has so far eluded precise definition. It was the aim of this study to unravel how B. dendrobatidis causes alterations that affect skin integrity. Exposure of Xenopus laevis skin to B. dendrobatidis zoospore supernatant using skin explants and Ussing chambers caused rapid disruption of intercellular junctions, demonstrated using histology and transmission electron microscopy. The loss of intercellular junctions led to detachment-induced cell apoptosis, or anoikis. The zoospore supernatant induced neither apoptosis nor necrosis in isolated primary keratinocytes of X. laevis. This supports the idea that the loss of cell contacts triggered apoptosis in the skin explants. Mass spectrometric analysis of the protein composition of the supernatant revealed a complex mixture, including several new virulence associated proteins, such as proteases, biofilm-associated proteins and a carotenoid ester lipase. Protease and lipase activity of the supernatant was confirmed with a protease and lipase assay.In conclusion, B. dendrobatidis zoospores produce a complex mixture of proteins that quickly disturbs epidermal intercellular junctions leading to anoikis in the anuran skin. The role of the identified proteins in this process remains to be determined.     

Modulation of thiol-disulfide oxidoreductases for increased production of disulfide-bond-containing proteins in Bacillus subtilis

Disulfide bonds are important for the correct folding, structural integrity, and activity of many biotechnologically relevant proteins. For synthesis and subsequent secretion of these proteins in bacteria, such as the well-known "cell factory" Bacillus subtilis, it is often the correct formation of disulfide bonds that is the greatest bottleneck. Degradation of inefficiently or incorrectly oxidized proteins and the requirement for costly and time-consuming reduction and oxidation steps in the downstream processing of the proteins still are major limitations for full exploitation of B. subtilis for biopharmaceutical production. Therefore, the present study was aimed at developing a novel in vivo strategy for improved production of secreted disulfide-bond-containing proteins. Three approaches were tested: depletion of the major cytoplasmic reductase TrxA; introduction of the heterologous oxidase DsbA from Staphylococcus carnosus; and addition of redox-active compounds to the growth medium. As shown using the disulfide-bond-containing molecule Escherichia coli PhoA as a model protein, combined use of these three approaches resulted in secretion of amounts of active PhoA that were approximately 3.5-fold larger than the amounts secreted by the parental strain B. subtilis 168. Our findings indicate that Bacillus strains with improved oxidizing properties can be engineered for biotechnological production of heterologous high-value proteins containing disulfide bonds.     

Cupiennius salei and Achaearanea tepidariorum: Spider models for investigating evolution and development

The spiders Cupiennius salei and Achaearanea tepidariorum are firmly established laboratory models that have already contributed greatly to answering evolutionary developmental questions. Here we appraise why these animals are such useful models from phylogeny, natural history and embryogenesis to the tools available for their manipulation. We then review recent studies of axis formation, segmentation, appendage development and neurogenesis in these spiders and how this has contributed to understanding the evolution of these processes. Furthermore, we discuss the potential of comparisons of silk production between Cupiennius and Achaearanea to investigate the origins and diversification of this evolutionary innovation. We suggest that further comparisons between these two spiders and other chelicerates will prove useful for understanding the evolution of development in metazoans.     

A perinatal nitric oxide donor increases renal vascular resistance and ameliorates hypertension and glomerular injury in adult fawn-hooded hypertensive rats

Enhancing perinatal nitric oxide (NO) availability persistently reduces blood pressure in spontaneously hypertensive rats. We hypothesize that this approach can be generalized to other models of genetic hypertension, for instance those associated with renal injury. Perinatal exposure to the NO donor molsidomine was studied in fawn-hooded hypertensive (FHH) rats, a model of mild hypertension, impaired preglomerular resistance, and progressive renal injury. Perinatal molsidomine increased urinary NO metabolite excretion at 8 wk of age, i.e., 4 wk after treatment was stopped (P < 0.05). Systolic blood pressure was persistently reduced after molsidomine (42-wk females: 118 +/- 3 vs. 141 +/- 5 and 36-wk males: 139 +/- 4 vs. 158 +/- 4 mmHg; both P < 0.001). Perinatal treatment decreased glomerular filtration rate (P < 0.05) and renal blood flow (P < 0.01) and increased renal vascular resistance (P < 0.05), without affecting filtration fraction, suggesting persistently increased preglomerular resistance. At 4 wk of age natriuresis was transiently increased by molsidomine (P < 0.05). Molsidomine decreased glomerulosclerosis (P < 0.05). Renal blood flow correlated positively with glomerulosclerosis in control (P < 0.001) but not in perinatally treated FHH rats. NO dependency of renal vascular resistance was increased by perinatal molsidomine. Perinatal enhancement of NO availability can ameliorate development of hypertension and renal injury in FHH rats. Paradoxically, glomerular protection by perinatal exposure to the NO donor molsidomine may be due to persistently increased preglomerular resistance. The mechanisms by which increased perinatal NO availability can persistently reprogram kidney function and ameliorate hypertension deserve further study.