The cysteine protease α-clostripain is not essential for the pathogenesis of Clostridium perfringens-mediated myonecrosis

Clostridium perfringens is the causative agent of clostridial myonecrosis or gas gangrene and produces many different extracellular toxins and enzymes, including the cysteine protease α-clostripain. Mutation of the α-clostripain structural gene, ccp, alters the turnover of secreted extracellular proteins in C. perfringens, but the role of α-clostripain in disease pathogenesis is not known. We insertionally inactivated the ccp gene C. perfringens strain 13 using TargeTron technology, constructing a strain that was no longer proteolytic on skim milk agar. Quantitative protease assays confirmed the absence of extracellular protease activity, which was restored by complementation with the wild-type ccp gene. The role of α-clostripain in virulence was assessed by analysing the isogenic wild-type, mutant and complemented strains in a mouse myonecrosis model. The results showed that although α-clostripain was the major extracellular protease, mutation of the ccp gene did not alter either the progression or the development of disease. These results do not rule out the possibility that this extracellular enzyme may still have a role in the early stages of the disease process.     

FcγRIIIa expression on monocytes in rheumatoid arthritis: role in immune-complex stimulated TNF production and non-response to methotrexate therapy

Objective

The expression of FcγRIIIa/CD16 may render monocytes targets for activation by IgG-containing immune complexes (IC). We investigated whether FcγRIIIa/CD16 was upregulated in rheumatoid arthritis (RA), associated with TNF production in response to IC-stimulation, and if this predicted response to methotrexate therapy.

Methods

FcγRIIIa/CD16 expression on CD14^low and CD14⁺⁺ monocytes was measured by flow cytometry in healthy controls and RA patients (early and long-standing disease). Intracellular TNF-staining was carried out after in vitro LPS or heat-aggregated immunoglobulin (HAG) activation. FcγRIIIa/CD16 expression pre- and post-steroid/methotrexate treatment was examined.

Results

Increased FcγRIIIa/CD16 expression on CD14⁺⁺ monocytes in long-standing RA patients compared to controls was demonstrated (p = 0.002) with intermediate levels in early-RA patients. HAG-induced TNF-production in RA patients was correlated with the percentage of CD14⁺⁺ monocytes expressing FcγRIIIa/CD16 (p<0.001). The percentage of CD14⁺⁺ monocytes expressing FcγRIIIa/CD16 at baseline in early DMARD-naïve RA patients was negatively correlated with DAS28-ESR improvement 14-weeks post-methotrexate therapy (p = 0.003) and was significantly increased in EULAR non-responders compared to moderate (p = 0.01) or good responders (p = 0.003). FcγRIIIa/CD16 expression was not correlated with age, presence of systemic inflammation or autoantibody titers.

Conclusion

Increased FcγRIIIa/CD16 expression on CD14⁺⁺ monocytes in RA may result in a cell that has increased responsiveness to IC-stimulation. This monocyte subset may contribute to non-response to methotrexate therapy.     

Clinical Utility of a Coronary Heart Disease Risk Prediction Gene Score in UK Healthy Middle Aged Men and in the Pakistani Population

BackgroundNumerous risk prediction algorithms based on conventional risk factors for Coronary Heart Disease (CHD) are available but provide only modest discrimination. The inclusion of genetic information may improve clinical utility.MethodsWe tested the use of two gene scores (GS) in the prospective second Northwick Park Heart Study (NPHSII) of 2775 healthy UK men (284 cases), and Pakistani case-control studies from Islamabad/Rawalpindi (321 cases/228 controls) and Lahore (414 cases/219 controls). The 19-SNP GS included SNPs in loci identified by GWAS and candidate gene studies, while the 13-SNP GS only included SNPs in loci identified by the CARDIoGRAMplusC4D consortium.ResultsIn NPHSII, the mean of both gene scores was higher in those who went on to develop CHD over 13.5 years of follow-up (19-SNP p=0.01, 13-SNP p=7x10^-3). In combination with the Framingham algorithm the GSs appeared to show improvement in discrimination (increase in area under the ROC curve, 19-SNP p=0.48, 13-SNP p=0.82) and risk classification (net reclassification improvement (NRI), 19-SNP p=0.28, 13-SNP p=0.42) compared to the Framingham algorithm alone, but these were not statistically significant. When considering only individuals who moved up a risk category with inclusion of the GS, the improvement in risk classification was statistically significant (19-SNP p=0.01, 13-SNP p=0.04). In the Pakistani samples, risk allele frequencies were significantly lower compared to NPHSII for 13/19 SNPs. In the Islamabad study, the mean gene score was higher in cases than controls only for the 13-SNP GS (2.24 v 2.34, p=0.04). There was no association with CHD and either score in the Lahore study.ConclusionThe performance of both GSs showed potential clinical utility in European men but much less utility in subjects from Pakistan, suggesting that a different set of risk loci or SNPs may be required for risk prediction in the South Asian population.     

Embryonic lethality leads to hybrid male inviability in hybrids between Drosophila melanogaster and D. santomea

The study of the morphological defects unique to interspecific hybrids can reveal which developmental pathways have diverged between species. Drosophila melanogaster and D. santomea diverged more than 10 million years ago, and when crossed produce sterile adult females. Adult hybrid males are absent from all interspecific crosses. We aimed to determine the fate of these hybrid males. To do so, we tracked the development of hybrid females and males using classic genetic markers and techniques. We found that hybrid males die predominantly as embryos with severe segment‐specification defects while a large proportion of hybrid females embryos hatch and survive to adulthood. In particular, we show that most male embryos show a characteristic abdominal ablation phenotype, not observed in either parental species. This suggests that sex‐specific embryonic developmental defects eliminate hybrid males in this interspecific cross. The study of the developmental abnormalities that occur in hybrids can lead to the understanding of cryptic molecular divergence between species sharing a conserved body plan.     

Vardenafil: structural basis for higher potency over sildenafil in inhibiting cGMP-specific phosphodiesterase-5 (PDE5)

Phosphodiesterase-5 (PDE5) inhibitors act by competing with the substrate, cGMP, for the catalytic site of the enzyme. Two commercialized PDE5 inhibitors, sildenafil and vardenafil, are being used to treat erectile dysfunction. These two compounds differ in the heterocyclic ring system used to mimic the purine ring of cGMP. They also differ in the substituent (ethyl/methyl) of a piperazine side chain. Although these are the only two structural differences, vardenafil has more than 20-fold greater potency than sildenafil for inhibiting purified PDE5. The molecular structural basis for the difference in potency of the two compounds was investigated by synthesizing an analog of sildenafil ("methyl-sildenafil") that contained the sildenafil ring system but with the appended ethyl group found in vardenafil, and an analog of vardenafil ("demethyl-vardenafil") that contained the vardenafil ring system but with the appended methyl group found in sildenafil. The IC50 of methyl-sildenafil for inhibiting PDE5 indicated that it was 64 times less potent than demethyl-vardenafil, which was similar to the finding that, based on IC50, sildenafil was 40 times less potent than vardenafil. Similarly, the EC50 of methyl-sildenafil for inhibiting [3H]vardenafil binding to PDE5 indicated that it was 84 times less potent than demethyl-vardenafil, while the EC50 for sildenafil indicated that it was 31 times less potent than vardenafil. It is concluded that the methyl/ethyl appended group on the piperazine moiety plays very little role in the difference in potency between sildenafil and vardenafil for inhibiting PDE5, whereas the differences in the ring systems play a critical role in higher potency of vardenafil over sildenafil.     

A simple cell motility assay demonstrates differential motility of human periodontal ligament fibroblasts, gingival fibroblasts, and pre-osteoblasts

During periodontal regeneration, multiple cell types can invade the wound site, thereby leading to repair. Cell motility requires interactions mediated by integrin receptors for the extracellular matrix (ECM), which might be useful in guiding specific cell populations into the periodontal defect. Our data demonstrate that fibroblasts exhibit differential motility when grown on ECM proteins. Specifically, gingival fibroblasts are twice as motile as periodontal ligament fibroblasts, whereas osteoblasts are essentially non-motile. Collagens promote the greatest motility of gingival fibroblasts in the following order: collagen III>collagen V>collagen I. Differences in motility do not correlate with cell proliferation or integrin expression. Osteoblasts display greater attachment to collagens than does either fibroblast population, but lower motility. Gingival fibroblast motility on collagen I is generally mediated by α2 integrins, whereas motility on collagen III involves α1 integrins. Other integrins (α10 or α11) may also contribute to gingival fibroblast motility. Thus, ECM proteins do indeed differentially promote the cell motility of periodontal cells. Because of their greater motility, gingival fibroblasts have more of a potential to invade periodontal wound sites and to contribute to regeneration. This finding may explain the formation of disorganized connective tissue masses rather than the occurrence of the true regeneration of the periodontium. This research was supported by the Louisiana Board of Regents through the Millennium Trust Health Excellence Fund, HEF-(2000-05)-04.     

Characterization of EVL-I as a protein kinase D substrate

EVL-I is a splice variant of EVL (Ena/VASP like protein), whose in vivo function and regulation are still poorly understood. We found that Protein Kinase D (PKD) interacts in vitro and in vivo with EVL-I and phosphorylates EVL-I in a 21 amino acid alternately-included insert in the EVH2 domain. Following knockdown of the capping protein CPβ and spreading on laminin, phosphorylated EVL-I can support filopodia formation and the phosphorylated EVL-I is localized at filopodial tips. Furthermore, we found that the lamellipodial localization of EVL-I is unaffected by phosphorylation, but that impairment of EVL-I phosphorylation is associated with ruffling of lamellipodia upon PDBu stimulation. Besides the lamellipodial and filopodial localization of phosphorylated EVL-I in fibroblasts, we determined that EVL-I is hyperphosphorylated and localized in the cell–cell contacts of certain breast cancer cells and mouse embryo keratinocytes. Taken together, our results show that phosphorylated EVL-I is present in lamellipodia, filopodia and cell–cell contacts and suggest the existence of signaling pathways that may affect EVL-I via phosphorylation of its EVH2 domain.     

Hominin occupations at the Dmanisi site, Georgia, Southern Caucasus: raw materials and technical behaviours of Europe's first hominins

Dmanisi is the oldest site outside of Africa that records unquestioned hominin occupations as well as the dispersal of hominins in Europe and Asia. The site has yielded large numbers of artefacts from several periods of hominin occupation. This analysis of Dmanisi stone tool technology includes a review of all the pieces recovered during the last 15 years of excavations. This lithic assemblage gives insights into the hominin behaviour at 1.7-1.8 Ma in Eurasia. Dmanisi hominins exploited local rocks derived from either nearby riverbeds or outcrops, and petrographic study provides data on patterns of stone procurement. Recent geological surveys and technological studies of the artefacts illustrate the roles of hominins in composing the assemblage. Dmanisi hominins selected two types of blanks, including cobbles and angular blocks, of basalt, andesite, and tuffs. Many complete cobbles, pebbles, and rolled blocks in basalt were unmodified, and geological analyses and surveys indicate that hominins brought manuports back to the site, suggesting a complex procurement strategy. Cores, flakes and debris show that all stages of flaking activity took place at the site. Numerous unifacial cores suggest that knapping was not very elaborate. Centripetal knapping is observed on some flake-cores. Knapping was influenced by the blank shape and natural angles. Most flaked objects were either cores or chopper-cores. Flakes predominate while flake tools are rare. The Dmanisi lithic assemblage is comparable to Oldowan sites in Africa in terms of reduction sequence, organisation of the removals, platform types, and the lack of retouched flakes. Dmanisi artefacts and may have been produced by the original hominins in Europe and Asia.     

Three novel pigmentation mutants generated by genome-wide random ENU mutagenesis in the mouse

Three mutant mice with pigmentation phenotypes were recovered from a genomewide random mouse chemical mutagenesis study. White toes (Whto; MGI:1861986), Belly spot and white toes (Bswt; MGI:2152776) and Dark footpads 2 (Dfp2; MGI:1861991) were identified following visual inspection of progeny from a male exposed to the point mutagen ethylnitrosourea (ENU). In order to rapidly localize the causative mutations, genome-wide linkage scans were performed on pooled DNA samples from backcross animals for each mutant line. Whto was mapped to proximal mouse chromosome (Mmu) 7 between Cen (the centromere) and D7Mit112 (8.0 cM from the centromere), Bswt was mapped to centric Mmul between D1Mit214 (32.1 cM) and D1Mit480 (32.8 cM) and Dfp2 was mapped to proximalMmu4 between Cen and D4Mit18 (5.2 cM). Whto, Bswt and Dfp2 may provide novel starting points in furthering the elucidation of genetic and biochemical pathways relevant to pigmentation and associated biological processes.