Residual active granzyme B in cathepsin C-null lymphocytes is sufficient for perforin-dependent target cell apoptosis

Cathepsin C activates serine proteases expressed in hematopoietic cells by cleaving an N-terminal dipeptide from the proenzyme upon granule packaging. The lymphocytes of cathepsin C-null mice are therefore proposed to totally lack granzyme B activity and perforin-dependent cytotoxicity. Surprisingly, we show, using live cell microscopy and other methodologies, that cells targeted by allogenic CD8(+) cytotoxic T lymphocyte (CTL) raised in cathepsin C-null mice die through perforin-dependent apoptosis indistinguishable from that induced by wild-type CTL. The cathepsin C-null CTL expressed reduced but still appreciable granzyme B activity, but minimal granzyme A activity. Also, in contrast to mice with inactivation of both their granzyme A/B genes, cathepsin C deficiency did not confer susceptibility to ectromelia virus infection in vivo. Overall, our results indicate that although cathepsin C clearly generates the majority of granzyme B activity, some is still generated in its absence, pointing to alternative mechanisms for granzyme B processing and activation. Cathepsin C deficiency also results in considerably milder immune deficiency than perforin or granzyme A/B deficiency.     

Cleavage of recombinant proteins at poly-His sequences by Co(II) and Cu(II)

Improved ways to cleave peptide chains at engineered sites easily and specifically would form useful tools for biochemical research. Uses of such methods include the activation or inactivation of enzymes or the removal of tags for enhancement of recombinant protein expression or tags used for purification of recombinant proteins. In this work we show by gel electrophoresis and mass spectroscopy that salts of Co(II) and Cu(II) can be used to cleave fusion proteins specifically at sites where sequences of His residues have been introduced by protein engineering. The His residues could be either consecutive or spaced with other amino acids in between. The cleavage reaction required the presence of low concentrations of ascorbate and in the case of Cu(II) also hydrogen peroxide. The amount of metal ions required for cleavage was very low; in the case of Cu(II) only one to two molar equivalents of Cu(II) to protein was required. In the case of Co(II), 10 molar equivalents gave optimal cleavage. The reaction occurred within minutes, at a wide pH range, and efficiently at temperatures ranging from 0 degrees C to 70 degrees C. The work described here can also have implications for understanding protein stability in vitro and in vivo.     

Population screening for coeliac disease in primary care by district nurses using a rapid antibody test: diagnostic accuracy and feasibility study

Objective To evaluate the feasibility and diagnostic accuracy of screening for coeliac disease by rapid detection of IgA antibodies to tissue transglutaminase performed in primary care.Design District nurses screened 6 year old children using rapid antibody testing of finger prick blood. They also collected capillary blood samples for laboratory determination of IgA and IgG antibodies to endomysium and IgA antibodies to tissue transglutaminase. Children with positive rapid test results were directly sent for biopsy of the small intestine.Setting Primary care in Jász-Nagykun-Szolnok county, Hungary.Participants 2690 children (77% of 6 year olds living in the county) and 120 nurses.Main outcome measures Positivity for antibodies to endomysium or transglutaminase in the laboratory and coeliac disease confirmed at biopsy.Results 37 children (1.4%, 95% confidence interval 0.9% to 1.8%) had biopsy confirmed coeliac disease. Only five of these children had been diagnosed clinically before screening. Rapid testing had a 78.1% sensitivity (70.0% to 89.3%) and 100% specificity (88.4% to 100%) for a final diagnosis of coeliac disease by biopsy. Sensitivity was 65.1% (50.2% to 77.6%) and specificity was 100% (99.8% to 100%) compared with combined results of IgA and IgG laboratory tests. Trained laboratory workers detected 30 of the 31 newly diagnosed IgA competent patients with the rapid test kit used blindly. Median time to biopsy after a positive rapid test result was significantly shorter (20 days, range 4-148) than after a positive laboratory result (142 days, 70-256; P<0.001). Children with coeliac disease detected at screening were smaller and had worse health status than their peers but they improved on a gluten-free diet.Conclusions A simple rapid antibody test enabled primary care nurses to detect patients with coeliac disease in the community who were not picked up in clinical care. Extra training is needed to improve sensitivity.     

Metabolic and phylogenetic analysis of microbial communities during phytoremediation of soil contaminated with weathered hydrocarbons and heavy metals

In the current study, the microbial ecology of weathered hydrocarbon and heavy metal contaminated soil undergoing phytoremediation was studied. The relationship of functional diversity, measured as carbon source utilisation in Biolog plates and extracellular enzymatic activities, and genetic diversity of bacteria was evaluated. Denaturing gradient gel electrophoresis was used for community analyses at the species level. Bulk soil and rhizosphere soil from pine and poplar plantations were analysed separately to determine if the plant rhizosphere impacted hydrocarbon degradation. Prevailing microbial communities in the field site were both genetically and metabolically diverse. Furthermore, both tree rhizosphere and fertilisation affected the compositions of these communities and increased activities of extracellular aminopeptidases. In addition, the abundance of alkane hydroxylase and naphthalene dioxygenase genes in the communities was low, but the prevalence of these genes was increased by the addition of bioavailable hydrocarbons. Tree rhizosphere communities had greater hydrocarbon degradation potential than those of bulk soil. Hydrocarbon utilising communities were dominated generally by the species Ralstonia eutropha and bacteria belonging to the genus Burkholderia. Despite the presence of viable hydrocarbon-degrading microbiota, decomposition of hydrocarbons from weathered hydrocarbon contaminated soil over four years, regardless of the presence of vegetation, was low in unfertilised soil. Compost addition enhanced the removal of hydrocarbons.     

Increased heat requirement for leaf flushing in temperate woody species over 1980–2012: effects of chilling,precipitation and insolation

Recent studies have revealed large unexplained variation in heat requirement‐based phenology models, resulting in large uncertainty when predicting ecosystem carbon and water balance responses to climate variability. Improving our understanding of the heat requirement for spring phenology is thus urgently needed. In this study, we estimated the species‐specific heat requirement for leaf flushing of 13 temperate woody species using long‐term phenological observations from Europe and North America. The species were defined as early and late flushing species according to the mean date of leaf flushing across all sites. Partial correlation analyses were applied to determine the temporal correlations between heat requirement and chilling accumulation, precipitation and insolation sum during dormancy. We found that the heat requirement for leaf flushing increased by almost 50% over the study period 1980–2012, with an average of 30 heat units per decade. This temporal increase in heat requirement was observed in all species, but was much larger for late than for early flushing species. Consistent with previous studies, we found that the heat requirement negatively correlates with chilling accumulation. Interestingly, after removing the variation induced by chilling accumulation, a predominantly positive partial correlation exists between heat requirement and precipitation sum, and a predominantly negative correlation between heat requirement and insolation sum. This suggests that besides the well‐known effect of chilling, the heat requirement for leaf flushing is also influenced by precipitation and insolation sum during dormancy. However, we hypothesize that the observed precipitation and insolation effects might be artefacts attributable to the inappropriate use of air temperature in the heat requirement quantification. Rather than air temperature, meristem temperature is probably the prominent driver of the leaf flushing process, but these data are not available. Further experimental research is thus needed to verify whether insolation and precipitation sums directly affect the heat requirement for leaf flushing.     

Identification of a common risk haplotype for canine idiopathic epilepsy in the ADAM23 gene

Background

Idiopathic epilepsy is a common neurological disease in human and domestic dogs but relatively few risk genes have been identified to date. The seizure characteristics, including focal and generalised seizures, are similar between the two species, with gene discovery facilitated by the reduced genetic heterogeneity of purebred dogs. We have recently identified a risk locus for idiopathic epilepsy in the Belgian Shepherd breed on a 4.4 megabase region on CFA37.

Results

We have expanded a previous study replicating the association with a combined analysis of 157 cases and 179 controls in three additional breeds: Schipperke, Finnish Spitz and Beagle (p_c = 2.9e–07, p_GWAS = 1.74E-02). A targeted resequencing of the 4.4 megabase region in twelve Belgian Shepherd cases and twelve controls with opposite haplotypes identified 37 case-specific variants within the ADAM23 gene. Twenty-seven variants were validated in 285 cases and 355 controls from four breeds, resulting in a strong replication of the ADAM23 locus (p_raw = 2.76e–15) and the identification of a common 28 kb-risk haplotype in all four breeds. Risk haplotype was present in frequencies of 0.49–0.7 in the breeds, suggesting that ADAM23 is a low penetrance risk gene for canine epilepsy.

Conclusions

These results implicate ADAM23 in common canine idiopathic epilepsy, although the causative variant remains yet to be identified. ADAM23 plays a role in synaptic transmission and interacts with known epilepsy genes, LGI1 and LGI2, and should be considered as a candidate gene for human epilepsies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1651-9) contains supplementary material, which is available to authorized users.     

Tree seasonality in a warming climate

Climate warming has increased researchers' interest in plant phenology and its modelling. Although the main focus is on projections of accelerated springtime phenological events, also a further extension of the growing season by delayed growth cessation is often projected. However, ecophysiological studies indicate that, for boreal and temperate trees, such generalisations are precluded owing to differential climatic conditions and inter- and intraspecific genetic differences. The annual cycle of these trees is an integrated system, where one phase affects subsequent phases, resulting in delayed impacts, which are only partially addressed in current ecophysiological models. Here, we outline an updated integrated conceptual model of the annual cycle by identifying ecophysiological phenomena that are particularly significant under climate warming.     

Gamma-irradiated influenza virus uniquely induces IFN-I mediated lymphocyte activation independent of the TLR7/MyD88 pathway

Background

We have shown previously in mice, that infection with live viruses, including influenza/A and Semliki Forest virus (SFV), induces systemic partial activation of lymphocytes, characterized by cell surface expression of CD69 and CD86, but not CD25. This partial lymphocytes activation is mediated by type-I interferons (IFN-I). Importantly, we have shown that γ-irradiated SFV does not induce IFN-I and the associated lymphocyte activation.

Principal Findings

Here we report that, in contrast to SFV, γ-irradiated influenza A virus elicits partial lymphocyte activation in vivo. Furthermore, we show that when using influenza viruses inactivated by a variety of methods (UV, ionising radiation and formalin treatment), as well as commercially available influenza vaccines, only γ-irradiated influenza virus is able to trigger IFN-I-dependent partial lymphocyte activation in the absence of the TLR7/MyD88 signalling pathways.

Conclusions

Our data suggest an important mechanism for the recognition of γ-irradiated influenza vaccine by cytosolic receptors, which correspond with the ability of γ-irradiated influenza virus to induce cross-reactive and cross-protective cytotoxic T cell responses.