Genome-based in silico identification of new Mycobacterium tuberculosis antigens activating polyfunctional CD8+ T cells in human tuberculosis

Although CD8(+) T cells help control Mycobacterium tuberculosis infection, their M. tuberculosis Ag repertoire, in vivo frequency, and functionality in human tuberculosis (TB) remains largely undefined. We have performed genome-based bioinformatics searches to identify new M. tuberculosis epitopes presented by major HLA class I supertypes A2, A3, and B7 (covering 80% of the human population). A total of 432 M. tuberculosis peptides predicted to bind to HLA-A*0201, HLA-A*0301, and HLA-B*0702 (representing the above supertypes) were synthesized and HLA-binding affinities determined. Peptide-specific CD8(+) T cell proliferation assays (CFSE dilution) in 41 M. tuberculosis-responsive donors identified 70 new M. tuberculosis epitopes. Using HLA/peptide tetramers for the 18 most prominently recognized HLA-A*0201-binding M. tuberculosis peptides, recognition by cured TB patients' CD8(+) T cells was validated for all 18 epitopes. Intracellular cytokine staining for IFN-γ, IL-2, and TNF-α revealed mono-, dual-, as well as triple-positive CD8(+) T cells, indicating these M. tuberculosis peptide-specific CD8(+) T cells were (poly)functional. Moreover, these T cells were primed during natural infection, because they were absent from M. tuberculosis-noninfected individuals. Control CMV peptide/HLA-A*0201 tetramers stained CD8(+) T cells in M. tuberculosis-infected and noninfected individuals equally, whereas Ebola peptide/HLA-A*0201 tetramers were negative. In conclusion, the M. tuberculosis-epitope/Ag repertoire for human CD8(+) T cells is much broader than hitherto suspected, and the newly identified M. tuberculosis Ags are recognized by (poly)functional CD8(+) T cells during control of infection. These results impact on TB-vaccine design and biomarker identification.     

The effect of exercise training on transverse tubules in normal, remodeled, and reverse remodeled hearts

The response of transverse (T)-tubules to exercise training in health and disease remains unclear. Therefore, we studied the effect of exercise training on the density and spacing of left ventricle cardiomyocyte T-tubules in normal and remodeled hearts that associate with detubulation, by confocal laser scanning microscopy. First, exercise training in normal rats increased cardiomyocyte volume by 16% (P < 0.01), with preserved T-tubule density. Thus, the T-tubules adapted to the physiologic hypertrophy. Next, we studied T-tubules in a rat model of metabolic syndrome with pressure overload-induced concentric left ventricle hypertrophy, evidenced by 15% (P < 0.01) increased cardiomyocyte size. These rats had only 85% (P < 0.01) of the T-tubule density of control rats. Exercise training further increased cardiomyocyte volume by 8% (P < 0.01); half to that in control rats, but the T-tubule density remained unchanged. Finally, post-myocardial infarction heart failure induced severe cardiac pathology, with a 70% (P < 0.01) increased cardiomyocyte volume that included both eccentric and concentric hypertrophy and 55% (P < 0.01) reduced T-tubule density. Exercise training reversed 50% (P < 0.01) of the pathologic hypertrophy, whereas the T-tubule density increased by 40% (P < 0.05) compared to sedentary heart failure, but remained at 60% of normal hearts (P < 0.01). Physiologic hypertrophy associated with conserved T-tubule spacing (~1.8-1.9 μm), whereas in pathologic hypertrophy, T-tubules appeared disorganized without regular spacing. In conclusion, cardiomyocytes maintain the relative T-tubule density during physiologic hypertrophy and after mild concentric pathologic hypertrophy, whereas after severe pathologic remodeling with a substantial loss of T-tubules; exercise training reverses the remodeling and partly corrects the T-tubule density.     

Full-length cardiac Na+/Ca2+ exchanger 1 protein is not phosphorylated by protein kinase A

The cardiac Na(+)/Ca(2+) exchanger 1 (NCX1) is an important regulator of intracellular Ca(2+) homeostasis and cardiac function. Several studies have indicated that NCX1 is phosphorylated by the cAMP-dependent protein kinase A (PKA) in vitro, which increases its activity. However, this finding is controversial and no phosphorylation site has so far been identified. Using bioinformatic analysis and peptide arrays, we screened NCX1 for putative PKA phosphorylation sites. Although several NCX1 synthetic peptides were phosphorylated by PKA in vitro, only one PKA site (threonine 731) was identified after mutational analysis. To further examine whether NCX1 protein could be PKA phosphorylated, wild-type and alanine-substituted NCX1-green fluorescent protein (GFP)-fusion proteins expressed in human embryonic kidney (HEK)293 cells were generated. No phosphorylation of full-length or calpain- or caspase-3 digested NCX1-GFP was observed with purified PKA-C and [γ-(32)P]ATP. Immunoblotting experiments with anti-PKA substrate and phosphothreonine-specific antibodies were further performed to investigate phosphorylation of endogenous NCX1. Phospho-NCX1 levels were also not increased after forskolin or isoproterenol treatment in vivo, in isolated neonatal cardiomyocytes, or in total heart homogenate. These data indicate that the novel in vitro PKA phosphorylation site is inaccessible in full-length as well as in calpain- or caspase-3 digested NCX1 protein, suggesting that NCX1 is not a direct target for PKA phosphorylation.     

Investigating the genome diversity of B. cereus and evolutionary aspects of B. anthracis emergence

Here we report the use of a multi-genome DNA microarray to investigate the genome diversity of Bacillus cereus group members and elucidate the events associated with the emergence of Bacillus anthracis the causative agent of anthrax—a lethal zoonotic disease. We initially performed directed genome sequencing of seven diverse B. cereus strains to identify novel sequences encoded in those genomes. The novel genes identified, combined with those publicly available, allowed the design of a “species” DNA microarray. Comparative genomic hybridization analyses of 41 strains indicate that substantial heterogeneity exists with respect to the genes comprising functional role categories. While the acquisition of the plasmid-encoded pathogenicity island (pXO1) and capsule genes (pXO2) represents a crucial landmark dictating the emergence of B. anthracis, the evolution of this species and its close relatives was associated with an overall shift in the fraction of genes devoted to energy metabolism, cellular processes, transport, as well as virulence.     

Retroposon insertions and the chronology of avian sex chromosome evolution

The vast majority of extant birds possess highly differentiated Z and W sex chromosomes. Nucleotide sequence data from gametologs (homologs on opposite sex chromosomes) suggest that this divergence occurred throughout early bird evolution via stepwise cessation of recombination between identical sex chromosomal regions. Here, we investigated avian sex chromosome differentiation from a novel perspective, using retroposon insertions and random insertions/deletions for the reconstruction of gametologous gene trees. Our data confirm that the CHD1Z/CHD1W genes differentiated in the ancestor of the neognaths, whereas the NIPBLZ/NIPBLW genes diverged in the neoavian ancestor and independently within Galloanserae. The divergence of the ATP5A1Z/ATP5A1W genes in galloanserans occurred independently in the chicken, the screamer, and the ancestor of duck-related birds. In Neoaves, this gene pair differentiated in each of the six sampled representatives, respectively. Additionally, three of our investigated loci can be utilized as universal, easy-to-use independent tools for molecular sexing of Neoaves or Neognathae.     

Rapid parallel adaptive radiations from a single hybridogenic ancestral population

The Alpine lake whitefish (Coregonus lavaretus) species complex is a classic example of a recent radiation, associated with colonization of the Alpine lakes following the glacial retreat (less than 15 kyr BP). They have formed a unique array of endemic lake flocks, each with one to six described sympatric species differing in morphology, diet and reproductive ecology. Here, we present a genomic investigation of the relationships between and within the lake flocks. Comparing the signal between over 1000 AFLP loci and mitochondrial control region sequence data, we use phylogenetic tree-based and population genetic methods to reconstruct the phylogenetic history of the group and to delineate the principal centres of genetic diversity within the radiation. We find significant cytonuclear discordance showing that the genomically monophyletic Alpine whitefish clade arose from a hybrid swarm of at least two glacial refugial lineages. Within this radiation, we find seven extant genetic clusters centred on seven lake systems. Most interestingly, we find evidence of sympatric speciation within and parallel evolution of equivalent phenotypes among these lake systems. However, we also find the genetic signature of human-mediated gene flow and diversity loss within many lakes, highlighting the fragility of recent radiations.     

Properties of the bubble protein, a defensin and an abundant component of a fungal exudate

Colonies of the ascomycete fungus Penicillium brevicompactum Dierckx produce bright yellow-green fluorescent exudate bubbles on its surface when grown on standard plant cell culture medium. According to SDS-PAGE analysis, the exudate is enriched in one protein, named bubble protein (BP). Detailed characteristics of BP are described, and also its corresponding genomic promoter and terminator sequences that flank sequences encoding signal peptide and a precursor sequence upstream of that of the mature protein. Following on previous work, the protein is now biochemically characterized. BP, the structure of which mainly consists of beta sheets, has four very stable disulfide bridges that resist standard procedures for reduction. With such traits, BP can now be categorized as a new member of the ever growing class of defensins. Indeed, the protein revealed anti-fungal effects as it inhibits growth of the yeast Saccharomyces cerevisiae in a dose-dependent manner. Structural classification places BP into the group of proteins with a knottin fold, founding the BP superfamily. Based on genomic alignments that revealed very high homology to four proteins of related fungi, a 3D structure prediction of the corresponding proteins was made. In addition, it was discovered that the closely related fungus Penicillium chrysogenum encodes a BP homolog – in addition to its PAF protein, which also is similar to BP – further suggesting that fungi may possess more than one defensin.     

Mapping out structural features in clinical care calling for ethical sensitivity: a theoretical approach to promote ethical competence in healthcare personnel and clinical ethical support services (CESS)

Clinical ethical support services (CESS) represent a multifaceted field of aims, consultancy models, and methodologies. Nevertheless, the overall aim of CESS can be summed up as contributing to healthcare of high ethical standards by improving ethically competent decision-making in clinical healthcare. In order to support clinical care adequately, CESS must pay systematic attention to all real-life ethical issues, including those which do not fall within the 'favourite' ethical issues of the day. In this paper we attempt to capture a comprehensive overview of categories of ethical tensions in clinical care. We present an analytical exposition of ethical structural features in judgement-based clinical care predicated on the assumption of the moral equality of human beings and the assessment of where healthcare contexts pose a challenge to achieving moral equality. The account and the emerging overview is worked out so that it can be easily contextualized with regards to national healthcare systems and specific branches of healthcare, as well as local healthcare institutions. By considering how the account and the overview can be applied to i) improve the ethical competence of healthcare personnel and consultants by broadening their sensitivity to ethical tensions, ii) identify neglected areas for ethical research, and iii) clarify the ethical responsibility of healthcare institutions' leadership, as well as specifying required institutionalized administration, we conclude that the proposed account should be considered useful for CESS.     

Anticancer double lipid prodrugs: liposomal preparation and characterization

The escape of encapsulated anticancer drugs from liposomes by passive diffusion often leads to suboptimal drug concentrations in the cancer tissue, therefore calling for effective trigger mechanisms to release the drug at the target. We investigated mixtures of lipid components that not only form stable liposomes, but also can be turned into active drugs by secretory phospholipase A? (sPLA?), an enzyme that is upregulated in various cancer cells, without the necessity for conventional liposome drug loading. The liposomes are composed of a novel lipid-based retinoid prodrug premixed with saturated phospholipids. The prodrug is found to be miscible with phospholipids, and the lipid mixtures are shown to form liposomes with the desired size distribution. The preparation procedure, phase behavior, and physicochemical properties of the formed liposomes are described as a function of lipid composition. We show that the premixing of the prodrug with phospholipids can be used to modify the physicochemical properties of liposomal formulations. The results should prove useful for further exploration of the potential for using these novel lipid prodrugs in liposomal formulations for cancer treatment.