Giant cell arteritis: how to diagnose?--a case report

We present a case of 77 years old male with suspected giant cell arteritis. With anamnesis, physical examination, immunological tests, Colour Doppler ultrasonography of superficial temporal artery and finally with patohistological analysis of temporal artery biopsy, we came to right diagnosis.     

Identification of a functionally essential amino acid for Arabidopsis cyclic nucleotide gated ion channels using the chimeric AtCNGC11/12 gene

We used the chimeric Arabidopsis cyclic nucleotide-gated ion channel AtCNGC11/12 to conduct a structure-function study of plant cyclic nucleotide-gated ion channels (CNGCs). AtCNGC11/12 induces multiple pathogen resistance responses in the Arabidopsis mutant constitutive expresser of PR genes 22 (cpr22). A genetic screen for mutants that suppress cpr22-conferred phenotypes identified an intragenic mutant, #73, which has a glutamate to lysine substitution (E519K) at the beginning of the eighth beta-sheet of the cyclic nucleotide-binding domain in AtCNGC11/12. The #73 mutant is morphologically identical to wild-type plants and has lost cpr22-related phenotypes including spontaneous cell death and enhanced pathogen resistance. Heterologous expression analysis using a K(+)-uptake-deficient yeast mutant revealed that this Glu519 is important for AtCNGC11/12 channel function, proving that the occurrence of cpr22 phenotypes requires active channel function of AtCNGC11/12. Additionally, Glu519 was also found to be important for the function of the wild-type channel AtCNGC12. Computational structural modeling and in vitro cAMP-binding assays suggest that Glu519 is a key residue for the structural stability of AtCNGCs and contributes to the interaction of the cyclic nucleotide-binding domain and the C-linker domain, rather than the binding of cAMP. Furthermore, a mutation in the alpha-subunit of the human cone receptor CNGA3 that causes total color blindness aligned well to the position of Glu519 in AtCNGC11/12. This suggests that AtCNGC11/12 suppressors could be a useful tool for discovering important residues not only for plant CNGCs but also for CNGCs in general.     

Interactions of DNA with giant liposomes

DNA interactions with the bilayers of cationic liposomes were studied using a novel model experiment: DNAs were locally injected by a micropipette to a part of a giant unilamellar vesicle. The resulting phenomena were directly observed in optical microscope. Giant unilamellar vesicles (GUVs), about 100 microm in diameter, made of phosphatidylcholines and up to 33 mol% of the natural bioactive cationic amphiphile sphingosine, were obtained by electroformation. The effects of DNAs of different length were tested: (i) 'short' DNAs-oligonucleotide 21b, and calf thymus 250 bp; (ii) 'long' DNAs-plasmid DNAs in super coil or liner form (between 2.7 and 8.0 kbp). DNAs were injected native, as well as marked with the fluorescent dye Hoechst. The resulting membrane topology transformations were monitored in phase contrast, while the DNA distribution was followed in fluorescence. DNA-induced endocytosis was observed due to the DNA/lipid membrane local interactions for all DNAs tested. Some of the DNA in the formed complex was associated with the induced endosomes, and some of it remained spread over the 'mother' GUV membrane for all DNAs tested, except for the longest one--the linear plasmid of 8 kbp. The last remained at the 'mother' GUV membrane and was not transported with the induced endosomes to the internal GUV space. Possible mechanisms for DNA/lipid membrane interaction were suggested. One of them involves DNA encapsulation within an inverted micelle included in the lipid membrane. The model observations could help in understanding events associated with interaction of DNA with biological membranes, as well as cationic liposomes/DNA complexes formation in gene transfer processes.     

Macroscopic consequences of the action of phospholipase C on giant unilamellar liposomes

Macroscopic consequences of the formation of diacylglycerol by phospholipase C (PC-PLC) in giant 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) unilamellar vesicles (GUVs, diameter 10-100 microm) were studied by phase contrast and fluorescence microscopy. PC-PLC caused a series of fast stepwise shrinkages of fluid SOPC GUVs, continuing until the vesicle disappeared beyond the optical resolution of the microscope. The presence of N-palmitoyl-sphingomyelin (mole fraction X = 0.25) in the GUVs did not affect the outcome of the PC-PLC reaction. In addition to hydrolysis, PC-PLC induced adhesion of vicinal vesicles. When multilamellar SOPC vesicles were used only a minor decrease in their diameter was evident suggesting that PC-PLC can exert its hydrolytic activity only in the outer monolayer. A series of stepwise shrinkages was observed also for 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) GUVs above their main phase transition temperature, T(m), i.e., when the bilayer is in the liquid crystalline state. However, this process was not observed for DMPC GUVs in the gel state, below T(m). These results are supported by the enhanced activity of PC-PLC upon exceeding T(m) of DMPC large unilamellar vesicles (diameter approximately 0.1 microm) used as a substrate. Studies on SOPC monolayers revealed that PC-PLC can exert its hydrolytic activity only at surface pressures below approximately 30 mN/m. Accordingly, the lack of changes in the gel state DMPC GUVs could be explained by the equilibrium lateral pressure in these vesicles exceeding this critical value.     

Characterization of the immunophenotypes and antigenomes of colorectal cancers reveals distinct tumor escape mechanisms and novel targets for immunotherapy

BackgroundWhile large-scale cancer genomic projects are comprehensively characterizing the mutational spectrum of various cancers, so far little attention has been devoted to either define the antigenicity of these mutations or to characterize the immune responses they elicit. Here we present a strategy to characterize the immunophenotypes and the antigen-ome of human colorectal cancer.ResultsWe apply our strategy to a large colorectal cancer cohort (n = 598) and show that subpopulations of tumor-infiltrating lymphocytes are associated with distinct molecular phenotypes. The characterization of the antigenome shows that a large number of cancer-germline antigens are expressed in all patients. In contrast, neo-antigens are rarely shared between patients, indicating that cancer vaccination requires individualized strategy. Analysis of the genetic basis of the tumors reveals distinct tumor escape mechanisms for the patient subgroups. Hypermutated tumors are depleted of immunosuppressive cells and show upregulation of immunoinhibitory molecules. Non-hypermutated tumors are enriched with immunosuppressive cells, and the expression of immunoinhibitors and MHC molecules is downregulated. Reconstruction of the interaction network of tumor-infiltrating lymphocytes and immunomodulatory molecules followed by a validation with 11 independent cohorts (n = 1,945) identifies BCMA as a novel druggable target. Finally, linear regression modeling identifies major determinants of tumor immunogenicity, which include well-characterized modulators as well as a novel candidate, CCR8, which is then tested in an orthologous immunodeficient mouse model.ConclusionsThe immunophenotypes of the tumors and the cancer antigenome remain widely unexplored, and our findings represent a step toward the development of personalized cancer immunotherapies.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0620-6) contains supplementary material, which is available to authorized users.     

Effect of UV Radiation and Other Abiotic Stress Factors on DNA of Different Wild Plant Species Grown in Three Successive Seasons in Alpine and Subalpine Regions

Plants in natural ecosystems are exposed to a combination of UV radiation, ionizing radiation (IR) and other abiotic factors. These factors change with the altitude. We investigated DNA alterations of some wild plants of different plant families in natural ecosystems at three altitudes in Rila Mountain, Bulgaria (1500, 1782, and 2925 m above sea level (a.s.l.) exposed to UV radiation, IR and other abiotic stresses, to assess the tolerance of plant species to the changing environmental conditions in three successive growth seasons. For this purpose, physicochemical, cytogenetic, and molecular methods were applied. DNA damage was assessed by micronucleus test and molecular method comet assay adapted and applied by us to wild plant species from Onagraceae, Rosaceae, Boraginaceae, Saxifragaceae, Orobanchaceae, Asteraceae and Poaceae families, growing at three different altitudes. Variability in the DNA sensitivity and the level of tolerance was observed among the plant species in response to combined abiotic factors assessed by induced DNA damage and gross beta activity. The studied representatives of Poaceae were less susceptible than the other studied species at all three altitudes and showed close level of DNA injuries to that of unaffected control plant grown in laboratory conditions. The lower levels of DNA damage of these wild plant species corresponded to their lower ability to accumulate radionuclides. There was a particularly pronounced low level of DNA injuries in the plant species at the highest altitude. The level of DNA damage showed correlation with the values of some abiotic environmental factors. The results would contribute to the elucidation of the extent of adaptation of plant species to the continuously changing environment and would be useful in selecting sensitive herbaceous monitor species for environmental impact assessment at mountain and alpine sites.     

Immunocytochemical demonstration of substance P in hamster Leydig cells during ontogenesis

The cellular localization of substance P immunoreactivity was demonstrated at the light microscopical level in the hamster testis during fetal and postnatal development. A selective immunostaining was observed both of fetal and adult generation of Leydig cells. The comparison of the immunocytochemical findings with the ultrastructural characteristics of Leydig cells provided evidence that Leydig cells besides their androgen-producing capacity also had an neuropeptide producing function. The possible role of substance P in the local paracrine control of gametogenesis was discussed.     

Molecular modeling of transmembrane helices 6 and 7 of the heptahelical lutropin receptor

In response to ligand binding and activating mutations, the lutropin receptor undergoes a conformational change to trigger a cellular response. D556 is the most common locus for naturally occurring activating mutations of the lutropin receptor, and a D556A mutant is shown to be constitutively active. A water-mediated proton transfer is postulated as part of the transmembrane signaling mechanism. Using energy minimization and ab initio calculations, a hydrogen bonding network involving a highly constrained water molecule(s) and D556 (helix 6) and N593/N597/Y601 (helix 7) is presented.     

Functional Differences of Invariant and Highly Conserved Residues in the Extracellular Domain of the Glycoprotein Hormone Receptors

Multiple interactions exist between human follicle-stimulating hormone (FSH) and the N-terminal hormone-binding fragment of the human FSH receptor (FSHR) extracellular domain (ECD). Binding of the other human glycoprotein hormones to their cognate human receptors (luteinizing hormone receptor (LHR) and thyroid-stimulating hormone receptor (TSHR)) was expected to be similar. This study focuses on amino acid residues in β-strands 2 (Lys⁷⁴), 4 (Tyr¹²⁴, Asn¹²⁹, and Thr¹³⁰), and 5 (Asp¹⁵⁰ and Asp¹⁵³) of the FSHR ECD identified in the human FSH·FSHR ECD crystal structure as contact sites with the common glycoprotein hormone α-subunit, and on noncontact residues in β-strands 2 (Ser⁷⁸) and 8 (Asp²²⁴ and Ser²²⁶) as controls. These nine residues are either invariant or highly conserved in LHR and TSHR. Mutagenesis and functional characterization of these residues in all three human receptors allowed an assessment of their contribution to binding and receptor activation. Surprisingly, the six reported α-subunit contact residues of the FSHR ECD could be replaced without significant loss of FSH binding, while cAMP signaling potency was diminished significantly with several replacements. Comparative studies of the homologous residues in LHR and TSHR revealed both similarities and differences. The results for FSH/FSHR were analyzed on the basis of the crystal structure of the FSH·FSHR ECD complex, and comparative modeling was used to generate structures for domains, proteins, and complexes for which no structures were available. Although structural information of hormone-receptor interaction allowed the identification of hormone-receptor contact sites, functional analysis of each contact site was necessary to assess its contribution to hormone binding and receptor activation.     

Haloferax volcanii PitA: an example of functional interaction between the Pfam chlorite dismutase and antibiotic biosynthesis monooxygenase families?

A curious fusion between chlorite dismutase-like and antibiotic biosynthesis monooxygenase-like domains within a single open reading frame has been revealed by both sequence homology and structural modeling in Haloferax volcanii PitA and its homologues in other halophilic archaea. While this fusion may reflect an environmental adaptation to life in hypersaline environments and hence one specific to haloarchaea, PitA and its homologues may represent a paradigm of biologically-relevant interplay between these two distinct activities in accordance with the Rosetta Stone approach.