Comparative genetic mapping reveals synteny and collinearity between the American cranberry and diploid blueberry genomes

Cranberry and blueberry are closely related and recently domesticated fruit crops in the genus Vaccinium. Both have a presumed American origin and likely evolved from a common ancestor; however, details of their adaptive radiation and the extent of their genomic divergence remains little understood. To better understand their evolutionary and genomic relationships, a set of 323 cross-transferable simple sequence repeat (SSR) markers were identified, added to existing marker datasets, and used to construct linkage maps for cranberry (582 SSRs) and an interspecific diploid blueberry population (V. darrowii x V. corymbosum) x V. corymbosum (409 markers, densest blueberry SSR map currently available). The maps allowed for the first comparative genetic mapping study in Vaccinium, and revealed a surprisingly high degree of macro-synteny and collinearity between the cranberry and blueberry genomes. Approximately 93% of the blueberry linkage map was collinear with cranberry, while the remaining 7% (66.3 cM) was spread across 15 non-collinear regions detected in eight of the 12 linkage groups. These observations suggest that large-scale genome differentiation between the cranberry and blueberry genomes has not occurred during their evolution, and that sequence information will be highly transferable between the species in future genetic research and breeding. Finally, the set of 323 cross-transferable SSRs and linkage maps they were used to construct can serve as a shared resource for the Vaccinium research community, enabling additional comparative mapping studies, the identification and transfer of quantitative trait loci and candidate genes between species, and future exploration of evolutionary relationships in Vaccinium.     

Chemotherapy and autophagy-mediated cell death in pancreatic cancer cells

Autophagy is an evolutionarily preserved degradation process of cytoplasmic cellular constituents and plays important physiological roles in human health and disease. It has been proposed that autophagy plays an important role both in tumor progression and in promotion of cancer cell death, although the molecular mechanisms responsible for this dual action of autophagy in cancer have not been elucidated. Pancreatic ductal adenocarcinoma is one of the most aggressive human malignancies with 2-3% five-year survival rate. Its poor prognosis has been attributed to the lack of specific symptoms and early detection tools, and its relatively refractory to traditional cytotoxic agents and radiotherapy. Experimental evidence pointed at autophagy as a pancreatic cancer cell mechanism to survive under adverse environmental conditions, or as a defective programmed cell death mechanism that favors pancreatic cancer cell resistance to treatment. Here, we consider several phenotypical alterations that have been related to increase or decrease the autophagic process in pancreatic tumor cells. We specially review autophagy as a cell death mechanism in response to chemotherapeutic drugs.     

Human papillomavirus type 16 E7 peptide-directed CD8+ T cells from patients with cervical cancer are cross-reactive with the coronavirus NS2 protein

Human papillomavirus type 16 (HPV16) E6 and E7 oncoproteins are required for cellular transformation and represent candidate targets for HPV-specific and major histocompatibility complex class I-restricted CD8(+)-T-cell responses in patients with cervical cancer. Recent evidence suggests that cross-reactivity represents the inherent nature of the T-cell repertoire. We identified HLA-A2 binding HPV16 E7 variant peptides from human, bacterial, or viral origin which are able to drive CD8(+)-T-cell responses directed against wild-type HPV16 E7 amino acid 11 to 19/20 (E7(11-19/20)) epitope YMLDLQPET(T) in vitro. CD8(+) T cells reacting to the HLA-A2-presented peptide from HPV16 E7(11-19(20)) recognized also the HLA-A2 binding peptide TMLDIQPED (amino acids 52 to 60) from the human coronavirus OC43 NS2 gene product. Establishment of coronavirus NS2-specific, HLA-A2-restricted CD8(+)-T-cell clones and ex vivo analysis of HPV16 E7 specific T cells obtained by HLA-A2 tetramer-guided sorting from PBL or tumor-infiltrating lymphocytes obtained from patients with cervical cancer showed that cross-reactivity with HPV16 E7(11-19(20)) and coronavirus NS2(52-60) represents a common feature of this antiviral immune response defined by cytokine production. Zero of 10 patients with carcinoma in situ neoplasia and 3 of 18 patients with cervical cancer showed > or =0.1% HPV16 E7-reactive T cells in CD8(+) peripheral blood lymphocytes. In vivo priming with HPV16 was confirmed in patients with cervical cancer or preinvasive HPV16-positive lesions using HLA-A2 tetramer complexes loaded with the E6-derived epitope KLPQLCTEL. In contrast, we could not detect E6-reactive T cells in healthy individuals. These data imply that the measurement of the HPV16 E7(11-19(20)) CD8(+)-T-cell response may reflect cross-reactivity with a common pathogen and that variant peptides may be employed to drive an effective cellular immune response against HPV.     

Carbon concentration and oxygen availability affect lipid and carotenoid production by carob pulp syrup‐grown Rhodosporidium toruloides NCYC 921

The simultaneous effect of oxygen availability and carbon source concentration on yeast lipid and carotenoid production has never been studied before. In this work, a Doehlert distribution design was used to study the simultaneous effect of carbon concentration and oxygen availability on Rhodosporidium toruloides NCYC 921 carotenoid and lipid production. A cheap industrial byproduct was used as carbon source (carob pulp syrup). A total sugar concentration of 106.3 g/L and a medium volume of 0.120 L induced the highest total carotenoid and total fatty acid productivities (4.60 μg/Lh and 0.029 g/Lh, respectively). Flow cytometry was used to assess yeast stress response under different cultivation conditions. The highest proportion of cells with permeabilised membrane (>20%) was induced when the cultivations were carried out at the highest sugar concentration studied (130.0 g/L) or when the culture reached the minimum final medium pH (4.60). The results showed that the total sugar concentration had a positive influence on the yeast biomass and carotenoid content, while the oxygen availability had little influence on the biomass concentration, but had a slight positive influence on the carotenoid content. Regarding the fatty acids, the two factors had a negative impact on the synthesis of these compounds.     

Impact of antigen presentation on TCR modulation and cytokine release: implications for detection and sorting of antigen-specific CD8+ T cells using HLA-A2 wild-type or HLA-A2 mutant tetrameric complexes

Soluble MHC class I molecules loaded with antigenic peptides are available either to detect and to enumerate or, alternatively, to sort and expand MHC class I-restricted and peptide-reactive T cells. A defined number of MHC class I/peptide complexes can now be implemented to measure T cell responses induced upon Ag-specific stimulation, including CD3/CD8/zeta-chain down-regulation, pattern, and quantity of cytokine secretion. As a paradigm, we analyzed the reactivity of a Melan-A/MART-1-specific and HLA-A2-restricted CD8(+) T cell clone to either soluble or solid-phase presented peptides, including the naturally processed and presented Melan-A/MART-1 peptide AAGIGILTV or the peptide analog ELAGIGILTV presented either by the HLA-A2 wild-type (wt) or mutant (alanineright arrowvaline aa 245) MHC class I molecule, which reduces engagement of the CD8 molecule with the HLA-A2 heavy chain. Soluble MHC class I complexes were used as either monomeric or tetrameric complexes. Soluble monomeric MHC class I complexes, loaded with the Melan-A/MART-1 peptide, resulted in CD3/CD8 and TCR zeta-chain down-regulation, but did not induce measurable cytokine release. In general, differences pertaining to CD3/CD8/zeta-chain regulation and cytokine release, including IL-2, IFN-gamma, and GM-CSF, were associated with 1) the format of Ag presentation (monomeric vs tetrameric MHC class I complexes), 2) wt vs mutant HLA-A2 molecules, and 3) the target Ag (wt vs analog peptide). These differences are to be considered if T cells are exposed to recombinant MHC class I Ags loaded with peptides implemented for detection, activation, or sorting of Ag-specific T cells.     

Protein Kinase Activity of in vitro Cultured Plant Cells in Relation to Growth and Starch Metabolism

Taking tetcyclacis, a norbornenodiazentine derivative, as an example, the influence of a growth retardant on the shoot growth of sunflower, soybean, and maize seedlings grown and treated in hydroculture was investigated. In detail, the reduction in the length of various shoot sections {epicotyl, 1st internode, leaf blade) caused by the retardant was studied. At low concentrations of the retardant (\lt10^-6 M) the shortening effects are substantially attributable to an influence on cell elongation, whereas cell division is inhibited as the concentration increases (τ10^-6 M). A comparison of the effects of tetcyclacis in cell suspension cultures of appropriate plant species showed that also in this system concentrations τ 10^-6 M inhibited cell division growth, i. e. there is comparability of plant/ cell culture regarding the retardant effect on cell division. In contrast to the intact plants, however, cell elongation appears to be of only subordinate importance for the growth of cell cultures, as it has been shown using parsley cell suspension cultures.It is discussed to what extent influencing the gibberellin or sterol biosynthesis by means of tetcyclacis provides an explanation for the concentration-dependent effect on the cell division and cell elongation processes.     

Effect of puromycin on cartilage proteoglycan structure and capacity to bind hyaluronic acid

Rat chondrosarcoma chondrocytes were cultured in the presence of puromycin to induce premature termination of core protein precursor. The structure and function of intracellular and extracellular proteoglycans were assessed by molecular sieve chromatography and polyacrylamide gel electrophoresis. [³H]Serine incorporation was maximally inhibited by 3 × 10^?4m puromycin but unaffected by 10 ^?5m puromycin. Proteoglycans synthesized in the presence of puromycin exhibited increased monomer size due to increased chondroitin sulfate chain size, typical of proteoglycans synthesized in the presence of protein synthesis inhibitors, but no loss in ability to bind to hyaluronic acid; and no loss in core protein size was observed after treatment with chondroitinase. These data suggest that chondrocytes select only completed or nearly completed core protein molecules to process into proteoglycans.     

Neurite Fasciculation Mediated by Complexes of Axonin-1 and Ng Cell Adhesion Molecule

Neural cell adhesion molecules composed of immunoglobulin and fibronectin type III-like domains have been implicated in cell adhesion, neurite outgrowth, and fasciculation. Axonin-1 and Ng cell adhesion molecule (NgCAM), two molecules with predominantly axonal expression exhibit homophilic interactions across the extracellular space (axonin- 1/axonin-1 and NgCAM/NgCAM) and a heterophilic interaction (axonin-1–NgCAM) that occurs exclusively in the plane of the same membrane (cis-interaction). Using domain deletion mutants we localized the NgCAM homophilic binding in the Ig domains 1-4 whereas heterophilic binding to axonin-1 was localized in the Ig domains 2-4 and the third FnIII domain. The NgCAM–NgCAM interaction could be established simultaneously with the axonin-1–NgCAM interaction. In contrast, the axonin-1–NgCAM interaction excluded axonin-1/axonin-1 binding. These results and the examination of the coclustering of axonin-1 and NgCAM at cell contacts, suggest that intercellular contact is mediated by a symmetric axonin-1₂/NgCAM₂ tetramer, in which homophilic NgCAM binding across the extracellular space occurs simultaneously with a cis-heterophilic interaction of axonin-1 and NgCAM. The enhanced neurite fasciculation after overexpression of NgCAM by adenoviral vectors indicates that NgCAM is the limiting component for the formation of the axonin-1₂/NgCAM₂ complexes and, thus, neurite fasciculation in DRG neurons.     

Geographic differences in genetic susceptibility to IgA nephropathy: GWAS replication study and geospatial risk analysis

IgA nephropathy (IgAN), major cause of kidney failure worldwide, is common in Asians, moderately prevalent in Europeans, and rare in Africans. It is not known if these differences represent variation in genes, environment, or ascertainment. In a recent GWAS, we localized five IgAN susceptibility loci on Chr.6p21 (HLA-DQB1/DRB1, PSMB9/TAP1, and DPA1/DPB2 loci), Chr.1q32 (CFHR3/R1 locus), and Chr.22q12 (HORMAD2 locus). These IgAN loci are associated with risk of other immune-mediated disorders such as type I diabetes, multiple sclerosis, or inflammatory bowel disease. We tested association of these loci in eight new independent cohorts of Asian, European, and African-American ancestry (N = 4,789), followed by meta-analysis with risk-score modeling in 12 cohorts (N = 10,755) and geospatial analysis in 85 world populations. Four susceptibility loci robustly replicated and all five loci were genome-wide significant in the combined cohort (P = 5×10⁻³²–3×10⁻¹⁰), with heterogeneity detected only at the PSMB9/TAP1 locus (I² = 0.60). Conditional analyses identified two new independent risk alleles within the HLA-DQB1/DRB1 locus, defining multiple risk and protective haplotypes within this interval. We also detected a significant genetic interaction, whereby the odds ratio for the HORMAD2 protective allele was reversed in homozygotes for a CFHR3/R1 deletion (P = 2.5×10⁻⁴). A seven–SNP genetic risk score, which explained 4.7% of overall IgAN risk, increased sharply with Eastward and Northward distance from Africa (r = 0.30, P = 3×10⁻¹²⁸). This model paralleled the known East–West gradient in disease risk. Moreover, the prediction of a South–North axis was confirmed by registry data showing that the prevalence of IgAN–attributable kidney failure is increased in Northern Europe, similar to multiple sclerosis and type I diabetes. Variation at IgAN susceptibility loci correlates with differences in disease prevalence among world populations. These findings inform genetic, biological, and epidemiological investigations of IgAN and permit cross-comparison with other complex traits that share genetic risk loci and geographic patterns with IgAN.     

Biofilm induced tolerance towards antimicrobial peptides

Increased tolerance to antimicrobial agents is thought to be an important feature of microbes growing in biofilms. We address the question of how biofilm organization affects antibiotic susceptibility. We established Escherichia coli biofilms with differential structural organization due to the presence of IncF plasmids expressing altered forms of the transfer pili in two different biofilm model systems. The mature biofilms were subsequently treated with two antibiotics with different molecular targets, the peptide antibiotic colistin and the fluoroquinolone ciprofloxacin. The dynamics of microbial killing were monitored by viable count determination, and confocal laser microscopy. Strains forming structurally organized biofilms show an increased bacterial survival when challenged with colistin, compared to strains forming unstructured biofilms. The increased survival is due to genetically regulated tolerant subpopulation formation and not caused by a general biofilm property. No significant difference in survival was detected when the strains were challenged with ciprofloxacin. Our data show that biofilm formation confers increased colistin tolerance to cells within the biofilm structure, but the protection is conditional being dependent on the structural organization of the biofilm, and the induction of specific tolerance mechanisms.