A stapled chromogranin A-derived peptide homes in on tumors that express αvβ6 or αvβ8 integrins

Rationale: The αvβ6- and αvβ8-integrins, two cell-adhesion receptors upregulated in many tumors and involved in the activation of the latency associated peptide (LAP)/TGFβ complex, represent potential targets for tumor imaging and therapy. We investigated the tumor-homing properties of a chromogranin A-derived peptide containing an RGDL motif followed by a chemically stapled alpha-helix (called “5a”), which selectively recognizes the LAP/TGFβ complex-binding site of αvβ6 and αvβ8.Methods: Peptide 5a was labeled with IRDye 800CW (a near-infrared fluorescent dye) or with ¹⁸F-NOTA (a label for positron emission tomography (PET)); the integrin-binding properties of free peptide and conjugates were then investigated using purified αvβ6/αvβ8 integrins and various αvβ6/αvβ8 single - or double-positive cancer cells; tumor-homing, biodistribution and imaging properties of the conjugates were investigated in subcutaneous and orthotopic αvβ6-positive carcinomas of the pancreas, and in mice bearing subcutaneous αvβ8-positive prostate tumors.Results: In vitro studies showed that 5a can bind both integrins with high affinity and inhibits cell-mediated TGFβ activation. The 5a-IRDye and 5a-NOTA conjugates could bind purified αvβ6/αvβ8 integrins with no loss of affinity compared to free peptide, and selectively recognized various αvβ6/αvβ8 single- or double-positive cancer cells, including cells from pancreatic carcinoma, melanoma, oral mucosa, bladder and prostate cancer. In vivo static and dynamic optical near-infrared and PET/CT imaging and biodistribution studies, performed in mice with subcutaneous and orthotopic αvβ6-positive carcinomas of the pancreas, showed high target-specific uptake of fluorescence- and radio-labeled peptide by tumors and low non-specific uptake in other organs and tissues, except for excretory organs. Significant target-specific uptake of fluorescence-labeled peptide was also observed in mice bearing αvβ8-positive prostate tumors.Conclusions: The results indicate that 5a can home to αvβ6- and/or αvβ8-positive tumors, suggesting that this peptide can be exploited as a ligand for delivering imaging or anticancer agents to αvβ6/αvβ8 single- or double-positive tumors, or as a tumor-homing inhibitor of these TGFβ activators.     

Craniometric variation of some Mediterranean and Atlantic populations of Stenella coeruleoalba (Mammalia,Delphinidae): A three‐dimensional geometric morphometric analysis

The variability of cranial features of Atlantic and Mediterranean samples of Stenella coeruleoalba was examined using a three‐dimensional geometric morphometric approach. Data were collected on 79 skulls from the upper and middle Mediterranean Sea, the Atlantic French coasts, and Scotland. Three‐dimensional x, y, and z coordinates of 27 landmarks were recorded on each left half skull using a Microscribe 3‐D digitizer. All configurations were rotated, centered, and scaled, and residuals from the mean configuration were analyzed through multivariate analyses of variance. Mahalanobis distances among populations were used to evaluate phenetic relationships. Consensus configurations were compared to visualize shape differences among samples. Analyses revealed significant differences among populations, a clear distinction of the Scottish coasts dolphins from the other samples, and a closer relationship of the dolphins from the French coasts to the Mediterranean populations than to the Scottish one. Shape differences are mainly concentrated in the rostral and in the occipital regions of the skull. Phylogenetic and adaptive factors were invoked as possible causes of the variation patterns.     

A genome-wide association analysis for porcine serum lipid traits reveals the existence of age-specific genetic determinants

Background

The genetic determinism of blood lipid concentrations, the main risk factor for atherosclerosis, is practically unknown in species other than human and mouse. Even in model organisms, little is known about how the genetic determinants of lipid traits are modulated by age-specific factors. To gain new insights into this issue, we have carried out a genome-wide association study (GWAS) for cholesterol (CHOL), triglyceride (TRIG) and low (LDL) and high (HDL) density lipoprotein concentrations measured in Duroc pigs at two time points (45 and 190 days).

Results

Analysis of data with mixed-model methods (EMMAX, GEMMA, GenABEL) and PLINK showed a low positional concordance between trait-associated regions (TARs) for serum lipids at 45 and 190 days. Besides, the proportion of phenotypic variance explained by SNPs at these two time points was also substantially different. The four analyses consistently detected two regions on SSC3 (124 Mb, CHOL and LDL at 190 days) and SSC6 (135 Mb, CHOL and TRIG at 190 days) with highly significant effects on the porcine blood lipid profile. Moreover, we have found that SNP variation within SSC3, SSC6, SSC10, SSC13 and SSC16 TARs is associated with the expression of several genes mapping to other chromosomes and related to lipid metabolism.

Conclusions

Our data demonstrate that the effects of genomic determinants influencing lipid concentrations in pigs, as well as the amount of phenotypic variance they explain, are influenced by age-related factors.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-758) contains supplementary material, which is available to authorized users.     

Lipid solvation effects contribute to the affinity of Gly-xxx-Gly motif-mediated helix-helix interactions

Interactions between transmembrane helices are mediated by the concave Gly-xxx-Gly motif surface. Whether Gly residues per se are sufficient for selection of this motif has not been established. Here, we used the in vivo TOXCAT assay to measure the relative affinities of all 18 combinations of Gly, Ala, and Ser "small-xxx-small" mutations in glycophorin A (GpA) and bacteriophage M13 major coat protein (MCP) homodimers. Affinity values were compared with the accessibility to a methylene-sized probe of the total surface area of each helix monomer as a measure of solvation by membrane components. A strong inverse correlation was found between nonpolar-group lipid accessibility and dimer affinity (R = 0.75 for GpA, p = 0.013, and R = 0.81 for MCP, p = 0.004), suggesting that lipid as a poor membrane protein solvent, conceptually analogous to water in soluble protein folding, can contribute to dimer stability and help to define helix-helix interfaces.     

The early light-inducible protein (ELIP) gene is expressed during the chloroplast-to-chromoplast transition in ripening tomato fruit

Chloroplast-to-chromoplast transitions during fruit ripening require massive transformation of the plastid internal membrane structure as the photosynthetic apparatus is disassembled. Early Light-Inducible Proteins (ELIPs) are known to accumulate in chloroplasts during thylakoid biogenesis and under stressful conditions. To determine if ELIP may also play a role in thylakoid disassembly during the chloroplast-to-chromoplast transition, ELIP mRNA expression was measured in tomato, Lycopersicon esculentum Mill. cv. Rutgers. An EST clone was identified in the Tomato Genome Project/Solanaceae Genomics Network database that has high sequence similarity with the amino acid sequence of Arabidopsis ELIP1 and ELIP2. It has complete identity in the two conserved regions of the protein. Genomic Southern blots indicate that the gene is a single copy in tomato. The genomic sequence shows the three-exon structure typical of ELIP sequences from other species. mRNA for this gene is barely detectable on northern blots from etiolated seedlings, but transiently accumulates to high levels 2 h after transfer to the light. Greenhouse-grown tomatoes were used to measure ELIP mRNA accumulation during fruit development and ripening. Tomato ELIP mRNA is detectable in all stages of fruit ripening, but is most abundant in the breaker/turning stage of development. A survey of tomato EST databases revealed that ELIP cDNA is also relatively abundant in developing flowers, which contain yellow chromoplasts. Combined, these results suggest that ELIP may play a newly-recognized role in the chloroplast-to-chromoplast transition process.     

Evidence of hepatitis E virus replication on cell cultures.

Several human and animal cell lines have been used to grow hepatitis E virus. The strain SAR-55 was adapted only on PLF/PLC/5 cell line without any visible cytopathic effect. The growth of the SAR-55 was monitored by examining the positive and the negative strands of HEV-RNA. Stool samples, obtained from hospitalised acute hepatitis patients at the Fever Hospital of Alexandria (Egypt), were used to confirm the susceptibility of PLF/PLC/5 cells. After more than one-week's cultivation, three stool samples out of 17 IgM anti-HEV positive and 1 from 52 IgG anti-HEV positive patients showed a specific RT-PCR amplification product. The nucleotide sequences of the methyltransferase region of the genome in the isolates revealed the maximum homology with Burma strain with several point mutations.     

Serum levels of anti-heat shock protein 27 antibodies in patients with chronic liver disease

Heat shock protein 27 (HSP27), an intracellular molecular chaperone, is involved in the pathogenesis of cancer by promoting both tumor cell proliferation and resistance to therapy. HSP27 is also present in the circulation and circulating HSP27 (sHSP27) can elicit an autoimmune response with production of antibodies. Levels of sHSP27 are enhanced in patients with hepatocellular carcinoma (HCC); it is, however, unknown whether changes in HSP27 antibody levels occur in patients with HCC and can be exploited as a circulating biomarker of HCC. Our aim was to assess the potential association between newly diagnosed HCC and serum anti-HSP27 antibody levels. In this cross-sectional study, anti-HSP27 antibody levels were measured in serum samples from 71 HCC patients, 80 subjects with chronic liver disease, and 38 control subjects by immunoenzymatic assay. Anti-HSP27 antibody levels did not differ significantly among groups. However, in patients with chronic active hepatitis/cirrhosis, anti-HSP27 levels were significantly higher in subjects with a positive history of alcoholism (p = 0.03). Our data do not support the hypothesis that anti-HSP27 antibody levels may help identify patients with HCC among subjects with chronic liver disease. However, our finding that alcohol-related liver disease is associated with higher anti-HSP27 levels is novel and deserves further investigations.     

Identification of differentially expressed microRNAs by microarray: a possible role for microRNA genes in pituitary adenomas

MicroRNAs (miRNAs) are small non-coding RNAs that control gene expression by targeting mRNA. It has been demonstrated that miRNA expression is altered in many human cancers, suggesting that they may play a role in human neoplasia. To determine whether miRNA expression is altered in pituitary adenomas, we analyzed the entire miRNAome in 32 pituitary adenomas and in 6 normal pituitary samples by microarray and by Real-Time PCR. Here, we show that 30 miRNAs are differentially expressed between normal pituitary and pituitary adenomas. Moreover, 24 miRNAs were identified as a predictive signature of pituitary adenoma and 29 miRNAs were able to predict pituitary adenoma histotype. miRNA expression could differentiate micro- from macro-adenomas and treated from non-treated patient samples. Several of the identified miRNAs are involved in cell proliferation and apoptosis, suggesting that their deregulated expression may be involved in pituitary tumorigenesis. Predictive miRNAs could be potentially useful diagnostic markers, improving the classification of pituitary adenomas.     

Pooled genome-wide analysis to identify novel risk loci for pediatric allergic asthma

Background

Genome-wide association studies of pooled DNA samples were shown to be a valuable tool to identify candidate SNPs associated to a phenotype. No such study was up to now applied to childhood allergic asthma, even if the very high complexity of asthma genetics is an appropriate field to explore the potential of pooled GWAS approach.

Methodology/Principal Findings

We performed a pooled GWAS and individual genotyping in 269 children with allergic respiratory diseases comparing allergic children with and without asthma. We used a modular approach to identify the most significant loci associated with asthma by combining silhouette statistics and physical distance method with cluster-adapted thresholding. We found 97% concordance between pooled GWAS and individual genotyping, with 36 out of 37 top-scoring SNPs significant at individual genotyping level. The most significant SNP is located inside the coding sequence of C5, an already identified asthma susceptibility gene, while the other loci regulate functions that are relevant to bronchial physiopathology, as immune- or inflammation-mediated mechanisms and airway smooth muscle contraction. Integration with gene expression data showed that almost half of the putative susceptibility genes are differentially expressed in experimental asthma mouse models.

Conclusion/Significance

Combined silhouette statistics and cluster-adapted physical distance threshold analysis of pooled GWAS data is an efficient method to identify candidate SNP associated to asthma development in an allergic pediatric population.