Bacterial product cantastim derived from Pseudomonas aeruginosa induces migration and maturation of dendritic cells

Dendritic cells (DCs) play a pivotal role in linking innate and adaptive immunity. Migration to the lymph nodes and maturation of DCs are crucial steps in the initiation of specific immune responses. The bacterial product CANTASTIM (CS) is a purified extract of Pseudomonas aeruginosa that induces non-specific protection against bacterial infection, enhances macrophage effector functions and modulates cytokines production. In this study, we used a mouse skin explant culture model and human monocyte-derived DCs to study the effect of CS on the migration and maturation of DCs, respectively. We noticed a significant increase in the number of DCs which migrated from the skin explants when CS was added to the culture medium. Also, CS was able to induce the expression of maturation-associated marker CD83 on human monocyte-derived DCs. DC-based tumor vaccines represent a promising approach for cancer immunotherapy and the migration rate and maturation state of DCs are important parameters for their clinical effectiveness. CS may be an attractive candidate to be tested for the production of DC-based vaccine.     

Systemic inflammatory markers in patients with aortic sclerosis

The aim of the study was to evaluate several mediators of inflammation in patients with aortic sclerosis in relation to severity of cardiovascular disease. Serum level of cytokines, soluble intracellular adhesion molecule 1, matrix metalloproteinase (MMP) 2 and 9 and their tissue inhibitor TIMP-1, were measured by ELISA and MMPs activity by zymography in 51 aortic sclerosis patients. The increase in MMPs expression positively correlated with their gelatinase activity; also there was a positive correlation between MMP-9 and TIMP-1 serum levels. Moreover, IL-6 concentration positively correlated with both serum level and activity of MMP-9. The level of IL-6 and IL-1Ra were higher in patients with a great burden of atherosclerosis. Noteworthy, statistically significant higher levels of IL-6 were noticed for patients with coronary artery disease. There was a significant increase in IL-6 serum level as well as a significant decrease in IL-1Ra for patients with a history of myocardial infarction. A trend toward higher concentration of inflammatory mediators was noticed in relation to the increase in severity of the aortic valve disease. Our results support the hypothesis of an "inflammatory pattern" associated with AS pathology and suggest the persistence of a chronic inflammation in patients who experienced acute coronary events.     

Chromosomal instability in Afrotheria: fragile sites,evolutionary breakpoints and phylogenetic inference from genome sequence assemblies

Background  

Extant placental mammals are divided into four major clades (Laurasiatheria, Supraprimates, Xenarthra and Afrotheria). Given that Afrotheria is generally thought to root the eutherian tree in phylogenetic analysis of large nuclear gene data sets, the study of the organization of the genomes of afrotherian species provides new insights into the dynamics of mammalian chromosomal evolution. Here we test if there are chromosomal bands with a high tendency to break and reorganize in Afrotheria, and by analyzing the expression of aphidicolin-induced common fragile sites in three afrotherian species, whether these are coincidental with recognized evolutionary breakpoints.     

Serotypes, virulence genes and intimin types of Shiga toxin (verocytotoxin)-producing Escherichia coli isolates from minced beef in Lugo (Spain) from 1995 through 2003

Background

The RNA-binding protein Hfq is involved in stress and virulence of several pathogens, probably due to its role as mediator in small RNA (sRNA)-mRNA interactions. In this study, we investigate the function of Hfq in the Gram-positive pathogen Staphylococcus aureus, by constructing hfq null mutant derivatives.

Results

We report that unexpectedly, in S. aureus, Hfq does not seem to play a crucial role in stress response, RNAIII or spa mRNA quantity and exoprotein expression, as tested in three virulent genetic backgrounds. Moreover, a global analysis of the RN6390 hfq mutant, which tests ~ 2000 phenotypes, supports our results concerning the non-implication of Hfq in stress response, and shows that Hfq is also not involved in resistance to several chemical agents and antibiotics and does not seem to be implicated in metabolic pathways.

Conclusion

Our data suggest that although sRNA-mRNA interactions in S. aureus are decisive for gene expression regulation, they do not require the RNA-chaperone protein Hfq. These interactions possibly require an RNA-chaperone protein other than Hfq, which remains to be found.     

ECERIFERUM2-LIKE Proteins Have Unique Biochemical and Physiological Functions in Very-Long-Chain Fatty Acid Elongation

The extension of very-long-chain fatty acids (VLCFAs) for the synthesis of specialized apoplastic lipids requires unique biochemical machinery. Condensing enzymes catalyze the first reaction in fatty acid elongation and determine the chain length of fatty acids accepted and produced by the fatty acid elongation complex. Although necessary for the elongation of all VLCFAs, known condensing enzymes cannot efficiently synthesize VLCFAs longer than 28 carbons, despite the prevalence of C28 to C34 acyl lipids in cuticular wax and the pollen coat. The eceriferum2 (cer2) mutant of Arabidopsis (Arabidopsis thaliana) was previously shown to have a specific deficiency in cuticular waxes longer than 28 carbons, and heterologous expression of CER2 in yeast (Saccharomyces cerevisiae) demonstrated that it can modify the acyl chain length produced by a condensing enzyme from 28 to 30 carbon atoms. Here, we report the physiological functions and biochemical specificities of the CER2 homologs CER2-LIKE1 and CER2-LIKE2 by mutant analysis and heterologous expression in yeast. We demonstrate that all three CER2-LIKEs function with the same small subset of condensing enzymes, and that they have different effects on the substrate specificity of the same condensing enzyme. Finally, we show that the changes in acyl chain length caused by each CER2-LIKE protein are of substantial importance for cuticle formation and pollen coat function.The extension of fatty acids to lengths greater than 28 carbons (C28) is an exceptional process in plant metabolism in that it requires unique biochemical machinery, and the elongation products are used for the synthesis of specialized plant metabolites. Derivatives of C30 to C34 fatty acids make up the bulk of plant cuticular wax, which coats all of a plant’s primary aerial surfaces. Cuticular wax serves as a barrier against transpirational water loss (Riederer and Schreiber, 2001) and protects the plant from both biotic (Eigenbrode, 1996) and abiotic (Grace and van Gardingen, 1996) stresses. C30 to C34 fatty acid-derived lipids are also components of the pollen coat, where they function in pollen hydration and germination on dry stigma (Elleman et al., 1992; Preuss et al., 1993).The core complex that elongates long-chain fatty acids (C16–C18) to very-long-chain fatty acids (VLCFAs; C20–C34) consists of four interacting proteins localized to the endoplasmic reticulum (ER). β-Keto-acyl-CoA synthases (KCSs), also known as condensing enzymes, catalyze the first reaction required for VLCFA elongation, condensing malonyl-CoA with an acyl-CoA (n) to produce a β-keto-acyl-CoA (n + 2). Condensation is both a specific and rate-limiting step in elongation (Millar and Kunst, 1997). Chain length specificity of KCSs is of particular importance because VLCFA length determines the downstream use of the fatty acid (for review, see Joubès et al., 2008; Haslam and Kunst, 2013a). There are two families of condensing enzymes in Arabidopsis (Arabidopsis thaliana). The ELONGATION-DEFECTIVE (ELO)-LIKE family is homologous to yeast (Saccharomyces cerevisiae) ELOs, and has putative functions in sphingolipid biosynthesis (Quist et al., 2009). Although our current understanding of plant ELO-LIKE physiological function and biochemical activity is limited, the mechanism of yeast Elo protein activity has been thoroughly investigated (Denic and Weissman, 2007). The FATTY ACID ELONGATION1 (FAE1)-type family is homologous to the first condensing enzyme identified in Arabidopsis, which is required for the synthesis of C20 to C22 VLCFAs in Arabidopsis oilseeds. Many of the 21 FAE1-type condensing enzymes of Arabidopsis have been characterized using reverse genetics and heterologous expression in yeast (Trenkamp et al., 2004; Blacklock and Jaworski, 2006; Paul et al., 2006; Tresch et al., 2012). This work has revealed the intriguing caveat that, although FAE1-type KCSs are involved in the synthesis of diverse downstream metabolites and use a broad range of acyl chain lengths, none are able to efficiently elongate VLCFAs beyond C28 (for review, see Haslam and Kunst, 2013a), which is essential for the production of cuticular wax components.Eceriferum2 (cer2) and glossy2 (gl2) mutants of Arabidopsis and Zea mays, respectively, are deficient in specific VLCFA-derived waxes longer than C28 (Bianchi et al., 1975; McNevin et al., 1993; Jenks et al., 1995). Both mutations were mapped to genes that do not resemble any component of the elongase complex (Tacke et al., 1995; Xia et al., 1996), but are homologous to the BAHD family of acyltransferases (St-Pierre et al., 1998). However, site-directed mutagenesis of conserved acyltransferase catalytic site amino acids in CER2 revealed that this motif is not required for CER2 function in cuticular wax synthesis (Haslam et al., 2012).CER6 is a condensing enzyme necessary for the accumulation of stem cuticular waxes in Arabidopsis, but when expressed in yeast, CER6 can only elongate VLCFAs to C28. When CER2 is expressed in yeast, it has no elongation activity. However, coexpression of CER2 and CER6 results in efficient production of C30 VLCFAs. Coexpression of CER2 with LfKCS45, a condensing enzyme from the crucifer Lesquerella fendleri that generates C28 and a small amount of C30 VLCFAs (Moon et al., 2004), does not alter product chain length (Haslam et al., 2012). Based on these observations, it was hypothesized that CER2 modifies the chain length specificity of the core elongase complex by interaction with specific KCS enzymes (Haslam et al., 2012).CER2 homologs are found in diverse flowering plant lineages, and many species have multiple CER2 homologs (Tuominen et al., 2011). A BLAST search of proteins from Arabidopsis identified two sequences with substantial similarity to CER2. {"type":"entrez-protein","attrs":{"text":"NP_193120","term_id":"15236357","term_text":"NP_193120"}}NP_193120 is 36% identical to CER2, and is encoded by the gene At4g13840. We named this gene CER2-LIKE1 (also known as CER26) (Pascal et al., 2013). {"type":"entrez-protein","attrs":{"text":"NP_566741","term_id":"18403923","term_text":"NP_566741"}}NP_566741 is 38% identical to CER2, and is encoded by the gene At3g23840. We named this gene CER2-LIKE2 (also named CER26-LIKE) (Pascal et al., 2013). Characterization of a cer2-like1 null mutant revealed a role for the CER2-LIKE1 protein in the elongation of leaf wax precursors beyond C30, analogous to the role of CER2 in C28 elongation in stems (Haslam et al., 2012; Pascal et al., 2013). cer2 cer2-like1 double mutants are deficient in the formation of wax components longer than C28 in both stems and leaves. As the cer2 single mutant has no leaf wax phenotype, the additive effect of these two mutations on leaf wax composition indicates that there is partial functional redundancy between the two genes.A comprehensive investigation of the biochemical and physiological functions of CER2-LIKE proteins is necessary. Beyond the value of knowing the specific roles of each homolog, such an investigation has potential to elucidate the nature of CER2-LIKE protein function. With this objective, we used our data to address the following questions: (1) Do CER2-LIKE proteins function with CER6 alone, or can they modify the activity of other FAE1-type condensing enzymes? (2) Do CER2-LIKE proteins have different effects on the substrate specificity of the same condensing enzyme, or is substrate specificity determined exclusively by the condensing enzyme? (3) What is the physiological relevance of the subtle changes in acyl lipid chain length that CER2-LIKE proteins induce?     

Use of targeted SNP selection for an improved anchoring of the melon (Cucumis melo L.) scaffold genome assembly

Background

The genome of the melon (Cucumis melo L.) double-haploid line DHL92 was recently sequenced, with 87.5 and 80.8% of the scaffold assembly anchored and oriented to the 12 linkage groups, respectively. However, insufficient marker coverage and a lack of recombination left several large, gene rich scaffolds unanchored, and some anchored scaffolds unoriented. To improve the anchoring and orientation of the melon genome assembly, we used resequencing data between the parental lines of DHL92 to develop a new set of SNP markers from unanchored scaffolds.

Results

A high-resolution genetic map composed of 580 SNPs was used to anchor 354.8 Mb of sequence, contained in 141 scaffolds (average size 2.5 Mb) and corresponding to 98.2% of the scaffold assembly, to the 12 melon chromosomes. Over 325.4 Mb (90%) of the assembly was oriented. The genetic map revealed regions of segregation distortion favoring SC alleles as well as recombination suppression regions coinciding with putative centromere, 45S, and 5S rDNA sites. New chromosome-scale pseudomolecules were created by incorporating to the previous v3.5 version an additional 38.3 Mb of anchored sequence representing 1,837 predicted genes contained in 55 scaffolds. Using fluorescent in situ hybridization (FISH) with BACs that produced chromosome-specific signals, melon chromosomes that correspond to the twelve linkage groups were identified, and a standardized karyotype of melon inbred line T111 was developed.

Conclusions

By utilizing resequencing data and targeted SNP selection combined with a large F2 mapping population, we significantly improved the quantity of anchored and oriented melon scaffold genome assembly. Using genome information combined with FISH mapping provided the first cytogenetic map of an inodorus melon type. With these results it was possible to make inferences on melon chromosome structure by relating zones of recombination suppression to centromeres and 45S and 5S heterochromatic regions. This study represents the first steps towards the integration of the high-resolution genetic and cytogenetic maps with the genomic sequence in melon that will provide more information on genome organization and allow for the improvement of the melon genome draft sequence.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-014-1196-3) contains supplementary material, which is available to authorized users.