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.     

Fuscoside E: a strong anti-inflammatory diterpene from Caribbean octocoral Eunicea fusca

The screen of 10 soft coral extracts collected from the Colombian Caribbean Sea in the TPA-induced ear edema model allowed us to identify Eunicea fusca extract among others as an interesting source of active compounds. The new diterpene, fuscoside E (1), along with the known fuscoside B (2), fuscol (3), (+)-germacrene D (4) and a mixture of six sterols (5-10), were isolated from this soft coral. Their structures were elucidated by 1D and 2D NMR spectroscopy techniques. Fuscoside E (1) absolute stereochemistry was determined by chiroptical methods. Fuscoside E (1) and B (2) showed strong anti-inflammatory in the above mentioned bioassay. Additionally, fuscoside E (1) and the sterol mixture (5-10) presented antifouling activity against bacterial strains involved in surface colonization.     

Inflammation and matrix remodeling during repair of ventilator-induced lung injury

High-pressure ventilation triggers different inflammatory and matrix remodeling responses within the lung. Although some of them may cause injury, the involvement of these mediators in repair is largely unknown. To identify mechanisms of repair after ventilator-induced lung injury (VILI), mice were randomly assigned to baseline conditions (no ventilation), injury [90 min of high-pressure ventilation without positive end-expiratory pressure (PEEP)], repair (injury followed by 4 h of low-pressure ventilation with PEEP), and ventilated controls (low-pressure ventilation with PEEP for 90 and 330 min). Histological injury and lung permeability increased during injury, but were partially reverted in the repair group. This was accompanied by a proinflammatory response, together with increases in TNF-α and IFN-γ, which returned to baseline during repair, and a decrease in IL-10. However, macrophage inflammatory protein-2 (MIP-2) and matrix metalloproteinases (MMP)-2 and -9 increased after injury and persisted in being elevated during repair. Mortality in the repair phase was 50%. Survivors showed increased cell proliferation, lower levels of collagen, and higher levels of MIP-2 and MMP-2. Pan-MMP or specific MMP-2 inhibition (but not MIP-2, TNF-α, or IL-4 inhibition) delayed epithelial repair in an in vitro wound model using murine or human alveolar cells cultured in the presence of bronchoalveolar lavage fluid from mice during the repair phase or from patients with acute respiratory distress syndrome, respectively. Similarly, MMP inhibition with doxycycline impaired lung repair after VILI in vivo. In conclusion, VILI can be reverted by normalizing ventilation pressures. An adequate inflammatory response and extracellular matrix remodeling are essential for recovery. MMP-2 could play a key role in epithelial repair after VILI and acute respiratory distress syndrome.     

Enriched sera protein profiling for detection of non-small cell lung cancer biomarkers

Background

Non Small Cell Lung Cancer (NSCLC) is the major cause of cancer related-death. Many patients receive diagnosis at advanced stage leading to a poor prognosis. At present, no satisfactory screening tests are available in clinical practice and the discovery and validation of new biomarkers is mandatory. Surface Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-ToF-MS) is a recent high-throughput technique used to detect new tumour markers. In this study we performed SELDI-ToF-MS analysis on serum samples treated with the ProteoMiner? kit, a combinatorial library of hexapeptide ligands coupled to beads, to reduce the wide dynamic range of protein concentration in the sample. Serum from 44 NSCLC patients and 19 healthy controls were analyzed with IMAC30-Cu and H50 ProteinChip Arrays.

Results

Comparing SELDI-ToF-MS protein profiles of NSCLC patients and healthy controls, 28 protein peaks were found significantly different (p < 0.05), and were used as predictors to build decision classification trees. This statistical analysis selected 10 protein peaks in the low-mass range (2-24 kDa) and 6 in the high-mass range (40-80 kDa). The classification models for the low-mass range had a sensitivity and specificity of 70.45% (31/44) and 68.42% (13/19) for IMAC30-Cu, and 72.73% (32/44) and 73.68% (14/19) for H50 ProteinChip Arrays.

Conclusions

These preliminary results suggest that SELDI-ToF-MS protein profiling of serum samples pretreated with ProteoMiner? can improve the discovery of protein peaks differentially expressed between NSCLC patients and healthy subjects, useful to build classification algorithms with high sensitivity and specificity. However, identification of the significantly different protein peaks needs further study in order to provide a better understanding of the biological nature of these potential biomarkers and their role in the underlying disease process.     

Microbiological quality of Pecorino Siciliano "primosale" cheese on retail sale in the street markets of Palermo, Italy

Pecorino Siciliano (PS) "primosale" is a traditional Sicilian fresh soft cheese made from sheep's milk. Short-ripening time and production from unpasteurized or raw milk can facilitate bacterial contamination of PS "primosale". The microbiological quality of "primosale" on retail sale in the street markets of Palermo, Italy was studied by detecting the common food pathogens Listeria monocytogenes and Staphylococcus aureus and indicator microorganisms, such as Escherichia coli, Enterobacteriaceae and Staphylococcaceae. In our study, 4% and 44% of the samples, respectively, did not comply with the acceptability levels fixed by European regulations for S. aureus and E. coli. A high contamination of bacteria belonging to Enterobacteriaceae and Staphylococcaceae was found in 42% and 50% of the cheeses analyzed, respectively. Such results indicate poor husbandry and poor hygiene practices during milk collection or preservation or during cheese production processes and handling. In addition, the retail sale conditions may have played a role in cheese contamination since a correlation was found between poor microbiological quality and some selling parameters. This study emphasizes the need to improve production hygiene throughout the PS food chain in line with the traditional cheese-making procedures. Labelling of PS with clear information on whether the cheese was prepared from raw milk also requires improvement.     

Bacterial extract cantastim activates macrophages via TLR-2

CANTASTIM is a second generation bacterial immunomodulator. The aim of this study was to examine the mechanism by which bacterial immunomodulator CANTASTIM induces production of inflammatory cytokines in monocytes/macrophages. Proinflammatory cytokines were induced in PMA-differentiated THP-1 cells by stimulation with TLR agonists and CANTASTIM in the presence or absence of anti-TLR blocking antibodies or isotype matched control antibodies. Also, RNA interference was used to knockdown TLR2 or TLR4 expression in PMA-differentiated THP-1 cells before stimulation. As expected, induction of TNF-alpha and IL-6 by TLR4 agonist LPS was inhibited in a significant manner by anti-TLR4 but not by anti-TLR2 antibody. Unexpectedly, treatment with anti-LR2 blocking antibody inhibited only IL-6 production induced by Pam3CSK4 while the level of TNF-alpha was unchanged. When cells were stimulated by TLR2 agonist heat-killed Listeria monocytogenes the release of TNF-alpha was significantly attenuated by anti-TLR2 antibodies. Silencing of TLR2 led to a statistically significant inhibition of TNF-alpha secretion induced by TLR2 agonist while siRNA silencing of TLR4 did not affect the response to TLR2 agonist. Cells exposed to CANTASTIM produced significant levels of pro-inflammatory cytokines but the levels were lower than LPS-stimulated cells. Production of both cytokines was inhibited by treatment with anti-TLR2 blocking antibody and not by anti-TLR4 antibody. Silencing of TLR2 led to a statistically significant inhibition of TNF-a secretion induced by CANTASTIM while silencing of TLR4 had no effect on the response to CANTASTIM. These results support the hypothesis that CANTASTIM may exert its immunomodulatory and adjuvant activities through interaction of its bacterial components with TLR2.     

A 5-formyltetrahydrofolate cycloligase paralog from all domains of life: comparative genomic and experimental evidence for a cryptic role in thiamin metabolism

A paralog (here termed COG0212) of the ATP-dependent folate salvage enzyme 5-formyltetrahydrofolate cycloligase (5-FCL) occurs in all domains of life and, although typically annotated as 5-FCL in pro- and eukaryotic genomes, is of unknown function. COG0212 is similar in overall structure to 5-FCL, particularly in the substrate binding region, and has distant similarity to other kinases. The Arabidopsis thaliana COG0212 protein was shown to be targeted to chloroplasts and to be required for embryo viability. Comparative genomic analysis revealed that a high proportion (19%) of archaeal and bacterial COG0212 genes are clustered on the chromosome with various genes implicated in thiamin metabolism or transport but showed no such association between COG0212 and folate metabolism. Consistent with the bioinformatic evidence for a role in thiamin metabolism, ablating COG0212 in the archaeon Haloferax volcanii caused accumulation of thiamin monophosphate. Biochemical and functional complementation tests of several known and hypothetical thiamin-related activities (involving thiamin, its breakdown products, and their phosphates) were, however, negative. Also consistent with the bioinformatic evidence, the COG0212 proteins from A. thaliana and prokaryote sources lacked 5-FCL activity in vitro and did not complement the growth defect or the characteristic 5-formyltetrahydrofolate accumulation of a 5-FCL-deficient (ΔygfA) Escherichia coli strain. We therefore propose (a) that COG0212 has an unrecognized yet sometimes crucial role in thiamin metabolism, most probably in salvage or detoxification, and (b) that is not a 5-FCL and should no longer be so annotated.     

Extreme climatic events change the dynamics and invasibility of semi-arid annual plant communities

Extreme climatic events represent disturbances that change the availability of resources. We studied their effects on annual plant assemblages in a semi-arid ecosystem in north-central Chile. We analysed 130 years of precipitation data using generalised extreme-value distribution to determine extreme events, and multivariate techniques to analyse 20 years of plant cover data of 34 native and 11 exotic species. Extreme drought resets the dynamics of the system and renders it susceptible to invasion. On the other hand, by favouring native annuals, moderately wet events change species composition and allow the community to be resilient to extreme drought. The probability of extreme drought has doubled over the last 50 years. Therefore, investigations on the interaction of climate change and biological invasions are relevant to determine the potential for future effects on the dynamics of semi-arid annual plant communities.