Flavonoids inhibit melanoma lung metastasis by impairing tumor cells endothelium interactions

Flavonoids comprise a class of low molecular weight compounds displaying a variety of biological activities including inhibition of tumor growth and metastasis. To gain insight into the mechanisms underlying metastasis inhibition, we have employed the B16-BL6 murine melanoma metastasis model. B57BL/6N mice were injected i.v. with tumor cells and Apigenin, Quercetin, or Tamoxifen, each at 50 mg/kg given i.p., and lung tumor cell colonies counted 14-6 days thereafter. Three different injection schedules were used for each drug: (a) daily injection, starting 24 h before injection of the tumor cells; (b) single dose, 24 h preceding tumor challenge; (c) daily injection, starting 24 h after the injection of the tumor cells. All three compounds significantly reduced tumor lung deposits (Apigenin = Quercetin > Tamoxifen). However, when treatment was delayed by 24 h after tumor cells (schedule c), multiple daily doses of Apigenin or Quercetin were less effective that a single dose of the same compound given 24 h before tumor challenge (schedule b). Apigenin and Quercetin, but not Tamoxifen, were found to inhibit VCAM-1 expression in a dose-dependent manner in HUVEC and in murine pulmonary endothelial cells. In ex vivo experiments, the number of tumor cells adhering to lung vessels was significantly diminished in animals treated with a single dose of Apigenin and Quercetin. These findings indicate that the inhibition of tumor cell metastasis by Apigenin or Quercetin may significantly depend on the ability of these compounds to alter the host's microenvironment, further substantiating the role of the intravascular processes in the metastatic cascade.     

Abnormal vasculature interferes with optic fissure closure in lmo2 mutant zebrafish embryos

Ocular coloboma is a potentially blinding congenital eye malformation caused by failure of optic fissure closure during early embryogenesis. The optic fissure is a ventral groove that forms during optic cup morphogenesis, and through which hyaloid artery and vein enter and leave the developing eye, respectively. After hyaloid artery and vein formation, the optic fissure closes around them. The mechanisms underlying optic fissure closure are poorly understood, and whether and how this process is influenced by hyaloid vessel development is unknown. Here we show that a loss-of-function mutation in lmo2, a gene specifically required for hematopoiesis and vascular development, results in failure of optic fissure closure in zebrafish. Analysis of ocular blood vessels in lmo2 mutants reveals that some vessels are severely dilated, including the hyaloid vein. Remarkably, reducing vessel size leads to rescue of optic fissure phenotype. Our results reveal a new mechanism leading to coloboma, whereby malformed blood vessels interfere with eye morphogenesis.     

Retrotransposon-based molecular markers for grapevine species and cultivars identification

Insertional polymorphisms of two copia-like (Vine-1, Tvv1) and one gypsy-like (Gret1) retrotransposon found in the grapevine genome were studied in 29 Vitis genotypes (Vitis arizonica, Vitis cinerea, Vitis labrusca, Vitis rupestis, Vitis rotundifolia, Vitis vinifera subsp. sylvestris and 23 V. vinifera subsp. sativa) using inter-retrotransposon amplified polymorphism (IRAP), retrotransposon-microsatellite amplified polymorphism (REMAP) and sequence-specific amplified polymorphism (SSAP) techniques. IRAP, REMAP and SSAP polymorphisms were compared with amplified fragment length polymorphism (AFLP), Inter-single sequence repeats (ISSR) and SSR polymorphisms by evaluating the information content, the number of loci simultaneously analysed per experiment, the effectiveness of the analyses in assessing the relationship between accessions and the number of loci needed to obtain a coefficient of variation of 10%. The UPGMA dendrograms of each molecular marker system were compared and the Mantel matrix correspondence test was applied. Furthermore, the corresponding insertion ages of the transposable elements were estimated for each retrotransposon subfamily analysed. The presence of Gret1, Tvv1 and Vine-1 retrotransposons in all analysed genotypes suggests that copia-like and gypsy-like retrotransposons are widespread in Vitis genus. The results indicate that these retrotransposons were active before Vitis speciation and contributed to Vitis genus evolution. IRAP, REMAP and SSAP markers allow the discrimination of Vitis species and V. vinifera subsp. sativa cultivars with certainty as has been shown with AFLP, ISSR and SSR analyses, but phylogenetic trees obtained by retrotransposon-based molecular markers polymorphisms show some significant differences in the allocation of the analysed accessions compare to those obtained by ISSR, AFLP and SSR molecular markers. The phylogenetic tree resulting from REMAP polymorphism appeared the most representative of the effective relationship between all analysed accessions.     

Using radon to quantify groundwater discharge and methane fluxes to a shallow,tundra lake on the Yukon-Kuskokwim Delta,Alaska

Northern lakes are a source of greenhouse gases to the atmosphere and contribute substantially to the global carbon budget. However, the sources of methane (CH₄) to northern lakes are poorly constrained limiting our ability to the assess impacts of future Arctic change. Here we present measurements of the natural groundwater tracer, radon, and CH₄ in a shallow lake on the Yukon-Kuskokwim Delta, AK and quantify groundwater discharge rates and fluxes of groundwater-derived CH₄. We found that groundwater was significantly enriched (2000%) in radon and CH₄ relative to lake water. Using a mass balance approach, we calculated average groundwater fluxes of 1.2 ± 0.6 and 4.3 ± 2.0 cm day⁻¹, respectively as conservative and upper limit estimates. Groundwater CH₄ fluxes were 7—24 mmol m⁻² day⁻¹ and significantly exceeded diffusive air–water CH₄ fluxes (1.3–2.3 mmol m⁻² day⁻¹) from the lake to the atmosphere, suggesting that groundwater is an important source of CH₄ to Arctic lakes and may drive observed CH₄ emissions. Isotopic signatures of CH₄ were depleted in groundwaters, consistent with microbial production. Higher methane concentrations in groundwater compared to other high latitude lakes were likely the source of the comparatively higher CH₄ diffusive fluxes, as compared to those reported previously in high latitude lakes. These findings indicate that deltaic lakes across warmer permafrost regions may act as important hotspots for CH₄ release across Arctic landscapes.

     

Network-based Analysis of Genome Wide Association Data Provides Novel Candidate Genes for Lipid and Lipoprotein Traits

Genome wide association studies (GWAS) identify susceptibility loci for complex traits, but do not identify particular genes of interest. Integration of functional and network information may help in overcoming this limitation and identifying new susceptibility loci. Using GWAS and comorbidity data, we present a network-based approach to predict candidate genes for lipid and lipoprotein traits. We apply a prediction pipeline incorporating interactome, co-expression, and comorbidity data to Global Lipids Genetics Consortium (GLGC) GWAS for four traits of interest, identifying phenotypically coherent modules. These modules provide insights regarding gene involvement in complex phenotypes with multiple susceptibility alleles and low effect sizes. To experimentally test our predictions, we selected four candidate genes and genotyped representative SNPs in the Malmö Diet and Cancer Cardiovascular Cohort. We found significant associations with LDL-C and total-cholesterol levels for a synonymous SNP (rs234706) in the cystathionine beta-synthase (CBS) gene (p = 1 × 10⁻⁵ and adjusted-p = 0.013, respectively). Further, liver samples taken from 206 patients revealed that patients with the minor allele of rs234706 had significant dysregulation of CBS (p = 0.04). Despite the known biological role of CBS in lipid metabolism, SNPs within the locus have not yet been identified in GWAS of lipoprotein traits. Thus, the GWAS-based Comorbidity Module (GCM) approach identifies candidate genes missed by GWAS studies, serving as a broadly applicable tool for the investigation of other complex disease phenotypes.Genome wide association studies (GWAS)¹ meta-analyses have pinpointed a number of new gene regions contributing to multifactorial diseases. GWAS typically find limited numbers of loci that contribute modestly to complex phenotypes (1), and GLGC meta-analysis of GWAS data has reached the limit of what can be expected (2) without the use of alternative strategies. Given that susceptibility loci for complex traits are unlikely to be randomly distributed in the genome (3), we might expect that the genes associated with a disease will be more likely to be present within the same pathways or functional groupings. In published cases, pathway based GWAS analysis provides an alternative approach to the dissection of complex disease traits (4, 5). In addition, nominal GWAS p values superimposed upon the human molecular network have been used to identify genes associated with multiple sclerosis (6), and the disease association protein–protein link evaluator (DAPPLE) has been used to find significant interactions among proteins encoded by genes in loci associated with other particular diseases (7). Other approaches incorporate heterogeneous molecular data such as linkage studies, cross species conservation measures, gene expression data and protein–protein interactions to better understand GWAS results (8, 9). Integrating molecular network information, pathway analyses, and GWAS data thus holds promise for identifying new susceptibility loci and improving the identification of relevant candidate genes.If a gene is involved in a specific functional process or disease, its molecular network neighbors might also be suspected to have some role (3). In line with this “local” hypothesis, proteins involved in the same disease show a high propensity to interact (10) or cluster together (11) with each other. Interactions between variations in multiple genes, each with strong or modest effects, perturbing the same pathways or modules, may govern complex traits (3, 6). The molecular triangulation (MT) algorithm can be applied to rank seed genes according to their common disease associated neighbors, assigning closer and more connected neighbors higher values (12). Interactions between modestly associated MT genes may be indicative of coherent disease pathways or of genes conferring susceptibility to disease in a coordinated manner. The jActiveModule method (13) combines seed gene scores with biologically relevant interactions to identify network modules where perturbations causative of disease are more likely to reside. Lastly, although not yet implemented at the module level, phenotypic coherence between interacting pairs of genes has been quantified using the combination of molecular level gene to disease relationships and Medicare comorbidity data (14, 15).We believe that GWAS significant SNPs and variants representing potential candidate genes can use the above strategies to reveal more about the missing heritability of complex phenotypes. The most important risk factors for coronary artery disease (CAD) include serum concentrations of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and triglycerides (TG). We present a GWAS-based meta-analysis Comorbid Module (GCM) approach that uses significant (p < 5 × 10⁻⁸) GWAS signals for these four traits in the context of molecular networks to prioritize modules of disease-associated candidate genes. We evaluate our approach experimentally through allelic association and genotyping within the Malmö Diet and Cancer Cardiovascular Cohort (MDC-CC) for SNPs representing top candidate genes.     

Temporal dynamics in the evolution of the sunflower genome as revealed by sequencing and annotation of three large genomic regions

Improved knowledge of genome composition, especially of its repetitive component, generates important informations in both theoretical and applied research. In this study, we provide the first insight into the local organization of the sunflower genome by sequencing and annotating 349,380 bp from 3 BAC clones, each including one single-copy gene. These analyses resulted in the identification of 11 putative gene sequences, 18 full-length LTR retrotransposons, 6 incomplete LTR retrotransposons, 2 non-autonomous LTR-retroelements (LINEs), 2 putative DNA transposons fragments and one putative helitron. Among LTR-retrotransposons, non-autonomous elements (the so-called LARDs), which do not carry any protein-encoding sequence, were discovered for the first time in the sunflower. The insertion time of intact retroelements was measured, based on sister LTRs divergence. All isolated elements were inserted relatively recently, especially those belonging to the Gypsy superfamily. Retrotransposon families related to those identified in the BAC clones are present also in other species of Helianthus, both annual and perennial, and even in other Asteraceae. In one of the three BAC clones, we found five copies of a lipid transfer protein (LTP) encoding gene within less than 100,000 bp, four of which are potentially functional. Two of these are interrupted by LTR retrotransposons, in the intron and in the coding sequence, respectively. The divergence between sister LTRs of the retrotransposons inserted within the genes indicates that LTP gene duplication started earlier than 1.749 MYRS ago. On the whole, the results reported in this study confirm that the sunflower is an excellent system to study transposons dynamics and evolution.     

Sequence and structural characterization of tbnat gene in isoniazid-resistant Mycobacterium tuberculosis: identification of new mutations

One of the key issues of current radiation research is the biological effect of low doses. Unfortunately, low dose science is hampered by the unavailability of easily performable, reliable and sensitive quantitative biomarkers suitable detecting low frequency alterations in irradiated cells. We applied a quantitative real time polymerase chain reaction (qRT-PCR) based protocol detecting common deletions (CD) in the mitochondrial genome to assess direct and non-targeted effects of radiation in human fibroblasts. In directly irradiated (IR) cells CD increased with dose and was higher in radiosensitive cells. Investigating conditioned medium-mediated bystander effects we demonstrated that low and high (0.1 and 2Gy) doses induced similar levels of bystander responses and found individual differences in human fibroblasts. The bystander response was not related to the radiosensitivity of the cells. The importance of signal sending donor and signal receiving target cells was investigated by placing conditioned medium from a bystander response positive cell line (F11-hTERT) to bystander negative cells (S1-hTERT) and vice versa. The data indicated that signal sending cells are more important in the medium-mediated bystander effect than recipients. Finally, we followed long term effects in immortalized radiation sensitive (S1-hTERT) and normal (F11-hTERT) fibroblasts up to 63 days after IR. In F11-hTERT cells CD level was increased until 35 days after IR then reduced back to control level by day 49. In S1-hTERT cells the increased CD level was also normalized by day 42, however a second wave of increased CD incidence appeared by day 49 which was maintained up to day 63 after IR. This second CD wave might be the indication of radiation-induced instability in the mitochondrial genome of S1-hTERT cells. The data demonstrated that measuring CD in mtDNA by qRT-PCR is a reliable and sensitive biomarker to estimate radiation-induced direct and non-targeted effects.