Comparative repeatome analysis on Triatoma infestans Andean and Non-Andean lineages, main vector of Chagas disease

Triatoma infestans is the most important Chagas disease vector in South America. Two main evolutionary lineages, named Andean and non-Andean, have been recognized by geographical distribution, phenetic and genetic characteristics. One of the main differences is the genomic size, varying over 30% in their haploid DNA content. Here we realize a genome wide analysis to compare the repetitive genome fraction (repeatome) between both lineages in order to identify the main repetitive DNA changes occurred during T. infestans differentiation process. RepeatExplorer analysis using Illumina reads showed that both lineages exhibit the same amount of non-repeat sequences, and that satellite DNA is by far the major component of repetitive DNA and the main responsible for the genome size differentiation between both lineages. We characterize 42 satellite DNA families, which are virtually all present in both lineages but with different amount in each lineage. Furthermore, chromosomal location of satellite DNA by fluorescence in situ hybridization showed that genomic variations in T. infestans are mainly due to satellite DNA families located on the heterochromatic regions. The results also show that many satDNA families are located on the euchromatic regions of the chromosomes

Holocentric chromosome evolution in kissing bugs (Hemiptera: Reduviidae: Triatominae): diversification of repeated sequences

Background: The analysis of the chromosomal and genome evolution in organisms with holocentric chromosomes is restricted by the lack of primary constriction or centromere. An interesting group is the hemipteran subfamily Triatominae, vectors of Chagas disease, which affects around 6 to 7 million people worldwide. This group exhibits extensive variability in the number and chromosomal location of repeated sequences such as heterochromatin and ribosomal genes. This paper tries to reveal the significant differences of the repeated sequences among Triatoma species through the use of genomic DNA probes. Methods: We analysed the chromosomal distribution and evolution of repeated sequences in Triatoma species by genomic in situ hybridization (GISH) using genomic DNA probes from two North American Triatoma species. These genomic probes were hybridized both on their own chromosomes and on other Triatoma species from North and South America, with different amounts and chromosome location of C-heterochromatin. The results were compared with those previously described using South American Triatoma genomic probes. Results: We observed two chromosomal hybridization patterns: (i) very intense hybridization signals concentrated on specific chromosomal regions or particular chromosomes; and (ii) lower intensity hybridization signals dispersed along all chromosomes. Self-GISH on T. rubrofasciata and T. dimidiata chromosomes presented strong hybridization signals on all C-heterochromatin regions. However, when we perform genomic cross-hybridizations, only strong signals are detected on the Y chromosome, leaving the C-heterochromatic autosomal regions unmarked. Conclusions: We confirm that repeated DNA of the Y chromosome is shared among Triatoma species and probably represents an ancestral character of the Triatomini tribe. On the contrary, autosomal heterochromatic regions are constituted by species-specific DNA repeats, most probably satDNA families, suggesting that Triatoma speciation involved the amplification of diverse types of autosomal repeats. Molecular characterization of principal repetitive DNAs seems to be an appropriate approach to infer evolutionary relationships in triatomines

Calcium-calmodulin-dependent protein kinase mediates the intracellular signalling pathways of cardiac apoptosis in mice with impaired glucose tolerance

The impact of cardiac apoptosis in pre-diabetic stages of diabetic cardiomyopathy is unknown. We show that myocytes from fructose-rich diet (FRD) animals exhibit arrhythmias produced by exacerbated Ca2+/calmodulin-protein kinase (CaMKII) activity, ryanodine receptor 2 (RyR2) phosphorylation and sarcoplasmic reticulum (SR) Ca2+ leak. We tested the hypothesis that this mechanism also underlies cardiac apoptosis in pre-diabetes.We generated a pre-diabetic model in FRD mice. FRD mice showed an increase in oxidative stress, hypertrophy and systolic dysfunction. FRD myocytes exhibited enhanced SR Ca2+ spontaneous events in the absence of SR Ca2+ load alterations vs. control-diet (CD) myocytes. In HEK293 cells, hyperglycaemia significantly enhanced [3H]ryanodine binding and CaMKII phosphorylation of RyR2-S2814 residue vs. normoglycaemia. CaMKII inhibition prevented hyperglycaemia-induced alterations. FRD also evoked cardiac apoptosis inWT mice vs. CD-WT mice. Co-treatment with the reactive oxygen species scavenger Tempol prevented FRD-induced apoptosis inWT mice. In contrast, FRD enhanced oxidative stress but not apoptosis in FRD-SR-AIP mice, in which a CaMKII inhibitor is targeted to the SR. FRD produced mitochondrial membrane depolarization inWT mice but not in S2814A mice, in which the CaMKII phosphorylation site on RyR2 was ablated. Furthermore, FRD decreased mitochondrial area, mean Feret diameter and mean SR–mitochondrial distance vs. CD-WT hearts. This remodelling was prevented in AC3I mice, with cardiac-targeted CaMKII inhibition. CaMKII phosphorylation of RyR2, SR Ca2+ leak and mitochondrial membrane depolarization are critically involved in the apoptotic pathway of the pre-diabetic heart. The FRD-induced decrease in SR–mitochondrial distance is likely to additionally favour Ca2+ transit between the two organelles.Facultad de Ciencias MédicasFacultad de Ciencias VeterinariasCentro de Investigaciones Cardiovasculare

Calcium-calmodulin-dependent protein kinase mediates the intracellular signalling pathways of cardiac apoptosis in mice with impaired glucose tolerance

The impact of cardiac apoptosis in pre-diabetic stages of diabetic cardiomyopathy is unknown. We show that myocytes from fructose-rich diet (FRD) animals exhibit arrhythmias produced by exacerbated Ca2+/calmodulin-protein kinase (CaMKII) activity, ryanodine receptor 2 (RyR2) phosphorylation and sarcoplasmic reticulum (SR) Ca2+ leak. We tested the hypothesis that this mechanism also underlies cardiac apoptosis in pre-diabetes.We generated a pre-diabetic model in FRD mice. FRD mice showed an increase in oxidative stress, hypertrophy and systolic dysfunction. FRD myocytes exhibited enhanced SR Ca2+ spontaneous events in the absence of SR Ca2+ load alterations vs. control-diet (CD) myocytes. In HEK293 cells, hyperglycaemia significantly enhanced [3H]ryanodine binding and CaMKII phosphorylation of RyR2-S2814 residue vs. normoglycaemia. CaMKII inhibition prevented hyperglycaemia-induced alterations. FRD also evoked cardiac apoptosis inWT mice vs. CD-WT mice. Co-treatment with the reactive oxygen species scavenger Tempol prevented FRD-induced apoptosis inWT mice. In contrast, FRD enhanced oxidative stress but not apoptosis in FRD-SR-AIP mice, in which a CaMKII inhibitor is targeted to the SR. FRD produced mitochondrial membrane depolarization inWT mice but not in S2814A mice, in which the CaMKII phosphorylation site on RyR2 was ablated. Furthermore, FRD decreased mitochondrial area, mean Feret diameter and mean SR–mitochondrial distance vs. CD-WT hearts. This remodelling was prevented in AC3I mice, with cardiac-targeted CaMKII inhibition. CaMKII phosphorylation of RyR2, SR Ca2+ leak and mitochondrial membrane depolarization are critically involved in the apoptotic pathway of the pre-diabetic heart. The FRD-induced decrease in SR–mitochondrial distance is likely to additionally favour Ca2+ transit between the two organelles.Facultad de Ciencias MédicasFacultad de Ciencias VeterinariasCentro de Investigaciones Cardiovasculare

Inflammatory stimuli alter bone marrow composition and compromise bone health in the malnourished host

Inflammation has a role in the pathogenesis of childhood malnutrition. We investigated the effect of malnutrition and inflammatory challenge on bone marrow composition and bone health. We studied an established murine model of moderate acute malnutrition at baseline and after acute inflammatory challenge with bacterial lipopolysaccharide (LPS), a surrogate of Gram-negative bacterial sepsis, or Leishmania donovani, the cause of visceral leishmaniasis. Both of these infections cause significant morbidity and mortality in malnourished children. Of the 2 stimuli, LPS caused more pronounced bone marrow changes that were amplified in malnourished mice. LPS challenge led to increased inflammatory cytokine expression (Il1b, Il6, and Tnf), inflammasome activation, and inflammatory monocyte accumulation in the bone marrow of malnourished mice. Depletion of inflammatory monocytes in Csfr1-LysMcre-DT malnourished mice significantly reduced the inflammasome activation and IL1-ß production after LPS challenge. The inflammatory challenge also led to increased expansion of mesenchymal stem cells (MSCs), bone marrow adiposity, and expression of genes (Pparg, Adipoq, and Srbp1) associated with adipogenesis in malnourished mice. This suggests that inflammatory challenge promotes differentiation of BM MSCs toward the adipocyte lineage rather than toward bone-forming osteoblasts in the malnourished host. Concurrent with this reduced osteoblastic potential there was an increase in bone-resorbing osteoclasts, enhanced osteoclast activity, upregulation of inflammatory genes, and IL-1B involved in osteoclast differentiation and activation. The resulting weakened bone formation and increased bone resorption would contribute to the bone fragility associated with malnutrition. Lastly, we evaluated the effect of replacing lipid rich in omega-6 fatty acids (corn oil) with lipid-rich in omega-3 fatty acids (fish oil) in the nutrient-deficient diet. LPS-challenged malnourished mice that received dietary fish oil showed decreased expression of inflammatory cytokines and Rankl and reduced osteoclast differentiation and activation in the bone marrow. This work demonstrates that the negative effect of inflammatory challenge on bone marrow is amplified in the malnourished host. Increasing dietary intake of omega-3 fatty acids may be a means to reduce inflammation and improve bone health in malnourished children

Calcium-calmodulin-dependent protein kinase mediates the intracellular signalling pathways of cardiac apoptosis in mice with impaired glucose tolerance

The impact of cardiac apoptosis in pre-diabetic stages of diabetic cardiomyopathy is unknown. We show that myocytes from fructose-rich diet (FRD) animals exhibit arrhythmias produced by exacerbated Ca2+/calmodulin-protein kinase (CaMKII) activity, ryanodine receptor 2 (RyR2) phosphorylation and sarcoplasmic reticulum (SR) Ca2+ leak. We tested the hypothesis that this mechanism also underlies cardiac apoptosis in pre-diabetes.We generated a pre-diabetic model in FRD mice. FRD mice showed an increase in oxidative stress, hypertrophy and systolic dysfunction. FRD myocytes exhibited enhanced SR Ca2+ spontaneous events in the absence of SR Ca2+ load alterations vs. control-diet (CD) myocytes. In HEK293 cells, hyperglycaemia significantly enhanced [3H]ryanodine binding and CaMKII phosphorylation of RyR2-S2814 residue vs. normoglycaemia. CaMKII inhibition prevented hyperglycaemia-induced alterations. FRD also evoked cardiac apoptosis inWT mice vs. CD-WT mice. Co-treatment with the reactive oxygen species scavenger Tempol prevented FRD-induced apoptosis inWT mice. In contrast, FRD enhanced oxidative stress but not apoptosis in FRD-SR-AIP mice, in which a CaMKII inhibitor is targeted to the SR. FRD produced mitochondrial membrane depolarization inWT mice but not in S2814A mice, in which the CaMKII phosphorylation site on RyR2 was ablated. Furthermore, FRD decreased mitochondrial area, mean Feret diameter and mean SR–mitochondrial distance vs. CD-WT hearts. This remodelling was prevented in AC3I mice, with cardiac-targeted CaMKII inhibition. CaMKII phosphorylation of RyR2, SR Ca2+ leak and mitochondrial membrane depolarization are critically involved in the apoptotic pathway of the pre-diabetic heart. The FRD-induced decrease in SR–mitochondrial distance is likely to additionally favour Ca2+ transit between the two organelles.Facultad de Ciencias MédicasFacultad de Ciencias VeterinariasCentro de Investigaciones Cardiovasculare

Tandemly repeated DNA families in the mouse genome

<p>Abstract</p> <p>Background</p> <p>Functional and morphological studies of tandem DNA repeats, that combine high portion of most genomes, are mostly limited due to the incomplete characterization of these genome elements. We report here a genome wide analysis of the large tandem repeats (TR) found in the mouse genome assemblies.</p> <p>Results</p> <p>Using a bioinformatics approach, we identified large TR with array size more than 3 kb in two mouse whole genome shotgun (WGS) assemblies. Large TR were classified based on sequence similarity, chromosome position, monomer length, array variability, and GC content; we identified four superfamilies, eight families, and 62 subfamilies - including 60 not previously described. 1) The superfamily of centromeric minor satellite is only found in the unassembled part of the reference genome. 2) The pericentromeric major satellite is the most abundant superfamily and reveals high order repeat structure. 3) Transposable elements related superfamily contains two families. 4) The superfamily of heterogeneous tandem repeats includes four families. One family is found only in the WGS, while two families represent tandem repeats with either single or multi locus location. Despite multi locus location, TRPC-21A-MM is placed into a separated family due to its abundance, strictly pericentromeric location, and resemblance to big human satellites.</p> <p>To confirm our data, we next performed <it>in situ </it>hybridization with three repeats from distinct families. TRPC-21A-MM probe hybridized to chromosomes 3 and 17, multi locus TR-22A-MM probe hybridized to ten chromosomes, and single locus TR-54B-MM probe hybridized with the long loops that emerge from chromosome ends. In addition to <it>in silico </it>predicted several extra-chromosomes were positive for TR by <it>in situ </it>analysis, potentially indicating inaccurate genome assembly of the heterochromatic genome regions.</p> <p>Conclusions</p> <p>Chromosome-specific TR had been predicted for mouse but no reliable cytogenetic probes were available before. We report new analysis that identified <it>in silico </it>and confirmed <it>in situ </it>3/17 chromosome-specific probe TRPC-21-MM. Thus, the new classification had proven to be useful tool for continuation of genome study, while annotated TR can be the valuable source of cytogenetic probes for chromosome recognition.</p

Fosmid library end sequencing reveals a rarely known genome structure of marine shrimp Penaeus monodon

<p>Abstract</p> <p>Background</p> <p>The black tiger shrimp (<it>Penaeus monodon</it>) is one of the most important aquaculture species in the world, representing the crustacean lineage which possesses the greatest species diversity among marine invertebrates. Yet, we barely know anything about their genomic structure. To understand the organization and evolution of the <it>P. monodon </it>genome, a fosmid library consisting of 288,000 colonies and was constructed, equivalent to 5.3-fold coverage of the 2.17 Gb genome. Approximately 11.1 Mb of fosmid end sequences (FESs) from 20,926 non-redundant reads representing 0.45% of the <it>P. monodon </it>genome were obtained for repetitive and protein-coding sequence analyses.</p> <p>Results</p> <p>We found that microsatellite sequences were highly abundant in the <it>P. monodon </it>genome, comprising 8.3% of the total length. The density and the average length of microsatellites were evidently higher in comparison to those of other taxa. AT-rich microsatellite motifs, especially poly (AT) and poly (AAT), were the most abundant. High abundance of microsatellite sequences were also found in the transcribed regions. Furthermore, <it>via </it>self-BlastN analysis we identified 103 novel repetitive element families which were categorized into four groups, <it>i.e</it>., 33 WSSV-like repeats, 14 retrotransposons, 5 gene-like repeats, and 51 unannotated repeats. Overall, various types of repeats comprise 51.18% of the <it>P. monodon </it>genome in length. Approximately 7.4% of the FESs contained protein-coding sequences, and the Inhibitor of Apoptosis Protein (IAP) gene and the Innexin 3 gene homologues appear to be present in high abundance in the <it>P. monodon </it>genome.</p> <p>Conclusions</p> <p>The redundancy of various repeat types in the <it>P. monodon </it>genome illustrates its highly repetitive nature. In particular, long and dense microsatellite sequences as well as abundant WSSV-like sequences highlight the uniqueness of genome organization of penaeid shrimp from those of other taxa. These results provide substantial improvement to our current knowledge not only for shrimp but also for marine crustaceans of large genome size.</p