Larisa Fedorova

Critical association of ncRNA with introns

It has been widely acknowledged that non-coding RNAs are master-regulators of genomic functions. However, the significance of the presence of ncRNA within introns has not received proper attention. ncRNA within introns are commonly produced through the post-splicing process and are specific signals of gene transcription events, impacting many other genes and modulating their expression. This study, along with the following discussion, details the association of thousands of ncRNAs—snoRNA, miRNA, siRNA, piRNA and long ncRNA—within human introns. We propose that such an association between human introns and ncRNAs has a pronounced synergistic effect with important implications for fine-tuning gene expression patterns across the entire genome.

Development of nonalcoholic steatohepatitis in insulin-resistant liver-specific S503A carcinoembryonic antigen-related cell adhesion molecule 1 mutant mice.

BACKGROUND & AIMS Liver-specific inactivation of carcinoembryonic antigen-related cell adhesion molecule 1 causes hyperinsulinemia and insulin resistance, which result from impaired insulin clearance, in liver-specific S503A carcinoembryonic antigen-related cell adhesion molecule 1 mutant mice (L-SACC1). These mice also develop steatosis. Because hepatic fat accumulation precedes hepatitis, lipid peroxidation, and apoptosis in the pathogenesis of nonalcoholic steatohepatitis (NASH), we investigated whether a high-fat diet, by causing inflammation, is sufficient to induce hepatitis and other features of NASH in L-SACC1 mice. METHODS L-SACC1 and wild-type mice were placed on a high-fat diet for 3 months, then several biochemical and histologic analyses were performed to investigate the NASH phenotype. RESULTS A high-fat diet caused hepatic macrosteatosis and hepatitis, characterized by increased hepatic tumor necrosis factor alpha levels and activation of the NF-kappaB pathway in L-SACC1 but not in wild-type mice. The high-fat diet also induced necrosis and apoptosis in the livers of the L-SACC1 mice. Insulin resistance in L-SACC1 fed a high-fat diet increased the hepatic procollagen protein level, suggesting a role in the development of fibrosis. CONCLUSIONS A high-fat diet induces key features of human NASH in insulin-resistant L-SACC1 mice, validating this model as a tool to study the molecular mechanisms of NASH.

Peroxisome Proliferator-Activated Receptor δ Agonist, HPP593, Prevents Renal Necrosis under Chronic Ischemia

The Goldblatt’s 2 kidney 1 clip (2K1C) rat animal model of renovascular hypertension is characterized by ischemic nephropathy of the clipped kidney. 2K1C rats were treated with a specific peroxisome proliferator-activated receptor δ (PPARδ) agonist, HPP593. Clipped kidneys from untreated rats developed tubular and glomerular necrosis and massive interstitial, periglomerular and perivascular fibrosis. HPP593 kidneys did not exhibit any histochemical features of necrosis; fibrotic lesions were present only in perivascular areas. Necrosis in the untreated clipped kidneys was associated with an increased oxidative stress, up regulation and mitochondrial translocation of the pro-death protein BNIP3 specifically in tubules. In the kidneys of HPP593-treated rats oxidative stress was attenuated and BNIP3 protein decreased notably in the mitochondrial fraction when compared to untreated animals. In untreated clipped kidneys, mitochondria were dysfunctional as revealed by perturbations in the levels of MCAD, COXIV, TFAM, and Parkin proteins and AMPK activation, while in HPP593-treated rats these proteins remained at the physiological levels. Nuclear amounts of oxidative stress-responsive proteins, NRF1 and NRF2 were below physiological levels in treated kidneys. Mitochondrial biogenesis and autophagy were inhibited similarly in both treated and untreated 2K1C kidneys as indicated by a decrease in PGC1-α and deficiency of the autophagy-essential proteins LC3-II and ATG5. However, HPP593 treatment resulted in increased accumulation of p62 protein, an autophagic substrate and an enhancer of NRF2 activity. Therefore, inhibition of BNIP3 activation by the preservation of mitochondrial function and control of oxidative stress by PPARδ is the most likely mechanism to account for the prevention of necrotic death in the kidney under conditions of persistent ischemia.

Down-regulation of the transcription factor snail in the placentas of patients with preeclampsia and in a rat model of preeclampsia

BackgroundPlacental malfunction in preeclampsia is believed to be a consequence of aberrant differentiation of trophoblast lineages and changes in utero-placental oxygenation. The transcription factor Snail, a master regulator molecule of epithelial-mesenchymal transition in embryonic development and in cancer, is shown to be involved in trophoblast differentiation as well. Moreover, Snail can be controlled by oxidative stress and hypoxia. Therefore, we examined the expression of Snail and its downstream target, e-cadherin, in human normal term, preterm and preeclamptic placentas, and in pregnant rats that developed preeclampsia-like symptoms in the response to a 20-fold increase in sodium intake.MethodsWestern blotting analysis was used for comparative expression of Snail and e- cadherin in total protein extracts. Placental cells expressing Snail and e-cadherin were identified by immunohistochemical double-labeling technique.ResultsThe levels of Snail protein were decreased in human preeclamptic placentas by 30% (p < 0.01) compared to normal term, and in the rat model by 40% (p < 0.001) compared to control placentas. In preterm placentas, the levels of Snail expression varied, yet there was a strong trend toward statistical significance between preterm and preeclamptic placentas. In humans, e-cadherin protein level was 30% higher in preeclamptic (p < 0.05) placentas and similarly, but not significantly (p = 0.1), high in the preterm placentas compared to normal term. In the rat model of preeclampsia, e-cadherin was increased by 60% (p < 0.01). Immunohistochemical examination of human placentas demonstrated Snail-positive staining in the nuclei of the villous trophoblasts and mesenchymal cells and in the invasive trophoblasts of the decidua. In the rat placenta, the majority of Snail positive cells were spongiotrophoblasts of the junctional zone, while in the labyrinth, Snail-positive sinusoidal giant trophoblasts cells were found in some focal areas located close to the junctional zone.ConclusionWe demonstrated that human preeclampsia and the salt-induced rat model of preeclampsia are associated with the reduced levels of Snail protein in placenta. Down-regulation of the transcription factor Snail in placental progenitor cell lineages, either by intrinsic defects and/or by extrinsic and maternal factors, may affect normal placenta development and function and thus contribute to the pathology of preeclampsia.

Mitochondrial impairment in the five-sixth nephrectomy model of chronic renal failure: proteomic approach

BackgroundKidney injuries provoke considerable adjustment of renal physiology, metabolism, and architecture to nephron loss. Despite remarkable regenerative capacity of the renal tissue, these adaptations often lead to tubular atrophy, interstial and glomerular scaring, and development of chronic kidney disease. The therapeutic strategies for prevention of the transition from acute kidney damage to a chronic condition are limited. The purpose of this study was to elucidate large-scale alterations of the renal cortex proteome in partially nephrecromized rats at an early stage of chronic kidney disease.MethodsSprague–Dawley 5/6 nephrectomized rats and sham-operated controls were sacrificed at day 28 post-surgery. To identify proteins with notable alteration of expression we applied a 2D-proteomics approach followed by mass-spectrometry. Altered expression of identified and related proteins was validated by Western blotting and immunohistochemistry.ResultsProteins with increased levels of expression after partial nephrectomy were albumin and vimentin. Proteins with decreased expression were metabolic or mitochondrial. Western blotting analysis showed that the renal cortex of nephrectomized rats expressed decreased amount (by ~50%) of proteins from the inner mitochondrial compartment - the beta-oxidation enzyme MCAD, the structural protein GRP-75, and the oxidative phosphorylation protein COXIV. Mitochondrial DNA copy number was decreased by 30% in the cortex of PNx rats. In contrast, the levels of an outer mitochondrial membrane protein, VDAC1, remained unchanged in remnant kidneys. Mitochondrial biogenesis was not altered after renal mass ablation as was indicated by unchanged levels of PPARγ and PGC1α proteins. Autophagy related protein Beclin 1 was up-regulated in remnant kidneys, however the level of LC3-II protein was unchanged. BNIP3 protein, which can initiate both mitochondrial autophagy and cell death, was up-regulated considerably in kidneys of nephrecomized rats.ConclusionsThe results of the study demonstrated that notable alterations in the renal cortex of 5/6 nephrectomized rats were associated with mitochondrial damage, however mitochondrial biogenesis and autophagy for replacement of damaged mitochondria were not stimulated. Accumulation of dysfunctional mitochondria after 5/6 nephrectomy may cause multiple adjustments in biosynthetic pathways, energy production, ROS signaling, and activation of pro-cell death regulatory pathways thus contributing to the development of chronic kidney disease.

Inference of distant genetic relations in humans using "1000 genomes".

Nucleotide sequence differences on the whole-genome scale have been computed for 1,092 people from 14 populations publicly available by the 1000 Genomes Project. Total number of differences in genetic variants between 96,464 human pairs has been calculated. The distributions of these differences for individuals within European, Asian, or African origin were characterized by narrow unimodal peaks with mean values of 3.8, 3.5, and 5.1 million, respectively, and standard deviations of 0.1–0.03 million. The total numbers of genomic differences between pairs of all known relatives were found to be significantly lower than their respective population means and in reverse proportion to the distance of their consanguinity. By counting the total number of genomic differences it is possible to infer familial relations for people that share down to 6% of common loci identical-by-descent. Detection of familial relations can be radically improved when only very rare genetic variants are taken into account. Counting of total number of shared very rare single nucleotide polymorphisms (SNPs) from whole-genome sequences allows establishing distant familial relations for persons with eighth and ninth degrees of relationship. Using this analysis we predicted 271 distant familial pairwise relations among 1,092 individuals that have not been declared by 1000 Genomes Project. Particularly, among 89 British and 97 Chinese individuals we found three British–Chinese pairs with distant genetic relationships. Individuals from these pairs share identical-by-descent DNA fragments that represent 0.001%, 0.004%, and 0.01% of their genomes. With affordable whole-genome sequencing techniques, very rare SNPs should become important genetic markers for familial relationships and population stratification.

Genome evolution by matrix algorithms: cellular automata approach to population genetics.

Mammalian genomes are replete with millions of polymorphic sites, among which those genetic variants that are colocated on the same chromosome and exist close to one another form blocks of closely linked mutations known as haplotypes. The linkage within haplotypes is constantly disrupted due to meiotic recombination events. Whole ensembles of such numerous haplotypes are subjected to evolutionary pressure, where mutations influence each other and should be considered as a whole entity—a gigantic matrix, unique for each individual genome. This idea was implemented into a computational approach, named Genome Evolution by Matrix Algorithms (GEMA) to model genomic changes taking into account all mutations in a population. GEMA has been tested for modeling of entire human chromosomes. The program can precisely mimic real biological processes that have influence on genome evolution such as: 1) Authentic arrangements of genes and functional genomic elements, 2) frequencies of various types of mutations in different nucleotide contexts, and 3) nonrandom distribution of meiotic recombination events along chromosomes. Computer modeling with GEMA has demonstrated that the number of meiotic recombination events per gamete is among the most crucial factors influencing population fitness. In humans, these recombinations create a gamete genome consisting on an average of 48 pieces of corresponding parental chromosomes. Such highly mosaic gamete structure allows preserving fitness of population under the intense influx of novel mutations (40 per individual) even when the number of mutations with deleterious effects is up to ten times more abundant than those with beneficial effects.

High-calorie diet exacerbates prostate neoplasia in mice with haploinsufficiency of Pten tumor suppressor gene

Objective Association between prostate cancer and obesity remains controversial. Allelic deletions of PTEN, a tumor suppressor gene, are common in prostate cancer in men. Monoallelic Pten deletion in mice causes low prostatic intraepithelial neoplasia (mPIN). This study tested the effect of a hypercaloric diet on prostate cancer in Pten+/− mice. Methods 1-month old mice were fed a high-calorie diet deriving 45% calories from fat for 3 and 6 months before prostate was analyzed histologically and biochemically for mPIN progression. Because Pten+/− mice are protected against diet-induced insulin resistance, we tested the role of insulin on cell growth in RWPE-1 normal human prostatic epithelial cells with siRNA knockdown of PTEN. Results In addition to activating PI3 kinase/Akt and Ras/MAPkinase pathways, high-calorie diet causes neoplastic progression, angiogenesis, inflammation and epithelial–mesenchymal transition. It also elevates the expression of fatty acid synthase (FAS), a lipogenic gene commonly elevated in progressive cancer. SiRNA-mediated downregulation of PTEN demonstrates increased cell growth and motility, and soft agar clonicity in addition to elevation in FAS in response to insulin in RWPE-1 normal human prostatic cells. Downregulating FAS in addition to PTEN, blunted the proliferative effect of insulin (and IL-6) in RWPE-1 cells. Conclusion High-calorie diet promotes prostate cancer progression in the genetically susceptible Pten haploinsufficient mouse while preserving insulin sensitivity. This appears to be partly due to increased inflammatory response to high-caloric intake in addition to increased ability of insulin to promote lipogenesis.

Bioinformatics analysis of plant orthologous introns: identification of an intronic tRNA-like sequence.

Orthologous introns have identical positions relative to the coding sequence in orthologous genes of different species. By analyzing the complete genomes of five plants we generated a database of 40,512 orthologous intron groups of dicotyledonous plants, 28,519 orthologous intron groups of angiosperms, and 15,726 of land plants (moss and angiosperms). Multiple sequence alignments of each orthologous intron group were obtained using the Mafft algorithm. The number of conserved regions in plant introns appeared to be hundreds of times fewer than that in mammals or vertebrates. Approximately three quarters of conserved intronic regions among angiosperms and dicots, in particular, correspond to alternatively-spliced exonic sequences. We registered only a handful of conserved intronic ncRNAs of flowering plants. However, the most evolutionarily conserved intronic region, which is ubiquitous for all plants examined in this study, including moss, possessed multiple structural features of tRNAs, which caused us to classify it as a putative tRNA-like ncRNA. Intronic sequences encoding tRNA-like structures are not unique to plants. Bioinformatics examination of the presence of tRNA inside introns revealed an unusually long-term association of four glycine tRNAs inside the Vac14 gene of fish, amniotes, and mammals.

Atlas of Cryptic Genetic Relatedness Among 1000 Human Genomes.

A novel computational method for detecting identical-by-descent (IBD) chromosomal segments between sequenced genomes is presented. It utilizes the distribution patterns of very rare genetic variants (vrGVs), which have minor allele frequencies <0.2%. Contrary to the existing probabilistic approaches our method is rather deterministic, because it considers a group of very rare events which cannot happen together only by chance. This method has been applied for exhaustive computational search of shared IBD segments among 1,092 sequenced individuals from 14 populations. It demonstrated that clusters of vrGVs are unique and powerful markers of genetic relatedness, that uncover IBD chromosomal segments between and within populations, irrespective of whether divergence was recent or occurred hundreds-to-thousands of years ago. We found that several IBD segments are shared by practically any possible pair of individuals belonging to the same population. Moreover, shared short IBD segments (median size 183 kb) were found in 10% of inter-continental human pairs, each comprising of a person from sub-Saharan Africa and a person from Southern Europe. The shortest shared IBD segments (median size 54 kb) were found in 0.42% of inter-continental pairs composed of individuals from Chinese/Japanese populations and Africans from Kenya and Nigeria. Knowledge of inheritance of IBD segments is important in clinical case–control and cohort studies, since unknown distant familial relationships could compromise interpretation of collected data. Clusters of vrGVs should be useful markers for familial relationship and common multifactorial disorders.