Search

Mesenchymal stem cells (MSC) attract an increasing amount of attention due to their unique therapeutic properties. Yet, MSC can undergo undesirable genetic and epigenetic changes during their propagation in vitro. In this study, we investigated whether polyploidy can compromise MSC oncological safety and therapeutic properties. For this purpose, we compared the impact of polyploidy on the transcriptome of cancer cells and MSC of various origins (bone marrow, placenta, and heart). First, we identified genes that are consistently ploidy-induced or ploidy-repressed through all comparisons. Then, we selected the master regulators using the protein interaction enrichment analysis (PIEA). The obtained ploidy-related gene signatures were verified using the data gained from polyploid and diploid populations of early cardiomyocytes (CARD) originating from iPSC. The multistep bioinformatic analysis applied to the cancer cells, MSC, and CARD indicated that polyploidy plays a pivotal role in driving the cell into hypertranscription. It was evident from the upregulation of gene modules implicated in housekeeping functions, stemness, unicellularity, DNA repair, and chromatin opening by means of histone acetylation operating via DNA damage associated with the NUA4/TIP60 complex. These features were complemented by the activation of the pathways implicated in centrosome maintenance and ciliogenesis and by the impairment of the pathways related to apoptosis, the circadian clock, and immunity. Overall, our findings suggest that, although polyploidy does not induce oncologic transformation of MSC, it might compromise their therapeutic properties because of global epigenetic changes and alterations in fundamental biological processes. The obtained results can contribute to the development and implementation of approaches enhancing the therapeutic properties of MSC by removing polyploid cells from the cell population.

Many cardiovascular diseases originate from growth retardation, inflammation, and malnutrition during early postnatal development. The nature of this phenomenon is not completely understood. Here we aimed to verify the hypothesis that systemic inflammation triggered by neonatal lactose intolerance (NLI) may exert long-term pathologic effects on cardiac developmental programs and cardiomyocyte transcriptome regulation. Using the rat model of NLI triggered by lactase overloading with lactose and the methods of cytophotometry, image analysis, and mRNA-seq, we evaluated cardiomyocyte ploidy, signs of DNA damage, and NLI-associated long-term transcriptomic changes of genes and gene modules that differed qualitatively (i.e., were switched on or switched off) in the experiment vs. the control. Our data indicated that NLI triggers the long-term animal growth retardation, cardiomyocyte hyperpolyploidy, and extensive transcriptomic rearrangements. Many of these rearrangements are known as manifestations of heart pathologies, including DNA and telomere instability, inflammation, fibrosis, and reactivation of fetal gene program. Moreover, bioinformatic analysis identified possible causes of these pathologic traits, including the impaired signaling via thyroid hormone, calcium, and glutathione. We also found transcriptomic manifestations of increased cardiomyocyte polyploidy, such as the induction of gene modules related to open chromatin, e.g., "negative regulation of chromosome organization", "transcription" and "ribosome biogenesis". These findings suggest that ploidy-related epigenetic alterations acquired in the neonatal period permanently rewire gene regulatory networks and alter cardiomyocyte transcriptome. Here we provided first evidence indicating that NLI can be an important trigger of developmental programming of adult cardiovascular disease. The obtained results can help to develop preventive strategies for reducing the NLI-associated adverse effects of inflammation on the developing cardiovascular system.

Using meta-analyses, we introduce a unicellular attractor (UCA) model integrating essential features of the 'atavistic reversal', 'cancer attractor', 'somatic mutation', 'genome chaos', and 'tissue organization field' theories. The 'atavistic reversal' theory is taken as a keystone. We propose a possible mechanism of this reversal, its refinement called 'gradual atavism', and evidence for the 'serial atavism' model. We showed the gradual core-to-periphery evolutionary growth of the human interactome resulting in the higher protein interaction density and global interactome centrality in the UC center. In addition, we revealed that UC genes are more actively expressed even in normal cells. The modeling of random walk along protein interaction trajectories demonstrated that random alterations in cellular networks, caused by genetic and epigenetic changes, can result in a further gradual activation of the UC center. These changes can be induced and accelerated by cellular stress that additionally activates UC genes (especially during cell proliferation), because the genes involved in cellular stress response and cell cycle are mostly of UC origin. The functional enrichment analysis showed that cancer cells demonstrate the hyperactivation of energetics and the suppression of multicellular genes involved in communication with the extracellular environment (especially immune surveillance). Collectively, these events can unleash selfish cell behavior aimed at survival at all means. All these changes are boosted by polyploidization. The UCA model may facilitate an understanding of oncogenesis and promote the development of therapeutic strategies.

The growth of complexity is an unsolved and underappreciated problem. We consider possible causes of this growth, hypotheses testing, molecular mechanisms, complexity measures, cases of simplification, and significance for biomedicine. We focus on a general ability of regulation, which is based on the growing information storage and processing capacities, as the main proxy of complexity. Natural selection is indifferent to complexity. However, complexification can be inferred from the same first principle, on which natural selection is founded. Natural selection depends on potentially unlimited reproduction under limited environmental conditions. Because of the demographic pressure, the simple ecological niches become fulfilled and diversified (due to species splitting and divergence). Diversification increases complexity of biocenoses. After the filling and diversification of simple niches, the more complex niches can arise. This is the 'atomic orbitals' (AO) model. Complexity has many shortcomings but it has an advantage. This advantage is ability to regulatory adaptation, including behavioral, formed in the evolution by means of genetic adaptation. Regulatory adaptation is much faster than genetic one because it is based on the information previously accumulated via genetic adaptation and learning. Regulatory adaptation further increases complexity of biocenoses. This is the 'regulatory advantage' (RA) model. The comparison of both models allows testable predictions. We focus on the animal evolution because of the appearance of higher regulatory level (nervous system), which is absent in other lineages, and relevance to humans (including biomedical aspects)., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The biogenetic law (recapitulation law) states that ontogenesis recapitulates phylogenesis. However, this law can be distorted by the modification of development. We showed the recapitulation of phylogenesis during the differentiation of various cell types, using a meta-analysis of human single-cell transcriptomes, with the control for cell cycle activity and the improved phylostratigraphy (gene dating). The multipotent progenitors, differentiated from pluripotent embryonic stem cells (ESC), showed the downregulation of unicellular (UC) genes and the upregulation of multicellular (MC) genes, but only in the case of those originating up to the Euteleostomi (bony vertebrates). This picture strikingly resembles the evolutionary profile of regulatory gene expansion due to gene duplication in the human genome. The recapitulation of phylogenesis in the induced pluripotent stem cells (iPSC) during their differentiation resembles the ESC pattern. The unipotent erythroblasts differentiating into erythrocytes showed the downregulation of UC genes and the upregulation of MC genes originating after the Euteleostomi. The MC interactome neighborhood of a protein encoded by a UC gene reverses the gene expression pattern. The functional analysis showed that the evolved environment of the UC proteins is typical for protein modifiers and signaling-related proteins. Besides a fundamental aspect, this approach can provide a unified framework for cancer biology and regenerative/rejuvenation medicine because oncogenesis can be defined as an atavistic reversal to a UC state, while regeneration and rejuvenation require an ontogenetic reversal.

Polyploid cells demonstrate biological plasticity and stress adaptation in evolution; development; and pathologies, including cardiovascular diseases, neurodegeneration, and cancer. The nature of ploidy-related advantages is still not completely understood. Here, we summarize the literature on molecular mechanisms underlying ploidy-related adaptive features. Polyploidy can regulate gene expression via chromatin opening, reawakening ancient evolutionary programs of embryonality. Chromatin opening switches on genes with bivalent chromatin domains that promote adaptation via rapid induction in response to signals of stress or morphogenesis. Therefore, stress-associated polyploidy can activate Myc proto-oncogenes, which further promote chromatin opening. Moreover, Myc proto-oncogenes can trigger polyploidization de novo and accelerate genome accumulation in already polyploid cells. As a result of these cooperative effects, polyploidy can increase the ability of cells to search for adaptive states of cellular programs through gene regulatory network rewiring. This ability is manifested in epigenetic plasticity associated with traits of stemness, unicellularity, flexible energy metabolism, and a complex system of DNA damage protection, combining primitive error-prone unicellular repair pathways, advanced error-free multicellular repair pathways, and DNA damage-buffering ability. These three features can be considered important components of the increased adaptability of polyploid cells. The evidence presented here contribute to the understanding of the nature of stress resistance associated with ploidy and may be useful in the development of new methods for the prevention and treatment of cardiovascular and oncological diseases.

DNA replication during cell proliferation is 'vertical' copying, which reproduces an initial amount of genetic information. Polyploidy, which results from whole-genome duplication, is a fundamental complement to vertical copying. Both organismal and cell polyploidy can emerge via premature cell cycle exit or via cell-cell fusion, the latter giving rise to polyploid hybrid organisms and epigenetic hybrids of somatic cells. Polyploidy-related increase in biological plasticity, adaptation, and stress resistance manifests in evolution, development, regeneration, aging, oncogenesis, and cardiovascular diseases. Despite the prevalence in nature and importance for medicine, agri- and aquaculture, biological processes and epigenetic mechanisms underlying these fundamental features largely remain unknown. The evolutionarily conserved features of polyploidy include activation of transcription, response to stress, DNA damage and hypoxia, and induction of programs of morphogenesis, unicellularity, and longevity, suggesting that these common features confer adaptive plasticity, viability, and stress resistance to polyploid cells and organisms. By increasing cell viability, polyploidization can provide survival under stressful conditions where diploid cells cannot survive. However, in somatic cells it occurs at the expense of specific function, thus promoting developmental programming of adult cardiovascular diseases and increasing the risk of cancer. Notably, genes arising via evolutionary polyploidization are heavily involved in cancer and other diseases. Ploidy-related changes of gene expression presumably originate from chromatin modifications and the derepression of bivalent genes. The provided evidence elucidates the role of polyploidy in evolution, development, aging, and carcinogenesis, and may contribute to the development of new strategies for promoting regeneration and preventing cardiovascular diseases and cancer.

Whole-genome duplication (WGD), or polyploidy, increases the amount of genetic information in the cell. WGDs of whole organisms are found in all branches of eukaryotes and act as a driving force of speciation, complication, and adaptations. Somatic-cell WGDs are observed in all types of tissues and can result from normal or altered ontogenetic programs, regeneration, pathological conditions, aging, malignancy, and metastasis. Despite the versatility of WGDs, their functional significance, general properties, and causes of their higher adaptive potential are unclear. Comparisons of whole-transcriptome data and information from various fields of molecular biology, genomics, and molecular medicine showed several common features for polyploidy of organisms and somatic and cancer cells, making it possible to understand what WGD properties lead to the emergence of an adaptive phenotype. The adaptation potential of WGDs may be associated with an increase in the complexity of the regulation of networks and signaling systems; a higher resistance to stress; and activation of ancient evolutionary programs of unicellularity and pathways of morphogenesis, survival, and life extension. A balance between the cell and organismal levels in controlling gene regulation may shift in stress towards the priority of cell survival, and the shift can lead to cardiovascular diseases and carcinogenesis. The presented information helps to understand how polyploidy creates new phenotypes and why it acts as a driving force of evolution and an important regulator of biological processes in somatic cells during ontogeny, pathogenesis, regeneration, and transformation.

The growth of complexity in evolution is a most intriguing phenomenon. Using gene phylostratigraphy, we showed this growth (as reflected in regulatory mechanisms) in the human genome, tracing the path from prokaryotes to hominids. Generally, the different regulatory gene families expanded at different times, yet only up to the Euteleostomi (bony vertebrates). The only exception was the expansion of transcription factors (TF) in placentals; however, we argue that this was not related to increase in general complexity. Surprisingly, although TF originated in the Prokaryota while chromatin appeared only in the Eukaryota, the expansion of epigenetic factors predated the expansion of TF. Signaling receptors, tumor suppressors, oncogenes, and aging- and disease-associated genes (indicating vulnerabilities in terms of complex organization and strongly enrichment in regulatory genes) also expanded only up to the Euteleostomi. The complexity-related gene properties (protein size, number of alternative splicing mRNA, length of untranslated mRNA, number of biological processes per gene, number of disordered regions in a protein, and density of TF-TF interactions) rose in multicellular organisms and declined after the Euteleostomi, and possibly earlier. At the same time, the speed of protein sequence evolution sharply increased in the genes that originated after the Euteleostomi. Thus, several lines of evidence indicate that molecular mechanisms of complexity growth were changing with time, and in the phyletic lineage leading to humans, the most salient shift occurred after the basic vertebrate body plan was fixed with bony skeleton. The obtained results can be useful for evolutionary medicine.

Background: Cancer stem cells' (CSCs) self-maintenance is regulated via the pluripotency pathways promoting the most aggressive tumor phenotype. This study aimed to use the activity of these pathways for the CSCs' subpopulation enrichment and separating cells characterized by the OCT4 and SOX2 expression., Methods: To select and analyze CSCs, we used the SORE6x lentiviral reporter plasmid for viral transduction of colon adenocarcinoma cells. Additionally, we assessed cell chemoresistance, clonogenic, invasive and migratory activity and the data of mRNA-seq and intrinsic disorder predisposition protein analysis (IDPPA)., Results: We obtained the line of CSC-like cells selected on the basis of the expression of the OCT4 and SOX2 stem cell factors. The enriched CSC-like subpopulation had increased chemoresistance as well as clonogenic and migration activities. The bioinformatic analysis of mRNA seq data identified the up-regulation of pluripotency, development, drug resistance and phototransduction pathways, and the downregulation of pathways related to proliferation, cell cycle, aging, and differentiation. IDPPA indicated that CSC-like cells are predisposed to increased intrinsic protein disorder., Conclusion: The use of the SORE6x reporter construct for CSCs enrichment allows us to obtain CSC-like population that can be used as a model to search for the new prognostic factors and potential therapeutic targets for colon cancer treatment.

Changes in gene expression play an important role in evolution and can be relevant to evolutionary medicine. In this work, a strong relationship was found between the statistical significance of evolutionary changes in the expression of orthologous genes in the five or six homologous mammalian tissues and the across-tissues unidirectionality of changes (i.e., they occur in the same direction in different tissues -- all upward or all downward). In the area of highly significant changes, the fraction of unidirectionally changed genes (UCG) was above 0.9 (random expectation is 0.03). This observation indicates that the most pronounced evolutionary changes in mammalian gene expression are systemic (i.e., they operate at the whole-organism level). The UCG are strongly enriched in the housekeeping genes. More specifically, in the human-chimpanzee comparison, the UCG are enriched in the pathways belonging to gene expression (translation is prominent), cell cycle control, ubiquitin-dependent protein degradation (mostly related to cell cycle control), apoptosis, and Parkinson's disease. In the human-macaque comparison, the two other neurodegenerative diseases (Alzheimer's and Huntington's) are added to the enriched pathways. The consolidation of gene expression changes at the level of pathways indicates that they are not neutral but functional. The systemic expression changes probably maintain the across-tissues balance of basic physiological processes in the course of evolution (e.g., during the movement along the fast-slow life axis). These results can be useful for understanding the variation in longevity and susceptibility to cancer and widespread neurodegenerative diseases. This approach can also guide the choice of prospective genes for studies aiming to decipher cis-regulatory code (the gene list is provided)., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Tumours were recently revealed to undergo a phylostratic and phenotypic shift to unicellularity. As well, aggressive tumours are characterized by an increased proportion of polyploid cells. In order to investigate a possible shared causation of these two features, we performed a comparative phylostratigraphic analysis of ploidy-related genes, obtained from transcriptomic data for polyploid and diploid human and mouse tissues using pairwise cross-species transcriptome comparison and principal component analysis. Our results indicate that polyploidy shifts the evolutionary age balance of the expressed genes from the late metazoan phylostrata towards the upregulation of unicellular and early metazoan phylostrata. The up-regulation of unicellular metabolic and drug-resistance pathways and the downregulation of pathways related to circadian clock were identified. This evolutionary shift was associated with the enrichment of ploidy with bivalent genes ( p < 10 -16 ). The protein interactome of activated bivalent genes revealed the increase of the connectivity of unicellulars and (early) multicellulars, while circadian regulators were depressed. The mutual polyploidy- c-MYC -bivalent genes-associated protein network was organized by gene-hubs engaged in both embryonic development and metastatic cancer including driver (proto)-oncogenes of viral origin. Our data suggest that, in cancer, the atavistic shift goes hand-in-hand with polyploidy and is driven by epigenetic mechanisms impinging on development-related bivalent genes.

The transcriptome consists of various gene modules that can be mutually dependent, and ignoring these dependencies may lead to misinterpretation. The most important problem is module dependence on cell-cycle activity. Using meta-analysis of over 30 000 single-cell transcriptomes, we show gene module dependencies on cell-cycle signature, which can be consistently observed in various normal and cancer cells. Transcript levels of receptors, plasma membrane, and differentiation-related genes are negatively regressed on cell-cycle signature. Pluripotency, stress response, DNA repair, chromatin remodeling, proteasomal protein degradation, protein network connectivity, and unicellular evolutionary origin are regressed positively. These effects cannot be explained by partial overlap of corresponding gene sets because they remain if the overlapped genes were removed. We propose a visual analysis of gene module-specific regression lines as complement to an uncurated enrichment analysis. The different lines for a same gene module indicate different cell conditions. The approach is tested on several problems (polyploidy, pluripotency, cancer, phylostratigraphy). Intriguingly, we found variation in cell-cycle activity, which is independent of cell progression through the cycle. The upregulation of G2/M checkpoint genes with downregulation of G2/M transition and cytokinesis is revealed in polyploid cells. A temporal increase in cell-cycle activity at transition from pluripotent to more differentiated state is found in human embryonic stem cells. The upregulation of unicellular interactome cluster in human cancers is shown in single cells with control for cell-cycle activity. The greater scatter around regression line in cancer cells suggests greater heterogeneity caused by deviation from a line of normal cells., (© 2020 Federation of European Biochemical Societies.)

The main contradiction of multicellularity (MCM) is between the unicellular (UC) and multicellular (MC) levels. In human interactome we revealed two giant clusters with MC and UC medians (and several smaller ones with MC medians). The enrichment of these clusters by phylostrata and by functions support the MC versus UC division. The total interactome and the giant clusters show a core-periphery evolutionary growth. From viewpoint of the MCM, the most important is the placement of genes, appearing at UC evolutionary stage, in the MC clusters. Thus, genes involved in vesicle-mediated transport, cell cycle, cellular responses to stress, post-translational modifications and many diseases appeared at UC evolutionary stage but are placed mostly in MC clusters. Genes downregulated with age are enriched in UC cluster, whereas the upregulated genes are preferentially placed in MC giant cluster. The tumor suppressor and pluripotency regulating pathways are also enriched in MC giant cluster. Therefore, this cluster probably operates as 'internal manager' constraining runaway unicellularity. The clusters have denser interactions within than between them, therefore they can serve as attractors (stable states of dynamic systems) of cellular programs. Importantly, the UC cluster have a higher inside/outside connection ratio compared with MC clusters, which suggests a stronger attractor effect and may explain why cells of MC organisms are prone to oncogenesis. The evolutionary clustering of human interactome elucidates the MC control over functions appearing at UC evolutionary stage and can build a framework for biosystems studies focusing on the interplay between UC and MC levels., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The prevalence of purifying selection in the nature suggests that larger organisms bear a higher number of slightly deleterious mutations because of smaller populations and therefore weaker selection. In this work redistribution of purifying selection in favor of information genes, pathways and processes was found in primates compared with treeshrew and rodents on the ground of genome-wide analysis. The genes which are more favored in primates belong mainly to regulation of gene expression and development, in treeshrew and rodents, to metabolism, transport, energetics, reproduction and olfaction. The former occur predominantly in the nucleus, the latter, in the cytoplasm and membranes. Thus, although purifying selection is on average weaker in the primates, it is stronger concentrated on the "information technology" of life (regulation of gene expression and development). Increased accuracy of information processes probably allows escaping "error catastrophes" in spite of more complex organization, larger body size and higher longevity., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Quiescence is the prevailing state of many cell types under homeostatic conditions. Yet, surprisingly, little is known about how quiescent cells respond to environmental challenges. The aim of the present study is to compare stress responses of cycling and quiescent mesenchymal stem cells (MSC). Human endometrial mesenchymal cells (eMSС) were employed as adult stem cells. eMSC quiescence was modeled by serum starvation. Sublethal heat shock (HS) was used as a stress factor. Both quiescent and cycling cells were heated at 45°C for 30 min and then returned to standard culture conditions for their recovery. HS response was monitored by DNA damage response, stress-induced premature senescence (SIPS), cell proliferation activity, and oxidative metabolism. It has been found that quiescent cells repair DNA more rapidly, resume proliferation, and undergo SIPS less than proliferating cells. HS-enforced ROS production in heated cycling cells was accompanied with increased expression of genes regulating redox-active proteins. Quiescent cells exposed to HS did not intensify the ROS production, and genes involved in antioxidant defense were mostly silent. Altogether, the results have shown that quiescent cells are more resistant to heat stress than cycling cells. Next-generation sequencing (NGS) demonstrates that HS-survived cells retain differentiation capacity and do not exhibit signs of spontaneous transformation.

Temperature is an important exogenous factor capable of leading to irreversible processes in the vital activity of cells. However, the long-term effects of heat shock (HS) on mesenchymal stromal cells (MSC) remain unstudied. We investigated the karyotype and DNA repair drivers and pathways in the human endometrium MSC (eMSC) survived progeny at passage 6 after sublethal heat stress (sublethal heat stress survived progeny (SHS-SP)). G-banding revealed an outbreak of random karyotype instability caused by chromosome breakages and aneuploidy. Molecular karyotyping confirmed the random nature of this instability. Transcriptome analysis found homologous recombination (HR) deficiency that most likely originated from the low thermostability of the AT-rich HR driving genes. SHS-SP protection from transformation is provided presumably by low oncogene expression maintained by tight co-regulation between thermosensitive HR drivers BRCA, ATM, ATR, and RAD51 (decreasing expression after SHS), and oncogenes mTOR, MDM2, KRAS, and EGFR. The cancer-related transcriptomic features previously identified in hTERT transformed MSC in culture were not found in SHS-SP, suggesting no traits of malignancy in them. The entrance of SHS-SP into replicative senescence after 25 passages confirms their mortality and absence of transformation features. Overall, our data indicate that SHS may trigger non-tumorigenic karyotypic instability due to HR deficiency and decrease of oncogene expression in progeny of SHS-survived MSC. These data can be helpful for the development of new therapeutic approaches in personalized medicine.

Cryptosporidiosis causes persistent diarrhoea in infants, immunocompromised patients and elderly persons. Long-term consequences of the disease include increased risk of malignancy, cardiomyopathy and gastrointestinal inflammation. This study aimed to investigate prolonged effects of cryptosporidiosis on innate immunity and growth in neonatal C3HA mice. The disease was challenged by Cryptosporidium parvum oocyst inoculation into 7-day-old animals. The mice whose intestine smears contained 3-5 or 6 and more oocysts per microscopic field at the day 5 after infection were considered as mildly or severely infected, correspondingly. To determine natural killer cell (NK) activity, we applied 3 H-uridine cytotoxic assay to the animals at 5-68 days after infection using K562 cells as targets. At severe infection, there was a statistically significant 1.5-2.0 fold decline of body mass, spleen mass and spleen cellularity that persisted in animals of all ages. Accordingly, NK cytotoxicity showed even more drastic drop reaching 2.7-3.0 folds that was statistically significant in all animals. At mild infection, the discovered effects were less pronounced and reached significance only in some age groups. Thus, our study provides evidence that NK cells show long-term cytotoxic activity decrease following Cryptosporidium infection in neonatal mice, particularly in severe disease., (© 2018 John Wiley & Sons Ltd.)

The AT-rich DNA is mostly associated with condensed chromatin, whereas the GC-rich sequence is preferably located in the dispersed chromatin. The AT-rich genes are prone to be tissue-specific (silenced in most tissues), while the GC-rich genes tend to be housekeeping (expressed in many tissues). This paper reports another important property of DNA base composition, which can affect repertoire of genes with high AT content. The GC-rich sequence is more liable to mutation. We found that Spearman correlation between human gene GC content and mutation probability is above 0.9. The change of base composition even in synonymous sites affects mutation probability of nonsynonymous sites and thus of encoded proteins. There is a unique type of housekeeping genes, which are especially unsafe when prone to mutation. Natural selection which usually removes deleterious mutations, in the case of these genes only increases the hazard because it can descend to suborganismal (cellular) level. These are cell cycle-related genes. In accordance with the proposed concept, they have low GC content of synonymous sites (despite them being housekeeping). The gene-centred protein interaction enrichment analysis (PIEA) showed the core clusters of genes whose interactants are modularly enriched in genes with AT-rich synonymous codons. This interconnected network is involved in double-strand break repair, DNA integrity checkpoints and chromosome pairing at mitosis. The damage of these genes results in genome and chromosome instability leading to cancer and other 'error catastrophes'. Reducing the nonsynonymous mutations, the usage of AT-rich synonymous codons can decrease probability of cancer by above 20-fold.

High temperature is a critical environmental and personal factor. Although heat shock is a well-studied biological phenomenon, hyperthermia response of stem cells is poorly understood. Previously, we demonstrated that sublethal heat shock induced premature senescence in human endometrial mesenchymal stem cells (eMSC). This study aimed to investigate the fate of eMSC-survived sublethal heat shock (SHS) with special emphasis on their genetic stability and possible malignant transformation using methods of classic and molecular karyotyping, next-generation sequencing, and transcriptome functional analysis. G-banding revealed random chromosome breakages and aneuploidy in the SHS-treated eMSC. Molecular karyotyping found no genomic imbalance in these cells. Gene module and protein interaction network analysis of mRNA sequencing data showed that compared to untreated cells, SHS-survived progeny revealed some difference in gene expression. However, no hallmarks of cancer were found. Our data identified downregulation of oncogenic signaling, upregulation of tumor-suppressing and prosenescence signaling, induction of mismatch, and excision DNA repair. The common feature of heated eMSC is the silence of MYC, AKT1/PKB oncogenes, and hTERT telomerase. Overall, our data indicate that despite genetic instability, SHS-survived eMSC do not undergo transformation. After long-term cultivation, these cells like their unheated counterparts enter replicative senescence and die.

The dependence of cancer on overexpressed c-MYC and its predisposition for polyploidy represents a double puzzle. We address this conundrum by cross-species transcription analysis of c-MYC interacting genes in polyploid vs. diploid tissues and cells, including human vs. mouse heart, mouse vs. human liver and purified 4n vs. 2n mouse decidua cells. Gene-by-gene transcriptome comparison and principal component analysis indicated that c-MYC interactants are significantly overrepresented among ploidy-associated genes. Protein interaction networks and gene module analysis revealed that the most upregulated genes relate to growth, stress response, proliferation, stemness and unicellularity, as well as to the pathways of cancer supported by MAPK and RAS coordinated pathways. A surprising feature was the up-regulation of epithelial-mesenchymal transition (EMT) modules embodied by the N-cadherin pathway and EMT regulators from SNAIL and TWIST families. Metabolic pathway analysis also revealed the EMT-linked features, such as global proteome remodeling, oxidative stress, DNA repair and Warburg-like energy metabolism. Genes associated with apoptosis, immunity, energy demand and tumour suppression were mostly down-regulated. Noteworthy, despite the association between polyploidy and ample features of cancer, polyploidy does not trigger it. Possibly it occurs because normal polyploidy does not go that far in embryonalisation and linked genome destabilisation. In general, the analysis of polyploid transcriptome explained the evolutionary relation of c-MYC and polyploidy to cancer.

Rodents include both the cancer-susceptible short-lived mouse and the two unrelated cancer-resistant long-lived mole-rats. In this work, their genomes were analyzed with the goal to reveal pathways enriched in genes, which are more similar between the mole-rats than between the mouse and the naked mole-rat. The pathways related to cell cycle control were prominent. They include external signal transduction and all cell cycle stages. There are several stem cell pathways among them. The other enriched pathways involve ubiquitin-dependent protein degradation, immunity, mRNA splicing, and apoptosis. The ubiquitin-dependent protein degradation is a core of network of enriched pathways. However, this phenomenon is not specific for the mouse and the mole-rats. The other muroid species show features similar to the mouse, whereas the non-muroid rodents and the human show features similar to the mole-rats. The higher ratio of non-synonymous to synonymous nucleotide substitutions (dN/dS) indicates the accelerated evolution of revealed pathways in the muroid rodents (except the blind mole-rat). Paradoxically, the dN/dS averaged over the whole genome is lower in the muroids, i.e., the purifying selection is generally stronger in them. In practical sense, these data suggest caveat for using muroid rodents (mouse, rat, and hamsters) as biomedical models of human conditions involving cell cycle and show the network of pathways where muroid genes are most different (compared with non-muroid) from human genes. The guinea pig is emphasized as a more suitable rodent model for biomedical research involving cell cycle.

Conservatism versus innovation is probably the most important dichotomy of all evolving systems. In molecular evolution the distinction between conservative (negative) selection, innovative (positive) selection and unconstrained evolution (drift) is usually ambiguous at the gene level. Only rare cases with the ratio of nonsynonymous to synonymous nucleotide substitutions above unity (dN/dS>1) are thought to be due to positive selection, whereas the lower dN/dS ratio may indicate negative selection in combination with drift. The density of the dN/dS ratio for orthologous genes forms a unimodal distribution where no particular regions can be discerned. Here it is shown that at the level of overrepresented pathways and processes the picture is strikingly different. The distribution is strongly polarized with a wide completely depressed middle part. This three-phase distribution is very robust. It is observed with various substitution models and remains at very low significance of overrepresentation (up to p<0.99). This fact suggests consolidation of either negative or positive selection but not of unconstrained evolution at the level of pathways/processes. The effect is demonstrated for different phylogenetic distances: from human to other primates, mammals and vertebrates. This approach suggests estimating the boundaries for conservative and innovative selection using the pathway/process level. Emphasizing the role of a critical mass of negatively or positively selected genes in a pathway/process, it can elucidate how the bridge between 'tinkering' at the gene level and 'design' at the higher levels is forming., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Design and development of highly sensitive method bioinformatics are important for investigation of casual relationships between epigenetic changes and gene activity. Cell polyploidy may trigger such changes. However, maintaining the balance of gene dosage, polyploidy may provide only a rather weak effect on their expression. Currently, there is no comprehensive and concordant data in regard to ploidy-associated transcriptomic changes. To find out how polypoidy affects gene activity, we have developed an integrative bioinformatic method of pairwise cross-species transcriptome analysis of mammalian tissues with various polyploidy degrees. The main benefit of this approach is its ability to separate species- and tissue-specific noises of evolutionary conserved effects. We demonstrat the application of the method for the analysis of gene modules and protein interactions networks coordinating programs of development, differentiation and pluripotency. The analysis was performed with transcriptomes of polyploid and diploid organs (human and mouse heart and liver). Our data indicate that ploidy-induced genes enrich Gene Ontology (GO) biological processes and KEGG pathways related to development, morphogenesis and stem cells biology (including Hippo, Pi3K, WNT, Hedgehog and TGF-β pathways) with higher degree than ploidy-inhibited genes. Thas, our data are the first to show that polyploidy may induce and coordinate developmental modules.

Paralog gene trees, which reflect the increase of genomic complexity in the evolution, can be complicated and ambiguous. A simpler complementary approach is analysis of density distribution of paralog pairs. It can reveal general features of genome evolution, which may be hidden in the forest of gene trees. It is known that distribution of human paralog pairs along the axis of protein divergence between pair members forms two main peaks. Here I show that there are three main peaks in the mouse genome. Thus, the multimodality of paralog pair distribution seems to be a fundamental feature of mammalian genomes. Despite the great diversity of domains presented in small amounts or in multidomain architectures with a few predominant domains, both in human and mouse the first peak consists mostly of gene pairs with zinc finger domains or olfactory receptor domain. In the mouse the olfactory receptor predominates, which stipulates the three-peak distribution (since in the olfactory receptors the second peak is closer to the first peak than in other genes). The mammalian-wide zinc finger orthologs are biased towards the second peak. Thus, the marsupial orthologs are nearly absent in the first peak of human and mouse. The gene pairs in the first peak show a lower ratio of nonsynonymous to synonymous substitutions, which suggests that their evolution is more constrained. The plausible explanation is that they are in subfunctionalization state (partition of initial function of ancestral gene), whereas the second peak contains gene pairs that are already in neofunctionalization state (acquiring of novel functions). These data suggest that the adaptive radiation of mammals was accompanied by a burst of duplication of zinc finger genes, which are located in the first (most recent) peak of paralog pairs., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Disturbances at the childhood age increase risk of the appearance of cardiovascular diseases decades later. The nature of this interconnection called ontogenetic programming is not completely understood. Valuable sources of knowledge about mechanisms of ontogenetic programming are data of interspecies study of biology of the body life cycles and of heart physiological capabilities. Taken into account the interspecies differences, these data allow finding the correct direction of experimental investigations. Results of studies of almost 100 homoiothermal species have shown the slow growth and a high loading on the heart at postnatal development to decrease its aerobic capability in adults. Basing on these data, we suggested that the neonatal gastroenteritis causing tachyarrhythmia, malabsorption, and the growth deceleration might lead to pathological changes in the heart. Our task was to evaluate the effect of cryptosporidial gastroenteritis of different degrees of severity on heart of the neonatal rats. By using methods of Real-Time PCR, immunocytochemistry, image analysis, and study of interatrial septum, we have established that a gradual increase of intensity of infestation with Cryptosporidium parvum oocytes causes sharp changes corresponding to the "all or nothing" response. At a weak infestation the interatrial septum was close (like in control), while significant changes in expression of isoforms of heavy chains of alpha- and beta-myosin were absent. At the intermediate and severe infestation, in the interatrial septum the foramen ovale was visualized and there were observed cardiac atrophy and a strong shift of ration of expression of myosin heavy chains toward the low-velocity beta chain. Thus, by disturbing the frequency-strength ratios and causing outflow of resources from the formed heart, the neonatal gastroenteritis produces pathological changes of the organ molecular and anatomical structures. Our results can be interest to evolutionary biologists and physicians, as they show importance of knowledge of evolutionary-comparative investigations for the search for novel risk factors of heart diseases and demonstrate interconnection between gastroenteritis, pathology of interatrial septum, and a change of composition of the main contractile proteins in cardiomyocytes.

Infectious gastroenteritis is one of the common causes of tachyarrythmia, malabsorbtion and growth retardation in children. Our recent studies have indicated that neonatal.cryptosporidial gastroenteritis is associated with long-term cardiomyocyte abnormalities. The aim of the present study was to find out how neonatal cryptosporidiosis of various severities affects cardiac anatomy and cardiomyocyte polyploidization, remodeling and HIF-1α expression. Using real-time PCR, cytometry, immunohistochemistry, image analysis and interatrial septum visual examination, we revealed that gradual increase in cryptosporidial invasion was associated with threshold changes. At weak parasitic infection, interatrial septum was entire and there was no statistically significant change in cardiomyocytes. At moderate and severe infection, all changes in cardiac anatomy and cardiomyocytes were statistically significant and demonstrated approximately similar degree. Compared to control, heart were atrophied and elongated, interatrial septum contained a small window (patentforamrn ovale), and cardiomyocytes lost protein, became elongated, thin and accumulated additional genomes. Also we found HIF-1α mRNA hyperexpression. Notable, the threshold response to gradual stimulus is an important criterion of development programming since such a response is commonly a consequence of abnormal anatomic structure formation and cell differentiation failure. Our results can be interesting for physicians because they indicate that even moderate cryptosporidiosis can be dangerous for neonatal heart and can trigger neonatal programming of cardiovascular pathology. Also, our results for the first time demonstrate the association between gastroenteritis, patent foramen ovale and cardiomyocyte malfunction.

Orthologs are genes from different genomes that originate from a common ancestor gene by speciation event. They are most similar by the structure of encoded proteins and therefore should have a similar function. Here I apply the principle used for detection of structural orthology for a genome-wide analysis of gene expression. For this purpose, I determine the mutual similarity rank in all-by-all comparison of among-tissues expression patterns. The expression of most part of human-mouse orthologs in homologous tissues is poorly correlated (average mutual coexpression rank is only 4835 out of 18,092). Genes from evolutionarily labile gene families, which experience rapid turnover of family composition, are among those with the strongest expression change. However, the revealed phenomenon is not limited to them. There is no or very weak relationship between protein sequence divergence and mutual coexpression rank. Also, generally there is no relationship between the ratio of nonsynonymous to synonymous nucleotide substitutions and coexpression rank. This relationship is tangible only within evolutionarily labile gene families. These results indicate that despite of a similar biochemical function of orthologs reflected in the conserved protein sequence, the physiological (systemic) context of this function can be changed. Also, these results suggest that gene biochemical function and its physiological role in the organism can evolve independently., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Proteome complexity increases in the evolution mostly by means of gene duplication followed by divergence. In this genome-scale study of human genome I show that density distribution of duplicate gene pairs along the axis of protein divergence between pair members forms two main peaks with a small peak and plateau before the first main peak. This picture indicates the existence of three evolutionary stages of duplicate gene evolution. The analysis of various functional parameters (gene expression level and breadth, transcription factor targets, protein interaction networks) suggests that subfunctionalization (partition of function) is a predominant mode of divergence in the first main peak, whereas neofunctionalization (acquiring of novel functions) prevails in the second main peak. The young duplicate pairs show a much higher expression level compared with singleton genes and more diverged duplicates, which indicates that requirement for high gene dosage is important for retention of duplicates just after the duplication event. Thus, a prevailing route of duplicate evolution seems to be the high gene dosage-subfunctionalization-neofunctionalization. This adaptationist model suggests that an organism is evolving in the direction of its most intensively used functions.

Retrospective epidemyological studies evidence that infant diseases leave survivors with an increased susceptibility to cardiovascular diseases in later life. At the same time, the mechanisms of this link remain poorly understood. Based on medical statistics reporting that infectious gastroenteritis is the most common cause of maladies in babies, infants and children, we analysed the effects of moderate cryptosporidial gastroenteritis on the heart and ventricular cardiomyocyte remodelling in rats of the first month of life. The disease was challenged by a worldwide human protozoic pathogen Cryptosporidium parvum (Apicomplexa, Sporozoa). The main symptoms manifested in the growth retardation moderate diarrhea. Using real-time PCR, cytophotometry, confocal microscopy and image analysis, we indicated that cryptosporidiosis was associated, with the atrophy heart and the elongation, narrowing, protein content decrease and hyperpolyploidization of cardiomyocytes and the moderate overexpression of hypoxia inducible factor 1alpha (HIF-1alpha) mRNA. Cardiomyocyte shape remodeling and heart atrophy presented in all age groups. The severity of these changes, hovewer, declined gradually from younger to older groups. In contrast, hyperpolyploidization and HIF-1alpha mRNA overexpression were registered mainly among animals aged between 6 and 13 days, and were barely detected and non-significant in older age groups. In the rat the time period covering 6-13 days after birth is known to coincide with the intensive cardiomyocyte polyploidization and the switch from proliferation to hypertrophy. Thus, our data indicate that neonatal cryptosporidiosis may be potential cardiovascular diseases risk factor and that one of the critical time windows for the growing heart covers the time period when cardiomyocyte undergo polyploidization.

The family of transcription factors with the C2H2 zinc finger domain is expanding in the evolution of vertebrates, reaching its highest numbers in the mammals. The question arises: whether an increased amount of these transcription factors is related to embryogenesis, nervous system, pathology or more of them are expressed in individual cells? Among mammals, the primates have a more complex anatomical structure than the rodents (e.g., brain). In this work, I show that a greater number of C2H2-ZF genes are expressed in the human cells than in the mouse cells. The effect is especially pronounced for C2H2-ZF genes accompanied with the KRAB domain. The relative difference between the numbers of C2H2-ZF(-KRAB) genes in the human and mouse cellular transcriptomes even exceeds their difference in the genomes (i.e. a greater subset of existing in the genome genes is expressed in the human cellular transcriptomes compared to the mouse transcriptomes). The evolutionary turnover of C2H2-ZF(-KRAB) genes acts in the direction of the revealed phenomenon, i.e. gene duplication and loss enhances the difference in the relative number of C2H2-ZF(-KRAB) genes between human and mouse cellular transcriptomes. A higher amount of these genes is expressed in the brain and embryonic cells (compared with other tissues), whereas a lower amount--in the cancer cells. It is specifically the C2H2-ZF transcription factors whose repertoire is poorer in the cancer and richer in the brain (other transcription factors taken together do not show this trend). These facts suggest that increase of anatomical complexity is accompanied by a more complex intracellular regulation involving these transcription factors. Malignization is associated with simplification of this regulation. These results agree with the known fact that human cells are more resistant to oncogenic transformation than mouse cells. The list of C2H2-ZF genes whose suppression might be involved in malignization is provided.

Cardiovascular diseases are the most common case of human death in developed countries. Thus, the discovering of their new risk factors is of primary importance. Based on epidemiology studies, vertebrate life-history traits comparison and cross-species cardiomyocyte transcriptome analysis, we suggest that one of these factors could be infectious gastroenteritis. This disease outflows recourses from cardio-vascular system and triggers pathological stimuli, like tachyarrhythmia, inflammation, malapsorption and energy depletion thereby disturbing cardiomyocyte metabolism and function. To test this hypothesis, we challenged gastroenteritis in neonatal rats with widespread human parasite Cryptosporidium parvum (Apicomplexa, Sporozoa). The results obtained by the methods of immunocytochemistry, quantitative morphometry and real-time PCR, indicate that moderate cryptosporidiosis lasting four days induces dramatic shift in myosin isoform expression ration toward isoform beta (with low ATPase activity) both at mRNA (by 1.7-4.5 folds) and protein (by 2.5-6 folds) levels. Antithetical manner of this shift and coherence between changes in mRNA and protein suggest that cryptosporidiosis affects all main steps of a complex myosin heavy chain regulatory network. Since the overexpression of myosin heavy chain beta (showing several times lower ATPase activity than myosin heavy chain alfa) is a generally accepted marker of human cardiac failure, we can consider cryptosporidial gastroenteritis as a new risk factor of cardiac contractile ability impairment. Our data can be interesting for clinicians.

Polyploid cells show great among-species and among-tissues diversity and relation to developmental mode, suggesting their importance in adaptive evolution and developmental programming. At the same time, excessive polyploidization is a hallmark of functional impairment, aging, growth disorders, and numerous pathologies including cancer and cardiac diseases. To shed light on this paradox and to find out how polyploidy contributes to organ functions, we review here the ploidy-associated shifts in activity of narrowly expressed (tissue specific) genes in human and mouse heart and liver, which have the reciprocal pattern of polyploidization. For this purpose, we use the modular biology approach and genome-scale cross-species comparison. It is evident from this review that heart and liver show similar traits in response to polyploidization. In both organs, polyploidy protects vitality (mainly due to the activation of sirtuin-mediated pathways), triggers the reserve adenosine-5'-triphosphate (ATP) production, and sustains tissue-specific functions by switching them to energy saving mode. In heart, the strongest effects consisted in the concerted up-regulation of contractile proteins and substitution of energy intensive proteins with energy economic ones. As a striking example, the energy intensive alpha myosin heavy chain (providing fast contraction) decreased its expression by a factor of 10, allowing a 270-fold increase of expression of beta myosin heavy chain (providing slow contraction), which has approximately threefold lower ATP-hydrolyzing activity. The liver showed the enhancement of immunity, reactive oxygen species and xenobiotic detoxication, and numerous metabolic adaptations to long-term energy depletion. Thus, somatic polyploidy may be an ingenious evolutionary instrument for fast adaptation to stress and new environments allowing trade-offs between high functional demand, stress, and energy depletion.

Using a new approach, I analysed human transcriptome coexpression network and revealed two large-scale nexuses. Besides gene coexpression, each nexus is characterized by a combination of gene evolutionary origin, function and among-tissues expression breadth. The first nexus contains mostly genes of pre-metazoan origin, which are widely expressed and have cell-centred functions. The second nexus is enriched in genes of metazoan origin, which are expressed more narrowly and have organism-centred functions. The revealed nexuses are supported by asymmetry in distribution of transcription factor targets between them. Within the metazoan nexus, there is a subnexus that is more pronounced in the nervous tissues and is enriched in gene regulatory complexity. It mostly contains genes related to nervous system, cell communication and multicellular organism processes and development. The revealed nexuses indicate a dichotomy in the transcriptional regulation and can provide a framework for further functional genomics studies., (Copyright 2010 Elsevier Inc. All rights reserved.)

Background: The aim of our study was to find out, whether cardiomyocyte genome duplication participates in developmental programming of adult hypertension and impaired heart aerobic capacity, and if it does, whether ploidy-related programming is reversible and what are the timeframes of the most critical window. For this propose we studied the effect of the well-known factors of programming, including growth retardation, infection, and cardiac overload on the level of neonatal cardiomyocyte ploidy, protein content and shape., Methods: Using the model of rat cryptosporidial gastroenteritis, we shifted the time point of infection day by day through the neonatal period and traced the immediate and postponed effects of disease on isolated cardiomyocyte ploidy, phenotype, and protein content., Results: We found that gastroenteritis caused cardiac atrophy and a burst-like premature genome accumulation, elongation, narrowing and protein loss in the cardiomyocytes. These changes resulted in sharp increase of DNA content at the expense of contractile proteins. We also revealed clear indications of critical window of heart development during the peak of cardiomyocyte transition from proliferation to hypertrophy. After the rehabilitation, the atrophy of heart and cardiomyocyte remodelling showed a conspicuous restoration, whereas the hyperpolyploidization did not regress. An irreversible manner of excessive genome duplication and its well-known ability to alter gene expression confirm our suggestion that it is implicated in the ontogenetic programming of heart development., Conclusion: We provided the first evidence that developmental programming can operate through cardiomyocyte genome duplication and that the critical window coincides with cell transition from proliferation to hypertrophy. Our data help determine the timing of critical window for human heart and would allow successful prevention of human cardiac abnormalities even before they become tangible., (Copyright (c) 2008 Elsevier Ireland Ltd. All rights reserved.)

Proteins encoded by highly expressed genes evolve more slowly. This correlation is thought to arise owing to purifying selection against toxicity of misfolded proteins (that should be more crucial for highly expressed genes). It is now widely accepted that this individual (by-gene) effect is a dominant cause in protein evolution. Here, I show that in mammals, the evolutionary rate of a protein is much more strongly related to the evolutionary rate of coexpressed proteins (and proteins of the same biological pathway) than to the expression level of its encoding gene. The complexity of gene regulation (estimated by the numbers of transcription factor targets and regulatory microRNA targets in the encoding gene) is another important cause, which is much stronger than gene expression level. Proteins encoded by complexly regulated genes evolve more slowly. The intronic length and the ratio of intronic to coding sequence lengths also correlate negatively with protein evolutionary rate (which contradicts the expectation from the negative link between expression level and evolutionary rate). One more important factor, which is much stronger than gene expression level, is evolutionary age. More recent proteins evolve faster, and expression level of an encoding gene becomes quite a minor cause in the evolution of mammal proteins of metazoan origin. These data suggest that, in contrast to a widespread opinion, systemic factors dominate mammal protein evolution.

Growth retardation, inflammation and cardiac overload in early childhood are linked with hypertension and infarction in adults. This link was termed as developmental programming. Exact mechanisms and critical time frames for development of the heart are still unknown. To elucidate these questions, we developed a model of moderate cryptosporidial gastroenteritis triggering main programming factors. Sliding the time point of infection day by day (from day 4 to day 18), we tested complete rat neonatal period. Also, we repeated all experiments 30 days after infection. Using methods of cytometry, immunocytochemistry and confocal microscopy, we compared sensitivity of ventricular cardiomyocyte shape, protein content and ploidy. Our data indicated that gastroenteritis lasting four days triggered cardiomyocyte atrophy, almost doubling cell length to width ratio, and premature and excessive polyploidization. Surprisingly, nucleus and cytoplasm reacted to the disease differently. Cardiomyocytes accumulated genomes only when the disease covered the time period between 6 and 14 days after birth, when cells substitute proliferative growth with hypertrophy. Contractile proteins and cell shape on the contrary, showed high sensitivity in the course of complete neonatal period. After restoration, ploidy did not regress, whereas cell shape and protein content revealed moderate restoration. Taking into account that somatic polyploidy is irreversible and that it alters global gene expression pattern, we may suggest that genome duplication is one of the instruments of developmental programming and that gastroenteritis is one if the triggers of this programming.

Excessive somatic polyploidy usually accompanies physiologic and pathologic overload and it is generally accepted as a symptom of pathology. At the same time, polyploidy cells exist in most fungal, plant, mollusk, fish, bird and mammalian tissues confirming their great evolutionary success. The secret of this success remains enigmatic. Since transcriptome rearrangements usually start with metabolic flux redistribution, we decided to investigate firstly the effects of polyploidy on cell metabolism. Using multitest approach of modular biology and databases Entrez Gene, RefSeq, GNF SymAtlas, Gene Ontology, KEGG, BioCarta; MsigDb, Reactome, GenMAPP, and HumanCyc, we performed detailed comparison of metabolic genes expression in human and mouse organs with reciprocal pattern of polyploidy (i. e. in the heart and in the liver). Pairwise criss-cross comparison of diploid vs. polyploid organs allowed removing species- and tissue-specific effects. From our results, polyploidy is associated with rearrangements of main metabolic pathways. We found deep depression of mitochondrial processes, features of autophagia, and increased carbohydrate degradation and lipid biosynthesis. Taken together, these changes pointed to the energy and oxygen deprivation. We also found clear indications of enhanced oxidative stress protection. The major of them are triggering of pentose-phosphate pathway, depression of mitochondria-cytoplasm electron shuttles, and impartment of electron flows across 1 (NADH dehydrogenase) and IV (cytochrome c-oxydase) breath complexes. We suggest that all these changes are necessary for the increase in metabolic plasticity and for the protection of replicating DNA from oxidative damage.

Whole-genome duplications are important for the growth of genome complexity. We investigated various factors involved in the evolution of yeast whole-genome duplicates (ohnologs) making emphasis on the analysis of protein interactions. We found that ohnologs have a lower number of protein interactions compared with small-scale duplicates and singletons (by about -40%). The loss of interactions was proportional to their initial number and independent of ohnolog position in the protein interaction network. A faster evolving member of an ohnolog pair has a lower number of interactions compared to its counterpart. The Gene Ontology mapping of non-overlapping and overlapping interactants of paired ohnologs reveals a sharp asymmetry in GO terms related to regulation. The fraction of these terms is much higher in non-overlapping interactants (compared to overlapping interactants and total dataset). Network clustering coefficient is lower in ohnologs, yet they show an increased density of protein interactions restricted within the whole ohnologs set. These facts suggest that subfunctionalization (or subneofunctionalization) reflected in the loss of protein interactions was a prevailing process in the divergence of ohnologs, which distinguishes them from small-scale duplicates. The loss of protein interactions was associated with the regulatory divergence between the members of an ohnolog pair. A small-scale modularity (reflected in clustering coefficient) probably was not important for ohnologs retention, yet a larger-scale modularity could be involved in their evolution.

It is shown here that in the yeast protein interaction network the global centrality measure (betweenness) depends on the protein evolutionary age (i.e., on historic contingency) more weakly than the local centrality measure (degree). This phenomenon is not observed in mutational duplication-and-divergence models. The network domains responsible for this difference deal with DNA/RNA information processing, regulation, and cell cycle. A selection vector can operate in these domains, which integrates the network activity and thus compensates for the process of mutational divergence.

The modular biology is supposed to be a bridge from the molecular to the systems biology. Using a new approach, it is shown here that the protein interaction networks of yeast Saccharomyces cerevisiae and bacteria Escherichia coli consist of two large-scale modularity layers, central and peripheral, separated by a zone of depressed modularity. This finding based on the analysis of network topology is further supported by the discovery that there are many more Gene Ontology categories (terms) and KEGG biochemical pathways that are overrepresented in the central and peripheral layers than in the intermediate zone. The categories of the central layer are mostly related to nuclear information processing, regulation and cell cycle, whereas the peripheral layer is dealing with various metabolic and energetic processes, transport and cell communication. A similar center-periphery polarization of modularity is found in the protein domain networks ('built-in interactome') and in a powergrid (as a non-biological example). These data suggest a 'polarized modularity' model of cellular networks where the central layer seems to be regulatory and to use information storage of the nucleus, whereas the peripheral layer seems devoted to more specialized tasks and environmental interactions, with a complex 'bus' between the layers.

Inflammation, malnutrition and growth retardation during critical time-windows of development play a powerful role in ontogenetic programming of the life-long risk to many adult diseases (including metabolic syndrome, obesity and diabetes). Cellular mechanisms and the accurate timing and duration of critical periods for the liver remain obscure. To resolve this problem, we developed a postnatal suckling-weanling rat model of mild, moderate, and acute gastroenteritis challenged by a protozoan parasitic spread throughout the whole world, namely Cryptosporidium parvum. The physiological state of the liver was evaluated by hepatocyte ploidy and protein content that were measured by cytophotometry and image analysis on isolated cells. Hepatocyte ploidy is known to irreversibly increase after stress and is associated with the decrease in liver physiological capacity. Hepatocyte hypertrophy reflects cell functional loading. From our results, cryptosporidiosis is able to provoke a burst in premature hepatocyte polyploidization and hypertrophy (in proportion to parasitic load), and thus plays an important role in epigenetic programming of hepatocyte structure and function. We revealed two sensitive periods in liver growth. The first period (the less sensitive) covers the time before the establishment of homoiothermy, i.e. 6-9 days after birth. The second period (the more sensitive) covers the time of weaning when the change of type of nutrition and the peak of hepatocyte polyploidization and differentiation occurs. Thus, our data provide direct evidence that phenomenon of ontogenetic programming is reflected at the cellular level.

We present a molecular and cellular phenomenon underlying the intriguing increase in phenotypic organizational complexity. For the same set of human-mouse orthologous genes (11 534 gene pairs) and homologous tissues (32 tissue pairs), human shows a greater fraction of tissue-specific genes and a greater ratio of the total expression of tissue-specific genes to housekeeping genes in each studied tissue, which suggests a generally higher level of evolutionary cell differentiation (specialization). This phenomenon is spectacularly more pronounced in those human tissues that are more directly involved in the increase of complexity, longevity and body size (i.e. it is reflected on the organismal level as well). Genes with a change in expression breadth show a greater human-mouse divergence of promoter regions and encoded proteins (i.e. the functional genomics data are supported by the structural analysis). Human also shows the higher expression of translation machinery. The upstream untranslated regions (5'UTRs) of human mRNAs are longer than mouse 5'UTRs (even after correction for the difference in genome sizes) and contain more uAUG codons, which suggest a more complex regulation at the translational level in human cells (and agrees well with the augmented cell specialization).

To elucidate the functional significance of genome multiplication in somatic tissues, we performed a large-scale analysis of ploidy-associated changes in expression of non-tissue-specific (i.e., broadly expressed) genes in the heart and liver of human and mouse (6585 homologous genes were analyzed). These species have inverse patterns of polyploidization in cardiomyocytes and hepatocytes. The between-species comparison of two pairs of homologous tissues with crisscross contrast in ploidy levels allows the removal of the effects of species and tissue specificity on the profile of gene activity. The different tests performed from the standpoint of modular biology revealed a consistent picture of ploidy-associated alteration in a wide range of functional gene groups. The major effects consisted of hypoxia-inducible factor-triggered changes in main cellular processes and signaling pathways, activation of defense against DNA lesions, acceleration of protein turnover and transcription, and the impairment of apoptosis, the immune response, and cytoskeleton maintenance. We also found a severe decline in aerobic respiration and stimulation of sugar and fatty acid metabolism. These metabolic rearrangements create a special type of metabolism that can be considered intermediate between aerobic and anaerobic. The metabolic and physiological changes revealed (reflected in the alteration of gene expression) help explain the unique ability of polyploid tissues to combine proliferation and differentiation, which are separated in diploid tissues. We argue that genome multiplication promotes cell survival and tissue regeneration under stressful conditions.

Introns are shorter in housekeeping genes than in tissue- or development-specific genes. Differing explanations have been offered for this phenomenon: selection for economy (in housekeeping genes), mutation bias or "genomic design." The large-scale implementation in this present paper of a rigorous local sequence alignment algorithm revealed an unprecedented fraction of evolutionarily conserved DNA in human-mouse introns ( approximately 60% of human and approximately 70% of mouse intron length remained after masking for lineage-specific repeats). The length distributions of both conserved and nonconserved regions are very broad but show peaks close to nucleosomal and di-nucleosomal DNA. Both the fraction of conserved sequence and its absolute length were higher in introns of tissue-specific genes than housekeeping genes. This difference remained after control for between-species identity of the conserved fraction, mutation rate, and GC content. In a more direct control, the product of the conserved sequence fraction and the between-species identity of this fraction (which can be considered to be the fraction of conserved nucleotides) was greater in introns of tissue-specific genes than housekeeping genes. Neither the fraction of intron length covered by repeats nor the balance of small insertions and deletions (indels) can explain the greater length of introns in tissue-specific genes. The length of the conserved intronic DNA in a gene is correlated with the number of functional domains in the protein encoded by that gene. These results suggest that the greater length of introns in tissue-specific genes is not due to selection for economy or mutation bias but instead is related to functional complexity (probably mediated by chromatin condensation), and that the evolution of the bulk of noncoding DNA is not completely neutral.

We show the negative link between genome size and metabolic intensity in tetrapods, using the heart index (relative heart mass) as a unified indicator of metabolic intensity in poikilothermal and homeothermal animals. We found two separate regression lines of heart index on genome size for reptiles-birds and amphibians-mammals (the slope of regression is steeper in reptiles-birds). We also show a negative correlation between GC content and nucleosome formation potential in vertebrate DNA, and, consistent with this relationship, a positive correlation between genome GC content and nuclear size (independent of genome size). It is known that there are two separate regression lines of genome GC content on genome size for reptiles-birds and amphibians-mammals: reptiles-birds have the relatively higher GC content (for their genome sizes) compared to amphibians-mammals. Our results suggest uniting all these data into one concept. The slope of negative regression between GC content and nucleosome formation potential is steeper in exons than in non-coding DNA (where nucleosome formation potential is generally higher), which indicates a special role of non-coding DNA for orderly chromatin organization. The chromatin condensation and nuclear size are supposed to be key parameters that accommodate the effects of both genome size and GC content and connect them with metabolic intensity. Our data suggest that the reptilian-birds clade evolved special relationships among these parameters, whereas mammals preserved the amphibian-like relationships. Surprisingly, mammals, although acquiring a more complex general organization, seem to retain certain genome-related properties that are similar to amphibians. At the same time, the slope of regression between nucleosome formation potential and GC content is steeper in poikilothermal than in homeothermal genomes, which suggests that mammals and birds acquired certain common features of genomic organization.

Human tissue-specific genes were reported to be longer than housekeeping genes (both in coding and intronic parts). The competing neutralist and adaptationist models were proposed to explain this observation. Here I show that in human genome the longest are genes with the intermediate expression pattern. From the standpoint of information theory, the regulation of such genes should be most complex. In the genomewide context, they are found here to have the higher informational load on all available levels: from participation in protein interaction networks, pathways and modules reflected in Gene Ontology categories through transcription factor regulatory sets and protein functional domains to amino acid tuples (words) in encoded proteins and nucleotide tuples in introns and promoter regions. Thus, the intermediately expressed genes have the higher functional and regulatory complexity that is reflected in their greater length (which is consistent with the 'genome design' model). The dichotomy of housekeeping versus tissue-specific entities is more pronounced on the modular level than on the molecular level. There are much lesser intermediate-specific modules (modules overrepresented in the intermediately expressed genes) than housekeeping or tissue-specific modules (normalized to gene number). The dichotomy of housekeeping versus tissue-specific genes and modules in multicellular organisms is probably caused by the burden of regulatory complexity acted on the intermediately expressed genes.

In this article, I show that, in the human genome, the GC content in genes (but not the CpG island in the promoter) is related to the maximum level of gene expression among tissues, whereas the promoter CpG island and gene CpG level are more strongly related to the breadth of expression among tissues. The relevance of gene GC content to expression cannot be a consequence (i.e. a byproduct) of transcription because it does not correlate with expression in the germline. The variation of GC content and CpG level can determine the characteristics of gene expression in a synergistic interplay with transcription-factor-binding sites (mediated by chromatin condensation).

Cell membrane-dependent chromatin condensation was studied by flow cytometry in erythrocytes of 36 species from six classes of vertebrates. A positive relationship was found between the degree of condensation and genome size. The distribution of variances among taxonomic levels is similar for both parameters. However, chromatin condensation varied relatively more at the lower taxonomic levels, which suggests that the degree of DNA packaging might serve for fine-tuning the 'skeletal' and/or 'buffering' function of noncoding DNA (although the range of this fine-tuning is smaller than the range of genome size changes). For two closely related amphibian species differing in genome size, change in chromatin condensation under the action of elevated extracellular salinity was investigated. Condensation was steadier and its reaction to changes in solvent composition was more inertial in the species with a larger genome, which is in agreement with the buffering function postulated for redundant DNA. The uppermost genome size in vertebrates (and in living beings in general) was updated using flow cytometry and was found to be about 80 pg (78,400 Mb). The widespread opinion that the largest genome occurs in unicellular organisms is rejected as being based on artifacts.

The nucleosome formation potential of introns, intergenic spacers and exons of human genes is shown here to negatively correlate with among-tissues breadth of gene expression. The nucleosome formation potential is also found to negatively correlate with the GC content of genomic sequences; the slope of regression line is steeper in exons compared with noncoding DNA (introns and intergenic spacers). The correlation with GC content is independent of sequence length; in turn, the nucleosome formation potential of introns and intergenic spacers positively (albeit weakly) correlates with sequence length independently of GC content. These findings help explain the functional significance of the isochores (regions differing in GC content) in the human genome as a result of optimization of genomic structure for epigenetic complexity and support the notion that noncoding DNA is important for orderly chromatin condensation and chromatin-mediated suppression of tissue-specific genes.