Large-scale Analyses of Disease Biomarkers and Apremilast Pharmacodynamic Effects.

Finding biomarkers that provide shared link between disease severity, drug-induced pharmacodynamic effects and response status in human trials can provide number of values for patient benefits: elucidating current therapeutic mechanism-of-action, and, back-translating to fast-track development of next-generation therapeutics. Both opportunities are predicated on proactive generation of human molecular profiles that capture longitudinal trajectories before and after pharmacological intervention. Here, we present the largest plasma proteomic biomarker dataset available to-date and the corresponding analyses from placebo-controlled Phase III clinical trials of the phosphodiesterase type 4 inhibitor apremilast in psoriasis (PSOR), psoriatic arthritis (PsA), and ankylosing spondylitis (AS) from 526 subjects overall. Using approximately 150 plasma analytes tracked across three time points, we identified IL-17A and KLK-7 as biomarkers for disease severity and apremilast pharmacodynamic effect in psoriasis patients. Combined decline rate of KLK-7, PEDF, MDC and ANGPTL4 by Week 16 represented biomarkers for the responder subgroup, shedding insights into therapeutic mechanisms. In ankylosing spondylitis patients, IL-6 and LRG-1 were identified as biomarkers with concordance to disease severity. Apremilast-induced LRG-1 increase was consistent with the overall lack of efficacy in ankylosing spondylitis. Taken together, these findings expanded the mechanistic knowledge base of apremilast and provided translational foundations to accelerate future efforts including compound differentiation, combination, and repurposing.

Computer Analysis of Glioma Transcriptome Profiling: Alternative Splicing Events.

Here we present the analysis of alternative splicing events on an example of glioblastoma cell culture samples using a set of computer tools in combination with database integration. The gene expression profiles of glioblastoma were obtained from cell culture samples of primary glioblastoma which were isolated and processed for RNA extraction. Transcriptome profiling of normal brain samples and glioblastoma were done by Illumina sequencing. The significant differentially expressed exon-level probes and their corresponding genes were identified using a combination of the splicing index method. Previous studies indicated that tumor-specific alternative splicing is important in the regulation of gene expression and corresponding protein functions during cancer development. Multiple alternative splicing transcripts have been identified as progression markers, including generalized splicing abnormalities and tumor- and stage-specific events. We used a set of computer tools which were recently applied to analysis of gene expression in laboratory animals to study differential splicing events. We found 69 transcripts that are differentially alternatively spliced. Three cancer-associated genes were considered in detail, in particular: APP (amyloid beta precursor protein), CASC4 (cancer susceptibility candidate 4) and TP53. Such alternative splicing opens new perspectives for cancer research.

Heterogeneity of neuroblastoma cell identity defined by transcriptional circuitries.

Neuroblastoma is a tumor of the peripheral sympathetic nervous system, derived from multipotent neural crest cells (NCCs). To define core regulatory circuitries (CRCs) controlling the gene expression program of neuroblastoma, we established and analyzed the neuroblastoma super-enhancer landscape. We discovered three types of identity in neuroblastoma cell lines: a sympathetic noradrenergic identity, defined by a CRC module including the PHOX2B, HAND2 and GATA3 transcription factors (TFs); an NCC-like identity, driven by a CRC module containing AP-1 TFs; and a mixed type, further deconvoluted at the single-cell level. Treatment of the mixed type with chemotherapeutic agents resulted in enrichment of NCC-like cells. The noradrenergic module was validated by ChIP-seq. Functional studies demonstrated dependency of neuroblastoma with noradrenergic identity on PHOX2B, evocative of lineage addiction. Most neuroblastoma primary tumors express TFs from the noradrenergic and NCC-like modules. Our data demonstrate a previously unknown aspect of tumor heterogeneity relevant for neuroblastoma treatment strategies.

The prevalence of familial hypercholesterolemia in the West Siberian region of the Russian Federation: A substudy of the ESSE-RF.

BACKGROUND: The prevalence of familial hypercholesterolemia (FH) in Russia has not previously been evaluated. The aim of our study was to investigate the prevalence of FH in the population of the West Siberian region of Russia, and then estimate the frequency of coronary artery disease (CAD) and treatment with cholesterol-lowering medication in FH patients. METHODS: The sample of our study consisted of participants from the population-based cohort of The Epidemiology of Cardiovascular Risk Factors and Diseases in Regions of the Russian Federation Study (ESSE-RF), conducted in the Tyumen and Kemerovo regions (1,630 and 1,622 people, respectively, aged 25-64). All participants who had LDL-cholesterol higher than 4.9 mmol/l and who had LDL-cholesterol less than or equal to 4.9 mmol/l but had statin therapy were examined and interviewed by experts in FH. RESULTS: The prevalence of patients with definite FH was 0.24% (one in 407) (95% confidence interval [CI]: 0.06%-0.42%), with probable FH was 0.68% (one in 148) (95% CI: 0.38%-0.98%), and with definite or probable FH combined was 0.92% (one in 108) (95% CI: 0.58%-1.26%). 40% (95% CI: 20.8%-59.2%) of patients with definite or probable FH had CAD. However, only 23% (95% CI: 6.3%-39.7%) of patients with definite or probable FH were on statins. The odds ratios for CAD and myocardial infarction (MI), adjusted for age, gender, region, smoking, hypertension, and diabetes mellitus, were 3.71 (95% CI: 1.58-8.72) (p = 0.003) and 4.06 (95% CI: 0.89-18.55) (р = 0.070) respectively for individuals with definite or probable FH relative to those who were unlikely to have FH. CONCLUSIONS: The prevalence of FH in Russia may be significantly higher than previously estimated. There is underdiagnosis and undertreatment of FH in Russia.

SITEX 2.0: Projections of protein functional sites on eukaryotic genes. Extension with orthologous genes.

Functional sites define the diversity of protein functions and are the central object of research of the structural and functional organization of proteins. The mechanisms underlying protein functional sites emergence and their variability during evolution are distinguished by duplication, shuffling, insertion and deletion of the exons in genes. The study of the correlation between a site structure and exon structure serves as the basis for the in-depth understanding of sites organization. In this regard, the development of programming resources that allow the realization of the mutual projection of exon structure of genes and primary and tertiary structures of encoded proteins is still the actual problem. Previously, we developed the SitEx system that provides information about protein and gene sequences with mapped exon borders and protein functional sites amino acid positions. The database included information on proteins with known 3D structure. However, data with respect to orthologs was not available. Therefore, we added the projection of sites positions to the exon structures of orthologs in SitEx 2.0. We implemented a search through database using site conservation variability and site discontinuity through exon structure. Inclusion of the information on orthologs allowed to expand the possibilities of SitEx usage for solving problems regarding the analysis of the structural and functional organization of proteins. Database URL: http://www-bionet.sscc.ru/sitex/ .

Metabolic model of central carbon and energy metabolisms of growing Arabidopsis thaliana in relation to sucrose translocation.

BACKGROUND: Sucrose translocation between plant tissues is crucial for growth, development and reproduction of plants. Systemic analysis of these metabolic and underlying regulatory processes allow a detailed understanding of carbon distribution within the plant and the formation of associated phenotypic traits. Sucrose translocation from 'source' tissues (e.g. mesophyll) to 'sink' tissues (e.g. root) is tightly bound to the proton gradient across the membranes. The plant sucrose transporters are grouped into efflux exporters (SWEET family) and proton-symport importers (SUC, STP families). To better understand regulation of sucrose export from source tissues and sucrose import into sink tissues, there is a need for a metabolic model that takes in account the tissue organisation of Arabidopsis thaliana with corresponding metabolic specificities of respective tissues in terms of sucrose and proton production/utilization. An ability of the model to operate under different light modes ('light' and 'dark') and correspondingly in different energy producing modes is particularly important in understanding regulatory modules. RESULTS: Here, we describe a multi-compartmental model consisting of a mesophyll cell with plastid and mitochondrion, a phloem cell, as well as a root cell with mitochondrion. In this model, the phloem was considered as a non-growing transport compartment, the mesophyll compartment was considered as both autotrophic (growing on CO2 under light) and heterotrophic (growing on starch in darkness), and the root was always considered as heterotrophic tissue dependent on sucrose supply from the mesophyll compartment. In total, the model includes 413 balanced compounds interconnected by 400 transformers. The structured metabolic model accounts for central carbon metabolism, photosynthesis, photorespiration, carbohydrate metabolism, energy and redox metabolisms, proton metabolism, biomass growth, nutrients uptake, proton gradient generation and sucrose translocation between tissues. Biochemical processes in the model were associated with gene-products (742 ORFs). Flux Balance Analysis (FBA) of the model resulted in balanced carbon, nitrogen, proton, energy and redox states under both light and dark conditions. The main H+-fluxes were reconstructed and their directions matched with proton-dependent sucrose translocation from 'source' to 'sink' under any light condition. CONCLUSIONS: The model quantified the translocation of sucrose between plant tissues in association with an integral balance of protons, which in turn is defined by operational modes of the energy metabolism.

Analysis of differential gene expression by RNA-seq data in brain areas of laboratory animals.

Computer analysis of gene expression in the nervous system plays a fundamental role in biology, genetics, and neurosciences. We studied molecular and genetic mechanisms of enhanced aggressiveness in comparison with tolerant behaviour using experimental animal models developed at the Institute of Cytology and Genetics SB RAS. Grey rats (Rattus norvegicus) have been subjected to selection during several generations in two directions – friendly, tolerant behaviour towards man (tame grey rats) and increased aggressive behaviour. We used samples from hypothalamus, mesencephalic tegmentum and periaqueductum grey matter from brain areas of grey rats genetically selected by behaviour in many generations. The set of computer tools and data processing pipelines helped to find genes and gene regulation patterns related to behaviour patterns. RNA - profiling experiments revealed the lists of differentially expressed genes in the contrast samples as well as differentially spliced isoforms. The gene ontology categories of protein transport, phosphoproteins, and nucleotide binding are presented together with categories of transmission of nerve impulses and neuron development were identified. Differential alternative splicing events found in the brain areas studied are statistically significant. We discuss role of alternative splicing events for neurospecific genes in behaviour patterns as well as extension of brain transcriptomics profiling.

Computer analysis of protein functional sites projection on exon structure of genes in Metazoa.

BACKGROUND: Study of the relationship between the structural and functional organization of proteins and their coding genes is necessary for an understanding of the evolution of molecular systems and can provide new knowledge for many applications for designing proteins with improved medical and biological properties. It is well known that the functional properties of proteins are determined by their functional sites. Functional sites are usually represented by a small number of amino acid residues that are distantly located from each other in the amino acid sequence. They are highly conserved within their functional group and vary significantly in structure between such groups. According to this facts analysis of the general properties of the structural organization of the functional sites at the protein level and, at the level of exon-intron structure of the coding gene is still an actual problem. RESULTS: One approach to this analysis is the projection of amino acid residue positions of the functional sites along with the exon boundaries to the gene structure. In this paper, we examined the discontinuity of the functional sites in the exon-intron structure of genes and the distribution of lengths and phases of the functional site encoding exons in vertebrate genes. We have shown that the DNA fragments coding the functional sites were in the same exons, or in close exons. The observed tendency to cluster the exons that code functional sites which could be considered as the unit of protein evolution. We studied the characteristics of the structure of the exon boundaries that code, and do not code, functional sites in 11 Metazoa species. This is accompanied by a reduced frequency of intercodon gaps (phase 0) in exons encoding the amino acid residue functional site, which may be evidence of the existence of evolutionary limitations to the exon shuffling. CONCLUSIONS: These results characterize the features of the coding exon-intron structure that affect the functionality of the encoded protein and allow a better understanding of the emergence of biological diversity.

SitEx: a computer system for analysis of projections of protein functional sites on eukaryotic genes.

Search of interrelationships between the structural-functional protein organization and exon structure of encoding gene provides insights into issues concerned with the function, origin and evolution of genes and proteins. The functions of proteins and their domains are defined mostly by functional sites. The relation of the exon-intron structure of the gene to the protein functional sites has been little studied. Development of resources containing data on projections of protein functional sites on eukaryotic genes is needed. We have developed SitEx, a database that contains information on functional site amino acid positions in the exon structure of encoding gene. SitEx is integrated with the BLAST and 3DExonScan programs. BLAST is used for searching sequence similarity between the query protein and polypeptides encoded by single exons stored in SitEx. The 3DExonScan program is used for searching for structural similarity of the given protein with these polypeptides using superimpositions. The developed computer system allows users to analyze the coding features of functional sites by taking into account the exon structure of the gene, to detect the exons involved in shuffling in protein evolution, also to design protein-engineering experiments. SitEx is accessible at http://www-bionet.sscc.ru/sitex/. Currently, it contains information about 9994 functional sites presented in 2021 proteins described in proteomes of 17 organisms.