V. V. Galatenko

High-throughput identification of reference genes for research and clinical RT-qPCR analysis of breast cancer samples.

BackgroundQuantification and normalization of RT-qPCR data critically depends on the expression of so called reference genes. Our goal was to develop a strategy for the selection of reference genes that utilizes microarray data analysis and combines known approaches for gene stability evaluation and to select a set of appropriate reference genes for research and clinical analysis of breast samples with different receptor and cancer status using this strategy.MethodsA preliminary search of reference genes was based on high-throughput analysis of microarray datasets. The final selection and validation of the candidate genes were based on the RT-qPCR data analysis using several known methods for expression stability evaluation: comparative ∆Ct method, geNorm, NormFinder and Haller equivalence test.ResultsA set of five reference genes was identified: ACTB, RPS23, HUWE1, EEF1A1 and SF3A1. The initial selection was based on the analysis of publically available well-annotated microarray datasets containing different breast cancers and normal breast epithelium from breast cancer patients and epithelium from cancer-free patients. The final selection and validation were performed using RT-qPCR data from 39 breast cancer biopsy samples. Three genes from the final set were identified by the means of microarray analysis and were novel in the context of breast cancer assay. We showed that the selected set of reference genes is more stable in comparison not only with individual genes, but also with a system of reference genes used in commercial OncotypeDX test.ConclusionA selection of reference genes for RT-qPCR can be efficiently performed by combining a preliminary search based on the high-throughput analysis of microarray datasets and final selection and validation based on the analysis of RT-qPCR data with a simultaneous examination of different expression stability measures. The identified set of reference genes proved to be less variable and thus potentially more efficient for research and clinical analysis of breast samples comparing to individual genes and the set of reference genes used in OncotypeDX assay.

Genome sequence of the pattern forming Paenibacillus vortex bacterium reveals potential for thriving in complex environments

BackgroundThe pattern-forming bacterium Paenibacillus vortex is notable for its advanced social behavior, which is reflected in development of colonies with highly intricate architectures. Prior to this study, only two other Paenibacillus species (Paenibacillus sp. JDR-2 and Paenibacillus larvae) have been sequenced. However, no genomic data is available on the Paenibacillus species with pattern-forming and complex social motility. Here we report the de novo genome sequence of this Gram-positive, soil-dwelling, sporulating bacterium.ResultsThe complete P. vortex genome was sequenced by a hybrid approach using 454 Life Sciences and Illumina, achieving a total of 289× coverage, with 99.8% sequence identity between the two methods. The sequencing results were validated using a custom designed Agilent microarray expression chip which represented the coding and the non-coding regions. Analysis of the P. vortex genome revealed 6,437 open reading frames (ORFs) and 73 non-coding RNA genes. Comparative genomic analysis with 500 complete bacterial genomes revealed exceptionally high number of two-component system (TCS) genes, transcription factors (TFs), transport and defense related genes. Additionally, we have identified genes involved in the production of antimicrobial compounds and extracellular degrading enzymes.ConclusionsThese findings suggest that P. vortex has advanced faculties to perceive and react to a wide range of signaling molecules and environmental conditions, which could be associated with its ability to reconfigure and replicate complex colony architectures. Additionally, P. vortex is likely to serve as a rich source of genes important for agricultural, medical and industrial applications and it has the potential to advance the study of social microbiology within Gram-positive bacteria.

miRNome of inflammatory breast cancer

BackgroundInflammatory breast cancer (IBC) is an extremely malignant form of breast cancer which can be easily misdiagnosed. Conclusive prognostic IBC molecular biomarkers which are also providing the perspectives for targeted therapy are lacking so far. The aim of this study was to reveal the IBC-specific miRNA expression profile and to evaluate its association with clinicopathological parameters.MethodsmiRNA expression profiles of 13 IBC and 17 non-IBC patients were characterized using comprehensive Affymetrix GeneChip miRNA 3.0 microarray platform. Bioinformatic analysis was used to reveal IBC-specific miRNAs, deregulated pathways and potential miRNA targets.Results31 differentially expressed miRNAs characterize IBC and mRNAs regulated by them and their associated pathways can functionally be attributed to IBC progression. In addition, a minimal predictive set of 4 miRNAs characteristic for the IBC phenotype and associated with the TP53 mutational status in breast cancer patients was identified.ConclusionsWe have characterized the complete miRNome of inflammatory breast cancer and found differentially expressed miRNAs which reliably classify the patients to IBC and non-IBC groups. We found that the mRNAs and pathways likely regulated by these miRNAs are highly relevant to cancer progression. Furthermore a minimal IBC-related predictive set of 4 miRNAs associated with the TP53 mutational status and survival for breast cancer patients was identified.

Genome Sequence of the Pattern-Forming Social Bacterium Paenibacillus dendritiformis C454 Chiral Morphotype

Paenibacillus dendritiformis is a Gram-positive, soil-dwelling, spore-forming social microorganism. An intriguing collective faculty of this strain is manifested by its ability to switch between different morphotypes, such as the branching (T) and the chiral (C) morphotypes. Here we report the 6.3-Mb draft genome sequence of the P. dendritiformis C454 chiral morphotype.

Highly informative marker sets consisting of genes with low individual degree of differential expression

Genes with significant differential expression are traditionally used to reveal the genetic background underlying phenotypic differences between cancer cells. We hypothesized that informative marker sets can be obtained by combining genes with a relatively low degree of individual differential expression. We developed a method for construction of highly informative gene combinations aimed at the maximization of the cumulative informative power and identified sets of 2–5 genes efficiently predicting recurrence for ER-positive breast cancer patients. The gene combinations constructed on the basis of microarray data were successfully applied to data acquired by RNA-seq. The developed method provides the basis for the generation of highly efficient prognostic and predictive gene signatures for cancer and other diseases. The identified gene sets can potentially reveal novel essential segments of gene interaction networks and pathways implied in cancer progression.

Evaluation of potential reference genes for qRT-PCR data normalization in HeLa cells

Reference genes selection is one of the most important stages in qPCR data normalization when a problem of quantitative determination of gene expression is addressed. Stability of gene expression level in all experimental conditions is a basic criterion for the reference gene selection. Over the past decade a lot of publications concerning validation methods of suitable reference genes appeared. In this paper, the main approaches (ΔCt, geNorm, qBase and Haller’s equivalence test) were applied for the reference genes identification in HeLa cell line which is one of the most popular cellular models. Expression stability of seven candidate genes (HPRT1, ACTB, GAPDH, RPS18, HSPC3, UBC and SDHA) was determined at standard conditions, under heat shock and during relaxation. The genes RPS18 and HSPC3 were chosen as reference after the combination of all the validation methods.

Changes in the Level of Circulating hsa-miR-297 and hsa-miR-19b-3p miRNA Are Associated with Generalization of Prostate Cancer

We performed diagnostic classification of plasma specimens from patients with non-metastatic and metastatic prostate cancer based on pairs of miRNA that have no individual diagnostic significance. Of 230 miRNA detected in plasma specimens, 3 pairs were diagnostically significant. The miRNA pair hsa-miR-19b-3p and hsa-miR-297 demonstrated highest sensitivity and specificity. Among common target genes of these miRNA, CFL2 gene associated with cell mobility was detected.

Novel biomarkers in cancer: The whole is greater than the sum of its parts.

The major issues hampering progress in the treatment of cancer patients are distant metastases and drug resistance to chemotherapy. Metastasis formation is a very complex process, and looking at gene signatures alone is not enough to get deep insight into it. This paper reviews traditional and novel approaches to identify gene signature biomarkers and intratumoural fluid pressure both as a novel way of creating predictive markers and as an obstacle to cancer therapy. Finally recently developed in vitro systems to predict the response of individual patient derived cancer explants to chemotherapy are discussed.

Tumour-like druggable gene expression pattern of CaCo2 cells in microfluidic chip

The human-on-chip technology provides an efficient basis for preclinical studies and has potentially a greater predictive power for human drug response than classical 2D cell culture. Here we report the expression profile of druggable genes in the human colon cancer cells CaCo2 in static culture and within a microfluidic chip. We identified gene expression pattern under flow to be closer to the one of CaCo2 primary xenograft tumours as compared to those cells grown without circulation. The obtained results indicate that a microenvironment connected to a circulation within a chip brings the cells closer to in vivo situation. Hence the human-on-chip technology is a more powerful tool for drug development than conventional 2D cell culture.

Intra- and interregional coregulation of opioid genes: broken symmetry in spinal circuits

Regulation of the formation and rewiring of neural circuits by neuropeptides may require coordinated production of these signaling molecules and their receptors that may be established at the transcriptional level. Here, we address this hypothesis by comparing absolute expression levels of opioid peptides with their receptors, the largest neuropeptide family, and by characterizing coexpression (transcriptionally coordinated) patterns of these genes. We demonstrated that expression patterns of opioid genes highly correlate within and across functionally and anatomically different areas. Opioid peptide genes, compared with their receptor genes, are transcribed at much greater absolute levels, which suggests formation of a neuropeptide cloud that covers the receptor‐expressed circuits. Surprisingly, we found that both expression levels and the proportion of opioid receptors are strongly lateralized in the spinal cord, interregional coexpression patterns are side specific, and intraregional coexpression profiles are affected differently by left‐ and right‐side unilateral body injury. We propose that opioid genes are regulated as interconnected components of the same molecular system distributed between distinct anatomic regions. The striking feature of this system is its asymmetric coexpression patterns, which suggest side‐specific regulation of selective neural circuits by opioid neurohormones.—Kononenko, O., Galatenko, V., Andersson, M., Bazov, I., Watanabe, H., Zhou, X. W., Iatsyshyna, A., Mityakina, I., Yakovleva, T., Sarkisyan, D., Ponomarev, I., Krishtal, O., Marklund, N., Tonevitsky, A., Adkins, D. L., Bakalkin, G. Intra‐ and interregional coregulation of opioid genes: broken symmetry in spinal circuits. FASEB J. 31, 1953–1963 (2017). www.fasebj.org