Publications by authors named "Alexander Tonevitsky"

79 Publications

Low expression of CD24 is associated with poor survival in colorectal cancer.

Biochimie 2021 Oct 9. Epub 2021 Oct 9.

Faculty of Biology and Biotechnology, HSE University, Moscow, Russia; SRC Bioclinicum, Moscow, Russia. Electronic address:

In this study we analyzed expression of CD24 in a cohort of colorectal cancer patients using immunohistochemistry staining of CD24. We found a significant association between absence or low expression of CD24 (10% of membranous and 55% of cytoplasmic staining) and shortened patient survival. Protein localization played a crucial role in the prognosis: membranous form was the major and prognostic one in primary tumors, while cytoplasmic expression was elevated in liver metastases compared to the primary tumors and contained prognostic information. Then, using The Cancer Genome Atlas Colon Adenocarcinoma (TCGA-COAD) RNA-seq data, we showed that CD24 mRNA level was two-fold decreased in primary colorectal cancers compared to adjacent normal mucosa. Like the protein staining data, ten percent of patients with the lowest mRNA expression levels of CD24 in primary tumors had reduced survival compared to the ones with higher expression. To explain these findings mechanistically, shRNA-mediated CD24 knockdown was performed in HT-29 colorectal cancer cells. It resulted in the increase of cell migration in vitro, no changes in proliferation and apoptosis, and a slight decrease in cell invasion. As increased cell migration is a hallmark of metastasis formation, this finding corroborates the association of a decreased CD24 expression with poor prognosis. Differential gene expression analysis revealed upregulation of genes involved in cell migration in the group of patients with low CD24 expression, including integrin subunit α3 and α3, β3 subunits of laminin 332. Further co-expression analysis identified SPI1, STAT1 and IRF1 transcription factors as putative master-regulators in this group.
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http://dx.doi.org/10.1016/j.biochi.2021.10.004DOI Listing
October 2021

Endocytosis and Transcytosis of SARS-CoV-2 Across the Intestinal Epithelium and Other Tissue Barriers.

Front Immunol 2021 7;12:636966. Epub 2021 Sep 7.

Laboratory of Microfluidic Technologies for Biomedicine, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow, Russia.

Since 2003, the world has been confronted with three new betacoronaviruses that cause human respiratory infections: SARS-CoV, which causes severe acute respiratory syndrome (SARS), MERS-CoV, which causes Middle East respiratory syndrome (MERS), and SARS-CoV-2, which causes Coronavirus Disease 2019 (COVID-19). The mechanisms of coronavirus transmission and dissemination in the human body determine the diagnostic and therapeutic strategies. An important problem is the possibility that viral particles overcome tissue barriers such as the intestine, respiratory tract, blood-brain barrier, and placenta. In this work, we will 1) consider the issue of endocytosis and the possibility of transcytosis and paracellular trafficking of coronaviruses across tissue barriers with an emphasis on the intestinal epithelium; 2) discuss the possibility of antibody-mediated transcytosis of opsonized viruses due to complexes of immunoglobulins with their receptors; 3) assess the possibility of the virus transfer into extracellular vesicles during intracellular transport; and 4) describe the clinical significance of these processes. Models of the intestinal epithelium and other barrier tissues for transcytosis studies will also be briefly characterized.
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http://dx.doi.org/10.3389/fimmu.2021.636966DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8452982PMC
October 2021

T-CoV: a comprehensive portal of HLA-peptide interactions affected by SARS-CoV-2 mutations.

Nucleic Acids Res 2021 Aug 16. Epub 2021 Aug 16.

Faculty of Biology and Biotechnology, HSE University, Moscow, Russia.

Rapidly appearing SARS-CoV-2 mutations can affect T cell epitopes, which can help the virus to evade either CD8 or CD4 T-cell responses. We developed T-cell COVID-19 Atlas (T-CoV, https://t-cov.hse.ru) - the comprehensive web portal, which allows one to analyze how SARS-CoV-2 mutations alter the presentation of viral peptides by HLA molecules. The data are presented for common virus variants and the most frequent HLA class I and class II alleles. Binding affinities of HLA molecules and viral peptides were assessed with accurate in silico methods. The obtained results highlight the importance of taking HLA alleles diversity into account: mutation-mediated alterations in HLA-peptide interactions were highly dependent on HLA alleles. For example, we found that the essential number of peptides tightly bound to HLA-B*07:02 in the reference Wuhan variant ceased to be tight binders for the Indian (Delta) and the UK (Alpha) variants. In summary, we believe that T-CoV will help researchers and clinicians to predict the susceptibility of individuals with different HLA genotypes to infection with variants of SARS-CoV-2 and/or forecast its severity.
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http://dx.doi.org/10.1093/nar/gkab701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8385993PMC
August 2021

Hairpin sequence and structure is associated with features of isomiR biogenesis.

RNA Biol 2021 Jul 21:1-9. Epub 2021 Jul 21.

Faculty of Biology and Biotechnology, HSE University, Moscow, Russia.

MiRNA isoforms (isomiRs) are single stranded small RNAs originating from the same pri-miRNA hairpin as a result of cleavage by Drosha and Dicer enzymes. Variations at the 5'-end of a miRNA alter the seed region of the molecule, thus affecting the targetome of the miRNA. In this manuscript, we analysed the distribution of miRNA cleavage positions across 31 different cancers using miRNA sequencing data of TCGA project. As a result, we found that the processing positions are not tissue specific and that all miRNAs could be correctly classified as ones exhibiting homogeneous or heterogeneous cleavage at one of the four cleavage sites. In 42% of cases (42 out of 100 miRNAs), we observed imprecise 5'-end Dicer cleavage, while this fraction was only 14% for Drosha (14 out of 99). To the contrary, almost all cleavage sites of 3'-ends (either Drosha or Dicer) were heterogeneous. With the use of only four nucleotides surrounding a 5'-end Dicer cleavage position we built a model which allowed us to distinguish between homogeneous and heterogeneous cleavage with the reliable quality (ROC AUC = 0.68). Finally, we showed the possible applications of the study by the analysis of two 5'-end isoforms originating from the same exogeneous shRNA hairpin. It turned out that the less expressed shRNA variant was functionally active, which led to the increased off-targeting. Thus, the obtained results could be applied to the design of shRNAs whose processing will result in a single 5'-variant.
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http://dx.doi.org/10.1080/15476286.2021.1952759DOI Listing
July 2021

Integrin alpha-V is an important driver in pancreatic adenocarcinoma progression.

J Exp Clin Cancer Res 2021 Jun 26;40(1):214. Epub 2021 Jun 26.

Institute of Anatomy and Experimental Morphology, University Medical-Center Hamburg-Eppendorf, Hamburg, Germany.

Background: Mesothelial E- and P-selectins substantially mediate the intraperitoneal spread of Pancreatic ductal adenocarcinoma (PDA) cells in xenograft models. In the absence of selectins in the host, the integrin subunit alpha-V (ITGAV, CD51) was upregulated in the remaining metastatic deposits. Here we present the first experimental study to investigate if ITGAV plays a functional role in PDA tumor growth and progression with a particular focus on intraperitoneal carcinomatosis.

Methods: Knockdown of ITGAV was generated using an RNA interference-mediated approach in two PDA cell lines. Tumor growth, intraperitoneal and distant metastasis were analyzed in a xenograft model. Cell lines were characterized in vitro. Gene expression of the xenograft tumors was analyzed. Patient samples were histologically classified and associations to survival were evaluated.

Results: The knockdown of ITGAV in PDA cells strongly reduces primary tumor growth, peritoneal carcinomatosis and spontaneous pulmonary metastasis. ITGAV activates latent TGF-β and thereby drives epithelial-mesenchymal transition. Combined depletion of ITGAV on the tumor cells and E- and P-selectins in the tumor-host synergistically almost abolishes intraperitoneal spread. Accordingly, high expression of ITGAV in PDA cells was associated with reduced survival in patients.

Conclusion: Combined depletion of ITGAV in PDA cells and E- and P-selectins in host mice massively suppresses intraperitoneal carcinomatosis of PDA cells xenografted into immunodeficient mice, confirming the hypothesis of a partly redundant adhesion cascade of metastasizing cancer cells. Our data strongly encourage developing novel therapeutic approaches for the combined targeting of E- and P-selectins and ITGAV in PDA.
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http://dx.doi.org/10.1186/s13046-021-01946-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8235815PMC
June 2021

Effect of the Expression of and Genes on the Metastatic Potential of Breast Cancer Cells.

Front Genet 2021 2;12:662843. Epub 2021 Jun 2.

Faculty of Biology and Biotechnologies, National Research University Higher School of Economics, Moscow, Russia.

Breast cancer (BC) is the leading cause of death from malignant neoplasms among women worldwide, and metastatic BC presents the biggest problems for treatment. Previously, it was shown that lower expression of and genes is associated with a higher risk of the formation of distant metastases in BC. In this work, we studied the change in phenotypical traits, as well as in the transcriptomic and proteomic profiles of BC cells as a result of the stable knockdown of and genes. The knockdown of and genes was found to lead to a strong increase in the expression of the matrix metalloproteinase (MMP) . These results were in good agreement with the correlation analysis of gene expression in tumor samples from patients and were additionally confirmed by zymography. The knockdown of and genes was also discovered to change the expression of a group of genes involved in the formation of intercellular contacts. In particular, the expression of the gene was markedly reduced, which also complies with the correlation analysis. The spheroid formation assay showed that intercellular adhesion decreased as a result of the knockdown of the and genes. Thus, the obtained data indicate that malignant breast tumors with reduced expression of the and genes can metastasize with a higher probability due to a more efficient invasion of tumor cells.
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http://dx.doi.org/10.3389/fgene.2021.662843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8206645PMC
June 2021

Hypoxia-Induced miR-148a Downregulation Contributes to Poor Survival in Colorectal Cancer.

Front Genet 2021 31;12:662468. Epub 2021 May 31.

Faculty of Biology and Biotechnology, HSE University, Moscow, Russia.

Hypoxia is an extensively investigated condition due to its contribution to various pathophysiological processes including cancer progression and metastasis formation. MicroRNAs (miRNAs) are well-known post-transcriptional gene expression regulators. However, their contribution to molecular response to hypoxia is highly dependent on cell/tissue types and causes of hypoxia. One of the most important examples is colorectal cancer, where no consensus on hypoxia-regulated miRNAs has been reached so far. In this work, we applied integrated mRNA and small RNA sequencing, followed by bioinformatics analysis, to study the landscape of hypoxia-induced miRNA and mRNA expression alterations in human colorectal cancer cell lines (HT-29 and Caco-2). A hypoxic microenvironment was chemically modeled using two different treatments: cobalt(II) chloride and oxyquinoline. Only one miRNA, hsa-miR-210-3p, was upregulated in all experimental conditions, while there were nine differentially expressed miRNAs under both treatments within the same cell line. Further bioinformatics analysis revealed a complex hypoxia-induced regulatory network: hypoxic downregulation of hsa-miR-148a-3p led to the upregulation of its two target genes, ITGA5 and PRNP, which was shown to be a factor contributing to tumor progression and poor survival in colorectal cancer patients.
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http://dx.doi.org/10.3389/fgene.2021.662468DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8202010PMC
May 2021

miRGTF-net: Integrative miRNA-gene-TF network analysis reveals key drivers of breast cancer recurrence.

PLoS One 2021 14;16(4):e0249424. Epub 2021 Apr 14.

Faculty of Biology and Biotechnology, HSE University, Moscow, Russia.

Analysis of regulatory networks is a powerful framework for identification and quantification of intracellular interactions. We introduce miRGTF-net, a novel tool for construction of miRNA-gene-TF networks. We consider multiple transcriptional and post-transcriptional interaction types, including regulation of gene and miRNA expression by transcription factors, gene silencing by miRNAs, and co-expression of host genes with their intronic miRNAs. The underlying algorithm uses information on experimentally validated interactions as well as integrative miRNA/mRNA expression profiles in a given set of samples. The latter ensures simultaneous tissue-specificity and biological validity of interactions. We applied miRGTF-net to paired miRNA/mRNA-sequencing data of breast cancer samples from The Cancer Genome Atlas (TCGA). Together with topological analysis of the constructed network we showed that considered players can form reliable prognostic gene signatures for ER-positive breast cancer. A number of signatures demonstrated remarkably high accuracy on transcriptomic data obtained by both microarrays and RNA sequencing from several independent patient cohorts. Furthermore, an essential part of prognostic genes were identified as direct targets of transcription factor E2F1. The putative interplay between estrogen receptor alpha and E2F1 was suggested as a potential recurrence factor in patients treated with tamoxifen. Source codes of miRGTF-net are available at GitHub (https://github.com/s-a-nersisyan/miRGTF-net).
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0249424PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8046230PMC
September 2021

Association of HLA Class I Genotypes With Severity of Coronavirus Disease-19.

Front Immunol 2021 23;12:641900. Epub 2021 Feb 23.

Faculty of Biology and Biotechnology, HSE University, Moscow, Russia.

Human leukocyte antigen (HLA) class I molecules play a crucial role in the development of a specific immune response to viral infections by presenting viral peptides at the cell surface where they will be further recognized by T cells. In the present manuscript, we explored whether HLA class I genotypes can be associated with the critical course of Coronavirus Disease-19 by searching possible connections between genotypes of deceased patients and their age at death. HLA-A, HLA-B, and HLA-C genotypes of = 111 deceased patients with COVID-19 (Moscow, Russia) and = 428 volunteers were identified with next-generation sequencing. Deceased patients were split into two groups according to age at the time of death: = 26 adult patients aged below 60 and = 85 elderly patients over 60. With the use of HLA class I genotypes, we developed a risk score (RS) which was associated with the ability to present severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) peptides by the HLA class I molecule set of an individual. The resulting RS was significantly higher in the group of deceased adults compared to elderly adults [ = 0.00348, area under the receiver operating characteristic curve ( = 0.68)]. In particular, presence of HLA-A01:01 allele was associated with high risk, while HLA-A02:01 and HLA-A03:01 mainly contributed to low risk. The analysis of patients with homozygosity strongly highlighted these results: homozygosity by HLA-A01:01 accompanied early deaths, while only one HLA-A02:01 homozygote died before 60 years of age. Application of the constructed RS model to an independent Spanish patients cohort ( = 45) revealed that the score was also associated with the severity of the disease. The obtained results suggest the important role of HLA class I peptide presentation in the development of a specific immune response to COVID-19.
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http://dx.doi.org/10.3389/fimmu.2021.641900DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7959787PMC
April 2021

HIF Prolyl Hydroxylase Inhibitors for COVID-19 Treatment: Pros and Cons.

Front Pharmacol 2020 29;11:621054. Epub 2021 Jan 29.

P. A. Hertsen Moscow Oncology Research Center, Branch of the National Medical Research Radiological Center, Ministry of Health of the Russian Federation, Moscow, Russia.

The review analyzes the potential advantages and problems associated with using HIF prolyl hydroxylase inhibitors as a treatment for COVID-19. HIF prolyl hydroxylase inhibitors are known to boost endogenous erythropoietin (Epo) and activate erythropoiesis by stabilizing and activating the hypoxia inducible factor (HIF). Recombinant Epo treatment has anti-inflammatory and healing properties, and thus, very likely, will be beneficial for moderate to severe cases of COVID-19. However, HIF PHD inhibition may have a significantly broader effect, in addition to stimulating the endogenous Epo production. The analysis of HIF target genes reveals that some HIF-targets, such as furin, could play a negative role with respect to viral entry. On the other hand, HIF prolyl hydroxylase inhibitors counteract ferroptosis, the process recently implicated in vessel damage during the later stages of COVID-19. Therefore, HIF prolyl hydroxylase inhibitors may serve as a promising treatment of COVID-19 complications, but they are unlikely to aid in the prevention of the initial stages of infection.
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http://dx.doi.org/10.3389/fphar.2020.621054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878396PMC
January 2021

Potential role of cellular miRNAs in coronavirus-host interplay.

PeerJ 2020 14;8:e9994. Epub 2020 Sep 14.

Faculty of Biology and Biotechnology, HSE University, Moscow, Russia.

Host miRNAs are known as important regulators of virus replication and pathogenesis. They can interact with various viruses through several possible mechanisms including direct binding of viral RNA. Identification of human miRNAs involved in coronavirus-host interplay becomes important due to the ongoing COVID-19 pandemic. In this article we performed computational prediction of high-confidence direct interactions between miRNAs and seven human coronavirus RNAs. As a result, we identified six miRNAs (miR-21-3p, miR-195-5p, miR-16-5p, miR-3065-5p, miR-424-5p and miR-421) with high binding probability across all analyzed viruses. Further bioinformatic analysis of binding sites revealed high conservativity of miRNA binding regions within RNAs of human coronaviruses and their strains. In order to discover the entire miRNA-virus interplay we further analyzed lungs miRNome of SARS-CoV infected mice using publicly available miRNA sequencing data. We found that miRNA miR-21-3p has the largest probability of binding the human coronavirus RNAs and being dramatically up-regulated in mouse lungs during infection induced by SARS-CoV.
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http://dx.doi.org/10.7717/peerj.9994DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7497610PMC
September 2020

Xenograft-derived mRNA/miR and protein interaction networks of systemic dissemination in human prostate cancer.

Eur J Cancer 2020 09 1;137:93-107. Epub 2020 Aug 1.

Institute of Anatomy and Experimental Morphology, University Medical Center, Hamburg-Eppendorf, Martinistrasse 52, Hamburg, Germany.

Background: Distant metastasis formation is the major clinical problem in prostate cancer (PCa) and the underlying mechanisms remain poorly understood. Our aim was to identify novel molecules that functionally contribute to human PCa systemic dissemination based on unbiased approaches.

Methods: We compared mRNA, microRNA (miR) and protein expression levels in established human PCa xenograft tumours with high (PC-3), moderate (VCaP) or weak (DU-145) spontaneous micrometastatic potential. By focussing on those mRNAs, miRs and proteins that were differentially regulated among the xenograft groups and known to interact with each other we constructed dissemination-related mRNA/miR and protein/miR networks. Next, we clinically and functionally validated our findings.

Results: Besides known determinants of PCa progression and/or metastasis, our interaction networks include several novel candidates. We observed a clear role of epithelial-to-mesenchymal transition (EMT) pathways for PCa dissemination, which was additionally confirmed by an independent human PCa model (ARCAP-E/-M). Two converging nodes, CD46 (decreasing with metastatic potential) and DDX21 (increasing with metastatic potential), were used to test the clinical relevance of the networks. Intriguingly, both network nodes consistently added prognostic information for patients with PCa whereas CD46 loss predicted poor outcome independent of established parameters. Accordingly, depletion of CD46 in weakly metastatic PCa cells induced EMT-like properties in vitro and spontaneous micrometastasis formation in vivo.

Conclusions: The clinical and functional relevance of the dissemination-related interaction networks shown here could be successfully validated by proof-of-principle experiments. Therefore, we suggest a direct pro-metastatic, clinically relevant role for the multiple novel candidates included in this study; these should be further exploited by future studies.
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http://dx.doi.org/10.1016/j.ejca.2020.06.025DOI Listing
September 2020

Integrative analysis of miRNA and mRNA sequencing data reveals potential regulatory mechanisms of ACE2 and TMPRSS2.

PLoS One 2020 29;15(7):e0235987. Epub 2020 Jul 29.

Faculty of Biology and Biotechnology, Higher School of Economics, Moscow, Russia.

Development of novel approaches for regulating the expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) is becoming increasingly important within the context of the ongoing COVID-19 pandemic since these enzymes play a crucial role in cell infection. In this work we searched for putative ACE2 and TMPRSS2 expression regulation networks mediated by various miRNA isoforms (isomiR) across different human organs using publicly available paired miRNA/mRNA-sequencing data from The Cancer Genome Atlas (TCGA) project. As a result, we identified several miRNA families targeting ACE2 and TMPRSS2 genes in multiple tissues. In particular, we found that lysine-specific demethylase 5B (JARID1B), encoded by the KDM5B gene, can indirectly affect ACE2 / TMPRSS2 expression by repressing transcription of hsa-let-7e / hsa-mir-125a and hsa-mir-141 / hsa-miR-200 miRNA families which are targeting these genes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0235987PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7390267PMC
August 2020

Knockdown of the α5 laminin chain affects differentiation of colorectal cancer cells and their sensitivity to chemotherapy.

Biochimie 2020 Jul 22;174:107-116. Epub 2020 Apr 22.

Faculty of Biology and Biotechnology, National Research University Higher School of Economics, Myasnitskaya str. 13/4, 117997, Moscow, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Miklukho-Maklaya str. 16/10, 117997, Moscow, Russia; Scientific Research Center Bioclinicum, Ugreshskaya str. 2/85, 115088, Moscow, Russia. Electronic address:

The interaction of tumor cells with the extracellular matrix (ECM) may affect the rate of cancer progression and metastasis. One of the major components of ECM are laminins, the heterotrimeric glycoproteins consisting of α-, β-, and γ-chains (αβγ). Laminins interact with their cell surface receptors and, thus, regulate multiple cellular processes. In this work, we demonstrate that shRNA-mediated knockdown of the α5 laminin chain results in Wnt- and mTORC1-dependent partial dedifferentiation of colorectal cancer cells. Furthermore, we showed that this dedifferentiation involved activation of ER-stress signaling, pathway promoting the sensitivity of cells to 5-fluorouracil.
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http://dx.doi.org/10.1016/j.biochi.2020.04.016DOI Listing
July 2020

Biology-inspired microphysiological systems to advance patient benefit and animal welfare in drug development

ALTEX 2020 28;37(3):365-394. Epub 2020 Feb 28.

National Center for Toxicological Research, FDA, Silver Spring, MD, USA.

The first microfluidic microphysiological systems (MPS) entered the academic scene more than 15 years ago and were considered an enabling technology to human (patho)biology in vitro and, therefore, provide alternative approaches to laboratory animals in pharmaceutical drug development and academic research. Nowadays, the field generates more than a thousand scientific publications per year. Despite the MPS hype in academia and by platform providers, which says this technology is about to reshape the entire in vitro culture landscape in basic and applied research, MPS approaches have neither been widely adopted by the pharmaceutical industry yet nor reached regulated drug authorization processes at all. Here, 46 leading experts from all stakeholders - academia, MPS supplier industry, pharmaceutical and consumer products industries, and leading regulatory agencies - worldwide have analyzed existing challenges and hurdles along the MPS-based assay life cycle in a second workshop of this kind in June 2019. They identified that the level of qualification of MPS-based assays for a given context of use and a communication gap between stakeholders are the major challenges for industrial adoption by end-users. Finally, a regulatory acceptance dilemma exists against that background. This t4 report elaborates on these findings in detail and summarizes solutions how to overcome the roadblocks. It provides recommendations and a roadmap towards regulatory accepted MPS-based models and assays for patients' benefit and further laboratory animal reduction in drug development. Finally, experts highlighted the potential of MPS-based human disease models to feedback into laboratory animal replacement in basic life science research.
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http://dx.doi.org/10.14573/altex.2001241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7863570PMC
July 2021

A Post-Processing Algorithm for miRNA Microarray Data.

Int J Mol Sci 2020 Feb 12;21(4). Epub 2020 Feb 12.

Faculty of Biology and Biotechnologies, Higher School of Economics, 117312 Moscow, Russia.

One of the main disadvantages of using DNA microarrays for miRNA expression profiling is the inability of adequate comparison of expression values across different miRNAs. This leads to a large amount of miRNAs with high scores which are actually not expressed in examined samples, i.e., false positives. We propose a post-processing algorithm which performs scoring of miRNAs in the results of microarray analysis based on expression values, time of discovery of miRNA, and correlation level between the expressions of miRNA and corresponding pre-miRNA in considered samples. The algorithm was successfully validated by the comparison of the results of its application to miRNA microarray breast tumor samples with publicly available miRNA-seq breast tumor data. Additionally, we obtained possible reasons why miRNA can appear as a false positive in microarray study using paired miRNA sequencing and array data. The use of DNA microarrays for estimating miRNA expression profile is limited by several factors. One of them consists of problems with comparing expression values of different miRNAs. In this work, we show that situation can be significantly improved if some additional information is taken into consideration in a comparison.
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http://dx.doi.org/10.3390/ijms21041228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072892PMC
February 2020

Impedance Spectroscopy as a Tool for Monitoring Performance in 3D Models of Epithelial Tissues.

Front Bioeng Biotechnol 2019 24;7:474. Epub 2020 Jan 24.

Faculty of Biology and Biotechnologies, Higher School of Economics, Moscow, Russia.

In contrast to traditional 2D cell cultures, both 3D models and organ-on-a-chip devices allow the study of the physiological responses of human cells. These models reconstruct human tissues in conditions closely resembling the body. Translation of these techniques into practice is hindered by associated labor costs, a need which may be remedied by automation. Impedance spectroscopy (IS) is a promising, automation-compatible label-free technology allowing to carry out a wide range of measurements both in real-time and as endpoints. IS has been applied to both the barrier cultures and the 3D constructs. Here we provide an overview of the impedance-based analysis in different setups and discuss its utility for organ-on-a-chip devices. Most attractive features of impedance-based assays are their compatibility with high-throughput format and supports for the measurements in real time with high temporal resolution, which allow tracing of the kinetics. As of now, IS-based techniques are not free of limitations, including imperfect understanding of the parameters that have their effects on the impedance, especially in 3D cell models, and relatively high cost of the consumables. Moreover, as the theory of IS stems from electromagnetic theory and is quite complex, work on popularization and explanation of the method for experimental biologists is required. It is expected that overcoming these limitations will lead to eventual establishing IS based systems as a standard for automated management of cell-based experiments in both academic and industry environments.
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http://dx.doi.org/10.3389/fbioe.2019.00474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6992543PMC
January 2020

-Regulating miR-4274 and Its Host Gene Play a Role in -Dependent Effects on Phenotype of Basal-Like Breast Cancer.

Front Mol Biosci 2019 8;6:122. Epub 2019 Nov 8.

Faculty of Biology and Biotechnologies, Higher School of Economics, Moscow, Russia.

Specificity of RNAi to selected target is challenged by off-target effects, both canonical and non-canonical. Notably, more than half of all human microRNAs are co-expressed with hosting them proteincoding genes. Here we dissect regulatory subnetwork centered on gene, which is associated with low proliferative state and high migratory activity of basal-like breast cancer. We inhibited expression of gene in a model cell line for basal-like breast carcinoma MDA-MB-231, then traced secondary and tertiary effects of this knockdown to , a laminin encoding gene that contributes to the phenotype of triple-negative breast cancer. -regulating miRNA miR-4274 and its host gene were highlighted as intermediate regulators of the expression levels of , which correlated in a basal-like breast carcinoma sample subset of TCGA to the levels of negatively. Overall, our study points that the secondary and tertiary layers of regulatory interactions are certainly underappreciated. As these types of molecular event may significantly contribute to the formation of the cell phenotypes after RNA interference based knockdowns, further studies of multilayered molecular networks affected by RNAi are warranted.
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http://dx.doi.org/10.3389/fmolb.2019.00122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6857517PMC
November 2019

Transcriptome of Extracellular Vesicles: State-of-the-Art.

Front Immunol 2019 28;10:202. Epub 2019 Feb 28.

Department of Cell Biology, Higher School of Economics, Moscow, Russia.

Exosomes and microvesicles are two major categories of extracellular vesicles (EVs) released by almost all cell types and are highly abundant in biological fluids. Both the molecular composition of EVs and their release are thought to be strictly regulated by external stimuli. Multiple studies have consistently demonstrated that EVs transfer proteins, lipids and RNA between various cell types, thus mediating intercellular communication, and signaling. Importantly, small non-coding RNAs within EVs are thought to be major contributors to the molecular events occurring in the recipient cell. Furthermore, RNA cargo in exosomes and microvesicles could hold tremendous potential as non-invasive biomarkers for multiple disorders, including pathologies of the immune system. This mini-review is aimed to provide the state-of-the-art in the EVs-associated RNA transcriptome field, as well as the comprehensive analysis of previous studies characterizing RNA content within EVs released by various cells using next-generation sequencing. Finally, we highlight the technical challenges associated with obtaining pure EVs and deep sequencing of the EV-associated RNAs.
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http://dx.doi.org/10.3389/fimmu.2019.00202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6404625PMC
January 2020

Towards embedding Caco-2 model of gut interface in a microfluidic device to enable multi-organ models for systems biology.

BMC Syst Biol 2019 03 5;13(Suppl 1):19. Epub 2019 Mar 5.

SRC BioClinicum, Moscow, Russia.

Background: A cancer cell line originating from human epithelial colorectal adenocarcinoma (Caco-2 cells) serves as a high capacity model for a preclinical screening of drugs. Recent need for incorporating barrier tissue into multi-organ chips calls for inclusion of Caco-2 cells into microperfused environment.

Results: This article describes a series of systems biology insights obtained from comparing Caco-2 models cells grown as conventional 2D layer and in a microfluidic chip. When basic electrical parameters of Caco-2 monolayers were evaluated using impedance spectrometry and MTT assays, no differences were noted. On the other hand, the microarray profiling of mRNAs and miRNAs revealed that grows on a microfluidic chip leads to the change in the production of specific miRNA, which regulate a set of genes for cell adhesion molecules (CAMs), and provide for more complete differentiation of Caco-2 monolayer. Moreover, the sets of miRNAs secreted at the apical surface of Caco-2 monolayers grown in conventional 2D culture and in microfluidic device differ.

Conclusions: When integrated into a multi-tissue platform, Caco-2 cells may aid in generating insights into complex pathophysiological processes, not possible to dissect in conventional cultures.
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http://dx.doi.org/10.1186/s12918-019-0686-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6399809PMC
March 2019

Specific refolding pathway of viscumin A chain in membrane-like medium reveals a possible mechanism of toxin entry into cell.

Sci Rep 2019 01 23;9(1):413. Epub 2019 Jan 23.

M.M. Shemyakin & Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya Street, 16/10, Moscow, 117997, Russia.

How is a water-soluble globular protein able to spontaneously cross a cellular membrane? It is commonly accepted that it undergoes significant structural rearrangements on the lipid-water interface, thus acquiring membrane binding and penetration ability. In this study molecular dynamics (MD) simulations have been used to explore large-scale conformational changes of the globular viscumin A chain in a complex environment - comprising urea and chloroform/methanol (CHCl/MeOH) mixture. Being well-packed in aqueous solution, viscumin A undergoes global structural rearrangements in both organic media. In urea, the protein is "swelling" and gradually loses its long-distance contacts, thus resembling the "molten globule" state. In CHCl/MeOH, viscumin A is in effect turned "inside out". This is accompanied with strengthening of the secondary structure and surface exposure of hydrophobic epitopes originally buried inside the globule. Resulting solvent-adapted models were further subjected to Monte Carlo simulations with an implicit hydrophobic slab membrane. In contrast to only a few point surface contacts in water and two short regions with weak protein-lipid interactions in urea, MD-derived structures in CHCl/MeOH reveal multiple determinants of membrane interaction. Consequently it is now possible to propose a specific pathway for the structural adaptation of viscumin A with respect to the cell membrane - a probable first step of its translocation into cytoplasmic targets.
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http://dx.doi.org/10.1038/s41598-018-36310-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6344525PMC
January 2019

Proteolytically released Lasso/teneurin-2 induces axonal attraction by interacting with latrophilin-1 on axonal growth cones.

Elife 2018 11 20;7. Epub 2018 Nov 20.

School of Pharmacy, University of Kent, Chatham, United Kingdom.

A presynaptic adhesion G-protein-coupled receptor, latrophilin-1, and a postsynaptic transmembrane protein, Lasso/teneurin-2, are implicated in trans-synaptic interaction that contributes to synapse formation. Surprisingly, during neuronal development, a substantial proportion of Lasso is released into the intercellular space by regulated proteolysis, potentially precluding its function in synaptogenesis. We found that released Lasso binds to cell-surface latrophilin-1 on axonal growth cones. Using microfluidic devices to create stable gradients of soluble Lasso, we show that it induces axonal attraction, without increasing neurite outgrowth. Using latrophilin-1 knockout in mice, we demonstrate that latrophilin-1 is required for this effect. After binding latrophilin-1, Lasso causes downstream signaling, which leads to an increase in cytosolic calcium and enhanced exocytosis, processes that are known to mediate growth cone steering. These findings reveal a novel mechanism of axonal pathfinding, whereby latrophilin-1 and Lasso mediate both short-range interaction that supports synaptogenesis, and long-range signaling that induces axonal attraction.
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http://dx.doi.org/10.7554/eLife.37935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6245728PMC
November 2018

Cell-Free Circulating Nucleic Acids as Early Biomarkers for NAFLD and NAFLD-Associated Disorders.

Front Physiol 2018 20;9:1256. Epub 2018 Sep 20.

Department of Cell Biology, Higher School of Economics, Moscow, Russia.

Non-alcoholic fatty liver disease (NAFLD) is the worldwide most common cause of chronic liver pathology, which prevalence strongly correlates with the increasing incidence of diabetes, obesity and metabolic syndrome in the general population. Simple steatosis, the earliest NAFLD stage, usually remains asymptomatic, and appropriate changes in the lifestyle, as well as the diet, can reverse the affected liver into the healthy state. The potential of simple steatosis to progress into severe fibrotic stages and to facilitate carcinogenesis necessitates timely NAFLD detection and risk stratification in community-based healthcare settings. Since their initial discovery a decade ago, extracellular circulating miRNAs have been found in all human biological fluids including blood and shown to hold great promises as non-invasive biomarkers. Normally, intracellular miRNAs participate in the regulation of gene expression, but once released by dying/dead cells they remain highly stable in the extracellular environment for prolonged periods. Therefore, circulating miRNA profiles can reflect the ongoing pathogenic processes in body's tissues and organs, and enable highly sensitive non-invasive diagnosis of multiple disorders. A non-urgent character of the NAFLD-related decision-making justifies the use of chronic liver diseases as an excellent test case for examining the practical utility of circulating miRNAs as biomarkers for longitudinal monitoring of human health. In this review, we summarize the state-of-the-art in the field of early diagnosis of NAFLD using circulating blood miRNAs, and stress the necessity of additional experimental validation of their diagnostic potential. We further emphasize on the potential diagnostics promises of other cell-free RNA species found in human biological fluids.
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http://dx.doi.org/10.3389/fphys.2018.01256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158334PMC
September 2018

In vitro and in silico liver models: Current trends, challenges and opportunities.

ALTEX 2018 28;35(3):397-412. Epub 2018 May 28.

Higher School of Economics, Moscow, Russia.

Most common drug development failures originate from either bioavailability problems, or unexpected toxic effects. The culprit is often the liver, which is responsible for biotransformation of a majority of xenobiotics. Liver may be modeled using "liver on a chip" devices, which may include established cell lines, primary human cells, and stem cell-derived hepatocyte-like cells. The choice of biological material along with its processing and maintenance greatly influence both the device performance and the resultant toxicity predictions. Impediments to the development of "liver on a chip" technology include the problems with standardization of cells, limitations imposed by culturing and the necessity to develop more complicated fluidic contours. Fortunately, recent breakthroughs in the development of cell-based reporters, including ones with fluorescent label, permits monitoring of the behavior of the cells embed into the "liver on a chip" devices. Finally, a set of computational approaches has been developed to model both particular toxic response and the homeostasis of human liver as a whole; these approaches pave a way to enhance the in silico stage of assessment for a potential toxicity.
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http://dx.doi.org/10.14573/altex.1803221DOI Listing
November 2018

Which cytochrome P450 metabolizes phenazepam? Step by step in silico, in vitro, and in vivo studies.

Drug Metab Pers Ther 2018 06;33(2):65-73

Russian Medical Academy of Continuous Professional Education, Moscow, Russian Federation.

Background: Phenazepam (bromdihydrochlorphenylbenzodiazepine) is the original Russian benzodiazepine tranquilizer belonging to 1,4-benzodiazepines. There is still limited knowledge about phenazepam's metabolic liver pathways and other pharmacokinetic features.

Methods: To determine phenazepam's metabolic pathways, the study was divided into three stages: in silico modeling, in vitro experiment (cell culture study), and in vivo confirmation. In silico modeling was performed on the specialized software PASS and GUSAR to evaluate phenazepam molecule affinity to different cytochromes. The in vitro study was performed using a hepatocytes' cell culture, cultivated in a microbioreactor to produce cytochrome P450 isoenzymes. The culture medium contained specific cytochrome P450 isoforms inhibitors and substrates (for CYP2C9, CYP3A4, CYP2C19, and CYP2B6) to determine the cytochrome that was responsible for phenazepam's metabolism. We also measured CYP3A activity using the 6-betahydroxycortisol/cortisol ratio in patients.

Results: According to in silico and in vitro analysis results, the most probable metabolizer of phenazepam is CYP3A4. By the in vivo study results, CYP3A activity decreased sufficiently (from 3.8 [95% CI: 2.94-4.65] to 2.79 [95% CI: 2.02-3.55], p=0.017) between the start and finish of treatment in patients who were prescribed just phenazepam.

Conclusions: Experimental in silico and in vivo studies confirmed that the original Russian benzodiazepine phenazepam was the substrate of CYP3A4 isoenzyme.
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http://dx.doi.org/10.1515/dmpt-2017-0036DOI Listing
June 2018

Cumulative prognostic power of laminin genes in colorectal cancer.

BMC Med Genomics 2018 02 13;11(Suppl 1). Epub 2018 Feb 13.

SRC Bioclinicum, Ugreshskaya str 2/85, 115088, Moscow, Russia.

Background: Laminins are a major family of extracellular matrix proteins and the main component of basement membranes. Laminins are involved in many if not all stages of cancer progression, and expression of laminin genes has prognostic value in various types of cancer, including colorectal. Only single laminin genes or components of a single laminin trimer with significant differential expression have been regarded as potential biomarkers to date.

Results: Here we compared prognostic power of classifiers constructed from sets of laminin genes with that of any single laminin gene. The analysis showed that cumulative prognostic power of sets of laminin genes was higher and was achieved already with pairs and triples of the genes. Interestingly, components of the pairs and the triples did not belong to any known laminin trimer, but, taken together with the gene weights, suggested higher LAMA4/LAMA5 expression ratio in patients with poor prognosis.

Conclusions: Analysis of the laminin expression profile rather than expression of the single genes or components of laminin trimers is useful for colorectal cancer prognosis in patients. High LAMA4/LAMA5 ratio is associated with increased permeability of basement membranes suggesting that basement membranes produced by colorectal tumors might be an important hindrance to their own dissemination in patients.
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http://dx.doi.org/10.1186/s12920-018-0332-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5836818PMC
February 2018

Knockdown of L1CAM significantly reduces metastasis in a xenograft model of human melanoma: L1CAM is a potential target for anti-melanoma therapy.

PLoS One 2018 12;13(2):e0192525. Epub 2018 Feb 12.

Institute of Anatomy and Experimental Morphology, University Cancer Center, University Medical-Center Hamburg-Eppendorf, Hamburg, Germany.

Finding additional functional targets for combination therapy could improve the outcome for melanoma patients. In a spontaneous metastasis xenograft model of human melanoma a shRNA mediated knockdown of L1CAM more than sevenfold reduced the number of lung metastases after the induction of subcutaneous tumors for two human melanoma cell lines (MeWo, MV3). Whole genome expression arrays of the initially L1CAM high MeWo subcutaneous tumors revealed unchanged or downregulated genes involved in epithelial to mesenchymal transition (EMT) except an upregulation of Jagged 1, indicating a compensatory change in Notch signaling especially as Jagged 1 expression showed an increase in MeWo L1CAM metastases and Jagged 1 was expressed in metastases of the initially L1CAM low MV3 cells as well. Expression of 17 genes showed concordant regulation for L1CAM knockdown tumors of both cell lines. The changes in gene expression indicated changes in the EMT network of the melanoma cells and an increase in p53/p21 and p38 activity contributing to the reduced metastatic potential of the L1CAM knockdowns. Taken together, these data make L1CAM a highly interesting therapeutic target to prevent further metastatic spread in melanoma patients.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0192525PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5809060PMC
April 2018

Comprehensive network of miRNA-induced intergenic interactions and a biological role of its core in cancer.

Sci Rep 2018 02 5;8(1):2418. Epub 2018 Feb 5.

SRC Bioclinicum, Ugreshskaya str. 2/85, 115088, Moscow, Russia.

MicroRNAs (miRNAs) are a family of short noncoding RNAs that posttranscriptionally regulate gene expression and play an important role in multiple cellular processes. A significant percentage of miRNAs are intragenic, which is often functionally related to their host genes playing either antagonistic or synergistic roles. In this study, we constructed and analyzed the entire network of intergenic interactions induced by intragenic miRNAs. We further focused on the core of this network, which was defined as a union of nontrivial strongly connected components, i.e., sets of nodes (genes) mutually connected via directed paths. Both the entire network and its core possessed statistically significant non-random properties. Specifically, genes forming the core had high expression levels and low expression variance. Furthermore, the network core did not split into separate components corresponding to individual signalling or metabolic pathways, but integrated genes involved in key cellular processes, including DNA replication, transcription, protein homeostasis and cell metabolism. We suggest that the network core, consisting of genes mutually regulated by their intragenic miRNAs, could coordinate adjacent pathways or homeostatic control circuits, serving as a horizontal inter-circuit link. Notably, expression patterns of these genes had an efficient prognostic potential for breast and colorectal cancer patients.
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http://dx.doi.org/10.1038/s41598-018-20215-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5799291PMC
February 2018

Selectin-independent adhesion during ovarian cancer metastasis.

Biochimie 2017 Nov 14;142:197-206. Epub 2017 Sep 14.

P. Herzen Moscow Oncology Research Institute, Moscow, 125284, Russia. Electronic address:

Purpose: Ovarian cancer (OvCa) progression mainly takes place by intraperitoneal spread. Adhesion of tumor cells to the mesothelial cells which form the inner surface of the peritoneum is a crucial step in this process. Cancer cells use in principle different molecules of the leukocyte adhesion cascade to facilitate adhesion. This cascade is initiated by selectin-ligand interactions followed by integrin - extracellular matrix protein interactions. Here we address the question whether all tumor cells predominantly employ selectin-dependent leukocyte-like adhesion cascade (SDAC) or whether they use integrin mediated adhesion for OvCa progression as well.

Methods: A comparative transcriptomic analysis of the human OvCa cell lines OVCAR8 and SKOV3 was performed. Intraperitoneal xenograft model of OVCAR8 cells was used to determine whether there is a correlation between SDAC gene expression and the metastatic potential of the control cells and the cells overexpressing c-Fos. Transcriptomic analysis of OVCAR8 and SKOV3 samples was performed using microarrays.

Results: One-third of the protein-coding genes involved in SDAC exhibited lower expression levels in OVCAR8 than in SKOV3 cells. In contrast to SKOV3 cells, c-Fos overexpression in OVCAR8 cells did not significantly influence the expression of SDAC genes. Intraperitoneal xenograft model of OVCAR8 cells unexpectedly demonstrated that the aggressiveness of OVCAR8 tumors was not depended on the c-Fos expression level and was comparable to that of SKOV3 control tumors. Gene expression analysis of tumors suggests that SKOV3-derived tumor progression was mainly depended on SDAC. Progression of OVCAR8 tumors relied on other cell adhesion molecules that do not interact with selectins.

Conclusions: High expression of c-Fos in ovarian cancer cells is not always associated with reduced metastatic potential. Low expression level of SDAC genes may not ensure low OvCa metastatic potential hence alternative adhesion mechanisms involving laminin-integrin interactions exist as well.
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http://dx.doi.org/10.1016/j.biochi.2017.09.009DOI Listing
November 2017

The Transcriptome of Type I Murine Astrocytes under Interferon-Gamma Exposure and Remyelination Stimulus.

Molecules 2017 May 15;22(5). Epub 2017 May 15.

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia.

Astrocytes are considered to be an important contributor to central nervous system (CNS) disorders, particularly multiple sclerosis. The transcriptome of these cells is greatly affected by cytokines released by lymphocytes, penetrating the blood-brain barrier-in particular, the classical pro-inflammatory cytokine interferon-gamma (IFNγ). We report here the transcriptomal profiling of astrocytes treated using IFNγ and benztropine, a putative remyelinization agent. Our findings indicate that the expression of genes involved in antigen processing and presentation in astrocytes are significantly upregulated upon IFNγ exposure, emphasizing the critical role of this cytokine in the redirection of immune response towards self-antigens. Data reported herein support previous observations that the IFNγ-induced JAK-STAT signaling pathway may be regarded as a valuable target for pharmaceutical interventions.
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http://dx.doi.org/10.3390/molecules22050808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6153759PMC
May 2017
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