Publications by authors named "Svetlana Lebedeva"

21 Publications

  • Page 1 of 1

The First Female Dry Immersion (NAIAD-2020): Design and Specifics of a 3-Day Study.

Front Physiol 2021 14;12:661959. Epub 2021 Jun 14.

Russian Federation State Scientific Center - Institute of Biomedical Problems of the Russian Academy of Sciences, Moscow, Russia.

This article describes procedures and some results of the first study of females undergoing 3-day Dry Immersion. The experiment "NAIAD-2020" was carried out at the Institute of Biomedical Problems (Moscow, Russia) with the participation of six healthy women volunteers (age 30.17 ± 5.5 years, height 1.66 ± 0.1 m, weight 62.05 ± 8.4 kg, BMI 22.39 ± 2.2 kg/m) with a natural menstrual cycle. During the study, a standard protocol was used, the same as for men, with a minimum period of time spent outside the immersion bath. Before, during and after Immersion, 22 experiments were carried out aimed at studying the neurophysiological, functional, metabolic and psychophysiological functions of the body, the results of which will be presented in future publications. The total time outside the bath for women did not exceed that for men. Systolic and diastolic pressure did not significantly change during the immersion. In the first 24 h after the end of the immersion, heart rate was significantly higher than the background values [(4,20) = 14.67; < 0.0001]. Changes in body temperature and water balance were consistent with the patterns found in men. No significant changes in height and weight were found during immersion. All women reported general discomfort and pain in the abdomen and back. The results of this study did not find significant risks to women's health and showed the feasibility of using this model of the effects of space flight in women of reproductive age.
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http://dx.doi.org/10.3389/fphys.2021.661959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8236811PMC
June 2021

Cytogenetic and molecular genetic methods for chromosomal translocations detection with reference to the gene.

Crit Rev Clin Lab Sci 2021 May 18;58(3):180-206. Epub 2020 Nov 18.

Department of Molecular Biology, Faculty of Biology, M.V. Lomonosov Moscow State University, Moscow, Russia.

Acute leukemias (ALs) are often associated with chromosomal translocations, in particular, gene rearrangements. Identification or confirmation of these translocations is carried out by a number of genetic and molecular methods, some of which are routinely used in clinical practice, while others are primarily used for research purposes. In the clinic, these methods serve to clarify diagnoses and monitor the course of disease and therapy. On the other hand, the identification of new translocations and the confirmation of known translocations are of key importance in the study of disease mechanisms and further molecular classification. There are multiple methods for the detection of rearrangements that differ in their principle of operation, the type of problem being solved, and the cost-result ratio. This review is intended to help researchers and clinicians studying AL and related chromosomal translocations to navigate this variety of methods. All methods considered in the review are grouped by their principle of action and include karyotyping, fluorescence in situ hybridization (FISH) with probes for whole chromosomes or individual loci, PCR and reverse transcription-based methods, and high-throughput sequencing. Another characteristic of the described methods is the type of problem being solved. This can be the discovery of new rearrangements, the determination of unknown partner genes participating in the rearrangement, or the confirmation of the proposed rearrangement between the two genes. We consider the specifics of the application, the basic principle of each method, and its pros and cons. To illustrate the application, examples of studying the rearrangements of the gene, one of the genes that are often rearranged in AL, are mentioned.
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http://dx.doi.org/10.1080/10408363.2020.1844135DOI Listing
May 2021

Quantification of NG2-positivity for the precise prediction of KMT2A gene rearrangements in childhood acute leukemia.

Genes Chromosomes Cancer 2021 Feb 20;60(2):88-99. Epub 2020 Nov 20.

Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russia.

It has long been known that there is a link between neuron glial antigen 2 (NG2) surface expression and KMT2A gene rearrangements in acute leukemia (AL). However, the exact levels of NG2 positivity that predict the presence of KMT2A rearrangement are not known. The current study focuses on a cohort of 505 pediatric AL patients who showed any level of positive NG2 expression (greater than 1% of cells) for whom comprehensive genetic data were available. NG2 expression was measured as either the percentage of positive cells or the number of molecules on the cell surface. KMT2A gene rearrangements were identified by FISH. The fusion partner was detected with RT-PCR, LDI-PCR or anchored multiplex PCR followed by high-throughput sequencing. KMT2A-positive samples comprised a substantial proportion of the NG2-positive cohort (180 of 505, 36%), with a total of 19 different types of translocation. Despite its occurrence in other AL genetic subgroups, NG2 expression was significantly increased in AL patients with KMT2A rearrangements in terms of both the cell percentage and number of molecules per cell. The threshold levels (TL) for NG2-positivity were established by ROC analysis of the whole cohort and separately for children less than 1 years old and older with lymphoblastic (ALL) and myeloid (AML) leukemia. The lowest TL was defined in infants with ALL (7%), while in older children, the threshold was higher (12%). In AML patients, the situation was reversed, with 28% NG2-positivity in infants and 14% in patients >1 year old. The defined TLs resulted in improved diagnostic performance compared to the conventional thresholds of 10% and 20% for all patient groups.
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http://dx.doi.org/10.1002/gcc.22915DOI Listing
February 2021

Quantification of Scheduling Impact on Safety and Efficacy Outcomes of Brain Metastasis Radio- and Immuno-Therapies: A Systematic Review and Meta-Analysis.

Front Oncol 2020 2;10:1609. Epub 2020 Sep 2.

M&S Decisions LLC, Moscow, Russia.

The goal of this quantitative research was to evaluate the impact of various factors (e.g., scheduling or radiotherapy (RT) type) on outcomes for RT vs. RT in combination with immune checkpoint inhibitors (ICI), in the treatment of brain metastases, via a meta-analysis. Clinical studies with at least one ICI+RT treatment combination arm with brain metastasis patients were identified via a systematic literature search. Data on 1-year overall survival (OS), 1-year local control (LC) and radionecrosis rate (RNR) were extracted; for combination studies which included an RT monotherapy arm, odds ratios (OR) for the aforementioned endpoints were additionally calculated and analyzed. Mixed-effects meta-analysis models were tested to evaluate impact on outcome, for different factors such as combination treatment scheduling and the type of ICI or RT used. 40 studies representing a total of 4,359 patients were identified. Higher 1-year OS was observed in ICI and RT combination vs. RT alone, with corresponding incidence rates of 59% [95% CI: 54-63%] vs. 32% [95% CI: 25-39%] ( < 0.001). Concurrent ICI and RT treatment was associated with significantly higher 1-year OS vs. sequential combinations: 68% [95% CI: 60-75%] vs. 54% [95% CI: 47-61%]. No statistically significant differences were observed in 1-year LC and RNR, when comparing combinations vs. RT monotherapies, with 1-year LC rates of 68% [95% CI: 40-90%] vs. 72% [95% CI: 63-80%] ( = 0.73) and RNR rates of 6% [95% CI: 2-13%] vs. 9% [95% CI: 5-14%] ( = 0.37). A comprehensive, study-level meta-analysis of brain metastasis disease treatments suggest that combinations of RT and ICI result in higher OS, yet comparable neurotoxicity profiles vs. RT alone, with a superiority of concurrent vs. sequential combination regimens. A similar meta-analysis using patient-level data from past trials, as well as future prospective randomized trials would help confirming these findings.
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http://dx.doi.org/10.3389/fonc.2020.01609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7492564PMC
September 2020

Vibrational spectroscopy and density of KO-BO-GeO glasses with variable B/Ge ratio.

Phys Chem Chem Phys 2019 Jun 4;21(23):12676-12684. Epub 2019 Jun 4.

Institute of Mineralogy SU FRC MG UB RAS, Miass 456317, Russia.

Glasses of the KO-BO-GeO system were studied by means of Raman and IR spectroscopy. The density of the samples was measured and the dependence of the molar volume and atomic density on composition was calculated. Curve-fitting of Raman spectra was applied to obtain a definition of the main structural units formed in the system. The conditions for highly-coordinated boron and germanium atoms were obtained. It was shown that potassium cations remain connected to germanate structural units at a B/Ge ratio of up to 1, whereas the explicit redistribution of borate and germanate structural groupings becomes most noticeable only at a B/Ge ratio > 2.
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http://dx.doi.org/10.1039/c9cp01374aDOI Listing
June 2019

Assessment of Psychosocial Functioning of Mothers of Children with Diabetes Mellitus Compared to Mothers of Healthy Children.

Biomed Res Int 2019 9;2019:6821575. Epub 2019 Apr 9.

Sechenov First Moscow State Medical University (Sechenov University), 119991, Moscow, Russia.

Diabetes mellitus (DM) is a chronic disease requiring changes in the behaviour of the entire family. The responsibility for implementing doctor's recommendations falls mainly upon the mother. The aim of this study is to assess the psychosocial functioning of mothers of children with DM compared to mothers of healthy children. The study involved 120 mothers: 60 with children with DM and 60 with healthy children. Data were collected using an original social-demographic questionnaire developed by the authors as well as Antonovsky's Sense of Coherence Scale (SOC-29), Schwarzer and Schultz's Berlin Social Support Scales (BSSS), Rosenberg's Self-Esteem Scale (SES), and Zigmond and Snaith's Hospital Anxiety and Depression Scale (HADS). The assessment scales were standardised and accredited by the Polish Psychological Association. The results suggest that DM in children has no effect on the psychosocial functioning of mothers regarding their self-esteem and sense of coherence. However, mothers of children with DM are well-prepared for living in a difficult situation. Social support offered to mothers of diabetic children helps them to maintain their psychosocial health.
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http://dx.doi.org/10.1155/2019/6821575DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6481141PMC
August 2019

Application of Acyzol in the Context of Zinc Deficiency and Perspectives.

Int J Mol Sci 2019 Apr 29;20(9). Epub 2019 Apr 29.

Department of Pharmacology and Pharmacy, Sechenov First Moscow State Medical University (Sechenov University), 8-2 Trubetskaya St., Moscow 119991, Russia.

Zinc is one of the most important essential trace elements. It is involved in more than 300 enzyme systems and is an indispensable participant in many biochemical processes. Zinc deficiency causes a number of disorders in the human body, the main ones being the delay of growth and puberty, immune disorders, and cognitive dysfunctions. There are over two billion people in the world suffering from zinc deficiency conditions. Acyzol, a zinc-containing medicine, developed as an antidote against carbon monoxide poisoning, demonstrates a wide range of pharmacological activities: Anti-inflammatory, reparative, detoxifying, immunomodulatory, bacteriostatic, hepatoprotective, adaptogenic, antioxidant, antihypoxic, and cardioprotective. The presence of zinc in the composition of Acyzol suggests the potential of the drug in the treatment and prevention of zinc deficiency conditions, such as Prasad's disease, immune system pathology, alopecia, allergodermatoses, prostate dysfunction, psoriasis, stomatitis, periodontitis, and delayed mental and physical development in children. Currently, the efficiency of Acyzol in the cases of zinc deficiency is shown in a large number of experimental studies. So, Acyzol can be used as a highly effective drug for pharmacologic therapy of a wide range of diseases and conditions and it opens up new perspectives in the treatment and prevention of zinc deficiency conditions.
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http://dx.doi.org/10.3390/ijms20092104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6539662PMC
April 2019

Global identification of functional microRNA-mRNA interactions in Drosophila.

Nat Commun 2019 04 9;10(1):1626. Epub 2019 Apr 9.

Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine, 13125, Berlin, Germany.

MicroRNAs (miRNAs) are key mediators of post-transcriptional gene expression silencing. So far, no comprehensive experimental annotation of functional miRNA target sites exists in Drosophila. Here, we generated a transcriptome-wide in vivo map of miRNA-mRNA interactions in Drosophila melanogaster, making use of single nucleotide resolution in Argonaute1 (AGO1) crosslinking and immunoprecipitation (CLIP) data. Absolute quantification of cellular miRNA levels presents the miRNA pool in Drosophila cell lines to be more diverse than previously reported. Benchmarking two CLIP approaches, we identify a similar predictive potential to unambiguously assign thousands of miRNA-mRNA pairs from AGO1 interaction data at unprecedented depth, achieving higher signal-to-noise ratios than with computational methods alone. Quantitative RNA-seq and sub-codon resolution ribosomal footprinting data upon AGO1 depletion enabled the determination of miRNA-mediated effects on target expression and translation. We thus provide the first comprehensive resource of miRNA target sites and their quantitative functional impact in Drosophila.
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http://dx.doi.org/10.1038/s41467-019-09586-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6456604PMC
April 2019

Acute myeloid leukemia with t(10;11)(p11-12;q23.3): Results of Russian Pediatric AML registration study.

Int J Lab Hematol 2019 Apr 9;41(2):287-292. Epub 2019 Jan 9.

Dmitry Rogachev National Medical and Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russia.

Introduction: Translocations involving the KMT2A gene (also known as MLL) are frequently diagnosed in pediatric acute leukemia cases with either lymphoblastic or myeloid origin. KMT2A is translocated to multiple partner genes, including MLLT10/AF10 localizing at chromosomal band 10p12. KMT2A-MLLT10 is one of the common chimeric genes diagnosed in acute leukemia with KMT2A rearrangement (8%), especially in acute myeloid leukemia (AML; 18%). MLLT10 is localized in very close proximity to two other KMT2A partner genes at 10p11-12-NEBL and ABI1, so they could not be distinguished by conventional cytogenetics.

Methods: In this work, we present a cohort of 28 patients enrolled into Russian Pediatric AML registration study carrying rearrangements between chromosomal regions 11q23.3 and 10p11-12. G-banding, FISH, reverse transcription PCR, and long-distance inverse PCR were used to characterize the KMT2A gene rearrangements in these patients.

Results: We demonstrate that 25 patients harbor the KMT2A-MLLT10 rearrangement, while three patients show the rare KMT2A rearrangements (2× KMT2A-NEBL; 1× KMT2A-ABI1).

Conclusions: Therefore, the combination of cytogenetic and molecular genetic methods is of high importance in diagnosing cases with t(10;11)(p11-12;q23.3).
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http://dx.doi.org/10.1111/ijlh.12969DOI Listing
April 2019

Deciphering human ribonucleoprotein regulatory networks.

Nucleic Acids Res 2019 01;47(2):570-581

Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine, Berlin, Germany.

RNA-binding proteins (RBPs) control and coordinate each stage in the life cycle of RNAs. Although in vivo binding sites of RBPs can now be determined genome-wide, most studies typically focused on individual RBPs. Here, we examined a large compendium of 114 high-quality transcriptome-wide in vivo RBP-RNA cross-linking interaction datasets generated by the same protocol in the same cell line and representing 64 distinct RBPs. Comparative analysis of categories of target RNA binding preference, sequence preference, and transcript region specificity was performed, and identified potential posttranscriptional regulatory modules, i.e. specific combinations of RBPs that bind to specific sets of RNAs and targeted regions. These regulatory modules represented functionally related proteins and exhibited distinct differences in RNA metabolism, expression variance, as well as subcellular localization. This integrative investigation of experimental RBP-RNA interaction evidence and RBP regulatory function in a human cell line will be a valuable resource for understanding the complexity of post-transcriptional regulation.
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http://dx.doi.org/10.1093/nar/gky1185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6344852PMC
January 2019

A case of pediatric acute myeloid leukemia with t(11;16)(q23;q24) leading to a novel KMT2A-USP10 fusion gene.

Genes Chromosomes Cancer 2018 10 14;57(10):522-524. Epub 2018 Aug 14.

Laboratory of Cytogenetics and Molecular Genetics, Dmitry Rogachev Research and Clinical Center for Pediatric Hematology, Oncology and Immunology, Moscow, Russia.

We present a leukemia case that exhibits a chromosomal translocation t(11;16)(q23;q23), which results in the expression of a novel KMT2A fusion gene. This novel fusion, KMT2A-USP10, was found in a relapse of acute myeloid leukaemia M5a. USP10 belongs to a protein family that deubiquitinates a distinct set of target proteins, and thus, increases the steady state protein levels of its target subproteome. One of the USP10 targets is TP53. Wildtype TP53 is usually rescued from proteasomal degradation by USP10. As most KMT2A leukemias display wildtype p53 alleles, one might argue that the disruption of an USP10 allele can be classified as a pro-oncogenic event.
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http://dx.doi.org/10.1002/gcc.22646DOI Listing
October 2018

Humoral and cellular immune responses to Yersinia pestis Pla antigen in humans immunized with live plague vaccine.

PLoS Negl Trop Dis 2018 06 11;12(6):e0006511. Epub 2018 Jun 11.

Department of Pathology, Department of Microbiology & Immunology, University of Texas Medical Branch, Galveston, Texas, United States of America.

Background: To establish correlates of human immunity to the live plague vaccine (LPV), we analyzed parameters of cellular and antibody response to the plasminogen activator Pla of Y. pestis. This outer membrane protease is an essential virulence factor that is steadily expressed by Y. pestis.

Methodology/principal Findings: PBMCs and sera were obtained from a cohort of naïve (n = 17) and LPV-vaccinated (n = 34) donors. Anti-Pla antibodies of different classes and IgG subclasses were determined by ELISA and immunoblotting. The analysis of antibody response was complicated with a strong reactivity of Pla with normal human sera. The linear Pla B-cell epitopes were mapped using a library of 15-mer overlapping peptides. Twelve peptides that reacted specifically with sera of vaccinated donors were found together with a major cross-reacting peptide IPNISPDSFTVAAST located at the N-terminus. PBMCs were stimulated with recombinant Pla followed by proliferative analysis and cytokine profiling. The T-cell recall response was pronounced in vaccinees less than a year post-immunization, and became Th17-polarized over time after many rounds of vaccination.

Conclusions/significance: The Pla protein can serve as a biomarker of successful vaccination with LPV. The diagnostic use of Pla will require elimination of cross-reactive parts of the antigen.
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http://dx.doi.org/10.1371/journal.pntd.0006511DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5995359PMC
June 2018

Successful retrograde recanalization of acute right dominant vertebral artery occlusion through the left posterior communicating artery in a patient with acute vertebrobasilar ischemic stroke.

Radiol Case Rep 2018 Apr 2;13(2):475-478. Epub 2018 Mar 2.

Regional cardiovascular center, City Hospital №40, Borisova str. 9, Sestroretsk, Saint-Petersburg, 197706, Russian Federation.

Advanced endovascular technology and techniques allow interventional radiologists to utilize novel ways of basilar artery recanalization in the setting of acute ischemic stroke, especially when routine approaches are not eligible. Several authors described nonstandard revascularization techniques in acute ischemic strokes due to basilar and middle cerebral arteries occlusions with full technical and clinical success. In this report, we present retrograde right vertebral artery recanalization using left posterior communicating artery for subsequent anterograde balloon angioplasty and stenting of a right vertebral artery ostium followed by full vertebrobasilar blood flow restoration. The case underscores the complexity of arterial thrombotic events, the beneficial role of endovascular intervention in vertebral occlusions and the necessity of prospective studies that identify optimal methods of treating vertebrobasilar stroke due to large vessel occlusions and their effectiveness and safety.
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http://dx.doi.org/10.1016/j.radcr.2018.02.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5906862PMC
April 2018

Characterization of genetic loss-of-function of Fus in zebrafish.

RNA Biol 2017 01 29;14(1):29-35. Epub 2016 Nov 29.

a Institute of Molecular Biology , Mainz , Germany.

The RNA-binding protein FUS is implicated in transcription, alternative splicing of neuronal genes and DNA repair. Mutations in FUS have been linked to human neurodegenerative diseases such as ALS (amyotrophic lateral sclerosis). We genetically disrupted fus in zebrafish (Danio rerio) using the CRISPR-Cas9 system. The fus knockout animals are fertile and did not show any distinctive phenotype. Mutation of fus induces mild changes in gene expression on the transcriptome and proteome level in the adult brain. We observed a significant influence of genetic background on gene expression and 3'UTR usage, which could mask the effects of loss of Fus. Unlike published fus morphants, maternal zygotic fus mutants do not show motoneuronal degeneration and exhibit normal locomotor activity.
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http://dx.doi.org/10.1080/15476286.2016.1256532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5270537PMC
January 2017

Profiling of RNA modifications by multiplexed stable isotope labelling.

Chem Commun (Camb) 2014 Apr 24;50(26):3516-8. Epub 2014 Feb 24.

Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, 5 Staudinger Weg, Mainz, D-55128, Germany.

The combination of (15)N/(13)C stable isotope labelling (SIL) and LC-MS/MS revealed a total of 52 modifications in RNA from E. coli and yeast, including 10 previously undescribed modifications. Two modifications, N-ribosylnicotinamide and 2-methylthioadenosine, were newly detected in species hitherto thought not to contain these modifications.
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http://dx.doi.org/10.1039/c3cc49114eDOI Listing
April 2014

Select microRNAs are essential for early development in the sea urchin.

Dev Biol 2012 Feb 3;362(1):104-13. Epub 2011 Dec 3.

Department of Molecular Biology, Cellular Biology and Biochemistry, Brown University, Providence, RI 02912, USA.

microRNAs (miRNAs) are small noncoding RNAs that mediate post-transcriptional gene regulation and have emerged as essential regulators of many developmental events. The transcriptional network during early embryogenesis of the purple sea urchin, Strongylocentrotus purpuratus, is well described and can serve as an excellent model to test functional contributions of miRNAs in embryogenesis. We examined the loss of function phenotypes of major components of the miRNA biogenesis pathway. Inhibition of de novo synthesis of Drosha and Dicer in the embryo led to consistent developmental defects, a failure to gastrulate, and embryonic lethality, including changes in the steady state levels of transcription factors and signaling molecules involved in germ layer specification. We annotated and profiled small RNA expression from the ovary and several early embryonic stages by deep sequencing followed by computational analysis. miRNAs as well as a large population of putative piRNAs (piwi-interacting RNAs) had dynamic accumulation profiles through early development. Defects in morphogenesis caused by loss of Drosha could be rescued with four miRNAs. Taken together our results indicate that post-transcriptional gene regulation directed by miRNAs is functionally important for early embryogenesis and is an integral part of the early embryonic gene regulatory network in S. purpuratus.
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http://dx.doi.org/10.1016/j.ydbio.2011.11.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3254792PMC
February 2012

Transcriptome-wide analysis of regulatory interactions of the RNA-binding protein HuR.

Mol Cell 2011 Aug 30;43(3):340-52. Epub 2011 Jun 30.

Laboratory of Systems Biology of Gene Regulatory Elements, Max Delbrück Center for Molecular Medicine, Berlin, Germany.

Posttranscriptional gene regulation relies on hundreds of RNA binding proteins (RBPs) but the function of most RBPs is unknown. The human RBP HuR/ELAVL1 is a conserved mRNA stability regulator. We used PAR-CLIP, a recently developed method based on RNA-protein crosslinking, to identify transcriptome-wide ∼26,000 HuR binding sites. These sites were on average highly conserved, enriched for HuR binding motifs and mainly located in 3' untranslated regions. Surprisingly, many sites were intronic, implicating HuR in mRNA processing. Upon HuR knockdown, mRNA levels and protein synthesis of thousands of target genes were downregulated, validating functionality. HuR and miRNA binding sites tended to reside nearby but generally did not overlap. Additionally, HuR knockdown triggered strong and specific upregulation of miR-7. In summary, we identified thousands of direct and functional HuR targets, found a human miRNA controlled by HuR, and propose a role for HuR in splicing.
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http://dx.doi.org/10.1016/j.molcel.2011.06.008DOI Listing
August 2011

High-resolution profiling and discovery of planarian small RNAs.

Proc Natl Acad Sci U S A 2009 Jul 29;106(28):11546-51. Epub 2009 Jun 29.

Max Delbrück Centrum für Molekulare Medizin, Robert-Rössle-Strasse 10, D-13125 Berlin-Buch, Germany.

Freshwater planarian flatworms possess uncanny regenerative capacities mediated by abundant and collectively totipotent adult stem cells. Key functions of these cells during regeneration and tissue homeostasis have been shown to depend on PIWI, a molecule required for Piwi-interacting RNA (piRNA) expression in planarians. Nevertheless, the full complement of piRNAs and microRNAs (miRNAs) in this organism has yet to be defined. Here we report on the large-scale cloning and sequencing of small RNAs from the planarian Schmidtea mediterranea, yielding altogether millions of sequenced, unique small RNAs. We show that piRNAs are in part organized in genomic clusters and that they share characteristic features with mammalian and fly piRNAs. We further identify 61 novel miRNA genes and thus double the number of known planarian miRNAs. Sequencing, as well as quantitative PCR of small RNAs, uncovered 10 miRNAs enriched in planarian stem cells. These miRNAs are down-regulated in animals in which stem cells have been abrogated by irradiation, and thus constitute miRNAs likely associated with specific stem-cell functions. Altogether, we present the first comprehensive small RNA analysis in animals belonging to the third animal superphylum, the Lophotrochozoa, and single out a number of miRNAs that may function in regeneration. Several of these miRNAs are deeply conserved in animals.
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http://dx.doi.org/10.1073/pnas.0905222106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2703670PMC
July 2009

Inhibition of DNA topoisomerase II may trigger illegitimate recombination in living cells: experiments with a model system.

J Cell Biochem 2006 Oct;99(2):598-608

Institute of Gene Biology, Russian Academy of Sciences, Vavilov str. 34/5, 119334 Moscow, Russia.

We have developed a plasmid test system to study recombination in vitro and in mammalian cells in vivo, and to analyze the possible role of DNA topoisomerase II. The system is based on a plasmid construct containing an inducible marker gene ccdB ("killer" (KIL) gene) whose product is lethal for bacterial cells, flanked by two different potentially recombinogenic elements. The plasmids were subjected to recombinogenic conditions in vitro or in vivo after transient transfection into COS-1 cells, and subsequently transformed into E. coli which was then grown in the presence of the ccdB gene inducer. Hence, all viable colonies contained recombinant plasmids since only recombination between the flanking regions could remove the KIL gene. Thus, it was possible to detect recombination events and to estimate their frequency. We found that the frequency of topoisomerase II-mediated recombination in vivo is significantly higher than in a minimal in vitro system. The presence of VM-26, an inhibitor of the religation step of the topoisomerase II reaction, increased the recombination frequency by 60%. We propose that cleavable complexes of topoisomerase II are either not religated, triggering error-prone repair of the DNA breaks, or are incorrectly religated resulting in strand exchange. We also studied the influence of sequences known to contain preferential breakpoints for recombination in vivo after chemotherapy with topoisomerase II-targeting drugs, but no preferential stimulation of recombination by these sequences was detected in this non-chromosomal context.
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http://dx.doi.org/10.1002/jcb.20938DOI Listing
October 2006

Impaired IL-4 and c-Maf expression and enhanced Th1-cell development in Vav1-deficient mice.

Blood 2005 Aug 21;106(4):1286-95. Epub 2005 Apr 21.

Division of Cell Biology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Dr, San Diego, CA 92121, USA.

Although c-Maf is crucial for Th2 differentiation and production of interleukin 4 (IL-4), its regulation is poorly understood. We report that Vav1-/- CD4+ T cells display deficient T-cell receptor (TCR)/CD28-induced IL-4 and c-Maf expression and, conversely, enhanced interferon gamma (IFN-gamma) production and T-bet expression (even when cultured under Th2-polarizing conditions), but intact expression of other Th2 cytokines and GATA-3. Up-regulation of c-Maf was dependent on Ca2+/nuclear factor of activated T cell (NFAT) and, together with IL-4 production, could be rescued in Vav1-/- T cells by Ca2+ ionophore. Deficient IL-4 production was restored by retrovirus-mediated Vav1 expression, but only partially by retroviral c-Maf expression. Similar IL-4 --> IFN-gamma skewing was observed in intact, antigen-primed Vav1-/- mice. Thus, Vav1 is selectively required for IL-4 and c-Maf expression, a requirement reflecting, at least in part, the dependence of c-Maf expression on Ca2+/NFAT signaling.
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http://dx.doi.org/10.1182/blood-2004-10-4074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1895205PMC
August 2005

SWAP-70-like adapter of T cells, an adapter protein that regulates early TCR-initiated signaling in Th2 lineage cells.

Immunity 2003 Mar;18(3):403-14

Division of Cell Biology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121, USA.

We describe the isolation of a protein, SWAP-70-like adapter of T cells (SLAT), which is expressed at high levels in thymocytes and differentiated Th2 cells. SLAT expression was upregulated in differentiating Th2 cells and downregulated in Th1 cells. Ectopic SLAT expression exerted positive or negative effects on IL-4 versus IFNgamma induction, respectively. TCR signaling induced translocation of SLAT to the immunological synapse and its association with ZAP-70 kinase. SLAT reduced the association of ZAP-70 with TCR-zeta and interfered with ZAP-70 but not Lck signaling. Consistent with these results, pharmacological inhibition of ZAP-70 also induced Th2 skewing. Thus, SLAT is a protein which plays a role in Th2 development and/or activation, perhaps by interfering with ZAP-70 signaling.
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http://dx.doi.org/10.1016/s1074-7613(03)00054-2DOI Listing
March 2003
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