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Developing pathogen-specific recombinant antibody fragments (especially nanobodies) is a very promising strategy for the treatment of infectious disease. Nanobodies have great potential for gene therapy application due to their single-gene nature. Historically, Mycoplasma hominis has not been considered pathogenic bacteria due to the lack of acute infection and partially due to multiple studies demonstrating high frequency of isolation of M. hominis samples from asymptomatic patients. However, recent studies on the role of latent M. hominis infection in oncologic transformation, especially prostate cancer, and reports that M. hominis infects Trichomonas and confers antibiotic resistance to Trichomonas, have generated new interest in this field. In the present study we have generated specific nanobody against M. hominis (aMh), for which the identified target is the ABC-transporter substrate-binding protein. aMh exhibits specific antibacterial action against M. hominis. In an attempt to improve the therapeutic properties, we have developed the adenoviral vectorbased gene therapy approach for passive immunization with nanobodies against M. hominis. For better penetration into the mucous layer of the genital tract, we fused aMh with the Fc-fragment of IgG. Application of this comprehensive approach with a single systemic administration of recombinant adenovirus expressing aMh-Fc demonstrated both prophylactic and therapeutic effects in a mouse model of genital M. hominis infection. © 2016 Burmistrova et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Schwartz Y.B.,Umeå University | Schwartz Y.B.,Rutgers University | Linder-Basso D.,Rutgers University | Kharchenko P.V.,Harvard University | And 25 more authors.
Genome Research | Year: 2012

Chromatin insulator elements and associated proteins have been proposed to partition eukaryotic genomes into sets of independently regulated domains. Here we test this hypothesis by quantitative genome-wide analysis of insulator protein binding to Drosophila chromatin. We find distinct combinatorial binding of insulator proteins to different classes of sites and uncover a novel type of insulator element that binds CP190 but not any other known insulator proteins. Functional characterization of different classes of binding sites indicates that only a small fraction act as robust insulators in standard enhancer-blocking assays. We show that insulators restrict the spreading of the H3K27me3 mark but only at a small number of Polycomb target regions and only to prevent repressive histone methylation within adjacent genes that are already transcriptionally inactive. RNAi knockdown of insulator proteins in cultured cells does not lead to major alterations in genome expression. Taken together, these observations argue against the concept of a genome partitioned by specialized boundary elements and suggest that insulators are reserved for specific regulation of selected genes. © 2012, Published by Cold Spring Harbor Laboratory Press.

In the Russian publication by Nesterenko et al. [Biofizika 57(4)], the general results and conclusions have been perverted by uncritically applying an absolutely invalid "unifying formula" proposed in a contemporary monograph from Russia's principal scientific publishing house [ISBN 978-5-02-035593-4]. On behalf of Nesterenko et al., I ask the readers to kindly ignore their "original" paper but take the fully revised English version [Biophysics 57 (4), 464] or contact the authors (tv-nesterenko@mail. ru, ubflab@ibp. ru). Here I briefly show the basic defects in the "novel means" of quantitative data analysis offered in that monograph, and use this deplorable occasion to touch on some more general problems regarding erroneous information in reputedly reputable sources. © 2012 Pleiades Publishing, Ltd.

Slastnikova T.A.,Moscow State University | Rosenkranz A.A.,Moscow State University | Gulak P.V.,Institute of Gene Biology | Schiffelers R.M.,University Utrecht | And 4 more authors.
International Journal of Nanomedicine | Year: 2012

Background: Modular nanotransporters (MNT) are recombinant multifunctional polypeptides created to exploit a cascade of cellular processes, initiated with membrane receptor recognition to deliver selective short-range and highly cytotoxic therapeutics to the cell nucleus. This research was designed for in vivo concept testing for this drug delivery platform using two modular nanotransporters, one targeted to the α-melanocyte-stimulating hormone (αMSH) receptor overexpressed on melanoma cells and the other to the epidermal growth factor (EGF) receptor overexpressed on several cancers, including glioblastoma, and head-and-neck and breast carcinoma cells.Methods: In vivo targeting of the modular nanotransporter was determined by immuno-fuorescence confocal laser scanning microscopy and by accumulation of 125I-labeled modular nanotransporters. The in vivo therapeutic effects of the modular nanotransporters were assessed by photodynamic therapy studies, given that the cytotoxicity of photosensitizers is critically dependent on their delivery to the cell nucleus.Results: Immunohistochemical analyses of tumor and neighboring normal tissues of mice injected with multifunctional nanotransporters demonstrated preferential uptake in tumor tissue, particularly in cell nuclei. With 125I-labeled MNT{αMSH}, optimal tumor:muscle and tumor:skin ratios of 8:1 and 9.8:1, respectively, were observed 3 hours after injection in B16-F1 melanoma-bearing mice. Treatment with bacteriochlorin p-MNT{αMSH} yielded 89%-98% tumor growth inhibition and a two-fold increase in survival for mice with B16-F1 and Cloudman S91 melanomas. Likewise, treatment of A431 human epidermoid carcinoma-bearing mice with chlorin e 6- MNT{EGF} resulted in 94% tumor growth inhibition compared with free chlorin e 6, with 75% of animals surviving at 3 months compared with 0% and 20% for untreated and free chlorin e 6-treated groups, respectively.Conclusion: The multifunctional nanotransporter approach provides a new in vivo functional platform for drug development that could, in principle, be applicable to any combination of cell surface receptor and agent (photosensitizers, oligonucleotides, radionuclides) requiring nuclear delivery to achieve maximum effectiveness. © 2012 Slastnikova et al.

Gavrilov A.,Institute of Gene Biology | Razin S.V.,Moscow State University | Cavalli G.,Institute of Human Genetics
Briefings in Functional Genomics | Year: 2015

Formaldehyde cross-linking is an important component of many technologies, including chromatin immunoprecipitation and chromosome conformation capture. The procedure remains empirical and poorly characterized, however, despite a long history of its use in research. Little is known about the specificity of in vivo cross-linking, its efficiency and chemical adducts induced by the procedure. It is time to search this black box. © The Author 2014. Published by Oxford University Press. All rights reserved.

PubMed | Russian National Research Medical University, Massachusetts Institute of Technology, Skolkovo Institute of Science and Technology, Moscow State University and 2 more.
Type: Journal Article | Journal: Genome research | Year: 2016

Recent advances enabled by the Hi-C technique have unraveled many principles of chromosomal folding that were subsequently linked to disease and gene regulation. In particular, Hi-C revealed that chromosomes of animals are organized into topologically associating domains (TADs), evolutionary conserved compact chromatin domains that influence gene expression. Mechanisms that underlie partitioning of the genome into TADs remain poorly understood. To explore principles of TAD folding in Drosophila melanogaster, we performed Hi-C and poly(A)(+) RNA-seq in four cell lines of various origins (S2, Kc167, DmBG3-c2, and OSC). Contrary to previous studies, we find that regions between TADs (i.e., the inter-TADs and TAD boundaries) in Drosophila are only weakly enriched with the insulator protein dCTCF, while another insulator protein Su(Hw) is preferentially present within TADs. However, Drosophila inter-TADs harbor active chromatin and constitutively transcribed (housekeeping) genes. Accordingly, we find that binding of insulator proteins dCTCF and Su(Hw) predicts TAD boundaries much worse than active chromatin marks do. Interestingly, inter-TADs correspond to decompacted inter-bands of polytene chromosomes, whereas TADs mostly correspond to densely packed bands. Collectively, our results suggest that TADs are condensed chromatin domains depleted in active chromatin marks, separated by regions of active chromatin. We propose the mechanism of TAD self-assembly based on the ability of nucleosomes from inactive chromatin to aggregate, and lack of this ability in acetylated nucleosomal arrays. Finally, we test this hypothesis by polymer simulations and find that TAD partitioning may be explained by different modes of inter-nucleosomal interactions for active and inactive chromatin.

PubMed | Institute of Gene Biology
Type: Journal Article | Journal: EJNMMI research | Year: 2012

This study evaluates the potential utility of a modular nanotransporter (MNT) for enhancing the nuclear delivery and cytotoxicity of the Auger electron emitter 125I in cancer cells that overexpress the epidermal growth factor receptor (EGFR).MNTs are recombinant multifunctional polypeptides that we have developed for achieving selective delivery of short-range therapeutics into cancer cells. MNTs contain functional modules for receptor binding, internalization, endosomal escape and nuclear translocation, thereby facilitating the transport of drugs from the cell surface to the nucleus. The MNT described herein utilized EGF as the targeting ligand and was labeled with 125I using N-succinimidyl-4-guanidinomethyl-3-[125I]iodobenzoate (SGMIB). Membrane binding, intracellular and nuclear accumulation kinetics, and clonogenic survival assays were performed using the EGFR-expressing A431 epidermoid carcinoma and D247 MG glioma cell lines.[125I]SGMIB-MNT bound to A431 and D247 MG cells with an affinity comparable to that of native EGF. More than 60% of internalized [125I]SGMIB-MNT radioactivity accumulated in the cell nuclei after a 1-h incubation. The cytotoxic effectiveness of [125I]SGMIB-MNT compared with 125I-labeled bovine serum albumin control was enhanced by a factor of 60 for D247 MG cells and more than 1,000-fold for A431 cells, which express higher levels of EGFR.MNT can be utilized to deliver 125I into the nuclei of cancer cells overexpressing EGFR, significantly enhancing cytotoxicity. Further evaluation of [125I]SGMIB-MNT as a targeted radiotherapeutic for EGFR-expressing cancer cells appears warranted.

News Article | March 25, 2016

Cellular stress is caused by heat, cold, lack of oxygen, changes in acidity level, inflammation, infection or toxins, irradiation with x-rays or ultraviolet light. Biologist Sergey Razin says, "We have demonstrated that acute heat stress triggers development of cellular senescence in normal and cancerous cells that are at an early S-phase of a cell cycle. We identified the mechanism by which heat stress induces cellular senescence. The reason for heat stress-induced senescence is persistent DNA damage response connected with the formation of difficult-to-repair, double-stranded DNA breaks." Using a wide range of methods, Russian scientists from MSU and Institute of Gene Biology, RAS, showed that a cell under stress is able to "break forks." This describes the structures in double-stranded DNA when the double helix is split so that each strand could serve as a template for the synthesis of a new DNA chain in cellular reproduction. This duplication of DNA is based on a complementary principle stating that each nucleotide—a 'letter' of a DNA being synthesized— is selected based on the type of nucleodide present in this position in the template chain. The researchers discovered that heat stress suppresses the activity of topoisomerase I, which relaxes DNA during replication by cutting one of the two strands. That leads to breaks in one strand, and when a replication fork reaches that spot, the other strand is also broken. When both strands are damaged, DNA is extremely difficult to repair. One more exciting outcomes of this study, according to Razin, is "a demonstration that genetically identical cells may differ dramatically both in resistance to exogenous stress factors and a type reaction to various stresses." Just as stress influences a person differently across a lifetime, cellular stress depends on the stage of the cell cycle, another subject addressed in the study. The lifetime of each somatic cell depends on its peculiarities—erythrocytes (biconcave red blood cells) live about 120 days, and epithelial cells lining the inside of the intestine about one to two days. Neurons and striated muscle tissue cells live just as long as the organism. Fast-living cells are constantly dividing to provide a sufficient replacements, while long-living cells almost never do. With all that diversity, somatic cells may be said to have four cell cylce phases: G1, S, G2 and mitosis, a division phase that results in building two identical daughter cells inheriting a chromatid—one half of a mother's chromosome. During the G1, pre-synthetic phase, cell growth occurs, and the cell is prepared for DNA doubling. Having received half of a chromosome, a cell needs to complete it in order to pass it to the next generation. This doubling synthetic phase happens during the S-phase. Accuracy in copying genetic information is under the strict control of the p53 protein. When a DNA strand is damaged, it boosts production of the p21 protein, which is connected to a complex of cyclin and cyclin-dependent kinases, responsible for initiating the next stage of the cycle. This delays the start of the S-phase, giving repair enzymes time to fix the damage. Then the G2 phase occurs, during which the cell grows and prepares itself for future division. At this stage, DNA is again subject to a mandatory inspection, and then mitosis begins. After mitosis, each of the newborn cells begins the G1 stage, and the cycle repeats. Some cells leave a row of divisions, hovering in the G0 phase, which, in a first approximation, is the G1 phase extended to an eternity; for the remaining cells, the cycle is also finite—after approximately 52 divisions, the cell ages, stops mitosis, and eventually dies. But when DNA in a cell is damaged so much that it is hard to repair, the cell cycle is terminated to avoid copying damaged genetic code, thereby creating generations of mutants, which leads to inflammation and the development of cancer tumors. "Based on cell reaction to heat stress, we formulated a model of cell senescence induction, which holds for many DNA-damaging agents. According to this model, any single- or double-stranded break happening at an early S-phase may initiate the cell senescence program," says Razin. The value of this research is ambiguous. On the one hand, scientists aspire to prevent the aging of normal cells to help them resist stress and function as long as possible. On the other hand, the controlled start of cellular senescence helps those cells deviating from the genetic program to die before becoming cancerous. That is why forcing defective cells to stop dividing and multiplying is vital for curing oncologic diseases. "Disclosure of the mechanisms of cellular senescence induced by a mild genotoxic (DNA-damaging) stress appears to be important both for understanding the reasons and mechanisms of aging, and a better understanding of cell response variability to exogenous and endogenous stress factors. This research also casts light on multiple previously unattended effects of DNA-damaging agents—for instance, camptothecin—which are often used in cancer therapy. Theoretically, the results of the study may form the basis for an optimization of existing protocols of simultaneous application of hyperthermia and chemotherapeutic agents for curing oncologic diseases," Razin concludes. More information: Artem K. Velichko et al. Mechanism of heat stress-induced cellular senescence elucidates the exclusive vulnerability of early S-phase cells to mild genotoxic stress, Nucleic Acids Research (2015). DOI: 10.1093/nar/gkv573

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