NEW POTENTIAL TREATMENT FOR BRAIN GLIOMA

According to the latest statistics, brain gliomas are the most common cause of death from CNS tumors. Brain gliomas are also ranked as the second (after stroke) cause of brain surgery The mortality rate from gliomas is high and sometimes reaches 80 %. It is because the tumor grows from undifferentiated cells, which causes its peracute development and malignant transformation. Symptoms of glioma occur at stages 3 and 4, when all treatment is symptomatic, and operations are palliative. In this regard, it is necessary to develop and introduce methods for non-surgical glioma treatment. These methods include the use of antisense oligonucleotides, optogenetics, and oncolytic viruses. The aim of antisense oligonucleotides is to replace a section in a glioma cell genome with a foreign one, which disrupts cell division and leads to apoptosis and necrosis of the entire tumor. Optogenetics excludes the introduction of substances into the body. It provides a certain light signal to glioma cells, which also suppresses the growth of an undifferentiated tumor. Oncolytic viruses are genetically modified viruses that identify tumor cells, penetrate into them and start a cascade of apoptotic reactions Despite all success, such methods are still studied at the laboratory level, their implementation in practical medicine is slow and cautious. However, insufficient knowledge retards the widespread use of potentially promising and effective drugs. Scientists around the world are developing methods to treat brain gliomas at different stages of their development. This article reflects modern achievements of scientists and neurosurgeons, describing new methods for brain glioma treatment. Key words: brain glioma, optogenetics, antisense oligonucleotides, oncolytic viruses, p53 gene. Согласно последним данным статистики, глиомы мозга являются наиболее частой причиной смертей от онкологии центральной нервной системы, а также занимают второе место по частоте как причина хирургических вмешательств на головной мозг, уступая инсультам. Смертность от глиом высока и порой достигает 80 %. Причина этого заключается в том, что опухоль растет из недифференцированных клеток, что обусловливает её молниеносный рост и быстрое озлокачествление. Симптомы глиомы возникают на 3–4 стадии развития, когда все лечение направлено на ликвидацию симптомов, а операции носят паллиативный характер. В связи с этим необходима разработка и внедрение методов по нехирургическому лечению глиом. Такими методами являются использование антисмысловых олигонуклеотидов, оптогенетика, применение онколитических вирусов. Суть использования антисмысловых олигонуклеотидов заключается в замене участка генома клетки глиомы на инородный, попавший извне, что нарушает деление клеток и приводит к апоптозу и некрозу всей опухоли. Оптогенетика исключает введение веществ в организм и заключается в подаче определенного светового сигнала на глиозные клетки, что также тормозит рост недифференцированной опухоли. Онколитические вирусы – это генномодифицированные вирусы, которые определяют опухолевые клетки, проникают в них и запускают каскад апоптотических реакций. Несмотря на все успехи, данные методы продолжают изучаться на уровне лабораторий, их внедрение в практическую медицину происходит медленно и со страхом. Однако недостаточная изученность тормозит широкое применение потенциально перспективных и эффективных лекарств. Учеными мира разрабатываются методы, позволяющие лечить глиомы мозга на разных стадиях их развития. Данная статья отображает современные достижения ученых и нейрохирургов в поисках возможности применения такого рода методов. Ключевые слова: глиома мозга, оптогенетика, антисмысловые олигонуклеотиды, онколитические вирусы, ген р53.

Next-Generation Vaccines Based on Self-Amplifying RNA

Recently, nucleic acid-based RNA and DNA vaccines have represented a better solution to avoid infectious diseases than “traditional” live and non-live vaccines. Synthetic RNA and DNA molecules allow scalable, rapid, and cell-free production of vaccines in response to an emerging disease such as the current COVID-19 pandemic. The development process begins with laboratory transcription of sequences encoding antigens, which are then formulated for delivery. The various potent of RNA over live and inactivated viruses are proven by advances in delivery approaches. These vaccines contain no infectious elements nor the risk of stable integration with the host cell genome compared to conventional vaccines. Conventional mRNA-based vaccines transfer genes of interest (GOI) of attenuated mRNA viruses to individual host cells. Synthetic mRNA in liposomes forms a modern, refined sample, resulting in a safer version of live attenuated RNA viruses. Self-amplifying RNA (saRNA) is a replicating version of mRNA-based vaccines that encode both (GOI) and viral replication machinery. saRNA is required at lower doses than conventional mRNA, which may improve immunization. Here we provide an overview of current mRNA vaccine approaches, summarize highlight challenges and recent successes, and offer perspectives on the future of mRNA vaccines.

Single cell genome sequencing of laboratory mouse microbiota improves taxonomic and functional resolution of this model microbial community

Laboratory mice are widely studied as models of mammalian biology, including the microbiota. However, much of the taxonomic and functional diversity of the mouse gut microbiome is missed in current metagenomic studies, because genome databases have not achieved a balanced representation of the diverse members of this ecosystem. Towards solving this problem, we used flow cytometry and low-coverage sequencing to capture the genomes of 764 single cells from the stool of three laboratory mice. From these, we generated 298 high-coverage microbial genome assemblies, which we annotated for open reading frames and phylogenetic placement. These genomes increase the gene catalog and phylogenetic breadth of the mouse microbiota, adding 135 novel species with the greatest increase in diversity to the Muribaculaceae and Bacteroidaceae families. This new diversity also improves the read mapping rate, taxonomic classifier performance, and gene detection rate of mouse stool metagenomes. The novel microbial functions revealed through our single-cell genomes highlight previously invisible pathways that may be important for life in the murine gastrointestinal tract.

The hidden genomic diversity of ciliated protists revealed by single-cell genome sequencing

Background Ciliated protists are a widely distributed, morphologically diverse, and genetically heterogeneous group of unicellular organisms, usually known for containing two types of nuclei: a transcribed polyploid macronucleus involved in gene expression and a silent diploid micronucleus responsible for transmission of genetic material during sexual reproduction and generation of the macronucleus. Although studies in a few species of culturable ciliated protists have revealed the highly dynamic nature of replicative and recombination events relating the micronucleus to the macronucleus, the broader understanding of the genomic diversity of ciliated protists, as well as their phylogenetic relationships and metabolic potential, has been hampered by the inability to culture numerous other species under laboratory conditions, as well as the presence of symbiotic bacteria and microalgae which provide a challenge for current sequencing technologies. Here, we optimized single-cell sequencing methods and associated data analyses, to effectively remove contamination by commensal bacteria, and generated high-quality genomes for a number of Euplotia species. Results We obtained eight high-quality Euplotia genomes by using single-cell genome sequencing techniques. The genomes have high genomic completeness, with sizes between 68 and 125 M and gene numbers between 14K and 25K. Through comparative genomic analysis, we found that there are a large number of gene expansion events in Euplotia genomes, and these expansions are closely related to the phenotypic evolution and specific environmental adaptations of individual species. We further found four distinct subgroups in the genus Euplotes, which exhibited considerable genetic distance and relative lack of conserved genomic syntenies. Comparative genomic analyses of Uronychia and its relatives revealed significant gene expansion associated with the ciliary movement machinery, which may be related to the unique and strong swimming ability. Conclusions We employed single-cell genomics to obtain eight ciliate genomes, characterized the underestimated genomic diversity of Euplotia, and determined the divergence time of representative species in this subclass for the first time. We also further investigated the extensive duplication events associated with speciation and environmental adaptation. This study provides a unique and valuable resource for understanding the evolutionary history and genetic diversity of ciliates.

Are HIV-1-Specific Antibody Levels Potentially Useful Laboratory Markers to Estimate HIV Reservoir Size? A Review

Despite the benefits achieved by the widespread availability of modern antiretroviral therapy (ART), HIV RNA integration into the host cell genome is responsible for the creation of latent HIV reservoirs, and represents a significant impediment to completely eliminating HIV infection in a patient via modern ART alone. Several methods to measure HIV reservoir size exist; however, simpler, cheaper, and faster tools are required in the quest for total HIV cure. Over the past few years, measurement of HIV-specific antibodies has evolved into a promising option for measuring HIV reservoir size, as they can be measured via simple, well-known techniques such as the western blot and enzyme-linked immunosorbent assay (ELISA). In this article, we re-visit the dynamic evolution of HIV-1-specific antibodies and the factors that may influence their levels in the circulation of HIV-positive individuals. Then, we describe the currently-known relationship between HIV-1-specific antibodies and HIV reservoir size based on study of data from contemporary literature published during the past 5 years. We conclude by highlighting current trends, and discussing the individual HIV-specific antibody that is likely to be the most reliable antibody for potential future utilization for quantification of HIV reservoir size.

Analysis of the Replication Mechanisms of the Human Papillomavirus Genomes

The life-cycle of human papillomaviruses (HPVs) includes three distinct phases of the viral genome replication. First, the viral genome is amplified in the infected cells, and this amplification is often accompanied by the oligomerization of the viral genomes. Second stage includes the replication of viral genomes in concert with the host cell genome. The viral genome is further amplified during the third stage of the viral-life cycle, which takes place only in the differentiated keratinocytes. We have previously shown that the HPV18 genomes utilize at least two distinct replication mechanisms during the initial amplification. One of these mechanisms is a well-described bidirectional replication via theta type of replication intermediates. The nature of another replication mechanism utilized by HPV18 involves most likely recombination-dependent replication. In this paper, we show that the usage of different replication mechanisms is a property shared also by other HPV types, namely HPV11 and HPV5. We further show that the emergence of the recombination dependent replication coincides with the oligomerization of the viral genomes and is dependent on the replicative DNA polymerases. We also show that the oligomeric genomes of HPV18 replicate almost exclusively using recombination dependent mechanism, whereas monomeric HPV31 genomes replicate bi-directionally during the maintenance phase of the viral life-cycle.

Whole chromosome loss and genomic instability in mouse embryos after CRISPR-Cas9 genome editing

Karyotype alterations have emerged as on-target complications from CRISPR-Cas9 genome editing. However, the events that lead to these karyotypic changes in embryos after Cas9-treatment remain unknown. Here, using imaging and single-cell genome sequencing of 8-cell stage embryos, we track both spontaneous and Cas9-induced karyotype aberrations through the first three divisions of embryonic development. We observe the generation of abnormal structures of the nucleus that arise as a consequence of errors in mitosis, including micronuclei and chromosome bridges, and determine their contribution to common karyotype aberrations including whole chromosome loss that has been recently reported after editing in embryos. Together, these data demonstrate that Cas9-mediated germline genome editing can lead to unwanted on-target side effects, including major chromosome structural alterations that can be propagated over several divisions of embryonic development.

Three Draft Single-Cell Genome Sequences of Novel SAR324 Strains Isolated from the Abyssopelagic Southern Ocean

SAR324 is a ubiquitous and phylogenetically distinct clade of Deltaproteobacteria in marine environments. Here, we present three single-cell amplified genome sequences from the SAR324 lineage, obtained from the abyssopelagic zone of the Indian sector of the Southern Ocean.

Reconstructing and counting genomic fragments through tagmentation-based haploid phasing

Single-cell sequencing provides a new level of granularity in studying the heterogeneous nature of cancer cells. For some cancers, this heterogeneity is the result of copy number changes of genes within the cellular genomes. The ability to accurately determine such copy number changes is critical in tracing and understanding tumorigenesis. Current single-cell genome sequencing methodologies infer copy numbers based on statistical approaches followed by rounding decimal numbers to integer values. Such methodologies are sample dependent, have varying calling sensitivities which heavily depend on the sample’s ploidy and are sensitive to noise in sequencing data. In this paper we have demonstrated the concept of integer-counting by using a novel bioinformatic algorithm built on our library construction chemistry in order to detect the discrete nature of the genome.

CRISPR/Cas9 ablation of integrated HIV-1 accumulates proviral DNA circles with reformed LTRs

Gene editing may be used to excise the human immunodeficiency virus type-1 (HIV-1) provirus from the host cell genome, possibly eradicating the infection. Here, using cells acutely or latently infected by HIV-1 and treated with long terminal repeat-targeting CRISPR/Cas9, we show that the excised HIV-1 provirus persists for a few weeks and may rearrange in circular molecules. Although circular proviral DNA is naturally formed during HIV-1 replication, we observed that gene-editing might increase proviral DNA circles with restored LTRs. These extrachromosomal elements were recovered and probed for residual activity through their re-transfection in uninfected cells. We discovered that they can be transcriptionally active in the presence of Tat and Rev. Although confirming that gene editing is a powerful tool to eradicate HIV-1 infection, this work highlights that, to achieve this goal, the LTRs must be cleaved in several pieces to avoid residual activity and minimize the risk of re-integration in a context of genomic instability, possibly caused by off-target activity of Cas9. IMPORTANCE Excision of HIV-1 provirus from host cell genome has proved feasible in vitro , and to some extent, in vivo. Among the different approaches, CRISPR/Cas9 is the most promising tool for gene editing. The present study underlines the remarkable effectiveness of CRISPR/Cas9 in removing the HIV-1 provirus from infected cells and investigates the fate of the excised HIV-1 genome. This study demonstrates that the free provirus may persist in the cell after editing and in appropriate circumstances may integrate back into the cell genome. As an episome, it might be transcriptionally active, especially in the presence of Tat and Rev. The persistence of HIV-1 episome was strongly decreased by gene editing with multiple targets. Although gene editing has the potential to eradicate HIV-1 infection, this work highlights a potential issue that warrants further investigation.