Molecular Genetic Manipulation Research Articles

Epigenetic control of plant senescence and cell death and its application in crop improvement.

Plant senescence is the last stage of plant development and a type of programmed cell death, occurring at a predictable time and cell. It involves the functional conversion from nutrient assimilation to nutrient remobilization, which substantially impacts plant architecture and plant biomass, crop quality, and horticultural ornamental traits. In past two decades, DNA damage was believed to be a main reason for cell senescence. Increasing evidence suggests that the alteration of epigenetic information is a contributing factor to cell senescence in organisms. In this review, we summarize the current research progresses of epigenetic and epitranscriptional mechanism involved in cell senescence of plant, at the regulatory level of DNA methylation, histone methylation and acetylation, chromatin remodeling, non-coding RNAs and RNA methylation. Furthermore, we discuss their molecular genetic manipulation and potential application in agriculture for crop improvement. Finally we point out the prospects of future research topics.

Recyclable CRISPR/Cas9-mediated gene disruption and deletions in Histoplasma.

Histoplasma is a primary fungal pathogen with the ability to infect otherwise healthy mammalian hosts, causing systemic and sometimes life-threatening disease. Thus far, molecular genetic manipulation of this organism has utilized RNA interference, random insertional mutagenesis, and a homologous recombination protocol that is highly variable and often inefficient. Targeted gene manipulations have been challenging due to poor rates of homologous recombination events in Histoplasma. Interrogation of the virulence strategies of this organism would be highly accelerated by a means of efficiently generating targeted mutations. We have developed a recyclable CRISPR/Cas9 system that can be used to introduce gene disruptions in Histoplasma with high efficiency, thereby allowing disruption of multiple genes.

Genetic and Environmental Factors Influencing the Production of Select Fungal Colorants: Challenges and Opportunities in Industrial Applications.

Natural colorants, mostly of plant and fungal origins, offer advantages over chemically synthetic colorants in terms of alleviating environmental pollution and promoting human health. The market value of natural colorants has been increasing significantly across the globe. Due to the ease of artificially culturing most fungi in the laboratory and in industrial settings, fungi have emerged as the organisms of choice for producing many natural colorants. Indeed, there is a wide variety of colorful fungi and a diversity in the structure and bioactivity of fungal colorants. Such broad diversities have spurred significant research efforts in fungi to search for natural alternatives to synthetic colorants. Here, we review recent research on the genetic and environmental factors influencing the production of three major types of natural fungal colorants: carotenoids, melanins, and polyketide-derived colorants. We highlight how molecular genetic studies and environmental condition manipulations are helping to overcome some of the challenges associated with value-added and large-scale productions of these colorants. We finish by discussing potential future trends, including synthetic biology approaches, in the commercial production of fungal colorants.

State-of-the-art review and update of in vivo models of necrotizing enterocolitis.

NEC remains one of the most common causes of mortality and morbidity in preterm infants. Animal models of necrotizing enterocolitis (NEC) have been crucial in improving our understanding of this devastating disease and identifying biochemical pathways with therapeutic potential. The pathogenesis of NEC remains incompletely understood, with no specific entity that unifies all infants that develop NEC. Therefore, investigators rely on animal models to manipulate variables and provide a means to test interventions, making them valuable tools to enhance our understanding and prevent and treat NEC. The advancements in molecular analytic tools, genetic manipulation, and imaging modalities and the emergence of scientific collaborations have given rise to unique perspectives and disease correlates, creating novel pathways of investigation. A critical review and understanding of the current phenotypic considerations of the highly relevant animal models of NEC are crucial to developing novel therapeutic and preventative strategies for NEC.

A Basic Guide to the Growth and Manipulation of the Blast Fungus, Magnaporthe oryzae.

The blast fungus, Magnaporthe oryzae, is a devastating plant pathogen that threatens global food security. The social and economic importance of blast disease has contributed to this filamentous fungus becoming a model organism for the study of host‐pathogen interactions. Availability of the complete genome sequences of many strains of the pathogen, as well as rice and wheat cultivars, coupled with the tractability of M. oryzae to classical and molecular genetic manipulation have contributed to its widespread study. Although M. oryzae has been extensively investigated for the past two decades, procedures for storing, maintaining, and manipulating the blast fungus in the laboratory had not been compiled and updated. As a consequence, there is considerable disparity in how the fungus is stored and manipulated between studies. In this article, we present a collection of protocols providing clear explanations of how to preserve filter stocks of M. oryzae; how to grow the fungus in both liquid and solid media; how to extract genomic DNA from fungal mycelium; how to induce appressorium formation on coverslips for visualization and tissue collection; and how to perform two distinct types of plant infection assay for virulence assessment. By sharing our most used laboratory procedures, we aim to address some of the knowledge gaps in current M. oryzae protocols and contribute to uniformity and robustness in studies by the Magnaporthe research community. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Storage of M. oryzae strains Basic Protocol 2: Revival and regular maintenance of M. oryzae cultures in solid medium Alternate Protocol 1: Regular maintenance of M. oryzae cultures in liquid medium Basic Protocol 3: Genomic DNA extraction from M. oryzae mycelium Alternate Protocol 2: Quick DNA extraction from M. oryzae mycelium Basic Protocol 4: M. oryzae induction of appressorium development on glass coverslips for microscopy Alternate Protocol 3: M. oryzae induction of appressorium development on glass coverslips for tissue collection Basic Protocol 5: M. oryzae rice infection assay through spray inoculation Alternate Protocol 4: M. oryzae leaf‐drop plant infection assay

YAP1 activation promotes epithelial-mesenchymal transition and cell survival of renal cell carcinoma cells under shear stress.

Renal cell carcinoma (RCC) is characterized by substantial vasculatures and increased fluid movement in tumor microenvironment, and the fluid shear stress modulates malignance, extravasation and metastatic seeding of tumor cells. However, the precise mechanism remains largely unclear. In this study, we found that low shear stress induced the Yes-associated protein (YAP1) activation and nuclear localization in RCC cells, as well as the downregulation of phosphorylated YAP1 at Ser127. Moreover, inhibition of ROCK or RhoA partially abolished YAP1 accumulation in the nucleus, and targeting YAP1 activation by small molecular inhibitor or genetic manipulation decreased the low shear stress-induced epithelial-mesenchymal transition (EMT) of RCC cells, and led to a decreased expression of N-cadherin as accompanied by downregulation of SNAIL1 and TWIST, accompanied by high shear stress-induced cell apoptosis. Salvianolic acid B, an aqueous component of danshen (Salvia miltiorrhiza), inhibited YAP1 and Hippo signaling activation, and abrogated low shear stress-induced EMT as a consequence. Taken together, our study suggests YAP1 is a fluid mechanosensor that transforms mechanical stimuli to cell signals, thereby facilitates anoikis resistance and tumor metastasis.

Robo2 Drives Target-Selective Peripheral Nerve Regeneration in Response to Glia-Derived Signals.

Peripheral nerves are divided into multiple branches leading to divergent synaptic targets. This poses a remarkable challenge for regenerating axons as they select their original trajectory at nerve branch-points. Despite implications for functional regeneration, the molecular mechanisms underlying target selectivity are not well characterized. Danio Rerio (zebrafish) motor nerves are composed of a ventral and a dorsal branch that diverge at a choice-point, and we have previously shown that regenerating axons faithfully select their original branch and targets. Here we identify robo2 as a key regulator of target-selective regeneration (sex of experimental subjects unknown). We demonstrate that robo2 function in regenerating axons is required and sufficient to drive target-selective regeneration, and that robo2 acts in response to glia located precisely where regenerating axons select the branch-specific trajectory to prevent and correct axonal errors. Combined, our results reveal a glia-derived mechanism that acts locally via axonal robo2 to promote target-selective regeneration.SIGNIFICANCE STATEMENT Despite its relevance for functional recovery, the molecular mechanisms that direct regenerating peripheral nerve axons toward their original targets are not well defined. Zebrafish spinal motor nerves are composed of a dorsal and a ventral branch that diverge at a stereotyped nerve branch-point, providing a unique opportunity to decipher the molecular mechanisms critical for target-selective regeneration. Using a combination of live cell imaging and molecular-genetic manipulations, we demonstrate that the robo2 guidance receptor is necessary and sufficient to promote target-selective regeneration. Moreover, we demonstrate that robo2 is part of a genetic pathway that generates transient, spatially restricted, and tightly coordinated signaling events that direct axons of the dorsal nerve branch toward their original, pre-injury targets.

Analysis of Codon Usage Patterns in Giardia duodenalis Based on Transcriptome Data from GiardiaDB.

Giardia duodenalis, a flagellated parasitic protozoan, the most common cause of parasite-induced diarrheal diseases worldwide. Codon usage bias (CUB) is an important evolutionary character in most species. However, G. duodenalis CUB remains unclear. Thus, this study analyzes codon usage patterns to assess the restriction factors and obtain useful information in shaping G. duodenalis CUB. The neutrality analysis result indicates that G. duodenalis has a wide GC3 distribution, which significantly correlates with GC12. ENC-plot result—suggesting that most genes were close to the expected curve with only a few strayed away points. This indicates that mutational pressure and natural selection played an important role in the development of CUB. The Parity Rule 2 plot (PR2) result demonstrates that the usage of GC and AT was out of proportion. Interestingly, we identified 26 optimal codons in the G. duodenalis genome, ending with G or C. In addition, GC content, gene expression, and protein size also influence G. duodenalis CUB formation. This study systematically analyzes G. duodenalis codon usage pattern and clarifies the mechanisms of G. duodenalis CUB. These results will be very useful to identify new genes, molecular genetic manipulation, and study of G. duodenalis evolution.

Advances and Perspectives for Genome Editing Tools of Corynebacterium glutamicum.

Corynebacterium glutamicum has been considered a promising synthetic biological platform for biomanufacturing and bioremediation. However, there are still some challenges in genetic manipulation of C. glutamicum. Recently, more and more genetic parts or elements (replicons, promoters, reporter genes, and selectable markers) have been mined, characterized, and applied. In addition, continuous improvement of classic molecular genetic manipulation techniques, such as allelic exchange via single/double-crossover, nuclease-mediated site-specific recombination, RecT-mediated single-chain recombination, actinophages integrase-mediated integration, and transposition mutation, has accelerated the molecular study of C. glutamicum. More importantly, emerging gene editing tools based on the CRISPR/Cas system is revolutionarily rewriting the pattern of genetic manipulation technology development for C. glutamicum, which made gene reprogramming, such as insertion, deletion, replacement, and point mutation, much more efficient and simpler. This review summarized the recent progress in molecular genetic manipulation technology development of C. glutamicum and discussed the bottlenecks and perspectives for future research of C. glutamicum as a distinctive microbial chassis.

Development of Genetic Modification Tools for Hanseniasporauvarum.

Apiculate yeasts belonging to the genus Hanseniaspora are commonly isolated from viticultural settings and often dominate the initial stages of grape must fermentations. Although considered spoilage yeasts, they are now increasingly becoming the focus of research, with several whole-genome sequencing studies published in recent years. However, tools for their molecular genetic manipulation are still lacking. Here, we report the development of a tool for the genetic modification of Hanseniaspora uvarum. This was employed for the disruption of the HuATF1 gene, which encodes a putative alcohol acetyltransferase involved in acetate ester formation. We generated a synthetic marker gene consisting of the HuTEF1 promoter controlling a hygromycin resistance open reading frame (ORF). This new marker gene was used in disruption cassettes containing long-flanking (1000 bp) homology regions to the target locus. By increasing the antibiotic concentration, transformants were obtained in which both alleles of the putative HuATF1 gene were deleted in a diploid H. uvarum strain. Phenotypic characterisation including fermentation in Müller-Thurgau must showed that the null mutant produced significantly less acetate ester, particularly ethyl acetate. This study marks the first steps in the development of gene modification tools and paves the road for functional gene analyses of this yeast.

Tissue-Specific Approaches Reveal Diverse Metabolic Functions of Rho-Kinase 1

Rho-kinase 1 (ROCK1) has been implicated in diverse metabolic functions throughout the body, with promising evidence identifying ROCK1 as a therapeutic target in diabetes and obesity. Considering these metabolic roles, several pharmacological inhibitors have been developed to elucidate the mechanisms underlying ROCK1 function. Y27632 and fasudil are two common ROCK1 inhibitors; however, they have varying non-specific selectivity to inhibit other AGC kinase subfamily members and whole-body pharmacological approaches lack tissue-specific insight. As a result, interpretation of studies with these inhibitors is difficult, and alternative approaches are needed to elucidate ROCK1’s tissue specific metabolic functions. Fortunately, recent technological advances utilizing molecular carriers or genetic manipulation have facilitated discovery of ROCK1’s tissue-specific mechanisms of action. In this article, we review the tissue-specific roles of ROCK1 in the regulation of energy balance and substrate utilization. We highlight prominent metabolic roles in liver, adipose, and skeletal muscle, in which ROCK1 regulates energy expenditure, glucose uptake, and lipid metabolism via inhibition of AMPK2α and paradoxical modulation of insulin signaling. Compared to ROCK1’s roles in peripheral tissues, we also describe contradictory functions of ROCK1 in the hypothalamus to increase energy expenditure and decrease food intake via leptin signaling. Furthermore, dysregulated ROCK1 activity in either of these tissues results in metabolic disease phenotypes. Overall, tissue-specific approaches have made great strides in deciphering the many critical metabolic functions of ROCK1 and, ultimately, may facilitate the development of novel treatments for metabolic disorders.

Advances in reverse genetics system of plant negative-strand RNA viruses

Negative-strand RNA (NSR) viruses contain not only medical important animal pathogens but also agricultural important plant pathogens. These NSR viruses cause deadliest diseases in humans, livestocks and agronomic crops and pose great threat to the health of human being and food security worldwide. The virus reverse genetics system is an important molecular genetic technology to study viral gene functions in virus infection cycle, disease pathology and virus-host interactions. For animal-infecting NSR viruses, the reverse genetics system has been established for more than twenty years. In contrast to the well-established reverse genetics systems for animal-infecting NSR viruses, establishment of such system for the plant-infecting NSR viruses turned out to be an extremely challenge work. Many research groups throughout the world have tried to use the similar strategies used for animal NSR viruses to establish reverse genetics systems for plant-infecting NSR viruses in the last twenty years but none of groups have succeeded. The absence of the reverse genetics system posed a major obstacle to molecular genetic manipulation and subsequent investigation of gene function and disease pathology for plant-infecting NSR viruses. Recently, big breakthroughs have been made in the establishment of the reverse genetics systems for plant NSR viruses by scientists from China and America, respectively. The systems were recently established for both nonsegmented and segmented plant NSR viruses. The reverse genetics system for a nonsegmented plant NSR virus was firstly developed for sonchus yellow net virus (SYNV), a non segmented nucleorhabdovirus NSR. Using the similar strategies as SYNV, barley yellow striate mosaic virus (BYSMV), a nonsegmented cytorhabdovirus NSR, was then succeeded in the establishment of reverse genetics system. The first reverse genetics system for a segmented plant NSR virus was developed using tomato spotted wilt virus (TSWV), a tospovirus with tripartite negative-stranded/ambisense RNA genomes. The reverse genetics system was also recently developed for rose rosette virus (RRV), an emaravirus with seven negative-sense mono-cistronic RNA genomes. The establishment of the reverse genetics system for plant NSR viruses typically involves two steps. The first step requires the construction of a mini-genome replication system. The viral genomic or antigenomic RNA was flanked by hammerhead and ribozyme to generate exact viral 5 ¢ and 3 ¢ end, and driven by 35S promoter. The mini-replicon construct was co-expressed with the constructs expressing nucleocapsid (N) protein, phosphoprotein (P; essential for rhabdovirus) and viral RNA-dependent RNA polymerase (RdRp). For the segmented plant NSR viruses such as TSWV, codon optimization and removal of intron-splicing sites of RdRp sequence are critical for the expression of a functional RdRp from 35S-driven constructs in planta . The second step involves the rescue of virus entirely from full-length infectious cDNA clones in plant. The recombinant virus can carry green fluorescence protein (GFP) gene reporter and infect Nicotiana benthamiana plant systemically. The establishment of reverse genetics for different plant-infecting NSR viruses now provided powerful systems to investigate all aspects of virus infection cycle and disease pathology. The recent breakthroughs of reverse genetics systems have opened a new era for the investigation of plant-infecting NSR viruses in the future.

Decoding the global outbreak of COVID-19: the nature is behind the scene.

The sudden emergence of SARS-CoV-2 causing the global pandemic is a major public health concern. Though the virus is considered as a novel entity, it is not a completely new member. It is just a new version of previously emerged human SARS corona virus. The rapid evolving nature by changing host body environment and extreme environmental stability, collectively makes SARS-CoV-2 into an extremely virulent genetic variant. The evolution of the virus has been occurred by the continuous process of molecular genetic manipulation, through mutation, deletion and genetic recombinationevents. Different host body environment acts as the supportive system for the pathogen which creates extreme selective pressure. By the process of genetic evolution the pathogen developes new characters. Then the new version of the virus has been naturally selected by susceptible human host and adapt itself inside the host body causing deadly effect. Moreover, extreme environmental stability helps in the process of viral survival outside the host and its transmission. Thus both the host body or internal environment and the external environment performs equally as a source, responsible for shaping the genetic evolution of the SARS-CoV-2 towards theCOVID-19 disease fitness in nature in a pandemic form.

Auxotrophic mutations of Trichophyton rubrum created by in vitro synthesized Cas9 ribonucleoprotein

BackgroundTrichophyton rubrum is an obligate human parasitic fungus and responsible for approximately 80–90% of dermatomycosis in human. Molecular genetic manipulations of this pathogen are challenging and available tools and protocols are only rudimentary. We adapt molecular genetics methods of well established fungal model organism, to knock out genes in T. rubrum. For the adaptation, crucial modifications are necessary. With the implementation of in vitro synthesized Cas9-sgRNA ribonucleoprotein complex, it is possible to adapt molecular genetic methods, to knock out genes in T. rubrum.ResultsThe gene knock-out method is based on integration of a selection marker into the target site, to interrupt the gene translation. The target gene gets preassigned by the homologous sequence of the in vitro synthesized Cas9-sgRNA ribonucleoprotein complex. To develop the method, we first isolated and characterized a T. rubrum strain with a high amount of microconidia. Next, we developed a transformation protocol, whereby the Cas9-sgRNA ribonucleoprotein gets delivered into the fungal protoplast by the PEG method.We knocked out the URA3 gene and resulted, as predicted, uracil auxotrophic strains. These strains can be used for specific gene knock-outs by reintegrating the URA3 fragment and selection on uracil lacking cultivation media. Exemplary, we knocked out the TRP3 gene and got the predicted phenotype, tryptophan auxotrophic strains. The mutation had been verified by sequencing.ConclusionsWe developed a method, based on in vitro synthesized Cas9-sgRNA ribonucleoprotein complex, for target specific gene knock-outs in T. rubrum. We knocked out the Ura3 gene and resulted uracil auxotrophic strains. These strains were used for target specific gene knock-outs by reintegrating the Ura3 fragment into the target gene site to interrupt the gene transcription. The developed method allows to adapt sophisticate gene manipulation methods of model fungal species to non-model species.

Protein Engineering of DNA-Dependent Enzymes.

Enzymes are extremely efficient natural catalysts of a variety of chemical reactions. Design of enzymes with new functions and properties has become one of the main goals of modern protein engineering. The field of protein engineering is growing intensively, and different strategies were developed for the creation of enzymes with new properties. While there is plenty of methods and instruments, all modern protein engineering strategies could be divided in two major groups, broadly based on the core ideas of rational design or directed evolution. DNA-dependent proteins present an important target of protein engineering due to their wide use in molecular cloning, bioanalytics, and genetic manipulations. Here we review examples of successful application of biochemical, structural and computational approaches belonging to both protein engineering strategies to create new proteins belonging to three important classes of DNA-dependent enzymes: CRISPR-associated nuclease Cas9, DNA polymerases, and DNA glycosylases. The review contains examples of successfully designed enzymes and discusses the most useful approaches in the engineering of these specific enzyme classes, problems restraining the development of this field, and future directions in the development and application of designed DNA-dependent enzymes.

Rescue of tomato spotted wilt virus entirely from complementary DNA clones

Negative-stranded/ambisense RNA viruses (NSVs) include not only dangerous pathogens of medical importance but also serious plant pathogens of agronomic importance. Tomato spotted wilt virus (TSWV) is one of the most important plant NSVs, infecting more than 1,000 plant species, and poses major threats to global food security. The segmented negative-stranded/ambisense RNA genomes of TSWV, however, have been a major obstacle to molecular genetic manipulation. In this study, we report the complete recovery of infectious TSWV entirely from complementary DNA (cDNA) clones. First, a replication- and transcription-competent minigenome replication system was established based on 35S-driven constructs of the S(-)-genomic (g) or S(+)-antigenomic (ag) RNA template, flanked by the 5' hammerhead and 3' ribozyme sequence of hepatitis delta virus, a nucleocapsid (N) protein gene and codon-optimized viral RNA-dependent RNA polymerase (RdRp) gene. Next, a movement-competent minigenome replication system was developed based on M(-)-gRNA, which was able to complement cell-to-cell and systemic movement of reconstituted ribonucleoprotein complexes (RNPs) of S RNA replicon. Finally, infectious TSWV and derivatives carrying eGFP reporters were rescued in planta via simultaneous expression of full-length cDNA constructs coding for S(+)-agRNA, M(-)-gRNA, and L(+)-agRNA in which the glycoprotein gene sequence of M(-)-gRNA was optimized. Viral rescue occurred with the addition of various RNAi suppressors including P19, HcPro, and γb, but TSWV NSs interfered with the rescue of genomic RNA. This reverse genetics system for TSWV now allows detailed molecular genetic analysis of all aspects of viral infection cycle and pathogenicity.

A script for initiating molecular biology studies with non-conventional yeasts based on Saccharomycopsis schoenii

Non-conventional yeasts (NCYs), i.e. all yeasts other than Saccharomyces cerevisiae, are emerging as novel production strains and gain more and more attention to exploit their unique properties. Yet, these yeasts can hardly compete against the advanced methodology and genetic tool kit available for exploiting and engineering S. cerevisiae. Currently, for many NCYs one has to start from scratch to initiate molecular genetic manipulations, which is often time consuming and not straight-forward. More so because utilization of S. cerevisiae tools based on short-flank mediated homologous recombination or plasmid biology are not readily applicable in NCYs. Here we present a script with discrete steps that will lead to the development of a basic and expandable molecular toolkit for ascomycetous NCYs and will allow genetic engineering of novel platform strains. For toolkit development the highly efficient in vivo recombination efficiency of S. cerevisiae is utilized in the generation and initial testing of tools. The basic toolkit includes promoters, reporter genes, selectable markers based on dominant antibiotic resistance genes and the generation of long-flanking homology disruption cassettes. The advantage of having pretested molecular tools that function in a heterologous host facilitate NCY strain manipulations. We demonstrate the usefulness of this script on Saccharomycopsis schoenii, a predator yeast with useful properties in fermentation and fungal biocontrol.

Lipid-Assisted Membrane Protein Folding and Topogenesis.

Due to the heterogenous lipid environment in which integral membrane proteins are embedded, they should follow a set of assembly rules, which govern transmembrane protein folding and topogenesis accordingly to a given lipid profile. Recombinant strains of bacteria have been engineered to have different membrane phospholipid compositions by molecular genetic manipulation of endogenous and foreign genes encoding lipid biosynthetic enzymes. Such strains provide a means to investigate the in vivo role of lipids in many different aspects of membrane function, folding and biogenesis. In vitro and in vivo studies established a function of lipids as molecular chaperones and topological determinants specifically assisting folding and topogenesis of membrane proteins. These results led to the extension of the Positive Inside Rule to Charge Balance Rule, which incorporates a role for lipid-protein interactions in determining membrane protein topological organization at the time of initial membrane insertion and dynamically after initial assembly. Membrane protein topogenesis appears to be a thermodynamically driven process in which lipid-protein interactions affect the potency of charged amino acid residues as topological signals. Dual topology for a membrane protein can be established during initial assembly where folding intermediates in multiple topological conformations are in rapid equilibrium (thus separated by a low activation energy), which is determined by the lipid environment. Post-assembly changes in lipid composition or post-translational modifications can trigger a reorganization of protein topology by inducing destabilization and refolding of a membrane protein. The lipid-dependent dynamic nature of membrane protein organization provides a novel means of regulating protein function.

Синтетическая биология – современные вызовы и проблемы биобезопасности

In 1980, German biochemist Barbara Hobom for the first time used the term “synthetic biology” when she described a transgenic bacterium constructed by use of DNA recombinant technology. Actually this direction has become active and has been widely developed only in the last decade. Today, this term is covered by a new branch of research which combines molecular biology and genetic manipulations for construction of new (yet not existing in nature) biologic functions and systems. The rapid growth of research in the field of molecular biology and genetics generates new trends, the development of which contributes to not only improving and expanding the possibilities of using its achievements for the benefit of humankind, but can also be used for illegal purposes. And one of the most vivid and promising trends is a synthetic biology, a new scientific direction in biology that deals with the design and creation of biological systems with prescribed properties and functions that have no analogs in nature. Theoretically, the modern possibilities of synthetic biology are limited only by the availability of a proper material base, the level of preparation and imagination of researchers. Base of state politics in area of chemical and biological safety focused on priority of directions, main tasks and mechanism for prevention of different chemical and biologic threats until 2025 was signed by the Russian president. The main purpose of these documents is a systematic decrease of negative impact of dangerous chemical and biological agents on humans and environment down to an appropriate level. The first Russian monitoring center was included in structure of All-Russian center for medicine of disaster Ministry of Health in January, 2019. The center will collect information about biological threats and analyze possible risks (including capabilities and achievements of synthetic biology). Existing international and Russian legal documents in bioethics issue are not harmonized, but provide a possibility to conduct clinical trials face I–III. Meanwhile, the necessity of further improvements of regulatory framework for the increasingly complex modern biomedical research involving the synthetic biology is obvious.

The Expanding Molecular Genetics Tool Kit in Chlamydia.

Chlamydia has emerged as an important model system for the study of host pathogen interactions, in part due to a resurgence in the development of tools for its molecular genetic manipulation. An additional tool, published by Keb et al. (G. Keb, R. Hayman, and K. A. Fields, J. Bacteriol. 200:e00479-18, 2018, https://doi.org/10.1128/JB.00479-18), now allows for custom genetic engineering of genomic regions that were traditionally recalcitrant to genetic manipulation, such as genes within operons. This new method will be an essential instrument for the elucidation of Chlamydia-host interactions.