Molecular Genetic Engineering Research Articles

Codon Usage Patterns Analysis of Camellia oleifera Based on Transcriptome Data

Camellia oleifera is an essential oil woody plant. The development of the camellia industry can relieve the a China’s pressure of importing edible oil. However, there are few studies on the codon usage bias (CUB) in C. oleifera genes. In this study, the codon usage patterns were analyzed by 35,178 reconstructed genes from the C. oleifera transcriptome. The mean GC and GC3 content of all transcripts was 51.14% and 55.43% respectively. A total of 18 optimal codons were identified, 15 of them ending with A or U. Which indicated the use of A/U codons was high frequency. Furthermore, the natural selection and mutational pressure, both influenced CUB in C. oleifera, however the natural selection was the most deciding factor. The nucleotide excretion of C. oleifera genome is rich. C. oleifera prefers A/U ending codons, and nature selection is one of the most important factors affecting CUB. This study will lay a theoretical foundation for the research of molecular evolution and genetic engineering in C. oleifera.

World practice of providing scientific advice on the development and authorisation of innovative medicines

Current challenges to healthcare, i.e. the emergence of new diseases, lack of therapies for known diseases and life-threatening conditions, identification of patients who do not respond to standard treatment, on the one hand, and the evolution of scientific understanding of disease processes, medicines, therapies, causes of treatment failures, and implementation in clinical practice of innovations related to molecular biology and genetic engineering, on the other hand, create conditions and opportunities for the development of innovative medicinal products. A relatively new class of medicines is based on human cells and tissues (the term used in Russian legislation is biomedical cell products, BCP). However, the inability to accurately predict the efficacy and financial rewards of such medicines for pharmaceutical companies, as well as significant labour and financial costs associated with their development and clinical use, hinder their entry into the market. The aim of the study was to analyse the foreign regulatory setting for the development and launch of human cell- and tissue-based products, as well as approaches of foreign regulatory authorities to scientific advice, which can be drawn upon by the Russian expert authority when providing advice to BCP developers. The paper summarises the results of analysis of regulations establishing the procedure for providing scientific advice by EU, USA, and Russian regulatory authorities, and analyses the advice provided for the human cell- and tissue-based products which are now authorised in the EU and USA. The analysis of advice provided by foreign regulatory authorities shows that the largest number of consultations were given for medicinal products based on genetically modified cells for the treatment of cancer and genetic diseases. The questions were mainly related to the contents of specifications for finished pharmaceutical products, safety evaluation, curtailing of preclinical studies due to the lack of relevant animal/disease models, the number of subjects and efficacy endpoints in clinical studies, assessment of the appearance of replication-competent retroviruses.

Strategies and tools for metabolic engineering in Bacillus subtilis

As a typical food safety industrial model strain, Bacillus subtilis has been widely used in the field of metabolic engineering due to its non-pathogenicity, strong ability of extracellular protein secretion and no obvious codon preference. In recent years, with the rapid development of molecular biology and genetic engineering technology, a variety of research strategies and tools have been used to construct B. subtilis chassis cells for efficient synthesis of biological products. This review introduces the research progress of B. subtilis from the aspects of promoter engineering, gene editing, genetic circuit, cofactor engineering and pathway enzyme assembly. Then, we also summarized the application of B. subtilis in the production of biological products. Finally, the future research directions of B. subtilis are prospected.

Screening of somatic mutations in the JAK2 and CALR genes by high-resolution melting curve analysis.

Somatic mutations associated with oncological diseases, including Ph-myeloproliferative neoplasms (Ph-MPN), are very diverse, occur with different frequencies and different allelic burden levels. Therefore, at the initial stage of performing molecular-genetic diagnostic procedures, it is desirable to be able to conduct screening tests in the laboratory. This is especially important when analyzing rare and diverse mutations. Analysis of high resolution melting curves (HRM analysis), which has high sensitivity and is suitable for screening all types of mutations, in a number of studies is proposed for the analysis of Ph-MPN associated mutations in the JAK2 and CALR genes. For analysis of somatic mutations in the majority of literature sources that we reviewed, the authors use the LightCycler (Roche) thermocycler and much rarely the CFX96 (Bio-Rad), which is often presented in Russian scientific and practical and medical organizations. The aim of the study was to screen the somatic JAK2 and CALR mutations by HRM analysis using the CFX96 thermocycler and the Precision Melt Analysis software (Bio-Rad, USA) for patients with Ph-MPN. In the present research, HRM analysis was conducted on the DNA samples from patients with mutations in the JAK2 or in the CALR gene. The Precision Melt Analysis software identified all variants of the analyzed mutations, both a single nucleotide substitution in the JAK2 gene (with allelic burden level in the range of 5-40%), and various indel mutations in the CALR gene (with allelic burden level in the range of 40-50%) Therefore, the HRM analysis that was conducted on the CFX96 allows screening of highly specific mutation for the diagnosis of Ph-MPN in the exon 14 of the JAK2 gene and in the exon 9 of the CALR gene. The inclusion of this screening research in the laboratory testing algorithm improves the efficiency and accessibility of molecular genetic technologies in the diagnosis of Ph-MPN.

ПРОБЛЕМА ГАЛИТОЗА В КОМПЛЕКСНОЙ ДИАГНОСТИКЕ И ЛЕЧЕНИИ ЗАБОЛЕВАНИЙ ПАРОДОНТА. ЛИТЕРАТУРНЫЙ ОБЗОР

Subject. Oral malodour (halitosis) in patients with periodontal disease.
 Objectives. To study and review the literature on local (dental) and general (internal diseases) factors causing halitosis in patients with periodontal disease.
 Conclusion. The present literature review provides a sufficient basis to state that the problem of halitosis is relevant; there is presented the overview of developed classifications and identified combinations of main causes of halitosis linking the extent of ozostomia to oral hygiene status and gingival hemorrhage. By modern molecular genetic technologies (polymerase chain reaction and 16S ribosomal RNA gene chipping), advances have been made in understanding the main role of uncultivated anaerobic oral pathogens associated with halitosis. The materials of this literature review help to get the complex idea of the problem of halitosis in patients with inflammatory periodontal disease and can be used by dental clinicians in health care practice.

Molecular Genetic Methods and Computer Technologies in the System of Epidemiological Surveillance of Salmonella Infection

Introduction: Salmonella infection occupies a leading position in the structure of acute intestinal infections caused by bacterial pathogens. In recent years, with the development of molecular genetic methods and introduction of techniques of computerized data processing, the improvement of the epidemiological surveillance system in the light of the world scientific achievements has become of particular importance. This review is aimed at presenting the history of developing molecular genetic methods and computer technologies in the study of Salmonella infection, and the update on the issue in the Russian Federation based on recent findings of the Research Institute of Epidemiology and Microbiology named after G.P. Somov, one of the leading Russian centers for the study of Salmonella. Materials and methods: We used databases of the Laboratory of Molecular Epidemiology of Somov Research Institute of Epidemiology and Microbiology, and did a literature search in the Web of Science, PubMed, Scopus, Elsevier, Springer, and Google Scholar. Results: Since the second half of the 1980s, the method of plasmid DNA analysis of bacteria of the Salmonella genus has been put into practice. Since 1990, this method has been the basis for microbiological molecular genetic monitoring of the pathogen. The geography of the studied strains, restricted to Primorsky Krai in the 1990s, already in the 2000s encompassed the Far Eastern and Siberian Federal Districts of the Russian Federation. The studies conducted by the Laboratory of Molecular Epidemiology helped improve the system of epidemiological surveillance of the causative agent of salmonellosis in the Far East and revealed the structure of the Salmonella population based on the analysis of plasmids contained in Salmonella strains. Conclusion: Several issues related to microbiology, epidemiology, clinical picture, and prevention of Salmonella infection were resolved during the comprehensive research. We discuss prospects for the development of molecular genetic methods and computer technologies in the study of Salmonella infection in the epidemiological surveillance system in the Russian Federation and emphasize the importance of whole-genome sequencing as a new “gold standard” in molecular epidemiology.

Pathogenetic Therapy of Epidermolysis Bullosa: Current State and Prospects.

Epidermolysis bullosa is a severe hereditary disease caused by mutations in genes encoding cutaneous basement membrane proteins. These mutations lead to dermal-epidermal junction failure and, as a result, to disturbances in the morphological integrity of the skin. Clinically, it manifests in the formation of blisters on the skin or mucosa that in some cases can turn into non-healing chronic wounds, which not only impairs patient's quality of life, but also is a live-threatening condition. Now, the main approaches in the treatment of epidermolysis bullosa are symptomatic therapy and palliative care, though they are little effective and are aimed at reducing the pain, but not to complete recovery. In light of this, the development of new treatment approaches aimed at correction of genetic defects is in progress. Various methods based on genetic engineering technologies, transplantation of autologous skin cells, progenitor skin cells, as well as hematopoietic and mesenchymal stem cells are studied. This review analyzes the pathogenetic methods developed for epidermolysis bullosa treatment based on the latest achievements of molecular genetics and cellular technologies, and discusses the prospects for the use of these technologies for the therapy of epidermolysis bullosa.

CORONAVIRUS DISEASE (COVID-19). CHALLENGES AND PROSPECTS OF SPECIFIC DIAGNOSTICS

The coronavirus disease (COVID-19) pandemic, which started in the late 2019 in China, has become an unforeseen challenge to the health care system of all the countries in the world. Establishment of a mass specific diagnostics of emergent infection caused by the coronavirus SARS-CoV-2 was one of the problems that needed immediate solution. This review presents the technologies used for the specific diagnostics of COVID-19. The advantages and limitations of the most common methodologies for detection of the pathogen or virus-specific antibodies are discussed. Detection of the virus genome fragments by reverse transcription and real-time polymerase chain reaction (rRT-PCR) allowed to achieve high accuracy of diagnosis. From the beginning of the pandemic, this method has been considered as the "gold" standard, despite the limitations associated with its high cost, complexity and the need for testing in specialized laboratories. Cheaper immunological methods have insufficient diagnostic efficiency and can be used only as complements to molecular testing. The review also presents promising methods of specific diagnostics of COVID-19 which are based on molecular genetic technologies, characterized by simplicity and rapidity, do not require expensive equipment and can be performed in points of care.

Primary biliary cholangitis

Primary biliary cholangitis (PBC), formerly known as primary biliary cirrhosis, is an organ-specific autoimmune disease predominantly affecting middle-aged women. It does not occur in children. PBC prevalence varies depending on the geographic location of the country. Over the past 30 years, there has been an increased incidence of PBC, while significant progress has been made in understanding the pathogenesis of PBC due to the development of innovative technologies in molecular biology, immunology and genetics. The presence of antimitochondrial antibodies and cholestasis on biochemical analysis is sufficient to make a diagnosis, without the need for liver biopsy. Small- and medium-sized bile ducts are the targets of PBC. In the first stage of the disease, granulomatous destruction of the bile ducts occurs; in the second stage, loss of bile ducts, their proliferation, increased size of the portal tracts with chronic inflammation; in the third stage - fibrosis with septal formation, loss of bile ducts and cholestasis; in the fourth stage - liver cirrhosis. Previously, the survival rate of PBC patients ranged from 7.5 to 16 years. However, it has improved significantly with ursodeoxycholic acid and obeticholic acid treatment. If there is no effect from treatment and end-stage liver failure sets in, liver transplantation is performed.

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.

MicroRNAs: Potential Targets for Developing Stress-Tolerant Crops.

Crop yield is challenged every year worldwide by changing climatic conditions. The forecasted climatic scenario urgently demands stress-tolerant crop varieties to feed the ever-increasing global population. Molecular breeding and genetic engineering approaches have been frequently exploited for developing crops with desired agronomic traits. Recently, microRNAs (miRNAs) have emerged as powerful molecules, which potentially serve as expression markers during stress conditions. The miRNAs are small non-coding endogenous RNAs, usually 20–24 nucleotides long, which mediate post-transcriptional gene silencing and fine-tune the regulation of many abiotic- and biotic-stress responsive genes in plants. The miRNAs usually function by specifically pairing with the target mRNAs, inducing their cleavage or repressing their translation. This review focuses on the exploration of the functional role of miRNAs in regulating plant responses to abiotic and biotic stresses. Moreover, a methodology is also discussed to mine stress-responsive miRNAs from the enormous amount of transcriptome data available in the public domain generated using next-generation sequencing (NGS). Considering the functional role of miRNAs in mediating stress responses, these molecules may be explored as novel targets for engineering stress-tolerant crop varieties.

MiRNAs: Tiny super-soldiers shaping the life of rice plants for facing “stress”-ful times

Abiotic stresses in different forms are a major cause of large-scale yield loss of agriculturally important crop plants including rice for a long time. Approaches to combat such adverse conditions are shifting from conventional methods to novel molecular biology techniques or genetic engineering mediated methods so as to improve yield attributes by increasing the stress tolerance of these crops. The microRNAs (miRNAs) are a group of small, intriguing molecule of about 22 nucleotides that plays a significant role not only in the regulation of gene expression but also takes part in transcriptional regulation and nutrient homeostasis in the plant system. Most importantly, it is estimated that the miRNAs are significantly involved in the mitigation of multiple plant abiotic stresses, thus a comprehensive understanding of miRNA-mediated abiotic stress mitigation may serve to develop suitable alternative strategies for abiotic stress alleviation in rice plants. Our study specifically deals with the role of different plant miRNAs in drought stress, heat and cold stress, salinity stress, metal(loid) stress (particularly arsenic and cadmium) and their function in regulation of phytohormones production. Moreover, this review is an in-depth compilation and analysis of works related to the stress mitigation role of miRNAs in plants, focusing on the potential mechanism by which these miRNAs work in different abiotic stresses by regulating different gene expression. Finally, we also suggest some of the possible miRNA targets that may be addressed in the near future, for the genetic improvement of important crops such as rice.

Design and evaluation of a custom 50K Infinium SNP array for egg-type chickens

With the development of molecular genetics and high-throughput sequencing technology, genotyping arrays consisting of large numbers of SNP have raised great interest in animal and plant research. However, the application of commercial chicken 600K SNP arrays has varied in different populations of egg-type chickens. Moreover, their genotyping cost is too high for large-scale population applications. Herein, we independently developed a custom Illumina 50K BeadChip, named PhenoixChip-I, for egg-type chickens based on SNP from 479 sequenced individuals in 7 lines. We filtered and selected SNP with stringent criteria, such as high polymorphism, genome coverage, design score, and priorities. Finally, a total of 43,681 effective SNP successfully genotyped were included on our custom array. Approximately 14K SNP were previously reported to be associated with important economic traits in egg-type chickens. Subsequently, we verified the applicability and efficiency of the PhenoixChip-I SNP array from many aspects, including evaluating its use scientific research (population structure analysis and genome-wide association study) and the poultry breeding industry (genomic selection). The findings in our study will play a crucial role in accelerating the genetic improvement of egg-type chickens.

The prospects for the creation of the National Center for Personalized Medicine of Endocrine Diseases

ФГБУ «НМИЦ эндокринологии» Минздрава России (НМИЦ эндокринологии) получил право реализовать программу развития Научного Центра Мирового Уровня «Национальный Центр Персонализированной медицины эндокринных заболеваний» (НЦПМЭЗ). Задачей НЦПМЭЗ станет не только создание системы персонализированного лечения, но и подготовка новых кадров для медицины. Фундаментальные исследования, проводимые на базе уже существующих институтов и лабораторий НМИЦ эндокринологии, будут расширены за счет создаваемых de novo лабораторий НЦПМЭЗ в соответствии с одобренным проектом. Данная публикация знакомит читателя с наиболее значимыми лабораториями, которые планируется создать в НЦПМЭЗ: лабораторией общей, молекулярной и популяционной генетики, биоинформатики, фармакогеномики, микробиоты, редактирования генома, математических и цифровых технологий, неинвазивных технологий диагностики эндокринопатий, клеточных технологий, искусственного интеллекта и принципиально новой, не имеющей аналогов, лабораторией метаболической визуализации и радиотераностики. Авторы надеются, что читатели одного из основных журналов для эндокринологов нашей страны примут активное участие в осуществлении планов НМИЦ эндокринологии, поскольку как у молодых, так и у опытных талантливых исследователей появится шанс стать частью команды Центра. Для выполнения амбициозных задач необходимо кардинально изменить мировоззрение врача в стране, разработать новую систему обучения за счет увеличения числа специалистов по медицинской генетике, транскриптомике, биостатистике и биоинформатике, работающих на стыке экспериментальной и клинической эндокринологии, что обеспечит транзит инновационных технологий в клиническую практику. Созданные лаборатории Научного Центра Мирового Уровня станут местом будничной работы нового поколения врачей, владеющих не только основами клинической работы, но и навыками фундаментальных исследований, что позволит им значительно улучшить методы прогнозирования, диагностики и лечения.

Consequences and Mitigation Strategies of Abiotic Stresses in Wheat (Triticum aestivum L.) under the Changing Climate

Wheat is one of the world’s most commonly consumed cereal grains. During abiotic stresses, the physiological and biochemical alterations in the cells reduce growth and development of plants that ultimately decrease the yield of wheat. Therefore, novel approaches are needed for sustainable wheat production under the changing climate to ensure food and nutritional security of the ever-increasing population of the world. There are two ways to alleviate the adverse effects of abiotic stresses in sustainable wheat production. These are (i) development of abiotic stress tolerant wheat cultivars by molecular breeding, speed breeding, genetic engineering, and/or gene editing approaches such as clustered regularly interspaced short palindromic repeats (CRISPR)-Cas toolkit, and (ii) application of improved agronomic, nano-based agricultural technology, and other climate-smart agricultural technologies. The development of stress-tolerant wheat cultivars by mobilizing global biodiversity and using molecular breeding, speed breeding, genetic engineering, and/or gene editing approaches such as CRISPR-Cas toolkit is considered the most promising ways for sustainable wheat production in the changing climate in major wheat-growing regions of the world. This comprehensive review updates the adverse effects of major abiotic stresses and discusses the potentials of some novel approaches such as molecular breeding, biotechnology and genetic-engineering, speed breeding, nanotechnology, and improved agronomic practices for sustainable wheat production in the changing climate.

High genetic similarity of Salmonella Enteritidis as a predominant serovar by an independent survey in 3 large-scale chicken farms in China

Salmonella Enteritidis (SE) are important zoonotic pathogens, and can be easily transferred to humans by contaminated animal products. Epidemic surveys of SE are necessary in current modern large-scale chicken farms. In this study, Salmonella strains were isolated from possibly infected samples collected at 3 independent farms, and their serotype, drug resistances, virulence genes, and genetic similarity were analyzed by molecular genetic analysis technologies including multilocus sequence typing (MLST), clustered regularly interspaced short palindromic repeats (CRISPR), pulsed-field gel electrophoresis (PFGE), and whole-genome sequencing (WGS). A total of 346 Salmonella strains were isolated from 3,598 samples (9.61%); 329 isolates were identified as SE (95.09%) and 308 isolates were multidrug resistant (93.62%). Virulotyping based on 6 virulence genes showed high similarity in SE isolates of each farm, with the exception of 2 isolates. All SE isolates were found to be the same ST11 type by MLST, and 22 strains of 150 SE isolates selected at random were found to belong to 1 cluster by PFGE and the same SET1 type by CRISPR. WGS results further revealed that these isolates belonged to the same clonal cluster, with high genetic similarity of 99.80 to 100.00%. All these results indicated that these SE isolates were overwhelmingly dominant and demonstrated high genetic similarity, which revealed that the same SE clone might be transmitted in these farms.

ВЛИЯНИЕ ПРЕБИОТИКА ВЕТЕЛАКТ НА МИКРОБИОТУ КИШЕЧНИКА КУР РОДИТЕЛЬСКОГО СТАДА

Using molecular genetic technologies, the positive effect of the prebiotic Vetelact on the intestinal microbiota of commercial laying hens has been shown.

Molecular Cloning and Genetic Engineering Design, Simulation and Management Software for Complex Synthetic Biology

Molecular Cloning and Genetic Engineering Design, Simulation and Management Software for Complex Synthetic Biology

Milestones in the History of Schizophrenia. A Comprehensive Chronology of Schizophrenia Research: What Do We Know and When Did We Know It

Milestones in the History of Schizophrenia. A Comprehensive Chronology of Schizophrenia Research: What Do We Know and When Did We Know It

Hemorrhagic fevers of viral nature. State of the problem and directions for creating effective means of prevention and treatment

Abstract. An attempt to summarize the data of available information materials on epidemiological aspects, the state and prospects of prevention and treatment of hemorrhagic fevers was. Hemorrhagic fevers of viral nature-zoonotic diseases caused by viruses containing ribonucleic acid are classified into 4 families: Arenaviridae, Bunyaviridae, Filoviridae and Flaviviridae. They are spread all over the world, and their pathogens are easily transmitted from person to person, thereby spreading quickly enough beyond the main focus of biological infection. That is why the causative agents of hemorrhagic fevers are regarded as highly contagious biological agents, and agents bioterrorism. Unfortunately, there are currently no effective means of specific prevention and treatment of these infections, and therapeutic measures are limited to the use of symptomatic means. In this regard, the search for substances with pronounced antiviral activity against pathogens of hemorrhagic fevers that can effectively protect against these infections, as well as prevent their occurrence and spread is one of the priority areas of research in modern Infectology, and with the involvement of modern achievements in the field of molecular Virology and genetic engineering. The data obtained in this regard allow a more in-depth understanding of the pathogenesis of hemorrhagic fevers, the mechanisms of interaction of the pathogen with the host at the cellular level, the mechanisms of intracellular replication of viruses, the formation of the hosts response to viral invasion and clinical manifestations of diseases.