Molecular Genetic Engineering Research Articles

Modern molecular genetic technologies for forming a list of representatives normal bird microflora

The results of the metagenomic analysis revealed a high phylogenetic and taxonomic diversity of the microbial community of both luminal and mucosal microflora of the bird intestinal microbiome. Analysis of the taxonomic structure of the compared microbiomes showed both similarities and differences in the taxonomic diversity and relative abundance (%) of bacteria of the compared microbiomes at the level of order, class, genus.At the type level, 24 taxonomic categories were identified in the luminal and mucosal intestinal microbiome of birds, at the class level 35, at the order level 75, at the family level 168, at the genus level 350.The main share of the microbial community of the luminal and mucosal microbiome was made up of microorganisms belonging to the Firmicutes, Proteobacteria, Actinobacteria Cyanobacteria, and Bacteroidetes phyla. Comparison of the biological diversity of the colonic and mucosal microbiomes at the phylum level showed that the relative value of Proteobacteria in relation to Firmicutes in the mucosal microbiome was 3.25 times higher, and the ratio of Firmicutes to Bacteroidetes in the colonic microbiome was 3.98 times higher. than in mucosal.At the genus level, differences in the composition of microbiomes were manifested by a greater relative share (in decreasing order) in the luminal microbiome of representatives of the genus Lactobacillus, Candidatus Arthromitus, Romboutsia, Gallibacterium, Campylobacter, Enterococcus, and representatives of the genus Acinetobacter, Staphylococcus, Bacillus and Bradyrhizobium in the mucosal microbiome. An in-depth study of the intestinal microbiota using cultivation techniques and high-tech methods for identifying microorganisms, taking into account the results of metagenomic analysis, can significantly expand the understanding of the species diversity of microbiomes and the functions of representatives of various taxa in maintaining bird health, as well as to form a census of representatives of normal bird microflora.

An R2R3-MYB network modulates stem strength by regulating lignin biosynthesis and secondary cell wall thickening in herbaceous peony.

Stem strength is an important agronomic trait affecting plant lodging, and plays an essential role in the quality and yield of plants. Thickened secondary cell walls in stems provide mechanical strength that allows plants to stand upright, but the regulatory mechanism of secondary cell wall thickening and stem strength in cut flowers remains unclear. In this study, first, a total of 11 non-redundant Paeonia lactiflora R2R3-MYBs related to stem strength were identified and isolated from cut-flower herbaceous peony, among which PlMYB43, PlMYB83 and PlMYB103 were the most upregulated differentially expressed genes. Then, the expression characteristics revealed that these three R2R3-MYBs were specifically expressed in stems and acted as transcriptional activators. Next, biological function verification showed that these P. lactiflora R2R3-MYBs positively regulated stem strength, secondary cell wall thickness and lignin deposition. Furthermore, yeast-one-hybrid and dual luciferase reporter assays demonstrated that they could bind to the promoter of caffeic acid O-methyltransferase gene (PlCOMT2) and/or laccase gene (PlLAC4), two key genes involved in lignin biosynthesis. In addition, the function of PlLAC4 in increasing lignin deposition was confirmed by virus-induced gene silencing and overexpression. Moreover, PlMYB83 could also act as a transcriptional activator of PlMYB43. The findings of the study propose a regulatory network of R2R3-MYBs modulating lignin biosynthesis and secondary cell wall thickening for improving stem lodging resistance, and provide a resource for molecular genetic engineering breeding of cut flowers.

Идентификация фитопатогенов сельскохозяйственных культур с применением генетических методов и технологий дистанционного зондирования Земли

This article presents an overview of the main molecular genetic methods and technologies of remote sensing used in the diagnosis of various diseases of economically valuable plant species and varieties. The introduction shows the main crops cultivated in Russia. The main phytopathogens considered on the example of wheat are also given. These include fungal diseases (various kinds of smut diseases, rust diseases, septiriosis spots, etc.), bacterial diseases, viral diseases and nematode diseases. In the chapter devoted to genetic research methods, both universal primers for fungal and bacterial infections and species-specific primers for phytopathogens for some economically valuable crops are considered. The chapter on remote sensing presents the main stages of identification of phytopathogens using remote sensing of the Earth. The methods used for analyzing remote sensing data of the Earth are also presented. The last chapter describes possible ways to combine genetic methods and remote sensing of the Earth to create libraries for assessing the areas of affected crops. The positive and negative sides of the genetic analysis of phytopathogens are described. The ways of leveling the negative sides using genetic analysis, involving methods of remote sensing of the Earth are proposed.

Using <i>Bacillus subtilis</i> as an oral vaccine carrier against <i>Streptococcus suis</i>

Due to the progressive growth of the bacteria caused by the widespread use of antibiotics, treatment of streptococcosis is becoming increasingly difficult. Reliable vaccination against Streptococcus suis is necessary. Modern molecular diagnostic and genetic engineering capabilities create prospects for direct cloning of the protective epitopes of the Lmb gene of the local S. suis strain into the proposed delivery system of the pig immune system antigen. Among oral vaccine carriers, Bacillus subtilis is recognized as a relatively environmentally friendly carrier with an efficient protein secretion system and adaptive metabolism capable of spore production under relatively harsh conditions. This spore property can be used to increase the stability and reusability of vaccines. The possibility of using the protective Lmb epitopes of S. suis in B. subtilis as a carrier of an oral recombinant vaccine against Streptococcus suis was studied. The nucleotide sequences of S. suis were obtained from the GenBank database after a preliminary analysis of literature data on the known protective antigens of S. suis of various serotypes. Nucleotide sequence analysis was performed using Unipro UGENE v. 43.0. The Immune Epitope Database (IEDB) was used to search for T (CTL and Th) and B dependent epitopes of the Lmb gene. A computer-designed vaccine in which localization of CTL, B, and Th epitopes is predicted is described. The results of cloning the sequence of the antigenically active epitope of the S. suis Lmb protein in B. subtilis for subsequent oral administration and study of changes in immunological reactions and adverse reactions in animals are described. The possibility to clone the epitopes of recombinant S. suis Lmb protein into the pBE-S polylinker vector was revealed. In the long term, it seems possible to create a new inexpensive and easy-to-use vaccine against S. suis that does not require injection.

IMPROVEMENT OF A SIMPLE AND EFFECTIVE TRANSFORMATION ASSAY FOR E COLI

Searching for efficient means by which bacteria uptake plasmid DNA is of a great practical importance in molecular biology and genetic engineering. We have described in this study five assays for preparing transformants E. coli cells (strain JM109) which are extremely efficient for plasmid uptake that typically yielded transformation efficiencies between 7.2×105 –1.9×107 colony-forming units per microgram (CFU/g) of plasmid DNA. Assay 05 with transformation efficiency 1.9×107 CFU/µg is the simplest and most effective. This assay was considered easy if compared with the original rapid colony transformation protocol described by Hanahan et al., 1991, in which they used transformation buffer (TFB) as transformation solution and the super-optimal broth with catabolite repression (SOC) media, these two are more complex if compared with CaCl2 solution and Luria Bertani (LB) media. One more point in favor of this protocol is its low time consumption. Transformation efficiency between 106 and 107 is not optimal but it is above average for molecular experiments. Therefore, it is possible that these assays may be widely applicable for bacterial transformations with plasmids and can be used as an inexpensive and reliable alternative to existing methods.

Імуногенетичні предиктори алергічного риніту

Abstract. The modern era of molecular-genetic technologies enables us to apply innovative developments in personalized medicine, phenotyping and endotyping of allergic diseases today. The article presents candidate genes that serve as molecular predictors of allergic rhinitis (AR). The presence of AR necessitates a significant increase in medical resources, and its treatment demands a multidisciplinary approach, considering the immunogenetic and pharmacogenetic, metabolic profile that correlates with the potential and effectiveness of rehabilitation for this cohort of patients. Key words: genes, genetic polymorphism, allergic rhinitis.

CRISPR-Cas Genome Editing for Horticultural Crops Improvement: Advantages and Prospects

Horticultural plants, in particular fruit trees, berry crops, and ornamentals, are valuable objects for studying their genetic and biochemical properties. Along with traditional methods of studying these plants, modern molecular genetic technologies are emerging, in particular genome editing using CRISPR/Cas9 nucleases. In this review, we have analyzed modern advances in genome editing of horticultural plants. To date, it has become possible to improve many plant characteristics using this technology, e.g., making plants resistant to biotic and abiotic stress factors, changing the time of flowering and fruit ripening, changing the growth characteristics of plants, as well as the taste properties of their fruits. CRISPR/Cas9 genome editing has been successfully carried out for many horticultural plants. Dozens of genes from these plants have been modified by means of genome editing technology. We have considered the main ways of delivering genetic constructs to plants as well as limitations that complicate the editing of target genes. The article reviews the prospects of using genome editing to improve the valuable properties of plants important to humans.

CACA guidelines for holistic integrative management of glioma

Glioma of the brain is a kind of tumor originating from neuroglial cells. It is the most common primary intracranial tumor, accounting for ~ 30% of all central nervous system tumors and 80% of malignant brain tumors. Glioma is characterized by high disability and recurrence rates. The disease seriously threatens the life of patients, affects their quality of life, and brings a heavy economic and psychological burden to patients, families, and society.With the progression of molecular genetic testing technology and the completion of various clinical trials, the classification scheme for glioma is increasingly well established. Diagnosis and treatment regimens, including traditional and new regimens, are becoming increasingly specialized and standardized. The purpose is to develop a clinical diagnosis and treatment guideline for glioma in the Chinese population suitable for Chinese doctors and the general population based on domestic and international glioma research progress. Thus, domestic practitioners in the field can obtain current information and provide better service to patients with glioma, promoting the development of domestic clinical medicine and basic research on glioma.

Research Progress on the Effect of Autolysis to Bacillus subtilis Fermentation Bioprocess

Bacillus subtilis is a gram-positive bacterium, a promising microorganism due to its strong extracellular protein secretion ability, non-toxic, and relatively mature industrial fermentation technology. However, cell autolysis during fermentation restricts the industrial application of B. subtilis. With the fast advancement of molecular biology and genetic engineering technology, various advanced procedures and gene editing tools have been used to successfully construct autolysis-resistant B. subtilis chassis cells to manufacture various biological products. This paper first analyses the causes of autolysis in B. subtilis from a mechanistic perspective and outlines various strategies to address autolysis in B. subtilis. Finally, potential strategies for solving the autolysis problem of B. subtilis are foreseen.

Ecological scenario of the plague microbe <i>Yersinia pestis</i> speciation underlying adequate molecular evolutionary model

It is known that the psychrophilic pseudotuberculosis microbe serotype 1 (Y. pseudotuberculosis 0:1b) causing Far East scarlet-like fever (FESLF) an intestinal infection found in a wide range of invertebrates and vertebrates inhabiting cold regions in the Northern and Central Asia as well as Far East is direct ancestor of the plague causative agent Yersinia pestis. However, the mechanism of Y. pestis speciation remains poorly elucidated. Numerous Y. pestis phylogenies created by using molecular genetic (MG) technologies are largely contradictory, being not in line with reliable data obtained by natural science approaches (e.g., ecology, epizootology, biogeography, and paleontology), which disagree with current evolutionary doctrine (synthetic theory of evolution). The MG approach provides no definitive answer to the questions of where, when, how, and under what circumstances the species Y. pestis arose. One of the reasons for such situation might be due to inadequacy of using the molecular evolutionary model for Y. pestis phylogenetics. Knowledge of the life cycles for the ancestral pseudotuberculosis and derivative plague microbes as well as related unique environmental features allows to create a reliable ecological model for the plague microbe evolution to be further used for assessing patterns of molecular variability and building proper molecular model that might be accepted for MG-reconstruction of plague microbe history. According to the ecological model, the species Y. pestis was formed in a tritopic manner (almost) simultaneously from FESLF clones (populations) in the three geographical populations of the Mongolian marmot-tarbagan (Marmota sibirica) and the flea Oropsylla silantiewi parasitizing on it. The inducer of speciation was coupled to the last maximum (Sartan) cooling in Central Asia occurred 2215 thousand years ago. Soil cooling and deep freezing resulted in altered behavior of the marmot flea larvae with emergence of facultative hematophagy, which, in turn, led to a unique traumatic (compared to routine alimentary) infection route of sleeping marmots with FESLF and, as a result, a unique way of Y. pestis speciation. The molecular model should predict a Y. pestis peripatric tritope speciation, existing numerous parallelisms in intraspecific variability associated with tritope speciation, and the quantum principle of speciation in the highly variable heterothermic (heteroimmune) stressful marmot-flea (Marmota sibirica Oropsylla silantiewi) host-vector environment involving stress-induced mutagenesis. Such molecular model of evolution may be useful for improving molecular methodology of phylogenetic constructions for a wide range of parasitic microorganisms.

Genome-Wide Association Study Reveals Novel QTLs and Candidate Genes for Grain Number in Rice.

Grain number per panicle (GNPP), determined mainly by panicle branching, is vital for rice yield. The dissection of the genetic basis underlying GNPP could help to improve rice yield. However, genetic resources, including quantitative trait loci (QTL) or genes for breeders to enhance rice GNPP, are still limited. Here, we conducted the genome-wide association study (GWAS) on the GNPP, primary branch number (PBN), and secondary branch number (SBN) of 468 rice accessions. We detected a total of 18 QTLs, including six for GNPP, six for PBN, and six for SBN, in the whole panel and the indica and japonica subpanels of 468 accessions. More importantly, qPSG1 was a common QTL for GNPP, PBN, and SBN and was demonstrated by chromosome segment substitution lines (CSSLs). Considering gene annotation, expression, and haplotype analysis, seven novel and strong GNPP-related candidate genes were mined from qPSG1. Our results provide clues to elucidate the molecular regulatory network of GNPP. The identified QTLs and candidate genes will contribute to the improvement of GNPP and rice yield via molecular marker-assisted selection (MAS) breeding and genetic engineering techniques.

Improving Stability and Specificity of CRISPR/Cas9 System by Selective Modification of Guide RNAs with 2'-fluoro and Locked Nucleic Acid Nucleotides.

The genome editing approach using the components of the CRISPR/Cas system has found wide application in molecular biology, fundamental medicine and genetic engineering. A promising method is to increase the efficacy and specificity of CRISPR/Cas-based genome editing systems by modifying their components. Here, we designed and chemically synthesized guide RNAs (crRNA, tracrRNA and sgRNA) containing modified nucleotides (2'-O-methyl, 2'-fluoro, LNA-locked nucleic acid) or deoxyribonucleotides in certain positions. We compared their resistance to nuclease digestion and examined the DNA cleavage efficacy of the CRISPR/Cas9 system guided by these modified guide RNAs. The replacement of ribonucleotides with 2'-fluoro modified or LNA nucleotides increased the lifetime of the crRNAs, while other types of modification did not change their nuclease resistance. Modification of crRNA or tracrRNA preserved the efficacy of the CRISPR/Cas9 system. Otherwise, the CRISPR/Cas9 systems with modified sgRNA showed a remarkable loss of DNA cleavage efficacy. The kinetic constant of DNA cleavage was higher for the system with 2'-fluoro modified crRNA. The 2'-modification of crRNA also decreased the off-target effect upon in vitro dsDNA cleavage.

Disposable, pressure-driven, and self-contained cartridge with pre-stored reagents for automated nucleic acid extraction

Nucleic acid extraction is vital in many applications such as molecular diagnostics, genetic engineering, and deoxyribonucleic acid (DNA) sequencing. Although recent advances in nucleic acid extraction have been made to improve the process using microfluidics, it is still necessary to make it easy for end-users by having pre-loaded reagents, eliminating pipetting between steps, and automating the process for point-of-care (POC) testing. Herein, we present a pressure-driven and self-contained cartridge with pre-stored reagents for automating nucleic acid purification. To reduce operational complexity, reagents were transferred through a microfluidic chip using pressurized air stored inside the cartridge instead of an external pump or valving system. After performing cell lysis, the cartridge was inserted into the device, and the nucleic acid was purified automatically within 3 min Escherichia coli (E. coli) O157:H7 DNA extracted by our device showed similar concentration, purity, and real-time polymerase chain reaction (qPCR) results as the conventional column-based nucleic acid extraction method. Our device achieved a detection limit of 103 CFU for E. coli DNA, which is the same as that obtained using the conventional solid-phase extraction method. This study introduces a novel approach for automating the sample preparation process, which can help in facilitating POC molecular diagnostics.

Inborn Errors of Metabolism Screening in Neonates: Current Perspective with Diagnosis and Therapy.

Inborn errors of metabolism (IEMs) are rare hereditary or acquired disorders resulting from an enzymatic deformity in biochemical and metabolic pathways influencing proteins, fats, carbohydrate metabolism, or hampered some organelle function. Even though individual IEMs are uncommon, together, they represent a diverse class of genetic diseases, with new issues and disease mechanisms being portrayed consistently. IEM includes the extraordinary multifaceted nature of the fundamental pathophysiology, biochemical diagnosis, molecular level investigation, and complex therapeutic choices. However, due to the molecular, biochemical, and clinical heterogeneity of IEM, screening alone will not detect and diagnose all illnesses included in newborn screening programs. Early diagnosis prevents the emergence of severe clinical symptoms in the majority of IEM cases, lowering morbidity and death. The appearance of IEM disease can vary from neonates to adult people, with the more serious conditions showing up in juvenile stages along with significant morbidity as well as mortality. Advances in understanding the physiological, biochemical, and molecular etiologies of numerous IEMs by means of modalities, for instance, the latest molecular-genetic technologies, genome engineering knowledge, entire exome sequencing, and metabolomics, have prompted remarkable advancement in detection and treatment in modern times. In this review, we analyze the biochemical basis of IEMs, clinical manifestations, the present status of screening, ongoing advances, and efficiency of diagnosis in treatment for IEMs, along with prospects for further exploration as well as innovation.

Assessment of Growth and Yield Performance of Wheat Genotypes (Triticum aestivum L) under Heat Stress Environment

An experiment was laid out with six wheat genotypes K-9162, K910-30, DBW-16, AAI-16, K-911, and NW-1014. This study was conducted during winter season 2020-2021 at the Student Instructional Farm and in the laboratory of Plant Molecular Biology and Genetic Engineering at Acharya Narendra Deva University of Agriculture & Technology, Kumarganj, Ayodhya –224 229 (U.P.) in Randomized Block Design (R.B.D.) The wheat genotypes were exposed to heat stress by delayed sowing of 60 days from the normal date of sowing (15 January 2021) so that reproductive stage of wheat can experience heat stress. The field preparation and normal agronomic practices were followed as per need of the crop. The wheat genotypes K-9162, K910-30 and K-911 showed less reduction in yield and yield components over the DBW-16, AAI-16 and NW-1014 wheat genotypes. Therefore, on the basis of less reduction in yield and yield components under heat stress, K-910-30, K-911 and K-9162 were showed the significant tolerant while the other genotypes like DBW-16, NW-1014 and AAI-16 were found high yielding under normal condition but also showed high reduction in yield under heat stress condition.

Development of SNP Markers from GWAS for Selecting Seed Coat and Aleurone Layers in Brown Rice (Oryza sativa L.).

Ninety-five percent of the general nutrients in rice are concentrated in the rice bran and germ, and many nutrients such as vitamins, minerals, dietary fiber, and essential fatty acids, as well as antioxidants such as tocopherol, are lost during milling. In this study, we investigated the thickness of seed coat and aleurone layers using a 294 rice core collection, and found candidate genes related to thickness of seed coat and aleurone layers, by performing a genome wide association study (GWAS) analysis using whole genome resequencing data. Two primer pairs that can be used as high-resolution melting (HRM) markers were developed. As a result of genotyping BC2F2 individuals derived from a cross between “Samgwang” and “Seolgaeng”, and using corresponding HRM markers, it was possible to finally develop HRM markers for selecting seed coat and aleurone layer thickness. This is expected to be used as basic data for the application of gene editing using CRISPR/Cas9 technology and for establishing a breeding strategy for high eating quality rice using molecular genetic technology.

COMPARATIVE ASSESSMENT OF CYTOGENETIC AND MOLECULAR APPROACHES FOR PRODUCT OF CONCEPTION: A RETROSPECTIVE STUDY OF 726 CASES

Spontaneous abortions occur approximately in 15 to 20% of pregnancies and chromosomal alterations are responsible for 50 to 70% of the cases. In the past, assessment of genetic abnormalities was limited to karyotype performed on placental or fetal tissue. However, advances in molecular genetic technologies now provide rich genetic information about the causes and risks for pregnancy loss. The main goal was to analyze the molecular and cytogenetic data obtained from miscarriage product, evaluated by four different techniques, to understand the advantages and limitations of each approach and have a conclusive result that assists in the investigation of the couple.

SYMPHONY consortium: Orchestrating personalized treatment for patients with bleeding disorders

BackgroundTreatment choices for individual patients with an inborn bleeding disorder are increasingly challenging due to increasing options and rising costs for society. We have initiated an integrated interdisciplinary national research program. ObjectivesThe SYMPHONY consortium strives to orchestrate personalized treatment in patients with an inborn bleeding disorder, by unraveling the mechanisms behind interindividual variations of bleeding phenotype. PatientsThe SYMPHONY consortium will investigate patients with an inborn bleeding disorder, both diagnosed and not yet diagnosed. ResultsResearch questions are categorized under the themes: (1) diagnosis, (2) treatment, and (3) fundamental research, and consist of work packages addressing specific domains. Importantly, collaborations between patients and talented researchers from different areas of expertise promise to augment the impact of the SYMPHONY consortium, leading to unique interactions and intellectual property. ConclusionsSYMPHONY will perform research on all aspects of care, treatment individualization in patients with inborn bleeding disorders, as well as diagnostic innovations and results of molecular genetics and cellular model technology with regard to the hemostatic process. We believe that these research investments will lead to health‐care innovations with long‐term clinical and societal impact.This consortium has been made possible by a governmental, competitive grant from the Netherlands Organization for Scientific Research (NWO) within the framework of the NWA‐ORC Call grant agreement NWA.1160.18.038.

Genetic diagnosis and treatment of hereditary renal tubular disease with hypokalemia and alkalosis.

Renal tubules play an important role in maintaining water, electrolyte, and acid-base balance. Renal tubule dysfunction can cause electrolyte disorders and acid-base imbalance. Clinically, hypokalemic renal tubular disease is the most common tubule disorder. With the development of molecular genetics and gene sequencing technology, hereditary renal tubular diseases have attracted attention, and an increasing number of pathogenic genes related to renal tubular diseases have been discovered and reported. Inherited renal tubular diseases mainly occur due to mutations in genes encoding various specific transporters or ion channels expressed on the tubular epithelial membrane, leading to dysfunctional renal tubular reabsorption, secretion, and excretion. An in-depth understanding of the molecular genetic basis of hereditary renal tubular disease will help to understand the physiological function of renal tubules, the mechanism by which the kidney maintains water, electrolyte, and acid-base balance, and the relationship between the kidney and other systems in the body. Meanwhile, understanding these diseases also improves our understanding of the pathogenesis of hypokalemia, alkalosis and other related diseases and ultimately promotes accurate diagnostics and effective disease treatment. The present review summarizes the most common hereditary renal tubular diseases (Bartter syndrome, Gitelman syndrome, EAST syndrome and Liddle syndrome) characterized by hypokalemia and alkalosis. Further detailed explanations are provided for pathogenic genes and functional proteins, clinical manifestations, intrinsic relationship between genotype and clinical phenotype, diagnostic clues, differential diagnosis, and treatment strategies for these diseases.

Postnatal neurogenesis in the human brain

Recently, a lot of data has been gathered which demonstrates neurogenesis in the brain of adult humans. In genetics, findings have been obtained that not only prove, but also elucidate the molecular mechanisms of neurogenesis. In some publications, however, morphology disputes neuronal renewal in adulthood. Therefore, this review presents the modern achievements of epigenetics, morphology, and physiology, which confirm and characterize postnatal neurogenesis in detail. We suggest that the introduction of molecular genetic technologies into morphological studies will be the starting point for the integration of these areas, complementing each other for the introduction of targeted therapy in clinical practice. Numerous evidence has been obtained of the presence of postnatal neurogenesis in adult humans in studies using bromodeoxyuridine, a carbon isotope of 14C, and 3H-thymidine, in comparative analyses of experimental data from animals. Neuronal stem cells, represented by radial glia present in the subventricular and subgranular zones of the human brain, are morphologically similar to neuroepithelial cells. They express marker proteins for astrocytes, which suggests that the proliferation of neuroglia found in adults can also indicate the regeneration of neurons. To prove this, further studies are required, with the exact identification of newly-formed cells, using specific molecular markers, and data from modern epigenetics. The integration of molecular genetic methods into morphology will facilitate not only the accurate determination of the classification of cells to a specific subpopulation but also to study the effects of various agents on the proliferation of neurons in the adult brain.