Molecular Biotechnology Research Articles

RAAV expressing recombinant antibody for emergency prevention and long-term prophylaxis of COVID-19.

Numerous agents for prophylaxis of SARS-CoV-2-induced diseases are currently registered for the clinical use. Formation of the immunity happens within several weeks following vaccine administration which is their key disadvantage. In contrast, drugs based on monoclonal antibodies, enable rapid passive immunization and therefore can be used for emergency pre- and post-exposure prophylaxis of COVID-19. However rapid elimination of antibody-based drugs from the circulation limits their usage for prolonged pre-exposure prophylaxis. In current work we developed a recombinant adeno-associated viral vector (rAAV), expressing a SARS-CoV-2 spike receptor-binding domain (RBD)-specific antibody P2C5 fused with a human IgG1 Fc fragment (P2C5-Fc) using methods of molecular biotechnology and bioprocessing. A P2C5-Fc antibody expressed by a proposed rAAV (rAAV-P2C5-Fc) was shown to circulate within more than 300 days in blood of transduced mice and protect animals from lethal SARS-CoV-2 virus (B.1.1.1 and Omicron BA.5 variants) lethal dose of 105 TCID50. In addition, rAAV-P2C5-Fc demonstrated 100% protective activity as emergency prevention and long-term prophylaxis, respectively. It was also demonstrated that high titers of neutralizing antibodies to the SARS-CoV-2 virus were detected in the blood serum of animals that received rAAV-P2C5-Fc for more than 10 months from the moment of administration.Our data therefore indicate applicability of an rAAV for passive immunization and induction of a rapid long-term protection against various SARS-CoV-2 variants.

Ceramide Synthase 1 Inhibits Brain Metastasis of Non-Small Cell Lung Cancer by Interacting with USP14 and Downregulating the PI3K/AKT/mTOR Signaling Pathway

Brain metastasis (BM) is common in patients with non-small cell lung cancer (NSCLC) and is associated with a poor prognosis. Ceramide synthase 1 (CERS1) participates in malignancy development, but its potential role in NSCLC BM remains unclear. This study aimed to explore the physiological effects and molecular mechanism of CERS1 in NSCLC BM. CERS1 expression was evaluated in NSCLC tissues and cell lines, and its physiological roles were subsequently explored in vivo and in vitro. Mass spectrometry and co-immunoprecipitation were performed to explore CERS1-interacting proteins. The associated signaling pathways of CERS1 in NSCLC BM were further investigated using bioinformatics analysis and molecular biotechnology. We demonstrated that CERS1 was significantly downregulated in NSCLC cell lines and BM tissues, and its upregulation was associated with better prognoses. In vitro, CERS1 overexpression inhibited cell migration, invasion, and the ability to penetrate the blood-brain barrier. Moreover, CERS1 interacted with ubiquitin-specific protease 14 (USP14) and inhibited BM progression by downregulating the PI3K/AKT/mTOR signaling pathway. Further, CERS1 expression substantially suppressed BM tumor formation in vivo. This study demonstrated that CERS1 plays a suppressor role in NSCLC BM by interacting with USP14 and downregulating the PI3K/AKT/mTOR signaling pathway, thereby serving as a novel therapeutic target for NSCLC BM.

Nanotechnology for Animal Sciences-New Insights and Pitfalls: A Review

Nanotechnology applications are increasingly recognized as potential tools in animal sciences, veterinary medicine, drug delivery, disease diagnosis and vaccine development. The nanoparticles and their therapeutic trends provide a realistic glimpse of current and future trends in veterinary diagnosis and medicine. Nanotechnology is ascribed to provide new horizons for applications in molecular biology and biotechnology by revolutionizing almost every field of veterinary or animal sciences by offering novel nano-scale tools and materials which can benefit society in all walks of life. The nanoparticles have been employed in diverse biological and non-biological functions. Nanomaterials can contribute to the farm animals for long-term sustainability by increasing the quality and quantity of safe, healthy and functional animal products. Although there are advantages while using nanoparticles to enhance performance and heal diseases in livestock, it is universally recognized that some nanoparticles can cause toxic and adverse effects on living creatures. Apart from that, the promise of nanoscience in veterinary medicine and livestock reproduction is yet to be explored. A few studies have been made on the uses of nanoparticles in veterinary medicine, livestock health and reproduction. Awareness is needed about the possible toxic effects and hazards, which could harm the living world. To improve the efficient and accurate diagnosis and treatment of animal diseases, more sophisticated research on nanomaterials and nanomedicines is required.

Comparative Analysis of DNA-Polymerases from Family A as a Tool to Search for Enzymes with New Properties

DNA-polymerases catalyze DNA synthesis during DNA replication, repair, and recombination. A number of DNA-polymerases, such as the Taq enzyme from Thermus aquaticus, are used in various applications of molecular biology and biotechnology, in particular as DNA amplification tools. However, the efficiency of these enzymes depends on factors such as DNA origin, primer composition, template length, GC-content, and the ability to form stable secondary structures. Such limitations in the use of currently known DNA-polymerases leave the problem of searching for new enzymes with improved properties. This review summarizes the main structural and molecular-kinetic features of the functioning of DNA-polymerases belonging to the structural family A, including Taq polymerase. A phylogenetic analysis of these enzymes was carried out, which made it possible to establish a highly conserved consensus sequence containing 62 amino acid residues distributed over the structure of the enzyme. A comparative analysis of these amino acid residues among poorly studied DNA-polymerases revealed 7 enzymes that potentially have the properties necessary for their use for DNA amplification.

Challenges of Scientific Research in Molecular Biotechnology in Yemen

Research is achieved for the sake of contribution towards science and getting information systematically, starting from the collection of data, passing through interpretation and final evaluation of these data and all that, in an organized manner which is called scientific research. Biotechnology includes the application both of older biological knowledge, which is based on structures, functioning, and development of animals and plants in their ecological setting, and of newer approaches, which are based on an understanding of genetics and on the cellular and subcellular processes. The development of biotechnology has given society an awesome new power and an equally awesome responsibility to use it wisely. Biotechnology is being applied in different areas of science such as agriculture, health, the environment, and many other aspects of science and industry. Most of these applications and developments are centered in the developed countries and the research is established by these notions. Currently, it has been obvious that biotechnology is crucial for notion’s economic growth and sustainable development. Yemen, as one of the most developing countries, is suffering to emerge molecular biotechnology research in universities and research units. This is because of many aspects that affect the soceity, goverment, and ultimatly the entire country, including society, human resources, facilities, infrastructure and environment resources, and stability. To conclude, the researchers in Yemen are surrounding by many challenges, which are starting in society and ending up in the laboratory. The research has very low priority on a long list of priorities such as food, stability, security, and healthcare. All the challenges can be solved, but it will need a more educated society and serious government policy toward research as a tool to build a strong society.

Stability and Molecular Characterization of Heat Tolerant Genotypes of Chickpea (Cicer arietinum L.)

Twenty eight diverse genotypes sown in three different dates were screened using thirty three SSR primers. Twelve morphological characters recorded. The current study was conducted at all India Coordinated Research Project on Chickpea at R.A.K., College of Agriculture, Sehore (M.P.) during Rabi 2020-21, and 2021-22. The molecular work was carried out at Plant Molecular Biology Laboratory, Department of Plant Molecular Biology and Biotechnology, College of Agriculture, Rajmata Vijayaraje Scindia Krishi Vishwa Vidyalaya, Gwalior (M.P.). The component G×E interaction were found significant for flower initiation, days to 50% flowering, days to pod initiation, plant height, days to maturity, number of pods per plant, number of empty pods per plant, number of seeds per plant, biological yield per plant, harvest index, 100 seed weight and seed yield per plant. The highest gene diversity was found in TA-135 (0.7474) followed by GAA-44 (0.7219), GAA-40 (0.7015), STMS-2 (0.6939), TA-71 (0.6709), NCPGR-1 (0.6403) and TA-18 (0.3648). Based on a dendrogram all the 28 genotypes were grouped into three major clusters, in which cluster I contained 2 genotypes, cluster II contained 5 genotypes and cluster III encompassed remaining 21 genotypes. Genotypes RVG 204, JG-14, and RVSSG-61 were found stable for favourable and unfavourable sowing conditions, while ICC-4958, JG-11, JG-12, RVG-203, RVG-204, RVSSG-52, JG-74, RVSSG-71 showed consistent performance during unfavourable sowing conditions for seed yield per plant. The important traits and marker based diversity and stability has been discussed in this research paper.

Heterologous mogrosides biosynthesis in cucumber and tomato by genetic manipulation

Mogrosides are widely used as high-value natural zero-calorie sweeteners that exhibit an array of biological activities and allow for vegetable flavour breeding by modern molecular biotechnology. In this study, we developed an In-fusion based gene stacking strategy for transgene stacking and a multi-gene vector harbouring 6 mogrosides biosynthesis genes and transformed it into Cucumis sativus and Lycopersicon esculentum. Here we show that transgenic cucumber can produce mogroside V and siamenoside I at 587 ng/g FW and 113 ng/g FW, respectively, and cultivated transgenic tomato with mogroside III. This study provides a strategy for vegetable flavour improvement, paving the way for heterologous biosynthesis of mogrosides.

Yuan Longping, a great world hunger fighter

Yuan Longping, a great world hunger fighter

Comparative Analysis of Family A DNA-Polymerases as a Searching Tool for Enzymes with New Properties

DNA polymerases catalyze DNA synthesis during DNA replication, repair, and recombination. A number of DNA polymerases, such as the Taq enzyme from Thermus aquaticus, are used in various applications of molecular biology and biotechnology, in particular as DNA amplification tools. However, the efficiency of these enzymes depends on factors such as DNA origin, primer composition, template length, GC-content, and the ability to form stable secondary structures. These limitations in the use of currently known DNA polymerases lead to the search for new enzymes with improved properties. This review summarizes the main structural and molecular-kinetic features of the functioning of DNA-polymerases belonging to structural family A, including Taq polymerase. A phylogenetic analysis of these enzymes was carried out, which made it possible to establish a highly conserved consensus sequence containing 62 amino acid residues distributed over the structure of the enzyme. A comparative analysis of these amino acid residues among poorly studied DNA-polymerases revealed 7 enzymes that potentially have the properties necessary for use in DNA amplification.

Archaea as a Model System for Molecular Biology and Biotechnology.

Archaea represents the third domain of life, displaying a closer relationship with eukaryotes than bacteria. These microorganisms are valuable model systems for molecular biology and biotechnology. In fact, nowadays, methanogens, halophiles, thermophilic euryarchaeota, and crenarchaeota are the four groups of archaea for which genetic systems have been well established, making them suitable as model systems and allowing for the increasing study of archaeal genes' functions. Furthermore, thermophiles are used to explore several aspects of archaeal biology, such as stress responses, DNA replication and repair, transcription, translation and its regulation mechanisms, CRISPR systems, and carbon and energy metabolism. Extremophilic archaea also represent a valuable source of new biomolecules for biological and biotechnological applications, and there is growing interest in the development of engineered strains. In this review, we report on some of the most important aspects of the use of archaea as a model system for genetic evolution, the development of genetic tools, and their application for the elucidation of the basal molecular mechanisms in this domain of life. Furthermore, an overview on the discovery of new enzymes of biotechnological interest from archaea thriving in extreme environments is reported.

Multi-macromolecular Extraction from Endosymbiotic Anthozoans.

Obligately symbiotic associations between reef-building corals (anthozoan cnidarians) and photosynthetically active dinoflagellates of the family Symbiodiniaceae comprise the functional basis of all coral reef ecosystems. Given the existential threats of global climate change toward these thermo-sensitive entities, there is an urgent need to better understand the physiological implications of changes in the abiotic milieu of scleractinian corals and their mutualistic algal endosymbionts. Although initially slow to leverage the immense breakthroughs in molecular biotechnology that have benefited humankind, coral biologists are making up for lost time in exploiting an array of ever-advancing molecular tools for answering key questions pertaining to the survival of corals in an ever-changing world. In order to comprehensively characterize the multi-omic landscape of the coral holobiont-the cnidarian host, its intracellular dinoflagellates, and a plethora of other microbial constituents-I introduce a series of protocols herein that yield large quantities of high-quality RNA, DNA, protein, lipids, and polar metabolites from a diverse array of reef corals and endosymbiotic sea anemones. Although numerous published articles in the invertebrate zoology field feature protocols that lead to sufficiently high yield of intact host coral macromolecules, through using the approach outlined herein one may simultaneously acquire a rich, multi-compartmental biochemical pool that truly reflects the complex and dynamic nature of these animal-plant chimeras.

Focused Engineering of Pyrrolysyl-tRNA Synthetase-Based Orthogonal Translation Systems for the Incorporation of Various Noncanonical Amino Acids.

The expansion of the genetic code has become a valuable tool for molecular biology, biochemistry, and biotechnology. The pyrrolysyl-tRNA synthetase (PylRS) variants with their cognate tRNAPyl derived from methanogenic archaea of the genus Methanosarcina are the most popular tools for ribosomally mediated site-specific and proteome-wide statistical incorporation of noncanonical amino acids (ncAAs) into proteins. The incorporation of ncAAs can be used for numerous biotechnological and even therapeutically relevant applications. Here we present a protocol of engineering PylRS for novel substrates with unique chemical functionalities. These functional groups can act as intrinsic probes, especially in complex biological environments such as mammalian cells, tissues, and even whole animals.

Gastroesophageal Reflux Caused Pharyngitis in Local Population A Comparative Clinical Study

Objective: In gastroesophageal reflux disease gastric contents caused erosive action in esophagus as well as in upper respiratory chamber which is a very common and painful medical complication in our population. Therefore investigation of GERD related risk factors are very important for health awareness. Study Design: A comparative clinical study. Population Sampling: In this study 2000 male and female individuals were selected with gastroesophageal reflux diseases from urban, suburban areas and rural areas. Sample Size: 2000 individuals collectively participated in present study, including 900 from urban areas, 853 from suburban areas, and 1010 from rural areas. Place and Duration: Current study was conducted in Department of Biochemistry Lahore Medical & Dental College Lahore, Indus Medical College Tando Muhammad khan Sindh and Institute of molecular Biology and Biotechnology (IMBB), The University of Lahore, Lahore Pakistan from February 2022 to July 2022. Methodology: In current study total 2000 individuals were selected and their interviewee conducted for the completion of questionnaire. The questionnaire was created based on lifestyle factors such as obesity, smoking, drinking, gastroesophageal reflux disease (GERD) frequency and severity in previous years, and history of respiratory and laryngopharyngeal disorders in the previous year. Results: The prevalence of heartburn, Regurgitation, Epigastric pain, Dyspepsia, Pharyngitis, Laryngitis, GRED Frequency, Smoking, Drinking alcohol and BMI among male and female individuals has significant (P > 0.05) with laryngopharyngeal, respiratory illnesses. The Correlation between percentage association of gastroesophageal reflux disease and laryngopharyngeal illness showed in table-3. The findings of this study were indicated a directly proportional significant (P>0.05) correlation of Pharyngitis and Laryngitis with GRED Frequency of individuals. Practical implication: Through current study health awareness can be delivered in the local population. Conclusion: In conclusion, gastroesophageal reflux disease is a substantial public health issue that affects a large portion of the adult population. The pathophysiology and etiology of gastroesophageal reflux disease are likely linked to a number of laryngopharyngeal, respiratory illnesses. Keywords: Esophagus, Gastroesophageal reflux diseases, Heartburn, Regurgitation and Retrosternal pain

Using loop‐mediated isothermal amplification combined with gold nanoparticles for optically rapid detection of shrimp Vibrio parahaemolyticus

AbstractShrimp is one of the most important aquatic products in the world. Early detection, prevention, and treatment of shrimp pathogens is important. One major shrimp pathogen, Vibrio parahaemolyticus causes acute hepatopancreatic necrosis disease (VPAHPND) has been studied, but research into pathogen detection needs to be strengthened. Therefore, we were targeting to VPAHPND and using emerging molecular biotechnologies to develop a method for pathogens detection for the shrimp industry. Gold nanoparticles (AuNPs) have an unique color change property. Free AuNPs in solution are the color of claret‐red, while the aggregated AuNPs appear slight purple. Based on the characteristics of visible color difference of AuNPs, we worked on establishing a simple, fast, and specific loop‐mediated isothermal amplification (LAMP) detection method for VPAHPND diagnosis using AuNPs. This method, amplified the target DNA of VPAHPND by LAMP and the DNA were directly added onto AuNPs solution for detection. The test results show a color change, so that the results can be visually inspected by naked eye. Using the methods, shrimp VPAHPND can be early detected and treated, boost the economic benefits, and efficiency of shrimp farming.

The 21st International Conference on Progress in Vaccination against Cancer (PIVAC-22), September 26-28, 2022, Turin, Italy.

The 21st international congress on Progress in Vaccination against Cancer (PIVAC-22, https://www.pivac22.it/) was held from September 26 to September 28, 2022, at the Molecular Biotechnology Center "Guido Tarone" in Turin, Italy. The meeting covered the most recent advances in the field of cancer immunotherapy, with a focus on the tumor microenvironment, and addressed how alterations to the microenvironment's molecular and metabolic features and the microbiota impact upon the response to immunotherapy.

Application of Molecular Biotechnology in Food Testing

Application of Molecular Biotechnology in Food Testing

Cu-Chitosan Nanoparticles Stimulated Defence Responses against Bacterial Pustule Disease in Soybean Crop

Aim: Assessment of Cu-chitosan nanoparticles on controlling Bacterial disease by enhancing the activity of different antioxidant and defence enzymes in soybean.
 Study Design: Randomized block Method
 Place and Duration of Study: Department of Molecular biology and Biotechnology (MBBT), RCA, Maharana Pratap University of Agriculture and Technology, Udaipur Sub humid southern plain region of India between 2017-2019.
 Methodology: We synthesized, characterized and tested the efficacy of Copper-chitosan Nanoparticles against Bacterial Pustule disease in Soybean. Activity of defence related enzymes [polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL)], and antioxidant [peroxidise (POD) and superoxide dismutase (SOD)] were estimated after treating the plants with various concentrations of Cu-chitosan nanoparticles.
 Results: Three treatments of Cu-chitosan treatments (0.02, 0.06 and 0.10%) were found most efficient in disease control in both laboratory and net house condition) experiments. Cu-chitosan Nanoparticles application to soybean plants significantly enhanced the activity of defence enzymes (PAL and PPO) and antioxidant enzymes (SOD and POD) from 1.1 to 2.29 folds.
 Conclusion: Cu-chitosan NPs developed in this study were effective in controlling disease by enhancing the activity of different defence related enzymes [polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL)], and antioxidant [peroxidise (POD) and superoxide dismutase (SOD)]. This could be an important nonmaterial research in the field of agriculture due to better compatibility of biodegradable Cu-chitosan nanoparticles with biological control. In this study, the effect of Cu-chitosan nanoparticles to boost defence responses against Xanthomonas axonopodis pv. Glycine were evaluated.

Современное состояние селекции и семеноводства хвойных пород

The article analyzes and summarizes information on breeding and seed production of the main forest-forming and commercial species for the countries of Northern Europe and Russia: Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies (L.) H. Karst). It is shown that in the last 40–50 years Sweden and Finland have completed the first (primary) cycle of plus-tree breeding, and the second cycle is being actively pursued. In terms of seed production, this means a gradual replacement of the 1st generation forest seed orchards with the forest seed orchards of enhanced genetic value (of 1.5 and 2nd generation). By the middle of the 21st century, the productivity of new artificial stands is expected to increase by 20–25 % in terms of stem wood stock due to breeding work. Successful and quite large-scale works were undertaken in plus trees phenotypic breeding and forest seed orchards (1st generation) planting in some regions of European Russia in the last third of the 20th century. However, even this first (primary) cycle of plus-tree breeding has not been fully completed. The latter makes it possible to lay out the necessary areas of multiplicative populations of increased genetic value – forest seed orchards of the 1.5 generation. Although this is impossible to achieve in many subjects of the Northwestern Federal District due to the insufficient number of selected plus trees and, most importantly, the extremely small area of progeny tests. At present, the vast majority of initially selected plus trees were not genetically tested, which hinders the possibility of further development of the plus-tree breeding system. Russia also lacks special breeding zoning of the main forest-forming species, providing a spatial organization of breeding work and the use of seeds from forest seed orchards. A federal program for the preservation and sustainable management of forest genetic resources of the Russian Federation, together with a number of subprograms for genetic research and breeding of the most important forest-forming species, should be developed to achieve progress in this direction, considering current advances in molecular genetics and biotechnology. Acknowledgments: Financial support of the research was provided by the federal budget for the state assignment of the Forest Research Institute and the Department of Multidisciplinary Scientific Research of Karelian Research Centre, Russian Academy of Sciences with partial support of the World-class Scientific and Educational Center “Russian Arctic: New Materials, Technologies and research Methods”. For citation: Raevsky B.V., Ignatenko R.V., Novichonok E.V., Prokopiuk V.M., Kuklina K.K. The Current State of Conifer Species Breeding and Seed Production. Lesnoy Zhurnal = Russian Forestry Journal, 2022, no. 6, pp. 9–37. (In Russ.). https://doi.org/10.37482/0536-1036-2022-6-9-37

The SHERLOCK Platform: An Insight into Advances in Viral Disease Diagnosis.

Persistence and prevalence of microbial diseases (pandemics, epidemics) is the most alarming threats to the human resulting in huge health and economic losses. Rapid detection and understanding of the disease dynamics by molecular biotechnology tools allow for robust reporting, treatment and control of diseases. As per WHO, the optimal diagnostic approach should be quick, specific, sensitive, without a stringed instrument, and low cost. The drawbacks of traditional detection techniques promote the use of CRISPR-mediated nucleic acid detection methods such as SHERLOCK as detection method. It takes advantage of the unexpected in vitro features of CRISPR-Cas system to develop field-deployable sensitive detection tools. Previously, CRISPR-mediated diagnostic methods have extensively been reviewed particularly for SARS-COV-2 detection, but it fails to provide the insight into advances of this technique. This study is the first attempt to review the advances of SHERLOCK approach as diagnostic tool for viral diseases detection. Variations of SHERLOCK mechanism for improved efficiency are discussed. Particularly integrated SHERLOCK approaches in terms of extraction-free assay and Bluetooth-enabled detection are reviewed to access their feasibility for the development of simpler and cost-effective diagnostic toolkits. Insight in to perks and limitations of diagnostic methods indicates its potential as ultimate diagnostic instrument for disease management.

Neurotoxicity mechanism of aconitine in HT22 cells studied by microfluidic chip-mass spectrometry

Aconitine, a common and main toxic component of Aconitum, is toxic to the central nervous system. However, the mechanism of aconitine neurotoxicity is not yet clear. In this work, we had the hypothesis that excitatory amino acids can trigger excitotoxicity as a pointcut to explore the mechanism of neurotoxicity induced by aconitine. HT22 cells were simulated by aconitine and the changes of target cell metabolites were real-time online investigated based on a microfluidic chip-mass spectrometry system. Meanwhile, to confirm the metabolic mechanism of aconitine toxicity on HT22 cells, the levels of lactate dehydrogenase, intracellular Ca2+, reactive oxygen species, glutathione and superoxide dismutase, and ratio of Bax/Bcl-2 protein were detected by molecular biotechnology. Integration of the detected results revealed that neurotoxicity induced by aconitine was associated with the process of excitotoxicity caused by glutamic acid and aspartic acid, which was followed by the accumulation of lactic acid and reduction of glucose. The surge of extracellular glutamic acid could further lead to a series of cascade reactions including intracellular Ca2+ overload and oxidative stress, and eventually result in cell apoptosis. In general, we illustrated a new mechanism of aconitine neurotoxicity and presented a novel analysis strategy that real-time online monitoring of cell metabolites can provide a new approach to mechanism analysis.