Institute for Biological Instrumentation

Vif and Vpr are HIV-1 accessory proteins that create optimal conditions for viral replication. They are considered as potential targets for the development of therapeutic agents. Natural amino acid substitutions in these proteins have previously been associated with disease progression. The aim of this study was to analyze the genetic diversity of Vif and Vpr in HIV-1 group M clades. A total of 5286 sequences were downloaded and analyzed. For 37 clades in group M, the consensus sequences, amino acid natural variation, and clade-specific amino acid residue substitutions (CSSs) were evaluated. Structural analysis and modeling of consensus sequences were performed for subtypes A1, B, C, and D. The average conservation degree in the HIV-1 group M was 86.4% for Vif and 91.3% for Vpr. In both proteins, the lowest amino acid diversity was observed in sub-subtype A6, and the highest in subtype B. In consensus sequences, the substitutions, which might influence pathogenesis, have been determined: in Vif—22H (11_cpx, 91_cpx) and 136P (A6, 01_AE, 15_01B, 59_01B, 89_BF1, 103_01B, 111_01C, 133_A6B), in Vpr—41N (06_cpx) and 55A (B, 07_BC, 35_01D, 56_cpx, 66_cpx, 66_BF1, 71_BF1, 85_BC, 137_0107). In functional motifs, CSSs associated with changes in the chemical properties of amino acid residues were noted. These findings could be taken into account for the development of therapeutic drugs in the future. No correlation was observed between the subtypes and the spatial organization of the oligomeric structures of Vif and Vpr. Using the structural analysis and modeling, it has been shown for the first time that Vif can interact with APOBEC3G as an oligomer.

Agricultural sector is undergoing a significant transformation to address the growing global demand for food while ensuring sustainability and minimizing environmental impact. Traditional farming methods, which rely heavily on manual processes and offer limited data insights, are increasingly inadequate for managing the complexities of modern agricultural needs. To overcome these challenges, the integration of advanced computing technologies specifically cloud, edge, and fog computing has emerged as a transformative solution in smart farming. This paper provides a comprehensive literature review on the role of these technologies in agriculture, focusing on their applications, benefits, challenges, and future directions. A proposed framework for integrating IoT, edge, fog, and cloud computing is presented, aiming to enhance productivity, resource efficiency, and environmental sustainability. The framework is evaluated through a case study conducted in the Maregaon District of Yavatmal (Maharashtra), demonstrating significant improvements in crop yields, resource utilization, and decision-making capabilities. The results highlight an accuracy of 95% in data collection and processing, a 20% increase in crop yields, and a 30% reduction in water usage. These findings underscore the potential of these technologies to revolutionize agriculture, ensuring food security and environmental sustainability in the face of growing global challenges.

L-methionine derivatives (L-methionine sulfoximine (MSX) and L-methionine sulfone (MSO)) are inhibitors of L-glutamine synthetase, which explains their toxic effect on cells. These derivatives are used in medical practice for treating inflammation, tuberculosis, and oncological diseases. Bacteria of the Salmonella, Pseudomonas , and Acinetobacter genera are resistant to the toxic effect of MSX and MSO due to the activity of L-methionine sulfoximine acetyltransferases (MSX-NAT). In this work, MSX-NAT from various Enterobacter species from the ESKAPE group of pathogens was analyzed. An analysis of multiple enzyme alignments revealed a high percentage of their sequence identity, despite the diversity of their polypeptide chain lengths. In addition, unique insertions in the N-terminal region of the proteins were found, the functions of which remain to be clarified.

Objective: The development of drought-tolerant genotypes of Quercus robur (“English” oak) is an important task in the context of climate change. This study aimed to analyze the transcriptomes of two oak genotypes with contrasting drought tolerance under normal water availability and water deficit conditions. Methods: Transcriptome samples were collected from two oak genotypes differing in drought tolerance. The study was conducted under two conditions: normal water availability (control) and water deficit (drought). Differentially expressed genes (DEGs) were identified to compare the responses between the two genotypes under both conditions. Results and Discussion: In genotype 1 (drought-tolerant), 70 DEGs were identified, with 42 upregulated and 28 downregulated. In genotype 2 (drought-sensitive), 97 DEGs were identified, with 37 upregulated and 60 downregulated. Genotype 1 exhibited a strong overrepresentation of GO terms related to abiotic stress response and changes in photosynthesis, suggesting a better adaptation to drought conditions. Genotype 2 showed DEGs primarily related to stress-induced regulation of hormone biosynthesis and stress response in the extracellular matrix, indicating a weaker drought response. An overlap analysis revealed one common gene with increased expression in both genotypes under drought: methylsterol monooxygenase, involved in steroid biosynthesis. Conclusions: The results provide insights into the molecular mechanisms behind drought tolerance in Quercus robur. The identification of differentially expressed genes could help in early identification of drought-tolerant genotypes, aiding in the development of oak varieties more suitable for future climate conditions.

Objective: Yeast Pichia pastoris (Komagataella phaffii) is widely used in food and pharmaceutical industries as microbial cell factories of recombinant proteins. Protease-activator of protein C (PAPC) of blood plasma from Aspergillus ochraceus VKM F-4104D micromycetes can potentially be introduced into therapeutic practice as a fibrinolytic drug and into diagnostic systems for blood coagulation analysis as the main component that activates protein C. The aim of this work was to construct a PAPC-producing strain based on P. pastoris and to demonstrate the effective production and secretion of the recombinant enzyme into the culture medium. Methods: We assembled a pD912-AFSnoEAEA-PAPC vector carrying the papc gene under a methanol-induced AOX1-promoter. This vector was used to transform P. pastoris yeast, and transformants were selected on a zeocin-containing medium. The clones most effectively producing the target enzyme were selected using an agar medium with casein and an analysis of the culture medium by SDS-PAGE. Results and Discussion: The selected clones secreted an active form of PAPC into the culture medium. The ability to production of the target enzyme remained after 60 generations. LC-MS analysis confirmed the presence of the enzyme in the culture medium and demonstrated that accumulation occurs in the mature active form. Conclusions: We demonstrated that P. pastoris system can be successfully used for the high-level expression of PAPC from A. ochraceus. The low amount of host cell proteins in the culture medium makes the subsequent purification of the target enzyme easier in comparison to bacterial expression systems. The obtained strain can be used for the further production of experimental industrial batches of the enzyme in biotechnological production facilities that support yeast fermentation.

Background. Methionine aminopeptidases (MAPs) are a class of enzymes that catalyze the removal of the N-terminal initiator methionine from a polypeptide chain. Bacterial MAPs are considered as targets for the development of broad-spectrum antibacterial drugs, and using MAPs in biotechnology necessitates the search for new MAPs and the study of their functioning and inhibition mechanisms.The aim of the study. To identify methionine aminopeptidase in the Thermus thermophilus genome (Tt-MAP) and to confirm its functional activity.Materials and methods. To identify Tt-MAP, we analyzed the Thermus thermophilus genome in the GeneBank database. Modern genetic engineering techniques (polymerase chain reaction, restriction, transformation, heterologous expression) were used to clone the putative open reading frame (ORF) encoding Tt-MAP in the pHUE vector. Various chromatography techniques (affinity, ion exchange, and size-exclusion) were used to obtain a purified enzyme preparation. The fluorogenic substrate L-methionine 7-amino-4-methylcoumarin (Met-AMC) was used to confirm the specific functional aminopeptidase activity of the enzyme.Results. An ORF encoding MAP was identified in the Thermus thermophilus bacterium genome. Oligonucleotide primers were designed based on the nucleotide sequence. The ORF was cloned in the vector, and the recombinant enzyme was produced in E. coli cells. A method for purifying the enzyme to a homogeneous state was developed using a series of sequential chromatographies, allowing up to 30 mg to be obtained from 1 liter of culture. Using the fluorogenic substrate Met-AMC, the specific functional activity of the enzyme was demonstrated (the enzyme cleaves methionine from the substrate).Conclusion. We have identified the Thermus thermophilus MAP and tested its functional activity. It has been shown that the ORF product TTHA1670 encodes a methionine-specific aminopeptidase, i. e. methionine aminopeptidase. The enzyme can be used in various fields of biotechnology and scientific research.

The pathogenic microorganism Staphylococcus aureus, one of the representatives of the staphylococcus family, is capable of developing resistance to antibiotics and antiseptics. The largest ribosomal protein, S1, from S. aureus contains four structural S1 domains, and short peptides synthesized based on its sequence have amyloidogenic and antimicrobial properties (antimicrobial peptides). In this work, in silico analysis of all available ribosomal protein S1 sequences from various S. aureus strains allowed us to identify residues that are characteristic of specific strains. Based on data from the I-Mutant service, changes in the stability of ribosomal protein S1 from various S. aureus strains were predicted. The results obtained will be used in the future for targeted mutations in the design of new antimicrobial peptides based on ribosomal protein S1.

This research aims to examine the effects of managerial ownership, financial distress, and earnings management on the integrity of financial reports. The population consists of all property and real estate companies listed on the IDX in 2018-2021. This research used purposive sampling for sampling, which resulted in a total of 10 companies being observed over a 4 year period, collecting a total of 40 research data. Research findings show that managerial ownership and financial distress have a significant and negative influence on the integrity of financial reports. On the other hand, there is no significant influence of earnings management on the integrity of financial statements. Overall, when these factors are evaluated, managerial ownership, financial distress and earnings management simultaneously have an influence on the integrity of financial statements