Oybek N. Ashirov

The Müllerian inhibiting substance (MIS) is a glycoprotein that belongs to the transforming growth factor-β family and is known for its crucial roles in embryonic development and cellular differentiation. Attempts to produce recombinant MIS using bacterial and yeast expression systems have faced challenges, including insufficient post-translational modifications and low expression efficiency. In this study, the MIS gene was successfully cloned into the pBacPAK8 transfer vector and expressed using the Bombyx mori baculovirus - insect cell expression system. Co-transfection of BMN1 insect cells with the recombinant plasmid and baculoviral DNA led to the successful generation of recombinant baculoviruses. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis and Western blot analyses of lysates from infected cells confirmed the expression of a ~70 kDa MIS protein with significant antigenicity. To the best of our knowledge, this is the first report describing the recombinant production of human MIS protein using the B. mori baculovirus system. This expression platform provides a promising approach for synthesizing structurally complex human proteins and lays the groundwork for future research and biomedical applications.

The paper was dedicated to the investigation of the insecticidal activities of recently prepared 1,3,4-oxadiazole compounds and their in vitro efficacy was determined with the help of cell lines taken out of Helicoverpa zea, Trichoplusia ni and Lymantria dispar. The MTT assay yielded results that showed that the compounds synthesized had insecticidal activities between 88.4 -97.2 - 100 µM that were similar to those of the commercial insecticide Imidacloprid 2F of 96.4 -98.3 - 100 µM. In silico testing showed that the newly prepared compounds bind to a variety of amino acid residues either with several interaction modes at the same time such as π-π stacking, hydrogen bonding, π-π anion interaction, and π-π alkyl interaction, specifically against acetylcholinesterase (AChE) of the insect species used. Since they are relatively harmless to the environment, these 1,3,4-oxadiazole derivatives have a potential as alternative agents in sustainable pests control in agriculture, which could be beneficial compared to the use of conventional insecticides. The results highlight the effectiveness of such compounds as effective agents against insect cell lines in agriculture. However, in order to completely prove their practical implementation, there is a need to conduct additional in vivo research and field tests are suggested to verify their effectiveness in real-world situations.

Derivatives of 1,2,4-triazole-3-thione exhibit a variety of biological activities, including antimicrobial (e.g., compounds 31d–k, 32d, 36f), antitumor (e.g., 71, 77a–c, 82g, 94h), anti-inflammatory, analgesic (100a, 102, 105), antidiabetic, and antioxidant (104, 138) activity. These compounds can be efficiently synthesized by classical methods (e.g., cyclization of thiosemicarbazides) and/or modern “green” approaches, which allow for obtaining target compounds in high yields (up to 96%). The presence of electron-donating groups (e.g., -OH, -OCH₃) enhances antimicrobial and antitumor activity. Substituents in the aromatic ring (e.g., NO₂, Cl) affect the ability to bind to biological targets such as DNA or enzymes. 1,2,4-triazole-3-thiones can also be used as fungicides and herbicides (e.g., 131), demonstrating high efficiency against phytopathogens. Thus, 1,2,4-triazole-3-thione derivatives are multifunctional compounds with high potential for the development of new drugs and agrochemicals. Their further study and modification can lead to the creation of more effective and safer drugs.

Derivatives of 1,2,4-triazole-3-thione exhibit a variety of biological activities, includ-ing: antimicrobial (e.g., compounds 31d-k, 32d, 36f), antitumor (e.g., 71, 77a-c, 82g, 94h), anti-inflammatory, analgesic (100a, 102, 105), antidiabetic and antioxidant (104, 138) activity. These compounds can be efficiently synthesized by classical methods (e.g., cyclization of thiosemicarbazides) and/or modern "green" approaches, which al-low obtaining target compounds in high yields (up to 96%). The presence of elec-tron-donating groups (e.g., -OH, -OCH₃) enhances antimicrobial and antitumor activi-ty. Substituents in the aromatic ring (e.g. NO₂, Cl) affect the ability to bind to biological targets such as DNA or enzymes. 1,2,4-triazole-3-thiones can also be used as fungicides and herbicides (e.g. 131), demonstrating high efficiency against phytopathogens. Thus, 1,2,4-triazole-3-thione derivatives are multifunctional compounds with high potential for the development of new drugs and agrochemicals. Their further study and modifi-cation can lead to the creation of more effective and safer drugs.

1,2,4-Triazole derivatives have a wide range of biological activities. The most well-known drug that contains 1,2,4-triazole as part of its structure is the nucleoside analogue ribavirin, an antiviral drug. Finding new nucleosides based on 1,2,4-triazole is a topical task. The aim of this study was to synthesize ribosides and deoxyribosides of 1,2,4-triazole-3-thione derivatives and test their antiviral activity against herpes simplex viruses. Three compounds from a series of synthesized mono- and disubstituted 1,2,4-triazole-3-thione derivatives were found to be substrates for E. coli purine nucleoside phosphorylase. Of six prepared nucleosides, the riboside and deoxyriboside of 3-phenacylthio-1,2,4-triazole were obtained at good yields. The yields of the disubstituted 1,2,4-triazol-3-thiones were low due to the effect of bulky substituents at the C3 and C5 positions on the selectivity of enzymatic glycosylation for one particular nitrogen atom in the triazole ring. The results of cytotoxic and antiviral studies on acyclovir-sensitive wild-type strain HSV-1/L2(TK+) and acyclovir-resistant strain (HSV-1/L2/R^ACV) in Vero E6 cell culture showed that the incorporation of a thiobutyl substituent into the C5 position of 3-phenyl-1,2,4-triazole results in a significant increase in the cytotoxicity of the base and antiviral activity. The highest antiviral activity was observed in the 3-phenacylthio-1-(β-D-ribofuranosyl)-1,2,4-triazole and 5-butylthio-1-(2-deoxy-β-D-ribofuranosyl)-3-phenyl-1,2,4-triazole nucleosides, with their selectivity indexes being significantly higher than that of ribavirin. It was also found that with the increasing lipophilicity of the nucleosides, the activity and toxicity of the tested compounds increased.

In this study, we aimed to isolate genomic DNA (gDNA) from a yeast cell using various methods and choose the cheapest, most convenient, and fastest method among them. For this purpose, based on a thorough review of the literature, three relatively optimal methods were selected based on the use of guanidine thiocyanate, lyticase enzyme, and lithium acetate/SDS reagents. In the research work, the disadvantages and advantages of these methods were studied and compared. The obtained results showed that the isolation method using the lyticase enzyme is not optimal, given the relatively long time and high cost of the enzyme used, the isolation method using lithium acetate/SDS reagents is much simpler, faster and cheaper, and the isolation method using the guanidine thiocyanate is recognized impractical due to poor isolation of genomic DNA. In addition, we also investigated the process of isolating genomic DNA from Candida albicans and molds other than Pichia pastoris. For this, only the lithium acetate/SDS method was used, and this method was shown to be able to isolate high quality genomic DNA from all types of yeasts and molds.

Background: SARS-CoV-2 is a virus responsible for the COVID-19 pandemic that began in late 2019. This pandemic has had a devastating impact worldwide, resulting in over 6.95 million deaths. The development of effective vaccines against the virus is crucial for preventing infection and reducing the severity of the disease. Objective: This study aimed to obtain the recombinant receptor-binding domain (RBD) and the nucleocapsid (N) proteins of SARS-CoV-2 as well as assess the immunogenicity of the combination of these recombinant proteins. Methods: The recombinant plasmids encoding the receptor-binding domain (RBD) of the spike protein of the Omicron variant and the nucleocapsid protein of SARS-CoV-2 were cloned into the yeast Pichia pastoris. The optimal fermentation conditions were established for recombinant P. pastoris strains. The methods for the isolation and purification of the target recombinant RBD and nucleocapsid proteins were developed. The immunogenicity of the purified recombinant proteins was evaluated by injecting them into mice and analyzing the specific IgG antibody responses using ELISA. Results: The study found that RBD and N proteins, as well as their combination, showed antigenic specificity and were highly immunogenic in mice. The immunogenicity was measured by determining the antibody titer, which represents the concentration of antibodies produced in response to the antigen. The antibody titers were 1:60000 for both RBD and N proteins, and 1:80000 for their combination. Conclusion: These findings suggest that the expressed proteins could be potential candidates for the development of vaccines or immunological diagnostic test kits for combatting or detecting the Omicron variant of SARS-CoV-2.

Purine nucleoside phosphorylase (PNP) is an enzyme involved in biosynthetic pathway of purine nucleosides. Purine nucleoside phosphorylase catalyzes the cleavage of the glycosidic bond of ribo- or deoxyribonucleosides to form the purine base and deoxyribose- or ribose-1-phosphate. The reversible reaction catalyzed by recombinant PNP of E. coli (EcPNP) has been successfully used for the synthesis of nucleoside analogues. The aim of this study was to clone and expression of recombinant PNP in Pichia pastoris. For expression of recombinant EcPNP in Pichia pastoris, a new recombinant plasmid DNA pPICZαA-PNP (4256 bp) containing the deoD gene (720 bp) amplified from E. coli strain RKMUz-221 has been cloned. The substrate specificity of the recombinant EcPNP expressed in yeast cells was studied by hydrolysis of inosine (9-beta-D-ribofuranosylhypoxanthine) to hypoxanthine and 2-deoxy-β-D-ribofuranosyl. The fraction containing the obtained recombinant EcPNP (≈28 kDa, as determined by SDS-PAGE) demonstrates significant hydrolytic activity, resulting in up to a 51% hydrolysis of inosine within a 5 h timeframe. These findings suggest that the recombinant enzyme holds a promise for applications in enzymatic transglycosylation of purine-type nucleosides.