Establishment of a Unified Register of Donor Sexual Gametes in the Republic of Kazakhstan.

OBJECTIVE: The purpose of this narrative review paper was to review the state and development of the field of donor gametes in Kazakhstan, compare its legislative and technical capabilities with other countries and identify key steps towards the establishment of a unified register of donor gametes in the Republic. MATERIALS AND METHODS: The narrative review paper conducted an analysis of scientific publications and legal documents to examine the implementation of Assisted Reproductive Technologies (ART), focusing on Donor Sexual Gametes (DSG), globally. It utilized medical publications from 2019 to 2023, legal acts, and recommendations from global health organizations to analyze eligibility criteria, legal regulations, and the social aspects of ART across different regions. RESULTS: In Kazakhstan, ART is regulated by legislation, with DSG procedures governed by age limits, medical screening, and restrictions on the number of children born from donated gametes. Worldwide, practices vary, but there is growing interest in establishing a unified register of reproductive donor material to enhance transparency and accountability. However, legal gaps and ethical considerations must be addressed. CONCLUSION: The study identifies gaps in Kazakhstan's legislation compared to Western countries, emphasizing the necessity for enhanced legal rights for donors and recipients, including options for anonymity. Ethical concerns highlight the importance of confidentiality and data security in accessing the donor registry. Overall, implementing such a register promises to enhance transparency, safety, and accountability in reproductive medicine.

A Comparative Study of New Fluorescent Anthraquinone and Benzanthrone α-Aminophosphonates: Synthesis, Spectroscopy, Toxicology, X-ray Crystallography, and Microscopy of Opisthorchis felineus.

In this research, we explore the synthesis of and characterize α-aminophosphonates derived from anthraquinone and benzanthrone, focusing on their fluorescence properties and potential applications in confocal laser scanning microscopy (CLSM). The synthesized compounds exhibit notable solvatochromic behavior, emitting fluorescence from green to red across various solvents. Spectroscopic analysis, including 1H-, 13C-, and 31P-NMR, FTIR, and mass spectrometry, confirms the chemical structures. The compounds' toxicity is evaluated using etiolated wheat sprouts, revealing varying degrees of impact on growth and oxidative damage. Furthermore, the study introduces these α-aminophosphonates for CLSM imaging of the parasitic flatworm Opisthorchis felineus, demonstrating their potential in visualizing biological specimens. Additionally, an X-ray crystallographic study of an anthraquinone α-aminophosphonate provides valuable structural insights.

Morphological and Molecular Characterization of Bacterial Pathogens Associated with Leaf Mottle of Sunflower in Northern Kazakhstan.

Leaf mottle is a serious disease in the common sunflower (Helianthus annuus L.), which affects plant growth and development and seed quality and yield. Over the past few years, the North Kazakhstan region, a sunflower-producing area in Kazakhstan, has been seriously affected by leaf mottle. Since 2021, symptomatic leaves have been collected from production areas of this base to determine the pathogens causing sunflower foliar diseases. One hundred bacterial strains were isolated, and two genera and five species were identified based on morphological characteristics, molecular genetics, and phylogenetic analysis (16S gene region). The genus Bacillus was represented by four species: Bacillus subtilis, B. megaterium, B. amyloliquefaciens, and B. flexus. The genus Paenibacillus was represented by one species, P. peoriae. Pathogenicity experiments showed that B. subtilis, B. megaterium, B. flexus, and P. peoriae could cause leaf mottle disease symptoms. However, disease symptoms caused by B. flexus were highly similar to those observed on infected leaves under natural conditions in the field. Therefore, these bacterial isolates were found to be the primary pathogens causing sunflower leaf mottle, and B. flexus was the most common and virulent pathogen in this study. In addition, this is the first report of B. megaterium, B. flexus, and P. peoriae as pathogens associated with sunflower leaf mottle in Kazakhstan.

Genetic identification of Trichinella species found in wild carnivores from the territory of Kazakhstan.

Trichinellosis, also called trichinosis, is a foodborne parasitic disease caused by eating raw or undercooked meat from animals infected with Trichinella spp. larvae and affects both animals and humans. Although on the territory of Kazakhstan, the species characteristics and prevalence of this helminth were studied back in the 90s, the data have not been updated since then. Given the above, our study was aimed at identifying Trichinella spp. using parasitological and molecular genetics methods. In our work, we studied 160 samples of muscle tissue of wild animals living in the natural zones of steppes and semi-deserts. Of the animals examined, 32 were positive for Trichinella spp., including 1 lynx (Lynx lynx), 17 wolves (Canis lupus), 11 foxes (Vulpes vulpes), 1 jackal (Canis aureus) and 2 corsac foxes (Vulpes corsac). Helminths were extracted using the digestion method. DNA was extracted using a Gene Jet commercial kit (Thermo Fisher Scientific, United Kingdom). For species identification a multiplex PCR, amplification of ESV, ITS1, and ITS2 genes regions was performed. After that, uniplex PCR was performed on the 5S rDNA and ITS1 genes region for sequencing analysis. The resulting sequences were subsequently used to construct a phylogenetic tree and the studied samples were identified as Trichinella nativa and Trichinella britovi. Thus, we can conclude that there is a circulation of two species of Trichinella in Kazakhstan, highlighting that constant control and monitoring of wild animals are necessary to prevent transmission and protect the health of people.

First report of stem rot of Adenium obesum and leaf spot of Persea americana caused by Aspergillus niger in Kazakhstan.

Adenium (Adenium obesum) and avocado (Persea americana) are commonly grown as exotic houseplants in city apartments of Kazakhstan. In April-May 2020, the wilting symptom was observed on the young stems of five 2-year-old A. obesum plants in a city apartment in Saryarqa District, Astana, Kazakhstan (71°25'E, 51°11'N). Leaves turned yellow and then dried up. Plants were completely wilted within 10 days (Fig. 1A). Similar symptoms were observed in newly grown A. obesum plants in November, 2021. At the same time, lesions were found on the leaves of three 3-month-old P. americana plants. Infected leaves displayed dry, dark-brown lesions and fell off easily (Fig. 2A). Both plants were cultivated side by side. The incidence of affected A. obesum was 80% out of 5 plants and P. americana was 100% out of 3 plants. To isolate the causal agent, the infected tissues from different leaves and stems of A. obesum and P. americana plants were cut into small pieces (5 × 5 mm), washed in 70% ethanol for 5 min, and then rinsed three times with sterile distilled water. Cut pieces were placed on potato dextrose agar (PDA) (Laboratorios Conda S.A., Spain) and incubated at 28°C for 7 days. Ten isolates were obtained from leaves and stems of the A. obesum and P. americana symptomatic samples. All fungal colonies were white initially, turned black gradually, reverse side light yellow (Fig. 1B and Fig. 2B), conidiophores biseriate with globose vesicles, conidia were spherical vesicles, light tan to black color, smooth-walled to roughened, and sizes ranged from 3.0 to 3.5 µm (n = 15) (Fig. 1C and Fig. 2C). These observations indicated that all the isolates resembled Aspergillus spp. (Bryan and Fennell 1965). DNA was extracted using the liquid nitrogen and phenol-chloroform extraction method (Butler 2012). A 526 bp product of the ITS region on rDNA and 568 bp product of the calmodulin protein-coding gene was amplified using following primer pairs ITS4/ITS5 (Abliz et al. 2003) and cmd5/cmd6, respectively (Hong et al. 2005). The PCR reaction was done under the following conditions: initial denaturation at 94°C for 5 min, 35 cycles at 95°C for 30 s to denature, 52°C for 40 s for annealing, and 72°C for 50 s for extension. A final extension step at 72°C for 7 min was also included. The sequencing was done using BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and the sequence was deposited in GenBank with accession nos. ON519078 (A. obesum ITS), ON519079 (P. americana ITS), OQ358173 (A. obesum calmodulin) and OQ358174 (P. americana calmodulin). These sequences were compared with other sequences of A. niger in GenBank using BLAST analysis (MG569619.1, MT588793.1, MH478660.1, MZ787576.1 and MW086485.1). The results showed that the sequences of ten isolates were identical and had 98-100% identity with those of Aspergillus niger (Fig. 3). The phylogenetic analysis was carried out with MEGA 11 (Tamura et al. 2021). To confirm the pathogenicity, three asymptomatic plants of each were inoculated with a suspension of conidia via pin-prick inoculation (1.0×106 conidia/ml; obtained from 2-week-old cultures). Control plants were inoculated with sterile distilled water. The inoculated plants were placed in climate chamber (BINDER, Germany) and incubated for 10 days at 28°C. Symptoms were developed in leaves of inoculated plants after 2 days in P. americana and after 5 days in A. obesum. Affected leaves turned yellow and their stems started drying. Symptoms of leaves were similar to those observed on naturally infected plants, while control plants remained asymptomatic. Re-isolation of the pathogen confirmed the presence of the A. niger pathogen. To our knowledge, this is the first report of A. niger causing stem rot of A. obesum and leaf spot of P. americana in Kazakhstan. Since different ornamentals are often planted together in gardens and nurseries, growers should be aware of potential transmission of A. niger among them. This finding provides a foundation to further investigate the biology and epidemiology of this disease so the developmentf diagnostic tools and management measures against it.

Genetic diversity of Echinococcus spp. in wild carnivorous animals in Kazakhstan.

Background and Aim: The study of Echinococcus infection among farm animals in Kazakhstan was carried out to monitor the invasion among livestock and map the data obtained. Unfortunately, there are only partial data on the study of echinococcosis among wild carnivores in Kazakhstan, which makes it difficult to conduct a comparative analysis of the epidemiological situation among wild animals. The present study aimed to estimate the genetic diversity of Echinococcus spp. (Leuckart, 1863) in Kazakhstan based on sequence analysis of cytochrome c oxidase subunit 1 (cox1) and dehydrogenase subunit 1 (nad1) of worms isolated from wild carnivorous animals wolf (Canis lupus), red fox (Vulpes vulpes) and corsac (Vulpes corsac). Materials and Methods: DNA from parasite tissue was used as a template for the amplification of the two mitochondrial genes cox1 and nad1. Sequencing was performed according to the manual for the Seq Studio Genetic Analyzer. The multiple alignments of obtained sequences were performed using the ClustalW algorithm in Mega (v.11) software. Alignments were exported as a Nexus extension and used as input for TCS v1.21 for the identification of haplotypes. The phylogenetic analysis was constructed according to the neighbor-joining method using Mega (v.11) software. Results: Analysis of the extensiveness of echinococcosis invasion showed that 6.3% were wolves, 18.2% were corsacs, and 85% were foxes. In total, 159 adults of Echinococcus spp. from the three species of animals in different parts of Kazakhstan were analyzed, and 17 individual biological samples were successfully sequenced. Sequence analysis of cox1 and nad1 genes revealed two types of echinococcosis - Echinococcus granulosus in red foxes and wolves, and Echinococcus multilocularis in corsacs. Sequencing of a portion of the mitochondrial genome made it possible to determine seven haplotypes of the pathogen in the studied samples of E. granulosus. Molecular analysis of cox1 and nad1 genes of E. multilocularis revealed three new haplotypes, which have significant variability compared with other studied Asian haplotypes. Conclusion: This study made it possible to fill the gaps in understanding the localization of the foci of the spread of the echinococcosis pathogen among the main wild carnivores and to determine the species reservoir of the pathogen in the greater territory of Kazakhstan.

First Record of Alternaria alternata causing necrosis of Thuja (Thuja occidentalis) in Kazakhstan.

Thuja is one of the ornamental plants used for landscaping parks and health resorts. The plant is distinguished by a pyramidal and conical crown shape and the presence of many thin branches with scale-shaped needles, green all year round. In addition, this plant has a number of antimicrobial properties, which affects the popularity of the plant in landscaping the health resort territory (Bakht et al. 2020, Chindyaeva et al. 2020). In January 2020, symptomatic Thuja plants were observed in Southern Kazakhstan. Symptoms included distortion of the crown. External examination of the trees revelaed the presence of white fluffy mycelium on Thuja branches. The branches acquired a yellow color with a necrotic lesion developing below the affected area. Samples of infected branches from different Thuja trees (n = 13) were collected. The infected branches were cut into small pieces (5 × 5 mm), washed in 70% ethanol for 30 min, and then rinsed three times with sterile distilled water. Later, these pieces were placed on Sabouraud's medium (Laboratorios Conda S.A., Spain) and incubated at 28°C for 7 days. Yellow-green colonies grew from the pieces of wood. The colonies had a light gray-whitish aerial mycelium. Conidia (n = 35) were pale to dark brown in color, irregular and ellipsoid to ovoid conical in shape. The size of the conidia varied from 5 to 25 µm × 6 to 12 µm (n = 40) with longitudinal and transverse septations. These morphological characters were previously described and corresponded to the Alternaria alternata (Simmons et al. 2007). Genomic DNA was extracted from mycelium using the liquid nitrogen and phenol-chloroform extraction method (Butler 2012). A 568 bp product of the Alt a1 gene and 472 bp product of the calmodulin protein-coding gene was amplified using following primer pairs Alt-for/Alt-rev (Hong et al. 2005) and CALDF1/CALDR1, respectively (Lawrence et al. 2013) (Integrated DNA Technologies, Inc., Coralville, IA, USA). The PCR reaction was done in a SimpliAmp thermal cycler (Applied biosystems, Waltham, MA, USA) under the following conditions: initial denaturation at 94 °C for 1 min, 35 cycles at 94°C for 30 s to denature, 57°C for 1 min for annealing, and 72°C for 1 min for extension. A final extension step at 72°C for 10 min was also included. The sequencing was done using BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and the sequence was deposited in GenBank with accession no. OL542696 calmodulin). These sequences were compared with other sequences in the GenBank by using the BLAST analysis (MZ222274.1 and MN473132.1). The phylogenetic analysis was carried out with MEGA 6 software (The Pennsylvania State University, University Park, PA, USA). To confirm the pathogenicity, 10 thuja branches from healthy trees from another area without visible pathologies were inoculated with a suspension of conidia (100 conidia/ml; obtained from 2-week-old cultures). Control samples were inoculated with sterile distilled water. The inoculated branches were placed in sterile plastic containers to maintain high humidity and incubated for 10 days at 28°C. After 7 days, irregular shaped lesions and fungal growth was observed at the site of inoculation. The affected area gradually increased in size with simioar symptomatology to that described above. Re-isolation of the pathogen and identification based on morphological features and sequencing confirmed the presence of the A. alternata pathogen. To our knowledge, this is the first report of A. alternata causing branches of thuja in Kazakhstan. Thuja is a rare plant species for this region; the cost and care are expensive. This case will allow timely diagnosis of the disease caused by Alternaria spp. in the future. It is necessary to develop preventive measures and a protocol for the treatment of thuja from a fungal infection. Bakht, J., 2020. Antibacterial activity of the crude extracts from medicinally important Thuja occidentalis. Pak J Pharm Sci. 33(2): 627-630. PMID: 32276908. Butler, J.M., 2012. Chapter 2 - DNA extraction methods. pp. 29-47 in Butler JM (Ed) Advanced topics in forensic DNA typing: Methodology. San Diego, Academic Press. doi: 10.1016/C2011-0-04189-3. Chindyaeva., L.N., et al. 2020. Comparative assessment of the phytoncidity of woody plants in the selection of species for landscaping: the possibility of use in sanatorium-and-spa practice. Vopr Kurortol Fizioter Lech Fiz Kult. 97(4): 44-51. Russian. doi: 10.17116/kurort20209704144. Hong, S.G. et al. 2005. Alt a1 allergen homologs from Alternaria and related taxa: analysis of phylogenetic content and secondary structure. Fungal Genet Biol 42:119-129. doi:10.1016/j.fgb.2004.10.009 Lawrence, D.P. et al. 2013. The sections of Alternaria: formalizing species-group concepts. Mycologia 105: 530-546. DOI: 10.3852/12-249. Simmons, E. G., 2007. Alternaria: An Identification Manual. CBS, Fungal Biodiversity Center, Utrecht, Netherlands.

Candida parapsilosis as a Causative Agent of Onychomycosis in Patient with Cirrhosis of the Liver.

We report the first case of non-dermatophytic onychomycosis of the toenail described in Kazakhstan caused by Candida parapsilosis. The biological properties of the strain were studied. C. parapsilosis forms white creamy colonies, smooth with focal wrinkles, and the reversum is light-yellow. The culture of C. parapsilosis is represented by a yeast form, characterized by the presence of round or cylindrical yeast cells with active budding. The strain has a high saccharolytic and urease activity and is indifferent to the sucrose and maltose. The C. parapsilosis strain was sensitive to polyene and azole antifungal agents. The highest sensitivity was found to ketoconazole, itraconazole and nystatin.

Biological properties of Phoma macrostoma related to non-dermatophyte onychomycosis.

We report a rare case of non-dermatophytic onychomycosis of the toenail caused by Phoma macrostoma. Was studied the biological properties of the strain isolated in Kazakhstan. P. macrostoma forms pink colonies, the reverzum is pink-orange. The mycelium is colorless, septate. The appearance of growth tubes from pycnidospores occurs within 12 hours, mycelial growth and branching after 18 hours, the appearance of pycnids is 48 hours. The saccharolytic and urease activity of the strain is low.

Formation of mammalian preribosomes proceeds from intermediate to composed state during ribosome maturation.

In eukaryotes, ribosome assembly is a rate-limiting step in ribosomal biogenesis that takes place in a distinctive subnuclear organelle, the nucleolus. How ribosomes get assembled at the nucleolar site by forming initial preribosomal complexes remains poorly characterized. In this study, using several human and murine cell lines, we developed a method for isolation of native mammalian preribosomal complexes by lysing cell nuclei through mild sonication. A sucrose gradient fractionation of the nuclear lysate resolved several ribonucleoprotein (RNP) complexes containing rRNAs and ribosomal proteins. Characterization of the RNP complexes with MS-based protein identification and Northern blotting-based rRNA detection approaches identified two types of preribosomes we named here as intermediate preribosomes (IPRibs) and composed preribosome (CPRib). IPRib complexes comprised large preribosomes (105S to 125S in size) containing the rRNA modification factors and premature rRNAs. We further observed that a distinctive CPRib complex consists of an 85S preribosome assembled with mature rRNAs and a ribosomal biogenesis factor, Ly1 antibody-reactive (LYAR), that does not associate with premature rRNAs and rRNA modification factors. rRNA-labeling experiments uncovered that IPRib assembly precedes CPRib complex formation. We also found that formation of the preribosomal complexes is nutrient-dependent because the abundances of IPRib and CPRib decreased substantially when cells were either deprived of amino acids or exposed to an mTOR kinase inhibitor. These findings indicate that preribosomes form via dynamic and nutrient-dependent processing events and progress from an intermediate to a composed state during ribosome maturation.

Opisthorchis felineus and Metorchis bilis Metacercariae in Cyprinid Fish Leuciscus idus in Nura-Sarysu River, Kazakhstan.

Aim of the present study was to provide presence of opisthorchiid metacercariae in cyprinid fish Leuciscus idus in Nura-Sarysu river, Kazakhstan. Infection rate of the ides by the metacercariae was 42%. The metacercariae, similar morphologically to those of the liver flukes, were found: elliptical in shape, 0.19-0.25×0.15-0.22 mm, oral and ventral suckers nearly equal size, and excretory bladder O-shape with black content, occupying posterior part of the body. The metacercariae were divided into 2 groups with differences in size and thickness of cyst wall. Adult flukes were recovered from the Syrian hamsters infected with the opisthorch metacercariae and identified with morphological characters to Opisthorchis felineus and Metorchis bilis. DNA sequences of ITS1, ITS2, and cox1 supported the taxonomic assignment.

Development of an ELISA using anti-idiotypic antibody for diagnosis of opisthorchiasis.

Monoclonal antibody specific for an epitope of cretory-secretory antigen protein of Opisthorchis felineus (Rivolta, 1884) (Trematoda: Opisthorchiidae) with a molecular weight of 28 kDa was used in a sandwich enzyme-linked immunosorbent assay (ELISA) for immobilisation of liver fluke specific antigen to the solid phase. Examination of human sera by this ELISA compared with commercial assays demonstrated that the monoclonal antibody epitope is located within this significant parasite protein. Anti-idiotypic antibody specific for the paratope of this monoclonal antibody was obtained by a hybridoma technique. Mimicking an epitope of excretory-secretory antigen of O. felineus, it had the capacity to bind specific antibody and elicit an antibody response. The value of anti-idiotypic antibody as a substitute for the liver fluke antigen was tested by ELISA using serum samples of infected dogs. Anti-idiotypic antibody proved to be of value in both an indirect-ELISA and a competitive-ELISA for diagnosis of opisthorchiasis. Mature trematodes were isolated from all infected animals. The faecal egg counts were negative in dogs with a relatively small number of parasites, despite finding antibodies in serum by ELISA. Substitution of parasite antigen with anti-idiotype avoids the use of experimental animals and also reduces time-consuming steps of antigen preparation.

The nuclear import of ribosomal proteins is regulated by mTOR.

Mechanistic target of rapamycin (mTOR) is a central component of the essential signaling pathway that regulates cell growth and proliferation by controlling anabolic processes in cells. mTOR exists in two distinct mTOR complexes known as mTORC1 and mTORC2 that reside mostly in cytoplasm. In our study, the biochemical characterization of mTOR led to discovery of its novel localization on nuclear envelope where it associates with a critical regulator of nuclear import Ran Binding Protein 2 (RanBP2). We show that association of mTOR with RanBP2 is dependent on the mTOR kinase activity that regulates the nuclear import of ribosomal proteins. The mTOR kinase inhibitors within thirty minutes caused a substantial decrease of ribosomal proteins in the nuclear but not cytoplasmic fraction. Detection of a nuclear accumulation of the GFP-tagged ribosomal protein rpL7a also indicated its dependence on the mTOR kinase activity. The nuclear abundance of ribosomal proteins was not affected by inhibition of mTOR Complex 1 (mTORC1) by rapamycin or deficiency of mTORC2, suggesting a distinctive role of the nuclear envelope mTOR complex in the nuclear import. Thus, we identified that mTOR in association with RanBP2 mediates the active nuclear import of ribosomal proteins.

Autoregulation of the mechanistic target of rapamycin (mTOR) complex 2 integrity is controlled by an ATP-dependent mechanism.

Nutrients are essential for living organisms because they fuel biological processes in cells. Cells monitor nutrient abundance and coordinate a ratio of anabolic and catabolic reactions. Mechanistic target of rapamycin (mTOR) signaling is the essential nutrient-sensing pathway that controls anabolic processes in cells. The central component of this pathway is mTOR, a highly conserved and essential protein kinase that exists in two distinct functional complexes. The nutrient-sensitive mTOR complex 1 (mTORC1) controls cell growth and cell size by phosphorylation of the regulators of protein synthesis S6K1 and 4EBP1, whereas its second complex, mTORC2, regulates cell proliferation by functioning as the regulatory kinase of Akt and other members of the AGC kinase family. The regulation of mTORC2 remains poorly characterized. Our study shows that the cellular ATP balance controls a basal kinase activity of mTORC2 that maintains the integrity of mTORC2 and phosphorylation of Akt on the turn motif Thr-450 site. We found that mTOR stabilizes SIN1 by phosphorylation of its hydrophobic and conserved Ser-260 site to maintain the integrity of mTORC2. The optimal kinase activity of mTORC2 requires a concentration of ATP above 1.2 mM and makes this kinase complex highly sensitive to ATP depletion. We found that not amino acid but glucose deprivation of cells or acute ATP depletion prevented the mTOR-dependent phosphorylation of SIN1 on Ser-260 and Akt on Thr-450. In a low glucose medium, the cells carrying a substitution of SIN1 with its phosphomimetic mutant show an increased rate of cell proliferation related to a higher abundance of mTORC2 and phosphorylation of Akt. Thus, the homeostatic ATP sensor mTOR controls the integrity of mTORC2 and phosphorylation of Akt on the turn motif site.