In this work we knocked out the LTS3 gene of the microalgae Chlamydomonas reinhardtii using the TIM technique optimized for the available equipment. We achieved transformation efficiency of 68.8%, knockout of this gene lead to the death of C. reinhardtii cells after several division cycles. The creation and study of genetically modified organisms in fundamental research allows a deeper understanding of the basic processes in the cells with the prospect of further applying this knowledge in practice. Microalgae are an interesting object for genetic engineering because of the great prospects for their application in biotechnology, but in almost every case it is necessary to develop new strategies and transformation methods for the introduction of genetic constructs into the cell. CRISPR/Cas revolutionized the field of genome editing due to its simplicity, efficiency and accuracy compared to previously used methods, which over time simplified the development of protocols [1]. Currently, the most effective method of transformation is TIM (Targeted Insertional Mutagenesis) [2], developed for the microalgae Chlamydomonas reinhardtii P.A. Dang. model object of photosynthesis genetics. To test and optimize the TIM technique [2] in our lab, we carried out a knockout of the LTS3 gene, a transcriptional activator of chlorophyll biosynthesis genes in heterotrophic conditions [3]. We used glass beads agitation and electroporation (Gene Pulser Xcell, Bio-Rad, USA) methods in order to introduce into C. reinhardtii cells of the CC-125 (wt, mt+) strain the ribonucleoprotein complex SpCas9/sgRNA and double-stranded donor DNA with paromomycin resistance gene. The effectiveness of transformation varied from 10.6% to 68.8%. Probably, the LTS3 gene product plays a key role in the pathway of chlorophyll biosynthesis, since its knockout led to the death of C. reinhardtii cells after several division cycles. The transformation protocol optimized for the equipment available in our lab can be further refined and used to study the functions of other C. reinhardtii genes.

The aim of the study was to determine the tropism of the human immunodeficiency virus in patients with virological failure of antiretroviral therapy (ART) from the Arkhangelsk Region based on the analysis of the env gene V3 loop nucleotide sequence.Materials and methods. We used blood plasma samples obtained from 76 HIV-infected persons from the Arkhangelsk Region with virological failure of antiretroviral therapy. The nucleotide sequences of the HIV env gene C2-V3-C3 region were studied by PCR followed by sequencing. The genotype of the studied strains was determined based on the analysis of their phylogenetic relations with reference sequences from the international GenBank database, as well as using specialized programs. To predict viral tropism, the Garrido rule and the online bioinformatic tool Geno2Pheno[coreceptor] were used. The Geno2Pheno[coreceptor] algorithm, determines the false positive rate (FPR) based on the analysis of the env gene V3 loop nucleotide sequence. Results and discussion. Significantly lower representation of R5X4/X4-tropic HIV variants in long-term infected persons with subsubtype A6 virus compared to subtype B virus has been shown. For all FPR cut-off algorithms, a significant correlation between subtype and HIV tropism was observed (p=0.0014 and p=0.013 for FPR 10 % and FPR 20 %, respectively). While among subtype B strains, at least 57 % were identified as R5X4/X4-tropic variants (for an FPR of 10 %), including two strains classified as X4-tropic; among HIV subsubtype A6 even at an FPR of 20 %, the frequency of R5X4/X4-tropic samples only slightly exceeded 22 %. It can be assumed that the dynamics of changes in HIV tropism depends on the virus subtype. Significant differences in the distribution of amino acid residues of the V3 region sequences in the examined group between R5-tropic and R5X4/X4-tropic strains of subsubtype A6 for positions 18 (χ2=7.616, p=0.0058), 21 (χ2=7.281, p=0.007), 24 (χ2=5.587, p=0.0181), and 34 (χ2=5.144, p=0.0233) have been demonstrated. Among the R5X4/X4-tropic strains of the A6 subsubtype, amino acid substitutions were registered at positions 6, 19, 21, 26, 29, 30, which were not found in the R5-tropic A6 strains. The high occurrence frequency of a number of mutations previously described as presumably associated with resistance to maraviroc and similar drugs may indicate a natural polymorphism characteristic of the A6 subsubtype, which does not correlate with resistance to CCR5 co-receptor antagonists.

The goal of this study is to describe the cyanolichen Peltigera praetextata ontogenesis stages and reveal some of their morphological, anatomical, and physiological features. This paper analyzes 300 P. praetextata thalli taken in 2019 from aspen trunks in the aspen-spruce communities of the middle taiga territory in Kivach Nature Reserve (62°15'15.9'N, 33°58'746.1"E) from permanent sample plots. The authors determined the ontogenetic state of each thallus based on morphology and anatomy data using the concept of a discrete approach to the description of ontogenesis (Suetina, 2001). They registered the following thalli parameters: length, width, presence and length of rhizines, presence and width of veins, presence of phyllidia and apothecia, number of notches and lobes, shape, edge curl, and surface tomentum. They studied the anatomical parameters (thallus total thickness, tomentum thickness, cortical layer, algal layer and medulla thickness, algae cell size) using sections of thalli samples in all ontogenetic states from the apical, medial, and basal parts in triplicate. They measured the content of photosynthetic pigments spectrophotometrically with preparation of alcoholic extracts. To assess the parameters of the water regime of thalli having different ontogenetic states, the authors calculated a specific thallus mass (STM) and water holding capacity (WHC). They made a statistical data measurement using one-way ANOVA and regression analysis. They also calculated the coefficient of variation (CV) to assess the variability of the features under study. The study identified four periods and 12 ontogenetic states of P. praetextata: latent (sp), pre-generative (pr, prt, j, iml, im2, im3, vl, v2), generative (g), and post-generative (ss, s). The values of the morphological and anatomical parameters of the thalli increase during their development. The early ontogenesis stages demonstrate the highest variability of the parameters. The highest variability of the thallus structures under study within the ontogenetic states is registered for the rhizine length, vein width, and medulla thickness, while the highest constancy is typical of the share of the algal layer in the thallus that varies within a very limited range (22-32%) and reaches the minimum values in the post-generative period. The mycobiont increases in volume through a medulla growth. The physiological characteristics of the P. praetextata thalli differ in various ontogenetic states, while the generative thalli characterized by a normal ratio of photosynthetic pigments, maximum water saturation, and maximum water holding capacity have the maximum values. Thus, at early development stages, the thalli demonstrate a lower ability to retain moisture and may represent a critical stage in a lichen life cycle. On the one hand, the revealed wide variability of the morphological and anatomical parameters of young thalli testifies to their high adaptability, but the physiological parameters of water saturation indicate the vulnerability of these stages. These parameters confirm the validity of the ontogenetic stages identified. The paper contains 5 Figures, 5 Tables and 55 References. The Authors declare no conflict of interest.

Introduction. As is currently known, the epidemic process in the Kaliningrad Region was mainly associated with the spread of the recombinant form of HIV-1 (CRF03_AB); however, regular HIV importations from other countries and continents has created favorable conditions for emergence and spread of various recombinant forms of the virus.The most complete information on the diversity of recombinant forms in the region is also necessary to understand the structure of drug resistance (DR). The aim of the study was to explore the HIV-1 genetic diversity in the Kaliningrad Region.Materials and methods. We studied 162 blood plasma samples obtained from patients from the Kaliningrad Region, both with confirmed virological failure of antiretroviral therapy (ART) and with newly diagnosed HIV infection. For reverse transcription and amplification of HIV genome fragments, diagnostic «AmpliSense HIVResist-Seq».Results and discussion. The various recombinants between subtypes A and B (74%) were predominant in study group: recombinant was between CRF03_AB and subtype A (33.95%) and CRF03_AB-like (13.58%) were the most common. Among the “pure” subtypes of the virus, subtype A6 (16.67%). The circulation of subtypes B (3.70%) and G (1.23%) was also noted.Ninety-six patients (59.26%) were identified with at least one mutation associated with antiretroviral (ARV) drug resistance.Conclusion. The observed diversity of subtypes and recombinant forms of the virus implies that the new recombinants are actively emerging in the studied region, both between existing recombinant forms and “pure” subtypes, as well as between “pure” subtypes.