CLARITY and Light-Sheet microscopy sample preparation in application to human cerebral organoids

Cerebral organoids are three-dimensional cell-culture systems that represent a unique experimental model reconstructing early events of human neurogenesis in vitro in health and various pathologies. The most commonly used approach to studying the morphological parameters of organoids is immunohistochemical analysis; therefore, the three-dimensional cytoarchitecture of organoids, such as neural networks or asymmetric internal organization, is difficult to reconstruct using routine approaches. Immunohistochemical analysis of biological objects is a universal method in biological research. One of the key stages of this method is the production of cryo- or paraffin serial sections of samples, which is a very laborious and time-consuming process. In addition, slices represent only a tiny part of the object under study; three-dimensional reconstruction from the obtained serial images is an extremely complex process and often requires expensive special programs for image processing. Unfortunately, staining and microscopic examination of samples are difficult due to their low permeability and a high level of autofluorescence. Tissue cleaning technologies combined with Light-Sheet microscopy allows these challenges to be overcome. CLARITY is one of the tissue preparation techniques that makes it possible to obtain opaque biological objects transparent while maintaining the integrity of their internal structures. This method is based on a special sample preparation, during which lipids are removed from cells and replaced with hydrogel compounds such as acrylamide, while proteins and nucleic acids remain intact. CLARITY provides researchers with a unique opportunity to study three-dimensional biological structures while preserving their internal organization, including whole animals or embryos, individual organs and artificially grown organoids, in particular cerebral organoids. This protocol summarizes an optimization of CLARITY conditions for human brain organoids and the preparation of Light-Sheet microscopy samples.

Taxonomic composition and biodiversity of the gut microbiome from patients with irritable bowel syndrome, ulcerative colitis, and asthma

To date, the association of an imbalance of the intestinal microbiota with various human diseases, including both diseases of the gastrointestinal tract and disorders of the immune system, has been shown. However, despite the huge amount of accumulated data, many key questions still remain unanswered. Given limited data on the composition of the gut microbiota in patients with ulcerative colitis (UC) and irritable bowel syndrome (IBS) from different parts of Siberia, as well as the lack of data on the gut microbiota of patients with bronchial asthma (BA), the aim of the study was to assess the biodiversity of the gut microbiota of patients with IBS, UC and BA in comparison with those of healthy volunteers (HV). In this study, a comparative assessment of the biodiversity and taxonomic structure of gut microbiome was conducted based on the sequencing of 16S rRNA genes obtained from fecal samples of patients with IBS, UC, BA and volunteers. Sequences of the Firmicutes and Bacteroidetes types dominated in all samples studied. The third most common in all samples were sequences of the Proteobacteria type, which contains pathogenic and opportunistic bacteria. Sequences of the Actinobacteria type were, on average, the fourth most common. The results showed the presence of dysbiosis in the samples from patients compared to the sample from HVs. The ratio of Firmicutes/Bacteroidetes was lower in the IBS and UC samples than in HV and higher the BA samples. In the samples from patients with intestinal diseases (IBS and UC), an increase in the proportion of sequences of the Bacteroidetes type and a decrease in the proportion of sequences of the Clostridia class, as well as the Ruminococcaceae, but not Erysipelotrichaceae family, were found. The IBS, UC, and BA samples had signif icantly more Proteobacteria sequences, including Methylobacterium, Sphingomonas, Parasutterella, Halomonas, Vibrio, as well as Escherichia spp. and Shigella spp. In the gut microbiota of adults with BA, a decrease in the proportion of Roseburia, Lachnospira, Veillonella sequences was detected, but the share of Faecalibacterium and Lactobacillus sequences was the same as in healthy individuals. A signif icant increase in the proportion of Halomonas and Vibrio sequences in the gut microbiota in patients with BA has been described for the f irst time.

Revealing the diversity of Fusarium micromycetes in agroecosystems of the North Caucasus plains for replenishing the State Collection of Phytopathogenic Microorganisms of the All-Russian Scientific Research Institute of a Phytopathology

In order to prevent crop yield losses from the most dangerous and economically important pathogenic organisms, it is necessary not only to monitor the virulence gene pool, but also to study the nature of pathogen variability and determine the potential for the emergence of new genes and races. This requires centralized collections of fungal cultures characterized by a set of stable strains to provide for phytopathological, immunological, breeding, genetic, toxicological, parasitological and other studies. The State Collection of Phytopathogenic Microorganisms of the ARSRIP is the State Depository of Phytopathogenic microorganisms that are non-pathogenic to humans or farmed animals. Currently, it has more than 4,500 accessions of plant pathogenic strains of fungi, oomycetes, bacteria, viruses, phytoplasmas, and the collection is updated annually. For this purpose, the study of the inter- and intraspecific genetic diversity of genus Fusarium was carried out in agricultural systems of the Krasnodar Territory. In 2020, the State Collection of Phytopathogenic Microorganisms was supplemented with 13 strains of Fusarium fungi isolated from tissues of winter wheat plants collected in several locations of the Krasnodar region. The complex of Fusarium fungi revealed on winter wheat usually included Fusarium oxysporum, F. culmorum, F. lolii, F. graminearum, F. fujikuroi, F. sporotrichioides, etc. The effect of the preceding crop on the frequency of Fusarium species isolated from winter wheat was observed. After series cloning of collected isolates, 21 strains of different fungal species characterized by stable morphology traits and known pathogenic and phytotoxic properties were selected for collection replenishment. Significant differences in pathogenic activity were revealed between fungi belonging to either the same or different species; the manifestation of this activity varied from the absence of any effect of spore suspensions on seedling development to a complete inhibition of their growth. The phytotoxic activity towards wheat seedlings varied from medium to high. Species possessing a high intensity of phytotoxic activities are the most dangerous for wheat, since they promote accumulation of dangerous phytotoxins in plant tissues.

Genetic polymorphism of local Abkhazian grape cultivars

Local grape cultivars from different countries of the world are an important part of the gene pool of this culture. Of particular interest are the genotypes of the most ancient regions of viticulture. The territories of the subtropical zone of Georgia and the central part of Abkhazia belong to one of the centers of origin of the cultural grapevine. The purpose of the work was to genotype native Abkhazian grape cultivars, to study their genetic diversity based on DNA profiling data and to compare them with the genotypes of local varieties of other viticultural regions. Samples of plants were taken on the territory of the Republic of Abkhazia in private farmsteads and in the collection of the agricultural firm “Vina i Vody Abkhazii“ (“Wines and Waters of Abkhazia”). The genotyping of the Abkhazian cultivars Avasirhva, Agbizh, Azhapsh, Azhizhkvakva, Azhikvaca, Atvizh, Atyrkuazh, Achkykazh, Kachich was carried out using 14 DNA markers, 9 of which are standard microsatellite markers recommended for the identification of grape varieties. To improve our knowledge about the sizes of the identified alleles, we used the DNA of grape cultivars with a known allelic composition at the analyzed loci. Statistical analysis of the data showed that the observed heterozygosity for the analyzed loci exceeded expected values, which indicates a genetic polymorphism of the studied sample of varieties. Evaluation of genetic similarity within the analyzed group based on the results of genotyping at 14 loci showed that the cultivars Kachich and Azhapsh differed from the other Abkhazian varieties. The obtained DNA profiles of the Abkhazian cultivars were checked for compliance with DNA-fingerprints of grape varieties in the Vitis International Variety Catalogue. The Georgian varieties Azhizhkvakva and Tsitska turned out to be synonyms according to DNA profiles, two varieties from the Database (Italian Albana bianca and Georgian Ojaleshi) have differences in DNA-fingerprints from the varieties Atyrkuazh and Azhikvatsa only in one allele, respectively. When comparing the identified Abkhazian grape genotypes, their difference from the sample of Dagestan, Don, Greek, Turkish, Italian, Spanish, and French varieties and genetic similarity with the genotypes of Georgian grapes were shown.

Assessment of genetic diversity and phylogenetic relationships in Black Pied cattle in the Novosibirsk Region using microsatellite markers

There are currently over a thousand indigenous cattle breeds well adapted to local habitat conditions thanks to their long history of evolution and breeding. Identification of the genetic variations controlling the adaptation of local cattle breeds for their further introduction into the genome of highly productive global breeds is a matter of great relevance. Studying individual populations of the same breed with the use of microsatellite markers makes it possible to assess their genetic diversity, relationships, and breed improvement potential. Although the Black Pied breed is the most common dairy cattle breed in Russia, there are only a few studies on genetic diversity in local Black Pied populations in some Russian regions. The goal of the present study was to analyze the genetic diversity in Black Pied cattle populations in the Novosibirsk Region and compare them with other Russian populations; to identify significantly divergent populations with a view to preserving them under the programs aimed at maintaining the genetic diversity of the domestic Black Pied breed. DNA samples from 4788 animals of the Black Pied breed from six breeding enterprises in the Novosibirsk Region have been studied using 11 microsatellite markers. No significant differences in genetic variability parameters were found between individual populations. Private alleles have been identified in five out of six populations. Five populations have shown inbreeding coefficient values (FIS) below zero, which indicates heterozygosity excess. The population distribution test, principal component analysis, FST and DEST values, cluster analysis, and phylogenetic analysis have revealed two populations genetically distinct from the others. Essentially, the genetic diversity parameters of the six studied Black Pied cattle populations from the Novosibirsk Region show no significant differences from other Russian populations of the breed. Excess heterozygosity is observed in most breeding enterprises, which is a sign of a low inbreeding rate. To maintain the genetic diversity of the Russian Black Pied cattle, we recommend focusing on the two populations with significant genetic distinctions from the others.

Prevalence of the polymorphic H-ficolin (FCN3) genes and mannosebinding lectin-associated serine protease-2 (MASP2) in indigenous populations from the Russian Arctic regions

Lectins, being the main proteins of the lectin pathway activating the complement system, are encoded by polymorphic genes, wherein point mutations cause the protein conformation and expression to change, which turns out to have an effect on the functionality and ability to respond to the pathogen. In the current study, largescale data on the population genotype distribution of the genes for H-ficolin FCN3 rs28357092 and mannose-binding lectin-associated serine protease MASP2 rs72550870 among the indigenous peoples of the Russian Arctic regions (Nenets, Dolgans and Nganasans, a mixed population and Russians: a total sample was about 1000 newborns) have been obtained for the first time. Genotyping was carried out using RT-PCR. The frequency of the homozygous variant del/del FCN3 rs28357092 associated with the total absence of the most powerful activator of the lectin complement pathway, N-ficolin, was revealed; 0 % in the Nenets, 0.8 % in the Dolgans and Nganasans, and 3.5 % among the Russians ( p < 0.01). Analysis of the prevalence of the MASP2 genotypes has shown the predominance of the homozygous variant AA in all studied populations, which agrees with the available world data. The heterozygous genotype AG rs72550870 associated with a reduced level of protease was found to occur rarely in the Nenets, Dolgans and Nganasans compared to newborns of Caucasoid origin from Krasnoyarsk: 0.5 % versus 3.3 %, respectively. Moreover, among 323 examined Nenets, one AG carrier was identified, whereas in Russians, 16 out of 242 examined newborns were found to be AG carriers ( p < 0.001). A homozygous variant (GG) in total absence of protease with impaired binding of both MBL and ficolins was not detected in any of the 980 examined newborns. An additional analysis of infectious morbidity in Arctic populations allows one to find phenotypic characteristics related to a high functional activity of the lectin pathway of complement activation as an most important factor for the first-line of anti-infectious defense, including such new viral diseases as COVID-19.

Putative regulatory functions of SNPs associated with bronchial asthma, arterial hypertension and their comorbid phenotype

Linkage disequilibrium (LD) of single nucleotide polymorphisms (SNPs) of TLR4/AL160272.2 (rs1927914, rs1928298, rs7038716, rs7026297, rs7025144) was estimated in the Slavs of West Siberia. We further investigated an association of SNPs in TLR4/AL160272.2 (rs1927914, rs7038716, rs7025144), SERPINA1 (rs1980616), ATXN2/BRAP (rs11065987), IL2RB (rs2284033), NT5C2 (rs11191582), CARD8 (rs11669386), ANG/RNASE4 (rs1010461), and ABTB2/ САТ (rs2022318) genes with bronchial asthma (BA), arterial hypertension (AH) and their comorbidity. Then, the disease-associated SNPs were annotated in silico in relation to their potential regulatory functions. Strong LD was detected between rs1928298 and rs1927914, as well as rs7026297 and rs7038716 in the Slavs of West Siberia. It was found that the rs1927914 G allele of the TLR4 gene and the rs1980616 C allele of the SERPINA1 gene are associated with the predisposition to BA. These SNPs can affect binding affinity of transcription factors of the Pou and Klf4 families, as well as the expression levels of the TLR4 and SERPINA1 genes. The rs11065987 allele A of the ATXN2/BRAP genes, the rs11669386 A allele of the CARD8 gene, the rs2284033 allele G of the IL2RB gene, and the rs11191582 allele G of the NT5C2 gene were associated with the risk of AH. These variants can alter binding affinity of the Hoxa9, Irf, RORalpha1 and HMG-IY transcription factors, as well as the expression levels of the ALDH2, CARD8, NT5C2, ARL3, and SFXN2 genes in blood cells/vessels/heart, respectively. The risk of developing a comorbid phenotype of AD and AH is associated with the A allele of rs7038716 and the T allele of rs7025144 of the TLR4/AL160272.2 genes, the A allele of rs1010461 of the ANG gene and the C allele of rs2022318 of the ABTB2/CAT genes. Variants rs7038716 and rs7025144 can change the expression levels of the TLR4 gene in blood cells, while rs1010461 and rs2022318 influence the expression levels of the ANG and RNASE4 genes as well as the CAT and ABTB2 genes in blood cells, lungs/vessels/heart.

The role of the KIBRA and APOE genes in developing spatial abilities in humans

In the contemporary high-tech society, spatial abilities predict individual life and professional success, especially in the STEM (Science, Technology, Engineering, and Mathematics) disciplines. According to neurobiological hypotheses, individual differences in cognitive abilities may be attributed to the functioning of genes involved in the regulation of neurogenesis and synaptic plasticity. In addition, genome-wide association studies identified rs17070145 located in the KIBRA gene, which was associated with individual differences in episodic memory. Considering a significant role of genetic and environmental components in cognitive functioning, the present study aimed to estimate the main effect of NGF (rs6330), NRXN1 (rs1045881, rs4971648), KIBRA (rs17070145), NRG1 (rs6994992), BDNF (rs6265), GRIN2B (rs3764030), APOE (rs7412, rs429358), and SNAP25 (rs363050) gene polymorphisms and to assess the effect of gene-environment interactions on individual differences in spatial ability in individuals without cognitive decline aged 18–25 years (N = 1011, 80 % women). Spatial abilities were measured using a battery of cognitive tests including the assessment of “3D shape rotation” (mental rotation). Multiple regression analysis, which was carried out in the total sample controlling for sex, ethnicity and the presence of the “risk” APOE ε4 allele, demonstrated the association of the rs17070145 Т-allele in the KIBRA gene with enhanced spatial ability (β = 1.32; pFDR = 0.037) compared to carriers of the rs17070145 CC-genotype. The analysis of gene-environment interactions revealed that nicotine smoking (β = 3.74; p = 0.010) and urban/rural residency in childhood (β = –6.94; p = 0.0002) modulated the association of KIBRA rs17070145 and АРОЕ (rs7412, rs429358) gene variants with individual differences in mental rotation, respectively. The data obtained confirm the effect of the KIBRA rs17070145 Т-allele on improved cognitive functioning and for the first time evidence the association of the mentioned genetic variant with spatial abilities in humans. A “protective” effect of the APOE ε2 allele on enhanced cognitive functioning is observed only under certain conditions related to childhood rearing.

The relationship between the genetic status of the Vrn-1 locus and the size of the root system in bread wheat (Triticum aestivum L.)

One of the main ways to fine-tune the adaptive potential of wheat cultivars is to regulate the timing of flowering using the genes of the Vrn-1 locus, which determines the type and rate of development. Recently, with the use of introgression and isogenic lines of bread wheat, it was shown that this locus is involved in the genetic control of root length and weight both under irrigation and drought conditions. It turned out that the VrnA1 gene is associated with a significant decrease in the size of the root system in a winter genotype. The Vrn-A1 gene had the strongest effect on the reduction of the root system in comparison with the homoeoallelic genes Vrn-B1 and Vrn-D1. The aim of this work was to determine whether the allelic composition of the genes at the Vrn-1 locus affects the root size in seven spring cultivars and in two lines of bread wheat differing in flowering time under conditions of normal watering and drought. The research was carried out in a hydroponic greenhouse; drought was created at the tillering stage. In this work, we have shown that early flowering wheat cultivars with the dominant Vrn-A1а allele have more lightweight and shorter roots under normal watering conditions compared to the late flowering carriers of the dominant homoeoalleles Vrn-B1 and Vrn-D1. In drought conditions, the root length decreased insignificantly, but the weight of the roots significantly decreased in all genotypes, with the exception of Diamant 2. It has been hypothesized that the level of the transcription factor VRN-1 at the onset of drought may affect the size of the root system. The large variability in root weight may indicate the participation, in addition to the Vrn-1 locus, of other gene networks in the formation of this trait. Breeders working to develop early maturing varieties should consider the possibility of reducing the root size, especially in arid conditions. A significant increase in the root size of line 821 with introgressions into chromosomes 2A, 2B, and 5A from T. timopheevii indicates the possibility of using congeners as a source of increasing the trait in wheat.

Genetic diversity of the Central European wild boar (Sus scrofa scrofa) population and domestic pig (Sus scrofa domesticus) breeds based on a microsatellite DNA locus

The results of studies of the genetic structure of the Central European wild boar (Sus scrofa scrofa) population and four breeds of domestic pigs (Duroc, Yorkshire, Large White and Landrace) bred in the Central Black Earth region of Russia are presented in this work. Based on 12 microsatellite loci, a significant ( p <0.05) decrease in the level of genetic variability in bred breeds was shown. The expected heterozygosity and Shannon index were as follows: in the wild boar, Ho = 0.763 ± 0.026, I = 1.717 ± 0.091; in the Duroc breed, Ho = 0.569 ± 0.068, I = 1.191 ± 0.157; in the Landrace, Ho = 0.618 ± 0.062, I = 1.201 ± 0.147; in the Large White, Ho = 0.680 ± 0.029, I = 1.362 ± 0.074; and in the Yorkshire, Ho = 0.642 ± 0.065, I = 1.287 ± 0.156. The results of checking genotypic Hardy–Weinberg equilibrium based on the G-test of maximum likelihood demonstrated that the overwhelming majority of loci in the wild boar population were in the state of said equilibrium. By contrast, in pig breed populations, some loci demonstrated a significant deviation from the indicated equilibrium. In addition, the Yorkshire, Large White, and Landrace populations had loci, for which the hypothesis of neutrality was reliably rejected based on the results of the Ewens–Watterson test. The revealed private alleles, characteristic of the wild boar and breeds, can later be used to identify them. The ordination of the centroids of different herds in the space of the first two principal coordinates based on the matrix of pairwise estimates of Nei’s genetic distances showed that the most distant populations are the Duroc and Boar breeds, and the most genetically close are the Yorkshire and Landrace breeds. The closest to the wild boar population was the Large White breed. The assessment of the effective size, carried out using the method based on the linkage disequilibrium and the molecular coancestry method, showed that in all studied groups, including the wild boar population, the effective size was less than 100 individuals. The low effective size of the wild boar population (Ne = 21.8, Neb = 4.0) is probably caused by the death and shooting of animals due to Pestis africana suum.