Expression Of Other Genes Research Articles

Tobacco rattle virus-induced VcANS gene silencing in blueberry fruit

Blueberry (Vaccinium corymbosum L.), a woody perennial bush in the genus Vaccinium, is an economically important and popular fruit crop worldwide. Development the superior cultivars, which including excellent fruit traits, not only means higher yielding and economic efficiency, but also produce fruit that to meet the preferences of different consumers. Excavating fruit quality-related genes, studying their functions, and using transgenic or molecular-assisted breeding are beneficial to the development of excellent blueberry varieties. Genetic transformation is an excellent way to study the function of genes in plants, however, it is a labor-intensive and time-consuming process to genetically transform many woody plants, including blueberry. Virus-induced gene silencing (VIGS) provides an efficient approach to knock-down the expression of target genes for functional analysis. In this study, tobacco rattle virus induced genes silencing (TRV-VIGS) was established in blueberry fruits using the VcANS gene as a reporter. The silenced sector of the skin of blueberry fruits injected with pTRV2 (plasmid Tobacco Rattle Virus, TRV-RNA2)::VcANS remained green or white at 25 days after agroinfiltration. In agroinfiltrated materials, the VcANS transcript levels were much lower in fruits with phenotypic changes (delayed color change) than in those infiltrated with the pTRV2 empty vector. Silencing of VcANS also affected the expression of other genes involved in the anthocyanin synthesis pathway. The experimental results support that VcANS can be used as an effective marker gene for VIGS system. In addition, the TRV-VIGS system has been successfully established in blueberry fruits, which provided an effective verification method for functional identification of unknown genes in blueberry fruits.

Effects of a Saccharomyces cerevisiae fermentation product-supplemented diet on fecal characteristics, oxidative stress, and blood gene expression of adult dogs undergoing transport stress.

Previously, a Saccharomyces cerevisiae fermentation product (SCFP) was shown to positively alter fecal microbiota, fecal metabolites, oxidative stress, and circulating immune cell function of adult dogs. The objective of this study was to measure the effects of SCFP on fecal characteristics, serum oxidative stress biomarkers, and whole blood gene expression of dogs undergoing transport stress. Sixteen adult pointer dogs [8M, 8F; mean age = 6.7 ± 2.1 yr; mean body weight (BW) = 25.5 ± 3.9 kg] were used in a randomized crossover design study. All dogs were fed a control diet for 4 wk, then randomly assigned to a control or SCFP-supplemented diet (formulated to include approximately 0.13% of the active SCFP ingredient) and fed to maintain BW for 11 wk. A 6-wk washout preceded the second 11-wk experimental period with dogs receiving opposite treatments. After 11 wk, fresh fecal and blood samples were collected before and after transport in a van for 45 min. Change from baseline data (i.e., before and after transport) were analyzed using the Mixed Models procedure of SAS 9.4, with P < 0.05 being significant and P < 0.10 being trends. Change in serum malondialdehyde concentrations increased (P < 0.05) and serum 8-isoprostane concentrations tended to increase (P < 0.10) in dogs fed SCFP, but decreased (P < 0.05) in control dogs after transport. Other serum markers were unaffected by diet during transport stress. Fecal dry matter percentage tended to be affected (P < 0.10) by diet during transport stress, being reduced in control dogs, but stable in dogs fed SCFP. Other fecal characteristics were unaffected by diet during transport stress. Genes associated with activation of innate immunity were impacted by diet in response to transport stress, with blood cyclooxygenase-2 and malondialdehyde mRNA expression being increased (P < 0.05) in control dogs, but stable or decreased in dogs fed SCFP. Expression of other genes was unaffected by diet during transport stress. These data suggest that the benefits of feeding a SCFP during transport stress may be mediated through suppression of innate immune cell activation.

Functional differentiation of bmp2a and bmp2b genes in zebrafish

Bone morphogenetic protein 2 plays an important role in the regulation of osteoblast proliferation and differentiation. Phylogenetic analysis showed that the bmp2 ortholog evolved from the same ancestral gene family in vertebrates and was duplicated in teleost, which were named bmp2a and bmp2b. The results of whole-mount in situ hybridization showed that the expression locations of bmp2a and bmp2b in zebrafish were different in different periods (24 hpf, 48 hpf, 72 hpf), which revealed potential functional differentiation between bmp2a and bmp2b. Phenotypic analysis showed that bmp2a mutations caused partial rib and vertebral deformities in zebrafish, while bmp2b−/− embryos died massively after 12 hpf due to abnormal somite formation. We further explored the expression pattern changes of genes (bmp2a, bmp2b, smad1, fgf4, runx2b, alp) related to skeletal development at different developmental stages (20 dpf, 60 dpf, 90 dpf) in wild-type and bmp2a−/− zebrafish. The results showed that the expression of runx2b in bmp2a−/− was significantly downregulated at three stages and the expression of other genes were significantly downregulated at 90 dpf compared with wild-type zebrafish. The study revealed functional differentiation of bmp2a and bmp2b in zebrafish embryonic and skeletal development.

RF13 | PMON142 Long Non-coding RNAs in Pituitary Differentiation and Endocrine Function

Abstract Mammalian genomes contain thousands of genes for long non-coding RNA (lncRNAs) that affect the expression of other genes through diverse mechanisms. The lncRNA transcripts are longer than 200 nucleotides and are often capped, spliced and polyadenylated, but they are not translated into proteins. Nuclear lncRNAs modify chromatin structure and transcription in trans or cis by interacting with the DNA, forming R-loops, and recruiting regulatory proteins. Not much is known about the role of lncRNA in pituitary gland differentiation and function, but there are several examples that provide a strong premise for investigating this class of transcripts more deeply. For example, the lncRNA MEG3 is a tumor suppressor that is silenced in non-functioning pituitary adenomas; and lncRNAs have been implicated in regulating the function of key transcription factors such as POU1F1, PITX1, and PITX2. To develop a research resource for the study of lncRNA, we used cell-type specific cre transgenes to tag individual adult pituitary cell types with fluorescent markers and enriched for thyrotropes, gonadotropes and somatotropes using fluorescence activated cell sorting. We determined the transcriptome of each cell population using RNA sequencing and mined the data for lncRNA. We detected hundreds of lncRNA in adult pituitary cells, and some were cell-type specific, and located in immediate proximity of genes that encode pituitary hormones or lineage-specific transcription factors. We discovered that the lncRNA Nr5a1os is enriched in gonadotropes, while Foxl2os is enriched in gonadotropes and thyrotropes. The lncRNA Platr9 is located on the opposite strand near Cga, which encodes the alpha subunit of thyrotropin and gonadotropin. We also mined transcriptome data from pituitary glands collected at embryonic days 12.5 and 14.5 and identified 351 unique lncRNAs, many of which are exclusively expressed during development. Among these is Six3os1, which is located upstream of Six3, a gene associated with holoprosencephaly and hypopituitarism. The location of many of these lncRNAs suggests that they may have a cis-acting regulatory role in pituitary development or physiological function. This research resource sets the foundation for examining the actions of lncRNAs on expression of nearby genes during development and in response to physiological demand. Future research will elucidate their mechanisms of action, determining whether they recruit regulatory proteins to the chromatin and/or form R-loops in these regions, and how this affects expression of the target gene(s). We expect these studies to further our understanding of pituitary biology. Presentation: Sunday, June 12, 2022 12:54 p.m. - 12:59 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

123 Effects of a Saccharomyces Cerevisiae Fermentation Product-Supplemented Diet on Fecal Characteristics, Oxidative Stress, and Blood Gene Expression of Adult Dogs Undergoing Transport Stress

Abstract Previously, a Saccharomyces cerevisiae fermentation product (SCFP) was shown to positively alter fecal microbiota, fecal metabolites, and circulating immune cell function of adult dogs. The objective of this study was to measure the effects of SCFP on fecal characteristics, serum oxidative stress biomarkers, and whole blood gene expression of adult dogs undergoing transport stress. Sixteen adult pointer dogs (8M, 8F; mean age = 8.35 y; mean BW = 27.13 kg) were used in a randomized crossover design study. All dogs were fed control diet for 4 wk, then randomly assigned to a control or SCFP-supplemented diet and fed to maintain BW for 11 wk. A 6-wk washout preceded the second 11-wk experimental period with dogs receiving opposite treatments. After 11 wk, fresh fecal and blood samples were collected before and after transport in a van for 45 min. Change from baseline data (i.e., before and after transport) were analyzed using the Mixed Models procedure of SAS 9.4, with P&amp;lt; 0.05 being significant and P&amp;lt; 0.10 being trends. Changes in serum malondialdehyde and 8-isoprostane:creatinine concentrations increased (P&amp;lt; 0.05) in dogs fed SCFP, but decreased in control dogs with transport stress. Other serum markers were unaffected by diet during transport stress. Fecal dry matter percentage tended to be affected (P&amp;lt; 0.10) by diet during transport stress, being reduced in control dogs, but stable in dogs fed SCFP. Other fecal characteristics were unaffected by diet during transport stress. Blood cyclooxgenase-2 and myeloperoxidase mRNA expression was affected (P&amp;lt; 0.05) by diet during transport stress, being increased in control dogs, but stable or decreased in dogs fed SCFP. Expression of other genes was unaffected by diet during transport stress. These data suggest that the benefits of feeding a SCFP in a novel transport stress model are mediated more so by suppressing innate immune cell activation than through decreasing lipid peroxidation.

PSXV-10 Poor Maternal Nutrition Impacts Mrna Expression of Epigenetic Factors in ram Offspring Liver

Abstract Poor maternal nutrition (over- and restricted-feeding) alters growth and metabolism in offspring after birth; however, the mechanisms are not well understood. Epigenetics, the heritable changes in gene expression without altering the DNA sequence, is a possible mechanism. We hypothesized that mRNA expression of epigenetic factors in livers of male offspring would be altered as a result of poor maternal diet during gestation. Multiparous Dorset ewes (n = 46) pregnant with twins were fed 100%, 60% or 140% of NRC requirements from d 30 ± 0.02 of gestation until parturition. Male offspring are referred to as CON (n=12), RES (n=21), and OVER (n=13), respectively. Ram lambs were euthanized at d 284±1.82, and liver tissue was immediately snap-frozen. RNA was isolated from liver and gene expression was quantified using real-time (RT)-PCR. Epigenetic factors of interest included DNA methyltransferases (DNMT; DNMT1, DNMT3B), ten eleven translocation enzyme 2 (TET2), lysine acetyltransferases (KAT; KAT1, KAT2B, KAT6A, KAT8), histone deacetylases (HDAC; HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC8, HDAC11, sirtuin1), and histone methyltransferases [ASH1 like histone lysine methyltransferase (ASH1L), euchromatic histone lysine methyltransferase (EHMT)1, EHMT2, SET domain bifurcated histone lysine methyltransferase 2 (SETDB2)]. Gene expression of ASH1L and KAT6A were decreased in liver tissue of RES compared with OVER by 42.8% and 59.7%, respectively (P≤0.017). Gene expression of DNMT1 and HDAC3 were decreased in RES compared with CON by 38.3% and 35.0%, respectively (P≤0.045). Gene expression of EHMT1 and HDAC8 were increased in OVER compared with CON by 19.3% and 37.4%, respectively (P≤0.045). There were no differences detected in expression of other genes (P≥0.060). Thus, maternal diet affects hepatic mRNA expression of specific epigenetic factors which may contribute to altered metabolism and liver function. Further investigation is warranted to determine how epigenetic modifications impact offspring growth and metabolism long-term.

Targeted transcript analysis in muscles from patients with genetically diverse congenital myopathies.

Congenital myopathies are a group of early onset muscle diseases of variable severity often with characteristic muscle biopsy findings and involvement of specific muscle types. The clinical diagnosis of patients typically relies on histopathological findings and is confirmed by genetic analysis. The most commonly mutated genes encode proteins involved in skeletal muscle excitation–contraction coupling, calcium regulation, sarcomeric proteins and thin–thick filament interaction. However, mutations in genes encoding proteins involved in other physiological functions (for example mutations in SELENON and MTM1, which encode for ubiquitously expressed proteins of low tissue specificity) have also been identified. This intriguing observation indicates that the presence of a genetic mutation impacts the expression of other genes whose product is important for skeletal muscle function. The aim of the present investigation was to verify if there are common changes in transcript and microRNA expression in muscles from patients with genetically heterogeneous congenital myopathies, focusing on genes encoding proteins involved in excitation–contraction coupling and calcium homeostasis, sarcomeric proteins, transcription factors and epigenetic enzymes. Our results identify RYR1, ATPB2B and miRNA-22 as common transcripts whose expression is decreased in muscles from congenital myopathy patients. The resulting protein deficiency may contribute to the muscle weakness observed in these patients. This study also provides information regarding potential biomarkers for monitoring disease progression and response to pharmacological treatments in patients with congenital myopathies.

The phenotype of the gut region is more stably retained than developmental stage in piglet intestinal organoids.

Intestinal organoids are innovative in vitro tools to study the digestive epithelium. The objective of this study was to generate jejunum and colon organoids from suckling and weaned piglets in order to determine the extent to which organoids retain a location-specific and a developmental stage-specific phenotype. Organoids were studied at three time points by gene expression profiling for comparison with the transcriptomic patterns observed in crypts in vivo. In addition, the gut microbiota and the metabolome were analyzed to characterize the luminal environment of epithelial cells at the origin of organoids. The location-specific expression of 60 genes differentially expressed between jejunum and colon crypts from suckling piglets was partially retained (48%) in the derived organoids at all time point. The regional expression of these genes was independent of luminal signals since the major differences in microbiota and metabolome observed in vivo between the jejunum and the colon were not reproduced in vitro. In contrast, the regional expression of other genes was erased in organoids. Moreover, the developmental stage-specific expression of 30 genes differentially expressed between the jejunum crypts of suckling and weaned piglets was not stably retained in the derived organoids. Differentiation of organoids was necessary to observe the regional expression of certain genes while it was not sufficient to reproduce developmental stage-specific expression patterns. In conclusion, piglet intestinal organoids retained a location-specific phenotype while the characteristics of developmental stage were erased in vitro. Reproducing more closely the luminal environment might help to increase the physiological relevance of intestinal organoids.

Functional analysis of ABCG2 gene in pigment transport of Neocaridina denticulata sinensis

Many strains of Neocaridina denticulata sinensis (cherry shrimp) possess vivid body colors.However, the molecular underpinnings of these various body colors are scarcely understood. To study the role of the ABCG2 gene in the pigmentation of cherry shrimp, four strains (red, yellow, blue, and wild strains) were sampled. The sequence and expression pattern of ABCG2in tissues and embryos were analyzed, and the distribution of ABCG2 was also explored via WFISH (whole mount flurescence in situ hybridization). And further, RNA interference (RNAi) was used to explore the role of ABCG2 in body color deposition. The results showed that the ABCG2 sequence contained the conserved motif of Walker A, Walker B, Q-loop, d-loop, and H-loop. In tissues, ABCG2 was highly expressed in the epidermis of the four strains. During development stages, it was first expressed at the cleavage stage, then decreased at the gastrula stage, with the lowest expression at the pre-nauplius stage. From the metanauplius stage, its expression level was significantly upregulated until it reached the highest level at the membrane-zoea stage. WFISH showed that ABCG2 was first expressed at the cleavage stage in all four strains, and its distribution was similar from the cleavage stage to the before-zoea stage. The strongest positive signals were observed at the membrane-zoea stage, which was consistent with the qPCR results. Moreover, at the membrane-zoea stage, the positive signals of the four strains were mainly distributed in the compound eye and appendages. RNAi knockdown of ABCG2 encumbered the development of compound eye pigment cells (CEPCs) and erythrophores. It had effects on the expressions of other genes related to body color. These results suggest that ABCG2 is involved in the development of compound eye and the proliferation of erythrophores. This study provides new ideas for the cultivation of novel body colors in cherry shrimp at the molecular level.

CircWalk: a novel approach to predict CircRNA-disease association based on heterogeneous network representation learning

BackgroundSeveral types of RNA in the cell are usually involved in biological processes with multiple functions. Coding RNAs code for proteins while non-coding RNAs regulate gene expression. Some single-strand RNAs can create a circular shape via the back splicing process and convert into a new type called circular RNA (circRNA). circRNAs are among the essential non-coding RNAs in the cell that involve multiple disorders. One of the critical functions of circRNAs is to regulate the expression of other genes through sponging micro RNAs (miRNAs) in diseases. This mechanism, known as the competing endogenous RNA (ceRNA) hypothesis, and additional information obtained from biological datasets can be used by computational approaches to predict novel associations between disease and circRNAs.ResultsWe applied multiple classifiers to validate the extracted features from the heterogeneous network and selected the most appropriate one based on some evaluation criteria. Then, the XGBoost is utilized in our pipeline to generate a novel approach, called CircWalk, to predict CircRNA-Disease associations. Our results demonstrate that CircWalk has reasonable accuracy and AUC compared with other state-of-the-art algorithms. We also use CircWalk to predict novel circRNAs associated with lung, gastric, and colorectal cancers as a case study. The results show that our approach can accurately detect novel circRNAs related to these diseases.ConclusionsConsidering the ceRNA hypothesis, we integrate multiple resources to construct a heterogeneous network from circRNAs, mRNAs, miRNAs, and diseases. Next, the DeepWalk algorithm is applied to the network to extract feature vectors for circRNAs and diseases. The extracted features are used to learn a classifier and generate a model to predict novel CircRNA-Disease associations. Our approach uses the concept of the ceRNA hypothesis and the miRNA sponge effect of circRNAs to predict their associations with diseases. Our results show that this outlook could help identify CircRNA-Disease associations more accurately.

Targeted Modulation of Interferon Response-Related Genes with IFN-Alpha/Lambda Inhibition.

Interferon (IFN) signaling resulting from external or internal inflammatory processes initiates the rapid release of cytokines and chemokines to target viral or bacterial invasion, as well as cancer and other diseases. Prolonged exposure to IFNs, or the overexpression of other cytokines, leads to immune exhaustion, enhancing inflammation and leading to the persistence of infection and promotion of disease. Hence, to control and stabilize an excessive immune response, approaches for the management of inflammation are required. The potential use of peptides as anti-inflammatory agents has been previously demonstrated. Our team discovered, and previously published, a 9-amino-acid cyclic peptide named ALOS4 which exhibits anti-cancer properties in vivo and in vitro. We suggested that the anti-cancer effect of ALOS4 arises from interaction with the immune system, possibly through the modulation of inflammatory processes. Here, we show that treatment with ALOS4 decreases basal cytokine levels in mice with chronic inflammation and prolongs the lifespan of mice with acute systemic inflammation induced by irradiation. We also show that pretreatment with ALOS4 reduces the expression of IFN alpha, IFN lambda, and selected interferon-response genes triggered by polyinosinic-polycytidylic acid (Poly I:C), a synthetic analog of viral double-stranded RNA, while upregulating the expression of other genes with antiviral activity. Hence, we conclude that ALOS4 does not prevent IFN signaling, but rather supports the antiviral response by upregulating the expression of interferon-response genes in an interferon-independent manner.

Abstract 4226: Effect of HDAC6 inhibition on the expression of immunomodulator genes in colorectal cancer cells

Abstract Cancer is one of the most frequent disease worldwide and currently, treatment failures are caused by pharmacological resistance or low success rate of conventional approaches. In particular, colorectal cancer (CRC) is one of the most common malignant neoplasm in Chile and according to the WHO, represents the second cause of death from tumors in the world, which is why there is a need to study new strategies for future therapies, like the immunotherapy aimed at inhibiting the immune checkpoints, as Programmed Death 1 (PD-1) receptor in T-cell, that interact with his ligand (PD-L1) in the tumor. However, there is a percentage of patients who don't respond to this therapy, making it necessary to search for alternative or adjuvant treatments. Histone deacetylase 6 (HDAC6), a mainly cytoplasmic protein, is involved in several cellular processes including the immune response, being involve in tumor progression. Specifically, it has been observed that HDAC6 stimulates STAT3 activity, a transcription factor involved in immunogenicity, which plays a role as a transcriptional inducer of different genes in these pathways, as PD-L1 (programmed death ligand 1). The over-expression of PD-L1 on some cancer cells promotes inhibition of T lymphocyte activation which reduces the immune response. Therefore, we will focus on pharmacological inhibition of HDAC6 in CRC cell due to its potential as adjuvants to avoid immunotolerance in cancer immunotherapy. Here, we investigate whether HDAC6 affects STAT3 activation in CRC cells, through treatments with Nexturastat A (NextA), a specific HDAC6 inhibitor, that indirectly, decreases the levels of activating post-translational modifications on STAT3 analyzed by Western blot and flow cytometry. In the same context, we analyze by RT-qPCR the decrease in the expression of IL-10 and PD-L1 after NextA treatment. Then to emulate an inflammatory environment, we treat the cells with Interleukin-6 (IL-6) and then with NextA, observing the changes described above over the STAT3 modification and activity. Finally, through a RNAseq assay with the last conditions, showed a reduced the expression of other genes involved in immunomodulatory pathways. These results suggest that treatments with specific HDAC6 inhibitors would reduce the functionality of STAT3 in CRC cells, impacting the expression of immunomodulator genes involved in the immune response, as IL-10, PD-L1 and others. Therefore, the use of specific HDAC6 inhibitors may be a reasonable and an interesting adjuvant strategy for help in checkpoint inhibitors immunotherapy, since HDAC6 inhibitors would indirectly decrease PD-L1 expression, one of the checkpoint inhibitors of immune system. Citation Format: Constanza Mardones, Camila Navarrete, Estrella Armijo, Fernando Rivas, Matias Ignacio Hepp. Effect of HDAC6 inhibition on the expression of immunomodulator genes in colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4226.

Impact of IsaA Gene Disruption: Decreasing Staphylococcal Biofilm and Alteration of Transcriptomic and Proteomic Profiles.

Staphylococcus aureus expresses diverse proteins at different stages of growth. The immunodominant staphylococcal antigen A (IsaA) is one of the proteins that is constitutively produced by S. aureus during colonisation and infection. SACOL2584 (or isaA) is the gene that encodes this protein. It has been suggested that IsaA can hydrolyse cell walls, and there is still need to study isaA gene disruption to analyse its impact on staphylococcal phenotypes and on alteration to its transcription and protein profiles. In the present study, the growth curve in RPMI medium (which mimics human plasma), autolytic activity, cell wall morphology, fibronectin and fibrinogen adhesion and biofilm formation of S. aureus SH1000 (wildtype) was compared to that of S. aureus MS001 (isaA mutant). RNA sequencing and liquid chromatography–tandem mass spectrometry were carried out on samples of both S. aureus strains taken during the exponential growth phase, followed by bioinformatics analysis. Disruption of isaA had no obvious effect on the growth curve and autolysis ability or thickness of cell walls, but this study revealed significant strength of fibronectin adherence in S. aureus MS001. In particular, the isaA mutant formed less biofilm than S. aureus SH1000. In addition, proteomics and transcriptomics showed that the adhesin/biofilm-related genes and hemolysin genes, such as sasF, sarX and hlgC, were consistently downregulated with isaA gene disruption. The majority of the upregulated genes or proteins in S. aureus MS001 were pur genes. Taken together, this study provides insight into how isaA disruption changes the expression of other genes and has implications regarding biofilm formation and biological processes.

Generating dynamic gene expression patterns without the need for regulatory circuits.

Synthetic biology has successfully advanced our ability to design and implement complex, time-varying genetic circuits to control the expression of recombinant proteins. However, these circuits typically require the production of regulatory genes whose only purpose is to coordinate expression of other genes. When designing very small genetic constructs, such as viral genomes, we may want to avoid introducing such auxiliary gene products while nevertheless encoding complex expression dynamics. To this end, here we demonstrate that varying only the placement and strengths of promoters, terminators, and RNase cleavage sites in a computational model of a bacteriophage genome is sufficient to achieve solutions to a variety of basic gene expression patterns. We discover these genetic solutions by computationally evolving genomes to reproduce desired gene expression time-course data. Our approach shows that non-trivial patterns can be evolved, including patterns where the relative ordering of genes by abundance changes over time. We find that some patterns are easier to evolve than others, and comparable expression patterns can be achieved via different genetic architectures. Our work opens up a novel avenue to genome engineering via fine-tuning the balance of gene expression and gene degradation rates.

Understanding the Mechanism of Dysglycemia in a Fanconi-Bickel Syndrome Patient.

Fanconi–Bickel Syndrome (FBS) is a rare disorder of carbohydrate metabolism that is characterized mainly by the accumulation of glycogen in the liver and kidney. It is inherited as an autosomal recessive disorder caused by mutations in the SLC2A2 gene, which encodes for GLUT2. Patients with FBS have dysglycemia but the molecular mechanisms of dysglycemia are still not clearly understood. Therefore, we aimed to understand the underlying molecular mechanisms of dysglycemia in a patient with FBS. Genomic DNA was isolated from a peripheral blood sample and analyzed by whole genome and Sanger sequencing. CRISPR-Cas9 was used to introduce a mutation that mimics the patient’s mutation in a human kidney cell line expressing GLUT2 (HEK293T). Mutant cells were used for molecular analysis to investigate the effects of the mutation on the expression and function of GLUT2, as well as the expression of other genes implicated in dysglycemia. The patient was found to have a homozygous nonsense mutation (c.901C>T, R301X) in the SLC2A2 gene. CRISPR-Cas9 successfully mimicked the patient’s mutation in HEK293T cells. The mutant cells showed overexpression of a dysfunctional GLUT2 protein, resulting in reduced glucose release activity and enhanced intracellular glucose accumulation. In addition, other glucose transporters (SGLT1 and SGLT2 in the kidney) were found to be induced in the mutant cells. These findings suggest the last loops (loops 9-12) of GLUT2 are essential for glucose transport activity and indicate that GLUT2 dysfunction is associated with dysglycemia in FBS.

Association of rs9939609 polymorphism in the FTO gene with features of androgen status in men

The purpose of the study was to investigate the association of carriage of rs9939609 polymorphism in the FTO gene with features of androgen status in men.Material and methods. The observational one-stage examination of 139 male patients aged 22 to 69 years, admitted to a therapeutic hospital in a planned manner has been carried out in the Clinic of the Federal Research Center for Fundamental and Translational Medicine in the period 2020–2021. All fasting men in the morning (8:00–8:30) were carried out of an anthropometric examination to determine physical development, sampling of venous blood from the cubital vein to assess the hormonal parameters of androgen status, and taking a sample of the buccal epithelium for molecular genetic research. Based on the carriage of the rs9939609 (T&gt;A) polymorphism of the FTO gene, the following groups (G) of patients were distinguished: G1 – wild-type homozygotes (WT/WT), G2 – heterozygotes (WT/A), G3 – homozygotes for the mutant allele (A/A).Results and discussion. The statistically significant differences were found in the levels of total testosterone between G1 and G3 (11.6 vs 14.5 nmol/L, p = 0.010), as well as free testosterone in G1 compared to G2 (0.233 vs 0.287 nmol/L, p = 0.012) and G3 (0.233 vs 0.321 nmol/L, p = 0.002) when analyzing the clinical-anthropometric and hormonal features of the androgen status of the examined male patients in the selected groups. Assessment of the association revealed statistically significant positive correlations between the carriage of the mutant allele and the levels of total (r = 0.247; p = 0.013) and free (r = 0.296; p = 0.003) testosterone, and negative - with the frequency of androgen deficiency according to the free testosterone criteria (r = –0.240; p = 0.016).Conclusions. It has been shown that the carriage of the A/A genotype of rs9939609 polymorphism of the FTO gene is associated with an increase in the levels of total and free testosterone, that indicates a protective effect against the development of androgen deficiency in men. The obtained results suggest that this effect is due to the epigenetic effect of the FTO gene protein product on the expression of other genes involved in the synthesis of androgens in the gonads, but also in the adrenal cortex since no association of the carriage of the rs9939609 polymorphism of the FTO gene with the DHEA-C level was found.

SRY-GENE IN THE PATHOGENESIS OF GONADAL DYSGENESIS. SWIER’S SYNDROME

Annotation: Swier's syndrome is a genetic disorder, a variant of hypogonadism with a 46 XY karyotype, in which complete or "pure" dysgenesis of the testicles is observed, a female phenotype is formed with a male genotype. It is a congenital disorder associated with the Y chromosome. Swier's syndrome is usually associated with a genetic variant of testicular dysgenesis associated with mutations in the SRY gene (Sex-determining Region Y) located on the Y-chromosome of most mammals, and is involved in the development of the male-type organism (locus Yp11.3). The SRY gene, localized on the short arm of the Y chromosome, is usually expressed by 7-8 weeks of embryogenesis, triggering a signal for the differentiation of Sertoli cells, germinal epithelium of the seminiferous tubules, and Leydig cells. During physiological embryogenesis, AMH (anti-Müllerian hormone) is released in Sertoli cells, leading to regression of the Müllerian ducts, and in Leydig cells stimulated by placental chorionic gonadotropin (CG), and then by their own luteinizing hormone (LH), an increasing amount of testosterone is secreted. The SRY gene encodes the protein TDF (testisdeterminingfactor), a transcription factor that regulates the expression of other genes, which in turn encodes transcription factors that initiate the development of the male reproductive system in the embryonic period. During spermatogenesis during meiosis, the Y chromosome can lose SRY by transferring this gene to the X chromosome. In the absence of full-fledged genetic material (in particular, the expression of the SRY gene), the formation of functionally active testicular tissue is impossible. The lack of secretion of anti-Müllerian hormone leads to the development of Müllerian ducts, from which the uterus, upper vagina and fallopian tubes are formed. Thus, despite the male 46 XY karyotype, the external and internal reproductive organs of the fetus are formed by the female. In this literature review, the defects of the genes responsible for the varieties and clinical manifestations of Swier's syndrome are analyzed.

Predicting miRNA-disease associations via layer attention graph convolutional network model

BackgroundMiRNA is a class of non-coding single-stranded RNA molecules with a length of approximately 22 nucleotides encoded by endogenous genes, which can regulate the expression of other genes. Therefore, it is very important to predict the associations between miRNA and disease. Predecessors developed a new prediction method of drug-disease association, and it achieved good results.MethodsIn this paper, we introduced the method of LAGCN to identify potential miRNA-disease associations. First, we integrate three associations into a heterogeneous network, such as the known miRNA-disease association, miRNA-miRNA similarities and disease-disease similarities, next we apply graph convolution network to learn the embedding of miRNA and disease. We use an attention mechanism to combine embedding from multiple convolution layers. Unobserved miRNA-disease associations are scored based on integrated embedding.ResultsAfter fivefold cross-validations, the value of AUC is reached 0.9091, which is higher than other prediction methods and baseline methods.ConclusionsIn this paper, we introduced the method of LAGCN to identify potential miRNA-disease associations. LAGCN has achieved good performance in predicting miRNA-disease associations, and it is superior to other association prediction methods and baseline methods.

Transcriptome Profiling of Developing Ovine Fat Tail Tissue Reveals an Important Role for MTFP1 in Regulation of Adipogenesis.

Fat-tail sheep exhibit a unique trait whereby substantial adipose tissue accumulates in the tail, a phenotype that is advantageous in many agroecological environments. In this study, we conducted histological assays, transcriptome analysis and functional assays to examine morphogenesis, characterize gene expression, and elucidate mechanisms that regulate fat tail development. We obtained the microstructure of tail before and after fat deposition, and demonstrated that measurable fat deposition occurred by the 80-day embryo (E80) stage, earlier than other tissues. Transcriptome profiling revealed 1,058 differentially expressed genes (DEGs) with six markedly different expression trends. GSEA enrichment and other downstream analyses showed important roles for genes and pathways involving in metabolism and that mitochondrial components were specifically overexpressed in the fat tail tissue of the 70-day embryo (E70). One hundred and eighty-three genes were further identified by leading edge gene analysis, among which, 17 genes have been reported in previous studies, including EEF1D, MTFP1, PPP1CA, PDGFD. Notably, the MTFP1 gene was highly correlated with the expression of other genes and with the highest enrichment score and gene expression change. Knockdown of MTFP1 in isolated adipose derived stem cells (ADSCs) inhibited cell proliferation and migration ability, besides, promoted the process of adipogenesis in vitro.

Changes in the testicular histomorphometry and their association with genes expression pattern of testes from birth to puberty in Beetal goat kids

In the present study, twenty-eight (n = 28) longitudinally rear male goat kids were slaughtered at monthly intervals (n = 4/group) till the 6th month of age; to examine the testicular development and establishment of spermatogenesis through histo-architecture along with mRNA expression of related genes. The results showed that seminiferous tubular diameter was linearly increased (P < 0.001) at birth (40 µm) to 6th months (200 µm). The epithelial height was 18.41 ± 0.65 µm at birth and 164 ± 2.82 µm at 6th months. Whereas, at 3rd months age greatest increase was observed (74.08 ± 1.66 µm), possibly due to a rapid increase in the numbers of spermatocytes and spermatids (4.0 ± 0.3 and 0.9 ± 0.04, respectively). At the same time point (4–6 months of age), a dramatic increase (P < 0.001) was observed in the mRNA level of genes expressed in haploid germ cells including Protamine (PRM1), Outer dense fiber protein (ODF2) and Stimulated by Retinoic Acid (STRA8). In addition, the maturation of Sertoli cells (SCs) was appeared to be completed at the start of 1st month (5.6 ± 0.5), which was further validated by sudden repression (P < 0.001) of SC-specific SOX9 and FSHR mRNA levels during this period. The spermatids that appeared at the end of the 3rd month reflect the start of spermatogenesis. However, a significant (P < 0.001) number of elongated spermatids and mature spermatozoa was observed at the end of 6 months, which indicated pubertal onset (9.8 ± 0.7 and 9.9 ± 0.9, respectively). The mRNA expression of other genes altered during this developmental phase was Heat-shock Protein 8 (HSPA8) and Aromatase (CYP19A1), both was important in sperm maturation and testosterone level. In conclusion, marked changes in cellular and molecular events were observed from the 3rd month; when active spermatogenesis initiated, and mature spermatozoa appeared in the 6th month at pubertal onset in Beetal bucks.