Changes of Transcriptomic Activity in Rat Brain Cells under the Influence of Synthetic Adrenocorticotropic Hormone-Like Peptides.

Synthetic peptides have a wide range of clinical effects. Of particular interest are peptides based on adrenocorticotropic hormone (ACTH) both as already used and as potential drugs for preventing consequences of cerebral ischemia. However, it is necessary to study influence of the peptide on the brain cells under normal physiological conditions, including understanding the risks of their use. Here, we used high-throughput RNA sequencing (RNA-Seq) to identify differentially expressed genes (DEGs) in the brain frontal cortex of rat receiving intraperitoneal administration of ACTH-like peptides ACTH(4-7)PGP (Semax) and ACTH(6-9)PGP, or saline. We identified 258 and 228 DEGs, respectively, with the fold change > 1.5 and Padj < 0.05 at 22.5 h after the first administration of Semax and ACTH(6-9)PGP. Metabolic pathways, characterizing both common and specific effects of the peptides on the transcriptome were identified. Both peptides predominantly caused decrease in expression of the genes associated with the immune system. At the same time, when comparing the effects of ACTH(6-9)PGP relative to Semax, DEGs were identified that characterized the main differences in the effects of the peptides. These genes were mostly downregulated and associated with neurosignaling systems and regulation of ion channels, thus characterizing differences in the effects of the peptides. Our data show how differences in the structure of ACTH derivatives are associated with the changes in the brain cell transcriptome following exposure to these related peptides. Furthermore, our results demonstrate that when studying influence of regulatory peptides on transcriptome under pathological conditions, it is necessary to take into account their actions under normal physiological conditions.

Human TRIM14 protects transgenic mice from influenza A viral infection without activation of other innate immunity pathways.

TRIM14 is an important component of innate immunity that defends organism from various viruses. It was shown that TRIM14 restricted influenza A virus (IAV) infection in cell cultures in an interferon-independent manner. However, it remained unclear whether TRIM14 affects IAV reproduction and immune system responses upon IAV infection in vivo. In order to investigate the effects of TRIM14 at the organismal level we generated transgenic mice overexpressing human TRIM14 gene. We found that IAV reproduction was strongly inhibited in lungs of transgenic mice, resulting in the increased survival of transgenic animals. Strikingly, upon IAV infection, the transcription of genes encoding interferons, IL-6, IL-1ß, and TNFα was notably weaker in lungs of transgenic animals than that in control mice, thus indicating the absence of significant induction of interferon and inflammatory responses. In spleen of transgenic mice, where TRIM14 was unexpectedly downregulated, upon IAV infection the transcription of genes encoding interferons was oppositely increased. Therefore, we demonstrated the key role of TRIM14 in anti-IAV protection in the model organism that is realized without noticeable activation of other innate immune system pathways.

The modern view on the role of glyprolines by metabolic syndrome.

The analysis of the literature data and own experimental studies on the effect of glyproline peptides on fat, carbohydrate metabolism, and hemostasis system when modeling metabolic syndrome in animals (rats) was carried out. Violations of fat and carbohydrate metabolism are characterized by hypercoagulation, increased glucose levels, dyslipidemia, and decreased rheological properties of blood. In this condition, the arginine- and lysine-containing glyprolines (PGP, PRP, RPGP, KKRRPGP, RKKRPGP, and KRRKPGP) at multiple (7 days) intranasal introduction had a complex effect on the hemostatic parameters, increasing antiplatelet, anticoagulant, and fibrinolytic activity of blood plasma. All the studied drugs also showed normoglycemic and normolipidemic effects and led to a slowdown or decrease in body weight growth. The analysis of the presented material allows us to speak about the prospects of using hit compounds as protective therapeutic agents. In the case of violations of fat and carbohydrate metabolism, regulatory glyprolines protect the body by displaying antithrombotic, hypolipidemic, and hypoglycemic properties.

Expression of the human TRIM14 and its mutant form (P207L) promotes apoptosis in transgenic loaches.

The tripartite-motif (TRIM)14 protein, one of the TRIM family members, was shown to participate in the antiviral and antibacterial defence. Besides, it appears to play an essential role in the processes of oncogenesis. In some types of human tumour cells, TRIM14 has been shown to inhibit apoptosis, while in others-the overexpression of TRIM14 promotes apoptosis. However, whether TRIM14 mediates apoptosis in the normal cells remains unknown. In the present study, we investigated the possible participation of the human TRIM14 gene and its mutant form (620C > T) in the induction of apoptosis in the transgenic larvae loach Misgurnus fossilis L. We observed that the expression of both forms of TRIM14 gene was accompanied by the increase of the frequency of pyknotic nuclei in fish embryos compared to control groups. Accordingly, using the TUNEL assay, the enhanced apoptosis was revealed upon expression of both forms of TRIM14 gene. The transcription of proapoptotic genes (bax, tp53, and casp9) was significantly increased in transgenic loaches expressing human wild-type TRIM14, but remained unchanged upon expression of its mutant form. In addition, the transcription of c-myc was upregulated in transgenic loaches expressing both forms. Thus, it can be assumed that during embryonic development TRIM14 has a proapoptotic effect on the cells via the activation of c-myc, tp53, and bax genes. Apparently, the mutant TRIM14 directs apoptosis via c-myc by p53-independent mechanism.

Dynamics of recombinant hG-CSF in transgenic goat: preliminary study in the founder during hormonally induced lactation.

This study aimed to characterize the dynamic of human granulocyte colony-stimulating factor (hG-CSF) during artificial lactation in a transgenic founder goat and to assess its potential ectopic expression and health. The female secreted 93.9 to 1,474.6 µg hG-CSF per mL of milk. Two peaks of serum hG-CSF (3,470 and 7,390 pg/mL) were detected in the first half of the lactation. Outside of the lactation, hG-CSF was absent from serum, indicating no ectopic expression. During the treatment to induce lactation, transgenic female presented increased neutrophil and lymphocyte blood counts when compared to nontransgenic female. Despite transient neutrophilia, serum biochemistry profiles indicated normal liver and renal functions. Thus, transgenic goat expressed hG-CSF in quantities sufficient for a commercial bioreactor and remained clinically healthy.

Insight into Glyproline Peptides' Activity through the Modulation of the Inflammatory and Neurosignaling Genetic Response Following Cerebral Ischemia-Reperfusion.

Glyprolines are Gly-Pro (GP)- or Pro-Gly (PG)-containing biogenic peptides. These peptides can act as neutrophil chemoattractants, or atheroprotective, anticoagulant, and neuroprotective agents. The Pro-Gly-Pro (PGP) tripeptide is an active factor of resistance to the biodegradation of peptide drugs. The synthetic Semax peptide, which includes Met-Glu-His-Phe (MEHF) fragments of adrenocorticotropic hormone and the C-terminal tripeptide PGP, serves as a neuroprotective drug for the treatment of ischemic stroke. Previously, we revealed that Semax mostly prevented the disruption of the gene expression pattern 24 h after a transient middle cerebral artery occlusion (tMCAO) in a rat brain model. The genes of this pattern were grouped into an inflammatory cluster (IC) and a neurotransmitter cluster (NC). Here, using real-time RT-PCR, the effect of other PGP-containing peptides, PGP and Pro-Gly-Pro-Leu (PGPL), on the expression of a number of genes in the IC and NC was studied 24 h after tMCAO. Both the PGP and PGPL peptides showed Semax-unlike effects, predominantly without changing gene expression 24 h after tMCAO. Moreover, there were IC genes (iL1b, iL6, and Socs3) for PGP, as well as IC (iL6, Ccl3, Socs3, and Fos) and NC genes (Cplx2, Neurod6, and Ptk2b) for PGPL, that significantly changed in expression levels after peptide administration compared to Semax treatment under tMCAO conditions. Furthermore, gene enrichment analysis was carried out, and a regulatory gene network was constructed. Thus, the spectra of the common and unique effects of the PGP, PGPL, and Semax peptides under ischemia-reperfusion were distinguished.

ACTH(6-9)-Pro-Gly-Pro ameliorates anxiety-like and depressive-like behaviour and gut mucosal microbiota composition in rats under conditions of chronic restraint stress.

The effects of the peptide ACTH(6-9)-Pro-Gly-Pro at doses of 5; 50; 500 µg/kg on the Wistar rats' behaviour and gut mucosal microbiota composition under conditions of chronic immobilization stress (CRS) were studied. CRS increased anxiety-like and depressive-like behaviour, disturbances in locomotor activity and gut dysbiosis. Administration of ACTH(6-9)-Pro-Gly-Pro showed many phenotypic results. Peptide demonstrated anti-depressant activity at doses of 5 and 500 µg/kg by a decrease in the total immobile time in the FST. ACTH(6-9)-Pro-Gly-Pro administered at a dose of 50 µg/kg resulted in an anxiolytic effect which is shown by an increase in the time in the open arms of EPM (p < 0.05) and a decrease in the time in the closed arms (p < 0.05). Moreover, the peptide led to a decrease in alpha- and beta-diversity of the gut microbiota (p < 0.01). Correlation and linear regression analysis demonstrated central mechanisms of ACTH(6-9)-Pro-Gly-Pro anxiolytic activity and both central and peripheral ones in an anti-depressant effect. In this way, peptide ACTH(6-9)-Pro-Gly-Pro could prevent the development of behavioural disturbances and gut dysbiosis caused by chronic restraint stress.

Human TAF-Iα promotes oncogenic transformation via enhancement of cell proliferation and suppression of apoptosis.

Template activating factor-I (TAF-I) is a multifunctional protein involved in various biological processes including the inhibition of histone acetylation, DNA replication, cell cycle regulation, and oncogenesis. Two main TAF-I isoforms with different N-termini, TAF-Iα and TAF-Iß (SET), are expressed in cells. There are numerous data about functional properties of TAF-Iß, whereas the effects of TAF-Iα remain largely unexplored. Here, we employed focus formation and cell proliferation assays, TUNEL staining, cytological analysis, and RT-qPCR to compare the effects of human TAF-Iα and TAF-Iß genes, transiently expressed in Rat2 cells and in Misgurnus fossilis loaches. We found that both TAF-I isoforms possessed equal oncogenic potential in these systems. Furthermore, an overexpression of human TAF-Iα and TAF-Iß in Rat2 cells promoted their proliferation. Accordingly, the mitotic index was increased in the transgenic loaches expressing human TAF-Iα or TAF-Iß. TUNEL assay as well as downregulation of p53 gene and upregulation of bcl-2 gene in these transgenic loaches demonstrated that both isoforms suppressed apoptosis. Thus, TAF-Iα isoform exerts the same oncogenic potential as TAF-Iß, likely by suppressing the apoptosis and promoting cell proliferation.

Peptide Selank Enhances the Effect of Diazepam in Reducing Anxiety in Unpredictable Chronic Mild Stress Conditions in Rats.

It was shown that the anxiolytic effect of Selank is comparable to that of classical benzodiazepine drugs and that the basis of their mechanism of action may be similar. These data suggest that the presence of Selank may change the action of classical benzodiazepine drugs. To test this hypothesis, we evaluated the anxiolytic activity of Selank and diazepam in rats both under conditions of unpredictable chronic mild stress and in its absence, after the individual and combined administration of these compounds using the elevated plus maze test. We found that, even in the absence of chronic stress, the administration of a course of test substances changed anxiety indicators toward their deterioration, but the changes after the administration of a course of Selank were less pronounced. In conditions of chronic stress, anxiety indicator values after the simultaneous use of diazepam and Selank did not differ from the respective values observed before chronic stress exposure. The data obtained indicate that the individual administration of Selank was the most effective in reducing elevated levels of anxiety, induced by the administration of a course of test substances, whereas the combination of diazepam with Selank was the most effective in reducing anxiety in unpredictable chronic mild stress conditions.

GABA, Selank, and Olanzapine Affect the Expression of Genes Involved in GABAergic Neurotransmission in IMR-32 Cells.

Clinical studies have shown that Selank had an anxiolytic effect comparable to that of classical benzodiazepine drugs, which can enhance the inhibitory effect of GABA by allosteric modulation of GABAA receptors. These data suggest that the molecular mechanism of the effect of Selank may also be related to its ability to affect the performance of the GABAergic system. To test this hypothesis, we studied the changes in expression of 84 genes involved in the functioning of the GABAergic system and in the processes of neurotransmission in the culture of neuroblastoma IMR-32 cells using qPCR method. As test substances, in addition to Selank, we selected the major GABAA receptor ligand, GABA, the atypical antipsychotic, olanzapine, and combinations of these compounds (Selank and GABA; Selank and olanzapine). We found no changes in the mRNA levels of the genes studied under the effect of Selank. The combined effect of GABA and Selank led to nearly complete suppression of changes in expression of genes in which mRNA levels changed under the effect of GABA. When Selank was used in conjunction with olanzapine, the expression alterations of more genes were observed compared with olanzapine alone. The data obtained indicate that Selank has no direct effect on the mRNA levels of the GABAergic system genes in neuroblastoma IMR-32 cells. At the same time, our results partially confirm the hypothesis that the peptide may affect the interaction of GABA with GABAA receptors. Our data also suggest that Selank may enhance the effect of olanzapine on the expression of the genes studied.

Semax, an analog of ACTH(4-10) with cognitive effects, regulates BDNF and trkB expression in the rat hippocampus.

The heptapeptide Semax (Met-Glu-His-Phe-Pro-Gly-Pro) is an analog of the adrenocorticotropin fragment (4-10) which after intranasal application has profound effects on learning and exerts marked neuroprotective activities. Here, we found that a single application of Semax (50 microg/kg body weight) results in a maximal 1.4-fold increase of BDNF protein levels accompanying with 1.6-fold increase of trkB tyrosine phosporylation levels, and a 3-fold and a 2-fold increase of exon III BDNF and trkB mRNA levels, respectively, in the rat hippocampus. Semax-treated animals showed a distinct increase in the number of conditioned avoidance reactions. We suggest that Semax affects cognitive brain functions by modulating the expression and the activation of the hippocampal BDNF/trkB system.

Selank Administration Affects the Expression of Some Genes Involved in GABAergic Neurotransmission.

Clinical studies have shown the similarity of the spectrum of physiological effects of Selank and classical benzodiazepines, such as diazepam and phenazepam. These data suggest that there is a similar basis of their mechanism of action. To test this hypothesis we studied the effect of Selank and GABA on the expression of genes involved in neurotransmission. We analyzed the expression of 84 genes involved in neurotransmission (e.g., major subunit of the GABA receptor, transporters, ion channels, dopamine, and serotonin receptors) in the frontal cortex of rats 1 and 3 h after the administration of Selank or GABA (300 µg/kg) using real-time PCR method. We found significant changes in the expression of 45 genes 1 h after the administration of the compounds. Three hours after Selank or GABA administration, 22 genes changed their expression. We found positive correlation between the changes in genes expression within 1 h after administration of Selank or GABA. Our results showed that Selank caused a number of alterations in the expression of genes involved in neurotransmission. The data obtained indicate that Selank is characterized by its complex effects on nerve cells, and one of its possible molecular mechanisms is associated with allosteric modulation of the GABAergic system.

The temporary dynamics of inflammation-related genes expression under tuftsin analog Selank action.

Previous studies have shown that synthetic tuftsin analogue Selank and its fragments cause a number of alterations in the expression of certain genes involved in inflammation in mouse spleen. In this work we studied the effect of Selank and its short fragment Gly-Pro on the temporary dynamics of C3, Casp1, Il2rg, and Xcr1 genes expression in mouse spleen after single intraperitoneal injection (100 µg/kg) of peptides using real-time PCR method. We found a significant 3-fold decrease in the C3 mRNA level just 30 min after Selank injection and similar alteration this gene mRNA level after Gly-Pro administration. A wave-like alteration in the Casp1 mRNA level was observed after Selank injection. We found a significant alteration in the mRNA level of the Il2rg gene at early time points after Selank and Gly-Pro administration and an almost equal reduction in the Xcr1 mRNA level 90 min after the administration of Selank and its fragment. Our results showed that, Selank and its short fragment Gly-Pro influence the expression of genes that mediate different types of immune responses, thereby maintaining the balance of the immune system. It should be noted that in most cases, there was a coincidence in the expression profiles of the studied genes after Selank and Gly-Pro administration. This might indicate an active contribution of the dipeptide to the final effect of Selank.

Methodological strategies for transgene copy number quantification in goats (Capra hircus) using real-time PCR.

Taking into account the importance of goats as transgenic models, as well as the rarity of copy number (CN) studies in farm animals, the present work aimed to evaluate methodological strategies for accurate and precise transgene CN quantification in goats using quantitative polymerase chain reaction (qPCR). Mouse and goat lines transgenic for human granulocyte-colony stimulating factor were used. After selecting the best genomic DNA extraction method to be applied in mouse and goat samples, intra-assay variations, accuracy and precision of CN quantifications were assessed. The optimized conditions were submitted to mathematical strategies and used to quantify CN in goat lines. The findings were as follows: validation of qPCR conditions is required, and amplification efficiency is the most important. Absolute and relative quantifications are able to produce similar results. For normalized absolute quantification, the same plasmid fragment used to generate goat lines must be mixed with wild-type goat genomic DNA, allowing the choice of an endogenous reference gene for data normalization. For relative quantifications, a resin-based genomic DNA extraction method is strongly recommended when using mouse tail tips as calibrators to avoid tissue-specific inhibitors. Efficient qPCR amplifications (≥95%) allow reliable CN measurements with SYBR technology. TaqMan must be used with caution in goats if the nucleotide sequence of the endogenous reference gene is not yet well understood. Adhering to these general guidelines can result in more exact CN determination in goats. Even when working under nonoptimal circumstances, if assays are performed that respect the minimum qPCR requirements, good estimations of transgene CN can be achieved.

Expression of inflammation-related genes in mouse spleen under tuftsin analog Selank.

Previous studies have shown that synthetic tuftsin analogue Selank causes a transcriptomic response in the rat hippocampus and in spleen cells and may participate in the regulation of inflammatory processes in the body. In this work we studied the effect of Selank and two of its fragments on the expression of genes involved in processes of inflammation. We analyzed the expression of 84 genes involved in processes of inflammation (e.g., chemokines, cytokines, and its receptors) in mouse spleen 6 and 24 h after Selank single intraperitoneal injection (100 µg/kg) using real-time PCR method. We found significant changes in the expression of 34 genes involved in inflammation processes. The detailed analysis of quantitative data showed that the Bcl6 gene, which plays a main role in the formation and development of the immune system, exhibited significant changes in its expression levels in response to injection of each of the peptides. Also, we observed expression changes for Bcl6 target and corepressor genes under the influence of Selank and its fragments. Our results showed that Selank and its fragments caused a number of alterations in the expression of genes involved in inflammation. The data obtained confirmed the participation of Selank in the processes of regulation of inflammation in the body. The complex biological effect of Selank may be partially determined by the systematic effect of this peptide on genomic expression.

Production of transgenic goat (Capra hircus) with human Granulocyte Colony Stimulating Factor (hG-CSF) gene in Brazil.

In order to produce transgenic goats with hG-CSF, a total of 24 adult Saanen and 48 adult undefined breed goats were used as donors and recipients, respectively. Donors were estrus-synchronized with vaginal sponges and superovulated by a treatment with 200 mg FSH given twice daily in decreasing doses over 3 days starting 48 h before sponge removal. Ovulation was induced by injecting 100 microg GnRH 36 h after sponge removal. The recipients also received an estrus synchronization treatment. Donors were mated with fertile Saanen bucks and, approximately 72 h after sponge removal, zygotes were recovered surgically by flushing oviducts. The recovered zygotes were briefly centrifuged to a reliable visualization of the pronuclei. The DNA construct containing hG-CSF gene flanked by goat and bovine alphas1-casein sequences was injected into pronuclei of 129 zygotes. The microinjected embryos (3-6 per female) were transferred to 27 recipients. Ten recipients became pregnant and 12 kids were born. One transgenic male founder was identified in the group of kids. This is the first report of a birth of a transgenic goat in Latin America.

Production of transgenic goat (Capra hircus) with human Granulocyte Colony Stimulating Factor (hG-CSF) gene in Brazil

In order to produce transgenic goats with hG-CSF, a total of 24 adult Saanen and 48 adult undefined breed goats were used as donors and recipients, respectively. Donors were estrus-synchronized with vaginal sponges and superovulated by a treatment with 200 mg FSH given twice daily in decreasing doses over 3 days starting 48 h before sponge removal. Ovulation was induced by injecting 100µg GnRH 36 h after sponge removal. The recipients also received an estrus synchronization treatment. Donors were mated with fertile Saanen bucks and, approximately 72 h after sponge removal, zygotes were recovered surgically by flushing oviducts. The recovered zygotes were briefly centrifuged to a reliable visualization of the pronuclei. The DNA construct containing hG-CSF gene flanked by goat and bovine alphas1-casein sequences was injected into pronuclei of 129 zygotes. The microinjected embryos (3-6 per female) were transferred to 27 recipients. Ten recipients became pregnant and 12 kids were born. One transgenic male founder was identified in the group of kids. This is the first report of a birth of a transgenic goat in Latin America.

A fim de produzir caprinos transgênicos para o hG-CSF, utilizou-se 24 cabras Saanen adultas e 48 cabras sem raça definida adultas como doadoras e receptoras, respectivamente. As doadoras tiveram o estro sincronizado por esponjas vaginais e foram superovuladas com 200 mg de FSH em doses decrescentes, duas vezes ao dia e iniciando 48 h antes da retirada da esponja. A ovulação foi induzida pela injeção de 100 µg de GnRH às 36 h após a retirada da esponja. As receptoras também receberam um tratamento de sincronização do estro. As doadoras foram cobertas por bodes Saanen férteis e, aproximadamente 72 h após a retirada da esponja, os zigotos foram colhidos cirurgicamente por lavagem dos ovidutos. Os zigotos colhidos foram rapidamente centrifugados para uma melhor visualização dos pró-núcleos. A construção de DNA, contendo o gene do hG-CSF flanqueado pelos genes caprino e bovino da alfas1-caseína, foi injetada em 129 embriões. Os embriões microinjetados (3 a 6 por receptora) foram transferidos para 27 receptoras que responderam ao tratamento. Dez receptoras ficaram gestantes e 12 crias foram produzidas. Um macho transgênico fundador foi identificado no grupo de crias nascidas. Este é o primeiro relato do nascimento de um caprino transgênico na América Latina.