Identification of Key TRIM Genes Involved in Response to Pseudomonas aeruginosa or Chlamydia spp. Infections in Human Cell Lines and in Mouse Organs.

Bacterial infections represent an unsolved problem today since bacteria can evade antibiotics and suppress the host's immune response. A family of TRIM proteins is known to play a role in antiviral defense. However, the data on the involvement of the corresponding genes in the antibacterial response are limited. Here, we used RT-qPCR to profile the transcript levels of TRIM genes, as well as interferons and inflammatory genes, in human cell lines (in vitro) and in mice (in vivo) after bacterial infections caused by Pseudomonas aeruginosa and Chlamydia spp. As a result, the genes were identified that are involved in the overall immune response and associated primarily with inflammation in human cells and in mouse organs when infected with both pathogens (TRIM7, 8, 14, 16, 17, 18, 19, 20, 21, 47, 68). TRIMs specific to the infection (TRIM59 for P. aeruginosa, TRIM67 for Chlamydia spp.) were revealed. Our findings can serve as a basis for further, more detailed studies on the mechanisms of the immune response to P. aeruginosa and Chlamydia spp. Studying the interaction between bacterial pathogens and the immune system contributes to the search for new ways to successfully fight bacterial infections.

Transcription of HOX Genes Is Significantly Increased during Neuronal Differentiation of iPSCs Derived from Patients with Parkinson's Disease.

Parkinson's disease (PD) is the most serious movement disorder, but the actual cause of this disease is still unknown. Induced pluripotent stem cell-derived neural cultures from PD patients carry the potential for experimental modeling of underlying molecular events. We analyzed the RNA-seq data of iPSC-derived neural precursor cells (NPCs) and terminally differentiated neurons (TDNs) from healthy donors (HD) and PD patients with mutations in PARK2 published previously. The high level of transcription of HOX family protein-coding genes and lncRNA transcribed from the HOX clusters was revealed in the neural cultures from PD patients, while in HD NPCs and TDNs, the majority of these genes were not expressed or slightly transcribed. The results of this analysis were generally confirmed by qPCR. The HOX paralogs in the 3' clusters were activated more strongly than the genes of the 5' cluster. The abnormal activation of the HOX gene program upon neuronal differentiation in the cells of PD patients raises the possibility that the abnormal expression of these key regulators of neuronal development impacts PD pathology. Further research is needed to investigate this hypothesis.

Activation of Embryonic Gene Transcription in Neural Precursor Cells Derived from the Induced Pluripotent Stem Cells of the Patients with Parkinson's Disease.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases in the world. Despite numerous studies, the causes of this pathology remain completely unknown. This is, among other things, due to the difficulty of obtaining biological material for analysis. Neural cell cultures derived from the induced pluripotent stem cells (IPSCs) provide a great potential for studying molecular events underlying the pathogenesis of PD. This paper presents the results of bioinformatic analysis of the data obtained using RNA-seq technology in the study of neural precursors (NP) derived from IPSCs of the healthy donors and patients with PD carrying various mutations that are commonly associated with familial PD. This analysis showed that the level of transcription of multiple genes actively expressed in the nervous system at the embryonic stage of development was significantly increased in the NP cells obtained from the patients with PD, unlike in the case of healthy donors. Bioinformatic data have been, in general, confirmed using real-time PCR. The obtained data suggest that one of the causes of PD may be the shift of the gene expression pattern in neuronal cells towards embryonic gene expression pattern (termed dematuration).

Overexpression of Parkin in the Neuronal Progenitor Cells from a Patient with Parkinson's Disease Shifts the Transcriptome Towards the Normal State.

Parkinson's disease (PD) is a neurodegenerative pathology caused by the progressive loss of dopaminergic neurons in the substantia nigra. Juvenile PD is known to be strongly associated with mutations in the PARK2 gene encoding E3 ubiquitin ligase Parkin. Despite numerous studies, molecular mechanisms that trigger PD remain largely unknown. Here, we compared the transcriptome of the neural progenitor (NP) cell line, derived from a PD patient with PARK2 mutation resulting in Parkin loss, with the transcriptome of the same NPs but expressing transgenic Parkin. We found that Parkin overexpression led to the substantial recovery of the transcriptome of NPs to a normal state indicating that alterations of transcription in PD-derived NPs were mainly caused by PARK2 mutations. Among genes significantly dysregulated in PD-derived NPs, 106 genes unambiguously restored their expression after reestablishing of the Parkin level. Based on the selected gene sets, we revealed the enriched Gene Ontology (GO) pathways including signaling, neurotransmitter transport and metabolism, response to stimulus, and apoptosis. Strikingly, dopamine receptor D4 that was previously associated with PD appears to be involved in the maximal number of GO-enriched pathways and therefore may be considered as a potential trigger of PD progression. Our findings may help in the screening for promising targets for PD treatment.

Composite Coatings Based on Recombinant Spidroins and Peptides with Motifs of the Extracellular Matrix Proteins Enhance Neuronal Differentiation of Neural Precursor Cells Derived from Human Induced Pluripotent Stem Cells.

The production and transplantation of functionally active human neurons is a promising approach to cell therapy. Biocompatible and biodegradable matrices that effectively promote the growth and directed differentiation of neural precursor cells (NPCs) into the desired neuronal types are very important. The aim of this study was to evaluate the suitability of novel composite coatings (CCs) containing recombinant spidroins (RSs) rS1/9 and rS2/12 in combination with recombinant fused proteins (FP) carrying bioactive motifs (BAP) of the extracellular matrix (ECM) proteins for the growth of NPCs derived from human induced pluripotent stem cells (iPSC) and their differentiation into neurons. NPCs were produced by the directed differentiation of human iPSCs. The growth and differentiation of NPCs cultured on different CC variants were compared with a Matrigel (MG) coating using qPCR analysis, immunocytochemical staining, and ELISA. An investigation revealed that the use of CCs consisting of a mixture of two RSs and FPs with different peptide motifs of ECMs increased the efficiency of obtaining neurons differentiated from iPSCs compared to Matrigel. CC consisting of two RSs and FPs with Arg-Gly-Asp-Ser (RGDS) and heparin binding peptide (HBP) is the most effective for the support of NPCs and their neuronal differentiation.

Glial Cultures Differentiated from iPSCs of Patients with PARK2-Associated Parkinson's Disease Demonstrate a Pro-Inflammatory Shift and Reduced Response to TNFα Stimulation.

Parkinson's disease (PD) is the second most common neurodegenerative diseases characterized by progressive loss of midbrain dopaminergic neurons in the substantia nigra. Mutations in the PARK2 gene are a frequent cause of familial forms of PD. Sustained chronic neuroinflammation in the central nervous system makes a significant contribution to neurodegeneration events. In response to inflammatory factors produced by activated microglia, astrocytes change their transcriptional programs and secretion profiles, thus acting as immunocompetent cells. Here, we investigated iPSC-derived glial cell cultures obtained from healthy donors (HD) and from PD patients with PARK2 mutations in resting state and upon stimulation by TNFα. The non-stimulated glia of PD patients demonstrated higher IL1B and IL6 expression levels and increased IL6 protein synthesis, while BDNF and GDNF expression was down-regulated when compared to that of the glial cells of HDs. In the presence of TNFα, all of the glial cultures displayed a multiplied expression of genes encoding inflammatory cytokines: TNFA, IL1B, and IL6, as well as IL6 protein synthesis, although PD glia responded to TNFα stimulation less strongly than HD glia. Our results demonstrated a pro-inflammatory shift, a suppression of the neuroprotective gene program, and some depletion of reactivity to TNFα in PARK2-deficient glia compared to glial cells of HDs.

Transcriptome datasets of neural progenitors and neurons differentiated from induced pluripotent stem cells of healthy donors and Parkinson's disease patients with mutations in the PARK2 gene.

Parkinson's disease (PD) is a complex systemic disorder caused by neurodegenerative processes in the brain that are mainly characterized by progressive loss of dopaminergic neurons in the substantia nigra. About 10% of PD cases have been linked to specific gene mutations (Zafar and Yaddanapudi, 2022) including the PARK2 gene that encodes a RING domain-containing E3 ubiquitin ligase Parkin. PD-Parkin patients have a younger onset, longer disease duration, and more severe clinical symptoms in comparison to PD patients with unknown causative PD mutations (Zhou et al., 2020). Induced pluripotent stem cells (iPSCs) are considered to be a powerful tool for disease modeling. To evaluate how mutations in PARK2 contribute to PD development, iPSC lines were obtained from three healthy donors and three PD patients with different mutations in the PARK2 gene. iPSC lines were differentiated consequently into neural progenitors (NPs) and then into terminally differentiated neurons (DNs). The data presented in this article were generated on an NextSeq 500 System (Illumina) and include transcriptome profiles for NPs and DNs of healthy donors and PD patients with mutations in the PARK2 gene. Top10 up- and down-regulated differentially expressed genes in NPs and DNs of patients with PD compared to healthy donors were also presented. A comparative transcriptome analysis of neuronal derivatives of healthy donors and PD patients allows to examine the contributions of the PARK2 gene mutations to PD pathogenesis.

Influence of N-Arachidonoyl Dopamine and N-Docosahexaenoyl Dopamine on the Expression of Neurotrophic Factors in Neuronal Differentiated Cultures of Human Induced Pluripotent Stem Cells under Conditions of Oxidative Stress.

Oxidative stress (OS) is implicated in the pathogenesis of several neurodegenerative diseases. We have previously shown that N-acyl dopamines (N-ADA and N-DDA) protect the neural cells of healthy donors and patients with Parkinson's disease from OS. In this study, we assessed the effects of N-acyl dopamines on the expression of neurotrophic factors in human-induced pluripotent stem cell-derived neuronal cultures enriched with dopaminergic neurons under conditions of OS induced by hydrogen peroxide. We showed that hydrogen peroxide treatment increased BDNF but not GDNF mRNA levels, while it did not affect the secretion of corresponding proteins into the culture medium of these cells. Application of N-acyl dopamines promoted BDNF release into the culture medium. Under conditions of OS, N-DDA also increased TRKB, TRKC and RET mRNA levels. Furthermore, N-acyl dopamines prevented cell death 24 h after OS induction and promoted the expression of antioxidant enzymes GPX1, GPX7, SOD1, SOD2 and CAT, as well as reduced the BAX/BCL2 mRNA ratio. These findings indicate that stimulation of the expression of neurotrophic factors and their receptors may underlie the neuroprotective effects of N-acyl dopamines in human neurons.

Gene Therapy for Critical Limb Ischemia: Per Aspera ad Astra.

Peripheral artery diseases remain a serious public health problem. Although there are many traditional methods for their treatment using conservative therapeutic techniques and surgery, gene therapy is an alternative and potentially more effective treatment option especially for "no-option" patients. This review treats the results of many years of research and application of gene therapy as an example of treatment of patients with critical limb ischemia. Data on successful and unsuccessful attempts to use this technology for treating this disease are presented. Trends in changing the paradigm of approaches to therapeutic angiogenesis are noted: from viral vectors to non-viral vectors, from gene transfer to the whole organism to targeted transfer to cells and tissues, from single-gene use to combination of genes; from DNA therapy to RNA therapy, from in vivo therapy to ex vivo therapy.

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.

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.

Neuroprotective and neurotoxic effects of endocannabinoid-like compounds, N-arachidonoyl dopamine and N-docosahexaenoyl dopamine in differentiated cultures of induced pluripotent stem cells derived from patients with Parkinson's disease.

The prominent protective effects in diverse neuron injury paradigms exerted by cannabinoids and in particular their endogenously produced species render the endocannabinoid system a promising molecular target in the treatment of neurodegenerative diseases. However, the effects of individual endocannabinoids in human cells remain poorly investigated. Neural derivatives of human induced pluripotent stem cells (iPSC) offer unique opportunities for studying the neuroprotective compounds and development of patient-specific treatment. For the first time the cytotoxic and neuroprotective effects endocannabinoids N-arachidonoyl dopamine (N-ADA) and N-docosahexaenoyl dopamine (N-DDA) were assessed in human neural progenitors and dopamine neurons derived from iPSCs of healthy donors and patients with Parkinson's disease. While the short-term treatment with the investigated compounds in 0.1-10 µM concentration range exerted no toxicity in these cell types, the long-term exposure to 0.1-5 µM N-ADA or N-DDA reduced the survival of human neural progenitors. At the same time, both N-ADA and N-DDA protected neural progenitors and terminally differentiated neurons both from healthy donors and patients with Parkinson's disease against oxidative stress induced by hydrogen peroxide. The observed dramatic difference in the mode of action of N-acyl dopamines points on the possible existence of novel pathogenic mechanism of neurodegeneration induced by prolonged uncompensated production of these substances within neuronal tissue and should also be considered as a precaution in the future development of N-acyl dopamine-based therapeutic drugs.

Estimation of the Mutagenic Potential of 8-Oxog in Nuclear Extracts of Mouse Cells Using the "Framed Mirror" Method.

We propose an improved earlier described "mirror" method for detecting in cell nuclear extracts mutations that arise in DNA during its replication due to the misincorporation of deoxyadenosine-5'-monophosphate (dAMP) opposite 7,8-dihydro-8-oxoguanine (8-oxoG). This method is based on the synthesis of a complementary chain ("mirror") by nuclear extracts of different mice organs on a template containing 8-oxoG and dideoxycytidine residue (ddC) at the 3'­end. The "mirror" was amplified by PCR using primers part of which was non-complementary to the template. It allowed obtaining the "framed mirror" products. The misincorporation of dAMP in "framed mirror" products forms an EcoRI restriction site. The restriction analysis of double-stranded "framed mirror" products allows a quantification of the mutation frequency in nuclear extracts. The data obtained show that the mutagenic potential of 8-oxoG markedly varied in different organs of adult mice and embryos.

Many Faces of TRIM Proteins on the Road from Pluripotency to Neurogenesis.

Tripartite motif (TRIM) proteins participate in numerous biological processes. They are the key players in immune system and are involved in the oncogenesis. Moreover, TRIMs are the highly conserved regulators of developmental pathways in both vertebrates and invertebrates. In particular, numerous data point to the participation of TRIMs in the determination of stem cell fate, as well as in the neurogenesis. TRIMs apply various mechanisms to perform their functions. Their common feature is the ability to ubiquitinate proteins mediated by the Really Interesting New Gene (RING) domain. Different C-terminal domains of TRIMs are involved in DNA and RNA binding, protein/protein interactions, and chromatin-mediated transcriptional regulation. Mutations and alterations of TRIM expression cause significant disturbances in the stem cells' self-renewal and neurogenesis, which result in the various pathologies of the nervous system (neurodegeneration, neuroinflammation, and malignant transformation). This review discusses the diverse molecular mechanisms of participation of TRIMs in stem cell maintenance and self-renewal as well as in neural differentiation processes and neuropathology.

A Hybrid Detection Method Based on Peroxidase-mediated Signal Amplification and Click Chemistry for Highly Sensitive Background-free Immunofluorescent Staining.

The copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is increasingly used for detection of various macromolecules and metabolites in biological samples. Here, we present a detailed analysis of the CuAAC reaction conditions in cells and tissue sections. Using the optimized CuAAC conditions, we have devised a highly sensitive immunostaining technique, based on the tyramide signal amplification/catalyzed reporter deposition (TSA/CARD) method with a novel alkyne tyramide substrate. The described method offers improved detection threshold compared to conventional immunofluorescent staining and produces significantly lower non-specific background than TSA/CARD with fluorescent tyramides.

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.

"Mirror" Method to Estimate Mutagenic Activity of DNA Lesions.

We propose an improved method for detecting mutations that arise in DNA due to misincorporations of deoxyadenosine-5'-monophosphate (dAMP) opposite 7,8-dihydro-8-oxoguanine (8-oxoG). The method is based on the synthesis of complementary chains ("mirror" products) using a template containing 8-oxoG. The misincorporation of dAMP in the "mirror" product forms EcoRI sites. The restriction analysis of double-stranded DNAs obtained by PCR of "mirror" product allows quantification of the mutagenesis frequency. In addition, single-strand conformational polymorphism (SSCP) analysis of the single-stranded "mirror" products shows that different DNA polymerases only incorporate dA or dC opposite 8-oxoG. The proposed approach used in developing this technique can be applied in the study of other lesions as well, both single and clustered.

Alternative splicing at exon 2 results in the loss of the catalytic activity of mouse DNA polymerase iota in vitro.

Y-family DNA polymerase iota (Pol ι) possesses both DNA polymerase and dRP lyase activities and was suggested to be involved in DNA translesion synthesis and base excision repair in mammals. The 129 strain of mice and its derivatives have a natural nonsense codon mutation in the second exon of the Pol ι gene resulting in truncation of the Pol ι protein. These mice were widely used as a Pol ι-null model for in vivo studies of the Pol ι function. However whether 129-derived strains of mice are fully deficient in the Pol ι functions was a subject of discussion since Pol ι mRNA undergoes alternative splicing at exon 2. Here we report purification of mouse Pol ι lacking the region encoded by exon 2, which includes several conserved residues involved in catalysis. We show that the deletion abrogates both the DNA polymerase and dRP lyase activities of Pol ι in the presence of either Mg2+ or Mn2+ ions. Thus, 129-derived strains of mice express catalytically inactive alternatively spliced Pol ι variant, whose cellular functions, if any exist, remain to be established.

Alterations in Synthesis and Repair of DNA during the Development of Loach Misgurnus fossilis.

Using a modified radiolabeled primer extension method (we named this modification misGvA-"misincorporation of G versus A") we have investigated the DNA synthesis and repair at early and late stages of development of loach Misgurnus fossilis. The misincorporation activity of DNA polymerase iota (Pol ι) in wild-type loach could not be detected by this method at any stage of loach development. In transgenic loach overexpressing human Pol ι we have shown that the bypassing of DNA synthesis arrest after incorporation of mismatched nucleotide by Pol ι (the T-stop) was not associated with this enzyme. Non-transgenic loach larvae are virtually lacking the capacity for error correction of DNA duplex containing a mismatched nucleotide. Such repair activity develops only in the adult fish. It appears that the initial stages of development are characterized by more intensive DNA synthesis, while in terminal stages the repair activities become more prominent. The misGvA approach clearly indicates substantial changes in the DNA synthesis intensity, although the role of particular replicative and repair DNA polymerases in this process requires further study.

Trim14 overexpression causes the same transcriptional changes in mouse embryonic stem cells and human HEK293 cells.

The trim14 (pub, KIAA0129) gene encodes the TRIM14 protein which is a member of the tripartite motif (TRIM) family. Previously, we revealed high expression levels of trim14 in HIV- or SIV-associated lymphomas and demonstrated the influence of trim14 on mesodermal differentiation of mouse embryonic stem cells (mESC). In the present work, to elucidate the role of trim14 in normal and pathological processes in the cell, we used two different types of cells transfected with trim14: mESC and human HEK293. Using subtractive hybridization and real-time PCR, we found a number of genes which expression was elevated in trim14-transfected mESC: hsp90ab1, prr13, pu.1, tnfrsf13c (baff-r), tnfrsf13b (taci), hlx1, hbp1, junb, and pdgfrb. A further analysis of the trim14-transfected mESC at the initial stage of differentiation (embryoid bodies (EB) formation) showed essential changes in the expression of these upregulated genes. The transfection of trim14 into HEK293 also induced an enhanced expression of the several genes upregulated in trim14-transfected mESC (hsp90ab1, prr13, pu.1, tnfrsf13c (baff-r), tnfrsf13b (taci), and hlx1). Summarizing, we found similar genes that participated in trim14-directed processes both in mESC and in HEK293. These results demonstrate the presence of the similar mechanism of trim14 gene action in different types of mammalian cells.