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Heart is a terminally differentiated organ almost unable to regenerate. The remodeling and hypertrophic growth are believed to be the main mechanisms of heart renovation after workloads or injury. Although there have been major advances in the identification of the genes and signaling pathways involved in mediating hypertrophy, further characterization of the underlying molecular mechanisms is needed due to the overall complexity of this process. Aim. The present work is an attempt to systematically assess the previous research on a β-catenin role in the heart muscle hypertrophy development. We hypothesized that β-catenin is a member of universal and conserved regulatory pathway for different tissues and species. To test this hypothesis, we performed the meta-analysis of experimental data available in different databases. Methods. The literature data were analyzed via Origin 8.0 using the simple regression and two-way ANOVA methods. Results. The results allowed selecting the most reproducible hypertrophy markers which were appropriate for the study of β-catenin function in the hypertrophy response (SERCA, actin DIF, Axin-2, c-myc, CD1, BNP, ANP and total protein/DNA index). The analysis shows that a decrease in the β-catenin expression has an ambiguous effect on heart hypertrophy. Conclusion. We have drawn interesting conclusions on the model and species-specific link between the β-catenin level and hypertrophy development, as well as between some hypertrophic markers and β-catenin expression on one hand, and hypertrophy development etc. on the other hand. The results also allowed selecting the most reproducible hypertrophy markers. [ABSTRACT FROM AUTHOR]

The β-catenin role in myocardium remodeling and hypertrophy development is the suβject of numerous and controversial investigations. Aim. To investigate the significance of cardiac aβlation of β-catenin for heart development using conditional knockout approach. Methods. Standard histological techniques (HE- and MT-staining) and quantitative RT-PCR were used. Results. Our data demonstrate that β-catenin haploinsufficiency in heart provokes upregulation of foetal genes program without visiβle morphological aβnormalities comparing to control groups of animals of the same age. Conclusions. Our data demonstrate that experimental conditions in this study provoke the delay in the development and growth of adult heart without visiβle morphological aβnormalities. [ABSTRACT FROM AUTHOR]

Aim. In our work we have focused on the embryonic cardiospecific ablation of alpha-E-catenin and its reflection on heart adult heart formation. Methods. We studied the significance of embryonic cardiospecific ablation of alpha-E-catenin for heart aging using the conditional knockout approach. We analyzed how alpha-E-catenin haploinsufficiency and homozygotic deletion affected the postnatal heart development and adult heart formation. For this we used measurement of the heart weight/body weight (HW/BW) and heart weight/tibia length (HW/TL) indices, hematoxylin-eosin staining, van Gieson staining, qPCR. Results. The alpha-E-catenin deletion leads to a shortened lifespan of the mutant mice. The mice with homozygotic deletion of alpha-E-catenin had mean survival 36.44 ± 2.62 weeks, haploinsufficiente mice – 38.11 ± 2.39 weeks, controls – 66.2 ± 4.1 weeks. The maximal lifespan in homozygotic mice was 48 weeks, in haploinsufficiente – 44 weeks, whereas in the control we observed more than 78 weeks. In our experiment we revealed the increasing of HW/ BW and HW/TL indices in the homozygotic and haploinsufficiente mice compared to the control mice. Using histological staining we found that the full ablation and deficiency of alpha-E-catenin led to the growing of interstitial fibrosis and cardiomiocyte disintegration. We analyzed the expression of target genes of the canonical WNT signaling and HIPPO-signaling. Conclusions. We have shown that embryonic cardiospecific deletion of one as well as both alleles of the alpha-E-catenin gene leads to the disorders of heart structure, which are typical for the dilated cardiomyopathy, ischemic heart disease, accompanied by fibrosis. As a result this violation of the heart tissue structure causes an early death of animals. We assume that the loss of alpha-Ecatenin leads to such dramatic consequences not only due to the violation of cells interactions, but to the deregulation of signalling pathways in cardiomyocytes, that should be clarified by further molecular genetics and physiological studies. [ABSTRACT FROM AUTHOR]

Aim. Though canonical WNT/β-catenin signaling is inhibited in adult heart it is well known that its activation is critical for heart adaptation to different changing conditions. The main aim of our work is to determine the significance of cardiac ablation of β-catenin during cardiogenesis for adult heart formation. Methods. Using conditional knock-out approaches we have generated mice with cardiospecific β-catenin haploinsufficiency. The routine PCR was used for genotyping of the experimental animals. The 1, 3 and 6 months old mice were used in our research. For morphological analysis the body weight/heart weight ratio was estimated. Standard histological (HE- and MT-staining) methods were implemented to analyze the paraffin sections. The quantitative RT-PCR was used for analyzing the level of ANP, BNP, β-MHC and α-MHC expression. The data were normalized to GAPDH gene expression. Results. In our research we focused on the role of β-catenin in adult myocardium functioning, hypertrophic response and adaptation to stress and aging. We have monitored the influence of embrionical β-catenin ablation in mice cardiomyocytes on heart development and canonical Wnt signaling activity. As a result we have revealed that β-catenin haploinsufficiency leads to fetal gene upregulation (ANP, BNP, β-MHC) in mice of 1 and 3 months age but without morphological abnormality of heart tissue. Meanwhile we have observed the tendency to heart development attenuation under β-catenin haploinsufficiency condition in adult mice (3 months). Analyzing the next time point (6 months) we have registered the decrease in expression of the hypertrophy response (ANP, BNP) genes in comparison to control mice of the same age group while β-MHC and α-MHC genes were still upregulated. Conclusion. We have observed that cardiac ablation of β-catenin during cardiogenesis leads to attenuation of adult heart development and fetal genes expression violation. [ABSTRACT FROM AUTHOR]

Importance: Alphavirus replicons are being developed as self-amplifying RNAs aimed at improving the efficacy of mRNA vaccines. These replicons are convenient for genetic manipulations and can express heterologous genetic information more efficiently and for a longer time than standard mRNAs. However, replicons mimic many aspects of viral replication in terms of induction of innate immune response, modification of cellular transcription and translation, and expression of nonstructural viral genes. Moreover, all replicons used in this study demonstrated expression of heterologous genes in cell- and replicon's origin-specific modes. Thus, many aspects of the interactions between replicons and the host remain insufficiently investigated, and further studies are needed to understand the biology of the replicons and their applicability for designing a new generation of mRNA vaccines. On the other hand, our data show that replicons are very flexible expression systems, and additional modifications may have strong positive impacts on protein expression., Competing Interests: The authors declare no conflict of interest.

Importance: Our study highlights the mechanisms behind the cell's resistance to stress granule (SG) formation after infection with Old World alphaviruses. Shortly after infection, the replication of these viruses hinders the cell's ability to form SGs, even when exposed to chemical inducers such as sodium arsenite. This resistance is primarily attributed to virus-induced transcriptional and translational shutoffs, rather than interactions between the viral nsP3 and the key components of SGs, G3BP1/2, or the ADP-ribosylhydrolase activity of nsP3 macro domain. While interactions between G3BPs and nsP3 are essential for the formation of viral replication complexes, their role in regulating SG development appears to be small, if any. Cells harboring replicating viruses or replicons with lower abilities to inhibit transcription and/or translation, but expressing wild-type nsP3, retain the ability for SG development. Understanding these mechanisms of regulation of SG formation contributes to our knowledge of viral replication and the intricate relationships between alphaviruses and host cells., Competing Interests: The authors declare no conflict of interest.

Replication of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strongly affects cellular metabolism and results in rapid development of the cytopathic effect (CPE). The hallmarks of virus-induced modifications are inhibition of translation of cellular mRNAs and redirection of the cellular translational machinery to the synthesis of virus-specific proteins. The multifunctional nonstructural protein 1 (nsp1) of SARS-CoV-2 is a major virulence factor and a key contributor to the development of translational shutoff. In this study, we applied a wide range of virological and structural approaches to further analyze nsp1 functions. The expression of this protein alone was found to be sufficient to cause CPE. However, we selected several nsp1 mutants exhibiting noncytopathic phenotypes. The attenuating mutations were detected in three clusters, located in the C-terminal helices, in one of the loops of the structured domain and in the junction of the disordered and structured fragment of nsp1. NMR-based analysis of the wild type nsp1 and its mutants did not confirm the existence of a stable β5-strand that was proposed by the X-ray structure. In solution, this protein appears to be present in a dynamic conformation, which is required for its functions in CPE development and viral replication. The NMR data also suggest a dynamic interaction between the N-terminal and C-terminal domains. The identified nsp1 mutations make this protein noncytotoxic and incapable of inducing translational shutoff, but they do not result in deleterious effects on viral cytopathogenicity. IMPORTANCE The nsp1 of SARS-CoV-2 is a multifunctional protein that modifies the intracellular environment for the needs of viral replication. It is responsible for the development of translational shutoff, and its expression alone is sufficient to cause a cytopathic effect (CPE). In this study, we selected a wide range of nsp1 mutants exhibiting noncytopathic phenotypes. The attenuating mutations, clustered in three different fragments of nsp1, were extensively characterized via virological and structural methods. Our data strongly suggest interactions between the nsp1 domains, which are required for the protein's functions in CPE development. Most of the mutations made nsp1 noncytotoxic and incapable of inducing translational shutoff. Most of them did not affect the viability of the viruses, but they did decrease the rates of replication in cells competent in type I IFN induction and signaling. These mutations, and their combinations, in particular, can be used for the development of SARS-CoV-2 variants with attenuated phenotypes.

Circulation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the human population leads to further viral evolution. The new variants that arise during this evolution are more infectious. Our data suggest that newer variants have shifted from utilizing both cathepsin/endosome- and TMPRSS2-mediated entry mechanisms to rely on a TMPRSS2-dependent entry pathway. Accordingly, only the early lineages of SARS-CoV-2 are capable of infecting and forming syncytia in Vero/ACE2 cells which lack TMPRSS2 expression. The presence of an intact multibasic furin cleavage site (FCS) in the S protein was a key requirement for cell-to-cell fusion. Deletion of FCS makes SARS-CoV-2 more infectious in vitro but renders it incapable of syncytium formation. Cell-to-cell fusion likely represents an alternative means of virus spread and is resistant to the presence of high levels of neutralizing monoclonal antibodies (MAbs) and immune sera in the media. In this study, we also noted that cells infected with SARS-CoV-2 with an intact FCS or alphavirus replicon expressing S protein (VEErep/S) released high levels of free S1 subunit. The released S1 is capable of activating the TLR4 receptor and inducing a pro-inflammatory response. Thus, S1 activation of TLR4 may be an important contributor to SARS-CoV-2-induced COVID-19 disease and needs to be considered in the design of COVID mRNA vaccines. Lastly, a VEErep/S-replicon was shown to produce large amounts of infectious, syncytium-forming pseudoviruses and thus could represent alternative experimental system for screening inhibitors of virus entry and syncytium formation. IMPORTANCE The results of this study demonstrate that the late lineages of SARS-CoV-2 evolved to more efficient use of the TMPRSS2-mediated entry pathway and gradually lost an ability to employ the cathepsins/endosome-mediated entry. The acquisition of a furin cleavage site (FCS) by SARS-CoV-2-specific S protein made the virus a potent producer of syncytia. Their formation is also determined by expression of ACE2 and TMPRSS2 and is resistant to neutralizing human MAbs and immune sera. Syncytium formation appears to be an alternative means of infection spread following the development of an adaptive immune response. Cells infected with SARS-CoV-2 with an intact FCS secrete high levels of the S1 subunit. The released S1 demonstrates an ability to activate the TLR4 receptor and induce pro-inflammatory cytokines, which represent a hallmark of SARS-CoV-2 pathogenesis. Alphavirus replicons encoding SARS-CoV-2 S protein cause spreading, syncytium-forming infection, and they can be applied as an experimental tool for studying the mechanism of syncytium formation.

One of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virulence factors is the ability to interact with high affinity to the ACE2 receptor, which mediates viral entry into cells. The results of our study demonstrate that within a few passages in cell culture, both the natural isolate of SARS-CoV-2 and the recombinant cDNA-derived variant acquire an additional ability to bind to heparan sulfate (HS). This promotes a primary attachment of viral particles to cells before their further interactions with the ACE2. Interaction with HS is acquired through multiple mechanisms. These include (i) accumulation of point mutations in the N-terminal domain (NTD) of the S protein, which increases the positive charge of the surface of this domain, (ii) insertions into the NTD of heterologous peptides containing positively charged amino acids, and (iii) mutation of the first amino acid downstream of the furin cleavage site. This last mutation affects S protein processing, transforms the unprocessed furin cleavage site into the heparin-binding peptide, and makes viruses less capable of syncytium formation. These viral adaptations result in higher affinity of viral particles to heparin, dramatic increase in plaque sizes, more efficient viral spread, higher infectious titers, and 2 orders of magnitude higher infectivity. The detected adaptations also suggest an active role of NTD in virus attachment and entry. As in the case of other RNA-positive (RNA + ) viruses, evolution to HS binding may result in virus attenuation in vivo . IMPORTANCE The spike protein of SARS-CoV-2 is a major determinant of viral pathogenesis. It mediates binding to the ACE2 receptor and, later, fusion of viral envelope and cellular membranes. The results of our study demonstrate that SARS-CoV-2 rapidly evolves during propagation in cultured cells. Its spike protein acquires mutations in the NTD and in the P1' position of the furin cleavage site (FCS). The amino acid substitutions or insertions of short peptides in NTD are closely located on the protein surface and increase its positive charge. They strongly increase affinity of the virus to heparan sulfate, make it dramatically more infectious for the cultured cells, and decrease the genome equivalent to PFU (GE/PFU) ratio by orders of magnitude. The S686G mutation also transforms the FCS into the heparin-binding peptide. Thus, the evolved SARS-CoV-2 variants efficiently use glycosaminoglycans on the cell surface for primary attachment before the high-affinity interaction of the spikes with the ACE2 receptor.

Chikungunya virus (CHIKV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. Within the last 2 decades, CHIKV has expanded its presence to both hemispheres and is currently circulating in both Old and New Worlds. Despite the severity and persistence of the arthritis it causes in humans, no approved vaccines or therapeutic means have been developed for CHIKV infection. Replication of alphaviruses, including CHIKV, is determined not only by their nonstructural proteins but also by a wide range of host factors, which are indispensable components of viral replication complexes (vRCs). Alphavirus nsP3s contain hypervariable domains (HVDs), which encode multiple motifs that drive recruitment of cell- and virus-specific host proteins into vRCs. Our previous data suggested that NAP1 family members are a group of host factors that may interact with CHIKV nsP3 HVD. In this study, we performed a detailed investigation of the NAP1 function in CHIKV replication in vertebrate cells. Our data demonstrate that (i) the NAP1-HVD interactions have strong stimulatory effects on CHIKV replication, (ii) both NAP1L1 and NAP1L4 interact with the CHIKV HVD, (iii) NAP1 family members interact with two motifs, which are located upstream and downstream of the G3BP-binding motifs of CHIKV HVD, (iv) NAP1 proteins interact only with a phosphorylated form of CHIKV HVD, and HVD phosphorylation is mediated by CK2 kinase, and (v) NAP1 and other families of host factors redundantly promote CHIKV replication and their bindings have additive stimulatory effects on viral replication. IMPORTANCE Cellular proteins play critical roles in the assembly of alphavirus replication complexes (vRCs). Their recruitment is determined by the viral nonstructural protein 3 (nsP3). This protein contains a long, disordered hypervariable domain (HVD), which encodes virus-specific combinations of short linear motifs interacting with host factors during vRC assembly. Our study defined the binding mechanism of NAP1 family members to CHIKV HVD and demonstrated a stimulatory effect of this interaction on viral replication. We show that interaction with NAP1L1 is mediated by two HVD motifs and requires phosphorylation of HVD by CK2 kinase. Based on the accumulated data, we present a map of the binding motifs of the critical host factors currently known to interact with CHIKV HVD. It can be used to manipulate cell specificity of viral replication and pathogenesis, and to develop a new generation of vaccine candidates.

Stromal interaction molecule (STIM) proteins play a crucial role in store-operated calcium entry (SOCE) as endoplasmic reticulum Ca 2+ sensors. In neurons, STIM2 was shown to have distinct functions from STIM1. However, its role in brain activity and behavior was not fully elucidated. The present study analyzed behavior in zebrafish ( Danio rerio ) that lacked stim2a . The mutant animals had no morphological abnormalities and were fertile. RNA-sequencing revealed alterations of the expression of transcription factor genes and several members of the calcium toolkit. Neuronal Ca 2+ activity was measured in vivo in neurons that expressed the GCaMP5G sensor. Optic tectum neurons in stim2a -/- fish had more frequent Ca 2+ signal oscillations compared with neurons in wildtype (WT) fish. We detected an increase in activity during the visual-motor response test, an increase in thigmotaxis in the open field test, and the disruption of phototaxis in the dark/light preference test in stim2a -/- mutants compared with WT. Both groups of animals reacted to glutamate and pentylenetetrazol with an increase in activity during the visual-motor response test, with no major differences between groups. Altogether, our results suggest that the hyperactive-like phenotype of stim2a -/- mutant zebrafish is caused by the dysregulation of Ca 2+ homeostasis and signaling.

In neurons, stromal interaction molecule (STIM) proteins regulate store-operated Ca 2+ entry (SOCE) and are involved in calcium signaling pathways. However, STIM activity in neurological diseases is unclear and should be clarified by studies that are performed in vivo rather than in cultured cells in vitro. The present study investigated the role of neuronal Stim2b protein in zebrafish. We generated stim2b knockout zebrafish, which were fertile and had a regular lifespan. Using various behavioral tests, we found that stim2b -/- zebrafish larvae were hyperactive compared with wild-type fish. The mutants exhibited increases in mobility and thigmotaxis and disruptions of phototaxis. They were also more sensitive to pentylenetetrazol and glutamate treatments. Using lightsheet microscopy, a higher average oscillation frequency and higher average amplitude of neuronal Ca 2+ oscillations were observed in stim2b -/- larvae. RNA sequencing detected upregulation of the annexin 3a and gpr39 genes and downregulation of the rrm2 , neuroguidin , and homer2 genes. The latter gene encodes a protein that is involved in several processes that are involved in Ca 2+ homeostasis in neurons, including metabotropic glutamate receptors. We propose that Stim2b deficiency in neurons dysregulates SOCE and triggers changes in gene expression, thereby causing abnormal behavior, such as hyperactivity and susceptibility to seizures.

Zebrafish are well-suited for in vivo calcium imaging because of the transparency of their larvae and the ability to express calcium probes in various cell subtypes. This model organism has been used extensively to study brain development, neuronal function, and network activity. However, only a few studies have investigated calcium homeostasis and signaling in zebrafish neurons, and little is known about the proteins that are involved in these processes. Using bioinformatics analysis and available databases, the present study identified 491 genes of the zebrafish Calcium Toolkit (CaTK). Using RNA-sequencing, we then evaluated the expression of these genes in the adult zebrafish brain and found 380 hits that belonged to the CaTK. Based on quantitative real-time polymerase chain reaction arrays, we estimated the relative mRNA levels in the brain of CaTK genes at two developmental stages. In both 5 dpf larvae and adult zebrafish, the highest relative expression was observed for tmbim4 , which encodes a Golgi membrane protein. The present data on CaTK genes will contribute to future applications of zebrafish as a model for in vivo and in vitro studies of Ca 2+ signaling., Competing Interests: The authors declare no conflict of interest.