We investigated the biochemical properties of the products of two paralogs, identified in Salmo salar. SsRidA-1 and SsRidA-2 complemented the growth defect of a Salmonella enterica ridA mutant, an in vivo model of 2AA stress. In vitro, both proteins hydrolyzed 2-imino acids (IA) to keto-acids and ammonia. SsRidA-1 was active on IA derived from nonpolar amino acids and poorly active or inactive on IA derived from other amino acids tested. In contrast, SsRidA-2 had a generally low catalytic efficiency, but showed a relatively higher activity with IA derived from L-Glu and aromatic amino acids. The crystal structures of SsRidA-1 and SsRidA-2 provided hints of the remarkably different conformational stability and substrate specificity. Overall, SsRidA-1 is similar to the mammalian orthologs whereas SsRidA-2 displays unique properties likely generated by functional specialization of a duplicated ancestral gene.Reactive Intermediate Deaminase (Rid) protein superfamily includes eight families among which the RidA is conserved in all domains of life. RidA proteins accelerate the deamination of the reactive 2-aminoacrylate (2AA), an enamine produced by some pyridoxal phosphate (PLP)-dependent enzymes. 2AA accumulation inhibits target enzymes with a detrimental impact on fitness. As a consequence of whole genome duplication, teleost fish have two  Cells protect their components using two strategies primarily aimed to (i) repair anomalous metabolites and reconvert them to the original form4, or (ii) pre-empt damage by converting the harmful compound into a benign one2. A demonstration of the biological relevance of this phenomenon is the increasing number of genes with previously unknown functions that encode enzymes involved in metabolite damage control across all living organisms5. A notable example is the Rid superfamily, known for many years as the YjgF/YER057c/UK114 family, for which a common in vitro activity as imine deaminase and involvement in a pre-empting mechanism has been elegantly demonstrated7. The term Rid (reactive intermediate deaminase) refers to the hydrolytic deamination of highly reactive and labile enamines/imines to α-ketoacid and ammonia, a reaction that would be otherwise relatively slow if left to occur spontaneously7.Metabolism is composed of different arrangements of enzyme-catalyzed reactions that convert a precursor into a product through a series of metabolic intermediates. Among hundreds of metabolites generated from catabolic and anabolic reactions, small molecules endowed with high reactivity and toxicity may arise and cause metabolite damage9. From the Rid1 to Rid3 families, a conserved arginine residue (Arg105) is believed to be sufficient to predict the imine hydrolyzing activity of the proteins and this residue is also shared by all RidA enzymes from Eukarya (Arg107). While Rid proteins from different families are normally observed in prokaryotes9, for still unclear reasons eukaryotes restricted their repertoire only to members of the RidA family. In most eukaryotes, RidA is a cytosolic enzyme encoded by a single-copy gene. However, a few notable exceptions have been described. For example, the yeast Saccharomyces cerevisiae, beside the canonical RidA (Hmf1p/Yil057cp), displays the presence of another paralogous protein (Mmf1p/Yil051cp) that is imported into the mitochondrion12. In plants , the RidA enzyme is localized in the chloroplast13. Interestingly, all teleosts (bony fish) have two RidA paralogs, most probably as a consequence of Clade-specific whole genome duplication (WGD), but neither of these paralogs has been characterized so far.The Rid superfamily is composed of 8 families: RidA is present in all domains of life whereas Rid1 to Rid7 families are unique to prokaryotes and found predominantly in bacteria1 for a review] and 2-aminocrotonate (2AC), the product of threonine dehydratase. The enamines 2AA and 2AC are believed to readily tautomerize to 2-iminopropionate (2-iminopyruvate) and 2-iminobutyrate, respectively. RidA accelerates the conversion of these compounds to the final products pyruvate and α-ketobutyrate, respectively, and ammonia. The conversion to benign products catalyzed by RidA enzymes constitutes a new paradigm in metabolite damage control1.The best-characterized function of RidA is the quenching of specific enamines. The most physiologically relevant compounds are 2-aminoacrylate (2AA), which is an obligate reaction product of the pyridoxal-5′-phosphate (PLP)-dependent serine dehydratases and cysteine desulfhydrases  and from mixed brain, kidney and spleen tissues (BT058640.1). This second RidA homolog matched to a locus on chromosome 14 (Chr ssa 14) indicating that it was the product of a single gene here named ridA-2.Two additional sequences were ascribable to a putative protein of 135 residues, hereafter referred to as ridA locus in a carefully selected collection of publicly available vertebrate genomes. A 1 MBp-chromosome region centered on the human ridA gene (for simplicity here indicated in lowercase) was analyzed. A remarkable conservation among ridA loci was observed , the genes surrounding human ridA showed a striking conservation in order and orientation within all the lineages having a single-copy ridA gene  and Orizia latipes (Japanese rice fish). Both loci showed only a partial conservation of gene content and order, most likely due to differential gene loss  conserved the typical order of the upstream genes but lacked all the downstream genes including ridA itself, suggesting that it was a remnant of extensive chromosomal rearrangements. By contrast, the other three loci contained a ridA gene. The ridA locus present on Chr ssa10 retained the human homologs matn2, pop1 and nipal2 around the ridA-1 gene, encompassing 7,674 bp and coding for SsRidA-1. The ridA gene on Chr ssa14, encompassing 11,465 bp, corresponded to ridA-2, and the associated genomic locus showed the lack of all the flanking genes except stk3. Both salmon ridA genes share an identical gene structure consisting of 6 exons and both genes are transcribed since full cDNA sequences were annotated as mentioned above. The third ridA-like gene resides in an unplaced scaffold (Unp) of 7,628 bp (NW_01235881), and has a single exon encoding a truncated RidA of 125 aminoacyl residues sharing only 41.47% identity with HsRidA protein. This anomalous sequence likely corresponds to an inactive retro-pseudogene since a corresponding cDNA sequence is not present in the database. Due to its anomalous features, it was excluded from subsequent analyses. Considerations regarding the relatively high variability in gene order and content compared to the archetypal human ridA locus are consistent with evolution by recombination and differential gene loss of these loci.An additional WGD event has been described in the common ancestor of the salmonid lineage, ~80 Mya after their divergence from the Esociformes fish order, and this justifies the presence of even four copies of a given gene in the salmonid speciesS. salar has two functionally intact paralogs, ridA-1 and ridA-2, residing in different chromosomes, namely Chr ssa10 (Gene ID: 106561137) and Chr ssa14 (Gene ID: 100195988). The two paralogous proteins, SsRidA-1 and SsRidA-2, share 77% amino acid identity and were the object of further studies.In conclusion, our BLASTP and comparative genomic analyses indicate that ). The frequency plot calculated from the multiple alignment using WebLogo33 showed the presence of several invariable residues in addition to the conserved R107, corresponding to R105 in bacteria , which were experimentally demonstrated to be catalytically active Fig. . The freria Fig. . The conSsRidA-1 and SsRidA-2 were cloned into pET-15b downstream to DNA regions encoding a 6xHis tag and a thrombin cleavage site. E. coli Rosetta (DE3) cells were transformed with the recombinant plasmids .To characterize the salmon RidA proteins, the CDS of 25. The activity of SsRidA-1 and SsRidA-2 on a broad spectrum of IA was compared to that of goat RidA (ChRidA), used as a reference being the best-characterized animal RidA25. Both SsRidA-1 and SsRidA-2 were active as imine deaminases but striking differences in substrate specificity between the isozymes were observed as highlighted in Fig. SsRidA-1 appeared to be active, like the goat enzyme, on IA derived from L-Ala, L-Met, L-Leu and L-Gln whereas SsRidA-2 was one to two orders of magnitude less active on these IA except for that derived from L-Ala, i.e. 2-iminopyruvate . The latter is the stable tautomer of 2AA, the common product of serine dehydratase and cysteine desulfhydrase reactions. All the RidA enzymes showed limited activity on IA derived from aromatic amino acids and also on L-dihydroxy-phenylalanine (L-DOPA), but SsRidA-2 displayed a weak preference for all these substrates except for L-Trp on which the goat enzyme showed a slightly higher activity . With respect to charged amino acids, no RidAs exhibited any significant activity in the presence of IA derived from basic amino acids and also from L-Asp. Interestingly, SsRidA-2 stood out revealing the highest level of activity for the IA derived from L-Glu  upon oxidation of the corresponding L-amino acidsGlu Fig. . These rSalmonella enterica, ridA mutants grown in minimal medium accumulate the reactive enamine 2-aminoacrylate (2AA), which is generated by the biosynthetic serine/threonine dehydratase  from the L-serine substrate7. In the presence of exogenous serine, 2AA damages a number of PLP-dependent enzymes, eliciting a severe growth defect21. To assess the ability of the two RidA orthologs from S. salar to deaminate enamines/imines in vivo, an S. enterica ridA mutant (DM14829) was transformed with plasmids pDM1616 or pDM1617 encoding SsRidA-1 or SsRidA-2, respectively  restored full growth to the S. enterica ridA mutant, with or without induction of the PBAD promoter by L-arabinose. In contrast, pDM1616 and pDM1617 allowed growth of the S. enterica ridA mutant, but only in the presence of exogenous L-arabinose. These data indicated that SsRidA-1 and SsRidA-2 have 2AA deaminase activity in vivo. The requirement for induction suggested that these proteins were less efficient 2AA deaminases than the S. enterica RidA enzyme. It is also possible that expression of the S. salar proteins was not as robust as that of S. enterica RidA from the pCV1 vector. However, since the synthetic genes were codon optimized for expression in S. enterica, this is considered unlikely. Protein stability may also account for the discrepancy between complementation constructs. Finally, there was a minor difference between SsRidA-1 and SsRidA-2, with expression of SsRidA-1 allowing slightly better growth of the S. enterica ridA mutant than SsRidA-2. These data suggest that 2AA could be a better substrate for SsRidA-1 than SsRidA-2, a hypothesis consistent with the observation that SsRidA-1 exhibited a higher 100/K50 than SsRidA-2  spectra with that of ChRidA. As shown in Fig. The secondary structures of S. salar RidA proteins was studied by monitoring the change in ellipticity at 220 nm, which reports on the α- helical content of the proteins, by varying the temperature from 20 to 98 °C. An almost flat trace was obtained for SsRidA-1, indicating that the protein conformation was not affected in this temperature range . This behavior has been previously reported also for ChRidA25, indicating that both goat RidA and salmon RidA-1 have a remarkable resistance to thermal unfolding. SsRidA-1 thermal stability was further investigated by repeating the experiment in the presence of increasing concentrations of urea . The melting temperature (Tm) in the absence of urea was extrapolated from the linear fitting of Tm as a function of urea concentration . The calculated value of 100.2 °C is slightly lower than that calculated for ChRidA (104 °C)25.The conformational stability of the SsRidA-2 showed a more canonical denaturation curve revealing a Tm of 65 °C . These results indicate that, despite the high similarity, SsRidA-1 and SsRidA-2 have very different conformational stability: the former is resistant to heat denaturation while the latter denatures at a temperature normally observed for mesophilic proteins34.On the other hand, ChRidA, were characterized by size-exclusion chromatography (SEC) combined with multi-angle light scattering (MALS) and dynamic light scattering (DLS), . Therefore, S. salar RidA-1 and RidA-2 assemble into homo-trimers, as previously reported for ChRidA and for other characterized orthologous proteins37. Moreover, the mass calculated across the protein peaks is constant suggesting that the samples are monodispersed with no evidence of dissociation equilibria affecting the trimeric association . To shed light on the cause of the lower elution volume of SsRidA-1 (11.13 mL) in comparison to SsRidA-2 (11.35 mL) and ChRidA (11.34 mL), the hydrodynamic radius (Rh) was calculated for each protein by DLS in the same chromatographic runs. As for the molar mass, also the Rh values of the three RidA forms are similar to each other  with an average value of 2.8 nm. These results rule out differences in shape or compactness of the proteins. Therefore, the different elution volume is most likely due to nonspecific electrostatic interactions between the proteins and the chromatographic matrix, being SsRidA-1 the only one having a net negative charge under the experimental condition used compared to the net positive charge of SsRidA-2 and ChRidA .CD analyses indicated that salmon RidA-2 is less stable than RidA-1. To analyze whether this could be ascribed to a different assembly in solution, the quaternary structure of the two isozymes, in comparison with S), Fig. . The molSsRidA-1 and SsRidA-2 quaternary structure assembly and stability were provided by native MS experiments. Specifically, the SsRidA-1 electrospray ionization (ESI) mass spectrum  from the monomer (permeate). No trace of protein was detectable in permeate whereas the monomer, dimer and trimer were still detectable by native MS in the retentate yielding a spectrum superimposable to that obtained for the unfractionated sample shown in Fig. SsRidA-2 monomer and dimer in Fig. SsRidA-2 trimer during MS analysis. Interestingly, no trace of monomeric or dimeric forms was found under the same experimental condition for SsRidA-1  values 0.73 Å/373 Cα and 0.75 Å/375 Cα] and with the structure of ChRidA 38. The two SsRidA-2 trimers are virtually identical  and also SsRidA-2 monomers are all well superposable . Monomeric SsRidA-1 and SsRidA-2 share the chorismate mutase-like fold common to all the members of the YjgF/YER057c/UK114 superfamily38: monomers fold in a single compact domain of one six-stranded β-sheet packed against two α-helices 37 37 Fig. . SsRidA-SsRidA-1 and SsRidA-2. SsRidA-1 is an efficient catalyst with hydrophobic IA and possesses a mainly hydrophobic 15–20 loop. On the other hand, SsRidA-2 has a positive charge in position 17 and, under our experimental conditions, showed a strong preference for the IA derived from L-Glu, while the lack of activity on the IA derived from L-Asp cannot be explained. These considerations suggest that the 15–20 loop is crucial in determining the distinct substrate specificity of RidA enzymes.The different chemical properties of the 15–20 loop help to rationalise the different substrate specificities of 25. Unlike most vertebrates, salmon and other teleosts contain two paralogous copies of the mammalian ridA gene. The high sequence similarity between SsRidA-1 and SsRidA-2 and the partial conservation in synteny between the underlying loci are largely consistent with their origin from a WGD event. However, considerations concerning sequence similarity, substrate specificity, conformational stability and structural features indicate that SsRidA-1 is the bona fide functional ortholog of the vertebrate RidA. The presence of RidA is consistent with the presence of L-serine dehydratase in salmon as annotated in Salmobase database.RidA activity is ubiquitous and the conservation from bacteria to mammals underlies its importance in preventing metabolic stressSsRidA-1 could play the predominant function in the deamination of IA derived from nonpolar amino acids  and be less active on IA derived from aromatic amino acids, substrates never tested before, and inactive on those derived from charged residues. In contrast, SsRidA-2 has a low catalytic efficiency with nonpolar substrates, but it has a high activity on the imino acid deriving from L-Glu indicating an opposite substrate preference with respect to SsRidA-1 and goat RidA. SsRidA-2 also displayed a relatively higher catalytic efficiency than other RidA on IA from aromatic amino acids, except the L-Trp derivative on which ChRidA is more active. These findings are important as they suggest that proteins of the RidA family could also be involved in the metabolism of L-Glu and aromatic amino acids or their derivatives among which are neurotransmitters and their precursors, i.e. L-Glu, 5-hydroxy tryptamine and L-DOPA. In this respect, it is worthwhile noting that in mammals, ridA gene expression is high in the liver, low in the kidney cortex and also in some areas of the brain except the cerebellum (http://genome.ucsc.edu). Finally, the two salmon RidA enzymes are endowed with activity that probably differs due to a specialization of the duplicated ridA gene that has been retained since, as suggested by genetic evidence, diploidy in salmon is not yet fully re-established29. However, hints to potential novel roles of RidA, other than protection from metabolic damage by 2AA derived from serine dehydratase and cysteine desulfhydrase, can emerge from our study.The striking different catalytic efficiencies with different IA substrates between the salmon RidAs suggest that these enzymes could play complementary functions in bony fish. Like the goat enzyme, S. salar and C. hircus and from the UCSC Genome browser for all the other species. Details concerning assembly versions and genomic annotations are reported in Table 41 were performed using the BLOSUM80 matrix42 and accepting only best reciprocal hits with an e-value ≤ 1e−5, which covered at least 50% of the protein length, and where “second-best” hits producing bit scores <90% of that associated with the best match. Putative orthologs were identified as best reciprocal BLAST hits by means of a custom Perl script. Synteny analyses were performed by cross-referencing putative orthologous proteins with the corresponding annotation in gtf format in order to derive genomic coordinates and the underlying gene structures, again by means of a custom Perl script.Complete proteomes were retrieved from NCBI GenBank for S. salar database at Salmobase 2.0  was performed to retrieve the corresponding cDNA sequences and genomic coordinates and sequences. The multiple alignment was done with Clustal Omega https://www.ebi.ac.uk/Tools/msa/clustalo/ and Box Shade https://embnet.vital-it.ch/software/BOX_doc.html using default parameters. The WebLogo was created using the software at https://weblogo.berkeley.edu.The sequences of RidA-like salmon proteins were retrieved by performing a BLASTP similarity search, applying the default parameters, in the salmon proteome using the human RidA as a query. A second similarity search using BLASTN or BLASTP in the Bacterial strains used in this work are listed in Table S. enterica serovar Typhimurium LT2 were used. Minimal medium was no-carbon E supplemented with 20 mM glycerol as the sole carbon source, 1 mM MgSO443, and trace minerals44. Difco nutrient broth (NB) (8 g/L) supplemented with 5 g/L NaCl was used as rich medium for S. enterica. Ampicillin was used in rich (150 μg/mL) and minimal (7.5 μg/mL) medium for plasmid maintenance, when appropriate.In the complementation study, derivatives of SsRidA-1and SsRidA-2 in E.coli were obtained by GENEWIZ GmbH  by cloning in the pET-15b vector double-digested with NdeI and XhoI, the synthesized DNA of the CDS. The fusion proteins carried at the N-terminus, the sequence MGSSHHHHHHSSGLVPR/GSH, comprising a 6xHis-tag and the cleavage site for thrombin (/) inside the thrombin recognition site (underlined). The proteins were expressed from E. coli strains harbouring the recombinant plasmids  and SsRidA-2 (UniprotKB C0H8l4) were codon optimized for expression in S. enterica LT2 using the online application OPTIMIZER (http://genomes.urv.es/OPTIMIZER/)46 and synthesized by GenScript , 47 for downstream use in complementation of an S. enterica ridA mutant by BspQI restriction cloning48. The resulting plasmids encoded SsRidA-1 (pDM1616) and SsRidA-2 (pDM1617) under the control of the arabinose-inducible PBAD promoter. pCV1-ridA (pDM1439), which harbored the ridA gene from S. enterica LT2, had been constructed previously23.For complementation tests, the CDS of see Fig. . Synthes25. The spectrophotometric assay measures the initial rate of formation of semicarbazone species derived from the spontaneous reaction of the IA with semicarbazide in the absence or presence of varying RidA concentrations. By accelerating the hydrolysis of IA to the corresponding ketoacid and ammonia, RidA subtracts IA to the reaction with semicarbazide leading to a decrease of the measured initial velocity of the reaction.The assay is based on the use of L-amino acid oxidases to generate IAs from appropriate amino acids25. The amount of the L-amino acid oxidase was adjusted in order to have similar rates of formation of IA with the different L-amino acids (~ 0.2–0.3 ΔA248/min). The reaction was started by the addition of the L-amino acid and monitored as described in25. The time-course of formation of the semicarbazone species was monitored as the increase of absorbance at 248 nm. The initial reaction velocity measured in the presence of a given concentration of RidA (vRidA) was then expressed as percent of the initial velocity measured in the absence of RidA (vo). The resulting residual velocity (v) values were then fitted with equation (1) using the GraFit 4.0 software (Erythacus Software Ltd) to calculate the K50 value and the associated error25.The reaction mixtures were set-up in quartz microcuvettes (10 mm light path) and were composed of 5 mM L-amino acid, except for the L-aromatic amino acids the concentration of which was 0.5 mM, 10 mM neutralized semicarbazide-HCl, catalase, 6.6 μg/ml, snake venom L-amino acid oxidase , showing 71% and 67% sequence identity with SsRidA-1 and SsRidA-2, respectively. Specifically, the monomeric template was used for SsRidA-1 and a whole trimeric ensemble for SsRidA-2.Both 55 and refinement with phenix.refine56. Ligand occupancy was estimated using a criterion based on keeping the atomic displacement parameters of each molecule comparable with those of the surrounding residues (in fully occupied sites). Water molecules were added with the program ARP/wARP57 and visually inspected with Coot55. For SsRidA-1 structure, in the last cycle of refinement, hydrogen atoms were added in calculated positions, contributing to the Fc calculation. Models were inspected and validated using Molprobity58; processing and refinement statistics are reported in Table 59.Initial molecular replacement solutions were subjected to subsequent cycles of manual building in CootSupplementary information.


The current coronavirus outbreak may provide an illustrative analogy for sustainability challenges, exemplifying how challenges such as climate change may become wicked problems demanding novel and drastic solution attempts. The current coronavirus pandemic is causing an unprecedented worldwide economic and social disruption. The rate of infected people is steadily on the rise (Dong et al. Both the coronavirus crisis and climate change, although occurring on different temporal scales, represent surprisingly similar problem and response dynamics. To this end, the coronavirus pandemic and climate change both feature traits that are characteristic of what has been termed “super wicked problems” (Levin et al. The coronavirus crisis is threatening to rapidly overwhelm the capacities of health care systems of severely affected countries around the globe . Disrupthttps://www.nature.com/nclimate]. Still, many experts agree that to date the global reaction to climate change has been insufficient and policy proposals would need to better account for the effect of culture as a mediator in societal reactions to climate change (Adger et al. It is becoming evident that the world community was ill prepared for the coronavirus pandemic, although experts flagged the problem early on (World Health Organization Responses to the coronavirus crisis demand urgent measures to minimize further spread, and despite unprecedented global measures already in place, halting the outbreak’s momentum is challenging. As with the coronavirus crisis, urgent actions to tackle anthropogenic climate change are now required as the impacts of changing climate are increasing in both scale and pace Pittock . The futBoth challenges demand solution-oriented action with multiple interventions that address the systemic causes of the crises. Furthermore, the changing nature of the crises means that a single specific solution may not be possible and interventions need to be continually adjusted and reassessed to changing conditions. Behavior change represents a crucial component in addressing both crises [Fischer ]. Arrest


Horse testis development and spermatogenesis are complex physiological processes.To study these processes, three immature and three mature testes were collected from the Mongolian horse, and six libraries were established using high-throughput RNA sequencing technology (RNA-Seq) to screen for genes related to testis development and spermatogenesis.A total of 16,237 upregulated genes and 8,641 downregulated genes were detected in the testis of the Mongolian horse. These genes play important roles in different developmental stages of spermatogenesis and testicular development. Five genes with alternative splicing events that may influence spermatogenesis and development of the testis were detected. GO (Gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses were performed for functional annotation of the differentially expressed genes. Pathways related to “spermatogenesis,” male gamete generation,” “spermatid development” and “oocyte meiosis” were significantly involved in different stages of testis development and spermatogenesis.Genes, pathways and alternative splicing events were identified with inferred functions in the process of spermatogenesis in the Mongolian horse. The identification of these differentially expressed genetic signatures improves our understanding of horse testis development and spermatogenesis. The mammalian testis is an important male reproductive organ that produces sperm and androgens. Spermatogenesis is a complex developmental process with a unique mechanism of cell division—meiosis—as a major defining event. Sperm development consists of three stages: 1) proliferation and differentiation of spermatogonia, 2) meiosis of spermatocytes, and (3) sperm maturation. Spermatogonial stem cells differentiate into spermatogonia and, with replication of their DNA content, give rise to primary spermatocytes. Secondary spermatocytes and spermatids are derived from the primary spermatocytes by DNA replication and tetraploid meiosis. Haploid sperm cells undergo at least four distinct morphological changes into sperm cells, namely chromatin condensation, acrosome formation, flagellar formation, and cytoplasmic reduction. In addition to spermatogenic cells, spermatogenesis also involves multiple somatic cells in testicular tissues, such as Sertoli and Leydig cells. In mammalian testes, spermatogenesis occurs within seminiferous tubules, where germ cells associate with Sertoli cells, which provide the appropriate microenvironment for spermatogenesis. Related genes in these cells play important roles in specific stages of germ cell development, and the transcription and translation of these genes vary in different stages of spermatogenesis [ prolifer meiosis RNA sequencing technology (RNA-Seq) technology, which provides a global view of gene expression profiles, has been developed for mapping and quantifying transcriptomes , 4. ThisThe Mongolian horse is one of the most famous native breeds in China, and in the world. It is a dual-purpose breed that can adapt to harsh climate and extensive breeding conditions. Normal development of the testis and spermatogenesis is extremely important to ensure high level semen production and protect population continuity, and genes that are important for different stages of testis development and spermatogenesis are likely to have roles in maintaining fertility. Unfortunately, the transcriptional expression of genes during testicular development and spermatogenesis in the horse is not well understood, thereby limiting the understanding of the mechanisms of deformation and maturation in spermatogenesis. This study aimed to study the transcriptome profiles of immature and mature male Mongolian horse testes utilizing high-throughput RNA-Seq technology and bioinformatics analysis. For this purpose, a testis transcript database was obtained, and DEGs and regulatory pathways were analyzed. This study provides new insights into gene expression and the regulatory mechanisms of horse testis development and spermatogenesis.To screen for genes related to testis development and spermiogenesis, a total of six libraries—three from immature colts  and three from mature stallions —were constructed and sequenced on the Illumina Hiseq platform. An overview of the information for these libraries is listed in Table E. caballus, which is the most accurate and commonly used reference genome for horses [The clean reads were aligned to the reference genome of r horses , 10, usihttp://rnaseq-mats.sourceforge.net/index.html): (1) SE: Skipped exon, (2) MXE: Mutually exclusive exon, (3) A5SS: Alternative 5′ splice site, (4) A3SS: Alternative 3′ splice site, and (5) RI: Retained intron. The kinds and quantities of AS events were counted, and the expression of each type of AS event was analyzed. The results indicate that numerous and varied AS events exist in the spermatogenesis process (Table Alternative splicing (AS) is a common occurrence in most eukaryotic cells and can result in the translation of different forms of proteins at various time points and environments, which increases the adaptability and physical fitness of the species. In our study, the AS events were classified into five kinds by rMATS  enrichment analysis was used to explore the functions of the DEGs in testis development. A total of 70 GO terms related to “biological processes,” “molecular functions,” and “cellular components” were significantly enriched across the ASM vs BSM. DEG groups Fig. . ApproxiP-values < 0.05. Six KEGG pathways were upregulated, including “ubiquitin-mediated proteolysis,” “protein processing in endoplasmic reticulum,” “RNA transport,” “metabolic pathways,” “oocyte meiosis,” and “cell cycle.” Four pathways were downregulated, including “ribosome,” “focal adhesion,” ‘proteoglycans in cancer,” and “ECM-receptor interaction”  pathway enrichment analysis was performed on these differently expressed genes with a cut-off criterion of corrected on” Fig. .Fig. 3SMEI1 (two different sites), EHMT2, BTRC, CLOCK, IQCG, ANAPC5 and SKP1, to validate the expression patterns by qRT-PCR. These genes belong to “oocyte meiosis” KEGG pathways and GO terms “spermatogenesis,” “male gamete generation,” and “spermatid development”  is regulated by 2 SE events as categorized under the GO terms “spermatogenesis,” “male gamete generation,” and “spermatid development”. The causality of the mutation was confirmed with a targeted null allele. Loss of IQCG disrupts spermiogenesis wherein tail formation is either incomplete or breaks apart from the sperm heads. Because IQ motif-containing genes typically regulate calmodulin, which in turn can impact the actin cytoskeleton, these findings suggest a potential role for localized calcium signaling in sperm flagellum morphogenesis, Ca2+ signaling is important for regulating sperm motility, most notably in the context of flagellar function and regulation [MEI1) gene also occurred in ASM. MEI1 was upregulated in ASM and is related to the GO terms “spermatogenesis,” “male gamete generation,” and “spermatid development.” MEI1 is expressed specifically in the testis and the first meiosis-specific mutation identified by forward genetic approaches in mammals. Mutations in the MEI1 gene disrupt meiosis during spermatogenesis, especially in Americans of European descent [Alternative splicing is a crucial mechanism for regulating gene expression and generating proteomic diversity. Recent estimates indicate that the expression of nearly 95% of human multi-exon genes involves AS , 12. In gulation –19. 11 S descent –22.SLC26A8, MEI1, EHMT2, DPY19L2, HIST1H2BA, CAPZA3, TSSK2, TSSK1B, KDM3A, ALMS1, and IQCG. Testis anion transporter 1(Tat1) is a protein that, in humans, is encoded by the SLC26A8 gene. SLC26A8 gene was localized to developing spermatocytes [DPY19L2 are associated with an infertility disorder known as spermatogenic failure type 9 (SPGF9) [TSSK2 is highly expressed in the testis and is required for the transformation of a ring-shaped structure around the base of the flagellum that originates from the chromatoid body [Other enriched genes in this study that belong to the “spermatogenesis,” “male gamete generation,” and “spermatid development” GO terms include atocytes –25. The atocytes . It acts (SPGF9) –35. Testoid body –41. The AURKA gene and is a member of a family of mitotic serine/threonine kinases [CCNB1 gene in humans [FBXO43), also called endogenous meiotic inhibitor 2 (EMI2), was originally identified in a yeast two-hybrid screen as a novel Xenopus polo-like kinase (Plx-1 ) [FBXO43 is also essential for spermatocytes to complete meiotic divisions in mice. In male FBXO43 knockout mice, spermatocytes cannot complete meiotic divisions, and spermatids are absent in the testes [In our study, six upregulated KEGG pathways were detected. Though the “oocyte meiosis” pathway is named according to its role in female fertility, genes detected in the pathway also regulated meiosis of sperm. Meiosis plays a critical role in the formation of sperm. A total genes in the oocyte meiosis pathway were detected, with functions that influence meiosis I and II. Cell division cycle 25C (CDC25C), which encode threonine/tyrosine phosphatases related with in meiotic cell cycle regulation, were found to be differentially localized in rat testicular germ cells during spermatogenesis . Aurora  kinases . This ge kinases , and its kinases . The G2/n humans . The CCNn humans . PLK1 isn humans . Meioticn humans , 50. F-b(Plx-1 ) , 52. FBXe testes .Our study focused on increasing understanding of the process of testis development and spermatogenesis in the Mongolian horse, which ultimately may lead to the development of approaches to improve fertility. Due to the lack of information available in regards to the genetics of horse spermatogenesis, we have inferred the function of certain DEGs according to available information in humans and mice. Furthermore, DEGs that have functions in development may not be directly applicable to many situations of infertility in the horse, such as azoospermia and asthenozoospermia.In our study, six mRNA libraries were constructed and sequenced from horse testes from ASM and BSM. A total of 16,237 upregulated and 8,641 downregulated genes were identified; most genes had functions within biological process categories and are known to participate in spermatogenesis and testis development. We analyzed a large number of genes related to spermatogenesis and development of the horse testis, including those that play important roles in different developmental stages. We also found that the AS events might influence the function of these genes and indirectly affect the spermatogenesis and development of the horse testis. These findings will help us to further understand how gene expression is regulated during testis development and spermatogenesis. Future research to evaluate the influence of AS events on testis development and spermatogenesis-related genes, or studies that combine other experimental results, such as miRNA and piRNA expression patterns, will provide more fundamental information in understanding the regulatory mechanisms of equine testis development and spermatogenesis at the molecular level.This study was approved by and conducted according to the guidelines of the Institutional Animal Ethics Committee and Animal Care Guidelines of the Inner Mongolia Agricultural University. The animals did not unnecessarily suffer at any stage of this study.We received permission to geld six healthy male Mongolian horses in Xilingol League, Inner Mongolia, China. The ages of the horses were determined based on physical examination of their teeth, as well as information from the owner. Three were sexually immature colts , between 11 and 13 months old. The other three were sexually mature stallions, between 3 and 4 years old . We surgically collected the testes of all six horses. The excised testes were stored in cryogenic vials with RNA/DNA sample protector (TaKaRa). Small aliquots (~ 5 g) from the testes of each animal from the parenchyma, including the seminiferous tubules and Leydig cells from the middle area, were immediately frozen in liquid nitrogen for quantitative real-time polymerase chain reaction (qRT-PCR) analysis. All horses were reared by their owner after our study.Total RNA was extracted using TRIzol™ Reagent (Invitrogen) according to the manufacturer’s protocols and resuspended in nuclease-free water. Then, the samples were treated with DNase I (Invitrogen) to eliminate any contaminating genomic DNA. RNA degradation and contamination were monitored on 1% agarose gels. RNA purity was assessed using the NanoPhotometer® spectrophotometer (IMPLEN). RNA concentrations were measured using the Qubit® RNA Assay Kit with the Qubit® 2.0 Fluorometer (Life Technologies). RNA integrity was assessed using the RNA Nano 6000 Assay Kit with the Bioanalyzer 2100 system (Agilent Technologies).Six libraries were constructed. The three immature testes libraries were designated BSM1, BSM2, and BSM3; and the three mature testes libraries were designated ASM1, ASM2, and ASM3. Approximately 3 μg RNA per sample were used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB) according to the manufacturer’s recommendations, and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-conjugated magnetic beads. Fragmentation was conducted using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First-strand cDNA was synthesized using random hexamer primer and M-MuLV reverse transcriptase (RNaseH-). Second-strand cDNA synthesis was subsequently performed using DNA polymerase I and RNase H. In the reaction buffer, dNTPs with dTTP were replaced by dUTP. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activity. After adenylation of the 3′ ends of the DNA fragments, NEBNext Adaptors with hairpin loop structure were ligated for hybridization. To select cDNA fragments of preferentially 150~200 bp in length, the libraries were purified with the AMPure XP system (Beckman Coulter). Then 3 μL of USER Enzyme (NEB) were used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min, followed by 5 min at 95 °C before PCR. PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer. Finally, the products were purified (AMPure XP system), and the library quality was assessed using the Agilent Bioanalyzer 2100 system.Clustering of the index-coded samples was performed on a cBot Cluster Generation System using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and 150 bp paired-end reads were generated.In order to guarantee data quality in our analysis, a Perl script was used to filter the Raw Data. The steps were as follows: The clean data were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data.www.ensembl.org. The index of the reference genome was built using Bowtie v2.2.3, and paired-end clean reads were aligned to the reference genome using TopHat v2.0.12 [The reference genome and gene model annotation files were directly downloaded from the  v2.0.12 , 55. Top v2.0.12 –58.HTSeq v0.6.1 was used to count the read numbers mapped to each gene . Then, tP-values were adjusted using the Benjamin and Hochberg’s approach for controlling the false discovery rate. Genes with an adjusted P-value < 0.05 found by DESeq were assigned as differentially expressed [Differential expression analysis of two stages  was performed using the DESeq R package (1.18.0). DESeq provides statistical routines for determining differential expression within digital gene expression data using a model based on the negative binomial distribution. The resulting xpressed , 62.P-value < 0.05 were considered significantly enriched DEGs [http://www.genome.jp/kegg/) [GO enrichment analysis of DEGs was implemented by the GOseq R package, in which the gene length bias was corrected. GO terms with corrected hed DEGs . KEGG isp/kegg/) . We usedp/kegg/) .Putative transcription factors were analyzed using the animal transcription factor database AnimalTFDB 2.0 . For genhttp://rnaseq-mats.sourceforge.net/index.html). The number of AS events in each sample was estimated separately.The Cufflinks v2.1.1 Reference Annotation Based Transcript (RABT) assembly method was used to construct and identify both known and novel transcripts from TopHat alignment results. Alternative splicing events were classified by the software rMATS  according to the manufacturer’s protocols. About 0.5 μg of total RNA was used as template to synthesize first-strand cDNA using a miScript II RT Kit (QIAGEN) according to the manufacturer’s protocols. The resultant cDNA was diluted to 0.1 μg/μL for further analysis by the MX3000P Real-Time PCR System (Agilent Technologies) using SYBR® Premix Ex Taq™ II(TaKaRa). β-actin was chosen as an internal reference gene to eliminate sample-to-sample variations based on evidence of its role as a constitutive house-keeping gene for qRT-PCR and its use in normalizing changes in specific gene expressions . The relAdditional file 1: Table S1. List of data output quality.Additional file 2: Table S2. Alignment of Reads and reference genome.Additional file 3: Table S3. Classification and quantity statistics of AS events.Additional file 4: Table S4. The results of qRT-PCR validation.


An in silico analysis showed that the slc4a2b is evolutionarily conserved, and we found that loss of function of slc4a2b resulted in a decreased number of HCs in zebrafish neuromasts due to increased HC apoptosis. Taken together, we conclude that slc4a2b plays a critical role in the development of HCs in zebrafish.Hair cells (HCs) function as important sensory receptors that can detect movement in their immediate environment. HCs in the inner ear can sense acoustic signals, while in aquatic vertebrates HCs can also detect movements, vibrations, and pressure gradients in the surrounding water. Many genes are responsible for the development of HCs, and developmental defects in HCs can lead to hearing loss and other sensory dysfunctions. Here, we found that the  Therefore, identifying deafness genes is crucial for protecting HCs from injury and thus for preventing and treating hereditary hearing loss.Mechanosensory hair cells (HCs) in the inner ear play a crucial role in sensing auditory and vestibular signals in the environment, and HC damage is a leading cause of hearing loss. Congenital hearing loss occurs in approximately 1/500 infants, and genetic factors account for at least 50% of cases [Ion homeostasis is essential for maintaining cell survival and proper cell function, and many hearing disorders are caused by inner ear HC ion homeostatic disruption. Therefore, membrane-associated ion channels, transporters, and exchangers are required for HC function, and loss of function of these proteins induces HC loss and thus leads to hearing deficits.SLC44A4 [slc26a2 [3−/Cl− anion exchangers (AEs) belong to the SLC4 family and include three primary members, namely AE1 (or SLC4A1), AE2 (or SLC4A2), and AE3 (or SLC4A3), each of which can exchange HCO3− for Cl− across the plasma membrane and thus play an important role in maintaining the intracellular pH (pHi), Cl− concentrations, and cell volume [SLC4A2 gene appears to be more widely distributed than the other two, and it has been shown to be expressed in the inner and outer HCs and in the supporting cells of rat and guinea pig cochleae [SLC4A2 is required for proper osteoclast differentiation and function in mice [SLC4A2 are associated with osteopetrosis in Red Angus cattle [3−/Cl− exchange activity is essential for calpain-dependent regulation of the actin cytoskeleton in osteoclasts [SLC4A2 in hearing has not been explored.The solute carrier (SLC) group of membrane transport proteins consists of more than 60 families and over 400 members. Mutations of some SLC genes have been shown to lead to hearing impairments or HC defects, for example, human SLC44A4  and zebr[slc26a2 . The HCOcochleae  and in t in mice , and muts cattle . SLC4A2-eoclasts , and SLCeoclasts , spermateoclasts , gastriceoclasts , and immeoclasts , 10. HowSLC4A2 genes in mammals, namely slc4a2a  and slc4a2b  [slc4a2a mRNA can be detected in the prospective midbrain as early as the five-somite stage, then later in the pronephric primordia and in the developing pronephric duct, where it persists through 72 hpf (hours post fertilization), and it mediates both Na-independent electroneutral Cl−/Cl− exchange and Cl−/HCO3− exchange [slc4a2b gene is expressed in the otic vesicle and lateral line neuromasts in zebrafish. An in silico analysis showed that the slc4a2b gene is highly evolutionarily conserved, and we further show that loss of function of slc4a2b resulted in decreased numbers of posterior lateral line neuromasts, HC clusters, and HCs due to increased HC apoptosis. Taken together, our findings highlight a critical role for the slc4a2b gene in HC development, and thus slc4a2b might be a newly identified deafness-related gene.In zebrafish, there are two unlinked genes that are equally closely related to the d ae2.2) , 15. slcexchange . We demoslc4a2b gene in zebrafish, we first analyzed the evolutionary features of the gene. As shown in slc4a2b gene has significant amino acid sequence similarities to other species. Moreover, the phyloP8way conservation score of slc4a2b is greater than more than half of the zebrafish protein-coding genes  [slc4a2b-specific morpholino-injected larvae (slc4a2b morphants (To determine the function of the 3c:mGFP)  in whichorphants ’, 3I decorphants ’, 3I.eya1 gene is a marker of lateral line neuromasts in zebrafish [eya1-specific WISH experiment provided powerful evidence that slc4a2b gene knockdown led to reduced numbers of neuromasts in zebrafish ;slc4a2bmut) using CRISPR/Cas9 gene editing technology with Tg(Brn3c:mGFP) transgenic zebrafish. As shown in slc4a2b gene was synthesized in vitro. After co-injection of slc4a2b-specific sgRNA and Cas9 mRNA, the zebrafish were grown and the genomic DNA was extracted for genotyping. The mutations were observed at the target site within the genome ;slc4a2bmut zebrafish was decreased significantly compared to controls, which was similar to what was seen in the slc4a2b morphants  zebrafish embryos. Zebrafish injected with slc4a2b mRNA had significantly higher slc4a2b expression level ;slc4a2bmut zebrafish indicating that loss of function of slc4a2b leads to HC apoptosis.To determine if apoptosis is the primary mechanism through which Zebrafish is an excellent animal model owing to its optical transparency, high rate of reproduction, ectogenesis, and known genome sequence that has high homology with the human genome. Zebrafish also have many organs and systems that are similar to those of mammals and humans. For example, the zebrafish lateral line system, which consists of a number of neuromasts that enable the fish to perceive changes in its surroundings, consists of HCs, supporting cells and mantle cells. These HCs have similar morphologies and functions in the transmission of sensory information as mammalian HCs. The zebrafish lateral line neuromast is thus a very useful model to study the factors that affect HC development and function because this knowledge is likely to be applicable to our understanding of how dysfunction of inner ear HCs in mammals leads to hearing loss.Recent decades have seen the identification of numerous genes that are responsible for hearing loss, and it is likely that there are still many deafness-related genes waiting to be discovered. Identifying such genes is a key step in a better understanding of the mechanisms of hearing loss and will form a fundamental basis for precise gene therapy in the future.Ion homeostasis is essential for cell survival and function, and because HCs detect mechanical signals and transform them into electrical signals, ion homeostasis also plays an indispensable role in hearing. In HCs, some membrane-located transport proteins, for example, ion channels, transporters, and exchangers, play key roles, and dysfunction of these proteins leads to disruption of ion homeostasis and ultimately to cell death and subsequent hearing impairment.3− and Cl−, and it ensures that the pHi is maintained within a narrow range. The zebrafish slc4a2b gene is a member of the slc4 gene family, but its role in zebrafish development has not been reported until now.The SLC protein family is made up of important solute carriers, and these proteins play vital roles in maintaining the physiology and function of numerous cell types. SLC4A2 is a sodium-independent anion exchanger of HCOslc4a2b gene. The phyloP8way conservation score analysis and the WISH experiment showed that the slc4a2b gene is highly conserved and is mainly expressed in the caudal vein, otic vesicle, and neuromasts at different developmental stages. We next examined the role of slc4a2b in zebrafish lateral line neuromast HC development using morpholino-mediated gene knockdown and CRISPR/Cas9-mediated gene editing methods. Loss of function analysis showed that the zebrafish slc4a2b gene is required for HC development. Further, we showed that the phenotype induced by slc4a2b gene deficiency can be rescued by slc4a2b-specific mRNA injection, and this verified the role of the slc4a2b gene. Finally, we sought to explain how slc4a2b gene defects can lead to decreased HCs. Staining for the apoptosis marker cleaved caspase-3 showed that slc4a2b deficiency results in HC apoptosis in the posterior lateral line neuromast.First, we analyzed the evolutionary conservation and expression pattern of the zebrafish slc4a2b gene deficiency can induce HC apoptosis mediated by caspase-3 activation in zebrafish.Apoptosis is one of the main types of cell death and is characterized by chromatin condensation and DNA degradation, and it can be divided into an extrinsic (or death receptor) and an intrinsic  pathway , 20. Apo+-ATPases are known to cause hearing loss in human deafness patients with distal tubular acidosis [gcm2, a global pH regulator, or raising fish in altered pHs have both been shown to decrease hair cell number and reduce hair cell function [Intracellular pH has a critical role in the maintenance of normal cell function and cell survival. pH regulation is important for hearing as mutation in Hacidosis . And it function , 50.slc4a2b gene is required for HC development in zebrafish and that slc4a2b gene deficiency leads to increased HC apoptosis. Given that SLC4A2 is involved in pHi regulation and that Slc4a2 deficiency alters pHi homeostasis in mice [slc4a2b gene defect is due to changes in intracellular physiological parameters such as pHi, but this needs to be confirmed in future studies.Taken together, these results indicate that the  in mice , 51, 52  in mice , 54, we SLC4A2 gene, the homologue of the zebrafish slc4a2b gene, is a potential deafness gene, and this should be confirmed in clinical studies.Zebrafish share approximately 70% of their genes with humans, and more than 80% of known human disease-related genes have a zebrafish counterpart . Based oTg(Brn3c:mGFP). In the latter, membrane-localized green fluorescent protein (GFP) is expressed specifically in the HCs [Two zebrafish lines were used in this study, the wild-type AB line and the transgenic line  the HCs . All of http://hgdownload.soe.ucsc.edu/goldenPath/danRer7/phyloP8way/vertebrate.phyloP8way.bw), where the 8 species used for the conservation score calculation are Homo sapiens (hg19), Mus musculus (mm9), Xenopus tropicalis (xenTro2), Tetraodon nigroviridis (tetNig2), Takifugu rubripes (fr2), Gasterosteus aculeatus (gasAcu1), Oryzias latipes (oryLat2), and Danio rerio (danRer7). The corresponding gene annotation gtf file was downloaded from Ensembl (ftp://ftp.ensembl.org/pub/release-74/gtf/danio_rerio/Danio_rerio.Zv9.74.gtf.gz). The gtf annotation file was converted into bed format using a homemade python script. The bigWigAverageOverBed software from the UCSC browser was used to calculate the average phyloP8way conservation score for each protein-coding gene (only considering the coding sequence regions). The phyloP8way conservation score density plot was drawn using an R script.The phyloP8way conservation score file for zebrafish (genome reference version: danRer7) was downloaded from the UCSC Browser (in situ hybridization (WISH) was performed according to standard procedures. First, a part of coding sequence of slc4a2b gene, amplified from zebrafish cDNA using the primers (5’-CAG CAT GGA TGA AGT GAC GG-3’ and 5’-CAG AAC CCT TGA CCA GCA TG-3’), was subcloned into the pGEM-T Easy vector, and then a gene-specific digoxigenin-labeled RNA probe was transcribed in vitro using the DIG RNA Labeling Kit (SP6&.T7)  following the manufacturer’s instructions. The pre-fixed embryos were incubated with the probe overnight at 4°C. Second, an alkaline phosphatase (AP)-conjugated antibody against digoxigenin  was used to detect the digoxigenin-labeled RNA probe. Finally, the AP-substrate NBT/BCIP solution  was added to the reaction system, and the development of color allowed the expression of the gene of interest to be visualized.The whole-mount slc4a2b gene-specific morpholino was 5’-TCA GGA CAC TGT GAA CCC GCT GAA C-3’, and it was obtained from Gene Tools, LLC. In this study, 2–3 nL of 0.3 mM morpholino oligo was microinjected into the embryos at the 1-cell stage. The morpholino blocks the translation of the Slc4a2b protein when the oligo binds to the slc4a2b mRNA.The oligo sequence of the slc4a2b gene mutant zebrafish, as described in our previous work [slc4a2b gene, and Cas9 mRNA was microinjected into each zebrafish embryo at the 1–2 cell stage. For the sgRNA synthesis, a forward primer (5’- TAA TAC GAC TCA CTA TAA GAC CAA CTC ACC AAC GCT GGT TTT AGA GCT AGA AAT AGC-3’), which contained a T7 promoter region and a slc4a2b gene-targeting region, and a universal reverse primer (5’-AAA AAA AGC ACC GAC TCG GTG CCA C-3’) were used in the PCR reaction with pT7 plasmid . For the Cas9 mRNA synthesis, a linearized pXT7-Cas9 plasmid was used as the template for transcription into Cas9 mRNA in vitro using the mMESSAGE mMACHINE® Kit .To generate the ous work , 2–3 nL  plasmid  as the tslc4a2b mRNA was co-injected into the embryos with the slc4a2b sgRNA. The mRNA was transcribed in vitro using the mMESSAGE mMACHINE® Kit . Briefly, approximately 3700 bp DNA fragments were synthesized by PCR using the primers slc4a2b-mRNA-EcoRI-F (5’-CGG AAT TCC GTG AGG TTA TGC TGC CCG TAA-3’) and slc4a2b-mRNA- XbaI-R (5’-GCT CTA GAG CTG ATA GCA GCT CAA ACG CTC-3’). The DNA fragments were subcloned into the pCS2+ vector, and the recombinant plasmid was linearized using the restriction endonuclease Not I and then transcribed into mRNA in vitro.In the rescue experiment, 2–3 nL of 50 ng/μL The vital dye FM1-43FX  was used to specifically label functional HCs in the neuromasts. First, the live zebrafish were anesthetized using MS-222 and then immersed into the labeling solution for 45 s at room temperature in the dark. Afterwards, the fish were rinsed three times using the egg water and then fixed for 2 hours at room temperature in 4% paraformaldehyde. After washing three times with PBS-T (0.1% Triton X-100 in PBS), 4',6-diamidino-2-phenylindole (DAPI) was added to label the nuclei.For immunofluorescence, the fish were anesthetized and then fixed using 4% paraformaldehyde. After washing three times with PBS-T, the fish were incubated in the antigen retrieval solution  for 15 min at 98°C. Nonspecific binding was then blocked with 10% donkey serum in PBS-T. Next, specific primary antibodies against GFP  and cleaved caspase-3  were added, and secondary antibodies were used to detect the primary antibodies.P < 0.05 was considered statistically significant. All data are presented as means with SEM, and all experiments were repeated at least three times.All data were analyzed using GraphPad Prism 8.0.2. Two-tailed, unpaired Student’s t-tests were used to determine statistical significance when comparing two groups. A value of All animal procedures were performed according to protocols approved by the Animal Care and Use Committee of Southeast University and Nantong University and were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize the number of animals used and to prevent their suffering.Supplementary Figures


RNA editing is a widespread post-transcriptional mechanism to introduce single nucleotide changes to RNA in human cancers. Here, we characterized the global RNA editing profiles of 373 hepatocellular carcinoma (HCC) and 50 adjacent normal liver samples from The Cancer Genome Atlas (TCGA) and revealed that most editing events tend to occur in minor percentage of samples with moderate editing degrees (20–30%). Moreover, these RNA editing prefer to be A-to-I RNA editing in protein coding genes, especially in 3′UTR regions. Considering the association between DNA mutation and RNA editing, our analysis found that RNA editing maybe a complementary event for DNA mutation of HCC risk genes in HCC patients. We next identified 454 HCC-related editing sites, and many locate on the same genes with the same editing patterns. The functional consequences of editing revealed 2,086 functional editing sites and demonstrated that most editing in coding regions are non-synonymous variations. Furthermore, our results showed that editing in the 3′UTR regions tend to influence miRNA–target binding, and the editing degree seems to be negatively correlated with gene expression. Finally, we found that 46 HCC-related editing sites with consequence are able to distinguish the prognosis differences of HCC patients, suggesting their clinical relevance. Together, our results highlight RNA editing as a valuable molecular resource for investigating HCC mechanisms and clinical treatments. Hepatocellular carcinoma (HCC) is a kind of malignant tumor with high mortality. It ranks third among all cancer-related mortality in the world. About 3% of the patients with cirrhosis can result in HCC, which is the most serious complication in chronic liver diseases . The higRNA editing is an important post-transcriptional modification event, which change genetic information at RNA level and generate results similar to DNA mutations, thus increasing the diversity of transcripts and proteomes . The mosRNA editing in normal cells are associated with adaptive evolution and cell development . ConversRecently, the incidence and progression of HCC were found associated with RNA editing events and may further help us reveal the pathogenic mechanisms underlying HCC. Chen et al. suggested that the hyper-editing event of AZIN1 results in a serine-to-glycine substitution at residue 367 of the AZIN1 protein, which may be a potential driver in the pathogenesis of HCC . Chan etRecently, The Cancer Genome Atlas (TCGA) project provides a large number of omics data of malignant tumors. Han et al. and Paz-Yaacov et al. identified RNA editing sites in multiple cancer types from TCGA, including HCC , 21. Howde novo identification of HCC-related RNA editing sites by integrating multiple omics data with bioinformatics methods, including genomic mutation, transcriptomic variation, and reference single nucleotide polymorphism (SNP) information, using 373 HCC tumor samples and 50 adjacent normals from TCGA. Here, the genomic distribution pattern of RNA editing events was described. We also deeply analyzed the influence of HCC-related RNA editing on downstream regulation system, including the effect on protein translation and miRNA regulation. Based on clinical information of HCC patients, our study further identified new biomarkers for clinical prognosis, which will promote the disclosure of molecular mechanisms of HCC from the perspective view of RNA editing.In our study, we intend to https://portal.gdc.cancer.gov/) research project   originally from The Cancer Genome Atlas  , includiAB-4052)  and the AB-4052) .http://genome.ucsc.edu/cgi-bin/hgTables) (http://software.broadinstitute.org/gsea/msigdb)  . In addi/msigdb) .We obtained the list of known tumor suppressive genes (TSGs) and oncogenic genes (OGs) from a previous study , which ivcf files were applied through five steps: (i) removing DNA mutation sites for each HCC sample; (ii) taking out all known SNPs in dbSNP version 137 or the 1000 Genome Project, and also insertion or deletion sites; (iii) further filtering sites to obtain editing sites with high confidence: if Fisher Strand (FS) >20, or Quality by Depth (QD) <2, or editing was supported <2 reads, or total coverage reads <10, these sites were removed, and we required at least 10% difference between the editing degrees of 90% quantile and 10% quantile across all samples; (iv) sites with 100% editing degree were also filtered, as 100% editing efficiency is thought to be unrealistic  keegtf files. If the RNA editing sites were simultaneously mapped to two strands, these sites were further removed. At last, we get 19,431 RNA editing sites for further analysis.Finally, we restricted editing sites to 46 human chromosomes. To get the exact RNA, which was edited, we used BEDtools to map the editing sites with gene annotation bowtie, processed by GATK4 with default parameters, with no matched DNA mutation datasets. Other pipelines were similar to the aforementioned method.The validated RNA-seq FASTQ datasets were aligned to the human reference genome (GRCh38) by p < 0.05), with HCC gain editing sites restricted to be no more than 5% editing sites in normal samples and HCC loss editing sites restricted to be no more than 5% editing sites in cancer samples. Dys-edit sites were just focused on the editing degree of 50 HCC cancer samples and mapped 50 normal samples, which were determined by two steps: (i) using the paired Student's t-test ; (ii) restricting editing sites to those having more than 0.25 editing degree change in at least two pairs of normal and cancer samples. The HCC dys-edit sites identified in the above two steps were changed in the same direction (simultaneously hyper-edited or hypo-edited in two steps). Finally, we identified 454 HCC-related RNA editing sites.HCC-related RNA editing sites include HCC gain, HCC loss, and significant dysregulated editing (dys-edit) sites. HCC gain or HCC loss editing sites were defined by Fisher's exact test . We focused on chemical and genetic perturbations (CGP), reactome pathways and biological processes (BP) of gene ontology (GO) originating from MsigDB.First, HCC-related RNA editing sites were mapped to gene name by BEDtools. Then, we performed functional enrichment analysis by hypergeometric test . The normalized gene expression levels of raw read counts were calculated using DESeq2 ; conversely, a minus sign indicated that an increased editing degree is associated with a decreased risk of survival (protective editing). More specifically, for editing sites, which are located in a gene region, we assigned a risk score to each HCC patient according to a combination of the product of the editing degree and gene expression, weighted by 1 or −1 according to the regression coefficients from the univariate Cox regression analysis mentioned above. The risk score for each patient was calculated as follows:i is 1 or −1 when the Cox regression coefficient of the editing site i is a positive or negative value, respectively. The n is the number of HCC-related RNA editing sites with consequence that are located in gene regions with an editing degree more than 0 in each sample. Expgene(i) is the expression level of gene in which the editing site i occurs. Editi represents the editing degree of the editing site i.where, βTo investigate whether risk scores are associated with tumor grades and tumor stages, we use the Wilcoxon rank-sum test. As the sample number with a tumor grade 4 or tumor stage 4 is too small, only 12 and 13, respectively, we only considered grade 1, grade 2, and grade 3 plus (including grade 3 and grade 4) for tumor grades and stage 1, stage 2, and stage 3 plus (including stage 3 and stage 4) for tumor stages. All patients were then classified into high-risk and low-risk groups using the median risk score as the cutoff point. The Kaplan–Meier method was further used to estimate the differences in overall survival time for these two patient groups (log-rank test). Patients having higher risk scores were expected to have poor survival outcomes.p < 0.05). The risk scores were calculated according to the mathematical formula above. For HBV/HCV infected patients or non-alcoholic fatty liver patients, we did a similar analysis mentioned above to obtain a risk score for each sample using three prognostic-related editing sites.We further focused on three prognostic-related editing sites whose editing degrees are significantly correlated with survival , grade, and stage of HCC patients, along with editing risk score.de novo methods for detecting RNA editing were developed recently    . Meanwhion test) . The obsp = 1.15e-05). Notably, ACOX1 was reported to play important roles in cancer development of HCC by stimulating hepatic fatty acid oxidation and H2O2 accumulation  especially for HCC gain and dys-edited sites. Interestingly, 73 genes were shared by at least two HCC-related RNA editing sites and accounted for 50% (202 editing sites of 404) . NotablyPrevious studies have suggested that RNA editing can be functional and involved in carcinoma initiation and progression by influencing amino acid encoding, alternative splicing, miRNA–target regulation, or expression changing of the corresponding gene , 39; Fig. Totallyp = 0.0003; p = 0.01) in the Cox multivariate analysis, after adjusting for patients' gender, age, BMI, tumor grades, and stages . As expected, we found that the risk scores measured by 46 HCC-related RNA editing sites with functional consequence correlated with tumor grades and stages in HCC patients, where higher risk scores in patients implicated higher tumor malignancy  . We furtd stages , which ip < 0.05; p = 0.03, data not shown). After adjusting for patients' gender, age, BMI, tumor grades, and stages, the risk score still has predictive power   , but notFurthermore, we dissected the function of these 46 HCC-related RNA editing sites with functional consequence. It is shown that they were widely involved in three reactome pathways: “metabolism of proteins,” “generic transcription pathway,” and “cell cycle” . The funHCC is a complex disease with poor prognosis and affected by multiple genetic alterations. DNA mutations are the most widely investigated driver events in all cancer types, including HCC. However, the mutation events are not presented in all HCC patients. Furthermore, we found that 98.51% mutation sites occurred in only a single HCC patient. Similar to the consequence of DNA mutation, RNA editing can also produce nucleotide variations at the RNA level. Importantly, the RNA editing seems to be a more sophisticated regulation, as the editing degree can be 0–100%, while DNA mutation should be mutation or not (0 or 1). Here we systematically identified RNA editing sites by integrating DNA mutation and SNP datasets, and found that HCC samples have a significantly high percentage of RNA editing sites compared with normal samples. We also observed RNA editing in 35 cases that lacked DNA mutations in HCC risk genes, and more edited risk genes occurred in mutated patients compared with patients who do not have any DNA mutation in the HCC risk gene. Thus, we proposed that RNA editing may be a risk factor to HCC, as a complementary event for DNA mutation in HCC risk genes.To further characterize the inferred RNA editing sites in our study, we compared the overlap of A-to-IRNA editing sites in gene regions identified by our study and the editing sites collected in the database of RADAR . We founPrevious studies focused on the effect of ADAR enzymes on RNA editing. Here we addressed another important question on whether these editing sites have an effect on the downstream regulation system. By considering the influence of RNA editing on coding sequence change, alternative splicing, miRNA–target regulation, and expression change, we identified 2,064 editing sites with functional consequence, which accounts for 10.62% of the total RNA editing sites . This percentage indicates that RNA editing events may play other important roles, such as the alteration of RNA-binding abilities, which were induced by RNA structure change, which was, in turn, induced by editing. Notably, we found a number of editing sites with miRNA–target regulation changes. More were identified when we set lower thresholds . If we dImportantly, we found 46 HCC-related RNA editing sites with functional consequence that can be used to predict the clinical outcome in HCC patients. In addition, they have independent predictive power after considering the gender, age, BMI, tumor grades, and stages. We identified three clinical prognostic-related editing sites, which can also provide predictive values in HBV-/HCV-infected patients and non-alcoholic fatty liver disease patients, including editing sites in ALDH2 , CNTNAP3B , and CBWD5 , of which ALDH2 is an HCC-related editing gene with high frequency (six HCC loss editing sites locates on the ALDH2 gene region). Interestingly, multiple studies revealed that ALDH2 is highly correlated with the pathogenic mechanism, risk, and survival of liver cancer patients, including HCC –50. Funchttps://portal.gdc.cancer.gov/) and ArrayExpress .The datasets analyzed in this study can be found in the database of Genotypes and Phenotypes (dbGaP) originated from The Cancer Genome Atlas (TCGA, ZB and JL conceived the study. ZB and JC processed the sequencing data. LW and FW analyzed the data. JC wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


ADAR and ADARb1). Deamination by ADAR and ADARb1 enzymes induces conformational changes in the 5-HT2CR that decrease the G-protein-coupling activity, agonist affinity, and thus serotonin signaling. Significantly, a single intra-CeA administration of a 5-HT2CR pharmacological antagonist produced a robust alleviation of PTSD-like behaviors (that was maintained for three weeks) as well as single systemic treatment. This work may suggest the way to a new avenue in the understanding of PTSD regulation.Post-traumatic-stress-disorder (PTSD) is a stress-related condition that may develop after exposure to a severe trauma-event. One of the core brain areas that is considered to be a key regulatory region of PTSD is the amygdala. Specifically, the central amygdala (CeA) is involved in emotion processing and associative fear learning memory, two main circuits involved in PTSD. Long term dysregulation of trauma-related emotional processing may be caused by neuroadaptations that affect gene expression. The adenosine-(A) to-inosine (I) RNA editing machinery is a post-transcriptional process that converts a genomic encoded A to I and is critical for normal brain function and development. Such editing has the potential to increase the transcriptome diversity, and disruption of this process has been linked to various central nervous system disorders. Here, we employed a unique animal model to examine the possibility that the RNA editing machinery is involved in PTSD. Detection of RNA editing specifically in the CeA revealed changes in the editing pattern of the 5-HT2C serotonin receptor (5-HT2CR) transcript accompanied by dynamic changes in the expression levels of the ADAR family enzymes ( Currently, PTSD is defined by the coexistence of four symptoms that may be evoked initially by the traumatic event itself2, but then may increase over time, in response to stress-associated cues, despite the absence of further exposure to stress3. Although the exact neuronal mechanism that underlies PTSD is yet to be discovered, a wealth of data concerning the biological circuits involved in fear and anxiety implicate the amygdala as a region that is central to these behaviors4. The amygdala has been shown to participate in the acquisition of conditioned fear paradigms in animal studies6 and in combat veterans, if this region is damaged, the development of PTSD is neutralized5.Post-traumatic stress disorder (PTSD) is a trauma- and stress-related disorder that may develop in survivors of a life-threatening traumatic event and can cause intense fear and a feeling of helplessness8. A number of animal studies involving fear learning reported that damage to the CeA causes a deficit in conditioned orientating and disrupts the fearful associative learning process9. These results have implicated the CeA as an important mediator in the physiological and behavioral expression of conditioned fear5. In particular, the serotonergic system that projects to the amygdala has been linked to PTSD and emotional regulation10 and has been implicated in the pathophysiology of mood and anxiety disorders12.Whereas the amygdala has a number of nuclei with diverse activities, the central nucleus of the amygdala (CeA) deserves special attention due to its role in mediating the response to negative states associated with stress14. Although neither a single gene nor a single signaling pathway region may entirely account for the development of a complex disease, low serotonergic activity and brain regional abnormalities in serotonin neurotransmission have been proposed as biological traits related to depression and suicidal behavior15. Among the serotonin receptor family, the serotonin 2c receptor (5-HT2CR) is of interest. Recent studies examining the effect of 5-HTR2C agonists showed that the CeA is highly sensitive to their effect on dopaminergic release, emphasizing the importance of this receptor located in the CeA in different neuropsychiatric diseases17. On the other hand, 5-HTR2C was suggested to play an important role in preventing repeated restraint stress in the amygdala18 and has been associated with regulation of mood, appetite, sleep, and sexual behavior20. Altered activity of the 5-HTR2C, a G-coupled protein receptor, has been reported in a variety of neuropsychiatric disorders21. For example, viral-overexpression of this receptor in the amygdala resulted in an anxiogenic effect22, which could be counteracted, at least in the short term, by the injection of a pharmacological 5-HT2CR antagonist directly into the amygdala23.Moreover, the CeA is known to be involved in an innate- and learned-fear as well as in the regulation of freezing behavior-responses3. Interestingly, the 5-HT2CR is a target of post-transcriptional adenosine-to-inosine (A-to-I) RNA editing25 carried-out by the adenosine deaminases acting on RNA enzymes (ADARs) family. The editing modifies an A to an I, leading to alterations in the amino acid sequence, and can generate a diversity of proteins that are different from those encoded in the genomic-DNA. It was previously shown to alter the 5-HT2CR affinity to its ligand through this machinery29. Nonetheless, the cellular and molecular targets in the CeA underlying the specific effect of RNA editing of the 5-HTR2C are still inconclusive30.However, the long term effects of exposure to a severe traumatic event, even in the absence of further stress, have not been much studied31, and the neuroadaptation dynamic of learning and memory may be responsible for the distressing memories of an emotionally traumatic event, we hypothesize that the regulation of gene expression in the CeA may contribute to the enduring plasticity of PTSD32. If 5-HT2CR- RNA editing in the CeA indeed plays a causal role in PTSD-like behavior, then targeting the dynamic changes in this pathway could be expected to influence the PTSD-like behavioral phenotype. A variety of experimental protocols have been designed to follow long-lasting responses to fear in rodents over periods of 24 h to 7 days post a single traumatic exposure39. Nonetheless, the vast majority of these protocol studies do not monitor the behavioral manifestation longer than the initial traumatic event41.Since PTSD is characterized as a memory disorder43, and in addition, provides the opportunity to examine the effects of the action of the RNA editing machinery on the 5-HT2CR longitudinally to exposure to a traumatic event. Specifically, we aim to test whether the CeA-ADAR enzymes are involved in PTSD-like susceptibility and resilience behaviors and to track any consequent changes in the A-to-I RNA editing patterns of the 5-HT2CR. Detection of such changes may reveal a role for the RNA editing machinery in PTSD-like susceptibility to a trauma-related memory.In the current study, we used an established animal model for the study of PTSD, which mimics the clinical expression of PTSD, including anxiety, social avoidance, and hyperarousal behaviorad libitum. All experiments were performed between 07:00 and 17:00, in daylight. All animal procedures were approved by the Bar-Ilan University Animal Care Committee and were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals.Adult male Sprague–Dawley rats  were housed under conditions of constant temperature (22 °C) and 50% humidity, with a 12-h light–12-h dark cycle. The rats were allowed to habituate to the animal house for one week before beginning the experiments. Rats were housed three per cage, where two of the animals were experimental rats, and the third was a companion rat. The same three rats remained together until the end of the study. Food and water were provided 42, consists of several stages spread over 8 weeks , to define the range for each basal behavioral parameter within the population. The upper and lower levels of this range were considered the boundaries of the ‘normal’ baseline. Deviations from this range were used to define PTSD-like behavior retrospectively (after the conclusion of the second reminder testing). According to the results, animals were categorized either as ‘susceptible’  or ‘resilient’ (exhibiting at least one behavior within baseline range)Brains were removed immediately after the second reminder and were placed in a perspex brain matrix and sliced into 1.0 mm segments. The CeA was punched using a 13G-14G microdissecting needle and was frozen at −70 °C until the RNA was extracted using the Total RNA Purification Micro Kit  according to the manufacturer’s instructions. Extracted RNA (2 µg) was treated with DNase and reverse-transcribed to generate cDNA .http://frodo.wi.mit.edu) and were tested for specificity and sensitivity by PCR before they were included in the primers set. Quantification of multiple RNA editing sites was followed by next-generation sequencing . The sequencing adaptors and tag barcodes that were attached to each PCR product (amplicon) were used to identify each sample by the sequence in the Fluidigm access-array library, prepared as described in the We used the UCSC genome browser Rat. 2004 (Baylor 3.4/rn4) assembly to identify any discrepancies between the RefSeq RNA data and the RNA sequencing output. For our focused screen, we employed a targeted-sequencing variation of next-generation sequencing (NGS) to generate and sequence multiple PCR amplicons from pre-determined genes, which contained the target editing site/s. The data obtained were screened for any A/G mismatches within the cDNA sequences. The signal strength of such mismatches was summed and scored according to the overall coverage as manifested by the output number of reads, and more importantly, by the percentage of A-to-G levels . The selective 5-HT2CR antagonist RS-102221  was injected  through the cannula 15 min before the third reminder , susceptible and resilient animals were anesthetized by intraperitoneal administration of ketamine hydrochloride (100 kg/mg) and xylazine (10 mg/kg). Each experimental group was randomly divided into two sub-groups, then the animals in each group were implanted bilaterally with a guide cannula (30 gauge) placed 1 mm above the CeA, sealed with a cannula dummy (Plastics One), and secured to the skull with screws and dental acrylic cement. The coordinates of the cannula, relative to BregmaCannula placement was verified by histological examinations of brain sections stained with propidium iodide (PI)  exposed to ‘trauma’ and three subsequent trauma-associated reminders  and susceptible (n = 25) animals42. After experiencing the traumatic event and the related reminders, susceptible animals showed an increase in freezing behavior over time in all three behavioral tests, while the resilient group did not. Analysis of all baseline behavioral samples revealed that the upper level for excluding outliers (95% confidence) in the exploration, social interaction, and hyperarousal conditions was twice the interquartile range. Animals in the susceptible and resilient groups showed a high within-group correlation , we discarded all the measurements with the cutoff of read coverage < 700 and A-to-I editing levels < 5%. Hence, only editing sites above this threshold and with a detectable signal in at least 5 animals in each group were selected for subsequent statistical analysis. The ten sites out of 48 that passed these criteria are located in the 5-HT2CR , Glutamate Ionotropic Receptor Kainate Type Subunit 2 , Calcium Voltage-Gated Channel Subunit Alpha1 D (CACNA1D- Chr16: 6068246 and 6068254), and Component of Oligomeric Golgi Complex 3 (Cog3- Chr15: 61477446).The CeA of animals retrospectively categorized as susceptible or resilient was punched immediately after the second trauma reminder (day35), and samples were subjected to the A-to-I RNA editing detection assay. Based on the results of Pinto and colleaguesp > 0.05, Fig. 47. The sites are located in the second intracellular loop of the G-protein coupled receptor and may, therefore, modulate the serotonin neurotransmission signaling cascade48. The edited isoforms were shown to have a robust reduction of the agonist-stimulated G-coupled protein compared to the non-edited form of the receptor49. Moreover, RNA editing also led to a loss of the active state of this receptor25 and a delay in agonist-stimulated calcium release in the fully edited isoforms27. The ability to regulate the RNA-editing of these five sites can, therefore, be expected to generate high diversity by generating up to 32 different mRNA transcripts that may then encode as many as 24 different protein isoforms that vary in their biochemical properties.Statistical analysis did not reveal any significant differences in the CeA-edited sites of susceptible compared to resilient animals  in the partially edited VNV isoform in the resilient group compared to the susceptible group   in the CeA did not uncover any significant site-specific differences between the susceptible and resilient animals Fig. . HoweverADAR family that regulate the RNA editing process  and the social interaction test demonstrated a highly correlative trend with the ADARb1 protein expression (r = −0.6 and p = 0.07).We next tested whether the observed higher frequency of the VNV isoform was associated with increased expression of the enzymes of the ess Fig. : ADARb1,54. Like editing, the antagonist might be expected to inhibit the activity of the receptor. Interestingly, treatment with RS-102221 significantly attenuated the freezing behavior in susceptible animals in all three behavioral scenarios compared with untreated susceptible controls  the RS-102221, a specific antagonist to the 5-HT2CR that was previously tested in a variety of mood disorders such as depression55. Behavior was measured immediately after the third reminder. The results indicated that RS-102221 treatment significantly attenuated freezing behaviors in susceptible animals compared to treated vehicle controls, immediately after the third reminder in all three behavioral tests , 30 min before the third trauma reminderADAR and ADARb1. Since no changes in the expression levels of 5-HT2CR transcript were shown, we believe that the behavioral profile of the resilient group, at least partly, stems from altering its editing. To emphasize the role of the observed changes, showing that high editing levels of the VNV isoform lead to resilience, we evaluated the behavioral response after an injection of a specific 5-HT2CR antagonist, RS-102221, directly into the CeA. The results showed that a single injection significantly attenuated the freezing response in all three behavioral situations tested. Interestingly, the relief of the stress response of susceptible animals was maintained for the long-term, when a following reminder of the trauma was tested. This treatment did not alter the behavior of the resilient group, suggesting that the effect was specific for susceptible animals, with no negative effects on animals that did not exhibit PTSD-like behavior.In the present study, we revealed that animals that were resistant to a traumatic event, compared with susceptible animals, had a significantly higher abundance of the VNV RNA edited isoform of the 5-HT2CR in the CeA. This alteration was associated with higher expression in protein levels of Moreover, systemic injection of RS-102221 attenuated the susceptible behavior profile in all the three examined tests, suggesting a translational approach to our results.17 and other studies showing that transgenic mice having only the fully edited VGV isoform of 5-HT2CR, which thereby overexpress the receptor in the brain, displayed greater fear expression, extensive fear extinction deficits, and fear generalization56. It is possible that editing of 5-HT2CR in different brain sites or different cell types might result in different apparent behavior. In our study, we focused on the amygdala. Nonetheless, tight cross-talk is found between this region and other related brain regions, such as the BNST41, parahippocampal gyrus, orbitofrontal cortex, sensorimotor cortex, the thalamus, and the anterior cingulate cortex43, that may differently respond to trauma. Modulating the 5-HT2CR activity in these regions exerts different downstream signaling pathways such as the brain-derived neurotrophic factor (BDNF)–tyrosine kinase B (TrkB) pathway, the glutamatergic N-methyl-D-aspartate (NMDA) receptor pathway, and the renin-angiotensin system pathway. These signaling pathways may have different effect PTSD-like display57.Importantly, our findings are not in accordance with studies that associate 5-HT2CR agonists with greater fear. Specifically, the effect of Lorcaserin and WAY-163909 on dopaminergic releaseAlternatively, it might be that the fully edited isoform functions differently than the partially edited one. Intriguingly, these data may suggest that the heritability of an editing setup may express a variety of functional intensities of the 5-HT2CR that are important to cope with stress. Thereby, dominance of edited VGV may be found in people that are more prone to develop PTSD when exposed to a trauma event. This should be verified in future studies.ADAR enzymes is not the only participant in anxiety-related disorders. Simmones and Karanovic have previously identified a role for ADARb1 and 5-HTR2C in the pathophysiology of major depressive suicide victims, thereby linking genetic and epigenetic factors to an elevated risk of suicide59.It is noteworthy that the 5-HT2CR regulated by 60. In this context, an increase in the VNV compared with the INI isoform, in the CeA, can cause a loss of 5-HT2CR activity by reducing the ligand efficacy of the G-coupled protein downstream. For this reason, 5-HT2CR antagonists are often used as pharmacological treatments for generalized anxiety disorders62 and were previously examined in a rat model for depression54 .The PTSD-like animal model used in this study generated a long-lasting susceptibility phenotypic behavior that was accompanied by the downregulation of ADAR enzymes combined with a decrease in the level of the VNV isoform of the 5-HT2CR. Our results demonstrate that blocking the 5-HT2CR by injecting RS-102221 antagonist into the CeA significantly attenuated the PTSD-like behavior of susceptible animals, supporting the hypothesis that the serotonin neurotransmission, via 5-HT2CR, plays a causal role in inducing anxiety-like and susceptibility behavior. Our observations are consistent with previous reports that highlighted the relationship between RNA editing of the 5-HT2CR and neuropsychiatric disorders, specifically with impaired serotonergic tone in PTSD. These studies showed that desensitization of the 5-HT2CR in serotonin transporter (SERT) knockout mice reduced the anxiety phenotype64. Further studies examining the specific cell type expressing the 5-HT2CR and other targets of ADARs may provide a deeper understanding of the mechanistic basis of PTSD. The present study explored other targets of ADARs, CACNA1D, and Grik2, but not Grik1.Taken together, our results support the suggestion of a causal role for the CeA as a critical brain region for the expression of PTSD-like behaviors and reveal the importance of specific 5-HT2CR -RNA editing as one molecule out of a wide range of targets with the potential to be edited. The specific cell type expressing the RNA editing changes of the 5-HT2CR was not examined. Therefore, a possible explanation for our results may suggest that the RNA editing changes observed in the resilience group may involve glutamatergic neurotransmission. As was previously reported, RS-102221 may target the 5-HT2C receptors present on glutamatergic cells, particularly of the CeA. Moreover, it is plausible that receptors other than 5-HT2CR could be altered by changes in ADARs’ expression and may participate in resilient behavior. These receptors may include glutamate receptors. This was previously reported in the study by Brande-Eilat and colleagues, showing that acquisition of conditioned freezing was associated with changes in expression levels of ADARs followed by RNA editing of glutamate ionotropic receptor kainate type subunit 1 (Grik1)65, that together provide a novel opportunity to converge on the role of RNA editing mechanism in the context of stress-related disorders. Findings from such models demonstrate the value of an unbiased, broad screening analysis of the RNA editing mechanism in different gene networks that may be reprogrammed in psychiatric diseases, by identifying a differential outcome in animals with PTSD-like susceptibility behavior compared to resilience. Our approach was to isolate candidate therapeutic targets by screening a wide variety of sites that could be involved in RNA editing and PTSD and examining the specific behavioral characteristics of each subject. Taken together, our results suggest a causal role for the 5-HT2CR and RNA editing as regulated by the ADAR enzymes in the CeA, for at least partly the expression of PTSD-like behavior. The relief produced by an antagonistic injection for this receptor in both the short and long term and when administered systemically after the traumatic event and its related reminders, suggests that this direction could have future therapeutic potential for PTSD patients.In Conclusion, the current study introduces a new approach to our understanding of PTSD. We took advantage of a unique PTSD-induced animal model, in comparison with genetic-modelssupplementary materials and methods


Coronavirus disease 2019 (COVID-19) is an unprecedented global crisis caused by a new strain of coronavirus called SARS-Cov 2 (Severe Acute Respiratory Coronavirus 2). It was first identified in the city of Wuhan, capital of Hubei Province in China and has rapidly spread across the world. On March 11, 2020 the disease was recognized as pandemic by the World Health Organization (WHO) . So far,In order to assess the implication of African countries in clinical trials it was imperative to conduct this study. We researched as of May 5, 2020 the ClinicalTrials.gov website to identify registered clinical trials on Coronavirus 2019 . We´ve uAlthough the pandemic started late in Africa, the current number of cases exceeds 30,000, representing almost half of the total cases detected in East and South Asia to date . The expThe authors declare no competing interests.