HB9e438::Venus, and proceeded to the genome-wide transcriptome analysis by RNA sequences. The results revealed the involvement of the pathology associated with synapses, epigenetics, and endoplasmic reticulum (ER) in SBMA. Notably, we demonstrated the involvement of the neuromuscular synapse via significant upregulation of Synaptotagmin, R-Spondin2 (RSPO2), and WNT ligands in motor neurons derived from SBMA patients, which are known to be associated with neuromuscular junction (NMJ) formation and acetylcholine receptor (AChR) clustering. These aberrant gene expression in neuromuscular synapses might represent a novel therapeutic target for SBMA.Spinal bulbar muscular atrophy (SBMA) is an adult-onset, slowly progressive motor neuron disease caused by abnormal CAG repeat expansion in the androgen receptor (AR) gene. Although ligand (testosterone)-dependent mutant AR aggregation has been shown to play important roles in motor neuronal degeneration by the analyses of transgenic mice models and in vitro cell culture models, the underlying disease mechanisms remain to be fully elucidated because of the discrepancy between model mice and SBMA patients. Thus, novel human disease models that recapitulate SBMA patients’ pathology more accurately are required for more precise pathophysiological analysis and the development of novel therapeutics. Here, we established disease specific iPSCs from four SBMA patients, and differentiated them into spinal motor neurons. To investigate motor neuron specific pathology, we purified iPSC-derived motor neurons using flow cytometry and cell sorting based on the motor neuron specific reporter,  Spinal bulbar muscular atrophy (SBMA) is an adult-onset slowly progressive lower motor neuron (MN) disease caused by abnormal CAG repeat expansion in the androgen receptor (AR) gene. SBMA is characterized by weakness and atrophy of limbs and bulbar muscles caused by the degeneration of spinal and bulbar MNs –3. ThrouInterestingly, recent analyses have shown that phenotypes and MN pathology of transgenic mice models were unexpectedly rescued by muscle-specific silencing of mutant AR, suggesting the involvement of muscle pathology in motor neuronal degeneration , 13. In Disease specific human induced pluripotent stem cells (hiPSCs) provide valuable disease models for neurodegenerative disorders , 16. The+-MNs of approximately 40–50%. Moreover, MNs could be enriched by flow cytometry and cell sorting with MN specific HB9e438::Venus reporter lentivirus [On the other hand, iPSC-derived disease models still have issues to overcome, including time and labor required for the establishment and differentiation of iPSCs, heterogeneities of differentiated neural cells, and large variations among iPSC clones. In particular, the phenotypes or molecular changes may be masked by variations of the differentiation efficiency among iPSC clones, indicating the necessity of a method to purify target cells. To overcome these issues, we previously established rapid, efficient, and simple MN differentiation system from hiPSCs, in which MN progenitors could be obtained within two weeks with the induction efficiency of HB9ntivirus .In this study, we established iPSCs from four SBMA patients, differentiated them into MNs, and investigated molecular pathology behind the motor neuronal degeneration in SBMA. We focused on common molecular changes among multiple iPSC clones derived from different patients. Differentiated MNs were purified using the lentiviral reporter system to overcome clonal variations of iPSCs, and to elucidate MN specific pathology. Furthermore, genome-wide transcriptome analyses by RNA sequences revealed the involvement of the key molecular pathology associated with synapses, epigenetics, and endoplasmic reticulum (ER) in SBMA. Pathology associated with neuromuscular synapses such as Synaptotagmin proteins, RSPO2, and WNT ligands were particularly highlighted, which may serve as novel therapeutic targets for SBMA.OCT4, SOX2, KLF4, L-MYC, LIN28, and shTP53 via episomal vectors. Three of the control iPSC clones, TIGE-9, YFE-16, and EKN3, have already been described previously [OCT4 and NANOG were also confirmed by quantitative RT-PCR, which showed comparable expression to KhES1 (human embryonic stem cells) [We first established iPSCs from skin fibroblasts isolated from four SBMA patients , and three healthy age-matched control adults    in all tTo determine the stability of expanded CAG repeat during reprogramming, we next examined the length of the CAG repeat in the AR gene by fragment analysis. As expected, the length of the CAG repeat in each iPSC clone was stable during reprogramming, except for SBMA1E-18, whose number of CAG repeat increased from 52 to 55 , which is a ligand of AR, to detect ligand-dependent pathology of SBMA. The cells were then detached and dissociated into single cells, and purified by flow cytometry and cell sorting based on HB9e438::Venus fluorescence  . AR expression was comparable in the NF and the HPF, consistent with the finding that AR expression was not specific to MNs.For the transcriptome analysis using purified/enriched populations of MNs, MNs were infected with the p-values less than 0.05 in NT, and 79 genes in HPF , Insulin-like growth factor-1 (IGF-1), Neurotrophin-3 , Glutamate ionotropic receptor delta type subunit 2 (GRID2), Glutamate ionotropic receptor kainate type subunit 1 (GRIK1), Glutamate metabotropic receptor 2 (GRM2), Regulator of G protein signaling 4 (RGS4) and Regulator of G protein signaling 5 (RGS5) are markedly upregulated, and Family with sequence similarity 135 member B (FAM135B) and Membrane-type frizzled-related protein (MFRP) are prominently downregulated, as shown in Fig. To investigate disease-associated genes in SBMA-MNs, total RNA was extracted from both unpurified MNs (NT) and purified MN fraction (HPF) from MNs derived from 4 SBMA-iPSCs and 4 control-iPSCs, and processed for RNA sequencing analysis. As a result, 119 genes showed adjusted To investigate important pathways and signals involved in SBMA pathology, we next performed gene set enrichment analysis (GSEA) . The datGO analysis revealed that the gene sets that were enriched in SBMA-MNs included gene sets related to synapse in both NT and HPF; those related to synaptic vesicles, calcium ions, and exocytosis in HPF Fig. a. PathwaAs for negatively enriched gene sets in SBMA-MNs in GO analysis, chromosome and ribosome related gene sets were occasionally detected in both NT and HPF. Furthermore, in HPF, in addition to the gene sets of mRNA catabolic processes and ribosomes, the gene sets of endoplasmic reticulum (ER) are positioned at higher ranks , which has previously been reported to be involved in SBMA pathogenesis [GRID2, GRIK1, R-spondin 2 (RSPO2), Synaptotagmin6 (SYT6), Synaptotagmin9 (SYT9), Wnt family member 2B (WNT2B) and Wnt family member 3A (WNT3A), most of which are involved in the synaptic functions in NMJs independent of agrin , which induces acetylcholine receptor (AChR) clustering at NMJs, did not show significant alteration  gene, and myotonic dystrophy (DM1), caused by CTG repeat expansion in the 3′ UTR of the DMPK gene, both of which exhibit somatic instability of the repeats, and significant anticipation [However, the consistency between the trinucleotide repeat instability in patients’ tissues and those in iPSCs is still not clear in other triplet repeat diseases, such as Huntington disease (HD), caused by CAG repeat expansion in the cipation . Althougcipation , 32, allcipation . Thus, tSince mutant AR is known to translocate into the nucleus of MNs in a ligand (testosterone)-dependent manner, causing transcriptional dysregulation and MN degeneration in SBMA , 8, iPSCCALCA and FAM135B, were also identified [CXCL14, IGF-1, and NTF3. CXCL14 is known to be upregulated in mice exhibiting similar phenotypes to ALS [IGF-1 is shown to suppress apoptosis of a mutant AR expressing MN cell line (MN-1) by phosphorylating AR via Akt, which inhibits ligand-dependent translocation of the mutant AR into the nucleus [IGF-1 rescued the phenotypes of SBMA model mice, which include mutant AR aggregations through Akt-dependent phosphorylation of the mutant AR [IGF-1 in SBMA-MNs could be attributable to the compensatory negative feedback of rescuing the pathology of SBMA. NTF3 (NT-3) is primarily known to be expressed and involved in the development of proprioceptive neurons in dorsal root ganglia [NTF3 in SBMA-MNs could induce increased synapse density or could be the result of compensatory negative feedback, associated with neuronal or neuromuscular synapse degeneration.Through RNA sequence analysis of four SBMA-MNs and four control-MNs, we identified 79 and 119 DEGs in purified (HPF) and unpurified (NT) MNs, respectively. Only 18 genes were commonly identified, suggesting the purification of MN enabled the detection of MN-specific pathology Fig. b. More ientified , 34, ind nucleus . IGF-1 a nucleus . Moreoveutant AR . Thus, t ganglia , 40. NT- ganglia , 42, and ganglia , 44. ThuAccording to GSEA, upregulation of (1) synapse-, (2) neurotransmitter-, (3) calcium-related exocytosis-, and (4) epigenetics-related gene sets, and downregulation of (5) ER-related gene sets were identified as involved in the pathogenesis of SBMA-MNs. Notably, in purified MNs (HPF), gene sets related to activated synapse function or those related to epigenetics were more significantly enriched in SBMA-MNs.Hes5 [Regarding epigenetics, the gene sets of high-CpG-density promoters (HCP) bearing the methylation mark at K4 and K27 (H3K4me2 and H3K27me3) were enriched in the HPF of the SBMA-MNs. Although abnormal histone modification has never been reported, DNA methyltransferase1 is known to be strongly expressed in SBMA model mice and patients’ spinal cords, which could be rescued by its inhibitor through the suppression of Hes5 . As the Hes5 , epigeneAs negatively enriched gene sets, ER-related gene sets are identified. ER plays crucial roles in the unfolded protein response (UPR), which is the ER protein quality control pathway. The deficiency of the UPR mediator, CHOP, was shown to activate macro-autophagy, which is a lysosomal protein quality control pathway and which accentuated phenotypes of SBMA model mice . MoreoveIGF-1, or the silencing of mutant AR, was shown to rescue the phenotypes of SBMA model mice, suggesting the involvement of skeletal muscles in the neurodegeneration in SBMA [GRID2, GRIK1, and GRM2 could be involved in glutamate toxicity in MNs, a phenomenon reported to occur in ALS. Upregulation of these genes in the SBMA-MNs suggests the involvement of similar glutamate toxicity in SBMA. Another example is that RGS proteins, including RGS4 and RGS5, negatively regulate G protein-coupled receptor (GPCR) signaling, and work in coordination to regulate key aspects of neurotransmitter release, synaptic transmission, and synaptic plasticity at neuronal synapses [RGS4 and RGS5 inactivate GPCR signaling, which could induce neurodegeneration [SYT6 and SYT9, plays important roles in calcium-dependent exocytosis of synaptic vesicles, and SYT9 has been shown to be involved in the pathology of ALS [Most highlighted differences between the SBMA- and control-MNs were within synapse-related gene sets. Recently, the skeletal muscle specific overexpression of  in SBMA , 13, 38. in SBMA , 48. Intsynapses . Increasneration . Moreovey of ALS .RSPO2 is highly expressed in MNs and directly binds to its receptor, leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) at the neuromuscular synapse. RSPO2 not only activates canonical Wnt pathways, but also promotes AChR clustering via muscle-specific tyrosine kinase (MuSK) and low-density lipoprotein receptor-related protein 4 (Lrp4) [WNT3A negatively regulate NMJ formation [RSPO2, and Wnt ligands in SBMA-MNs, could induce abnormal neuromuscular synapse formation, or could be caused by compensatory negative feedback of pathological synaptic degeneration.As for the neuromuscular synapse formation, 4 (Lrp4) , 53. Sim4 (Lrp4) –56, but ormation . Thus, iIn this study, SBMA disease-specific iPSCs provided a valuable disease model that enabled elucidation of previously unknown pathologies underlying neurodegeneration. Comprehensive transcriptome analysis using purified MNs revealed MN-specific and patient-specific pathologies that have never been investigated in detail. Notably, this analysis further highlighted synaptic pathologies in SBMA. In contrast to most of the previous studies which focused on muscular pathology causing non-cell autonomous neurodegeneration and NMJ dysfunctions, we demonstrated that neuromuscular synapses and synaptic functions of NMJs could be affected by MN pathology in SBMA, and also identified alteration of gene expressions in MNs, such as Synaptotagmin, RSPO2, and WNT ligands. Through these analysis, identification of novel therapeutic targets focusing on neuromuscular synapses is expected.5 HDFs were transfected with 1 μg of each of the following: pCXLE-hOCT3/4-shp53 (OCT4 and shTP53), pCXLE-hSK (SOX2 and KLF4), and pCXLE-hUL . Plasmid transfection was performed using the Neon transfection system . After 6 days, the cells were harvested and plated on mitomycin-C-treated SNL murine fibroblast feeder cells in 0.1% gelatin-coated tissue culture dishes in human fibroblast medium. On the next day, the medium was changed to standard human embryonic stem cell (hESC) medium containing DMEM/F-12 , 20% knockout serum replacement (KSR) , 2 mM L-glutamine, 1% non-essential amino acids (NEAA) , 0.1 mM 2-mercaptoethanol (2-ME) , 0.5% penicillin/streptomycin, and 4 ng/mL recombinant human fibroblast growth factor-2 (FGF-2) . When the colonies had grown to a sufficiently large size, they were picked and expanded in the same way as hESCs and hiPSCs. The properties of the established iPSC clones were evaluated, as described previously  were obtained from 4 Japanese SBMA patients and 3 controls: a 36-year-old Japanese male from the Japanese Collection of Research Bioresources (JCRB) Cell Bank TIG114), a 24-year-old Japanese male (YF), and a 39-year-old Japanese male (KN). HDFs were cultured in DMEM , 10% fetal bovine serum , 2 mM L-glutamine, and 1% penicillin/streptomycin. Then, 6 × 1014, a 24-2, and 20% KSR) and cultured in suspension in bacteriological dishes in standard hESC medium, after the removal of SNL feeder cells, with incubation for 1–2 h in gelatin-coated dishes. On day 1, the medium was changed to human embryoid body (hEB) medium containing DMEM/F-12, 5% KSR, 2 mM L-glutamine, 1% NEAA, and 0.1 mM 2-ME with 300 nM LDN-193189 , 3 μM SB431542 , and 3 μM CHIR99021 . On day 2, the medium was changed to fresh hEB medium containing 300 nM LDN-193189, 3 μM SB431542, and 3 μM CHIR99021, and 1 μM retinoic acid (RA) . From day 4 to day 14, hEBs were cultured in hEB medium containing 1 μM RA and 1 μM purmorphamine , and the medium was changed every 2–3 days. On day 14, hEBs were enzymatically dissociated into single cells using TrypLE Select . The dissociated cells were plated on poly-l-ornithine (PO) and recombinant mouse Laminin , or growth-factor-reduced Matrigel -coated dishes at a density of 5 × 104–1 × 105 cells/cm2 and cultured in motor neuron medium (MNM) consisting of media hormone mix (MHM) or KBM Neural Stem Cell medium  [2 atmosphere. 10 nM DHT  was added to MN culture at the day of lentiviral infection (4 days after monolayer differentiation). Half of the medium was changed every 2–3 days.iPSCs were differentiated into spinal MNs, as previously described . iPSCs w, Japan)  supplemeCells were fixed in 4% paraformaldehyde for 15–25 min at room temperature. After blocking in blocking buffer (PBS containing 10% FBS and 0.3% Triton X-100), the cells were incubated with primary antibodies overnight at 4 °C. The cells were then rinsed with PBS three times and incubated with Alexa 488-, Alexa 555-, or Alexa 647- conjugated secondary antibodies (Thermo Fisher Scientific) for 1 h at room temperature. Nuclei were stained with 10 μg/ml Hoechst 33258 . The cells were then rinsed with PBS three times, mounted on slides, and examined using IX83 . The primary antibodies used in these analyses were listed in Additional file 5–5 × 105 cells were injected into the testes of NOD/ SCID mice . At 8–10 weeks after injection, the tumors were dissected and fixed with PBS containing 4% PFA. Paraffin-embedded tissue was sliced and stained with hematoxylin and eosin. Images were obtained using a BZ-9000 microscope .Each iPSC clone was harvested in dissociation solution, collected into tubes, and centrifuged, and the resulting pellets were suspended in hESC medium with 10 μΜ Y-27632 , which is a Rho-associated coiled-coil forming kinase (ROCK) inhibitor. Then, 1 × 10DNA was extracted using DNeasy Blood & Tissue kits . PCR amplification of the CAG repeat in the AR gene was performed using a fluorescent-labeled forward primer (5′-TCCAGAATCTGTTCCAGAGCGTGC-3′) and an unlabeled reverse primer (5′-GCTGTGAAGGTTGCTGTTCCTCAT-3′). Detailed PCR conditions were described previously . For detβ-ACTIN mRNA. The primer sequences and PCR cycling conditions are listed in Additional file RNA was isolated using a RNeasy mini kit  and then converted into cDNA using SuperScript III reverse transcriptase  and Oligo dT primers as described previously , 59. ReaTM 293T cells cultured in DMEM supplemented with 10% FBS in 150 mm dishes were transfected with 16 μg of pSIN2-HB9e438-βglo-Venus (a variant of yellow fluorescent protein (YFP) with fast and efficient maturation [HB9e438::Venus reporter lentivirus. Lentiviral infection was performed on the day 4 of monolayer MN differentiation. For lentiviral infection, HB9e438::Venus in Opti-MEM  was added to a MN culture, followed by incubation for 2 h, after which the total medium was changed to MNM.Lenti-Xturation ) or pSINHB9e438::Venus lentivirus using Tissue Dissociation Kits (Miltenyi Biotec) according to the manufacturer’s instructions. The dissociated cells (5 × 104–1 × 105 cells) were suspended in 50–100 μl of Hanks’ balanced salt solution (HBSS)  containing 2% FBS, 10 mM HEPES, and 1 μg/ml 7-AAD. The cells were then analyzed and sorted based on the expression of the HB9e438::Venus reporter using a FACSAria III cell sorter .For flow cytometric analysis and cell sorting, iPSC-derived MNs were dissociated 4 weeks after infection with the http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm) [http://jtreeview.sourceforge.net/) [The yield and quality control of total RNA were measured using Nano Drop 2000c  and Agilent RNA6000 Nano Kit , respectively. The 2100 Bioanalyzer system  was used to qualify RNA integrity number (RIN). RNA samples with sufficient RIN values (more than 9.2) were subjected to the generation of mRNA libraries using Illumina TruSeq protocols for poly-A selection, fragmentation, and adaptor ligation, according to the manufacturer’s instructions (TruSeq RNA Sample Prep Kit v2). Quantification of libraries was performed using Qubit3.0 (Thermo Fisher), Agilent 2200 TapeStaition System (Agilent), and qPCR analysis by Kapa Library Quantification Kit (TakaRa). The multiplexed libraries were sequenced as 75 nt pair end runs on an Illumina NextSeq500 system . Sequence reads were mapped to the reference human genome (GRCh38/hg38) using STAR . We then excluded reads mapped to rRNA and tRNA regions. Annotation of the rRNA and tRNA regions were obtained from the UCSC Table Browser. Read counts of transcripts (feature counts ) were caare.htm)  by average.net/) .http://www.broadinstitute.org/gsea/index.jsp. The expression profile data were analyzed using GSEA 4.0.1. The gene sets were downloaded from the Molecular Signatures Database (MSigDB) and C2  and Canonical pathway (CP)) and C5 (Gene ontology gene sets) were used for the GSEA.GSEA is a computational method that determines if a priori defined set of genes show statistically significant, concordant differences between two biological states , and is p-value for a false discovery rate (FDR) correction was performed by the Benjamini–Hochberg (B-H) method. The gene set enrichment analysis was performed using the Fisher’s exact test and corrected with the B-H FDR.For the statistical analysis in the quantitative RT-PCR, either the Student’s t-test or the Welch’s t-test was used. For the differential analysis of mRNAs between control and SBMA MNs using DESeq2, adjusted Additional file 1: Figure S1. Evaluation of established iPSCs. Related to Fig. Additional file 2: Figure S2. Karyotype analysis of the established iPSC clones via G-banding analysis. Related to Fig. Additional file 3: Table S1. DEGs in heatmap (NT). Related to Fig. Table S2. DEGs in heatmap (HPF). Related to Fig. Table S3. Profiles of GSEA. Table S4. Gene ontology terms (down in NT). List of GO terms negatively enriched in SBMA-MNs of NT (FDR < 0.2). Table S5. Gene ontology terms (down in HPF). List of GO terms negatively enriched in SBMA-MNs of HPF (FDR < 0.01). Table S6. Pathway terms (down in NT). List of pathway terms negatively enriched in SBMA-MNs of NT (FDR < 0.01). Table S7. Pathway terms (down in HPF). List of pathway terms negatively enriched in SBMA-MNs of HPF (FDR < 0.01). Table S8. The extracted upregulated genes in SBMA-MNs in GSEA. List of 96 genes that were significantly upregulated in enriched GO and pathway terms with p < 0.05 and significantly higher (> 2-fold) expression levels in SBMA-MNs than in control-MNs. Table S9. Primer sequences and cycling conditions for quantitative RT-PCR. Table S10. Antibodies for immunocytochemistry.


On-CXC1 and On-CXC2) were identified at both the genomic and proteomic levels. A southern blot analysis and comparison searching in Ensembl confirmed the typical structure of the CXC chemokine genes and provided evidence for unusual mechanisms used to generate the two different CXC chemokine transcripts that have not been reported in other vertebrate species so far. The expression levels of On-CXC1 and On-CXC2 were analyzed by quantitative real-time PCR. These two mRNAs were detected in various tissues of normal Nile tilapia, especially in the spleen, heart, and head kidney, indicating a homeostatic function in immunosurveillance. A time-course experiment clearly demonstrated that these two transcripts were effectively enhanced in the head kidney, spleen and trunk kidney of Nile tilapia 6, 12 and 24 h after injection with Streptococcus agalactiae but were down-regulated in all tested tissues at 48 h, reflecting the fact that they have short half-lives during the crucial response to pathogens that is characteristic of CXC chemokine genes in other vertebrates. Functional analyses obviously exhibited that these two CXC chemokines at concentrations of 1–10 μg strongly inactivated S. agalactiae and Flavobacterium columnare and effectively induced phagocytosis of leukocytes in vitro.Two CXC chemokines in Nile tilapia ( Streptococcus agalactiae  is a common pathogenic bacterium that is a major cause of streptococcosis in Nile tilapia in Thailand. S. agalactiae is a beta-hemolytic, Gram-positive, non-motile, non-acid-fast and non-spore-forming bacterium. The clinical signs of S. agalactiae in Nile tilapia include septicemia, exophthalmia, corneal opacity, melanosis, swimming abnormalities, swelling and hemorrhage of the internal organs [Flavobacterium columnare was also observed to kill large numbers of Nile tilapia of various sizes. Thus, the diseases caused by these bacteria have a large impact on Nile tilapia cultures and are major problems for Nile tilapia farming in Thailand. Most farmers use a variety of chemicals and drugs to control this disease. However, the application of drugs and chemicals directly affects consumer health and the aquatic environment. To minimize drug usage, it is important to understand the fish immune system and consequently develop prophylactic or therapeutic strategies applicable to Nile tilapia aquaculture.Tilapia is one of the most popular fish cultured in global aquaculture after carp and salmonid fish. Since the late 1990s, the large-scale production and international trade of Nile tilapia has grown rapidly, and this fish has become an important commodity in international markets. The global production of farmed tilapia reached 4.6 and 6.5 million metric tons in 2013 and 2019, respectively [Melanogrammus aeglefinus) and Atlantic cod (Gadus morhua) [Lampetra fluviatilis), which is a jawless fish (agnathan) [In all vertebrates, including fish, innate and adaptive immune responses protect against diseases . Innate  morhua)  are exce morhua) . Althoug morhua) . Among t morhua) . In fishgnathan) . Interesgnathan) . The putgnathan) ,25. Addignathan) ,32,33,34I values), which are attracted and incorporated into negatively charged bacterial membranes, causing membrane disruption and loss of intracellular constituents [Antimicrobial peptides (AMPs) are effective components of the innate immune system for providing an essential first-line defense against a wide range of microorganisms  ,36. Genetituents . Over thtituents , catfishtituents  and chantituents , severalwww.ensembl.org) and PCR was conducted to elucidate the full CXC gene organization in the Nile tilapia genome. The expression of these CXC chemokine mRNAs in various tissues of normal fish and in fish inoculated with S. agalactiae at different concentrations was analyzed using semi-quantitative RT-PCR and quantitative real-time PCR. Two recombinant CXC proteins were produced and tested for their biological functions regarding the chemotactic induction of phagocytosis and antimicrobial killing of pathogenic bacteria that caused severe disease outbreaks in Nile tilapia.In the present study, two different CXC chemokines of the Nile tilapia were successfully cloned and characterized at both the genomic and proteomic levels. A southern blot analysis was employed to estimate the copy number of CXC chemokine genes in the Nile tilapia genome. The analysis of the genomic segment using the Ensembl Nile tilapia genomic databases  were obtained from Manit Genetics Co., Ltd.,  and was acclimatized under laboratory conditions at the laboratory of Aquatic Animal Health Management, Department of Aquaculture, Faculty of Fisheries, Kasetsart University . Subsequently, the experimental fish was maintained in an 80-L glass tank containing freshwater with an aeration system and fed twice a day with commercial feed at 5% body weight. The protocol was approved by the Animal Ethics Committee, Kasetsart University, Thailand (ethics ID: ACKU61-FIS-004).After acclimating for 7 days, the experimental fish was sacrificed, and the head kidney and spleen were collected. Total RNA was isolated from these two organs using TRIzol reagent . Then, a QuickPrep Micro mRNA Purification kit  was used to isolate the mRNA following the manufacturer’s instructions, and the samples were stored at −80 °C until use.In this experiment, 0.5 and 0.5 μg of mRNA from the spleen and head kidney, respectively, was pooled and used as a template to synthesize the 3′ and 5′ first-strand cDNAs using a SMARTer™ RACE cDNA amplification kit  following the company’s protocol.On-CXC1 and On-CXC2. Based on comparisons with published sequences using the BLAST programs from the GenBank database (http://www.ncbi.nlm.nih.gov), it was clearly demonstrated that EST clone HK10 contained the full sequence of On-CXC1 and clone HK737 possessed a partial nucleotide sequence of On-CXC2.Using the isolated mRNA, two uncharacterized cDNAs of the EST clones HK10 (FF279523) and HK737 (FF280148), which are homologous to the CXC chemokines of other vertebrate species, were obtained from the head kidney cDNA library of Nile tilapia . These fTaq buffer, 0.5 μL 10 mM dNTP mix (2.5 mM each), 0.5 μL (2.5 unit) TaKaRa LA Taq DNA Polymerase, 2.5 μL 10× universal primer mix, 0.4 μM UPM-long, 2 μM UPM-short , cloned into the pGEM-T Easy vector , and then ligated and transformed into Escherichia coli (strain JM109) following the manufacturer’s instructions. Then, plasmid was extracted from the positive clones using a Plasmid DNA Extraction Manual Kit . Finally, nucleotide sequencing of the target plasmid in both the 5′ and 3′ directions was conducted using the primers M13F and M13R (http://www.ncbi.nlm.nih.gov) were used to assess homology against all matched sequences in the database when the E-value was less than 1 × 10−4 [http://www.ncbi.nl.nih.gov/gorf/gorf.html). The signal peptide of each CXC protein was analyzed using SignalP software .The PCR products from and M13R  by Macro1 × 10−4 . The cDNhttp://bitincka.com/ledion/matgat/). The deduced amino acid sequences were aligned and compared with other known CXC chemokine genes of various vertebrate species using the CLUSTAL W program  [http://www.ebi.ac.uk/Tools/clustalw/index.html) [The identity and similarity of both nucleotide and amino acid sequences of Nile tilapia CXC cDNA were evaluated against CXC cDNAs of other vertebrates using the MatGAT  version 2.02 (ex.html) . A phyloex.html)   , and a pex.html) .®-1077 . The two-layer mixture was centrifuged at 400× g for 30 min at 25 °C in a swing rotor centrifuge, and 3 mL of the opaque interface was aspirated and moved into a new conical centrifuge tube and 3 mL of phosphate-buffered saline  was added. The PBL solution was gently mixed and 3 times of centrifugation at 250× g for 10 min were made. One millimeter of TRIzol reagent  was added to dissolve the PBL pellet. One hundred milligrams of each tissue in 1 mL TRIzol reagent was carried out as previously described. The contaminating genomic DNA in total RNA samples were treated with DNase I . To synthesize first-strand cDNA, a RevertAid First Strand cDNA Synthesis Kit  was applied in a 200-µL test tube using 1 µg of total RNA from each tissue and an oligo (dT) 18 primer, followed by incubation at 70 °C for 5 min. The first-strand cDNA was carefully synthesized based on the recommendation of the company’s protocol and kept at −20 °C until used.In this part, total RNA isolated from the brain, gills, gonad, heart, head kidney, intestine, liver, muscle, skin, spleen, stomach, trunk kidney and peripheral blood leukocytes (PBLs) of the healthy fish were used for using TRIzol reagent  according to the manufacturer’s protocol. For PBL isolation, 1.5 mL of whole blood was withdrawn from the caudal vein with a sterile syringe coated with 50 IU heparin solution. The unclotted blood was diluted with 1.5 mL of RPMI medium. This component was further put into a 15-mL conical tube containing 3 mL HistopaqueOn-CXC1, and CXC-2F New and CXC-2R New for On-CXC2  were used in this assay. Fish were weekly acclimatized in a 250-L fiberglass tank supplemented with fully oxygenated freshwater and were fed a commercial feed at 5% body weight twice a day. At the end of acclimatization, fish were divided into two major groups, where 15 fish for each group were moved into an 80-L glass tank containing clean freshwater with an excellent aeration system. The remaining three fish were used as an initial control group.S. agalactiae virulent strain (SAAQH001) was obtained from infected Nile tilapia grown in the central region of Thailand. A single colony of S. agalactiae was inoculated in 10 mL of tryptic soy broth  and incubated in a shaker water bath at 35 °C for 18–24 h. Then, the bacterial cells were collected by centrifugation at 800× g for 15 min and washed twice with sterile 0.85% NaCl. The bacterial pellet was then re-suspended in sterile 0.85% NaCl. The S. agalactiae concentration was adjusted to 1 × 109 CFU/mL with an optical density at 560 nm of 0.2 and was further serially diluted to reach the target concentration of 1 × 103 CFU/mL.The 3 and 1 × 109 CFU/mL of S. agalactiae prepared in 2.7.2, respectively. The three fish in the control group were injected with 0.1 mL of 0.85% NaCl via the same route as the bacterial injection groups. At 6 h after injection, the head kidney, spleen and trunk kidney were collected from three fish in each group. Additionally, at 12, 24, 48 h and 7 days after injection, the head kidney, spleen and trunk kidney were collected from three fish in each bacterial injected group.In this step, all fish in the first and second groups were intraperitoneally injected with 0.1 mL bacterial suspension containing 1 × 10Total RNA and first-strand cDNA of the head kidney, spleen and trunk kidney were extracted and synthesized from three fish in each group in 2.7.1 at different time points using the same method described previously.On-CXC1 amplification or CXC-2F New and CXC-2R New for On-CXC2 amplification  for the CXC chemokine and β-actin genes was calculated, and the standard curve was used to determine their starting copy number. This method is based on equal PCR efficiencies for the target and internal control mRNA. The relative copy number of target mRNA was calculated according to the 2-∆∆CT method [T) between the CXC chemokine and β-actin mRNAs of each reaction was used to normalize the level of mRNA and determine their relative expression.First-strand cDNAs from the head kidney, spleen and trunk kidney of the control fish and infected fish from fication . The CXCfication . The PCRT method . FinallyS. agalactiae at different time points using the expression level of the fish at the initial time as a calibrator. Subsequently, relative expression ratios of CXC chemokine genes at different time courses were analyzed using a one-way analysis of variance (ANOVA). The significance of the means of relative expression ratios was determined using Duncan’s new multiple range test (DMRT) method. Differences were considered significant when the p-value was less than 0.05.The relative expression ratio of the CXC chemokine mRNAs was calculated in all tested tissues of Nile tilapia in response to EcoRI and PstI restriction enzymes following the manufacturer’s protocol . The digested DNA was separated on a 1.5% agarose gel in 1× TBE buffer at 50 V. The standard size was based on the reference of a lambda (λ) Hind III DNA marker. The digested DNA was transferred to a nitrocellulose membrane by the capillary blotting method with 20× SSC  using the methods described by Sambrook and Russell [A DNeasy Blood and Tissue Kit ) was used to extract genomic DNA from the whole blood of three healthy fish according to the manufacturer’s instructions. Ten micrograms of DNA from each fish as separately used and digested with the  Russell . FinallyOn-CXC1 (The primers CXC-1FSB and CXC-1RSB were used to PCR amplify a specific probe for On-CXC1 . The PCROn-CXC1 and On-CXC2) from the present study were assessed using the Ensembl Nile tilapia genomic databases (www.ensembl.org). BLAST searches were conducted to localize the putative CXC chemokine genes in the Nile tilapia genome. In addition, genomic DNA was extracted from the spleen of a healthy fish as described in On-CXC1) were used to clarify the unclear sequence information found in the Ensembl database. Genomic DNA was used as template for PCR amplification. The PCR analysis was performed with the same conditions described in ® Gel Extraction Kit , ligated into the pGEM-T Easy vector and transformed into JM109 competent cells. Positive colonies were screened by PCR analysis and sequenced as described in The full-length Nile tilapia CXC chemokine sequences  (® Gel Extraction Kit (QIAgen®), ligated into the pGEM T-Easy vector, and transformed into JM109 competent cells as described in Nde I and Xho I) to confirm the appearance of the inserted DNAs. The obtained plasmid was sequenced to confirm correct in-frame insertion. The recombinant plasmid was further used for Nde I and Xho I restriction enzyme digestion, the digested DNA fragments were purified and ligated into the pET28b(+) expression vector and transformed into JM109 competent cells, and the transformed bacteria were grown at 37 °C in LB broth containing 100 mg/mL of kanamycin. The positive clones were extracted and transformed into E. coli BL21 (DE3) competent cells for protein expression. After induction, recombinant proteins (rOn-CXC1 and rOn-CXC2), containing a His tags at both the N- and C-termini were carefully purified using a previously described protocol [The cDNAs encoding the mature On-CXC2)  with theprotocol .8 beads/mL and 5 × 106 phagocytes/mL, respectively, with RPMI 1640 medium. Three experimental groups were created for each recombinant protein. For the first and second groups, the latex beads were separately incubated with 10 and 100 µg/mL, respectively, of rOn-CXC1and rOn-CXC2 in RPMI medium, while the latex beads in the third (control) group were incubated with RPMI 1640 medium. These groups were run in triplicate. The phagocytic cells adhering to the surface of the cover slip were incubated with the latex beads at different concentrations of protein at room temperature for 1.5 h to allow latex-bead particle uptake. Attached cells and beads were further stained with Diff-Quick staining dye , and at least 200 cells were counted under 100× light microscopy. The percentage of phagocytosis (PA) and the phagocytic index (PI) were calculated according to the formulas below. Statistical analyses of these two parameters were conducted as described above.A phagocytosis assay was employed for analyzing the biological activity of the proteins based on the method described by , with apS. agalactiae and F. columnare at the same concentrations. F. columnare was prepared in Shieh’s broth based on the protocol described by [In this experiment, evaluation of the phagocytic activity of PBLs of Nile tilapia mostly followed the protocol described in ribed by . PhagocyOn-CXC were serially diluted from 10 to 0.02 μg, and bacterial concentrations of 1 × 105 CFU were used. Wells that only contained broth medium and bacteria were provided as a negative control. After incubation at 30 °C for 24 h, the lowest concentration that caused complete inhibition indicated by clear broth was defined as the MIC. Growth of bacteria in each well was measured at 0, 1, 3, 6, 12, 18 and 24 h with an iMarkTM Microplate Absorbance Reader (Bio-Rad) at 600 nm. During these periods, morphological changes of bacterial cells exposed with 1.25 and 10 mg/mL of each CXC protein were stained using Gram’s staining methods and visualized at 1, 3 and 6 h by light microscopy  under 1000× magnification.The two pathogenic bacteria from On-CXC1 (KR673910) and comprised 1346 bp, including a 408 bp open reading frame (ORF). The predicted amino acid sequence of On-CXC1 had 135 amino acid residues and contained a 116 bp 5′ untranslated region (UTR) and an 822 bp 3′UTR. The 3′UTR contained 4 mRNA instability motifs (ATTTA) and 7 different polyadenylation signals, including ACTAAA, TATAAA and five AATAAA sequences (On-CXC2 (KR673911), and its full-length cDNA was 653 bp, with a 321-bp ORF. The predicted amino acid sequence of On-CXC2 had 106 amino acid residues. The complete cDNA of On-CXC2 had a 53 bp 5′UTR and a 279 bp 3′UTR. The 3′UTR contained 2 mRNA instability motifs (ATTTA and ATTTTA) and 5 different polyadenylation signals, including one AAAAAG, one AATAAA, one AATAGA and two AAAACA sequences  that is found in well-known CXC chemokines from other vertebrate species  and valine , were observed. Structural analysis of the predicted amino acid sequence of the On-CXC1 and On-CXC2 polypeptides showed that these proteins had a molecular weight and isoelectric point (pI) of 15.12 kDa and 9.56 for On-CXC1 and 12.11 kDa and 9.76 for On-CXC2, respectively.In addition, putative signal peptides of both  species . A singlOn-CXC1 and On-CXC2 had significant amino acid identity and similarity, with scores of 73.7% and 76.3%, respectively  CXC chemokine, which was located in a novel CXC subgroup [The evolutionary relationship of the Nile tilapia CXC chemokine proteins with other known vertebrate CXC chemokines was analyzed using previously reported sequence data . There wsubgroup . Moreovewww.ensembl.org). The results showed that the On-CXC genes matched two different locations in the Nile tilapia genome, scaffolds AERX01074721  . InteresOn-CXC1 and On-CXC2 mRNA expression in Nile tilapia in response to two concentrations of S. agalactiae at different time points. In the head kidney, On-CXC1 mRNA was significantly increased in fish injected with 1 × 109 CFU/mL of S. agalactiae at 6 and 12 h and at days 1 and 2, with relative expression of 2.26 ± 0.24, 3.56 ± 0.22, 2.64 ± 0.46 and 2.78 ± 0.15, respectively, compared to the control (p < 0.05). However, at day 7, both concentrations of bacteria significantly decreased mRNA levels of On-CXC1 to 0.54 ± 0.22 and 0.48 ± 0.14 compared with the control (p < 0.05)   A. In the at 12 h B. Simila < 0.05) C.On-CXC2, at 6 h, 12 h and day 1, the two concentrations of S. agalactiae significantly induced mRNA transcript levels in the head kidney compared with other groups, with levels of 4.64 ± 0.48 and 6.48 ± 0.56, 6.26 ± 0.12 and 6.67 ± 0.22, and 2.52 ± 0.22 and 8.42 ± 0.46, respectively (p < 0.05). However, at day 7, these bacterial concentrations resulted in significantly lower expression levels than the control with levels of 0.24 ± 0.08 and 0.18 ± 0.14, respectively (p < 0.05)  . The higher concentration of S. agalactiae effectively increased the On-CXC2 mRNA level compared to the control only at 6 and 12 h with 2.45 ± 0.16 and 7.45 ± 0.27, respectively (p < 0.05) (For  < 0.05) A. In the < 0.05) B. In the < 0.05) C.On-CXC1 and rOn-CXC2 were successfully produced as soluble forms. The purified rOn-CXC1   (The rng cells . The PA  < 0.05) A. Simila < 0.05) B.On-CXC1 and rOn-CXC2 protein-treated PBLs. Several engulfed streptococci and long, rod-shaped cells were observed. Unexpectedly, morphologically changed cells were normally found to be engulfed, especially at 3 and 6 h. Swollen streptococci and mono- and diplococcus of S. agalactiae , creating a typical CXC motif. The presence of putative signal peptides in both On-CXC1 and On-CXC2 suggests that Nile tilapia CXC chemokines are secreted molecules, as reported in other vertebrate chemokines.In the present study, two full-length cDNAs encoding Nile tilapia CXC chemokines were successfully cloned and named On-CXC1 had 4 exons and 3 introns, which is consistent with previous studies in humans, catfish, rainbow trout and soft-shelled turtles [On-CXC2 was generated by a specific process from a pseudogene fragment containing Ψ exons 3-4 and some part (exons 1 and 2) of the functional On-CXC1 gene. This pattern has not been previously reported in any vertebrates. Gene duplication of the pseudo ΨCXC gene fragments may be specific to Nile tilapia or other teleosts that occurred during their evolution and is important for generating diverse gene products and modulating gene expression and function in vertebrate species, including teleost fish. In recent years, alternative splicing analyses of CXC chemokine genes have been reported in several fish genomes, such as the zebrafish, medaka, fugu, channel catfish and stickleback [On-CXC2 in this study may be a unique process that additionally helps Nile tilapia to produce other forms of CXC molecules in association with invading pathogen defense mechanisms. Further study is needed to elucidate the associated mechanism.Information from the Nile tilapia genome and sequence analysis of the obtained cDNAs clearly showed that  turtles   were also identified in the 3′UTR of pression . It suggpression , catfishpression , Japanespression , and larpression . Essentipression . This suStructurally, the Nile tilapia CXC chemokine molecules found in this study lacked an ELR motif immediately preceding the CXC motif. Therefore, they were classified into the non-ELR subgroup and identified as novel CXC chemokines. Unlike IL-8 molecules in mammals and avian species, the Nile tilapia CXC chemokines in the present study and other fish CXC chemokines, with the exception of haddock and Atlantic cod CXC molecules, lack an ELR motif immediately preceding the CXC motif. Several studies have identified the ELR motif in various species and shown that ELR-containing CXC chemokines attract neutrophils, whereas non-ELR-containing chemokines mainly attract lymphocytes and monocytes ,17. HoweOn-CXC1 and On-CXC2 were placed into a novel CXC subgroup. Therefore, they were grouped with the Mandarin fish, rock bream and Atlantic halibut CXC chemokines and clustered with the closely related Mandarin fish CXC chemokines. In addition, the novel CXC chemokine subgroup was strictly separated into a distinct clade compared to related genes in other teleosts and higher vertebrates. Therefore, they may be derived from different evolutionary events than those of other teleosts and higher vertebrates. This information may provide further insight into the evolutionary relationship between chemokines of Nile tilapia and other species.The amino acid sequence and phylogenetic analysis of the 64 CXC chemokine members of fish and higher vertebrates indicated separately clustering of 10 different subgroups. Both On-CXC1 and On-CXC2 was observed in the spleen, which is one of the most important hematopoietic tissues. These results suggest that the spleen is an important site of lymphocytes and macrophages, which are sources of CXC chemokines under normal conditions.In this study, the gene expression profiles of the Nile tilapia CXC chemokines were analyzed by qRT-PCR. The qRT-PCR results demonstrated that both transcripts were constitutively expressed at different levels in all tested tissues of normal fish. A constitutive expression pattern of CXC chemokine genes has also been found in several fish, such as catfish , JapanesOn-CXC transcripts were observed in all the tested tissues of normal fish. This result may have been due to the ability of the On-CXC1 and On-CXC2 that continuously modulate gene expression and function under normal conditions [On-CXC1 and On-CXC2) to understand the regulation of this mechanism.Interestingly, slightly different levels of the two different nditions . TherefoS. agalactiae, real-time PCR was used to quantify Nile tilapia CXC mRNA expression in the immune-related organs of the fish, including head kidney, spleen and trunk kidney. The kidney (anterior and posterior) and spleen are the largest lymphoid organs in teleost fish and are the source of macrophages involved in the phagocytosis of bacteria in innate immunity. Therefore, the basal expression of cytokines and chemokines is most prominent in these tissues. The result from this study showed that On-CXC1 and On-CXC2 mRNA were rapidly up-regulated to different levels in all tested tissues during the first 6–12 h after injection with 1 × 103 and 1 × 109 CFU/mL of S. agalactiae. During this time, the CXC chemokines might be vigorously produced by monocytes and macrophages in the infected site to eliminate foreign antigens; there, they function as the immunomodulators of leukocytes. Similar to other fish, non-ELR-containing CXC chemokines appear to be regulated by bacterial stimuli. The elevated expression of non-ELR-containing CXC chemokines following induction by different pathogens or immune-enhancing components has been reported in several teleost species, such as catfish challenged with Edwardsiella ictaruli [Upon induction with ictaruli , common ictaruli  and rockictaruli .S. agalactiae in this study indicated their important role as chemoattractants that recruit inflammatory cells to the infected site.In this study, the Nile tilapia CXC chemokine genes were abundantly expressed in the spleen and trunk kidney, which are classified as secondary lymphoid tissues in teleosts. Generally, streptococcosis targets the brain and eyes as well as the spleen and kidney, which contain a large number of immune cells, including lymphocytes and professional macrophages . Therefo9 CFU/mL of S. agalactiae was shown to be a potent inducer of CXC chemokines in Nile tilapia, while the concentration of 1 × 103 CFU/mL showed a lesser degree of induction. These results indicate that 1 × 109 CFU/mL of S. agalactiae strongly stimulates CXC chemokine expression compared to lower concentrations of pathogenic bacteria. This could be due to the speed of disease progression between different concentrations of an invading pathogen.Based on the dose-dependent induction, the concentration of 1 × 10On-CXC1 and On-CXC2 were observed in this study. This was especially apparent in the trunk kidney, where 1 × 103 CFU/mL of S. agalactiae induced higher On-CXC2 mRNA levels from 6 h to 7 days compared to 1 × 109 CFU/mL of S. agalactiae. This may have been due to different mature mRNA isoforms generated by the alternative splicing mechanism, which resulted in the appearance of different functions or expressions in different cell types or tissues. Many recent studies have reported that alternative splicing is the major mechanism for producing diverse gene products and modulating gene expression [On-CXC2 was more sensitive to both lower and higher concentrations of S. agalactiae at 12 h than On-CXC1 in all tested tissues, and it also sensitively responded to low doses of bacteria in the trunk kidney from days 1–7, suggesting that the shorter CXC protein may have specific responses to the concentration of stimulated bacteria, organs and reaction times.Moreover, different expression patterns of pression . In catfpression  resultedpression ,58, simipression . Additiowww.ensembl.org) and PCR amplification were conducted to elucidate the genomic organization of these genes. These results clearly confirmed the presence of the chemokine gene components and pseudogenes involved in the novel mechanisms of CXC production in Nile tilapia. Generally, the alternative splicing is an important mechanism for generating diverse gene products and modulating gene expression and functions in eukaryotic species, including some CXC chemokines of teleosts. However, the mechanism used to create the On-CXC2 was not observed to produce the CXC proteins in other vertebrates. Obviously, the genomic organization of Nile tilapia CXC chemokines, which contain four exons and three introns, was consistent with well-defined fish CXC chemokine gene structure, and the splice sites for the first two introns have also been conserved. However, the process used to generate the On-CXC2 by using pseudogene fragments and the functional On-CXC1 gene and the variation in a splice site in the third intron of the On-CXC1 gene may reflect a divergence event that led to differences in the structure and size of these genes. Further information from other teleosts may aid in supporting and elucidating this unusual process to diversify of CXC genes and its transcriptional and translational processes.Southern blot analysis and further analyses using the Ensembl project  and -negative (F. columnare) bacteria in a dose-dependent manner. Interestingly, the chemokines showed slightly stronger antimicrobial activity against F. columnare compared with S. agalactiae. Our findings are consistent with several reports where LPS, lipid A, or lipoteichoic acid could be a target for the bactericidal activity of AMPs [In the current study, the capacities of Nile tilapia CXC chemokines to mediate antimicrobial activity against pathogenic bacteria were analyzed. The results showed that both r of AMPs ,65. This of AMPs ,67,68, w of AMPs .S. agalactiae and F. columnare cells were obviously different from the untreated group, suggesting that Nile tilapia CXC chemokines may attach to the negative charges on bacterial cell walls to damage bacterial cell membranes and release cell fluid. The results in this experiment revealed the correlation with antimicrobial activity, which showed a decline in bacterial cell growth in the CXC-treated group compared with the untreated group. Following exposure to rOnCXC1 and rOnCXC2, obvious morphological changes occur in F. columnare cells. At 10 µg/mL, elongated cells and shortened cells were observed for short periods, and lysed cells were observed after exposure for long (3–6 h) periods. In the case of S. agalactiae, the bacterial cells changed in shape (swelling or irregular shape) and clumped together, changes that were associated with cell membrane destruction and loss of intracellular constituents. These findings suggested that different types of bacteria may affect different reactions of the CXC chemokines.Due to the electrostatic interactions between cationic chemokines and the anionic bacterial cell membrane, the morphological changes of In summary, analyses of the molecular immunology and immunological effects of bacterial infection on Nile tilapia CXC genes contribute to our understanding of fish immune responses. These data provide basic molecular information useful for further investigations of the function of CXC chemokine genes in the Nile tilapia immune system and the evolutionary relationships of important immune-related genes among vertebrates. Information obtained in the present study clearly indicates a dual function of Nile tilapia CXC chemokines as the mediators of antimicrobial activity. Additionally, the chemokines could initially promote leukocyte migration to sites of infection and simultaneously kill pathogenic bacteria. Hence, these properties of CXC chemokines will facilitate bacterial clearance and an enhanced immune responses in fish, which will be useful for developing effective strategies, such as highly potent vaccine adjuvants for disease prevention from the harmful pathogenic bacteria that are now causing mass mortality in the Nile tilapia industry.


Culture with the osteogenic induction medium containing 10 μg/mL GOQDs induced calcium nodule formation, increased alkaline phosphatase (ALP) activity, and upregulated SHED mRNA and protein levels of OCN, RUNX2, COL I, and β-catenin. With the addition of Dickkopf 1 (DKK-1) or β-catenin knockdown, expression levels of the above mRNAs and proteins were decreased in GOQD-treated SHEDs. In summary, at a concentration of 10 μg/mL, GOQDs promote SHED proliferation and osteogenic differentiation via the Wnt/β-catenin signaling pathway. This work provides new ideas and fundamental information on interactions between GOQDs and SHEDs that are relevant for the biomedical engineering field.Graphene oxide quantum dots (GOQDs) are a carbon nanomaterial with broad potential for application in the field of nanomaterial biomedicine. Stem cells from human exfoliated deciduous teeth (SHEDs) play an important role in tissue engineering and regenerative medicine. This study investigated the effects of GOQDs on SHED osteogenic differentiation. GOQDs were synthesized; then, the proliferation of SHEDs incubated in GOQDs at different concentrations was evaluated; and the live cells were observed. We observed that live SHEDs incubated in GOQDs emitted green fluorescence in the absence of chemical dyes, and 1, 10, and 50  A growing number of recent studies have focused on bone tissue engineering technologies to address these issues. This refers to the technique of bone regeneration and repair of bone defects by inducing osteogenic differentiation in stem cells, accurately forming the desired tissue for the defect  patient –4. It isμg/mL GOQDs promoted SHED proliferation [In 2003, Miura and colleagues isolated a type of cell from the pulp of deciduous teeth that is highly self-renewable and has extensive proliferation and multidirectional differentiation potential that they called SHEDs . There aferation .β-catenin signaling pathway, which called for further investigation.GOQDs are a carbon nanomaterial of several nanometers per dot and have important properties of quantum confinement effect, stable photoluminescence (PL), and good biocompatibility . For theβ-catenin signaling pathway is a highly evolutionarily conserved pathway involved in regulating multiple biological processes [β-catenin in the cytoplasm and its subsequent translocation to the nucleus are important signs of pathway activation, which leads to the transcription of proteins in the Wnt/β-catenin pathway and downstream activation of target genes [β-catenin signaling in bone tissue, and it has been shown to regulate osteogenic differentiation in human dental pulp stem cells (hDPSCs). Jiang et al. [kif3a in hDPSCs was knocked down and Wnt3a was added to the osteogenic induction medium, the expression of the osteoblastic marker ALP and the Wnt/β-catenin signaling pathway marker β-catenin were significantly increased. This suggested that kif3a might promote osteogenic differentiation of hDPSCs. Li et al. [β-catenin expression, thus inhibiting SHED osteogenic differentiation. Liu et al. [β-catenin can act as a downstream regulatory protein for telomerase reverse transcriptase. They found that low-concentration acetylsalicylic acid upregulated Wnt/β-catenin pathway protein expression by improving the activity of telomerase reverse transcriptase and enhancing β-catenin expression, ultimately promoting SHED osteogenic differentiation. Our group has found that the expression trend for β-catenin following GOQD treatment is the same as that for osteoblastic markers, which induce osteogenic differentiation in SHEDs [The Wnt/rocesses . The proet genes . Many reg et al.  found thi et al.  demonstru et al.  indicatein SHEDs .β-catenin signaling pathway. DKK-1 was used to inhibit the Wnt/β-catenin signaling pathway, and β-catenin was knocked down to verify the mechanism by which GOQDs induce SHED osteogenic differentiation. We hypothesized that at a concentration of 10 μg/mL, GOQDs would promote osteogenic differentiation via the Wnt/β-catenin signaling pathway.In the present study, we investigated the effects of GOQDs on SHED osteogenic differentiation and explored the underlying mechanism. First, we prepared GOQDs using a bottom-up method ; then, w2O) were put into a beaker and heated to 200°C for 2–3 h. During the heating process, the CA was dissolved, and the colorless liquid changed to yellow and finally to orange, indicating GOQD formation. Next, the orange liquid was dialyzed in a 500 Da molecular weight dialysis bag for 48 h to remove CA, and then the liquid was freeze-dried to yield solid GOQDs, which were dissolved in tri-distilled water to obtain a GOQD suspension at a concentration of 1 mg/mL and sterilized with a 0.22 μm filter membrane .GOQDs were prepared by directly pyrolyzing citric acid  using the bottom-up method described previously . In detaThe nanosize morphologies of the GOQDs were characterized by transmission electron microscopy  at an acceleration voltage of 300 kV. The compositions of GOQDs were characterized by Fourier transform infrared spectrometry . The ultraviolet- (UV-) visible spectra of GOQDs were measured on a Lambda 950 spectrophotometer . The fluorescence spectra of GOQDs under 365 nm excitation wavelengths were measured on a Cary Eclipse .3 cells/well on 96-well plates, and then the SHEDs were treated with GOQDs at concentrations of 1, 10, 50, 100, and 200 μg/mL. After 1, 3, 5, and 7 days, the culture medium was replaced with 10 μL CCK-8 solution and 100 μL DMEM, and the plates were incubated at 37°C for 1 h. The optical density (OD) at 450 nm was measured using a microplate reader .Ethics committee approval was provided by the School of Stomatology, Sun Yat-sen University. SHEDs were isolated and identified as described previously . The cyt4 cells/dish, then treated with GOQDs at concentrations of 1, 10, and 50 μg/mL. After 3 days, SHEDs were washed with phosphate-buffered saline  and imaged.The morphology of SHEDs treated with GOQDs was observed via confocal laser scanning microscopy . SHEDs were plated on Petri dishes at a concentration of 1 × 10μM dexamethasone , 10 mM β-glycerophosphate (Sigma-Aldrich), and 50 μM ascorbic acid (Sigma-Aldrich), with 1, 10, or 50 μg/mL GOQDs, for 14 days. After culturing, the cells were fixed in 4% paraformaldehyde for 30 min, rinsed with PBS, stained with alizarin red  for 30 min at room temperature, and washed with PBS. Finally, mineralized nodules were photographed with an inverted microscope . The control group was incubated in OIM without GOQDs.SHEDs were seeded in six-well plates; upon reaching 70–80% confluence, they were treated with the osteogenic induction medium (OIM) created from DMEM with 10% FBS, 1% P/S, 1 μg/mL GOQDs for 3 and 7 days. The control group was incubated with OIM without GOQDs. ALP activity was determined using an ALP assay kit , according to the manufacturer's instructions.SHEDs were seeded in 24-well plates, and when 70–80% confluence was reached, they were cultured in OIM with 1, 10, or 50 μg/mL GOQDs for 7 and 14 days. The control group was incubated in OIM without GOQDs. Total RNA was extracted using the TRIzol reagent , following the manufacturer's protocol. PrimeScript™ RT Master Mix  was used to convert messenger RNA (mRNA) into complementary DNA (cDNA). Then, the cDNA was quantified using qRT-PCR, which was performed on a LightCycler 480 Detection System  with an SYBR Green Kit (Roche) using gene-specific primers. The primer sequences are shown in SHEDs were seeded in six-well plates, and when they reached 70–80% confluence, they were cultured in OIM, with 1, 10, or 50 μg/mL GOQDs for 14 days. The control group was incubated in OIM without GOQDs. Briefly, the radioimmunoprecipitation assay buffer  containing 1 mmol/L protease inhibitor cocktail  was used to lyse cells, a bicinchoninic acid (BCA) assay kit (CWBIO) was used to measure protein concentrations, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis  was used to separate proteins that were then transferred onto polyvinylidene fluoride membranes (Millipore). Next, 5% fat-free milk in Tris-buffered saline with Tween  was used to block membranes for 1 h at room temperature, which were then incubated with primary antibodies  and anti-collagen I (COL I), 1 : 500, Abcam, Cambridge, UK; anti-runt-related transcription factor-2 (RUNX2), anti-β-catenin, and anti-GAPDH, 1 : 1000, Cell Signaling Technology (CST), Danvers, MA, USA) for 18 h at 4°C. Finally, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibodies , and bands were detected with an enhanced chemiluminescence detection system (Millipore) and quantified using ImageJ v1.47 software . GAPDH was used as an internal control.SHEDs were seeded in six-well plates, and when they reached 70–80% confluence, they were cultured in OIM with 1, 10, or 50 μg/mL to induce osteogenic differentiation for the next experiment. SHEDs were seeded in six-well plates, and when they reached 70–80% confluence, they were cultured in OIM with GOQDs and with or without 100 ng/mL DKK-1. The cells were divided into three groups: control , DKK-1 , and GOQD (SHEDs cultured in OIM and GOQDs). The effects of DKK-1 relating to the GOQD-mediated induction of osteogenic differentiation in SHEDs were confirmed via qRT-PCR and WB assays.After the above steps, SHEDs were treated with GOQDs at a concentration of 10 β-catenin small interfering RNA (siRNA) lentivirus . siRNA against β-catenin GTATTTGAAGTATACCATA and a nonspecific shRNA construct were designed and cloned onto an hU6-MCS-CMV-EGFP vector. Control viruses that contained the EGFP tag  were also provided. After incubation for 10 h at 37°C and 5% CO2, the supernatant was removed and replaced with a culture medium for 3 days. Infected cells were imaged with a fluorescence inverted microscope (Zeiss), and the knockdown effect was confirmed via qRT-PCR and WB assays. SHEDs infected with β-catenin recombinant lentiviruses or empty lentiviral vectors were defined as the β-catenin-KD group and the control-si group, respectively.SHEDs were seeded into a culture flask, and when they reached 50% confluence, they were treated with DMEM containing enhanced green fluorescence protein-labeled (EGFP) μg/mL GOQDs to induce osteogenic differentiation for the next experiment. They were seeded in six-well plates, and upon reaching 70–80% confluence, they were cultured in OIM that included GOQDs. The cells were independently divided into three groups: control-si , GOQD-si (SHEDs infected with control viruses and cultured in OIM and GOQDs), and β-catenin-si (SHEDs infected with β-catenin recombinant lentiviruses and cultured in OIM and GOQDs). The effects of β-catenin on GOQD-mediated induction of osteogenic differentiation in SHEDs were confirmed via qRT-PCR and WB assays.SHEDs were treated with 10 p < 0.05. SPSS 20.0 software  was used for all statistical analyses.The data are shown as means ± standard deviations. One-way analysis of variance was used to perform statistical analyses, and the Bonferroni method was used for multiple comparisons. Differences were considered significant at −1 and 1390 cm−1 were observed due to stretching in hydroxyl groups (–OH) and stretching vibrations in carboxyl groups (C=O) at 1690 cm−1 and hydrocarbon groups (C–H) at 2880 cm−1. As shown in Figures GOQD morphology was observed using TEM ; they weμg/mL GOQDs significantly promoted SHED proliferation at days 3, 5, and 7. At the highest concentration of 200 μg/mL, GOQDs slightly suppressed cell viability . Therefore, we chose the 1, 10, and 50 μg/mL concentrations of GOQDs to perform the following assays.CCK-8 assays were used to evaluate SHED viability . The resIn vitro cell images are shown in μg/mL, SHEDs emitted green fluorescence, predominantly in the cytoplasm, under illumination at a wavelength of 488 nm. SHEDs that were not exposed to GOQDs and imaged under the same conditions did not show any fluorescence. In all groups, SHEDs showed a normal spindle-like or fibroblast-like morphology.μg/mL GOQD groups was greater than that in the control group, and it was less evident in the 50 μg/mL GOQD group. Calcium nodule formation in the 1 μg/mL group was greater than that in the 50 μg/mL group but less than that in the 10 μg/mL group. The highest level of mineralization was observed in the 10 μg/mL group.Calcium nodules were formed in all four groups after osteogenic induction for 14 days . Calciumμg/mL GOQD group was significantly higher than that in the other groups. ALP activity was increased in the 1 and 10 μg/mL GOQD groups compared to the control group, and it was significantly decreased in the 50 μg/mL GOQD group.After osteogenic differentiation of SHEDs for 3 and 7 days , ALP actOCN, RUNX2, and COL I and the Wnt/β-catenin signaling pathway-related mRNA β-catenin were higher in the 1 and 10 μg/mL GOQD groups and lower in the 50 μg/mL GOQD group. After 14 days of induction  used by Qiu et al. [μg/mL did not significantly inhibit mesenchymal stem cell (MSC) proliferation.A cytotoxicity assessment was conducted to determine the appropriate concentration of GOQDs to induce SHED proliferation. Cell growth curves are often used to measure logarithmic growth, which reflects the effects of materials on cell viability . A cell u et al. . Their gin vivo optical imaging [Many researchers are currently employing GOQDs in cell imaging , 28, rea imaging . Zhang e imaging  demonstrμg/mL GOQD group  were better able to induce osteogenic differentiation in bone MSCs than 50 μg/mL N-GQDs, matching the optimal concentration of GOQDs found in this study.To investigate further, qRT-PCR and WB assays were used to measure the mRNA and protein expression of osteogenic-related markers. OCN is a matrix protein expressed in the early stages of mineralization and highly expressed in the late stages of osteoblastic differentiation, and it plays an important role in bone tissue mineralization . RUNX2 iu et al.  found thg et al.  found thβ-catenin signaling pathway-related factor, β-catenin, were in accordance with those of OCN, RUNX2, and COL I. Upregulated β-catenin and its nuclear translocation are considered markers of Wnt/β-catenin pathway activation [β-catenin signaling pathway might regulate the GOQD-mediated induction of osteogenic differentiation in SHEDs. DKK-1 is an inhibitor of the Wnt/β-catenin signaling pathway [β-catenin in the cytoplasm and blocking signal transduction of the Wnt/β-catenin signaling pathway [μg/mL GOQD group to verify whether this could antagonize GOQD-induced osteogenic differentiation in SHEDs and preliminarily explored how the Wnt/β-catenin signaling pathway regulates this process. The results are shown in β-catenin expression, thus antagonizing the ability of GOQDs to induce osteogenic differentiation in SHEDs. To confirm this, β-catenin was silenced (β-catenin knockdown on SHED osteogenic differentiation are shown in β-Catenin knockdown reduced OCN, RUNX2, and COL I at the mRNA and protein levels, which supports our assessment of the effects of DKK-1.The mRNA and protein expression trends of the Wnt/tivation , 23. The pathway , which c pathway , 42. In silenced , and thein vitro results demonstrated that GOQDs promote SHED proliferation and osteogenic differentiation in a concentration-dependent manner and that the Wnt/β-catenin signaling pathway is activated during this process. However, further in vivo research is necessary to verify these results.Finally, the β-catenin signaling pathway is involved in regulating these effects. This work provides new ideas and fundamental information on the interactions between GOQDs and SHEDs that are relevant for the biomedical engineering field.GOQDs promote concentration-dependent proliferation and osteogenic differentiation of SHEDs, and the Wnt/


Protein methyl transferases play critical roles in numerous regulatory pathways that underlie cancer development, progression and therapy-response. Here we discuss the function of PRMT5, a member of the nine-member PRMT family, in controlling oncogenic processes including tumor intrinsic, as well as extrinsic microenvironmental signaling pathways. We discuss PRMT5 effect on histone methylation and methylation of regulatory proteins including those involved in RNA splicing, cell cycle, cell death and metabolic signaling. In all, we highlight the importance of PRMT5 regulation and function in cancer, which provide the foundation for therapeutic modalities targeting PRMT5. Posttranslational modifications (PTMs) are critical for proteome diversification. Protein modification on one or multiple sites can determine its conformation, subcellular localization, interaction with other proteins, stability, and/or activity. Such PTMs are mediated by diverse enzymatic processes, among them, phosphorylation, acetylation, ubiquitination, methylation and hydroxylation, and in turn reversed by enzymes that antagonize them, such as phosphatases, deubiquitinating enzymes, deacetylases and demethylases, to name a few.Schizosaccharomyces pombe) and Hsl7p . PRMT5 was later characterized as a distinct type of mammalian protein arginine N-methyltransferase [Protein methylation on arginine residue was initially reported in late 1960s and early 1970s . The firnsferase .Arginine-methylated proteins function in a number of key cellular processes required for maintenance of tissue homeostasis as well as diseases phenotypes. Use of both genetic (KO (knockout) mouse models) and pharmacological  tools has established the importance of arginine methylation in stem cell activity, development, neurodegenerative disease and cancer (reviewed in ). Among G-monomethylarginine (MMA) and asymmetric ω-NG, NG – asymmetric dimethylarginine (aDMA). The type II PRMTs PRMT5 and 9 catalyze MMA and ω-NG, NG – symmetric dimethylarginine (sDMA). PRMT7, a type III PRMT, catalyzes only MMA. These modifications elicit a steric effect and change hydrogen bonding interaction of the methylated side chain, in turn altering molecular characteristics and function of the modified protein.PRMT family enzymes serve as “writers” of PTM, catalyzing three distinct types of methylation. In all, a methyl (-CH3) group from the methyl donor S-adenosylmethionine (SAM or AdoMet) is transferred to a guanidinium nitrogen of arginine on a target protein, generating a methylated guanidinium moiety and S-adenosylhomocysteine (SAH or AdoHcy), which is salvaged and re-used for methionine biosynthesis. In human cells, nine PRMTs catalyze three distinct methylation reactions. Type I PRMTs, including PRMT1, 2, 3, 6, 8, and CARM1 (PRMT4), catalyze ω-NPRMT5 forms a unique hetero-octameric complex, which is composed of four PRMT5 proteins plus four essential cofactors, MEP50 (methylosome protein 50)/WDR77 (WD repeat domain 77). The unique N-terminal TIM barrel structure of the PRMT5 monomer enables formation of PRMT5 tetramer at the center of octameric complex and subsequent decoration of the PRMT5 tetramer with four MEP50 molecules , 12. TheThe C-terminal catalytic domain of PRMT5 consists of two domains, the Rossam fold and β-barrel domains, required for binding cofactor  and substrate, respectively. The structural restraints of substrate binding dictate preferential methylation of glycine-rich sequences, which allow conformational freedom of polypeptide chain to form a sharp β-turn . Indeed,PRMT5 methylation of proteins is implicated in control of normal and pathological conditions, among them development, tissue homeostasis and cancer. Global PRMT5 KO in mice results in embryonic lethality , indicatTable 1). The intrinsic roles of PRMT5 in cancer could be marked by altered expression of PRMT5 or of its adaptor proteins or by altered availability of factors that control its catalytic activity. Such deregulation of PRMT5 catalytic activity is often reflected by arginine methylation of target proteins functioning in either epigenetic regulation of gene expression, splicing, or signal transduction.PRMT5 plays a complex role in oncogenesis, as it is known to control expression of genes implicated in both tumor promotion and suppression .Arginine methylation of histone tails is an epigenetic modification catalyzed by PRMT writers that controls gene expression. Type I or type II PRMTs introduce MMA, aDMA or sDMA to arginine residues in histone tails, enabling recruitment of specific reader(s) of those modified arginine residues, along with other chromatin remodeling modifiers. This in turn determines histone marks leading to gene activation or repression  PRMT5 repression of miR-29b upregulates FLT3 –24.While H3R8me2s and H4R3me2s are largely considered repressive marks, they are also implicated in transcriptional activation of some genes, as has been shown for FGFR3 and eIF4E expression in colorectal cancers , and AR H4R3 methylation by PRMT5 (H4R3me2s) was also linked with gene repression, through histone ubiquitination or DNA methylation. PRMT5 methylation of H4R3me2s results in its interaction with PHF1 (plant homeodomain (PHD) finger protein-1) through the N-terminal PHD domain, while its second PHD motif binds DDB1, a component of CRL4B-Ring E3 ligase complex. By bridging DDB1 to methylated histones PHF1 enables the monoubiquitination of H2AK119, which is essential to maintain PcG (polycomb group)-target gene repressive identity , 30. PRMFigure 1) [In contrast, PRMT5 methylation on H3R2 is generally associated with transcriptional activation of target genes. Notably, H3R2 can be methylated by PRMT5 or PRMT6, resulting in three types of methylation, H3R2me1, H3R2me2a or H3R2me2s. H3R2me2s, catalyzed by PRMT5, recruits WDR5 (a reader with a tudor domain recognizing sDMA) along with the MLL-family coactivator complex, resulting in H3K4me3, indicative of an active promoter, in the euchromatic loci –34. On tigure 1) –37. Struigure 1) . Genotoxigure 1) . PRMT5 figure 1) .Figure 2). STRAP, a factor recruited to the p53 complex during the DNA damage response (DDR), recruits PRMT5 and facilitates arginine methylation within the p53 oligomerization domain, decreasing p53 oligomerization which in turn increases nuclear retention of p53, with a concomitant increase in the expression of its target genes p21 and PUMA [Several non-histone proteins with tumor promoting or suppressing functions have been identified as PRMT5 substrates  [PRMT5 methylation (sDMA) of arginine 30 in the DNA-binding domain of p65/RelA, a subunit of the NF-κB transcription factor, activates NF-κB transcriptional activity via enhancing its DNA binding affinity . Conversma (HCC) . PRMT5 mma (HCC) . Mechanima (HCC) . PRMT5 mma (HCC) . Our stuma (HCC) .Growing evidence supports a crucial role of constitutive and alternative RNA splicing in control of genes driving cancer phenotypes , 55. ThuFigure 3) [Early studies identified PRMT5 as part of the 20S methylosome containing MEP50, pICln, SmD1, SmD3 and SmB, in which the three Sm proteins were methylated by PRMT5 (igure 3) , 59. Metigure 3) , 59, 60.igure 3) –63.In mouse neural progenitor cells, PRMT5 deletion causes defects in splicing of mRNAs with weak 5′ donor sites. Such defects take place in alternative splicing of Mdm4 in the absence of PRMT5, resulting in a shorter and less stable Mdm4 mRNA that activates the p53 pathway . Indeed,Notably, cyclinD1/CDK4 also induces MEP50 phosphorylation which increases PRMT5 activity , and corFigure 3).PRMT5 control of splicing fidelity is also a factor in c-Myc-driven lymphomagenesis. Gene sets enriched in Eµ-myc B cell tumors include transcripts associated with snRNP biogenesis, RNA processing and RNA splicing, among them PRMT5. Myc- or PRMT5-depletion resulted in aberrant splicing (either exon skipping or retained introns) of genes associated with cell cycle arrest or apoptosis . ConsistTIP60/KAT5, a histone lysine acetyltransferase that drives homologous recombination (HR) DNA repair, is also regulated by PRMT5-mediated alternative splicing. In response to DNA damage, PRMT5 facilitates alternative splicing of TIP60/KAT pre-mRNA to a TIP60a isoform with higher H4 lysine acetylase activity, which ensures error-free HR DNA repair and maintenance of genome integrity in hematopoietic cells .In addition to PRMT5 methylation that alters its transcriptional activity, E2F1 is also implicated in regulation of alternative splicing following PRMT5-methylation. E2F1 methylation promotes recruitment of p100/TSN as well as the snRNP spliceosome, which regulates splicing of E2F1 targets , 71. Furin vitro and in vivo growth of tumor cells more effectively than treatment with either alone [Given that numerous splicing-related proteins are methylated on arginine, it is not surprising that PRMT inhibition evokes defects in splicing fidelity of genes critical to cancer , 56, 57.er alone , 75. Proer alone , 75.Tumor cells control protein translation as a means to adapt to tumor intrinsic and extrinsic stress conditions. PRMT5 reportedly interacts with and modifies (sDMA) RPS10  which serves ribosome assembly. Such modification facilitates RPS10's ability to interact with NPM1 , which ensures proper ribosome assembly, and general protein synthesis, supporting tumor cell proliferation . hnRNPA1In addition to transcriptional activation of FGFR genes by PRMT5 in lung and colon cancers , 78, 79,Figure 4) [Arginine methylation of EGFR by PRMT5 occurs on a residue proximal to the auto-phosphorylated tyrosine following EGF-stimulation and thereby facilitates SHP recruitment and reduces ERK-activation and proliferation/migration of breast cancer cells . Along tigure 4) . These sBy contrast, PDGFR (platelet derived growth factor receptor) signaling in oligodendrocyte progenitor cells is positively regulated by PRMT5-methylation . PRMT5-mNF-κB plays a crucial role tumorigenesis and is modified by PRMT5 in response to diverse cellular signals. As discussed above, PRMT5 activates NF-κB by direct methylation of the p65 subunit . PRMT5 aTGFβ signaling can both up- or down-regulate target genes involved in growth and the epithelial-mesenchymal-transition. SKI, a SMAD-interacting transcriptional repressor, is methylated by the PRMT5-MEP50-SHARPIN complex, which fine-tunes transcriptional regulation of TGFβ signaling on a specific subset of genes .Figure 5).The DDR plays crucial roles in maintaining genomic stability by activating cell cycle checkpoints and DNA repair processes in response to anti-cancer therapies, including ionizing radiation and chemotherapy –87. ProtPRMT5 regulates expression of cell cycle/apoptosis-associated genes by modifying p53, E2F-1 and KLF4 during the DDR. PRMT5 controls H2AX protein availability by transcriptional activation of RNF168, an E3 ligase introducing K-63 ubiquitination on N-terminal K13, thereby protecting H2AX from Smurf2-mediated K-48 degradative ubiquitination on C-terminal K119. Consequently, PRMT5 ensures formation of phosphorylated H2AX (γH2AX) foci at DNA double strand break (DSB) for recruiting DNA repair proteins .PRMT5 also directly modifies and controls components of DDR-associated pathways. For maturation of Okazaki fragments during replication, FEN1, a special DNA structure-specific flap endonuclease, which is important for DNA replication and repair, is recruited to replication foci by binding to PCNA (proliferating cell nuclear antigen). PRMT5 methylation of FEN1 at R192 facilitates its interaction with PCNA by inhibiting FEN1 phosphorylation at S187 by CDK1/cyclin A or CDK2/cyclin E, ensuring interaction between FEN1 and PCNA for intact DNA replication and DNA repair processes at replication and repair foci . Rad9, aTop1 (DNA topoisomerase 1) relieves DNA supercoiling during replication and transcription by forming a transient and reversible protein-ssDNA-linked structure, Top1cc (Top1 cleavage complex), that is hydrolyzed by TDP1 (tyrosyl-DNA phosphodiesterase 1). Unresolved Top1cc complexes often cause DNA DSBs and the DDR upon replication or transcription. PRMT5-catalyzed methylation of two arginines in TDP1 directly enhances TDP1 catalytic activity and promotes formation of repair foci containing the XRCC-1 complex at Top1cc-induced DNA damaged sites by facilitating interaction with XRCC1, and thereby resolving trapped Top1cc .Growing evidence points to a link between genetic and epigenetic alterations in oncogenesis and anti-tumor immune responses. PRMT5 depletion antagonizes melanoma growth in immunocompetent but not immunocompromised mice whereas PRMT5 overexpression accelerates tumor growth. Increased abundance of infiltrated immune cells seen upon PRMT5 inhibition coincides with enhanced anti-tumor immunity . PRMT5 cNotably, PRMT5 plays several roles in hematopoiesis as in the development, activation and differentiation of diverse immune cells. Conditional PRMT5 knockout in hematopoietic cells causes bone marrow (BM) aplasia, due to loss of hematopoietic progenitor cells that exhibit severe defects in cytokine signaling . ProteomParadoxically, PRMT5 is also required for the suppressive function of regulatory T cells (Treg), which is essential to suppress autoimmunity. Mice with conditional PRMT5 knockout in Treg cells develop severe scurfy-like autoimmunity. Treg cells lacking PRMT5 or treated with PRMT5 inhibitors show loss of immunosuppressive function due to the loss of R51 methylation in FOXP3, a master regulator of Treg . NotablyFigure 6).PRMT5 expression is upregulated in most cancer types, and in most cases such upregulation is associated with poor patient survival. Given that genetic alterations  are rare in PRMT5 genes, epigenetic control of PRMT5 in cancer emerges as a key player in the control of its expression and activity , a chaperone-dependent E3 ligase functioning in protein-folding-associated degradation, reportedly promotes the proteasomal degradation of PRMT5 following treatment with the HSP90 inhibitor 17-AAG . LINC011PRMT5 activity is itself regulated by multiple PTMs including phosphorylation/dephosphorylation, methylation, acetylation and protein-protein interactions. The JAK2 mutation JAKV617F, found in most non-CML MPN (myeloproliferative neoplasia), facilitates JAK2 interaction with and phosphorylation of PRMT5, which attenuates methylase activity by preventing interaction with MEP50 . A threoPRMT5 recruitment to a given substrate resulting in its methylation depends on its associated adaptor proteins, which define its conformation, activity and subcellular localization. PRMT5 interaction with MEP50/WDR77 in hetero-octameric complex increases its stability and activity . PRMT5 mPRMT5 activity is also limited by the endogenous inhibitor, 5′methylthioadenosine (MTA), a byproduct of the polyamine synthesis pathway, which is metabolized by methylthioadenosine phosphorylase (MTAP). However, due to proximity to CDKN2A on Chr9p21, MTAP is co-deleted with CDKN2A in 15-40% of human cancers, resulting in accumulation of unmetabolized MTA and attenuation of endogenous PRMT5 activity. Accordingly, MTAP loss confers vulnerability to further inhibition of PRMT5 or of type I PRMTs , 125–128Given that PRMT5 participates in a wide range of physiological processes, crucial for maintenance of cancer phenotypes, perturbation of PRMT5 is expected to provide novel means to treat cancer. Along these lines, PRMT5 plays an important role in embryonic stem and tissue specific stem/precursor cells , 130, anSignificantly, PRMT5 inhibition in established tumors could disable mechanisms protecting tumor cells from DNA damaging stress brought on by radiotherapy and chemotherapy. Indeed, PRMT5 depletion/inhibition sensitizes tumor cells to drugs inducing the DDR, exemplified by cytarabine , PARP iP95H) or treatment with an SF3B1 inhibitor renders AML cells vulnerable to PRMT5 and/or PRMT1 inhibition [Emerging evidence suggests that disruption of splicing fidelity by PRMT5 depletion/inhibition could have an anti-cancer effect , 130. Inhibition . Likewishibition .The unprecedent success of ICT is hampered by limited responses seen in a fraction of patients or by development of resistance. PRMT5 activity suppresses anti-tumor immune responses by supporting tumor intrinsic mechanisms allowing immune evasion, Treg function, and repression of IFN-1 and MHCI pathways , 103, prIdentification of novel biomarkers is important to allow stratification of patients for precision anti-cancer treatment and improve therapeutic efficacy. The presence of SF mutations and MTAP homozygous deletion represents examples of biomarkers that aid in patient stratification for targeting PRMT5 , 125–127The first in-class PRMT5 inhibitor, EPZ015666, was reported in 2015 . EPZ0156PRMT5 regulates numerous cellular pathways but exerts major effects on histone function in transcription and RNA splicing. PRMT5 directly methylates many proteins to control their subcellular localization, protein-protein interactions, stability or activity. Many of these contribute to oncogenic transformation, and thus evaluation of potential PRMT5 inhibitors is warranted. Given the importance of PRMT5 in oncogenesis, deciphering mechanisms underlying PRMT5 expression, activity as well as subcellular localization will advance the development of advanced PRMT5-related therapeutic modalities. Thus far, researchers have largely focused on PRMT5 control of tumor intrinsic changes; however, PRMT5 effects on the tumor microenvironment, and on immune cells in particular, should be assessed in future studies. Another current knowledge gap is understanding dynamic cross talk between different PRMT family members, as exemplified by recent studies showing that PRMT1 and PRMT5 modification of a single protein may regulate opposing functions. Importantly, inhibition of one PRMT member may impact activity of another, as shown for increased PRMT5 methylation upon PRMT1 inhibition. Potential interplay among family members is critical to understand when using selective inhibitors, and in some cases, may justify targeting multiple PRMT members. Lastly, PRMT5 activity is reportedly downregulated in 30% of human cancers, due to its negative regulation by MTA, which is enhanced upon MTAP-co-deletion with CDKN2A. The notion that genetic-based stratification for PRMT5 inhibition can help map tumors vulnerable to PRMT5 targeting remains attractive and deserves to be further assessed.