The mitochondrial targeting domain (MTD) of Noxa contributes to its mitochondrial localization and to apoptosis induction. As a peptide, MTD fused with octa-arginine (R8), a CPP, induces necrosis related to intracellular calcium influx and destruction of mitochondria and endoplasmic reticulum. We searched for homologs of MTD, and compared their cell killing capability when fused with R8. Three of the seven peptides triggered cell death with similar mechanisms. The comparative analysis of peptide sequences showed that four amino acid sites of MTD are critical in regulating necrosis, suggesting the potential to generate artificial, adjustable cytotoxic peptides, which could be effective medicines for many diseases. Thus, homologs functionality could hint to the functions of their belonging proteins. Noxa protein belongs to the Bcl-2 family and consists only of a Bcl-2 homology 3 (BH3) domain as well as a mitochondrial targeting domain (MTD) , 2. In r2The MTD, in addition to its targeting function, strongly induces necrotic cell death with its flanking region  or cell-penetrating peptide, octa-arginine R8) , 7. With6 , 7. WitBnip1 MTD-like motif (B1MLM), derived from another BH3-only protein, Bnip1, also induces cell death in the same manner . Bnip1 iIn this study, we searched for more MTD homologs and analyzed their sequences and cell death activity. Some of them showed strong cytotoxicity accompanied by intracellular calcium influx, formation of membrane blebs, generation of mitochondrial ROS, and ER destruction. Analysis of their sequences evidenced several critical amino acid sites, and suggested that artificially produced cytotoxic MTD peptides could control cytotoxicity. Moreover, it showed the possibility that proteins containing these homologs may be involved in cell death processes.2 atmosphere.HeLa cells were purchased from the Korean Cell Line Bank (Korea) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum and 1× penicillin-streptomycin  at 37°C in a humidified 5% COAll the peptides in All the sequences of homologs were aligned using Clustal Omega and ClustalW base/residue numbering. The sequences were sorted according to their percent identity with MTD. Each amino acid was colored according to the ClustalX color scheme.HeLa cells were cultured in 96-well plates to 90% confluency and treated with peptides mixed in phenol red-free DMEM for 1 h. After incubation, MTS solution  was added and the absorbance of each well was measured at 450 nm.2, 0.41 mM MgSO4, 5.33 mM KCl, 0.44 mM KH2PO4, 4.17 mM NaHCO3, 137.93 mM NaCl, 0.34 mM Na2HPO4, 5.56 mM D-glucose, and 1.26 mM CaCl2; Gibco-Thermo Fisher) and treated for 10 min. The cells were washed with pre-warmed HBSS buffer and treated the peptides. Immediately after the treatment, we obtained time-lapse images using an argon laser scanning confocal microscope (Leica TCS SP5 Microsystems of KBSI Gwangju Center) at 5-s intervals, 488 nm excitation, to visualize Fluo-4-AM and MitoSOX.HeLa cells were cultured on a Lab-Tek Chamber glass slide to 60% confluency. Fluo-4 AM  and MitoSOX , used to monitor intracellular calcium and mitochondrial ROS, respectively, were mixed in HBSS buffer  to HeLa cells and measured the intensity of Fluo-4 using confocal microscopy with argon laser. Treatment with R8:SDAD1 to HeLa cells showed a pattern most similar to that of R8:MTD . ImmediaIn addition to MTD-induced cell death, mitochondrial ROS formation plays an important role in other cell death mechanisms, such as apoptosis and necrosis. Therefore, we assumed that these cytotoxic MTD homologs might also induce mitochondrial ROS formation. To measure the mitochondrial ROS level, we treated HeLa cells with the mitochondrial ROS indicator MitoSOX and the cytotoxic MTD homologs, and then measured the fluorescence intensity using time-lapse confocal microscopy. Cytotoxic MTD homologs like R8:M-SDAD1 , R8:M-TRIn a previous study, we showed that R8:MTD induced the disruption of ER and mitochondria, as the binding target of MTD is VDAC, and VDAC is also expressed on the ER and mitochondria membranes . TherefoThe many homologs of Noxa protein MTD show different cytotoxicity. In addition to previously reported leucine (fifth and ninth) residues, phenylalanine (tenth) seems to be critical in maintaining cytotoxicity as well. R8:M-SDAD1, R8:M-TRRAD, and R8:M-ST3GAL3 showed strong cytotoxicity, with a mechanism, which may be the same as that adopted by R8:MTD. The intracellular calcium spike, mitochondrial ROS formation, and ER disruption induced by R8:M-SDAD1, R8:M-TRRAD, and R8:M-ST3GAL3 in HeLa cells were inhibited by DIDS. These results imply that these homologs could bind to VDACs as well. Amino acid replacement studies to investigate the peptide sequences of the homologs may be helpful to synthesize cytotoxic peptides and artificially control their cytotoxicity for medical applications, e.g., medicines for cancer or other diseases. Furthermore, the possibility of MTD homologs binding to mitochondria and ER could be a new path to reveal other protein functions, such as induction of cell death.


Magnaporthae oryzae (M. oryzae) is the most destructive disease of rice worldwide. In this study, one hundred and two isolates of M. oryzae were collected from rice (Oryzae sativa L.) from 2001 to 2017, and six rice varieties with resistance genes Pizt, Pish, Pik, Pib, and Pi2 were used in a genome-wide association study to identify pathogenicity loci in M. oryzae. Genome-wide association analysis was performed using 5338 single nucleotide polymorphism (SNPs) and phenotypic data of neck blast screening by TASSEL software together with haplotype block and SNP effect analysis. Twenty-seven significant SNPs were identified on chromosomes 1, 2, 3, 4, 5, 6, and 7. Many predicted genes (820 genes) were found in the target regions of six rice varieties. Most of these genes are described as putative uncharacterized proteins, however, some genes were reported related to virulence in M. oryzae. Moreover, this study revealed that R genes, Pik, Pish, and Pi2, were broad-spectrum resistant against neck blast disease caused by Thai blast isolate. Haplotype analysis revealed that the combination of the favorable alleles causing reduced virulence of isolates against IRBLz5-CA carrying Pi2 gene contributes 69% of the phenotypic variation in pathogenicity. The target regions and information are useful to develop marker-specific genes to classify blast fungal isolates and select appropriate resistance genes for rice cultivation and improvement. Magnaporthe oryzae asexual stage (anamorph Pyricularia oryzae (P. oryzae) sexual stage) is the most destructive rice (Oryza sativa L.) disease worldwide. Leaf blast and neck blast are the two rice blast pathosystems caused by the same fungus [Avr) in the pathogen and a matching resistance gene (R) in the host [Avr protein or effector molecule secreted by a pathogen can directly/indirectly trigger a specific molecule in the plant to initiate a resistance pathway [Avr genes is crucial to understand the resistance process and the potential utility of resistance genes. Currently, more than 40 Avr genes have been identified in rice blast fungus and only 11 Avr genes have been cloned, such as PWL1, PWL2, Avr-Piz-t, Avr-Pita, ACE1, Avr-Pia, Avr-Pib, Avr1-CO39, Avr-Pik, Avr-Pii, Avr-Pi9, and Avr-Pi54 [Avr genes have been identified by QTL mapping approaches and are specific to leaf blast disease.Rice is an important food crop and is very susceptible to diseases like blast, which is a known threat to global food security. This disease alone is responsible for approximately 30% of global yield loss . Rice ble fungus . Rice ble fungus ,5. Underthe host . The Avr pathway . TherefoAvr-Pi54 . Most AvXa43(t) novel resistance gene for bacterial blight in rice [AvrPi7, 2, 3, 4, and 7 in M. oryzae against leaf blast disease [Pi24 [Pb1 gene was susceptible at a young stage and resistant at reproductive stage [Genome-wide association studies can be a powerful tool to identify candidate alleles that control quantitative and qualitative traits in plants, microbes, and animals . GWAS ha in rice  and resi in rice . It has  disease . Pathogese [Pi24 . The ricve stage . TherefoM. oryzae corresponding to resistance genes for neck blast disease in the six cultivars, by analyzing the association between genetic variation and disease response. This study will provide more information to understand the genetics controlling the virulence of the fungus and will help breeders to select the appropriate R genes to be used in breeding programs to improve varieties with broad spectrum and durable resistance to rice blast.In this study, we aimed to determine the response of six rice cultivars, IRBLzt-T, IRBLsh-S, IRBLk-K, IRBLb-B, IRBLz5-CA, and Lijiangxintuangheigu (LTH)  against blast fungus and identify SNPs associated with pathogenicity genes in M. oryzae collected from several locations in Thailand between 2001–2017 were used for whole genome re-sequencing and disease screening on rice ; the genetic background of developed rice IRBL was used as universal susceptible check for disease evaluation.Six rice varieties were used for neck blast disease screening. Five rice varieties containing different blast resistance genes were developed by the International Rice Research Institute (IRRI)  includinM. oryzae isolates70-15, assembly version MG8, (release-51: http://fungi.ensembl.org/Magnaporthe_oryzae/nfo/Index accessed on 20 September 2019).Genomic DNA was extracted by using a high purity PCR template preparation kit  from mycelia that were cultured and collected . Measure4 conidia/mL. Inoculation was carried out by injecting 1 mL per panicle at the reproductive stage–when the panicle emerges 1 to 3 cm above the flag leaf– using a syringe. Three replications of 10 plants per replicate were inoculated. The disease was assessed at 14 days after inoculation using a scale of 0, 1, 3, 5, 7, and 9 (0 is highly resistant and 9 is highly susceptible) [Neck blast disease inoculation was performed under greenhouse condition. Blast isolates were cultured for seven days at 25 °C on rice polish agar (RPA) medium, . The mycelia were scraped, and sporulation was induced under blacklight cabinet for two days. Then, clean water was added to collect the conidia. The suspension of conidia was adjusted to a concentration of 1 × 10eptible) . The diseptible) . 2 correlation (indep-pairwise) more than 0.3 in every 50 kb were filtered out. After filtering, linkage disequilibrium (LD) was analyzed in PLINK version 1.9. The association analysis was performed by TASSEL version 5.7.2 using 5338 selected SNPs and infection phenotypes on the six rice varieties. SNPs above a threshold of −log10 (P) ≥ 3.0 were considered significantly associated with the six traits.Whole genome re-sequencing was performed across all blast isolates by MGIseq200, and the sequences were then compared with the reference sequence–isolate 70-15–and SNPs were called to constitute the genotype data. A total of 310,530 SNPs were filtered using PLINK version 1.9, by eliminating genotypes with >10% missing data and rare SNPs with <5% minor allele frequencies (MAF). Subsequently, the genotype was pruned based on linkage disequilibrium. By using PLINK version 1.9, SNPs having rFrom the GWAS results, significantly associated SNPs were observed for their linkage disequilibrium blocks and haplotypes. Haploview software was implemented with the solid spine method for defining blocks in each chromosome . After bM. oryzae (MG8) (https://fungi.ensembl.org accessed on 1 April 2021), UniProtKB (www.uniprot.org accessed on 26 November 2021), and PHI-base (www.phi-base.org accessed on 27 November 2021).In identifying candidate genes, linkage disequilibrium (LD) decay was considered. LD decay was analyzed by PopLDdecay software , and thePish), IRBLk-KA (Pik) and IRBLz5-CA (Pi2) were the top three broad-spectrum resistant (BSR) rice with levels of over 90%, while LTH had the lowest BSR at 60.71% . The population structure of M. oryzae was divided into four groups   . Most of and G4) , IRBLk-KThere were 10 significant SNPs on chromosomes 1, 2, 3, 4, 5 and 7 for IRBLsh-S, 8 SNPs on chromosomes 1, 2, 3, 5, and 6 for IRBLz5-CA, 5 SNPs on chromosomes 1, 2, and 6 for IRBLk-KA, 1 SNP on chromosome 3 for IRBLzt-T, 1 SNP each on chromosome 6 for IRBLb-B and LTH .Pizt), IRBLsh-S (Pish), IRBLk-KA (Pik), IRBLb-B (Pib), Lijiangxintuangheigu (LTH) (Sck), and IRBLz5-CA (Pi2), were used in the analysis. SNPs associated with average disease score were detected on chromosomes 1, 2, 3, 4, 5, 6, and 7 using MLM analysis -C6 fungal-type domain-containing protein. This gene functions to reduce virulence in IRBLz5-CA.SNP-trait association analysis was performed using TASSEL v.5 to identify pathogenicity genes. Disease reactions on six rice varieties carrying different blast resistance genes, IRBLzt-T  = 20.04, p = 0.001) and TTAC (t (50) = 20.04, p = 0.001) was statistically significant.Haplotype analysis of four SNPs for IRBLz5-CA revealed that alleles C, T, A, and T from MG2_5441644, MG3_1645410, MG6_703373, and MG6_957987 contributed 18%, 28%, 14%, and 16% of PVE, which signals reduced virulence of the isolates against IRBLz5-CA carrying Pi2 gene . OverallM. oryzae and Oryza stiva interaction has been shown to be a classic gene system [Avr) may be important for plant–pathogen interactions. In rice, the interaction between host plant and pathogen M. oryzae is well documented as a gene-for-gene system. In this study, we used a differential system consisting of five differential R genes and a universal susceptible LTH for the identification of the pathogenicity specific to neck blast resistance gene under greenhouse condition. Based on the disease score, IRBLzt-T and IRBLb-B–having Pizt and Pib R genes–and LTH were significantly susceptible to neck blast, and in rice variety F145-2 containing Pi-b gene showed that the resistance was broken down and caused leaf blast disease; thus, Pi-b gene may not be recommended in Thailand. The susceptibility of the Pib gene has been shown in the monogenic IRBLb-B as well as LTH when tested in three field blast nurseries in Korea [R genes, Pish, Pik, and Pi2 showed strong resistance to leaf blast diseases not only in the greenhouse but also under field conditions [Pish, Pik, and Pi2 or Piz5 R genes showed resistance against neck blast in many rice growing regions using different isolates. Therefore, these varieties can be useful to help breeders to select resistant R genes for breeding programs in Thailand. In a previous study, R genes ), Pi9, Pish, Pib, Piz-5 or Pi2, Pita, Pita-2, and Pi19) were recommended to be chosen for developing rice potentially resistant to blast disease breeding programs in Thailand [Pi1, Piz-5, and Pita-2 genes have been reported to be effectively resistant to blast disease in Thailand [The e system , and a min Korea  and in iin Korea . Among tnditions ,29. For Thailand . Pi1, PiThailand . These iM. oryzae can be divided into four groups, as shown in Pik, Pish, and Pi2 maybe suitable in the northeast region of Thailand. On the other hand, Pib and Pizt may not show broad resistance in the northeast [Pish and Pi7. Jao Hom Nin had been used in breeding programs in Thailand by introgressing QTL for blast resistance found in Jao Hom Nin to the rice cultivar RD6 [Pib and Pizt may not show broad resistance to blast in northeastern Thailand. Although there is a tendency for the blast isolates from the northeast to be grouped in G3 and G4, the rest of the isolates were mixed in every group and therefore cannot be distinguished by regional origin indicating low relationship between year of collection, location, and genotypic data. However, more investigation is needed to gain more information in the mixture of isolates in clusters G1 and G2. Thailand is known as an origin and place of high diversity of M. oryzae [M. Oryzae, requires two mating types: MAT1-1 and MAT1-2. Additionally, it was observed in north, northeast, and central Thailand that most of the isolates were of the same mating type (MAT1-2) [The blast isolate population of ortheast , as it hivar RD6 . Unfortu. oryzae . However(MAT1-2) ,35. M. oryzae that correspond to resistance genes for neck blast disease in six rice cultivars, by analyzing the associations between genetic variation of 102 isolates and their disease response on rice varieties. The analysis was performed with IRBLzt-T, IRBLsh-S, IRBLk-KA, IRBLb-B, IRBLz5-CA, and LTH . A number of SNPs (5338) was used in genome wide association, and we found the significant SNPs linked to Avr genes on chromosomes 1, 2, 3, 4, 5, 6, and 7. Some of the chromosomes corresponded to leaf blast QTL on chromosomes 1, 2, 3, 4, and 7 [In this study, we aimed to identify SNPs associated with pathogenicity genes in 4, and 7 , except MoPLC2 (Phi ID: 2018) in which the mutant phenotype shows loss of pathogenicity. The C2 domain in MoPLC2 is involved in Ca2+-dependent membrane binding. The hyphal growth rate of MoPLC2 mutant was normal other than the conidial production being reduced, which probably explains the loss of pathogenicity [Zn2Cys6 transcription factor and is responsible for reduced virulence in mutant phenotype. The Zn2Cys6 transcription factor in M. oryzae is essential in fungal development, and is involved in pathogenicity at multiple developmental stages [M. oryzae. The information from this study will be useful to narrow down and develop specific primers in the future. The gene MGG_05332 has phosphatidylinositol phospholipase C activity related with intracellular signal transduction. This gene was found between MG3-5427965 to MG3-5431132 on chromosome 3, and about 29 Kb away from the GWAS significant SNP at position 5461007 for the variety IRBLz5-CA. From the Phi database (Phi-base.org), gene MGG_05332 is also known as genicity . This col stages . This stR genes to identify resistance and susceptible haplotypes [For haplotypes in relation to phenotype, there is lack of samples for some haplotypes, such as in IRBLsh-S, for which some allele combinations  were not identified. By using more varieties and isolates, the separation of the phenotype from its haplotypes should be guaranteed. It was determined from plotypes ,45,46, wplotypes . In thisR genes, Pik, Pish, and Pi2 were broad spectrum resistance against 90% of Thai blast isolates inoculated. Moreover, these genes were the most resistant when M. oryzae isolates were grouped based on the genotypic data. This is the first gene identification study for pathogenicity in M. oryzae for neck blast disease on rice. GWAS and haplotype analyses resulted in the identification of 27 significant SNP positions on chromosomes 1 to 7 associated with pathogenicity of blast disease fungus. Haplotype analysis revealed that specific allele combinations reduced fungus virulence and therefore resulted in increasing blast resistance in rice. Two genes, MGG_05332 and MGG_17623, were reported to be related to virulence in M. oryzae and located close to significant SNPs in IRBLz5-CA, MG3-5461007, and MG6-957987 on chromosomes 3 and 6, respectively. Therefore, these genes should be useful for rice blast disease resistance breeding programs.Neck blast screening in this study revealed that


Fluorescent in situ hybridization indicated germline-specific localization of lnc196 and lnc304 in the cytoplasm and nucleus of spermatogonia, spermatocyte, and occyte, and they were co-localized with vasa in the cytoplasm of the spermatogonia. By contrast, lnc172 was localized in the cytoplasm of male germline, myoid cells and ovarian somatic cells. Loss- and gain-of-function experiments demonstrated that knockdown and PGC-specific overexpression of lnc304 as well as universal overexpression of lnc172 significantly disrupted PGC development. In summary, the present study revealed the lncRNA profile of zebrafish PGC and identified two novel lncRNAs associated with PGC development, providing new insights for understanding the regulatory mechanism of PGC development.Long noncoding RNAs (lncRNAs) are regulatory transcripts in various biological processes. However, the role of lncRNAs in germline development remains poorly understood, especially for fish primordial germ cell (PGC) development. In this study, the lncRNA profile of zebrafish PGC was revealed by single cell RNA-sequencing and bioinformatic prediction. We established the regulation network of lncRNA-mRNA associated with PGC development, from which we identified three novel lncRNAs— Drosophila melanogaster, PGCs migrate across the midgut to the mesoderm towards the somatic gonadal precursors [Drosophila melanogaster [Primordial germ cells (PGCs), the precursors of germline, segregate at early embryonic development and migrate towards the genital ridge, where they proliferate and differentiate into gonocytes that give rise to gametes ,2,3. Theecursors . In addiecursors . Fish PGecursors . The cheecursors . The G pnogaster  and chemnogaster ,12. Xenopus, the Xlsirts, a group of non-translatable repeat transcripts, are indispensable for the translocation of Vg1 and other heterologous RNAs [Drosophila, the lncRNA-pgc is required for RNA pol II–dependent transcriptional silence in PGCs via blocking the transition from preinitiation to transcriptional elongation [In lower animals, including teleost fish, PGC development is dependent on molecules that reside within the maternally inherited germ plasm, the non-membrane phase-separated organelle condensates made of RNAs and proteins (RNPs) ,14,15, wous RNAs ,31. In Dongation ,33. In mongation , the maiongation , the posongation . Howeverlnc172 and lnc304, associated with PGC migration in zebrafish.Single-cell RNA sequencing (scRNA-seq) is facilitated to investigate the transcriptome landscape of stem cell homeostasis ,38,39. ITg (kop: EGFP-3′UTR-nanos) zebrafish of AB genetic background were obtained from the China zebrafish resource center . Zebrafish (Danio rerio) were raised and maintained at 28 °C with 10 h (h) darkness and 14 h light. The embryos were incubated in Ringer’s solution in 28 °C incubator . All the procedures with zebrafish were approved by the Ethics Committee of Sun Yat-sen University.http://zfin.org/zf_info/zfbook/zfbk.html, accessed on 1 January 2022). The de-chorion embryos were disassociated to a single cell in a deyolking buffer , rinsed twice in a washing buffer , and resuspended in nuclease-free PBS. Single GFP-labelled PGC was collected with a capillary tube under a fluorescence microscope . The collected single PGC was frozen in each tube containing the lysis buffer for single-cell RNA library construction from Annoroad gene technology Co. Ltd. . A total of 30 PGCs were collected in 30 tubes. After quality checking, 3 of the 30 samples were used for scRNA-seq independently.All the embryos were collected after natural spawning and staged as previously reported . GFP-labThe collected cells were directly amplified to construct the cDNA library by the Smart-Seq2 method  and werehttps://github.com/tanghaibao/Goatools, accessed on 1 January 2022).Known lncRNAs were obtained by blasting to known lncRNA databases . To obtain putative novel lncRNAs, potential coding transcripts were removed by three types of forecasting software including the coding potential calculator, pfam-scan, and Coding-Non-Coding-Index as previously described . The cisTM reverse transcription mix, random primer . qPCR was performed on the Roche Light Cycler 480II . The thermal cycling conditions were as previously described [ef1a and rpl13a were used as reference genes to calculate gene expression with 2-ΔΔCt method. All the data were represented with mean ± SD from three independent experiments in triplicates. The primers used in this study are listed in To validate the scRNA transcriptome, strand cDNA libraries of singe PGC were first constructed with a single cell sequence−specific amplification kit . The total RNA of adult tissues and embryos were extracted using a TRIzol reagent , and the RNAs were reverse transcribed with GoScriptescribed . ef1a anlnc196, lnc304, and lnc172 were amplified with a pair of primers whose reverse primer contained the core sequence of T7 promoter at the 5′ end .For lncRNA synthesis, full-length DNA fragments of lncRNA were inserted into the pCS2+ vector with poly A terminator removed by double digestion of Xho I and Not I enzymes. For PGC-specific overexpression of lncRNAs, the 3′UTR of lnc196, lnc304, and lnc172 ASOs and control ASO were injected into 1-cell stage Tg (kop: EGFP-UTR-nanos3) embryos, respectively. The injected embryos were collected for qPCR analysis and tracing PGC development. For overexpression, 200 pg lncRNAs were injected to 1-cell stage embryos, and embryos injected with 200 pg rfp mRNA were used as control. Rescue experiments were performed by co-injection of 200 pg lncRNAs and 10 pM corresponding ASOs into 1-cell stage Tg embryos. All the experiments were performed in more than three independent experiments in triplicate. Each independent experiment was performed in triplicate.Sequence-specific antisense oligonucleotides (ASOs) were synthesized . Different dosages of The PGC development was examined at 24 hpf under the stereo fluorescence microscope . The standard for the phenotype of PGC mislocalization was described as in the previous report . The phep < 0.5 was considered a statistically significant difference.The statistics were calculated and analyzed with SPSS version 20. The qPCR results were analyzed by Student’s t-test, and the phenotypes in the different experimental groups were analyzed by one-way ANOVA. Tg kop: egfp−UTR−nos3) at 5 h post fertilization (hpf) as previously described [ca15b, nanos3, dnd1, gra, and tdrd7a and two mitochondria components, mt-nd4 and mt-nd5  family, piwi-like RNA-mediated gene silencing 1 (piwil1), chemokine (C-X-C motif) receptor 7 (cxcr7), gamma-glutamyltransferase 1a (ggt1), regulator of G protein signaling 14a (rgs14a), and nanos homolog 1 (nos1)  . From tha-biased A. Because testes B–D. Duri epiboly E–G.lnc172, lnc196, and lnc304 in zebrafish gonads by fluorescent in situ hybridization (FISH). In mature testes, lnc196 and lnc304 were localized in the nucleus and cytoplasm of spermatogonia and spermatocytes, and their signals gradually weakened from the spermatogonia over primary spermatocyte to secondary spermatocyte   . By contoid cell G–H. In as oocyte A–F, and tic cell G–H. The lnc172, lnc196, and lnc304 in PGC development, we performed loss of- and gain-of-function experiments by injection of sequence-specific antisense oligonucleotides (ASO) and lncRNAs, respectively. The qPCR results indicated that injection of ASOs significantly decreased endogenous lncRNAs (lnc172 or lnc196. The ASO- injected embryos are shown at 24 h post fertilization (hpf) in lnc304 ASO-injected embryos  that will be specifically overexpressed in the PGCs because of somatic clearance by the miR-430 from gastrulation [lnc304 (lnc304-nos3 3′utr injected embryos (lnc172-nos3-3′utr-injected embryos (lnc172 injected embryos required somatic overexpression of lnc172. These results indicate different ways lnc304 and lnc172 function in the regulation of PGC development: PGC-specific overexpression of lnc304 and soma overexpression of lnc172 disrupted the PGC development.Universal overexpression of rulation . Compare [lnc304 A–C, the  embryos D,G. By c embryos E,F,H, sulnc172 and lnc304, which play important roles in PGC development. To the best of our knowledge, it is the first report about the function of lncRNAs in the PGC development of fish.In the present study, we revealed lncRNA profiles of zebrafish PGC, established the regulation network of lncRNA-mRNA associated with PGC, and found two novel lncRNAs Zebrafish PGCs specify at ~3 hpf, polarize at ~4.5 hpf, initiate to migrate at ~5 hpf, and reach to the genital ridge and stop migration at 24 hpf . Genome-piwil2, dazap1/2, tdrd1/2/3/5/6/7a/12/15, and tdrdkh. Piwil2 interacts with the piRNAs, which is critical for maintaining transposon silence in zebrafish PGC [rgs14a, cxcr7a, and hmgcr [lnc196, lnc304, and lnc172, which were predicted to regulate piwil2, nr0b1, and tdrd12, respectively. However, whether other lncRNAs involved in the regulation network might be associated with PGC development and the interaction among these lncRNAs and PGC- associated genes is a question that should be investigated in the future.In the lncRNA-mRNA regulation network, there were 12 known RNPs including fish PGC . The gerfish PGC ,50. DAZ fish PGC ,51,52. Tnd hmgcr ,19,53,54lnc172, lnc196, and lnc304 in embryos and gonads and observed strong signals in the gonads (lnc196 and lnc304 were co-localized with vasa in the spermatogonia, and the signals weakened from the spermatogonia to the secondary spermatocyte, which is consistent with the expression pattern of fish germ plasm components [lnc196 and lnc304 in male germline and pre-vitellogenic oocyte indicated that they might be associated with chromatin structure and transcriptional regulation. lnc172 was a highly expressed myoid cell and ovarian somatic cell, which are the gonadal somatic cells that play important role in germline self-renewal, differentiation, and maintenance via sexual hormones synthesis and response [lnc172, lnc196, and lnc304 might play important roles during gametogenesis.Generally, nucleus-localized lncRNAs are associated with the regulation of chromatin topology and neighboring genes ,56,57, ae gonads , but we mponents ,61,62. Tresponse ,64,65. Isdf1, cxcr4, and cxcr7 destroy directional migration, leading to PGCs dispersed in the trunk [lnc304 and universal overexpression are probably due to the somatic overexpression of lnc172, resulting in PGC scatted in the tail, somite, eyes, and brain (cxcr4 morpholino-injected embryos. These results demonstrated that proper expression of lncRNAs in soma and PGC is important for directing PGC migration towards the genital ridge. However, the detail process and mechanism of how lnc172 and lnc304 regulate PGC need to be further investigated. Zebrafish PGCs employ “amoeboid migration” to migrate towards the genital ridge, which is fine-tuned by intrinsic factors and the surrounding niche ,66. The he trunk ,69,70. Ind brain , similarIn summary, the present study provided lncRNA profiles of zebrafish PGC and identified two novel lncRNA critical in directing PGC migration, which is helpful for understanding the role of lncRNA in PGC development.


GZMB, NRP1, PDCD1, FASLG, and TNFRS18), regulatory molecules of Tregs suppressive function , and Treg-associated transcription factors  in 52 GV patients and 48 controls by real-time PCR (qPCR). We found significantly reduced GZMB, NRP1, SERPINB9, and ITPR1 transcripts in GV Tregs compared to controls . There were 0.44-, 0.45-, 0.32-, and 0.54-fold decrease in GZMB, NRP1, SERPINB9, and ITPR1 transcripts in GV Tregs. Additionally, disease activity and severity-based analyses revealed significantly decreased GZMB (p = 0.019 and 0.034), SERPINB9 (p = 0.031 and p = 0.035), and ITPR1 (p = 0.0003 and p = 0.034) transcripts in active vitiligo and severe GV patients' Tregs. Interestingly, we found a positive correlation for ITPR1 with GZMB  and SERPINB9  transcripts in GV Tregs. Moreover, we found positive correlation for percentage Treg mediated suppression of CD4+ and CD8+T cells with ITPR1 , GZMB , NRP1 , and SERPINB9  in GV Tregs. Further, calcium treatment of Tregs resulted into significantly increased ITPR1, SERPINB9, and GZMB transcripts in GV Tregs . Overall, our results for the first time revealed the crucial role of GZMB, NRP1, SERPINB9, and ITPR1 transcripts in decreased Treg suppressive capacity leading to GV pathogenesis, progression, and severity. In addition, our study highlighted that ITPR1 might be linked with decreased GZMB and NRP1 expression in GV Tregs. Moreover, our study for the first time suggest that increased SERPINB9 transcripts may lead to endogenous granzyme B-mediated Tregs apoptosis, and calcium treatment of Tregs may improve the Treg suppressive capacity. These findings may further aid in development of Treg-based therapeutics for GV.Generalized vitiligo (GV) is an autoimmune skin disease characterized by bilateral white patches over the entire body. Regulatory T cells (Tregs) maintain peripheral tolerance; however, they are found to be reduced in numbers and function in vitiligo patients. The exact mechanism for reduced Treg suppressive capacity is unknown. Therefore, we aimed to assess transcript levels of Tregs-associated immunosuppressive genes ( Further, the inherent levels of ITPR1, GZMB, NRP1, and SERPINB9 transcripts in Tregs of GV were correlated with the Treg suppressive capacity by Spearman's rank correlation analysis.The isolated Tregs population was divided into two fractions. The inherent levels of CT values in GV patients and controls for relative mRNA expression analysis, disease activity analysis, disease severity analysis, age of onset analysis, and gender-based analysis was carried out using nonparametric Mann–Whitney U test. The 2ΔΔCT- analysis was carried out to calculate the fold difference in gene expression. The statistical analysis was carried out using GraphPad prism software . p ≤ 0.05 was considered statistically significant.The comparison of mean ∆GZMB, NRP1, PDCD1, FASLG, and TNFRS18) were assessed in 52 GV patients and 48 controls using nonparametric Mann–Whitney U test after normalization with GAPDH expression. We found significantly reduced transcript levels for GZMB and NRP1 expression in GV Tregs compared to control Tregs  were assessed in 52 GV patients and 48 controls using nonparametric Mann–Whitney U test after normalization with GAPDH expression. Our study suggested significant decrease in transcript levels of SERPINB9 and ITPR1 for GV Tregs compared to controls' Tregs  were assessed in 52 GV patients and 48 controls using nonparametric Mann–Whitney U test after normalization with GAPDH expression. We did not find any significant difference for transcript levels of GATA2, GATA3, RUNX1, STAT3, and STAT5 in GV Tregs when compared to controls' Tregs  and NRP1  transcripts in Tregs of GV patients , NRP1 , SERPINB9 , and ITPR1  and percentage Treg-mediated suppression of CD8+ T cells with GZMB , NRP1 , SERPINB9 , and ITPR1  in GV patients  governs the release of calcium from the endoplasmic reticulum . The exa pathway . FinallyITPR1 transcripts may lead to impaired Treg activation and suppressive function, we assessed the mRNA expression of Treg-associated suppressive molecules GZMB, NRP1, PDCD1, FASLG, and TNFRS18 in GV patients. Studies have suggested that Tregs control the immune response by granzyme B-dependent cytotoxicity [GZMB transcripts in GV patients. Moreover, we found that the reduced GZMB transcripts were associated with GV disease severity and disease progression, suggesting the crucial role of GZMB expression in Tregs function. Furthermore, we found a positive correlation between GZMB transcripts and ITPR1 transcripts. Therefore, our study suggests that impaired ITPR1 transcripts might lead to reduced Treg intracellular calcium levels leading to impaired NFAT signalling pathway in GV  and epigenetic changes in GZMB promoter must be explored as they might be responsible for the decreased GZMB expression.As altered toxicity . Upon TCtoxicity , 35. Howay in GV . Additiopromoter . Therefoon in GV . HoweverNRP1 transcripts in GV patients, suggesting for the crucial role of NRP1 in reduced Treg suppressive function leading to GV pathogenesis. Moreover, we found a positive correlation of NRP1 with ITPR1 transcripts. Although the link between ITPR1-calcium-NFAT signalling pathway and NRP1 is unknown, previous studies suggest that NRP1 expression is accompanied with high levels of NFATc1 transcript expression [ITPR1-calcium-NFAT signalling pathway might be involved in reduced NRP1 transcripts which led to impaired Treg suppressive function in GV patients . NRP-1 is constitutively expressed on the surface of Tregs and mediates prolonged binding with immature dendritic cells , 37. Antpression . Additiopression . Overallpatients . HoweverSERPINB9 in GV Tregs. SERPINB9 is an endogenous granzyme B inhibitor [SERPINB9 [SERPINB9 transcripts in GV Tregs. Moreover, we found a significant association of SERPINB9 transcripts with GV disease severity and disease progression. Additionally, previous studies have suggested significantly decreased Treg cells in vitiligo patients [SERPINB9 transcripts can lead to Treg cells apoptosis, resulting into decreased Tregs number, thereby contributing to GV pathogenesis, progression, and severity. However, future studies must confirm these findings by studying granzyme caspases-mediated apoptosis pathway in Tregs of GV patients.Furthermore, we assessed the expression levels of nhibitor , 40. AftSERPINB9 . PreviouSERPINB9 . Interespatients , 14, 15,GATA2, GATA3, RUNX1, STAT3, and STAT5) in GV patients. The GATA transcription factors are zinc finger motif DNA-binding proteins, and they have a crucial role in Treg function [RUNX1 is a runt-related transcription factor, and it plays a crucial role in generation and function of Treg cells [STAT3 decreases the Tregs generation [STAT5 plays a crucial role in sustaining FOXP3 expression in Tregs [GATA2, GATA3, RUNX1, STAT3, and STAT5) with GV pathogenesis, progression, and severity.Next, we aimed to study the key Treg-associated transcription factors (function , 27. Genfunction . Additioeg cells . Moreoveeg cells , 41. Genneration . Moreovein vitro Treg suppression assay in GV patients [in vitro Treg-mediated suppression of CD4+ and CD8+ T cells in GV patients [ITPR1, GZMB, NRP1, and SERPINB9 on in vitro Treg suppressive capacity, we carried out correlation analysis. Interestingly, we found a positive correlation for Treg-mediated suppression of CD4+ and CD8+ T cells with ITPR1, GZMB, NRP1, and SERPINB9 transcripts in GV patients' Tregs. Therefore, our results suggest that decreased ITPR1, GZMB, NRP1, and SERPINB9 transcripts might impair Treg suppressive function, resulting in widespread activation of CD4+ and CD8+ T cells, which could lead to GV pathogenesis (Previously, we had studied the patients . Our stupatients . To studogenesis .ORAI1 after calcium treatment [ITPR1 is crucial for the release of calcium in Tregs, we studied the transcript levels of ITPR1 in calcium-treated Tregs of GV patients and controls. Interestingly, our study suggested an increase in mRNA expression of ITPR1 in Tregs after the calcium treatment (ITPR1 after the calcium treatment may further lead to enhanced Treg suppressive activity, we accessed the transcript levels of GZMB and SERPINB9 in calcium-treated Tregs of GV patients. Our study suggested that the calcium treatment increased the expression levels of GZMB and SERPINB9 in GV Tregs (Figures 2+-calmodulin-calcineurin-NFAT signalling pathway may be a potent therapeutic target for GV.Our previous study suggested that calcium treatment of Tregs increased the intracellular calcium influx in Tregs of GV patients, due to increased expression of calcium ion channel gene reatment . Moreovereatment . As ITPRreatment . As the  Figures . Our resGZMB, NRP1, SERPINB9, and ITPR1 transcripts in reduced Treg-mediated suppression of CD4+ and CD8+ T cells which lead to GV pathogenesis, progression, and severity. Moreover, our study highlighted that ITPR1 might be responsible for decreased GZMB and NRP1 transcripts in GV Tregs. Furthermore, our study revealed that the increased SERPINB9 transcripts might result in endogenous granzyme B-mediated Tregs apoptosis, and the Treg suppressive capacity can be enhanced after calcium treatment in Tregs. These findings may aid in development of Treg-based therapeutics for GV; however, in vivo studies must be carried out to validate the role of GZMB, NRP1, SERPINB9, and ITPR1 in Treg-mediated GV pathogenesis.Overall, our results for the first time suggest the crucial involvement of


Federation International of Gynaecology and Obstetrics (FIGO) classification from 2018. Management was divided into Surgery, Radiotherapy, and Chemotherapy.Cervical cancer mostly caused by Human Papilloma Virus. Staging and therapy have been extensively studied, and highly correlated with the cellular development of oncogenesis. Mutation was caused by E6 and E7 oncoprotein, also inactivation of 2 tumor suppressor factors (pRB and p53). P53 also regulated MMP1, which dysregulation of MMP transcription would promote tumor metastasis, because of its role in extracellular matrix degradation in tumor invasion. Clinical staging of Cervical Cancer was based on  •Metalloproteinase from a matrix, known as Matrixin or MMP , was part of sub-group of zinc-endoproteinase, produced by soft tissue.•This enzyme then involved in an important cascade that resulted in soft tissue degradation, either physiologically or pathologically.•Matrix metalloproteinases (MMPs) have two opposite functions, which are promoting and inhibition of cancer.•Physiologically, MMP expression was very low or even zero in all of human's tissues. It only increased in reactive or reparative condition. Anatomically, cervix is one third lower portion of uterus, cylindrical, protruding, and connected with vagina through external orifice of the uterus. Risk of cancer could be caused by genetic factors, bad lifestyles habit, less hygiene, and sexually active with multiple partners  see Tab, Pic 4TaMore than 2 million of ≥15 years old women are at risk of cervical cancer. 527624 are diagnosed each year with 265672 death. Cervical cancer is 4th most common cancer in women, and second highest in women between age 15–44. In Asia alone, the incidence was 284823. Whereas Southeast region had 9082 for age 15 to 39, 32892 cases for age 40–64, and 8581 cases for women above 65 year of age. Indonesia ranked 4th highest case in Southeast Asia, after Cambodia, Myanmar, and Thailand. Around 7.9% (528000) new cases dan 7.5% (266000) death are reported in 2012. More than 85% of cervical cancer cases and 87% of deaths are reported in the less developed countries. It was the second most cause of death after breast cancer, mostly due to lower early-detection rate of precancerous lesion ).).32]).a.Vaginal bleedingb.Increasing and bad odor leukorrheac.Pain: usually in intermediate and advanced stage, or infected neoplasm. Located at lower abdomen, gluteal region, or sacrococcygeal. Lower abdominal pain could indicate infection, water accumulation, or pus in the uterine cavity, causing uterus contraction and pain. Intermittent pain could be caused by tumor compression or invasion that obstruct or dilate ureter. Hydronephrosis might cause low back pain, lower extremities, gluteal, or sacrum pain; also due to tumor pressure to nerve of pelvic cavity region.d.Urinary tract symptoms (often due to infection): incontinence, urgency, dysuria. With cancer progression to bladder, hematuria and pyuria developed, even cysto-vaginal fistula. When cardinal ligament or ureter was invaded, hydronephrosis and uremia ensued.e.Digestive problems: lesion could spread to cardinal or sacral ligament, put pressure on rectum, causing obstipation; even invaded rectum and lead to hematochezia and rectovaginal fistula.f.Systemic symptoms: weakness, lethargy, fever, weight loss, anemia, and edema.Gold standard of Cervical Cancer diagnosis is based on histopathological examination of cervical biopsy. Early stage of cancer usually symptomatic, but some of the common symptoms were:a.VaginFederation International Of Gynaecology And Obstetrics (FIGO) classification from 2018 [Staging is important in determining disease spread, prognosis, management plan, and comparing therapeutic methods. Clinical staging was based on rom 2018 .Squamous carcinoma were the most common types, which were accounted for ± 90%, adenocarcinoma 5%, and others 5% .1.SurgeryAfter diagnosis was confirmed by histopathology and the stage was set by clinical manifestation and radiology, management was started based on the location, tumor size, stage, patient's age, general conditions, and fertility reservation.2.RadiotherapySurgery could stand as curative and palliative care. Curative is therapy to eliminate causes and clinical manifestarion of the diseases. Whereas palliative is a way to correct patient's condition with or without eliminating the cause. Radical hysterectomy has goals to remove uterus and cervix, parametrium, paracolpium, and vagina. It was usually done at the early stage, which was IA until IIA (FIGO Classification).3.ChemotherapyRadiation could destroy cancer cells in the cervix, parametrium, pelvic wall, and lymph nodes. It was recommended for stage IIB, III, and IV. Like surgery, it also has a curative and palliative purposes. In curative, radiation destroy metastatic cancer cells in lymph nodes while preserves as much as possible healthy tissues in rectum, urinary bladder, small bowel, and ureter. It was done in the stage IIB until IIIC, or even at the earlier stage when there was contraindication to surgery or as an adjuvant therapy after surgery with high re-occurrence rate. When cancer has spread outside pelvic wall, palliative radiation was given selectively in stage IVA. Radiation used high energy ray to destroy and hamper cancer growth. Side effect would be rectal and vaginal irritation, damage to urinary bladder, rectum, and ovarium.Route of administration of chemotherapy as a management of cancer, could be oral, intravenous, intraperitoneal, or intramuscular. The primary goals were to kill cancer cells, hamper growth, or as a radiosensitiser. Based on cancer type and stage, it could act as curative  or palliative.Study by Rameri (2017) with neoadjuvant chemotherapy in cervical cancer stage IB2-IVA showed that the overall recurrency rate (ORR) was not that different than the cases with definitive treatment ,37.Purposes of neoadjuvant chemotherapy were to decrease tumor size to facilitate surgery, decrease recurrence rate and increase survival rate. But it was controversial, based on the study. In patient that resistant to chemotherapy, it would delay the definitive therapy. Study in Prague also showed that complete response rate to neoadjuvant chemotherapy was 12.6% with progressive rate of 6%, thus it was very important to have a certain marker that could show which patient might be resistant to chemotherapy .Clinical factors such as age, haemoglobin level (Hb), tumor size, histopathology cancer type, and differentiation degree, would affect chemotherapy response, as well as angiogenesis factors as a response to hypoxia in cervical cancer .Hypoxia was reported to play a role in resistance to chemotherapy and radiotherapy. Hypoxic cell released HIF (hypoxia inducible factor), which in turn induce angiogenesis factors, such Vascular Endothelial Growth Factor (VEGF), angiopoietin, angiogenin, and Platelet Derived Growth Factor (PDGF). Then they activated endothelial protease, proliferation, and migration, as well as reducing apoptosis activity .As cancer spread in the advanced phase, chemotherapy act more in palliative care, to preserve patient's quality of life. Combination chemotherapy was used in metastatic diseases as single agent would not give satisfied effect. Most common combination were platinum and taxane.1Stage 0: 100% of patients will recover.2Stage 1: divided into IA and IB. IA has 5-years survival rate of 95%. As for stage IB, 5-years survival rate were 70–90%. Women with cancer in lymph node were not included.3Stage 2: divided into 2A and 2B. 2A has 5-years survival rate of 70–90%. And 5-years survival rate of stage 2B were 60–65%.4Stage 3: 5-years survival rate were 30–50%.5Stage 4: 5-years survival rate were 20–30%.Cervical Cancer has a bad prognosis, because 85–90% were diagnosed at invasive, advanced, or even terminal stage. Parameter in determining prognostic factors were clinical and histopathological, such as: general condition, staging, primary tumor size, cell types, and Broders differentiation degree. Generally, 5-years survival rate for Stage I was more than 90%, 60–80% for Stage II, around 50% for stage III, to less than 30% for stage IV.1.2Metalloproteinase from a matrix, known as Matrixin or MMP , was part of sub-group of zinc-endoproteinase, produced by soft tissue. This enzyme then involved in an important cascade that resulted in soft tissue degradation, either physiologically or pathologically. Decrease of extracellular matrix (ECM) was part of an important step in invasion and metastasis of neoplasm. This resulted from activation of matrix metalloproteinase (MMPs) that degraded protein component of ECM, followed by other physiological process, such as angiogenesis, apoptosis, and new soft tissue production. All these would support the development of cancer .MMP also known to be correlated with formation and development of squamous cell carcinoma (SCC). MMP gene involved in SSC, includes MMP1, 2, 3, 7, 9, 10, 11, 12, 13, which over-expressed in the SCC tissues, compared to normal tissue. Each of those genes could be classified into different categories. MMP1 was part of collagenase in interstitial .As interstitial collagenase, abnormal expression of MMP1 was seen in the development of cancer. Over-expression was clearly detected in several cancer cases, and highly correlated with prognosis. Besides, MMP1 also promoted angiogenesis by activating protease-activated receptor1 in endothelial .MMP1 that's produced by tumor cells contributed functionally in hematogenous spread of SCC. It induced vascular permeability through activation of endothelial Protease Activated Receptor (PAR)-1, thus made invasion and metastasis possible .A.cleaving insulin-growth-factor-binding protein (IGF-BP), releasing IGF; through transmembrane precursor growth factors including growth factor-α (TGF-α); and by regulating extracellular matrix, indirectly increase interaction between extracellular matrix and integrins. At the other hand, MMP could also slowed down cancer growth through Transforming growth factor-β (TGF-β) from latent TGF-β complexes.MMPs promote cancer growth by B.death receptor FAS. But MMPs could also cause apoptosis, by changing extracellular matrix composition, that influence integrin signal.MMP increase cancer survival by IGF initiation through FAS ligand pathway (FASL), that play a role in C.vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2), and TGF-β. These stimulated endothelial proliferation and migration. In addition, MMPs also induce cancer invasion through extracellular matrix structural component, such as: Collagen type I (Col-I) and IV (Col-IV) as well as fibrin. Collagen played role as pro-angiogenesis by binding with integrin αvβ3. At the other hand, MMPs also have a role as anti-angiogenesis through plasminogen and Col-XVIII, producing angiostatin and endostatin factors. MMPs participate in urokinase-type plasminogen activator receptor (uPAR) on cell surface.MMPs promote angiogenesis by increasing bioavailability of pro-angiogenesis D.MMPs regulate invasion by degrading extracellular matrix structural component, especially through laminin 5 (Lam-5) pathway, CD44 molecule adhesion pathway, and E cadherin (E-cad). In addition, cancer cell invasion needs MMP-9 to CD44 migration. But MMPs could inhibit metastasis through CXCL12 pathway. On the contrary, chemokine of CXC family could promote breast cancer metastasis.E.epithelial-to-mesenchymal transition (EMT), correlated with malignancy through molecular pathway E-cad dan TGF-β. MMPs increased differentiation by changing extracellular matrix component and integrin signal.MMPs promote F.α1-proteinase inhibitor (α1-PI), decreasing cancer sensitivity toward natural killer cells; and split CC and CXC chemokine family, that help cancer cells slip away from leukocytes.Cellular inflammation was the main key in MMPs involvement in cancer progress because it also inhibits immune reaction towards cancer cells. MMPs broke down interleukin-2-α (IL-2Rα) receptor on T-lymphocytes, which in turn inhibit T-lymphocytes proliferation; releasing TGF-β, which is an important suppressor of T-cell reaction against cancer cell; cleave Matrix metalloproteinases (MMPs) have two opposite functions, which are promoting and inhibition of cancer.Multiple factors contributed to the development of oncogenesis. Changes in signalling pathways, accompanied by genetic instability and mutation werw caused by high-risk Human Papilloma Virus (HPV) infection (E6 and E7 oncoprotein) . Besidestrans-activation of cellular protein needed for viral DNA replication. Persistent high-risk HPV infection induce HPV integration into the host genome, causing overexpression of E6 and E7. Interaction with DNMT in turn, caused deviation of tumor suppressor gene metilation. This is the main factors in carcinogenesis [E6 was able to degrade p53 by direct contact with E6AP ubiquitin ligase, thus inhibiting p53-dependant signal and contributing to tumorigenesis. E7 could interact with retinoblastoma protein groups  and disrupt E2F groups of transcription factors. These unplanned interactions caused ogenesis .E6 effect in multiple pathways of carcinogenesis would influence initiation, progression, and metastasis. E6 activated PIK3 through AKT pathway. It also had effect on PTEN by activation PDZ protein, which in turn increase pAKT and cell proliferation. These indicated increase in ribosomal protein S6 kinase  Akt phosE7 interaction with HDAC resulted in chromosome remodelling and genome instability. E7 also activated PIK3 or AKT pathway. It correlated with its ability to bind and activate Rb protein, and in turn correlate with HPV-induced high-grade Intraepithelial squamous lesion .Cell cycles depend on cyclin protein and Cyclin-dependent kinase (CDKs). Cyclin regulate CDKs. CDKs became active when it binds with cyclin and formed complexes. Cyclin was classified into A, B, D and E, each played a role in different point of cell cycles. Cyclin D were synthesized at the beginning of G1 phase, bound with CDK4 and CDK6. At the end of G1, Cyclin E were synthesized and bound with CDK2. When 3 complexes were formed, cell enter S phase. This holds important role in the initiation of DNA replication  and cell cycle transition . Cyclin In transcription regulation, Cyclin E has a receptor site for transcription factor E2F. This induced transcriptional repression by binding with large complexes containing E2F4, DP1, and protein socket that repressed Cyclin E expression till the end of G1 phase. pRb could also formed large repressor complexes with Histone Deacetylase (HDAC) and mammalian complex component hSW1/snf, like SNF-2(BRG1 and hbrm), start at the end of S phase to G1 phase ).hSW - SNF complex interacted with cyclin E and modulated BRG1 ability to restrain cell growth  This helpRb phosphorylation by cyclin D/CDK4 cancelling its interaction with HDAC and cyclin E transactivation, ending G1 phase Cyclin A and CDK1 transcription were withheld by pRb and hSW/SNF complexes. Cyclin E/CDK2 could phosphorylate pRb or hSW/SNF components when cyclin E concentration were high enough and pRb and hSW/SNF interaction was disrupted. Disregulation of each transcription complexes happened in cancer cell. Studies showed that there's overexpression of Cyclin E when Rb was inactivated in HPV16 infection with E7 oncogene  AlthoughCancer was mentioned as a disease caused by dysregulation of cell proliferation  Cell proP53 also regulated MMP1 (147)→57, which were a zinc-bonded endopeptidase in human. Dysregulation of MMP transcription would promote tumor metastasis , becausePhysiologically, MMP expression was very low or even zero in all of human's tissues. It only increased in reactive or reparative condition  MMP funcMMP holds an important role in tumor growth development. Specifically, MMP digested molecule on cell surface that mediated cell adhesion with other cells or ECM. Thus promoting cancer cell penetration in hematogenous and lymphatic spread, then metastasis .1.Extracellular domain, incl. connective tissue domain, rich in cysteine and fibronectin type III2.Transmembrane domain3.Intracellular domainEph receptors were consist of 3 parts , which wUnlike Eph, Ephrin-A didn't have intracellular domain that attached to cell-membrane through glycosyl lipo-inositol groups . Eph recAbnormal expression of Eph and Ephrin in tumor cells correlated with cancer growth, metastasis, and tumor spread, as well as host survival (180)(181). ,85, The


Lung cancer has the world’s second highest cancer incidence and second highest cancer-related mortality rate. However, the mechanism underlying non-small cell lung cancer (NSCLC) remained to be unclear. Overall, this study for the first time revealed Stress Granule Regulators were mutated and dysregulated in NSCLC samples by analyzing TCGA database. Moreover, three subtypes of NSCLC were identified based on the expression levels of Stress Granule Regulators. Patients in cluster 2 showed a higher survival rate than those in clusters 1 and 3. Bioinformatics analysis indicated the cell cycle, mTOR signaling pathway, EGFR signaling, PI3K/Akt signaling and DNA damage repair signaling were significantly related to molecular subtypes. Moreover, we performed a prediction analysis of the response to the inhibitors against the aforementioned signaling. Our results showed patients in C2 NSCLC had the highest sensitivity to MK.2206 (AKT.inhibitor) and Rapamycin (mTOR inhibitor). Patients in C3 NSCLC had the highest sensitivity for Temsirolimus , BIBW2992 , Erlotinib , PD.0332991 (CDK4/6 inhibitor), CGP.60474 (CDK inhibitor), and Gefitinib . Moreover, our results showed patients in C1 NSCLC had the highest sensitivity to AKT.inhibitor, AZD6482 (PI3K inhibitor). To evaluate the response to immune therapy of different subtypes, we analyzed the tumor immune inflation, immune regulators expression, and TIDE score in different SG related subtypes. These results showed that C2 and C3 may be more sensitive to immune therapy. To better predict the prognosis of NSCLC, we analyzed the correlation between stress granule regulator expression and overall survival time in NSCLC and constructed a Stress Granule Score including EIF2S1, CTSG, EIF4G1, IGF2BP1, PABPC1 to predict the prognosis of NSCLC. Overall, this study for the first time uncovers the effect of stress particles on drug response, immune response, and prognosis, laying a new theoretical foundation for the NSCLC prognosis and treatment. Lung cancer has the world’s second highest cancer incidence and second highest cancer-related mortality rate . After aWhen cells are subjected to external stressors such as hypoxia, oxidative stress, arsenic acid virus infection, and so on, mRNA translation stops and stress granules (SR) are formed, which are found in the cytoplasm to protect cells and help cells adapt to their surroundings . When thThe researchers analyzed 503 NSCLC samples from the TCGA database and discovered three subtypes. In terms of patient prognosis, treatment responsiveness, and immune cell infiltration, we discovered that these three subgroups were very inconsistent. For that purpose, we devised a scoring system that allowed us to quantify the number of stress particles in individual patients and examine the link between stress particles and biological parameters. According to the findings, stress particles were linked to several cancer indicators, immunological infiltration, treatment sensitivity, and patient survival, according to the findings. This is the first study to thoroughly uncover the critical function of stress particles in NSCLC, laying a new theoretical foundation for the disease’s molecular process and treatment.https://portal.gdc.gov/Cancer.gov) database was used to get genome-wide expression data. The Molecular Signature Database was used to obtain a database of stress- granules-related genes (SGRGs) (MSigDB) (TCGA ((MSigDB) .p-value of less than 0.05.Furthermore, the limma R package was used to identify DEGs in order to acquire putative key genes in BMs. DEGs were screened using the following criteria: |fold change| > 1.5 and a p 0.01), 138 DEGs were chosen for further investigation. These 138 genes have been labeled as “subtype-specific genes.”First, using univariate Cox regression  was used for the pathway enrichment analysis. An adjusted p value of 0.05 was considered statistically significant.The DAVID database , which was defined as the stress granule score:The optimal cutoff values derived by the survminer R program were used to separate NSCLC samples into high and low stress particle score groups. To further examine the model’s predictive performance, we ran Kaplan-Meier analyses of stress particle scores in NSCLC and generated hazard ratios (HRs) using univariate and multivariate Cox regression analysis. We used ROC curves to fit the Stress Granule score to significant clinical measures acquired through multivariate Cox regression and to assess the Stress Granule score’s accuracy. Logistic regression analysis of multivariate fit was performed using the R program “glmnet,” and the ROC curve was plotted using the R package “timeROC”.We applied correlation analysis to discover the relationship between stress particle scores and 28 immune cell types in order to study the link between stress particle scores and biological parameters.Based on the publicly available pharmacogenomics database GDSC , we predThe purpose of this study was to look at the roles of stress granule regulators in NSCLC. The workflow for the analysis of this study was presented in Also, we analyzed the expression patterns of 87 stress granule regulators in NSCLC using TCGA databases. In NSCLC samples, STYXL1, ATXN2L, UBAP2L, GRB7, DDX25, CTSG, SSB, CAPRIN1, CIRBP, DCP1A, ELAVL1, TIA1, EIF4G1, PRRC2C, MAPT, DHX9, OGFOD1, DDX3X, CSDE1, HABP4, PRKAA2, CELF1, DYRK3, DDX1 were significantly dysregulated in NSCLC. These results demonstrated that stress granule regulators have a crucial role in NSCLC .Furthermore, protein–protein interaction networks revealed that these stress granule-related proteins commonly interacted with one another . SeveralFor the current investigation, a total of 503 NSCLC samples were analyzed, along with complete survival data. We identified 87 differentially expressed genes (DEGs) within clusters using univariate Cox regression analysis to create a Stress Granule subtype with prognostic significance. Following that, we performed secondary clustering, which revealed that the optimal number of clusters was 3, as determined by CDF curves . Unsuperp = 0.762).Furthermore, we compared the three NSCLC subtypes in terms of gender, smoking status, M stage, T stage, N stage, grade, metastasis status, and race . ClinicaIn order to understand the potential mechanisms underlying the effect of stress granules on NSCLC progression, we analyzed the DEGs between different stress granule subtypes in NSCLC. In total, 913 DEGs, including 476 upregulated and 437 downregulated genes, were identified in cluster1 compared to cluster2 NSCLC samples . 913 DEGTo further determine the biological functions of the DEGs, KEGG enrichment analysis was performed using DAVID. As shown in The bioinformatics analysis showed the cell cycle, mTOR signaling pathway, EGFR signaling, PI3KAkt signaling and DNA damage repair signaling were significantly related to molecular subtypes . UnderstFrom the TCGA dataset, we created a heatmap to display the relative abundance of immune invading cell subpopulations . Accordi+ nave, common lymphoid progenitor, and T cell CD4+ Th2 cells are highly enriched in the C1 and C3 subtypes of NSCLC. C2 had high inflation levels of T cell CD4+ nave, T cell CD4+ central memory, T cell CD4+ effector memory, and endothelial cells. However, C3 had high inflation levels of myeloid dendritic cells, T cell CD8+, T cell CD8+ central memory, Myeloid dendritic cell activated, hematopoietic stem cells, macrophages, macrophagage M1, macrophagage M2, B cell memory, T cell NK, plasmacytoid dendritic cell, and B cell plasma. Very interestingly, the immune score analysis showed immune score, stroma score, and microenvironment score were highest in C3 NSCLC samples and lowest in C1 NSCLC samples.As present in We also analyzed the expression levels of immune regulators among these subtypes of NSCLC. The results showed CD274 was significantly up-regulated in C1 compared to C2 and C3 . These rThe TIDE score was calculated in the TCGA LUAD cohort to predict immune treatment response. According to the findings, the TIDE score was significantly lower in the C2 and C3 subtypes of NSCLC than in the C1 subtype of NSCLC . AccordiTo better predict the prognosis of NSCLC, we analyzed the correlation between stress granule regulator expression and overall survival time in NSCLC. The higher levels of SYMPK, BICD1, IGF2BP1, CSDE1, DDX1, YBX1, PABPC1, MOV10, VCP, HSF1, ELAVL1, SSB, EIF4E, G3BP1, EIF2S1, KPNB1, EIF4G1 are related to shorter OS time in patients with NSCLC. However, higher levels of CTSG, RBPMS, RBFOX1, and HABP4 are linked to a prolonged OS duration in individuals with NSCLC .We created a to better predict prognosis and guide tailored treatment . IndividIn conclusion, our research identifies three stress granule-associated NSCLC subgroups with distinct prognostic, gender, mutational, and immunoinfiltrating characteristics. To quantify stress particle levels in individual tumors, we built a risk model termed the stress particle score; the stress particle score demonstrated considerable advantages in predicting prognosis and immunotherapy response. Our research for the first time discovered the roles of stress granule associated molecular subtypes in prediction of drug response, immune response, and prognosis in NSCLC, laying a new theoretical foundation for the NSCLC prognosis and treatment.In this study, we analyzed the expression and mutation pattern of stress granule regulators. We found the majority of stress granule regulators were amplified, deleted, or mutated in NSCLC, with UBAP2L was the most frequently mutated genes. Also, we revealed UBAP2L was significantly up-regulated in NSCLC. UBAP2L has been shown to be overexpressed in several tumors, such as liver carcinoma , breast in vitro and drive tumor cells to join the apoptotic program  and Rapamycin (mTOR inhibitor) with the lowest IC 50 value. Patients in C3 NSCLC had the highest sensitivity for Temsirolimus , BIBW2992 , Erlotinib , PD.0332991 (CDK4/6 inhibitor), CGP.60474 (CDK inhibitor), and Gefitinib  with the lowest IC 50 value. Moreover, our results showed patients in C1 NSCLC had the highest sensitivity to AKT.inhibitor, AZD6482 (PI3K inhibitor) . The ideIn recent years, immunotherapy represented by immune checkpoint inhibitors has significantly improved the survival of patients with advanced lung cancer and changed the treatment mode for lung cancer . With th+ T cells can predict the efficacy of immunotherapy in multiple tumor types, including immunotherapy alone and immunocombination therapy  and Rapamycin (mTOR inhibitor). Patients in C3 NSCLC had the highest sensitivity for Temsirolimus , BIBW2992 , Erlotinib , PD.0332991 (CDK4/6 inhibitor), CGP.60474 (CDK inhibitor), and Gefitinib . Moreover, our results showed patients in C1 NSCLC had the highest sensitivity to AKT inhibitor, AZD6482 (PI3K inhibitor). To evaluate the response to immune therapy of different subtypes, we analyzed the tumor immune inflation, immune regulators expression, and TIDE score in different SG related subtypes. These results showed that C2 and C3 may be more sensitive to immune therapy. To better predict the prognosis of NSCLC, we analyzed the correlation between stress granule regulator expression and overall survival time in NSCLC and constructed a Stress Granule Score including EIF2S1, CTSG, EIF4G1, IGF2BP1, PABPC1 to predict the prognosis of NSCLC. Overall, This study for the first time uncover the effect of stress particles on drug response, immune response, and prognosis, laying a new theoretical foundation for the NSCLC prognosis and treatment.


FUS1/TUSC2 (FUSion1/TUmor Suppressor Candidate 2) is a tumor suppressor gene (TSG) originally described as a member of the TSG cluster from human 3p21.3 chromosomal region frequently deleted in lung cancer. Its role as a TSG in lung, breast, bone, and other cancers was demonstrated by several groups, but molecular mechanisms of its activities are starting to unveil lately. They suggest that Fus1-dependent mechanisms are relevant in etiologies of diseases beyond cancer, such as chronic inflammation, bacterial and viral infections, premature aging, and geriatric diseases. Here, we revisit the discovery of FUS1 gene in the context of tumor initiation and progression, and review 20 years of research into FUS1 functions and its molecular, structural, and biological aspects that have led to its use in clinical trials and gene therapy. We present a data-driven view on how interactions of Fus1 with the mitochondrial Ca2+ (mitoCa2+) transport machinery maintain cellular Ca2+ homeostasis and control cell apoptosis and senescence. This Fus1-mediated cellular homeostasis is at the crux of tumor suppressor, anti-inflammatory and anti-aging activities. Loss of TSG functions in malignant cells occurs at the DNA, transcriptional and post-transcriptional levels and may be achieved via different mechanisms. According to the Knudson’s two-hit paradigm , first hit comes from the loss of heterozygosity (LOH) due to deletion or loss-of-function mutation in one of two TSG alleles that can be inherited as a recessive mutation. Second hit causes a biallelic mutation eliminating the function of the remaining TSG allele. This transforms the cell towards malignancy. The effect of haploinsufficiency, when mutation of a single gene copy is sufficient for malignant transformation, has been described for some TSGs  , 832+ tre1) Fig.  84, 85], 852+ troverload . Howevertivation . MICU1 h content .Fig. 3Ro2+ uptake. This is especially important as tumor cells experience increased ROS production. Interestingly, MCU contains ROS sensing cysteines in its N-terminus. Their oxidation promotes MCUcx assembly, persistent channel activity, and mitoCa2+ overload following by opening the permeability transition pore, which triggers cell death , a mitochondrial form of Na+/Ca2+ exchanger (mNCX), and Ca2+-binding EF-hand motif containing LETM1, a 2H+/Ca2+ exchanger   Figs.  and 4C. via the SH2-domain . This process should activate mitophagy, but in Fus1−/− mice it rather leads to accumulation of nonfunctional mitochondria. Fus1−/− cochlear cells displayed a major down-regulation in the expression of PTEN-induced kinase-1 (PINK1) .Overexpression of blockers , 140. REblockers . RecentlThus, studies initiated about 40 years ago to dissect chromosomal aberrations in lung cancer cells culminated in a new promising gene therapy aimed to battle most aggressive lung cancer stages. In addition, Fus1/Tusc2-mediated anti-inflammatory and anti-aging activities provide avenues for development of new approaches to fight conditions of chronic inflammation, infections, premature aging and geriartic diseases.


Patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) seropositive for autoantibodies against nodal and paranodal proteins display distinct clinical presentations. In the latest study, CIDP with autoantibodies against paranodal proteins was defined as autoimmune nodopathy (AN). We herein present a case of 39-year-old male with anti- neurofascin (NF) 155 and NF186 IgG4 antibody with gait disturbance and tremor, who was followed up for 4 months and demonstrated clinical improvements after apparently effective rituximab therapy. In addition, a literature review was conducted to investigate the clinical characteristics of anti-NF155/NF186-positive AN. In recent years, some chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) specific biomarkers have been discovered successively, including antibodies such as anti-neurofascin (NF)155 ,3,4,5, aAnti-NF155 IgG4 antibody had been found in patients with AN, these patients were associated with younger age of onset, higher frequency of drop foot, gait disturbance, sensory ataxia, tremor, distal acquired demyelinating symmetric (DADS) neuropathy phenotype, higher cerebrospinal fluid (CSF) protein levels, and nerve conduction studies (NCS) showed that the distal and F-wave latencies were more significantly prolonged, as well as the effects of intravenous immunoglobulin (IVIG) was poor ,17,18.Meanwhile, antibodies against NF186 were found in less than 3% of patients with AN ,20,21. DHere, we firstly report the presence with nodo/paranodal protein NF155/NF186 IgG4 antibodies in a Chinese patient with AN and summarize the clinical characteristics, electrophysiological, pathological description and following therapy.6/L, and he was diagnosed as CIDP and treated with 4.3 g of methylprednisolone (500 mg × 5 days + 240 mg × 5 days + 120 mg × 5 days). After treatment, the numbness of limbs improved better than before. On 22 May 2020, He went to Ruijin Hospital Luwan branch and received 800 mg cyclophosphamide pulse therapy. After that, the patient received cyclophosphamide 400–800 mg pulse therapy every 2 months . He appeared bilateral upper limb’s tremor in October 2020. Since onset, he has normal urination and defecation, and no significant changes in body weight.A 39-year-old man presented with an 18-month history of numbness of limbs and 17-month distal weakness of lower limbs to our hospital in May, 2021. In October 2019, he felt numbness at the end of the fingers and toes, and more severe in the lower limbs. After one month, the numbness gradually developed to the bilateral limbs and appeared the lower extremity weakness. So, he went to the local hospital in November 2019. The nerve conduction velocity (NCV) showed that the conduction function of the common peroneal nerve and tibial nerve was decreased. CSF indicated albuminocytologic dissociation. He was diagnosed as Guillain-Barre syndrome (GBS). But the patient refused IVIG treatment for economic factor. Finally, he received steroid pulse therapy and the symptoms were improved after the treatment. In January 2020, he appeared to gait instability, accompanied with a feeling of stepping on cotton. In March 2020, He found bilateral lower limbs began to become thinner, and he went to the local hospital to receive IVIG treatment, but the symptoms were not ameliorated. In April 2020, he went to the Ruijin Hospital Luwan branch, the CSF showed albuminocytologic dissociation, Pandy’s test was strong positive (+++), protein was 2447 mg/L, the leukocyte count was 1.3 × 10He denied family history, past history and personal history. There was no additional history of fever, trauma, infection, or vaccination. There was no obvious abnormality in the physical examination of the internal medicine system.Neurological examinations showed low frequency tremor at both arms, ataxic-stepping gait, the muscle strength of bilateral upper limbs was grade V, the muscle strength of bilateral lower limbs was grade V-, bilateral lower limbs tendon reflexes were diminished, muscle atrophy could be seen in bilateral lower limbs, with high arch feet, deep sensory impairment, and decreased acupuncture sensation on both sides of the ankle joints below 2–3 transverse fingers. Bilateral finger-nose test was negative, bilateral heel-knee-shin test was positive, Romberg’s sign was positive, straight line walking test was positive, and there was no abnormality in other neurological examination.2O. Routine CSF analysis revealed that the leukocyte count was 1 × 106/L, Pandy’s test was positive (++), protein was 1773.56 mg/L, chloride was 136 mmol /L, glucose was 3.38 mmol/L, albumin quotient (index) was 40 and IgG oligoclonal bands (OCB) was negative. Then, a series of antibodies of noda-paranodopathy diseases were screened by cell-based assay (CBA) for both serum and CSF. The results showed that the antibodies against NF155 IgG4 (serum 1:1000 and CSF 1:100) and NF186 IgG4 (serum 1:100 and CSF 1:100) were positive for both serum and CSF . There was no obvious abnormality in the routine electroencephalogram (EEG). Subsequently, lumbar puncture was performed with the CSF pressure was 170 mmH and CSF , while aA nerve biopsy of the left sural nerve showed no obvious abnormality in the density of myelinated nerve fibers, and no significant demyelination A–C.2) with improvement of motor and sensory deficit. A follow-up at 4 months demonstrated a marked improvement. He was able to run about 100 m, and said that the distal paraesthesias of lower limbs were much better than before. Repeat serum and CSF antibody tests showed that the antibodies against NF155 IgG4 and NF186 IgG4 in serum were both 1:1000, in CSF were both 1:10 , which are in line with the previous studies ,23,24. HAmong anti-NF155 IgG4 antibody patients, histological examinations of the biopsied sural nerves revealed subperineurial edema and occasional paranodal demyelination, but no vasculitis, inflammatory cell infiltrates, or onion bulbs . Even yeThe neuroimaging features of anti-NF186 AN have not yet been clarified. Only one study  performeThe so-called anti-pan-NF-associated neuropathies caused by the co-existence of anti-NF186/140 and NF155 antibodies were extremely rare and caused life-threatening symptoms such as almost locked-in syndromes ,19,20,28In this case, the symptoms were quite mild and revealed an effective clinical response to IVIG, so no further immunotherapy was required. Fels et al.  describeIn summary, the antibody mentioned above is one of the specific biomarkers of AN discovered in recent years. When severe peripheral nerve damage occurs, especially accompanied by tremor, ataxia, and poor response to IVIG treatment, it should be considered that this subtype may be possible, and relevant antibodies should be tested in time. At present, the knowledge and understanding of anti-NF155/NF-186 IgG4 antibody-positive AN is still relatively less in China. So, we strive to achieve early diagnosis and provide more active and targeted treatment and prevention of recurrence and aggravation as soon as possible.


And there are 3 SNP peaks located on the Chr 4, 6, and 7, which could be the “hot spots” regions for mining and identifying candidate genes for salt tolerance. Furthermore, 616 unique candidate genes were screened surrounding the significant SNPs, which are associated with transport proteins, protein kinases, binding proteins, and other proteins of unknown function. Meanwhile, transcriptomic analysis (RNA-Seq) was carried out to compare the salt-tolerant (CM72) and salt-sensitive (Gairdner) genotypes subjected to salt stress. And there was a greater accumulation of differentially expressed genes(DEGs) in Gairdner compared to CM72, mainly enriched in metabolic pathway, biosynthesis of secondary metabolites, photosynthesis, signal transduction,emphasizing the different transcriptional response in both genotypes following salt exposure. Combined GWAS and RNA-Seq analysis revealed 5 promising salt-responding genes  from the hot spot regions, which were verified between the salt-tolerant and salt-sensitive varieties by qRT-PCR. In all, these results provide candidate SNPs and genes responsible for salinity responding in barley, and a new idea for studying such genetic basis in similar crops.Salt stress is one of the main abiotic stresses affecting crop yield and quality. Barley has strong salt tolerance, however, the underlying genetic basis is not fully clear, especially in the seedling stage. This study examined the ionic changes in barley core germplasms under the control and salt conditions. Genome-wide association study (GWAS) analysis revealed 54 significant SNPs from a pool of 25,342 SNPs distributed in 7 chromosomes (Chr) of the Illumina Barley 50K SNP array. These SNPs are associated with ion homeostasis traits, sodium (Na Though + in plant tissues, on the one hand, causes osmotic stress, making it more difficult for the root system to absorb water, resulting in accelerated leaf senescence and death. On the other hand, it leads to a serious imbalance of intracellular ions, which affects the absorption of nutrients such as K, N, P, Ca, Mg, and Fe, resulting in plant physiology and metabolic disorders  (+ in vacuoles by Na+/H+ antiporter (NHXs)  signal pathway  , indicatth (PCD) . To counth (PCD) , such asr (NHXs)  and effl pathway . High-afg of Na+ . In addig of Na+ . In addig of Na+ , NO  is an ancient cereal crop considered to be the world’s most tolerant crop to salt, drought, low temperature, and cold stresses, which can be used as a model crop for studying the molecular regulation mechanism of salt tolerance . At presGossypium hirsutum L.)  can find SNPs positioned across the genome as molecular markers and, therefore, is an efficient and convenient tool to dissect the genetic basis of complex traits in the plant, such as Arabidopsis , rice (Oryza sativa), maize (Zea mays), wheat (Triticum aestivum), and cotton (utum L.) . In barlutum L.) . Sbei etutum L.) , HKT1;5 utum L.) . Mwando utum L.) , the firutum L.) , which c+/K+ balance in barley leaves. The results might provide useful candidate SNPs and genes for high-quality barley salt-tolerance breeding and for understanding the mechanisms for salt tolerance in barley.Accordingly, this study identified the natural allelic variation in the barley genome and candidate genes that are significantly associated with salt tolerance. Here, we performed a GWAS using a natural population of 288 barley core collections and the Illumina Barley 50K SNP array based on ion traits for salt tolerance. In addition, we performed transcriptome analysis and qRT-PCR to verify the candidate genes identified by GWAS. In total, we identified 54 SNPs and 5 putative candidate genes associated with Na22.1To evaluate salinity tolerance, a natural population consisting of a global barley core collection of 288 barley accessions (149 two-rowed and 139 six-rowed) collected from different countries or regions was used in this study. Among the accessions, 129 were from different provinces in China, 156 were from Australia, Germany, Japan, and other countries, and 3 were from unknown geographical sources  were transferred to a turnover box containing modified Hoagland’s nutrient solution as described by 2.3+ and K+ contents in dried leaves were determined using an ICP-OES/MS spectrometer as described by + content, K+content, and Na+/K+ ratio. Statistical analysis, Pearson correlation between traits, ANOVA, and principal component analysis were performed by SPSS 20 software.To estimate salt tolerance in barley, data of seedlings phenotypes were collected and statistically analyzed after 3 weeks of different treatment conditions. After 3 weeks of salt treatment, the sampled seedlings were rinsed with deionized water and dried with absorbent papers. Immediately after sample collection, plant height, fresh and dry weights, and other phenotypes were measured. Na2.4The genomic DNA was extracted from young leaf tissues for genotyping using the CTAB method . The bar2.5http://taylor0.biology.ucla.Edu/structureHarvester/), when △K achieved maximum value, the optimal K was the number of clusters. Principal component analysis (PCA) was used to assess the population structure by GAPIT software  between pairwise SNPs (MAF > 0.05) was estimated using the PopLDdecay software (10 (P) ≥-log10 (1/n), where n denotes the total filtered SNPs.The LD parameter (software . The ass2.7http://floresta.eead.csic.es/barleymap/) and Gramene (http://ensembl.gramene.org/Hordeum_vulgare/Info/Index) using the Morex genome. GO (Gene Ontology) and KEGG PATHWAY enrichment analyses were performed through the GENE ONTOLOGY (http://geneontology.org/).Candidate genes located within and/or adjacent to the associated SNPs were identified through the Barleymap (2.8https://webblast.ipk-gatersleben.de/barley_ibsc/downloads/) by HISAT2 tools software. Only reads with a perfect match or one mismatch were further analyzed and annotated based on the reference genome. All the downstream analyses were based on clean data with high quality. The original datas of the transcription sequencing were submitted to SRA database at NCBI (accession number: PRJNA866193 and PRJNA892763).The seeds of salt-tolerant barley CM72 (C) and salt-sensitive barley Gairdner (G) were grown in a greenhouse, in Nanjing, China. Seedlings were collected after 0h, 3h, 12h, and 48 h with three biological replicates under 300 mM salt concentration and frozen in liquid nitrogen. Total RNA was extracted from the leaf samples using the EASY spin Plant RNA kit , and sequencing libraries were generated using NEBNextR UltraTM Directional RNA Library Prep Kit for IlluminaR  following manufacturer’s recommendations. The libraries were sequenced on an Illumina Hiseq Xten platform and paired-end reads were generated. Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. Then the clean reads were mapped to the reference genome sequence (2 (foldchange (FC))| ≥1 were set as the threshold by DESeq2 softwore (Gene expression levels were estimated by fragments per kilobase of transcript per million fragments mapped (FPKM). The false discovery rate (FDR) < 0.05 and |logsoftwore  for signsoftwore  software2.9Six salt-tolerant and six salt-sensitive varieties were selected and grown in the greenhouse for 48 h under 300 mM salt stress, among them, CM72 and Gairdner were used as reference varieties  and 6.8 μL ddH33.1To find the new salt-tolerant variety of barley, a set of 288 barley germplasms from all over the world were examined for salt tolerance at the seedling stage. After salt treatment (300 mM NaCl) for 3 weeks, the barley genotypes showed typical symptoms of salt damage, such as stunting, leaf wilting, and yellowing  and Na+/K+ ratio  from 5.46 to 48.66 mg/g (7.92-fold) and 0.09 to 2.25 (23.52-fold), respectively. Meanwhile, K+ content  decreased significantly from 61.36 to 22.75 mg/g showing about a 62.92% reduction (+ (R-Na+), K+ (R-K+), and Na+/K+ (R-Na+/K+), which can be used to evaluate salt tolerance, ranged from 2.95 to 33.13, 0.21 to 0.66, and 6.42 to 115.75, respectively  and a positive correlation was also found between K+ and morphological traits (except YLN)  ≥ 4.40) located mainly on Chr 2, 4, 5, 6, and 7. Some of these were detected repeatedly, indicating their true association with traits  were detected on Chr 2 located at 6.193 and 647.542 Mb, respectively. There were 6 SNPs associated with Na+ content (CK-Na+), including 2 on Chr 5 and 4 on Chr 6, respectively. Under salt treatment, 20 significant SNPs were identified including 7 on Chr 4 and 9 on Chr 7 for Salt-Na+, which were nearby on their respective chromosomes (10 (P)=6.55) was found at 639.94 Mb on Chr 4 =7.35).Under control treatment, we detected 8 significant SNPs that correlated with ion-related traits  = 6.77) located at 639.75 Mb. However, we did not find any SNP sites significantly associated with Na+/K+ ratio under control treatment (CK-Na+/K+), Salt-K+, and R-K+  between the eight associated SNPs (+ of Hap1 (45.58 mg/g) was less than that of Hap2  was 26.21% lower than that of Hap2 , which is involved in the ionic stress signaling pathway , Huntington’s disease  and so on  and RNA glycosylase activity (GO:0030597) with the common four genes were the most significantly enriched categories  aligned with the specified reference genome and led to the identification of 90,591 genes  enrichment analysis is performed to determine the potential function of DEGs in response to salt stress. The results of GO enrichment analysis indicated that in terms of cellular component the enriched same GO terms were cell, cell part, membrane, organelle in both CM72 and Gairdner. GO molecular function analysis suggested that the DEGs mainly richer in binding, catalytic activity, structural molecule activity, transporter activity in both CM72 and Gairdner. Within the biological process, the mainly enriched GO terms were biological regulation, cellular process, metabolic process, response to stimulus) in both CM72 and Gairdner  were identified for ion traits, and there were three SNP peaks located on Chr 4, 6, and 7  from the hot spot regions as protein kinases and transporters were identified by GWAS and RNA-Seq integrating analysis , which was a down-regulated gene by RNA-seq, consistent with the previous findings that HKT negatively regulates salt tolerance when HKT;5 was knockdown, Na+ translocation from roots to shoots decrease, and increases in K+/Na+ in barley  were not identified as common candidates , which is an important enzyme localized in cytoplasm as well as stroma of chloroplast in photosynthetic carbon metabolism, glycolysis, gluconeogenesis, and reductive pentose phosphate cycle  to transport H+ in the cytoplasm to the vacuole, and plasma membrane ATPase  to transport the H+ in the cytoplasm to the outside of the cell to maintain cellular pH homeostasis  family protein with molecular function of metal ion binding (GO:0046872) that homologous to Arabidopsis synaptotagmin-3. In Arabidopsis, the stress-induced SYT3 is an endoplasmic reticulum-plasma membrane (ER-PM) tether that also participates in maintaining PM integrity along with SYT1 , salt, and drought stresses  with ATP binding (GO:0005524), is involved in ion homeostasis, ROS scavenging, and osmotic regulation, and have a dual role in regulating both plant architecture and salt stress responses (HORVU5Hr1G124880 encodes the MATE efflux family protein with antiporter activity (GO:0015297) which modulates ABA sensitivity and increases tolerance to drought, salt, and cold stress with high production of antioxidant enzymes and significantly reduced levels of oxidants in plants (HORVU7Hr1G101590 and HORVU7Hr1G103510 encode Cytochrome P450 superfamily protein and transducin/WD40 repeat-like superfamily protein (WD40), respectively. Both of these gene products participate in response to salt stress  that may be related to ion balance in leaves from phenotypic and haplotype screening. This study provides novel SNPs and candidate genes for the study of salt tolerance in barley. Our results provide a basis for breeding new salt-tolerant barley varieties with marker-assisted selection.The data presented in the study are deposited in the SRA database at NCBI repository, accession number PRJNA866193 and PRJNA892763 for RNA-seq data; and European Variation Archive repository for SNP data, accession number PRJEB59041.TX and WY conceived and designed the idea. TX, SM, XZ, JD, YZ, and XY performed the experiments. TX and WY analyzed the data. TX wrote and revised the manuscript. SM, XZ, JD, YZ, XY, and WY critically revised the article. All authors contributed to the article and approved the submitted version.