DMC-siERCC2 hybrid nanoparticle enhances TRAIL sensitivity by inducing cell cycle arrest for glioblastoma treatment.

ERCC2 plays a pivotal role in DNA damage repair, however, its specific function in cancer remains elusive. In this study, we made a significant breakthrough by discovering a substantial upregulation of ERCC2 expression in glioblastoma (GBM) tumor tissue. Moreover, elevated levels of ERCC2 expression were closely associated with poor prognosis. Further investigation into the effects of ERCC2 on GBM revealed that suppressing its expression significantly inhibited malignant growth and migration of GBM cells, while overexpression of ERCC2 promoted tumor cell growth. Through mechanistic studies, we elucidated that inhibiting ERCC2 led to cell cycle arrest in the G0/G1 phase by blocking the CDK2/CDK4/CDK6/Cyclin D1/Cyclin D3 pathway. Notably, we also discovered a direct link between ERCC2 and CDK4, a critical protein in cell cycle regulation. Additionally, we explored the potential of TRAIL, a low-toxicity death ligand cytokine with anticancer properties. Despite the typical resistance of GBM cells to TRAIL, tumor cells undergoing cell cycle arrest exhibited significantly enhanced sensitivity to TRAIL. Therefore, we devised a combination strategy, employing TRAIL with the nanoparticle DMC-siERCC2, which effectively suppressed the GBM cell proliferation and induced apoptosis. In summary, our study suggests that targeting ERCC2 holds promise as a therapeutic approach to GBM treatment.

Imaging and Liquid Biopsy for Distinguishing True Progression From Pseudoprogression in Gliomas, Current Advances and Challenges.

RATIONALE AND OBJECTIVES: Gliomas are aggressive brain tumors with a poor prognosis. Assessing treatment response is challenging because magnetic resonance imaging (MRI) may not distinguish true progression (TP) from pseudoprogression (PsP). This review aims to discuss imaging techniques and liquid biopsies used to distinguish TP from PsP. MATERIALS AND METHODS: This review synthesizes existing literature to examine advances in imaging techniques, such as magnetic resonance diffusion imaging (MRDI), perfusion-weighted imaging (PWI) MRI, and liquid biopsies, for identifying TP or PsP through tumor markers and tissue characteristics. RESULTS: Advanced imaging techniques, including MRDI and PWI MRI, have proven effective in delineating tumor tissue properties, offering valuable insights into glioma behavior. Similarly, liquid biopsy has emerged as a potent tool for identifying tumor-derived markers in biofluids, offering a non-invasive glimpse into tumor evolution. Despite their promise, these methodologies grapple with significant challenges. Their sensitivity remains inconsistent, complicating the accurate differentiation between TP and PSP. Furthermore, the absence of standardized protocols across platforms impedes the reliability of comparisons, while inherent biological variability adds complexity to data interpretation. CONCLUSION: Their potential applications have been highlighted, but gaps remain before routine clinical use. Further research is needed to develop and validate these promising methods for distinguishing TP from PsP in gliomas.

Lactobacillus plantarum L168 improves hyperoxia-induced pulmonary inflammation and hypoalveolarization in a rat model of bronchopulmonary dysplasia.

Alteration of gut microbiota can affect chronic lung diseases, such as asthma and chronic obstructive pulmonary disease, through abnormal immune and inflammatory responses. Previous studies have shown a feasible connection between gut microbiota and bronchopulmonary dysplasia (BPD) in preterm infants. However, whether BPD can be ameliorated by restoring the gut microbiota remains unclear. In preterm infants with BPD, we found variance in the diversity and structure of gut microbiota. Similarly, BPD rats showed gut dysbiosis, characterized by a deficiency of Lactobacillus, which was abundant in normal rats. We therefore explored the effect and potential mechanism of action of a probiotic strain, Lactobacillus plantarum L168, in improving BPD. The BPD rats were treated with L. plantarum L168 by gavage for 2 weeks, and the effect was evaluated by lung histopathology, lung function, and serum inflammatory markers. Subsequently, we observed reduced lung injury and improved lung development in BPD rats exposed to L. plantarum L168. Further evaluation revealed that L. plantarum L168 improved intestinal permeability in BPD rats. Serum metabolomics showed altered inflammation-associated metabolites following L. plantarum L168 intervention, notably a marked increase in anti-inflammatory metabolites. In agreement with the metabolites analysis, RNA-seq analysis of the intestine and lung showed that inflammation and immune-related genes were down-regulated. Based on the information from RNA-seq, we validated that L. plantarum L168 might improve BPD relating to down-regulation of TLR4 /NF-κB /CCL4 pathway. Together, our findings suggest the potential of L. plantarum L168 to provide probiotic-based therapeutic strategies for BPD.

Efficacy of fecal microbiota transplantation in patients with Parkinson's disease: clinical trial results from a randomized, placebo-controlled design.

The occurrence and development of Parkinson's disease (PD) have been demonstrated to be related to gut dysbiosis, however, the impact of fecal microbiota transplantation (FMT) on microbiota engraftment in PD patients is uncertain. We performed a randomized, placebo-controlled trial at the Department of Neurology, Army Medical University Southwest Hospital in China (ChiCTR1900021405) from February 2019 to December 2019. Fifty-six participants with mild to moderate PD (Hoehn-Yahr stage 1-3) were randomly assigned to the FMT and placebo group, 27 patients in the FMT group and 27 in the placebo group completed the whole trial. During the follow-up, no severe adverse effect was observed, and patients with FMT treatment showed significant improvement in PD-related autonomic symptoms compared with the placebo group at the end of this trial (MDS-UPDRS total score, group×time effect, B = -6.56 [-12.98, -0.13], P < 0.05). Additionally, FMT improved gastrointestinal disorders and a marked increase in the complexity of the microecological system in patients. This study demonstrated that FMT through oral administration is clinically feasible and has the potential to improve the effectiveness of current medications in the clinical symptoms of PD patients.

The involvement of transient receptor potential channels in mast cell activation by microbubbles.

This study was to explore the activation of mast cells by microbubbles, with the focus on transient receptor potential (TRP) channels mediated degranulation and calcium influx. Bone marrow-derived mast cells (BMMCs) were primarily obtained from femurs in mice and induced differentiation for 4 weeks. After the purity identification, BMMCs were contacted by homogeneous microbubbles with the diameter of 1 mm for 1 h. ß-hexosaminidase and histamine levels in supernatants were assessed by enzyme-linked immunosorbent assay (ELISA) and the CD63 expression was tested by flow cytometry. The intracellular calcium binding with Fluo-4 AM dyes in BMMCs was observed under the fluorescence microscope and the mean fluorescence intensity was quantitatively measured by flow cytometry. ß-hexosaminidase release, histamine concentration, CD63 expression and calcium influx were significantly increased in BMMCs group upon microbubble stimulation compared to the control groups. After preconditioning with the available inhibitors and microbubble contact, only transient receptor potential vanilloid 1 (TRPV1) and TRPV4 inhibitors robustly suppressed the microbubble-induced degranulation. Likewise, the elevated fluorescence intensity of cytosolic calcium level was also significantly weaken. The results demonstrated microbubble stimulus effectively promoted BMMCs degranulation, which could be substantially restrained by inhibitors targeted for blocking TRPV1 or TRPV4 channel. The alternation of intracellular calcium level in BMMCs was consistent with the changes of degranulation capacity. It's suggested that the activation of BMMCs by microbubbles may involve specific TRP calcium dependent channels.

Assembly of 43 human Y chromosomes reveals extensive complexity and variation.

The prevalence of highly repetitive sequences within the human Y chromosome has prevented its complete assembly to date1 and led to its systematic omission from genomic analyses. Here we present de novo assemblies of 43 Y chromosomes spanning 182,900 years of human evolution and report considerable diversity in size and structure. Half of the male-specific euchromatic region is subject to large inversions with a greater than twofold higher recurrence rate compared with all other chromosomes2. Ampliconic sequences associated with these inversions show differing mutation rates that are sequence context dependent, and some ampliconic genes exhibit evidence for concerted evolution with the acquisition and purging of lineage-specific pseudogenes. The largest heterochromatic region in the human genome, Yq12, is composed of alternating repeat arrays that show extensive variation in the number, size and distribution, but retain a 1:1 copy-number ratio. Finally, our data suggest that the boundary between the recombining pseudoautosomal region 1 and the non-recombining portions of the X and Y chromosomes lies 500 kb away from the currently established1 boundary. The availability of fully sequence-resolved Y chromosomes from multiple individuals provides a unique opportunity for identifying new associations of traits with specific Y-chromosomal variants and garnering insights into the evolution and function of complex regions of the human genome.

Characterization of Early Gonadal Differentiation and Induction of Sex Reversal in the Rock Bream Oplegnathus fasciatus.

Rock bream (Oplegnathus fasciatus) is a typical fish that has a unique multiple sex chromosome system (♀X1X1X2X2/♂X1X2Y). We examined the early gonadal development in rock bream via continuous histological observations of the gonads at 40-120 days post hatching (dph). The fish was identified as a typical gonochorist, and female gonads were found to differentiate earlier than male gonads. The ovarian cavity of the female was initially observed at 80 dph, whereas the efferent duct of the male was not observed until 100 dph. Immunofluorescence with the vasa-antibody revealed that germ cells were predominantly distributed around the ovarian cavity in females and on the edge of the gonad in males during the early stages of sex differentiation. Sex reversal was induced via the oral administration of letrozole (LTZ), 17α-methyltestosterone (MT), and 17ß-estradiol (E2), respectively, during the labile period of gonadal development. LTZ and MT induced 100% masculinization of genotype-females, whereas E2 induced only 50-60% feminization of genotype-males. Such findings suggest that the fish retained high sexual plasticity despite the existence of the neo-Y chromosome. MT and E2 had negative effect on fish growth, whereas LTZ did not exert such side effect. LTZ and MT could accelerate gonadal development in sex-reversed genotype-males, whereas E2 inhibited gonadal development in genotype-females of rock bream. These findings provide a basis for further research on the mechanisms of sex determination and differentiation in fishes with X1X2Y sex chromosome system and provide a sex reversal protocol for rock bream.

Lactobacillus plantarum-derived indole-3-lactic acid ameliorates colorectal tumorigenesis via epigenetic regulation of CD8+ T cell immunity.

Previous studies have shown that Lactobacillus species play a role in ameliorating colorectal cancer (CRC) in a mouse model. However, the underlying mechanisms remain largely unknown. Here, we found that administration of a probiotic strain, Lactobacillus plantarum L168 and its metabolite, indole-3-lactic acid, ameliorated intestinal inflammation, tumor growth, and gut dysbiosis. Mechanistically, we indicated that indole-3-lactic acid accelerated IL12a production in dendritic cells by enhancing H3K27ac binding at the enhancer regions of IL12a that contributed to priming CD8+ T cell immunity against tumor growth. Furthermore, indole-3-lactic acid was found to transcriptionally inhibit Saa3 expression related to cholesterol metabolism of CD8+ T cells through changing chromatin accessibility and subsequent enhancing function of tumor-infiltrating CD8+ T cells. Together, our findings provide new insights into the epigenetic regulation of probiotics-mediated anti-tumor immunity and suggest the potential of L. plantarum L168 and indole-3-lactic acid to develop therapeutic strategies for patients with CRC.

Discovery of a first-in-class orally available HBV cccDNA inhibitor.

BACKGROUND & AIMS: The persistence of covalently closed circular DNA (cccDNA) in infected hepatocytes is the major barrier preventing viral eradication with existing therapies in patients with chronic hepatitis B. Therapeutic agents that can eliminate cccDNA are urgently needed to achieve viral eradication and thus HBV cure. METHODS: A phenotypic assay with HBV-infected primary human hepatocytes (PHHs) was employed to screen for novel cccDNA inhibitors. A HBVcircle mouse model and a uPA-SCID (urokinase-type plasminogen activator-severe combined immunodeficiency) humanized liver mouse model were used to evaluate the anti-HBV efficacy of the discovered cccDNA inhibitors. RESULTS: Potent and dose-dependent reductions in extracellular HBV DNA, HBsAg, and HBeAg levels were achieved upon the initiation of ccc_R08 treatment two days after the HBV infection of PHHs. More importantly, the level of cccDNA was specifically reduced by ccc_R08, while it did not obviously affect mitochondrial DNA. Additionally, ccc_R08 showed no significant cytotoxicity in PHHs or in multiple proliferating cell lines. The twice daily oral administration of ccc_R08 to HBVcircle model mice, which contained surrogate cccDNA molecules, significantly decreased the serum levels of HBV DNA and antigens, and these effects were sustained during the off-treatment follow-up period. Moreover, at the end of follow-up, the levels of surrogate cccDNA molecules in the livers of ccc_R08-treated HBVcircle mice were reduced to below the lower limit of quantification. CONCLUSIONS: We have discovered a small-molecule cccDNA inhibitor that reduces HBV cccDNA levels. cccDNA inhibitors potentially represent a new approach to completely cure patients chronically infected with HBV. IMPACT AND IMPLICATIONS: Covalently closed circular DNA (cccDNA) persistence in HBV-infected hepatocytes is the root cause of chronic hepatitis B. We discovered a novel small-molecule cccDNA inhibitor that can specifically reduce cccDNA levels in HBV-infected hepatocytes. This type of molecule could offer a new approach to completely cure patients chronically infected with HBV.

IKBIP, a novel glioblastoma biomarker, maintains abnormal proliferation of tumor cells by inhibiting the ubiquitination and degradation of CDK4.

Sustained proliferative signaling is a crucial hallmark and therapeutic target in glioblastoma (GBM); however, new intrinsic regulators and their underlying mechanisms remain to be elucidated. In this study, I kappa B kinase interacting protein (IKBIP) was identified to be correlated with the progression of GBM by analysis of The Cancer Genome Atlas (TCGA) data. TCGA database analysis indicated that higher IKBIP expression was associated with high tumor grade and poor prognosis in GBM patients, and these correlations were subsequently validated in clinical samples. IKBIP knockdown induced G1/S arrest by blocking the Cyclin D1/CDK4/CDK6/CDK2 pathway. Our results showed that IKBIP may bind directly to CDK4, a key cell cycle checkpoint protein, and prevent its ubiquitination-mediated degradation in GBM cells. An in vivo study confirmed that IKBIP knockdown strongly suppressed cell proliferation and tumor growth and prolonged survival in a mouse xenograft model established with human GBM cells. In conclusion, IKBIP functions as a novel driver of GBM by binding and stabilizing the CDK4 protein. IKBIP could be a potential therapeutic target in GBM.

Discovery of Novel cccDNA Reducers toward the Cure of Hepatitis B Virus Infection.

Chronic hepatitis B virus (HBV) infection is a worldwide disease that causes thousands of deaths per year. Currently, there is no therapeutic that can completely cure already infected HBV patients due to the inability of humans to eliminate covalently closed circular DNA (cccDNA), which serves as the template to (re)initiate an infection even after prolonged viral suppression. Through phenotypic screening, we discovered xanthone series hits as novel HBV cccDNA reducers, and subsequent structure optimization led to the identification of a lead compound with improved antiviral activity and pharmacokinetic profiles. A representative compound 59 demonstrated good potency and oral bioavailability with no cellular toxicity. In an HBVcircle mouse model, compound 59 showed excellent efficacy in significantly reducing HBV antigens, DNA, and intrahepatic cccDNA levels.

Comparative Transcriptome Analyses Characterize Expression Signatures Among Males, Females, Neo-Males, and Gynogenetic Females in the Yellow Drum (Nibea albiflora).

The yellow drum (Nibea albiflora) is one of the most important marine economic fish in China, and its sexually dimorphic growth makes it preferable for mono-sex culture. Although gynogenesis and neo-male induction techniques have been established, the molecular pathways and regulatory mechanisms of sex determination and maintenance in gynogenetic females and neo-males remains far from fully understood. In this study, the gene expression profiles were investigated in the gonads and brains of wild-type male, wild-type female, neo-male, and gynogenetic female yellow drum using comparative transcriptome analyses. Generally, a total of 52,999 novel transcripts were obtained in RNA-seq, of which 45,651 were isoforms of known protein-coding genes, 1,358 novel protein-coding genes, and 5,990 long non-coding RNAs. We found that the differences between wild-type males and neo-males and between wild-type females and gynogenetic females were relatively small at both the histological and transcriptomic levels, indicating that artificial gynogenesis or hormonal sex reversal may have minimal effects on normal female or male life function, respectively. In the brain, pathways such as "Oocyte meiosis", "Cell cycle", and "Riboflavin metabolism" were found to be significantly enriched. In the gonads, pathways such as "Prolactin signaling pathway", "PPAR signaling pathway", "Cholesterol metabolism", and "Jak-STAT signaling pathway" were found to play important roles in maintaining the regular proliferation and differentiation of females and males in yellow drum. In particular, we found that zp4 might be an effective molecular marker to differentiate between gynogenetic and normal females owing to its unique expression pattern. The results of this study may help to elucidate the molecular mechanisms involved in sex maintenance in the gonads and brain and provide basic data for genetic breeding of the yellow drum.

Recurrent inversion polymorphisms in humans associate with genetic instability and genomic disorders.

Unlike copy number variants (CNVs), inversions remain an underexplored genetic variation class. By integrating multiple genomic technologies, we discover 729 inversions in 41 human genomes. Approximately 85% of inversions <2 kbp form by twin-priming during L1 retrotransposition; 80% of the larger inversions are balanced and affect twice as many nucleotides as CNVs. Balanced inversions show an excess of common variants, and 72% are flanked by segmental duplications (SDs) or retrotransposons. Since flanking repeats promote non-allelic homologous recombination, we developed complementary approaches to identify recurrent inversion formation. We describe 40 recurrent inversions encompassing 0.6% of the genome, showing inversion rates up to 2.7 × 10-4 per locus per generation. Recurrent inversions exhibit a sex-chromosomal bias and co-localize with genomic disorder critical regions. We propose that inversion recurrence results in an elevated number of heterozygous carriers and structural SD diversity, which increases mutability in the population and predisposes specific haplotypes to disease-causing CNVs.

Rapid detection of InDel within the KDM3B gene in five sheep breeds using the mathematical expectation (ME) method.

Lysine demethylase 3B (KDM3B), a candidate gene associated with bone formation and growth, and differentiation of osteoblast, might affect the animal growth traits. Herein, the insertion/deletion (InDel) of the KDM3B gene was quickly detected in 882 sheep from five breeds using the mathematical expectation (ME) method. The results showed that there were two genotypes of 7-bp variation in KDM3B, including II (insertion/insertion) and ID (insertion/deletion), and the frequency of two genotypes varied among the five sheep breeds. Association analysis results demonstrated that the 7-bp indel was significantly associated with chest depth of LFT sheep (P = 0.012), and body weight (P = 0.006), body height (P = 0.030), chest depth (P = 0.043), chest circumference (P = 0.016), abdominal width (P = 0.035) and height at hip cross (P = 0.022) in LXBH sheep. Moreover, II genotype was the predominant genotype with excellent consistency in sheep growth traits (P < 0.05). Collectively, the above results suggest that this locus can be used as an effective molecular marker to improve the sheep growth traits and provide a scientific basis for the development of sheep breeding.

SvAnna: efficient and accurate pathogenicity prediction of coding and regulatory structural variants in long-read genome sequencing.

Structural variants (SVs) are implicated in the etiology of Mendelian diseases but have been systematically underascertained owing to sequencing technology limitations. Long-read sequencing enables comprehensive detection of SVs, but approaches for prioritization of candidate SVs are needed. Structural variant Annotation and analysis (SvAnna) assesses all classes of SVs and their intersection with transcripts and regulatory sequences, relating predicted effects on gene function with clinical phenotype data. SvAnna places 87% of deleterious SVs in the top ten ranks. The interpretable prioritizations offered by SvAnna will facilitate the widespread adoption of long-read sequencing in diagnostic genomics. SvAnna is available at https://github.com/TheJacksonLaboratory/SvAnn a .

Sex-Specific Genomic Region Identification and Molecular Sex Marker Development of Rock Bream (Oplegnathus fasciatus).

Rock bream (Oplegnathus fasciatus) is a valuable commercial marine teleost species, which exhibits sexual dimorphism in growth performance. However, the absence of a rapid and cost-effective sex identification method based on sex-specific genetic marker has impeded study on sex determination mechanisms and breeding applications. In the present study, we firstly developed the PCR method for identifying potential sex-specific sequences in Oplegnathus fasciatus with the next-generation sequencing. Sex-specific genomic regions/loci for sex determination were discovered on Chr2 and Chr6 by genome-wide association analysis, sequencing depth, and heterozygosity comparison between females and males. Candidate sex-determining genes (CCDC63, ITR, WNT4) were furtherly detected in transcriptome data of testes and ovaries. Taken together, a male-specific 34-bp deletion on the Chr2 was identified and developed into molecular marker of sex for O. fasciatus. After validation in individuals with known phenotypic sexes, the accuracy was 100%. This study gives an insight into the mechanism of sex determination in O. fasciatus, and the gender marker is crucial both for future genomic research and for development of efficient and sustainable aquaculture practice.

JAX-CNV: A Whole-genome Sequencing-based Algorithm for Copy Number Detection at Clinical Grade Level.

We aimed to develop a whole-genome sequencing (WGS)-based copy number variant (CNV) calling algorithm with the potential of replacing chromosomal microarray assay (CMA) for clinical diagnosis. JAX-CNV is thus developed for CNV detection from WGS data. The performance of this CNV calling algorithm was evaluated in a blinded manner on 31 samples and compared to the 112 CNVs reported by clinically validated CMAs for these 31 samples. The result showed that JAX-CNV recalled 100% of these CNVs. Besides, JAX-CNV identified an average of 30 CNVs per individual, respresenting an approximately seven-fold increase compared to calls of clinically validated CMAs. Experimental validation of 24 randomly selected CNVs showed one false positive, i.e., a false discovery rate (FDR) of 4.17%. A robustness test on lower-coverage data revealed a 100% sensitivity for CNVs larger than 300 kb (the current threshold for College of American Pathologists) down to 10× coverage. For CNVs larger than 50 kb, sensitivities were 100% for coverages deeper than 20×, 97% for 15×, and 95% for 10×. We developed a WGS-based CNV pipeline, including this newly developed CNV caller JAX-CNV, and found it capable of detecting CMA-reported CNVs at a sensitivity of 100% with about a FDR of 4%. We propose that JAX-CNV could be further examined in a multi-institutional study to justify the transition of first-tier genetic testing from CMAs to WGS. JAX-CNV is available at https://github.com/TheJacksonLaboratory/JAX-CNV.

Mako: A Graph-based Pattern Growth Approach to Detect Complex Structural Variants.

Complex structural variants (CSVs) are genomic alterations that have more than two breakpoints and are considered as the simultaneous occurrence of simple structural variants. However, detecting the compounded mutational signals of CSVs is challenging through a commonly used model-match strategy. As a result, there has been limited progress for CSV discovery compared with simple structural variants. Here, we systematically analyzed the multi-breakpoint connection feature of CSVs, and proposed Mako, utilizing a bottom-up guided model-free strategy, to detect CSVs from paired-end short-read sequencing. Specifically, we implemented a graph-based pattern growth approach, where the graph depicts potential breakpoint connections, and pattern growth enables CSV detection without pre-defined models. Comprehensive evaluations on both simulated and real datasets revealed that Mako outperformed other algorithms. Notably, validation rates of CSVs on real data based on experimental and computational validations as well as manual inspections are around 70%, where the medians of experimental and computational breakpoint shift are 13 bp and 26 bp, respectively. Moreover, the Mako CSV subgraph effectively characterized the breakpoint connections of a CSV event and uncovered a total of 15 CSV types, including two novel types of adjacent segment swap and tandem dispersed duplication. Further analysis of these CSVs also revealed the impact of sequence homology on the formation of CSVs. Mako is publicly available at https://github.com/xjtu-omics/Mako.

Identification and Characterization of Dimorphic Expression of Sex-Related Genes in Rock Bream, a Fish With Multiple Sex Chromosomes.

The rock bream (Oplegnathus fasciatus) is a typical fish with a unique multiple sex chromosome system. In this study, we investigated the gene expression profiling in the gonads and brains of both males and females using RNA-Seq to identify sex-related genes and pathways. In accordance with the dimorphic expression profiles, combined with Gene ontology and KEGG enrichment analyses, a number of potential genes and pathways associated with sex determination were obtained from transcriptional analysis, especially some sex-biased genes and pathways. Next, we selected 18 candidate genes and analyzed their expression in different tissues and developmental stages. We found that the expression levels of Amh, Dmrt1, Sox9, Dmrtb1, and Nanos2 were significantly higher in the testis than those in the ovary or other tissues, whereas the expression levels of ZP4, Bouncer, RNF208, FoxH1, and TOB were significantly higher in the ovary than those in the testis. Furthermore, the expression levels of these genes in different developmental stages of gonads also showed sexually dimorphic patterns, suggesting that they might play important roles during gonadal development. These genes are useful markers for investigating sex determination and differentiation in rock bream. The findings of this study can provide insights into the molecular mechanisms of sex determination and differentiation in fish with multiple sex chromosome systems.