Alkenyl oxindole is a novel PROTAC moiety that recruits the CRL4DCAF11 E3 ubiquitin ligase complex for targeted protein degradation.

Alkenyl oxindoles have been characterized as autophagosome-tethering compounds (ATTECs), which can target mutant huntingtin protein (mHTT) for lysosomal degradation. In order to expand the application of alkenyl oxindoles for targeted protein degradation, we designed and synthesized a series of heterobifunctional compounds by conjugating different alkenyl oxindoles with bromodomain-containing protein 4 (BRD4) inhibitor JQ1. Through structure-activity relationship study, we successfully developed JQ1-alkenyl oxindole conjugates that potently degrade BRD4. Unexpectedly, we found that these molecules degrade BRD4 through the ubiquitin-proteasome system, rather than the autophagy-lysosomal pathway. Using pooled CRISPR interference (CRISPRi) screening, we revealed that JQ1-alkenyl oxindole conjugates recruit the E3 ubiquitin ligase complex CRL4DCAF11 for substrate degradation. Furthermore, we validated the most potent heterobifunctional molecule HL435 as a promising drug-like lead compound to exert antitumor activity both in vitro and in a mouse xenograft tumor model. Our research provides new employable proteolysis targeting chimera (PROTAC) moieties for targeted protein degradation, providing new possibilities for drug discovery.

Genome-wide identification and expression analysis of the WNK kinase gene family in soybean.

WNK kinases are a unique class of serine/threonine protein kinases that lack a conserved catalytic lysine residue in the kinase domain, hence the name WNK (with no K, i.e., lysine). WNK kinases are involved in various physiological processes in plants, such as circadian rhythm, flowering time, and stress responses. In this study, we identified 26 WNK genes in soybean and analyzed their phylogenetic relationships, gene structures, chromosomal distribution, cis-regulatory elements, expression patterns, and conserved protein motifs. The soybean WNK genes were unevenly distributed on 15 chromosomes and underwent 21 segmental duplication events during evolution. We detected 14 types of cis-regulatory elements in the promoters of the WNK genes, indicating their potential involvement in different signaling pathways. The transcriptome database revealed tissue-specific and salt stress-responsive expression of WNK genes in soybean, the second of which was confirmed by salt treatments and qRT-PCR analysis. We found that most WNK genes were significantly up-regulated by salt stress within 3 h in both roots and leaves, except for WNK5, which showed a distinct expression pattern. Our findings provide valuable insights into the molecular characteristics and evolutionary history of the soybean WNK gene family and lay a foundation for further analysis of WNK gene functions in soybean. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01440-5.

Physicochemical Synergistic Separator Coating Induces Uniform and Rapid Deposition of Li and Zn Ions.

Li and Zn metal batteries are the most promising candidates to replace conventional Li-ion batteries. However, a series of issues, especially dendrites caused by uneven deposition of cations during charge-discharge cycles, hinder their practical application. Here, we proposed a facile separator modification method which combines physical and chemical forces to regulate uniform and rapid deposition of both Li+ and Zn2+. Physically, the electronegativity of modified separators drives rapid transport of metal ions via a surface diffusion mode. Chemically, the polar surface functional groups on coated separators induce uniform deposition of metal ions so that the dendrite growth is effectively inhibited. As a result, the Li and Zn metal anodes employing modified separators can cycle stably for over 1000 h under a large current density of 10 mA cm-2.

The Biosynthesis Process of Small RNA and Its Pivotal Roles in Plant Development.

In the realm of plant biology, small RNAs (sRNAs) are imperative in the orchestration of gene expression, playing pivotal roles across a spectrum of developmental sequences and responses to environmental stressors. The biosynthetic cascade of sRNAs is characterized by an elaborate network of enzymatic pathways that meticulously process double-stranded RNA (dsRNA) precursors into sRNA molecules, typically 20 to 30 nucleotides in length. These sRNAs, chiefly microRNAs (miRNAs) and small interfering RNAs (siRNAs), are integral in guiding the RNA-induced silencing complex (RISC) to selectively target messenger RNAs (mRNAs) for post-transcriptional modulation. This regulation is achieved either through the targeted cleavage or the suppression of translational efficiency of the mRNAs. In plant development, sRNAs are integral to the modulation of key pathways that govern growth patterns, organ differentiation, and developmental timing. The biogenesis of sRNA itself is a fine-tuned process, beginning with transcription and proceeding through a series of processing steps involving Dicer-like enzymes and RNA-binding proteins. Recent advances in the field have illuminated the complex processes underlying the generation and function of small RNAs (sRNAs), including the identification of new sRNA categories and the clarification of their involvement in the intercommunication among diverse regulatory pathways. This review endeavors to evaluate the contemporary comprehension of sRNA biosynthesis and to underscore the pivotal role these molecules play in directing the intricate performance of plant developmental processes.

Turn "Waste" Into Wealth: MoO2 @coal Gangue Electrocatalyst with Amorphous/Crystalline Heterostructure for Efficient Li-O2 Batteries.

Unreasonable accumulation of coal gangue in mining area has become the major source of global pollution. Probing the high-valued utilization of coal gangue has become a key approach to address the problem. Herein, a promising catalyst of MoO2 @coal gangue with amorphous/crystalline heterostructure derived from mine solid waste, which acts as an efficient cathode for Li-O2 batteries is first reported. Impressively, the as-prepared catalyst exhibits a favorable initial discharge capacity of 9748 mAh g-1 and promising long-term cyclic stability over 2200 h. Experimental results coupled with density functional theory (DFT) analysis reveal that the synergistic interaction between high-activity MoO2 and stable SiO2 , unique amorphous/crystalline heterostructure and the modified interfacial adsorption of LiO2 intermediate are critical factors in promoting the electrochemical performance. This work provides a new insight to design marked electrocatalysts by mine solid waste for Li-O2 batteries.

Genome-Wide Identification and Characterization of the Soybean Snf2 Gene Family and Expression Response to Rhizobia.

Sucrose nonfermenting 2 (Snf2) family proteins are the core component of chromatin remodeling complexes that can alter chromatin structure and nucleosome position by utilizing the energy of ATP, playing a vital role in transcription regulation, DNA replication, and DNA damage repair. Snf2 family proteins have been characterized in various species including plants, and they have been found to regulate development and stress responses in Arabidopsis. Soybean (Glycine max) is an important food and economic crop worldwide, unlike other non-leguminous crops, soybeans can form a symbiotic relationship with rhizobia for biological nitrogen fixation. However, little is known about Snf2 family proteins in soybean. In this study, we identified 66 Snf2 family genes in soybean that could be classified into six groups like Arabidopsis, unevenly distributed on 20 soybean chromosomes. Phylogenetic analysis with Arabidopsis revealed that these 66 Snf2 family genes could be divided into 18 subfamilies. Collinear analysis showed that segmental duplication was the main mechanism for expansion of Snf2 genes rather than tandem repeats. Further evolutionary analysis indicated that the duplicated gene pairs had undergone purifying selection. All Snf2 proteins contained seven domains, and each Snf2 protein had at least one SNF2_N domain and one Helicase_C domain. Promoter analysis revealed that most Snf2 genes had cis-elements associated with jasmonic acid, abscisic acid, and nodule specificity in their promoter regions. Microarray data and real-time quantitative PCR (qPCR) analysis revealed that the expression profiles of most Snf2 family genes were detected in both root and nodule tissues, and some of them were found to be significantly downregulated after rhizobial infection. In this study, we conducted a comprehensive analysis of the soybean Snf2 family genes and demonstrated their responsiveness to Rhizobia infection. This provides insight into the potential roles of Snf2 family genes in soybean symbiotic nodulation.

Production, purification, and functional properties of microbial fibrinolytic enzymes produced by microorganism obtained from soy-based fermented foods: developments and challenges.

According to the World Health Organization, cardiovascular disease (CVD) has become a major cause of chronic illness around the globe. It has been reported that soy-based fermented food (SFF) is very effective in preventing thrombus (one of the most important contributing factors to CVD), which are mainly attributed to the bioactive substances, especially the fibrinolytic enzymes (FE) generated by microorganisms during the fermentation process of soybean food. This paper therefore mainly reviewed the microbial fibrinolytic enzymes (MFE) from SFF. We first discuss the use of microbial fermentation to produce FE, with an emphasis on the strains involved. The production, purification, physicochemical properties, structure-functional attributes, functional properties and possible application of MFE from SFF are then discussed. Finally, current limitations and future perspectives for the production, purification, and the practical application of MFE are discussed. MFE from SFF pose multiple health benefits, including thrombolysis, antihypertension, anti-inflammatory, anti-hyperlipidemia, anticancer, neuroprotective, antiviral and other activities. Therefore, they exhibit great potential for functional foods and nutraceutical applications, especially foods with CVDs prevention potential.

The Coxiella burnetii QpH1 plasmid is a virulence factor for colonizing bone marrow-derived murine macrophages.

Coxiella burnetii strains carry one of four large, conserved, autonomously replicating plasmids (QpH1, QpRS, QpDV, and QpDG) or a QpRS-like chromosomally integrated sequence of unknown function. Here we report the characterization of the QpH1 plasmid of C. burnetii Nine Mile phase II by making QpH1-deficient strains. A shuttle vector pQGK containing the CBUA0036-0039a region (predicted as being required for the QpH1 maintenance) was constructed. The pQGK vector can be stably transformed into the Nine Mile II and maintained at a similar low copy like QpH1. Importantly, transformation with pQGK cured the endogenous QpH1 due to plasmid incompatibility. Compared to a Nine Mile II transformant of a RSF1010-ori based vector, the pQGK transformant shows a similar growth curve in both axenic media and Buffalo green monkey kidney cells, a variable growth defect in macrophage-like THP-1 cells depending on the origin of inoculum, and dramatically reduced ability of colonizing wild-type bone marrow-derived murine macrophages. Furthermore, we found CBUA0037-0039 ORFs are essential for plasmid maintenance, and CBUA0037-0038 ORFs account for plasmid compatibility. And plasmid-deficient C. burnetii can be isolated by using CBUA0037 or -0038 deletion vectors. Furthermore, QpH1-deficient C. burnetii strains caused a lesser extent of splenomegaly in SCID mice but, intriguingly, they had significant growth in SCID mouse-sourced macrophages. Taken together, our data suggest that QpH1 encodes factor(s) essential for colonizing murine, not human, macrophages. This study suggests a critical role of QpH1 for C. burnetii persistence in rodents and expands the toolkit for the genetic studies in C. burnetii Author summary All C. burnetii isolates carry one of four large, conserved, autonomously replicating plasmids or a plasmid-like chromosomally integrated sequence. The plasmid is a candidate virulence factor of unknown function. Here we describe the construction of novel shuttle vectors that allow making plasmid-deficient C. burnetii mutants. With this plasmid-curing approach, we characterized the role of the QpH1 plasmid in in vitro and in vivo C. burnetii infection models. We found that the plasmid plays a critical role for C. burnetii growth in murine macrophages. Our work suggests an essential role of the QpH1 plasmid for the acquisition of colonizing capability in rodents by C. burnetii This study represents a major step toward unravelling the mystery of the C. burnetii cryptic plasmids.

A Self-Jet Vapor-Phase Growth of 3D FeNi@NCNT Clusters as Efficient Oxygen Electrocatalysts for Zinc-Air Batteries.

Development of highly active, robust electrocatalysts to accelerate the sluggish oxygen reduction reaction (ORR) and oxygen evolution reaction (OER) is crucial and challenging for the practical application of metal-air batteries. In this effort, a novel and facile self-jet vapor-phase growth approach is developed, from which highly dispersive FeNi alloy nanoparticles (NPs) encapsulated in N-doped carbon nanotubes (NCNT) grown on a cotton pad (FeNi@NCNT-CP) can be fabricated. The as-prepared FeNi@NCNT-CP clusters exhibit superior bifunctional catalytic activity, with a high half-wave potential of 0.85 V toward ORR and a low potential of 1.59 V at 10 mA cm-2 toward OER. Specifically, owing to the synergistic effects of FeNi alloy NPs and NCNT, FeNi@NCNT-CP clusters deliver excellent stability, demonstrating a small potential gap of 0.73 V between ORR and OER after operation for 10 000 cycles. Furthermore, FeNi@NCNT-CP serves as a cost-effective, superior catalyst for the cathode of a rechargeable Zn-air battery, outperforming a catalyst mixture of expensive Pt/C and IrO2 . FeNi@NCNT-CP provides a maximum power density of 200 mW cm-2 and a cycling stability of up to 250 h. This contribution provides new prospects to prepare non-noble electrocatalysts for metal-air battery cathodes.

Redox-Mediated Endocytosis of a Receptor-Like Kinase during Distal Stem Cell Differentiation Depends on Its Tumor Necrosis Factor Receptor Domain.

Cellular redox status plays critical roles in cell division and differentiation, but the underlying mechanism is unclear. Here we explored the effect of redox status on stem cell identity in distal stem cells (DSCs) of Arabidopsis (Arabidopsis thaliana) roots. Treatment with the reductive reagent glutathione and the oxidative reagent H2O2 inhibited DSC differentiation, as did endogenously altering reactive oxygen species production via various mutations. This suggests that both highly reductive and oxidative environments inhibit specification of stem cell identity. In our observations of mutant components of the CLAVATA3/ENDOSPERM SURROUNDING REGION 40 (CLE40)-ARABIDOPSIS CRINKLY4 (ACR4)/CLAVATA1 (CLV1)-WUSCHEL RELATED HOMEOBOX5 (WOX5) module, both reductive and oxidative reagents influenced DSC differentiation in wox5-1 and clv1-1, but not in acr4-2 or cle40 mutant plants. The stability of the receptor-like kinase ACR4 is modulated by redox status through endocytosis in root tips. ACR4 with multiple Cys mutations in the tumor necrosis factor receptor (TNFR) extracellular domain failed to undergo endocytosis. ACR4 with a complete deletion of the TNFR domain was localized directly to endosomes, bypassing the plasma membrane. Both mutations affected DSC differentiation, but not seed filling. Conversely, the intracellular domain of the ACR4 protein is partially required for seed filling, but not for DSC differentiation. Our study uncovers an important biological role of the TNFR domain in redox-mediated endocytosis of ACR4 in root DSC differentiation.

Neuropharmacological effects of Aconiti Lateralis Radix Praeparata.

Aconiti Lateralis Radix Praeparata (Fuzi in Chinese), which are the lateral roots of Aconitum Carmichaelii Debx, is widely used in China to treat many neurological diseases. Fuzi, in its various forms, has many neuropharmacological effects. It can act as an analgesic and help with depression, epilepsy, and dementia. However, the neuropharmacological effects of Aconiti Lateralis Radix Praeparata are seldom comprehensively reviewed. In this review, the neuropharmacological activities of some components contained in Aconiti Lateralis Radix Praeparata are considered. These include aconitine, mesaconitine, hypaconitine, total alkaloid, polysaccharide-1, benzoylmesaconine, fuziline, songorine, and napelline. We also specifically discuss the antidepressant effects of total alkaloids and polysaccharide-1. This review may provide a theoretical basis for further utilization of Aconiti Lateralis Radix Praeparata for diseases that affect the central nervous system.

Neuropharmacological Effects of Mesaconitine: Evidence from Molecular and Cellular Basis of Neural Circuit.

Mesaconitine (MA), a diester-diterpenoid alkaloid in aconite roots, is considered to be one of the most important bioactive ingredients. In this review, we summarized its neuropharmacological effects, including analgesic effects and antiepileptiform effects. Mesaconitine can act on the central noradrenergic system and the serotonin system; behaving like the norepinephrine reuptake inhibitors and tricyclic antidepressants that increase norepinephrine levels in stress-induced depression. Therefore, the possible perspectives for future studies on the depression of MA were also discussed as well. The pharmacological effect of MA on depression is worthy of further study.

Long non-coding RNA LINC00152 promotes cell growth and invasion of papillary thyroid carcinoma by regulating the miR-497/BDNF axis.

Long intergenic non-coding RNA 152 (LINC00152) was reported to be tightly linked to tumorigenesis and progression in multiple cancers. However, its biological role and modulatory mechanism in papillary thyroid carcinoma (PTC) has not been elucidated. In this study, we determined the expression levels of LINC00152 in PTC tissues and cell lines by quantitative real time polymerase chain reaction (qRT-PCR). Cell proliferation, colony formation, migration, and invasion were measured by a Cell Counting Kit-8 assay, colony formation analysis, wound healing, and transwell invasion assay, respectively. A luciferase reporter assay and qRT-PCR were used to determine whether LINC00152 interacts with miR-497 directly. We established a xenograft mouse model to examine the underlying molecular mechanism and effect of LINC00152 on tumor growth in vivo. We found that LINC00152 expression was significantly increased in PTC tissues and derived cell lines. LINC00152 knockdown significantly inhibited proliferation, colony formation, migration, and invasion in vitro, and impaired tumor growth in vivo. We revealed that LINC00152 functioned as a competing endogenous RNA to the miR-497 sponge, downregulating its downstream target brain-derived neurotrophic factor (BDNF), which is an oncogene in thyroid cancer. These findings suggest that LINC00152 is responsible for PTC cell proliferation and invasion and exerts its function by regulating the miR-497/BDNF axis.

Prevalence characteristics of single and multiple HPV infections in women with cervical cancer and precancerous lesions in Beijing, China.

We assessed the prevalence characteristics of single and multiple high-risk human papillomavirus (HR-HPV) infections. A total of 1783 women who underwent colposcopy and cervical biopsy for abnormal ThinPrep Cytology Test and/or HR-HPV subtype genotyping results were enrolled in the study. Among the participants, 770 were diagnosed with cervicitis, 395 with cervical intraepithelial neoplasia grade 1 (CIN1), 542 with CIN2-3, and 76 with squamous cell carcinoma (SCC), with HR-HPV infection rates of 75.8%, 85.8%, 95.9%, and 88.4%, respectively. The prevalence of total and multiple HR-HPV infections exhibited a bimodal age distribution with a peak at ≤25 years, a decline with age and a second peak at ≥55 years, whereas single HR-HPV infections exhibited one peak from 35 to 44 years. The four most dominant HPV genotypes were HPV 16 (29.5%), 52 (15.0%), 58 (14.2%), and 18 (10.4%). In total, 67.0%, 70.4%, and 82.1% of patients with CIN1, CIN2-3, and SCC, respectively, had a single HR-HPV infection, which increased significantly with the aggravation of the cervical lesion grade (P = 0.045). Patients with a single HPV 16 infection had higher incidences of CIN2+ (62.2%) than those with multiple HPV 16 infections (52.4%) (P = 0.021). Patients coinfected with HPV 16 had higher CIN2+ incidence than those with single HPV 52, 31, 33, 35, 39, 45, 51, 56, or 59 infections (P < 0.001). This study provided baseline data on the prevalence characteristics of single and multiple HR-HPV infections in women attending a gynecological outpatient clinic in Beijing.

Design, synthesis and evaluation of 2-amino-imidazol-4-one derivatives as potent ß-site amyloid precursor protein cleaving enzyme 1 (BACE-1) inhibitors.

Inhibition of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) to prevent brain ß-amyloid (Aß) peptide's formation is a potential effective approach to treat Alzheimer's disease. In this report we described a structure-based optimization of a series of BACE1 inhibitors derived from an iminopyrimidinone scaffold W-41 (IC50 = 7.1 µM) by Wyeth, which had good selectivity and brain permeability but low activity. The results showed that occupying the S3 cavity of BACE1 enzyme could be an effective strategy to increase the biological activity, and five compounds exhibited stronger inhibitory activity and higher liposolubility than W-41, with L-5 was the most potent inhibitor against BACE1 (IC50 = 0.12 µM, logP = 2.49).

Recognition of the toxicity of aristolochic acid.

WHAT IS KNOWN AND OBJECTIVE: Aristolochic acid (AA) is an abundant compound in Aristolochia plants and various natural herbs. In the 1990s, a slimming formula used in Belgium that contains Aristolochia fangchi was reported to cause kidney damage and bladder cancer, and aristolochic acid nephropathy (AAN) is now well recognized worldwide. In October 2017, researchers reported an AA signature that is closely associated with hepatocellular carcinoma (HCC) worldwide. COMMENT: There are differing opinions on the toxicity of AA, and different countries have taken different measures to address the issue. There is a lack of clarity on the causal role of AA in hepatocarcinogenesis and on the potential underlying mechanisms for the reported nephrotoxicity and carcinogenicity. The toxicity of AA differs depending on gender and age, and other risk factors that could explain the variability in the toxicity of AA remain to be identified. WHAT IS NEW AND CONCLUSION: Whether preparations containing AA, such as many Chinese medicines, should be used remains controversial, and this issue warrants further investigation before definite conclusions can be drawn.

Molecular Cloning and Sexually Dimorphic Expression Analysis of nanos2 in the Sea Urchin, Mesocentrotus nudus.

Sea urchin (Mesocentrotus nudus) is an economically important mariculture species in China and the gonads are the solely edible parts to human. The molecular mechanisms of gonad development have attracted increasing attention in recent years. Although the nanos2 gene has been identified as a germ cell marker in several invertebrates, little is known about nanos2 in adult sea urchins. Hereinto, we report the characterization of Mnnano2, an M. nudus nanos2 homology gene. Mnnanos2 is a maternal factor and can be detected continuously during embryogenesis and early ontogeny. Real-time quantitative PCR (RT-qPCR) and section in situ hybridization (ISH) analysis revealed a dynamic and sexually dimorphic expression pattern of Mnnano2 in the gonads. Its expression reached the maximal level at Stage 2 along with the gonad development in both ovary and testis. In the ovary, Mnnanos2 is specifically expressed in germ cells. In contrast, Mnnanos2 is expressed in both nutritive phagocytes (NP) cells and male germ cells in testis. Moreover, knocking down of Mnnanos2 by means of RNA interference (RNAi) reduced nanos2 and boule expression but conversely increased the expression of foxl2. Therefore, our data suggest that Mnnanos2 may serve as a female germ cell marker during gametogenesis and provide chances to uncover its function in adult sea urchin.

Divergent Expression Patterns and Function of Two cxcr4 Paralogs in Hermaphroditic Epinephelus coioides.

Chemokine receptor Cxcr4 evolved two paralogs in the teleost lineage. However, cxcr4a and cxcr4b have been characterized only in a few species. In this study, we identified two cxcr4 paralogs from the orange-spotted grouper, Epinephelus coioides. The phylogenetic relationship and gene structure and synteny suggest that the duplicated cxcr4a/b should result from the teleost-specific genome duplication (Ts3R). The teleost cxcr4 gene clusters in two paralogous chromosomes exhibit a complementary gene loss/retention pattern. Ec_cxcr4a and Ec_cxcr4b show differential and biased expression patterns in grouper adult tissue, gonads, and embryos at different stages. During embryogenesis, Ec_cxcr4a/b are abundantly transcribed from the neurula stage and mainly expressed in the neural plate and sensory organs, indicating their roles in neurogenesis. Ec_Cxcr4a and Ec_Cxcr4b possess different chemotactic migratory abilities from the human SDF-1α, Ec_Cxcl12a, and Ec_Cxcl12b. Moreover, we uncovered the N-terminus and TM5 domain as the key elements for specific ligand⁻receptor recognition of Ec_Cxcr4a-Ec_Cxcl12b and Ec_Cxcr4b-Ec_Cxcl12a. Based on the biased and divergent expression patterns of Eccxcr4a/b, and specific ligand⁻receptor recognition of Ec_Cxcl12a/b⁻Ec_Cxcr4b/a, the current study provides a paradigm of sub-functionalization of two teleost paralogs after Ts3R.

Divergent Expression Patterns and Function Implications of Four nanos Genes in a Hermaphroditic Fish, Epinephelus coioides.

Multiple nanos genes have been characterized in several fishes, but the functional implications of their various expression patterns remain unclear. In this study, we identified and characterized four nanos genes from a hermaphroditic fish orange-spotted grouper, Epinephelus coioides. Ecnanos1a and Ecnanos1b show divergent expression patterns, and the dynamic expression change of Ecnanos1a in pituitaries during sex change is associated with testis differentiation and spermatogenesis. Ecnanos2 and Ecnanos3 might be germline stem cells (GSCs) and primordial germ cells (PGCs)-specific markers, respectively. Significantly, Ecnanos3 3'-untranslated region (UTR) is necessary for PGC specific expression, where a non-canonical "GCACGTTT" sequence is required for miR-430-mediated repression of Ecnanos3 RNA. Furthermore, grouper Dead end (Dnd) can relieve miR-430 repression in PGCs by associating with a 23 bp U-rich region (URR) in Ecnanos3 3'-UTR. The current study revealed the functional association of multiple nanos genes with PGC formation and germ cell development in orange-spotted grouper, and opened up new possibilities for developing biotechnologies through utilizing the associations between Ecnanos3 and PGCs or between Ecnanos2 and GSCs in the hermaphroditic fish.