Microglial phagocytosis is crucial for maintaining central nervous system (CNS) homeostasis, a process that depends on normal lysosomal acidification and is precisely regulated by vacuolar-type ATPase (V-ATPase). While mutations in the V-ATPase a3 subunit (encoded by the tcirg1b gene in zebrafish) are a major cause of human malignant osteopetrosis, the subunit's function in the CNS remains unknown. To investigate the role of the V-ATPase a3 subunit in the zebrafish CNS, we generated a tcirg1b knockout model. Although mutant zebrafish displayed no early neuronal defects, adult brains exhibited significant pathological alterations and behavioral abnormalities. Previous data showed that loss of tcirg1b resulted in enlarged microglia, suggesting potential functional alterations. In this study, transcriptome sequencing analysis of zebrafish macrophages revealed that phagosome formation and intracellular pH regulation pathway genes were significantly down-regulated. Functional analysis confirmed that V-ATPase a3 subunit deficiency impairs lysosomal acidification and digestive function in microglia, leading to the accumulation of apoptotic cell debris and TMR-dextran. Notably, specific restoration of tcirg1b expression in microglia successfully rescued the behavioral phenotypes of mutants, suggesting that the regulation of CNS homeostasis by the V-ATPase a3 subunit is primarily mediated through microglia. In summary, this study provides the first in vivo evidence that tcirg1b deficiency disrupts microglial function, thereby indirectly leading to an imbalance in CNS homeostasis. Our findings reveal a key role for the V-ATPase a3 subunit in regulating neural homeostasis and offer a new theoretical framework for studying the mechanisms of neurological diseases.

Somatic mutations in the ASXL1 gene, frequently found in myeloid malignancies, often generate C-terminal truncations and lead to abnormal protein accumulation. However, the molecular mechanisms regulating ASXL1 protein stability remain incompletely understood. Using a P2A peptide-based dual-reporter system, we demonstrate that truncated ASXL1 lacking the C-terminal domain (CTD) exhibits enhanced stability due to reduced ubiquitin-proteasome-mediated degradation. Dissection of the CTD revealed both destabilizing (IDR-1) and stabilizing (PEST) elements, indicating a complex sequence-encoded regulation of ASXL1 turnover. We identify Host Cell Factor 1 (HCF-1) as a cofactor that binds to the C-terminal ~200 amino acids of ASXL1 and promotes its turnover in a proteasome-dependent manner. Deletion of this region abrogates HCF-1 binding and stabilizes ASXL1. Although their binding sites are spatially distinct, HCF-1 and BAP1 show reciprocal antagonism in association with ASXL1, consistent with indirect coupling in complex assembly. Our findings identify a degron-like regulatory region within the ASXL1 CTD that integrates structural disorder and cofactor interactions to fine-tune protein stability, providing mechanistic insight into how truncating mutations dysregulate ASXL1 homeostasis.

Hematopoietic stem and progenitor cells (HSPCs) sustain blood cell production by balancing self-renewal and differentiation. While regulatory networks of transcription factors are well established during development of these cells, intrinsic cytoskeletal elements remain unclear. Here we show that the gamma-tubulin ring complex (γ-TuRC), a key regulator of microtubule nucleation, is essential for HSPC expansion in zebrafish. Forward genetic screening identifies the zebrafish smu1347 mutant, which exhibits HSPC exhaustion during definitive hematopoiesis. Positional cloning reveals a nonsense mutation in the tubgcp6 gene, encoding a core component of γ-TuRC, as responsible for the smu1347 phenotype. Mutation of Tubgcp6 causes mitotic arrest, disorganized spindle formation, and increased p53-dependent apoptosis. Time-lapse imaging and lineage tracing further demonstrate that Tubgcp6-deficient HSPCs preferentially undergo symmetric differentiation rather than self-renewal. Disrupting other γ-TuRC subunits (Tubgcp3, Tubgcp4, Tubgcp5) produces similar hematopoietic defects, underscoring the importance of intact microtubule nucleation for stem cell maintenance. These findings identify γ-TuRC-mediated microtubule organization as a critical regulator of HSPC fate and suggest that Tubgcp6 may represent a potential therapeutic target for bone marrow failure syndromes and stem cell exhaustion disorders.

The brown planthopper (BPH), Nilaparvata lugens (Stål), is a major rice pest across Asia. Control of N. lugens has been mainly relied on chemical insecticides. However, overuse has contributed to emergence of high-level insecticide resistances, particularly neonicotinoids like dinotefuran. Glutathione S-transferases (GSTs) play crucial roles in detoxifying insecticides involved in metabolic resistance. Here, we aimed to investigate the transcriptional regulation involved in the GST-mediated resistance to dinotefuran in N. lugens. GST gene expression and enzyme activity were found to be significantly up-regulated in field populations of N. lugens, and dinotefuran was identified as the key selection pressure by insecticide screening. RNA interference results showed a significant functional redundancy between the GSTm1 and GSTs2 genes, and resistance to dinotefuran was substantially compromised only upon co-silencing, whereas individual knockdown triggered reciprocal compensatory up-regulation. Analysis of the GSTm1 and GSTs2 promoter region revealed that the genes was individually regulated by two newly identified transcription factors related to resistance with different roles: BarH1 (BH1) acted as a repressor of GSTm1, while zen2 as an activator of GSTs2. This specific interaction was confirmed by the combination of promoter analysis, reporter gene experiments, site-directed mutagenesis, and electrophoretic mobility shift assays. In addition, treatment with dinotefuran inhibited expression of BH1 and induced zen2. Silencing BH1 led to higher expression of GSTm1 and increased resistance, whereas knocking down zen2 resulted in lower expression of GSTs2 and restored insecticide susceptibility. These findings greatly improve the understanding of the transcriptional regulation mechanisms of GST genes in insecticide resistance.

The molecular mechanisms by which insects perceive and behaviorally adapt to host plant nutritional variation constitute a fundamental question in insect-plant coevolution. The brown planthopper (Nilaparvata lugens, BPH), a monophagous pest of rice (Oryza sativa), exhibits striking wing dimorphism that directly governs its outbreak patterns: long-winged morphs (LW) initiate migration to escape low-quality hosts, while short-winged morphs (SW) drive local population explosions, thriving in high-quality hosts. However, the mechanism underlying this migration-residence behavior in response to host plant senescence remains unclear. Here, we identified early 4th and early 5th instars as sensitive stages in response to the yellow-ripe rice, a phenological stage marking the onset of host plant senescence that must be promptly avoided. High-performance liquid chromatography (HPLC) revealed that serotonin (5-HT) levels were significantly increased in BPHs during the sensitive stages. Exogenous administration of 5-HT and its precursor 5-hydroxytryptophan (5-HTP) significantly promoted long-winged morph development, whereas pharmacological inhibition with α-methyltryptophan (AMTP, a serotonin synthesis inhibitor) attenuated the wing dimorphism response to yellow-ripe rice. Identification and functional analysis of 5-HT synthesis pathway enzymes suggested that tryptophan hydroxylase (NlTRH) and aromatic L-amino acid decarboxylase (NlAADC) regulated the expression of insulin-like peptide 3 (NlIlp3), subsequently regulating the expression of insulin receptors 1 and 2 (NlInR1, NlInR2), which control wing dimorphism. In contrast, phenylalanine hydroxylase (NlPAH) showed no involvement. This study highlights the vital role of serotonin in wing dimorphism of BPH in response to host plant senescence and offers new targets for sustainable control of the pest.

Mast cells originate from mammalian myeloid hematopoiesis and exert a crucial role in inflammation and allergies. In recent years, studies have reported that carboxypeptidase A5 (cpa5) can serve as a marker for zebrafish mast cells. However, the absence of systematic experimental validation limited understanding of the origin, molecular characteristics, and function of mast cells. To address this gap, we initially confirmed zebrafish mast cell like cells in hematopoietic and peripheral tissues via staining and cytomorphological identification. Subsequently, spatiotemporal analysis and Tg(cpa5:GFP) sorting showed cpa5 mainly in mature neutrophils, rarely in mast cell like cells. Finally, gene knockout experiments revealed genetic compensation between cpa1 and cpa5 within the same gene family, and we further generated cpa1-/-cpa5-/- double mutant zebrafish. Experiment showed that in cpa1-/-cpa5-/-, the number of neutrophils was reduced while their functional properties remained intact; the number of mast cell like cells increased but their functions were impaired. This study revises the current understanding of cpa5 as a specific mast cell marker, explores its role in neutrophils and mast cells development, and thereby offers novel insights into zebrafish immune system.

Acute monocytic leukemia (AML-M5) is a type of acute myeloid leukemia, characterized by a dominance of monocytes in the bone marrow and peripheral blood. AML-M5 exhibits a poor prognosis compared to other AML subtypes. Despite clinical recognition, current research on AML-M5 remains relatively limited, and its underlying pathogenic mechanisms are not yet fully understood. In this study, we uncover a distinct and heightened expression of CBX4, a core component of PRC1, in the peripheral blood of individuals diagnosed with AML-M5. By generating cbx4 overexpression transgenic and deleted mutant zebrafish lines, we observe elevated cbx4 expression in monocyte/macrophage, selectively modulating their production during zebrafish hematopoiesis. Notably, aging zebrafish with cbx4 overexpression exhibit a progression to AML-M5-like hematopoiesis. Further mechanistic analyses reveal that Cbx4 regulates the fate of monocyte/macrophage lineage by suppressing runx1 expression. This suppression is achieved through the recruitment of HDAC to the runx1 promoter via cbx4, resulting in the down-regulation of the H3K27 acetylation level of runx1. These findings offer novel insights, providing potential avenues for risk assessment and molecular diagnosis of AML-M5 leukemia. Moreover, CBX4 emerges as a promising target for the diagnosis and treatment of AML-M5 leukemia.

Environmental estrogenic compounds are increasingly recognized as neurotoxicants, yet their effects on microglia intracellular homeostasis in vivo remain poorly understood. Using zebrafish larvae as a live imaging model, we identify the synthetic estrogen mestranol as a potent inducer of a reversible lysosomal storage-like state in microglia. Mestranol exposure causes pronounced microglia hypertrophy and reduces neutral red staining, without affecting microglia number or neuronal apoptosis. Functional analyses show that mestranol-treated microglia retain phagocytic capacity toward apoptotic neurons and bacterial particles but fail to efficiently digest internalized cargo, leading to intracellular accumulation. Although acidic vesicles and protease‑associated compartments expand and disperse throughout the cytoplasm, lysosomal degradative efficiency is markedly impaired, suggesting that cargo digestion is compromised despite the formation of acidic phagolysosomal compartments. Transcriptomic profiling of flow‑sorted macrophage/microglia populations reveals coordinated down‑regulation of lysosomal-phagosomal and immune gene networks, including MIT/TFE family transcription factors (TFEB/TFEC) and immune master regulators such as SPI1, IRFs, BATF, MAFB, and RUNX3. Overexpression of TFEC partially rescues both estradiol and mestranol-induced microglia hypertrophy and neutral red loss, although the rescue is only partial, indicating a more complex mechanism involving additional TFEC-independent pathways. Notably, microglia lysosomal dysfunction and hypertrophy are reversible upon drug withdrawal. Together, our findings demonstrate that mestranol induces an acquired and regulatable lysosomal stress state in microglia, characterized by immune transcriptional suppression and impaired intracellular digestion. This work establishes a live, reversible in vivo model for studying estrogen‑associated neuroimmunotoxicity and microglia vulnerability to lysosomal stress.

At present, the application of CRISPR/Cas9 technology for genetic manipulation in insects is predominantly concentrated on Diptera model species, including Drosophila and mosquitoes. In contrast, non-model insects such as the brown planthoppers (BPH, Nilaparvata lugens), a major insect pest of rice, have received less attention in genetic manipulation due to insufficient tools. Here, the analysis of spatiotemporal expression patterns revealed that β2-tubulin in BPH (NlB2t) was predominantly concentrated in male adults and male testis, exhibiting high expression levels. Knockdown of NlB2t expression by using RNAi resulted in the obstruction of male testis development. Mating between the RNAi-treated males and wild-type females led to a notable reduction in the number of eggs laid and the hatching rate of those eggs by 58.2% and 50.6%, respectively. The longevity of RNAi males significantly increased, and females that had previously mated with RNAi males exhibited a diminished inclination for re-mating with wild-type males. The dual-luciferase reporter assay demonstrated robust promoter activity in the upstream 943 bp of NlB2t, capable of driving Cas9 protein expression in vivo and effectively inducing target gene knockout. These findings elucidated that NlB2t may be a key gene in BPH male testis development and reproduction, as a promising target for sterilization. Its upstream promoter serves as a germline promoter, significantly facilitating the development of genetic control tools based on CRISPR/Cas9 technology in BPH.

Primitive hematopoiesis is a crucial process in the organism, responsible for the transportation of oxygen and nutrients during early embryonic stages and laying the foundation for the immune system. During primitive hematopoiesis, hematopoietic-related transcription factors and their cofactors interact to form a complex regulatory network that controls the process of primitive hematopoiesis. Among the bHLH transcription factor family, SCL and LYL1 are key factors in embryonic hematopoiesis. SCL is responsible for initiating primitive hematopoiesis, while LYL1, a paralog of SCL, compensates for the hematopoietic impact of SCL deficiency in adulthood. However, the role of LYL1 in primitive hematopoiesis remains unclear. This study, through analysis of zebrafish blood cell scRNA-seq data, discovered high expression of CABZ01066694.1 in hematopoietic stem/progenitor cells. Sequence alignment revealed it as a short transcript of the lyl1 gene. Subsequently, using 5'RACE and sequencing, the study confirmed the existence of both long (lyl1f) and short (lyl1s) transcripts of lyl1 in zebrafish and humans, similar to mice. Further analysis of scRNA-seq and RNA-seq data from public databases showed that in zebrafish primitive hematopoietic cells, lyl1 primarily transcribes lyl1s. Finally, using Morpholino technology to knock down lyl1f and lyl1s separately, it was found that knocking down lyl1s hindered the production of primitive myeloid progenitors and primitive granulocytes, whereas knocking down lyl1f promoted the production of primitive macrophages. In conclusion, this study demonstrates the existence of long and short transcripts of lyl1 in zebrafish and humans, with distinct roles in regulating primitive myelopoiesis, providing new insights into the regulation of primitive hematopoiesis.

RNA interference (RNAi) is a powerful tool for regulating gene expression and pest control. However, its application is hindered by off-target effects that can lead to adverse effects in both gene function studies and RNA biopesticide development. To address this challenge, a tool was developed. The dsRNA Off-target Minimisation Generator (dsOMG) website (https://dsomg.sysu.edu.cn/) employs all possible siRNAs derived from dicing the input sequence as queries to perform searches against the NCBI Transcript Reference Sequences (refseq_RNA) database, which encompasses 29,964 organisms, using BLAST-based alignment strategies to predict potential off-target effects. The website was developed using Java Spring Boot, while Vue 3.0, and Element UI were used to create a dynamic front-end interface. dsRNA synthesis templates can be swiftly obtained using dsOMG by executing a seven-step process tailored for both the "Gene function study" module and the "RNA biopesticide development" module. Re-evaluation with dsOMG revealed the intraspecific off-target risks associated with eight genes as well as the interspecific off-target risks of two commercial dsRNA products. Overall, dsOMG facilitates RNAi-based gene function studies across diverse organisms and provides practical tools to support RNA biopesticide development, thereby contributing to establishing the theoretical foundation for future safety assessment strategies of RNA biopesticides.

Approximately 30% of patients with myelodysplastic syndrome (MDS) progress to secondary acute myeloid leukemia (sAML) via accumulating gene mutations. Genomic analyses reveal a complex interplay among mutant genes, with co-occurring and mutually exclusive patterns. Hyperactivation of c-MYB and deficiency of PU.1 have been linked to myeloid disorders. We report a case of AML with concurrent PU.1 and c-MYB mutations, exhibiting early onset, high blast count, chemo-resistance, indicating high-risk features, along with elevated Pelger-Huët anomaly (PHA). However, the synergistic mechanism of c-MYB and PU.1 in sAML remains unclear. Using c-Myb-hyperactivation and Pu.1-deficient double-strain (c-mybhyper;pu.1G242D/G242D) zebrafish, we investigated MDS/sAML progression. Surprisingly, the double mutant exhibited a distinct type of neutrophil resembling clinical PHA cells and demonstrated a higher rate of MDS/sAML transformation. Further expression analysis revealed reduced lmnb1 expression in double-mutant zebrafish. Knockdown of lmnb1 resulted in PHA and increased blast cells, while overexpression of lmnb1 in c-mybhyper;pu.1G242D/G242D reduced PHA cell level. This suggests that c-Myb hyperactivation and Pu.1 deficiency synergistically reduce lmnb1 expression, inducing the development of PHA-like neutrophils and promoting MDS/sAML progression in zebrafish. Moreover, coadministration of cell cycle inhibitor cytarabine (Ara-C) and the differential inducer all-trans retinoic acid (ATRA) could effectively relieve the neutrophil expansion and PHA symptoms in c-mybhyper;pu.1G242D/G242D zebrafish. Our findings revealed that c-Myb hyperactivation and Pu.1 deficiency played a synergistic role in sAML development and suggests a phenotypic association between the emergence of PH-like cells and the transformation to sAML. Furthermore, c-mybhyper;pu.1G242D/G242D zebrafish might serve as a suitable sAML model for drug screening.

Hypertrophic cardiomyopathy (HCM) is a common inherited cardiac disorder often linked to mutations in various genes, including junctophilin-2 (JPH2), a key non-sarcomeric protein essential for forming junctional membrane complexes between the plasma membrane (PM) and endoplasmic/sarcoplasmic reticulum (ER/SR). Deciphering the molecular mechanisms behind these mutations is crucial for the development of effective therapies. In this study, we investigate the pathological effects of the HCM-associated S165F mutation in JPH2. Through integrated structural and biochemical analyses, we demonstrate that the S165F mutation induces an unintended intramolecular interaction in JPH2, disrupting its normal interaction with CaV1.2. This mutation compromises ER-PM junctions and disrupts Ca2+ signaling, leading to cellular hypertrophy in COS7 and H9c2 cell models. Furthermore, zebrafish overexpressing JPH2_S165F exhibit cardiac dysfunction, including pericardial edema and reduced heart rate. Notably, treatment with the ryanodine receptor agonist PCB-95 ameliorates these phenotypes, underscoring its potential clinical relevance. These findings offer new insights into the molecular mechanisms of JPH2-related HCM and provide a foundation for exploring novel therapeutic interventions. Mechanistic analysis of the S165F mutation in Junctophilin-2 reveals aberrant intramolecular binding that disrupts CaV1.2 coupling and Ca²⁺ signaling, driving hypertrophic cardiomyopathy phenotypes in zebrafish rescued by PCB-95.

Acute promyelocytic leukemia (APL) is driven by the specific fusion gene PML-RARA produced by chromosomal translocation. Three classic isoforms, L, V, and S, are found in more than 95% of APL patients. However, atypical PML-RARA isoforms are usually associated with uncertain disease progression and treatment prognosis. Recently, we found a novel PML-RARA isoform (named PA) in a patient with atypical clinical characteristics of APL. In order to provide valuable insights for clinical treatment, we constructed the novel PML-RARA isoform zebrafish model for all-trans retinoic acid (ATRA) treatment experiments and comparison with classical isoforms. We found that the effect of PA PML-RARA on the expression of neutrophil-related genes was comparable with classical isoforms and ATRA treatment worked successfully in the zebrafish model. Sequence and structure analysis of the PA protein confirmed its similarity to classical isoforms and the fusion site of PA PML-RARA did not affect the ATRA binding site. As expected, the patient achieved complete remission within two months of treatment with ATRA in combination with arsenic trioxide (ATO) and had a favorable prognosis during the three-year follow-up. Our study highlights the accuracy and efficacy of the PML-RARA zebrafish model in combination with protein structure prediction in support of clinical treatment strategies.

Microglia,as the resident immune cells of the central nervous system(CNS),maintain neural homeostasis by orchestrating the clearance of necrotic neurons,cellular debris,and pathogens through phagocytosis.This intracellular process involves a dynamic cascade of protein-mediated events,including cargo recognition and phagosome formation,phagosome maturation,and lysosomal degradation.Phagosome maturation critically relies on V-ATPase-driven acidification,with proton transport efficiency predominantly determined by its a subunit.In mammals,the V-ATPase a subunit has four isoforms(a1,a2,a3 and a4),whereas zebrafish(Danio rerio)possess only the three isoforms(a1,a2 and a3),with marked differences observed in their targeted cell types and suborganellar localization patterns.Notably,the V-ATPase a3 subunit is primarily localized on osteoclast lysosomes,facilitating bone resorption by acidification.The preliminary studies have revealed its conserved lysosomal localization in zebrafish microglia;however,its functional role in microglial development and regulatory mechanisms underlying phagosome maturation remain elusive.To address this knowledge gap,we employed a V-ATPase a3 subunit-deficient(tcirg1b-/-)zebrafish model,integrating whole-mount in situ hybridization,immunofluorescence,co-immunoprecipitation(Co-IP),and apoptosis assays.Key findings include:The a3 subunit is expressed during early zebrafish development;a3 subunit deficiency caused abnormal phagosome accumulation,microglial swelling and decreased activity,showing an"indigestion-like"phenotype;By exogenous labeling of late phagosomes and immunofluorescence staining,it was confirmed that the defect of V-ATPase a3 subunit impaired late phagosome-lysosome fusion in microglia;Co-IP demonstrated direct binding between the a3 subunit and Rab7.Rab7 knockdown phenocopied the"indigestion"morphology,suggesting a cooperative role in phagosome degradation.These results demonstrate that the V-ATPase a3 subunit regulates microglial phagosome maturation by mediating Rab7-dependent phagolysosomal fusion.In conclusion,this study not only elucidates the molecular mechanism underlying microglial phagocytic capacity but also provides novel insights into the evolutionarily conserved roles of V-ATPase isoforms in cellular clearance pathways.

Insecticide resistance in pest control poses a threat to agricultural production and human health. Numerous insect species express genes coding for detoxification enzymes that have broad substrate promiscuity thus conferring resistance to various insecticides. However, whether the homologs of these genes play similar roles in resistance phenotypes of closely related species remains largely unclear. Therefore, this study compares the resistance profiles of three major rice planthopper species (Delphacidae) (Laodelphax striatellus, Nilaparvata lugens, and Sogatella furcifera) based on the metabolic activity of their cytochrome P450s. Genome-wide analyses resulted in 68, 70, and 64 P450 genes in L. striatellus, N. lugens, and S. furcifera, respectively. Phylogenetic analyses among these genes found that most resistance-related genes in one species had homologs in other planthopper species. The most resistance-relevant orthogroup (CYP6ERs) showed higher evolutionary instability than most other groups. RNAi and in vitro metabolism assays revealed that CYP6ERs confers more divergent insecticide resistance profiles among planthopper species than the other two major resistance-related P450 subfamilies (CYP6AYs and CYP4C61s). Alphafold-based structural predictions and alignments suggested that P450 orthogroups with higher phylogenetic instability tended to have less structural similarities, resulting in more divergent metabolic profiles. This relationship was also in silico validated on Aphidae aphids and Lepidoptera noctuids. This study proposes combined phylogenetic and toxicogenomic analyses for understanding CYPome-based insecticide resistance convergency and divergency among closely related pests. These findings may improve the accuracy and rationality of chemical pest control.

Insects, the most numerous and diverse group of animal species on Earth, have important interactions with humans through providing resources, transmitting diseases and damaging agricultural cultivars. Cytochrome P450 monooxygenases (P450s) are one of the most important protein families in insects implicated in the endogenous metabolism and detoxification of xenobiotics, including allelochemicals, insecticides and environmental pollutants. To better understand the evolution and function of insect P450s and support the development and application of insecticides for pest control, an integrated bioinformatics platform is highly desirable. Here, we present the Insect Cytochrome P450 database (ICPD, http://www.insectp450.net/), which contains 66,477 P450s collected from public databases and predicted from the genomes of 682 insect species using a standardised bioinformatics protocol. Phylogenetic relationships between P450 genes are constructed for each species. The structures of all P450 proteins in the database are predicted using ESMFold, then visualised using WeView. Web services, such as BLAST, homogeneous modelling and molecular docking, are provided for determining the catalytic activities of P450 proteins. The ICPD will facilitate systematic investigations of the evolution and functions of the complete insect P450 complement, and represents a powerful tool for guiding insecticide design and application.

RNA interference (RNAi) studies for biopesticide development have been extensively performed. However, an ongoing summary of such studies to promote the development of RNA biopesticides is required. Data were collected from 74 RNAi studies for the development of biopesticides targeting Nilaparvata lugens, a serious insect pest of rice, from 2010 to March 2023. Regression analysis and conditional inference tree were used to explore the factors affecting the effect and off-target risk of RNAi in pest control. The results showed that days after treatment (DAT) and gene type were important factors influencing the RNAi effect, and 100% of the studied housekeeping genes had a high-lethal effect when DAT >= 7 days. dsRNA dose had a significant positive impact on RNAi effect. In the range of 200 to 887 bp, dsRNA length had no significant impact on RNAi effect. The proportion of conserved domains (PCD) had no significant impact on RNAi effect and off-target risk. To advance the development of RNA biopesticides, it is essential to minimize the off-target risk of dsRNA and to develop dsRNA delivery methods suitable for field management.

Objective Junctophilin-2 (JPH2) is an essential structural protein that maintains junctional membrane complexes (JMCs) in cardiomyocytes by tethering the plasma membrane to the sarcoplasmic reticulum, thereby facilitating excitation-contraction (E-C) coupling. Mutations in JPH2 have been associated with hypertrophic cardiomyopathy (HCM), but the molecular mechanisms governing its membrane-binding properties and the functional relevance of its membrane occupation and recognition nexus (MORN) repeat motifs remain incompletely understood. This study aimed to elucidate the structural basis of JPH2 membrane association and its implications for HCM pathogenesis. Methods A recombinant N-terminal fragment of mouse JPH2 (residues 1-440), encompassing the MORN repeats and an adjacent helical region, was purified under near-physiological buffer conditions. X-ray crystallography was employed to determine the structure of the JPH2 MORN-Helix domain. Sequence conservation analysis across species and junctophilin isoforms was performed to assess the evolutionary conservation of key structural features. Functional membrane-binding assays were conducted using liposome co-sedimentation and cell-based localization studies in COS7 and HeLa cells. In addition, site-directed mutagenesis targeting positively charged residues and known HCM-associated mutations, including R347C, was used to evaluate their effects on membrane interaction and subcellular localization. Results The crystal structure of the mouse JPH2 MORN-Helix domain was resolved at 2.6 & Aring;, revealing a compact, elongated architecture consisting of multiple tandem MORN motifs arranged in a curved configuration, forming a continuous hydrophobic core stabilized by alternating aromatic residues. A C-terminal alpha-helix further reinforced structural integrity. Conservation analysis identified the inner groove of the MORN array as a highly conserved surface, suggesting its role as a protein-binding interface. A flexible linker segment enriched in positively charged residues, located adjacent to the MORN motifs, was found to mediate direct electrostatic interactions with negatively charged phospholipid membranes. Functional assays demonstrated that mutation of these basic residues impaired membrane association, while the HCM-linked R347C mutation completely abolished membrane localization in cellular assays, despite preserving the overall MORN-Helix fold in structural modeling. Conclusion This study provides structural insight into the membrane-binding mechanism of the cardiomyocyte-specific protein JPH2, highlighting the dual roles of its MORN-Helix domain in membrane anchoring and protein interactions. The findings clarify the structural basis for membrane targeting via a positively charged linker and demonstrate that disruption of this interaction-such as that caused by the R347C mutation-likely contributes to HCM pathogenesis. These results not only enhance current understanding of JPH2 function in cardiac E-C coupling but also offer a structural framework for future investigations into the assembly and regulation of JMCs in both physiological and disease contexts.