Search Results (359)

The combination of Chinsurah Boro II (BT)-type cytoplasmic male sterility (CMS) and Rf1, the main fertility restorer gene (Rf) for CMS-BT, has been extensively utilized for the production of three-line commercial japonica hybrid seeds. The identification of new Rf genes holds significance for the breeding of BT-type restorer lines, aiming to enhance the heterosis level of BT-type japonica hybrids. In the present study, ‘02428’, a wide-compatibility japonica variety, was observed to partially restore fertility to BT-type CMS lines. Genetic analysis revealed that ‘02428’ carries a dominant Rf gene, Rf21(t), responsible for the fertility restoration of BT-type CMS lines. Leveraging bulked segregant analysis (BSA) resequencing technology and molecular markers, the Rf21(t) locus was identified, and mapped within a candidate interval of 6–12.5 Mb on chromosome 2. Using the iso-cytoplasmic restorer populations, Rf21(t) was ultimately mapped to an interval of approximately 77 kb, encompassing 12 predicted genes, including LOC_Os02g17360, encoding a PPR-domain-containing protein and LOC_Os02g17380 (Rf2), a cloned Rf for Lead-rice-type CMS. A comparative sequence analysis, gene expression profiling and gene knockout experiments confirmed that LOC_Os02g17360 and LOC_Os02g17380 are the most likely candidates of Rf21(t). Furthermore, Rf21(t) showed the dosage effect on the fertility restoration of BT-type CMS lines. This newly identified Rf21(t) represents a valuable genetic resource for the breeding of BT-type japonica restorer lines. Our findings offer practical insights for breeders interested in advancing BT-type japonica hybrid development. Full article

The basic leucine zipper (bZIP) transcription factors are involved in a wide range of physiological processes in plants, including hormone signaling, stress responses, and growth and development regulation. They play a key role in abscisic acid (ABA)-mediated immune regulation. However, the immune-related function of OsbZIP76 in rice remains poorly understood. In this study, we generated OsbZIP76 knockout (KO) lines using CRISPR/Cas9-mediated genome editing and examined their phenotypic responses to the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo) and the fungal pathogen Magnaporthe oryzae. The KO lines showed increased susceptibility to both pathogens compared to wild-type (WT) plants. Furthermore, qRT-PCR analysis revealed that, upon pathogen infection, the expression of pathogenesis-related genes such as PR1a, PR5, and NPR1 was significantly suppressed in the KO lines. ABA treatment experiments showed that KO lines were hypersensitive to exogenous ABA, indicating a role for OsbZIP76 in ABA perception and signaling. Notably, the expression of the OsbZIP76 gene itself was strongly induced by both ABA treatment and pathogen infection, supporting its role as a positive regulator in ABA-associated immune signaling. Overall, this study demonstrates that OsbZIP76 functions as an important immune regulator by integrating defense gene expression with ABA signaling, providing new insights into the molecular crosstalk between hormonal signaling and pathogen defense mechanisms. Full article

Background/Objectives: 5-Aminolevulinic acid (5-ALA) is a biosynthetic precursor of heme that induces heme oxygenase-1 (HO-1). Therapeutic induction of HO-1 has shown effectiveness in various autoimmune disease models, including type 1 diabetes (T1D). However, the efficacy of 5-ALA as an HO-1 inducer in T1D models remains unexplored. This study aimed to investigate the therapeutic efficacy of oral 5-ALA administration in preventing autoimmune diabetes development in nonobese diabetic (NOD) mice. Methods: We evaluated diabetes incidence, levels of insulin autoantibody, and severity of insulitis in 5-ALA-treated and control NOD mice. HO-1 expression of dendritic cells in the pancreatic islets and spleen of 5-ALA-treated NOD mice was measured. The IFN-γ/IL-17 of islet-infiltrating T cells and IL-10/IL-12 productions of dendritic cells in the spleen of 5-ALA-treated NOD mice were assessed. We stimulated islet antigen-specific CD4⁺ T cells with islet antigen-pulsed dendritic cells in the presence of 5-ALA and examined the proliferation of the T cells. Finally, we adoptively transferred islet antigen-specific CD4⁺ T cells into 5-ALA-treated, immunodeficient NOD-Rag1 knockout mice, and diabetes incidence in recipients was determined. Results: Oral 5-ALA treatment did not significantly impact diabetes incidence, levels of insulin autoantibody, and insulitis. No significant difference was observed in HO-1 expression in dendritic cells and cytokine production of T cells and dendritic cells. Similarly, there was no significant difference in the proliferation of islet antigen-specific CD4⁺ T cells in vitro and diabetes induction in transfer experiments. Conclusions: Oral administration of 5-ALA has a limited effect on suppressing the development of autoimmune diabetes in NOD mice. Full article

Flavonoid synthases (FNSs) are key enzymes catalyzing the conversion of flavanones to flavonoids, yet their functions in citrus remain functionally uncharacterized. In this study, we identified three FNSII genes in the citrus genome. Phylogenetic analysis revealed that citrus FNSII genes share the closest evolutionary distance with apple FNSII genes. Chromosomal localization demonstrated that the three FNSII genes are distributed across two out of nine chromosomes. Gene structure analysis indicated that the majority of motifs within these three FNSII genes are highly conserved. We cloned a gene called CitFNSII-1 from citrus. Transient overexpression of CitFNSII-1 in citrus leaves significantly increased flavonoid content, while simultaneous virus-induced silencing of CitFNSII-1 led to synchronously and significantly reduced gene expression levels and flavonoid content in citrus seedlings. Through the Agrobacterium rhizogenes-mediated genetic transformation system, overexpression of CitFNSII-1 was found to markedly enhance flavonoid accumulation in hairy roots, whereas knockout of CitFNSII-1 resulted in a significant decrease in flavonoid content in hairy roots. Further experiments verified an interaction between CitFNSII-1 and the Chalcone isomerase-1 (CHI-1) protein. The results demonstrated that the flavonoid accumulation patterns of CHI-1 and CitFNSII-1 are highly similar. In conclusion, this study advances the understanding of the flavonoid biosynthesis pathway in citrus and provides a theoretical foundation for molecular breeding strategies in citrus. Full article

Background/Objectives: Non-melanoma skin cancer (NMSC) is among the most common cancers in the United States, with solar ultraviolet (UV) radiation being a primary etiological factor. T-LAK cell-originated protein kinase (TOPK), a serine/threonine kinase activated by solar UV, has been implicated in skin carcinogenesis. This study aimed to investigate the mechanistic role of TOPK in solar UV-induced skin damage and tumor development. Methods: RNA sequencing (RNA-seq) was performed on skin tissues from wild-type (WT) and TOPK knockout (KO) mice, with or without solar UV exposure, to identify TOPK-regulated genes and pathways. Follow-up experiments using Western blotting, immunofluorescence, and luciferase assays were conducted in vitro and in vivo. Functional assays included 3D spheroid and Transwell co-culture systems involving cutaneous squamous cell carcinoma (cSCC) and fibroblast cells. Results: TOPK deletion altered gene expression profiles and inhibited solar UV-induced activation of multiple signaling pathways, including cytokine–cytokine receptor interaction, PI3K/AKT, MAPKs, PKG, cAMP, and calcium signaling. RNA-seq and protein analyses identified interleukin-19 (IL19) as a key downstream effector suppressed by TOPK deletion. In cSCC and fibroblast cells, TOPK knockdown reduced IL19 expression and secretion. IL19 promoted cSCC growth and activated PI3K/AKT, ERK, and TOPK pathways. Additionally, chronic TGFβ exposure increased IL19 expression and activated fibroblasts, as indicated by elevated αSMA and FAPα levels. Conclusions: These findings establish TOPK as a central regulator of solar UV-induced skin carcinogenesis, partially via modulation of IL19 signaling and fibroblast activation. Targeting TOPK may offer a novel strategy for the prevention and treatment of NMSC. Full article

Mitogen-Activated Protein Kinase Kinase Kinase 1 (MAP3K1) is a key signaling molecule essential for eyelid closure during embryogenesis. In mice, Map3k1 knockout leads to a fully penetrant eye-open at birth (EOB) phenotype due to disrupted MAPK signaling, abnormal epithelial differentiation, and morphogenesis. To further explore the roles of MAP3K1 in ocular development, we generated a Cre-inducible gain-of-function transgenic mouse, designated as Map3k1^TG, and crossed it with Lens epithelial (Le)-Cre mice to drive MAP3K1 overexpression in developing ocular surface epithelium (OSE). Map3k1^TG;Le-Cre embryos exhibited ocular defects including premature eyelid closure, lens degeneration, and corneal edema. While corneal epithelial differentiation remained intact, the lens epithelium degenerated with lens formation compromised. Eyelid epithelium was markedly thickened, containing cells with aberrant keratin (K)14/K10 co-expression. Genetic rescue experiments revealed that Map3k1^TG;Le-Cre restored eyelid closure in Map3k1 knockout mice, whereas MAP3K1 deficiency attenuated the epithelial thickening caused by transgene expression. Mechanistically, MAP3K1 overexpression enhanced c-Jun phosphorylation in vivo and activated JNK-c-Jun, WNT, TGFβ, and Notch signaling and promoted keratinocyte proliferation and migration in vitro. These findings highlight a dose-sensitive role for MAP3K1 in regulating epithelial proliferation, differentiation, and morphogenesis during eyelid development. Full article

Here, we describe the effects of double knockout (KO) of the cbs and cse genes, which are responsible for H₂S synthesis through the transsulfuration pathway, and KO of the sulfurtransferase gene (dtst1) in Drosophila melanogaster females. The analysis of H₂S production in flies showed minimal levels in the double- and triple-knockout strains. The double- (cbs-/-; cse-/-) and triple- (cbs-/-; cse-/-; dtst-/-) KO flies exhibited a shortened lifespan and reduced fecundity, and showed dramatic changes in Malpighian tubule morphology. The transcriptomic analysis revealed a profound increase in the expression levels of several genes involved in excretory system function in the double-KO and especially the triple-KO flies. Importantly, major groups of differentially expressed genes (DEGs) in the whole bodies of females and ovaries of KO strains included genes responsible for detoxification, reproduction, mitochondrial activity, excretion, cell migration, and muscle system function. The reduced fecundity observed in the double- and triple-KO flies correlated with pronounced changes in the ovarian transcriptome. At the same time, the single knockout of dtst1 increased the flies’ fecundity and lifespan. Our experiments exploring unique Drosophila strains with KO of major H₂S-related genes revealed several new pathways controlled by this ancient adaptogenic system that is involved in various human diseases and aging. Full article

Hedgehog (HH) signaling plays important roles in the development of the nervous system (Sonic hedgehog), bone, cartilage (Indian Hedgehog) and testis (Desert Hedgehog). Research on HH and testes has mostly been conducted in HH-knockout mice and rats, etc. The relationship between HH and cellular junctions has mostly been found in the nervous system and intestine. However, few research studies concerning the link between HH signaling and cell junctions in testis function have been reported. We identified the members of HH signaling that are involved in Eriocheir sinensis testes: HH, Smoothen, Patched, Kif27 and Ci. HH has only one homolog in E. sinensis and is expressed in several types of germ cells in the testes. We found that Kif27 colocalized with Ci in the testes. The knockdown of HH induced enlarged interstitial spaces of the seminiferous tubules. A biotin–streptavidin immunofluorescence experiment indicated that the hemolymph–testis barrier (HTB) was disrupted. Western blot results showed that pinin, HH signaling and cell proliferation- and apoptosis-related protein levels were downregulated. Further immunofluorescent results showed the dislocation of several junction proteins, the abnormality of F-actin and the slowdown of germ cell proliferation and apoptosis. While β-catenin entered the spermatocyte nucleus, it did not activate Wnt-β-catenin signaling, which indicated that the disturbance of the cell cycle in germ cells was not caused by Wnt-β-catenin signaling. In summary, HH signaling plays some roles beyond our understanding in the regulation of the HTB and the germ cell cycle in E. sinensis testes. Full article

Gnathodiaphyseal dysplasia (GDD) is a rare autosomal dominant genetic disease, mainly characterized by enlargement of the mandible, osteosclerosis, and frequent fracture of tubular bone. GDD is caused by heterozygous mutations in Anoctamin 5 (ANO5). We have previously generated an Ano5 knockout (KO) mice model and validated the phenotypes consistent with GDD patients, including enhanced bone formation and alkaline phosphatase (ALP) activity. Experiments have identified that Ano5 deficiency elevated the osteogenesis of calvaria-derived osteoblasts (mCOBs). In this study, we found that Ano5 deficiency notably inhibited miR-34c-5p expression. Krüppel-Like Factor 4 (Klf4), a target gene of miR-34c-5p confirmed by dual luciferase reporter assay, was up-regulated in Ano5^−/− mCOBs, accompanied by activated downstream canonical Wnt/β-catenin signaling and increased expression of β-catenin. Overexpression of miR-34c-5p in Ano5^−/− mCOBs inhibited osteogenic capacity by suppressing proliferative capacity, osteoblast-related factor levels, ALP activity, and matrix calcification through regulating KLF4/β-catenin signaling axis. Furthermore, miR-34c-5p adeno-associated virus (AAV) treatment in vivo rescued the abnormally thickened cortical bone and enhanced biomechanical properties in Ano5^−/− mice. Importantly, the serum level of P1NP, a marker of bone formation, was also significantly declined. We conclude that dysregulation of miR-34c-5p contributes to the enhanced osteogenesis in GDD by excessive activation of KLF4/β-catenin signaling axis under Ano5-deficient conditions. This study elucidates the pathogenesis of GDD and provides novel insights into the therapeutic strategies. Full article

Background: Lactate dehydrogenase (LDH) activity, producing high levels of lactate from pyruvate in cancer cells, is often associated with poor patient prognosis. We previously showed enhanced LDH/lactate levels in estrogen receptor (ER) compared to ER + breast cancer cells; lactate or pyruvate supplementation to ER + cells significantly enhanced their motile ability, while LDHB gene knockout (KO) or treatment with LDH inhibitors reduced the motility of the highly aggressive ER breast cancer cells. Aims: To investigate the molecular mechanisms by which lactate, LDHB KO, or treatment with LDH inhibitors can modulate the motile capabilities of breast cancer cell lines. Methods: KO experiments were performed using siRNA, and global expression was determined by proteomic profiling with Proteome Profiler Human XL Oncology arrays, Western blot, and immunofluorescence. Results: Lactate supplementation to ER + breast cancer cells enhanced expression of vimentin, N-cadherin, and snail, while reducing the expression of JAM-A, E-cadherin, and nectin-4. This expression profile was reversed with LDHB KO in ER cells. LDHB KO, or treatment with LDH inhibitors in ER cells, also reduced the expression of IL-6, IL-8, and MMP-2. The expressions of other markers such as PECAM-1, CCL20, and ENPP-2 were differentially modulated with LDH B KO in de novo ER cells (MDA-MB-231) vs. those that had ER knockout (pII). Conclusions: Our data show a novel role for lactate in modulating the EMT status in breast cancer cells and highlight the important role of lactate in breast cancer motility in part through modulating EMT status and the expression profile of cytokines, adhesion molecules, MMP-2, and nectin-4. Full article

MSTN has been used as a candidate gene in the genetics, breeding, and improvement of animal breeds. However, the possible mechanism by which the MSTN gene regulates muscle development through PSMA6 is not well understood. Previous methylome and transcriptome sequencing analyses of gluteal muscle tissues from MSTN+/−Luxi cattle and wild-type Luxi cattle identified that the PSMA6 gene exhibited a negative correlation between methylation levels and transcriptional activity. To investigate whether MSTN expression regulates PSMA6 gene expression, we examined the effects of MSTN on DNA methyltransferases (DNMT1, DNMT2, DNMT3A, and DNMT3B) and DNA demethylases (TET1, TET2, and TET3). Additionally, chromatin immunoprecipitation (ChIP) assays were performed to detect the binding interaction between PSMA6 and TET2. In this paper, we first established an MSTN knockdown cellular model to preliminarily validate its regulatory effect on PSMA6 expression. Subsequently, the developmental impact of PSMA6 on bovine skeletal muscle satellite cells was further investigated through both knockdown and overexpression of the PSMA6 gene. Furthermore, we examined changes in the expression of key components of the AKT/mTOR signaling pathway to elucidate the mechanisms underlying the PSMA6-mediated regulation of satellite cell development. The results demonstrate that myostatin (MSTN) inhibition significantly decreased proteasome 20S subunit alpha-6 (PSMA6) gene expression, while increasing demethylase expression, particularly ten-eleven translocation-2 (TET2), which exhibited the most pronounced changes. During the cell proliferation stage, the markers Paired Box 7 (PAX7) and Ki-67 exhibited no significant changes, whereas the PSMA6 gene was either overexpressed or disrupted. Conversely, PSMA6 overexpression altered the myogenic differentiation markers, causing the differential regulation of myosin heavy chain (MyHC) and myogenin (MyoG) expression, with MyHC upregulation and concurrent MyoG downregulation. PSMA6 gene overexpression led to the downregulation of AKT1 and Rac1, as well as the activation of the AKT/mTOR pathway, including key factors such as mTOR, p-mTOR, RPS6, p-RPS6, and RhoA. PSMA6 interference resulted in the downregulation of p-mTOR and the upregulation of p-RPS6. Gene expression profiling in our study revealed that the myostatin (MSTN) knockout model significantly reduced the transcriptional levels of the proteasome α6 subunit (PSMA6) (p < 0.05), with the regulatory intensity showing a significant negative correlation with MSTN expression. This molecular evidence substantiates a negative regulatory axis between MSTN and PSMA6. Functional experiments demonstrated that PSMA6 overexpression specifically enhanced myotube formation rates in bovine skeletal muscle satellite cells, whereas siRNA-mediated PSMA6 knockdown exhibited no significant effects on cellular proliferation, indicating the functional specificity of this gene in myogenic differentiation. Mechanistic investigations further revealed that PSMA6 activates the canonical AKT/mTOR signaling transduction cascade through the phosphorylation of AKT and its downstream effector mTOR, thereby mediating the expression of myogenic regulatory factors MyoD and myogenin. Collectively, these findings demonstrate that MSTN deficiency alleviates the transcriptional repression of PSMA6, remodels skeletal muscle differentiation-associated signaling networks, and ultimately drives the directional differentiation of satellite cells toward myofiber specification. Full article

Microcephaly with pontine and cerebellar hypoplasia (MICPCH) syndrome is a severe neurodevelopmental disorder caused by a deficiency in the X-linked gene calcium/calmodulin-dependent serine protein kinase (CASK). A better understanding of the role of CASK in the pathophysiology of neurodevelopmental disorders may provide insights into novel therapeutic and diagnostic strategies for MICPCH syndrome and other neurodegenerative diseases. To investigate this, we generated CASK knockout (KO) cerebellar granule (CG) cell culture from CASK floxed (CASK^flox/flox) mice by infecting lentiviruses expressing codon-improved Cre recombinase (iCre). We performed RNA-sequencing (RNA-seq) on these cells and found that CASK-KO CG cells underwent apoptosis by activating intracellular Jun N-terminal kinase (JNK) signaling and upregulating reactive oxygen species (ROS)-related gene expression. We also performed mouse gait analysis and limb clasping behavior experiments on trans-heterozygous CASK-KO and Hprt-eGFP (CASK^+/- Hprt^eGFP/+) mice. The CASK^+/- Hprt^eGFP/+ mice exhibited cerebellar ataxic phenotypes as judged by the scores of these experiments compared to the CASK wild-type control (CASK^+/+ Hprt^eGFP/+) mice. Interestingly, the administration of the JNK inhibitor, JNK-IN-8, in CASK-KO CG cell cultures increased CG cell survival by reducing ROS generation. Moreover, injection of JNK-IN-8 into the cerebellum of CASK^+/- Hprt^eGFP/+ mice suppressed CG cell death and alleviated cerebellar ataxic phenotypes in vivo. In conclusion, JNK-IN-8 suppresses the cell death and activation of the ROS pathway in CASK-KO CG cells in both in vitro and in vivo models, suggesting its potential as a therapeutic strategy for cerebellar neurodegeneration in MICPCH syndrome. Full article

The amplitude of the phasic output of thyrotropin-releasing hormone (TRH) into the hypothalamus–pituitary portal capillaries is likely controlled by the TRH-degrading ectoenzyme (TRH-DE) expressed on the surface of median eminence (ME) β2-tanycytes. To extend this hypothesis, we performed experiments on adult rodents reared in standard conditions. TRH-DE was close to the putative sites of TRH release in the male rat external layer of the ME. In global Trhde knockout mice, basal hypothalamus–pituitary–thyroid (HPT) axis parameters were not altered but we detected an increased vimentin (a tanycyte marker) positive coverage of the portal vessels. We then overexpressed TRH-DE or a dominant negative isoform by microinjection of adeno-associated virus 1 (AAV1) vectors into the third ventricle of adult male rats. Two weeks after microinjection, cold-stress-induced serum TSH concentration was decreased if ME TRH-DE activity had been enhanced. However, the long-term modification of TRH-DE activity in the ME had only a small impact on basal serum TSH concentration but increased Trhr expression in the anterior pituitary of animals transduced with AAV1-TRH-DE. Thus, long-term modifications of ME TRH-DE activity lead to limited changes in serum TSH concentration in adult rodents reared in standard conditions, possibly because of adaptations of TRH communication in the ME and/or anterior pituitary. Full article

In eutherian mammals, embryo implantation is a critical process for a successful pregnancy. In mice, the activation of the leukemia inhibitory factor (LIF) receptor–STAT3 signaling axis induces embryo adhesion and decidualization. The LIF receptor is believed to function as a heterodimer composed of LIFR (encoded by Lifr) and GP130 (encoded by Il6st); however, their distinct expression patterns in the uterine epithelium immediately prior to implantation suggest divergent functional roles. In this study, we generated uterine epithelium-specific Lifr knockout (Lifr eKO) mice and conducted a comprehensive gene expression analysis of the endometrium before implantation. We compared these results with those from uterine epithelium-specific Gp130 knockout (Gp130 eKO) mice. Similarly to Gp130 eKO mice, Lifr eKO mice were completely infertile. We identified 299 genes with expression changes greater than twofold following gene deletion; among these, 31 genes were downregulated and 57 genes were upregulated in both eKO models. Many of the downregulated genes were previously implicated in uterine function. Hub gene analysis identified Erbb2 and c-Fos as key regulators in both models. Further experiments using an ERBB2 inhibitor suggested that LIFR–ERBB2-mediated signaling plays a crucial role in embryo implantation. Full article

Dynamin-related protein 1 (Drp1) is a crucial player in mitochondrial fission and liver function. The interactions between mitochondria, endoplasmic reticulum (ER), and lipid droplets (LDs) are fundamental for lipid metabolism. This study utilized liver-specific Drp1 knockout (Drp1LiKO) mice to investigate the effects of Drp1 deficiency on organelle interactions, metabolism, and inflammation. Our analysis revealed disrupted interactions between mitochondria and LDs, as well as altered interactions among ER, mitochondria, and LDs in Drp1LiKO mice. Through mass spectrometry and microarray analysis, we identified changes in lipid profiles and perturbed expression of lipid metabolism genes in the livers of Drp1LiKO mice. Further in vitro experiments using primary hepatocytes from Drp1LiKO mice confirmed disturbances in lipid metabolism and increased inflammation. These findings highlight the critical involvement of Drp1 in regulating organelle interactions for efficient lipid metabolism and overall liver health. Targeting Drp1-mediated organelle interactions may offer potential for developing therapies for liver diseases associated with disrupted lipid metabolism. Full article