RESUMEN
Strawberry is considered as a model plant for studying the ripening of abscisic acid (ABA)-regulated non-climacteric fruits, a process in which sugar plays a fundamental role, while how ABA regulates sugar accumulation remains unclear. This study provides a direct line of physiological, biochemical, and molecular evidence that ABA signaling regulates sugar accumulation via the FaRIPK1-FaTCP7-FaSTP13/FaSPT signaling pathway. Herein, FaRIPK1, a red-initial protein kinase 1 previously identified in strawberry fruit, not only interacted with the transcription factor FaTCP7 (TEOSINTE BRANCHEN 1, CYCLOIDEA, and PCF) but also phosphorylated the critical Ser89 and Thr93 sites of FaTCP7, which negatively regulated strawberry fruit ripening, as evidenced by the transient overexpression (OE) and virus-induced gene silencing transgenic system. Furthermore, the DAP-seq experiments revealed that FvTCP7 bound the motif "GTGGNNCCCNC" in the promoters of two sugar transporter genes, FaSTP13 (sugar transport protein 13) and FaSPT (sugar phosphate/phosphate translocator), inhibiting their transcription activities as determined by the electrophoretic mobility shift assay, yeast one-hybrid, and dual-luciferase reporter assays. The downregulated FaSTP13 and FaSPT transcripts in the FaTCP7-OE fruit resulted in a reduction in soluble sugar content. Consistently, the yeast absorption test revealed that the two transporters had hexose transport activity. Especially, the phosphorylation-inhibited binding of FaTCP7 to the promoters of FaSTP13 and FaSPT could result in the release of their transcriptional activities. In addition, the phosphomimetic form FaTCP7S89D or FaTCP7T93D could rescue the phenotype of FaTCP7-OE fruits. Importantly, exogenous ABA treatment enhanced the FaRIPK1-FaTCP7 interaction. Overall, we found direct evidence that ABA signaling controls sugar accumulation during strawberry fruit ripening via the "FaRIPK1-FaTCP7-FaSTP13/FaSPT" module.
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Ácido Abscísico , Fragaria , Frutas , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas , Ácido Abscísico/metabolismo , Frutas/genética , Frutas/metabolismo , Frutas/crecimiento & desarrollo , Fragaria/genética , Fragaria/metabolismo , Fragaria/crecimiento & desarrollo , Fragaria/fisiología , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Transducción de Señal , Azúcares/metabolismo , Fosforilación , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Reguladores del Crecimiento de las Plantas/metabolismo , Plantas Modificadas GenéticamenteRESUMEN
Early vascularization plays an essential role during the whole process in bone regeneration because of the function of secreting cytokines, transporting nutrients and metabolic wastes. As the preliminary basis of bone repair, angiogenesis is regulated by immune cells represented by macrophages to a great extent. However, with the discovery of the endolymphatic circulation system inside bone tissue, the role of vascularization became complicated and confusing. Herein, we developed a macrophage/lymphatic endothelial cells (LECs)/human umbilical vein endothelial cells (HUVECs) co-culture system to evaluate the effect of macrophage treated lymphatic endothelial cells on angiogenesis in vitro and in vivo. In this study, we collected the medium from macrophage (CM) for LECs culture. We found that CM2 could promote the expression of LECs markers and migration ability, which indicated the enhanced lymphogenesis. In addition, the medium from LECs was collected for culturing HUVECs. The CM2-treated LECs showed superior angiogenesis property including the migration capacity and expression of angiogenetic markers, which suggested the superior vascularization. Rat femoral condyle defect model was applied to confirm the hypothesis in vivo. Generally, M2-macrophage treated LECs showed prominent angiogenetic potential coupling with osteogenesis.
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Técnicas de Cocultivo , Células Endoteliales de la Vena Umbilical Humana , Macrófagos , Neovascularización Fisiológica , Osteogénesis , Humanos , Animales , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Macrófagos/metabolismo , Ratas , Células Endoteliales/metabolismo , Movimiento Celular , Ratas Sprague-Dawley , Regeneración Ósea/fisiología , Ratones , Células Cultivadas , Masculino , AngiogénesisRESUMEN
Plant vacuoles serve as the primary intracellular compartments for inorganic phosphate (Pi) storage. Passage of Pi across vacuolar membranes plays a critical role in buffering the cytoplasmic Pi level against fluctuations of external Pi and metabolic activities. To gain new insights into the proteins and processes, vacuolar Pi level regulated by vacuolar phosphate transporter 1 (VPT1) in Arabidopsis, we carried out tandem mass tag labeling proteome and phosphoproteome profiling of Arabidopsis WT and vpt1 loss-of-function mutant plants. The vpt1 mutant had a marked reduced vacuolar Pi level and a slight increased cytosol Pi level. The mutant was stunted as reflected in the reduction of the fresh weight compared with WT plants and bolting earlier under normal growth conditions in soil. Over 5566 proteins and 7965 phosphopeptides were quantified. About 146 and 83 proteins were significantly changed at protein abundance or site-specific phosphorylation levels, but only six proteins were shared between them. Functional enrichment analysis revealed that the changes of Pi states in vpt1 are associated with photosynthesis, translation, RNA splicing, and defense response, consistent with similar studies in Arabidopsis. Except for PAP26, EIN2, and KIN10, which were reported to be associated with phosphate starvation signal, we also found that many differential proteins involved in abscisic acid signaling, such as CARK1, SnRK1, and AREB3, were significantly changed in vpt1. Our study illuminates several new aspects of the phosphate response and identifies important targets for further investigation and potential crop improvement.
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Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Fosfatos/metabolismo , Proteoma/metabolismo , Vacuolas/metabolismo , Proteínas de Transporte de Fosfato/genética , Proteínas de Transporte de Fosfato/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Strawberry (Fragaria×ananassa) is a model plant for studying non-climacteric fruit ripening regulated by abscisic acid (ABA). However, the signaling of ABA in the regulation of fruit coloration is not fully understood. Here, a transcription factor FabHLH3 key to fruit coloration is identified by yeast two hybrid library screening using FaSnRK2.6 as a bait, an ABA core signaling component negative to ripening. Indeed, this interaction is also confirmed by firefly luciferase complementation assay and pull-down assay. RT-qPCR and Western blotting analysis confirm FabHLH3 is expressed ubiquitously in strawberry and stably during fruit development. Manipulating both FabHLH3 and FaSnRK2.6 expression by overexpression and interference demonstrates that FabHLH3 and FaSnRK2.6 promote and inhibit strawberry fruit coloration, respectively, using the marker gene FaUFGT, key to anthocyanin biosynthesis. FaSnRK2.6 can phosphorylate FabHLH3, which promotes FaUFGT expression by the directly binding to its promoter. The phosphorylation inhibits the binding of FabHLH3 to FaUFGT promoter, consequently suppressing FaUFGT expression. Altogether, FaSnRK2.6, a negative kinase in ripening, interacts with and phosphorylates FabHLH3 to suppress FaUFGT expression. With the increase of ABA content in strawberry fruit ripening, the expression of FaSnRK2.6 decreased, which released FabHLH3 transcription activity and enhanced FaUFGT expression, finally promoting the coloration. Thus, our findings fill a gap how FaSnRK2.6 negatively regulates strawberry fruit coloration and ripening by FabHLH3.
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Sertoli cells are essential for testis development and normal spermatogenesis by providing support and nutrients. Pre-messenger RNA (pre-mRNA) processing is the basic mechanism required for gene expression, and members of the serine/arginine-rich protein (SR) family are key components of the machines that perform these basic processing events. Serine/arginine-rich splicing factor 2 (SRSF2) is an important member of the SR family; however, the physiological functions of SRSF2 in Sertoli cells are still unclear. Here, we found that SRSF2 was localized in the nuclei of Sertoli and germ cells in male mice at all stages by breeding Amh-Cre mice obtained with Srsf2-specific knockout in Sertoli cells to define the function of SRSF2 in Sertoli cells. The experimental results showed that specific deletion of SRSF2 impaired fetal Sertoli cell proliferation and induced abnormal apoptosis and severe DNA damage in seminiferous tubules, resulting in severe testicular dysplasia, seminiferous tubule atrophy, and almost no normal seminiferous tubules at postnatal day 14. Eventually, these changes resulted in failure to produce normal sperm and infertility. Further RNA-seq results showed that many key genes related to proliferation and apoptosis were downregulated; Racgap1 mRNA undergoes exon skipping. Thus, SRSF2-dependent Sertoli cells are essential for testicular development and male reproduction.
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Semen , Células de Sertoli , Animales , Masculino , Ratones , Arginina/metabolismo , ARN Mensajero/metabolismo , Células de Sertoli/metabolismo , Espermatogénesis/fisiología , Testículo/metabolismoRESUMEN
BACKGROUND: Ovarian folliculogenesis is a tightly regulated process leading to the formation of functional oocytes and involving successive quality control mechanisms that monitor chromosomal DNA integrity and meiotic recombination. A number of factors and mechanisms have been suggested to be involved in folliculogenesis and associated with premature ovarian insufficiency, including abnormal alternative splicing (AS) of pre-mRNAs. Serine/arginine-rich splicing factor 1 (SRSF1; previously SF2/ASF) is a pivotal posttranscriptional regulator of gene expression in various biological processes. However, the physiological roles and mechanism of SRSF1 action in mouse early-stage oocytes remain elusive. Here, we show that SRSF1 is essential for primordial follicle formation and number determination during meiotic prophase I. RESULTS: The conditional knockout (cKO) of Srsf1 in mouse oocytes impairs primordial follicle formation and leads to primary ovarian insufficiency (POI). Oocyte-specific genes that regulate primordial follicle formation (e.g., Lhx8, Nobox, Sohlh1, Sohlh2, Figla, Kit, Jag1, and Rac1) are suppressed in newborn Stra8-GFPCre Srsf1Fl/Fl mouse ovaries. However, meiotic defects are the leading cause of abnormal primordial follicle formation. Immunofluorescence analyses suggest that failed synapsis and an inability to undergo recombination result in fewer homologous DNA crossovers (COs) in the Srsf1 cKO mouse ovaries. Moreover, SRSF1 directly binds and regulates the expression of the POI-related genes Six6os1 and Msh5 via AS to implement the meiotic prophase I program. CONCLUSIONS: Altogether, our data reveal the critical role of an SRSF1-mediated posttranscriptional regulatory mechanism in the mouse oocyte meiotic prophase I program, providing a framework to elucidate the molecular mechanisms of the posttranscriptional network underlying primordial follicle formation.
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Meiosis , Profase Meiótica I , Factores de Empalme Serina-Arginina , Animales , Femenino , Ratones , Empalme Alternativo , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Meiosis/genética , Oocitos , Ovario , Factores de Empalme Serina-Arginina/genéticaRESUMEN
Global climate change-caused drought stress, high temperatures and other extreme weather profoundly impact plant growth and development, restricting sustainable crop production. To cope with various environmental stimuli, plants can optimize the opening and closing of stomata to balance CO2 uptake for photosynthesis and water loss from leaves. Guard cells perceive and integrate various signals to adjust stomatal pores through turgor pressure regulation. Molecular mechanisms and signaling networks underlying the stomatal movements in response to environmental stresses have been extensively studied and elucidated. This review focuses on the molecular mechanisms of stomatal movements mediated by abscisic acid, light, CO2 , reactive oxygen species, pathogens, temperature, and other phytohormones. We discussed the significance of elucidating the integrative mechanisms that regulate stomatal movements in helping design smart crops with enhanced water use efficiency and resilience in a climate-changing world.
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Reguladores del Crecimiento de las Plantas , Estomas de Plantas , Estomas de Plantas/fisiología , Ácido Abscísico , Hojas de la Planta/fisiología , Plantas , Agua/fisiologíaRESUMEN
BACKGROUND: Volatile components are important secondary metabolites essential to fruit aroma quality, thus, in the past decades many studies have been extensively performed in clarifying fruit aroma formation. However, aroma components and biosynthesis in the fruit of Binzi (Malus pumila × Malus asiatica), an old local species with attractive aroma remain unknown. RESULTS: We investigated two Binzi cultivars, 'Xiangbinzi' (here named high-fragrant Binzi, 'HFBZ') and 'Hulabin' (here named low-fragrant Binzi, 'LFBZ') by monitoring the variation of volatiles and their precursors by Gas Chromatography-Mass Spectrometer (GC-MS), as well as their related genes by RNA-seq during post-harvest ripening. We firstly confirmed that 'HFBZ' and 'LFBZ' fruit showed respiratory climacteric by detecting respiratory rate and ethylene emission during post-harvest; found that esters were the major aroma components in 'HFBZ' fruit, and hexyl 2-methylbutyrate was responsible for the 'fruity' note and most potent aroma component, followed by ethyl acetate, ethyl butanoate, (E)-2-hexenal, and 1-hexanol. Regarding aroma synthesis, fatty acid metabolism seemed to be more important than amino acid metabolism for aroma synthesis in 'HFBZ' fruit. Based on RNA-seq and quantitative reverse transcription PCR (RT-qPCR), LOX2a, LOX5a, ADH1, and AAT1 genes are pointed to the LOX pathway, which may play a vital role in the aroma formation of 'HFBZ' fruit. CONCLUSION: Our study firstly investigated the aroma components and related genes of Binzi fruit, and provided an insight into the fragrant nature of Malus species.
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Malus , Compuestos Orgánicos Volátiles , Malus/genética , Odorantes/análisis , Frutas/metabolismo , Ésteres/metabolismo , Cromatografía de Gases , Compuestos Orgánicos Volátiles/metabolismoRESUMEN
The mechanisms that balance plant growth and stress responses are poorly understood, but they appear to involve abscisic acid (ABA) signaling mediated by protein kinases. Here, to explore these mechanisms, we examined the responses of Arabidopsis thaliana protein kinase mutants to ABA treatment. We found that mutants of BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) were hypersensitive to the effects of ABA on both seed germination and primary root growth. The kinase OPEN STOMATA 1 (OST1) was more highly activated by ABA in bak1 mutant than the wild type. BAK1 was not activated by ABA treatment in the dominant negative mutant abi1-1 or the pyr1 pyl4 pyl5 pyl8 quadruple mutant, but it was more highly activated by this treatment in the abi1-2 abi2-2 hab1-1 loss-of-function triple mutant than the wild type. BAK1 phosphorylates OST1 T146 and inhibits its activity. Genetic analyses suggested that BAK1 acts at or upstream of core components in the ABA signaling pathway, including PYLs, PP2Cs, and SnRK2s, during seed germination and primary root growth. Although the upstream brassinosteroid (BR) signaling components BAK1 and BR INSENSITIVE 1 (BRI1) positively regulate ABA-induced stomatal closure, mutations affecting downstream components of BR signaling, including BRASSINOSTEROID-SIGNALING KINASEs (BSKs) and BRASSINOSTEROID-INSENSITIVE 2 (BIN2), did not affect ABA-mediated stomatal movement. Thus, our study uncovered an important role of BAK1 in negatively regulating ABA signaling during seed germination and primary root growth, but positively modulating ABA-induced stomatal closure, thus optimizing the plant growth under drought stress.
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Proteínas de Arabidopsis , Arabidopsis , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Brasinoesteroides/metabolismo , Regulación de la Expresión Génica de las Plantas , Mutación/genética , Estomas de Plantas/fisiología , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Protein kinases are major players in various signal transduction pathways. Understanding the molecular mechanisms behind plant responses to biotic and abiotic stresses has become critical for developing and breeding climate-resilient crops. In this review, we summarize recent progress on understanding plant drought, salt, and cold stress responses, with a focus on signal perception and transduction by different protein kinases, especially sucrose nonfermenting1 (SNF1)-related protein kinases (SnRKs), mitogen-activated protein kinase (MAPK) cascades, calcium-dependent protein kinases (CDPKs/CPKs), and receptor-like kinases (RLKs). We also discuss future challenges in these research fields.
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Quinasa 2 de Adhesión Focal/metabolismo , Sequías , Quinasa 2 de Adhesión Focal/genética , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Both plant receptor-like protein kinases (RLKs) and ubiquitin-mediated proteolysis play crucial roles in plant responses to drought stress. However, the mechanism by which E3 ubiquitin ligases modulate RLKs is poorly understood. In this study, we showed that Arabidopsis PLANT U-BOX PROTEIN 11 (PUB11), an E3 ubiquitin ligase, negatively regulates abscisic acid (ABA)-mediated drought responses. PUB11 interacts with and ubiquitinates two receptor-like protein kinases, LEUCINE RICH REPEAT PROTEIN 1 (LRR1) and KINASE 7 (KIN7), and mediates their degradation during plant responses to drought stress in vitro and in vivo. pub11 mutants were more tolerant, whereas lrr1 and kin7 mutants were more sensitive, to drought stress than the wild type. Genetic analyses show that the pub11 lrr1 kin7 triple mutant exhibited similar drought sensitivity as the lrr1 kin7 double mutant, placing PUB11 upstream of the two RLKs. Abscisic acid and drought treatment promoted the accumulation of PUB11, which likely accelerates LRR1 and KIN7 degradation. Together, our results reveal that PUB11 negatively regulates plant responses to drought stress by destabilizing the LRR1 and KIN7 RLKs.
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Adaptación Fisiológica , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Sequías , Proteínas de Unión a Fosfato/metabolismo , Proteínas Quinasas/metabolismo , Proteolisis , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Arabidopsis/genética , Mutación/genética , Unión Proteica , Estrés Fisiológico , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Di-(2-ethylhexyl) phthalate (DEHP) is a typical endocrine disrupting chemical with relatively high concentrations in agricultural soils of China. Here, a rhizobox experiment was conducted to investigate the variations in microbial community and DEHP dissipation among different soil rhizospheric compartments between low (Fengyousimiao) and high (Peizataifeng) DEHP-accumulating cultivars of rice (Oryza sativa L.) grown in DEHP spiked soil (0, 20, 100â¯mg/kg). The dissipation rates of DEHP in rhizospheric soils of Peizataifeng were generally significantly higher than those of Fengyousimiao, with the highest removal rate in 0-2â¯mm rhizosphere. The results of Illumina-HiSeq high-throughput sequencing revealed that both bacterial and fungal diversity and community structure were significantly different in rhizospheric soils of the two cultivars. DEHP dissipation rates in 0-2â¯mm rhizosphere of Peizataifeng were positively correlated with bacterial and fungal diversity. The relative abundance of DEHP-degrading bacterial genera Acinetobacter, Pseudomonas and Bacillus of Peizataifeng was generally higher than those in the same rhizospheric compartment of Fengyousimiao in DEHP treatments, resulting in different rhizospheric DEHP dissipation. Cultivation of Peizataifeng in agricultural soil is promising to facilitate DEHP dissipation and ensure safety of agricultural products.
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Dietilhexil Ftalato/análisis , Oryza/microbiología , Rizosfera , Microbiología del Suelo , Contaminantes del Suelo/análisis , Suelo/química , Agricultura , China , Dietilhexil Ftalato/química , Dietilhexil Ftalato/metabolismo , Oryza/química , Oryza/metabolismo , Ácidos Ftálicos , Contaminantes del Suelo/químicaRESUMEN
Glaesserella parasuis (GPS) can cause severe systemic inflammation in pigs, resulting in huge economic losses to the pig industry. At present, no effective method is available for the prevention and control of GPS infection. Molecular breeding for disease resistance is imminent, but disease-resistance genes have not been identified. To study the mechanism of systemic acute inflammation caused by GPS, we established three in vitro infection models (3D4/21 cells, PK15 cells, and PAVEC cells) according to its infection path. There was no significant difference in apoptosis among the three kinds of cells after 12 h of continuous GPS stimulation, while inflammatory factors were significantly upregulated. Subsequent transcriptome analysis revealed 1969, 1207, and 3564 differentially expressed genes (DEGs) in 3D4/21 cells, PK15 cells, and PAVEC cells, respectively, after GPS infection. Many of the DEGs were predicted to be associated with inflammatory responses (C3, CD44, etc.); cell proliferation, growth and apoptosis; gene expression; and protein phosphorylation. Key signaling pathways, including S100 family signaling, bacteria and virus recognition, and pathogen-induced cytokine storm signaling, were enriched based on Ingenuity Pathway Analysis (IPA). Furthermore, a total of three putative transmembrane receptors and two putative G-protein-coupled receptors, namely F3, ICAM1, PLAUR, ACKR3, and GPRC5A, were identified by IPA among the three types of cells. ACKR3 and GPRC5A play pivotal roles in bacterial adhesion, invasion, host immune response and inflammatory response through the S100 family signaling pathway. Our findings provide new insights into the pathological mechanisms underlying systemic inflammation caused by GPS infection in pigs, and they lay a foundation for further research on disease-resistance breeding to GPS.
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Haemophilus parasuis , Inflamación , Transducción de Señal , Enfermedades de los Porcinos , Animales , Porcinos , Haemophilus parasuis/genética , Haemophilus parasuis/patogenicidad , Transducción de Señal/genética , Inflamación/genética , Inflamación/microbiología , Enfermedades de los Porcinos/microbiología , Enfermedades de los Porcinos/genética , Enfermedades de los Porcinos/inmunología , Infecciones por Haemophilus/veterinaria , Infecciones por Haemophilus/genética , Infecciones por Haemophilus/microbiología , Infecciones por Haemophilus/inmunología , Transcriptoma/genética , Perfilación de la Expresión Génica , Línea Celular , Apoptosis/genéticaRESUMEN
Glaesserella parasuis (G. parasuis) causes systemic infection in pigs, but its effects on skeletal muscle and underlying mechanisms are poorly understood. We investigated G. parasuis infection in colostrum-deprived piglets, observing decreased daily weight gain and upregulation of inflammatory factors in skeletal muscle. Muscle fiber area and diameter were significantly reduced in the treated group (n = 3) compared to the control group (n = 3), accompanied by increased expression of FOXO1, FBXO32, TRIM63, CTSL, and BNIP3. Based on mRNA and microRNA (miRNA) sequencing, we identified 1642 differentially expressed (DE) mRNAs and 19 known DE miRNAs in skeletal muscle tissues between the two groups. We predicted target genes with opposite expression patterns to the 19 miRNAs and found significant enrichment and activation of the FoxO signaling pathway. We found that the upregulated core effectors FOXO1 and FOXO4 were targeted by downregulated ssc-miR-486, ssc-miR-370, ssc-miR-615, and ssc-miR-224. Further investigation showed that their downstream upregulated genes involved in protein degradation were also targeted by the downregulated ssc-miR-370, ssc-miR-615, ssc-miR-194a-5p, and ssc-miR-194b-5p. These findings suggest that G. parasuis infection causes skeletal muscle atrophy in piglets through accelerated protein degradation mediated by the "miRNAs-FOXO1/4" axis, while further research is necessary to validate the regulatory relationships. Our results provide new insights into the understanding of systemic inflammation growth mechanisms caused by G. parasuis and the role of miRNAs in bacterial infection pathogenesis.
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MicroARNs , Porcinos/genética , Animales , MicroARNs/genética , MicroARNs/metabolismo , ARN Mensajero/genética , Perfilación de la Expresión Génica , Músculo Esquelético/metabolismo , Fibras Musculares Esqueléticas/metabolismoRESUMEN
Skin hyperpigmentation is a worldwide condition associated with augmented melanogenesis. However, conventional therapies often entail various adverse effects. Here, we explore the safety range and depigmentary effects of polysaccharides extract of Tricholoma matsutake (PETM) in an in vitro model and further evaluated its efficacy at the clinical level. An induced-melanogenesis model was established by treating B16-F10 melanoma cells with 8-methoxypsoralen (8-MOP). Effects of PETM on cell viability and melanin content were examined and compared to a commonly used depigmentary agent, α-arbutin. Expressions of key melanogenic factors and upstream signaling pathway were analysed by quantitative PCR and western blot. Moreover, a placebo-controlled clinical study involving Chinese females with skin hyperpigmentation was conducted to measure the efficacy of PETM on improving facial pigmented spots, melanin index, and individual typology angle (ITA°). Results demonstrated that PETM (up to 0.5 mg/mL) had little effect on the viability and motility of B16-F10 cells. Notably, it significantly suppressed the melanin content and expressions of key melanogenic factors induced by 8-MOP in B16-F10 melanoma cells. Western blotting results revealed that PETM inhibited melanogenesis by inactivating c-Jun N-terminal kinase (JNK), and this inhibitory role could be rescued by JNK agonist treatment. Clinical findings showed that PETM treatment resulted in a significant reduction of facial hyperpigmented spot, decreased melanin index, and improved ITA° value compared to the placebo-control group. In conclusion, these in vitro and clinical evidence demonstrated the safety and depigmentary efficacy of PETM, a novel polysaccharide agent. The distinct mechanism of action of PETM on melanogenic signaling pathway positions it as a promising agent for developing alternative therapies.
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Spermatogonial stem cells (SSCs) are essential for continuous spermatogenesis and male fertility. The underlying mechanisms of alternative splicing (AS) in mouse SSCs are still largely unclear. We demonstrated that SRSF1 is essential for gene expression and splicing in mouse SSCs. Crosslinking immunoprecipitation and sequencing data revealed that spermatogonia-related genes (e.g. Plzf, Id4, Setdb1, Stra8, Tial1/Tiar, Bcas2, Ddx5, Srsf10, Uhrf1, and Bud31) were bound by SRSF1 in the mouse testes. Specific deletion of Srsf1 in mouse germ cells impairs homing of precursor SSCs leading to male infertility. Whole-mount staining data showed the absence of germ cells in the testes of adult conditional knockout (cKO) mice, which indicates Sertoli cell-only syndrome in cKO mice. The expression of spermatogonia-related genes (e.g. Gfra1, Pou5f1, Plzf, Dnd1, Stra8, and Taf4b) was significantly reduced in the testes of cKO mice. Moreover, multiomics analysis suggests that SRSF1 may affect survival of spermatogonia by directly binding and regulating Tial1/Tiar expression through AS. In addition, immunoprecipitation mass spectrometry and co-immunoprecipitation data showed that SRSF1 interacts with RNA splicing-related proteins (e.g. SART1, RBM15, and SRSF10). Collectively, our data reveal the critical role of SRSF1 in spermatogonia survival, which may provide a framework to elucidate the molecular mechanisms of the posttranscriptional network underlying homing of precursor SSCs.
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Espermatogonias , Testículo , Animales , Masculino , Ratones , Proteínas de Ciclo Celular/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Represoras/metabolismo , Factores de Empalme de ARN/metabolismo , Factores de Empalme Serina-Arginina/genética , Factores de Empalme Serina-Arginina/metabolismo , Espermatogénesis/genética , Espermatogonias/metabolismo , Células Madre/metabolismo , Testículo/metabolismoRESUMEN
Vacuolar Phosphate Transporter1 (VPT1)-mediated phosphate uptake in the vacuoles is essential to plant development and fruit ripening. Interestingly, here we find that the VPT1 may transport sugar in response to soluble sugar status of fruits. The VvVPT1 protein isolated from grape (Vitis vinifera) berries was tonoplast-localized and contains SPX (Syg1/Pho81/XPR1) and MFS (major facilitator superfamily) domains. Its mRNA expression was significantly increased during fruit ripening and induced by sucrose. Functional analyses based on transient transgenic systems in grape berry showed that VvVPT1 positively regulated berry ripening and significantly affected hexose contents, fruit firmness, and ripening-related gene expression. The VPT1 proteins (Grape VvVPT1, strawberry FaVPT1, and Arabidopsis AtVPT1) all showed low affinity for phosphate verified in yeast system, while they appear different in sugar transport capacity, consistent with fruit sugar status. Thus, our findings reveal a role for VPT1 in fruit ripening, associated to its SPX and MFS domains in direct transport of soluble sugar available into the vacuole, and open potential avenues for genetic improvement in fleshy fruit.
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Background: Previous models for differentiating benign and malignant thyroid nodules(TN) have predominantly focused on the characteristics of the nodules themselves, without considering the specific features of the thyroid gland(TG) in patients with Hashimoto's thyroiditis(HT). In this study, we analyzed the clinical and ultrasound radiomics(USR) features of TN in patients with HT and constructed a model for differentiating benign and malignant nodules specifically in this population. Methods: We retrospectively collected clinical and ultrasound data from 227 patients with TN and concomitant HT(161 for training, 66 for testing). Two experienced sonographers delineated the TG and TN regions, and USR features were extracted using Python. Lasso regression and logistic analysis were employed to select relevant USR features and clinical data to construct the model for differentiating benign and malignant TN. The performance of the model was evaluated using area under the curve(AUC), calibration curves, and decision curve analysis(DCA). Results: A total of 1,162 USR features were extracted from TN and the TG in the 227 patients with HT. Lasso regression identified 14 features, which were used to construct the TN score, TG score, and TN+TG score. Univariate analysis identified six clinical predictors: TI-RADS, echoic type, aspect ratio, boundary, calcification, and thyroid function. Multivariable analysis revealed that incorporating USR scores improved the performance of the model for differentiating benign and malignant TN in patients with HT. Specifically, the TN+TG score resulted in the highest increase in AUC(from 0.83 to 0.94) in the clinical prediction model. Calibration curves and DCA demonstrated higher accuracy and net benefit for the TN+TG+clinical model. Conclusion: USR features of both the TG and TN can be utilized for differentiating benign and malignant TN in patients with HT. These findings highlight the importance of considering the entire TG in the evaluation of TN in HT patients, providing valuable insights for clinical decision-making in this population.
Asunto(s)
Enfermedad de Hashimoto , Nódulo Tiroideo , Humanos , Nódulo Tiroideo/patología , Estudios Retrospectivos , Modelos Estadísticos , Pronóstico , Enfermedad de Hashimoto/complicacionesRESUMEN
BACKGROUND: Granulosa cell proliferation and differentiation are essential for follicle development. Breast cancer amplified sequence 2 (BCAS2) is necessary for spermatogenesis, oocyte development, and maintaining the genome integrity of early embryos in mice. However, the function of BCAS2 in granulosa cells is still unknown. RESULTS: We show that conditional disruption of Bcas2 in granulosa cells caused follicle development failure; the ratio of the positive cells of the cell proliferation markers PCNA and Ki67 were unchanged in granulosa cells. Specific deletion of Bcas2 caused a decrease in the BrdU-positive cell ratio, cell cycle arrest, DNA damage, and an increase in apoptosis in granulosa cells, and RPA1 was abnormally stained in granulosa cells. RNA-seq results revealed that knockout of Bcas2 results in unusual expression of cellular senescence genes. BCAS2 participated in the PRP19 complex to mediate alternative splicing (AS) of E2f3 and Flt3l mRNA to inhibit the cell cycle. Knockout of Bcas2 resulted in a significant decrease in the ratio of BrdU-positive cells in the human granulosa-like tumour (KGN) cell line. CONCLUSIONS: Our results suggest that BCAS2 may influence the proliferation and survival of granulosa cells through regulating pre-mRNA splicing of E2f3 and Flt3l by forming the splicing complex with CDC5L and PRP19.
Asunto(s)
Empalme Alternativo , Factores de Transcripción , Masculino , Femenino , Humanos , Animales , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Bromodesoxiuridina/metabolismo , Ratones Noqueados , Factores de Transcripción/genética , Células de la Granulosa/metabolismo , Supervivencia Celular/genética , Proteínas de Neoplasias/genética , Proteínas de Unión al ARN/genética , Proteínas de Ciclo Celular/genéticaRESUMEN
Insulin secreted by pancreatic ß cells is essential for maintaining blood glucose levels. Diabetes is caused primarily by a loss of ß cells or impairment of ß-cell function. A previous whole-transcriptome analysis of islets from a type 2 diabetes group and a control group showed that a splicing disorder occurred in approximately 25% of splicing events. Breast carcinoma amplified sequence 2 (BCAS2) is a spliceosome component whose function in islet ß cells is unclear. Here, we report that knockdown of Bcas2 decreased glucose- and KCl-stimulated insulin secretion in the NIT-1 cell line. Pancreas weight, glucose tolerance, and insulin sensitivity were measured in normal chow-fed Bcas2 f/f-ßKO mice, and ß-cell mass and islet size were analyzed by immunohistochemistry. Glucose intolerance developed in Bcas2 f/f-ßKO mice, but there were no significant differences in pancreas weight, insulin sensitivity, ß-cell mass, or islet size. Furthermore, observation of glucose-stimulated insulin secretion and insulin secretion granules in normal chow-fed mice revealed that the insulin level in serum and the number of insulin secretion granules were decreased in Bcas2 f/f-ßKO mice. These differences were related to abnormal splicing of Syt7 and Tcf7l2 pre-mRNA. Taken together, these results demonstrate that BCAS2 is involved in alternative splicing during insulin synthesis and secretion.