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RNA-RNA interactions play a crucial role in regulating gene expression and various biological processes, but identifying these interactions on a transcriptomic scale remains a challenge. To address this, we have developed a new biochemical technique called pCp-biotin labelled RNA hybrid and ultraviolet crosslinking and immunoprecipitation (lhCLIP) that enables the transcriptome-wide identification of intra- and intermolecular RNA-RNA interactions mediated by a specific RNA-binding protein (RBP). Using lhCLIP, we have uncovered a diverse landscape of intermolecular RNA interactions recognized by hnRNPK in human cells, involving all major classes of noncoding RNAs (ncRNAs) and mRNA. Notably, hnRNPK selectively binds with snRNA U4, U11, and U12, and shapes the secondary structure of these snRNAs, which may impact RNA splicing. Our study demonstrates the potential of lhCLIP as a user-friendly and widely applicable method for discovering RNA-RNA interactions mediated by a particular protein of interest and provides a valuable tool for further investigating the role of RBPs in gene expression and biological processes.
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ARN Nuclear Pequeño , ARN , Humanos , ARN/genética , ARN/metabolismo , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Empalme del ARN/genética , ARN no Traducido/genética , ARN Mensajero/metabolismoRESUMEN
OBJECTIVE: Marginal alveolar bone loss is one of the key features of periodontitis and can be observed via panoramic radiographs. This study aimed to establish a cascading learning method with deep learning (DL) for precise radiographic bone loss (RBL) measurements at specific tooth positions. MATERIALS AND METHODS: Through the design of two tasks for tooth position recognition and tooth semantic segmentation using the SegFormer model, specific tooth's crown, intrabony portion, and suprabony portion of the roots were obtained. The RBL was subsequently measured by length through these three areas using the principal component analysis (PCA) principal axis. RESULTS: The average intersection over union (IoU) for the tooth position recognition task was 0.8906, with an F1-score of 0.9338. The average IoU for the tooth semantic segmentation task was 0.8465, with an F1-score of 0.9138. When the two tasks were combined, the average IoU was 0.7889, with an F1-score of 0.8674. The correlation coefficient between the RBL prediction results based on the PCA principal axis and the clinicians' measurements exceeded 0.85. Compared to those of the other two methods, the average precision of the predicted RBL was 0.7722, the average sensitivity was 0.7416, and the average F1-score was 0.7444. CONCLUSIONS: The method for predicting RBL using DL and PCA produced promising results, offering rapid and reliable auxiliary information for future periodontal disease diagnosis. CLINICAL RELEVANCE: Precise RBL measurements are important for periodontal diagnosis. The proposed RBL-SF can measure RBL at specific tooth positions and assign the bone loss stage. The ability of the RBL-SF to measure RBL at specific tooth positions can guide clinicians to a certain extent in the accurate diagnosis of periodontitis.
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Pérdida de Hueso Alveolar , Periodontitis , Diente , Humanos , Pérdida de Hueso Alveolar/diagnóstico por imagen , Proceso Alveolar , Periodontitis/diagnóstico por imagen , Corona del DienteRESUMEN
MAIN CONCLUSION: Two trans-isopentenyl diphosphate synthase and one squalene synthase genes were identified and proved to be involved in the triterpenoid biosynthesis in Platycodon grandiflorus. Platycodon grandiflorus is a commonly used traditional Chinese medicine. The main bioactive compounds of P. grandiflorus are triterpenoid saponins. The biosynthetic pathway of triterpenoid saponins in P. grandiflorus has been preliminarily explored. However, limited functional information on related genes has been reported. A total of three trans-isopentenyl diphosphate synthases (trans-IDSs) genes (PgFPPS, PgGGPPS1 and PgGGPPS2) and one squalene synthase (SQS) gene (PgSQS) in P. grandiflorus were screened and identified from transcriptome dataset. Subcellular localization of the proteins was defined based on the analysis of GFP-tagged. The activity of genes was verified in Escherichia coli, demonstrating that recombinant PgFPPS catalysed the production of farnesyl diphosphate. PgGGPPS1 produced geranylgeranyl diphosphate, whereas PgGGPPS2 did not exhibit catalytic activity. By structural identification of encoding genes, a transmembrane region was found at the C-terminus of the PgSQS gene, which produced an insoluble protein when expressed in E. coli but showed no apparent effect on the enzyme function. Furthermore, some triterpenoid saponin synthesis-related genes were discovered by combining the component content and the gene expression assays at the five growth stages of P. grandiflorus seedlings. The accumulation of active components in P. grandiflorus was closely associated with the expression level of genes related to the synthesis pathway.
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Platycodon , Saponinas , Farnesil Difosfato Farnesil Transferasa/genética , Platycodon/genética , Escherichia coli/genética , Saponinas/genéticaRESUMEN
MAIN CONCLUSION: Comprehensive transcriptome analysis of different Platycodon grandiflorus tissues discovered genes related to triterpenoid saponin biosynthesis. Platycodon grandiflorus (Jacq.) A. DC. (P. grandiflorus), a traditional Chinese medicine, contains considerable triterpenoid saponins with broad pharmacological activities. Triterpenoid saponins are the major components of P. grandiflorus. Here, single-molecule real-time and next-generation sequencing technologies were combined to comprehensively analyse the transcriptome and identify genes involved in triterpenoid saponin biosynthesis in P. grandiflorus. We quantified four saponins in P. grandiflorus and found that their total content was highest in the roots and lowest in the stems and leaves. A total of 173,354 non-redundant transcripts were generated from the PacBio platform, and three full-length transcripts of ß-amyrin synthase, the key synthase of ß-amyrin, were identified. A total of 132,610 clean reads obtained from the DNBSEQ platform were utilised to explore key genes related to the triterpenoid saponin biosynthetic pathway in P. grandiflorus, and 96 differentially expressed genes were selected as candidates. The expression levels of these genes were verified by quantitative real-time PCR. Our reliable transcriptome data provide valuable information on the related biosynthesis pathway and may provide insights into the molecular mechanisms of triterpenoid saponin biosynthesis in P. grandiflorus.
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Platycodon , Saponinas , Triterpenos , Perfilación de la Expresión Génica , Platycodon/genética , TranscriptomaRESUMEN
MAIN CONCLUSION: Two squalene synthase genes AlSQS1 and AlSQS2 were isolated from Atractylodes lancea and functionally characterized using in vitro enzymatic reactions. Atractylodes lancea is a traditional herb used for the treatment of rheumatic diseases, gastric disorders, and influenza. Its major active ingredients include sesquiterpenoids and triterpenes. Squalene synthase (SQS; EC 2.5.1.21) catalyzes the first enzymatic step in the central isoprenoid pathway towards sterol and triterpenoid biosynthesis. In this study, we aimed to investigate two SQSs from A. lancea using cloning and in vitro enzymatic characterization. Bioinformatics and phylogenetic analyses revealed that the AlSQSs exhibited high homology with other plant SQSs. Furthermore, AlSQS1 was observed to be localized in both the nucleus and cytoplasm, whereas AlSQS2 was localized in the cytoplasm and endoplasmic reticulum. To obtain soluble recombinant enzymes, AlSQS1 and AlSQS2 were successfully expressed as glutathione S-transferase (GST)-tagged fusion proteins in Escherichia coli Transetta (DE3). Approximately 68 kDa recombinant proteins were obtained using GST-tag affinity chromatography and Western blot analysis. Results of the in vitro enzymatic reactions established that both AlSQS1 and AlSQS2 were functional, which verifies their catalytic ability in converting two farnesyl pyrophosphates to squalene. The expression patterns of AlSQS and selected terpenoid genes were also investigated in two A. lancea chemotypes using available RNA sequencing data. AlSQS1 and AlSQS2, which showed relatively similar expression in the three tissues, were more highly expressed in the stems than in the leaves and rhizomes. Methyl jasmonate (MeJA) was used as an elicitor to analyze the expression profiles of AlSQSs. The results of qRT-PCR analysis revealed that the gene expression of AlSQS1 and AlSQS2 plummeted at lowest value at 12 h and reached its peak at 24 h. This study is the first report on the cloning, characterization, and expression of SQSs in A. lancea. Therefore, our findings contribute novel insights that may be useful for future studies regarding terpenoid biosynthesis in A. lancea.
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Atractylodes , Farnesil Difosfato Farnesil Transferasa , Atractylodes/enzimología , Atractylodes/genética , Clonación Molecular , Farnesil Difosfato Farnesil Transferasa/genética , Genes de Plantas , Filogenia , Análisis de Secuencia de ARN , EscualenoRESUMEN
In this study, the gene encoding the key enzyme 3-ketoacyl-CoA thiolase(KAT) in the fatty acid ß-oxidation pathway of Atractylodes lancea was cloned. Meanwhile, bioinformatics analysis, prokaryotic expression and gene expression analysis were carried out, which laid a foundation for the study of fatty acid ß-oxidation mechanism of A. lancea. The full-length sequence of the gene was cloned by RT-PCR with the specific primers designed according to the sequence information of KAT gene in the transcriptomic data of A. lancea and designated as AIKAT(GenBank accession number MW665111). The results showed that the open reading frame(ORF) of AIKAT was 1 323 bp, encoding 440 amino acid. The deduced protein had a theoretical molecular weight of 46 344.36 and an isoelectric point of 8.92. AIKAT was predicted to be a stable alkaline protein without transmembrane segment. The secondary structure of AIKAT was predicted to be mainly composed of α-helix. The tertiary structure of AIKAT protein was predicted by homology modeling method. Homologous alignment revealed that AIKAT shared high sequence identity with the KAT proteins(AaKAT2, CcKAT2, RgKAT and AtKAT, respectively) of Artemisia annua, Cynara cardunculus var. scolymus, Rehmannia glutinosa and Arabidopsis thaliana. The phylogenetic analysis showed that AIKAT clustered with CcKAT2, confirming the homology of 3-ketoacyl-CoA thiolase genes in Compositae. The prokaryotic expression vector pET-32 a-AIKAT was constructed and transformed into Escherichia coli BL21(DE3) for protein expression. The target protein was successfully expressed as a soluble protein of about 64 kDa. A real-time quantitative PCR analysis was performed to profile the AIKAT expression in different tissues of A. lancea. The results demonstrated that the expression level of AIKAT was the highest in rhizome, followed by that in leaves and stems. In this study, the full-length cDNA of AIKAT was cloned and expressed in E. coli BL21(DE3), and qRT-PCR showed the differential expression of this gene in different tissues, which laid a foundation for further research on the molecular mechanism of fatty acid ß-oxidation in A. lancea.
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Atractylodes , Secuencia de Aminoácidos , Atractylodes/genética , Clonación Molecular , Coenzima A , Escherichia coli/genética , FilogeniaRESUMEN
High fructose intake is a risk factor for liver fibrosis. Polydatin is a main constituent of the rhizome of Polygonum cuspidatum, which has been used in traditional Chinese medicine to treat liver fibrosis. However, the underlying mechanisms of fructose-driven liver fibrosis as well as the actions of polydatin are not fully understood. In this study, fructose was found to promote zinc finger E-box binding homeobox 1 (ZEB1) nuclear translocation, decrease microRNA-203 (miR-203) expression, increase survivin, activate transforming growth factor ß1 (TGF-ß1)/Smad signalling, down-regulate E-cadherin, and up-regulate fibroblast specific protein 1 (FSP1), vimentin, N-cadherin and collagen I (COL1A1) in rat livers and BRL-3A cells, in parallel with fructose-induced liver fibrosis. Furthermore, ZEB1 nuclear translocation-mediated miR-203 low-expression was found to target survivin to activate TGF-ß1/Smad signalling, causing the EMT in fructose-exposed BRL-3A cells. Polydatin antagonized ZEB1 nuclear translocation to up-regulate miR-203, subsequently blocked survivin-activated TGF-ß1/Smad signalling, which were consistent with its protection against fructose-induced EMT and liver fibrosis. These results suggest that ZEB1 nuclear translocation may play an essential role in fructose-induced EMT in liver fibrosis by targeting survivin to activate TGF-ß1/Smad signalling. The suppression of ZEB1 nuclear translocation by polydatin may be a novel strategy for attenuating the EMT in liver fibrosis associated with high fructose diet.
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Transición Epitelial-Mesenquimal , Glucósidos/farmacología , Cirrosis Hepática/metabolismo , Estilbenos/farmacología , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo , Transporte Activo de Núcleo Celular , Animales , Cadherinas/metabolismo , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Fructosa , Cirrosis Hepática/inducido químicamente , Masculino , MicroARNs/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
We present here the recent advances in exploring new techniques related to interferometric synthetic aperture radar (InSAR) signal and data processing and applications.
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Depression is a common mental disorder in modern society. A traditional Chinese medicine Huanglian-Wendan decoction with potential anti-inflammation is used as a clinical antidepressant. Our previous study showed central and peripheral inflammatory responses in a rat model of depression developed by chronic unpredictable mild stress (CUMS). Here, we investigated the anti-inflammatory activity and mechanism of Huanglian-Wendan decoction in CUMS rats. LC-MS/MS and HPLC were performed to determine the major compounds in water extract of this decoction. This study showed that Huanglian-Wendan decoction significantly increased sucrose consumption and reduced serum levels of interleukin-1 beta (IL-1ß), IL-6, and alanine aminotransferase (ALT) in CUMS rats. Moreover, this decoction inhibited nuclear entry of nuclear factor-kappa B (NF-κB) with the reduction of phosphorylated protein of NF-κB (p-NF-κB) and inhibitor of NF-κB alpha (p-IκBα) and downregulated protein of nod-like receptor family pyrin domain-containing 3 (NLRP3), apoptosis-associated speck-like protein containing CARD (ASC), cysteinyl aspartate-specific proteinase-1 (Caspase-1), and IL-1ß in liver and brain regions of CUMS rats. These findings demonstrated that Huanglian-Wendan decoction had antidepressant activity with hepatoprotection in CUMS rats coinciding with its anti-inflammation in both periphery and central. The inhibitory modulation of NF-κB and NLRP3 inflammasome activation by Huanglian-Wendan decoction may mediate its antidepressant action.
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Encéfalo/metabolismo , Medicamentos Herbarios Chinos/farmacología , Inflamasomas/efectos de los fármacos , Inflamasomas/metabolismo , Hígado/metabolismo , FN-kappa B/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Animales , Western Blotting , Encéfalo/efectos de los fármacos , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Hígado/efectos de los fármacos , Masculino , RatasRESUMEN
Rock-mineral spectrum is a mixture of varied mineral spectra, through which we can obtain information about its components quickly and conveniently without any damage to the sample. Empirical mode decomposition (EMD) cannot directly decompose source signals from information of the mixture, and independent component analysis (ICA) requires the number of mixed signals to be no less than the number of source signals. Combining these two methods, mixed signals can be decomposed using EMD method to obtain intrinsic mode function (IMF), while certain IMFs together with mixed signals can be used as input data matrix of ICA to obtain the source signals. This method overcomes the shortcomings of IMF and ICA. Studies have shown that, the higher content of source signals contained in the mixed signal, the better estimation can be obtained through EMD and ICA. The number of IMFs that participate in ICA decomposition determines the number of approximation of source signals. The accuracy of source signal estimation increases with the correlation coefficient between IMF and mixed signals. By applying this method to quantitative analysis of rock-mineral spectrum, information of the component minerals in rock-mineral can be obtained, which improves the efficiency of component analysis in detecting rock-minerals outside.
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The WRKY family of transcription factors (TFs) is an important gene family involved in abiotic stress responses. Although the roles of WRKY TFs in plant abiotic stress responses are well studied, little is known about the stress-induced changes in WRKY family in Platycodon grandiflorus. 42 PgWRKY genes in seven subgroups were identified in the P. grandiflorus genome. The content of eight platycodins in P. grandiflorus was investigated under cold, heat, and drought stresses. Platycodin D levels significantly increased under three abiotic stresses, while the content changes of other platycodins varied. Transcriptome analysis showed that different WRKY family members exhibited varied expression patterns under different abiotic stresses. PgWRKY20, PgWRKY26, and PgWRKY39 were identified as three key candidates for temperature and drought stress responses, and were cloned and analysed for sequence characteristics, gene structure, subcellular localisation, and expression patterns. The RT-qPCR results showed that PgWRKY26 expression significantly increased after heat stress for 48 h, cold stress for 6 h, and drought stress for 2 d (DS_2 d). The PgWRKY39 expression level significantly increased at DS_2 d. This study provides a theoretical basis for clarifying the molecular mechanism of the abiotic stress responses of the WRKY gene family in P. grandiflorus.
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Platycodon , Platycodon/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estrés Fisiológico/genética , Genoma de Planta , Respuesta al Choque por Frío , Perfilación de la Expresión Génica/métodos , Filogenia , Regulación de la Expresión Génica de las PlantasRESUMEN
Introduction: The uridine diphosphate (UDP)-glycosyltransferase (UGT) family is the largest glycosyltransferase family, which is involved in the biosynthesis of natural plant products and response to abiotic stress. UGT has been studied in many medicinal plants, but there are few reports on Platycodon grandiflorus. This study is devoted to genome-wide analysis of UGT family and identification of UGT genes involved in drought stress of Platycodon grandiflorus (PgUGTs). Methods: The genome data of Platycodon grandiflorus was used for genome-wide identification of PgUGTs, online website and bioinformatics analysis software was used to conduct bioinformatics analysis of PgUGT genes and the genes highly responsive to drought stress were screened out by qRT-PCR, these genes were cloned and conducted bioinformatics analysis. Results: A total of 75 PgUGT genes were identified in P.grandiflorus genome and clustered into 14 subgroups. The PgUGTs were distributed on nine chromosomes, containing multiple cis-acting elements and 22 pairs of duplicate genes were identified. Protein-protein interaction analysis was performed to predict the interaction between PgUGT proteins. Additionally, six genes were upregulated after 3d under drought stress and three genes (PGrchr09G0563, PGrchr06G0523, PGrchr06G1266) responded significantly to drought stress, as confirmed by qRT-PCR. This was especially true for PGrchr06G1266, the expression of which increased 16.21-fold after 3d of treatment. We cloned and conducted bioinformatics analysis of three candidate genes, both of which contained conserved motifs and several cis-acting elements related to stress response, PGrchr06G1266 contained the most elements. Discussion: PgGT1 was confirmed to catalyze the C-3 position of platycodin D and only eight amino acids showed differences between gene PGr008G1527 and PgGT1, which means PGr008G1527 may be able to catalyze the C-3 position of platycodin D in the same manner as PgGT1. Seven genes were highly expressed in the roots, stems, and leaves, these genes may play important roles in the development of the roots, stems, and leaves of P. grandiflorus. Three genes were highly responsive to drought stress, among which the expression of PGrchr06G1266 was increased 16.21-fold after 3d of drought stress treatment, indicating that PGrchr06G1266 plays an important role in drought stress tolerance. To summarize, this study laied the foundation to better understand the molecular bases of responses to drought stress and the biosynthesis of platycodin.
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Head and neck cancers (HNCs) account for ~4% of all cancers in North America and encompass cancers affecting the oral cavity, pharynx, larynx, sinuses, nasal cavity, and salivary glands. The anatomical complexity of the head and neck region, characterized by highly perfused and innervated structures, presents challenges in the early diagnosis and treatment of these cancers. The utilization of sub-microliter volumes and the unique phenomenon associated with microscale fluid dynamics have facilitated the development of microfluidic platforms for studying complex biological systems. The advent of on-chip microfluidics has significantly impacted the diagnosis and treatment strategies of HNC. Sensor-based microfluidics and point-of-care devices have improved the detection and monitoring of cancer biomarkers using biological specimens like saliva, urine, blood, and serum. Additionally, tumor-on-a-chip platforms have allowed the creation of patient-specific cancer models on a chip, enabling the development of personalized treatments through high-throughput screening of drugs. In this review, we first focus on how microfluidics enable the development of an enhanced, functional drug screening process for targeted treatment in HNCs. We then discuss current advances in microfluidic platforms for biomarker sensing and early detection, followed by on-chip modeling of HNC to evaluate treatment response. Finally, we address the practical challenges that hinder the clinical translation of these microfluidic advances.
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Atractylodes lancea (Thunb.) DC. is an important medicinal plant mainly distributed in China. A. lancea is rich in volatile oils and has a significant effect on various diseases, including coronavirus disease 2019 (COVID-19). Based on the signature constituents of volatile oils, A. lancea is divided into two chemotypes: the Dabieshan and Maoshan chemotype. Gas chromatography-mass spectrometry (GC-MS) results revealed that the hinesol and ß-eudesmol contents in the Dabieshan chemotype were higher than those in the Maoshan chemotype. Next-generation sequencing (NGS) and single-molecule real-time (SMRT) sequencing technologies were combined to investigate the molecular mechanisms of sesquiterpenoid biosynthesis in A. lancea. A total of 42 differentially expressed genes (DEGs) for terpenoid biosynthesis were identified in the two chemotype groups, and nine full-length terpene synthase (TPS) genes were identified. Subcellular localization revealed that AlTPS1 and AlTPS2 proteins were localized in the nucleus and endoplasmic reticulum. They use FPP as a substrate to generate sesquiterpenoids. AlTPS1 catalyzes biosynthesis of elemol while AlTPS2 is observed to perform ß-farnesene synthase activity. This study provides information for understanding the differences in the accumulation of terpenoids in two chemotypes of A. lancea and lays a foundation for further elucidation of the molecular mechanism of sesquiterpenoid biosynthesis.
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Atractylodes , COVID-19 , Aceites Volátiles , Sesquiterpenos , Atractylodes/química , Sesquiterpenos/metabolismo , Aceites Volátiles/química , Perfilación de la Expresión GénicaRESUMEN
Various culture conditions by small molecules have been explored to extend pluripotency of stem cells, but their impacts on cell fate in vivo remain elusive. We systematically compared the effects of various culture conditions on the pluripotency and cell fate in vivo of mouse embryonic stem cells (ESCs) by tetraploid embryo complementation assay. Conventional ESC cultures in serum/LIF-based condition produced complete ESC mice and also the survival to adulthood at the highest rates of all other chemical-based cultures. Moreover, long-term examination of the survived ESC mice demonstrated that conventional ESC cultures did not lead to visible abnormality for up to 1.5-2 years, whereas the prolonged chemical-based cultures developed retroperitoneal atypical teratomas or leiomyomas. The chemical-based cultures exhibited transcriptomes and epigenomes that typically differed from those of conventional ESC cultures. Our results warrant further refinement of culture conditions in promoting the pluripotency and safety of ESCs in future applications.
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Células Madre Pluripotentes , Teratoma , Ratones , Animales , Células Madre Embrionarias de Ratones/patología , Células Cultivadas , Células Madre Embrionarias , Teratoma/patología , Diferenciación CelularRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Herbal formulas have unique efficacy and are of great significance to the theory and practice of Chinese medicine and are therefore gaining increasing attention in research. Painong powder (PNS), composed of Aurantii fructus immaturus (Zhishi in Chinese, ZS), Paeoniae Radix Alba (Baishao in Chinese, BS), and Platycodonis Radix (Jiegeng in Chinese, JG), has remarkable effects on the detoxification and discharge of pus. JG is traditionally used to treat pulmonary carbuncles and is considered a 'medicinal guide'. According to the composition theory of prescriptions, JG is an 'assistant and guide' medicine. The role of JG as an adjuvant has gained increasing attention. AIM OF THE STUDY: The study was designed to prove the efficacy of PNS in ulcerative colitis (UC) and to study the role of JG in PNS via pharmacodynamic, pharmacokinetic, and tissue distribution analyses. MATERIALS AND METHODS: For the pharmacodynamic study, the UC rat model was induced using 5% trinitrobenzene sulfonic acid (TNBS). The results of the macroscopic characterization, histological analysis, and cytokine levels, including those of tumour necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and nuclear factor-kappa B (NF-κB), were integrated to evaluate the treatment of UC with PNS. In addition, an LC-MS/MS method was established and validated to analyze the blood pharmacokinetic parameters and tissue distribution of naringin and paeoniflorin. RESULTS: After the administration of high-dose PNS, the UC rats showed amelioration of macroscopic damage at the lesion site. The cytokine levels in the plasma, colon, and lung tissues were also decreased. The pharmacokinetic parameters showed that compared with UC rats administered with PNS-JG, those administered with PNS showed an increase in the AUC, MRT, and Tmax of naringin and paeoniflorin, and a decrease in their clearance rate. Furthermore, naringin and paeoniflorin had higher concentrations in the colon and lung tissues in the normal and model groups administered with PNS than in those administered with PNS-JG. CONCLUSIONS: PNS was shown to have marked therapeutic efficacy against TNBS-induced UC in rats. The effect of JG in PNS was reflected by the differences in the pharmacokinetic parameters and tissue distribution of the active components, providing valuable information for the clinical application of PNS in the treatment of UC. However, knowledge about how JG works as an adjuvant medicine in PNS is still lacking.
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Campanulaceae , Colitis Ulcerosa , Medicamentos Herbarios Chinos , Fitoterapia , Animales , Ratas , Área Bajo la Curva , Campanulaceae/química , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/tratamiento farmacológico , Citocinas/genética , Citocinas/metabolismo , Medicamentos Herbarios Chinos/farmacocinética , Medicamentos Herbarios Chinos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Semivida , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Polvos , Ratas Sprague-Dawley , Distribución Tisular , Ácido Trinitrobencenosulfónico/toxicidadRESUMEN
Platycodon grandiflorus, a perennial flowering plant widely distributed in China and South Korea, is an excellent resource for both food and medicine. The main active compounds of P. grandiflorus are triterpenoid saponins. WRKY transcription factors (TFs) are among the largest gene families in plants and play an important role in regulating plant terpenoid accumulation, physiological metabolism, and stress response. Numerous studies have been reported on other medicinal plants; however, little is known about WRKY genes in P. grandiflorus. In this study, 27 PgWRKYs were identified in the P. grandiflorus transcriptome. Phylogenetic analysis showed that PgWRKY genes were clustered into three main groups and five subgroups. Transcriptome analysis showed that the PgWRKY gene expression patterns in different tissues differed between those in Tongcheng City (Southern Anhui) and Taihe County (Northern Anhui). Gene expression analysis based on RNA sequencing and qRT-PCR analysis showed that most PgWRKY genes were expressed after induction with methyl jasmonate (MeJA). Co-expressing PgWRKY genes with triterpenoid biosynthesis pathway genes revealed four PgWRKY genes that may have functions in triterpenoid biosynthesis. Additionally, functional annotation and protein-protein interaction analysis of PgWRKY proteins were performed to predict their roles in potential regulatory networks. Thus, we systematically analyzed the structure, evolution, and expression patterns of PgWRKY genes to provide an important theoretical basis for further exploring the molecular basis and regulatory mechanism of WRKY TFs in triterpenoid biosynthesis.
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Platycodon , Triterpenos , Acetatos , Ciclopentanos , Regulación de la Expresión Génica de las Plantas/genética , Oxilipinas , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Platycodon/genética , Platycodon/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcriptoma/genéticaRESUMEN
Totipotent cells have more robust developmental potency than any other cell types, giving rise to both embryonic and extraembryonic tissues. Stable totipotent cell cultures and deciphering the principles of totipotency regulation would be invaluable to understand cell plasticity and lineage segregation in early development. Our approach of remodeling the pericentromeric heterochromatin and re-establishing the totipotency-specific broad H3K4me3 domains promotes the pluri-to-totipotency transition. Our protocol establishes a closer match of mouse 2-cell (2C) embryos than any other 2C-like cells. These totipotent-like stem cells (TLSCs) are stable in culture and possess unique molecular features of the mouse 2C embryo. Functionally, TLSCs are competent for germline transmission and give rise to both embryonic and extraembryonic lineages at high frequency. Therefore, TLSCs represent a highly valuable cell type for studies of totipotency and embryology.
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Ensamble y Desensamble de Cromatina , Células Madre Totipotentes , Animales , Diferenciación Celular , Plasticidad de la Célula , Cromatina/metabolismo , Embrión de Mamíferos , RatonesRESUMEN
Platycodon grandiflorus is a well-known and widely distributed traditional herbal medicine and functional food in Asia, with triterpenoids as the main bioactive component in its roots. Acetyl-CoA C-acetyltransferase (AACT) is the initiation enzyme in the mevalonate pathway and plays an important role in the biosynthesis of terpenoids. OBJECTIVE: The objective of this study was to clone and identify the PgAACT function in P. grandiflorus. METHODS: The full-length sequence of PgAACT genes was isolated and cloned from P. grandiflorus by polymerase chain reaction (PCR). The recombinant plasmid was constructed using the pET-32a vector and expressed in E. coli Transetta (DE3) cells. Subcellular localization of AACT was observed in the epidermal cells of N. tabacum. Quantitative reverse transcription-PCR (qRT-PCR) was used to identify the PgAACT gene transcription levels. After MeJA treatment, the changes in AACT gene expression were observed, and UHPLC-Q-Exactive Orbitrap MS/MS was used to detect the changes in P. grandiflorus saponins. RESULTS: In this study, two full-length cDNAs encoding AACT1 (PgAACT1) and AACT2 (PgAACT2) were isolated and cloned from P. grandiflorus. The deduced PgAACT1 and PgAACT2 proteins contain 408 and 416 amino acids, respectively. The recombinant vectors were constructed, and the protein expression was improved by optimizing the reaction conditions. Sodium dodecyl sulphate-polycrylamide gel electrophloresis and western blot analysis showed that the PgAACT genes were successfully expressed, with molecular weights of the recombinant proteins of 61 and 63 kDa, respectively. Subcellular localization showed that the PgAACT genes were localized in the cytoplasm. Tissue specificity analysis of P. grandiflorus from different habitats showed that PgAACT genes were expressed in the roots, stems, and leaves. After MeJA treatment, the expression level of PgAACT genes and the content of total saponins of P. grandiflorus were significantly increased, suggesting that PgAACT genes play an important role in regulating plant defense systems. CONCLUSION: Cloning, expression, and functional analysis of PgAACT1 and PgAACT2 will be helpful in understanding the role of these two genes in terpene biosynthesis.
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Platycodon , Saponinas , Platycodon/genética , Platycodon/metabolismo , Acetil-CoA C-Acetiltransferasa/genética , Acetil-CoA C-Acetiltransferasa/metabolismo , ADN Complementario/genética , Regulación de la Expresión Génica de las Plantas , Escherichia coli/genética , Espectrometría de Masas en Tándem , Clonación Molecular , TerpenosRESUMEN
In human embryos, major zygotic genome activation (ZGA) initiates at the eight-cell (8C) stage. Abnormal ZGA leads to developmental defects and even contributes to the failure of human blastocyst formation or implantation. An in vitro cell model mimicking human 8C blastomeres would be invaluable to understanding the mechanisms regulating key biological events during early human development. Using the non-canonical promoter of LEUTX that putatively regulates human ZGA, we developed an 8C::mCherry reporter, which specifically marks the 8C state, to isolate rare 8C-like cells (8CLCs) from human preimplantation epiblast-like stem cells. The 8CLCs express a panel of human ZGA genes and have a unique transcriptome resembling that of the human 8C embryo. Using the 8C::mCherry reporter, we further optimize the chemical-based culture condition to increase and maintain the 8CLC population. Functionally, 8CLCs can self-organize to form blastocyst-like structures. The discovery and maintenance of 8CLCs provide an opportunity to recapitulate early human development.