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Biochemical processes often require spatial regulation and specific microenvironments. The general lack of organelles in bacteria limits the potential of bioengineering complex intracellular reactions. Here, we demonstrate synthetic membraneless organelles in Escherichia coli termed transcriptionally engineered addressable RNA solvent droplets (TEARS). TEARS are assembled from RNA-binding protein recruiting domains fused to poly-CAG repeats that spontaneously drive liquid-liquid phase separation from the bulk cytoplasm. Targeting TEARS with fluorescent proteins revealed multilayered structures with composition and reaction robustness governed by non-equilibrium dynamics. We show that TEARS provide organelle-like bioprocess isolation for sequestering biochemical pathways, controlling metabolic branch points, buffering mRNA translation rates, and scaffolding protein-protein interactions. We anticipate TEARS to be a simple and versatile tool for spatially controlling E. coli biochemistry. Particularly, the modular design of TEARS enables applications without expression fine-tuning, simplifying the design-build-test cycle of bioengineering.
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Escherichia coli , Organelas , Escherichia coli/genética , Organelas/metabolismo , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Solventes/análise , Solventes/metabolismoRESUMO
Phase separation is a vital mechanism that mediates the formation of biomolecular condensates and their functions. Necroptosis is a lytic form of programmed cell death mediated by RIPK1, RIPK3, and MLKL downstream of TNFR1 and has been implicated in mediating many human diseases. However, whether necroptosis is regulated by phase separation is not yet known. Here, we show that upon the induction of necroptosis and recruitment by the adaptor protein TAX1BP1, PARP5A and its binding partner RNF146 form liquid-like condensates by multivalent interactions to perform poly ADP-ribosylation (PARylation) and PARylation-dependent ubiquitination (PARdU) of activated RIPK1 in mouse embryonic fibroblasts. We show that PARdU predominantly occurs on the K376 residue of mouse RIPK1, which promotes proteasomal degradation of kinase-activated RIPK1 to restrain necroptosis. Our data demonstrate that PARdU on K376 of mouse RIPK1 provides an alternative cell death checkpoint mediated by phase separation-dependent control of necroptosis by PARP5A and RNF146.
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Necroptose , Separação de Fases , Animais , Camundongos , Apoptose/fisiologia , Morte Celular , Fibroblastos/metabolismo , Necroptose/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
An Arabidopsis (Arabidopsis thaliana) mitogen-activated protein kinase (MAPK) cascade composed of YODA (YDA)-MKK4/MKK5-MPK3/MPK6 plays an essential role downstream of the ERECTA (ER)/ER-LIKE (ERL) receptor complex in regulating stomatal development in the leaf epidermis. STOMAGEN (STO), a peptide ligand produced in mesophyll cells, competes with EPIDERMAL PATTERNING FACTOR2 (EPF2) for binding ER/ERL receptors to promote stomatal formation. In this study, we found that activation of MPK3/MPK6 suppresses STO expression. Using MUTE and STO promoters that confer epidermis- and mesophyll-specific expression, respectively, we generated lines with cell-specific activation and suppression of MPK3/MPK6. The activation or suppression of MPK3/MPK6 in either epidermis or mesophyll cells is sufficient to alter stomatal differentiation. Epistatic analyses demonstrated that STO overexpression can rescue the suppression of stomatal formation conferred by the mesophyll-specific expression of the constitutively active MKK4DD or MKK5DD, but not by the epidermis-specific expression of these constitutively active MKKs. These data suggest that STO is downstream of MPK3/MPK6 in mesophyll cells, but upstream of MPK3/MPK6 in epidermal cells in stomatal development signaling. This function of the MPK3/MPK6 cascade allows it to coordinate plant epidermis development based on its activity in mesophyll cells during leaf development.
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The maternal-to-zygotic transition (MZT) is a conserved and fundamental process during which the maternal environment is converted to an environment of embryonic-driven development through dramatic reprogramming. However, how maternally supplied transcripts are dynamically regulated during MZT remains largely unknown. Herein, through genome-wide profiling of RNA 5-methylcytosine (m5C) modification in zebrafish early embryos, we found that m5C-modified maternal mRNAs display higher stability than non-m5C-modified mRNAs during MZT. We discovered that Y-box binding protein 1 (Ybx1) preferentially recognizes m5C-modified mRNAs through π-π interactions with a key residue, Trp45, in Ybx1's cold shock domain (CSD), which plays essential roles in maternal mRNA stability and early embryogenesis of zebrafish. Together with the mRNA stabilizer Pabpc1a, Ybx1 promotes the stability of its target mRNAs in an m5C-dependent manner. Our study demonstrates an unexpected mechanism of RNA m5C-regulated maternal mRNA stabilization during zebrafish MZT, highlighting the critical role of m5C mRNA modification in early development.
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5-Metilcitosina/metabolismo , Embrião não Mamífero/embriologia , Desenvolvimento Embrionário/fisiologia , Estabilidade de RNA/fisiologia , RNA Mensageiro Estocado/metabolismo , Peixe-Zebra/embriologia , Animais , Células HeLa , Humanos , Camundongos , RNA Mensageiro Estocado/genética , Peixe-Zebra/genéticaRESUMO
Cell death in human diseases is often a consequence of disrupted cellular homeostasis. If cell death is prevented without restoring cellular homeostasis, it may lead to a persistent dysfunctional and pathological state. Although mechanisms of cell death have been thoroughly investigated1-3, it remains unclear how homeostasis can be restored after inhibition of cell death. Here we identify TRADD4-6, an adaptor protein, as a direct regulator of both cellular homeostasis and apoptosis. TRADD modulates cellular homeostasis by inhibiting K63-linked ubiquitination of beclin 1 mediated by TRAF2, cIAP1 and cIAP2, thereby reducing autophagy. TRADD deficiency inhibits RIPK1-dependent extrinsic apoptosis and proteasomal stress-induced intrinsic apoptosis. We also show that the small molecules ICCB-19 and Apt-1 bind to a pocket on the N-terminal TRAF2-binding domain of TRADD (TRADD-N), which interacts with the C-terminal domain (TRADD-C) and TRAF2 to modulate the ubiquitination of RIPK1 and beclin 1. Inhibition of TRADD by ICCB-19 or Apt-1 blocks apoptosis and restores cellular homeostasis by activating autophagy in cells with accumulated mutant tau, α-synuclein, or huntingtin. Treatment with Apt-1 restored proteostasis and inhibited cell death in a mouse model of proteinopathy induced by mutant tau(P301S). We conclude that pharmacological targeting of TRADD may represent a promising strategy for inhibiting cell death and restoring homeostasis to treat human diseases.
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Apoptose/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Proteína de Domínio de Morte Associada a Receptor de TNF/antagonistas & inibidores , Proteína de Domínio de Morte Associada a Receptor de TNF/metabolismo , Animais , Autofagia/efeitos dos fármacos , Proteína 3 com Repetições IAP de Baculovírus/metabolismo , Proteína Beclina-1/química , Proteína Beclina-1/metabolismo , Bortezomib/antagonistas & inibidores , Bortezomib/farmacologia , Linhagem Celular , Humanos , Proteína Huntingtina/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Masculino , Camundongos , Modelos Moleculares , Emaranhados Neurofibrilares/metabolismo , Proteoma/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/química , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína de Domínio de Morte Associada a Receptor de TNF/química , Proteína de Domínio de Morte Associada a Receptor de TNF/deficiência , Fator 2 Associado a Receptor de TNF/metabolismo , Ubiquitinação , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismoRESUMO
TAK1 is a key modulator of both NF-κB signaling and RIPK1. In TNF signaling pathway, activation of TAK1 directly mediates the phosphorylation of IKK complex and RIPK1. In a search for small molecule activators of RIPK1-mediated necroptosis, we found R406/R788, two small molecule analogs that could promote sustained activation of TAK1. Treatment with R406 sensitized cells to TNF-mediated necroptosis and RIPK1-dependent apoptosis by promoting sustained RIPK1 activation. Using click chemistry and multiple biochemical binding assays, we showed that treatment with R406 promotes the activation of TAK1 by directly binding to TAK1, independent of its original target Syk kinase. Treatment with R406 promoted the ubiquitination of TAK1 and the interaction of activated TAK1 with ubiquitinated RIPK1. Finally, we showed that R406/R788 could promote the cancer-killing activities of TRAIL in vitro and in mouse models. Our studies demonstrate the possibility of developing small molecule TAK1 activators to potentiate the effect of TRAIL as anticancer therapies.
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Apoptose , Neoplasias , Animais , Camundongos , Morte Celular , Citosol , Neoplasias/tratamento farmacológico , Neoplasias/genética , UbiquitinaçãoRESUMO
LUBAC-mediated linear ubiquitination plays a pivotal role in regulation of cell death and inflammatory pathways. Genetic deficiency in LUBAC components leads to severe immune dysfunction or embryonic lethality. LUBAC has been extensively studied for its role in mediating TNF signaling. However, Tnfr1 knockout is not able to fully rescue the embryonic lethality of LUBAC deficiency, suggesting that LUBAC may modify additional key cellular substrates in promoting cell survival. GPx4 is an important selenoprotein involved in regulating cellular redox homeostasis in defense against lipid peroxidation-mediated cell death known as ferroptosis. Here we demonstrate that LUBAC deficiency sensitizes to ferroptosis by promoting GPx4 degradation and downstream lipid peroxidation. LUBAC binds and stabilizes GPx4 by modulating its linear ubiquitination both in normal condition and under oxidative stress. Our findings identify GPx4 as a key substrate of LUBAC and a previously unrecognized role of LUBAC-mediated linear ubiquitination in regulating cellular redox status and cell death.
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Receptores Tipo I de Fatores de Necrose Tumoral , Ubiquitina , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , NF-kappa B/metabolismo , UbiquitinaçãoRESUMO
BACKGROUND: Cotton is a major world cash crop and an important source of natural fiber, oil, and protein. Drought stress is becoming a restrictive factor affecting cotton production. To facilitate the development of drought-tolerant cotton varieties, it is necessary to study the molecular mechanism of drought stress response by exploring key drought-resistant genes and related regulatory factors. RESULTS: In this study, two cotton varieties, ZY007 (drought-sensitive) and ZY168 (drought-tolerant), showing obvious phenotypic differences under drought stress, were selected. A total of 25,898 drought-induced genes were identified, exhibiting significant enrichment in pathways related to plant stress responses. Under drought induction, At subgenome expression bias was observed at the whole-genome level, which may be due to stronger inhibition of Dt subgenome expression. A gene co-expression module that was significantly associated with drought resistance was identified. About 90% of topologically associating domain (TAD) boundaries were stable, and 6613 TAD variation events were identified between the two varieties under drought. We identified 92 genes in ZY007 and 98 in ZY168 related to chromatin 3D structural variation and induced by drought stress. These genes are closely linked to the cotton response to drought stress through canonical hormone-responsive pathways, modulation of kinase and phosphatase activities, facilitation of calcium ion transport, and other related molecular mechanisms. CONCLUSIONS: These results lay a foundation for elucidating the molecular mechanism of the cotton drought response and provide important regulatory locus and gene resources for the future molecular breeding of drought-resistant cotton varieties.
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Cromatina , Secas , Regulação da Expressão Gênica de Plantas , Gossypium , Gossypium/genética , Gossypium/fisiologia , Cromatina/metabolismo , Estresse Fisiológico/genética , Genes de PlantasRESUMO
BACKGROUND: The expression of Egl-9 family hypoxia-inducible factor 3 (EGLN3) is notably decreased in various malignancies, including gastric cancer (GC). While the predominant focus has been on the hydroxylase activity of EGLN3 for its antitumour effects, recent findings have suggested nonenzymatic roles for EGLN3. METHODS: This study assessed the clinical significance of EGLN3 expression in GC and explored the connection between EGLN3 DNA promoter methylation and transcriptional silencing. To investigate the effect of EGLN3 on GC cells, a gain-of-function strategy was adopted. RNA sequencing was conducted to identify the key effector molecules and signalling pathways associated with EGLN3. RESULTS: EGLN3 expression was significantly reduced in GC tissues, correlating with poorer patient prognosis. EGLN3 hypermethylation disrupts transcriptional equilibrium, contributing to deeper tumour invasion and lymph node metastasis, thus exacerbating GC progression. Conversely, restoration of EGLN3 expression in GC cells substantially inhibited cell proliferation and metastasis. EGLN3 was also found to impede the malignant progression of GC cells by downregulating Jumonji C domain-containing protein 8-mediated activation of the NF-κB pathway, independent of its hydroxylase activity. CONCLUSIONS: EGLN3 has the potential to hinder the spread of GC cells through a nonenzymatic mechanism, thereby shedding light on the complex nature of GC progression.
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NF-kappa B , Neoplasias Gástricas , Humanos , NF-kappa B/genética , NF-kappa B/metabolismo , Neoplasias Gástricas/patologia , Transdução de Sinais/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Oxigenases de Função Mista/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismoRESUMO
PURPOSE: Inflammatory breast cancer (IBC), a rare and highly aggressive form of breast cancer, accounts for 10% of breast cancer-related deaths. Previous omics studies of IBC have focused solely on one of genomics or transcriptomics and did not discover common differences that could distinguish IBC from non-IBC. METHODS: Seventeen IBC patients and five non-IBC patients as well as additional thirty-three Asian breast cancer samples from TCGA-BRCA were included for the study. We performed whole-exon sequencing (WES) to investigate different somatic genomic alterations, copy number variants, and large structural variants between IBC and non-IBC. Bulk RNA sequencing (RNA-seq) was performed to examine the differentially expressed genes, pathway enrichment, and gene fusions. WES and RNA-seq data were further investigated in combination to discover genes that were dysregulated in both genomics and transcriptomics. RESULTS: Copy number variation analysis identified 10 cytobands that showed higher frequency in IBC. Structural variation analysis showed more frequent deletions in IBC. Pathway enrichment and immune infiltration analysis indicated increased immune activation in IBC samples. Gene fusions including CTSC-RAB38 were found to be more common in IBC. We demonstrated more commonly dysregulated RAS pathway in IBC according to both WES and RNA-seq. Inhibitors targeting RAS signaling and its downstream pathways were predicted to possess promising effects in IBC treatment. CONCLUSION: We discovered differences unique in Asian women that could potentially explain IBC etiology and presented RAS signaling pathway as a potential therapeutic target in IBC treatment.
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Precise integration of diverse therapeutic approaches into nanomaterials is the key to the development of multimodal synergistic cancer therapy. In this work, tadpole-like carbon nanotubes with Fe nanoparticle encapsulated at the head and Zn single-atom anchored on the body (Fe@CNT-Zn) is precisely designed and facilely prepared via one-pot carbonization. In vitro studies revealed the integration of chemotherapy (CT), chemodynamic therapy (CDT), photothermal therapy (PTT), and photodynamic therapy (PDT) in Fe@CNT-Zn as well as the near-infrared light (NIR)-responsive cascade therapeutic efficacy. Furthermore, in vivo studies demonstrated the NIR-triggered cascade-amplifying synergistic cancer therapy in a B16 tumor-bearing mouse model. The results not only showcased the Fe@CNT-Zn as a potential tetramodal therapeutic platform, but also demonstrated a proof-of-concept on metal-organic framework-based "one stone for multiple birds" strategy for in situ functionalization of carbon materials.
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Acute graft-versus-host disease (aGVHD) is primarily driven by allogeneic donor T cells associated with an altered composition of the host gut microbiome and its metabolites. The severity of aGVHD after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is not solely determined by the host and donor characteristics; however, the underlying mechanisms remain unclear. Using single-cell RNA sequencing, we decoded the immune cell atlas of 12 patients who underwent allo-HSCT: six with aGVHD and six with non-aGVHD. We performed a fecal microbiota (16SrRNA sequencing) analysis to investigate the fecal bacterial composition of 82 patients: 30 with aGVHD and 52 with non-aGVHD. Fecal samples from these patients were analyzed for bile acid metabolism. Through multi-omic analysis, we identified a feedback loop involving "immune cell-gut microbes-bile acid metabolites" contributing to heightened immune responses in patients with aGVHD. The dysbiosis of the gut microbiota and disruption of bile acid metabolism contributed to an exaggerated interleukin-1 mediated immune response. Our findings suggest that resistin and defensins are crucial in mitigating against aGVHD. Therefore, a comprehensive multi-omic atlas incorporating immune cells, gut microbes, and bile acid metabolites was developed in this study and used to propose novel, non-immunosuppressive approaches to prevent aGVHD.
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Ácidos e Sais Biliares , Fezes , Microbioma Gastrointestinal , Doença Enxerto-Hospedeiro , Ácidos e Sais Biliares/metabolismo , Humanos , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/microbiologia , Microbioma Gastrointestinal/imunologia , Feminino , Masculino , Fezes/microbiologia , Pessoa de Meia-Idade , Doença Aguda , Adulto , Retroalimentação Fisiológica , Imunidade , Metabolômica , Transplante de Células-Tronco Hematopoéticas , MultiômicaRESUMO
Semiconductor quantum dots (QDs) have recently caused a stir as a promising and powerful lighting material applied in real-time fluorescence detection, display, and imaging. Photonic nanostructures are well suited for enhancing photoluminescence (PL) due to their ability to tailor the electromagnetic field, which raises both radiative and nonradiative decay rate of QDs nearby. However, several proposed structures with a complicated manufacturing process or low PL enhancement hinder their application and commercialization. Here, we present two kinds of dual-resonance gratings to effectively improve PL enhancement and propose a facile fabrication method based on holographic lithography. A maximum of 220-fold PL enhancement from CdSe/CdS/ZnS QDs are realized on 1D Al-coated photoresist (PR) gratings, where dual resonance bands are excited to simultaneously overlap the absorption and emission bands of QDs, much larger than those of some reported structures. Giant PL enhancement realized by cost-effective method further suggests the potential of better developing the nanostructure to QD-based optical and optoelectronic devices.
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BACKGROUND: Neuroepithelial transforming gene 1 (NET1) is a RhoA subfamily guanine nucleotide exchange factor that governs a wide array of biological processes. However, its roles in meiotic oocyte remain unclear. We herein demonstrated that the NET1-HACE1-RAC1 pathway mediates meiotic defects in the progression of oocyte maturation. METHODS: NET1 was reduced using a specific small interfering RNA in mouse oocytes. Spindle assembly, chromosomal alignment, the actin cap, and chromosomal spreads were visualized by immunostaining and analyzed under confocal microscopy. We also applied mass spectroscopy, and western blot analysis for this investigation. RESULTS: Our results revealed that NET1 was localized to the nucleus at the GV stage, and that after GVBD, NET1 was localized to the cytoplasm and predominantly distributed around the chromosomes, commensurate with meiotic progression. NET1 resided in the cytoplasm and significantly accumulated on the spindle at the MI and MII stages. Mouse oocytes depleted of Net1 exhibited aberrant first polar body extrusion and asymmetric division defects. We also determined that Net1 depletion resulted in reduced RAC1 protein expression in mouse oocytes, and that NET1 protected RAC1 from degradation by HACE1, and it was essential for actin dynamics and meiotic spindle formation. Importantly, exogenous RAC1 expression in Net1-depleted oocytes significantly rescued these defects. CONCLUSIONS: Our results suggest that NET1 exhibits multiple roles in spindle stability and actin dynamics during mouse oocyte meiosis.
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Actinas , Fuso Acromático , Animais , Camundongos , Actinas/metabolismo , Meiose , Oncogenes , Oócitos/metabolismo , Fuso Acromático/metabolismoRESUMO
BACKGROUND AND AIMS: Lymph node metastasis significantly affects the prognosis of early gastric cancer patients. EUS plays a crucial role in the preoperative assessment of early gastric cancer. This study evaluated the efficacy of EUS in identifying lymph node metastasis in early gastric cancer patients and developed a risk score model to aid in choosing the best treatment options. METHODS: We retrospectively analyzed the effectiveness of EUS for detecting lymph node metastasis in early gastric cancer patients. A risk score model for predicting lymph node metastasis preoperatively was created using independent risk factors identified through binary logistic regression analysis and subsequently validated. Receiver operating characteristic curves were generated for both the development and validation cohorts. RESULTS: The overall accuracy of EUS in identifying lymph node metastasis was 85.3%, although its sensitivity (29.2%) and positive predictive value (38.7%) were relatively low. Patients were categorized based on preoperative risk factors for lymph node metastasis, including tumor size of ≥20 mm, lymph nodes of ≥10 mm, body mass index of ≥24 kg/m2, and lymph node metastasis on CT scans. A 7-point risk score model was developed to assess the likelihood of lymph node metastasis. The areas under the receiver operating characteristic curve for the development and validation sets were 0.842 and 0.837, respectively, with sensitivities of 64% and 79%, respectively. CONCLUSIONS: We developed a practical risk score model based on preoperative factors to help EUS predict lymph node metastasis in early gastric cancer patients, guiding the selection of optimal treatment approaches for these patients.
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OBJECTIVES: This study aimed to develop the scoring functions for the recently developed value assessment framework (VAF) for China, which comprises 12 attributes. METHODS: We implemented a factorial survey among Chinese healthcare stakeholders from July to September 2022. A total of 240 hypothetical drug value profiles described by the VAF were grouped into 60 blocks and randomly assigned to respondents. Each respondent was assigned with 1 block, each presented in 3 disease scenarios of different levels of severity. For each profile, respondents were asked to assess the drug's value on a scale from 0 (lowest) to 10 (highest) and make 1 of the 3 insurance recommendations: cover, to be negotiated for coverage, or reject. Linear and logistic mixed-effects models were used to develop scoring functions for aggregating the value attributes. RESULTS: A total of 365 respondents participated in the survey. 3968 responses from 331 respondents were included in the analysis. Most of the included respondents were under 45 (n = 256, 77.3%), females (n = 208, 62.8%), living in urban areas (n = 296, 89.4%), and with a bachelor's degree or higher (n = 303, 91.5%). Health benefits and safety carried more weights than other attributes in the scoring functions across disease scenarios. The value and probability of entering negotiation or receiving insurance coverage for the attribute profiles for severe/critical disease were higher than for mild/moderate disease. CONCLUSIONS: The scoring functions of the VAF can be used to assess the value of a drug and its probability of entering negotiation or receiving insurance coverage in China.
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Atenção à Saúde , Cobertura do Seguro , Feminino , Humanos , Inquéritos e Questionários , Probabilidade , ChinaRESUMO
ABSTRACT: Cardiovascular disease (CVD) is the leading cause of morbidity and mortality globally. CVD and kidney disease are closely related, with kidney injury increasing CVD mortality. The pathogenesis of cardiovascular and renal diseases involves complex and diverse interactions between multiple extracellular and intracellular signaling molecules, among which transient receptor potential vanilloid 1 (TRPV1)/transient receptor potential ankyrin 1 (TRPA1) channels have received increasing attention. TRPV1 belongs to the vanilloid receptor subtype family of transient receptor potential ion channels, and TRPA1 belongs to the transient receptor potential channel superfamily. TRPV1/TRPA1 are jointly involved in the management of cardiovascular and renal diseases and play important roles in regulating vascular tension, promoting angiogenesis, antifibrosis, anti-inflammation, and antioxidation. The mechanism of TRPV1/TRPA1 is mainly related to regulation of intracellular calcium influx and release of nitric oxide and calcitonin gene-related peptide. Therefore, this study takes the TRPV1/TRPA1 channel as the research object, analyzes and summarizes the process and mechanism of TRPV1/TRPA1 affecting cardiovascular and renal diseases, and lays a foundation for the treatment of cardiorenal diseases.
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Doenças Cardiovasculares , Nefropatias , Transdução de Sinais , Canal de Cátion TRPA1 , Canais de Cátion TRPV , Humanos , Canal de Cátion TRPA1/metabolismo , Canais de Cátion TRPV/metabolismo , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/fisiopatologia , Animais , Nefropatias/metabolismo , Nefropatias/fisiopatologia , Sistema Cardiovascular/metabolismo , Sistema Cardiovascular/fisiopatologia , Sistema Cardiovascular/efeitos dos fármacos , Rim/metabolismo , Rim/fisiopatologia , Sinalização do Cálcio/efeitos dos fármacos , Fármacos Cardiovasculares/uso terapêutico , Fármacos Cardiovasculares/farmacologiaRESUMO
TRIM family proteins are widely found in multicellular organisms and are involved in a wide range of life activities, and also act as crucial regulators in the antiviral natural immune response. This study aimed to reveal the molecular mechanism of rainbow trout TRIM protein in the anti-IHNV process. The results demonstrated that 99.1 % homology between the rainbow trout and the chinook salmon (Oncorhynchus tshawytscha) TRIM32. When rainbow trout were infected with IHNV, the TRIM32 was highly expressed in the gill, spleen, kidney and blood. Meanwhile, rainbow trout TRIM32 has E3 ubiquitin ligase activity and undergoes K29-linked polyubiquitination modifications dependent on the RING structural domain was determined by immunoprecipitation. TRIM32 could interact with the NV protein of IHNV and degrade NV protein through the ubiquitin-proteasome pathway, and was also able to activate NF-κB transcription, thereby inhibiting the replication of IHNV. Moreover, the results of the animal studies showed that the survival rate of rainbow trout overexpressing TRIM32 was 70.2 % which was significantly higher than that of the control group, and stimulating the body to produce high levels of IgM when the host was infected with the virus. In addition, TRIM32 can activate the NF-κB signalling pathway and participate in the antiviral natural immune response. The results of this study will help us to understand the molecular mechanism of TRIM protein resistance in rainbow trout, and provide new ideas for disease resistance breeding, vaccine development and immune formulation development in rainbow trout.
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The bridged polycyclic sesquiterpenoids derived from sativene, isosativene, and longifolene have unique structures, and many chemical synthesis approaches with at least 10 steps have been reported. However, their biosynthetic pathway remains undescribed. A minimal biosynthetic gene cluster (BGC), named bip, encoding a sesquiterpene cyclase (BipA) and a cytochrome P450 (BipB) is characterized to produce such complex sesquiterpenoids with multiple carbon skeletons based on enzymatic assays, heterologous expression, and precursor experiments. BipA is demonstrated as a versatile cyclase with (-)-sativene as the dominant product and (-)-isosativene and (-)-longifolene as minor ones. BipB is capable of hydroxylating different enantiomeric sesquiterpenes, such as (-)-longifolene and (+)-longifolene, at C-15 and C-14 in turn. The C-15- or both C-15- and C-14-hydroxylated products are then further oxidized by unclustered oxidases, resulting in a structurally diverse array of sesquiterpenoids. Bioinformatic analysis reveals the BipB homologues as a discrete clade of fungal sesquiterpene P450s. These findings elucidate the concise and divergent biosynthesis of such intricate bridged polycyclic sesquiterpenoids, offer valuable biocatalysts for biotransformation, and highlight the distinct biosynthetic strategy employed by nature compared to chemical synthesis.
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Sistema Enzimático do Citocromo P-450 , Família Multigênica , Estrutura Molecular , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sesquiterpenos/metabolismo , Sesquiterpenos/química , Vias Biossintéticas/genética , Sesquiterpenos Policíclicos/química , Sesquiterpenos Policíclicos/metabolismo , EstereoisomerismoRESUMO
Aberrantly activated mTOR signaling pathway is commonly found in malignancies including gastric cancer (GC). DEPTOR, as a naturally occurred inhibitor of mTOR, functions in the pro- or anti-tumor manner depending on distinct tumor contexts. However, the roles of DEPTOR in GC remain largely unknown. In this study, DEPTOR expression was identified to be significantly decreased in GC tissues compared with matched normal gastric tissues, and reduced DEPTOR level was indicative of poor prognosis in patients. Restored DEPTOR expression inhibited the propagation in AGS and NCI-N87 cells, whose DEPTOR levels are low, via deactivating mTOR signaling pathway. Likewise, cabergoline (CAB) attenuated the proliferation in AGS and NCI-N87 cells via partially rescuing DEPTOR protein level. Targeted metabolomics analysis showed that several key metabolites, such as l-serine, significantly changed in AGS cells with DEPTOR restoration. These results revealed the anti-proliferation function of DEPTOR in GC cells, suggesting that restored DEPTOR expression using CAB may be a potential therapeutic approach for patients with GC.