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Maintenance of proper levels of the methyl donor S-adenosylmethionine (SAM) is critical for a wide variety of biological processes. We demonstrate that the N6-adenosine methyltransferase METTL16 regulates expression of human MAT2A, which encodes the SAM synthetase expressed in most cells. Upon SAM depletion by methionine starvation, cells induce MAT2A expression by enhanced splicing of a retained intron. Induction requires METTL16 and its methylation substrate, a vertebrate conserved hairpin (hp1) in the MAT2A 3' UTR. Increasing METTL16 occupancy on the MAT2A 3' UTR is sufficient to induce efficient splicing. We propose that, under SAM-limiting conditions, METTL16 occupancy on hp1 increases due to inefficient enzymatic turnover, which promotes MAT2A splicing. We further show that METTL16 is the long-unknown methyltransferase for the U6 spliceosomal small nuclear RNA (snRNA). These observations suggest that the conserved U6 snRNA methyltransferase evolved an additional function in vertebrates to regulate SAM homeostasis.
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Íntrons , Metionina Adenosiltransferase/genética , Metiltransferases/metabolismo , Splicing de RNA , S-Adenosilmetionina/metabolismo , Animais , Sequência de Bases , Regulação Enzimológica da Expressão Gênica , Células HEK293 , Humanos , Sequências Repetidas Invertidas , Metionina Adenosiltransferase/química , Metilação , Metiltransferases/química , Schizosaccharomyces/metabolismoRESUMO
Long noncoding RNAs (lncRNAs) have emerged as regulators of diverse biological processes. Here, we describe the initial functional analysis of a poorly characterized human lncRNA (LINC00657) that is induced after DNA damage, which we termed "noncoding RNA activated by DNA damage", or NORAD. NORAD is highly conserved and abundant, with expression levels of approximately 500-1,000 copies per cell. Remarkably, inactivation of NORAD triggers dramatic aneuploidy in previously karyotypically stable cell lines. NORAD maintains genomic stability by sequestering PUMILIO proteins, which repress the stability and translation of mRNAs to which they bind. In the absence of NORAD, PUMILIO proteins drive chromosomal instability by hyperactively repressing mitotic, DNA repair, and DNA replication factors. These findings introduce a mechanism that regulates the activity of a deeply conserved and highly dosage-sensitive family of RNA binding proteins and reveal unanticipated roles for a lncRNA and PUMILIO proteins in the maintenance of genomic stability.
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Instabilidade Genômica , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Sequência de Bases , Instabilidade Cromossômica , Células HCT116 , Humanos , Camundongos , Ploidias , RNA Longo não Codificante/química , RNA Longo não Codificante/genéticaRESUMO
Loss of function of the DIS3L2 exoribonuclease is associated with Wilms tumor and the Perlman congenital overgrowth syndrome. LIN28, a Wilms tumor oncoprotein, triggers the DIS3L2-mediated degradation of the precursor of let-7, a microRNA that inhibits Wilms tumor development. These observations have led to speculation that DIS3L2-mediated tumor suppression is attributable to let-7 regulation. Here we examine new DIS3L2-deficient cell lines and mouse models, demonstrating that DIS3L2 loss has no effect on mature let-7 levels. Rather, analysis of Dis3l2-null nephron progenitor cells, a potential cell of origin of Wilms tumors, reveals up-regulation of Igf2, a growth-promoting gene strongly associated with Wilms tumorigenesis. These findings nominate a new potential mechanism underlying the pathology associated with DIS3L2 deficiency.
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Exorribonucleases/genética , Macrossomia Fetal/genética , Fator de Crescimento Insulin-Like II/genética , Regulação para Cima , Tumor de Wilms/genética , Animais , Linhagem Celular , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Mutação , Néfrons/citologia , Néfrons/fisiopatologia , Células-TroncoRESUMO
Direct reprogramming of fibroblasts to cardiomyocytes represents a potential means of restoring cardiac function following myocardial injury. AKT1 in the presence of four cardiogenic transcription factors, GATA4, HAND2, MEF2C, and TBX5 (AGHMT), efficiently induces the cardiac gene program in mouse embryonic fibroblasts but not adult fibroblasts. To identify additional regulators of adult cardiac reprogramming, we performed an unbiased screen of transcription factors and cytokines for those that might enhance or suppress the cardiogenic activity of AGHMT in adult mouse fibroblasts. Among a collection of inducers and repressors of cardiac reprogramming, we discovered that the zinc finger transcription factor 281 (ZNF281) potently stimulates cardiac reprogramming by genome-wide association with GATA4 on cardiac enhancers. Concomitantly, ZNF281 suppresses expression of genes associated with inflammatory signaling, suggesting the antagonistic convergence of cardiac and inflammatory transcriptional programs. Consistent with an inhibitory influence of inflammatory pathways on cardiac reprogramming, blockade of these pathways with anti-inflammatory drugs or components of the nucleosome remodeling deacetylase (NuRD) complex, which associate with ZNF281, stimulates cardiac gene expression. We conclude that ZNF281 acts at a nexus of cardiac and inflammatory gene programs, which exert opposing influences on fibroblast to cardiac reprogramming.
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Reprogramação Celular/genética , Regulação da Expressão Gênica/genética , Fatores de Transcrição/metabolismo , Anti-Inflamatórios/farmacologia , Reprogramação Celular/efeitos dos fármacos , Fibroblastos/fisiologia , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Estudo de Associação Genômica Ampla , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Proteínas Repressoras , TranscriptomaRESUMO
The simultaneous discrimination of multiple homologous sequences faces challenges due to the high similarity of sequences and the complexity of the discrimination system in most reported works. Herein, a simple and ingenious analysis method was developed to identify eight miRNAs of the let-7 family by combining logic gates and entropy-driven catalytic (EDC)-based lanthanide labeling inductively coupled plasma mass spectrometry (ICP-MS) technology. Specifically, eight miRNAs were first divided into four types according to the difference of bases in the domains 2 and 3 on sequences. To identify the type of targets, a DNA logic gate was constructed with two strand displacement reactions on magnetic beads that could be initiated by different types of targets. Based on the difference of the output signals after two strand displacement reactions, the type of targets was distinguished preliminarily. Then, the discrimination of a specific target was achieved with EDC-based lanthanide labeling ICP-MS detection. By labeling the different magnetic probes with different elemental tags, a specific element signal released from magnetic beads after EDC could be detected by ICP-MS, and therefore, simultaneous detection of homologous sequences was completed. This work provided a novel and simple method for highly specific identification of homologous sequences with the assistance of a logic gate and can promote further development of elemental labeling ICP-MS in the field of multiple analysis.
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Microfluidic chips have emerged as a promising tool for sorting and enriching circulating tumor cells (CTCs) in blood, while the efficacy and purity of CTC sorting greatly depend on chip design. Herein, a novel cascaded phase-transfer microfluidic chip was developed for high-efficiency sorting, purification, release, and detection of MCF-7 cells (as a model CTC) in blood samples. MCF-7 cells were specifically captured by EpCAM aptamer-modified magnetic beads and then introduced into the designed cascaded phase-transfer microfluidic chip that consisted of three functional regions (sorting, purification, and release zone). In the sorting zone, the MCF-7 cells moved toward the inner wall of the channel and entered the purification zone for primary separation from white blood cells; in the purification zone, the MCF-7 cells were transferred to the phosphate-buffered saline flow under the interaction of Dean forces and central magnetic force, achieving high purification of MCF-7 cells from blood samples; in the release zone, MCF-7 cells were further transferred into the nuclease solution and fixed in groove by the strong magnetic force and hydrodynamic force, and the continuously flowing nuclease solution cleaved the aptamer on the trapped MCF-7 cells, causing gentle release of MCF-7 cells for subsequent inductively coupled plasma mass spectrometry (ICP-MS) detection or further cultivation. By measurement of the endogenous element Zn in the cells using ICP-MS for cell counting, an average cell recovery of 84% for MCF-7 cells was obtained in spiked blood samples. The developed method was applied in the analysis of real blood samples from healthy people and breast cancer patients, and CTCs were successfully detected in all tested patient samples (16/16). Additionally, the removal of the magnetic probes on the cell surface significantly improved cell viability up to 99.3%. Therefore, the developed cascaded phase-transfer microfluidic chip ICP-MS system possessed high integration for CTCs analysis with high cell viability, cell recovery, and purity, showing great advantages in early clinical cancer diagnosis.
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Técnicas Analíticas Microfluídicas , Células Neoplásicas Circulantes , Humanos , Células Neoplásicas Circulantes/patologia , Microfluídica , Separação Celular/métodos , Linhagem Celular Tumoral , Técnicas Analíticas Microfluídicas/métodos , Fenômenos MagnéticosRESUMO
Circulating tumor cells (CTCs) are recognized as promising targets for liquid biopsy, which play an important role in early diagnosis and efficacy monitoring of cancer. However, due to the extreme scarcity of CTCs and partial size overlap between CTCs and white blood cells (WBCs), the separation and detection of CTCs from blood remain a big challenge. To address this issue, we fabricated a microfluidic chip by integrating a passive contraction-expansion array (CEA) inertial sorting zone and an active magnetophoresis zone with the trapezoidal groove and online coupled it with inductively coupled plasma mass spectrometry (ICP-MS) for rapid separation and precise detection of MCF-7 cells (as a model CTC) in blood samples. In the integrated microfluidic chip, most of the small-sized WBCs can be rapidly removed in the circular CEA inertial sorter, while the rest of the magnetically labeled WBCs can be further captured in the trapezoidal groove under the magnetic field. As a result, the rapid separation of MCF-7 cells from blood samples was achieved with an average recovery of 91.6% at a sample flow rate of 200 µL min-1. The developed online integrated inertial-magnetophoresis microfluidic chip-ICP-MS system has been applied for the detection of CTCs in real clinical blood samples with a fast analysis speed (5 min per 1 mL blood). CTCs were detected in all 24 blood samples from patients with different types of cancer, exhibiting excellent application potential in clinical diagnosis.
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A central aim of community ecology is to understand how local species diversity is shaped. Agricultural activities are reshaping and filtering soil biodiversity and communities; however, ecological processes that structure agricultural communities have often overlooked the role of the regional species pool, mainly owing to the lack of large datasets across several regions. Here, we conducted a soil survey of 941 plots of agricultural and adjacent natural ecosystems (e.g., forest, wetland, grassland, and desert) in 38 regions across diverse climatic and soil gradients to evaluate whether the regional species pool of soil microbes from adjacent natural ecosystems is important in shaping agricultural soil microbial diversity and completeness. Using a framework of multiscales community assembly, we revealed that the regional species pool was an important predictor of agricultural bacterial diversity and explained a unique variation that cannot be predicted by historical legacy, large-scale environmental factors, and local community assembly processes. Moreover, the species pool effects were associated with microbial dormancy potential, where taxa with higher dormancy potential exhibited stronger species pool effects. Bacterial diversity in regions with higher agricultural intensity was more influenced by species pool effects than that in regions with low intensity, indicating that the maintenance of agricultural biodiversity in high-intensity regions strongly depends on species present in the surrounding landscape. Models for community completeness indicated the positive effect of regional species pool, further implying the community unsaturation and increased potential in bacterial diversity of agricultural ecosystems. Overall, our study reveals the indubitable role of regional species pool from adjacent natural ecosystems in predicting bacterial diversity, which has useful implication for biodiversity management and conservation in agricultural systems.
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Bactérias , Ecossistema , Biodiversidade , Solo/química , Florestas , Microbiologia do SoloRESUMO
Depression is a mental illness frequently accompanied by disordered energy metabolism. A dysregulated hypothalamus pituitary adrenal axis response with aberrant glucocorticoids (GCs) release is often observed in patients with depression. However, the associated etiology between GCs and brain energy metabolism remains poorly understood. Here, using metabolomic analysis, we showed that the tricarboxylic acid (TCA) cycle was inhibited in chronic social defeat stress (CSDS)-exposed mice and patients with first-episode depression. Decreased mitochondrial oxidative phosphorylation was concomitant with the impairment of the TCA cycle. In parallel, the activity of pyruvate dehydrogenase (PDH), the gatekeeper of mitochondrial TCA flux, was suppressed, which is associated with the CSDS-induced neuronal pyruvate dehydrogenase kinase 2 (PDK2) expression and consequently enhanced PDH phosphorylation. Considering the well-acknowledged role of GCs in energy metabolism, we further demonstrated that glucocorticoid receptors (GR) stimulated PDK2 expression by directly binding to its promoter region. Meanwhile, silencing PDK2 abrogated glucocorticoid-induced PDH inhibition, restored the neuronal oxidative phosphorylation, and improved the flux of isotope-labeled carbon (U-13C] glucose) into the TCA cycle. Additionally, in vivo, pharmacological inhibition and neuron-specific silencing of GR or PDK2 restored CSDS-induced PDH phosphorylation and exerted antidepressant activities against chronic stress exposure. Taken together, our findings reveal a novel mechanism of depression manifestation, whereby elevated GCs levels regulate PDK2 transcription via GR, thereby impairing brain energy metabolism and contributing to the onset of this condition.
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Metabolismo Energético , Complexo Piruvato Desidrogenase , Humanos , Camundongos , Animais , Complexo Piruvato Desidrogenase/metabolismo , Ciclo do Ácido Cítrico/fisiologia , Encéfalo/metabolismo , FosforilaçãoRESUMO
Antineoplastic agents that target tubulin have shown efficacy as chemotherapeutic drugs, yet they are often constrained by multidrug resistance (MDR) and unwanted side effects. A multi-targeted strategy demonstrates great potency in reducing toxicity and enhancing efficacy and provides an alternative way for attenuating MDR. In this study, a series of dual-targeted anti-cancer agents based on indole-chalcone derivatives and the camptothecin (CPT) scaffold were synthesized. Among them, 14-1 demonstrated superior anti-proliferative activity than its precursor 13-1, CPT or their physical mixtures against tested cancer cells, including multidrug-resistant variants, while exhibited moderate cytotoxicity toward human normal cells. Mechanistic studies revealed that 14-1 acted as a glutathione-responsive prodrug, inducing apoptosis by substantially enhancing intracellular uptake of CPT, inhibiting tubulin polymerization, increasing the accumulation of intracellular reactive oxygen species, and initiating a mitochondrion-dependent apoptotic pathway. Moreover, 14-1 notably induced autophagy and suppressed topoisomerase I activity to further promote apoptosis. Importantly, 14-1 displayed potent inhibitory effect on tumor growth in paclitaxel (PTX)-resistant colorectal cancer (HCT-116/PTX) xenograft models without inducing obvious toxicity compared with CPT- or combo-treated group. These results suggest that 14-1 holds promise as a novel candidate for anti-cancer therapy, particularly in PTX-resistant cancers.
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Antineoplásicos , Chalconas , Neoplasias do Colo , Pró-Fármacos , Humanos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Camptotecina/farmacologia , Linhagem Celular Tumoral , Chalconas/farmacologia , Neoplasias do Colo/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Glutationa , Paclitaxel/farmacologia , Pró-Fármacos/farmacologia , Tubulina (Proteína)/farmacologia , Autofagia/efeitos dos fármacosRESUMO
BACKGROUND Lateral malleolus fractures, typically from trauma, sports, or accidents, are common, with supination external rotation (SER) injuries being most prevalent. SER injuries involve complex joint mechanics and often necessitate surgical intervention for instability. This study compares intramedullary nail and plate fixation for fibula fractures in SER type IV ankle injuries, considering their biomechanical properties and influence on fracture healing. MATERIAL AND METHODS A prospective, randomized study was conducted between January 2021 and December 2021. A total of 81 patients with SER injuries were included in the study. Surgical procedures were performed using either intramedullary nails or plates. The following parameters were recorded and analyzed: postoperative complications, operation times, bone healing times, American Orthopaedic Foot & Ankle Society (AOFAS) scores, visual analog scale (VAS) scores for pain, and ankle range of motion. RESULTS Out of the 81 cases, 42 were treated with intramedullary nails, while 39 received plate fixation. Statistical analysis revealed a significantly lower rate of postoperative complications in the intramedullary nail group than in the the plate fixation group (9.52% vs 30.77%, P<0.0164). However, there were no significant differences between the 2 groups in terms of operation time, bone healing time, AOFAS scores, VAS scores, and functional evaluations (P>0.05). CONCLUSIONS Plate fixation and intramedullary nail fixation are effective techniques for treating fibula fractures in SER type IV injuries. However, intramedullary nail fixation demonstrates a lower rate of complications. Therefore, intramedullary nails may be preferable to plate fixation for the management of fibula fractures in SER type IV ankle injuries.
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Fraturas do Tornozelo , Traumatismos do Tornozelo , Fraturas da Fíbula , Fixação Intramedular de Fraturas , Humanos , Supinação , Estudos Prospectivos , Traumatismos do Tornozelo/cirurgia , Fixação Intramedular de Fraturas/métodos , Fraturas do Tornozelo/cirurgia , Fixação Interna de Fraturas/métodos , Placas Ósseas , Complicações Pós-Operatórias , Pinos Ortopédicos , Resultado do TratamentoRESUMO
OBJECTIVES: Ultrasound-targeted microbubble disruption (UTMD) is a widely used technique to improve the differentiation and proliferation capacity of mesenchymal stem cells (MSCs), but the optimal therapeutic parameters for UTMD are unclear. In this study, we aimed to find the appropriate peak negative pressure (PNP), which is a key parameter for enhancing the stemness properties and proliferation of MSCs. METHODS: Experiments were performed in UTMD group, ultrasound (US) group under different PNP exposure conditions (0.5, 1.0, and 1.5 MPa), and control group. Apoptosis safety was analyzed by flow cytometry and MSC proliferation was measured at 12, 24, and 36 hours after irradiation by cell counting kit 8. The expression of the stemness genes NANOG, OCT-4, and SOX-2 were determined by enzyme-linked immunosorbent assay (ELISA) or reverse transcription polymerase chain reaction. RESULTS: The results showed that the 1.5 MPa UTMD-treated group had the highest proliferation capacity of MSCs at 24 hours. ELISA or quantitative reverse transcription polymerase chain reaction results showed that UTMD treatment of the 1.5 MPa group significantly upregulated the expression of the stemness genes NANOG, SOX-2, and OCT-4. CONCLUSIONS: In conclusion, the appropriate peak PNP value of UTMD was 1.5 MPa, and 1.5 MPa-mediated UTMD group obviously promoted MSCs proliferation and maintained stemness by upregulating the expression of stemness genes.
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Proliferação de Células , Células-Tronco Mesenquimais , Microbolhas , Regulação para Cima , Células Cultivadas , Ondas Ultrassônicas , Animais , Células da Medula ÓsseaRESUMO
Neutrophil extracellular traps (NETs) are implicated in gastric cancer (GC) growth, metastatic dissemination, cancer-associated thrombosis, etc. This work is conducted to elucidate the heterogeneity of NETs in GC. The transcriptome heterogeneity of NETs is investigated in TCGA-STAD via a consensus clustering algorithm, with subsequent external verification in the GSE88433 and GSE88437 cohorts. Clinical and molecular traits, the immune microenvironment, and drug response are characterized in the identified NET-based clusters. Based upon the feature genes of NETs, a classifier is built for estimating NET-based clusters via machine learning. Multiple experiments are utilized to verify the expressions and implications of the feature genes in GC. A novel NET-based classification system is proposed for reflecting the heterogeneity of NETs in GC. Two NET-based clusters have unique and heterogeneous clinical and molecular features, immune microenvironments, and responses to targeted therapy and immunotherapy. A logistic regression model reliably differentiates the NET-based clusters. The feature genes C5AR1, CSF1R, CSF2RB, CYBB, HCK, ITGB2, LILRB2, MNDA, MPEG1, PLEK, SRGN, and STAB1 are proven to be aberrantly expressed in GC cells. Specific knockdown of C5AR1 effectively hinders GC cell growth and elicits intracellular ROS accumulation. In addition, its suppression suppresses the aggressiveness and EMT phenotype of GC cells. In all, NETs are the main contributors to intratumoral heterogeneity and differential drug sensitivity in GC, and C5AR1â has been shown to trigger GC growth and metastatic spread. These findings collectively provide a theoretical basis for the use of anti-NETs in GC treatment.
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Armadilhas Extracelulares , Neoplasias Gástricas , Humanos , Armadilhas Extracelulares/metabolismo , Neutrófilos , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Fenótipo , Microambiente Tumoral/genéticaRESUMO
Beclin 1, an autophagy and haploinsufficient tumor-suppressor protein, is frequently monoallelically deleted in breast and ovarian cancers. However, the precise mechanisms by which Beclin 1 inhibits tumor growth remain largely unknown. To address this question, we performed a genome-wide CRISPR/Cas9 screen in MCF7 breast cancer cells to identify genes whose loss of function reverse Beclin 1-dependent inhibition of cellular proliferation. Small guide RNAs targeting CDH1 and CTNNA1, tumor-suppressor genes that encode cadherin/catenin complex members E-cadherin and alpha-catenin, respectively, were highly enriched in the screen. CRISPR/Cas9-mediated knockout of CDH1 or CTNNA1 reversed Beclin 1-dependent suppression of breast cancer cell proliferation and anchorage-independent growth. Moreover, deletion of CDH1 or CTNNA1 inhibited the tumor-suppressor effects of Beclin 1 in breast cancer xenografts. Enforced Beclin 1 expression in MCF7 cells and tumor xenografts increased cell surface localization of E-cadherin and decreased expression of mesenchymal markers and beta-catenin/Wnt target genes. Furthermore, CRISPR/Cas9-mediated knockout of BECN1 and the autophagy class III phosphatidylinositol kinase complex 2 (PI3KC3-C2) gene, UVRAG, but not PI3KC3-C1-specific ATG14 or other autophagy genes ATG13, ATG5, or ATG7, resulted in decreased E-cadherin plasma membrane and increased cytoplasmic E-cadherin localization. Taken together, these data reveal previously unrecognized cooperation between Beclin 1 and E-cadherin-mediated tumor suppression in breast cancer cells.
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Proteína Beclina-1/metabolismo , Neoplasias da Mama/metabolismo , Caderinas/metabolismo , Genes Supressores de Tumor , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Proteínas Relacionadas à Autofagia/metabolismo , Neoplasias da Mama/patologia , Sistemas CRISPR-Cas/genética , Membrana Celular/metabolismo , Proliferação de Células/genética , Feminino , Genoma Humano , Humanos , Interferons/metabolismo , Células MCF-7 , Camundongos Endogâmicos NOD , Camundongos SCID , Transporte Proteico , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , alfa Catenina/metabolismoRESUMO
CONTEXT: Podophyllotoxin (PPT) derivatives, used in cancer therapy, require development toward enhanced efficacy and reduced toxicity. OBJECTIVE: This study synthesizes PPT derivatives to assess their anticancer activities. MATERIALS AND METHODS: Compounds E1-E16 antiproliferative activity was tested against four human cancer cell lines (H446, MCF-7, HeLa, A549) and two normal cell lines (L02, BEAS-2B) using the CCK-8 assay. The effects of compound E5 on A549 cell growth were evaluated through molecular docking, in vitro assays (flow cytometry, wound healing, Transwell, colony formation, Western blot), and in vivo tests in female BALB/c nude mice treated with E5 (2 and 4 mg/kg). E5 (4 mg/kg) significantly reduced xenograft tumor growth compared to the DMSO control group. RESULTS: Among the 16 PPT derivatives tested for cytotoxicity, E5 exhibited potent effects against A549 cells (IC50: 0.35 ± 0.13 µM) and exceeded the reference drugs PPT and etoposide to inhibit the growth of xenograft tumours. E5-induced cell cycle arrest in the S and G2/M phases accelerated tubulin depolymerization and triggered apoptosis and mitochondrial depolarization while regulating the expression of apoptosis-related proteins and effectively inhibited cell migration and invasion, suggesting a potential to limit metastasis. Molecular docking showed binding of E5 to tubulin at the colchicine site and to Akt, with a consequent down-regulation of PI3K/Akt pathway proteins. DISCUSSION AND CONCLUSIONS: This research lays the groundwork for advancing cancer treatment through developing and using PPT derivatives. The encouraging results associated with E5 call for extended research and clinical validation, leading to novel and more effective cancer therapies.
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Antineoplásicos , Podofilotoxina , Camundongos , Animais , Humanos , Feminino , Podofilotoxina/farmacologia , Podofilotoxina/química , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/farmacologia , Simulação de Acoplamento Molecular , Camundongos Nus , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Ensaios de Seleção de Medicamentos Antitumorais , Antineoplásicos/farmacologia , Antineoplásicos/química , Proliferação de Células , Linhagem Celular Tumoral , Apoptose , Moduladores de Tubulina/farmacologia , Moduladores de Tubulina/químicaRESUMO
Circulating tumor cell (CTC) detection is essential for early cancer diagnosis and evaluating treatment efficacy. Despite the growing interest in isolating CTCs and further quantifying surface biomarkers at the single-cell level, highly efficient separation of rare CTCs from massive blood cells is still a big challenge. Here, we developed an all-in-one microfluidic chip system for the immunolabeling, magnetic separation, and focusing of HepG2 cells (as a CTC model) and online combined it with single cell-inductively coupled plasma mass spectrometry (SC-ICP-MS) for quantitative analysis of the asialoglycoprotein receptor (ASGPR) on single HepG2 cells. Lanthanide-labeled anti-ASGPR monoclonal antibody and antiepithelial cell adhesion molecule-modified magnetic beads were prepared as signal and magnetic probes, respectively. Target cells were highly efficiently labeled with signal and magnetic probes in the mixing zone of the microfluidic chip and then focused and sorted in the separation zone by specific magnetic separation techniques to avoid matrix contamination. The average cell recovery of HepG2 cells was derived to be 94.1 ± 5.7% with high separation efficiency and purity. The sorted cells with signal probes were detected for enumeration and quantification of ASGPR on their surface by SC-ICP-MS. The developed method showed good specificity and high sensitivity, detecting an average of (1.0 ± 0.2) × 105 ASGPR molecules per cell surface. This method can be used for absolute quantitative analysis of ASGPR on the surface of single hepatocellular carcinoma cells in real-world samples, providing a highly efficient analytical platform for studying targeted drug delivery in cancer therapy.
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Microfluídica , Células Neoplásicas Circulantes , Humanos , Linhagem Celular , Membrana Celular , Espectrometria de MassasRESUMO
The processes governing soil bacteria biogeography are still not fully understood. It remains unknown how the importance of environmental filtering and dispersal differs between bacterial taxonomic and functional biogeography, and whether their importance is scale-dependent. We sampled soils across the Tibet plateau, with distances among plots ranging from 20 m to 1550 km. Taxonomic composition of bacterial community was characterized by 16S amplicon sequencing and functional community composition by qPCR targeting 9 functional groups involved in N dynamics. Factors representing climate, soil and plant community were measured to assess different facets of environmental dissimilarity. Both bacterial taxonomic and functional dissimilarities were more related to abiotic dissimilarity than biotic (vegetation) dissimilarity or distance. Taxonomic dissimilarity was mostly explained by differences in soil pH and mean annual temperature (MAT), while functional dissimilarity was linked to differences in soil N and P availabilities and N:P ratio. Soil pH and MAT remained the main determinants of taxonomic dissimilarity across spatial scales. In contrast, the explanatory variables of N-related functional dissimilarity varied across the scales, with soil moisture and organic matter having the highest role across short distances (<~330 km), and available P, N:P ratio and distance being important over long distances (>~660 km). Our results demonstrate how biodiversity dimension (taxonomic versus functional aspects) and spatial scale influence the factors driving soil bacterial biogeography.
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Microbiologia do Solo , Solo , Tibet , Solo/química , Bactérias/genética , Biodiversidade , PlantasRESUMO
Rhizosphere microbes play key roles in plant growth and productivity in agricultural systems. One of the critical issues is revealing the interaction of agricultural management (M) and rhizosphere selection effects (R) on soil microbial communities, root exudates and plant productivity. Through a field management experiment, we found that bacteria were more sensitive to the M × R interaction effect than fungi, and the positive effect of rhizosphere bacterial diversity on plant biomass existed in the bacterial three two-tillage system. In addition, inoculation experiments demonstrated that the nitrogen cycle-related isolate Stenotrophomonas could promote plant growth and alter the activities of extracellular enzymes N-acetyl- d-glucosaminidase and leucine aminopeptidase in rhizosphere soil. Microbe-metabolites network analysis revealed that hubnodes Burkholderia-Caballeronia-Paraburkholderia and Pseudomonas were recruited by specific root metabolites under the M × R interaction effect, and the inoculation of 10 rhizosphere-matched isolates further proved that these microbes could promote the growth of soybean seedlings. Kyoto Encyclopaedia of Genes and Genomes pathway analysis indicated that the growth-promoting mechanisms of these beneficial genera were closely related to metabolic pathways such as amino acid metabolism, melatonin biosynthesis, aerobactin biosynthesis and so on. This study provides field observation and experimental evidence to reveal the close relationship between beneficial rhizosphere microbes and plant productivity under the M × R interaction effect.
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BACKGROUND: Novel ADC drugs provide a new therapeutic strategy for gastric cancer.The present study aimed to analyze the clinical efficacy and drug toxicities of disitamab vedotin (RC48) plus immune checkpoint inhibitors(ICIs) and RC48 as third-line therapies and beyond for advanced and metastatic gastric cancer patients. METHODS: This was an observational multicenter real-world study.From August 2021 to January 2022,patients with HER2-positive or HER2-low advanced and metastatic gastric cancer and failed from two or more lines of prior therapy were enrolled and treated with RC48 plus ICIs or RC48. In this study, progression free survival(PFS) was the primary end point. Other evaluation indicators were objective response rate(ORR),disease control rate(DCR),overall survival(OS) and drug toxicities. RESULTS: 45 patients were enrolled,of which 25 patients received RC48 plus ICIs,20 patients received RC48.Patients who received RC48 plus ICIs obtained better ORR (36.0% vs. 10.0%, P = 0.044) and DCR (80.0% vs. 50.0%, P = 0.034) compared with RC48,and simultaneously,the median PFS in RC48 plus ICIs group were superior to RC48 group(6.2 m vs. 3.9 m).The median OS was not reached.No statistically differences were found between HER2-positive and HER2-low group with respect to ORR (27.3% vs. 16.7%, P = 0.464),DCR (66.7% vs. 66.7%, P = 1.000),median PFS(5.7 m vs. 4.3 m, P = 0.299).The most common adverse events (AEs) were decreased white blood count,decreased neutrophil count,fatigue,hypoaesthesia and alopecia.Grade 3-4 AEs occurred in 7(35.0%) patients of RC48 group and 10(40.0%) patients of RC48 plus ICIs group,respectively. CONCLUSION: Compared with RC48 monotherapy, ICIs plus RC48 demonstrated superior third-line and beyond therapeutic efficacy for HER2-positive or HER2-low advanced and metastatic gastric cancer patients with manageable safety.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Neoplasias Esplênicas , Neoplasias Gástricas , Humanos , Inibidores de Checkpoint Imunológico , Neoplasias Gástricas/tratamento farmacológico , AlopeciaRESUMO
MicroRNAs (miRNAs) perform critical functions in normal physiology and disease by associating with Argonaute proteins and downregulating partially complementary messenger RNAs (mRNAs). Here we use clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) genome-wide loss-of-function screening coupled with a fluorescent reporter of miRNA activity in human cells to identify new regulators of the miRNA pathway. By using iterative rounds of screening, we reveal a novel mechanism whereby target engagement by Argonaute 2 (AGO2) triggers its hierarchical, multi-site phosphorylation by CSNK1A1 on a set of highly conserved residues (S824-S834), followed by rapid dephosphorylation by the ANKRD52-PPP6C phosphatase complex. Although genetic and biochemical studies demonstrate that AGO2 phosphorylation on these residues inhibits target mRNA binding, inactivation of this phosphorylation cycle globally impairs miRNA-mediated silencing. Analysis of the transcriptome-wide binding profile of non-phosphorylatable AGO2 reveals a pronounced expansion of the target repertoire bound at steady-state, effectively reducing the active pool of AGO2 on a per-target basis. These findings support a model in which an AGO2 phosphorylation cycle stimulated by target engagement regulates miRNA:target interactions to maintain the global efficiency of miRNA-mediated silencing.