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An effective tool to assess embryo quality in the assisted reproduction clinical practice will enhance successful implantation rates and mitigate high risks of multiple pregnancies. Potential biomarkers secreted into culture medium (CM) during embryo development enable rapid and noninvasive methods of assessing embryo quality. However, small volumes, low biomolecule concentrations, and impurity interference collectively preclude the identification of quality-related biomarkers in single blastocyst CM. Here, we developed a noninvasive trace multiomics approach to screen for potential markers in individual human blastocyst CM. We collected 84 CM samples and divided them into high-quality (HQ) and low-quality (LQ) groups. We evaluated the differentially expressed proteins (DEPs) and metabolites (DEMs) in HQ and LQ CM. A total of 504 proteins and 189 metabolites were detected in individual blastocyst CM. Moreover, 9 DEPs and 32 DEMs were identified in different quality embryo CM. We also categorized HQ embryos into positive implantation (PI) and negative implantation (NI) groups based on ultrasound findings on day 28. We identified 41 DEPs and 4 DEMs associated with clinical implantation outcomes in morphologically HQ embryos using a multiomics analysis approach. This study provides a noninvasive multiomics analysis technique and identifies potential biomarkers for clinical embryo developmental quality assessment.
Assuntos
Biomarcadores , Meios de Cultura , Metabolômica , Proteômica , Humanos , Biomarcadores/metabolismo , Biomarcadores/análise , Proteômica/métodos , Metabolômica/métodos , Meios de Cultura/química , Meios de Cultura/metabolismo , Blastocisto/metabolismo , Blastocisto/citologia , Técnicas de Cultura Embrionária , Embrião de Mamíferos/metabolismo , MultiômicaRESUMO
Tumor-derived extracellular vesicles (EVs) carry tumor-specific proteins and RNAs, thus becoming prevalent targets for early cancer diagnosis. However, low expression of EV cargos and insufficient diagnostic power of individual biomarkers hindered EVs application in clinical practice. Herein, we propose a multiplex Codetection platform of proteins and RNAs (Co-PAR) for EVs. Co-PAR adopted a pair of antibody-DNA probes to recognize the same target protein, which in turn formed a double-stranded DNA. Thus, the target protein could be quantified by detecting the double-stranded DNA via qPCR. Meanwhile, qRT-PCR simultaneously quantified the target RNAs. Thus, with a regular qPCR instrument, Co-PAR enabled the codetection of multiplex proteins and RNAs, with the sensitivity of 102 EVs/µL (targeting CD63) and 1 EV/µL (targeting snRNA U6). We analyzed the coexpressions of three protein markers (CD63, GPC-1, HER2) and three RNA markers (snRNA U6, GPC-1 mRNA, miR-10b) on EVs from three pancreatic cell lines and 30 human plasma samples using Co-PAR. The diagnostic accuracy of the 6-biomarker combination reached 92.9%, which was at least 6.2% higher than that of 3-biomarker combinations and at least 13.5% higher than that of 6 single biomarkers. Co-PAR, as a multiparameter detection platform for EVs, has great potential in early disease diagnosis.
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Biomarcadores Tumorais , Detecção Precoce de Câncer , Vesículas Extracelulares , Neoplasias Pancreáticas , Humanos , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/metabolismo , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/análise , RNA/análise , Linhagem Celular TumoralRESUMO
Appropriate DNA end synapsis, regulated by core components of the synaptic complex including KU70-KU80, LIG4, XRCC4, and XLF, is central to non-homologous end joining (NHEJ) repair of chromatinized DNA double-strand breaks (DSBs). However, it remains enigmatic whether chromatin modifications can influence the formation of NHEJ synaptic complex at DNA ends, and if so, how this is achieved. Here, we report that the mitotic deacetylase complex (MiDAC) serves as a key regulator of DNA end synapsis during NHEJ repair in mammalian cells. Mechanistically, MiDAC removes combinatorial acetyl marks on histone H2A (H2AK5acK9ac) around DSB-proximal chromatin, suppressing hyperaccumulation of bromodomain-containing protein BRD4 that would otherwise undergo liquid-liquid phase separation with KU80 and prevent the proper installation of LIG4-XRCC4-XLF onto DSB ends. This study provides mechanistic insight into the control of NHEJ synaptic complex assembly by a specific chromatin signature and highlights the critical role of H2A hypoacetylation in restraining unscheduled compartmentalization of DNA repair machinery.
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Cromatina , Proteínas Nucleares , Animais , Cromatina/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , DNA/genética , Reparo do DNA por Junção de Extremidades , Histonas/genética , Histonas/metabolismo , Pareamento Cromossômico , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Mamíferos/metabolismoRESUMO
BACKGROUND: Abdominal aortic aneurysm (AAA) is a potentially life-threatening vascular condition, but approved medical therapies to prevent AAA progression and rupture are currently lacking. Sphingolipid metabolism disorders are associated with the occurrence and development of AAA. It has been discovered that ganglioside GM3, a sialic acid-containing type of glycosphingolipid, plays a protective role in atherosclerosis, which is an important risk factor for AAA; however, the potential contribution of GM3 to AAA development has not been investigated. METHODS: We performed a metabolomics study to evaluated GM3 level in plasma of human patients with AAA. We profiled GM3 synthase (ST3GAL5) expression in the mouse model of aneurysm and human AAA tissues through Western blotting and immunofluorescence staining. RNA sequencing, affinity purification and mass spectrometry, proteomic analysis, surface plasmon resonance analysis, and functional studies were used to dissect the molecular mechanism of GM3-regulating ferroptosis. We conditionally deleted and overexpressed St3gal5 in smooth muscle cells (SMCs) in vivo to investigate its role in AAA. RESULTS: We found significantly reduced plasma levels of GM3 in human patients with AAA. GM3 content and ST3GAL5 expression were decreased in abdominal aortic vascular SMCs in patients with AAA and an AAA mouse model. RNA sequencing analysis showed that ST3GAL5 silencing in human aortic SMCs induced ferroptosis. We showed that GM3 interacted directly with the extracellular domain of TFR1 (transferrin receptor 1), a cell membrane protein critical for cellular iron uptake, and disrupted its interaction with holo-transferrin. SMC-specific St3gal5 knockout exacerbated iron accumulation at lesion sites and significantly promoted AAA development in mice, whereas GM3 supplementation suppressed lipid peroxidation, reduced iron deposition in aortic vascular SMCs, and markedly decreased AAA incidence. CONCLUSIONS: Together, these results suggest that GM3 dysregulation promotes ferroptosis of vascular SMCs in AAA. Furthermore, GM3 may constitute a new therapeutic target for AAA.
Assuntos
Aneurisma da Aorta Abdominal , Ferroptose , Humanos , Camundongos , Animais , Gangliosídeo G(M3)/metabolismo , Proteômica , Músculo Liso Vascular/metabolismo , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/prevenção & controle , Aneurisma da Aorta Abdominal/metabolismo , Ferro , Miócitos de Músculo Liso/metabolismo , Modelos Animais de DoençasRESUMO
The oil spreading technique relies on biosurfactant to reduce the surface tension of an oil film and form an oil spreading ring in the center, and then judges the content of biosurfactant according to the diameter of the spreading ring. However, the instability and large errors of the traditional oil spreading technique limit its further application. In this paper, we modified the traditional oil spreading technique by optimizing the oily material, image acquisition and calculation method, which improves the accuracy and stability of the quantification of biosurfactant. We screened lipopeptides and glycolipid biosurfactants for rapid and quantitative analysis of biosurfactant concentrations. By selecting areas by color done by the software to modify image acquisition, the results showed that the modified oil spreading technique has a good quantitative effect, reflected in the concentration of biosurfactant being proportional to the diameter of the sample droplet. More importantly, using the pixel ratio method instead of the diameter measurement method to optimize the calculation method, the region selection was more exact, and the accuracy of the data results was high, and the calculation efficiency was improved significantly. Finally, the contents of rhamnolipid and lipopeptide in oilfield water samples were judged by the modified oil spreading technique, the relative errors were analyzed according to the different substances as the standard, and the quantitative measurement and analysis of oilfield water samples (the produced water of Zhan 3-X24 and the injected water of the estuary oil production plant) were realized. The study provides a new perspective on the accuracy and stability of the method in the quantification of biosurfactant, and provided some theoretical and data support for the study of the microbial oil displacement technology mechanism.
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The efficiency of homologous recombination (HR) in the repair of DNA double-strand breaks (DSBs) is closely associated with genome stability and tumor response to chemotherapy. While many factors have been functionally characterized in HR, such as TOPBP1, their precise regulation remains unclear. Here, we report that TOPBP1 interacts with the RNA-binding protein HTATSF1 in a cell-cycle- and phosphorylation-dependent manner. Mechanistically, CK2 phosphorylates HTATSF1 to facilitate binding to TOPBP1, which promotes S-phase-specific TOPBP1 recruitment to damaged chromatin and subsequent RPA/RAD51-dependent HR, genome integrity, and cancer-cell viability. The localization of HTATSF1-TOPBP1 to DSBs is potentially independent of the transcription-coupled RNA-binding and processing capacity of HTATSF1 but rather relies on the recognition of poly(ADP-ribosyl)ated RPA by HTATSF1, which can be blunted with PARP inhibitors. Together, our study provides a mechanistic insight into TOPBP1 loading at HR-prone DSB sites via HTATSF1 and reveals how RPA-RAD51 exchange is tuned by a PARylation-phosphorylation cascade.
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Poli ADP Ribosilação , Rad51 Recombinase , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Recombinação Homóloga/genética , Fosforilação , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismoRESUMO
BACKGROUND: Single-stranded DNA (ssDNA) coated with replication protein A (RPA) acts as a key platform for the recruitment and exchange of genome maintenance factors in DNA damage response. Yet, how the formation of the ssDNA-RPA intermediate is regulated remains elusive. RESULTS: Here, we report that the lamin-associated protein LAP2α is physically associated with RPA, and LAP2α preferentially facilitates RPA deposition on damaged chromatin via physical contacts between LAP2α and RPA1. Importantly, LAP2α-promoted RPA binding to ssDNA plays a critical role in protection of replication forks, activation of ATR, and repair of damaged DNA. We further demonstrate that the preference of LAP2α-promoted RPA loading on damaged chromatin depends on poly ADP-ribose polymerase PARP1, but not poly(ADP-ribosyl)ation. CONCLUSIONS: Our study provides mechanistic insight into RPA deposition in response to DNA damage and reveals a genome protection role of LAP2α.
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Cromatina , Proteína de Replicação A , Dano ao DNA , Reparo do DNA , Replicação do DNA , DNA de Cadeia Simples , Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/genética , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismoRESUMO
BACKGROUND: Metabolic syndrome is related to cardiovascular diseases, which is attributed in part, to arterial stiffness; however, the mechanisms remain unclear. The present study aimed to investigate the molecular mechanisms of metabolic syndrome-induced arterial stiffness and to identify new therapeutic targets. METHODS: Arterial stiffness was induced by high-fat/high-sucrose diet in mice, which was quantified by Doppler ultrasound. Four-dimensional label-free quantitative proteomic analysis, affinity purification and mass spectrometry, and immunoprecipitation and GST (glutathione S-transferase) pull-down experiments were performed to explore the mechanism of YAP (Yes-associated protein)-mediated TGF (transforming growth factor) ß pathway activation. RESULTS: YAP protein was upregulated in the aortic tunica media of mice fed a high-fat/high-sucrose diet for 2 weeks and precedes arterial stiffness. Smooth muscle cell-specific YAP knockdown attenuated high-fat/high-sucrose diet-induced arterial stiffness and activation of TGFß-Smad2/3 signaling pathway in arteries. By contrast, Myh11CreERT2-YapTg mice exhibited exacerbated high-fat/high-sucrose diet-induced arterial stiffness and enhanced TGFß-activated Smad2/3 phosphorylation in arteries. PPM1B (protein phosphatase, Mg2+/Mn2+-dependent 1B) was identified as a YAP-bound phosphatase that translocates into the nucleus to dephosphorylate Smads (mothers against decapentaplegic homologs) in response to TGFß. This process was inhibited by YAP through removal of the K63-linked ubiquitin chain of PPM1B at K326. CONCLUSIONS: This study provides a new mechanism by which smooth muscle cell YAP regulates the TGFß pathway and a potential therapeutic target in metabolic syndrome-associated arterial stiffness.
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Síndrome Metabólica , Rigidez Vascular , Animais , Dieta Hiperlipídica/efeitos adversos , Camundongos , Proteômica , Sacarose , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Rigidez Vascular/fisiologia , Proteínas de Sinalização YAPRESUMO
The DNA damage response (DDR) pathway generally protects against genome instability, and defects in DDR have been exploited therapeutically in cancer treatment. We have reported that histone demethylase PHF8 demethylates TOPBP1 K118 mono-methylation (K118me1) to drive the activation of ATR kinase, one of the master regulators of replication stress. However, whether dysregulation of this physiological signalling is involved in tumorigenesis remains unknown. Here, we showed PHF8-promoted TOPBP1 demethylation is clinically associated with breast tumorigenesis and patient survival. Mammary gland tumors from Phf8 knockout mice grow slowly and exhibit higher level of K118me1, lower ATR activity, and increased chromosomal instability. Importantly, we found that disruption of PHF8-TOPBP1 axis suppresses breast tumorigenesis and creates a breast tumor-specific vulnerability to PARP inhibitor (PARPi) and platinum drug. CRISPR/Cas9 mutation modelling of the deleted or truncated mutation of PHF8 in clinical tumor samples demonstrated breast tumor cells expressing the mimetic variants are more vulnerable to PARPi. Together, our study supports the pursuit of PHF8-TOPBP1 signalling pathway as promising avenues for targeted therapies of PHF8-TOPBP1 proficient tumors, and provides proof-of-concept evidence for loss-of-function of PHF8 as a therapeutic indicator of PARPis.
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Neoplasias da Mama/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histona Desmetilases/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Feminino , Instabilidade Genômica/efeitos dos fármacos , Instabilidade Genômica/fisiologia , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Polylactic acid (PLA)/nano-TiO2(TiO2 NPs)/Graphene oxide (GO) nano-fibrous films were prepared by ultrasonic assisted electrostatic spinning technology, and the effects of TiO2 NPs:GO mass ratio and ultrasonic power on film morphology and mechanical, thermal, barrier and antibacterial properties were investigated. The addition of TiO2 NPs and GO can significantly increase the tensile strength and elongation at the break of PLA nano-fibrous films, and improve the water barrier properties of the nano-fibrous films. The antibacterial experiment showed that the inhibition rates of the nano-fibrous films against Escherichia coli and Staphylococcus aureus after 24 h exposure to UV irradiation reached 94.4 ± 1.8% and 92.6 ± 1.7% At the same time, the fresh-keeping packaging experiment of green peppers at room temperature, through the determination of hardness, soluble solids, chlorophyll content to determine the degree of decay of green pepper, it showed that PLA/TiO2 NPs/GO nano-fibrous films can better maintain the sensory quality of green peppers, delay the rate of spoilage of green peppers, and prolong the preservation period of green peppers.
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Antibacterianos/química , Antibacterianos/farmacologia , Capsicum/microbiologia , Grafite/química , Nanopartículas/química , Poliésteres/química , Titânio/química , Escherichia coli/efeitos dos fármacos , Conservação de Alimentos/métodos , Nanocompostos/química , Staphylococcus aureus/efeitos dos fármacosRESUMO
Rationale: A number of guanine nucleotide exchange factors (GEFs) including epithelial cell transforming factor ECT2 are believed to drive carcinogenesis through activating distinct oncogenic GTPases. Yet, whether GEF-independent activity of ECT2 also plays a role in tumorigenesis remains unclear. Methods: Immunohistochemical (IHC) staining, colony formation and xenograft assays were used to examine the role of ECT2 in breast carcinogenesis. Co-immunoprecipitation, immunofluorescent stainings, in vivo deubiquitination and in vitro deubiquitination experiments were performed to examine the physical and functional interaction between ECT2 and ubiquitin-specific protease USP7. High-throughput RNA sequencing, quantitative reverse transcription-PCR and Western blotting were employed to investigate the biological significance of the interplay between ECT2 and USP7. Results: We report that ECT2 plays a tumor-promoting role in breast cancer, and GEF activity-deficient ECT2 is able to alleviate ECT2 depletion associated growth defects in breast cancer cells. Mechanistically, we demonstrated that ECT2 physically interacts with ubiquitin-specific protease USP7 and functionally facilitates USP7 intermolecular self-association, -deubiquitination and -stabilization in a GEF activity-independent manner. USP7 in turn, deubiquitinates and stabilizes ECT2, resulting in a feedforward regulatory circuit that ultimately sustains the expression of oncogenic protein MDM2. Conclusion: Our study uncovers a GEF-independent role of ECT2 in promoting survival of breast cancer cells, provides a molecular insight for the reciprocal regulation of ECT2 and USP7, and supports the pursuit of ECT2/USP7 as potential targets for breast cancer intervention.