RESUMO
BACKGROUND: Human papillomavirus (HPV) infection has become an important etiological driver of oropharyngeal squamous cell carcinoma (OPSCC), leading to unique tumor characteristics. However, the interplay between HPV-associated tumor cells and tumor microenvironment (TME) remains an enigma. METHODS: We performed a single-cell RNA-sequencing (scRNA-seq) on HPV-positive (HPV+) and HPV-negative (HPVâ) OPSCC tumors, each for three samples, and one normal tonsil tissue. Ex vivo validation assays including immunofluorescence staining, cell line co-culture, and flow cytometry analysis were used to test specific subtypes of HPV+ tumor cells and their communications with T cells. RESULTS: Through a comprehensive single-cell transcriptome analysis, we uncover the distinct transcriptional signatures between HPV+ and HPVâ OPSCC. Specifically, HPV+ OPSCC tumor cells manifest an enhanced interferon response and elevated expression of the major histocompatibility complex II (MHC-II), potentially bolstering tumor recognition and immune response. Furthermore, we identify a CXCL13+CD4+ T cell subset that exhibits dual features of both follicular and pro-inflammatory helper T cells. Noteworthily, HPV+ OPSCC tumor cells embrace extensive intercellular communications with CXCL13+CD4+ T cells. Interaction with HPV+ OPSCC tumor cells amplifies CXCL13 and IFNγ release in CD4+T cells, fostering a pro-inflammatory TME. Additionally, HPV+ tumor cells expressing high MHC-II and CXCL13+CD4+ T cell prevalence are indicative of favorable overall survival rates in OPSCC patients. CONCLUSIONS: Together, our study underscores a synergistic inflammatory immune response orchestrated by highly immunogenic tumor cells and CXCL13+CD4+ T cells in HPV+ OPSCC, offering useful insights into strategy development for patient stratification and effective immunotherapy in OPSCC.
Assuntos
Linfócitos T CD4-Positivos , Quimiocina CXCL13 , Imunoterapia , Neoplasias Orofaríngeas , Infecções por Papillomavirus , Microambiente Tumoral , Humanos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Quimiocina CXCL13/metabolismo , Quimiocina CXCL13/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Imunoterapia/métodos , Ativação Linfocitária , Neoplasias Orofaríngeas/imunologia , Neoplasias Orofaríngeas/virologia , Neoplasias Orofaríngeas/terapia , Papillomaviridae , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/virologia , Infecções por Papillomavirus/complicaçõesRESUMO
BACKGROUND: The fat mass and obesity-associated protein (FTO) is an RNA demethylase that contributes to several physiological processes. Nonetheless, the impact of FTO on bone remodeling in the midpalatal suture while undergoing rapid maxillary expansion (RME) remains unclear. METHODS: First, to explore the expression of FTO in the midpalatal suture during RME, six rats were randomly divided into two groups: Expansion group and Sham group (springs without being activated). Then, suture mesenchymal stem cells (SuSCs) were isolated as in vitro model. The expression of FTO was knocked down by small interfering RNA to study the effect of FTO on the osteogenic differentiation of SuSCs. Finally, to evaluate the function of FTO in the process of bone remodeling in the midpalatal suture, ten rats were randomly divided into two groups: FB23-2 group (10 µM, a small molecule inhibitor of FTO) and DMSO group (control). RESULTS: Increased arch width and higher expression of OCN and FTO in the midpalatal area were observed in expansion group (Pâ <â .05). In the in vitro model, the mRNA expression levels of Runx2, Bmp2, Col1a1, Spp1, and Tnfrsf11b were decreased (Pâ <â .05) upon knocking down FTO. Additionally, the protein levels of RUNX2 and OPN were also decreased (Pâ <â 0.05). Adding FB23-2, a small-molecule inhibitor targeting FTO, to the medium of SuSCs caused a decrease in the mRNA expression levels of Runx2, Bmp2, Col1a1, Spp1, and Tnfrsf11b (Pâ <â 0.05). There was a statistically significant difference in evaluating the expression of OCN and OPN on the palatal suture between the FB23-2 and DMSO groups (Pâ <â .05). LIMITATION: The molecular mechanisms by which FTO regulates SuSCs osteogenesis remain to be elucidated. The FTO conditional knock out mouse model can be established to further elucidate the role of FTO during RME. CONCLUSION: FTO contributes to the osteogenic differentiation of SuSCs and plays a promoting role in midpalatal suture bone remodeling during the RME.
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Dioxigenase FTO Dependente de alfa-Cetoglutarato , Técnica de Expansão Palatina , Animais , Ratos , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Remodelação Óssea , Subunidade alfa 1 de Fator de Ligação ao Core , Dimetil Sulfóxido , Osteogênese , RNA MensageiroRESUMO
Although similar to trunk and limb skeletal muscles, masticatory muscles are believed as unique in both developmental origins and myogenesis. Gαi2 has been demonstrated to promote muscle hypertrophy and muscle satellite cell differentiation in limb muscles. However, the effect of Gαi2 on masticatory muscles is still unexplored. This study aimed to identify the role of Gαi2 in the proliferation and differentiation of masticatory muscle satellite cells, further exploring the metabolic mechanism of masticatory muscles. The proliferation rate, myotube size, fusion index of masticatory muscle satellite cells and Pax7, Myf5, MyoD, Tcf21 and Musculin expressions were significantly decreased by Gαi2 knockdown, while in cells infected with AdV4-Gαi2, the proliferation rate, myotube size, fusion index and Tbx1 expression were significantly increased. Masticatory muscle satellite cells also displayed phenotype transformation as Gαi2 changed. In addition, Gαi2 altered myosin heavy chain (MyHC) isoforms of myotubes with less MyHC-2A expression in siGαi2 group and more MyHC-slow expression in AdV4-Gαi2 group. In conclusion, Gαi2 could positively affect the adult myogenesis of masticatory muscle satellite cells and maintain the superiority of MyHC-slow. Masticatory muscle satellite cells may have their unique Gαi2-regulated myogenic transcriptional networks, although they may share some common characteristics with trunk and limb muscles.
Assuntos
Células Satélites de Músculo Esquelético , Células Satélites de Músculo Esquelético/metabolismo , Células Cultivadas , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Cadeias Pesadas de Miosina/genética , Diferenciação Celular/genética , Desenvolvimento Muscular/genéticaRESUMO
OBJECTIVE: Apical periodontitis is the most frequently occurring pathological lesion. Fat mass and obesity-associated protein (Fto) is the first identified RNA N6-methyladenosine demethylase. However, whether Fto regulates apical periodontitis remains unclear. This study aimed to explore the mechanisms of Fto in the tumor necrosis factor-α (TNF-α)-induced inflammatory response. MATERIALS AND METHODS: We established an apical periodontitis model. An immortalized cementoblast cell line (OCCM-30) cells were exposed to TNF-α. Fto, Il6, Mcp1, and Mmp9 expressions were assessed by qRT-PCR. We knocked down Fto using lentiviruses and detected TNF-α-induced inflammation-related gene expressions and mRNA stability. RESULTS: Mice with apical periodontitis showed downregulation of Fto expression. OCCM-30 cells exposed to TNF-α showed an upregulation of inflammation-related genes with a decrease in Fto. Furthermore, knockdown of Fto promoted the expressions of Il6, Mcp1, and Mmp9 in TNF-α-treated OCCM-30 cells as compared with negative control cells, whereas it did not affect the mRNA stability. Interestingly, Fto knockdown activated the p65, p38, and ERK1/2 pathways, and it slightly activated the JNK signaling pathway after TNF-α administration in OCCM-30 cells. CONCLUSION: A TNF-α-induced decrease in the expression of Fto might play a critical role in the inflammatory response in cementoblasts, and knockdown of Fto might upregulate the inflammatory response.
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Cemento Dentário , Periodontite Periapical , Camundongos , Animais , Fator de Necrose Tumoral alfa/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , NF-kappa B/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Interleucina-6/metabolismo , Inflamação/metabolismo , Periodontite Periapical/metabolismoRESUMO
BACKGROUND AND OBJECTIVES: Peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, a master regulator of mitochondrial biogenesis and oxidative metabolism, has been associated with many inflammatory diseases. However, little is known about the function and mechanism of PGC-1α in cementoblasts under periodontitis. Our study aimed to investigate the effects of PGC-1α in immortalized cementoblast cell line OCCM-30 under TNF-α stimulation. MATERIALS AND METHODS: OCCM-30 cells were cultured and exposed to TNF-α, and PGC-1α expression was assessed by Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Chemical inhibitors targeting various signaling pathways including NF-κB, p38 MAPK, Akt, and p53 were used to identify the regulatory mechanism involved. ZLN005 was used to upregulate PGC-1α and the subsequent alteration of inflammatory cytokines expression under TNF-α stimulation were examined by qRT-PCR and Elisa. PGC-1α siRNA was employed to further verify the role of PGC-1α in inflammatory response. Dual-reporter gene assays were performed to examine the transcriptional activity of p65, and the phosphorylation level of p65 was evaluated by western blotting. Immunofluorescence assays and nuclear and cytoplasmic extractions were performed to check the nuclear translocation of p65. Coimmunoprecipitation studies were also performed to check whether there is direct binding between p65 and PGC-1α. RESULTS: TNF-α suppressed PGC-1α expression in OCCM-30 cells. Blocking p38 MAPK pathways restored the expression of PGC-1α. ZLN005 can upregulate PGC-1α in OCCM-30 cells. The upregulation of PGC-1α by ZLN005 inhibited TNF-α-induced proinflammatory cytokine expression, which was impaired by the transfection of PGC-1α siRNA. Knocking down PGC-1α also partially restored the ZLN005-decreased transcriptional activity of p65. However, the phosphorylation level and nuclear translocation of p65 were not significantly affected by PGC-1α. It was found that p65 was bound to PGC-1α in OCCM-30 cells stimulated by TNF-α, and the binding was increased upon ZLN005 treatment. CONCLUSIONS: PGC-1α can attenuate TNF-α-induced inflammatory responses in OCCM-30 cells.
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NF-kappa B , Fator de Necrose Tumoral alfa , NF-kappa B/metabolismo , RNA Interferente Pequeno , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
OBJECTIVE: Cementum which is a layer of thin and bone-like mineralised tissue covering tooth root surface is deposited and mineralised by cementoblasts. Recent studies suggested long noncoding RNA H19 (H19) promotes osteoblast differentiation and matrix mineralisation, however, the effect of H19 on cementoblasts remains unknown. This study aimed to clarify the regulatory effects of H19 on cementoblast differentiation, mineralisation, and proliferation. MATERIAL AND METHODS: An immortalised murine cementoblast cell line OCCM-30 was used in this study. H19 expression was examined by real-time quantitative polymerase chain reaction (RT-qPCR) during OCCM-30 cell differentiation. OCCM-30 cells were transfected with lentivirus or siRNA to up-regulate or down-regulate H19, then the levels of runt-related transcription factor 2 (Runx2), osterix (Sp7), alkaline phosphatase (Alpl), bone sialoprotein (Ibsp), osteocalcin (Bglap) were tested by RT-qPCR or western blot. Alizarin red staining, ALP activity assay and MTS assay were performed to determine the mineralisation and proliferation ability of OCCM-30 cells. RESULTS: H19 was dramatically increased during OCCM-30 cell differentiation. Overexpression of H19 increased the levels of Runx2, Sp7, Alpl, Ibsp, and Bglap and enhanced ALP activity and the formation of mineral nodules. While down-regulation of H19 suppressed the above cementoblast differentiation genes and inhibited ALP activity and mineral nodule formation. However, the proliferation of OCCM-30 cells was not affected. CONCLUSIONS: H19 promotes the differentiation and mineralisation of cementoblasts without affecting cell proliferation.
Assuntos
Cemento Dentário , RNA Longo não Codificante , Animais , Diferenciação Celular , Proliferação de Células , Sialoproteína de Ligação à Integrina , Camundongos , RNA Longo não Codificante/genéticaRESUMO
Bioorthogonal chemistry has been considered as a powerful tool for biomolecule labeling due to its site specificity, moderate reaction conditions, high yield, and simple post-treatment. Covalent coupling is commonly used to modify quantum dots (QDs) with bioorthogonal functional group (azide or cycloalkyne), but it has a negative effect in the decrease of QDs' quantum yield and stability and increase of QDs' hydrodynamic diameter. To overcome these disadvantages, we propose a novel method for the preparation of two kinds of clickable QDs by the strong interaction of -SH with metal ions. One system involves azide-DNA-functionalized QDs, which are used for bioconjugation with dibenzocyclooctyne (DBCO)-modified glucose oxidase (GOx) to form a GOx-QDs complex. After bioconjugation, the stability of QDs was improved, and the activity of GOx was also enhanced. The GOx-QDs complex was used for rapid detection of blood glucose by spectroscopy, naked eye, and paper-based analytical devices. The second system involves DBCO-DNA-functionalized QDs, which are used for an in situ bioorthogonal labeling of HeLa cells through metabolic oligosaccharide engineering. Therefore, these clickable QDs based on DNA functionalization can be applied for rapid and effective labeling of biomolecules of interest.
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Técnicas Biossensoriais/métodos , Pontos Quânticos , Glicemia , Compostos de Cádmio/química , Diabetes Mellitus/sangue , Glucose/química , Glucose/metabolismo , Células HeLa , Humanos , Telúrio/química , Zinco/químicaRESUMO
When dealing with infectious pathogens, the risk of contamination or infection in the process of detecting them is nonnegligible. Separation-free detection will be beneficial in operation and safety. In this work, we proposed a DNAzyme walker for homogeneous and isothermal detection of enterovirus. The DNAzyme is divided into two inactivate subunits. When the subunit-conjugated antibody binds to the target virus, the activity of the DNAzyme recovers as a result of spatial proximity. The walker propels, and the fluorescence recovers. The final fluorescence intensity of the reaction mixture is related to the concentration of the target virus. The detection limit of this proposed method is 6.6 × 104 copies/mL for EV71 and 4.3 × 104 copies/mL for CVB3, respectively. Besides, this method was applied in detection of EV71 in clinical samples with a satisfactory result. The entire experiment is easy to operate, and the proposed method has great potential for practical use.
Assuntos
DNA Catalítico , Enterovirus Humano A , Enterovirus , Antígenos Virais , FluorescênciaRESUMO
BACKGROUND AND OBJECTIVE: Cementum is a part of the periodontium and anchors periodontal ligaments to the alveolar bone. Cementoblasts are responsible for the cementum formation via matrix deposition and subsequently mineralization. Thus, exploring novel mechanisms underlying the function of cementoblast contributes to the treatment of cementum damage. Recently, circRNA Lrp6 (circLRP6) has been of interest due to its active role in cell differentiation, but its potential role in cementoblast differentiation remains unclear. Herein, we attempted to elucidate the role of circLRP6 in cementoblast differentiation and clarify any associated mechanisms. MATERIAL AND METHODS: The mRNA expressions of circLRP6, miR-145a-5p, zinc finger E-box binding homeobox 2 (Zeb2), runt-related transcription factor 2 (Runx2), osteopontin (Opn), and bone sialoprotein (Bsp) were evaluated by qRT-PCR. The protein levels of Zeb2 were measured by Western blot. Bioinformatic analysis and dual-luciferase reporter assays were used to test the potential binding targets of miR-145a-5p. The differentiation potentials of the cementoblasts were assessed by Alkaline phosphatase (ALP) staining, ALP activity assay, Alizarin red S (ARS) staining, and quantification. RESULTS: In this study, circLRP6 was significantly upregulated in cementoblast differentiation. Furthermore, circLRP6 knockdown inhibited ALP levels, reduced calcium nodule formation and the expression of Runx2, Opn, and Bsp. Mechanically, bioinformatic analysis and dual-luciferase reporter assays confirmed miR-145a-5p was a potential binding target of circLRP6. miR-145a-5p can negatively regulate cementoblast differentiation. Subsequently, bioinformatic analysis and dual-luciferase reporter assays confirmed Zeb2 was a potential miR-145a-5p target. miR-145a-5p overexpression resulted in a downregulation of Zeb2. Furthermore, Zeb2 inhibition partially reversed the effect of circLRP6 during cementoblast differentiation. CONCLUSION: Taken together, circLRP6 appears to modulate cementoblast differentiation by antagonizing the function of miR-145a-5p, thereby increasing Zeb2. This study serves as a stepping stone for the potential development of an approach to promote cementum formation.
Assuntos
Cemento Dentário , MicroRNAs , Diferenciação Celular , MicroRNAs/genética , Ligamento Periodontal , RNA CircularRESUMO
Fluorescent labeling and dynamic tracking is a powerful tool for exploring virus infection mechanisms. However, for small-sized viruses, virus tracking studies are usually hindered by a lack of appropriate labeling methods that do not dampen virus yield or infectivity. Here, we report a universal strategy for labeling viruses with chemical dyes and Quantum dots (QDs). Enterovirus 71 (EV71) was produced in a cell line that stably expresses a mutant methionyl-tRNA synthetase (MetRS), which can charge azidonorleucine (ANL) to the methionine sites of viral proteins during translation. Then, the ANL-containing virus was easily labeled with DBCO-AF647 and DBCO-QDs. The labeled virus shows sufficient yield and no obvious decrease in infectivity and can be used for imaging the virus entry process. Using the labeled EV71, different functions of scavenger receptor class B, member 2 (SCARB2), and heparan sulfate (HS) in EV71 infection were comparatively studied. The cell entry process of a strong HS-binding EV71 strain was investigated by real-time dynamic visualization of EV71-QDs in living cells. Taken together, our study described a universal biocompatible virus labeling method, visualized the dynamic viral entry process, and reported details of the receptor usage of EV71.
Assuntos
Enterovirus/metabolismo , Pontos Quânticos/química , Receptores Virais/metabolismo , Animais , Azidas , Linhagem Celular , Chlorocebus aethiops , Enterovirus/genética , Enterovirus Humano A/genética , Enterovirus Humano A/metabolismo , Células HeLa , Humanos , Norleucina/análogos & derivados , Receptores Depuradores/metabolismo , Células Vero , Proteínas Virais , Internalização do VírusRESUMO
Idiopathic pulmonary fibrosis (IPF) is a progressive disease characterized by excessive deposition of extracellular matrix (ECM) and chronic inflammation with limited therapeutic options. Psoralen, a major active component extracted from Psoralea corylifolia L. seed, has several biological effects. However, the role of psoralen in IPF is still unclear. Here, we hypothesized that psoralen played an essential role in IPF in the inhibition of fibroblast proliferation and inflammatory response. A murine model of IPF was established by injecting bleomycin (BLM) intratracheally, and psoralen was administered for 14 days from the 7th to 21st day after BLM injection. Our results demonstrated that psoralen treatment reduced body weight loss and improved the survival rate of mice with IPF. Histological and immunofluorescent examination showed that psoralen alleviated BLM-induced lung parenchymal inflammatory and fibrotic alteration. Furthermore, psoralen inhibited proliferation and collagen synthesis of mouse fibroblasts and partially reversed BLM-induced expression of α-smooth muscle actin at both the tissue and cell level. Moreover, psoralen decreased the expression of transforming growth factor-ß1, interleukin-1ß, and tumor necrosis factor-α in the lungs of BLM-stimulated mice. Our results reveale for the first time that psoralen exerts therapeutic effects against IPF in a BLM-induced murine model.
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OBJECTIVE: miR-146a is widely induced during the immune response. However, little is known about the biogenesis, function and mechanism of miR-146a in cementoblasts during the pathogenesis of periodontitis. This study aimed to investigate the effects of miR-146a in murine cementoblast-derived OCCM-30 cells following IL-1ß stimulation. MATERIALS AND METHODS: OCCM-30 cells were cultured and exposed to IL-1ß. IL-6, IL-1ß and TNF-α, and miR-146a-5p expression was assessed by qRT-PCR. Mimics/inhibitors were transiently transfected into cells to determine the function of miR-146a-5p. Signalling pathways including p38 MAPK, ERK1/2 and NF-κB were studied by using specific inhibitors. The indicated proteins were measured by Western blot analysis and ELISA. RESULTS: In IL-1ß-stimulated OCCM-30 cells, the expression levels of miR-146a-5p along with IL-6 and IL-1ß increased in a time-dependent manner. The ERK1/2, p38 MAPK and NF-κB pathway were activated upon IL-1ß stimulation. Blocking the NF-κB pathway decreased IL-6, IL-1ß and miR-146a-5p expression. The overexpression of miR-146a-5p reduced IL-6 and IL-1ß expression, while the inhibition of miR-146a-5p increased IL-6 and IL-1ß expression in IL-1ß-treated OCCM-30 cells. miR-146a-5p attenuated IL-6 and IL-1ß expression via the IRAK1/TRAF6 pathway. CONCLUSION: This study suggested that miR-146a-5p attenuates IL-1ß-induced inflammatory factors in cementoblast-derived cell line.
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OBJECTIVE: Irisin is a newly identified exercise-induced myokine which can affect glucose metabolism and cortical bone mass and strength. However, the influence of irisin on cementoblasts remains largely unknown. MATERIAL AND METHODS: An immortalized mouse cementoblast cell line OCCM-30 was used in this study. Cementoblast differentiation markers and PGC-1α in cells cultured with mineral induction medium were evaluated by qRT-PCR. Cementoblast mineralization was evaluated by alizarin red staining. Differentiation markers and the activity of p38 MAPK pathway under irisin stimulation were assessed by qRT-PCR or Western blot analysis. p38 MAPK pathway inhibitor SB203580 or p38 siRNA was used to further identify the regulatory mechanism. Cell proliferation treated with irisin was examined by CCK-8 method. RESULTS: The expression of Runx2, osterix, ALP, and PGC-1α was up-regulated consistently under mineral induction. The formation of mineralized nodules was increased by irisin. Runx2, osterix, ALP, and osteocalcin were obviously up-regulated under irisin stimulation as well as the activity of p38 MAPK pathway. When pretreated with SB203580 or p38 siRNA before irisin stimulation, the irisin-induced differentiation was distinctly suppressed. OCCM-30 cell proliferation was enhanced when treated with high-dose irisin for long time. CONCLUSION: Irisin can promote the differentiation of cementoblasts via p38 MAPK pathway.
Assuntos
Diferenciação Celular , Cemento Dentário , Fibronectinas , Proteínas Quinases p38 Ativadas por Mitógeno , Animais , Proliferação de Células , Fibronectinas/farmacologia , CamundongosRESUMO
Detection of viruses with high sensitivity is critical for the prevention and treatment of the related disease. Two homogeneous target-induced cascade amplification methods were proposed for the detection of enterovirus 71 and coxsackievirus B3. These methods both employ DNAzyme but differ in the way in which the DNAzyme is amplified. In the hybridization chain reaction (HCR)-based strategy, the DNAzyme is assembled by hairpin DNA strands, while in the rolling circle amplification (RCA)-based strategy, the DNAzyme is synthesized by the polymerase. On the basis of the virion structure, we investigated the effects of using only VP1-antibody or VP1-antibody and VP2-antibody on the detection. And the combination of two kinds of antibodies was found to further improve the performance of the detection. Subsequently, the simultaneous detection of EV71 and CVB3 was achieved by the RCA-based strategy. And the proposed methods were also applied in clinical samples analysis with a satisfactory result, showing great potential for applications in virus detection.
Assuntos
DNA Catalítico/biossíntese , Enterovirus Humano A/isolamento & purificação , Enterovirus Humano B/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Anticorpos Antivirais , DNA Catalítico/metabolismo , HumanosRESUMO
In this work, a three-dimensional DNA machine based on the isothermal strand-displacement polymerase reaction (ISDPR) has been constructed. The walking behavior of a DNA walker on the obstructive surface of magnetic beads has also been studied by adding different nucleic acid blocks. The "leg" of the DNA walker could hybridize with a hairpin structure DNA named H1 and lead to the opening of it. And the newly exposed stem would interact with a primer. A strand exchange has happened with the assistance of polymerase and dNTPs, so that the "leg" has been displaced and the DNA walker could be pushed to move on the surface. But the nucleic acid blocks could increase steric hindrance and obstruct this process, which is similar to the behavior of human beings walking on craggy paths. Through changing these blocks, such as the structure, the amount, and the length of blocks, the movement of the DNA walker has been controlled. What's more, the results of its application for DNA detection are satisfactory. The limit of detection is 21.6 pM. Also, this method has been successfully applied in complex biological samples. Graphical abstract á .
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DNA/análise , DNA/química , Imãs , Primers do DNA/química , Eletroforese em Gel de Ágar , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/métodos , Conformação de Ácido NucleicoRESUMO
A fluorometric method is described for the determination of the tumor biomarker mucin 1 (MUC1). It is based on signal amplification of the hybridization chain reaction (HCR), and the interaction between a luminescent ruthenium(II) complex and CdZnTeS quantum dots (QDs). If MUC1 bind to the biotin-labeled aptamer, it will initiate HCR with hairpins H1 and H2 to form a long-range dsDNA. The long nucleic acid chains are then linked on the surface of streptavidin-modified magnetic microparticles (MMPs) through streptavidin-biotin interaction. The luminescent ruthenium(II) complex is then embedded in the long dsDNA linked to the MMPs. Hence, there is little Ru complex in the supernatant after magnetic separation, and the fluorescence of the CdZnTeS QDs (best measured at excitation/emission wavelengths of 350/530 nm) is only slightly quenched. In the absence of target, the fluorescence of the CdZnTeS QDs is strongly quenched. Fluorescence increases linearly in the 0.2-100 ng·mL-1 MUC1 concentration range, and the LOD is 0.13 ng·mL-1 (at S/N = 3). The method was applied to the determination of MUC1 in spiked human serum samples. Graphical abstract A fluorometric turn-on aptasensor for mucin 1 is described that is based on the interaction between a Ru(II) complex and quantum dots (QDs). The detection system includes biotin-labeled aptamer-H0, hairpins H1 and H2, streptavidin-modified magnetic microparticles (MMPs), Ru(bpy)2(dppx)2+ and CdZnTeS QDs.
Assuntos
Aptâmeros de Nucleotídeos/química , Complexos de Coordenação/química , Corantes Fluorescentes/química , Mucina-1/sangue , Pontos Quânticos/química , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Técnicas Biossensoriais/métodos , DNA/química , DNA/genética , Humanos , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico , Hibridização de Ácido Nucleico , Rutênio/química , Espectrometria de Fluorescência/métodosRESUMO
Autophagy is a self-digestive process that eliminates impaired or aged proteins and potentially toxic intracellular components to maintain homeostasis. We previously demonstrated that TNF-α played a critical role in cementoblast differentiation, mineralization and apoptosis; however, the effect of TNF-α on cementoblast autophagy has remained unclear. In this study, an elevated immunofluorescence signal of LC3B and autophagic vacuoles, autophagosomes and autolysosomes were detected under TNF-α stimulation in OCCM-30 cells. Autophagy-related genes and proteins, Beclin-1, LC3A and Atg-5, were significantly upregulated by TNF-α in a time- and concentration-dependent manner. During this process, the activity of Stat3 was dramatically enhanced and when the activity of Stat3 was blocked by either a specific chemical inhibitor or siRNA transfection before TNF-α stimulation, the TNF-α-induced upregulation of autophagy-related genes and proteins was strongly inhibited. Our results suggest that TNF-α induced autophagy in cementoblasts was dependent, or partially dependent on the activity of Stat3 signaling pathway.
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Autofagia/efeitos dos fármacos , Cemento Dentário/citologia , Cemento Dentário/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/farmacologia , Animais , Linhagem Celular , Cemento Dentário/efeitos dos fármacos , Cemento Dentário/ultraestrutura , CamundongosRESUMO
The uncertainties of China's gross primary productivity (GPP) estimates by global data-oriented products and ecosystem models justify a development of high-resolution data-oriented GPP dataset over China. We applied a machine learning algorithm developing a new GPP dataset for China with 0.1° spatial resolution and monthly temporal frequency based on eddy flux measurements from 40 sites in China and surrounding countries, most of which have not been explored in previous global GPP datasets. According to our estimates, mean annual GPP over China is 6.62 ± 0.23 PgC/year during 1982-2015 with a clear gradient from southeast to northwest. The trend of GPP estimated by this study (0.020 ± 0.002 PgC/year2 from 1982 to 2015) is almost two times of that estimated by the previous global dataset. The GPP increment is widely spread with 60% area showing significant increasing trend (p < .05), except for Inner Mongolia. Most ecosystem models overestimated the GPP magnitudes but underestimated the temporal trend of GPP. The monsoon affected eastern China, in particular the area surrounding Qinling Mountain, seems having larger contribution to interannual variability (IAV) of China's GPP than the semiarid northwestern China and Tibetan Plateau. At country scale, temperature is the dominant climatic driver for IAV of GPP. The area where IAV of GPP dominated by temperature is about 42%, while precipitation and solar radiation dominate 31% and 27% respectively over semiarid area and cold-wet area. Such spatial pattern was generally consistent with global GPP dataset, except over the Tibetan Plateau and northeastern forests, but not captured by most ecosystem models, highlighting future research needs to improve the modeling of ecosystem response to climate variations.
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Agricultura/tendências , Mudança Climática , Ecossistema , China , Temperatura , IncertezaRESUMO
In this work, a simple and novel ratiometric fluorescence method based on ROX-DNA-functionalized CdZnTeS quantum dots (QDs) was developed for the detection of dopamine (DA) and tyrosinase (TYR). A ratiometric fluorescent probe was constructed by binding phosphorothioate DNA to the metal ions of QDs, which is a modification-free and low-cost method. DA was easily oxidized to DA quinone under the catalysis of TYR by dissolved O2, which effectively quenched the fluorescence of the QDs. Strong linear correlations were achieved for TYR in the range of 10.0-100.0 ng mL-1 and for DA in the range of 10.0-1000.0 nM. The limit of detection was estimated to be as low as 1.05 ng mL-1 for TYR and 1.93 nM for DA. Moreover, various colors were displayed in the course of detection, which could be observed by the naked eye. Therefore, an on-site and sensitive fluorescence method for the visual detection of DA and TYR can be developed. In addition, the findings revealed the potential applicability of the ratiometric fluorescent probe for the detection of DA and TYR in human serum. This ratiometric fluorescence method is not only sensitive and selective but also rapid and convenient for the detection of the analytes without sophisticated instrumentation.
Assuntos
Técnicas Biossensoriais/métodos , Dopamina/sangue , Corantes Fluorescentes/química , Monofenol Mono-Oxigenase/sangue , Cádmio/química , DNA/química , Corantes Fluorescentes/síntese química , Humanos , Limite de Detecção , Tamanho da Partícula , Pontos Quânticos/química , Sulfetos/química , Telúrio/química , Zinco/químicaRESUMO
OBJECTIVES: Thoracoscopic segmentectomy is the recommended treatment option for small peripheral pulmonary nodules. To assess the ability of preoperative three-dimensional (3D) reconstruction computed tomography (CT) to shorten the operative time and improve perioperative outcomes in thoracoscopic segmentectomy compared with standard chest CT, we conducted this randomized controlled trial. METHODS: The DRIVATS study was a multicentre, randomized controlled trial conducted in 3 hospitals between July 2019 and November 2023. Patients with small peripheral pulmonary nodules not reaching segment borders were randomized in a 1:1 ratio to receive either 3D reconstruction CT or standard chest CT before thoracoscopic segmentectomy. The primary end-point was operative time. The secondary end-points included incidence of postoperative complications, intraoperative blood loss and operative accident event. RESULTS: A total of 191 patients were enrolled in this study: 95 in the 3D reconstruction CT group and 96 in the standard chest CT group. All patients underwent thoracoscopic segmentectomy except for 1 patient in the standard chest CT group who received a wedge resection. There is no significant difference in operative time between the 3D reconstruction CT group (median, 100 min [interquartile range (IQR), 85-120]) and the standard chest CT group (median, 100 min [IQR, 81-140]) (P = 0.82). Only 1 intraoperative complication occurred in the standard chest CT group. No significant difference was observed in the incidence of postoperative complications between the 2 groups (P = 0.52). Other perioperative outcomes were also similar. CONCLUSIONS: In patients with small peripheral pulmonary nodules not reaching segment borders, the use of 3D reconstruction CT in thoracoscopic segmentectomy was feasible, but it did not result in significant differences in operative time or perioperative outcomes compared to standard chest CT.