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With significant advancements in understanding gene functions and therapy, the potential misuse of gene technologies, particularly in the context of sports through gene doping (GD), has come to the forefront. This raises concerns regarding the need for point-of-care testing of various GD candidates to counter illicit practices in sports. However, current GD detection techniques, such as PCR, lack the portability required for on-site multiplexed detection. In this study, we introduce an integrated microfluidics-based chip for multiplexed gene doping detection, termed MGD-Chip. Through the strategic design of hydrophilic and hydrophobic channels, MGD-Chip enables the RPA and CRISPR-Cas12a assays to be sequentially performed on the device, ensuring minimal interference and cross-contamination. Six potential GD candidates were selected and successfully tested simultaneously on the platform within 1 h. Demonstrating exceptional specificity, the platform achieved a detection sensitivity of 0.1 nM for unamplified target plasmids and 1 aM for amplified ones. Validation using mouse models established by injecting IGFI and EPO transgenes confirmed the platform's efficacy in detecting gene doping in real samples. This technology, capable of detecting multiple targets using portable elements, holds promise for real-time GD detection at sports events, offering a rapid, highly sensitive, and user-friendly solution to uphold the integrity of sports competitions.
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Técnicas Biosensibles , Sistemas CRISPR-Cas , Doping en los Deportes , Interacciones Hidrofóbicas e Hidrofílicas , Dispositivos Laboratorio en un Chip , Sistemas CRISPR-Cas/genética , Doping en los Deportes/prevención & control , Animales , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Ratones , Humanos , Eritropoyetina/genética , Eritropoyetina/análisis , Diseño de Equipo , Proteínas Asociadas a CRISPR/genética , Proteínas Bacterianas , EndodesoxirribonucleasasRESUMEN
Bone metastasis is a lethal consequence of breast cancer. Here we used single-cell transcriptomics to investigate the molecular mechanisms underlying bone metastasis colonization-the rate-limiting step in the metastatic cascade. We identified that lymphotoxin-ß (LTß) is highly expressed in tumour cells within the bone microenvironment and this expression is associated with poor bone metastasis-free survival. LTß promotes tumour cell colonization and outgrowth in multiple breast cancer models. Mechanistically, tumour-derived LTß activates osteoblasts through nuclear factor-κB2 signalling to secrete CCL2/5, which facilitates tumour cell adhesion to osteoblasts and accelerates osteoclastogenesis, leading to bone metastasis progression. Blocking LTß signalling with a decoy receptor significantly suppressed bone metastasis in vivo, whereas clinical sample analysis revealed significantly higher LTß expression in bone metastases than in primary tumours. Our findings highlight LTß as a bone niche-induced factor that promotes tumour cell colonization and osteolytic outgrowth and underscore its potential as a therapeutic target for patients with bone metastatic disease.
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The cure rates for osteosarcoma have remained unchanged in the past three decades, especially for patients with pulmonary metastasis. Thus, a new and effective treatment for metastatic osteosarcoma is urgently needed. Anlotinib has been reported to have antitumor effects on advanced osteosarcoma. However, both the effect of anlotinib on autophagy in osteosarcoma and the mechanism of anlotinib-mediated autophagy in pulmonary metastasis are unclear. The effect of anlotinib treatment on the metastasis of osteosarcoma was investigated by transwell assays, wound healing assays, and animal experiments. Related proteins were detected by western blotting after anlotinib treatment, ATG5 silencing, or ATG5 overexpression. Immunofluorescence staining and transmission electron microscopy were used to detect alterations in autophagy and the cytoskeleton. Anlotinib inhibited the migration and invasion of osteosarcoma cells but promoted autophagy and increased ATG5 expression. Furthermore, the decreases in invasion and migration induced by anlotinib treatment were enhanced by ATG5 silencing. In addition, Y-27632 inhibited cytoskeletal rearrangement, which was rescued by ATG5 overexpression. ATG5 overexpression enhanced epithelial-mesenchymal transition (EMT). Mechanistically, anlotinib-induced autophagy promoted migration and invasion by activating EMT and cytoskeletal rearrangement through ATG5 both in vitro and in vivo. Our results demonstrated that anlotinib can induce protective autophagy in osteosarcoma cells and that inhibition of anlotinib-induced autophagy enhanced the inhibitory effects of anlotinib on osteosarcoma metastasis. Thus, the therapeutic effect of anlotinib treatment can be improved by combination treatment with autophagy inhibitors, which provides a new direction for the treatment of metastatic osteosarcoma.
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Neoplasias Óseas , Indoles , Neoplasias Pulmonares , Osteosarcoma , Quinolinas , Animales , Humanos , Proliferación Celular , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/patología , Autofagia , Transición Epitelial-Mesenquimal , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/patología , Citoesqueleto/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proteína 5 Relacionada con la Autofagia/farmacología , Proteína 5 Relacionada con la Autofagia/uso terapéuticoRESUMEN
In this study, we investigated Cas13a's efficacy in trans-cleaving RNA G-quadruplexes (rG4s) as an alternative to ssRNA reporters in CRISPR-Cas13a diagnostics. Our findings demonstrate enhanced efficiency due to the structural arrangement of rG4s. Implementing a simplified CRISPR-Cas13a system based on rG4, we identified SARS-CoV-2 infections in 25 patient samples within 1 hour without target pre-amplification.
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COVID-19 , G-Cuádruplex , Humanos , ARN/genética , ARN/química , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , COVID-19/diagnósticoRESUMEN
The emergence of graphene quantum dots (GQDs) expands the use of graphene derivatives in nanomedicine for its direct therapeutic applications in treating neurodegeneration, inflammation, metabolic dysfunction, and among others. Nevertheless, the biosafety assessment of GQDs remains deficient mostly because of the diverse surface characteristics of the nanoparticles. Our prior work demonstrated that GQDs can induce strong thigmotactic effects in zebrafish larvae over a wide range of concentrations, yet the underlying metabolic mechanisms remain largely unknown. In this study, we conducted a further exploration about graphene oxide quantum dots (GOQDs) for its potential neurotoxic effect on the behaviors of zebrafish larvae by combining neurotransmitter-targeted metabolomics with locomotion analysis. After continuous exposure to a concentration gradient of GOQDs (12.5 - 25 - 50 - 100 - 200 µg/mL) for 7 days, the thigmotactic activities of zebrafish larvae were observed across all exposure concentrations relative to the control group, while the basal locomotor activities, including distance moved and average velocity, were significantly changed by low concentrations of GOQDs. Targeted metabolomics was performed using zebrafish larvae at 7 days post-fertilization (dpf) that were exposed to 12.5 and 200 µg/mL, both of which were found to perturb the kynurenine pathway by regulating the levels of kynurenine, 3-hydroxyanthranilic acid (3-HAA), and quinolinic acid (QA). Furthermore, the thigmotaxis of larval fish induced by GOQDs during exposure could be counteracted by supplementing Ro-61-8048, an agonist acting on kynurenine 3-monooxygenase (KMO). In conclusion, our study establishes the involvement of the kynurenine pathway in GOQDs-induced thigmotaxis, which is independent of the transcriptional modulation of glutamate receptor families.
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Grafito , Puntos Cuánticos , Animales , Pez Cebra , Grafito/toxicidad , Puntos Cuánticos/toxicidad , Quinurenina/farmacología , LarvaRESUMEN
Abstract The cure rates for osteosarcoma have remained unchanged in the past three decades, especially for patients with pulmonary metastasis. Thus, a new and effective treatment for metastatic osteosarcoma is urgently needed. Anlotinib has been reported to have antitumor effects on advanced osteosarcoma. However, both the effect of anlotinib on autophagy in osteosarcoma and the mechanism of anlotinib-mediated autophagy in pulmonary metastasis are unclear. The effect of anlotinib treatment on the metastasis of osteosarcoma was investigated by transwell assays, wound healing assays, and animal experiments. Related proteins were detected by western blotting after anlotinib treatment, ATG5 silencing, or ATG5 overexpression. Immunofluorescence staining and transmission electron microscopy were used to detect alterations in autophagy and the cytoskeleton. Anlotinib inhibited the migration and invasion of osteosarcoma cells but promoted autophagy and increased ATG5 expression. Furthermore, the decreases in invasion and migration induced by anlotinib treatment were enhanced by ATG5 silencing. In addition, Y-27632 inhibited cytoskeletal rearrangement, which was rescued by ATG5 overexpression. ATG5 overexpression enhanced epithelial-mesenchymal transition (EMT). Mechanistically, anlotinib-induced autophagy promoted migration and invasion by activating EMT and cytoskeletal rearrangement through ATG5 both in vitro and in vivo. Our results demonstrated that anlotinib can induce protective autophagy in osteosarcoma cells and that inhibition of anlotinib-induced autophagy enhanced the inhibitory effects of anlotinib on osteosarcoma metastasis. Thus, the therapeutic effect of anlotinib treatment can be improved by combination treatment with autophagy inhibitors, which provides a new direction for the treatment of metastatic osteosarcoma.
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Photodynamic therapy (PDT) has shown great potential for tumor treatment as the method is noninvasive, highly selective, and causes minimal side effects. However, conventional type II PDT, which relies on 1O2, presents poor therapeutic efficacy for hypoxic tumors due to its reliance on oxygen. Here, CeO2/Ti3C2-MXene (CeO2@MXene) hybrids were successfully designed by growing CeO2in situ using Ti3C2-MXene (MXene) nanosheets. CeO2@MXene serves as a reduction-oxidation (REDOX) center due to the presence of Ce in the lattice of CeO2 nanoparticles. This REDOX center reacts with H2O2 to generate oxygen and weakens the hypoxic tumor cell environment, achieving type II PDT. At the same time, many other ROS (such as â O2- and â OH) can be produced via a type I photodynamic mechanism (electron transfer process). The CeO2@MXene heterojunction performs nanoenzymatic functions for synergistic type I and type II PDT, which improves cancer treatment.
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Neoplasias Óseas , Nitritos , Osteosarcoma , Elementos de Transición , Humanos , Peróxido de Hidrógeno , Hipoxia , OxígenoRESUMEN
The majority of patients with late-stage breast cancer develop distal bone metastases. The bone microenvironment can affect response to therapy, and uncovering the underlying mechanisms could help identify improved strategies for treating bone metastatic breast cancer. Here, we observed that osteoclasts reduced the sensitivity of breast cancer cells to DNA damaging agents, including cisplatin and the PARP inhibitor (PARPi) olaparib. Metabolic profiling identified elevated glutamine production by osteoclasts. Glutamine supplementation enhanced the survival of breast cancer cells treated with DNA damaging agents, while blocking glutamine uptake increased sensitivity and suppressed bone metastasis. GPX4, the critical enzyme responsible for glutathione oxidation, was upregulated in cancer cells following PARPi treatment through stress-induced ATF4-dependent transcriptional programming. Increased glutamine uptake and GPX4 upregulation concertedly enhanced glutathione metabolism in cancer cells to help neutralize oxidative stress and generate PARPi resistance. Analysis of paired patient samples of primary breast tumors and bone metastases revealed significant induction of GPX4 in bone metastases. Combination therapy utilizing PARPi and zoledronate, which blocks osteoclast activity and thereby reduces the microenvironmental glutamine supply, generated a synergistic effect in reducing bone metastasis. These results identify a role for glutamine production by bone-resident cells in supporting metastatic cancer cells to overcome oxidative stress and develop resistance to DNA-damaging therapies. SIGNIFICANCE: Metabolic interaction between osteoclasts and tumor cells contributes to resistance to DNA-damaging agents, which can be blocked by combination treatment with PARP and osteoclast inhibitors to reduce bone metastatic burden.
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Neoplasias Óseas , Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/patología , Osteoclastos/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Glutamina/farmacología , Neoplasias Óseas/secundario , ADN , Glutatión , Línea Celular Tumoral , Microambiente TumoralRESUMEN
Two-dimensional (2D) nanomaterials as drug carriers and photosensitizers have emerged as a promising antitumor strategy. However, our understanding of 2D antitumor nanomaterials is limited to intrinsic properties or additive modification of different materials. Subtractive structural engineering of 2D nanomaterials for better antitumor efficacy is largely overlooked. Here, subtractively engineered 2D MXenes with uniformly distributed nanopores are synthesized. The nanoporous defects endowed MXene with enhanced surface plasmon resonance effect for better optical absorbance performance and strong exciton-phonon coupling for higher photothermal conversion efficiency. In addition, porous structure improves the binding ability between drug and unsaturated bonds, thus promoting drug-loading capacity and reducing uncontrolled drug release. Furthermore, the porous structure provides adhesion sites for filopodia, thereby promoting the cellular internalization of the drug. Clinically, osteosarcoma is the most common bone malignancy routinely treated with doxorubicin-based chemotherapy. There have been no significant treatment advances in the past decade. As a proof-of-concept, nanoporous MXene loaded with doxorubicin is developed for treating human osteosarcoma cells. The porous MXene platform results in a higher amount of doxorubicin-loading, faster near-infrared (NIR)-controlled doxorubicin release, higher photothermal efficacy under NIR irradiation, and increased cell adhesion and internalization. This facile method pioneers a new paradigm for enhancing 2D material functions and is attractive for tumor treatment.
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Neoplasias Óseas , Nanoporos , Osteosarcoma , Humanos , Nanomedicina , Doxorrubicina/farmacología , Doxorrubicina/química , Osteosarcoma/tratamiento farmacológico , Fototerapia , Línea Celular TumoralRESUMEN
Graphene quantum dots (GQDs) recently gain much attention for its medicinal values in treating diseases such as neurodegeneration and inflammations. However, owing to the high permeability of GQDs across the blood-brain barrier, whether its retention in the central nervous system (CNS) perturbs neurobehaviors remains less reported. In the study, the locomotion of zebrafish larvae (Danio rerio) was fully evaluated when administrated by two GQDs in a concentration gradient, respectively as reduced-GQDs (R-GQDs): 150, 300, 600, 1200, and 2400 g/L, and graphene oxide QDs (GOQDs): 60, 120, 240, 480, and 960 g/L. After exposure, the larvae were kept for locomotion analysis within one week's depuration. Substantial data showed that the basal locomotor activity of zebrafish larvae was not significantly changed by both two GQDs at low concentrations while weakened greatly with the increase of concentrations, and the total ATP levels of zebrafish larvae were also found to decrease significantly when exposed to the highest concentrations of GQDs. Next, the thigmotactic effect was observed to be remarkably induced in larvae by both two GQDs at any concentrations during exposure, and remained strong in larvae treated by high concentrations of R-GQDs after 7 days' depuration. To be noted, we found that GQDs affected the synaptic plasticity via downregulating the mRNA levels of NMDA and AMPA receptor family members as well as the total glutamine levels in zebrafish larvae. Together, our study presented robust data underlying the locomotor abnormalities aroused by GQDs in zebrafish larvae and indicated the potential adverse effects of GQDs on synaptic plasticity.
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Grafito , Puntos Cuánticos , Animales , Pez Cebra , Puntos Cuánticos/toxicidad , Grafito/toxicidad , Larva , Plasticidad NeuronalRESUMEN
BACKGROUND: Pulmonary metastasis is the main cause of poor prognosis in osteosarcoma. Sialic acid-bound immunoglobulin lectin 15 (Siglec-15) has been demonstrated to be obviously correlated with pulmonary metastasis in osteosarcoma patients. However, the effect of Siglec-15 on autophagy in osteosarcoma remains unclear, while the role and mechanism of Siglec-15-related autophagy in lung metastasis also remain unknown. METHODS: The expression levels of Siglec-15 and Beclin-1 were detected in osteosarcoma tissues using immunohistochemistry (IHC). The effect of Siglec-15 on metastasis was investigated using Transwell, wound healing and animal experiments with osteosarcoma cells. Corresponding proteins were confirmed using Western blotting when Siglec-15 or Beclin-1 was silenced or overexpressed. Changes in autophagy and the cytoskeleton were detected using immunofluorescence and transmission electron microscopy. RESULTS: Siglec-15 and Beclin-1 expression was evaluated both in lung metastases and in patients who presented with pulmonary metastasis of osteosarcoma. Immunoprecipitation experiments revealed that Siglec-15 interacts directly with Beclin-1, an important autophagic protein. Moreover, loss of Siglec-15 distinctly inhibited autophagy and reduced Beclin-1/ATG14 expression. The decreased invasion and migration caused by Siglec-15 silencing could be reversed by Beclin-1 overexpression. Additionally, autophagy can promote the epithelial-mesenchymal transition (EMT) and affect cytoskeletal rearrangement, which was confirmed by overexpression or silencing of Beclin-1. CONCLUSIONS: These findings confirmed the role of Siglec-15 in the regulation of autophagy and elaborated the relationship and mechanisms between autophagy and the metastasis of osteosarcoma cells.
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GQDs show great potential in drug carriers, bioimaging, biosensors, theranostics, and are recently reported as promising therapeutic agents to treat amyloid-related diseases such as Parkinson's disease and inflammations such as colitis. However, current toxicity data about GQDs based on in vivo toxicity assessments remain scarce. In the study, we examined the mRNA expression changes of zebrafish embryos exposed to four types of GQDs, including raw graphene quantum dots (R-GQDs), graphene oxide quantum dots (GOQDs), carboxyl GQDs (C-GQDs), and aminated GQDs (A-GQDs). Firstly, we treated embryos with the four GQDs at three concentrations (50, 100, and 200 µg/mL), and found that only A-GQDs caused embryonic developmental arrest at 100 and 200 µg/mL with significantly decreased survival rates and heartbeat rates, as well as the elevated malformation rates. Next, we analyzed the mRNA sequencing data acquired from zebrafish embryos exposed to the four GQDs for 7 days at 100 µg/mL, and found that all GQDs can act on potassium (K+) and calcium (Ca2+) channels, and spliceosomes with varying degrees of regulatory effects. Compared to other GQDs, A-GQDs can strongly perturb the anticoagulant protein C (PC) pathway via activating most genes associated with complement and coagulation system, cell adhesion molecules (CAMs), and MAPK. In conclusion, this study provided substantial transcriptomic data underlying the common signaling pathways induced by various types of GQDs and pointed out the specific toxicity of A-GQDs on hemostatic system.
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Grafito , Puntos Cuánticos , Animales , Grafito/toxicidad , Puntos Cuánticos/toxicidad , ARN Mensajero/genética , Transcriptoma , Pez Cebra/genéticaRESUMEN
This study aimed to evaluate the anti-pancreatic cancer effects of flavonoids in Plumula Nelumbinis. High-performance liquid chromatography/quadrupole time-of-flight mass spectrometry showed that apiin, rhoifolin, and vitexin were three principal components in total flavonoids derived from Plumula Nelumbinis, with vitexin being the most abundant component. Cell viability assay revealed that apiin, rhoifolin, and vitexin could inhibit proliferation of PANC-1 and ASPC-1, with rhoifolin showing the maximum inhibitory effect. Rhoifolin inhibited cell proliferation and promoted apoptosis of pancreatic cancer cells, which was associated with up-regulated JNK and p-JNK as well as down-regulated p-AKT. Rhoifolin also inhibited cell migration and invasion, and increased the antioxidant capacity in PANC-1 and ASPC-1. Besides, AKT activator (SC79) or JNK inhibitor (SP600125) effectively reversed the anticancer effects of rhoifolin in pancreatic cancer. Quantitative proteomics analysis showed that rhoifolin altered proteomic profiles in pancreatic cancer cells. Western blot analysis showed that rhoifolin down-regulated transforming growth factor beta 2 (TGF-ß2), the regulator of proteoglycan synthesis, with the concomitant down-regulation of phosphorylated SMAD family member 2 (SMAD2), the downstream effector of TGF-ß2. In conclusion, rhoifolin regulates the AKT/JNK/caspase-3 and TGF-ß2/SMAD2 signaling pathways, which may contribute to its anti-pancreatic cancer effects.
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Sistema de Señalización de MAP Quinasas , Neoplasias Pancreáticas , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Disacáridos , Flavonoides , Glicósidos , Humanos , Neoplasias Pancreáticas/tratamiento farmacológico , Proteómica , Proteínas Proto-Oncogénicas c-aktRESUMEN
PURPOSE: Sialic acid-bound immunoglobulin lectin 15 (Siglec-15) plays a crucial role in many kinds of tumors. The relationship between Siglec-15 and the prognosis of osteosarcoma patients and its role in the apoptosis and pyroptosis of osteosarcoma cells are not sufficiently understood. Our study aimed to investigate the function of Siglec-15 in osteosarcoma cells and its effect on tumor cell proliferation, apoptosis and pyroptosis. MATERIALS AND METHODS: The Siglec-15 expression in pathological sections of osteosarcoma patients was analyzed and the overall survival time related to the expression of Siglec-15 was further analyzed. Next, we detected the expression of Siglec-15 in osteosarcoma cells and downregulated the expression of Siglec-15 by small interfering RNA (siRNA). The proliferation, apoptosis and pyroptosis of osteosarcoma cells were studied by proliferation and apoptosis kits and Western blotting. Furthermore, the Siglec-15 signaling pathway was examined, which may be involved in the observed cellular effects. RESULTS: We demonstrated the expression of Siglec-15 in osteosarcoma cells. SiRNA-mediated downregulation of Siglec-15 was successful. We found that knockdown of Siglec-15 in osteosarcoma cell lines significantly inhibited proliferation while promoting apoptosis. Further investigation showed that the expression of proliferation-related proteins was downregulated and that apoptosis- and pyroptosis-related proteins were upregulated. In addition, we found that Siglec-15 may inhibit proliferation while inducing apoptosis and pyroptosis via the (Signal Transducer and Activator of Transcription 3) STAT3/Bcl-2 pathway in osteosarcoma. CONCLUSIONS: In this study, we demonstrated that the ablation of Siglec-15 in osteosarcoma inhibited proliferation and promoted apoptosis and pyroptosis by targeting the Siglec-15/STAT3/Bcl-2 pathway.
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Neoplasias Óseas , Osteosarcoma , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico , Humanos , Apoptosis/genética , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Línea Celular Tumoral , Proliferación Celular/genética , Ácido N-Acetilneuramínico , Osteosarcoma/genética , Osteosarcoma/metabolismo , Osteosarcoma/patología , Piroptosis , ARN Interferente Pequeño/genética , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/genética , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismoRESUMEN
OBJECTIVE: To investigate the effects of bilateral early breast sucking and unilateral early breast sucking within 2 h after delivery on lactation, breast distending pain and postpartum lochia. METHODS: The clinical data of 128 parturients were analyzed retrospectively. According to the different ways of early breast suction, the parturients were divided into control group (n=64) and observation group (n=64). Among them, unilateral early sucking was performed within 2 h after delivery in the control group, while bilateral early sucking was carried out in the observation group. We compared the lactation, breast distending pain, postpartum lochia, nutritional status, coagulation index, complications and breast feeding success rate. RESULTS: The lactation amount of the observation group was higher than that of the control group on the 3rd, 5th and 7th day after delivery; the VAS scores showed opposite trends; the amount of lochia in the observation group was less than that in the control group; the ALb, PA and Hb levels in the observation group were higher on the 7th day after delivery. Compared with the control group, the FIB, PT and APTT time was shorter and the D-D level was higher; the incidence of postpartum hemorrhage, galactostasis and postpartum depression in the observation group was lower, and the success rate of feeding on the 1st, 3rd and 7th day after delivery was higher in the observation group seven days after intervention. CONCLUSION: Bilateral early breast sucking within 2 h after delivery is helpful to increase lactation and reduce breast distending pain and postpartum lochia. It also promotes maternal nutritional status and coagulation function, reduces the incidence of postpartum complications, and improves the breastfeeding rate.
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BACKGROUND: Recent studies indicated that exosomal programmed death-ligand 1 (PD-L1) derived from cancers could induce immunosuppression and tumor pathogenesis. However, it is unclear how exosomes influence osteosarcoma (OS) progression and whether PD-L1 also exists in serum exosomes (Sr-exosomes) of patients with osteosarcoma. We examined serum exosomes from 70 OS patients, 9 patients with benign tumors and 22 healthy donors. OS-derived exosomes were functionally evaluated in vivo and in vitro. RESULTS: The characteristics of exosomes derived from OS patient serum and OS cell lines were confirmed by several methods. We found OS patients had a higher level of exosomal PD-L1 compared to healthy donors. Meanwhile, OS patients with pulmonary metastasis also showed a relatively higher level of exosomal PD-L1 than patients without metastasis. Next, bioinformatic analysis demonstrated that Sr-exosomes isolated from OS patients may involve in the important process of immune function and cancer pathogenesis for OS patients. Co-expression network centered with PD-L1 among Sr-exosomal differently expressed mRNA demonstrated exosomal N-cadherin had a close relationship with exosomal PD-L1 expression. Then, we confirmed higher level of Sr-exosomal N-cadherin in OS patients with pulmonary metastasis compared to ones without metastasis. Furthermore, we elucidated osteosarcoma-derived exosomes and exosomal-PD-L1 promoted the pulmonary metastasis in metastatic models. ROC (Receiver Operating Characteristic Curve) analysis showed AUC (Area Under Curve) of 0.823 for exosomal PD-L1, 0.806 for exosomal N-cadherin and 0.817 for exosomal N-cadherin/E-cadherin to distinguish OS patients with pulmonary metastasis from ones without metastasis. CONCLUSIONS: Osteosarcoma stimulates pulmonary metastasis by releasing exosomes, that carry PD-L1 and N-cadherin. Detection of exosomal PD-L1 and N-cadherin from serum of OS patients may predict pulmonary metastasis progression for OS patients.
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Antígeno B7-H1/sangre , Biomarcadores de Tumor/sangre , Cadherinas/sangre , Exosomas/química , Neoplasias Pulmonares/diagnóstico , Metástasis de la Neoplasia/diagnóstico , Osteosarcoma/metabolismo , Animales , Antígeno B7-H1/genética , Cadherinas/genética , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Biología Computacional , Progresión de la Enfermedad , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Pulmonares/secundario , Ratones Endogámicos BALB C , ARN Mensajero/metabolismo , Distribución Tisular , Cicatrización de HeridasRESUMEN
The survival rate of osteosarcoma, the most prevalent primary bone tumor, has not been effectively improved in the last 30 years. Hence, new treatments and drugs are urgently needed. Antiangiogenic therapy and immunotherapy have good antitumor effects in many kinds of tumors. It is hypothesized that there may be a synergistic effect between immune checkpoint inhibitors and antiangiogenic therapy. Nevertheless, its potential mechanism is still unclear. Vascular endothelial growth factor receptor-2 (VEGFR2) expression was detected by immunohistochemistry in 18 paired osteosarcoma tissues. Moreover, we investigated the effects of apatinib treatment and VEGFR2 knockdown on osteosarcoma as well as the relevant underlying mechanism. Immunohistochemistry assays showed that, compared with that in primary osteosarcoma, VEGFR2 expression was higher in lung metastases. VEGFR2 was positively correlated with PD-L2 expression in osteosarcoma lung metastasis. Transwell assays indicated that VEGFR2 inhibition reduced osteosarcoma cell metastatic abilities in vitro. We also demonstrated that VEGFR2 inhibition downregulated the STAT3 and RhoA-ROCK-LIMK2 pathways, thereby attenuating migration and invasion. Additionally, VEGFR2 inhibition targeted STAT3, through which it reduced PD-L2 expression in osteosarcoma cells. VEGFR2 inhibition markedly attenuated osteosarcoma lung metastatic ability in vivo. In this study, we presented the pro-metastatic functional mechanism of VEGFR2 in osteosarcoma. VEGFR2 inhibition exhibits antitumor effects through antiangiogenic effects and inhibition of immune escape, which possibly provides potential clinical treatment for metastatic osteosarcoma.
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PURPOSE: To compare the impact of visual field (VF) testing on intraocular pressure (IOP) change trends between healthy subjects and glaucoma patients. METHODS: We recruited healthy volunteer subjects who did not have previous ocular diseases and open-angle glaucoma patients who were medically controlled well. IOP in both eyes of each participant was measured by using a noncontact tonometer at five time points: before, immediately after (0 minute), and 10, 30, and 60 minutes after the standard automated perimetry. Repeated measures ANOVA was used to analyze the effect of VF testing on IOP change trends in healthy and glaucoma eyes. RESULTS: Forty healthy subjects (80 eyes) and 31 open-angle glaucoma patients (62 eyes) were included for the study. The baseline IOP of healthy and glaucoma eyes was 16.11 ± 3.01 mmHg and 15.78 ± 3.57 mmHg, respectively. After the VF testing, the IOP in healthy eyes was decreased by 1.5% at 0 minute, 6.5% at 10 minutes (P < 0.001), 6.6% at 30 minutes (P < 0.001), and 7.0% at 1 hour (P < 0.001), indicating that this reduction was sustained for at least 1 hour. However, the IOP in glaucoma eyes was increased by 12.7% at 0 minute (P < 0.001) and, then, returned towards initial values 1 hour after the VF testing. CONCLUSIONS: IOP change trends after VF field testing between healthy subjects and glaucoma patients were quite different. VF testing led to a mild and relatively sustained IOP decrease in healthy subjects, whereas IOP in open-angle glaucoma patients tended to significantly increase immediately after VF testing and, then, returned to pretest values after 1 hour. These findings indicate that the factors of VF testing should be considered in the clinical IOP measurements.