Newly graphene/polypyrrole (rGO/PPy) modified carbon felt as bio-cathode in bio-electrochemical systems (BESs) achieving complete denitrification.

Nitrate reduction in bio-electrochemical systems (BESs) has attracted wide attention due to its low sludge yields and cost-efficiency advantages. However, the high resistance of traditional electrodes is considered to limit the denitrification performance of BESs. Herein, a new graphene/polypyrrole (rGO/PPy) modified electrode is fabricated via one-step electrodeposition and used as cathode in BES for improving nitrate removal from wastewater. The formation and morphological results support the successful formation of rGO/PPy nanohybrids and confirm the part covalent bonding of Py into GO honeycomb lattices to form a three-dimensional cross-linked spatial structure. The electrochemical tests indicate that the rGO/PPy electrode outperforms the unmodified electrode due to the 3.9-fold increase in electrochemical active surface area and 6.9-fold decrease in the charge transfer resistance (Rct). Batch denitrification activity tests demonstrate that the BES equipped with modified rGO/PPy biocathode could not only achieve the full denitrification efficiency of 100% with energy recovery (15.9 × 10-2 ± 0.14 A/m2), but also favor microbial attach and growth with improved biocompatible surface. This work provides a feasible electrochemical route to fabricate and design a high-performance bioelectrode to enhance denitrification in BESs.

Bidirectional Enhancement of Nitrogen Removal by Indigenous Synergetic Microalgal-Bacterial Consortia in Harsh Low-C/N Wastewater.

Conventional microalgal-bacterial consortia have limited capacity to treat low-C/N wastewater due to carbon limitation and single nitrogen (N) removal mode. In this work, indigenous synergetic microalgal-bacterial consortia with high N removal performance and bidirectional interaction were successful in treating rare earth tailing wastewaters with low-C/N. Ammonia removal reached 0.89 mg N L-1 h-1, 1.84-fold more efficient than a common microalgal-bacterial system. Metagenomics-based metabolic reconstruction revealed bidirectional microalgal-bacterial interactions. The presence of microalgae increased the abundance of bacterial N-related genes by 1.5- to 57-fold. Similarly, the presence of bacteria increased the abundance of microalgal N assimilation by 2.5- to 15.8-fold. Furthermore, nine bacterial species were isolated, and the bidirectional promotion of N removal by the microalgal-bacterial system was verified. The mechanism of microalgal N assimilation enhanced by indole-3-acetic acid was revealed. In addition, the bidirectional mode of the system ensured the scavenging of toxic byproducts from nitrate metabolism to maintain the stability of the system. Collectively, the bidirectional enhancement system of synergetic microalgae-bacteria was established as an effective N removal strategy to broaden the stable application of this system for the effective treatment of low C/N ratio wastewater.

HMOX1-overexpressing mesenchymal stem cell-derived exosomes facilitate diabetic wound healing by promoting angiogenesis and fibroblast function.

Many scholars have suggested that exosomes (Exos) can carry active molecules to induce angiogenesis and thus accelerate diabetic wound healing. Heme oxygenase-1 (HO-1) encoded by the gene HMOX1 promotes wound healing in DM by enhancing angiogenesis. Nevertheless, whether HMOX1 regulates wound healing in DM through mesenchymal stem cell-derived exosomes (MSC-Exos) remains to be further explored. The primary isolated- and cultured-cells expressed MSC-specific marker proteins, and had low immunogenicity and multi-differentiation potential, which means that MSCs were successfully isolated in this study. Notably, HO-1 protein expression was significantly higher in Exo-HMOX1 than in Exos, indicating that HMOX1 could be delivered to Exos as an MSCs-secreted protein. After verifying the -Exo structure, fibroblasts, keratinocytes, and human umbilical vein endothelial cells (HUVECs) were incubated with Exo-HMOX1 or Exo, and the findings displayed that Exo-HMOX1 introduction promoted the proliferation and migration of fibroblasts, keratinocytes and the angiogenic ability of HUVECs in vitro study. After establishing diabetic wound model mice, PBS, Exo, and Exo-HMOX1 were subcutaneously injected into multiple sites on the 1st, 3rd, 7th, and 14th day, DM injected with Exo-HMOX1 showed faster wound healing, re-epithelialization, collagen deposition, and angiogenesis than those in PBS and Exo groups in vitro study. In summary, Exo-HMOX1 could enhance the activity of fibroblasts, keratinocytes, and HUVEC, and accelerate wound healing by promoting angiogenesis in DM.

Buffered loofah supported Microalgae-Bacteria symbiotic (MBS) system for enhanced nitrogen removal from rare earth element tailings (REEs) wastewater: Performance and functional gene analysis.

Rare earth element tailings (REEs) wastewater, which has the characteristics of high ammonia nitrogen (NH4+-N) and low COD. It can cause eutrophication and biotoxicity in water which is produced in high volumes, requiring treatment before final disposal. Microalgae-Bacteria symbiotic (MBS) system can be applied in REEs wastewater, but its low extent of nitrogen removal and instability limit its application. By adding biodegradable carrier as both carbon source and carrier, the system can be stabilized and the efficiency can be improved. In this work, the extent of NH4+-N removal reached 100% within 24 h in a MBS system after adding loofah under optimal conditions, and the removal rate reached 127.6 mg NH4+-N·L-1·d-1. In addition, the carbon release from loofah in 3 d reached 408.7 mg/L, which could be used as a carbon source to support denitrification. During 90 d of operation of the MBS system loaded with loofah, the effluent NH4+-N was less than 15 mg/L. At phylum level, Proteobacteria were dominant which accounted for 78.2%. Functional gene analysis showed that enhancement of microalgae assimilation was the main factor affecting NH4+-N removal. This work expands our understanding of the enhanced role of carbon-based carriers in the denitrification of REEs wastewater.

A pilot scale study on the treatment of rare earth tailings (REEs) wastewater with low C/N ratio using microalgae photobioreactor.

Microalgae appear to be a promising and ecologically safe way for nutrients removal from rare earth tailings (REEs) wastewater with CO2 fixation and added benefits of resource recovery and recycling. In this study, a pilot scale (50 L) co-flocculating microalgae photobioreactor (Ma-PBR) as constructed and operated for 140 days to treat REEs wastewater with low C/N ratio of 0.51-0.56. The removal rate of ammonia nitrogen (NH4+-N) reached 88.04% and the effluent residual concentration was as low as 9.91 mg/L that have met the Emission Standards of Pollutants from Rare Earths Industry (GB 26451-2011). Timely supplementation of trace elements was necessary to maintain the activity of microalgae and then prolonged the operation time. The dominant phyla in co-flocculating microalgae was Chlorophyta, the relative abundance of which was higher than 80%. Tetradesmus belonging to Chlorophyceae was the dominant genus with relative abundance of 80.35%. The results provided a practical support for the scaling-up of Ma-PBR to treat REEs wastewater.

Enhanced ammonia nitrogen removal from actual rare earth element tailings (REEs) wastewater by microalgae-bacteria symbiosis system (MBS): Ratio optimization of microalgae to bacteria and mechanism analysis.

Microalgae-bacteria symbiosis system (MBS) appear to be a promising way for treating the rare earth elements (REEs) wastewater due to the natural symbiotic interactions between microalgae and bacteria. Herein, we investigated the effect of different inoculation ratios of microalgae and bacteria including 3:1 (MB_1), 1:1 (MB_2) and 1:3 (MB_3) on NH4+-N removal from REEs wastewater and analyzed the corresponding biological mechanism. The NH4+-N removal rate with MB_3 reached 17.69 ± 0.45 mg NH4+-N/L d-1, which was 2.58 times higher than that in single microalgae system. The results were further verified in continuous feeding photobioreactors and kept stable for 100 days. Metagenomic analysis revealed that the abundance of genes related to microalgae assimilation increased by 14 %-50 % in answer to photosynthesis and NH4+-N absorption, while that related to nitrification apparently dropped, indicating that MBS was a sustainable method capable of enhancing NH4+-N removal from REEs wastewater.

Sorafenib inhibits doxorubicin-induced PD-L1 upregulation to improve immunosuppressive microenvironment in Osteosarcoma.

PURPOSE: Although undergoing conventional chemotherapy significantly improves the prognosis of Osteosarcoma, chemoresistance and failure of therapy is still a significant challenge. Furthermore, conventional chemotherapy, like doxorubicin, would upregulate the expression of programmed death-ligand 1 (PD-L1) which caused an immunosuppressive microenvironment and unsatisfied treatment result in Osteosarcoma. Thus, it is urgent to explore a strategy to overcome this disadvantage. METHODS: Human Osteosarcoma cell line MG63 and mouse Osteosarcoma cell line K7 were included in this study. Subcutaneous tumor model was used by injection of K7 cells in BALB/C mice to test the effect of doxorubicin and sorafenib on tumor growth. PD-L1 expression was tested in vitro (flow cytometry, western blot and PCR) and in vivo (flow cytometry and immunohistochemistry). Proportion of immune cells (CD4, CD8, Tregs, and cytotoxic T lymphocytes) in vivo was analyzed with flow cytometry. RESULTS: Combination of sorafenib and doxorubicin inhibited tumor growth significantly in vivo. Doxorubicin increased PD-L1 expression in vitro and in vivo, while sorafenib inhibited doxorubicin-induced PD-L1 upregulation in vitro and in vivo. Proportion of interferon-γ-secreting CD8 + T lymphocytes in tumor tissue was increased significantly when sorafenib was combined with doxorubicin, while proportion of CD4, CD8, and Tregs was not significantly changed. Extracellular signal-regulated kinases (ERK) pathway could be one of the key mechanisms by which doxorubicin induced upregulation of PD-L1 in Osteosarcoma cells. CONCLUSION: Combination of sorafenib and conventional chemotherapeutic reagents is a potent strategy to improve treatment effectiveness by modulating tumor microenvironment in Osteosarcoma through increasing proportion of cytotoxic T lymphocytes.

Superenhancers activate the autophagy-related genes Beclin1 and LC3B to drive metastasis and drug resistance in osteosarcoma.

Metastasis and drug resistance are the leading causes of poor prognosis in patients with osteosarcoma. Identifying the relevant factors that drive metastasis and drug resistance is the key to improving the therapeutic outcome of osteosarcoma. Here, we reported that autophagy was highly activated in metastatic osteosarcoma. We found increased autophagolysosomes in metastatic osteosarcoma cell lines by using electron microscopy, Western blot, and immunofluorescence experiments. We further examined the expression of the autophagy-related genes Beclin1 and LC3B in 82 patients through immunohistochemistry and found that Beclin1 and LC3B were highly related to unfavorable prognosis of osteosarcoma. Knockdown of Beclin1 and LC3B reduced invasion, metastasis, and proliferation in metastatic osteosarcoma cells. In vitro and in vivo studies also demonstrated that inhibiting by 3-MA inhibited cell growth and metastasis. Moreover, we demonstrated that autophagy-related genes were activated by SEs and that the inhibition of SEs by JQ-1 decreased the metastasis of osteosarcoma. Overall, our findings highlighted the association of autophagy with osteosarcoma progression and shed new light on autophagy-targeting therapy for osteosarcoma.

Microalgae-bacteria tandem-type ponds simultaneously removing ammonia nitrogen and phosphorus towards municipal wastewater advanced treatment.

Low C/N municipal wastewater is difficult to be treated effectively via traditional biological methods, leading to concentrations of pollutants in effluent far exceeding increasingly strict standards. In this work, we propose a novel microalgae-bacteria tandem-type process to simultaneously remove ammonia nitrogen (NH4+-N) and phosphorus (P) from municipal wastewater. A 4.5 L microalgae-bacteria tandem-type reactor was constructed and operated stably for 40 days. The removal efficiencies of NH4+-N and P reached 97.5% and 92.9%, respectively, effluent concentrations were 0.53 and 0.17 mg/L on average, which met the Environmental quality standards for surface water in China (GB 3838-2002). Remarkably, microalgae ponds accounted for 69.3% and 76.3% of the overall NH4+-N and P removal via microalgae assimilation. Furthermore, 16 S rRNA gene amplicon sequencing revealed the abundance of bacteria changed, suggesting that the presence of microalgae leads to some species extinction and low-abundance bacteria increase. This work demonstrated that the microalgae-bacteria tandem-type processes can be efficient and widely applied in the advanced treatment of municipal wastewater.

DNA Base Pairing-Inspired Supramolecular Nanodrug Camouflaged by Cancer-Cell Membrane for Osteosarcoma Treatment.

Osteosarcoma (OS) is one of the most common bone malignant tumors which mainly develops in adolescents. Although neoadjuvant chemotherapy has improved the prognosis of patients, numerous chemotherapeutic challenges still limit their use. Here, inspired by the Watson-Crick base pairing in nucleic acids, hydrophobic (methotrexate) and hydrophilic (floxuridine) chemo-drugs are mixed and self-assembled into M:F nanoparticles (M:F NPs) through molecular recognition. Then, the obtained NPs are co-extruded with membranes derived from OS cells to form cancer-cell membrane-coated NPs (CCNPs). With protected membranes at the outer layer, CCNPs are highly stable in both physiological and weak acid tumor conditions and possess homologous tumor targeted capability. Furthermore, the proteomic analysis first identifies over 400 proteins reserved in CCNPs, most of them participating in tumor cell targeting and adhesion processes. In vitro studies reveal that CCNPs significantly inhibit the PI3K/AKT/mTOR pathway, which promotes cell apoptosis and cell cycle arrest. More importantly, cell membrane camouflage significantly prolongs the circulation half-life of CCNPs, elevates the drug accumulation at tumor sites, and promotes anti-tumor efficacy in vivo. As a convenient and effective strategy to construct a biomimetic NP with high drug loading ratio, the CCNPs provide new potentials for precise and synergistic antitumor treatment.

Construction and validation of a novel apoptosis-associated prognostic signature related to osteosarcoma metastasis and immune infiltration.

BACKGROUND: Apoptosis played vital roles in the formation and progression of osteosarcoma. However, no studies elucidated the prognostic relationships between apoptosis-associated genes (AAGs) and osteosarcoma. METHODS: The differentially expressed genes associated with osteosarcoma metastasis and apoptosis were identified from GEO and MSigDB databases. The apoptosis-associated prognostic signature was established through univariate and multivariate cox regression analyses. The Kaplan-Meier (KM) survival curve, ROC curve and nomogram were constructed to investigate the predictive value of this signature. CIBERSORT algorithm and ssGSEA were used to explore the relationships between immune infiltration and AAG signature. The above results were validated in another GEO dataset and the expression of AAGs was also validated in osteosarcoma patient samples by immunohistochemistry. RESULTS: HSPB1 and IER3 were involved in AAG signature. In training and validation datasets, apoptosis-associated risk scores were negatively related to patient survival rates and the AAG signature was regarded as the independent prognostic factor. ROC and calibration curves demonstrated the signature and nomogram were reliable. GSEA revealed the signature related to immune-associated pathways. ssGSEA indicated that one immune cell and three immune functions were significantly dysregulated. The immunohistochemistry analyses of patients' samples revealed that AAGs were significantly differently expressed between metastasis and non-metastasis osteosarcomas. CONCLUSIONS: The present study identified and validated a novel apoptosis-associated prognostic signature related to osteosarcoma metastasis. It could serve as the potential biomarker and therapeutic targets for osteosarcoma in the future.

Thrombospondin-1 induced programmed death-ligand 1-mediated immunosuppression by activating the STAT3 pathway in osteosarcoma.

Thrombospondin-1 (TSP1) is generally assumed to suppress the growth of osteosarcoma through inhibiting angiogenesis; however, it is unclear whether TSP1 could affect the antitumor immunity against osteosarcoma. We aimed to explore the immune-related tumor-promoting effects of TSP1 and decipher its underlying mechanism. First, we identified that TSP1 regulated programmed death-ligand 1 (PD-L1) expression, which was related to the CD8+ T cells anergy in osteosarcoma cells. The exact role of PD-L1 in the immunosuppressive effect of TSP1 was then further confirmed by the addition of the PD-L1 neutralizing Ab. With the addition of PD-L1 neutralizing Abs during cocultivation, the inhibition of CD8+ T cells was abolished to a certain extent. Further mechanistic investigations showed that TSP1-induced PD-L1 upregulation was achieved by activation of the signal transducer and activator of transcription 3 (STAT3) pathway. In vivo experiments also indicated that TSP1 overexpression could promote the growth of primary lesions, whereas TSP1 knockdown effectively inhibits the growth of the primary lesion as well as lung metastasis by restoring the antitumor immunity. Thrombospondin-1 knockdown combined with PD-L1 neutralizing Ab achieved a more pronounced antitumor effect. Taken together, our study showed that TSP1 upregulates PD-L1 by activating the STAT3 pathway and, therefore, impairs the antitumor immunity against osteosarcoma.

The Outcome of Discontinuing Tyrosine Kinase Inhibitors in Advanced Sarcoma Following a Favorable Tumor Response to Antiangiogenics Therapy.

(1) Background: The use of antiangiogenic TKIs (AA-TKIs) has recently emerged as a major paradigm shift in the treatment of advanced sarcoma. However, the feasibility of drug holidays for patients demonstrating a very favorable response remains unknown. (2) Methods: We aim to explore the outcomes of patients with advanced sarcoma who discontinued AA-TKIs after a (near-) complete remission or were long-term responders. Patients with advanced disease were included if they had bilateral or multiple lung metastases, extrapulmonary recurrence, a short disease-free interval, etc., at the initiation of AA-TKIs. (3) Results: A total of 22 patients with AA-TKI discontinuation were analyzed, with a median follow-up of 22.3 months post-discontinuation. Prior to discontinuation, there were four drug-induced complete remissions (CRs), twelve surgical CRs, and six long-term responders. Disease progression was observed in 17/22 (77.3%) patients, with a median of 4.2 months. However, since the majority were still sensitive to the original AA-TKIs and amenable to a second surgical remission, 7 out of these 17 patients achieved a second CR after disease progression and were thus considered as relapse-free post-discontinuation (pd-RFS). Therefore, the pd-RFS and post-discontinuation overall survival (pd-OS) in the last follow-up were 12/22 (54.5%) and 16/22 (72.7%), respectively. Remarkably, surgical CR and drug tapering off (versus abrupt stopping) were associated with a greater pd-RFS and pd-OS (p < 0.05). Furthermore, higher necrosis rates (p = 0.040) and lower neutrophil-to-lymphocyte ratios (NLR) (p = 0.060) before discontinuation tend to have a better pd-RFS. (4) Conclusions: Our results suggest that AA-TKI discontinuation with a taper-off strategy might be safe and feasible in highly selected patients with advanced sarcoma. Surgical CR, NLR, and tumor necrosis rates before discontinuation were potential biomarkers for AA-TKI withdrawal.

Exploration and Validation of a Novel Inflammatory Response-Associated Gene Signature to Predict Osteosarcoma Prognosis and Immune Infiltration.

BACKGROUND: Inflammatory response took part in the progression of tumor and was regarded as the hallmark of cancer. However, the prognostic relationship between osteosarcoma and inflammatory response-associated genes (IRGs) was unclear. This research aimed to explore the correlations between osteosarcoma prognosis and IRG signature. METHODS: The inflammatory response-associated differentially expressed messenger RNAs (DEmRNAs) were screened out through Gene Expression Omnibus (GEO) and Molecular Signature Database (MSigDB) databases. Univariate and multivariate cox regression analyses were utilized to construct the IRG signature. The prognostic value of signature was investigated through Kaplan-Meier (KM) survival curve and nomogram. DEmRNAs among high and low inflammatory response-associated risks were identified and functional enrichment analyses were conducted. ESTIMATE, CIBERSORT and single-sample gene set enrichment analyses (ssGSEA) were implied to reveal the alterations in immune infiltration. All the above results were validated in Target database. The expression of IRGs was also validated in different cell lines by quantitative real-time PCR (qRT-PCR) and osteosarcoma patient samples by immunohistochemistry. RESULTS: The IRG signature that consisted of two genes (MYC, CLEC5A) was established. In training and validation datasets, patients with lower risk scores survived longer and the IRG signature was confirmed as the independent prognostic factor in osteosarcoma. The nomogram was constructed and the calibration curves demonstrated the reliability of this model. Functional analysis of risk score-associated DEmRNAs indicated that immune-related pathways and functions were significantly enriched. ssGSEA revealed that 14 immune cells and 11 immune functions were significantly dysregulated. The qRT-PCR results indicated IRGs were significantly differently expressed in osteosarcoma and osteoblast cell lines. The immunohistochemistry analyses of patients' samples revealed the same result. CONCLUSION: The novel osteosarcoma inflammatory response-associated prognostic signature was established and validated in this study. This model could serve as the biomarker and therapeutic target for osteosarcoma in the future.

Development and Validation of a Hypoxia-Associated Prognostic Signature Related to Osteosarcoma Metastasis and Immune Infiltration.

BACKGROUND: Increasing evidence has shown that hypoxia microenvironment relates to tumor initiation and progression. However, no studies focus on the application of hypoxia-associated genes in predicting osteosarcoma patients' prognosis. This research aims to identify the hypoxia-associated genes related to osteosarcoma metastasis and construct a gene signature to predict osteosarcoma prognosis. METHODS: The differentially expressed messenger RNAs (DEmRNAs) related to osteosarcoma metastasis were identified from Therapeutically Applicable Research to Generate Effective Treatments (Target) database. Univariate and multivariate cox regression analyses were performed to develop the hypoxia-associated prognostic signature. The Kaplan-Meier (KM) survival analyses of patients with high and low hypoxia risk scores were conducted. The nomogram was constructed and the gene signature was validated in the external Gene Expression Omnibus (GEO) cohort. Single-sample gene set enrichment analysis (ssGSEA) was conducted to investigate the relationships between immune infiltration and gene signature. RESULTS: Two genes, including decorin (DCN) and prolyl 4-hydroxylase subunit alpha 1 (P4HA1), were involved in the hypoxia-associated gene signature. In training and testing datasets, patients with high-risk scores showed lower survival rates and the gene signature was identified as the independent prognostic factor. Receiver operating characteristic (ROC) curves demonstrated the robustness of signature. Functional analyses of DEmRNAs among high- and low-risk groups revealed that immune-associated functions and pathways were significantly enriched. Furthermore, ssGSEA showed that five immune cells (DCs, macrophages, neutrophils, pDCs, and TIL) and three immune features (CCR, APC co inhibition, and Check-point) were down-regulated in the high-risk group. CONCLUSION: The current study established and validated a novel hypoxia-associated gene signature in osteosarcoma. It could act as a prognostic biomarker and serve as therapeutic guidance in clinical applications.

Targeting autophagy enhances atezolizumab-induced mitochondria-related apoptosis in osteosarcoma.

In this study, we identified the multifaceted effects of atezolizumab, a specific monoclonal antibody against PD-L1, in tumor suppression except for restoring antitumor immunity, and investigated the promising ways to improve its efficacy. Atezolizumab could inhibit the proliferation and induce immune-independent apoptosis of osteosarcoma cells. With further exploration, we found that atezolizumab could impair mitochondria of osteosarcoma cells, resulting in increased release of reactive oxygen species and cytochrome-c, eventually leading to mitochondrial-related apoptosis via activating JNK pathway. Nevertheless, the excessive release of reactive oxygen species also activated the protective autophagy of osteosarcoma cells. Therefore, when we combined atezolizumab with autophagy inhibitors, the cytotoxic effect of atezolizumab on osteosarcoma cells was significantly enhanced in vitro. Further in vivo experiments also confirmed that atezolizumab combined with chloroquine achieved the most significant antitumor effect. Taken together, our study indicates that atezolizumab can induce mitochondrial-related apoptosis and protective autophagy independently of the immune system, and targeting autophagy is a promising combinatorial approach to amplify its cytotoxicity.

Development and analysis of long non-coding RNA-associated competing endogenous RNA network for osteosarcoma metastasis.

BACKGROUND: Osteosarcoma is the primary bone malignant neoplasm that often develops metastasis. Increasing evidences have shown that non-coding RNAs (ncRNAs) relate to the progression of osteosarcoma. However, the ncRNAs' roles in osteosarcoma metastasis are still unknown. METHODS: Differentially expressed (DE) RNAs were identified from Gene Expression Omnibus (GEO) database. Protein-protein interaction (PPI) of DE messenger RNAs (DEmRNAs) was built through STRING database. The target mRNAs and long ncRNAs (lncRNAs) of microRNAs (miRNA) were predicted through miRDB, Targetscan and Genecode databases, which then cross-checked with previously obtained DERNAs to construct competing endogenous RNA (ceRNA) network. All networks were visualized via Cytoscape and the hub RNAs were screened out through Cytoscape plug-in Cytohubba. The gene functional and pathway analyses were performed through DAVID and MirPath databases. The survival analyses of hub RNAs were obtained through Kaplan-Meier (KM) survival curves. RESULTS: Five hundred sixty-four DEmRNAs, 16 DElncRNAs and 22 DEmiRNAs were screened out. GO functional and KEGG pathway analyses showed that DERNAs were significantly associated with tumor metastasis. The ceRNA network including 6 lncRNAs, 55 mRNAs and 20 miRNAs were constructed and the top 10 hub RNAs were obtained. Above all, PI3K/AKT signaling pathway was identified as the most important osteosarcoma metastasis-associated pathway and its hub ceRNA module was constructed. The survival analyses showed that the RNAs in hub ceRNA module closely related to osteosarcoma patients' prognosis. CONCLUSIONS: The current study provided a new perspective on osteosarcoma metastasis. More importantly, the RNAs in hub ceRNA module might act as the novel therapeutic targets and prognostic factors for osteosarcoma patients.

A novel in vivo mouse intervertebral disc degeneration model induced by compressive suture.

Intervertebral disc degeneration (IDD) is the root cause of many musculoskeletal disorders of the spine. However, the etiology of IDD is complex and still not well understood. Animal models of IDD would be useful in deciphering the underlying mechanisms. But the existing animal models have their limitations. Therefore, to establish a novel mouse model that can simulate the human IDD process in vivo, we proposed to carefully circumcise the 2 mm-wide tail skin and then compressively sutured the defect with a simple end-to-end suture to exert excessive pressure on the disc. After 1-week, 2-week, and 4-week compression, the mice were sacrificed and the intervertebral discs were harvested for tissue analysis. The radiological, morphological, and molecular modifications of intervertebral discs were measured to characterize this model. Radiologically, the water content of the intervertebral disc decreased significantly after 2-week compression. Morphologically, the nucleus pulposus showed a decrease in volume and the number of notochordal cells. The compressive suture also broke the balance between anabolic and catabolic enzymes in nucleus pulposus, which led to the remodeling of the extracellular matrix in nucleus pulposus as the content of aggrecan and collagen II decreased. The compressive suture could induce intervertebral discs degeneration in a more reasonable way, which was solely influenced by mechanical loading, as the mice caudal vertebrae still moved freely after the operation. This kind of animal model could be adapted as a reliable in vivo mouse IDD model for the research regarding the etiology and treatments of IDD.

Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples.

BACKGROUND: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. METHODS: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. RESULTS: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. CONCLUSIONS: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research.

MicroRNA-200a induces immunosuppression by promoting PTEN-mediated PD-L1 upregulation in osteosarcoma.

In this study, we identified microRNAs that regulate the expression of programmed death-ligand 1(PD-L1) in osteosarcoma and investigated their role in PD-L1-targeted immunotherapy. MicroRNA sequencing analysis showed that the expression of PD-L1 is regulated by microRNA-200a in U2OS, 143B, and K7 osteosarcoma cells. MicroRNA-200a overexpression induced the upregulation of PD-L1 in the osteosarcoma cells. CD8+ T cells co-cultured with microRNA-200a-overexpressing osteosarcoma cells showed reduced survival, proliferation, and secretion of granzyme B and perforin. The same phenomenon was also observed in the K7-derived syngeneic mouse model, as microRNA-200a promoted tumor growth by increasing the percentage of Foxp3+ regulatory T lymphocytes while reducing the proportions of CD4+, CD8+, and IFN-γ+ cytotoxic T lymphocytes. But microRNA-200a overexpression group was also more responsive to PD-L1-targeted immunotherapy than the controls. In addition, the tumor tissues from 32 osteosarcoma patients showed that high expression of microRNA-200a and PD-L1 was associated with poor tumor necrosis rate after chemotherapy. Moreover, we confirmed that tensin homolog deleted on chromosome ten (PTEN) could act as the target gene for microRNA-200a during the upregulation of PD-L1. Thus, our findings provide important and novel insight into a regulatory axis involving microRNA-200a/PTEN/ PD-L1 axis, which determines osteosarcoma growth and the efficacy of PD-L1-targeted immunotherapy.