Ammonia nitrogen exposure caused structural damages to gill mitochondria of clam Ruditapes philippinarum.

Ammonia nitrogen has been one of the key pollution indicators along the Chinese coastline for quite a few years. Our previous studies have proved that ammonia nitrogen is harmful for Ruditapes philippinarum clam in several aspects. Environmental concentrations of ammonia nitrogen were found to significantly decrease ATP contents and disturb ATP metabolism, in addition to reducing the potential across the mitochondrial membrane in clam gill tissues. Accordingly, mitochondrion is considered as one of the target organelles of ammonia nitrogen toxicity in clams. However, there is a lack of direct evidence to prove it. In order to reveal detail information of ammonia nitrogen toxicity on clam mitochondria and screen the related biomarker to indicate ammonia nitrogen pollution, mitochondrial parameters in gill tissues including swelling, mtDNA copy number and marker enzyme (succinic dehydrogenase, SDH) activity were measured after the clams were exposed to 0.1 mg/L and 0.5 mg/L ammonia nitrogen for 3 days and 21 days, respectively. Moreover, adverse effects of ammonia nitrogen exposure on clam mitochondrial ultra-structures, mitochondrial swelling and division were also discriminated under transmission electron microscope (TEM). Final results showed that ammonia nitrogen exposure to both concentrations significantly induced mitochondrial swelling, reduced the number of mitochondria and messed their normal structure, decreased the number of mtDNA copies and down-regulated SDH activity, all in a concentration and duration dependent manner. So, the present study helps us to better understand the structural damage of ammonia nitrogen on mitochondria in clam gill cells and provides fundamental data for ammonia nitrogen control in aquaculture.

LncRNA MIR4435-2HG potentiates the proliferation and invasion of glioblastoma cells via modulating miR-1224-5p/TGFBR2 axis.

Glioblastoma (GBM) belongs to the high-grade (IV) gliomas with extremely poor prognosis. Accumulating evidence uncovered the key roles of long non-coding RNAs (lncRNAs) in GBM development. This study aimed to determine the biological actions and the clinical relevance of lncRNA MIR4435-2 Host Gene (MIR4435-2HG) in GBM. Data from GEPIA database showed that MIR4435-2HG was up-regulated in GBM tissues and high expression of MIR4435-2HG correlated with shorter overall survival of GBM patients. Further experimental assays verified the up-regulation of MIR4435-2HG in GBM tissues and cell lines. In vitro cell studies and in vivo animal studies showed that knockdown of MIR4435-2HG resulted in the inhibition of GBM cell proliferation and invasion and in vivo tumour growth, while MIR4435-2HG overexpression driven GBM progression. Furthermore, MIR44435-2HG was found to sponge miR-1224-5p and suppress miR-1224-5p expression; overexpression of miR-1224-5p attenuated the enhancement in GBM cell proliferation and invasion induced by MIR4435-2HG overexpression. In a subsequent study, miR-1224-5p was found to target transforming growth factor-beta receptor type 2 (TGFBR2) and repressed TGFBR2 expression, and in vitro assays showed that miR-1224-5p exerted tumour-suppressive effects via targeting TGFBR2. More importantly, TGFRB2 knockdown antagonized hyper-proliferation and invasion of GBM cells with MIR4435-2HG overexpression. Clinically, the down-regulation of miR-1224-5p and up-regulation of TGFBR2 were verified in the GBM clinical samples. Taken together, the present study suggests the oncogenic role of MIR4435-2HG in GBM and underlies the key function of MIR4435-2HG-driven GBM progression via targeting miR-1224-5p/TGFBR2 axis.

Low p21 level is necessary for the suppressive effects of micoRNA-31 on glioma cell migration and invasion.

MicroRNAs (miRNAs), a kind of endogenous non-coding RNAs, regulate gene expression through binding to the 3'-untranslational region (UTR) of target messenger RNAs (mRNAs) and act as endogenous agents of RNA interference, resulting in either mRNA degradation or translational repression. MiR-31 has been demonstrated to be associated with the development and progression of glioma. However, the underlying molecular mechanism remains largely unclear. In the present study, we demonstrated that miR-31 only inhibited the cell migration and invasion, as well as the expression of a known miR-31 target oncogene radixin, in U251 glioma cells that expressed low level of p21; however, miR-31 showed no above effects on glioma SHG44 cells that highly expressed p21. Moreover, upregulation of p21 in U251 cells reversed the suppressive effects of miR-31 on the cell migration and invasion, suggesting that low p21 level is necessary for the miR-31-mediated inhibitory effects on glioma. Furthermore, analysis for 35 glioma specimens showed that the expression of radixin was negatively correlated with the miR-31 level in glioma tissues with low p21 expression; however, no such correlation was found in glioma tissues with high p21 level, further supporting that the low p21 level is necessary for the suppressive effect of miR-31 on the expression of its target oncogenes. In summary, our study demonstrates that the suppressive effect of miR-31 on glioma cell migration and invasion is p21-dependent, and suggests that miR-31 may be used for the treatment of patients with p21-deficent glioma.

ALOX5 contributes to glioma progression by promoting 5-HETE-mediated immunosuppressive M2 polarization and PD-L1 expression of glioma-associated microglia/macrophages.

BACKGROUND: Oxylipin metabolism plays an essential role in glioma progression and immune modulation in the tumor microenvironment. Lipid metabolic reprogramming has been linked to macrophage remodeling, while the understanding of oxylipins and their catalyzed enzymes lipoxygenases in the regulation of glioma-associated microglia/macrophages (GAMs) remains largely unexplored. METHODS: To explore the pathophysiological relevance of oxylipin in human glioma, we performed Ultra-high performance liquid chromatography-MS/MS (UHPLC-MS/MS) analysis in human glioma and non-tumor brain tissues. To comprehensively investigate the role of arachidonate lipoxygenase 5 (ALOX5) in glioma, we performed in vivo bioluminescent imaging, immunofluorescence staining and flow cytometry analysis on tumors from orthotopic glioma-bearing mice. We developed an ALOX5-targeted nanobody, and tested its anti-glioma efficacy of combination therapy with α-programmed cell death protein-1 (PD-1). RESULTS: In this study, we found that ALOX5 and its oxylipin 5-hydroxyeicosatetraenoic acid (5-HETE) are upregulated in glioma, accumulating programmed death-ligand 1 (PD-L1)+ M2-GAMs and orchestrating an immunosuppressive tumor microenvironment. Mechanistically, 5-HETE derived from ALOX5-overexpressing glioma cells, promotes GAMs migration, PD-L1 expression, and M2 polarization by facilitating nuclear translocation of nuclear factor erythroid 2-related factor 2. Additionally, a nanobody targeting ALOX5 is developed that markedly suppresses 5-HETE efflux from glioma cells, attenuates M2 polarization of GAMs, and consequently ameliorates glioma progression. Furthermore, the combination therapy of the ALOX5-targeted nanobody plus α-PD-1 exhibits superior anti-glioma efficacy. CONCLUSIONS: Our findings reveal a pivotal role of the ALOX5/5-HETE axis in regulating GAMs and highlight the ALOX5-targeted nanobody as a potential therapeutic agent, which could potentiate immune checkpoint therapy for glioma.

Rational protein engineering of a ketoacids decarboxylase for efficient production of 1,2,4-butanetriol from arabinose.

BACKGROUND: Lignocellulose, the most abundant non-edible feedstock on Earth, holds substantial potential for eco-friendly chemicals, fuels, and pharmaceuticals production. Glucose, xylose, and arabinose are primary components in lignocellulose, and their efficient conversion into high-value products is vital for economic viability. While glucose and xylose have been explored for such purpose, arabinose has been relatively overlooked. RESULTS: This study demonstrates a microbial platform for producing 1,2,4-butanetriol (BTO) from arabinose, a versatile compound with diverse applications in military, polymer, rubber and pharmaceutical industries. The screening of the key pathway enzyme, keto acids decarboxylase, facilitated the production of 276.7 mg/L of BTO from arabinose in Escherichia coli. Through protein engineering of the rate-limiting enzyme KivD, which involved reducing the size of the binding pocket to accommodate a smaller substrate, its activity improved threefold, resulting in an increase in the BTO titer to 475.1 mg/L. Additionally, modular optimization was employed to adjust the expression levels of pathway genes, further enhancing BTO production to 705.1 mg/L. CONCLUSION: The present study showcases a promising microbial platform for sustainable BTO production from arabinose. These works widen the spectrum of potential lignocellulosic products and lays the foundation for comprehensive utilization of lignocellulosic components.

The Mining Method of Ideological and Political Elements in University Public Mental Health Courses Based on Artificial Intelligence Technology.

Artificial intelligence technology has become an important part of the development of Internet technology. Artificial intelligence technology can help colleges and universities optimize the network ideological and political teaching system. Artificial intelligence technology provides more accurate data resources and rich and reliable educational technology means for online public mental health education in colleges and universities. This paper comprehensively uses a variety of methods such as qualitative and quantitative analysis, case and empirical analysis, literature analysis, and artificial intelligence technology. Artificial intelligence technology has been closely integrated with online public mental health education in colleges and universities. The model systematically analyzes the optimization methods of artificial intelligence technology methods for the online public mental health education system in colleges and universities, and constructs an innovation system for online public mental health education in colleges and universities. Based on the comprehensive analysis of artificial intelligence and public health in colleges and universities, this paper further proposes the application of artificial intelligence technology in online public mental health education in colleges and universities. On this basis, the model conducts an in-depth analysis of artificial intelligence technology and the online public mental health innovation system. The model supports the development of ideological and political teaching in colleges and universities through various forms such as idea innovation, path innovation, carrier innovation, and mechanism innovation.

Modifications of calcium metabolism and apoptosis after ammonia nitrogen exposure imply a tumorous fate in clam Ruditapes philippinarum?

Ammonia nitrogen (NH3N) is a kind of toxic inorganic nitrogen that has been a great ecological stressor to the marine organisms for quite a few years in Chinese coastal area. Toxic mechanism of ammonia nitrogen on marine bivalve is not well elucidated, especially in calcium metabolism and apoptosis. In the present study, clams Ruditapes philippinarum were used as the experiment animals to receive NH3N exposure with environmental concentrations for 21 days. Results showed that NH3N exposure induced ROS production, decreased Ca2+ concentration, and increased caspase 3 activities in the clam gill cells. In addition, three kinds of Ca2+ exchanger genes, e. g. Na+/K+/Ca2+ exchanger 2, Na+/Ca2+ exchanger 3 and monovalent cation/H+ antiporter, exhibited significant increments in transcription to eliminate intracellular Ca2+. Besides, NH3N exposure significantly increased mRNA expression levels of key anti-apoptotic regulator Bcl-2 genes (Bcl2-1 and Bcl2-1), which would inhibit the apoptosis degree in gill cells. Taken together, increased Ca2+-extrusion and apoptosis inhibition would act cooperatively to alleviate the apoptosis degree and extend the lifespan, so that some kind of tumor might develop in oxidative damaged gill cells after NH3N exposure. Therefore, it is predicted that NH3N exposure will probably bring the clam R. philippinarum a tumorous fate, which will be a great challenge for the healthy development of molluscs aquaculture under the current pollution condition. In addition, caspase 3 activity and mRNA expression levels of Bcl2-2 and Na+/Ca2+ exchanger 3 could be used as potential clam biomarkers to indicate NH3N pollution.

A facile method for anti-cancer drug encapsulation into polymersomes with a core-satellite structure.

Polymersomes possess the self-assembly vesicular structure similar to liposomes. Although a variety of comparisons between polymersomes and liposomes in the aspects of physical properties, preparation and applications have been elaborated in many studies, few focus on their differences in drug encapsulation, delivery and release in vitro and in vivo. In the present work, we have provided a modified direct hydration method to encapsulate anti-cancer drug paclitaxel (PTX) into PEG-b-PCL constituted polymersomes (PTX@PS). In addition to advantages including narrow particle size distribution, high colloid stability and moderate drug-loading efficiency, we find that the loaded drug aggregate in small clusters and reside through the polymersome membrane, representing a unique core-satellite structure which might facilitate the sustained drug release. Compared with commercial liposomal PTX formulation (Lipusu®), PTX@PS exhibited superb tumor cell killing ability underlain by multiple pro-apoptotic mechanisms. Moreover, endocytic process of PTX@PS significantly inhibits drug transporter P-gp expression which could be largely activated by free drug diffusion. In glioma mice models, it has also confirmed that PTX@PS remarkably eradicate tumors, which renders polymersomes as a promising alternative to liposomes as drug carriers in clinic.

miR-18a promotes glioblastoma development by down-regulating ALOXE3-mediated ferroptotic and anti-migration activities.

The development of glioblastoma (GBM) is typically accompanied by marked changes in lipid metabolism. Oxylipins and their catalyzed enzymes lipoxygenases (LOXs) have been shown to participate in the development of cancers via multiple pathways, while the understanding of LOXs in GBM remains enigmatic. Thus, we aimed to explore the expression and functional roles of LOXs in the development of GBM. Here we showed that ALOXE3 was markedly down-regulated in human GBM. Knockdown of ALOXE3 in GBM cells fostered the orthotopic tumor growth and shortened lifespan in mice. ALOXE3 deficiency rendered GBM cells resistant to p53-SLC7A11 dependent ferroptosis, promoting GBM cell survival. Mechanistically, miR-18a directly targeted ALOXE3 and suppressed its expression and functions in GBM cells. Furthermore, ALOXE3 silencing promoted 12-hydroxyeicosatetraenoic acids (12-HETE) secretion from GBM cells, in turn, 12-HETE enhanced migration of GBM cells by activating Gs-protein-coupled receptor (GsPCR)-PI3K-Akt pathway in an autocrine manner. Altogether, miR-18a/ALOXE3 axis exerts tumor promoting functions by regulating ferroptosis and migration of GBM cells. Targeting miR-18a/ALOXE3 axis may provide novel therapeutic approaches for GBM treatment.

Synergism between cryoablation and GM-CSF: enhanced immune function of splenic dendritic cells in mice with glioma.

Glioma is the most common malignant primary brain tumor, and it has a poor prognosis. Studies have shown that cryoablation can activate antitumor immunoeffects by promoting the augmentation of dendritic cells (DCs). Granulocyte macrophage colony-stimulating factor (GM-CSF) has been shown to be useful for immunotherapy against glioma because it can stimulate DCs to present tumor antigen. Previous studies have shown that cryoablation and GM-CSF can exert antitumor effects. To test the hypothesis that combined therapy with cryoablation and GM-CSF for glioma could synergistically improve specific antiglioma immunity in mice, we tested the validity of this assumption in a murine subcutaneous GL261 glioma model. C57BL/6 mice with subcutaneous GL261 glioma were created and divided into four groups: no treatment, GM-CSF injection, cryoablation treatment, and GM-CSF and cryoablation combined treatment (n=20 in each group). Serial immune indicators were detected at sequential time points during treatment. Compared with the other groups, in the combined treatment group, DCs were more activated and their numbers were markedly upregulated, the secretion of interferon-γ from Th1 cells of mice spleen was increased, and the cytolytic activity of CD8 CTLs exerted a more significant cytotoxic effect on GL261 glioma cells (P<0.05 for all). Furthermore, these changes peaked on the 7th day after treatment, and then gradually reduced, until the 21st day; these changes were higher than those at pretreatment (P<0.05). It is concluded that combined therapy with argon-helium cryoablation and GM-CSF could synergistically enhance the activation of DCs and induce a robust tumor-specific immunologic response in glioma-bearing mice.

Immune responsive gene 1, a novel oncogene, increases the growth and tumorigenicity of glioma.

Immune responsive gene 1 (IRG1) is highly expressed in mammalian macrophages during inflammation. However, the role of IRG1 in tumorigenesis remains unclear. In the present study, we aimed to clarify the epigenetic regulation and biological functions of IRG1 in glioma. We found that the expression level of IRG1 influenced the WHO stage in 140 glioma patients. Overexpression of IRG1 increased the growth, invasion, and tumorigenesis in U251 and SHG-44 glioma cells both in vitro and in vivo. Suppression of IRG1 expression by si-IRG1 decreased the levels of cell cycle regulatory proteins, namely, E2F1, p21, CDK4, CDK6 and cyclin D1. Knockdown of IRG1 expression by RNA interference increased E-cadherin expression and decreased the amounts of snail and vimentin. Furthermore, the suppression of IRG1 expression inhibited the expression of NF-κB and STAT3, suggesting a role of IRG1 in regulating the genes associated with these factors and thereby contributing to a decrease in glioma cell proliferation, migration and invasion. Collectively, our findings revealed that IRG1 is a candidate oncogene that is amplified in glioma and is involved in novel mechanisms that influence glioma pathogenesis.

In-situ administration of dendritic cells following argon-helium cryosurgery enhances specific antiglioma immunity in mice.

Dendritic cells (DCs) are highly specialized antigen-presenting cells that play a key role in the activation of naive T cells. With an aim to explore whether in-situ administration of DCs following argon-helium cryosurgery could enhance specific antiglioma immunity in mice, we evaluated the validity of this approach in a murine subcutaneous GL261 glioma model. C57BL/6 mice models bearing subcutaneous GL261 glioma were established and then divided into four groups, namely, no-treatment group (n=14), DC group (n=14), cryosurgery group (n=15), and cryosurgery+DC group (n=15). Compared with the other groups, cryosurgery combined with DCs injection reduced tumor sizes and significantly prolonged survival. In addition, the combined treatment resulted in significantly increasing percentages of CD3, CD3CD4 cells, the ratio of CD3CD4/CD3CD8, and the level of serum interleukin-12 10 days after treatments. Furthermore, in the combined treatment group, Th1 cells were significantly higher than those in the other groups, and the splenic cytotoxic T lymphocyte of mice showed significantly increasing specific cytotoxicity against GL261 cells. These results indicated that in addition to the destruction of tumor, cryosurgery combined with DCs injection enhanced systemic antitumor immunity, suggesting the potential usefulness of the combined treatment in the clinical management of gliomas.

[Individualized therapy and outcomes of microsurgery, radiotherapy, and chemotherapy for astrocytoma].

BACKGROUND & OBJECTIVE: Astrocytomas, constitute about 75% of neuroepithelial tumors, is one of the most common primary tumors in central nervous system with fairly high incidence and poor prognosis. Individualized multimodality is the hope for improving prognosis of patients with astrocytoma. This study was designed to investigate the efficiency of individualized treatment of microsurgery, radiotherapy, and chemotherapy for 62 patients with astrocytoma. METHODS: Sixty-two patients with astrocytoma in study group were treated with individualized multimodality of microsurgery, postoperative radiotherapy, and/or postoperative chemotherapy according to in vitro sensitivity assay. After microsurgery, 59 patients accepted radiotherapy, 46 patients received chemotherapy. Fifty patients with astrocytoma in control group were treated with conventional treatment of surgery, chemotherapy, and radiotherapy. After surgery, 31 patients received radiotherapy following by BCNU chemotherapy, while 19 patients accepted BCNU chemotherapy following radiotherapy. Pathologic diagnosis of patients in study group were 19 cases of grade, 32 cases of grade III, and 11 cases of grade IV; in control group were 13 cases of grade II, 28 cases of grade III, and 9 cases of grade IV. Mean follow-up time were 25.8 months, and the outcome was evaluated by MRI, KPS, and survival rate. RESULTS: Tumor total resection rate in study group was 67.7%, while that in control group was 58.0%. There was no significant difference of KPS and survival rate in patients with low-grade astrocytoma between 2 groups, while the outcome of patients with malignant astrocytoma was significantly improved by individualized treatment. In study group, 2-year expectant survival rate of patients with astrocytoma of grade III, and grade IV were 93.7%, and 36.3%, while in control group were 67.5%, and 22.2% (P< 0.05). In glioblastoma patients, median survival time of study group was 18.68 months, while that of control group was 12.83 months (P< 0.01). CONCLUSION: Individualized microsurgery may improve the total resection of astrocytoma, and benefit to postoperative treatment.Individualized radiotherapy/chemotherapy may prevent patients from some complications. Individualized management may improve prognosis of patients with astrocytoma, particularly malignant astrocytoma.