PRRSV non-structural protein 5 inhibits antiviral innate immunity by degrading multiple proteins of RLR signaling pathway through FAM134B-mediated ER-phagy.

Viruses employ various evasion strategies to establish prolonged infection, with evasion of innate immunity being particularly crucial. Porcine reproductive and respiratory syndrome virus (PRRSV) is a significant pathogen in swine industry, characterized by reproductive failures in sows and respiratory distress in pigs of all ages, leading to substantial economic losses globally. In this study, we found that the non-structural protein 5 (Nsp5) of PRRSV antagonizes innate immune responses via inhibiting the expression of type I interferon (IFN-I) and IFN-stimulated genes (ISGs), which is achieved by degrading multiple proteins of RIG-I-like receptor (RLR) signaling pathway (RIG-I, MDA5, MAVS, TBK1, IRF3, and IRF7). Furthermore, we showed that PRRSV Nsp5 is located in endoplasmic reticulum (ER), where it promotes accumulation of RLR signaling pathway proteins. Further data demonstrated that Nsp5 activates reticulophagy (ER-phagy), which is responsible for the degradation of RLR signaling pathway proteins and IFN-I production. Mechanistically, Nsp5 interacts with one of the ER-phagy receptor family with sequence similarity 134 member B (FAM134B), promoting the oligomerization of FAM134B. These findings elucidate a novel mechanism by which PRRSV utilizes FAM134B-mediated ER-phagy to elude host antiviral immunity.IMPORTANCEInnate immunity is the first line of host defense against viral infections. Therefore, viruses developed numerous mechanisms to evade the host innate immune responses for their own benefit. PRRSV, one of the most important endemic swine viruses, poses a significant threat to the swine industry worldwide. Here, we demonstrate for the first time that PRRSV utilizes its non-structural protein Nsp5 to degrade multiple proteins of RLR signaling pathways, which play important roles in IFN-I production. Moreover, FAM134B-mediated ER-phagy was further proved to be responsible for the protein's degradation. Our study highlights the critical role of ER-phagy in immune evasion of PRRSV to favor replication and provides new insights into the prevention and control of PRRSV.

The stigma of patients with chronic insomnia: a clinical study.

BACKGROUND: The objective of this study was to explore the stigma and related influencing factors in individuals with chronic insomnia disorder (CID). METHODS: A total of 70 CID patients and 70 healthy controls (CON) were enrolled in the study. All subjects completed the assessments of sleep, emotion, and cognition. Their stigma and life quality were measured using the Chronic Stigma Scale and the 36-Item Short-Form Health Survey (SF-36). RESULTS: The ratio of individuals with stigma was significantly different between CID and CON groups (C2 = 35.6, p < 0.001). Compared with the CON group, the CID group had higher scores for total stigma (U = 662.0, p < 0.001), internalized stigma (U = 593.0, p < 0.001), enacted stigma (U = 1568.0, p < 0.001), PSQI (U = 2485.0, p < 0.001) and HAMD-17 (U = 69.5, p < 0.001) as well as lower scores for MoCA-C (U = 3997.5, p < 0.001) and most items of SF-36. Partial correlation analysis showed that different items of the Chronic Stigma Scale were positively correlated with illness duration, PSQI and HAMD-17 scores, while negatively correlated with one or more items of the SF-36. Multivariate regression analysis showed that illness duration and the Mental Health domain of the SF-36 were independent risk factors for one or more items of stigma in CID patients. CONCLUSION: Patients with CID have an increased risk of stigma. Moreover, illness duration and Mental Health may be primary factors related to stigma.

Decreased Serum Exosomal miR-152-3p Contributes to the Progression of Acute Ischemic Stroke.

BACKGROUND: The current study aims to investigate the expression and potential role of exosome-derived miR-152-3p in acute ischemic stroke (AIS) patients. METHODS: Exosomes were isolated from AIS patients and healthy controls. The level of exosome miR-152-3p was examined using RT-PCR. Receiver operating characteristic (ROC) curves were used to assess exosome miR-152-3p as a biomarker, and the area under the curve (AUC) was reported. RESULTS: Our data showed that the expression of serum exosome miR-152-3p in patients with AIS was significantly lower than in healthy controls. In contrast to those with NIHSS score < 7, the level of exosome miR-152-3p was significantly reduced in AIS patients with NIHSS score ≥ 7, indicating that the decrease of exosome miR-152-3p level is significantly related to the severity of endothelial injury. Moreover, the lowest level of exosome miR-152-3p was found in large-artery atherosclerosis (LAA) patients compared to that in small-vessel occlusion (SAA), cardioembolism (CE) and stroke of undetermined etiology (SUE) group. In addition, exosome miR-152-3p level was significantly lower in acute phase than in chronic phase. ROC curve showed that the AUC of exosome miR-152-3p level was 0.935, which indicated that exosome miR-152-3p level could distinguish AIS patients from non-healthy controls. CONCLUSIONS: In summary, exosome miR-152-3p is a risk factor of cerebral infarction. Enhancing the expression of exosome miR-152-3p in the circulating system may be a promising strategy for the prevention and treatment of AIS.

Downregulation of miR-205 in migrating epithelial tongue facilitates skin wound re-epithelialization by derepressing ITGA5.

Keratinocyte migration is essential for re-epithelialization during skin wound healing, but the molecular mechanisms regulating this cellular response remain to be completely clarified. Here we show that keratinocyte-specific miR-205 is significantly downregulated in the leading edge of the migrating epithelial tongue after skin injury in mice. In HaCaT keratinocytes, miR-205 could be downregulated by TGF-ß1 stimulation. And similar to the effect of TGF-ß1, miR-205 knockdown could promote keratinocyte migration in wound scratch model in vitro. Furthermore, topical inhibition of miR-205 by administrating Pluronic gel containing antagomir-205 could accelerate re-epithelialization in mouse skin wound model in vivo. Moreover, we identified integrin alpha 5 (ITGA5) as one key functional miR-205 target in the re-epithelialization process and epidermal downregulation of miR-205 may desilence ITGA5 to promote keratinocyte migration. And knockdown of ITGA5 would abolish the pro-migratory effects of miR-205 inhibition in vitro. What's more, we found dysregulation of miR-205 and its target ITGA5 in epidermis of clinical chronic wound samples with persistence of high level miR-205 and absence of ITGA5. Our findings indicate that downregulation of miR-205 in the leading migrating keratinocytes is critical for re-epithelialization and miR-205 may be a potential therapeutic target for chronic wounds.

Cell Density-Dependent Upregulation of PDCD4 in Keratinocytes and Its Implications for Epidermal Homeostasis and Repair.

Programmed cell death 4 (PDCD4) is one multi-functional tumor suppressor inhibiting neoplastic transformation and tumor invasion. The role of PDCD4 in tumorigenesis has attracted more attention and has been systematically elucidated in cutaneous tumors. However, the normal biological function of PDCD4 in skin is still unclear. In this study, for the first time, we find that tumor suppressor PDCD4 is uniquely induced in a cell density-dependent manner in keratinocytes. To determine the potential role of PDCD4 in keratinocyte cell biology, we show that knockdown of PDCD4 by siRNAs can promote cell proliferation in lower cell density and partially impair contact inhibition in confluent HaCaT cells, indicating that PDCD4 serves as an important regulator of keratinocytes proliferation and contact inhibition in vitro. Further, knockdown of PDCD4 can induce upregulation of cyclin D1, one key regulator of the cell cycle. Furthermore, the expression patterns of PDCD4 in normal skin, different hair cycles and the process of wound healing are described in detail in vivo, which suggest a steady-state regulatory role of PDCD4 in epidermal homeostasis and wound healing. These findings provide a novel molecular mechanism for keratinocytes' biology and indicate that PDCD4 plays a role in epidermal homeostasis.

miR-198 Represses the Proliferation of HaCaT Cells by Targeting Cyclin D2.

BACKGROUND: MiR-198 has been considered as an inhibitor of cell proliferation, invasion, migration and a promoter of apoptosis in most cancer cells, while its effect on non-cancer cells is poorly understood. METHODS: The effect of miR-198 transfection on HaCaT cell proliferation was firstly detected using Cell Count Kit-8 and the cell cycle progression was analyzed by flow cytometry. Using bioinformatics analyses and luciferase assay, a new target of miR-198 was searched and identified. Then, the effect of the new target gene of miR-198 on cell proliferation and cell cycle was also detected. RESULTS: Here we showed that miR-198 directly bound to the 3'-UTR of CCND2 mRNA, which was a key regulator in cell cycle progression. Overexpressed miR-198 repressed CCND2 expression at mRNA and protein levels and subsequently led to cell proliferation inhibition and cell cycle arrest in the G1 phase. Transfection ofSiCCND2 in HaCaT cells showed similar inhibitory effects on cell proliferation and cell cycle progression. CONCLUSION: In conclusion, we have identified that miR-198 inhibited HaCaT cell proliferation by directly targeting CCND2.

P53-participated cellular and molecular responses to irradiation are cell differentiation-determined in murine intestinal epithelium.

AIM: Cells respond differently to DNA damaging agents, which may related to cell context and differentiation status. The aim of present study was to observe the cellular and molecular responses of cells in different differentiation status to ionizing irradiation (IR). METHODS: Crypt-villus unit of murine small intestine was adopted as a cell differentiation model. DNA damage responses (DDRs) of crypt and villus were observed 1-24 h after 12 Gy IR using gene expression microarray analysis, immunohistochemical staining, Western blotting and Electrophoretic Mobility Shift Assay. RESULTS: Microarray analysis revealed that most differentially expressed genes were related to p53 signaling pathway in crypt 4h after IR and in both crypt and villus 24h after IR. In crypt stem cells/progenitor cells, H2AX was phosphorylated and dephosphorylated quickly, Ki67 attenuated, cell apoptosis enhanced, phosphorylated P53 increased and translocated into nuclear with the ability to bind p53-specific sequence. In upper crypt (transit amplifying cells) and crypt-villus junction, cells kept survive and proliferate as indicated by retained Ki67 expression, suppressed p53 activation, and rare apoptosis. CONCLUSIONS: DDRs varied with cell differentiation status and cell function in small intestinal epithelium. P53 signaling pathway could be an important regulatory mechanism of DDRs.

Electroreductive Ring-Opening Carboxylation of 1,3-Oxazolidin-2-ones with CO2 for Accessing ß-Amino Acids.

Electrocarboxylation of the C(sp3)-O bond in 1,3-oxazolidin-2-ones with CO2 to achieve ß-amino acids is developed. The C-O bond in substrates can be selectively cleaved via the single electron transfer on the surface of a cathode or through a CO2• - intermediate under additive-free conditions. A great diversity of ß-amino acids can be obtained in a moderate to excellent yield and readily converted to various biologically active compounds.

miR-21 regulates skin wound healing by targeting multiple aspects of the healing process.

With the clarification of the important roles of microRNAs (miRNAs) in diverse physiologic and pathologic processes, the effects of miRNAs in wound healing have attracted more attention recently. However, the global pattern of miRNA expression in wound tissue is still unknown. In the present study, we depicted the miRNA profile and identified at least 54 miRNAs, including miR-21, changed for more than twofold at the stage of granulation formation during wound healing. These miRNAs were closely related to the major events of wound healing, including cell migration and proliferation, angiogenesis, and matrix remolding. Furthermore, we found that miR-21 was up-regulated after skin injury, mainly in activated and migrating epithelial cells of epidermis and mesenchymal cells of dermis. Locally antagonizing miR-21 by directly injecting antagomir to wound edge caused significant delay of wound closure with impaired collagen deposition. Unexpectedly, we found wounds treated with miR-21 antagomir had an obvious defect in wound contraction at an early stage of wound healing. The significant role of miR-21 in wound contraction was further confirmed by in vivo gain-of-function and in vitro loss-of-function experiments. In conclusion, the present study has for the first time depicted miRNA profiling of wound healing and demonstrated the involvement of miR-21 in regulating the wound contraction and collagen deposition. These results suggest that miR-21 may be a new medical target in skin wound manipulation.

Targeted Vezf1-null mutation impairs vascular structure formation during embryonic stem cell differentiation.

OBJECTIVE: Vezf1 encodes an early zinc finger transcription factor that is essential for normal vascular development and functions in a dose-dependent manner. Here, we investigated the role of Vezf1 during processes of endothelial cell differentiation and maturation by studying mutant Vezf1 embryonic stem (ES) cells using the in vitro embryoid body differentiation model and the in vivo teratocarcinoma model. METHODS AND RESULTS: Vezf1-/- ES cell-derived embryoid bodies failed to form a well-organized vascular network and showed dramatic vascular sprouting defects. Our results indicate that the retinol pathway is an important mediator of Vezf1 function and that loss of Vezf1 results in reduced retinol/vitamin A signaling and aberrant extracellular matrix (ECM) formation. Unexpectedly, we also uncovered defects during in vitro differentiation of Vezf1-/- ES cells along hematopoietic cell lineages. Vezf1-/- ES cell-derived teratocarcinomas were able to spontaneously differentiate into cell types of all 3 germ layers. However, histological and immunohistochemical examination of these tumors showed decreased cell proliferation, delayed differentiation, and large foci of cells with extensive deposition of ECM. Embryoid bodies and teratocarcinomas derived from heterozygous ES cells displayed an intermediate phenotype. CONCLUSIONS: Together, these results suggest that Vezf1 is involved in early differentiation processes of the vasculature by regulating cell differentiation, proliferation, and ECM distribution and deposition.

Induced endothelial differentiation of cells from a murine embryonic mesenchymal cell line C3H/10T1/2 by angiogenic factors in vitro.

A murine embryonic mesenchymal cell line C3H/10T1/2 possesses the potential to differentiate into multiple cell phenotypes and has been recognized as multipotent mesenchymal stem cells, but no in vitro model of its endothelial differentiation has been established and the effect of angiogenic factors on the differentiation is unknown. The aim of the present study was to evaluate the role of angiogenic factors in inducing endothelial differentiation of C3H/10T1/2 cells in vitro. C3H/10T1/2 cells were treated with angiogenic factors, VEGF (10 ng/mL) and bFGF (5 ng/mL). At specified time points, cells were subjected to morphological study, immunofluorescence staining, RT-PCR, LDL-uptake tests and 3-D culture for the examination of the structural and functional characteristics of endothelial cells. Classic cobblestone-like growth pattern appeared at 6 day of the induced differentiation. Immunofluorescence staining and RT-PCR analyses revealed that the induced cells exhibited endothelial cell-specific markers such as CD31, von Willebrand factor, Flk1, Flt1, VE-cadherin, Tie2, EphrinB2 and Vezf1 at 9 day. The induced C3H/10T1/2 cells exhibited functional characteristics of the mature endothelial phenotype, such as uptake of acetylated low-density lipoproteins (Ac-LDL) and formation of capillary-like structures in three-dimensional culture. At 9 day, Weibel-Palade bodies were observed under a transmission electron microscope. This study demonstrates, for the first time, endothelial differentiation of C3H/10T1/2 cells induced by angiogenic factors, VEGF and bFGF, and confirms the multipotential differentiation ability. This in vitro model is useful for investigating the molecular events in endothelial differentiation of mesenchymal stem cells.

Progress in research on radiation combined injury in China.

The significant feature of radiation combined injury is the occurrence of a combined effect. For decades our institute has focused on studying the key complications of radiation-burn injury, including shock, suppression of hematopoiesis and immunity, gastrointestinal damage and local refractory wound healing. Here we summarize recent advancements in elucidating the mechanisms of and potential treatments for radiation combined injury. Concerning the suppression and regeneration of hematopoiesis in radiation combined injury, mechanisms of megakaryocyte damage have been elucidated and a new type of fusion protein stimulating thrombopoiesis has been developed and is being tested in animals. With regard to the damage and repair of intestinal epithelium, the important molecular mechanisms of radiation combined injury have been clarified, and new measures to prevent and treat gastrointestinal tract injury are proposed. With respect to the difficulties encountered in wound healing, the underlying causes of radiation combined injury have been proposed, and some potential methods to accelerate wound closure are under study. Systemic experiments have been done to determine the appropriate time for eschar excision and skin grafting, and the results provided significant insight into clinical treatment of the injury. In the search for early therapeutic regimens for severe burns and radiation combined injury to prevent deterioration of injuries and to improve survival, cervical sympathetic ganglion block was used for the treatment of animals with radiation combined injury and had significant benefits. These research advancements have potential for application in on-site emergency rescue and in-hospital treatment of radiation combined injury.

A molecular mimic demonstrates that phosphorylated human prolactin is a potent anti-angiogenic hormone.

S179D prolactin (PRL) is an experimentally useful mimic of naturally phosphorylated human prolactin. S179D PRL, but not unmodified PRL, was found to be anti-angiogenic in both the chorioallantoic membrane and corneal assays. Further investigation using human endothelial in vitro models showed reduced cell number, reduced tubule formation in Matrigel, and reduced migration and invasion, as a function of treatment with S179D PRL. Analysis of growth factors in human endothelial cells in response to S179D PRL showed: a decreased expression or release of endogenous PRL, heme-oxygenase-1, basic fibroblast growth factor (bFGF), angiogenin, epidermal growth factor and vascular endothelial growth factor; and an increased expression of inhibitors of matrix metalloproteases. S179D PRL also blocked signaling from bFGF in these cells. We conclude that this molecular mimic of a pituitary hormone is a potent anti-angiogenic protein, partly as a result of its ability to reduce utilization of several well-established endothelial autocrine growth loops, partly by its ability to block signaling from bFGF and partly because of its ability to decrease endothelial migration. These findings suggest that circulating levels of phosphorylated PRL may influence the progression of cancer and, furthermore, that S179D PRL may be a useful anti-angiogenic therapeutic.

Non-cultured dermal-derived mesenchymal cells attenuate sepsis induced by cecal ligation and puncture in mice.

Sepsis remains a threat to critically ill patients and carries a high morbidity and mortality. Cell-based therapies have risen in prominence in recent years. Dermal-derived mesenchymal cells (DMCs) are attractive as one of the abundant sources from which to isolate mesenchymal cells for therapeutic applications and can be easily accessed with minimal harm to the donor. In this study, we described for the first time the use of non-cultured DMCs for treating sepsis in a cecal ligation and puncture (CLP) mouse model and investigated their immunomodulatory effects. We found that non-cultured DMCs administration provides a beneficial effect to improve survival in CLP-induced sepsis. This effect is partly mediated by the ability of DMCs to home to sites of injury, to reduce the inflammatory response, to inhibit apoptosis, and to stimulate macrophage migration and phagocytosis. Our further findings suggest that DMCs treatment modulates the beneficial cytoprotective effects exhibited during sepsis, at least in part, by altering miRNA expression. These discoveries provide important evidence that non-cultured DMCs therapy has a specific anti-inflammatory effect on sepsis, and provide the basis for the development of a new therapeutic strategy for managing clinical sepsis.

Small molecular neutral microcrystalline iridium(III) complexes as promising molecular oxygen sensors.

Small molecular neutral Ir(III) complexes have been demonstrated to be promising self-inclusive microcrystalline thin-film oxygen sensors with relatively high sensitivity (Ksv = 6.41), good stability, and linear Stern-Volmer behavior (R(2) = 0.9979).

The predictive effect of overexpressed miR-34a on good survival of cancer patients: a systematic review and meta-analysis.

BACKGROUND: MicroRNA-34a (miR-34a) is a potential prognostic factor for survival in patients with several types of cancer according to previous clinical researches. We conducted a systematic review and meta-analysis to summarize the significance of increased miR-34a expression in the prognosis of patients' overall survival. MATERIALS AND METHODS: The present systematic review and meta-analysis of 15 researches included 2,597 patients. Overexpression of miR-34a may predict good overall survival ([OS], HR =0.76, 95% confidence interval: 0.55-1.06, P=0.105), but the effect was not significant enough. Subgroup analysis results showed miR-34a was an ideal predictor for digestive system cancer (OS, HR =0.50, 95% confidence interval: 0.25-0.99, P=0.048). The predictive effects of elevated expression of miR-34a on the OS of untreated and treated patients were not of obvious differences. CONCLUSION: This systematic review and meta-analysis showed that miR-34a has a predictive effect on overall survival of patients with digestive system cancer.

[Determination of zearalenone and its metabolites in grains by capillary electrophoresis].

OBJECTIVE: To establish a method for the determination of zearalenone and its metabolites in grains by capillary electrophoresis. METHODS: The samples were extracted with liquid-liquid partition and ultrasonic extraction, cleaned up with C18 cartridges, then determined by micellar electrokinetic capillary chromatography (MEKC). RESULTS: The limits of detection for Zearalenone (ZON), alpha-Zearalenol (alpha-ZOL), beta-Zearalenol (beta-ZOL), Aflatoxin B1 (AFT B1) and Ochratoxin A (Och A) were 0.0084, 0.081, 0.14, 0.0016 and 0.031 microgram/ml, respectively. Recoveries of all compounds were in the range of 77.9%-103.1%. Relative standard deviations (RSDs) were 0.63%-1.98%. CONCLUSION: This sensitive and accurate method can be used in the determination of zearalenone and its metabolites in grains.

Elevated expression of miR-210 predicts poor survival of cancer patients: a systematic review and meta-analysis.

BACKGROUND: MiRNAs are important regulators of different biological processes, including tumorigenesis. MiR-210 is a potential prognostic factor for survival in patients with cancer according to previous clinical researches. We conducted a systematic review and meta-analysis to summarize the significance of increased miR-210 expression in the prognosis of indicated cancers. METHODOLOGY/PRINCIPAL FINDINGS: The present systematic review and meta-analysis of 16 researches included 1809 patients with 7 different types of cancers from 7 countries, and aimed to explore the association between miR-210 expression and the survival of cancer patients. Over-expression of miR-210 may predict poor overall survival (OS, HR = 1.33, 95% CI: 0.85-2.09, P = 0.210), but the effect was not significant. While the predictive effect on disease-free survival (DFS, HR = 1.89, 95% CI: 1.30-2.74, P = 0.001), progression-free survival (PFS, HR = 1.20, 95% CI: 1.05-1.38, P = 0.007) and relapse-free survival(RFS, HR = 4.42, 95% CI: 2.14-9.15, P = 0.000) for patients with breast cancer, primary head and neck squamous cell carcinoma (HNSCC), renal cancer, soft-tissue sarcoma, pediatric osteosarcoma, bladder cancer or glioblastoma was certain. Subgroup analysis showed the limited predictive effect of over-expressed miR-210 on breast cancer OS (HR = 1.63, 95% CI: 0.47-5.67, P = 0.443), breast cancer DFS (HR = 2.03, 95% CI: 0.90-4.57, P = 0.088), sarcoma OS (HR = 1.24, 95% CI: 0.20-7.89, P = 0.818) and renal cancer OS (HR = 1.16, 95% CI: 0.27-4.94, P = 0.842). CONCLUSIONS/SIGNIFICANCE: This systematic review and meta-analysis suggests that miR-210 has a predictive effect on survival of patients with studied cancer types as indexed by disease-free survival, progression-free survival and relapse-free survival. While the predictive effect on overall survival, breast cancer overall survival, breast cancer disease-free survival, sarcoma overall survival and renal cancer overall survival was not statistically significant.

Hyperplasia of pericytes is one of the main characteristics of microvascular architecture in malignant glioma.

OBJECTIVES: To investigate the role of pericytes in constructing the malformed microvessels (MVs) and participating microvascular architecture heterogeneity of glioma. METHODS: Forty human glioma tissue samples (WHO grade II-IV) were included in present study. Observation of blood vessel patterns, quantitative analysis of endothelial cells (ECs)- and pericyte-labeled MVs and comparison between malignant grades based on single- or double-immunohistochemical staining. The MV number density (MVND), microvascular pericyte number density (MPND), and microvascular pericyte area density (MPAD) were calculated. The expression of PDGFß was also scored after immunostaining. RESULTS: In grade II glioma, most of tumor MVs were the thin-wall CD34+ vessels with near normal morphology. In addition to thin-wall CD34+ MVs, more thick-wall MVs were found in grade III glioma, which often showed α-SMA positive. Most of MVs in grade IV glioma were in the form of plexus, curled cell cords and glomeruloid microvascular proliferation while the α-SMA+ cells were the main components. The MVs usually showed disordered arrangement, loose connection and active cell proliferation as shown by Ki67 and α-SMA coexpression. With the increase of glioma grades, the α-SMA+ MVND, CD34+ MVND and MPND were significantly augmented although the increase of CD34+ MVND but not MPAD was statistically insignificant between grade III and IV. It was interesting that some vessel-like structures only consist of α-SMA+ cells, assuming the guiding role of pericytes in angiogenesis. The expression level of PDGFß was upregulated and directly correlated with the MPND in different glioma grades. CONCLUSION: Hyperplasia of pericytes was one of the significant characteristics of malignant glioma and locally proliferated pericytes were the main constituent of MVs in high grade glioma. The pathological characteristics of pericytes could be used as indexes of malignant grades of glioma.