High-throughput screening identifies ibuprofen as an sEV PD-L1 inhibitor for synergistic cancer immunotherapy.

Programmed death-ligand 1 (PD-L1) on tumor-derived small extracellular vesicles (sEVs) limits therapeutic effectiveness by interacting with the PD-1 receptor on host immune cells. Targeting the secretion of sEV PD-L1 has emerged as a promising strategy to enhance immunotherapy. However, the lack of small-molecule inhibitors poses a challenge for clinical translation. In this study, we developed a target and phenotype dual-driven high-throughput screening strategy that combined virtual screening with nanoflow-based experimental verification. We identified ibuprofen (IBP) as a novel inhibitor that effectively targeted sEV PD-L1 secretion. IBP disrupted the biogenesis and secretion of PD-L1+ sEVs in tumor cells by physically interacting with a critical regulator of sEV biogenesis, hepatocyte growth factor-regulated tyrosine kinase substrate. Notably, the mechanism of action of IBP is distinct from its commonly known targets, cyclooxygenases. Administration of IBP stimulated antitumor immunity and enhanced the efficacy of anti-PD-1 therapy in melanoma and oral squamous cell carcinoma mouse models. To address potential adverse effects, we further developed an IBP gel for topical application, which demonstrated remarkable therapeutic efficacy when combined with anti-PD-1 treatment. The discovery of this specific small inhibitor provides a promising avenue for establishing durable, systemic antitumor immunity.

Systemic development of wheat-Thinopyrum elongatum translocation lines and their deployment in wheat breeding for Fusarium head blight resistance.

Fusarium head blight (FHB), mainly caused by Fusarium graminearum, is one of the most destructive diseases of wheat (Triticum aestivum) around the world. FHB causes significant yield losses and reduces grain quality. The lack of resistance resources is a major bottleneck for wheat FHB resistance breeding. As a wheat relative, Thinopyrum elongatum contains many genes that can be used for wheat improvement. Although the novel gene Fhb-7EL was mapped on chromosome 7EL of Th. elongatum, successful transfer of the FHB resistance gene into commercial wheat varieties has not been reported. In this study, we developed 836 wheat-Th. elongatum translocation lines of various types by irradiating the pollen of the wheat-Th. elongatum addition line CS-7EL at the flowering stage, among which 81 were identified as resistant to FHB. By backcrossing the FHB-resistant lines with the main cultivar Jimai 22, three wheat-Th. elongatum translocation lines, Zhongke 1878, Zhongke 166, and Zhongke 545, were successfully applied in wheat breeding without yield penalty. Combining karyotype and phenotype analyses, we mapped the Fhb-7EL gene to the distal end of chromosome 7EL. Five molecular markers linked with the FHB resistance interval were developed, which facilitates molecular marker-assisted breeding. Altogether, we successfully applied alien chromatin with FHB resistance from Th. elongatum in wheat breeding without yield penalty. These newly developed FHB-resistant wheat-Th. elongatum translocation lines, Zhongke 1878, Zhongke 166, and Zhongke 545, can be used as novel resistance resources for wheat breeding.

Tribofilm Formation of Zinc Dialkyldithiophosphate as Oil Additives Impregnated in Porous Polyimide under Different Contacts.

To improve the tribological properties of porous polyimide (PPI), ZDDP-mixed PAO4 was impregnated in PPI (denoted as ZPPI), and the tribological properties of ZPPI under single- and double-contacts were investigated. In the single-contact of ZPPI-steel, a rough and thick tribofilm was formed on the steel ball, which could protect the steel surface but resulted in large fluctuations in the friction coefficient. In the double-contact of ZPPI-steel-steel, ZDDP formed a uniform and thinner tribofilm on steel surfaces, leading to a lower friction. ZDDP could inhibit the formation of iron oxides significantly in the double-contact, while the antioxidant effect of ZDDP in the single-contact of ZPPI-steel was not obvious. ZnS and ZnO generated from ZDDP were adsorbed in the ZPPI pores, which aggravated the blackening of the ZPPI worn surface.

Immunosuppressive effect of small extracellular vesicle PD-L1 is restricted by co-expression of CD80.

BACKGROUND: The PD-L1 on tumor cell-derived small extracellular vesicles (sEVs) can suppress the proliferation and cytokine production of T cells. However, PD-L1 can also be expressed by non-tumor cells. The present study is designed to test whether immunocytes release immunosuppressive PD-L1-positive sEVs. METHODS: sEVs were isolated from different clinical samples of head and neck squamous cell carcinoma (HNSCC) patients, the level and cellular origins of PD-L1-positive sEVs were assessed. Co-expression of CD80 on PD-L1-positive sEVs was examined to evaluate the immunosuppressive and tumor-promotive effects. RESULTS: PD-L1-positive sEVs in HNSCC patients had various cellular origins, including tumor cell, T cell, B cell, dendritic cell and monocyte/macrophage. However, PD-L1-positive sEVs derived from immune cells did not exert immunosuppressive functions due to the co-expression of CD80. It was verified that co-expression of CD80 disrupted the binding of sEV PD-L1 to its receptor PD-1 on T cells and attenuated the immunosuppression mediated by sEV PD-L1 both in vitro and in vivo. CONCLUSION: The study suggests that PD-L1-positive sEVs have the cellular origin and functional heterogeneity. Co-expression of CD80 could restrict the immunosuppressive effect of sEV PD-L1. A greater understanding of PD-L1-positive sEV subsets is required to further improve their clinical application.

The predictive value and correlation of ß-catenin, CMTM6, and PD-L1 expression in colorectal cancer.

CMTM6 is a major regulator of PD-L1 expression. Aberrant Wnt pathway signaling occurs in most sporadic colorectal cancers (CRC). However, the significance and correlation of ß-catenin, CMTM6, and PD-L1 immunohistochemical expression in CRC is still unknown and need to be further verified. We evaluated the expression levels of ß-catenin, CMTM6, PD-L1, and MMR (mismatch repair) proteins by immunohistochemistry in CRC tissue microarray (TMA), and evaluated the association among ß-catenin, CMTM6, PD-L1 expression, MMR status, and clinicopathological features in 704 CRC patients. Positive expression of PD-L1 in tumor cells (TC) is associated with more frequent dMMR (mismatch repair deficient) status, CMTM6 expression, right colon, and younger CRC patients. The expression of PD-L1 in tumor-infiltrating immune cells (IC) is associated with a higher frequency of adenocarcinoma, ß-catenin, and CMTM6 expression. In univariate analysis, age, histological subtype, histologic grade, lymphatic metastasis, TNM stage, MMR status, and expression of PD-L1 protein in IC were significantly associated with the overall survival. In multivariate analysis, age, histologic grade, TNM stage, MMR status, and expression of PD-L1 protein in IC were independent prognostic factors. The overall survival of the adjuvant chemotherapy group was significantly higher than those non-chemotherapy in TNM stage III-IV CRC patients, but no significant overall survival improvement was found in the positive PD-L1 in TC, positive PD-L1 in IC, positive CMTM6, low ß-catenin expression, or dMMR status subgroups. Expression of CMTM6 and PD-L1 in CRC are positively associated with ß-catenin and reliable biomarkers for the prediction of responding to chemotherapy. The expression of ß-catenin/CMTM6/PD-L1 and MMR status may be valuable biomarkers for guiding different treatment strategies in CRC patients.

The important role of the receptor for activated C kinase 1 (RACK1) in nasopharyngeal carcinoma progression.

BACKGROUND: The receptor for activated C kinase 1 (RACK1) is involved in various cancers, but its roles in nasopharyngeal carcinoma (NPC) have not yet been fully elucidated. METHODS: Initially, RACK1 expression was analyzed by immunohistochemistry in NPC and normal nasopharyngeal (NP) tissues. It was also detected by qPCR and Western blot in NPC cells. Confocal microscope and immunofluorescence were performed to detect the subcellular compartmentalization of RACK1. Subsequently, after up- or down-regulating RACK1 in NPC cells, cell proliferation and migration/invasion were tested using in vitro assays including MTT, EdU, colony formation, Transwell and Boyden assays. Furthermore, several key molecules were detected by Western blot to explore underlying mechanism. Finally, clinical samples were analyzed to confirm the relationship between RACK1 expression and clinical features. RESULTS: Receptor for activated C kinase 1 expression was much higher in NPC than NP tissues. And RACK1 was mainly located in the cytoplasm. Overexpression of RACK1 promoted NPC cell proliferation and metastasis/invasion, whereas depletion of this protein suppressed NPC cell proliferation and metastasis/invasion. Mechanistically, RACK1 deprivation obviously suppressed the activation of Akt and FAK, suggesting the PI3K/Akt/FAK pathway as one of functional mechanisms of RACK1 in NPC. Furthermore, clinical sample analysis indicated a positive correlation between in vivo expression of RACK1 with lymph node invasion and clinical stage of NPC. CONCLUSION: Our results demonstrate that RACK1 protein plays an important role in NPC development and progression. The upregulation of RACK1 can promote the proliferation and invasion of NPC by regulating the PI3K/Akt/FAK signal pathway. Thus, this study contributes to the discovery of a potential therapeutic target for NPC.

The expression of CXCL13 and its relation to unfavorable clinical characteristics in young breast cancer.

BACKGROUND: Young breast cancer occupies a higher and higher proportion of breast cancer, especially in Asia, and is associated with a more unfavorable prognosis compared with the disease arising in older women. However, the poor prognosis of young breast cancer cannot be fully explained by the clinical and molecular factors. METHODS: This study investigated 1125 Chinese breast cancer patients diagnosed from 2009 to 2013. A data mining of gene expression profiles was performed for the young and older breast cancer patients, identifying significantly differentially expressed genes. Quantitative RT-PCR, Western blotting and immunohistochemistry assay were carried out for the clinical sample validations. RESULTS: The investigation firstly displayed that young patients (≤45 years) accounted for 47.6 % (535/1125) of breast cancer, and clinically associated with some unfavorable factors related to poor prognosis, such as invasive pathological type, high tumor grade, lymph node positive, ER negative and triple-negative subtype. Subsequently, 553 significantly differentially expressed genes were identified by the data mining. Of them, a set of genes related to immune function were observed to be up-regulated in young patients with breast cancer. Impressively, the CXCL13 (C-X-C motif chemokine 13) expression level showed the most significant difference (FC = 2.64, P = 8.2 × 10(-4)). Furthermore, the validations with clinical samples and correlation analysis demonstrated that CXCL13 was indeed highly expressed in young breast cancer and closely associated with some prognostic factors including lymph node positive and ER negative. CONCLUSION: This is the first to indicate the clinical relevance of CXCL13 to young breast cancer and represents a potential therapeutic target for young breast cancer.

TGFßR2 is a major target of miR-93 in nasopharyngeal carcinoma aggressiveness.

BACKGROUND: MiR-17-92 cluster and its paralogues have emerged as crucial regulators of many oncogenes and tumor suppressors. Transforming growth factor-ß receptor II (TGFßR2), as an important tumor suppressor, is involved in various cancer types. However, it is in cancer that only two miRNAs of this cluster and its paralogues have been reported so far to regulate TGFßR2. MiR-93 is oncogenic, but its targetome in cancer has not been fully defined. The role of miR-93 in nasopharyngeal carcinoma (NPC) still remains largely unknown. METHODS: We firstly evaluated the clinical signature of TGFßR2 down-regulation in clinical samples, and next used a miRNA expression profiling analysis followed by multi-validations, including Luciferase reporter assay, to identify miRNAs targeting TGFßR2 in NPC. In vitro and in vivo studies were performed to further investigate the effects of miRNA-mediated TGFßR2 down-regulation on NPC aggressiveness. Finally, mechanism studies were conducted to explore the associated pathway and genes influenced by this miRNA-mediated TGFßR2 down-regulation. RESULTS: TGFßR2 was down-regulated in more than 50% of NPC patients. It is an unfavorable prognosis factor contributing to clinical NPC aggressiveness. A cluster set of 4 TGFßR2-associated miRNAs was identified; they are all from miR-17-92 cluster and its paralogues, of which miR-93 was one of the most significant miRNAs, directly targeting TGFßR2, promoting cell proliferation, invasion and metastasis in vitro and in vivo. Moreover, miR-93 resulted in the attenuation of Smad-dependent TGF-ß signaling and the activation of PI3K/Akt pathway by suppressing TGFßR2, further promoting NPC cell uncontrolled growth, invasion, metastasis and EMT-like process. Impressively, the knockdown of TGFßR2 by siRNA displayed a consentaneous phenocopy with the effect of miR-93 in NPC cells, supporting TGFßR2 is a major target of miR-93. Our findings were also substantiated by investigation of the clinical signatures of miR-93 and TGFßR2 in NPC. CONCLUSION: The present study reports an involvement of miR-93-mediated TGFßR2 down-regulation in NPC aggressiveness, thus giving extended insights into molecular mechanisms underlying cancer aggressiveness. Approaches aimed at blocking miR-93 may serve as a promising therapeutic strategy for treating NPC patients.

Driver gene alterations in NSCLC patients in southern China and their correlation with clinicopathologic characteristics.

Introduction: In this study, we aimed to explore the relationship between clinicopathological features and driver gene changes in Chinese NSCLC patients. Methods: Amplification refractory mutation system PCR was used to detect the aberrations of 10 driver oncogenes in 851 Chinese NSCLC patients, and their correlation with clinicopathological characteristics was also analyzed. Moreover, three models of logistic regression were used to analyze the association between histopathology and EGFR or KRAS mutations. Results: The top two most frequently aberrant target oncogenes were EGFR (48.06%) and KRAS (9.51%). These were followed by ALK (5.41%), HER2 (2.35%), MET (2.23%), RET (2.11%), ROS1 (1.88%), BRAF (0.47%), NRAS (0.24%), and PIK3CA (0.12%). Additionally, 11 (1.29%) patients had synchronous gene alterations in two genes. The main EGFR mutations were exon 21 L858R and exon 19-Del, which accounted for 45.97% and 42.79% of all EGFR mutations, respectively. Logistic regression analysis showed that the frequency of EGFR mutations was positively correlated with women, non-smokers, lung adenocarcinoma, and invasive non-mucinous adenocarcinoma (IA), and negatively correlated with solid nodule, micro-invasive adenocarcinoma, and solid-predominant adenocarcinoma. KRAS mutations were positively associated with men and longer tumor long diameters and negatively correlated with lung adenocarcinoma (P < 0.05 for all). Conclusion: Our findings suggest that the EGFR mutation frequency was higher in women, non-smokers, lung adenocarcinoma, and the IA subtype in lung adenocarcinoma patients, while the KRAS mutation rate was higher in men and patients with longer tumor long diameter and lower in lung adenocarcinoma patients.

The Predictive Value of CD3+/CD8+ Lymphocyte Infiltration and PD-L1 Expression in Colorectal Cancer.

AIM: The immune system plays an important role in tumor development and treatment. In this study, we aimed to determine the relationships among the expressions of PD-L1, CD3, CD8, MMR proteins, clinicopathological features, and prognosis of CRC. METHODS: Immunohistochemistry was used to determine the expression of PD-L1, CD3, and CD8 in 771 patients with CRC. RESULTS: The expression of PD-L1 in TC was related to the right colon, adenocarcinoma, and dMMR, and in IC, it was related to younger CRC patients and the TNM stage. The expression of CD3 and CD8 in tumor-infiltrating lymphocytes was related to lymph node metastasis and the TNM stage. The expression of PD-L1 in TC and IC was correlated with the infiltration of CD3+ and CD8+ lymphocytes. Univariate survival analysis showed that the expression of PD-L1 in TC, IC, and dMMR was related to a better prognosis. Multivariate survival analysis showed that age, TNM stage, and dMMR were independent prognostic factors for CRC. The OS of the chemotherapy was significantly higher than that of the non-chemotherapy in III-IV TNM stage patients; CRC patients with positive PD-L1 expression in TC or IC and dMMR did not benefit from chemotherapy. CONCLUSIONS: PD-L1 expression in TC and IC was closely related to the density of CD3 and CD8 infiltration in tumor-infiltrating lymphocytes. The expression of CD3 and CD8 in tumor-infiltrating lymphocytes and the expression of PD-L1 in IC were linked to the TNM stage of CRC patients. PD-L1 expression in TC and IC and MMR status may act as an important biomarker for guiding the postoperative treatment of III-IV TNM stage CRC patients.

Stabilizing and upregulating Axin with tankyrase inhibitor reverses 5-fluorouracil chemoresistance and proliferation by targeting the WNT/caveolin-1 axis in colorectal cancer cells.

Chemoresistance is a main obstacle for colorectal cancer treatment. In this study, we evaluated the effects and mechanisms of the WNT/ß-catenin signaling pathway on the chemoresistance of SW480 and SW620 colorectal cancer cells. The activity of ß-catenin was activated/inhibited by the small molecule compound GSK-3 inhibitor 6-bromo-indirubin-3'-oxime and the tankyrase inhibitor XAV939. The downstream target genes of the WNT/ß-catenin signaling pathway were screened using a cDNA microarray and bioinformatics analysis. Apoptosis induced by 5-Fu, cell cycle distribution and expression levels of WNT/ß-catenin/TCF12/caveolin-1 and multidrug resistance proteins were examed by flow cytometry and western blot after ß-catenin activation/inhibition and caveolin-1 overexpression/interference. The effect and mechanism of XAV939 on proliferation and apoptosis induced by 5-Fu in xenograft tumors of nude mice were evaluated by immunohistochemistry and TUNEL staining. 6-Bromo-indirubin-3'-oxime treatment increased ß-catenin expression by regulating GSK-3ß phosphorylation, accompanied by upregulation of TCF12, caveolin-1, P-gp, and MRP2 and downregulation of apoptosis induced by 5-Fu. Conversely, XAV939 treatment decreased ß-catenin expression by upregulating Axin, accompanied by downregulation of TCF12, Caveolin-1, P-gp, and MRP2 and upregulation of apoptosis induced by 5-Fu. The caveolin-1 gene was identified as an important downstream gene of the WNT/ß-catenin signaling pathway. Caveolin-1 overexpression upregulated ß-catenin expression, increased P-gp and MRP2 expression and decreased apoptosis induced by 5-Fu; conversely, caveolin-1 interference caused the opposite effects. In addition, in vivo experiments showed that XAV939 treatment reduced ß-catenin expression, increased apoptosis induced by 5-Fu and repressed xenograft tumor growth. Our findings suggested that inhibition of WNT/ß-catenin/TCF12/caveolin-1 provides a new promising therapeutic strategy for colorectal cancer treatment.

Bioinspired gelatin nano-film implanted into composite scaffold exhibiting both expandable adhesion and enhanced proliferation.

Tissue engineering technology provides a new treatment to the cartilage damage. Recent progress has focused on coating strategies with the printed scaffold surface, using various materials such as bioactive nanocomposites. However, the fracture and exfoliation of printed scaffolds remain challenges due to their poor adhesion on smooth substrates. These limitations can be offset by developing a versatile film. Here, inspired by the mechanism of the wet adhesion of snails, we introduced a biomimetic nanoscale gelatin film between a smooth conductive slide and a scaffold, which enhanced early cell adhesion rates through water absorption, swelling and adhesion. A bionic technique of preparing gelatin nanofilms and PVP/PCL 3D scaffolds, which involved E-Jet atomization deposition and E-Jet printing techniques based on the electrohydrodynamic effect, was investigated. It is found that the composite scaffold with 400 nm gelatin nanofilm significantly enhances cell attachment (from 62 % to 87 %) and proliferation (increased 6.5 times in 7 days). Collectively, this study highlights the combination of biomimetic nanoscale adhesive film in promoting cell adhesion and cartilage differentiation, which benefiting from water absorption and swelling of gelatin nanofilm. This work provides a new idea for the potential application in the orthopedics field.

Biomimetic Water-Repelling Surfaces with Robustly Flexible Structures.

Biomimetic liquid-repelling surfaces have been the subject of considerable scientific research and technological application. To design such surfaces, a flexibility-based oscillation strategy has been shown to resolve the problem of liquid-surface positioning encountered by the previous, rigidity-based asymmetry strategy; however, its usage is limited by weak mechanical robustness and confined repellency enhancement. Here, we design a flexible surface comprising mesoscale heads and microscale spring sets, in analogy to the mushroomlike geometry discovered on springtail cuticles, and then realize this through three-dimensional projection microstereolithography. Such a surface exhibits strong mechanical robustness against ubiquitous normal and shear compression and even endures tribological friction. Simultaneously, the surface elevates water repellency for impacting droplets by enhancing impalement resistance and reducing contact time, partially reaching an improvement of ∼80% via structural tilting movements. This is the first demonstration of flexible interfacial structures to robustly endure tribological friction as well as to promote water repellency, approaching real-world applications of water repelling. Also, a flexibility gradient is created on the surface to directionally manipulate droplets, paving the way for droplet transport.

[Evaluation of pre-harvest sprouting resistance of wheat varieties from different ecological regions.] / 不同生态区小麦品种的穗发芽抗性评价.

The pre-harvest sprouting (PHS) resistance of 137 wheat varieties from different regions was evaluated and the relative germination index (RGI) was calculated. The relationships between PHS and grain quality traits, amylase activity and related gene expression level of these varieties were analyzed. The results showed that wheat varieties from the middle and lower reaches of Yangtze River Valley winter wheat region had the lowest RGI value and the highest ratio of resistant pre-harvest sprouting wheat varieties, followed by the varieties from the upper reach of Yangtze River Valley winter wheat region and from the Yellow and Huai River Valley. Red-grain wheat had lower RGI than white-grain wheat. RGI was positively correlated with seed length, seed width, and spikelet number, but not correlated with other grain traits (panicle type, ear color, ear length, and spikelet density, grain per spike and 1000-grain weight). RGI was negatively associated with the test weight, dough development time, and flour yield, but not with other quality indices (protein content, wet gluten content, water absorption, stability time, sedimentation, extension area, extensibility and max resistance). Amylase activity of different varieties increased with seed imbibition time. RGI was positively associated with α-amylase activity after germinating for 24-72 hours. The cluster analysis results of resistant varieties were consistent with the PHS resistance evaluation after 48 hours. RGI was positively associated with the related gene expression with seed imbibition time.

Lnc-M2 controls M2 macrophage differentiation via the PKA/CREB pathway.

Long noncoding RNAs (lncRNAs) play an indispensable role in the process of M1 macrophage via regulating the development of macrophages and their responses to bacterial pathogens and viral infections. However, there are few studies on the lncRNA-mediated functions and regulatory mechanisms of M2 macrophage polarization. In this study, we found a number of differentially expressed lncRNAs between human monocyte derived M0 and M2 macrophages according to array analysis and quantitative polymerase chain reaction (qPCR) validation. The lncRNA RP11-389C8.2 (we named lnc-M2 in this study) was observed to be highly expressed in M2 macrophages. In Situ Localization and Quantification Analysis showed that lnc-M2 was expressed in the nucleus and cytosolic compartments of M2 macrophages. Notably, lnc-M2 knockdown enhanced the phagocytic ability of M2 macrophages. Ulteriorly, the results of RNA-Protein interaction experiments indicated that protein kinase A (PKA) was a lnc-M2 associated RNA-binding protein (RBP). Western blot showed that phosphorylated cAMP response element binding protein (p-CREB), a well-known key downstream transcription factor of PKA, was lowly phosphorylated in lnc-M2-silencing M2 macrophages. Furthermore, we found that transcriptional factor Signal Transducer And Activator Of Transcription 3 (STAT3) promoted lnc-M2 transcription along with the up-regulation of epigenetic histone modification markers at the lnc-M2 promoter locus, indicating that STAT3 activated lnc-M2 and eventually facilitated the process of M2 macrophage differentiation via the PKA/CREB pathway. Collectively, our date provide evidence that the transcription factor STAT3 can promote the transcription of lnc-M2 and facilitated the process of M2 macrophage differentiation via the PKA/CREB pathway. This study highlights a novel mechanism underlying the M2 macrophage differentiation.

Author Correction: Epstein-Barr virus-encoded microRNA BART1 induces tumour metastasis by regulating PTEN-dependent pathways in nasopharyngeal carcinoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Low concentration trifluoperazine promotes proliferation and reduces calcium-dependent apoptosis in glioma cells.

Glioma patients constitute the greatest percentage of depressed neoplasm patients. These patients often require antidepressant treatment, but the effect of antidepressant drugs on glioma cells requires further evaluation. In the present study, we evaluated the effect of trifluoperazine (TFP) on the proliferation and apoptosis of glioma cells. Transcriptomic and bioinformatics analysis results suggested that antidepressant drugs, especially TFP, may upregulate the drug-resistant ability of glioma cells. A low concentration of TFP upregulated the viability of glioma cells. Colony formation and EdU assays confirmed that TFP treatment accelerates glioma cell proliferation, but no significant difference was found in the cell cycle distribution of glioma cells after treatment with TFP or control. Flow cytometry and TUNEL staining results suggested that TFP treatment decreased apoptosis in glioma cells. In addition, TFP treatment downregulated the intracellular Ca2+ concentration of glioma cells. In vivo experimental results indicated that TFP treatment promoted proliferation and reduced apoptosis in xenograft tumours in nude mice. Taken together, our results suggest that a low concentration of TFP promotes proliferation and reduces apoptosis in glioma cells both in vitro and in vivo. The potential harmful effects of antidepressant drugs on gliomas require further evaluation before their use in glioma patients.

Chinese Wheat Mosaic Virus-Induced Gene Silencing in Monocots and Dicots at Low Temperature.

Virus-induced gene silencing (VIGS) is an important tool for functional genomics studies in plants. With this method, it is possible to target most endogenous genes and downregulate the messenger RNA (mRNA) in a sequence-specific manner. Chinese wheat mosaic virus (CWMV) has a bipartite, single-strand positive RNA genome, and can infect both wheat and Nicotiana benthamiana, and the optimal temperature for systemic infection in plants is 17°C. To assess the potential of the virus as a vector for gene silencing at low temperature, a fragment of the N. benthamiana or wheat phytoene desaturase (PDS) gene was expressed from a modified CWMV RNA2 clone and the resulting photo bleaching in infected plants was used as a reporter for silencing. Downregulation of PDS mRNA was also measured by quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR). In experiments using fragments of PDS ranging from 500 to 1500 nucleotides, insert length influenced the stability and the efficiency of VIGS. The CWMV induced silencing system was also used to suppress miR165/166 and miR3134a through expression of miRNA target mimics. The relative expression levels of mature miR165/166 and miR3134a decreased whereas the transcript levels of their target genes increased. Interestingly, we also found the CWMV-induced silencing system was more efficient compare with the vector based on Barley stripe mosaic virus (BSMV) or Foxtail mosaic virus (FoMV) in wheat or the vector based on TRV in N. benthamiana at 17°C. In summary, the CWMV vector is effective in silencing endogenous genes and miRNAs at 17°C, thereby providing a powerful tool for gene function analysis in both N. benthamiana and wheat at low temperature.