Post-translational Control of the Temporal Dynamics of Transcription Factor Activity Regulates Neurogenesis.

Neurogenesis is initiated by the transient expression of the highly conserved proneural proteins, bHLH transcriptional regulators. Here, we discover a conserved post-translational switch governing the duration of proneural protein activity that is required for proper neuronal development. Phosphorylation of a single Serine at the same position in Scute and Atonal proneural proteins governs the transition from active to inactive forms by regulating DNA binding. The equivalent Neurogenin2 Threonine also regulates DNA binding and proneural activity in the developing mammalian neocortex. Using genome editing in Drosophila, we show that Atonal outlives its mRNA but is inactivated by phosphorylation. Inhibiting the phosphorylation of the conserved proneural Serine causes quantitative changes in expression dynamics and target gene expression resulting in neuronal number and fate defects. Strikingly, even a subtle change from Serine to Threonine appears to shift the duration of Atonal activity in vivo, resulting in neuronal fate defects.

The Drosophila neurogenin Tap functionally interacts with the Wnt-PCP pathway to regulate neuronal extension and guidance.

The neurogenin (Ngn) transcription factors control early neurogenesis and neurite outgrowth in mammalian cortex. In contrast to their proneural activity, their function in neurite growth is poorly understood. Drosophila has a single predicted Ngn homolog, Tap, of unknown function. Here we show that Tap is not a proneural protein in Drosophila but is required for proper axonal growth and guidance of neurons of the mushroom body, a neuropile required for associative learning and memory. Genetic and expression analyses suggest that Tap inhibits excessive axonal growth by fine regulation of the levels of the Wnt signaling adaptor protein Dishevelled.

An ultrasensitive and simple assay for the Hepatitis C virus using a reduced graphene oxide-assisted hybridization chain reaction.

Hepatitis C virus (HCV) is a major cause of chronic liver disease, which affects 2-3% of the world population. Until now, the early detection of HCV has been a great challenge, especially for those who live in developing countries. In this study, we developed a novel and ultrasensitive assay for the detection of HCV RNA based on the reduced graphene oxide nanosheets (rGONS) and hybridization chain reaction (HCR) amplification technique. This detection system contains a pair of single fluorophore-labeled hairpin probes that can freely exist in the solution in the absence of target RNA. The introduction of target RNA can robustly trigger a HCR with the two probes and produce long nanowires containing a double-stranded structure. The weak adsorption to rGONS makes the long nanowires emit a strong fluorescence. Using this enzyme-free amplification strategy, we developed a new method for the HCV RNA assay with a detection limit of 10 fM, which is far more sensitive than the common GO-based fluorescence method. Furthermore, the new method exhibits high selectivity for the discrimination of perfectly complementary and mismatched sequences. Finally, the new method was successfully used as a HCV RNA assay in biological samples with a strong anti-interference capability in complicated environments. In summary, these remarkable characteristics of the new method highlight its potential use in a clinical sample primary screening.

Fluorometric determination of RNase H via a DNAzyme conjugated to reduced graphene oxide, and its application to screening for inhibitors and activators.

A new fluorometric method is delineated for the detection of RNase H activity by combining DNAzyme with reduced graphene oxide (rGO). In the absence of RNase H, the fluorescence of FAM-labeled probe is quenched due to the strong adsorption on the rGO. The presence of RNase H can release the active DNAzyme from the DNA-RNA chimeric strand. This triggers the cleavage of the signal probe at the rA site with the help of the cofactor Mg2+. The recycle cleavage can directly result in the amplified signal emitted by the FAM-labeled short fragment. The method allows the activity of RNase H to be detected in a linear range of 0.01 to 5 U·mL-1. The detection limit of 0.018 U·mL-1 is calculated by the principle of three-time standard deviation over the blank signal. Then, RNase H-targeting natural compounds were screened for their inhibitory action. Among the investigated compounds, five were screened as RNase H inhibitors in a concentration-dependent manner, and 4 compounds were identified as activators. Finally, the method was reliably used for discriminating the difference of RNase H activity in human serum. It is found that RNase H activity was upregulated in patients with hepatitis C virus infection. Graphical abstract The schematic presentation of rGO-DNAzyme-based RNase H detection. RNase H triggers the active DNAzyme releasing from the DNA-RNA chimeric strand, which can cleavage probes to FAM-labeled short fragments and make the fluorescence signal cycle amplified.

Neurogenins in brain development and disease: an overview.

The production of neurons, astrocytes and oligodendrocytes is regulated by a group of transcription factors, which determine cell fates and specify subtype identities in the nervous system. Here we focus on profiling the distinct roles of Neurogenin (Ngn or Neurog) family members during the neuronal development. Ngn proteins are tightly regulated to be expressed at defined times and positions of different progenitor cell pools. In addition to their well-elucidated proneural function, Ngn proteins play various critical roles to specify or maintain cell fate and regulate neurite outgrowth and targeting in the central nervous system. Finally, Ngns have been associated with neuronal disorders. Therefore understanding the function and regulation of Ngns will not only improve the understanding of the molecular mechanism underlying the development of nervous system, but may also provide insight into neuronal disease.

Identifying α-KG-dependent prognostic signature for lower-grade glioma based on transcriptome profiles.

The inhibition of alpha-ketoglutarate (α-KG)-dependent dioxygenases is thought to contribute to isocitrate dehydrogenase (IDH) mutation-derived malignancy. Herein, we aim to thoroughly investigate the expression pattern and prognostic significance of genes encoding α-KG-dependent enzymes for lower-grade glioma (LGG) patients. In this retrospective study, a total of 775 LGG patients were enrolled. The generalized linear model, least absolute shrinkage and selection operator Cox regression, and nomogram were applied to identify the enzyme-based signature. With the use of gene set enrichment analysis and Gene Ontology, the probable molecular abnormalities underlying high-risk patients were investigated. By comprehensively analyzing mRNA data, we observed that 41 genes were differentially expressed between IDHMUT and IDHWT LGG patients. A risk signature comprising 10 genes, which could divide samples into high- and low-risk groups of distinct prognoses, was developed and independently validated. This enzyme-based signature was indicative of a more malignant phenotype. The nomogram model incorporating the risk signature, molecular biomarkers, and clinicopathological parameters proved the incremental utility of the α-KG-dependent signature by achieving a more accurate prediction impact. Our study demonstrates that the α-KG-dependent enzyme-encoding genes were differentially expressed in relation to the IDH phenotype and may serve as a promising indicator for clinical outcomes of LGG patients.

The viral ankyrin repeat protein (ORF124L) from infectious spleen and kidney necrosis virus attenuates nuclear factor-κB activation and interacts with IκB kinase ß.

The ankyrin (ANK) repeat is one of the most common protein-protein interaction motifs, found predominantly in eukaryotes and bacteria, but the functions of the ANK repeat are rarely researched in animal viruses, with the exception of poxviruses. Infectious spleen and kidney necrosis virus (ISKNV) is a typical member of the genus Megalocytivirus in the family Iridoviridae and is a causative agent of epizootics in fish. The genome of ISKNV contains four putative viral ANK (vANK) repeat proteins and their functions remain largely unknown. In the present study, it was found that ORF124L, a vANK repeat protein in ISKNV, encodes a protein of 274 aa with three ANK repeats. Transcription of ORF124L was detected at 12 h post-infection (p.i.) and reached a peak at 40 h p.i. ORF124L was found to localize to both the nucleus and the cytoplasm in mandarin fish fry cells. ISKNV ORF124L interacted with the mandarin fish IκB kinase ß protein (scIKKß), and attenuated tumour necrosis factor alpha (TNF-α)- or phorbol myristate acetate (PMA)-induced activity of a nuclear factor κB (NF-κB)-luciferase reporter but did not interfere with the activity of an activator protein 1 (AP-1)-luciferase reporter. Phosphorylation of IκBα and nuclear translocation of NF-κB were also impaired by ISKNV ORF124L. In summary, ORF124L was identified as a vANK repeat protein and its role in inhibition of TNF-α-induced NF-κB signalling was investigated through interaction with the mandarin fish IKKß. This work may help to improve our understanding of the function of fish iridovirus ANK repeat proteins.

miR-30c-5p inhibits glioma proliferation and invasion via targeting Bcl2.

BACKGROUND: Glioma is a highly malignant brain tumor, characterized by the poor prognosis and high recurrence rates. Previous studies have confirmed that miRNA-30c-5p is closely associated with tumor cell biological properties. The present study explored the biological role of miR-30c-5p in human glioma malignant behavior and underlying mechanisms. METHODS: Levels of miR-30c-5p were detected in glioma tissues and adjacent normal tissues. Two glioma cell lines including U87 and U251 were transfected with miR-30c-5p mimic or inhibitors. Cell proliferation was evaluated by MTT assay and colony formation assay. Cell apoptosis and invasive potential of glioma cells were assessed by flow cytometry and transwell assays, respectively. Luciferase reporter assay was performed to validate the target gene of miR-30c-5p. RESULTS: Levels of miR-30c-5p were dramatically decreased in glioma tissues as compared to the adjacent normal tissues. Upregulation of miR-30c-5p significantly suppressed cell growth and colony formation, and induced apoptosis in glioma cells. In contrast, inhibition of miR-30c-5p promoted the proliferation and inhibited apoptosis in tumor cells. Furthermore, miR-30c-5p strongly suppresses the invasion of glioma cells. Western blot showed that Bcl-2 was significantly decreased following treatment with miR-30c-5p mimics and increased after miR-30c-5p inhibitor treatment. Moreover, luciferase reporter assays indicated that transfection of miR-30c-5p led to a marked reduction of luciferase activity, but had no effect on Bcl-2 3'-UTR mutated fragment. Mechanically, miR-30c-5p promoted the activation of caspase 3 and caspase 9 in glioma cells. Furthermore, miR-30c-5p promoted apoptosis and inhibited colony formation and migration, and knockdown of Bcl2 further increased the number of apoptotic cells and suppressed colony formation and migration of glioma cells. By contrast, miR-30c-5p inhibitors decreased apoptosis and increased colony formation and migration, and restored Bcl2 expression further suppressed glioma cell apoptosis and enhanced colony formation and migration. CONCLUSIONS: These results demonstrated that miR-30c-5p regulated growth, apoptosis and migration in glioma cells by targeting Bcl2, suggesting that miR-30c-5p might serve as a novel target for glioma therapy.

The B7-H4 gene induces immune escape partly via upregulating the PD-1/Stat3 pathway in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is associated with high mortality rates worldwide. The costimulatory molecule, B7-H4, a member of the B7 family, plays an important role in immune regulation, mainly by inhibiting the proliferation of T cells to achieve a negative regulatory T cell immune response. The mechanism of action of B7-H4 in non-small cell lung cancer is unknown at present. Tumor tissues from 71 patients subjected to radical pneumonectomy were examined, along with NSCLC cells and BALB/c mice. Among the 71 NSCLC cases, overall and recurrence-free survival rates were significantly lower in those displaying high B7-H4 expression. Mechanistic analyses showed that B7-H4 promoted the growth and metastasis of non-small cell lung cancer tumor tissues in mice through effects on CD8+ T cell apoptosis. Data from western blot experiments further suggested that B7-H4 induced CD8+ T cell death, both in vitro and in vivo, and affecting the PD-1/Stat3 pathway and promoting immune escape of tumor cells. Our collective findings support the potential utility of B7-H4 gene expression as a marker of NSCLC prognosis and provide a novel strategy for targeted therapy.

LncRNA SNHG6 acts as a prognostic factor to regulate cell proliferation in glioma through targeting p21.

It has been certified that long noncoding RNAs (lncRNAs) are crucial regulators in the progression of various human cancers. snoRNA host gene 6 (SNHG6) has been uncovered to affect the initial stage and tumorigenesis of hepatocellular carcinoma. Nevertheless, the expression pattern and biological role of SNHG6 in glioma still need to be investigated. The study aims to investigate the expression pattern, biological role and the potential mechanism of SNHG6 in glioma. In this study, the high expression of SNHG6 was tested in both glioma tissues and glioma cells. The correlation between expression levels of SNHG6 and the overall survival of glioma patients was demonstrated by using Kaplan Meier method analysis. Next, gain of function assays revealed that overexpression of SNHG6 can promote the formation of malignant phenotype of 1800 cell. However, results of loss-of-function assays revealed that silenced SNHG6 exerted the inhibitory function on glioma cell growth. Flow cytometric analysis was performed in glioma cells to detect the anti-oncogenic effects of silenced SNHG6 on cell cycle and apoptosis. Finally, we identified that p21 was involved in glioma cell proliferation after SNHG6 was downregulated. Taken together, we concluded that SNHG6 is a regulator and a potential therapeutic target in glioma.

Keyhole Approach for Clipping Intracranial Aneurysm: Comparison of Supraorbital and Pterional Keyhole Approach.

OBJECTIVE: The aim of this research was to compare the functional outcome and safety between supraorbital keyhole approach (SKA) and pterional keyhole approach (PKA) for clipping intracranial aneurysms. METHODS: This is a retrospective study involving 318 patients with a total of 365 aneurysms who underwent keyhole surgery, comprising 195 cases in SKA group and 123 cases in PKA group. The outcome measures include Glasgow Outcome Scale, complete clipping rate, adverse events incidence, operation view angle, working distance, and surgical incision condition. RESULTS: Of a total of 356 aneurysms that were clipped and 9 trapped, no significant difference was observed in Glasgow Outcome Scale score, adverse events incidence, or complete clipping rate between the SKA and PKA groups. The distance from skin incision to anterior clinoid process was 5.87 ± 0.24 cm in SKA and 5.12 ± 0.27 cm in PKA. The operation view angle (from midline to the operating channel in sagittal plane) was 30°-40° in the SKA group and 60°-68° in the PKA group. CONCLUSIONS: Our research demonstrates that both SKA and PKA are safe and effective for most anterior circulation aneurysms and parts of posterior circulation aneurysms. The SKA exposures aneurysm better on deep and sagittal directions and is more suitable for clipping aneurysms by the contralateral approach due to the short distance. The PKA has a good exposure on the neck of aneurysm with dorsal direction of parent artery and can be used to evacuate hematoma in the temporal lobe when clipping the aneurysm. Integrating multimodal 3-dimensional images could help neurosurgeon in selecting an appropriate and effective approach.

Actein inhibits glioma growth via a mitochondria-mediated pathway.

Previous studies indicate that the triterpene glycoside Actein from the herb black cohosh inhibits growth of human breast cancer cells. This study sought to investigate the effects of Actein on glioma cell growth and explore the potential mechanisms. Our results showed that administration of Actein significantly inhibited glioma cell viability in a dose- and time-dependent manner. Actein also increasingly inhibited the colony formation processes in glioma U87 cells and U251 cells. Administration of Actein also induced mitochondria-related apoptosis by increasing expression of pro-apoptotic factors Bax, cleaved caspase-3, cleaved caspase-9 and cleaved poly (ADP-ribose) polymerase 1 (PARP1) as well as decreasing anti-apoptotic Bcl-2 expression in U87 cells and U251 cells. In a xenograft model of glioma, Actein suppressed tumor growth and consistently induced cell apoptosis with the same mechanisms observed in vitro. In all, this study is the first report to address the growth inhibitory effects of Actein on glioma growth and propose that mitochondria-mediated apoptosis pathway may underlie the biological activities of Actein in glioma. Our study suggests that administration of Actein may serve as a potent therapeutic strategy for treatment of glioma.

Nuclear paraspeckle assembly transcript 1 promotes the metastasis and epithelial-mesenchymal transition of hepatoblastoma cells by inhibiting miR-129-5p.

The abnormal expression of nuclear paraspeckle assembly transcript 1 (NEAT1) may serve critical functions for the development and progression of various types of human tumor. However, the expression and biological function of NEAT1 in hepatoblastoma (HB) and the underlying mechanisms for the function of NEAT1 in HB remain largely uncharacterized. In the present study, the results of reverse transcription-quantitative polymerase chain reaction revealed that the expression of NEAT1 was significantly elevated in HB tissues. HB tissues with metastasis also exhibited significantly increased levels of NEAT1 compared with tissues without metastasis. The biological functions of NEAT1 were then assessed using gain-/loss-of-function studies. The results of in vitro assays revealed that inhibiting NEAT1 expression reduced the migration and invasion of HepG2 cells. By contrast, the induced expression of NEAT1 exhibited the opposite effect. The present study also demonstrated that the inhibition of NEAT1 expression prevented the epithelial-mesenchymal transition of HepG2 cells, whereas forced expression of NEAT1 exhibited the opposite effect. In addition, it was confirmed that NEAT1 could modulate the expression of microRNA (miR)-129-5p in HepG2 cells, and that NEAT1 may exert its effect on the metastatic behaviors and epithelial-mesenchymal transition of HepG2 cells by inhibiting miR-129-5p. In conclusion, the present study indicated that NEAT1 expression was aberrantly increased in HB and that it may promote the metastasis of HB cells by inhibiting miR-129-5p. Targeting NEAT1 may potentially be a novel therapeutic option for treating patients with HB.

Histone deacetylase 5 promotes Wilms' tumor cell proliferation through the upregulation of c-Met.

The histone deacetylase (HDAC) family is comprised of enzymes, which are involved in modulating the majority of critical cellular processes, including transcriptional regulation, apoptosis, proliferation and cell cycle progression. However, the biological function of HDAC5 in Wilms' tumor remains to be fully elucidated. The present study aimed to investigate the expression and function of HDAC5 in Wilm's tumor. It was demonstrated that the mRNA and protein levels of HDAC5 were upregulated in human Wilms' tumor tissues. Overexpression of HDAC5 in G401 cells was observed to significantly promote cellular proliferation, as demonstrated by the results of an MTT assay and bromodeoxyuridine incorporation assay. By contrast, HDAC5 knockdown using small interfering RNA suppressed the proliferation of the G401 cells. At the molecular level, the present study demonstrated that HDAC5 promoted the expression of c­Met, which has been previously identified as an oncogene. In addition, downregulation of c­Met inhibited the proliferative effects of HDAC5 in human Wilms' tumor cells. Taken together, these results suggested that HDAC5 promotes cellular proliferation through the upregulation of c­Met, and may provide a novel therapeutic target for the treatment of patients with Wilms' tumor.

Stat3 inhibits WTX expression through up-regulation of microRNA-370 in Wilms tumor.

Wilms tumor (WT) is a genetically heterogeneous childhood kidney tumor. Several genetic mutations have been identified in WT patients, including inactivation of WTX, somatic stabilizing CTNNB1, and p53 mutations. However, the molecular mechanisms in tumorigenesis remain largely unexplored. Stat3 is a transcription factor that can promote oncogenesis. Stat3 activation is commonly viewed as crucial for multiple tumor proliferation and metastasis. We show that Stat3 is highly activated in Wilms tumor tissues compared to those in adjacent tissues. IL-6 treatment or transfection of a constitutively activated Stat3 in G401 cells promotes cell proliferation. At the molecular level, we further elucidate that Stat3 inhibits WTX expression through up-regulation of microRNA-370. Our results suggest that Stat3/miR-370/WTX regulatory axis might be a critical mechanism in Wilms tumor cells.

Molecular cloning of IKKß from the mandarin fish Siniperca chuatsi and its up-regulation in cells by ISKNV infection.

The IκB kinase ß (IKKß) plays crucial roles in regulating activation of nuclear factor-kappa B (NF-κB) in response to proinflammatory factors and microbial and viral infections. Here, we report the cloning of an IKKß cDNA (named SicIKKß) from the mandarin fish Siniperca chuatsi. The full-length cDNA is 4052bp and contains an ORF that encodes a predicted protein of 743-amino acid residues. The deduced amino acid sequence of SicIKKß has the same domain organization as human IKKß, which consists of a serine/threonine kinase domain, a leucine zipper motif and a putative helix-loop-helix motif. Quantitative RT-PCR showed that SicIKKß was ubiquitously expressed in tissues of mandarin fish, and its expression in mandarin fish fry (MFF-1) cells was up-regulated during the course of ISKNV infection.