Glioma Single-Cell Biomechanical Analysis by Cyclic Conical Constricted Microfluidics.

Determining the grade of glioma is a critical step in choosing patients' treatment plans in clinical practices. The pathological diagnosis of patient's glioma samples requires extensive staining and imaging procedures, which are expensive and time-consuming. Current advanced uniform-width-constriction-channel-based microfluidics have proven to be effective in distinguishing cancer cells from normal tissues, such as breast cancer, ovarian cancer, prostate cancer, etc. However, the uniform-width-constriction channels can result in low yields on glioma cells with irregular morphologies and high heterogeneity. In this research, we presented an innovative cyclic conical constricted (CCC) microfluidic device to better differentiate glioma cells from normal glial cells. Compared with the widely used uniform-width-constriction microchannels, the new CCC configuration forces single cells to deform gradually and obtains the biophysical attributes from each deformation. The human-derived glioma cell lines U-87 and U-251, as well as the human-derived normal glial astrocyte cell line HA-1800 were selected as the proof of concept. The results showed that CCC channels can effectively obtain the biomechanical characteristics of different 12-25 µm glial cell lines. The patient glioma samples with WHO grades II, III, and IV were tested by CCC channels and compared between Elastic Net (ENet) and Lasso analysis. The results demonstrated that CCC channels and the ENet can successfully select critical biomechanical parameters to differentiate the grades of single-glioma cells. This CCC device can be potentially further applied to the extensive family of brain tumors at the single-cell level.

EN1 Regulates Cell Growth and Proliferation in Human Glioma Cells via Hedgehog Signaling.

Glioblastoma is an aggressive cancer of the nervous system that accounts for the majority of brain cancer-related deaths. Through cross-species transcriptome studies, we found that Engrailed 1 (EN1) is highly expressed in serum-free cultured glioma cells as well as glioma tissues, and increased expression level predicts a worse prognosis. EN1 controls glioma cell proliferation, colony formation, migration, and tumorigenic capacity in vivo. It also influences sensitivity of glioma cells to γ-ray irradiation by regulating intracellular ROS levels. Mechanistically, EN1 influences Hedgehog signaling by regulating the level of Gli1 as well as primary cilia length and the primary cilia transport-related protein TULP3. In conclusion, we demonstrate that EN1 acts as an oncogenic regulator that contributes to glioblastoma pathogenesis and could serve as a diagnostic/prognostic marker and therapeutic target for glioblastoma.

Xylarodons A and B, new hexaketides from the endophytic fungus Xylaria sp. SC1440.

Two new hexaketides, xylarodons B (1) and C (2), were isolated from solid cultures of the endophytic fungus Xylaria sp. SC1440. The structures of these compounds were elucidated on the basis of detailed 1D, 2D NMR, and HRESIMS analysis. Their absolute configurations were established by experimental and TDDFT calculated ECD spectra. The isolated compounds were evaluated for cytotoxic and tyrosinase inhibitory activity.

S-adenosylmethionine reduces the inhibitory effect of Aß on BDNF expression through decreasing methylation level of BDNF exon â £ in rats.

The structure of brain-derived neurotrophic factor (BDNF) gene is complex, which is composed of eight non-coding exons and one coding exon, each of them has its own unique promoter. Multiple BDNF transcripts have distinct functional properties and epigenetic modulation of BDNF gene transcription is implicated in the neurological disorders. In the present study, rat models with amyloid-ß (Aß) intrahippocampal injection and PC12 cells were used to explore the role of DNA methylation in the promoters of BDNF exon Ⅳ and exon Ⅵ in BDNF suppression caused by Aß. We found that Aß inhibited BDNF expression accompanying with hypermethylation in BDNF exon Ⅳ promoter, meanwhile, S-adenosylmethionine (SAM), primary methyl donor, reversed the low BDNF expression through demethylation in BDNF exon Ⅳ promoter. No methylation change was observed in BDNF exon Ⅵ promoter. The alteration of DNA methylation caused by Aß or SAM was mediated by DNA methyltransferase 3A (DNMT3A). These data suggest that methylation change in BDNF exon Ⅳ is involved in the regulation of BDNF expression by Aß or SAM, and further support the view of specific epigenetic modifications of a certain BDNF gene transcript.

SQSTM1/ p62 oligomerization contributes to Aß-induced inhibition of Nrf2 signaling.

SQSTM1/p62, also known as sequestosome 1 (SQSTM1) or p62, is an intracellular protein induced by stress and functions as an adaptor molecule in diverse cellular processes. Oxidative damage induced by overproduction of amyloid-ß (Aß) and the impairment of endogenous antioxidant Nrf2 signaling have been documented in the brains of Alzheimer's disease (AD) patients. The causes of the inactivation of Nrf2 signaling under Aß-induced oxidative stress are unclear, and p62 might be involved in this process. In this study, APP/PS1 transgenic mice, Aß intrahippocampal injection rat model, and SH-SY5Y cells were used to reveal that the alterations in the oligomeric state of p62 participated in the regulation of Nrf2 signaling under Aß insult. The present in vivo and in vitro studies revealed that short-term treatment of Aß activated Nrf2 signaling, while long-term Aß treatment inhibited it through either canonical or noncanonical Nrf2 activation pathway. p62 oligomerization was largely attenuated under long-term Aß treatment. The reduction of p62 oligomerization weakened p62 sequestration to Keap1, leading to Nrf2 signaling inhibition. Our findings provide a better understanding of p62-mediated modulation on Nrf2 activity and highlight a potential therapeutic target of p62 in AD.

S-Adenosylmethionine Alleviates Amyloid-ß-Induced Neural Injury by Enhancing Trans-Sulfuration Pathway Activity in Astrocytes.

BACKGROUND: Glutathione (GSH) is an important endogenous antioxidant protecting cells from oxidative injury. Cysteine (Cys), the substrate limiting the production of GSH, is mainly generated from the trans-sulfuration pathway. S-adenosylmethionine (SAM) is a critical molecule produced in the methionine cycle and can be utilized by the trans-sulfuration pathway. Reductions in GSH and SAM as well as dysfunction in the trans-sulfuration pathway have been documented in the brains of Alzheimer's disease (AD) patients. Our previous in vivo study revealed that SAM administration attenuated oxidative stress induced by amyloid-ß (Aß) through the enhancement of GSH. OBJECTIVE: To investigate the effect of Aß-induced oxidative stress on the trans-sulfuration pathway in astrocytes and neurons, respectively, and the protective effect of SAM on neurons. METHODS: APP/PS1 transgenic mice and the primary cultured astrocytes, neurons, and HT22 cells were used in the current study. RESULTS: SAM could rescue the low trans-sulfuration pathway activity induced by Aß only in astrocytes, accompanying with increasing levels of Cys and GSH. The decrease of cellular viability of neurons caused by Aß was greatly reversed when co-cultured with astrocytes with SAM intervention. Meanwhile, SAM improved cognitive performance in APP/PS1 mice. CONCLUSION: In terms of astrocyte protection from oxidative stress, SAM might be a potent antioxidant in the therapy of AD patients.

Responsive Expression of MafF to ß-Amyloid-Induced Oxidative Stress.

The small musculoaponeurotic fibrosarcoma (sMaf) proteins MafF, MafG, and MafK are basic region leucine zipper- (bZIP-) type transcription factors and display tissue- or stimulus-specific expression patterns. As the oxidative stress reactive proteins, sMafs are implicated in various neurological disorders. In the present study, the expressions of sMafs were investigated across five databases gathering transcriptomic data from 74 Alzheimer's disease (AD) patients and 66 controls in the Gene Expression Omnibus (GEO) database. The expression of MafF was increased in the hippocampus of AD patients, which was negatively correlated with the expression of the glutamate cysteine ligase catalytic subunit (GCLC). Furthermore, MafF was significantly increased in patients with Braak stage V-VI, compared to those with Braak stage III-IV. ß-Amyloid (Aß), a strong inducer of oxidative stress, plays a crucial role in the pathogenesis of AD. The responsive expressions of sMafs to Aß-induced oxidative stress were studied in the APP/PS1 mouse model of AD, Aß intrahippocampal injection rats, and several human cell lines from different tissue origins. This study revealed that only the induction of MafF was accompanied with reduction of GCLC and glutathione (GSH). MafF knockdown suppressed the increase of GSH induced by Aß. Among sMafs, MafF is the most responsive to Aß-induced oxidative stress and might potentiate the inhibition of antioxidation. These results provide a better understanding of sMaf modulation in AD and highlight MafF as a potential therapeutic target in AD.

The lncRNA H19 binding to let-7b promotes hippocampal glial cell activation and epileptic seizures by targeting Stat3 in a rat model of temporal lobe epilepsy.

OBJECTIVES: Glial cell activation contributes to the inflammatory response and occurrence of epilepsy. Our preliminary study demonstrated that the long non-coding RNA, H19, promotes hippocampal glial cell activation during epileptogenesis. However, the precise mechanisms underlying this effect remain unclear. MATERIALS AND METHODS: H19 and let-7b were overexpressed or silenced using an adeno-associated viral vector in vivo. Their expression in a kainic acid-induced epilepsy model was evaluated by real-time quantitative PCR, fluorescence in situ hybridization, and cytoplasmic and nuclear RNA isolation. A dual-luciferase reporter assay was used to evaluate the direct binding of let-7b to its target genes and H19. Western blot, video camera monitoring and Morris water maze were performed to confirm the role of H19 and let7b on epileptogenesis. RESULTS: H19 was increased in rat hippocampus neurons after status epilepticus, which might be due to epileptic seizure-induced hypoxia. Increased H19 aggravated the epileptic seizures, memory impairment and mossy fibre sprouting of the epileptic rats. H19 could competitively bind to let-7b to suppress its expression. Overexpression of let-7b inhibited hippocampal glial cell activation, inflammatory response and epileptic seizures by targeting Stat3. Moreover, overexpressed H19 reversed the inhibitory effect of let-7b on glial cell activation. CONCLUSIONS: LncRNA H19 could competitively bind to let-7b to promote hippocampal glial cell activation and epileptic seizures by targeting Stat3 in a rat model of temporal lobe epilepsy.

Integrated transcriptome expression profiling reveals a novel lncRNA associated with L-DOPA-induced dyskinesia in a rat model of Parkinson's disease.

Levodopa-induced dyskinesia (LID) is a common complication of chronic dopamine replacement therapy in the treatment of Parkinson's disease (PD). Long noncoding RNAs regulate gene expression and participate in many biological processes. However, the role of long noncoding RNAs in LID is not well understood. In the present study, we examined the lncRNA transcriptome profile of a rat model of PD and LID by RNA sequence and got a subset of lncRNAs, which were gradually decreased during the development of PD and LID. We further identified a previously uncharacterized long noncoding RNA, NONRATT023402.2, and its target genes glutathione S-transferase omega (Gsto)2 and prostaglandin E receptor (Ptger)3. All of them were decreased in the PD and LID rats as shown by quantitative real-time PCR, fluorescence in situ hybridization and western blotting. Pearson's correlation analysis showed that their expression was positively correlated with the dyskinesia score of LID rats. In vitro experiments by small interfering RNA confirmed that slicing NONRATT023402 inhibited Gsto2 and Ptger3 and promoted the inflammatory response. These results demonstrate that NONRATT023402.2 may have inhibitive effects on the development of PD and LID.

Xylaropyrones B and C, new γ-pyrones from the endophytic fungus Xylaria sp. SC1440.

Two new γ-pyrones, xylaropyrones B (1) and C (2), together with three known compounds, xylaropyrone (3), annularin A (4) and annularin C (5), were isolated from solid cultures of the endophytic fungus Xylaria sp. SC1440. The structures of these compounds were determined mainly by analysis of their NMR spectroscopic data. The relative configurations of 1 and 2 were assigned on the basis of J-based configurational analysis, and the absolute configurations were established by experimental and TDDFT calculated ECD spectra. The isolated compounds were evaluated for cytotoxic and tyrosinase inhibitory activity.