Identification of glycerophospholipids using self-built recognition software based on positive and negative ion high-resolution mass spectrometric fragmentation experiments.

Glycerophospholipids (GPs) have a wide variety and complex structure, which makes their identification challenging. Our software affords a novel tool for the automated identification of non-target GPs in biological mixtures. Here, we explored the multi-stage fragmentation processes of GPs in positive and negative ion modes, and then constructed multi-stage fragment ion databases. This database includes 8214 simulated GP molecules from a random combination of fatty acids corresponding to 42,439 self-built predicted multi-stage fragment ions in positive ion mode and 31,487 self-built predicted multi-stage fragment ions in negative ion mode (MS ≤ 3). The automatic GP identification (AGPI) software can screen out GP candidates utilizing the MS1 accurate mass. The isomers of fatty acid chains and the phosphoryl head group can be distinguished using the MS2 and MS3 fragment spectra in positive-ion and negative-ion modes. All of the selected 45 GP standards were putatively identified using AGPI software; however, there were false positives because the software cannot distinguish positional isomers of fatty acids. Therefore, the AGPI software could be applied to identify GPs in samples, such as cancer cells; we successfully identified 41 GPs in cancer cells.

Hydroxychloroquine alleviates the neurotoxicity induced by anti-ribosomal P antibodies.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by a wide spectrum of autoantibodies, among which anti-ribosomal P (anti-P) antibodies are considered to be closely related to the neuropsychiatric SLE (NPSLE). Hydroxychloroquine (HCQ) has been proven to be effective against a variety of autoimmune diseases and is an essential drug for the treatment of SLE. In this study, we investigated the effects of anti-ribosomal P (anti-P) antibodies on neural cells and determined whether hydroxychloroquine (HCQ) influenced the anti-P antibodies-induced changes. The results showed that the binding of anti-P antibodies with mouse neuroblastoma- 2a (N2a) cells and rat primary neurons resulted in elevated intracellular calcium levels, inducing decreased cell viability and cell apoptosis. These inhibitory effects were alleviated by HCQ in a concentration-dependent manner by reducing the intracellular calcium levels and modulating the expression of apoptotic proteins. In summary, our study demonstrates that anti-P antibodies induce neural cell damage. HCQ could ease the damage effects and may play a neuroprotective role in NPSLE.

Cathepsin C aggravates neuroinflammation via promoting production of CCL2 and CXCL2 in glial cells and neurons in a cryogenic brain lesion.

OBJECTIVE: Chemokines regulate infiltration of immune cells to brain in inflammation. Cathepsin C (CatC), a lysosomal protease, has been found to participate in neuroinflammation. However, how CatC affects chemokines expression in neuroinflammation triggered by traumatic brain injury (TBI) remains unclear. Here, we investigated the effects of CatC on chemokines and neuroinflammation in TBI. METHODS: The present study used CatC knockdown (KD) and overexpression (OE) mice to generate cryogenic brain lesion model and determined effects of CatC on expression of chemokines CCL2, CCL5 and CXCL2 and infiltration of immune cells in acute and chronic phases of the lesion. Further, cellular sources of various chemokines were demonstrated in vitro. Values were compared with wild type (WT) mice. RESULTS: The results found that 6 h after lesion, CatC expression,IL-1ß and TNF-α mRNA and protein expression were strongly induced in the lesions; CCL2 and CXCL2 mRNA and protein expression were increased in CatC OE mice, while decreased in CatC KD mice. On the 3rd day after lesion, macrophages and neutrophils were mainly infiltrated to the lesions. Simultaneously, Iba-1+ cells in CatC OE mice were increased, while MPO + cells in CatC KD mice were decreased. In contrast, on the 28th day after lesion, a few lymphocytes were infiltrated surrounding new blood vessels. CatC OE mice showed larger volumes of scar areas, higher expression of CCL2,CXCL2,IL-1ß,TNF-α,IL-6 and iNOS, as well as stronger GFAP+ and Iba-1+ signals, while CatC KD mice had reversed effects. No significant differences of CCL5 expression were found in various genotype mice. Further, in vitro study demonstrated CatC-induced expression of CCL2 were mainly derived from microglia and neurons, while CXCL2 derived from microglia and astrocytes. CONCLUSION: Our data indicate that CatC aggravates neuroinflammation via promoting production of CCL2 and CXCL2 in glial cells and neurons in a cryogenic brain lesion, providing potential cellular and molecular targets for future intervention of TBI and other neuroinflammatory diseases.

Comprehensive pathway-related genes signature for prognosis and recurrence of ovarian cancer.

BACKGROUND: Ovarian cancer (OC) is a highly malignant disease with a poor prognosis and high recurrence rate. At present, there is no accurate strategy to predict the prognosis and recurrence of OC. The aim of this study was to identify gene-based signatures to predict OC prognosis and recurrence. METHODS: mRNA expression profiles and corresponding clinical information regarding OC were collected from The Cancer Genome Atlas (TCGA) database. Gene set enrichment analysis (GSEA) and LASSO analysis were performed, and Kaplan-Meier curves, time-dependent ROC curves, and nomograms were constructed using R software and GraphPad Prism7. RESULTS: We first identified several key signalling pathways that affected ovarian tumorigenesis by GSEA. We then established a nine-gene-based signature for overall survival (OS) and a five-gene-based-signature for relapse-free survival (RFS) using LASSO Cox regression analysis of the TCGA dataset and validated the prognostic value of these signatures in independent GEO datasets. We also confirmed that these signatures were independent risk factors for OS and RFS by multivariate Cox analysis. Time-dependent ROC analysis showed that the AUC values for OS and RFS were 0.640, 0.663, 0.758, and 0.891, and 0.638, 0.722, 0.813, and 0.972 at 1, 3, 5, and 10 years, respectively. The results of the nomogram analysis demonstrated that combining two signatures with the TNM staging system and tumour status yielded better predictive ability. CONCLUSION: In conclusion, the two-gene-based signatures established in this study may serve as novel and independent prognostic indicators for OS and RFS.

Behavioral, inflammatory and neurochemical disturbances in LPS and UCMS-induced mouse models of depression.

The immuno-inflammatory activation triggered by various stresses play an important role in pathophysiology of depression. The immune responses display differential pathological characters in different stresses. However, comparative data and analysis on behavioural, inflammatory and neurochemical changes in different stress-induced depression is limited. To imitate different stressful situations, in this study, mice were subjected to a single injection of LPS (0.5 mg/kg, i.p.) and UCMS (4 week period), respectively. LPS-stressed mice showed more immobility time in FST and TST, as well as more time in periphery in OFT than UCMS-stressed mice. Further, LPS-stressed mice showed robuster expression and release of TNF-α, IL-1ß and IL-6 in serum and depression-related brain areas (prefrontal cortex, hippocampus and striatum) as compared to UCMS-stressed mice. The ELISA results showed that IDO expression was significantly increased following LPS and UCMS stresses, but more increased IDO expression was observed in prefrontal cortex and hippocampus of LPS-stressed mice. The decrease of 5-HT and BDNF was detected only in hippocampus of LPS-stressed mice, but in overall all the brain areas assessed in UCMS-stressed mice as compared to control. The data indicate that LPS induced more severe depressive-like behaviours and robuster immune activation than UCMS. Our study strongly imply that hippocampus is relatively more vulnerable to acute inflammatory challenge in depression, while chronic psychological stress is more likely to cause the multidimensional symptoms of clinical depression. Our findings provide more insight into pathophysiology in various stress-induced depression and also implicate a potential suitability of different stress models.

MiR-181a enhances drug sensitivity in mitoxantone-resistant breast cancer cells by targeting breast cancer resistance protein (BCRP/ABCG2).

Breast cancer resistance protein (BCRP)/ATP-binding cassette subfamily G member 2 (ABCG2) mediates multidrug resistance (MDR) in breast cancers. In this study, we aimed to investigate the role of microRNAs in regulation of BCRP expression and BCRP-mediated drug resistance in breast cancer cells. Microarray analysis was performed to determine the differential expression patterns of miRNAs that target BCRP between the MX-resistant breast cancer cell line MCF-7/MX and its parental MX-sensitive cell line MCF-7. MiR-181a was found to be the most significantly down-regulated miRNA in MCF-7/MX cells. Luciferase activity assay showed that miR-181a mimics inhibited BCRP expression by targeting the 3' untranslated region (UTR) of the BCRP mRNA. Overexpression of miR-181a down-regulated BCRP expression, and sensitized MX-resistant MCF-7/MX cells to MX. In a nude mouse xenograft model, intratumoral injection of miR-181a mimics inhibited BCRP expression, and enhanced the antitumor activity of MX. In addition, miR-181a inhibitors up-regulated BCRP expression, and rendered MX-sensitive MCF-7 cells resistant to MX. These findings suggest that miR-181a regulates BCRP expression via binding to the 3'-UTR of BCRP mRNA. MiR-181a is critical for regulation of BCRP-mediated resistance to MX. MiR-181a may be a potential target for preventing and reversing drug resistance in breast cancer.