GPR37 expression as a prognostic marker in gliomas: a bioinformatics-based analysis.

BACKGROUND: Gliomas are the most frequently diagnosed primary brain tumors, and are associated with multiple molecular aberrations during their development and progression. GPR37 is an orphan G protein-coupled receptor (GPCR) that is implicated in different physiological pathways in the brain, and has been linked to various malignancies. The aim of this study was to explore the relationship between GPR37 gene expression and the clinicopathological factors, patient prognosis, tumor-infiltrating immune cell signature GSEA and methylation levels in glioma. METHODS: We explored the diagnostic value, clinical relevance, and molecular function of GPR37 in glioma using TCGA, STRING, cBioPortal, Tumor Immunity Estimation Resource (TIMER) database and MethSurv databases. Besides, the "ssGSEA" algorithm was conducted to estimate immune cells infiltration abundance, with 'ggplot2' package visualizing the results. Immunohistochemical staining of clinical samples were used to verify the speculations of bioinformatics analysis. RESULTS: GPR37 expression was significantly higher in the glioma tissues compared to the normal brain tissues, and was linked to poor prognosis. Functional annotation of GPR37 showed enrichment of ether lipid metabolism, fat digestion and absorption, and histidine metabolism. In addition, GSEA showed that GPR37 was positively correlated to the positive regulation of macrophage derived foam cell differentiation, negative regulation of T cell receptor signaling pathway, neuroactive ligand receptor interaction, calcium signaling pathway, and negatively associated with immunoglobulin complex, immunoglobulin complex circulating, ribosome and spliceosome mediated by circulating immunoglobulin etc. TIMER2.0 and ssGSEA showed that GPR37 expression was significantly associated with the infiltration of T cells, CD8 T cell, eosinophils, macrophages, neutrophils, NK CD56dim cells, NK cells, plasmacytoid DCs (pDCs), T helper cells and T effector memory (Tem) cells. In addition, high GPR37 expression was positively correlated with increased infiltration of M2 macrophages, which in turn was associated with poor prognosis. Furthermore, GPR37 was positively correlated with various immune checkpoints (ICPs). Finally, hypomethylation of the GPR37 promoter was associated with its high expression levels and poor prognosis in glioma. CONCLUSION: GPR37 had diagnostic and prognostic value in glioma. The possible biological mechanisms of GPR37 provide novel insights into the clinical diagnosis and treatment of glioma.

Elevated LILRB1 expression predicts poor prognosis and is associated with tumor immune infiltration in patients with glioma.

BACKGROUND: Leukocyte immunoglobulin-like receptor subfamily B1 (LILRB1) is regarded as an inhibitory molecule. However, the importance of LILRB1 expression in glioma has not yet been determined. This investigation examined the immunological signature, clinicopathological importance and prognostic value of LILRB1 expression in glioma. METHODS: We used data from the UCSC XENA database, the Cancer Genome Atlas (TCGA) database, the Chinese Glioma Genome Atlas (CGGA) database, the STRING database, the MEXPRESS database and our clinical glioma samples to perform bioinformatic analysis and used vitro experiments to examine the predictive value and potential biological roles of LILRB1 in glioma. RESULTS: Higher LILRB1 expression was considerably present in the higher WHO grade glioma group and was linked to a poorer prognosis in patients with glioma. Gene set enrichment analysis (GSEA) revealed that LILRB1 was positively correlated with the JAK/STAT signaling pathway. LILRB1 combined with tumor mutational burden (TMB) and microsatellite instability (MSI) may be a promising indicator for the effectiveness of immunotherapy in patients with glioma. Increased LILRB1 expression was positively linked with the hypomethylation, M2 macrophage infiltration, immune checkpoints (ICPs) and M2 macrophage makers. Univariate and multivariate Cox regression analyses determined that increased LILRB1 expression was a standalone causal factor for glioma. Vitro experiments determined that LILRB1 positively enhanced the proliferation, migration and invasion in glioma cells. MRI images demonstrated that higher LILRB1 expression was related with larger tumor volume in patients with glioma. CONCLUSION: Dysregulation of LILRB1 in glioma is correlated with immune infiltration and is a standalone causal factor for glioma.

Screening of high-efficiency and low-toxicity antitumor active components in Macleaya cordata seeds based on the competitive effect of drugs on double targets by a new laminar flow chip.

It is urgent to obtain targeted drugs that selectively bind to pathological targets rather than physiological targets in the early stage of drug screening. G-Quadruplex has become one of the important targets in the development of anti-tumor drugs. However, drugs that target quadruplexes may also bind to dsDNA, which may lead to adverse reactions. In this study, a new three-phase laminar flow chip was constructed to enable the multi-components of a traditional Chinese medicine extract to dynamically and competitively bind with G-quadruplex DNA (on target) and double-stranded DNA (off target), so as to select high-efficiency and low-toxicity anti-tumor drugs. The results showed that there were five compounds in the extracts of Macleaya cordata seeds that exhibited obvious differences in binding to the two targets. Furthermore, the binding constants and modes of four identified alkaloids as they bound to two DNA targets were verified by fluorescence spectra and molecular docking methods. The toxicity to HepG2 and LO2 cells from the four alkaloids was also compared. The results showed that sanguinarine and chelerythrine could be used as candidate drugs with stronger binding to HT24 than DNA26. The chip can also be used for other types of double-target screening of other traditional Chinese medicine extracts or compound libraries.

Simultaneous determination of free and total paclitaxel in blood in a three-phase laminar flow microchip.

In this study, a three-phase laminar flow microfluidic chip (TPL chip) combined with HPLC was developed for monitoring free and total concentrations of paclitaxel (PTX) in blood simultaneously. A diluted whole blood sample (aqueous phase) was introduced into the chip, ethyl acetate (organic phase) was introduced into the chip for extraction, and an interphase was used to prevent the blood sample from coming into direct contact with the organic phase. Because only free drug can quantitatively diffuse into the organic extraction phase and the free drug fraction has a linear relationship with the dilution factor of blood, both the free and total drug concentrations can be obtained by detecting the concentration of paclitaxel in the organic extraction phase. The governing factor such as flow rate for extraction was optimized. Docetaxel was used as an internal standard. The reliability of the quantitative diffusion of molecules in the TPL chip was proved by the methodological investigation of PTX in PBS sample, which showed a good linearity in the concentration range of 0.5 - 100 µg/mL and a detection limit of 7 ng/mL. Good repeatibilities for retention time (RSD of PTX is 1.23%, docetaxel is 1.14%, n = 5) and peak area ratio of PTX to docetaxel (RSD is 4.38%) were obtained. For blood sample analysis, only 100 µL of sample was needed and whole pretreatment was finished in 35 min, and a recovery of 94~117% were obtained. The provided method showed advantages in fast analysis speed, minimum sample handing, and potential ability of automation, and integration.

Inhibition of HDAC4 Attenuated JNK/c-Jun-Dependent Neuronal Apoptosis and Early Brain Injury Following Subarachnoid Hemorrhage by Transcriptionally Suppressing MKK7.

The c-Jun N-terminal kinase (JNK)/c-Jun cascade-dependent neuronal apoptosis has been identified as a central element for early brain injury (EBI) following subarachnoid hemorrhage (SAH), but the molecular mechanisms underlying this process are still thoroughly undefined to date. In this study, we found that pan-histone deacetylase (HDAC) inhibition by TSA, SAHA, VPA, and M344 led to a remarkable decrease in the phosphorylation of JNK and c-Jun, concomitant with a significant abrogation of apoptosis caused by potassium deprivation in cultured cerebellar granule neurons (CGNs). Further investigation showed that these effects resulted from HDAC inhibition-induced transcriptional suppression of MKK7, a well-known upstream kinase of JNK. Using small interference RNAs (siRNAs) to silence the respective HDAC members, HDAC4 was screened to be required for MKK7 transcription and JNK/c-Jun activation. LMK235, a specific HDAC4 inhibitor, dose-dependently suppressed MKK7 transcription and JNK/c-Jun activity. Functionally, HDAC4 inhibition via knockdown or LMK235 significantly rescued CGN apoptosis induced by potassium deprivation. Moreover, administration of LMK235 remarkably ameliorated the EBI process in SAH rats, associated with an obvious reduction in MKK7 transcription, JNK/c-Jun activity, and neuronal apoptosis. Collectively, the findings provide new insights into the molecular mechanism of neuronal apoptosis regarding HDAC4 in the selective regulation of MKK7 transcription and JNK/c-Jun activity. HDAC4 inhibition could be a potential alternative to prevent MKK7/JNK/c-Jun axis-mediated nervous disorders, including SAH-caused EBI.

Utility of in vitro and in vivo systems for studying the permeability of capsaicin and nonivamide through different intestinal regions.

1. The present study determined and compared the permeability of capsaicin and nonivamide along the length of the intestine in rats. Accordingly, the purpose was to evaluate this synthetic analog as a clinical substitute for capsaicin.. 2. Permeabilities of capsaicin and nonivamide were measured in experiments utilizing Ussing chambers and in vivo methods. Capsaicin concentrations were examined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). 3. Both capsaicin (0.80 × 10-6 cm/s) and nonivamide (0.22 × 10-6 cm/s, p > 0.05) had poor permeabilities across the jejunal membrane. The permeability of nonivamide (10.12 × 10-6 cm/s) was significantly greater than that of capsaicin (5.34 × 10-6 cm/s, p < 0.05) across the iliac membrane. In contrast, the permeability of nonivamide (8.42 × 10-6 cm/s) across the colonic membrane was markedly lower than that of capsaicin (14.48 × 10-6 cm/s, p < 0.05). In accordance with the in vitro study, the drug concentration-time curve of nonivamide was significantly higher in the ileum (F = 14.18, p < 0.05) but lower in the colon (F = 11.86, p < 0.05) compared with capsaicin. 4. The results demonstrate that capsaicin and nonivamide exhibit varying permeabilities across several different intestinal tissues. The relevance of such extended investigations to healthcare is underscored by the lower cost of nonivamide versus capsaicin, along with potential application in prevention and management of the disease.

Integration of laminar flow extraction and capillary electrophoretic separation in one microfluidic chip for detection of plant alkaloids in blood samples.

The design, construction and testing for integration of liquid-liquid extraction (EX) and capillary electrophoretic (CE) separation on one glass microchip was reported. In this EX-CE chip, a 1.5 cm-long and 200 µm-wide EX channel was used for extraction based on the two-phase laminar flow, followed by a single-cross CE unit for on-line analysis without any auxiliary devices. One side of the EX channel surface for the organic solvent phase was selectively modified to be hydrophobic while the surface of the other side for the aqueous phase remained hydrophilic, and the extraction product reservoir is also used as the sample reservoir for the subsequent chip separation in the CE channel. With the surface-directed liquid flow behavior and liquid level adjustment in various reservoirs of the EX-CE chip, no disturbance occurred between the extraction (EX) and capillary electrophoretic (CE) units. A small heating block was placed under the chip to accelerate solvent evaporation after liquid-liquid extraction. Sanguinarine (SAN), a plant alkaloid, was used as a model analyte to evaluate the performance of the EX-CE chip. The influences of organic solvent type and liquid flow speed on the extraction efficiency were investigated. Rhodamine 123 (Rh123) was used as an internal standard for quantification of Sanguinarine (SAN) in a physiological buffer (e.g. PBS) or blood samples. A good linearity in the concentration range of 0.05 µg mL-1 to 0.1 mg mL-1 for SAN in PBS was obtained, with the detection limit of 0.5 ng mL-1. Good repeatibilities for migration times (RSD of SAN is 0.63%, Rh123 is 0.91%, n = 5) and peak area ratio of SAN to Rh123 (RSD is 1.3%, n = 5) were obtained. For blood sample analysis, only 20 µL of sample was needed, and the whole analysis was finished in 17 min. In addition to the advantages in fast analysis speed, minimum sample handling, potential automation, the reported method showed an on-line sample pre-concentration capability.