Paraventricular thalamus-insular cortex circuit mediates colorectal visceral pain induced by neonatal colonic inflammation in mice.

AIMS: Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder, but its pathogenesis remains incompletely understood, particularly the involvements of central nervous system sensitization in colorectal visceral pain. Our study was to investigate whether the paraventricular thalamus (PVT) projected to the insular cortex (IC) to regulate colorectal visceral pain in neonatal colonic inflammation (NCI) mice and underlying mechanisms. METHODS: We applied optogenetic, chemogenetic, or pharmacological approaches to manipulate the glutamatergicPVT-IC pathway. Fiber photometry was used to assess neuronal activity. Electromyography activities in response to colorectal distension (CRD) were measured to evaluate the colorectal visceral pain. RESULTS: NCI enhanced c-Fos expression and calcium activity upon CRD in the ICGlu, and optogenetic manipulation of them altered colorectal visceral pain responses accordingly. Viral tracing indicated that the PVTGlu projected to the ICGlu. Optogenetic manipulation of PVTGlu changed colorectal visceral pain responses. Furthermore, selective optogenetic modulation of PVT projections in the IC influenced colorectal visceral pain, which was reversed by chemogenetic manipulation of downstream ICGlu. CONCLUSIONS: This study identified a novel PVT-IC neural circuit playing a critical role in colorectal visceral pain in a mouse model of IBS.

Rab27a-mediated exosome secretion in anterior cingulate cortex contributes to colorectal visceral pain in adult mice with neonatal maternal deprivation.

Chronic visceral pain is a common symptom of irritable bowel syndrome (IBS). Exosomes are involved in the development of pain. Rab27a can mediate the release of exosomes. The purpose of this study is to investigate how Rab27a-mediated exosome secretion in the anterior cingulate cortex (ACC) regulates visceral hyperalgesia induced with neonatal maternal deprivation (NMD) in adult mice. The colorectal distension method was adopted to measure visceral pain. The BCA protein assay kit was applied to detect the exosome protein concentration. Western blotting, quantitative PCR, and immunofluorescence technique were adopted to detect the expression of Rab27a and the markers of exosomes. Exosomes extracted from ACC were more in NMD mice than in control (CON) mice. Injection of the exosome-specific inhibitor GW4869 in ACC attenuated colorectal visceral pain of NMD mice. Injection of NMD-derived exosomes produced colorectal visceral pain in CON mice. Rab27a was upregulated in ACC of NMD mice. Rab27a was highly expressed in ACC neurons of NMD mice, rather than astrocytes and microglia. Injection of Rab27a-siRNA reduced the release of exosomes and attenuated the colorectal visceral pain in NMD mice. This study suggested that overexpression of Rab27a increased exosome secretion in ACC neurons, thus contributing to visceral hyperalgesia in NMD mice.NEW & NOTEWORTHY This work demonstrated that the expression of Rab27a in the anterior cingulate cortex was upregulated, which mediated multivesicular bodies trafficking to the plasma membrane and led to the increased release of neuronal exosomes, thus contributing to colorectal visceral pain in neonatal maternal deprivation (NMD) mice. Blocking the release of exosomes or downregulation of Rab27a could alleviate colorectal visceral pain in NMD mice. These data may provide a promising strategy for the treatment of visceral pain in irritable bowel syndrome patients.

Bibliometric and visualized analysis of research relating to minimally invasive spine surgery reported over the period 2000-2022.

Background: Since entering the 21st century, there has been an increasing interest in minimally invasive surgery for spinal diseases, which has led to the continued development of minimally invasive spine surgery (MISS), with major breakthroughs in technology and technical skills. However, in recent years, there is little relevant research using bibliometrics to analyze the field of MISS research. The purpose of this study is to sort out the publication situation and topic trends of articles in the field of MISS research from the perspective of bibliometrics. Methods: The articles and reviews related to MISS from 2000 to 2022 were retrieved and downloaded from the Web of Science Core Collection (WOSCC). Visualization and knowledge mapping were performed using three bibliometric tools, including online bibliometric platform, CiteSpace and VOSviewer software. Curve fitting and correlation analysis were performed using Microsoft Excel software. The global research publication output, contributions of countries, institutions, authors, and journals, average citations per item (ACI), Hirsch index (H-index), research hot keywords, etc., in this field were analyzed. Results: A total of 2384 papers were retrieved, including 2135 original papers and 249 review papers. In the past 22 years, the number of annual publications of MISS research has shown a steady growth trend. China contributed the most papers, and the United States ranked second, but the United States had the highest total citations, and H-index value. The most prolific institutions were Soochow University, Capital Medical University and Wooridul Spine Hospital. In this field, Professors Lee SH, Ahn Y and Yang HL have made significant achievements. However, there is relatively little international collaboration between institutions or researchers. World Neurosurgery is the most published journal on MISS research. According to the keyword co-occurrence analysis, recent keywords mainly focus on researches on minimally invasive modalities, techniques and prognosis, while on the keyword analysis of the ongoing bursts, percutaneous transforaminal endoscopic discectomy, lumbar diskectomy, spinal stenosis, recompression, diskectomy, endoscopic spine surgery, laminectomy, transforaminal lumbar interbody fusion, etc., will likely continue to be a research hotspot in the near future. Conclusion: Looking at the temporal trend in the number of publications per year, the number of publications for the MISS study will increase in the near future. China has the highest number of publications, but the US has the highest quality publications. International cooperation needs to be further strengthened. Our findings can provide useful information for the academic community and identify possible research fronts and hotspots in the coming years.

Outcomes of ABO-incompatible liver transplantation in end-stage liver disease patients co-infected with hepatitis B and human immunodeficiency virus.

BACKGROUND: Human immunodeficiency virus (HIV)-positive patients coinfected with hepatitis B virus (HBV) are eligible for liver transplantation (LT) in Africa and Southeast Asia, particularly China. However, the outcome of HIV-HBV coinfected patients referred for ABO-incompatible LT (ABOi-LT) is unknown. AIM: To clarify the outcome of ABOi-LT for HIV-HBV coinfected patients with end-stage liver disease (ESLD). METHODS: We report on two Chinese HIV-HBV coinfected patients with ESLD who underwent A to O brain-dead donor LT and reviewed the literature on HIV-HBV coinfected patients treated with ABO-compatible LT. The pretransplantation HIV viral load was undetectable, with no active opportunistic infections. Induction therapy consisted of two sessions of plasmapheresis and a single dose of rituximab in two split doses, followed by an intraoperative regimen of intravenous immunoglobulin, methylprednisolone, and basiliximab. Post-transplant maintenance immunosuppressive agents consisted of tacrolimus and mycophenolate mofetil, and prednisone. RESULTS: At the intermediate-term follow-up, patients showed undetectable HIV viral load, CD4(+) T cell counts greater than 150 cells/µL, no HBV recurrence, and stable liver function. A liver allograft biopsy showed no evidence of acute cellular rejection. Both patients survived at 36-42 mo of follow-up. CONCLUSION: This is the first report of ABOi-LT in HIV-HBV recipients with good intermediate-term outcomes, suggesting that ABOi-LT may be feasible and safe for HIV-HBV coinfected patients with ESLD.

Transcriptional investigation of the toxic mechanisms of perfluorooctane sulfonate in rats based on an RNA-Seq approach.

Perfluorooctane sulfonate (PFOS) was widely used in industrial applications before it was listed as a persistent organic pollutant by the Conference of the Parties in the Stockholm Convention in 2009. Although the potential toxicity of PFOS has been studied, its toxic mechanisms remain largely undefined. Here, we investigated novel hub genes and pathways affected by PFOS to gain new conceptions of the toxic mechanisms of PFOS. Reduced body weight gain and abnormal ultra-structures in the liver and kidney tissues were spotted in PFOS-exposed rats, indicating successful establishment of the PFOS-exposed rat model. The transcriptomic alterations of blood samples upon PFOS exposure were analysed using RNA-Seq. GO analysis indicates that the differentially expressed gene-enriched GO terms are related to metabolism, cellular processes, and biological regulation. Kyoto encyclopaedia of gene and genomes (KEGG) and gene set enrichment analysis (GSEA) were conducted to identify six key pathways: spliceosome, B cell receptor signalling pathway, acute myeloid leukaemia, protein processing in the endoplasmic reticulum, NF-kappa B signalling pathway, and Fc gamma R-mediated phagocytosis. The top 10 hub genes were screened from a protein-protein interaction network and verified via quantitative real-time polymerase chain reaction. The overall pathway network and hub genes may provide new insights into the toxic mechanisms of PFOS exposure states.

Folic acid attenuates chronic visceral pain by reducing clostridiales abundance and hydrogen sulfide production.

Irritable bowel syndrome (IBS) related chronic visceral pain affects 20% of people worldwide. The treatment options are very limited. Although the scholarly reviews have appraised the potential effects of the intestinal microbiota on intestinal motility and sensation, the exact mechanism of intestinal microbiota in IBS-like chronic visceral pain remains largely unclear. The purpose of this study is to investigate whether Folic Acid (FA) attenuated visceral pain and its possible mechanisms. Chronic visceral hyperalgesia was induced in rats by neonatal colonic inflammation (NCI). 16S rDNA analysis of fecal samples from human subjects and rats was performed. Patch clamp recording was used to determine synaptic transmission of colonic-related spinal dorsal horn. Alpha diversity of intestinal flora was increased in patients with IBS, as well as the obviously increased abundance of Clostridiales order (a main bacteria producing hydrogen sulfide). The hydrogen sulfide content was positive correlation with visceral pain score in patients with IBS. Consistently, NCI increased Clostridiales frequency and hydrogen sulfide content in feces of adult rats. Notably, the concentration of FA was markedly decreased in peripheral blood of IBS patients compared with non-IBS human subjects. FA supplement alleviated chronic visceral pain and normalized the Clostridiales frequency in NCI rats. In addition, FA supplement significantly reduced the frequency of sEPSCs of neurons in the spinal dorsal horn of NCI rats. Folic Acid treatment attenuated chronic visceral pain of NCI rats through reducing hydrogen sulfide production from Clostridiales in intestine.

Wide-Scope Multi-residue analysis of pesticides in beef by gas chromatography coupled with quadrupole Orbitrap mass spectrometry.

Increasing pesticide contamination in foods of animal origin has made the wide-scope multi-residue analysis of pesticides an international concern. By using 191 pesticides, this study investigates a sensitive and reliable method for multi-residue analysis of pesticides in beef to determine the extent of the application of this method. The QuEChERS method was employed to extract and purify the pesticides as C18 was utilized as the absorbents. Then, the purified pesticides were analysed using gas chromatography - quadrupole orbitrap mass spectrometry (GC-Q-Orbitrap-MS). The validation test results revealed that this method was satisfactorily sensitive since its screening detection limit (SDL) ranged from 0.2 to 100 µg∙kg-1. The recovery tests implemented at three spiking levels, namely 100, 200, and 500 µg∙kg-1, generated the results of 71.95 %-113.97 %, while the intra- and inter-day precisions were 0.27 %-17.94 %, indicating that this method had excellent accuracy and precision.

A national-scale distribution of organochlorine pesticides (OCPs) in cropland soils and major types of food crops in China: Co-occurrence and associated risks.

Organochlorine pesticides (OCPs) are persistent organic pollutants (POPs) that resist degradation in the environment. OCPs remain detectable in cropland systems in China. However, spatial distribution of OCPs across China and associated ecological and health risks, as well as the relationship between levels of OCPs in cropland soils and crops, remain to be elucidated. To fill these gaps, we conducted a national-scale characterization of 19 individual OCPs in cropland soils and food crops including cereals and legumes in China, which were on-spot sampled simultaneously. Sparse canonical correlation analysis was employed to investigate the co-occurrence of OCPs in cropland soils and corresponding food crops. The ecological soil screening levels and risk quotient method were adopted for ecological and health risk assessment, respectively. Dichlorodiphenyltrichloroethanes (DDTs) were dominant in cropland systems, with its levels ranging up to 337 and 22.8 µg/kg in cropland soils and food crops, respectively. The mean ∑OCP levels in cropland soils varied from below the limit of detection to 337 µg/kg. Peanuts were the most contaminated crop, in which endosulfans and hexachlorobenzene (HCB) were co-occurrent with those in cropland soils (correlation coefficient R = 0.999 and 0.947, respectively). Besides, lindane and ß-endosulfan in rice were co-occurrent with those in cropland soils (R = 0.810 and 0.868, respectively). The componential ratio analysis indicated fresh inputs of technical DDT, lindane, chlordane, endosulfan, HCB and aldrin. Among these pesticides, ecological impacts of DDTs, lindane, aldrin and ß-endosulfan could be expected. Human health risk assessment suggested that daily consumption of the OCP-contaminated food crops raises a health concern especially for male teens. It is concluded that OCPs remain present in cropland systems in China at levels that raise a concern for both environment and human health.

A large geographic-scale characterization of organochlorine pesticides (OCPs) in surface sediments and multiple aquatic foods of inland freshwater aquaculture ponds in China: Co-occurrence, source and risk assessment.

Inland freshwater aquaculture ponds (IFAPs) represent the key component of the global lentic freshwater environment and are increasingly important for global aquaculture production, yet the occurrence of organochlorine pesticides (OCPs) in these pond systems remains largely unknown. Here, we characterized the residual concentrations of 19 individual OCPs in sediments and in cultured fish and crustacean species (crabs, shrimp, crayfish and lobster), which were on-spot sampled from the IFAPs at a large region-scale in China. The total OCP levels in sediments varied dramatically between regions. Crabs presented the greatest OCP contamination among the studied species. Dichlorodiphenyltrichloroethanes (DDTs) was the dominating contaminant in sediments and crabs and its stable degradation products 4,4'-DDE and 4,4'-DDD were co-occurrent between these two compartments. The diagnostic ratio analysis indicated fresh inputs of DDTs, lindane and aldrin in multiple regions, which may be resulted from agricultural soil erosion, surface runoff and local anthropogenic activities. Ecological impacts of these pesticides could be expected at some sites due to their levels in sediments above the risk level. Risk assessment based on the OCP levels corrected by the cooking loss revealed that daily consumption of the IFAPs-derived aquatic foods may pose carcinogenic risks in humans.

Wide-scope multi-residue analysis of pesticides in beef by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Increasing pesticide contamination in foods of animal origin has made the wide-scope multi-residue analysis of pesticides an international concern. In this study, a sensitive and reliable multi-residue pesticide analysis method for beef was developed with the use of 129 pesticides to demonstrate the full scope of the method. The pesticides were extracted and purified using a modified QuEChERS technique and analysed using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. An orthogonal array design was employed for optimisation of the method, significantly scientising and simplifying the process. Method validation revealed satisfactory sensitivity with limits of quantification values ranging from 0.003 to 11.37 µg∙kg-1. Matrix effects ranged from 83.85% to 120.66%. Recovery at three spiking levels (20, 50, and 100 µg∙kg-1) were 70.51-128.12% and the intra-day and inter-day precisions were less than 20%, indicating good accuracy and precision.

Computer-aided diagnosis of isocitrate dehydrogenase genotypes in glioblastomas from radiomic patterns.

World Health Organization tumor classifications of the central nervous system differentiate glioblastoma multiforme (GBM) into wild-type (WT) and mutant isocitrate dehydrogenase (IDH) genotypes. This study proposes a noninvasive computer-aided diagnosis to interpret the status of IDH in glioblastomas from transformed magnetic resonance imaging patterns. The collected image database was composed of 32 WT and 7 mutant IDH cases. For each image, a ranklet transformation which changed the original pixel values into relative coefficients was 1st applied to reduce the effects of different scanning parameters and machines on the underlying patterns. Extracting various textural features from the transformed ranklet images and combining them in a logistic regression classifier allowed an IDH prediction. We achieved an accuracy of 90%, a sensitivity of 57%, and a specificity of 97%. Four of the selected textural features in the classifier (homogeneity, difference entropy, information measure of correlation, and inverse difference normalized) were significant (P < .05), and the other 2 were close to being significant (P = .06). The proposed computer-aided diagnosis system based on radiomic textural features from ranklet-transformed images using relative rankings of pixel values as intensity-invariant coefficients is a promising noninvasive solution to provide recommendations about the IDH status in GBM across different healthcare institutions.

Multi-residue analysis of 126 pesticides in chicken muscle by ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry.

Ensuring food safety requires effective methods for the simultaneous analysis of pesticide residues in animal-origin foods. A sensitive and reliable method for the multi-residue analysis of 126 pesticides in chicken muscle was developed using a modified QuEChERS technique and ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. A new enhanced matrix removal-lipid material was used as the QuEChERS dispersive purification adsorbent. Method validation revealed satisfactory linearities with correlation coefficients higher than 0.994 for all pesticides. All limits of quantification were below 10 µg/kg, except in the cases of oxadiazon, clomazone, and lactofen. The matrix effects were nearly negligible, ranging from 73.09% to 125.22%. The recoveries at three spiked levels (50, 100, and 200 µg/kg) were 71.38-120.43% and the intra-day and inter-day precisions were lower than 10%, indicating the good accuracy and precision of the established method.

Single-Molecule Kinetic Fingerprinting for the Ultrasensitive Detection of Small Molecules with Aptasensors.

Aptamers have emerged as promising molecular tools for small-molecule analyte sensing. However, the performance of such aptasensors is generally limited by leakage since it has been difficult to completely suppress signal in the absence of analyte, resulting in a compromise between sensitivity and specificity. Here, we describe a methodology for the ultrasensitive detection of analytes combining aptasensors with single-molecule kinetic fingerprinting. A short, fluorescently labeled DNA probe is utilized to detect the structural changes upon ligand binding to the designed hairpin-shaped aptasensor probe. The Poisson statistics of binding and dissociation events of the DNA probe to single surface-immobilized aptasensor molecules is monitored by total internal reflection fluorescence microscopy, permitting the high-accuracy discrimination of the ligand bound and ligand-free states, resulting in zero background. The programmable dynamics of the hairpin enables fine-tuning of the hybridization kinetics of the fluorescent probe, rendering the acquisition time sufficiently flexible to optimize discrimination. Remarkable detection limits are achieved for a diverse set of analytes when spiked into chicken meat extract: the nucleotide adenosine (0.3 pM), the insecticide acetamiprid (0.35 pM), and the dioxin-like toxin PCB-77 (0.72 pM), which is superior to recently reported aptasensors. Our generalizable method significantly improves the performance of aptasensors, with the potential to extend to other molecular biomarkers.

Background-Free Imaging of a Viral Capsid Proteins Coated Anisotropic Nanoparticle on a Living Cell Membrane with Dark-Field Optical Microscopy.

Exploring the diffusion dynamics of a viral capsid proteins (VCP)-functionalized nanocarrier on a living cell membrane could provide much kinetic information for the better understanding of their biological functionality. Gold nanoparticles are an excellent core material of nanocarriers because of the good biocompatibility as well as versatile surface chemistry. However, due to the strong scattering background from subcellular organelles, it is a grand challenge to selectively image an individual nanocarrier on a living cell membrane. In this work, we demonstrated a convenient strategy to effectively screen the scattering background from living cells for single-particle imaging with a polarization-resolved dual-channel imaging module. By taking advantage of the polarization of anisotropic gold nanoparticles (gold nanorods, GNRs), the signals from cell components could be counteracted after subtracting the sequential images one by one, while those transiently rotating GNRs on the cell membrane still exist in the processed image. In contrast to the previously reported methods, this method does not require a complicated optical setup alignment and sophisticated digital image analysis process. According to the single-particle imaging results, the majority of VCP-GNRs were anchoring on the cell membrane with confined diffusion. Interestingly, on further inspection of the diffusion trajectories, the particles displayed anomalous confined diffusion with randomly distributed large walking steps during the whole track. Non-Gaussian step distribution was noted, indicating heterogeneous binding and desorption processes on the cell membrane. As a consequence of the robust background screening capability, this approach would find broad applications for single-particle imaging under a noisy environment, e.g., living cells.

Anterior vs conventional approach right hepatic resection for large hepatocellular carcinoma: A systematic review and meta-analysis.

AIM: To compare the clinical outcomes of right hepatectomy for large hepatocellular carcinoma via the anterior and conventional approach. METHODS: We comprehensively performed an electronic search of PubMed, EMBASE, and the Cochrane Library for randomized controlled trials (RCTs) or controlled clinical trials (CCTs) published between January 2000 and May 2017 concerning the anterior approach (AA) and the conventional approach (CA) to right hepatectomy. Studies that met the inclusion criteria were included, and their outcome analyses were further assessed using a fixed or random effects model. RESULTS: This analysis included 2297 patients enrolled in 16 studies (3 RCTs and 13 CTTs). Intraoperative blood loss [weighted mean difference = -255.21; 95% confidence interval (95%CI): -371.3 to -139.12; P < 0.0001], intraoperative blood transfusion [odds ratio (OR) = 0.42; 95%CI: 0.29-0.61; P < 0.0001], mortality (OR = 0.59; 95%CI: 0.38-0.92; P = 0.02), morbidity (OR = 0.77; 95%CI: 0.62-0.95; P = 0.01), and recurrence rate (OR = 0.62; 95%CI: 0.47-0.83; P = 0.001) were significantly reduced in the AA group. Patients in the AA group had better overall survival (hazard ratio [HR] = 0.71; 95%CI: 0.50-1.00; P = 0.05) and disease-free survival (HR = 0.67; 95%CI: 0.58-0.79; P < 0.0001) than those in the CA group. CONCLUSION: The AA is safe and effective for right hepatectomy for large hepatocellular carcinoma and could accelerate postoperative recovery and achieve better survival outcomes than the CA.

Neonatal Colonic Inflammation Increases Spinal Transmission and Cystathionine ß-Synthetase Expression in Spinal Dorsal Horn of Rats with Visceral Hypersensitivity.

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder characterized by chronic abdominal pain and alteration of bowel movements. The pathogenesis of visceral hypersensitivity in IBS patients remains largely unknown. Hydrogen sulfide (H2S) is reported to play an important role in development of visceral hyperalgesia. However, the role of H2S at spinal dorsal horn level remains elusive in visceral hypersensitivity. The aim of this study is designed to investigate how H2S takes part in visceral hypersensitivity of adult rats with neonatal colonic inflammation (NCI). Visceral hypersensitivity was induced by neonatal colonic injection of diluted acetic acid. Expression of an endogenous H2S synthesizing enzyme cystathionine ß-synthetase (CBS) was determined by Western blot. Excitability and synaptic transmission of neurons in the substantia gelatinosa (SG) of spinal cord was recorded by patch clamping. Here, we showed that expression of CBS in the spinal dorsal horn was significantly upregulated in NCI rats. The frequency of glutamatergic synaptic activities in SG was markedly enhanced in NCI rats when compared with control rats. Application of NaHS increased the frequency of both spontaneous and miniature excitatory post-synaptic currents of SG neurons in control rats through a presynaptic mechanism. In contrast, application of AOAA, an inhibitor of CBS, dramatically suppressed the frequency of glutamatergic synaptic activities of SG neurons of NCI rats. Importantly, intrathecal injection of AOAA remarkably attenuated visceral hypersensitivity of NCI rats. These results suggest that H2S modulates pain signaling likely through a presynaptic mechanism in SG of spinal dorsal horn, thus providing a potential therapeutic strategy for treatment for chronic visceral pain in patients with IBS.

Fatty acid profiling of blood cell membranes by gas chromatography with mass spectrometry.

Fatty acids, which are well-known for their influence on human metabolism and signal transduction, are also a substantial component of cellular membranes and regulate the basic properties and functions of membranes. Owing to their multiple functions, fatty acid profiles of cell membranes are of great interest to those who are studying the relationship between membrane biochemical compositions and functions. A HCl-catalyzed derivation method and a gas chromatography with mass spectrometry analysis method were developed to accurately profile the fatty acids in cell membranes of erythrocytes, leukocytes, and platelets. The detection limits of all 35 fatty acids ranged from 0.58 to 22 ng/mL and the limits of quantitation were between 2.1 and 72 ng/mL. Finally, the established method was used to profile the membrane fatty acids of 44 healthy volunteers from the north and south of China. Results revealed significant differences in the fatty acid profiles from the two regions, particularly those of the erythrocytes. This technique may be applied to cell membrane studies to generate new biological hypotheses concerning fatty acid composition and membrane functions as well as to construct related disease profiles.

High-throughput intracellular pteridinic profiling by liquid chromatography-quadrupole time-of-flight mass spectrometry.

Pteridines are a diverse family of endogenous metabolites that may serve as useful diagnostic biomarkers for disease. While many preparative and analytical techniques have been described for analysis of selected pteridines in biological fluids, broad intracellular pteridine detection remains a significant analytical challenge. In this study, a novel, specific and sensitive extraction and high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOF MS) method was developed to simultaneously quantify seven intracellular pteridines and monitor 18 additional, naturally-occurring intracellular pteridines. The newly developed method was validated through evaluation of spiked recoveries (84.5-109.4%), reproducibility (2.1-5.4% RSD), method detection limits (0.1-3.0 µg L(-1)) and limits of quantitation (0.1-1 µg L(-1)), and finally application to non-small cell lung cancer A549 cells. Twenty-three pteridine derivatives were successfully detected from cell lysates with an average RSD of 12% among culture replicates. Quantified intracellular pteridine levels ranged from 1 to 1000 nM in good agreement with previous studies. Finally, this technique may be applied to cellular studies to generate new biological hypotheses concerning pteridine physiological and pathological functions as well as to discovery new pteridine-based biomarkers.

[Targeted therapy of malignant glioma by a chimeric fusion toxin composed of IL-13 and Pseudomonas exotoxin in vitro].

OBJECTIVE: To examine the antitumor activity of IL-13PE38 on solid malignant glioma cells in vitro and to investigate its relationship between the antitumor activity of IL-13PE38 and the expression level of IL-13Rα2 in malignant glioma. METHODS: Ten fresh tissues of anaplastic glioma and 11 fresh tissues of glioblastoma multiforme were obtained during craniotomy at First Affiliated Hospital, Wenzhou Medical College between June 2009 and December 2010. All fresh glioma cells were cultured in vitro with IL-13PE38. Then the cytotoxicity of IL-13PE38 was determined by colorimetric MTS proliferation assay and the SR (survival rate) calculated. The expression level of IL-13Rα2 was studied by immunohistochemical SABC method in 21 cases of malignant glioma. And the value of integrated optical density (IOD) was examined by computer assisted pathological image analysis system. The correlation between the IOD of IL-13Rα2 and the SR of malignant glioma cells was also studied. RESULTS: (1) There were strongly positive expression of IL-13Rα2 in most cases (19/21, 90%). And the expression level of IL-13Rα2 in glioblastoma multiforme was higher than the expression level of IL-13Rα2 in anaplastic glioma (P < 0.05). (2) As the fresh malignant glioma cells were cultured with IL-13PE38 of same concentration, the number of surviving cells decreased in different degrees. The survival rate of 14 cases were < 70% and 9 cases < 50%. And the survival rate of anaplastic glioma cells was higher than that of glioblastoma multiforme cells (P < 0.05). (3) The IOD of IL-13Rα2 and the SR of malignant glioma cells were strongly negatively correlated (r = -0.093, P < 0.01). CONCLUSION: A low concentration of IL-13PE38 shows a high level of cytotoxicity for solid malignant glioma cells. And its cytotoxic efficiency depends on the expression level of IL-13Rα2.