[Determination of indigo and brilliant blue in different types of food products by high performance liquid chromatography with solid phase extraction].

OBJECTIVE: To develop and validate a solid phase extraction-high performance liquid chromatographic( SPE-HPLC) method for the simultaneous determination of indigo and brilliant blue in different types of food products. METHODS: The artificial colors in food products were extracted by acetonitrile / water and purified by WAX SPE cartridges, The separation was achieved using a Waters Symmetry C_(18)( 5 µm, 4. 6 mm × 250 mm) column and a binary gradient mobile phase of methanol and 0. 02 mol/L ammonium acetate solution, detected by HPLC-PDA. RESULTS: The validated analytical method showed that there was a good linearity in the range of 0. 05- 20. 00 µg/mL for both indigo and brilliant blue( r > 0. 999). The lowest detection limits of indigo and brilliant blue were 0. 04 and 0. 02 mg/kg, respectively. The average recoveries were among 81. 8%- 101. 1%, with relative standard deviation( RSD) of 2. 1%- 4. 9%( n =6) for both artificial colors. CONCLUSION: The method has high selectivity, high sensitivity, good recovery and reproducibility. It is suitable to simultaneously monitor indigo and brilliant blue in several types of food products based on the food classification system of GB 2760-2014.

Urinary concentrations of fungicide carbendazim's metabolite and associations with oxidative stress biomarkers in young children.

Carbendazim (CBDZ) is the most widely used fungicide in China. It is ubiquitous in environment and can induce oxidative stress in mammals, while data on occurrence of its metabolite in human urine are scarce, and the relationship between CBDZ and oxidative stress biomarkers (OSBs) in young children has not been examined. The aim of this study was to measure the concentrations of methyl 5-hydroxy-2-benzimidazolecarbamate (5-HBC, the main metabolite of CBDZ in urine) in 390 urine samples collected from 130 healthy young (< 6.6 years old) children from Shenzhen and Wuhan, in south and central China, respectively, and to evaluate the associations of 5-HBC with three selected OSBs (4-HNEMA, 8-OHG, and 8-OHdG, for lipid, RNA, and DNA, respectively). 5-HBC was found in 99.2% of the urine samples at concentrations ranging from below the method detection limit (< 0.005 ng/mL) to 10.9 ng/mL (median: 0.11 ng/mL). Moderate inter-day reproducibility was found for specific gravity-adjusted 5-HBC concentrations (intraclass correlation coefficient: 0.50). The urinary 5-HBC concentrations were significantly and positively associated with 4-HNEMA (p < 0.01). An interquartile range increase in urinary 5-HBC concentrations was associated with a 42.1% increase in 4-HNEMA, which implied that CBDZ exposure might be associated with lipid peroxidation in young children without occupational exposure. As far as we know, this pilot study is the first to report urinary 5-HBC and its associations with OSBs in children.

Dissection of the bed nucleus of the stria terminalis neuronal subtypes in feeding regulation.

The bed nucleus of the stria terminalis (BNST) plays an important role in feeding regulation through projections to other brain areas. However, whether functional distinctions exist within different BNST cells is not clear. Here, we found optogenetic activation of LH-projecting BNST neurons induced aversion and significantly reduced consumption of normal chow but not high-fat diets (HFD). In contrast, photoactivation of vlPAG-projecting BNST neurons induced place preference and promoted HFD intake, without affecting normal chow consumption. Moreover, optogenetic silencing of LH-projecting BNST neurons reduced the consumption of normal chow in fasted mice, while photoinhibition of vlPAG-projecting BNST neurons decreased the consumption of HFD in both fed and fasted mice. We then labeled the LH- and vlPAG-projecting BNST neurons using retroAAV-GFP and retroAAV-mCherry, respectively, and found these two populations of neurons have different anatomical distribution and electrophysiological properties. Taken together, we identified vlPAG-projecting and LH-projecting BNST neurons are two distinct populations of cells with significant differences in functional and anatomic characteristics.

Inhibition of TANK-binding kinase1 attenuates the astrocyte-mediated neuroinflammatory response through YAP signaling after spinal cord injury.

AIMS: TANK-binding kinase 1 (TBK1) is involved in regulating the pathological process of a variety of inflammatory diseases in the central nervous system. However, its role and underlying molecular mechanisms in spinal cord injury (SCI) remain largely unknown. METHODS: We employed the TBK1 inhibitor amlexanox (ALX) to address this question. An in vivo clip-compressive SCI model and in vitro lipopolysaccharide (LPS)-induced astrocyte inflammation model were established to examine the effects of TBK1 inhibition on the expression of proinflammatory cytokines. RESULTS: In this study, we found that TBK1 and TBK1-medicated innate immune pathways, such as TBK1/IRF3 and noncanonical NF-κB signaling, were activated in astrocytes and neurons after SCI. Furthermore, inhibition of TBK1 by ALX alleviated neuroinflammation response, reduced the loss of motor neurons, and improved the functional recovery after SCI. Mechanistically, inhibition of TBK1 activity promoted the activation of the noncanonical NF-κB signaling pathway and inhibited p-IRF3 activity in LPS-induced astrocytes, and the TBK1 activity was required for astrocytic activation through yes-associated protein (YAP) signaling after SCI and in LPS-induced astrocytes inflammation model. CONCLUSION: TBK1-medicated innate immune pathway in astrocytes through YAP signaling plays an important role in the pathogenesis of SCI and inhibition of TBK1 may be a potential therapeutic drug for SCI.

The application of amine-terminated silicon quantum dots on the imaging of human serum proteins after polyacrylamide gel electrophoresis (PAGE).

Novel amine-terminated silicon (Si) quantum dots (QDs) were synthesized and applied for the detection of human serum proteins on gels directly after polyacrylamide gel electrophoresis (PAGE). The diameter of these stable amine-terminated Si QDs was in the range of 0.5-2.0 nm. In this study, the fluorescent imaging conditions, such as the buffer solution, pH value, buffer concentration and quantity of Si QDs, were optimized and the possible mechanisms of Si QDs-protein interaction were analyzed. The mode of Si QDs and human serum albumin association was found to occur by hydrogen bond interactions; this was probably attributed to the interaction between the amino group of amine-terminated Si QDs and the carboxyl group of proteins. Meanwhile, human serum proteins separated by native 1D and native 2D electrophoresis were detected by Si QD-based fluorescent imaging. Some proteins, such as isoform 1 of α-1-antitrypsin, complement C3 (Fragment) and hemopexin, which were identified by mass spectrometry (MS), were easily detected by using Si QDs, but not with CBB-R250 staining. The Si QDs-based fluorescent imaging technique with high resolution is a sensitive and dependable method for direct detection of human serum proteins, and has enormous potential in clinical diagnosis.

Atractylon inhibits the tumorigenesis of glioblastoma through SIRT3 signaling.

Glioblastoma (GBM) is the most common primary malignant brain tumor. Although there are various treatments for glioblastoma including surgery, radiotherapy, systemic therapy (chemotherapy and targeted therapy) and supportive therapy, the overall prognosis remains poor and the long-term survival rate is very low. Atractylon, a bioactive compound extracted from the Chinese herb Atractylodes lancea (Thunb.) DC. or Atractylodes chinensis (DC.) Koidz., has been reported to induce apoptosis and suppress metastasis in hepatic cancer cells. However, the roles and mechanisms of atractylon in GBM cells remain unknown. In the present study, we aimed to evaluate the effects of atractylon on the anti-tumorigenesis properties of GBM. Firstly, results of CCK8, colony formation, cell proliferation, and flow cytometry assays showed that atractylon inhibited the proliferation of GBM cells by arresting cells at the G1 phase of cell cycle. In addition, atractylon suppressed the migration and induced apoptosis of GBM cells. Mechanistically, atractylon treatment caused a significant up-regulation of sirtuin 3 (SIRT3, a tumor suppressor) mRNA and protein in GBM cells. Furthermore, inhibition of SIRT3 by the selective SIRT3 inhibitor 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP) partially restored the anti-proliferation and migration effects of atractylon in GBM cells. Finally, atractylon treatment also inhibited the in vivo growth of GBM cells in xenograft models through SIRT3 activation. Taken together, these results reveal a previously unknown role of atractylon in inhibiting GBM in vitro and in vivo through up-regulating SIRT3, which suggests novel strategies for the treatment of GBM.

Fraxinellone inhibits progression of glioblastoma via regulating the SIRT3 signaling pathway.

Glioblastoma (GBM) is the most prevalent type of adult primary brain tumor and chemotherapy of GBM was limited by drug-resistance. Fraxinellone is a tetrahydro-benzofuranone derivative with various pharmacological activities. However, the pharmacological effects of fraxinellone on GBM remains largely unknown. Here, we found that fraxinellone inhibited the proliferation and growth of GBM cells in a dose-dependent manner in vitro. Subsequently, we found that fraxinellone suppressed the migration and induced apoptosis of GBM cells in vitro. Using western blot and immunostaining, we further found that fraxinellone downregulated the expressions of sirtuin 3 (SIRT3), and superoxide dismutase 2 (SOD2), a downstream of SIRT3 in GBM cells. Meanwhile, reactive oxygen species (ROS) were increased in these fraxinellone-treated GBM cells. Interestingly, overexpression of SIRT3 (SIRT3-OE) indeed partially restored the inhibition of both cell proliferation and migration of GBM cells induced by fraxinellone. Finally, we found that fraxinellone could inhibit the growth of GBM in xenograft model through the inactivation of SIRT3 signaling pathway. Taken together, these results suggest that fraxinellone suppressed the growth and migration of GBM cells by downregulating SIRT3 signaling in vitro, and inhibited the tumorigenesis of GBMs in vivo.

Hyperprogression, a challenge of PD-1/PD-L1 inhibitors treatments: potential mechanisms and coping strategies.

Immunotherapy is now a mainstay in cancer treatments. Programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) immune checkpoint inhibitor (ICI) therapies have opened up a new venue of advanced cancer immunotherapy. However, hyperprogressive disease (HPD) induced by PD-1/PD-L1 inhibitors caused a significant decrease in the overall survival (OS) of the patients, which compromise the efficacy of PD-1/PD-L1 inhibitors. Therefore, HPD has become an urgent issue to be addressed in the clinical uses of PD-1/PD-L1 inhibitors. The mechanisms of HPD remain unclear, and possible predictive factors of HPD are not well understood. In this review, we summarized the potential mechanisms of HPD and coping strategies that can effectively reduce the occurrence and development of HPD.

Irigenin inhibits glioblastoma progression through suppressing YAP/ß-catenin signaling.

Glioblastoma (GBM) is the most malignant glioma in brain tumors with low survival and high recurrence rate. Irigenin, as an isoflavone compound extracted from Shegan, has shown many pharmacological functions such as antioxidant, anti-inflammatory and anti-tumor. However, the effects of irigenin on GBM cells and the related molecular mechanisms remain unexplored. In this study, we found that irigenin inhibited the proliferation of GBM cells in a dose-dependent manner by several assays in vitro. Subsequently, we found that irigenin arrested cell cycle at G2/M phase and induced apoptosis of GBM cells in vitro. In addition, irigenin inhibited the migration of GBM cells. Mechanically, we found that irigenin treatment decreased the expression of YAP (yes-associated protein), suppressed ß-catenin signaling. Furthermore, overexpression of YAP partially restored the anti-tumor effects of irigenin on GBM cells in vitro. Finally, we found that irigenin inhibited the growth of tumor in GBM xenograft mice model through inactivation of YAP. Taken together, these results suggest that irigenin exerts its anticancer effects on GBM via inhibiting YAP/ß-catenin signaling, which may provide a new strategy for the treatment of GBM.

Microglia-Specific Expression of HEXA and HEXB Leads to Poor Prognosis in Glioblastoma Patients.

Glioblastoma multiforme (GBM) is one of the deadliest cancers in brain. There have been few treatment advances for GBM despite increasing scientific understanding of this disease. ß-hexosaminidase (Hex) is an important enzyme system in human body, encoded by two genes, HEXA and HEXB, are closely related to central nervous system (CNS) diseases such as Sandhoff disease (SD) and Tay-Sachs disease (TSD). However, the expression pattern and function of HEXA and HEXB in GBM remains unclear. Here, we found that both the mRNA and protein expression levels of HEXA and HEXB were significantly upregulated in GBM patient samples. The results from single-cell RNA-sequencing (scRNA-seq) database and double immunostaining showed that HEXA and HEXB were specifically expressed in microglia in GBM patient samples. Furthermore, our in vitro experiments revealed that conditioned media from HEXA and HEXB knockdown-microglia cells could inhibit the proliferation and migration of GBM cells. Finally, according to survival analysis based on online database, higher expression of HEXA and HEXB was associated with poor prognosis in GBM patients. In conclusion, these results suggest that microglial HEXA and HEXB play fundamental role in GBM progression, and they will be potential biomarkers for GBM.

TEMED enhanced photoluminescent imaging detection of proteins in human serum using quantum dots after PAGE.

In this paper, the development of a novel enhanced photoluminescent (PL) imaging method for human serum proteins detection after polyacrylamide gel electrophoresis (PAGE) is described. Thioglycolic acid (TGA)-capped CdTe QDs and enhanced reagent tetramethylethylenediamine (TEMED) have been introduced, resulting in direct detection of various proteins in native polyacrylamide gels and expanded application scope to SDS gels. Some relatively low-abundance proteins such as Zinc-α(2)-glycoprotein (ZAG) and α(2)-HS-glycoprotein (α(2)-HSG) are easily detected by TEMED enhanced PL imaging and identified by MS and MS/MS techniques. In the present study, the PL imaging conditions such as QDs concentration, alkali concentration, and enhanced reagents are optimized and the possible mechanisms are analyzed. The sensitivity of TEMED enhanced PL imaging is satisfying, with a linear range of 11.7-375 ng for ferritin, comparing with 46.9-375 ng in CBB-R250 staining and 23.4-375 ng in direct PL imaging. As a novel PL imaging detection method that is simple, fast, and sensitive, it shows great analytical potential in proteome research and in biochemistry.

A plasma sputtering decoration route to producing thickness-tunable ZnO/TiO(2) core/shell nanorod arrays.

We report a radio frequency magnetron sputtering method for producing TiO(2) shell coatings directly on the surface of ZnO nanorod arrays. ZnO nanorod arrays were firstly fabricated on transparent conducting oxide substrates by a hydrothermal route, and subsequently decorated with TiO(2) by a plasma sputtering deposition process. The core/shell nanorods have single-crystal ZnO cores and anatase TiO(2) shells. The shells are homogeneously coated onto the whole ZnO nanorods without thickness change. This approach enables us to tailor the thickness of the TiO(2) shell for desired photovoltaic applications on a one-nanometer scale. The function of the TiO(2) shell as a blocking layer for increasing charge separation and suppression of the surface recombination was tested in dye-sensitized solar cells. The enhanced photocurrent and open-circuit voltage gave rise to increased photovoltaic efficiency and decreased dark current, indicating successful functioning of the TiO(2) shell.

Fast haptoglobin phenotyping based on microchip electrophoresis.

A new and fast method for haptoglobin phenotyping was developed based on microchip electrophoresis with laser induced fluorescence detection. Haptoglobin phenotypes 1-1 and 2-2 were labeled with fluorescein isothiocyanate. The analyses were performed on glass microchip which was simply treated with sodium dodecyl sulfate. After the optimization of the separation conditions, Hp 1-1 and Hp 2-2 could be differentiated in 150s and the detection limits for Hp 1-1 and Hp 2-2 were 0.39 and 0.62 µg/mL, respectively. Finally, the method was applied to human serum samples from healthy people and liver cancer patients. A decrease in Hp concentration for liver cancer patients was confirmed. Featuring high efficiency, speed, simplicity, the method reveals great potentials for the diagnosis of diseases and proteome research.

On-line microheterogeneity analysis and rapid phenotyping of haptoglobin by capillary electrophoresis using sodium dodecyl sulfate as additive.

To improve the detection sensitivity and determine phenotypes of haptoglobin (Hp), a prefilling technique was developed and tested in capillary electrophoresis (CE) with UV-vis absorbance detection. Adding 0.01% sodium dodecyl sulfate (SDS) to the protein sample and 0.1% SDS to the prefilling buffer solution, on-line stacking and microheterogeneity separation of Hp were achieved. In addition, the influences of pH, buffer concentration, sample and prefilling buffer SDS concentration upon resolution were examined. Under optimized conditions, Hp-microheterogeneity was well resolved and two phenotypes of Hp (Hp 1-1 and Hp 2-2) were differentiated. This method was applied to the analysis of sera from normal individuals and beta-Thalassemia patients. After the depletion of albumin (HSA) and immunoglobulin G (IgG), this method allowed to determine two phenotypes in different individuals and to detect the decrease of Hp in beta-Thalassemia patients. Featuring high efficiency, speed and simplicity, the proposed method shows great potential for use in clinical diagnosis and proteome research.

Enhanced separation of seven quinolones by capillary electrophoresis with silica nanoparticles as additive.

This paper describes the enhanced separation of lomefloxacin, sparfloxacin, fleroxacin, norfloxacin, ofloxacin, gatifloxacin and pazufloxacin by capillary zone electrophoresis (CZE) using silica nanoparticles (SiNPs) as running buffer additive. The impact of SiNPs concentration on the resolution and selectivity of separation was investigated and a given value of SiNPs was finally chosen under the optimum conditions. The addition of the SiNPs to the running buffer enabled electroosmotic flow (EOF) decrease and permitted full interaction between SiNPs and analytes. The influence of separation voltage, pH and buffer concentration on the separation in the presence of SiNPs was examined. Interactions between drugs and nanoparticles during the separation are discussed; the determination of interaction constants is also achieved. A good resolution of seven quinolones was obtained within 15 min in a 50 cm effective length fused-silica capillary at a separation voltage of +10 kV in a 12 mM disodium tetraborate-phosphate buffer (pH 9.08) containing 5.2 microgmL(-1) SiNPs.