Quantitative analysis of vitamin D using m/MALDI-TOF mass spectrometry based on a parylene matrix chip

Vitamin D deficiency is associated with various disorders and is diagnosed based on the concentration of 25-hydroxy vitamin D3 (25(OH)D3) in serum. The parylene matrix chip was fabricated to reduce the matrix background noise, and the homogenous distribution of the matrix was retained for the quantitative analysis of 25(OH)D3. The Amplex Red assay was performed to confirm that the sample-matrix mixing zone of the parylene matrix chip was formed below the surface of the parylene-N film. The homogeneous distribution of the matrix was verified from the fluorescence image. For effective analysis using a parylene matrix chip, 25(OH)D3 was modified through the nucleophilic addition of betaine aldehyde (BA) to form a hemiacetal salt. Such modified 25(OH)D3 with a positive charge from BA could be effectively analyzed using MALDI-TOF mass spectrometry. Serum 25(OH)D3 was extracted by liquid–liquid extraction (LLE) and quantified using MALDI-TOF mass spectrometry based on the parylene matrix chip. The intensity of the mass peak of 25(OH)D3 was linearly correlated (r2 = 0.992) with the concentration of 25(OH)D3 spiked in serum, and the LOD was 0.0056 pmol/μL. Energy drinks and vitamin D3 tablets were also employed for the real sample analysis. Finally, the results of the chemiluminescence binding assay and MALDI-TOF mass spectrometry were statistically analyzed to determine the applicability of the method using the Bland–Altman test and Passing–Bablok regression.

Abstract P212: Peroxiredoxin-4 And Dopamine D5 Receptor Interact To Reduce Oxidative Stress And Inflammation In The Kidney

Peroxiredoxin-4 (PRDX4), an endoplasmic reticulum peroxiredoxin protein, plays a protective role against oxidative stress and inflammation by reducing hydrogen peroxide to water. The dopamine D5 receptor (D 5 R) is also important in protecting against oxidative stress, but the interaction between PRDX4 and D 5 R in regulating oxidative stress in the kidney is not known. In D 5 R-HEK 293 cells, fenoldopam (FEN, 25 nM/12 hr, n=4), a D1-likereceptor agonist, increased PRDX4 protein expression (1.92±0.12-fold over basal level, n=4), mainly in non-lipid rafts (LRs: 24.9±11.4%, non-LRs: 75.1±11.4%, baseline; LRs: 30.9±13.9%, non-LRs: 174.1±16.7%, FEN). FEN also increased the co-immunoprecipitation of D 5 R and PRDX4 and their colocalization, particularly in the endoplasmic reticulum. In human renal proximal tubule cells (hRPTCs), FEN (25 nM/12 hr, n=3) increased PRDX4 and D 5 R interaction in non-LRs, also. Si-RNA silencing of PRDX4 increased reactive oxygen species (ROS) production and impaired the inhibitory effect of FEN on ROS production (scrambled siRNA: 100.0±11.6% and 65.4±5.6% for Vehicle (Veh) and FEN, respectively; PRDX4 siRNA: 147.7±11.8% and 134.8±11.2% for Veh and FEN, respectively, n=4/group) detected by Amplex Red. In addition in both D 5 R-HEK 293 and hRPTCs, siRNA silencing of PRDX4 increased the production of interleukin-1β (26.88±3.8 and 46.40±4.2 pg/mL [n=3, D 5 R-HEK 293]; 15.87±1.2 and 37.9±1.4 pg/mL [n=3 in hRPTCs]), tumor necrosis factor (131.7±6.5 and 271.2±18.1 pg/mL [n=4, D 5 R-HEK 293]; 108.8±11.8 and 240.1±13.7 pg/mL [n=4 in hRPTCs]), and caspase-12 (15.21±3.8 and 40.78±4.3 ng/mL [n=4,n D 5 R-HEK 293]; 8.8±1.1 and 27.9±2.0 ng/mL [n=4, hRPTCs]). Furthermore, the protein expression of D 5 R was decreased in PRDX4 siRNA-treated D 5 R-HEK293 (~41.2%, n=3) and hRPTCs (~39.6%, n=3). PRDX4 protein was also reduced in the kidney homogenates from D 5 R -/- mice (WT: 1.00±0.18, n=5; D 5 R -/- : 0.686±0.14, n=4; P<0.05). Taken together, PRDX4 interacts with D 5 R to decrease oxidative stress and inflammation in the kidney.

A Label-free Optical Sensing Platform for Beta-glucosidase Activity Using Protein-inorganic Hybrid Nanoflowers

In this work, a convenient, label-free and dual-signal readout optical sensing platform for the sensitively and selectively determination of beta-glucosidase (β-Glu) activity was reported using protein-inorganic hybrid nanoflowers [BSA-Cu3(PO4)2·3H2O] possessing peroxidase-mimicking activity. The nanoflowers (NFs) were facilely synthesized through a self-assembled synthesis strategy at room temperature. The as-prepared NFs could catalytically convert the colorless and non-fluorescent Amplex Red into colored and highly fluorescent resorufin in the presence of hydrogen peroxide via electron transfer process. β-Glu could hydrolyze cyanogenic glycoside, using amygdalin (Amy) as a model, into cyanide ions (CN-), which can subsequently efficiently suppress the catalytic activity of NFs, accompanied with the fluorescence decrease and the color fading. The concentration of CN- was controlled by β-Glu-triggered enzymatic reaction of Amy. Thus, a sensing system was established for fluorescent and visual determination of β-Glu activity. Under the optimum conditions, the present fluorescent and visual bimodal sensing platform exhibited good sensitivity for β-Glu activity assay with a detection limit of 0.33 U·L-1. The sensing platform was further applied to determinate β-Glu in real samples and satisfactory results were attained. Additionally, the label-free optical sensing system can potentially be a promising candidate for β-Glu inhibitors screening.

A proposed enzyme-linked hydrogen peroxide radical assay system (HORAS)

Reactive Oxygen Species (ROS) are chemically reactive molecules that contain oxygen. ROS are naturally generated as a byproduct during mitochondrial oxidative metabolism as well as by cellular responses to a variety of inflammatory stimuli. Intracellularly formed ROS plays an important role in maintaining homeostasis and in cell signaling but, ROS are challenging to quantify. Phagocytic cells such as macrophages may produce H2O2 during the action of bacterial engulfment. Here UV-Vis versus LC-ESI-MS detection methods for an enzyme-linked, cellular assay of H2O2 production in cultured macrophages are compared. In the presence of Horseradish Peroxidase (HRP), Amplex Red (AR) reacts with H2O2 in a 1:1 stoichiometry to produce the red-fluorescent oxidation product resorufin that can be measured by UV/Vis at an absorbance of 570 nm or by LC-ESI-MS at 214 m/z [M+H]+. RAW 264.7 macrophages were stimulated by microscopic foreign particles, with the addition of 0.1mM of Amplex Red substrate and 10 ng/mL of HRP to the cellular media to enzymatically detect H2O2 production. The oxidation product resorufin can be detected by the colorimetric method as low as 50 pmol while liquid chromatography with electrospray ionization and mass spectrometry (LC-ESI-MS) was able to detect as little as 0.2 pmol in vitro. Thus, it was possible to measure low levels of H2O2 released by cells using an enzyme coupled cellular assay with LC-ESI-MS.

A proposed enzyme-linked hydrogen peroxide radical assay system (HORAS)

Reactive Oxygen Species (ROS) are chemically reactive molecules that contain oxygen. ROS are naturally generated as a byproduct during mitochondrial oxidative metabolism as well as by cellular responses to a variety of inflammatory stimuli. Intracellularly formed ROS plays an important role in maintaining homeostasis and in cell signaling but, ROS are challenging to quantify. Phagocytic cells such as macrophages may produce H2O2 during the action of bacterial engulfment. Here UV-Vis versus LC-ESI-MS detection methods for an enzyme-linked, cellular assay of H2O2 production in cultured macrophages are compared. In the presence of Horseradish Peroxidase (HRP), Amplex Red (AR) reacts with H2O2 in a 1:1 stoichiometry to produce the red-fluorescent oxidation product resorufin that can be measured by UV/Vis at an absorbance of 570 nm or by LC-ESI-MS at 214 m/z [M+H]+. RAW 264.7 macrophages were stimulated by microscopic foreign particles, with the addition of 0.1mM of Amplex Red substrate and 10 ng/mL of HRP to the cellular media to enzymatically detect H2O2 production. The oxidation product resorufin can be detected by the colorimetric method as low as 50 pmol while liquid chromatography with electrospray ionization and mass spectrometry (LC-ESI-MS) was able to detect as little as 0.2 pmol in vitro. Thus, it was possible to measure low levels of H2O2 released by cells using an enzyme coupled cellular assay with LC-ESI-MS.

An artificial cell containing cyanobacteria for endosymbiosis mimicking

The bottom-up constructed artificial cells help to understand the cell working mechanism and provide the evolution clues for organisms. Cyanobacteria are believed to be the ancestors of chloroplasts according to endosymbiosis theory. Herein we demonstrate an artificial cell containing cyanobacteria to mimic endosymbiosis phenomenon. The cyanobacteria sustainably produce glucose molecules by converting light energy into chemical energy. Two downstream “metabolic” pathways starting from glucose molecules are investigated. One involves enzyme cascade reaction to produce H2O2 (assisted by glucose oxidase) first, followed by converting Amplex red to resorufin (assisted by horseradish peroxidase). The more biological one involves nicotinamide adenine dinucleotide (NADH) production in the presence of NAD+ and glucose dehydrogenase. Further, NADH molecules are oxidized into NAD+ by pyruvate catalyzed by lactate dehydrogenase, meanwhile, lactate is obtained. Therefore, the sustainable cascade cycling of NADH/NAD+ is built. The artificial cells built here simulate the endosymbiosis phenomenon, meanwhile pave the way for investigating more complicated sustainable energy supplied metabolism inside artificial cells.

Photooxidation-induced fluorescence amplification system for an ultra-sensitive enzyme-linked immunosorbent assay (ELISA)

This report suggests a method of enhancing the sensitivity of chemifluorescence-based ELISA, using photooxidation-induced fluorescence amplification (PIFA). The PIFA utilized autocatalytic photooxidation of the chemifluorescent substrate, 10-acetyl 3,7-dihydroxyphenoxazine (ADHP, Amplex Red) to amplify the fluorescent product resorufin, initially oxidized by horse radish peroxidase (HRP). As the amplification rate is proportional to the initial level of resorufin, the level of antigen labeled by HRP is quantified by analyzing the profile of fluorescence intensity. The normalized profile was interpolated into an autocatalysis model, and the rate of increase at half-maximum time was quantified by the use of an amplification index (AI). The lower limit of detection, for resorufin or HRP, was less than one-tenth that of the plate reader. It requires only slight modification of the fluorescence reader and is fully compatible with conventional or commercial ELISA. When it is applied to a commercial ELISA kit for the detection of amyloid beta, it is verified that the PIFA assay enhanced the detection sensitivity by more than a factor of 10 and was compatible with a conventional 96-well ELISA assay kit. We anticipate this PIFA assay to be used in research for the detection of low levels of proteins and for the early diagnosis of various diseases with rare protein biomarkers, at ultra-low (pg/mL) concentrations.

INFLAMMATORY CYTOKINES AND MICROBIAL LIGANDS AND METABOLITES INTERACT TO MODULATE DUOX2 EXPRESSION AND ACTIVITY

Background The pathogenesis of inflammatory bowel diseases (IBD) is characterized by a dysregulated crosstalk between the host and the microbiome that leads to the development of inflammation and dysbiosis. Dysbiosis in IBD involves an expansion of Proteobacteria and a reduction of Firmicutes, particularly of butyrate-producing species such as Faecalibacterium prausnitzii. The epithelial NADPH oxidase dual oxidase 2 (DUOX2), which prevents bacterial colonization of the mucosa through the production of hydrogen peroxide (H2O2), is the only gene consistently altered in IBD patients before the onset of disease. However, the involvement of DUOX2 in IBD is not well understood. We aimed to define how inflammation and the microbiota regulate DUOX2 activity. Methods C57BI/6J males raised in specific-pathogen free (SPF) and germ-free (GF) conditions underwent a model of dextran sulfate sodium (DSS)-induced colitis for 6 days. We obtained colon specimens for histopathology and isolation of colon epithelial cells (CEC). We stimulated colonoids from wild-type (WT), toll-like receptor 4-KO (Tlr4-KO), and Duox2-KO mice with IFNγ, heat-killed adherent invasive Escherichia coli (AIEC) strain LF82, and heat-killed F. prausnitzii strain A2-165 and determined expression of Duox2, Duoxa2, and production of H2O2. We also treated colonoids with butyrate, a microbial metabolite with anti-inflammatory properties. Extracellular H2O2 production was analyzed by means of the Amplex Red assay, whereas gene expression was determined by qPCR. Results SPF mice undergoing DSS-induced colitis developed overt inflammation that was accompanied by upregulation of Duox2 and Duoxa2, as well as increased production of H2O2 in freshly isolated CECs. DSS-treated GF mice developed a mild inflammation that also caused increased H2O2 production and Duoxa2 upregulation. WT colonoid stimulation with IFNγ and the Crohn’s disease-associated pathobiont AIEC induced Duox2 and Duoxa2 expression, whereas heat-killed F. prausnitzii did not. Similarly, both IFNγ and AIEC promoted epithelial production of H2O2 in WT colonoids but not Duox2-KO colonoids, indicating that epithelial release of H2O2 in response to these stimuli is mediated by DUOX2. Response to AIEC additionally required functional TLR4. Although heat-killed F. prausnitzii did not alter H2O2 production, its metabolite butyrate caused a significant blockade in the release of H2O2 in response to both IFNγ and AIEC. Conclusions Our results show that both inflammation and pathobionts induce the expression and activity of DUOX2, which begets more inflammation. We posit that specific depletion of pathobionts or restitution with butyrate-producing bacteria such as F. prausnitzii may be beneficial in IBD.

Light-Induced Oxidase Activity of DNAzyme-Modified Quantum Dots

Here, we report the synthesis of a quantum dot (QD)-DNA covalent conjugate to be used as an H2O2-free DNAzyme system with oxidase activity. Amino-coupling conjugation was carried out between amino-modified oligonucleotides (CatG4-NH2) and carboxylated quantum dots (CdTe@COOH QDs). The obtained products were characterized by spectroscopic methods (UV-Vis, fluorescence, circular dichroizm (CD), and IR) and the transmission electron microscopy (TEM) technique. A QD-DNA system with a low polydispersity and high stability in aqueous solutions was successfully obtained. The catalytic activity of the QD-DNA conjugate was examined with Amplex Red and ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate)) indicators using reactive oxygen species (ROS) generated by visible light irradiation. The synthesized QD-DNAzyme exhibited enhanced catalytic activity compared with the reference system (a mixture of QDs and DNAzyme). This proved the assumption that the covalent attachment of DNAzyme to the surface of QD resulted in a beneficial effect on its catalytic activity. The results proved that the QD-DNAzyme system can be used for generation of the signal by light irradiation. The light-induced oxidase activity of the conjugate was demonstrated, proving that the QD-DNAzyme system can be useful for the development of new cellular bioassays, e.g., for the determination of oxygen radical scavengers.