Hydrogen Sulfide Promotes Postnatal Cardiomyocyte Proliferation by Upregulating SIRT1 Signaling Pathway.

Hydrogen sulfide (H2S) has been identified as a novel gasotransmitter and a substantial antioxidant that can activate various cellular targets to regulate physiological and pathological processes in mammals. However, under physiological conditions, it remains unclear whether it is involved in regulating cardiomyocyte (CM) proliferation during postnatal development in mice. This study mainly aimed to evaluate the role of H2S in postnatal CM proliferation and its regulating molecular mechanisms. We found that sodium hydrosulfide (NaHS, the most widely used H2S donor, 50-200 µM) increased neonatal mouse primary CM proliferation in a dose-dependent manner in vitro. Consistently, exogenous administration of H2S also promoted CM proliferation and increased the total number of CMs at postnatal 7 and 14 days in vivo. Moreover, we observed that the protein expression of SIRT1 was significantly upregulated after NaHS treatment. Inhibition of SIRT1 with EX-527 or si-SIRT1 decreased CM proliferation, while enhancement of the activation of SIRT1 with SRT1720 promoted CM proliferation. Meanwhile, pharmacological and genetic blocking of SIRT1 repressed the effect of NaHS on CM proliferation. Taken together, these results reveal that H2S plays a promotional role in proliferation of CMs in vivo and in vitro and SIRT1 is required for H2S-mediated CM proliferation, which indicates that H2S may be a potential modulator for heart development in postnatal time window.

Evaluation of Longitudinal Pain Study in Sickle Cell Disease (ELIPSIS) by patient-reported outcomes, actigraphy, and biomarkers.

Clinical trials in sickle cell disease (SCD) often focus on health care utilization for painful vaso-occlusive crises (VOCs). However, no objective, quantifiable pain biomarkers exist, pain is not specific to VOCs, health care utilization varies between patients, unreported at-home VOCs likely contribute to long-term outcomes, and patient-reported outcomes are seldom considered. This noninterventional, longitudinal, 6-month study aimed to develop tools to identify VOCs in SCD patients with or without health care utilization. Participants wore an actigraph device, tracking sleep and activity. Patients with SCD used an electronic patient-reported outcome (ePRO) tool to collect data on pain, medication, fatigue, and daily function. Patients self-reported when they experienced VOC pain (VOC day). Biomarkers were collected every 3 weeks (non-VOC). Self-reported VOCs triggered at-home or in-hospital blood collection. The study enrolled 37 participants with SCD; 35 completed the study. Participants reported 114 VOC events and 346 VOC days, of which 62.3% and 78.3%, respectively, were self-treated at home. The ePRO and actigraphy captured end points of pain, functionality, fatigue, activity, and sleep; each was significantly altered on VOC days compared with non-VOC days. Biomarkers collected at home or in the hospital on VOC days were significantly altered compared with non-VOC baseline values, including leukocyte-platelet aggregates, microfluidic-based blood cell adhesion, interleukin-6, C-reactive protein, interleukin-10, tumor necrosis factor-α, and thrombin-antithrombin. The Evaluation of Longitudinal Pain Study in Sickle Cell Disease (ELIPSIS) trial shows the feasibility of accurately monitoring out-of-hospital pain by using patient-reported VOC days as potential end points for clinical trials in SCD; it describes the changes in biomarkers and activity measured by actigraphy that may enable improved identification and assessment of VOCs.

A new spectrophotometric method for uric acid detection based on copper doped mimic peroxidase.

Cascade reactions catalyzed by natural uricase and mimic peroxidase (MPOD) have been applied for uric acid (UA) detection. However, the optimal catalytic activity of MPOD is mostly in acidic conditions (pH 2-5), mismatching the optimal catalytic alkaline environment of uricase. In this paper, using CuSO4 and urea as raw materials, a MPOD with high catalytic activity in alkaline environment was synthesized by hydrothermal method. Then, based on coupling reaction of uricase/UA/MPOD/guaiacol (GA) system, a novel spectrophotometric method was established to detect 5-60 µmol/L UA (limit of detection = 3.14 µmol/L (S/N = 3)) and accurately quantified serum UA (275.6 ± 39.9 µmol/L, n = 5) with 95-105% of standard addition recovery. The results were consistent with commercial UA kit (p > 0.05). The MPOD could replace natural POD to reduce the cost of UA detection due to simple preparation and cheap raw materials, and is expected to achieve the specific detection of some substances, like glucose and cholesterol, combined with glucose oxidase and cholesterol oxidase.

Changes and clinical significance of serum MMP-9, TIMP-1, COX-2, and immune levels in patients with asthma.

OBJECTIVE: To detect serum metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinases (TIMP-1), cyclooxygenase-2 (COX-2), and T helper cells 1-T helper cells 2 (Th1-Th2) levels in asthma patients and assess their clinical significance. METHODS: A total of 72 patients experiencing acute asthma (acute group), 66 stable asthma patients (stable group), and 60 healthy volunteers (control group) were included in this study. The levels of TIMP-1, COX-2, and Th1-Th2 in patients with acute asthma were measured following treatment with budesonide aerosol inhalation. In addition, the levels of MMP-9, TIMP-1, COX-2 and Th1-Th2 were compared in patients with different severity of acute asthma before and after treatment. RESULTS: The serum levels of MMP-9, TIMP-1, and COX-2 showed an increasing trend in the control, stable, and acute groups, while levels of Th1-Th2 showed a sequential decreasing trend, and the differences were statistically significant. Comparison of lung function indexes among the three groups of patients established a negative correlation between serum MMP-9 and its forced vital capacity% predicted (FEV%pred), TIMP-1, and COX-2, and FEV%pred and forced expiratory volume in 1 s-forced vital capacity (FEV1/FVC) levels, but a positive correlation between Th1-Th2 and FEV1/FVC levels in the acute group. A significant difference was observed on comparing the levels of serum MMP-9, TIMP-1, COX-2, and Th1-Th2 in patients with different conditions in the acute group. Specifically, as the condition worsened, a significant increase in serum MMP-9, TIMP-1, and COX-2 levels but a significant decrease in Th1-Th2 levels was observed. After treatment, we observed a significant decrease in serum MMP-9, TIMP-1, and COX-2 levels but a significant increase in Th1-Th2 levels in the acute group. CONCLUSION: The serum levels of MMP-9, TIMP-1, COX-2, and Th1-Th2 are valuable indicators reflecting the condition of asthma patients and could be considered promising clinical monitoring indicators.

Longitudinal assessment of adhesion to vascular cell adhesion molecule-1 at steady state and during vaso-occlusive crises in sickle cell disease.

Sickle cell disease (SCD) is characterized by frequent and unpredictable vaso-occlusive crises (VOCs). Sickle erythrocytes (SSRBCs) contribute to VOCs by participating in a series of adhesive events with blood cells and the vascular endothelium. Adhesion assays have been used to evaluate the relationship between SSRBC adhesion and SCD severity. We developed a standardized, clinical flow adhesion assay of whole blood to vascular cell adhesion molecule (FA-WB-VCAM). The objective of this study was to assess the variability and clinical predictive value of FA-WB-VCAM in a six-month longitudinal, observational study (ELIPSIS) in SCD subjects during at-home, steady-state and self-reported VOCs, and following VOC resolution. We observed a strong relationship between FA-WB-VCAM and SCD severity. Adhesion indices were significantly lower in SCD subjects on hydroxycarbamide and increased during VOCs; at-home VOCs had significantly higher FA-WB-VCAM than steady-state and contact VOCs. SCD subjects with a high frequency of self-reported VOCs had a pro-adhesive phenotype at steady state and were stratified into a high-adhesive phenotype cohort; two years prospectively we observed a higher frequency of VOCs in the high-adhesion cohort. This study supports stratifying SCD subjects based on steady-state FA-WB-VCAM and suggests that FA-WB-VCAM may be a plausible surrogate end-point for SCD severity.

Flow adhesion of whole blood to P-selectin: a prognostic biomarker for vaso-occlusive crisis in sickle cell disease.

Blood cell adhesion to P-selectin and vascular cell adhesion molecule-1 (VCAM-1) contributes to the pathophysiology of vaso-occlusion crisis (VOC) events in individuals with sickle cell disease (SCD). We evaluated the use of standardized flow adhesion biomarkers in a six-month, 35-subjects longitudinal study (ELIPSIS). Flow adhesion of whole blood on P-selectin (FA-WB-Psel) and VCAM1 (FA-WB-VCAM), and of isolated white blood cells on P-selectin (FA-WBC-Psel) and VCAM-1 (FA-WBC-VCAM) were elevated on VOC days compared with non-VOC days, but only FA-WB-Psel reached statistical significance (P = 0·015). Optimal cut-off values were established with Cox regression models for FA-WB-Psel [46 cells/mm²; hazard ratio (HR): 2·3; 95% confidence interval (CI):1·4-4·0; P = 0·01] and FA-WB-VCAM (408 cells/mm², HR:1·8; 95% CI: 0·9-3·45; P = 0·01). A combined (FA-WB-Psel and FA-WB-VCAM) multimarker risk score was also significantly (P = 0·0006) correlated with VOC risk that was two-fold higher for intermediate and 5·64-fold higher for high score. The concordance (C)-index for the multimarker score was 0·63 in the six-month period (95% CI: 0·56-0·70), indicating a better ability to distinguish patient risk of VOC, compared to individual biomarkers FA-WB-VCAM (C-index: 0·57; 95% CI: 0·49-0·65) or FA-WB-Psel (C-index: 0·58; 95% CI: 0·53-0·62). The presented multimarker score can be used to risk-stratify individuals with SCD during their steady state into low, intermediate, and high-risk strata for self-reported VOCs. Such risk stratification could help focus healthcare resources more efficiently to maintiain health, personalize treatment selection to each patient's individual needs, and potentially reduce healthcare costs.

Synthesis optimization of rich-urea carbon-dots and application in the determination of H2S in rich- and barren-liquids of desulphurizing solutions.

In the research of carbon dots (CDs) containing various nitrogen sources, it was first found that urea/citric acid-CDs showed a selective discolouration reaction with sulphide ions. Therefore, by optimizing various synthesis and detection conditions of the CDs determining sulfur ions, such as the raw material ratio, temperature, time, pH, and oxidation atmosphere in the CD synthesis, a discolour CD-probe method for trace-level sulphide ions was developed. The method is environmentally friendly, shows two linear-response ranges in 0.050-1.0 mg L-1 (A = -0.0827c + 0.8366) and 1.0-15 mg L-1 S2- (A = -0.0209c + 0.7587) and can be used for the high and low concentration quantification of sulphide in various wastewaters. Subsequently, in order to realize the separation and detection of sulphide ions in wastewaters or rich- and barren-liquids containing N-methyldiethanolamine and other substances in desulphurizing solutions, an automatic pretreatment system was also established.

[Microassay of High-Risk Human Papillomavirus Genotype Based on R6G-ddATP/SNaPshot-Gel Fluorescence Method].

OBJECTIVE: R6G-ddATP was used as a dideoxy fluorescence substrate to establish the single base end extension (SNaPShot)-gel fluorescence method for the rapid detection of the genotypes of three high-risk human papillomaviruses (HR-HPV) ( HPV18, HPV33 and HPV35) genotypes. METHODS: HPV quality control products were used as as samples, and R6G-ddATP dideoxy fluorescence reagent was used as substrate. Firstly, HPV was amplified by using universal primers to obtain the first round of amplified products, which were purified and used as templates for subsequent SNaPShot reactions. Then, specific one-step extension primers were used to perform SNaPShot reaction to generate R6G-fluorescence-labeled DNA extension products. The product was subjected to agarose gel electrophoresis, the results of which were observed under a Gel Imager, and the HPV genotyping was done with different one-step extension primers. Each sample was tested three times and the results were compared with DNA sequencing results. RESULTS: The preferred annealing temperature for SNaPShot reaction is 55 ℃. Three HPV genotypes were examined by R6G-ddATP/SNaPShot gel fluorescence assay under optimal conditions, and the results were consistent with DNA sequencing results. CONCLUSION: The R6G-ddATP/SNaPShot-gel fluorescence method for the micro-detection methods of three HR-HPV genotypes was successfully established and can be used for rapid detection of HPV genotypes.

Cross-sectional analysis of adhesion in individuals with sickle cell disease using a standardized whole blood adhesion bioassay to VCAM-1.

Sickle cell disease (SCD) is characterized by frequent and unpredictable vaso-occlusive episodes (VOEs) that lead to severe pain, organ damage, and early death. Lack of reliable biomarkers that objectively define VOEs remains a critical barrier to improving the care for SCD patients. VOEs result from a complex interplay of cell-cell interactions that promote micro-vascular occlusion. Earlier studies demonstrated that sickle erythrocytes are more adherent than non-sickle erythrocytes and established a direct link between adhesion and frequency of VOEs. We developed a standardized, flow-based adhesion bioassay to assess the adhesive properties of SCD blood samples. The current study provides a cross-sectional analysis of steady state adhesion in SCD patients presenting at monthly out-patient hematology visits. Steady state adhesion varied from patient-to-patient. Adhesion positively correlated with reticulocyte percent and WBC count although there was no significant relationship between adhesion and platelets or hemoglobin in this study. Additionally, steady state adhesion indices were significantly lower in SCD subjects receiving hydroxyurea therapy when compared to the untreated group. The well-plate based microfluidic flow adhesion bioassay described in this report may provide a platform to identify SCD subjects with severe disease phenotypes, predict impending VOEs, and monitor response to current and developing therapies.

Simultaneous quantification of peroxidase and ascorbic acid in biosamples with an automatic system based on a Fe(iii)/methylthymol blue-carbon dot simulative enzyme.

Peroxidase (POD) and ascorbic acid (AsA) usually coexist in organisms to synergistically protect them from reactive oxygen damage, and their contents undergo dynamic changes under different physiological conditions. What's more, the response of POD-catalytic activity in spectrophotometry has to be corrected using the content of concomitant AsA because we found that there is an extinction reaction between AsA and chromogenic products obtained from POD catalysis. With these implications, by skilfully using the chromogenic and the extinction phenomena in the guaiacol/POD/H2O2 reaction, an automatic analysis system for simultaneous quantification of POD (73-440 U L-1) and AsA (4-60 mg L-1) was successfully established based on flow injection analysis (FIA). Furthermore, under acidic conditions (0.5 mol L-1 of HCl), hydrothermal synthesis (250 °C for 1 h) was used for synthesizing new carbon dots (sPOD-CDs) of methylthymol blue (0.08 g L-1)/FeCl3 (0.8 g L-1), which is a simulative enzyme for POD, and it was first used for catalyzing the guaiacol/H2O2 reaction within the FIA system to replace natural HRP in the extinction reaction. This sPOD-CD solution has no background absorption and its concentration shows excellent correlation with simulative POD-activity. Finally, after optimization, this FIA system was utilized to testify that the reducibility of AsA is due to ascorbate ions and to determine POD and AsA in some plant samples. The standard addition recovery experiment showed that there was no interference from the matrix in real samples (recoveries: 95%-105%), and the obtained POD and AsA results were also consistent with the reference experiments (relative deviation ≤ 2.80%, t-test ≥ 0.07). The proposed FIA system is characterized by high sample-throughput (40 samples per h), better repeatability (relative standard deviation ≤ 1.4%), etc.

A novel immobilization fluorescence capillary analysis method and its applications.

Fluorescence capillary analysis (FCA) realizes trace-level analysis of micro-volume samples; it is easy to operate, extremely low in analytical cost and can significantly lessen environmental pollution from analytical chemistry waste. FCA has the characteristics of green analytical chemistry and has been applied in clinical, biochemical, pharmaceutical, food safety and other fields. FCA basically involves a micro-volume glass capillary, a capillary holder and an ordinary fluorescence detector. The capillary is not only a container for chemical reaction and detection but also functions as a carrier to immobilize enzymes, gene probes or reagents; it can be used repeatedly or can be disposable. In analysis, the capillary which is modified with functional reagents sucks in a measured liquid for the reaction and is then inserted into the holder within the fluorescent detector for measurement. The immobilized FCA method has been successfully used in the determination of reduced coenzyme I, ethanol in liqueur, lactic acid in dairy products, pyruvic acid and glucose in serum, trace-level sulfated bile acid in urine, the ratio of pyruvic/lactic acid in serum, and pyruvic acid in cells as well as in DNA end-labeling and dyeing methods. Further, FCA can also be extended to capillary arrays to complete multipurpose simultaneous determinations and can be combined with mobile phones as fluorescence detectors for use in mobile health analytical technology. FCA will produce considerable social benefits in medicine, pharmacy, fermentation of food, environmental protection and other fields. Therefore, the relevant contents are presented in this tutorial review.

[Microanalysis of Acetaldehyde Dehydrogenase 2 Genotype Based on SNaPShot-FCA].

OBJECTIVE: To establish SNaPShot-fluorescence capillary analysis (SNaPShot-FCA) assay for rapid detection of the genotype of aldehyde dehydrogenase 2 gene (ALDH2) rs671 locus. METHODS: The genomic DNA was extracted from peripheral blood cells. Using R6G-ddATP and cy5-ddGTP as fluorescent substrates, the ALDH2 gene was amplified by SNaPShot to generate DNA products with different fluorescent dyes at the 3' end. FCA was used to detect the products separated by agarose gel electrophoresis and recovered by gel recovery kit, and the genetype of ALDH2 polymorphism was analyzed by fluorescence spectrum. The samples were tested three times repeatedly and compared with the results of DNA sequencing. RESULTS: The optimal concentrations of R6G-ddATP and cy5-ddGTP were 1.4 µmol/L and 8.0 µmol/L, respectively. 106 samples were tested for ALDH2 genotype by SNaPShot-FCA under optimal conditions, including 67 of wild type (GG), 38 of hybrid type (AG), and 1 of mutant type (AA), which were consistent with the sequencing results. CONCLUSION: This study successfully established the SNaPShot-FCA for the micro-detection of ALDH2 genotype for the rapid screening and identification of ALDH2 gene.

Development of a fluorescent capillary biosensor based on self-assembled AuNP/LDH for micro-volume intracellular pyruvate.

Herein, a fluorescent capillary biosensor was developed for quantifying micro-volume intracellular pyruvate (PA), in which AuNPs and lactate dehydrogenase (LDH) were modified on the inner surface of an amination capillary (20 µL) via a self-assembly technique. The PA concentration was quantified by the change in the value of the fluorescence of NADH after sucking a mixed solution of the sample and NADH into the biosensor. This study investigated factors including the degree of protonation of the amino groups on the surface of the capillary, the AuNP concentration and time for self-assembly, the activity concentration and time for the LDH self-assembly, the flow rate and acidity for LDH immobilization, pH, temperature, and reaction time for the NADH/PA/LDH reaction system. Under the optimized conditions, the linear response range of the biosensor towards PA was 2.5-120 µmol L-1, in which the determination limit and detection limit were 2.5 and 0.75 µmol L-1, respectively. The biosensor could be reused more than 41 times when its relative standard deviation (RSD) was controlled at less than 1.5%. At room temperature (approximately 25 °C), the intracellular PA in the erythrocyte of a healthy person was measured using the biosensor, and the PA content was observed to be 241.76 ± 68.05 µmol L-1 (n = 8). The standard addition recovery was 95-106%. Employment of the AuNPs in the PA biosensor not only improved the affinity of the immobilized LDH towards PA and its stability, but also significantly enhanced the service life of the PA biosensor.

Research on a New Micro-Volume Fluorescence Capillary Biosensor Assay for Sequentially Quantifying Pyruvate and Lactate.

It was studied that making conditions of a micro-volume fluorescence capillary biosensor for determining pyruvate (PA) and lactate (LA). The biosensor made under the optimized conditions could be used for sequential quantifications of LA in the range 0.10-1.2 mM and PA in 4-120 µM, and its recovery for PA and LA was in a satisfactory range 97-106% for human serum samples, with detection limits of 0.023 mM for LA (RSD < 1.89%, n = 11) and 0.87 µM for PA (RSD < 1.70%, n = 11). The new assay possessed these advantages that the LDH immobilizing on capillary realized the reuse of expensive enzyme in fluorospectrophotometry, and the consumption of serum samples or chemical reagents decreased to 9 µL in per assay, and the analytes no needed to preseparation, and it also are accurate and reliable. Consequently, the fluorescence capillary biosensor should have a good prospect in assaying PA and LA or LA/PA ratios for clinical medicines or biology field. The optimization conditions and parameters obtained in this study have also a certain guiding significance for the development of biochip based on glass substrate.

Expression, purification and functional characterization of a novel 3α-hydroxysteroid dehydrogenase from Pseudomonas aeruginosa.

3α-Hydroxysteroid dehydrogenase (3α-HSD) catalyzes the oxidation of the 3-hydroxyl group of steroids. The enzymatic conversion is a critical step in the enzymatic assay of urinary sulfated bile acids (SBAs), which is a valuable diagnosis index of hepatobiliary diseases. However, the source of 3α-HSD for clinical applications is limited. In this study, an open reading frame (ORF) encoding a novel 3α-HSD was successfully cloned from Pseudomonas aeruginosa and expressed in Escherichia coli BL21 (DE3). The recombinant protein was purified by immobilized metal ion affinity chromatography. Enzyme characterization studies revealed that the protein has 3α-HSD activity and the Km value for sodium cholate is 1.06 mmol L(-1). More than 60% relative enzyme activity was observed in a wide range of pH and temperature, with an optimum pH at 8.0 and an optimum temperature at 30°C. The enzyme's good thermostability under 40°C would be favorable in clinical applications. Ion interference experiments indicated that Zn(2+) was an activating cofactor which increased the enzyme activity 1.75-fold. With the favorable characteristics mentioned above, the new 3α-HSD is a promising enzyme for clinical applications. More importantly, the present work is the first report on a 3α-HSD from P. aeruginosa.

Tracking of Stem Cells in Chronic Liver Diseases: Current Trends and Developments.

Stem cell therapy holds great promise for future clinical practice for treatment of advanced liver diseases. However, the fate of stem cells after transplantation, including the distribution, viability, and the cell clearance, has not been fully elucidated. Herein, recent advances regarding the imaging tools for stem cells tracking mainly in chronic liver diseases with the advantages and disadvantages of each approach have been described. Magnetic resonance imaging is a promising clinical imaging modality due to non-radioactivity, excellent penetrability, and high spatial resolution. Fluorescence imaging and radionuclide imaging demonstrate relatively increased sensitivity, with the latter excelling in real-time monitoring. Reporter genes specialize in long-term tracing. Nevertheless, the disadvantages of low sensitivity, radiation, exogenous gene risk are inevitably present in each of these means, respectively. In this review, we aim to comprehensively evaluate the current state of methods for tracking of stem cell, highlighting their strengths and weaknesses, and providing insights into their future potential. Multimodality imaging strategies may overcome the inherent limitations of single-modality imaging by combining the strengths of different imaging techniques to provide more comprehensive information in the clinical setting.

Cytochrome c oxidase subunit 4 isoform 2-knockout mice show reduced enzyme activity, airway hyporeactivity, and lung pathology.

Cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial electron transport chain. The purpose of this study was to analyze the function of lung-specific cytochrome c oxidase subunit 4 isoform 2 (COX4i2) in vitro and in COX4i2-knockout mice in vivo. COX was isolated from cow lung and liver as control and functionally analyzed. COX4i2-knockout mice were generated and the effect of the gene knockout was determined, including COX activity, tissue energy levels, noninvasive and invasive lung function, and lung pathology. These studies were complemented by a comprehensive functional screen performed at the German Mouse Clinic (Neuherberg, Germany). We show that isolated cow lung COX containing COX4i2 is about twice as active (88 and 102% increased activity in the presence of allosteric activator ADP and inhibitor ATP, respectively) as liver COX, which lacks COX4i2. In COX4i2-knockout mice, lung COX activity and cellular ATP levels were significantly reduced (-50 and -29%, respectively). Knockout mice showed decreased airway responsiveness (60% reduced P(enh) and 58% reduced airway resistance upon challenge with 25 and 100 mg methacholine, respectively), and they developed a lung pathology deteriorating with age that included the appearance of Charcot-Leyden crystals. In addition, there was an interesting sex-specific phenotype, in which the knockout females showed reduced lean mass (-12%), reduced total oxygen consumption rate (-8%), improved glucose tolerance, and reduced grip force (-14%) compared to wild-type females. Our data suggest that high activity lung COX is a central determinant of airway function and is required for maximal airway responsiveness and healthy lung function. Since airway constriction requires energy, we propose a model in which reduced tissue ATP levels explain protection from airway hyperresponsiveness, i.e., absence of COX4i2 leads to reduced lung COX activity and ATP levels, which results in impaired airway constriction and thus reduced airway responsiveness; long-term lung pathology develops in the knockout mice due to impairment of energy-costly lung maintenance processes; and therefore, we propose mitochondrial oxidative phosphorylation as a novel target for the treatment of respiratory diseases, such as asthma.

On-line monitoring of a recirculating-flow fluorescent capillary system for exploring the interaction mechanism of carbon dots/metal ions.

The interaction mechanism between carbon dots (CDs) and metal ions is essential for optimizing their design, synthesis, and application. However, it must be accurately distinguished and quantified because of CDs' complex structure, composition, and coexisting various response mechanisms or products. Herein, a recirculating-flow fluorescence capillary analysis (RF-FCA) system was developed to online monitor the fluorescence kinetics of CDs interacting with metal ions. The fluorescence kinetics of purification and dissociation of CDs/metal ion complexes were easy to monitor online by integrating immobilized CDs and RF-FCA. Here, CDs derived from citric acid and ethylenediamine were used as a model system. We found that the fluorescence of CDs is quenched by Cu(II) and Hg(II) only through the formation of a coordination complex, by Cr(VI) only through the inner filtering effect, and by Fe(III) through the above two mechanisms. Then the kinetics of the competitive interaction between metal ions were used to address the difference of binding sites on CDs with metal ions, wherein Hg(II) was bound to other sites of CDs besides the same sites of CDs with Fe(III) and Cu(II). Finally, from the fluorescence kinetics of fluorescent molecules in the CD structure with metal ions, the difference was due to the presence of two fluorescent centers in the carbon core and molecular state in the CDs. Therefore, the RF-FCA system can distinguish and quantify the interaction mechanism between metal ions and CDs effectively and accurately and be a potential detection or performance characterization method.

Characterization and Immunogenicity of Recombinant A. flavus Uox Modified by Co/EDTA Carbon Dots.

BACKGROUND: Uricase (Uox) is a major drug in gout and a supplementary drug in cancer treatment. Because allergic reactions caused by Uox limit its clinical application,10% Co/EDTA was used to chemically modify Uox from A. flavus to reduce its immunogenicity. METHODS: The immunogenicity of Uox and 10% Co/EDTA-Uox was examined by determining the antibody titer and concentration of IL-2, IL-6, IL-10, and TNF-ß in quail and rat serum. Moreover, we examined the pharmacokinetics of 10% Co/EDTA-Uox in rats and acute toxicity in mice. RESULTS: The concentration of UA decreased from 771.85 ±180.99 to 299.47 ±20.37 µmoL/L（p<0.01） in the hyperuricemia model of quails injected by 10% Co/EDTA-Uox. Two-way immuno-diffusion electrophoresis revealed that 10% Co/EDTA-Uox did not produce antibody, whereas the antibody titer against Uox was 1:16. The concentrations of four cytokines in the 10% Co/EDTA-Uox group were significantly lower than in Uox group (p < 0.01)； The titer of IgG and IgM against 10% Co/EDTA-Uox was significantly lower than that against Uox at different serum dilutions (p < 0.0001). The pharmacokinetic data indicated that the half-life time of 10% Co/EDTA- Uox( 69.315h) was significantly longer than that of Uox(13.4 h)（p<0.01）. The tissue section of the liver, heart, kidney, and spleen revealed no toxicity in Uox and 10% Co/EDTA- Uox groups. CONCLUSION: 10% Co/EDTA-Uox possesses little immunogenicity, a long half-life time, and a highly efficient degradation of UA.