Wild Heterotrophic Nitrifying Strain Pseudomonas BT1 Isolated from Kitchen Waste Sludge Restores Ammonia Nitrogen Removal in a Sewage Treatment Plant Shocked by Thiourea.

Thiourea is used in agriculture and industry as a metal scavenger, synthetic intermediate, and nitrification inhibitor. However, in wastewater, it can inhibit the nitrification process and induce the collapse of the nitrification system. In such a case, ammonia-oxidizing bacteria (AOB) lose their ability to remove ammonia. We investigated the nitrification system of a 60,000-t/d municipal sewage treatment plant in Nanjing, which collapsed after receiving 5-15 ppm (5-15 mg/L) thiourea. Ammonia nitrogen removal quickly recovered to more than 95% after inoculation with 10 t high-efficiency nitrification sludge, which was collected from a kitchen waste treatment plant. A heterotrophic nitrification strain was isolated from the inoculated sludge and identified as wild Pseudomonas by 16S rDNA sequencing and named "BT1." Based on thiourea tolerance tests, BT1 can tolerate a thiourea content of more than 500 ppm. For comparison, the in situ process was imitated by the simulation system, and the wastewater shocked by 10 ppm thiourea could still meet the emission standard after adding 1% (V/V) BT1. High-throughput sequencing analysis was applied to study microbial succession during thiourea shock loading. The results showed that Hydrogenophaga and Thiobacillus grew with the growth of BT1. Pseudomonas BT1 was used for a 6,000-t/d printed circuit board (PCB) wastewater treatment system, the nitrification system returned to normal in 15 days, and the degradation rate stabilized at more than 95%.

Prediction and characterization of the linear IgE epitopes for the major soybean allergen ß-conglycinin using immunoinformatics tools.

The α subunit of ß-conglycinin is a major allergen in soybean. The objective of this study was to predict and identify the linear immunoglobulin (Ig)E epitopes of the soybean α subunit of ß-conglycinin. Three immunoinformatics tools were used to predict the potential epitopes and were confirmed by dot-blot inhibition using sera from soybean allergic subjects. As a result, 15 peptides were predicted and assembled by solid-phase synthesis. Eleven epitopes were identified by the dot-blot inhibition test. Moreover, peptide 3 had IgE binding capability with all sera(5/5) tested, while peptide 1, 4, 6, 8 and12 could bind to 4/5 of the sera samples. Secondary structure prediction of peptide 3 and circular dichroism test validated that the structure of peptide 3 was a random coil.

Electrochemical detection of peanut allergen Ara h 1 using a sensitive DNA biosensor based on stem-loop probe.

A novel electrochemical DNA sensor was developed by using a stem-loop probe for peanut allergen Ara h 1 detection. The probe was modified with a thiol at its 5' end and a biotin at its 3' end. The biotin-tagged "molecular beacon"-like probe was attached to the surface of a gold electrode to form a stem-loop structure by self-assembly through facile gold-thiol affinity. 6-Mercaptohexanol (MCH) was used to cover the remnant bare region. The stem--loop probe was "closed" when the target was absent, and then the hybridization of the target induced the conformational change to "open", along with the biotin at its 3' end moved away from the electrode surface. The probe conformational change process was verified by circular dichroism (CD); meanwhile, electron-transfer efficiency changes between probe and electrode were proved by electrochemical impedance spectroscopy (EIS). The detection limit of this method was 0.35 fM with the linear response ranging from 10(-15) to 10(-10) M. Moreover, a complementary target could be discriminated from one-base mismatch and noncomplementarity. The proposed strategy has been successfully applied to detect Ara h 1 in the peanut DNA extracts of peanut milk beverage, and the concentration of it was 3.2 × 10(-13) mol/L.

Acute activation of acid ceramidase affects cytokine-induced cytotoxicity in rat islet beta-cells.

Ceramidase hydrolyzes ceramide and produces sphingosine as a substrate of sphingosine kinase (SPHK), which transforms sphingosine to sphingosine-1-phosphate. It has been reported that cytokines elicit SPHK activation in rat beta-cells. As a sphingosine provider, ceramidase should also be activated. In our previous work, we showed that the increase in mRNA and protein levels in cytokine-treated INS-1 rat beta-cells resulted in chronic activation of neutral ceramidase. Here we found that acid ceramidase (AC) is activated by cytokines at an early stage via tyrosine phosphorylation. In addition, basal AC activity was first detected in INS-1 cells and isolated rat islets, and cytokine-induced cell growth was significantly repressed when AC was pharmacologically inhibited.

Tollip attenuated the hypertrophic response of cardiomyocytes induced by IL-1beta.

We examined the role of Tollip in the hypertrophic response of cardiomyocytes. C57BL/6 mice were subjected to transverse aortic constriction (TAC) for 2 weeks and age-matched sham surgical operated mice served as control. TAC significantly reduced the association of Tollip with IRAK-1 by 66.4 percent and increased NF-kappaB binding activity by 86.5 percent and the levels of phospho-p38 by 114.6 percent in the myocardium compared with sham control, respectively. In vitro experiments showed that IL-1beta stimulation also significantly reduced the association of Tollip with IRAK-1 and increased NF-kappaB binding activity in neonatal cardiomyocytes. Tollip overexpression by transfection of cardiac myocytes significantly attenuated the IL-1beta-induced hypertrophic response of cardiac myocytes as evidenced by reduced cell size (16.4 percent) and decreased ANP expression (33.3 percent). Overexpression of Tollip also reduced NF-kappaB binding activity by 30.7 percent and phospho-p38 by 47.1 percent, respectively. The results suggest that Tollip could be a negative regulator during the development of cardiac hypertrophy. The negative regulation of cardiac hypertrophy by Tollip may involve downregulation of the MyD88-dependent NF-kappaB activation pathway.

Transcription factor GATA-4 is involved in erythropoietin-induced cardioprotection against myocardial ischemia/reperfusion injury.

BACKGROUND: Erythropoietin (EPO) can reduce myocardial ischemia/reperfusion (I/R) injury. However, the cellular mechanisms have not been elucidated entirely. The present study was to investigate whether transcription factor GATA-4 could be involved in EPO-induced cardioprotection when it was administered after ischemia, immediately before reperfusion. METHODS AND RESULTS: Male Balb/c mice treated with or without EPO were subjected to ischemia (45 min) followed by reperfusion (4 h). TTC staining showed that the infarct size in EPO-treated mice was significantly reduced compared with untreated I/R mice (P<0.05). Echocardiography examination suggested that EPO administration significantly improved cardiac function following I/R. TUNEL assay indicated that EPO treatment decreased apoptosis. EPO administration also significantly increased the level of nuclear GATA-4 phosphorylation in the myocardium which was positively correlated with the reduction of myocardial infarction. In vitro hypoxia/re-oxygenation study showed that EPO treatment increased the levels of phospho-GATA-4 and decreased cardiomyocyte apoptosis. More significantly, blocking GATA-4 by transfection of a dominant-negative form of GATA-4 (dnGATA-4) abolished EPO-induced cardioprotective effects. CONCLUSION: EPO administration after ischemia, just before reperfusion induced cardioprotection and stimulated GATA-4 phosphorylation. Activation of GATA-4 may be one of the mechanisms by which EPO induced protection against myocardial I/R injury.

Chronic activation of neutral ceramidase protects beta-cells against cytokine-induced apoptosis.

AIM: To investigate the activity and expression of neutral ceramidase (N-CDase) in the insulin-secreting cell line INS-1 and its role in the cellular response to cytokines. METHODS: HPLC, Western blotting, and quantitative real-time PCR were performed to detect the activity and expression of N-CDase in INS-1 cells treated with a cytokine mixture (5 ng/mL interleukin-1beta, 10 ng/mL TNF-alpha, and 50 ng/mL interferon-gamma). The expression and activity of N-CDase in the INS-1 cells were specifically inhibited using N-CDase-siRNA transfection. Annexin V-fluorescein- isothiocyanate/propidium iodide flow cytometry was used to assess apoptosis in the INS-1 cells. RESULTS: The INS-1 cells exhibited some basal N-CDase activity, and cytokines induced a time-dependent delay in the activation of NCDase. As a result, the activation of N-CDase was first detectable at 8 h after stimulation. It peaked at 16 h and remained elevated at 24 h. Cytokines also upregulated the mRNA and protein expression of N-CDase in the INS-1 cells. Furthermore, when N-CDase activity was inhibited by RNA interference, cytokine-induced apoptosis in the INS-1 cells was markedly increased. CONCLUSION: The N-CDase pathway is active in INS-1 cells, and the chronic activation of N-CDase is involved in the pathological response of beta-cells to cytokines, potentially providing protection against cytokine toxicity.