Identification of molecular basis that underlie enzymatic specificity of AzoRo from Rhodococcus opacus 1CP: A potential NADH:quinone oxidoreductase.

Azo dyes are important to various industries such as textile industries. However, these dyes are known to comprise toxic, mutagenic, and carcinogenic representatives. Several approaches have already been employed to mitigate the problem such as the use of enzymes. Azoreductases have been well-studied in its capability to reduce azo dyes. AzoRo from Rhodococcus opacus 1CP has been found to be accepting only methyl red as a substrate, surmising that the enzyme may have a narrow active site. To determine the active site configuration of AzoRo at atomic level and identify the key residues involved in substrate binding and enzyme specificity, we have determined the crystal structure of holo-AzoRo and employed a rational design approach to generate AzoRo variants. The results reported here show that AzoRo has a different configuration of the active site when compared with other bacterial NAD(P)H azoreductases, having other key residues playing a role in the substrate binding and restricting the enzyme activity towards different azo dyes. Moreover, it was observed that AzoRo has only about 50% coupling yield to methyl red and p-benzoquinone - giving rise to the possibility that NADH oxidation still occurs even during catalysis. Results also showed that AzoRo is more active and more efficient towards quinones (about four times higher than methyl red).

Risk factors for instent restenosis of sirolimus-coated stents in coronary intervention for patients with unstable angina.

To investigate the instent restenosis rate of sirolimus-coated stents in percutaneous coronary intervention (PCI) and risk factors for in-stent restenosis, patients with unstable angina (UA) caused by coronary artery stenosis were enrolled, and all clinical and imaging data were analyzed. Among 143 enrolled patients with UA aged 35-83 (mean 60.9 ± 10.0) years enrolled, there were 114 (79.7%) male and 29 (20.3%) female patients. Arterial stenosis was present in one coronary artery in 6 (4.2%) patients, in two coronary arteries in 20 (14.0%) patients, in three arteries in 116 (81.1%), and in four coronary arteries in 1 (0.7%) patient. Stenting was successfully performed in all (100%) patients, and 181 stents were deployed. The quantitative flow ratio (QFR) was 0.92 ± 0.03 (range 0.84-0.96) immediately after stenting, and the TIMI was grade 3 in all patients. The diameter of the stents deployed ranged 2.25-4 mm (mean 3.04 ± 0.44) with a length ranging 10 mm to 104 mm (mean 32.73 ± 15.5). Follow-up angiography was performed in all patients with a duration of 1-92 (mean 15.0 ± 18.8) months. Instent restenosis ≥ 50% occurred in 25 (17.5%) patients. In univariate logistic regression analysis, significant (P < 0.05) risk factors for instent restenosis ≥ 50% were QFR (OR 0.036, 95% CI 0.13-0.97), stent diameter (OR 0.43, 95% CI 0.18-0.92), hypertension (OR 3.16, 95% CI 1.02-9.82), smoking (OR 0.31, 95% CI 0.11-0.89), and neutrophil count (OR 2.22, 95% CI 1.10-5.44). In multivariate analysis, QFR (OR 0.02, 95% CI 0.002-0.19), stent diameter (OR 0.06, 95% CI 0.005-0.59), hypertension (OR 6.75, 95% CI 1.83-35.72) and neutrophil count (OR 276.07, 95% CI 12.32-10,959.95) were significant (P < 0.05) independent risk factors for instent restenosis ≥ 50%. In conclusion, certain instent restenosis rates occurs after the sirolimus-eluted coronary stent deployment for the treatment of coronary artery stenosis in patients with UA, and quantitative flow ratio after stenting, stent diameter, hypertension, and neutrophil count are significant risk factors for instent restenosis of the sirolimus-coated stents in coronary intervention.

Research on health risk assessment of heavy metals in soil based on multi-factor source apportionment: A case study in Guangdong Province, China.

Environmental problems caused by heavy metal pollution in soil have attracted widespread attention worldwide. Identifying and quantifying the heavy metal pollution sources and risks is crucial for subsequent soil management. In this study, an integrated source-risk method for source apportionment and risk assessment based on the PMF model, the geodetector model and the health risk assessment model (HRA) was proposed and applied. Analysis of Hg, As, Pb, Cd, Cu, Ni, Cr, and Zn in 208 topsoils showed that the average contents of eight heavy metals were 1.87-5.86 times greater than corresponding background values, among which Cd and As were relatively high, which were higher than the specified soil risk screening values, high-value areas of heavy metals are mainly concentrated in the central part of the study area. The source apportionment showed that the accumulation of heavy metals was affected by five sources: atmospheric deposition (16.3 %), natural sources (33.1 %), industrial activities dominated by metal mining (15.1 %), industrial activities dominated by metal smelting (12.6 %) and traffic sources (22.9 %). The results of the health risk assessment showed that the carcinogenic risks (adult: 4.74E-05, children: 7.41E-05) of heavy metals in soil to the study population were both acceptable, the non-carcinogenic risk of adult (THI = 0.277) was within the limit, while the non-carcinogenic risk of children (THI = 1.70) was higher than the limit value. Ingestion (89.5 %-95.9 %) contributed the greatest health risk among all exposure routes. Source 3 (arsenic-related industrial activities dominated by metal mining) contributed the most to the HI and CRI of adults and children (all above 50 %), therefore, in the formulation stage of soil management strategy in this area, priority should be given to the control and management of this pollution source. These results can provide more detailed support for environmental protection departments to propose targeted soil pollution control measures.

[Source Apportionment and Potential Ecological Risk Assessment of Soil Heavy Metals in Typical Industrial and Mining Towns in North China].

To understand the status of heavy metals in soils of typical industrial and mining towns and quantitatively analyze the potential sources, the contents of seven heavy metals (Cd, As, Pb, Cr, Cu, Ni, and Zn) in 150 surface soils in Xuanhua District, Zhangjiakou City, Hebei Province were collected and examined. The geoaccumulation index and potential ecological risk index methods were used to evaluate the heavy metal pollution status and potential ecological risk. Principal component analysis (PCA) and the positive matrix factorization (PMF) model were used to comprehensively analyze the pollution sources of seven heavy metals, and geostatistics was used to identify the high contribution areas of potential sources. The results revealed that:① the average values of heavy metals in the study area ranged from 0.23-103.34 mg·kg-1, among which the average contents of Cd, Pb, Cu, and Zn were higher than the soil background value of Hebei Province. ② The results of the geoaccumulation and potential ecological risk indices demonstrated that the degree of pollution of the seven heavy metals was in the following order:Cd>Pb>Cu>Zn>Ni>As>Cr, the content of Cd in 16% sites was above a moderate pollution level, and the potential ecological risk of heavy metals in more than 95% sites was at a light risk level. ③ The main sources of accumulation of the seven heavy metals in the study area were combined sources of industry and traffic, natural sources, and agricultural sources, with their contribution rates of 33.1%, 48.7%, and 18.2%, respectively. Among them, Cd, Pb, Cu, and Zn were primarily affected by the combined sources of industry and transportation; Cr, Ni, and As were mainly affected by natural sources, whereas Cd and some As were affected by agricultural sources. The organic combination of PCA, PMF model, and geostatistical methods confirmed the results of each analysis, which increased the reliability of the analytical results of heavy metal sources.

Prediction of high-risk areas of soil heavy metal pollution with multiple factors on a large scale in industrial agglomeration areas.

Heavy metals in soil are a great threat to ecosystems and human health. The rapid development of industrialization has created a serious risk of heavy metal pollution in soil. The study took the industrial-intensive Dahetan subbasin as the typical area. The factors and interactions that affected the distribution of soil heavy metals (Cd, Hg, As, Pb and Cr) in the typical area were explored based on the Geodetector model. The analysis results were extended to predict high-risk areas of heavy metal pollution in the soil in the Xiangjiang River basin. The results showed that Cd, As and Pb were significantly affected by local industrial and mining activities, and Hg and Cr were primarily affected by natural factors, such as pH and soil type. Compared to a single factor, the interaction between factors had a greater impact on the concentration of heavy metals. The high-risk areas of soil heavy metal pollution in the Xiangjiang River basin were primarily concentrated in the upper reaches and middle reaches.Significant overlapping of high-risk areas of multiple heavy metals occurred in the west, middle and south of the basin. The spatial visualization of the high-risk areas was realized, and the influence of several factors was integrated via layer superposition. This study proposes a new idea to predict the high-risk areas of soil pollution in large-scale areas to provide a reference for the regional prevention and control of soil pollution.

Changing the electron donor improves azoreductase dye degrading activity at neutral pH.

The oxygen-insensitive azoreductase AzoRo originating from Rhodococcus opacus 1CP was found to be most active at low pH (ca. 4) and high temperature (ca. 50°C). AzoRo is not an efficient biocatalyst when used at low pH due to stability problems. To overcome this issue, we discovered that AzoRo accepts an alternative electron donor, 1-benzyl-1,4-dihydronicotinamide (BNAH), which allows fast turnover at neutral pH. In order to screen this nicotinamide coenzyme mimic as a source of electrons, AzoRo-catalysed reactions were run under neutral conditions, under which typically slow rates are observed with NADH. For the reduction of 1 azo bond by azoreductases 2mol nicotinamide coenzyme are needed. AzoRo displayed Methyl Red (MR) reduction activities with NADH and NADPH of 5.49±0.14Umg-1 and 4.96±0.25Umg-1, respectively, whereas with BNAH it displayed 17.01±0.74Umg-1 (following BNAH oxidation) and 7.16±0.06Umg-1 (following MR reduction). Binding of BNAH to AzoRo was determined with a Km of 18.75±2.45µM (BNAH oxidation) and 12.45±0.47µM (MR reduction). In order to show applicability of this system an upscaled reaction was performed using 78.6µg of purified AzoRo to convert 2.96µmol of MR (total reaction volume: 40ml) within a 1h reaction.

Prognostic significance of human papillomavirus viral load in correlation with different therapeutic modalities in cervical cancer patients.

PURPOSE: High-risk human papillomavirus (HR-HPV) infections were the causal factor in the development of cervical cancer, but the significance of HPV viral load in the prediction of the response to current therapeutic approaches had not reached consensus. The present study was performed to assess the high risk HPV viral load of cervical cancer patients who underwent radiotherapy alone or in combination with chemotherapy or hyperthermotherapy or both in correlation to long-term survival. METHODS: 116 cervical cancer patients were recruited and assigned into four groups of different therapeutic modalities. The prevalent high risk types of HPV 16, 18, 58 were detected by type specific in situ hybridization (ISH), and HPV mRNA was detected by RNA scope assay using RNA scope 2.0 FFPE Reagent Kit. Semi-quantification of the HR-HPV viral load was measured based on the intensity of ISH signal captured from the tumor nests in the grey scale. RESULTS: The HR-HPV viral load had a significant negative correlation with survival (rs=-0.368, P=0.001). The 15-year survival rate of low viral load group was 68.18%, moderate viral load group was 52.17%, and high viral load group was 34.69% (P=0.001). HPV mRNA expression was strongly consistent with HPV viral load. The 15-year survival rates of different therapeutic groups were 39.29%, 58.62%, 50.00%, 55.17%, respectively (P=0.545). Combinatorial treatment modalities improved the actual survival, which demonstrated no significant difference among 5, 10 and 15 years comparison. Cox regression analysis showed that the relative risk of death was obviously higher in the HPV 18 single positive group and high HPV viral load group. CONCLUSIONS: The semi-quantitive viral load assessment in situ is a feasible approach in clinical practice. The more the HPV viral load was, the worse the survival of patients would be. The combinational treatments were in favor of the disease-stabilization.