Discriminative detection of soman or VX exposure using europium chelated microparticle-based immunofluorescence microfluidic chip.

The retrospective detection of organophosphorus nerve agents (OPNAs) exposure has been achieved by the off-site analysis of OPNA-human serum albumin (HSA) adducts using mass spectrometry-based detection approaches. However, few specific methods are accessible for on-site detection. To address this, a novel immunofluorescence microfluidic chip (IFMC) testing system combining europium chelated microparticle (EuCM) with self-driven microfluidic chip assay has been established to unambiguously determine soman (GD) and VX exposure within 20 min, respectively. The detection system was based on the principle of indirect competitive enzyme-linked immunosorbent assay. The specific monoclonal antibodies that respectively recognized the phosphonylated tyrosine 411 of GD-HSA and VX-HSA adducts were labeled by EuCM to capture corresponding adducts in the exposed samples. The phosphonylated peptides in the test line and goat-anti-rabbit antibody in the control line were utilized to bind the EuCM-labeled antibodies for signal exhibition. The developed IFMC chip could discriminatively detect exposed HSA adducts with high specificity, demonstrating a low limit of detection at exposure concentrations of 0.5 × 10-6 mol/L VX and 1.0 × 10-6 mol/L GD. The exposed serum samples can be qualitatively detected following an additional pretreatment procedure. This is a novel rapid detection system capable of discriminating GD and VX exposure, providing an alternative method for rapidly identifying OPNA exposure.

Screening of monoclonal antibodies against specific phosphonylation sites and analysis of serum samples exposed to soman and VX using an indirect competitive enzyme-linked immunosorbent assay.

Organophosphorus nerve agents (OPNAs) covalently bind to tyrosine 411 of human serum albumin (HSA) and the formed adducts are stable biomarkers of OPNA exposure. The detection of these adducts has been limited to mass spectrometry techniques combined with protein digestion. Here, we developed indirect competitive ELISA (icELISA) methods to verify OPNA exposure by the detection of OPNA-phosphonylated adducts at tyrosine 411 residue (OPNA-HSA adducts), in which monoclonal antibodies (mAbs) against phosphonylation sites at tyrosine 411 were introduced. The two mAbs were prepared by the fourth generation of rabbit mAb technology using the phosphonylated peptides of LVRY(GD or VX)TKKVPQC as the haptens. These mAbs were screened using our developed competitive ELISA method and then selected based on their individual affinity and selectivity. As a result, we obtained two mAbs that recognized the HSA Tyr 411 adduct of GD (mAb-5G2) or VX (mAb-12B9), respectively. They shared the highest affinity exhibiting a Kd value of about 10-6 mol/L of the OPNA exposure concentration. They also had remarkable selectivity, which could especially recognize their individual OPNA-HSA adducts in a native state but did not recognize other OPNA-HSAs and unadducted HSAs. Especially for mAb-12B9, it could clearly distinguish VX-HSA and GB-HSA between which there was only one alkyl difference in their phosphonyl portion of the adducted sites. The two mAbs were then used to build the icELISA method for analysis of the serum samples exposed to OPNA. It was found that the detectable lowest GD- and VX-exposed concentrations in serum samples were respectively 1.0 × 10-6 mol/L and 10.0 × 10-6 mol/L. This study provides one novel approach and strategy for the retrospective detection of OPNA exposure, and the two mAbs have great potential to be extended for point-of-care testing of OPNA intoxication.

The growth and uptake of Ga and In of rice (Oryza sative L.) seedlings as affected by Ga and In concentrations in hydroponic cultures.

Limited information is available on the effects of gallium (Ga) and indium (In) on the growth of paddy rice. The Ga and In are emerging contaminants and widely used in high-tech industries nowadays. Understanding the toxicity and accumulation of Ga and In by rice plants is important for reducing the effect on rice production and exposure risk to human by rice consumption. Therefore, this study investigates the effect of Ga and In on the growth of rice seedlings and examines the accumulation and distribution of those elements in plant tissues. Hydroponic cultures were conducted in phytotron glasshouse with controlled temperature and relative humidity conditions, and the rice seedlings were treated with different levels of Ga and In in the nutrient solutions. The growth index and the concentrations of Ga and In in roots and shoots of rice seedlings were measured after harvesting. A significant increase in growth index with increasing Ga concentrations in culture solutions (<10mgGaL-1) was observed. In addition, the uptake of N, K, Mg, Ca, Mn by rice plants was also enhanced by Ga. However, the growth inhibition were observed while the In concentrations higher than 0.08mgL-1, and the nutrients accumulated in rice plants were also significant decreased after In treatments. Based on the dose-response curve, we observed that the EC10 (effective concentration resulting in 10% growth inhibition) value for In treatment was 0.17mgL-1. The results of plant analysis indicated that the roots were the dominant sink of Ga and In in rice seedlings, and it was also found that the capability of translocation of Ga from roots to shoots were higher than In. In addition, it was also found that the PT10 (threshold concentration of phytotoxicity resulting in 10% growth retardation) values based on shoot height and total biomass for In were 15.4 and 10.6µgplant-1, respectively. The beneficial effects on the plant growth of rice seedlings were found by the addition of Ga in culture solutions. In contrast, the In treatments led to growth inhibition of rice seedlings. There were differences in the phytotoxicity, uptake, and translocation of the two emerging contaminants in rice seedlings.

Revealing nitrogenous VX metabolites and the whole-molecule VX metabolism in the urine of guinea pigs.

VX, a well-known organophosphorus nerve agent (OPNA), poses a significant threat to public safety if employed by terrorists. Obtaining complete metabolites is critical to unequivocally confirm its alleged use/exposure and elucidate its whole-molecular metabolism. However, the nitrogenous VX metabolites containing 2-diisopropylaminoethyl moiety from urinary excretion remain unknown. Therefore, this study applied a newly developed untargeted workflow platform to discover and identify them using VX-exposed guinea pigs as animal models. 2-(N,N-diisopropylamino)ethanesulfonic acid (DiPSA) was revealed as a novel nitrogenous VX metabolite in urine, and 2-(Diisopropylaminoethyl) methyl sulfide (DAEMS) was confirmed as another in plasma, indicating that VX metabolism differed between urine and plasma. It is the first report of a nitrogenous VX metabolite in urine and a complete elucidation of the VX metabolic pathway. DiPSA was evaluated as an excellent VX exposure biomarker. The whole-molecule VX metabolism in urine was characterized entirely for the first time via the simultaneous quantification of DiPSA and two known P-based biomarkers. About 52.1% and 32.4% of VX were excreted in urine as P-based and nitrogenous biomarkers within 24 h. These findings provide valuable insights into the unambiguous detection of OPNA exposure/intoxication and human and environmental exposure risk assessment.

Simultaneous solvent extraction and quantification of eleven amine compounds related to Chemical Weapon Convention in soils via hydrophilic interaction liquid chromatography-tandem mass spectrometry.

The detection of Chemical Weapon Convention (CWC)-related amine compounds including the precursors or degradation products of V-type organophosphorus nerve agent, nitrogen mustard and 3-quinuclidinyl benzilate is an important aspect for verifying their intact chemical warfare agents. This work focuses on the development of a novel formulation for the simultaneous solvent extraction of eleven CWC-related amine compounds, from the four-type soil matrices including environmental standard soil, sand, clay, and loam. Extracts were well separated on the hydrophilic interaction liquid chromatography (HILIC) and then detected by MS/MS multiple reaction monitoring mode. The type and component of solvent mixtures were optimized to cover a wide range of polarity over all eleven amine compounds with high extraction efficiencies. Extraction parameters, such as the proportion of methanol, water and NH4OH, the times and the period of extraction, and volumes of extraction solution were optimized. The results indicated that a mixed solvent of methanol/water (44:53, v/v) in 3.0% NH4OH was the optimal formulation for extraction of all 11 analytes with high mean extraction recoveries (64.4-96.1%). Specificity and sensitivity were well improved by the good separation of 11 analytes from four-type soil matrices using these optimized HILIC parameters. This method was fully validated for each analyte in four soil matrices. The linear range of 11 analytes was 0.50/0.75-500 ng·g-1 with correlation coefficient (R2) ≥0.990, and intra/inter-day accuracies were 70.3-125% with relative standard deviation (RSD) ≤19.3%. Limit of detection (LOD) of 11 analytes ranged from 0.01 to 0.5 ng·g-1, which was far lower than those reported in previous studies. The built method accomplishes simultaneously quantitative and trace measurement of all eleven CWC-related amine compounds within a single solvent extraction and detection. It only takes a small amount of soil samples and possesses the highest sensitivity over all previous methods. This study provides an optional recommended operating procedure for determination of CWC-related amine compounds in four typical types of complex soils during chemical weapons verification.

[Change of unsaturated fatty acids in hippocampus of mice exposed to lead].

OBJECTIVE: To study possible impairment mechanisms of learning and memory abilities from unsaturated fatty acids in hippocampus of mice exposed to lead. METHODS: Forty-eight healthy mice were divided into 4 groups: low dose (0.625 g/L), middle dose (1.250 g/L) and high dose (2.500 g/L) of lead solution in diet and control group (distilled water). The mice in treatment groups were fed with lead solution every day while the mice in control group were fed with distilled water for 50 days. After learning and memory abilities were measured, the mice were killed and contents of oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3), arachidonic acid (AA,C20:4), eicosapentaenoic acid (EPA,C20:5) and docosahexaenoic acid (DHA, C22:6 ) in hippocampus of mice were determined by high performance liquid chromatography (HPLC). RESULTS: (1) In the four training days, the mice treated with lead in the middle dose group and high dose group significantly increased the escape latencies compared with the mice treated with distilled water (P<0.05), and on the 4th day, the low dosage mice's escape latencies were delayed (P<0.05). The escape latencies of the 1st, 2nd, 3rd and 4th day had significantly positive linear relation with lead dose. Their relative coefficient in turn is r=0.973, 0.985, 0.929 and 0.936, indicating that lead harmed spatial memory of mice in Morris water maze (MWM). (2) The contents of C18:2 and AA were obviously enhanced in hippocampus of middle and high dosage (P<0.05); while there was evident decrease in the contents of C18:3, EPA and DHA (P<0.05); the content of C18:1 was decreased significantly in high dosage group (P<0.01). The mice's escape latencies had significantly negative linear relation with contents of C18:1, C18:3, EPA and DHA, while there was positive linear relation significantly with contents of C18:2 and AA. Their relative coefficient in turn was r=-0.901, -0.914, -0.893, -0.855, 0.936, 0.727. CONCLUSION: Lead interferes with the metabolism of hippocampus fatty acids and affects membrane function in hippocampus of mice, which might contribute to change of the synthesis, metabolism and release of central neurotransmitter and decrease of the learning and memory abilities.

[Treatment of calcaneal fracture by closed reduction and minimally invasive plate fixation assisted with bidirectional distractor distraction].

OBJECTIVE: To investigate clinical effects of calcaneal fracture with closed reduction and minimally invasive plate fixation assisted with bidirectional distractor distraction. METHODS: From September 2015 to October 2016, 11 male patients(13 feet) with calcaneal fractures treated with bidirectional distractor distraction assisted with minimally invasive plate fixation were retrospectively studied. They were aged from 24 to 57 years old with an average of 36.4 years old;8 feet were type IIand 5 feet were type III according to Sanders classification. Postoperative incision, fracture healing, Böhler angle, Gissane angle were observed and Maryland scoring system was used to evaluate clinical effects. RESULTS: All fractures healed well without incision inflammation and incision disunion. All patients were followed up from 12 to 15 months with an average of 13.5 months. Böhler angle were improved from (9.6±7.3)° before operation to (20.2±4.6) ° at 1 year after operation, and had statistical meaning; Gissane angle increased from (92.7 ±8.5)° before operation to (121.7 ±7.6) ° at 1 year after operation. Maryland score at 1 year after operation was 88.79±8.25, and 11 feet got excellent results and 2 feet moderate. CONCLUSIONS: Bidirectional distractor distraction assisted with minimally invasive plate fixation could effectively fix calcaneal fractures, reduce postoperative complications, and get satisfied results of postoperative images and functional recovery. It is one of effective methods for treating Sanders II and III calcaneal fractures.

Arsenic accumulation and speciation in rice grains influenced by arsenic phytotoxicity and rice genotypes grown in arsenic-elevated paddy soils.

Rice consumption is a major route of As exposure to human for the population of worldwide. This study investigates the effect of phytotoxicity and rice genotypes on the content and speciation of As in rice grains grown in different levels of As-elevated paddy soils from Taiwan. Three levels of As-elevated soils and six rice genotypes commonly planted in Taiwan were used for this study. The results indicate that As contents in grains of rice is not proportional to soil As concentrations and they were equal or higher in indica genotypes than japonica genotypes used in this study. It was also found that the As phytotoxicity not only reducing the grain yields but also the As concentrations in grain of rice. The predominant As species found in rice grains were dimethylarsinic acid (DMA) and arsenite. The concentrations of DMA increased with total As concentrations, wherggeas the arsenite remained in a narrow range from 0.1 to 0.3 mg kg(-1). Because of the lower toxicity of DMA than inorganic As species, the health risks may not be increased through consumption of rice even when total As content in the grains is increased.

[Preparation and characterization the polyclonal antibody of the nonstructural protein of human highly pathogenic H5N1 avian influenza viruses].

OBJECTIVE: Of this study was to prepare high sensitivity and high specificity of highly pathogenic H5N1 subtype avian influenza virus NS1 protein antibody and a preliminary assessment of its potency. METHODS: Construct pET-28a (+) recombinant vector containing the H5N1 subtype of avian influenza virus NS1 sequences of E. coli BL21 (DE3), induced expression of NS1 protein, NS1 recombinant protein was obtained by Ni-NTA column purified by affinity chromatography, and SDS-PAGE and Western Blot analysis. Purified protein antigen to immunize New Zealand white rabbits, obtained rabbit anti-NS1 serum, affinity-purified polyclonal antibodies. Using ELISA and Western Blot analysis of purified antibody titer and specificity. RESULTS: NS1 fusion protein was highly expressed in a purity of greater than 90%, with the fusion protein was used to immunize New Zealand white rabbits anti-NS1 polyclonal antibody titer of 1:80 000, and specific recognition of the H5N1 subtype of avian influenza virus NS1 protein. CONCLUSIONS: NS1 polyclonal antibodies to NS1 recombinant protein purified antigen, with better potency and specificity, and to prepare the conditions for the development of the H5N1 subtype of avian influenza virus detection kit.