Innovative non-targeted screening approach using High-resolution mass spectrometry for the screening of organic chemicals and identification of specific tracers of soil and dust exposure in children.

Environmental contamination through direct contact, ingestion and inhalation are common routes of children's exposure to chemicals, in which through indoor and outdoor activities associated with common hand-to-mouth, touching objects, and behavioral tendencies, children can be susceptible and vulnerable to organic contaminants in the environment. The objectives of this study were the screening and identification of a wide range of organic contaminants in indoor dust, soil, food, drinking water, and urine matrices (N = 439), prioritizing chemicals to assess children's environmental exposure, and selection of unique tracers of soil and dust ingestion in young children by non-targeted analysis (NTA) using Q-Exactive Orbitrap followed data processing by the Compound Discoverer (v3.3, SP2). Chemical features were first prioritized based on their predominant abundance (peak area>500,000), detection frequency (in >50% of the samples), available information on their uses and potential toxicological effects. Specific tracers of soil and dust exposure in children were selected in this study including Tripropyl citrate and 4-Dodecylbenzenesulfonic acid. The criteria for selection of the tracers were based on their higher abundance, detection frequency, unique functional uses, measurable amounts in urine (suitable biomarker), and with information on gastrointestinal absorption, metabolism, and excretion, and were further confirmed by authentic standards. We are proposing for the first time suitable unique tracers for dust ingestion by children.

A computational framework to explore large-scale biosynthetic diversity.

Genome mining has become a key technology to exploit natural product diversity. Although initially performed on a single-genome basis, the process is now being scaled up to mine entire genera, strain collections and microbiomes. However, no bioinformatic framework is currently available for effectively analyzing datasets of this size and complexity. In the present study, a streamlined computational workflow is provided, consisting of two new software tools: the 'biosynthetic gene similarity clustering and prospecting engine' (BiG-SCAPE), which facilitates fast and interactive sequence similarity network analysis of biosynthetic gene clusters and gene cluster families; and the 'core analysis of syntenic orthologues to prioritize natural product gene clusters' (CORASON), which elucidates phylogenetic relationships within and across these families. BiG-SCAPE is validated by correlating its output to metabolomic data across 363 actinobacterial strains and the discovery potential of CORASON is demonstrated by comprehensively mapping biosynthetic diversity across a range of detoxin/rimosamide-related gene cluster families, culminating in the characterization of seven detoxin analogues.

Endothelial Cell Culture Under Perfusion On A Polyester-Toner Microfluidic Device.

This study presents an inexpensive and easy way to produce a microfluidic device that mimics a blood vessel, serving as a start point for cell culture under perfusion, cardiovascular research, and toxicological studies. Endpoint assays (i.e., MTT reduction and NO assays) were used and revealed that the components making up the microchip, which is made of polyester and toner (PT), did not induce cell death or nitric oxide (NO) production. Applying oxygen plasma and fibronectin improved the adhesion and proliferation endothelial cell along the microchannel. As expected, these treatments showed an increase in vascular endothelial growth factor (VEGF-A) concentration profiles, which is correlated with adherence and cell proliferation, thus promoting endothelialization of the device for neovascularization. Regardless the simplicity of the device, our "vein-on-a-chip" mimetic has a potential to serve as a powerful tool for those that demand a rapid microfabrication method in cell biology or organ-on-a-chip research.

Capillary electrophoresis coupled to contactless conductivity detection for analysis of amino acids of agricultural interest in composting.

Composting is a sustainable approach to manage animal and vegetal waste generated in the Fundação Parque Zoológico de São Paulo. The resulting compost is often used in ZOO's premises as an organic fertilizer for the production of vegetables, which is further used to feed the animals. The composting product provides many forms of mineral and also amino acids (AA) that are absorbed by plants as nutrients. Since most amino acids absorb only slightly or not at all in the UV wavelengths, we developed a method for the determination of AA of agricultural interest in the composting samples. Due to the complexity of samples, we used ion exchange chromatography for the purification of AA prior to analysis. The proposed CZE-C4 D method allowed a separation of the AA in a short analysis time (less than 3.0 min), with great linearity (with R2 ranging from 0.993 to 0.998). Using a BGE of 10 mmol/L TEA, reduction of high-frequency noise and lower baseline fluctuations were obtained. The LOQ for the five AA were around 35 µmol/L, and were adequate for our purpose. In addition, the method showed good precision (RSD of peak area and migration time less than 1.55 and 1.16%, respectively).

Recombinant hepatitis C virus-envelope protein 2 interactions with low-density lipoprotein/CD81 receptors

Hepatitis C virus (HCV) envelope protein 2 (E2) is involved in viral binding to host cells. The aim of this work was to produce recombinant E2B and E2Y HCV proteins in Escherichia coli and Pichia pastoris, respectively, and to study their interactions with low-density lipoprotein receptor (LDLr) and CD81 in human umbilical vein endothelial cells (HUVEC) and the ECV304 bladder carcinoma cell line. To investigate the effects of human LDL and differences in protein structure (glycosylated or not) on binding efficiency, the recombinant proteins were either associated or not associated with lipoproteins before being assayed. The immunoreactivity of the recombinant proteins was analysed using pooled serum samples that were either positive or negative for hepatitis C. The cells were immunophenotyped by LDLr and CD81 using flow cytometry. Binding and binding inhibition assays were performed in the presence of LDL, foetal bovine serum (FCS) and specific antibodies. The results revealed that binding was reduced in the absence of FCS, but that the addition of human LDL rescued and increased binding capacity. In HUVEC cells, the use of antibodies to block LDLr led to a significant reduction in the binding of E2B and E2Y. CD81 antibodies did not affect E2B and E2Y binding. In ECV304 cells, blocking LDLr and CD81 produced similar effects, but they were not as marked as those that were observed in HUVEC cells. In conclusion, recombinant HCV E2 is dependent on LDL for its ability to bind to LDLr in HUVEC and ECV304 cells. These findings are relevant because E2 acts to anchor HCV to host cells; therefore, high blood levels of LDL could enhance viral infectivity in chronic hepatitis C patients.

Recombinant hepatitis C virus-envelope protein 2 interactions with low-density lipoprotein/CD81 receptors.

Hepatitis C virus (HCV) envelope protein 2 (E2) is involved in viral binding to host cells. The aim of this work was to produce recombinant E2B and E2Y HCV proteins in Escherichia coli and Pichia pastoris, respectively, and to study their interactions with low-density lipoprotein receptor (LDLr) and CD81 in human umbilical vein endothelial cells (HUVEC) and the ECV304 bladder carcinoma cell line. To investigate the effects of human LDL and differences in protein structure (glycosylated or not) on binding efficiency, the recombinant proteins were either associated or not associated with lipoproteins before being assayed. The immunoreactivity of the recombinant proteins was analysed using pooled serum samples that were either positive or negative for hepatitis C. The cells were immunophenotyped by LDLr and CD81 using flow cytometry. Binding and binding inhibition assays were performed in the presence of LDL, foetal bovine serum (FCS) and specific antibodies. The results revealed that binding was reduced in the absence of FCS, but that the addition of human LDL rescued and increased binding capacity. In HUVEC cells, the use of antibodies to block LDLr led to a significant reduction in the binding of E2B and E2Y. CD81 antibodies did not affect E2B and E2Y binding. In ECV304 cells, blocking LDLr and CD81 produced similar effects, but they were not as marked as those that were observed in HUVEC cells. In conclusion, recombinant HCV E2 is dependent on LDL for its ability to bind to LDLr in HUVEC and ECV304 cells. These findings are relevant because E2 acts to anchor HCV to host cells; therefore, high blood levels of LDL could enhance viral infectivity in chronic hepatitis C patients.

Lethal effects of abamectin on the aquatic organisms Daphnia similis, Chironomus xanthus and Danio rerio.

Abamectin is used as an acaricide and insecticide for fruits, vegetables and ornamental plants, as well as a parasiticide for animals. One of the major problems of applying pesticides to crops is the likelihood of contaminating aquatic ecosystems by drift or runoff. Therefore, toxicity tests in the laboratory are important tools to predict the effects of chemical substances in aquatic ecosystems. The aim of this study was to assess the potential hazards of abamectin to the freshwater biota and consequently the possible losses of ecological services in contaminated water bodies. For this purpose, we identified the toxicity of abamectin on daphnids, insects and fish. Abamectin was highly toxic, with an EC(50) 48 h for Daphnia similis of 5.1 ng L(-1), LC(50) 96 h for Chironomus xanthus of 2.67 µg L(-1) and LC(50) 48 h for Danio rerio of 33 µg L(-1).

Development of methodology for determination of pesticides residue in water by SPE/HPLC/DAD.

To boost crop yield, sugarcane growers are using increasing amounts of pesticides to combat insects and weeds. But residues of these compounds can pollute water resources, such as lakes, rivers and aquifers. The present paper reports the results of a study of water samples from the Feijão River, which is the source of drinking water for the city of São Carlos, São Paulo, Brazil. The samples were evaluated for the presence of four leading pesticides--ametryn, atrazine, diuron and fipronil--used on sugarcane, the dominant culture in the region. The samples were obtained from three points along the river: the headwaters, along the middle course of the river and just before the municipal water intake station. The pesticides were extracted from the water samples by solid-phase extraction (SPE) and then analyzed by liquid chromatography with diode array detection (LC-DAD). The analytical method was validated by traditional methods, obtaining recovery values between 90 and 95%, with precision deviations inferior to 2.56%, correlation coefficients above 0.99 and detection and quantification limits varying from 0.02 to 0.05 mg L(-1) and 0.07 to 0.17 mg L(-1), respectively. No presence of residues of the pesticides was detected in the samples, considering the detection limits of the method employed.