Prediction of Pesticide Interactions with Proteins Involved in Human Reproduction by Using a Virtual Screening Approach: A Case Study of Famoxadone Binding CRBP-III and Izumo.

In recent years, the awareness that pesticides can have other effects apart from generic toxicity is growing. In particular, several pieces of evidence highlight their influence on human fertility. In this study, we investigated, by a virtual screening approach, the binding between pesticides and proteins present in human gametes or associated with reproduction, in order to identify new interactions that could affect human fertility. To this aim, we prepared ligand (pesticides) and receptor (proteins) 3D structure datasets from online structural databases (such as PubChem and RCSB), and performed a virtual screening analysis using Autodock Vina. In the comparison of the predicted interactions, we found that famoxadone was predicted to bind Cellular Retinol Binding Protein-III in the retinol-binding site with a better minimum energy value of -10.4 Kcal/mol and an RMSD of 3.77 with respect to retinol (-7.1 Kcal/mol). In addition to a similar network of interactions, famoxadone binding is more stabilized by additional hydrophobic patches including L20, V29, A33, F57, L117, and L118 amino acid residues and hydrogen bonds with Y19 and K40. These results support a possible competitive effect of famoxadone on retinol binding with impacts on the ability of developing the cardiac tissue, in accordance with the literature data on zebrafish embryos. Moreover, famoxadone binds, with a minimum energy value between -8.3 and -8.0 Kcal/mol, to the IZUMO Sperm-Egg Fusion Protein, interacting with a network of polar and hydrophobic amino acid residues in the cavity between the 4HB and Ig-like domains. This binding is more stabilized by a predicted hydrogen bond with the N185 residue of the protein. A hindrance in this position can probably affect the conformational change for JUNO binding, avoiding the gamete membrane fusion to form the zygote. This work opens new interesting perspectives of study on the effects of pesticides on fertility, extending the knowledge to other typologies of interaction which can affect different steps of the reproductive process.

A new biomarker panel of ultraconserved long non-coding RNAs for bladder cancer prognosis by a machine learning based methodology.

BACKGROUND: Recent studies have indicated that a special class of long non-coding RNAs (lncRNAs), namely Transcribed-Ultraconservative Regions are transcribed from specific DNA regions (T-UCRs), 100[Formula: see text] conserved in human, mouse, and rat genomes. This is noticeable, as lncRNAs are usually poorly conserved. Despite their peculiarities, T-UCRs remain very understudied in many diseases, including cancer and, yet, it is known that dysregulation of T-UCRs is associated with cancer as well as with human neurological, cardiovascular, and developmental pathologies. We have recently reported the T-UCR uc.8+ as a potential prognostic biomarker in bladder cancer. RESULTS: The aim of this work is to develop a methodology, based on machine learning techniques, for the selection of a predictive signature panel for bladder cancer onset. To this end, we analyzed the expression profiles of T-UCRs from surgically removed normal and bladder cancer tissues, by using custom expression microarray. Bladder tissue samples from 24 bladder cancer patients (12 Low Grade and 12 High Grade), with complete clinical data, and 17 control samples from normal bladder epithelium were analysed. After the selection of preferentially expressed and statistically significant T-UCRs, we adopted an ensemble of statistical and machine learning based approaches (i.e., logistic regression, Random Forest, XGBoost and LASSO) for ranking the most important diagnostic molecules. We identified a signature panel of 13 selected T-UCRs with altered expression profiles in cancer, able to efficiently discriminate between normal and bladder cancer patient samples. Also, using this signature panel, we classified bladder cancer patients in four groups, each characterized by a different survival extent. As expected, the group including only Low Grade bladder cancer patients had greater overall survival than patients with the majority of High Grade bladder cancer. However, a specific signature of deregulated T-UCRs identifies sub-types of bladder cancer patients with different prognosis regardless of the bladder cancer Grade. CONCLUSIONS: Here we present the results for the classification of bladder cancer (Low and High Grade) patient samples and normal bladder epithelium controls by using a machine learning application. The T-UCR's panel can be used for learning an eXplainable Artificial Intelligent model and develop a robust decision support system for bladder cancer early diagnosis providing urinary T-UCRs data of new patients. The use of this system instead of the current methodology will result in a non-invasive approach, reducing uncomfortable procedures (such as cystoscopy) for the patients. Overall, these results raise the possibility of new automatic systems, which could help the RNA-based prognosis and/or the cancer therapy in bladder cancer patients, and demonstrate the successful application of Artificial Intelligence to the definition of an independent prognostic biomarker panel.

Emerging RNA-Based Therapeutic and Diagnostic Options: Recent Advances and Future Challenges in Genitourinary Cancers.

Renal cell carcinoma, bladder cancer, and prostate cancer are the most widespread genitourinary tumors. Their treatment and diagnosis have significantly evolved over recent years, due to an increasing understanding of oncogenic factors and the molecular mechanisms involved. Using sophisticated genome sequencing technologies, the non-coding RNAs, such as microRNAs, long non-coding RNAs, and circular RNAs, have all been implicated in the occurrence and progression of genitourinary cancers. Interestingly, DNA, protein, and RNA interactions with lncRNAs and other biological macromolecules drive some of these cancer phenotypes. Studies on the molecular mechanisms of lncRNAs have identified new functional markers that could be potentially useful as biomarkers for effective diagnosis and/or as targets for therapeutic intervention. This review focuses on the mechanisms underlying abnormal lncRNA expression in genitourinary tumors and discusses their role in diagnostics, prognosis, and treatment.

Risk assessment of honey bee stressors based on in silico analysis of molecular interactions.

A global decline of the honey bee Apis mellifera has been observed in the last decades. This pollinator plays a fundamental role in food production and the economy in Europe. The decline of honey bee colonies is linked to several stressors, including pesticides. The current pesticide risk assessment of honey bees in Europe focuses on lethal effects and lacks reflection on sublethal effects. A better understanding of the consequences that exposure to these chemicals has on honey bees is still needed. In this context, the aim of this European Food Risk Assessment Fellowship Programme fellowship project has been to use in silico methodologies, such as virtual screening, as a first step to identify possible interactions at the molecular level between A. mellifera proteins and pesticide ligands. For this purpose, a docking study of the proteins from A. mellifera and pesticide ligands extracted from online databases has been performed by using the software Autodock Vina. The results obtained were a ranking based on the predicted affinity of the pesticides for specific and non-specific binding sites on bee macromolecules. These results were compared with data obtained from the literature and linked to potential sublethal effects. Finally, a risk assessment analysis of the identified molecular stressors of honey bees was performed. The results of this study are considered a starting point to identify new sources of possible stress for honey bees and thereby contribute to the overall understanding of the honey bee decline.

Advances in Cancer Diagnosis: Bio-Electrochemical and Biophysical Characterizations of Cancer Cells.

Cancer is a worldwide leading cause of death, and it is projected that newly diagnosed cases globally will reach 27.5 million each year by 2040. Cancers (malignant tumors), unlike benign tumors are characterized by structural and functional dedifferentiation (anaplasia), breaching of the basement membrane, spreading to adjacent tissues (invasiveness), and the capability to spread to distant sites (metastasis). In the cancer biology research field, understanding and characterizing cancer metastasis as well as features of cell death (apoptosis) is considered a technically challenging subject of study and clinically is very critical and necessary. Therefore, in addition to the cytochemical methods traditionally used, novel biophysical and bioelectrochemical techniques (e.g., cyclic voltammetry and electrochemical impedance spectroscopy), atomic force microscopy, and electron microscopic methods are increasingly being deployed to better understand these processes. Implementing those methods at the preclinical level enables the rapid screening of new anticancer drugs with understanding of their central mechanism for cancer therapy. In this review, principles and basic concepts of new techniques suggested for metastasis, and apoptosis examinations for research purposes are introduced, along with examples of each technique. From our recommendations, the privilege of combining the bio-electrochemical and biosensing techniques with the conventional cytochemical methods either for research or for biomedical diagnosis should be emphasized.

Altered Expression of Protamine-like and Their DNA Binding Induced by Cr(VI): A Possible Risk to Spermatogenesis?

Chromium (VI) is the most dangerous oxidation state among the stable forms of chromium. In this work, we evaluated the effect of exposing Mytilus galloprovincialis for 24 h to 1, 10, and 100 nM chromium (VI) on the properties of Protamine-like (PLs) and their gene levels in the gonads. Specifically, we analyzed, by AU-PAGE and SDS-PAGE, PLs extracted from unexposed and exposed mussels. In addition, via EMSA, we evaluated the ability of PLs to bind DNA and also verified their potential to protect DNA from oxidative damage. Finally, we assessed possible alterations in gonadal expression of mt10, hsp70, and genes encoding for PLs-II/PL-IV and PL-III. We found that for all experimental approaches the most relevant alterations occurred after exposure to 1 nM Cr(VI). In particular, a comigration of PL-II with PL-III was observed by SDS-PAGE; and a reduced ability of PLs to bind and protect DNA from oxidative damage was recorded. This dose of chromium (VI) exposure was also the one that produced the greatest alterations in the expression of both mt10 and PL-II/PL-IV encoding genes. All of these changes suggest that this dose of chromium (VI) exposure could affect the reproductive health of Mytilus galloprovincialis.

Monitoring of pesticide amount in water and drinkable food by a fluorescence-based biosensor.

The identification of pollutants is crucial to protect water resources and ensure food safety. The available analytical methodologies allow reliable detection of organic pollutants such as pesticides; however, there is the need for faster, direct and continuous methodologies for real-time monitoring of pesticides. Fluorescent-based biosensors have been recently proposed as a valid alternative due to their advantage of being easy, cheap and specific. In this context, the aim of the present EU-FORA fellowship programme was to develop and apply a fluorescence-based biosensing device for the detection of organophosphate (OP) pesticides in water samples and drinkable food. The study was addressed using a mutant of the thermostable esterase-2 from Alicyclobacillus acidocaldarius (EST2-S35C) as a bioreceptor for OP pesticides. The use of EST2 involves some significant advantages including specificity and affinity towards OPs, and high stability over time in a different range of temperatures and pH. The protein was labelled to the fluorescent probe IAEDANS and fluorescence measurements of quenching in solution and in immobilised form were performed. The results showed good stability and sensitivity, reaching low limits of detection and quantification and a constant signal intensity over time. The addition of paraoxon quenched the fluorescence of the complex, reaching a plateau at 100 pmol paraoxon. The decrease of enzymatic activity of EST2-S35C-IAEDANS in the presence of paraoxon correlated the inhibition of the labelled enzyme with the decrease in fluorescence. The results from the application of the biosensor with real samples showed a decrease in fluorescence in surface water samples, contaminated by OPs. The use of the developed fluorescence-based biosensor demonstrated its applicability for real samples monitoring and could ensure the production of large amounts of data in a short period of time which can be used to address environmental and food safety risk assessment.

Monitoring of pesticide amount in fruit and vegetables by a fluorescence-based sensor.

Faster, sensitive and real-time methods for detecting organophosphate (OP) pesticides are urged for in situ monitoring of these widely spread contaminants. For this reason, several efforts have been addressed for the development of performant biosensors. The thermostable enzyme esterase-2 from Alicyclobacillus acidocaldarius (EST2), with a lipase-like Ser-His-Asp catalytic triad with a high affinity to OPs, is a promising candidate as a bioreceptor for biosensor development. Within this EU-FORA fellowship project, two different components of the biosensor were evaluated: (i) the use of the enzymatic bioreceptor in solution or immobilised in a solid membrane; (ii) the measurement of fluorescence quenching by direct measurement of the fluorescence probe intensity signal or by fluorescence resonance energy transfer (FRET) from the tryptophans located in the catalytic site of the enzyme to a binded fluorescence probe. Fluorescence spectroscopy is among the most used techniques in analytical chemistry laboratories, mainly due to its high sensitivity and simplicity. To this aim, the developed IAEDANS-labelled EST2-S35C mutant has been used. Fluorometric measurements with both methods showed linearity with increased EST2-S35C concentrations. No significant interference on FRET measurements was observed due to changes in medium pH or due to the addition of other organic components (glucose, ascorbic acid, yeast extract). Both methods presented similar sensitivity towards detecting OPs, with fluorescence quenching due to the presence of paraoxon at environmentally relevant concentrations from 0.09 µM. The obtained results are of high relevance to further development of biosensors for the pesticide monitoring that: (i) decrease the expenses of the analysis; (ii) simplify the procedures for pesticide detection; (iii) reduce the time of response. Furthermore, the use of biosensors for pesticides real-time and in situ detection of pesticides promises to increase the number of samples analysed, providing a larger amount of data for food safety risk assessment.

CD33 rs2455069 SNP: Correlation with Alzheimer's Disease and Hypothesis of Functional Role.

The CD33 gene encodes for a member of the sialic-acid-binding immunoglobulin-type lectin (Siglec) family, and is one of the top-ranked Alzheimer's disease (AD) risk genes identified by genome-wide association studies (GWAS). Many CD33 polymorphisms are associated with an increased risk of AD, but the function and potential mechanism of many CD33 single-nucleotide polymorphisms (SNPs) in promoting AD have yet to be elucidated. We recently identified the CD33 SNP rs2455069-A>G (R69G) in a familial form of dementia. Here, we demonstrate an association between the G allele of the rs2455069 gene variant and the presence of AD in a cohort of 195 patients from southern Italy. We carried out in silico analysis of the 3D structures of CD33 carrying the identified SNP to provide insights into its functional effect. Structural models of the CD33 variant carrying the R69G amino acid change were compared to the CD33 wild type, and used for the docking analysis using sialic acid as the ligand. Our analysis demonstrated that the CD33-R69G variant may bind sialic acid at additional binding sites compared to the wild type, thus potentially increasing its affinity/specificity for this molecule. Our results led to a new hypothesis of rs2455069-A>G SNP as a risk factor for AD, suggesting that a long-term cumulative effect of the CD33-R69G variant results from the binding of sialic acid, acting as an enhancer of the CD33 inhibitory effects on amyloid plaque degradation.

A FRET Approach to Detect Paraoxon among Organophosphate Pesticides Using a Fluorescent Biosensor.

The development of faster, sensitive and real-time methods for detecting organophosphate (OP) pesticides is of utmost priority in the in situ monitoring of these widespread compounds. Research on enzyme-based biosensors is increasing, and a promising candidate as a bioreceptor is the thermostable enzyme esterase-2 from Alicyclobacillus acidocaldarius (EST2), with a lipase-like Ser-His-Asp catalytic triad with a high affinity for OPs. This study aimed to evaluate the applicability of Förster resonance energy transfer (FRET) as a sensitive and reliable method to quantify OPs at environmentally relevant concentrations. For this purpose, the previously developed IAEDANS-labelled EST2-S35C mutant was used, in which tryptophan and IAEDANS fluorophores are the donor and the acceptor, respectively. Fluorometric measurements showed linearity with increased EST2-S35C concentrations. No significant interference was observed in the FRET measurements due to changes in the pH of the medium or the addition of other organic components (glucose, ascorbic acid or yeast extract). Fluorescence quenching due to the presence of paraoxon was observed at concentrations as low as 2 nM, which are considered harmful for the ecosystem. These results pave the way for further experiments encompassing more complex matrices.

A 3D printable adapter for solid-state fluorescence measurements: the case of an immobilized enzymatic bioreceptor for organophosphate pesticides detection.

The widespread use of pesticides in the last decades and their accumulation into the environment gave rise to major environmental and human health concerns. To address this topic, the scientific community pointed out the need to develop methodologies to detect and measure the presence of pesticides in different matrices. Biosensors have been recently explored as fast, easy, and sensitive methods for direct organophosphate pesticides monitoring. Thus, the present work aimed at designing and testing a 3D printed adapter useful on different equipment, and a membrane support to immobilize the esterase-2 from Alicyclobacillus acidocaldarius (EST2) bioreceptor. The latter is labelled with the IAEDANS, a bright fluorescent probe. EST2 was selected since it shows a high specificity toward paraoxon. Our results showed good stability and replicability, with an increasing linear fluorescent intensity recorded from 15 to 150 pmol of labelled EST2. Linearity of data was also observed when using the immobilized labelled EST2 to detect increasing amounts of paraoxon, with a limit of detection (LOD) of 0.09 pmol. This LOD value reveals the high sensitivity of our membrane support when mounted on the 3D adapter, comparable to modern methods using robotic workstations. Notably, the use of an independent support significantly simplified the manipulation of the membrane during experimental procedures and enabled it to match the specificities of different systems. In sum, this work emphasizes the advantages of using 3D printed accessories adapted to respond to the newest research needs.

Selymatra: A web application for protein-profiling analysis of mass spectra.

Surface enhanced laser desorption/ionization-time of flight (SELDI-TOF) mass spectrometry is a variant of the matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. It is used in many cases especially for the analysis of protein profiling and for preliminary screening of biomarkers in complex samples. Unfortunately, these analyses are time consuming and protein identification is generally strictly limited. SELDI-TOF analysis of mass spectra (SELYMATRA) is a web application (WA) developed to reduce these limitations by (i) automating the identification processes and (ii) introducing the possibility to predict proteins in complex mixtures from cells and tissues. The WA architectural pattern is the model-view-controller, commonly used in software development. The WA compares the mass value between two mass spectra (sample vs. control) to extract differences, and, according to the set parameters, it queries a local database to predict most likely proteins based on their masses and different expression amplification. The WA was validated in a cellular model overexpressing a tagged NURR1 receptor, being able to recognize the tagged protein in the profiling of transformed cells. A help page, including a description of parameters for WA use, is available on the website.

Microbial Electrochemical Systems: Principles, Construction and Biosensing Applications.

Microbial electrochemical systems are a fast emerging technology that use microorganisms to harvest the chemical energy from bioorganic materials to produce electrical power. Due to their flexibility and the wide variety of materials that can be used as a source, these devices show promise for applications in many fields including energy, environment and sensing. Microbial electrochemical systems rely on the integration of microbial cells, bioelectrochemistry, material science and electrochemical technologies to achieve effective conversion of the chemical energy stored in organic materials into electrical power. Therefore, the interaction between microorganisms and electrodes and their operation at physiological important potentials are critical for their development. This article provides an overview of the principles and applications of microbial electrochemical systems, their development status and potential for implementation in the biosensing field. It also provides a discussion of the recent developments in the selection of electrode materials to improve electron transfer using nanomaterials along with challenges for achieving practical implementation, and examples of applications in the biosensing field.

Subcellular Localization of uc.8+ as a Prognostic Biomarker in Bladder Cancer Tissue.

Non-coding RNA transcripts originating from Ultraconserved Regions (UCRs) have tissue-specific expression and play relevant roles in the pathophysiology of multiple cancer types. Among them, we recently identified and characterized the ultra-conserved-transcript-8+ (uc.8+), whose levels correlate with grading and staging of bladder cancer. Here, to validate uc.8+ as a potential biomarker in bladder cancer, we assessed its expression and subcellular localization by using tissue microarray on 73 human bladder cancer specimens. We quantified uc.8+ by in-situ hybridization and correlated its expression levels with clinical characteristics and patient survival. The analysis of subcellular localization indicated the simultaneous presence of uc.8+ in the cytoplasm and nucleus of cells from the Low-Grade group, whereas a prevalent cytoplasmic localization was observed in samples from the High-Grade group, supporting the hypothesis of uc.8+ nuclear-to-cytoplasmic translocation in most malignant tumor forms. Moreover, analysis of uc.8+ expression and subcellular localization in tumor-surrounding stroma revealed a marked down-regulation of uc.8+ levels compared to the paired (adjacent) tumor region. Finally, deep machine-learning approaches identified nucleotide sequences associated with uc.8+ localization in nucleus and/or cytoplasm, allowing to predict possible RNA binding proteins associated with uc.8+, recognizing also sequences involved in mRNA cytoplasm-translocation. Our model suggests uc.8+ subcellular localization as a potential prognostic biomarker for bladder cancer.

Point-of-Care Diagnostics of COVID-19: From Current Work to Future Perspectives.

Coronaviruses have received global concern since 2003, when an outbreak caused by SARS-CoV emerged in China. Later on, in 2012, the Middle-East respiratory syndrome spread in Saudi Arabia, caused by MERS-CoV. Currently, the global crisis is caused by the pandemic SARS-CoV-2, which belongs to the same lineage of SARS-CoV. In response to the urgent need of diagnostic tools, several lab-based and biosensing techniques have been proposed so far. Five main areas have been individuated and discussed in terms of their strengths and weaknesses. The cell-culture detection and the microneutralization tests are still considered highly reliable methods. The genetic screening, featuring the well-established Real-time polymerase chain reaction (RT-PCR), represents the gold standard for virus detection in nasopharyngeal swabs. On the other side, immunoassays were developed, either by screening/antigen recognition of IgM/IgG or by detecting the whole virus, in blood and sera. Next, proteomic mass-spectrometry (MS)-based methodologies have also been proposed for the analysis of swab samples. Finally, virus-biosensing devices were efficiently designed. Both electrochemical immunosensors and eye-based technologies have been described, showing detection times lower than 10 min after swab introduction. Alternative to swab-based techniques, lateral flow point-of-care immunoassays are already commercially available for the analysis of blood samples. Such biosensing devices hold the advantage of being portable for on-site testing in hospitals, airports, and hotspots, virtually without any sample treatment or complicated lab precautions.

Forty years of study on the thermostable ß-glycosidase from S. solfataricus: Production, biochemical characterization and biotechnological applications.

The aim of this paper is to make the point on the fortieth years study on the ß-glycosidase from Sulfolobus solfataricus. This enzyme represents one of the thermophilic biocatalysts, which is more extensively studied as witnessed by the numerous literature reports available since 1980. Comprehensive biochemical studies highlighted its broad substrate specificity for ß-d-galacto-, gluco-, and fuco-sides and also showed its remarkable exo-glucosidase and transglycosidase activities. The enzyme demonstrated to be active and stable over a wide range of temperature and pHs, withstanding to several drastic conditions comprising solvents and detergents. Over the years, a great deal of studies were focused on its homotetrameric tridimensional structure, elucidating several structural features involved in the enzyme stability, such as ion pairs and post-translational modifications. Several ß-glycosidase mutants were produced in the years in order to understand its peculiar behavior in extreme conditions and/or to improve its functional properties. The ß-glycosidase overproduction was also afforded reporting numerous studies dealing with its production in the mesophilic host Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, and Lactococcus lactis. Relevant applications in food, beverages, bioenergy, pharmaceuticals, and nutraceutical fields of this enzyme, both in free and immobilized forms, highlighted its biotechnological relevance.

Discovery of the Involvement in DNA Oxidative Damage of Human Sperm Nuclear Basic Proteins of Healthy Young Men Living in Polluted Areas.

DNA oxidative damage is one of the main concerns being implicated in severe cell alterations, promoting different types of human disorders and diseases. For their characteristics, male gametes are the most sensitive cells to the accumulation of damaged DNA. We have recently reported the relevance of arginine residues in the Cu(II)-induced DNA breakage of sperm H1 histones. In this work, we have extended our previous findings investigating the involvement of human sperm nuclear basic proteins on DNA oxidative damage in healthy males presenting copper and chromium excess in their semen. We found in 84% of those males an altered protamines/histones ratio and a different DNA binding mode even for those presenting a canonical protamines/histones ratio. Furthermore, all the sperm nuclear basic proteins from these samples that resulted were involved in DNA oxidative damage, supporting the idea that these proteins could promote the Fenton reaction in DNA proximity by increasing the availability of these metals near the binding surface of DNA. In conclusion, our study reveals a new and unexpected behavior of human sperm nuclear basic proteins in oxidative DNA damage, providing new insights for understanding the mechanisms related to processes in which oxidative DNA damage is implicated.

Highly Sensitive Detection of Chemically Modified Thio-Organophosphates by an Enzymatic Biosensing Device: An Automated Robotic Approach.

Pesticides represent some of the most common man-made chemicals in the world. Despite their unquestionable utility in the agricultural field and in the prevention of pest infestation in public areas of cities, pesticides and their biotransformation products are toxic to the environment and hazardous to human health. Esterase-based biosensors represent a viable alternative to the expensive and time-consuming systems currently used for their detection. In this work, we used the esterase-2 from Alicyclobacillus acidocaldarius as bioreceptor for a biosensing device based on an automated robotic approach. Coupling the robotic system with a fluorescence inhibition assay, in only 30 s of enzymatic assay, we accomplished the detection limit of 10 pmol for 11 chemically oxidized thio-organophosphates in solution. In addition, we observed differences in the shape of the inhibition curves determined measuring the decrease of esterase-2 residual activity over time. These differences could be used for the characterization and identification of thio-organophosphate pesticides, leading to a pseudo fingerprinting for each of these compounds. This research represents a starting point to develop technologies for automated screening of toxic compounds in samples from industrial sectors, such as the food industry, and for environmental monitoring.

Determination of Picomolar Concentrations of Paraoxon in Human Urine by Fluorescence-Based Enzymatic Assay.

Organophosphate (OP) pesticides are widely used in the agricultural field and in the prevention of pest infestation in private and public areas of cities. Despite their unquestionable utility, several of these compounds demonstrate toxic effects to the environment and human health. In particular, the occurrence of some organophosphate pesticides is correlated to the incidence of nervous system disorders, especially in children. The detection of pesticide residues in the human body represents an important task to preserve human health. In our work we propose the use of esterase-based biosensors as a viable alternative to the expensive and time-consuming systems currently used for their detection in human fluids. Using the esterase-2 activity, coupled with a fluorescence inhibition assay, we are able to detect very low concentration levels of diethyl (4-nitrophenyl) phosphate (paraoxon) in the range of the femtomole (fmol). Method robustness tests indicate the stability of esterase-2 in a diluted solution of 4% human urine, and we are able to accurately determine concentration levels of paraoxon in the range from 0.1 to 2 picomoles (pmol). The system sensitivity for OP detection is calculated at 524 ± 14.15 fmol of paraoxon recognized at 10% of inhibition, with an estimated limit of quantification of 262 ± 8.12 pmol mL-1. These values are comparable with the most recent analysis methods based on mass spectrometry carried out on human samples for pesticide detection. This research represents a starting point to develop cheap and fast testing methods for a rapid screening of toxic substances in human samples.

Molecular effects of copper on the reproductive system of mytilus galloprovincialis.

This study aims to assess the effects induced by 24 hr exposure to a subtoxic copper concentration on the reproductive system (gonads, spermatozoa, and protamine-like [PL] proteins) of Mytilus galloprovincialis. Inductively coupled plasma-mass spectrometry indicated accumulation of this metal in gonads, spermatozoa, and PL proteins of exposed mussels. Further, real-time polymerase chain reaction analyses showed altered expression levels of mt10 and PL proteins genes in spermatozoa and gonads, respectively, of exposed mussels. Protamine-like proteins, which represent the main basic component of sperm chromatin of this organism, showed a higher DNA binding affinity and a different DNA binding mode in exposed mussels. Moreover, an increased amount of NaCl was required for the release from sperm nuclei of PL-III, the main PL protein component. Finally, PL proteins extracted from exposed mussels promoted DNA oxidative damage in the presence of H 2 O 2. These results demonstrate that the tolerable copper amount could also affect the properties of PL proteins and determine the negative effects on the reproductive system of this organism. These analyses could be useful to develop quick and efficient chromatin-based genotoxicity tests for pollution biomonitoring programs.