Electrosynthesized poly(o-aminophenol) films as biomimetic coatings for dopamine detection on Pt substrates

Dopamine (DA) is a neurotransmitter, and its levels in the human body are associated with serious diseases. The need for a suitable detection method in medical practice has encouraged the development of electrochemical sensors that take advantage of DA electroactivity. Molecularly imprinted polymers (MIPs) are biomimetic materials able to selectively recognize target analytes. A novel MIP sensor for DA is proposed here based on a thin film of poly(o-aminophenol) electrosynthesized on bare Pt. A fast and easy method for executing the procedure for MIP deposition has been developed based on mild experimental conditions that are able to prevent electrode fouling from DA oxidation products. The MIP exhibited a limit of detection of 0.65 µM, and appreciable reproducibility and stability. The high recognition capability of poly(o-aminophenol) towards DA allowed for the achievement of notable selectivity: ascorbic acid, uric acid, serotonin, and tyramine did not interfere with DA detection, even at higher concentrations. The proposed sensor was successfully applied for DA detection in urine samples, showing good recovery

Biosensore amperometrico per la L-lisina basato su co-crosslinking di lisina-ossidasi su elettrodi di Pt modificati con polipirrolo overossidato

La determinazione dell’aminoacido essenziale L-lisina è di particolare interesse in campo biochimico, biotecnologico ed alimentare poichè, ad es., i suoi livelli sono in genere associabili, rispettivamente, a talune disfunzioni patologiche ed alle qualità nutrizionali di un prodotto alimentare. In questo contesto, l’impiego di metodi enzimatici specifici, basati su elettrodi ad enzima immobilizzato, costituisce certamente una valida alternativa alle metodiche analitiche convenzionali. Diversi biosensori per la determinazione della lisina sono descritti in letteratura. White e Guilbault [1] hanno esplorato la determinazione potenziometrica della lisina accoppiando elettrodi a CO2 con la lisina-decarbossilasi mentre Dempsey et al. [2] hanno sviluppato un biosensore amperometrico immobilizzando la lisina-deidrogenasi su elettrodi di Pt. Sfortunatamente, il metodo potenziometrico è limitato dall’interferenza dovuta alla CO2 atmosferica che ne riduce notevolmente la sensibilità mentre il secondo necessita la presenza dell’NAD+ come cofattore e di un mediatore in soluzione. Conseguentemente, l’approccio più efficiente risulta essere quello amperometrico basato sull’impiego della L-lisina-α-ossidasi che catalizza l’ossidazione dell’aminoacido ad α-cheto-ε-aminocaproato, ione ammonio ed acqua ossigenata. Nella realizzazione di un biosensore è preliminare lo sviluppo e l’ottimizzazione di una metodica di immobilizzazione enzimatica efficiente, in grado di assicurare un notevole grado di stabilità dell’enzima immobilizzato ed elevate attività. Diverse tecniche di immobilizzazione sono state esplorate per la lisina ossidasi quali ad es. l’immobilizzazione covalente su nylon [3] o membrane preattivate [4], l’intrappolamento in collagene [5], il crosslinking [6] o il co-crosslinking [7] dell’enzima su elettrodi di Pt modificati con 1,2-diaminobenzene. In particolare, il co-crosslinking è una tecnica di immobilizzazione particolarmente versatile e vantaggiosa in quanto applicabile ad una vasta gamma di enzimi e facilmente adattabile alle geometrie elettrodiche usualmente utilizzate nei rivelatori elettrochimici in batch ed in flusso. Biosensori per colina ed acetilcolina [8], ad es., sono stati realizzati immobilizzando il sistema bienzimatico acetilcolinesterasi/colina ossidasi su elettrodi di Pt mediante co-crosslinking con albumina di siero bovina e glutaraldeide; ancora, lo stesso approccio ha permesso la realizzazione di un biosensore interference and fouling-free per il glucosio [9] basato su un doppio strato costituito da glucosio ossidasi co-crosslinked e polipirrolo overossidato. Nel laboratorio degli autori è stato di recente messo a punto un nuovo biosensore per la determinazione della lisina in campioni di interesse farmacologico ed alimentare basato su co-crosslinking della lisina ossidasi con una proteina inerte quale l’albumina di siero bovino e la glutaraldeide come crosslinker. Uno studio delle concentrazioni ottimali di enzima, proteina inerte e crosslinker ha permesso la realizzazione su elettrodi di Pt di una membrana ad elevata attività enzimatica ed al tempo stesso meccanicamente stabile tanto da permetterne l’applicazione per analisi in flusso. Il sensore così realizzato ha evidenziato un valore di sensibilità relativamente elevato, pari a circa 1.4 µA/mM mm2, un range lineare esteso sino a circa 0.6 mM, un breve tempo di risposta (6-7 secondi) ed una stabilità tale da consentire un impiego in continuo per più di 40 giorni senza apprezzabile variazione della sensibilità. Una caratterizzazione elettroanalitica del biosensore ha consentito di stimare un valore di KM apparente pari a circa 2.1 ± 0.2 mM ed, al tempo stesso, ha evidenziato uno spiccato controllo diffusivo, difficilmente riscontrabile nei dispositivi basati sulla immobilizzazione elettrochimica. In particolare, questo studio ha messo in evidenza la possibilità di modulare il comportamento cinetico dell’elettrodo da diffusivo a enzimatico variando il pH dell’elettrolita di supporto. E’ possibile quindi nel presente caso ottimizzare le performances del biosensore senza modificare variabili complesse quali la concentrazione dell’enzima immobilizzato e lo spessore e la permeabilità al substrato della membrana enzimatica. Nonostante gli enzimi siano notoriamente specifici nei confronti di un singolo substrato, la lisina-ossidasi catalizza in soluzione, seppur con rese inferiori, anche l’ossidazione di altri aminoacidi [10], quali l’ornitina, la fenilalanina e l’arginina, che potrebbero interferire nella determinazione della lisina. Sebbene l’approccio più usuale per ovviare a questo inconveniente sia quello di selezionare la fonte dell’enzima [7] in base alla sua maggiore specificità, il presente studio ha evidenziato che un opportuno controllo cinetico del sensore, variando il pH e/o la velocità di flusso, permette con un comune enzima commerciale specificità ottimali se non migliori a quelle riscontrabili con enzimi selezionati da fonti opportune. La codeposizione sull’elettrodo di un polimero permselettivo elettrosintetizzato [9], ha infine permesso la realizzazione di un biosensore per L-lisina, scevro da interferenza ed avvelenamento, tramite elettrodeposizione di una membrana polimerica permselettiva basata su polipirrolo overossidato. Riferimenti 1. W. C. White, G. Guilbault, Anal. Chem., 50 (1978) 1481 2. E. Dempsey, J. Wang, V. Wollenberg, M. Ozsos, M. R. Smith, Biosensors and Bioelectronics, 7 (1992) 323 3. E. Vrbovà e M. Marek, Anal. Chim. Acta, 239 (1990) 263 4. M. G. Lavagnini, D. Moscone, G. Palleschi, D. Compagnone, C. Cremisini, Talanta, 40(8) (1993) 1301 5. E. Vrbovà e M. Marek, Collect. Czech. Chem. Commun., 55 (1990) 2568 6. A. Curulli, S. Kelly, C. O’Sullivan, G. G. Guilbaut, G. Palleschi, Biosensors and Bioelectronics 13 (1998) 1245 7. S. C. Kelly, P. J. O’Connell, C. K. O’Sullivan, G. G. Guilbaut, , Anal. Chim. Acta, 412 (2000) 111 8. A. Guerrieri, G. E. De Benedetto, F. Palmisano, P. G. Zambonin, Analyst, 120 (1995) 2731 9. A. Guerrieri, G. E. De Benedetto, F. Palmisano, P. G. Zambonin, Biosensors & Bioelectronics, 13 (1) (1998) 103 10. H. Kusakabe, K. Kodama, A. Kuminaka, H. Yoshimo, H. Misono, K. Soda, J. Biol. Chem., 255 (1980) 97

Validation of an analytical method for nitrite and nitrate determination in meat foods for infants by ion chromatography with conductivity detection

Nitrate and nitrite as sodium or potassium salts are usually added to meat products to develop the characteristic flavor, to inhibit the growth of microorganisms (particularly Clostridium botulinum), and effectively control rancidity by inhibiting lipid oxidation. However, both nitrate and nitrite ions need to be monitored for ensuring the quality and safety of cured meats. In this work, for the first time the content of nitrite and nitrate ions in homogenized meat samples of baby foods was determined by a validated method based on ion chromatography (IC) coupled with conductivity detection. Recoveries of nitrate and nitrite ions in meat samples were not lower than 84 ± 6%. The detection limits of nitrate and nitrite were 0.08 mg L−1 and 0.13 mg L−1, respectively. Five commercial samples of homogenized meat, namely lamb, rabbit, chicken, veal, and beef, for infant feeding were investigated; while nitrite content was below the detection limit, nitrate ranged from 10.7 to 21.0 mg kg−1. The results indicated that nitrate contents were below the European (EU) fixed value of 200 mg kg−1, and an acceptable daily intake of 3.7 mg kg−1 was estimated

An Interplay between a Face-Centred Composite Experimental Design and Solid-Phase Microextraction for Wine Aroma GC/MS Analysis

For oenological products, most of the intrinsic and extrinsic drivers of perceived quality are associated with specific aromatic profiles. Aromatic diversity has been recognized as a central element in perceived quality as it is able to transmit the complex interactions between grape variety, geographical characteristics, and viticultural and winemaking practices, including the fermentative process. A comprehensive characterization of flavour compounds by headspace solid-phase microextraction (HS-SPME) and gas chromatography coupled to mass spectrometric analysis is often needed in order to ascertain the quality of wine. HS-SPME requires a proper optimization that can be achieved through an adequate experimental design. Here, a HS-SPME/GC-MS based method was developed to investigate the volatile compounds of wine samples obtained by laboratory-scale fermentations. This was performed by inoculating a commercial Saccharomyces cerevisiae strain, which is used both as single starter and as mixed starter, with an indigenous Hanseniaspora osmophila strain. The experimental conditions of HS-SPME (extraction temperature and time) were optimized by applying a face-centred composite experimental design. Up to 95% of the total variance was explained by the proposed model. The optimized method allowed us to confirm the usefulness of combining the inoculation of grapes with selected yeast strains in co-culture situations in order to improve the wine bouquet

Development and Validation of a Reversed-Phase HPLC Method with UV Detection for the Determination of L-Dopa in Vicia faba L. Broad Beans

L-Dopa (LD), a substance used medically in the treatment of Parkinson's disease, is found in several natural products, such as Vicia faba L., also known as broad beans. Due to its low chemical stability, LD analysis in plant matrices requires an appropriate optimization of the chosen analytical method to obtain reliable results. This work proposes an HPLC-UV method, validated according to EURACHEM guidelines as regards linearity, limits of detection and quantification, precision, accuracy, and matrix effect. The LD extraction was studied by evaluating its aqueous stability over 3 months. The best chromatographic conditions were found by systematically testing several C-18 stationary phases and acidic mobile phases. In addition, the assessment of the best storage treatment of Vicia faba L. broad beans able to preserve a high LD content was performed. The best LD determination conditions include sun-drying storage, extraction in HCl 0.1 M, chromatographic separation with a Discovery C-18 column, 250 x 4.6 mm, 5 mu m particle size, and 99% formic acid 0.2% v/v and 1% methanol as the mobile phase. The optimized method proposed here overcomes the problems linked to LD stability and separation, thus contributing to the improvement of its analytical determination

Frequent 4EBP1 Amplification Induces Synthetic Dependence on FGFR Signaling in Cancer

Simple Summary Our work establishes that amplification of 4EBP1, as a part of Chr. 8p11, creates a synthetic dependency on FGFR1 signaling in cancer. 4EBP1 is phosphorylated by FGFR1 and PI3K signaling, and accordingly cancer with 4EBP1-FGFR1 amplification is more sensitive to FGFR1 and PI3K inhibition due to inhibition of 4EBP1 phosphorylation. Moreover, we characterize the translational targets of 4EBP1 and identify that 4EBP1 specifically regulates the translation of genes involved in insulin signaling, glucose metabolism, and the inositol pathway that plays a role in cancer progression. The eIF4E translation initiation factor has oncogenic properties and concordantly, the inhibitory eIF4E-binding protein (4EBP1) is considered a tumor suppressor. The exact molecular effects of 4EBP1 activation in cancer are still unknown. Surprisingly, 4EBP1 is a target of genomic copy number gains (Chr. 8p11) in breast and lung cancer. We noticed that 4EBP1 gains are genetically linked to gains in neighboring genes, including WHSC1L1 and FGFR1. Our results show that FGFR1 gains act to attenuate the function of 4EBP1 via PI3K-mediated phosphorylation at Thr37/46, Ser65, and Thr70 sites. This implies that not 4EBP1 but instead FGFR1 is the genetic target of Chr. 8p11 gains in breast and lung cancer. Accordingly, these tumors show increased sensitivity to FGFR1 and PI3K inhibition, and this is a therapeutic vulnerability through restoring the tumor-suppressive function of 4EBP1. Ribosome profiling reveals genes involved in insulin signaling, glucose metabolism, and the inositol pathway to be the relevant translational targets of 4EBP1. These mRNAs are among the top 200 translation targets and are highly enriched for structure and sequence motifs in their 5 ' UTR, which depends on the 4EBP1-EIF4E activity. In summary, we identified the translational targets of 4EBP1-EIF4E that facilitate the tumor suppressor function of 4EBP1 in cancer

Sweet basil functional quality as shaped by genotype and macronutrient concentration reciprocal action

Basil (Ocimum basilicum L.) is among the most widespread aromatic plants due to its versatility of use and its beneficial health properties. This aromatic plant thrives in hydroponics, which is a valid tool to improve the production and functional quality of crops, but nevertheless, it offers the possibility to de-seasonalize production. A floating raft system was adopted to test the production and quality potential during autumn season of three different genotypes of Genovese basil (Aroma 2, Eleonora and Italiano Classico) grown in three nutrient solutions with crescent electrical conductivity (EC: 1, 2 and 3 dS m−1). The aromatic and phenolic profiles were determined by GC/MS and HPLC analysis, respectively. The combination Aroma 2 and the EC 2 dS m−1 resulted in the highest production, both in terms of fresh weight and dry biomass. The 2 dS m−1 treatment determined the major phenolic content, 44%, compared to the other two EC. Italiano Classico showed a higher total polyphenolic content in addition to a different aromatic profile compared to the other cultivars, characterized by a higher percentage of Eucalyptol (+37%) and Eugenol (+107%) and a lower percentage of linalool (−44%). Correct management of the nutritional solution combined with adequate genetic material managed an improvement in the production and the obtainment of the desired aromatic and phenolic profiles

Fibronectin rescues estrogen receptor α from lysosomal degradation in breast cancer cells

Estrogen receptor α (ERα) is expressed in tissues as diverse as brains and mammary glands. In breast cancer, ERα is a key regulator of tumor progression. Therefore, understanding what activates ERα is critical for cancer treatment in particular and cell biology in general. Using biochemical approaches and superresolution microscopy, we show that estrogen drives membrane ERα into endosomes in breast cancer cells and that its fate is determined by the presence of fibronectin (FN) in the extracellular matrix; it is trafficked to lysosomes in the absence of FN and avoids the lysosomal compartment in its presence. In this context, FN prolongs ERα half-life and strengthens its transcriptional activity. We show that ERα is associated with β1-integrin at the membrane, and this integrin follows the same endocytosis and subcellular trafficking pathway triggered by estrogen. Moreover, ERα+ vesicles are present within human breast tissues, and colocalization with β1-integrin is detected primarily in tumors. Our work unravels a key, clinically relevant mechanism of microenvironmental regulation of ERα signaling.Fil: Sampayo, Rocío Guadalupe. Universidad Nacional de San Martin. Instituto de Nanosistemas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Toscani, Andrés Martin. Universidad Nacional de Luján; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Rubashkin, Matthew G.. University of California; Estados UnidosFil: Thi, Kate. Lawrence Berkeley National Laboratory; Estados UnidosFil: Masullo, Luciano Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Violi, Ianina Lucila. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; ArgentinaFil: Lakins, Jonathon N.. University of California; Estados UnidosFil: Caceres, Alfredo Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Hines, William C.. Lawrence Berkeley National Laboratory; Estados UnidosFil: Coluccio Leskow, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad Nacional de Luján; ArgentinaFil: Stefani, Fernando Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Chialvo, Dante Renato. Universidad de Buenos Aires; Argentina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro Internacional de Estudios Avanzados; ArgentinaFil: Bissell, Mina J.. Lawrence Berkeley National Laboratory; Estados UnidosFil: Weaver, Valerie M.. University of California; Estados UnidosFil: Simian, Marina. Universidad Nacional de San Martin. Instituto de Nanosistemas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentin

Solving Nonlinear Parabolic Equations by a Strongly Implicit Finite-Difference Scheme

We discuss the numerical solution of nonlinear parabolic partial differential equations, exhibiting finite speed of propagation, via a strongly implicit finite-difference scheme with formal truncation error $\mathcal{O}\left[(\Delta x)^2 + (\Delta t)^2 \right]$. Our application of interest is the spreading of viscous gravity currents in the study of which these type of differential equations arise. Viscous gravity currents are low Reynolds number (viscous forces dominate inertial forces) flow phenomena in which a dense, viscous fluid displaces a lighter (usually immiscible) fluid. The fluids may be confined by the sidewalls of a channel or propagate in an unconfined two-dimensional (or axisymmetric three-dimensional) geometry. Under the lubrication approximation, the mathematical description of the spreading of these fluids reduces to solving the so-called thin-film equation for the current's shape $h(x,t)$. To solve such nonlinear parabolic equations we propose a finite-difference scheme based on the Crank--Nicolson idea. We implement the scheme for problems involving a single spatial coordinate (i.e., two-dimensional, axisymmetric or spherically-symmetric three-dimensional currents) on an equispaced but staggered grid. We benchmark the scheme against analytical solutions and highlight its strong numerical stability by specifically considering the spreading of non-Newtonian power-law fluids in a variable-width confined channel-like geometry (a "Hele-Shaw cell") subject to a given mass conservation/balance constraint. We show that this constraint can be implemented by re-expressing it as nonlinear flux boundary conditions on the domain's endpoints. Then, we show numerically that the scheme achieves its full second-order accuracy in space and time. We also highlight through numerical simulations how the proposed scheme accurately respects the mass conservation/balance constraint.Comment: 36 pages, 9 figures, Springer book class; v2 includes improvements and corrections; to appear as a contribution in "Applied Wave Mathematics II

Comprehensive Genetic Landscape of Uveal Melanoma by Whole-Genome Sequencing.

Uveal melanoma (UM) is a rare intraocular tumor that, similar to cutaneous melanoma, originates from melanocytes. To gain insights into its genetics, we performed whole-genome sequencing at very deep coverage of tumor-control pairs in 33 samples (24 primary and 9 metastases). Genome-wide, the number of coding mutations was rather low (only 17 variants per tumor on average; range 7-28), thus radically different from cutaneous melanoma, where hundreds of exonic DNA insults are usually detected. Furthermore, no UV light-induced mutational signature was identified. Recurrent coding mutations were found in the known UM drivers GNAQ, GNA11, BAP1, EIF1AX, and SF3B1. Other genes, i.e., TP53BP1, CSMD1, TTC28, DLK2, and KTN1, were also found to harbor somatic mutations in more than one individual, possibly indicating a previously undescribed association with UM pathogenesis. De novo assembly of unmatched reads from non-coding DNA revealed peculiar copy-number variations defining specific UM subtypes, which in turn could be associated with metastatic transformation. Mutational-driven comparison with other tumor types showed that UM is very similar to pediatric tumors, characterized by very few somatic insults and, possibly, important epigenetic changes. Through the analysis of whole-genome sequencing data, our findings shed new light on the molecular genetics of uveal melanoma, delineating it as an atypical tumor of the adult for which somatic events other than mutations in exonic DNA shape its genetic landscape and define its metastatic potential