Sustainable Copper Electrochemical Stripping onto a Paper-Based Substrate for Clinical Application

The electroanalytical field has exploited great advantages in using paper-based substrates, even if the word “paper” might be general. In fact, the mainly adopted paper-based substrates are often chromatographic and office ones. They are characterized by the following main features (and drawbacks): chromatographic paper is well-established for storing reagents/treating samples, but the sensitivity compared to traditional screen-printed ones is lower (due to porosity), while office paper represents a sustainable alternative to plastic (with similar sensitivity), but its porosity is not enough to load reagents. To overcome the limitations that might arise due to the adoption of a type of individual paper-based substrate, herein, we describe for the first time the development of a two-dimensional merged paper-based device for electrochemical copper ion detection in serum. In this work, we report a novel configuration to produce an integrated all-in-one electrochemical device, in which no additional working medium has to be added by the end user and the sensitivity can be tuned by rapid preconcentration on porous paper, with the advantage of making the platform adaptable to real matrix scenarios. The novel architecture has been obtained by combining office paper to screen-print a sustainable and robust electrochemical strip and a chromatographic disk to (1) store the reagents, (2) collect real samples, and (3) preconcentrate the analyte of interest. The novel sensing platform has allowed us to obtain a detection limit for copper ions down to 4 ppb in all the solutions that have been investigated, namely, standard solutions and serum, and a repeatability of ca. 10% has been obtained. Inductively coupled plasma-mass spectrometry measurements confirmed the satisfactory correlation

sj-docx-1-nmi-10.1177_11786388211065372 – Supplemental material for Modulation of Human Hydrogen Sulfide Metabolism by Micronutrients, Preliminary Data

Supplemental material, sj-docx-1-nmi-10.1177_11786388211065372 for Modulation of Human Hydrogen Sulfide Metabolism by Micronutrients, Preliminary Data by Maurizio Dattilo, Carolina Fontanarosa, Michele Spinelli, Vittorio Bini and Angela Amoresano in Nutrition and Metabolic Insights</p

Image1_The antimicrobial peptide Magainin-2 interacts with BamA impairing folding of E. coli membrane proteins.JPEG

Antimicrobial peptides (AMPs) are a unique and diverse group of molecules endowed with a broad spectrum of antibiotics properties that are being considered as new alternative therapeutic agents. Most of these peptides are membrane-active molecules, killing bacteria by membrane disruption. However, recently an increasing number of AMPs was shown to enter bacterial cells and target intracellular processes fundamental for bacterial life. In this paper we investigated the mechanism of action of Maganin-2 (Mag-2), a well-known antimicrobial peptide isolated from the African clawed frog Xenopus laevis, by functional proteomic approaches. Several proteins belonging to E. coli macromolecular membrane complexes were identified as Mag-2 putative interactors. Among these, we focused our attention on BamA a membrane protein belonging to the BAM complex responsible for the folding and insertion of nascent β-barrel Outer Membrane Proteins (OMPs) in the outer membrane. In silico predictions by molecular modelling, in vitro fluorescence binding and Light Scattering experiments carried out using a recombinant form of BamA confirmed the formation of a stable Mag-2/BamA complex and indicated a high affinity of the peptide for BamA. Functional implications of this interactions were investigated by two alternative and complementary approaches. The amount of outer membrane proteins OmpA and OmpF produced in E. coli following Mag-2 incubation were evaluated by both western blot analysis and quantitative tandem mass spectrometry in Multiple Reaction Monitoring scan mode. In both experiments a gradual decrease in outer membrane proteins production with time was observed as a consequence of Mag-2 treatment. These results suggested BamA as a possible good target for the rational design of new antibiotics since this protein is responsible for a crucial biological event of bacterial life and is absent in humans.</p

Synthesis and Proteomic Activity Evaluation of a new Isotope-Coded Affinity Tagging (ICAT) Reagent

During recent years, quantitative proteome profiling has taken advantage of incorporating the traditional stable isotope dilution analysis into global scale or discovery-based proteomic experiments that use mass spectrometers as detectors to allow the pairwise study of differently expressed proteins. Quantitative protein analysis by means of the isotope-coded affinity tag (ICAT) method and tandem mass spectrometry (MS) enables the pairwise comparison of protein expression levels in biological samples. Herein, a modified ICAT reagent, named BAA-ICAT (beta-alanine-arm-ICAT) in which the polyether linker is replaced by a more water-soluble polyamide one, was investigated

LC–MS/MS-Based Quantification Method of Polyphenols for Valorization of Ancient Apple Cultivars from Cilento

Safeguarding the biodiversity of plant species is of fundamental importance for their defense against pests and diseases even through the maintenance and dissemination of ancient agricultural traditions rooted within the small rural environments. The investigation area of the current research covered some municipalities belonging to the “Parco Nazionale del Cilento e Vallo di Diano” including the sub-mountainous part of “Comunità Montana del Vallo di Diano (Salerno, Campania)”. Fifteen ancient apple varieties were collected from local communities to be analyzed and compared to some commercially available apples. To this aim, a Folin–Ciocâlteu assay was preliminarily used to measure the total polyphenol content in both ancient and commercial apple cultivars. Then, a liquid chromatography−tandem mass spectrometry (LC–MS/MS) analysis in the multiple reaction monitoring (MRM) ion mode was then implemented to detect and quantify specific polyphenols and to obtain a molecular comparison of a wide panel of polyphenols. The main finding of the present work pointed out that ancient apple cultivars are richer than commercial ones in anthocyanins, dihydrochalcones, and chlorogenic acid, whose beneficial effects on health are widely known. Thus, the safeguarding of these ancient varieties is greatly encouraged for the richness of polyphenols crucial both for the defense of plants from insects and for remarkable nutraceutical properties, in addition to the need for germplasm conservation as a source of genetic variability

Identification of ΔNp63α Protein Interactions by Mass Spectrometry

p63, a transcription factor related to the p53 tumor suppressor, plays a key role in epidermal differentiation and limb development. The gene has two distinct promoters that allow the formation of proteins that either contain (TA) or lack (ΔN) a transactivation domain. ΔNp63α is the most widely expressed isoform, at all stages of development and in adult tissues. It supports the regenerative capacity of basal keratinocytes and its upregulation is a hallmark of human squamous carcinomas. To get insight into the complex biology of ΔNp63α, we set out to identify ΔNp63α interacting proteins by co-immunoprecipitation in mammalian cells and mass spectrometry analysis. A total of 49 potential ΔNp63α binding proteins, including several heterogeneous ribonucleoproteins (hnRNPs), were identified. Integration of the proteomic data with a Human Coexpression Network highlighted 5 putative p63 protein interactors whose expression is significantly comodulated with p63: hnRNPA/B, hnRNPK, hnRNPQ, FUS/TLS and Keratin 5. hnRNPA/B was already described as a p63 partner, but the others were novel. Interaction of ΔNp63α with hnRNPQ, hnRNPK and FUS/TLS was confirmed by reciprocal co-immunoprecipitations in human keratinocytes. The finding that ΔNp63α exists in complexes with several RNA-binding proteins lays the premises for the analysis of the role of ΔNp63α in mRNA metabolism and transport

Identification of ΔNp63α Protein Interactions by Mass Spectrometry

p63, a transcription factor related to the p53 tumor suppressor, plays a key role in epidermal differentiation and limb development. The gene has two distinct promoters that allow the formation of proteins that either contain (TA) or lack (ΔN) a transactivation domain. ΔNp63α is the most widely expressed isoform, at all stages of development and in adult tissues. It supports the regenerative capacity of basal keratinocytes and its upregulation is a hallmark of human squamous carcinomas. To get insight into the complex biology of ΔNp63α, we set out to identify ΔNp63α interacting proteins by co-immunoprecipitation in mammalian cells and mass spectrometry analysis. A total of 49 potential ΔNp63α binding proteins, including several heterogeneous ribonucleoproteins (hnRNPs), were identified. Integration of the proteomic data with a Human Coexpression Network highlighted 5 putative p63 protein interactors whose expression is significantly comodulated with p63: hnRNPA/B, hnRNPK, hnRNPQ, FUS/TLS and Keratin 5. hnRNPA/B was already described as a p63 partner, but the others were novel. Interaction of ΔNp63α with hnRNPQ, hnRNPK and FUS/TLS was confirmed by reciprocal co-immunoprecipitations in human keratinocytes. The finding that ΔNp63α exists in complexes with several RNA-binding proteins lays the premises for the analysis of the role of ΔNp63α in mRNA metabolism and transport

Multiple Reaction Monitoring Tandem Mass Spectrometry Approach for the Identification of Biological Fluids at Crime Scene Investigations

Knowledge of the nature of biofluids at a crime scene is just as important as DNA test to link the nature of the biofluid, the criminal act, and the dynamics of the crime. Identification of methods currently used for each biological fluid (blood, semen, saliva, urine) suffer from several limitations including instability of assayed biomolecules, and low selectivity and specificity; as an example of the latter issue, it is not possible to discriminate between alpha-amylase 1 (present in saliva) and alpha-amylase 2 (present in semen and vaginal secretion. In this context, the aim of the work has been to provide a predictive protein signature characteristic of each biofluid by the recognition of specific peptides unique for each protein in a single analysis. A panel of four protein biomarkers for blood, four for saliva, five for semen, and two for urine has been monitored has been monitored by using a single multiple reaction monitoring (MRM)-based method targeting concomitantly 46 different peptides. Then, The optimized method allows four biological matrices to be identified when present on their own or in 50:50 mixture with another biofluid. Finally, a valid strategy combining both DNA analysis and liquid chromatographic-tandem mass spectrometric multiple reaction monitoring (LC-MS-MRM) identification of biofluids on the same sample has been demonstrated to be particularly effective in forensic investigation of real trace evidence collected at a crime scene

LC–MS/MS-Based Quantification Method of Polyphenols for Valorization of Ancient Apple Cultivars from Cilento

Safeguarding the biodiversity of plant species is of fundamental importance for their defense against pests and diseases even through the maintenance and dissemination of ancient agricultural traditions rooted within the small rural environments. The investigation area of the current research covered some municipalities belonging to the “Parco Nazionale del Cilento e Vallo di Diano” including the sub-mountainous part of “Comunità Montana del Vallo di Diano (Salerno, Campania)”. Fifteen ancient apple varieties were collected from local communities to be analyzed and compared to some commercially available apples. To this aim, a Folin–Ciocâlteu assay was preliminarily used to measure the total polyphenol content in both ancient and commercial apple cultivars. Then, a liquid chromatography−tandem mass spectrometry (LC–MS/MS) analysis in the multiple reaction monitoring (MRM) ion mode was then implemented to detect and quantify specific polyphenols and to obtain a molecular comparison of a wide panel of polyphenols. The main finding of the present work pointed out that ancient apple cultivars are richer than commercial ones in anthocyanins, dihydrochalcones, and chlorogenic acid, whose beneficial effects on health are widely known. Thus, the safeguarding of these ancient varieties is greatly encouraged for the richness of polyphenols crucial both for the defense of plants from insects and for remarkable nutraceutical properties, in addition to the need for germplasm conservation as a source of genetic variability

Western blot analysis.

<p>Western blot analysis of proteins extracted from mature spores of wild type (PY79, lane 1), <i>ΔcotGΔcotH</i> (AZ603, lane 2), <i>ΔcotGΔcotH amyE::cotGcotH</i> (AZ608, lane 3), <i>ΔcotGΔcotH amyE::cotG_stopcotH</i> (AZ604, lane 4), <i>cotH::spc</i> (ER220, lane 5) and <i>ΔcotGΔcotH amyE::cotG</i> (AZ607, lane 6 of panel B) strains. For CotA and CotB detection (panel A) the proteins have been extracted by SDS treatment while for CotC and CotU detection (panel B) the NaOH treatment has been used. Proteins (25 µg) were reacted with CotA, CotB and CotC specific rabbit antibodies and then with peroxidase-conjugated secondary antibodies and visualized by the Pierce method. The estimated size of CotB, CotC and CotU is indicated.</p