Reactions of medicinal gold(III) compounds with proteins and peptides explored by electrospray ionization mass spectrometry and complementary biophysical methods

Electrospray ionization mass spectrometry (ESI MS) is a powerful investigative tool to analyze the reactions of metallodrugs with proteins and peptides and characterize the resulting adducts. Here, we have applied this type of approach to four experimental anticancer gold(III) compounds for which extensive biological and mechanistic data had previously been gathered, namely, Auoxo6, Au2phen, AuL12, and Aubipyc. These gold(III) compounds were reacted with two representative proteins, i.e., human serum albumin (HSA) and human carbonic anhydrase I (hCA I), and with the C-terminal dodecapeptide of thioredoxin reductase. ESI MS analysis allowed us to elucidate the nature of the resulting metal–protein adducts from which the main features of the occurring metallodrug–protein reactions can be inferred. In selected cases, MS data were integrated and supported by independent 1HNMR and UV–Vis absorption measurements to gain an overall description of the occurring processes. From data analysis, it emerges that most of the investigated gold(III) complexes, endowed with an appreciable oxidizing character, undergo quite facile reduction to gold(I); the resulting gold(I) species tightly associate with the above proteins/peptides with a remarkable selectivity for free cysteine residues. In contrast, in the case of the less-oxidizing Aubipyc complex, the gold(III) oxidation state is conserved, and a gold(III) fragment still containing the original ligand is found to be associated with the target proteins. It is notable that the C-terminal dodecapeptide of thioredoxin reductase containing the characteristic –Gly–Cys–Sec–Gly metal-binding motif is able in all cases to trigger gold(III)-to-gold(I) reduction. Our investigation allowed us to identify in detail the nature of the gold fragments that ultimately bind the protein targets and determine the exact binding stoichiometry; some insight on the reaction kinetics was also gained. Notably, a few clear correlations could be established between the structure of the metal complexes and the nature of the resulting protein adducts. The mechanistic implications of these findings are analyzed and thoroughly discussed. Overall, the present results set the stage to better understand the real target biomolecules of these gold compounds and elucidate at the atomic level their interaction modes with proteins and peptides

Influences of postharvest storage and processing techniques on antioxidant and nutraceutical properties of rubus idaeus l.: A mini-review

The growth of agricultural mechanization has promoted an increase in raspberry production, and for this reason, the best postharvest storage and processing techniques capable of maintaining the health beneficial properties of these perishable berry fruits have been widely studied. Indeed, raspberries are a rich source of bioactive chemical compounds (e.g., ellagitannins, anthocyanins, and ascorbic acid), but these can be altered by postharvest storage and processing techniques before consumption. Although there are clear differences in storage times and techniques, the content of bioactive chemical compounds is relatively stable with some minor changes in ascorbic acid or anthocyanin content during cold (5◦C) or frozen storage. In the literature, processing techniques such as juicing or drying have negatively affected the content of bioactive chemical compounds. Among drying techniques, hot air (oven) drying is the process that alters the content of bioactive chemical compounds the most. For this reason, new drying technologies such as microwave and heat pumps have been developed. These novel techniques are more successful in retaining bioactive chemical compounds with respect to conventional hot air drying. This mini-review surveys recent literature concerning the effects of postharvest storage and processing techniques on raspberry bioactive chemical compound content

Reading a GEM with a VLSI pixel ASIC used as a direct charge collecting anode

In MicroPattern Gas Detectors (MPGD) when the pixel size is below 100 micron and the number of pixels is large (above 1000) it is virtually impossible to use the conventional PCB read-out approach to bring the signal charge from the individual pixel to the external electronics chain. For this reason a custom CMOS array of 2101 active pixels with 80 micron pitch, directly used as the charge collecting anode of a GEM amplifying structure, has been developed and built. Each charge collecting pad, hexagonally shaped, realized using the top metal layer of a deep submicron VLSI technology is individually connected to a full electronics chain (pre-amplifier, shaping-amplifier, sample and hold, multiplexer) which is built immediately below it by using the remaining five active layers. The GEM and the drift electrode window are assembled directly over the chip so the ASIC itself becomes the pixelized anode of a MicroPattern Gas Detector. With this approach, for the first time, gas detectors have reached the level of integration and resolution typical of solid state pixel detectors. Results from the first tests of this new read-out concept are presented. An Astronomical X-Ray Polarimetry application is also discussed.Comment: 11 pages, 14 figures, presented at the Xth Vienna Conference on Instrumentation (Vienna, February 16-21 2004). For a higher resolution paper contact [email protected]

Plasduino: an inexpensive, general purpose data acquisition framework for educational experiments

Based on the Arduino development platform, Plasduino is an open-source data acquisition framework specifically designed for educational physics experiments. The source code, schematics and documentation are in the public domain under a GPL license and the system, streamlined for low cost and ease of use, can be replicated on the scale of a typical didactic lab with minimal effort. We describe the basic architecture of the system and illustrate its potential with some real-life examples.Comment: 11 pages, 10 figures, presented at the XCIX conference of the Societ\`a Italiana di Fisic

Nutritional and nutraceutical properties of raw and traditionally obtained flour from chestnut fruit grown in Tuscany

The study of local chestnut and traditional techniques related to their use and consumption are considered of primary importance to promote their nutritional/nutraceutical values. Fruit of four local chestnut cultivars (‘Carpinese’, ‘Pontecosi’, ‘Capannaccia’ and ‘Morona’) from Garfagnana (Italy) were analysed under nutritional and antioxidant aspects and compared with their flour obtained through a traditional thermal-drying process. Raw fruit contained significative amounts of P, K and Mg (~ 149, 1960 and 50 mg 100 g−1 dry weight, respectively) and they were characterised by a good moisture content (~ 49%) and starch (~ 50 g 100 g−1 dw). The traditional thermal-drying processes affected the carbohydrate content of dried chestnut showing a higher sucrose and lower starch content as compared to raw fruits. Traditional thermal-drying processes negatively influenced also total phenol content (TP) and total antioxidant activity: flours from all cultivars contained lower amounts of TP than raw fruit except for ‘Morona’ in which these compounds remained unchanged. This study provides new useful information about the evaluation of nutritional and nutraceutical characteristics of Tuscany local chestnuts and the effects of a traditional thermal-drying processing method, helping consumers and producers to valorise these “forest products”

Food-Specific IgG4 Antibody-Guided Exclusion Diet Improves Conditions of Patients with Chronic Pain

Introduction Chronic pain is related to gastrointestinal (GI) functions because food components affect inflammation and pain through their action on the GI immune and/or neural system and because many analgesics interact with the gut to alter its structure and function. Immunoglobulin G4 (IgG4) are food-specific antibodies resulting from exposure of the gut immune system to nutrients. High IgG4 levels have been found to be associated with inflammation. Methods IgG4 were determined (both with the rapid test and enzyme-linked immunosorbent assay, ELISA) in men and women outpatients with chronic pain. All subjects were asked to exclude for 4 weeks all foods to which they had high blood levels of IgG4 antibodies. Pain and quality of life questionnaires were administered before (visit 1) and after (visit 2) the personalized exclusion diet period. Visual analogue scale (VAS), Italian Pain Questionnaire (QUID) and Margolis (MA) questionnaires were administered to determine pain intensity, pain features and pain extent, while Short Form Health Survey (SF-36) and Profile of Mood States (POMS) were used to test the quality of life and mood state. The nutritional status was evaluated in all subjects. Subject groups were women of reproductive age (pre-MW), women in menopause for at least 1 year (MW) and men. Results Fifty-four subjects with chronic pain (n = 12 neuropathic, n = 14 diffuse pain, n = 11 headache, n = 17 low back pain) completed the two visits and the 1-month exclusion diet. At visit 1, 47 (87%) subjects showed medium/high levels of IgG4 to at least one food. The foods showing the highest IgG4 values were eggs, dairy products, cereals and dried fruit. At visit 2, IgG4 levels were decreased, increased or unchanged. In all groups, the 4-week exclusion diet resulted in a significant reduction in all pain measures and an improvement of quality of life parameters. In particular, at visit 2, the VAS score determined in the morning decreased by more than 50%. Conclusions A food elimination diet based on IgG4 antibody levels may be effective in reducing pain and improving quality of life in patients with chronic pain

Versatile electrical stimulator for cardiac tissue engineering—Investigation of charge-balanced monophasic and biphasic electrical stimulations

The application of biomimetic physical stimuli replicating the in vivo dynamic microenvironment is crucial for the in vitro development of functional cardiac tissues. In particular, pulsed electrical stimulation (ES) has been shown to improve the functional properties of in vitro cultured cardiomyocytes. However, commercially available electrical stimulators are expensive and cumbersome devices while customized solutions often allow limited parameter tunability, constraining the investigation of different ES protocols. The goal of this study was to develop a versatile compact electrical stimulator (ELETTRA) for biomimetic cardiac tissue engineering approaches, designed for delivering controlled parallelizable ES at a competitive cost. ELETTRA is based on an open-source micro-controller running custom software and is combinable with different cell/tissue culture set-ups, allowing simultaneously testing different ES patterns on multiple samples. In particular, customized culture chambers were appositely designed and manufactured for investigating the influence of monophasic and biphasic pulsed ES on cardiac cell monolayers. Finite element analysis was performed for characterizing the spatial distributions of the electrical field and the current density within the culture chamber. Performance tests confirmed the accuracy, compliance, and reliability of the ES parameters delivered by ELETTRA. Biological tests were performed on neonatal rat cardiac cells, electrically stimulated for 4 days, by comparing, for the first time, the monophasic waveform (electric field = 5 V/cm) to biphasic waveforms by matching either the absolute value of the electric field variation (biphasic ES at ±2.5 V/cm) or the total delivered charge (biphasic ES at ±5 V/cm). Findings suggested that monophasic ES at 5 V/cm and, particularly, charge-balanced biphasic ES at ±5 V/cm were effective in enhancing electrical functionality of stimulated cardiac cells and in promoting synchronous contraction

The effects of two gold-N-heterocyclic carbene (NHC) complexes in ovarian cancer cells: a redox proteomic study

Purpose: Ovarian cancer is the fifth leading cause of cancer-related deaths in women. Standard treatment consists of tumor debulking surgery followed by platinum and paclitaxel chemotherapy; yet, despite the initial response, about 70-75% of patients develop resistance to chemotherapy. Gold compounds represent a family of very promising anticancer drugs. Among them, we previously investigated the cytotoxic and pro-apoptotic properties of Au(NHC) and Au(NHC)2PF6, i.e., a monocarbene gold(I) complex and the corresponding bis(carbene) complex. Gold compounds are known to alter the redox state of cells interacting with free cysteine and selenocysteine residues of several proteins. Herein, a redox proteomic study has been carried out to elucidate the mechanisms of cytotoxicity in A2780 human ovarian cancer cells. Methods: A biotinylated iodoacetamide labeling method coupled with mass spectrometry was used to identify oxidation-sensitive protein cysteines. Results: Gold carbene complexes cause extensive oxidation of several cellular proteins; many affected proteins belong to two major functional classes: carbohydrate metabolism, and cytoskeleton organization/cell adhesion. Among the affected proteins, Glyceraldehyde-3-phosphate dehydrogenase inhibition was proved by enzymatic assays and by ESI-MS studies. We also found that Au(NHC)2PF6 inhibits mitochondrial respiration impairing complex I function. Concerning the oxidized cytoskeletal proteins, gold binding to the free cysteines of actin was demonstrated by ESI-MS analysis. Notably, both gold compounds affected cell migration and invasion. Conclusions: In this study, we deepened the mode of action of Au(NHC) and Au(NHC)2PF6, identifying common cellular targets but confirming their different influence on the mitochondrial function

Anticancer effects against colorectal cancer models of chloro(triethylphosphine)gold(I) encapsulated in PLGA–PEG nanoparticles

Chloro(triethylphosphine)gold(I), (Et(3)PAuCl hereafter), is an Auranofin (AF)-related compound showing very similar biological and pharmacological properties. Like AF, Et(3)PAuCl exhibits potent antiproliferative properties in vitro toward a variety of cancer cell lines and is a promising anticancer drug candidate. We wondered whether Et(3)PAuCl encapsulation might lead to an improved pharmacological profile also considering the likely reduction of unwanted side-reactions that are responsible for adverse effects and for drug inactivation. Et(3)PAuCl was encapsulated in biocompatible PLGA–PEG nanoparticles (NPs) and the new formulation evaluated in colorectal HCT-116 cancer cells in comparison to the free gold complex. Notably, encapsulated Et(3)PAuCl (nano-Et(3)PAuCl hereafter) mostly retains the cellular properties of the free gold complex and elicits even greater cytotoxic effects in colorectal cancer (CRC) cells, mediated by apoptosis and autophagy. Moreover, a remarkable inhibition of two crucial signaling pathways, i.e. ERK and AKT, by nano-Et(3)PAuCl, was clearly documented. The implications of these findings are discussed. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10534-021-00313-0