IMMUNOREACT 7: Regular aspirin use is associated with immune surveillance activation in colorectal cancer.

BACKGROUND: Long-term daily use of aspirin reduces incidence and mortality due to colorectal cancer (CRC). This study aimed to analyze the effect of aspirin on the tumor microenvironment, systemic immunity, and on the healthy mucosa surrounding cancer. METHODS: Patients with a diagnosis of CRC operated on from 2015 to 2019 were retrospectively analyzed (METACCRE cohort). Expression of mRNA of immune surveillance-related genes (PD-L1, CD80, CD86, HLA I, and HLA II) in CRC primary cells treated with aspirin were extracted from Gene Expression Omnibus-deposited public database (GSE76583). The experiment was replicated in cell lines. The mucosal immune microenvironment of a subgroup of patients participating in the IMMUNOREACT1 (ClinicalTrials.gov NCT04915326) project was analyzed with immunohistochemistry and flow cytometry. RESULTS: In the METACCRE Cohort, 12% of 238 patients analyzed were aspirin users. Nodal metastasis was significantly less frequent (p = .008) and tumor-infiltrating lymphocyte infiltration was higher (p = .02) among aspirin users. In the CRC primary cells and selected cell lines, CD80 mRNA expression was increased following aspirin treatment (p = .001). In the healthy mucosa surrounding rectal cancer, the ratio of CD8/CD3 and epithelial cells expressing CD80 was higher in aspirin users (p = .027 and p = .034, respectively). CONCLUSIONS: These data suggested that regular aspirin use may have an active role in enhancing immunosurveillance against CRC.

Role of mass spectrometry in the study of interactions between amylin and metal ions.

Amylin (islet amyloid polypeptide [IAPP]) is a neuroendocrine hormone synthesized with insulin in the beta cells of pancreatic islets. The two hormones act in different ways: in fact insulin triggers glucose uptake in muscle and liver cells, removing glucose from the bloodstream and making it available for energy use and storage, while amylin regulates glucose homeostasis. Aside these positive physiological aspects, human amyloid polypeptide (hIAPP) readily forms amyloid in vitro. Amyloids are aggregates of proteins and in the human body amyloids are considered responsible of the development of various diseases. These aspects have been widely described and discussed in literature and to give a view of the highly complexity of this biochemical behavior the different physical, chemical, biological and medical aspects are shortly described in this review. It is strongly affected by the presence on metal ions, responsible for or inhibiting the formation of fibrils. Mass spectrometry resulted (and still results) to be a particularly powerful tool to obtain valid and effective experimental data to describe the hIAPP behavior. Aside classical approaches devoted to investigation on metal ion-hIAPP structures, which reflects on the identification of metal-protein interaction site(s) and of possible metal-induced conformational changes of the protein, interesting results have been obtained by ion mobility mass spectrometry, giving, on the basis of collisional cross-section data, information on both the oligomerization processes and the conformation changes. Laser ablation electrospray ionization-ion mobility spectrometry-mass spectrometry (LAESI-IMS-MS), allowed to obtain information on the binding stoichiometry, complex dissociation constant, and the oxidation state of the copper for the amylin-copper interaction. Alternatively to inorganic ions, small organic molecules have been tested by ESI-IMS-MS as inhibitor of amyloid assembly. Also in this case the obtained data demonstrate the validity of the ESI-IMS-MS approach as a high-throughput screen for inhibitors of amyloid assembly, providing valid information concerning the identity of the interacting species, the nature of binding and the effect of the ligand on protein aggregation. Effects of Cu2+ and Zn2+ ions in the degradation of human and murine IAPP by insulin-degrading enzyme were studied by liquid chromatography/mass spectrometry (LC/MS). The literature data show that mass spectrometry is a highly valid and effective tool in the study of the amylin behavior, so to individuate medical strategies to avoid the undesired formation of amyloids in in vivo conditions.

Liquid electron ionization-mass spectrometry as a novel strategy for integrating normal-phase liquid chromatography with low and high-resolution mass spectrometry.

Normal-phase liquid chromatography (NPLC) plays a pivotal role in the rapid separation of non-polar compounds, facilitating isomer separation and finding applications in various crucial areas where aprotic solvents are necessary. Similar to reversed-phase liquid chromatography (RPLC), NPLC requires a robust and sensitive detector to unequivocally identify the analytes, such as a mass spectrometer. However, coupling NPLC with mass spectrometry (MS) poses challenges due to the incompatibility between the non-polar solvents used as the mobile phase and the primary ionization techniques employed in MS. Several analytical methods have been developed to combine NPLC with electrospray ionization (ESI), but these methods are restricted to the analysis of polar compounds. In most cases, atmospheric pressure chemical ionization (APCI) becomes necessary to expand the range of analysis applications. To overcome these limitations and fully realize the potential of NPLC-MS coupling, a technique termed liquid electron ionization-mass spectrometry (LEI-MS) can be used. LEI-MS offers a straightforward solution by enabling the effective coupling of NPLC with both low and high-resolution MS. LEI allows for the comprehensive analysis of non-polar compounds and provides a powerful tool for isomer separation and precise identification of analytes. Optimal separations, mass spectral qualities, and matches with the NIST library were obtained in both configurations, demonstrating the potential of the proposed approach.

Mass Spectrometry Investigation of Some ATP-Binding Cassette (ABC) Proteins.

Drug resistance remains one of the main causes of poor outcome in cancer therapy. It is also becoming evident that drug resistance to both chemotherapy and to antibiotics is driven by more than one mechanism. So far, there are at least eight recognized mechanisms behind such resistance. In this review, we choose to discuss one of these mechanisms, which is known to be partially driven by a class of transmembrane proteins known as ATP-binding cassette (ABC) transporters. In normal tissues, ABC transporters protect the cells from the toxic effects of xenobiotics, whereas in tumor cells, they reduce the intracellular concentrations of anticancer drugs, which ultimately leads to the emergence of multidrug resistance (MDR). A deeper understanding of the structures and the biology of these proteins is central to current efforts to circumvent resistance to both chemotherapy, targeted therapy, and antibiotics. Understanding the biology and the function of these proteins requires detailed structural and conformational information for this class of membrane proteins. For many years, such structural information has been mainly provided by X-ray crystallography and cryo-electron microscopy. More recently, mass spectrometry-based methods assumed an important role in the area of structural and conformational characterization of this class of proteins. The contribution of this technique to structural biology has been enhanced by its combination with liquid chromatography and ion mobility, as well as more refined labelling protocols and the use of more efficient fragmentation methods, which allow the detection and localization of labile post-translational modifications. In this review, we discuss the contribution of mass spectrometry to efforts to characterize some members of the ATP-binding cassette (ABC) proteins and why such a contribution is relevant to efforts to clarify the link between the overexpression of these proteins and the most widespread mechanism of chemoresistance.

An integrated multiomics analysis of rectal cancer patients identified POU2F3 as a putative druggable target and entinostat as a cytotoxic enhancer of 5-fluorouracil.

Rectal cancer (RC) accounts for one-third of colorectal cancers (CRC), and 40% of these are locally advanced rectal cancers (LARC). The use of neoadjuvant chemoradiotherapy (nCRT) significantly reduces the rate of local recurrence compared to adjuvant therapy or surgery alone. However, after nCRT, up to 40%-60% of patients show a poor pathological response, while only about 20% achieve a pathological complete response. In this scenario, the identification of novel predictors of tumor response to nCRT is urgently needed to reduce LARC mortality and to spare poorly responding patients from unnecessary treatments. Therefore, by combining gene and microRNA expression datasets with proteomic data from LARC patients, we developed an integrated network centered on seven hub-genes putatively involved in the response to nCRT. In an independent validation cohort of LARC patients, we confirmed that differential expression of NFKB1, TRAF6 and STAT3 is correlated with the response to nCRT. In addition, the functional enrichment analysis also revealed that these genes are strongly related to hallmarks of cancer and inflammation, whose dysfunction may causatively affect LARC patient's response to nCRT. Furthermore, by constructing the transcription factor-module network, we hypothesized a protective role of POU2F3 gene, which could be used as a new drug target in LARC patients. Finally, we identified and tested in vitro entinostat, a histone deacetylase inhibitor, as a chemical compound that could be combined with a classical therapeutic regimen in order to design more efficient therapeutic strategies in LARC management.

Smartphone-Based Electrochemical Biosensor for On-Site Nutritional Quality Assessment of Coffee Blends.

This work aimed to develop an easy-to-use smartphone-based electrochemical biosensor to quickly assess a coffee blend's total polyphenols (Phs) content at the industrial and individual levels. The device is based on a commercial carbon-based screen-printed electrode (SPE) modified with multi-walled carbon nanotubes (CNTs) and gold nanoparticles (GNPs). At the same time, the biological recognition element, Laccase from Trametes versicolor, TvLac, was immobilized on the sensor surface by using glutaraldehyde (GA) as a cross-linking agent. The platform was electrochemically characterized to ascertain the influence of the SPE surface modification on its performance. The working electrode (WE) surface morphology characterization was obtained by scanning electron microscopy (SEM) and Fourier-transform infrared (FT-IR) imaging. All the measurements were carried out with a micro-potentiostat, the Sensit Smart by PalmSens, connected to a smartphone. The developed biosensor provided a sensitivity of 0.12 µA/µM, a linear response ranging from 5 to 70 µM, and a lower detection limit (LOD) of 2.99 µM. Afterward, the biosensor was tested for quantifying the total Phs content in coffee blends, evaluating the influence of both the variety and the roasting degree. The smartphone-based electrochemical biosensor's performance was validated through the Folin-Ciocâlteu standard method.

Mass Spectrometry Contribution to Pediatric Cancers Research.

For over four decades, mass spectrometry-based methods have provided a wealth of information relevant to various challenges in the field of cancers research. These challenges included identification and validation of novel biomarkers for various diseases, in particular for various forms of cancer. These biomarkers serve various objectives including monitoring patient response to the various forms of therapy, differentiating subgroups of the same type of cancer, and providing proteomic data to complement datasets generated by genomic, epigenetic, and transcriptomic methods. The same proteomic data can be used to provide prognostic information and could guide scientists and medics to new and innovative targeted therapies The past decade has seen a rapid emergence of epigenetics as a major contributor to carcinogenesis. This development has given a fresh momentum to MS-based proteomics, which demonstrated to be an unrivalled tool for the analyses of protein post-translational modifications associated with chromatin modifications. In particular, high-resolution mass spectrometry has been recently used for systematic quantification of chromatin modifications. Data generated by this approach are central in the search for new therapies for various forms of cancer and will help in attempts to decipher antitumor drug resistance. To appreciate the contribution of mass spectrometry-based proteomics to biomarkers discovery and to our understanding of mechanisms behind the initiation and progression of various forms of cancer, a number of recent investigations are discussed. These investigations also include results provided by two-dimensional gel electrophoresis combined with mass spectrometry.

Mass Spectrometry-Based Proteomics: Analyses Related to Drug-Resistance and Disease Biomarkers.

Mass spectrometry-based proteomics is a key player in research efforts to characterize aberrant epigenetic alterations, including histone post-translational modifications and DNA methylation. Data generated by this approach complements and enrich datasets generated by genomic, epigenetic and transcriptomics approaches. These combined datasets can provide much-needed information on various mechanisms responsible for drug resistance, the discovery and validation of potential biomarkers for different diseases, the identification of signaling pathways, and genes and enzymes to be targeted by future therapies. The increasing use of high-resolution, high-accuracy mass spectrometers combined with more refined protein labeling and enrichment procedures enhanced the role of this approach in the investigation of these epigenetic modifications. In this review, we discuss recent MS-based studies, which are contributing to current research efforts to understand certain mechanisms behind drug resistance to therapy. We also discuss how these MS-based analyses are contributing to biomarkers discovery and validation.

Polysulfide Speciation and Migration in Catholyte Lithium-Sulfur Cells.

Semi-liquid catholyte Lithium-Sulfur (Li-S) cells have shown to be a promising path to realize high energy density energy storage devices. In general, Li-S cells rely on the conversion of elemental sulfur to soluble polysulfide species. In the case of catholyte cells, the active material is added through polysulfide species dissolved in the electrolyte. Herein, we use operando Raman spectroscopy to track the speciation and migration of polysulfides in the catholyte to shed light on the processes taking place. Combined with ex-situ surface and electrochemical analysis we show that the migration of polysulfides is central in order to maximize the performance in terms of capacity (active material utilization) as well as interphase stability on the Li-metal anode during cycling. More specifically we show that using a catholyte where the polysulfides have the dual roles of active material and conducting species, e. g. no traditional Li-salt (such as LiTFSI) is present, results in a higher mobility and faster migration of polysulfides. We also reveal how the formation of long chain polysulfides in the catholyte is delayed during charge as a result of rapid formation and migration of shorter chain species, beneficial for reaching higher capacities. However, the depletion of ionic species during the last stage of charge, due to the conversion to and precipitation of elemental sulfur on the cathode support, results in polarization of the cell before full conversion can be achieved.

Critical Role of Functional Groups Containing N, S, and O on Graphene Surface for Stable and Fast Charging Li-S Batteries.

Lithium-sulfur (Li-S) batteries are considered one of the most promising energy storage technologies, possibly replacing the state-of-the-art lithium-ion (Li-ion) batteries owing to their high energy density, low cost, and eco-compatibility. However, the migration of high-order lithium polysulfides (LiPs) to the lithium surface and the sluggish electrochemical kinetics pose challenges to their commercialization. The interactions between the cathode and LiPs can be enhanced by the doping of the carbon host with heteroatoms, however with relatively low doping content (<10%) in the bulk of the carbon, which can hardly interact with LiPs at the host surface. In this study, the grafting of versatile functional groups with designable properties (e.g., catalytic effects) directly on the surface of the carbon host is proposed to enhance interactions with LiPs. As model systems, benzene groups containing N/O and S/O atoms are vertically grafted and uniformly distributed on the surface of expanded reduced graphene oxide, fostering a stable interface between the cathode and LiPs. The combination of experiments and density functional theory calculations demonstrate improvements in chemical interactions between graphene and LiPs, with an enhancement in the electrochemical kinetics, power, and energy densities.

MASS SPECTROMETRY FOR A HOLISTIC VIEW OF NATURAL EXTRACTS OF PHYTOTHERAPEUTIC INTEREST.

In the study of natural products new strategies which favor a holistic approach, integrating the traditional reductionist methods usually employed, have been proposed. In this frame, the studies carried out by us in the last decade show that fingerprints, mainly obtained by electrospray ionization mass spectrometry (ESI-MS), lead to the characterization of natural extracts from different botanical species but also of phytotherapeutic products constituted by mixtures of extracts from different plants. Laser desorption ionization and matrix-assisted laser desorption ionization techniques were also employed and by the use of different matrices some complementary results were achieved. Results obtained by standard spectrophotometric and liquid chromatography methods were compared with those achieved by direct infusion of the extract in ESI-MS conditions, indicating an excellent agreement between the two approaches. The findings of these researches were considered in the frame of complex systems theory, investigating how relationships between a system's parts can give rise to its collective behaviors and how the system interacts and forms relationships with its environment. In this view, the peculiar pharmacological behavior of biologically active natural compounds can be justified by the occurrence of molecular interactions due to the high complexity of the natural matrix. Some of these interactions have been widely studied in the case of green tea extracts (GTEs) proving unequivocally the presence of caffeine/catechin complexes in GTE samples. The presence of bimolecular complexes has been observed also in the case of Ceylon tea and Mate extracts. These data indicate that the formation of complexes in natural extracts is a common behavior and their presence must be considered in the description of natural extracts and, consequently, in their biological activity. ©2020 John Wiley & Sons Ltd. Mass Spec Rev.

BTK inhibitors synergise with 5-FU to treat drug-resistant TP53-null colon cancers.

Colorectal cancer (CRC) is the fourth cause of death from cancer worldwide mainly due to the high incidence of drug-resistance. During a screen for new actionable targets in drug-resistant tumours we recently identified p65BTK - a novel oncogenic isoform of Bruton's tyrosine kinase. Studying three different cohorts of patients here we show that p65BTK expression correlates with histotype and cancer progression. Using drug-resistant TP53-null colon cancer cells as a model we demonstrated that p65BTK silencing or chemical inhibition overcame the 5-fluorouracil resistance of CRC cell lines and patient-derived organoids and significantly reduced the growth of xenografted tumours. Mechanistically, we show that blocking p65BTK in drug-resistant cells abolished a 5-FU-elicited TGFB1 protective response and triggered E2F-dependent apoptosis. Taken together, our data demonstrated that targeting p65BTK restores the apoptotic response to chemotherapy of drug-resistant CRCs and gives a proof-of-concept for suggesting the use of BTK inhibitors in combination with 5-FU as a novel therapeutic approach in CRC patients. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The Combination of 2D Layered Graphene Oxide and 3D Porous Cellulose Heterogeneous Membranes for Nanofluidic Osmotic Power Generation.

Salinity gradient energy, as a type of blue energy, is a promising sustainable energy source. Its energy conversion efficiency is significantly determined by the selective membranes. Recently, nanofluidic membrane made by two-dimensional (2D) nanomaterials (e.g., graphene) with densely packed nanochannels has been considered as a high-efficient membrane in the osmotic power generation research field. Herein, the graphene oxide-cellulose acetate (GO-CA) heterogeneous membrane was assembled by combining a porous CA membrane and a layered GO membrane; the combination of 2D nanochannels and 3D porous structures make it show high surface-charge-governed property and excellent ion transport stability, resulting in an efficient osmotic power harvesting. A power density of about 0.13 W/m2 is achieved for the sea-river mimicking system and up to 0.55 W/m2 at a 500-fold salinity gradient. With different functions, the CA and GO membranes served as ion storage layer and ion selection layer, respectively. The GO-CA heterogeneous membrane open a promising avenue for fabrication of porous and layered platform for wide potential applications, such as sustainable power generation, water purification, and seawater desalination.

miR-27a is a master regulator of metabolic reprogramming and chemoresistance in colorectal cancer.

BACKGROUND: Metabolic reprogramming towards aerobic glycolysis in cancer supports unrestricted cell proliferation, survival and chemoresistance. The molecular bases of these processes are still undefined. Recent reports suggest crucial roles for microRNAs. Here, we provide new evidence of the implication of miR-27a in modulating colorectal cancer (CRC) metabolism and chemoresistance. METHODS: A survey of miR-27a expression profile in TCGA-COAD dataset revealed that miR-27a-overexpressing CRCs are enriched in gene signatures of mitochondrial dysfunction, deregulated oxidative phosphorylation, mTOR activation and reduced chemosensitivity. The same pathways were analysed in cell lines in which we modified miR-27a levels. The response to chemotherapy was investigated in an independent cohort and cell lines. RESULTS: miR-27a upregulation in vitro associated with impaired oxidative phosphorylation, overall mitochondrial activities and slight influence on glycolysis. miR-27a hampered AMPK, enhanced mTOR signalling and acted in concert with oncogenes and tumour cell metabolic regulators to force an aerobic glycolytic metabolism supporting biomass production, unrestricted growth and chemoresistance. This latter association was confirmed in our cohort of patients and cell lines. CONCLUSIONS: We disclose an unprecedented role for miR-27a as a master regulator of cancer metabolism reprogramming that impinges on CRC response to chemotherapy, underscoring its theragnostic properties.

Correction: miR-27a is a master regulator of metabolic reprogramming and chemoresistance in colorectal cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Designing Highly Conductive Functional Groups Improving Guest-Host Interactions in Li/S Batteries.

Li-sulfur batteries are of great interest due to their potential to surpass the energy densities of other battery types, but the low electronic conductivity of both sulfur and its discharge products requires the use of a conductive host material. The most common is the use of different porous carbons which normally are hydrophobic and hardly retain the polar discharge products of the Li/S reaction, such as Li2 S and lithium polysulfides (LiPs), at the working electrode. Functionalized hosts have been proposed as a strategy to improve LiPs interactions, including the use of heteroatom doping, organic frameworks, metals, metal oxides, sulfide particles, and conductive polymers. Despite demonstrating an improved cycle life, the functionalized structures often have an intrinsic limitation related to a low electronic conductivity resulting in slow kinetics and poor rate capability of Li/S cells. Herein, recent research trends aimed at designing sulfur electrodes with highly conductive functional groups on nanostructured hosts surface are reviewed. The main concepts, key developments, and parameters for building 3D hosts architectures that enable fast charge rates and long cycle life at high sulfur loadings are discussed.

The role of mass spectrometry in studies of glycation processes and diabetes management.

In the last decade, mass spectrometry has been widely employed in the study of diabetes. This was mainly due to the development of new, highly sensitive, and specific methods representing powerful tools to go deep into the biochemical and pathogenetic processes typical of the disease. The aim of this review is to give a panorama of the scientifically valid results obtained in this contest. The recent studies on glycation processes, in particular those devoted to the mechanism of production and to the reactivity of advanced glycation end products (AGEs, AGE peptides, glyoxal, methylglyoxal, dicarbonyl compounds) allowed to obtain a different view on short and long term complications of diabetes. These results have been employed in the research of effective markers and mass spectrometry represented a precious tool allowing the monitoring of diabetic nephropathy, cardiovascular complications, and gestational diabetes. The same approaches have been employed to monitor the non-insulinic diabetes pharmacological treatments, as well as in the discovery and characterization of antidiabetic agents from natural products. © 2018 Wiley Periodicals, Inc. Mass Spec Rev 38:112-146, 2019.

Correction to: Assessment of intratumor immune-microenvironment in colorectal cancers with extranodal extension of nodal metastases.

[This corrects the article DOI: 10.1186/s12935-018-0634-8.].

New Mass Spectrometric Approaches for the Quantitative Evaluation of Anticancer Drug Levels in Treated Patients.

Alternatively to the well-consolidated liquid chromatography coupled to tandem mass spectrometry approach used for the evaluation of anticancer drug concentrations in treated patients, new mass spectrometric methods have been proposed and tested recently. They exhibited faster analysis time and, at first sight, simpler instrumental approaches. However, results obtained by these methods require an in-depth evaluation, because of their strong dependence on the experimental set-up. In this short review, the quantification of irinotecan, sunitinib, and 6-α-hydroxy paclitaxel (the main metabolite of paclitaxel) by laser desorption ionization techniques (matrix-assisted laser desorption/ionization, nanostructure-assisted laser desorption/ionization, and surface-assisted laser desorption/ionization) is reported and discussed, showing the advantages but also the drawbacks of the methods. The matrix-assisted laser desorption/ionization approach led to the most reliable results, and the cross-validation for the quantitative analysis of irinotecan indicates that this method can be fruitfully used for therapeutic drug monitoring and pharmacokinetic studies. Another recently proposed technique, paper spray mass spectrometry, has been tested for the quantitative measurement of imatinib in plasma samples. Even if the approach is, at first sight, really simple, the parameterization of the analytical and instrumental aspects has required many efforts to reach satisfactory results. What it should be expected in the future is the evaluation of these methods, not only in scientific environments dedicated to instrument development, but also in clinical chemistry laboratories, to evaluate their effectiveness and to give new and valid tools for TDM and for other qualitative or quantitative measurements of biomedical interest.

Preclinical three-dimensional colorectal cancer model: The next generation of in vitro drug efficacy evaluation.

Colorectal cancer (CRC), the third most common cancer diagnosed in both men and women in the United States, shows a highly ineffective therapeutic management. In these years neither substantial improvements nor new therapeutic approaches have been provided to patients. Performing the early lead discovery phases of new cancer drugs in cellular models, resembling as far as possible the real in vivo tumor environment, may be more effective in predicting their future success in the later clinical phases. In this review, we critically describe the most representative bioengineered models for anticancer drug screening in CRC from the conventional two-dimensional models to the new-generation three-dimensional scaffold-based ones. The scaffold aims to replace the extracellular matrix, thus influencing the biomechanical, biochemical, and biological properties of cells and tissues. In this scenario, we believe that reconstitution of tumor condition is mandatory for an alternative in vitro methods to study cancer development and therapeutic strategies.