Publications by authors named "Cristina Airoldi"

63 Publications

Cross-Linking Effects Dictate the Preference of Galectins to Bind LacNAc-Decorated HPMA Copolymers.

Int J Mol Sci 2021 Jun 1;22(11). Epub 2021 Jun 1.

CIC bioGUNE, Basque Research and Technology Alliance, BRTA, Bizkaia Technology Park, 48162 Derio, Bizkaia, Spain.

The interaction of multi-LacNAc (Galβ1-4GlcNAc)-containing -(2-hydroxypropyl) methacrylamide (HPMA) copolymers with human galectin-1 (Gal-1) and the carbohydrate recognition domain (CRD) of human galectin-3 (Gal-3) was analyzed using NMR methods in addition to cryo-electron-microscopy and dynamic light scattering (DLS) experiments. The interaction with individual LacNAc-containing components of the polymer was studied for comparison purposes. For Gal-3 CRD, the NMR data suggest a canonical interaction of the individual small-molecule bi- and trivalent ligands with the lectin binding site and better affinity for the trivalent arrangement due to statistical effects. For the glycopolymers, the interaction was stronger, although no evidence for forming a large supramolecule was obtained. In contrast, for Gal-1, the results indicate the formation of large cross-linked supramolecules in the presence of multivalent LacNAc entities for both the individual building blocks and the polymers. Interestingly, the bivalent and trivalent presentation of LacNAc in the polymer did not produce such an increase, indicating that the multivalency provided by the polymer is sufficient for triggering an efficient binding between the glycopolymer and Gal-1. This hypothesis was further demonstrated by electron microscopy and DLS methods.
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http://dx.doi.org/10.3390/ijms22116000DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8199549PMC
June 2021

On-cell saturation transfer difference NMR for the identification of FimH ligands and inhibitors.

Bioorg Chem 2021 07 29;112:104876. Epub 2021 Mar 29.

BioOrg NMR Lab, Department of Biotechnology and Biosciences, University of Milano-Bicocca, P.zza della Scienza, 2, 20126 Milan, Italy. Electronic address:

We describe the development of an on-cell NMR method for the rapid screening of FimH ligands and the structural identification of ligand binding epitopes. FimH is a mannose-binding bacterial adhesin expressed at the apical end of type 1 pili of uropathogenic bacterial strains and responsible for their d-mannose sensitive adhesion to host mammalian epithelial cells. Because of these properties, FimH is a key virulence factor and an attractive therapeutic target for urinary tract infection. We prepared synthetic d-mannose decorated dendrimers, we tested their ability to prevent the FimH-mediated yeast agglutination, and thus we used the compounds showing the best inhibitory activity as models of FimH multivalent ligands to set up our NMR methodology. Our experimental protocol, based on on-cell STD NMR techniques, is a suitable tool for the screening and the epitope mapping of FimH ligands aimed at the development of new antiadhesive and diagnostic tools against urinary tract infection pathogens. Notably, the study is carried out in a physiological environment, i.e. at the surface of living pathogen cells expressing FimH.
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http://dx.doi.org/10.1016/j.bioorg.2021.104876DOI Listing
July 2021

The Multi-Level Mechanism of Action of a Pan-Ras Inhibitor Explains its Antiproliferative Activity on Cetuximab-Resistant Cancer Cells.

Front Mol Biosci 2021 17;8:625979. Epub 2021 Feb 17.

Department of Biotechnology and Biosciences, University of Milan-Bicocca, Milan, Italy.

Ras oncoproteins play a crucial role in the onset, maintenance, and progression of the most common and deadly human cancers. Despite extensive research efforts, only a few mutant-specific Ras inhibitors have been reported. We show that cmp4-previously identified as a water-soluble Ras inhibitor- targets multiple steps in the activation and downstream signaling of different Ras mutants and isoforms. Binding of this pan-Ras inhibitor to an extended Switch II pocket on HRas and KRas proteins induces a conformational change that down-regulates intrinsic and GEF-mediated nucleotide dissociation and exchange and effector binding. A mathematical model of the Ras activation cycle predicts that the inhibitor severely reduces the proliferation of different Ras-driven cancer cells, effectively cooperating with Cetuximab to reduce proliferation even of Cetuximab-resistant cancer cell lines. Experimental data confirm the model prediction, indicating that the pan-Ras inhibitor is an appropriate candidate for medicinal chemistry efforts tailored at improving its currently unsatisfactory affinity.
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http://dx.doi.org/10.3389/fmolb.2021.625979DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7925909PMC
February 2021

Targeting GRP receptor: Design, synthesis and preliminary biological characterization of new non-peptide antagonists of bombesin.

Bioorg Chem 2021 04 17;109:104739. Epub 2021 Feb 17.

Department of Biotechnology and Biosciences, University of Milano - Bicocca, P.zza della Scienza 2, 20126 Milan, Italy; Milan Center for Neuroscience, University of Milano-Bicocca, P.zza dell'Ateneo Nuovo 1, 20126 Milano, Italy. Electronic address:

We report the rational design, synthesis, and in vitro preliminary evaluation of a new small library of non-peptide ligands of Gastrin Releasing Peptide Receptor (GRP-R), able to antagonize its natural ligand bombesin (BN) in the nanomolar range of concentration. GRP-R is a transmembrane G-protein coupled receptor promoting the stimulation of cancer cell proliferation. Being overexpressed on the surface of different human cancer cell lines, GRP-R is ideal for the selective delivery to tumor cells of both anticancer drug and diagnostic devices. What makes very challenging the design of non-peptide BN analogues is that the 3D structure of the GRP-R is not available, which is the case for many membrane-bound receptors. Thus, the design of GRP-R ligands has to be based on the structure of its natural ligands, BN and GRP. We recently mapped the BN binding epitope by NMR and here we exploited the same spectroscopy, combined with MD, to define BN conformation in proximity of biological membranes, where the interaction with GRP-R takes place. The gained structural information was used to identify a rigid C-galactosidic scaffold able to support pharmacophore groups mimicking the BN key residues' side chains in a suitable manner for binding to GRP-R. Our BN antagonists represent hit compounds for the rational design and synthesis of new ligands and modulators of GRP-R. The further optimization of the pharmacophore groups will allow to increase the biological activity. Due to their favorable chemical properties and stability, they could be employed for the active receptor-mediated targeting of GRP-R positive tumors.
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http://dx.doi.org/10.1016/j.bioorg.2021.104739DOI Listing
April 2021

Synthesis, Molecular Modeling and Biological Evaluation of Metabolically Stable Analogues of the Endogenous Fatty Acid Amide Palmitoylethanolamide.

Int J Mol Sci 2020 Nov 28;21(23). Epub 2020 Nov 28.

Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy.

Palmitoylethanolamide (PEA) belongs to the class of -acylethanolamine and is an endogenous lipid potentially useful in a wide range of therapeutic areas; products containing PEA are licensed for use in humans as a nutraceutical, a food supplement, or food for medical purposes for its analgesic and anti-inflammatory properties demonstrating efficacy and tolerability. However, the exogenously administered PEA is rapidly inactivated; in this process, fatty acid amide hydrolase (FAAH) plays a key role both in hepatic metabolism and in intracellular degradation. So, the aim of the present study was the design and synthesis of PEA analogues that are more resistant to FAAH-mediated hydrolysis. A small library of PEA analogues was designed and tested by molecular docking and density functional theory calculations to find the more stable analogue. The computational investigation identified RePEA as the best candidate in terms of both synthetic accessibility and metabolic stability to FAAH-mediated hydrolysis. The selected compound was synthesized and assayed ex vivo to monitor FAAH-mediated hydrolysis and to confirm its anti-inflammatory properties. H-NMR spectroscopy performed on membrane samples containing FAAH in integral membrane protein demonstrated that RePEA is not processed by FAAH, in contrast with PEA. Moreover, RePEA retains PEA's ability to inhibit LPS-induced cytokine release in both murine N9 microglial cells and human PMA-THP-1 cells.
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http://dx.doi.org/10.3390/ijms21239074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7730713PMC
November 2020

Structural Basis of Inhibition of the Pioneer Transcription Factor NF-Y by Suramin.

Cells 2020 10 29;9(11). Epub 2020 Oct 29.

Department of Biosciences, University of Milano, Via Celoria 26, 20133 Milano, Italy.

NF-Y is a transcription factor (TF) comprising three subunits (NF-YA, NF-YB, NF-YC) that binds with high specificity to the CCAAT sequence, a widespread regulatory element in gene promoters of prosurvival, cell-cycle-promoting, and metabolic genes. Tumor cells undergo "metabolic rewiring" through overexpression of genes involved in such pathways, many of which are under NF-Y control. In addition, NF-YA appears to be overexpressed in many tumor types. Thus, limiting NF-Y activity may represent a desirable anti-cancer strategy, which is an ongoing field of research. With virtual-screening docking simulations on a library of pharmacologically active compounds, we identified suramin as a potential NF-Y inhibitor. We focused on suramin given its high water-solubility that is an important factor for in vitro testing, since NF-Y is sensitive to DMSO. By electrophoretic mobility shift assays (EMSA), isothermal titration calorimetry (ITC), STD NMR, X-ray crystallography, and molecular dynamics (MD) simulations, we showed that suramin binds to the histone fold domains (HFDs) of NF-Y, preventing DNA-binding. Our analyses, provide atomic-level detail on the interaction between suramin and NF-Y and reveal a region of the protein, nearby the suramin-binding site and poorly conserved in other HFD-containing TFs, that may represent a promising starting point for rational design of more specific and potent inhibitors with potential therapeutic applications.
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http://dx.doi.org/10.3390/cells9112370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7692634PMC
October 2020

NMR-based Lavado cocoa chemical characterization and comparison with fermented cocoa varieties: Insights on cocoa's anti-amyloidogenic activity.

Food Chem 2021 Mar 1;341(Pt 2):128249. Epub 2020 Oct 1.

BioOrgNMR Lab, Department of Biotechnologies and Biosciences, University of Milano-Bicocca, P.zza della Scienza 2, 20126 Milan, Italy; Milan Center for Neuroscience (NeuroMI), University of Milano-Bicocca, P.zza dell'Ateneo Nuovo 1, 20126 Milano Italy. Electronic address:

The metabolic profile of Lavado cocoa was characterized for the first time by NMR spectroscopy, then compared with the profiles of fermented and processed varieties, Natural and commercial cocoa. The significant difference in the contents of theobromine and flavanols prompted us to examine the cocoa varieties to seek correlations between these metabolite concentrations and the anti-amyloidogenic activity reported for cocoa in the literature. We combined NMR spectroscopy, preparative reversed-phase (RP) chromatography, atomic force microscopy, in vitro biochemical and cell assays, to investigate and compare the anti-amyloidogenic properties of extracts and fractions enriched in different metabolite classes. Lavado variety was the most active and the catechins and theobromine were the chemical components of cocoa hindering Aβ peptide on-pathway aggregation and toxicity in a human neuroblastoma SH-SY5Y cell line.
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http://dx.doi.org/10.1016/j.foodchem.2020.128249DOI Listing
March 2021

Correction to: Different phytotoxic effect of Lolium multiflorum Lam. leaves against Echinochloa oryzoides (Ard.) Fritsch and Oryza sativa L.

Environ Sci Pollut Res Int 2020 10;27(28):35870

Department of Agricultural and Environmental Sciences, Università degli Studi di Milano, Via G. Celoria 2, 20133, Milan, Italy.

In the title, it should be Oryza instead of Oriza.
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http://dx.doi.org/10.1007/s11356-020-10140-4DOI Listing
October 2020

Effectiveness of Vigna unguiculata seed extracts in preventing colorectal cancer.

Food Funct 2020 Jul 26;11(7):5853-5865. Epub 2020 Jun 26.

Department of Biotechnology and Biosciences, University of Milan-Bicocca, Piazza della Scienza 2, 20126, Milano, Italy.

Colorectal cancer (CRC) is one of the most common types of cancer, especially in Western countries, and its incidence rate is increasing every year. In this study, for the first time Vigna unguiculata L. Walp. (cowpea) water boiled seed extracts were found to reduce the viability of different colorectal cancer (CRC) cell lines, such as E705, DiFi and SW480 and the proliferation of Caco-2 line too, without affecting CCD841 healthy cell line. Furthermore, the extracts showed the ability to reduce the level of Epidermal Growth Factor Receptor (EGFR) phosphorylation in E705, DiFi and SW480 cell lines and to lower the EC50 of a CRC common drug, cetuximab, on E705 and DiFi lines from 161.7 ng mL to 0.06 ng mL and from 49.5 ng mL to 0.2 ng mL respectively. The extract was characterized in its protein and metabolite profiles by tandem mass spectrometry and H-NMR analyses. A Bowman-Birk protease inhibitor was identified within the protein fraction and was supposed to be the main active component. These findings confirm the importance of a legume-based diet to prevent the outbreak of many CRC and to reduce the amount of drug administered during a therapeutic cycle.
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http://dx.doi.org/10.1039/d0fo00913jDOI Listing
July 2020

Different phytotoxic effect of Lolium multiflorum Lam. leaves against Echinochloa oryzoides (Ard.) Fritsch and Oryza sativa L.

Environ Sci Pollut Res Int 2020 09 11;27(26):33204-33214. Epub 2020 Jun 11.

Department of Agricultural and Environmental Sciences, Università degli Studi di Milano, Via G. Celoria 2, 20133, Milan, Italy.

Rice cultivation, particularly prone to weed issues, requires practices able to effectively control them, however reducing the use of herbicides, responsible for damage to human health and ecosystem sustainability. Alternative strategies for weed management can be based on plant-plant interaction phenomena. In this context, a group of organic farmers has developed a pragmatic approach for weed containment using Lolium multiflorum Lam. as a cover crop before rice. The present study aimed to confirm the farmer field observations reporting a preferential inhibitory effect of L. multiflorum on Echinochloa oryzoides (Ard.) Fritsch, one of the most yield-damaging rice weed, compared with Oryza sativa L. The study showed that L. multiflorum was able to significantly reduce the seed germination of E. oryzoides. It was found to be more susceptible than O. sativa both to the effect of the aqueous extract and powder of L. multiflorum leaves (23-79% vs. 3-57% and 26-100% vs. 23-31%, respectively). In addition, the leaf extract was able to affect E. oryzoides growth starting from 20% concentration both in relation to the root and shoot length while O. sativa exhibited differences compared with the control only under the influence of extract 50%. The L. multiflorum leaf characterization by NMR and UPLC-HR-MS analyses led to the identification of 35 compounds including several polyphenols, glycosyl flavonoids and glycosyl terpenoids, as well as different amino acids and organic acids. Some of them (e.g. protocatechuic and gallic acids) are already known as allelochemicals confirming that L. multiflorum is a source of plant growth inhibitors.
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http://dx.doi.org/10.1007/s11356-020-09573-8DOI Listing
September 2020

Metabolomic profiling of beers: Combining H NMR spectroscopy and chemometric approaches to discriminate craft and industrial products.

Food Chem 2020 Oct 15;327:127025. Epub 2020 May 15.

BioOrgNMR Lab, Department of Biotechnology and Biosciences, University of Milano-Bicocca, P.zza della Scienza 2, 20126 Milan, Italy. Electronic address:

The authentication and traceability of craft beers is an important issue for both beer consumers and producers. Reliable analytical methods able to identify and discriminate products are needed to protect the craft brew market against fraud and counterfeit. Here, H NMR analysis of 31 beer samples, differing for beer style and brewing method (craft or industrial) was combined with multivariate statistical analysis, following both an untargeted and a targeted approach. NMR-based analysis of beer samples was sped developing a specific protocol enabling the automatic identification and quantification of metabolites in approximately thirty seconds per spectrum. A clear discrimination was achieved by exploiting H NMR analysis and multivariate chemometric methods and the targeted approach identified the metabolites responsible for the segregation. Overall, this study reports an analytical approach addressing beer traceability and is the starting point for the development of a standardized protocol for the discrimination of industrial and craft beers.
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http://dx.doi.org/10.1016/j.foodchem.2020.127025DOI Listing
October 2020

On-cell saturation transfer difference NMR study of Bombesin binding to GRP receptor.

Bioorg Chem 2020 06 18;99:103861. Epub 2020 Apr 18.

Department of Biotechnology and Biosciences, University of Milano-Bicocca, P.zza della Scienza 2, 20126 Milan, Italy. Electronic address:

We report the NMR characterization of the molecular interaction between Gastrin Releasing Peptide Receptor (GRP-R) and its natural ligand bombesin (BN). GRP-R is a transmembrane G-protein coupled receptor promoting the stimulation of cancer cell proliferation; in addition, being overexpressed on the surface of different human cancer cell lines, it is ideal for the development of new strategies for the selective targeted delivery of anticancer drugs and diagnostic devices to tumor cells. However, the design of new GRP-R binders requires structural information on receptor interaction with its natural ligands. The experimental protocol presented herein, based on on-cell STD NMR techniques, is a powerful tool for the screening and the epitope mapping of GRP-R ligands aimed at the development of new anticancer and diagnostic tools. Notably, the study can be carried out in a physiological environment, at the surface of tumoral cells overespressing GRP-R. Moreover, to the best of our knowledge, this is the first example of an NMR experiment able to detect and investigate the structural determinants of BN/GRP-R interaction.
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http://dx.doi.org/10.1016/j.bioorg.2020.103861DOI Listing
June 2020

A community-built calibration system: The case study of quantification of metabolites in grape juice by qNMR spectroscopy.

Talanta 2020 Jul 18;214:120855. Epub 2020 Feb 18.

The United States Pharmacopeial Convention (USP), 12601 Twinbrook Parkway, Rockville, MD, 20852-1790, USA.

Nuclear Magnetic Resonance (NMR) is an analytical technique extensively used in almost every chemical laboratory for structural identification. This technique provides statistically equivalent signals in spite of using spectrometer with different hardware features and is successfully used for the traceability and quantification of analytes in food samples. Nevertheless, to date only a few internationally agreed guidelines have been reported on the use of NMR for quantitative analysis. The main goal of the present study is to provide a methodological pipeline to assess the reproducibility of NMR data produced for a given matrix by spectrometers from different manufacturers, with different magnetic field strengths, age and hardware configurations. The results have been analyzed through a sequence of chemometric tests to generate a community-built calibration system which was used to verify the performance of the spectrometers and the reproducibility of the predicted sample concentrations.
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http://dx.doi.org/10.1016/j.talanta.2020.120855DOI Listing
July 2020

Anticancer Effects of Wild Mountain Extract in Adrenocortical Tumor Cell Models.

Front Pharmacol 2019 10;10:1647. Epub 2020 Feb 10.

Endocrinology Unit, Department of Medicine (DIMED), University of Padova, Padova, Italy.

Mint [ (L.) Hudson] is an aromatic plant that belongs to Lamiaceae family. It is traditionally used as herbal tea in Europe, Australia and North Africa and shows numerous pharmacological effects, such as spasmolytic, antioxidant, antimicrobial and anti-hemolytic. Recently, its antiproliferative role has been suggested in a small number of tumor cell models, but no data are available on adrenocortical carcinoma, a malignancy with a survival rate at 5 years of 20%-30% which frequently metastasize. This work aimed to study the effects of L. crude extract (ME) on two adrenocortical tumor cell models (H295R and SW13 cells). Chemical composition of ME was assessed by gas-chromatography/mass spectrometry and NMR spectroscopy analysis. Brine shrimp lethality assay showed ME effects at >0.5 µg/µl (p < 0.05). Cell viability and vitality were determined by MTT, SRB, and trypan blue assays in H295R and SW13 cells. The anti-proliferative effects of ME were more evident in SW13 cells at 72 h (ME > 0.5 µg/µl, p < 0.05). Combination of ME with mitotane (approved drug for adrenocortical carcinoma) seemed not to reinforce the efficacy of the herb. As control, human fibroblasts were treated with ME with no effect on cell viability. Clonogenic assay was concordant with previous cell viability tests (ME > 0.5 µg/µl, p < 0.05), while Wright staining demonstrated the presence of both necrotic and apoptotic cells. Cell cycle analysis showed a strong increase in subG0/G1 phase, related to cell death. Furthermore, MAPK and PI3k/Akt pathways were modulated by Western blot analysis when treating cells with ME alone or combined with mitotane. The crude methanolic extract of wild mountain mint can decrease cell viability, vitality and survival of adrenocortical tumor cell models, in particular of SW13 cells. These data show the potential anticancer effects of ME, still more work is needed to corroborate these findings.
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http://dx.doi.org/10.3389/fphar.2019.01647DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7025550PMC
February 2020

Targeting Bacterial Biofilm: A New LecA Multivalent Ligand with Inhibitory Activity.

Chembiochem 2019 12 24;20(23):2911-2915. Epub 2019 Sep 24.

Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126, Milano, Italy.

Biofilm formation by bacterial pathogens is a hallmark of chronic infections and is associated to increased antibiotic tolerance that makes pathogens difficult to eradicate with conventional antibiotic therapies. Infections caused by Pseudomonas aeruginosa are of great concern, especially for immunocompromised and cystic fibrosis patients. P. aeruginosa lectins LecA and LecB are virulence factors and play a key role in establishing biofilm; therefore, inhibition of the function of these proteins has potential in dismantling the bacterium from the protective biofilm environment and in restoring the activity of antibiotics. Here, we report the NMR characterization of the binding of a galactose-based dendrimer (Gal18) to LecA. Moreover, we demonstrate the activity of the Gal18 molecule in inhibiting P. aeruginosa biofilm formation in vitro.
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http://dx.doi.org/10.1002/cbic.201900383DOI Listing
December 2019

Glycan Carriers As Glycotools for Medicinal Chemistry Applications.

Curr Med Chem 2019 ;26(35):6349-6398

Department of Biotechnology and Biosciences, University of Milano-Bicocca Milano, Italy.

Carbohydrates are one of the most powerful and versatile classes of biomolecules that nature uses to regulate organisms' biochemistry, modulating plenty of signaling events within cells, triggering a plethora of physiological and pathological cellular behaviors. In this framework, glycan carrier systems or carbohydrate-decorated materials constitute interesting and relevant tools for medicinal chemistry applications. In the last few decades, efforts have been focused, among others, on the development of multivalent glycoconjugates, biosensors, glycoarrays, carbohydrate-decorated biomaterials for regenerative medicine, and glyconanoparticles. This review aims to provide the reader with a general overview of the different carbohydrate carrier systems that have been developed as tools in different medicinal chemistry approaches relying on carbohydrate-protein interactions. Given the extent of this topic, the present review will focus on selected examples that highlight the advancements and potentialities offered by this specific area of research, rather than being an exhaustive literature survey of any specific glyco-functionalized system.
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http://dx.doi.org/10.2174/0929867326666190104164653DOI Listing
December 2019

Coffee variety, origin and extraction procedure: Implications for coffee beneficial effects on human health.

Food Chem 2019 Apr 12;278:47-55. Epub 2018 Nov 12.

Department of Biotechnologies and Biosciences, University of Milano-Bicocca, P.zza della Scienza 2, 20126 Milan, Italy. Electronic address:

We set up an efficient protocol for the rapid analysis of NMR spectra of green and roasted coffee extracts, enabling the automatic identification and quantification of metabolites in approximately two minutes per spectrum. This method allowed for the metabolic profiling and the subsequent evaluation of the content of bioactive compounds and antioxidant activity of coffee samples, depending on their species (Arabica and Robusta), geographical origin and extraction procedure (hydroalcoholic, espresso and moka). The hydroalcoholic extraction is the most efficient method in terms of yields of low molecular weight compounds (in particular chlorogenic acids), while moka extraction provides the highest amounts of melanoidins. Moreover, that the ratio between health-giving compounds (chlorogenic acids, trigonelline and choline) and caffeine is higher in Arabica coffees. The data collected provide useful insights for the selection of coffee raw material to be used in the preparation of coffee-based dietary supplements, nutraceuticals and functional beverages.
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http://dx.doi.org/10.1016/j.foodchem.2018.11.063DOI Listing
April 2019

Methacycline displays a strong efficacy in reducing toxicity in a SCA3 Caenorhabditis elegans model.

Biochim Biophys Acta Gen Subj 2019 02 27;1863(2):279-290. Epub 2018 Oct 27.

Department of Biotechnologies and Biosciences, University of Milano-Bicocca, 20126 Milan, Italy; Milan Center of Neuroscience (NeuroMI), 20126 Milan, Italy. Electronic address:

Background: We have previously demonstrated the neuroprotective activity of tetracycline on a Spinocerebellar Ataxia 3 nematode model. Here, we present the screening of a small library of tetracycline congeners in order to identify the most effective compound in preventing ataxin-3 aggregation.

Methods: We performed the assays on the Josephin Domain as it is directly involved in the onset of fibrillation. We used thioflavin T and solubility assays to spot out the most effective tetracycline congeners; Fourier transform infrared and NMR spectroscopies to characterize their mode of action. We employed an ataxic Caenorhabditis elegans model to evaluate the pharmacological efficacy of tetracycline congeners.

Results: Methacycline was identified as the most effective compound. Like tetracycline, methacycline neither significantly affected the aggregation kinetics nor did it change the secondary structures of the final aggregates but increased the solubility of the aggregated species. Saturation transfer NMR experiments demonstrated methacycline capability to only bind the oligomeric species of Josephin Domain. Competition assays also showed that methacycline binds to the Josephin Domain more tightly than tetracycline. The treatment with methacycline induced a significant improvement in motility and locomotion of the transgenic C. elegans without changing its lifespan. The efficacy was distinctly stronger than that of tetracycline. Noteworthy, unlike tetracycline, methacycline was able to retard aging-related decline in motility of even the healthy worms used.

Conclusions: The apparent absence of toxic effects displayed by methacycline, along with its stronger efficacy in contrasting expanded ataxin-3 toxicity, makes it a possible candidate for a chronic treatment of the disease.
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http://dx.doi.org/10.1016/j.bbagen.2018.10.008DOI Listing
February 2019

bioNMR-based identification of natural anti-Aβ compounds in Peucedanum ostruthium.

Bioorg Chem 2019 03 12;83:76-86. Epub 2018 Oct 12.

Department of Biotechnology and Biosciences, University of Milano - Bicocca, P.zza della Scienza 2, 20126 Milan, Italy; NeuroMI, Milan Center for Neuroscience, University of Milano - Bicocca, 20126 Milano, Italy. Electronic address:

The growing interest in medicinal plants for the identification of new bioactive compounds and the formulation of new nutraceuticals and drugs prompted us to develop a powerful experimental approach allowing the detailed metabolic profiling of complex plant extracts, the identification of ligands of macromolecular targets of biomedical relevance and a preliminary characterization of their biological activity. To this end, we selected Peucedanum ostruthium, a plant traditionally employed in Austria and Italy for its several potential therapeutic applications, as case study. We combined the use of NMR and UPLC-HR-MS for the identification of the metabolites present in its leaves and rhizome extracts. Due to the significant content of polyphenols, particularly chlorogenic acids, recently identified as anti-amyloidogenic compounds, polyphenols-enriched fractions were prepared and tested for their ability to prevent Aβ1-42 peptide aggregation and neurotoxicity in a neuronal human cell line. STD-NMR experiments allowed the detailed identification of Aβ oligomers' ligands responsible for the anti-amyloidogenic activity. These data provide experimental protocols and structural information suitable for the development of innovative molecular tools for prevention, therapy and diagnosis of Alzheimer's disease.
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http://dx.doi.org/10.1016/j.bioorg.2018.10.016DOI Listing
March 2019

Methionine supplementation stimulates mitochondrial respiration.

Biochim Biophys Acta Mol Cell Res 2018 12 2;1865(12):1901-1913. Epub 2018 Oct 2.

Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy; SYSBIO, Centre of Systems Biology, Milan, Italy. Electronic address:

Mitochondria play essential metabolic functions in eukaryotes. Although their major role is the generation of energy in the form of ATP, they are also involved in maintenance of cellular redox state, conversion and biosynthesis of metabolites and signal transduction. Most mitochondrial functions are conserved in eukaryotic systems and mitochondrial dysfunctions trigger several human diseases. By using multi-omics approach, we investigate the effect of methionine supplementation on yeast cellular metabolism, considering its role in the regulation of key cellular processes. Methionine supplementation induces an up-regulation of proteins related to mitochondrial functions such as TCA cycle, electron transport chain and respiration, combined with an enhancement of mitochondrial pyruvate uptake and TCA cycle activity. This metabolic signature is more noticeable in cells lacking Snf1/AMPK, the conserved signalling regulator of energy homeostasis. Remarkably, snf1Δ cells strongly depend on mitochondrial respiration and suppression of pyruvate transport is detrimental for this mutant in methionine condition, indicating that respiration mostly relies on pyruvate flux into mitochondrial pathways. These data provide new insights into the regulation of mitochondrial metabolism and extends our understanding on the role of methionine in regulating energy signalling pathways.
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http://dx.doi.org/10.1016/j.bbamcr.2018.09.007DOI Listing
December 2018

Structure-Activity Relationship in Monosaccharide-Based Toll-Like Receptor 4 (TLR4) Antagonists.

J Med Chem 2018 04 12;61(7):2895-2909. Epub 2018 Mar 12.

Department of Biotechnology and Biosciences , University of Milano-Bicocca , Piazza della Scienza, 2 , 20126 Milano , Italy.

The structure-activity relationship was investigated in a series of synthetic TLR4 antagonists formed by a glucosamine core linked to two phosphate esters and two linear carbon chains. Molecular modeling showed that the compounds with 10, 12, and 14 carbons chains are associated with higher stabilization of the MD-2/TLR4 antagonist conformation than in the case of the C16 variant. Binding experiments with human MD-2 showed that the C12 and C14 variants have higher affinity than C10, while the C16 variant did not interact with the protein. The molecules, with the exception of the C16 variant, inhibited the LPS-stimulated TLR4 signal in human and murine cells, and the antagonist potency mirrored the MD-2 affinity calculated from in vitro binding experiments. Fourier-transform infrared, nuclear magnetic resonance, and small angle X-ray scattering measurements suggested that the aggregation state in aqueous solution depends on fatty acid chain lengths and that this property can influence TLR4 activity in this series of compounds.
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http://dx.doi.org/10.1021/acs.jmedchem.7b01803DOI Listing
April 2018

NMR-driven identification of anti-amyloidogenic compounds in green and roasted coffee extracts.

Food Chem 2018 Jun 12;252:171-180. Epub 2018 Jan 12.

Department of Biotechnologies and Biosciences, University of Milano-Bicocca, P.zza della Scienza 2, 20126 Milan, Italy; Milan Center of Neuroscience (NeuroMI), 20126 Milano Italy. Electronic address:

To identify food and beverages that provide the regular intake of natural compounds capable of interfering with toxic amyloidogenic aggregates, we developed an experimental protocol that combines NMR spectroscopy and atomic force microscopy, in vitro biochemical and cell assays to detect anti-Aβ molecules in natural edible matrices. We applied this approach to investigate the potential anti-amyloidogenic properties of coffee and its molecular constituents. Our data showed that green and roasted coffee extracts and their main components, 5-O-caffeoylquinic acid and melanoidins, can hinder Aβ on-pathway aggregation and toxicity in a human neuroblastoma SH-SY5Y cell line. Coffee extracts and melanoidins also counteract hydrogen peroxide- and rotenone-induced cytotoxicity and modulate some autophagic pathways in the same cell line.
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http://dx.doi.org/10.1016/j.foodchem.2018.01.075DOI Listing
June 2018

Flavonoids in the Treatment of Alzheimer's and Other Neurodegenerative Diseases.

Curr Med Chem 2018 ;25(27):3228-3246

Department of Biotechnology and Bioscience, University of Milano-Bicocca, Milan, Italy.

Flavonoids are phytochemicals present in almost all terrestrial plants and, as a consequence, in plant-based foods, and thus consumed by humans through diet. Recent evidences suggest that several flavonoids have positive effects against dementia and Alzheimer's disease, reversing age-related declines in neurocognitive performances. In this review, we provide a general classification of natural and synthetic flavonoids, a description of their physico-chemical properties, in particular their redox properties and stability, and an extensive overview about their biological activities and structure-activity relationship in the field of neurodegenerative diseases. In addition, a section will be dedicated to the synthetic strategies for the preparation of bioactive derivatives. This information will be essential for the design and development of new drugs that can improve brain functions.
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http://dx.doi.org/10.2174/0929867325666180209132125DOI Listing
September 2018

HIV-1 matrix protein p17 misfolding forms toxic amyloidogenic assemblies that induce neurocognitive disorders.

Sci Rep 2017 09 4;7(1):10313. Epub 2017 Sep 4.

Department of Molecular Biochemistry and Pharmacology, IRCCS- Istituto di Ricerche Farmacologiche "Mario Negri", Via G. La Masa 19, 20156, Milano, Italy.

Human immunodeficiency virus type-1 (HIV-1)-associated neurocognitive disorder (HAND) remains an important neurological manifestation that adversely affects a patient's quality of life. HIV-1 matrix protein p17 (p17) has been detected in autoptic brain tissue of HAND individuals who presented early with severe AIDS encephalopathy. We hypothesised that the ability of p17 to misfold may result in the generation of toxic assemblies in the brain and may be relevant for HAND pathogenesis. A multidisciplinary integrated approach has been applied to determine the ability of p17 to form soluble amyloidogenic assemblies in vitro. To provide new information into the potential pathogenic role of soluble p17 species in HAND, their toxicological capability was evaluated in vivo. In C. elegans, capable of recognising toxic assemblies of amyloidogenic proteins, p17 induces a specific toxic effect which can be counteracted by tetracyclines, drugs able to hinder the formation of large oligomers and consequently amyloid fibrils. The intrahippocampal injection of p17 in mice reduces their cognitive function and induces behavioral deficiencies. These findings offer a new way of thinking about the possible cause of neurodegeneration in HIV-1-seropositive patients, which engages the ability of p17 to form soluble toxic assemblies.
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http://dx.doi.org/10.1038/s41598-017-10875-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5583282PMC
September 2017

Natural Compounds in Cancer Prevention: Effects of Coffee Extracts and Their Main Polyphenolic Component, 5-O-Caffeoylquinic Acid, on Oncogenic Ras Proteins.

Chem Asian J 2017 Sep 29;12(18):2457-2466. Epub 2017 Aug 29.

Department of Biotechnology and Biosciences, Università degli Studi di Milano-Bicocca, Piazza della Scienza 2, 20126, Milan, Italy.

Recent epidemiological studies have demonstrated that the consumption of healthy foods that are particularly rich in polyphenols might reduce the incidence of cancer and neurodegenerative diseases. In particular, chlorogenic acids (CGAs) occur ubiquitously in food and represent the most abundant polyphenols in the human diet. A number of beneficial biological effects of CGAs, such as anti-inflammatory activity, anti-carcinogenic activity, and protection against neurodegenerative diseases, have been reported. However, the molecular mechanisms at the base of these biological activities have not yet been investigated in depth. By combining NMR spectroscopy, molecular docking, surface plasmon resonance and ex vivo assays of the Ras-dependent breast cancer cell line MDA-MB-231, we contribute to the elucidation of the molecular basis of the activity of CGAs and natural extracts from green and roasted coffee beans as chemoprotective dietary supplements.
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http://dx.doi.org/10.1002/asia.201700844DOI Listing
September 2017

Epigallocatechin-3-gallate and related phenol compounds redirect the amyloidogenic aggregation pathway of ataxin-3 towards non-toxic aggregates and prevent toxicity in neural cells and Caenorhabditis elegans animal model.

Hum Mol Genet 2017 09;26(17):3271-3284

Department of Biotechnologies and Biosciences, University of Milano-Bicocca, 20126 Milan, Italy.

The protein ataxin-3 (ATX3) triggers an amyloid-related neurodegenerative disease when its polyglutamine stretch is expanded beyond a critical threshold. We formerly demonstrated that the polyphenol epigallocatechin-3-gallate (EGCG) could redirect amyloid aggregation of a full-length, expanded ATX3 (ATX3-Q55) towards non-toxic, soluble, SDS-resistant aggregates. Here, we have characterized other related phenol compounds, although smaller in size, i.e. (-)-epigallocatechin gallate (EGC), and gallic acid (GA). We analysed the aggregation pattern of ATX3-Q55 and of the N-terminal globular Josephin domain (JD) by assessing the time course of the soluble protein, as well its structural features by FTIR and AFM, in the presence and the absence of the mentioned compounds. All of them redirected the aggregation pattern towards soluble, SDS-resistant aggregates. They also prevented the appearance of ordered side-chain hydrogen bonding in ATX3-Q55, which is the hallmark of polyQ-related amyloids. Molecular docking analyses on the JD highlighted three interacting regions, including the central, aggregation-prone one. All three compounds bound to each of them, although with different patterns. This might account for their capability to prevent amyloidogenesis. Saturation transfer difference NMR experiments also confirmed EGCG and EGC binding to monomeric JD. ATX3-Q55 pre-incubation with any of the three compounds prevented its calcium-influx-mediated cytotoxicity towards neural cells. Finally, all the phenols significantly reduced toxicity in a transgenic Caenorhabditis elegans strain expressing an expanded ATX3. Overall, our results show that the three polyphenols act in a substantially similar manner. GA, however, might be more suitable for antiamyloid treatments due to its simpler structure and higher chemical stability.
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http://dx.doi.org/10.1093/hmg/ddx211DOI Listing
September 2017

H NMR To Evaluate the Metabolome of Bronchoalveolar Lavage Fluid (BALf) in Bronchiolitis Obliterans Syndrome (BOS): Toward the Development of a New Approach for Biomarker Identification.

J Proteome Res 2017 04 14;16(4):1669-1682. Epub 2017 Mar 14.

Department of Biotechnology and Biosciences, University of Milano-Bicocca , 20126 Milan, Italy.

This report describes the application of NMR spectroscopy to the profiling of metabolites in bronchoalveolar lavage fluid (BALf) of lung transplant recipients without bronchiolitis obliterans syndrome (BOS) (stable, S, n = 10), and with BOS at different degrees of severity (BOS 0p, n = 10; BOS I, n = 10). Through the fine-tuning of a number of parameters concerning both sample preparation/processing and variations of spectra acquisition modes, an efficient and reproducible protocol was designed for the screening of metabolites in a pulmonary fluid that should reflect the status of airway inflammation/injury. Exploiting the combination of mono- and bidimensional NMR experiments, 38 polar metabolites, including amino acids, Krebs cycle intermediates, mono- and disaccharides, nucleotides, and phospholipid precursors, were unequivocally identified. To determine which signature could be correlated with the onset of BOS, the metabolites' content of the above recipients was analyzed by multivariate (PCA and OPLS-DA) statistical methods. PCA analysis (almost) totally differentiated S from BOS I, and this discrimination was significantly improved by the application of OPLS-DA, whose model was characterized by excellent fit and prediction values (R = 0.99 and Q = 0.88). The analysis of S vs BOS 0p and of BOS 0p vs BOS I samples showed a clear discrimination of considered cohorts, although with a poorer efficiency compared to those measured for S vs BOS I patients. The data shown in this work assess the suitability of the NMR approach in monitoring different pathological lung conditions.
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http://dx.doi.org/10.1021/acs.jproteome.6b01038DOI Listing
April 2017

Flavonoids and Their Glycosides as Anti-amyloidogenic Compounds: Aβ1-42 Interaction Studies to Gain New Insights into Their Potential for Alzheimer's Disease Prevention and Therapy.

Chem Asian J 2017 Jan 5;12(1):67-75. Epub 2016 Dec 5.

Department of Biotecnology and Bioscience, University of Milano-Bicocca, Piazza della Scienza 2, I-20126, Milan, Italy.

Combining NMR spectroscopy, transmission electron microscopy, biochemical and in vitro toxicity assays, we characterized the effect of flavonoid glycosylation, a chemical modification found very frequently in nature, on their ability to recognize and bind Aβ1-42 oligomers, preventing their aggregation and their neurotoxicity. Our data allow the elucidation of their structure-activity relationships, showing that glycosylation has a modest impact on flavonoid affinity for Aβ oligomers but, at the same time, increases both solubility and chemical stability, thus promoting their beneficial properties against Alzheimer's disease (AD). As flavonoids and their glycosides are widely available in natural foods, our results provide important information for the evaluation of the role of a flavonoid-rich diet for the prevention of AD. In addition, the structural data collected can be exploited for the rational design of more potent Aβ oligomer inhibitors, useful for the development of new therapies against AD.
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http://dx.doi.org/10.1002/asia.201601291DOI Listing
January 2017

H NMR To Explore the Metabolome of Exhaled Breath Condensate in α-Antitrypsin Deficient Patients: A Pilot Study.

J Proteome Res 2016 12 5;15(12):4569-4578. Epub 2016 Oct 5.

Department of Pulmonology, Leiden University Medical Center , 2333 Leiden, The Netherlands.

The metabolomic analysis of exhaled breath condensate (EBC) may provide insights on both the pathology of pulmonary disorders and the response to therapy. This pilot study describes the ability of nuclear magnetic resonance (NMR)-based metabolomics to discriminate α1-antitrypsin deficient (AATD)-patients, who were diagnosed with moderate to severe emphysema, from healthy individuals. Comparative analysis of samples from these two homogeneous cohorts of individuals resulted in the generation of NMR profiles that were different from both a qualitative and a quantitative point-of-view. Among the identified metabolites that separated patients from controls, acetoin, propionate, acetate, and propane-1,2 diol were those presenting the biggest difference. Unambiguous confirmation that the two groups could be completely differentiated on the basis of their metabolite content came from the application of univariate and multivariate statistical analysis (principal component analysis, partial least squares discriminant analysis (PLS-DA), and orthogonal PLS-DA). MetaboAnalyst 3.0 platform, used to define a relationship among metabolites, allowed us to observe that pyruvate metabolism is the most-involved pathway, most of metabolites being originated from pyruvate. These preliminary data suggest that NMR, with its ability to differentiate the metabolic fingerprint of EBC of AATD patients from that of healthy controls, has a potential "clinical applicability" in this area.
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http://dx.doi.org/10.1021/acs.jproteome.6b00648DOI Listing
December 2016

Protein Kinase A Activation Promotes Cancer Cell Resistance to Glucose Starvation and Anoikis.

PLoS Genet 2016 Mar 15;12(3):e1005931. Epub 2016 Mar 15.

Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy.

Cancer cells often rely on glycolysis to obtain energy and support anabolic growth. Several studies showed that glycolytic cells are susceptible to cell death when subjected to low glucose availability or to lack of glucose. However, some cancer cells, including glycolytic ones, can efficiently acquire higher tolerance to glucose depletion, leading to their survival and aggressiveness. Although increased resistance to glucose starvation has been shown to be a consequence of signaling pathways and compensatory metabolic routes activation, the full repertoire of the underlying molecular alterations remain elusive. Using omics and computational analyses, we found that cyclic adenosine monophosphate-Protein Kinase A (cAMP-PKA) axis activation is fundamental for cancer cell resistance to glucose starvation and anoikis. Notably, here we show that such a PKA-dependent survival is mediated by parallel activation of autophagy and glutamine utilization that in concert concur to attenuate the endoplasmic reticulum (ER) stress and to sustain cell anabolism. Indeed, the inhibition of PKA-mediated autophagy or glutamine metabolism increased the level of cell death, suggesting that the induction of autophagy and metabolic rewiring by PKA is important for cancer cellular survival under glucose starvation. Importantly, both processes actively participate to cancer cell survival mediated by suspension-activated PKA as well. In addition we identify also a PKA/Src mechanism capable to protect cancer cells from anoikis. Our results reveal for the first time the role of the versatile PKA in cancer cells survival under chronic glucose starvation and anoikis and may be a novel potential target for cancer treatment.
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http://dx.doi.org/10.1371/journal.pgen.1005931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4792400PMC
March 2016
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