Detection of Tumor-Associated Autoantibodies in the Sera of Pancreatic Cancer Patients Using Engineered MUC1 Glycopeptide Nanoparticle Probes.

Pancreatic cancer is one of the deadliest cancers worldwide, mainly due to late diagnosis. Therefore, there is an urgent need for novel diagnostic approaches to identify the disease as early as possible. We have developed a diagnostic assay for pancreatic cancer based on the detection of naturally occurring tumor associated autoantibodies against Mucin-1 (MUC1) using engineered glycopeptides on nanoparticle probes. We used a structure-guided approach to develop unnatural glycopeptides as model antigens for tumor-associated MUC1. We designed a collection of 13 glycopeptides to bind either SM3 or 5E5, two monoclonal antibodies with distinct epitopes known to recognize tumor associated MUC1. Glycopeptide binding to SM3 or 5E5 was confirmed by surface plasmon resonance and rationalized by molecular dynamics simulations. These model antigens were conjugated to gold nanoparticles and used in a dot-blot assay to detect autoantibodies in serum samples from pancreatic cancer patients and healthy volunteers. Nanoparticle probes with glycopeptides displaying the SM3 epitope did not have diagnostic potential. Instead, nanoparticle probes displaying glycopeptides with high affinity for 5E5 could discriminate between cancer patients and healthy controls. Remarkably, the best-discriminating probes show significantly better true and false positive rates than the current clinical biomarkers CA19-9 and carcinoembryonic antigen (CEA).

Niosomes as Biocompatible Scaffolds for the Multivalent Presentation of Tumor-Associated Antigens (TACAs) to the Immune System.

Fully synthetic tumor-associated carbohydrate antigen (TACA)-based vaccines are a promising strategy to treat cancer. To overcome the intrinsic low immunogenicity of TACAs, the choice of the antigens' analogues and multivalent presentation have been proved to be successful. Here, we present the preparation, characterization, and in vitro screening of niosomes displaying multiple copies of the mucin antigen TnThr (niosomes-7) or of TnThr mimetic 1 (niosomes-2). Unprecedentedly, structural differences, likely related to the carbohydrate portions, were observed for the two colloidal systems. Both niosomal systems are stable, nontoxic and endowed with promising immunogenic properties.

Polysaccharides' Structures and Functions in Biofilm Architecture of Antimicrobial-Resistant (AMR) Pathogens.

Bacteria and fungi have developed resistance to the existing therapies such as antibiotics and antifungal drugs, and multiple mechanisms are mediating this resistance. Among these, the formation of an extracellular matrix embedding different bacterial cells, called biofilm, is an effective strategy through which bacterial and fungal cells are establishing a relationship in a unique environment. The biofilm provides them the possibility to transfer genes conferring resistance, to prevent them from desiccation and to impede the penetration of antibiotics or antifungal drugs. Biofilms are formed of several constituents including extracellular DNA, proteins and polysaccharides. Depending on the bacteria, different polysaccharides form the biofilm matrix in different microorganisms, some of them involved in the first stage of cells' attachment to surfaces and to each other, and some responsible for giving the biofilm structure resistance and stability. In this review, we describe the structure and the role of different polysaccharides in bacterial and fungal biofilms, we revise the analytical methods to characterize them quantitatively and qualitatively and finally we provide an overview of potential new antimicrobial therapies able to inhibit biofilm formation by targeting exopolysaccharides.

Conformationally Constrained Sialyl Analogues as New Potential Binders of h-CD22.

Here, two conformationally constrained sialyl analogues were synthesized and characterized in their interaction with the inhibitory Siglec, human CD22 (h-CD22). An orthogonal approach, including biophysical assays (SPR and fluorescence), ligand-based NMR techniques, and molecular modelling, was employed to disentangle the interaction mechanisms at a molecular level. The results showed that the Sialyl-TnThr antigen analogue represents a promising scaffold for the design of novel h-CD22 inhibitors. Our findings also suggest that the introduction of a biphenyl moiety at position 9 of the sialic acid hampers canonical accommodation of the ligand in the protein binding pocket, even though the affinity with respect to the natural ligand is increased. Our results address the search for novel modifications of the Neu5Ac-α(2-6)-Gal epitope, outline new insights for the design and synthesis of high-affinity h-CD22 ligands, and offer novel prospects for therapeutic intervention to prevent autoimmune diseases and B-cell malignancies.

A Sulfonated Tweezer-Shaped Receptor Selectively Recognizes Caffeine in Water.

The selective recognition of caffeine in water among structurally related xanthines and purine or pyrimidine bases was achieved by a simple tweezer-shaped receptor featuring sulfonate hydrosolubilizing groups. The remarkable affinity for caffeine, among the highest reported thus far in the literature and larger than that shown by adenosine receptors of all subtypes, stems from a synergistic combination of hydrogen bonding, CH-π, and π-stacking interactions.

Probing the Efficiency of 13-Pyridylalkyl Berberine Derivatives to Human Telomeric G-Quadruplexes Binding: Spectroscopic, Solid State and In Silico Analysis.

The interaction between the series of berberine derivatives 1-5 (NAX071, NAX120, NAX075, NAX077 and NAX079) and human telomeric G-quadruplexes (G4), which are able to inhibit the Telomerase enzyme's activity in malignant cells, was investigated. The derivatives bear a pyridine moiety connected by a hydrocarbon linker of varying length (n = 1-5, with n number of aliphatic carbon atoms) to the C13 position of the parent berberine. As for the G4s, both bimolecular 5'-TAGGGTTAGGGT-3' (Tel12) and monomolecular 5'-TAGGGTTAGGGTTAGGGTTAGGG-3' (Tel23) DNA oligonucleotides were considered. Spectrophotometric titrations, melting tests, X-ray diffraction solid state analysis and in silico molecular dynamics (MD) simulations were used to describe the different systems. The results were compared in search of structure-activity relationships. The analysis pointed out the formation of 1:1 complexes between Tel12 and all ligands, whereas both 1:1 and 2:1 ligand/G4 stoichiometries were found for the adduct formed by NAX071 (n = 1). Tel12, with tetrads free from the hindrance by the loop, showed a higher affinity. The details of the different binding geometries were discussed, highlighting the importance of H-bonds given by the berberine benzodioxole group and a correlation between the strength of binding and the hydrocarbon linker length. Theoretical (MD) and experimental (X-ray) structural studies evidence the possibility for the berberine core to interact with one or both G4 strands, depending on the constraints given by the linker length, thus affecting the G4 stabilization effect.

Association between Ideal Cardiovascular Health and aortic stiffness in Italian adolescents. The MACISTE study.

BACKGROUND AND AIMS: Ideal Cardiovascular Health (ICH), defined as optimal levels of cardiovascular (CV) health factors and behaviors, has been reported to be very low in adults and children, with consequent several negative health outcomes and higher CV risk. The present study investigated the burden of ICH among Italian adolescents and its association with carotid-femoral pulse wave velocity (cf-PWV). METHODS AND RESULTS: 387 healthy adolescents (mean age 17.1 ± 1.4 years) attending the "G. Donatelli" High School in Terni, Italy, were evaluated. ICH was assessed through clinical evaluation, laboratory measures and interviewer-administered questionnaires. Cf-PWV was measured by arterial tonometry (SphygmoCor). For each ICH metric, a score of 2 was assigned for ideal levels, 1 for intermediate, and 0 for poor. All subjects showed at least one ICH metric, whereas none showed all ICH 7 metrics. The average number of ICH metrics was 4.3 ± 1.1. The highest rates were observed for fasting blood glucose (98%), whereas an ideal healthy diet was achieved only by 8% of subjects. The Cf-PWV was inversely and linearly associated with the sum of ICH metrics (p = 0.03) and the ICH score (p < 0.01). At the multivariate analysis, the association between ICH score and cf-PWV remained significant after adjustment for age, sex, heart rate, mean arterial pressure and other confounders (p = 0.04). CONCLUSION: ICH is relatively uncommon among Italian adolescents and inversely related to cf-PWV. Our results showed a detrimental association between CV unhealthy factors and behaviors with increased aortic stiffness, which starts developing at an early stage of the lifespan.

Synthesis of an STnThr analogue, structurally based on a TnThr antigen mimetic.

The monosaccharide Tn and the disaccharide STn are tumor antigens with similar structures and common biosynthetic pathways. Both are always over-expressed simultaneously on tumor cell surfaces. We report herein the efficient synthesis of the STnThr antigen analogue 2, featuring the immunogenic TnThr mimetic 1 aglycon. Analogously to the native STn, 2 is recognized by the influenza N1 neuraminidase. A model of the N1·2 complex showed the sialyl moiety of 2 well nested in the active site pocket, with docking unaffected by the rigid aglycon. The analogue 2 is, therefore, in association with mimetic 1, a good determinant for the design of new multiantigen cancer vaccines.

Effect of structure levels on surface-enhanced Raman scattering of human telomeric G-quadruplexes in diluted and crowded media.

Human telomeric G-quadruplexes are emerging targets in anticancer drug discovery since they are able to efficiently inhibit telomerase, an enzyme which is greatly involved in telomere instability and immortalization process in malignant cells. G-quadruplex (G4) DNA is highly polymorphic and can adopt different topologies upon addition of electrolytes, additives, and ligands. The study of G-quadruplex forms under various conditions, however, might be quite challenging. In this work, surface-enhanced Raman scattering (SERS) spectroscopy has been applied to study G-quadruplexes formed by human telomeric sequences, d[A3G3(TTAGGG)3A2] (Tel26) and d[(TTAGGG)4T2] (wtTel26), under dilute and crowding conditions. The SERS spectra distinctive of hybrid-1 and hybrid-2 G-quadruplexes of Tel26 and wtTel26, respectively, were observed for the sequences folded in the presence of K+ ions (110 mM) in a buffered solution, representing the diluted medium. Polyethylene glycol (5, 10, 15, 20, and 40% v/v PEG) was used to create a molecular-crowded environment, resulting in the formation of the parallel G-quadruplexes of both studied human telomeric sequences. Despite extensive overlap by the crowding agent bands, the SERS spectral features indicative of parallel G4 form of Tel26 were recognized. The obtained results implied that SERS of G-quadruplexes reflected not only the primary structure of the studied human telomeric sequence, including its nucleobase composition and sequence, but also its secondary structure in the sense of Hoogsteen hydrogen bonds responsible for the guanine tetrad formation, and finally its tertiary structure, defining a three-dimensional DNA shape, positioned close to the enhancing metallic surface. Graphical abstract.

Induction of a Four-Way Junction Structure in the DNA Palindromic Hexanucleotide 5'-d(CGTACG)-3' by a Mononuclear Platinum Complex.

Four-way junctions (4WJs) are supramolecular DNA assemblies comprising four interacting DNA strands that in biology are involved in DNA-damage repair. In this study, a new mononuclear platinum(II) complex 1 was prepared that is capable of driving the crystallization of the DNA oligomer 5'-d(CGTACG)-3' specifically into a 4WJ-like motif. In the crystal structure of the 1-CGTACG adduct, the distorted-square-planar platinum complex binds to the core of the 4WJ-like motif through π-π stacking and hydrogen bonding, without forming any platinum-nitrogen coordination bonds. Our observations suggest that the specific molecular properties of the metal complex are crucially responsible for triggering the selective assembly of this peculiar DNA superstructure.

[Au(9-methylcaffein-8-ylidene)2 ]+ /DNA Tel23 System: Solution, Computational, and Biological Studies.

Physicochemical methods have been used to investigate interactions occurring in solution between the dicarbene gold(I) complex [Au(9-methylcaffein-8-ylidene)2 ]BF4 (AuNHC) and a human telomeric DNA sequence, namely Tel23. Circular dichroism measurements allow identification of the conformational changes experienced by Tel23 upon interaction with AuNHC, and the respective binding stoichiometries and constants were determined. Computational studies provide a good link between previous crystallographic results of the same system and the present solution data, offering an exhaustive description of the inherent noncovalent metallodrug-DNA interactions. Remarkably, we found that a preformed AuNHC/Tel23 adduct is capable of producing strong and selective inhibition of the enzyme telomerase. The latter feature is mechanistically relevant and might account for the conspicuous in vitro anticancer properties of the investigated dicarbene gold(I) complex.

Solution NMR Structure of a Ligand/Hybrid-2-G-Quadruplex Complex Reveals Rearrangements that Affect Ligand Binding.

Telomeric G-quadruplexes have recently emerged as drug targets in cancer research. Herein, we present the first NMR structure of a telomeric DNA G-quadruplex that adopts the biologically relevant hybrid-2 conformation in a ligand-bound state. We solved the complex with a metalorganic gold(III) ligand that stabilizes G-quadruplexes. Analysis of the free and bound structures reveals structural changes in the capping region of the G-quadruplex. The ligand is sandwiched between one terminal G-tetrad and a flanking nucleotide. This complex structure involves a major structural rearrangement compared to the free G-quadruplex structure as observed for other G-quadruplexes in different conformations, invalidating simple docking approaches to ligand-G-quadruplex structure determination.

Determinants for Tight and Selective Binding of a Medicinal Dicarbene Gold(I) Complex to a Telomeric DNA G-Quadruplex: a Joint ESI MS and XRD Investigation.

The dicarbene gold(I) complex [Au(9-methylcaffein-8-ylidene)2 ]BF4 is an exceptional organometallic compound of profound interest as a prospective anticancer agent. This gold(I) complex was previously reported to be highly cytotoxic toward various cancer cell lines in vitro and behaves as a selective G-quadruplex stabilizer. Interactions of the gold complex with various telomeric DNA models have been analyzed by a combined ESI MS and X-ray diffraction (XRD) approach. ESI MS measurements confirmed formation of stable adducts between the intact gold(I) complex and Tel 23 DNA sequence. The crystal structure of the adduct formed between [Au(9-methylcaffein-8-ylidene)2 ](+) and Tel 23 DNA G-quadruplex was solved. Tel 23 maintains a characteristic propeller conformation while binding three gold(I) dicarbene moieties at two distinct sites. Stacking interactions appear to drive noncovalent binding of the gold(I) complex. The structural basis for tight gold(I) complex/G-quadruplex recognition and its selectivity are described.

Structurally Constrained MUC1-Tn Mimetic Antigen as Template for Molecularly Imprinted Polymers (MIPs): A Promising Tool for Cancer Diagnostics.

Abnormal glycoconjugates have distinctly been recognized as potential biomarkers for cancer diagnosis. A great deal of attention has been focused on Tn antigen, an oversimplified mucin-1 O-glycan, over-expressed in different cancers. Herein, we investigate the possibility to replace the use of anti-Tn monoclonal antibodies with an innovative class of catecholamine-based Molecularly Imprinted Polymers (MIPs), emerging in recent years as promising tools for bioanalytical applications. MIPs are synthetic receptors characterized by high sensitivity and specificity towards the imprinted target. Here, original polynorepinephrine-based MIPs coupled to Surface Plasmon Resonance biosensing for Tn antigen recognition are reported. We have verified the imprinting and binding capacity of these MIPs towards very small antigenic entities, represented by the natural Tn antigen and the TnThr mimetic 1 (conjugated to BSA or linked to a MUC1 hexapeptide analogue), and compared the biosensor performances with an anti-Tn monoclonal antibody. The results clearly display the effectiveness of the pursued imprinting strategies.

Recent advances and future perspectives on carbohydrate-based cancer vaccines and therapeutics.

Carbohydrates are abundantly expressed on the surface of both eukaryotic and prokaryotic cells, often as post translational modifications of proteins. Glycoproteins are recognized by the immune system and can trigger both innate and humoral responses. This feature has been harnessed to generate vaccines against polysaccharide-encapsulated bacteria such as Streptococcus pneumoniae, Hemophilus influenzae type b and Neisseria meningitidis. In cancer, glycosylation plays a pivotal role in malignancy development and progression. Since glycans are specifically expressed on the surface of tumor cells, they have been targeted for the discovery of anticancer preventive and therapeutic treatments, such as vaccines and monoclonal antibodies. Despite the various efforts made over the last years, resulting in a series of clinical studies, attempts of vaccination with carbohydrate-based candidates have proven unsuccessful, primarily due to the immune tolerance often associated with these glycans. New strategies are thus deployed to enhance carbohydrate-based cancer vaccines. Moreover, lessons learned from glycan immunobiology paved the way to the development of new monoclonal antibodies specifically designed to recognize cancer-bound carbohydrates and induce tumor cell killing. Herein we provide an overview of the immunological principles behind the immune response towards glycans and glycoconjugates and the approaches exploited at both preclinical and clinical level to target cancer-associated glycans for the development of vaccines and therapeutic monoclonal antibodies. We also discuss gaps and opportunities to successfully advance glycan-directed cancer therapies, which could provide patients with innovative and effective treatments.

Sustainability of Rearing System Using Multicriteria Analysis: Application in Commercial Poultry Production.

The aim of the present study was to develop a multicriteria model for the comparison of three commercial poultry farms: organic with Ross 308 genotype (OR), organic with Naked Neck genotype (ONN) and a conventional system (C), which represents the most common commercial farming system. A model based on multicriteria decision analysis was developed, considering for the first time the One Welfare approach in an operational manner, including three dimensions: human, environmental and animal welfare. The three alternatives demonstrated different performances, according to the different dimensions considered. In particular, the two organic systems performed better for human welfare and animal welfare, with relevant differences due to the genetic strains used. Conventional rearing performed better for the environment index due to the method chosen. The multicriteria analysis showed that the organic system performed better overall than the conventional system. In particular, the use of an adapted Slow Growing (SG) strain positively affected the final rank, mainly by reducing welfare problems and producing good economic and social performance. The stability of the results was verified by performing a sensitivity analysis, specifically a weight stability analysis, which confirmed the strength of results.

Correction to "Pyridine Derivative of the Natural Alkaloid Berberine as Human Telomeric G4-DNA Binder: A Solution and Solid-State Study".

[This corrects the article DOI: 10.1021/acsmedchemlett.9b00516.].

Pyridine Derivative of the Natural Alkaloid Berberine as Human Telomeric G4-DNA Binder: A Solution and Solid-State Study.

Telomerase is an enzyme deputed to the maintenance of eukaryotic chromosomes; however, its overexpression is a recognized hallmark of many cancer forms. A viable route for the inhibition of telomerase in malignant cells is the stabilization of G-quadruplex structures (G4) at the 3' overhang of telomeres. Berberine has shown in this regard valuable G4 binding properties together with a significant anticancer activity and telomerase inhibition effects. Here, we focused on a berberine derivative featuring a pyridine containing side group at the 13th position. Such modification actually improves the binding toward telomeric G-quadruplexes and establishes a degree of selectivity in the interaction with different sequences. Moreover, the X-ray crystal structure obtained for the complex formed by the ligand and a bimolecular human telomeric quadruplex affords a better understanding of the 13-berberine derivatives behavior with telomeric G4 and allows to draw useful insights for the future design of derivatives with remarkable anticancer properties.

A Structurally Simple Vaccine Candidate Reduces Progression and Dissemination of Triple-Negative Breast Cancer.

The Tn antigen is a well-known tumor-associated carbohydrate determinant, often incorporated in glycopeptides to develop cancer vaccines. Herein, four copies of a conformationally constrained mimetic of the antigen TnThr (GalNAc-Thr) were conjugated to the adjuvant CRM197, a protein licensed for human use. The resulting vaccine candidate, mime[4]CRM elicited a robust immune response in a triple-negative breast cancer mouse model, correlated with high frequency of CD4+ T cells and low frequency of M2-type macrophages, which reduces tumor progression and lung metastasis growth. Mime[4]CRM-mediated activation of human dendritic cells is reported, and the proliferation of mime[4]CRM-specific T cells, in cancer tissue and peripheral blood of patients with breast cancer, is demonstrated. The locked conformation of the TnThr mimetic and a proper presentation on the surface of CRM197 may explain the binding of the conjugate to the anti-Tn antibody Tn218 and its efficacy to fight cancer cells in mice.

Tn antigen analogues: the synthetic way to "upgrade" an attracting tumour associated carbohydrate antigen (TACA).

In the last two decades, the paramount importance of Tumor Associated Carbohydrate Antigens (TACAs) as targets for anticancer vaccine development has been firmly assessed. The Tn antigen is an ideal target for immunotherapy, in that it is masked on normal cells, but exposed on cancer cells. However, it is difficult to elicit an effective and long-lasting response against Tn antigen and other TACAs. Here we report on the Tn antigen analogues developed to boost the latent Tn immune response. Hopefully, the results reported herein will be of help for the rational design of effective TACA-based immunostimulants.