New Aß(1-42) ligands from anti-amyloid antibodies: Design, synthesis, and structural interaction.

Alzheimer's disease (AD), is the most common neurodegenerative disorder of the aging population resulting in progressive cognitive and functional decline. Accumulation of amyloid plaques around neuronal cells is considered a critical pathogenetic event and, in most cases, a hallmark of the pathology. In the attempt to identify anti-AD drug candidates, hundreds of molecules targeting Aß peptides have been screened. Peptide molecules have been widely explored, appreciating chemical stability, biocompatibility, and low production cost. More recently, many anti-Aß(1-42) monoclonal antibodies have been developed, given the excellent potential of immunotherapy for treating or preventing AD. Antibodies are versatile ligands that bind a large variety of molecules with high affinity and specificity; however, their extensive therapeutic application is complex and requires huge economic investments. Novel approaches to identify alternative antibody formats are considered with great interest. In this context, taking advantage of the favorable peptide properties and the availability of Aß-antibodies structural data, we followed an innovative research approach to identify short peptide sequences on the model of the binding sites of Aß(1-42)/antibodies. WAibH and SYSTPGK were designed as mimics of solanezumab and aducanumab, respectively. Circular dichroism and nuclear magnetic resonance analysis reveal that the antibody-derived peptides interact with Aß(1-42) in the soluble monomeric form. Moreover, AFM microscopy imaging shows that WAibH and SYSTPGK are capable of controlling the Aß(1-42) aggregation. The strategy to identify WAibH and SYSTPGK is innovative and can be widely applied for new anti-Aß antibody mimicking peptides.

Exploring the Early Stages of the Amyloid Aß(1-42) Peptide Aggregation Process: An NMR Study.

Alzheimer's disease (AD) is a neurodegenerative pathology characterized by the presence of neurofibrillary tangles and amyloid plaques, the latter mainly composed of Aß(1-40) and Aß(1-42) peptides. The control of the Aß aggregation process as a therapeutic strategy for AD has prompted the interest to investigate the conformation of the Aß peptides, taking advantage of computational and experimental techniques. Mixtures composed of systematically different proportions of HFIP and water have been used to monitor, by NMR, the conformational transition of the Aß(1-42) from soluble α-helical structure to ß-sheet aggregates. In the previous studies, 50/50 HFIP/water proportion emerged as the solution condition where the first evident Aß(1-42) conformational changes occur. In the hypothesis that this solvent reproduces the best condition to catch transitional helical-ß-sheet Aß(1-42) conformations, in this study, we report an extensive NMR conformational analysis of Aß(1-42) in 50/50 HFIP/water v/v. Aß(1-42) structure was solved by us, giving evidence that the evolution of Aß(1-42) peptide from helical to the ß-sheet may follow unexpected routes. Molecular dynamics simulations confirm that the structural model we calculated represents a starting condition for amyloid fibrils formation.

Effect of Very-Low-Calorie Ketogenic Diet on Psoriasis Patients: A Nuclear Magnetic Resonance-Based Metabolomic Study.

Psoriasis is an inflammatory disease of the epidermis based on an immunological mechanism involving Langerhans cells and T lymphocytes that produce pro-inflammatory cytokines. Genetic factors, environmental factors, and improper nutrition are considered triggers of the disease. Numerous studies have reported that in a high number of patients, psoriasis is associated with obesity. Excess adipose tissue, typical of obesity, causes a systemic inflammatory status coming from the inflammatory active adipose tissue; therefore, weight reduction is a strategy to fight this pro-inflammatory state. This study aimed to evaluate how a nutritional regimen based on a ketogenic diet influenced the clinical parameters, metabolic profile, and inflammatory state of psoriasis patients. To this end, 30 psoriasis patients were subjected to a ketogenic nutritional regimen and monitored for 4 weeks by evaluating the clinical data, biochemical and clinical parameters, NMR metabolomic profile, and IL-2, IL-1ß, TNF-α, IFN-γ, and IL-4 concentrations before and after the nutritional regimen. Our data show that a low-calorie ketogenic diet can be considered a successful strategy and therapeutic option to gain an improvement in psoriasis-related dysmetabolism, with significant correction of the full metabolic and inflammatory status.

In Amyotrophic Lateral Sclerosis Blood Cytokines Are Altered, but Do Not Correlate with Changes in Brain Topology.

Aim: The present study aims at investigating the possible correlation between peripheral markers of inflammation and brain networks. Introduction: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease dominated by progressive motor impairment. Among the complex mechanisms contributing to the pathogenesis of the disease, neuroinflammation, which is associated with altered circulating cytokine levels, is suggested to play a prominent role. Methods: Based on magnetoencephalography data, we estimated topological properties of the brain networks in ALS patients and healthy controls. Subsequently, the blood levels of a subset of cytokines were assayed. Finally, we modeled the brain topological features in the function of the cytokine levels. Results: Significant differences were found in the levels of the cytokines interleukin (IL)-4, IL-1ß, and interferon-gamma (IFN-γ) between patients and controls. In particular, IL-4 and IL-1ß levels increased in ALS patients, while the IFN-γ level was higher in healthy controls. We also detected modifications in brain global topological parameters in terms of hyperconnectedness. Despite both blood cytokines and brain topology being altered in ALS patients, such changes do not appear to be in a direct relationship. Conclusion: Our results would be in line with the idea that topological changes relate to neurodegenerative processes. However, the absence of correlation between blood cytokines and topological parameters of brain networks does not preclude that inflammatory processes contribute to the alterations of the brain networks. Impact statement The progression of amyotrophic lateral sclerosis entails both neurodegenerative and inflammatory processes. Furthermore, disease progression induces global modifications of the brain networks, with advanced stages showing a more compact, hyperconnected network topology. The pathophysiological processes underlying topological changes are unknown. In this article, we hypothesized that the global inflammatory profile would relate to the topological alterations. Our results showed that this is not the case, as modeling the topological properties as a function of the inflammatory state did not yield good predictions. Hence, our results suggest that topological changes might directly relate to neurodegenerative processes instead.

Binding of the Anti-FIV Peptide C8 to Differently Charged Membrane Models: From First Docking to Membrane Tubulation.

Gp36 is the virus envelope glycoproteins catalyzing the fusion of the feline immunodeficiency virus with the host cells. The peptide C8 is a tryptophan-rich peptide corresponding to the fragment 770W-I777 of gp36 exerting antiviral activity by binding the membrane cell and inhibiting the virus entry. Several factors, including the membrane surface charge, regulate the binding of C8 to the lipid membrane. Based on the evidence that imperceptible variation of membrane charge may induce a dramatic effect in several critical biological events, in the present work we investigate the effect induced by systematic variation of charge in phospholipid bilayers on the aptitude of C8 to interact with lipid membranes, the tendency of C8 to assume specific conformational states and the re-organization of the lipid bilayer upon the interaction with C8. Accordingly, employing a bottom-up multiscale protocol, including CD, NMR, ESR spectroscopy, atomistic molecular dynamics simulations, and confocal microscopy, we studied C8 in six membrane models composed of different ratios of zwitterionic/negatively charged phospholipids. Our data show that charge content modulates C8-membrane binding with significant effects on the peptide conformations. C8 in micelle solution or in SUV formed by DPC or DOPC zwitterionic phospholipids assumes regular ß-turn structures that are progressively destabilized as the concentration of negatively charged SDS or DOPG phospholipids exceed 40%. Interaction of C8 with zwitterionic membrane surface is mediated by Trp1 and Trp4 that are deepened in the membrane, forming H-bonds and cation-π interactions with the DOPC polar heads. Additional stabilizing salt bridge interactions involve Glu2 and Asp3. MD and ESR data show that the C8-membrane affinity increases as the concentration of zwitterionic phospholipid increases. In the lipid membrane characterized by an excess of zwitterionic phospholipids, C8 is adsorbed at the membrane interface, inducing a stiffening of the outer region of the DOPC bilayer. However, the bound of C8 significantly perturbs the whole organization of lipid bilayer resulting in membrane remodeling. These events, measurable as a variation of the bilayer thickness, are the onset mechanism of the membrane fusion and vesicle tubulation observed in confocal microscopy by imaging zwitterionic MLVs in the presence of C8 peptide.

NMR Structure of the FIV gp36 C-Terminal Heptad Repeat and Membrane-Proximal External Region.

Feline immunodeficiency virus (FIV), a lentivirus causing an immunodeficiency syndrome in cats, represents a relevant model of pre-screening therapies for human immunodeficiency virus (HIV). The envelope glycoproteins gp36 in FIV and gp41 in HIV mediate the fusion of the virus with the host cell membrane. They have a common structural framework in the C-terminal region that includes a Trp-rich membrane-proximal external region (MPER) and a C-terminal heptad repeat (CHR). MPER is essential for the correct positioning of gp36 on the lipid membrane, whereas CHR is essential for the stabilization of the low-energy six-helical bundle (6HB) that is necessary for the fusion of the virus envelope with the cell membrane. Conformational data for gp36 are missing, and several aspects of the MPER structure of different lentiviruses are still debated. In the present work, we report the structural investigation of a gp36 construct that includes the MPER and part of the CHR domain (737-786gp36 CHR-MPER). Using 2D and 3D homo and heteronuclear NMR spectra on 15N and 13C double-labelled samples, we solved the NMR structure in micelles composed of dodecyl phosphocholine (DPC) and sodium dodecyl sulfate (SDS) 90/10 M: M. The structure of 737-786gp36 CHR-MPER is characterized by a helix-turn-helix motif, with a regular α-helix and a moderately flexible 310 helix, characterizing the CHR and the MPER domains, respectively. The two helices are linked by a flexible loop regulating their orientation at a ~43° angle. We investigated the positioning of 737-786gp36 CHR-MPER on the lipid membrane using spin label-enhanced NMR and ESR spectroscopies. On a different scale, using confocal microscopy imaging, we studied the effect of 737-786gp36 CHR-MPER on 1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPC/DOPG) multilamellar vesicles (MLVs). This effect results in membrane budding and tubulation that is reminiscent of a membrane-plasticizing role that is typical of MPER domains during the event in which the virus envelope merges with the host cell membrane.

NMR for screening and a biochemical assay: Identification of new FPPS inhibitors exerting anticancer activity.

Farnesyl pyrophosphate synthase (FPPS) is a crucial enzyme for the synthesis of isoprenoids and the key target of nitrogen-containing bisphosphonates (N-BPs). N-BPs are potent and selective FPPS inhibitors that are used in the treatment of bone-related diseases, but have poor pharmacokinetic properties. Given the key role played by FPPS in many cancer-related pathways and the pharmacokinetic limits of N-BPs, hundreds of molecules have been screened to identify new FPPS inhibitors characterized by improved drug-like properties that are useful for broader therapeutic applications in solid, non-skeletal tumours. We have previously shown that N6-isopentenyladenosine (i6A) and its related compound N6-benzyladenosine (2) exert anti-glioma activity by interfering with the mevalonate pathway and inhibiting FPPS. Here, we report the design and synthesis of a panel of N6-benzyladenosine derivatives (compounds 2a-m) incorporating different chemical moieties on the benzyl ring. Compounds 2a-m show in vitro antiproliferative activity in U87MG glioma cells and, analogous to the bisphosphonate FPPS inhibitors, exhibit immunogenic properties in ex vivo γδ T cells from stimulated peripheral blood mononuclear cells (PBMCs). Using saturation transfer difference (STD) and quantitative 1H nuclear magnetic resonance (NMR) experiments, we found that 2f, the N6-benzyladenosine analogue that includes a tertbutyl moiety in the para position of the benzyl ring, is endowed with increased FPPS binding and inhibition compared to the parent compounds i6A and 2. N6-benzyladenosine derivatives, characterized by structural features that are significantly different from those of N-BPs, have been confirmed to be promising chemical scaffolds for the development of non N-BP FPPS inhibitors, exerting combined cytotoxic and immunostimulatory activities.

High-resolution magic angle spinning nuclear magnetic resonance (HR-MAS-NMR) as quick and direct insight of almonds.

Almonds are the tasty seeds of Prunus dulcis plants globally appreciated for the pleasant palatability and remarkable nutritional value, therefore it is very spread as snack and as basic ingredient of the confectionery products. The HR-MAS-NMR is a simple spectroscopy able to directly and quickly explore the chemical composition of powdered seed samples dispersed in D2O. 1H spectra witness the remarkable presence of triglyceride fatty esters together with sucrose; other minor water soluble metabolites are also detectable. This very rough approach is effectively providing chemical profiles featuring almond samples. In this analysis we were able to statistically distinguish the "Avola" almonds from other marketed products submitted to the same analysis. This is just a first investigation based on the main compounds but it might pave the way toward the quantitative evaluation of many other compounds in the almond therefore implementing the HR-MAS-NMR knowledge of these precious seeds.

Structural basis of antiviral activity of peptides from MPER of FIV gp36.

Feline immunodeficiency virus (FIV) is a naturally occurring Lentivirus causing acquired immunodeficiency syndrome in felines. It is considered a useful non-primate model to study HIV infection, and to test anti-HIV vaccine. Similarly to HIV, FIV enters cells via a mechanism involving a surface glycoprotein named gp36. C8 is a short synthetic peptide corresponding to the residues 770WEDWVGWI777 of gp36 membrane proximal external region (MPER). It elicits antiviral activity by inhibiting the fusion of the FIV and host cell membrane. C8 is endowed with evident membrane binding property, inducing alteration of the phospholipid bilayer and membrane fusion. The presence and the position of tryptophan residues in C8 are important for antiviral activity: the C8 derivative C6a, obtained by truncating the N-terminal 770WE771 residues, exhibits conserved antiviral activity, while the C8 derivative C6b, derived from the truncation of the C-terminal 776WI777, is nearly inactive. To elucidate the structural factors that induce the different activity profiles of C6a and C6b, in spite of their similarity, we investigated the structural behaviour of the two peptides in membrane mimicking environments. Conformational data on the short peptides C6a and C6b, matched to those of their parent peptide C8, allow describing a pharmacophore model of antiviral fusion inhibitors. This includes the essential structural motifs to design new simplified molecules overcoming the pharmacokinetic and high cost limitations affecting the antiviral entry inhibitors that currently are in therapy.

Saliva of patients affected by salivary gland tumour: An NMR metabolomics analysis.

Cancers affecting the salivary glands have been an increasing incidence. Salivary gland cancer is not detected until it reaches an advanced stage, which would generally result in a poor prognosis and survival rate. Therefore, early detection as well as the screening of high risk populations with precancerous lesions remains an unmet medical need. In the present work, we present a NMR-based metabolomic study of the saliva of patients suffering from salivary gland tumours. Analysis of data was done using a combined approach based on PRICONA quantitative analysis and statistical multivariate analysis. Interestingly, both the analytical methods indicate that individuals affected by parotid tumour have a characteristic metabolomic profile characterized by abnormalities in the concentration of several aminoacids. Among these the most significant are those relative to Alanine and Leucine suggestive of an alteration in the metabolic pathways of glycogenic aminoacids and ketone bodies. Our data, describing the preliminary metabolomics fingerprint of parotid tumour, are consistent with the recent view that oncogenic signalling corresponds to alteration in the metabolism of nutrient pull (Vander Heiden et al., 2009), rather than to a single metabolite.

Circulating microRNAs are deregulated in overweight/obese children: preliminary results of the I.Family study.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs involved in the modulation of gene expression and in the control of numerous cell functions. Alterations of miRNA patterns frequently occur in cancer and metabolic disorders, including obesity. Recent studies showed remarkable stability of miRNAs in both plasma and serum making them suitable as potential circulating biomarkers for a variety of diseases and conditions. The aim of this study was to assess the profile of circulating miRNAs expressed in plasma samples of overweight or obese (OW/Ob) and normal weight (NW) prepubertal children from a European cohort (www.ifamilystudy.eu). The project, aimed to assess the determinants of eating behavior in children and adolescents of eight European countries, is built on the IDEFICS cohort (www.ideficsstudy.eu), established in 2006. Among the participants of the I.Family Italian Cohort, ten OW/Ob (age 10.7 ± 1.5 years, BMI 31.6 ± 4.3 kg/m(2)) and ten NW (age 10.5 ± 2.7 years, BMI 16.4 ± 1.7 kg/m(2)) children were selected for the study. Gene arrays were employed to differentially screen the expression of 372 miRNAs in pooled plasma samples. Deregulated miRNAs (p < 0.05) were further validated in the individual samples using a real-time PCR (RT-qPCR) approach. RESULTS: Using a significance threshold of p < 0.05 and a fold-change threshold of ± 4.0, we preliminarily identified in the pooled samples eight miRNAs that differed between the OW/Ob and NW groups. The validation by RT-qPCR in the individual plasma samples showed a twofold upregulation of miR-31-5p, a threefold upregulation of miR-2355-5p, and a 0.5-fold downregulation of miR-206 in OW/Ob as compared with NW. The molecular functions of these differentially expressed plasma miRNAs as well as their expected mRNA targets were predicted by bioinformatics tools. CONCLUSIONS: This pilot study shows that three circulating miRNAs are differentially regulated in OW/Ob as compared with NW children. Although causal pathways cannot be firmly inferred by these results, that deserve confirmation in larger samples, it is conceivable that circulating miRNAs may be novel biomarkers of obesity and related metabolic disturbances.

Celiac disease: role of intestinal compartments in the mucosal immune response.

Different approaches have been used to study the pattern of cytokines in celiac disease (CD). Laser capture microdissection (LCM) is a powerful tool for the isolation of specific tissue compartments. We aimed to investigate the mucosal immune response that takes place in different intestinal compartments of CD patients, dissected by LCM, analyzing cytokine expression profile. Frozen section of jejunum was obtained from 15 untreated CD and 15 control. Surface epithelium and lamina propria compartment were isolated by LCM. RNA from each LCM sample was extracted and, after a retrotranscription step, messenger RNA levels for MxA, IL-15, TNF-α, IFN-γ, IL-17α, IL-21, IL-10, and TGF-ß were determined by quantitative reverse transcriptase-PCR. Increased gene expression levels of MxA, IL-15, TNF-α, IL-10, and TGF-ß was observed in the surface epithelium of untreated CD with respect to control. Furthermore, all the cytokines investigated were upregulated in the lamina propria of untreated CD as compared to control. Within the untreated CD group the expression of IL-15 was higher, in the surface epithelium than in the lamina propria, whereas the expression levels of IL-17 and IL-21 were higher in the lamina propria than in the surface epithelium. Finally, high levels of IL-10 and TGF-ß were detected in both compartments of untreated CD biopsies. In CD, surface epithelium and lamina propria compartments, play a prominent role in determining innate and adaptive immunity, respectively. Conversely, surface epithelium and lamina propria produce high levels of anti-inflammatory cytokines, suggesting that both compartments are involved in the immunoregulatory response.

Nuclear aggregates of polyamines in a radiation-induced DNA damage model.

Polyamines (PA) are believed to protect DNA minimizing the effect of radiation damage either by inducing DNA compaction and aggregation or acting as scavengers of free radicals. Using an in vitro pDNA double strand breakage assay based on gel electrophoretic mobility, we compared the protective capability of PA against γ-radiation with that of compounds generated by the supramolecular self-assembly of nuclear polyamines and phosphates, named Nuclear Aggregates of Polyamines (NAPs). Both unassembled PA and in vitro produced NAPs (ivNAPs) were ineffective in conferring pDNA protection at the sub-mM concentration. Single PA showed an appreciable protective effect only at high (mM) concentrations. However, concentrations of spermine (4+) within a critical range (0.481 mM) induced pDNA precipitation, an event that was not observed with NAPs-pDNA interaction. We conclude that the interaction of individual PA is ineffective to assure DNA protection, simultaneously preserving the flexibility and charge density of the double strand. Furthermore, data obtained by testing polyamine and ivNAPS with the current radiation-induced DNA damage model support the concept that PA-phosphate aggregates are the only forms through which PA interact with DNA.