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Abstract: In recent years, the scientific community has been trying to tackle different diseases by using unifying and holistic approaches based on the concept that it is possible to target apparently very different diseases under a comprehensive general scheme. In other words, various different diseases have been grouped together under the label of “conformational diseases”, because the triggering cause for each malady is the misfolding of a specific protein, whose dyshomeostasis and accumulation cause all the other downhill biomolecular events characteristic of each different disease. In a parallel manner, analytical techniques have developed to investigate protein misfolding and accumulation, so as to give a valid technical support to the investigation of conformational diseases. In this scenario, surface plasmon resonance (SPR) has widely contributed to study many different aspects correlated to conformational diseases, offering the advantages of real time investigations, use of small amounts of biological materials and possibility to mimic the cellular environments without recurring to the use of fluorescent tags. In this review, after a brief introduction about conformational diseases and the SPR technique, a thorough description of the various uses of SPR to investigate the biomolecular mechanisms involved in these diseases is given in order to provide the reader with an exhaustive list as well as a critical perspective of the use of SPR for such topic. The case of Alzheimer’s disease is discussed at a deeper level. We hope that this work will make the reader aware of all the possible SPR experimental approaches, which can be used to develop new possible therapeutic strategies to tackle conformational diseases.

Surface functionalization with biological molecules, such as peptides or proteins, is a very promising method for developing new biomaterials with many potential applications. However, due to their chemical complexity, the characterization of biological materials is often a very challenging task. In this context, time-of-flight secondary ion mass spectrometry is a very helpful characterization tool due to its ability to provide very detailed spatially resolved chemical information of the topmost layer. The peculiar emission/ion formation mechanisms involved in ToF-SIMS analysis often do not allow the detection of the molecular ion of proteins and peptides, providing a rich fragmentation pattern, which is difficult to be related to the surface composition using a univariate approach, due to the relevant number of peaks in the SIMS spectra of peptides and proteins and the slight differences in intensities between different samples. Therefore, we used multivariate analysis to extract the information contained in the ToF-SIMS spectra of four peptides with high amino acid sequence similarity along the peptide chain. The reference peptide (TAT1) is a 12-unit sequence of six amino acids (GRKKRRQRRRPS). The other three peptides have been obtained by inserting a bAla-H dipeptide (carnosine) in three different positions inside the TAT1 chain, namely, GRKKRRQRRRPS-bAla-H (TAT1-Car), bAla-HGRKKRRQRRRPS (Car-TAT1), and GRKKRRQ-bAla-H-RRRPS (T-Car-T). We show that these peptides can be distinguished by ToF-SIMS combined with multivariate data analysis.

The diffuse and renewed use of silver as antimicrobial agent has caused the development of resistance to silver ions in some bacterial strains, posing a serious threat for health systems. In order to cast light on the mechanistic features of resistance, here, we aimed to understand how silver interacts with the periplasmic metal-binding protein SilE which is engaged in bacterial silver detoxification. This aim was addressed by studying two peptide portions of SilE sequence (SP2 and SP3) that contain the putative motifs involved in Ag+ binding. We demonstrate that SP2 model peptide is involved in silver binding through its histidine and methionine residues in the two HXXM binding sites. In particular, the first binding site is supposed to bind the Ag+ ion in a linear fashion, while the second binding site complexes the silver ion in a distorted trigonal planar fashion. We propose a model where the SP2 peptide binds two silver ions when the concentration ratio Ag+/SP2 is ≥10.0. We also suggest that the two binding sites of SP2 have different affinity for silver. This evidence comes from the change in the path direction of the Nuclear Magnetic Resonance (NMR) cross-peaks upon the addition of Ag+. Here, we report the conformational changes of SilE model peptides occurring upon silver binding, monitored at a deep level of molecular details. This was addressed by a multifaceted approach, combining NMR, circular dichroism, and mass spectrometry experiments.

The possibility to monitor peptide and protein aggregation is of paramount importance in the so-called conformational diseases, as the understanding of many physiological pathways, as well as pathological processes involved in the development of such diseases, depends very much on the actual possibility to monitor biomolecule oligomeric distribution and aggregation. In this work, we report a novel experimental method to monitor protein aggregation, based on the change of the fluorescent properties of carbon dots upon protein binding. The results obtained in the case of insulin with this newly proposed experimental approach are compared with those obtained with other common experimental techniques normally used for the same purpose (circular dichroism, DLS, PICUP and ThT fluorescence). The greatest advantage of the hereby presented methodology over all the other experimental methods considered is the possibility to monitor the initial stages of insulin aggregation under the different experimental conditions sampled and the absence of possible disturbances and/or molecular probes during the aggregation process. Graphical abstract

l-Carnosine is an endogenous dipeptide that has high potential for therapeutic purposes, being an antioxidant with metal chelating, anti-aggregating, anti-inflammatory, and neuroprotective properties. Despite its potential therapeutic values, the biomolecular mechanisms involved in neuroprotection are not fully understood. Here, we demonstrate, at chemical and biochemical levels, that insulin-degrading enzyme plays a pivotal role in carnosine neuroprotection.

Insulin-degrading enzyme (IDE) is a highly conserved zinc metallopeptidase and is capable to catalytically cleave several substrates besides insulin, playing a pivotal role in several different biochemical pathways. Although its mechanism of action has been widely investigated, many conundrums still remain, hindering the possibility to rationally design specific modulators which could have important therapeutical applications in several diseases such as diabetes and Alzheimer's disease. In this scenario, we have developed a novel surface plasmon resonance (SPR) method which allows for directly measuring the enzyme cooperativity for the binding of insulin in the presence of different IDE activity modulators: carnosine, ATP, and EDTA. Results indicate that both positive and negative modulations of the IDE activity can be correlated to an increase and a decrease of the measured Hill coefficient, respectively, giving a new insight into the IDE activity mechanism. The use of the IDE R767A mutant for which oligomerization is hindered confirmed that the positive allosteric modulation of IDE by carnosine is due to a change in the enzyme oligomeric state occurring also for the enzyme immobilized on the gold SPR chip.

In this study, the cyclic compression and crush behavior of chiral auxetic lattice structures produced from titanium alloy (Ti6Al4V) metallic powder using electron beam melting (EBM) additive manufacturing technology is investigated numerically and experimentally. For material characterization and understanding the material behavior of EBM printed parts, tensile and three-point flexural tests were conducted. Log signals produced during the EBM process were investigated to confirm the stability of process and the health of the produced parts. Furthermore, a compressive cyclic load profile was applied to the EBM printed chiral units having two different thicknesses to track their Poisson’s ratios and displacement limits under large displacements in the absence of degradation, permanent deformations and failures. Chiral units were also crushed to investigate the effect of failure and deformation mechanisms on the energy absorption characteristics. Moreover, a surface roughness study was conducted due to high surface roughness of EBM printed parts, and an equation is offered to define load-carrying effective areas to prevent misleading cross-section measurements. In compliance with the equation and tensile test results, a constitutive equation was formed and used after a selection and calibration process to verify the numerical model for optimum topology design and mechanical performance forecasting using a non-linear computational model with failure analysis. As a result, the cyclic compression and crush numerical analyses of EBM printed Ti6Al4V chiral cells were validated with the experimental results. It was shown that the constitutive equation of EBM printed as-built parts was extracted accurately considering the build orientation and surface roughness profile. Besides, the cyclic compressive and crush behavior of chiral units were investigated. The regions of the chiral units prone to prematurely fail under crush loads were determined, and deformation modes were investigated to increase the energy absorption abilities.

Dipyridamole is currently used as a medication that inhibits blood clot formation and it is also investigated in the context of neurodegenerative and other amyloid related diseases. Here, we propose this molecule as a new diagnostic tool to follow the aggregation properties of three different amyloidogenic proteins tested (insulin, amylin and amyloid β peptide 1-40). Results show that dipyridamole is sensitive to early stage amyloid formation undetected by thioflavin T, giving a different response for the aggregation of the three different proteins. In addition, we show that dipyridamole is also able to enhance ubiquitin chain growth, paving the way to its potential application as therapeutic agent in neurodegenerative diseases.

Aβ (1-40) can transfer from the aqueous phase to the bilayer and thus form stable ion-channel-like pores where the protein has alpha-helical conformation. The stability of the pores is due to the presence of the GXXXG motif. It has been reported that these ion-channel-like pores are stabilized by a Cα―H···O hydrogen bond that is established between a glycine of the GXXXG sequence of an alpha-helix and another amino acid of a vicinal alpha-helix. However, conflicting data are reported in the literature. Some authors have suggested that hydrogen bonding does not have a stabilizing function. Here we synthesized pentapeptides having a GXXXG motif to explore its role in pore stability. We used molecular dynamics simulations, quantum mechanics, and experimental biophysical techniques to determine whether hydrogen bonding was formed and had a stabilizing function in ion-channel-like structures. Starting from our previous molecular dynamics data, molecular quantum mechanics simulations, and ATR data showed that a stable ion-channel-like pore formed and a band centered at 2910 cm−1 was attributed to the interaction between Gly 7 of an alpha-helix and Asp 23 of a vicinal alpha-helix.

Ubiquitin Proteasome System ( UPS) is an adaptable and finely tuned system that sustains proteostasis network under a large variety of physiopathological conditions. Its dysregulation is often associated with the onset and progression of human diseases; hence, UPS modulation has emerged as a promising new avenue for the development of treatments of several relevant pathologies, such as cancer and neurodegeneration. The clinical interest in proteasome inhibition has considerably increased after the FDA approval in 2003 of bortezomib for relapsed/refractory multiple myeloma, which is now used in the front-line setting. Thereafter, two other proteasome inhibitors (carfilzomib and ixazomib), designed to overcome resistance to bortezomib, have been approved for treatment-experienced patients, and a variety of novel inhibitors are currently under preclinical and clinical investigation not only for haematological malignancies but also for solid tumours. However, since UPS collapse leads to toxic misfolded proteins accumulation, proteasome is attracting even more interest as a target for the care of neurodegenerative diseases, which are sustained by UPS impairment. Thus, conceptually, proteasome activation represents an innovative and largely unexplored target for drug development. According to a multidisciplinary approach, spanning from chemistry, biochemistry, molecular biology to pharmacology, this review will summarize the most recent available literature regarding different aspects of proteasome biology, focusing on structure, function and regulation of proteasome in physiological and pathological processes, mostly cancer and neurodegenerative diseases, connecting biochemical features and clinical studies of proteasome targeting drugs. (C) 2020 Elsevier Inc. All rights reserved.

Introduced in 2019, the Reddito di cittadinanza (RdC) has replaced the Reddito di inclusione (ReI) as a universal minimum income scheme in Italy. In this paper, we use BIMic, the Bank of Italy’s static (non-behavioural) microsimulation model, to measure the effects of the RdC in terms of inequality reduction and, as a novel contribution, of absolute poverty alleviation. Our results, which do not account for behavioural responses to policy changes, show that the RdC is effective in reducing inequality, and attenuating the incidence, and even more so the intensity, of absolute poverty. We also document how certain features of the design of this benefit affect the distribution of these effects across the population. For this purpose, we simulate two hypothetical changes to the current design of the RdC: one that directs more resources to large households with minors (on average more in need than other households) and the other that takes into account the differences in the cost of living according to geographical areas and municipality size.

Rett Syndrome (RTT) is a rare X-linked neurodevelopmental disorder which affects about 1: 10000 live births. In >95% of subjects RTT is caused by a mutation in Methyl-CpG binding protein-2 (MECP2) gene, which encodes for a transcription regulator with pleiotropic genetic/epigenetic activities. The molecular mechanisms underscoring the phenotypic alteration of RTT are largely unknown and this has impaired the development of therapeutic approaches to alleviate signs and symptoms during disease progression. A defective proteasome biogenesis into two skin primary fibroblasts isolated from RTT subjects harbouring non-sense (early-truncating) MeCP2 mutations (i.e., R190fs and R255X) is herewith reported. Proteasome is the proteolytic machinery of Ubiquitin Proteasome System (UPS), a pathway of overwhelming relevance for post-mitotic cells metabolism. Molecular, transcription and proteomic analyses indicate that MeCP2 mutations down-regulate the expression of one proteasome subunit, ?7, and of two chaperones, PAC1 and PAC2, which bind each other in the earliest step of proteasome biogenesis. Furthermore, this molecular alteration recapitulates in neuron-like SH-SY5Y cells upon silencing of MeCP2 expression, envisaging a general significance of this transcription regulator in proteasome biogenesis.

Since the inception of membrane-based CO2 separation technology in the late 1970s, a large number of material studies have been performed to improve the material properties of membrane materials and tremendous progress has been made with regard to CO2 separation to increase the trade-off between permeability and selectivity, known as the “Robeson upper bound.” In membrane science, material properties and membrane properties are related but completely different, especially in thin-film composite (TFC) membranes. Compared to the large number of material studies, few studies have been performed on membrane properties, and very few membranes have been successfully commercialized because an extremely thin selective layer is required in CO2/N2 separation membranes for high CO2 permeance; however, defects are easily generated, deteriorating gas transport properties on a large-scale. In this study, we theoretically analyzed the effect of defects on the gas transport properties of a TFC membrane and introduced a method to control defects during the preparation of large-scale membranes. This work will provide a facile and practical approach to preparing TFC membranes and will be helpful toward extending material studies to real membrane demonstrations.

The interaction of insulin-degrading enzyme (IDE) with the main intracellular proteasome assemblies (i.e, 30S, 26S and 20S) was analyzed by enzymatic activity, mass spectrometry and native gel electrophoresis. IDE was mainly detected in association with assemblies with at least one free 20S end and biochemical investigations suggest that IDE competes with the 19S in vitro. IDE directly binds the 20S and affects its proteolytic activities in a bimodal fashion, very similar in human and yeast 20S, inhibiting at (IDE) 30 nM and activating at (IDE) 30 nM. Only an activating effect is observed in a yeast mutant locked in the "open" conformation (i.e., the alpha-3 Delta N 20S), envisaging a possible role of IDE as modulator of the 20S "open"-"closed" allosteric equilibrium. Protein-protein docking in silico proposes that the interaction between IDE and the 20S could involve the C-term helix of the 20S alpha-3 subunit which regulates the gate opening of the 20S.

The actual surface termination and lateral size of a nanomaterial is crucial in its interaction with biomolecules at the aqueous interface. Graphene oxide (GO) nanosheets have been demonstrated as promising nanoplatform for both diagnostic and therapeutic applications. To this respect, 'smart' GO nanocarriers have been obtained by the surface functionalisation with polymers sensitive, e.g., to pH, as the polyacrylate (PAA) case. In this work, hybrid GO/PAA samples prepared respectively at low (GOPAAthin) or high (GOPAAthick) monomer grafting ratio, were scrutinised both theoretically, by molecular dynamic calculations, and experimentally by a multitechnique approach, including spectroscopic (UV-visible, fluorescence, Raman, Attenuated-total reflectance-Fourier transformed infrared and X-ray photoelectron spectroscopies), spectrometric (time-of-flight secondary ion and electrospray ionisation mass spectrometries) and microscopic (atomic force and confocal microscopies) methods. The actual surface termination, evaluated in terms of the relative ratio between polar and dispersive groups at the surface of the GO/polymer systems, was found to correlate with the average orientation of hydrophilic/hydrophobic domains of albumin, used as model protein. Moreover, the comparison among GO, GO-PAAthin and GO-PAAthick in the optical response at the interface with aqueous solutions, both at acid and at physiological pH, showed that the hybrid GO-polymer platform could be suitable not only to exploit a pH-triggered drug release but also for a modulation of the GO intrinsic emission properties. Energy transfer experiments on the GO/polymer oxide/fluorescein-labelled albumin/doxorubicin assembly showed significant differences for GO and GO-PAA samples, thus demonstrating the occurrence of different electronic processes at the hybrid nano-bio-interfaces. Confocal microscopy studies of cellular uptake in neuroblastoma cells confirmed the promising potentialities of the developed nanoplatform for applications at the biointerface.

Defective DNA damage response (DDR) is frequently associated with tumorigenesis. Abrogation of DDR leads to genomic instability, which is one of the most common characteristics of human cancers. TP53 mutations with gain-of-function activity are associated with tumors under high replicative stress, high genomic instability, and reduced patient survival. The BRCA1 and RAD17 genes encode two pivotal DNA repair proteins required for proper cell-cycle regulation and maintenance of genomic stability. We initially evaluated whether miR-205-5p, a microRNA (miRNA) highly expressed in head and neck squamous cell carcinoma (HNSCC), targeted BRCA1 and RAD17 expression. We found that, in vitro and in vivo, BRCA1 and RAD17 are targets of miR-205-5p in HNSCC, leading to inefficient DNA repair and increased chromosomal instability. Conversely, miR-205-5p downregulation increased BRCA1 and RAD17 messenger RNA (mRNA) levels, leading to a reduction in in vivo tumor growth. Interestingly, miR-205-5p expression was significantly anti-correlated with BRCA1 and RAD17 targets. Furthermore, we documented that miR-205-5p expression was higher in tumoral and peritumoral HNSCC tissues than non-tumoral tissues in patients exhibiting reduced local recurrence-free survival. Collectively, these findings unveil miR-205-5p&rsquo, s notable role in determining genomic instability in HNSCC through its selective targeting of BRCA1 and RAD17 gene expression. High miR-205-5p levels in the peritumoral tissues might be relevant for the early detection of minimal residual disease and pre-cancer molecular alterations involved in tumor development.

Background Over the past decade, newly designed cancer therapies have not significantly improved the survival of patients diagnosed with Malignant Pleural Mesothelioma (MPM). Among a limited number of genes that are frequently mutated in MPM several of them encode proteins that belong to the HIPPO tumor suppressor pathway. Methods The anticancer effects of the top flower standardized extract of Filipendula vulgaris (Dropwort) were characterized in “in vitro” and “in vivo” models of MPM. At the molecular level, two “omic” approaches were used to investigate Dropwort anticancer mechanism of action: a metabolomic profiling and a phosphoarray analysis. Results We found that Dropwort significantly reduced cell proliferation, viability, migration and in vivo tumor growth of MPM cell lines. Notably, Dropwort affected viability of tumor-initiating MPM cells and synergized with Cisplatin and Pemetrexed in vitro. Metabolomic profiling revealed that Dropwort treatment affected both glycolysis/tricarboxylic acid cycle as for the decreased consumption of glucose, pyruvate, succinate and acetate, and the lipid metabolism. We also document that Dropwort exerted its anticancer effects, at least partially, promoting YAP and TAZ protein ubiquitination. Conclusions Our findings reveal that Dropwort is a promising source of natural compound(s) for targeting the HIPPO pathway with chemo-preventive and anticancer implications for MPM management. Electronic supplementary material The online version of this article (10.1186/s13046-019-1352-3) contains supplementary material, which is available to authorized users.

Appetite for ubiquitin: a gushy travel companion in the intracellular journey of the amyloid β peptide., Several lines of evidence point to a compromised proteostasis associated with a reduction of the Ubiquitin Proteasome System (UPS) activity in patients affected by Alzheimer's Disease (AD) and suggest that the amyloid β peptide (Aβ) is an important player in the game. Inspired also by many reports, underlining the presence of ubiquitin (Ub) in the amyloid plaques of AD brains, here we set out to test whether Ub may bind the Aβ peptide and have any effect on its clearance pathways. By using an integrated array of MALDI-TOF/UPLC-HRMS, fluorescence, NMR, SPR, Microscale Thermophoresis (MST) and molecular dynamics studies, we consistently demonstrated that Aβ40 binds Ub with a 1 : 1 stoichiometry and Kd in the high micromolar range. In particular, we show that the N-terminal domain of the Aβ peptide (through residues D1, E3 and R5) interacts with the C-terminal tail of Ub (involving residues K63 and E64), inducing the central region of Aβ (14HQKLVFFAEDVGSNK28) to adopt a mixed α-helix/β-turn structure. ELISA assays, carried out in neuroblastoma cell lysates, suggest that Aβ competitively binds Ub also in the presence of the entire pool of cytosolic Ub binding proteins. Ub-bound Aβ has a lower tendency to aggregate into amyloid-like fibrils and is more slowly degraded by the Insulin Degrading Enzyme (IDE). Finally, we observe that the water soluble fragment Aβ1–16 significantly inhibits Ub chain growth reactions. These results evidence how the non-covalent interaction between Aβ peptides and Ub may have relevant effects on the regulation of the upstream events of the UPS and pave the way to future in vivo studies addressing the role played by Aβ peptide in the malfunction of proteome maintenance occurring in AD.

When lipid membranes containing ω-6 polyunsaturated fatty acyl chains are subjected to oxidative stress, one of the reaction products is 4-hydroxy-2-nonenal (HNE)-a chemically reactive short chain alkenal that can covalently modify proteins. The ubiquitin proteasome system is involved in the clearing of proteins modified by oxidation products such as HNE, but the chemical structure, stability and function of ubiquitin may be impaired by HNE modification. To evaluate this possibility, the susceptibility of ubiquitin to modification by HNE has been characterized over a range of concentrations where ubiquitin forms non-covalent oligomers. Results indicate that HNE modifies ubiquitin at only two of the many possible sites, and that HNE modification at these two sites alters the ubiquitin oligomerization equilibrium. These results suggest that any role ubiquitin may have in clearing proteins damaged by oxidative stress may itself be impaired by oxidative lipid degradation products. Copyright © 2016 John Wiley & Sons, Ltd.

Despite the enormous number of therapeutic advances in medicine, nowadays many diseases are still incurable, mainly due to the lack of knowledge of the pathological biochemical pathways triggering those diseases. For this reason, it is compulsory for the scientific community to investigate and unveil the biomolecular mechanisms responsible for the development of those diseases, such as Alzheimer’s disease and diabetes, which are widespread all over the world. In this scenario, it is of paramount importance to develop new analytical techniques and experimental procedures that are capable to make the above-mentioned investigations feasible. These new methods should allow easy performable analysis carried out in a label-free environment, in order to give reliable answers to specific biochemical questions. A recent paper published on Bioscience Reports by Ivancic et al. (https://doi.org/10.1042/BSR20181416) proposes a new analytical technique capable to reveal some mechanistic insights into the regulation of insulin-degrading enzyme (IDE), a protein involved in the above-mentioned diseases. IDE is a multifaceted enzyme having different and not well-defined roles in the cell, but it is primarily a proteolytic enzyme capable to degrade several different amyloidogenic substrates involved in different diseases. Moreover, many molecules are responsible for IDE activity modulation so that understanding how IDE activity is regulated represents a very challenging analytical task. The new analytical approach proposed by Ivancic et al. reports on the possibility to study IDE activity in an unbiased and label-free manner, representing a valid alternative assay for the investigation of any proteases degradative activity.

Invited for this month's cover are the collaborating groups at the University of Calabria (Unical, Italy)-Department of Chemistry and Chemical Technologies (CTC) and Department of Pharmacy and Health and Nutritional Sciences (PHNS), at the National Research Council (CNR, Italy), Institute on Membrane Technology (ITM), and at the Karlsruhe University of Applied Sciences (HSKA, Germany), Institute of Applied Research (IAR), which cooperated in the framework of the European Union's Horizon 2020 project "VicInAqua". The front cover shows the structure of an acryloxyalkyltrithylammonium (AATEA) cation, characterized by a polymerizable end, a long-chain alkyl linker, and a quaternary ammonium moiety. The bromide salts (AATEABs) have been synthesized by a two-step procedure carried out entirely under air without any need for chromatographic purifications. Some AATEABs have shown significant antimicrobial activity and represent useful precursors for the preparation of polymeric films with antimicrobial properties. Read the full text of the article at 10.1002/cplu.201700194.

The AMP-activated protein kinase (AMPK) is a heterotrimeric protein complex that is an important sensor of cellular energy status. Reduced expression of the AMPK β1 isoform has been linked to reduced survival in different cancers, but whether this accelerates tumor progression and the potential mechanism mediating these effects are not known. Furthermore, it is unknown whether AMPK β1 is implicated in tumorigenesis, and if so, what tissues may be most sensitive. In the current study, we find that in the absence of the tumor suppressor p53, germline genetic deletion of AMPK β1 accelerates the appearance of a T-cell lymphoma that reduces lifespan compared to p53 deficiency alone. This increased tumorigenesis is linked to increases in interleukin-1β (IL1β), reductions in acetyl-CoA carboxylase (ACC) phosphorylation, and elevated lipogenesis. Collectively, these data indicate that reductions in the AMPK β1 subunit accelerate the development of T-cell lymphoma, suggesting that therapies targeting this AMPK subunit or inhibiting lipogenesis may be effective for limiting the proliferation of p53-mutant tumors.

Insulin-degrading enzyme (IDE) is a ubiquitous zinc peptidase of the inverzincin family, which has been initially discovered as the enzyme responsible for insulin catabolism; therefore, its involvement in the onset of diabetes has been largely investigated. However, further studies on IDE unraveled its ability to degrade several other polypeptides, such as β-amyloid, amylin, and glucagon, envisaging the possible implication of IDE dys-regulation in the “aggregopathies” and, in particular, in neurodegenerative diseases. Over the last decade, a novel scenario on IDE biology has emerged, pointing out a multi-functional role of this enzyme in several basic cellular processes. In particular, latest advances indicate that IDE behaves as a heat shock protein and modulates the ubiquitin–proteasome system, suggesting a major implication in proteins turnover and cell homeostasis. In addition, recent observations have highlighted that the regulation of glucose metabolism by IDE is not merely based on its largely proposed role in the degradation of insulin in vivo. There is increasing evidence that improper IDE function, regulation, or trafficking might contribute to the etiology of metabolic diseases. In addition, the enzymatic activity of IDE is affected by metals levels, thus suggesting a role also in the metal homeostasis (metallostasis), which is thought to be tightly linked to the malfunction of the “quality control” machinery of the cell. Focusing on the physiological role of IDE, we will address a comprehensive vision of the very complex scenario in which IDE takes part, outlining its crucial role in interconnecting several relevant cellular processes.

Neoadjuvant chemotherapy has been successfully tested in several bulky solid tumors, but it has not been utilized in advanced cutaneous melanoma, primarily because effec- tive medical treatments for this disease have been lacking. However, with the development of new immunotherapies (monoclonal antibodies specific for cytotoxic T lymphocyte-associated antigen 4 (anti-CTLA-4) and programmed death protein-1 (anti-PD1)) and small molecules interfering with intracellular pathways (anti-BRAF and mitogen-activated protein kinase kinase (anti- MEK)) the use of this approach is becoming a viable treatment strategy for locally advanced melanoma. The neoadjuvant setting provides a double opportunity for a better knowledge of these drugs: a short-term evaluation of their intrinsic activity, and a deeper analysis of their action and resistance-induction mechanisms. BRAF inhibitors seem to be ideal candidates for the neoadjuvant setting, because of their prompt, repeatedly confirmed response in V600E BRAF-mutant metastatic melanoma. In this report we summarize studies focused on the neo- adjuvant use of traditional medical treatments in advanced melanoma and anecdotal cases of this approach with the use of biologic therapies. Moreover, we discuss our experience with neoadjuvant targeted therapy as a priming for radical surgery in a patient with BRAF V600E mutation-positive advanced melanoma.

Melatonin has been known to be a chemopreventive agent since its levels inversely correlate with the risk of developing cancer. We have recently shown that melatonin induces p38-dependent phosphorylation of both p53 and histone H2AX. This is associated with a p53-mediated increase in repair of both endogenous and chemotherapy-induced DNA damage. In addition, the inhibition of p38 activities impairs melatonin's capability to induce a p53-dependent DNA damage response and thus its ability to maintain genome integrity. Since melatonin-induced p53 phosphorylation requires an intact p38 phosphorylation cascade and p38 can be activated by G proteins, we supposed that melatonin's activities could be mediated by its G-protein-coupled membrane receptors, MT1 and MT2. Here, we show that the activation of the p53-dependent DNA damage response by melatonin is indeed mediated by MT1 and MT2. As a result, the absence of either receptor impairs melatonin's ability to reduce both cell proliferation and clonogenic potential of cancer cells. In addition, this causes an impairment of the p53-dependent DNA damage response. By providing molecular insight, our findings might have translational impact, suggesting the involvement of melatonin receptors in tumorigenesis.

The study of metal-protein interactions is an expanding field of research investigated by bioinorganic chemists as it has wide applications in biological systems. Very recently, it has been reported that it is possible to study metal-protein interactions by immobilizing biomolecules on metal surfaces and applying experimental approaches based on plasmonics which have usually been used to investigate protein-protein interactions. This is possible because the electronic structure of metals generates plasmons whose properties can be exploited to obtain information from biomolecules that interact not only with other molecules but also with ions in solution. One major challenge of such approaches is to immobilize the protein to be studied on a metal surface with preserved native structure. This review reports and discusses all the works that deal with such an expanding new field of application of plasmonics with specific attention to surface plasmon resonance, highlighting the advantages and drawbacks of such approaches in comparison with other experimental techniques traditionally used to study metal-protein interactions.

Accumulation of neurotoxic amyloid-beta peptide (A beta) and alteration of metal homeostasis (metallostasis) in the brain are two main factors that have been very often associated with neurodegenerative diseases, such as Alzheimer's disease (AD). A beta is constantly produced from the amyloid-precursor-protein APP precursor and immediately catabolized under normal conditions, whereas dysmetabolism of A beta and/or metal ions seems to lead to a pathological deposition. Although insulin-degrading enzyme (IDE) is the main metalloprotease involved in A beta degradation in the brain being up-regulated in some areas of AD brains, the role of IDE for the onset and development of AD is far from being under-stood. Moreover, the biomolecular mechanisms involved in the recognition and interaction between IDE and its substrates are still obscure. In spite of the important role of metals (such as copper, aluminum, and zinc), which has brought us to propose a "metal hypothesis of AD", a targeted study of the effect of metallostasis on IDE activity has never been carried out. In this work, we have investigated the role that various metal ions (i.e., Cu2+, Cu+, Zn2+, Ag+, and Al3+) play in modulating the interaction between IDE and two A beta peptide fragments, namely A beta(1-16) and A beta(16-28). It was therefore possible to identify the direct effect that such metal ions have on IDE structure and enzymatic activity without interferences caused by metal-induced substrate modifications. Mass spectrometry and kinetic studies revealed that, among all the metal ions tested, only Cu2+, Cu+, and Ag+ have an inhibitory effect on IDE activity. Moreover, the inhibition of copper(II) is reversed by adding zinc(II), whereas the monovalent cations affect the enzyme activity irreversibly. The molecular basis of their action on the enzyme is also discussed on the basis of computational investigations.

To evaluate prostate cancer (PCa) detection after repeated negative saturation biopsy, 75 patients, aged 53-78 years, underwent transurethral resection of prostate (TURP) because of persistent suspicion of cancer; median PSA was 11.8 ng ml(-1) and 58 men complained lower urinary tract symptoms (LUTS). In 12 (16%) and 3 (4%) men a T1a and T1b PCa was found with median PSA and Gleason score equal to 14.2 vs 23.6 ng ml(-1) and 5.6 vs 7 ng ml(-1). In case of persistent suspicion of PCa after repeated negative saturation biopsy, TURP may be proposed, aside from the coexistence of LUTS, to rule out a PCa, in younger patients with high PSA values (or =20 ng ml(-1)).

Amyloid β-peptides (Aβs) are involved in several neuropathological conditions such as Alzheimer's disease and considerable experimental evidences have emerged indicating that different proteases play a major role in regulating the accumulation of Aβs in the brain. Particularly, insulin-degrading enzyme (IDE) has been shown to degrade Aβs at different cleavage sites, but the experimental results reported in the literature and obtained by mass spectrometry methods are somehow fragmentary. The detection of Aβs is often complicated by solubility issues, oxidation artifacts and spontaneous aggregation/cleavage and, in order to rationalize the different reported results, we analyzed Aβs solutions by three different MS approaches: matrix assisted laser desorption ionization-time of flight (MALDI-TOF), atmospheric pressure (AP) MALDI ion trap and electrospray ionization (ESI) ion trap. Differences in the obtained results are discussed and ESI is chosen as the most suitable MS method for Aβs detection. Finally, cleavage sites produced by interaction of Aβs with IDE are identified, two of which had never been reported in the literature.

Matrix metalloproteinases (MMPs) are cell-secreted soluble and membrane-tethered enzymes that are capable of degrading extracellular matrix proteins, but also can process a number of bioactive molecules. They are involved in the cleavage of cell surface receptors, but are also thought to play a major role on cell behaviors as well as in diverse physiological and pathological processes, including embryonic development, wound repair, inflammatory diseases, and cancer. For these reasons, it is obvious that a control over MMPs activity is highly desirable. Consequently, the frantic search for new inhibitors has been coupled to the development of high-throughput methods able to rapidly screen the effect of possible MMP inhibitors on the activity of these enzymes. In this scenario, solid-state-based methods play a major role because of their compatibility with array formats that are able to extract more information from smaller sample volumes and offer some important advantages that are not available in the standard solution assays. In this work, the catalytic domain of MMP-12 was immobilized on a gold substrate and the surface coverage was measured by FT-SPR experiments. A new experimental procedure was developed to freeze-dry the anchored molecules and their activity was measured by ESI-MS. The kinetics parameters obtained for the immobilized enzyme are in good accordance with those reported for similar systems in solution. Inhibition of the immobilized molecules was also carried out, demonstrating the applicability of the method for rapid screening of MMP inhibitors.

Carbon-13 MAS refocused INADEQUATE experiments have been carried out on the polymer phase of CsC 6 0 and provide a complete and unambiguous assignment of the high-resolution carbon-13 MAS spectrum. This allows experimental Knight shifts for each carbon site on the fullerene molecule to be compared with calculated hyperfine coupling constants. The comparison supports a model for the electronic structure which predicts high electron density away from the linkages between fullerene molecules with electron transport dominated by interchain hopping.

Thioflavin T (ThioT) fluorescence is commonly used to quantify amyloid formation in vivo and in vitro, but its binding mode and basis for fibril selectivity is not understood, and there is no consensus on the relationship between fluorescence intensity and fibril mass. To determine whether the rate of fibril formation affected ThioT fluorescence, fibrils of amyloid beta (Aβ) proteins were formed at different concentrations of the monomeric protein. The intensity of ThioT fluorescence increased with the monomer concentration during fibril formation. A similar dependence on monomer concentration was observed for insulin fibrils. The affinity of the dye for Aβ fibrils formed at different rates was assessed by surface-plasmon resonance (SPR), indicating that fibrils grown from more concentrated monomer solutions bound larger amounts of ThioT per mass of Aβ, consistent with the steady-state fluorescence results. However, the dissociation constants and the affinity of individual binding sites were independent of the monomer concentration of Aβ proteins. The orientation of ThioT in Aβ fibrils was evaluated with polarized fluorescence microscopy, and found to be aligned toward the long axis of the fibril. On the basis of these results, it is proposed that ThioT binds to localized polymorphic sites along the length of a fibril, rather than to well-defined binding sites in a mature fibril with regular structure.

Amylin is a 37-residue peptide hormone produced by the islet beta-cells of pancreas and the formation of amylin aggregates is strongly associated with beta-cell degeneration in type 2 diabetes, as demonstrated by more than 95% of patients exhibiting amylin amyloid upon autopsy. It is widely recognized that metal ions such as copper(II) have been implicated in the aggregation process of amyloidogenic peptides such as Ab and alpha-synuclein and there is evidence that amylin self-assembly is also largely affected by copper(II). For this reason, in this work, the role of copper(II) in the aggregation of amylin has been investigated by several different experimental approaches. Mass spectrometric investigations show that copper(II) induces significant changes in the amylin structure, which decrease the protein fibrillogenesis as observed by ThT measurements. Accordingly, solid-state NMR experiments together with computational analysis carried out on a model amylin fragment confirmed the non-fibrillogenic nature of the copper(II) induced aggregated structure. Finally, the presence of copper(II) is also shown to have a major influence on amylin proneness to be degraded by proteases and cytotoxicity studies on different cell cultures are reported.

We review polarization splitting and rotating photonic devices based on silicon-on-insulator technology platform, focusing on their performance and design criteria. In addition, we present a theoretical investigation and optimization of some rotator and splitter architectures to be employed for polarization diversity circuits. In this context, fabrication tolerances and their influences on device performance are theoretically estimated by rigorous simulations too.

The term “cryptome” refers to the subset of cryptic peptides with bioactivities that are often unpredictable and very different from the parent protein. These cryptic peptides are generated by proteolytic cleavage of proteases, whose identification in vivo can be very challenging. In this work, we show that insulin-degrading enzyme (IDE) is able to degrade specific amino acid sequences present in the neuropeptide pro-NPFFA (NPFF precursor), generating some cryptic peptides that are also observed after incubation with rat brain cortex homogenate. The reported experimental findings support the increasingly accredited hypothesis, according to which, due to its wide substrate selectivity, IDE is involved in a wide variety of physiopathological processes.

Thyroid carcinomas, even when well differentiated, usually appear as hypofunctioning at scintigraphy. We report a case of an aggressive insular thyroid carcinoma presenting as an autonomously functioning thyroid nodule and causing severe thyrotoxicosis. The tumor was metastatic to a cervical lymph node and both lungs. An activating mutation of the TSH receptor gene in both the primary tumor and the lymph node metastasis was found, due to a base substitution at codon 633 (normal guanine at position 1896 replaced by cytosine CAC for GAC causing aspartic acid substitution by histidine). Other known oncogenes (gsp, ras, PTC/ret, trk, met, and p53) were not involved. This is the first description of an activating TSH receptor mutation in a thyroid hyperfunctioning carcinoma in which an aggressive malignant phenotype coexisted with activation of the cAMP cascade and differentiated thyroid functions.