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The recent derivation, based on pure quantum chemistry (QC) first-principles, of the pseudocontact shifts (PCSs) caused by a paramagnetic metal center on far away nuclei has cast doubts on the validity of the semiempirical (SE) theory, predicting PCSs to arise from the metal magnetic susceptibility anisotropy. The SE theory has been used and applied countless times, especially in the last 2 decades, to obtain structural information on proteins containing paramagnetic metal ions. We show here that the QC and SE predictions can be directly tested against experiments, provided a suitable macromolecular system is used. The SE approach yields a good prediction of the experimental PCSs while the QC one does not. It appears that the classic theory is able to grasp satisfactorily the underlying physics.

Data integration in structural biology has become a paradigm for the characterization of biomolecular systems, and it is now accepted that combining different techniques can fill the gaps in each other's blind spots. In this frame, one of the combinations, which we have implemented in REFMAC-NMR, is residual dipolar couplings from NMR together with experimental data from X-ray diffraction. The first are exquisitely sensitive to the local details but does not give any information about overall shape, whereas the latter encodes more the information about the overall shape but at the same time tends to miss the local details even at the highest resolutions. Once crystals are obtained, it is often rather easy to obtain a complete X-ray dataset, however it is time-consuming to obtain an exhaustive NMR dataset. Here, we discuss the effect of including a-priori knowledge on the properties of the system to reduce the number of experimental data needed to obtain a more complete picture. We thus introduce a set of new features of REFMAC-NMR that allow for improved handling of RDC data for multidomain proteins and multisubunit biomolecular complexes, and encompasses the use of pseudo-contact shifts as an additional source of NMR-based information. The new feature may either help in improving the refinement, or assist in spotting differences between the crystal and the solution data. We show three different examples where NMR and X-ray data can be reconciled to a unique structural model without invoking mobility.

The heterogeneous composition of vaccine formulations and the relatively low concentration make the characterization of the protein antigens extremely challenging. Aluminum-containing adjuvants have been used to enhance the immune response of several antigens over the last 90 years and still remain the most commonly used. Here, we show that solid-state NMR and isotope labeling methods can be used to characterize the structural features of the protein antigen component of vaccines and to investigate the preservation of the folding state of proteins adsorbed on Alum hydroxide matrix, providing the way to identify the regions of the protein that are mainly affected by the presence of the inorganic matrix. l-Asparaginase from E. coli has been used as a pilot model of protein antigen. This methodology can find application in several steps of the vaccine development pipeline, from the antigen optimization, through the design of vaccine formulation, up to stability studies and manufacturing process., Competing Interests: Competing interestsF.B. is an employee of the GSK group of companies. All other authors declare no competing interests.

α-Synuclein (α-syn) is found to be naturally present in biofluids such as cerebrospinal fluid (CSF) and serum. Human serum albumin (HSA) is the most abundant protein found in these biofluids, which, beyond transporting hormones and drugs, also exerts a chaperone-like activity binding other proteins in blood and inhibiting their aggregation. Contrasting results are reported in the literature about the effects of albumin on α-syn aggregation. We characterized the binding region of HSA on α-syn by high-field solution NMR spectroscopy and the effect of HSA on α-syn aggregation by thioflavin-T (ThT) fluorescence under both low-ionic-strength and physiological conditions at the albumin concentration in serum and CSF. We found that HSA, at the concentration found in human serum, slows the aggregation of α-syn significantly. α-Syn interacts with HSA in an ionic strength- and pH-dependent manner. The binding is driven by hydrophobic interactions at the N-terminus under physiological experimental conditions and by electrostatic interactions at the C-terminus at low ionic strength. This work provides novel information about the proteostasis of α-syn in biofluids and supports the hypothesis of a chaperone-like behavior of HSA.

Effective cancer therapy largely depends on inducing apoptosis in cancer cells via chemotherapy and/or radiation. Monitoring apoptosis in real-time provides invaluable information for evaluating cancer therapy response and screening preclinical anticancer drugs. In this work, we describe the design, synthesis, characterization, and in vitro evaluation of caspase probe 1 (CP1), a bimodal fluorescence-magnetic resonance (FL-MR) probe that exhibits simultaneous FL-MR turn-on response to caspase-3/7. Both caspases exist as inactive zymogens in normal cells but are activated during apoptosis and are unique biomarkers for this process. CP1 has three distinct components: a DOTA-Gd(III) chelate that provides the MR signal enhancement, tetraphenylethylene as the aggregation induced emission luminogen (AIEgen), and DEVD peptide which is a substrate for caspase-3/7. In response to caspase-3/7, the water-soluble peptide DEVD is cleaved and the remaining Gd(III)-AIEgen (Gad-AIE) conjugate aggregates leading to increased FL-MR signals. CP1 exhibited sensitive and selective dual FL-MR turn-on response to caspase-3/7 in vitro and was successfully tested by fluorescence imaging of apoptotic cells. Remarkably, we were able to use the FL response of CP1 to quantify the exact concentrations of inactive and active agents and accurately predict the MR signal in vitro. We have demonstrated that the aggregation-driven FL-MR probe design is a unique method for MR signal quantification. This probe design platform can be adapted for a variety of different imaging targets, opening new and exciting avenues for multimodal molecular imaging.

Pseudocontact shifts are traditionally described as a function of the anisotropy of the paramagnetic susceptibility tensor, according to the semiempirical theory mainly developed by Kurland and McGarvey [J. Magn. Reson. 2, 286-301 (1970)]. The paramagnetic susceptibility tensor is required to be symmetric. Applying point-dipole approximation to the quantum chemistry theory of hyperfine shift, pseudocontact shifts are found to scale with a non-symmetric tensor that differs by a factor g T /g e from the paramagnetic susceptibility tensor derived within the semiempirical framework. We analyze the foundations of the Kurland-McGarvey pseudocontact shift expression and recall that it is inherently based on the Russell-Saunders (LS) coupling approximation for the spin-orbit coupling. We show that the difference between the semiempirical and quantum chemistry pseudocontact shift expressions arises directly from the different treatment of the orbital contribution to the hyperfine coupling.

Solid state NMR (SSNMR) has earned a substantial success in the characterization of paramagnetic systems over the last decades. Nowadays, the resolution and sensitivity of solid state NMR in biological molecules has improved significantly and these advancements can be translated into the study of paramagnetic biomolecules. However, the electronic properties of different metal centers affect the quality of their SSNMR spectra differently, and not all systems turn out to be equally easy to approach by this technique. In this review we will try to give an overview of the properties of different paramagnetic centers and how they can be used to increase the chances of experimental success., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Few studies are available on metabolic changes in liver injuries and this is the first metabolomic study evaluating a group of HCV-positive patients, before and after viral eradication via DAA IFN-free regimens, using 1 H-NMR to characterize and compare their serum fingerprints to naïve HBV-patients and healthy donors. The investigation clearly shows differences in the metabolomic profile of HCV patients before and after effective DAA treatment. Significant changes in metabolites levels in patients undergoing therapy suggest alterations in several metabolic pathways. It has been shown that 1 H-NMR fingerprinting approach is an optimal technique in predicting the specific infection and the healthy status of studied subjects (Monte-Carlo cross validated accuracies: 86% in the HCV vs HBV model, 98.7% in the HCV vs HC model). Metabolite data collected support the hypothesis that the HCV virus induces glycolysis over oxidative phosphorylation in a similar manner to the Warburg effect in cancer, moreover our results have demonstrated a different action of the two viruses on cellular metabolism, corroborating the hypothesis that the metabolic perturbation on patients could be attributed to a direct role in viral infection. This metabolomic study has revealed some alteration in metabolites for the first time (2-oxoglutarate and 3-hydroxybutrate) concerning the HCV-infection model that could explain several extrahepatic manifestations associated with such an infection.

Primary biliary cholangitis (PBC) is a chronic autoimmune liver disease characterized by ongoing inflammatory destruction of the interlobular bile ducts, eventually leading to chronic cholestasis and biliary cirrhosis. This study primarily aims to define the metabolomic signature of PBC after comparison with healthy controls (HC). Second, it aims to evaluate the possible metabolic association between PBC and celiac disease (CD), an immune-mediated disorder frequently associated with PBC. Serum and urine samples from 20 PBC, 21 CD, and 19 sex-matched HC subjects were collected. 1 H nuclear magnetic resonance (NMR) spectra for all samples were acquired, and multivariate statistics were used to evaluate the differences among the three groups and to provide information about the involved metabolites. The classification accuracies to discriminate PBC and HC groups were 78.9-84.6% for serum and 76.9% for urine. In comparison to HC, PBC patient sera were characterized by altered levels ( p value <0.05) of pyruvate, citrate, glutamate, glutamine, serine, tyrosine, phenylalanine, and lactate. PBC patient urine showed lower levels ( p value <0.05) of trigonelline and hippurate with respect to HC. Furthermore, the NMR metabolomic fingerprint was able to cluster PBC with respect to CD patients, and the classification accuracies in the discriminations between these groups were 81.9-91.7% for serum and 77.7% for urine. Our results show that PBC displays a unique metabolomic fingerprint, which led to speculation about an impaired energy metabolism, probably associated with an altered gut microbiota. PBC and CD showed two distinct metabolic fingerprints. These data could provide clues for the comprehension of the PBC pathogenetic mechanisms and the detection of novel therapeutic targets.

Resonance assignment and structural characterization of pharmacologically relevant proteins promise to improve understanding and safety of these proteins by rational design. However, the PEG coating that is used to evade the immune system also causes these molecules to "evade" the standard structural biology methodologies. We here demonstrate that it is possible to obtain the resonance assignment and a reliable structural model of large PEGylated proteins through an integrated approach encompassing NMR and X-ray crystallography., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Partial symmetry, i.e., the presence of more than one molecule in the asymmetric unit of a crystal, is a relatively rare phenomenon in small-molecule crystallography, but is quite common in protein crystallography, where it is typically known as non-crystallographic symmetry (NCS). Several papers in literature propose molecular determinants such as crystal contacts, thermal factors, or TLS parameters as an explanation for the phenomenon of intrinsic asymmetry among molecules that are in principle equivalent. Nevertheless, are all of the above determinants the cause or are they rather the effect? In the general frame of the NCS often observed in crystals of biomolecules, this paper deals with nickel(II)-substituted human carbonic anhydrase(II) (hCAII) and its SAD structure determination at the nickel edge. The structure revealed two non-equivalent molecules in the asymmetric unit, the presence of a secondary nickel-binding site at the N-terminus of both molecules (which had never been found before in the nickel-substituted enzyme) and two different coordination geometries of the active site nickel (hexa-coordinated in one molecule and mainly penta-coordinated in the other). The above-mentioned standard molecular crystallographic determinants of this asymmetry are analyzed and presented in detail for this particular case. From these considerations, we speculate on the existence of a fundamental, although yet unknown, common cause for the partial symmetry that is so often encountered in X-ray structures of biomolecules.

Metabolomics deals with the whole ensemble of metabolites (the metabolome). As one of the -omic sciences, it relates to biology, physiology, pathology and medicine; but metabolites are chemical entities, small organic molecules or inorganic ions. Therefore, their proper identification and quantitation in complex biological matrices requires a solid chemical ground. With respect to for example, DNA, metabolites are much more prone to oxidation or enzymatic degradation: we can reconstruct large parts of a mammoth's genome from a small specimen, but we are unable to do the same with its metabolome, which was probably largely degraded a few hours after the animal's death. Thus, we need standard operating procedures, good chemical skills in sample preparation for storage and subsequent analysis, accurate analytical procedures, a broad knowledge of chemometrics and advanced statistical tools, and a good knowledge of at least one of the two metabolomic techniques, MS or NMR. All these skills are traditionally cultivated by chemists. Here we focus on metabolomics from the chemical standpoint and restrict ourselves to NMR. From the analytical point of view, NMR has pros and cons but does provide a peculiar holistic perspective that may speak for its future adoption as a population-wide health screening technique., (© 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Background: Risk stratification and management of acute myocardial infarction patients continue to be challenging despite considerable efforts made in the last decades by many clinicians and researchers. The aim of this study was to investigate the metabolomic fingerprint of acute myocardial infarction using nuclear magnetic resonance spectroscopy on patient serum samples and to evaluate the possible role of metabolomics in the prognostic stratification of acute myocardial infarction patients., Methods: In total, 978 acute myocardial infarction patients were enrolled in this study; of these, 146 died and 832 survived during 2 years of follow-up after the acute myocardial infarction. Serum samples were analyzed via high-resolution 1 H-nuclear magnetic resonance spectroscopy and the spectra were used to characterize the metabolic fingerprint of patients. Multivariate statistics were used to create a prognostic model for the prediction of death within 2 years after the cardiovascular event., Results: In the training set, metabolomics showed significant differential clustering of the two outcomes cohorts. A prognostic risk model predicted death with 76.9% sensitivity, 79.5% specificity, and 78.2% accuracy, and an area under the receiver operating characteristics curve of 0.859. These results were reproduced in the validation set, obtaining 72.6% sensitivity, 72.6% specificity, and 72.6% accuracy. Cox models were used to compare the known prognostic factors (for example, Global Registry of Acute Coronary Events score, age, sex, Killip class) with the metabolomic random forest risk score. In the univariate analysis, many prognostic factors were statistically associated with the outcomes; among them, the random forest score calculated from the nuclear magnetic resonance data showed a statistically relevant hazard ratio of 6.45 (p = 2.16×10 -16 ). Moreover, in the multivariate regression only age, dyslipidemia, previous cerebrovascular disease, Killip class, and random forest score remained statistically significant, demonstrating their independence from the other variables., Conclusions: For the first time, metabolomic profiling technologies were used to discriminate between patients with different outcomes after an acute myocardial infarction. These technologies seem to be a valid and accurate addition to standard stratification based on clinical and biohumoral parameters.

Scope: According to Eurostat 2016, approximately 119 million European citizens live at-risk-of-poverty (ROP). This subpopulation is highly diverse by ethnicity, age, and culture in the different EU states, but they all have in common a low income that could represent an increased risk of nutrient deficiencies due to poor nutritional habits. This study aims to investigate the human urine metabolome in the search of common biomarkers representing dietary deficiencies amongst European populations at ROP., Methods and Results: 2732 urine samples were collected from 1391 subjects across five different European countries, including the United Kingdom, Finland, Italy, Lithuania, and Serbia, and analyzed using 1 H-NMR spectroscopy. The resulting urine metabolome data were explored according to study design factors including economic status, country, and gender., Conclusion: Partitioning of the effects derived from the study design factors using ANOVA-simultaneous component analysis (ASCA) revealed that country and gender effects were responsible for most of the systematic variation. The effect of economic status was, as expected, much weaker than country and gender, but more pronounced in Lithuania than in other countries. Citrate and hippurate were among the most powerful ROP biomarkers. The possible relationship between these markers and nutritional deficiencies amongst the ROP population is discussed., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Matrix metalloproteinases hydrolyze proteins and glycoproteins forming the extracellular matrix, cytokines and growth factors released in the extracellular space, and membrane-bound receptors on the outer cell membrane. The pathological relevance of MMPs has prompted the structural and functional characterization of these enzymes and the development of synthetic inhibitors as possible drug candidates. Recent studies have provided a better understanding of the substrate preference of the different members of the family, and structural data on the mechanism by which these enzymes hydrolyze the substrates. Here, we report the recent advancements in the understanding of the mechanism of collagenolysis and elastolysis, and we discuss the perspectives of new therapeutic strategies for targeting MMPs., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

This paper takes the cue from the previously solved crystal structure of human apo-S100Z and compares it with that of the calcium-bound S100Z from zebrafish in order to stress, for this particular S100, the significant role of the presence of calcium in promoting supramolecular assemblies with likely biological meaning. This consideration is then expanded through a wider review on analogous situations concerning all other S100s for which there is crystallographic o biochemical evidence of how the presence of calcium promotes the formation of quaternary complexes.The paper also deals with some considerations on the quality of the crystals obtained for the solved members of this family and on the need for experimental phasing for solving some of the structures where the good general sequence homology among the members of the family would have suggested molecular replacement (MR) as the easiest way to solve them.These considerations, along with the PCA analysis carried out on all the known S100s, further demonstrate that calcium plays a fundamental role in triggering quaternary structure formation for several members of this family of proteins.

Protein glycosylation is the most complex post-translational modification process. More than 50 % of human cells proteins are glycosylated, whereas bacteria such as E. coli do not have this modification machinery. Indeed, the carbohydrate residues in natural proteins affect their folding, immunogenicity, and stability toward proteases, besides controlling biological properties and activities. It is therefore important to introduce such structural modification in bioengineered proteins lacking the presence of carbohydrate residues. This is not trivial as it requires reagents and conditions compatible with the protein's stability and reactivity. This work reports on the introduction of lactose moieties in two natural proteins, namely ubiquitin (Ub) and l-asparaginase II (ANSII). The synthetic route employed is based on the sulfur(VI) fluoride exchange (SuFEx) coupling of a lactose tethered arylfluorosulfate (Lact-Ar-OSO 2 F) with the ϵ-NH 2 group of lysine residues of the proteins. This metal-free click SuFEx reaction relies on the properties of the fluorosulfate employed, which is easily prepared in multigram scale from available precursors and reacts chemoselectively with the ϵ-NH 2 group of lysine residues under mild conditions. Thus, iterative couplings of Lact-Ar-OSO 2 F to Ub and ANSII, afforded multiple glycosylations of these proteins so that up to three and four Lact-Ar-OSO 2 groups were introduced in Ub and ANSII, respectively, via the formation of a sulfamoyl (OSO 2 -NH) linkage., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Intrinsically disordered proteins (IDPs) experience a diverse spectrum of motions that are difficult to characterize with a single experimental technique. Herein we combine high- and low-field nuclear spin relaxation, nanosecond fluorescence correlation spectroscopy (nsFCS), and long molecular dynamics simulations of alpha-synuclein, an IDP involved in Parkinson disease, to obtain a comprehensive picture of its conformational dynamics. The combined analysis shows that fast motions below 2 ns caused by local dihedral angle fluctuations and conformational sampling within and between Ramachandran substates decorrelate most of the backbone N-H orientational memory. However, slow motions with correlation times of up to ca. 13 ns from segmental dynamics are present throughout the alpha-synuclein chain, in particular in its C-terminal domain, and global chain reconfiguration occurs on a timescale of ca. 60 ns. Our study demonstrates a powerful strategy to determine residue-specific protein dynamics in IDPs at different time and length scales., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The properties of the conformational landscape of a biomolecule are of capital importance to understand its function. It is widely accepted that a statistical ensemble is far more representative than a single structure, especially for proteins with disordered regions. While experimental data provide the most important handle on the conformational variability that the system is experiencing, they usually report on either time or ensemble averages. Since the available conformations largely outnumber the (independent) available experimental data, the latter can be equally well reproduced by a variety of ensembles. We have proposed the Maximum Occurrence (MaxOcc) approach to provide an upper bound of the statistical weight of each conformation. This method is expected to converge towards the true statistical weights by increasing the number of independent experimental datasets. In this paper we explore the ability of DEER (Double Electron Electron Resonance) data, which report on the distance distribution between two spin labels attached to a biomolecule, to restrain the MaxOcc values and its complementarity to previously introduced experimental techniques such as NMR and Small-Angle X-ray Scattering. We here present the case of Ca2+ bound calmodulin (CaM) as a test case and show that DEER data impose a sizeable reduction of the conformational space described by high MaxOcc conformations.

Purpose: To propose an MRI quality assurance procedure that can be used for routine controls and multi-centre comparison of different MR-scanners for quantitative diffusion-weighted imaging (DWI)., Materials and Methods: 44 MR-scanners with different field strengths (1 T, 1.5 T and 3 T) were included in the study. DWI acquisitions (b-value range 0-1000 s/mm 2 ), with three different orthogonal diffusion gradient directions, were performed for each MR-scanner. All DWI acquisitions were performed by using a standard spherical plastic doped water phantom. Phantom solution ADC value and its dependence with temperature was measured using a DOSY sequence on a 600 MHz NMR spectrometer. Apparent diffusion coefficient (ADC) along each diffusion gradient direction and mean ADC were estimated, both at magnet isocentre and in six different position 50 mm away from isocentre, along positive and negative AP, RL and HF directions., Results: A good agreement was found between the nominal and measured mean ADC at isocentre: more than 90% of mean ADC measurements were within 5% from the nominal value, and the highest deviation was 11.3%. Away from isocentre, the effect of the diffusion gradient direction on ADC estimation was larger than 5% in 47% of included scanners and a spatial non uniformity larger than 5% was reported in 13% of centres., Conclusion: ADC accuracy and spatial uniformity can vary appreciably depending on MR scanner model, sequence implementation (i.e. gradient diffusion direction) and hardware characteristics. The DWI quality assurance protocol proposed in this study can be employed in order to assess the accuracy and spatial uniformity of estimated ADC values, in single- as well as multi-centre studies., (Copyright © 2018. Published by Elsevier Ltd.)

A fast and reproducible protocol for milk Nuclear Magnetic Resonance (NMR) metabolomic fingerprinting was developed, allowing for an accurate discrimination among milk samples from large-scale distribution, as well as among milk sample from different farms located in the same restricted geographical area. Seasonal variations in milk composition and correlations with cows' nutritional patterns are also assessed, underlining relationships between feeding and metabolites. The most important difference was related to the use of silage feeding. This finding is relevant to assess the suitability of milk for different dairy products. A prominent example is parmesan cheese, the preparation protocol of which excludes milk from silage-fed cows., (Copyright © 2018. Published by Elsevier Ltd.)

Precision medicine may significantly contribute to rapid disease diagnosis and targeted therapy, but relies on the availability of detailed, subject specific, clinical information. Proton nuclear magnetic resonance (¹H⁻NMR) spectroscopy of body fluids can extract individual metabolic fingerprints. Herein, we studied 64 patients admitted to the Florence main hospital emergency room with severe abdominal pain. A blood sample was drawn from each patient at admission, and the corresponding sera underwent ¹H⁻NMR metabolomics fingerprinting. Unsupervised Principal Component Analysis (PCA) analysis showed a significant discrimination between a group of patients with symptoms of upper abdominal pain and a second group consisting of patients with diffuse abdominal/intestinal pain. Prompted by this observation, supervised statistical analysis (Orthogonal Partial Least Squares⁻Discriminant Analysis (OPLS-DA)) showed a very good discrimination (>90%) between the two groups of symptoms. This is a surprising finding, given that neither of the two symptoms points directly to a specific disease among those studied here. Actually herein, upper abdominal pain may result from either symptomatic gallstones, cholecystitis, or pancreatitis, while diffuse abdominal/intestinal pain may result from either intestinal ischemia, strangulated obstruction, or mechanical obstruction. Although limited by the small number of samples from each of these six conditions, discrimination of these diseases was attempted. In the first symptom group, >70% discrimination accuracy was obtained among symptomatic gallstones, pancreatitis, and cholecystitis, while for the second symptom group >85% classification accuracy was obtained for intestinal ischemia, strangulated obstruction, and mechanical obstruction. No single metabolite stands up as a possible biomarker for any of these diseases, while the contribution of the whole ¹H⁻NMR serum fingerprint seems to be a promising candidate, to be confirmed on larger cohorts, as a first-line discriminator for these diseases.

Many bacteria, often associated with eukaryotic hosts and of relevance for biotechnological applications, harbor a multipartite genome composed of more than one replicon. Biotechnologically relevant phenotypes are often encoded by genes residing on the secondary replicons. A synthetic biology approach to developing enhanced strains for biotechnological purposes could therefore involve merging pieces or entire replicons from multiple strains into a single genome. Here we report the creation of a genomic hybrid strain in a model multipartite genome species, the plant-symbiotic bacterium Sinorhizobium meliloti. We term this strain as cis-hybrid, since it is produced by genomic material coming from the same species' pangenome. In particular, we moved the secondary replicon pSymA (accounting for nearly 20% of total genome content) from a donor S. meliloti strain to an acceptor strain. The cis-hybrid strain was screened for a panel of complex phenotypes (carbon/nitrogen utilization phenotypes, intra- and extracellular metabolomes, symbiosis, and various microbiological tests). Additionally, metabolic network reconstruction and constraint-based modeling were employed for in silico prediction of metabolic flux reorganization. Phenotypes of the cis-hybrid strain were in good agreement with those of both parental strains. Interestingly, the symbiotic phenotype showed a marked cultivar-specific improvement with the cis-hybrid strains compared to both parental strains. These results provide a proof-of-principle for the feasibility of genome-wide replicon-based remodelling of bacterial strains for improved biotechnological applications in precision agriculture.

Biophysical parameters can accelerate drug development; e.g., rigid ligands may reduce entropic penalty and improve binding affinity. We studied systematically the impact of ligand rigidification on thermodynamics using a series of fasudil derivatives inhibiting protein kinase A by crystallography, isothermal titration calorimetry, nuclear magnetic resonance, and molecular dynamics simulations. The ligands varied in their internal degrees of freedom but conserve the number of heteroatoms. Counterintuitively, the most flexible ligand displays the entropically most favored binding. As experiment shows, this cannot be explained by higher residual flexibility of ligand, protein, or formed complex nor by a deviating or increased release of water molecules upon complex formation. NMR and crystal structures show no differences in flexibility and water release, although strong ligand-induced adaptations are observed. Instead, the flexible ligand entraps more efficiently water molecules in solution prior to protein binding, and by release of these waters, the favored entropic binding is observed.

The aggregation of Aβ1-40 was monitored by solution NMR, which showed a trend complementary to the one observed by ThT-fluorescence. The NMR data support a kinetic model where Aβ1-40 initially aggregates with the reversible formation of oligomeric species, which then irreversibly convert into fibrils.

Cellular uptake of human H-ferritin loaded with 50 or 350 iron ions results in significant cytotoxicity on HeLa cells at submicromolar concentrations. Conversely, Horse Spleen Ferritin, that can be considered a model of L-cages, as it contains only about 10% of H subunits, even when loaded with 1000 iron ions, is toxic only at >1 order of magnitude higher protein concentrations. We propose here that the different cytotoxicity of the two ferritin cages originates from the presence in H-ferritin of a pool of non-biomineralized iron ions bound at the ferroxidase catalytic sites of H-ferritin subunits. This iron pool is readily released during the endosomal-mediated H-ferritin internalization., Competing Interests: CONFLICTS OF INTEREST All authors declare no conflicts of interest.

In the fight against Glioblastoma Multiforme, recent literature data have highlighted that integrin α5β1 and p53 are part of convergent pathways in the control of glioma apoptosis. This observation prompted us to seek a molecule able to simultaneously modulate both target families. Analyzing the results of a previous virtual screening against murine double minute 2 protein (MDM2), we envisaged that Arg-Gly-Asp (RGD)-mimetic molecules could be inhibitors of MDM2/4. Herein, we present the discovery of compound 7, which inhibits both MDM2/4 and α5β1/αvβ3 integrins. A lead optimization campaign was carried out on 7 with the aim to preserve the activities on integrins while improving those on MDM proteins. Compound 9 turned out to be a potent MDM2/4 and α5β1/αvβ3 blocker. In p53-wild type glioma cells, 9 arrested cell cycle and proliferation and strongly reduced cell invasiveness, emerging as the first molecule of a novel class of integrin/MDM inhibitors, which might be especially useful in subpopulations of patients with glioblastoma expressing a functional p53 concomitantly with a high level of α5β1 integrin.

Breast cancer (BC) is a heterogeneous disease which has been characterised and stratified by many platforms such as clinicopathological risk factors, genomic assays, computer generated models, and various "-omic" technologies. Genomic, proteomic and transcriptomic analysis in breast cancer research is well established, and metabolomics, which can be considered a downstream manifestation of the former disciplines, is of growing interest. The past decade has seen significant progress made within the field of clinical metabolomic BC research, with several groups demonstrating results with significant promise in the setting of BC screening and biological characterisation, as well as future potential for prognostic metabolomic biomarkers., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A fundamental challenge in the design of bioresponsive (or bioactivated) Gd III -based magnetic resonance (MR) imaging probes is the considerable background signal present in the "preactivated" state that arises from outer-sphere relaxation processes. When sufficient concentrations of a bioresponsive agent are present (i.e., a detectable signal in the image), the inner- and outer-sphere contributions to r 1 may be misinterpreted to conclude that the agent has been activated, when it has not. Of the several parameters that determine the observed MR signal of an agent, only the electron relaxation time ( T 1e ) impacts both the inner- and outer-sphere relaxation. Therefore, strategies to minimize this background signal must be developed to create a near zero-background (or truly "off" state) of the agent. Here, we demonstrate that intramolecular magnetic exchange coupling when Gd III is coupled to a paramagnetic transition metal provides a means to overcome the contribution of second- and outer-sphere contributions to the observed relaxivity. We have prepared a series of complexes with the general formula LMLn(μ-O 2 CCH 3 )(O 2 CCH 3 ) 2 (M = Co, Cu, Zn). Solid-state magnetic susceptibility measurements reveal significant magnetic coupling between Gd III and the transition metal ion. Nuclear magnetic relaxation dispersion (NMRD) analysis confirms that the observed differences in relaxivity are associated with the modulation of T 1e at Gd III . These results clearly demonstrate that magnetic exchange coupling between Gd III and a transition metal ion can provide a significant decrease in T 1e (and therefore the relaxivity of Gd III ). This design strategy is being exploited to prepare new generations of preclinical bioresponsive MR imaging probes with near zero-background.

Active bioinspired materials are appealing biotechnological targets, and their study is gaining momentum. These materials, which comprise of an inorganic matrix and one or more biomolecules, are extremely variable and therefore may result difficult to characterize in their intimate structure. In this work we have prepared a hydroxyapatite-l-asparaginase composite, with the perspective of using it in acute leukemia treatment. We demonstrate that the use of electron microscopy and powder X-ray diffraction, combined with the atomic-resolution information coming from solid-state NMR, allows us to understand the topology of the material and how the different components interplay to obtain an active composite.

NMR (nuclear magnetic resonance) investigation through the exploitation of paramagnetic effects is passing from an approach limited to few specialists in the field to a generally applicable method that must be considered, especially for the characterization of systems hardly affordable with other techniques. This is mostly due to the fact that paramagnetic data are long range in nature, thus providing information for the structural and dynamic characterization of complex biomolecular architectures in their native environment. On the other hand, this information usually needs to be complemented by data from other sources. Integration of paramagnetic NMR with other techniques, and the development of protocols for a joint analysis of all available data, is fundamental for achieving a comprehensive characterization of complex biological systems. We describe here a few examples of the new possibilities offered by paramagnetic data used in integrated structural approaches., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society and the Royal Society of Biology.)

Background: Prospective pharmacological studies on breathomics profiles in COPD patients have not been previously reported. We assessed the effects of treatment and withdrawal of an extrafine inhaled corticosteroid (ICS)-long-acting β 2 -agonist (LABA) fixed dose combination (FDC) using a multidimensional classification model including breathomics. Methods: A pilot, proof-of-concept, pharmacological study was undertaken in 14 COPD patients on maintenance treatment with inhaled fluticasone propionate/salmeterol (500/50 μg b.i.d.) for at least 8 weeks (visit 1). Patients received 2-week treatment with inhaled beclomethasone dipropionate/formoterol (100/6 μg b.i.d.) (visit 2), 4-week treatment with formoterol alone (6 μg b.i.d.) (visit 3), and 4-week treatment with beclomethasone/formoterol (100/6 μg b.i.d.) (visit 4). Exhaled breath analysis with two e-noses, based on different technologies, and exhaled breath condensate (EBC) NMR-based metabolomics were performed. Sputum cell counts, sputum supernatant and EBC prostaglandin E 2 (PGE 2 ) and 15-F 2t -isoprostane, fraction of exhaled nitric oxide, and spirometry were measured. Results: Compared with formoterol alone, EBC acetate and sputum PGE 2 , reflecting airway inflammation, were reduced after 4-week beclomethasone/formoterol. Three independent breathomics techniques showed that extrafine beclomethasone/formoterol short-term treatment was associated with different breathprints compared with regular fluticasone propionate/salmeterol. Either ICS/LABA FDC vs. formoterol alone was associated with increased pre-bronchodilator FEF 25-75% and FEV 1 /FVC ( P = 0.008-0.029). The multidimensional model distinguished fluticasone propionate/salmeterol vs. beclomethasone/formoterol, fluticasone propionate/salmeterol vs. formoterol, and formoterol vs. beclomethasone/formoterol (accuracy > 70%, P < 0.01). Conclusions: Breathomics could be used for assessing ICS treatment and withdrawal in COPD patients. Large, controlled, prospective pharmacological trials are required to clarify the biological implications of breathomics changes. EUDRACT number: 2012-001749-42.

The human antigen R (HuR) is an RNA-binding protein known to modulate the expression of target mRNA coding for proteins involved in inflammation, tumorigenesis, and stress responses and is a valuable drug target. We previously found that dihydrotanshinone-I (DHTS, 1) prevents the association of HuR with its RNA substrate, thus imparing its function. Herein, inspired by DHTS structure, we designed and synthesized an array of ortho-quinones (tanshinone mimics) using a function-oriented synthetic approach. Among others, compound 6a and 6n turned out to be more effective than 1, showing a nanomolar K i and disrupting HuR binding to RNA in cells. A combined approach of NMR titration and molecular dynamics (MD) simulations suggests that 6a stabilizes HuR in a peculiar closed conformation, which is incompatible with RNA binding. Alpha screen and RNA-electrophoretic mobility shift assays (REMSA) data on newly synthesized compounds allowed, for the first time, the generation of structure activity relationships (SARs), thus providing a solid background for the generation of highly effective HuR disruptors.

Down syndrome (DS) is caused by the presence of a supernumerary copy of the human chromosome 21 (Hsa21) and is the most frequent genetic cause of intellectual disability (ID). Key traits of DS are the distinctive facies and cognitive impairment. We conducted for the first time an analysis of the Nuclear Magnetic Resonance (NMR)-detectable part of the metabolome in plasma and urine samples, studying 67 subjects with DS and 29 normal subjects as controls selected among DS siblings. Multivariate analysis of the NMR metabolomic profiles showed a clear discrimination (up to of 80% accuracy) between the DS and the control groups. The univariate analysis of plasma and urine revealed a significant alteration for some interesting metabolites. Remarkably, most of the altered concentrations were consistent with the 3:2 gene dosage model, suggesting effects caused by the presence of three copies of Hsa21 rather than two: DS/normal ratio in plasma was 1.23 (pyruvate), 1.47 (succinate), 1.39 (fumarate), 1.33 (lactate), 1.4 (formate). Several significantly altered metabolites are produced at the beginning or during the Krebs cycle. Accounting for sex, age and fasting state did not significantly affect the main result of both multivariate and univariate analysis.

We have recently proposed a novel drug discovery approach based on biophysical screening of focused positional scanning libraries in which each element of the library contained a common binding moiety for the given target or class of targets. In this Letter, we report on the implementation of this approach to target metal containing proteins. In our implementation, we first derived a focused positional scanning combinatorial library of peptide mimetics (of approximately 100,000 compounds) in which each element of the library contained the metal-chelating moiety hydroxamic acid at the C-terminal. Screening of this library by nuclear magnetic resonance spectroscopy in solution allowed the identification of a novel and selective compound series targeting MMP-12. The data supported that our general approach, perhaps applied using other metal chelating agents or other initial binding fragments, may result very effective in deriving novel and selective agents against metalloenzyme., Competing Interests: The authors declare no competing financial interest.

The [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii has been studied using 1 H NMR spectroscopy identifying the paramagnetically shifted 1 H resonances associated with both the [4Fe-4S] H and the [2Fe] H subclusters of the active site "H-cluster". The signal pattern of the unmaturated HydA1 containing only [4Fe-4S] H is reminiscent of bacterial-type ferredoxins. The spectra of maturated HydA1, with a complete H-cluster in the active H ox and the CO-inhibited H ox -CO state, reveal additional upfield and downfield shifted 1 H resonances originating from the four methylene protons of the azadithiolate ligand in the [2Fe] H subsite. The two axial protons are affected by positive spin density, while the two equatorial protons experience negative spin density. These protons can be used as important probes sensing the effects of ligand-binding to the catalytic site of the H-cluster.

In the era of precision medicine, the analysis of simple information like sex and age can increase the potential to better diagnose and treat conditions that occur more frequently in one of the two sexes, present sex-specific symptoms and outcomes, or are characteristic of a specific age group. We present here a study of the association networks constructed from an array of 22 plasma metabolites measured on a cohort of 844 healthy blood donors. Through differential network analysis we show that specific association networks can be associated with sex and age: Different connectivity patterns were observed, suggesting sex-related variability in several metabolic pathways (branched-chain amino acids, ketone bodies, and propanoate metabolism). Reduction in metabolite hub connectivity was also found to be associated with age in both sex groups. Network analysis was complemented with standard univariate and multivariate statistical analysis that revealed age- and sex-specific metabolic signatures. Our results demonstrate that the characterization of metabolite-metabolite association networks is a promising and powerful tool to investigate the human phenotype at a molecular level.

NMR is a widely used analytical technique with a growing number of repositories available. As a result, demands for a vendor-agnostic, open data format for long-term archiving of NMR data have emerged with the aim to ease and encourage sharing, comparison, and reuse of NMR data. Here we present nmrML, an open XML-based exchange and storage format for NMR spectral data. The nmrML format is intended to be fully compatible with existing NMR data for chemical, biochemical, and metabolomics experiments. nmrML can capture raw NMR data, spectral data acquisition parameters, and where available spectral metadata, such as chemical structures associated with spectral assignments. The nmrML format is compatible with pure-compound NMR data for reference spectral libraries as well as NMR data from complex biomixtures, i.e., metabolomics experiments. To facilitate format conversions, we provide nmrML converters for Bruker, JEOL and Agilent/Varian vendor formats. In addition, easy-to-use Web-based spectral viewing, processing, and spectral assignment tools that read and write nmrML have been developed. Software libraries and Web services for data validation are available for tool developers and end-users. The nmrML format has already been adopted for capturing and disseminating NMR data for small molecules by several open source data processing tools and metabolomics reference spectral libraries, e.g., serving as storage format for the MetaboLights data repository. The nmrML open access data standard has been endorsed by the Metabolomics Standards Initiative (MSI), and we here encourage user participation and feedback to increase usability and make it a successful standard.

Paramagnetic NMR data can be profitably incorporated in structural refinement protocols of metalloproteins or metal-substituted proteins, mostly as distance or angle restraints. However, they could in principle provide much more information, because the magnetic susceptibility of a paramagnetic metal ion is largely determined by its coordination sphere. This information can in turn be used to evaluate changes occurring in the coordination sphere of the metal when ligands (e.g.: inhibitors) are bound to the protein. This gives an experimental handle on the molecular structure in the vicinity of the metal which falls in the so-called blind sphere. The magnetic susceptibility anisotropy tensors of cobalt(II) and nickel(II) ions bound to human carbonic anhydrase II in free and inhibited forms have been determined. The change of the magnetic susceptibility anisotropy is directly linked to the binding mode of different ligands in the active site of the enzyme. Indication about the metal coordination sphere in the presence of an inhibitor in pharmaceutically relevant proteins could be important in the design of selective drugs with a structure-based approach.

Background: Natural products offer a wide range of biological activities, but they are not easily integrated in the drug discovery pipeline, because of their inherent scaffold intricacy and the associated complexity in their synthetic chemistry. Enzymes may be used to perform regioselective and stereoselective incorporation of functional groups in the natural product core, avoiding harsh reaction conditions, several protection/deprotection and purification steps., Methods: Herein, we developed a three step protocol carried out inside an NMR-tube. 1st-step: STD-NMR was used to predict the: i) capacity of natural products as enzyme substrates and ii) possible regioselectivity of the biotransformations. 2nd-step: The real-time formation of multiple-biotransformation products in the NMR-tube bioreactor was monitored in-situ. 3rd-step: STD-NMR was applied in the mixture of the biotransformed products to screen ligands for protein targets., Results: Herein, we developed a simple and time-effective process, the "NMR-tube bioreactor", that is able to: (i) predict which component of a mixture of natural products can be enzymatically transformed, (ii) monitor in situ the transformation efficacy and regioselectivity in crude extracts and multiple substrate biotransformations without fractionation and (iii) simultaneously screen for interactions of the biotransformation products with pharmaceutical protein targets., Conclusions: We have developed a green, time-, and cost-effective process that provide a simple route from natural products to lead compounds for drug discovery., General Significanse: This process can speed up the most crucial steps in the early drug discovery process, and reduce the chemical manipulations usually involved in the pipeline, improving the environmental compatibility., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The development of a methodology for the structural characterization at atomic detail of proteins conjugated to nanoparticles would be a breakthrough in nanotechnology. Solution and solid-state NMR spectroscopies are currently used to investigate molecules and peptides grafted onto nanoparticles, but the strategies used so far fall short in the application to proteins, which represent a thrilling development in theranostics. We here demonstrate the feasibility of highly-resolved multidimensional heteronuclear spectra of a large protein assembly conjugated to PEGylated gold nanoparticles. The spectra have been obtained by direct proton detection under fast MAS and allow for both a fast fingerprinting for the assessment of the preservation of the native fold and for resonance assignment. We thus demonstrate that the structural characterization and the application of the structure-based methodologies to proteins bound to gold nanoparticles is feasible and potentially extensible to other hybrid protein-nanomaterials.

A new version of the program PARAssign has been evaluated for assignment of NMR resonances of the 76 methyl groups in leucines, isoleucines and valines in a 25 kDa protein, using only the structure of the protein and pseudocontact shifts (PCS) generated with a lanthanoid tag at up to three attachment sites. The number of reliable assignments depends strongly on two factors. The principle axes of the magnetic susceptibility tensors of the paramagnetic centers should not be parallel so as to avoid correlated PCS. Second, the fraction of resonances in the spectrum of a paramagnetic sample that can be paired with the diamagnetic counterparts is critical for the assignment. With the data from two tag positions a reliable assignment could be obtained for 60% of the methyl groups and for many of the remaining resonances the number of possible assignments is limited to two or three. With a single tag, reliable assignments can be obtained for methyl groups with large PCS near the tag. It is concluded that assignment of methyl group resonances by paramagnetic tagging can be particularly useful in combination with some additional data, such as from mutagenesis or NOE-based experiments. Approaches to yield the best assignment results with PCS generating tags are discussed.

The NMR chemical shifts of a substance in a complex mixture strongly depend on the composition of the mixture itself, as many weak interactions occur that are hardly predictable. Chemical shift variability is the major obstacle to automatically assigning, and subsequently quantitating, metabolite signals in body fluids, particularly urine. Here we demonstrate that the chemical shifts of signals in urine are actually predictable. This is achieved by constructing ca. 4000 artificial mixtures where the concentrations of 52 most abundant urine metabolites-including 11 inorganic ions-are varied, to sparsely but efficiently populate an N-dimensional concentration matrix. A strong relationship is established between the concentration matrix and the chemical shift matrix, so that chemical shifts of > 90 metabolite signals can be accurately predicted in real urine samples. The concentrations of the invisible inorganic ions are also accurately predicted, along with those of albumin and of several other abundant urine components.

Carbohydrate-based vaccines are among the safest and most effective vaccines and represent potent tools for prevention of life-threatening bacterial infectious diseases, like meningitis and pneumonia. The chemical conjugation of a weak antigen to protein as a source of T-cell epitopes generates a glycoconjugate vaccine that results more immunogenic. Several methods have been used so far to characterize the resulting polysaccharide-protein conjugates. However, a reduced number of methodologies has been proposed for measuring the degree of saccharide conjugation at the possible protein sites. Here we show that detailed information on large proteins conjugated with large polysaccharides can be achieved by a combination of solution and solid-state NMR spectroscopy. As a test case, a large protein assembly, l-asparaginase II, has been conjugated with Neisseria meningitidis serogroup C capsular polysaccharide and the pattern and degree of conjugation were determined., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The function of p53 protein, also known as "genome guardian", might be impaired by the overexpression of its primary cellular inhibitor, the murine double minute 2 protein (MDM2). However, the recent finding that MDM2-selective inhibitors induce high levels of its homologue MDM4, prompt us to identify, through a receptor-based virtual screening on an in house database, dual MDM2/MDM4 binders. Compound 1 turned out to possess an IC 50 of 93.7 and of 4.6 nM on MDM2 and MDM4, respectively. A series of compounds were synthesized to optimize its activity on MDM2. As a result, compound 12 showed low nanomolar IC 50 for both targets. NMR studies confirmed the pocket of binding of 12 as predicted by the Glide docking software. Notably, 12 was able to cause concentration-dependent inhibition of cell proliferation, yielding an IC 50 value of 356 ± 21 nM in neuroblastoma SHSY5Y cells and proved even to efficiently block cancer stem cell growth.

The Human antigen R protein (HuR) is an RNA-binding protein that recognizes U/AU-rich elements in diverse RNAs through two RNA-recognition motifs, RRM1 and RRM2, and post-transcriptionally regulates the fate of target RNAs. The natural product dihydrotanshinone-I (DHTS) prevents the association of HuR and target RNAs in vitro and in cultured cells by interfering with the binding of HuR to RNA. Here, we report the structural determinants of the interaction between DHTS and HuR and the impact of DHTS on HuR binding to target mRNAs transcriptome-wide. NMR titration and Molecular Dynamics simulation identified the residues within RRM1 and RRM2 responsible for the interaction between DHTS and HuR. RNA Electromobility Shifts and Alpha Screen Assays showed that DHTS interacts with HuR through the same binding regions as target RNAs, stabilizing HuR in a locked conformation that hampers RNA binding competitively. HuR ribonucleoprotein immunoprecipitation followed by microarray (RIP-chip) analysis showed that DHTS treatment of HeLa cells paradoxically enriched HuR binding to mRNAs with longer 3'UTR and with higher density of U/AU-rich elements, suggesting that DHTS inhibits the association of HuR to weaker target mRNAs. In vivo, DHTS potently inhibited xenograft tumor growth in a HuR-dependent model without systemic toxicity., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The effects arising in NMR spectroscopy because of the presence of unpaired electrons, collectively referred to as "paramagnetic NMR" have attracted increasing attention over the last decades. From the standpoint of the structural and mechanistic biology, paramagnetic NMR provides long range restraints that can be used to assess the accuracy of crystal structures in solution and to improve them by simultaneous refinements through NMR and X-ray data. These restraints also provide information on structure rearrangements and conformational variability in biomolecular systems. Theoretical improvements in quantum chemistry calculations can nowadays allow for accurate calculations of the paramagnetic data from a molecular structural model, thus providing a tool to refine the metal coordination environment by matching the paramagnetic effects observed far away from the metal. Furthermore, the availability of an improved technology (higher fields and faster magic angle spinning) has promoted paramagnetic NMR applications in the fast-growing area of biomolecular solid-state NMR. Major improvements in dynamic nuclear polarization have been recently achieved, especially through the exploitation of the Overhauser effect occurring through the contact-driven relaxation mechanism: the very large enhancement of the 13 C signal observed in a variety of liquid organic compounds at high fields is expected to open up new perspectives for applications of solution NMR., (Copyright © 2017 Elsevier Inc. All rights reserved.)