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Guar gum (GG) and Guar gum/borax (GGb) hydrogels are studied by means of rheology, Low Field Nuclear Magnetic Resonance (LF NMR) and model drug release tests. These three approaches are used to estimate the mesh size (ζ) of the polymeric network. A comparison with similar Scleroglucan systems is carried out. In the case of GGb, the rheological and Low Field NMR estimations of ζ lead to comparable results, while the drug release approach seems to underestimate ζ. Such discrepancy is attributed to the viscous effect of some polymeric chains that, although bound to the network to one end, can freely fluctuate among meshes. The viscous drag exerted by these chains slows down drug diffusion through the polymeric network. A proof for this hypothesis is given by the case of Scleroglucan gel, where the viscous contribution is not so significant and a good agreement between the rheological and release test approaches was found.

Intrinsically disordered proteins (IDPs) perform a broad range of functions in biology, suggesting that the ability to design IDPs could help expand the repertoire of proteins with novel functions. Computational design of IDPs with specific conformational properties has, however, been difficult because of their substantial dynamics and structural complexity. We describe a general algorithm for designing IDPs with specific structural properties. We demonstrate the power of the algorithm by generating variants of naturally occurring IDPs that differ in compaction, long-range contacts, and propensity to phase separate. We experimentally tested and validated our designs and analyzed the sequence features that determine conformations. We show how our results are captured by a machine learning model, enabling us to speed up the algorithm. Our work expands the toolbox for computational protein design and will facilitate the design of proteins whose functions exploit the many properties afforded by protein disorder.

Förster resonance energy transfer (FRET) is a widely-used and versatile technique for the structural characterization of biomolecules. Here, we introduce FRETpredict, an easy-to-use Python software to predict FRET efficiencies from ensembles of protein conformations. FRETpredict uses a rotamer library approach to describe the FRET probes covalently bound to the protein. The software efficiently and flexibly operates on large conformational ensembles such as those generated by molecular dynamics simulations to facilitate the validation or refinement of molecular models and the interpretation of experimental data. We provide access to rotamer libraries for many commonly used dyes and linkers and describe a general methodology to generate new rotamer libraries for FRET probes. We demonstrate the performance and accuracy of the software for different types of systems: a rigid peptide (polyproline 11), an intrinsically disordered protein (ACTR), and three folded proteins (HiSiaP, SBD2, and MalE). FRETpredict is open source (GPLv3) and is available at github.com/KULL-Centre/FRETpredict and as a Python PyPI package at pypi.org/project/FRETpredict ., (© 2024. The Author(s).)

Intrinsically disordered proteins and regions (collectively, IDRs) are pervasive across proteomes in all kingdoms of life, help to shape biological functions and are involved in numerous diseases. IDRs populate a diverse set of transiently formed structures and defy conventional sequence-structure-function relationships 1 . Developments in protein science have made it possible to predict the three-dimensional structures of folded proteins at the proteome scale 2 . By contrast, there is a lack of knowledge about the conformational properties of IDRs, partly because the sequences of disordered proteins are poorly conserved and also because only a few of these proteins have been characterized experimentally. The inability to predict structural properties of IDRs across the proteome has limited our understanding of the functional roles of IDRs and how evolution shapes them. As a supplement to previous structural studies of individual IDRs 3 , we developed an efficient molecular model to generate conformational ensembles of IDRs and thereby to predict their conformational properties from sequences 4,5 . Here we use this model to simulate nearly all of the IDRs in the human proteome. Examining conformational ensembles of 28,058 IDRs, we show how chain compaction is correlated with cellular function and localization. We provide insights into how sequence features relate to chain compaction and, using a machine-learning model trained on our simulation data, show the conservation of conformational properties across orthologues. Our results recapitulate observations from previous studies of individual protein systems and exemplify how to link-at the proteome scale-conformational ensembles with cellular function and localization, amino acid sequence, evolutionary conservation and disease variants. Our freely available database of conformational properties will encourage further experimental investigation and enable the generation of hypotheses about the biological roles and evolution of IDRs., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The ionizable-lipid component of RNA-containing nanoparticles controls the pH-dependent behavior necessary for an efficient delivery of the cargo-the so-called endosomal escape. However, it is still an empirical exercise to identify optimally performing lipids. Here, we study two well-known ionizable lipids, DLin-MC3-DMA and DLin-DMA using a combination of experiments, multiscale computer simulations, and electrostatic theory. All-atom molecular dynamics simulations, and experimentally measured polar headgroup p K a values, are used to develop a coarse-grained representation of the lipids, which enables the investigation of the pH-dependent behavior of lipid nanoparticles (LNPs) through Monte Carlo simulations, in the absence and presence of RNA molecules. Our results show that the charge state of the lipids is determined by the interplay between lipid shape and headgroup chemistry, providing an explanation for the similar pH-dependent ionization state observed for lipids with headgroup p K a values about one-pH-unit apart. The pH dependence of lipid ionization is significantly influenced by the presence of RNA, whereby charge neutrality is achieved by imparting a finite and constant charge per lipid at intermediate pH values. The simulation results are experimentally supported by measurements of α-carbon 13 C-NMR chemical shifts for eGFP mRNA LNPs of both DLin-MC3-DMA and DLin-DMA at various pH conditions. Further, we evaluate the applicability of a mean-field Poisson-Boltzmann theory to capture these phenomena., Competing Interests: Competing interests statement:A.D., G.G., M.Y.A., D.U., J.U., and L.L. were employed by AstraZeneca R&D Gothenburg at the time of this work.

We present a floristic-vegetational study on a plant community dominated by Geranium argenteum in the Sibillini Mountains (Central Apennines), at the southern limit of its distribution in the Apennines. It is a rare pioneer community located at an elevation of about 2100 m a.s.l. with northern exposure on the fractured rocky ridges and at the edges of the rocky detrital valleys on lithosol, with a prolonged presence of snowpack and gelifraction processes. The results of the phytosociological analysis allow us to propose the new Festuco italicae-Geranietum argentei association referred to as the Leontopodio nivalis-Elynion myosuroidis alliance ( Carici rupestris-Kobresietea bellardii class). The comparison with the Alpine and the Northern Apennines phytocoenoses characterized by Geranium argenteum allows us to provide a new interpretation of the syntaxonomical framework concerning the Geranium argenteum communities within its Alpine-Apennine range in light of the new data presented in this paper. The new Festuco italicae-Geranietum argentei association represents a further contribution to the knowledge of the relict alpine vegetation of the Leontopodio nivalis-Elynion myosuroidis alliance in the Sibillini Mountains and thus in the Central Apennines. Finally, habitat monitoring will be essential for assessing the impacts of climate change on this fragile and narrowly restricted plant community.

A considerable number of lytic polysaccharide monooxygenases (LPMOs) and other carbohydrate-active enzymes are modular, with catalytic domains being tethered to additional domains, such as carbohydrate-binding modules, by flexible linkers. While such linkers may affect the structure, function, and stability of the enzyme, their roles remain largely enigmatic, as do the reasons for natural variation in length and sequence. Here, we have explored linker functionality using the two-domain cellulose-active ScLPMO10C from Streptomyces coelicolor as a model system. In addition to investigating the WT enzyme, we engineered three linker variants to address the impact of both length and sequence and characterized these using small-angle X-ray scattering, NMR, molecular dynamics simulations, and functional assays. The resulting data revealed that, in the case of ScLPMO10C, linker length is the main determinant of linker conformation and enzyme performance. Both the WT and a serine-rich variant, which have the same linker length, demonstrated better performance compared with those with either a shorter linker or a longer linker. A highlight of our findings was the substantial thermostability observed in the serine-rich variant. Importantly, the linker affects thermal unfolding behavior and enzyme stability. In particular, unfolding studies show that the two domains unfold independently when mixed, whereas the full-length enzyme shows one cooperative unfolding transition, meaning that the impact of linkers in biomass-processing enzymes is more complex than mere structural tethering., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

A causal effect can be defined as a comparison of outcomes that result from two or more alternative actions, with only one of the action-outcome pairs actually being observed. In healthcare, the gold standard for causal effect measurements is randomized controlled trials (RCTs), in which a target population is explicitly defined and each study sample is randomly assigned to either the treatment or control cohorts. The great potential to derive actionable insights from causal relationships has led to a growing body of machine-learning research applying causal effect estimators to observational data in the fields of healthcare, education, and economics. The primary difference between causal effect studies utilizing observational data and RCTs is that for observational data, the study occurs after the treatment, and therefore we do not have control over the treatment assignment mechanism. This can lead to massive differences in covariate distributions between control and treatment samples, making a comparison of causal effects confounded and unreliable. Classical approaches have sought to solve this problem piecemeal, first by predicting treatment assignment and then treatment effect separately. Recent work extended part of these approaches to a new family of representation-learning algorithms, showing that the upper bound of the expected treatment effect estimation error is determined by two factors: the outcome generalization-error of the representation and the distance between the treated and control distributions induced by the representation. To achieve minimal dissimilarity in learning such distributions, in this work we propose a specific auto-balancing, self-supervised objective. Experiments on real and benchmark datasets revealed that our approach consistently produced less biased estimates than previously published state-of-the-art methods. We demonstrate that the reduction in error can be directly attributed to the ability to learn representations that explicitly reduce such dissimilarity; further, in case of violations of the positivity assumption (frequent in observational data), we show our approach performs significantly better than the previous state of the art. Thus, by learning representations that induce similar distributions of the treated and control cohorts, we present evidence to support the error bound dissimilarity hypothesis as well as providing a new state-of-the-art model for causal effect estimation., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Gino Tesei reports a relationship with Elevance Health Inc that includes: employment and equity or stocks. Stefanos Giampanis reports a relationship with Elevance Health Inc that includes: employment and equity or stocks. Jingpu Shi reports a relationship with Elevance Health Inc that includes: employment and equity or stocks. Beau Norgeot reports a relationship with Elevance Health Inc that includes: employment and equity or stocks., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The formation and viscoelastic properties of condensates of intrinsically disordered proteins (IDPs) is dictated by amino acid sequence and solution conditions. Because of the involvement of biomolecular condensates in cell physiology and disease, advancing our understanding of the relationship between protein sequence and phase separation (PS) may have important implications in the formulation of new therapeutic hypotheses. Here, we present CALVADOS 2, a coarse-grained model of IDPs that accurately predicts conformational properties and propensities to undergo PS for diverse sequences and solution conditions. In particular, we systematically study the effect of varying the range of the nonionic interactions and use our findings to improve the temperature scale of the model. We further optimize the residue-specific model parameters against experimental data on the conformational properties of 55 proteins, while also leveraging 70 hydrophobicity scales from the literature to avoid overfitting the training data. Extensive testing shows that the model accurately predicts chain compaction and PS propensity for sequences of diverse length and charge patterning, as well as at different temperatures and salt concentrations., Competing Interests: No competing interests were disclosed., (Copyright: © 2023 Tesei G and Lindorff-Larsen K.)

Purpose:  i) To assess the inter- and intra-observer reliability of ultrasound (US) in the evaluation of the hyaline cartilage (HC) of the metacarpal head (MH) in patients with rheumatoid arthritis (RA) and in healthy subjects (HS) both qualitatively and quantitatively. ii) To calculate the smallest detectable difference (SDD) of the MH cartilage thickness measurement. iii) To correlate the qualitative scoring system and the quantitative assessment., Materials and Methods:  US examination was performed on 280 MHs of 20 patients with RA and 15 HS using a very high frequency probe (up to 22 MHz). HC status was evaluated both qualitatively (using a five-grade scoring system) and quantitatively (using the average value of the longitudinal and transverse measures). The HC of MHs from II to V metacarpophalangeal joint of both hands were scanned independently on the same day by two rheumatologists to assess inter-observer reliability. All subjects were re-examined using the same scanning protocol and the same US setting by one sonographer after a week to assess intra-observer reliability., Results:  The inter-observer agreement and intra-observer agreement were moderate to substantial (k = 0.66 and k = 0.73) for the qualitative scoring system and high (ICC = 0.93 and ICC = 0.94) for the quantitative assessment. The SDD of the MH cartilage thickness measurement was 0.09 mm. A significant correlation between the two scoring systems was found (r = -0.35; p < 0.001)., Conclusion:  The present study describes the main methodological issues of HC assessment. Using a standardized protocol, both the qualitative and the quantitative scoring systems can be reliable., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Past anthropogenic disturbances lowered the altitudinal distribution of the Mediterranean Fagus sylvatica forests below 2,000 m a.s.l. Accordingly, our current understanding of the southern distribution range of F. sylvatica forests is restricted to managed stands below this elevation, neglecting relic forests growing above. This study has shed light on the structure and species assemblage of an unmanaged relict subalpine F. sylvatica stand growing within the core of its southernmost glacial refugia and at its highest species range elevation limit (2,140 m a.s.l.) in southern Apennines (Italy). Here, tree biometric attributes and understory species abundances were assessed in eight permanent plots systematically positioned from 1,650 to 2,130 m a.s.l. In the subalpine belt, F. sylvatica had formed a dense clonal stem population that was layered downward on the steepest slopes. The density and spatial aggregation of the stems were increased, while their stature and crown size were decreased. Above 2,000 m, changes in tree growth patterns, from upright single-stemmed to procumbent multi-stemmed, and canopy layer architecture, with crowns packed and closer to the floor, were allowed for the persistence of understory herbaceous species of biogeographic interest. Clonal layering represents an adaptive regeneration strategy for the subalpine belt environmental constraints not previously recognized in managed Mediterranean F. sylvatica forests. The clonal structure and unique species assemblage of this relic forest highlight the value of its inclusion in the priority areas networks, representing a long-term management strategy of emblematic glacial and microclimatic refugia., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Saulino, Rita, Allegrezza, Zotti, Mogavero, Tesei, Montecchiari, Allevato, Borghetti, Bonanomi and Saracino.)

In the past decade, there has been exponentially growing interest in the use of observational data collected as a part of routine healthcare practice to determine the effect of a treatment with causal inference models. Validation of these models, however, has been a challenge because the ground truth is unknown: only one treatment-outcome pair for each person can be observed. There have been multiple efforts to fill this void using synthetic data where the ground truth can be generated. However, to date, these datasets have been severely limited in their utility either by being modeled after small non-representative patient populations, being dissimilar to real target populations, or only providing known effects for two cohorts (treated vs. control). In this work, we produced a large-scale and realistic synthetic dataset that provides ground truth effects for over 10 hypertension treatments on blood pressure outcomes. The synthetic dataset was created by modeling a nationwide cohort of more than 580, 000 hypertension patient data including each person's multi-year history of diagnoses, medications, and laboratory values. We designed a data generation process by combining an adapted ADS-GAN model for fictitious patient information generation and a neural network for treatment outcome generation. Wasserstein distance of 0.35 demonstrates that our synthetic data follows a nearly identical joint distribution to the patient cohort used to generate the data. Patient privacy was a primary concern for this study; the ϵ-identifiability metric, which estimates the probability of actual patients being identified, is 0.008%, ensuring that our synthetic data cannot be used to identify any actual patients. To demonstrate its usage, we tested the bias in causal effect estimation of four well-established models using this dataset. The approach we used can be readily extended to other types of diseases in the clinical domain, and to datasets in other domains as well., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Shi, Wang, Tesei and Norgeot.)

Coarse-grained molecular dynamics simulations are a useful tool to determine conformational ensembles of proteins. Here, we show that the coarse-grained force field Martini 3 underestimates the global dimensions of intrinsically disordered proteins (IDPs) and multidomain proteins when compared with small-angle X-ray scattering (SAXS) data and that increasing the strength of protein-water interactions favors more expanded conformations. We find that increasing the strength of interactions between protein and water by ca. 10% results in improved agreement with the SAXS data for IDPs and multidomain proteins. We also show that this correction results in a more accurate description of self-association of IDPs and folded proteins and better agreement with paramagnetic relaxation enhancement data for most IDPs. While simulations with this revised force field still show deviations to experiments for some systems, our results suggest that it is overall a substantial improvement for coarse-grained simulations of soluble proteins.

Many intrinsically disordered proteins (IDPs) may undergo liquid-liquid phase separation (LLPS) and participate in the formation of membraneless organelles in the cell, thereby contributing to the regulation and compartmentalization of intracellular biochemical reactions. The phase behavior of IDPs is sequence dependent, and its investigation through molecular simulations requires protein models that combine computational efficiency with an accurate description of intramolecular and intermolecular interactions. We developed a general coarse-grained model of IDPs, with residue-level detail, based on an extensive set of experimental data on single-chain properties. Ensemble-averaged experimental observables are predicted from molecular simulations, and a data-driven parameter-learning procedure is used to identify the residue-specific model parameters that minimize the discrepancy between predictions and experiments. The model accurately reproduces the experimentally observed conformational propensities of a set of IDPs. Through two-body as well as large-scale molecular simulations, we show that the optimization of the intramolecular interactions results in improved predictions of protein self-association and LLPS., Competing Interests: The authors declare no competing interest.

Objective: Osteoporosis (OP) can complicate the course of rheumatic musculoskeletal diseases (RMDs) and connective tissue diseases (CTDs). Denosumab, a monoclonal antibody against RANK-L, showed beneficial effect in rheumatoid arthritis in inhibiting radiographic progression and erosive burden. We tested the efficacy, safety, and persistence on the treatment of the combination of biologic disease-modifying antirheumatic drugs (bDMARDs)/denosumab versus bDMARD in patients with RMD and CTD., Methods: This is a retrospective evaluation of a single center, including patients with RMD/CTD (including rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, systemic sclerosis, and overlap syndromes) treatment with bDMARD/denosumab, compared to age, gender, disease, bDMARD, and conventional synthetic disease-modifying antirheumatic drugs-matched controls., Results: Twenty-eight bDMARD/denosumab patients and 49 bDMARD patients were eligible. Despite a statistically significant difference during the first-year efficacy (due to the different baseline timepoint), there was no difference in the efficacy profile in the second year of treatment and in the safety profile (including local, systemic, and serious adverse events). Moreover, no statistically significant difference in the persistence of bDMARD treatment over 2 years of evaluation was found. The combination of bDMARD and denosumab was not an independent predictor of disease flare or bDMARD treatment withdrawal., Conclusion: The combination of bDMARD and denosumab does not alter the efficacy and the safety profile of the bDMARD in patients with RMD/CTD. Future studies verifying the radiological disease inhibition could support denosumab use in RMD/CTD other than rheumatoid arthritis, when complicated by OP.

Background/objective: The aims of this study were to describe in detail the ultrasound (US)-guided procedures in our daily rheumatology practice, to evaluate the feasibility and accuracy of US-guided procedures, and to test the efficacy of our disinfection protocol in preventing infectious complications., Methods: This was a cross-sectional and monocentric study. Information regarding patients' age, sex, body mass index, rheumatic disease, US pathological findings, aspirated and/or injected anatomical site, US equipment (ie, probe type and frequency), and needle type was consecutively collected for each US-guided procedure in a third-level rheumatology center., Results: A total of 643 US-guided procedures were performed, with a mean of 5.2 procedures per working-day. In 94.2% of the patients, only one procedure was carried out, whereas in 5.8%, more than one. The mean time was 7 ± 2.5 minutes. Ultrasound-guided procedures were highly accurate (accuracy rate higher than 95%) and safe (adverse events were reported in 0.8%). Our disinfection protocol was effective in preventing infectious complications. Probes with frequency values between 8 to 13 MHz and 20-, 21-, and 22-gauge needles were the most frequently used at shoulder, knee, wrist, elbow, and ankle level. High-frequency linear probes (ranging between 18 and 22 MHz) and 23- and 25-gauge needles were used for injecting small joints of the hands and feet. Convex low-frequency probe (2-7 MHz) and 18- and 20-gauge needles were the most used for performing hip joint aspirations and/or injections., Conclusions: This study reports useful information for setting up a service providing US-guided procedures in rheumatology and supports the feasibility, accuracy, and safety of US-guided procedures., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Ultrasound (US) is useful in monitoring RA patients, with the US7 score allowing grey-scale and power-Doppler (PD) semi-quantitative evaluation of synovitis and teno-synovitis. We evaluated real-life efficacy and safety of Baricitinib, an oral selective JAK1-2 inhibitor, in RA patients using clinical, clinimetric, and US assessments., Methods: Disease activity score in 28 joints calculated with C-reactive protein (DAS28-CRP), disease activity score in 28 joints calculated with erythrocyte sedimentation rate (DAS28-ESR), clinical disease activity index (CDAI), simplified disease activity index (SDAI), visual analogue scale (VAS)-pain, health assessment questionnaire (HAQ), COCHIN scale, adverse events (AE), concomitant medications, laboratory parameters, and US7 were performed/recorded at baseline, 1, 3, and 6 months in RA patients starting Baricitinib. Responder/non-responder status was determined according to the EULAR Response Criteria at 3 months. SDAI clinical remission or low disease activity (LDA) were calculated at 3 and 6 months., Results: In 43 enrolled patients, a significant improvement in disease activity and US7 components (except tendon PD) and a reduction of steroid dosage were observed. Responders at 3 months showed a significantly higher reduction of CDAI, SDAI, COCHIN scale, VAS-pain, and US7 synovialPD, compared with non-responders. At 3 and 6 months, remission/LDA was achieved by 12.8/53.8% and 21.6/51.3% patients, respectively. The csDMARD co-treatment was independently associated with remission/LDA at 3 months. Safety-related drop-outs were in line with literature data. The steroid dosage was associated with AE development at 6 months., Conclusion: The real-life data, also obtained with US evaluation, confirmed the Baricitinib efficacy in RA disease control, as well as the utility of assessment during the follow up of disease activity., Competing Interests: Conflict of interest statement: Cosimo Bruni reports consultancy fee from Actelion, Eli Lilly; grants from Gruppo Italiano Lotta alla Sclerodermia (GILS), Fondazione Italiana Ricerca sull’Artrite (FIRA), European Scleroderma Trial and Research (EUSTAR), Foundation for Research in Rheumatology (FOREUM), Italian Society of Rheumatology (SIR), outside the submitted work. Marco Matucci-Cerinic reports grant and personal fees from Actelion, personal fees from Biogen, personal fees from Bayer, personal fees from Boehringer Ingelheim, personal fees from CSL Behring, personal fees from Eli-Lilly, outside the submitted work. GT, LC, FN, RT, LT, MC, FB, GF: no conflict of interest to declare., (© The Author(s), 2021.)

Objective: The aim of the present retrospective observational study was to evaluate the change of Renal Resistive Index (RRI) over time (ΔRRI) and under treatment in patients with systemic sclerosis (SSc) as well as to correlate these changes with disease complications., Methods: Two hundred thirty patients [29 male, median age 57 (IQR 48-67) yrs] were enrolled. At baseline and follow-up (3.43, IQR 2.81-4.45 yrs), we collected the following data: disease variables, nailfold videocapillaroscopy (NVC) pattern, forced vital capacity (FVC), diffusing lung capacity for carbon monoxide (DLCO), systolic pulmonary arterial pressure (sPAP), presence of interstitial lung disease, RRI, evaluation of glomerular filtration rate, and new onset of pulmonary arterial hypertension (PAH)., Results: RRI value is high in SSc patients with digital ulcers and anticentromere antibodies, active and late NVC patterns, and limited cutaneous SSc. A significant correlation was observed between ΔRRI and ΔsPAP (R = 0.17, P = 0.02), with statistically higher ΔRRI (0.08 ± 0.02 vs 0.03 ± 0.05, P = 0.04) in patients complicated by PAH onset. No other new-onset complication was associated with ΔRRI. The receiver-operating characteristic curve analysis confirmed the predictive role of ΔRRI in development of new PAH (area under the curve 0.84, 95% CI 0.75-0.93, P = 0.02). In patients with SSc never exposed to sildenafil, ΔRRI was higher (0.04 ± 0.05) compared to both patients exposed to sildenafil during the study period (0.01 ± 0.05, P = 0.03) or in those exposed at the time of baseline evaluation (0.00 ± 0.05, P = 0.01)., Conclusion: RRI and its variation in time are a reliable marker of SSc-related vasculopathy, both in renal and extrarenal compartments., (Copyright © 2021 by the Journal of Rheumatology.)

Owing to their plasticity, intrinsically disordered and multidomain proteins require descriptions based on multiple conformations, thus calling for techniques and analysis tools that are capable of dealing with conformational ensembles rather than a single protein structure. Here, we introduce DEER-PREdict, a software program to predict Double Electron-Electron Resonance distance distributions as well as Paramagnetic Relaxation Enhancement rates from ensembles of protein conformations. DEER-PREdict uses an established rotamer library approach to describe the paramagnetic probes which are bound covalently to the protein.DEER-PREdict has been designed to operate efficiently on large conformational ensembles, such as those generated by molecular dynamics simulation, to facilitate the validation or refinement of molecular models as well as the interpretation of experimental data. The performance and accuracy of the software is demonstrated with experimentally characterized protein systems: HIV-1 protease, T4 Lysozyme and Acyl-CoA-binding protein. DEER-PREdict is open source (GPLv3) and available at github.com/KULL-Centre/DEERpredict and as a Python PyPI package pypi.org/project/DEERPREdict., Competing Interests: The authors have declared that no competing interests exist.

Enterohemorrhagic and enteropathogenic Escherichia coli are among the most important food-borne pathogens, posing a global health threat. The virulence factor intimin is essential for the attachment of pathogenic E. coli to the intestinal host cell. Intimin consists of four extracellular bacterial immunoglobulin-like (Big) domains, D00-D2, extending into the fifth lectin subdomain (D3) that binds to the Tir-receptor on the host cell. Here, we present the crystal structures of the elusive D00-D0 domains at 1.5 Å and D0-D1 at 1.8 Å resolution, which confirms that the passenger of intimin has five distinct domains. We describe that D00-D0 exhibits a higher degree of rigidity and D00 likely functions as a juncture domain at the outer membrane-extracellular medium interface. We conclude that D00 is a unique Big domain with a specific topology likely found in a broad range of other inverse autotransporters. The accumulated data allows us to model the complete passenger of intimin and propose functionality to the Big domains, D00-D0-D1, extending directly from the membrane.

Ailanthus altissima is an invasive alien species (IAS) present throughout Europe and included in the list of alien species of Union concern. In sub-Mediterranean areas of central Italy, there is a lack of knowledge about this invasive species and its interactions with the native forest ecosystems. We aim to find what are the main differences in vegetation structure and floristic diversity between A. altissima forests and native forests through the assessment of the principal ecological parameters that differ between the forest types. We performed 38 phytosociological relevés and sampling of ecological parameters in A. altissima forest communities and neighboring native forests. We analyzed how species richness, diversity, life forms, life strategies, structural characteristics, and ecological parameters changed in A. altissima forests compared with native ones. We found that in A. altissima forests, there is a shift in herbaceous layer richness, with a higher presence of annual ruderal herbs and the absence of herbaceous species linked to the forest environment. The ecological parameters that diverge from the native forests were total nitrogen, total carbon, and C/N ratio. A. altissima forest communities could threaten the biodiversity of the native forest ecosystems in the sub-Mediterranean landscape, favoring ruderal species and inhibiting the presence of typical forest species.

Species coexistence in grasslands is regulated by several environmental factors and interactions with the soil microbial community. Here, the development of the Basidiomycetes fungus Agaricus arvensis, forming fairy rings, in a species-rich Mediterranean grassland, is described. Effects of the mycelial front on plants, fungi and bacteria were assessed by vegetation survey and next generation sequencing approaches. Our results showed a fungal-dependent shift in the community structure operated by a wave-like spread of fairy rings that decreased plant, fungal and bacterial diversity, indicating a detrimental effect of fairy rings on most species. The fairy rings induced successional processes in plants that enhanced the replacement of a community dominated by perennial plants with short-living and fast-growing plant species. In parallel, fungal and bacterial communities showed evident differences in species composition with several taxa associated within distinct sampling zone across the fairy rings. Notably, bacteria belonging to the Burkholderia genus and fungi of the genus Trichoderma increased in response to the advancing mycelium of A. arvensis. The profound changes in community composition and the overall increase in taxa diversity at ecosystemic scale suggest that fairy ring-forming fungi may act as ecosystem engineer species in Mediterranean grasslands., (© 2020 The Authors. New Phytologist © 2020 New Phytologist Trust.)

Nanodiscs are membrane mimetics that consist of a protein belt surrounding a lipid bilayer, and are broadly used for characterization of membrane proteins. Here, we investigate the structure, dynamics and biophysical properties of two small nanodiscs, MSP1D1ΔH5 and ΔH4H5. We combine our SAXS and SANS experiments with molecular dynamics simulations and previously obtained NMR and EPR data to derive and validate a conformational ensemble that represents the structure and dynamics of the nanodisc. We find that it displays conformational heterogeneity with various elliptical shapes, and with substantial differences in lipid ordering in the centre and rim of the discs. Together, our results reconcile previous apparently conflicting observations about the shape of nanodiscs, and pave the way for future integrative studies of larger complex systems such as membrane proteins embedded in nanodiscs., Competing Interests: TB, VH, LK, SM, NJ, GT, SB, BS, LA, KL No competing interests declared, (© 2020, Bengtsen et al.)

Background: Renal Resistive Index (RRI), reflects changes in both renal vascular and tubular-interstitial compartments and in systemic vascular compliance related to age and comorbidities., Objectives: a) To investigate determinants of RRI in SSc population, b) its association with SSc-related features and c) to test its prognostic impact on organ specific worsening or death., Methods: 380 SSc patients ≥18 years were enrolled after giving informed consent. Baseline data on RRI, laboratory, instrumental and therapeutic features were retrospectively collected. Age-SSc adjusted cut-offs were created by dividing the population in age quartiles and considering RRI values >75th percentile as pathologic. Clinical follow-up was performed until last available visit or the development/worsening of specific internal organ involvement or death., Results: RRI was independently predicted by age and systolic pulmonary arterial pressure on Echo. Therefore, we created Age-SSc adjusted pathologic RRI cut-offs, which were significantly associated with various disease related skin and lung fibrotic manifestations, as well as vasculopathic complications. After a mean follow-up of 3.6 ± 2.6 years, RRI was one of the independent predictors (together with modified Rodnan skin score, interstitial lung disease, presence of dyspnoea and late nailfold-videocapillaroscopy pattern) for mortality, with 0.68 as best cut-off (sensitivity 88.5%, specificity 50.9%)., Conclusion: If corroborated, Renal Resistive Index cut-offs might be used to evaluate renal and extrarenal involvement in SSc and could serve as predictors of mortality., (Copyright © 2019 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.)

Molecular-level computer simulations of peptide aggregation, translocation, and protonation at and in biomembranes are impeded by the large time and length scales involved. We present a computationally efficient, coarse-grained, and solvent-free model for the interaction between lipid bilayers and peptides. The model combines an accurate description of mechanical membrane properties with a new granular representation of the dielectric mismatch between lipids and the aqueous phase. All-atom force fields can be easily mapped onto the coarse-grained model, and parameters for coarse-grained monopeptides accurately extrapolate to membrane permeation free energies for the corresponding dipeptides and tripeptides. Acid-base equilibria of titratable amino acid residues are further studied using a constant-pH ensemble, capturing protonation state changes upon membrane translocation. Important differences between histidine, lysine, and arginine are observed, which are in good agreement with experimental observations.

It is a textbook knowledge that charges of the same polarity repel each other. For two monovalent ions in the gas phase at a close contact this repulsive interaction amounts to hundreds of kilojoules per mole. In aqueous solutions, however, this Coulomb repulsion is strongly attenuated by a factor equal to the dielectric constant of the medium. The residual repulsion, which now amounts only to units of kilojoules per mole, may be in principle offset by attractive interactions. Probably the smallest cationic pair, where a combination of dispersion and cavitation forces overwhelms the Coulomb repulsion, consists of two guanidinium ions in water. Indeed, by a combination of molecular dynamics with electronic structure calculations and electrophoretic, as well as spectroscopic, experiments, we have demonstrated that aqueous guanidinium cations form (weakly) thermodynamically stable like-charge ion pairs. The importance of pairing of guanidinium cations in aqueous solutions goes beyond a mere physical curiosity, since it has significant biochemical implications. Guanidinium chloride is known to be an efficient and flexible protein denaturant. This is due to the ability of the orientationally amphiphilic guanidinium cations to disrupt various secondary structural motifs of proteins by pairing promiscuously with both hydrophobic and hydrophilic groups, including guanidinium-containing side chains of arginines. The fact that the cationic guanidinium moiety forms the dominant part of the arginine side chain implies that the like-charge ion pairing may also play a role for interactions between peptides and proteins. Indeed, arginine-arginine pairing has been frequently found in structural protein databases. In particular, when strengthened by a presence of negatively charged glutamate, aspartate, or C-terminal carboxylic groups, this binding motif helps to stabilize peptide or protein dimers and is also found in or near active sites of several enzymes. The like-charge pairing of the guanidinium side-chain groups may also hold the key to the understanding of the arginine "magic", that is, the extraordinary ability of arginine-rich polypeptides to passively penetrate across cellular membranes. Unlike polylysines, which are also highly cationic but lack the ease in crossing membranes, polyarginines do not exhibit mutual repulsion. Instead, they accumulate at the membrane, weaken it, and might eventually cross in a concerted, "train-like" manner. This behavior of arginine-rich cell penetrating peptides can be exploited when devising smart strategies how to deliver in a targeted way molecular cargos into the cell.

The large and sparsely hydrated thiocyanate anion, SCN - , plays a prominent role in the study of specific ion effects in biological, colloid, and atmospheric chemistry due to its extreme position in the Hofmeister series. Using atomistic modeling of aqueous SCN - solutions, we provide novel insight at the molecular scale into the experimentally observed differences in ion pairing, clustering, reorientation dynamics, mutual diffusion, and solubility between the sodium, Na + , and the potassium, K + , salt. Compared to KSCN, NaSCN has a less pronounced tendency to ion pairing; nevertheless, at high salt concentrations, we observe a strong attraction between Na + cations and the nitrogen end of SCN - , resulting in larger and more closely packed ion clusters. To accurately model aqueous SCN - solutions in computer simulations, we develop a thermodynamically consistent force field rooted in quantum-chemical calculations and refined using the Kirkwood-Buff theory. The force field is compatible with the extended simple point charge and three-point optimal point charge classical water models and reproduces experimental activity derivatives and air-water surface tension for a wide range of salt concentrations.

Amyloid aggregates are associated with a range of human neurodegenerative disorders, and it has been shown that neurotoxicity is dependent on aggregate size. Combining molecular simulation with analytical theory, a predictive model is proposed for the adsorption of amyloid aggregates onto oppositely charged surfaces, where the interaction is governed by an interplay between electrostatic attraction and entropic repulsion. Predictions are experimentally validated against quartz crystal microbalance-dissipation experiments of amyloid beta peptides and fragmented fibrils in the presence of a supported lipid bilayer. Assuming amyloids as rigid, elongated particles, we observe nonmonotonic trends for the extent of adsorption with respect to aggregate size and preferential adsorption of smaller aggregates over larger ones. Our findings describe a general phenomenon with implications for stiff polyions and rodlike particles that are electrostatically attracted to a surface.

The term spondyloarthritis (SpA) represents a condition characterised by a broad spectrum of clinical manifestations, laboratory abnormalities and imaging features; in particular, SpA is an inflammatory condition in which both peripheral and axial joints might be affected. The majority of people with this disease have either psoriatic arthritis or axial spondyloarthritis, which includes ankylosing spondylitis. Less common subgroups are enteropathic SpA, which is associated with inflammatory bowel diseases (Crohn's disease and ulcerative colitis), reactive arthritis, which can occur in people following gastrointestinal or genitourinary infections and undifferentiated SpA, that does not meet the diagnostic criteria of the other subgroups at onset, but that may evolve to do so later. Very interestingly, much of the emerging data show how SpA, during its course, tends to associate with the development of some comorbidities; in particular, with cardiovascular diseases, diabetes mellitus, osteoporosis and depressive disorders. Healthcare professionals in non-specialist settings do not always recognise the signs and symptoms of SpA, particularly spinal symptoms, which may be mistakenly attributed to other causes of low back pain, thus leading to significant delays in diagnosis and treatment of the disease itself and of its related comorbidities, with consequent disease progression and disability, compromising the health-related quality of life of patients. In this paper we reviewed the literature of the past year (Medline search of articles published from 1st March 2016 to 28th February 2017) with the aim of approaching the spectrum of SpA from some different points of view, to try to give the reader an insight into this clinically challenging group of rheumatic pathologies.

Small-angle X-ray scattering (SAXS) measurements reveal a striking difference in intermolecular interactions between two short highly charged peptides-deca-arginine (R10) and deca-lysine (K10). Comparison of SAXS curves at high and low salt concentration shows that R10 self-associates, while interactions between K10 chains are purely repulsive. The self-association of R10 is stronger at lower ionic strengths, indicating that the attraction between R10 molecules has an important electrostatic component. SAXS data are complemented by NMR measurements and potentials of mean force between the peptides, calculated by means of umbrella-sampling molecular dynamics (MD) simulations. All-atom MD simulations elucidate the origin of the R10-R10 attraction by providing structural information on the dimeric state. The last two C-terminal residues of R10 constitute an adhesive patch formed by stacking of the side chains of two arginine residues and by salt bridges formed between the like-charge ion pair and the C-terminal carboxyl groups. A statistical analysis of the Protein Data Bank reveals that this mode of interaction is a common feature in proteins., Competing Interests: The authors declare no conflict of interest.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with a highly variable course and prognosis. The management of the disease is still a clinical challenge for the treating physicians as many aspects regarding the disease pathogenesis, clinical picture and outcomes remain to be elucidated. New and interesting data are emerging; here the recent literature on SLE pathogenesis, clinical and laboratory aspects, as well as treatments and comorbidities, are reviewed and the main findings summarised in order to provide a bird's eye on the relevant papers on these topics.

Purpose: Regenerative scaffold-based procedures are emerging as a potential therapeutic option for the treatment of chondral and osteochondral lesions. In general, we can summarize most of the recent developments to reach clinical application into 2 major trends: the use of different cell sources or the application of biomaterials as a cell-free approach. The aim of this systematic review was to analyze both preclinical and clinical studies on these new trends to understand how the available evidence supports the use of cell sources or justifies the cell-free approach for the scaffold-based treatment of cartilage lesions., Methods: The research was performed using the PubMed database by looking at studies published in the English language referring to chondral or osteochondral defect repair with scaffold-based procedures until the end of 2013. The following strings were used: ("cartilage"[MeSH] AND "tissue scaffolds"[MeSH])., Results: The search showed an increasing number of published articles each year for both scaffold-based approaches, identifying a total of 305 articles. Among clinical trials, 116 used cell-based scaffold treatments and 11 used scaffolds alone. In the preclinical setting, a scaffold/cell combination was the most used treatment approach (133 v 45 articles), with mesenchymal stem cells (MSCs) being the favorite cell type. Bone marrow was the most used cell source, but other sources are gaining interest. Among articles comparing scaffolds with or without cells, the majority reported superior results for cells (71 of 89 articles). In the clinical setting, most of the articles analyzed chondrocyte-based scaffolds, with only 7 studies using MSCs; all cells were from bone marrow. Despite the lower number of articles, cell-free scaffolds are gaining popularity, with a recent increase in published studies showing promising results., Conclusions: This systematic review underlined the difficulties in understanding the real need for cells to increase the scaffold-based cartilage healing potential because of the heterogeneity of products used as well as the design of the published studies. Scaffold and cell combinations were the most investigated option in the preclinical setting, showing generally superior results, but in the clinical setting, both strategies remain used. In particular, although chondrocytes are the most commonly used cell type, research showed increasing interest in the potential of MSCs for cartilage regeneration. However, the difficulties in managing cell cultures, together with the development of a new generation of materials able to exploit the intrinsic tissue regeneration ability, justifies the clinical use of cell-free scaffolds, with increasing literature and promising preliminary results., Level of Evidence: Level IV, systematic review of Level I to IV studies., (Copyright © 2015 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.)

Background: Chronic Achilles tendinopathy is responsible for a severe reduction in physical performance and persistent pain. There is currently a number of therapeutic options and the local administration of growth factors is an emerging treatment strategy. In particular, platelet-rich plasma (PRP) is a widely used way to provide a local regenerative stimulus for tendon healing. The aim of this study was to document the mid-term results obtained after treating recalcitrant Achilles tendinopathy with injections of high concentrate, leucocyte-rich PRP., Materials and Methods: Twenty-seven patients (mean age: 44.6 years; 22 men and 5 women) affected by chronic mid-portion Achilles tendinopathy (7 bilateral, for a total of 34 tendons), refractory to previous treatments, were enrolled. Patients were treated with three ultrasound-guided intra-tendinous injections of PRP at 2-week intervals. Patients were prospectively evaluated at baseline, and then at 2, 6, and up to a mean of 54.1 months of follow-up (minimum 30 months), using the following tools: Blanzina, VISA-A, EQ-VAS for general health, and Tegner scores., Results: The VISA-A score showed a significant improvement: the baseline score of 49.9±18.1 increased to 62.9±19.8 at 2 months (p=0.002), with a further improvement at 6 months (84.3±17.1, p<0.0005), and stable results at 4.5 years (90.0±13.9). The EQ-VAS score also showed a similar positive trend. An evaluation of the activity level confirmed these findings, showing a significant improvement in the Tegner score over time (p=0.017 for the final evaluation). The longer duration of symptoms before treatment was associated with a slower return to sport (p=0.041)., Discussion: PRP injections produced good overall results for the treatment of chronic recalcitrant Achilles tendinopathy with a stable outcome up to a medium-term follow-up. Longer symptom duration was related with a more difficult return to sporting activity.

Poly(vinyl alcohol) (PVA) is an amphiphilic macromolecule with surfactant activity. The peculiar behavior of this polymer at the water-air interface is at the basis of its use as material for hydrated microdevices, films, and nanofibers. This work aims to investigate the behavior of PVA and water within the surface domain of highly diluted aqueous solutions by means of atomistic molecular dynamics simulations. Monodisperse atactic oligomers of 30 residues were distributed within water slabs in a vacuum box and allowed to diffuse toward the surface. After equilibration, structural features and dynamical properties of polymer chains and water in the interfacial domains were analyzed as a function of PVA surface concentration at 293 K. Surface pressure values obtained from simulations are in agreement with experimental values at corresponding polymer specific surface areas. In the explored concentration range of 6-34 μmol of residues/m(2), the chains display a transition between two states. At lower surface concentrations, elongated, quite rigid structures are adsorbed on the surface, whereas partially submerged globular aggregates, locally covered by thin water layers, are formed at higher surface concentrations. At PVA concentrations higher than about 20 μmol of residues/m(2), the percolation of chain aggregates over the interface plane produces a surface-confined polymer network with stable pores filled by water molecules. A substantial slowing of polymer and water dynamics in the interfacial domains is highlighted by the mean squared displacement time behavior of terminal residues and the interaction time of PVA-water hydrogen bonding. The diffusion coefficient of water and lifetime of hydrogen bonds between solvent molecules are halved and doubled, respectively, at the interface with the highest polymer concentration. The attenuation of water and polymer mobility concur to stabilize PVA hydrated networks in contact with air.

Molecular dynamics (MD) simulations of a poly(vinyl alcohol), PVA, oligomer in very diluted aqueous solution have been carried out, at the 2-fold aim to investigate structural and dynamical features of this system and to comparatively test the robustness of G45A3 and G45A4 GROMOS force fields in modeling PVA in aqueous environment. An atactic PVA chain of 30 repeating units in water at concentration of 3% (w/w) has been simulated at 293, 303, and 323 K. The trajectory analysis has focused on the temperature influence on chain size, conformational properties and intramolecular hydrogen bonding of PVA. The PVA-water interaction and the polymer induced modifications of water properties have been also investigated. Simulation results have been compared with available experimental data on PVA aqueous solutions, in order to evaluate the reliability of these force fields in the MD simulation of PVA-based systems in aqueous environment. Findings obtained from the simulations with the G45A4 force field have shown a better agreement with experimental results and have highlighted peculiar structural and dynamical characteristics of PVA in dilute aqueous solution. In particular, the simulation has shown a heterogeneity in the overall chain structure, not affected by temperature, and the absence of intrachain hydrogen bonds between nonadjacent residues. The trajectory analysis has revealed that the polymer influence on water structure and dynamics involves about six water molecules per PVA residue.

The relationship between plasma atrial natriuretic factor (ANF), blood pressure (BP), age, plasma renin activity (PRA) and urinary sodium excretion was studied in 64 normal subjects (mean age 48.7 +/- 2.1 yrs; BP: 126.5 +/- 1.6/79.5 +/- 0.9 mmHg) and in 104 untreated uncomplicated essential hypertensives (50.8 +/- 1.1 yrs; BP: 164.7 +/- 1.6/105.2 +/- 0.6 mmHg). ANF was measured by radioimmunoassay after extraction on C18 columns. ANF was significantly higher in the hypertensives than in the normal subjects (37.1 +/- 1.2 vs 29.7 +/- 1.5 pg/ml, P less than 0.01). In normals plasma ANF was significantly correlated with age (r = 0.72, P less than 0.001), Na excretion (r = 0.42, P less than 0.001) and PRA (r = -0.71, P less than 0.001) whereas in the hypertensives ANF plasma levels correlated only with systolic (r = 0.46, P less than 0.001) and diastolic (r = 0.51, P less than 0.001) BP. In addition in hypertensive patients, by multivariate linear regression analysis, a significant correlation was found between age, known duration of hypertension and plasma ANF. The partial correlation coefficient between duration of hypertension and plasma ANF was highly significant (r = 0.80, P less than 0.001). These findings suggest that in essential hypertension the level of arterial BP is a main determinant of the ANF plasma values offsetting the ability of other physiological factors to regulate plasma ANF levels.