Heterogeneity of determining disease severity, clinical course and outcomes in systemic sclerosis-associated interstitial lung disease: a systematic literature review.

Objective The course of systemic sclerosis-associated interstitial lung disease (SSc-ILD) is highly variable and different from continuously progressive idiopathic pulmonary fibrosis (IPF). Most proposed definitions of progressive pulmonary fibrosis or SSc-ILD severity are based on the research data from patients with IPF and are not validated for patients with SSc-ILD. Our study aimed to gather the current evidence for severity, progression and outcomes of SSc-ILD.Methods A systematic literature review to search for definitions of severity, progression and outcomes recorded for SSc-ILD was performed according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines in Medline, Embase, Web of Science and Cochrane Library up to 1 August 2023.Results A total of 9054 papers were reviewed and 342 were finally included. The most frequent tools used for the definition of SSc-ILD progression and severity were combined changes of carbon monoxide diffusing capacity (DLCO) and forced vital capacity (FVC), isolated FVC or DLCO changes, high-resolution CT (HRCT) extension and composite algorithms including pulmonary function test, clinical signs and HRCT data. Mortality was the most frequently reported long-term event, both from all causes or ILD related.Conclusions The studies presenting definitions of SSc-ILD 'progression', 'severity' and 'outcome' show a large heterogeneity. These results emphasise the need for developing a standardised, consensus definition of severe SSc-ILD, to link a disease specific definition of progression as a surrogate outcome for clinical trials and clinical practice.PROSPERO registration number CRD42022379254.Cite Now.

Oxidases as Oxygen Scavengers in Hypoxic Conditions: A Kinetic Model.

A simple kinetic model allowed for the description of the observed decay of the oxygen content in hypoxic aqueous samples with and without headspace, in the presence of glucose oxidase (Glucox) or laccase and their substrates (glucose for Glucox and ABTS for Laccase). The experimental tests involved both the direct measurement of the oxygen content with a fluorescence-based probe and the indirect stopped-flow spectroscopic detection of colored compounds generated from suitable chromogenic reagents. The complete depletion of dissolved oxygen occurred in the no-headspace samples, whereas some residual oxygen remained in a steady state in the samples with headspace. Simple pseudo-first-order kinetics was adequate to describe the behavior of the system, as long as oxygen was the rate-limiting compound, i.e., in the presence of excess substrates. The values of the kinetic constants drawn from best-fit routines of the data from both experimental approaches were quite comparable. The oxygen residues in the samples with headspace seemed related to the low solubility of O2 in the aqueous phase, especially if compared with the large amount of oxygen in the headspace. The extent of such residue decreased by increasing the concentration of the enzyme. The kinetic model proposed in this paper can be of help in assembling suitable sensors to be used for food safety and quality control.

Potential Role of JAK Inhibitors in the Treatment of Systemic Sclerosis-Associated Interstitial Lung Disease: A Narrative Review from Pathogenesis to Real-Life Data.

BACKGROUND: Systemic sclerosis-associated interstitial lung disease (SSc-ILD) is one of the most relevant complications of SSc and the major cause of death. The pathogenesis of SSc-ILD involves a complex interplay of multiple cell types and different molecular pathways, with both inflammation and fibrosis as pathological hallmarks. To date, there are no treatments able to target both components of the disease. Janus kinase inhibitors (JAKinibs) represent an interesting therapeutic option because they exert both anti-inflammatory and anti-fibrotic properties. METHODS: Here, we performed a narrative review concerning the potential role of JAKinibs in SSc-ILD to define the state of art and to evaluate the pathogenetic rationale behind this type of treatment. RESULTS: Currently, few studies investigated SSc-ILD response to JAKinibs treatment. Data were analyzed from three clinical studies and four case reports and progression of SSc-ILD was not evident in 93.5% of patients treated with JAKinibs. CONCLUSIONS: Available evidence of efficacy of JAKinibs in SSc-ILD is sparse but promising. JAKinibs could be an interesting treatment in SSc-ILD because of their potential inhibition of the fibrotic processes combined with their anti-inflammatory action. Moreover, JAKinibs were also shown in some studies to have a potential effect on pulmonary arterial hypertension (PAH), another threatening complication in SSc. More data are necessary to define JAKinibs role in SSc-ILD treatment.

Dietary Polyphenols and In Vitro Intestinal Fructose Uptake and Transport: A Systematic Literature Review.

Recent evidence links chronic consumption of large amounts of fructose (FRU) with several non-communicable disease. After ingestion, dietary FRU is absorbed into the intestinal tract by glucose transporter (GLUT) 5 and transported to the portal vein via GLUT2. GLUT2 is primarily localized on the basolateral membrane, but GLUT2 may be dislocated post-prandially from the basolateral membrane of intestinal cells to the apical one. Polyphenols (PP) are plant secondary metabolites that exert hypoglycemic properties by modulating intracellular insulin signaling pathways and by inhibiting intestinal enzymes and transporters. Post-prandially, PP may reach high concentrations in the gut lumen, making the inhibition of FRU absorption a prime target for exploring the effects of PP on FRU metabolism. Herein, we have systematically reviewed studies on the effect of PP and PP-rich products on FRU uptake and transport in intestinal cells. In spite of expectations, the very different experimental conditions in the various individual studies do not allow definitive conclusions to be drawn. Future investigations should rely on standardized conditions in order to obtain comparable results that allow a credible rating of polyphenols and polyphenol-rich products as inhibitors of fructose uptake.

The Use and Utility of Machine Learning in Achieving Precision Medicine in Systemic Sclerosis: A Narrative Review.

BACKGROUND: Systemic sclerosis (SSc) is a rare connective tissue disease that can affect different organs and has extremely heterogenous presentations. This complexity makes it difficult to perform an early diagnosis and a subsequent subclassification of the disease. This hinders a personalized approach in clinical practice. In this context, machine learning (ML), a branch of artificial intelligence (AI), is able to recognize relationships in data and predict outcomes. METHODS: Here, we performed a narrative review concerning the application of ML in SSc to define the state of art and evaluate its role in a precision medicine context. RESULTS: Currently, ML has been used to stratify SSc patients and identify those at high risk of severe complications. Additionally, ML may be useful in the early detection of organ involvement. Furthermore, ML might have a role in target therapy approach and in predicting drug response. CONCLUSION: Available evidence about the utility of ML in SSc is sparse but promising. Future improvements in this field could result in a big step toward precision medicine. Further research is needed to define ML application in clinical practice.

Protein interactions in the biological assembly of iron-sulfur clusters in Escherichia coli: Molecular and mechanistic aspects of the earliest assembly steps.

This contribution focuses on the earliest steps of the assembly of FeS clusters and their insertion into acceptor apoproteins, that call for transient formation of a 2Fe2S cluster on a scaffold protein from sulfide and iron salts. For the sake of simplicity, this report is essentially limited to the Escherichia coli isc-encoded proteins and does not take into account agents that modulate the enzymatic synthesis of sulfide by protein in the same operon or the redox events associated with both sulfide generation and conversion of 2Fe2S structures in clusters of higher nuclearity. Therefore, the results discussed here are based on chemical reconstitution systems using inorganic sulfide, ferric salts, and excess thiols. This simplification offers the possibility to address some mechanistic issues related to the role of protein/protein interaction as for modulating: (a) the rate of cluster assembly on scaffold proteins; (b) the stability of the cluster on the scaffold protein; and (c) the rate of transfer to acceptor apoproteins as also influenced by the acceptor concentration. The emerging picture highlights the mechanistic versatility of the systems, that is discussed in terms of the capability of such an apparently simple combination of proteins to cope with various physiological situation. The hypothetical mechanism presented here may represent an additional way of modulating the rate and outcome of the overall process while avoiding potential toxicity issues.

Beta-Lactoglobulin as a Model Food Protein: How to Promote, Prevent, and Exploit Its Unfolding Processes.

Bovine milk beta-lactoglobulin (BLG) is a small whey protein that is a common ingredient in many foods. Many of the properties of BLG relevant to the food industry are related to its unfolding processes induced by physical or chemical treatments. Unfolding occurs through a number of individual steps, generating transient intermediates through reversible and irreversible modifications. The rate of formation of these intermediates and of their further evolution into different structures often dictates the outcome of a given process. This report addresses the main structural features of the BLG unfolding intermediates under conditions that may facilitate or impair their formation in response to chemical or physical denaturing agents. In consideration of the short lifespan of the transient species generated upon unfolding, this review also discusses how various methodological approaches may be adapted in exploring the process-dependent structural modifications of BLG from a kinetic and/or a thermodynamic standpoint. Some of the conceptual and methodological approaches presented and discussed in this review can provide hints for improving the understanding of transient conformers formation by proteins present in other food systems, as well as when other physical or chemical denaturing agents are acting on proteins much different from BLG in complex food systems.

Dipeptidyl peptidase 4/CD26 expression in human idiopathic inflammatory myopathies reveals skeletal muscle injury and vascular inflammation.

OBJECTIVES: We performed a retrospective and prospective observational study to investigate whether the T lymphocyte activation antigen dipeptidyl peptidase 4 (DPP4)/CD26 is expressed in the skeletal muscle of patients with idiopathic inflammatory myopathies (IIM) and whether its expression offers clues to understand the events taking place in the tissue. METHODS: CD26 expression in the muscle, evaluated by immunofluorescence, was assessed in 32 patients with IIM and 5 healthy controls and compared among patients with dermatomyositis (DM), immune-mediated necrotising myopathy (IMNM), inclusion body myositis (IBM), and polymyositis (PM). The relationship of CD26 expression and localization with clinical, serological and histological features was determined. RESULTS: CD26 is selectively expressed in the skeletal muscle of patients with IIM. The highest levels of CD26 are found in the skeletal muscle from patients with DM and in particular in those characterized by tissue necrosis and vascular inflammation. CD26 expression is associated with decreased muscle performance and independently predicts the number of treatments before reaching disease stabilization or improvement (odds ratio, OR=1.2, p<0.05). CONCLUSIONS: CD26 is expressed in the IIM skeletal muscle and may represent a target of molecular intervention for patients with treatment-refractory myositis.

The Yin-Yang Pharmacomicrobiomics on Treatment Response in Inflammatory Arthritides: A Narrative Review.

(1) Background: Gut microbiota (GM) is the set of microorganisms inhabiting the gastroenteric tract that seems to have a role in the pathogenesis of rheumatic diseases. Recently, many authors proved that GM may influence pharmacodynamics and pharmacokinetics of several drugs with complex interactions that are studied by the growing field of pharmacomicrobiomics. The aim of this review is to highlight current evidence on pharmacomicrobiomics applied to the main treatments of Rheumatoid Arthritis and Spondyloarthritis in order to maximize therapeutic success, in the framework of Personalized Medicine. (2) Methods: We performed a narrative review concerning pharmacomicrobiomics in inflammatory arthritides. We evaluated the influence of gut microbiota on treatment response of conventional Disease Modifying Anti-Rheumatic drugs (cDMARDs) (Methotrexate and Leflunomide) and biological Disease Modifying Anti-Rheumatic drugs (bDMARDs) (Tumor necrosis factor inhibitors, Interleukin-17 inhibitors, Interleukin 12/23 inhibitors, Abatacept, Janus Kinase inhibitors and Rituximab). (3) Results: We found a great amount of studies concerning Methotrexate and Tumor Necrosis Inhibitors (TNFi). Conversely, fewer data were available about Interleukin-17 inhibitors (IL-17i) and Interleukin 12/23 inhibitors (IL-12/23i), while none was identified for Janus Kinase Inhibitors (JAKi), Tocilizumab, Abatacept and Rituximab. We observed that microbiota and drugs are influenced in a mutual and reciprocal way. Indeed, microbiota seems to influence therapeutic response and efficacy, whereas in the other hand, drugs may restore healthy microbiota. (4) Conclusions: Future improvement in pharmacomicrobiomics could help to detect an effective biomarker able to guide treatment choice and optimize management of inflammatory arthritides.

Circular Dichroism to Probe the Synthesis, Transfer, and Stability of Fe-S Clusters.

All Fe-S proteins are characterized by distinctive circular dichroism (CD) features in the visible region of the spectrum due to chiral interaction between the cluster itself and the protein backbone. Therefore, the presence of a CD signal in the visible region relates to the presence of the cluster, whereas the disappearance of the signal refers to cluster breakdown or redox changes. The position of the CD features in the spectrum and the intensity of individual components of the CD signal show great variations among different Fe-S proteins. This feature can provide information on transfer processes between proteins, as well as on possible changes in cluster nuclearity. This method can also be used to detect changes in the chemical nature or spatial organization of cluster ligands that may be concurrent with cluster transfer and associated events.

Monitoring the carryover of egg proteins in pasta making to support allergen risk management.

Egg proteins are among the major food allergens. Very often, the same pasta-making plants are used for industrial production of egg-based pasta (EBP) and semolina-only pasta (SP), so that residual egg proteins may be present in SP. This calls for defining the amount of semolina pasta that should be discarded when switching production lines. In this study, the egg proteins content was measured in pasta samples taken at various times after switching production lines from EBP to SP Both long and short pasta shapes were sampled before and after a drying step. Protocols meant to circumvent the difficulties associated with detecting egg proteins in a complex matrix after processing were set up for using commercial ELISA kits to monitor the disappearance of egg proteins from the products. The use of both denaturants and disulphide reductants to solubilise egg proteins was found to be mandatory, as verified by ovalbumin detection by ELISA and by using mass spectrometry to assess residual egg white lysozyme. Appropriate sample preparation protocols were used to monitor the progressive disappearance of egg proteins in the products when shifting production lines in an industrial pasta plant, providing a basis for credible, reliable, and consistent self-control procedures. For lines with a production capacity of 2200-2400 kg h-1, the amount of material to be discarded to ensure that products meet the strictest analytical requirements has been found to be around 2000-3000 kg (for long pasta) and 3000-4000 kg (for short pasta).

Redox Titration of Flavoproteins: An Overview.

Redox titration of flavoproteins allows to detect and analyze (1) the determinants of the stabilization of individual redox forms of the flavin by the protein; (2) the binding of the redox-active cofactor to the protein; (3) the effects of other components of the systems (such as micro- or macromolecular interactors) on parameters 1 and 2; (4) the pattern of electron flow to and from the flavin cofactor to other redox-active chemical species, including those present in the protein itself or in its physiological partners. This overview presents and discusses the fundamentals of the methodological approaches most commonly used for these purposes, and illustrates how data may be obtained in a reliable way, and how they can be read and interpreted.

Begelomab for severe refractory dermatomyositis: A case report.

RATIONALE: Severe refractory idiopathic inflammatory myopathy (IIM) represents a challenge for the clinician. The lack of efficacy of available tools reflects our incomplete insight into the molecular events sustaining the inflammatory tissue damage in these patients. We present the first case of refractory IIM treated with anti-dipeptidyl peptidase-4 (DPP-4)/cluster of differentiation 26 (CD26) monoclonal antibody. PATIENT CONCERNS: A 55-year old man presented with proximal muscle weakness, diffuse erythematous skin lesions which rapidly evolved into ulcerations, dysphagia and dysphonia. DIAGNOSIS: Increased serum creatine kinase levels and histological findings at muscle and skin biopsies were compatible with the diagnosis of dermatomyositis (DM). Several lines of treatment failed to control the disease including steroids, mycophenolate mofetil, tacrolimus, intravenous immunoglobulins and rituximab. Despite therapy, the patient also had recurrent intestinal vasculitis causing bowel perforation. Concurrently, DPP-4/CD26 expression in the patient's skin and skeletal muscle was observed. INTERVENTIONS: The patient was treated with begelomab, a murine immunoglobulin G2b monoclonal antibody against DPP-4/CD26. OUTCOMES: Dysphagia, skin lesions and intestinal vasculitis resolved and the patient experienced a significant improvement of his quality of life. CONCLUSION: Blockade of DPP-4/CD26, which is expressed on T cells and mediates T cell activation and function, is safe and might be effective in patients with refractory DM.

Cefoxitin treatment of MRSA leads to a shift in the IL-12/IL-23 production pattern in dendritic cells by a mechanism involving changes in the MAPK signaling.

Methicillin resistant Staphylococcus aureus (MRSA) constitute a serious health care problem worldwide. This study addresses the effect of ß-lactam treatment on the ability of clinically relevant MRSA strains to induce IL-12 and IL-23. MRSA strains induced a dose-dependent IL-12 response in murine bone-marrow-derived dendritic cells that was dependent on endocytosis and acidic degradation. Facilitated induction of IL-12 (but not of IL-23) called for activation of the MAP kinase JNK, and was suppressed by p38. Compromised peptidoglycan structure in cefoxitin-treated bacteria - as denoted by increased sensitivity to mutanolysin -caused a shift from IL-12 towards IL-23. Moreover, cefoxitin treatment of MRSA led to a p38 MAPK-dependent early up-regulation of Dual Specificity Phosphatase (DUSP)-1. Compared to common MRSA, characteristics associated with a persister phenotype increased intracellular survival and upon cefoxitin treatment, the peptidoglycan was not equally compromised and the cytokine induction still required phagosomal acidification. Together, these data demonstrate that ß-lactam treatment changes the MRSA-induced IL-12/IL-23 pattern determined by the activation of JNK and p38. We suggest that accelerated endosomal degradation of the peptidoglycan in cefoxitin-treated MRSA leads to an early expression of DUSP-1 and accordingly, a reduction in the IL-12/IL-23 ratio in dendritic cells. This may influence the clearance of S. aureus.

Bio-Functional and Structural Properties of Pasta Enriched with a Debranning Fraction from Purple Wheat.

A colored and fiber-rich fraction from the debranning of purple wheat was incorporated at 25% into semolina- and flour-based pasta produced on a pilot-plant scale, with the aim of increasing anthocyanin and total phenolic content with respect to pasta obtained from whole pigmented grains. The debranning fraction impaired the formation of disulfide-stabilized protein networks in semolina-based systems. Recovery of phenolics was impaired by the pasta making process, and cooking decreased the phenolic content in both enriched samples. Cooking-related losses in anthocyanins and total phenolics were similar, but anthocyanins in the cooked semolina-based pasta were around 20% of what was expected from the formulation. HPLC (High Performance Liquid Chromatography) profiling of phenolics was carried out on extracts from either type of enriched pasta both before and after cooking and indicate possible preferential retention of specific compounds in each type of enriched pasta. Extracts from cooked samples of either enriched pasta were tested as inhibitors of enzymes involved in glucose metabolism and uptake, as well as for their capacity of suppressing the response to inflammatory stimuli. Results of both biological tests indicate that the phenolics in extracts from both cooked pasta samples had inhibitory capacities higher than extracts of the original debranning fraction at identical concentrations of total bioactives.

Inhibition of Pancreatic α-amylase by Resveratrol Derivatives: Biological Activity and Molecular Modelling Evidence for Cooperativity between Viniferin Enantiomers.

To improve the current understanding of the role of stilbenoids in the management of diabetes, the inhibition of the pancreatic α-amylase by resveratrol derivatives was investigated. To approach in a systematic way, the mechanistic and structural aspects of the interaction, potential bioactive agents were prepared as single molecules, that were used for the biological evaluation of the determinants of inhibitory binding. Some dimeric stilbenoids-in particular, viniferin isomers- were found to be better than the reference drug acarbose in inhibiting the pancreatic α-amylase. Racemic mixtures of viniferins were more effective inhibitors than the respective isolated pure enantiomers at an equivalent total concentration, and displayed cooperative effects not observed with the individual enantiomers. The molecular docking analysis provided a thermodynamics-based rationale for the measured inhibitory ability and for the observed synergistic effects. Indeed, the binding of additional ligands on the surface of the alpha-amylase was found to decrease the dissociation constant of inhibitors bound to the active site of the enzyme, thus providing a mechanistic rationale for the observed inhibitory synergies.

Effects of starch addition on the activity and specificity of food-grade lipases.

Lipases are surface-active enzymes, acting on their substrates at the polar/nonpolar interface in emulsions. This study was aimed to test whether their activity, specificity, and the rates of formation/degradation of the various hydrolysis intermediates (i.e., mono- and diglycerides of interest as surface-active agents) could be modulated by adhesion of the triglyceride substrates as a thin layer on the surface of solids. These hypotheses were tested by using an array of food-grade lipases used in bakery, testing various types of starch as the "solid" phase. Starch-dependent increase in the hydrolysis rate was tested by pH-stat techniques on pure triglycerides and on food-grade oils in diluted emulsions. Starch-related improvements in the rate of fatty acids release were most evident at temperatures above 40 °C, and when using maize starch instead of wheat starch. Starch-dependent changes in the nature of the hydrolysis products were tested by chromatographic profiling of ethyl ether extracts from aqueous slurries containing up to 33% fat and 33% starch. Accumulation of mono- and diglycerides as hydrolysis intermediates was found to be modulated by the type of oil being used, by the reaction conditions, as well as by the enzyme nature and amount.

Greetings from foodland: Teaching biochemistry to BS students in food-related courses in Italy.

Biochemistry has always been a mandatory topic within BS courses aimed at food science students at the University of Milan, namely: Food Science & Technology and Catering Sciences. Addressing biochemistry topics in this peculiar setting requires: (i) specific focus on topics that are seldom considered in courses offered in bio-medical curricula; (ii) close integration with other area disciplines, such as food biotechnology; (iii) ad hoc design of laboratory classes; and (iv) an array of elective courses covering specific aspects of biochemistry. In this context, for example, protein chemistry is presented by using food proteins of known structure and discussed in terms of structural features in the raw materials and of structural and chemical modifications occurring upon processing. Along the same lines, metabolic pathways and their regulation are presented starting from widespread metabolism-related issues and to issues related to food safety (including food allergies and intolerances). A similar "hands on" approach is used for laboratory classes, that cover about one third of total credits and are aimed at providing fundamental-type information by analyzing practical situations in the food chain. In spite of their inherent complexity and volume, biochemistry courses score very well with the students in mandatory anonymous surveys. Our approach to biochemistry courses seems to help the students in "visualizing" the practical implications of concepts acquired in other courses within their curricula. The students' appreciation is confirmed by the sizeable attendance to elective and specialized biochemical-themed courses. © 2019 International Union of Biochemistry and Molecular Biology, 47(4):394-403, 2019.

Surface Layer of Lactobacillus helveticus MIMLh5 Promotes Endocytosis by Dendritic Cells.

Surface layers (S-layers) are proteinaceous arrays covering the cell walls of numerous bacteria. Their suggested properties, such as interactions with the host immune system, have been only poorly described. Here, we aimed to elucidate the role of the S-layer from the probiotic bacterial strain Lactobacillus helveticus MIMLh5 in the stimulation of murine bone-marrow-derived dendritic cells (DCs). MIMLh5 induced greater production of interferon beta (IFN-ß), interleukin 10 (IL-10), and IL-12p70, compared to S-layer-depleted MIMLh5 (naked MIMLh5 [n-MIMLh5]), whereas the isolated S-layer was a poor immunostimulator. No differences in the production of tumor necrosis factor alpha (TNF-α) or IL-1ß were found. Inhibition of the mitogen-activated protein kinases JNK1/2, p38, and ERK1/2 modified IL-12p70 production similarly in MIMLh5 and n-MIMLh5, suggesting the induction of the same signaling pathways by the two bacterial preparations. Treatment of DCs with cytochalasin D to inhibit endocytosis before the addition of fluorescently labeled MIMLh5 cells led to a dramatic reduction in the proportion of fluorescence-positive DCs and decreased IL-12 production. Endocytosis and IL-12 production were only marginally affected by cytochalasin D pretreatment when fluorescently labeled n-MIMLh5 was used. Treatment of DCs with fluorescently labeled S-layer-coated polystyrene beads (Sl-beads) resulted in much greater uptake of beads, compared to noncoated beads. Prestimulation of DCs with cytochalasin D reduced the uptake of Sl-beads more than plain beads. These findings indicate that the S-layer plays a role in the endocytosis of MIMLh5 by DCs. In conclusion, this study provides evidence that the S-layer of L. helveticus MIMLh5 is involved in endocytosis of the bacterium, which is important for strong Th1-inducing cytokine production.IMPORTANCE Beneficial microbes may positively affect host physiology at various levels, e.g., by participating in immune system maturation and modulation, boosting defenses and dampening reactions, thus affecting the whole homeostasis. As a consequence, the use of probiotics is increasingly regarded as suitable for more extended applications for health maintenance, not only microbiota balancing. This implies a deep knowledge of the mechanisms and molecules involved in host-microbe interactions, for the final purpose of fine tuning the choice of a probiotic strain for a specific outcome. With this aim, studies targeted to the description of strain-related immunomodulatory effects and the identification of bacterial molecules responsible for specific responses are indispensable. This study provides new insights in the characterization of the food-origin probiotic bacterium L. helveticus MIMLh5 and its S-layer protein as a driver for the cross-talk with DCs.

Topological features of the intermolecular contacts in gluten-forming proteins: Exploring a novel methodological approach based on gold nanoparticles.

This work introduces a novel methodological approach to study both the geometry of complex protein networks and the nature of the interacting proteins. This approach is based on the high reactivity of Au+ ions on the surface of gold nanoparticles (AuNPs) towards thiols, that allows fast formation of a covalent bond between accessible protein thiols and AuNPs. In the case of the durum wheat semolina used in the exploratory studies reported here, the nature of proteins covalently bound to AuNPs is expected to be affected by both the compactness of the protein network and by the AuNPs size. Simple centrifugation procedures allowed recovery of the protein-loaded AuNPs that remained soluble, and the protein(s) covalently bound on the surface of soluble AuNP were identified by MS analysis of their proteolytic fragments. Gluten-forming proteins were found to be bound to soluble AuNPs only when detergents or chaotropes were added to the semolina/AuNPs suspension at room temperature. AuNPs-bound proteins also included gluten-forming proteins with no reported free thiols, suggesting that they are piggybacked on other thiol-containing gluten-forming proteins via disulfide bonds already present in the otherwise untreated semolina. The potential of this approach is discussed in terms of the possibility of developing a methodology suitable for further clarification of the geometrical features of protein networks, of the nature of the involved proteins, and of the type of interaction they establish, as well as any modifications of these features upon processing.