To investigate thiol-disulfide interchange reactions in heated milk yielding non-native intramolecular rearranged and intermolecular cross-linked proteins, a proteomic study based on nanoLC-ESI-Q-Orbitrap-MS/MS and dedicated bioinformatics was accomplished. Raw milk samples heated for different times and various commercial dairy products were analyzed. Qualitative experiments on tryptic digests of resolved protein mixtures assigned the corresponding disulfide-linked peptides. Results confirmed the limited data available on few milk proteins, generated the widest inventory of components (63 in number) involved in thiol-disulfide exchange processes, and provided novel structural information on S-S-bridged molecules. Quantitative experiments on unresolved protein mixtures from both sample typologies estimated the population of molecules associated with thiol-disulfide reshuffling processes. Disulfide-linked peptides associated with native intramolecular S-S bonds generally showed a progressive reduction depending on heating time/harshness, whereas those related to specific non-native intramolecular/intermolecular ones showed an opposite quantitative trend. This was associated with a temperature-dependent augmented reactivity of definite native protein thiols and S-S bridges, which determined the formation of non-native rearranged monomers and cross-linked oligomers. Results provided novel information for possibly linking the nature and extent of thiol-disulfide exchange reactions in heated milk proteins to the corresponding functional and technological characteristics, with possible implications on food digestibility, allergenicity, and bioactivity.

Tuberous sclerosis complex (TSC) is a rare, multisystem genetic disorder that leads to the development of benign tumors in multiple organs and neurological symptoms. TSC clinical manifestations show a great heterogenicity, with most patients presenting severe neuropsychiatric and neurological disorders. TSC is caused by loss-of-function mutations in either Tsc1 or Tsc2 genes, leading to overexpression of the mechanistic target of rapamycin (mTOR) and, consequently, abnormal cellular growth, proliferation and differentiation as well as to cell migration defects. Beside the growing interest, TSC remains a disorder poorly understood, with limited perspectives in the field of therapeutic strategies. Here we used murine postnatal subventricular zone (SVZ) neural stem progenitor cells (NSPCs) deficient of Tsc1 gene as a TSC model to unravel novel molecular aspects of the pathophysiology of this disease. 2D-DIGE-based proteomic analysis detected 55 differently represented spots in Tsc1-deficient cells, compared to wild-type counterparts, which were associated with 36 protein entries after corresponding trypsinolysis and nanoLC-ESI-Q-Orbitrap-MS/MS analysis. Proteomic results were validated using various experimental approaches. Bioinformatics associated differently represented proteins with oxidative stress and redox pathways, methylglyoxal biosynthesis, myelin sheath, protein S-nitrosylation and carbohydrate metabolism. Because most of these cellular pathways have already been linked to TSC features, these results were useful to clarify some molecular aspects of TSC etiopathogenesis and suggested novel promising therapeutic protein targets. SIGNIFICANCE: Tuberous Sclerosis Complex (TSC) is a multisystemic disorder caused by inactivating mutations of TSC1 or TSC2 genes, which induce overactivation of the mTOR component. The molecular mechanisms underlying the pathogenesis of TSC remain unclear, probably due to complexity of mTOR signaling network. To have a picture of protein abundance changes occurring in TSC disorder, murine postnatal subventricular zone (SVZ) neural stem progenitor cells (NSPCs) deficient of Tsc1 gene were used as a model of disease. Thus, Tsc1-deficient SVZ NSPCs and wild-type cells were comparatively evaluated by proteomics. This analysis evidenced changes in the abundance of proteins involved in oxidative/nitrosative stress, cytoskeleton remodelling, neurotransmission, neurogenesis and carbohydrate metabolism. These proteins might clarify novel molecular aspects of TSC etiopathogenesis and constitute putative molecular targets for novel therapeutic management of TSC-related disorders.

In recent years, the restoration of metal(loid) polluted soils through the combined use of plants-amendments-microorganisms is a strategy that is receiving great attention. However, the molecular processes underlying this synergy are not fully understood. Thus, the aim of this work was to provide insight into the biological mechanisms used by Arabidopsis thaliana to grow in soil contaminated by arsenic and lead and amended with biochar and/or Bacillus sp. inoculum. To accomplish this goal, a pot experiment was set up and the effects of the biochar amendment and the bacterial isolate were evaluated, both alone and in combination. The effects of the plant-biochar-bacteria synergy were assessed on soil physicochemical characteristics, plant growth and ability to stabilize or accumulate metal(loid)s. In addition, a bioinformatics-assisted proteomics approach was used to understand the molecular processes underlying A. thaliana growth in the different tested conditions. Results showed that the use of biochar and/or Bacillus inoculum resulted in improvements in soil properties and plant growth. Bioinformatics-assisted proteomic analysis showed that, on the one hand, the use of biochar alone led to an over-representation of proteins involved in nutrient metabolism providing plants with essential nutrients for growth. However, biochar alone induced plant defense mechanism dysfunction and increased susceptibility to pathogen attack. On the other hand, the use of bacterial inoculation helped plants to grow thanks to the activation of molecular pathways involved in the defense against biotic stress. Only the combined use of biochar and bacteria ensured the correct balance between molecular processes associated with growth and metal(loid) stress response in Arabidopsis plants.

Background: Various bioactive peptides are present in foods and food protein hydrolysates, or are generated in the stomach/intestine of organisms after digestion of dietary proteins. Those resisting gastrointestinal degradation can exert local effects in the gut or systemic effects in the organism body as result of their transport across the intestinal epithelium in the bloodstream, and subsequent adsorption in various organs. For most of these molecules, no concentration data regarding body fluids/tissues are available; this information is essential to rationalize their bioavailability and putative bioactivity. Scope and approach: The main purpose of this study is to provide an exhaustive overview of the bioactive food-derived peptides identified in the gastrointestinal tract, blood, body tissues, urine, breastmilk and feces of animal models or humans fed specific diets, as well as a description of the adsorption mechanisms and metabolic processes eventually affecting their fate. Untargeted and targeted peptidomic methods used for their quali-quantitative description are also reported, together with recent technological advances that have partially solved various analytical challenges in this research field and have disclosed future promising scenarios in nutrition and physiology. Key findings and conclusions: Available information emphasizes that organism tissues/body fluids are pervaded of food-derived species resulting from the digestion of dietary proteins, including some already proved having a specific biological activity. For some for which blood concentration was measured, ascertained data highlight levels in the nanomolar range, which are lower than those generally used for in vitro functional assays. Conversely, few peptides have shown concentration values compatible with a substantial molecular bioavailability and a putative bioactivity. Thus, it remains uncertain if the presence of bioactive food-derived peptides in the body fluids/tissues can be associated with a significant functional effect. Accordingly, the actual study of these exogenous peptides in the human body is more relevant than ever, with the ultimate aim of tangling the complex relationship between diet and health.

Chemical communication in elephants has been well studied at the chemical and behavioural levels. Pheromones have been identified in the Asian elephant (Elephas maximus), including (Z)-7-dodecenyl acetate and frontalin, and their specific effects on the sexual behaviour of elephants have been accurately documented. In contrast, our knowledge on the proteins mediating detection of pheromones in elephants remains poor and superficial, with only three annotated and reliable entries in sequence databases, two of them being odorant-binding proteins (OBPs), and the third a member of von Ebner's gland (VEG) proteins. Proteomic analysis of trunk wash extract from African elephant (Loxodonta africana) identified one of the OBPs (LafrOBP1) as the main component. We therefore expressed LafrOBP1 and its Asian elephant orthologue in yeast Pichia pastoris and found that both recombinant proteins, as well as the natural LafrOBP1 are tuned to (Z)-7-dodecenyl acetate, but have no affinity for frontalin. Both the natural and recombinant LafrOBP1 carry post-translational modifications such as O-glycosylation, phosphorylation and acetylation, but as these modifications affect only a very small amount of the protein, we cannot establish their potential effects on the ligand-binding properties of OBP1.

Recent findings have demonstrated that mitochondria can be transferred between cells to control metabolic homeostasis. Although the mitochondria of brown adipocytes comprise a large component of the cell volume and undergo reorganization to sustain thermogenesis, it remains unclear whether an intercellular mitochondrial transfer occurs in brown adipose tissue (BAT) and regulates adaptive thermogenesis. Herein, we demonstrated that thermogenically stressed brown adipocytes release extracellular vesicles (EVs) that contain oxidatively damaged mitochondrial parts to avoid failure of the thermogenic program. When re-uptaken by parental brown adipocytes, mitochondria-derived EVs reduced peroxisome proliferator-activated receptor-? signaling and the levels of mitochondrial proteins, including UCP1. Their removal via the phagocytic activity of BAT-resident macrophages is instrumental in preserving BAT physiology. Depletion of macrophages in vivo causes the abnormal accumulation of extracellular mitochondrial vesicles in BAT, impairing the thermogenic response to cold exposure. These findings reveal a homeostatic role of tissue-resident macrophages in the mitochondrial quality control of BAT.

Pheromonal communication is widespread among living organisms, but in apes and particularly in humans there is currently no strong evidence for such phenomenon. Among primates, lemurs use pheromones to communicate within members of the same species, whereas in some monkeys such capabilities seem to be lost. Chemical communication in humans appears to be impaired by the lack or malfunctioning of biochemical tools and anatomical structures mediating detection of pheromones. Here, we report on a pheromone-carrier protein (SAL) adopting a "reverse chemical ecology" approach to get insights on the structures of potential pheromones in a representative species of lemurs (Microcebus murinus) known to use pheromones, Old-World monkeys (Cercocebus atys) for which chemical communication has been observed, and humans (Homo sapiens), where pheromones and chemical communication are still questioned. We have expressed the SAL orthologous proteins of these primate species, after reconstructing the gene encoding the human SAL, which is disrupted due to a single base mutation preventing its translation into RNA. Ligand-binding experiments with the recombinant SALs revealed macrocyclic ketones and lactones as the best ligands for all three proteins, suggesting cyclopentadecanone, pentadecanolide, and closely related compounds as the best candidates for potential pheromones. Such hypothesis agrees with the presence of a chemical very similar to hexadecanolide in the gland secretions of Mandrillus sphinx, a species closely related to C. atys. Our results indicate that the function of this carrier protein has not changed much during evolution from lemurs to humans, although its physiological role has been certainly impaired in humans.

Streptomyces coelicolor is a model organism for the study of Streptomyces, a genus of Gram-positive bacteria that undergoes a complex life cycle and produces a broad repertoire of bioactive metabolites and extracellular enzymes. This study investigated the production and characterization of membrane vesicles (MVs) in liquid cultures of S. coelicolor M145 from a structural and biochemical point of view; this was achieved by combining microscopic, physical and -omics analyses. Two main populations of MVs, with different size and cargo, were isolated and purified. S. coelicolor MV cargo was determined being complex and containing different kinds of proteins and metabolites. In particular, a whole of 166 proteins involved in cell metabolism/differentiation, molecular processing/transport, and stress response was identified in MVs, the latter functional class being also important for bacterial morpho-physiological differentiation. A subset of these proteins was protected from degradation following treatment of MVs with proteinase K, indicating their localization inside the vesicles. Moreover, S. coelicolor MVs contained an array of metabolites, such as antibiotics, vitamins, amino acids and components of carbon metabolism. In conclusion, this analysis provides detailed information on S. coelicolor MVs under basal conditions and corresponding content, which may be useful in a next future to elucidate vesicle biogenesis and functions. Importance Streptomycetes are widely distributed in nature, and they are characterized by a complex life cycle that involves morphological differentiation. They are very relevant in industry because they produce about a half of the antibiotics used clinically and other important pharmaceutical products having natural origin. Streptomyces coelicolor is a model organism for the study of bacterial differentiation and bioactive molecule production. S. coelicolor produces extracellular vesicles carrying many molecules such as proteins and metabolites, including antibiotics. The elucidation of S. coelicolor extracellular vesicle cargo will help to understand different aspects of streptomycete physiology, such as cell communication during differentiation and response to environmental stimuli. Moreover, the capability of carrying different kind of biomolecules opens up new biotechnological possibilities related to drug delivery. Indeed, the decoding of molecular mechanisms involved in cargo selection may lead to the customization of the content of extracellular vesicles.

Various protein cross-linking reactions leading to molecular polymerization and covalent aggregates have been described in processed foods. They are an undesired side effect of processes designed to reduce bacterial load, extend shelf life, and modify technological properties, as well as being an expected result of treatments designed to modify raw material texture and function. Although the formation of these products is known to affect the sensory and technological properties of foods, the corresponding cross-linking reactions and resulting protein polymers have not yet undergone detailed molecular characterization. This is essential for describing how their generation can be related to food processing conditions and quality parameters. Due to the complex structure of cross-linked species, bottom-up proteomic procedures developed to characterize various amino acid modifications associated with food processing conditions currently offer a limited molecular description of bridged peptide structures. Recent progress in cross-linking mass spectrometry for the topological characterization of protein complexes has facilitated the development of various proteomic methods and bioinformatic tools for unveiling bridged species, which can now also be used for the detailed molecular characterization of polymeric cross-linked products in processed foods. We here examine their benefits and limitations in terms of evaluating cross-linked food proteins and propose future scenarios for application in foodomics. They offer potential for understanding the protein cross-linking formation mechanisms in processed foods, and how the inherent beneficial properties of treated foodstuffs can be preserved or enhanced.

Metal(loid)s are toxic to animal life, human health, and plants, therefore, their removal from polluted areas is imperative in order to minimize their impact on the ecosystems. The use of plant-amendment-microorganism synergy is a promising option, but not yet fully explored, to manage lands contaminated with metal(loid)s. However, molecular factors and mechanisms underlying this interaction are also almost unknown. The aim of the present study was to characterize Arabidopsis thaliana growth and response on arsenic and lead polluted soil. To accomplish this aim, a pot experiment was performed testing the effect of biochar and/or autochthonous metal(loid) resistant Bacillus isolate on physico-chemical soil properties and on plant growth and metal(loid) uptake/intake. Furthermore, bioinformatics-assisted proteomics approach was used to understand common and specific mechanisms regulating plant growth and metal(loid) tolerance in tested conditions. Results showed that biochar and/or Bacillus induced significant and positive effects on soil properties, increasing pH, Ctot, Ntot and Ptot concentrations and decreasing nutrients (Nav and Pav), As and Pb availability. Plant growth was also enhanced by addition of biochar and/or inoculum, reaching the maximum when biochar and bacteria were combined. The deciphering of molecular mechanisms revealed that combination of biochar and bacterial inoculation mitigate Arabidopsis growth and defense tradeoff and underline the great potential of plant-biochar-inoculum synergic application in more effective and large scale-up phytostabilizing systems.

Background: The human SH3 domain Binding Glutamic acid Rich Like 3 (SH3BGRL3) gene is highly conserved in phylogeny and widely expressed in human tissues. However, its function is largely undetermined. The protein was found to be overexpressed in several tumors, and recent work suggested a possible relationship with EGFR family members. We aimed at further highlighting on these issues and investigated SH3BGRL3 molecular interactions and its role in cellular migration ability. Results: We first engineered the ErbB2-overexpressing SKBR3 cells to express exogenous SH3BGRL3, as well as wild type Myo1c or different deletion mutants. Confocal microscopy analysis indicated that SH3BGRL3 co-localized with Myo1c and ErbB2 at plasma membranes. However, co-immunoprecipitation assays and mass spectrometry demonstrated that SH3BGRL3 did not directly bind ErbB2, but specifically recognized Myo1c, on its IQ-bearing neck region. Importantly, the interaction with Myo1c was Ca2+-dependent. A role for SH3BGRL3 in cell migration was also assessed, as RNA interference of SH3BGRL3 in MDA-MB-231 cells, used as a classical migration model, remarkably impaired the migration ability of these cells. On the other side, its over-expression increased cell motility. Conclusion: The results of this study provide insights for the formulation of novel hypotheses on the putative role of SH3BGRL3 protein in the regulation of myosin-cytoskeleton dialog and in cell migration. It could be envisaged the SH3BGRL3-Myo1c interaction as a regulation mechanism for cytoskeleton dynamics. It is well known that, at low Ca2+ concentrations, the IQ domains of Myo1c are bound by calmodulin. Here we found that binding of Myo1c to SH3BGRL3 requires instead the presence of Ca2+. Thus, it could be hypothesized that Myo1c conformation may be modulated by Ca2+-driven mechanisms that involve alternative binding by calmodulin or SH3BGRL3, for the regulation of cytoskeletal activity.

Spider mites are one of the major agricultural pests, feeding on a large variety of plants. As a contribution to understanding chemical communication in these arthropods, we have characterized a recently discovered class of odorant-binding proteins (OBPs) in Tetranychus urticae. As in other species of Chelicerata, the four OBPs of T. urticae contain six conserved cysteines paired in a pattern (C1-C6, C2-C3, C4-C5) differing from that of insect counterparts (C1-C3, C2-C5, C4-C6). Proteomic analysis uncovered a second family of OBPs, including twelve members that are likely to be unique to T. urticae. A three-dimensional model of TurtOBP1, built on the recent X-ray structure of Varroa destructor OBP1, shows protein folding different from that of insect OBPs, although with some common features. Ligand-binding experiments indicated some affinity to coniferyl aldehyde, but specific ligands may still need to be found among very large molecules, as suggested by the size of the binding pocket.

Odorant-binding proteins (OBPs), as they occur in insects, form a distinct class of proteins that apparently has no closely related representatives in other animals. However, ticks, mites, spiders and millipedes contain genes encoding proteins with sequence similarity to insect OBPs. In this work, we have explored the structure and function of such non-insect OBPs in the mite Varroa destructor, a major pest of honey bee. Varroa OBPs present six cysteines paired into three disulphide bridges, but with positions in the sequence and connections different from those of their insect counterparts. VdesOBP1 structure was determined in two closely related crystal forms and appears to be a monomer. Its structure assembles five ?-helices linked by three disulphide bridges, one of them exhibiting a different connection as compared to their insect counterparts. Comparison with classical OBPs reveals that the second of the six ?-helices is lacking in VdesOBP1. Ligand-binding experiments revealed molecules able to bind only specific OBPs with a moderate affinity, suggesting that either optimal ligands have still to be identified, or post-translational modifications present in the native proteins may be essential for modulating binding activity, or else these OBPs might represent a failed attempt in evolution and are not used by the mites.

Environmental conditions and timing of egg storage highly affect raw material quality. Aging and endogenous processing of constituent proteins can determine important changes in specific functions and technological properties of inner egg compartments. We here used integrated peptidomic procedures to identify peptide markers of egg freshness. At first, peptides extracted from egg white and yolk plasma taken from eggs stored for different times were subjected to a label-free untargeted quantitation procedure based on nanoLC-ESI-Q-Orbitrap-MS/MS, which identified 836 and 1974 unique variable molecules, respectively. By applying stringent criteria for filtering data, 30 and 66 putative egg aging markers were selected for egg white and yolk plasma, respectively. Proposed molecules were then validated through a targeted label-free parallel reaction monitoring procedure based on nanoLC-ESI-Q-Orbitrap-MS/MS, confirming quantitative trends for 19 and 25 peptides in egg white and yolk plasma, respectively, and generating a robust panel of egg storage markers. Quantitative results reflected physico-chemical phenomena occurring in egg compartments during storage and offered essential information for the development of novel control procedures to assess quality features of fresh/stored raw material.

Metal(loid)s are toxic to animal life, human health and plants; therefore, their removal from polluted areas is imperative in order to minimize their impact on the ecosystems. The use of plant-amendment-microorganism synergy is a promising option, but not yet fully explored, to manage lands contaminated with metal(loid)s. However, molecular factors and mechanisms underlying this interaction are almost unknown. The aim of the present study was to evaluate the effects of amendments and bacteria, both alone and in combination, on Arabidopsis thaliana grown on arsenic and lead polluted soils. To accomplish this aim, a pot experiment was performed testing the effect of biochar and/or autochthonous metal(loid) resistant Bacillus isolates on physico-chemical soil properties and on plant growth and metal(loid) uptake/intake. Furthermore, bioinformatics-assisted proteomics was used to understand common and specific mechanisms regulating plant growth and metal(loid) tolerance in tested conditions. Results showed that biochar and/or Bacillus induced significant and positive effects on soil properties, increasing pH, Ctot, Ntot and Ptot concentrations and decreasing nutrients (Nav and Pav), As and Pb availability. Plant growth was also enhanced by addition of biochar and/or bacterial inoculum, reaching the maximum when biochar and microorganism were combined. The deciphering of molecular mechanisms revealed that combination of biochar and bacterial inoculation mitigate Arabidopsis growth and defense tradeoff, and underline the great potential of plant-biochar-inoculum synergic application in more effective and large scale-up phytostabilizing systems.

In actinomycetes, antibiotic production is often associated with a morpho-physiological differentiation program that is regulated by complex molecular and metabolic networks. Many aspects of these regulatory circuits have been already elucidated and many others still deserve further investigations. In this regard, the possible role of many small open reading frames (smORFs) in actinomycete morpho-physiological differentiation is still elusive. In Streptomyces coelicolor, inactivation of the smORF trpM (SCO2038) - whose product modulates L-tryptophan biosynthesis - impairs production of antibiotics and morphological differentiation. Indeed, it was demonstrated that TrpM is able to interact with PepA (SCO2179), a putative cytosol aminopeptidase playing a key role in antibiotic production and sporulation. In this work, a S. coelicolor trpM knock-in (Sco-trpMKI) mutant strain was generated by cloning trpM into overexpressing vector to further investigate the role of trpM in actinomycete growth and morpho-physiological differentiation. Results highlighted that trpM: (i) stimulates growth and actinorhodin (ACT) production; (ii) decreases calcium-dependent antibiotic (CDA) production; (iii) has no effect on undecylprodigiosin production. Metabolic pathways influenced by trpM knock-in were investigated by combining two-difference in gel electrophoresis/nanoliquid chromatography coupled to electrospray linear ion trap tandem mass spectrometry (2D-DIGE/nanoLC-ESI-LIT-MS/MS) and by LC-ESI-MS/MS procedures, respectively. These analyses demonstrated that over-expression of trpM causes an over-representation of factors involved in protein synthesis and nucleotide metabolism as well as a down-representation of proteins involved in central carbon and amino acid metabolism. At the metabolic level, this corresponded to a differential accumulation pattern of different amino acids - including aromatic ones but tryptophan - and central carbon intermediates. PepA was also down-represented in Sco-trpMKI. The latter was produced as recombinant His-tagged protein and was originally proven having the predicted aminopeptidase activity. Altogether, these results highlight the stimulatory effect of trpM in S. coelicolor growth and ACT biosynthesis, which are elicited through the modulation of various metabolic pathways and PepA representation, further confirming the complexity of regulatory networks that control antibiotic production in actinomycetes.

Chicken egg white is a raw material broadly utilized as additive for the preparation of food and cosmetoceutical products. To describe its molecular properties, various proteomic investigations were performed in the last decade characterizing highly abundant components. No peptidomic counterparts were accomplished so far; scientific literature only reports on the characterization of specific bioactive peptides or preparations from egg white and its hydrolysates, which was performed through dedicated functional assays. In this study, a broad description of the egg white peptidome at 24, 336 and 672 h after laying was achieved using three peptide extraction procedures, which were combined with MALDI-TOF-TOF-MS and nanoLC-ESI-Q-Orbitrap-MS/MS analyses. In the whole, 506 peptides were characterized; they mostly resulted from the physiological degradation of intact proteins following the activity of endoprotease ArgC-, trypsin- and plasmin-like enzymes. Eventual detection of peptide post-translational modifications also provided structural information on parental proteins. When analyzed by bioinformatics and/or compared with literature data, identified peptides allowed recognizing a number of protein fragments associated with different hypothetical biological activities. These results confirmed previous observations regarding functional characteristics of egg white unfractionated preparations or purified molecules, emphasizing the useful application of this raw material in human nutrition and cosmetics. Finally, a comparative label-free peptidomic evaluation of samples stored for different times under refrigeration identified 31 peptides showing significant quantitative changes during storage. BIOLOGICAL SIGNIFICANCE: This study provided the largest inventory of peptides described in chicken egg while so far. In addition, it identified a number of protein fragments associated with hypothetical antihypertensive, antioxidant, antiinflammatory, antimicrobial, anticancer, antiviral, antibiofilm, calcium-binding, antidiabetic, antithrombotic, adipogenic differentiating, stimulating/immunostimulating, hormonal, lipid-binding and cell adhesion-affecting activities. These results confirmed previous observations regarding functional characteristics of egg white unfractionated preparations or purified molecules, emphasizing the useful application of this raw material in human nutrition and cosmetics.

There is currently a large amount of research being done into the phytoremediation of polluted soils. Plant installation in contaminated soils may require the application of soil amendments, such as biochar, compost and/or iron grit, which can improve the soil conditions and reduce the metal (loid) phytoavailability and mobility. The beneficial effects of these amendments on soil properties, plant growth and metal (loid) accumulation ability have already been described, although their effect on the plants response machinery has been poorly studied. This study aimed to assess the effect of these amendments on Salix viminalis growth and metal (loid) accumulation, as well as elucidating associated molecular mechanisms. The results showed that the amendment applications improved plant growth by three fold, except for the biochar plus iron combination. It also revealed that metal (loid)s were not effectively translocated from the roots to the shoots (translocation factors <1), their bioaccumulation peaked in the roots, and increased in the presence of iron-based amendments. Corresponding proteomic profiles revealed 34 protein spots differentially represented and suggested that plants counteracted metal (loid)-induced oxidative stress after the addition of biochar and/or compost by eliciting proper defense and signaling pathways, and by redirecting the metabolic fluxes towards primary and secondary metabolism. However, they did highlight the occurrence of oxidative stress markers when the biochar plus iron amendment was applied, which could be both the cause and result of protein degradation impairment.

Carbonyl reductase 1 (CBR1) is an NADP-dependent enzyme that exerts a detoxifying role, which catalyses the transformation of carbonyl-containing compounds. The ability of CBR1 to act on adducts between glutathione and lipid peroxidation derived aldehydes has recently been reported. In the present study, exploiting mass spectrometry and fluorescence spectroscopy, evidence is shown that CBR1 is able to retain NADP(H) at the active site even after extensive dialysis, and that this retention may also occur when the enzyme is performing catalysis. This property, together with the multi-substrate specificity of CBR1 in both directions of red/ox reactions, generates inter-conversion red/ox cycles. This particular feature of CBR1, in the case of the transformation of 3-glutathionyl, 4-hydroxynonanal (GSHNE), which is a key substrate of the enzyme in detoxification, supports the disproportionation reaction of GSHNE without any apparent exchange of the cofactor with the solution. The importance of the cofactor as a prosthetic group for other dehydrogenases exerting a detoxification role is discussed.

Osteochondrosis is a failure of the endochondral ossification that affects developing joints in humans and several animal species. It is a localized idiopathic joint disorder characterized by focal chondronecrosis and growing cartilage retention, which can lead to the formation of fissures, subchondral bone cysts, or intra-articular fragments. Osteochondrosis is a complex multifactorial disease associated with extracellular matrix alterations and failure in chondrocyte differentiation, mainly due to genetic, biochemical, and nutritional factors, as well as traumas. This study describes the main proteomic alterations occurring in chondrocytes isolated from osteochondrotic cartilage fragments. A comparative analysis performed on equine osteochondrotic and healthy chondrocytes showed 26 protein species as differentially represented. In particular, quantitative changes in the extracellular matrix, cytoskeletal and chaperone proteins, and in cell adhesion and signaling molecules were observed in osteochondrotic cells, compared to healthy controls. Functional group analysis annotated most of these proteins in "growth plate and cartilage development", while others were included in "glycolysis and gluconeogenesis", "positive regulation of protein import", "cell-cell adhesion mediator activity", and "mitochondrion nucleoid". These results may help to clarify some chondrocyte functional alterations that may play a significant role in determining the onset and progression of equine osteochondrosis and, being related, of human juvenile osteochondrosis.