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Abstract Background Colorectal cancer (CRC) risk is strongly affected by dietary habits with red and processed meat increasing risk, and foods rich in dietary fibres considered protective. Dietary habits also shape gut microbiota, but the role of the combination between diet, the gut microbiota, and the metabolite profile on CRC risk is still missing an unequivocal characterisation. Methods To investigate how gut microbiota affects diet-associated CRC risk, we fed Apc-mutated PIRC rats and azoxymethane (AOM)-induced rats the following diets: a high-risk red/processed meat-based diet (MBD), a normalised risk diet (MBD with α-tocopherol, MBDT), a low-risk pesco-vegetarian diet (PVD), and control diet. We then conducted faecal microbiota transplantation (FMT) from PIRC rats to germ-free rats treated with AOM and fed a standard diet for 3 months. We analysed multiple tumour markers and assessed the variations in the faecal microbiota using 16S rRNA gene sequencing together with targeted- and untargeted-metabolomics analyses. Results In both animal models, the PVD group exhibited significantly lower colon tumorigenesis than the MBD ones, consistent with various CRC biomarkers. Faecal microbiota and its metabolites also revealed significant diet-dependent profiles. Intriguingly, when faeces from PIRC rats fed these diets were transplanted into germ-free rats, those transplanted with MBD faeces developed a higher number of preneoplastic lesions together with distinctive diet-related bacterial and metabolic profiles. PVD determines a selection of nine taxonomic markers mainly belonging to Lachnospiraceae and Prevotellaceae families exclusively associated with at least two different animal models, and within these, four taxonomic markers were shared across all the three animal models. An inverse correlation between nonconjugated bile acids and bacterial genera mainly belonging to the Lachnospiraceae and Prevotellaceae families (representative of the PVD group) was present, suggesting a potential mechanism of action for the protective effect of these genera against CRC. Conclusions These results highlight the protective effects of PVD while reaffirming the carcinogenic properties of MBD diets. In germ-free rats, FMT induced changes reminiscent of dietary effects, including heightened preneoplastic lesions in MBD rats and the transmission of specific diet-related bacterial and metabolic profiles. Importantly, to the best of our knowledge, this is the first study showing that diet-associated cancer risk can be transferred with faeces, establishing gut microbiota as a determinant of diet-associated CRC risk. Therefore, this study marks the pioneering demonstration of faecal transfer as a means of conveying diet-related cancer risk, firmly establishing the gut microbiota as a pivotal factor in diet-associated CRC susceptibility. Video Abstract

Abstract Biostimulants are heterogeneous products designed to support plant development and to improve the yield and quality of crops. Here, we focused on the effects of triacontanol, a promising biostimulant found in cuticle waxes, on tomato growth and productivity. We examined various phenological traits related to vegetative growth, flowering and fruit yield, the metabolic profile of fruits, and the response of triacontanol-treated plants to salt stress. Additionally, a proteomic analysis was conducted to clarify the molecular mechanisms underlying triacontanol action. Triacontanol application induced advanced and increased blooming without affecting plant growth. Biochemical analyses of fruits showed minimal changes in nutritional properties. The treatment also increased the germination rate of seeds by altering hormone homeostasis and reduced salt stress-induced damage. Proteomics analysis of leaves revealed that triacontanol increased the abundance of proteins related to development and abiotic stress, while down-regulating proteins involved in biotic stress resistance. The proteome of the fruits was not significantly affected by triacontanol, confirming that biostimulation did not alter the nutritional properties of fruits. Overall, our findings provide evidence of the effects of triacontanol on growth, development, and stress tolerance, shedding light on its mechanism of action and providing new insights into its potential in agricultural practices.

Salinity stress represents a key factor for global agriculture. Plants can respond to salinity stress by adapting their physiology in different ways with the aim of limiting reductions in growth and development. Importantly, moisture retention capacity, permeability and nutrient availability of substrates represent critical variables for plants as they may further influence the effect of osmotic stress. Here, a multidisciplinary approach was applied to evaluate the role of two different substrates, peat and perlite, on 2-year-old potted cuttings of Olea europaea (cultivar Arbequina) under different salinity stress conditions (0, 100 and 200 mM NaCl). Biometric and physiological data indicate that plants potted in perlite (AP) generally present lower growth and photosynthetic rates when compared with peat (AS) in combination with salinity stress. Ion measurements indicate a rise in Na+ accumulation with increasing stress severity, which alters the ion ratio in both substrates. In addition, differences occurred in polyphenol contents, with a general increase in quinic acid and rutin contents in AS and AP samples, respectively. Metabolomic and biometric data were also coupled with metabarcoding analysis, which indicates that the moderate salinity treatment (100 mM NaCl, T100) reshaped the endophytic community of plants grown on both substrates. Taken together, the data suggest that the strategy used by a glycophytic species such as the olive tree to cope with salinity stress seems to be highly related to availability of water and nutrients. The lack of both may be simulated by perlite, enhancing the effect of salinity stress response in woody plants. Lastly, applying the beneficial endophytic bacterial taxa identified here could represent a step forward in increasing plant defence and nutrient uptake and reducing inputs for modern and more sustainable agriculture.

Abstract Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer in the adult population. Late diagnosis, resistance to therapeutics and recurrence of metastatic lesions account for the highest mortality rate among kidney cancer patients. Identifying novel biomarkers for early cancer detection and elucidating the mechanisms underlying ccRCC will provide clues to treat this aggressive malignant tumor. Here, we report that the ubiquitin ligase praja2 forms a complex with-and ubiquitylates the AP2 adapter complex, contributing to receptor endocytosis and clearance. In human RCC tissues and cells, downregulation of praja2 by oncogenic miRNAs (oncomiRs) and the proteasome markedly impairs endocytosis and clearance of the epidermal growth factor receptor (EGFR), and amplifies downstream mitogenic and proliferative signaling. Restoring praja2 levels in RCC cells downregulates EGFR, rewires cancer cell metabolism and ultimately inhibits tumor cell growth and metastasis. Accordingly, genetic ablation of praja2 in mice upregulates RTKs (i.e. EGFR and VEGFR) and induces epithelial and vascular alterations in the kidney tissue. In summary, our findings identify a regulatory loop between oncomiRs and the ubiquitin proteasome system that finely controls RTKs endocytosis and clearance, positively impacting mitogenic signaling and kidney cancer growth.

Dietary patterns high in fat and sugar promote brain dysfunction, but the preventive efficacy of probiotics has been little explored. This study investigated whether the administration of Limosilactobacillus reuteri DSM 17938 (L.reuteri; 108 CFU/day) counteracts cognitive dysfunction and brain redox unbalance induced in male Wistar rats fed a western diet (WD) for 8 weeks.The results showed that L. reuteri prevented the WD-associated impairment in brain memory function, as evaluated by NOR test, and consistently increased specific molecular markers of synaptic plasticity. The condition of oxidative stress induced by WD, namely enhanced oxidation to proteins and lipids, increased oxidants-producing enzymes as well as reduced nuclear Nrf2 and Nrf2-dependent antioxidant enzymes, was ameliorated by the probiotic. Unexpectedly, mitochondria displayed a diet-induced increase in oxidative capacity, not modified by the probiotic. A protective effect of the probiotic on gut absorption of fructose and its further delivery to brain was evidenced as mechanism involved in cortex redox homeostasis. These findings evidence the potential utility of L. reuteri against WD-induced brain dysfunction and implicate the Nrf2 pathway as a potential mechanism for this effect.

Cryptosporidium parvum is a common cause of a zoonotic disease and a main cause of diarrhea in newborns. Effective drugs or vaccines are still lacking. Oocyst is the infective form of the parasite; after its ingestion, the oocyst excysts and releases four sporozoites into the host intestine that rapidly attack the enterocytes. The membrane protein CpRom1 is a large rhomboid protease that is expressed by sporozoites and recognized as antigen by the host immune system. In this study, we observed the release of CpRom1 with extracellular vesicles (EVs) that was not previously described. To investigate this phenomenon, we isolated and resolved EVs from the excystation medium by differential ultracentrifugation. Fluorescence flow cytometry and transmission electron microscopy (TEM) experiments identified two types of sporozoite-derived vesicles: large extracellular vesicles (LEVs) and small extracellular vesicles (SEVs). Nanoparticle tracking analysis (NTA) revealed mode diameter of 181 nm for LEVs and 105 nm for SEVs, respectively. Immunodetection experiments proved the presence of CpRom1 and the Golgi protein CpGRASP in LEVs, while immune-electron microscopy trials demonstrated the localization of CpRom1 on the LEVs surface. TEM and scanning electron microscopy (SEM) showed that LEVs were generated by means of the budding of the outer membrane of sporozoites; conversely, the origin of SEVs remained uncertain. Distinct protein compositions were observed between LEVs and SEVs as evidenced by their corresponding electrophoretic profiles. Indeed, a dedicated proteomic analysis identified 5 and 16 proteins unique for LEVs and SEVs, respectively. Overall, 60 proteins were identified in the proteome of both types of vesicles and most of these proteins (48 in number) were already identified in the molecular cargo of extracellular vesicles from other organisms. Noteworthy, we identified 12 proteins unique to Cryptosporidium spp. and this last group included the immunodominant parasite antigen glycoprotein GP60, which is one of the most abundant proteins in both LEVs and SEVs.

Susceptibility to proteolytic activity is a critical limitation for food-derived peptides possibly influencing human physiological processes. This study explores the ex vivo stability and degradation kinetics of the milk-derived opioid peptide β-casomorphin-7 (BCM7) in human blood. Blood specimens collected from three healthy volunteers were individually spiked with synthetic BCM7 and sampled at seven time points over 2 h. Liquid chromatography-electrospray-high resolution tandem mass spectrometry was used to monitor the stability of BCM7 and the formation of its hydrolytic fragments. Human plasma peptidases rapidly hydrolyzed BCM7 generating inactive peptides with similar sharp degradation kinetics across the blood of different individuals. The estimated plasma half-life (t1/2) value of BCM7 ranged from 35 to 40 min. The peptide degradation pattern pointed to prolyl oligopeptidase, prolidase, and dipeptidyl peptidase as the primary enzyme candidates responsible for BCM7 hydrolysis. Overall, the findings of this study suggest that BCM7 cannot exert systemic effects in humans.

Maillard reaction readily takes place in dairy products because of the association between thermal treatments, extended storage and the matrix composition. Along with the impairment of protein digestion, the formation of glycation and α-dicarbonyl compounds is a concern for quality attributes of whey proteins when used as ingredients. In this paper, we outline the capacity of brewer's spent grain melanoidins in reducing the accumulation of α-dicarbonyl compounds, thus controlling the formation of dietary advanced glycation end-products in accelerated shelf life at 35 °C. Results revealed that brewer's spent grain melanoidins targeted methylglyoxal and glyoxal reactivity leading to the reduction of N-ε-carboxymethyllysine and methylglyoxal-hydroimidazolone up to 27 and 60%, respectively. We here describe that the presence of melanoidins is instrumental in limiting the undesired effects of α-dicarbonyl compounds on whey proteins.

Edible plant and fruit-derived nanovesicles (NVs) are membrane-enclosed particles with round-shape morphology and signaling functions, which resemble mammalian cell-derived extracellular vesicles. These NVs can transmit cross-kingdom signals as they contain bioactive molecules and exert biological effects on mammalian cells. Their properties and stability in the gastrointestinal tract suggest NVs as a promising nutraceutical tool. In this study, we have demonstrated for the first time the presence of NVs in olive vegetation water (OVW), a waste by-product generated during olive oil production. Biophysical characterization by scanning electron microscopy, cryo-transmission electron microscopy, and nanoparticle tracking analysis revealed the presence in OVW of NVs having size and morphology similar to that of vesicles isolated from edible plants. Integrated lipidomic, metabolomic, and proteomic analyses showed that OVW-NVs carry a set of lipids, metabolites and proteins which have recognized antioxidant and anti-inflammatory activities. The nature of biomolecules identified in OVW-NVs suggests that these vesicles could exert beneficial effects on mammalian cells and could be used in the nutraceutical and food industries. The successful isolation of OVW-NVs and the characterization of their features strengthen the idea that agricultural waste might represent a source of NVs having features similar to NVs isolated from edible plants/fruits.

Abstract FKBP51 plays a relevant role in sustaining cancer cells, particularly melanoma. This cochaperone participates in several signaling pathways. FKBP51 forms a complex with Akt and PHLPP, which is reported to dephosphorylate Akt. Given the recent discovery of a spliced FKBP51 isoform, in this paper, we interrogate the canonical and spliced isoforms in regulation of Akt activation. We show that the TPR domain of FKBP51 mediates Akt ubiquitination at K63, which is an essential step for Akt activation. The spliced FKBP51, lacking such domain, cannot link K63-Ub residues to Akt. Unexpectedly, PHLPP silencing does not foster phosphorylation of Akt, and its overexpression even induces phosphorylation of Akt. PHLPP stabilizes levels of E3-ubiquitin ligase TRAF6 and supports K63-ubiquitination of Akt. The interactome profile of FKBP51 from melanoma cells highlights a relevant role for PHLPP in improving oncogenic hallmarks, particularly, cell proliferation.

Several carbonic anhydrase (CA, EC 4.2.1.1) isoforms play an essential role in processes connected to tumorigenesis, as they efficiently accelerate the hydration of carbon dioxide to bicarbonate and proton. In this context, examples are CA IX and CA XII, which were proved to be upregulated in many solid malignancies. On the other hand, cancer and the immune system are inextricably linked, and targeting the immune checkpoints recently was shown to efficiently improve the treatment of malignancies. In this study, we have investigated the expression of CA isoforms in tumour-infiltrating lymphocytes (TILs) that, according to the immunosurveillance theory, were suggested to have a crucial role in the development of colorectal cancer (CRC). T lymphocytes isolated from healthy surrounding mucosa showed a higher CA activity compared to those present in tumour and peripheral blood in the same patients. CA I and II were confirmed as enzyme isoforms involved in the process, as determined by proteomic analysis of corresponding TIL samples. These preliminary findings suggest a dysregulation of the local immune response in the CRC tissues and a loss of effective anticancer mechanisms mediated by CAs therein.

Abstract 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.

Cervical cancer (CC) is one of the most frequent cancers in women worldwide. The epithelial-mesenchymal transition (EMT) and the extracellular release of TGF-β are phenomena typically associated with different tumorigenic processes, including tumour cell proliferation and metastatization. Specific human microRNAs (miRNAs; miRs) involved in these tumorigenic processes have been identified, becoming important diagnostic and prognostic markers, and even potential therapeutic targets. In parallel, different studies have also shown that plant miRNAs can mediate a cross-kingdom regulation (CKR) of mammalian genes and modulate host's gene expression under pathological conditions, restoring the regulatory activity of endogenous miRNAs lost in cancer. In our previous studies, the miRNome from Moringa oleifera Lam. (henceforth moringa or mol) has been sequenced, showing the presence of several conserved miRNAs in the plant kingdom, whose ability to differentially regulate proliferation and apoptosis in healthy and cancer cells has been demonstrated. Furthermore, the effects of mol-miR treatment on tumorigenesis and EMT have been proved in liver tumour cells. According to these premises, we here investigated the proteomic profile of CC-derived HeLa cells exposed to a mol-miRNA pool, demonstrating the down-representation of specific factors involved in tumorigenesis. The treatment with plant miRs was able to modulate proteins involved in several biological processes linked to EMT. Furthermore, it reduced the expression of TGF-β and significantly inhibited cell motility, as observed following Scratch test and cell viability measurements, with a significant increase of apoptotic events. In conclusion, our results suggest and pave the way for the development of new potential therapeutic approaches based on CKR mediated by plant miRNAs for contrasting human cervical cancer, even in the form of adjuvants to classic treatments for limiting their side effects.

Abstract Glioblastoma multiforme (GBM) is the most frequent and aggressive form of primary brain tumor in the adult population; its high recurrence rate and resistance to current therapeutics urgently demand a better therapy. Regulation of protein stability by the ubiquitin proteasome system (UPS) represents an important control mechanism of cell growth. UPS deregulation is mechanistically linked to the development and progression of a variety of human cancers, including GBM. Thus, the UPS represents a potentially valuable target for GBM treatment. Using an integrated approach that includes proteomics, transcriptomics and metabolic profiling, we identify praja2, a RING E3 ubiquitin ligase, as the key component of a signaling network that regulates GBM cell growth and metabolism. Praja2 is preferentially expressed in primary GBM lesions expressing the wild-type isocitrate dehydrogenase 1 gene (IDH1). Mechanistically, we found that praja2 ubiquitylates and degrades the kinase suppressor of Ras 2 (KSR2). As a consequence, praja2 restrains the activity of downstream AMP-dependent protein kinase in GBM cells and attenuates the oxidative metabolism. Delivery in the brain of siRNA targeting praja2 by transferrin-targeted self-assembling nanoparticles (SANPs) prevented KSR2 degradation and inhibited GBM growth, reducing the size of the tumor and prolonging the survival rate of treated mice. These data identify praja2 as an essential regulator of cancer cell metabolism, and as a potential therapeutic target to suppress GBM growth.

Warm-blooded animals may have Malassezia pachydermatis on healthy skin, but changes in the skin microenvironment or host defences induce this opportunistic commensal to become pathogenic. Malassezia infections in humans and animals are commonly treated with azole antifungals. Fungistatic treatments, together with their long-term use, contribute to the selection and the establishment of drug-resistant fungi. To counteract this rising problem, researchers must find new antifungal drugs and enhance drug resistance management strategies. Cyclic adenosine monophosphate, adenylyl cyclase, and bicarbonate have been found to promote fungal virulence, adhesion, hydrolase synthesis, and host cell death. The CO2/HCO3-/pH-sensing in fungi is triggered by HCO3- produced by metalloenzymes carbonic anhydrases (CAs, EC 4.2.1.1). It has been demonstrated that the growth of M. globosa can be inhibited in vivo by primary sulphonamides, which are the typical CA inhibitors. Here, we report the cloning, purification, and characterisation of the β-CA (MpaCA) from the pathogenic fungus M. pachydermatis, which is homologous to the enzyme encoded in the genome of M. globosa and M. restricta, that are responsible for dandruff and seborrhoeic dermatitis. Fungal CAs could be thus considered a new pharmacological target for combating fungal infections and drug resistance developed by most fungi to the already used drugs.

Together with phenological and genomic approaches, gel-based and label-free proteomic as well metabolomic procedures were separately applied to plants to highlight differences between ecotypes, to estimate genetic variability within/between organism populations, or to characterize specific mutants/genetically modified lines at metabolic level. To investigate the possible use of tandem mass tag (TMT)-based quantitative proteomics in the above-mentioned contexts and based on the absence of combined proteo-metabolomic studies on Diospyros kaki cultivars, we here applied integrated proteomic and metabolomic approaches to fruits from Italian persimmon ecotypes with the aim to characterize plant phenotypic diversity at molecular level. We identified 2255 proteins in fruits, assigning 102 differentially represented components between cultivars, including some related to pomological, nutritional and allergenic characteristics. Thirty-three polyphenols were also identified and quantified, which belong to hydroxybenzoic acid, flavanol, hydroxycinnamic acid, flavonol, flavanone and dihydrochalcone sub-classes. Heat-map representation of quantitative proteomic and metabolomic results highlighted compound representation differences in various accessions, whose elaboration through Euclidean distance functions and other linkage methods defined dendrograms establishing phenotypic relationships between cultivars. Principal component analysis of proteomic and metabolomic data provided clear information on phenotypic differences/similarities between persimmon accessions. Coherent cultivar association results were observed between proteomic and metabolomic data, emphasizing the utility of integrating combined omic approaches to identify and validate phenotypic relationships between ecotypes, and to estimate corresponding variability and distance. Accordingly, this study describes an original, combined approach to outline phenotypic signatures in persimmon cultivars, which may be used for a further characterization of other ecotypes of the same species and an improved description of nutritional characteristics of corresponding fruits.