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Vision in vertebrates is mediated by the eye, a complex organ with developmental and functional similarities to the central nervous system. Eye proteomics has emerged as a powerful tool for investigating ocular function and disease mechanisms, including neurodegeneration and ocular toxicity. The zebrafish ( Danio rerio ) is a well-established model in biomedical research, including ophthalmology, due to its highly developed visual system, rapid eye maturation, and genetic homology with humans. Building on previous findings that thermal stress can affect neural tissues, this study investigates whether prolonged exposure to non-optimal temperatures also impacts the zebrafish eye proteome. Adult zebrafish were maintained for 21 days at elevated (34 °C), control (26 °C), or low (18 °C) temperatures, and eye proteomes were analysed by tandem mass spectrometry. Our results reveal that both low and high temperatures induce distinct alterations in the expression of proteins involved in critical eye processes. Notably, high-temperature exposure modulates pathways such as sirtuin signalling while downregulating proteins involved in oxidative phosphorylation, electron transport, and ATP synthesis, alongside decreased expression of proteins central to visual phototransduction. These data indicate that environmental temperature can directly impact eye protein homeostasis, supporting a potential role for the thermal stress in ocular dysfunction.

Replacing cereals with food leftovers could reduce feed-food competition and keep nutrients and energy in the food chain. Former food products (FFPs) are industrial food leftovers no more intended for human but still suitable as alternative and sustainable feedstuffs for monogastric. In this study, omics approaches were applied to evaluate the impact of dietary FFPs on pig liver proteome and plasma peptidome. Thirty-six Swiss Large White male castrated pigs were randomly assigned to three dietary treatments [control (CTR), 30% CTR replaced with salty FFP (SA), 30% CTR replaced with sugary FFP (SU)] from the start of the growing phase (22.4 ± 1.7 kg) until slaughtering (110 ± 3 kg). The low number of differentially regulated proteins in each comparison matrix (SA/SU vs. CTR) and the lack of metabolic interaction indicated a marginal impact on hepatic lipid metabolism. The plasma peptidomics investigation showed low variability between the peptidome of the three dietary groups and identified three possible bioactive peptides in the SA group associated with anti-hypertension and vascular homeostasis regulation. To conclude, the limited modulation of liver proteome and plasma peptidome by the SA and SU diets strenghtened the idea of reusing FFPs as feed ingredients to make pig production more sustainable.

The conservation of local chicken breeds is essential to safeguard genetic biodiversity and promote sustainable poultry production. Sperm cryopreservation is a key tool for the long-term maintenance of genetic diversity by enabling the storage of male gametes from endangered or valuable breeders for future use in conservation and breeding programs. However, significant variability in semen quality and fertility across breeds limits the effectiveness of cryopreservation protocols. This study aimed to explore the relationship between sperm protein composition and semen quality in five Italian chicken breeds, Bionda Piemontese (BP), Bianca di Saluzzo (BS), Mericanel della Brianza (MB), Pepoi (Pe), and Robusta Maculata (R), known for distinct reproductive traits. Semen samples were analyzed for volume, concentration, membrane integrity, and sperm motility, and for sperm proteomes by a label-free shotgun proteomics approach, to characterize potential molecular pathways associated with semen quality. Significant inter-breed differences were observed in semen parameters; Pe roosters showed the highest semen volume and concentration, but lower values in kinematic traits, including curvilinear velocity (VCL), straight-line velocity (VSL), and average path velocity (VAP). RM and MB exhibited the most favorable sperm membrane integrity, progressive sperm motility and sperm kinematic profile, with high VCL, VSL, VAP, linearity and wobble, despite lower semen volume. The remaining breeds showed intermediate values across most traits. Overall, results suggest breed-specific patterns and a potential trade-off between semen quantity and sperm motion efficiency. Proteomic analysis showed that proteins involved in energy metabolism, cytoskeletal dynamics, and membrane integrity were differentially abundant across breeds and correlated with specific semen traits. Gene Set Enrichment Analysis revealed enrichment of pathways such as the HSP90 chaperone cycle, TP53 transcriptional regulation, and insulin-like growth factor signaling in association with sperm motility and quality. Our findings demonstrate that sperm proteomes are associated with breed-specific fertility traits. This study provides new insights into the molecular basis of semen quality variability and biological conservation of avian genetic resources.

The flavoenzyme d-amino acid oxidase (DAAO) is deputed to the degradation of d-enantiomers of amino acids. DAAO plays various relevant physiological roles in different organisms and tissues. Thus, it has been recently suggested that the goblet cells of the mucosal epithelia secrete into the lumen of intestine, a processed and active form of DAAO that uses the intestinal d-amino acids to generate hydrogen peroxide (H2O2), an immune messenger that helps fighting gut pathogens, and by doing so controls the homeostasis of gut microbiota. Here, we show that the DAAO form lacking the 1–16 amino acid residues (the putative secretion signal) is unstable and inactive, and that DAAO is present in the epithelial layer and the mucosa of mouse gut, where it is largely proteolyzed. In silico predicted DAAO-derived antimicrobial peptides show activity against various Gram-positive and Gram-negative bacteria but not on Lactobacilli species, which represent the commensal microbiota. Peptidomic analysis reveals the presence of such peptides in the mucosal fraction. Collectively, we identify a novel mechanism for gut microbiota selection implying DAAO-derived antimicrobial peptides which are generated by intestinal proteases and that are secreted in the gut lumen. In conclusion, we herein report an additional, ancillary role for mammalian DAAO, unrelated to its enzymatic activity.

In recent decades, the food system has been faced with the significant problem of increasing food waste. Therefore, the feed industry, supported by scientific research, is attempting to valorise the use of discarded biomass as co-products for the livestock sector, in line with EU objectives. In parallel, the search for functional products that can ensure animal health and performances is a common fundamental goal for both animal husbandry and feeding. In this context, camelina cake (CAMC), cardoon cake (CC) and cardoon meal (CM), due valuable nutritional profile, represent prospective alternatives. Therefore, the aim of this work was to investigate the antioxidant activity of CAMC, CC and CM following in vitro digestion using 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), Ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) assays. Total phenolic content (TPC) and angiotensin converting enzyme (ACE) inhibitory activity, actively involved in modulating antioxidant properties, were also studied. Further, a peptidomic analysis was adopted to substantiate the presence of bioactive peptides after in vitro digestion. The results obtained confirmed an interesting nutritional profile of CAMC, CC and CM and relevant antioxidant and ACE inhibitory activities. In particular, considering antioxidant profile, CM and CC revealed a significantly higher (10969.80 ± 18.93 mg TE/100 g and 10451.40 ± 149.17 mg TE/100 g, respectively; p  < 0.05) ABTS value than CAMC (9511.18 ± 315.29 mg TE/100 g); a trend also confirmed with the FRAP assay (306.74 ± 5.68 mg FeSO 4 /100 g; 272.84 ± 11.02 mg FeSO 4 /100 g; 103.84 ± 3.27 mg FeSO 4 /100 g, for CC, CM and CAMC, respectively). Similar results were obtained for TPC, demonstrating the involvement of phenols in modulating antioxidant activity. Finally, CAMC was found to have a higher ACE inhibitory activity (40.34 ± 10.11%) than the other matrices. Furthermore, potentially bioactive peptides associated with ACE inhibitory, anti-hypertensive, anti-cancer, antimicrobial, antiviral, antithrombotic, DPP-IV inhibitory and PEP-inhibitory activities were identified in CAMC. This profile was broader than that of CC and CM. The presence of such peptides corroborates the antioxidant and ACE profile of the sample. Although the data obtained report the important antioxidant profile of CAMC, CC, and CM and support their possible use, future investigations, particularly in vivo trials will be critical to evaluate and further investigate their effects on the health and performance of farm animals.

Astrocytes are the major source of L-serine (L-Ser) in the brain: the glycolytic intermediate D-3-phosphoglycerate is converted into L-Ser through the phosphorylated pathway (PP) made up of three enzymes, phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase (PSAT) and phosphoserine phosphatase (PSP), recently proposed to generate a metabolic assembly named serinosome. In the central nervous system, L-Ser is used for a number of functions, including the synthesis of glycine (Gly) and D-serine (D-Ser), the two key NMDAR co-agonists. Here, we used iPSC-derived human astrocytes as a cellular model to evaluate the impact on cell metabolism of the overexpression of each of the three enzymes of the PP as GFP-tagged proteins. The subcellular cytosolic localization of PP enzymes remains unchanged compared to endogenous proteins, while the complex formation is increased in all cases. Notably, among the factors involved, the overexpression of PHGDH appears to play a pivotal role in promoting the serinosome assembly and/or stabilization, highlighting the critical importance of this multi-domain protein. Particularly, the overexpression of each enzyme of the PP alters the cellular metabolism in a specific way. The L-Ser and Gly levels increase more in PHGDH overexpressing cells, in agreement with the known kinetics of the PP. A consistent increase in the TCA cycle, as well as in mitochondrial activities, serine-glycine-one carbon pathway, asparagine, arginine, purine and pyrimidines metabolism is also observed. Peculiar alterations are observed when each enzyme of the PP is overexpressed, strongly supporting the use of human iPSC-derived astrocytes overexpressing the PP pathway enzymes as a valuable cellular model for understanding how Ser glial metabolism occurs in a non-tumor system under both physiological and pathological conditions.

Embryonic and pluripotent stem cells hold great promise in generating β-cells for both replacing medicine and novel therapeutic discoveries in diabetes mellitus. However, their differentiation in vitro is still inefficient, and functional studies reveal that most of these β-like cells still fail to fully mirror the adult β-cell physiology. For their proper growth and functioning, β-cells require a very specific environment, the islet niche, which provides a myriad of chemical and physical signals. While the nature and effects of chemical stimuli have been widely characterized, less is known about the mechanical signals. We here review the current status of knowledge of biophysical cues provided by the niche where β-cells normally live and differentiate, and we underline the possible machinery designated for mechanotransduction in β-cells. Although the regulatory mechanisms remain poorly understood, the analysis reveals that β-cells are equipped with all mechanosensors and signaling proteins actively involved in mechanotransduction in other cell types, and they respond to mechanical cues by changing their behavior. By engineering microenvironments mirroring the biophysical niche properties it is possible to elucidate the β-cell mechanotransductive-regulatory mechanisms and to harness them for the promotion of β-cell differentiation capacity in vitro.

Background Dairy cow milking practices require cleaning and disinfection of the teat skin before and after milking to ensure the safety and quality of milk and prevent intramammary infections. Antimicrobial proteins of natural origin can be valuable alternatives to traditional disinfectants. In a recent field trial, we demonstrated that a teat dip based on a nisin A-producing Lactococcus cremoris (L) had comparable efficacy to conventional iodophor dip (C) in preventing dairy cow mastitis. Here, we present the differential shotgun proteomics investigation of the milk collected during the trial. Methods Four groups of quarter milk samples with low (LSCC) and high somatic cell count (HSCC) collected at the beginning (T0) and end (TF) of the trial were analyzed for a total of 28 LSCC (14 LSCC T0 and 14 LSCC TF) and 12 HSCC (6 HSCC T0 and 6 HSCC TF) samples. Milk proteins were digested into peptides, separated by nanoHPLC, and analyzed by tandem mass spectrometry (LC-MS/MS) on an Orbitrap Fusion Tribrid mass spectrometer. The proteins were identified with MaxQuant and interaction networks of the differential proteins were investigated with STRING. The proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD045030. Results In healthy milk (LSCC), we detected 90 and 80 differential proteins at T0 and TF, respectively. At TF, the Lactococcus group showed higher levels of antimicrobial proteins. In mastitis milk (HSCC), we detected 88 and 106 differential proteins at T0 and TF, respectively. In the Lactococcus group, 14 proteins with antimicrobial and immune defense functions were enriched at TF vs. 4 proteins at T0. Cathelicidins were among the most relevant enriched proteins. Western immunoblotting validation confirmed the differential abundance. Conclusions At T0, the proteomic differences observed in healthy milk of the two groups were most likely dependent on physiological variation. On the other hand, antimicrobial and immune defense functions were higher in the milk of cows with mammary gland inflammation of the Lactococcus -treated group. Among other factors, the immunostimulatory action of nisin A might be considered as a contributor.

Mass spectrometry (MS)-based proteomics has recently attracted the attention from forensic pathologists. This work is the first report of the development of a shotgun bottom-up proteomic approach based on rapid protein extraction and nano-liquid chromatography/high-resolution mass spectrometry applied to full-thickness human skin for the differential analysis of normal and ecchymotic tissues to identify new biomarkers for bruise characterization and dating. We identified around 2000 proteins from each pooled extract. The method showed excellent precision on independent replicates, with Pearson correlation coefficients always higher than 95%. Glycophorin A, a known biomarker of vital wounds from immunochemical studies, was identified only in ecchymotic tissues, as confirmed by Western blotting analysis. This finding suggests that this protein can be used as a MS-detectable biomarker of wound vitality. By focusing on skin samples from individuals with known wound dating, besides Glycophorin A, other proteins differentially expressed in ecchymotic samples and dependant on wound age were identified, although further analysis on larger datasets are needed to validate these findings. This study paves the way for an in-depth investigation of the potential of MS-based techniques for wound examination in forensic pathology, overcoming the limitations of immunochemical assays.

Global warming is driving significant changes in aquatic ecosystems, where temperature fluctuations influence biological processes across multiple levels of organisation. As ectothermic organisms, fish are particularly susceptible, with even minor thermal shifts affecting their metabolism, behaviour, and overall fitness. Understanding these responses is essential for evaluating the ecological and evolutionary consequences of climate change. This study investigates the effects of acute (4-day) and chronic (21-day) exposure to three temperature regimes—18 °C (low), 26 °C (control), and 34 °C (high)—on the spatio-temporal shoaling behaviour of adult zebrafish (Danio rerio). Groups of four fish were tested for six minutes in water maintained at the same temperature as their prior acclimation. Shoaling behaviour was assessed by analysing shoal structure—encompassing shoal dimensions and cohesion—as well as spatial positioning. Parameters measured included inter-fish distance, shoal volume, shoal area, homogeneity index, distance to the centroid, and the shoal’s vertical and horizontal distribution. Results revealed complex behavioural changes influenced by both temperature and duration of exposure. At 18 °C, zebrafish showed a marked preference for the bottom zone and exhibited no significant temporal modulation in exploratory behaviour—patterns indicative of heightened anxiety-like responses. In contrast, exposure to 34 °C resulted in increased shoal cohesion, particularly under chronic conditions, and a progressive increase in environmental exploration over the six-minute test period. This enhancement in exploratory activity was especially evident when compared to the first minute of the test and was characterised by greater vertical movement—reflected in the increased use of the upper zone—and broader horizontal exploration, including more frequent occupation of peripheral areas. These findings align with previous research linking thermal variation to neurobiological and proteomic alterations in zebrafish. By elucidating how temperature modulates social behaviour in ectotherms, this study offers valuable insights into the potential behavioural impacts of climate change on aquatic ecosystems.