The Neurotoxic Effect of Environmental Temperature Variation in Adult Zebrafish (Danio rerio).

Neurotoxicity consists of the altered functionality of the nervous system caused by exposure to chemical agents or altered chemical-physical parameters. The neurotoxic effect can be evaluated from the molecular to the behavioural level. The zebrafish Danio rerio is a model organism used in many research fields, including ecotoxicology and neurotoxicology. Recent studies by our research group have demonstrated that the exposure of adult zebrafish to low (18 °C) or high (34 °C) temperatures alters their brain proteome and fish behaviour compared to control (26 °C). These results showed that thermal variation alters the functionality of the nervous system, suggesting a temperature-induced neurotoxic effect. To demonstrate that temperature variation can be counted among the factors that generate neurotoxicity, eight different protein datasets, previously published by our research group, were subjected to new analyses using an integrated proteomic approach by means of the Ingenuity Pathway Analysis (IPA) software (Release December 2022). The datasets consist of brain proteome analyses of wild type adult zebrafish kept at three different temperatures (18 °C, 26 °C, and 34 °C) for 4 days (acute) or 21 days (chronic treatment), and of BDNF+/- and BDNF-/- zebrafish kept at 26 °C or 34 °C for 21 days. The results (a) demonstrate that thermal alterations generate an effect that can be defined as neurotoxic (p value ≤ 0.05, activation Z score ≤ -2 or ≥2), (b) identify 16 proteins that can be used as hallmarks of the neurotoxic processes common to all the treatments applied and (c) provide three protein panels (p value ≤ 0.05) related to 18 °C, 34 °C, and BDNF depletion that can be linked to anxiety-like or boldness behaviour upon these treatments.

Molecular response of Sargassum vulgare to acidification at volcanic CO2 vents: Insights from proteomic and metabolite analyses.

Ocean acidification is impacting marine life all over the world. Understanding how species can cope with the changes in seawater carbonate chemistry represents a challenging issue. We addressed this topic using underwater CO2 vents that naturally acidify some marine areas off the island of Ischia. In the most acidified area of the vents, having a mean pH value of 6.7, comparable to far-future predicted acidification scenarios (by 2300), the biomass is dominated by the brown alga Sargassum vulgare. The novelty of the present study is the characterization of the S. vulgare proteome together with metabolite analyses to identify the key proteins, metabolites, and pathways affected by ocean acidification. A total of 367 and 387 proteins were identified in populations grown at pH that approximates the current global average (8.1) and acidified sites, respectively. Analysis of their relative abundance revealed that 304 proteins are present in samples from both sites: 111 proteins are either higher or exclusively present under acidified conditions, whereas 120 proteins are either lower or present only under control conditions. Functionally, under acidification, a decrease in proteins related to translation and post-translational processes and an increase of proteins involved in photosynthesis, glycolysis, oxidation-reduction processes, and protein folding were observed. In addition, small-molecule metabolism was affected, leading to a decrease of some fatty acids and antioxidant compounds under acidification. Overall, the results obtained by proteins and metabolites analyses, integrated with previous transcriptomic, physiological, and biochemical studies, allowed us to delineate the molecular strategies adopted by S. vulgare to grow in future acidified environments, including an increase of proteins involved in energetic metabolism, oxidation-reduction processes, and protein folding at the expense of proteins involved in translation and post-translational processes.

A Wide-Proteome Analysis to Identify Molecular Pathways Involved in Kidney Response to High-Fat Diet in Mice.

The etiopathogenesis of obesity-related chronic kidney disease (CKD) is still scarcely understood. To this aim, we assessed the effect of high-fat diet (HF) on molecular pathways leading to organ damage, steatosis, and fibrosis. Six-week-old male C57BL/6N mice were fed HF diet or normal chow for 20 weeks. Kidneys were collected for genomic, proteomic, histological studies, and lipid quantification. The main findings were as follows: (1) HF diet activated specific pathways leading to fibrosis and increased fatty acid metabolism; (2) HF diet promoted a metabolic shift of lipid metabolism from peroxisomes to mitochondria; (3) no signs of lipid accumulation and/or fibrosis were observed, histologically; (4) the early signs of kidney damage seemed to be related to changes in membrane protein expression; (5) the proto-oncogene MYC was one of the upstream transcriptional regulators of changes occurring in protein expression. These results demonstrated the potential usefulness of specific selected molecules as early markers of renal injury in HF, while histomorphological changes become visible later in obesity-related CDK. The integration of these information with data from biological fluids could help the identification of biomarkers useful for the early detection and prevention of tissue damage in clinical practice.

Brain Proteome and Behavioural Analysis in Wild Type, BDNF+/- and BDNF-/- Adult Zebrafish (Danio rerio) Exposed to Two Different Temperatures.

Experimental evidence suggests that environmental stress conditions can alter the expression of BDNF and that the expression of this neurotrophin influences behavioural responses in mammalian models. It has been recently demonstrated that exposure to 34 °C for 21 days alters the brain proteome and behaviour in zebrafish. The aim of this work was to investigate the role of BDNF in the nervous system of adult zebrafish under control and heat treatment conditions. For this purpose, zebrafish from three different genotypes (wild type, heterozygous BDNF+/- and knock out BDNF-/-) were kept for 21 days at 26 °C or 34 °C and then euthanized for brain molecular analyses or subjected to behavioural tests (Y-maze test, novel tank test, light and dark test, social preference test, mirror biting test) for assessing behavioural aspects such as boldness, anxiety, social preference, aggressive behaviour, interest for the novel environment and exploration. qRT-PCR analysis showed the reduction of gene expression of BDNF and its receptors after heat treatment in wild type zebrafish. Moreover, proteomic analysis and behavioural tests showed genotype- and temperature-dependent effects on brain proteome and behavioural responding. Overall, the absent expression of BDNF in KO alters (1) the brain proteome by reducing the expression of proteins involved in synapse functioning and neurotransmitter-mediated transduction; (2) the behaviour, which can be interpreted as bolder and less anxious and (3) the cellular and behavioural response to thermal treatment.

Covalent inhibition of P. falciparum ferredoxin-NADP+ reductase: Exploring alternative strategies for the development of new antimalarial drugs.

The protozoan Plasmodium falciparum is the main aetiological agent of tropical malaria. Characteristic of the phylum is the presence of a plastid-like organelle which hosts several homologs of plant proteins, including a ferredoxin (PfFd) and its NADPH-dependent reductase (PfFNR). The PfFNR/PfFd redox system is essential for the parasite, while mammals share no homologous proteins, making the enzyme an attractive target for novel and much needed antimalarial drugs. Based on previous findings, three chemically reactive residues important for PfFNR activity were identified: namely, the active-site Cys99, responsible for hydride transfer; Cys284, whose oxidation leads to an inactive dimeric form of the protein; and His286, which is involved in NADPH binding. These amino acid residues were probed by several residue-specific reagents and the two cysteines were shown to be promising targets for covalent inhibition. The quantitative and qualitative description of the reactivity of few compounds, including a repurposed drug, set the bases for the development of more potent and specific antimalarial leads.

Glycosylation Tunes Neuroserpin Physiological and Pathological Properties.

Neuroserpin (NS) is a member of the serine protease inhibitors superfamily. Specific point mutations are responsible for its accumulation in the endoplasmic reticulum of neurons that leads to a pathological condition named familial encephalopathy with neuroserpin inclusion bodies (FENIB). Wild-type NS presents two N-glycosylation chains and does not form polymers in vivo, while non-glycosylated NS causes aberrant polymer accumulation in cell models. To date, all in vitro studies have been conducted on bacterially expressed NS, de facto neglecting the role of glycosylation in the biochemical properties of NS. Here, we report the expression and purification of human glycosylated NS (gNS) using a novel eukaryotic expression system, LEXSY. Our results confirm the correct N-glycosylation of wild-type gNS. The fold and stability of gNS are not altered compared to bacterially expressed NS, as demonstrated by the circular dichroism and intrinsic tryptophan fluorescence assays. Intriguingly, gNS displays a remarkably reduced polymerisation propensity compared to non-glycosylated NS, in keeping with what was previously observed for wild-type NS in vivo and in cell models. Thus, our results support the relevance of gNS as a new in vitro tool to study the molecular bases of FENIB.

High-Resolution Mass Spectrometry-Based Approaches for the Detection and Quantification of Peptidase Activity in Plasma.

Proteomic technologies have identified 234 peptidases in plasma but little quantitative information about the proteolytic activity has been uncovered. In this study, the substrate profile of plasma proteases was evaluated using two nano-LC-ESI-MS/MS methods. Multiplex substrate profiling by mass spectrometry (MSP-MS) quantifies plasma protease activity in vitro using a global and unbiased library of synthetic peptide reporter substrates, and shotgun peptidomics quantifies protein degradation products that have been generated in vivo by proteases. The two approaches gave complementary results since they both highlight key peptidase activities in plasma including amino- and carboxypeptidases with different substrate specificity profiles. These assays provide a significant advantage over traditional approaches, such as fluorogenic peptide reporter substrates, because they can detect active plasma proteases in a global and unbiased manner, in comparison to detecting select proteases using specific reporter substrates. We discovered that plasma proteins are cleaved by endoproteases and these peptide products are subsequently degraded by amino- and carboxypeptidases. The exopeptidases are more active and stable in plasma and therefore were found to be the most active proteases in the in vitro assay. The protocols presented here set the groundwork for studies to evaluate changes in plasma proteolytic activity in shock.

Proteomic profile of maternal-aged blastocoel fluid suggests a novel role for ubiquitin system in blastocyst quality.

PURPOSE: The etiology of maternal aging, a common cause of female factor infertility and a rate-limiting step in vitro fertilization (IVF) success, remains still unclear. Proteomic changes responsible for the impaired successful pregnancy outcome after IVF with aged blastocysts have not been yet evaluated. The objective of this prospective study was to employ proteomic techniques and bioinformatic tools to enlight differences at the protein level in blastocoel fluid of aged and younger woman. METHODS: Protein composition of human blastocoel fluid isolated by micromanipulation from 46 blastocysts of women aged <37 years (group A) and 29 of women aged ≥37 years (group B) have been identified by a shotgun proteomic approach based on high-resolution nano-liquid chromatography electrospray-ionization-tandem mass spectrometry (nLC-ESI-MS/MS) using label free for the relative quantification of their expression levels. RESULTS: The proteomic analysis leads to the identification and quantification of 148 proteins; 132 and 116 proteins were identified in groups A and B, respectively. Interestingly, the identified proteins are mainly involved in processes aimed at fine tuning embryo implantation and development. Among the 100 proteins commonly expressed in both groups, 17 proteins are upregulated and 44 downregulated in group B compared to group A. Overall, the analysis identified 33 proteins, which were increased or present only in B while 76 were decreased in B or present only in A. CONCLUSIONS: Data revealed that maternal aging mainly affects blastocyst survival and implantation through unbalancing the equilibrium of the ubiquitin system known to play a crucial role in fine-tuning several aspects required to ensure successful pregnancy outcome.

Snf1 Phosphorylates Adenylate Cyclase and Negatively Regulates Protein Kinase A-dependent Transcription in Saccharomyces cerevisiae.

In eukaryotes, nutrient availability and metabolism are coordinated by sensing mechanisms and signaling pathways, which influence a broad set of cellular functions such as transcription and metabolic pathways to match environmental conditions. In yeast, PKA is activated in the presence of high glucose concentrations, favoring fast nutrient utilization, shutting down stress responses, and boosting growth. On the contrary, Snf1/AMPK is activated in the presence of low glucose or alternative carbon sources, thus promoting an energy saving program through transcriptional activation and phosphorylation of metabolic enzymes. The PKA and Snf1/AMPK pathways share common downstream targets. Moreover, PKA has been reported to negatively influence the activation of Snf1/AMPK. We report a new cross-talk mechanism with a Snf1-dependent regulation of the PKA pathway. We show that Snf1 and adenylate cyclase (Cyr1) interact in a nutrient-independent manner. Moreover, we identify Cyr1 as a Snf1 substrate and show that Snf1 activation state influences Cyr1 phosphorylation pattern, cAMP intracellular levels, and PKA-dependent transcription.

NEU3 activity enhances EGFR activation without affecting EGFR expression and acts on its sialylation levels.

Several studies performed over the last decade have focused on the role of sialylation in the progression of cancer and, in particular, on the association between deregulation of sialidases and tumorigenic transformation. The plasma membrane-associated sialidase NEU3 is often deregulated in colorectal cancer (CRC), and it was shown that this enzyme co-immunoprecipitates in HeLa cells with epidermal growth factor receptor (EGFR), the molecular target of most recent monoclonal antibody-based therapies against CRC. To investigate the role of NEU3 sialidase on EGFR deregulation in CRC, we first collected data on NEU3 gene expression levels from a library of commercial colon cell lines, demonstrating that NEU3 transcription is upregulated in these cell lines. We also found EGFR to be hyperphosphorylated in all cell lines, with the exception of SW620 cells and the CCD841 normal intestinal cell line. By comparing the effects induced by overexpression of either the wild-type or the inactive mutant form of NEU3 on EGFR, we demonstrated that the active form of NEU3 enhanced receptor activation without affecting EGFR mRNA or protein expression. Moreover, through western blots and mass spectrometry analysis, we found that EGFR immunoprecipitated from cells overexpressing active NEU3, unlike the receptor from mock cells and cells overexpressing inactive NEU3, is desialylated. On the whole, our data demonstrate that, besides the already reported indirect EGFR activation through GM3, sialidase NEU3 could also play a role on EGFR activation through its desialylation.

Antioxidant capacity and peptidomic analysis of in vitro digested Camelina sativa L. Crantz and Cynara cardunculus co-products.

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 FeSO4/100 g; 272.84 ± 11.02 mg FeSO4/100 g; 103.84 ± 3.27 mg FeSO4/100 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.

Nitric oxide synthase mediates PC12 differentiation induced by the surface topography of nanostructured TiO2.

BACKGROUND: Substrate nanoscale topography influences cell proliferation and differentiation through mechanisms that are at present poorly understood. In particular the molecular mechanism through which cells 'sense' and adapt to the substrate and activate specific intracellular signals, influencing cells survival and behavior, remains to be clarified. RESULTS: To characterize these processes at the molecular level we studied the differentiation of PC12 cells on nanostructured TiO2 films obtained by supersonic cluster beam deposition.Our findings indicate that, in PC12 cells grown without Nerve Growth Factor (NGF), the roughness of nanostructured TiO2 triggers neuritogenesis by activating the expression of nitric oxide synthase (NOS) and the phospho-extracellular signal-regulated kinase 1/2 (pERK1/2) signaling. Differentiation is associated with an increase in protein nitration as observed in PC12 cells grown on flat surfaces in the presence of NGF. We demonstrate that cell differentiation and protein nitration induced by topography are not specific for PC12 cells but can be regarded as generalized effects produced by the substrate on different neuronal-like cell types, as shown by growing the human neuroblastoma SH-SY5Y cell line on nanostructured TiO2. CONCLUSION: Our data provide the evidence that the nitric oxide (NO) signal cascade is involved in the differentiation process induced by nanotopography, adding new information on the mechanism and proteins involved in the neuritogenesis triggered by the surface properties.

Response to microplastic exposure: An exploration into the sea urchin immune cell proteome.

It is now known that the Mediterranean Sea currently is one of the major hotspot for microplastics (MPs; < 5 mm) pollution and that the risks will be even more pronounced in the coming years. Thus, the in-depth study of the mechanisms underlying the MPs toxicity in key Mediterranean organisms, subjected to high anthropic pressures, has become a categorical imperative to pursue. Here, we explore for the first time the sea urchins immune cells profile combined to their proteome upon in vivo exposure (72 h) to different concentrations of polystyrene-microbeads (micro-PS) starting from relevant environmental concentrations (10, 50, 103, 104 MP/L). Every 24 h, immunological parameters were monitored. After 72 h, the abundance of MPs was examined in various organs and coelomocytes were collected for proteomic analysis based on a shotgun label free proteomic approach. While sea urchins treated with the lowest concentration tested (10 and 50 micro-PS/L) did not show the presence of micro-PS in any tissue, in the specimens exposed to the highest concentration (103 and 104 micro-PS) there was an internalisation of 9.75 ± 2.75 and 113.75 ± 34.5 MP/g, respectively. Proteomic analyses revealed that MPs exposure altered coelomocytes protein profile not only compared to the control group but also among the different micro-PS concentrations and these variations are micro-PS concentration dependent. The proteins exclusively expressed in the coelomocytes of specimens exposed to MPs are mainly metabolite interconversion enzymes, involved in cellular processes, indicating a severe alteration of the cellular metabolic pathways. Overall, these findings provide new insights on the mode of action of MPs in the sea urchin immune cells both at the molecular and cellular level.

Serine metabolism during differentiation of human iPSC-derived astrocytes.

Astrocytes are essential players in development and functions, being particularly relevant as regulators of brain energy metabolism, ionic homeostasis and synaptic transmission. They are also the major source of l-serine in the brain, which is synthesized from the glycolytic intermediate 3-phosphoglycerate through the phosphorylated pathway. l-Serine is the precursor of the two main co-agonists of the N-methyl-d-aspartate receptor, glycine and d-serine. Strikingly, dysfunctions in both l- and d-serine metabolism are associated with neurological and psychiatric disorders. Here, we exploited a differentiation protocol, based on the generation of human mature astrocytes from neural stem cells, and investigated the modification of the proteomic and metabolomic profile during the differentiation process. We show that differentiated astrocytes are more similar to mature rather than to reactive ones, and that axogenesis and pyrimidine metabolism increase up to 30 days along with the folate cycle and sphingolipid metabolism. Consistent with the proliferation and cellular maturation processes that are taking place, also the intracellular levels of l-serine, glycine, threonine, l- and d-aspartate (which level is unexpectedly higher than that of d-serine) show the same biosynthetic time course. A significant utilization of l-serine from the medium is apparent while glycine is first consumed and then released with a peak at 30 days, parallel to its intracellular level. These results underline how metabolism changes during astrocyte differentiation, highlight that d-serine synthesis is restricted in differentiated astrocytes and provide a valuable model for developing potential novel therapeutic approaches to address brain diseases, especially the ones related to serine metabolism alterations.

Mass spectrometry-based proteomic strategy for ecchymotic skin examination in forensic pathology.

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.

Ovothiol ensures the correct developmental programme of the sea urchin Paracentrotus lividus embryo.

Ovothiols are π-methyl-5-thiohistidines produced in great amounts in sea urchin eggs, where they can act as protective agents against the oxidative burst at fertilization and environmental stressors during development. Here we examined the biological relevance of ovothiol during the embryogenesis of the sea urchin Paracentrotus lividus by assessing the localization of the key biosynthetic enzyme OvoA, both at transcript and protein level, and perturbing its protein translation by morpholino antisense oligonucleotide-mediated knockdown experiments. In addition, we explored the possible involvement of ovothiol in the inflammatory response by assessing ovoA gene expression and protein localization following exposure to bacterial lipopolysaccharide. The results of the present study suggest that ovothiol may be a key regulator of cell proliferation in early developing embryos. Moreover, the localization of OvoA in key larval cells and tissues, in control and inflammatory conditions, suggests that ovothiol may ensure larval skeleton formation and mediate inflammatory processes triggered by bacterial infection. This work significantly contributes to the understanding of the biological function of ovothiols in marine organisms, and may provide new inspiration for the identification of the biological activities of ovothiols in humans, considering the pharmacological potential of these molecules.

Phosphoproteomic mapping of CCR5 and ACKR2 signaling properties.

ACKR2 is an atypical chemokine receptor which is structurally uncoupled from G proteins and is unable to activate signaling pathways used by conventional chemokine receptors to promote cell migration. Nonetheless, ACKR2 regulates inflammatory and immune responses by shaping chemokine gradients in tissues via scavenging inflammatory chemokines. To investigate the signaling pathways downstream to ACKR2, a quantitative SILAC-based phosphoproteomic analysis coupled with a systems biology approach with network analysis, was carried out on a HEK293 cell model expressing either ACKR2 or its conventional counterpart CCR5. The model was stimulated with the common agonist CCL3L1 for short (3 min) and long (30 min) durations. As expected, many of the identified proteins are known to participate in conventional signal transduction pathways and in the regulation of cytoskeleton dynamics. However, our analyses revealed unique phosphorylation and network signatures, suggesting roles for ACKR2 other than its scavenger activity. In conclusion, the mapping of phosphorylation events at a holistic level indicated that conventional and atypical chemokine receptors differ in signaling properties. This provides an unprecedented level of detail in chemokine receptor signaling and identifying potential targets for the regulation of ACKR2 and CCR5 function.

Insulin and serine metabolism as sex-specific hallmarks of Alzheimer's disease in the human hippocampus.

Healthy aging is an ambitious aspiration for humans, but neurodegenerative disorders, such as Alzheimer's disease (AD), strongly affect quality of life. Using an integrated omics approach, we investigate alterations in the molecular composition of postmortem hippocampus samples of healthy persons and individuals with AD. Profound differences are apparent between control and AD male and female cohorts in terms of up- and downregulated metabolic pathways. A decrease in the insulin response is evident in AD when comparing the female with the male group. The serine metabolism (linked to the glycolytic pathway and generating the N-methyl-D-aspartate [NMDA] receptor coagonist D-serine) is also significantly modulated: the D-Ser/total serine ratio represents a way to counteract age-related cognitive decline in healthy men and during AD onset in women. These results show how AD changes and, in certain respects, almost reverses sex-specific proteomic and metabolomic profiles, highlighting how different pathophysiological mechanisms are active in men and women.

Protein pattern of Xenopus laevis embryos grown in simulated microgravity.

Numerous studies indicate that microgravity affects cell growth and differentiation in many living organisms, and various processes are modified when cells are placed under conditions of weightlessness. However, until now, there is no coherent explanation for these observations, and little information is available concerning the biomolecules involved. Our aim has been to investigate the protein pattern of Xenopus laevis embryos exposed to simulated microgravity during the first 6 days of development. A proteomic approach was applied to compare the protein profiles of Xenopus embryos developed in simulated microgravity and in normal conditions. Attention was focused on embryos that do not present visible malformations in order to investigate if weightlessness has effects at protein level in the absence of macroscopic alterations. The data presented strongly suggest that some of the major components of the cytoskeleton vary in such conditions. Three major findings are described for the first time: (i) the expression of important factors involved in the organization and stabilization of the cytoskeleton, such as Arp (actin-related protein) 3 and stathmin, is heavily affected by microgravity; (ii) the amount of the two major cytoskeletal proteins, actin and tubulin, do not change in such conditions; however, (iii) an increase in the tyrosine nitration of these two proteins can be detected. The data suggest that, in the absence of morphological alterations, simulated microgravity affects the intracellular movement system of cells by altering cytoskeletal proteins heavily involved in the regulation of cytoskeleton remodelling.

Multi-omic analyses in Abyssinian cats with primary renal amyloid deposits.

The amyloidoses constitute a group of diseases occurring in humans and animals that are characterized by abnormal deposits of aggregated proteins in organs, affecting their structure and function. In the Abyssinian cat breed, a familial form of renal amyloidosis has been described. In this study, multi-omics analyses were applied and integrated to explore some aspects of the unknown pathogenetic processes in cats. Whole-genome sequences of two affected Abyssinians and 195 controls of other breeds (part of the 99 Lives initiative) were screened to prioritize potential disease-associated variants. Proteome and miRNAome from formalin-fixed paraffin-embedded kidney specimens of fully necropsied Abyssinian cats, three affected and three non-amyloidosis-affected were characterized. While the trigger of the disorder remains unclear, overall, (i) 35,960 genomic variants were detected; (ii) 215 and 56 proteins were identified as exclusive or overexpressed in the affected and control kidneys, respectively; (iii) 60 miRNAs were differentially expressed, 20 of which are newly described. With omics data integration, the general conclusions are: (i) the familial amyloid renal form in Abyssinians is not a simple monogenic trait; (ii) amyloid deposition is not triggered by mutated amyloidogenic proteins but is a mix of proteins codified by wild-type genes; (iii) the form is biochemically classifiable as AA amyloidosis.