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Acetic acid bacteria (AAB) and other members of the complex microbiotas, whose activity is essential for vinegar production, display biodiversity and richness that is difficult to study in depth due to their highly selective culture conditions. In recent years, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has emerged as a powerful tool for rapidly identifying thousands of proteins present in microbial communities, offering broader precision and coverage. In this work, a novel method based on LC-MS/MS was established and developed from previous studies. This methodology was tested in three studies, enabling the characterization of three submerged acetification profiles using innovative raw materials (synthetic alcohol medium, fine wine, and craft beer) while working in a semicontinuous mode. The biodiversity of existing microorganisms was clarified, and both the predominant taxa ( Komagataeibacter , Acetobacter , Gluconacetobacter , and Gluconobacter ) and others never detected in these media ( Asaia and Bombella , among others) were identified. The key functions and adaptive metabolic strategies were determined using comparative studies, mainly those related to cellular material biosynthesis, energy-associated pathways, and cellular detoxification processes. This study provides the groundwork for a highly reliable and reproducible method for the characterization of microbial profiles in the vinegar industry.

Pancreatic cancer is currently the third leading cause of cancer death in the United States. The clinical hallmarks of this disease include abdominal pain that radiates to the back, the presence of a hypoenhancing intrapancreatic lesion on imaging, and widespread liver metastases. Technologies such as tissue clearing and three-dimensional (3D) reconstruction of digitized serially sectioned hematoxylin and eosin-stained slides can be used to visualize large (up to 2- to 3-centimeter cube) tissues at cellular resolution. When applied to human pancreatic cancers, these 3D visualization techniques have provided novel insights into the basis of a number of the clinical characteristics of this disease. Here, we describe the clinical features of pancreatic cancer, review techniques for clearing and the 3D reconstruction of digitized microscope slides, and provide examples that illustrate how 3D visualization of human pancreatic cancer at the microscopic level has revealed features not apparent in 2D microscopy and, in so doing, has closed the gap between bench and bedside. Compared with animal models and 2D microscopy, studies of human tissues in 3D can reveal the difference between what can happen and what does happen in human cancers., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Microorganisms play unique, essential and integral roles in the biosphere. This work aims to assess the utility of soil's metaomics for environmental diagnosis. Doñana National Park (DNP) was selected as a natural lab since it contains a strictly protected core that is surrounded by numerous threats of pollution. Culture-independent high-throughput molecular tools were used to evaluate the alterations of the global structure and metabolic activities of the microbiome. 16S rRNA sequencing shows lower bacterial abundance and diversity in areas historically exposed to contamination that surround DNP. For metaproteomics, an innovative post-alkaline protein extraction protocol was developed. After NaOH treatment, successive washing with Tris-HCl buffer supplemented with glycerol was essential to eliminate interferences. Starting from soils with different physicochemical characteristics, the method renders proteins with a remarkable resolution on SDS-PAGE gels. The proteins extracted were analysed by using an in-house database constructed from the rRNA data. LC-MS/MS analysis identified 2182 non-redundant proteins with 135 showing significant differences in relative abundance in the soils around DNP. Relevant global biological processes were altered in response to the environmental changes, such as protective and antioxidant mechanisms, translation, folding and homeostasis of proteins, membrane transport and aerobic respiratory metabolism., (© 2021 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Kefir is a fermented dairy product, associated to health benefits because of being a probiotic and due to the presence of molecules with biological activity. In this work, we have profiled the peptide composition of goat milk kefir at three different fermentation times using a peptidomics approach, in order to study changes in peptide concentrations and patterns of protein digestion throughout the fermentation time. We identified 2328 unique peptides corresponding to 22 protein annotations, with a maximum of peptides found after 24 h fermentation. We established different digestion patterns according to the nature of the proteins, and quantified the changes in the peptides appearing in all the fermentation times. We also identified 11 peptides that matched exactly to sequences with biological activity in databases, almost all of them belonging to caseins. This is the most comprehensive proteomic analysis of goat milk kefir to date., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Natural environments are receiving an increasing number of contaminants. Therefore, the evaluation and identification of early responses to pollution in these complex habitats is an urgent and challenging task. Doñana National Park (DNP, SW Spain) has been widely used as a model area for environmental studies because, despite its strictly protected core, it is surrounded by numerous threat sources from agricultural, mining and industrial activities. Since many pollutants often induce oxidative stress, redox proteomics was used to detect redox-based variations within the proteome of Mus spretus mice captured in DNP and the surrounding areas. Functional analysis showed that most differentially oxidized proteins are involved in the maintenance of homeostasis, by eliciting mechanisms to respond to toxic substances and oxidative stress, such as antioxidant and biotransformation processes, immune and inflammatory responses, and blood coagulation. Furthermore, changes in the overall protein abundance were also analysed by label-free quantitative proteomics. The upregulation of phase I and II biotransformation enzymes in mice from Lucio del Palacio may be an alert for organic pollution in the area located at the heart of DNP. Metabolic processes involved in protein turnover (proteolysis, amino acid catabolism, new protein biosynthesis and folding) were activated in response to oxidative damage to these biomolecules. Consequently, aerobic respiratory metabolism increased to address the greater ATP demands. Alterations of cholesterol metabolism that could cause hepatic steatosis were also detected. The proteomic detection of globally altered metabolic and physiological processes offers a complete view of the main biological changes caused by environmental pollution in complex habitats., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The major derivate of DDT, 1,1-dichloro-2,2-bis (p-chlorophenyl) ethylene (p,p´-DDE), is a persistent pollutant previously associated with oxidative stress. Additionally, p,p´-DDE has been linked to several metabolic alterations related to sexual function in rodents. In this study, we analysed the effects of a non-lethal p,p´-DDE dose to Mus spretus mice in testes, focusing on oxidative damage to biomolecules, defence mechanisms against oxidative stress and post-translational protein modifications. No increase in lipid or DNA oxidation was observed, although antioxidative enzymatic defences and redox status of glutathione were altered in several ways. Global protein carbonylation and phosphorylation were significantly reduced in testes from p,p´-DDE-exposed mice; however, the total redox state of Cys thiols did not exhibit a defined pattern. We analysed the reversible redox state of specific Cys residues in detail with differential isotopic labelling and a shotgun labelling-based MS/MS proteomic approach for identification and quantification of altered peptides. Our results show that Cys residues are significantly affected by p,p´-DDE in several proteins related to oxidative stress and/or male fertility, particularly those participating in fertilization, sperm capacitation and blood coagulation. These molecular changes could explain the sexual abnormalities previously described in p,p´-DDE exposed organisms., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Fish skin mucus is the first line of defense against infections and it discriminates between pathogenic and commensal bacterial strains. Mucus composition varies amongst fish species and is influenced by endogenous and exogenous factors. This study describes the first proteome map of the epidermal mucus of farmed gilthead seabream (Sparus aurata). We used an integrative proteomic approach by combining a label-free procedure (LC-MS/MS) with the classical 2-DE-PMF-MS/MS methodology. The identified mucosal proteins were clustered in four groups according to their biological functions. Structural proteins (actins, keratins, tubulins, tropomyosin, cofilin-2 and filamin-A) and metabolic proteins (ribosomal proteins, proteasomal subunits, NACA, VCP, histones, NDPK, transferrin, glycolytic enzymes, ATP synthase components, beta-globin, Apo-A1 and FABP7) were the best represented functional categories. We also found proteins involved in stress response (WAP65, HSPC70, Cu,Zn-SOD, and PRDX1 and PRDX2) and signal transduction (PP2A 65kDa regulatory subunit, 14-3-3 protein beta/alpha, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, RhoGDI and PEBP1). Most of the identified proteins address different aspects of the innate immune response. Additionally, we analyzed bacterial peptides identified in the skin mucus of healthy S. aurata. These results revealed that genera belonging to the Lactobacillales order constitute the most abundant microorganism populations in this habitat., Biological Significance: This work shows that proteomic methods can be used to characterize fish skin mucus. Using a coupled approach of LC-MS/MS and a 2-DE-PMF-MS/MS, we have obtained the first comprehensive view of the skin mucosal proteome of S. aurata, a fish species that is economically relevant for Mediterranean aquaculture. We identified a panel of proteins involved in a variety of biological functions, particularly in the innate immune response. Furthermore, to our knowledge, this is the first time a proteomic approach has been used to examine the microbiota in the skin mucus of a fish species. Overall, these results support further immunological researches in S. aurata and are relevant for the culture of this important fish species., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The oxidation and reduction of cysteine thiols are thought to be a major mechanism for redox regulation. The aim of this study was to identify proteins with reactive thiols and determine their oxidation profiles under oxidative stress induced by simultaneous silencing of antioxidant defences (peroxiredoxin-1, peroxiredoxin-3, and the catalytic subunit of the glutamate-cysteine ligase), and/or treatment with glucose oxidase (GO). Using an approach that combined the labelling of reversibly oxidised cysteines, 2-DE protein separation and MS analysis, we identified 26 proteins with cysteines prone to reversible oxidation belonging to different functional classes. Among these proteins are those that have not been previously recognised as reversible oxidation targets, including cytoplasmic aspartate aminotransferase, proteasome subunit alpha type-6, heterogeneous nuclear ribonucleoproteins isoA2/B1 and A/B, and histidine triad nucleotide-binding protein 1. We provide the first evidence of reversible oxidation for specific cysteines, including Cys112 and Cys146 in glutamate dehydrogenase 1, Cys17 in actins, Cys5 in protein disulfide-isomerase A3, and Cys267 in the heat shock cognate 71 kDa protein. Silencing induced lower oxidative stress than GO treatment. Nevertheless, we detected some proteins particularly sensitive to oxidation by silencing. We hypothesised that these proteins may play a role in regulatory mechanisms by redox stress., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Yeast Grx2 plays a role in the antioxidant glutathione linked defense acting on the redox status of protein cysteines, but the exact action or its specificity is not known. Moreover, it localizes in cytosol and mitochondria where it can exert different functions. To search for functions of Grx2 we determined the differential "Thiolic Redox Proteome" of control and peroxide-treated yeast mutant cells lacking the gene for Grx2 or expressing Grx2 exclusively in the mitochondria. Forty-two proteins have been identified that have alternative redox oxidation states as a consequence of Grx2 absence from the cell or expression in the mitochondria and absence from the cytosol. The precise cysteine residues affected have been mapped for each protein. One target protein, Rib3p, which has as yet an undefined function in respiration, was confirmed to have its Cys56 reversibly S-glutathionylated in vitro in a Grx2p dependent process. Grx2-dependent redox changes in key enzymes of glutamate consuming amino acid biosynthetic pathways could favor glutathione biosynthesis. Other target proteins are involved in membrane fusion, cell wall structure and ribosome assembly, but others are of unknown function. These results provide clues on the metabolic hot spots of redox regulatory mechanisms., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Maslinic acid (MA) is a pentacyclic triterpene used as a feed additive to stimulate growth, protein-turnover rates, and hyperplasia in fish. To further our understanding of cellular mechanisms underlying the action of MA, we have used 2-DE coupled with MS to identify proteins differentially expressed in the livers of juvenile gilthead sea bream (Sparus aurata) grown under fish-farm conditions and fed with a 100 mg/kg MA-enriched diet (MA(100)). After the comparison of the protein profiles from MA(100) fed fish and from control, 49 protein spots were found to be altered in abundance (≥2-fold). Analysis by MALDI-TOF/TOF allowed the unambiguous identification of 29 spots, corresponding to 19 different proteins. These proteins were: phosphoglucomutase, phosphoglucose isomerase, S-adenosyl methionine-dependent methyltransferase class I, aldehyde dehydrogenase, catalase, 6-phosphogluconate dehydrogenase, fumarylacetoacetate hydrolase, 4-hydroxyphenylpyruvic dioxygenase, methylmalonate-semialdehyde dehydrogenase, lysozyme, urate oxidase, elongation factor 2, 60 kDa heat-shock protein, 58 kDa glucose-regulated protein, cytokeratin E7, type-II keratin, intermediate filament proteins, 17-β-hydroxysteroid dehydrogenase type 4, and kinase suppressor of Ras1. Western blot analysis of kinase suppressor of Ras1, glucose 6-phosphate dehydrogenase, elongation factor 2, 60 kDa heat-shock protein, and catalase supported the proteome evidence. Based on the changes found in the protein-expression levels of these proteins, we proposed a cellular-signalling pathway to explain the hepatic-cell response to the intake of a diet containing MA., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: Rivastigmine transdermal patches for the treatment of Alzheimer's disease (AD) have potential benefits compared to capsules because of their sustained absorption through the skin, good local tolerability and reduction of gastrointestinal problems., Purpose: To assess gastrointestinal and skin tolerability and the need for optimal dose titration of rivastigmine transdermal patches in Alzheimer's disease patients previously treated with oral rivastigmine., Patients and Methods: A multicenter, randomized, open-label study including patients with mild to moderate AD (DSM-IV) previously treated with rivastigmine capsules (6-12 mg/day) was conducted. Patients were randomized to: continue with capsules for 3 months (n=49) or switch to rivastigmine patch without titration (9.5mg/day for 3 months; n=48), or switch to rivastigmine patch with titration (4.6 mg/day for 1 month followed by 9.5mg/day for 2 months, n=43)., Results: Incidence of gastrointestinal adverse events was 6.1% in the group treated orally and 4.2% in the group treated with non-titrated patches (P=.908). Skin tolerability was good (n=15, 16.7%) without any serious adverse events registered. Patch treatment was considered very easy to use by 72% of patients compared with 30% in the group with oral treatment (P=.0005). 60% of patients were satisfied with the patch, while only 14% were satisfied with capsules (P<.0001)., Conclusions: Rivastigmine patches have a tolerability profile similar to that shown by capsules, but are associated with greater patient satisfaction., (Copyright © 2010 Sociedad Española de Neurología. Published by Elsevier Espana. All rights reserved.)

We evaluated the suitability of high-throughput proteomic methods to monitor terrestrial ecosystems. Free-living Mus spretus from three sites along the "Domingo Rubio" (DR) stream were compared with mice from Doñana Biological Reserve ("Santa Olalla" lagoon (SOL) negative control), using specimens from an industrial settlement (phosphogypsum stacks (PS)) and rice fields ("Matochal" rice fields (ARZ)) as positive controls. Our 2-DE analysis showed 36 spots with significantly altered expression. Sixteen were identified by MALDI-TOF-PMF and peptide matching with Mus musculus databases. Identified proteins play different roles: cytoskeletal dynamics, proteolysis, biotransformation, oxidative-stress adaptation, and metabolism. Animals from different polluted environments showed contrasting differences in their proteomes, with specific increases and decreases in selected groups of proteins that seem to be co-ordinately regulated. Proteomic data were consistent with metal biomonitoring and conventional biomarker responses, indicating that DR (and PS/ARZ) animals sustained a heavier pollutant burden than SOL specimens and suffered a chronic oxidative stress. Whereas some protein expression differences may protect mice from pollutant toxicity, others should make them more susceptible. Transcript expression signatures agree with the documented lack of correlation between mRNA and protein levels. Nonetheless, a positive significant correlation was found between the gpx1 mRNA molecules and the intensity of one of the two identified GPX1 isospots.

Background: Alternative splicing is a widespread mechanism of gene expression regulation. Previous analyses based on conventional RT-PCR reported the presence of an unspliced c-fos transcript in several mammalian systems. Compared to the well-defined knowledge on the alternative splicing of fosB, the physiological relevance of the unspliced c-fos transcript in regulating c-fos expression remains largely unknown. This work aimed to investigate the functional significance of the alternative splicing c-fos pre-mRNA., Results: A set of primers was designed to demonstrate that, whereas introns 1 and 2 are regularly spliced from primary c-fos transcript, intron 3 remains unspliced in part of total transcript molecules. Here, the two species are referred to as c-fos-2 (+ intron 3) and spliced c-fos (- intron 3) transcripts. Then, we used a quantitatively rigorous approach based on real-time PCR to provide, for the first time, the actual steady-state copy numbers of the two c-fos transcripts. We tested how the mouse-organ context and mouse-gestational age, the synthesis and turnover rates of the investigated transcripts, and the serum stimulation of quiescent cells modulate their absolute-expression profiles. Intron 3 generates an in-frame premature termination codon that predicts the synthesis of a truncated c-Fos protein. This prediction was evaluated by immunoaffinity chromatography purification of c-Fos proteins., Conclusion: We demonstrate that: (i) The c-fos-2 transcript is ubiquitously synthesized either in vivo or in vitro, in amounts that are higher or similar to those of mRNAs coding for other Fos family members, like FosB, DeltaFosB, Fra-1 or Fra-2. (ii) Intron 3 confers to c-fos-2 an outstanding destabilizing effect of about 6-fold. (iii) Major determinant of c-fos-2 steady-state levels in cultured cells is its remarkably high rate of synthesis. (iv) Rapid changes in the synthesis and/or degradation rates of both c-fos transcripts in serum-stimulated cells give rise to rapid and transient changes in their relative proportions. Taken as a whole, these findings suggest a co-ordinated fine-tune of the two c-fos transcript species, supporting the notion that the alternative processing of the precursor mRNA might be physiologically relevant. Moreover, we detected a c-Fos immunoreactive species corresponding in mobility to the predicted truncated variant.

Thioredoxins function as general protein disulphide reductases. Mammalian male germ cells are equipped with a set of three testis-specific thioredoxins (named Sptrx-1, -2, and -3, respectively) that are expressed either in different structures within the sperm cell or at different stages of sperm development. Previous studies based on qualitative northern-blot and in situ hybridization analyses restricted the presence of Sptrx mRNAs to adult testis, but nothing is known about their transcriptional regulation or relative expression levels in this tissue. In this report, we investigate the transcriptional profiles of the mouse Sptrx genes in terms of the germ cell-specific regulation by promoter analysis in GC-2spd(ts) cells. Besides, we perform a comprehensive quantification of the Sptrx mRNA molecules by real-time PCR in whole-animal experiments. By these means, we show that transcription is differentially regulated for each Sptrx gene and identify the 5'-flanking regions anticipated to contain the cis-regulatory elements responsible, at least in part, for the transcriptional silencing and/or activation of the Sptrx genes. In addition, we show remarkable age-associated variations between the Sptrx mRNA expression patterns.