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Background & Aims: Long-term effects of coronavirus disease 2019 (long COVID) develop in a substantial number of people following an acute COVID-19 episode. Red beetroot juice may have positive effects on multiple pathways involved in long COVID. The aim of this pilot study was to explore the impact of beetroot juice supplementation on physical function, gut microbiota, and systemic inflammation in adults with long COVID., Methods: A single-center, double-blind, placebo-controlled randomized trial was conducted to test the effects of 14 days of beetroot juice supplementation, rich in nitrates and betalains, on functional and biological outcomes in adults aged between 20 and 60 years with long COVID. Participants were randomized 1:1 to receive either daily oral supplementation with 200 mL beetroot juice (∼600 mg nitrate) or placebo (∼60 mg nitrate) for 14 days. The primary endpoint was the change from baseline to day 14 in a fatigue resistance test. Secondary outcomes included the distance walked on the 6-min walk test, handgrip strength, and flow-mediated dilation. Secondary endpoints also included changes from baseline in circulating inflammatory mediators and metagenomic and fecal water metabolomic profiles. Partial least squares discriminant analysis (PLS-DA) models were built to evaluate the differences in biological variables associated with the interventions., Results: Thirty-one participants were randomized in the study. Twenty-five of them (median (interquartile range) age 40 (10), 14 [56 %] women), received either beetroot juice (15) or placebo (10) and completed the study. At 14 days, fatigue resistance significantly improved from baseline (mean difference [standard error]: +21.8 [3.7] s; p < 0.001) with no significant differences between intervention groups. A significant increase from baseline in the distance walked on the 6-min walk test was observed (mean difference [standard error]: +30.0 [9.4] m; p = 0.03), which was not different between groups. Flow-mediated dilation did not differ between participants who received beetroot juice and those on placebo. PLS-DA models allowed correct classification of participants with 92.2 ± 4.4 % accuracy. Those who ingested red beetroot juice had a greater abundance of bacteria with well-known beneficial effects, including Akkermansia, Oscillospira, Prevotella, Roseburia, Ruminococcaceae, and Turicibacter, compared with placebo. Participants allocated to beetroot juice supplementation were also characterized by significantly higher levels of fecal nicotinate, trimethylamine, and markers of beetroot juice intake (e.g., 5,6-dihydroxyindole). Finally, higher levels of interferon gamma and macrophage inflammatory protein-1β were found in participants who consumed beetroot juice., Conclusion: Beetroot juice supplementation for two weeks did not to induce significant improvements in functional outcomes in adults with long COVID compared with placebo. Beneficial effects were observed in both gut microbiota composition (i.e., increase in probiotic species) and inflammatory mediators., Trial Registration: Trial was registered under ClinicalTrials.gov. Identifier no. NCT06535165., Competing Interests: Conflict of interest A.T. and W.A. are employees of Aureli Mario S.S. Agricola, which donated both beetroot juice and placebo for the study., (Copyright © 2024 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

A clinical report of otomyiasis in a 1-year-old girl is reported. A III instar larva of Sarcophaga sp. was microscopically identified and Sarcophaga (Liopygia) argyrostoma (Diptera, Sarcophagidae) was suspected. A molecular method targeting a fragment of the cox1 gene was used to confirm the identity of the specimen. Although myiases are not frequent manifestations in otolaryngology, they should arouse the attention of doctors, social workers and parents dealing with disabled people, the elderly and children. This contribution also highlights the need of combining microscopy and molecular tools to achieve a correct and reliable identification of the specimen/s., (Copyright © 2022 Elsevier B.V. All rights reserved.)

During the last decade, the evidences on the relationship between neurodevelopmental disorders and the microbial communities of the intestinal tract have considerably grown. Particularly, the role of gut microbiota (GM) ecology and predicted functions in Autism Spectrum Disorders (ASD) has been especially investigated by 16S rRNA targeted and shotgun metagenomics, trying to assess disease signature and their correlation with cognitive impairment or gastrointestinal (GI) manifestations of the disease. Herein we present a metaproteomic approach to point out the microbial gene expression profiles, their functional annotations, and the taxonomic distribution of gut microbial communities in ASD children. We pursued a LC-MS/MS based investigation, to compare the GM profiles of patients with those of their respective relatives and aged-matched controls, providing a quantitative evaluation of bacterial metaproteins by SWATH analysis. All data were managed by a multiple step bioinformatic pipeline, including network analysis. In particular, comparing ASD subjects with CTRLs, up-regulation was found for some metaproteins associated with Clostridia and with carbohydrate metabolism (glyceraldehyde-3-phosphate and glutamate dehydrogenases), while down-regulation was observed for others associated with Bacteroidia (SusC and SusD family together with the TonB dependent receptor). Moreover, network analysis highlighted specific microbial correlations among ASD subgroups characterized by different functioning levels and GI symptoms. SIGNIFICANCE: To the best of our knowledge, this study represents the first metaproteomic investigation on the gut microbiota of ASD children compared with relatives and age-matched CTRLs. Remarkably, the applied SWATH methodology allowed the attribution of differentially regulated functions to specific microbial taxa, offering a novel and complementary point of view with respect to previous studies., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Gut microbiota exerts a crucial role in gastrointestinal (GI) and extra-intestinal (EI) disorders. In this context, Akkermansia muciniphila is pivotal for the maintenance of host health and has been correlated with several disorders., Aim: To explore the potential role of A. muciniphila as common dysbiotic marker linked to the disease status., Methods: A cohort of patients affected by GI and EI disorders was enrolled and compared to healthy controls (CTRLs). A targeted-metagenomics approach combined to unsupervised cluster and machine learning (ML) analyses provided microbiota signatures., Results: Microbiota composition was associated to disease phenotype, therapies, diet and anthropometric features, identifying phenotype and therapies as the most impacting variables on microbiota ecology. Unsupervised cluster analyses identified one cluster composed by the majority of patients. DESeq2 algorithm identified ten microbial discriminatory features of patients and CTRLs clusters. Among these microbes, Akkermansia muciniphila resulted the discriminating ML node between patients and CTRLs, independently of specific GI/EI disease or confounding effects. A. muciniphila decrease represented a transversal signature of gut microbiota alteration, showing also an inverse correlation with α-diversity., Conclusion: Overall, A. muciniphila decline may have a crucial role in affecting microbial ecology and in discriminating patients from healthy subjects. Its grading may be considered as a gut dysbiosis feature associated to disease-related microbiota profile., Competing Interests: Declaration of Competing Interest Authors do not have any competing interests to declare., (Copyright © 2020. Published by Elsevier Ltd.)

Food allergy is the disease where the immune system is elicited by antigens in food. Although innocuous for immune-tolerant individuals, an ever-growing number of food allergenic people are being registered worldwide. To date, no treatment to cure food allergy is available and the disease management relies on the careful exclusion of the allergenic food from the diet of the allergic individuals. Great efforts are ongoing to clarify the allergenic mechanisms of the diverse allergenic proteins of food origin, aimed to both designing suitable therapies and for a timely and precise diagnosis of the allergic condition. Among the other omics sciences, mass spectrometry (MS)-based proteomics is gaining a steadily increasing interest by the whole scientific community acknowledged its high versatility. In the present work, the latest proteomics based-studies on allergenic proteins are reviewed to provide guidance on the different MS-based methodologies adopted in the research on food allergens. Our review points to highlight the strengths of the MS-based proteomics and how these have been exploited to address specific research questions. Also, the most common drawbacks encountered in a proteomic study are discussed, providing an overview that helps novel researchers in choosing the more suitable experimental workflow. SIGNIFICANCE: Wide wealth of knowledge arising from the various MS-based proteomic investigations is improving our understanding of food allergy through molecular characterization of food allergens. The present work reviews the key aspects to be evaluated while investigating food allergens by means of MS-based proteomics and provide guidance to the novel research groups approaching to the fascinating world of MS-based food allergens detection., Competing Interests: Declaration of Competing Interest The authors declare that there exist no conflicts of interest with regard to the present study., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Breastfeeding is nowadays known to be one of the most critical factors contributing to the development of an efficient immune system. In the last decade, a consistent number of pieces of evidence demonstrated the relationship between a healthy organism and its gut microbiota. However, this link is still not fully understood and requires further investigation. We recently adopted a murine model to describe the impact of either maternal milk or parental genetic background, on the composition of the gut microbial population in the first weeks of life. A metaproteomic approach to such complex environments is a big challenge that requires a strong effort in both data production and analysis, including the set-up of dedicated multitasking bioinformatics pipelines. Herein we present an LC-MS/MS based investigation to monitor mouse gut microbiota in the early life, aiming at characterizing its functions and metabolic activities together with a taxonomic description in terms of operational taxonomic units. We provided a quantitative evaluation of bacterial metaproteins, taking into account differential expression results in relation to the functional and taxonomic classification, particularly with proteins from orthologues groups. This allowed the reduction of the bias arising from the presence of a high number of shared peptides, and proteins, among different bacterial species. We also focused on host mucosal proteome and its modulation, according to different microbiota composition. SIGNIFICANCE: This paper would represent a reference work for investigations on gut microbiota in early life, from both a microbiological and a functional proteomic point of view. We focused on the shaping of the mouse gut microbiota in dependence on the feeding modality, defining a reliable taxonomic description, highlighting some functional characteristics of the microbial community, and performing a first quantitative evaluation by data independent analysis in metaproteomics., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Growing evidence supports the potential role of intestinal microbiota in the pathophysiology of inflammatory bowel diseases (IBD) even if the literature does not reveal uniform alterations. The aim of the study was to evaluate the mucosal (MM) and faecal microbiota (FM) composition in a cohort of IBD patients compared to healthy controls (CTRLs)., Methods: Faecal and mucosal samples were collected from 14 IBD patients and 11 CTRLs. The V1-V3 region of 16S rRNA locus was amplified on a 454-Junior Genome Sequencer. Reads were grouped into operational taxonomic units (OTUs) at a sequence similarity level of 97% for taxonomic assignment, and aligned for OTUs matching against Greengenes database., Results: Irrespective of disease localization and activity, in the MM of IBD patients a statistically significant increase of Proteobacteria (especially Enterobacteriaceae, Acidaminococcus, Veillonella dispar) and decrease of Firmicutes (especially Roseburia and Faecalibacterium prausnitzii) and Actinobacteria was found compared to CTRLs. In the colon district some specific bacterial biomarkers were identified: Enterobacteriaceae for IBD stools, Bacteroides for IBD biopsies, Mogibacteriaceae, Ruminococcaceae and Prevotella for CTRL stools, Ruminococcaceae for CTRL biopsies., Conclusions: The profiles of FM were more similar to CTRLs, suggesting that microbiota adhering to the gut mucosa better discriminates patients from controls, with the identification of some interesting biomarkers., (Copyright © 2018 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

The main aim of this work was to evaluate, at pilot scale in an industrial environment, the effects of the biocontrol agent Lactococcus lactis CBM21 and thyme essential oil compared to chlorine, used in the washing step of fresh-cut lamb's lettuce, on the microbiota and its changes in relation to the time of storage. The modification of the microbial population was studied through pyrosequencing in addition to the traditional plate counts. In addition, the volatile molecule and sensory profiles were evaluated during the storage. The results showed no significant differences in terms of total aerobic mesophilic cell loads in relation to the washing solution adopted. However, the pyrosequencing data permitted to identify the genera and species able to dominate the spoilage associations over storage in relation to the treatment applied. Also, the analyses of the volatile molecule profiles of the samples during storage allowed the identification of specific molecules as markers of the spoilage for each different treatment. The sensory analyses after 3 and 5 days of storage showed the preference of the panelists for samples washed with the combination thyme EO and the biocontrol agent. These samples were preferred for attributes such as flavor, acceptability and overall quality. These results highlighted the effect of the innovative washing solutions on the quality of lettuce through the shift of microbiota towards genera and species with lower potential in decreasing the sensory properties of the product., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

To investigate the prevalence of protozoan contamination by Giardia duodenalis, Cryptosporidium spp., Toxoplasma gondii and Cyclospora cayetanensis, in 'ready to eat' (RTE) salads on sale in Italy, 648 packages were purchased from industrial and local brands. Nine individual packages from each brand were collected per month, pooled and subjected to microscopy and molecular analyses. Microscopic examination of 864 slides detected Cryptosporidium spp. but also Blastocystis hominis and Dientamoeba fragilis. Molecular tools identified G. duodenalis assemblage A, Cryptosporidium parvum and Cryptosporidium ubiquitum, T. gondii Type I and C. cayetanensis. B. hominis and D. fragilis were also molecularly confirmed. The overall prevalence of each protozoan species was 0.6% for G. duodenalis, 0.8% for T. gondii, 0.9% for Cryptosporidium spp., and 1.3% for C. cayetanensis, while prevalence for B. hominis was 0.5% and for D. fragilis 0.2%. Microscopy and/or molecular tools revealed that 4.2% of the samples were contaminated by at least one protozoan species, and 0.6% of samples presented contamination by two protozoan species, with a number of oocysts ranging from 62 to 554 per g of vegetable matter for T. gondii, and 46 to 1.580 for C. cayetanensis. This is Europe's first large-scale study on the presence of protozoans in packaged salads, and shows that RTE sanitation processes do not guarantee a product free from protozoans of fecal origin., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Unlabelled: The functional complexity of human gut microbiota and its relationship with host physiology and environmental modulating factors, offers the opportunity to investigate (i) the host and microbiota role in organism-environment relationship; (ii) the individual functional diversity and response to environmental stimuli (exposome); (iii) the host genome and microbiota metagenomes' modifications by diet-mediated epigenomic controls (nutriepigenomics); and (iv) the genotype-phenotype "trajectories" under physiological and disease constraints. Systems biology-based approaches aim at integrating biological data at cellular, tissue and organ organization levels, using computational modeling to interpret diseases' physiopathological mechanisms (i.e., onset and progression). Proteomics improves the existing gene models by profiling molecular phenotypes at protein abundance level, by analyzing post-translational modifications and protein-protein interactions and providing specific pathway information, hence contributing to functional molecular networks. Transcriptomics and metabolomics may determine host ad microbiota changes induced by food ingredients at molecular level, complementing functional genomics and proteomics data. Since foodomics is an -omic wide methodology may feed back all integrative data to foster the omics-based systems medicine field. Hence, coupled to ecological genomics of gut microbial communities, foodomics may highlight health benefits from nutrients, dissecting diet-induced gut microbiota eubiosis mechanisms and significantly contributing to understand and prevent complex disease phenotypes., Biological Significance: Besides transcriptomics and proteomics there is a growing interest in applying metabolic profiling to food science for the development of functional foods. Indeed, one of the biggest challenges of modern nutrition is to propose a healthy diet to populations worldwide, intrinsically respecting the high inter-individual variability, driven by complex host/nutrients/microbiota/environment interactions. Therefore, metabolic profiling can assist at various levels for the development of functional foods, starting from screening for food composition to identification of new biomarkers to trace food intake. This current approach can support diet intervention strategies, epidemiological studies, and controlling of metabolic disorders worldwide spreading, hence ensuring healthy aging. With high-throughput molecular technologies driving foodomics, studying bidirectional interactions of host-microbial co-metabolism, innate immune development, dysfunctional nutrient absorption and processing, complex signaling pathways involved in nutritional metabolism, is now likely. In all cases, as microbiome pipeline efforts continue, it is possible that enhanced standardized protocols can be developed, which may lead to new testable biological and clinical hypotheses. This Review provides a comprehensive update on the current state-of-the-art of the integrated -omics route in food, microbiota and host co-metabolism studies, which may revolutionize the design of new dietary intervention strategies., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Escherichia coli (E. coli) urinary tract infections (UTIs) are becoming a serious problem both for pets and humans (zoonosis) due to the close contact and to the increasing resistance to antibiotics. This study has been performed in order to unravel the mechanism of induced enrofloxacin resistance in canine E. coli isolates that represent a good tool to study this pathology. The isolated E. coli has been induced with enrofloxacin and studied through 2D DIGE and shotgun MS. Discovered differentially expressed proteins are principally involved in antibiotic resistance and linked to oxidative stress response, to DNA protection and to membrane permeability. Moreover, since enrofloxacin is an inhibitor of DNA gyrase, the overexpression of DNA starvation/stationary phase protection protein (Dsp) could be a central point to discover the mechanism of this clone to counteract the effects of enrofloxacin. In parallel, the dramatic decrease of the synthesis of the outer membrane protein W, which represents one of the main gates for enrofloxacin entrance, could explain additional mechanism of E. coli defense against this antibiotic. All 2D DIGE and MS data have been deposited into the ProteomeXchange Consortium with identifier PXD002000 and DOI http://dx.doi.org/10.6019/PXD002000. This article is part of a Special Issue entitled: HUPO 2014., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In order to characterize newborn mouse gut microbiota phylotypes in very early-life stages, an original metaproteomic pipeline, based on LC-MS(2)-spectra and Mascot driven NCBI non-redundant repository database interrogation was developed. An original computational analysis assisted in the generation of a taxonomic gut architecture from protein hits to operational taxonomic units (OTUs) and related functional categories. Regardless of the mouse's genetic background, a prevalence of Firmicutes (Lactobacillaceae) and Proteobacteria (Enterobacteriaceae) was observed among the entire Eubacteria taxonomic node. However, a higher abundance of Firmicutes was retrieved for Balb/c gut microbiota compared to Rag2(ko) mice, the latter was mainly characterized by a Proteobacteria enriched microbiota. The metaproteomic-obtained OTUs were supported, for the identification (ID) of the cultivable bacteria fraction, corroborated by axenic culture-based MALDI-TOF MS IDs. Particularly, functional analysis of Rag2(ko) mice gut microbiota proteins revealed the presence of abundant glutathione, riboflavin metabolism and pentose phosphate pathway components, possibly related to genetic background. The metaproteomic pipeline herein presented may represent a useful tool to investigate the highly debated onset of the human gut microbiota in the first days of life, when the bacterial composition, despite its very low diversity (complexity), is still very far from an exhaustive description and other complex microbial consortia., Biological Significance: The manuscript deals with a "frontier" topic regarding the study of the gut microbiota and the application of a metaproteomic pipeline to unveil the complexity of this fascinating ecosystem at the very early stages of life. Indeed during these phases, its diversity is very low but the bacterial content is highly "instable", and the relative balance between mucosal and fecal bacteria starts its dynamics of "fight" to get homeostasis. However, in the neonatal period, especially immediately after birth, a comprehensive description of this microbial eco-organ is still lacking, while it should be mandatory to highlight its first mechanisms of homeostasis and perturbation, while it co-develops with and within the host species. In order to unravel its low but almost unknown microbial community multiplicity, the newborn mouse gut, characterized by a "very" low complexity, was herein selected as model to design a LC-MS(2)-based shotgun metaproteomic approach, potentially suitable to study onset and shaping in human newborns. A microbiological semi-automatic computational analysis was performed to infer gut phylotypes; such as proof of evidence, related OTUs were compared to axenic-culture-based MALDI-TOF MS IDs showing consistency at family and phyla levels for the bacterial cultivable fraction. This article is part of a Special Issue entitled: Trends in Microbial Proteomics., (© 2013. Published by Elsevier B.V. All rights reserved.)

The establishment of gut microbiota immediately after birth is modulated by different mechanisms that can be considered specific determinants of temporal and spatial variability. Over the last few years, molecular methods have been offering a complementary support to the classical microbiology, often underpowered by its inability to provide unbiased representation of gut microbiota. The advent of high-throughput-omics-based methods has opened new avenues in the knowledge of the gut ecosystem by shedding light on its shape and modulation. Such methods may unveil taxa distribution, role and density of microbial habitants, hence highlighting individual phenotyping (physiological traits) and their relationship with gut dysbiosis, inflammation processes, metabolic disorders (pathological conditions). Synergic meta-omics or "systems biology"-based approaches may concur in providing advanced information on microbiota establishment and pathogen control. During early-life stages this massive amount of data may provide gut microbiota descriptive and functional charts which can be exploited to perform a good practice in childcare and pediatrics, thus providing nutraceutical benefits and endorsing healthy development and aging. This article is part of a Special Issue entitled: Translational Proteomics., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Major changes in medical, intensive care and organ transplantation practices are drastically increasing the risk of fungal opportunistic infections. We designed and set-up a MALDI-TOF MS-based assay to identify the most isolated and emerging therapy-refractory/uncommon fungi from cystic fibrosis (CF) and immunocompromised patients. Two-hundred and thirty isolates from 10 different genera (Aspergillus, Emericella, Fusarium, Geosmithia, Neosartorya, Penicillium, Pseudallescheria, Scedosporium, Talaromyces, Fomitopsis), investigated during routine diagnostic efforts, were correlated to 22 laboratory-adapted reference MALDI-TOF MS "proteomic phenotypes". A growth time-course at 30°C on Sabouraud agar medium was performed for the 22 "phenotypes" at 48, 72, 96 and 120h points. The best peptide extraction conditions for full recovery of conidia- or asci-producing multihyphal morph structures and the highest intra- and inter-class profiling correlation were identified for the 120h point spectra dataset, from which an engineered library derived (pre-analytical phase). Fingerprinting classifiers, selected by Wilcoxon/Kruskal-Wallis algorithm, were computed by Genetic Algorithm, Support Vector Machine, Supervised Neuronal Network and Quick Classifier model construction. MS identification (ID) of clinical isolates was referred to genotyping (GT) and, retrospectively, compared to routine morphotyping (MT) IDs (analytical phase). Proteomic phenotyping is revolutionizing diagnostic mycology as fully reflecting species/morph varieties but often overcoming taxonomic hindrance., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Omega-amino acid monooxygenases (EC 1.14.13.-), catalysing the formation of hydroxamate precursors of microbial siderophores (e.g., pyoverdine), have so far eluded structural and biochemical characterisation. Here, the expression of recombinant L-ornithine-Ndelta-oxygenase (PvdA) from Pseudomonas aeruginosa PAO1 is reported. A library of eight monoclonal antibodies (MAbs) directed against PvdA has been generated. Two MAb families recognising the N- and C-terminal regions of PvdA were identified. The MAbs made it possible to demonstrate that 45-48 kDa PvdA homologues are expressed in response to iron limitation by different species and strains of fluorescent pseudomonads. Despite the different degrees in sequence similarity between P. aeruginosa PvdA and putative homologues from Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae, Burkholderia cepacia, and Ralstonia solanacearum, in silico domain scanning predicts an impressive conservation of putative cofactor and substrate binding domains. The MAb library was also used to monitor PvdA expression during the transition of P. aeruginosa from iron-sufficient to iron-deficient growth.