The metaproteome of the gut microbiota in pediatric patients affected by COVID-19.

Introduction: The gut microbiota (GM) play a significant role in the infectivity and severity of COVID-19 infection. However, the available literature primarily focuses on adult patients and it is known that the microbiota undergoes changes throughout the lifespan, with significant alterations occurring during infancy and subsequently stabilizing during adulthood. Moreover, children have exhibited milder symptoms of COVID-19 disease, which has been associated with the abundance of certain protective bacteria. Here, we examine the metaproteome of pediatric patients to uncover the biological mechanisms that underlie this protective effect of the GM. Methods: We performed nanoliquid chromatography coupled with tandem mass spectrometry on a high resolution analytical platform, resulting in label free quantification of bacterial protein groups (PGs), along with functional annotations via COG and KEGG databases by MetaLab-MAG. Additionally, taxonomic assignment was possible through the use of the lowest common ancestor algorithm provided by Unipept software. Results: A COVID-19 GM functional dissimilarity respect to healthy subjects was identified by univariate analysis. The alteration in COVID-19 GM function is primarily based on bacterial pathways that predominantly involve metabolic processes, such as those related to tryptophan, butanoate, fatty acid, and bile acid biosynthesis, as well as antibiotic resistance and virulence. Discussion: These findings highlight the mechanisms by which the pediatric GM could contribute to protection against the more severe manifestations of the disease in children. Uncovering these mechanisms can, therefore, have important implications in the discovery of novel adjuvant therapies for severe COVID-19.

Characterization of Extracellular Vesicles in Osteoporotic Patients Compared to Osteopenic and Healthy Controls.

Extracellular vesicles (EVs) are mediators of a range of pathological conditions. However, their role in bone loss disease has not been well understood. In this study we characterized plasma EVs of 54 osteoporotic (OP) postmenopausal women compared to 48 osteopenic (OPN) and 44 healthy controls (CN), and we investigated their effects on osteoclasts and osteoblasts. We found no differences between the three groups in terms of anthropometric measurements and biochemical evaluation of serum calcium, phosphate, creatinine, PTH, 25-hydroxy vitamin D and bone biomarkers, except for an increase of CTX level in OP group. FACS analysis revealed that OP patients presented a significantly increased number of EVs and RANKL+ EVs compared with both CN and OPN subjects. Total EVs are negatively associated with the lumbar spine T-score and femoral neck T-score. Only in the OPN patients we observed a positive association between the total number of EVs and RANKL+ EVs with the serum RANKL. In vitro studies revealed that OP EVs supported osteoclastogenesis of healthy donor peripheral blood mononuclear cells at the same level observed following RANKL and M-CSF treatment, reduced the ability of mesenchymal stem cells to differentiate into osteoblasts, while inducing an increase of OSTERIX and RANKL expression in mature osteoblasts. The analysis of miRNome revealed that miR-1246 and miR-1224-5p were the most upregulated and downregulated in OP EVs; the modulated EV-miRNAs in OP and OPN compared to CN are related to osteoclast differentiation, interleukin-13 production and regulation of canonical WNT pathway. A proteomic comparison between OPN and CN EVs evidenced a decrease in fibrinogen, vitronectin, and clusterin and an increase in coagulation factors and apolipoprotein, which was also upregulated in OP EVs. Interestingly, an increase in RANKL+ EVs and exosomal miR-1246 was also observed in samples from patients affected by Gorham-Stout disease, suggesting that EVs could be good candidate as bone loss disease biomarkers. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Intestinal Permeability and Dysbiosis in Female Patients with Recurrent Cystitis: A Pilot Study.

Recurrent cystitis (RC) is a common disease, especially in females. Anatomical, behavioral and genetic predisposing factors are associated with the ascending retrograde route, which often causes bladder infections. RC seems to be mainly caused by agents derived from the intestinal microbiota, and most frequently by Escherichia coli. Intestinal contiguity contributes to the etiopathogenesis of RC and an alteration in intestinal permeability could have a major role in RC. The aim of this pilot study is to assess gut microbiome dysbiosis and intestinal permeability in female patients with RC. Patients with RC (n = 16) were enrolled and compared with healthy female subjects (n = 15) and patients with chronic gastrointestinal (GI) disorders (n = 238). We calculated the Acute Cystitis Symptom Score/Urinary Tract Infection Symptom Assessment (ACSS/UTISA) and Gastrointestinal Symptom Rating Scale (GSRS) scores and evaluated intestinal permeability and the fecal microbiome in the first two cohorts. Patients with RC showed an increased prevalence of gastrointestinal symptoms compared with healthy controls. Of the patients with RC, 88% showed an increased intestinal permeability with reduced biodiversity of gut microbiota compared to healthy controls, and 68% of the RC patients had a final diagnosis of gastrointestinal disease. Similarly, GI patients reported a higher incidence of urinary symptoms with a diagnosis of RC in 20%. Gut barrier impairment seems to play a major role in the pathogenesis of RC. Further studies are necessary to elucidate the role of microbiota and intestinal permeability in urinary tract infections.

Proteomics and Metabolomics Approaches towards a Functional Insight onto AUTISM Spectrum Disorders: Phenotype Stratification and Biomarker Discovery.

Autism spectrum disorders (ASDs) are neurodevelopmental disorders characterized by behavioral alterations and currently affect about 1% of children. Significant genetic factors and mechanisms underline the causation of ASD. Indeed, many affected individuals are diagnosed with chromosomal abnormalities, submicroscopic deletions or duplications, single-gene disorders or variants. However, a range of metabolic abnormalities has been highlighted in many patients, by identifying biofluid metabolome and proteome profiles potentially usable as ASD biomarkers. Indeed, next-generation sequencing and other omics platforms, including proteomics and metabolomics, have uncovered early age disease biomarkers which may lead to novel diagnostic tools and treatment targets that may vary from patient to patient depending on the specific genomic and other omics findings. The progressive identification of new proteins and metabolites acting as biomarker candidates, combined with patient genetic and clinical data and environmental factors, including microbiota, would bring us towards advanced clinical decision support systems (CDSSs) assisted by machine learning models for advanced ASD-personalized medicine. Herein, we will discuss novel computational solutions to evaluate new proteome and metabolome ASD biomarker candidates, in terms of their recurrence in the reviewed literature and laboratory medicine feasibility. Moreover, the way to exploit CDSS, performed by artificial intelligence, is presented as an effective tool to integrate omics data to electronic health/medical records (EHR/EMR), hopefully acting as added value in the near future for the clinical management of ASD.

Spleen development is modulated by neonatal gut microbiota.

The full development of the mammalian immune system occurs after birth upon exposure to non self-antigens. The gut is the first site of bacterial colonization where it is crucial to create the appropriate microenvironment able to balance effector or tolerogenic responses to external stimuli. It is a well-established fact that at mucosal sites bacteria play a key role in developing the immune system but we ignore how colonising bacteria impact the maturation of the spleen. Here we addressed this issue. Taking advantage of the fact that milk SIgA regulates bacterial colonization of the newborn intestine, we generated immunocompetent mice born either from IgA pro-efficient or IgA deficient females. Having demonstrated that SIgA in maternal milk modulates neonatal gut microbiota by promoting an increased diversity of the colonizing species we also found that immunocompetent pups, not exposed to milk SIgA, fail to properly develop the FDC network and primary follicles in the spleen compromising the response to T-dependent antigens. The presence of a less diverse microbiota with a higher representation of pathogenic species leads to a fast replenishment of the marginal zone and the IgM plasma cell compartment of the spleen as well as IgA plasma cells in the gut.

Unravelling the effect of clostridia spores and lysozyme on microbiota dynamics in Grana Padano cheese: A metaproteomics approach.

UNLABELLED: Grana Padano is a typical Italian Protected Designation of Origin (PDO) hard cheese largely consumed all over the world. The major problem during its production is represented by late blowing. Clostridia are gasogen bacteria responsible of the swelling during ripening, and they are partially counteracted by the use of egg white lysozyme as additive. In this work was applied, for the first time in cheese, a metaproteomic approach that identified the functional dynamics of microbial consortia in relation to the number of clostridial spores and lysozyme treatment using experimental samples of Grana Padano cheese. We used a combined custom BLAST+/MEGAN/STAMP approach to obtain a global taxonomic view associated to low and high clostridial spores cheese without and with lysozyme. Main differences were highlighted in the bacilli class. Functional analysis with SEED provided a deep view into several metabolic pathways, highlighting the subsystems "amino acid and derivatives" and "clustering-based subsystem" as the targeted subsystems during lysozyme treatment in the high spore group. In these subsystems, acetate kinase from clostridia was one of the main enzymes affected by the lysozyme treatment. BIOLOGICAL SIGNIFICANCE: Metaproteomics is a very promising and useful technique in the control of food safety and quality, from fresh products until 'ready to eat' food. Tools able to identify at molecular level the dynamic fingerprinting of food microbiota could be of great help to improve food safety and quality.

A metaproteomic pipeline to identify newborn mouse gut phylotypes.

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.

Enantioselective supramolecular carriers for nucleoside drugs. A thermodynamic and kinetic gas phase investigation.

The enantioselective interactions between chiral tetra-amidic receptors and nucleosides have been investigated by the ESI-IT-MS and ESI-FT-ICR-MS methodologies. Configurational effects on the CID fragmentation of diastereomeric [M(H)(2)•H•A](+) aggregates (A = 2'-deoxycytidine dC, citarabine (ara-C) were found to be mostly offset by isotope effect in [S(X)(2)•H•A](+) (X = H, D) differently from the results obtained on the analogues (A = cytidine C and gemcitabine G). This result points the involvement of two different nucleoside/tetraamide isoforms. The structural differences of the [M(H)(2)•H•A](+) (A = C and G) complexes vs. the [M(H)(2)•H•A](+) (dC and ara-C) ones is fully confirmed by the kinetics of their uptake of the 2-aminobutane enantiomers, measured by FT-ICR mass spectrometry. Indeed, uptake of the 2-aminobutane enantiomers by [M(H)(n)•H•A](+) (n = 1,2; A = dC and ara-C) complexes is reversible, while that by [M(H)(n)•H•A](+) (n = 1,2; A = C and G) is not. The most encouraging result concerning the measured fragmentation and kinetic differences between C and ara-C, that are just epimers, indicates the possibility to subtly modulate the non-covalent drug/receptor interactions, through the electronic properties of the 2'-substituent on the nucleoside furanose ring, and furthermore on its three-dimensional position.

The dynamic chromatographic behavior of tri-o-thymotide on HPLC chiral stationary phases.

The interconverting stereoisomers of tri-o-thymotide have been separated by HPLC on chiral stationary phases and the temperature dependence of the dynamic chromatographic patterns has been interpreted in terms of exchange between enantiomeric propeller and helical conformations. Computed low energy structures and CD spectra were used in absolute configuration assignment.

The "bridge" game: role of the fourth player in chiral recognition.

A new team player: The "three-point interaction" model, which is usually employed to rationalize chiral recognition, does not account for the amazing enantioselectivity measured for the receptors of many proteic acceptors. Gas-phase experiments have indicated that at least a fourth "player" must be considered: the rigidity that a receptor opposes to distortions of its cavity resulting from noncovalent interactions with a chiral molecule (see picture).

Proteomics investigation of human platelets in healthy donors and cystic fibrosis patients by shotgun nUPLC-MSE and 2DE: a comparative study.

Platelets are of pathophysiological relevance in haemostasis, wound repair, inflammation and cardiovascular disease. We have shown that human platelets express a biologically active Cystic Fibrosis Transmembrane Conductance Regulator, which is dysfunctional in Cystic Fibrosis (CF) patients, and regulate platelet responses related to inflammation and its resolution. In order to further elucidate platelet involvement in CF inflammation, we pursued a comparative proteomic analysis of cells from healthy donors and CF patients, in association with a non-supervised comparative analysis of the Gene Ontology. Our results, showing changes in the integrin signalling in CF, support a pro-inflammatory profile of CF platelets.

Proteomics investigation of human platelets by shotgun nUPLC-MSE and 2DE experimental strategies: a comparative study.

BACKGROUND: Platelets, the smallest human blood cells component, have a key role in the control of haemostasis and thrombosis but they have also been shown to be implicated in a number of different pathological states because of their involvement also in the process of inflammation end its resolution. Their peculiar anucleated morphology render the proteomics an intriguing approach to understand their biology. Given the high impact of platelet in different diseases we have started a systematic investigation of protein repertoire in controlled platelet preparation. MATERIAL AND METHODS: Platelets have been extracted from blood of healthy donors (n=6) collected by venipuncture in Vacutainer. The quality of the preparation was assessed by observation and enumeration in a Bürker chamber with a conventional tissue culture microscope. To characterize human platelets proteome we analysed the pool of purified platelets combining two proteomic approaches: 2-DE separation combined with Mass Spectrometry and nanoscale ultra performances LC-MS(E) shotgun proteomics experiments. RESULTS: The 2D gel analysis leads an average of 1900 protein spots, after the filtering of "noise" and "false positive" spots, over 500 were selected to be eligible for further analysis given their optimal spot quality value. To perform the analysis by ion accounting shotgun proteomic approach, based on nano ultra performance liquid chromatography (nUPLC) coupled to MS(E) processing of continuum LC-MS data, the same pool of samples was subject to liquid phase tryptic digestion and the peptide obtained used for the experiments. All the data obtained were analysed using ProteinLynx GlobalServer v2.3 (PLGS, Waters). Three analytical replicates run were acquire in high/low energy modes and associated to a human protein database returning the identification of 100 distinct genes. Comparative analysis of the Gene Ontology has been performed to evaluate the differential functional representation of the molecular repertoire investigated with these two orthogonal approaches. DISCUSSION: The overall molecular function classification revealed differences between the two proteomic approaches. In particular, we found significant differences in cytoskeletal proteins (19.65% 2-DE versus 45.60 Shotgun) and receptors (0,92% 2-DE versus 6.90% Shotgun).

Towards enzyme-like enantioselectivity in the gas phase: conformational control of selectivity in chiral macrocyclic dimers.

Conformational factors in self-assembled chiral tetraamide macrocycles control their gas-phase enantioselectivity towards the ethyl ester of naphthylalanine to levels typical of enzymes.

Dynamic HPLC of stereolabile iron(II) complexes on chiral stationary phases.

A series of chiral tris-(1,10)-phenanthroline iron(II) complexes have been resolved by HPLC on chiral stationary phases based on either cellulose tris-(3,5-dimethylphenylcarbamate) or teicoplanin. At sub ambient temperatures, baseline separation of the enantiomers was observed for five different iron(II) complexes featuring substituted phenanthroline ligands. Dynamic HPLC profiles were observed near or above room temperature, indicating on-column Delta/Lambda enantiomerization. Rate constants for the Delta/Lambda interconversion in free solution and during chromatography were obtained by thermal racemization experiments and by computer simulation of the HPLC dynamic plots, respectively.

Enantiodiscrimination of bilirubin-IXalpha enantiomers in biomembrane models: has chirality a role in bilirubin toxicity?

Simple biomembrane models, namely micellar aggregates formed by enantiopure sodium N-acylprolinates, are able to convert the racemic mixture of bilirubin-IXalpha into an enantiomerically enriched mixture, thus suggesting a possible role of chirality in bilirubin toxicity due to the perturbation of neuron membrane dynamics. The length of alkyl chain does not influence the extent of equilibrium displacement, however, it affects the conformation of bilirubin, thus confirming the role of lipid structure in the membrane/bilirubin interaction, and suggesting a non-superficial main site of association.

Methyl ether derivatives of p-tert-Butyl[3.1.3.1]homooxacalixarene. Formation, structure, and complexes with quaternary ammonium ions.

[structure: see text] The whole set (five compounds) of partially O-methylated products of p-tert-butyl[3.1.3.1]homooxacalixarene, currently named p-tert-butyltetrahomodioxacalix[4]arene, have been prepared. Their structure has been investigated in solution through NMR techniques and in the solid state by single-crystal X-ray diffraction. A systematic investigation, extended to the parent tetraphenol and to the tetramethyl ether derivative, has been carried out on the complexation of tetramethylammonium, acetylcholine, N-methylpyridinium, and tetraethylammonium picrate in CDCl3. The observed trends in the binding and in the selectivity of the strictly related hosts could be analyzed on the basis of the varying importance of intramolecular hydrogen bonding and its effects on the conformation of the free and of the complexed ligands. On increasing the number of methyl ether functions, the cone conformation appears to be relatively less stable but deeper, so small organic cations can be more effectively encircled.

5,5'-Di-tert-butyl-2,2'-dihydroxy-3,3'-methylenedibenzaldehyde and 6,6'-di-tert-butyl-8,8'-methylenebis(spiro[4H-1,3-benzodioxin-2,1'-cyclohexane]).

Two related compounds containing p-tert-butyl-o-methylene-linked phenol or phenol-derived subunits are described, namely 5,5'-di-tert-butyl-2,2'-dihydroxy-3,3'-methylenedibenzaldehyde, C(23)H(28)O(4), (I), and 6,6'-di-tert-butyl-8,8'-methylenebis(spiro[4H-1,3-benzodioxin-2,1'-cyclohexane]), C(35)H(48)O(4), (II). Both compounds adopt a 'butterfly' shape, with the two phenol or phenol-derived O atoms in distal positions. Phenol and aldehyde groups in (I) are involved in intramolecular hydrogen bonds and the two dioxin rings in (II) are in distorted half-chair conformations.