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1.
Front Immunol ; 15: 1404209, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39035000

RESUMO

The recent development of single cell sequencing technologies has revolutionized the state-of-art of cell biology, allowing the simultaneous measurement of thousands of genes in single cells. This technology has been applied to study the transcriptome of single cells in homeostasis and also in response to pathogenic exposure, greatly increasing our knowledge of the immune response to infectious agents. Yet the number of these studies performed in aquacultured fish species is still very limited. Thus, in the current study, we have used the 10x Genomics single cell RNA sequencing technology to study the response of rainbow trout (Oncorhynchus mykiss) peripheral blood leukocytes (PBLs) to infectious pancreatic necrosis virus (IPNV), an important trout pathogen. The study allowed us to obtain a transcriptomic profile of 12 transcriptionally distinct leukocyte cell subpopulations that included four different subsets of B cells, T cells, monocytes, two populations of dendritic-like cells (DCs), hematopoietic progenitor cells, non-specific cytotoxic cells (NCC), neutrophils and thrombocytes. The transcriptional pattern of these leukocyte subpopulations was compared in PBL cultures that had been exposed in vitro to IPNV for 24 h and mock-infected cultures. Our results revealed that monocytes and neutrophils showed the highest number of upregulated protein-coding genes in response to IPNV. Interestingly, IgM+IgD+ and IgT+ B cells also upregulated an important number of genes to the virus, but a much fainter response was observed in ccl4 + or plasma-like cells (irf4 + cells). A substantial number of protein-coding genes and genes coding for ribosomal proteins were also transcriptionally upregulated in response to IPNV in T cells and thrombocytes. Interestingly, although genes coding for ribosomal proteins were regulated in all affected PBL subpopulations, the number of such genes transcriptionally regulated was higher in IgM+IgD+ and IgT+ B cells. A further analysis dissected which of the regulated genes were common and which were specific to the different cell clusters, identifying eight genes that were transcriptionally upregulated in all the affected groups. The data provided constitutes a comprehensive transcriptional perspective of how the different leukocyte populations present in blood respond to an early viral encounter in fish.


Assuntos
Infecções por Birnaviridae , Doenças dos Peixes , Vírus da Necrose Pancreática Infecciosa , Leucócitos , Oncorhynchus mykiss , Análise de Célula Única , Animais , Oncorhynchus mykiss/imunologia , Oncorhynchus mykiss/virologia , Vírus da Necrose Pancreática Infecciosa/imunologia , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/veterinária , Infecções por Birnaviridae/virologia , Análise de Célula Única/métodos , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Leucócitos/imunologia , Leucócitos/virologia , Transcriptoma , Perfilação da Expressão Gênica/métodos
2.
Food Res Int ; 169: 112887, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37254335

RESUMO

Mass spectrometry has become the technique of choice for the assessment of a high variety of molecules in complex food matrices. It is best suited for monitoring the evolution of digestive processes in vivo and in vitro. However, considering the variety of equipment available in different laboratories and the diversity of sample preparation methods, instrumental settings for data acquisition, statistical evaluations, and interpretations of results, it is difficult to predict a priori the ideal parameters for optimal results. The present work addressed this uncertainty by executing an inter-laboratory study with samples collected during in vitro digestion and presenting an overview of the state-of-the-art mass spectrometry applications and analytical capabilities available for studying food digestion. Three representative high-protein foods - skim milk powder (SMP), cooked chicken breast and tofu - were digested according to the static INFOGEST protocol with sample collection at five different time points during gastric and intestinal digestion. Ten laboratories analysed all digesta with their in-house equipment and applying theirconventional workflow. The compiled results demonstrate in general, that soy proteins had a slower gastric digestion and the presence of longer peptide sequences in the intestinal phase compared to SMP or chicken proteins, suggesting a higher resistance to the digestion of soy proteins. Differences in results among the various laboratories were attributed more to the peptide selection criteria than to the individual analytical platforms. Overall, the combination of mass spectrometry techniques with suitable methodological and statistical approaches is adequate for contributing to the characterisation of the recently defined digestome.


Assuntos
Digestão , Proteínas de Soja , Animais , Proteínas de Soja/metabolismo , Leite/química , Peptídeos/análise , Espectrometria de Massas
3.
Nutrients ; 15(5)2023 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-36904065

RESUMO

It is known that casein hydrolysis accelerates gastrointestinal transit in comparison to intact casein, although the effect of the protein hydrolysis on the composition of the digests is not fully understood. The aim of this work is to characterize, at the peptidome level, duodenal digests from pigs, as a model of human digestion, fed with micellar casein and a previously described casein hydrolysate. In addition, in parallel experiments, plasma amino acid levels were quantified. A slower transit of nitrogen to the duodenum was found when the animals received micellar casein. Duodenal digests from casein contained a wider range of peptide sizes and a higher number of peptides above five amino acids long in comparison with the digests from the hydrolysate. The peptide profile was markedly different, and although ß-casomorphin-7 precursors were also found in hydrolysate samples, other opioid sequences were more abundant in the casein digests. Within the same substrate, the evolution of the peptide pattern at different time points showed minimal changes, suggesting that the protein degradation rate relies more on the gastrointestinal location than on digestion time. Higher plasma concentrations of methionine, valine, lysine and amino acid metabolites were found in animals fed with the hydrolysate at short times (<200 min). The duodenal peptide profiles were evaluated with discriminant analysis tools specific for peptidomics to identify sequence differences between both substrates that can be used for future human physiological and metabolic studies.


Assuntos
Aminoácidos , Caseínas , Suínos , Humanos , Animais , Caseínas/metabolismo , Aminoácidos/metabolismo , Peptídeos/metabolismo , Trato Gastrointestinal/metabolismo
4.
Foods ; 10(4)2021 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-33919642

RESUMO

Human milk proteins have shown to vary in concentration and distribution through lactation. However, while some regulatory components, such as hormones, have shown associations with regard to the mothers' body mass index, there is limited information on the possible influence of this condition on the whole protein distribution. The objective of this study was to evaluate the protein profile of human milk from normal weight and overweight or obese mothers to identify differences in protein expression in colostrum, transitional and mature milk. The mass spectrometry analysis showed the ability to class with a high degree of confidence the lactation state and the milk profile according to the mother's condition. Individual milk samples were subjected to a digestion in vitro model that takes into account the specificities of the gastrointestinal conditions of full-term newborn infants. The digestion products were compared with available data from the digestive contents in newborns. The behavior of the most abundant proteins and the overall peptide generation and survival, showed good correspondence with in vivo data.

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