Sarcopenic metabolomic profile reflected a sarcopenic phenotype associated with amino acid and essential fatty acid changes.

INTRODUCTION: Although sarcopenia greatly affects health and quality of life in older people, its pathophysiological causes are not fully elucidated. To face this challenge, omics technologies can be used. The metabolome gives a vision of the interaction between the genome and the environment through metabolic networks, thus contributing in clarifying the pathophysiology of the sarcopenic phenotype. OBJECTIVES: The main goal of this study was to compare the plasma metabolome of sarcopenic and non-sarcopenic older people. METHODS: Cross-sectional study of 20 sarcopenic and 21 non-sarcopenic older subjects with available frozen plasma samples. Non-targeted metabolomic study by ultra-high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) analysis with later bioinformatics data analysis. Once the significantly different metabolites were identified, the KEGG database was used on them to establish which were the metabolic pathways mainly involved. RESULTS: From 657 features identified, 210 showed significant differences between the study groups, and 30 had a FoldChangeLog2 > 2. The most interesting metabolic pathways found with the KEGG database were the biosynthesis of amino acids, arginine and proline metabolism, the biosynthesis of alkaloids derived from ornithine, linoleic acid metabolism, and the biosynthesis of unsaturated fatty acids. CONCLUSIONS: The study results allowed us to confirm that the concept of "sarcopenic phenotype" is also witnessed at the plasma metabolite levels. The non-targeted metabolomics study can open a wide view of the sarcopenic features changes at the plasma level, which would be linked to the sarcopenic physiopathological alterations.

Characterization and pathogenicity of Vibrio splendidus strains associated with massive mortalities of commercial hatchery-reared larvae of scallop Argopecten purpuratus (Lamarck, 1819).

Three strains (VPAP16, VPAP18 and VPAP23 strains) were isolated as the most predominant organisms from 3 different episodes of massive mortalities of larval cultures of the Chilean scallop Argopecten purpuratus occurred in different commercial hatcheries located in northern Chile. The main aims of this study were to identify the pathogenic strains and investigate their pathogenic activity. Based on selected phenotypic features and sequence identity of the 16S rRNA gene and the housekeeping gene, RNA polymerase α-chain rpoA, all pathogenic strains were identified as Vibrio splendidus. Healthy 10-day-old scallop larvae cultures exhibited mortality percentages of 69.61±3.35%, 79.78±6.11% and 61.73±3.71% after 48 h when were inoculated with 1×10(6) CFU (colony forming units)mL(-1) of VPAP16, VPAP18 and VPAP23 strains, respectively, and evidenced that concentrations ⩾10(4) CFU mL(-1) would probably be detrimental for the larval culture. The main clinical signs observed in challenged larvae for 24h were bacterial swarms on the margins of the larvae, extension and disruption of the velum, detachment of velum cilia cells and digestive tissue necrosis. Otherwise, challenge assays using pathogenic strains stained with 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein hydrochloride (5-DTAF)evidenced that after 1h stained bacteria were detected in high density in the digestive gland and the margin of the shell. When larval cultures were inoculated with cell-free extracellular products (ECP) of V. splendidus strains, exhibited larval mortalities higher than 70% (VPAP16), 80% (VPAP18) and 50% (VPAP23) after 24 h, even when ECP were treated with proteinase K or heat, indicating that extracellular pathogenic activity is mainly mediated by non-proteic thermostable compounds. In this study all Koch's postulates were fulfilled and it was demonstrated for the first time the pathogenic activity of V. splendidus strains on reared-larvae of scallop A. purpuratus and prompt the necessity to maintain this species at concentrations lower than 10(4) CFU mL(-1) to avoid episodes of mass mortalities in scallop hatcheries.

Debaryomyces hansenii and Rhodotorula mucilaginosa comprised the yeast core gut microbiota of wild and reared carnivorous salmonids, croaker and yellowtail.

This is the first study using molecular and culture-based methods aimed at investigating the composition of the intestinal yeast microbiota of wild and reared carnivorous salmonids, croaker and yellowtail, to characterize their cores and to evaluate the enzymatic activities of the cultivated yeast. Among 103 samples from salmonids, croaker and yellowtail, yeast were detected in 85.4%, with 43 species identified. The core of reared fish was composed of eight species, in contrast to the wild fish core, which consisted of two species: Debaryomyces hansenii and Rhodotorula mucilaginosa. Despite the smaller diversity of the wild fish core, similar enzymatic profiles were detected for the species from the wild and reared cores. For principal component analysis, samples grouped together independently of host species, domestication status and location. A high proportion of yeast produced aminopeptidases and lipases, which may be explained by the high proportion of protein and lipids in the carnivorous diet. This study reveals the presence of a yeast community in the fish gut that appears to be strongly shaped by a carnivorous diet. Yeast in the gut increases the repertoire of microorganisms interacting with the host intestine, which could influence health and disease.

Lysin and Lytic Phages Reduce Vibrio Counts in Live Feed and Fish Larvae.

Vibrio species are naturally found in estuarine and marine ecosystems, but are also recognized as significant human enteropathogens, often linked to seafood-related illnesses. In aquaculture settings, Vibrio poses a substantial risk of infectious diseases, resulting in considerable stock losses and prompting the use of antimicrobials. However, this practice contributes to the proliferation of antimicrobial-resistant (AMR) bacteria and resistance genes. Our investigation aimed to explore the potential of biological agents such as bacteriophage CH20 and endolysin LysVPp1 in reducing Vibrio bacterial loads in both rotifer and fish larvae. LysVPp1's lytic activity was assessed by measuring absorbance reduction against various pathogenic Vibrio strains. Phage CH20 exhibited a limited host range, affecting only Vibrio alginolyticus GV09, a highly pathogenic strain. Both CH20 and LysVPp1 were evaluated for their effectiveness in reducing Vibrio load in rotifers or fish larvae through short-setting bioassays. Our results demonstrated the significant lytic effect of endolysin LysVPp1 on strains of Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio splendidus. Furthermore, we have showcased the feasibility of reducing the load of pathogenic Vibrio in live feed and fish larvae by using a non-antibiotic-based approach, such as lytic phage and endolysin LysVPp1, thus contributing to the progress of a sustainable aquaculture from a One Health perspective.

Effect of Dietary Carbohydrate-to-Protein Ratio on Gut Microbiota in Atlantic Salmon (Salmo salar).

Atlantic salmon (Salmo salar) is a carnivorous fish species whose productive performance tends to be suboptimal when fed low-cost carbohydrate rich meals. It is of interest to study the dynamics of gut microbiota communities in salmonids fed high carbohydrate diets since gut microbes are referred to as key players that influence the metabolism and physiology of the host. A study was conducted to determine the effect of feeding a high carbohydrate diet to Atlantic salmon in gut microbiota communities. A medium carbohydrate (15% wheat starch)/medium protein (MC/MP) diet or a high carbohydrate (30% wheat starch)/low protein (HC/LP) diet was fed to triplicate tanks (28 fish each) during four weeks. We conducted an in-depth characterization of the distal intestine digesta microbiota using high-throughput sequencing of the V4 region of the 16S rRNA gene. Firmicutes, Actinobacteria and Proteobacteria were the major phyla determined in either experimental group. Phylum Planctomycetes, class Planctomycetia, order Planctomycetales and genus Lactococcus were significantly more abundant in fish fed the HC/LP diet compared with fish fed the MC/MP diet. Our study suggests feeding a carbohydrate rich meal to salmon exerts a low impact on the structure of gut microbial communities, affecting mostly low-abundance bacteria capable of metabolizing anaerobically carbohydrates as a major energy-yielding substrate.

In-depth analysis of swim bladder-associated microbiota in rainbow trout (Oncorhynchus mykiss).

Our knowledge regarding microbiota associated with the swim bladder of physostomous, fish with the swim bladder connected to the esophagus via the pneumatic duct, remains largely unknown. The goal of this study was to conduct the first in-depth characterization of the swim bladder-associated microbiota using high-throughput sequencing of the V4 region of the 16 S rRNA gene in rainbow trout (Oncorhynchus mykiss). We observed major differences in bacterial communities composition between swim bladder-associated microbiota and distal intestine digesta microbiota in fish. Whilst bacteria genera, such as Cohnella, Lactococcus and Mycoplasma were more abundant in swim bladder-associated microbiota, Citrobacter, Rhodobacter and Clavibacter were more abundant in distal intestine digesta microbiota. The presumptive metabolic function analysis (PICRUSt) revealed several metabolic pathways to be more abundant in the swim bladder-associated microbiota, including metabolism of carbohydrates, nucleotides and lipoic acid as well as oxidative phosphorylation, cell growth, translation, replication and repair. Distal intestine digesta microbiota showed greater abundance of nitrogen metabolism, amino acid metabolism, biosynthesis of unsaturated fatty acids and bacterial secretion system. We demonstrated swim bladder harbors a unique microbiota, which composition and metabolic function differ from microbiota associated with the gut in fish.

Fasting Upregulates npy, agrp, and ghsr Without Increasing Ghrelin Levels in Zebrafish (Danio rerio) Larvae.

Food intake in fish and mammals is orchestrated by hypothalamic crosstalk between orexigenic (food intake stimulation) and anorexigenic (food intake inhibition) signals. Some of these signals are released by peripheral tissues that are associated with energy homeostasis or nutrient availability. During the fish larva stage, orexigenic stimulation plays a critical role in individual viability. The goal of this study was to assess the mRNA levels of the main neuropeptides involved in food intake regulation (npy, agrp, carppt, and pomc), in concert with the mRNA levels and peptide levels of ghrelin, under a fasting intervention at the larval stage in zebrafish (Danio rerio). Prior to the fasting intervention, the zebrafish larva cohort was reared for 20 days post fertilization (dpf) and then randomly divided into two groups of 20 individuals. One group was subjected to a fasting intervention for 5 days (fasted group), and the other group was fed normally (fed group); this experimental protocol was performed twice independently. At the end of the fasting period, individuals from each experimental group were divided into different analysis groups, for evaluations such as relative gene expression, immunohistochemistry, and liquid chromatography coupled to nano high-resolution mass spectrometry (nLC-HRMS) analyses. The relative expression levels of the following genes were assessed: neuropeptide Y (npy), agouti-related peptide (agrp), proopiomelanocortin (pomc), cocaine and amphetamine-regulated transcript (cartpt), ghrelin (ghrl), ghrelin O-acyltransferase (mboat4), growth hormone secretagogue receptor (ghsr), and glucokinase (gck). In the fasted group, significant upregulation of orexigenic peptides (npy - agrp) and ghsr was observed, which was associated with significant downregulation of gck. The anorexigenic peptides (pomc and cartpt) did not show any significant modulation between the groups, similar to mboat4. Contrary to what was expected, the relative mRNA upregulation of the orexigenic peptides observed in the fasted experimental group could not be associated with significant ghrelin modulation as assessed by three different approaches: qPCR (relative gene expression of ghrelin), nLC-HRMS (des-acyl-ghrelin levels), and immunohistochemistry (integrated optical density of prepropeptides in intestinal and hepatopancreas tissues). Our results demonstrate that zebrafish larvae at 25 dpf exhibit suitable modulation of the relative mRNA levels of orexigenic peptides (npy and agrp) in response to fasting intervention; nevertheless, ghrelin was not coregulated by fasting. Therefore, it can be suggested that ghrelin is not an essential peptide for an increase in appetite in the zebrafish larva stage. These results give rise to new questions about food intake regulation factors in the early stages of fish.

Performance of Debaryomyces hansenii as a Diet for Rotifers for Feeding Zebrafish Larvae.

The zebrafish larval stage is a critical moment due to high mortality rates associated with inadequate supplies of nutritional requirements. Larval feeding has important challenges associated with such factors as small mouth gape (≈100 µm), the low activity of digestive enzymes, and the intake of live food. A common zebrafish live food at the onset of exogenous feeding is rotifers, mainly Brachionus plicatilis. These rotifers should be fed with other microorganisms such as microalgae or yeast, mostly from the Saccharomyces genus. In the laboratory, the culture of microalgae is more expensive than the culture of yeast. The aim of this study was to evaluate the performance of Debaryomyces hansenii as a diet for rotifers in comparison to a microalgae-based diet (Rotigrow®). To achieve this aim, we assessed the rotifer total protein content, the rotifers fatty acid profile, zebrafish larval growth performance, the expression of key growth, and endocrine appetite regulation genes. The total protein and fatty acids content were similar in both rotifer cultures, averaging 35% of dry matter (DM) and 18% of DM, respectively. Interestingly, the fatty acids profile showed differences between the two rotifer cultures: omega-3 fatty acids were only observed in the Microalgae/rotifer, whereas, omega-6 fatty acids presented similar levels in both rotifer cultures. No differences were observed in the larval body length distribution or mortalities between the rotifer cultures. However, gh, igf-1, and cck gene expression showed significantly higher upregulation in zebrafish fed the Microalgae/rotifer diet compared with those fed the Debaryomyces/rotifer diet. In conclusion, D. hansenii could be an alternative diet for rotifer used as a live food in zebrafish larvae at the onset of exogenous feeding. The gene responses observed in this work open up the opportunity to study the effect of omega-3 supply on growth regulation in zebrafish.

Genome Sequence of a Lactococcus lactis Strain Isolated from Salmonid Intestinal Microbiota.

Lactococcus lactis is a common inhabitant of the intestinal microbiota of salmonids, especially those in aquaculture systems. Here, we present a genome sequence of a Lactococcus lactis strain isolated from the intestinal contents of rainbow trout reared in Chile.

Effect of the dietary inclusion of soybean components on the innate immune system in zebrafish.

Some components of plant-based meals, such as saponins and vegetal proteins, have been proposed as inducers of intestinal inflammation in some fish. However, the molecular and cellular bases for this phenomenon have not been reported. In this work, zebrafish were used as a model to evaluate the effects of individual soybean meal components, such as saponins and soy proteins. Zebrafish larvae fed a fish meal feed containing soy components were assessed according to low and high inclusion levels. The granulocytes associated with the digestive tract and the induction of genes related to the immune system were quantitated as markers of the effects of the dietary components. A significant increase in the number of granulocytes was observed after feeding fish diets containing high saponin or soy protein contents. These dietary components also induced the expression of genes related to the innate immune system, including myeloid-specific peroxidase, as well as the complement protein and cytokines. These results reveal the influence of dietary components on the stimulation of the immune system. These observations could be significant to understanding the contributions of saponin and soy protein to the onset of enteritis in aqua-cultured fish, and this knowledge may aid in defining the role of the innate immune system in other inflammatory diseases involving dietary components in mammals.

Reduction of soybean meal non-starch polysaccharides and α-galactosides by solid-state fermentation using cellulolytic bacteria obtained from different environments.

Soybean meal (SBM) is an important protein source in animal feed. However, the levels of SBM inclusion are restricted in some animal species by the presence of antinutritional factors (ANFs), including non-starch polysaccharides (NSPs) and α-galactosides (GOSs). The aim of this study was to reduce the soybean meal NSPs and GOSs by solid-state fermentation (SSF) using a combination of cellulolytic bacteria isolated from different environments (termites, earthworms, corn silage and bovine ruminal content). To analyse the key enzymatic activities, the isolates were grown in minimal media containing NSPs extracted from SBM. The selected bacterial strains belonged to the genera Streptomyces, Cohnella and Cellulosimicrobium. SSF resulted in a reduction of nearly 24% in the total NSPs, 83% of stachyose and 69% of raffinose and an increase in the protein content. These results suggest that cellulolytic bacteria-based SSF processing facilitates SBM nutritional improvement. In addition, the use of fermented SBM in animal diets can be recommended.

PCR-TTGE analysis of 16S rRNA from rainbow trout (Oncorhynchus mykiss) gut microbiota reveals host-specific communities of active bacteria.

This study assessed the relative contributions of host genetics and diet in shaping the gut microbiota of rainbow trout. Full sibling fish from four unrelated families, each consisting of individuals derived from the mating of one male and one female belonging to a breeding program, were fed diets containing either vegetable proteins or vegetable oils for two months in comparison to a control diet consisting of only fish protein and fish oil. Two parallel approaches were applied on the same samples: transcriptionally active bacterial populations were examined based on RNA analysis and were compared with bacterial populations obtained from DNA analysis. Comparison of temporal temperature gradient gel electrophoresis (TTGE) profiles from DNA and RNA showed important differences, indicating that active bacterial populations were better described by RNA analysis. Results showed that some bacterial groups were significantly (P<0.05) associated with specific families, indicating that microbiota composition may be influenced by the host. In addition, the effect of diet on microbiota composition was dependent on the trout family.

Molecular analysis of intestinal microbiota of rainbow trout (Oncorhynchus mykiss).

The aim of this study was to evaluate different molecular tools based on the 16S rRNA gene, internal transcribed spacer, and the rpoB gene to examine the bacterial populations present in juvenile rainbow trout intestines. DNA was extracted from both pooled intestinal samples and bacterial strains. Genes were PCR-amplified and analysed using both temporal temperature gradient gel electrophoresis (TTGE) and restriction fragment length polymorphism methods. Because of the high cultivability of the samples, representative bacterial strains were retrieved and we compared the profiles obtained from isolated bacteria with the profile of total bacteria from intestinal contents. Direct analysis based on rpoB-TTGE revealed a simple bacterial composition with two to four bands per sample, while the 16S rRNA gene-TTGE showed multiple bands and comigration for a few species. Sequencing of the 16S rRNA gene- and rpoB-TTGE bands revealed that the intestinal microbiota was dominated by Lactococcus lactis, Citrobacter gillenii, Kluyvera intermedia, Obesumbacterium proteus, and Shewanella marinus. In contrast to 16S rRNA gene-TTGE, rpoB-TTGE profiles derived from bacterial strains produced one band per species. Because the single-copy state of rpoB leads to a single band in TTGE, the rpoB gene is a promising molecular marker for investigating the bacterial community of the rainbow trout intestinal microbiota.

Oxytetracycline treatment reduces bacterial diversity of intestinal microbiota of Atlantic salmon.

The effect of oxytetracycline (OTC) treatment on intestinal bacterial populations in juvenile Atlantic salmon Salmo salar was evaluated. Oxytetracycline was administered by way of medicated feed to fish held in experimental tanks. Restriction fragment length polymorphism and sequencing of 16S rDNA from isolates were used to analyze the intestinal microbiota before, during, and after OTC administration. The microbiota from untreated fish was more diverse, consisting mainly of Pseudomonas, Acinetobacter, Bacillus, Flavobacterium, Psycrobacter, and Brevundimonas spp. In contrast, the microbiota of the OTC-treated group was characterized by lower diversity and consisted only of Aeromonas, clustering with A. sobria and A. salmonicida. Antibiotic-resistant isolates were identified as Aeromonas spp.; sequencing the resistance determinant showed it to be the tetE gene. Overall, OTC treatment changed the composition of the intestinal microbiota of Atlantic salmon, as evidenced by a reduction in bacterial diversity. These results support the current concern that antibiotic treatment can facilitate the proliferation of opportunistic bacteria by eradicating competing microorganisms.

Enfermedad de Von Willebrand en obstetricia: experiencia clínica / Von Willebrand's disease in obstetrics: clinical experience

Se presentan 6 pacientes embarazadas afectadas por la enfermedad de Von Willebrand; se analizan las pruebas de laboratorio efectuadas y se comentan los resultados obtenidos. Se efectúan algunas proposiciones para el manejo de esta afección en la paciente embarazada