RESUMEN
The enzymatic repertoire of starter cultures belonging to the Lactococcus genus determines various important characteristics of fermented dairy products but might change in response to the substantial environmental changes in the manufacturing process. Assessing bacterial proteome adaptation in dairy and other food environments is challenging due to the high matrix-protein concentration and is even further complicated in particularly cheese by the high fat concentrations, the semi-solid state of that matrix, and the non-growing state of the bacteria. Here, we present bacterial harvesting and processing procedures that enable reproducible, high-resolution proteome determination in lactococcal cultures harvested from laboratory media, milk, and miniature Gouda cheese. Comparative proteome analysis of Lactococcus cremoris NCDO712 grown in laboratory medium and milk revealed proteome adaptations that predominantly reflect the differential (micro-)nutrient availability in these two environments. Additionally, the drastic environmental changes during cheese manufacturing only elicited subtle changes in the L. cremoris NCDO712 proteome, including modified expression levels of enzymes involved in flavour formation. The technical advances we describe offer novel opportunities to evaluate bacterial proteomes in relation to their performance in complex, protein- and/or fat-rich food matrices and highlight the potential of steering starter culture performance by preculture condition adjustments.
Asunto(s)
Queso , Productos Lácteos Cultivados , Lactococcus lactis , Animales , Proteoma/metabolismo , Fermentación , Queso/microbiología , Leche/microbiología , Lactococcus lactis/genética , Lactococcus lactis/metabolismoRESUMEN
IMPORTANCE: The availability of nutrients to microorganisms varies considerably between different environments, and changes can occur rapidly. As a general rule, a fast growth rate-typically growth on glucose-is associated with the repression of other carbohydrate utilization genes, but it is not clear to what extent catabolite repression is exerted by other sugars. We investigated the hierarchy of sugar utilization after substrate transitions in Lactococcus cremoris. For this, we determined the proteome and carbohydrate utilization capacity after growth on different sugars. The results show that the preparedness of cells for the utilization of "slower" sugars is not strictly determined by the growth rate. The data point to individual proteins relevant for various sugar transitions and suggest that the evolutionary history of the organism might be responsible for deviations from a strictly growth rate-related sugar catabolization hierarchy.
Asunto(s)
Carbohidratos , Azúcares , Glucosa/metabolismoRESUMEN
Butyrate in human milk (HM) has been suggested to reduce excessive weight and adipo-sity gains during infancy. However, HM butyrate's origins, determinants, and its influencing mechanism on weight gain are not completely understood. These were studied in the prospective longitudinal Cambridge Baby Growth and Breastfeeding Study (CBGS-BF), in which infants (n = 59) were exclusively breastfed for at least 6 weeks. Infant growth (birth, 2 weeks, 6 weeks, 3 months, 6 months, and 12 months) and HM butyrate concentrations (2 weeks, 6 weeks, 3 months, and 6 months) were measured. At age 6 weeks, HM intake volume was measured by deuterium-labelled water technique and HM microbiota by 16S sequencing. Cross-sectionally at 6 weeks, HM butyrate was associated with HM microbiota composition (p = 0.036) although no association with the abundance of typical butyrate producers was detected. In longitudinal analyses across all time points, HM butyrate concentrations were overall negatively associated with infant weight and adiposity, and associations were stronger at younger infant ages. HM butyrate concentration was also inversely correlated with HM intake volume, supporting a possible mechanism whereby butyrate might reduce infant growth via appetite regulation and modulation of HM intake.
Asunto(s)
Microbiota , Leche Humana , Femenino , Humanos , Lactante , Butiratos , Estudios Prospectivos , Lactancia Materna , Aumento de PesoRESUMEN
Growth patterns of breastfed infants show substantial inter-individual differences, partly influenced by breast milk (BM) nutritional composition. However, BM nutritional composition does not accurately indicate BM nutrient intakes. This study aimed to examine the associations between both BM intake volumes and macronutrient intakes with infant growth. Mother-infant dyads (n 94) were recruited into the Cambridge Baby Growth and Breastfeeding Study (CBGS-BF) from a single maternity hospital at birth; all infants received exclusive breast-feeding (EBF) for at least 6 weeks. Infant weight, length and skinfolds thicknesses (adiposity) were repeatedly measured from birth to 12 months. Post-feed BM samples were collected at 6 weeks to measure TAG (fat), lactose (carbohydrate) (both by 1H-NMR) and protein concentrations (Dumas method). BM intake volume was estimated from seventy infants between 4 and 6 weeks using dose-to-the-mother deuterium oxide (2H2O) turnover. In the full cohort and among sixty infants who received EBF for 3+ months, higher BM intake at 6 weeks was associated with initial faster growth between 0 and 6 weeks (ß + se 3·58 + 0·47 for weight and 4·53 + 0·6 for adiposity gains, both P < 0·0001) but subsequent slower growth between 3 and 12 months (ß + se - 2·27 + 0·7 for weight and -2·65 + 0·69 for adiposity gains, both P < 0·005). BM carbohydrate and protein intakes at 4-6 weeks were positively associated with early (0-6 weeks) but tended to be negatively related with later (3-12 months) adiposity gains, while BM fat intake showed no association, suggesting that carbohydrate and protein intakes may have more functional relevance to later infant growth and adiposity.
Asunto(s)
Lactancia Materna , Leche Humana , Recién Nacido , Humanos , Lactante , Femenino , Embarazo , Leche Humana/química , Fenómenos Fisiológicos Nutricionales del Lactante , Obesidad , Ingestión de Alimentos , Carbohidratos/análisisRESUMEN
Human milk oligosaccharides (HMOs) act as a vital role in the development of infant's gut microbiome and immune function. This study aimed to measure 12 oligosaccharides in milk from Chinese donors (n = 203), and evaluated the influences of multiple factors on the HMOs profiles. The results indicated that concentrations of 6'-sialyllactose were the highest among 12 oligosaccharides (2.31 ± 0.81 g/L). HMOs concentrations varied depending on geographical location. Latitude was observed to be related to concentrations of Lacto-N-neohexaose, lacto-N-fucopentaose III, 3'-sialyllactose (r = -0.67, r = +0.63 and r = +0.50, respectively). Environmental factors like seasons correlated with lacto-N-difucohexaose â ¡, Lacto-N-neohexaose and 2'-fucosyllactose (r = -0.47, r = -0.4, r = -0.35, respectively). Several HMOs concentrations were correlated with maternal diet. As a consequence, the HMOs profiles measured were influenced by geographical, environmental, maternal anthropometric as well as dietary factors.
Asunto(s)
Leche Humana , Oligosacáridos , China , Dieta , Humanos , LactanteRESUMEN
The aim of this study was to use fecal metabolite profiling to evaluate the effects of contrasting sanitary conditions and the associated subclinical health status of pigs. We analyzed fecal metabolite profiles by nuclear magnetic resonance (1H NMR) from pigs aged 14 and 22 weeks. Pigs kept under low and high sanitary conditions differed in fecal metabolites related to the degradation of dietary starch, metabolism of the gut microbiome, and degradation of components of animal (host) origin. The metabolites that differed significantly (FDR < 0.1) were from metabolic processes involved in either maintaining nutrient digestive capacity, including purine metabolism, energy metabolism, bile acid breakdown and recycling, or immune system metabolism. The results show that the fecal metabolite profiles reflect the sanitary conditions under which the pigs are kept. The fecal metabolite profiles closely resembled the profiles of metabolites found in the colon of pigs. Fecal valerate and kynurenic acid could potentially be used as "non-invasive" biomarkers of immune or inflammatory status that could form the basis for monitoring subclinical health status in pigs.
RESUMEN
Argonaute proteins use single-stranded RNA or DNA guides to target complementary nucleic acids. This allows eukaryotic Argonaute proteins to mediate RNA interference and long prokaryotic Argonaute proteins to interfere with invading nucleic acids. The function and mechanisms of the phylogenetically distinct short prokaryotic Argonaute proteins remain poorly understood. We demonstrate that short prokaryotic Argonaute and the associated TIR-APAZ (SPARTA) proteins form heterodimeric complexes. Upon guide RNA-mediated target DNA binding, four SPARTA heterodimers form oligomers in which TIR domain-mediated NAD(P)ase activity is unleashed. When expressed in Escherichia coli, SPARTA is activated in the presence of highly transcribed multicopy plasmid DNA, which causes cell death through NAD(P)+ depletion. This results in the removal of plasmid-invaded cells from bacterial cultures. Furthermore, we show that SPARTA can be repurposed for the programmable detection of DNA sequences. In conclusion, our work identifies SPARTA as a prokaryotic immune system that reduces cell viability upon RNA-guided detection of invading DNA.
Asunto(s)
Proteínas Argonautas , Células Procariotas/fisiología , Proteínas Argonautas/metabolismo , ADN/metabolismo , Células Procariotas/citología , Células Procariotas/metabolismo , ARN Guía de KinetoplastidaRESUMEN
The use of herbal supplements for improved sexual performance is a common practice amongst the youth and some senior citizens in Ghana. These products are considered 'natural' and greatly preferred over synthetic alternatives due to the assurance of little to no adverse effects by producers. However, the high rate of adulteration often compromises their safety. Forty herbal supplements, of which 25 were previously shown to result in medium to high intake of phosphodiesterase type-5 (PDE-5) inhibitors using a PDE-Glo bioassay, were further investigated using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to examine the reliability of the bioassay and whether the observed higher responses could be ascribed to inherent plant constituents or adulterants. Results showed significant amounts of vardenafil, tadalafil and especially sildenafil, in 2, 1 and 10 samples, respectively, with total concentration levels resulting in estimated daily intakes (EDIs) above 25 mg sildenafil equivalents with six supplements even having EDIs above 100 mg sildenafil equivalents. Only one sample contained a natural ingredient (icariin), but its concentration (0.013 mg g-1) was too low to explain the observed potency in the bioassay. The estimated concentrations of PDE-5 inhibitors in 35 supplements, according to the bioassay, were in line with those of the LC-MS/MS analysis. However, discrepancies were observed for five supplements. Further examination of one of the latter supplements using the PDE-Glo bioassay to select the positive fraction and further examination with LC-MS/MS and 1H-NMR revealed the presence of hydroxythiohomosildenafil, a sildenafil analogue not yet included in the liquid chromatography-mass spectrometry reference library. This study demonstrates the significance of applying a tiered approach, where the use of a bioassay is followed by chemical analysis of bioactive samples in order to identify unknown bioactive compounds.
Asunto(s)
Inhibidores de Fosfodiesterasa 5 , Espectrometría de Masas en Tándem , Cromatografía Liquida , Suplementos Dietéticos/análisis , Cromatografía de Gases y Espectrometría de Masas , Inhibidores de Fosfodiesterasa 5/análisis , Hidrolasas Diéster Fosfóricas , Reproducibilidad de los Resultados , Citrato de Sildenafil/análisisRESUMEN
Twenty medicinal plants with previously established anti-viral activity against a wild-type RVFV were further investigated using bio-chemometric and analytical techniques. The aim being to identify compounds common in plants with anti-RVFV activity, potentially being the major contributors to the anti-viral effect. Proton nuclear magnetic resonance (1H NMR) spectroscopy coupled with multivariate data analysis (MVDA) was applied to characterize metabolite profiles of twenty antiviral medicinal plants. Discrimination and prediction of metabolome data of active anti-RVFV from the less-active samples was assessed using the multivariate statistical models by constructing a robust principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) regression model. Annotation of metabolites in the samples with higher activity were performed by Chenomx software and the compounds confirmed using Ultra-High-Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (UHPLC-qTOF-MS). Both the PCA and OPLS-DA score plots showed clustering of samples; however, the OPLS-DA plot indicated a clear separation among active and less-active samples. Metabolic biomarkers were screened by p-value < 0.05 and variable importance in the projection (VIP) value >1 and S-plot. Among active samples, the most prominent metabolites putatively identified by NMR include trigonelline, vanillic acid, fumarate, chlorogenic acid, ferulate, and formate. The presence of the compounds were confirmed by UHPLC-qTOF-MS, and two hydroxylated fatty acids were additionally detected indicated by peaks at m/z 293.2116 and m/z 295.2274 13S-Hydroxy-9Z,11E,15Z-octadecatrienoic acid and 13-Hydroxy-9Z,11E-octadecadienoic acid were annotated for the first time in all the antiviral active samples and are considered potential metabolites responsible for the antiviral activity. The study provides a metabolomic profile of anti-RVFV plant extracts and report for the first time the presence of hydroxylated fatty acids 13S-Hydroxy-9Z,11E,15Z-octadecatrienoic acid and 13-Hydroxy-9Z,11E-octadecadienoic acid, present in all the tested medicinal plants with high anti-RVFV activity and is a potential target for the future development of antiviral therapeutic agents.
RESUMEN
Human milk is a dynamic biofluid, and its detailed composition receives increasing attention. While most studies focus on changes over time or differences between maternal characteristics, interindividual variation receives little attention. Nevertheless, a comprehensive insight into this can help interpret human milk studies and help human milk banks provide targeted milk for recipients. This study aimed to map interindividual variation in the human milk proteome, peptidome, and metabolome and to investigate possible explanations for this variation. A set of 286 milk samples was collected from 29 mothers in the third month postpartum. Samples were pooled per mother, and proteins, peptides, and metabolites were analyzed. A substantial coefficient of variation (>100%) was observed for 4.6% and 36.2% of the proteins and peptides, respectively. In addition, using weighted correlation network analysis (WGCNA), 5 protein and 11 peptide clusters were obtained, showing distinct characteristics. With this, several associations were found between the different data sets and with specific sample characteristics. This study provides insight into the dynamics of human milk protein, peptide, and metabolite composition. In addition, it will support future studies that evaluate the effect size of a parameter of interest by enabling a comparison with natural variability.
Asunto(s)
Leche Humana , Proteoma , Femenino , Humanos , Metaboloma , Proteínas de la Leche/metabolismo , Leche Humana/química , Péptidos/análisis , Proteoma/análisisRESUMEN
Growth and nutrition during early life have been strongly linked to future health and metabolic risks. The Cambridge Baby Growth Study (CBGS), a longitudinal birth cohort of 2229 mother-infant pairs, was set up in 2001 to investigate early life determinant factors of infant growth and body composition in the UK setting. To carry out extensive profiling of breastmilk intakes and composition in relation to infancy growth, the Cambridge Baby Growth and Breastfeeding Study (CBGS-BF) was established upon the original CBGS. The strict inclusion criteria were applied, focusing on a normal birth weight vaginally delivered infant cohort born of healthy and non-obese mothers. Crucially, only infants who were exclusively breastfed for the first 6 weeks of life were retained in the analysed study sample. At each visit from birth, 2 weeks, 6 weeks, and then at 3, 6, 12, 24, and 36 months, longitudinal anthropometric measurements and blood spot collections were conducted. Infant body composition was assessed using air displacement plethysmography (ADP) at 6 weeks and 3 months of age. Breast milk was collected for macronutrients and human milk oligosaccharides (HMO) measurements. Breast milk intake volume was also estimated, as well as sterile breastmilk and infant stool collection for microbiome study.
Asunto(s)
Lactancia Materna , Desarrollo Infantil , Leche Humana , Valor Nutritivo , Adiposidad , Factores de Edad , Estatura , Preescolar , Inglaterra , Femenino , Microbioma Gastrointestinal , Cabeza/crecimiento & desarrollo , Humanos , Lactante , Fenómenos Fisiológicos Nutricionales del Lactante , Recién Nacido , Masculino , Leche Humana/química , Leche Humana/microbiología , Estado Nutricional , Factores de Tiempo , Circunferencia de la Cintura , Aumento de PesoRESUMEN
Fructoselysine is formed upon heating during processing of food products, and being a key intermediate in advanced glycation end product formation considered to be potentially hazardous to human health. Human gut microbes can degrade fructoselysine to yield the short chain fatty acid butyrate. However, quantitative information on these biochemical reactions is lacking, and interindividual differences therein are not well established. Anaerobic incubations with pooled and individual human fecal slurries were optimized and applied to derive quantitative kinetic information for these biochemical reactions. Of 16 individuals tested, 11 were fructoselysine metabolizers, with Vmax, Km and kcat-values varying up to 14.6-fold, 9.5-fold, and 4.4-fold, respectively. Following fructoselysine exposure, 10 of these 11 metabolizers produced significantly increased butyrate concentrations, varying up to 8.6-fold. Bacterial taxonomic profiling of the fecal samples revealed differential abundant taxa for these reactions (e.g. families Ruminococcaceae, Christenellaceae), and Ruminococcus_1 showed the strongest correlation with fructoselysine degradation and butyrate production (ρ ≥ 0.8). This study highlights substantial interindividual differences in gut microbial degradation of fructoselysine. The presented method allows for quantification of gut microbial degradation kinetics for foodborne xenobiotics, and interindividual differences therein, which can be used to refine prediction of internal exposure.
Asunto(s)
Heces/microbiología , Lisina/análogos & derivados , Adulto , Variación Biológica Poblacional , Ácidos Grasos Volátiles/metabolismo , Femenino , Microbioma Gastrointestinal/genética , Humanos , Lisina/metabolismo , Masculino , Persona de Mediana Edad , ARN Ribosómico 16S , Adulto JovenRESUMEN
To establish the effect of the presence of milk serum proteins on heat-induced changes to lactoferrin, lactoferrin alone, and lactoferrin mixed with either milk serum or ß-lactoglobulin was heated at 65 °C, 70 °C and 75 °C for 30 min. After heating, the effect of milk serum proteins on aggregation of lactoferrin was characterized, after which the effect of such aggregation on digestion and bacteriostatic capacity of lactoferrin were determined. The presence of milk serum proteins accelerated the aggregation of lactoferrin during heating through thiol/disulphide interchange. Lactoferrin also formed disulphide-linked aggregates when it was heated with ß-lactoglobulin. Protein aggregates formed at 75 °C were much more resistant to infant digestion, causing decreased peptide release from lactoferrin. Heating lactoferrin and milk serum proteins together accelerated the loss of bacteriostatic activity upon heating. In conclusion, heat-induced aggregation of lactoferrin with milk serum proteins affected both its digestion and its bacteriostatic activity.
Asunto(s)
Lactoferrina/química , Lactoferrina/farmacocinética , Proteínas de la Leche/química , Animales , Antibacterianos/farmacología , Digestión , Jugo Gástrico , Calor , Humanos , Lactoglobulinas/química , Leche/química , Tamaño de la Partícula , Proteína de Suero de Leche/químicaRESUMEN
To quantify interindividual differences in the human intestinal microbial metabolism of (-)-epicatechin (EC), in vitro anaerobic incubations with fecal inocula from 24 healthy donors were conducted. EC-derived colonic microbial metabolites were qualitatively and quantitively analyzed by liquid chromatography triple quadrupole mass spectrometry (LC-TQ-MS) and liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS). Quantitative microbiota characterization was achieved by 16S rRNA analysis. The results obtained show 1-(3',4'-dihydroxyphenyl)-3-(2â³,4â³,6â³-dihydroxyphenyl)-2-propanol (3,4-diHPP-2-ol) and 5-(3',4'-dihydroxyphenyl)-γ-valerolactone (3,4-diHPV) to be key intermediate microbial metabolites of EC and also revealed the substantial interindividual differences in both the rate of EC conversion and the time-dependent EC metabolite pattern. Furthermore, substantial differences in microbiota composition among different individuals were detected. Correlations between specific microbial phylotypes and formation of certain metabolites were established. It is concluded that interindividual differences in the intestinal microbial metabolism of EC may contribute to interindividual differences in potential health effects of EC-abundant dietary foods or drinks.
RESUMEN
Differences in sanitary conditions, as model to induce differences in subclinical immune stimulation, affect the growth performance and nutrient metabolism in pigs. The objective of the present study was to evaluate the colonic microbiota and the colonic and systemic metabolome of female pigs differing in health status induced by sanitary conditions. We analyzed blood and colon digesta metabolite profiles using Nuclear Magnetic Resonance (1H NMR) and Triple quadrupole mass spectrometry, as well as colonic microbiota profiles. 1H NMR is a quantitative metabolomics technique applicable to biological samples. Weaned piglets of 4 weeks of age were kept under high or low sanitary conditions for the first 9 weeks of life. The microbiota diversity in colon digesta was higher in pigs subjected to low sanitary conditions (n = 18 per treatment group). The abundance of 34 bacterial genera was higher in colon digesta of low sanitary condition pigs, while colon digesta of high sanitary status pigs showed a higher abundance for four bacterial groups including the Megasphaera genus (p < 0.003) involved in lactate fermentation. Metabolite profiles (n = 18 per treatment group) in blood were different between both groups of pigs. These different profiles suggested changes in general nutrient metabolism, and more specifically in amino acid metabolism. Moreover, differences in compounds related to the immune system and responses to stress were observed. Microbiome-specific metabolites in blood were also affected by sanitary status of the pigs. We conclude that the microbiome composition in colon and the systemic metabolite profiles are affected by sanitary conditions and related to suboptimal health. These data are useful for exploring further relationships between health, metabolic status and performance and for the identification of biomarkers related to health (indices) and performance.
RESUMEN
Burkea africana is a leguminous tree used for medicinal purposes, growing in clusters, on soils impoverished from most nutrients. The study aimed to determine the factors responsible for successful reproduction and establishment of the B. africana trees in nature, as all efforts for commercial production has been proven unsuccessful. An investigation was carried out to determine the metabolomic profile, chemical composition, and microbial composition of the soils where B. africana grows (Burkea soil) versus the soil where it does not grow (non-Burkea soil). 1H-NMR metabolomic analysis showed different metabolites in the respective soils. Trehalose and betaine, as well as a choline-like and carnitine-like compound, were found to be in higher concentration in Burkea soils, whereas, acetate, lactate, and formate were concentrated in non-Burkea soils. Liquid Chromatography-Mass Spectrometry analysis revealed the presence of numerous amino acids such as aspartic acid and glutamine to be higher in Burkea soils. Since it was previously suggested that the soil microbial diversity is the major driver for establishment and survival of seedlings in nature, Deoxyribonucleic acid (DNA) was extracted and a BLAST analysis conducted for species identification. Penicillium species was found to be highly prevalent and discriminant between the two soils, associated with the Burkea soils. No differences in the bacterial composition of Burkea and non-Burkea soils were observed. The variances in fungal composition suggests that species supremacy play a role in development of B. africana trees and is responsible for creating a supporting environment for natural establishment and survival of seedlings.
RESUMEN
Bacterial lipoproteins are well-recognized microorganism-associated molecular patterns, which interact with Toll-like receptor (TLR) 2, an important pattern recognition receptor of the host innate immune system. Lipoproteins are conjugated with two- or three-acyl chains (di- or tri-acyl), which is essential for appropriate anchoring in the cell membrane as well as for the interaction with TLR2. Lipoproteins have mostly been studied in pathogens and have established roles in various biological processes, such as nutrient import, cell wall cross-linking and remodeling, and host-cell interaction. By contrast, information on the role of lipoproteins in the physiology and host interaction of probiotic bacteria is scarce. By deletion of lgt, encoding prolipoprotein diacylglyceryl transferase, responsible for lipidation of lipoprotein precursors, we investigated the roles of the collective group of lipoproteins in the physiology of the probiotic model strain Lactobacillus plantarum WCFS1 using proteomic analysis of secreted proteins. To investigate the consequences of the lgt mutation in host-cell interaction, the capacity of mutant and wild-type bacteria to stimulate TLR2 signaling and inflammatory responses was compared using (reporter-) cell-based models. These experiments exemplified the critical contribution of the acyl chains of lipoproteins in immunomodulation. To the best of our knowledge, this is the first study that investigated collective lipoprotein functions in a model strain for probiotic lactobacilli, and we show that the lipoproteins in L. plantarum WCFS1 are critical drivers of anti-inflammatory host responses toward this strain.
RESUMEN
The formation and repair of N2-(trans-isosafrol-3'-yl)-2'-deoxyguanosine (S-3'-N2-dG) DNA adduct derived from the spice and herbal alkenylbenzene constituent safrole were investigated. DNA adduct formation and repair were studied in vitro and using molecular dynamics (MD) simulations. DNA adduct formation was quantified using liquid chromatography-mass spectrometry (LCMS) in wild type and NER (nucleotide excision repair) deficient CHO cells and also in HepaRG cells and primary rat hepatocytes after different periods of repair following exposure to safrole or 1'-hydroxysafrole (1'-OH safrole). The slower repair of the DNA adducts found in NER deficient cells compared to that in CHO wild type cells indicates a role for NER in repair of S-3'-N2-dG DNA adducts. However, DNA repair in liver cell models appeared to be limited, with over 90% of the adducts remaining even after 24 or 48 h recovery. In our further studies, MD simulations indicated that S-3'-N2-dG adduct formation causes only subtle changes in the DNA structure, potentially explaining inefficient activation of NER. Inefficiency of NER mediated repair of S-3'-N2-dG adducts points at persistence and potential bioaccumulation of safrole DNA adducts upon daily dietary exposure.
Asunto(s)
Aductos de ADN/química , Simulación de Dinámica Molecular , Safrol/química , Animales , Células Cultivadas , Reparación del ADN , Humanos , RatasRESUMEN
Dairy cows can experience a negative energy balance (NEB) in early lactation when feed intake is too low to meet the energy requirements for body maintenance and milk production. Metabolic changes occur in mammary gland cells of animals experiencing a negative energy balance. We studied these metabolic changes in milk samples from dairy cows in relation to energy balance status using liquid chromatography-mass spectrometry (QQQ-LC-MS) and nuclear magnetic resonance (1H NMR). NMR and LC-MS techniques are complementary techniques that enabled a comprehensive overview of milk metabolites in our study. Energy balance and milk samples were obtained from 87 dairy cows. A total of 55 milk metabolites were reliably detected, of which 15 metabolites were positively correlated to energy balance and 20 were negatively correlated to energy balance. Cows in NEB produced more milk with increased milk fat yield and higher concentrations of citrate, cis-aconitate, creatinine, glycine, phosphocreatine, galactose-1-phosphate, glucose-1-phosphate, UDP-N-acetyl-galactosamine, UDP-N-acetyl-glucosamine, and phosphocholine but lower concentrations of choline, ethanolamine, fucose, N-acetyl-neuraminic acid, N-acetyl-glucosamine, and N-acetyl-galactosamine. During NEB, we observed an increased leakage of cellular content, increased synthesis of nucleic acids and cell membrane phospholipids, an increase in one-carbon metabolic processes, and an increase in lipid-triglyceride anabolism. Overall, both apoptosis combined with cellular renewal is paramount in the mammary gland in cows in NEB.
Asunto(s)
Lactancia , Leche , Animales , Bovinos , Dieta , Metabolismo Energético , Femenino , Metabolómica , TriglicéridosRESUMEN
The emergence of intestinal organoids, as a stem cell-based self-renewable model system, has led to many studies on intestinal development and cell-cell signaling. However, potential issues regarding the phenotypic stability and reproducibility of the methodology during culture still needs to be addressed for different organoids. Here we investigated the transcriptomes of jejunum organoids derived from the same pig as well as batch-to-batch variation of organoids derived from different pigs over long-term passage. The set of genes expressed in organoids closely resembled that of the tissue of origin, including small intestine specific genes, for at least 17 passages. Minor differences in gene expression were observed between individual organoid cultures. In contrast, most small intestine-specific genes were not expressed in the jejunum cell line IPEC-J2, which also showed gene expression consistent with cancer phenotypes. We conclude that intestinal organoids provide a robust and stable model for translational research with clear advantages over transformed cells.