Defining the Ceramide Composition of Bovine and Human Milk Gangliosides by Direct Infusion ESI-CID Tandem Mass Spectrometry of Native and Permethylated Molecular Species.

Gangliosides are glycosphingolipids composed of an oligosaccharide that contains one or more sialic acid residues and is linked to a ceramide, a lipid composed of a long chain base (LCB) that bears an amide-linked fatty acyl group (FA). The ceramide portions of gangliosides are embedded in cell membranes; the exposed glycans interact with the extracellular environment. Gangliosides play a myriad of roles in activities such as cell-cell communication, formation of lipid rafts, cellular adhesion, calcium homeostasis, host-pathogen interaction, and viral invasion. Although the epitopes responsible for the interactions of gangliosides are located in the glycan, the epitope presentation is strongly influenced by the orientation of the attached ceramide within the lipid membrane, a feature that depends on the details of its structure, that is, the specific LCB and FA. Since the identities of both the glycan and the ceramide affect the activity of gangliosides, it is important to characterize the individual intact molecular forms. We report here a mass spectrometry-based method that combines the information gained from low-energy collision-induced dissociation (CID) measurements for the determination of the glycan with tandem mass spectra obtained at stepped higher-energy CID for the detailed characterization of the LCB and FA components of intact gangliosides. We provide results from applications of this method to the analysis of gangliosides present in bovine and human milk in order to demonstrate the assignment of LCB and FA for intact gangliosides and differential detection of isomeric ceramide structures.

Potential sex differences in human milk fatty acids and their association with atopic dermatitis: Results of the Ulm SPATZ health study.

BACKGROUND: Polyunsaturated fatty acids (PUFAs) in human milk are essential in immune system maturation and might play a role in the development of allergic conditions, such as atopic dermatitis (AD) in infants. Immune system responses are modulated by sex, but data on the sex-specific associations with PUFAs are limited. We therefore explored sex-specific differences in human milk PUFAs and their association with AD up to 2 years. METHODS: PUFAs were measured in human milk samples from the Ulm SPATZ Health Study at 6 weeks (n = 512) and 6 months (n = 367). Associations with AD up to 2 years were evaluated using crude and multivariable logistic regression. Interactions between infant sex and PUFAs were explored by including the product term. RESULTS: No significant associations were observed with 6-week data. At 6 months, the median relative proportion of docosahexaenoic acid (DHA) was significantly higher in milk for female than male infants (p = .001). Female infants whose milk was lower in quintile proportions of alpha-linolenic acid (ALA) at 6 months had lower odds of AD compared to males [first vs. fifth quintile OR (95% confidence interval): 0.13 (0.02, 0.66), p = .02]. This interaction was not significant when correcting for multiple testing (α threshold: p = .004). No other statistically significant associations were observed. CONCLUSION: Individual quintile PUFA proportions in human milk were not associated with AD, overall and in a sex-specific manner. More comprehensive and statistically powered longitudinal studies are needed to determine whether potential sex differences in human milk, if any, could be of clinical relevance for infants including the investigation of mediating factors.

Brain simulation as a cloud service: The Virtual Brain on EBRAINS.

The Virtual Brain (TVB) is now available as open-source services on the cloud research platform EBRAINS (ebrains.eu). It offers software for constructing, simulating and analysing brain network models including the TVB simulator; magnetic resonance imaging (MRI) processing pipelines to extract structural and functional brain networks; combined simulation of large-scale brain networks with small-scale spiking networks; automatic conversion of user-specified model equations into fast simulation code; simulation-ready brain models of patients and healthy volunteers; Bayesian parameter optimization in epilepsy patient models; data and software for mouse brain simulation; and extensive educational material. TVB cloud services facilitate reproducible online collaboration and discovery of data assets, models, and software embedded in scalable and secure workflows, a precondition for research on large cohort data sets, better generalizability, and clinical translation.

Human milk oligosaccharide profiles and child atopic dermatitis up to 2 years of age: The Ulm SPATZ Health Study.

BACKGROUND: Human milk oligosaccharides (HMOs) have several biological functions. Yet, very few studies have investigated the effect of HMOs on the development of allergies and even fewer on their specific associations with atopic dermatitis (AD) during early childhood. OBJECTIVE: This study investigated whether individual HMO concentrations, measured at two time points of lactation, were associated with reported diagnosis of AD in children up to two years of age. METHOD: Outcome data were available for HMOs measured in human milk samples collected at 6 weeks (n = 534) and 6 months (n = 356) of lactation. Associations of HMOs with AD, ascertained from parents and pediatricians at ages one and two years, were assessed in crude and adjusted logistic regression models. RESULTS: Few associations were statistically significant at the conventional level (p < .05), for example, 6-week Lacto-N-neotetraose with 2-year AD [OR 95%CI: 0.82 (0.66, 1.00)] and 6-month 3'-sialyllactose among non-secretor mothers with 1-year AD [2.59 (1.53, 6.81)]. Importantly, accounting for multiple testing, these and all further associations were not statistically significant (all p > .0031, which is the threshold for statistical significance after correction for multiple testing). CONCLUSION: Our findings suggest that the intake of different levels (or even absence) of the individual HMOs measured at 6 weeks and 6 months of lactation, in the current study, is not significantly associated with the development of AD in early childhood. Given the exploratory nature of our study and the limited sample size, these results should be interpreted with caution. The specific HMOs for which we show plausible associations at conventional level may warrant further research and investigation.

Dietary Intake of Chinese Lactating Women Is Associated with the Fatty Acid Profile of Their Milk.

INTRODUCTION: The present study aimed to explore the relationship between the dietary intake and the human milk (HM) fatty acid (FA) profile of Chinese lactating women. METHODS: HM samples and food records were obtained from 122 Chinese women over 5 visits between 0 and 51 days postpartum. Adjusted multiple regression was performed to explore associations between maternal dietary intakes of energy, macronutrients, FAs and foods, and the HM FA profile. Analyses were performed separately for colostrum and mature milk. RESULTS: Dietary intakes of total polyunsaturated FAs (PUFAs), eicosapentaenoic acid (EPA), and docosahexaenoic acid were positively associated with the HM contents of PUFAs, omega-6 (n-6) PUFAs, and linoleic acid (LA), and the intakes of n-3 PUFAs and α-linolenic acid (ALA) were negatively associated with saturated FA levels in HM. Associations were stronger for mature milk. Intakes of milk/dairy, meat/poultry, and eggs were negatively associated with n-6 PUFAs, LA, and EPA in mature milk, whereas the opposite was seen for fish/shrimp. Positive associations were also found between fish/shrimp and total and n-3 PUFAs in mature milk. CONCLUSION: The HM FA profile of Chinese women is associated with their diet, and in particular with their FA intake. Tailored nutritional advice based on HM FA composition may optimize HM FA profile and thereby contribute to healthy infant development.

Personalized Profiling Reveals Donor- and Lactation-Specific Trends in the Human Milk Proteome and Peptidome.

BACKGROUND: Human milk is the most genuine form of personalized nutrition, whereby its nutritional and bioactive constituents support the changing needs of the growing infant. Personalized proteome profiling strategies may provide insights into maternal-infant relationships. Proteins and endogenous peptides in human milk play an important role as nutrients for growth and have distinct functionality such as immune defense. Comprehensive monitoring of all of the human milk proteinaceous components, including endogenous peptides, is required to fully understand the changing role of the human milk proteome throughout lactation. OBJECTIVE: We aimed to investigate the personalized nature of the human milk proteome and peptidome for individual mother-infant dyads. METHODS: Two individual healthy milk donors, aged 29 and 32 y and both of a normal BMI, were longitudinally observed over weeks 1, 2, 3, 4, 6, 8, 10, 12, and 16 postpartum. Milk collection was standardized. Comprehensive variations in the human milk proteinaceous components were assessed using quantitative LC-MS/MS methods. RESULTS: We longitudinally profiled the concentrations of >1300 milk proteins and 2000 endogenous milk peptides spanning 16 wk of lactation for 2 individual donors. We observed many gradual and alike changes in both donors related to temporal effects, for instance early lactation was marked by high concentrations of proteins and peptides involved in lactose synthesis and immune development. Uniquely, in 1 of the 2 donors, we observed a substantial anomaly in the milk composition, exclusively at week 6, likely indicating a response to inflammation and/or infection. CONCLUSIONS: Here, we provide a resource for characterizing the lactational changes in the human milk proteome, encompassing thousands of proteins and endogenous peptides. Further, we demonstrate the feasibility and benefit of personalized profiling to monitor the influence of milk on the development of the newborn, as well as the health status of each individual mother-infant pair.

Dietary Intake of Breastfeeding Mothers in Developed Countries: A Systematic Review and Results of the MEDIDIET Study.

BACKGROUND: Lactation is a demanding period for women, and a good nutrition is crucial for optimal health of mothers and infants. OBJECTIVES: To provide new data and summarize the overall evidence on maternal nutrient intakes during lactation in developed countries, we present a systematic review (SR) of the literature and concurrently original results of the Italian MEDIDIET study. We compared nutrient intakes with dietary reference values (DRVs) proposed by the European Food Safety Authority. METHODS: Studies were identified searching PubMed/Embase databases up to February 2020. Observational studies reporting at least energy and macronutrient intakes of healthy breastfeeding mothers who followed non-restricted and non-specific diets were included. Studies on populations with severe nutritional deficiencies were excluded. The MEDIDIET study enrolled 300 healthy breastfeeding mothers at 6 ± 1 wk postpartum. Usual diet was concomitantly evaluated through a validated and reproducible FFQ. Nutrient intakes were estimated using a food composition database. RESULTS: Twenty-eight articles regarding 32 distinct study populations were included. Maternal nutrient intakes were generally in agreement across studies included in the SR and conforming to DRVs. Within micronutrients, vitamin D intake was below the recommendation. In the MEDIDIET study, mean intakes of energy (1950 ± 445 kcal/d), carbohydrates (270 ± 20.1 g/d), proteins (87.8 ± 20.1 g/d), and fats (65.6 ± 18.9 g/d) were similar to those observed in the SR. Moreover, observed intakes seemed to reflect the typical Mediterranean diet, with low intakes of carbohydrates, SFAs, and PUFAs and high intakes of MUFAs and vitamins. Conversely, protein intake was mainly derived from animal sources. CONCLUSIONS: This SR showed that nutrient intakes of breastfeeding mothers in developed countries are generally in line with DRVs despite different dietary patterns worldwide. Some nutritional deficiencies emerged, highlighting the need for additional nutritional advice. Mothers participating in the MEDIDIET study showed a nutritional profile in agreement with the Mediterranean diet.

Changes in human milk fatty acid composition and maternal lifestyle-related factors over a decade: a comparison between the two Ulm Birth Cohort Studies.

Human milk fatty acid composition varies during lactation and is influenced by maternal diet, maternal lifestyle-related factors and genetic background. This is one of the first studies to investigate a period effect, that is, the impact of lifestyle-related changes on human milk fatty acid composition, in two different cohorts. Lactating women were recruited from the general population a decade apart in Ulm, Germany, using similar methodology. Human milk samples collected 6 weeks postpartum were analysed (Ulm Birth Cohort Study (UBCS (2000)), n 567; Ulm SPATZ Health Study (SPATZ (2012)), n 458). Centred log ratio transformation was applied to fatty acid data. Principal component analysis was used to determine study-dependent fatty acid profiles. A general linear model was used to determine the study (or period) effect on fatty acid profiles adjusting for duration of gestation, age, education, delivery mode, smoking and pre-pregnancy BMI. Two principal components were retained (PC1 and PC2). PC1 was associated with UBCS, while PC2 was associated with SPATZ. PC1 comprised high SFA, and low MUFA, n-6 and n-3 long-chain PUFA (LCPUFA). The inverse was true for PC2. Although human milk remains a source of essential fatty acids, infants could be at risk of inadequate n-3 and n-6 LCPUFA intake through human milk. The differences in the human milk fatty acid profiles also reflect changes in maternal dietary habits in the more recent cohort, which may comprise lower intakes of dietary trans-fatty acids and SFA and higher intakes of vegetable oils.

Optimization of a human milk-directed quantitative sIgA ELISA method substantiated by mass spectrometry.

Immunoglobulins are the primary protective products in human milk and are responsible for transferring maternal pathogen memory to the infant, providing protection by binding to recognized pathogens and inhibiting virulence. To better understand potentially protective/anti-infective compounds in human milk, the establishment of human milk-tailored analytical approaches is crucial, as most contemporary analytical methods have been optimized for plasma or serum. One of the most prominent immunoglobulins in human milk is secretory immunoglobulin A (sIgA), which may be relevant for the protection of breastfed infants from harmful pathogens. Advanced sIgA detection methods can help monitor the immune status and development of the mother-infant dyad. We therefore developed an enzyme-linked immunosorbent assay (ELISA) sIgA method for the quantitative analysis of IgA plus secretory component (SC), validated with sIgA standards and substantiated by mass spectrometry (MS)-based proteomics. A very strong correlation was observed between the MS-detected IgA1 and the human milk-specific sIgA ELISA (r = 0.82). Overall, the MS data indicate that the developed human milk sIgA ELISA does not differentiate between sIgA1 and sIgA2 and is, therefore, a reflection of total sIgA. Furthermore, our MS data and the human milk-derived sIgA ELISA data are better correlated than data derived from a standard serum IgA ELISA kit (relative to MS IgA1 r = 0.82 and r = 0.42, respectively). We therefore propose our human milk-specific sIgA ELISA as an ideal quantitative indicator of total sIgA with advantages over current serum IgA ELISA kits.

Monitoring Human Milk ß-Casein Phosphorylation and O-Glycosylation Over Lactation Reveals Distinct Differences between the Proteome and Endogenous Peptidome.

Human milk is a vital biofluid containing a myriad of molecular components to ensure an infant's best start at a healthy life. One key component of human milk is ß-casein, a protein which is not only a structural constituent of casein micelles but also a source of bioactive, often antimicrobial, peptides contributing to milk's endogenous peptidome. Importantly, post-translational modifications (PTMs) like phosphorylation and glycosylation typically affect the function of proteins and peptides; however, here our understanding of ß-casein is critically limited. To uncover the scope of proteoforms and endogenous peptidoforms we utilized mass spectrometry (LC-MS/MS) to achieve in-depth longitudinal profiling of ß-casein from human milk, studying two donors across 16 weeks of lactation. We not only observed changes in ß-casein's known protein and endogenous peptide phosphorylation, but also in previously unexplored O-glycosylation. This newly discovered PTM of ß-casein may be important as it resides on known ß-casein-derived antimicrobial peptide sequences.

Research and Practice of AI Ethics: A Case Study Approach Juxtaposing Academic Discourse with Organisational Reality.

This study investigates the ethical use of Big Data and Artificial Intelligence (AI) technologies (BD + AI)-using an empirical approach. The paper categorises the current literature and presents a multi-case study of 'on-the-ground' ethical issues that uses qualitative tools to analyse findings from ten targeted case-studies from a range of domains. The analysis coalesces identified singular ethical issues, (from the literature), into clusters to offer a comparison with the proposed classification in the literature. The results show that despite the variety of different social domains, fields, and applications of AI, there is overlap and correlation between the organisations' ethical concerns. This more detailed understanding of ethics in AI + BD is required to ensure that the multitude of suggested ways of addressing them can be targeted and succeed in mitigating the pertinent ethical issues that are often discussed in the literature.

Quantitative Longitudinal Inventory of the N-Glycoproteome of Human Milk from a Single Donor Reveals the Highly Variable Repertoire and Dynamic Site-Specific Changes.

Protein N-glycosylation on human milk proteins assists in protecting an infant's health and functions among others as competitive inhibitors of pathogen binding and immunomodulators. Due to the individual uniqueness of each mother's milk and the overall complexity and temporal changes of protein N-glycosylation, analysis of the human milk N-glycoproteome requires longitudinal personalized approaches, providing protein- and N-site-specific quantitative information. Here, we describe an automated platform using hydrophilic-interaction chromatography (HILIC)-based cartridges enabling the proteome-wide monitoring of intact N-glycopeptides using just a digest of 150 µg of breast milk protein. We were able to map around 1700 glycopeptides from 110 glycoproteins covering 191 glycosites, of which 43 sites have not been previously reported with experimental evidence. We next quantified 287 of these glycopeptides originating from 50 glycoproteins using a targeted proteomics approach. Although each glycoprotein, N-glycosylation site, and attached glycan revealed distinct dynamic changes, we did observe a few general trends. For instance, fucosylation, especially terminal fucosylation, increased across the lactation period. Building on the improved glycoproteomics approach outlined above, future studies are warranted to reveal the potential impact of the observed glycosylation microheterogeneity on the healthy development of infants.

Human milk oligosaccharides promote immune tolerance via direct interactions with human dendritic cells.

Human milk oligosaccharides (HMOS) are a complex mixture of bioactive components supporting the immune development of breastfed-infants. Dendritic cells (DCs) play a central role in the regulation of immune responses, being specialized in antigen presentation and driving T-cell priming as well as differentiation. However, little is known about the direct effects of HMOS on human DC phenotypes and functions. Here, we report that HMOS mixture isolated from pooled human milk, induced semi-maturation of human monocytes-derived DCs (moDCs), and elevated levels of IL-10, IL-27 and IL-6 but not IL-12p70 and TNF-α. Consistently, HMOS-conditioned human moDCs promoted Treg generation from naïve CD4+ T cells. Interestingly, HMOS limited LPS-induced maturation of human moDCs, while maintained IL-10 and IL-27 secretion and reduced LPS-induced production of IL-12p70, IL-6 and TNF-α. Furthermore, HMOS+LPS-stimulated DCs induced a higher frequency of Tregs and increased IL-10 production, while a reduction in Tbet+Th1 frequency and IFN-γ production was detected as compared to LPS-DCs. The regulatory effects of HMOS seemed to be mediated by interactions of HMOS with receptors, including but not limited to TLR4 and DC-SIGN on human moDCs. In conclusion, HMOS contain tolerogenic factors influencing human moDCs and thereby modulating the development of the neonatal immune system.

Targeted LC-ESI-MS2 characterization of human milk oligosaccharide diversity at 6 to 16 weeks post-partum reveals clear staging effects and distinctive milk groups.

Many molecular components in human milk (HM), such as human milk oligosaccharides (HMOs), assist in the healthy development of infants. It has been hypothesized that the functional benefits of HM may be highly dependent on the abundance and individual fine structures of contained HMOs and that distinctive HM groups can be defined by their HMO profiles. However, the structural diversity and abundances of individual HMOs may also vary between milk donors and at different stages of lactations. Improvements in efficiency and selectivity of quantitative HMO analysis are essential to further expand our understanding about the impact of HMO variations on healthy early life development. Hence, we applied here a targeted, highly selective, and semi-quantitative LC-ESI-MS2 approach by analyzing 2 × 30 mature human milk samples collected at 6 and 16 weeks post-partum. The analytical approach covered the most abundant HMOs up to hexasaccharides and, for the first time, also assigned blood group A and B tetrasaccharides. Principal component analysis (PCA) was employed and allowed for automatic grouping and assignment of human milk samples to four human milk groups which are related to the maternal Secretor (Se) and Lewis (Le) genotypes. We found that HMO diversity varied significantly between these four HM groups. Variations were driven by HMOs being either dependent or independent of maternal genetic Se and Le status. We found preliminary evidence for an additional HM subgroup within the Se- and Le-positive HM group I. Furthermore, the abundances of 6 distinct HMO structures (including 6'-SL and 3-FL) changed significantly with progression of lactation. Graphical abstract.

Ethical and Social Aspects of Neurorobotics.

The interdisciplinary field of neurorobotics looks to neuroscience to overcome the limitations of modern robotics technology, to robotics to advance our understanding of the neural system's inner workings, and to information technology to develop tools that support those complementary endeavours. The development of these technologies is still at an early stage, which makes them an ideal candidate for proactive and anticipatory ethical reflection. This article explains the current state of neurorobotics development within the Human Brain Project, originating from a close collaboration between the scientific and technical experts who drive neurorobotics innovation, and the humanities and social sciences scholars who provide contextualising and reflective capabilities. This article discusses some of the ethical issues which can reasonably be expected. On this basis, the article explores possible gaps identified within this collaborative, ethical reflection that calls for attention to ensure that the development of neurorobotics is ethically sound and socially acceptable and desirable.

Discovery and Quantification of Nonhuman Proteins in Human Milk.

The question whether and which nonhuman peptides or proteins are present in human milk was raised many decades ago. However, due to cross-reactivity or nonspecific antibody recognition, the accuracy of detection by immunochemical methods has been a concern. Additionally, the relative low-abundance of nonhuman peptides/proteins in the complex milk sample makes them a challenging target to detect. Here, by deep proteome profiling, we detected several nonhuman peptides, which could be grouped as nonhuman proteins. We next estimated their concentration in human milk by combining data-dependent shotgun proteomics and parallel reaction monitoring. First, we fractionated human milk at the protein level and were able to detect 1577 human proteins. Additionally, we identified 109 nonhuman peptides, of which 71 were grouped into 36 nonhuman proteins. In the next step, we targeted 37 nonhuman peptides and nine of them could be repeatedly quantified in human milk samples. Peptides/proteins originating from bovine milk products were the dominant nonhuman proteins observed, notably bovine caseins (α-S1-, α-S2-, ß-, κ-caseins) and ß-lactoglobulin. The method we present here can be expanded to investigate more about nonhuman peptides and proteins in human milk and give a better understanding of how human milk plays a role in allergy prevention.

Soluble CD14 concentration in human breast milk and its potential role in child atopic dermatitis: Results of the Ulm Birth Cohort Studies.

BACKGROUND: Soluble CD14 (sCD14) is one of many factors in human breast milk which may influence programming of the immune response in the breastfed child. Although previous studies have mostly found little association between sCD14 concentration in breast milk and atopic outcomes, recent evidence continues to support a role of sCD14 in immune-related disease. OBJECTIVE: We aimed to clarify whether an association exists between sCD14 concentration in human breast milk (m-sCD14) and child atopic dermatitis (AD) diagnosis by age 3 years within the context of two large birth cohorts. METHODS: Data were obtained from the Ulm Birth Cohort Study (UBCS) and the Ulm SPATZ Health Study, methodologically similar birth cohort studies, each consisting of approximately 1000 newborns and their mothers recruited from the general population shortly after delivery in Ulm, Southern Germany, respectively, from 11/2000 to 11/2001 and 04/2012 to 05/2013. sCD14 concentrations were measured by different ELISAs (UBCS: IBL, SPATZ: R&D) in breast milk samples collected at 6 weeks post-delivery in both studies and additionally at 6 months and 1 year in SPATZ. Children's AD diagnosis was assessed using parent and paediatrician reports at 1, 2 and 3 years of age. RESULTS: Complete exposure and outcome data were available for 659 UBCS and 489 SPATZ children. In both cohorts, sCD14 concentration was significantly associated with breastfeeding frequency (P < 0.01). We observed no association between m-sCD14 concentration and child AD diagnosis in either study. CONCLUSIONS: Our results do not support an association between sCD14 concentration in mature breast milk and AD among breastfed children.

The Combination of 2'-Fucosyllactose with Short-Chain Galacto-Oligosaccharides and Long-Chain Fructo-Oligosaccharides that Enhance Influenza Vaccine Responses Is Associated with Mucosal Immune Regulation in Mice.

BACKGROUND: A critical role for host-microbe interactions and establishment of vaccine responses has been postulated. Human milk oligosaccharides, of which 2'-fucosyllactose (2'FL) is the most prevalent, are known to alter host-associated microbial communities and play a critical role in the immunologic development of breastfed infants. OBJECTIVES: Dietary supplementation with a combination of 2'FL and prebiotic short-chain (sc) galacto-oligosaccharides (GOS) and long-chain (lc) fructo-oligosaccharides (FOS) was employed to examine human milk oligosaccharide effects on immune responsiveness, within a murine influenza vaccination model. METHODS: Female mice (6 wk old, C57Bl/6JOlaHsd) were fed either control diet (CON) or scGOS/lcFOS/2'FL-containing diet (GF2F) for 45 d. After starting dietary intervention (day 14), mice received a primary influenza vaccination (day 0) followed by a booster vaccination (day 21), after which ear challenges were conducted to measure vaccine-specific delayed type hypersensitivity (DTH). Serum immunoglobulin (Ig) levels, fecal and cecal microbial community structure, short-chain fatty acids, host intestinal gene expression and cellular responses in the mesenteric lymph nodes (MLNs) were also measured. RESULTS: Relative to CON, mice fed the GF2F diet had increased influenza vaccine-specific DTH responses (79.3%; P < 0.01), higher levels of both IgG1 (3.2-fold; P < 0.05) and IgG2a (1.2-fold; P < 0.05) in serum, and greater percentages of activated B cells (0.3%; P < 0.05), regulatory T cells (1.64%; P < 0.05), and T-helper 1 cells (2.2%; P < 0.05) in their MLNs. GF2F-fed mice had elevated cecal butyric (P < 0.05) and propionic (P < 0.05) acid levels relative to CON, which correlated to DTH responses (R2 = 0.22; P = 0.05 and R2 = 0.39; P < 0.01, respectively). Specific fecal microbial taxa altered in GF2F diet fed mice relative to CON were significantly correlated with the DTH response and IgG2a level increases. CONCLUSIONS: Dietary GF2F improved influenza vaccine-specific T-helper 1 responses and B cell activation in MLNs and enhanced systemic IgG1 and IgG2a concentrations in mice. These immunologic changes are correlated with microbial community structure and metabolites.

Label-free targeted LC-ESI-MS2 analysis of human milk oligosaccharides (HMOS) and related human milk groups with enhanced structural selectivity.

Human milk (HM) supports the healthy development of neonates and exerts many of its beneficial effects via contained free human milk oligosaccharides (HMOS). These HMOS exhibit a complexity and structural diversity that pose a significant analytical challenge. A detailed characterization of HMOS is essential as every individual structure may have a different function/activity. Certain HMOS isomers may even fundamentally differ in their biological function, and especially their characterization by LC or LC-MS is often impaired by co-elution phenomena. Thus, more efficient analytical methodologies with enhanced structural selectivity are required. Therefore, we developed a negative ion mode LC-ESI-MS2 approach featuring straightforward sample preparation, environmentally friendly EtOH gradient elution, and enhanced, semiquantitative characterization of distinct native HMOS by multiple reaction monitoring (MRM). Our MRM-LC-MS setup takes advantage of highly selective, glycan configuration-dependent collision-induced dissociation (CID) fragments to identify individual neutral and acidic HMOS. Notably, many human milk oligosaccharide isomers could be distinguished in a retention time-independent manner. This contrasts with other contemporary MRM approaches relying on rather unspecific MRM transitions. Our method was used to determine the most abundant human milk tri-, tetra-, penta-, and hexaoses semiquantitatively in a single LC-MS assay. Detected HMO structures included fucosyllactoses (e.g., 2'-FL), lacto-N-difucotetraose (LDFT), lacto-N-tetraoses (LNTs), lacto-N-fucopentaoses (e.g., LNFP I, LNFP II and III), lacto-N-difucohexaoses (LNDFHs) as well as sialyllactoses (SLs) and tentatively assigned blood group A and B tetrasaccharides from which correct human milk type assignment could be also demonstrated. Correctness of milk typing was validated for milk groups I-IV by high pressure anion exchange chromatography (HPAEC) coupled to pulsed amperometric detection (HPAEC-PAD). Graphical Abstract ᅟ.

Toward an efficient workflow for the analysis of the human milk peptidome.

There is a growing interest for investigating endogenous peptides from human biofluids which may provide yet unknown functional benefits or provide an early indication of disease states as potential biomarkers. A major technical bottleneck in the investigation of endogenous peptides from body fluids, e.g., serum, urine, saliva, and milk, is that each of these fluids seems to require unique workflows for peptide extraction and analysis. Thus, protocols optimized for serum cannot be directly translated to milk. One biofluid that is readily available, but which has not been extensively explored, is human milk, whose analysis could contribute to our understanding of the immune development of the newborn infant. Due to the occurrence of highly abundant lipids, proteins, and saccharides, milk peptidomics requires dedicated sample preparation steps. The aim of this study was to develop a time and cost-efficient workflow for the analysis of the human milk peptidome, for which we compared peptide extraction methodologies and peptide fragmentation methods. A method using strong acid protein precipitation and analysis by collision-induced dissociation fragmentation was found to be superior to all other test methods, allowing us qualitative and quantitative detection of about 4000 endogenous human milk peptides in a total analysis time of just 18 h.