Human milk oligosaccharides: every baby needs a sugar mama

Human milk oligosaccharides (HMOs) are a family of structurally diverse unconjugated glycans that are highly abundant in and unique to human milk. Originally, HMOs were discovered as a prebiotic "bifidus factor" that serves as a metabolic substrate for desired bacteria and shapes an intestinal microbiota composition with health benefits for the breast-fed neonate. Today, HMOs are known to be more than just "food for bugs". An accumulating body of evidence suggests that HMOs are antiadhesive antimicrobials that serve as soluble decoy receptors, prevent pathogen attachment to infant mucosal surfaces and lower the risk for viral, bacterial and protozoan parasite infections. In addition, HMOs may modulate epithelial and immune cell responses, reduce excessive mucosal leukocyte infiltration and activation, lower the risk for necrotizing enterocolitis and provide the infant with sialic acid as a potentially essential nutrient for brain development and cognition. Most data, however, stem from in vitro, ex vivo or animal studies and occasionally from association studies in mother-infant cohorts. Powered, randomized and controlled intervention studies will be needed to confirm relevance for human neonates. The first part of this review introduces the pioneers in HMO research, outlines HMO structural diversity and describes what is known about HMO biosynthesis in the mother's mammary gland and their metabolism in the breast-fed infant. The second part highlights the postulated beneficial effects of HMO for the breast-fed neonate, compares HMOs with oligosaccharides in the milk of other mammals and in infant formula and summarizes the current roadblocks and future opportunities for HMO research.     

Conformational analysis of the milk oligosaccharides

Possible conformations of lacto-N-tetraose, lacto-N-neotetraose, related disaccharides, and other milk oligosaccharides have been studied by an energy-minimization procedure using empirical potential functions. Lacto-N-tetraose favors a “curved” conformation, while lacto-N-neotetraose favors an approximately “straight” conformation. These two conformations differ mainly in the position of the terminal galactose residue with respect to the rest of the molecule. This difference explains the greater strength of lacto-N-neotetraose compared with lacto-N-tetraose in its ability to inhibit the cross-reaction of blood group P1 fractions with Type XIV pneumococcal antipolysaccharide. Although the favored conformation of lacto-N-tetraose (inactive) agrees with the model proposed by the earlier workers, that for lacto-N-neotetraose (active) differs. The favored conformations for the disaccharides galactose-β(1-4)-N-acetylglucosamine, galactose-β(1-3)-N-acetylglucosamine, and lactose are similar in overall shape, differing only in the nature and orientation of the side groups. This explains their nearly equal inhibitory activity. These theoretical models also explain the increased activity of lacto-N-fucopentaose I over that of lacto-N-tetraose and the relative activities of the substituted lactoses. The present studies suggest that it is the overall shape of the molecule which is important for activity, rather than the terminal β(1-4)-linked galactose residue alone.     

Evolutionary glycomics: characterization of milk oligosaccharides in primates

Free oligosaccharides are abundant components of mammalian milk and have primary roles as prebiotic compounds, in immune defense, and in brain development. A mass spectrometry-based technique is applied to profile milk oligosaccharides from apes (chimpanzee, gorilla, and siamang), new world monkeys (golden lion tamarin and common marmoset), and an old world monkey (rhesus). The purpose of this study is to evaluate the patterns of primate milk oligosaccharide composition from a phylogenetic perspective to assess the extent to which the compositions of HMOs derives from ancestral primate patterns as opposed to more recent evolutionary events. Milk oligosaccharides were quantitated by nanoflow liquid chromatography on chip-based devices. The relative abundances of fucosylated and sialylated milk oligosaccharides in primates were also determined. For a systematic and comprehensive study of evolutionary patterns of milk oligosaccharides, cluster analysis of primate milk was performed using the chromatographic profile. In general, the oligosaccharides in primate milk, including humans, are more complex and exhibit greater diversity compared to the ones in nonprimate milk. A detailed comparison of the oligosaccharides across evolution revealed nonsequential developmental pattern, that is, that primate milk oligosaccharides do not necessarily cluster according to the primate phylogeny. This report represents the first comprehensive and quantitative effort to profile and elucidate the structures of free milk oligosaccharides so that they can be related to glycan function in different primates.     

Capillary affinophoresis as a versatile tool for the study of biomolecular interactions: a mini-review

Combination of capillary electrophoresis and bioaffinity interaction gave rise to powerful research tools for analyzing molecular recognition. They take advantages of the electrophoretic behavior of the complex formed between a target biomolecule and a specially designed mobile ligand molecule (affinophore or affinity probe), and enable detection of complex formation, determination of the equilibrium constants and stoichiometry, etc.     

Exploring purine N7 interactions via atomic mutagenesis: the group I ribozyme as a case study

Atomic mutagenesis has emerged as a powerful tool to unravel specific interactions in complex RNA molecules. An early extensive study of analogs of the exogenous guanosine nucleophile in group I intron self-splicing by Bass and Cech demonstrated structure-function relationships analogous to those seen for protein ligands and provided strong evidence for a well-formed substrate binding site made of RNA. Subsequent functional and structural studies have confirmed these interacting sites and extended our understanding of them, with one notable exception. Whereas 7-methyl guanosine did not affect reactivity in the original study, a subsequent study revealed a deleterious effect of the seemingly more conservative 7-deaza substitution. Here we investigate this paradox, studying these and other analogs with the more thoroughly characterized ribozyme derived from the Tetrahymena group I intron. We found that the 7-deaza substitution lowers binding by ~20-fold, relative to the cognate exogenous guanosine nucleophile, whereas binding and reaction with 7-methyl and 8-aza-7-deaza substitutions have no effect. These and additional results suggest that there is no functionally important contact between the N7 atom of the exogenous guanosine and the ribozyme. Rather, they are consistent with indirect effects introduced by the N7 substitution on stacking interactions and/or solvation that are important for binding. The set of analogs used herein should be valuable in deciphering nucleic acid interactions and how they change through reaction cycles for other RNAs and RNA/protein complexes.     

Human milk oligosaccharides: the novel modulator of intestinal microbiota

Human milk, which nourishes the early infants, is a source of bioactive components for the infant growth, development and commensal formulation as well. Human milk oligosaccharide is a group of complex and diverse glycans that is apparently not absorbed in human gastrointestinal tract. Although most mammalian milk contains oligosaccharides, oligosaccharides in human milk exhibit unique features in terms of their types, amounts, sizes, and functionalities. In addition to the prevention of infectious bacteria and the development of early immune system, human milk oligosaccharides are able to facilitate the healthy intestinal microbiota. Bifidobacterial intestinal microbiota appears to be established by the unilateral interaction between milk oligosaccharides, human intestinal activity and commensals. Digestibility, membrane transportation and catabolic activity by bacteria and intestinal epithelial cells, all of which are linked to the structural of human milk oligosaccharides, are crucial in determining intestinal microbiota. [BMB Reports 2012; 45(8): 433-441].     

Ranking the selectivity of PubChem screening hits by activity-based protein profiling: MMP13 as a case study

High-throughput screening (HTS) has become an integral part of academic and industrial efforts aimed at developing new chemical probes and drugs. These screens typically generate several 'hits', or lead active compounds, that must be prioritized for follow-up medicinal chemistry studies. Among primary considerations for ranking lead compounds is selectivity for the intended target, especially among mechanistically related proteins. Here, we show how the chemical proteomic technology activity-based protein profiling (ABPP) can serve as a universal assay to rank HTS hits based on their selectivity across many members of an enzyme superfamily. As a case study, four metalloproteinase-13 (MMP13) inhibitors of similar potency originating from a publically supported HTS and reported in PubChem were tested by ABPP for selectivity against a panel of 27 diverse metalloproteases. The inhibitors could be readily separated into two groups: (1) those that were active against several metalloproteases and (2) those that showed high selectivity for MMP13. The latter set of inhibitors was thereby designated as more suitable for future medicinal chemistry optimization. We anticipate that ABPP will find general utility as a platform to rank the selectivity of lead compounds emerging from HTS assays for a wide variety of enzymes.     

Analysis and role of oligosaccharides in milk

Milk is an important fluid in glycobiology because it contains a number of short carbohydrate chains either free or as glycoconjugates. These compounds as a class are the most abundant component and benefit the infant by developing and maintaining the infant's gut flora. New and emerging methods for oligosaccharide analysis have been developed to study milk. These methods allow for the rapid profiling of oligosaccharide mixtures with quantitation. With these tools, the role of oligosaccharide in milk is being understood. They further point to how oligosaccharide analysis can be performed, which until now has been very difficult and have lagged significantly those of other biopolymers. [BMB Reports 2012; 45(8): 442-451].     

Characterization of oligosaccharides in milk of a mink, Mustela vison

Carbohydrates were extracted from a sample of milk from a mink, Mustela vison (Family Mustelidae). Free neutral and acidic oligosaccharides were isolated from the carbohydrate fraction and their chemical structures were compared with those of white-nosed coati (Nasua narica, Procyonidae) and harbour seal (Phoca vitulina, Phocidae) that we had studied previously. The ratio of free lactose to milk oligosaccharides was similar to that in milk of the white-nosed coati; in both species, this ratio was much lower than that in the milk of most eutherians. The neutral oligosaccharides of mink milk had alpha(1-3)-linked Gal or alpha(1-2)-linked Fuc residues at their non-reducing ends, as in the neutral oligosaccharides of white-nosed coati milk. Some of the neutral and acidic oligosaccharides, determined here, had been found also in harbour seal milk, but the harbour seal oligosaccharides did not contain alpha(1-3)-linked Gal residues.     

Caenorhabditis elegans as a host for the study of host-pathogen interactions

Recently, pathogenicity models that involve the killing of the genetically tractable nematode Caenorhabditis elegans by human pathogens have been developed. From the perspective of the pathogen, the advantage of these models is that thousands of mutagenized bacterial clones can be individually screened for avirulent mutants on separate petri plates seeded with C. elegans. The advantages of using C. elegans to study host responses to pathogen attack are the extensive genetic and genomic resources available and the relative ease of identifying C. elegans mutants that exhibit altered susceptibility to pathogen attack. The use of Caenorhabditis elegans as the host for a variety of human pathogens is discussed.     

GFP technology for the study of biocontrol agents in tritrophic interactions: a case study with Pseudozyma flocculosa

GFP technology was applied to the biocontrol agent (BCA) Pseudozyma flocculosa to study its development and interactions at the tritrophic level plant-powdery mildew-BCA. Transformation experiments with GFP led to the production of a strongly fluorescent strain, Act-4, that displayed biocontrol traits typical of P. flocculosa WT. Following inundative applications, growth of P. flocculosa Act-4 was closely and almost exclusively associated with the colonies of the pathogen regardless of the powdery mildew species or the host plant tested. Development of P. flocculosa Act-4 on control leaves alone was extremely limited 24 h after its application and was typical of the epiphytic growth characterizing this type of yeast-like fungus. Based on the strong correlation between the colonization pattern of the different powdery mildew species tested and the presence of P. flocculosa Act-4, as determined by its fluorescence, it seems that growth of the BCA is dependant on the presence of powdery mildews. These results demonstrate that the GFP technology can be used to study plant-pathogen-BCA interactions and fulfill a wide array of purposes ranging from fundamental observations of the biocontrol behavior of a BCA to very applied ones serving some of the requirements for the registration of BCA's such as defining their environmental fate.     

Iron dynamics in the rhizosphere as a case study for analyzing interactions between soils,plants and microbes

Plant and Soil - Iron is an essential element for plants and microbes. However, in most cultivated soils, the concentration of iron available for these living organisms is very low because its...     

Chitosan as a lipid binder: a langmuir monolayer study of chitosan-lipid interactions

Owing to its distinct chemico-biological properties, chitosan, a cationic biopolymer, offers a great potential in multifarious bioapplications. One such application is as a dietary antilipidemic supplement to be used to reduce obesity/overweight and to lower cholesterol. The lipid-binding efficiency of chitosan, however, remains debatable. Accordingly, in this study we investigated the interactions of chitosan with selected lipids, cholesterol and fatty acids, the latter including saturated (stearic acid) and unsaturated (oleic, linoleic, alpha-linolenic) acids. The experiments were performed with the Langmuir monolayer technique, in which surface pressure-area isotherms were recorded for the lipid monolayers spread on the acetate buffer pH 4.0 subphase in the absence and presence of chitosan. We found that the presence of chitosan in the subphase strongly influenced the shape and location of the isotherms, proving that there existed attractions between chitosan and lipid molecules. The attractions were revealed by changes of the molecular organization of the monolayers. The common feature of these changes was that all the monolayers studied underwent expansion, in each case reaching saturation with increasing chitosan concentration. In agreement with the lipid molecular structures, the highest expansions were observed for the most unsaturated fatty acids, linoleic and alpha-linolenic, the lowest for stearic acid, with oleic acid and cholesterol being the intermediate cases. By contrast, the main distinguishing feature of these changes was that, although none of the monolayers studied changed its state when completely saturated with chitosan, compared to the parent ones the compactness of the monolayers was modified. The solid monolayers of stearic acid and cholesterol were loosened, whereas those of all the unsaturated acids, liquid in nature, were tightened. On the basis of these results we tentatively propose a mechanism of the chitosan action that includes both electrostatic and hydrophobic lipid-chitosan interactions as well as hydrogen bonding between them.     

Human milk oligosaccharides promote the growth of staphylococci

Human milk oligosaccharides (HMO), which constitute a major component of human milk, promote the growth of particular bacterial species in the infant's gastrointestinal tract. We hypothesized that HMO also interact with the bacterial communities present in human milk. To test this hypothesis, two experiments were conducted. First, milk samples were collected from healthy women (n = 16); culture-independent analysis of the bacterial communities was performed, HMO content was analyzed, and the relation between these factors was investigated. A positive correlation was observed between the relative abundance of Staphylococcus and total HMO content (r = 0.66). In a follow-up study, we conducted a series of in vitro growth curve experiments utilizing Staphylococcus aureus or Staphylococcus epidermidis and HMO isolated from human milk. HMO exhibited stimulatory effects on bacterial growth under various nutritional conditions. Analysis of culture supernatants from these experiments revealed that HMO did not measurably disappear from the culture medium, indicating that the growth-enhancing effects were not a result of bacterial metabolism of the HMO. Instead, stimulation of growth caused greater utilization of amino acids in minimal medium. Collectively, the data provide evidence that HMO may promote the growth of Staphylococcus species in the lactating mammary gland.     

Proton-nuclear magnetic resonance study of peracetylated derivatives of ten oligosaccharides isolated from human milk

Proton-nuclear magnetic resonance (NMR) spectra of peracetylated derivatives of ten structurally related oligosaccharides isolated from human milk were measured for solutions in CDCl3 at 360 MHz. The following oligosaccharides were investigated: Gal beta 1 leads to 4Glc-ol (1), GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc-ol (2), Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc-ol (3), Gal beta 1 leads to 3GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc-ol (4), Gal beta 1 leads to 3GlcNAc(4 comes from 1Fuc alpha) beta 1 leads to 3Gal beta 1 leads to 4Glc-ol (5), Fuc alpha 1 leads to 2Gal beta 1 leads to 3GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc-ol (6), Fuc alpha 1 leads to 2Gal beta 1 leads to 3GlcNAc(4 comes from 1Fuc alpha)beta 1 leads to 3Gal beta 1 leads to 4Glc-ol (7), Fuc alpha 1 leads to 2Gal beta 1 leads to 4Glc-ol(3 comes from 1Fuc alpha) (8), and a 1:3 mixture of Fuc alpha 1 leads to 2Gal beta 1 leads to 4Glc-ol (9) and Gal beta 1 leads to 4Glc-ol(3 comes from 1Fuc alpha) (10). Owing to the strong downfield shifts of the resonances of protons linked to acetoxylated carbons, the problems of signal overlap are less severe and the spin systems of all constituent sugar residues can be assigned fully. The sites of glycosidic linkage can be recognized by the high-field position of the signals of protons linked to those sites; for example, type 1 (Gal beta 1 leads to 3GlcNAc) and type 2(Gal beta 1 leads to 4GlcNAc) saccharide chains can be distinguished. The sequence can be established by observing a nuclear Overhauser effect involving the anomomeric and the aglyconic proton.     

The interactions of neuropeptides with membrane model systems: a case study.

The interactions between the positively charged neuropeptides substance P (SP), bradykinin (BK), and zwitterionic Met-enkephalin (ME) neuropeptides, and negatively charged SDS and zwitterionic lysophosphatidylcholine (LPC) membrane model systems, have been investigated using one- and two-dimensional nmr experiments. Proton longitudinal relaxation studies were used to characterize these interactions as intrinsic or extrinsic. An extrinsic interaction are similar to those observed for extrinsic membrane proteins. An intrinsic interaction are similar to those observed for intrinsic membrane proteins, and would require that the hydrophobic residues penetrate or insert into the hydrophobic core of the membrane. The interactions between both SP and BK and SDS, based on nmr results, may be characterized as intrinsic, and the interaction between ME and SDS may be characterized as extrinsic. Two-dimensional nuclear Overhauser enhancement spectroscopy experiments proved the insertion of the phenylalanine residues on both SP and BK into the hydrophobic core of SDS micelles. The interaction between SP and BK with LPC based on nmr results are characterized as extrinsic, with the interaction between ME and SDS characterized as weakly intrinsic.     

Chemiluminometric β-galactosidase detection as a basis for a tetracycline screening test in milk

The observation that tetracyclines inhibit the biosynthesis of β-galactosidase in Escherichia coli to a greater extent than other antibacterials was exploited for the development of a chemiluminometric method to detect residues of this class of antibiotics in milk. The procedure involves the incubation of a milk sample with 10⁷ CFU/ml of an E. coli strain in the presence of IPTG, an inducer of β-galactosidase, and of EGTA, a chelator of calcium ions, followed by a 1000-fold dilution and measurement of the residual enzymatic activity using the chemiluminogenic substrate Galacton. Chemiluminometry proved an essential tool in this procedure because the extensive dilution of the sample, necessary to avoid light quenching by turbidity, results in an insufficient level of β-galactosidase activity to be measurable by colorimetry. This tetracycline galactosidase (TG) test has been validated and compared in the field to existing commercial screening assays for antibiotics. Its detection limit for tetracyclines ranges between 40 and 65 μg/kg, which is below the European maximum residue limit (MRL = 100 μg/kg) in milk. No other antibacterials, at concentrations commonly expected in milk, were found to interfere with the TG test. Strategies to avoid false positive reactions possibly arising from very high somatic cell counts will be reported elsewhere. © 1998 John Wiley & Sons, Ltd.     

Human oral cavity as a model for the study of genome-genome interactions

The enormous diversity of culturable bacteria within the oral microbial community coupled with experimental accessibility renders the human oral cavity a valuable model to investigate genome-genome interactions. The complex interactions of oral bacteria result in the formation of biofilms on the surfaces of the oral cavity. One mechanism thought to be important in biofilm formation is the coaggregation of bacterial partners. In this paper, we examine the role of coaggregation in oral biofilms and develop protocols to elucidate the spatial organization of bacterial species retained within oral biofilms. To explore these issues, we have employed two experimental systems: the saliva-coated flowcell and the retrievable enamel chip. From flowcell studies, we have determined that coaggregation can greatly influence the ability of an oral bacterial species to grow and be retained within the developing biofilm. To examine the spatial architecture of oral biofilms, fluorescent in situ hybridization protocols were developed that successfully target specific members of the oral microbial community. Together, these approaches provide insight into the development of oral biofilms and expand our understanding of genome-genome interactions.     

Dominant network interactions are not correlated with resource availability: a case study using mistletoe host interactions

Network theory in ecology has been central to understanding species co‐occurrence patterns, specialization and community stability. However, network theory has traditionally focused on the ‘higher’ trophic level where exploitation of network ‘partners’ (i.e. individual interactions in response to resource availability) have remained underappreciated. In this study we tested how clumping and host availability influenced mistletoe–host interactions in a semi‐arid woodland, central Australia. We used a hierarchical approach that evaluated individual interactions by modifying the traditional randomization technique to simulate clumping and host exploitation. Using published literature we then compared our results with mistletoes from other genera. We found that mistletoes clump on fewer trees than predicted, even though interaction strength was no different from random expectations, and we found no evidence that common trees were heavily infected as predicted by the host availability hypothesis. The rate of host exploitation (measured as the proportion of trees infected) in semi‐arid Australia is similar to that for mistletoe genera in other parts of the world. We hypothesize that specific host trees act as a focal point for infection that facilitates the spread and overall population size of mistletoes. Overall our results indicate that resources, such as the number of trees in a mistletoe network, are less important than clumping of individual plants. We suggest that exploitation of available resources may play a similar role in other networks that extend beyond antagonistic relationships such as parasite or herbivore interactions.