Effects of addition of 2-fucosyllactose to infant formula on growth and specific pathways of utilization by Bifidobacterium in healthy term infants.

Oligosaccharides in human milk support health via intestinal microbiome. We studied effects of addition of 2-fucosyllactose (2'FL) to the infant formula on infant growth, occurrence of adverse events (AE), and infant microbiome, including expression of microbial genes that metabolize 2'FL. Our hypothesis was that while 2'FL would not affect growth, it would cause changes in microbiome metabolism. In a double-blinded randomized controlled study fashion, the infant formula ± 2'FL or human milk was fed to healthy term infants for 16 weeks. Fecal samples obtained at baseline and week 16 were analyzed for microbial populations, metagenomic species concept (MGS), and genetics of gut metabolic modules (GMMs). There were no effects of addition of 2'FL on growth or AEs. There were no significant differences by feeding group in MGS richness or Shannon diversity at baseline, but formula groups each had significantly greater richness (p < 0.05) and diversity (p < 0.05) after 16 weeks of feeding than the breastfed group. While two glycosyl hydrolase (GH) families (GH42 and GH112) were significantly increased, two other GH families (GH20 and GH2) were significantly decreased in the test formula group compared to the control formula group; although modest, addition of 2'FL resulted in changes in microbiome in the direction of breastfed infants, consistent with internal metabolism of HMOs by Bifidobacterium.

Determination of Casein Allergens in Extensively Hydrolyzed Casein Infant Formula by Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: The use of hypoallergenic infant formulas and the need for reliable tests to determine the presence of residual antigens have increased in parallel. OBJECTIVE: An LC-MS method for quantitation of casein was validated using incurred samples and a matrix-matched external standard curve. METHOD: Powdered infant formula samples were extracted in a buffer of sodium deoxycholate and ammonium bicarbonate at 60°C and filtered through 7 kDa desalting columns. Samples were digested overnight with trypsin and precipitated with acid prior to analysis of marker peptides by tandem mass spectrometry. RESULTS: Based on three marker peptides, the linear range for casein was 1.8-42 µg/g of powdered infant formula with an LOQ of 1.8 µg/g. The determination coefficients (R2) for each curve were ≥0.99 for casein peptides. Method repeatability was ≤22% RSD and intermediate precision was ≤23% RSD; recovery of casein from incurred material (2-20 µg/g) ranged from 78% to 118%. CONCLUSIONS: An LC-MS/MS method was developed and validated for confirmation of casein allergens in hypoallergenic infant formula. HIGHLIGHTS: A method was developed to accurately and reliably quantify casein allergens in extensively hydrolyzed casein infant formula by LC-MS without the need for custom peptide standards.

Modifications of human histone H3 variants during mitosis.

Phosphorylation of histone H3 is a hallmark event in mitosis and is associated with chromosome condensation. Here, we use a combination of immobilized metal affinity chromatography and tandem mass spectrometry to characterize post-translational modifications associated with phosphorylation on the N-terminal tails of histone H3 variants purified from mitotically arrested HeLa cells. Modifications observed in vivo on lysine residues adjacent to phosphorylated Ser and Thr provide support for the existence of the "methyl/phos", binary-switch hypothesis [Fischle, W., Wang, Y., and Allis, C. D. (2003) Nature 425, 475-479]. ELISA with antibodies selective for H3 at Ser10, Ser28, and Thr3 show reduced activity when adjacent Lys residues are modified. When used together, mass spectrometry and immunoassay methods provide a powerful approach for elucidation of the histone code and identification of histone post-translational modifications that occur during mitosis and other specific cellular events.

Characterization of phosphorylation sites on histone H1 isoforms by tandem mass spectrometry.

Histone H1 isoforms isolated from asynchronously grown HeLa cells were subjected to enzymatic digestion and analyzed by nano-flow reversed-phase high performance liquid chromatography (RP-HPLC) tandem mass spectrometry (MS/MS) on both quadrupole ion trap and linear quadrupole ion trap-Fourier transform ion cyclotron resonance mass spectrometers. We have observed all five major isoforms of histone H1 (H1.1, H1.2, H1.3, H1.4, and H1.5) as well as a lesser studied H1, isoform H1.X. MS/MS experiments confirmed N-terminal acetylation on all isoforms plus a single internal acetylation site. Immobilized metal affinity chromatography in combination with tandem mass spectrometry was utilized to identify 19 phosphorylation sites on the five major H1 isoforms plus H1.X. Fourteen of these phosphorylation sites were located on peptides containing the cyclin dependent kinase (CDK) consensus motif (S/T)-P-X-Z (where X is any amino acid and Z is a basic amino acid). Five phosphorylation sites were identified in regions that did not fit the consensus CDK motif. One of these phosphorylation sites was found on the serine residue on the H1.4 peptide KARKSAGAAKR. The adjacent lysine residue to the phosphoserine was also shown to be methylated. This finding raises the question of whether the hypothesized "methyl/phos" switch could be extended to linker histones, and not exclusive to core histones.

The enhancement of histone H4 and H2A serine 1 phosphorylation during mitosis and S-phase is evolutionarily conserved.

Histone phosphorylation has long been associated with condensed mitotic chromatin; however, the functional roles of these modifications are not yet understood. Histones H1 and H3 are highly phosphorylated from late G2 through telophase in many organisms, and have been implicated in chromatin condensation and sister chromatid segregation. However, mutational analyses in yeast and biochemical experiments with Xenopus extracts have demonstrated that phosphorylation of H1 and H3 is not essential for such processes. In this study, we investigated additional histone phosphorylation events that may have redundant functions to H1 and H3 phosphorylation during mitosis. We developed an antibody to H4 and H2A that are phosphorylated at their respective serine 1 (S1) residues and found that H4S1/H2AS1 are highly phosphorylated in the mitotic chromatin of worm, fly, and mammals. Mitotic H4/H2A phosphorylation has similar timing and localization as H3 phosphorylation, and closely correlates with the chromatin condensation events during mitosis. We also detected a lower level of H4/H2A phosphorylation in 5-bromo-2-deoxyuridine-positive S-phase cells, which corroborates earlier studies that identified H4S1 phosphorylation on newly synthesized histones during S-phase. The evolutionarily conserved phosphorylation of H4/H2A during the cell cycle suggests that they may have a dual purpose in chromatin condensation during mitosis and histone deposition during S-phase.

Histone methyltransferases direct different degrees of methylation to define distinct chromatin domains.

The functional significance of mono-, di-, and trimethylation of lysine residues within histone proteins remains unclear. Antibodies developed to selectively recognize each of these methylated states at histone H3 lysine 9 (H3 Lys9) demonstrated that mono- and dimethylation localized specifically to silent domains within euchromatin. In contrast, trimethylated H3 Lys9 was enriched at pericentric heterochromatin. Enzymes known to methylate H3 Lys9 displayed remarkably different enzymatic properties in vivo. G9a was responsible for all detectable H3 Lys9 dimethylation and a significant amount of monomethylation within silent euchromatin. In contrast, Suv39h1 and Suv39h2 directed H3 Lys9 trimethylation specifically at pericentric heterochromatin. Thus, different methylated states of H3 Lys9 are directed by specific histone methyltransferases to "mark" distinct domains of silent chromatin.