16S rRNA gene-based profiling of the human infant gut microbiota is strongly influenced by sample processing and PCR primer choice

Acknowledgements The authors acknowledge the assistance of Grietje Holtrop (RINH-BioSS) with the statistical analysis of the data and the Wellcome Trust Sanger Institute’s 454 pyrosequencing team for generating 16S rRNA gene data. AWW, PS and JP received core funding support from the Wellcome Trust [grant number 098051]. AWW, JCM, HJF and KPS are funded by the Scottish Government (SG-RESAS).Peer reviewedPublisher PD

Gastrointestinal presentation of kawasaki disease: A red flag for severe disease?

Background Kawasaki disease (KD) is a febrile systemic vasculitis of unknown etiology and the main cause of acquired heart disease among children in the developed world. To date, abdominal involvement at presentation is not recognized as a risk factor for a more severe form of the disease. Objective To evaluate whether presenting abdominal manifestations identify a group at major risk for Intravenous immunoglobulin (IVIG)-resistance and coronary lesions. Methods Retrospective study of KD patients diagnosed between 2000 and 2015 in 13 pediatric units in Italy. Patients were divided into 2 groups according to the presence or absence of abdominal manifestations at onset. We compared their demographic and clinical data, IVIG-responsiveness, coronary ectasia/aneurysms, laboratory findings from the acute and subacute phases. Results 302 patients (181 boys) were enrolled: 106 patients with, and 196 patients without presenting abdominal features. Seasonality was different between the groups (p = 0.034). Patients with abdominal manifestations were younger (p = 0.006) and more frequently underwent delayed treatment (p = 0.014). In the acute phase, patients with abdominal presentation had higher platelet counts (PLT) (p = 0.042) and lower albuminemia (p = 0.009), while, in the subacute phase, they had higher white blood cell counts (WBC) and PLT (p = 0.002 and p < 0.005, respectively) and lower red blood cell counts (RBC) and hemoglobin (Hb) (p = 0.031 and p 0.009). Moreover, the above mentioned group was more likely to be IVIG-resistant (p < 0.005) and have coronary aneurysms (p = 0.007). In the multivariate analysis, presenting abdominal manifestations, age younger than 6 months, IVIG- resistance, delayed treatment and albumin concentration in the acute phase were independent risk factors for coronary aneurysms (respectively p<0.005, <0.005, = 0.005 and 0.009). Conclusions This is the first multicenter report demonstrating that presenting gastrointestinal features in KD identify patients at higher risk for IVIG-resistance and for the development of coronary aneurysms in a predominantly Caucasian population

Serum HMGB1 levels are independently associated with glucose clamp-derived measures of insulin resistance in women with PCOS

Purpose: PCOS is associated with low grade inflammation which could play a role in insulin resistance and ovarian dysfunction. Preliminary findings suggested that serum levels of HMGB1, a cytokine involved in inflammation, might be altered in women with PCOS. Primary aim of this study was to assess whether HMGB1 serum concentrations are associated with PCOS and with the state of insulin resistance of these women. Methods: Sixty women with PCOS, selected to have a similar proportion of subjects with altered or normal insulin sensitivity, and 29 healthy controls were studied. Serum HMGB1 levels were compared in subgroups of PCOS women and controls. In PCOS women, insulin sensitivity was assessed by the glucose clamp technique and HMGB1 was measured at baseline and after acute hyperinsulinemia. Results: HMGB1 levels were similar in women with PCOS and controls and no elements used for diagnosing PCOS were associated with serum HMGB1. However, HMGB1 concentrations were higher in insulin-resistant vs insulin-sensitive PCOS women (p = 0.017), and inversely associated with insulin-induced total and non-oxidative glucose metabolism. In both subgroups of PCOS women, serum HMBG1 levels significantly increased after acute hyperinsulinemia. Conclusions: These data suggest that HMGB1 levels are not associated with PCOS per se, but with insulin resistance. Further research should establish the underlying nature of this relationship, and whether this protein might play a role in the metabolic complications of PCOS

First foods and gut microbes

The establishment of the human gut microbiota in early life has been associated with later health and disease. During the 1st months after birth, the microbial composition in the gut is known to be affected by the mode of delivery, use of antibiotics, geographical location and type of feeding (breast/formula). Consequently, the neonatal period and early infancy has attracted much attention. However, after this first period the gut microbial composition continues to develop until the age of 3 years, and these 1st years have been designated “a window of opportunity” for microbial modulation. The beginning and end of this window is currently debated, but it likely coincides with the complementary feeding period, marking the gradual transition from milk-based infant feeding to family diet usually occurring between 6 and 24 months. Furthermore, the ‘first 1000 days,’ i.e., the period from conception until age 2 years, are generally recognized to be of particular importance for the healthy development of children. While dietary changes are known to affect the adult gut microbiota, there is a gap in our knowledge on how the introduction of new dietary components into the diet of infants/young children affects the gut microbiota development. This perspective paper summarizes the currently very few studies addressing the effects of complementary diet on gut microbiota, and highlights the recent finding that transition to family foods greatly impacts the development of gut microbial diversity. Further, we discuss potential impacts on child health and the need for further studies on this important topic

Tracking the oxidative kinetics of carbohydrates, amino acids and fatty acids in the house sparrow using exhaled \u3csup\u3e13\u3c/sup\u3eCO\u3csub\u3e2\u3c/sub\u3e

Clinicians commonly measure the 13CO2 in exhaled breath samples following administration of a metabolic tracer (breath testing) to diagnose certain infections and metabolic disorders. We believe that breath testing can become a powerful tool to investigate novel questions about the influence of ecological and physiological factors on the oxidative fates of exogenous nutrients. Here we examined several predictions regarding the oxidative kinetics of specific carbohydrates, amino acids and fatty acids in a dietary generalist, the house sparrow (Passer domesticus). After administering postprandial birds with 20 mg of one of seven 13C-labeled tracers, we measured rates of 13CO2 production every 15 min over 2 h. We found that sparrows oxidized exogenous amino acids far more rapidly than carbohydrates or fatty acids, and that different tracers belonging to the same class of physiological fuels had unique oxidative kinetics. Glycine had a mean maximum rate of oxidation (2021 nmol min−1) that was significantly higher than that of leucine (351 nmol min−1), supporting our prediction that nonessential amino acids are oxidized more rapidly than essential amino acids. Exogenous glucose and fructose were oxidized to a similar extent (5.9% of dose), but the time required to reach maximum rates of oxidation was longer for fructose. The maximum rates of oxidation were significantly higher when exogenous glucose was administered as an aqueous solution (122 nmol min−1), rather than as an oil suspension (93 nmol min−1), supporting our prediction that exogenous lipids negatively influence rates of exogenous glucose oxidation. Dietary fatty acids had the lowest maximum rates of oxidation (2-6 nmol min−1), and differed significantly in the extent to which each was oxidized, with 0.73%, 0.63% and 0.21% of palmitic, oleic and stearic acid tracers oxidized, respectively

Seafood inclusion ion early years' feeding : a comparison of commercial products to home-cooking

Background and Aims Under-exposure to seafood during early years feeding, when taste and food acceptance is developed, may impact on the future development of a healthy diet. The aim of this study was to investigate the inclusion of seafood in commercial baby food products and baby and toddler cookbooks, and the occurrence of beneficiary and cautionary information on seafood in the cookbooks. Methods A survey was conducted of all commercial pre-prepared baby food main-meal products in Scotland from September-December 2012. The primary food type within each product, (vegetables, poultry, meat, and seafood), nutritional composition, and ingredient contribution were collected. A survey of Amazon’s top 20 best-selling baby and toddler cookbooks was conducted in June 2013. The types and varieties of the different food types cited in addition to recipes, beneficiary claims and cautionary information was recorded. Results Seafood (n=13 (3.8%)) was significantly underrepresented as a main-meal product compared to poultry (103 (30.2%)), meat (121 (35.5%)) and vegetables (104 (30.5%)). Similarly, seafood-based main-meal recipes were significantly lower than vegetable recipes however were not significantly different to poultry and meat recipes. Cautionary claims in the cookbooks were significantly higher for seafood than other food types. Conclusions Parents who predominantly wean their infant using commercial products are may face challenges in sourcing a suitable range of products to enable the inclusion of seafood. Parents who predominantly home-cook have greater exposure to seafood in recipes however, this may be counteracted by the prominence of negative seafood messages, deterring them from including this healthful food into the diet of their infant.Publisher PDFNon peer reviewe

Site, rate and extent of starch digestion in weaning infants

BACKGROUND The colon is believed to salvage energy from unabsorbed starch especially when the capacity of the small intestine to digest it is limited. The extent to which this occurs is not known.AIMS The aim of this thesis was to determine site and relative extent of starch digestion and fermentation in young children using the individual and combined approaches of stable isotope breath tests and in vitro stool fermentation models.STABLE ISOTOPE BREATH TEST METHODS Thirteen children (10m, 3f), median (range) age 11.8 mo (7.6 -22.7 mo), took a starchy breakfast containing ¹³C labelled wheat flour following an overnight fast. Duplicate breath samples were obtained before breakfast and every 30 min for 12 h. Breath ¹³CO₂ enrichment was measured by isotope ratio mass spectrometry and results were expressed as percentage dose recovered (PDR) for each 30 min. PDR data were analysed and mathematically curve fitted either assuming a constant estimate of CO₂ production rate or adjusted for physical activity.STABLE ISOTOPE BREATH TEST RESULTS Mean ± SD cumulative ¹³C PDR (cPDR) at 12 h was 21.3% ± 8.4% for unadjusted data and 26.5% ± 11.6% for adjusted data. A composite fit of two curves fitted significantly better than a single curve. Curve fitting allowed estimation of cPDRs of small intestine (17.5% ± 6.5% and 22.7% ± 9.3% for unadjusted and adjusted data respectively) and colon (4.6% ± 2.9% and 6.3% ± 5.4 %). From these results it is speculated that the colon may account for up to 20% of starch digestion in young children.IN VITRO COLONIC FERMENTATION METHODS A simulated colonic environment was used to account for the fate of raw and cooked starch that was fermented in the colon of young children. A slurry was prepared from faecal samples of 6 infants (7 - 10 mo), 6 toddlers (16 - 21 mo) and 7 adults (24 - 56 years). Each slurry was anaerobically incubated with raw or cooked maize starch in MacCartney bottles in a shaking water bath. Parallel incubations were stopped at 4 and 24 h. The headspace gas volume was analysed for CO₂ and methane. The culture supernatant was analyzed for the volatile short chain fatty acids acetate, propionate and butyrate (SCFA), lactate and residual starch.IN VITRO COLONIC FERMENTATION RESULTS There was a decreasing trend of SCFA production with age at 4 h which was not evident at 24 h. At 4 h, toddler stools produced the most CO₂ followed by infants and then adults, but this trend was not seen at 24 h. Methane was detected in 3 adults only. Lactate was detected mainly at 4 h in children only. The production of SCFA at 4 h generally declined with age but the differences at 24 h were less marked, suggesting fermentation is a more rapid process in young children than in adults. A highly efficient energy salvage process may take place in the colon of young children.CALCULATIONS USING BOTH DATA SETS AND CONCLUSIONS Using data from studies described in both parts of the dissertation, it has been possible to derive stoichiometric equations for the whole gut digestion of starch, and thereby calculate its potential energy. There are a number of limitations to the methodology and from assumptions that have been made, but this provides an attractive means to calculate relative roles of small intestine and colon to starch digestion in young children which in turn may form the scientific basis for nutritional advice given to mothers

Vectorial targeting of an endogenous apical membrane sialoglycoprotein and uvomorulin in MDCK cells.

We studied the cell-surface delivery pathways of newly synthesized membrane glycoproteins in MDCK cells and for this purpose we characterized an endogenous apical integral membrane glycoprotein. By combining a pulse-chase protocol with domain-selective cell-surface biotinylation, immune precipitation, and streptavidin-agarose precipitation (Le Bivic et al. 1989. Proc. Natl. Acad. Sci USA. 86:9313-9317), we followed the appearance at the cell surface of a major apical sialoglycoprotein, gp114, a basolateral protein, uvomorulin, and a transcytosing protein, the polyimmunoglobulin receptor (pIg-R). We determined that both gp114 and uvomorulin appeared to be delivered directly to their respective surface, with mistargeting levels of 8 and 2%, respectively. Using the same technique, the pIg-R was first detected on the basolateral domain and then on the apical domain, to be finally released into the apical medium, as described (Mostov, K. E., and D. L. Deitcher. 1986. Cell. 46:613-621). To directly determine whether the gp114 pool present on the basolateral surface was a precursor of the apical gp114, we compared it with the equivalent pIg-R pool, by labeling with sulfo-NHS-SS-biotin, a cleavable, tight junction-impermeable probe, and by following the fraction of this probe that became resistant to basal glutathione and accessible to apical glutathione during incubation at 37 degrees C. We found that, contrary to pIg-R, basolateral gp114 was poorly endocytosed and was not transcytosed to the apical side. These results demonstrate that an endogenous apical integral membrane glycoprotein of Madin-Darby canine kidney cells is sorted intracellularly and is vectorially targeted to the apical surface