Establishment of Intestinal Microbiota during Early Life: a Longitudinal,Explorative Study of a Large Cohort of Danish Infants

Fecal samples were obtained from a cohort of 330 healthy Danish infants at 9, 18, and 36 months after birth, enabling characterization of interbacterial relationships by use of quantitative PCR targeting 31 selected bacterial 16S rRNA gene targets representing different phylogenetic levels. Nutritional parameters and measures of growth and body composition were determined and investigated in relation to the observed development in microbiota composition. We found that significant changes in the gut microbiota occurred, particularly from age 9 to 18 months, when cessation of breastfeeding and introduction of a complementary feeding induce replacement of a microbiota characterized by lactobacilli, bifidobacteria, and Enterobacteriaceae with a microbiota dominated by Clostridium spp. and Bacteroides spp. Classification of samples by a proxy enterotype based on the relative levels of Bacteroides spp. and Prevotella spp. showed that enterotype establishment occurs between 9 and 36 months. Thirty percent of the individuals shifted enterotype between 18 and 36 months. The composition of the microbiota was most pronouncedly influenced by the time of cessation of breastfeeding. From 9 to 18 months, a positive correlation was observed between the increase in body mass index and the increase of the short-chain-fatty-acid-producing clostridia, the Clostridum leptum group, and Eubacterium hallii. Considering previously established positive associations between rapid infant weight gain, early breastfeeding discontinuation, and later-life obesity, the corresponding microbial findings seen here warrant attention.     

Foraging ecology and spatial behaviour of the red fox (Vulpes vulpes) in a wet grassland ecosystem

We investigated diet composition, habitat selection and spatial behaviour of the red fox (Vulpes vulpes) in relation to the availability of wader nests in a coastal polder area in southwest Denmark. The predatory role of the red fox in wet grassland ecosystems has profound implications for conservation status of declining populations of grassland breeding waders. However, few studies have focussed on the foraging ecology and behaviour of the red fox in these landscapes. Faecal analyses revealed that fox diet consisted of birds (43 % of prey remains?/?32 % of biomass), rodents (39 %?/?21 %), sheep (mainly as carrion, 14 %?/?41 %) and lagomorphs (4 %?/?7 %). Charadriiformes (including waders) comprised 3–12 % of prey remains throughout the year. Telemetry data and spotlight counts indicated that foxes did not select areas with high densities of breeding waders, suggesting that foxes did not target wader nests while foraging. Foxes maintained stable home ranges throughout their lives, indicating that the area sustained a permanent fox population all year round. The population densities, estimated from spotlight surveys, were 0.74 visible foxes km^?2 (95 % CI; 0.34–1.61) on the preferred breeding habitat for waders and 1.21 km^?2 in other open habitats such as cultivated fields. Our results indicate that red fox predation on wader nests is incidental, consistent with the notion that red foxes are generalist predators that opportunistically subsist on many prey groups.     

Characterization of an immunodominant cancer-specific O-glycopeptide epitope in murine podoplanin (OTS8)

Auto-antibodies induced by cancer represent promising sensitive biomarkers and probes to identify immunotherapeutic targets without immunological tolerance. Surprisingly few epitopes for such auto-antibodies have been identified to date. Recently, a cancer-specific syngeneic murine monoclonal antibody 237, developed to a spontaneous murine fibrosarcoma, was shown to be directed to murine podoplanin (OTS8) with truncated Tn O-glycans. Our understanding of such cancer-specific auto-antibodies to truncated glycoforms of glycoproteins is limited. Here we have investigated immunogenicity of a chemoenzymatically produced Tn-glycopeptide derived from the putative murine podoplanin O-glycopeptide epitope. We found that the Tn O-glycopeptide was highly immunogenic in mice and produced a Tn-glycoform specific response with no reactivity against unglycosylated peptides or the O-glycopeptide with extended O-glycan (STn and T glycoforms). The immunodominant epitope was strictly dependent on the peptide sequence, required Tn at a specific single Thr residue (Thr⁷⁷), and antibodies to the epitope were not found in naive mice. We further tested a Tn O-glycopeptide library derived from human podoplanin by microarray analysis and demonstrated that the epitope was not conserved in man. We also tested human cancer sera for potential auto-antibodies to similar epitopes, but did not detect such antibodies to the Tn-library of podoplanin. The reagents and methods developed will be valuable for further studies of the nature and timing of induction of auto-antibodies to distinct O-glycopeptide epitopes induced by cancer. The results demonstrate that truncated O-glycopeptides constitute highly distinct antibody epitopes with great potential as targets for biomarkers and immunotherapeutics.     

Inhibition of plasminogen activator inhibitor-1 binding to endocytosis receptors of the low-density-lipoprotein receptor family by a peptide isolated from a phage display library

The functions of the serpin PAI-1 (plasminogen activator inhibitor-1) are based on molecular interactions with its target proteases uPA and tPA (urokinase-type and tissue-type plasminogen activator respectively), with vitronectin and with endocytosis receptors of the low-density-lipoprotein family. Understanding the significance of these interactions would be facilitated by the ability to block them individually. Using phage display, we have identified the disulfide-constrained peptide motif CFGWC with affinity for natural human PAI-1. The three-dimensional structure of a peptide containing this motif (DVPCFGWCQDA) was determined by liquid-state NMR spectroscopy. A binding site in the so-called flexible joint region of PAI-1 was suggested by molecular modelling and validated through binding studies with various competitors and site-directed mutagenesis of PAI-1. The peptide with an N-terminal biotin inhibited the binding of the uPA-PAI-1 complex to the endocytosis receptors low-density-lipoprotein-receptor-related protein 1A (LRP-1A) and very-low-density-lipoprotein receptor (VLDLR) in vitro and inhibited endocytosis of the uPA-PAI-1 complex in U937 cells. We conclude that the isolated peptide represents a novel approach to pharmacological interference with the functions of PAI-1 based on inhibition of one specific molecular interaction.     

Isotopic niche differs between seal and fish‐eating killer whales (Orcinus orca) in northern Norway

Ecological diversity has been reported for killer whales (Orcinus orca) throughout the North Atlantic but patterns of prey specialization have remained poorly understood. We quantify interindividual dietary variations in killer whales (n = 38) sampled throughout the year in 2017–2018 in northern Norway using stable isotopic nitrogen (δ¹⁵N: ¹⁵N/¹⁴N) and carbon (δ¹³C: ¹³C/¹²C) ratios. A Gaussian mixture model assigned sampled individuals to three differentiated clusters, characterized by disparate nonoverlapping isotopic niches, that were consistent with predatory field observations: seal‐eaters, herring‐eaters, and lumpfish‐eaters. Seal‐eaters showed higher δ¹⁵N values (mean ± SD: 12.6 ± 0.3‰, range = 12.3–13.2‰, n = 10) compared to herring‐eaters (mean ± SD: 11.7 ± 0.2‰, range = 11.4–11.9‰, n = 19) and lumpfish‐eaters (mean ± SD: 11.6 ± 0.2‰, range = 11.3–11.9, n = 9). Elevated δ¹⁵N values for seal‐eaters, regardless of sampling season, confirmed feeding at high trophic levels throughout the year. However, a wide isotopic niche and low measured δ¹⁵N values in the seal‐eaters, compared to that of whales that would eat solely seals (δ_N‐measured = 12.6 vs. δ_N‐expected = 15.5), indicated a diverse diet that includes both fish and mammal prey. A narrow niche for killer whales sampled at herring and lumpfish seasonal grounds supported seasonal prey specialization reflective of local peaks in prey abundance for the two fish‐eating groups. Our results, thus, show differences in prey specialization within this killer whale population in Norway and that the episodic observations of killer whales feeding on prey other than fish are a consistent behavior, as reflected in different isotopic niches between seal and fish‐eating individuals.     

Enhancing pollination is more effective than increased conventional agriculture inputs for improving watermelon yields

Agricultural practices to improve yields in small‐scale farms in Africa usually focus on improving growing conditions for the crops by applying fertilizers, irrigation, and/or pesticides. This may, however, have limited effect on yield if the availability of effective pollinators is too low. In this study, we established an experiment to test whether soil fertility, soil moisture, and/or pollination was limiting watermelon (Citrullus lanatus) yields in Northern Tanzania. We subjected the experimental field to common farming practices while we treated selected plants with extrafertilizer applications, increased irrigation and/or extra pollination in a three‐way factorial experiment. One week before harvest, we assessed yield from each plant, quantified as the number of mature fruits and their weights. We also assessed fruit shape since this may affect the market price. For the first fruit ripening on each plant, we also assessed sugar content (brix) and flesh color as measures of fruit quality for human consumption. Extra pollination significantly increased the probability of a plant producing a second fruit of a size the farmer could sell at the market, and also the fruit sugar content, whereas additional fertilizer applications or increased irrigation did not improve yields. In addition, we did not find significant effects of increased fertilizer or watering on fruit sugar, weight, or color. We concluded that, insufficient pollination is limiting watermelon yields in our experiment and we suggest that this may be a common situation in sub‐Saharan Africa. It is therefore critically important that small‐scale farmers understand the role of pollinators and understand their importance for agricultural production. Agricultural policies to improve yields in developing countries should therefore also include measures to improve pollination services by giving education and advisory services to farmers on how to develop pollinator‐friendly habitats in agricultural landscapes.     

Measurements of total and regional body composition in preschool children: A comparison of MRI,DXA, and anthropometric data

Objective:

There are clear sex differences in the distribution of visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) in adults, with males having more VAT and less SAT than females. This study assessed whether these differences between the sexes were already present in preschool children. It also evaluated which measures of body composition were most appropriate for assessing abdominal obesity in this age group.

Design and Methods:

One‐hundred and five children (57 boys and 48 girls) participated in the study. Body composition was measured using dual‐energy X‐ray absorptiometry (DXA). Weight, height, and waist circumference (WC) were also recorded. Magnetic resonance imaging (MRI) of the entire abdomen using sixteen 10‐mm‐thick T₁‐weighted slices was performed in a subgroup of 48 children (30 boys and 18 girls); SAT and VAT volumes were measured using semiautomated segmentation.

Results:

Boys had significantly more VAT than girls (0.17 versus 0.10 l, P < 0.001). Results showed that VAT correlated significantly with all measurements of anthropometry (P < 0.01) after adjusting for SAT and for total fat mass measured with DXA. The mean limits of agreement between DXA and MRI regarding truncal FM were calculated to be ?11.4 (range ?17.8 to ?3.6), using a Bland–Altman plot.

Conclusion:

Sex differences in adipose tissue distribution are apparent at an early age. MRI is the best method with which to study abdominal fat distribution in young children.

     

The unfolded protein response to endoplasmic reticulum stress in cultured astrocytes and rat brain during experimental diabetes

Oxidative-nitrosative stress and inflammatory responses are associated with endoplasmic reticulum (ER) stress in diabetic retinopathy, raising the possibility that disturbances in ER protein processing may contribute to CNS dysfunction in diabetics. Upregulation of the unfolded protein response (UPR) is a homeostatic response to accumulation of abnormal proteins in the ER, and the present study tested the hypothesis that the UPR is upregulated in two models for diabetes, cultured astrocytes grown in 25 mmol/L glucose for up to 4 weeks and brain of streptozotocin (STZ)-treated rats with diabetes for 1–7 months. Markers associated with translational blockade (phospho-eIF2α and apoptosis (CHOP), inflammatory response (inducible nitric oxide synthase, iNOS), and nitrosative stress (nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase, GAPDH) were not detected in either model. Nrf2 was present in nuclei of low- and high-glucose cultures, consistent with oxidative stress. Astrocytic ATF4 expression was not altered by culture glucose concentration, whereas phospho-IRE and ATF6 levels were higher in low- compared with high-glucose cultures. The glucose-regulated chaperones, GRP78 and GRP94, were also expressed at higher levels in low- than high-glucose cultures, probably due to recurrent glucose depletion between feeding cycles. In STZ-rat cerebral cortex, ATF4 level was transiently reduced at 4 months, and p-IRE levels were transiently elevated at 3 months. However, GRP78 and GRP94 expression was not upregulated, and iNOS, amyloid-β, and nuclear accumulation of GAPDH were not evident in STZ-diabetic brain. High-glucose cultured astrocytes and STZ-diabetic brain are relatively resistant to diabetes-induced ER stress, in sharp contrast with cultured retinal Müller cells and diabetic rodent retina.