Maturation of White Adipose Tissue Function in C57BL/6j Mice From Weaning to Young Adulthood.

White adipose tissue (WAT) distribution and WAT mitochondrial function contribute to total body metabolic health throughout life. Nutritional interventions starting in the postweaning period may impact later life WAT health and function. We therefore assessed changes in mitochondrial density and function markers in WAT depots of young mice. Inguinal (ING), epididymal (EPI) and retroperitoneal (RP) WAT of 21, 42 and 98 days old C57BL/6j mice was collected. Mitochondrial density [citrate synthase (CS), mtDNA] and function [subunits of oxidative phosphorylation complexes (OXPHOS)] markers were analyzed, together with gene expression of browning markers (Ucp1, Cidea). mRNA of ING WAT of 21 and 98 old mice was sequenced to further investigate functional changes of the mitochondria and alterations in cell populations. CS levels decreased significantly over time in all depots. ING showed most pronounced changes, including significantly decreased levels of OXPHOS complex I, II, and III subunits and gene expression of Ucp1 (PN21-42 and PN42-98) and Cidea (PN42-98). White adipocyte markers were higher at PN98 in ING WAT. Analyses of RNA sequence data showed that the mitochondrial functional profile changed over time from "growth-supporting" mitochondria focused on ATP production (and dissipation), to more steady-state mitochondria with more diverse functions and higher biosynthesis. Mitochondrial density and energy metabolism markers declined in all three depots over time after weaning. This was most pronounced in ING WAT and associated with reduced browning markers, increased whitening and an altered metabolism. In particular the PN21-42 period may provide a time window to study mitochondrial adaptation and effects of nutritional exposures relevant for later life metabolic health.

Specific synbiotics in early life protect against diet-induced obesity in adult mice.

AIMS: The metabolic state of human adults is associated with their gut microbiome. The symbiosis between host and microbiome is initiated at birth, and early life microbiome perturbation can disturb health throughout life. Here, we determined how beneficial microbiome interventions in early life affect metabolic health in adulthood. METHODS: Postnatal diets were supplemented with either prebiotics (scGOS/lcFOS) or synbiotics (scGOS/lcFOS with Bifidobacterium breve M-16 V) until post-natal (PN) day 42 in a well-established rodent model for nutritional programming. Mice were subsequently challenged with a high-fat Western-style diet (WSD) for 8 weeks. Body weight and composition were monitored, as was gut microbiota composition at PN21, 42 and 98. Markers of glucose homeostasis, lipid metabolism and host transcriptomics of 6 target tissues were determined in adulthood (PN98). RESULTS: Early life synbiotics protected mice against WSD-induced excessive fat accumulation throughout life, replicable in 2 independent European animal facilities. Adult insulin sensitivity and dyslipidaemia were improved and most pronounced changes in gene expression were observed in the ileum. We observed subtle changes in faecal microbiota composition, both in early life and in adulthood, including increased abundance of Bifidobacterium. Microbiota transplantation using samples collected from synbiotics-supplemented adolescent mice at PN42 to age-matched germ-free recipients did not transfer the beneficial phenotype, indicating that synbiotics-modified microbiota at PN42 is not sufficient to transfer long-lasting protection of metabolic health status. CONCLUSION: Together, these findings show the potential and importance of timing of synbiotic interventions in early life during crucial microbiota development as a preventive measure to lower the risk of obesity and improve metabolic health throughout life.

Supramolecular structure of dietary fat in early life modulates expression of markers for mitochondrial content and capacity in adipose tissue of adult mice.

BACKGROUND: Previous studies have shown that early life nutrition can modulate the development of white adipose tissue and thereby affect the risk on obesity and metabolic disease later in life. For instance, postnatal feeding with a concept infant milk formula with large, phospholipid coated lipid droplets (Concept, Nuturis®), resulted in reduced adiposity in adult mice. The present study investigated whether differences in cell energy metabolism, using markers of mitochondrial content and capacity, may contribute to the observed effects. METHODS: C57Bl/6j male mice were exposed to a rodent diet containing the Concept (Concept) or standard (CTRL) infant milk formula from postnatal day 16 until postnatal day 42, followed by a western style diet challenge until postnatal day 98. Markers for mitochondrial content and capacity were analyzed in retroperitoneal white adipose tissue and gene expression of metabolic markers was measured in both retroperitoneal white adipose tissue and muscle tibialis (M. tibialis) at postnatal day 98. RESULTS: In retroperitoneal white adipose tissue, the Concept group showed higher citrate synthase activity and mitochondrial DNA expression compared to the CTRL group (p < 0.05). In addition, protein expression of mitochondrial cytochrome c oxidase subunit I of the oxidative phosphorylation pathway/cascade was increased in the Concept group compared to CTRL (p < 0.05). In the M. tibialis, gene expression of uncoupling protein 3 was higher in the Concept compared to the CTRL group. Other gene and protein expression markers for mitochondrial oxidative capacity were not different between groups. CONCLUSION: Postnatal feeding with large, phospholipid coated lipid droplets generating a different supramolecular structure of dietary lipids enhances adult gene and protein expression of specific mitochondrial oxidative capacity markers, indicative of increased substrate oxidation in white adipose tissue and skeletal muscle. Although functional mitochondrial capacity was not measured, these results may suggest that adaptations in mitochondrial function via early feeding with a more physiological structure of dietary lipids, could underlie the observed beneficial effects on later life adiposity.

Milk fat globule membrane coating of large lipid droplets in the diet of young mice prevents body fat accumulation in adulthood.

Epidemiological studies have demonstrated protective effects of breast-feeding on childhood obesity. Differences between human milk and infant milk formula (IMF) in dietary lipid structure may contribute to this effect. In our mouse model, feeding a diet containing large lipid droplets coated with phospholipids (PL) (Nuturis®; PL of milk fat globule membrane (MFGM) fraction origin) in early life protected against excessive body fat accumulation following a diet challenge in adult life. We now set out to determine the relevance of increased droplet size and/or MFGM lipid droplet coating to the observed anti-obesogenic effects in adult life. From day 16 to 42, male mouse pups were exposed to diets with small (S) or large (L) lipid droplets (0·3 v. 2·9 µm average mode diameter, respectively), either without MFGM or with MFGM coating around the lipid droplet, resulting in four groups: S (control diet), L, Scoating and Lcoating (Nuturis® IMF diet). Mice were subsequently challenged with a Western-style diet until dissection at postnatal day 98. A non-challenged group served as reference (REF). We repeatedly determined body composition between postnatal day 42 and 98. At day 98 plasma and gene expression measurements were performed. Only the Nuturis® IMF diet (Lcoating) in early life containing MFGM-coated large lipid droplets reduced body fat mass to a level comparable with the REF group. These data support the notion that the structural aspects of lipids in human milk, for example, both lipid droplet size as well as the MFGM coating, may contribute to its reported protective effect against obesity in later life.

Spatiotemporal progression of localized bacterial peritonitis before and after open abdomen lavage monitored by in vivo bioluminescent imaging.

BACKGROUND: Bacterial peritonitis is a life-threatening abdominal infection associated with high morbidity and mortality. The rat is a popular animal model for studying peritonitis and its treatment, but longitudinal monitoring of the progression of peritonitis in live animals has been impossible until now and thus required a large number of animals. Our objective was to develop a noninvasive in vivo imaging technique to monitor the spatiotemporal spread of bacterial peritonitis. METHODS: Peritonitis was induced in 8 immunocompetent male Wistar rats by placing fibrin clots containing 5x10(8) cells of both Bacteroides fragilis (American Type Tissue Culture [ATCC)] 25,285 and bioluminescent Escherichia coli Xen14. After 1 or 2 days, infected clots were removed and open abdomen lavage was performed. In vivo bioluminescent imaging was used to monitor the spread of peritonitis. RESULTS: Bioluminescent in vivo imaging showed an increase in the area of spread, and the number of E. coli tripled into the rat's abdominal cavity on day 1 after clot insertion; however, on day 2, encapsulation of the clot confined bacterial spread. Bioluminescent E. coli respread over the peritoneal cavity after lavage; within 10 days, however, in vivo imaging showed a decrease of 3-4 orders of magnitude in bacterial load. CONCLUSION: Bioluminescent in vivo imaging can be effectively used to monitor the spatiotemporal behavior of the peritonitis during 3 different stages of the disease process: initiation, treatment, and follow-up. Imaging allows researchers to repeatedly image the same animal, thereby reducing variability and providing greater confidence in determining treatment efficacies for therapeutic interventions using a small number of animals.

Synthesis and biological evaluation of asymmetric gramicidin S analogues containing modified D-phenylalanine residues.

The synthesis of new analogues of the cationic antimicrobial peptide gramicidin S, having a modified D-phenylalanine residue, their antibacterial properties against several gram positive and negative strains, as well as their hemolytic activity is reported.

Influence of a chitosan on oral bacterial adhesion and growth in vitro.

Generally, mechanical plaque control without chemical support is insufficient to prevent oral diseases, and an ongoing quest exists for new antimicrobials for use in oral healthcare. Chitosans are polycationic, naturally occurring antimicrobials that are rapidly finding their way into oral healthcare. In this study we determined the effects of pellicle treatment with chitosan on bacterial adhesion and growth. Chitosan caused a reduction in bacterial adhesion and was responsible for bacterial death upon contact compared with a buffer control. Exposure of adhering bacteria to a chitosan solution or a buffer control did not cause detachment, but the chitosan solution left a small proportion of the adhering bacteria alive. Growth after exposure to chitosan was similar to (Streptococcus mutans ATCC700610, Streptococcus oralis HM1, Streptococcus sobrinus HG1025) or less than (S. mutans NS, Actinomyces naeslundii HM1) that of the control, while biofilm viability after chitosan treatment was lower than that of the control, except for S. oralis HM1. Therefore, chitosan is a promising antimicrobial for use in oral healthcare.

Chitosan adsorption to salivary pellicles.

The salivary pellicle is a negatively charged protein film, to which oral bacteria readily adhere. Chitosans are cationic biomolecules with known antimicrobial properties that can be modified in different ways to enhance its antimicrobial activity. Here, we determined the changes in surface chemical composition using X-ray photoelectron spectroscopy (XPS), in hydrophobicity by analyzing water contact angles, in charge through measuring streaming potentials, and evaluated morphology using atomic force microscopy (AFM), of salivary pellicles upon adsorption of different chitosans. The adsorption of chitosans to pellicles was chemically evident from altered carbon functionalities and the presence of an N(1s) peak at 401.1 eV as a result of protonated amines in XPS. Chitosan adsorption made the pellicle (zeta potential of untreated pellicles 29 mV) positively charged and more hydrophobic. A chemically modified chitosan (CL) and an unmodified chitosan (UC) caused aggregation of adsorbed salivary proteins, and AFM revealed clumps of protein after treatment with these chitosans, yielding an increase in pellicle surface roughness from 5.1 nm to between 16.3 and 35.6 nm for CL and UC, respectively. In summary, chitosans have a clear tendency to adsorb to salivary pellicles with a profound effect on the surface properties of the pellicle. Therefore, chitosans may provide anchoring molecules to affix antimicrobials to pellicle surfaces.

Prevention of pin tract infection in external stainless steel fixator frames using electric current in a goat model.

Pin tract infections of external fixators used in orthopaedic reconstructive bone surgery are serious complications that can eventually lead to periostitis and osteomyelitis. In vitro experiments have demonstrated that bacteria adhering to stainless steel in a biofilm mode of growth detach under the influence of small electric currents, while remaining bacteria become less viable upon current application. Therefore, we have investigated whether a 100microA electric current can prevent signs of clinical infection around percutaneous pins, implanted in the tibia of goats. Three pins were inserted into the lateral right tibia of nine goats, of which one served for additional frame support. Two pins were infected with a Staphylococcus epidermidis strain of which one pin was subjected to electric current, while the other pin was used as control. Pin sites were examined daily. The wound electrical resistance decreased with worsening of the infection from a dry condition to a purulent stage. After 21 days, animals were sacrificed and the pins taken out. Infection developed in 89% of the control pin sites, whereas only 11% of the pin sites in the current group showed infection. These results show that infection of percutaneous pin sites of external fixators in reconstructive bone surgery can be prevented by the application of a small DC electric current.

Removal of two waterborne pathogenic bacterial strains by activated carbon particles prior to and after charge modification.

Waterborne diseases constitute a threat to public health despite costly treatment measures aimed at removing pathogenic microorganisms from potable water supplies. This paper compared the removal of Raoultella terrigena ATCC 33257 and Escherichia coli ATCC 25922 by negatively and positively charged types of activated carbon particles. Both strains display bimodal negative zeta-potential distributions in stabilized water. Carbon particles were suspended to an equivalent external geometric surface area of 700 cm2 in 250 mL of a bacterial suspension, with shaking. Samples were taken after different durations for plate counting. Initial removal rates were less elevated for the positively charged carbon particle than expected, yielding the conclusion that bacterial adhesion under shaking is mass-transport limited. After 360 min, however, the log-reduction of the more negatively charged R. terrigena in suspension was largest for the positively charged carbon particles as compared with the negatively charged ones, although conditioning in ultrapure or tap water of positively charged carbon particles for 21 days eliminated the favorable effect of the positive charge due to counterion adsorption from the water. Removal of the less negatively charged E. coli was less affected by aging of the (positively charged) carbon particles, confirming the role of electrostatic interactions in bacterial removal by activated carbon particles. The microporous, negatively charged coconut carbon performed less than the mesoporous, positively charged carbon particle prior to conditioning but did not suffer from loss of effect after conditioning in ultrapure or tap water.