Structure and Function of Bovine Whey Derived Oligosaccharides Showing Synbiotic Epithelial Barrier Protective Properties.

Commensal gut microbiota and probiotics have numerous effects on the host's metabolic and protective systems, which occur primarily through the intestinal epithelial cell interface. Prebiotics, like galacto-oligosaccharides (GOS) are widely used to modulate their function and abundance. However, important structure-function relations may exist, requiring a detailed structural characterization. Here, we detailed the structural characterization of bovine whey derived oligosaccharide preparations enriched with GOS or not, dubbed GOS-enriched milk oligosaccharides (GMOS) or MOS, respectively. We explore GMOS's and MOS's potential to improve intestinal epithelial barrier function, assessed in a model based on barrier disruptive effects of the Clostridioides difficile toxin A. GMOS and MOS contain mainly GOS species composed of ß1-6- and ß1-3-linked galactoses, and 3'- and 6'-sialyllactose. Both GMOS and MOS, combined with lactobacilli, like Lactobacillus rhamnosus (LPR, NCC4007), gave synergistic epithelial barrier protection, while no such effect was observed with Bifidobacterium longum (BL NCC3001), Escherichia coli (Nissle) or fructo-oligosaccharides. Mechanistically, for barrier protection with MOS, (i) viable LPR was required, (ii) acidification of growth medium was not enough, (iii) LPR did not directly neutralize toxin A, and (iv) physical proximity of LPR with the intestinal epithelial cells was necessary. This is the first study, highlighting the importance of structure-function specificity and the necessity of the simultaneous presence of prebiotic, probiotic and host cell interactions required for a biological effect.

Randomised clinical trial: Bifidobacterium lactis NCC2818 probiotic vs placebo, and impact on gut transit time, symptoms, and gut microbiology in chronic constipation.

BACKGROUND: Constipation is a prevalent gastrointestinal disorder. Patient dissatisfaction with prescribed medications is common, and there is need for alternative management strategies. Evidence shows that Bifidobacterium species may be beneficial in constipation. AIM: To investigate changes in physiological and clinical measures of gut function in patients with chronic constipation following the consumption of Bifidobacterium lactis NCC2818, compared to placebo. METHODS: Participants were randomised to a 4-week supplementation with B. lactis NCC2818 (1.5 x 1010 CFU/d) or placebo. Gut transit time was measured using a radio-opaque marker, while symptoms and quality of life were assessed using validated questionnaires. Gut microbiota composition was assessed using quantitative polymerase chain reaction. Analysis of covariance was used for normally distributed variables, and Mann-Whitney test for non-normally distributed variables. RESULTS: Seventy-five participants were randomised. There was no significant difference between the probiotic and placebo groups in gut transit time change from baseline to week 2 (-11.7 hours, SD 33.0 hours vs -12.9 hours, SD 33.6 hours; P = 0.863) or to week 4 (-20.4 hours, SD 32.5 h vs -8.7 hours, SD 33.8 hours; P = 0.103). There were also no improvements in stool output, symptoms, or quality of life. No differences were found in Bifidobacterium concentrations between the probiotic and placebo groups at week 4 (9.5 log10 /g dry faeces, SD 0.3 vs 9.4 log10 /g, SD 1.0; P = 0.509). CONCLUSIONS: Bifidobacterium lactis NCC2818 was not effective in the management of mild chronic constipation. This study highlights the importance of further studies and their publication to better understand the strain-specific effects of probiotics.

Short-Term Daily Intake of Polydextrose Fiber Does Not Shorten Intestinal Transit Time in Constipated Adults: A Randomized Controlled Trial.

Chronic constipation (CC) remains a common gastrointestinal (GI) disorder that conveys a substantial healthcare burden. Expert guidelines recommend increasing fiber intake, yet the clinical evidence to support this needs strengthening for specific fibers. The aim was to evaluate changes in intestinal transit time and GI symptoms in CC patients who consumed polydextrose. In a randomized, double-blind, placebo-controlled trial, 128 adults with CC received 8 g or 12 g polydextrose, or placebo, daily for 4 weeks. Transit time, as primary outcome, was assessed by radiopaque marker distribution after 2-weeks intervention. Bowel habits, GI symptoms and quality of life (QOL) were assessed by questionnaire, including the Patient-Assessment of Constipation (PAC) Symptoms (SYM), and PAC-QOL. Following 2-weeks intervention, no reduction was seen in transit time in any group and following 2- or 4-weeks intervention, no improvements were seen in stool frequency or consistency in any group. After 2-weeks intervention with 8 g/day polydextrose an improvement was seen in the PAC-SYM rectal score (p = 0.041). After 4-weeks intervention both rectal (p = 0.049) and stool (p = 0.029) scores improved while improvement in the QOL satisfaction score did not reach significance (p = 0.071). Overall, the results suggest that 2-weeks consumption of 8 or 12 g/day polydextrose does not significantly improve physiological measures of gut function in CC adults. Longer term consumption may improve clinical measures, but further studies will be required to substantiate this.

Sialic acid utilization.

Early postnatal development encounters milk as a key environmental variable and yet the sole nutrient source. One evolutionary conserved constituent of milk is sialic acid, which is generally displayed on glycoconjugates and free glycans. During early postnatal development, high sialic acid need was proposed to be unmet by the endogenous sialic acid synthetic capacity. Hence, milk sialic acid was proposed to serve as a conditional nutrient for the newborn. In the elderly, at the other end of ontogeny, decreased sialylation in the brain, saliva, and immune system is observed. Analogous to the neonatal situation, the endogenous synthetic capacity may be unable to keep up with the need in this age group. The data discussed here propose a functional dietary role of sialic acid as a building block for sialylation and beyond.

Sialic acid utilisation and synthesis in the neonatal rat revisited.

BACKGROUND: Milk is the sole source of nutrients for neonatal mammals and is generally considered to have co-evolved with the developmental needs of the suckling newborn. One evolutionary conserved constituent of milk and present on many glycoconjugates is sialic acid. The brain and colon are major sites of sialic acid display and together with the liver also of synthesis. METHODOLOGY/PRINCIPAL FINDINGS: In this study we examined in rats the relationship between the sialic acid content of milk and the uptake, utilization and synthesis of sialic acid in suckling pups. In rat milk sialic acid was found primarily as 3' sialyllactose and at highest levels between 3 and 10 days postpartum and that decreased towards weaning. In the liver of suckling pups sialic acid synthesis paralleled the increase in milk sialic acid reaching and keeping maximum activity from postnatal day 5 onwards. In the colon, gene expression profiles suggested that a switch from sialic acid uptake and catabolism towards sialic acid synthesis and utilization occurred that mirrored the change of sialic acid in milk from high to low expression. In brain sialic acid related gene expression profiles did not change to any great extent during the suckling period. CONCLUSIONS/SIGNIFICANCE: Our results support the views that (i) when milk sialic acid levels are high, in the colon this sialic acid is catabolized to GlcNAc that in turn may be used as such or used as substrate for sialic acid synthesis and (ii) when milk sialic acid levels are low the endogenous sialic acid synthetic machinery in colon is activated.

Yeast, beef and pork extracts counteract Clostridium difficile toxin A enterotoxicity.

Clostridium difficile is responsible for a large proportion of nosocomial cases of antibiotic-associated diarrhoea and pseudomembranous colitis. The present study provides evidence that yeast, beef and pork extracts, ingredients commonly used to grow bacteria, can counteract C. difficile toxin A enterotoxicity in vitro and in vivo. In model intestinal epithelial cells the individual extracts could prevent the toxin A-induced decrease in epithelial barrier function and partially prevented actin disaggregation and cell rounding. Mice with ad libitum access to individual extracts for 1 week had almost complete reduction in toxin A-induced fluid secretion in intestinal loops. Concomitantly, the toxin A-induced expression of the essential proinflammatory mediator Cox-2 was normalized. Moreover this protective effect was also seen when mice received only two doses of extract by intragastric gavage within 1 week. These results show that yeast, beef and pork extracts have the potential to counteract the intestinal pathogenesis triggered by C. difficile toxin A.

Xenopus polo-like kinase Plx1 regulates XErp1, a novel inhibitor of APC/C activity.

Metaphase-to-anaphase transition is a fundamental step in cell cycle progression where duplicated sister-chromatids segregate to the future daughter cells. The anaphase-promoting complex/cyclosome (APC/C) is a highly regulated ubiquitin-ligase that triggers anaphase onset and mitotic exit by targeting securin and mitotic cyclins for destruction. It was previously shown that the Xenopus polo-like kinase Plx1 is essential to activate APC/C upon release from cytostatic factor (CSF) arrest in Xenopus egg extract. Although the mechanism by which Plx1 regulates APC/C activation remained unclear, the existence of a putative APC/C inhibitor was postulated whose activity would be neutralized by Plx1 upon CSF release. Here we identify XErp1, a novel Plx1-regulated inhibitor of APC/C activity, and we demonstrate that XErp1 is required to prevent anaphase onset in CSF-arrested Xenopus egg extract. Inactivation of XErp1 leads to premature APC/C activation. Conversely, addition of excess XErp1 to Xenopus egg extract prevents APC/C activation. Plx1 phosphorylates XErp1 in vitro at a site that targets XErp1 for degradation upon CSF release. Thus, our data lead to a model of APC/C activation in Xenopus egg extract in which Plx1 targets the APC/C inhibitor XErp1 for degradation.

The forkhead-associated domain protein Cep170 interacts with Polo-like kinase 1 and serves as a marker for mature centrioles.

We report the characterization of Cep170, a forkhead-associated (FHA) domain protein of previously unknown function. Cep170 was identified in a yeast two-hybrid screen for interactors of Polo-like kinase 1 (Plk1). In human cells, Cep170 is constantly expressed throughout the cell cycle but phosphorylated during mitosis. It interacts with Plk1 in vivo and can be phosphorylated by Plk1 in vitro, suggesting that it is a physiological substrate of this kinase. Both overexpression and small interfering RNA (siRNA)-mediated depletion studies suggest a role for Cep170 in microtuble organization and cell morphology. Cep170 associates with centrosomes during interphase and with spindle microtubules during mitosis. As shown by immunoelectron microscopy, Cep170 associates with subdistal appendages, typical of the mature mother centriole. Thus, anti-Cep170 antibodies stain only one centriole during G1, S, and early G2, but two centrioles during late G2 phase of the cell cycle. We show that Cep170 labeling can be used to discriminate bona fide centriole overduplication from centriole amplification that results from aborted cell division.

Polo-like kinase 1 regulates Nlp, a centrosome protein involved in microtubule nucleation.

In animal cells, most microtubules are nucleated at centrosomes. At the onset of mitosis, centrosomes undergo a structural reorganization, termed maturation, which leads to increased microtubule nucleation activity. Centrosome maturation is regulated by several kinases, including Polo-like kinase 1 (Plk1). Here, we identify a centrosomal Plk1 substrate, termed Nlp (ninein-like protein), whose properties suggest an important role in microtubule organization. Nlp interacts with two components of the gamma-tubulin ring complex and stimulates microtubule nucleation. Plk1 phosphorylates Nlp and disrupts both its centrosome association and its gamma-tubulin interaction. Overexpression of an Nlp mutant lacking Plk1 phosphorylation sites severely disturbs mitotic spindle formation. We propose that Nlp plays an important role in microtubule organization during interphase, and that the activation of Plk1 at the onset of mitosis triggers the displacement of Nlp from the centrosome, allowing the establishment of a mitotic scaffold with enhanced microtubule nucleation activity.