Metabolism of soluble proteins by rumen microorganisms and the influence of condensed tannins on nitrogen solubility and degradation

The amino acid requirements of ruminants are met by two sources; microbes leaving the rumen and dietary protein escaping fermentation in the rumen. Much ruminant research has therefore focused on improving amino acid supply to the duodenum by increasing both microbial protein synthesis and escape of feed proteins from the rumen. The escape of dietary protein is dependent on the degradation characteristics and retention time in the rumen. The overall aims of this thesis were to increase the knowledge of ruminal degradation of buffer soluble proteins and to investigate the effect of condensed tannins on the degradation. The thesis examines the effect of trichloroacetic acid, perchloric acid, and tungstic acid on detection and recovery of feed peptides and chemically-defined peptides. Twenty-five feeds were screened for buffer soluble protein N. In vitro ruminal degradation rates of buffer soluble proteins were estimated in 11 of these feeds. Buffer soluble protein from peas or cold-pressed rapeseed cake was given as a pulse dose together with a liquid marker to lactating dairy cows, to investigate rumen in vivo degradation rates. The concentration of condensed tannins was determined for birdsfoot trefoil (Lotus corniculatus L.), big trefoil (L. uliginosus Schkur.) and sainfoin (Onobrychis viciifolia Scop.) and related to nitrogen solubility in fresh-frozen and ensiled material and ruminal in vitro degradation. The different protein precipitants did not alter detection of peptides formed in a ruminal in vitro system. The recovery of an eight-residue peptide was 0.66, 0.88, and 0.91 for tungstic acid, perchloric acid, and trichloroacetic acid, respectively. The content of soluble protein N in feeds ranged from 0 to 874 g/kg buffer soluble N with the highest contents for lupine, peas, and cold-pressed rapeseed cake. Fractional degradation rates determined in vitro for soluble protein ranged from 0.18 (linseed cake) to 1.0 /h (casein). Soluble protein from soybean meal, peas, and lupine were degraded at intermediate rates. Soluble proteins given as a pulse dose were rapidly degraded in vivo. Results were disturbed by slow mixing in the rumen. In several cases, concentration of the liquid marker was higher after 1 h than 0.5 h post dosing. Based on two experiments and a total of nine varieties of birdsfoot trefoil, the concentration of condensed tannins determined by a radial diffusion method ranged from 3 to 17 g/kg DM. The tannin content for sainfoin and big trefoil was 21.6 (maximum concentration) and 24.8 g/kg DM, respectively. Buffer N solubility and ruminal in vitro N degradability were negatively correlated to tannin content. Tannin content and wilting time cumulatively reduced buffer soluble nitrogen in birdsfoot trefoil silage. This thesis shows that there is no distinct cut-off for peptide size when using different protein precipitation agents. Soluble proteins degrade at different rates depending on source. As they are not instantly degraded, they must contribute to the dietary amino acid supply of the ruminant. The in sacco method, which assumes complete degradation of soluble proteins should not be used for feeds high in soluble proteins

Granitic groundwater colloids sampling and characterisation: the strategy for artefact elimination

Colloids were separated by submicro-filtration of granitic groundwater samples collected at-line under in-situ thermodynamic conditions after down-hole groundwater sampling and transfer at the well head. The methodology avoids the generation of artefacts produced by pH changes due to CO2 exchange, yielding potential carbonate precipitation, or by O2 contamination yielding oxidized insoluble phases. The enhanced pressure and the anoxic conditions are also maintained through the filtering procedure. This procedure was carried out after a period of regular sampling of groundwater pumped to the ground surface and continuous on-line long-term measurements (weeks, months) of chemical and physical parameters in the unbroken sample water both at the ground surface and at depth down-hole. Colloid samples were characterized on the submicro-filtration membrane by scanning electron microscopy. Under deep granite groundwater conditions, natural colloids occur sparsely. The colloid concentration was determined C col ∼1 and ∼50μg L−1 for sizes ranging from 50 to 200nm or n col ∼ 3.9 × 109 and 47 × 109 L−1 for sizes larger than 50nm for KFM11A, Forsmark, and KLX17A, Laxemar, Oskarshamn, respectively, Sweden. These colloids are expected to be clay particles with an average size smaller than 200nm for the Na-Ca-Cl and Na-Cl groundwaters (pH 7.6 and 8.00, ionic strength ∼10−1 and ∼10−2 mol L−1, respectively, for KFM11A and KLX17A), the colloid concentrations were comparable with values previously reported in the literatur

Collecting evidence from distributed sources to evaluate railway suicide and trespass prevention measures

It can be difficult to select from available safety preventative measures, especially where there is limited evidence of effectiveness in different contexts. This paper describes application of a method to identify and evaluate wide-ranging preventative measures for rail suicide and trespass fatalities. Evidence from literature and industry sources was collated and reviewed in a two stage process to achieve consensus among experts on the likely effects of the measures and factors influencing their implementation. Multiple evaluation criteria were used to examine the measures from different perspectives. Fencing, awareness campaigns and different types of organisational initiatives were recommended for further testing. This is the first time evidence has been collected internationally across such a range of preventative measures. Commentary is provided on using this type of approach to select safety measures from a pool of prevention options, including how re-framing the scope of the exercise could identify alternative options for prevention. Practitioner summary: The findings give insight to how different measures work in different ways and how industry can consider this in strategic initiatives. The method could be used in future studies with different frames of reference (e.g. different timescales, level of ambition and safety context e.g. railway crossings or highway fatalities)

Self-management of first trimester medical termination of pregnancy: a qualitative study of women's experiences

Objective: To explore the experiences of women in Scotland who return home to complete medical termination of pregnancy (TOP) ≤63 days of gestation, after being administered with mifepristone and misoprostol at an NHS TOP clinic. Design: Qualitative interview study. Setting: One National Health Service health board (administrative) area in Scotland. Population or Sample: Women in Scotland who had undergone medical TOP ≤63 days, and self-managed passing the pregnancy at home; recruited from three clinics in on NHS health board area between January and July 2014. Methods: In-depth, semi-structured interviews with 44 women in Scotland who had recently undergone TOP ≤63 days of gestation, and who returned home to pass the pregnancy. Data were analysed thematically using an approach informed by the Framework method. Main Outcome Measures: Women's experiences of self-management of TOP ≤63 days of gestation. Results: Key themes emerging from the analysis related to self-administration of misoprostol in clinic; reasons for choosing home self-management; facilitation of self-management and expectation-setting; experiences of getting home; self-managing and monitoring treatment progress; support for self-management (in person and remotely); and pregnancy self-testing to confirm completion. Conclusions: Participants primarily found self-administration of misoprostol and home self-management to be acceptable and/or preferable, particularly where this was experienced as a decision made jointly with health professionals. The way in which home self-management is presented to women at clinic requires ongoing attention. Women could benefit from the option of home administration of misoprostol

The sunburn response in human skin is characterized by sequential eicosanoid profiles that may mediate its early and late phases.

yesSunburn is a commonly occurring acute inflammatory process, with dermal vasodilatation and leukocyte infiltration as central features. Ultraviolet (UV) B-induced hydrolysis of membrane phospholipids releases polyunsaturated fatty acids and their subsequent metabolism by cyclooxygenases (COX) and lipoxygenases (LOX) may produce potent eicosanoid mediators modulating different stages of the inflammation. Our objective was to identify candidate eicosanoids formed during the sunburn reaction in relation to its clinical and histological course. We exposed skin of healthy humans (n=32) to UVB and for 72h examined (i) expression of pro- and anti-inflammatory eicosanoids using LC/ESI-MS/MS and (ii) immunohistochemical expression of COX-2, 12-LOX, 15-LOX and leucocyte markers, while (iii) quantifying clinical erythema. We show that vasodilatory prostaglandins (PG)E2, PGF2¿ and PGE3 accompany the erythema in the first 24-48h, associated with increased COX-2 expression at 24h. Novel, potent leukocyte chemoattractants 11-, 12- and 8-monohydroxy-eicosatetraenoic acid (-HETE) are elevated from 4-72h, in association with peak dermal neutrophil influx at 24h, and increased dermal CD3+ lymphocytes and 12- and 15-LOX expression from 24-72h. Anti-inflammatory metabolite 15-HETE shows later expression, peaking at 72h. Sunburn is characterized by overlapping phases of increases in COX products followed by LOX products that may regulate subsequent events and ultimately its resolution.The Wellcome Trus

Role of Ca2+-activated K+ channels and Na+-K+-ATPase in prostaglandin E1- and E2-induced inhibition of the adrenergic response in human vas deferens

We studied the role of K+ channels and Na+,K+-ATPase in the presynaptic inhibitory effects of prostaglandin E1 (PGE1) and PGE2 on the adrenergic responses of human vas deferens. Furthermore, we determined the effects of increasing extracellular K+ concentrations ([K+]o) and inhibition of Na+,K+- ATPase on neurogenic and norepinephrine-induced contractile responses. Ring segments of the epididymal part of the vas deferens were taken from 45 elective vasectomies and mounted in organ baths for isometric recording of tension. The neuromodulatory effects of PGEs were tested in the presence of K+ channel blockers. PGE1 and PGE2 (10_8 to 10_6 M) induced inhibition of adrenergic contractions. The presence of tetraethylammonium (10_3 M), charybdotoxin (10_7 M), or iberiotoxin(10_7 M), prevented the inhibitory effects of PGE1 and PGE2 on the adrenergic contraction. Botx glibenclamide (10_5 M) and apamin (10_6 M) failed to antagonize PGE1 and PGE2 effects. Raising the [K+]o from 15.8 mM to 25.8 mM caused inhibition of the neurogenic contractions. Ouabain at a concentration insufficient to alter the resting tension (10_6 M) increased contractions induced by electrical stimulation but did not alter the contractions to norepinephrine. The inhibition of neurogenic responses induced PGE1, PGE2 and increased extracellular concentration of K+ was almost completely prevented by ouabain (10_6 M). The results demonstrate that PGE1 and PGE2 inhibit adrenergic responses by a prejunctional mechanism that involves the activation of large-conductance Ca2+- activated K+ channels and Na+,K+-ATPas

Signaling via β2 Integrins Triggers Neutrophil-Dependent Alteration in Endothelial Barrier Function

Activation of polymorphonuclear leukocytes (PMNs) and adhesion to the endothelial lining is a major cause of edema formation. Although known to be dependent on the function of β2 integrins (CD11/CD18), the precise mechanisms by which adherent PMNs may impair endothelial barrier capacity remain unclear. Here, the role of transmembrane signaling by β2 integrins in PMN-induced alterations in tight junctional permeability of cultured endothelial cell (EC) monolayers was investigated. PMN activation, in the absence of proinflammatory stimuli, was accomplished through antibody cross-linking of CD11b/CD18, mimicking adhesion-dependent receptor engagement. CD18 cross-linking in PMNs added to the EC monolayer provoked a prompt increase in EC permeability that coincided with a rise in EC cytosolic free Ca2+ and rearrangement of actin filaments, events similar to those evoked by chemoattractant PMN activation. Cell-free supernatant obtained after CD18 cross-linking in suspended PMNs triggered an EC response indistinguishable from that induced by direct PMN activation, and caused clear-cut venular plasma leakage when added to the hamster cheek pouch in vivo preparation. The PMN-evoked EC response was specific to β2 integrin engagement inasmuch as antibody cross-linking of l-selectin or CD44 was without effect on EC function. Our data demonstrate a causal link between outside-in signaling by β2 integrins and the capacity of PMNs to induce alterations in vascular permeability, and suggest a paracrine mechanism that involves PMN-derived cationic protein(s) in the cellular crosstalk between PMNs and ECs