Somatic Cell Counts (Leucocyte Counts) A Standard of Milk Acceptability

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Collagen cross-linking: insights on the evolution of metazoan extracellular matrix

Collagens constitute a large family of extracellular matrix (ECM) proteins that play a fundamental role in supporting the structure of various tissues in multicellular animals. The mechanical strength of fibrillar collagens is highly dependent on the formation of covalent cross-links between individual fibrils, a process initiated by the enzymatic action of members of the lysyl oxidase (LOX) family. Fibrillar collagens are present in a wide variety of animals, therefore often being associated with metazoan evolution, where the emergence of an ancestral collagen chain has been proposed to lead to the formation of different clades. While LOX-generated collagen cross-linking metabolites have been detected in different metazoan families, there is limited information about when and how collagen acquired this particular modification. By analyzing telopeptide and helical sequences, we identified highly conserved, potential cross-linking sites throughout the metazoan tree of life. Based on this analysis, we propose that they have importantly contributed to the formation and further expansion of fibrillar collagens

Structural constraints on the evolution of the collagen fibril: convergence on a 1014-residue COL domain.

Type I collagen is the fundamental component of the extracellular matrix. Its α1 gene is the direct descendant of ancestral fibrillar collagen and contains 57 exons encoding the rod-like triple-helical COL domain. We trace the evolution of the COL domain from a primordial collagen 18 residues in length to its present 1014 residues, the limit of its possible length. In order to maintain and improve the essential structural features of collagen during evolution, exons can be added or extended only in permitted, non-random increments that preserve the position of spatially sensitive cross-linkage sites. Such sites cannot be maintained unless the twist of the triple helix is close to 30 amino acids per turn. Inspection of the gene structure of other long structural proteins, fibronectin and titin, suggests that their evolution might have been subject to similar constraints.The work was funded by a project grant to D.A.S. and R.W.F. from British Heart Foundation (PG/08/011/24416). R.W.F. was supported by BHF Programme grant nos. (RG/09/003/27122 and RG/15/4/31268).This is the final version of the article. It first appeared from Royal Society Publishing via http://dx.doi.org/10.1098/rsob.14022

Explosibility of Coarse Biomass Powders

Pulverised biomass is being used in electric power generation, either co-fired with coal or increasingly as 100% biomass. However, there is minimal information in the literature on the mechanism of flame propagation in pulverised biomass. In the present work the explosion technique was used to obtain fundamental information on the rate of flame propagation, the lean limits of flame propagation and related explosion characteristics of coarse biomass. A large part of this research involved the modification of the ISO 1m3 method to enable it to be used with coarse fibrous biomass powders. The technique that worked was to follow the Hartmann method and place the dust inside the vessel using a hemispherical bowl and then disperse this dust with a blast of air. This was demonstrated to work with coarse woody biomass and the calibration was established using cornflour and referenced to the standard method. The MEC and Kst for dusts were shown to have a dependence on the particle size. However, very coarse particles still propagated a flame, with no evidence that this was due to preferentially burning of the finer particles. Biomass particles of 300-500µm were shown to be flammable, i.e. as large as kerosene mist and large than coal particles will propagate a flame. For coarse woody biomass the Kst values were very low <20 bar m/s in many cases, but the peak pressure was high and hence the explosion would destroy biomass handling plant. This work found that the unburnt material was compressed into a layer against the wall of the vessel ahead of the flame front, thus preventing it from interacting with the flame front. It was postulated that large particles lagged the main flame due to interaction with the explosion induced wind. This led to large particles being pyrolysed behind the flame front and then to arrive last at the wall and so appear as on outer pyrolysed layer on the material compressed against the wall. This explanation also enabled an explanation to be given for the very rich mixtures that could burn with dusts than could not burn if the material was a gas

LipidFinder: a computational workflow for discovery of lipids identifies eicosanoid-phosphoinositides in platelets

Accurate and high-quality curation of lipidomic datasets generated from plasma, cells, or tissues is becoming essential for cell biology investigations and biomarker discovery for personalized medicine. However, a major challenge lies in removing artifacts otherwise mistakenly interpreted as real lipids from large mass spectrometry files (>60 K features), while retaining genuine ions in the dataset. This requires powerful informatics tools; however, available workflows have not been tailored specifically for lipidomics, particularly discovery research. We designed LipidFinder, an open-source Python workflow. An algorithm is included that optimizes analysis based on users’ own data, and outputs are screened against online databases and categorized into LIPID MAPS classes. LipidFinder outperformed three widely used metabolomics packages using data from human platelets. We show a family of three 12-hydroxyeicosatetraenoic acid phosphoinositides (16:0/, 18:1/, 18:0/12-HETE-PI) generated by thrombin-activated platelets, indicating crosstalk between eicosanoid and phosphoinositide pathways in human cells. The software is available on GitHub (https://github.com/cjbrasher/LipidFinder), with full userguides

Hydroxyproline Ring Pucker Causes Frustration of Helix Parameters in the Collagen Triple Helix.

Collagens, the most abundant proteins in mammals, are defined by their triple-helical structures and distinctive Gly-Xaa-Yaa repeating sequence, where Xaa is often proline and Yaa, hydroxyproline (Hyp/O). It is known that hydroxyproline in the Yaa position stabilises the triple helix, and that lack of proline hydroxylation in vivo leads to dysfunctional collagen extracellular matrix assembly, due to a range of factors such as a change in hydration properties. In addition, we note that in model peptides, when Yaa is unmodified proline, the Xaa proline has a strong propensity to adopt an endo ring conformation, whilst when Yaa is hydroxyproline, the Xaa proline adopts a range of endo and exo conformations. Here we use a combination of solid-state NMR spectroscopy and potential energy landscape modelling of synthetic triple-helical collagen peptides to understand this effect. We show that hydroxylation of the Yaa proline causes the Xaa proline ring conformation to become metastable, which in turn confers flexibility on the triple helix.The authors acknowledge BBSRC grant number BB/G021392/1 (MJD, DGR), EPSRC DTA studentship and Doctoral Prize (WYC), British Heart Foundation RG/09/003/27122 and PG/08/011/24416 (RWF, DB, DAS), Wellcome Trust 094470/Z/10/Z (RWF, DB, DAS), ERC and EPSRC (DJW).This is the final version of the article. It first appeared from NPG via http://dx.doi.org/10.1038/srep1255

The properties conferred upon triple-helical collagen-mimetic peptides by the presence of cysteine residues

AbstractRecently, the ability of polymeric collagen-like peptides to regulate cell behavior has generated great interest. A triple-helical peptide known as collagen-related peptide (CRP) contains the sequence (Gly-Pro-Hyp)10. With Gly-Pro-Cys triplets appended to both of its termini, designated CRPcys, chemical cross-linking using heterobifunctional reagents generates CRPcys-XL, a potent, widely used, polymeric agonist for platelet Glycoprotein VI, whereas non-cross-linked, monomeric CRPcys antagonizes Glycoprotein VI. Here, we describe how cysteine in these triplets may also undergo random air-induced oxidation, especially upon prolonged storage or repeated freeze–thawing, to form disulphide bonds, resulting in a lesser degree of polymerization than with chemical cross-linking. We investigated the monomeric and polymeric states of these and other cysteine-containing collagen-derived peptides, using gel filtration and dynamic light scattering, allowing the size of a CRP-XL aggregate to be estimated. The effect of cysteine thiols upon peptide adsorption to surfaces and subsequent platelet responses was investigated. This demonstrated that cysteine is required for strong binding to glass coverslips and to plastic plates used in ELISA assays

The recognition of collagen and triple-helical toolkit peptides by MMP-13: sequence specificity for binding and cleavage.

Remodeling of collagen by matrix metalloproteinases (MMPs) is crucial to tissue homeostasis and repair. MMP-13 is a collagenase with a substrate preference for collagen II over collagens I and III. It recognizes a specific, well-known site in the tropocollagen molecule where its binding locally perturbs the triple helix, allowing the catalytic domain of the active enzyme to cleave the collagen α chains sequentially, at Gly(775)-Leu(776) in collagen II. However, the specific residues upon which collagen recognition depends within and surrounding this locus have not been systematically mapped. Using our triple-helical peptide Collagen Toolkit libraries in solid-phase binding assays, we found that MMP-13 shows little affinity for Collagen Toolkit III, but binds selectively to two triple-helical peptides of Toolkit II. We have identified the residues required for the adhesion of both proMMP-13 and MMP-13 to one of these, Toolkit peptide II-44, which contains the canonical collagenase cleavage site. MMP-13 was unable to bind to a linear peptide of the same sequence as II-44. We also discovered a second binding site near the N terminus of collagen II (starting at helix residue 127) in Toolkit peptide II-8. The pattern of binding of the free hemopexin domain of MMP-13 was similar to that of the full-length enzyme, but the free catalytic subunit bound none of our peptides. The susceptibility of Toolkit peptides to proteolysis in solution was independent of the very specific recognition of immobilized peptides by MMP-13; the enzyme proved able to cleave a range of dissolved collagen peptides.This work was supported by a British Heart Foundation programme grant, RG/009/003/27122, and peptide synthesis, by grants from Medical Research Council and Wellcome Trust.This is the author accepted manuscript. The final version can be found on the publisher's website at: http://www.jbc.org/content/early/2014/07/09/jbc.M114.58344

Proline provides site-specific flexibility for in vivo collagen.

Fibrillar collagens have mechanical and biological roles, providing tissues with both tensile strength and cell binding sites which allow molecular interactions with cell-surface receptors such as integrins. A key question is: how do collagens allow tissue flexibility whilst maintaining well-defined ligand binding sites? Here we show that proline residues in collagen glycine-proline-hydroxyproline (Gly-Pro-Hyp) triplets provide local conformational flexibility, which in turn confers well-defined, low energy molecular compression-extension and bending, by employing two-dimensional 13C-13C correlation NMR spectroscopy on 13C-labelled intact ex vivo bone and in vitro osteoblast extracellular matrix. We also find that the positions of Gly-Pro-Hyp triplets are highly conserved between animal species, and are spatially clustered in the currently-accepted model of molecular ordering in collagen type I fibrils. We propose that the Gly-Pro-Hyp triplets in fibrillar collagens provide fibril "expansion joints" to maintain molecular ordering within the fibril, thereby preserving the structural integrity of ligand binding sites.BBSRC, EPSRC, Raymond and Beverly Sackler Fund for Physics of Medicine, Wellcome Trust, ER

Impaired platelet-dependent thrombin generation associated with thrombocytopenia is improved by prothrombin complex concentrates in vitro.

BACKGROUND: Impaired thrombin generation (TG) in patients with acquired coagulopathy, is due to low coagulation factors and thrombocytopenia. The latter is typically treated with platelet transfusions and the former with plasma and occasionally with prothrombin complex concentrates (PCCs). We hypothesized that manipulating the concentrations of coagulation factors might result in restoration of platelet-dependent TG over and above that of simple replacement therapy. OBJECTIVE: To investigate the influence of PCCs on impaired TG secondary to thrombocytopenia. METHODS: TG was evaluated by thrombin generation assay using a thrombocytopenia model in which normal plasma samples with varying platelet counts (20-300 × 109/L) were spiked with PCCs (25%-150% increase in plasma PCC levels). RESULTS: PCCs and platelets significantly increased TG in a dose-dependent manner in vitro. Two-way repeated measures of analysis of variance showed variance in peak height, area under the curve, time to peak, and velocity. This variance explained, respectively, by levels of PCC was 47, 59, 25 and 53%; by platelet count was 45, 28, 44, and 14%; by the combination was 80, 67, 70, and 62% variance; and a combination with additional interaction was 91, 84, 76, and 68%. TG at a platelet count 40 × 109/L with an approximate 25% increase in PCC concentration was similar to TG at 150 × 109/L. Similarly, patient samples spiked ex vivo with PCCs also showed highly significant improvements in TG. CONCLUSIONS: Impaired TG of thrombocytopenia is improved by PCCs, supporting the need for additional studies in complex coagulopathies characterized by mild to moderate thrombocytopenia and abnormal coagulation