Polyphosphate delays fibrin polymerisation and alters the mechanical properties of the fibrin network

Summary Polyphosphate (polyP) binds to fibrin(ogen) and alters fibrin structure, generating a heterogeneous network composed of ‘knots’ interspersed by large pores. Here we show platelet-derived polyP elicits similar structural changes in fibrin and examine the mechanism by which polyP alters fibrin structure. Polymerisation of fibrinogen with thrombin and CaCl2 was studied using spinning disk confocal (SDC) microscopy. PolyP delayed fibrin polymerisation generating shorter protofibrils emanating from a nucleus-type structure. Consistent with this, cascade blue-polyP accumulated in fibrin ‘knots’. Protofibril formation was visualized by atomic force microscopy (AFM) ± polyP. In the presence of polyP abundant monomers of longer length were visualised by AFM, suggesting that polyP binds to monomeric fibrin. Shorter oligomers form in the presence of polyP, consistent with the stunted protofibrils visualised by SDC microscopy. We examined whether these structural changes induced by polyP alter fibrin’s viscoelastic properties by rheometry. PolyP reduced the stiffness (G’) and ability of the fibrin network to deform plastically G”, but to different extents. Consequently, the relative plastic component (loss tangent (G”/G’)) was 61 % higher implying that networks containing polyP are less stiff and more plastic. Local rheological measurements, performed using magnetic tweezers, indicate that the fibrin dense knots are stiffer and more plastic, reflecting the heterogeneity of the network. Our data show that polyP impedes fibrin polymerisation, stunting protofibril growth producing ‘knotted’ regions, which are rich in fibrin and polyP. Consequently, the mechanical properties of the fibrin network are altered resulting in clots with overall reduced stiffness and increased ability to deform plastically. Supplementary Material to this article is available online at www.thrombosis-online.com

Establishing race-, gender- and age-specific reference intervals for pyridoxal 5’-phosphate in the NHANES population to better identify adult hypophosphatasia

Introduction Bisphosphonate treatment in adults with hypophosphatasia (HPP) may increase fracture risk. PLP is a useful marker in biochemically differentiating HPP from osteoporosis in adults. In order to identify elevated PLP, robust reference intervals are needed which are calculated in a large, representative sample population. Methods Complete data from 9069 individuals (ages 20–80, 50.6% female) from two years of the NHANES Survey (2007–2008 and 2009–2010) were investigated. Differences in PLP in the presence of four factors; inflammation (CRP ≥5.0 mg/L), low ALP (<36 IU/L), chronic kidney disease (eGFR <60 mL/min/1.732), and daily vitamin B6 supplementation, were investigated. Race, gender and age differences in PLP were then investigated; 95% reference intervals were calculated that reflected these differences. Results Inflammation and chronic kidney disease were associated with lower PLP (p < .0001 and p = .0005 respectively), while low ALP and vitamin B6 supplementation were associated with higher PLP (both p < .0001). Individuals were excluded based on the presence of these factors; a reference interval population (n = 4463) was established. There were significant differences in PLP depending on race and gender (p < .0001) Increasing age was correlated with decreasing PLP (spearman's rho −0.204, p < .0001). Race- and gender-specific 95% reference intervals were calculated. In male patients, these were also calculated according to age groups: young and older adults (ages 20–49 years and ≥50 years respectively). Conclusions In order to identify adult hypophosphatasia based on elevated PLP, considerations must be made depending on the race, gender and age of the individual. Factors associated with significant differences in PLP must also be considered when assessing biochemical measurements

Genetic Background Can Result in a Marked or Minimal Effect of Gene Knockout (GPR55 and CB2 Receptor) in Experimental Autoimmune Encephalomyelitis Models of Multiple Sclerosis

PMCID: PMC379391

Specifying content and mechanisms of change in interventions to change professionals’ practice : an illustration from the Good Goals study in occupational therapy

PMID: 23078918 [PubMed - indexed for MEDLINE] PMCID: PMC3502268 Free PMC Article The study was funded by the Chief Scientist Office of the Scottish Government Health Directorates (ref: CZF/1/38). The views expressed in this paper are those of the authors. The funder was not involved in the conduct of the study or preparation of the manuscript.Peer reviewedPublisher PD

A cardinal role for cathepsin D in co-ordinating the host-mediated apoptosis of macrophages and killing of pneumococci

The bactericidal function of macrophages against pneumococci is enhanced by their apoptotic demise, which is controlled by the anti-apoptotic protein Mcl-1. Here, we show that lysosomal membrane permeabilization (LMP) and cytosolic translocation of activated cathepsin D occur prior to activation of a mitochondrial pathway of macrophage apoptosis. Pharmacological inhibition or knockout of cathepsin D during pneumococcal infection blocked macrophage apoptosis. As a result of cathepsin D activation, Mcl-1 interacted with its ubiquitin ligase Mule and expression declined. Inhibition of cathepsin D had no effect on early bacterial killing but inhibited the late phase of apoptosis-associated killing of pneumococci in vitro. Mice bearing a cathepsin D-/- hematopoietic system demonstrated reduced macrophage apoptosis in vivo, with decreased clearance of pneumococci and enhanced recruitment of neutrophils to control pulmonary infection. These findings establish an unexpected role for a cathepsin D-mediated lysosomal pathway of apoptosis in pulmonary host defense and underscore the importance of apoptosis-associated microbial killing to macrophage function

A direct physical interaction between Nanog and Sox2 regulates embryonic stem cell self-renewal

Embryonic stem (ES) cell self-renewal efficiency is determined by the Nanog protein level. However, the protein partners of Nanog that function to direct self-renewal are unclear. Here, we identify a Nanog interactome of over 130 proteins including transcription factors, chromatin modifying complexes, phosphorylation and ubiquitination enzymes, basal transcriptional machinery members, and RNA processing factors. Sox2 was identified as a robust interacting partner of Nanog. The purified Nanog–Sox2 complex identified a DNA recognition sequence present in multiple overlapping Nanog/Sox2 ChIP-Seq data sets. The Nanog tryptophan repeat region is necessary and sufficient for interaction with Sox2, with tryptophan residues required. In Sox2, tyrosine to alanine mutations within a triple-repeat motif (S X T/S Y) abrogates the Nanog–Sox2 interaction, alters expression of genes associated with the Nanog-Sox2 cognate sequence, and reduces the ability of Sox2 to rescue ES cell differentiation induced by endogenous Sox2 deletion. Substitution of the tyrosines with phenylalanine rescues both the Sox2–Nanog interaction and efficient self-renewal. These results suggest that aromatic stacking of Nanog tryptophans and Sox2 tyrosines mediates an interaction central to ES cell self-renewal

High Fat Diet Prevents Over-Crowding Induced Decrease of Sex Ratio in Mice

Adaptive theory predicts that mothers would be advantaged by adjusting the sex ratio of their offspring in relation to their offspring's future reproductive success. In the present study, we tested the effect of housing mice under crowded condition on the sex ratio and whether the fat content of the diet has any influence on the outcome of pregnancies. Three-week-old mice were placed on the control diet (NFD) for 3 weeks. Thereafter the mice were allotted randomly to two groups of 7 cages each with 4, 6, 8, 10, 12, 14, and 16 mice in every cage to create increasing crowding gradient and fed either NFD or high fat diet (HFD). After 4 weeks, dams were bred and outcomes of pregnancy were analyzed. The average dam body weight (DBW) at conception, litter size (LS) and SR were significantly higher in HFD fed dams. Further, male biased litters declined with increasing crowding in NFD group but not in HFD. The LS and SR in NFD declined significantly with increasing crowding, whereas only LS was reduced in HFD group. We conclude that female mice housed under overcrowding conditions shift offspring SR in favor of daughters in consistent with the TW hypothesis and high fat diet reduces this influence of overcrowding

Hydroxyl radical-aided thermal pretreatment of algal biomass for enhanced biodegradability

BACKGROUND: Algal biomass, known as a potential feedstock for biofuel production, has cell wall structures that differ from terrestrial biomass. The existing methods for processing algae are limited to conventional pretreatments for terrestrial biomass. RESULTS: In this study, we investigated a novel hydroxyl radical-aided approach for pretreating different types of algal biomass. In this process, hydroxyl radicals formed by a Fenton system were employed in combination with heating to alter the crystalline structure and hydrogen bonds of cellulose in the algal biomass. FeSO(4) and H(2)O(2) at low concentrations were employed to initiate the formation of hydroxyl radicals. This method releases trapped polysaccharides in algal cell walls and converts them into fermentable sugars. The effects of temperature, time, and hydroxyl radical concentration were analyzed. The optimal pretreatment condition [100 °C, 30 min, and 5.3 mM H(2)O(2) (determined FeSO(4) concentration of 11.9 mM)] was identified using a central composite design. Complete (100 %) carbohydrate recovery was achieved with some algal biomass without formation of inhibitors such as hydroxymethylfurfural and furfural as by-products. Both microalgal and macroalgal biomasses showed higher enzymatic digestibility of cellulose conversion (>80 %) after the milder pretreatment condition. CONCLUSION: Hydroxyl radical-aided thermal pretreatment was used as a novel method to convert the carbohydrates in the algal cell wall into simple sugars. Overall, this method increased the amount of glucose released from the algal biomass. Overall, enhanced algal biomass digestibility was demonstrated with the proposed pretreatment process. The new pretreatment requires low concentration of chemical solvents and milder temperature conditions, which can prevent the toxic and corrosive effects that typically result from conventional pretreatments. Our data showed that the advantages of the new pretreatment include higher carbohydrate recovery, no inhibitor production, and lower energy consumption. The new pretreatment development mimicking natural system could be useful for biochemical conversion of algal biomass to fuels and chemicals. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13068-015-0372-2) contains supplementary material, which is available to authorized users

Exercise and postprandial lipemia: effects on vascular health in inactive adults

Background: There is evidence to suggest that postprandial lipemia are is linked to the impairment of endothelial function, which is characterized by an imbalance between the actions of vasodilators and vasoconstrictors. The aim of this study was to determine the effects of a 12-week high-intensity training (HIT) and moderate continuous training (MCT) protocol on postprandial lipemia, vascular function and arterial stiffness in inactive adults after high-fat meal (HFM) ingestion. Methods: A randomized clinical trial was conducted in 20 healthy, inactive adults (31.6 ± 7.1 years). Participants followed the two exercise protocols for 12 weeks. To induce a state of postprandial lipemia (PPL), all subjects received a HFM. Endothelial function was measured using flow-mediated vasodilation (FMD), normalized brachial artery FMD (nFMD), aortic pulse wave velocity (PWV) and augmentation index (AIx). Plasma total cholesterol, high-density lipoprotein cholesterol (HDL-c), triglycerides and glucose were also measured. Results: The effects of a HFM were evaluated in a fasted state and 60, 120, 180, and 240 min postprandially. A significant decrease in serum glucose between 0 min (fasted state) and 120 min postprandially was found in the HIT group (P= 0. 035). Likewise, FMD (%) was significantly different between the fasted state and 60 min after a HFM in the HIT group (P = 0.042). The total cholesterol response expressed as area under curve (AUC)(0–240) was lower following HIT than following MCT, but no significant differences were observed (8%, P > 0.05). Similarly, triglycerides AUC(0–240) was also lower after HIT compared with MCT, which trended towards significance (24%, P= 0.076). The AUC(0–240) for the glucose response was significantly lower following HIT than MCT (10%, P = 0.008). FMD and nFMD AUC(0–240) were significantly higher following HIT than following MCT (46.9%, P = 0.021 and 67.3%, P = 0.009, respectively). PWV AUC(0–240) did not differ following between the two exercise groups (2.3%, P > 0.05). Conclusions: Supervised exercise training mitigates endothelial dysfunction and glucose response induced by PPL. Exercise intensity plays an important role in these protective effects, and medium-term HIT may be more effective than MCT in reducing postprandial glucose levels and attenuating vascular impairment.This study as funded in part by the Center for Studies on Measurement of Physical Activity, School of Medicine and Health Sciences, Universidad del Rosario (Code N° FIUR DN-BG001). We declare that the results of the study are presented clearly, honestly, and without fabrication, falsification, or appropriate data manipulatio

Expression of M. tuberculosis-induced suppressor of cytokine signaling (SOCS) 1, SOCS3, FoxP3 and secretion of IL-6 associates with differing clinical severity of tuberculosis

Background Appropriate immune activation of T cells and macrophages is central for the control of Mycobacterium tuberculosis infections. IFN-γ stimulated responses are lowered in tuberculosis (TB), while expression of Suppressor of Cytokine Signaling (SOCS) molecules – 1 and 3 and CD4+CD25+FoxP3+T regulatory cells is increased. Here we investigated the association of these molecules in regard to clinical severity of TB. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from patients with pulmonary TB (PTB, n = 33), extra-pulmonary TB (ETB, n = 33) and healthy endemic controls (EC, n = 15). Cases were classified as moderately advanced or far advanced PTB, and less severe or severe disseminated ETB. M. tuberculosis -stimulated IFN-γ, SOCS1, SOCS3 and FoxP3 gene expression and secretion of Th1 and Th2 cytokines was measured. Statistical analysis was performed using Mann–Whitney U, Wilcoxon Rank and Kruskal Wallis non-parametric tests. Results In un-stimulated PBMCs, IL-6 (p = 0.018) and IL-10 (p = 0.013) secretion levels were increased in PTB while IL-10 was also increased in ETB (p = 0.003), all in comparison with EC. M. tuberculosis-stimulated IL-6 (p = 0.003) was lowered in ETB as compared with EC. SOCS1 mRNA expression in M. tuberculosis stimulated PBMCs levels in moderately advanced PTB (p = 0.022), far advanced (p = 0.014) PTB, and severe ETB (p = 0.009) were raised as compared with EC. On the other hand, SOCS1 mRNA titers were reduced in less severe ETB, in comparison with severe ETB (p = 0.027) and far advanced PTB (p = 0.016). SOCS3 mRNA accumulation was reduced in far advanced PTB (p = 0.007) and FoxP3 mRNA expression was increased in less severe ETB as compared with EC (p = 0.017). Conclusions The lowered SOCS1 mRNA levels in patients with less severe extra-pulmonary TB as compared to those with more severe ETB and PTB may lead to elevated IFN-γ pathway gene expression in the latter group. As localized ETB has shown to be associated with more effective Th1 immunity and adaptive responses, this suggests a role for SOCS1 in determining disease outcome in extra-pulmonary TB