GPVI and CLEC-2

The glycoprotein VI (GPVI)-FcR gamma-chain complex initiates powerful activation of platelets by the subendothelial matrix proteins collagen and laminin, which are exposed following vessel damage. Initiation of platelet activation is through an immunoreceptor tyrosine-based activation motif (ITAM). C-type lectin receptor 2 (CLEC-2), following engagement by its endogenous ligand, podoplanin, also mediates powerful platelet activation through Src and Syk kinases, but regulates Syk through a novel dimerization mechanism via a single YxxL motif known as a hemITAM. This chapter compares the signaling pathways of both receptors and their role in hemostasis and thrombosis. Platelets are also increasingly implicated in processes beyond hemostasis and thrombosis. One such process is the efficient separation of the lymphatic and blood vasculatures, which is dependent on CLEC-2-mediated platelet activation.

Platelet actin nodules are podosome-like structures dependent on Wiskott-Aldrich syndrome protein and ARP2/3 complex

The actin nodule is a novel F-actin structure present in platelets during early spreading. However, only limited detail is known regarding nodule organization and function. Here we use electron microscopy, SIM and dSTORM super-resolution, and live-cell TIRF microscopy to characterize the structural organization and signalling pathways associated with nodule formation. Nodules are composed of up to four actin-rich structures linked together by actin bundles. They are enriched in the adhesion-related proteins talin and vinculin, have a central core of tyrosine phosphorylated proteins and are depleted of integrins at the plasma membrane. Nodule formation is dependent on Wiskott-Aldrich syndrome protein (WASp) and the ARP2/3 complex. WASp(-/-) mouse blood displays impaired platelet aggregate formation at arteriolar shear rates. We propose actin nodules are platelet podosome-related structures required for platelet-platelet interaction and their absence contributes to the bleeding diathesis of Wiskott-Aldrich syndrome.

GPVI and CLEC-2 in hemostasis and vascular integrity

The glycoprotein VI (GPVI)-FcR gamma-chain complex initiates powerful activation of platelets by the subendothelial matrix proteins collagen and laminin through an immunoreceptor tyrosine-based activation motif (ITAM)-regulated signaling pathway. ITAMs are characterized by two YxxL sequences separated by 6-12 amino acids and are found associated with several classes of immunoglobulin (Ig) and C-type lectin receptors in hematopoietic cells, including Fc receptors. Cross-linking of the Ig GPVI leads to phosphorylation of two conserved tyrosines in the FcR gamma-chain ITAM by Src family tyrosine kinases, followed by binding and activation of the tandem SH2 domain-containing Syk tyrosine kinase and stimulation of a downstream signaling cascade that culminates in activation of phospholipase Cgamma2 (PLCgamma2). In contrast, the C-type lectin receptor CLEC-2 mediates powerful platelet activation through Src and Syk kinases, but regulates Syk through a novel dimerization mechanism via a single YxxL motif known as a hemITAM. CLEC-2 is a receptor for podoplanin, which is expressed at high levels in several tissues, including type 1 lung alveolar cells, lymphatic endothelial cells, kidney podocytes and some tumors, but is absent from vascular endothelial cells and platelets. In this article, we compare the mechanism of platelet activation by GPVI and CLEC-2 and consider their functional roles in hemostasis and other vascular processes, including maintenance of vascular integrity, angiogenesis and lymphogenesis.

Manipulation of milk fatty acid composition in lactating cows: opportunities and challenges

Public health policies recommend a population wide decrease in the consumption of saturated fatty acids (SFA) to lower the incidence of cardiovascular and metabolic diseases. In most developed countries, milk and dairy products are the major source of SFA in the human diet. Altering milk fat composition offers the opportunity to lower the consumption of SFA without requiring a change in eating habits. Supplementing the diet of lactating cows with oilseeds, plant oils and marine lipids can be used to replace the SFA in milk fat with monounsaturated fatty acids (MUFA), and to a lesser extent, polyunsaturated fatty acids (PUFA). Due to ruminal metabolism, the decreases in milk SFA are also accompanied by increases in trans fatty acids (TFA), including conjugated isomers. The potential to lower SFA, enrich cis MUFA and PUFA, and alter the abundance and distribution of individual TFA in milk differs according to oil source, form of lipid supplement and degree of oilseed processing, and the influence of other components in the diet. The present review summarises recent evidence on changes in milk fat composition that can be achieved using dietary lipid supplements and highlights the challenges to commercial production of modified milk and dairy products. A meta-analysis on the effects of oilseeds on milk fatty acid composition is also presented.

Construction contracts: law and management. 5th edition

The fifth edition of this best-selling textbook has been thoroughly revised to provide the most up-to-date and comprehensive coverage of the legislation, administration and management of construction contracts. It now includes comparison of working with JCT, NEC3 and FIDIC contracts, throughout. In line with new thinking in construction management research, this authoritative guide is essential reading for every construction undergraduate and is an extremely useful source of reference for practitioners.

Intracellular trafficking, localization, and mobilization of platelet-borne thiol isomerases

OBJECTIVE: Thiol isomerases facilitate protein folding in the endoplasmic reticulum, and several of these enzymes, including protein disulfide isomerase and ERp57, are mobilized to the surface of activated platelets, where they influence platelet aggregation, blood coagulation, and thrombus formation. In this study, we examined the synthesis and trafficking of thiol isomerases in megakaryocytes, determined their subcellular localization in platelets, and identified the cellular events responsible for their movement to the platelet surface on activation. APPROACH AND RESULTS: Immunofluorescence microscopy imaging was used to localize protein disulfide isomerase and ERp57 in murine and human megakaryocytes at various developmental stages. Immunofluorescence microscopy and subcellular fractionation analysis were used to localize these proteins in platelets to a compartment distinct from known secretory vesicles that overlaps with an inner cell-surface membrane region defined by the endoplasmic/sarcoplasmic reticulum proteins calnexin and sarco/endoplasmic reticulum calcium ATPase 3. Immunofluorescence microscopy and flow cytometry were used to monitor thiol isomerase mobilization in activated platelets in the presence and absence of actin polymerization (inhibited by latrunculin) and in the presence or absence of membrane fusion mediated by Munc13-4 (absent in platelets from Unc13dJinx mice). CONCLUSIONS: Platelet-borne thiol isomerases are trafficked independently of secretory granule contents in megakaryocytes and become concentrated in a subcellular compartment near the inner surface of the platelet outer membrane corresponding to the sarco/endoplasmic reticulum of these cells. Thiol isomerases are mobilized to the surface of activated platelets via a process that requires actin polymerization but not soluble N-ethylmaleimide-sensitive fusion protein attachment receptor/Munc13-4-dependent vesicular-plasma membrane fusion.

Nurturing bilingual learners: challenges and concerns in Singapore

Singapore’s bilingual policy legitimises English not only as the language of governmental administration and interethnic communication, but also as the medium of instruction in all schools on all levels and across all subjects except mother tongues (MTs). As a result of these politics of language recognition, a visible shift has occurred in all ethnic groups away from MTs towards English. To rectify the language shift situation, the government has emphasised that developing bilingualism and raising bilingual children should begin in preschools. In this paper, we examine two top-down official documents: Review of Mother Tongue Languages Report, issued in 2011, and Nurturing Early Learners Framework for Mother Tongue Languages, developed in 2013. Attempting to identify some of the complex factors that influence language shift, we present an intertextual analysis of the Report and the curriculum Framework. In doing so, we compare the consistencies and locate the implicit inconsistencies in the policy position on bilingual education in preschools. We conclude the article by outlining the implications for changing the current bilingual educational models and providing teacher training programmes that maximise the learning opportunities of young bilingual learners.

Interaction between FTO gene variants and lifestyle factors on metabolic traits in an Asian Indian population

Background Lifestyle factors such as diet and physical activity have been shown to modify the association between fat mass and obesity–associated (FTO) gene variants and metabolic traits in several populations; however, there are no gene-lifestyle interaction studies, to date, among Asian Indians living in India. In this study, we examined whether dietary factors and physical activity modified the association between two FTO single nucleotide polymorphisms (rs8050136 and rs11076023) (SNPs) and obesity traits and type 2 diabetes (T2D). Methods The study included 734 unrelated T2D and 884 normal glucose-tolerant (NGT) participants randomly selected from the urban component of the Chennai Urban Rural Epidemiology Study (CURES). Dietary intakes were assessed using a validated interviewer administered semi-quantitative food frequency questionnaire (FFQ). Physical activity was based upon the self-report. Interaction analyses were performed by including the interaction terms in the linear/logistic regression model. Results There was a significant interaction between SNP rs8050136 and carbohydrate intake (% energy) (Pinteraction = 0.04), where the ‘A’ allele carriers had 2.46 times increased risk of obesity than those with ‘CC’ genotype (P = 3.0 × 10−5) among individuals in the highest tertile of carbohydrate intake (% energy, 71 %). A significant interaction was also observed between SNP rs11076023 and dietary fibre intake (Pinteraction = 0.0008), where individuals with AA genotype who are in the 3rd tertile of dietary fibre intake had 1.62 cm lower waist circumference than those with ‘T’ allele carriers (P = 0.02). Furthermore, among those who were physically inactive, the ‘A’ allele carriers of the SNP rs8050136 had 1.89 times increased risk of obesity than those with ‘CC’ genotype (P = 4.0 × 10−5). Conclusions This is the first study to provide evidence for a gene-diet and gene-physical activity interaction on obesity and T2D in an Asian Indian population. Our findings suggest that the association between FTO SNPs and obesity might be influenced by carbohydrate and dietary fibre intake and physical inactivity. Further understanding of how FTO gene influences obesity and T2D through dietary and exercise interventions is warranted to advance the development of behavioral intervention and personalised lifestyle strategies, which could reduce the risk of metabolic diseases in this Asian Indian population.

Calcium Hydroxide Promotes Cementogenesis and Induces Cementoblastic Differentiation of Mesenchymal Periodontal Ligament Cells in a CEMP1-and ERK-Dependent Manner

Periodontal tissue engineering is a complex process requiring the regeneration of bone, cementum, and periodontal ligament (PDL). Since cementum regeneration is poorly understood, we used a dog model of dental pulpal necrosis and in vitro cellular wounding and mineralization assays to determine the mechanism of action of calcium hydroxide, Ca(OH)(2), in cementogenesis. Laser capture microdissection (LCM) followed by qRT-PCR were used to assay responses of periapical tissues to Ca(OH)(2) treatment. Additionally, viability, proliferation, migration, and mineralization responses of human mesenchymal PDL cells to Ca(OH)(2) were assayed. Finally, biochemical inhibitors and siRNA were used to investigate Ca(OH)(2)-mediated signaling in PDL cell differentiation. In vivo, Ca(OH)(2)-treated teeth formed a neocementum in a STRO-1- and cementum protein-1 (CEMP1)-positive cellular environment. LCM-harvested tissues adjacent to the neocementum exhibited higher mRNA levels for CEMP1, integrin-binding sialoprotein, and Runx2 than central PDL cells. In vitro, Ca(OH)(2) and CEMP1 promoted STRO-1-positive cell proliferation, migration, and wound closure. Ca(OH)(2) stimulated expression of the cementum-specific proteins CEMP1 and PTPLA/CAP in an ERK-dependent manner. Lastly, Ca(OH)(2) stimulated mineralization by CEMP1-positive cells. Blocking CEMP1 and ERK function abolished Ca(OH)(2)-induced mineralization, confirming a role for CEMP1 and ERK in the process. Ca(OH)(2) promotes cementogenesis and recruits STRO-1-positive mesenchymal PDL cells to undergo cementoblastic differentiation and mineralization via a CEMP1- and ERK-dependent pathway.

Crossover study of diets enriched with virgin olive oil, walnuts or almonds. Effects on lipids and other cardiovascular risk markers

Background and aims: Virgin olive oil (VOO) and nuts are basic components of the Mediterranean diet, a heart-healthy dietary pattern. Nuts have well known cholesterol lowering effects, while evidence is unclear for VOO. We designed a study in hypercholesterolemic patients to assess the effects on serum lipids and other intermediate markers of cardiovascular risk of replacing 40% of the fat in the background diet with VOO, walnuts or almonds. Methods and Results: After a 4 week run-in period with a healthy diet, eligible candidates were randomized into three diet sequences in a crossover design, with a common background diet enriched with VOO, walnuts or almonds, lasting 4 weeks each. Outcomes were changes of serum lipids and oxidation and inflammation markers, measured by standard methods. Plasma fatty acids were determined by gas chromatography to assess compliance. In 18 participants completing the study (9 women, mean age 56 y, BMI 25.7 kg/m(2)), LDL-cholesterol was reduced from baseline by 7.3%, 10.8% and 13.4% after the VOO, walnut and almond diets, respectively (P = 0.001, Friedman test). Total cholesterol and LDL/HDL ratios decreased in parallel. LDL-cholesterol decreases were greater than predicted from dietary fatty acid and cholesterol exchanges among diets. No changes of other lipid fractions, oxidation analytes or inflammatory biomarkers were observed. Plasma fatty acid changes after each diet sequence supported good compliance.

High intake of fruits and vegetables predicts weight loss in Brazilian overweight adults

To determine whether changes in dietary intakes predict weight loss, we studied 80 overweight adults who attended a nutritional counseling program during 6 months of follow-up at a primary health care center in Brazil. Habitual diet was assessed using a validated food frequency questionnaire at baseline and after 6 months. The mean age (+/-SD) of the participants was 46.5 +/- 9.5 years, and their mean body mass index was 29 +/- 3 kg/m(2) at baseline. After 6 months, the differences in body weight and fruit/vegetable intake were -1.4 +/- 3 kg and 109 +/- 320 g daily, respectively. Using multiple linear regression models adjusted for age, sex, changes in walking time, and total energy intake, the increased intake of dietary fiber from fruits/vegetables was associated with a greater weight loss (beta 1 [95% confidence interval (CI)] = -0.180 [-0.269, -0.0911) after 6 months of follow-up. Similar results were observed for increased intake of vegetables (beta 1 [95% CI] = -0.00497 [-0.008, -0.0021) and fruits (beta 1 [95% CI] = -0.00290 [-0.005, -0.001]) as predictors of weight loss. The increase of 100 g/d of vegetables and fruits represented a body weight loss of 500 and 300 g after 6 months, respectively (P <.05). Our findings support the relevance of increased intakes of fruits and vegetables that may help avoid weight gain in overweight adults. (C) 2008 Elsevier Inc. All rights reserved.

Under-reporting of energy intake is more prevalent in a healthy dietary pattern cluster

The aim of the present study was to determine whether under-reporting rates vary between dietary pattern Clusters. Subjects were sixty-five Brazilian women. During 3 weeks, anthropometric data were collected. total energy expenditure (TEE) was determined by the doubly labelled water method and diet Was Measured. Energy intake (El) and the daily frequency of consumption per 1000 kJ of twenty-two food groups were obtained from a FFQ. These frequencies were entered into a Cluster analysis procedure in order to obtain dietary patterns. Under-reporters were defined Lis those who did not lose more than 1 kg of body weight during the study and presented EI:TEE less than 0.82. Three dietary pattern clusters were identified and named according to their most recurrent food groups: sweet foods (SW). starchy foods (ST) and health), (H). Subjects from the healthy cluster had the lowest mean EI:TEE (SW = 0.86, ST = 0.71 and H = 0.58: P = 0.003) and EI - TEE (SW = -0.49 MJ, ST = - 3.20 MJ and H = -5.09 MJ; P = 0.008). The proportion of Under-reporters was 45.2 (95 % CI 35.5, 55.0) % in the SW Cluster: 58.3 (95 % CI 48.6, 68.0) % in the ST Cluster and 70.0 (95 % CI 61.0, 79) % in the H cluster (P=0.34). Thus, in Brazilian women, Under-reporting of El is not uniformly distributed among, dietary pattern clusters and tends to be more severe among subjects from the healthy cluster. This cluster is more consistent with both dietary guidelines and with what lay individuals usually consider `healthy eating`.

Intermittent Therapy with 1,25 Vitamin D and Calcitonin Prevents Cyclosporin-Induced Alveolar Bone Loss in Rats

Bone loss associated with cyclosporin A (CsA) therapy can result in serious morbidity to patients. Intermittent administration of 1,25 Vitamin D and calcitonin reduces osteopenia in a murine model of postmenopausal osteoporosis. The purpose of this study was to evaluate the effects of this therapeutic approach on CsA-induced alveolar bone loss in rats. Forty male Wistar rats were allocated to four experimental groups according to the treatment received during 8 weeks: (1) CsA (10 mg/kg/day, s.c.); (2) 1,25 Vitamin D (2 mu g/kg, p.o.; in weeks 1, 3, 5, and 7) plus calcitonin (2 mu g/kg, i.p.; in weeks 2, 4, 6, and 8); (3) CsA concurrently with intermittent 1,25 Vitamin D and calcitonin administration; and (4) the control treatment group (vehicle). At the end of the 8-week treatment period, serum concentrations of bone-specific alkaline phosphatase, tartrate-resistant acid phosphatase (TRAP-5b), osteocalcin, interleukin (IL)-1 beta, IL-6, and tumor necrosis factor alpha (TNF-alpha) were measured and an analysis of bone volume, bone surface, number of osteoblasts, and osteoclasts was performed. CsA administration resulted in significant alveolar bone resorption, as assessed by a lower bone volume and an increased number of osteoclasts, and increased serum bone-specific alkaline phosphatase, TRAP-5b, IL-1 beta, IL-6, and TNF-alpha concentrations. The intermittent administration of calcitriol and calcitonin prevented the CsA-induced osteopenic changes and the increased serum concentrations of TRAP-5b and inflammatory cytokines. Intermittent calcitriol/calcitonin therapy prevents CsA-induced alveolar bone loss in rats and normalizes the production of associated inflammatory mediators.

Insulin temporal sensitivity and its signaling pathway in the rat pineal gland

Aims: In our previous work, we reported that the insulin potentiating effect on melatonin synthesis is regulated by a post-transcriptional mechanism. However, the major proteins of the insulin signaling pathway (ISP) and the possible pathway component recruited on the potentiating effect of insulin had not been characterized. A second question raised was whether windows of sensitivity to insulin exist in the pineal gland due to insulin rhythmic secretion pattern. Main methods: Melatonin content from norepinephrine(NE)-synchronized pineal gland cultures was quantified by high performance liquid chromatography with electrochemical detection and arylalkylamine-N-acetyltransferase (AANAT) activity was assayed by radiometry. Immunoblotting and immunoprecipitation techniques were performed to establish the ISP proteins expression and the formation of 14-3-3: AANAT complex, respectively. Key findings: The temporal insulin susceptibility protocol revealed two periods of insulin potentiating effect, one at the beginning and another one at the end of the in vitro induced ""night"". In some Timed-insulin Stimulation (TSs), insulin also promoted a reduction on melatonin synthesis, showing its dual action in cultured pineal glands. The major ISP components, such as IR beta, IGF-1R, IRS-1, IRS-2 and PI3K(p85), as well tyrosine phosphorylation of pp85 were characterized within pineal glands. Insulin is not involved in the 14-3-3:AANAT complex formation. The blockage of PI3K by LY 294002 reduced melatonin synthesis and AANAT activity. Significance: The present study demonstrated windows of differential insulin sensitivity, a functional ISP and the PI3K-dependent insulin potentiating effect on NE-mediated melatonin synthesis, supporting the hypothesis of a crosstalk between noradrenergic and insulin pathways in the rat pineal gland. (C) 2010 Elsevier Inc. All rights reserved.

Utp25p, a nucleolar Saccharomyces cerevisiae protein, interacts with U3 snoRNP subunits and affects processing of the 35S pre-rRNA

In eukaryotes, pre-rRNA processing depends on a large number of nonribosomal trans-acting factors that form intriguingly organized complexes. Two intermediate complexes, pre-40S and pre-60S, are formed at the early stages of 35S pre-rRNA processing and give rise to the mature ribosome subunits. Each of these complexes contains specific pre-rRNAs, some ribosomal proteins and processing factors. The novel yeast protein Utp25p has previously been identified in the nucleolus, an indication that this protein could be involved in ribosome biogenesis. Here we show that Utp25p interacts with the SSU processome proteins Sas10p and Mpp10p, and affects 18S rRNA maturation. Depletion of Utp25p leads to accumulation of the pre-rRNA 35S and the aberrant rRNA 23S, and to a severe reduction in 40S ribosomal subunit levels. Our results indicate that Utp25p is a novel SSU processome subunit involved in pre-40S maturation.